Thesis Report On: "Unveiling Chemical and Pharmacological Studies To Provide New Insights On Hibiscus Macrophyllus

Thesis Report
On
“Unveiling chemical and pharmacological studies to provide
new insights on Hibiscus macrophyllus”
GONO BISHWABIDYALAY
Submitted By
Name Exam Roll Registration No.
Ila Mahjabin 2321 G.Pharma-3035/19
Maria Rahman 2322 G.Pharma-3036/19
Shazedul 2335 G.Pharma-3049/19
IslamAfroza Islam 2336 G.Pharma-3050/19
MD. Jahidul Islam 2339 G.Pharma-3053/19
Supervised by
Md. Golam Mostofa
Lecturer
Department of Pharmacy
Gono Bishwabidyalay
B.Pharm (35th Batch)
A dissertation submitted to the Department of Pharmacy, Gono
Bishwabidyalay in the partial fulfillment of the requirements for the
Award of the Degree, Bachelor of Pharmacy (Honors)
Department of Pharmacy
Gono Bishwabidyalay
Savar, Dhaka-1344.
DECLARATION BY THE CANDIDATE
We hereby declare that this dissertation entitled “Unveiling

chemical and pharmacological studies to provide new
insights on Hibiscus macrophyllus” is an authentic and genuine
research work carried out by us under the guidance of Md.
Golam Mostofa, Department of Pharmacy, Gono
Bishwabidyalay, Savar, Dhaka-1344.
Declared By
Ila Mahjabin
Exam Roll: 2321
Reg. No: G.Pharma- 3035/19
Maria Rahman
Exam Roll: 2322
Reg. No: G.Pharma -3036/19
Shazedul Islam
Exam Roll: 2335
Afroza Islam
Exam Roll: 2336
MD. Jahidul Islam

Exam Roll: 2339
i
ACKNOWLEDGMENT
All praises to Almighty Allah who give us strength, patience, and ability to
perform our research work successfully.
We would like to express our best regards, heartfelt gratefulness, deep

appreciation to our honorable, amicable, dynamic, and beloved supervisor, Md.
Golam Mostofa, Lecturer, Department of Pharmacy, Gono Bishwabidyalay for
his dedicated supervision, proficient guidance, priceless suggestions, generous
help, encouragement, and co-operation throughout the entire period of our
research work as well as to prepare this dissertation. We are indebted a lot to him
and we feel proud for giving us such an opportunity to work in close association
with him.
We would like to offer extraordinary indebtedness and gratitude to Md.

Khaleqeuzzaman, Head of the Dept., Department of Pharmacy, Gono
Bishwabidyalay, for his valuable suggestions, inspiration, and support during our
research work.
We are indeed grateful to our entire Lab Officer and Lab Assistants, Department
of Pharmacy, Gono Bishwabidyalay, for their cordial co-operation during our
research work.
Enormous thanks to the Department of Microbiology, Gono Bishwabidyalay, for

helping us with their expertise in microbial assays.
Finally, we would like to express our indebted gratitude to our parents and family
ii
members who inspired and supported us throughout our research work.
LIST OF CONTENT
CHAPTER ONE: GENERAL INTRODUCTION 1-29

1.1 Oxidative Stress (OS) 1
1.2 Free Radical 1-2
1.2.1 Classification of Free Radical 2
1.2.2 Mechanism of Formation of Free Radicals 2-3
1.2.3 Production of Free Radical in The Human Body 3-4
1.2.4 Mechanism of Disease Formation by Free 4
Radicals
1.3 Leading Cause of Death 4-5
1.4 Oxidative Stress and Human Disease 5-6
1.4.1 Oxidative Stress and Cancer 6
1.5 Cancer 6-7
1.5.1 Causes of Cancer 7-8
1.5.2 Oxidative Stress Develops Cancer 8-9
1.6 Oxidative Stress and Cardiovascular Disease 9-10
1.7 Oxidative Stress and Diabetes 10-11
1.8 Oxidative Stress prevention and treatment 11-12
1.9 Preventing of oxidative stress 13
1.10 Antioxidant 13
1.10.1 Types of Antioxidants 13-14
1.10.2 Antioxidant Defense System 14
1.10.3 Mechanism of action of Antioxidants 14-15
1.10.4 Plant source of Antioxidant 15-16
1.10.5 Benefits of Natural Antioxidants 16-18
1.10.6 Antioxidants and Cancer 18-19
1.11 Plants as a source of drugs in Cancer Therapy 19-20
1.12 Description of Plant Investigated 21
1.12.1 The Plant Family: Malvaceae 21-24
1.13 Objective 24-25
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1.14 Present Study Protocol 25-26
1.15 Reference 26-29
CHAPTER TWO: LITERATURE REVIEW 30-72
2.1 Chemical work 30
2.1.1 Alkaloids 30-31
2.1.2 Amino acids 31
2.1.3 Anthocyanin 31
2.1.4 Organic Acids 31-32
2.1.5 Amide 32
2.1.6 Anthraquinones 32
2.1.7 Carbohydrates 32
2.1.8 Cardiac glycosides 33
2.1.9 Coumarins 33
2.1.10 Diterpenes 33
2.1.11 Flavonoiods 33-34
2.1.12 Glycosides 34
2.1.13 Gums 34
2.1.14 Mucilage 35
2.1.15 Phenolic compounds 35
2.1.16 Phytosterols 35-36
2.1.17 Pigments 36
2.1.18 Proteins 36
2.1.19 Quinones 36
2.1.20 Reducing sugar 36
2.1.21 Saponins 36-37
2.1.22 Steroid 37
2.1.23 Tannins 37-38
2.1.24 Terpenoids 38
2.1.25 Triterpenoids 39
2.2 Pharmacological Activities 39
2.2.1 Anti-inflammatory activities 39-40
2.2.2 Anti-diarrheal activities 40-41
2.2.3 Analgesic Activity 41
2.2.4 CNS activity 42
2.2.5 Antibacterial activity 42-45
2.2.6 Antioxidant activity 46-49
2.2.7 Antifungal activity 49
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2.2.8 Anti-cancerous activity 49-50
2.2.9 Effect on lipid metabolism 50-51
2.2.10 Hepato-protective activity 51-52
2.2.11 Haemato-toxicity 52
2.2.12 Anti-diabetic activity 52-53
2.2.13 Anti-hypertensive activity 53
2.2.14 Anthelmintic activity 53
2.3 Reference 53-72
CHAPTER THEREE: METHODS AND MATERIALS 73-99
3.1 Methods and Materials 73
3.1.1 General Methods 73
3.1.2 Collection and Proper Identification of Plant 73
3.1.3 Preparation of Plant Materials 73
3.1.4 Extraction 73
3.1.5 Cold Extraction 73-74
3.1.6 Hot Extraction 74
3.2 Chemical Studies on H. macrophyllus 74
3.2.1 Collection and Identification of Plant 74-75
3.2.2 Preparation of Plants Barks 75
3.2.3 Extraction of Plants Barks 76
3.3 Qualitative Phytochemical Analysis 77
3.3.1 Test for Alkaloids 77
3.3.2 Test for Glycosides 77
3.3.3 Test for Saponins 77
3.3.4 Test for Steroids 77
3.3.5 Test for Tannins 77
3.4 Quantitative Phytochemical Analysis 78
3.4.1.1 Determination of Total Flavonoids 78
3.4.1.2 Principle 78
3.4.1.3 Materials and Apparatus 78
3.4.1.4 Experimental Procedure 78-79
3.4.2.1 Determination of Total Phenolics 79
3.4.2.4 Experimental Procedure 80
3.5 In-Vitro Antioxidant Activity 80
3.5.1.1 Determination of Total Antioxidant Capacity 80
v
3.5.2.1 Reducing Power Assessment 81
3.5.2.2 Principle 81-82
3.5.3.1 DPPH Free Radical Scavenging Assay 83
3.6 In-Vivo Biological Activity 84
3.6.1 Evaluation of Cytotoxic Activity by Brine 84
Shrimp Lethality Bioassay
3.6.1.1 Principle 84-85
3.6.1.2 Materials and Apparatus 85-86
3.6.1.3 Preparation of Brine Water 86
3.6.1.4 Hatching of Brine Shrimp Eggs 86
3.6.1.5 Preparation of the Test Sample 86
3.6.1.6 Preparation of Control Groups 86
3.6.1.7 Preparation of the Positive Control Group 87
3.6.1.8 Preparation of the Negative Control Group 87
3.6.1.9 Application of Brine Shrimp Nauplii 87
3.6.1.10 Counting of Nauplii 87
3.6.1.11 Analysis of Data 88
3.6.2 Antibacterial Screening 88-89
3.6.2.1 Principle of Disc Diffusion Assay Method 89
3.6.2.2.1 Test Materials Used for the Study 90
3.6.2.2.2 Materials and Apparatus 90
3.6.2.2.3 Test Organisms 90-91
3.6.2.3 Sterilization Procedure 91
3.6.2.4 Culture Media 91-92
3.6.2.5 Preparation of Medium 92
3.6.2.6 Preparation of Subculture 92
3.6.2.7 Preparation of Test Plates 92-93
3.6.2.8 Preparation of Discs and Test Samples 93
3.6.2.9 Preparation of Discs, Diffusion and Incubation 93-94
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3.6.2.10 Measurement of Zone of Inhibition 94
3.6.3 Evaluation of Anthelmintic Activity 94
3.6.3.1 Materials and Apparatus 94-95
3.6.4 Thrombolytic Activity of Plant Extracts 96
3.7 Reference 97-99
CHAPTER FOUR: RESULTS 100-123
4.1 Chemical Studies 100
4.1.1 Preparation of Crude Extract 100
4.1.2 Qualitative Phytochemical Analysis 100
4.3 Quantitative Analysis 101
4.3.1 Determination of Total Phenolics 101-103
4.3.2 Determination of Total Flavonoids 103-105
4.4 In-vitro Antioxidant Activity 105
4.4.1 Total Antioxidant Activity 105-108
4.4.2 Reducing Power Assessment 108-111
4.4.3 DPPH Free Radical Scavenging Assay 111-114
4.5 Evaluation of Cytotoxic Activity by Brine Shrimp Lethality 114-117
Bioassay
4.6 Evaluation of Anthelmintic Activity 117-121
4.7 In-Vivo Antibacterial Activity 121
4.7.1 Determination of Zone of Inhibition 121-122
4.8 Evaluation of Thrombolytic Activity 122-123
CHAPTER FIVE: DISCUSSION 125
5.1 Discussion 125
5.1.1 Qualitative Phytochemical Analysis 125
5.1.2 Phenolic and Flavonoid Contents 126
5.1.3 Total Antioxidant Capacity 126-127
5.1.4 Reducing Power Assessment 127
5.1.5 DPPH Free Radical Scavenging Activity 127-128
5.1.6 Cytotoxic Effect on Brine Shrimp 128
5.1.7 Evaluation of Anthelmintic 129
5.1.8 Antibacterial Screening 129
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5.1.9 Thrombolytic Activity 129-130
CHAPTER SIX: SUMMAR 133
6.1 Summary 133
ABBREVIATION
FDA Food And Drug Administration
AA Ascorbic Acid
GAE Gallic Acid Equivalent
CME Cold Methanolic Extract
HME Hot Methanolic Extract
CHF Chloroform Fraction
AQF Aqueous Fraction
EAF Ethyl Acetate Fraction
BHT Butylate Hydroxy Toluene
DPPH 1,1- diphenyl-2-picrylhydrazyl
Conc. Concentration
IC50 Half Maximal Inhibitory Concentration
FCR Folin-ciocalteu Reagent
FeCl3 Ferric Chloride
FeSO4 Ferrous Sulphate
GA Gallic Acid
KCl Potassium Chloride
K2HPO4 Dipotassium Hydrogen Phosphate
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KH2PO4 Potassium Dihydrogen Phosphate
H. macrophyllus Hibiscus macrophyllus
RT Room Temperature
NaCl Sodium chloride
ºC Degree Celsius
STD Standard Deviation
UV Ultraviolet
µg Microgram
ml Milliliter
mM Millimolar
μL Micro Liter
Abstract
Plant is the great source of supplement and drugs to cure many life-threatening diseases. The
goal of the current study was to unveil the chemical and pharmacological activity of Hibiscus
macrophyllus (H. macrophyllus) barks. For this purpose, the barks of H. macrophyllus were
identified, collected, dried, ground into coarse powder, and then extracted with methanol using
cold extraction method and hot extraction method to produce distinct extractives, CME and
HME, respectively.
Using a conventional UV-spectroscopic approach, the flavonoids and polyphenols content as

well as the antioxidant activity of the various extractives were assessed. The cytotoxic effect of
H. macrophyllus was evaluated by brine shrimp lethality bioassay. The thrombolytic activity in
terms of in vitro clot lysis was performed
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Polyphenols and flavonoids are the good source of antioxidant. At a concentration of 25µg/ml,
the phenolic contents of CME and HME were found to be 27.48 and 29.01 mg GAE per gm of
dried sample, respectively. At a concentration of 100µg/ml, the flavonoid and phenolic contents
of CME and HME were found to be 19.96 and 35.53 mg QE per gm of dried sample,
respectively. The extracts also demonstrated the presence of alkaloids, glycosides, saponins,
steroids, and tannins.
Both HME and CME demonstrated the highest levels of DPPH radical scavenging activity, with
IC50 values of 12 and 31 µg/ml for DPPH, respectively, compared to 15 µg/ml for standard BHT
in DPPH. According to the IC50 value, the potency of the examined extractives can be summed
up as HME> CME for DPPH. In case of total antioxidant activity, the HME and CME showed
the following order: AA > CME > HME. On the other hand, ferric reducing capacity of HME
and CME showed the following order: AA > CME > HME by considering the DPPH radical
scavenging activity, total antioxidant activity and ferric reducing capacity, CME possessed more
antioxidant compounds than HME.
The brine shrimp lethality test was also used to assess the cytotoxic potential of H. macrophyllus.
For CME and HME, respectively, the LD50 values for all the extracts were 151.46 and 200
µg/ml. Based on the LD50, the extracts are in the following order: VCS > CME > HME.
Atherothrombotic diseases are serious consequences of the thrombus fromed in the blood vessels
which can be dissolved by thrombolytic agents. The HME and CME of H. macrophyllus
displayed the thrombolytic activity on human blood. HME and CME both possessed
thrombolytic activity which was concentration dependant.The percentage of clot lysis of HME
and CME were 25.5 and 24.84, respectively at concentration, 0.5 mg/ml where that of negative
control was 1.3.
No extract of barks of H. macrophyllus had antibacterial action against both the tested Gram-
positive and Gram-negative bacterial strains at 100 µg/disc, 200 µg/disc and 400 µg/disc.
For anthelmintic activity test, all extracts of H. macrophyllus were also taken into consideration.
x
At 20 mg/ml, 50 mg/ml and 100 mg/ml, both extracts demonstrated outstanding anthelmintic
activity against earth worms. For this test, Albendazole used as the standard. The time of
paralysis of Albendazole, HME and CME were 4.55, 2.21 and 3.05 minutes, respectively where
the time of death were 6.39, 4.05 and 5.17 minutes, respectively at concentration, 100 mg/ml.
HME and CME had more activity than Albendazole.
Taken all the results together, our findings suggest that H. macrophyllus barks is a good source
of antioxidant, cytotoxic, thrombolytic and antiworm compounds. Therefore, use of H.
macrophyllus barks would be a potential candidate for the prevention and treatment of cancer,
Atherothrombotic diseases and parasitic disorders.
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Chapter One (General Introduction)
IiInIntroduction)
1. General Introduction
1.1 Oxidative Stress (OS)
OS is essentially an imbalance between the body's capacity to neutralize or detoxify the

damaging effects of free radicals through the action of antioxidants and the generation of free
radicals (Subramaniam et al., 2020). Because oxygen free radicals destroy biological molecules
like lipids, proteins, and DNA, OS is bad. Through the generation of reactive oxygen species
(ROS), such as peroxides and free radicals that harm all cell components, disturbances in the
normal redox state of tissues can have harmful effects. The high amounts of ROS produce
necrosis by lipid peroxidation, DNA oxidation, or protein oxidation. This damage results in ATP
depletion, which prevents regulated apoptosis and forces the cell to just disintegrate (Evans et al.,
2015). However, OS also plays a crucial part in the control of intracellular signal transduction
and physiologic adaptability (Yoshikawa & Naito, 2002).
1.2 Free Radical
Any molecular species that has an unpaired electron in an atomic orbital and is capable of
existing independently is referred to as a free radical. Most radicals share a few basic
characteristics that are caused by the presence of an unpaired electron. Numerous radicals are
very reactive and insecure. They can act as oxidants or reductants since they can either donate an
electron to or take one from other molecules (Cheeseman & Slater, 1993). In various disease
conditions, the hydroxyl radical, superoxide anion radical, hydrogen peroxide, oxygen singlet,
hypochlorite, nitric oxide radical, and peroxynitrite radical are the most significant oxygen-
containing free radicals.
These are extremely reactive substances that can damage biologically important components like
DNA, proteins, carbohydrates, and lipids in cell membranes and the nucleus (Young &
Woodside, 2001). Important macromolecules are attacked by radicals, which damages cells and
disturbs homeostasis. Free radicals can attack every type of molecule in the body. Lipids, nucleic
acids, and proteins are the main targets among them. Such typical alterations with aging are
1
IiInIntroduction)
pretty common to everybody, and free radical responses are expected to create progressive
detrimental changes that build with age throughout the body. However, patterns driven by
heredity and environmental variables that control free radical damage are superimposed on this
universal pattern. At specific ages defined by genetic and environmental factors, these appear as
illnesses.
Two leading causes of death are cancer and atherosclerosis, both of which are prominent "free
radical" diseases (Lobo et al., 2010)
1.2.1 Classification of Free Radical
According to the classification proposed, the radicals may be divided into
I. Primary (superoxide, semi quinones and nitric oxide),
II. Secondary (hydroxyl and lipid radicals) and
III. Tertiary (radicals of antioxidants).
The primary radicals are formed by enzymatic systems and perform biologically important
functions. The secondary radicals are formed from hydroperoxides in the reactions of divalent
iron ions and damage to cell structures. (Vladimirov, 1998)
1.2.2 Mechanism of Formation of Free Radicals
Free radicals are the natural byproducts of chemical processes, such as metabolism. Food,
medicine, air, water can generate free radicals. Free radicals can be formed by three ways-
❖ By hemolytic cleavage of covalent bond of normal molecule with each fragment

retaining one paired of electrons
X:Y X* + Y*
❖ By the loss of single electron from normal molecule
X:Y X+ + Y-
2
IiInIntroduction)
❖ By addition of single electron to normal molecule
X:Y X-`
A radical might donate its unpaired electron to other molecules. It might take electron from other
molecules in order to pair or it might simply join to the molecule and thus proceed as a chain
reaction and produce a free radical.
1.2.3 Production of Free Radical in the Human Body
Free radicals and other reactive oxygen species (ROS) are produced by the body's normal, vital
metabolic processes or by external factors like exposure to Xrays, ozone, tobacco smoke, air poll
utants, and industrial chemicals. Both enzymatic and nonenzymatic reactions result in the consta
nt formation of free radicals within the cells.
Free radicals are produced by enzymatic processes such as those in the respiratory chain, phagoc
ytosis, prostaglandin synthesis, and the cytochrome P450 system (Liu et al., 1999; Lobo et al., 20
10).Both ionizing reactions and nonenzymatic reactions involving oxygen and organic compoun
ds can result in the formation of free radicals.
Several internal sources of free radicals include
 Mitochondria
 Xanthine oxidase
 Peroxisomes
 Inflammation
 Phagocytosis
 Exercise
 Ischemia/reperfusion injury
 Some externally generated sources of free radicals are:
 Cigarette smoke
 Environmental pollutants
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IiInIntroduction)
 Radiation
 Certain drugs, pesticides
 Industrial solvents
 Ozone (Lobo et al., 2010
1.2.4 Mechanism of Disease Formation by Free Radicals
Fig.1.1: Mechanism of disease formation by free radicals & other chemicals. (Modification of
Sharma 2014)
1.3 Leading Cause of Death

WHO estimates that 56 million people worldwide would pass away from various diseases in
2019. The first main cause of death among them is CVD. According to estimates, 17.9 million
deaths worldwide in 2019 were attributable to CVDs, or 32% of all fatalities. Heart attack and
stroke deaths accounted for 85% of these fatalities. The majority of CVD fatalities occur in low-
and middle-income nations. According to GBD, the number of common cases of all CVD nearly
doubled from 271 million in 1990 to 523 million in 2019. (Roth et al., 2020). 10.8 million People
died from cancer, which is the second largest cause of mortality, according to GLOBOCAN.
With an anticipated 1.8 million fatalities (18%), lung cancer continued to be the most common
4
IiInIntroduction)
type of cancer. Colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast (6.9%)
cancers were the next most common types of cancer. In 2040, 28.4 million new cases of cancer
are anticipated worldwide, a 47% increase from 2020. (Sung et al., 2021). 3.97 million People
have respiratory diseases, 2.49 million have lower respiratory infections, 1.55 million have
diabetes, and 1.2 million die from Alzheimer's disease per year (Naghavi et al., 2017). 8.7
million People die prematurely each year due to tobacco smoking, according to the Global
Burden of Disease research. These projections, which apply to fatalities in the year 2019 as of
June 2021, are the most recent ones. 7.7 million of those fatalities are related to smoking, and 1.3
million of those deaths are caused by secondhand smoke exposure in non-smokers. The two
leading causes of mortality in Bangladesh are cancer and respiratory disorders, which together
account for nearly 25% of all fatalities. According to the Bangladesh Department of Statistics, a
total of 8, 54,253 persons passed away in 2020 from a variety of reasons, with cardiac arrest
accounting for about 21.1% of those deaths (BBS). According to information provided by the
BBS (Heart Attacks the Most Frequent Cause of Death in Bangladesh Last Year: BBS | The
Daily Star, 2021), 1, 80,408 persons have died from heart attacks. According to VA, ischemic
heart disease is the main factor of adult male deaths in Bangladesh, accounting for roughly 50%
of deaths.
More fatalities than the next two causes, chronic respiratory illness and stroke. Ischemic heart
disease, stroke, and chronic respiratory illness account for a combined 62% and 60% of all adult
mortality in men and women, respectively. In the top 15 causes for both sexes, diabetes, chronic
renal disease, cirrhosis, and pneumonia are all listed. The top 15 leading causes of death for men
include traffic accidents (3%), lung cancer (3%), prostate cancer (2%), esophageal cancer (1%),
and tuberculosis (TB) (1%), but not for women, who are more at risk from falls (2%),
diarrhea/dysentery (2%), maternal (2%), breast cancer (2%), and cervical cancer (2%) (Shawon
et al., 2021). Diabetes is one of the leading causes of mortality in Bangladesh, where it is thought
to be responsible for 3% of all fatalities. It's interesting to note that Bangladesh had a sharp rise
in the number of diabetes cases in the beginning of the twenty-first century, which prompted
health officials to devise strategies to slow the disease's spread. One of Bangladesh's top health
concerns right now is diabetes (The 10 Leading Causes of Death in Bangladesh - WorldAtlas,
2018.).
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1.4 Oxidative Stress and Human Disease

Cancers, atherosclerosis, inflammatory conditions, aging, hemochromatosis, AIDS, emphysema,
organ transplantation, gastric ulcers, hypertension, preeclampsia, neurological disorders
(Alzheimer's disease, Parkinson's disease, muscular dystrophy), and neurological disorders
(Alzheimer's disease, Parkinson's disease, muscular dystrophy) have all been linked to oxidative
stress as life-threatening conditions (Lobo et al., 2010).
1.4.1 Oxidative Stress and Cancer

In vivo, a variety of reactive species (RS) are produced, many of which are potent oxidizers that
can harm DNA and other macromolecules. The increased risk of cancer development in older
people may be explained by the 'normal' rates of RS generation. Greater RS formation can
promote the development of malignancy. In fact, deletion of several antioxidant defense enzymes
increases oxidative damage levels and aids in the development of age-related cancer in animals.
The primary focus of this explanation has been on the direct oxidative harm that particular RS,
such the hydroxyl radical (OH•), cause to DNA. Yet, malignancy does not always result from
elevated amounts of DNA base oxidation products such (8-hydroxy-2′-deoxyguanosine), even
though malignant tumors frequently have elevated levels of DNA base oxidation. Hence, RS's
additional actions—possibly including those that affect p53, cell proliferation, invasiveness, and
metastasis—must also be significant. Malignancy is predisposed to by chronic inflammation,
although the role of RS in this is likely complex because RS can occasionally function as anti-
inflammatory drugs (Barry Halliwell, 2007).
1.5 Cancer
Cancer is the second-leading cause of death in the world. Accounting for nearly 10 million
deaths in 2020 (International Agency for Research on Cancer; 2020). According to GLOBOCAN
2020 over 19.3 million new cases of cancer will be diagnosed annually and will be died over
10.0 million a year (Sung et al., 2021). Cancer is the malignant growth due to uncontrolled cell
division which can invade nearby parts of the body as well as spread distant parts of the body
(known as metastasis) through blood stream (López-Lázaro, 2018). It is now used as a general
6
IiInIntroduction)
term for over a hundred diseases characterized by the uncontrolled, abnormal growth of cells. It
is also known as malignant neoplasia (Magrath, 1989). Breast cancer (2.26 million new cases),
lung (2.21 million), colon and rectum (1.93 million), prostate (1.41 million), non-melanoma skin
(1.20 million), and stomach cancer (1.20 million) were the most prevalent cancers in 2020. (1.09
million). Lung (1.80 million), colon and rectum (0.91 million), liver (0.83 million), stomach
(0.77 million), and breast (0.77 million) were the cancers that killed the most people in 2020.
(0.68 million). At some point in their lives, about 39.5% of men and women will receive a cancer
diagnosis (based on 2015–2017 data). According to estimates, 16,850 kids and teenagers
between the ages of 0 and 19 will be diagnosed with cancer in 2020, and 1,730 of them will pass
away from it (International Agency for Research on Cancer). According to predictions, there will
be an estimated 11.4 million cancer deaths worldwide in 2030. The most prevalent cancers in
Bangladesh are cervical, breast, and esophageal cancers. Nonetheless, the most common causes
of cancer-related death are esophageal, lung, and pharyngeal malignancies. Cancer cases are
predicted to increase, from 136,719 in 2015 to 250,726 in 2035.
Fig.1.2: Trends in the incidence of New Cancer Case in Bangladesh: 2015- 2035 (Source:
Cancer on the Global Stage: Incidence and Cancer-Related Mortality in Bangladesh - The
ASCO Post)
1.5.1Causes of Cancer
Cancer is a complicated set of diseases with numerous potential causes, including environmental
variables like ultraviolet light, pollution, and hepatitis B as well as chemicals that cause cancer,
known as carcinogens, such as tobacco smoke, chemotherapy, asbestos, benzene, and vinyl
7
IiInIntroduction)
chloride. The world's leading preventable cause of cancer is smoking (William C. Shiel Jr.,
2017). After smoking, being overweight and obese is the second-leading avoidable cause of
cancer. About 5-10% of cancer occurrences can be linked to genetic abnormalities, whereas the
rest 90–95% have their roots in the environment and way of life (Hahn & Weinberg, 2002).
(Czene & Hemminki, 2002). Cigarette smoking, alcohol consumption, diet (fried foods, red
meat), sun exposure, exposure to pollutants from the environment, infections, oxidative stress,
obesity, and inactivity are among the lifestyle factors. According to the data, nearly 25–30% of
all cancer-related deaths are linked to tobacco use, 30–35% to diet, 15% to infections like
Helicobacter pylori, hepatitis B, hepatitis C, human papillomavirus infection, Epstein-Barr virus,
and HIV, and the remaining percentage is linked to other factors like radiation, stress, physical
activity, environmental pollutants, etc (Anand et al., 2008).
Fig.1.3: Causes of cancer

Source: What are the main causes of cancer? (researchgate.net)
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1.5.2 Oxidative Stress Develops Cancer
As oxidative stress has been linked to the onset and progression of cancer by increasing DNA
damage, genomic variability, and cell proliferation, antioxidants may be able to affect
carcinogenesis (Mileo & Miccadei, 2016).
Fig.1.4: Mechanism of development of Cancer by OS
Source:
https://www.researchgate.net/publication/40696720_Oxidative_Stress_and_Oxidat
ive_Damage_in_Carcinogenesis.
1.6 Oxidative Stress and Cardiovascular Disease
A class of diseases known as cardiovascular disease (CVD) affects the heart or blood vessels.
Coronary artery disease (CAD) such as angina and myocardial infarction is a kind of CVD
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(commonly known as a heart attack). Other CVDs consist of Cardiomyopathy, abnormal,
abnormal, heart disease, peripheral artery disease, thromboembolic disease, venous thrombosis,
stroke, heart failure, hypertensive heart disease, rheumatic heart disease, and (Wang et al., 2016).
Across the entire world, cardiovascular diseases (CVDs) are the main cause of death. According
to estimates, 17.9 million deaths worldwide in 2019 were attributable to CVDs, or 32% of all
fatalities. Heart attack and stroke deaths accounted for 85% of these fatalities. The majority of
CVD fatalities occur in low- and middle-income nations. In 2019, non-communicable illnesses
caused 17 million premature deaths (before the age of 70), and 38% of those fatalities were
attributable to CVDs. By addressing behavioral risk factors like tobacco use, unhealthy eating
and obesity, inactivity and problematic alcohol consumption, the majority of cardiovascular
illnesses can be avoided (Mileo & Miccadei, 2016).
1.7 Oxidative Stress and Diabetes

Hyperglycemia, a condition in which blood glucose levels are too high, and inadequate insulin
production or action, both occur in people with diabetes mellitus, a set of metabolic illnesses
(Maritim et al., 2003). A protein (hormone) called insulin is produced by the beta cells of the
pancreas in response to a variety of stimuli, including glucose, sulphonyl urea, and arginine, but
glucose is the primary regulator (Joshi et al., 2007). Long-term blood glucose increase is linked
to macro- and microvascular problems that can include renal disease, heart disease, stroke, and
blindness. In addition to hyperglycemia, oxidative stress and hyperlipidemia also play significant
roles in the etiology of diabetes and increase the risk of complications. The human body is
constantly exposed to various substances that produce reactive species known as free radicals
(ROS/RNS), which by The oxidation of cellular machinery is brought on by the transfer of their
free unpaired electron. The body possesses endogenous antioxidant systems or it acquires
exogenous antioxidants from the diet to combat the harmful effects of such species. These
antioxidants neutralize the harmful species and maintain the body's homeostasis. Any imbalance
between RS and antioxidants results in "oxidative stress," a situation that leads to the emergence
of pathological conditions, one of which is diabetes.
The majority of research suggests that oxidative stress plays a role in the etiology of diabetes
through changes in enzymatic systems, lipid peroxidation, poor glutathione metabolism, and
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decreased levels of vitamin C. Several biomarkers of oxidative stress in diabetes mellitus include
lipids, proteins, DNA damage, glutathione, catalane, and superoxide dismutase. Diabetes
problems brought on by oxidative stress may include stroke, neuropathy, retinopathy, and
nephropathy (Asmat et al., 2016). The anticipated prevalence of diabetes worldwide in 2019 is
9.3% (463 million people), and it is expected to increase to 10.2% (578 million) by 2030 and
10.9% (700 million) by 2045. Urban places (10.8%) have a higher incidence than rural ones
(7.2%), while high-income countries (10.4%) have a higher prevalence than low-income ones
(4.0%). One in two (50.1%) diabetics are unaware that they have the disease. Impairment in
glucose tolerance is predicted to affect 7.5% (374 million) people worldwide in 2019 and 8.0%
(454 million) people by 2030 and 8.6% (548 million) people by 2045. (Saeedi et al., 2019).
1.8 Oxidative Stress Prevention and Treatment
The leading global cause of illness and mortality is cardiovascular disease. Cardiovascular
illnesses are caused by cellular aberrations, pathological circumstances, redox dysregulation, and
a dyshomeostasis of inflammation. (33) There is still no safe and efficient approach for their
prevention or treatment after years of continuous research. Recently, molecular hydrogen has
been studied in both preclinical and clinical studies on a variety of diseases linked to oxidative
and inflammatory stress, including radiation-induced heart disease, ischemia-reperfusion injury,
myocardial and brain infarction, heart storage, heart transplantation, etc.
Inhalation, drinking hydrogen-rich water and injection of hydrogen-rich saline are the three main
ways that hydrogen is delivered. Pro-inflammatory cytokines, excessive ROS generation, and the
activation of the antioxidant transcription factor Nrf2 are all suppressed as a result of the
positively modulated signal transmission and gene expression. The precise methods of action of
H2, despite the fact that it appears to be a significant biological molecule with anti-oxidant, anti-
inflammatory, and anti-apoptotic actions, are yet unknown. Although no clinical harm has been
observed, some evidence points to H2 having a minor hormetic-like impact, which probably
mediates some of its advantages. The molecular information, along with the pre-clinical and
clinical trials, point to H2 as a potential treatment for disorders like ROS/inflammation-induced
cardiotoxicity.
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Fig. 1.5: Production of ROS
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Fig. 1.6: Mechanisms of molecular hydrogen action in condition of increased oxidative stress.
1.9 Preventing of Oxidative stress
Increasing levels of endogenous and exogenous antioxidants, reducing exposure to

environmental contaminants with oxidizing qualities, or decreasing the creation of oxidative
stress through stabilizing mitochondrial energy production and efficiency are three ways to
reduce oxidative stress.
There are two approaches to affect endogenous oxidative stress: by preventing the generation of
ROS or by quenching ROS with antioxidants (Poljsak, 2011).
1.10 Antioxidant
An antioxidant is a molecule that is stable enough to give an electron to an out-of-control free

radical and neutralize it, so lowering the radical's potential for harm. These antioxidants' primary
ability to scavenge free radicals helps them delay or reduce cellular damage (B. Halliwell, 1995;
Kebede & Admassu, 2019). These antioxidants with low molecular weight can safely interact
with free radicals to stop the chain reaction before important molecules are harmed. Some of
these antioxidants, like glutathione and uric acid, are created by the body's regular metabolic
processes (Kumar, 2014; Shi et al., 1999). The diet also contains some softer antioxidants.
Although the body has multiple enzyme systems that scavenge free radicals, vitamin E (-
tocopherol), vitamin C (ascorbic acid), and beta-carotene are the main micronutrient (vitamin)
antioxidants (Balakrishnan et al., 2014; Levine et al., 1999). These micronutrients must be
obtained through diet because the body cannot produce them (Kebede & Admassu, 2019).
1.10.1 Types of Anti-oxidant

There are two types of antioxidants in the human body that is enzymatic and non-enzymatic
antioxidant. These are as follows
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Fig. 1.7: Types of antioxidants
1.10.2 Antioxidant Defense System
In addition to scavenging free radicals, antioxidants also function as hydrogen donors, electron
donors, peroxide decomposers, quenchers of singlet oxygen, enzyme inhibitors, synergists and
metal-chelating agents. In the intracellular and extracellular environment, antioxidants that are
both enzymatic and non-enzymatic are present to detoxify ROS (Lobo et al., 2010).
1.10.3 Mechanism of Action of Antioxidants
For antioxidants, two main modes of action have been proposed (Rice- Evans & Diplock, 1993).
The first process breaks a chain by giving a free radical present in the system an electron that is
donated by the main antioxidant. The second process includes quenching a chain-initiating
catalyst to remove ROS/RNS initiators (secondary antioxidants). Co-antioxidants, electron
donation, metal ion chelation, or gene expression regulation are just a few of the ways that
antioxidants can affect biological systems (Krinsky, 1992).
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Fig. 1.8: Mechanism of action of antioxidants (Modification of Sharma 2014)
1.10.4 Plant Source of Antioxidant

Foods and medicines, especially those containing oils and fats, frequently use synthetic and
natural dietary antioxidants to prevent oxidation. Several synthetic phenolic antioxidants exist,
including butylated hydroxytoluene. As two notable instances, butylated hydroxytoluene (BHT)
and butylated hydroxyanisole (BHA) (Lobo et al., 2010).
Antioxidants are abundant in plant-based meals. They are most prevalent in fruits, vegetables,
whole grains, nuts, and some types of meat, chicken, and fish.
Good sources of specific antioxidants include:
 Allium sulfur compounds – leeks, onions and garlic

 Anthocyanins – eggplant, grapes and berries
 Beta-carotene – pumpkin, mangoes, apricots, carrots, spinach and parsley
 Catechins – red wine and tea
 Copper – seafood, lean meat, milk and nuts
 Cryptoxanthins – red capsicum, pumpkin and mangoes
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 Flavonoids – tea, green tea, citrus fruits, red wine, onion and apples
 Indoles – cruciferous vegetables such as broccoli, cabbage and cauliflower
 Isoflavonoids – soybeans, tofu, lentils, peas and milk
 Lignans – sesame seeds, bran, whole grains and vegetables
 Lutein – green, leafy vegetables like spinach, and corn
 Lycopene – tomatoes, pink grapefruit and watermelon
 Manganese – seafood, lean meat, milk and nuts
 Polyphenols – thyme and oregano
 Selenium – seafood, offal, lean meat and whole grains
 Vitamin A – liver, sweet potatoes, carrots, milk, and egg yolks
 Vitamin C – oranges, blackcurrants, kiwifruit, mangoes, broccoli, spinach, capsicum
and strawberries
 Vitamin E – vegetable oils (such as wheatgerm oil), avocados, nuts, seeds and whole
grains
 Zinc – seafood, lean meat, milk and nuts
 Zoochemical – red meat, offal and fish. Also derived from the plants that animals eat.
1.10.5 Benefits of Natural Antioxidants

Consumers are increasingly searching for innovative, healthier, and higher-quality solutions as
eating habits change and develop. This indicates that people are becoming more conscious of the
substances in the food they consume and more accustomed to terms like preservatives, additives,
and antioxidants. They try to eat healthier every day in order to maintain their health
As a result of society's demand for higher quality in their daily dietary intake, natural
antioxidants have surpassed synthetic ones in this context and within the field of food additives.
Also, synthetic antioxidants are facing an increasing number of issues, making it advantageous to
switch to natural antioxidants.
Yet even so, why pick natural antioxidants to keep dietary lipids stable? What distinguishes them
from synthetic ones and what makes them superior? Despite the fact that the idea of natural
antioxidants is inherently different, we will outline some of the ways in which these additions
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improve food production.
There are nine benefits of using natural antioxidants when producing food for humans.
1. They work incredibly well as antioxidants: The primary justification is its superior antioxidant
capacity. The oxidation of fats can be delayed by several natural antioxidants using various
techniques, such as oxygen extinction, free radical reduction, or antioxidant regeneration, often
with better outcomes than synthetic antioxidants.
2. They better support food production processes and protect the finished product more: Natural
antioxidants are less volatile and more stable at high temperatures due to their composition and
chemical properties, which means that they support food production processes like frying,
cooking, and baking better and protect the finished product more. Carry Through is the term for
the ability to "survive" the thermal process and remain in the finished product.
The fact that natural antioxidants are frequently more soluble than synthetic antioxidants is
another advantage of using them. Due to the fact that an antioxidant can only stop the
autoxidation process. It's crucial to make sure that its dispersion is as uniform as feasible in the
lipid medium.
3. There are soluble in oil and soluble in water: On the other hand, there are natural antioxidants
that are soluble in oil and soluble in water, meaning that there are various solutions based on the
manufacturing process and the product to which they will be added. Moreover, prepackaged
solutions with ingredients that, for instance, enable the conversion of a liposoluble antioxidant
into a water-dispersible substance are readily available on the market.
4. They are legal everywhere in the world: Another benefit of natural antioxidants is that they are
not subject to the same restrictions or bans on use as synthetic antioxidants that have been set by
regulatory bodies like the FDA or EFSA.
5. They have no impact on the color, flavor, or odor of the finished product: It is important to
note that natural antioxidants like tocopherols and ascorbic acid, when used in low amounts,
have no impact on the color, flavor, or odor of the finished product.
6. Another characteristic that sets natural antioxidants apart is the fact that some of them, like
tocopherols, are acceptable for use in organic foods. This is because they are only used in small
dosages, which ensures that they stay within the parameters set for this kind of permitted product
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in other natural substances.
7. Certain combinations are more effective than they are alone: Moreover, different
combinations of natural antioxidants are frequently found on the market. Synergism is the
process through which two or more antioxidants operate better together than the quantitative
equivalent of any one of them working alone. The combination of tocopherols with rosemary
extract is one example of this.
8. Because they are healthier and more natural, they bring value: Finally, consumer demand has
increased. A natural, balanced diet is becoming more and more in demand as society shifts to
healthier lifestyle choices. As opposed to synthetic antioxidants, the usage of these antioxidants
in food production adds value.
1.10.6 Antioxidant and Cancer

Antioxidants have the ability to scavenge free radicals created by OS and donate electrons to
them. Excessive free radical production occurs during OS-induced carcinogenesis. These free
radicals rob electrons from DNA, mutating the molecule. A lot of malignancies are brought on
by aberrant proteins, which this mutation may produce. Antioxidants lessen the OS-induced
carcinogenesis by giving or sharing their extra electrons with free radicals. By inhibiting the free
radicals that damage DNA, -carotene's antioxidant action may help prevent cancer. Thus, -
carotene's photoprotective qualities may offer defense against UV light-induced carcinogenesis.
Immuno-enhancement of -carotene may aid in the prevention of cancer. By changing how
carcinogens are metabolized by the liver, -carotene may also have anti-carcinogenic effects (Van
Poppel & Goldbohm, 1995).
Vitamin C may aid in the prevention of cancer (Glatthaar et al., 1986). Antioxidant effects,
inhibiting the development of nitrosamines, enhancing the immunological response, and
speeding up the detoxification of liver enzymes are some of the potential mechanisms through
which vitamin C may promote carcinogenesis. By enhancing humoral antibody defense,
bacterial infection resistance, cell-mediated immunity, the production of T-lymphocytes' m-
necrosis factor, inhibiting the formation of mutagens, repairing DNA membranes, and
preventing the development of micro cell lines, vitamin E, a crucial antioxidant, contributes to
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immune competence (Sokol, 1988). As a result, vitamin E may be helpful in the fight against
cancer as it suppresses carcinogenesis by boosting the immune system. The combination of the
three aforementioned antioxidants revealed the greatest decrease in the risk of developing
cardiac cancer (Ashok & Ali, 1999).
Fig.1.9: Mechanism of free radical scavenging by antioxidants.
1.11 Plants as a Source of Drugs in Cancer Therapy

The primary natural supply of medications for many ailments, including cancer, is plant life. The
following list includes a few chemicals that could make good anticancer medication candidates.
Table 1.1: List of some anticancer drugs obtained from plants
Drug/Chemical Action Plant Source

Camptothecin Anticancerous Camptotheca acuminate
Colchiceine amide Colchicum autumnale
Antitumor agent
(Autumn crocus)
Colchicum autumnale
Demecolcine Antitumor agent (Autumn crocus)
Podophyllum peltatum
Etoposide Antitumor agent (mayapple)
Anticancer, antitumor Camptotheca acuminate
Irinotecan agent
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Tabebuia species
Lapachol Anticancer, antitumor
(Trumpet tree)
Monocrotaline Topical antitumor agent Crotalaria sessiliflora

Antitumor, anticancer Podophyllum peltatum
Podophyllotoxin agent (mayapple)
Taxus brevifolia (Pacific yew)
Taxol Antitumor agent
Podophyllum peltatum
Teniposide Antitumor agent
(mayapple)
Antitumor, anticancer Camptotheca acuminate
Topotecan agent
Antitumor, Catharanthus roseus
Vinblastine Antileukemic agent (Madagascar periwinkle)
Antitumor, Catharanthus roseus
Vincristine Antileukemic agent (Madagascar periwinkle)
Seven plant-derived medications for the treatment of cancer were recently approved by the Food
and Drug Administration (FDA). Following are comprehensive descriptions of various
medications:
Nowadays, Taxol/Paclitaxel, a substance derived from the Taxus brevifolia, is the medicine of
choice for treating a variety of tumorous malignancies, including breast cancer.
During the 1950s, the use of the chemical vinblastine, which is derived from the Catharanthus
roseus plant, has raised the survival rate of childhood leukemias by 80%.
Another anti-leukemic medication derived from Catharanthus roseus is vincristine.
Topotecan: Topotecan is a plant alkaloid analog derived from Camptotheca acuminate that has
been given FDA approval for the treatment of small cell lung cancer and ovarian cancer. At
present, it is under investigation clinically for the treatment of several types of cancer by alone or
combination with other anticancer drugs.
Irinotecan is a chemically similar plant alkaloid that can be found in the Camptotheca acuminata
plant. The FDA has authorized it for the treatments of Trial trials are now being conducted for a
number of malignancies, including metastatic colorectal cancer.
Etoposide: Epipodophyllotoxin, a plant chemical derived from Podophyllum peltatum, is the
source of etoposide, a semi-synthetic derivative.
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Teniposide: A semi-synthetic derivative of a substance derived from Podophyllum peltatum is
teniposide.
The aforementioned evidence has shown that plants are an important source of possible cancer
inhibitors.
1.12 Description of Plant Investigated

1.12.1 The Plant Family: Malvaceae
Malvaceae, or the mallows, is a family of flowering plants estimated to contain 244 genera with

4225 known species of herbs, shrubs and trees, distributed primarily tropical areas in the world.
Well-known members of economic importance include okra, cotton, cacao and durian. There are
also some general containing familiar ornamentals, such as Alcea (hollyhock), Malva (mallow),
and Tilia (lime or linden tree). The largest genre a in terms of number of species
include Hibiscus (300 species), Sterculia (250 species), Dombeya (250 species), Pavonia (200
species) and Sida (200 species).
Botanical Features of Hibiscus macrophyllus:
Scientific Name: Hibiscus macrophyllus
Common Name: Khasiaudal, Kashipala.
Vernacular Name: Largeleaf rosemallow
Synonymous Name:
 Hibiscus barbatus Noronha
 Hibiscus setosus Roxb.
 Hibiscus vestitus Griff.
 Hibiscus vulpinus Reinw. ex Blume
 Pariti macrophyllum (Roxb. ex Hornem.) G.Don
 Talipariti macrophyllum (Roxb. ex Hornem.) Fryxell
 Triplochiton spathacea Alef
Scientific Classification:
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Kingdom: Plantae
Division: Tracheophyta
Subdivision: Angiosperms
Class: Eudicops
Superorder: Rosids
Order: Malvales
Suborder: Hibisceae
Family: Malvaceae
Subfamily: Malvoideae
Genus: Hibiscus
Species: Hibiscus macrophyllus.
(Hibiscus macrophyllus. Species, 2022)
Habit: Tree
Habitat : Hill Top
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Figure: Hibiscus macrophyllus
Habit: Tree
Habitat: Hill top
Shape and Size: 3 to 15 meters tall
Plant Part: Leaves, Flower, Fruits, Bark, Seed.
Leaves: Leaf blades broadly ovate, 5-40 cm long, 4.5-50 cm wide, basally deeply cordate, the
margin crenulate to subentire, apically abruptly acuminate, palmately 7-9-nerved, discolorous,
coarsely tomentose, more densely so and yellowish beneath, with a nectary on each principal
vein beneath positioned 1/3-2/3 the distance from base to apex, the nectary often obscured by
pubescence; petioles 17-35 cm long, with pubescence like stem (especially basally); stipules 3-
11 cm long, 1.5-3 cm wide, ovate to scimitar-shaped, sessile and amplexicaul, hirsute (especially
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externally), deciduous, leaving annular scars.
Pedicel: Pedicels axillary, solitary (or sometimes in 2-flowered sympodia), presented
horizontally, 1-5 cm long, densely hirsute, the subtending leaves reduced to 6-8 cm long;
involucel ca. 2.5 cm long, the 8-10 elements nearly distinct, lanceolate or ligulate, ca. 3 mm
wide, yellowish hirsute: calyx 2.5-3 cm long, slightly exceeding the involucel, more than half-
divided, the 5 lobes each 3-nerved, less densely hirsute than involucel, nectaries absent; petals 4-
6 cm long, 2-4 cm wide, yellow with dark red basal spot (fading purplish), glabrous within,
externally hirsutulous; staminal column 3-4 cm long, glabrous, yellowish, filamentiferous
throughout, the filaments 2-8 mm long; anthers yellow, 1.5 mm long; style exceeding staminal
column by ca. 8 mm, dividing into 5 purplish red and pilose branches, the stigmas 1.5 mm wide
(apparently clavate), minutely pilose.
Capsule: Capsules erect, obovoid, 2.5-3.5 cm long, 18-20 mm in diameter, 5-locular, apiculate
or beaked, shaggy-hirsute;
Flowers: Multi-flowered cymes, 30 cm long, 7-9 cm wide and spiny pollen. The flowers are
commonly borne in definite or indefinite axillary inflorescences, which are often reduced to a
single flower, but may also be cauliflorous, oppositifolious, or terminal. They often bear
supernumerary bracts in the structure of a bicolor unit. They can be unisexual or bisexual, and
are generally actinomorphic, often associated with conspicuous bracts, forming an epicalyx.
They generally have five valvate sepals, most frequently basally connate, with five
imbricate petals. The stamens are five to numerous, and connate at least at their bases, but often
forming a tube around the pistils. The pistils are composed of two to many connate carpels.
The ovary is superior, with axial placentation, with capitate or lobed stigma. The flowers
have nectaries made of many tightly packed glandular hairs, usually positioned on the sepals.
Fruits: Capsule oblong, 2-3 cm wide, densely scabrous hirsute. Schizocarps or nuts.
Barks: Gray-white bark.
Seeds: Numerous, reniform, 4 mm long, with 3 mm ferruginous hairs along edges.
Distribution:
Global Distribution:
India (Assam), Bangladesh, Indo-China, Thailand, Peninsular Malaysia, Sumatra, Java and
Borneo (Kalimantan, Sabah); B
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Flowering and Fruiting: March to May
Cultivation:
It is found in humid, semi-arid and arid habitats, the principal absence being from the polar,
tundra and taiga zones. (Alpine species are confined to the Andes.) Species of Malvaceae range
from ephemeral herbs to tall rain-forest trees.
Propagation: Seed
Uses:
Hibiscus is used;
 For treating loss of appetite,
 Colds,
 Heart and nerve diseases,
 Upper respiratory tract pain and swelling (inflammation),
 Fluid retention, stomach irritation, and disorders of circulation;
 For dissolving phlegm; as a gentle laxative; and as a diuretic to increase urine output.
 Preventing renal stone formation, as well as its respiratory and sedative effects.
 Used in the preparation of jams, jellies, and cold and warm teas and drinks.
1.13 Objective
Chronic disorders include cancer, cardiovascular diseases, diabetes, cancer, and neurological
diseases all have oxidative stress as a major contributor to their etiology. Long-term exposure to
pro-oxidant substances at elevated levels can lead to structural flaws in mitochondrial DNA as
well as functional changes in a number of enzymes and cellular components, which can lead to
abnormalities in gene expression. The current way of life, which includes processed foods,
exposure to numerous chemicals, and inactivity all contribute significantly to the development of
oxidative stress (Sharifi-Rad et al., 2020).
Reactive oxygen species may contribute to carcinogenesis in OS by upregulating oncogenes
(such as Bcr-Abl, BCL-2, RAS, Myc, etc.) and downregulating tumor suppressor genes (e.g.,
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BRCA1, BRCA2, Rb, p53, etc.).
If the cellular antioxidant defense mechanism is compromised, these genes' alterations in
expression become more pronounced (Chow 2010).
Due to the inclusion of phytochemicals including polyphenols, carotenoids, vitamin E, and
vitamin C, botanical supplements with increased antioxidants can provide protection by reducing
OS (Zhang et al., 2015).
Plants have historically been very rich sources of bioactive chemicals and provide medicines for
the treatment and prevention of numerous illnesses, including cancer. Along with its long-
standing usage in cancer care, a number of novel plant-derived substances, including vinblastine,
vincristine, etoposide, teniposide, taxol, Taxotere, topotecan, etc., have recently received
approval for use as cancer treatments.
Although these medications can help with cancer, they cannot cure it. They are also exceedingly
expensive and have serious side effects that can be fatal. Also, the majority of cancer patients in
our nation are unable to afford the costs of this treatment. This has rekindled interest in the
search for a novel anticancer medication derived from plants that has little harmful effects on
healthy cells. In order to discover the chemicals from the bioactive component of H.
macrophyllus barks, this study was created.
1.14 Present Study Protocol

The present study focused on the chemical and biological investigations of barks of Hibiscus
macrophyllus which includes-
1. Collection and identification of barks of Hibiscus macrophyllus from Madabkunda Eco Park,
Shylet.
2. Dried and crushed into a fine powder of barks of H. macrophyllus.
3. Chemical investigation:
 Cold and Hot extraction of barks with methanol.
 Qualitative phytochemical analysis of the extract was performed by Lead acetate
test, Frothing test, Libermann-Burchard’s test, Keller- Killani Test, Color test.
• Determination of total phenolic and flavonoid contents
1. Biological investigation:
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 Determination of DPPH radical scavenging activity.
 Determination of total antioxidant activity.
 Determination of ferric reducing antioxidant capacity.
 Evaluation of in vitro cytotoxic effect on brine shrimp nauplii.
 Determination of anthelminthic activity.
 Determination of thrombolytic activity.
 Antibacterial study.
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Chapter Two (Literature Review)
2. Literature Review
2.1 Chemical Work
The genus Hibiscus belongs to the mallow family, Malvaceae comprising of about 275 species
growing in tropical and sub-tropical areas. The various species of genus Hibiscus have been used
as traditional medicine all over the world. There are numerous reports of their traditional
medicinal uses in various countries like Bangladesh, India, Nigeria, China, and Sri Lankan etc. to
cure various ailments such as hypertension, cardiac diseases, stomach-ache, urine problems, skin
diseases and many more. Based on the historical knowledge, various pharmacological and
phytochemical studies on some species of the genus Hibiscus have been done. Nevertheless,
there are no up-to-date articles published which can provide an overview of pharmacological
effects of the genus Hibiscus. Therefore, the main objective of the review article is to provide a
systematic comprehensive summary of traditional uses, phytochemistry and pharmacology and
toxicology of the genus Hibiscus and to build up a correlation between its traditional
ethnobotanical uses and pharmacological activities so as to find some advanced research
opportunities in this field. The given information on the ethnobotanical uses, phytoconstituents
and various medicinal properties of the genus Hibiscus was gathered from the online scientific
databases through search in Google, Google Scholar, ScienceDirect, NCBI, PubMed, Springer
Link, and Research Gate by using some keywords as. Besides these websites other sources such
as published literature and unpublished ongoing thesis and dissertation were also consulted.
Previously conducted research revealed that the genus contains good amount of
phytoconstituents such as antioxidants, phytosterols, saponins, lignin, essential oils, glycosides,
and anthocyanins etc. Presence of these bioactive compounds in the crude extracts of the plants
make it suitable for various medicinal properties like anti-cancer, anti-inflammatory, anti-
diabetic, anti-obesity, anti-ulcer, hypersensitive, hypolipidemic, hepatoprotective,
nephroprotective and many more.
2.1.1 Alkaloids
Abdul-Awal et al., 2016 confirmed the presence of alkaloids in the ethanolic extract of barks of
H. tiliaceus.
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Bindu Ch et al., 2019 showed the presence of alkaloids in the methanolic extract of H. hirtus
leaves.
Cn et al., 2015 revealed the presence of alkaloids in the ethanolic and methanolic extract of
H. sabdariffa.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of alkaloids in the methanolic extract
of leaves of H. radiatus.
N et al., 2015 displayed the presence of alkaloids in the ethanolic extract of leaves and stem of
H. hispidissimus Griffith.
Olivia et al., 2021 reported the presence of alkaloids in the methanolic extract of H. asper leaves.
2.1.2 Amino acids
Bindu Ch et al., 2019 showed the presence of amino acids in the methanolic extract of H. hirtus
leaves.
Enemali Shaibu & Koma Okwute, 2021 expressed the presence of amino acids in the methanolic
extract of leaves of H. radiatus.
S. Patel & Adhav, 2016 revealed the presence of amino acid in the ethanolic extract of
flowers and leaves of H. rosa-sinensis Linn.
2.1.3 Anthocyanin
Amrhein & Frank, 2017; Barve et al., 2010; Ishikura, 2014 showed that the presence of
anthocyanin in the aqueous, methanol and chloroform extract of red petals, petals and leaves of
H. mutabilis respectively.
Adhirajan et al., 2003; Ajay et al., 2007; Bhakta & Das, 2017; Gauthaman et al., 2006 confirmed
the presence of anthocyanin in the aqueous and chloroform extract of leaves, aerial part, flowers
and calyces of H. rosa sinensis respectively.
Hida et al., 2007; Williamson et al., 2013 revealed the presence of anthocyanin in the aqueous
and methanolic extract of calyces and flowers of H. sabdariffa respectively.
Lowry, 1976; Shimokawa et al., 2015 reported the presence of anthocyanin in the aqueous and
methanolic extract of flowers and petals of H. tilliaceus respectively.
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2.1.4 Organic acids
Da-Costa-Rocha et al., 2014; Eggensperger & Wilker, 1996; Lin et al., 2012 exhibited the
presence of organic acids in the aqueous extract of calyces, leaves and seeds of H. sabdariffa
respectively.
Kobayashi, 1976; Y. N. Singh et al., 1984; Whistler, 1985 showed the presence of organic acids
in the aqueous and methanolic extract of calyces, leaves and flowers of H. tilliaceus respectively.
Chen et al., 2006; Wang et al., 2011 displayed the presence of organic acids in the aqueous
extract of leaves and wood of H. tilliaceus respectively.
2.1.5 Amide
Chen et al., 2006 showed that the presence of amide in the aqueous extract of wood of H.
tilliaceus.
2.1.6 Anthraquinones
N et al., 2015 reported the presence of anthraquinones in the ethanolic extract of flower bud of
S. Patel & Adhav, 2016 showed the presence of anthraquinones in the ethanolic extract of
flowers and leaves of H. rosa-sinensis Linn.
Tiwari et al., 2015 expressed the presence of anthraquinones in the ethanolic extract of whole
plants of H. rosa-sinensis Linn.
2.1.7 Carbohydrates
Babu & Lakshmana, 2018 confirmed the presence of carbohydrates in the chloroform and
ethanolic extracts of the whole plant of H. platanifolius.
Bindu Ch et al., 2019 revealed the presence of carbohydrates in the methanolic extract of H.
hirtus leaves.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of carbohydrates in the methanolic
Priyanka et al., 2022 displayed the presence of carbohydrate in the ethanolic extract of leaves of
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H. hirtus L.
S. Patel & Adhav, 2016 reported the presence of carbohydrates in the ethanolic extract of flowers
and leaves of H. rosa-sinensis Linn.
2.1.8 Cardiac glycosides
N et al., 2015 showed the presence of cardiac glycosides in the ethanolic extract of leaf, stem and
flower bud of H. hispidissimus Griffith.
2.1.9 Coumarins
Manikandan S and Asha B, 2019 revealed the presence of coumarins in the aqueous and
ethanolic extracts of H. cannabinus leaves.
N et al., 2015 showed the presence of coumarins in the ethanolic extract of H. hispidissimus
Griffith leaves.
2.1.10 Diterpenes
N et al., 2015 reported the presence of diterpenes in the ethanolic extract of leaves, stem and
flower bud of H. Hispidissimus Griffith.
2.1.11 Flavonoids
Bindu Ch et al., 2019 showed the presence of flavonoids in the methanolic extract of H. hirtus
leaves.
Babu & Lakshmana, 2018 confirmed the presence of flavonoids in the chloroform extract,
ethanolic extract and methanolic extract of the whole plant of H. platanifolius.
Cn et al., 2015 revealed the presence of flavonoid in the ethanolic extract, methanolic extract of
samples of H. sabdariffa.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of flavonoids in the methanol
Manikandan S and Asha B, 2019 revealed the presence of flavonoids in the water and ethanolic
extracts of H. cannabinus leaves.
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Mekar Saptarini et al., 2016 showed the presence of flavonoid in the ethanolic extract of leaves
of H. hirtus L. respectively.
Nishitha et al., 2018 showed the presence of flavonoids in the ethyl acetate extract of fresh
flowers of H. vitifolius.
N et al., 2015 showed the presence of flavonoids in the ethanolic extracts of the stem and flower
bud of H. hispidissimus Griffith.
Olivia et al., 2021 identified the presence of flavonoids of methanol fraction of H. asper leaves.
S. Patel & Adhav, 2016 showed the presence of flavonoids in the ethanolic extract of flowers and
leaves off H. rosa-sinensis Linn.
Rathi et al., 2003 confirmed the presence of flavonoids in the ethanolic extract of roots of H.
aculeatus.
Umachigi et al., 2014 revealed the presence of flavonoids in the methanolic extract of H.
Syriacus leaves.
2.1.12 Glycosides
Bindu Ch et al., 2019 showed the presence of glycosides in the methanolic extract of H. hirtus
leaves.
Cn et al., 2015 revealed the presence of glycoside in the distilled water, ethanol extract, methanol
extract, petroleum ether extract, ethyl acetate extract of H. sabdariffa respectively.
N et al., 2015 showed the presence of glycosides in the ethanolic extract of leaf, stem, flower bud
of H. Hispidissimus Griffith.
Priyanka et al., 2022 showed the presence of glycosides in the ethanolic extract of leaves of H.
hirtus L. respectively.
Olivia et al., 2021 identified the presence of glycosides of methanol fraction of H. asper leaves.
S. Patel & Adhav, 2016 showed the presence of glycosides in the ethanolic extract of flowers and
2.1.13 Gums
Abdul-Awal et al., 2016 confirmed the presence of gums in the ethanolic extract of barks of H.
tiliaceus.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of gums in the methanol extract of
34
leaves of H. radiatus.
S. Patel & Adhav, 2016 showed the presence of Gums in the ethanolic extract of flowers and
2.1.14 Mucilage
Enemali Shaibu & Koma Okwute, 2021 showed the presence of mucilage in the methanol extract
S. Patel & Adhav, 2016 showed the presence of mucilage in the ethanolic extract of flowers and
2.1.15 Phenolic Compounds
Bindu Ch et al., 2019 showed the presence of phenolic compounds in the methanolic extract of
H. hirtus leaves.
Cn et al., 2015 revealed the presence of phenol in the distilled water, methanol extract of samples
of H. sabdariffa.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of Phenolic compounds in the
methanol extract of leaves of H. radiatus.
Manikandan S and Asha B, 2019 revealed the presence of phenols in the water, ethanol,
chloroform, ethyl acetate, petroleum ether extracts of H. cannabinus leaves.
Nishitha et al., 2018 showed the presence of phenolic compounds in the ethyl acetate extract of
fresh flowers of H. Vitifolius.
N et al., 2015 representing the presence of phenolic compounds in the ethanolic extract of stem
of H. hispidissimus Griffith.
Olivia et al., 2021 identified the presence of phenols of methanol fraction of H. asper leaves.
S. Patel & Adhav, 2016 showed the presence of phenolic compounds in the ethanolic extract of
flowers and leaves off H. rosa sinensis Linn.
Umachigi et al., 2014 revealed the presence of phenols in the methanolic extract of H. syriacus
leaves.
Vilela et al., 2018 detected the phenolic compounds of ethanolic extracts of H. aceuteatus leaves.
35
2.1.16 Phytosterols
Enemali Shaibu & Koma Okwute, 2021 showed the presence of phytosterols in the methanol
N et al., 2015 showed the presence of phytosterols in the ethanolic extract of stem and flower
S. Patel & Adhav, 2016 showed the presence of phytosterols in the ethanolic extract of flowers
and leaves off H. rosa-sinensis Linn.
2.1.17 Pigments
N et al., 2015 representing the presence of various pigments like chlorophyll a and b in the
ethanolic extract of stem of H. hispidissimus Griffith.
2.1.18 Proteins
Bindu Ch et al., 2019 showed the presence of proteins in the methanolic extract of H. hirtus
leaves.
S. Patel & Adhav, 2016 showed the presence of protein in the ethanolic extract of flowers and
2.1.19 Quinones
Manikandan S and Asha B, 2019 revealed the presence of Quinones in the water, ethanol,
chloroform, ethyl acetate and petroleum ether extracts of H. cannabinus leaves respectively.
N et al., 2015 showed the presence of Quinones in the ethanolic extract of leaf of H.
hispidissimus Griffith.
2.1.20 Reducing Sugar
Abdul-Awal et al., 2016 confirmed the presence of reducing sugars in the ethanolic extract of
barks of H. tiliaceus.
Abdul-Awal et al., 2016 confirmed the presence of reducing sugars in the ethanolic extract of
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barks of H. tiliaceus.
2.1.21 Saponins
Cn et al., 2015 revealed the presence of saponin in the distilled water, ethanol extract, petroleum
ether extract, ethyl acetate extract of samples of H. sabdariffa respectively.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of saponins in the methanol extract
Manikandan S and Asha B, 2019 revealed the presence of saponins in the ethanol and petroleum
ether extracts of H. cannabinus leaves.
N et al., 2015 showed the presence of saponins by the foam test in the ethanolic extract of leaf of
Olivia et al., 2021 identified the presence of saponins from methanol fraction of H. asper leaves.
Rathi et al., 2003 confirmed the presence of saponins in the ethanolic extract of roots of H.
aculeatus.
2.1.22 Steroid
Bindu Ch et al., 2019 showed the presence of steroids in the methanolic extract of H. hirtus
leaves.
Manikandan S and Asha B, 2019 revealed the presence of steroids in the ethanolic extracts of H.
cannabinus leaves.
Nishitha et al., 2018 showed the presence of steroids in the ethyl acetate extract of fresh flowers
of H. vitifolius.
N et al., 2015 showed the presence of steroids in the ethanolic extract of stem of H.
Priyanka et al., 2022 confirmed the presence of steroid in the ethanolic extract of leaves of H.
hirtus L. respectively.
Olivia et al., 2021 identified the presence of steroids from the GC-MS analysis of methanol
fraction of H. asper leaves.
Rathi et al., 2003 confirmed the presence of Steroids in the ethanolic extract of roots of H.
aculeatus.
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2.1.23 Tannins
Abdul-Awal et al., 2016 confirmed the presence of tannins in the ethanolic extract of barks and
leaves of H. tiliaceus.
Bindu Ch et al., 2019 showed the presence of tannins in the methanolic extract of H. hirtus
leaves.
Cn et al., 2015 revealed the presence of tannin in the petroleum ether extract, ethyl acetate
extract of samples of H. sabdariffa.
Enemali Shaibu & Koma Okwute, 2021 showed the presence of tannins in the methanol extract
Manikandan S and Asha B, 2019 revealed the presence of tannins in the water, ethanol,
chloroform and petroleum ether extracts of H. cannabinus leaves.
Nishitha et al., 2018 showed the presence of tannins in the Ethyl acetate extract of fresh flowers
of H. vitifolius.
N et al., 2015 showed the presence of tannins in the ethanolic extract of leaf, stem and flower
Olivia et al., 2021 identified the presence of tannins from the GC-MS analysis of methanol
Priyanka et al., 2022 confirmed the presence of tannin in the ethanolic extract of H. hirtus L.
respectively.
S. Patel & Adhav, 2016 showed the presence of tannins in the ethanolic extract of flowers and
Rathi et al., 2003 confirmed the presence of tannins in the ethanolic extract of roots of H.
aculeatus.
2.1.24 Terpenoids
Enemali Shaibu & Koma Okwute, 2021 showed the presence of terpenoid in the methanol
Manikandan S and Asha B, 2019 revealed the presence of terpenoids in the ethanolic extracts of
38
H. cannabinus leaves.
N et al., 2015 showed the presence of terpenoids in the ethanolic extract of stem and flower bud
of H. hispidissimus Griffith.
Olivia et al., 2021 identified the presence of terpenoids from the GC-MS analysis of methanol
S. Patel & Adhav, 2016 showed the presence of terpenoids in the ethanolic extract of flowers
and leaves off H. rosa-sinensis Linn.
Rathi et al., 2003 confirmed the presence of terpenoids in the ethanolic extract of roots of H.
aculeatus.
2.1.25 Triterpenoids
Babu & Lakshmana, 2018 confirmed the presence of triterpenoids ethanolic extract of the whole
plant of H. platanifolius.
Nishitha et al., 2018 showed the presence of triterpenoids in the ethyl acetate extract of fresh
flowers of H. vitifolius.
N et al., 2015 showed the presence of triterpenoids in the ethanolic extract of leaf and stem of H.
2.2 Pharmacological activities
Besides being eye-catching morphologically, pharmacological activities of Hibiscus are also

great source of attraction. The plant shows antibacterial, anti-fungal, anti-inflammatory, anti-
cancerous, anti-hyperepidemic, anti-glycemic activities along with various other health related
benefits like effect on lipid metabolism, anti-hypertensive effects, effects on Hai growth and
anti-analgesic activities.
2.2.1 Anti-inflammatory activities
al Faruq et al., 2018 confirmed that the leaves extracts of H. surattensis possesses significant
anti-inflammatory (mild to moderate) activity.
39
Awad et al., 2014 reported that Petroleum ether extract of H. sabdarriffa seeds have significant
anti-inflammatory activities in acute and chronic anti-inflammatory models.
Begum et al., 2018 reported that ethanolic extract of roots of H. rosa sinensis Linn has
significant anti-inflammatory effects on SD rats.
Bhangale et al., 2015 reported that the ethyl acetate extract of H. cannabinus Linn seed has anti-
inflammatory activity.
Boré et al., 1993 reported that the methanolic extract of H. rosa- sinensis leaves showed
significant anti-inflammatory activity on rats.
Chan et al., 2016 reported that the methanol, petroleum ether, and chloroform leaf extracts of H.
tiliaceus possess anti-inflammatory activity.
Foyet, Abdou, et al., 2011 reported that the aqueous and methanolic extracts of H. asper Hook. f.
Leaves possess significant anti-inflammatory activity.
M. K. Ali et al., 2011 reported that the ethanolic calyx extract of H. sabdariffa Linn. showed the
presence of Antinociceptive and anti-inflammatory activity in mice.
Meraiyebu et al., 2013 reported that the Methanolic Extract of H. sabdariffa showed the presence
of significant anti-inflammatory activity on adult Wister rat.
Park et al., 2022 reported that the young leaves of H. syriacus possessed anti-inflammatory
effect.
Prabhakaran et al., 2019 reported that the ethyl acetate extract of the flower H. vitifolius L. had
anti-inflammatory activity.
Priyanka et al., 2022 reported that the ethanolic extracts of H. hirtus L. leaves had strong anti-
inflammatory activities.
Raduan et al., 2013 reported that the ethanolic extract of flower and leaf of H. rosa-sinensis var
alba (white Hibiscus) and H. rosa-sinensis L. (red Hibiscus) possessed significant anti-
inflammatory activity.
Shaibu & Okwute, 2021 revealed that the methanolic extract of H. radiatus leaves possessed
significant anti-inflammatory activity.
Shaikh et al., 2016 reported that the methanolic and aqueous extract of H. cannabinus leaves
showed significant anti-inflammatory activity.
Tambe & Bhambar, 2014 reported that the petroleum ether, chloroform, ethyl acetate and ethanol
extracts of leaves of H. cannabinus Linn possessed significant anti-inflammatory effects.
40
2.2.2 Anti-diarrhoeal activities
al Faruq et al., 2018 revealed that the leaf extracts of H. surattensis possesses significant anti-
diarrheal activity.
M. K. Ali et al., 2011 reported that the ethanolic calyx extract of H. sabdariffa Linn showed the
presence of antidiarrheal activity in mice.
Ateufack et al., 2014 confirmed that the aqueous and methanolic extracts of H. asper leave
possess significant antidiarrheal activity.
Babu et al., 2022 expressed that the ethanolic extract of whole plant of H. platanifolius possesses
antidiarrheal activity.
2.2.3. Analgesic activity
Abdul-Awal et al., 2016 reported that the ethanol extract of leaf and bark of H. tiliaceus
possesses analgesic activity.
M. K. Ali et al., 2011 confirmed that the ethanolic calyx extract of H. sabdariffa Linn. showed
the presence of Antinociceptive activity in Mice.
al Faruq et al., 2018 revealed that the leaf extracts of H. surattensis possesses significant
analgesic activity.
Awad et al., 2014 displayed that Petroleum ether extract of H. sabdarriffa seeds have significant
analgesic activity.
Begum et al., 2018 revealed that ethanolic extract of roots of H. rosa sinensis Linn possess
remarkable analgesic effects.
Bhangale et al., 2015 showed that the ethyl acetate extract of H. cannabinus Linn seed has
central and peripheral analgesic activity.
Boré et al., 1993 expressed that the methanolic extract of H. rosa- sinensis leaves showed
significant analgesic activity on rats.
Chan et al., 2016 exhibited that the methanol, petroleum ether, and chloroform leaf extracts of H.
tiliaceus possesses analgesic activity.
Ghogare et al., 2007 reported that the petroleum ether, ethyl acetate, and methanol extract of H.
41
mutabilis bark showed the significant analgesic activity.

Patil et al., 2016 confirmed that the aqueous extract of leaves of H. sabdariffa in albino rats
showed the presence of significant analgesic activity.
Sawarkar et al., 2009 revealed the Aqueous and alcoholic extracts of H. rosa-sinensis Linn
produced significant analgesic activity of the leaves of the plant.
Shaibu & Okwute, 2021 showed that the methanolic extract of H. radiatus leaves possesses
significant analgesic activity.
Tambe & Bhambar, 2014 reported that the petroleum ether, chloroform, ethyl acetate and ethanol
extracts of leaves of H. cannabinus possess well analgesic effects.
2.2.4. CNS activity
Abdul-Awal et al., 2016 reported that the ethanol extract of leaf and bark of H. tiliaceus possess
CNS activity.
Amos et al., 2003 showed that the aqueous Extract of H. sabdariffa showed the presence of
sedative in nature with possible neuroleptic properties.
Begum & Younus, 2018 displayed that the ethanol extract of H. rosa sinensis roots shows CNS
activity.
Foyet, Hritcu, et al., 2011 expressed that the methanolic extract of H. asper leaves showed the
presence of significant anxiolytic and antidepressant activity.
(Girme, 2013) exhibited that the petroleum ether, ethyl acetate and methanol extracts of H.
mutabilis bark has good CNS depressant activity.
Shewale et al., 2012 revealed that the methanolic extract of H. rosa-sinensis flowers possess
potential antidepressant (CNS) activity.
2.2.5. Anti-bacterial activity
Abdallah, 2016 reported that the methanol extract of H. Sabdariffa Calyces shows antibacterial
activity against Escherichia coli, Salmonella enteric, Klebsiella pneumonia, Proteus vulgaris,
and Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus
42
cereus respectively.
Abdul-Awal et al., 2016 showed that the ethanolic extract of H. tiliaceus Leaves and bark shows
antibacterial activity against Staphylococcus aureus, S. epidermidis, S. saprophyticus, S.
pyogenes, Plesiomonas shigelloides, Shigella dysenteriae, S. flexneri, S. boydii, S. sonnei,
Pseudomonas aeruginosa, Vibrio cholera, and Salmonella typhi respectively.
Adamu & Ngwu, 2015 displayed that the methanolic extract of H. Sabdariffa leaves shows
antibacterial activity against S. typhi, E. coli and S. aureus respectively.
Adebisi & Ojokoh, 2011 reported that the methanol, water extract of H. sabdariffa green and red
calyx shows antibacterial activity against Bacillus cereus, Streptococcus faecalis, Clostridium
sporogeneses, Micrococcus luteus, E. coli Pseudomonas aeruginosa, Klebsiella pneumonia,
Serrita marcessens, Proteus vulgaris and Proteus rettgeri respectively.
Andriani et al., 2017 expressed that the methanol and chloroform, methanol and ethyl acetate
fractions extract of H. tiliaceus fruits, leaves and twigs shows antibacterial activity against
Pseudomonasa erouginosa respectively.
Ajoku et al., 2015 reported that the Hexane, Ethyl acetate and Methanol extract of H. Sabdariffa
Calyx shows antibacterial activity against E. coli, S. aureus, Pseudomonas aeroginosa,
Salmonella typhi, and Bacillus subtilis respectively.
Boadi, 2014 exhibited that the petroleum ether, ethyl-acetate, methanol and water extract of H.
Sabdariffa Calyx shows antibacterial activity against Staphylococcus aureus, Bacillus subtilis,
Klebsiella pneumonia and Escherichia coli respectively.
Chan et al., 2016 presented that the methanolic and ethanolic extract of H. tiliaceus leaves
showed a well antibacterial activities against Bacillus cereus, Micrococcus luteus,
Staphylococcus aureus, and S. aureus, E. coli and Salmonella paratyphi respectively.
Edema & Alaga, 2012 reported that the aqueous and ethanolic extract of H. sabdariffa L. Calyces
shows antibacterial activity against E. coli, Staphylococcu saureus, Salmonella typhi, Shigella
dysenteries, and Streptococcus mutans.
Fullerton et al., 2011 displayed that the aqueous methanol extract of H. Sabdariffa calycers
shows antibacterial activity against E. Coli.
Garbi et al., 2012 reported that the methanolic extract of H. Sabdariffa calyx shows antibacterial
activity against Cornybacterium diptheria, S. aureus, Enterococcus faeclis, Listeria
monocytogenes, B. cereus, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa,
43
Serratia marcescens, E. coli and Klebsiella.

Hajera Khatun et al., 2011 expressed that the methanol extract of H. Sabdariffa fruits shows
antibacterial activity against Sarcina lutea, Shigella dysenteriae, E. coli, Shigella boydii,
Bacillus subtilis, B. megaterium, B. anthracis, B. cereus and P. Aeruginosa.
Jung et al., 2013 revealed that the water and ethanolic extract of H. Sabdariffa Calyces shows
antibacterial activity against Bacillus subtilis, S. aureus and E. Coli.
Kehinde Nathaniel & E, 2015 reported that the Aqueous ethanol, hexane and methanol extract of
H. Sabdariffa Seed coats shows antibacterial activity against E. coli, S. aureus, S. pneumoniae,
K. aerogenes, S. species and P. aeruginosa.
Khairul Bariyyah et al., 2019 displayed that the methanolic extract of H. Sabdariffa flower
shows antibacterial activity against Aeromonas hydrophilia.
Linn. et al., 2015 expressed that the aqueous and hydro ethanol extract of H. Sabdariffa Calyx
shows antibacterial activity against E. coli, S. aureus, P. aeruginosa and B. subtilis.
M. Zhang et al., 2011 reported that the ethanolic extract of H. Sabdariffa leaves shows
antibacterial activity against Listeria monocytogenes, S.typhimurium and E. Coli.
Priyanka et al., 2022 displayed that the ethanolic extract of H. hirtus L. leaves shows
antibacterial activity against E. coli and Staphylococcus.
Punasiya et al., 2014 manifested that the petroleum ether, benzene, chloroform, methanol and
aqueous extract of H. syriacus leaves shows antibacterial activity against Bacillus cereus,
Staphylococcus epidermis, Kleibsiella pneumonia and Bacillus subtilis.
R. Patel et al., 2012 demonstrated that the hexane, ethylacetate, methanol and aqueous extract of
Hibiscus rosa-sinensis leaves show antibacterial activity against Staphylococcus auresus,
B.subtilis, Stretpomyces alboniger, Micrococcus luteus, S. epidermis, Pseudomonas aeruginosa
and Bordetella bronchiseptica respectively.
Radha et al., 2017 and Amita et al., 2018 indicated that the aqueous and ethanolic extract of
Hibiscus rosa-sinensis leaves shows antibacterial activity against Aeromonas hydrophila.
Ruban & Gajalakshmi, 2012 displayed that the methanolic and ethanolic extract of Hibiscus
rosa-sinensis flower shows antibacterial activity against S. aureus, Streptococcus sp., B.subtillis,
E. coli, Salmonella sp and P. aeruginosa.
S. Das, 2014 asserted that the petroleum ether and ethanolic extract of H. Sabdariffa Calyces
shows antibacterial activity against Kleibsella pneumoniae, Strephyllococcus aureus, B.cerus and
44
Lactobacillus brevis respectively.

Salman & Tajaldeen, 2018 exhibited that the hot and cold aqueous extract of H. Sabdariffa
calyces shows antibacterial activity against S. aureus.
Sekar. et al., 2015 reported that the methanol extract of H. Sabdariffa leaves, and fruits shows
antibacterial activity against Streptococcus mutans, Staphylococcus aureus, Escherichia coli and
Pseudomonas aeruginosa respectively.
Shaibu & Okwute, 2021 revealed that the methanolic extract of Hibiscus radiatus leaves showed
a significant antibacterial property against Staphylococcus aureus, Bacillus subtilis and E. coli
respectively.
Singh et al., 2017 reported that the aqueous and ethanol extract of Hibiscus rosa-sinensis leaves
shows antibacterial activity against P. aeruginosa and A. hydrophilla respectively.
S. Singh et al., 2019 expressed that the methanol and ethanol extract of Hibiscus rosa-sinensis
flower shows antibacterial activity against Klebsiella pneumoniae, Escherichia coli, Bacillus
subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella sp. respectively.
SIVAN KUMAR & ANNA SHEBA L, 2019 reported that the aqueous methanol extract of H.
Sabdariffa Leaves and stem shows antibacterial activity against Staphylococcus aureus,
Sobhy et al., 2017 exhibited that the ethanol, methanol, aqueous and ethylacetate extract of H.
rosa-sinensis flower shows antibacterial activity against E. coli, P. aeruginosa and S. aureus
respectively.
Sulaiman et al., 2014 displayed that the aqueous extract of H. Sabdariffa leaves shows
antibacterial activity against E. coli, Klebsiella pneumonia, Staphylococcus aureaus and
Thiripurasudari et al., 2015 manifested that the phosphate buffer extract of H. Sabdariffa leaves
and seeds shows antibacterial activity against Staphylococcus sp.
Tyagi et al., 2017 reported that the deionized water extract of H. rosa-sinensis Leaves and Silver
and gold nanoparticles shows antibacterial activity against Pseudomonas aeruginosa, Bacillus
subtilis, Micrococccus luteus, S.aureus, S. epidermidis, Enterobacter aerogenes, E. coli, S.
pneumoniae and Aeromonas hydrophila respectively.
U. Tiwari et al., 2015 demonstrated that the methanolic extract of H. rosa-sinensis leaves and
flower shows antibacterial activity against E. coli and S. aureus.
45
Udo et al., 2016 reported that the aqueous methanolic extract of H. rosa-sinensis leaves and
flower shows antibacterial activity against Bacillus subtilis and S. aureus respectively.
Vastrad & Byadgi, 2018 displayed that the ethanol, methanol and distilled water extract of H.
rosa-sinensis leaves shows antibacterial activity against S. aureus and E. coli respectively.
Vijayakumar et al., 2018 reported that the methanol, water and ethyl acetate extract of H. rosa-
sinensis flower shows antibacterial activity against E. coli, B. subtilis and S. aureus.
Youns et al., 2018 expressed that the methanolic extract of H. Sabdariffa calyces shows
antibacterial activity against E. coli, P. aeruginosa, K. pneumonia, S. typhi, B. subtilis and S.
aureus respectively.
2.2.6. Antioxidant activity
Abdoulaye et al., 2018 reported that the Water and ethanol extract of H. acetosella steam
showed a significant range of antioxidant activity against DPPH radical.
al Faruq et al., 2018 confirmed that the leaf extracts of H. surattensis possesses significant anti-
oxidant (moderate) activity against DPPH radical.
Afify & Hassan, 2016 displayed that the water, ethanol, and absolute ethanol extract of H. rosa-
sinensis flower showed a significant range of antioxidant activity to DPPH, nitric oxide,
hydroxyl radical scavenging activity.
B. Zhang et al., 2014 demonstrated that the methanolic extract of H. sabdariffa flower showed a
significant range of antioxidant activity against DPPH and ABTS assay.
Falade et al., 2010 exhibited that the methanolic extract of H. rosa-sinensis flower showed the
significant range of antioxidant activity against DPPH radical.
Formagio et al., 2015 revealed that the methanolic extract of H. sabdariffa leaves and calyx
showed a significant range of antioxidant activity against DPPH.
Garg et al., 2012 asserted that the aqueous and methanol extract of H. rosa-sinensis stem and
leaves showed a significant range of antioxidant activity to DPPH reduction assay, scavenging of
SO, H2O2 and NO, reducing power, FRAP assay.
Ghaffar & El-Elaimy., 2016 expressed that the 70% ethanol/water extract of H. rosa-sinensis
leaves showed a significant range of antioxidant activity to Butylated hydroxyl toluene (BHT),
Ascorbic acid (ASA).
46
Ghosh & Dutta, 2017 displayed that the ethanolic extract of H. rosa-sinensis flower showed a
significant range of antioxidant activity to Hydrogen peroxide scavenging assay.
Guleria et al., 2015 confirmed that the methanolic extract of H. rosa-sinensis corolla and calyx
showed a significant range of antioxidant activity to Ferric ion Reducing Power Assay and Nitric
Oxide Scavenging assay.
I. Ghosh et al., 2015 manifested that the ethanolic extract of H. Sabdariffa L. calyx showed a
significant range of antioxidant activity against FRAP and DPPH.
Islam et al., 2018 demonstrated that the methanolic extract of H. sabdariffa L. of bark showed a
significant range of antioxidant activity against DPPH and ABTS.
Jamini et al., 2019 exhibited that the methanolic extract of H. sabdariffa calyx showed a
significant range of antioxidant activity against DPPH.
Jung et al., 2013 displayed that the water and ethanol extract of H. sabdariffa calyces showed a
Lade et al., 2022 manifested that the methanolic extract of H. sabdariffa calyces showed a
Linn. et al., 2015 asserted that the aqueous and hydro ethanol extract of H. sabdariffa calyx
showed a significant range of antioxidant activity against DPPH and hydroxyl radical.
M. Kumar et al., 2012 revealed that the aqueous, percent ethanol, ethyl acetate fraction extract of
H. sabdariffa leaves showed a significant range of antioxidant activity against DPPH.
M. Zhang et al., 2011 reported that the ethanolic extract of H. sabdariffa leaves showed a
Mandade et al., 2011 exhibited that the ethanol extract of H. rosa-sinensis leaves showed a
significant range of antioxidant activity against ferric thio cyanate, Hydrogen peroxide
scavenging, DPPH, ABTS radicals’ cations and Super oxide an ion radical scavenging by
riboflavin methionine illuminates' system.
Mondal et al., 2016 confirmed that the ethanolic extract of H. rosa-sinensis leaves showed a
significant range of antioxidant activity to DPPH, Nitric oxide, Superoxide radical.
Nangui Abrogoua et al., 2015 expressed that the methanolic extract of H. sabdariffa calyx and
callus showed a significant range of antioxidant activity against DPPH.
Obouayeba et al., 2014 reported that the methanolic extract of H. Sabdariffa petal showed a
47
Pillai & Mini, 2014 displayed that the ethanol and ethyl acetate fraction extract of H. rosa-
sinensis petals showed a significant range of antioxidant activity against DPPH radical.
Prasanna, 2017 demonstrated that the aqueous and ethanol extract of H. rosa-sinensis leaves
showed a significant range of antioxidant activity to DPPH, NO, FRAP and H2O2.
Purushothaman et al., 2016 exhibited that the methanolic extract of H. rosa-sinensis flower
showed a significant range of antioxidant activity to DPPH, hydrogen peroxide and superoxide
radical scavenging activity.
Rm & Nair, 2018 manifested that the leaves extract of H. rosa-sinensis leaves showed a
significant range of antioxidant activity to FRAP, DPPH, hydroxyl, superoxide, nitric oxide and
hydrogen peroxide scavenging assay.
Rusmini et al., 2019 asserted that the ethanolic extract of H. cannabinus leaves showed a
Ryu et al., 2017 reported that the water extract of H. cannabinus leaves, bark, flowers and seeds
S. Das, 2014 revealed that the ethanol, methanol, petroleum ether and aqueous extract of H.
sabdariffa calyces showed a significant range of antioxidant activity against DPPH.
Saravanan et al., 2011 expressed that the ethanol and aqueous extract of H. platanifolius leaves
showed a significant range of antioxidant activity by reducing power and hydrogen peroxide
scavenging assay DPPH.
Sankaran & Vadivel, 2011 confirmed that the ethanolic extract of H. rosa-sinensis flower
showed a significant range of antioxidant activity to SOD, GPx, CAT.
Sigei et al., 2017 displayed that the methanolic extract of H. sabdariffa calyces showed a
Sheth & Subrata De., 2012 exhibited that the methanolic extract of H. rosa-sinensis flowers
S. Singh et al., 2019 manifested that the methanol and ethanol extract of H. rosa-sinensis flower
Sirag et al., 2014 asserted that the ethanolic extract of H. Sabdariffa L. calyx showed a
Subhaswaraj et al., 2017 revealed that the ethanolic extract of H. sabdariffa leaves showed a
48
T & A, 2018 reported that the methanol extract of H. asper leaves and calyx showed a significant
range of antioxidant activity against DPPH radical.
Umachig et al., 2008 expressed that the methanolic extract of H. syriacus L. leaves showed a
significant range of antioxidant activity against DPPH, Superoxide radical scavenging activity in
NBT system, reducing power and Inhibition of lipid peroxidation induced by TBARS in liver
homogenate.
V. Kumar et al., 2013 confirmed that the aqueous extract of H. rosa-sinensis root and leaves
showed a significant range of antioxidant activity to Super oxide anions and Hydroxyl radicals.
Wahid et al., 2019 displayed that the ethanol extract of H. rosa-sinensis flower showed a
Widowati et al., 2017 demonstrated that the methanolic extract of H. sabdariffa flower showed a
significant range of antioxidant activity against DPPH, ABTS, FRAP assay.
Zahid et al., 2014 exhibited that the methanolic extract of H. schizopetalus flower and leaves
showed a significant range of antioxidant activity against DPPH, DPPH, Nitric oxide, Nitric
oxide.
2.2.7. Anti-fungal activity
Ajoku et al., 2016 displayed that the hexane, ethylacetate, methanolic extract of H. sabdariffa
calyx showed significant range of antifungal activity against Candida albicans.
(Durga et al., 2018) demonstrated that the acetone extract of H. rosa-sinensis flower showed
significant level of antifungal activity against Candida albicans, Aspergillus niger, Tricoderma
viride and Rhizopus microsporous.
Edema & Alaga, 2012 exhibited that the ethanolic extract of H. sabdariffa calyces showed
significant range of antifungal activity against Candida albicans.
Jang et al., 2012 asserted that the methanolic extract of H. syriacus root showed significant range
of antifungal activity against Trichophyton mentagrophytes.
L. Das et al., 2015 revealed that the aqueous, ethanol, methanol extract of H. rosa-sinensis leaves
showed maximum antifungal activity against Trichophyton rubrum and Candida albicans
respectively.
Lade et al., 2022 expressed that the methanolic extract of H. sabdariffa calyces showed
49
significant range of antifungal activity against Candida albicans.

Linn. et al., 2015 confirmed that the aqueous and hydroethanol extract of H. sabdariffa calyx
showed significant range of antifungal activity against Candida albicans.
2.2.8. Anti-cancerous activity
Abdul-Awal et al., 2016 displayed that the leaves and bark extract of H. tiliaceus showed
cytotoxic effect on Brine shrimp.
Alam et al., 2018 reported that the aerial parts extract of H. calyphyllus, H. deflersii, H.
micranthus showed Anti-cancer activity on HepG2, MCF-7 cell lines.
B. Zhang et al., 2014 demonstrated that the Seeds extract of H. sabdariffa L. exhibited Anti-
tumour activity against Human cervical hela cells.
Cheng et al., 2012 expressed that the acetone extract of H. syriacus root and bark showed
significant Anti proliferative effect on Human lung cancer cells.
Durga et al., 2018 manifested that the flower extract of H. rosa-sinensis exhibited potent anti-
cancer activity against hela cell lines.
Formagio et al., 2015 revelead that the methanol extract of H. sabdariffa leaves and calyx
showed significant Anti-tumor activity on Leukaemia line k-562.
Hajera Khatun et al., 2011 asserted that the fruits extract of H. sabdariffa showed Cytotoxicity
activity through Brine shrimp lethality bioassay.
Hosseini et al., 2017) reveled that the seeds extract of H. sabdariffa showed Cytotoxicity on
H9c2 cardiomyoblast cells.
Maciel et al., 2018 expressed that the Calyx extract of H. sabdariffa showed significant Anti-
proliferative activity on Caco-2, hepG-2, HCT8 and A549 cell lines.
Nishitha et al., 2018 confirmed that the flowers extract of H. vitifolius showed significant
cytotoxic activity against hela cell lines.
Sangeetha & Ananthi, 2018 displayed that the leaves extract of H. sabdariffa showed significant
cytotoxic activity on HepG2 cell lines.
Wong et al., 2014 demonstrated that the seeds extract and seed oil of H. Sabdariffa exhibited in
vitro cytotoxic activity on human cancer cell lines.
50
2.2.9 Effect on Lipid Metabolism
Bhasker et al., 2011 reported that the flower extract of H. rosa-sinensis showed hypoglycemic
and Hypolipidemic activity on Rats.
Biswas & Souza, 2014 displayed that the flower extract of H. rosa-sinensis has significant
Hypolipidemic activity on Albino Wistar Rats.
Gaffer et al., 2014 demonstrated that the calyxes’ extract of H. sabdariffa showed significant
Hypolipidemic effect on Albino Rats.
Gomathi et al., 2008 expressed that the Flower petals extract of H. rosa-sinensis has Lipid
lowering effect on Albino Rats.
Gosain et al., 2010 exhibited that the leaves extract of H. sabdariffa showed significant
Hypolipidemic activity on Hyperlipidemic Rats.
Mishra et al., 2011 mamifested that the methanolic extract of H. rosa-sinensis leaves showed
Anti-hyperlipidaemic activity on Mice.
Ndarubu et al., 2019 expressed that the leaves extract of H. sabdariffa showed Hypoglycaemic
and Hypolipidemic effects on Rats.
Ojiako et al., 2013 confirmed that the leaves extract of H. rosa-sinensis observed
Hyperlipidaemic activity in case of diabetic rabbits.
Peng et al., 2011 reveled that the calyces extract of H. sabdariffa showed significant
Hyperglycaemia, Hyperinsulinemia, and Hyperlipidaemia activity on Rats.
Sankaran & Vadivel, 2011 asserted that the flowers extract of H. rosa-sinensis showed
Hypoglycaemic and hypolipidemic activity on Rats.
Saravanan et al., 2011 reported that the leaves extract of H. platanifolius showed Hypoglycemic
and hypolipidemic activity on Rats.
Sikarwar & Patil, 2011 displayed that the flowers extract of H. rosa-sinensis showed
Antihyperlipidemic activity on Rats.
Zahid et al., 2014 exhibited that the flowers and leaves extract of H. schizopetalus Hook showed
significant Hypolipidemic effect on Rats.
Zaki et al., 2017 displayed that the leaves extract of H. rosa-sinensis showed significant
Hypoglycaemic effect on Rats.
51
2.2.10. Hepato-protective activity
Abubakar et al., 2010 demonstrated that the calyx extract of H. sabdariffa showed significant
Hepato-renaltoxicity on Albino rats.
Alqasoumi & Alsheikh, 2008 exhibited that the pods extract of H. esculentus showed Hepato-
protective activity on Wistar albino Rats.
El-Sayed, 2018 asserted that the leaves extract of H. rosa-sinensis showed Hepato-protective
activity on Albino Rats.
Joshua et al., 2017 reveled that the leaves extract of H. sabdariffa showed Hepato-curative effect
on Albino Rats.
Olanrewaju et al., 2017 exhibited that the leaves extract of H. sabdariffa showed ethanol induced
Hepatotoxicity on Rats.
Rajani et al., 2017 reported that the leaves extract of H. sabdariffa showed significant Hepato-
protective activity on Rats.
Samuel et al., 2012 confirmed that the root extract of H. vitifolius showed significant Hepato-
toxicity activity on Albino rats.
2.2.11. Haemato-toxicity
Agbor et al., 2005 reported that the leaves extract of H. cannabinus showed significant
Haematinic activity after extract administration on Rats.
Famurewa et al., 2015 expressed that the calyxes’ extract of H. sabdariffa showed significant
Haemato-toxicity after extract administration on Rats.
Kumar Meena et al., 2007 demonstrated that the flowers extract of H. rosa-sinensis exhibited
significant Haemato-protective activity after extract administration on Rats.
Purushothaman et al., 2016 reveled that the flowers extract of H. rosa-sinensis exhibited
significant Anti-haemolytic activity on Venous blood samples.
2.2.12. Anti-diabetic activity
52
Afiune et al., 2017 reported that the flower extract of H. rosa-sinensis showed significant effect
on diabetes after extract administration on Rats.
Anandhi et al., 2013 asserted that the leaves extract of H. rosa-sinensis showed significant Anti-
diabetic activity after extract administration on Rats.
Chan et al., 2016 exhibited that the methanol flower extract of H. tiliaceus shows anti-diabetic
activity on diabetic Wistar Rats.
Chan et al., 2016 reveled that the methanol flower extract of H. mutabilis shows anti-diabetic
activity on diabetic Wistar Rats.
Nirosha et al., 2014 manifested that the leaves extract of H. syriacus showed significant Anti-
diabetic activity after extract administration on Rats.
Ojewumi & Kadiri, 2013 confirmed that the leaves, stem and roots extract of H. sabdariffa
showed significant Anti-diabetic activity after extract administration on Rats.
Raghavendra et al., 2016 exhibited that the ethanolic extract of H. platanifolius stem showed
significant Anti-diabetic activity after extract administration on rats.
2.2.13 Anti-hypertensive activity
Cn et al., 2015 reported that the Calyces extract of H. sabdariffa showed significant Anti-
hypertensive activity after extract administration on Rats.
Eyitayo Balogun et al., 2019 demonstrated that the leaves extract of H. sabdariffa showed
significant Anti-hypertensive activity after extract administration on Wistar Rats.
2.2.14 Anthelmintic activity
R., 2015 reported that the ethanolic extract of leaves of H. Cannabis L showed the potent anti-
helminthic activity.
S et al., 2013 confirmed that the petroleum ether, ethanol, methanol, ethyl acetate and distilled
water extracts of leaves of H. rosa sinensis showed the strong anti-helminthic activity.
Bindu Ch et al., 2019 demonstrated that the methanolic extract of leaves of H. hirtus. L showed
the significant anti-helminthic activity.
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Chapter Three: (Methods & Materials)
3.1 Methods and Materials

3.1.1 General Methods
The following three major steps in the chemical investigation of a plant are:
1. Collection and proper identification.
2. Preparation of plant sample.
3. Extraction.
3.1.2 Collection and Proper Identification of Plant

Either the whole plant or various parts of the plant, such as: barks, leaves, roots, flowers or fruits
must be collected from a legitimate source and identified by an expert taxonomist.
3.1.3 Preparation of Plant Material

Generally, the plant part is collected and washed up with tap water in fresh condition. It was cut
into small pieces, dried in direct sunlight and then dried in an oven at 35°- 40°C to make it
suitable for grinding purpose. The coarse powder is stored in an airtight container for future use.
3.1.4 Extraction
Extraction can be done in two ways, such as:
1. Cold Extraction and
2. Hot Extraction.
3.1.5 Cold Extraction
Organic compounds present in plant material can
be isolated using suitable solvent or solvent
systems in which the desired compounds are
soluble. Selection of solvent or solvents actually
depends on the type of compounds required to
isolate. It is supposed to be assumed that polar
solvents are likely to dissolve polar compounds
and non-polar solvents arelikely to dissolve non-
Fig. 3.1: Rotary Evaporator
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polar compounds. For cold extraction, the plant material are dipped into the appropriate solvent
at room temperature (RT) and allowed to stand for stand for several days with occasional
shaking. When maximum concentration of the compounds in the solvent is achieved, the solvent
is separated from the powdered plant materials by filtration. Evaporation of the solvent in
vacuum affords a crude mixture of the soluble compounds in that particular solvent. Separation
is then performed using various techniques of chromatography (Harwood et al., 1989).
3.1.6 Hot Extraction

In hot extraction process, the sample can be extracted at
elevated temperature (usually reflux pump). In actual
practice, the extraction from solids is often tedious and
requires thorough contact and heating with the solvent. This
is done in a special apparatus, the Soxhlet extractor. The
apparatus ensures maximum extraction with a limited
quantity of solvent. Evaporation of the solvent under reduce
pressure affords a crude mixture of the compound dissolve
in those particular solvents and proceeds for separation
using various techniques of chromatography
with a limited quantity of solvent. Evaporation of the
solvent under reduces pressure affords a crude mixture of
the compound dissolve in those particular solvents and Fig. 3.1: Rotary Evaporator
solvent
proceeds for separation using various techniques of
chromatography.
3.2 Chemical Studies on Hibiscus Macrophyllus

3.2.1 Collection and Identification of Plant
Barks of H. macrophyllus were collected from Madhabkundo Eco Park, Bangladesh in 15 th
September, 2022 and were identified by a Principle Experimental Officer, Dept. of Botany, JU
university. where a voucher specimen (10106) is deposited.
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Fig.3.3: Barks of H. macrophyllus
3.2.2 Preparation of Plant’s Barks
Collected barks were washed properly with fresh tap water to remove dirty materials and were
shade dried for several days with occasional sun drying. These were then dried in an oven for 24
hours at considerably low temperature for better grinding. The dried materials were ground into
coarse powder by a grinding machine in the Department of Pharmacy, Gono Bishwabidyalay,
Bangladesh and stored at RT for future use.
Fig.3.4: Powder of H. macrophyllus Barks
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3.2.3 Extraction of Plant’s Barks

Extraction was performed according to Alma et al., 2002. About 200 gm. of powdered barks of
H. macrophyllus was taken in an amber colored extraction bottle (2.5 liters capacity) and soaked
the leaves with 100% methanol (1.5 L × 5 times). The sealed bottle was occasional shaking and
stirring. The final extract was filtered separately through cotton and then Whatman No.1 filter
papers followed by concentrated with a rotary evaporator under vacuum pressure at 40°C to
afford 8.36 gm barks extract and from soxhlet extractor we get 20.52 gm of hot extracts.
Figure: 3.5: Extraction process of H. macrophyllus
3.3 Qualitative Phytochemical Analysis

3.3.1 Test for Alkaloids
Mayer’s Test: 1 mL of extract was taken and placed into a test tube. Then 1 mL of potassium
mercuric iodide solution (Mayer’s reagent) was added and shaken. Emergence of whitish or
cream precipitate implies the presence of alkaloids.
3.3.2Test for Glycosides

Keller-Kiliani Test: About 2 ml of each extract was taken in different test tubes followed by 1
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mL of glacial acetic acid containing trace amount of FeC l3 and 1 mL of concentrated H2SO4 were
added to the extract carefully. A reddish-brown color is formed at the junction of two layer and
the upper layer turns bluish green in presence of glycosides.
3.3.3 Test for Saponins
Frothing test: Ten (10) mg of each extract was taken in different test tubes and shaken vigorously
with 5 mL of water. Production of persistent foam indicates the presence of saponins.
3.3.4 Test for Steroids

Liebermann-Burchard’s Test: Each extract was dissolved in 1 mL of chloroform. 2 mL acetic
anhydride and 1 mL concentrated sulfuric acid were added. Formation of greenish color solution
indicates the presence of steroids.
3.3.5 Test for Tannins

Lead acetate test: About 5 mL of an aqueous solution of different fractions was taken in different
test tubes and few drops of 1% solution of lead acetate were added to the test tube. A yellow or
red precipitate was the indication for the presence of tannins.
3.4 Quantitative Phytochemical Analysis

3.4.1.1 Determination of Total Flavonoids
The content of total flavonoids of HME and CME of H. macrophyllus were determined by
aluminum chloride colorimetric method according to Zhisen et al., 1999. Quercetin was used as
standard and the total flavonoids content of the extractives were expressed as mg of QE/gm. Of
dried extract.
3.4.1.2 Principle
The content of total flavonoids in different extractives was determined by the well-known
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aluminum chloride-colorimetric method. In this method, aluminum chloride formed complex

with hydroxyl groups of flavonoids present in the samples. This complex has the maximum
absorbance at 510 nm.
3.4.1.3 Materials and Apparatus

• Aluminum chloride
• Sodium nitrate
• Sodium hydroxide
• Methanol
• Quercetin
• Micropipette (10-100 µL)
• Micropipette (100-1000 µL)
• Pipette (1-10 mL)
• UV-spectrophotometer
3.4.1.4 Experimental Procedure

1. 0.5 ml of plant extract or standard of different concentrations were taken in different test
tubes.
2. 1.5 ml of methanol was added in each test tube.
3. 100 µL of 10% AlCl3 was added in each test tube
4. 100 µL of 1M of potassium acetate (CH3COOK) was added to the mixture.
5. 2.8 ml of distilled water was added in each of the test tube.
6. Stand for 30 minutes at room temperature to complete the reaction.
6. Then the absorbance of the solution of each test tube was measured at 510 nm using a
spectrophotometer against blank.
7. A typical blank solution contained all reagents except plant extract and/or standard solution.
8. The total content of flavonoid compounds in methanolic extract and its four fractions was
calculated as Quercetin equivalent (QE) by the following formula equation:
C = (𝑥×𝑉)/M
Where
C = total content of flavonoid compounds as mg QE in each gram of dried extract.
x = QE concentration in mg/ml present in that particular sample concentration.
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V = Final volume of the solution in ml

M = Mass of the sample in final solution in gm
3.4.2.1 Determination of Total Phenolics

The content of total phenolic of hot and cold extracts of Hibiscus macrophyllus were determined
according to Wolfe et al., 2003 in which Folin-ciocalteu reagent (FCR) was used as oxidizing
agent and Gallic Acid (GA) was used as standard.
3.4.2.2 Principle
The content of total phenolic compounds of different extracts of the plant was determined by
FCR. The FCR actually measured a sample’s reducing capacity. The exact chemical nature of the
FCR is not known, but it is believed to contain heteropolyphosphotunstates molybdates.
Sequences of reversible one or two electron reduction reactions lead to blue species, possibly
(PMW11O40)4. In essence, it is believed that the molybdenum is easier to be reduced in the
complex and electron-transfer reaction occurs between reductants and Mo (VI):

a. Folin-ciocalteu reagent
b. Sodium carbonate
c. Methanol
d. Gallic acid
e. Micropipette (10-100 µL)
f. Micropipette (100-1000 µL)
g. Pipette (1-10 ml)
h. UV-spectrophotometer

1. 0.4 ml of plant extract or standard of different concentrations were taken in different test
tubes.
2. 2.0 ml of FCR (Diluted 10 times with de-ionize water) reagent solution was added into each
test tube.
3. 3.0 ml of sodium carbonate (7.5%) solution was added into each test tube.
4. Placed in dark place for 30 minutes to complete the reaction.
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6. A typical blank solution contained all reagents except plant extracts and/or standard solution.
7. The total content of phenolic compounds in methanolic extracts and its four fractions was
calculated as Gallic acid equivalent (GAE) by the following formula:
C = (𝑥×𝑉)/M
Where
C = total content of phenolic compounds as mg GAE in each Gram of dried extract.
x = GAE concentration in mg/mL present in that particular Sample concentration.
V = Final volume of the solution in ml.
M = Mass of the sample in final solution in gm
3.5 In-Vitro Antioxidant Activity

3.5.1.1 Determination of Total Antioxidant Capacity
The total antioxidant capacity of the total antioxidant capacity of HME and CME of H.
macrophyllus determined according to Prieto et al., 1999 with some modifications.
3.5.1.2 Principle
The phosphomolybdenum method usually detects antioxidants, such as ascorbic acid, some
phenolics, α-tocopherol and carotenoids. The phosphomolybdenum method was based on the
reduction of Mo (VI) to Mo (V) by the antioxidant compounds and subsequent formation of a
green phosphate/Mo (V) complex at acidic ph. In essence, it is believed that the molybdenum is
easier to be reduced in the complex and electron-transfer reaction occurs between reductants and
Mo (VI) and the formation of a green phosphate/Mo (V) complex with a maximal absorption at
695 nm.
Mo (VI) + e  Mo (V)

a. Sulfuric acid
b. Sodium phosphate
c. Ammonium molybdate
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d. Ascorbic acid
e. Methanol
f. Water bath
g. Micropipette (10-100 µL)
h. Micropipette (100-1000 µL)
i. Pipette (1-10 ml)
j. UV-spectrophotometer

1. 0.5 mL of plant extract or standard of different concentrations was taken in different test
tubes.
2. 3 mL of reaction mixture containing 0.6 M sulfuric acid, 28 mm sodium phosphate and 1%
ammonium molybdate was added into each test tube.
3. The test tubes were incubated at 95°C for 10 minutes to complete the reaction.
spectrophotometer against blank after cooling at room temperature.
5. A typical blank solution contained the same solution mixture without plant extracts and/or
standard and it was incubated under the same conditions as the rest of the sample solution.
3.5.2.1 Reducing Power Assessment

The Fe3+ reducing power of methanolic hot and cold extractives of barks of H. macrophyllus
was evaluated by the method of Oyaizu 1986.
3.5.2.2 Principle
In this assay, the yellow color of the test solution changes to various shades of green and blue
depending on the reducing power of antioxidant samples. The reducing capacity of a compound
may serve as a significant indicator of its potential antioxidant activity. The presence of
reductants, such as antioxidant substances in the samples causes the reduction of the Fe 3+-
ferricyanide complex to the ferrous form by donating an electron. The amount of Fe2+ complex
can then be monitored by measuring the formation of Perl’s Prussian blue at 700 nm.
Fe3+-ferricyanide + e Fe2+-ferricyanide
81

a. Potassium ferricyanide
b. Trichloro acetic acid, TCA
c. Ferric chloride
d. Phosphate buffer
e. Ascorbic acid
f. Water bath
g. Centrifuge machine
h. Micropipette (10-100 µL)
i. Micropipette (100-1000 µL)
j. Pipette (1-10 mL)
k. UV spectrophotometer

1. 0.25 ml of plant extract or standard of different concentrations was taken in different test
tubes.
2. 0.625 ml of potassium buffer (0.2 M, pH 6.6) and 0.625 mL of 1% potassium ferricyanide
[K3Fe (CN)6], solution was added into each test tube.
3. All the test tubes were incubated for 20 minutes at 50°C to complete the reaction.
4. 0.625 mL of TCA, 10% solution was added into each test tube.
5. All the test tubes were centrifuged at 3000 rpm for 10 min.
6. 1.8 mL supernatant was withdrawn from the mixture of each test tubes and mix with 1.8 mL
of distilled water.
7. 0.36 mL of 0.1% ferric chloride (FeCl3) solution was added to each diluted reaction mixture.
3.5.3.1 DPPH (1, 1-diphenyl-2-picrylhydrazyl) Free Radical

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Scavenging Assay
DPPH was used to evaluate the free radical scavenging activity of various compounds and
medicinal plants (Blois 1958; Desmarchelier et al., 1997).
3.5.3.2 Principle
The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used to evaluate the free
radical scavenging capacity of antioxidants. DPPH free radical is reduced to the corresponding
hydrazine when it reacts with hydrogen donors. DPPH can make stable free radicals in aqueous
or methanol solution. With this method, it is possible to determine the antiradical power of an
antioxidant activity by measurement of the decrease in the absorbance of DPPH at 517 nm.
Resulting from a color change from purple to yellow, the absorbance decreased when the DPPH
was scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH
molecule. In the radical form, this molecule had an absorbance at 517 nm which disappeared
after acceptance of an electron or hydrogen radical from an antioxidant compound to become a
stable diamagnetic molecule.

a. DPPH
b. Methanol
c. Butylated hydroxy toluene (BHT)
d. Micropipette (10-100 µL)
e. Micropipette (100-1000 µL)
f. Pipette (1-10 mL)
g. UV spectrophotometer

1. 1.6 mL of methanol solution of plant extracts or standard at different concentrations were
taken in different test tubes.
2. 2.4 mL of methanol solution of 0.1 mm DPPH was added into each test tube.
3. The test tubes were incubated at RT for 30 minutes in dark place to complete the reaction.
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spectrophotometer.
6. The percentage (%) scavenging activity of DPPH radicals was calculated from the
following equation
% I = {(Ac-As)/Ac} ×100
Where
Ac= Absorbance of the control
As= Absorbance of the extract/ standard
7. Then all the % scavenging were plotted against concentration, and from the graph IC50 was
calculated.
3.6 In-Vivo Biological Activity

3.6.1 Evaluation of Cytotoxic Activity by Brine Shrimp Lethality
Bioassay
3.6.1.1 Principle
Brine shrimp lethality bioassay is a recent development in the bioassay for the bioactive
compounds (McLaughlin et al., 1992; Meyer et al., 1982; Persoone et al., 1980; McLaughlin et
al., 1988; Chatterjee et al., 1975). Natural products (extracts, fractions and pure compounds) can
be tested for their bioactivity by this method. Here the simple zoological organism (brine shrimp
nauplii) is used as a convenient monitor for screening and fractionation in the discovery of new
bioactive natural products. This bioassay indicates cytotoxicity as well as a wide range of
pharmacological activities of the compounds as cytotoxic property may be due to the presence of
phytochemicals such as saponins, triterpenes, tannins and polyphenolic compounds in the
extract (Armania et al., 2013) and this phytochemical compounds exist in plants exhibited anti-
tumorigenic effects via multiple anticancer pathways such as by interaction with key enzymes in
cellular signaling pathways, cell cycle, apoptosis and metastasis etc., (Zhiyu et al., 2012; Man et
al., 2010; Lamoral-Theys et al., 2010; Kuttan et al., 2007). It is reported that polyphenolic
compounds have antioxidative properties and cytotoxic activity are correlated with the
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antioxidant properties. As multiple mechanisms account for polyphenol-induced cytotoxicity but

SARs between polyphenols and cytotoxicity are not well understood (Mitsuhashi et al., 2008).
The brine shrimp assay has advantages of being rapid (24 hours), inexpensive and simple (as no
aseptic technique is required). It easily utilizes a large number of organisms for statistical
validation and requires no special equipment and relatively small amount of sample is sufficient.
Moreover, it does not require animal serum, as it is needed for the determination of cytotoxicity.
In previous test all the extracts of H. macrophyllus showed the remarkable phenolic compounds
and antioxidant properties. Therefore, all the extracts of H. macrophyllus were further
investigating whether it has capability to show cytotoxic effect against brine shrimp nauplii or
not.
a. Artemia salina leach (brine shrimp eggs)

b. Sea salt (non-ionized, NaCl) (Merck, India)
c. Small tank with perforated dividing dam to hatch the shrimp
d. Lamp to attract the nauplii
e. Pipette (1 mL and 5 ml)
f. Micropipette (10-100 μl adjustable)
g. Glass vials (5 ml)
h. Magnifying glass
i. Test samples of the experimental plant
j. Micropipette (1-10 μl adjustable)
k. Vincristine sulphate
l. DMSO (Dimethyl sulfoxide)
3.6.1.3 Preparation of Brine Water

38 grams of sea-salt (non-ionized NaCl) was dissolved in 1 liter of sterilized distilled water and
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then filtered off to get clear solution.
3.6.1.4 Hatching of Brine Shrimp Eggs

1 liter of seawater (brine water) was taken in the small tank, and 1.5 gm of shrimp eggs were
added to one side of the divided tank. Constant oxygen supply at constant temperature (around
37°C) was carried out during the hatching time. The eggs were allowed for two days to hatch
and mature as nauplii (larvae). The hatched shrimps were attracted to the lamp on the other side
of the divided tank through the perforated dam. These nauplii were taken for this bioassay.
3.6.1.5 Preparation of the Test Sample

Samples that were taken for the operation were the CME and HME of barks of H. macrophyllus.
All the samples (2 mg each) were initially dissolved in 400 μl DMSO to get a concentration of 5
μg/mL for each of the samples, which was used as stock solution. Seven doses (12.5, 25, 50, 100,
200, 400, 800 μg/mL) of each sample were used for the cytotoxicity test of brine shrimp nauplii.
With the help of a micropipette 1.25, 2.5, 5, 10, 20, 40 and 80 μl of each sample were transferred
from the stock solution in 7 different test tubes. Brine water was added to each test tube making
the volume up to 5 ml. The final concentration of the samples in these test tubes becomes 12.5,
25, 50, 100, 200, 400, 800 μg/mL. For each concentration, the concentration of DMSO in this
test tube should not exceed 50 μg/mL of brine as because above this concentration cytotoxicity
may arise.
3.6.1.6 Preparation of Control Groups

The control groups are used in cytotoxicity study to validate the test method and ensure that the
results obtained are only due to the activity of the test agents and the effects of the other possible
factors are nullified. Usually, two types of control groups are used.
1. Positive control (Vincristine Sulfate)
2. Negative control (DMSO only)
3.6.1.7 Preparation of the Positive Control Group

In cytotoxicity study, a widely accepted cytotoxic agent Vincristine Sulfate was used as positive
control and the result of the test agents (samples) was compared with the result obtained for the
positive control. In the present study, Vincristine Sulfate was used as the positive control. 1 mg
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of Vincristine Sulfate was dissolved in 0.3125 μl of DMSO to get a concentration of 5 μg/μl.

This was used as stock solution of Vincristine Sulfate. The final concentration of Vincristine
Sulfate in the test tube were adjusted at 0.157, 0.313, 0.625, 1.25, 2.5, 5 and 10 μg/mL
concentrations using the same method used for the samples with the help of micropipette.
3.6.1.8 Preparation of the Negative Control Group

In the negative control group 2.5, 5, 10, 20 and 30 μl of DMSO were added to each of the
remarked glass test tube containing 5 ml of simulated sea water, and 10 shrimp nauplii were used
as control groups. If the brine shrimp nauplii in this test tube show a rapid mortality rate, then
the test is considered as in valid as the nauplii died due to some reason other than the
cytotoxicity of the sample.
3.6.1.9 Application of Brine Shrimp Nauplii

Hundred vials were taken for eight samples and two control groups in different concentrations.
With the help of a dropper, 10 living nauplii were transferred to each of the test tube containing
sea water up to 5 ml. Then with the help of micropipette, specific volume of samples transferred
from the stock solution to the vials to get final concentration of 12.5, 25, 50, 100, 200 and 400,
800 μg/mL. The concentration of DMSO in this test tube should not exceed 50 μg/mL of brine as
because above this concentration cytotoxicity due to DMSO may arise. A magnifying glass was
used for convenient counting of the nauplii. As counting of 10 nauplii was not being possible
accurately, therefore a range of 9-12 nauplii were taken for this study.
3.6.1.10 Counting of Nauplii

After 24-hours of incubation, the test tubes were observed using a magnifying glass and the
numbers of survivors in each test tube were counted and the results were noted. From this data,
the percentage of mortality of the nauplii was calculated at each concentration by the following
formula:
% Mortality = {(Number-Dead)/ Number} × 100
3.6.1.11 Analysis of the Data

The dose of mortality was analyzed by using graphical method. The percentage of mortality was
plotted against respective concentrations used and from the graph effective dose, IC 50 was
calculated. This represents the concentration at which 50% of the nauplii died after a certain
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exposure time.
3.6.2 Antibacterial Screening

The antibacterial screening of an agent is essential to ascertain its spectrum against various types
of pathogenic organisms. Antibacterial activity of any plant can be detected by observing the
growth response of various microorganisms to the plant extract, which is placed in contact with
them. Antibacterial activity was observed against both Gram-positive and Gram-negative
bacteria. In general, antibacterial screening is undertaken in two phases.
A primary qualitative assay to detect the presence or absence of activity and a secondary assay
which quantities the relative potency, expressed as the minimum inhibitory concentration (MIC)
value.
The primary assay can be done in three ways such as-

1. Disc diffusion assay method
2. Dilution method
3. Bioautographic method
Among these methods, the disc diffusion assay method (Bauer et al., 1966) is widely acceptable
for the preliminary evaluation of antibacterial activity. It uses different concentrations of the
agents absorbed on sterile filter paper discs.
There is no standardized method for expressing the results of antibacterial screening. Some
investigators use the diameter of the zone of inhibition or the minimum weight of extract that
inhibits the growth of a microorganism. Disk diffusion is essentially a qualitative or semi
quantitative test indicating the sensitivity or resistance of microorganisms to the test material.
However, no distinction between bacteriostatic and bactericidal activity can be made by this
method (Ronald 1982). The principal factors that determine the size of the zone of inhibition are
1. Intrinsic antimicrobial susceptibility of the test sample

2. Growth rate of the test organisms
3. Diffusion rate of the test sample
4. Concentration of test organisms inoculated in the medium
5. Concentration of test sample per disc and
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6. Thickness of the medium in the petri dishes.
3.6.2.1 Principle of Disc Diffusion Assay Method

Disc diffusion assay method is based on the ability of antibiotics to diffuse from a confined
source through the nutrient agar gel and create a concentration gradient. If the agar is seeded or
streaked with a sensitive organism, a zone of inhibition will result where the concentration
exceeds the MIC for the particular organism.
In this method, measured amount of the test samples is dissolved in definite volumes of solvent
to give solutions of known concentrations (μg/mL). Then sterile filter paper discs having 5 mm
in diameter are impregnated with known amounts of the test substances and dried. These test
material discs as well as standard antibiotic discs are placed on plates containing a suitable
medium (nutrient agar) seeded with the test organisms. These plates are kept at 4° C for 24 hours
to allow maximum diffusion. A number of events take place simultaneously which includes:
1. The dried discs absorb water from the agar medium, and the material under test is dissolved.
2. The test material diffuses from the surrounding medium according to the physical law that
controls the diffusion of molecules through agar gel.
3. There is a gradual change of test material concentration in the agar surrounding each disc.
The plates are then kept in an incubator at 37° C for 12-18 hours to allow the growth of the
organisms. If the test material has antibacterial activity, it will inhibit the growth of
microorganisms, giving clear, distinct zone called “Zone of Inhibition”. The antibacterial activity
of the test agent is determined by measuring the diameter of the zone of inhibition in term of mm
(The World of Plants, 1980)

3.6.2.2.1 Test Materials Used for the Study
In our present study, the antibacterial activity of the HME and CME of H. macrophyllus was
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investigated in comparison with a standard antibiotic cefuroxime (5 μg/discs).
3.6.2.2.2 Materials and Apparatus

a. Filter paper disks (5 mm in diameter)
b. Test tubes
c. Petri dishes (120 mm in diameter)
d. Sterile forceps
e. Sterile cotton
f. Inoculating loop
g. Bunsen burner
h. Micropipette (10-100 l)
i. Laminar air flow unit
j. Autoclave
k. Incubator
l. Punch machine
m. Beakers
n. Nutrient agar media
o. Alcohol (95%)
p. Methanol
q. Vials
3.6.2.2.3 Test Organisms

Both Gram-positive and Gram-negative bacterial strains taken for the test were listed in the
Table-3.1. These organisms are available in the Microbiological Research Laboratory at the
Department of Pharmacy. The pure culture of which was previously collected from the
Microbiology Department, Gono Bishwabidyalay, Savar, Bangladesh.
Table 3.1: List of tested bacteria
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Gram-positive Gram-negative
1. Bacillus subtilis 1. Escherichia coli
2. Staphylococcus aureus 2. Salmonella
3. Klebsiella sp
3.6.2.3 Sterilization Procedure

Antibacterial screening was done in laminar hood and all types of other precautions were highly
maintained to avoid any contamination of the organisms under test. UV light has switched on
before one hour working in laminar hood to avoid any accidental contamination. Petri dishes and
other glassware were sterilized by autoclaving at 121° C and a pressure of 15-lbs./sq. inch for 20
mins. Blank discs were first kept in a coverer petri dish and then subjected to dry by heat
sterilization at 180° C for 1 hour. Later they were kept in laminar hood under UV light for 30
mins
3.6.2.4 Culture Media

The following media are usually used to demonstrate the antibacterial activity and to make
subculture of the test organisms.
a. Nutrient agar medium

b. Nutrient broth medium
c. Mueller - Hinton medium
d. Tryptic soy broth medium
e. Trypticase soy agar medium
Among these, the nutrient agar medium is most frequently used and the composition of nutrient
agar medium is given below in Table 3.2.
Table 3.2: Composition of nutrient agar media
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Ingredients Amount
Bacteriopeptone 0.5 gm
Sodium chloride 0.5 gm
Bacto yeast extract 1.0 gm
Bacto agar 2.0 gm
Distilled water 100 mL
PH 7.2 ± 0.1 at 25° C
3.6.2.5 Preparation of Medium

Prepared nutrient agar medium (composition mentioned above) was collected from the store and
was used for antibacterial screening. To prepare required volume of this medium, 28 gm of the
prepared medium was dissolved in distilled water (q.s. to 1000 mL) as directed on the level of
the pack. It was then heated in water bath to dissolve the agar until a transparent solution was
obtained.
3.6.2.6 Preparation of Subculture

The media prepared were then dispensed in 20 mL and 5 mL, to prepare plates and slants,
respectively in a number of clean test tubes. The slants were used for making fresh culture of
microorganisms, which in turn was used for sensitivity tests. The tubes were then plugged with
cotton and sterilized in an autoclave at a temperature of 121° C and pressure of 15 lbs./sq. inch
for 15 mins. With the help of an inoculation loop, the test organisms were transferred from the
pure culture to the agar slants in a laminar airflow unit. The inoculated slants were then
incubated at 37° C for 24 hours to assure the growth of test organisms. This culture was used
within one week.
3.6.2.7 Preparation of Test Plates

The test organism was transferred from the subculture to the test tube containing 20 mL
autoclaved medium with the help of an inoculating loop in an aseptic area. The test tube was
shaken by rotation to get a uniform suspension of the organism. The bacterial suspension was
92
immediately transferred to the sterile petri dishes in an aseptic area and was rotate several times,
first clockwise and then anti- clockwise to assure homogeneous distribution of the test
organisms. The depth of media into each petri dish (120 mm diameter) was approximately 4 mm.
After that the medium was cooled at RT, and then it was stored in a refrigerator at 4° C.
3.6.2.8 Preparation of Discs and Test Samples
 Preparation of Discs
Three types of discs were prepared for antibacterial screening. These are
a. Sample discs
b. Standard discs
c. Control/ Blank discs
a. Sample discs: Sterilized filter paper discs having 5 mm in diameter were prepared with
the help of punch machine and were taken in a blank Petri dish. Sample solution of desired
concentration was applied on the discs with the help of a micropipette in an aseptic
condition.
b. Standard discs: These are used to compare the antibacterial activity of test materials. In
our study, cefuroxime (50 g/disc) standard disk was used as a reference standard.
c. Blank discs: These were used as negative control to ensure that the residual solvent and
the filter paper were not active themselves.
➢ Preparation of test samples
a. Test sample of HME 10 mg of hot extracts of Hibiscus macrophyllus were dissolved in 10

mL ethanol in different vial giving the concentration of 200 µg/disc, 400 µg/disc & 100
µg/disc respectively.
b. Test sample of Cold extracts: 10 mg of cold extracts of Hibiscus macrophyllus were
dissolved in 10 mL ethanol in different vial giving the concentration of 200 µg/disc, 400
µg/disc & 100 µg/disc respectively.
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3.6.2.9 Placement of the Discs, Diffusion and Incubation

The sample discs impregnated separately with the test materials and standard antibiotic discs
were placed gently on the solidified agar plates, freshly seeded with the test organisms with the
help of a sterile forceps to assure complete contact with the surface of the medium. The discs
were placed in such that the disc was no closer than 15 mm to the edge of the plate and far
enough apart to prevent overlapping the zones of inhibition. The plates were then inverted and
kept in a refrigerator for about 24 hours at 4° C. This is sufficient time for the material to diffuse
a considerable area of the medium. Finally, the plates were incubated at 37° C for 12-18 hours.
3.6.2.10 Measurement of Zone of Inhibition

After 12 hours of incubation, the antibacterial activity of the test agents was determined by
measuring the diameter of the zones of inhibition in cm with a transparent scale and compared
with the standard disc.
3.6.3 Evaluation of Anthelmintic Activity

Some helminths infections such as filariasis have solely a few therapeutic modalities nowadays.
The continuous and frequent dependency on a small range of compounds has led to drug
resistance in several helminthic strains. Additionally, treating with albendazole led to several
side effects, where the individual’s reported allergy, nervous system symptoms, GIT
disturbances and allergic phenomena. Some anthelmintic medication, like praziquantel and
albendazole, are contraindicated in lactating and pregnant women. These medications are also
contraindicated in patients suffering from hepatitis and in children below 2 years of age.
The anthelmintic assay was performed in vitro using adult earthworm that is P. Megascolex,
since it has anatomical and physiological similarities with the intestinal round worm of human
beings for the preliminary analysis of anthelmintic activity.

Experimental animals Collection: The worms Collection Indianearthworm P.
megascolex were collected from the Garden soil (wet soil) located Sharifbag near our University
campus. Adult earthworms (Pheretima megascolex) were used to study anthelmintic activity.
94
The earthworms were collected, washed with normal saline to remove all fecal matter. The
earthworms of 5-8 cm in length and 0.2-0.3 cm in width were used for all experimental protocol.
Drugs and chemicals:

a. Hot and Cold extracts of Hibiscus macrophyllus
b. Standard Albendazole
c. NaCl
d. Methanol
e. DMF (Dimethyl formamide)
Apparatus:
a. Test tubes
b. Petri dishes (120 mm in diameter)
c. Pipette (1 mL and 5 mL)
d. Micropipette (10-100 μl adjustable)

1. Earth worms of nearly equal size (6cm ± 1) were selected randomly for present study
2. The worms were acclimatized to the laboratory condition before experimentation
3. The earthworms were divided into three groups of six earth worms each.
4. Six earthworms of nearly equal size were placed in standard drug solution and test
compound’s solutions at room temperature.
5. Normal saline used as control.
6. Standard drug and test
7. Compounds 10mg, 50mg and 100mg were dissolved in minimum quantity (2 ml) of dimethyl
formamide (DMF) and adjusted the volume up to 15 ml with normal saline solution.
8. The test compounds and standard were evaluated by the time taken for complete paralysis
and death of earthworms.
9. The mean lethal time for each test compound was recorded and compared with standard
drug.
10. The time taken by worms to become motionless was noted as paralysis time
95
11. To ascertain the death of the motionless worms were frequently applied with external stimuli,
which stimulate and induce movement in the worms, if alive.
12. The mean lethal time and paralysis time of the earthworms for different test compounds and
standard drug were tabulated in the results section respectively.
3.6.4 Thrombolytic activity of Plant extracts

Various methods were developed to measure the clot lysis activity of thrombolytic drugs. The
best way to study thrombolytic drugs is through in vitro clot lysis model. Earlier Basta et al
worked on artificial clots and used ultrasound methods to measure the thrombolytic activity of
streptokinase. Several other models have been reported which either uses complicated
mathematical or computing skills, but all these methods are very costly and not affordable in
developing countries. The above-mentioned problems demand a need of simple and cost
effective clot lytic model for measurement of clot lysis activity of thrombolytic drugs. In the
present study venous blood was taken. Plant extracts were taken as a positive control and water
was taken as a negative control. We can calculate the percentage of clot lysis by the following
formula:
% Of clot lysis= {(Weight of the clot before lysis-weight of clot after
lysis)/weight of the clot before lysis} X 100
Specimen
Venous blood was drawn from healthy human volunteers (n=20) without a history of oral
contraceptive or anticoagulant therapy (using a protocol approved by our Institutional Ethics
Committee). 500 μl of blood was transferred to each of the previously weighed microcentrifuge
tubes to form clot.
Apparatus:
a. Micropipette (10-100 μl)
b. Micropipette (100-1000 μl)
c. Test tubes
d. Sterile syringe
e. Micro-centrifuge tubes
96
f. Water bath
g. Cotton bud
h. Beaker
i. Conical flask
3.6.4.2 Experimental Procedure:

1.5 ml of venous blood taken in a test tube
2. Each micro-centrifuge tube contain 500 μl of venous blood
3. Add 200 μl of 2% CaCl2 solution
4. Mixed well
5. Incubate the micro-centrifuge tubes at 37° C for 45 Minutes
6. After the Clot formation serum was removed without disturbing the clot
7. Take the weight of the colt before lysis (Clot weight=weight of the tube containing the clot
before lysis-weight of the tube)
8. Plant extracts were added as a positive control as (Venous blood: Plant Extracts) = (4:4, 4:3,
4:2, 4:1)
9. Water (4:4) was added as a negative control.
10. Incubate the micro-centrifuge at 37° C for 90 minutes
11. After the lysis of the clot fluid was removed
12. Take the weight of the clot after lysis (Clot weight=weight of the tube containing the clot
after lysis-weight of the tube)
3.7 Reference
Armania, N., Yazan, L., Musa, S.N., Ismail, I.S., Foo, J.B., Chan, K.W., Noreen, H., Hisyam,
A.H., Zulfahmi, S., & Ismail, M. (2013). Dillenia suffruticosa exhibited antioxidant and
cytotoxic activity through induction of apoptosis and G2/M cell cycle arrest. Journal of
Ethnopharmacology. 146, 525-535.
Bauer, A.W., Kirby, W.M., Sherris, J.C., & Turck, M. (1966). Antibiotic susceptibility testing by
a standardized single disk method. American Journal of Clinical Pathology. 45(4), 493-496.
Blois, M.S., (1958). Antioxidant determinations by the use of a stable free radical. Nature. 1199–
1200.
97
Chatterjee, K.D., (1975). Parasitology in relation to clinical medicine. 10th ed. 54- 59.
Desmarchelier, C., Bermudez, M.J.N., Coussio, J., Ciccia, G., & Boveris, A. (1997). Antioxidant
and prooxidant activities in aqueous extract of Argentine plants. International Journal of
Pharmacognosy. 35, 116–120.
Harwood, Laurence, M., Moody, & Christopher J. (1989). Experimental organic chemistry:
Principles and Practice (Illustrated ed.). 47–51.
Kuttan, G., Kumar, K.B., Guruvayoorappan, C., & Kuttan, R. (2007). Antitumor, anti-invasion,
and antimetastatic effects of curcumin. Advances in Experimental Medicine and Biology. 595,
173-184.
Lamoral-Theys, D., Pottier, L., Dufrasne, F., Neve, J., Dubois, J., Kornienko, A., Kiss, R., &
Ingrassia, L. (2010). Natural polyphenols that display anticancer properties through inhibition of
kinase activity. Current Medicinal Chemistry. 17, 812-825.
Man, S., Gao, W., Zhang, Y., Huang, L., & Liu, C. (2010). Chemical study and medical
application of saponins as anti-cancer agents. Fitoterapia. 81, 703- 714.
McLaughlin, J.L. (1988). Brine shrimp and crown gall tumors: Simple bioassays for the
discovery of plant antitumor agents. Proceedings, NH workshop, bioassay for discovery of
antitumor and antiviral agent from natural sources. Bethesda. 534, 112-137.
McLaughlin, J.L. (1991). Bench-top bioassay for the discovery of bioactive compound in higher
plants. Brenesic. 34, 1-14.
Meyer, B.N., Ferringni, N.R., Putnam, J.E., Lacobsen, L.B., Nichois, D.E., & Mclaughlin, J.L.
(1982). Brine shrimp: a convenient general bioassay for active constituents. Planta Medica.
45(5), 31-34.
Mitsuhashi, S., Saito, A., Nakajima, N., Shima, H., & Ubukata, M. (2008). Pyrogallol Structure
in Polyphenols is Involved in Apoptosis-induction on HEK293T and K562 Cells. Molecules. 13,
2998-3006.
Oyaizu, M. (1986). Studies on products of browning reactions: antioxidant activities of products
of browning reaction prepared from glucose amine. The Japanese Journal of Nutrition and
Dietetics. 44, 307–315.
Persoone, G. (1980). Proceeding of the international symposium on brine shrimp,Artemia salina.
Universa press, Witteren, Belgium. 1-3.
98
Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant
capacity through the formation of a phosphomolybdenum complex: specific application to the
determination of vitamin E. Analytical Biochemistry. 269, 337–341.
Ronald, R. (1982) Antibiotics-An Introduction. F. Hoffman La Roche & Co.Basle, Switzerland.
70-71.
Vanwagenen, B.C., Larsen, R., Cardellina, J.H., Randazzo, D., Lidert, Z.C., & Swithenbank, C.
(1993). Ulosantoin, a potent insecticide from the sponge Ulosa ruetzleri. Journal of Organic
Chemistry. 58(2), 335-337.
Wolfe, K., Wu, X., & Liu, R.H. (2003). Antioxidant activity of apple peels.Journal of
Agricultural and Food Chemistry. 51(3), 609–614.
Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid contents in
mulberry and their scavenging effects on superoxide radicals. Food Chemistry. 64, 555–559.
Zhiyu, W., Yue, C., Neng, W., Mei, W.D., Wei, L.Y., Feng, H., Gang, S.J., Po, Y.D., Yuan,
G.X., & Jian-Ping, C. (2012). Dioscin induces cancer cell apoptosis through elevated oxidative
stress mediated by downregulation of peroxiredoxins. Cancer Biology & Therapy. 13(3), 138
99
Chapter Four (Results)
4.1 Chemical Studies

4.1.1 Preparation of Crude Extract
Barks of H. macrophyllus was collected from Madabkundo Eco Park and washed with fresh tap
water to remove dirty materials and were dried for several days with occasional sun
shade drying. These were finally dried in an oven for 24 hours at considerably 30˚C
temperature for better grinding. The dried materials were ground into coarse powder
by a grinding machine in the Department of Pharmacy, Gono Bishwabidyalay, Savar,
Dhaka, Bangladesh and stored in air tight container at RT for future use.
About 200 gm of powdered barks of H. macrophyllus was taken in an amber colored extraction
bottle (2.5 liters capacity) and soaked the materials with 100% methanol (1.5 L × 5 times) for 3
days. After extraction of it, 8.36 gm crude methanolic barks extract was obtained.
About 600 gm of powdered barks of H. macrophyllus was taken for hot extraction into soxhlet
extractor with sufficient quantitiy of methanol and its was kept for 20 days and collected the
extracts frequently after a certain preiods of time. After the extraction 20.64 gm of crude
methanolic barks extract was collected.
4.1.2 Qualitative Phytochemical Analysis

The preliminary phytochemical screening of different extractives was done to ascertain the
presence or absence of bioactive components. The presence or absence of saponins, tannins,
glycosides, steroids, alkaloids, carbohydrate and protein has been shown qualitatively in the
Table 4.1.
Table 4.1: Results of qualitative tests of HME and CME of H. macrophyllus.
Phytochemical Test CME HME
Saponins + +
Tannins + +
Glycosides + +
Steroids + +
100
Alkaloids + +
Here,
+ = Present = - Not present
4.3 Quantitative Analysis

4.3.1 Determination of Total Phenolics
The content of total phenolics of HME and CME of H. macrophyllus barks was determined
using FCR. The content of phenolics of the extractives was calculated on the basis of
the standard curve for GA (Gallic Acid) as shown in Table 4.2 and in Fig. 4.1. The
results were expressed as mg of GAE/gm of dried extractives.
Table 4.2: Absorbance of GA (standard) at different concentrations after treatment with FCR
Concentration Absorbance Mean±STD

(µg/ml)
a b c
1.5625 0.303 0.331 0.349 0.33± 0.009
3.125 0.508 0.535 0.541 0.53 ± 0.005
6.25 0.889 0.93 0.971 0.93 ± 0.019
12.5 1.656 1.669 1.744 1.69 ± 0.031
25 3.37 3.39 3.47 33.41 ± 0.033
101
Standard Curve of Galic Acid

4
3.5
Absorbance at 760 nm
3 f(x) = 0.131148100358423 x + 0.106569444444445

R² = 0.999330478785525
2.5
2
1.5
1
0.5
0
0 5 10 15 20 25 30
Concentration (µg/ml)
Fig. 4.1: Standard curve of GA for the determination of total phenolics

The results of phenolics content of HME CME and extracts of H. macrophyllus were
shown in Table 4. And Fig:4.2
Table 4.3: Determination of total phenolic contents of HME and CME extracts of H.
macrophyllus
Mg
Sample Conc. Absorbance mg GAE/gm of dried G
name (µg/ml) A
E
/
g
m
a b c a b c o
f
s
a
m
p
l
e
(
M
e
a
n
102
±
S
T
D
)
HME 25 0.202 0.197 0.206 29.11 27.58 30.33 29.01±1.12
CME 25 0.196 0.193 0.201 27.28 26.36 28.8 27.48±1.00
Total Phenolic Contents HME and CME of Hibiscus

macrophyllus
29.5
29
mg GAE/gm of dried sample
28.5
28
27.5
27
26.5
HME CME
Name of the samples
Fig. 4.2: Determination of total phenolic contents of HME and CME

of H. macrophyllus.
Here, HME and CME are representing as Hot and Cold Extracts of H. macrophyllus
respectively. Values are mean of triplicate experiments and represented as (Mean).
The highest phenolic content was found in HME Extracts of H. macrophyllus (29.01
mg of GAE/gm of dried extract) at a concentration of 25 µg/mL followed by Cold
Extracts of H. macrophyllus (27.48 mg of GAE / gm of dried extract) at same
concentrations. To compare the phenolic content among the HME and CME, it was
observed that HME had a slightly larger amount of phenolic content than that of
103
CME.
4.3.2 Determination of Total Flavonoids
The content of total flavonoids of HME and CME of H. macrophyllus were determined using
well known aluminum chloride colorimetric method using Quercetin as standard. The
flavonoids content of the extractives was calculated on the basis of the standard curve
for Quercetin (shown in Table 4.4 and in Fig. 4.3) and the results were expressed as
mg of QE/gm of extractives.
Table 4.4: Absorbance of Quercetin (standard) at different concentrations for quantitative

determination of total flavonoids.
Conc. Absorbance
(µg/ml) Mean±STD
a b C
1.5625 0.1 0.133 0.137 0.12 ± 0.01
3.125 0.167 0.213 0.146 0.18 ± 0.02
6.25 0.314 0.462 0.327 0.37 ± 0.06
12.5 0.577 0.885 0.551 0.67 ± 0.15
25 1.549 1.901 0.8 1.42 ± 0.45
50 2.714 2.883 1.734 2.44 ± 0.51
104
Standard Curve of Quercetin

3
2.5
f(x) = 0.0486891257995736 x + 0.0678606965174129

R² = 0.994066019545241
2
1.5
0.5
0
0 10 20 30 40 50 60
Concentration (µg/ml)
Fig. 4.3: Standard curve of Quercetin for the determination of total flavonoids
Table 4.5: Determination of total flavonoids contents of HME and CME of H. macrophyllus
mg QE/gm Of
Sample Conc. Absorbance mg QE/gm of dried sample
name (µg/ml) (Mean±STD)
a b c a b c
HME 100 0.253 0.239 0.227 38.26 35.39 32.93 35.53±2.178
CME 100 0.159 0.169 0.163 19 21.05 19.82 19.96±0.842
105
Total Flvonoid Contents HME and CME of Hibiscus

macrophyllus
40
35
mg QE/gm of dried sample
30
25
20
15
10
5
0
HME CME
Name of the samples
Fig. 4.4: Determination of total flavonoids contents of HME and CME of H. macrophyllus
The flavonoids content of HME and CME of H. macrophyllus (shown in Table 4.5 and Fig:4.4)
were 35.53 and 19.96 mg of QE/gm of dried extractives at a concentration of 100 µg/mL,
respectively. Comparing the total flavonoids content among the two extracts (HME and CME); it
was observed that HME of H. macrophyllus contained highest amounts of flavonoids. CME
contain almost half amount less compared to HME extracts.
4.4 In-vitro Antioxidant Activity\

4.4.1 Total Antioxidant Activity
The total antioxidant activity of HME and CME of H. macrophyllus was assessed by
phosphomolybdenum method, based on the reduction of Mo (V1) to Mo (V) by the
standard and the formation of green phosphate/ Mo (v) complex with a maximal
absorption at 695 nm. Total antioxidant activity of HME and CME and standard
Ascorbic Acid was depicted in Table 4.6 and in Fig. 4.5
Table 4.6: Determination of total antioxidant activity of Ascorbic Acid at different

concentrations.
106
Conc.(µg/mL) Absorbance
Mean±STD
a b c
1.5625 0.151 0.154 0.162 0.16 ± 0.004
3.125 0.201 0.194 0.184 0.19 ± 0.006
6.25 0.302 0.278 0.337 0.31 ± 0.024
12.5 0.472 0.448 0.457 0.46 ± 0.009
25 0.886 0.831 0.823 0.85 ± 0.028
50 1.792 1.884 1.781 1.82 ± 0.046
100 3.987 3.836 3.843 3.89 ± 0.069
Total Antioxidant Capacity of Ascorbic Acid

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 10 20 30 40 50 60 70 80 90 100
Concentration (µg/mL)
Fig. 4.5: Total antioxidant activity of Ascorbic acid at different concentrations.

Table 4.7: Determination of total antioxidant activity of HME and CME of H. macrophyllus at
different concentrations.
Sample Conc.(µg/mL) Absorbance Mean±STD

a b c
1.5625 0.119 0.124 0.121 0.12± 0.002
3.125 0.142 0.135 0.13 0.14± 0.005
6.25 0.16 0.151 0.167 0.16± 0.006
107
HME 12.50 0.196 0.186 0.191 0.191± 0.004

25 0.256 0.269 0.264 0.263± 0.005
50 0.397 0.414 0.393 0.401± 0.009
100 0.626 0.638 0.605 0.623 ± 0.012
1.5625 0.135 0.143 0.142 0.140±0.003
3.125 0.153 0.157 0.158 0.156±0.002
CME 6.25 0.198 0.176 0.173 0.182±0.011
12.5 0.219 0.221 0.227 0.222±0.003
25 0.355 0.348 0.351 0.351±0.002
50 0.531 0.513 0.553 0.532±0.016
100 0.737 0.743 0.747 0.742±0.004
Total Antioxidant Capacity

4.5
4
3.5
3
2.5 HME
CME
2 AA
1.5
1
0.5
0
0 20 40 60 80 100 120
Fig. 4.6a: Comparison of total antioxidant activity of HME and CME of H. macrophyllus
with Ascorbic acid.
108
Comparison of total antioxidant activity of HME and CME

with Ascorbic acid
4.5
4
3.5
Mean Absorbance
3
2.5
2 Series 1
1.5
1
0.5
0
AA CME HME
Name of the Sample
Fig. 4.6b: Comparison of total antioxidant activity of HME and CME of H. macrophyllus
with Ascorbic acid
Here, AA= Ascorbic Acid.

The total antioxidant activity of HME and CME of H.macrophyllus were showed in Table
4.7 and Fig. 4.6. Among the two extracts CME had the highest total antioxidant
activity (absorbance of 0.742) followed by HME (absorbance of 0.623) at a
concentration of 100 µg/mL. Comparing the results, it was found that the HME
and CME extracts have quite good but not significant activity comparing to the
antioxidant activity with that of the standard, AA. Our results demonstrated that
both the HME and CME had appreciable antioxidant activity, which was
concentration dependent. The antioxidant activity of HME and CME of Hibiscus
macrophyllus and AA exhibited the following order:
AA>CME>HME
4.4.2 Reducing Power Assessment

The Fe3+ reducing power of HME and CME of H. macrophyllus and standard, Ascorbic
acid was determined by the method described by Oyaizu (1986) with slight
109
modification. The reductive capabilities of HME and CME and AA were shown
in Table 4.8 and 4.9, and in Fig. 4.7 and 4.8
Table 4.8: Determination of Fe3+ reducing power capacity of Ascorbic Acid at different
concentrations.
Conc.(µg/mL) Absorbance Mean±STD

a b c
1.5625 0.13 0.141 0.151 0.141±0.008
3.125 0.171 0.183 0.176 0.177±0.004
6.25 0.235 0.261 0.251 0.249±0.009
12.5 0.32 0.386 0.352 0.353±0.023
25 0.4 0.57 0.48 0.483±0.06
50 0.72 0.85 0.76 0.776±0.047
100 1.45 1.57 1.41 1.476±0.065
Reducing Power Capacity of AA

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Fig. 4.7: Comparison of reducing power capacity of AA at different concentrations.
The ferric reducing capacity of HME and CME of H. macrophyllus was also investigated that
shown here under:
110
Table 4.9: Determination of Fe3+ reducing power capacity of HME and CME at different
concentrations.
Sample Conc. (µg/mL) absorbance Mean±STD

a b
1.5625 0.041 0.035 0.038±0.003
3.125 0.064 0.061 0.062±0.001
6.25 0.117 0.112 0.114±0.002
HME 12.5 0.205 0.213 0.209±0.004
25 0.312 0.319 0.315±0.003
50 0.562 0.551 0.556±0.005
100 0.932 0.943 0.937± 0.005
1.5625 0.089 0.091 0.090±0.001
3.125 0.114 0.119 0.116±0.002
6.25 0.163 0.166 0.164±0.001
CME 12.5 0.255 0.261 0.258±0.003
25 0.367 0.358 0.363±0.004
50 0.588 0.591 0.589±0.001
100 1.123 1.128 1.125±0.002
Among the fractions, the absorbance (1.125) of CME was quite close to absorbance (1.476)
standard, AA at a concentration of 100 µg/mL and both the HME and CME show the
similar reducing capacity comparing to each other. These results demonstrated that
HME and CME of H. macrophyllus had signification reducing capacity, which was
concentration dependent. The reducing power of different extractives and AA showed
the following order:
AA>CME>HME
111
Reducing Power Capability

1.6
1.4
absorbance at 700 nm
1.2
1 HME
0.8 CME
0.6 AA
0.4
0.2
0
0 20 40 60 80 100 120
Conc. .(µg/mL)
F
ig. 4.8a: Comparison of reducing power capacity of HME and CME of H. macrophyllus and AA
at different concentration
Comparison of reducing power capacity of HME and CME and AA

1.6
1.4
Mean Absorbance
1.2
1
0.8
Series 1
0.6
0.4
0.2
0
AA CME HME
Name of the Sample
Fig.
4.8b: Comparison of reducing power capacity of HME and CME of H. macrophyllus and AA at
different concentration
4.4.3 DPPH Free Radical Scavenging Assay

The antioxidant activity of the extractives of H. macrophyllus bark was evaluated by the widely
112
used and most reliable DPPH radical scavenging assay method developed by Blois
(1958). The method is based on the ability of the extractives to scavenge the stable
DPPH radical that contains an odd electron. This radical absorbance at 517 nm and
decolorizes after neutralization by the antioxidants. The activity is increased by
increasing the concentration of the sample extractives. The results of DPPH radical
scavenging assay by standard BHT (butylated hydroxytoluene) were given in Table
4.10 and in Fig. 4.10. The scavenging activity of the IC50 values of BHT 15 µg/mL)
Table 4.10: Determination of DPPH free radical scavenging activity of BHT at different
concentrations.
Conc. % Of scavenging % Of IC50 value
(µg/mL) scavenging (µg/mL)
a b c
(Mean±STD)
6.25 28.23 28.45 28.52 28.4±0.12
12.5 45.32 45.56 45.62 45.51±0.12
15
25 66.54 66.72 66.34 66.53±0.15
50 79.56 79.63 79.12 79.44±0.22
100 86.95 86.61 86.23 86.59±0.29
200 90.22 90.32 90.51 90.35±0.12
The scavenging activity of HME and CME of H. macrophyllus was also determined. The results
of DPPH radical scavenging assays of these two extracts and BHT were given in Table
4.11 and in Fig. 4.11.
113
Standard curve of BHT

100
90
80
70
% of scavenging
60
50
40
30
20
10
0
0 50 100 150 200 250
conc. (µg/mL)
Fig. 4.11: Standard curve of BHT

Table 4.11: Determination of DPPH free radical scavenging activity of HME and CME of H.
macrophyllus at different concentrations.
Sample Conc. % Of Scavenging % of IC50value

(µg/mL) Scavenging
a B c
(Mean±STD)
6.25 30.97 30.33 31.17 30.82±0.35
12.5 57.78 58.11 57.37 57.75±0.31
CME
25 78.62 78.10 77.96 78.23±0.28 12
50 80.97 81.31 80.53 80.94±0.32
100 83.51 82.86 83.23 83.2±0.26
200 84.61 84.95 84.12 84.56±0.34
Sample Conc. % of Scavenging % Of IC50value

scavenging
a B c
(Mean±STD
)
6.25 5.25 5.57 5.11 5.31±0.19
12.5 21.56 21.78 21.17 21.51±0.25
114
25 42.75 42.85 42.37 42.66±0.21 31

HME 50 74.46 74.12 74.73 74.46±0.25
100 77.17 77.53 77.41 77.37±0.15
200 81.71 81.27 81.86 81.61±0.25
Taking absorbance at 517 nm and decolorizes after neutralization by the antioxidants. The activity
is increased by increasing the concentration of the sample extractives.
The results of DPPH radical scavenging assay by CME, HME and standard BHT (butylated
hydroxytoluene) were given in Table 4.10 and in Fig. 4.11. The scavenging activity of the
CME was similar to that of BHT (standard). The IC50 values of BHT, HME and CME
were (15 g/mL, 31 and 12) respectively.
DPPH Free Radical Scavenging Activity

35
30
25
IC50 value
20
IC50 value
15
10
0
BHT HME CME
Name of sample
Fig. 4.12: IC50 (g/mL) values of different extractives of B. insigne leaves and standard, BHT for
DPPH free radical scavenging.
4.5. Evaluation of Cytotoxic Activity by Brine Shrimp Lethality

Bioassay
The cytotoxicity of HME and CME of H. macrophyllus was studied by using Brine Shrimp
115
lethality bioassay (McLughilin et al., 1992; Meyer et al., 1982; Persoone et al., 1980;
McLaughlin et al., 1988; Chatterjee et al., 1975). The cytotoxicity of HME and CME
was studied by using Brine Shrimp lethality bioassay. Here the effect of each
extractive at different concentrations (12.5-800µg/ml) on the mortality of nauplii was
measured. The effect of different Concentrations of VCS (standard) and extractives
on nauplii mortality is presented in Table 4.12 and 4.13 and in Fig. 4.12 and 4.13. The
VCS showed mortality of nauplii when the concentration was lowered gradually from
800 g/ml (100 % mortality) to 6.25 g/ml (16.67 % mortality). The LC50 of VCS
was found to be 138.62 g/ml.
Table 4.12: Effect of VCS (standard) on mortality of Brine Shrimp nauplii at different
concentrations.
LC50
Sample Conc. (µg/mL) Number Dead Live % Mortality
(µg/mL)
0.15625 12 2 11 16.67
0.3125 10 3 7 30.00
0.625 11 4 7 36.36 1.75

VCS
1.25 11 5 6 45.45
2.5 14 8 6 57.14
5 13 10 3 76.92
10 14 14 0 100
116
% Mortality
120
100
80
% Mortality
60
40
20
0
0 1 2 3 4 5 6 7 8 9 10
Conc. (µg/mL)
Fig. 4.12: Effects of VCS (standard) on the mortality of Brine Shrimp nauplii at different
concentrations.
Table 4.13: Effect of HME and CME of H. macrophyllus on mortality of Brine Shrimp nauplii at
different concentrations.
Conc. % LC50
Sample (µg/mL) Number Dead Live
Mortality (µg/ml)
12.5 10 1 9 10
25 10 2 8 20
50 11 3 8 27.27
HME 100 11 5 6 45.45 200.00
200 10 5 5 50
400 11 9 2 81.81
800 10 10 0 100
CME 12.5 10 2 8 20.00 151.46
25 11 4 7 36.36
50 10 4 6 40.00
100 11 5 6 45.45
117
200 11 7 4 63.63
400 10 8 2 80.00
800 12 11 1 91.66
Among the both extractives, the CME showed most potent activity with LC 50 value 151.46
g/ml. The LC50 values of HME are 200 g/ml, respectively. The lower LC50 means
higher toxicity. Our results demonstrated that the both extractives of H. macrophyllus
had significant cytotoxic activity. The cytotoxic activity of both extractives of H.
macrophyllus and VCS exhibited the following order:
VCS > CME> HME
LC50 (mg/mL) values of different extractives of H. macrophyllus

250
200
150
(µg/ml)
LC50
Series 1
100
50
0
VCS CME HME
Name of the samples
Fig. 4.13: LC50 (g/mL) values of different extractives of H. macrophyllus and standard, VCS for
Brine Shrimp lethality Bioassay.
4.6 Evaluation of Anthelminthic Activity

The HME and CME of H. macrophyllus was evaluated for Anthelminthic activity by Earth
118
Worm model at 20, 50 and 100 mg/ml. Standard was taken as Albendazole. Control
was taken as Saline and DMF. The results were shown in (Table 4.14 and 4.15)
(Figures 4.14 and 4.15).
Time in Minutes
Conc.(mg/ml)
Albendazole HME CME
20 12.34 7.56 8.27
50 8.13 5.12 6.29
100 4.55 2.21 3.05
Table4.14: Paralysis time of Earth worms by the effects of HME and CME of H. macrophyllus
and Standard Albendazole
Paralysis time of Albendazole and Extracts

14
12
10 Albendazole
Time in Minutes
8 HME
CME
6
0
10 20 30 40 50 60 70 80 90 100 110
Concentration (mg/ml)
Fi
gure 4.14a: Paralysis time of Earth worms by the effects of Albendazole and HME and CME of
H. macrophyllus
119
Paralysis time of Albendazole and Extracts

5
4.5
4
Paralysis Time
3.5
3
2.5 Minutes
2
1.5
1
0.5
0
Albendazole HME CME
Name of the Sample
Figure 4.14b: Paralysis time of Earth worms by the effect of Albendazole and HME and CME of
H. macrophyllus
Table4.15: Death time for HME and CME of H. macrophyllus and Standard Albendazole
Time in Minutes
Conc.(mg/ml)
Albendazole HME CME
20 16.42 10.34 12.09
50 11.07 7.13 8.41
100 6.39 4.03 5.17
120
Death time of Albendazole and Extract

18
16
14
12
Time in Minutes
10
Albendazole
HME
8 CME
6
0
10 20 30 40 50 60 70 80 90 100 110
Concentration (mg/ml)
Figure 4.15a: Death time of Earth worms by the effects of Albendazole, HME and CME of H.
macrophyllus
Death time of Albendazole and Extract

7
5
Death Time
4
Minutes
3
0
Albendazole HME CME
Name of the sample

Figur
e 4.15b: Death time of Earth worms by the effects of Albendazole, HME and CME of H.
macrophyllus
121
As shown in the results HME of H. macrophyllus showed potent activity when compared to
the CME of the plant H. macrophyllus and the two Extracts are exhibiting
considerable activity (dose dependent) when compared with reference standard
Albendazole. The present research work showed the validity and the clinical use of
HME and CME of H. macrophyllus in the control of anthelminthic activity. However,
further investigation required for chemical and pharmacological properties.
4.7 In-Vitro Antibacterial Activity

4.7.1 Determination of Zone of Inhibition
The antibacterial potential of plant extracts was evaluated according to their zone of inhibition
against various pathogens and the results (zone of inhibition) were compared with the
activity of the standards; Cefuroxime (50 μg/disc). No extract showed activity at 100
µg/disc, 200 µg/disc and 400 µg/disc against both the tested Gram- positive and
Gram-negative bacterial strain is shown in table 4.16.
Name of Standard HME CME
Organism
Bacillus subtilis - - -
Staphylococcus 2.2cm - -
aureus
Klebsiella sp. - - -
Escherichia coli 2cm - -
Salmonella - - -
Table 4.16. Antibacterial activity (zone of inhibition, cm) of HME and CME of
H. macrophyllus at 100 μg/disc, 200 μg/disc and 400 μg/disc and standard at 50
μg/disc.
122
\
Fig4.16 : Antimicrobial test of HME of H. macrophyllus at different concentration
Fig 4.17: Antibacterial activity of CME of H. macrophyllus at different concentration.
4.8 Evaluation of Thrombolytic activity

The HME and CME of the plant H. macrophyllus were evaluated for thrombolytic activity.
Negative Control was taken as water. The results were shown in (Tables:4.17) (Figures 4.18).
Tables: 4.17: Thrombolytic activity of HME and CME of H. macrophyllus

Weight of clot (mg)
Sample Conc.(mg/ml) % of clot lysis
Before lysis After lysis
0.125 0.5443 0.4843 6%
0.25 0.5544 0.4272 12.72%
HME
0.375 0.5514 0.3677 18.37%
0.5 0.5563 0.3013 25.5%
0.125 0.5627 0.4858 7.69%
0.25 0.5597 0.4366 12.31%
CME
0.375 0.5552 0.3721 18.31 %
0.5 0.5606 0.3122 24.84%
Negative control 0 0.5512 0.5382 1.3%
123
Thrombolytic activity of Extracts

0.5 mg/ml 0.375 mg/ml 0.25 mg/ml 0.125 mg/ml water
25.50% 24.84%
18.37% 18.31%
% of Clot lysis
12.72% 12.31%
7.69%
6.00%
1.30% 1.30%
HME CME
Fig
ures 4.18: Percentage of Clot lysis of blood samples of normal subjects by different
concentrations of HME and CME of H. macrophyllus.
The results of effective clot lysis percentage by methanolic extract of H. macrophyllus at four
concen-trations and negative control (water) are given in Table 4.17. The percentage of clot lysis
was 26% to 6% when addition of 0.5 mg/ml, 0.375 mg/ml, 0.25 mg/ml and 0.125 mg/ml conc.
Of HME and CME and water used as a negative control. Negative control showed percentage of
clot lysis very negligible 1.30%. On the basis of these results, it’s shown that four
concentrations (0.5 mg/ml, 0.375mg/ml, 0.25 mg/ml and 0.125 mg/ml) of crude methanolic
extracts induced highly significant (p<0.001) clot lysis activity in vitro of 26%, 18.37%, 12.72
%, and 6% respectively in HME and 24.84%, 18.31%, 12.31%, 7.69% in CME respectively
when comparing with 1.30% clot lysis of water considered as a negative control.
4.9 Reference
Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical. Nature.
181,1199–1200.
Chatterjee, K.D. (1975). Parasitology in relation to clinical medicine. 10th ed. 54- 59.
McLaughlin, J.L. (1988). Brine shrimp and crown gall tumors: Simple bioassays for
124
the discovery of plant antitumor agents. Proceedings, NH workshop, bioassay for

discovery of antitumor and antiviral agent from natural sources. Bethesda. 534, 112-
137.
McLughilin, J.L. (1991). Bench-top bioassay for the discovery of bioactive compound
in higher plants. Brenesic. 34, 1-14.
Meyer, B.N., Ferringni, N.R., Putnam, J.E., Lacobsen, L.B., Nichois, D.E., & Mclaughlin, J.L.
(1982). Brine shrimp: a convenient general bioassay for active constituents. Planta Medica.
45(5), 31-34.
Oyaizu, M. (1986). Studies on products of browning reactions: antioxidant activities

of products of browning reaction prepared from glucose amine. The Japanese Journal
of Nutrition and Dietetics. 44, 307–315.
Persoone, G. (1980). Proceeding of the international symposium on brine shrimp, Artemia

salina. Universa press, Witteren, Belgium. 1-3.
Vanwagenen, B.C., Larsen, R., Cardellina, J.H., Randazzo, D., Lidert, Z.C., &
Swithenbank, C. (1993). Ulosantoin, a potent insecticide from the sponge Ulosa
ruetzleri. Journal of Organic Chemistry. 58(2), 335-337.
125
Chapter Five (Discussion)
5.1 Discussion
Accumulating evidence suggests that many dangerous pathophysiological processes, such as
cancer, diabetes, cardiovascular and neurodegenerative diseases, are associated with the
accumulation of unstable free radicals due to lacking of enough antioxidants and higher
oxidative stress (Gilgun-Sherki et al., 2002; Sharifi-Rad et al., 2020). These unstable radicals
have the tendency to become stable through electron pairing with biological macromolecules
such as proteins, lipids, and DNA in healthy human cells, thus causing protein and DNA damage
(Gilgun-Sherki et al., 2002). Therefore, dietary intake of antioxidants is imperative to protect
cells from damage by scavenging the unstable free radicals (Rahman et al., 2015).
Plants have long been a source of exogenous (i.e., dietary) antioxidants (ARA Rima et al., 2021;
Halliwell, 2007). It is believed that two-thirds of the world's plant species have medicinal
importance, and almost all of these have excellent antioxidant potential (Krishnaiah et al., 2011).
For assessment of antioxidant potential of compounds, a single assay method is not sufficient.
Furthermore, different antioxidant assays vary in terms of assay principle and experimental
conditions. For instant, some methods use organic radical producers e.g., DPPH and some use
metal ions for oxidation e.g., FRAC assay technique. The time factor associated with their
chemical reactions to produce free radicals by oxidation reaction also different from each other.
Since the procedure and experimental conditions are different for different techniques, the
various antioxidants are considered as a control for different assay techniques according to their
rate and time of scavenging. In addition, antioxidants could be polar e.g., phenolics, flavonoids
etc. or non-polar e.g., vitamin E in nature and they can act as radical scavenger by electron
donating mechanism or by hydrogen donating mechanism. Therefore, different coantrol
antioxidants (e.g.,BHT,Quercetin, Ascorbic Acid) were used for different antioxidant assays
(Rahmanet al., 2015).
5.1.1 Qualitative Phytochemical Analysis
Our preliminary phytochemical screening of HME and CME of H.macrophyllus showed the
presence of saponins, tannins, glycosides, alkaloids and carbohydrate. Alkaloids and tannins
were present in large amount in the hot and cold extract of H.macrophyllus. These biologically
125
active constituents provided us beneficial health effect by different mechanisms. Plants

possessing saponins have anti-inflammatory, antifungal, antibacterial, anti-parasitic, anti-cancer,
and antiviral activities (Mugford & Osbourn, 2013; Podolak et al., 2010; Sparg et al., 2004).
Tannins exert several pharmacological effects, including antioxidant and free radical scavenging
activity as well as antimicrobial, anti‐cancer, antinutritional, antibacterial, antidiarrheal,
anti-inflammatory and cardio‐protective properties. They also seem to exert beneficial effects on
metabolic disorders and prevent the onset of several oxidative stress‐relate (Smeriglio et al.,
2017; Sparg et al., 2004).
5.1.2 Phenolic and Flavonoid Contents
Polyphenols are naturally occurring compounds found largely in the fruits, vegetables, cereals
and beverages. Over 8,000 polyphenolic compounds and more than 6,000 flavonoids were
reported in various plants (Amaral et al., 2019). Polyphenols are generally involved in defense
against ultraviolet radiation or aggression by pathogens. Polyphenols may be classified into
different groups as a function of the number of phenol rings. The main classes include phenolics,
flavonoids, stilbenes and lignans. Polyphenols present in plants offered some protection against
development of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative
diseases (Pandey & Rizvi, 2009).
Our results showed that the HME and CME of H. macrophyllus contain significant amount of
phenolics. The higher amount of phenolics (mg of GAE / gm of dried extract) was found in the
hot (29.006 mg) and in cold (27.48 mg),
at a concentration of 25 µg/ml (Table 4.3 and Fig. 4.3). These data indicated that HME and CME
of H. macrophyllus barks is a potential source for phenolic compounds.
Flavonoids, another class of bioactive polyphenols, are reported to have potent antioxidant
potential (Raj et al., 1999). The flavonoids content (mg of Quercetin/gm dried extract) was
found in the CME (19.96 mg) at a concentration of 100 µg/mL. Among the both extracts (Table
4.5, Fig no. 4.5), the highest amount was found in HME (35.53 mg) at the same concentration
(100 µg/mL). This result demonstrated that the H. macrophyllus is a potent source of
antioxidants.
126
5.1.3 Total Antioxidant Capacity
The antioxidant potential of the HME and CME of H. macrophyllus was estimated from their
ability to reduce the reduction of Mo (VI) to Mo (V) by the antioxidant-enriched fractions and
subsequent formation of a green phosphate/Mo (V) complex at acidic pH. Antioxidant activity
depends on the presence of its bio-active compounds mainly polyphenols, carotenoids, and
vitamin E and C (Oktay et al., 2003). This suggests that the concentration of the bioactive
compounds present in the extract is important to showing antioxidant activity. Thus, higher
concentration of bioactive compounds in the extracts shows higher antioxidant activity. In this
study, the antioxidant capacity (in terms of absorbance at 695 nm) of the extractives was in the
range of 0.12–0.742 at the concentration ranges from 1.5625-100 µg/mL. All the extracts
showed good antioxidant activities that gradually increase with increasing concentration (Table
4.7 and Fig 4.7). Our results suggest that the antioxidant capacity can be attributed to the
extractive’s chemical composition and polyphenol contents.
5.1.4 Reducing Power Assessment
Reducing power is also widely used in evaluating antioxidant activity of plant polyphenols. The
reducing power is generally associated with the presence of reductants, which exert antioxidant
action by breaking the free radical chains by donating a hydrogen atom. In this assay, the
presence of reductants in the antioxidant sample reduces Fe 3+/ferricyanide complex to the
Fe2+/ferrous form. Thus, the reducing power of the sample can be monitored by measuring the
formation of Perl’s Prussian blue at 700 nm (Oktay et al., 2003). In this study, the iron reducing
capacity of the HME and CME was estimated from their ability to reduce the Fe 3+/ferricyanide
complex to the ferrous form by donating an electron. The reducing ability of the extracts (in
terms of absorbance at 700 nm) was in the range of 0.038 to 1.125 at the concentration ranges
from 1.5625 to 100 µg/mL. All the extracts showed a good reducing power capacity, which was
concentration-dependent (Table 4.9 and Fig 4.9). We assumed that the antioxidant activity and
the reducing power capacity of the extracts was likely due to the presence of polyphenols, which
127
can act as free radical scavenger by donating an electron.
5.1.5 DPPH Free Radical Scavenging Activity
The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability
(Baumann et al., 1980). Radical scavenging activities are very important to prevent the
deleterious role of free radicals in different diseases, including cancer. DPPH free radical
scavenging is an accepted mechanism for screening the antioxidant activity of barks extracts. In
the DPPH assay, violet color DPPH solution is reduced to yellow colored product,
diphenylpicryl hydrazine, by the addition of the extract in a concentration dependent manner.
This method has been used extensively to predict antioxidant activities because of the relatively
short time required for analysis. Among the extracts, the highest scavenging activity was found
in CME (IC50 value of 12 g/mL) when compared to standard BHT (IC50 of 15 g/mL) (Table
4.11 and Fig 4.11). It has been reported that polyphenol contents scavenge the DPPH radicals by
their hydrogen donating ability (Baumann et al., 1980; Huang et al., 2005). The results obtained
in this study suggest that all the extracts of H. macrophyllus showed radical scavenging activity
by their electron transfer ability. It was reported that the total polyphenols content and radical
scavenging antioxidant activity are highly correlated (Huang et al., 2005).
5.1.6 Cytotoxic Effect on Brine Shrimp
The Cytotoxicity of Hot and cold extract of H. macrophyllus was evaluated by brine shrimp
lethality bioassay. All the extracts showed cytotoxic effect. The LC 50 values of HME and CME
were found to be 200, 151 μg/ml, respectively (Fig. 4.13). Among the both extractives, CME
Extract showed the most cytotoxic activity. The LC50 value of standard VCS was found to be
1.75 μg/ml. The lower LC50 means higher toxicity. On the basis of our results obtained from the
present study, it can be concluded that H. macrophyllus barks extracts possessed remarkable
cytotoxic activities and it reported that cytotoxic property may be due to the presence of
phytochemicals such as saponins and polyphenolic compounds in the extract (Kumar et al.,
2021) and this phytochemical compounds exist in plants exhibited anti-tumorigenic effects via
128
multiple anticancer pathways such as by interaction with key enzymes in cellular signaling
pathways, cell cycle, apoptosis and metastasis etc. (Manzione et al., 2020). Present work was a
preliminary effort which will require further detailed investigation, including characterization of
active compounds.
5.1.7 Evaluation of Anthelmintic
In present study, the barks extract of H. macrophyllus possess significant activity due to the
present of absent responsible phytochemicals. It was observed from the study that, the barks
extract demonstrated anthelmintic activity. The barks extract of H. macrophyllus exhibited
significant dose dependent anthelmintic activity in earthworms in comparison to that of the
standard of albendazole. The findings of this test results revealed that, the extract exhibited not
only paralysis but also death of earthworms and the calculated time for paralysis and death of
earthworms were inversely proportional to the barks extract concentration. Previous studies data
reported that, the presence of phenol, tannins, alkaloids, and terpenoids may be responsible for
exhibit anthelmintic activity (Doughari, 2006; Salhan et al., 2011). In accordance with these
studies, those compounds are present in the HME and CME of H. macrophyllus. That’s why it
exhibited good anthelmintic activity.
5.1.8 Antibacterial Screening
Antibacterial activity was studied using disk diffusion assay method. The HME and CME of H.
macrophyllus had no antibacterial activity against both the tested Gram-positive and Gram-
negative bacterial strain at 100 μg/disc, 200 μg/disc and 400 μg/disc (Table 4.16 and Fig 4.16
and 4.17).
5.1.9 Thrombolytic activity
The phytochemical screening report confirmed that the presence of saponin, tannin and
alkaloids as a natural product in the methanolic extract of H. macrophyllus. From the previous
study, it was reported the presence of phytochemicals in the plant extract having active role in
129
the management of various diseases (Yadav and Agarwal, 2010). This study displays the in
vitro thrombolytic potential of crude methanolic extract H. macrophyllus barks using human
blood. The results show very good thrombolytic activity. This is an important finding which
may have important implications in cardiovascular health (Hossain et al., 2014) because blood
clot formation is considered to be a critical event in which the damaged expanses of the
endothelial cell surface or blood vessel are clogged by the deposition of fibrin, platelets and
tissue factor (Furie and Furie, 2008). In addition, this finding may indicate the possibility of
developing novel thrombolytic agents from the barks of the plant. It was reported that the
presence of phytochemicals like saponin, tannin and alkaloids in the plant extract are the
probable reason for demonstrating the thrombolytic activity (Bhowmick et al., 2014;
Chowdhury et al., 2011). In present study, the barks extract of H. macrophyllus possess
significant thrombolytic activity due to the presence of responsible phytochemicals.
5.2 Reference
Amaral, R. G., Santos, S. A. dos, Andrade, L. N., Severino, P., & Carvalho, A. A. (2019).
Natural Products as Treatment against Cancer: A Historical and Current Vision. Clinics in
Oncology, 4(1562), 1–5.
ARA Rima, R., Ara Rima, R., Islam Sagor, S., & Anjum, A. (2021). In vitro antioxidant
activities of the roots of Coffea benghalensis B Heyne ex Schult. Growing in Bangladesh.
Journal of Pharmacognosy and Phytochemistry, 10(2). www.phytojournal.com
Baumann, J., Wurm, G., & Bruchhausen, F. V. (1980). [Prostaglandin synthetase inhibition by
flavonoids and phenolic compounds in relation to their O2--scavenging properties (author’s

transl)]. Archiv Der Pharmazie, 313(4), 330–337.
Fuchs-Tarlovsky, V. (2013). Role of antioxidants in cancer therapy. Nutrition (Burbank, Los

Angeles County, Calif.), 29(1), 15–21.
Gilgun-Sherki, Y., Rosenbaum, Z., Melamed, E., & Offen, D. (2002). Antioxidant therapy in
acute central nervous system injury: Current state. Pharmacological Reviews, 54(2), 271–284.
130
Halliwell, B. (2007). Oxidative stress and cancer: have we moved forward?

Biochemical Journal, 401(1), 1–11.
Huang, D., Boxin, O. U., & Prior, R. L. (2005). The Chemistry behind Antioxidant Capacity
Assays. Journal of Agricultural and Food Chemistry, 53(6), 1841–1856.
Krishnaiah, D., Sarbatly, R., & Nithyanandam, R. (2011). A review of the antioxidant potential
of medicinal plant species. Food and Bioproducts Processing, 89(3), 217–233.
Kumar, M., Changan, S., Tomar, M., Prajapati, U., Saurabh, V., Hasan, M., Sasi, M.,
Maheshwari, C., Singh, S., Dhumal, S., Radha, Thakur, M., Punia, S., Satankar, V., Amarowicz,
R., & Mekhemar, M. (2021). Custard apple (Annona squamosa l.) leaves: Nutritional
composition, phytochemical profile, and health-promoting biological activities. Biomolecules,
11(5), 614–636.
Manzione, M. G., Martorell, M., Sharopov, F., Bhat, N. G., Kumar, N. V. A., Fokou, P. V. T., &
Pezzani, R. (2020). Phytochemical and pharmacological properties of asperuloside, a systematic
review. European Journal of Pharmacology, 883, 173344.
Mugford, S. T., & Osbourn, A. (2013). Saponin Synthesis and Function. Isoprenoid
Synthesis in Plants and Microorganisms, 405-424.
Oktay, M., Gülçin, I., & Küfrevioǧlu, Ö. I. (2003). Determination of in vitro antioxidant activity
of fennel (Foeniculum vulgare) seed extracts. LWT - Food Science and Technology, 36(2), 263–
271.
Orumwensodia, K. O., Uadia, P. O., & Choudhary, M. I. (2021). Phytotoxicity, cytotoxicity and
chemical composition of H. macrophyllus Stem bark. Clinical Phytoscience 2021 7:1, 7(1), 1–9.
Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidantsin human health
and disease. Oxidative Medicine and Cellular Longevity, 2(5), 270.
Podolak, I., Galanty, A., & Sobolewska, D. (2010). Saponins as cytotoxic agents: a review.
Phytochemistry Reviews: Proceedings of the Phytochemical Society of Europe, 9(3), 425–474.
Rahman, M. M., Islam, M. B., Biswas, M., & Khurshid Alam, A. H. M. (2015). In vitro
antioxidant and free radical scavenging activity of different parts of Tabebuia pallida growing in
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Bangladesh. BMC Research Notes, 8(1), 1-9.
Sharifi-Rad, M., Anil Kumar, N. V., Zucca, P., Varoni, E. M., Dini, L., Panzarini, E., Rajkovic,
J., Tsouh Fokou, P. V., Azzini, E., Peluso, I., Prakash Mishra, A., Nigam, M., El Rayess, Y.,
Beyrouthy, M. El, Polito, L., Iriti, M., Martins, N., Martorell, M., Docea, A. O., Sharifi-Rad, J.
(2020). Lifestyle, Oxidative Stress, and Antioxidants: Back and Forth in the Pathophysiology of
Chronic Diseases. Frontiers in Physiology, 11, 694.
Smeriglio, A., Barreca, D., Bellocco, E., & Trombetta, D. (2017). Proanthocyanidins and
hydrolysable tannins: occurrence, dietary intake and pharmacological effects. British Journal of
Pharmacology, 174(11), 1244–1262.
Sparg, S. G., Light, M. E., & Van Staden, J. (2004). Biological activities and distribution of plant
saponins. Journal of Ethnopharmacology, 94(2–3), 219–243.
Sundaram, S., & Prabhakaran, J. (2017). Effect of 1-methylcyclopropene (1- MCP ) on volatile
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Yang, M. T., Kuo, T. F., Chung, K. F., Liang, Y. C., Yang, C. W., Lin, C. Y.,Feng, C. S., Chen,
Z. W., Lee, T. H., Hsiao, C. L., & Yang, W. C. (2020).
132
133
Chapter Six (Summary)
6.1 Summary
The main purpose of this study was to investigate the phytochemical and pharmacological
activity of the barks of H. macrophyllus.
Phytochemical screening of different extracts confirmed the presence of saponins, tannins,

glycoside, steroids and alkaloids and in the barks of H. macrophyllus. The phenolic
contents of HME and CME are 29.01 and 27.48 mg GAE per gm of dried sample.
The flavonoids contents of HME and CME are 35.53 and 19.96 mg QE per gm of
dried sample. The order of polyphenolic contents of different extractives were HME >
CME. The order of flavonoid contents of different extractives were HME > CME.
The total antioxidant activity of different extractives and standard, AA showed the
order as AA > CME > HME. The reducing power of different extractives and
standard AA showed the order as AA > CME > HME. The DPPH scavenging activity
of different extractives of H. macrophyllus and standards with their IC50 (15 µg/mL)
exhibited the order as CME > BHT > HME. In the evaluation of In-Vitro cytotoxic
effect on brine shrimp nauplii showed that the order of cytotoxicity of HME and CME
and VCS of was VCS > CME > HME. The CME and HME of H. macrophyllus barks
had no antibacterial activity against both the tested Gram-positive and Gram-negative
bacterial strain at 100 μg/disc, 200 μg/disc and 400 μg/disc. In the anthelminthic
activity both CME and HME showed significant activity by comparing with the
standard albendazole and the activity followed as HME > CME > STD. In the
thrombolytic study both the HME and CME showed similar activity.
Finally, we can summarize our findings that the barks of H. macrophyllus is a good source
of antioxidants and had possessed polyphenolics, antioxidant, free radical
scavenging, cytotoxic, thrombolytic and anthelmintic activity. Further studies
are needed to identify the most pharmacologic activity.
133

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We hereby declare that this dissertation entitled “Unveiling

MD. Jahidul Islam

We would like to express our best regards, heartfelt gratefulness, deep

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Enormous thanks to the Department of Microbiology, Gono Bishwabidyalay, for

CHAPTER ONE: GENERAL INTRODUCTION 1-29

GAE Gallic Acid Equivalent

CME Cold Methanolic Extract

HME Hot Methanolic Extract

CHF Chloroform Fraction

AQF Aqueous Fraction

EAF Ethyl Acetate Fraction

BHT Butylate Hydroxy Toluene

DPPH 1,1- diphenyl-2-picrylhydrazyl

IC50 Half Maximal Inhibitory Concentration

FCR Folin-ciocalteu Reagent

FeCl3 Ferric Chloride

FeSO4 Ferrous Sulphate

KCl Potassium Chloride

K2HPO4 Dipotassium Hydrogen Phosphate

H. macrophyllus Hibiscus macrophyllus

NaCl Sodium chloride

STD Standard Deviation

Using a conventional UV-spectroscopic approach, the flavonoids and polyphenols content as

1.1 Oxidative Stress (OS)

OS is essentially an imbalance between the body's capacity to neutralize or detoxify the

1.2 Free Radical

1.2.1 Classification of Free Radical

According to the classification proposed, the radicals may be divided into

I. Primary (superoxide, semi quinones and nitric oxide),

II. Secondary (hydroxyl and lipid radicals) and

III. Tertiary (radicals of antioxidants).

1.2.2 Mechanism of Formation of Free Radicals

❖ By hemolytic cleavage of covalent bond of normal molecule with each fragment

1.2.3 Production of Free Radical in the Human Body

 Some externally generated sources of free radicals are:

 Certain drugs, pesticides

 Ozone (Lobo et al., 2010

1.2.4 Mechanism of Disease Formation by Free Radicals

1.3 Leading Cause of Death

1.4 Oxidative Stress and Human Disease

1.4.1 Oxidative Stress and Cancer

Fig.1.3: Causes of cancer

Fig.1.4: Mechanism of development of Cancer by OS

1.6 Oxidative Stress and Cardiovascular Disease

1.7 Oxidative Stress and Diabetes

1.8 Oxidative Stress Prevention and Treatment

Fig. 1.5: Production of ROS

Increasing levels of endogenous and exogenous antioxidants, reducing exposure to

An antioxidant is a molecule that is stable enough to give an electron to an out-of-control free

1.10.1 Types of Anti-oxidant

Fig. 1.7: Types of antioxidants

1.10.2 Antioxidant Defense System

1.10.3 Mechanism of Action of Antioxidants

Fig. 1.8: Mechanism of action of antioxidants (Modification of Sharma 2014)

1.10.4 Plant Source of Antioxidant

Good sources of specific antioxidants include:

 Allium sulfur compounds – leeks, onions and garlic

1.10.5 Benefits of Natural Antioxidants

1.10.6 Antioxidant and Cancer

Fig.1.9: Mechanism of free radical scavenging by antioxidants.

1.11 Plants as a Source of Drugs in Cancer Therapy