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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
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.
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
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].
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.
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
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
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
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
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
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
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
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-
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.
Fig. 3.
Fig. 3.
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
Area [mVs]
Height [mV]
Area [%]
Height [%]
W05 [min]
No peak to report B
1 2 3
1 2 3 4 5
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
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.
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.
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.
References
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