PHARMACOLOGICAL AND NANOBIOTECHNOLOGICAL ASPECTS OF GYMNEMA SYLVESTRE

Somendra Kumar, Dinesh Kumar, Motiram Sahu and Anil Kumar Department of Biotechnology, Govt. V.Y.T. PG. Autonomous College, Durg, Chhattisgarh, 491001, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 25 August 2020 Final Accepted: 28 September 2020 Published: October 2020


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Nowadays,in developing countries the herbal medications are becoming prominent due to better result, easy availabilityto common individuals, low side effects,quality, safe use, and wide pharmacological applications as compared to marketed drugs and more potential in health problem treatment, thus the demands for herbal medications have drastically increased (Vikram et al., 2014). Many plant species contain many valuable bioactive compounds which are isolated and administrated directly as drugs, lead compounds and have pharmacological importance. Traditional approaches derived from medicinal plants provide a natural key to unlock many healthrelated complications (Tiwari et al., 2014).
India has enriched with indigenousmedicinal flora that has been used in Indian traditional medications from the prehistoric era for the treatment of several human ailments. Herbal plants are the major source of drugs in the Indian medicine system and also in other prehistoric systems in the world (Jayachitra and Muniyandi, 2016). Phytoconstituents of herbal plants possess active compounds widely used in drug discovery (Subramaniyan and Srinivasan, 2014).
The present work is focused on traditionally utilized a valuable medicinal plantGymnemasylvestre with an overview of its phytochemical investigation and green synthesis of silver nanoparticles.Gymnemasylvestre a potent antidiabetic plant, used in homoeopathy, folk and ayurvedic system of medicine, which belongs to family Asclepiadaceae, a vulnerable species, commonly known as Periploca of the woods in English,Madhunashini in Sanskrit andGurmar in Hindi which means "destroyer of sugar" (Mitraet al., 1995;Kumar, 2015).Gymnemasylvestre is also being widely used forthe treatment of eye complaints, inflammations, asthma and snake bite by tribal population (Anonymous, 1956;Selvanayagamet al., 1995).
National Botanical Research Institute, Lucknow, India conducted a floristic study in 7 districts of Chhattisgarh state and enlisted Gymnemasylvestre R. Br. among 44 other species in the list of Endangered Taxa of Chhattisgarh state, India (CGMPB, Raipur, Chhattisgarh, 2017). In present scenario the field of nanotechnology is the most dynamic section of research in material sciences and engineering for the synthesis of nanoparticles and that field is emerging significantly all over the world. Nanoparticles expresses completely novel and improved properties into particular features such as shape, size (1 to 100 nm) and structure.

Taxonomy of the plant Taxonomical classification of
In recent years, green nanotechnology attracted many researchers from various field like physics, chemistry, material science, engineering, medicine and biotechnology. Nanotechnology involves in the process of production, manipulation and also the size of the material ranging from less than a micron to that of an individual atom. The physical, chemical and biological process is the wide variety of method used for the synthesis of nanoparticle (Sastry et al., 2003). Bio-nanotechnology is an emerging branch of science that combines physical and chemical approaches with biological principles to produce nano-sized elements with specific functions.
Silver nanoparticles have an important role in the field of nanotechnology because of their incomparable biological, chemical and physical properties. In recent year, various reliable and eco-accommodating efforts and technology were made to synthesis green methods for synthesis of nanoparticles to avoid hazardous by-products for a reduced level of pollution. Gardea-Torresdeyet al.,in 2003 first time utilized plant sourceAlfalfa sprouts for the formation of metallic nanoparticles and first time described plant material based green approach of synthesis of silver nanoparticles (Rafiqueet al., 2017).Silver nanoparticles have an extensive superstructure which responsible for the vital biochemical activity, catalytic properties and atomic nature that is compared with large elements having the same chemical compositions (Xu et al., 2006).

Chemical reagents
For present work, analytical grade biochemicals were used and purchased from HI-MEDIA Pvt. Ltd., Mumbai.

Collection of plant materials
Based on the ethnopharmacological records, an important Indian traditional medicinal plant Gymnemasylvestrewas selected for the study. This plant was collected from Chhattisgarh State Medicinal Plant Board Raipur, Chhattisgarh during February 2020.Department of Botany, Government V.Y.T. PG. Autonomous. College, Durg, Chhattisgarh, identified the plant material. The plant samples leafand stem of Gymnemasylvestrewere rinsed thrice with distilled water followed by double distilled water to remove the dust particles and other contaminants then after thatshade dried at 25-30 ℃ for two weeks followed by the grinding process and the fine powder was kept in the well labelled air-tight glass bottles in darkness at -20℃ until further use.

Plant extract preparation
The powdered plant materials from leaf and stem (150 gm each) were extracted successively with six solvents likepetroleum ether, benzene, chloroform, methanol and distilled water (aqueous) using Soxhlet apparatus at 60 to 85 ℃ for 6 to 10 hours to extract the polar and non-polar compounds (Elgorashi and Staden, 2004). Each sample extract was filtered with the help of Whatman filter paper (No.1) and then kept in sterile air-tight glass bottles for further study.

Qualitative phytochemical analysis
Qualitative phytochemical analysis was carried out for the detection of important phytoconstituents like-alkaloid, cardiac glycosides, flavonoids, saponins,steroids, tannins, terpenoids, protein, phenol and carbohydrates in the plant sample extracts using the following standard protocols with some modifications (Harborne, 1998; Trease, 2002).
Test for alkaloids 0.5 ml extract, 3 ml of methanol, 300 µl of acetic acid were mixed and then few drops of ammonium hydroxide solution was added. Formation of precipitate showed the presence of alkaloids in the sample extract.
Test for cardiac glycosides 0.5 ml of extract, 0.2 ml glacial acetic acid and dropwise 3.5% ferric chloride were added then layered with 1 ml of conc. sulfuric acid. The formation of a reddish-brown ring at the interface indicated the presence of cardiac glycosides in the sample extract.
Test for flavonoids 0.5 ml plant extract, 5 ml distilled water were mixed in the test tube then it was filtered. 5ml of dilute ammonium solution and then conc. sulfuric acid was added. The formation of yellow colour indicated the presence of flavonoids in the sample extract.
Test for saponin 2.5 ml of extract, 5 ml of distilled water were added and then solution was vigorously shaken and the formation of bubbles or stable persistence of foam indicated the presence of saponins in sample extract.
Test for steroids 0.5 ml of extract, 3 ml of chloroform was added and then the solution was filtered then 2 ml of conc. sulfuric acid was added to the filtrate. The formation of a reddish-brown ring at the interface indicated the presence of steroids in the sample extract.
Test for tannins 0.5 ml of extract, 5 ml of distilled water were added than 1% ferric chloride was added. The appearance of deep green coloration indicated the presence of tannin in the sample extract.
Test for terpenoids 0.5 ml extract, 2 ml of chloroform were mixed then a few drops of concentrated sulfuric acid was added. The appearance of the reddish-brown colour at the interface showed the presence of terpenoids in the sample extract.

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Test for phenols 1 ml extract, 2 ml of distilled water weremixed then 2 drops of 10% FeCl 3 was added. The appearance of a blue or green colour indicated the phenol group present in the sample extract.
Test for protein 1 ml extract,500µL of copper sulphate solution, 500µL of 5% sodium hydroxide solution were mixed. The appearance of purple violet colour indicated the presence of protein in the plant extract.
Test for carbohydrates 1-2 ml extract, 1-2 drop of ɑ-naphthalol was added and then the 2 ml of conc. sulphuric acid was added. The appearance of a violet ring indicated the presence of sugar in the plant extract.

Thin Layer Chromatographicanalysis of plant extract
Thin-layer chromatography was performed to detect the chemical profile of different extract of Gymnemasylvestre.
The TLC plates were preparedaccording to standard protocols as slightly modified, 25 g of silica gel-G was mixed with 50 ml of distilled water and with the help of spreader slurry formed uniformly spread over TLC plates with a thickness of 0.25 mm. Asa stationary phase,TLC plates were allowed to dry at room temperature and then plates were incubated in an oven at 100ºC for 2 hrs. for activation. The mobile phase consists of the solvent system (selected based onthe presence of phytochemicals).A soft pencil was used for TLC platemarking. Prepared plant extracts were loaded on the activated TLC plates on a marked spot by using the applicator and then allowed to run for 2 hrs (Harborne, 1998 and Mishraet al., 2014).The TLC spots were exposed in UV-illuminator to analyse the migration of band patterns and the Retention factor values (Rf) of bands were calculated and noted using the following formula-Synthesis of silver nanoparticlesfromGymnemasylvestre 10 ml of the stem aqueous extract of Gymnemasylvestrewas added into 90 ml of an aqueous solution of 3mM silver nitrate. The mixture was incubated in a water bath (Yorco Universal) for 30 minutes at 50°C and exposed to a range of controlled temperatures for 24hours. The appearance of brown colour in solution showed the formation of Gymnemasylvestresilver nanoparticles. The solution of silver nanoparticles was then kept in dark and dried in hot air oven to make a crystallized form for further analysis and preserved at 4 °C for further use (Suprajaet al., 2017).

Characterization of silver nanoparticle UV-Visible Spectroscopy analysis of synthesized nanoparticles
The synthesis of Gymnemasylvestresilver nanoparticles was confirmed using UV-Visible Spectrophotometer (Systronics UV-VIS Spectrophotometer 117) with a resolutionbetween 200 and 700 nm(Suprajaet al., 2017).

FTIR analysis for synthesized nanoparticles
Fourier transforms infrared (FTIR) spectroscopy is a chemical analytical tool used to detect the possible functional groups present in Gymnemasylvestre silver nanoparticlesbased on the peak value in the region of infrared radiation. The major function of FTIR spectroscopy was to determine the nature of the association of Phyto-molecules or Phytoextracts with nanoparticles. Infra-red (IR) analysis of Gymnemasylvestre silver nanoparticles was done with the help ofInfra-red Spectrophotometer (BRUKER, ALPHA II, ECO ATR) at the Department of Chemistry, Govt. V.Y.T. PG. Autonomous College, Durg, Chhattisgarh, India.

X-Ray Diffraction analysis
Silver nanoparticles were harvested and its crystalline structure characterized by XRD analysis. X-Ray Diffraction (XRD) toolwas used to evaluate the crystalline nature of synthesized silver nanoparticles of Gymnemasylvestre. The XRD pattern was recorded using computer-controlled XRD-system and analysed through PAN Analytical software.The crystallite domain size or silver nanoparticles was calculated from the width of the XRD peaks by using the following Debye-Scherrer formula-

Qualitative phytochemical analysis
Phytochemical study of medicinal plant is very useful for the investigation of many biologically active bioactive compounds that contribute to the colour, flavour and other features of leaves, flowers, fruits and stems (Singh and Deo, 2014). Leaf and stem extracts of Gymnemasylvestre were screened for the qualitative presence of major active ingredients such as alkaloids, cardiac glycosides, flavonoids, saponins, steroids, tannins, terpenoids, phenols, protein and carbohydrates according to the standard method.
Saponin was found in all the sample extract. Tannin was found only in the aqueous leaf extract. The steroid was present in aqueous, methanol and petroleum ether leaf and stem extract. Flavonoid was found in Aqueous, methanol and petroleum ether leaf and stem extract along withbenzene stem extract. Alkaloids werefound in aqueous and benzene leaf and stem extracts along with chloroform leaf extract. Cardiac glycoside wasfound in aqueous, chloroform and petroleum ether leaf and stem extract along with methanol stem extract. Terpenoid was found in aqueous, methanol and chloroform leaf extract along with methanol and petroleum ether stem extract. Phenol was present in methanol and benzene leaf extract. Carbohydrate was found in methanol leaf and stem extract. Protein was found in the aqueous stem, leaf and stem methanol extract (Fig-02; Table No-01).
Many investigations reported these phytoconstituents to have many therapeutic applications, so it is expected that these species have many medicinal uses. Our findings are comparable to those obtained by Singh

Synthesis of silver nanoparticles fromGymnemasylvestre
The silver nanoparticles were synthesized using an aqueous extract of stem of Gymnemasylvestre. UV-Visible spectral analysis showed an absorbance peak at 430 nm with special reference to the excitation of surfaces plasmon vibration by silver nanoparticles. FTIR analysis of nanoparticles revealed the presence of molecular functional groups such as amides, amines, phenolic compounds, and alkenes. These phytochemicals act capping and stabilizing agents for silver nanoparticles.XRD patterns also suggested the occurrence of crystalline silver ions.

UV-Visible analysis of synthesizedGymnemasylvestreAgNPs
UV-vis absorption spectra of colloidal solution of stem aqueous extract and silver nitrate solution have been depicted inFig-04.Originally the silver nitrate solution was colourless but after addition of aqueous stem extract, it turned as brown. Change in colour of the solution from colourless to brown strongly indicated the synthesis of silver nanoparticles in stem extract medium. The colour of the solution was varied with the increasing incubation time as colourless, yellowish-brown, brown and deep brown. The increase of incubation period increases the reduction rate of silver ions, this gives a higher concentration of silver nanoparticles have a deep brown solution. A single and strong absorption spectrum at 419 nm is characteristics surface plasmon resonance (SPR) peak of silver nanoparticles and hence it confirmed their formation. The strong and sharp absorption spectra are because of surface plasmon vibrations of silver nanoparticles in the visible region.

FTIR Spectral analysis of GymnemasylvestreAgNPs
FTIR spectrum is a very useful tool for the discovery of the possible bio-molecules interactions in the silver nanoparticles of stem extract of Gymnemasylvestre. FTIR spectrum was applied in the range of 400 -4000 cm -1 .The wide-ranging infrared spectrum of aqueous stem extract of Gymnemasylvestrehas been presented in Fig-05& Table  No Our results from FTIR spectral analysis suggested that phytochemicals like-flavonoids, terpenoids and polyphenols are found in the stem extract were mainly responsible for the reduction process of silver ions into nanoscale silver particles and protein present in extract could act as capping agent that help in the stabilization the silver nanoparticles of stem extract of Gymnemasylvestre. Previous research works made bySuprajaet al., 2017 and Gomathiet al., 2020 may be correlative with our observations and also supported our findings.

XRD analysis ofGymnemasylvestreAgNPs
For confirming the synthesis of silver nanoparticle as a crystalline form, the XRD study was performed with the diffraction from 10° to80° at 2θ angles. Figure 06 showed the XRD graph of Gymnemasylvestre silver nanoparticles. X-Ray Diffraction study provides various size-dependent features of crystalline nature of silver nanoparticles likeheight, width and peak position. Four different and important characteristic peaks were observed at the 2θ of 23.59°, 27.99°, 32.26° and 46.23° that correspond to (548), (967), (2425) and (1123)heights indicating that SNPs are highly crystalline respectively. In XRD pattern analysis a peak at 32.26°exhibited the synthesis of pure silver nanoparticles.Crystalline size of the silver nanoparticles was determined by the help of Scherrer's formula and foundto 39.5 nm.
Similarly, Suprajaet al., 2017 characterized silver nanoparticle with the help of XRD analysis and reported that silver nanoparticles synthesized from leaves extract of Gymnemasylvestre were highly crystalline. Investigation 908 revealed that five important and different characteristic peaks were observedat the 2θ of38.6°, 45.8°, 58.7°, 64.8° and 78.6°correspond to(111), (200), (220), (311) and (222) respectively.Gomathiet al., 2020studied XRD crystalline analysis of silver nanoparticle of Gymnemasylvestreand observed three different and important characteristic peaks at the 2θ of45, 65and 77correspond to(200), (220) and(222) planes of Ag crystal. The study revealed that the mean particle size of silver nanoparticles decreases from 28 nm to23 nm abouta rise in extract volume from 5 to 15 ml.Pingaleet al., 2018 performed the plant-mediated synthesis of silver nanoparticles from aqueous extract of stem and root of Gymnemasylvestre and found that the diffraction peaks of therange of 2θ (20-80°) correspond to (111) (200) (220) and (311) planes and average crystalline size of silver nanoparticles was 25.3 nm. Thus by and large our findings are affirmative to the previous author and authenticate the pharmaceutical features of Gymnemasylvestrefor further pharmacological applications.   Methanol stem extract 2 0.90 3.
Benzene stem Extract 2 0.33  2929.17 C-H stretching of methylene groups of the protein or alkenes 3.
1630.05 C=C stretching of alkene and amide 4.
1334.03 C-N stretching of amines and amides 5.
1190.83 C-N stretching of amines and amides 6.

Conclusion:-
In the present study, we have successfully screened qualitative phytochemical analysis and synthesized the green silver nanoparticles from Gymnemasylvestre stem extract.The phytochemical analysis of stem and leaves extract showed the presence of important secondary metabolites such as alkaloids,cardiac glycosides,flavonoids,saponins,steroids,tannins, terpenoids, carbohydrates,phenols and proteins.The biologically synthesized silver nanoparticleswere characterized through UV-Visible Spectroscopy, FTIR analysis, andXRD analysis. Bioactive secondary metabolites found in the aqueous extract of Gymnemasylvestrehave to reduce and caping properties that's why aqueous extract is suitable for green synthesis of silver nanoparticles within small times of 30 minutes. UV-Visible spectrum analysis confirmed the optical properties of nanoparticles based on surface plasmon resonance (SPR)responsible for the colour change of the medium. FTIR analysis confirmed the presence of specific functional groups such as alkenes, amines, amides, alcohols, phenols, aromatic and halo compounds.

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Crystalline nature and size of silver nanoparticles were determined by the help of XRD pattern. XRD analysis showed the size of silver nanoparticles was 39.5 nm obtained by Scherrer's formula.