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In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate β-cells regeneration and anti-diabetic activity in Wistar rats
Phytomedicine 17 (2010) 1033–1039
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Short communication
In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate -cells regeneration and anti-diabetic activity in Wistar rats A. Bakrudeen Ali Ahmed a,c,∗ , A.S. Rao b , M.V. Rao a a
Department of Plant Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India c Department of Chemistry, Pukyong National University, Busan, South Korea b
a r t i c l e Keywords: Gymnema sylvestre Gymnemic acid Growth curve Organ weight Liver glycogen -Cell regeneration
i n f o
a b s t r a c t A methanol extract of Gymnema sylvestre leaf and callus showed anti-diabetic activities through regenerating -cells. Optimum callus was developed under stress conditions of blue light with 2,4-D (1.5 mg/l) and KN (0.5 mg/l), which induced maximum biomass of green compact callus at 45 days, as determined by growth curve analysis. Leaf and optimum callus extracts contains gymnemic acid, which was analyzed using TLC, HPTLC and HPLC methods. The research reported here deals with leaf and callus extracts of G. sylvestre, which significantly increase the weight of the whole body, liver, pancreas and liver glycogen content in alloxan-induced diabetic rats (Wistar rats). The gymnemic acid of leaf and callus extracts significantly increases the regeneration of -cells in treated rats, when compared with the standard diabetic rats. It could have potential as a pharmaceutical drug for insulin-dependent diabetes mellitus (IDDM). © 2010 Elsevier GmbH. All rights reserved.
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 insulin-producing -cells in the pancreas, resulting in low or no production of insulin, a hormone necessary for survival (International Expert Committee 2009). Gymnema sylvestre (syn. Periploca sylvestris Retz) 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 (Mitra et al., 1995). The extract of G. sylvestre plays a major role in blood glucose homeostasis through increased serum insulin level through regeneration of the endocrine pancreas (Shanmugasundaram et al., 1983; Shanmugasundaram et al., 1990). G. sylvestre occurs mainly in the Deccan peninsula of western India, Tropical Africa, Vietnam, Malaysia, Srilanka and is widely available in Japan, Germany and the USA as a health food (Ye et al., 2000). Within the last 10 years, a number of Gymnema products, including Gymnema capsules, Gymnema tea, Bioshape® , and Diaxinol® have appeared on the world market.
In the past few decades, secondary metabolite production from plant tissue culture has been identified as a tremendous resource for new drug development and clinical research in the fields of pharmacology and medicine. Plant cell culture extracts have also been used widely in the form of fractions and isolated compounds as potential bioactive molecules (Sokmen et al., 1999). In vitro developed callus tends to produce various active compounds, including gymnemic acid and gymnemagenin (Kanetkar et al., 2006). However, external factors like phytohormone, shaking speeds, pH, and medium play an important role in gymnemic acid production in suspension cultures (Devi et al., 2006). In addition, sucrose, inoculum density, auxins, and aeration also play a very crucial role in the production of gymnemic acid through bioreactor-dependent cell growth (Lee et al., 2006). The present studies were undertaken to identify the G. sylvestre leaf and callus extract molecules that contribute to or promote -cell regeneration and anti-diabetic effects. In addition, we have characterized the gymnemic acid role in anti-diabetic experiments; it was found that pancreas weight and glycogen content were increased in the liver of alloxan-induced diabetic Wistar rats. Furthermore, an emphasis is laid on -cell regeneration and the determinants implicated. Materials and methods Plant material and sterilization
∗ Corresponding author at: Marine Bioprocess Research Center, Department of Chemistry, Pukyong National University, Busan, South Korea. Tel.: +82 51 629 7098; fax: +82 51 629 7099. E-mail address: [email protected] (A.B.A. Ahmed). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.019
G. sylvestre plants (GS) were collected from the Pachamalai hills, and maintained in the plant science garden of the Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Leaf explants
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were washed with tap water, Teepol solution, then 70% ethanol for 30 min and 0.1% HgCl2 for 2 min. Prior to inoculation, explants were washed several times in sterile distilled water.
Pvt. Ltd., India) (200 mg/kg body wt./day/rat) given (P.O.) daily (Shanmugasundaram et al., 1990; Ahmed et al., 2008).
Callus induction
Experimental animal
Leaf explants of G. sylvestre were grown in MS medium (Murashige and Skoog, 1962) supplemented with 2,4-D (1.5 mg/l) and KN (0.5 mg/l). Callus culture was maintained at 25 ± 2 ◦ C, 16 h/8 h (light/dark) photoperiod with 25 mol m−2 s−1 of light intensity (Ahmed et al., 2009a).
All experiments were approved by Institutional Animal Ethics Committee (IAEC) procedure guidelines of CPCSEA. Male and female (8–9-month old) Wistar rats (body weight 200 ± 25 g each) were maintained at Bharathidasan University, Tiruchirappalli, on standard pellet diet (Hindustan Lever Ltd., Bangalore, India) with free access to tap water.
Callus developed under stress condition Callus cultures were maintained under different stress conditions for gymnemic acid production (Ahmed et al., 2009a). The protocol was as follows: different color light (blue, red, green and white fluorescent tubes); temperature (20 ◦ C, 25 ± 2 ◦ C, 30 ◦ C and 35 ◦ C); photoperiod (4 h/20 h, 8 h/16 h, 12 h/12 h, 20 h/4 h light/dark), sucrose (2%, 4%, 5%, 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 Plant growth regulators (PGRs) with stress treatment callus fresh and dry weight were determined at 15, 25, 35, 45 and 55 days. Maximum biomass (fresh, dry weight) and green compact callus were noted for all treatments. Phytochemical studies on leaf and in vitro callus G. sylvestre dried leaves and in vitro callus were extracted with methanol 5 times (Rehman et al., 2003). The collected methanol extract was centrifuged at 5000 × g for 10 min at room temperature, then the methanol supernatant carefully pipetted out into fresh eppendorf tubes without disturbing the interphase residues. Green-color supernatant (20 l) was screened using TLC and HPTLC with standard gymnemic acid (Prof. Kazuko Yoshikawa, Kyoto Pharmaceutical University, Japan) for gymnemic acid quantification (Ahmed et al., 2009a). HPTLC was performed using a Camag HPTLC system (CAMAG, Switzerland) equipped with a sample applicator Linomat IV, TLC scanner III and integration software CAT 4.0. The mobile phase of Isopropyl alcohol:chloroform:methanol:acetic acid (5:3:1:0.5) was suitable for separation of gymnemic acid and scanned at 200 nm, respectively.
Chemical preparation and induction of diabetes Alloxan monohydrate (Otta kemi, Mumbai, India) (100 mg/kg body wt./rat) was dissolved in distilled water and injected intraperitoneally (i.p.). The experimental rats entered a diabetic state after 72 h, then the Wistar rats were divided into groups for treatment with G. sylvestre leaf and callus extracts. Therapeutic human insulin (Torrent Pharmaceuticals Ltd., Mehsana, India) mixture was shaken well and injected daily (4 U/kg body wt.) subcutaneously as a diabetic standard (Chattopadhyay 1998).
Instruments and methods The body weight, liver and pancreas weights of the experimental Wistar rats were checked in fundamental weight instruments. Liver glycogen (Raghuramalu et al., 1983) and pancreas were removed for further analysis. The removed pancreas was fixed in dewaxed and hydrated paraffin for micro section (6 m thickness), then stained with hematocylin and eosin. In addition, stained pancreas was washed with Gomori’s aldehyde fuchsin for determination of -cells in Langerhans islets (Gomori, 1950) as described below. For -cells determination, the 0.5 g basic fuchsin was dissolved in 70% alcohol, to which were added 1.0 ml paraldehyde with 1.5 ml conc. HCl. Embedding hydrated paraffin sections were treated with Lugol iodine solution for 30 min, then bleached using 5% sodium thiosulfate for 2 min followed by washing of the section for 5 min in running tap water for oxidation. The -cells were rinsed with 95% and 70% alcohol for 10 min, and then washed with distilled water followed by Ehrlich’s hematoxylin for 4 min and counter stain for 45 s, respectively. Finally, the stain coupling jar was treated with aldehyde fuchsin for 2–10 min. The end point of -cells stood out clearly in dark purple against a colorless background.
HPLC studies in callus and leaf extracts Gymnemic acid was screened in leaf and 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 gymnemic acid separation, the following systems and protocols were used: water HPLC system (Shimadzu model, Japan), 510 pump, 7725 Rheodyne injector, C18 (ODS) reversed-phase column, water 486 UV detector, with mobile phasewater/methanol (35:65, HPLC grade), 0.1% acetic acid, sample applied: 20 l, flow rate: 1 ml/min, read at 230 nm, on UV detector. Preparation of GS leaf and callus extracts In vivo Gymnema leaves extract (GLE) and in vitro callus extracts GCE1 –GCE6 were prepared by the method reported previously (Ahmed et al., 2008). The extracted sample was evaporated and dissolved in saline water (Sterile NS saline, Baxter
Treatment groups Group I (control): free access to tap water. Groups II–X: diabetes-induced animals were divided into 9 groups administered saline, 4 U/kg body wt. of insulin/day, 200 mg/kg body wt. (rat)/day GLE and GCE1 –GCE6 , respectively. The experimental Wistar rats were sacrificed at 10, 20 and 30 days. The following treatment groups were used in this experiment: Group II – diabetic control (Alloxan); Group III – Diabetic standard (insulin); Group IV – diabetic rats + G. sylvestre leaf extract; Group V (GCE1 ) – blue light with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); Group VI (GCE2 ) – 5% sucrose with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); Group VII (GCE3 ) – 12 h photoperiod MS + with 2,4-D (1.5 mg/l) + KN (0.5 mg/l), Group VIII (GCE4 ) – 3 mM NH4 NO3 with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l), Group IX (GCE5 ) – MS + 2,4D (1.5 mg/l) + KN (0.5 mg/l) and Group X (GCE6 ) – MS + NAA (1.0 mg/l) + KN (1.5 mg/l), respectively.
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Statistical analysis
Results and discussion
Only data which showed an advantageous effect were included in tables and presented in mean ± SE (replicated 3 times). Experimental design was completely random and factorial with control, alloxan alone, alloxan with insulin, alloxan with leaf and alloxan with callus extracts. The data were subjected to analysis of variance and mean separation was carried out using Duncan’s multiplerange test (DMRT) at 5% level significance (Gomez and Gomez, 1976).
Callus initiation under stress conditions
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In vitro callus failed without PGRs in MS medium, while different media (MS, B5, SH, WPM) with PGRs affected the callus initiation in leaf explants (data not shown). Trial of auxins alone, and auxins with cytokinins combinations showed callus induction, among which 2,4-D (1.5 mg/l) with KN (0.5 mg/l) induced green compact callus with maximum dry weight at 45 days.
Fig. 1. (A) Effect of different color lights with (OPGRs) optimum plants growth regulators (MS + 2,4-D) + KN (0.5 mg/l). ( ) Blue lights + OPGRs; ( ) green lights + OPGRs; ( ) red lights + OPGRs; ( ) dark (without lights) + OPGRs; ( ) white lights + OPGRs. (B) Effect of different temperature with optimum plant growth regulators (OPGRs) (MS + 2,4D) (1.5 mg/l) + KN (0.5 mg/l); () 20 ◦ C + OPGRs; ( ) 30 ◦ C + OPGRs; ( ) 35 ◦ C + OPGRs; ( ) 25 ± 2 ◦ C + OPGRs. (C) Effect of different photoperiod with optimum plant growth regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 4 h + OPGRs; ( ) 8 h OPGRs; ( ) 12 h OPGRs; ( ) 16 h (control) + OPGRs; ( ) 20 h + OPGRs; ( ) 24 h + OPGRs; ( ) dark + OPGRs. (D) Effect of different concentration carbon source (sucrose) with optimum plant growth regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 2% sucrose + OPGRs; ( ) 3% sucrose (control) OPGRs; ( ) 4% sucrose + OPGRs; ( ) 5% sucrose + OPGRs; ( ) 6% sucrose + OPGRs. (E) Effect of ammonia nitrate concentration (NH4 NO3 ) with (OPGRs) optimum plant growth regulators (MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); ( ) 0 mM NH4 NO3 + OPGRs (control) () 1 mM NH4 NO3 + OPGRs; ( ) 2 mM ) 3 mM NH4 NO3 + OPGRs; ( ) 4 mM NH4 NO3 + OPGRs. (For interpretation of the references to color in this figure legend, the reader is referred to the NH4 NO3 + OPGRs; ( web version of the article.)
Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not significantly different at 5% probability level according to DMRT. a GCE1 – blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 – 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 – 12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE4 – 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE5 – MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 – MS + NAA (1.0 mg/l) + KN (1.5 mg/l).
2.12c 2.26ab 2.35a 1.90bc 1.94b 2.17a 2.00bc 2.17b 2.39a 1.90c 2.04ab 2.15a 1.76a 1.54b 1.35bc Liver glycogen (mg/g) 10th 2.65c 20th 2.74ab 30th 2.82a
2.15bc 2.24b 2.56a
1.95bc 2.10b 2.32a
2.10c 2.35ab 2.47a
2.16bc 2.25b 2.46a
8.4c 8.7ab 9.0a 8.1bc 8.4b 8.9a 7.9ab 7.5c 8.0a 8.0c 8.4ab 8.7a 7.8a 7.5ab 7.0c Liver weight (g/animal) 10th 8.0c 20th 8.5ab 30th 9.0a
8.0c 8.2ab 8.4a
8.5bc 8.7b 9.0a
7.6c 8.0ab 8.3a
7.6b 7.2bc 8.2a
1.14c 1.20ab 1.25a 1.30c 1.35ab 1.37a 1.28bc 1.30b 1.39a 1.10c 1.31ab 1.35a 1.35ab 1.20c 1.42a 1.29bc 1.30b 1.37a 1.10c 1.19ab 1.25a 1.28c 1.37ab 1.40a 1.16a 1.05b 0.95bc Pancreas weight (g/animal) 10th 1.30bc 20th 1.35b 30th 1.42a
208.0bc 199.0fg 205.0f 220.0a 200.0de 196.6fg 205.0c 215.0a 208.0bc 196.0f 195.0fg 215.0a 203.0a 191.6c 172.0e 162.0fg Body weight (g/animal) 0 200.0d 10th 200.0de 20th 225.0bc 30th 240.0a
223.3c 213.3g 222.5cd 235.0a
226.6e 226.6e 237.5c 240.0a
220.0ed 213.0ef 225.0c 245.0a
206.0cd 200.0e 215.0b 220.0a
Group IX alloxan + GCE5 Group VIII alloxan + GCE4 Group VII alloxan + GCE3 Group VI alloxan + GCE2 Group V alloxan + GCE1 Group IV alloxan + leaf Group III alloxan + insulin Group II alloxan Group I (control) Days
Table 1 Body weight (g) of Wistar rats with leaf and callus (methanol) extracts (GSC1 –GSC6 )a of G. sylvestre.
193.0e 190.0g 205.0b 210.0a
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Group X Alloxan + GCE6
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Kumar et al. (2002) reported MS medium with PGRs were suitable for callus proliferation of G. sylvestre, but callus proliferation in other auxins with cytokinins concentration was not pursued, because of their poor quality and production (data not shown). Recently 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 (Ahmed et al., 2009a). Stress treatment and callus growth curve were screened at different days (15, 25, 35, 45 and 55 days). Lag phase (15–25 days), callus initiation and proliferation were observed. 25–35 days (exponential phase), biomass and green compact callus increased significantly. However, stationary phase at 45 days, maximum biomass and green compact callus were shown. Decline phase, the nature and biomass of callus were significantly reduced as compared to other phases. In Asclepiadaceae species, the stationary phase was observed at 22 days; at this phase cryptosin was isolated from Cryptolepis buchanani (Venkateswara et al., 1987) and gagaminine from Cynanchum wilfordii (Shin et al., 2003). Optimized concentration was maintained under stress conditions; leaf explants under blue light, 5% sucrose induced the maximum biomass with green compact callus, then photoperiod, temperature and ammonium nitrate concentrations (Fig. 1A–E). Photoperiod and temperature affected physical appearance, 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. Blue light revealed green compact callus with maximum biomass at 45 days. Cryptochrome showed a top peak at 450 nm, a wavelength close to the blue light; under these conditions, more Pr transformed into Pfr for phenyletanoid glycosides production (Ouyang et al., 2003). Zhao et al. (2001) reported that blue light stimulates jaceosidin production in Saussurea medusa.
TLC, HPTLC, HPLC studies Methanol supernatants of G. sylvestre leaf and in vitro calluses were screened using TLC and HPTLC. After chromatogram, samples were dried and sprayed with specific reagent (vanillin sulfuric acid reagent) at room temperature for detection of gymnemic acid. Standard gymnemic acid showed as a single brown band; however, the callus extract displayed additional brown bands with Rf value (0.44) greater than that of gymnemic acid (0.43). In HPTLC analysis, methanol solvent was run up to 80 mm and scan chromatograms at 200 nm under UV reflectance mode. Gymnemic acid content leaf and callus extract data were compared with standard gymnemic acid. The calibration curve was linear; the correlation coefficient indicated good linearity between concentration and area. To ascertain peak purity of test sample, we compared in vivo and in vitro methanol extract reflectance spectra with standard, which provides test sample purity (data not shown). Gymnestrogenin (triterpenoids) determine in G. sylvestre through HPTLC at 293 nm (Puratchimani and Jha, 2004). For HPLC analysis, leaf and callus methanol extracts (20 l) were uploaded in HPLC system to quantify gymnemic acid under retention time. UV spectrophotometer peak area data were compared with standard gymnemic acid. The gymnemic acid content was increased in leaf, GSC1 and GSC3 over other callus extracts (data not shown). Imoto et al., 1991 reported that a methanol extract contains gymnemic acid through HPLC. Many authors had isolated and identified gymnemic acid earlier. In 1989, Yoshikawa and coworkers isolated gymnemic acids from a hot water extract of G. sylvestre, which they named gymnemic acids I, II, III, IV, V, VI and VII, respectively, and evaluated using HPLC (Sugihara et al., 2000).
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Fig. 2. Histologic examination of pancreatic -cells in alloxan treated animals. (A) Pancreas of normal rats. Note abundant patches if -cells (arrows) in purple color (200×); (B) pancreas of alloxan treated rats, hardly and -cells noticed, all islets degenerated (200×); (C) pancreas of alloxan treated rats, after insulin treatments showed abundant patches of -cells (arrows) in purple color (200×); (D) pancreas treated rats, after G. sylvestre leaf extract (200×); (E) pancreas treated rats, after G. sylvestre callus extract 1 (200×); (F) G. sylvestre callus extract 2 (200×); (G) G. sylvestre callus extract 3 (200×); (H) G. sylvestre callus extract 4 (200×); (I) G. sylvestre callus extract 5 (200×); (J) G. sylvestre callus extract 6 (200×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Body, liver, pancreas weight and liver glycogen As shown in Table 1, the long-term antihyperglycemic effect was examined for GS leaf and callus extracts. The whole body, liver and pancreas weights were reduced in the diabetic control group after 30 days, although oral administration of 200 mg/kg body wt. GS leaf and callus extracts significantly increased and maintained the body liver and pancreas weights in the 4 U/kg body wt. insulin group Wistar rats. We have recently published a review of the pharmacological activities, a phytochemical investigation and in vitro studies of G. sylvestre (Ahmed et al., 2009b). Liver glycogen was significantly decreased in diabetic control rats, whereas with leaf and callus extracts of GS, liver glycogen significantly increased (Table 1). Restoration of hepatic glycogen by GS leaf and callus could improve the insulin secretion or inhibition of glucose-6-phosphatase in liver, and has prevented the conversion of glucose 6-phosphate to glucose (Shanmugasundaram et al., 1990). Shanmugasundaram et al. (1983) reported that G. sylvestre restored glycogen and enzymes in diabetic rabbit liver after 24 weeks of treatment. We earlier reported that leaf and callus
extracts of G. sylvestre reduced blood sugar and lipid profiles such as cholesterol, triglyceride, HDL, LDL, VLDL in alloxan-induced diabetic Wistar rats (Ahmed et al., 2008). Shanmugasundaram et al. (1990) reported aqueous extract of G. sylvestre leaf (200 mg/kg body wt.) normalized blood sugar level in STZ-induced diabetic rats. However, gymnemic acid from G. sylvestre leaf regulated hyperglycemia (Gholap and Kar 2005) and an aqueous extract maintained the blood glucose level in normal Wistar rats (Rafiullah et al., 2006). Histological examinations of pancreas Fig. 2A–J depicts the histological examination (200×) of pancreatic -cells in control, diabetic control, diabetic standard, GS leaf and callus extracts groups. Histological pancreas islets were checked separately in hematoxylin and eosin with aldehyde fuchsin section. In hematoxylin and eosin sections, the degree of atrophy, hydropic degeneration, necrosis, hyalinization, or fibrosis was checked (data not shown), while in aldehyde fuchsin sections, the -cells percentage was recorded based on atrophy cells, and the -granules were increased or decreased at the 10th, 20th and 30th
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Table 2 Leaf and callus extractsa of Gymnema sylvestre regenerate the pancreatic -cells in alloxan-induced diabetic Wistar rats. Treatment group
-Cell regeneration frequency (%) 10th day
Group I (normal) Group II (diabetic control) Group III (insulin) Group IV (leaf) Group V (callus extract 1) Group VI (callus extract 2) Group VII (callus extract 3) Group VIII (callus extract 4) Group IX (callus extract 5) Group X (callus extract 6)
81.0 16.0 38.0 49.0 53.3 42.3 51.3 30.3 28.0 27.0
± ± ± ± ± ± ± ± ± ±
2.82a 2.16j 2.62b 4.54e 8.07c 5.89ef 3.13cd 2.12g 6.01gh 3.77i
20th day 83.6 12.3 41.0 58.6 50.0 47.0 55.0 32.6 31.6 34.0
± ± ± ± ± ± ± ± ± ±
4.27a 1.44j 1.69ab 1.18c 0.94e 3.85ef 4.54cd 6.15gh 4.11i 3.39g
30th day 80.0 8.6 45.5 66.6 59.6 54.3 57.6 39.0 45.0 37.6
± ± ± ± ± ± ± ± ± ±
2.62ab 1.65j 2.82a 2.88c 4.09cd 4.57ef 3.47e 4.54gh 1.88g 0.98i
Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not significantly different at 5% probability level according to DMRT. a GCE1 – blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 – 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 –12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE4 – 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE5 –MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 – MS + NAA (1.0 mg/l) + KN (1.5 mg/l).
days (Table 2). In electron microscopic analysis, the pancreatic cells islets showed no pathological alternation in control Wistar rats. The nucleus, nuclear envelope, mitochondria, endoplasmic reticulum and Golgi complex of -cells were normal (Fig. 2A). However, 80% of -cells contained a number of secretory granules, which had a space between core and membranes diffusely distributed in cytoplasm at end of experiment. The secretory granules showed moderate homogenous (or) slightly heterogenous electron density and single layered membrane (Fig. 2A). Electron microscope analysis of -cells in diabetic standard rats (alloxan alone) displayed the loss of nuclear envelope, mitochondrial vacuolization, swelling and dilatation of endoplasmic reticulum (Fig. 2B). During alloxan treatment, -cytotoxin causes a massive destruction of -cells in islets of Langerhans, and reduces the -cells and insulin release (Colca et al., 1983). Diabetic standard (insulin), GS leaf and callus extracts treated groups showed significant increase in the secretary granule of -cells and reached to control level (Fig. 2D–J). The -cells were apparently normal in the architecture of the nucleus, which revealed that gymnemic acid prevented the -cells damage as compared to control rats. But low gymnemic acid content callus extracts decreased the secretory granules and losses of nuclear envelope, mitochondrial vacuolization, swelling and dilation of the endoplasmic reticulum in diabetic rats. Degenerative changes in pancreatic -cells were minimized and normal morphology maintained in diabetic rats by administration of gymnemic acid content extracts as evidenced in histopathological examination. Pancreas hematoxylin/eosin with aldehyde fuchsin stains displayed the ruptured islets and decreased -cells in diabetic control rats, whereas leaf and callus extracts regenerated the -cells frequency in diabetic standard (Fig. 2C; 45.5%), GS leaf extract (Fig. 2D; 66.6%), leaf callus extract 1 (Fig. 2E; 59.6%), callus extract 2 (Fig. 2F; 54.3%), callus extract 3 (Fig. 2G; 57.6%), callus extract 4 (Fig. 2H; 39.0%), callus extract 5 (Fig. 2I; 45.0%) and callus extract 6 (Fig. 2J; 37.6%) (Table 2). In treatment pancreatic cells, AMP was activated and the protein kinases showed a unique function or connecting cellular energy status to the ability of insulin synthesis and secretion of -cells (Da Silva Xavier et al., 2000). Alcohol extract of G. sylvestre stimulated the insulin secretion in islets of Langerhans and pancreatic -cell lines (Persaud et al., 1999). Srivastava et al. (1986) reported that pancreas tissue was completely destroyed in alloxan treatment, after G. sylvestre leaf extract regenerated the prolonged survival and adaptogenic activity. However, a smaller dose of STZ (45 mg/kg body wt.) led to damage of the pancreatic acinar cells and reversed by Aegle marmelos (Arumugam et al., 2008). Successful -cell regeneration was achieved using diabetic medicinal plants such as Momordica charantia, Beta vulgaris, Catharanthus roseus, and Eugenia jambolana (Das et al., 1996; Sharma et al., 2006).
In conclusion, the leaf and callus extracts of G. sylvestre have antihyperglycemic effects as determined using alloxan-induced diabetic Wistar rats. Our experimental data suggested that alloxan administered to Wistar rats through intraperitoneal route exhibits diabetic activity. We conclude that the effect of G. sylvestre leaf and callus extracts as strong herbal remedies is confirmed in this study, and suggest that they may be capable of fully restoring pancreatic -cells function and thus curing type I diabetes. Studies of G. sylvestre callus extract have revealed how -cells may be formed and regenerated in vitro and could provide further direction for potential drug development for IDDM treatment. References Ahmed, A.B.A., Rao, A.S., Rao, M.V., 2009a. In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R.Br. Ex. Roemer and Schultes through callus culture under stress conditions. Methods Mol. Biol. 547, 93–105. Ahmed, A.B.A., Komalavalli, N., Muthukumar, M., Benjamin, J.H.F., Rao, A.S., Kim, S.K., Rao, M.V., 2009b. Pharmacological activities, phytochemical investigations and in vitro studies of Gymnema sylvestre R.Br.—a historical review. Comprehensive Bioactive Natural products–Potential & Challenges, vol. 1, pp. 75–99. Ahmed, A.B.A., Rao, A.S., Rao, M.V., 2008. Role of in vivo and in vitro callus of Gymnema sylvestre (Retz) R.Br. Ex. Roemer & Schultes in maintaining the normal levels of blood glucose and lipid profile in diabetic Wistar rats. Biomedicine 28, 134–138. Arumugam, S., Kavimani, S., Ahmed, A.B.A., Kadalmani, B., Akbarsha, M.A., Rao, M.V., 2008. Antidiabetic activity of leaf and callus extracts of Aegle marmelos in rabbit. Sci. Asia 34, 317–321. Chattopadhyay, R.R., 1998. Possible mechanism of antihyperglycemic effect of Gymnema sylvestre leaf extract. Part I. Gen. Pharmacol. 31, 495–496. Colca, J.R., Kotagel, N., Brooks, C.L., Lacy, P.E., Landt, M., McDanleld, M.L., 1983. Alloxan inhibition of Ca2+ and calmodulin dependent protein kinase in pancreatic islets. J. Biochem. 225, 7260–7263. Das, A.V., Padayatti, P.S., Paulose, C.S., 1996. Effect of leaf extract of Aegle marmelos (L.) on histological and ultra structural changes in tissues of streptozotocin induced diabetic rats. Indian J. Exp. Biol. 34, 341–345. Devi, C.S., Murugesh, S., Srinivasan, V.M., 2006. Gymnemic acid production in suspension cell cultures of Gymnema sylvestre. J. Appl. Sci. 6, 2263–2268. Da Silva Xavier, G., Leclerc, I., Salt, I.P., Doiron, B., Hardie, D.G., Kahn, A., Rutter, G.A., 2000. Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc. Natl. Acad. Sci. U.S.A. 97, 4023–4028. Gholap, S., Kar, A., 2005. Gymnemic acids from Gymnema sylvestre potentially regulates dexamethasone induced hyperglycemia in mice. Pharm. Biol. 43, 192–195. Gomez, K.A., Gomez, A.A., 1976. Statistical Procedures for Agricultural Research with Emphasis on Rice. International Rice Research Institute, Los Bans, IRRI Publ, Philippines, pp. 264. Gomori, G., 1950. Aldehyde-fuchsin: a new stain for elastic tissue. Am. J. Clin. Pathol. 20, 665. Imoto, T., Yamamoto, F., Miyasaka, A., Hantano, H., 1991. High-performance liquid chromatography-atmospheric pressure ionization mass spectrometry of gymnemic acids. J. Chromatogr. 557, 383–389. International Expert Committee, 2009. Report on the role of the A1C assay in the diagnosis of diabetes. Diabetes Care 32, 1–8. Kanetkar, P.V., Singhal, R.S., Laddha, K.S., Kamat, M.Y., 2006. Extraction and quantification of gymnemic acids through gymnemagenin from callus cultures of Gymnema sylvestre. Phytochem. Anal. 17, 409–413. Kumar, H.G., Murthy, H.N., Paek, K.Y., 2002. Somatic embryogenesis in Gymnema sylvestre. Plant Cell Tiss. Org. Cult. 71, 85–88. Lee, E.J., Mobin, M., Hahn, E.J., Peak, K.Y., 2006. Effects of sucrose, inoculum density, auxins, and aeration volume on cell growth of Gymnema sylvestre. J. Plant Biotechnol. 49, 427–431.
A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 1033–1039 Mitra, S.K., Gopumadhavan, S., Muralidhar, T.S., Anturlikar, S.D., Sujatha, M.B., 1995. Effect of D-400 herbomineral preparation on lipid profile glycated haemoglobin and glucose tolerance in streptozotocin induced diabetes in rats. Indian J. Exp. Biol. 33, 798–800. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497. Ouyang, J., Wang, X., Zhao, B., Wang, Y., 2003. Light intensity and spectral quality influencing the callus growth of Cistanche deserticola and biosynthesis of phenylethanoid glycosides. Plant Sci. 165, 657–661. Puratchimani, V., Jha, S., 2004. Standardisation of Gymnema sylvestre R.Br., with reference of gymnemagenin by high-performance thin-layer chromatography. Phytochem. Anal. 15, 164–166. Persaud, S.J., Al-Majed, H., Raman, A., Jones, P.M., 1999. Gymnema sylvestre stimulates insulin releases in vitro by increased membrane permeability. J. Endocrinol. 163, 207–212. Rafiullah, M.R.M., Siddiqui, A.W., Mir, S.R., Ali, M., Pillai, K.K., Singh, S., 2006. Antidiabetic activity of some Indian medicinal plants. Pharm. Biol. 44, 95– 99. Raghuramalu, N., Nair, M.K., Sundaram, S.K., 1983. A manual of laboratory techniques, Hyderabad. Natl. Inst. Nutr., 96–98. Rehman, R.U., Israr, M., Srivastava, P.S., Bansal, K.C., Abdin, M.Z., 2003. In vitro regeneration of witloof chicory (Cichorium intybus L.) from leaf explants and accumulation of esculin. In Vitro Cell. Dev. Biol. 39, 142–146. Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., 2006. Antihyperglycemic effect of the fruit-pulp of Eugenia jambolana in experimental diabetes mellitus. J. Ethnopharmacol. 104, 367–373.
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Shanmugasundaram, E.R.B., Leela, K.G., Radha, K.S., Rajendran, V.M., 1990. Possible regeneration of the islets of Langerhans in streptozotocin—diabetic rats given Gymnema sylvestre leaf extracts. J. Ethnopharmacol. 30, 265–279. Shanmugasundaram, K.R., Panneerselvam, C., Samudram, P., Shanmugasundaram, E.R.B., 1983. Enzyme changes and glucose utilization in diabetic rabbits the effects of Gymnema sylvestre, R.Br. J. Ethnopharmacol. 7, 205–234. Shin, G., Chil, M., Lee, D., 2003. Comparative study of the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture of Cynanchum wilfordii. Biol. Pharm. Bull. 26, 1321–1325. Sokmen, A., Jones, B.M., Erturk, M., 1999. Antimicrobial activity of extracts from the cell cultures of some Turkish medicinal plants. Phytother. Res. 13, 355–357. Srivastava, Y., Bhatt, H.V., Jhala, C.I., 1986. Oral Gymnema sylvestre leaf extracts inducing protracted longevity and hypoglycemia in alloxan diabetic rats: Review and experimental study. Int. J. Crude. Drug. Res. 24, 171–176. Sugihara, Y., Nojima, H., Matsuda, H., Murakami, T., Yoshikawa, M., Kimura, I., 2000. Antihyperglycemic effects of gymnemic acid IV, a compound derived from Gymnema sylvestre leaves in streptozotocin-diabetic mice. J. Asian Nat. Prod. Res. 2, 321–327. Venkateswara, R, Sankara Rao, K., Vaidyanathan, C.S., 1987. Cryptosin-a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem & Schult. Plant Cell Rep. 6, 291–293. Ye, W.C., Zhang, Q., Liu, X., Che, C., Zhao, S., 2000. Oleanane saponins from Gymnema sylvestre. Phytochemistry 53, 893–899. Zhao, D., Xing, J., Li, M., Lu, D., Zhao, Q., 2001. Optimization of growth and Jaceosidin in callus and cell suspension of Saussurea medusa. Plant Cell Tiss. Org. Cult. 67, 227–234.
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Short communication
In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate -cells regeneration and anti-diabetic activity in Wistar rats A. Bakrudeen Ali Ahmed a,c,∗ , A.S. Rao b , M.V. Rao a a
Department of Plant Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India c Department of Chemistry, Pukyong National University, Busan, South Korea b
a r t i c l e Keywords: Gymnema sylvestre Gymnemic acid Growth curve Organ weight Liver glycogen -Cell regeneration
i n f o
a b s t r a c t A methanol extract of Gymnema sylvestre leaf and callus showed anti-diabetic activities through regenerating -cells. Optimum callus was developed under stress conditions of blue light with 2,4-D (1.5 mg/l) and KN (0.5 mg/l), which induced maximum biomass of green compact callus at 45 days, as determined by growth curve analysis. Leaf and optimum callus extracts contains gymnemic acid, which was analyzed using TLC, HPTLC and HPLC methods. The research reported here deals with leaf and callus extracts of G. sylvestre, which significantly increase the weight of the whole body, liver, pancreas and liver glycogen content in alloxan-induced diabetic rats (Wistar rats). The gymnemic acid of leaf and callus extracts significantly increases the regeneration of -cells in treated rats, when compared with the standard diabetic rats. It could have potential as a pharmaceutical drug for insulin-dependent diabetes mellitus (IDDM). © 2010 Elsevier GmbH. All rights reserved.
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 insulin-producing -cells in the pancreas, resulting in low or no production of insulin, a hormone necessary for survival (International Expert Committee 2009). Gymnema sylvestre (syn. Periploca sylvestris Retz) 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 (Mitra et al., 1995). The extract of G. sylvestre plays a major role in blood glucose homeostasis through increased serum insulin level through regeneration of the endocrine pancreas (Shanmugasundaram et al., 1983; Shanmugasundaram et al., 1990). G. sylvestre occurs mainly in the Deccan peninsula of western India, Tropical Africa, Vietnam, Malaysia, Srilanka and is widely available in Japan, Germany and the USA as a health food (Ye et al., 2000). Within the last 10 years, a number of Gymnema products, including Gymnema capsules, Gymnema tea, Bioshape® , and Diaxinol® have appeared on the world market.
In the past few decades, secondary metabolite production from plant tissue culture has been identified as a tremendous resource for new drug development and clinical research in the fields of pharmacology and medicine. Plant cell culture extracts have also been used widely in the form of fractions and isolated compounds as potential bioactive molecules (Sokmen et al., 1999). In vitro developed callus tends to produce various active compounds, including gymnemic acid and gymnemagenin (Kanetkar et al., 2006). However, external factors like phytohormone, shaking speeds, pH, and medium play an important role in gymnemic acid production in suspension cultures (Devi et al., 2006). In addition, sucrose, inoculum density, auxins, and aeration also play a very crucial role in the production of gymnemic acid through bioreactor-dependent cell growth (Lee et al., 2006). The present studies were undertaken to identify the G. sylvestre leaf and callus extract molecules that contribute to or promote -cell regeneration and anti-diabetic effects. In addition, we have characterized the gymnemic acid role in anti-diabetic experiments; it was found that pancreas weight and glycogen content were increased in the liver of alloxan-induced diabetic Wistar rats. Furthermore, an emphasis is laid on -cell regeneration and the determinants implicated. Materials and methods Plant material and sterilization
∗ Corresponding author at: Marine Bioprocess Research Center, Department of Chemistry, Pukyong National University, Busan, South Korea. Tel.: +82 51 629 7098; fax: +82 51 629 7099. E-mail address: [email protected] (A.B.A. Ahmed). 0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.03.019
G. sylvestre plants (GS) were collected from the Pachamalai hills, and maintained in the plant science garden of the Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Leaf explants
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were washed with tap water, Teepol solution, then 70% ethanol for 30 min and 0.1% HgCl2 for 2 min. Prior to inoculation, explants were washed several times in sterile distilled water.
Pvt. Ltd., India) (200 mg/kg body wt./day/rat) given (P.O.) daily (Shanmugasundaram et al., 1990; Ahmed et al., 2008).
Callus induction
Experimental animal
Leaf explants of G. sylvestre were grown in MS medium (Murashige and Skoog, 1962) supplemented with 2,4-D (1.5 mg/l) and KN (0.5 mg/l). Callus culture was maintained at 25 ± 2 ◦ C, 16 h/8 h (light/dark) photoperiod with 25 mol m−2 s−1 of light intensity (Ahmed et al., 2009a).
All experiments were approved by Institutional Animal Ethics Committee (IAEC) procedure guidelines of CPCSEA. Male and female (8–9-month old) Wistar rats (body weight 200 ± 25 g each) were maintained at Bharathidasan University, Tiruchirappalli, on standard pellet diet (Hindustan Lever Ltd., Bangalore, India) with free access to tap water.
Callus developed under stress condition Callus cultures were maintained under different stress conditions for gymnemic acid production (Ahmed et al., 2009a). The protocol was as follows: different color light (blue, red, green and white fluorescent tubes); temperature (20 ◦ C, 25 ± 2 ◦ C, 30 ◦ C and 35 ◦ C); photoperiod (4 h/20 h, 8 h/16 h, 12 h/12 h, 20 h/4 h light/dark), sucrose (2%, 4%, 5%, 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 Plant growth regulators (PGRs) with stress treatment callus fresh and dry weight were determined at 15, 25, 35, 45 and 55 days. Maximum biomass (fresh, dry weight) and green compact callus were noted for all treatments. Phytochemical studies on leaf and in vitro callus G. sylvestre dried leaves and in vitro callus were extracted with methanol 5 times (Rehman et al., 2003). The collected methanol extract was centrifuged at 5000 × g for 10 min at room temperature, then the methanol supernatant carefully pipetted out into fresh eppendorf tubes without disturbing the interphase residues. Green-color supernatant (20 l) was screened using TLC and HPTLC with standard gymnemic acid (Prof. Kazuko Yoshikawa, Kyoto Pharmaceutical University, Japan) for gymnemic acid quantification (Ahmed et al., 2009a). HPTLC was performed using a Camag HPTLC system (CAMAG, Switzerland) equipped with a sample applicator Linomat IV, TLC scanner III and integration software CAT 4.0. The mobile phase of Isopropyl alcohol:chloroform:methanol:acetic acid (5:3:1:0.5) was suitable for separation of gymnemic acid and scanned at 200 nm, respectively.
Chemical preparation and induction of diabetes Alloxan monohydrate (Otta kemi, Mumbai, India) (100 mg/kg body wt./rat) was dissolved in distilled water and injected intraperitoneally (i.p.). The experimental rats entered a diabetic state after 72 h, then the Wistar rats were divided into groups for treatment with G. sylvestre leaf and callus extracts. Therapeutic human insulin (Torrent Pharmaceuticals Ltd., Mehsana, India) mixture was shaken well and injected daily (4 U/kg body wt.) subcutaneously as a diabetic standard (Chattopadhyay 1998).
Instruments and methods The body weight, liver and pancreas weights of the experimental Wistar rats were checked in fundamental weight instruments. Liver glycogen (Raghuramalu et al., 1983) and pancreas were removed for further analysis. The removed pancreas was fixed in dewaxed and hydrated paraffin for micro section (6 m thickness), then stained with hematocylin and eosin. In addition, stained pancreas was washed with Gomori’s aldehyde fuchsin for determination of -cells in Langerhans islets (Gomori, 1950) as described below. For -cells determination, the 0.5 g basic fuchsin was dissolved in 70% alcohol, to which were added 1.0 ml paraldehyde with 1.5 ml conc. HCl. Embedding hydrated paraffin sections were treated with Lugol iodine solution for 30 min, then bleached using 5% sodium thiosulfate for 2 min followed by washing of the section for 5 min in running tap water for oxidation. The -cells were rinsed with 95% and 70% alcohol for 10 min, and then washed with distilled water followed by Ehrlich’s hematoxylin for 4 min and counter stain for 45 s, respectively. Finally, the stain coupling jar was treated with aldehyde fuchsin for 2–10 min. The end point of -cells stood out clearly in dark purple against a colorless background.
HPLC studies in callus and leaf extracts Gymnemic acid was screened in leaf and 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 gymnemic acid separation, the following systems and protocols were used: water HPLC system (Shimadzu model, Japan), 510 pump, 7725 Rheodyne injector, C18 (ODS) reversed-phase column, water 486 UV detector, with mobile phasewater/methanol (35:65, HPLC grade), 0.1% acetic acid, sample applied: 20 l, flow rate: 1 ml/min, read at 230 nm, on UV detector. Preparation of GS leaf and callus extracts In vivo Gymnema leaves extract (GLE) and in vitro callus extracts GCE1 –GCE6 were prepared by the method reported previously (Ahmed et al., 2008). The extracted sample was evaporated and dissolved in saline water (Sterile NS saline, Baxter
Treatment groups Group I (control): free access to tap water. Groups II–X: diabetes-induced animals were divided into 9 groups administered saline, 4 U/kg body wt. of insulin/day, 200 mg/kg body wt. (rat)/day GLE and GCE1 –GCE6 , respectively. The experimental Wistar rats were sacrificed at 10, 20 and 30 days. The following treatment groups were used in this experiment: Group II – diabetic control (Alloxan); Group III – Diabetic standard (insulin); Group IV – diabetic rats + G. sylvestre leaf extract; Group V (GCE1 ) – blue light with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); Group VI (GCE2 ) – 5% sucrose with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); Group VII (GCE3 ) – 12 h photoperiod MS + with 2,4-D (1.5 mg/l) + KN (0.5 mg/l), Group VIII (GCE4 ) – 3 mM NH4 NO3 with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l), Group IX (GCE5 ) – MS + 2,4D (1.5 mg/l) + KN (0.5 mg/l) and Group X (GCE6 ) – MS + NAA (1.0 mg/l) + KN (1.5 mg/l), respectively.
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Statistical analysis
Results and discussion
Only data which showed an advantageous effect were included in tables and presented in mean ± SE (replicated 3 times). Experimental design was completely random and factorial with control, alloxan alone, alloxan with insulin, alloxan with leaf and alloxan with callus extracts. The data were subjected to analysis of variance and mean separation was carried out using Duncan’s multiplerange test (DMRT) at 5% level significance (Gomez and Gomez, 1976).
Callus initiation under stress conditions
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In vitro callus failed without PGRs in MS medium, while different media (MS, B5, SH, WPM) with PGRs affected the callus initiation in leaf explants (data not shown). Trial of auxins alone, and auxins with cytokinins combinations showed callus induction, among which 2,4-D (1.5 mg/l) with KN (0.5 mg/l) induced green compact callus with maximum dry weight at 45 days.
Fig. 1. (A) Effect of different color lights with (OPGRs) optimum plants growth regulators (MS + 2,4-D) + KN (0.5 mg/l). ( ) Blue lights + OPGRs; ( ) green lights + OPGRs; ( ) red lights + OPGRs; ( ) dark (without lights) + OPGRs; ( ) white lights + OPGRs. (B) Effect of different temperature with optimum plant growth regulators (OPGRs) (MS + 2,4D) (1.5 mg/l) + KN (0.5 mg/l); () 20 ◦ C + OPGRs; ( ) 30 ◦ C + OPGRs; ( ) 35 ◦ C + OPGRs; ( ) 25 ± 2 ◦ C + OPGRs. (C) Effect of different photoperiod with optimum plant growth regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 4 h + OPGRs; ( ) 8 h OPGRs; ( ) 12 h OPGRs; ( ) 16 h (control) + OPGRs; ( ) 20 h + OPGRs; ( ) 24 h + OPGRs; ( ) dark + OPGRs. (D) Effect of different concentration carbon source (sucrose) with optimum plant growth regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 2% sucrose + OPGRs; ( ) 3% sucrose (control) OPGRs; ( ) 4% sucrose + OPGRs; ( ) 5% sucrose + OPGRs; ( ) 6% sucrose + OPGRs. (E) Effect of ammonia nitrate concentration (NH4 NO3 ) with (OPGRs) optimum plant growth regulators (MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); ( ) 0 mM NH4 NO3 + OPGRs (control) () 1 mM NH4 NO3 + OPGRs; ( ) 2 mM ) 3 mM NH4 NO3 + OPGRs; ( ) 4 mM NH4 NO3 + OPGRs. (For interpretation of the references to color in this figure legend, the reader is referred to the NH4 NO3 + OPGRs; ( web version of the article.)
Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not significantly different at 5% probability level according to DMRT. a GCE1 – blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 – 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 – 12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE4 – 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE5 – MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 – MS + NAA (1.0 mg/l) + KN (1.5 mg/l).
2.12c 2.26ab 2.35a 1.90bc 1.94b 2.17a 2.00bc 2.17b 2.39a 1.90c 2.04ab 2.15a 1.76a 1.54b 1.35bc Liver glycogen (mg/g) 10th 2.65c 20th 2.74ab 30th 2.82a
2.15bc 2.24b 2.56a
1.95bc 2.10b 2.32a
2.10c 2.35ab 2.47a
2.16bc 2.25b 2.46a
8.4c 8.7ab 9.0a 8.1bc 8.4b 8.9a 7.9ab 7.5c 8.0a 8.0c 8.4ab 8.7a 7.8a 7.5ab 7.0c Liver weight (g/animal) 10th 8.0c 20th 8.5ab 30th 9.0a
8.0c 8.2ab 8.4a
8.5bc 8.7b 9.0a
7.6c 8.0ab 8.3a
7.6b 7.2bc 8.2a
1.14c 1.20ab 1.25a 1.30c 1.35ab 1.37a 1.28bc 1.30b 1.39a 1.10c 1.31ab 1.35a 1.35ab 1.20c 1.42a 1.29bc 1.30b 1.37a 1.10c 1.19ab 1.25a 1.28c 1.37ab 1.40a 1.16a 1.05b 0.95bc Pancreas weight (g/animal) 10th 1.30bc 20th 1.35b 30th 1.42a
208.0bc 199.0fg 205.0f 220.0a 200.0de 196.6fg 205.0c 215.0a 208.0bc 196.0f 195.0fg 215.0a 203.0a 191.6c 172.0e 162.0fg Body weight (g/animal) 0 200.0d 10th 200.0de 20th 225.0bc 30th 240.0a
223.3c 213.3g 222.5cd 235.0a
226.6e 226.6e 237.5c 240.0a
220.0ed 213.0ef 225.0c 245.0a
206.0cd 200.0e 215.0b 220.0a
Group IX alloxan + GCE5 Group VIII alloxan + GCE4 Group VII alloxan + GCE3 Group VI alloxan + GCE2 Group V alloxan + GCE1 Group IV alloxan + leaf Group III alloxan + insulin Group II alloxan Group I (control) Days
Table 1 Body weight (g) of Wistar rats with leaf and callus (methanol) extracts (GSC1 –GSC6 )a of G. sylvestre.
193.0e 190.0g 205.0b 210.0a
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Group X Alloxan + GCE6
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Kumar et al. (2002) reported MS medium with PGRs were suitable for callus proliferation of G. sylvestre, but callus proliferation in other auxins with cytokinins concentration was not pursued, because of their poor quality and production (data not shown). Recently 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 (Ahmed et al., 2009a). Stress treatment and callus growth curve were screened at different days (15, 25, 35, 45 and 55 days). Lag phase (15–25 days), callus initiation and proliferation were observed. 25–35 days (exponential phase), biomass and green compact callus increased significantly. However, stationary phase at 45 days, maximum biomass and green compact callus were shown. Decline phase, the nature and biomass of callus were significantly reduced as compared to other phases. In Asclepiadaceae species, the stationary phase was observed at 22 days; at this phase cryptosin was isolated from Cryptolepis buchanani (Venkateswara et al., 1987) and gagaminine from Cynanchum wilfordii (Shin et al., 2003). Optimized concentration was maintained under stress conditions; leaf explants under blue light, 5% sucrose induced the maximum biomass with green compact callus, then photoperiod, temperature and ammonium nitrate concentrations (Fig. 1A–E). Photoperiod and temperature affected physical appearance, 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. Blue light revealed green compact callus with maximum biomass at 45 days. Cryptochrome showed a top peak at 450 nm, a wavelength close to the blue light; under these conditions, more Pr transformed into Pfr for phenyletanoid glycosides production (Ouyang et al., 2003). Zhao et al. (2001) reported that blue light stimulates jaceosidin production in Saussurea medusa.
TLC, HPTLC, HPLC studies Methanol supernatants of G. sylvestre leaf and in vitro calluses were screened using TLC and HPTLC. After chromatogram, samples were dried and sprayed with specific reagent (vanillin sulfuric acid reagent) at room temperature for detection of gymnemic acid. Standard gymnemic acid showed as a single brown band; however, the callus extract displayed additional brown bands with Rf value (0.44) greater than that of gymnemic acid (0.43). In HPTLC analysis, methanol solvent was run up to 80 mm and scan chromatograms at 200 nm under UV reflectance mode. Gymnemic acid content leaf and callus extract data were compared with standard gymnemic acid. The calibration curve was linear; the correlation coefficient indicated good linearity between concentration and area. To ascertain peak purity of test sample, we compared in vivo and in vitro methanol extract reflectance spectra with standard, which provides test sample purity (data not shown). Gymnestrogenin (triterpenoids) determine in G. sylvestre through HPTLC at 293 nm (Puratchimani and Jha, 2004). For HPLC analysis, leaf and callus methanol extracts (20 l) were uploaded in HPLC system to quantify gymnemic acid under retention time. UV spectrophotometer peak area data were compared with standard gymnemic acid. The gymnemic acid content was increased in leaf, GSC1 and GSC3 over other callus extracts (data not shown). Imoto et al., 1991 reported that a methanol extract contains gymnemic acid through HPLC. Many authors had isolated and identified gymnemic acid earlier. In 1989, Yoshikawa and coworkers isolated gymnemic acids from a hot water extract of G. sylvestre, which they named gymnemic acids I, II, III, IV, V, VI and VII, respectively, and evaluated using HPLC (Sugihara et al., 2000).
A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 1033–1039
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Fig. 2. Histologic examination of pancreatic -cells in alloxan treated animals. (A) Pancreas of normal rats. Note abundant patches if -cells (arrows) in purple color (200×); (B) pancreas of alloxan treated rats, hardly and -cells noticed, all islets degenerated (200×); (C) pancreas of alloxan treated rats, after insulin treatments showed abundant patches of -cells (arrows) in purple color (200×); (D) pancreas treated rats, after G. sylvestre leaf extract (200×); (E) pancreas treated rats, after G. sylvestre callus extract 1 (200×); (F) G. sylvestre callus extract 2 (200×); (G) G. sylvestre callus extract 3 (200×); (H) G. sylvestre callus extract 4 (200×); (I) G. sylvestre callus extract 5 (200×); (J) G. sylvestre callus extract 6 (200×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Body, liver, pancreas weight and liver glycogen As shown in Table 1, the long-term antihyperglycemic effect was examined for GS leaf and callus extracts. The whole body, liver and pancreas weights were reduced in the diabetic control group after 30 days, although oral administration of 200 mg/kg body wt. GS leaf and callus extracts significantly increased and maintained the body liver and pancreas weights in the 4 U/kg body wt. insulin group Wistar rats. We have recently published a review of the pharmacological activities, a phytochemical investigation and in vitro studies of G. sylvestre (Ahmed et al., 2009b). Liver glycogen was significantly decreased in diabetic control rats, whereas with leaf and callus extracts of GS, liver glycogen significantly increased (Table 1). Restoration of hepatic glycogen by GS leaf and callus could improve the insulin secretion or inhibition of glucose-6-phosphatase in liver, and has prevented the conversion of glucose 6-phosphate to glucose (Shanmugasundaram et al., 1990). Shanmugasundaram et al. (1983) reported that G. sylvestre restored glycogen and enzymes in diabetic rabbit liver after 24 weeks of treatment. We earlier reported that leaf and callus
extracts of G. sylvestre reduced blood sugar and lipid profiles such as cholesterol, triglyceride, HDL, LDL, VLDL in alloxan-induced diabetic Wistar rats (Ahmed et al., 2008). Shanmugasundaram et al. (1990) reported aqueous extract of G. sylvestre leaf (200 mg/kg body wt.) normalized blood sugar level in STZ-induced diabetic rats. However, gymnemic acid from G. sylvestre leaf regulated hyperglycemia (Gholap and Kar 2005) and an aqueous extract maintained the blood glucose level in normal Wistar rats (Rafiullah et al., 2006). Histological examinations of pancreas Fig. 2A–J depicts the histological examination (200×) of pancreatic -cells in control, diabetic control, diabetic standard, GS leaf and callus extracts groups. Histological pancreas islets were checked separately in hematoxylin and eosin with aldehyde fuchsin section. In hematoxylin and eosin sections, the degree of atrophy, hydropic degeneration, necrosis, hyalinization, or fibrosis was checked (data not shown), while in aldehyde fuchsin sections, the -cells percentage was recorded based on atrophy cells, and the -granules were increased or decreased at the 10th, 20th and 30th
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Table 2 Leaf and callus extractsa of Gymnema sylvestre regenerate the pancreatic -cells in alloxan-induced diabetic Wistar rats. Treatment group
-Cell regeneration frequency (%) 10th day
Group I (normal) Group II (diabetic control) Group III (insulin) Group IV (leaf) Group V (callus extract 1) Group VI (callus extract 2) Group VII (callus extract 3) Group VIII (callus extract 4) Group IX (callus extract 5) Group X (callus extract 6)
81.0 16.0 38.0 49.0 53.3 42.3 51.3 30.3 28.0 27.0
± ± ± ± ± ± ± ± ± ±
2.82a 2.16j 2.62b 4.54e 8.07c 5.89ef 3.13cd 2.12g 6.01gh 3.77i
20th day 83.6 12.3 41.0 58.6 50.0 47.0 55.0 32.6 31.6 34.0
± ± ± ± ± ± ± ± ± ±
4.27a 1.44j 1.69ab 1.18c 0.94e 3.85ef 4.54cd 6.15gh 4.11i 3.39g
30th day 80.0 8.6 45.5 66.6 59.6 54.3 57.6 39.0 45.0 37.6
± ± ± ± ± ± ± ± ± ±
2.62ab 1.65j 2.82a 2.88c 4.09cd 4.57ef 3.47e 4.54gh 1.88g 0.98i
Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not significantly different at 5% probability level according to DMRT. a GCE1 – blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 – 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 –12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE4 – 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE5 –MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 – MS + NAA (1.0 mg/l) + KN (1.5 mg/l).
days (Table 2). In electron microscopic analysis, the pancreatic cells islets showed no pathological alternation in control Wistar rats. The nucleus, nuclear envelope, mitochondria, endoplasmic reticulum and Golgi complex of -cells were normal (Fig. 2A). However, 80% of -cells contained a number of secretory granules, which had a space between core and membranes diffusely distributed in cytoplasm at end of experiment. The secretory granules showed moderate homogenous (or) slightly heterogenous electron density and single layered membrane (Fig. 2A). Electron microscope analysis of -cells in diabetic standard rats (alloxan alone) displayed the loss of nuclear envelope, mitochondrial vacuolization, swelling and dilatation of endoplasmic reticulum (Fig. 2B). During alloxan treatment, -cytotoxin causes a massive destruction of -cells in islets of Langerhans, and reduces the -cells and insulin release (Colca et al., 1983). Diabetic standard (insulin), GS leaf and callus extracts treated groups showed significant increase in the secretary granule of -cells and reached to control level (Fig. 2D–J). The -cells were apparently normal in the architecture of the nucleus, which revealed that gymnemic acid prevented the -cells damage as compared to control rats. But low gymnemic acid content callus extracts decreased the secretory granules and losses of nuclear envelope, mitochondrial vacuolization, swelling and dilation of the endoplasmic reticulum in diabetic rats. Degenerative changes in pancreatic -cells were minimized and normal morphology maintained in diabetic rats by administration of gymnemic acid content extracts as evidenced in histopathological examination. Pancreas hematoxylin/eosin with aldehyde fuchsin stains displayed the ruptured islets and decreased -cells in diabetic control rats, whereas leaf and callus extracts regenerated the -cells frequency in diabetic standard (Fig. 2C; 45.5%), GS leaf extract (Fig. 2D; 66.6%), leaf callus extract 1 (Fig. 2E; 59.6%), callus extract 2 (Fig. 2F; 54.3%), callus extract 3 (Fig. 2G; 57.6%), callus extract 4 (Fig. 2H; 39.0%), callus extract 5 (Fig. 2I; 45.0%) and callus extract 6 (Fig. 2J; 37.6%) (Table 2). In treatment pancreatic cells, AMP was activated and the protein kinases showed a unique function or connecting cellular energy status to the ability of insulin synthesis and secretion of -cells (Da Silva Xavier et al., 2000). Alcohol extract of G. sylvestre stimulated the insulin secretion in islets of Langerhans and pancreatic -cell lines (Persaud et al., 1999). Srivastava et al. (1986) reported that pancreas tissue was completely destroyed in alloxan treatment, after G. sylvestre leaf extract regenerated the prolonged survival and adaptogenic activity. However, a smaller dose of STZ (45 mg/kg body wt.) led to damage of the pancreatic acinar cells and reversed by Aegle marmelos (Arumugam et al., 2008). Successful -cell regeneration was achieved using diabetic medicinal plants such as Momordica charantia, Beta vulgaris, Catharanthus roseus, and Eugenia jambolana (Das et al., 1996; Sharma et al., 2006).
In conclusion, the leaf and callus extracts of G. sylvestre have antihyperglycemic effects as determined using alloxan-induced diabetic Wistar rats. Our experimental data suggested that alloxan administered to Wistar rats through intraperitoneal route exhibits diabetic activity. We conclude that the effect of G. sylvestre leaf and callus extracts as strong herbal remedies is confirmed in this study, and suggest that they may be capable of fully restoring pancreatic -cells function and thus curing type I diabetes. Studies of G. sylvestre callus extract have revealed how -cells may be formed and regenerated in vitro and could provide further direction for potential drug development for IDDM treatment. References Ahmed, A.B.A., Rao, A.S., Rao, M.V., 2009a. In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R.Br. Ex. Roemer and Schultes through callus culture under stress conditions. Methods Mol. Biol. 547, 93–105. Ahmed, A.B.A., Komalavalli, N., Muthukumar, M., Benjamin, J.H.F., Rao, A.S., Kim, S.K., Rao, M.V., 2009b. Pharmacological activities, phytochemical investigations and in vitro studies of Gymnema sylvestre R.Br.—a historical review. Comprehensive Bioactive Natural products–Potential & Challenges, vol. 1, pp. 75–99. Ahmed, A.B.A., Rao, A.S., Rao, M.V., 2008. Role of in vivo and in vitro callus of Gymnema sylvestre (Retz) R.Br. Ex. Roemer & Schultes in maintaining the normal levels of blood glucose and lipid profile in diabetic Wistar rats. Biomedicine 28, 134–138. Arumugam, S., Kavimani, S., Ahmed, A.B.A., Kadalmani, B., Akbarsha, M.A., Rao, M.V., 2008. Antidiabetic activity of leaf and callus extracts of Aegle marmelos in rabbit. Sci. Asia 34, 317–321. Chattopadhyay, R.R., 1998. Possible mechanism of antihyperglycemic effect of Gymnema sylvestre leaf extract. Part I. Gen. Pharmacol. 31, 495–496. Colca, J.R., Kotagel, N., Brooks, C.L., Lacy, P.E., Landt, M., McDanleld, M.L., 1983. Alloxan inhibition of Ca2+ and calmodulin dependent protein kinase in pancreatic islets. J. Biochem. 225, 7260–7263. Das, A.V., Padayatti, P.S., Paulose, C.S., 1996. Effect of leaf extract of Aegle marmelos (L.) on histological and ultra structural changes in tissues of streptozotocin induced diabetic rats. Indian J. Exp. Biol. 34, 341–345. Devi, C.S., Murugesh, S., Srinivasan, V.M., 2006. Gymnemic acid production in suspension cell cultures of Gymnema sylvestre. J. Appl. Sci. 6, 2263–2268. Da Silva Xavier, G., Leclerc, I., Salt, I.P., Doiron, B., Hardie, D.G., Kahn, A., Rutter, G.A., 2000. Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc. Natl. Acad. Sci. U.S.A. 97, 4023–4028. Gholap, S., Kar, A., 2005. Gymnemic acids from Gymnema sylvestre potentially regulates dexamethasone induced hyperglycemia in mice. Pharm. Biol. 43, 192–195. Gomez, K.A., Gomez, A.A., 1976. Statistical Procedures for Agricultural Research with Emphasis on Rice. International Rice Research Institute, Los Bans, IRRI Publ, Philippines, pp. 264. Gomori, G., 1950. Aldehyde-fuchsin: a new stain for elastic tissue. Am. J. Clin. Pathol. 20, 665. Imoto, T., Yamamoto, F., Miyasaka, A., Hantano, H., 1991. High-performance liquid chromatography-atmospheric pressure ionization mass spectrometry of gymnemic acids. J. Chromatogr. 557, 383–389. International Expert Committee, 2009. Report on the role of the A1C assay in the diagnosis of diabetes. Diabetes Care 32, 1–8. Kanetkar, P.V., Singhal, R.S., Laddha, K.S., Kamat, M.Y., 2006. Extraction and quantification of gymnemic acids through gymnemagenin from callus cultures of Gymnema sylvestre. Phytochem. Anal. 17, 409–413. Kumar, H.G., Murthy, H.N., Paek, K.Y., 2002. Somatic embryogenesis in Gymnema sylvestre. Plant Cell Tiss. Org. Cult. 71, 85–88. Lee, E.J., Mobin, M., Hahn, E.J., Peak, K.Y., 2006. Effects of sucrose, inoculum density, auxins, and aeration volume on cell growth of Gymnema sylvestre. J. Plant Biotechnol. 49, 427–431.
A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 1033–1039 Mitra, S.K., Gopumadhavan, S., Muralidhar, T.S., Anturlikar, S.D., Sujatha, M.B., 1995. Effect of D-400 herbomineral preparation on lipid profile glycated haemoglobin and glucose tolerance in streptozotocin induced diabetes in rats. Indian J. Exp. Biol. 33, 798–800. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497. Ouyang, J., Wang, X., Zhao, B., Wang, Y., 2003. Light intensity and spectral quality influencing the callus growth of Cistanche deserticola and biosynthesis of phenylethanoid glycosides. Plant Sci. 165, 657–661. Puratchimani, V., Jha, S., 2004. Standardisation of Gymnema sylvestre R.Br., with reference of gymnemagenin by high-performance thin-layer chromatography. Phytochem. Anal. 15, 164–166. Persaud, S.J., Al-Majed, H., Raman, A., Jones, P.M., 1999. Gymnema sylvestre stimulates insulin releases in vitro by increased membrane permeability. J. Endocrinol. 163, 207–212. Rafiullah, M.R.M., Siddiqui, A.W., Mir, S.R., Ali, M., Pillai, K.K., Singh, S., 2006. Antidiabetic activity of some Indian medicinal plants. Pharm. Biol. 44, 95– 99. Raghuramalu, N., Nair, M.K., Sundaram, S.K., 1983. A manual of laboratory techniques, Hyderabad. Natl. Inst. Nutr., 96–98. Rehman, R.U., Israr, M., Srivastava, P.S., Bansal, K.C., Abdin, M.Z., 2003. In vitro regeneration of witloof chicory (Cichorium intybus L.) from leaf explants and accumulation of esculin. In Vitro Cell. Dev. Biol. 39, 142–146. Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., 2006. Antihyperglycemic effect of the fruit-pulp of Eugenia jambolana in experimental diabetes mellitus. J. Ethnopharmacol. 104, 367–373.
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Shanmugasundaram, E.R.B., Leela, K.G., Radha, K.S., Rajendran, V.M., 1990. Possible regeneration of the islets of Langerhans in streptozotocin—diabetic rats given Gymnema sylvestre leaf extracts. J. Ethnopharmacol. 30, 265–279. Shanmugasundaram, K.R., Panneerselvam, C., Samudram, P., Shanmugasundaram, E.R.B., 1983. Enzyme changes and glucose utilization in diabetic rabbits the effects of Gymnema sylvestre, R.Br. J. Ethnopharmacol. 7, 205–234. Shin, G., Chil, M., Lee, D., 2003. Comparative study of the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture of Cynanchum wilfordii. Biol. Pharm. Bull. 26, 1321–1325. Sokmen, A., Jones, B.M., Erturk, M., 1999. Antimicrobial activity of extracts from the cell cultures of some Turkish medicinal plants. Phytother. Res. 13, 355–357. Srivastava, Y., Bhatt, H.V., Jhala, C.I., 1986. Oral Gymnema sylvestre leaf extracts inducing protracted longevity and hypoglycemia in alloxan diabetic rats: Review and experimental study. Int. J. Crude. Drug. Res. 24, 171–176. Sugihara, Y., Nojima, H., Matsuda, H., Murakami, T., Yoshikawa, M., Kimura, I., 2000. Antihyperglycemic effects of gymnemic acid IV, a compound derived from Gymnema sylvestre leaves in streptozotocin-diabetic mice. J. Asian Nat. Prod. Res. 2, 321–327. Venkateswara, R, Sankara Rao, K., Vaidyanathan, C.S., 1987. Cryptosin-a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem & Schult. Plant Cell Rep. 6, 291–293. Ye, W.C., Zhang, Q., Liu, X., Che, C., Zhao, S., 2000. Oleanane saponins from Gymnema sylvestre. Phytochemistry 53, 893–899. Zhao, D., Xing, J., Li, M., Lu, D., Zhao, Q., 2001. Optimization of growth and Jaceosidin in callus and cell suspension of Saussurea medusa. Plant Cell Tiss. Org. Cult. 67, 227–234.