INTRODUCTION :

Human beings have depended on nature for their simple

requirements as being the sources for the medicines , shelters , food stuffs ,
and also fragrances , clothing , flavours, fertilizers and means of transportation
throughout the ages. For the large proportions of world’s population medicinal
plants continue to show a dominant role in the healthcare system and this is
mainly true in developing countries, where herbal medicine has the continuous
history of them long use. The development and recognition of medicinal and
financial aids of these plants are on rise in both industrialized and developing
nation. ( Geneba et al.,1998 ).

The foundations of typical traditional systems of medicine for thousands of

years that have been in existence have formed from plants. The plants remain
to their offer mankind with new medicines. Some of the beneficial properties
ascribed to plants have recognised to be flawed and medicinal plant treatment
is based on the experimental findings of hundreds to thousands of years. The
earliest reports carved on clay tablets in cuneiform date from about 2600 BC
are from Mesopotamia ; Then among the materials that were used were oils of
Commiphora species (Myrrh) , Cedrus species (Cedar) and alsoGlycyrrhiza glabra
( Licorice ) , Papaver somniferum ( Poppy juice ) and Cupressus sempervirens
(Cypress) are still used today for the cure of diseases extending from colds and
coughs to inflammation and parasitic infections.( A. G. Fakim 2006 ).

The traditional medicine practice is widespread in China, India, Japan, Pakistan,

SriLanka and Thailand and so on.About 40% of the total medicinal consumption
is attributed to traditional tribal medicines alone by China.
Herbal medicine has its roots in every culture around the world. There are the
many different systems of the traditional medicine, and the philosophy and
practices of each are influenced by social conditions, environment and the
geographic location, but these systems all agree on a holistic approach to life.
Well-known systems of herbal medicine like Traditional Chinese Medicine and
Ayurvedic Medicine believe in their only central idea that there should be an
emphasis on health rather than on disease. By using healing herbs, people can
thrive and focus on their overall conditions, rather than on a particular ailment
that is typically arises from a lack of the equilibrium of the mind alsobody and
environment. Herbal medicine has its origins in ancient cultures. It involves the
medicinal use of the plants to treat a disease and enhance general health and
wellbeing. ( Tapsell LC, Hemphill I, Cobiac L et al.., 2006 ).

Millions of dollars have recently been invested in the looking for promising
medicinal herbs. These substantial research investments in traditional herbal
medicine are still relatively modest when they are to compared to the overall
pharmaceutical industry, but it proves that researchers are beginning to steer
away from conventional drug development and look toward more alternative
and also natural forms of the treatment.Natural plant products have been used
throughout human history for various purposes. In fact, written records of the
use of the herbal medicine date back more than 5,000 years, and for much of
history, herbal medicine was the only medicine.Today, plants are being used to
treat a number of health concerns and conditions, including allergies, arthritis,
migraines, fatigue, skin infections, wounds, burns, gastrointestinal issues and
even cancer - proving that it’s true that food is medicine. These herbs are less
expensive and also more they’re a safer means of treatment than conventional
medications, which is why so many people are choosing to go back to this type
of traditional idea of medicine. ( Dr. Susan Sam .., 2019 )
In Thailand , herbal medicines make use of legumes encountered also in the
Caesalpiniaceae, the Fabaceae, and the Mimosaceae. It is estimated that mid
-90s , more than US $ 2.5 billion have resulted from the sales of the herbal
medicines. Then the herbal medicinal preparations are more in demand than
mainstream pharmaceutical products in Japan. ( Refaz Ahmad Dar et al.., 2017 ).

BOTANICAL NAME : Hemidesmus Indicus

Kingdom : plantae Order : Gentianales

Clade : Tracheophytes Family : Apocynaceae

Clade : Angiosperms Genus : Hemidesmus

Clade : Eudicots. Species : H. Indicus

Clade : Asterids
INTRODUCTION OF PLANT :

Hemidesmus Indicus (L.) R. Br. Ex schult is known as

Indian Sarsaparilla , country sarsaparilla and false sarsaparilla in the English.
Anantmula , Dhavalasariva , Gopa , Gopakanya , Gopabandhu , Gopavali , Gopi,
Kerala, Krishodari, Lata, Nagajihva, sariva, Sugandha and Shgandhi in Sanskrit;
Magrabu and Hindisalsa in Hindi ; Salsa in Urdu ; Anantmul in Assamese and
Bengali; Anantveland Upalsari in Gujrati; Upalsan in Marathi; Onotomulo and
Sugudimalo in Oriya; Hemidesmus indicus, Naruninti, Narunentiand Narunari in
Malayalam; Namdaberu and Sogadaberu Kannada; Hemidesmus indicus in Tamil;
Sugandhipala in Telegu and Ushba in Persian ( Kirtikar and Basu, 1984; Warrier
et al., 1994; ).

The plant is taxonomically different from “true” Sarsaparilla (Smilaxfebrifuga,

family). H. indicus, earlier placed in the family Asclepiadaceae, is now placed in
the family Apocynaceae following phylogenetic classifications. Periploca indica
L. is considered as the widely accepted synonym for the plant species. Roots of
H. indicas has been used as tonic, diuretic and alterative. Historically, the plant
wasInstruduced into Great Britain from India.Initially it was employed under the
name of Smilax aspera for some time ( Remington and Wood ., 1918 ).

In the Ayurveda , the plant is used in the treatment of fever, stress, topical
wound , bone loss , low body weight andpsoriasis. It also is One of the Rasayana
plants of Ayurveda. H.indicus was introduced in European medicine in year 1831.
Besides, Ayurvedic literature also depicts its use as anti-atherogenic,anti-anti-
atheroge spasmodic, memory enhancing, immunopotentiating and also anti-
inflammatory agents .(Fulzele et al., 2002 ).
The plant is marketed in the USA under a variety of the commonly names
including Anantamul, Indian Sarsaparilla, and Sariva in the forms of polyherbal
formulations, as topically applied oils or creams, and as oral tablets which have
been reviewed elaborately by Weissner (2014).H. indicus continues to be used
as a traditional remedy for the dysentery and diarrhea and infections , skin
disease , menorrhagia , post - partum recovery , nephrit ic complaints , diabetes,
stomach - ache , gastrointestinal disorders , jaundice , fever , cough , headache,
pain,inflammation, edema, mouth sores,and also toothache, gonorrhea,syphilis,
impotence, snakebite, scorpion sting, leucoderma, psoriasis and rheumatism.It
is also being used as blood purifier, body coolant, appetizer, as well as health
and vitality promoter (Dey and De et al., 2012; ).

Various bioactive phytochemicals such as 2-hydroxy-4- methoxybenzoic acid

(HMBA) , 2-hydroxy-4-methoxybenzaldehyde (MBALD) , 4- hydroxy-3-methoxy
benzaldehyde (vanillin) , 3-hydroxy-4- methoxybenzaldehyde , lupeol acetate ,
hindicusine and di-O-acetylhindicusine and β-amyrin palm itate were reported
from the plant . One of the Rasayana plants of Ayurveda. i. e., H. indicus was
introduced in European medicineFew poly herbal formulations containing this
plant have shown anti- ulcerogenic, cytotoxic and anti-tubercular activities .
The plant extract and formulations from exhibit anti-arthritic, mosquitocidal,
hepatoprotective, anti-cancer, anti - mutagenic, anti - acne, radioprotective,
and also anti- thrombotic, neuroprotective, cardioprotective, anti-diarrheal,
nephroprotective, and H. indicus continues to be used as a more traditional
remedy for the dysentery and diarrhea and infections also the ulcerogenic,
genotoxic, anti-inflammatory, wound healing, diuretic, anti-pyretic, anti-
asthmatic, and also immunomodulatory, antimicrobial, anti-angiogenic and anti
-osteoclastic properties.( Maha lingam et al., 2008; ).
USAGE OF OTHER PARTS OF THE PLANT :

In addition to the roots, other parts of the Hemidesmus indicus plant,

such as its leaves and stems, are also used in traditional medicine. For example :

LEAVES : The leaves of the Hemidesmus indicus are believed to have

the medicinal properties and are used in the treatment of various ailments,
such as the skin diseases, fever, and cough. Hemidesmus indicus is believed to
be a classic home-remedy to help relive the body pain, tackle urinary infection
and constipation. With many more benefits of consuming Hemidesmus
indicus, it also serves as a great beverage that can be best enjoyed with a
dash of lemon.

STEMS : The stems of the Hemidesmus indicus are used to make a

cooling and also refreshing beverage that is believed to have a soothing effect
on the body. This beverage commonly known as Hemidesmus indicus Sarbath
in all the southern side India and it also collected and prepared by boiling the
stem extract in water along with sugar and other flavoring.

It is important to note that the use of different parts of Hemidesmus indicus

for medicinal purposes has not been extensively scientifically tested , and its
the efficacy and safety may vary depending on the individual.As with any
herbal remedy,it is the always advisable to the consult with a qualified
healthcare professional before using Hemidesmus indicus or its parts for all
the medical purposes. This Hemidesmus indicus syrup is an effective way to
treat our stomach disorders and digestion problems without swallowing bitter
pills. For centuries, Hemidesmus indicus sharbat is consumed to purify the
blood; thus adding to the many medicinal benefits that come with it. Tackling
indigestion with bitter pills can be very distasteful. Substituting those with a
glass of Hemidesmus indicus sharbat is said to do wonders. Hemidesmus
indicus is also said to help with skin problems including psoriasis, acne, rashes,
and syphilis.( sethi.., 2006 ).
VERNACULAR NAMES :

• Botanical Name: Hemidesmus indicus

• Tamil Name: Nannari

• English Name: Indian Sarsaparilla

• Hindi Name: Anantmool, Kapuri

• Kannada Name: Sogade Beru

• Malayalam Name: Naruninti

• Telugu Name: Sugandhipala, muttavapylagamu

• Marathi Name: upalsari, uparsal ( Bhopal.., 2012 ).

MORPHOLOGY :

HABIT - Perennial pros trate or twining shrub.root stock is woody.

STEM - Slender,terete,glabrous or pubescent,striate,thickened at the nodes.

LEAVES - simple , opposite ,bvaroiable , elliptic - oblong to the linear

lanceolate,glabrous,darkgreen,often variegated with white above,pale and
sometimes,silvery white andpubescent beneath,reticulate venation.

INFLORESCENCE - sub sessile cyme.

FLOWER - pedicel short,clothed with numerous bracts.

FRUIT - cylindric,tapering to a point a the apex,straight or sometimes slightly

Curved striate glabrous.

SEED - 6 to 8 mm long,ovate-oblong,,black com is silvery white,25 cm long.

(Jagtap and signh..,1999 ).
DISTRIBUTION :

This plant is grows in mesophytic and semi dry conditions in the

plains. This plant is found in all over Asia and in Madhya Pradesh, West Bengal
and Karnataka. Hemidesmus indicus (L.) R.Br. is established in all over southern
Asia. However it is also originated in India , wherever it is still primarily found
growing wildly. Hemidesmus indicus (L.) R.Br. is produce in Malaysia, Indonesia
and Srilanka. It is old medicinal plant, plant parts has been exported to world.
Hemidesmus indicus grows in the plains and to an altitude of 600m semi-dry
to mesophytic conditions. (Sasidharan and Siddique et al., 2004 ).

It is found widely throughout India precisely from Gangetic plains to eastwards

of Bengal, Sundarbans, Assam, and different locations of central, western, and
southern parts of India. It is usually seen in deciduous forests and uncultivated
lands, and hedges. Other than India it is also reported to be found in Sri Lanka,
Pakistan, Iran, Bangladesh, Moluccas, and also Malaysia. Hemidesmus Indicus is
widely distributed in places like India, Pakistan, Sri Lanka, Bangladesh, Iran, Iraq
and also in Indonesia. It is also cultivated at altitude of 600 m. ( Nandy et al.,
2020 )

It is originated from India where it is still primarily found growing wildly. H.

indicus is a prostrate or semi-erect shrub found throughout India from upper
Gangetic plains, eastwards to Assam and also throughout Central, Western and
Southern India upto an elevation of 600 m. It is also known to grow in Malaysia,
Indonesia, Pakistan, Bangladesh and Sri Lanka. The different vernacular names
known are Ananta-mula, Ananthamoola, Asclepiaspseudosarsa, and Country
Sarasaparilla, Durivel, East Indian Sarsaparilla, Eternroot,Karibandha, Magrabu,
Muttavapulagamu, Naga-jihva, Naruninti, Nunnari, Nunnerroot, Onontomulo,
Periplocaindica, Sariva, etc.., ( N.Sasidharan et al.., 2004 ).
 Hemidesmus Indicus Distribution In The World

PHYTOCHEMICAL CONSTITUENTS OF THE HEMIDESMUS INDICUS :

 AIM AND OBJECTIVE

AIM :

To study the Qualitative analysis of phytochemical compounds,

Antimicrobial Activity, Antioxidant and Quantitative analysis by Thin layer
chromatography of Hemidesmus indicus root extract.

OBJECTIVE :

1. To obtain the root extract of Hemidesmus Indicus using Methanol

solvent.

2. To perform phytochemical analysis using the root extracts.

3. The extracts were used for antibacterial activity against selected

pathogens E.coli, staphylococcus aureus.

4. To study antioxidant activity by DPPH radical scavenging assay .

5. Quantitative analysis by Thin layer chromatography.

REVIEW OF LITERATURE :

The study stated that provides all standardized data about a

medicinal plant as a ready reckoner reference to herbal manufacturers will
greatly contribute to preparing standardized herbal medicines with global
acceptability. In view of this the present review to document the data on
various standardization parameters reported for the medicinal plant
Hemidesmus indicus (Hemidesmus indicus), a plant well known in Indian
traditional system of medicine.( G Renukaadevi and Shubashini K Sripathi et al..,
June 2022 ) .

The study on Hemidesmus indicus roots are very slender with a diameter less
than 1cm and they possess a characteristic pleasant smell with a sweetish taste.
In the transverse section there are plenty of starch grains prismatic crystals of
calcium oxalate crystals and lactiferous ducts are seen in the cortex portion
which forms the major identifying features. Three types of vessels are found in
the powder microscopy. These results suggest that Hemidesmus indicus var.
pubescens root was able to attenuate Dextran Sodium Sulfate induced ethanol
augmented ulcerative colitis indicating its anti-ulcerative colitis effects. The
study suggested that Among all the compounds 2-hydroxy4- methoxy
benzaldehyde (MBALD) was found to be the major chemical entity. Further, RP-
HPLC analysis was carried out to decipher the amount of MBALD present in
roots of two morphologically distinct H. indicus plants were found to be 0.1827
and 0.1537 mg/gram of tissue of slender and long leafed and broad-leafed
plants, respectively. (Satish et al.., 2018 ).

The present review covers recent work on H. indicus which have not received
adequate coverage in the reviews on this plant so far.Modern searches for
bioactive molecules typically make use of sophisticated bioassays and bioassay
-guided fractionation of medicinal plants used by traditional healers. A good
number of potent drugs and a large number of therapeutic leads and many
new pharmacologically active constituents have been developed fro The
manufacture of morphine on industrial scale.( Aravind ramasamy et al.., 2017 ).

From the study we get to know, The plant parts, roots and rhizome have been
utilized for hundreds of years in Ayurvedic medication for relieving countless
diseases. Many of reported studies highlighted the potential pharmacological
properties of H. indicus like anti - cataractous, antidiarrhoeal, etc. The present
work aims overall for the Ayurvedic and modern therapeutical information of
Hemidesmus indicus with various reported for the Ayurvedic literature and the
scientific pharmacological studies. This plant is a good source of the different
bioactive chemica l compound like Hemidesmin-1 and Hemidesmin -2, αamyrin,
β-amyrin, lupeol acetate, and also β- sitosterol, hemidesmol and hemidesterol
which were responsible for many of the pharmacological activities. This review
aims at providing an up-to-date overview of the comprehensive account of
the phytochemical investigation, therapeutic potential and pharmacological
studies of Hemidesmus indicus. ( Alka mehta et al.., 2012 )

The study says that this plant has been used since a long time to cure a lot of
disease.Traditionally used of this plant has been done in Srilanka, India and
country since very long time. It is a very important medicinal plant in term of
human being, so we are studying its review of literature. From this study we
came to know The most useful part of the Hemidesmus indicus activity like
antimicrobial activity, anti- enterobacterial activity activity, natriuretic and
saliuretic activity,renoprotective activity venom activity.(shifali Takun et al ..,
2021 )
The plant is found to be grown in uncultivated lands and deciduous forests, so
this highly valuable plant species is needed to be most conserved and also to
ensure their protection from illegal harvesting. This review is carried out about
the pharmacology, phytochemical composition, ethnomedicinal use, and also
the application of the plant biotechnology techniques for the conservation of
the Hemidesmus indicus. In the following study, ethanolic extracts of roots of
Hemidesmus indicus and leaves of Simarouba glauca were evaluated for their
antimicrobial activity against Staphylococcus aureus, Escherichia coli and also
Bacillus subtilis by the well- diffusion method. The minimum inhibitory of the
concentration was determined. Further, the isolation and purification of the
constituents responsible are under process. ( Harish Moorthy et al., 2021 )

The present study suggest that the Hemidesmus indicus has protective activity
against arthritis and the activity might be attributed to presence of terpenoid
in the hydroalcoholic extract , as well as in ethyl acetate fraction. Collectively,
H.indicus could act as a disease of modifying the therapeutic in pharmaceutical
industries by utilizing it as alternative therapy for the management of oxidative
stress and its related disorders. ( Mamatha et al .., 2018 )

One of the biggest issues faced in today’s health - care and medicine is the
threat posed by the rapidly emerging drug-resistant microorganisms. Certain
Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, etc. are
showing resistance against most of the known antibiotics. These organisms
being pathogenic need to be contained, and prevented from causing infections.
Since the ‘ Drugs of last Resort ’ are also no longer effective, there is an urgent
necessity to search for equally effective therapeutics. In the following study
ethanolic extracts to roots of Hemidesmus indicus and leaves of Simarouba
glauca were evaluated for their antimicrobial activity against Staphylococcus
aureus, Escherichia coli and the Bacillus subtilis by well-diffusion method. The
minimum inhibitory concentration .further, the isolation and purification of the
constituents responsible are under process. ( Sanita das et al .., 2011 )

The susceptibility of the bacterial pathogens was in the order of S. aureus, K.

pneumoniae and P. aeruginosa. The antimicrobial activity of plant extracts was
synergistic with antibiotics tested. Results of the present study suggest that
the aqueous extracts of H. indicus, F. bengalensis and P. marspium roxb has
significant antibacterial activity against the pathogenic bacteria.The extract
protected the bone marrow cells from c isplatin-induced genotoxicity in an
inverse dose-dependent manner. ( Kannabiran et al., 2009)

However, the extract was cytotoxic at all doses. But, under split dose regime it
conferred a higher level of genoprotection and was not cytotoxic at the lower
two doses.The concern caused by the increasing the rate of antibiotic resistant
strains of the microorganisms requires immediate alternative solutions. Plant
extracts have been used for a very long time and have great potential as anti-
bacterial agents. In this study,The Ethanolic extracts of root of Hemidesmus
indicus and leaves of the Simarouba glauca were prepared . Hemidesmus
indicus and leaves of Simarouba glauca were evaluated for their antimicrobial
activity against Staphylococcus aureus, Escherichia coli and the Bacillus
subtilis by well-diffusion method .This plant has the ability to synthesize a wide
variety of the chemical compounds like terpenoids, saponin, glycosides,
flavonoids, tannin sterols and volatile oil that are used to perform an
important biological function. Several studied are being carried towards its
activities more like an antioxidant , antiacne , antibacterial , and antifungal, and
antimicrobial activity. ( Growrinair et al .., 2022)
 MATERIALS AND METHODS

COLLECTION OF SAMPLE :

The genuine sample of Hemidesmus indicus was collected

from market of chennai.

Hemidesmus Indicus Root Powder

SAMPLE PREPARATION :

Fresh roots were air dried and coarsely powdered. Obtained

powder was soaked in a conical flask with methanol and water on the basis of
increasing polarity of solvents and kept in a mechanical shaker ( at the room
temperature) for 72 h followed by filtration with Whatman filter paper. Later on,
under a high pressure , each filtered extract was concentrated by the rotary
evaporator at a maximal temperature of 35 °C. All the extracts were protected
from light and stored at 4 °C for further use. ( Mohan et al.., 2015 ).
 Hemidesmus Indicus Root Extract

PHYTOCHEMICAL ANALYSIS :

TEST FOR PHYTOCHEMICALS :

Test for flavonoids :

1 g of the powdered dried leaves of each specimen was boiled

with 10 ml of distilled water for 5 minutes and filtered while hot. Few drops of
20 % sodium hydroxide solution were added to the 1 ml of the cooled filtrate. A
change to theyellow colour which on addition of acid changed to the colorless
solution depicted the presence of flavonoids.
Test for tannins :

1 g of each powdered sample was separately boiled with 20 ml distilled

water for five minutes in a water bath and was filtered while hot 1 ml of cool
filtrate was distilled to the 5 ml with distilled water and a few drops (2-3)of 10 %
ferric chloride were observed for any formation of precipitates and any color
change. A bluish-black or brownish green precipitate indicated the presence of
tannins.

Test for alkaloids :

1 g of powdered sample of each specimen was separately boiled with

distilled water and 10 ml hydrochloric acid on a water bath and filtered. The pH
of the filtrate was adjusted with ammonia to about 6-7. A very small quantity of
the following reagents was added separately to about 0.5 ml of the filtrate in a
different test tube and also observed. Picric acid solution. 10% tannic solution.
Mayer’s reagent ( Potassium mercuric iodide solution ). The test tubes were
observed for coloured precipitates or turbidity.

Test for Cardiac glycosides (Keller Kelliani’s test) :

5ml of each extract was treated with 2ml of glacial acetic acid in a test
tube and a drop of ferric chloride solution was added to it. This was carefully
underlayed with 1ml concentrated sulphuric acid. A brown ring at the interface
indicated the presence of deoxysugar characteristic of cardiac glycosides. A
violet ring may appear below the ring while in the acetic acid layer, a greenish
ring may form.
Test for Phenols (Ferric chloride test) :

A fraction of the extracts was treated with aqueous 5% ferric chloride

and observed for formation of deep blue or black colour.

Test for Quinones :

A small amount of extract was treated with concentrated HCL and

observed for the formation of yellow precipitate (or coloration).

Test for carbohydrate (Molisch's Test) :

Mix the extract with 2ml of Molisch‟s reagent and shake the mixture
properly. After that, add 2ml of concentrated H2So4 along the side of the tube.
Appearance of a violet ring at the interphase indicates the presence of the
carbohydrate.

Test for Triterpenoids ( Salkowski Test ) :

To the extract, add 2ml of chloroform and 3ml of concentrated H2SO4.

Formation of yellow ring at the interface of the two liquids that turns reddish
brown colour.

Test for Sterols ( Salkowski’s Test ) :

One ml of extract was treated with 2 ml of chloroform and equal amount

of concentrated sulphuric acid was added, upper layer is turns to red indicates
the presence of the sterols and steroids.
ANTI BACTERIAL ACTIVITY :

The Hemidesmus Indicus root extracts was tested against gram

+ve and gram–ve bacterial for their antimicrobial activities.

MATERIALS REQUIRED :

1) Fresh solvent extracts

2) Disc

3) Petri plates

4) Swabs and Test tubes

5) Bacteria culture

6) Inoculation loop

7) Muller Hinton agar, Nutrient agar

ANTI BACTERIAL ACTIVITY ASSAY :

No of samples:

No of Microorganisms: 2

1) E.Coli

2) staphylococcus aureus

Antibiotic agent : Gentamicin , Tetracycline . ( Ratha et al.., 2012 ).

PREPARATION OF BACTERIAL MEDIUM :

The mediums were taken in the different conical flasks and sterilized by
autoclaving at 121 ̊ C for 15 minutes, as per the manufacturer specification.
After sterilization, the agar medium pour in to sterile petri plates immediately
to avoid solidification within conical flasks.( Remington.., 2000 ).

Agar well Diffusion method :

Antibacterial activity of root extract of Hemidesmus indicus

was carried out using the agar well diffusion method[14]. The solidified nutrient
agar in the petri plates was inoculated by the dispensing the inoculum using
sterilized cotton swabs which is used as previously immersed in the inoculum
containing test tube and spread evenly onto the solidified agar medium. Five
wells were created in each plate with the help of a sterile well-borer of 8 mm
diameter. The root extract was then poured into each well containing 250, 375,
500 and 625 μg/mL concentrations And All the plates with extract loaded wells
were incubated at 37º C for 24 h and the antibacterial activity was assessed by
measuring the diameter of the inhibition zone formed around the well.
Tetracycline (25 µg) was used as positive control.( Gennars.., 2000 ).

Anti-oxidant activity :

The in vitro and in vivo antioxidant potential of root bark

of Hemidesmusindicus R.Br.was evaluated for the radical scavenging activity by
DPPH reduction, superoxide radical scavenging activity in riboflavin/light/NBT
system (nitro blue tetrazolium), nitric oxide (NO) radical scavenging activity in
sodium nitroprusside/Greiss reagent system and inhibition of lipid peroxidation
induced by iron - ADP - ascorbate in liver homogenate and phenyl hydrazine
induced haemolysis in erythrocyte mem-brane stabilization study. The extract
was found to have different levels of antioxidant properties in the models
tested. In scavenging DPPH and superoxide radicals, its activity was intense,
while in the scavenging NO radical, it was moderate. It also inhibited lipid
peroxidation of thd liver homogenate and also the haemolysis induced by
phenylhydrazine confirming the membrane stabilization activity. Whereas,
Phytochemicals like flavonoids, and polyphenols, terpenoids, cumarins and
glycosides have antioxidant properties. Evalution of antioxidant activity of
methanolic extract of H. indicus root bark in vitro and ex-vivo models is done.
70% methanolic extract of H. indicus root, which contain large amout of
flavonoids and phenolic compounds, exhibit high antioxidant and free radical
scavenging activities. It also chelates iron and has reducing power. These in
vitro assays indicate that the extract contains constituents that can be a
significant source of natural antioxidant. ( chenhy.., 1995 ).

DPPH radical scavenging assay :

The antioxidant activity of root extract of Hemidesmus

indicus was measured based on the scavenging activity of the stable 1, 1-
diphenyl 2-picrylhydrazyl (DPPH) free radical [10]. One mL of 0.1 mM DPPH
solution in methanol was mixed with 1 mL of various concentrations (20-120
μg/mL) of root extract. The mixture was then allowed to stand for 30 min
incubation in dark. One mL methanol mixed with 1 mL DPPH solution was used
as control. The decrease in absorbance was measured at 517 nm using UV-Vis
Spectrophotometer. ( chittibabu.., 2019 ).

The percentage of inhibition was calculated as: %

Control-sample
% of DPPH˙ radical inhibition = ------------------- × 100.

Control
MATERIALS REQUIRED :

Hemidesmus indicus root extracts ( methanol )

Test tubes, pipettes,test tube rack etc.

CHEMICAL :

❖ 1,1 – diphenylhydrazyl (DPPH)

❖ Butylated Hydroxy Toluene (BHT)

❖ Methanol

❖ Dimethyl sulphoxide

REAGENTS :

❖ DMSO-Di methyl sulfoxide

❖ DPPH-1 mg/ml in methanol

❖ BHT(standard)-1.6mg/ml in methanol

❖ Sample-desired concentration from 1 mg/ml-max of 5 mg/ml

(in methanol/DMSO).

METHODOLOGY :

PROCEDURE :

1. Aliquot 3.7 ml of absolute methanol in all test tubes.

2. The 3.8ml of absolute methanol was added to blank

3. Add 100µl of BHT to tube marked as standard and 100µl of the respective
samples to all other tubes marked as tests.
4. The 200µl of DPPH reagent was added to all the test tubes including blank.

5. Incubate all test tubes at room temperature in dark condition for 30 minutes.

6. The absorbance of all samples was read at 517nm.

QUANTITATIVE ANALYSIS

Thin layer chromatography :

Thin layer chromatography (TLC) was carried out for root

extract of Hemidesmus indicus in silica gel precoated TLC aluminium sheets
(Merck/60 F254). The root extract was spotted at 0.3 mm above from all of the
bottom of the TLC plate (1.5x5 cm). The spotted TLC plate was placed in a 100
mL beaker containing the solvent mixture of the toluene: methanal (1:1) and the
chromatogram was developed. The spots were visualized in UV light chamber
at 254 nm as well as under iodine. The Rf values of coloured spots were
calculated [15]. ( stahl.., 2005 ).

Distance travelled by the solute

Rf value = ------------------------------------
Distance travelled by the solvent

RESULT AND DISCUSSION

PHYTOCHEMICAL ANALYSIS :

The phytochemical analysis of methanol root extract of

H. indicus showed the presence (+) of alkaloids, terpenoids, phenolic
compounds root, flavonoids, glycosides and saponins. ( Raaman.., 2006 ).
PHYTOCHEMICAL TEST FOR HEMIDESMUS INDICUS ROOT
METHANOL EXTRACT :

1. NaoH

2. Salkowski

3. Conc. H2So4

4. Keller kellani

5. Braymer

6. KoH

7. Ferric chloride

8. Picric

9. Molisch

10. 10% NaoH

11. Copper acetate

 S.NO METHANOL EXTRACT RESULTS

1. Cumarins +

2. Phytosterols +

3. Flavonoids +

4. Cardiac glycosides +

5. Tannins +

6. Quinones +

7. Phenols +

8. Alkaloids +

9. Carbohydrates +

10. Reducing sugar +

11. Diterpenes +

ANTIBACTERIAL ACTIVITY :

The methanol root extract of H. indicus were investigated for in

vitro antibacterial activity against Gram-positive bacteria ( Staphylococcus
aureus) and Gram-negative bacteria (Escherichia coli ). The antibacterial
sensitivity of the crude extract and their potency were assessed quantitatively
by measuring the diameter of clear zone in cultures in petri plates. The
antibacterial activity of these extracts could be correlated to the presence of
secondary metabolites such as flavonoids, phenolic compounds, terpenoids,
tannin and alkaloids that adversely affect the growth. ( Arunesh.., 2016 ).
Antibacterial activity of the methanol root extract of H. indicus on the various
pathogenic organisms.

S.
No ORGANISMS STANDARD
ZONE OF INHIBITION

(mm) (Tetracycline)

250 µg 375 µg 500 µg 600 µg

1. Staphylococcus aureus 10 11 12 16 16

2. Escherichia colicol 12 13 14 15 14
 Antibacterial Activity
18
16
14
Zone of inhibition(mm)

12
10
8
6
4
2
0
250 500 Standard
CONCENTRATION (µg/ml)
STAPHYLOCOCCUS AUREUS

ANTIOXIDANT ACTIVITY :

DPPH radical scavenging assay :

The ability of methanol root extract of H. indicus to scavenge

free radicals was assessed by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical.
The maximum DPPH˙ radical scavenging activity was 64.31±4.50 at 120µg/mL
concentration. The root extract of H. indicus demonstrated high capacity for
scavenging free radicals by reducing the stable purple colour DPPH (1, 1-
diphenyl-2- picrylhydrazyl) radical to yellow colour 1,1-diphenyl-2-
picrylhydrazine and the reducing capacity increased with increasing
concentration of the extract. The IC50 was 37.44 μg/mL concentration and was
compared with standard ascorbic acid. ( Sivaraj..., 2018 ).
 S. No CONCENTRATION % of Inhibition*

( µg/ mL)

1. 20 46.80

2. 40 53.42

3. 60 56.55

4. 80 58.37

5. 100 61.51

6. 120 64.31

DPPH
70

60

50
% Inhibition

40

30

20

10

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

DPPH
Thin Layer Chromatography :

Thin layer chromatography analysis was carried out in the solvent

system of Methanol: isopropyl alcohol with the ratio of 1:1. The separated
compounds in TLC were showed in Figure,( saraswati.., 2019 ).

Compounds separated by Thin Layer Chromatography

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