BIOACTIVE AND THERAPEUTIC POTENTIAL OF

Syzygium cumini L. FRUIT

By

Seyede Sayame Bokhari

2013-ag-1762
M.Sc. Biochemistry (UAF)

A Thesis submitted in partial fulfillment of the requirements for the degree

of

MASTER OF PHILOSOPHY
In
BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE
FAISALABAD, PAKISTAN, 2017
 DECLARATION

I hereby declare that the contents of the thesis, Bioactive and therapeutic potential of

Syzygium cumini L. fruit is the product of my own research and no part has been copied
from any published source (except the references, standard mathematical or genetic
models/equations/formulate/protocols etc.). I further declare that this work has not been
submitted for the award of any other diploma/ degree.

Seyede Sayame Bokhari

2013-ag-1762
To,
The Controller of Examination,
University of Agriculture,
Faisalabad.

We, the supervisory committee, certify that contents and form of thesis submitted by Seyede
Sayame Bokhari, Regd. No. 2013-ag-1762, have been found satisfactory and recommend
that it be processed for evaluation by the external examiner(s) for award of degree.

SUPERVISORY COMMITTEE

1) Supervisor ___________________________
Dr. Fatma Hussain

2) Member ___________________________
Dr. Muhammad Shahid

3) Member ___________________________
Dr. Junaid Ali Khan
 DEDICATED TO

ALMIGHTY ALLAH
WHO gave me health and ability to do work.

HAZRAT MUHAMMAD
(PEACE BE UPON HIM)

Who conveyed the message of ALLAH to mankind so that

mankind may follow the right path.

MY LOVING & CARING PARENTS,

Who supported me with affectionate and prayers

AND

MY SWEET SISTERS
 ACKNOWLEDGEMENT
In the name of Allah who is the most omnipotence and most omnipresence

If oceans turn into ink and all of the woods become pens, even then the praises of “ ALLAH”
Almighty cannot be expressed. He is the one who created the universe and knows whatever is
there in it, hidden or evident and who bestowed upon me the intellectual ability and wisdom
to search for its secrets. I offer my humble thanks to Allah Almighty for bestowing upon me
the potential and ability for successful accomplishment of this research work. About all I am
indebted to Almighty Allah, Lord of my life and everything in the universe and His Holy
Prophet, Hazrat Muhammad (P.B.U.H) who is forever model of guidance and knowledge
for humanity and whose blessings enabled me to perceive and suit higher ideas of life.

I deem it utmost pleasure to avail the opportunity to express the heartiest gratitude and deep
sense of devotion to my worthy supervisor, Dr. Fatma Hussain, Department of Biochemistry,
University of agriculture, Faisalabad for her kind guidance, assistance and endorsement
from the very early stage of this research as well as giving me extraordinary experiences
throughout the research work.
I wish to record my sincere appreciation to the members of my supervisory committee, Dr.
Muhammad Shahid and Dr. Junaid Ali Khan, (Institute of Pharmacy, Physiology and
Pharmacology, Faculty of Veterinary Sciences) for their keen interest, dynamic supervision,
and valuable comments, scholastic and constructive suggestions throughout my research
work.
Friends are the valuable assets of one’s life, right from the memorable cups of tea to the late-
night chats and gossips have really strengthened me a lot and helped me attain waypoints in
the best doable fashion especially to Abida Irshad, Hina Zafar and Sajila Sharif.
I extend my obligations to my amorous Parents (Syed Mohammad Usman and Masooma
Goodarzi) and Sisters (Saira and Sara), without whose moral and financial support, I
wouldn’t have been at this position today. I can simply say that whatever I am today it is all
due to prayers of my Parents.
 Seyede Sayame Bokhari
CONTENTS

CHAPTER PAGE
TITLE
NO. NO.

i. INTRODUCTION 01

ii. REVIEW OF LITERATURE 13

iii. MATERIALS AND METHODS 28

iv. RESULTS AND DISCUSSION 58

v. SUMMARY 86

88
LITERATURE CITED
 LIST OF TABLES

TABLE PAGE
TABLE TITLE
NO. NO.

3.3 Fractionation solvents 30

Schematic representation of recipe to perform

3.4 39
Ames test

Mean ± SD absorbance values of TPC and

4.1 58
TFC

4.2 ANOVA for total phenolic content 59

4.3 ANOVA for total flavonoid content 59

4.4 Mean ± SD values for DPPH assay 61

4.5 ANOVA for DPPH activity 62

4.6 Antioxidant contents and activity 63

4.7 Biofilm inhibition (quantitative) 64

4.8 Mean ± SD values for antiglycation activity 65

 Mean ± SD values of three days intervals
4.9 66
antiglycation activity

4.10 ANOVA for antiglycation activity 67

4.11 Percentage of antiglycation activity 69

4.12 AChE and alpha-glucosidase inhibition assays 70

4.13 Absorbance values for alpha-amylase activity 71

4.14 ANOVA for alpha-amylase inhibitory analysis 72

Percentage of alpha-amylase inhibitory

4.15 74
activity

4.16 Ames assay 75

4.17 % mortality of brine shrimp larvae 77

4.18 DNA-damage protection activity 78

4.19 Hemolytic and thrombolytic activity 79

4.20 In vivo biochemical analysis 86

LIST OF FIGURES
FIGURE PAGE
FIGURE TITLE
NO. NO.

Different growth stages and parts of S. cumini

1.1 09
plant

Structures of some phytochemicals present in

1.2 12
different parts of S. cumini plant

3.1 S. cumini fruit at lab. 29

3.2 Dried fruit and powder 30

3.3 Extract and fractionation 30

3.4 Standard curve for total phenolic content 32

3.5 Determination of TPC 32

3.6 Standard curve for total flavonoid content 33

3.7 Determination of TFC 33

3.8 DPPH scavenging assay 34

3.9 Antiglycation assay 36

3.10 AChE inhibition assay 37

3.11 Alpha-glucosidase inhibition assay 38

3.12 Alpha-amylase inhibition assay 38

3.13 Various steps of thrombolytic assay 45

4.1 Graphical representation for TPC values 60

4.2 Graphical representation for TFC values 61

Graphical representation of mean values for

4.3 63
DPPH activity

4.4 Qualitative assay of biofilm inhibition 65

Graphical comparison of antiglycation activity

4.5 67
on different days

Graphical representation of time dependent

4.6 71
alpha-amylase inhibitory activity

4.7 Ames assay test plates 74

Agarose gel electrophoresis of DNA-damage

4.8 77
protection assay

ABSTRACT
 To investigate bioactive and therapeutic potential of Syzygium cumini fruit in
different fractions, samples were prepared in seven solvents viz., methanol, ethanol, n-
butanol, n-hexane, chloroform, ethyl acetate and aqueous. Antioxidant profile was assessed
by determination of DPPH (2,2-diphenyl-l-picrylhydrazyl) assay, total flavonoid content
(TFC) and total phenolic content (TPC). Antidiabetic potential was determined by glycation,
acetylcholinesteras, alpha-glucosidase and alpha-amylase inhibition assays. Cytotoxicity was
confirmed by hemolytic and DNA-damage protection assays. Mutagenicity was tested by
brine shrimp and Ames assays. Thrombolytic and antibiofilm activities were also assessed.
Methanol extract exhibited the highest TPC (13 ± 0.030 g gallic acid equivalent /100 g dry
weight) and TFC (25 ± 0.030 g catechin equivalent /100 g dry weight) whereas highest
DPPH scavenging activity (48%) was of ethyl acetate fraction. Maximum growth restraint
was of chloroform fraction against Pasteurella multocida (82.31%) and Staphylococcus
aureus (47.64%). Chloroform fraction showed maximum glycation inhibition (55%). n-
butanol and ethanolic fractions were most potent inhibitors of acetylcholinesterase, alpha-
glucosidase and alpha-amylase with 13.33, 44 and 80.1% inhibitions respectively. The
mortality percentage of brine shrimp shown by the fractions of fruit of S. cumini in this study
ranged between 0-90 with highly active aqueous fraction that caused great toxicity to brine
shrimp larvae and the safest fraction was ethanol with no toxic effect. Percent hemolysis was
in the range of 8-30% and ethyl acetate fraction showed the highest value. All extracts
prevented DNA-damage effectively. Chloroform fraction showed the highest (23%)
thrombolytic efficacy. In vivo biochemical analysis was done by using commercially
available kits In vivo biochemical analysis of antidiabetic, antioxidant, antihyperlipidemic,
hepatoprotective and renoprotective was done by using commercially available kits. The
plant extract had significant effect on antidiabetic parameters such as serum glucose and
insulin and glycated hemoglobin levels. Antioxidant activity of the extract was non-
significant with respect to SOD, GPx and lipid peroxidation level but for catalase activity it
was significant. The extract significantly affected lipid profile of the rats by reverting total
cholesterol, LDL, triglycerides and VLDL to normal values. There was a notable change in
values of ALP and ALT activities in the rats, on treatment with S. cumini extract and it did
not affect the activities of AST, GGT and total protein level markedly. The findings of this
study revealed that different fruit extracts of S. cumini had significant pharmacological
properties especially with respect to diabetes mellitus and there is a need for further
investigation of therapeutic potentials possessed by this plant.
CHAPTER 1

INTRODUCTION

Human beings and animals have been related with plants clearly since the beginning
of the life on the earth. People used their botanical intelligence for systematization of the
medicinal native plants of an area and they established the renowned traditional medicine
systems namely Tibetan, Chinese, Unani and Ayurvedic of Asia, the Amazonian and the
Native American of South and North America and some local systems of Africa. There exist
about 7 billion people and 250,000 plants in planet earth. Plants lived on earth for so many
years before humans and they differ from humans in that plants do not need humans and they
can live without them but humans need plants for food and medicine when the people
became alert of insecurity and side effects of man-made drugs on human health (Sharma et
al., 2012) besides the need of oxygen and food provided by plants (Mamedov, 2012).

Plants have been used by people as natural sources for food and medicine since
thousands years ago. The use of plants and their products as food and drug sources is
increasing as they are cheap specially for people of developing countries (Hossain et al.,
2016), easily accessible and contain secure and safe biologically active compounds and have
no or less side effects on humans (Saravanan and Pari, 2008; Duraiswamy et al., 2016).

As per a report by WHO (World Health Organization), 80% of the world’s populace
rely largely on traditional medication and utilization of plant extracts or their active
compounds to cure diseases (Sharma et al., 2016). Homeopathy, aromatherapy, herbal
therapy, flora-therapy and alternative medicine are other terms given to this type of medicine
(Hossain et al., 2016).

About 35,000 to 70,000 plant species have been used as therapeutic agents and it
corresponds to 14 to 28% of 250,000 species expected to be found everywhere in the world
which is corresponding to 35 to 70% of all plant species used medically, worldwide. More
than 50 drugs used often are originated from tropical plants in today’s global market
(Mamedov, 2012).

1
 The world’s maximum biodiversity is present in Brazil and it accounts for over about
20% of all identified species.This country presents the greatest number of plants with more
than 55000 defined species which is equivalent to 22% of the entire plants present in the
world. This biodiversity contributes to a large usage of medicinal plants as sources of
medicines. Almost 80% of the population of Brazil depends nearly totally on medicines
prepared from natural sources and uses only 37% of the drugs produced commercially.
Therefore, phytotherapics favoured a cheaper and shorter production entering the market,
since the use of medicinal plants do not require strict control of quality concerning efficacy
and safety in comparison with synthetic drugs (Filho et al., 2015).

Different plants that are used for the medical cure purpose have been developed in a
very ancient era. The eldest written record of use of medicinal plants for drugs preparation
has been discovered on an almost five thousands years old Sumerian stone piece in Nagpur.
It contained twelve recipes for medicine preparation with refer to more than 250 different
alkaloids containing plants such as henbane, mandrake and poppy (Petrovska, 2012).

Archaeological discoveries in an old interment place in Iraq revealed the usage of

numerous plants such as marshmallow groundsel, yarrow, hyacinth, ephedra and thistle that
are still used in traditional medicine today. According to ancient Babylon, priests were used
to practice medical treatments. Many evidences of the use of herbs as medicines have been
found in several archaeological sites. Clay blocks with medical prescriptions written on them,
have been found in some babylonian and Sumerian sites. King of Babylon had been recorded
the signs of the usage of medicinal plants such as henbane, cassia, varieties of mint and
liquorice which are still being used in traditional medicine in the laws of Hammurabi, in
1728 B.C.E,. Around 400 Assyrian medications prepared from mineral and plant sources are
identified today. Resin extracted from pine is a renowned Assyrian medication used
internally for therapy of diseases of liver and kidney and externally as a muscle pain reliever.
The Assyrian had enough knowledge about the medicinal properties of mandrake and seeds
of poppy. The medical instructions including the components and quantities for the
preparstion of medicines, written on papyri were found in Egypt. The papyrus of Ebers is the
oldest found medical document which was discovered during 19th century, in Egypt and is

2
dated to 16th century. This document holds 877 remedies and prescriptions including many
spice plants such as frankincense, fennel and myrrh (Yaniv, 2014).

Emperor of China, Shen Nung Circa wrote a book named “Pen T’Sao” on grasses and
roots in 2500 B.C., containing 365 drugs made from dried portions of medicinal plants that
are still being used such as camphor, the great yellow gentian, rhei rhisoma, podophyllum,
jimson weed, theae folium, ginseng, ephedra and bark of cinnamon. Holy books of India
called Vedas contain remedies using medicinal plants grown mostly in areas of that country
(Petrovska, 2012).

For almost thousand years after Vedas, there was found no evidence on the
development of ethnobotanical sciences in India as this country has a long rich cultural and
biological diversity. Later, very important workings were done on Indian system of
medicines in the age of the Charak and Susruta (2500 B.C. to 600 B.C.) written to pre-
Buddhist period. After that, importance of the ethnobotany studies were again realized and
continued during the British period and since then many workers have attempted to find out
the ethnobotanical sources of medicinal plants through the help of folk healer.
Ethnomedicine practice is mainly based on tradition and orally used different kinds of plants
and plant parts. Generally, indigenous botanical medicinal practices are very popular in third
world countries, where modern and sophisticated health services are limited (Kalita et al.,
2015).

Essential oils (EOs) are not completely insoluble in water and can be dissolved in
water to a very low extent. They often are fragrant and have a unique taste due to which,
large amounts of them can be used for the production of perfumes and in flavoring industry.
EOs are generally extracted by fragrance extraction methods such as cold pressing or
maceration and steam distillation. EOs are often complex mixtures of hundreds of bioactive
volatile aromatic components because of which they possess biological activities. Mostly
they contain alcohols, terpenes such as monoterpenes, sesqui- and diterpenes, esters, acids,
aldehydes, epoxides, ketones, sulfides, and amines. Generally the constituents of EOs are
divided into two groups of terpenes and aromatic compounds.

3
 Composition of the EOs of plants depends upon the part of the plant used to extract
the oil such as roots, stems, leaves, flowers, fruits, pericarp, seed, wood and bark (Calo et al.,
2015). Altitude is a factor that affects the components of medicinally used plants such as
EOs. The concentration of EOs of plants living at high altitudes is more than that of those
present in lower altitudes (Mamedov, 2012).

Medicinal plants contain various bioactive compounds working in harmony together

and each of them can affect human body directly or indirectly. Direct effect is based on
pharmacological activity of bioactive components in the plant and indirect effect is related to
the interaction of that plant with different plants and drugs used. Exploration of medicinal
plants to treat outbreak ailments should be of the plants living in the geographic area, where
those ailments were arisen. The disease might exist there for thousands of years and spread of
disease should be controlled with medicinal plants native to such areas (Mamedov, 2012).

Diabetes mellitus, a long-lasting metabolic sickness is more prevalent in developing

countries and is considered to be 7th cause of mortality worldwide (Bhuyan et al., 2010)
while on estimation, every 5 seconds a person is diagnosed as diabetic anywhere in the world
and one dies of this disease every 10 seconds (Preeti et al., 2014). It is distinguished by high
blood glucose concentration and its urinary loss known as glucosuria with elevated urine
productivity (Kadali et al., 2016) following little or no insulin secretion or its resistance and
disturbs carbohydrate, lipid and protein metabolism. Impaired carbohydrate metabolism and
constant effort of physiological system to maintain the metabolism of carbohydrate put a
strain on endocrine system. Continuous weakening of endocrine control worsens the
metabolic troubles leading primarily to hyperglycemia (Akuodor et al., 2014).

Increased buildup of AGEs (advanced glycation end products) have been contributed
to the complications associated with DM. AGEs formation and protein glycation are
accompanied by free radical formation through glycated proteins and auto-oxidation of
glucose. The glycated end-products interact with signaling cell surface receptors, resulting in
oxidative stress, inflammation and vascular dysfunction that together generate diabetic
complications. The increasing oxidative stress caused by hyperglycemia deshapens the
structure of erythrocytes and disturbs their function leading to great fragility due to
membrane lipid peroxidation and reduced levels of antioxidant enzymes (Tupe et al., 2015).

4
 Hyperglycemia in association with hyperlipidemia, glycosuria, polyuria, polydipsia,
negative nitrogen balance, ketonemia, polyphagia and weight gain can lead to failure of
several organs such as the eyes, nerves, kidneys, blood vessels and heart and create health
problems such as hepatopathy, nephropathy, retinopathy, cardiomyopathy atherosclerosis and
coronary heart diseases (Rekha et al., 2010; Behera et al., 2014).

Neuropathy of diabetes causes prickling or stinging sensation and pain in the feet and
this altered sensation also increases the risk of skin damage. Vascular complications in the
legs put up the risk of diabetic foot ulcers, the diabetes-related foot problems which are
difficult to treat and may require amputation (Nasri et al., 2015).

In most studies, lifestyle and dietary issues increase chances of occurrence of DM

(Mustafa et al., 2016). Diabetes of type 1 or insulin-dependent DM is caused by the
autoimmune mediated (Yaseen et al., 2015) loss of pancreatic β-cells leading to low levels or
lack of insulin and type 2 or non-insulin-dependent DM with hereditary background, is
recognized by insulin resistance and declined insulin secretion (Shikha et al., 2011). Type 2
is most common, counting about 90-95% of all diabetic cases. WHO stated that
approximately 4% of the world’s populace had suffered from diabetes in 1995 with 5.4%
increase, by 2025 (Awasthi et al., 2016). Around 90% of populace of the world are tolerating
type 2 DM (Tarafdar et al., 2014).

As of late, manufactured hypoglycemic medications and other drugs used to treat

diabetes have their particular toxicity and danger for instance, hepatorenal and hypoglycemic
instabilities (Mahomoodally et al., 2016) thus it is important using parts of plants to make
natural medicines and to guarantee safety of therapy (Sharafeldin and Rizvi, 2015). There are
countless plants recorded to be good to prepare drugs for treating diabetes among which
Syzygium cumini is verified theoretically to be efficient as antidiabetic plant (Katiyar et al.,
2016).

Syzygium cumini (L.) skeels. also called with substitutes Eugenia cumini, Eugenia
jambolana, Eugenia djouant, Myrtus cumini, Calyptranthes jambolana and Eugenia
caryophyllifolia (Rodrigues et al., 2016) is named ‘Brahaspati’ in Sanskrit language and

5
commonly named as black plum, java plum, jamaica plum, portuguese plum, jamun and
indian blackberrys. It belongs the family Myrtaceae (Myrtle) (Ramya et al., 2012).

The Myrtaceae family is comprised of about 133 genera and 3800 species. It is
mostly diverse in Southeast Asia, Australia and southern temperate to tropical areas of
America, but it showes lower diversity in Africa. Family is distinguishd by a group of
characteristics such as presence of oil glands on the entire leaf, numerous stamens, half
inferior or inferior ovary, vestured pits on xylem vessels and internal phloem. This family has
been recently classified as two subfamilies, namely Leptospermoideae (capsular-fruited) and
Myrtoideae (fleshy-fruited) (Wilson et al., 2001). Family Myrtaceae is economically
important all over the world for some of its plants such as Psidium which is the eatable fruit
of guava, spices such as cloves, the dried Syzygium aromaticum flower buds used for food
applications, bottle brushes (Callis temon), myrtle (Myrtus communis) and Eucalyptus that
are cultivated locally as ornamentals e.g., avenue trees (Stephen, 2012). Plants of this family
can be cultivated in waterlogged areas and are introduced to reserve forests to reduce the
water contents of the soil or water tables in such forests (Orwa et al., 2009; Al-Edany and Al-
Saadi, 2012).

A brief history about classification of Eugenia and Syzygium genera described in

Myrtaceae family explains that because of the wide range of differences between the
members of this genus, botanists were not satisfied with its classification. Niedenzu
reclassified the genus most comprehensively in 1893. He established several separate genera
based on the characters of the flower mainly. His system lacked distinct generic limits and it
wasn’t acceptable by many botanists who considered it in a broader sense and merged the
separated genera to the single genus of Eugenia. Many species fell under genus Eugenia that
made Merrill and Perry classify the genus based on seed structure. According to Merrill and
Perry species of Eugenia were characterized by fruits with easily crushable pericarps and free
seed having joined cotyledons and smooth testa but Syzygium, a separate genus was
distinguished by fruits that were not breakable easily and the seeds contained two separable
cotyledons. But in 1949, Henderson did not accept seed specifications as good generic
characters to make distinct genera. Finally, Ingle and Dadswell in 1953 studied wood
anatomy of several plants belonging to Myrtaceae family and suggested that this

6
characteristic is good enough to split Eugenia into two separate genera Eugenia-A and
Eugenia-B consisting of genera Acmena, Cleistocalyx and Syzygium (Wilson, 1957).

The genus Syzygium belonging to family Myrtaceae is naturally present in tropical

areas, specifically those of Australia and America. It is distributed worldwide but unevenly in
tropics and subtropical regions. This genus contains about 1100 species with a range
extending from Madagascar and Africa through the pacific. It is highly diversed in
Northeastern Australia and Malaysia. Plants in this genus are reported to contain volatile oils
for which they have been used in medicine (Ayyanar and Subash-Babu, 2012). Syzygium has
been derived from the Greek word ‘syzgios’ meaning paired, due to the twigs and leaves that
grow at the same point in various species (Orwa et al., 2009).

Syzygium cumini, a large perennial tree is native to Asian countries like India, Burma,
Bangladesh, Nepal, Sri Lanka, Indonesia, Pakistan and Malaysia. It is also grown in some
warm parts of America, Eastern Africa and Madagascar. According to history, the tree is
worshiped in Bhuddism so it is cultivated close to hindu holy places and is supposed to be
revered by lord Krishna. Botanically, the plant has a straight trunk with the bark that is
grayish brown growing up to 15-30 m tall, greenish-white flowers and narrow leaves. Bark is
1.0 to 2.5 cm thick, dark grey or brown, smooth and has bitter taste. The fruits are oval with a
large central seed, developing in May to June and are like large berries and form clusters of
4-20. They are green at first, turn pink and become purple-black when ripe and give their
color to the tongue when eaten. They have a sweetish-sour taste and are rich source of
vitamins A and C (Ramya et al., 2012). S. cumini at different stages of growth has been
depicted in fig. 1.1(a-g).

Scientific classification of Syzygium cumini

Kingdom: Plantae
Class: Magnliopsida
Order: Myrtales
Family: Myrtaceae
Genus: Syzygium
Species: Cumini

7
 Jamun juice of good quality is excellent for preparing syrup, squash and sherbet.
Squash in India is prepared by the juice pressed out from cooked crushed fruits at 140 °F (for
5 to 10 minutes) and is mixed with sugar, sodium benzoate, citric acid and water as
preservative agent. Jamuns of good quality and suitable size, with a sub-acid or sweet taste
and minimum acidity are great raw materials for making tarts, jam and sauces. Acerbic fruits
are soaked in salty water or pricked by being rubbed with salt and left for almost one hour to
improve taste. The low-quality fruits can be used to make juice that is often comparable to
juice prepared from grapes. It is better not to squeeze the fruits for extracting juice from
cooked jamuns as it lessens astringency. Jamuns having white flesh contain enough pectin to
make a good jelly by shortening the cooking time. Commonly, fruits with purple flesh are
deficient in pectin but yield colored jelly which needs to be mixed with commercial jelling
agents or combined with fruits rich in pectin, such as ketembillas and unripe guavas. In Goa
and Philippines, jamun is a brilliant material for the production of wine. Fermented fruits
have also been used to make Brandy and jambava, a distilled spirit. A pure purple vinegar
with enjoyable aroma and mild taste is also extensively produced from jamun fruits
throughout India (Swami et al., 2012).

Syzygium cumini tree grows rapidly and can provide excellent firewood and charcoal.
Its wood with specific gravity of 0.77, burns very good and produces about 4800 kcal/kg
energy. It is resistant to termites, durable in water and inspite of being tough to work, it gets
cut well and is used for construction, boat and bridges building, making musical instruments
especially guitars, furniture, cart wheels, tool handles, chest plywood, troughs, well curbs,
agricultural tools, sleepers, as pillars for shafts and galleries in mines and commercial tea.
The lumber is reddish-brown or reddish-grey. Well-grained heartwood is applied in external
woodworks and carpentry. Bark is used in tanning industry and releases a brown dye that is
used to color the fishnets (Orwa et al., 2009).

8
 a) b)

c) d)

e) f) g)

Fig. 1.1: a) Seeds, b) Small young plant, c) Mature tree, d) Flowers, e) unripe fruits,

f) Ripe fruits in a bunch and g) Bark

Being a medicinal plant, nearly all portions of this plant can be used to manufacture
medicine. As per Ayurveda, bark aids in digestion, has wound healing properties and is also
used to cure ulcer, bronchitis, asthma, sore throat, blood impurities, thirst and dysentery.

9
Seeds are used as diuretic agents and can slow down diabetes. Jamun is believed to be semen
promoting, haematinic and thermoregulant (Ramya et al., 2012).

It is reported that soft tissue of jamun fruit is nutritive and holds several minerals such
as phosphorous, calcium, sodium, potassium, zinc and iron, carbohydrates like sucrose,
glucose, galactose and raffinose and free amino acids like cysteine and alanine. Leaves
contain acylated flavonol glycosides, esterase and galloyl carboxylase and can be used for
contraction of vagina after delivery and reduction of mucus and odor. The powder prepared
from peel of juman can also be used to produce colorant for food and pharmaceuticals. The
phytochemicals of this plant are numerous anthocyanins, flavonoids, glycosides, saponins,
betulinic acid, friedelin, ellagic acid, gallic acid, kaempferol, myricetin (a flavonol),
gallocatechin, tannins and eugenin (Tavares et al., 2016) accountable for antidiabetic,
antiamnesic, antioxidant, antihyperlipidemic, antihypolipidemic, anticancer, antiallergic,
antiinflammatory, antimicrobial, antifertility, anticlastogenic, antinociceptive, antidiarrhoeal,
antiscorbutic, anti-HIV, antiradiation, antiperoxidase, piles curing and gastroprotective
effects (Rafiqkhan and Viswambharan, 2015). Alkaloids are also important phytochemicals
that can be used for the preparation of vaccines against viruses (Islam et al., 2015).

Flavonoid Anthocyanin

Betulinic acid Eugenin

10
 Ellagic acid Gallic acid

Friedelin Glycoside

Gallocatechin Ferulic acid

Myricetin Kaempferol

Malvidin Dephinidin

11
 Caffeic acid Saponin

Petunidin Mycaminose

Tannic acid

Fig. 1.2: Structures of some phytochemicals present in different parts of S. cumini plant

12
CHAPTER 2

REVIEW OF LITERATURE

Antidiabetic activity

Diabetes mellitus is a group of ailments knwon by elevated level of glucose in blood

or hyperglycemia, hyperlipidemia, impaired metabolism of carbohaydrates, lipids and
proteins accomplishing many other complications fatal if remain untreated (Akuodor et al.,
2014).

Hypoglycemic potential of different portions of seeds of jamun such as entire seed,

seed coat and kernel was estimated using glibenclamide as control. Ethanol extract of kernel
lowered urea, blood glucose and cholesterol significantly. It improved glucose tolerance,
liver glycogen and total protein level. The ethanol extract also reduced glutamate
oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activity in
rats. The entire seed was a moderate hypoglycemic agent and seed coat had no effect (Ravi et
al., 2004).

India is ranked amongst the countries with high occurrence of diabetes and its
treatment with no side effects is of concern. Herbal remedies help reduce the side effects of
synthetic drugs but they need proper standardization of phytochemicals to be proved as
bioactive compounds and preparation of proper doses and the clinical use of the
phytoconstituents is of concern. The number of phytochemicals, binding machinery of
secondary metabolites of S. cumini and its antidiabetic action in vitro, were investigated. In
silico study showed that ellagic acid can possibly regulate the activity of carbohydrate
metabolizing enzymes with extra affinity for those requiring minor binding energies which
are alpha-glucosidase, beta-glucosidase, alpha-amylase, glucokinase and glycogen synthase
kinase. In vitro assay of alpha-amylase hindrance revealed that the extract in ethanol was best
(73.33%) inhibitor comparing with acarbose, the standard drug (65.99%). Thin layer
chromatography analysis revealed that this extract contained 5 different components,
amongst which ellagic acid was most effective (Bansode et al., 2016).

13
 S. cumini, used for treatment of DM in indian traditional medication, was studied by
scientists during last 125 years and near hundred reports were written on its effective
influences on diabetes prior to insulin discovery (Helmstädter, 2008). Powdered pulp
possessed coloring potential and proanthocyanidin component active against pancreatic
lipase, alpha-amylase and alpha-glucosidase (Srivastava et al., 2012; Borges et al., 2016).

S. cumini ethanol extract decrased blood glucose and high insulin level (preventing
hyperglycemia), glucose tolerance, total proteins, high density lipoprotein (HDL) cholesterol
and liver glycogen in STZ-induced rats in experiments arranged by Bhuyan et al. (2010).

Aqueous and methanolic extracts of barks, roots, seeds and leaves of S. cumini at
concentrations of 50 and 100 mg/kg given to alloxan-mediated diabetic male rats, for 3
weeks, showed hypoglycemic effect and significantly decreased the blood glucose (Deb et
al., 2013). The hypoglycemic ability may be due to elevated action of cathepsin-B that makes
insulin from proinsulin, stimulating its discharge (Jadhav et al., 2009).

The phytochemical mycaminose, discovered in plant seed extract was responsible for
reduction of the glucose level in blood of STZ-induced rats (Kumar et al., 2008; Elavarasi et
al., 2013; Gurjar et al., 2016).

To investigate the antihyperglycemic potential of seeds aqueous extract on STZ-

nicotinamide induced diabetes in albino rats, they were fed on bread, gram and pulses. Two
of the test groups were injected single dose of nicotinamide and streptozotocin (120 and 60
mg/kg body weight) intraperitoneally after 15 minutes. A normal and test group were
administered by extract (400 mg/kg body weight) later during the experiment period. After 4
weeks, aqueous extract could prevent hyperglycemia induced by streptozotocin and
nicotinamide (Sarma, 2014).

Effect on hypertension

Hypertension by clinical definition is a blood pressure level at which the organization

of treatment lowers the blood pressure-related mortality and morbidity. A new classification
suggests a criteria for defining normal blood pressure as the ratio of systolic blood pressure
to dyastolic blood pressure (S.B.P./D.B.P.) less than 120/80, pre-hypertension as

14
S.B.P./D.B.P. equals 120-139/80-89, hypertension stage I as S.B.P./D.B.P. equals 140-
159/90-99, hypertension stage II as S.B.P./D.B.P. ≥ 160/100 and isolated systolic
hypertension as S.B.P. ≥ 140 and D.B.P. less than 90 which is frequent among elderly.
Universality of hypertension is almost 1.5-2.0 times greater in patients with DM than in an
appropriately matched nondiabetic population. Mechanism, onset and prevalence of
hypertension differ in type 1 and 2 diabetes. Type 1 patients generally dont present with
hypertension at the time of diagnosis; Blood pressure rises after the onset of renal
insufficiency and hypertension then worsens the condition to renal failure but Type 2 patients
are usually hypertensive when diagnosed as diabetics. Many factors such as obesity,
advanced age of diagnosis and renal insufficiency contribute to hypertension in type 2 DM,
at the time of diagnosis. Hyperglycemia in diabetic patients increases total concentration of
replaceable sodium in body, which can lead to accumulation of ECF (extracellular fluid) and
increased volume of plasma. It has been reported that insulin resistance and hyperinsulinemia
may be in accordance with hypertension maintenance because of sodium retention promotion
and enhancement of activity of sympathetic nervous system. Chronic hypertension may lead
to many adverse effects on major organ systems in body, necessitating its effective control.
Many patients of type 2 diabetes are dually diagnosed as hypertensive and diabetics from the
beginning and hyperglycemia in patients of type 2 contributes to development of
hypertension, treatment of hyperglycemia might produce beneficial effect on hypertension.
To study the effect of jamun seed powder on blood pressure of type 2 diabetes, 99 patients
were arbitrarily divided. Group-A including 50 patients was supplemented with 10 g/day
jamun seed powder and group-B with 49 was given placebo powder. S.B.P. and D.B.P. were
noted at baseline, 30th, 60th and 90th days. The groups were compared and it was observed
that mean S.B.P. of group-A reduced significantly from 152 ± 17.14 mmHg to 145 ± 15.47
mmHg, 143 ± 14.41 mmHg and 140 ± 13.23 mmHg after supplementation with jamun seed
powder for 30, 60 and 90 days respectively. Mean D.B.P. of group-A reduced significantly
from 91 ± 13.57 mmHg to 86 ± 11.60 mmHg and 85 ± 3.91 mmHg after 60 and 90 days
respectively. No notable change was detected in S.B.P. and D.B.P. of group-B. It was
concluded that supplementation with jamun seed powder significantly lowers the blood
pressure of patients besides its well-known antihyperglycemic effects. Thus it helps in
improving overall health of diabetic patients (Sidana and Singh, 2016).

15
Antioxidant activity

Reactive oxygen species (ROS) like O 2−, OH−, 1O2 and H2O2 contain reactive oxygen
atoms and are harmful to DNA, proteins and lipids (Mohamed et al., 2013). These are
produced in the normal metabolic activities of the cells and are involved in metabolic
diseases such as AIDS, arthritis, cancer, liver disorders and DM and antioxidant remedy is
helps their treatment (Singh et al., 2016). Widely utilized antioxidants are tert-
butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene (BHT) (El-Anany
and Ali, 2013) and phenolic antioxidants containing benzene ring with as a minimum, an
attached hydroxyl group (ArOH) that break the oxidative chain reactions. The antioxidants
act through two mechanisms called as hydrogen atom donator or HAT in which a hydrogen
atom is removed from antioxidant by free radical making it a radical. In this way, the free
radical is converted to a more stable product and oxidation is prevented. In the other
mechanism known as single electron transfer (SET), The donation of an electron to free
radical by antioxidant takes place that makes the antioxidant a positively charged radical
preventing the oxidative chain reactions by reduction of the oxidized intermediates to stable
form. There are several antioxidant assay methods on the basis of so called mechanisms. The
classification under HAT includes total reactive antioxidant potential (TRAP) and oxygen
radical antioxidant capacity assays. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical
scavenging, 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS •+), ferric
thiocyanate (FTC) and FRAP (ferric reducing antioxidant power) fall in the SET category
(Siti-Azima et al., 2013).

Researches done in last twenty years had shown that jamun have excellent complex
of natural antioxidant compounds such as anthocyanins, ellagic acid and glucosidejamboline.
Major anthocyanins are malvidin, cyanidine, petunidin and glucoside. Other phytonutrients
such as flavonoids and ferulic acid, a type of phenolic acids are also present in jamun fruit
and seed that have anticancer, antiaging and antioxidant capabilities (Shah et al., 2012; Raza
et al., 2015).

To evaluate the nutritional benefits of powder of jamun pulp even after being dried in
altered drying conditions, quantitative analysis of significant phytochemicals and antioxidant
ability was performed by standard methods and DPPH assay. Total phenols, anthocyanins
16
and flavonoid contents of each dried sample were altered at different temperatures. In freeze
dryer at −40 °C, the highest TPC (total phenolic content) estimated by Folin-Ciocalteau
reagent was 13.99 mg gallic acid equivalent over g dry weight (GAE)/g, TFC (total flavonoid
content) measured by AlCl3 technique was 104.8 mg quercetin equivalent (QE)/g and
concentration of anthocyanins was 7.56 mg/g showing that freeze-dried powder was most
potent antioxidant. The lowest values for TPC, TFC and anthocyanins were 7.6 mg GAE/g,
34.05 mg QE/g and 1.43 mg/g in cross flow dryer, respectively. In brief, S. cumini is an
underutilized fruit possessing many potent pharmaceutical properties (Jebitta and Allwin,
2016).

Bioactive compounds such as TFC, TPC and anthocyanin contents of seed and fruit
were quantified. Ethanol and methanol extracts of dehydrated fruit and seed of jamun were
prepared by common solvent extraction method. Aqueous extract was prepared for making
comparison between efficiencies of other extracts. Phytochemical screening of ethanol and
methanol extracts revealed that exract prepared in ethanol was the best at 45 minutes. The
extract of fruit contained maximum TPC, TFC and anthocyanins while, maximum phenolics
and flavonoids in seed extract were different and seeds contained no anthocyanins. Bioactive
phenolics, flavonoids and anthocyanins make S. cumini a good source of antioxidant
components (Raza et al., 2015).

Antioxidant and hypoglycemic activities of jamun pulp aqueous extract was

examined in STZ-caused diabetic wistar female rats. An STZ injection after 3 days, increased
levels of glucose to 230 mg/dL. Administration of extract at two different doses, significantly
(p<0.001) lowered the levels of blood glucose and prevented body weight loss depending on
dose used. The aqueous extract reduced hydroperoxides, lipid peroxidation and free radicals
formation in the liver of the rats by increasing the levels of glutathione-S-transferase,
superoxide dismutase (SOD), glutahione peroxidase (GPx), catalase (CAT) and reduced
glutathione (GSH) (Rekha et al., 2008).

Hydromethanolic extracts from some bangladeshi plants were investigated for

antioxidant potential by DPPH assay. Among them, S. cumini showed the highest DPPH
scavenging capacity with IC50 4.25 g/mL as compared to that displayed by ascorbic acid, the
control antioxidant, depending on different doses (Hasan et al., 2009). Among leaf methanol

17
extract and its aqueous, chloroform, n-hexane and ethyl acetate fractions, the last fraction
was strong antioxidant due to having phenolic compounds (Pradhan, 2016).

Diabetic rats induced by streptozotocin were administered with powdered seed and
peel of S. cumini orally for twenty eight days. Rats were slaughtered on day 28 and their
kidney, pancreas and liver were examined to estimate the levels of GPx, CAT, SOD, lipid
peroxidation and GSH. Finally, it was concluded that powder of seed and peel raised the
levels of said enzymes (Pradhan and Pradhan, 2015).

Antioxidant activity of chapatti made from whole wheat flour supplemented with
jamun pulp at different levels was evaluated by Kapoor et al. (2015). Jamun pulp
supplementation increased the concentrations of bioactive compounds such as anthocyanins
and phenols in chapattis. TPC and antioxidant ability incresed by 99.73 and 44.38%
respectively. Anthocyanins were not observed in control chapatti but the range was 1.41-2.64
mg/100 g content in jamun supplemented chapatti for 5-15% supplementation levels.
Chapattis exhibited full puffing at supplementation. The supplementation also improved
crude fiber content by 13.77%. The study revealed that supplementation of wheat flour with
jamun pulp improved nutritional and antioxidant status of chapatti.

Ethanol, ether, ethyl acetate, chloroform, hexane and aqueous extracts of fruit of S.
cumini were evaluted for antioxidant and anticancer potentials by DPPH method and
leukemia cancer cells viability. The results indicated that the ethanol extract was most potent
antioxidant and antileukemia agent. Spectroscopic investigation of active components
isolated from ethanol extract revealed that fruit extracts of S. cumini contained sterols such as
ɣ-sitosterol and phenolics such as kaempferol 7-O-methylether responsible for such activities
(Afify et al., 2011).

Belapurkar and Goyal (2014) examined methanolic extract containing flavonoids,

alkaloids, tannins, saponins and glycosides. TPC and TFC were shown to be 88 ± 5.25
mg/mL and 54.5 ± 9.64 mg/mL, taking quercetin as standard. Antioxidant potential was also
estimated by DPPH assay with ascorbic acid as control and 100-500 µg/mL dose of extract
was in the range of 82.75-99.89. Percent radical scavenging of extract was shown to be
higher than the activity of the standard for the whole range of concentrations.

18
 Eshwarappa et al. (2014) studied the antioxidant profile of leaf gall extracts of S.
cumini by using DPPH, hydroxyl scavenging, FRAP and nitric oxide scavenging methods. In
all methods, the methanol extract exhibited greater antioxidant power than the standard,
ascorbic acid and it contained maximum TPC and TFC. Flavonoids, phenolics, terpenoids,
reducing sugars and phytosterols were the compounds identified in both extracts. It was
suggested that the named compounds were responsible for higher level of antioxidant activity
exhibited by the extract. These results can justify the use of the plant leaf gall extract as a
good source of medicine to overcome the oxidative problems caused by many oxidants
produced naturally during metabolic reactions or those produced in the environment.

Antihyperlipidemic activity

Lipid profile variations are common problems in DM (Ravi et al., 2005).

Antihyperlipidemic effect of S. cumini seed extract was investigated on hyperlipidemia in
rats induced by high cholesterol diet adding 2% coconut oil, 2% cholesterol and 1% sodium
cholate in chow diet for one week. Treatment with extract significantly decreased serum
triglycerides, VLDL (very low density lipoprotein), cholesterol, LDL (low density
lipoprotein) and increased the HDL/HDL ratio. It was concluded that antihyperlipidemic
activity of alcholic extract of jamun might be due to possessing flavonoids, alkaloids,
saponins, phenols, tannis and triterpenoids found in initial phytochemical screening (Modi et
al., 2009).

Different extracts of seed of jamun, hydroalcoholic extract of seed kernel, aqueous

extract of its fruit and administration of an Indian herbal formulation, Dihar containing S.
cumini were reported to lower cholesterol, triacylglycerol, LDL and elevated HDL level at
different doses in diabetic models. Antihyperlipidemic capacity of the mentioned extracts
made from different parts of plant was believed to be due to availability of flavonoids in
extracts and the inhibitory effect of these compounds on enzyme HMG-CoA (3-hydroxy-3-
methyl-glutaryl-CoA) reductase that is involved in synthesis of cholesterol. The mechanism
of inhibitory action of these compounds is by increasing the expression of cyclic adenosine
monophosphate (cAMP)-dependent phosphokinase, the enzyme responsible for inhibition of
HMG-CoA reductase. Another reason for such activity shown by extracts can be the
reduction in cholesterol absorption in the intestine along with incresed clearance rate of free
19
fatty acids and triacylglycerols following by the betterment of action of insulin in th diabetic
models (Chagas et al., 2015).

Hepatoprotective activity

Liver is the site for metabolism of endogenous compounds and neutralization of toxic
substances, chemicals, drugs and xenobiotics. The contaminated food and water are also
detoxified by liver biotransformation enzymes (Islam et al., 2015). The excessive intake of
toxins and activities of the enzymes and infections lead to hepatic stress, impaired liver
function and various liver diseases. Hepatotoxic agents react with the cellular components
and damage the cells causing necrosis and fibrosis. Hepatic disturbances are major causes of
high rate of mortality in comparison with diseases related to other organs or body parts.
Synthetic drugs used to cure hepatic concerns have certain side effects on body. S. cumini is
believed to be liver stimulant in Unani medicine system and can be used to treat liver related
diseases.
Hepatoprotective activity of seed aqueous extract was evaluated on STZ-induced
diabetic rats by dividing them into different groups of control, diabetic control, group given
standard hypoglycemic drug orally and two other groups administered with two different
doses of extract. After 3 days, the rats with level of higher than 200 mg/dL of glucose were
considered as diabetics. These rats were divided in to different groups further considered as
diabetic control and three other groups receiving their drugs for 120 days. On 120th day,
blood samples were collected and liver markers, alanine transaminase (ALT), alkaline
phosphatase (ALP), aspartate transaminase (AST) and ɣ-glutamyltranspeptidase (GGT) were
measured by auto-analyzer. Results indicated an increase in the markers in diabetic control
group and other groups showed significant reduction of the enzymes. Protective effect shown
by high dose extract of seed was greater than that of low dose. In conclusion, jamun seed
powder aqueous extract had hepatoprotective capability (Behera et al., 2014).
Salim and Paarakh (2009) evaluated hepatoprotective potential of leaf and fruit peel
aqueous extracts of S. cumini containing sophronitis coccinea anthocyanin against
hepatotoxicity from carbon tetrachloride in rats. Aqueous extract lowered the levels of SGOT
and SGPT. Anthocyanin being non-toxic at concentrations of 50-500 ppm suppressed 54%,
lactate dehydrogenase (LDH) leakage induced by CCl 4 at 50 ppm and improved cell viability

20
(39%). It also increased GSH level and GPx activity two-folds. Results revealed that the
extract of fruit peel is responsible for protection against oxidative damage caused by CCl 4
induced in hepatocytes of rats.
The discovery of cisplatin, cis-Cl2H6N2Pt or CDDP was a start point triggering the use
of platinum (II) and different metal containing compounds as possible anticancer drugs.
Cisplatin is a commonly used drug for cancer chemotherapy. Cisplatin effect on biochemical
and histopathological factors and improving effects of aqueous extract of jamun seeds in
wistar male rats were investigated. Four different groups of adult rats were made as control
receiving normal saline and groups receiving single cisplatin intraperitoneal injection (7
mg/kg body weight), aqueous extract of seed (400 mg/kg body weight) one day before
injection orally for one week and aqueous seed extract (400 mg/kg body weight) only.
Exposure to cisplatin can lead to opposing effects on hematological hepatotoxicity involving
erythrocytes. Cisplatin administration leads to decrease in levels of enzymatic and
nonenzymatic antioxidants. Treatment with seed extract regulated the levels of all the
biochemical and hematological factors. These outcomes highpoint the importance of S.
cumini aqueous seed extract as protective agent for cisplatin-induced hepatotoxicity
(Maheswari and Manohari, 2015).
Renoprotective activity

Nephrotoxicity is a damage to the kidneys that results from exposure to toxic

substances and drugs such as antibiotics, analgesics, contrast agents, aminoglycosides and
amphotericin-B, the drugs that damage the kidneys directly. The kidneys are the vital organs
of the urinary system that purify the blood by removing wastes and excess substances from it
and excreting them in the urine. The kidneys filter about 180 L/day of blood that is about
four times the amount that passes through other organs. Because of this high volume of blood
passing the kidneys, they are more often exposed to toxic chemicals in the blood and are very
prone to injury from those substances (Kuttiappan et al., 2015).

Roughly, nineteen million of adults suffer from chronic kidney diseases and 80000
individuals on estimation are diagnosed with chronic kidney failure in India every year.
Renal replacement is the only way to cure end-stage failure of kidneys till date and if kidneys
can’t be replaced, dialysis is an alternative which unluckily is harshly limited by some

21
limitations like high expenditure. Various therapeutic medicines such as chemotherapeutic
compounds like cisplatin, aminoglycoside antibiotics and chemicals such as CCl 4, sodium
oxalate, ethylene glycol and heavy metals like mercury, arsenic, cadmium and lead can
unfavorably affect kidneys leading to nephritic syndrome, chronic interstitial nephritis and
acute renal failure. Despite the limitations of clinical use of cisplatin in cancer therapy due to
its harmful side effects like nephrotoxicity, this drug has been applied for the treatment of
numerous cancerous or noncancerous tumors such as uterine cervix carcinomas, lung, breast
and testis.

Almost 1/3rd of patients under treatment with cisplatin have suffered from clinical
nephrotoxicity, expressed by elevated serum creatinine, impaired electrolyte levels in serum
and decreased glomerular filtration rate. Though the mechanisms of nephrotoxicity by
cisplatin aren’t fully recognized, many researches have reported contribution of the active
free radicals such as ROS, abnormal function of mitochondria, reduction of antioxidant
potential and activation of necrotic or apoptotic programmed cell death pathways.
Nephroprotective agents are the compounds that are active against nephrotoxicity.
Pharmaceutical plants show protective activities due to containing various active principles
and chemical components (Janakiraman and Jayaprakash, 2015).

Renal failure is of two types of acute and chronic. The first is recognized by
significant deterioration of excretory function of kidneys taking place for hours or days and
high concentration of creatinine or urea in plasma is a mark for its clinical detection. This
type can occur as an isolated problem, but it commonly arises during setting of circulatory
disruption related with serious disease, trauma or surgery; transient renal dysfunction.
Chronic type is the systemic and metabolic clinical condition which is the consequence of a
significant, slow and irreversible decline of excretory and homeostatic functions of kidneys
(Swati and Rajashekhar, 2015).

As a nephroprotective agent, fruit powder of S. cumini was taken one spoon with
water two times a day for fifteen days for treatment of kidney stones and it was effective
(Kuttiappan et al., 2015). Ethanolic extract of fruits of S. cumini at doses 250 and 500 mg/kg
were examined on cisplatin-induced nephrotoxicity (6 mg/kg) in albino rats.
Nephroprotective activity of plant was determined by estimating the levels of serum

22
creatinine, serum total proteins, urinary protein, blood urea nitrogen and lipid peroxidation in
kidneys. Cisplatin increased the level of serum markers, the excretion of protein in the urine
and renal malondialdehyde level and lowered the creatinine clearance rate. Ethanol extract of
fruits of S. cumini significantly reversed the effects of cisplatin, dose dependently (Adikay et
al., 2010).

As per report by Ayyanar et al. (2013), S. cumini fruit aqueous extract consumed
orally by diabetic rats prevented elevation of urine volume, albumin excretion into urine,
renal hypertrophy and cataract. Jamun also avoided the progress of atherosclerosis due to
containing oleanolic acid that can prevent free radicals formation. The ethanol extract
enhanced kidney failure by reducing urea (Sharafeldin and Rizvi, 2015)

S. cumini played an effective role against acetaminophen-induced damage to organs.

The markers of liver and kidney such as ALT, AST, ALP, uric acid and creatinine were
measured by Sagor et al. (2016) and in conclusion, 1% seed in food, lessened extracellular
matrix deposition, inflammatory penetration, iron overload and collagen secretion caused by
acetaminophen in kidney and liver and it could be contributed to the antioxidant potential of
this plant.

One of the renal disorders is the formation of kidney stones and calcium oxalate is a
type of stone forming in the kidneys. To evaluate the therapeutic potential of S. cumini in this
case, the alcoholic extract of flower at different doses was given to ethylene glycol-induced
model rats in order to reduce the growth of stones in the kidneys of hyperoxaluria rats. The
result indicated significant decrease in kidney stones and serum and urine levels of
creatinine, calcium, phosphorous and urea. The result was compared with that of furosemide
used as standard drug (Khan, 2016).
Antimicrobial activity
Prabhakaran et al. (2011) revealed extracts of S. cumini are rich in flavonoids,
carbohydrates, phenols and tannins. Especially leaf ethanolic extract and aqueous extract of
seeds were found to act very effectively against various Gram-positive and Gram-negative
bacterial strains.

23
 Extracts of S. cumini leaf prepared in petroleum ether, methanol and water by using
different concentrations (15, 10 and 5%) were tested for antimicrobial activity. The in vitro
bioassay was designed to inhabit the growth of four types of bacteria, namely Escherichia
coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subitils and two types of
fungi, Candida albicans and Aspergillus niger. The extracts of leaf showed different
activities against the selected bacteria at all concentration levels, by using cup plate diffusion
method and extract of petroleum ether was active against E. coli only. Methanol extract
showed variable activities against bacteria in different concentrations, especially 15%
concentration showed high activity against B. subtilis and aqueous extract showed variable
activities against most of the tested bacteria. E. coli strain was found to be sensitive to all
concentrations (Elfadil et al., 2015).
Stem and leaf extracts of jamun had antibacterial property against all used bacteria.
But maximum inhibition zone (25 mm) was observed against Roultella plantikola and
minimum (14 mm) against P. aeruginosa by using fruit extract. Inhibition zones of 25 mm
and 14 mm were also observed by using leaf and fruit extracts against R. plantikola. These
extracts showed maximum and minimum zones of inhibition (18 mm and 7 mm) against
strains of fungi viz. Penicillium chrysogenum and C. albicans respectively (Pareek et al.,
2015).
Antimicrobial property of seed extracts of some plants namely S. cumini, Phoenix
sylvestris, Annona squamosal, Manilkara zapota and Tamarindus indica prepared by
microwave aided extraction method was investigated against yeasts, mold and an anaerobic
bacterium by broth dilution test and minimum inhibitory concentration was calculated.
Methanol extract of S. cumini and T. indica inhibited almost 60% of test microorganisms
(Ramanuj et al., 2012).
Antibacterial effect of the extracts and EOs of jamun leaf prepared in chloroform,
petroleum ether, methanol and ethyl acetate was assessed against Serratia marcescens, E.
coli, Bacillus cereus, S. aureus, Enterobacter faecalis, Proteus vulgaris, Salmonella
paratyphi, Klebsiella pneumoniae and P. aeruginosa by agar well diffusion method. Leaf
extracts and EOs exhibited significant inhibition against all strains comparable with that of
synthetic antibiotics like gentamicin sulphate and ciprofloxacin examined under same

24
conditions. The results showed that the methanolic extract of leaf and its EOs can be counted
as good source of antimicrobials applied for drug production (Reddy and Jose, 2013).
The antimicrobial potential of extracts of barks, pulps, fruits and leaves of S. cumini
was examined against some bacterial strains by disc diffusion method. Extracts exhibited
inhibitory behavior against growth of S. aureus, B. subtilis and B. cereus (Gram-positive
bacteria). Gram-negatives such as Vibrio cholera, Shigella flexneri and P. aeruginosa also
showed no resistance to the extracts. The bark and leaf extracts were more potent
antimicrobials than the seed and pulp extracts (Mubassara et al., 2015). S. cumini ethanol
extract was also most active against Corynebacterium diphtheria, S. aureus and
Staphylococcus epidermidis amongst other tested plants (Sesbania grandiflora, Senna alata,
Tabernaemontana divaricate and Acacia farnesiana) (Mueller et al., 2015).

Enzyme inhibitory activity

Maximum inhibition of porcine pancreatic alpha-amylase was exhibited by aqueous
extract of seeds of S. cumini. The inhibitors of alpha-amylase were isolated from seed extract
into fractions by preparative thin layer chromatography and different R f (Retardation factor)
values were calculated. The fraction having R f value in range of 0.285-0.43 was found to be
most effective inhibitor which was analyzed through LC-MS. It was concluded that isolated
components from seed extract were identified as 3,5,7,4`-tetrahydroxy flavanone and
betulinic acid. Dixon plot revealed the noncompetitive nature of the inhibition (Karthic et al.,
2008).

The inhibitory activity of extracts of seed kernel of S. cumini on alpha-glucosidase

activity from Saccharomyces cerevisiae, Bacillus stearothermophilus and rat intestine were
examined. Kernel extracts were found as most potent inhibitors of α-glucosidase from B.
stearothermophilus and acetone extract was a potent inhibitor of this enzyme when tested in
vivo using Goto–Kakizaki rats (Shinde et al., 2008).

Studying jamun seed aqueous extract effect on the activity of enzymes having role in
the functions of lymphocytes, Bellé et al. (2012) took lymphocytes of healthy donors and
exposed them to different concentrations (10, 30 and 100 mg/mL) of the extract for the
periods of 4 and 6 hours and then evaluated the activities of enzymes acetylcholinesterase
(AChE), adenosine deaminase (ADA) and dipeptidylpeptidase IV (DPP IV). They also
25
checked CD26 expression and cellular viability. As the result, the extract inhibited the
enzymes ADA and DPP IV without altering protein CD26 expression depending on the time
of their exposition to the extract. The extract did not affect cell viability or the activity of
AChE. These outcomes indicated that the aqueous seed extract possesses immunomodulatory
properties by inhibiting the DPP IV-ADA complex pathway.

Anticancer activity

Methanolic extract of jamun fruit pulp with different concentrations was examined in
vitro to study its cytotoxicity to MCF-7 cells of human breast cancer using the micro-culture
tetrazolium assay. Trypan blue dye exclusion technique was applied to determine percent cell
viability. The assay indicated that viability of cell cultures was reduced and inhibited to
different extents in the existence of concentrations of extract (Tripathy and Pradhan, 2015).

Antiinflammatory potential

Muruganandan et al. (2001) studied the antiinflammatory effect of ethanolic extract

of bark in rats divided into three different groups of acute, subacute and chronic
inflammation phases. Animals were administered with the extract of dose of 10.125 g/kg
with no sign of toxicity. The extract showed significant antiinflammatory potential. No
gastric lesion was induced in rats by the extract in acute and chronic ulcerogenic trials. The
study revealed that extract of S. cumini bark possesses strong antiinflammatory activity
against several inflammation phases with no side effects on gastric mucosa.

According to Sehwag and Das (2014) ethyl acetate and ethanol extracts of jamun seed
exhibited effective antiinflammatory activity against paw oedema in wistar rats induced by
carrageenan. Aqueous and methanolic extracts also reduced oedema by 69 and 48%
respectively which was a good result as compared to 75% reduction by diclofenac sodium as
a synthetic drug at lower dose. Chloroform extract of jamun showed a notable reduction of
oedema induced by kaolin as well. Another inflammatory disease called arthritis, the
inflammation in joints was also prevented by methanolic extract of seed. This extract had
significant effect on Freund’s adjuvant induced arthritis and it was reported that the

26
inhibition of the disease was directly proportional to the dose of extract when it was
administered orally.

27
CHAPTER 3

MATERIALS AND METHODS

3.1 Plan of work

The purpose of study was to estimate in vitro and in vivo biochemical assays of S.
cumini. Research work was accomplished in the following laboratories:

 Clinico-Medical Biochemistry Laboratory, Department of Biochemistry, University

of Agriculture, Faisalabad.

 Medicinal Biochemistry Laboratory, Department of Biochemistry, University of

Agriculture, Faisalabad, Pakistan.

The work plan has been discussed under the following headings:

3.1.1 Collection and identification of plant materials

3.1.2 Preparation and fractionation of plant extracts

3.1.3 In vitro biochemical analysis of the plant extracts

3.1.3.1 Antioxidant profile of the plant extracts

3.1.3.2 Biofilm formation inhibitory activity of the plant extracts

3.1.3.3 Antidiabetic evaluation of the plant extracts

3.1.3.4 Mutagenicity of the plant extracts

3.1.3.5 Cytotoxic activity of the plant extracts

3.1.3.6 Thrombolytic assay of the plant extracts

3.1.4.2 In vivo biochemical analysis of the plant extracts

28
 3.1.4.2.1 Antidiabetic potential

3.1.4.2.2 Antioxidant profile

3.1.4.2.3 Antihyperlipidemic potential

3.1.4.2.4 Hepatoprotective potential

3.1.4.2.5 Renoprotective potential

3.1.5 Statistical analysis

3.1.1 Collection and identification of plant material

Plant: Syzygium cumini (black plum) fruit was randomly collected from local market of
Faisalabad. Plant was identified and authenticated at the Department of Botany, University of
Agriculture, Faisalabad, Pakistan.

Fig. 3.1: S. cumini fruit at lab.

3.1.2 Preparation and fractionation of plant extracts
The chosen part of plant was washed, dried, ground to a fine powder and stored in a
clean sealed container. The extract of powdered material was prepared trice in methanol
being kept in the solvent for intervals of 72 hours at normal temperature. The solvent was
evaporated on water bath to make the extract viscous then dried and kept at −4 °C. Then it
was dissolved in distilled water for fractionation in some polarity based solvents viz., n-
hexane, n-butanol, chloroform, ethyl acetate, ethanol, methanol and aqueous using separating
funnel (Mehmood et al., 2012).

29
 a) b)

Fig. 3.2: a) Dried fruit of S. cumini and b) Powder

a) b)

Fig. 3.3: a) Methanol extract and b) Fractionation process

The solvents used with their densities are mentioned below.

Table 3.3: Fractionation solvents

Solvents Density (g/mL)

n-hexane 0.66

n-butanol 0.81

Chloroform 1.50

Ethyl acetate 0.89

Ethanol 0.79

30
 Methanol 0.79

Distilled water 0.998

Fractionation yields were: n-hexane (120 g), n-butanol (20 g), chloroform (60 g),
ethyl acetate (5 g), ethanol (60 g), methanol (85 g) and aqueous (10 g) fractions.
3.1.3 In vitro biochemical analysis
3.1.3.1 Antioxidant profile

The antioxidant profile of S. cumini was assessed by following methods:

3.1.3.1.1: Total Phenolic Content (TPC)

3.1.3.1.2: Total Flavonoid Content (TFC)

3.1.3.1.3: DPPH radical scavenging capacity

3.1.3.1.1 Total Phenolic Content (TPC)

The concentration of phenolics was determined by Folin-Ciocalteu reagent as

described by Chahardehi et al. (2009). Folin-Ciocalteau reagent is made from oxidation of
phenols in the mixture of phosphomolybdic acid and phosphotungstic acid and its reduction
to form oxides of molybdenum and tungsten with blue color. The produced blue color during
reduction shows maximum absorption at wavelength 750 nm which is related to the total
amount of phenolics present. Briefly, distilled water (1.58 mL), test samples (20 µL) and
diluted reagent (10%; 100 µL) were mixed and 3 mL Na2CO3 (1% w/v) was added after 3
minutes and incubated for half an hour. Absorbance was read at 750 nm. Results were
calculated as mg GAE/g.

31
 Fig. 3.4: Standard curve for total phenolic content

Fig. 3.5: Determination of TPC

3.1.3.1.2 Total Flavonoid Content (TFC)

AlCl3 colorimetric technique was undertaken to determine flavonoid content as

described by Siddique et al. (2010). The elementary principle of AlCl3 colorimetric technique
is the production of acid stable complexes of AlCl 3 with Carbon-3 or 5 hydroxyl groups and
Carbon-4 keto group in flavonols and flavones. It also makes easily breakable acid
complexes with ortho-dihydroxyl groups on flavonoids A- or B-rings (Kalita et al., 2013).
Briefly, we mixed test samples (50 µL each), 160 µL NaNO2 and 1.26 mL distilled water in
test tubes and left for 10 minutes. 300 µL of 10% AlCl3 was added to the mixtures following
the addition of 1 mL NaOH after 5 minutes and incubated for almost 20 minutes. The

32
absorbance was read at about 510 nm with a double beam UV/Visible spectrophotometer and
compared with a standard curve.

Fig. 3.6: Standard curve for total flavonoid content

Fig. 3.7: Determination of TFC

3.1.3.1.3 DPPH radical scavenging assay

According to Souri et al. (2008), antioxidant capacity was determined on the basis of
2,2-diphenyl-l-picrylhydrazyl DPPH scavenging ability. The principle assumes a hydrogen
donor as an antioxidant and is founded on DPPH reduction in the existence of that hydrogen.
Extracts lessen the color intensity of DPPH due to capacity of hydrogen donation. DPPH is a
stable purple nitrogen containing free radical (DPPH •) showing maximum absorption at 517
nm. As it accepts an electron from an antioxidant present in its solution, it disappears and its

33
absorption reduces to yellow which is an indicator of antioxidant activity of test sample
(Kalita et al., 2013). 1 mL solution of DPPH (0.004 mg DPPH in 15 mL methanol) was
added to 1 µL extract solution and covered by aluminum foil for 35 minutes. All estimations
were done trice. The radical scavenging capacities were expressed as percent inhibition,
calculated as: % DPPH scavenging = [A (control) – A (sample) /A (control)] × 100, where, A (control) was
absorbance of control and A (sample) was e of test samples. A curve of % inhibition against
concentration was plotted and IC50 (half-maximal inhibitory concentration) was determined.

Fig. 3.8: DPPH scavenging assay

3.1.3.2 Biofilm formation inhibition assay

The biofilm quantitative and qualitative inhibition assays were conducted as per the
schemes designed by Anjum et al. (2014) and Dheepa et al. (2011).

3.1.3.2.1 Quantitative biofilm inhibition assay by microtitre plate method

Nutrient broth, plant extracts and bacterial strains namely S. aureus (Gram-positive)
and Pasteurella multocida (Gram-negative) of 100 µL each were taken in 96-wells flat
bottomed plastic tissue culture microliter plate and incubated aerobically at 37 °C overnight.
Next day, plates were washed with sterile phosphate buffer saline, three times and shook to
remove unattached bacteria. The adhered bacteria were fixed with 99% methanol. Methanol
was discarded and plates were air dried after 15 minutes. 100 µL of 50% crystal violet stain
was added and extra stain was rinsed off with tap water. After that plates were dried and the
dye was solubilized with 100 µL, glacial acetic acid (33% v/v) per well. Absorbance was
read by plate reader (BioTek, USA) at 630 nm as described by Anjum et al. (2014).

34
Ampicillin with nutrient broth was taken as positive control while nutrient broth with
bacterial strains was used as negative control. Percent inhibitions were calculated as: %
inhibition = 100 × [A (control) – A (sample) / A (control)]

3.1.3.2.2 Qualitative biofilm formation inhibition assay

Using the method of Dheepa et al. (2011), nutrient broth was poured on the glass
slides and inoculated with clean culture of bacterial strain a night at 37 °C. Then 2% crystal
violet stain was put on slides for 10 minutes and they were washed. Nutrient broth and
inoculum together were taken as negative controls and ampicillin was taken as positive
control. The prepared extract/fractions were examined for their inhibitory effect on formation
of microbial biofilm under microscope.

3.1.3.3 Antidiabetic evaluation

3.1.3.3.1 Antiglycation potential

Antiglycation process was assayed as stated by Matsuda et al. (2003). The reaction
solution of 100 mg D-glucose and 10 mg bovine serum albumin (BSA) in 67 mM sodium
phosphate buffer (pH 7.2), 150 µL each was kept at 37 °C for 3, 7 and 11 days. Then
absorbance of 0.2 mL diluted reaction solution was read at 370 nm as excitation wavelength
and at 440 nm as emission wavelength by spectrophotometer (BMS UV-2600, Japan). The
solution with no D-glucose was used as blank. Metformin was used as a reference compound.

Calculation of % inhibition:

% inhibition = [A (control) − A (test) / A (control)] × 100

 A (test) = Experimental value
 A (control) = Control value

35
 Fig. 3.9: Antiglycation assay
3.1.3.3.2 Enzyme inhibition assay
Enzyme inhibitory assay was performed by estimating AChE, alpha-glucosidase and
alpha-amylase inhibitory activity.
3.1.3.3.2.1 AChE inhibition assay
AChE restraint was assessed following the scheme of Rahman et al. (2005)
3.1.3.3.2.1.1 Preparation of reagents
3.1.3.3.2.1.1.1 Phosphate buffer-1 (0.1 M) (for enzyme and test)
15.6 g of Na2HPO4.2H2O dissolved in 750 mL distilled water and checked for the pH
to be adjusted to 8 by NaOH solution. After that made volume 1 liter by distilled water and
stored at 4 °C.
3.1.3.3.2.1.1.2 Phosphate buffer-2 (0.1 M) (for Ellman’s reagent)
15.6 g of Na2HPO4.2H2O dissolved in 750 mL distilled water and checked for the pH
to be adjusted to 7 by adding NaOH solution. After that made volume 1 liter by distilled
water and stored at 4 °C.
3.1.3.3.2.1.1.3 Buffered Ellman’s reagent (DTNB)
39.6 g of Ellman’s reagent and 15 mg NaHCO 3 were dissolved in 10 mL phosphate
buffer-2 solution. This solution was stored at 4 °C.
3.1.3.3.2.1.1.4 Acetylcholine iodide (substrate)
We dissolved 108.35 mg of acetylcholine iodide in distilled water (5 mL) and stored
at 4 °C.

36
3.1.3.3.2.1.1.5 Acetylcholinesterase (enzyme)
Enzyme solution was made by taking the enzyme in phosphate buffer-1, setting its
concentration about 0.0025 U/mL and this enzyme solution was kept in iced water bath at 5
°C.
Following the method, phosphate buffer-1 (2.81 mL), 30 µL extracts, AChE stock
solution (30 µL) and 100 µL 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) stock solution were
mixed and incubated for 5-10 minutes at 25 °C. After incubation, 30 µL of substrate stock
solution was added and absorbance was read at 412 nm. As positive physostigmine (0.441 g
amigra tablet dissolved in 10 mL dis. H2O) and as negative control methanol were used.
Percent inhibition was calculated as: % inhibition = [A (control) – A (sample) /A (control)] × 100

Fig. 3.10: AChE inhibition assay

3.1.3.3.2.2 Alpha-glucosidase inhibition assay

This was assayed by chromogenic method as explained by Apostolidis et al. (2006).

Briefly, 100 µL sample solution and (500 µL) α-glucosidase solution in 0.1 M phosphate
buffer (pH 6.9; 1.0 U/mL) were poured in test tubes and incubated at normal temperature for
15 minutes. Then 500 µL 5 mM solution of p-nitrophenyl-α-D-glucopyranoside in same
buffer was mixed. The mixtures were incubated for 5 minutes and absorbance was read at
405 nm using microplate reader. 1000 µL of acarbose and methanol were considered as
positive and negative controls respectively and experiment was performed in duplicate.
Percent inhibition was calculated as: % inhibition = (1− A (sample) /A (control)) × 100

37
 Fig. 3.11: Alpha-glucosidase inhibition assay
3.1.3.3.2.3 Alpha-amylase inhibition assay
This hindrance potential was assayed as performed by Apostolidis et al. (2006).
Briefly, 100 μL of 20% (v/v) extracts and standard acarbose and 100 μL of solution of amylase
in 0.02 M sodium phosphate buffer (pH 6.9; 0.5 mg/mL) were kept at room temperature for 10
minutes. After preincubation, 500 μL 0.5% solution of starch was added and incubated for 30
minutes. 1 mL of reagent 3,5 dinitrosalicylic acid stopped the reaction, held in water bath.
Distilled water (10 mL) was added to reaction mixture and absorbance was read against blank at
540 nm. % inhibition = A (control) –A (sample) /A (control) × 100 where, A (control) = Absorbance of
control and A (sample) = Absorbance of test samples

Fig. 3.12: Alpha-amylase inhibition assay

3.1.3.4 Mutagenicity/Carcinogenicity assay

3.1.3.4.1 Ames test

The explained protocol was followed to test the mutagenicity/carcinogenicity of S.

cumini fruit extracts.

38
3.1.3.4.1.1 Muta-Chromoplate kit

Mutagenicity assessment was done by commercially available kit, Muta-Chromoplate

from Environmental Biodetection Products Incorporation (EBPI, Ontario, Canada). Ames
bacterial reverse-mutation test (Ames et al., 1975) was applied and completed in liquid
culture medium known as fluctuation test.

3.1.3.4.1.2 Test bacterial strains

Cat no. TA98 and Cat no. TA100 were two mutant strains of Salmonella typhimurium
taken from EBPI were sustained on nutrient agar at 4 ºC ± 1 ºC. Strains were inoculated and
incubated in nutrient broth at 37 ºC for a night before test.

Procedure

The examination was done in microtiter plate as stated by Maron and Ames in 1983.
Plates were kept at 37 °C for 72 hours, covered with plastic pouches. The test plates were
read against blank plate with wells colored purple indicating no mutation. The plates,
standard and background were observed for turbid, partial yellow and full yellow wells
(positive) and purple wells (negative). Test samples were counted as toxic to strains if all
wells remained purple. An extract was counted as mutagenic if the number of positive wells
in related plates was more than positive wells in the background plate.

3.1.3.4.1.4 Scheme of test

The amounts of solutions of test samples and chemicals along with negative and
positive standards were employed according to the scheme given below.

Table 3.4: Schematic representation of recipe to perform Ames test

39
 Treatment Plant Reagent Deionized Test
Standards
(Microtiter plate) extract mixture water strain

Blank ---- ---- 2.5 17.5 ----

Background I for S.
---- ---- 2.5 17.5 0.005
typhymorium TA100

Background II for S.
---- ---- 2.5 17.5 0.005
typhymorium TA98

Standard mutagen for

S. typhymorium 0.1 ---- 2.5 17.5 0.005
TA100

Standard mutagen for

0.1 ---- 2.5 17.5 0.005
S. typhymorium TA98

Test sample 1
---- 0.005 2.5 17.5 0.005
(methanol)

Test sample 2
---- 0.005 2.5 17.5 0.005
(ethanol)

Test sample 3 (n-

---- 0.005 2.5 17.5 0.005
butanol)

Test sample 4 (n- 0.005 2.5 17.5 0.005

----
hexane)
Test sample 5 (ethyl 0.005 2.5 17.5 0.005
----
acetate)
Test sample 6 0.005 2.5 17.5 0.005
----
(chloroform)
40
Test sample 7 0.005 2.5 17.5 0.005
----
(aqueous)
All concentrations were taken in µL
3.1.3.4.2 Brine shrimp test
This test was conducted and prohibit analysis was done by following the technique of
Meyer et al. (1982). Ten shrimp and 5 mL non-natural sea water were poured in each sample
vessel by a reusable pipette (Scientific Products, diSPo pipettes). The number of nauplii was
macroscopically countable in stem of the pipette tip exposed to bright background. Shrimp
were fed with droplets of dry suspension of 3 mg red star yeast in 5 mL non-natural sea
water. The vessels were kept under light. After 6 and 24 hours, the survivors were counted
with the help of a magnifying glass of 3X and the percentage of deaths at control and
different doses of extracts were determined. The counts after one night were more valuable.
In case of occurrence of deaths in control, the data were made precise applying Abbott’s
formula: % deaths = (test – control /control) × 100
3.1.3.5 Cytotoxic activity
3.1.3.5.1 Hemolytic assay
The cytotoxicity of the extracts and polar fractions of S. cumini was evaluated by
hemolytic activity following the method of Powell et al. (2000). Blood samples were
centrifuged at 1500 rpm three minutes to distinct the plasma from cellular portion of blood.
Plasma was discarded and the pellets of red blood cells (RBCs) were rinsed with 5 mL
chilled sterile isotonic phosphate buffer saline (PBS; pH 7.4), three times and centrifuged
again at 1500 rpm for 5 minutes and a suspension of cells in normal saline was prepared. The
rinsed RBCs were calculated on heamocytometer, maintaining the RBCs amount to 7.068 ×
108 cells/mL. Then 180 µL diluted blood cell suspension and 20 µL of each test sample was
taken in eppendorfs and left for 40 minutes at 37 °C. Tubes were disturbed after 15 minutes,
cooled and centrifuged for 6 minutes followed by taking supernatant (100 µL) and diluting
with PBS (900 mL). 200 µL of cold contents of tubes was poured in ELISA microtiter plate.
For the test, 0.1% triton X-100 was considered as positive control and PBS as negative
control. Absorbance was read at 576 nm by BioTeK, µ Quant (BioTek, Winooski, VT,
USA).
Percent hemolytic inhibition was calculated by given formula:

% Hemolysis = (A (sample) – A (negative control) /A (positive control) – A (negative control)) × 100

41
3.1.3.5.2 DNA-damage and protective assay

The pBR 322/CT-DNA was left unprotected from H 2O2 and UV and DNA-damage
protection was performed with optimized plant extracts following the technique stated by
Ruma et al. (2013)

3.1.3.5.2.1 Preparation of reagents

3.1.3.5.2.1.1 Preparation of 50 mM phosphate (PO4) buffer

Said buffer was made by 100 mL solution of 0.55 g Na 2HPO4 and 0.18 g NaH2PO4 in
distilled water.

3.1.3.5.2.1.2 Preparation of DNA solution

Two folds dilution of 0.5 µg/μL of CT-DNA to 0.5 µg/3 µL was made by addition of
50 mM phosphate buffer of pH 7.4 in micro-eppendorf tubes.

3.1.3.5.2.1.3 Preparation of 30% H2O2

30% solution of H2O2 was made from its 36% solution.

3.1.3.5.2.1.4 Reaction mixture preparation

Approximately, 3 µL of diluted CT-DNA and 20 µL stock solution of the test

samples, 4 µL of 30% H2O2 and 3 µL of Tris base-acetic acid-EDTA (TAE) buffer were
transferred to the micro-eppendorf tubes.

3.1.3.5.2.1.5 Control 1 preparation

Diluted CT-DNA (10 µL) and 12 µL of phosphate buffer were taken in micro-
eppendorf tubes.

3.1.3.5.2.1.6 Control 2 preparation

10 µL of diluted CT-DNA and 12 µL of H2O2 and FeSO4 were added in micro-

eppendorf tubes.

42
Procedure

TAE buffer (1X) was made by dissolution of 50X buffer (10 mL) in distilled water
(490 mL) and agarose gel solution (1%) in buffer was made by dissolution of agarose (1 g) in
1X TAE buffer (100 mL). Agarose solution was warmed up in oven for 1 minute to make a
homogenous mixture and cooled to suitable temperature. 20 µL of stain ethedium bromide
was added to agarose solution, shaken and poured in gel tray of electrophoresis system to be
solidified for 30 minutes. Then the same buffer as gel running buffer was poured in tray to
the level at which electrodes both were dipped in it. The samples and reaction mixture along
with 3 µL bromophenol blue as tracking dye were incubated and loaded in each well made
by a comb. Reaction mixtures were run horizontally in columns dipped in buffer at 100 volts
for 40 minutes.

The photographs of gel were made under UV by gel document system (SynGene,
England). various antioxidant treatments, negative and positive controls and a molecular
marker were loaded in wells, for each run. The positive control contained plasmid DNA and
Fenton’s reagent whereas the negative control was only the plasmid DNA. The results were
analyzed by trans-illuminator.

3.1.3.6 Thrombolytic assay

Thrombolytic activity was investigated as described by Ali et al. (2014).

3.1.3.6.1 Collection of blood samples

Samples were taken from blood laboratory of University of Agriculture, Faisalabad.

The samples were collected from the veins of healthy helpers. 500 µL of blood samples was
poured in the preweighed micro-centrifuge tubes.

3.1.3.6.2 Optimization of blood samples conditions

As the normal body temperature is 37 °C therefore, it was too important to optimize the
blood samples temperature by keeping them in the incubator at 37 °C for 50 minutes.

43
3.1.3.6.3 Formation of blood clots

Micro-centrifuge tubes containing blood samples were incubated straight for clot
formation for about 45 minutes and the serum accumulated above optimized blood clots was
removed carefully with the help of a micropipette. Then the tubes were weighed and the
variance between the weights of empty tubes (W t) and the weights of tubes containing clots
(Wtb) determined the weights of blood clots (Wb).

3.1.3.6.4 Biochemical process for clot lysis

100 µL of test samples were added to the blood clots in properly labeled micro-
centrifuge tubes. Citric acid was the positive control and tubes were incubated, inverted in
the stand at 37 °C for the lysis of clots (90 minutes).

3.1.3.6.5 Inspection of clot lysis

The fluid accumulated in the lids of the tubes was then removed carefully with the help
of micropipette and tubes were weighed. The difference of the weights of empty tubes from
the weights of tubes containing clots after lytic process (W tl) showed the weights of clots
lyzed (Wl). Test was done in triplicate. Clot lyses percentage was calculated as under:

Wb = Wtb – Wt

Wl = Wtl – Wt

% lysis = Wl / Wb ×100

a) b)

44
 c) d)

Fig. 3.13: Various steps of thrombolytic assay: a) Weighing the blood clots,

b) Centrifugation, c) Incubation in water bath and d) Serum removal

3.1.4 In vivo study

In vivo study was carried out at animal house, National Institute of Food Science and
Technology, University of Agriculture, Faisalabad.
3.1.4.1 Experimental design
3.1.4.1.1 Experimental animals and induction of diabetes
Healthy male rats of weights 200-250 g were bought from National Institute of Health
(NIH), Islamabad, Pakistan.
Rats were divided into random groups, housed and adapted in clean and dry cages
and in well-ventilated animal house for 7 days before commencement of experiment. The
animals were nourished with water and pellet diet as ad libitum. DM was induced in selected
groups by administrating 5% normal saline solution of alloxan monohydrate at 80 mg/kg
body weight into marginal ear vein. Level of fasting blood glucose was checked on different
days until observation of stable hyperglycemia. The rats with levels of greater than 150
mg/dL for fasting blood glucose were diabetic.
3.1.4.1.2 Study groups
Division of animals to different groups was as follow:
Group 1: Control group (Non- diabetic)
Group 2: Control group (Diabetic group)
Group 3: Diabetic group that received S. cumini
Group 4: Diabetic group that received herbal formulation
Group 5: Diabetic group that received synthetic drug, glibenclamide

45
 Treatment was started after induction of diabetes and continued for 21 days. Pre-
treatment and post-treatment body weights were noted. By the end of treatment, rats were
slaughtered under sodium pentobarbitone anesthesia according to guidelines. Sample blood
were taken to analyze biochemically during pre- and post-treatment phases (Rekha et al.,
2008).
3.1.4.2 Biochemical analysis
3.1.4.2.1 Antidiabetic potential
Glycated hemoglobin level, serum glucose and insulin levels were assessed by using
the commercially available kits.
3.1.4.2.1.1 Determination of glycated hemoglobin content
Glycated hemoglobin level was measured by using the commercially available kit
(Boric Acid Affinity Chromatography; Wuxi BioHermes Bio & Medical Technology Co.,
Ltd.).
Principle
The A1C EZ® HbA1c analysis system product utilizes a boric acid affinity
chromatography method to quantitatively measure the percentage of glycohemoglobin A1c
(HbA1c) in total hemoglobin. A solid phase separation matrix is implemented as a membrane
modified chemically to hold both boronate groups and negatively charged groups. positively
charged non-glycohemoglobin and glycohemoglobin attach to negatively charged groups in
the acidic pH buffer drifting through the matrix but in basic pH, the hemoglobin gives up its
charge and releases from ionic binding. When pH of buffer is basic, boronate binds cis-diols
as well as the glucose attached to glycohemoglobin keeping the glycohemoglobin in the
matrix. The analyzer uses an optical reflectance measurement technique to measure the
membrane and quantify the hemoglobin present in the matrix under every condition. The
ratio of the glycohemoglobin to the total hemoglobin was calculated and reported as %
HbA1c units.
Sample requirements
Blood samples from the finger were collected in EDTA-coated tubes and placed at
room temperature for more than 10 minutes to prevent thawing, prior to test. The calibration
code chip was removed from the test kit package, inserted into the slot at the top right-side of
the analyzer. The analyzer was turned on and it was confirmed the test code displayed on the

46
screen matched with the test code included in the kit. One test strip was taken out of the test
strip vial or foil pouch and inserted into the test strip slot in the analyzer with the notched end
of the strip towards the front and the bottom sides of the strip with circular-hole face down
according to the voice prompt. Three drops of buffer-A were applied to the buffer port. After
the reaction was finished according to the procedures, the reaction time was set. Blood
sample was collected by sampler by touching its thread to the finger blood or venous blood
and held for three seconds. The sampler thread was put into the test strip blood application
area and held for three seconds to allow the blood sample to transfer to the test strip. The
analyzer sent out three consecutive “di” prompts. After the prompt, sampler was removed
from the test strip. After the reaction was finished according to the procedures, the reaction
time was set and next step started. Two drops of buffer-B were applied to the buffer port and
the reaction time was set again. After the reaction was finished, the analyzer displayed and
announced the test (HbA1c) value.
3.1.4.2.1.2 Determination of serum glucose level
GOD-PAP scheme, an enzymatic colorimetric assay (liquicolor) was applied for
determination of glucose level in serum.
Method

Glucose is calculated following glucose oxidase mediated oxidation process that

produces H2O2. Under catalytic act of peroxidase (POD), the produced H 2O2 reacts with
phenol and 4-aminoantipyrine to quinoneimine, a reddish violet dye as indicator. 200 or 10
µL sample or standard (glucose) and 2000 or 1000 µL reagent (sample and blank), were kept
at 25 or 37 °C for 15 or 5 minutes, respectively and taken into cuvette (optical path 1 cm).
Absorbance was read at 500 nm within 1 hour.

Calculation of glucose concentration:

C = 100 × ΔA (sample) /ΔA (STD) [mg/dL] or

C = 5.55 × ΔA (sample) /ΔA (STD) [mM]

47
3.1.4.2.1.3 Determination of serum insulin level (Insulin ELISA)

Principle

It is a double-site solid form of enzyme immunoassay, with principle of direct

sandwich system consisting of two antibodies of a single clone fixed against isolated
antigenic determining factors over insulin. During incubation, insulin interacts with enzymes
(horseradish peroxidase, HRP)-conjugated anti-insulin antibody and anti-insulin antibody
attached to the micro-titer well. Rinsing removes unattached antibody labeled with enzyme.
The bound HRP-complex is distinguished by reacting with TMB (3,3',5,5'-
Tetramethylbenzidine) substrate. The colorimetric endpoint in process is reached by addition
of an acid and read by ELISA reader.

Procedure

Before starting the assay, all reagents were kept at 25 °C and mixed gently before use.
The required number of covered strips was set in the holder. 25 µL of control, patient’s sera
and insulin standards were pipetted into appropriate wells. 100 µL working conjugate of
insulin-enzyme was taken in wells, mixed well for 15 seconds and incubated for 1 hour at 18-
26 °C. The wells were rinsed thrice with 1X buffer (300 µL). Blotting on porous papers was
done. 100 µL TMB was added and incubated in wells for 15 minutes at normal temperature.
Stop solution (50 µL) was added and plates were shaken for mixing the solution. Absorbance
was measured at 450 nm in 15 minutes after addition of stop solution.

Calculation of results

The standard curve was drawn as follow:

1. Value of insulin standard in each vessel and on every kit, was checked. This value might
be different from portion to portion.

2. Absorbance for insulin standards were taken on vertical axis and concentrations of insulin
standard in µLU/mL on horizontal axis and plotted.

3. The absorbance for every unknown sample and controls was read from constructed curve.

48
4. Values being above the maximum point of standard were rechecked after the dilution with
standard "0".

3.1.4.2.2 Antioxidant profile

Superoxide dismutase, lipid peroxidation, glutathione peroxidase and catalase activity
were determined by using the commercially available kits.
3.1.4.2.2.1 Determination of SOD activity
Activity of SOD was assessed by using the technique explained by Pari and Latha
(2004). The principle of assay is inhibiting the production of phenazine methosulfate,
nicotinamide adenine di-nucleotide and amino blue tetrazolium formazan. 50% inhibition of
nitroblue tetrazolium reduction/min/mg protein was the expression of single unit of enzyme.
3.1.4.2.2.2 Estimation of lipid peroxidation
It was colorimetrically assessed by hydroperoxides and TBARS (thiobarbituric acid
reactive substances) as per the technique of Pari and Latha (2004). Briefly, tissue
homogenate in Tris HCl buffer at pH 7.5 (0.1 mL) was mixed with TBA-HCl-TCA reagent
(2 mL) (thiobarbituric acid 0.37%, trichloroacetic acid 15% and HCl (0.25 N) at 1:1:1 ratio),
put on water bath for 10 minutes and cooled. Absorbance of supernatant was read at 535 nm.
Same amount of tissue homogenate and Fox reagent (0.9 mL) consisting of 9.8 mg
ammonium iron (II) sulphate, 88 mg BHT and 7.6 mg xylenol orange added to methanol (90
mL) and 250 mM sulphuric acid (10 mL) and left for incubation at 37 °C for 35 minutes. The
developed color was colorimetrically measured at 560 nm. Concentration of hydroperoxides
was calculated as mM/100 g tissue.
3.1.4.2.2.3 Estimation of GPx activity
Activity of GPx was determined by the technique explained by Pari and Latha (2004).
Reaction mixture containing 0.4 M Tris HCl buffer of pH 7.0 (0.2 mL), tissue homogenate
(0.2 mL) homogenized in same buffer, 10 mM sodium azide (0.1 mL), 0.2 mM H 2O2 (0.1
mL) and 0.2 mL glutathione was left for incubation at 37 °C for 15 minutes. After adding
10% TCA (0.4 mL), mixture was centrifuged. The supernatant was tested for glutathione
concentration by Ellman’s reagent made from 19.8 mg DTNB dissolved in 0.1% sodium
nitrate (100 mL). Absorbance was checked at 412 nm. Activity of GPx was calculated as µg
of GSH consumed/min/mg of protein.

49
3.1.4.2.2.4 Determination of CAT activity
This was estimated at 620 nm colorimetrically and calculated as µmoles of H 2O2
utilized/min/mg of protein as done by Pari and Latha (2004). The reaction in 1.5 mL mixture
containing tissue homogenate or supernatant (0.1 mL), 2 M H 2O2 (0.4 mL) and 1 mL 0.01 M
phosphate buffer (pH 7) was caught by adding 2.0 mL of reagent dichromate-acetic acid.
3.1.4.2.3 Antihyperlipidemic potential
Serum lipid profile of rats including total cholesterol, HDL, LDL, VLDL and
triglycerides was estimated by using commercially available kits.
3.1.4.2.3.1 Total cholesterol estimation (liquicolor)
Level of total cholesterol was measured by method of CHOD-PAP which is a
colorimetric enzymatic test for cholesterol with lipid clearing factor. The absorbance was
determined at 500 nm.
Method
The cholesterol is measured after oxidation and enzymatic hydrolysis. Quinoneimine,
the indicator is produced from H 2O2 and 4-aminiphenazone in the availability of phenol and
peroxidase.
Calculation of the cholesterol concentration

1. With factor

Wavelength C [mM] C [mg/dL]

500 nm 14.3 × A 553 × A

2. With standard
C = 5.17 × ΔA (sample) /ΔA (STD) [mM] or

C = 200 × ΔA (sample) /ΔA (STD) [mg/dL]

3.1.4.2.3.2 HDL, LDL and VLDL estimation

The cholesterol level was determined by using cholesterol determination enzymatic

kit.
Principle

50
 LDL, VLDL and chylomicrons are precipitated by addition of 1.4 mM
phosphotungstic acid and 8.6 mM magnesium chloride (providing Mg ++ ions) to solution of
sample. Supernatant will contain HDL only, after centrifugation.
Precipitation
Sample (200 µL) and precipitation reagent (500 µL) were incubated together for 15
minutes and centrifuged for 20 minutes at 2500 g. Clear supernatant (0.1 mL) was shifted to
precipitation solution for cholesterol determination within 2 hours after centrifugation. 100
µL sample supernatant with 1000 µL cholesterol reagent and 100 µL standard with 1000 µL
reagent were together incubated for 5 or 15 minutes at 37 or 25 °C. Absorbance was read
against blank value within 45 minutes.
Cholesterol (HDL-C) was measured by kit method (Ecoline Diasys GmbH Merck, Germany)
as: HDL concentration of unknown sample = (C (standard) /A (standard)) × A (unknown sample)

Concentration of the standard equals that of total cholesterol in its standard solution.
LDL-C concentrations were calculated as:

Total cholesterol – VLDL-C – HDL-C (Friedewald et al., 1972) whereas, VLDL-cholesterol

was calculated as: Triglyceride/5 (DeLong et al., 1986).

3.1.4.2.3.3 Estimation of triglycerides

Principle

The estimation of triglycerides is done after cleavage by lipoprotein-lipase enzyme.

The quinoneimine indicator forms from 4-Cholrophenol and 4-Aminoantipyrine by H 2O2 and
catalytic act of peroxidase.

Procedure

Reagents

50 mM Pipes buffer at pH 7.0, p-chlorophenol (2.7 mM), Mg2+ (14.8 mM), ATP (3.15
mM), GK (glycerokinase) ≥ 500 U/L, lipoprotein-lipase (LPL) ≥ 2000 U/L, peroxidase ≥
500U/L, 4-aminoantipyrine (0.31 mM), glycerol-3-phosphateoxidase (GPO) ≥ 4000 U/L,
standard solution (200 mg/dL) and potassium ferrocyanide (10 µM).

51
 10 µL sample, 1000 µL reagent solution and 1000 µL of sample/standard were
incubated for 5 minutes at 37 °C. Absorbance was checked at 500 nm.

Triglycerides concentration was calculated as:

Triglyceride concentration of unknown sample = C (standard) /A (standard) × A (unknown sample)

3.1.4.2.4 Hepatoprotective potential

Serum ASAT, ALAT, ALP, GGT and total protein were measured by using
commercially available kits.
3.1.4.2.4.1 Determination of serum ASAT (GOT)
The assay was performed with and without presence of pyridoxal-5-phosphate (P-5P),
the indicative reagent for in vitro quantitative determination of ASAT in plasma or serum on
photometric systems method, the adjusted UV-test designed by International Federation of
Clinical Chemistry and Laboratory Medicine (IFCC).
Principle
Adding solution of 0.09 mM P-5P in 0.7 mM Good’s buffer at pH 9.6 stabilizes
transaminases and prevents low values obtained falsely in the samples of inadequate
endogenous P-5P, for example from intensive care patients and those with liver disease and
myocardial infarction.
For determination with P-5P, 1 part of P-5P was mixed with 100 parts of reagent 1
comprising of Tris (80 mM; pH 7.65), MDH (malate dehydrogenase) ≥ 600U/L, L-aspartate
(240 mM) and LDH ≥ 900 U/L (for example, 100 µL P-5P + 10 mL R1).
For determination without P-5P, 4 parts R1 were mixed with 1 part of reagent 2
comprising of 0.18 mM NADH and 12 mM 2-oxoglutarate (for example, 20 mL R1 + 5 mL
R2, Mixture of reagents 1 and 2 is called monoreagent). To check the absorbance of
substrate, 100 µL sample and reagent 1 (1000 µL) were incubated for 5 minutes, followed by
addition of 250 µL reagent 2 and absorbance was read against air after 1 minute. To read
sample absorbance at 340 nm within 3 minutes, 100 µL sample and 1000 µL monoreagent
were mixed.

Calculation

52
 ΔA/min was determined and multiplied by factors for substrate (2143) and sample
(1745).

ΔA/min × factor = ASAT activity [U/L]

3.1.4.2.4.2 Determination of serum ALAT (GPT)

The assay uses the same principle explained for the determination of ASAT activity.
For determination with P-5P, 1 part of P-5P was mixed with 100 parts of reagent 1
made from Tris (pH 7.15; 100 mM), LDH ≥ 1700 U/L and L-alanine (500 mM).
For determination without P-5P, 4 parts of R1 were mixed with 1 part of R2 made
from 2-oxoglutarate (15 mM) and NADH (0.18 mM) as monoreagent.
For substrate, 100 µL sample and 1000 µL reagent 1 were incubated at 37 °C for 5
minutes, then 250 µL reagent 2 was added. Absorbance was checked at 340 nm against air
after 1 minute and 1, 2 and 3 minutes thereafter.
For sample, 100 µL sample and 1000 µL monoreagent were mixed and absorbance
was read after 1 minute.
Calculation
From values of absorbance, ΔA/min was calculated and multiplied by the matching
factor for substrate (2143) and sample (1745).

ΔA/min × factor = ALAT activity [U/L]

3.1.4.2.4.3 Determination of ALP
Method
Adjusted standard method of kinetic photometric as per the German society of
clinical chemistry (DGKC) was undertaken. Absorbance was taken against air at 400-420 nm
at 25 °C, 30 °C or 37 °C.
Sample was prepared by mixing 4 parts of 1.2 M diethanolamine (pH 9.8) and 0.6
mM magnesium chloride and 1 part of p-nitrophenylphosphate (50 mM) as monoreagent.
For sample or calibrator, 20 µL of reagent 1 was incubated for approximately 1
minute, reagent 2 (250 µL) was added and absorbance was measured after 1 minute.
For sample or calibrator, 20 µL, monoreagent 1000 µL were mixed and absorbance
was read after 1 minute.

53
Calculation

1. With factor

ΔA/min was calculated from absorbance readings and was multiplied by the corresponding
factor for substrate (3433) and sample (2757):

ΔA/min × factor = ALP activity [U/L]

2. With calibrator

ALP [U/L] = (ΔA/min (sample)) /ΔA/min (calibrator)) × C (calibrator) [U/L]

3.1.4.2.4.4 Determination of GGT level
Serum GGT level was assessed by using the commercially available kit (carboxy
substrate method).
Reaction principle

ɣ-glutamyltranspeptidase catalyzes transferring of an amino group between

glycylglycine and L-γ-glutamyl-3-carboxy-4-nitroanilide to produce 5-amino-2-
nitrobenzoate and L-γ-glutamylglycylglycine. The formation rate of 5-amino-2-nitrobenzoate
is quantified as an intensification in absorbance that is proportional to GGT activity in the
sample.

The working reagent was a substrate tablet dissolved in 2.2 mL buffer reagent and
kept at 2-8 °C. 1 mL working reagent was pipetted and incubated into a clean dry test tube
labeled as Test (T) at 37 °C for 1 minute following the addition of 0.1 mL sample, mixed
well and after 1 minute, absorbance (A) was read at 405 nm and repeated after every 1, 2 and
3 minutes. Mean absorbance was calculated as change in absorbance value per minute
(ΔA/min.). GGT activity in U/L = ΔA/min. × 1158

3.1.4.2.4.5 Estimation of total protein

Total protein was determined by the technique of Bradford, (1976).
Reaction principle
The Bradford technique is based on binding of Coomassie blue G250 dye to protein.
Dye in free form can be present in three ionic states with different pK a values of 1.82, 12.4

54
and 1.15 (Walker, 2002). Out of three charged states of the dye predominating in the assay
acidic solution of reagent, the cationic green and red forms have maximum absorbance at 650
and 470 nm, respectively. Contrarily, blue anionic form that binds to protein, shows
maximum absorbances at 590 nm. Thus, the amount of protein can be determined by
estimating the amount of dye in the ionic blue form. It is generally accomplished by reading
the absorbance of solution at 595 nm.
Procedure
Two kinds of assay are explained here as standard and microassay, which are
appropriate to measure between 10-100 and 1-10 μg of protein, respectively.
Standard assay method
Between 10-100 μg protein was pipetted in 100 μL sample into a vessel. Duplications
of each sample were prepared and 10-100 μL standard solution of 1 mg/mL γ-globulin were
pipetted in tubes and diluted to 100 μL adding distilled water for the calibration curve. 100
μL of water was pipetted in another tube to make blank. 5 mL protein reagent was added to
tubes and mixed well by gentle vortex or inversion, preventing foaming. The A 595 of
standards and samples against the blank between 2 minutes and 1 hour after mixing was
measured.
Microassay method
This assay is subtler and beneficial when the quantity of the unidentified protein is
low or limited.
Samples duplicated between 1-10 μg protein in 100 μL volume were pipetted in
polyethylene microfuge tubes of 1.5 mL capacity. For the calibration curve, duplications of
10 to 100 μL standard solutions of 100 μg/mL γ-globulin were pipetted in tubes with final
volumes adjusted to 100 μL adding distilled water. Water (100 μL) was pipetted in another
tube for blank. 1 mL protein reagent was added and mixed slowly. Absorbance was read
between 2 minutes and 1 hour after adding the protein reagent.
33.1.4.2.5 Renoprotective potential
Serum urea, creatinine, urinary albumin and total protein were determined by using
commercial kits.
3.1.4.2.5.1 Determination of serum urea

55
 Level of serum urea was assessed using the commercially available kit (UV test,
glutamate dehydrogenase (GLDH) method)
Principle
The reduction in absorbance of NADH per unit time during reaction of urea is proportional to
concentration of urea.

Method
To prepare sample, 4 parts of R1 made of ADP (0.7 mM), 2-oxoglutarate (9 mM),
GLDH ≥ 1KU/L, urease ≥ 30 KU/L and NADH (0.3 mM) was mixed with 1 part of R2 (Tris,
PH 7.7; 100 mM) as monoreagent and left for at least 15 minutes at 15-25 °C before use. The
method was optimized for 2-point kinetic measurement.
For substrate, 10 µL sample/standard and 1000 µL reagent 1 were mixed and 1000
µL reagent 1 as blank were incubated for 5 minutes and 250 µL reagent 2 was added to both.
For sample, 1000 µL monoreagent was as blank and for sample/standard, 1000 µL
monoreagent and 10 µL sample were mixed and incubated for approximately 1 minute at
25/30 °C or approximately 40 seconds at 37 °C. Absorbance was read as A 1 and after further
1 minute, A2. ΔA = [(A1 – A2) (sample or standard)] – [(A1 – A2) (blank)]
Calculation with standard or calibrator

Urea (mg/dL) = ΔA (sample) /ΔA (standard) (mM) × C (standard) (mg/dL)

3.1.4.2.5.2 Determination of creatinine

Principle
A yellowish orange compound is formed from creatinine with picric acid in basic
solution. A prescription of protein does not occur at low concentration of picric acid used in
this method. The color intensity and concentration of the dye produced over specific reaction
times is a determinant of the concentration of creatinine. Late secondary reactions do not
cause interference due to the quick reaction between creatinine and picric acid.
Sample preparation

56
 Serum, heparin, plasma, urine, premixture containing 8.73 mM picric acid as R1 and
R2 consisting of disodium phosphate (12.5 mM), sodium hydroxide (312.5 mM) and
creatinine (20 mg/dL, 2.0 mg/dL and 177 µM) as standard were prepared.
Procedure with premixture
Reagent 1 and 2 in a ratio of 1+1, 50 µL serum or 1+99 diluted urine, 50 µL standard
and working reagent consisting of 500 µL sample and 500 µL standard were mixed and
absorbance of the sample (As1) and standard (Ast1) after 1 minute was measured at 492, 500 or
505 nm. Exactly, 2 minutes after the first reading the absorbance was redetermined as A s2 and
Ast2. Concentration of serum creatinine was calculated as under:
Creatinine concentration:
[mg/dL] = As2 − As1 /Ast2 − Ast1 × 2.00
[µM] = As2 − As1 /Ast2 − Ast1 × 177.0
3.1.4.2.5.3 Quantitative determination of urinary albumin
Urinary albumin was assessed by bromcresol green colorimetric method.
Principle
Albumin with bromcresol green at a little acidic pH produces a yellow-green to
green-blue color change in indicator. The color intensification is relative to the concentration
of albumin in sample.
Reagents
0.12 mM bromcresol green (pH 4.2) and 5 g/dL aqueous primary standard solution of
albumin
Procedure
The spectrophotometer was adjusted to zero using distilled water. 1.0 mL bromcresol
green reagent as blank, 1.0 mL reagent with 5 µL standard as standard and 1.0 mL reagent
with 5 µL sample were taken into cuvettes and incubated for 10 minutes at 15-25 °C.
Absorbance (A) of samples and standard was read at 630 nm. Concentration of albumin was
calculated as:
A (sample) /A (standard) × 5C (standard) = g/dL albumin
Conversion factor: g/dL × 144.9 = µmol
3.1.4.2.5.4 Estimation of total protein
Total protein was determined by the technique of Bradford, (1976), explained earlier.

57
3.1.5 Statistical analysis
The data was analyzed by ANOVA that is used for the comparison of more than two
population means simultaneously (Montgomery, 2008).

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Antioxidant constituents and activity
Plants can prevent oxidative stress by producing secondary metabolites such as
flavonoids, tannins, nitric acid, polyphenols and phenols as natural antioxidants that scavenge
free radicals and inhibit progression of degenerative ailments. The most effective agents are
believed to be phenolics and flavonoids found in seeds, fruits and herbs (Nair et al., 2013). In
the present research, all phenolics of S. cumini fractions were separated by Folin-Ciocalteu
technique because of its feasibility, low connection, power and speed (Sultan et al., 2014)
and results were calculated as g equivalent of gallic acid/ 100 g dry weight. Total flavonoid
contents were evaluated by AlCl3 colorimetric method and results were calculated as g
equivalent of catechin / 100 g of sample dry weight.
The absorbance readings for TPC and TFC of S. cumini as mean ± SD of triplicate
qualities are displayed in table 4.1.
Table 4.1: Mean ± SD absorbance values of TPC and TFC

Extract/Fractions TPC TFC

Methanol 0.673 ± 0.030 B

0.688 ± 0.030 AB

Ethanol 0.649 ± 0.015 B

0.647 ± 0.022 ABC

58
 n-butanol 0.400 ± 0.010 D
0.427 ± 0.015 D

n-hexane 0.544 ± 0.020 C

0.439 ± 0.019 D

Chloroform 0.490 ± 0.023 CD

0.562 ± 0.014 C

Ethyl acetate 0.429 ± 0.012 D

0.415 ± 0.012 D

Aqueous 0.540 ± 0.021 C

0.608 ± 0.010 BC

Means that share same letters are non-significant statistically (P>0.05).

Table 4.2: ANOVA for total phenolic content

Source of Degrees of
Sum of squares Mean squares F-value
variation freedom

Treatment 7 0.323874 0.046268 34.25**

Error 16 0.021615 0.001351

Total 23 0.345489

** = Highly significant (P<0.01)

Table 4.3: ANOVA for total flavonoid content

Degrees of
Source of variation Sum of squares Mean squares F-value
freedom
Treatment 7 0.373546 0.053364 37.26**
Error 16 0.022916 0.001432
Total 23 0.396462

** = Highly significant (P<0.01)

According to data provided in table 4.6, TPC and TFC were in the range of 6 ± 0.012-
13 ± 0.030 g GAE/100 g and 14 ± 0.010-25 ± 0.030 g catechin equivalent (CE)/100 g
respectively. Methanol was the most active solvent in extracting maximum TPC and TFC.

59
For other fractions TPC in a decreasing order was: ethanol > aqueous > chloroform > n-
butanol > n-hexane > ethyl acetate and the decreasing trend of TFC values was: ethanol > n-
hexane > aqueous > ethyl acetate > chloroform > n-butanol. Previous examination of S.
cumini methanolic extract indicated TPC of 88 ± 5.25 mg/mL and TFC of 54.5 ± 9.64
mg/mL (Belapurkar and Goyal, 2014). An earlier report indicated TPC and TFC values of
43.64 mg GAE/g and 17.02 mg QE/g respectively (Siti-Azima et al., 2013).

0.9

0.8

0.7

0.6
TPC antioxidant activity

0.5

0.4

0.3

0.2

0.1

0
ne

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lac
Co

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bu

et

Et
Aq

n-

lo

M
hy

n-
Ch
Et

Samples

Fig. 4.1: Graphical representation for TPC values

Graphical illustration for TPC values showed most amazing qualities of phenolic
content for methanol, ethanol and aqueous extracts. Although other solvents contained
certain amounts of phenolic content but values were less than that of methanol, ethanol and
aqueous fractions. The control showed highest activity as compared to all other fractions.

60
 12

10

TFC antioxidant activity 8

0
ne

rm
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s
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l
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ro

no

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lac
Co

ro

bu

et

Et
Aq

n-

lo

M
hy

n-
Ch
Et

Samples

Fig. 4.2: Graphical representation for TFC values

Graphical demonstration for TFC values indicated highest flavonoid content for
methanol and ethanol extracts. Other solvents contained less flavonoids.

The mean absorbance values for DPPH scavenging activity are listed in table 4.4.

Table 4.4: Mean ± SD values for DPPH assay

Extract/Fractions Mean ± SD

Methanol 0.583 ± 0.026 BC

Ethanol 0.638 ± 0.033 B

n-butanol 0.497 ± 0.021 CD

n-hexane 0.440 ± 0.022 D

61
 Chloroform 0.590 ± 0.012 BC

Ethyl acetate 0.418 ± 0.021 D

Aqueous 0.458 ± 0.022 D

Control 0.780 ± 0.023 A

Means with similar alphabets are non-significant statistically (P>0.01).

Table 4.5: ANOVA for DPPH activity

Mean
Source of variation Degrees of freedom Sum of squares F-value
squares

Treatment 7 0.314926 0.044989 33.08**

Error 16 0.021763 0.001360

Total 23 0.336689

** = Highly significant (P<0.01)

Percent DPPH activity was in the range of 21-48% (table 4.6). Ethyl acetate fraction
was most potent scavenger of DPPH. The % activity for other fractions in descending order
was: n-hexane > aqueous > n-butanol > methanol > chloroform > ethanol. A previous
experiment obtained IC50 of 4.25 µg/mL (90%) for hydromethanol extract of S. cumini (Hasan
et al., 2009). Similarly, in another study conducted by Banerjee et al. (2005), aqueous extract
showed IC50 value of 168 µg/mL. Many previous studies proved that polyphenols, the major
antioxidant components of S. cumini extracts can actively donate an electron or hydrogen
atom to free radicals and prevent the oxidative stress (Perera et al., 2014).

62
Table 4.6: Antioxidant contents and activity

TPC TFC
Extract/Fractions % DPPH scavenging
g GAE/100 g g CE/100 g
Methanol 13 25 27
Ethanol 12 23 21

n-butanol 8 14 37
n-hexane 7 21.9 45
Chloroform 9 17 25
Ethyl acetate 6 20 48
Aqueous 11 21.8 43
BHT (control) - - 90
Data represented as mean percentage. TPC is total phenolic content expressed as g gallic acid
equivalent (GAE)/100 g sample dry weight; TFC is total flavonoid content expressed as g
catechin equivalent (CE)/100 g sample dry weight; DPPH: 2,2-diphenyl-l-picrylhydrazyl,
BHT: Butylated hydroxytoluene

12

10

8
DPPH antioxidant activity

0
rm
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s
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Et
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Aq

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n-
Ch
Et

Samples

Fig. 4.3: Graphical representation of mean values for DPPH activity

63
 Graphical illustration for values of DPPH showed the most remarkable qualities for
concentrates of chloroform, ethanol and methanol comparing with values showed for the
other fractions.

4.2 Biofilm inhibitory assay

Growth inhibitory effects on two bacterial strains were determined and results are
shown in table 4.7. The micrographs of maximum and minimum inhibitory effects are
displayed in fig. 4.4 (a-d). Chloroform fraction exhibited highest inhibition against both
strains and the lowest inhibitions were shown by aqueous and methanolic fractions against P.
multocida and S. aureus respectively. Result in decreasing trend for P. multocida was: n-
hexane > n-butanol > methanol > ethyl acetate > ethanol and for S. aureus was: ethanol >
aqueous > n-hexane > n-butanol > ethyl acetate. Methanol extract inhibited K. pneumoniae
(Gram-negative) by 79.94%, previously (Gopu et al., 2015). According to another research,
ethanolic extract of S. cumini fruit exhibited noble antimicrobial activity against S. aureus, K.
pneumoniae and E. coli with minimum inhibitory concentration and inhibition zone in the
range 0.5-2.5 mg/mL and 14-23 mm, respectively (Singh et al., 2016). The bacterial growth
inhibitory effect of test samples might be due to the significant role of delocalized electrons
and availability of hydroxyl groups in phenolic contents present in extracts (Bag et al., 2012).

Table 4.7: Biofilm inhibition (quantitative)

Extract/ (Pasteurella multocida) (Staphylococcus aureus)

Fractions
% Inhibition % Inhibition

Methanol 69.36 8.76

Ethanol 55.78 42.24
n-butanol 76.42 21.91
n-hexane 77.15 30.78
Chloroform 82.31 47.64
Ethyl acetate 56.10 16.06
Aqueous 47.57 33.48
Ampicillin 83.78 82.24

Data represented as percentage. Ampicillin: positive control

64
 a b

c d
Fig. 4.4: a) (Qualitative assay) inhibition of P. multocida by chloroform fraction (max.),
b) Inhibition of P. multocida by aqueous fraction (min.), c) Inhibition of S. aureus by
chloroform fraction (max.), d) Inhibition of S. aureus by methanol fraction (min.)
4.3 Antidiabetic evaluation
Antidiabetic potential was evaluated by testing the extracts for their potential to
constrain glycation process or enzyme activity. Glycation happens when sugars like glucose
and fructose bind the proteins and lipids and create ketoamines (Kazeem et al., 2012). Alpha-
amylase catalyzes the breakdown of sugars in human body and its restraint is likewise
entirely supportive to treat diabetes.
4.3.1 Antiglycation activity
The mean values for absorbance of antiglycation activity are displayed in table 4.8.

Table 4.8: Mean ± SD values for antiglycation activity

Extract/Fractions Mean ± SD

Methanol 0.589 ± 0.025 CD

Ethanol 0.590 ± 0.025 CD

n-butanol 0.602 ± 0.015 CD

n-hexane 0.742 ± 0.021 B

65
 Chloroform 0.428 ± 0.010 EF

Ethyl acetate 0.506 ± 0.017 DE

Aqueous 0.676 ± 0.026 BC

Metformin 0.360 ± 0.0112 F

Control 0.879 ± 0.023 A

Means with similar alphabets are non-significant statistically (P>0.05).

Table 4.9: Mean ± SD values of three days intervals antiglycation activity
Sr. No. Days Mean ± SE

1 3rd 0.579 ± 0.044 A

2 7th 0.572 ± 0.055 AB

3 11th 0.538 ± 0.055 B
Means with same alphabets are non-significant statistically (P>0.05).
Table 4.10: ANOVA for antiglycation activity
Degrees of
Source of variation Sum of squares Mean squares F-value
freedom

Days 2 0.010628 0.005314 3.69*

Treatment 10 0.789345 0.078935 54.82**

Error 20 0.028796 0.001440

Total 32 0.828770

* = Significant (P<0.05); ** = Highly significant (P<0.01)

Fig. 4.5: Graphical comparison of antiglycation activity on different days

66
 The activity 12
was checked after
third, seventh and 10
eleventh days with
most amazing 8

Antiglycation activity
potential for third
day. 6
Table 4.11:
Percentage of 4
antiglycation
activity 2
Time
Extract/Fractions rd
0 3 Day 7th Day 11th Day
3rd 7th 11th
Methanol day day day 37 38
37
Days
Ethanol 35 32
27
n-butanol 33 30
24
n-hexane 23 27
17
Chloroform 56 55
51
Ethyl acetate 47 47
38
Aqueous 17 15
14
Metformin 57 62 63
Data represented as percentage. Metformin: positive control
According to table 4.11, at the end of 11 th day, chloroform fraction exhibited highest
(55%) antiglycation effect. The results for other fractions in decreasing order were: ethyl
acetate > methanol > ethanol > n-butanol > n-hexane > aqueous. A study found inhibition of
7% by aqueous S. cumini seed extract (Tupe et al., 2015). It was earlier stated that organic
fractions of seed repressed glycation by 78.14% (Deve et al., 2014). The variation of results

67
of this study and previous studies might be due to different solvents and different extraction
methods used.
4.3.2 Enzyme inhibitory potential
The data for acetylcholinesterase and alpha-glucosidase inhibition percentage is
summarized in table 4.12.
4.3.2.1 Acetylcholinesterase inhibition assay
In AChE inhibition assay, ethanolic fraction showed the highest inhibition by 13.33%
and the lowest activity was of chloroform fraction. The trend in ascending order for other
fractions was: chloroform < n-butanol < aqueous < ethyl acetate < n-hexane < methanol.
Previously, ethyl acetate, n-hexane and methanol extracts of S. cumini inhibited AChE with
IC50 values of more than 50 µg/mL (Darusman et al., 2013). Analogous inferences
documented in 2011 that leaf extract reduced the activity of AChE in RBCs of type 2
diabetes, effectively (Bona et al., 2011). Methanolic extract could act as antamnesic agent for
the treatment of spatial memory impairments in rats. The extracts of plant act as antamnesic
agents because of containing flavonoids and tannins that have inhibitory effect on AChE
enzyme (Alikatte et al., 2012).
4.3.2.2 Alpha-glucosidase inhibition assay
The highest and significant inhibitory effect on alpha-glucosidase was exerted by n-
butanol fraction showing 80.1% restraint and in contrast fraction of n-hexane did not inhibit
this enzyme. The ascending trend of inhibition for all fractions was as: n-hexane < aqueous <
methanol < ethanol < chloroform < ethyl acetate < n-butanol.
Inhibitory ability of ethanol, ethyl acetate, acetone and 1-butanol fractions of S.
cumini seed kernel against alpha-glucosidase from different sources was calculated in the
range of 24.6 ± 0.7 to 2.8 ± 0.1 µg/mL as reported by Shinde et al. (2008).

Table 4.12: AChE and alpha-glucosidase inhibition assays

% Inhibition
Extract/Fractions
AChE activity α-glucosidase activity

68
 Methanol 11.48 59.7

Ethanol 13.33 61.9

n-butanol 2.52 80.1

n-hexane 7.94 0

Chloroform 1.05 63.1

Ethyl acetate 5.87 79.7

Aqueous 5.61 16.5

Acarbose - 78.9

Physostigmine 59.51 -

Data represented as percentage. Positive controls: acarbose (α-glucosidase inhibitory assay)

and physostigmine (AChE inhibitory assay)
4.3.2.3 Alpha-amylase inhibition assay
The absorbance values for alpha-amylase inhibitory activity are shown in table 4.13.

69
Table 4.13: Absorbance values for alpha-amylase activity

Time (minute)
Extract/
Fraction
30 60 90 120 Mean

0.581 ± 0.015a-d 0.438 ± 0.017g-j 0.327 ± 0.017k-h 0.315 ± 0.015k 0.415 ± 0.031E
Methanol

0.610 ± 0.021abc 0.501 ± 0.08c-h 0.493 ± 0.021d-j 0.368 ± 0.019jk 0.493 ± 0.025BC
Ethanol

0.559 ± 0.026a-f 0.487 ± 0.028d-i 0.468 ± 0.020e-j 0.387 ± 0.022ijk 0.475 ± 0.024BCD
n-butanol

0.619 ± 0.020ab 0.544 ± 0.019a-g 0.461 ± 0.020e-j 0.410 ± 0.014h-k 0.509 ± 0.027B
n-hexane

0.559 ± 0.021a-e 0.547 ± 0.020a-g 0.456 ± 0.021e-j 0.401 ± 0.017h-k 0.491 ± 0.021BC
Chloroform

0.511 ± 0.015b-h 0.504 ± 0.024c-h 0.485 ± 0.022d-i 0.455 ± 0.024e-j 0.489 ± 0.012BCD
Ethyl acetate

0.630 ± 0.021a 0.608 ± 0.010abc 0.542 ± 0.020a-g 0.538 ± 0.030a-g 0.580 ± 0.017A
Aqueous

70
 0.534 ± 0.015a-g 0.469 ± 0.020e-j 0.462 ± 0.020e-j 0.314 ± 0.012k 0.445 ± 0.026DE
Control

0.566 ± 0.014A 0.505 ± 0.015B 0.465 ± 0.015C 0.407 ± 0.017D

Mean -----

71
 Enzyme restraint activity assay was time dependent and in triplicate at distinctive
time intervals of 30, 60, 90 and 120 minutes. All fractions showed different activities at said
time intervals.

Table 4.14: ANOVA for alpha-amylase inhibitory analysis

Degrees of Mean
Source of variation Sum of squares F-value
freedom squares

Time 0.12195 106.49**

9
Treatment 3 0.365877 21.81**
0.02497
Time x Treatment 8 0.199809 5.11**
6
Error 24 0.140391
0.00585
Total 72 0.082458
0
107 0.788535
0.00114
5

** = Highly significant (P<0.01)

72
 0.7

0.6

0.5
Enzyme inhibitory analysis
0.4

0.3
30 Minute
0.2 60 Minute
90 Minute
0.1 120 Minute

Aq r
te

Ch ane

l
ol

l
s
rm

no
no

ro
to

ou
he utan
ta

nt
fo

bi

ha
ha
ue
x
ce

Co
he

hi
ro

Et
et
b
lA

In
lo
n-

M
n-
hy

tic
Et

nt
Sy

Treatment

Fig. 4.6: Graphical representation of time dependent alpha-amylase inhibitory activity

n-hexane and aqueous fractions showed wonderful alpha-amylase inhibitory effect.

Both fractions inhibited alpha-amylase near to synthetic inhibitor but n-butanol and ethyl
acetate fractions showed the least alpha-amylase hindrance.

Table 4.15: Percentage of alpha-amylase inhibitory activity

Time
Extract/Fractions
30min 60min 90min 120min

Methanol 8 7 13 23

Ethanol 17 14 8 13

n-butanol 18 25 17 44

73
 n-hexane 4 18 10 32

Chloroform 10 19 12 27

Ethyl acetate 8 28 39 41

Aqueous 3 12 17 25

Glucobay 26 31 14 17

Data represented as percentage. Glucobay: positive control

At 120 minutes of incubation, most effective inhibitor of alpha-amylase was n-
butanol fraction with 44% inhibition. The percent inhibition of other fractions in ascending
order was: ethanol < aqueous < methanol < chloroform < n-hexane < ethyl acetate.
Previously, Bansode et al. (2016) reported the value of 73.33% inhibition by S. cumini
ethanol extract. Methanol and aqueous seed extracts significantly inhibited alpha-amylase
activity from 50 to 94.70% (Singh and Marar, 2011). The antidiabetic capacity of S. cumini
extracts is attributable to phenolics, flavonoids, myricetin, luteolin, quercetin and tannic acid
that can inhibit production of AGEs and activity of alpha-amylase (Singh and Marar, 2011;
Perera et al., 2013).
4.4 Mutagenicity/Carcinogenicity assay

4.4.1 Ames test

The results of mutagenicity assessment are depicted in figure 4.7 and summarized in
table 4.16. All the tested fractions had reverent effect on TA100 strain and among those
tested in TA98 strain, ethyl acetate fraction was found to be non-mutagenic while the rest of
fractions had mutagenic effect with ethanol fraction as strongest and aqueous as weakest one.
The decreasing order of mutagenicity for other test samples was: n-hexane > n-butanol >
methanol > chloroform.

74
 Similarly, Vicentini et al. (2001) testing mutagenicity of herbal tea prepared from
leaves of S. cumini showed that it did not affect the cell cycle of Allium cepa used as vegetal
test system. The tea also caused no alteration in the cycle of cell division and the number of
chromosomal damage in the bone marrow cells of rats.

An earlier experiment indicated that S. cumini fruit extracts had antimutagenic ability
and decreased the formation of micronucleated polychromatic erythrocytes, diminishing
mutagenic effect of mitomycin C (Lim-Sylianco et al., 1986).

a) b)

c) d)

e) f)

75
 g) h)

i) j)

k) l)

Fig. 4.7: a) Background plate for TA98, b) Background plate for TA100, c) Standard
mutagen (K2Cr2O7) plate TA98, d) Standard mutagen (NaN3) plate TA100, e-h) Test
plates for TA98 and i-l) Test plates for TA100, both with methanol, ethanol, n-butanol,
n-hexane, chloroform, ethyl acetate and aqueous fractions (1-7), respectively

Table 4.16: Ames assay

No. of +ive No. of +ive

wells/total no. Result wells/total no. Result
Extract/Fractions
of wells interpretation of wells interpretation
(TA98) (TA100)
Methanol 82/96 Mutagenic 96/96 Mutagenic

76
Ethanol 90/96 Mutagenic 96/96 Mutagenic
n-butanol 86/96 Mutagenic 96/96 Mutagenic
n-hexane 88/96 Mutagenic 96/96 Mutagenic
Chloroform 74/96 Mutagenic 96/96 Mutagenic
Ethyl acetate 22/96 Non-mutagenic 96/96 Mutagenic
Aqueous 70/96 Mutagenic 96/96 Mutagenic
Standard 84/96 Mutagenic 85/96 Mutagenic
Background 6/96 Non-mutagenic 15/96 Non-mutagenic

4.4.2 Brine shrimp assay

The results of brine shrimp assay have been summarized in table 4.17.

Table 4.17: Percent mortality of brine shrimp larvae after treating with various
fractions
Number of
Number of brine
surviving brine
Extract/Fractions shrimp larvae % Mortality
shrimp larvae
Used
(after 24 hours)
Methanol 10 7 30
Ethanol 10 10 0
n-butanol 10 2 80
n-hexane 10 5 50
Chloroform 10 4 60
Ethyl acetate 10 6 40
Aqueous 10 1 90
Control (Distilled 10 10 0
water)

The decreasing order of % mortality for all fractions was: aqueous > n-butanol >
chloroform > n-hexane > ethyl acetate > methanol > ethanol. The mortality percentage
shown by the fractions of fruit of S. cumini in this study ranges between 0-90 with highly
active fraction of aqueous that caused great toxicity to brine shrimp larvae and the safest
fraction was the ethanol against larvae with no toxic effect while Paul et al. (2011) indicated
that ethanolic extract of S. cumini leaf with % mortality of 30-61.90% is safe to be used in
vivo for treatment purposes. Freeze-dried methanolic extracts prepared from leaves and
branches of S. cumini had no toxic impact on brine shrimp larvae (0% lethality) when tested

77
by Manawaty et al. (2013) and this proved that these parts of plant are not toxic. According
to these results, it can be concluded that brine shrimp cytotoxicity test is a suitable simple
tool that can be applied to discover the toxicity of great number of drugs and plant extracts.

4.5 Cytotoxic potential

Cytotoxic assay was carried out by examination of DNA protecting and hemolytic
activities of the fractions.

4.5.1 Hemolytic activity

The highest hemolytic potential (30%) was found with ethyl acetate fraction and the
lowest (8%) was of aqueous fraction (table 4.19). Metabolites of S. cumini caused no damage
to erythrocytes membranes, thus having no cytotoxic effects on RBCs. The ascending order
of % hemolysis was: aqueous < methanol < ethanol < n-butanol < chloroform < n-hexane <
ethyl acetate. Methanol extract of S. cumini seeds showed strong hemolysis and membrane
stabilizing property as reported by Saha et al. (2013). On the other hand, ethyl acetate and
methanolic extracts were found to be nonhemolytic (Bag et al., 2012; Tripathy et al., 2016).
The antigenotoxic and antihemolyzing properties of S. cumini are correlated with the
antioxidant potential possessed by the plant phytoconstituents (Arun et al., 2011; Saha et al.,
2013). Being nonhemolytic, S. cumini is believed to contain no cardiac saponins,
phlobatannins, alkaloids and glycosides that can destroy RBCs (Tripathy et al., 2016).

4.5.2 DNA-damage and protective assay

According to the results displayed in fig. 4.8 (b) and table 4.18, all extracts except
ethanol protected DNA against H2O2-induced damage. The outcomes of this study indicated
that secondary metabolites can possibly counteract H 2O2 damaging effect on DNA by
scavenging free radicals. Ethanol and aqueous extracts of S. cumini seed effectively protected
pBR322 DNA against hydroxyl radical induced breaks in DNA strands (Arun et al., 2011)
and leaf extract protected DNA damage in cultured ɣ-irradiated splenocytes of rats in
previous experiment conducted by other researchers (Jagetia et al., 2012).

78
 Fig. 4.8: Agarose gel electrophoresis of DNA-damage protection assay

a) Lane1: ladder, Lane 2 and 3: positive and negative control (FeSO 4), Lane 4 and 5:
negative controls (H2O2), b) Lane 1-7: plasmid DNA + aqueous, methanol, n-butanol,
chloroform, n-hexane, ethyl acetate and ethanol fractions, respectively + H2O2

Table 4.18: DNA-damage protection activity

Lane
Solvents DNA-damage protection
No.

1 Aqueous + Plasmid DNA no DNA damaged

2 Methanol + Plasmid DNA no DNA damaged

3 n-butanol + Plasmid DNA no DNA damaged

Chloroform + Plasmid
4 no DNA damaged
DNA

n-hexane + Plasmid DNA

5 no DNA damaged
Ethyl acetate + Plasmid
6 DNA no DNA damaged

Ethanol + Plasmid DNA

7 DNA damaged

79
4.6 Thrombolytic activity

Clot formation in the vessels is a serious problem. It disturbs flow of blood, blocks
blood vessels and leads to necrosis of body tissues by reducing blood and oxygen supply to
the tissues (Sakib et al., 2015). Thrombolytic potential was in the range 10.2-23% (table
4.19) and chloroform fraction showed the highest percent thrombolysis. Clot lysis percentage
increased in the order: ethanol < n-hexane < methanol < ethyl acetate < aqueous < n-butanol
< chloroform. To date, no relevant analysis on S. cumini is available in literature. Syzygium
fruticosum aqueous, CCl4, petroleum ether and chloroform extracts exhibited thrombolytic
activity in the range 33.46-62.51% (Chadni et al., 2014). Chloroform fraction of stems of
Glycosmis arborea dissolved blood clots by 36.50% (Hossain et al., 2012).

Table 4.19: Hemolytic and thrombolytic activity

Percentage
Extract/Fractions
Hemolysis Thrombolysis

Methanol 11 13.45

Ethanol 11.89 10.2

n-butanol 12.3 21

n-hexane 23 12.75

Chloroform 14 23

Ethyl acetate 30 16.87

Aqueous 8 19.3

Triton X-100 72 -
- 91
Streptokinase

80
Data represented as percentage. Triton X-100 and streptokinase: Positive control

81
In vivo biochemical analysis

The results of evaluated biochemical parameters considered in current research in animal models are summarized in table 4.20.

Table 4.20: The values of different in vivo parameters

Group 5
Group 3 Group 4
Sr. Group 1 Group 2 Diabetic group
In vivo parameters Diabetic group (S. Diabetic group
No. Diabetic Non-diabetic (herbal
cumini extract) (glibenclamide)
formulation)
Antidiabetic activity
1 Serum glucose (mg/dL) 265.6 ± 30.26 99.5 ± 13.12 150.8 ± 30.45* 140.1 ± 39.47** 265.4 ± 24.39 NS
2 Serum insulin (µL/mL) 4 ± 0.3 12 ± 1.2 9.5 ± 1.9* 9.3 ± 1.7** 4.0 ± 0.9 NS
3 HbA1c (%) 10.6 ± 0.7 5.5 ± 0.6 7.9 ± 1.7* 6.9 ± 0.7** 9.5 ± 1.1 NS
Antioxidant activity
1 SOD (Units/mg protein) 3.70 ± 0.19 9.20 ± 0.42 4.07 ± 0.30 NS 6.43 ± 0.42** 3.60 ± 0.35 NS
Glutathione peroxidase
2 (Red) (Units/mg 5.44 ± 0.28 9.43 ± 0.43 6.06 ± 0.30 NS 8.52 ± 0.43** 5.15 ± 0.19 NS
protein)
Catalase (Units/mg
3 42.20 ± 2.75 83.33 ± 2.93 65.22 ± 2.16* 71.30 ± 3.60** 42.39 ± 5.01 NS
protein)
Lipid peroxidation
4 1.77 ± 0.08 0.85 ± 0.03 1.50 ± 0.33NS 0.96 ± 0.07** 1.89 ± 0.10 NS
(mM/100g of tissue)
Antihyperlipidemic activity
1 Triglycerides (mg/dL) 166.81 ± 4.33 89.15 ± 6.37 135.32 ± 10.2* 78.32 ± 5.7** 146.03 ± 10.5
Total cholesterol
2 210.2 ± 4.77 54.67 ± 7.64 180.13 ± 1.49* 123.75 ± 2.80** 186.42 ± 4.17*
(mg/dL)
3 HDL (mg/dL) 43.86 ± 3.65 27.99 ± 7.21 40.12 ± 1.5 NS 51.54 ± 1.67 47.98 ± 3.70
4 LDL (mg/dL) 132.98 ± 0.99 8.85 ± 5.61 105.17 ± 1.71* 56.55 ± 2.97** 109.24 ± 1.82
5 VLDL (mg/dL) 17.65 ± 1.73 31.66 ± 2.16 20.26 ± 1.41 17.4 ± 0.4 22.75 ± 1.77

82
Hepatoprotective activity
Aspartate transaminase
1 92.60 ± 3.93 24.80 ± 1.24 65.39 ± 3.55 31.29 ± 2.10** 77.01 ± 1.05
(IU/dL)
Alanine transaminase
2 91.60 ± 3.19 31.20 ± 2.82 75.00 ± 3.76* 43.15 ± 2.50** 81.06 ± 2.5
(IU/dL)
Alkaline phosphatase
3 770 ± 55.60 326.3 ± 43.40 689.3 ± 46.41* 321 ± 45.33** 789 ± 45.05
(IU/dL)
ɣ-glutamyl
4 16.5 ± 1.95 2.00 ± 0.45 13 ± 0.74 5.67 ± 0.56** 17.07 ± 1.45
transpeptidase (IU/dL)
5 Total protein (IU) 59.33 ± 2.09 63.5 ± 1.41 65.31 ± 1.22 62.17 ± 2.19 61.11 ± 1.03
Renoprotective activity
1 Urea (mg/dL) 70 ± 7.3 18.5 ± 1.5 52.8 ± 4.5 30.25 ± 1.27** 72 ± 5.2
2 Creatinine (mg/dL) 0.93 ± 0.14 0.55 ± 0.018 0.80 ± 0.25 0.80 ± 0.24** 0.99 ± 0.21
3 Urinary albumin (g/dL) 2.3 ± 0.1 5.0 ± 0.02 5.6 ± 0.04 4.2 ± 0.1 1.9 ± 0.01
4 Total protein (g/dL) 4.4 ± 0.03 8.0 ± 0.1 8.4 ± 0.01 6.5 ± 0.2 4.5 ± 0.02
Data expressed as mean or percentage ± SEM of triplicate measurements for groups of seven animals each

* Significant (P<0.05) as compared to diabetic control

** Highly significant P<0.05 as compared to diabetic control

NS: non-significant

TG: triglyceride; TC: total cholesterol

83
Antidiabetic activity

The data obtained for serum glucose, insulin and glycated hemoglobin levels in
respective groups of test animal models have been presented in table 4.20.

Level of serum glucose was considerably high (265.6 ± 30.26 mg/dL) in control
alloxan-induced diabetic group in comparison with normal group of rats. Administration of
extracts of S. cumini significantly at P<0.05, reduced serum glucose level to 150.8 ± 30.45
mg/dL as compared to control diabetic group. A previous study showed that oral utilization
of methanolic and ethyl acetate fractions of seed of S. cumini for 15 days, made significant
(P<0.05) reductions to 182.85 ± 4.58, 154.85 ± 10.24, 192.03 ± 5.80 and 178.14 ± 9.30
mg/dL in blood glucose levels of STZ-induced diabetic rat models at two different
concentrations (200 and 400 mg/kg body weight) respectively (Kumar et al., 2008).
Similarly, Ali et al. (2016) reported that methanol and aqueous extracts of S. cumini bark had
significant antidiabetic effect on alloxan-induced diabetic rats by reducing the blood glucose
level in the range 249.04 ± 3.89-180.21 ± 8.68 mg/dL from first to 15 th day of dose
dependent administration of the extracts. Whole seed and seed kernel also reduced blood
glucose of diabetic rats from 100.2 ± 3.9 to 89.2 ± 3.9 mg/dL at significance level of p<0.001
in study of Ravi et al. (2004). The results of previous study support those found in this work,
which means the extracts of different parts of jamun have effective antidiabetic effect.

In present research, the level of serum insulin was very low (4 ± 0.3 µL/mL) in
diabetic group in comparison normal group but treatment with extracts of S. cumini could
revert the level back to nearly normal (9.5 ± 1.9 µL/mL) at significant level of P<0.05. A 200
mg/kg dose of aqueous extract of S. cumini pulp increased serum insulin level of STZ-
induced diabetic rat models to a near normal level (25 µL/mL) (Rekha et al., 2010). Serum
insulin level was reverted to near normal level of 1.35 ± 0.08 IU/mL on treating with S.
cumini seed extract (Sharafeldin and Rizvi, 2015).

Glycated hemoglobin level in diabetic group (control) used in this study was 10.6 ±
0.7% and it got decreased to 7.9 ± 1.7%; p<0.05 that shows the significant antiglycation
effect of fractions of jamun fruit in vivo. Sharafeldin and Rizvi, (2015) found significant

84
(p<0.001) reduction to 9.56 ± 0.19% in glycosylated hemoglobin level of diabetic rats in
their study.

Antioxidant activity

According to data provided in table 4.20, there was a high reduction in levels of
antioxidant enzymes, SOD and GPx caused by alloxan induction of diabetes in the rats in
comparison with normal group and extract of jamun did not affect these two parameters well
as it changed the levels of these enzymes very slightly from 3.70 ± 0.19 and 5.44 ± 0.28 to
4.07 ± 0.30 and 6.06 ± 0.30 Units/mg protein respectively when administered to rats while
level of CAT improved significantly (p<0.05; 65.22 ± 2.16 Units/mg protein). Lipid
peroxidation also was not affected too much (1.50 ± 0.33 mM/100g of tissue) by plant extract
in this research.

Antioxidant treating of STZ-induced diabetic rats with S. cumini seed extract by

Sharafeldin and Rizvi, (2015) reversed the levels of SOD, CAT and GPx and TBARS, upto
105.33 U/mg protein, 26.38 µM of H2O2/min/mg proteins, 27.42 µg of GSH utilized/min/mg
proteins and 5.75 µM of malondialdehyde/min/mg protein which was a significant (p<0.001)
effect while earlier, SOD, CAT and GPx levels had been increased to 4.87 ± 0.25 U/mg
protein, 36.38 ± 7.3 nmoles H2O2 decomposed/min/mg protein, 6.25 ± 0.25 μ moles GSH
oxidized/min/mg protein and level of TBARS and hydroperoxides decreased to the range 1.4
and 98 mM/100 g wet tissue by S. cumini pulp aqueous extract in the study conducted by
Rekha et al. (2008) dose dependently. The results shown in these studies are similar to those
found in present study to some extent. Rekha et al. also reported different significant effects
of the same extract at 200 mg/kg body weight on SOD, CAT, TBARS and hydroperoxides
levels in 2010, as 5.62 ± 0.28 U/mg protein, 37.94 ± 4.8 n moles of H 2O2
decomposed/min/mg protein and 1.0 and 75 mM/100 g wet tissue.

Antihyperlipidemic activity

Induction of diabetes in rats notably increased the levels of triglycerides, total

cholesterol, HDL, LDL and VLDL as compared to control normal group in this study.
Administration of S. cumini extract effectively reversed this effect of alloxan and showed the

85
results as 135.32 ± 10.2, 180.13 ± 1.49, 40.12 ± 1.5, 105.17 ± 1.71 and 20.26 ± 1.41 mg/dL for
levels and concentrations of lipid profile parameters in diabetic rats.

Lipid profile of high cholesterol fed diet rats was changed significantly (p<0.05) on
treating rats with seed ethanolic extract of S. cumini to be consisted of cholesterol (282.02 ±
6.38), triglyceride (125.89 ± 05.23), HDL-C (1.54 ± 0.89), LDL (321.17 ± 26.10) and VLDL
(25.17 ± 0.50 mg/dL) in experiment designed by Modi et al. (2009).

Sanches et al. (2016) recorded values of 120 and 70 mg/dL for triglycerides and total
cholesterol level in the serum of obese rats which were significant (p<0.05) comparing with
control obese rats.

Diet containing ethanolic extracts of seed and fruit of jamun significantly reduced the
serum cholesterol level from 80.45 ± 3.22 to 75.55 ± 3.02 mg/dL and 80.9 ± 3.21 to 77.35 ±
3.09 mg/dL, serum LDL level from 30.53 ± 1.19 to 28.29 ± 1.10 mg/dL and from 31.04 ±
1.17 to 29.10 ± 1.13 mg/dL and triglycerides from 65.95 ± 2.63 to 62.96 ± 2.51 mg/dL and
from 65.57 ± 2.67 to 63.56 ± 2.59 mg/dL and increased HDL from 37.05 ± 1.48 to 37.80 ±
1.53 mg/dL and 36.92 ± 1.47 to 37.54 ± 1.51 mg/dL, in the respective groups (Raza et al.,
2015).

In an experiment designed by Ravi et al. (2005), the serum levels of lipid profile
parameters of diabetic rats treated with S. cumini seed kernel aqueous extract were significantly
(p<0.05) changed to 85.3 ± 4.5 for total cholesterol, 89.2 ± 7.2 for triglycerides, 20 for HDL, 50
for LDL and 18 mg/dL for VLDL. This effect proves that S. cumini can be used as an
antihyperlipidemic agent.

Hepatoprotective activity

The markers of hepatic function such as AST, ALT, ALP, GGT and total protein were
increased to notable levels following the induction of diabetes in rats in present research.
Treatment with different extract of S. cumini caused significant reduction in activities of named
enzymes and level of total protein proving to have no hepatotoxic effect but to be a
hepatoprotective agent. The values for these markers are as 65.39 ± 3.55, 75.00 ± 3.76, 689.3 ±
46.41, 13 ± 0.74 IU/dL and 65.31 ± 1.22 IU, respectively. The levels of liver enzymes, AST,
ALT and ALP were reduced to 277.55 ± 0.106-5.08%, 74.542 ± 0.141-5.03% and 835.466 ±

86
0.079-0.85% IU/L on oral treatment with S. cumini seed extract in streptozotocin-nicotinamide-
induced rats by Sarma, (2014). These results are comparable with those of present study. Das
and Sarma, (2009) induced hepatotoxicity in rats by paracetamol and treated them with extract
of S. cumini to examine its hepatoprotective effect as the result of which they found that the
increased activities of liver enzymes and total protein level were significantly (p<0.01) back to
near normal values as 120 ± 1.16 U/L, 42 ± 1.00, 20 ± 1.65 KAU and 4 ± 0.10 g% respectively
in comparison with paracetamol-treated group.

To understand the effect of jamun juice on levels of AST, ALT and ALP, rats were
administered with S. cumini fruit juice. Levels of these liver markers were decreased slightly, in
the ranges 44.1 ± 0.33-43.5 ± 0.37, 29.3 ± 1.47-27.8 ± 0.45 and 60.6 ± 0.57-63.7 ± 0.48 (El-
Anany and Ali, 2013).

Behera et al. (2014) examined seed aqueous extract of S. cumini for its hepatoprotective
ability and it was found very effective in this respect. The extract at two different doses
prevented the increase in levels of hepatic biomarkers due to streptozotocin by keeping levels of
enzymes AST, ALT, ALP and GGT near the normal levels of 54.50 ± 13.13, 46.00 ± 5.48,
92.17 ± 1.72 and 19.67 ± 3.50 (250 mg/kg dose) and 40.00 ± 2.19, 37.33 ± 3.88, 83.00 ± 5.51
and 15.00 ± 2.19 IU/L (500 mg/kg dose) respectively.

The levels of liver function markers were lower in seed extract treated groups at
significant level of p<0.05 than those in CCl4-toxic group. Level of AST was reduced to 151.67
± 8.16 and 132.50 ± 8.22 U/L, that of ALT to 91.34 ± 9.46 and 80.34 ± 2.43 U/L and that for
ALP to 26.34 ± 3.73 and 22.34 ± 2.07 U/L under the influence of two different concentrations
of extract as a hepatoprotective substance (Islam et al., 2015).

Renoprotective activity

The level of serum creatinine, urea, urinary albumin and total protein of diabetic control
group were different from those in normal control group which changed to nearly their normal
levels on treatment with S. cumini extract.

Sarma, (2014) examined the influence of extract of S. cumini seed on serum urea and
creatinine levels in STZ-nicotinamide-induced diabetic albino rats and it was found that the
extract reversed the effect of STZ-nicotinamide with urea and creatinine values of 18.040 ±

87
0.034-16.29% and 1.93 ± 0.014-45.48% mg/dL respectively while same parameters in cisplatin
induced kidney failure in rats were evaluated by Adikay et al. (2010) to be 62.20 ± 0.21 and 1.9
± 0.22 mg/dL under influence of ethanol extract of S. cumini fruit. El-Anany and Ali in 2013
showed that fruit juice of S. cumini at different concentrations is beneficial for the health of rats
by changing the value of serum urea in the range 30.1 ± 0.01-29.7 ± 0.51.

88
CHAPTER 5 SUMMARY

Present research work was designed to investigate bioactive and therapeutic potential
of Syzygium cumini fruit in different fractions. Syzygium cumini (Linn.), commonly known as
black plum, member of the family Myrtaceae is a large perennial tree native to Asian
countries like India, Burma, Bangladesh, Nepal, Sri Lanka, Indonesia, Pakistan and
Malaysia.

It is reported that soft tissue of jamun fruit is nutritive and holds several minerals such
as phosphorous, calcium, sodium, potassium, zinc and iron, carbohydrates like sucrose,
glucose, galactose and raffinose and free amino acids like cysteine and alanine. Leaves
contain acylated flavonol glycosides, esterase and galloyl carboxylase and can be used for
contraction of vagina after delivery and reduction of mucus and odor. The powder prepared
from peel of juman can also be used to produce colorant for food and pharmaceuticals.

For analysis of S. cumini fruit extract and fractions in this research, samples were
prepared in seven solvents viz., methanol, ethanol, n-butanol, n-hexane, chloroform, ethyl
acetate and aqueous. Antioxidant activity was analyzed by determination of total phenolic
content (TPC) using Folin-Ciocalteu reagent, total flavonoid content (TFC) by aluminum
chloride colorimetric method and antioxidant capacity by DPPH (2,2-diphenyl-l-
picrylhydrazyl) scavenging assay. Antibiofilm activity was evaluated by microtiter dish assay
and antidiabetic potential was determined by glycation, acetylcholinesterase, alpha-
glucosidase and alpha-amylase inhibitory assays. Cytotoxic activity was confirmed by
hemolytic and a DNA-damage protection assay as well as thrombolytic activity was assessed.
Methanol extract exhibited the highest TPC (13 ± 0.030 g gallic acid equivalent /100 g dry
weight) and TFC (25 ± 0.030 g catechin equivalent /100 g dry weight) whereas highest
DPPH scavenging activity (48%) was of ethyl acetate fraction. Maximum growth restraint
was of chloroform fraction against Pasteurella multocida (82.31%) and Staphylococcus
aureus (47.64%). Chloroform fraction showed maximum glycation inhibition (55%). n-
butanol and ethanolic fractions were most potent inhibitors of acetylcholinesterase, alpha-
glucosidase and alpha-amylase with 13.33, 44 and 80.1% inhibitions respectively. The

89
mortality percentage of brine shrimp shown by the fractions of fruit of S. cumini in this study
ranged between 0-90 with highly active aqueous fraction at caused great toxicity to brine
shrimp larvae and the safest fraction was ethanol against larvae with no toxic effect. Percent
hemolysis was in the range of 8-30% and ethyl acetate fraction showed the highest value. All
extracts prevented DNA-damage effectively. Chloroform fraction showed the highest (23%)
thrombolytic efficacy. In vivo biochemical analysis of antidiabetic, antioxidant,
antihyperlipidemic, hepatoprotective and renoprotective was done by using commercially
available kits. The plant extract had significant effect on antidiabetic parameters such as
serum glucose and insulin and glycated hemoglobin levels. Antioxidant activity of the extract
was non-significant with respect to SOD, GPx and lipid peroxidation level but for catalase
activity it was significant. The extract significantly affected lipid profile of the rats by
reverting back the levels of total cholesterol, triglycerides, LDL and VLDL to the normal
values but its influence on HDL level was not significant. There was a significant change in
the values for ALT and ALP activities in the rats, on treatment with S. cumini extract and it
did not affect the activities of AST, GGT and total protein level markedly. The
administration of extract in rats to change the concentrations of renal biomarkers such as
urea, creatinine, urinary albumin and total protein was not too successful in the experiment of
this research. The results of this study revealed that different fruit extracts of S. cumini had
significant pharmacological properties especially with respect to diabetes mellitus and there
is a need for further investigation of therapeutic potentials possessed by this plant.

90
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