Acta Chromatographica DOI: 10.1556/AChrom.25.2013.2.

10

HPTLC/HPLC and Gravimetric Methodology for the Identification and Quantification of Gymnemic Acid from Gymnema sylvestre Methanolic Extracts
A.B.A. AHMED1, 3*, A.S. RAO2, M.V. RAO1, AND R.M. TAHA3
Department 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: dr.bakrudeenaliahmed@yahoo.co.in
1

Summary. Gymnemic acid (GA), a well known anti-diabetic compound has been detected in methanol extracts of intact leaves and in vitro callus cultures derived from leaf explants of Gymnema sylvestre. Callus biomass was developed in MS medium with optimum plant growth regulators (OPGRs) of 2,4-D (1.5 mg L1) + KN (0.5 mg L1) under abiotic stress conditions at 45 days determined by growth curve analysis. GA detection and quantification were carried out using thin-layer chromatography (TLC), highperformance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), and gravimetric techniques. GA detection peak area and their absorption spectra were evaluated through HPTLC and HPLC with the standard GA. Quantification of GA had showed the linearity, accuracy, robustness and precision by HPLC. GA content was significantly higher in gravimetric method than HPLC. All these methods were found to be simple, accurate, selective and rapid and could be successfully applied for the determination of GA. It could have potential as a pharmaceutical drug for Type 1 diabetes mellitus (IDDM) and obesity. Key Words: Gymnema sylvestre, gymnemic acid (GA), abiotic stress, HPTLC, HPLC, gravimetric method

Introduction
Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM), is a common pediatric chronic disease, affecting an increasing number of children every year. IDDM occurs due to autoimmune destruction of insulinproducing -cells in the pancreas, resulting in low or no production of insulin, a hormone necessary for survival [1]. According to World Health Organization, obesity has reached epidemic proportions globally, with at least 2.6 million people dying each year as a result of being overweight or obese [2].
02312522 2012 Akadmiai Kiad, Budapest

A.B.A. Ahmed et al.

Gymnema sylvestre (syn. Periploca sylvestris Retz) is a woody climber belonging 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 homeopathic 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 Teas and Gymnema chewing gum are being made from G. sylvestre leaves and promoted as a natural method for controlling obesity, to increase the insulin secretion via pancreatic beta cells regeneration and to deterimine anti-sweet activity on tongue [6]. It mainly occurs in the Deccan peninsula of western India, tropical Africa, Vietnam, Malaysia, and Sri Lanka [7]. Several products, under the brand names such as Body Slatto Tea, Gymnema, Gymnema Tea, Gymnema Diet, Sugar Off and Pilisoft are commercially available in markets of Japan, Germany and USA as health foods and cosmetics. Plant cell culture extracts have also been used widely in the form of fractions and isolated compounds as potential bioactive molecules [8]. Gymnemic acid (GA), mixture triterpene saponins, was discovered in 1847 to temporarily reduce the sweet taste of sugar in humans [9]. In vitro developed 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 in suspension cultures [11]. In addition, sucrose, inoculums density, auxins and aeration also play a very crucial role in the production of GA through bioreactor-dependent cell growth [12]. We have recently published the in vitro GA production [13, 14], and given biological actions of anti-diabetes and regenerated pancreatic cells in Wistar rats [15, 16]. The chromatographic techniques such as thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) and gravimetric methods are helpful for quantification of GA. The present report is advancement over the earlier protocol [13] because it describes the establishment of in vitro callus from leaf explants of G. sylvestre and the enhancement of GA using various types of abiotic stress factors and quantified GA via TLC, HPTLC, HPLC and gravimetric method.

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 Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Leaf explants were washed 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 in sterile 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: standard gymnemic 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

A.B.A. Ahmed et al.

Chemicals and Reagents

A gymnemic acid mixture was made from G. sylvestre leaves and in vitro callus extracts. Vanillin sulphuric acid reagent was freshly prepared for determination of GA (1 g of vanillin was dissolved in 90 ethanol, to this 5 mL of acetic anhydride and 5 mL of sulphuric acid were added). The authentic GA standard (1 mg mL1 methanol) was a gift from Prof. Kazuko Yoshikawa, Kyoto Pharmaceutical University, Japan. All other chemicals were of reagent grade and purchased from Qualigen Chemical Co. and Sigma Chemical Co., India.

Callus Induction
Explants (leaf) of G. sylvestre were grown in MS medium [17] supplemented with auxins [IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), NAA (1-naphthaleneacetic acid), 2,4-D (2,4-dicholorophenoxyacetic acid): 0.5 5.0 mg L1], cytokinins [BA (6-benzylaminopurine), KN (6-furfurylaminopurine): 0.22.0 mg L1] and adenine sulphate (Ads) (515 mg L1), respectively [13].

Callus Developed under Stress Conditions

The initiated callus cultures were maintained under different abiotic stress conditions for GA enhancement [13]. The protocol was developed as follows: 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%) and ammonium nitrate (1 mM, 2 mM, 3 mM, and 4 mM). Optimum callus biomass was determined using growth curve analysis, in all treatments.

In Vitro Callus Growth Curve

Callus cultures were optimized and evaluated quantitatively for their nature, biomass and GA content at the end of their respective growth cycle (0 15, 1525, 2535, 3545, and 4555 days), when treating with various combinations of PGRs. At regular interval for all the treatments, each callus was harvested by careful separation from media using metal spatulas, and fresh and dry weight was promptly recorded.

HPTLC/HPLC and Gravimetric Methodology

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 powdered intact leaves and in vitro callus were extracted with methanol 5 times [18]. The collected methanol extract was centrifuged at 5000 g for 10 min at room temperature, then the methanol supernatant was carefully pipetted out into fresh eppendrof tubes without disturbing the inter-phase residues. Green-color supernatant (20 L) was used for the estimation of GA in the sample 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 applied on TLC plate along with standard GA was developed in air tight chamber containing isopropyl alcoholchloroformmethanolacetic acid (5:3:1:0.5; v/v). After the chromatographic run was over, the chromatogram was dried at 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 extracts of intact leaf and in vitro callus (20 L) were applied in Camag (CAMAG, Switzerland) HPTLC system assisted with sample applicator Linomat 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 of the sorbent is narrower than for conventional TLC layers. EMD silica gel 60 F254 fluorescent TLC plate and developed in a TLC chamber using the appropriate mobile phase. Ten samples were applied on each plate at a start line 8 mm from the bottom, including nine lanes of in vitro callus and intact leaves with reference GA (20 L). The mobile phase of isopropyl alcohol chloroformmethanolacetic acid (5:3:1:0.5; v/v) was allowed to run up to 80 mm for separation of GA at a wavelength of 200 nm by use of TLC Scanner 3; integration and quantification were performed using CAT 4.0 software. The optimized chamber and the mobile phase were maintained at room temperature (30C) for 30 min at relative humidity of 55 5%. In addition, the in vitro optimum callus was screened on different periods (015, 1525, 2535, 3545, and 4555 days) through the HPTLC for standardization of growth curve analysis. A digital camera with manual exposure set-

A.B.A. Ahmed et al.

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 from the TLC plates for each picture taken. Essentially, TLC analyzer is a stimulated TLC scanner: TLC scanner pans across an HPTLC plate with a beam of light emitted through an adjustable slit. In contrast, a digital image is made up of many rows and columns of dots called pixels. Thus, a digital image is essentially a matrix numbers and TLC analyzer virtually pans across the 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 100 = = Sample apply HPTLC mg 1000 mg 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 separation, the following systems and protocols were used: water HPLC system (Shimadzu model, Japan), two 510 pumps, 7725 Rheodyne auto injector, a DUG-12 A degasser, SCL-10Avp system controller, C18 (ODS) reversedphase column (150 mm 4.6 mm i.d., 5 m particle size) and water 486 UV detector (all from Shimadzu, Kyoto, Japan). The mobile phase consisted of 0.1% acetic acid; watermethanol (v/v) (35:65, HPLC grade) delivered at a constant flow rate of 1 mL min1. For identification and quantification of GA content samples, the respective retention time (RT) and peak area data from the 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

HPTLC/HPLC and Gravimetric Methodology

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 on a 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 the pH was adjusted to 7.58.5 with 11% KOH. This solution was dissolved with 50% (v/v) ethanol and filtered. After that, weigh 3.00 g of the crude extract into a beaker. Dissolved in 50 mL of distilled water, filter and to the filtrate add 10% hydrochloric acid till pH 1.5. Allow to stand for 30 min at room temperature. Above samples were filtered on Whatman No. 1 filter paper and then washed with 20 mL distilled water and discard the filtrate. Collect the precipitate and dissolve in 20 mL 80% (v/v) methanol. Combine the filtrate and washing, evaporate in pre-weighed beaker and dry in oven under vacuum at 70 C to a constant weight. Weigh and calculate the percentage of total GA [19]. The amount of GA was expressed in the percent of dry weight.

Results and Discussion

Callus Induction
In past few decades, secondary metabolites production from plant tissue culture has been identified as a tremendous resource for new drug development and clinical research in the field of pharmacology and medicine. Callus induction was obtained in MS medium supplemented with auxins and 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 maximum biomass was determined by growth curve analysis between 3545 days of stationary phase [16], whereas other auxins and cytokinins combinations had induced the green friable, brown friable, white friable and white watery callus. Adenine sulphate was added to the OPGRs, concentration induced the green compact callus and biomass drastically reduced in leaf explants of G. sylvestre (Table I). In Asclepiadaceae, Shin et al. (2003) reported that the active secondary metabolites (gagaminine) was induced at stationary phase of Cynanchum wilfordii [20].

A.B.A. Ahmed et al. Table I. In vitro production of gymnemic acid determined in callus (dry weight) by TLC and HPTLC MS + plant growth regulators (mg L1) Standard gymnemic acid Intact leaf In vitro callus NAA (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) Dry weight biomass (mg L1) 116 112 110 129 128 118 139 144 TLC Rfvalue 0.44 0.41 0.39 0.42 0.41 0.42 0.40 0.37 0.40 0.43 Start 0.36 0.34 0.40 0.36 0.34 0.35 0.35 0.38 0.34 0.35 HPTLC Rf-value Middle 0.42 0.38 0.41 0.38 0.37 0.38 0.39 0.39 0.38 0.38 End 0.46 0.42 0.42 0.39 0.39 0.41 0.42 0.42 0.42 0.42 Gymnemic acid content (mg g1) 19.75 00.38 00.92 00.48 04.81 08.32 00.94 11.32 12.77

117

0.44

0.38

0.40

0.42

00.58

114

0.39

0.36

0.37

0.38

00.25

105

0.41

0.36

0.39

0.40

00.38

102

0.39

0.36

0.37

0.38

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 cytokinins were suitable for callus production [14]. Fig. 2AD and Table II described the stress 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, 25 35, 3545, and 4555 days). In lag phase (015 days), in vitro callus growth was slowed at first; the biomass was drastically reduced than other phase and

HPTLC/HPLC and Gravimetric Methodology

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

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the GA content was absent (Fig. 2A). In log phase (1525 days), callus initiation 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 stationary phase of 3545 days, maximum biomass with green compact callus was shown and GA quantity was 12.77 mg g1. In decline phase (45 55 days), the biomass and GA content (8.47 mg g1) significantly reduced than stationary phase determined by HPTLC (Fig. 2D; Table II). We have recently reported that the stationary phase methanol callus extracts had reduced the blood sugar and maintained the lipid profile level in alloxan induced diabetic Wistar rats [15].
Table II. Gymnemic acid production determined by growth curve analysis on different days through HPTLC MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1)/ growth curve analysis 015 days (Lag phase) 1525 days (Log phase) 2535 days (Exponential phase) 3545 days (Stationary phase) 4555 days (Decline phase) Biomass (D.W.) (mg L1) 47 85 119 144 122 Rf-value Start 0.33 0.36 0.35 0.35 Middle 0.38 0.38 0.38 0.39 End 0.45 0.42 0.42 0.44 Gymnemic acid (mg g1) 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 metabolites [21]. OPGRs were maintained under abiotic stress conditions of blue light, 5% sucrose, 12-h photoperiod induced the maximum biomass and green compact callus (Fig. 1B, E, and F), than red light, 4% sucrose and 3 mM ammonium nitrate (Fig. 1C, D, and G). Many papers point out that light may inhibit or stimulate the production of secondary metabolites in callus culture. First, light provides the energy to the plant cells through photosynthesis. Second, light is a signal received by photoreceptor to regulate the growth, differentiation and metabolism [22]. Hence, the white light induced green compact callus and maximum biomass than in callus of green and red light.

HPTLC/HPLC and Gravimetric Methodology

Table III depicts that the MS medium supplemented with OPGRs contains the 5% sucrose that induced the green compact callus and GA production in leaf explants of G. sylvestre. Hence, 2%, 4% and 6% sucrose had reduced biomass and GA content, which showed the light green friable and green friable callus (data not shown). In case of 12-h photoperiod with OPGRs induced GA accumulation was determined at stationary phase of 3545 days. However, the 4-h, 8-h, 20-h, 24-h, and 16-h (control) photoperiod reduced biomass and GA production than 12-h photoperiod (data not shown). Temperature stress had affected physical appearance of the callus, producing white watery and white friable callus (data not shown). These calluses were stored for a long time, and the media turned brown in color. It is obvious that we found OPGRs with 3 mM NH4NO3 increased the biomass and GA, whereas all other NH4NO3 concentrations (1 mM, 2 mM and 4 mM) reduced the GA content at 3545 days of stationary phase (Table III).
Table III. In vitro production of gymnemic acid through abiotic stress conditions determined by HPLC and gravimetric analysis Biomass (D.W.) (mg L1) 139 144 173 164 152 159 136 122 116 Gymnemic acid (mg g1) GravimetHPLC ric 19.52 11.04 12.22 53.94 33.39 17.34 26.27 02.90 08.92 03.07 23.27 12.42 14.65 58.28 35.40 19.10 26.86 03.55 10.36 5.72

Treatment in vitro callus

Intact leaf In 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 stress MS + 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% sucrose MS + 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 temperature MS + 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

TLC and HPTLC Studies in Leaf and Callus Extracts

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

A.B.A. Ahmed et al.

values to those of the standard GA (Fig 1K and L; Table I). In some cases, the Rf values of the callus extracts were slightly higher. HPTLC offers several advantages, such as facilitating automatic application, scanning in situ, small quantity of mobile phase, and lower analysis time and cost per analysis [23]. Several callus extracts can be run simultaneously using a small quantity of mobile phase. Furthermore, the developed TLC plates can be scanned for several times with same or different parameters as mentioned earlier. The concentrates (10) callus extract samples are analyzed in one run; this method proves to be very sensitive, relatively fast, inexpensive and suitable for therapeutic drug monitoring and pharmacokinetic 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 and not integrated to the base line, we made the following adjustment such as silica gel granular materials, multiple separation (the single time run multiple samples) and two-dimensional process were done. TLC analyzer shows 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 black and white image density (a densitogram). It also finds the maximum and minimum values for these same variables. GA purity was confirmed in the intact leaf and callus extracts by recording the absorption spectra developed in start, middle and end of the peak. Standard GA was shown the single peak at different time intervals of the experiment (Fig. 3A). TLC analyzer automatically produces multi-spectral scans from an image; however, multi-spectral scans can also be produced using almost any image-editing software by simply reading the pixel brightness values using the eyedropper tool then plotting those values in a graphics program. In fact, EMD Silica Gel 60F 254 fluorescent TLC was developed using an image-editing program, but TLC analyzer saves a great deal of time. The intact leaf and callus extracts sample curve was linear; the correlation coefficient has good linearity between concentration and area, it could be helpful to calculate the GA amount in the respectable sample (Fig. 3BN). Green friable callus was induced 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 and cytokinins combination (Fig. 3CD). Hence, NAA and 2,4-D combined with cytokinins callus extracts increased the GA content (Fig. 3EI). In this controversial, MS medium supplemented with OPGRs only has produced the maximum biomass and GA than auxins and cytokinins combinations in 35 45 days of stationary phase (Table I; Fig. 3J). However, the OPGRs were combined with adenine sulphate, and the biomass and GA content, drastically reduced, was determined by HPTLC (Table I; Fig. 3KN). GA content was increased in MS medium with auxins and cytokinins concentrations de-

HPTLC/HPLC and Gravimetric Methodology

rived from leaf explants of G. sylvestre determined by HPTLC [25]. All calibration curves in this research were produced by plotting the peak optical density for each of the same concentration of the different samples. TLC analyzer automatically outputs the peak optical density calculated in the manner.
A B

Fig. 3.

A.B.A. Ahmed et al.

Fig. 3.

HPTLC/HPLC and Gravimetric Methodology

Fig. 3.

A.B.A. Ahmed et al.

Fig. 3. HPTLC analysis of gymnemic acid determined in methanol extracts of Gymnema 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 L1); E. MS + 2,4-D (1.5 mg L1) + BA (0.5 mg L1); F. MS + NAA (1.0 mg L1) + BA (0.5 mg L1); G. MS + NAA (1.0 mg L1) + KN (1.0 mg L1); H. MS + NAA (1.0 mg L1) + BA (1.0 mg L1); I. MS + NAA (1.0 mg L1) + KN (1.5 mg L1); J. MS + 2,4-D (1.5 mg L1) + KN (0.5 mg L1) (OPGRs); K. MS + NAA (1.5 mg L1) + 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 L1) + BA (1.0 mg L1) + 5 mg L1 Ads; N. MS + 2,4-D (1.0 mg L1) + KN (1.0 mg L1) + 5 mg L1 Ads

HPLC Studies in Leaf and Callus Extracts

For HPLC analysis, leaf and callus methanol extracts (20 L) were uploaded in HPLC system to quantify GA under retention time (5 min). UV spectrophotometer peak area data were compared with standard gymnemic acid (Table III). Gymnemagenin and GA were significantly increased in callus culture through leaf explants of G. sylvestre [1016]. In the present study, maximum GA production was observed in MS medium supplemented with OPGRs under blue light induced 4.4 fold as compared with white fluorescent light and out of which 2.8 fold was found in intact leaves determined by HPLC analysis. We have recently published a review of pharmacological activities, 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 through single peak at initial, middle, and end of the HPLC experiment (Fig. 4BD).

HPTLC/HPLC and Gravimetric Methodology

Reten. Time [min]

Area [mVs]

Height [mV]

Area [%]

Height [%]

W05 [min]

No peak to report B

Reten. Time [min] 2.997 Total

Area [mVs] 42.609 42.609

Height [mV] 2.463 2.463 Fig. 4.

Area [%] 100.0 100.0

Height [%] 100.0 100.0

W05 [min] 0.29

A.B.A. Ahmed et al.

Reten. Time [min] 3.010 Total

Area [mVs] 39.420 39.420

Height [mV] 2.353 2.353

Area [%] 100.0 100.0

Height [%] 100.0 100.0

Reten. Time [min] 3.013 Total

Area [mVs] 43.211 43.211

Height [mV] 2.353 2.353 Fig. 4.

Area [%] 100.0 100.0

Height [%] 100.0 100.0

W05 [min] 0.29

HPTLC/HPLC and Gravimetric Methodology

1 2 3

Reten. Time [min] 2.667 3.003 3.333 Total

Area [mVs] 628.235 416.005 818.611 1862.851

Height [mV] 27.617 24.000 16.749 68.366

Area [%] 33.7 22.3 43.9 100.0

Height [%] 40.4 35.1 24.5 100.0

W05 [min] 0.34 0.34 0.64

1 2 3 4 5

Reten. Time [min] 2.267 2.999 3.383 3.747 9.030

Area [mVs] 99.868 235.271 109.091 497.750 6.800

Height [mV] 6.730 12.794 7.454 8.396 0.295

Area [%] 10.5 24.8 11.5 52.5 0.7

Height [%] 18.9 35.9 20.9 23.5 0.8

Fig. 4. HPLC analysis of gymnemic acid determined in methanol extracts of G. sylvestre leaf 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 L 1 ) + KN (1.5 mg L 1 ); 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

A.B.A. Ahmed et al.

Imoto et al. (1991) reported that GA content was confirmed by HPLC in methanol leaf extracts of G. sylvestre [27]. GA quantification was done in the respectable leaf and in vitro callus methanol extracts of G. sylvestre. Fig. 4E described that the GA content was increased in intact leaf explants (19.52 mg g1) compared to in vitro callus culture of MS medium supplemented with NAA (1.0 mg L1) + KN (1.5 mg L1) (11.04 mg g1) (Fig. 4F) and OPGRS (2,4-D 1.5 mg L1 + KN 0.5 mg L1; 12.22 mg g1; Fig. 4G). Many authors had isolated and identified GA earlier in leaf explants of methanol extracts. In 1989, Yoshikawa and co-workers isolated GAs from a hot water extract of G. sylvestre, which they named GA, I, II, III, IV, V, VI, and VII, respectively, and evaluated using HPLC [28, 29]. The above mentioned OPGRs sample kept under abiotic stress conditions and the developed callus methanol extracts were further analyzed (Fig. 4HN). Blue light with OPGRs was induced the maximum GA (53.94 mg g1) (Fig. 4H) than 5% sucrose treatment (33.39 mg g1) (Fig. 4I) followed by 3 mM NH4NO3 (19.10 mg g1) (Fig. 4J). However, other physical stress conditions GA content was reduced 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 (2.9 mg g1) (Fig. 4L). In case of dark treatment, GA content was absent. We have previously reported that this in vitro abiotic stress callus of G. sylvestre significantly increased the pancreatic -cells and maintained the body weight, pancreas weight, liver weight and liver glycogen level in alloxan induced diabetic Wistar rats [16].

Linearity
As per the ICH guidelines, validation parameters such as linearity, accuracy, precision, and robustness were checked. The linearity of the method was determined at three concentrations (1030 g mL1) of GA. Twenty micrograms per milliliter GA results show that an excellent correlation exists between response factor and concentration of GA within the concentration range indicated above.

Accuracy and Precision

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 80, 100, and 120%, and the percentage recovery was calculated. Our studies recovery was within the range of 100 2% which indicates accuracy of the method. The precision of the method was demonstrated by interday and intraday variation studies. In the intraday studies, three repeated injections of standard and sample solution were made in a day and the response factor of GA peaks and per-

HPTLC/HPLC and Gravimetric Methodology

centage were calculated. In the interday variation studies, three repeated injections of standard and sample solutions were made on three consecutive days and response factor of GA peak and percentage were calculated (data not shown). Intra- and interday accuracy were established from quality control standards by evaluating nominal and mean measured concentrations of quality control standards which were compared and expressed as % difference (diff. %). Diff. % was calculated using the formula: Diff.% [(mean measured concentration nominal concentration)/nominal concentration] 100.

Robustness
Wavelength (200 nm) of GA compound was studied showing a sufficient absorption, 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 analytical time and improve the sensitivity and peak shape of GA, a gradient, characterized by a decreased amount of acetic acid (0.1%) was applied before the elution of GA. However, GA is eluted isocratically in order to guarantee robustness.

Gravimetric Analysis in Leaf and Callus Extracts

Table III described 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 identification of plant natural products were done in HPLC than other methods, because HPLC method has the good selectivity, sensitivity of detection, together 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 for extraction of the GA. The GA can be determined on abiotic stress treatment callus 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 of poly-herbal combinations and compared with respective biomarker compounds [31].

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 the abiotic stress conditions determined by HPLC and gravimetric methods. With this method, we hope that more people can be promoted and start the GA bioequivalence study in the future. Thus, it appears that blue light stress has to be used as a tool for enhancing GA accumulation in the in vitro callus culture [32]. We suggest that the GA content intact leaf and in vitro callus could potentially regulate pancreatic -cells for IDDM (insulin-dependent diabetes mellitus) [16]. The proposed RP-HPLC and HPTLC methods for the estimation of GA in intact leaf and in vitro callus are selective and sensitive than gravimetric method. GA has UV absorbing molecules with specific chromophores in their structures that absorb at a particular wavelength, and this fact has been successfully employed for their quantitative determination by UV spectrophotometric method. The development of a rapid, sensitive and accurate analytical method for routine quantitative determination of samples will reduce unnecessary tedious sample preparations and cost of materials and labor.

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