hptlc/hplc and gravimetric methodology for the identification and quantification of gymnemic acid...

7/28/2019 HPTLC/HPLC and Gravimetric methodology for the identification and quantification of gymnemic acid from Gymne

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Acta ChromatographicaDOI: 10.1556/AChrom.25.2013.2.10

02312522 2012 Akadmiai Kiad, Budapest

HPTLC/HPLC and Gravimetric Methodology for

the Identification and Quantification ofGymnemic Acid from Gymnema sylvestreMethanolic Extracts

A.B.A. AHMED1, 3*, A.S. RAO2, M.V. RAO1, AND R.M. TAHA3

1Department of Plant Science, Bharathidasan University, Tiruchirappalli, 620 024,Tamil Nadu, India

2Department of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024,Tamil Nadu, India

3Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603,Kuala Lumpur, Malaysia

E-mail: [email protected]

Summary. Gymnemic acid (GA), a well known anti-diabetic compound has been de-tected in methanol extracts of intact leaves and in vitro callus cultures derived from leafexplants of Gymnema sylvestre.Callus biomass was developed in MS medium with opti-mum plant growth regulators (OPGRs) of 2,4-D (1.5 mg L1) + KN (0.5 mg L1) underabiotic stress conditions at 45 days determined by growth curve analysis. GA detectionand quantification were carried out using thin-layer chromatography (TLC), high-

performance thin-layer chromatography (HPTLC), high-performance liquid chromatog-raphy (HPLC), and gravimetric techniques. GA detection peak area and their absorptionspectra were evaluated through HPTLC and HPLC with the standard GA. Quantifica-tion of GA had showed the linearity, accuracy, robustness and precision by HPLC. GAcontent was significantly higher in gravimetric method than HPLC. All these methodswere found to be simple, accurate, selective and rapid and could be successfully appliedfor the determination of GA. It could have potential as a pharmaceutical drug for Type 1diabetes mellitus (IDDM) and obesity.

Key Words: Gymnema sylvestre, gymnemic acid (GA), abiotic stress, HPTLC, HPLC, gra-

vimetric method

Introduction

Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM), is a com-mon pediatric chronic disease, affecting an increasing number of childrenevery year. IDDM occurs due to autoimmune destruction of insulin-producing -cells in the pancreas, resulting in low or no production of insu-lin, a hormone necessary for survival [1]. According to World Health Or-

ganization, obesity has reached epidemic proportions globally, with at least2.6 million people dying each year as a result of being overweight orobese [2].


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Gymnema sylvestre (syn. Periploca sylvestris Retz) is a woody climber be-longing to the Asclepiadaceae family. It is a traditional medicinal plant,with reported use as a remedy for diabetes mellitus, stomachic and diuretic

problems. The plant extracts are also used in folk, ayurvedic and homeo-pathic systems of medicine [3]. The extract of G. sylvestre plays a major role

in blood glucose homeostasis through increased serum insulin level via -cells regeneration of the endocrine pancreas [4, 5]. In Japan, Gymnema Teasand Gymnema chewing gum are being made from G. sylvestre leaves andpromoted as a natural method for controlling obesity, to increase the insulinsecretion via pancreatic beta cells regeneration and to deterimine anti-sweetactivity on tongue [6]. It mainly occurs in the Deccan peninsula of westernIndia, tropical Africa, Vietnam, Malaysia, and Sri Lanka [7]. Several prod-

ucts, under the brand names such as Body Slatto Tea, Gymnema,Gymnema Tea, Gymnema Diet, Sugar Off and Pilisoft are commer-cially available in markets of Japan, Germany and USA as health foods andcosmetics.

Plant cell culture extracts have also been used widely in the form offractions and isolated compounds as potential bioactive molecules [8].Gymnemic acid (GA), mixture triterpene saponins, was discovered in 1847to temporarily reduce the sweet taste of sugar in humans [9]. In vitro devel-oped callus trends to produce various bioactive compounds, including GA

and gymnemagenin [10]. However, external factors like phytohormone,shaking speeds, pH and medium play important roles in GA production insuspension cultures [11]. In addition, sucrose, inoculums density, auxinsand aeration also play a very crucial role in the production of GA throughbioreactor-dependent cell growth [12]. We have recently published the invi-tro GA production [13, 14], and given biological actions of anti-diabetes and

regenerated pancreatic cells in Wistar rats [15, 16]. The chromatographictechniques such as thin-layer chromatography (TLC), high-performancethin-layer chromatography (HPTLC), high-performance liquid chromatog-raphy (HPLC) and gravimetric methods are helpful for quantification ofGA. The present report is advancement over the earlier protocol [13] be-cause it describes the establishment of in vitro callus from leaf explants of G.sylvestre and the enhancement of GA using various types of abiotic stressfactors and quantified GA via TLC, HPTLC, HPLC and gravimetric method.


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HPTLC/HPLC and Gravimetric Methodology

Experimental

Plant Material

G. sylvestre plants (GS) were collected from the Pachamalai hills (Fig. 1A)and maintained in the Department of Plant science garden of the Bharathi-dasan University, Tiruchirappalli, Tamil Nadu, India. Leaf explants werewashed with tap water, Teepol solution, then 70% ethanol for 30 s and 0.1%HgCl2 for 2 min. Prior to inoculation, explants were washed several times insterile distilled water [15].

Fig. 1. Gymnemic acid in in vitro abiotic stress with OPGRs [MS + 2,4D(1.5 mg L1) + KN (0.5 mg L1)] and intact leaf of Gymnema sylvestre analyzed through

TLC. A. Habit with twig and flower; B. blue light + OPGRs; C. red light + OPGRs;D. 4% sucrose + OPGRs; E. 5% sucrose + OPGRs; F. 12-h photoperiod + OPGRs;

G. 3 mM NH4NO3 + OPGRs; H. dried callus (before and after methanol extraction);

I 1, 2, 3: IBA (white friable callus); J 1, 2, 3, 4: IAA (white watery callus); K 1: standardgymnemic acid; K 2, 3: 2,4-D and NAA (green compact callus); L 1: NAA + KN(green compact callus); L 2: 2,4-D + KN (green compact callus); L 3: intact leaf


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Chemicals and Reagents

A gymnemic acid mixture was made from G. sylvestre leaves and in vitro cal-

lus extracts. Vanillin sulphuric acid reagent was freshly prepared for deter-mination of GA (1 g of vanillin was dissolved in 90 ethanol, to this 5 mL ofacetic anhydride and 5 mL of sulphuric acid were added). The authentic GAstandard (1 mg mL1 methanol) was a gift from Prof. Kazuko Yoshikawa,Kyoto Pharmaceutical University, Japan. All other chemicals were of re-agent grade and purchased from Qualigen Chemical Co. and Sigma Chemi-cal Co., India.

Callus Induction

Explants (leaf) of G. sylvestre were grown in MS medium [17] supplementedwith auxins [IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), NAA(1-naphthaleneacetic acid), 2,4-D (2,4-dicholorophenoxyacetic acid): 0.55.0 mg L1], cytokinins [BA (6-benzylaminopurine), KN (6-furfurylami-nopurine): 0.22.0 mg L1] and adenine sulphate (Ads) (515 mg L1), re-spectively [13].

Callus Developed under Stress Conditions

The initiated callus cultures were maintained under different abiotic stressconditions for GA enhancement [13]. The protocol was developed as fol-lows: different color light (blue, red, green, and white fluorescent tubes);temperature (20C, 25C, 30C, and 35C); photoperiod (4 h/20 h, 8 h/16 h,12 h/12 h, and 20 h/4 h light/dark), sucrose (2%, 4%, 5%, and 6%) andammonium nitrate (1 mM, 2 mM, 3 mM, and 4 mM). Optimum callus bio-mass was determined using growth curve analysis, in all treatments.

In Vitro Callus Growth Curve

Callus cultures were optimized and evaluated quantitatively for their na-ture, biomass and GA content at the end of their respective growth cycle (015, 1525, 2535, 3545, and 4555 days), when treating with various combi-nations of PGRs. At regular interval for all the treatments, each callus washarvested by careful separation from media using metal spatulas, and freshand dry weight was promptly recorded.


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Sample Preparation

G. sylvestre intact leaves were dried at room temperature, and the in vitro

callus was dried at 40C (Fig. 1H). Suitable amounts (500 mg) of the pow-dered intact leaves and in vitro callus were extracted with methanol 5 times

[18]. The collected methanol extract was centrifuged at 5000 g for 10 minat room temperature, then the methanol supernatant was carefully pipettedout into fresh eppendrof tubes without disturbing the inter-phase residues.

Green-color supernatant (20 L) was used for the estimation of GA in thesample by TLC, HPTLC, and HPLC.

TLC Analysis in Leaf and Callus

The reaction products were spotted on preparative silica gel plate activated

at 110C for 30 min. The intact leaf and in vitro callus extracts were appliedon TLC plate along with standard GA was developed in air tight chambercontaining isopropyl alcoholchloroformmethanolacetic acid (5:3:1:0.5;v/v). After the chromatographic run was over, the chromatogram was driedat room temperature and sprayed with vanillin sulphuric acid reagent(freshly prepared) for detection of GA.

HPTLC Analysis in Leaf and Callus

HPTLC (high-performance thin layer chromatography), the methanol ex-

tracts of intact leaf and in vitro callus (20 L) were applied in Camag(CAMAG, Switzerland) HPTLC system assisted with sample applicator Li-nomat IV for quantification of GA. HPTLC plates are characterized by

smaller particles (10 m), thinner layers (150 m) and smaller plates

(10 m developing distance). In addition, the particle size distribution ofthe sorbent is narrower than for conventional TLC layers. EMD silica gel 60F254 fluorescent TLC plate and developed in a TLC chamber using the ap-propriate mobile phase. Ten samples were applied on each plate at a startline 8 mm from the bottom, including nine lanes of in vitro callus and intactleaves with reference GA (20 L). The mobile phase of isopropyl alcoholchloroformmethanolacetic acid (5:3:1:0.5; v/v) was allowed to run up to80 mm for separation of GA at a wavelength of 200 nm by use of TLC Scan-ner 3; integration and quantification were performed using CAT 4.0 soft-ware. The optimized chamber and the mobile phase were maintained atroom temperature (30C) for 30 min at relative humidity of 55 5%. In addi-tion, the in vitro optimum callus was screened on different periods (015,1525, 2535, 3545, and 4555 days) through the HPTLC for standardiza-tion of growth curve analysis. A digital camera with manual exposure set-


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tings (Nikon D1X) and a 254-nanometer UV lamp (Mineral Light UVS-11)were attached to the stand so that they were the same distance away fromthe TLC plates for each picture taken. Essentially, TLC analyzer is a stimu-

lated TLC scanner: TLC scanner pans across an HPTLC plate with a beam oflight emitted through an adjustable slit. In contrast, a digital image is madeup of many rows and columns of dots called pixels. Thus, a digital imageis essentially a matrix numbers and TLC analyzer virtually pans acrossthe matrix, combining moving averages to create a graph.

Calculation

Sample Peak Area Conc. of Std (g) Volume of dilute

Standard Peak Area Volume of dilute Conc. of sample (mg)

Std apply HPTLC g 1000 mg 100 = =

Sample apply HPTLC mg 1000 gm

HPLC Studies in Leaf and Callus

GA was screened in intact leaf and in vitro callus (1 g dry wt.) extracts by

above procedure. After centrifugation, an aliquot of methanol supernatant(4 mL) was evaporated and dried. The residue (ca, 6 mg) was dissolved in

MeOH (5 mL), and injected into an HPLC column (20 L). For GA separa-tion, the following systems and protocols were used: water HPLC system(Shimadzu model, Japan), two 510 pumps, 7725 Rheodyne auto injector, aDUG-12 A degasser, SCL-10Avp system controller, C18 (ODS) reversed-

phase column (150 mm 4.6 mm i.d., 5 m particle size) and water 486 UVdetector (all from Shimadzu, Kyoto, Japan). The mobile phase consisted of0.1% acetic acid; watermethanol (v/v) (35:65, HPLC grade) delivered at a

constant flow rate of 1 mL min1

. For identification and quantification of GAcontent samples, the respective retention time (RT) and peak area data fromthe calibration curve were analyzed. Each sample was injected three times.

Calculation

Std. Conc. (g) Test of compound peak (mV) Volume of extract (mL) =Std. peak area (mV) 0.02 (volume in mL injected) 500 mg of sample

g 1000 mg= =

Mg 1000 gm


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Gravimetric Analysis in Leaf and Callus

For gravimetric analysis, 500 mg of G. sylvestre leaves powder was dissolved

in 10 mL of 50% (v/v) ethanol, then 2 mL of KOH was added and heated ona boiling water bath under reflex for an hour and then cooled. To this,1.8 mL of 12N HCL was added and heated on water bath. After cooling thepH was adjusted to 7.58.5 with 11% KOH. This solution was dissolvedwith 50% (v/v) ethanol and filtered. After that, weigh 3.00 g of the crude ex-tract into a beaker. Dissolved in 50 mL of distilled water, filter and to the fil-trate add 10% hydrochloric acid till pH 1.5. Allow to stand for 30 min atroom temperature. Above samples were filtered on Whatman No. 1 filterpaper and then washed with 20 mL distilled water and discard the filtrate.

Collect the precipitate and dissolve in 20 mL 80% (v/v) methanol. Combinethe filtrate and washing, evaporate in pre-weighed beaker and dry in ovenunder vacuum at 70 C to a constant weight. Weigh and calculate the per-centage of total GA [19]. The amount of GA was expressed in the percent ofdry weight.

Results and Discussion

Callus Induction

In past few decades, secondary metabolites production from plant tissueculture has been identified as a tremendous resource for new drug devel-opment and clinical research in the field of pharmacology and medicine.Callus induction was obtained in MS medium supplemented with auxinsand cytokinins in leaf explants of G. sylvestre. MS with 2,4-D (1.5 mg L1)and KN (0.5 mg L1) had induced the green compact callus, and maximumbiomass was determined by growth curve analysis between 3545 days ofstationary phase [16], whereas other auxins and cytokinins combinationshad induced the green friable, brown friable, white friable and white waterycallus. Adenine sulphate was added to the OPGRs, concentration inducedthe green compact callus and biomass drastically reduced in leaf explants ofG. sylvestre (Table I). In Asclepiadaceae, Shin et al. (2003) reported that theactive secondary metabolites (gagaminine) was induced at stationary phaseof Cynanchum wilfordii [20].


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Table I. In vitro production of gymnemic acid determined in callus (dry weight) byTLC and HPTLC

TLC HPTLC

Rf-valueMS + plant growthregulators (mg L1)

Dry

weightbiomass(mg L1)

Rf-value Start Middle End

Gymnemic

acidcontent(mg g1)

Standard gymnemic acid 0.44 0.36 0.42 0.46

Intact leaf 0.41 0.34 0.38 0.42 19.75

In vitro callusNAA (1.0 mg L1)2,4-D (1.5 mg L1)

2,4-D (1.5 mg L1) + BA(0.5 mg L1)

NAA (1.0 mg L1) + BA(0.5 mg L1)

NAA (1.0 mg L1) + KN(1.0 mg L1)

NAA (1.0 mg L1) + BA(1.0 mg L1)

NAA (1.0 mg L1) + KN(1.5 mg L1)

2,4-D (1.5 mg L1) + KN(0.5 mg L1)

NAA (1.5 mg L1) + BA(0.5 mg L1) + Ads

(5.0 mg L1)NAA (1.5 mg L1) + KN

(1.0 mg L1) + Ads(5.0 mg L1)

2,4-D (1.5 mg L1) + BA(1.0 mg L1) + Ads

(5.0 mg L1)2,4-D (1.0 mg L1) + KN

(1.0 mg L1

) + Ads(5.0 mg L1)

116112

110

129

128

118

139

144

117

114

105

102

0.390.42

0.41

0.42

0.40

0.37

0.40

0.43

0.44

0.39

0.41

0.39

0.400.36

0.34

0.35

0.35

0.38

0.34

0.35

0.38

0.36

0.36

0.36

0.410.38

0.37

0.38

0.39

0.39

0.38

0.38

0.40

0.37

0.39

0.37

0.420.39

0.39

0.41

0.42

0.42

0.42

0.42

0.42

0.38

0.40

0.38

00.3800.92

00.48

04.81

08.32

00.94

11.32

12.77

00.58

00.25

00.38

00.35

Callus Growth Curve Analysis

We have reported on callus production in different media such as MS, SH,WPM and B5 media, among which MS media with auxins and cytokininswere suitable for callus production [14]. Fig. 2AD and Table IIdescribed thestress treatment, and callus growth curve of OPGRs [2,4-D (1.5 mg

L1) + KN (0.5 mg L1)] were screened on different periods (015, 1525, 2535, 3545, and 4555 days). In lag phase (015 days), in vitro callus growthwas slowed at first; the biomass was drastically reduced than other phase and


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Fig. 2. HPTLC analysis of gymnemic acid in different days of optimum in vitro callus(MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) extracts of G. sylvestre. A. 015 days;

B. 1525 days; C. 2535 days; D. 4555 days

A B

C D


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the GA content was absent (Fig. 2A). In log phase (1525 days), callus initia-tion and proliferation and GA production (0.88 mg g1) were observed (Fig.2B; Table II). In 2535 days (exponential phase), biomass and green compact

callus increased the GA content (3.39 mg g1) (Fig. 2C). However, in the sta-tionary phase of 3545 days, maximum biomass with green compact calluswas shown and GA quantity was 12.77 mg g1. In decline phase (4555 days), the biomass and GA content (8.47 mg g1) significantly reducedthan stationary phase determined by HPTLC (Fig. 2D; Table II). We have re-cently reported that the stationary phase methanol callus extracts had re-duced the blood sugar and maintained the lipid profile level in alloxan in-duced diabetic Wistar rats [15].

Table II. Gymnemic acid production determined by growth curve analysis on differentdays through HPTLC

Rf-valueMS + 2,4-D(1.5 mg L1) + KN

(0.5 mg L1)/growth curve analysis

Biomass(D.W.)

(mg L1) Start Middle End

Gymnemic acid(mg g1)

015 days (Lag phase)1525 days (Log phase)2535 days (Exponen-

tial phase)3545 days (Stationary

phase)4555 days (Decline

phase)

4785

119

144

122

0.33

0.36

0.35

0.35

0.38

0.38

0.38

0.39

0.45

0.42

0.42

0.44

0.88

3.39

12.77

8.47

Callus Induction Under Stress Conditions

It is well known that the diverse external factors, such as the temperature,light, pH and salt concentration influence the production of secondary me-tabolites [21]. OPGRs were maintained under abiotic stress conditions ofblue light, 5% sucrose, 12-h photoperiod induced the maximum biomassand green compact callus (Fig. 1B, E, and F), than red light, 4% sucrose and 3mM ammonium nitrate (Fig. 1C, D, and G). Many papers point out that lightmay inhibit or stimulate the production of secondary metabolites in callusculture. First, light provides the energy to the plant cells through photosyn-thesis. Second, light is a signal received by photoreceptor to regulate thegrowth, differentiation and metabolism [22]. Hence, the white light inducedgreen compact callus and maximum biomass than in callus of green and redlight.


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Table IIIdepicts that the MS medium supplemented with OPGRs con-tains the 5% sucrose that induced the green compact callus and GA produc-tion in leaf explants of G. sylvestre. Hence, 2%, 4% and 6% sucrose had re-

duced biomass and GA content, which showed the light green friable andgreen friable callus (data not shown). In case of 12-h photoperiod withOPGRs induced GA accumulation was determined at stationary phase of3545 days. However, the 4-h, 8-h, 20-h, 24-h, and 16-h (control) photope-riod reduced biomass and GA production than 12-h photoperiod (data notshown). Temperature stress had affected physical appearance of the callus,producing white watery and white friable callus (data not shown). Thesecalluses were stored for a long time, and the media turned brown in color. Itis obvious that we found OPGRs with 3 mM NH4NO3 increased the biomass

and GA, whereas all other NH4NO3 concentrations (1 mM, 2 mM and4 mM) reduced the GA content at 3545 days of stationary phase(Table III).

Table III. In vitro production of gymnemic acid through abiotic stress conditionsdetermined by HPLC and gravimetric analysis

Gymnemic acid(mg g1)

Treatment in vitro callusBiomass(D.W.)

(mg L1

) HPLC

Gravimet-

ric

Intact leafIn vitro callus

MS + NAA (1.0 mg L1) + KN (1.5 mg L1)MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1)

Abiotic stressMS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + blue light

MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + 5% sucroseMS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + 3mM NH4NO3

MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + 12-h photoperiod

MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + 30C temperatureMS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + red light

MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) + green light

139144

173164152159

136122116

19.52

11.0412.22

53.9433.3917.3426.27

02.9008.9203.07

23.27

12.4214.65

58.2835.4019.1026.86

03.5510.36

5.72

TLC and HPTLC Studies in Leaf and Callus Extracts

For TLC separation, the intact leaf and callus extracts reaction mixtureswere applied to the plates after concentrating. IAA and IBA induced callusextracts did not show the brown band in TLC and HPTLC analysis (Fig. 1Iand J), when spraying the vanillin sulphuric acid reagent, whereas the aux-ins combined with cytokinins induced green compact and their methanolextracts brown bands could be unequivocally identified by relating the Rf


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values to those of the standard GA (Fig 1K and L; Table I). In some cases, theRf values of the callus extracts were slightly higher.

HPTLC offers several advantages, such as facilitating automatic appli-

cation, scanning in situ, small quantity of mobile phase, and lower analysistime and cost per analysis [23]. Several callus extracts can be run simultane-ously using a small quantity of mobile phase. Furthermore, the developedTLC plates can be scanned for several times with same or different parame-

ters as mentioned earlier. The concentrates (10) callus extract samples areanalyzed in one run; this method proves to be very sensitive, relatively fast,inexpensive and suitable for therapeutic drug monitoring and pharmacoki-netic studies. The chromatography developing time was shorter in HPTLC(6 min) than in TLC (40 min) of the mobile phase of isopropyl alcohol

chloroformmethanolacetic acid (5:3:1:0.5; v/v). In the sample clarity andnot integrated to the base line, we made the following adjustment such assilica gel granular materials, multiple separation (the single time runmultiple samples) and two-dimensional process were done. TLC analyzershows the path of the scan on the image and creates graphs of the red,green, and blue components (a multiple spectral scan) as well as the blackand white image density (a densitogram). It also finds the maximum andminimum values for these same variables. GA purity was confirmed in theintact leaf and callus extracts by recording the absorption spectra developed

in start, middle and end of the peak. Standard GA was shown the singlepeak at different time intervals of the experiment (Fig. 3A). TLC analyzerautomatically produces multi-spectral scans from an image; however,multi-spectral scans can also be produced using almost any image-editingsoftware by simply reading the pixel brightness values using the eyedrop-per tool then plotting those values in a graphics program. In fact, EMD Sil-ica Gel 60F 254 fluorescent TLC was developed using an image-editing pro-gram, but TLC analyzer saves a great deal of time. The intact leaf and callusextracts sample curve was linear; the correlation coefficient has good linear-ity between concentration and area, it could be helpful to calculate the GAamount in the respectable sample (Fig. 3BN). Green friable callus was in-duced in MS medium supplemented with NAA (1.0 mg L1) and 2,4-D(1.5 mg L1), and the GA content was drastically reduced than auxins andcytokinins combination (Fig. 3CD). Hence, NAA and 2,4-D combined withcytokinins callus extracts increased the GA content (Fig. 3EI). In this con-troversial, MS medium supplemented with OPGRs only has produced themaximum biomass and GA than auxins and cytokinins combinations in 3545 days of stationary phase (Table I; Fig. 3J). However, the OPGRs werecombined with adenine sulphate, and the biomass and GA content, drasti-cally reduced, was determined by HPTLC (Table I; Fig. 3KN). GA contentwas increased in MS medium with auxins and cytokinins concentrations de-


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rived from leaf explants of G. sylvestre determined by HPTLC [25]. All cali-bration curves in this research were produced by plotting the peak opticaldensity for each of the same concentration of the different samples. TLC

analyzer automatically outputs the peak optical density calculated in themanner.

Fig. 3.

A

C D

B


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Fig. 3.

E F

G H


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Fig. 3.

K L

JI


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Fig. 3. HPTLC analysis of gymnemic acid determined in methanol extracts ofGymnema sylvestre

intact leaf and in vitro callus extracts. A. Standard gymnemic acid; B. Intact leaf; C. MS + NAA

(1.0 mg L1); D. MS + 2,4-D (1.5 mg L

1); E. MS + 2,4-D (1.5 mg L

1) + BA (0.5 mg L

1);

F. MS + NAA (1.0 mg L1) + BA (0.5 mg L

1); G. MS + NAA (1.0 mg L

1) + KN (1.0 mg L

1);

H. MS + NAA (1.0 mg L1) + BA (1.0 mg L

1); I. MS + NAA (1.0 mg L

1) + KN (1.5 mg L

1);

J. MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L

1) (OPGRs); K. MS + NAA (1.5 mg L

1) + BA

(0.5 mg L1) + 5 mg L1 Ads; L. MS + NAA (1.5 mg L1) + KN (1.0 mg L1) + 5 mg L1 Ads; M.MS medium + 2,4-D (1.5 mg L

1) + BA (1.0 mg L

1) + 5 mg L

1Ads; N. MS + 2,4-D (1.0 mg

L1) + KN (1.0 mg L

1) + 5 mg L

1Ads

HPLC Studies in Leaf and Callus Extracts

For HPLC analysis, leaf and callus methanol extracts (20 L) were uploadedin HPLC system to quantify GA under retention time (5 min). UV spectro-photometer peak area data were compared with standard gymnemic acid

(Table III). Gymnemagenin and GA were significantly increased in callusculture through leaf explants of G. sylvestre [1016]. In the present study,maximum GA production was observed in MS medium supplemented withOPGRs under blue light induced 4.4 fold as compared with white fluores-cent light and out of which 2.8 fold was found in intact leaves determinedby HPLC analysis. We have recently published a review of pharmacologicalactivities, a phytochemical investigation and in vitro studies of G. sylvestre[26].

HPLC mobile phase [0.1% acetic acid; watermethanol (v/v) (35:65,

HPLC grade)] purity was analyzed, without sample and standard of GA(Fig. 4A). Standard GA stability and impurity were characterized throughsingle peak at initial, middle, and end of the HPLC experiment (Fig. 4BD).

M N


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Reten. Time[min]

Area

[mVs]Height[mV]

Area[%]

Height[%]

W05[min]

No peak to report

Reten.

Time

[min]

Area

[mVs]

Height

[mV]

Area

[%]

Height

[%]

W05

[min]

1 2.997 42.609 2.463 100.0 100.0 0.29

Total 42.609 2.463 100.0 100.0

Fig. 4.

A

B


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A.B.A. Ahmed et al.

Reten. Time

[min]

Area

[mVs]

Height

[mV]

Area

[%]

Height

[%]

1 3.010 39.420 2.353 100.0 100.0

Total 39.420 2.353 100.0 100.0

Reten. Time

[min]

Area

[mVs]

Height

[mV]

Area

[%]

Height

[%]

W05

[min]

1 3.013 43.211 2.353 100.0 100.0 0.29

Total 43.211 2.353 100.0 100.0

Fig. 4.

C

D


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Reten. Time

[min]

Area

[mVs]

Height

[mV]

Area

[%]

Height

[%]

W05

[min]

1 2.667 628.235 27.617 33.7 40.4 0.34

2 3.003 416.005 24.000 22.3 35.1 0.34

3 3.333 818.611 16.749 43.9 24.5 0.64

Total 1862.851 68.366 100.0 100.0

Reten. Time[min]

Area[mVs]

Height[mV]

Area[%]

Height[%]

1 2.267 99.868 6.730 10.5 18.9

2 2.999 235.271 12.794 24.8 35.9

3 3.383 109.091 7.454 11.5 20.9

4 3.747 497.750 8.396 52.5 23.5

5 9.030 6.800 0.295 0.7 0.8

Fig. 4. HPLC analysis of gymnemic acid determined in methanol extracts of G. sylvestreleaf and abiotic in vitro callus extracts. A. Mobile phase without sample and standard;B, C, and D. standard gymnemic acid (B. before start experiment; C. middle experiment;

D. end experiment); E. intact leaf; F. MS + NAA (1.0 mg L1) + KN (1.5 mg L1);G. MS + OPGRs; H. MS + OPGRs + blue light; I. MS + OPGRs + 5% sucrose;

J. MS + OPGRs + 3mM NH4NO3; K. MS + OPGRs + 12-h photoperiod; L. MS + OPGRs +30C; M. MS + OPGRs + red light; N. MS + OPGRs + green light

E

F


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A.B.A. Ahmed et al.

Imoto et al. (1991) reported that GA content was confirmed by HPLC inmethanol leaf extracts of G. sylvestre [27]. GA quantification was done in therespectable leaf and in vitro callus methanol extracts of G. sylvestre. Fig. 4E

described that the GA content was increased in intact leaf explants (19.52mg g1) compared to in vitro callus culture of MS medium supplementedwith NAA (1.0 mg L1) + KN (1.5 mg L1) (11.04 mg g1) (Fig. 4F) andOPGRS (2,4-D 1.5 mg L1 + KN 0.5 mg L1; 12.22 mg g1; Fig. 4G). Many au-thors had isolated and identified GA earlier in leaf explants of methanol ex-tracts. In 1989, Yoshikawa and co-workers isolated GAs from a hot waterextract of G. sylvestre, which they named GA, I, II, III, IV, V, VI, and VII, re-spectively, and evaluated using HPLC [28, 29]. The above mentionedOPGRs sample kept under abiotic stress conditions and the developed cal-

lus methanol extracts were further analyzed (Fig. 4HN). Blue light withOPGRs wasinduced the maximum GA (53.94mg g1) (Fig. 4H) than 5% su-crose treatment (33.39 mg g1) (Fig. 4I) followed by 3 mM NH4NO3 (19.10mg g1) (Fig. 4J). However, other physical stress conditions GA content wasreduced in this order 12-h photoperiod (26.27 mg g1) (Fig. 4K), red light

(8.90 mg g1) (Fig. 4M) green light (5.72 mg g1) (Fig. 4N) and 30C (2.9mg g1) (Fig. 4L). In case of dark treatment, GA content was absent. We havepreviously reported that this in vitro abiotic stress callus of G. sylvestre sig-

nificantly increased the pancreatic -cells and maintained the body weight,

pancreas weight, liver weight and liver glycogen level in alloxan induceddiabetic Wistar rats [16].

Linearity

As per the ICH guidelines, validation parameters such as linearity, accu-racy, precision, and robustness were checked. The linearity of the method

was determined at three concentrations (1030 g mL1) of GA. Twenty mi-crograms per milliliter GA results show that an excellent correlation exists

between response factor and concentration of GA within the concentrationrange indicated above.

Accuracy and Precision

The accuracy of the method was determined by recovery experiments. Therecovery studies were carried out at three levels of 80, 100, and 120%, andthe percentage recovery was calculated. Our studies recovery was withinthe range of 100 2% which indicates accuracy of the method. The precision

of the method was demonstrated by interday and intraday variation stud-ies. In the intraday studies, three repeated injections of standard and samplesolution were made in a day and the response factor of GA peaks and per-


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centage were calculated. In the interday variation studies, three repeated in-jections of standard and sample solutions were made on three consecutivedays and response factor of GA peak and percentage were calculated (data

not shown). Intra- and interday accuracy were established from quality con-trol standards by evaluating nominal and mean measured concentrations ofquality control standards which were compared and expressed as % differ-ence (diff. %). Diff. % was calculated using the formula: Diff.% [(meanmeasured concentration nominal concentration)/nominal concentra-

tion] 100.

Robustness

Wavelength (200 nm) of GA compound was studied showing a sufficientabsorption, and an overloading of the column can be avoided. Adding 0.2%acetic acid gave a rather good separation of GA. In order to shorten the ana-lytical time and improve the sensitivity and peak shape of GA, a gradient,characterized by a decreased amount of acetic acid (0.1%) was applied be-fore the elution of GA. However, GA is eluted isocratically in order to guar-antee robustness.

Gravimetric Analysis in Leaf and Callus Extracts

Table IIIdescribed the quantification of GA in leaf and callus extracts of G.sylvestre by gravimetric method. GA quantification (based on mass of solid)had significantly higher than HPLC since efficient quantification and identi-fication of plant natural products were done in HPLC than other methods,because HPLC method has the good selectivity, sensitivity of detection, to-gether with the capability of providing on-line structural information [30].One of the most difficult parts during the method development was to

achieve a high and reproducible recovery from the solvent which is used forextraction of the GA. The GA can be determined on abiotic stress treatmentcallus of blue light stress (58.28 mg g1) which was greater than 5% sucrose(35.40 mg g1), 12-h photoperiod (26.86 mg g1), intact leaf (23.27 mg g1),3 mM NH4NO3 (19.10 mg g1) and 30C (03.55 mg g1) (Table III). Joshi et al.(2007) reported that gravimetric methods have many remarks than HPLC ofpoly-herbal combinations and compared with respective biomarker com-pounds [31].


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A.B.A. Ahmed et al.

Conclusion

We conclude that GA was produced from the leaf explants of G. sylvestre

maintained in MS medium with OPGRs and further enhanced under theabiotic stress conditions determined by HPLC and gravimetric methods.With this method, we hope that more people can be promoted and start theGA bioequivalence study in the future. Thus, it appears that blue light stresshas to be used as a tool for enhancing GA accumulation in the in vitro callusculture [32]. We suggest that the GA content intact leaf and in vitro callus

could potentially regulate pancreatic -cells for IDDM (insulin-dependentdiabetes mellitus) [16]. The proposed RP-HPLC and HPTLC methods forthe estimation of GA in intact leaf and in vitro callus are selective and sensi-

tive than gravimetric method. GA has UV absorbing molecules with specificchromophores in their structures that absorb at a particular wavelength,and this fact has been successfully employed for their quantitative determi-nation by UV spectrophotometric method. The development of a rapid, sen-sitive and accurate analytical method for routine quantitative determinationof samples will reduce unnecessary tedious sample preparations and cost ofmaterials and labor.

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[32] A. Bakrudeen Ali Ahmed, A.S. Rao, M.V. Rao, and R.M. Taha, Agro Food Ind HiTec., 23, 34 (2012)Accepted by MWH

hptlc/hplc and gravimetric methodology for the identification and quantification of gymnemic acid...

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