chromatography based metabolomics and in silico gymnema … · 2019. 7. 30. · researcharticle...

Research ArticleChromatography Based Metabolomics and In Silico Screening ofGymnema sylvestre Leaf Extract for Its Antidiabetic Potential

Shabana Parveen,1 Mohd Hafizur Rehman Ansari,2 Rabea Parveen,1 Washim Khan,2

Sayeed Ahmad ,2 and Syed Akhtar Husain1

1Human Genetics Laboratory, Department of Bioscience, Jamia Millia Islamia, New Delhi 110025, India2Bioactive Natural Product Laboratory, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

Correspondence should be addressed to Sayeed Ahmad; sahmad [email protected]

Received 10 June 2018; Revised 3 October 2018; Accepted 6 December 2018; Published 6 January 2019

Academic Editor: Mohammed S. Ali-Shtayeh

Copyright © 2019 Shabana Parveen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Gymnema sylvestre, popularly known as gurmar, is extensively used in traditional systems of medicine for diabetes, stomachailments, liver diseases, and cardiac disorders. Dried leaf powder of G. sylvestre was extracted through soxhlation using 70% (v/v)alcohol. The hydroalcoholic extract was concentrated to 1/4th of its volume and basified to isolate gymnemic acid enriched extractusing chloroform. The isolated extract was checked for its antioxidant potential against 1, 1-diphenyl-2-picryl-hydrazyl (DPPH),which showed scavenging activity of 82.31% at 80 𝜇g/mL of extract. Quality control analysis of the extract was carried out by TLC.Chloroform andmethanol (9.5:0.5, v/v) were used as a solvent system and separated compoundswere detected at 254 and 366 nm.Atotal of 13 metabolites were separated. However, major peaks were at Rf 0.12, 0.69, 0.79, and 0.85. Further, UPLC-MS fingerprintingof the extract was done using acetonitrile and 0.5% formic acid in water as mobile phase in gradient elution mode. A total of 21metabolites were separated and tentatively identified from the database. Deacyl gymnemic acid and quercetin are the two majormetabolites found in the extract. Gymnemic acid, deacyl gymnemic acid, and quercetin were docked with ten different proteinsassociated with glucose metabolism, transport, and glucose utilization. It has been observed that gymnemic acid was more potentthan deacyl gymnemic acid in terms of binding affinity towards proteins and showed a favorable interaction with amino acidresidues at the active site. Thus, the present study gives an insight of identified metabolites with protein interaction and a reasonfor the hypoglycemic potential of deacyl gymnemic acid enriched extract, which can be further explored for in vitro and in vivostudies to establish its phytopharmacological and therapeutic effect.

1. Introduction

Diabetes mellitus (DM) describes a metabolic disorder char-acterized by a deficiency in insulin production and its actionor both [1]. It is thriving distributed in nearly all countriesand constantly increases in numbers and implication, asvarying quality of life lead to reduced physical activity andincreased obesity in populations. That leads to prolongedhyperglycemia with variabilities in most metabolic processesinside the human body [2]. As per global concern WorldHealth Organization (WHO), 347 million people worldwideare suffering from DM, with the estimate that it will be theseventh leading cause of death in 2030. A total of 1.5 milliondeaths are directly triggered by diabetes in 2012. It was the

eighth leading cause of death among both sexes and the fifthleading cause of death in women. When chewed, the freshleaves of G. sylvestre have the outstanding property of para-lyzing the sense of sweet taste substance for few times. Thegymnemic acid molecules in terms of atomic arrangementsare analogous to that of glucose molecules. These types ofmolecules fill the receptor location on the taste buds therebystopping its activation by sugar molecules existing in thefood. This, up-to-date study showed that the most generalmedicinal plants with remarkable antidiabetic importance interms of their mechanism and modes of action together withthe methodology part used for their quality, safety, and effi-cacy assessment to explore the biological standardization ofthousands of traditionally used medicinal plants both in vitro

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019, Article ID 7523159, 14 pageshttps://doi.org/10.1155/2019/7523159

http://orcid.org/0000-0003-1573-152Xhttps://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://doi.org/10.1155/2019/7523159

2 Evidence-Based Complementary and Alternative Medicine

and in vivo metabolomics approach with chromatographicprofiling to assess the claimed activity with the aim of findingpotent antidiabetic markers from the natural resources [3].In Indian systems of medicine, i.e., Ayurveda, the G. sylvestreprominently used in the therapy of dyspepsia, constipation,and hyperglycemia [4] hemorrhoids, jaundice vesicle, renalcalculi, asthma, cardiopathy [5] amenorrhea, bronchitis, andleukoderma [6, 7]. The ethanolic extract of G. sylvestreleaves showed the presence of eleven different isoforms ofgymnemic acidswith differentmolecular weights (gymnemicacid I to gymnemic acid XI). The major phytoconstituentsfound in G. sylvestre are gymnemic acid (GA), gudmarine,and saponins. Gymnemic acid is a pentacyclic triterpenoid,the main active principle displaying antidiabetic activity [6].The plant derived extract of G. sylvestre has already reportedto have direct insulinotropic activities on 𝛽 cells and isolatedislets of human in vitro [8]. Moreover, antidiabetic potential,G. sylvestre, has the capability of total cholesterol and lowertriglyceride in serum and its antiatherosclerotic potentialwere almost similar to that of a standard lipid-lowering agentclofibrate. Some studies reflected the ability of G. sylvestre toinhibit the formation of advanced glycation end products andsorbitol accumulation [9].

Due to the presence of specific metabolites, it has beenused for different therapeutic purposes. Furthermore, theseherbal materials have the significant application for variousphytopharmacological applications. Several herbal prepara-tions containing the dried leaves of G. sylvestre or its extractare being used for various therapeutic purposes. These plantmaterials are being used in traditional system of medicine fordifferent disease especially in diabetes. Antidiabetic potentialof G. sylvestre leaves has been reported but its metabolomiccharacterization has not been fully explored. Less scientificdata are available on the bioactive metabolites responsible forits antidiabetic activity. In our study, we have qualitativelyanalyzed the number and category of metabolites present inextract through LC-MS and identified the bioactive metabo-lites through in silico screening. Further, we have tested thathydroalcoholic extract has been tested for its antidiabeticpotential using in vitro and ex vivo approaches. In this contextour study provides solid scientific evidence in support of itsantidiabetic activity. We have authenticated and extracted theleaves of G. sylvestre.

2. Methodology

2.1. Plant Material and Extract Preparation. The leaves of G.sylvestre obtained from Botanical Garden of Jamia Hamdard,New Delhi, and authenticated as per the standard protocolspecified in Ayurvedic Pharmacopoeia. The authenticatedplant materials have been deposited in the Bioactive NaturalProduct Laboratory for future reference with a voucher spec-imen number JH/SPER/BNPL/Shabana/2014/GS. The plantsample was washed, shade dried, and coarsely powdered.The powdered drug materials (200 g) of G. sylvestre weredefatted with petroleum ether and extracted through soxh-lation using 70% (v/v) alcohol for 24 h. The hydroalcoholicextract was concentrated to 1/4th of its volume by rotary

vaporization under reduced pressure.The extract was filteredand subjected to basify to isolate gymnemic acid enrichedextract using chloroform. The extractive value and % yield ofextract were calculated and stored at 4∘C for bioactivity andquantitative analysis.

2.2. Total Phenolic and Flavonoid Content. Through theFolin-Ciocalteu method, the total phenolic content in thehydroalcoholic extract ofG. sylvestrewas determined accord-ing to the procedure described in the literature [10]. Differentconcentrations of gallic acid solutions (as the standard equiv-alent of phenol) were used for establishing the calibrationcurvewhichwas further used for the determination of phenolcontent. All the experiments were carried out in triplicate.The obtained regression equation from the calibration plotwas used for the determination of total phenolic content andexpressed as mg of gallic acid equivalent per gram of extract.Aluminum chloride (AlCl

3) colorimetric method was used

for the determination of total flavonoid content [10]. Thetotal flavonoid content in the hydroalcoholic extract wascalculated from a calibration curve of standard (rutin) byusing its different dilutions concentrations ranging from 10to 100𝜇g/mL. The total flavonoid content was expressed asmg/g of rutin equivalent.

2.3. Determination of Antioxidant Potential. The antioxidantpotential of the extract was determined by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. The stock solution of differentconcentrations of extract (10, 20, and 40 𝜇g/mL) was mixedwith 1.0mL of a methanolic solution of DPPH (1.0 𝜇g/mL)and incubated for 30min in dark at room temperature.Then, the absorbance was recorded at 517 nm using a UV-visible spectrophotometer. Trolox was used as standards forcomparison.

2.4. In Vitro Carbohydrate Digesting Enzyme Inhibition Assay.For the determination of the 𝛼-amylase inhibitory potentialof extract, previously developed method was followed [10].Briefly, accurately weighed 5mg of enzyme was dissolved in10mL of 20mM phosphate buffer (pH 6.9) at 37∘C, whilethe extract was dissolved in dimethylsulfoxide and diluted inphosphate buffer. Different concentration of extract rangingfrom 100 to 1000𝜇g/mL was used for the determination of𝛼-amylase inhibition potential. One mL of diluted extractand 1.0mL of enzyme solutions (0.5mg/mL) were mixedtogether and incubated at room temperature for 30 min. Afterthe completion of incubation, 1.0mL of 0.5% (w/v) starchsolution was added to the mixture and then again kept for10min at room temperature. About 2.0mL of dinitrosalicylicacid was added to the reaction mixture and heated in boilingwater for 5min to stop the previously ongoing reaction. Theresulting mixture was cooled and absorbance was measuredcalorimetrically at 565 nm.

For the determination of 𝛼-glucosidase inhibitory poten-tial of extract, previously reported procedure [10] wasfollowed. Extract prepared for 𝛼-amylase assay was usedfor 𝛼-glucosidase assay also. While an enzyme solution(1.0U/mL) was prepared in 10mM phosphate buffer (pH

Evidence-Based Complementary and Alternative Medicine 3

6.8). Briefly, the 100 𝜇L of diluted extract and 200𝜇L ofenzyme solution were incubated at 37∘C for ten minutes.Then, 100𝜇L of p-nitrophenyl-𝛼-D glucopyranoside (PNPG)solution (5.0mM) in 10mM phosphate buffer (pH 6.8) wereadded to start the reaction and the mixture was incubatedat 37∘C for 30min. Then the reaction was stopped after theaddition of 2.0mL of 0.1M sodium carbonate (Na

2CO3).

Finally, the absorbance was recorded at 405 nm of the yellowcolored p-nitrophenol freely released from p-nitrophenyl-𝛼-D-glucopyranoside. Acarbosewas used as the positive controland the results were expressed as the inhibition rate (%) ofenzymatic activity and figured by the beneath equation:

% Inhibition of enzyme activity

= (Abscontrol − Abssample) ×100

Abscontrol

(1)

where Abscontrol is the OD of reaction without extract orstandard and Abssample is the OD of the reaction of withextract or standard.

2.5. ExVivoGlucose Uptake. The inhibition assay of intestinalglucose uptake was determined in rat hemidiaphragm. Aboutovernight fasted rats were used for this assay. Animals wereeuthanized by anesthesia and dissected to isolate diaphragm.It was immediately dipped in the ice-cold Krebs-Henseleitbuffer which was previously equilibrated with 95% oxygen,5% carbon dioxide in a cylindrical vessel of organ bath. Theextract was added in the same compartment and incubated atroom temperature for one hour. Further, glucose solution wasadded and again incubated for 30min at room temperature.After the completion of incubation, a sample from thesupernatant was collected and the unabsorbed glucose wasestimated using glucose estimation kit which was commer-cially utilized. Extracts with different concentrations rangingfrom 25 to 100𝜇g/mL were used. Inhibition of glucose wasmeasured by the following formula:

Inhibition of glucose = (𝐶2 − 𝐶1)𝑔 of hemidiaphragm (2)

where C2 and C1 were final and initial glucose concentra-tion after the incubation.

Skeletal muscle was isolated from the dissected animalsand used for glucose uptake assay. The previously standard-ized protocol was used to check the effect of the extracton glucose uptake in isolated rat skeletal muscle [11]. Itwas immediately dipped in ice-cold Kreb’s buffer which waspreviously equilibrated with 95% oxygen and 5% carbondioxide in a cylindrical vessel of organ bath. The musclewas with buffer for three times. The extract was added inthe same compartment and incubated at room temperaturefor one hour. Further, glucose solution (20mM) was addedand again incubated for 30min at room temperature. Incontrol reaction, only glucose was added while, in caseof test reaction, extract with different concentration (25-100𝜇g/mL) was used separately. Metformin standard wasused as positive control. Before and after the completionof incubation, 1.0mL of supernatant was collected and the

unabsorbed glucose was estimated using glucose estimationkit which was commercially utilized. The amount of glucoseuptake by per gram of muscle was measured by the followingformula:

Muscle glucose uptake = (𝐶1 − 𝐶2)𝑔 of muscle tissue (3)

where C1 and C2 are the concentrations of glucose before andafter incubation, respectively.

2.6. Effect of Extract on Glucose Uptake in Yeast Cells. Com-mercially utilized baker’s yeast (Saccharomyces cerevisiae) wasobtained from Institute of Microbial Technology, Chandi-garh, India, and used for this assay. The obtained microbeswere subcultured in potato dextrose agarmediumand furthersuspension culture was prepared in potato dextrose broth.The culture medium was centrifuged at 3,000×g for 5minand the cell pellet was washed in distilled water until thesupernatant fluids were clear. About 10% v/v of cell pelletsuspension was prepared with the supernatant fluid. OnemL of the glucose solution was added with 1mL of extractand incubated at 37∘C for 10min. The reaction was startedby adding 100𝜇L of yeast suspension to the above mixture.The resulting reaction mixture was vortexed and incubatedat 37∘C for 60min. After the completion of the mixture, itwas centrifuged and the glucose concentration was measuredfrom the supernatant [10]. Different concentrations of glucose(5, 10, and 25mM) and extract (250, 500, 750, and 1000𝜇g)were tested. The percentage increase of glucose in yeast cellwas determined using the following equation:

Percentage increase in glucose uptake

= (Abssample − Abscontrol) ×100

Abssample

(4)

where Abscontrol is the reaction without extract and Abssampleis the reaction with extract.

2.7. TLC Fingerprinting of Extract. The hydroalcoholicextract was dissolved in HPLC grade methanol and filteredthrough 0.25𝜇M membrane filter. The chromatographyanalysis was performed on aluminum TLC plates coatedwith 0.2𝜇M layers of silica gel 60F

254(Merck Millipore,

Germany). Samples were applied with 4.0mm wide bandand 8.3mm gap between each band by the use of a LinomatV sample applicator (CAMAG, Switzerland). The sampleconcentration was 10mg/Ml and 5.0𝜇L sample was appliedwith a constant sample application rate of 5.0 𝜇L. Forthe best separation of metabolites, chloroform: methanol(95:05, %v/v) was used as a mobile phase and the platewas developed in a 20 × 10 cm twin-trough glass chamberwith linear ascending mode up to 80mm. Further, the platewas removed from chamber and air dried. The developedplate was scanned at two different wavelengths, i.e., 254 and366nm with a TLC scanner III (CAMAG, Switzerland) withslit dimension of 4.0 × 0.30mm, and the scanning speed was10mm/s.The sample application and scanning were operated


by winCats software. The developed method was validated asper the ICH guidelines for quality control of herbal drugs andbotanicals [12, 13]. The peak areas of triplicate samples wereused for analyzing the metabolic diversity of hydroalcoholicextract.

2.8. Ultraperformance Liquid Chromatography-Mass Spec-trometry Analysis of the Extract. Water’s ACQUITY UPLC�system (Serial No. #F09 UPB 920M; Model code # UPB;Waters Corp., MA, USA) equipped with a binary solventdelivery system, column manager, an auto sampler, and atunable MS detector (Serial No # JAA 272; Synapt; Waters,Manchester, UK) was used for UPLC-MS analysis of extract.The extract was chromatographically separated in previouslydegassed mobile phase consisting of 0.5% v/v formic acidin water (A) and acetonitrile (B) in gradient elution mode(initially 100%A and hold for 5min; further, decreased to 5%A in 20min). Water’s ACQUITY UPLC� BEH C18 (100.0 ×2.1mm × 1.7 𝜇m) column was used and flow rate of mobilephase was 0.4 mL/min. The column manager and samplemanager temperature were set to 35 ± 2∘C and 25 ± 2∘C,respectively. About 10 𝜇L of sample was injected with the splitmode of 5:1 with the help of autoinjector and the pressure ofthe system was set to 15000 psi.

The separated metabolites were detected by MS detectoron a quadruple orthogonal acceleration time of flight tan-dem mass spectrometer (Waters Q-TOF Premier TM). Thenebulizer gas and cone gas were set to 500 L/h and 50 L/h,respectively. The source temperature of MS detector was setto 100∘C. The capillary voltage and cone voltage were set to3.0 kV and 40 kV, respectively. For collision of ion, argongas was used at a pressure of 5.3 × 10−5 Torr. The Q-TOFPremier� was operated in scan mode with resolution over8500 mass with 1.0min scan time and 0.02 s interscan delay.Both UPLC and the mass detector were operated by usingMass Lynx V 4.1 software incorporated with the instrument.The separated compounds were identified based on their m/zvalue through literature survey [10].

2.9. In Silico Screening. To understand the binding mecha-nisms of active constituents of gymnema leaves, molecularmodelling studies were accomplished for deaclgymnemicacid, gymnemic acid, quercetin, and the aglycone moietygymnemagenin with target proteins by the mopac 6 soft-ware package (Stewart Computational Chemistry, ColoradoSprings, USA). Different proteins were presumed to interactwith targeted molecules (deaclgymnemic acid, gymnemicacid, quercetin, and gymnemagenin). Nine different pro-teins such as (dipeptidyl peptidase, glucokinase, glutaminefructose-6-phosphate amidotransferase, AMPkinase, GLUT-2, stearoyl-coA desaturase, GLUT-4, sulfonylurea receptor,and mitochondrial Na+/K+ exchanger) were selected fordocking analysis. All the docking calculations were achievedon different protein models. In AutoDock tools, solvationparameters, essential hydrogen atoms, and Kollman unitedatom type charges were added. Autogrid program wasemployed for generation of affinity (grid) maps of × Å gridpoints and 0.375 Å spacing. Van der Waals and the electro-static terms were generated by AutoDock parameter set and

distance dependent dielectric functions, respectively. Simula-tions of docking were executed using the Lamarckian geneticalgorithm and the Solis andWets local search method. Initialposition, orientation, and torsions of the ligand moleculeshave been randomly selected. During docking, all rotatabletorsions were released. A translational step of 0.2 Å was used,whereas 5 quaternion and torsion steps were utilized in eachsearch. In each docking experiment, two different runs wereset and it was terminated after the assessment of maximum250000 energy was reached. The structure of molecules inmol format was generated in the CDX format using thetool ChemDraw Ultra 7.0.1 (CambridgeSoft Corporation,Cambridge, USA) and transformed to input ligand format(pdb) for docking by OpenBabel version 2.3.2 Open Babel:An open chemical toolbox (Journal of Cheminformatics 2011,3:33).

3. Results

The dried leaves of G. sylvestre were defatted through petether (60-80∘C). The hydroalcoholic extract (70%) was pre-pared from soxhlation process. The percentage yield of 70%hydroalcoholic extract was found to be 24.30%w/w. Theextract was filtered and basified to isolate the gymnemicacid enriched fraction by successive solvent selection processusing chloroform with 1.31%w/w yield. Further, the extractwas dried and stored at 4∘C until use.

3.1. Phenolic and Flavonoid Content of Extract. The totalphenolic and flavonoid content of the hydroalcoholic extractwas determined from the calibration curve of gallic acid(r2 = 0.998) and rutin (r2 = 0.989), respectively. The totalphenolic content was found to be 29.36 mg of gallic acidequivalents per gram of extract, while flavonoid contentwas 18.65 mg of rutin equivalents per gram of extract. Thisextract is enriched with phenols and especially flavonoidswhich are mainly responsible for the antioxidant potentialof extract. The antioxidant activity was due to the presenceof free hydroxyl group present in flavonoids of extract. Freehydroxyl group scavenges the free radicals caused betterantioxidant potential. The antioxidant activity of flavonoids,which include flavones, flavanols, and condensed tannins,depends on the presence of free (OH) hydroxyl groups, espe-cially 3-OH, since the present report of antioxidant activityof hydroalcoholic extract suggesting a complete profilingvia phytochemical and metabolomics profiling needs to bedone to identify the other active phenolic and flavonoidcomponents in the field of drug discovery and development.

3.2. Antioxidant Potential. Due to the simplicity in a bio-chemical reaction, the free radical scavenging activity ofany plant extract is commonly used to determine by DPPHradical. Hydroalcoholic extracts are the rich sources for phe-nols and flavonoids, which are having redox properties withantioxidant potential. Our phytochemical screening revealedthat it has the major abundance of phenolic and flavonoidmetabolites. The hydroalcoholic extract had clearly shownthe strong antioxidant potential against all free radicals. The


25 g/mL 50 g/mL 100 g/mLExtract concentration

-amylase-glucosidase

0

10

20

30

40

50

60

70

80

90%

of i

nhib

ition

Figure 1: Carbohydrate digesting enzyme (𝛼-amylase and 𝛼-glucosidase) inhibition potential of G. sylvestre extract.

results clearly showed that DPPH scavenging activity was14.84 ± 0.12, 21.14 ± 0.20, and 34.36 ± 0.45 of mMTR equiva-lent, at a concentration of 10, 20, and 40 𝜇g/mL of G. Sylvestreextract, respectively, while that of the control, i.e., ascorbicacid was 41.36mMTRwhen the concentration was 35𝜇g/mL.The antioxidant potential which was equivalent to DPPHscavenging was increased with respect to the extract concen-tration and it was increased to a concentration of 40 𝜇g/mL.Beyond the level of extract used, no increment in antioxidantpotential was observed. The hydroalcoholic extract enrichedwith flavonoids which have the potential to scavenge thefree radicals associated with different ailments and biologicalcycle, singlet oxygen, and other oxidizing molecules. Apartfrom the scavenging the free radicals, flavonoids suppressthe production of reactive oxygen species, quenched thetrace elements, and upregulate antioxidant defenses whichare directly involved in the production of free radical. Similaractions were also reported in extract enriched with phenoliccontent.

3.3. Carbohydrate Digesting Enzyme Inhibition Potential.The different concentration of 25𝜇g/mL, 50 𝜇g/mL, and100𝜇g/mL of hydroalcoholic extract clearly showed that the% inhibition of 𝛼-amylase and 𝛼-glucosidase activity con-sistently increases with concentration-dependent manner,respectively.The hydroalcoholic extract has clearly shown theremarkable potential of carbohydrate-digesting enzyme at aspecific concentration. Carbohydrate digesting enzyme (𝛼-glucosidase) inhibitory potential significantly increased withincrease in the concentration of hydroalcoholic extract. Thecarbohydrate enzyme inhibition potential of extract has beenshown in Figure 1.

3.4. Yeast Cell Uptake. By facilitated diffusion process inbaker’s yeast follows glucose transport process. The processin which glucose was uptaken by skeletal muscle was similar

10 mmol/L20 mmol/L30 mmol/L

0

50

100

150

200

250

% in

crea

se o

f glu

cose

upt

ake

0.1 0.2 0.3 0.4 0.5 0.60Extract concentration (mg/mL)

Figure 2: Effect of extract on glucose uptake in yeast cell uptake.

to the process glucose transport in a yeast cell. After spe-cific incubation time period in experimental medium, theamount of the glucose remained in the process of glucoseuptake by the yeast cell. It consistently increased in a dose-dependent manner. Figure 2 clearly shows that the percentincreases in glucose uptake in yeast cells at different glucoseconcentrations, i.e., 10mmol/L, 20mmol/L, and 30mmol/Lwith respect to concentrations of extract. A concentration-dependent glucose uptake was increased in a yeast cell in thepresence of extract. In case of positive control, metforminwas used and it also showed the increased glucose uptakein yeast cell. However, hydroalcoholic extract showed greatereffectiveness in glucose uptake by yeast cells as comparedto positive control. Glucose uptake in yeast cell followeddiffusion process which was similar to glucose uptake inskeletal muscle. Thus, these results indicate that hydroal-coholic extract will increase the glucose uptake in skeletalmuscle or it will cause an increment in peripheral glucoseutilization, while a significant difference in glucose uptake inyeast cell was observed when it was incubated with extract ascompared to yeast cell incubated with metformin.

3.5. Ex Vivo Antidiabetic Potential. Generally, researchershave used the isolated diaphragm to check the effect ofmetabolites on the intestinal glucose inhibition. In our study,we have checked the effect of hydroalcoholic extract of G.sylvestre leaves on intestinal glucose absorption. A markeddecrease in glucose absorption in the intestine by extract wasrecorded as compared to control. A dose-dependent glucoseabsorption inhibition was recorded (Figure 3). A maximumglucose absorption inhibition (76.25%) has been observedat a concentration of 100𝜇g/mL of extract. Similar resultswere obtained in glucose uptake assay in skeletal muscle.Theeffects of the extract on glucose uptake in isolated rat skeletalmuscle are shown in Figure 3. A maximum 28.6% glucoseuptake was increased at a dose of 100 𝜇g/mL of extract ascompared to control.


25 50 100 MetforminExtract concentration (g/mL)

% Inhibition intestinal glucose uptake% Increase skeletal muscle glucose uptake

0

10

20

30

40

50

60

70

80

90

% C

hang

e

Figure 3: Effect of extract on intestinal absorption inhibition andpercentage glucose uptake in skeletal muscle.

Table 1: TLC fingerprinting of hydroalcoholic extract of G. sylvestreleaves.

S. No. RfArea%

At 254nm At 366nm1. 0.01 0.98 1.102. 0.03 - 1.303. 0.05 2.20 1.964. 0.07 - 20.235. 0.12 40.31 22.516. 0.16 3.00 2.547. 0.23 2.49 2.228. 0.25 1.25 -9. 0.31 2.14 2.9810. 0.53 - 1.9711. 0.60 3.28 8.5312. 0.69 13.70 23.6213. 0.79 14.72 8.7914. 0.85 9.99 2.2515. 0.91 5.94 -Total 15 12 13

3.6. TLC Fingerprinting Analysis. For TLC fingerprintinganalysis, hydroalcoholic extract was dissolved in methanol,filtered, and analyzed through 0.25𝜇M membrane filter.Chromatographic separation was performed by using chlo-roform: methanol: formic acid, 9:5:0.5, v/v/v, as the mobilephase. The developed plate was scanned at two differentwavelengths, 254 nm and 366nm (Figure 4). The TLC anal-ysis of hydroalcoholic extract clearly showed the separationof total 15 metabolites. Scanning at 254 and 366nm, anumber of compounds analyzed are 12 and 13, respectively(Table 1). However, major components in terms of peaks areain chromatogram were found at Rf 0.12 (40.3%), 0.69 (13.7%),0.79 (14.72%), 0.85 (9.99%)and 0.91 (5.94%) at 254 nm.Whileat 366 nm, major compounds found at Rf 0.07 (20.23%), 0.12

(22.51%), 0.60 (8.53%), 0.69 (23.62%), and 0.79 (8.79%). Onemajor peak was found at Rf 0.07 (20.23%) while scanning at366 nm but this peak was not found at 254 nm. Similarly, onepeak at Rf 0.91 (5.94%) was found when scanned at 254 nmbut found absent at 366 nm.

3.7. UPLC-MS Analysis. All the metabolites present in thehydroalcoholic extract were dissolved inmethanol and, basedon this assumption, the methanolic solution was analyzedthroughUPLC-MS for their completemetabolic profiling andidentification of diversity of metabolites. Table 2 summarizesall the metabolites characterized in G. sylvestre extract elutedat different retention times, experimental m/z, and tentativenamewith nature of compounds. A total of 58most abundantmetabolites were analyzed and identified through m/z valueand from literature survey. By comparing chromatogram ofblank with the chromatogram of extract, a clear and contrastchromatogram was observed based on their retention time(Figure 5). LC-MS fingerprinting of the G. sylvestre extractwas done using 0.5% v/v formic acid in water (A) and ace-tonitrile (B) as mobile phase in gradient elution mode. A totalof 58 metabolites were separated and tentatively identifiedfrom the database. Deacyl gymnemic acid and quercetin werethe two major metabolites found in the extract with m/zvalue of quercetin, conderitol, deacylgymnemic acid, niacin,ascorbic acid, rutin, kaempferol, niacin, D-quercetin, andstigmasterol. Mass spectra of major metabolites have beenshown in Figure 6.

Major groups of tentatively identified metabolites arealkaloids (25.8%), amino acids (1.7%), coumarins (5.2%), fattyacids (5.2%), flavonoids (25.8%), glycosides (5.2%), lignans(3.5%), lipids (3.4%), nucleosides (5.2%), phenols (5.2%),terpenoids (13.7%), and vitamins (5.2%) (Figure 7). Ourinvestigations revealed that the hydroalcoholic composedof major phenolic and flavonoid metabolites which areresponsible for its therapeutic potential. Thus, for the analysisof low and high abundant metabolites with wide polarityrange, UPLC-MS method seems to be the best method ofanalysis.

3.8. In Silico Screening. Gymnemic acid, deacyl gymnemicacid, and quercetin were docked with ten different proteinsassociated with glucose metabolism, transport, and glucoseutilization. The docking of tested metabolites with targetedproteins was therefore performed, and corresponding fitnessscores were determined. High fitness scored metaboliteswere subjected to the elucidation of their interaction surfaceand total intermolecular energy with targeted moleculesand proteins separately [14]. Four ligands (deacyl gymnemicacid, gymnemagenin, gymnemic acid, and quercetin) wereselected for this study. All these metabolites showed goodinteraction with glucose transporter and deacylgymnemicacid showed better affinity as compared to other metabolites.Almost all the compounds showed good affinity and theestimated free energy of ligand-protein interaction was found< -5.0 for the receptor proteins in this interaction indicatingthat affinity of these proteins towards targeted metabolitesmight be changed after oral administration of extract. It


Table2:UPL

C-MSfin

gerprin

tingprofi

leof

hydroalcoh

olicextracto

fG.sylvestre.

Metab

olite

sm/z

Nam

eClassof

compo

und

Reference

M1

350.15

And

rographo

lide

Labd

anetriterpeno

idBM

L80745

M2

367.2

3Cu

rcum

inFlavon

oid

TY00

0081

M3

457.17

(-)-Ep

igallocatechin

gallate

Polyph

enolcds

TY00

0083

M4

293.2

Gingerol

Terpenoids

CO00

0211

M5

267.14

3-Hydroxy-3-m

etho

xyflavone

Flavon

oids

BML8

0385

M6

351.2

3Ajmalicine

Indo

lealkaloids

FIO00

001

M7

277.2

Ascorbica

cidmagnesiu

mph

osph

ate

Vitamin

PubC

hem

CID:10

1614363

M8

147.14

Con

duritol

Polyph

enolcds

PubC

hem

CID:136345

M9

365.21

Isop

entenyl-A

denine-7-glucosid

ePy

ridinea

lkaloids

CE00

0239

M10

251.15

(3aR

)-(+)-Sclareolide

Terpenoids

BML8

0075

M11

303.07

Dihydroqu

ercetin

Flavon

oids

BML8

1120

M12

349.2

2Strychnine

NOxide

Labd

aned

iterpenoid

CO00

0416

M13

275.3

Eserine

KO00

8958

M14

323.2

Quinine

Quinidine

alkaloid

BML82035

M15

230.27

7-Diethylam

ino-4-methylcou

marin

Cou

marin

SM884302

M16

335.24

Berberine

Isoq

uino

linea

lkaloids

KO00

8886

M17

316.31

Capillarisin

Sesquiterpno

ids

TY00

0038

M18

336.24

Lobelin

ePiperid

inea

lkaloid

BML8

1620

M19

365.17

Isop

entenyl-A

denine-7-glucosid

ePy

ridinea

lkaloids

CE00

0239

M20

413.29

S,R-Noscapine

Benzyliso

quinolinea

lkaloid

CE00

0163

M21

291.18

Karanjin

Steroidalalkaloid

BML8

1520

M22

277.2

4Lino

lenica

cid

Steroidalalkaloid

BML8

1605

M23

333.22

Strychnine

Labd

aned

iterpenoid

WA00

0648

M24

302.33

Hesperetin

Flavon

oids

BML8

1380

M25

337.2

58-Geranyloxypsoralen

Furano

coum

arin

BML806

40M26

318.32

Myricetin

Flavon

oids

TY00

0149

M27

275.22

Eserine

KO00

8958

M28

301.16

Hem

atoxylin

Neoflavono

ids

BML8

1375

M29

319.2

5Cop

tisine

Benzo[c]ph

enanthrid

inea

lkaloids

TY00

0106

M30

149.0

5Methion

ine

Sulphu

rcon

tainingam

inoacid

CE00

0452


Table2:Con

tinued.

Metab

olite

sm/z

Nam

eClassof

compo

und

Reference

M31

382.46

Dihydrozeatin-9-beta-D-glucosid

ePu

rinen

ucleosidee

PR020117

M32

279.2

5Lino

leicacid

Unsaturated

fatty

acids

EQ331601

M33

365.29

Isop

entenyl-A

denine-7-glucosid

e-[d6]

Pyrid

inea

lkaloids

CE00

0594

M34

444.43

Bufotalin

(Sapon

in)

Sapo

nin

TY00

0016

M35

124.12

Orcinol

Flavon

oids

BML8

1850

M36

151.15

Cathine

Phenylprop

anes

EQ333501

M37

165.14

D-Q

uercito

lFlavon

oids

PubC

hem

CID:441437

M38

177.0

9Ascorbica

cid

Vitamin

PubC

hem

CID:546

7006

7M39

207.16

Anthraquino

nePu

bChem

CID:6780

M40

251.16

7-Hydroxy-3-m

ethylflavon

eFlavon

oids

BML8

0610

M41

274.31

Phloretin

Dihydrochalchon

e(Ph

enolclass)

TY00

0158

M42

301.18

Quercetin

Flavon

oids

CE00

0168

M43

335.25

Senecion

ine

Pyrrolidizinea

lkaloids

FIO00235

M44

351.2

54-Methylumbelliferylglucuronide

Benzop

yran

alkaloids

CE00

0020

M45

353.26

Chelidon

ine

Phenyliso

quinolinea

lkaloids

CE00

0133

M46

353.3

Asarin

inBe

nzofuran

type

lignan

BML8

0780

M47

413.28

Stigmasterol

Benzofuran

type

lignan

PubC

hem

CID:5280794

M48

429.4

Ono

nin

Isofl

avon

oid

PR0200

43M49

523.39

1-Stearoylglyceroph

osph

ocho

line

Glyceroph

osph

olipids

MT0

00126

M50

549.4

4Quercetin-3-(6-m

alon

yl)-glucoside

Flavon

oid

PR101032

M51

579.4

5Naringin

Flavon

oid

CE00

0186

M52

593.3

Kaem

pferol-3-O

-𝛽-glucopyrano

syl-7

-O-𝛼-rhamno

pyrano

side

Glyceroph

osph

olipids

PR101010

M53

639.3

4Dem

etho

xycentaureidin

7-O-rutinoside

Flavon

oids

BML8

1075

M54

682.4

Deacylgym

nemicacid

IITriterpenoidalsapon

ins

PubC

hem

CID:44144

284

M55

693.52

Rutin

3-m

alon

ate

Flavon

oids

PubC

hem

CID:10556617

M56

763.56

Gym

nemicacid

IVTriterpenoidalsapon

ins

PubC

hem

CID:14264

063

M57

835.59

Triacylglycerol16:0-16:0-18:0

Unsaturated

fatty

acids

UT0

00540

M58

877.5

1Triacylglycerol18:2-18:2-18:2

Unsaturated

fatty

acids

UT0

00521


0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Rf

100

200

300

400

500

600

0

111098

7654

3

21

AU

(a)

11

10

987

6

54

1

100

200

300

400

500

0

450

350

250

150

50

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90Rf

23

AU

12

13

(b)

Figure 4: TLC chromatogram of extract scanned at (a) 254 nm and (b) 366nm.

2.33349.157635

3.91274.3015051

4.56335.245720

4.93333.2312209

6.15333.246073

8.01301.1712214

8.11319.2510127

9.12321.2745105

Time

%

Time Area%1.00 1.541.89 1.772.33 4.342.60 1.022.74 0.202.82 0.263.08 1.333.21 1.663.35 3.573.49 0.693.57 0.843.66 0.933.77 1.88391 8.554.07 0.454.17 0.384.28 0.824.34 0.054.51 1.014.56 3.254.65 1.704.79 2.764.93 6.944.99 1.165.09 0.375.24 0.925.36 0.765.41 0.335.50 0.576.15 3.456.36 1.646.50 0.986.63 0.667.0 2.95

7.32 0.327.42 1.197.52 0.488.01 6.948.11 5.759.12 2562

GM 1 1: TOF MS ES+BPI6.75e5Area

Figure 5: UPLC-MS chromatogram of extract.

has been observed that gymnemic acid was more potentthan deacyl gymnemic acid in terms of binding affinitytowards proteins and showed a favorable interaction withamino acid residues at the active site. Docking summary ofmetabolites docked with proteins associated with diabeteshas been shown in Table 3. Some of the major protein

interactions are shown in Figure 8. Thus, the present insilico screening study gives an insight of tentatively identifiedmetabolites with a specific protein. The data obtained fromin silico screening will help to define/predict the mechanismbehind the antidiabetic potential of metabolites present inthe extract ofG. sylvestre through ligand-receptor interaction


100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100

0

%

100

0

%

100

0

%

100

0

%

100

0

%

100

0

%

100

0

%

GM1 644 (9.117) Cm (640:650)

GM1 573 (8.113) Cm (565:577)

GM1 348 (4.931) Cm (334:348)

GM1 322 (4.563) Cm (315:324)

GM1 277 (3.927) Cm (275:284)

GM1 235 (3.333) Cm (216:235)

GM1 164 (2.329) Cm (162:168)

321.2668

322.2704393.3207 413.2883

1: TOF MS ES+3.49e6

124.1157 149.0529 256.2919 279.2597

301.1679 319.2507

333.2654 365.2902 382.4635 444.4390

1: TOF MS ES+1.12e6

124.1159 151.1513 275.2278

302.3313

318.3255

333.2283337.2592

365.1700413.2960

1: TOF MS ES+7.46e5

124.1162 277.2432316.3101

335.2077335.2440

336.2474 365.1702413.2983

1: TOF MS ES+5.61e5

230.2768

274.3019

275.3046 323.2085 351.2379

1: TOF MS ES+1.23e6

124.1163 147.1475 191.1741 246.2723251.1541 277.2060 303.0775 349.2231

351.2389

365.2178367.2315 457.1772

1: TOF MS ES+5.02e5

267.1495 293.2003

349.1512

350.1547367.2337 457.1769

1: TOF MS ES+5.00e5

Figure 6: Mass spectrum of major abundant metabolites analyzed through UPLC-MS.

Alkaloid24%

Amino acid2%

Coumarin5%

Fatty acid5%

Flavonoid25%

Glycoside5%

Lignan3%

Lipids3%

Nucleosides5%

Phenol5%

Terpenoid13%

Vitamin5%

Figure 7: Categorization of analysed metabolites.


(a)

Phe 325 (A)

Tyr 545 (A)

Ser 358 (A)

Tyr 583 (A)

Arg 427 (A)

Tyr 456 (A)Arg 354 (A)

(b)

Figure 8: (a) 3D and (b) 2D interaction of deacyl gymnemic acid with DPP4.

based on binding energies or fitness score [15]. Further, amolecular-based study is required for confirmation of theabove-proposed mechanism.

4. Discussion

The use of medicinal plants and traditional medicine indeveloped and developing countries is becoming popular asa medical alternative in the treatment of various ailmentsincluding diabetes. It has been known that oxidative damageis associated with a number of disease processes and possiblemechanism including diabetes mellitus. In the present study,the hydroalcoholic extract from G. sylvestre leaf exhibitedpotent antioxidant activities and increased the activity ofenzymes beneficial in the prevention of diabetes.

From ethnopharmacological relevance point of view, theextract of G. sylvestre has been used for therapeutic purposesand current scientific data are available for its several othertherapeutic actions such as antioxidant, anticancer, anti-inflammatory, antidiabetic, hypolipidemic, and hypotensive[7]. Our study clearly demonstrated that the hydroalcoholicextract of G. sylvestre was utilized with a well-defined lowdose as mentioned in Ayurved Pharmacopeia for its antidi-abetic potential.

We have obtained more yield of hydroalcoholic extractas compared by using general extraction procedure throughmaceration with water yielding and this can be used inindustrial scale also. The total amount of phenolic andflavonoid content in the extracts from G. sylvestre leaf wasdetermined. Gallic acid and rutin were used to express thephenolic and flavonoid content, respectively. The content ofphenolic and flavonoids found in natural plants is knownto have a number of beneficial health effects associated withnatural antioxidants suppressing the LDL cholesterol oxida-tion [16] and decreasing the risk of heart disorder [17]. Thehydroalcoholic extract showed potent antioxidant potentialand is expressed asTrolox equivalent.Themetabolites presentin the extract were able to scavenge the DPPH and weobtained the similar type of activities as ascorbic acid showed.The results obtained from antioxidant assay indicate that

hydroalcoholic extract of G. sylvestre leaf contains potentantioxidants potential. It has earlier been proved that plantsrich in polyphenolic compounds, such as phenolic acids andflavonoids, possess outstanding antioxidant activities [18].

The results clearly showed that the in vitro 𝛼-amylaseand 𝛼 glucosidase inhibition test displayed that the hydroal-coholic extract of G. sylvestre had a potent inhibitory effectand it showed better activity as compared to acarbose asa standard drug. This in vitro finding suggested that thehydroalcoholic extract of G. sylvestre can be able to sig-nificantly reduce the postprandial level by inhibiting theactivity of 𝛼-amylase and 𝛼-glucosidase, which are importantenzymes in the digestion of the complex carbohydrates intoabsorbable monosaccharides in the food. Glucose uptake inyeast cell follows the passive diffusion as similar to glucoseuptake in human skeletal muscle [19]. If any extract is ableto increase glucose uptake in yeast cell which will also beable to increase glucose uptake in skeletal muscle [20]. Thehydroalcoholic extractwas able to increase the glucose uptakein yeast cell more than two hundred percent as compared tocontrol condition without having extract (Figure 2).Thus thisyeast cell uptake is an evidence of the antidiabetic potentialof hydroalcoholic extract. Glucose uptake in yeast cell hasbeen done very first time for G. sylvestre extract. This resultwas further supported by ex vivo glucose uptake assay. Thehydroalcoholic extractwas able to increase the glucose uptakein skeletal muscle isolated from rats. In the presence ofextract, a significant increase in glucose uptake was observedas compared to normal conditions (Figure 3). On the otherhand, we have checked the effect of the hydroalcoholic extracton glucose absorption inhibition in intestine. We found thatthe extract was able to inhibit the glucose absorption inintestine. From both the ex vivo experiments suggest that theextract is able to increase the peripheral glucose utilizationand also decrease the glucose absorption. Thus, it will bemore powerful antidiabetic medicine for the patients whoare suffering from type 2 diabetes [21]. This ex vivo studyprovided the biological environment and gives the possiblemechanism of the hydroalcoholic extract of G. sylvestre forits antidiabetic potential.


Table3:Docking

summaryof

major

abun

dant

analysed

metabolitesw

ithdifferent

proteins

associated

with

diabetes.

Major

metab

olite

sPa

rameters

P-1

P-2

P-3

P-4

P-5

P-6

P-7

P-8

P-9

Deacylgym

nemicacid

Free

energy

(kcal/m

ol)

-6.5

359.4

-13.17

-9.22

27.07

-8.83

-11.0

8-5.57

31.67

InteractSurfa

ce599.5

614.3

963.6

859.5

778.9

1079.2

1032.1

669.8

698.1

Interm

olecular

Energy

(kcal/m

ol)

-7.94

59.1

-13.97

-10.36

26.35

-8.94

-11.7

7-6.96

19.28

Gym

nemagen

inFree

energy

(kcal/m

ol)

-8.49

-2.56

59.4

-10.85

19.98

-8.91

-10.81

-7.59

-8.39

InteractSurfa

ce593.8

646.1

614.3

733.8

481.6

626.8

689.5

558.3

501.2

Interm

olecular

Energy

(kcal/m

ol)

-8.79

-2.86

59.1

-11.15

+19.6

9-9.21

-11.11

-7.89

-8.69

Gym

nemicacid

Free

energy

(kcal/m

ol)

-7.78

-4.29

-9.89

-16.59

-12.49

-9.44

-13.92

-7.97

-3.77

InteractSurfa

ce582.2

718.1

559.8

824.1

596.3

977.7

819.5

633.3

594.3

Interm

olecular

Energy

(kcal/m

ol)

-9.74

-4.15

-9.06

-17.8

5-14.13

-11.4

7-15.94

-9.6

-2.61

Quercetin

Free

energy

(kcal/m

ol)

-5.6

-3.76

-7.46

-7.77

-7.18

-6.08

-8.82

-7.40

-6.28

InteractSurfa

ce562.5

602.1

613.0

662.4

630.2

520.6

734.0

644.6

624.4

Interm

olecular

Energy

(kcal/m

ol)

-5.9

-4.06

-7.76

-8.07

-7.48

-6.38

-9.12

-7.70

-6.58

P-1:DPP

4;P-

2:glutam

ine–fructose-6-pho

sphatetransaminase;P-

3:AMP-activ

ated

proteinkinase;P

-4:G

LUT2;P-

5:SC

D1;P-

6:GLU

T4;P-

7:sulfo

nylureareceptor;P

-8:11-b

etahydroxysteroid

dehydrogenase

type

1;P-

9:sodium

/potassiu

m/calcium

exchanger.


Quality control analysis is one of the major concerns forherbal formulation. TLC fingerprinting is usually used to getthe metabolite patterns of any extract so that we can identifythe extract in future. If any extract will have the same TLCpattern, it must show the same biological activity and weknow its TLC fingerprint. In this context, we have developedthe TLC method and analyzed our extract. The developedmethod was reproducible and we have total 13 metabolitespresent in the hydroalcoholic extract (Figure 4). This TLCfingerprint can be used for its quality control analysis andregulatory bodies to assure its quality and safety [12]. Further,to identify the different metabolites present in the extract,we performed UPLC-MS analysis. UPLC-MS is the mostpowerful tool for the identification of polar and nonpolarmetabolites. In our experiment, we chromatographicallyseparated and tentatively identified based on their mass.Total 58 metabolites were analyzed and identified throughUPLC-MS (Table 2). Further, identified metabolites werecategorized (Figure 7). HPTLC fingerprinting and LC-MSanalysis identify the constituents present in the hydroal-coholic extract which usually polar secondary metabolitessuch as glycosides, phenols, nucleosides, terpenoids, vita-mins, lipids lignans, fatty acids, saponin, and tannins andsome primary metabolites such as glycosides, vitamins, andproteins. Metabolomics is a useful and powerful tool forthe chemical and pharmacological standardization of plantextract [22] and it has the potential to make a revolution inresearch of natural product and to advance the scientificallydevelopment of herbal based medicine. Metabolomes ofsome important medicinal plants are particularly a valuablenatural resource for the evidence-based development ofnew nutraceuticals and phytotherapeuticals. This is the firsttime we identified the metabolites present in hydroalco-holic extract which were further categorized. This studywill be helpful for the researchers who are working inG. sylvestre.

In order to predict the mechanism, we have checked theaffinity of major metabolites present in the extract with theproteins associated with glucose biosynthesis, metabolism,and utilization. We have selected total four major metabolitespresent in extract and identified through UPLC-MS. Thesemetabolites were docked with protein and we have analyzedtheir affinity (Table 3). It has found that gymnemic acid ismore potent than deacyl gymnemic acid in terms of affinitytowards selected protein. It was observed that almost allcompounds are with low binding energies and clearly showsthat every compound for the enzyme was found with goodaffinity. In silico study of responsible metabolites gives aninsight of tentatively identified metabolites with a specificprotein. The in silico approach can be used to model theinteraction and binding energy between a small molecule andprotein at the atomic level, which allows us to characterize thebehavior of metabolites in the binding site of target proteinsas well as elucidate the preliminary mechanism of action ofmolecules.

In summary, we chromatographically characterized thehydroalcoholic extract of G. sylvestre leaves extract and wehave tested its antidiabetic potential by using in vitro, ex vivo,in silico, and metabolomics approaches. The results of the

study will be helpful for the development of phytopharma-ceuticals which can be used for the management of diabetes.

5. Conclusion

The present study substantiated the hypoglycemic potentialof G. sylvestre leaves, which has been used since long forthe management of diabetes. The hydroalcoholic extractexhibited the hypoglycemic activity by increasing the glu-cose uptake in skeletal muscle, inhibiting intestinal glucoseabsorption, and by scavenging redox molecule. In silico studypredicted the mechanism behind the antidiabetic potentialof extract. However, a molecular level study is needed tobe performed for better clarification and providing morescientific data. Thus, hydroalcoholic extract enriched withgymnemic acid and deacylgymnemic acid can be explored forthe development of phytopharmaceuticals.

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

Theauthors wish to confirm that there are no known conflictsof interest associated with this manuscript.

Authors’ Contributions

Sayeed Ahmad and Syed Akhtar Husain equally contributedto this paper. The authors are responsible for the content andwriting of the paper.

Acknowledgments

The authors would like to acknowledge the University GrantsCommission (UGC), New Delhi, for providing Fellowship toShabana Parveen to carry out the research work.

References

[1] A. N. Singab and F. S. Youssef, “Medicinal plants with potentialantidiabetic activity and their assessment,”Medicinal and Aro-matic Plants, vol. 3, no. 1, pp. 1–12, 2014.

[2] J. E. Shaw, R. A. Sicree, and P. Z. Zimmet, “Global estimates ofthe prevalence of diabetes for 2010 and 2030,”Diabetes Researchand Clinical Practice, vol. 87, no. 1, pp. 4–14, 2010.

[3] I. B. Abdel-Farid,M. G. Sheded, and E. A. Mohamed, “Metabo-lomic profiling and antioxidant activity of someAcacia species,”Saudi Journal of Biological Sciences, vol. 21, no. 5, pp. 400–408,2014.

[4] J. Hess, J. W. Kadereit, and P. Vargas, “The colonization his-tory of Olea europaea L. in Macaronesia based on internaltranscribed spacer 1 (ITS-1) sequences, randomly amplifiedpolymorphic DNAs (RAPD), and intersimple sequence repeats(ISSR),”Molecular Ecology, vol. 9, no. 7, pp. 857–868, 2000.

[5] D. C. Joshi, P. K. Shrotria, R. Singh, M. K. Srivastava, and H.S. Chawla, “Assessment of RAPD and ISSR marker systems


for establishing distinctiveness of forage Sorghum (Sorghumbicolor L. Moench) varieties as additional descriptors forplant variety protection,” Indian Journal of Genetics and PlantBreeding, vol. 71, no. 1, pp. 25–36, 2011.

[6] A. K. Verma, S. S. Dhawan, S. Singh, K. A. Bharati, and Jyotsana,“Genetic and chemical profiling of Gymnema sylvestre acces-sions from central India: its implication for quality control andtherapeutic potential of plant,” Pharmacognosy Magazine, vol.12, supplement 4, pp. S407–S413, 2016.

[7] P. Tiwari, B. N.Mishra, andN. S. Sangwan, “Phytochemical andpharmacological properties ofGymnema sylvestre: an importantmedicinal plant,” BioMed Research International, vol. 2014,Article ID 830285, 18 pages, 2014.

[8] A. Al-Romaiyan, A. J. King, S. J. Persaud, and P. M. Jones, “Anovel extract of Gymnema sylvestre improves glucose tolerancein vivo and stimulates insulin secretion and synthesis in vitro,”Phytotherapy Research, vol. 27, no. 7, pp. 1006–1011, 2013.

[9] L. Kishore and R. Singh, “Protective effect of Gymnemasylvestre L. against advanced glycation end-product, sorbitolaccumulation and aldose reductase activity in HomoeopathicFormulation,” Indian Journal of Research in Homoeopathy, vol.9, no. 4, article 240, 2015.

[10] W. Khan, R. Parveen, K. Chester, S. Parveen, and S. Ahmad,“Hypoglycemic potential of aqueous extract ofMoringa oleiferaleaf and in vivo GC-MSmetabolomics,” Frontiers in Pharmacol-ogy, vol. 8, pp. 1–16, 2017.

[11] C. I. Chukwuma andM. S. Islam, “Effects of xylitol on carbohy-drate digesting enzymes activity, intestinal glucose absorptionand muscle glucose uptake: a multi-mode study,” Food andFunction, vol. 6, no. 3, pp. 955–962, 2015.

[12] M. N. Mallick, M. Singh, R. Parveen et al., “HPTLC analysisof bioactivity guided anticancer enriched fraction of hydroal-coholic extract of Picrorhiza kurroa,” BioMed Research Interna-tional, vol. 2015, Article ID 513875, 18 pages, 2015.

[13] M. Singh, E. T. Tamboli, Y. T. Kamal, W. Ahmad, S. H.Ansari, and S. Ahmad, “Quality control and in vitro antioxidantpotential of Coriandrum sativum Linn.,” Journal of Pharmacyand Bioallied Sciences, vol. 7, no. 4, pp. 280–283, 2015.

[14] R. B. Kumar andM. X. Suresh, “A computational perspective ofmolecular interactions through virtual screening, pharmacoki-netic and dynamic prediction on ribosome toxin A chain andinhibitors of Ricinus communis,” Pharmacognosy Research, vol.4, no. 1, pp. 2–10, 2012.

[15] D. B. Kitchen,H. Decornez, J. R. Furr, and J. Bajorath, “Dockingand scoring in virtual screening for drug discovery: methodsand applications,” Nature Reviews Drug Discovery, vol. 3, no. 11,pp. 935–949, 2004.

[16] A. S. Meyer, O.-S. Yi, D. A. Pearson, A. L. Waterhouse, andE. N. Frankel, “Inhibition of human low-density lipoproteinoxidation in relation to composition of phenolic antioxidantsin grapes (Vitis vinifera),” Journal of Agricultural and FoodChemistry, vol. 45, no. 5, pp. 1638–1643, 1997.

[17] P. Tiwari, R. S. Sangwan, Asha et al., “Molecular cloningand biochemical characterization of a recombinant sterol 3-O-glucosyltransferase fromGymnema sylvestre R.Br. catalyzingbiosynthesis of steryl glucosides,” BioMed Research Interna-tional, vol. 2014, Article ID 934351, 14 pages, 2014.

[18] R. A. Larson, “The antioxidants of higher plants,” Phytochem-istry, vol. 27, no. 4, pp. 969–978, 1988.

[19] V. P. Cirillo, “Mechanism of glucose transport across the yeastcell membrane,” Journal of Bacteriology, vol. 84, pp. 485–491,1962.

[20] I. C. Selvaraj, “In vitro investigation of antidiabetic potential ofselected traditional medicinal plants,” International Journal ofPharmacognosy and Phytochemical Research, vol. 6, no. 4, pp.856–861, 2014.

[21] G. Jiang and B. B. Zhang, “Glucagon and regulation ofglucose metabolism,” The American Journal of Physiology—Endocrinology & Metabolism, vol. 284, no. 4, pp. E671–E678,2003.

[22] G. Ulrich-Merzenich, H. Zeitler, D. Jobst, D. Panek, H. Vetter,and H. Wagner, “Application of the “-Omic-” technologies inphytomedicine,” Journal of Natural Remedies, vol. 7, no. 1, pp.1–18, 2007.

Stem Cells International

Hindawiwww.hindawi.com Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation http://www.hindawi.com Volume 2013Hindawiwww.hindawi.com

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwww.hindawi.com Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwww.hindawi.com

Submit your manuscripts atwww.hindawi.com
https://www.hindawi.com/journals/sci/https://www.hindawi.com/journals/mi/https://www.hindawi.com/journals/ije/https://www.hindawi.com/journals/dm/https://www.hindawi.com/journals/bmri/https://www.hindawi.com/journals/jo/https://www.hindawi.com/journals/omcl/https://www.hindawi.com/journals/ppar/https://www.hindawi.com/journals/tswj/https://www.hindawi.com/journals/jir/https://www.hindawi.com/journals/jobe/https://www.hindawi.com/journals/cmmm/https://www.hindawi.com/journals/bn/https://www.hindawi.com/journals/joph/https://www.hindawi.com/journals/jdr/https://www.hindawi.com/journals/art/https://www.hindawi.com/journals/grp/https://www.hindawi.com/journals/pd/https://www.hindawi.com/journals/ecam/https://www.hindawi.com/https://www.hindawi.com/

chromatography based metabolomics and in silico gymnema … · 2019. 7. 30. · researcharticle...

Documents