PHYTOCHEMICAL INVESTIGATION AND

BIOLOGICAL CHARACTERIZATION OF
ETHANOLIC EXTRACT OF EVOLVULUS
ALSINOIDES
THESIS SUBMITTED TO HINDUSTAN INSTITUTE OF TECHNOLOGY AND
SCIENCE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF THE DEGREE OF

BACHELOR OF TECHNOLOGY

IN

BIOTECHNOLOGY
Submitted by
ANANYA SARKAR
Reg No: 17107002
DEPARTMENT OF CHEMICAL ENGINEERING

PADUR, CHENNAI 603103

MAY 2022

i
 BONAFIDE CERTIFICATE

Certified that this project report “PHYTOCHEMICAL

INVESTIGATION AND BIOLOGICAL CHARACTERIZATION OF
ETHANOLIC EXTRACT OF EVOLVULUS ALSINOIDS” is the
Bonafide work of “ANANYA SARKAR (17107002)” who carried out the
project work under my supervision. Certified further that to the best of my
knowledge the work reported here does not form part of any other project/
research work on the basis of which a degree or award was conferred on an
earlier occasion on this or any other candidate.

HEAD OF THE DEPARTMENT SUPERVISOR

DR. B. VIVEKANANDAN DR. R. ANITHA

Associate Professor & Head, Asst. Professor (S.G),
Department of Chemical Engineering, Department of Chemical Engineering,
Hindustan Institute of Technology Hindustan Institute of Technology
and Science. and Science.

INTERNAL EXAMINER EXTERNAL EXAMINER

Name: Name:
Designation: Designation:

ii
ANANYA SARKAR

17107002

Bachelor of technology

Department of Chemical Engineering

HITS, Padur.

DECLARATION

I hereby declare that the thesis entitled “PHYTOCHEMICAL INVESTIGATION AND

BIOLOGICAL CHARACTERIZATION OF ETHANOLIC EXTRACT OF
Evolvulus alsinoides” submitted by me to Hindustan Institution of Technology & Science,
for the award of the degree of Bachelor of Technology in Chemical Engineering is a record
of Bonafide research work carried out by me under the guidance of Dr. R. Anitha during
the period of 8 semester and has not formed the basis for the award of any degree, diploma,
associateship, fellowship, titles in this or any other University or other similar institution
of higher learning.

Place: Chennai ANANYA SARKAR

Date:

DR. R ANITHA

(Supervisor)

iii
 ACKNOWLEDGEMENT

First and foremost, I would like to thank the Almighty for showering his blessings, wisdom,
physical and mental strength on me to complete the research work.
I sincerely thank Dr. (Mrs.) Elizabeth Verghese, Chancellor, Dr. Anand Jacob Verghese,
Pro Chancellor, Dr. Aby Sam, Director, Mr. Ashok Verghese, Director, for providing me
with an opportunity to do research in this esteemed institution.
I wish to express my sincere thanks to Vice Chancellor, Pro Vice- Chancellor, Registrar
Controller of Examinations, Director (Research) and Dean (Research), Hindustan
Institute of Technology and Science, Chennai, for their guidance and support.
I am also indebted to Dr. B. Vivekanandan, HOD, Department of Chemical Engineering
for his guidance and support which enabled me to complete my project.
I would like to thank my internal guide Dr. R. Anitha for continually guiding and actively
participating in my project, giving valuable suggestion to complete my project.

Last but not least, I deeply indebted to my parents who have been my greatest support
while I worked day and night for the project to make it a success.

ANANYA SARKAR

iv
 TABLE OF CONTENT

SL. NO CONTENT PAGE NO.

ABSTRACT vii
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATION xi
1. INTRODUCTION 1
1.1 Medicinal properties
2. LITERATURE REVIEW 4
3. MATERIAL AND METHOD 9
3.1 Preparation of extract 9
3.2 Qualitative phytochemical analysis 9
3.2.1 Determination of total phenols 9
3.2.2 Determination of total flavonoids 9
3.2.3 Estimation of total tannin content 10
3.3 Qualitative analysis of phytochemicals 10
3.4 In vitro assay 11
3.4.1 DPPH˙ radical scavenging activity 11
3.4.2 Superoxide (O2˙-) radical scavenging activity 12
3.4.3 Phosphomolybdenum reduction assay 12
3.4.4 Ferric (Fe3+) reducing power assay 12
3.5 Antibacterial activity 13
3.5.1 Nutrient broth agar medium 13
3.6 Antifungal activity 13
3.6.1 Potato dextrose agar medium 14
3.7 Agar well diffusion method 14

v
3.8 Thin layer chromatography 15
3.9 Anti-inflammatory activity 15
3.9.1 Membrane stabilization assay 15
3.10 Gas-chromatography -Mass spectrometry (GC-MS) 17
3.11 Statistical Analysis 17
4. RESULTS AND DISCUSSION 18
4.1 Phytochemicals Analysis 18
4.2 Total Phenols And Flavonoids And Tannins 19
4.3 DPPH˙ Radical Scavenging Assay 20
4.4 Superoxide (O2˙-) Radical Scavenging Activity 21
4.5 Phosphomolybdenum Reduction Assay 23
4.6 Ferric (Fe3+) Reducing Power Assay 24
4.7 Antibacterial Activity 25
4.8 Antifungal Activity 27
4.9 Thin Layer Chromatography 28
4.10 Anti-Inflammatory Activity 29
4.11 Protein Denaturation 30
4.12 Gas-Chromatography -Mass Spectrometry (GC-MS) 31
5. CONCLUSION 34
6. REFERENCES 35

vi
 ABSTRACT

Shankhpushpi, Vishnukrantha, Vishnu-kranta, Vishukarandi, Morning-glory, and

slender dwarf morning-glory are all names for Evolvulus alsinoides, a flowering
plant in the Convolvulaceae family. It boosts intelligence, improves memory and
retention, relieves stress, anxiety, depression, and confusion, and promotes restful
sleep. It's an annual/perennial herb with prostrate branches that extend outward in
all directions. The root system is extensive and complex. Leaflets are 2.5-5 cm long,
elliptical to oblong in shape, and rounded at the base. White appressed and long
spreading hairs cover the leaves abundantly. Seedlings appear in nature after 2-3
showers in July and August. The purpose of this study was to determine the
antioxidant, anti-inflammatory, and antibacterial effects of an ethanol extract of
Evolvulus alsinoides, as well as to use GC-MS to identify active compounds. The
antioxidant potential of an ethanol extract of Evolvulus alsinoides was assessed
using a variety of antioxidant tests. The maximum DPPH radical scavenging activity
of ethanol extracts was 53.19±0.56% at 600µg/ml concentration. The IC50 values
of DPPH˙ radical scavenging activity was 557.62 µg/mL concentration. The
maximum superoxide radical scavenging activity of Evolvulus alsinoides extract
was 85.46±0.92% at 120 µg/mL concentration and the IC50 was 48.75 µg/mL
concentration. The maximum Fe3+ reduction and Mo6+ reduction was
75.87±0.48%and 30.82±1.26%at 12 0µg/mL concentration and the RC50 were
194.18µg/mL and 41.32µg/mL concentration respectively. Antibacterial and
antifungal activity was carried out by agar well diffusion method with maximum
zone of inhibition (ZOI) of 18 mm against Staphylococcus aureus and 12 mm against
Candida albicans respectively at a concentration of 1000 µg/ml. Furthermore, the
ethanolic extract exhibited anti-inflammatory activity with maximum haemolytic
inhibition at 1000 µg/mL with 50.94 ± 0.04 % (IC50 98.15 µg/mL). Maximum

vii
Protein denaturation for the same extract was noticed to be 32.22 ± 0.18 % at 20
µg/ml (IC50 31.03 µg/ml), which was time and dose-dependent. Hence, the present
study left enough background for future research on the ethanolic extract of
Evolvulus alsinoides for subsequent identification, purification and isolation of
compounds responsible for these activities.

Keywords: Evolvulus alsinoides, Antibacterial activity, Antioxidant activity, Anti-

inflammatory, DPPH, GC-MS.

viii
 LIST OF TABLES

SL. No TITLE PAGE NO.

1. Qualitative analysis of ethanol extract of Evolvulus 18
alsinoides
2. Quantitative estimations of ethanol extract of Evolvulus 20
alsinoides
3. DPPH˙ radical scavenging activity of ethanol extract of 20
Evolvulus alsinoides
4. Superoxide (O2˙-) radical scavenging assay of ethanol 22
extract of Evolvulus alsinoides
5. Phospho-molybdenum reduction activity of ethanol 23
extract of Evolvulus alsinoides
6. Ferric (Fe3+) reducing power activity of ethanol extract 24
of Evolvulus alsinoides
7. Antimicrobial activity of ethanol extract of Evolvulus 27
alsinoides
8. Antifungal activity of ethanol extract of Evolvulus 28
alsinoides
10. Haemolytic inhibition activity of ethanol extract of 29
Evolvulus alsinoides
11. Protein denaturation activity of ethanol extract of 30
Evolvulus alsinoides
12. GCMS analysis of of ethanol extract of Evolvulus 31
alsinoides

ix
 LIST OF FIGURES

SL. NO TITLE PAGE NO

1.1 Evolvulus alsinoides 2
3.1 DPPH˙ radical scavenging activity of ethanol extract of 21
Evolvulus alsinoides
3.2 Superoxide (O2˙-) radical ethanol extract of Evolvulus 22
alsinoides
3.3 Phosphomolybdenum reducing power activity of ethanol 23
extract of Evolvulus alsinoides
3.4 Ferric (Fe3+) reducing power activity of ethanol extract of 25
Evolvulus alsinoides
3.5 Antimicrobial activity of ethanol extract of Evolvulus 26
alsinoides
3.6 Antifungal activity of ethanol extract Evolvulus alsinoides 28
3.7 Compounds separated by Thin Layer Chromatography 29
3.8 Haemolytic inhibition activity of ethanol extract of 30
Evolvulus alsinoides
3.9 Protein denaturation activity of ethanol extract of Evolvulus 30
alsinoides
3.10 GCMS Analysis 31

x
 LIST OF ABBREVIATION

ABBREVIATION EXPLAINATION

DPPH 1, 1- di-phenyl 2-picrylhydrazyl

EDTA Ethylenediamine tetraacetic acid

STZ Streptozotocin

TLC Thin layer chromatography

GC-MS Gas chromatography–Mass Spectrometry

NBT Nitroblue tetrazolium

xi
 1. INTRODUCTION

After the planet became responsive to the adverse effects and limitations of artificial
medication, the standard system of treatment is gaining favor. Plants are used as therapeutic
agents in each un-ionized (Unani, Ayurveda) and unstructured forms since times of yore.
Shankhupushpi (Evolvulus alsinoides Linn.) is one such powerful herb that has been
employed by physicians from times of yore. In Unani literature, it's considered a memory
attention and has been used as a rejuvenator, anti-aging, mental stimulant, and ataractic
drug. The Indian assemblage accepts all components of Evolvulus alsinoides for
therapeutic functions [1]. The potential of Ayurveda herbs profit healthfully and it looks
doubtless exploring that medicinal potential is increasing recently. Plant-based medicine is
gaining a lot of interest these days, thanks to the emergence of fresh ways to chemical
characterization and medical inquisitions. For a long time, healthy plants have been prized
for their ability to cure and relieve pain. Plants that are beneficial to one's health are
frequently used for their healing capabilities. Herbs of many types are utilised in
remarkable folk medicines, and they have a long history of being beneficial in traditional
cures. In vitro screening methods also add to the necessity for very important primary
inquisition, which is necessary to obtain desirable plant extracts with promising and useful
properties for future chemical and medical analysis. [2].

Evolvulus alsinoides is a flat perennial herb with a little branching wood rootstock.
They square measure they're capsulate with plentiful branches that are annual and on the
far side thirty cm long. The branches embrace long hairs and often prostrate. Evolvulus
alsinoides (Figure 1.1) incorporates tiny leaves that square measure elliptic in form. They're
acute, sensitive, and thickly bushy, and every component of this plant is used in Ayurvedic
medicines to treat cough, cold, and fever. They're also utilised to treat inflammation-related
neurodegenerative disorders. The use of this plant in the treatment of Cupid's itch is also
recommended [3]. Azoospermia, inotropic, and anti-inflammatory properties have also
been discovered. This plant not only has these effects, but it also has anti-hemorrhagic and

1
anti-oxidant properties. This herb was used in ancient medicines as a brain tonic, and it has
been proven in preclinical studies in recent years. This plant is most commonly used to
treat bronchial asthma and memory loss, two of the most common neurological illnesses.
The most remarkable attribute of E. alsinoides is that it enhances memory and
intellectuality.

Taxonomy

Kingdom: Plantae

Phylum: Tracheophytes

Class: Angiospermae

Category: Eudicots

Order: Solanales

Family: Convolvulaceae

Genus: Evolvulus

Species: Evolvulus alsinoides

Figure 1.1: Evolvulus alsinoides

2
1.1 Medicinal properties

The bitter herb is used as an alternative, anthelminthic, anti-diarrheal, bitter,

febrifuge, tonic, and vermifuge in its whole. It is used as an infusion to treat digestive
disorders for which it is considered to be a powerful remedy - particularly dysentery. It is
used to treat fevers, nervous debility, memory loss, as well as syphilis, scrofula, and other
diseases when combined with cumin and milk. A decoction is used to treat gonorrhea [4].
The plant's infusion is used to cure syphilis, scrofula, and snake bites. Hair development is
aided by the application of an oil infusion. To heal wounds, powdered leaves are used
topically. The crushed leaves are placed to swollen glands in the neck as a poultice. The
leaves are used to make cigarettes that are smoked to treat bronchitis and asthma. Flavonols
and saponins are said to be present in the plant [5] and ergot alkaloids are amides of the
indole derivative D-lysergic acid, which is biosynthetically produced from the amino acid
tryptophan, which accumulate in the plant's cultured tissues.

Although the sclerotium of the fungus Claviceps purpurea or related fungi is the
most well-known source of ergot alkaloids, many lysergic acid alkaloids have also been
discovered from members of the Convolvulaceae family. The whole plant's ethanol extract
has anti-ulcer and anti-catatonic properties. The perfume houses employ the fragrant smoke
from burning leaves. Alternaria brassicae, Alternaria brassicicola, and Fusarium
oxysporum spore germination and mycelial growth were prevented by a water extract of
the corolla.

3
 2. LITERATURE REVIEW

Sethiya et al., (2009) characterized that the Shankhpushpi is an Ayurvedic medication

known for its effects on the central nervous system, particularly in terms of memory and
intelligence [6]. Ayurvedic and other Sanskrit literature revealed the existence of four
separate plant species known as Shankhpushpi, which are employed in a variety of
Ayurvedic prescriptions documented in ancient books, either alone or in conjunction with
other herbs. Convulvulus pluricaulis Choisy. (Convulvulaceae), Evolvulus alsinoides Linn.
(Convulvulaceae), Clitoria ternatea Linn. (Papilionaceae), and Canscora decussata Schult
are among the botanicals found in the sources (Gentianaceae). A review of the available
scientific information in terms of pharmacological characteristics, chemical constituents,
and pharmacological activities, as well as preclinical and clinical applications of
controversial Shankhpushpi sources has been prepared with the goal of reviewing scientific
work on Shankhpushpi. It could provide differentiation parameters and allow for a better
understanding of medication action variations through the utilization of different botanical
sources.

Mehla et al., (2020) demonstrated that cognitive impairment is a serious health issue
that is linked to ageing, stress, hypertension, and different neurodegenerative conditions
such as Parkinson's disease and epilepsy [7]. The subject of this review is Alzheimer's
disease (AD), which is the leading cause of cognitive impairment. Progressive memory
loss, linguistic deficiencies, depression, agitation, mood problems, and psychosis are all
symptoms. Although cholinergic dysfunction, -amyloid plaques, and neurofibrillary tangle
formation are hallmarks of Alzheimer's disease, it is also linked to dys-regulation of other
neurotransmitters, elevated levels of advanced glycation end products, oxidative damage,
neuro-inflammation, genetic, and environmental factors.

On one hand, this complex etio-pathology makes a response to commonly used

drugs such as donepezil, rivastigmine, galantamine and memantine less predictable and
often unsatisfactory. On the other hand, it supports the use of herbal medicines due to their
nonspecific antioxidant and anti-inflammatory activity and specific cholinesterase
4
inhibitory activity. The popularity of herbal medicines is also increasing due to their
perceived effectiveness, safety and affordability. In the present article, the experimental
and clinical evidence have been reviewed for various Indian herbal medicines such
as Centella asiatica, Bacopa monnieri, Curcuma longa, Clitoria ternatea, Withania
somnifera, Celastrus paniculatus, Evolvulus alsinoides, Desmodium gangeticum, Eclipta
alba, Moringa oleifera and Convolvulus pluricaulis, which have shown potential in
cognitive impairment. Some commonly available herbal formulations for memory
impairment in India have also been reviewed.

Gomathi et al., (2014) studied the accumulation of free radicals in the body causes
a variety of oxidative stress-related illnesses [8]. Endogenous systems, exposure to diverse
physiochemical situations, and disease states all produce free radicals. For proper
physiological function, there must be a balance between free radicals and antioxidants.
Many studies are being conducted around the world in order to discover natural
antioxidants derived from plants. The primary ingredients of medicinal plants are studied
using FTIR spectroscopy, which is a fast and effective analytical approach. The therapeutic
qualities of the chemical compounds of the plants were identified and monitored. The
purpose of this study was to determine the antioxidant activity of an ethanolic extract of
Evolvulus alsinoides in vitro, as well as to conduct an FTIR spectroscopic analysis.

Nahata et al., (2010) characterized that the whole plant of 'Shankhpushpi' has been
used professionally for ages in the Ayurvedic school of medicine for its memory
potentiating, anxiolytic, and tranquillizing characteristics [9]. The purpose of this study
was to see how Evolvulus alsinoides (EA), also known as Shankhpushpi, affected learning
and memory in mice. Nootropic activities, such as Cook and Weidley's pole climbing
apparatus, passive avoidance paradigms, and active avoidance tests, were employed to
assess learning and memory. The memory-improving properties of EA's ethanol extract, as
well as its ethyl acetate and aqueous fractions, were studied. Separate groups of rats
received two doses of the ethanol extract, ethyl acetate, and aqueous fractions (100 and 200
mg/kg p.o.). In rats, both dosages of all EA extracts increased learning and memory

5
significantly. Furthermore, the amnesia caused by scopolamine (0.3 mg/kg i.p.) was
significantly reversed at these levels. In the step-down and shuttle-box avoidance
paradigms, nootropic activity was compared using piracetam as the reference, and both
showed substantial memory boosting effects.

Austin et al., (2008) observed that the people in the Indian subcontinent call
Evolvulus alsinoides shankhapushpi and vishnukranti, two Sanskrit-based common names
[10]. These are pre-European names for a medicinal American species that has been
brought into the area. The exact date of introduction is unknown, however it most likely
occurred in the 1500s or 1600s. The alien plant Evolvulus alsinoides was adopted, given
an ancient Indian name, and introduced into some Old World pharmacopoeias, according
to an examination of its relationships, geographic distribution, names in Asia, medical uses,
and chemical and laboratory analysis. The herb was reportedly used in remedies not just
because it reminded people of specific elements of their gods and goddesses, but also
because the chemicals it contained were beneficial against certain ailments.

Ambika et al., (2017) explained that the plant-derived compounds are becoming
increasingly important in the treatment of a variety of diseases. Many Convolvulaceae
plants include chemicals that have been shown to have wound-healing and anti-diabetic
properties [11]. Such chemicals can be utilised as part of diabetic wound healing
treatments, as well as in combination with antimicrobial therapy to lower the risk of drug
resistance and allergic responses. Nanoparticles and adhesive patches, for example, are
new ways for producing herbal formulations that can increase the delivery of plant-based
medicinal ingredients.

Recent Advances: Merremia tridentata, Argyreia speciosa, and Ipomoea batatas have
anti-diabetic and wound-healing properties, whereas Evolvulus alsinoides, Evolvulus
nummularius, Argyreia cuneata, and Ipomoea carnea have wound-healing properties.

6
Critical Issues: Drug resistance is a serious issue with antimicrobial therapy, and it can
have an impact on wound healing. Phyto-constituents can help with healing and lessen the
need for antibiotics.

Future Directions: Plants of the Convolvulaceae family have long been used for
antimicrobial, anti-diabetic, and wound-healing purposes, and all of the plants chosen for
this study had antimicrobial, anti-diabetic, and wound-healing qualities. Detailed
phytochemical research on these plants could aid in the development of new wound-
healing treatments.

Mehla et al., (2012) presented Evolvulus alsinoides, also known as Shankpushapi,

is a commonly used traditional medicine for enhancing memory [12]. They evaluated the
in vitro free radical scavenging and enzymes (acetyl-cholinesterase, butyrylcholinestrase,
glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase
(PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)) inhibitory
activities of aqueous and hydro-alcoholic extracts of E. alsinoides [13]. Hydro-alcoholic
extract of E. alsinoides demonstrated more free radical scavenging activity as compared to
aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β,
ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous
extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as
compared to aqueous extract but no significant difference in phenolic content of the two
extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and
500mg/kg, p.o. was selected for in vivo study in intra-cerebro ventricularly injected
streptozotocin (STZ) induced cognitive impairment in male Wistar rats.

Elevated plus maze, passive avoidance and Morris water maze were used for
assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative
stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and
superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II)
expression were studied in cerebral cortex and hippocampus of rat brain at the end of the
study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced

7
cognitive impairment by reducing the oxidative stress, improving cholinergic function and
preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer
potential of hydro-alcoholic extract of E. alsinoides [14].

Gomathi et al., (2012) studied that many modern medications are based on plants
and plant-based products, and they are currently in use for a variety of disorders [15]. The
goal of this study was to look into the biological contents and finger printing of the
ethanolic extract of Evolvulus alsinoides using high performance thin layer
chromatography (HPTLC). Standard techniques were used to screen for phytochemicals,
and an HPTLC method was developed to assess alkaloids, flavonoids, and phenolic
compounds in the ethanolic extract of Evolvulus alsinoides. Ethanol removed more
secondary metabolites than other solvents, according to preliminary phytochemical
screening. In the ethanolic extract, HPTLC fingerprinting revealed the presence of different
alkaloids, flavonoids, and phenols (quercetin). Due to the presence of phenolic compounds,
Evolvulus alsinoides may serve as a source of powerful antioxidants that may be used in
the prevention of many diseases such as cancer, diabetes, and cardiovascular disorders.

In the pharmaceutical business and plant systematic investigations, the HPTLC

finger print of Evolvulus alsinoides may be beneficial in distinguishing the species from
adulterants and acting as a biochemical identifier for this medicinally important plant.

8
 3. MATERIALS AND METHODS

3.1 PREPARATION OF EXTRACT

Evolvulus alsinoides was found in a commercial market in Chennai, Tamil Nadu,

India. About 15g of Evolvulus alsinoides was weighed and soaked in 250 mL of ethanol
for 48 hours.

3.2 QUALITATIVE PHYTOCHEMICAL ANALYSIS

Preliminary phytochemical screening was performed on the extract of Evolvulus

alsinoides using particular reagents, as per conventional procedures.

3.2.1 Determination of total phenols

The total phenolic compounds were determined using the Folin-Ciocalteau reagent
technique with minor modifications. One hundred µL of ethanol extract (1mg/mL) of
Evolvulus alsinoides was mixed with 900 µL of 1 mL Folin-Ciocalteau reagent and
methanol (1:10 diluted with distilled water). After 5 minutes, 1 mL of 20% Na2CO3
solution was added. The mixture was then incubated for 30 minutes at room temperature
in the dark. At 765 nm, the absorbance was measured using a UV-VIS spectrophotometer.
Gallic acid equivalent (µg/mg of extract), a frequently used standard, was utilised to
quantify total phenolic content.

3.2.2 Determination of total flavonoids

The total flavonoid content of Evolvulus alsinoides ethanol extract was measured
using a slightly modified aluminium chloride colorimetric method as described by Liu et
al [16]. 500µL litres of methanol were combined with 500µL of extract (1 mg/mL). Then,
1 mL of 5% sodium nitrite solution and 1 mL of 10% aluminium chloride solution were
added and shaken well. Finally, 100 µL of 1 M NaOH solution was added. It was incubated
at room temperature for 30 minutes. The absorbance was measured at 510 nm. The result
was represented as quercetin equivalent (µg/mg of extract).

9
3.2.3 Estimation of total tannin content

`Folin - Ciocalteu was used to determine the tannins. About 0.1 mL of the sample
extract was added to 900 µL of distilled water and 1 mL of Folin - Ciocalteu phenol reagent,
1 mL of 35% sodium carbonate solution was added and diluted to 10 mL with distilled
water. A series of tannic acid reference standard solutions (20, 40, 60, 80, 100 g/ ml) were
made in the same way as stated earlier and stored at room temperature for 30 minutes. A UV/
Visible spectrophotometer was used to determine the absorbance of test and standard solutions at
700 nm. The tannin concentration was measured in mg of tannic acid equivalents per mg of extract
(µg/mg) [17].

3.3 QUALITATIVE ANALYSIS OF PHYTOCHEMICALS

Standard procedures were used to screen the extracts for the presence of several
phytochemicals (Raaman, 2006) [18].

Alkaloids: The extract (50 mg) was mixed with 1 mL dilute Hcl. Mayer's reagent was
applied to this filtrate drop by drop, and the presence of creamy white precipitate was
detected.

Glycosides: 3 mL chloroform was added to 2 mL extract and mixed. After that, the
chloroform layer was separated, a 10% ammonia solution was added, and the pink colour
development was noticed.

Saponins: The extract (50 mg) was mixed with 10 mL distilled water, vigorously shaken
for 5 minutes, and foam formation was observed.

Phenols: The extract (50 mg) was combined with 5 mL distilled water and 0.5 mL ferric
chloride solution (5%) to make a solution. The presence of phenols was indicated by a dark
green colour.

10
Flavonoids: 2 mL sodium hydroxide solution was added to 5 mL extract, and the presence
of yellow colour was noted. After adding strong sulfuric acid drop by drop, the yellow
colour vanished.

Terpenoids: 2 mL chloroform was added to a 5 mL extract. When strong sulphuric acid

was added, a reddish-brown colour layer appeared.

Steroids: 1 mL chloroform was mixed with 2 mL extract. The blue green colour was
achieved by adding 0.5 mL acetic anhydride and 1 mL concentrated sulphuric acid.

Tannins: A few drops of 1% gelatine solution and 10% NaCl solution were added to a 2
mL extract. The presence of tannins was detected by the precipitation of gelatin.

Carbohydrate: 2 drops of alpha-naphthol are added to 2ml of extract and shaken. After
that, a few drops of concentrated H2SO4 are added. Formation of violet rings.

Quinones: Add con. H2SO4 to 2 mL of extract. Formation of brown colour

3.4 IN VITRO ANTIOXIDANT ASSAYS

3.4.1 DPPH˙ radical scavenging activity

The antioxidant activity of an ethanol extract of E. alsinoides was determined using

the scavenging activity of the stable 1, 1- di-phenyl 2-picrylhydrazyl (DPPH) free radical.
1 mL of 0.1 mM DPPH solution in methanol was mixed with 1 mL of varied quantities of
plant extracts (100-600 µg/mL). The mixture was then incubated in the dark for 30 minutes.
The reference standard for this study was ascorbic acid [19]. As a control, 1 mL methanol
and 1 mL DPPH solution were utilized. The decrease in absorbance was measured using
at 517 nm. The percentage of inhibition was calculated using the following formula:

11
3.4.2 Superoxide radical scavenging activity

The riboflavin-EDTA-NBT technique was used to test superoxide radical

scavenging activity. Different amounts of ethanol extract of E. alsinoides, 50 mM
phosphate buffer (pH 7.6), 1.5 mM riboflavin, 12 mM EDTA, and 50 mM NBT were added
in that order to the reaction mixture. The reaction was started by illuminating the reaction
mixture for 90 seconds [20]. The absorbance was measured at 590 nm immediately after
illumination. As a positive control, ascorbic acid was employed.

% of superoxide radical inhibition = Control – Sample ×100

Control

3.4.3 Phosphomolybdenum reduction assay

The antioxidant capacity of E. alsinoides ethanol extract was determined as

described by Prieto et al. At concentrations ranging from 50 to 300 µg/mL, the extract was
combined with a reagent solution containing ammonium molybdate (4 mM), sodium
phosphate (28 mM), and sulphuric acid (600 mM) for 90 minutes, the reaction mixture was
incubated in a water bath at 90°C. The colored complex's absorbance was measured at 695
nm. Ascorbic acid was used as the standard reference. The percentage of inhibition was
calculated using the following formula:

% of Phosphomolybdenum radical inhibition = Sample– Control ×100

Sample

3.4.4 Ferric (Fe 3+) reducing power assay

The reducing power of E. alsinoides ethanol extract was determined using a slightly
modified version of Yen and Chen's technique. 1 mL plant extract at various concentrations
(50-300 µg/mL) was combined with 1 mL phosphate buffer (0.2 M, pH 6.6) and 1 mL
potassium ferricyanide [K3Fe (CN) 6] at 1% (w/v). The mixtures were then incubated for
20 minutes at 50°C. Each combination received one mL of 10% (w/v) tri-chloro acetic
acid. The absorbance was measured at 700 nm using a Spectrophotometer after adding 1

12
mL of 0.1 percent (w/v) FeCl3 to the 1 mL mixture [21]. The standard reference was
ascorbic acid. The percentage of inhibition was calculated using the following formula:

% of Fe3+ radical inhibition = Sample – Control ×100

Sample

3.5 ANTIBACTERIAL ACTIVITY

Microbial strains
Gram-positive bacteria like Bacillus subtilis, Micrococcus luteus, and
Staphylococcus aureus, as well as Gram-negative bacteria like Escherichia coli and
Pseudomonas aeruginosas, were utilized to test antibacterial activities.
Reference and control

The standard reference was decided to be tetracycline. The controls were made up
of solidified agar that had been soaked in solvent and in which the test chemicals were
soluble.

Aseptic conditions

The aseptic chamber (1.3m x 1.6m x 0.6m), which is a wooden box with a door, was
cleaned with 70% ethanol and irradiated with short wave UV light (from lamp).
3.5.1 Nutrient broth agar medium
The nutrient broth agar medium was made according to normal procedures (peptone
5 g, yeast 3 g, NaCl 5 g, distilled water 1000 mL, agar 20 g) and suspended in 125 mL
distilled water in a 250 mL conical flask, swirled, boiled to dissolve, and then autoclaved
at 15 pounds and 121°C for 15 minutes. In the aseptic laminar chamber, the heated medium
was put into sterile Petri plates. For 15 minutes, the medium was allowed to solidify.
3.6 ANTIFUNGAL ACTIVITY
Candida albicans, Candida tropicalis and Candida krusei were utilised to test antifungal
activities.

13
Reference and control

The standard reference was decided to be fluconazole. The controls were made up
of solidified agar that had been soaked in solvent and in which the test chemicals were
soluble.

3.6.1 Potato dextrose agar medium

The potato dextrose agar medium was made according to normal procedures (potato
200 g, dextrose 20 g, Ph 6.5, distilled water 1000 mL, agar 20 g) then suspended in 150
mL distilled water in a 250 mL conical flask, swirled, boiled to dissolve, and then
autoclaved at 15 pounds and 121°C for 15 minutes. In the aseptic Laminar chamber, the
heated medium was put into sterile Petri plates. For 15 minutes, the medium was allowed
to solidify.
3.7 AGAR WELL DIFFUSION METHOD
The agar well diffusion method was used to test the antibacterial activity of an
ethanol extract of E. alsinoides. The inoculum was dispensed using sterilised cotton swabs
that were previously submerged in the inoculum containing test tube and dispersed equally
across the solidified nutrient agar medium in the petri plates. A sterile well-borer with an
8 mm diameter was used to make five wells in each plate. The root tuber extract was then
poured into each well with concentrations of 250, 500, and 1000 µg/mL. The antibacterial
activity of all the plates with extract filled wells was measured by measuring the diameter
of the inhibition zone generated around the well after 24 hours of incubation at 37 ̊C.
Tetracycline (25 µg) was used as positive control [22].
The antifungal activity of E.alsinoides ethanol extract was tested using the agar well
diffusion method. The inoculum was dispensed using sterile cotton swabs that had been
submerged in the inoculum containing test tube and dispersed equally across the solidified
nutrient agar medium in the petri plates. A sterile well-borer with an 8 mm diameter was
used to make five wells in each plate. The root tuber extract was then poured into each well
containing 250, 500 and 1000 μg/mL concentrations. The antibacterial activity of all the
plates with extract filled wells was measured by measuring the diameter of the inhibition

14
zone generated around the well after 24 hours of incubation at 37ºC.Fluconazole (25 µg)
was used as positive control.
3.8 THIN LAYER CHROMATOGRAPHY

Merck TLC aluminium sheets, silica gel 60 F254 (20 x 20 cm), preloaded plates
were used to chromatograph the ethanol extract of Evolvulus alsinoides. The extract was
spotted about 0.3 mm above the TLC plate's bottom. The chromatogram was created using
a solvent system that was adequate for the job [23]. UV light at 254 nm was used to see the
dots. The coloured spots' Rf values were recorded. Rf values were determined and the ratio
in which separate bands emerged was optimized.

Calculation of Rf value:
Rf value = Distance travelled by the solute
Distance travelled by the solvent

3.9 ANTI-INFLAMMATORY ACTIVITY

3.9.1 Membrane stabilization assay

Preparation of Red Blood cells (RBCs) suspension

Blood was drawn from a healthy human volunteer who had not taken any NSAIDs (Non-
Steroidal Anti-inflammatory Drugs) for two weeks prior to the experiment and transferred
to centrifuge tubes (contained anticoagulant EDTA)[24]. The blood was cleaned three
times in a 10 mM sodium phosphate buffer containing an isotonic buffer solution (154 mM
NaCl) (pH 7.4). Each time, the blood was centrifuged for 10 minutes at 3000 rpm.

Heat induced haemolysis

The reaction combination (2mL) comprised of 1 mL test sample at various

concentrations (100–500 µg/mL) and 1 mL 10 percent RBC suspension; the control test
tube contained simply saline instead of test sample. For 30 minutes, all centrifuge tubes
holding reaction mixture were placed in a water bath at 56 ̊C. The tubes were cooled under

15
running tap water at the end of the incubation [25]. The reaction mixture was centrifuged
for 5 minutes at 2500 rpm, and the supernatants' absorbance was measured at 560 nm.
Aspirin was used as a standard drug.

The Percentage inhibition of Haemolysis was calculated as follows:

Percentage of inhibition = (Abs control –Abs sample) / Abs control X 100

3.10 GAS CHROMATOGRAPHY–MASS SPECTROMETRY (GC–MS)

For GC-MS analysis, the samples were injected into a HP-5 column (30 m X 0.25
mm i.d with 0.25 μm film thickness), Agilent technologies 6890 N JEOL GC Mate II GC-
MS model. Following chromatographic conditions were used: Helium as carrier gas, flow
rate of 1 mL/min; and the injector was operated at 200°C and column oven temperature
was programmed as 50-250°C at a rate of 10°C/min injection mode. Following MS
conditions were used: ionization voltage of 70 eV; ion source temperature of 250°C;
interface temperature of 250°C; mass range of 50-600 mass units[26].

Identification of components

For the interpretation of the mass spectrum of the GC-MS, the National Institute of
Standards and Technology (NIST) database with over 62,000 patterns was employed [27].
The unknown component's mass spectrum was compared to the spectrum of known
components contained in the NIST library.

16
3.11 STATISTICAL ANALYSIS

The data were expressed as mean ± standard deviation of three parallel readings.
The IC50 value was calculated using linear regression analysis.

17
 4. RESULT AND DISCUSSION

4.1 PHYTOCHEMICAL ANALYSIS

The phytochemical analysis of rhizome ethanol extract of Evolvulus alsinoides

showed the presence of terpenoids, flavonoids, phenolic compounds, glycosides and
saponins, alkaloids, quinones, carbohydrate, tannins and steroids.
Table 1: Qualitative analysis of ethanol extract of Evolvulus alsinoides
S. No Phytochemicals Tests Results
1 Alkaloids 1.Dragendorff’s Test +
2.Hager’s Test
2. Terpenoids CHCl3 + conc. H2SO4 +
3. Flavonoids NaOH solution +
4. Phenols FeCl3 solution +
5. Glycosides killer -killani test +
6. Saponins Foam test +
7. Quinones Conc. H2SO4 +
8. Carbohydrate Molische test +

9. Tannins Lead Acetate test +

10. Steroids Libermann +

Bunchard’s test

18
4.2 TOTAL PHENOLS AND FLAVONOIDS AND TANNINS

Oxidative stress is considered to be substantial, if not crucial, in the initiation and

development of many diseases, including: inflammation, autoimmune diseases, cataract,
cancer, Parkinson’s disease, arteriosclerosis and aging. Oxidative stress plays a role in heart
diseases, neurodegenerative diseases, cancer and in the aging process [28]. Phenolic
compounds are physiologically active against herbivores or pathogens, used as
insecticides, fungicides or pharmaceuticals and is supported by increasing evidence
suggesting that oxidative damage plays a role in the development of chronic, age-related
degenerative diseases, and that dietary antioxidants lower the risk of disease. Flavonoids
can prevent injury caused by free radicals in various ways and one way is the direct
scavenging of free radicals. Flavonoids are oxidised by radicals, resulting in a more stable,
less-reactive radical. In other words, flavonoids stabilize the reactive oxygen species by
reacting with the reactive compound of the radical.

Because of the high reactivity of the hydroxyl group of the flavonoids, radicals are
made inactive. Antioxidants are specific compounds that protect human, animal and plant
cells against the damaging effects of free radicals (reactive oxygen species, ROS). An
imbalance between antioxidants and free radicals results in oxidative stress, may lead to
cellular damage. At present, most antioxidants are manufactured synthetically, belonging
to the class of synthetic antioxidants. The main disadvantage of synthetic antioxidants is
the side effects when consumed in vivo (Chen et al., 1992) [29]. The phenol and flavonoid
compounds quantified in the rhizome ethanol extract of Evolvulus alsinoides seemed to be
responsible for the antioxidant activity. The total phenol content was 0.875±1.14 µg/mg of
GAE and the total flavonoid content was 451.92±0.33 µg/mg of QE in the extract. These
results provide a comprehensive profile of the antioxidant activity of ethanol extract of
Evolvulus alsinoides with respect to their phenols and flavonoids content.

19
 Table 2: Quantitative estimations of ethanol extract of Evolvulus alsinoides

S. No Phytochemicals Amount
(µg/mg)
1. Phenols 0.875
2. Flavonoids 451.92
3. Tannins 0.564

4.3 DPPH˙ RADICAL SCAVENGING ASSAY

One of the most used antioxidant assays is DPPH free radical scavenging. The
DPPH radical scavenging assay is a de-colorization assay that measures antioxidants ability
to directly scavenge DPPH radicals by detecting their absorbance with a spectrophotometer
at 517 nm. Using the 1, 1-diphenyl-2-picrylhydrazyl radical, the ability of an ethanol
extract of Evolvulus alsinoides to scavenge free radicals generated was tested (DPPH) [30].
At 600 g/mL concentration, the highest DPPH radical scavenging activity was 53.19±0.56.
Ethanol extract of Evolvulus alsinoides reducing the stable DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical to the yellow colored 1,1-diphenyl-2-picrylhydrazine, the extract
revealed a high capacity for scavenging free radicals, and the reducing capacity improved
with increasing concentration of the extract. The IC50 was found to be 557.62 μg/mL
concentration and was compared with standard (Ascorbic acid, IC50 = 11.98 μg/mL
concentration).
Table 3: DPPH˙ radical scavenging activity of ethanol extract of Evolvulus alsinoides

S. No. Concentration % inhibition of ethanol

(µg/ml) extract
1 Control 0.00
2 100 0.84±0.48
3 200 14.3±0.41
4 300 21.56±0.21

20
 5 400 31.16±0.33
6 500 44.34±0.38
7 600 53.19±0.56

60

50 DPPH…
% of Inhibition

40

30

20

10

0
100 200 300 400 500 600
Concentration(µg/mL)

Figure 3.1: DPPH˙ radical scavenging activity of ethanol extract of Evolvulus

alsinoides

4.4 SUPEROXIDE (O2˙-) RADICAL SCAVENGING ACTIVITY ETHANOL

EXTRACT OF EVOLVULUS ALSINOIDES
Superoxide anion is also very harmful to cellular components and their effects can
be magnified because it produces other kinds of free radicals and oxidizing agents.
Flavonoids are effective antioxidants, mainly because they scavenge superoxide anions.
Superoxide anions derived from dissolved oxygen by the riboflavin-light-NBT system will
reduce NBT in this system. In this method, superoxide anion reduces the yellow dye
(NBT2+) to blue formazan, which is measured at 590 nm in UV-Vis spectrophotometer
[31]. Antioxidants are able to inhibit the blue NBT formation and the decrease of
absorbance with antioxidants indicates the consumption of superoxide anion in the reaction
mixture. The maximum superoxide radical scavenging activity of Evolvulus alsinoides

21
was 85.46±0.92 at 120 µg/mL concentration and the IC50 was 48.75 µg/mL concentration.
It was compared with the standard of ascorbic acid (IC50 = 9.65 μg/mL concentration).

Table 4: Superoxide (O2˙-) radical scavenging assay of ethanol extract of Evolvulus

alsinoides

S. No. Concentration % inhibition of ethanol

(µg/ml) extract
1 Control 0.00
2 20 11.95±1.60
3 40 12.82±0.82
4 60 61.53±0.52
5 80 61.24±1.54
6 100 71.79±0.61
7 120 85.46±0.92

90
80 Superoxide Radicle
70
% Of Inhibtion

60
50
40
30
20
10
0
20 40 60 80 100 120
Concentration (µg/mL)

Figure 3.2: Superoxide (O2˙-) radical ethanol extract of Evolvulus alsinoides

22
4.5 PHOSPHO-MOLYBDENUM REDUCTION ACTIVITY ETHANOL
EXTRACT OF EVOLVULUS ALSINOIDES

The total antioxidant activity of was measured by phospho-molybdenum reduction

method which is based on the reduction of Mo (VI) to Mo(V) by the formation of green
phosphate/Mo (V) complex at acidic pH, with a maximum absorption at 695 nm. The
maximum phospho-molybdenum reduction was 58.10±0.95 % at 120 µg/mL concentration
and the RC50 was 46.97 µg/mL concentration. It was compared with the standard ascorbic
acid (RC50 = 6.34 μg/mL concentration). PM assay is a quantitative method to investigate
the reduction reaction rate among antioxidant, oxidant and molybdenum ligand. It involves
in thermally generating auto-oxidation during prolonged incubation period at higher
temperature.

Table 5: Phospho-molybdenum reduction activity of ethanol extract of Evolvulus

alsinoides

S. No. Concentration % inhibition of

(µg/ml) ethanol extract
1 Control 0.00
2 20 36.72±1.66
3 40 42.58±1.45
4 60 58.10±0.95
5 80 67.53±0.35
6 100 71.42±0.58
7 120 75.87±0.48

23
 80
70
% of Reduction
60 Phosphomolybdenum
Reduction
50
40
30
20
10
0
20 40 60 80 100 120
Concentration (µg/mL)

Figure 3.3: Phospho-molybdenum reducing power activity of ethanol extract of

Evolvulus alsinoides

4.6. FERRIC (FE3+) REDUCING POWER ACTIVITY OF ETHANOL

EXTRACT OF EVOLVULUS ALSINOIDES

The reducing power assay was carried out by the reduction of Fe3+ to Fe2+ by the
ethanol extract of Evolvulus alsinoides and the subsequent formation of ferro-ferric
complex. The reduction ability increases with increase in concentration of the extract. The
maximum Fe3+ reduction was 30.82±1.26% at 60 µg/mL concentration and the RC50 was
194.18µg/mL concentration. It was compared with the standard ascorbic acid (RC50 = 7.72
μg/mL concentration). Also in this assay, higher absorbance of the reaction mixture
indicates higher reduction potential. The reducing capacity of ethanol extract poses as a
significant indicator of its potential antioxidant activity. The reducing capacity of the
extract was performed using Fe3+ to Fe2+ reduction assay as the yellow colour changes to
green or blue colour depending on the concentration of antioxidants. The antioxidants such
as phenolic acids sand flavonoids were present, considerable amount in ethanol extract of
Evolvulus alsinoides and showed the reducing capacity in a concentration dependent
manner.

24
Table 6: Ferric (Fe3+) reducing power activity of ethanol extract of Evolvulus
alsinoides

S. Concentration % inhibition of ethanol

No. (µg/ml) extract
1 Control 0.00
2 20 17.98±1.32
3 40 18.17±1.61
4 60 18.21±1.38
5 80 21.48±1.32
6 100 29.62±1.10
7 120 30.82±1.26

35
Fe3+ Reducing Power
30

25
% Of Reduction

20

15

10

0
20 40 60 80 100 120
Concentration(µg/mL)

Figure 3.4: Ferric (Fe3+) reducing power activity of ethanol extract of Evolvulus
alsinoides

4.7 ANTIBACTERIAL ACTIVITY

The ethanol extract of Evolvulus alsinoides were investigated for in vitro

antibacterial activity against microorganism including Gram-positive bacteria (Bacillus
25
subtilis, Micrococcus luteus, Staphylococcus aureus) and Gram-negative bacteria
(Escherichia coli, Pseudomonas aeruginosas). The antibacterial sensitivity and potency of
the crude extracts were quantified by measuring the diameter of the clear zone in petri plate
cultures. At a dosage of 1000 µg/mL, the greatest zone of inhibition for Staphylococcus
aureus was 18 mm. The antibacterial activity may be due to the presence of secondary
metabolites such as phenolic compounds, terpenoids, tannin and alkaloids that adversely
affect the growth of microbes.

Staphylococcus aureus Bacillus subtilis Micrococcus luteus

Pseudomonas aeruginosas Escherichia coli

Figure 3.5: Antibacterial activity of ethanol extract of Evolvulus alsinoides

26
 Table 7: Antibacterial activity of ethanol extract of Evolvulus alsinoides

S. No Organisms Zone of inhibition Standard

mm (Tetracycline)
250 500 1000
µg µg µg
1 Bacillus subtilis - - - -

2 Micrococcus luteus - - - 27

3 Staphylococcus aureus 14 16 18 32
.
4 Escherichia coli 13 15 17 28

5 Pseudomonas - - - 18
aeruginosas

4.8 ANTIFUNGAL ACTIVITY

The ethanol extract of Evolvulus alsinoides were investigated for in vitro antifungal
activity against microorganism including fungus (Candida albicans, Candida tropicalis
and Candida krusei). The antifungal sensitivity and potency of the crude extracts were
quantified by measuring the diameter of the clear zone in petri plate cultures. At a dosage
of 1000 µg/mL, the greatest zone of inhibition for Candida albicans was 12 mm. Secondary
metabolites such as phenolic chemicals, terpenoids, tannin, and alkaloids, which inhibit
microbe growth, may be responsible for the antifungal activity.

27
 Candida albicans Candida tropicalis Candida krusei

Figure 3.6. Antifungal activity ethanol extract Evolvulus alsinoides.

Table 8: Antifungal activity of ethanol extract of Evolvulus alsinoides

S. No Organisms Zone of inhibition Standard

mm (Fluconazoles)
250 500 1000
µg µg µg
1 Candida albicans - - 12 -

2 Candida tropicalis - - 11 10

3 Candida krusei - - 11 -

4.9 THIN LAYER CHROMATOGRAPHY

Methanol: Chloroform at a ratio of 1.5:0.5 was used as the solvent system for thin layer
chromatography analysis. The separated compounds in TLC were showed in Figure 3.8.

28
 0.51 0.51

0.43 0.43

0.41
0.39
0.41 0.39

Figure 3.8. Compounds separated by Thin Layer Chromatography

4.10 ANTI-INFLAMMATORY

Table 9: Haemolytic inhibition activity of ethanol extract of Evolvulus alsinoides

S. Concentration % of inhibition
No
1 20 11.28±1.46
2 40 13.09±4.59
3 60 18.81±2.73
4 80 43.38±0.25
5 100 50.94±0.04
6 120 56.60±0.25

29
 HAEMOLYTIC INHIBITION
60

50

% OF INHIBITION
40

30

20

10

0
1 2 3 4 5 6
CONCENTRATION

Figure 3.9. Haemolytic inhibition activity of ethanol extract of Evolvulus alsinoides

4.11 PROTEIN DENATURATION

Table 10. Protein denaturation activity of ethanol extract of Evolvulus alsinoides

S. No Concentration % inhibition
1 20 32.22±0.18
2 40 79.37±0.61
3 60 79.18±0.95
4 80 81.58±0.64
5 100 84.53±0.48
6 120 94.47±0.49

30
 PROTEIN DENATURATION
100
90
80

% OF INHIBITION
70
60
50
40
30
20
10
0
1 2 3 4 5 6
CONCENTRATION /ML)

Figure 3.10: Protein denaturation activity of ethanol extract of Evolvulus alsinoides

4.12 GC-MS ANALYSIS

GC-MS analysis of ethanol extract of Evolvulus alsinoides was showed in Table 11.
An antioxidant compounds flavone compound (5,7-dihydroxy-3-phenylchromen-4-one)
and phytol were eluted and recorded.

Figure 3.11. GCMS Analysis

31
 Table 11: GCMS analysis of ethanol extract of Evolvulus alsinoides

S. Compound name RT Compound structure Mol. Mol. Activity

No weight formula
g/mol
1
Antimicrobial and
Terrein 154.164 C8H10O3 antitumor (Jyoti
12.77 Goutam et al.,2017)

222.24 C15H10O2 -
2. Flavone 15.05

Antibacterial and
antifungal (Hakan
3. 4H-1- 17.03 238.24 C15H10O3 Goker et al.,2005)
Benzopyran-4-
one, 7-hydroxy-2-
phenyl
Antioxidant, anti-
4. n-Hexadecanoic 17.87 256.430 C16H32O2 inflammatory
acid (Vasudevan Aparna
et al.,2012)

2-Hexadecanoic Antioxidant (Y Yang

5. acid, 2,3- 19 296.5 C19H36O2 et al.,2018)
dimethyl-,methyl
ester, [E]
Antimicrobial (V
6. 10-Heneicosene 19.8 Vanitha et al., 2020)
[c,t] 294.6 C21H42

Methyl eicosa-
7. 5,8,11,14,17- 20.58 316.5 C21H32O2 -
pentaenoate

32
8. 1,4-Diphenethyl- 21.6
2,6- 322.401 C20H22N2O2 -
piperazinedione

Antimicrobial (G
Methoxyacetic Raju et al.,2014)
9. acid, heptadecyl 23.43 328.5 C20H40O3
ester

33
 5. CONCLUSION

Antioxidants are chemicals that, when present at low quantities, considerably delay or
prevent the oxidation of an oxidisable substrate. Antioxidants can be found in abundance
in plants. According to the findings of this study, the ethanol extract of Evolvulus alsinoides
possesses substantial antioxidant properties that help to prevent the damaging effects of
free radicals. The findings of this study suggest that Evolvulus alsinoides could be used as
an antioxidant.

Antibacterial activity is most important characteristic of medical textiles, to provide

adequate protection against microorganisms, biological fluids, and aerosols, as well as
disease transmission. The results of the present study indicate that ethanol extract Evolvulus
alsinoides has significant antibacterial activities to protection against microorganisms.
Antifungal activity is done to guide the treatment of fungal diseases, and to identify
resistance to antifungals. The results of the present study indicate that ethanol extract
Evolvulus alsinoides has significant antifungal activities to protection against fungus.

The anti-inflammatory activity was exhibited by plant extract shows a positive

prospect for the treatment of inflammation using natural products.

34
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