Faculty of Science and Engineering

Department of Pharmacy

Project Report On:

Study of In vitro thrombolytic, membrane stability and antioxidant potential bark of Xylia
Xylocarpa.

Submitted by–

ID No. P-153015

Session: Autumn-2015 to Spring-2019

Department of Pharmacy, IIUC.

A project paper submitted to the department for the partial fulfillment of the degree of
Bachelor of pharmacy (Honors).

A project paper submitted to the Department of Pharmacy, Faculty of Science and

Engineering, International Islamic University Chittagong (IIUC) in partial fulfillment of the
requirements for the degree of Bachelor of Pharmacy (Honors).

1
 ID No: P-153015 Session: Autumn-2015 to Spring -2019 Semester: 8th

External Member
Project Defense Board
……………………… Spring 2019

Mohammed Abu Sayeed

Associate Professor, Department of Pharmacy
Internal Member
International Islamic University Chittagong

DR. Md . Areeful Haqe

Assistant Professor, Department of Pharmacy
Internal Member
International Islamic University Chittagong

Prof. Dr. Abdur Rashid Extern Project Defense Board

University Dhaka External Member

A.T.M .Mostafa Kamal

Assistant Professor &Chairman Chairman
Department of Pharmacy Project Defense Board
International Islamic University Chittagong Spring 2019

DEDICATED

To

My beloved
2
 Parents &Teachers.

Without their Care, affection

And Prayers

I would never succeed.

Acknowledgements
.

First and foremost, I am so grateful to Almighty Allah (SWT), most gracious, most merciful
for giving me strength, patience and ability to perform this project work successfully. I would
like to express my best regards, profound gratitude, and deep appreciation to my honorable
and beloved supervisor A.S.M. Ali Reza Assistant Professor and coordinator, Department of
Pharmacy, International Islamic University Chittagong, for her competent supervision,

3
scientific and inspiring guidance, valuable suggestion, and constructive criticism of my
project work and in preparing this dissertation.
It’s a greater pleasure for me to express my heartfelt thanks to Prof. Dr. Mir Ezharul
Hossain, Dr. Mohammed Akter Sayeed, Associate Professor,Mohammad Abu Sayeed,
Mohammad Tarek, Assistant Professor, &Md. Masudur Rahman Mamun,Assistant
Professorof , Department of Pharmacy for their support during the entire project work the
Department of Pharmacy, IIUC, who were kind enough for sparing their precious time to
arrange the project tools.
I show my gratitude to my classmates, especially to osman sorwer for her cordial helps. I
also say thanks to all the staffs, Lab attendants, & Lab technician of Department of Pharmacy
IIUC, for their help & sincere co-operation.
Finally, I wish to express my sincere gratitude and the obligation to my parents most
especially my dear mother who supports me every step in my life & given constant
encouragement and never-ending affection and blessings.

Abstract
Aim & Objective:
The present study describes of medicinal uses of Xylia Xylocarpa Bark for the selected area
for Thrombolytic and Membrane stability test assay, and In Vitro Antioxidant” of the
methanolic extract.
In-vitro Antioxidant activity of the plant extract was determined by the DDPH free radical
scavenging assay, Reducing sugar, flavonoid assay.
Background
Medicinal plants very important and survival competitive the drug concentration for
development of new drugs. To separate any plant possessing medicinal multiplication,
accurately scientific screening is uniqe. Specifically several type of plants are known to have
various proficiency for treatment others types of diseases. If the right plant is known for

4
healing a particul and other disease, try to should be made to isolate the bioactive lead
molecule(s) from the plant. About 30 some communities are living at others parts of
Bangladesh. The Garo is one of them and sacrifice as an ethnic group of ‘Tibbeti Borman,
belonging to the Mon-golian human race. They are now living in Mymensingh, Tangail,
Netrokona, Sylhet and Sunamgonj districts. They have their own curative practices involving
many medicinal plants as evident from some ethno botanical surveys. This plants can be
methodically appraise to explore their healing efficiency. With this plant view, Xylia
Xylocarpa
Results:
After test result the plant of Xylia Xylocarpa from bark and it gives other effect and activity.
specifically The thrombolytic study, crude methanol extract of rhizom and its aqueous
fraction manifested remarkable it present the effect of clot lysis. The antioxidant assays,
crude methanol extracts of the tested plant parts of the bark and their aqueous fractions
developed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) effect, and decreasing efficacy
activity are the present is so much activity and most effected of anticancer and finally tested
the antioxidant flavonoid scavenging activity are the present this plant positive and proper
intentness. except, this extractives also shown substantial ferric reducing potential in ferric
reducing antioxidant power (FRAP) assay .The membrane stabilizing assay, crude methanol
extracts of rhizomes and fertile foliage fronds and their haemolysis fractions were found to
be very effective for stabilizing erythrocyte membrane in hypotonic solution.. Crude
methanol extracts of the plant parts and their aqueous fractions were also found rich in
phenolic.
Conclusion:The result indicates that, Xylia Xylocarpa have favorable antioxidant and
possess upgrade prevent oxidation, activity .According to the study of the results of this plant
extract may be uses of natural compound.
Keywords:
Xylia Xylocarpa ,In vitro thrombolytic activity ,DPPH scavenging assay, Reducing sugar,
Flavonoid assay activity and Membrane stability.

5
 Contents Page number

Chapter 01: Introduction

1.0.Introduction 12-20

1.1.Thrombolytic 16-17

1.2. In-vitro Antioxidant activity 17-18

1.2.1..Antioxidant 17-18
1.2.2. Types of antioxidant 18-18

1.2.3. Natural antioxidant 18-18

1.2.4. Mechanism action of antioxidant 18-19
1.2.5. Industrial uses of antioxidant 19-19

6
1.3.In-vitro Membrane stability 19-20

Chapter02:Plant Profile 21-23

2.1. Name of the plant 21-21

2.1.1. Synonyms of Xylia Xylocarpa 21-21

2.1.2. Local name 21-21

2.1.3. Taxonomy of the plant 21-21

2.1.4. Used part 22-22

2.1.5. Distribution 22-22

2.1.6. Description 22-23

Chapter 03:Materials And Methods

3.1. Collection of Plant Material and proper identification 24-24

3.2. Preparation of plant material 24-24
3.3. Extraction 24-24
3.4. Reagent and Equipment 24-25
3.4.1. Drugs and Chemicals 24-24

3.4.2. Tools and machines 24-25

3.4.3.In vitro Thrombolytic Activity 25-27
3.5. In vitro antioxidant assay 28 -34

3.5.1. DPPH Free Radical Scavenging Assay 28-28

3.5.1.1. Principle 28-28

3.5.1.3. Preparation of reagents 29-29

3.5.1.4. Procedure 29-30

7
3.6. Determination of Reducing Sugar assay 30-30

3.6.2.1. Principle 30-30

30-31
3.6.2.2. Reagents and materials

3.6.2.3. Preparation of reagents 31-32

32-32
3.6.2.4. Experimental procedure

3.7. Total Flavonoid Content 32-32

3.7.1. Material for Total flavonoid content 32-33

3.7.2. Preparationof Reagents 33-33

3.7.3. Method 33-34

3.8. In vitro Membrane stability 34-34

3.8.1. Principle 34-34

3.8.2. Materials and apparatus 34-35

3.8.3. Exprimental procedure 36-36

Chapter 04: Results

4.1. In vitro Thrombolytic Activity 37-37

4.2. In-vitro antioxidant activity 38-38

4.2.1. DPPH Free Radical Scavenging Assay 38-38

4.2.2. Determination of Reducing Sugar 39-39

4.2.3. Total Flavonoids Content 40 41

4.3. In vitro Membrane Stability 42-43

Chapter 05: Discussions 44-45

Chapter 06: Conclusion 45-45

References 46-47

8
 List of Figure

Figure No. Title of the Figure Page No.

Figure-01 Bark of Xylia Xylocarpa 23-23

Figure-02 Thrombolytic activity of Xylia Xylocarpa 38-38

Figure-03 DPPH radical scavenging activity of Xylia 39-39

Xylocarpa Concentration vs. scavenging effect
Figure-04 Calibration curve of ascorbic acid standard 40-40

Figure-05 Calibration curve of Qutercetin acid standard 41-41

Figure-06 Membrane stability of Xylia Xylocarpa 43-43

List of Table
Table No. Title of the Tables Page No.

Table-01 Thrombolytic activity of Xylia Xylocarpa 37-37

Table-02 DPPH radical Scavenging assay for Xylia 38-38

Xylocarpa
Table-03 DPPH radical Scavenging assay for standard 39-39
Ascorbic Acid
Table-04 Total Reducing sugar content activity of Xylia 39-39
Xylocarpa
Table-05 Absorbance Quercetin acid (Standard 40-41

Table 06 Total flavonoid content activity of Xylia 41-41

Xylocarpa
Table 07 Membrane stability of Xylia Xylocarpa 42-42

9
Table-08 Absorbance erythrocyte membrane 42-43

Chapter :01

10
1.1. Introduction:

Plant is one kind of Extensive number of organisms within the biological kingdom Plantae.
Ordinarily these species are Deliberated a little amount of agility and manufacture their
own food. They comprise a host of familiar organisms including trees, forbs, shrubs, grasses,
vines, ferns, and mosses. The plant connotate a tax on with feature of multicellularity,
cellstructure with walls bearing cellulose, and organisms capable of photosynthesis [1].The
plant and Xylia Xylocarpa traditionally used in the Bark Thickness at Breast Height (mm)
Height
It is also known as loha kat in Bengali. Basically medicinal Plant has been formulated
organs, contains substance that can be used for therapeutic index which is a forerunner for
synthesis of useful drugs[2].The plants express the therapeutic properties and gives
pharmacological effects on the human and animal body are generally designated as
“Medicinal Plants” are no morphological characteristics in the medicinal plants growing with
them.
 Medicinal plants are a momentous natural wealth.
 It is extensively are used in industrial sector.
 They are eagerly available and commonly used.
 They play is momentous a role in health care services.
 They reveal therapeutic properties which important raw materials for the manufacture
of traditional and modern medicine.

1.1.1. Short History of Medicinal Plant

Plants were used for medicinal purposes well before prehistoric times. Ancient Unani
manuscripts mentioned herbal use in Egyptian papyrus and Chinese writings. There is
evidence of herbs being used as medicine by Unani Hakims, Indian Vaids, and European and
Mediterranean cultures for over 4000 years. Indigenous cultures for example in Rome, Egypt,
Iran, Africa, and America used herbs in their rituals of healing, while other traditional
medical systems such as Unani, Ayurveda, and Chinese Medicine were systematically used
for herbal therapies. India among ancient civilisations was known to be a rich repository of
medicinal plants. India's forest is the main repository of large numbers of medicinal and
aromatic plants, largely collected as raw materials for manufacturing drugs and perfumery.
About 8,000 herbal remedies have been codified in INDIA AYUSH programmes. Siddha,
Ayurveda, Unani, Folk and

11
Exporting medicinal plants into other countries can acquire a small amount of foreign
particles.

They play a crucial role in a country's economy.

The various chemicals work simultaneously within the body and produce progressive healing
within the tissues of the body.

Research widely used for the discovery of medicine.

New drug concentration for new deasease is being given to medicinal plant.
It features a pro Traditional medicine as native or folk medicine is also known. It produced
unscientific wisdom highlighting modern medicine. Cultivation known as conventional
medicines includes herbal, Ayurvedic, Siddha medicine, Unani, medicine, Islamic medicine,
traditional Chinese medicine, Mati, Ifá, traditional African medicine and other pseudo-
medical wisdom and study throughout the universe[3].
We examined in these practices that medicinal plants are most inclinable for research and
knowledge of health. WHO (World Health Organization) recently reported that 80% of
people around the world rely on herbal medicines for some part of their primary health care
needs. Around 21,000 plant species have the ability to be used as medicinal plants according
to WHO.
1.1.2. Importance of Medicinal Plants
Medicinal plant treatment is considered to be very safe, since there are no or minimal side
effects. These remedies are in sync with nature, which represents the greatest benefit. The
golden truth is, herbal treatment use is independent of any age group and gender.
The ancient scholars claimed only that herbs were remedies to treat a number of problems
and diseases related to health. They performed thorough study of the same, experimented to
arrive at correct conclusions about the efficacy of various herbs having medicinal value. Most
of the medications developed as such are free from side effects or reactions. This is why
herbal therapy is becoming increasingly popular all over the globe.
Medicinal plants like Aloe, Tulsi, Neem, Turmeric and Ginger cure multiple common
ailments. Those are considered in many parts of the country as home remedies. It is known
that many consumers use Basil (Tulsi) in their daily lives to make medicines, black tea, pooja
and other activities. Besides the medicinal uses, the herbs are also used in natural dyeing, pest
control, food, perfume, tea, etc. Various types of medicinal plants / herbs are used in many

12
countries to keep the ants, flies, mice and flee away from homes and offices. Now a few days
of medicinal herbs are important sources for pharmaceutical production.
Recipes for the treatment of common ailments such as diarrhoea, constipation, hypertension,
low sperm count, dysentery and poor penile erection, piles, coated tongue, menstrual
disorders, bronchial asthma, leucorrhoea and fevers are provided quite effectively by the
traditional medicine practitioners.
There has been a tremendous increase in herbal medicine use over the past two decades; but
there is still a significant lack of research data in this field.

1.1.2. Medicinal Plants in Bangladesh

In our country there are approximately 500 medicinal plants. They have a lot of diversity.
They possess diversity in scale, form, habitat, power of adaptation, flowering, season of
development, effect on human life. We can easily detect them for their diversity as
individuals. We can utilize them for their diversity in several purposes. Main of these is
disease control. This medicinal plants produce most of the antibiotics such as subtilin,
polimixin, penicillin etc. 7 Each group of plants has its own diversity and this diversity is of
particular importance.
1.1.3.Medicinal plants and the Bangladesh Economy

It is estimated that some 12,500 tons of Bangladesh-produced dried medicinal plant material
are sold. These products are worth some Tk 255 million ($4.5 million) for the rural economy,
and at the factory / wholesale rate around Tk.330 M ($5.8 M). The 5,000 tons of imported
medicinal plants cost around Tk 480 m ($8 m). There are believed to be around 350
interdistrict beparis served by 6,000 to 10,000 local collectors, pikers and growers. There are
reportedly around 200 Unani and 200 Ayuverdic registered factories in total 14, plus some 70
homeopathic factories (Dixie et al., 2005).Future medicinal plant prospects in Bangladesh.

Recently, the Bangladeshi government developed development strategies acknowledging the

potential role of traditional healers (Baiddyas & Kabiraj) in rural health care, and proposed
required guidance in a 2008 draft national health policy. Parliament's review of this now. In
addition to this initiative, the government has already established positions for 45 medical
officers named alternative medicines (AMC) in 45 healthcare complexes in Thana to align
this branch with modern healthcare systems. In many Thana Health Complexes a
demonstration plot with essential and commonly used medicinal plants has already been
13
developed for people to be aware of the importance of MP in conventional health care
systems. These state initiatives are generalized and do not generally simply reflect the field
practitioners ' specific demands, as there is essentially no controlling body or structure to
track the entire process. The poor performance in this sector is also responsible for weak
linkages between research organizations and state forest department.

1.1.4. Information Sources of Medicinal Plants

There are many information sources of medicinal plants some of which are described below
in a nutshell.
A. Medical Botany: These are published books and periodicals ascribing the native flora of
various regions of the world and the medicinal uses for each plant. It may also include the
synonyms of plants and the constituents. e.g. Flora of Saudi Arabia (Dr. Mijahid)
B. Official Books: Almost all the countries have their own official books for the medicinals
used in health care system. All the Pharmacopoeias like IP, BP, USP, BPC, Biological
Abstracts, Chemical Abstracts, Merck Index etc.
C. Reputed Books on Medicinal Plants: e.g. Materia Medica, Ibn Sina (Avicina), Al-Antaki,
ESCOP (European Scientific Cooperative for Phytotherapy), Monographs etc.
D. Ethnobotany: This means the study of plants in their relationship to human. Many reports
describing the habitual use and relationship of man and the surrounding flora are available.
E. Herbaria (herbarium): Herbarium is a representative of whole plant or organ of plant
which is preserved to provide a reference specimen when required.
F. Phytopharmacological Surveys: These are the surveys which are concerned with the
biological activities of plant extracts or constituents and they are available in specialized
periodicals or books.
G. Information from Personnel: The sources may be trustful patient, data from hospital,
gardener, agriculture department, Microbiology department etc.
H. Journals: Journals on medicinal plant research may be monthly, quarterly or annually. Few
examples of reputed journals are Planata Medica, Phytochemistry, Ethnopharmacology,
Natural Products, Pharmaceutical Biology etc
I. Online Medicinal Plant Databases: Prelude Medicinal Plants database (Africa), Rain Tree
(Amazon), Chinese Medicinal Plants Database (China), Encyclopedia of Indian Medicinal
Plants (India) are few examples of online Medicinal Plant Databases.

14
1.1. 5. In vitro Thrombolytic Activity:
Thrombolysis, also known as thrombolytic therapy, is a treatment for dissolving harmful clots
in the blood vessels, increasing blood flow and preventing tissue and organs from being
impaired. Thrombolysis may require the injection of clot-busting drugs via an intravenous
line (IV) or a long catheter that delivers drugs directly to the blockage site. Like other
developing countries, thromboembolic disorders are a major cause of morbidity and mortality
in Bangladesh.[1 ] Alteplase, anistreplase, streptokinase, urokinase, and tissue plasminogen
activator are widely used thrombolytic agents for dissolving clots. [2] All current
thrombolytic agents still have major drawbacks, including the need for optimum effectiveness
of large doses, minimal fibrin sensitivity and propensity to bleed. ]. It has been observed in
recent years that heart disease is increasing to a large extent, and the side effects of synthetic
drugs are becoming an ever-increasing therapeutic issue. Quick all of the available
thrombolytic agents still have major deficiencies[5 ]. According to one of the studies, about
30% of pharmaceuticals are made from plants around the world and are considered less
harmful and less free from side effects than synthetic ones. It is therefore necessary to
determine the healthy, little or no side-effective herbal drugs, because natural products of
higher plants can provide a new source of thrombolytic agents and antimitotic agents.

1.1.6. In-vitro Antioxidant activity:

Various methods such as DPPH free radical scavenging assay, phenol, flavonoid assay, have

calculated the antioxidant function of plant extracts. They dissolved the compounds in

methanol. Excessive free radical development directly destroys biological molecules such as

DNA, protein, lipid, carbohydrate etc. An antioxidant can inhibit oxidation of other

molecules. Oxidation is a chemical reaction in which electrons or hydrogen are transferred

from one substance to another. In order to help decide in a cell it generated free radicals and

these radicals. It can occasionally cause cell damage or death[4].

1.1.6.1. Antioxidant
Antioxidants are compounds which inhibit oxidation and cause many damage to organisms in
the cells. Antioxidant such as thiols or ascorbic acid, and the oxidative stress balance.
Internally, it produced antioxidant overlap between plants and animals, such as glutathione
and co-enzymes. Antioxidants are those substances that prevented oxidation and stored foods.
It can prevent unstable molecules which produced certain disease.

15
 Oxidation reaction can form free radicals and these reactions can cause damage or
death the cells.
 Antioxidants terminate these chain reactions by removing free radical intermediates
and inhibit other oxidation reactions.
 For example: superoxide, hydroxyl and peroxide are attach to the free radicals that
helps to prevents cell destruction.

1.1.6.2. Types of antioxidant:

There are three primary antioxidants found in nature. These are given below:
 Phytochemical
 Vitamin
 Enzyme
Phytochemicals are antioxidants which protect against free radicals used by plants. The
human body contains no antioxidant vitamins, and is a daily intake. Common antioxidant
vitamins include vitamin A, vitamin C, vitamins E and folic acid. Enzymes come from
protein and minerals, and are synthesized in the body of man. The antioxidant in cocoa and
dark chocolate has been associated with health benefits such as reduced risk factors for heart
disease, lower inflammation and lower blood pressure.
1.1.6.3. Natural antioxidant
There are three classes of natural antioxidants: vitamins, carotenoids, and phenolic
compounds. It can be contained in particles from food such as fruits and vegetables.
Glutathione is the most powerful and important antioxidant in body tissues which helps to
determine. It also makes fat-soluble vitamin E or vitamin C. Antioxidants are a set of
chemical substances which, through food or supplements, inhibit oxidation and the enzyme
system. It prevents degenerative illnesses like dementia and Alzheimer's disease. Coenzyme
is a product that is anti-aging and useful for health care. It plays a role in regulating the
absorption of cellular energy and inhibiting oxidation properties. It was synthesized by our
body through certain foods, such as meat, fish, oilseeds and spinach. During high fat meals, it
is easily absorbed into body function.
1.1.6.4. Mechanism action of antioxidant
Antioxidants are effected by several common mechanisms, including scavenging species that
initiate peroxidation, quenching single-point oxygen, chelating metals, breaking free radical
chain reactions, and reducing O2 concentration. Antioxidant molecules are not all equally

16
powerful in reacting to these varied mechanisms. Phenolic acids, for example, are effective at
trapping free radicals but not as good at chelating metals while flavonoids can do both
efficiently–scavenge free radicals and chelate metals[5].
Antioxidant helps the human body protect cells from free radicals formation. They produced
natural products, minerals, vitamins etc.

1.1.6.5. Industrial uses of antioxidant

 It is widely used in industrial products such as rubbers, plastic.
 Commonly used as stabilizers in fuels and lubricants to prevent oxidation.
 In gasoline to prevent polymerization that leads to the formation of engine-fouling
residues.
 Antioxidant enzymes like superoxide dismutase, catalase prevent oxidation by chain
initiation.
 It prevent damage and loss of strength.
 Used in food and cosmetic.

1.1.7. In-vitro Membrane Stability Activity

Freshly collected into k3 EDTA (F.L. Medical s.r.l. torreglia Italy) tubes were two milliliters
of blood from healthy volunteers and patients with serologically confirmed acute dengue viral
infections. All samples of blood were kept at 4 ° C 24 hours before use (Awe, Makinde et al.
2009). An aliquot of 1.0 ml micro-centrifuge tubes, at 2500 rpm for 5 min, was centrifuge
and the supernatant drained. The cell suspension was washed for 5 min at 2500 rpm with
sterile saline solution (0.89 per cent w / v NaCl) and centrifuge. This was repeated three times
until the supernatant was transparent and colorless, and measured the volume of the packed
cell (PVC). The cellular portion was reconstituted with phosphate buffered saline (10 Mm,
pH 7.4) at a 40 percent suspension (v / v) and used in the assay.(Ranasinghe, Ranasinghe et
al, 2012).

Preparation of Human Red Blood Cells Suspension

Crisp whole human blood was collected and mixed with equivalent volume of sanitized
Alsever arrangement (2% dextrose, 0.8% sodium citrate, 0.05% citrus extract, and 0.42%
sodium chloride in water). The blood was centrifuged for 10 min at 3000 rpm and stuffed

17
cells were washed with iso saline several times (0.85 per cent pH 7.2). Blood volume has
been measured and reconstituted as 10 per cent v / v iso saline suspension (Chippada, Volluri
et al. 2011)

Chapter: 02

2. Plant profile

2.1. Name of the plant: Xylia xylocarpa.

18
 2.1.1. Synonyms of Xylia xylocarpa
 Acacia xylocarpa (Roxb.) Willd.

 Inga   xylocarpa (Roxb.) DC.

 Mimosa  Xylocarpa Roxb.

 Xylia dolabriformis Benth

2.1.2. Local name

This tree is found in South and Southeast Asia; it is known as Pyinkado (Burmese: in
Myanmar, Căm xe in Vietnam, Sokram in Cambodia and Jamba in Karnataka (India). It has
also been planted in certain parts of East Africa. Xylia xylocarpa 

2.1.3. Taxonomy of the plant

Kingdom : Plantae

Order: Fabales

Family: Fabaceae

Subfamily: Caesalpinioideae

Genus: Xylia

2.1.4. Used part: Bark

19
2.1.5. Distribution
Forest of Chittagong and Chittagong Hill Tracts area .
Originally, it was derived from the Society Islands and it is tropical countries. It following
ranges are indicated:
 Bangladesh
 India
 Myanmar
 Vietnam
 Combodia.
 Karnataka
 East africa

2.1.6. Description
Xylia xylocarpa is a species of plant in the  family  Fabaceae,  This perennial tree is
very conspicuous in the flowering season owing to its bright yellow flowers.
X. xylocarpa  produces hardwood, and in Vietnam it is classified as an 'ironwood' with
its name referring to use in traditional cart making. The cross-section of a trunk has a
distinctive yellowish-white and thick outer layer, with a crimson-dark core of fine
grain and high density (1.15 with 15% moisture content). The wood pulp may be
used for making wrapping paper .

20
 Figure1.1. Xylia Xylocarpa (Bark)

Xylia xylocarpa is a deciduous tree with a dense crown usually growing 20 - 40

Meters tall, though on dry and poor sites the tree may be smaller and the bole
crooked The bole is usually straight and cylindrical, sometimes with small buttresses.
It can be unbranched for 12 meters or more and around 40 - 60cm in diameter.

Chapter : 03

3. Material and Method

3.1. Collection of Plant Material and proper identification

Bark of the plant Xylia Xylocarpa collected from Hathazari area, Chittagong, Bangladesh in
the month of November 2019.

3.2. Preparation of plant material

 Firstly the plants are collected in fresh state.

21
 Then these are cut into small pieces if necessary, to make it suitable for grinding
purpose.
 The Bark were dried for a period of 20 days under shade and ground.
 The materials are grinded into coarse powder with the help of a grinder and stored in
an air tight container for further use.

3.3. Extraction
Extraction is a separation process which determined acute measuring phases:
 Liquid-liquid extraction which used to separate compound or metal complexes, water
and organic solvent.
 Solid- solid extraction which is a sample preparation and separated from one
compound from other compounds according to physical or chemical properties.
 Acid- base extraction which is a procedure that using sequential liquid- liquid
extraction to purify acid and base on their chemical properties.
 Occasional shaking stirring
 Filtered through a cotton plug followed by Whitman filter paper number.
 The solvent was evaporated with water bath to yield viscous mass.
 The viscous mass was kept at room temperature under a ceiling fan to get a dried
extract.
 The prepared extract kept for further pharmacological screening [10].

3.4. Reagent and Equipment

3.4.1. Drugs and Chemicals
 Streptokinase
 DMSO
 Methanol
 Ascorbic acid

3.4.2. Tools and machines

 Blender
 Water bath
 Incubator

22
 Beaker
 Test tubes
 Micropipette
 Aluminum foil
 Feeding gavage
 Syringes
 Eppendorf tube
 Price tag
 Vials
 UV Spectrophotometer

3.5.In vitro Thrombolytic Activity:

3.5.1: Principle:
Thrombus (blood clot) developed in the circulatory system due to failure of hemostasis
causes vascular blockage and leads to serious consequences in thrombolytic diseases such as
acute myocardial or cerebral infarction which may cause death . Thrombolytic drugs are used
to dissolve blood clots in a procedure termed thrombolysis . Alteplase, anistreplase,
streptokinase, urokinase and tissue plasminogen activator (tPA) are commonly used
thrombolytic agents to dissolve clots. Heparin and Aspirin are only moderately efficient for
acceleration of lysis and prevention of reocclusion, but are safe. Continued investigation in
this area will provide new insights and promote progress towards the development of the
ideal thrombolytic activity which are characterized by maximal coronary arteral
thrombolysis with minimal bleeding. Selective third generation thrombolytic activity such as
monoteplase, tenecteplase, reteplase etc.
3.5.2. Chemicals and Materials
 Streptokinase (STK) vials of 1500000 I.U
 5 blood (5 mL) sample drawn from healthy volunteers
 Plant extract
 Epindrop
 Micropipette
3.5.3. Sample preparation

23
The crude extract was suspended in 10 mL distilled water and shaken vigorously on a vortex
mixer. Then the suspension was kept overnight and decanted to remove the soluble
supernatant, which was filtered through a filter paper. The solution was then ready for in vitro
evaluation of clot lysis activity.
3.5.4. Streptokinase (SK) solution preparation
To the commercially available lyophilized SK vial (Polamin Werk GmbH, Herdecke,
Germany) of 15,00,000 I.U., 5 ml sterile distilled water was added and mixed properly. This
suspension was used as a stock from which 100 μl (30,000 I.U) was used for in vitro
thrombolysis.
3.5.5. Specimen
Whole blood (5 mL) was drawn from healthy human volunteers (n=05) without a history of
oral contraceptive or anticoagulant therapy. 500 μL of blood was transferred to each of the
five previously weighed alpine tubes to form clots.
3.5.6. Thrombolytic assay
Experiments for clot lysis were carried as reported earlier. Venous blood drawn from healthy
volunteers was transferred in different preweighed sterile eppendorf tubes (500 μL/tube) and
incubated at 37 °C for 45 minutes. After clot formation, serum was completely removed
(aspirated out without disturbing the clot formed). Each tube having clot was again weighed
to determine the clot weight (clot weight = weight of clot containing tube-weight of tube
alone). Each eppendorf tube containing clot was properly labeled and 100 μl of plant extract
was added to the tubes. All the tubes were then incubated at 37 °C for 90 minutes and
observed for clot lysis. After incubation, fluid obtained was removed and tubes were again
weighed to observe the difference in weight after clot disruption. Difference in weight taken
before and after clot lysis was expressed as percentage of clot lysis. Streptokinase and water
were used as positive and negative control, respectively.
% clot lysis = (Weight of the lysis clot /Weight of clot before lysis) × 100.

15) Concentration 250 µmL Preparation- take 2.5 mL Solution from concentration 500 µmL
in test tube +2.5 mL Distilled water
16) Concentration 125 µmL Preparation- take 2.5 ml Solution from concentration 250 µmL
in test tube +2.5 mL Distilled water
17) Similar way, we can prepare 62.5 concentration test tube.
18) All the test-tube incubated 37 degree Temperature for 30 minutes.
19) After Incubation centrifuge at the 3000rpm for 15 minutes (without Control 1 & 2).
24
20) Then Serum is Collected Observe absorbance at 560 nm.
21) Collect the Value of Abs.

Extract Absorbance−Control absorbance

22) % of stabilizing activity ¿ x100
Control 1 Absorbance Value

3.7. In vitro Antioxidant Assay:

A number of assays were used for measuring the antioxidant potential of the test samples.
Depending on the mechanism, methods for the evaluation of antioxidant activities of the
extracts were divided into two categories:
1. Methods determining the ability of test samples to donate an electron to any electron
acceptor.
2. Methods which determining the ability of a sample to inhibit the enzymes, which
produce reactive oxygen species [11].

3.6.1. DPPH Scavenging Assay

Barca et al described a procedure that facilitates the determination of sample’s ability to free
radical scavenging measuring the change in absorbance of DPPH (2,2-diphenyl-1-
picrylhydrazyl) at 517 nm by the spectrophotometer.

3.6.1.1. Principle
(2,2-diphenyl-1-picrylhydrazyl) is widely used as a free radical which can determine the
scavenging capacity of a corresponding antioxidant. DPPH free radical is produced to the
corresponding hydrazine when it reacts with hydrogen donors . DPPH produces free radicals
that stay stable in the methanol or aqueous solution.

25
This process effects the absorbance data with gradually increasing amount of sample solution
having antioxidant properties, absorbance falls gradually. As the redox reaction proceeds,
color of the solution turns from purple to yellow.
The absorbance is taken at 517 nm by spectrophotometer and the serial sample concentration
gives different data accordingly. The data having lower concentration, higher absorbance and
higher concentration and lower absorbance are further calculated to determine the antioxidant
properties.
3.6.1.2. Reagent and Apparatus
 DPPH
 Methanol
 Ascorbic acid as standard
 Test tubes
 Pipette
 Beaker
 Spectrophotometer
3.6.1.3. Preparation of Reagents
Preparation of DPPH solution (0.004% w/v)
4 mg/0.004 gm DPPH was dissolved in 100 mL of methanol (95%) in order to prepare
0.004% (w/v) solution and kept in dark room.
Preparation of Standard Ascorbic acid solution (0.05%)
2 mg of ascorbic acid was dissolved into 4 mL of methanol. Thus, the concentration of the
solution was 0.05% (w/v) or 500 µg/mL which is called stock solution. Then serial dilution
was performed in order to prepare different concentration solution (250 µg/mL, 125 µg/mL,
62.5 µg/mL, 31.25 µg/mL and 15.63 µg/mL).
Preparation of Stock Solution
2 mg of plant extract was dissolved into 4 mL of methanol. Thus, the concentration of the
solution was 500 µg/mL. Then serial dilution was performed in order to prepare different
concentration solutions (250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL and 15.63
µg/mL).
Preparation of Control
For negative control, 3 mL of DPPH was used. Methanol was used as blank solution.
3.6.1.4. Experiment procedure
 2 mL solution of plant extract at different concentrations in the test tubes.

26
 3 mL methanol solution of DPPH was added into the test tubes.
 The test tubes were incubated at room temperature in dark environment for 30
minutes.
 Then the absorbance of the solution was measured at 517 nm using spectrophotometer
against blank.
 A typical blank solution contained all reagents except plant extract or standard
solution.
 The percentage (%) of inhibition activity was calculated from thr following equation:
%I= [(A0-A1)/A0]×100
Where,
A0= The absorbance of control
A1= The absorbance of the extract/standard
Then, % inhibitions were plotted against concentration and from the graph IC50.
3.6.2. Reducing Power Capacity
The reducing power of Xylia Xylocarpa was evaluated by Oyaizu method.
3.6.2.1. Principle
In this test, the yellow color of the test solution is converted into green or blue as the
oxidation goes on. The color however determines the degree of reaction occurred by the
antioxidant substance. This color also works as an indicator while Fe 3+ (-ferricyanide)
reduced into Fe2+ ferrous by reducing agent. The amount of Fe 2+ can be measured by taking
the absorbance of Perl’s Prussian blue at 700 nm.
Fe3+ + (-ferricyanide) + e Fe2+ + (-ferricyanide)
3.6.2.2. Reagents and Materials
 Potassium ferricyanide [K2Fe(CN)6]
 Trichloro acetic acid, Ferric chloride [FeCl3]
 Phosphate Buffer [K2HPO4 + KH2PO4]
 Ascorbic acid
 Water bath
 Centrifuge machine
 Pipette
 UV spectrophotometer
3.6.2.3. Preparation of Reagents
Preparation of 0.2 M, (pH 6.6) phosphate buffer

27
3.48 g DI potassium phosphate (K2HPO4) and 2.72 g monopotassium phosphate (KH2PO4)
was taken in a 100 mL volumetric flask and small amount of distilled water was added and
dissolved in it. The final volume was made up to the mark by adding required amount of
distilled water.

Preparation of 1% potassium ferricyanide

1 gm of potassium ferricyanide was taken in a 100 mL volumetric flask and small amount of
distilled water was added and dissolved in it. The final volume was made up to the mark by
adding required amount of distilled water.

Preparation of 10% trichloroacetic acid (TCA)

10 gm of trichloroacetic acid was taken in a 100 mL volumetric flask and small amount of
distilled water was added and dissolved in it. The final volume was made up to the mark by
adding required amount of distilled water.

Preparation of 0.1% ferric chloride (FeCl3)

0.1 gm/100 mg of ferric chloride was taken in a 100 mL volumetric flask and small amount
of distilled water was added and dissolved in it. The final volume was made up to the mark
by adding required amount of distilled water.

Preparation of standard
2 mg of ascorbic acid was mixed with 4 ml of methanol, so the concentration of the solution
was 500 µg/mL. This is called the stock solution. The serial dilution was performed in order
to prepare different concentrated solution ( 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25
µg/mL, 15.625 µg/mL).

Preparation of test sample

2 mg of plant extract was mixed with 4 ml of methanol, so the concentration of the solution
was 500 µg/mL. This is called the stock solution. The serial dilution was performed in order
to prepare different concentrated solution (250 µg/ml, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL,
15.625 µg/mL).

Preparation of blank

28
Blank consists of all the reagents, except for the test sample or standard is substituted by 1
mL of methanol.

3.6.2.4. Experiment Procedure

 2 mL of test sample or standard (at different concentrations 500 to 15.625 µg/mL)

was mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2.5
mL, 1%).
 The mixture was incubated for 20 min at 50oC to complete the reaction.
 A portion (2.5 mL) of tri chloro acetic acid TCA (10%) was added to the mixture,
which was then centrifuged at 3000 rpm for 10 min.
 The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and
FeC13 (0.5 ml, 0.1%).
 Then the absorbance was measured at 700 nm by using UV-visible spectrophotometer
against blank.

3.6.3. Determination of Total Flavonoid Content (TFC)

3.6.3.1. Principle
The content of total flavonoid content in the sample determined by aluminium chloride color
metric method .flavonoid are natural antioxidants found in plant as phytochemical the
hydroxyl group present in the flavonoid forms complex with the aluminium.The absorbance
is taken in 420 nm by using spectrophotometer.

3.6.3.2. Reagent and apparatus

 Aluminium chloride (Alcl3 )

 Potassium acetate (1M)
 Methanol

29
 Quercetin (Reagent grade)
 Micropipette (10-100)
 Pipette (1-10) and UV spectroscopy.

3.6.3.3. Preparation of reagents

Preparation of 10 % AlCl3: 10 gm of AlCl3 was taken in a volumetric flask and small amount
of distilled water added up to 100 mL .

Preparation of 1M potassium acetate:

9.815 gm of potassium acetate was taken in a volumetric and distilled water added up to
100mL .The concentration is 1M.
Preparation of standard:
1mg per mL quercetin was required to prepare a solution which have the concentration of
100mg/ml. Serial dilution was performed to prepared different concentration solution
(12.5mg/mL.100mg/mL.

Preparation of blank solution:

Blank solution is prepared by adding all the reagents except extract and standard which is
replaced by o.25mL of ethanol.
Preparation of extract solution: Extract solution was prepared by the concentration of
1000mg/mL.

3.6.3.4. Experimental procedure:

1) 1 mL of standard of different concentration was taken in test tube.

2) 3 mL of methanol was added into the test tube.
3) 200µL of 10%aluminium chloride was taken in the test tube.
4) 200 µL 1Mpotassium acetated was added in the test tube.
5) 5.6 mL of distilled water was added into the test tube.
6) The test was then incubated in room temperature for 30 minutes to complete the
reaction.
7) The absorbance was taken against blank in 415 nm in UV spectrophotometer a typical
blank contained all reagents except plant extract or standard solution

30
 C =(c ×V)

Where,
C= Total content of flavonoid compounds ,mg/g plant extract ,in Quercetin equivalent
(QE)
C =The concentration of gallic acid established from the calibration curve ,mg/ml
V =The volume of extract, mL.
m =The weight of pure plant extract, gm.

m= Weight of the fraction

3.7. In Vitro Membrane Stability:

The method of Sadique et al. (1989) as modified by Oyedapo and Famurewa (1995) and
Oyedapoetal. (2004) was employed in the membrane stabilizing activity assay.
3.7.1:Instrument:
1) Centrifuge Machine
2) 7 Test tube
3) 1 Pipette
4) Glass Rod
5) Cose Tape
6) Spectrophotometer and Cell
7) Vials
8) Test tube stand
9) Incubator -30 minutes, 37 Degree temperature
10) 5 Beak

3.7.2: Materials:
1) Alsever Solution (100mL)
Dextrose-2gm
Na-Citrate-0.8gm
Citric Acid-0.5gm

31
 NaCl -0.42%
Distilled water-Up to 100mL
2. Iso Saline - (100mL)
NaCl -0.85gm
Distilled Water Upto100 mL
Maintain PH -7.2
3. Hypo saline-(100 mL)
NaCl -0.36gm
Distilled Water Up to 100mL
4. .Phosphate Buffer-(100mL)
Na2HPO4-0.26gm
NaH2PO4 -0.14gm
Distilled water Up to 100mL
5. RBC Solution Preparation:
Blood Sample at least 2 mL
10 mL RBC is Dissolve in=100 mL Distilled Water
1 “ “ “ “ “=100/10 “ “
2 l“ “ “ “ “=10*2 “ “
=20 “ “
So, 18mL Distilled water+2mLBlood Solution=20mL RBC Solution
6. Extract Solution:
10 mg extract +10 mL Distilled water

3.7.3. Procedure:
1) 4 Solution need to be Prepared in 4 Separate Beaker (Alsever/Isosaline/Hypo
saline/Phosphate Buffer).
2) Alsever solution prepared immediately before blood Collection.
3) Then RBC Solution needs to be Prepare.
4) Same amount of RBC Solution + Same amount of Alsever solution need to take in
Centrifuge tube. It needs to Centrifuge at 3000 rpm for 15 minutes. then withdrawn Serum
level and add same amount of Isosaline (same as Serum level). then again centrifuge the
mixture at 3000 rpm for 15 minutes.
5) After that again withdrawn Serum level and add same amount of Isosaline (same as Serum
level).then again centrifuge the mixture at 3000 rpm for 15 minutes.

32
6) Total 4/5 times needs to repeats the process.
7) Then RBC needs to preserve in Refrigerator at 4 Degree Temperature.
8) Two Control Needs to prepared in Two Separate Test tube.(e.g Control 1,Control 2)
9) CONTROL 1 Contain - 2 mL Hypo saline, 1 mL phosphate Buffer, 1 ml Extract solution
(total 4 ml solution).
10) CONTROL 2 Contain - 2 ml Hypo saline, 1 mL phosphate Buffer, 1 ml Iso saline 1 mL
Extract solution (total 5 mL solution).

11) 5 test tube needs to mark at concentration 1000, 500, 250, 125, 62.5uml on test tube wall.

12)1000 µmL needs to take in test tube-1ml RBC solution, 1 mL Extract Solution, 2 mL
Hypo saline, 1 mL phosphate Buffer (total solution 5 mL).

13) By using Serial Dilution prepare concentration 1000, 500, 250, 125, 62.5µmL.

14) Concentration 500 umL Preparation- take 2.5 mL Solution from concentration 1000 µmL
in test tube +2.5 mL Distilled water

Chapter: 04
4. Result
4.1. Thrombolytic activity:
Addition of 100 μL SK, a positive control (15,00,000 I.U.) to the clots along with 90 minutes
of incubation at 37°C, showed 42.52% clot lysis. Clots when treated with 100 μL sterile
distilled water (negative control) showed only negligible clot lysis (2.68%). The in-vitro
thrombolytic activity study revealed that Xylia Xylocarpa showed 14.572%. This activity
shown the table 1.
Table.1: Clot lysis by saline water and methanolic extract of Xylia Xylocarpacopared with
streptokinase

33
 Empty Weight of Weight of Weight of Final Percentage Mean
weight clot with clot, clot with weight of (%) of clot (%)
of tube, tube, C=B-A tube after lysis clot, lysis
A B (gm) lysis, E=B-D
(gm) (gm) D (gm)
(gm)

0.78 1.00 0.22 0.99 0.01 4.55

0.80 1.14 0.34 1.13 0.01 2.94

0.79 1.02 0.23 1.00 0.02 8.7 14.572

0.82 1.22 0.4 1.10 0.12 30

0.79 1.14 0.3 1.06 0.08 26.67

Group % of clot lysis

Streptokinase 42.52
Extract 14.57
Distilled water 2.68

% of clot lysis
50
42.52
40

30 % of clot lysis
20 14.57
10
2.68
0
Streptokinase Extract Distilled water

Figure.2: Clot lysis by distilled water and methanol extract of Xylia xylocarpa compared

with Streptokinase.

34
4.2. In-vitro Antioxidant Activity:

4.2.1. DPPH Free Radical Scavenging Assay:

The Results for the DPPH free radical scavenging activity of methanol extract of shown in
(Figure-3). The extract showed a dose dependent radical scavenging effect in DPPH assay.
The half inhibition concentration (IC50) for free radicals achieved by the extract is 47.27
μg/ml which is statistically significant compared to that (IC 50290.209 μg/ml) of ascorbic acid
which is shown in (Table-2&3) So, comparison with the ascorbic acid, it is clear that plant
extracts possess anti radical activity.

Table.2: DPPH free radical scavenging of ascorbic acid

Serial Concentration Absorbance % inhibition of ascorbic

No. (µg/mL) (nm) acid IC50
1 25 0.53 21.73
2 50 0.34 49.59
3 100 0.26 62.78 92.63
4 200 0.18 74.17
5 400 0.12 82.46
Table.3: DPPH free radical scavenging of methanolic extract of Xylia xylocarpa

Serial Concentration Absorbance % inhibition of crude

IC50
No. (µg/mL) (nm) extract
1 25 0.42 19.33  
2 50 0.32 35.8  
3 100 0.23 53.2 140.34
4 200 0.15 70.6  
5 400 0.09 82.2  

35
 90
80 f(x) = 0.13
0.15 x + 38.05
28.7
70 R² = 0.69
0.83
60
% inhibition

50 % inhibition of ascorbic
40 acid
30 Linear (% inhibition of
20 ascorbic acid)
10
0
0 50 100 150 200 250 300 350 400 450
Concentration (µg/mL)

Figure .3: DPPH free radical scavenging of Xylia Xylocarpa in comparison to ascorbic
acid (standard).

4.2.2. Reducing sugar assay:

In reducing power assay, the obtained result showed the proportional increase in reducing
power with the increase of extract concentration which shown in figure-4 .The absorbance
Xylia Xylocarpa is increasing when concentration is increasing which indicates the increase
of reducing power .Various absorbance different is given in table

Table.4: Effect of reducing sugar capacity of Xylia Xylocarpa

Concentration
Absorbance of Ascorbic acid (nm) Absorbance of Extract (nm)
(µg/mL)
31.25 0.43 1.023
62.5 0.72 1.153
125 1.01 2.472
250 1.35 2.5
500 1.59 3

3.5
3 f(x) = 0 x + 1.27
R² = 0.72
Absorbance (nm)

2.5
Absorbance of Ascorbic acid
2 (nm)
1.5 f(x) = 0 x + 0.58 Linear (Absorbance of
1 R² = 0.85 Ascorbic acid (nm))
Absorbance of Extract (nm)
0.5
Linear (Absorbance of Extract
0 (nm))
0 100 200 300 400 500 600
Concentration (µg/mL)

36
Figure.4 :Reducing power capacity of methanolic extract of Xylia Xylocarpa compared with

ascorbic acid.

4.2.3 .Total flavonoid content (TFC):

Plant flavonoid is a phytochemical responsible for antioxidant activity .The assay will

determine the flavonoid content of the test sample in compare with Quercetin .The content

will be seen table .And with the help of the diagram the content of flavonoid is of Xylia

Xylocarpa established in 86.811 mg/g .The flvonoid figure-4 shown .

A= (c×V)/m
Where,
A= Total phenol content (mg/g, quercetin equivalent)
c= The concentration of quercetin established from the calibration curve (mg/mL)
V= The volume of extract (m L)
m= The weight of pure plant extract (g)

Table.5: Quercetin Absorbance

Concentration (µg/mL) Absorbance (nm)

12.5 0.116

25 0.196

50 0.344

100 0.995

37
 Calibration curve of Quercetin
1.2
1
Absorbance (nm)

0.8 f(x) = 0.01 x − 0.06

R² = 0.97
0.6 Abs(nm)
Linear (Abs(nm))
0.4
0.2
0
0 20 40 60 80 100 120
Concentration (µg/mL)

Figure 5: Calibration curve of Quercetin

Table 6: Data of determination of total flavonoid content of methanolic extract of Xylia

Xylocarpa .

Sample Weight of Absorbanc QE QE TFC as QE Mean

solution dry extract e Conc. Conc. A=(c×v)/m (mg/gm)
(µg/mL) per mL (nm) C C (mg/gm)
m(mg) (µg/mL) (mg/mL)
1000 0.001 0.631 124.314 0.124 124.314
1000 0.001 0.628 67.814 0.068 67.814 86.811
1000 0.001 0.633 68.304 0.068 68.304

4.3. In-vitro membrane stability

Membrane stabilizing activity of methanolic extract of Xylia XylocarpaIn the study of
membrane stabilization activity, the methanol extract at different concentrations of 1000, 500,
250, 125 and 62.5μg/mL protected significantly (p<0.0001) the erythrocyte membrane
against lysis induced by hypotonic solution. The extract showed 61.41%, 54.74%, 38.95%,
35.09%, 30.53% inhibition of hypotonic solution induced hemolysis when compared with the
standard.

% inhibition of heamolysis =100 –{(Test solution –Drug control )/Blood control}×100

38
Table.7: Percentage of inhibition of haemolysis by methanolic extract of Xylia Xylocarpa.
Serial Concentrations Test solution Drug control Blood Result (%)
Number (mg/mL) Abs(nm) Abs(nm) control
abs(nm)
1 1000 0.327 0.217 61.41

2 500 0.292 0.163 54.74

3 250 0.267 0.093 0.285 38.95
4 125 0.244 0.059 35.09
5 62.5 0.219 0.021 30.53
Table 08: Membrane stabilizing activity of Xylia Xyliacarpa

Concentrations (mg/mL) Standard(% of Extract(% of inhibitition of

Inhibition of haemolysis)
haemolysis)
1000 91.25 61.41
500 86.16 54.74
250 81.36 38.95
125 77.69 35.09
62/5 72.23 30.53

100
90
80 72.23
70 61.41
60 54.74 Standeard(% of Inhibition of
50 haemolysis)
38.95
40 35.09 Extract(% of inhibitition of
30.53 haemolysis)
30
20
10
0
1000 500 250 125 62/5

Figure 06: Membrane stabilizing activity of Xylia Xylocarpa.

39
 Chapter: 05

Discussion

The significant thrombolytic ability of the plant Xylia Xylocarpa dissolves antioxidant clot
lysis which is very effective. The most successful assay in plant operations is antioxidant
assaying. Antioxidants are often good for prevention of health and illnesses. These potent
substances mostly come from fresh fruits and vegetables. It is beneficial to our members of
the body and strengthened cells, which work effectively against harmful organisms. The
antioxidants are molecules which stimulate the body. The advantages of organic antioxidants
are very important for good health, because they can cause a wide range of diseases and
chronic diseases if free radicals are left unchallenged. Natural antioxidants are therefore most
beneficial for the antioxidants because of their reduced side effects. Inflammation is a
biologic state of great complexity. Chronic inflammation may include aging, cancer,
adipogénesis, diabetes, cardiovascular problems, lung disease, etc. Studies of the stability of
the membranes are useful in determining plant biological activity. The selective index is an
important measure for distinguishing biologically active substances & negligible. To
counteract the complications generated by free radicals, antioxidants might be very
beneficial. Different fractions of Xylia Xylocarpa were subjected to DPPH and hydrogen
peroxide free radical scavenging assays, where crude methanol extracts and their aqueous
fractions scavenged the free radicals evidently (Figs.3 and 4On the other hand, the
decoloration in the ABTS assay shows the ability of an antioxidant species to contribute
electrons or hydrogen atoms to deactivate the radical ABTS cells. Aqueous fractions and

40
crude methanol extracts again showed significant radical ABTS scavenging capability (Fig.
6). FRAP assay, however, tests the reduction potential, where a compound exerts its effect by
adding hydrogen atom to the complex of ferric tripyridyltriazine and interferes with the
radical chain reaction. Xylia Xylocarpa a is commonly used to treat jaundice which may be
due to this fern's antioxidant ability. In the worsening of diabetic complications a higher level
of free radicals and oxidative stress has a vital role to play. Hence the use of antioxidants
decreases oxidative stress and helps in the treatment of diabetes The Garo healers use Xylia
Xylocarpa to treat diabetes mellitus, wound healing, diarrhea which is very natural in the
light of this fern's antioxidant capacity. Antioxidant treatment was reported very promising
for quick alleviation of respiratory infection and inflammation. Rhizomes and fronds of Xylia
Xylocarpa are also locally used for treating birth grith breast length and ntural compound,
which might be assisted with the antioxidant potential of this plant as evident from the
present study along with its antibacterial potential as reported earlier.

41
 Chapter: 06

6. Conclusion
This study suggests that methanolic extract of Xylia Xylocarpa exhibit moderate
thrombolytic clot dissolve lysis and antioxidant activities which proves the presence of
responsible bioactive compound in Xylia Xylocarpa. And finally the test result to the
membrane stability is the given perfectly effect compare to the standard substance .All
pharmacological profile were not performed for single plant improvement on such techniques
advancement in the procedure careful and systemic methods requires new supplementary
approaches. Crude methanol extracts and petroleum ether fractions of rhizomes and fertile
foliage fronds were very promising to show membrane stabilizing anti inflammatory
potential. Besides, crude methanol extracts and aqueous fractions were very active for
displaying thrombolytic activity. In addition, aqueous fractions and crude methanol extracts
of both parts of the plant were very capable to scavenge the free radicals and reduce oxidized
materials, which might be attributed to the high level of antioxidant reducing sugar ,DPPH
and flavonoid contents of the polar extractives. The outcomes of the present study add some
values to the reported healing traits of this fern. Overall, the medicinal potential of this plant
can be considered very substantial. Further comprehensive investigations are required to
isolate bioactive compound(s) and know the underlying mechanisms. Herbal drug developers
might also use these information for appropriate safety and efficacy trial in future.

42
 Chapter: 07

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