Chapter 1:

Introduction

1
Introduction

The short-lived, unpaired electrons can be found in the outer orbits of reactive substances known
as free radical and are also capable of independent existence (1).They can function as an oxidant
or a reductant by either transferring an electron to another molecule or taking one away from it.
These radicals are therefore quite reactive. Certain oxygen species, referred to as reactive oxygen
species (ROS), are not reactive in their native state but are able to produce free radicals (2). The
free radicals and the non-reactive radicals are two major groups of components under which ROS
are categorised. The superoxide radical (O2 •), hydroxyl radical (OH), singlet oxygen ( 1O2), non-
radical H2O2, and peroxynitrite (ONOO-)—are the most significant free radicals. The non-
reactive radicals, on the other hand, are a class of substances that do not constitute radicals yet are
incredibly reactive or readily transform into reactive species. Hypochlorous acid (HClO), H 2O2,
organic peroxides, aldehydes, O3, and O2 are a few examples of these compounds (2).

Biological molecules like fats, nucleic acid, proteins and carbohydrates that are present within the
body can be attacked easily by ROS. Although some of these ROS have beneficial effects on cell
physiology in vivo, but they may also harm DNA and cell membranes by causing oxidation, that
can lead to diseases(3).

Reactive species are detrimental because they are radicals which can produce more radicals or
have a larger potential for oxidation. The fundamental reason why reactive species have harmful
effects is due to this. Since the interplay between reactive oxygen species and the structure of
biomolecules resembles a chain process involving radicals, oxidative damage similarly resembles
in the form of chains. New reactive species are created as a result of this chain reaction, which in
turn damages other biomolecules(4,5).

Free radicals; They are produced by the autooxidation of small molecules, enzymes, and proteins,
mitochondrial electron transport systems, peroxisomes, the plasma membrane, and oxidative
stressors(6). Cells continuously produce free radicals as a result of enzymatic and non-enzymatic
processes. The respiratory chain and the cytochrome P-450 system are the some of the examples
of enzymatic reactions that serve as a source of production of free radicals(7–9). The non-
enzymatic reactions of oxygen can also produce free radicals.

2
Some self-produced free radical sources are- Xanthine oxidase and mitochondria, arachidonate
highways; phagocytosis; inflammation; peroxisomes , exercise, injury from ischemia and
reperfusion(9).

Reactive oxygen species (ROS) and the harm they inflict can be avoided by a variety of defence
mechanisms. The term "antioxidant defence systems" or simply "antioxidants" refers to these
mechanisms(10).

Mammalian cells have radical detoxification defence mechanisms. SOD, CAT, and GPX are
important enzymes that eliminate harmful peroxides. In addition to antioxidant enzymes, non
enzymatic compounds such as thioredoxin, thiols, and disulfide-bonding are also crucial
components of antioxidant defence mechanisms. Some of the molecules, such α-tocopherol, β -
carotene, and ascorbic acid, as well as some micronutrient components like zinc and selenium,
are exogenous in nature and derived from food(11).

Antioxidants acts in four different ways.

It has a scavenging effect on free oxygen radicals by retaining them or changing them into new,
weaker molecules. These effects are produced by tracheobronchial mucus, antioxidants, and tiny
compounds. The inhibitory effect interacts with free oxygen radicals and transfers the element
hydrogen to them, reducing their activity or inactivating them. Flavonoids and vitamins both
have such effect,

Binding free oxygen radicals has the effect of breaking their chains, and stopping their functions
is known as the "chain-breaking effect."Minerals, haemoglobin, and ceruloplasmin exhibit chain-
breaking effects,

Restoring the damage or injury generated due to free radicals is a restorative effect.(10)

There are two types of antioxidants: endogenous and exogenous. Endogenous antioxidant
includes enzyme and non-enzymatic. On the other hand, exogenous antioxidant are used in
vitamins, drugs and food. By associating with free radicals, antioxidants interact along with them
and it prevents them from harming cells. Due to the decreased risk of cells developing
abnormally and eventually creating tumors, as well as the decreased risk of cell apoptosis, these

3
characteristics increase the possibility of living a healthier and longer life with less negative
effects on ageing(12).

An excess of free radicals posses risk to the body. They are also required for the body to be able
to perceive its own functioning and to be protected from illnesses. The body maintains a very
delicate balance to manage free radicals. There are some mechanisms that prevents, eliminates, or
minimizes oxidative cell damage. Antioxidants are substances that inactivate oxidants directly.
The four main ways in which all antioxidants exert their effects are as follows-(11)

Suppressive effect, Chain-breaking effect, Collector effect and Restorative effect.

Oxidative stress is a condition that develops when the concentration of ROS is higher than the
concentration of antioxidant neutralising species. Atherosclerosis, cancer, malaria, neurological
illnesses including Alzheimer’s, Parkinson’s, Huntington’s have all been linked to oxidative
stress(13). Free radicals such as malondialdehyde (MDA have been found to be more prevelant in
Alzheimer’s patient.(14) According to research, Obesity, hypertension, diabetes, dyslipidemia,
and other metabolic syndrome-related disorders, as well as cardiovascular diseases are all
influenced by oxidative stress(15,16). Cancer is another illness associated to ROS, it have been
proven to activate oncogenes such as transcription factors Jun and Fos, the over-expression of
which is directly linked to lung cancer(17). In lung tumours, ROS can alter the tumour suppressor
gene p53, causing it to lose its apoptotic function and operate as an oncogene.

Plants have been consumed for a long time as food because they are full of vitamins and other
nutrients that are beneficial for the body. Through these applications, the idea of plants as a
source of antioxidants has recently come into greater focus because oxidative stress is now
considered as a major contributor to majority of human diseases and because the antioxidant
defence system in humans is usually insufficient to combat the level of free radicals in the body.
As a result, plants have drawn a lot of attention as a source of antioxidants(15).

Plants contain both enzymatic and non-enzymatic antioxidant defence systems to shield them
from free radicals, just like other humans. Because they lack an immune system, unlike
mammals, plants use their antioxidant defence system to defend themselves against microbial

4
diseases and animal herbivores. This could be the cause for the presence of antioxidants in plants.
Moreover, these phytochemicals also act as a barrier against environmental stress(16).

The biodiversity found in mountains is distinctive. Compared to lowland plants, the majority of
high altitude plant species are solitary and have a constrained range of niche habitats (17). As a
result, the plant populations of the highlands have more endemism than the plant population of
the lowlands(18,19). Due to their limited altitudinal distribution ranges, endemic species are
frequently at danger of extinction at high altitudes(20,21). High altitudes habitats are primarily
impacted by climate limitations and most plants only thrive close to their climatic limits(22,23).

It is anticipated that authentic, dependable medications made from plants grown in their original
habitats will have adequate and ideal chemical compositions.(24)In a study, the antimicrobial
effectiveness of Saturejathymbra which is commonly known as savory of Crete were collected
from various altitudes and tested against a variety of pathogens, plants from low altitude showed
better antifungal activity, whereas those growing at a high altitude showed better antibacterial
activity. This is just an example of how the potency of plant extracts can be impacted by
altitude(25).

In the tropics and subtropics, the genus Hibiscus (Malvaceae) comprises of around 275 species.
Most of the Hibiscus species have an impressive colour pattern with the base of the corolla
forming deep-coloured heart. Hibiscus has paired stipules and simple, lobed, alternating, or spiral
leaves. Flowers have a cup-shaped calyx, five petals connected at the base, a style with many
stamens, and five hairy lobes on the stigma and are radially symmetrical(26).

All around the world, medicinal plants have been utilized traditionally to treat and prevent
disease. Rural regions still use a variety of locally produced drugs as household remedies for
various illnesses and herbal medicines plays significant role in these places. As a result, the
Hibiscus genus could be a fantastic natural source for accelerating the development of novel
drugs as well as an affordable way to treat cancer and other ailments in the developing world(27).

Hibiscus rosa-sinensis is indigenous to China and India. It is found widely in Brazil that is
utilized for urban landscaping, as a living fence, also as an ornamental plant known as China
rose, rose mallow, Chinese hibiscus or shoe flower. It is used in several home remedies. They

5
also possess therapeutic qualities like antibacterial, antioxidant, and antipyretic properties.
Moreover, it is known to have radical scavenging action(28).

Plant produce anti-oxidative phenolic compounds in order to shield themselves from oxidative
damage caused by ultraviolet (UV) exposure. Increased UV-B radiation stimulates the production
of phenolic compounds such as flavanoids in plant tissues. These compounds serve as filters that
effectively absorb and reduce UV flux to plant tissues. Enzymes that are involved in phenolic
synthesis are generated in higher volumes or exhibit increased activity. When exposed to the
condition of elevated light, leaves of tropical forest plants produce more antioxidants(29).

Low temperatures have also been shown to increase PAL synthesis in plants, resulting in
increased phenolic production, including flavanoids. Even in the absence of UV-B radiation,
lower temperatures increase phenolic biosynthesis in some plant species(30). Flavanoids are
widely recognized in human diets as antioxidants with health promoting properties. They also
perform a variety of biological functions, such as interacting with plants and their
environment(31).

Higher altitudes and coastal areas receive more UV radiation, so one would expect that coastal
and highland plants have stronger antioxidant qualities. However there have been a few studies
that compare antioxidant properties between highland and lowland plant populations, as well as
coastal and inland plant populations(26). And according to a study, Hibiscus rosa-sinensis
populations in the highlands had higher antioxidant potential than those in the lowlands(26).

In view of these, the present study has been carried out on DPPH radical scavenging assay,and
total antioxidant capacity, to understand the property of antioxidant as well as the altitudinal
variation of H. rosa-sinensis leaves and flowers that are collected from two different altitudes
(Guwahati and Gangtok).

6
 Chapter 2:

Review of Literature

7
Review of literature

Till date, many elements such as radiation, environmental pollution such as air pollution & water
pollution etc, pesticides, developing technology, stress in living cells all contribute to the
generation of free radicals in the body. Free radicals are extremely reactive forms of oxygen that
cause havoc in the cells of organisms. (10). There are compelling evidence suggests that the
accumulation of free radicals contributes to the development of many harmful patho-
physiological processes, includes cancer, diabetes, cardiovascular disease and neurodegenerative
diseases(2). According to research, free radicals have a substantial impact on ageing, free radical
damage can be prevented with sufficient antioxidant defence, and consuming the right amount of
antioxidant nutrients can improve quality of life(10).

Figure 1 Age-related Oxidative Stress and Antioxidant Balance(32)

Reactive oxygen species (ROS), an inevitable by-product of aerobic respiration, are necessary
and advantageous for regular cell signalling. Reactive nitrogen species (RNS) can also have
physiological benefits. Antioxidants may effectively neutralize excess ROS/RNS in a cell that is
functioning normally. The excessive production of reactive species such as superoxide(O 2),

8
hydroxyl radical(*OH), peroxynitrite(ONOO-), H 2O2, hydroperoxides(ROOH), 1O2, reactive lipid
aldehydes, and reactive nitric oxide, in combination with low antioxidant levels in the body, may
result in oxidative damage to the cellular components such as lipid, protein, DNA. This
phenomena is experienced by most of the elderly people, and ultimately contributes to age-
related disorders. Significant studies has shown that how important it is to consume antioxidant
from dietary nutrients to replenish the body’s low level of antioxidants, notably endogenous
antioxidants like glutathione and coenzyme Q1o (Figure 1)(32).

2.1. History:

Figure 2 The Discovery of Organic Free Radicals by Moses Gomberg(33)

Guyton de Morveau coined the term “radical” in 1786, and Gay-Lussac, Liebig, and Berzelius
later used it to describe atomic groups that were found unchanged in many of the substances. The
term was first used by Liebig and Wohler, who reported in a paper they published in 1832 that
the radical that gave rise to the formula C 14H10O2 remained unchanged throughout the various
transformations of the bitter almond essence and its derivatives are chlorine and bromine(33).

Evidence for the isolation of organic free radicals with measured lifetime was only discovered at
the beginning of the 20th century as a result of Gomberg’s production of the triphenylmethyl
radical(C6H5)3C(34).

In the beginning of the 2oth century, the concept of free radicals emerged in chemistry, where
chemists initially classified them as intermediate organic and inorganic molecules with a number

9
of possible definitions. On the basis of Daniel Gilbert and Rebecca Gersham’s work which was
hypothesized in the year 1954 that these radicals not only played a significant biological roles
contexts but were also responsible of several harmful cell activities(35). And a clear
understanding of these radicals was then put forth. After that, in 1956, Herman Denham added
that these reactive species might be crucial to physiological processes, particularly in the process
of ageing(36). Numerous investigations and research projects were inspired by this free radical
theory of ageing hypothesis, which greatly advanced knowledge of radicals and related species
including reactive nitrogen species(RNS), reactive oxygen species(ROS), and non-radical
reactive species(37).

It has been hypothesised that Helicobacter pylori infection in the human stomach causes
increased production of ROS and RNS, which in turn leads to the development of gastric cancer.
Urolithiasis is caused by excessive ROS in human kidneys(38). Additionally it has been claimed
that ROS harms the cellular components of cartilage, causing osteoarthritis, and has been shown
to be responsible for harming the pancreatic islets cells(39,40)(41).

Medicinal plants have been used for a variety of ailments throughout history. The plants' ability
to synthesise chemical compounds that are critical in the prevention of numerous diseases such as
cancer and diabetes has led to their designation as therapeutic plants. At least 12,000 of these
compounds have been identified as of currently, accounting for fewer than 10% of all known
compounds(42,43).

Three important sectors of medicinal plants can be thought of and considered as:

(a) Modern drugs that employ between 30 to 35 medicinal herbs;

(b) Ayurveda, Siddha, Unani, Amchi, and Tibetan systems of medicine, which employ 1,200-
2,000 different medicinal plants, are among the organized.
(c) Local health rituals that are derived from customs followed by villagers and tribal people
that use more than 8,000 different types of medicinal plants for the purpose of basic
health care(44).

10
2.2. Importance of medicinal plants:

Figure 3 Health Benefits of Herbs and Species (45)

In different parts of the world, plants have continued to serve crucial roles in sustaining human
life(46). These includes medicinal plants which contains elements that have pharmacological
effects as well as therapeutic properties on the human body. Generally medicinal plants produce
and accumulate secondary metabolites naturally for example alkaloids, sterols, terpenes,
flavonoids, saponins, glycosides and tannins, while their extracts have found use in the
pharmaceutical, nutraceutical as well as in the other chemical industry(47).

Since the dawn of time, many studies have been conducted all over the world to confirm their
efficacy, and some of the results have prompted the development of plant-based medications(48).

On May 30, 2006, a Presidential Initiative Committee on the Development, Promotion, and
Commercialization of Nigerian Herbal Medicinal Products was established, with the goal of
selling medicinal plants and their by-products for US$1 billion in Nigeria within ten years(49).

Furthermore, as the cost of preserving one's own health has risen, herbal therapies have gained
appeal in these civilizations for the treatment of minor ailments. In fact, there is a substantial risk
that many therapeutic plants will become extinct or lose genetic diversity as a result of market
and public demand, which has been extremely strong(48).

2.3. Prevention of disease through medicinal plants:

11
Approaches to Prevent Communicable Diseases- The prevention of communicable illnesses relies
heavily on three key strategies i.e. immunization, outbreak investigations, and surveillance. Even
while it may seem that medicinal plants play a small part in these methods, a number of these
plants and the traditional medicines made from them have been utilized to improve the immune
system’s response to a number of disease agents(50).

Approaches to Prevent Non-Communicable Diseases- The WHO 2008 to 2013 Action plan for
the Global Strategy for the Prevention and Control of Non-Communicable Diseases outlined an
intersectoral, multi-level plan to slow the rising prevalence of Non-Communicable Diseases
around the world with a special focus on the low and medium income country. The broad
objectives of the plan were to:

1) Map the emerging Non-Communicable Diseases epidemic and identify its social,
economic, behavioral, and political determinants;
2) Reduce the level of exposure of individuals and the population to the common
modifiable risk factors, such as tobacco use, an unhealthy diet, and physical
inactivity;
3) And improve the quality of care for those with Non-Communicable Diseases by
creating evidence based-norms, standards, and guidelines for cost-effective
interventions(51).

Figure 4 Risk Factors for Non-communicable Diseases in a Casual Chain(51)

12
Especially in the early stages, Medicinal Plants play specialized roles in enhancing access to
healthcare for those who have NCDs and in the management of the biological risk factors for
NCDs.

Approaches of Common Factor- The goal of the common risk factor approach is to bring together
various health promoters who are trying to eliminate common risk factors as a means of disease
prevention. For instance, a poor diet can result in dental decay, cancer, diabetes, obesity and other
diseases. As a result, with diet serving as a unifying subject, nutritionists, diabetologists,
oncologists and dental professionals can work together. A modified version of this strategy can
be a helpful tool for enlisting the support of other health promoters, combating various illness
types, and spreading the ideas of medicinal plants. By collaborating with other organizations,
particularly, suitable medicinal herbs can be taken into the diets to reduce disease and suffering.
With this strategy, people promoting the use of medicinal plants will be able to work with others
who are promoting health in order to combat conditions like HIV/AIDS, cancer, diabetes,
malaria, cardiovascular disease, tuberculosis, dental and dermatological disorders (52).

Figure 5 The Common Factor Approach(52)

In conclusion, from the analysis of the existing approaches of disease prevention, it can be
concluded that medicinal plants plays a significant role in the prevention of disease, and that their

13
use and promotion are compatible with the methods now in use. To appropriately identify,
recognize and position medicinal plants, however, deliberate steps need to be made in the
development and use of these methods.

2.4. Medicinal plants in correlation with diseases:

Use of medicinal plants Yasukawa (2012) examined the chemopreventive properties of natural
sources, foods, supplements, crude pharmaceuticals and Kampo medications. He noted that one
of the most essential public health initiatives at the moment is cancer chemoprevention. The
multistage carcinogenesis theory are- initiation, promotion, and progression, which is the
foundation of the majority of research on cancer prevention(53,54).

Azadirachta indica (Family Meliaceae) Neem- Terpenoids and steroids, among more than 60
other types of biochemicals, have been extracted in pure form from this plant. According to Paul
et al. in 2011, the anticancer activities of the plant have mostly been investigated in terms of its
apoptotic, preventative, and protective actions against different forms of cancer and their
molecular pathways. About 15-20% of all breast tumors are triple-negative breast cancers
(TNBC), and these tumors are frequently aggressive and extensively metastatic. But TNBCs have
few available treatment choices, which is unfortunate(56).

Moringa pergrina (Moringaceae)- M. peregrina has reportedly been shown to produce

hypoglycemic effects in the past. In streptozocin diabetic rats, the hydroalcoholic extract fraction
of M. peregrine seeds and aerial parts was found to have antidiabetic activity(58).

Cynomorium coccineum (Cynomoriaceae)- Pharmacological investigations revealed that the

Cynomorium plants have a variety of biological properties, some of which were already known
from traditional medicine, such as antioxidant, immunity-boosting, antidiabetic, neuroprotective,
and other bioactivities(59).

Ducrosiaanethifolia (Apiaceae)- The major isolation furanocoumarins from D. anethifoliaand

their ethanolic extract showed concentration-dependent inhibition of the carbohydrate
metabolizing enzymes- amylase, glucosidase and galactosidase in vitro.

14
Excoecariaagallocha LinnMangrove plant- According to a study, E. agallocha methanolic
extract contains a number of antioxidant compounds that can effectively scavenge ROS. Because
of its high flavonoid concentration, it possesses great radical scavenging action and the potential
to prevent oxidative DNA damage. Flavonoids have a number of medicinal benefits, which
includes antioxidant, anticancer, antiviral and anti-inflammatory properties(60).

2.5. The phytochemistry of Hibiscus rosa-sinensis:

Figure 6 Hibiscus rosa-sinensis

Due to the rising disease burden, nowadays natural plant products are being utilised extensively.
Worldwide, there is a large population of the plant H. rosa-sinensis Linn. The Indian traditional
system of medicine has employed its leaves, barks, roots and flowers to cure a variety of diseases.

Many research investigations conducted in the animal models, examined that the leaves and
flowers of Hibiscus rosa-sinensis has its anti-diabetic and antioxidant properties. In the case of
medicine, red-flowered variety are preferred more.

According to the study, it was reported that it contains total tannins, total steroid content and total
aklanoids and saponins(61,62). Along with these, there are other substances present like vitamins
as well as the cyclopeptide alkaloid cyanidine chloride, quercetin, and hentriacontane. Taraxeryl
acetate, stigmasterol, β-sitosterol, and other three cyclopropane chemicals and their derivatives
are all found in the leaves and stems. Cyanidin diglucoside, flavonoids and vitamins are all
present in hibiscus flower. And the roots of H. rosa-sinensis contain triterpenoids, saponins,
mucilage, flavonoids, phenolic compounds, glucose and glycosidase(63).

15
2.6. Classification:

Kingdom- Plantae-Plants

Subkingdom- Tracheobionta-Vascular plants

Superdivision- Spermatophyta-Seed plants

Division- Magnoliophyta-Flowering plants

Class- Magnoliopsia-Dicotyledons

Subclass- Dilleniidae

Order- Malvales

Family- Malvaceae-Mallow family

Genus- Hibiscus L

Species- Hibiscus rosa-sinensis L

2.7. Traditional Applications of Hibiscus rosa-sinensis:

Hibiscus flowers and leaves are used for contraception, abortion, infertility treatment,
menorrhagia, bronchitis, emmenagogue therapy, cough, and diuretic in India(64). Hibiscus
flowers have long been used in sachets and perfumes throughout Africa and its surrounding
tropical regions. In sections of Northern Nigeria, hibiscus has traditionally been used to cure
constipation. The leaves are traditionally used as emollients and aperients to cure constipation,
skin problems, and burning sensations(65).

The plant, used in Egypt is said to be diuretic in nature, cures cardiac and nervous ailments.
Hibiscus leaves are commonly used as an anti-diarrheal, in Japan. In Iran, sour tea is used to
alleviate hypertension. Hibiscus flowers are frequently used in the preparation of herbal tea, in
Western countries. People in Thailand drink Roselle juice to satisfy their thirst(61).

16
Table 1 Therapeutic applications of the plant H. rosa-sinensis

Sr Therapeutic Parts Model Medium Dose Activity Reference

l study used
1. Antioxidant Leaf Extraction of Strong scavenging (66)
activity polysaccharide activities invitro
2. In-vitro Stem Methanol and Invitro antioxidant (67)
activity and and distilled water and phytochemical
phytochemical leaves analysis
analysis
3. Antioxidant Leaves Ethanol water Lipid peroxidation (68)
activity extract (LPO) and protein
oxidation (PO)
4. Antioxidant Root Male Methanolic 100mg, Restoration of (69)
Wister extract 200mg antioxidant enzyme
Rat and and treatment in
300mg/ VCMs and tongue
kg/bw protrusions.
5. Antioxidant Leaves NOD Ethanolic 100 and Effect on islets of (70)
activity MIce extract 200 Langerhans and
mg/kg antioxidant activity
body in non-obese
weight diabetic (NOD)
mouse

17
Srl Therapeutic Parts Model Medium Dose Activity Reference
study used
6. Hypolipidemic Roots Charles Ethanolic 500 Inhibitory effect (71)
activity Foster extract mg/kg of hepatic
male /bw steatosis
rats (hypolipidemic
activity)
7. Antimicrobial Leaves, Ethanol 50 and Agar-well (72)
activity Flowes extract 100 diffusion method
mg/well
8. Medicinal Flower Adult Aqueous 500 Reversible (73)
properties swiss extract mg/kg of suppression of
albino BW spermatogenesis,
mice cholesterol level
and glucose level
9. Cytotoxic Leaf Leukae Petroleum Possess potentials (74)
activity and mic ether, ethyl as effective
stem cell acetate and cytotoxic agents
line(K- methanol against K- 562
562) extracts cells
10. Cardioprotecti Flowers Male 80% ethanol 90, 180 Positive inotropic (75)
ve effects adult reconstituted and 360 effect, and
Wistar in water μg/ml cardioprotection
rats
(23027
0 g)

18
2.8. Mountain Plants and the Influence of Climates:

Climate change is thought to be particularly sensitive to temperature-limited habitats. As a result

of this warming, for instance, plant species and communities in high mountains will be forced
upward in elevation and may be eradicated if already at mountain summits. If evidence of a
persistent alteration in summit floras were to be found, this would show that global warming is
already having a large ecological impact. Although some preliminary observations have recently
been presented4, no firm evidence has yet been revealed. In the summer of 1992, data was
collected on the condition of the flora at 26 summits higher than 3,000 metres in the heart of the
Alps, and we compared the present-day records of vascular plant species' cover and abundance
with earlier ones. Over the last few decades, species richness has increased and is more
prominent in at lower elevations (figure 6).

Figure 7 The Abundance of Species Recorded in the Past and Present at Nival Summits in the
Alps(20)

19
The abundance of species recorded in the past and present at nival summits in the Alps is plotted
versus elevation. Rare species were given a weight of 0.25, while common species were given a
weight of 0.5, and abundant species were given a weight of 1.0. due to the relatively high
likelihood that rare species were missed by the original authors, they were downweighted. 24
summits with siliceous bedrock are visible. The historical documents range in age from 40 to 90
years. Since humans have very little of an impact on the majority of summits under consideration,
it is inconsequential(20).

However, a comparison of the fitted regression lines for species-richness with altitude shows that
there is an overall tendency towards an increase in the alpine-nival flora. The nival vegetation
above the confined alpine grasslands generally exhibits an exponential reduction in species
richness with altitude(20).

2.9. Altitudinal Variations of Different Species of Hibiscus:

H. mutabilis leaves are broadly oval, with five triangular lobes. Morning flowers are white,
afternoon flowers are pink, and night flowers are red. H. sabdariffa leaves has large oval leaf
with 3-5 lobes and reddish stalks. Pinkish flowers appear in the morning and orange in the
evening. H. taiwanensis has finely serrated leaves and yellow blossoms with a dark brown centre.
Heart-shaped leaves and bell-shaped blooms with a maroon heart and stigma characterise H.
tiliaceus. The blossoms are yellow in the morning and orange-red in the evening. The leaves of
H. rosa-sinensis are oblong with serrated margins. crimson flowers with a long and slender style,
yellow anthers, and a crimson stigma. H. schizopetalus resembles H. rosa-sinensis in appearance
and the flowers are red.

In a study, which was carried out in Malaysia, found that based on the AOP of the leaves and
flowers of various hibiscus species tested are classified into three groups. H. tiliaceus species
with equivalent values in leaves and flowers, H. mutabilis and H. sabdariffa species with
significantly higher values in leaves than flowers, and H. taiwanensis, H. rosa-sinensis, and H.
schizopetalus species with significantly higher values in flowers than leaves(26).

The AOP of leaves from two highland populations and two lowland populations of Hibiscus rosa-
sinensis from the same mountain range were also compared. Highland populations were

20
discovered to be much higher than lowland populations by analysing the four populations
studied.(26).

21
Chapter 3:

Methodology

22
Methodology

3.1. Sample collection:

Leaves and Flowers of Hibiscus rosa-sinensis were collected from Panbazar, Assam, Guwahati
(55m from sea level) on 4thJanuary, 2023 at 8:30am and from Lumsey, Tadong, Gangtok (1650m
from sea level) on 2nd February, 2023 at 6:45am. The voucher specimens were deposited in the
Department of Medical Biotechnology for future reference.

Separately, plant materials were cleansed with water to eliminate dirt and other pollutants before
being shade-dried and/or air-dried for several days with periodic sun-drying. The dried materials
were stored at RT in preparation for future usage.

23
(a) (b)

(c) (d)

Figure 8. Sample collection (a) Collection of sample (b) Sample washed and kept for drying
(c) Air-dried for several days (d) Dried sample

24
3.2. Preparation of extract:

Dried sample was then coarsely powdered in a grinder and stored it in a neatly labeled container
and are subjected to maceration. Maceration is a process in which coarsely powdered drug
material, such as flowers, leaves, or stem bark, is placed within a container and menstruum is
poured on top until the plant material is thoroughly covered. About 10g of leaves and 5g of
flowers of each powdered plant materials was weighed in an electronic weighing balance and
then was taken in conical flask and soaked with 50ml of methanol. The sealed conical flask were
kept for 72 hours and it was occasionally subjected to rotator shaker. After three days, the
extracts were filtered individually using a fresh cotton cloth and then using Whatman No.1 filter
papers. The filtrates were concentrated in waterbath at 45°C. And then to get the concentrated
extract lyophilizer was used.

(a) (b)

25
 (c) (d)

(e) (f)

Figure 9 Preparation of extract (a) Weighed each powdered plant containing in beaker in an
electronic weighing balance (b) Powdered sample was taken in a conical flask and soaked it in
methanol (c) The sealed conical flask was occasionally subjected to rotator shaker for 72 hours
(d) After three days, the extracts were filtered through cotton cloth and then with whatman
no.1 filter paper (e) Then the filtrates were concentrated in waterbath at 45°C (f). To get the
extract more concentrated, freeze-drying i.e. lyophilizer was used.

26
3.3. Materials Required:

3.3.1. Chemicals: (Aakar Biotechnology Pvt. Ltd.)

i. Dimethyl sulfoxide (DMSO)/ DM Water,

ii. Methanol,
iii. Distilled Water,
iv. 2,2-diphenylpicrylhydrazyl (DPPH) Reagent (50X),
v. Ascorbic Acid,
vi. Cupric Chloride,
vii. Ammonium Acetate,
viii. Neocuproine (50X),
ix. Trolox

3.3.2. Equipments and glasswares:

i. Beaker (Borosil),
ii. Pipette,
iii. Pipette tips (Tarsons),
iv. Conical Flask (Borosil),
v. Falcon tube,
vi. Centrifuge tubes (Tarsons),
vii. Measuring Cylinder,
viii. Spatula,
ix. Funnel
x. Test tubes (Rimless)
xi. Microtitre plate

3.3.3. Instruments:

i. Lyophilizer (Zebra),

27
 ii. Waterbath (Remi),
iii. Electronic Weighing Balance (Denver),
iv. Microplate Reader

3.4. Groups for the bioassays:

Set 1- Negative Control

Set 2- Blank for Negative Control

Set 3- Treatment Group

Set 4- Blank for Treatment Group

3.5. DPPH radical scavenging assay:

The ability of the plant extractive to donate hydrogen atoms was assessed using the
decolorization of a methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In methanol
solution, DPPH creates a violet or purple colour that fades to varying hues of yellow in the
presence of antioxidants.

3.5.1. Preparation of plant extract:

Guwahati Leaves: 10g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 300mg/ml, and it was converted to 10mg/ml.

Table 2 Reference Chart for preparation of different concentration of Guwahati leave extract

Srl Concentrations Serial Dilution

1. 10mg/ml 33.33μl of stock solution + 966.67μl of Methanol
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of Methanol
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of Methanol
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of Methanol
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of Methanol
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of Methanol

28
Guwahati Flowers: 5g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 200mg/ml, and it was converted to 10mg/ml.

Table 3 Reference Chart for preparation of different concentration of Guwahati flower extract

Srl Concentrations Serial Dilution

1. 10mg/ml 50μl of stock solution + 950μl of Methanol
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of Methanol
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of Methanol
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of Methanol
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of Methanol
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of Methanol

Gangtok Leaves: 5g of lyophilized product was weighed and dissolved in 10% DMSO. From the
prepared stock solution of 233mg/ml, and it was converted to 10mg/ml.

Table 4 Reference Chart for preparation of different concentration of Gangtok leave extract

Srl Concentrations Serial Dilution

1. 10mg/ml 42,91μl of stock solution + 957.09μl of Methanol
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of Methanol
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of Methanol
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of Methanol
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of Methanol
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of Methanol

29
Gangtok Flowers: 10g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 1000mg/ml, and it was converted to 10mg/ml.

Table 5 Reference Chart for preparation of different concentration of Gangtok flower extract

Srl Concentrations Serial Dilution

1. 10mg/ml 20μl of stock solution + 989μl of Methanol
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of Methanol
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of Methanol
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of Methanol
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of Methanol
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of Methanol

3.5.2. Solutions Preparation-

DPPH Stock Solution: Prepared the DPPH 50X stock by adding 1.4ml solvent i.e. methanol to
the tube labeled as DPPH stoch (50X). Dissolved completely before use.

DPPH Working Solution: Prepared the 50ml DPPH 1X working solution by adding 1ml DPPH
stock to 49ml of solvent i.e. methanol.

Standard solution: No dilution required. Stock concentration of the Ascorbic Acid solution is
1mg/ml (5.6 mM).

3.5.3. Experimental Procedure-

1. Added 10μl of different stock dilutions of the test sample/ or the standard to each wells
labeled for set 3 & 4. Added 10μl of solvent i.e. methanol to each wells labeled for set 1
& 2.
2. Added 200μl of DPPH Reagent to each wells for the set number 1 & 3 and added 200μl
solvent i.e. methanol to the each wells for the set number 2 & 4.

30
 3. Incubated themicrotest plate for 30 minutes in the dark at room temperature.
4. Measured the absorbance at 540nm with microplate reader.
The experiment was repeated three times at each concentration. Percentage DPPH radical
scavenging assay was calculated by the following equation:

DPPH inhibition % = ¿ x 100

Where,
Abscontrol= Absorbance of control
Abssample= Absorbance of sample/standard

Then the % of inhibition was plotted against concentration, and from the graph IC 50 was
calculated.

IC50 = [ Concentration of tested agent ×50

% inhibition ]

31
3.6. Determination of total antioxidant capacity (CUPRAC) assay:

CUPric Reducing Antioxidant Capacity, or CUPRAC is a new technique called assay permits the
assessment of both hydrophilic and hydrophobic substances Total Antioxidant Capacity (TAC).
Antioxidants can be oxidized by the primary reagent, copper (II)-neocuproine (2,0-dimethyl-1,
10-phenanthroline), producing a coloured end result. By increasing the reagent’s redox potential,
the chelation with neocuproine makes it possible for a quicker reaction.

Light blue CUPRAC reagent Yellow-orange product

3.6.1. Preparation of plant extract-

Guwahati Leaves: 10g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 300mg/ml, and it was converted to 10mg/ml.

Table 6 Reference Chart for preparation of different concentration of Guwahati leave extract

Srl Concentrations Serial Dilution

1. 10mg/ml 33.33μl of stock solution + 966.67μl of DM Water
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of DM Water
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of DM Water
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of DM Water
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of DM Water
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of DM Water

32
Guwahati Flowers: 5g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 200mg/ml, and it was converted to 10mg/ml.

Table 7 Reference Chart for preparation of different concentration of Guwahati flower extract

Srl Concentrations Serial Dilution

1. 10mg/ml 50μl of stock solution + 950μl of DM Water
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of DM Water
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of DM Water
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of DM Water
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of DM Water
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of DM Water

Gangtok Leaves: 5g of lyophilized product was weighed and dissolved in 10% DMSO. From the
prepared stock solution of 233mg/ml, and it was converted to 10mg/ml.

Table 8 Reference Chart for preparation of different concentration of Gangtok leave extract

Srl Concentrations Serial Dilution

1. 10mg/ml 42.91μl of stock solution + 957.09μl of DM Water
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of DM Water
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of DM Water
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of DM Water
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of DM Water
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of DM Water

33
Gangtok Flowers: 10g of lyophilized product was weighed and dissolved in 10% DMSO. From
the prepared stock solution of 1000mg/ml, and it was converted to 10mg/ml.

Table 9 Reference Chart for preparation of different concentration of Gangtok flower extract

Srl Concentrations Serial Dilution

1. 10mg/ml 20μl of stock solution + 989μl of DM Water
2. 5mg/ml 100μl solution from 10mg/ml + 100μl of DM Water
3. 2.5mg/ml 100μl solution from 5mg/ml + 100μl of DM Water
4. 1.25mg/ml 100μl solution from 2.5mg/ml + 100μl of DM Water
5. 0.625mg/ml 100μl solution from 1.25mg/ml + 100μl of DM Water
6. 0.312mg/ml 100μl solution from 0.625mg/ml + 100μl of DM Water

3.6.2. Solutions Preparation-

Neocuproine Working Solution: Prepared 5ml of neocuproine working solution by adding 100μ
of neocuproine to 4.9ml of DM water.

CUPRAC Reagent: For 15ml of CUPRAC reagent, mixed 5ml of Cupric Chloride, 5ml of
Ammonium Acetate and 5ml of neocuproine working solution. Mixed it properly and prepared
just before use.

Standard: No dilution required, stock concentration of the Trolox solution is 1mM.

3.6.3. Experimental Procedure-

1. Added 10μl of different stock dilutions of the test sample/ or the standard to each wells
labeled for set 3 & 4. And added 10μl of solvent i.e. DM water to the each wells labeled
for set 1 & 2.

34
 2. Added 200μl of CUPRAC Reagent to each wells for the set number 1 & 3 and added
200μl solvent i.e. DM water to the each wells for the set number 2 & 4.
3. Incubated the microtest plate for 30 minutes.
4. Measured the absorbance at 450nm with microplate reader.
The experiment was repeated three times at each concentration. Reducing activity can be
calculated with the following equation:

%Reduction power = ¿ x 100

Where,
Abscontrol=Absorbance without sample
Abssample= Absorbance of sample/standard

The calculated values were transformed into a linear equation (Y = bX + a). The IC 50 value was
obtained from the calculation when the % reduction was 50%.

IC50 =[ Concentration of tested agent ×50

% inhibition ]

35
Figure 11. Result of Total actioxidant capacity (CUPRAC) assay

36
Chapter 4:

Result

37
Result

The present study aimed to evaluate the antioxidant activity of Hibiscus rosa-sinensis grown in
different altitude by total antioxidant capacity (CUPRAC) assay and DPPH radical scavenging
assay.

4.1. Yield percentage: The collected sample was processed (washing, maceration
filtration, and concentration) followed by lyophilization to obtain the final extract of plant
product. The lyophilized product was then quantified for its yield. The final obtained
yield percentage of the crude (W/W) from H. rosa-sinensis, after methanolic leaves and
flowers extract was- Guwahati leaves (3%), Guwahati flowers (2%), Gangtok leaves
(2.33%) and Gangtok flowers (10%). It was highest in Gangtok flower.

Table 10. Yeild rate of plant extract (% W/W)

Sample Extract Percentage yield of extract

(Methanol) (H. rosa-sinensis)
Guwahati Leaves 3%

Guwahati Flowers 2%

Gangtok Leaves 2.33%

Gangtok Flowers 10%

38
 4.2. DPPH Radical Scavenging Assay: In DPPH radical scavenging assay, the
methanolic leaves and flowers extract showed dose dependent manner inhibition activity.
The percentage of inhibition of H. rosa-sinensis of highland i.e. Gangtok (leaves) was
highest at 10mg/ml with maximum percentage 37.46±5.49, as compared to the lowland
i.e. Guwahati (leaves) with maximum percentage 16.26±5.72. And the percentage
inhibition of H. rosa-sinensis of lowland i.e. Guwahati (flowers) was highest at 10mg/ml
with maximum percentage 95.61±4.75, as compared to the highland i.e. Gangtok
(flowers) with maximum percentage 46.90±5.43 (Table 11). Between all the samples of
lowland and highland, Guwahati (flowers) has exhibited an exceptional antioxidant
property. And Guwahati (leaves) has the lowest antioxidant potential out of all other
sample. The antioxidant property of the samples was predicted and it is arranged in
ascending order below –

Ghy (F) ˃ Gtk (F) ˃ Gtk (L) ˃ Ghy (L)

3.2mg/ml ˃ 10.69mg/ml ˃ 14.39mg/ml ˃ 33.5mg/ml

The standard drug (Ascorbic acid) shows the highest DPPH radical scavenging assay at 100µg/ml
concentration i.e 97.12±0.56 and the IC50 value of the standard (ascorbic acid) is 2.31µg/ml
(Table 12)

39
Table 11. IC50 value for DPPH radical scavenging assay by H. rosa-sinensis leaves and flowers

Sample Percentage of DPPH inhibition (%) IC 50

s 10mg/ml 5mg/ml 2.5mg/ml 1.25mg/ml 0.625mg/ml 0.312mg/ml (mg/mL)

Ghy (L) 16.26±5.72 8.74±1.83 4.66±1.34 3.34±2.32 3.22±2.04 2.06±2.05 33.5

33.71±5.2
Ghy (F) 95.61±4.75 85.27±1.66 57.31±3.55 19.14±3.38 12.98±3.14 3.2
8

Gtk (L) 37.46±5.49 13.95±2.65 8.52±2.65 8.79±4.42 4.59±1.30 2.76±2.67 14.39

10.82±4.2
Gtk (F) 46.90±5.43 24.90±3.32 15.99±3.31 6.28±3.94 3.54±2.34 10.69
1

Table 12 IC50 value for DPPH radical scavenging assay of standard (ascorbic acid)

Percentage of DPPH inhibition (%) IC 50

Samples
100µg/ml 50µg/ml 25µg/ml 12.5µg/ml 6.25µg/ml 3.125µg/ml (µg/mL)

68.20±2.9
Std 97.12±0.56 89.22±1.61 80.49±1.49 41.13±2.82 31.14±4.93 2.31
0

40
 Average % inhibation (b) Average % inhibation
120.00 120.00
100.00 f(x) = 0.586231819234818 x + 48.6482593484632 100.00
R² = 0.667935428445529
80.00 80.00
60.00 60.00
40.00 40.00
20.00 20.00
f(x) = 1.44354523078113 x + 1.6417956126509
0.00 0.00 R² = 20.993964765707411
0 20 40 60 80 100 120 0 4 6 8 10 12

(c) (d
Average % inhibation ) Average % inhibation
120.00 120.00

100.00 f(x) = 8.53441092349924 x + 22.6682019639351 100.00

R² = 0.83916099664608 80.00
80.00
60.00 60.00

40.00 40.00
f(x) = 3.35836747344382 x + 1.6602224119458
20.00 20.00 R² = 0.954705630217673
0.00 0.00
0 2 4 6 8 10 12 0 2 4 6 8 10 12

(e)
Average % inhibation
120.00

100.00

80.00

60.00

40.00 f(x) = 4.31175533102916 x + 3.92387827205514

R² = 0.994244996653201
20.00

0.00
0 2 4 6 8 10 12

Figure 12. Antioxidant activity curve of DPPH radical scavenging assay (a) Ascorbic
acid (standard) (b) Guwahati leaves (c) Guwahati flowers (d) Gangtok leaves (e)
Gangtok flowers

41
 DPPH Inhibition (%)
120.00

100.00

80.00

60.00

40.00

20.00

0.00
10mg/ml 5mg/ml 2.5mg/ml 1.25mg/ml 0.625mg/ml 0.312mg/ml

33.5 GHY (L) 33.5 GTK (L) 33.5 GHY (F) 33.5 GTK (F)

Figure 13. Percentage inhibition of DPPH radical scavenging assay

(GHY- Guwahati, GTK- Gangtok)

4.3. Total Antioxidant Capacity (CUPRAC) Assay: In total antioxidant capacity

(CUPRAC) assay, the methanolic extract shows that an increase in absorbance implies an
increase in reducing power. The percentage of reducing power of H. rosa-sinensis of
lowland i.e. Guwahati (leaves) was highest at 10mg/ml with maximum percentage of
88.02±0.19 as compared to the highland i.e. Gangtok (leaves) with maximum percentage
85.76±1.10. And the percentage inhibition of H. rosa-sinensis of lowland i.e. Guwahati
(flowers) was highest at 10mg/ml with maximum percentage 99.99±0.00 , as compared to
the highland i.e. Gangtok (flowers) with maximum percentage 92.08±1.02 (Table 13).
Between all the samples of lowland and highland, Guwahati (flowers) has exhibited an
exceptional antioxidant property. And Gangtok (leaves) has the lowest antioxidant
potential out of all other sample. The antioxidant property of the samples was predicted
and it is arranged in ascending order below –
Ghy (F) ˃ Ghy (L) ˃ Gtk (F) ˃ Gtk (L)
-1.65mg/ml ˃ 0.71mg/ml ˃ 0.79mg/ml˃ 3.47

42
The standard drug (Ascorbic acid) shows the highest total antioxidant capacity (CUPRAC) assay
at 100µM/ml concentration i.e 74.49±2.25 and the IC50 value of the standard (ascorbic acid) is
36.14µM/ml (Table 14).

Table 13 IC50 value for total antioxidant capacity (CUPRAC) assay by H. rosa-sinensis leaves
and flowers

Percentage of TAC Reduction (%) IC 50

Sample
1.25mg/ 0.625mg/ 0.312mg/ (mg/mL
s 10mg/ml 5mg/ml 2.5mg/ml
ml ml ml )

88.02±0.1 84.07±0.9 74.56±1.8 50.96±4.6

Ghy (L) 49.13±5.58 31.73±7.94 0.71
9 9 0 3

99.99±0.0 89.79±0.3 83.45±0.7 78.17±1.2

Ghy (F) 67.17±1.23 28.08±2.80 -1.65
0 8 4 5

85.76±1.1 77.31±2.0 65.13±1.3 30.90±2.0

Gtk (L) 20.36±1.67 12.38±4.50 3.47
0 4 9 9

92.08±1.0 86.74±1.6 77.90±2.8 62.18±6.2

Gtk (F) 42.17±6.46 24.80±3.03 0.79
2 4 1 5

Table 14 IC50 value for total antioxidant capacity (CUPRAC) assay of standard (trolox)

Percentage of TAC Reduction (%) IC 50

Sample
12.5µM/ 6.25µM/ 3.125µM/ (µM/mL
s 100µM/ml 50µM/ml 25µM/ml
ml ml ml )

74.49±2.2 59.92±6.9 51.21±2.2

Std 42.53±1.57 36.48±2.16 26.63±5.06 36.14
5 0 3

43
 Average % inhibation (b) Average % inhibation
120.00 100.00
90.00 f(x) = 5.0862740932753 x + 46.3903311628022
100.00 80.00 R² = 0.705636074679676
80.00 70.00
f(x) = 0.438308928990911 x + 34.1624826216314 60.00
60.00 R² = 0.896740544324611 50.00
40.00
40.00 30.00
20.00 20.00
10.00
0.00 0.00
0 20 40 60 80 100 120 0 2 4 6 8 10 12

(c) (d)
Average % inhibation Average % inhibation
120.00 100.00
90.00 f(x) = 7.34446635655909 x + 24.5430798026188
100.00 f(x) = 4.95487078313191 x + 58.1844700272167 80.00 R² = 0.756174506469964
R² = 0.529392513773324 70.00
80.00
60.00
60.00 50.00
40.00
40.00 30.00
20.00 20.00
10.00
0.00 0.00
0 2 4 6 8 10 12 0 2 4 6 8 10 12

(e) Average % inhibation

100.00
90.00 f(x) = 5.74917625745823 x + 45.4480525034664
80.00 R² = 0.646005402785563
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 2 4 6 8 10 12

Figure 14. Antioxidant activity curve of total antioxidant capacity (CUPRAC)

scavenging assay (a) Trolox (standard) (b) Guwahati leaves (c) Guwahati flowers (d)
Gangtok leaves (e) Gangtok flowers

44
 TAC Reduction(%)
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
10mg/ml 5mg/ml 2.5mg/ml 1.25mg/ml 0.625mg/ml 0.312mg/ml

GHY (L) GTK (L) GHY (F) GTK (F)

Figure 15. Percentage reduction of total antioxidant capacity (CUPRAC)

assay

45
Chapter 5:

Discussion

46
Discussion

For thousands of years, plants have served as the foundation for sophisticated traditional
medicine practises practised by people in China, India, and other nations. The Artharvaveda, the
basis for Ayurvedic medicine in India, clay tablets in Mesopotamia, and the Ebar Papyrus in
Egypt contain some of the earliest records of the use of plants as pharmaceuticals. Herbalism is
growing more popular as an alternative medicine since it lowers the cost of healthcare for
customers. (76).

Hibiscus rosa sinensis, often known as Chinesis Hibiscus or Tropical Hibiscus. Hibiscus rosa-
sinensis, a Chinese native, is a potent medicinal plant. It is a common perennial shrub in Indian
gardens. In addition, it is widely used as a hedge or fence plant. It is also found at altitudes
ranging from sea level to 1200 m above sea level(77–79).

Hibiscus rosa-sinensis flower decoctions are used as aphrodisiacs, for menorrhagia, uterine
bleeding, and for fertility control in India and Vanuatu. It has anti-complementary, antidiarrhetic
and antiphologistic properties. Hibiscus rosa-sinensis flower has antispermatogenic, and
anticonvulsant properties as well as antitumor, antihypertensive, antioxidant, and antiammonemic
properties. Leaves and flowers have hypoglycemia action as well(73).

In-vitro studies of Hibiscus flowers, leaves, and stem extract indicated strong antioxidant activity
that reduced the risk of numerous diseases. Microorganism control, sporadic outbreaks of
epidemics caused by drug-resistant bacteria and microbes all represent a significant risk to public
health. Hibiscus flower and leaf extract demonstrated antibacterial activity against several
pathogenic bacteria, the risk of many infectious illnesses hence gets reduces.

Diabetes is becoming more common over the world. Several research investigations have found
that medicinal herbs can lesses the risk of diabetes. Various animal experiments revealed an anti-
diabetic effect when different parts of the Hibiscus plant were administered. Hibiscus leaf extract
also demonstrated anti-tumor properties in vitro(80).

A gastrointestinal condition characterised by an inflammatory breach in the skin or mucus

membrane lining the alimentary canal is known as an ulcer. Ulcers can be caused by irregular

47
eating habits, stress, and continuous drug usage. Ulcers are treated using a variety of medicinal
herbs. Various solvent extracts of Hibiscus roots were discovered to have antiulcer activity in
albino rats.(81).

Traditional remedies have been developed as pharmacological drugs to treat liver cirrhosis,
according to hepatology study. In albino rats, different doses of Hibiscus flower extract
demonstrated hepatoprotective properties. In male swiss albino mice, a hydroalcoholic extract of
Hibiscus leaves inhibited pro-inflammatory mediators such as NO and TNF-, implying that it is
responsible for the healing effect of colitis (82).

Hibiscus rosa-sinensis could be utilised as a protein bank in grazing, sown in strips with grasses,
or in silvopastoralsystems as animal feed. Furthermore, it can be utilised as cut and carry feed,
hay, silage, or dehydrate and is best grown vigorously with high plant densities(83).

H. rosa-sinensis leaves have a high concentration of phenolic compounds and flavonoids,

resulting in high antioxidant activity. This medicinal herb has been utilised to induce
miscarriages and to accelerate placental evacuation following animal births. In the future
research, it is critical to determine when the forage consumption of H. rosa-sinensis causes
hormonal alterations and reproductive harm in fertile females(85,86).

Another study looked at the effects of H. rosa-sinensis leaves on coughing in guinea pigs. In
addition, Ali et al. (89)and Mondal et al.(90) discovered that aqueous and ethanolic extracts of H.
rosa-sinensis leaves have a significant healing effect in laboratory mice(91).

In a study of methanol extracts produced from the flowers of H. rosa-sinensis L. were tested for
antibacterial activity against microorganisms such as E. coli, Streptococcus pyogenes, and
Staphylococcus aureus. H. rosa-sinensis antibacterial capability was demonstrated in all flower
methanol extracts at high concentrations.

Total antioxidant activity of plant extracts cannot be measured using a single method due to the
complex composition of phytochemicals as well as oxidative processes. The goal of this study
was to evaluate the in vitro antioxidant activity of methanolic from H. rosa-sinensis leaves and
flowers utilising a range of testing methods. In recent years, there has been a lot of focus on the
antioxidant properties of plant-derived dietary constituents of food(92).

48
The antioxidant potential of H. rosa-sinensis methanolic extracts of two different altitude was
estimated based on CUPRAC Antioxidant Capacity Assay which involves a redox reduction
between the CUPRAC reagent and antioxidants present in the sample that include a leading thiol
group, such as glutathione.

Reducing power is also commonly employed to assess the antioxidant activity of plant
polyphenols. Reductants, which have an antioxidant effect by breaking free radical chains by
donating a hydrogen atom, are frequently associated with the existence of reducing power. The
iron reduction capacity of H. rosa-sinensis methanolic extracts was calculated in this study based
on their ability to donate an electron to decrease the Fe3+-ferricyanide complex to the ferrous
form(67).

The ability of antioxidants to donate hydrogen is thought to be responsible for their anti-DPPH
actions. Radical scavenging actions are crucial for reducing free radical damage in a variety of
diseases, including cancer. The DPPH free radical scavenging mechanism is a well-established
approach for evaluating the antioxidant capabilities of plant extracts(93). This method has been
widely used to predict antioxidant activity because to the short time required for analysis..

The statistical analysis of this study have shown that the leaves of Gangtok has the highest
antioxidant activity compared to the leaves of Guwahati. And the flowers of Guwahati exhibited
the maximum antioxidant potential in comparison to the Gangtok flowers.

49
Chapter 6:

Conclusion

50
Conclusion

Medicinal plants have been utilised in medicine since the beginning of mankind. Surprisingly,
several medicinal plants have high levels of antioxidants other than vitamin C and vitamin E.
Medicinal plants have a bright future and the majority of them have not yet been studied for their
medicinal properties. Among all other medicinal plants, H. rosa-sinensis also posses high amount
of antioxidant property. The findings of this study support the view, that the methanol extract of
H. rosa-sinensis flower are promising sources of antioxidants value than the leaves and may be
effective as disease prevention agents in some cases. Methanol extract exhibited the best yield
and antioxidant activity at high concentrations, implying that it might be used to isolate diverse
active pharmaceutical value compounds. The current data indicate that methanol is an effective
solvent for extracting antioxidant compounds.

51
Chapter 7:

Reference

52
References

1. Atasoy N, Yücel UM, Atasoy N, Yücel UM. Antioxidants from Plant Sources and Free
Radicals. Reactive Oxygen Species. IntechOpen; 2021 [cited 2022 Dec 1].

2. Engwa GA. Free Radicals and the Role of Plant Phytochemicals as Antioxidants Against
Oxidative Stress-Related Diseases. In: Asao T, Asaduzzaman M, editors. Phytochemicals -
Source of Antioxidants and Role in Disease Prevention. InTech; 2018 [cited 2023 May 11].

3. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and
antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol.
2007;39(1):44–84.

4. Diplock AT, Charleux JL, Crozier-Willi G, Kok FJ, Rice-Evans C, Roberfroid M, et al.
Functional food science and defence against reactive oxidative species. Br J Nutr. 1998
Aug;80 Suppl 1:S77-112.

5. Dogra V, Kim C. Singlet Oxygen Metabolism: From Genesis to Signaling. Front Plant Sci.
2019;10:1640.

6. Free Radicals - Physiopedia. [cited 2023 May 11].

7. Fimognari C. Role of Oxidative RNA Damage in Chronic-Degenerative Diseases. Oxid Med

Cell Longev. 2015;2015:358713.

8. Liu TZ, Stern A, Morrow JD. The isoprostanes: unique bioactive products of lipid
peroxidation. An overview. J Biomed Sci. 1998;5(6):415–20.

9. Free radicals, antioxidants and functional foods: Impact on human health - PMC . [cited 2023
May 11].

10. Atasoy N, Mercan Yücel U. Antioxidants from Plant Sources and Free Radicals. In:
Ahmad R, editor. Biochemistry. IntechOpen; 2022 [cited 2023 May 11].

11. Halliwell, B. and Gutteridge, J.M.C. (1999) Free Radicals in Biology and Medicine. In
Halliwell, B. and Gutteridge, J.M.C., Eds., Free Radicals in Biology and Medicine, 3rd
Edition, Oxford University Press, Oxford, 1-25. - References - Scientific Research Publishing .
[cited 2023 May 11].

12. Papas AM. Antioxidant Status, Diet, Nutrition, and Health. CRC Press; 2019. 491 p.

13. Chaitanya V, Pathan A, Mazumdar SS, Chakravarthi GP, Parine N, Bobbarala V. Role of
oxidative stress in human health: an overview. J Pharm Res. 2010 Jan 1;3:1330–3.

14. Selley M, Close D, Stern S. The effect of increased concentrations of homocysteine on the
concentration of (E)-4-hydroxy-2-nonenal in the plasma and cerebrospinal fluid of patients
with Alzheimer’s disease. Neurobiol Aging. 2002 May 1;23:383–8.

53
15. Baud L, Ardaillou R. Reactive oxygen species: production and role in the kidney. Am J
Physiol. 1986 Nov;251(5 Pt 2):F765-776.

16. Kasote DM, Katyare SS, Hegde MV, Bae H. Significance of Antioxidant Potential of
Plants and its Relevance to Therapeutic Applications. Int J Biol Sci. 2015 Jun 11;11(8):982–
91.

17. Williams SE, Williams YM, VanDerWal J, Isaac JL, Shoo LP, Johnson CN. Ecological
specialization and population size in a biodiversity hotspot: how rare species avoid extinction.
Proc Natl Acad Sci U S A. 2009 Nov 17;106 Suppl 2(Suppl 2):19737–41.

18. Alhaithloul HAS. Impact of Combined Heat and Drought Stress on the Potential Growth
Responses of the Desert Grass Artemisia sieberi alba: Relation to Biochemical and Molecular
Adaptation. Plants Basel Switz. 2019 Oct 15;8(10):416.

19. Habib N, Ali Q, Ali S, Javed MT, Zulqurnain Haider M, Perveen R, et al. Use of Nitric
Oxide and Hydrogen Peroxide for Better Yield of Wheat (Triticum aestivum L.) under Water
Deficit Conditions: Growth, Osmoregulation, and Antioxidative Defense Mechanism. Plants
Basel Switz. 2020 Feb 22;9(2):285.

20. Grabherr G, Gottfried M, Paull H. Climate effects on mountain plants. Nature. 1994 Jun
9;369(6480):448.

21. Moustafa-Farag M, Almoneafy A, Mahmoud A, Elkelish A, Arnao MB, Li L, et al.

Melatonin and Its Protective Role against Biotic Stress Impacts on Plants. Biomolecules. 2019
Dec 28;10(1):54.

22. Population structure and ecological role of Moringa peregrina (Forssk.) Fiori. at its
northwestern range edge in the Hajar Mountains: Plant Biosystems - An International Journal
Dealing with all Aspects of Plant Biology: Vol 151, No 1. [cited 2023 May 11].

23. Elkelish AA, Alhaithloul HAS, Qari SH, Soliman MH, Hasanuzzaman M. Pretreatment
with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato
seedlings by modulating physiological, biochemical, and molecular mechanisms. Environ Exp
Bot. 2020 Mar 1;171:103946.

24. Dong J, Ma X, Wei Q, Peng S, Zhang S. Effects of growing location on the contents of
secondary metabolites in the leaves of four selected superior clones of Eucommia ulmoides.
Ind Crops Prod. 2011 Nov 1;34(3):1607–14.

25. Altitude impact on the chemical profile and biological activities of Satureja thymbra L.
essential oil | BMC Complementary Medicine and Therapies | Full Text. [cited 2023 May 11].

26. Wong S, Lim Y, Chan E. ANTIOXIDANT PROPERTIES OF HIBISCUS: SPECIES

VARIATION, ALTITUDINAL CHANGE, COASTAL INFLUENCE AND FLORAL
COLOUR CHANGE. J Trop For Sci. 2009;

54
27. Jamwal M, Puri S, Radha R, Sharma N, Prakash S, Kumar V, et al. Effect of altitudinal
variations on biological activities of Justicia adhatoda L. growing wildly in Western
Himalayas: An in vitro assessment. J Appl Pharm Sci. 2022 Aug 4;156–64.

28. Nade VS, Kanhere SV, Kawale LA, Yadav AV. Cognitive enhancing and antioxidant
activity of ethyl acetate soluble fraction of the methanol extract of Hibiscus rosa sinensis in
scopolamine-induced amnesia. Indian J Pharmacol. 2011 Apr;43(2):137–42.

29. Frankel S, Berenbaum M. Effects of light regime on antioxidant content of foliage in a

tropical forest community. Biotropica. 1999 Sep;31(3):422–9.

30. Rai N, Neugart S, Schröter D, Lindfors AV, Aphalo PJ. Responses of flavonoids to solar
UV radiation and gradual soil drying in two Medicago truncatula accessions. Photochem
Photobiol Sci. 2023 Mar 30 [cited 2023 May 12];

31. The flavonoid biosynthetic pathway in plants: Function and evolution - Koes - 1994 -
BioEssays - Wiley Online Library. [cited 2023 May 12].

32. Tan BL, Norhaizan ME, Liew WPP, Sulaiman Rahman H. Antioxidant and Oxidative
Stress: A Mutual Interplay in Age-Related Diseases. Front Pharmacol. 2018 Oct 16;9:1162.

33. Di Meo S, Venditti P. Evolution of the Knowledge of Free Radicals and Other Oxidants.
Oxid Med Cell Longev. 2020 Apr 23;2020:9829176.

34. Gomberg M. AN INSTANCE OF TRIVALENT CARBON: TRIPHENYLMETHYL.

ACS Publications. American Chemical Society; 2002 [cited 2023 May 13].

35. Gilbert DL. Perspective on the History of Oxygen and Life. In: Gilbert DL, editor.
Oxygen and Living Processes. New York, NY: Springer; 1981. p. 1–43. (Topics in
Environmental Physiology and Medicine).

36. Aging: A Theory Based on Free Radical and Radiation Chemistry | Journal of
Gerontology | Oxford Academic [Internet]. [cited 2023 May 12]. Available from:
https://academic.oup.com/geronj/article-abstract/11/3/298/616585?redirectedFrom=fulltext

37. The aging process. | PNAS. [cited 2023 May 12].

38. (PDF) Oxidative stress and human health | Taibur Rahman - Academia.edu. [cited 2023
May 12].

39. Yudoh K, Nguyen van T, Nakamura H, Hongo-Masuko K, Kato T, Nishioka K. Potential

involvement of oxidative stress in cartilage senescence and development of osteoarthritis:
oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte
function. Arthritis Res Ther. 2005;7(2):R380-391.

55
40. Hayden MR, Tyagi SC. Neural redox stress and remodeling in metabolic syndrome, type
2 diabetes mellitus, and diabetic neuropathy. Med Sci Monit Int Med J Exp Clin Res. 2004
Dec;10(12):RA291-307.

41. Verzola D, Bertolotto MB, Villaggio B, Ottonello L, Dallegri F, Salvatore F, et al.

Oxidative stress mediates apoptotic changes induced by hyperglycemia in human tubular
kidney cells. J Am Soc Nephrol JASN. 2004 Jan;15 Suppl 1:S85-87.

42. Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M, et al. Health
benefits of herbs and spices: the past, the present, the future. Med J Aust. 2006 Aug
21;185(S4):S1–24.

43. Lai PK, Roy J. Antimicrobial and chemopreventive properties of herbs and spices. Curr
Med Chem. 2004 Jun;11(11):1451–60.

44. (PDF) PHARMACOLOGY OF MEDICINAL PLANTS AND NATURAL PRODUCTS |

Chaitanya Bhatt - Academia.edu. [cited 2023 May 16].

45. Health Benefits of Herbs & Spices. Organic Facts. [cited 2023 May 24].

46. Karou SD, Tchacondo T, Ilboudo DP, Simpore J. Sub-Saharan Rubiaceae: a review of
their traditional uses, phytochemistry and biological activities. Pak J Biol Sci PJBS. 2011 Feb
1;14(3):149–69.

47. Wyk BE van, Oudtshoorn B van, Gericke N. Medicinal plants of South Africa. Med
Plants South Afr . 1997 [cited 2023 May 12];

48. Sofowora A, Ogunbodede E, Onayade A. The Role and Place of Medicinal Plants in the
Strategies for Disease Prevention. Afr J Tradit Complement Altern Med. 2013 Aug
12;10(5):210–29.

49. Hoareau L, DaSilva EJ. Medicinal Plants: A Re-emerging Health Aid. Electron J
Biotechnol. 1999 Aug;2(2):3–4.

50. Pierro FD, Rapacioli G, Ferrara T, Togni S. Use of a standardized extract from Echinacea
angustifolia (Polinacea[R]) for the prevention of respiratory tract infections. Altern Med Rev.
2012 Mar 1;17(1):36–42.

51. Towards the Discovery of Novel Phytochemicals for Disease Prevention from Native
Australian Plants: An Ethnobotanical Approach | Asia Pacific Journal of Clinical Nutrition.
[cited 2023 May 13].

52. Sheiham A, Watt RG. The common risk factor approach: a rational basis for promoting
oral health. Community Dent Oral Epidemiol. 2000 Dec;28(6):399–406.

53. Facts and theories concerning the mechanisms of carcinogenesis - Pitot - 1991 - The
FASEB Journal - Wiley Online Library. [cited 2023 May 13].

56
54. Yasukawa K. Medicinal and Edible Plants as Cancer Preventive Agents. In: Drug
Discovery Research in Pharmacognosy. IntechOpen; 2012 [cited 2023 May 13].

55. Phytosterol Pygeum africanum regulates prostate cancer in vitro and in vivo |
SpringerLink . [cited 2023 May 13].

56. Anticancer biology of Azadirachta indica L (neem): A mini review: Cancer Biology &
Therapy: Vol 12, No 6 . [cited 2023 May 13].

57. Zhou K, Raffoul JJ. Potential Anticancer Properties of Grape Antioxidants. J Oncol.
2012;2012:803294.

58. Senthilkumar A, Karuvantevida N, Rastrelli L, Kurup SS, Cheruth AJ. Traditional Uses,
Pharmacological Efficacy, and Phytochemistry of Moringa peregrina (Forssk.) Fiori. —A
Review. Front Pharmacol. 2018 May 11;9:465.

59. Meng HC, Wang S, Li Y, Kuang YY, Ma CM. Chemical constituents and pharmacologic
actions of Cynomorium plants. Chin J Nat Med. 2013 Jul 1;11(4):321–9.

60. Poorna CA, Resmi MS, Soniya EV, Poorna CA, Resmi MS, Soniya EV. In vitro
Antioxidant Analysis and the DNA Damage Protective Activity of Leaf Extract of the
Excoecaria agallocha Linn Mangrove Plant. In: Agricultural Chemistry [Internet]. IntechOpen;
2013 [cited 2023 May 16]. Available from: https://www.intechopen.com/chapters/43053

61. Khristi V, Patel V. THERAPEUTIC POTENTIAL OF HIBISCUS ROSA SINENSIS: A

REVIEW. Int J Nutr Diet. 2017 Jan 4;4:105–23.

62. Jadhav VM, Thorat RM, Kadam VJ, Sathe NS. Traditional medicinal uses of Hibiscus
rosa-sinensis. J Pharm Res. 2009;2(8):1220–2.

63. Kumari AVAG, Palavesam A, Sunilson JAJ, Anandarajagopal K, Vignesh M, Parkavi J.

Preliminary phytochemical and antiulcer studies of Hibiscus rosa sinensis Linn. root extracts.
Int J Green Pharm IJGP . 2010 [cited 2023 May 13];4(1).

64. Jadhav VM, Thorat RM, Sathe VJ. Traditional medicinal uses of Hibiscus rosa-sinensis. J
Pharm Res. 2009 Jan 1;2.

65. Kirtikar KR. Indian Medicinal Plants: By K.R. Kirtikar, B.D. Basu, and An I.C.S. In 4
volumes. 2nd ed. Edited, revised, enlarged, and mostly rewritten by E. Blatter, J.F. Caius, and
K.S. Mhaskar. Allahabad: Lalit Mohan Basu; 1935.

66. Afshari K, Samavati V, Shahidi SA. Ultrasonic-assisted extraction and in-vitro

antioxidant activity of polysaccharide from Hibiscus leaf. Int J Biol Macromol. 2015 Mar
1;74:558–67.

57
67. Garg D, Shaikh A, Muley A, Marar T. In-vitro antioxidant activity and phytochemical
analysis in extracts of Hibiscus rosa-sinensis stem and leaves. Free Rad Antiox. 2012 Jul
1;2:41.

68. Ghaffar and El-Elaimy - In vitro, antioxidant and scavenging activities of.pdf . [cited
2023 May 24].

69. Nade et al. - 2009 - Effect of Hibiscus rosa sinensis on reserpine-indu.pdf. [cited 2023
May 24].

70. Moqbel FS, Naik PR, Najma HM, Selvaraj S. Antidiabetic properties of Hibiscus rosa
sinensis L. leaf extract fractions on nonobese diabetic (NOD) mouse. Indian J Exp Biol. 2011
Jan;49(1):24–9.

71. Kumar V, Singh P, Chander R, Mahdi F, Singh S, Singh R, et al. Hypolipidemic activity
of Hibiscus rosa sinensis root in rats. Indian J Biochem Biophys. 2009 Dec;46(6):507–10.

72. Uddin B, Hossan T, Paul S, Ahmed T, Nahar T, Ahmed S. Antibacterial activity of the
ethanol extracts of Hibiscus rosa-sinensis leaves and flowers against clinical isolates of
bacteria. Bangladesh J Life Sci 2010 222 65-73. 2010 Dec 1;22:65–73.

73. Mishra N, Tandon VL, Munjal A. Evaluation of Medicinal Properties of Hibiscus rosa
sinensis in Male Swiss Albino Mice. In 2010 [cited 2023 May 24].

74. Arullappan S, Muhamad S, Zakaria Z. Cytotoxic Activity of the Leaf and Stem Extracts
of Hibiscus rosa sinensis (Malvaceae) against Leukaemic Cell Line (K-562). Trop J Pharm
Res. 2013 Oct 29;12(5):743–6.

75. Khandelwal VKM, Balaraman R, Pancza D, Ravingerová T. Hemidesmus indicus and

Hibiscus rosa-sinensis Affect Ischemia Reperfusion Injury in Isolated Rat Hearts. Evid-Based
Complement Altern Med ECAM. 2011;2011:802937.

76. Medicinal Plants & Their Importance as Alternative Medicine | I was a born genius, but
education ruined me . [cited 2023 May 13].

77. Cruz Hernández A, Hernández Sanchez D, Gómez-Vázquez A, Govea-Luciano A, Pinos-

Rodríguez JM, Álvarez González CA, et al. Tannin concentration and degradation rate in vitro
of Morus alba and Hibiscus rosa-sinensis. Acta Univ. 2019 [cited 2023 May 25];29.

78. Cuéllar ND, Arrieta Herrera JM. Evaluating Physiological Responses to Hibiscus rosa-
sinensis L. under Conditions of Direct Planting and Nursery. Rev Corpoica Cienc Tecnol
Agropecu . 2010 [cited 2023 May 25];

79. Cu N, Fn M. Anatomical features of the roots and leaves of Hibiscus rosa sinensis and
Abelmoschus esculenta.

58
80. Mamun A, Islam S, Alam K, Rahman A, Rahman A, Rashid M. Effects of Ethanolic
Extract of Hibiscus rosa-sinensis Leaves on Alloxan-Induced Diabetes with Dyslipidemia in
Rats. Bangladesh Pharm J. 2013 Apr 10;16.

81. K PK, A A, G R, D S, Ch GK, Nl lakshmi S. Gastroprotective effect of flower extracts of

Hibiscus rosa sinensis against acute gastric lesion models in rodents. J Pharmacogn
Phytochem. 2014;3(3):137–45.

82. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Gosavi TP, Badole SL, et al. Effect of
hydroalcoholic extract of Hibiscus rosa sinensis Linn. leaves in experimental colitis in rats.
Asian Pac J Trop Biomed. 2012 May;2(5):337–44.

83. Utilization of Ramon (Brosimum alicastrum Sw.) and Cayena (Hibiscus rosa-sinensis L.)
to fed rabbits | Request PDF. [cited 2023 May 25].

84. Missoum A. An update review on Hibiscus rosa sinensis phytochemistry and medicinal
uses. J Ayurvedic Herb Med. 2018 Aug 31;4:135–46.

85. Garg D, Shaikh A, Muley A, Marar T. In-vitro antioxidant activity and phytochemical
analysis in extracts of Hibiscus rosa-sinensis stem and leaves. Free Rad Antiox. 2012 Jul
1;2:41.

86. Ghaffar FRA, El-Elaimy IA. In vitro, antioxidant and scavenging activities of Hibiscus
rosa sinensis crude extract. J Appl Pharm Sci.

87. Nivsarkar M, Patel M, Padh H, Bapu C, Shrivastava N. Blastocyst implantation failure in

mice due to “nonreceptive endometrium”: endometrial alterations by Hibiscus rosa-sinensis
leaf extract. Contraception. 2005 Mar;71(3):227–30.

88. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Gosavi TP, Badole SL, et al. Effect of
hydroalcoholic extract of Hibiscus rosa sinensis Linn. leaves in experimental colitis in rats.
Asian Pac J Trop Biomed. 2012 May;2(5):337–44.

89. Evaluation of Hibiscus rosa-sinensis leaves extracts as wound healing promoter on rats |
IEEE Conference Publication | IEEE Xplore [Internet]. [cited 2023 May 25].

90. Mondal DS, Ghosh D, Nuni S, Ganapaty S. Evaluation of Antioxidant, Toxicological and
wound healing Properties of Hibiscus rosa-sinensis L. (Malvaceae) ethanolic leaves extract on
different Experimental animal models. Indian J Pharm Educ Res. 2016 Nov 1;50:620–37.

91. (PDF) Preclinical investigation of anti-tussive activity of Hibiscus rosa-sinensis on guinea

pig [Internet]. [cited 2023 May 25].

92. Gülçin I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006 Jan
18;78(8):803–11.

59
93. Rahman MdM, Islam MdB, Biswas M, Khurshid Alam AHM. In vitro antioxidant and
free radical scavenging activity of different parts of Tabebuia pallida growing in Bangladesh.
BMC Res Notes. 2015 Dec;8(1):621.

60
Chapter 8:

Annexure

61
Annexure

62
63
64