Journal of Ethnopharmacology 153 (2014) 917–921
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Efficacy of ginseng adventitious root extract on hyperglycemia in streptozotocin-induced diabetic rats Hosakatte Niranjana Murthy a,b,n, Vijayalaxmi S. Dandin b, Eun Jung Lee c, Kee Yoeup Paek a,c,n a
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Botany, Karnatak University, Dharwad 580003, India c Cheongsol Biotech Co. Ltd., Industry Academic Cooperation Foundation Agribusiness Incubator Center, 205, Chungbuk National University, Cheongju 361-763, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 December 2013 Received in revised form 23 March 2014 Accepted 28 March 2014 Available online 5 April 2014
Ethnopharmacological relevance: Ginseng has various bioactive effects on human health including its potential activity of improving the glucose homeostasis and insulin sensitivity. Materials and methods: Tissue culture raised mountain ginseng adventitious root (TCMGARs) extract enriched with ginsenosides was used as experimental material. Streptozotocin-induced diabetic ‘Sprague Dawley’ male rats were used as experimental systems and were fed with Tissue culture raised mountain ginseng adventitious root extract. Field cultivated Korean ginseng root extract fed rats were used as positive control and several indices such as body weight, blood glucose level and other serological indicators were tested. Results: Chemical profile showed TCMGARs were rich in varied ginsenosides especially Rb1, Rb2, Rc, Rd, Rg3, and Rh2 when compared to field cultivated Korean ginseng. TCMGARs extract at dosage levels of 250 and 500 mg/kg body weight significantly lowered the blood glucose, total cholesterol and triglyceride content in streptozotocin-induced diabetic rats. Conclusion: The data of in vivo experiments on anti-glycemic effects of TCMGARs proves their efficacy and also their use as dietary supplement. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antidiabetic Blood glucose Dietary supplement Ginsenosides
1. Introduction Diabetes mellitus is a metabolic disease resulting due to the reduced insulin secretion by pancreas or due to a low biological activity of the insulin secreted and is classified as insulindependent (type 1) and insulin independent type (type 2) (Geroge and Ludvik, 2000). Panax ginseng C.A. Meyer (Korean ginseng) is traditionally used for treating hyperglycemia including diabetes mellitus (Ivorra et al., 1989; Attele et al., 1999) and it is recognized officially as one of the herbal drug ingredients in China for treating diabetes mellitus (Jia et al., 2003). Ginseng is available in commercial market in two forms namely red ginseng and white ginseng. The roots of Panax ginseng is steamed and dried to prepare red ginseng, while the peeled roots dried without steaming are designated as white ginseng. The major components of
n Corresponding authors at: Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel: þ82 43 266 3245; fax: þ 82 43 266 3246. E-mail addresses: [email protected] (H.N. Murthy), [email protected] (K.Y. Paek).
http://dx.doi.org/10.1016/j.jep.2014.03.062 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
ginseng are triterpenoidal dammarane glycosides (saponins) called ginsenosides. More than 30 kinds of ginsenosides have been reported and they have been named as ‘Rx’ based on their mobility on thin layer chromatographic plates, with polarity decreasing from index ‘a’ to ‘h’. They differ from one another by the type of sugar moieties, their number and the site of attachment (Park et al., 2005). Ginsenosides are classified into three groups by their structure i.e., Rb group (protopanaxadiols including Rb1, Rb2, Rc and Rd, etc.), the Rg group (protopanaxatriols including Rg1, Re, Rf, and Rg2, etc.) and the Ro group (Oleanolic acid; Park et al., 2005). It was reported that diol-type ginsenosides such as Rb1, Rb2, Rc, Rd, Rg3 and Rh2 are having anti-diabetic activities (Suda et al., 2000) and ginsenoside Rh2 increases insulin secretion in streptozotocin-induced diabetic rats to decrease the blood glucose concentration (Lai et al., 2006). Recently, microbial fermentation methods have been introduced and red ginseng powder was fermented using micro-organisms such as lactic acid bacteria to transform ginsenosides such as Rb1, Rb2, RC and Rd into readily absorbable forms (Bae et al., 2004; Trinh et al., 2007; Kim et al., 2010) and these fermented red ginseng powders and extracts were
918
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
also made available in the market. Simultaneously, a group of scientists have induced adventitious roots from 100 year old mountain ginseng and called them as ‘tissue cultured mountain ginseng adventitious roots’ (TCMGARs), and also adopted methyl jasmonate elicitation strategy to overproduce pharmacologically useful ginsenosides (Paek et al., 2009). TCMGARs were reported to be rich in diol-group of ginsenosides (Sivakumar et al., 2005) and were proved as ‘biosafe’ through toxicological evaluations (Sivakumar et al., 2006). The aims of the study were to investigate the efficacy of TCMGARs, the evaluation of antidiabetic effect of TCMGARs following the administration of TCMGARs extracts in streptozotocin (STZ) induced diabetic rats and the examination of body weight, blood glucose level and other serological indicators. Chemical profiling of ginsenosides present in TCMGARs as well as field cultivated Korean ginseng was also carried out before their administration for antidiabetic evaluations.
2. Materials and methods 2.1. Material Tissue cultured mountain ginseng adventitious roots (TCMGARs) and freshly harvested field cultivated ginseng roots (positive control) were dried (forced air drying at 50 1C), powdered and soaked in 50% aqueous ethanol for 10 days at 25 1C and filtered. The solution evaporated in vacuo gave a semi-gelatinous extract and yielded the crude ginsenosides of 128.51 mg/g and 100.08 mg/g from ginseng adventitious roots and cultivated ginseng roots respectively. 2.2. Determination of ginsenoside content The chemical profiling of ginsenosides in these two materials was carried out by high pressure liquid chromatography (HPLC) at the Korean Food Research Institute, Sungnam-Si, Republic of Korea (File no. AO2012-06-26-200). Extraction and analysis of ginsenosides were carried out using the protocol by Yu et al. (2002). The ginsenoside fractions were analyzed using HPLC system (Shimadzu, Kyoto) consisting of 10AT pump, 10AXL autosampler, SPDM10A photodiode array detector, and CTO-10A column oven, 5 μM Lichrosorb column (250 4.6 mm2) (Altech, Deerfild, IL), and a C18 guard column, at 40 1C. The eluted peaks were detected at 203 nm and quantified against external standards of ginsenosides, Rf, Rb2, Rd, (Karl Roth, Germany), Re, Rg1, Rg2, Rh1, Rh2, Rb1, Rb3, Rc and Rg2 (Wako, Osaka, Japan). The mobile phase was a gradient elution of water (A) and acetonitrile (B), commencing with 20% B, rising to 22% B after 20 min then to 46% after 45 min and 55% B after 50 min. 2.3. Experimental animals Experiments were carried out in Biotoxtech Laboratory, South Korea on Sprague Dawley male rats. All the experimental animals were supplemented with a standard diet and were maintained in a room kept under environmentally controlled conditions of 2471 1C and 12:12 h light/dark cycles. The animals had free access to water and a standard diet [a normal laboratory commercial stock diet containing 16% protein, 56% carbohydrate and 8% fat (w/w)]. 2.4. Oral glucose tolerance test Blood glucose levels of normal rats kept under fasting for at least 12 h were determined. Then the rats were orally administered TCMGARs (125, 250 and 500 mg/kg body weight) or field cultivated Korean ginseng extract (250 mg/kg body weight) dissolved in distilled water. For the control group, an equal amount of normal saline was
given. Subsequently, oral dose of 40% glucose was administered to all the experimental group of rats at 1 g/kg rate and later blood samples were collected at 30, 60 and 120 min post-glucose administration to record the variation in blood glucose levels. 2.5. Induction of diabetes in experimental rats In the treatment studies, animals were divided into six groups of seven animals in each (G). All animals were of seven weeks old and the average body weight was 305 g. Group G1 consisted of normal rats and which served as a control and the remaining five groups consisted of the rats induced with diabetes. The G2 was streptozotocin control (diabetes control) and G3, G4 and G5 groups were included with diabetic rats which were fed with diet containing tissue cultured mountain ginseng extract at a dose of 125, 250 and 500 mg/kg body weight respectively. Group G6 had diabetic rats which were fed with a diet containing field cultivated Korean ginseng extract of 250 mg/kg body weight. All rats were kept on observation for four weeks and further used for biochemical analysis. For the induction of diabetes, rats that had undergone one week adjustment period were kept under fasting for a minimum of 12 h, and the intra-peritoneal injection of streptozotocin (STZ) diluted in 0.01 M citrate buffer was administered at 50 mg/kg of body weight. In the normal control group, the same concentration of normal saline (without streptozotocin) was used for intra peritoneal injection. Blood collected from the veins of tail region was used to confirm the induction of diabetes, and the rats with fasting blood glucose levels of about 200 mg/dL were used for further experiments. 2.6. Analysis of body weight, blood glucose, and other biochemical tests Increase in body weight was measured every week at the same time in a study period of four weeks. Alterations in blood glucose levels during the feeding period were measured at weekly intervals using a blood glucose monitoring system (ACCU-CHEK Sensor; Roche Diagnostics GmbH, Mannheim, Germany), and blood was collected from the veins of tail region of the rats which were kept on fasting for over 12 h. The total triglyceride level and cholesterol contents were measured using a triglyceride measuring kit and a total cholesterol content measuring kit respectively (Asan Pharmaceuticals, Whasung, Korea). 2.7. Statistical analysis The results were statistically analyzed by using analysis of variance (ANOVA) followed by a Student's t-test and all the values are expressed as mean with standard errors.
3. Results and discussion 3.1. Ginsenoside content in TCMGARs and Korean ginseng The details of ginsenosides present in TCMGARs and field cultivated Korean ginseng are presented in Fig. 1 and Table 1. The TCMGARs possessed higher amounts of Rb1, Rb2, Rc, Rd, Re, Rg1and Rf ginsenosides when compared to field cultivated Korean ginseng, additionally, ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2 which were lacking in the extracts of field cultivated Korean ginseng were abundant in the TCMGARs. The higher accumulation of ginsenosides and other novel ginsenosides is obvious because of the influence of methyl jasmonate elicitation during the cultivation of TCMGARs but it was not observed with field cultivated Korean ginseng (Paek et al., 2009). It is reported that methyl
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
919
Fig. 1. HPLC chromatogram of field cultivated ginseng (above) and tissue cultured mountain ginseng adventitious roots (below).
Table 1 Ginsenoside contents of tissue cultured mountain ginseng adventitious roots in comparison with cultivated Korean ginseng.a Ginsenoside
Rb1 Rb2 Rb3 Rc Rd Re Rg1 Rg2 Rg3 Rh1 Rh2 Rf
Content (mg/g dry weight) TCMGARs
Korean ginseng
4.1 2.3 0.9 2.0 4.2 0.2 0.19 0.38 11.2 0.49 3.8 1.64
0.05 0.06 – 0.03 0.06 0.24 0.17 – – – – 0.03
a Analysis was carried out by the Korea Food Research Institute, Sungnam-si, Republic of Korea.
jasmonate stimulates the ginsenoside biosynthetic pathway thus enhancing the accumulation of ginsenosides up to 5–7 fold in TCMGARs (Yu et al., 2002; Kim et al., 2004). In ginseng, the dioltype of ginsenosides such as Rb1, Rb2, Rc, Rd, Rg3 and Rh2 exhibit anti-diabetic activity (Suda et al., 2000), and the ginsenoside Rh2 increases the insulin secretion in STZ-induced diabetic rats to reduce the blood glucose level (Lai et al., 2006). As TCMGARs possess higher concentrations of Rb1, Rb2, Rc, Rd, Rg3 and Rh2 ginsenosides, we had expected a greater impact of TCMGARs extracts in combating STZ-induced hyperglycemic activity in the present studies. Efforts were also made in the past for the enhancement in accumulation of diol-type of ginsenosides in red ginseng by the fermentation technology i.e., fermentation of red ginseng powder with intestinal bacteria such as Lactobacillus fermentum and also improved accumulation of Rg3 and Rh2 type of ginsenosides has been reported (Kim et al., 2010). 3.2. Oral glucose tolerance test in experimental rats The oral glucose tolerance tests in the rats administered with TCMGARs extracts showed a gradual decrease in the glucose levels
Fig. 2. Oral glucose tolerance test: effect of tissue cultured mountain ginseng adventitious root extract (TCMAGR) and field cultivated Korean ginseng (KG) extract feeding. In the oral glucose tolerance test, glucose levels were estimated followed by the oral administration of 1 g/kg D-(þ )-glucose at indicated time points. Control group (Control), TCMGAR 125, TCMGAR 250, TCMGAR 500, KG 250. Mean 7standard error (n¼ 7).
with the increase in time duration as well as with an increase in the dosage levels of TCMGARs extract (Fig. 2). The blood glucose level was highest at 30 min after glucose administration, thereafter showed a decrease in blood glucose levels. The groups fed with TCMGARs extract as well as Korean ginseng showed a significant reduction in the blood glucose levels when compared to the group that was administered with glucose alone and the one without sample administration. Relatively, the groups fed with 250 and 500 mg/kg TCMGARs extract demonstrated a greater reduction in blood glucose level than that of groups fed with 125 mg/kg. At 120 min post-glucose administration, the blood glucose levels were reduced to almost the level before fasting, regardless of the sample concentration used. These experimental results clearly demonstrate the effects of TCMGARs extracts as well as Korean ginseng extracts in the improvement of glucose metabolism.
920
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
Table 2 Effects of tissue cultured mountain ginseng adventitious root (TCMGR) extracts on total cholesterol and triglyceride contents of plasma in streptozotocin-induced rats for 4 weeks. Group
Dose (mg/kg)
G1
0
G2 STZ treated
STZ control
G3 STZþTCMGARa
125
G4 STZþ TCMGAR G5 STZþ TCMGAR G6 STZþ Korean ginseng
250
500
250
Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N
Total cholesterol (mg/dL)
Triglyceride (mg/dL)
25.4 3.2 7 44.3n 6.8 7 39.7 2.9 7 32.4† 7.5 7 31.4† 3.1 7 39.9 7.4 7
43.3 4.8 7 60.5n 5.4 7 28.1† 4.8 7 33.2† 4.8 7 23.5† 3.8 7 29.5† 3.3 7
Fig. 3. Effect of tissue cultured mountain ginseng adventitious root (TCMAGR) extract on body weight in STZ-induced diabetic rats after four weeks of treatment. G, normal control; G2, STZ control, G3, diabetic group fed with 125 mg/kg of TCMGARs extract; G4, diabetic group fed with 250 mg/kg of TCMGARs extract; G5, diabetic group fed with 500 mg/kg of TCMGARs extract; G6, diabetic group fed with 250 mg/kg field cultivated Korean ginseng extract. *Po 0.05 vs normal control, P o 0.05 vs STZ control.
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR—Tissue cultured mountain ginseng adventitious root extract. Po 0.05 vs normal control. Po 0.05 vs STZ control.
3.3. Anti-diabetic property of TCMGARs STZ treatment of rats leads to selective destruction of the insulin secreting β-cells of the pancreas resulting in diabetes mellitus (Gilman et al., 1990). The symptoms of diabetes mellitus are polydipsia, polyphagia, polyuria and the most significant noticeable morphological change is decrease in body weight (Kim et al., 2010). All these symptoms were observed with STZ treated rats in the present studies. Changes in the body weight of rats with STZ-induced diabetes and after feeding for four weeks with TCMGARs as well as field cultivated Korean ginseng are shown in Table 2. Consistent increase in body weight over a time period was observed with the control group of normal rats. There was a significant decrease in body weight in STZ-induced group of rats (Fig. 3). The body weight of the TCMGARs extractadministered group was decreased when compared to the normal group, but it was significantly higher than the body weight of STZ control groups especially with G4 and G5 (i.e., the groups of rats administered with 250 and 500 mg/kg of TCMGARs). However, there was no improvement in the body weight of STZ-induced diabetic rats administered with 125 mg/kg TCMGARs extract and 250 mg/kg Korean ginseng extract. It was reported that the inefficient energy production during glucose metabolism affects the growth and development in diabetic individuals (Park and Cho, 2006), which leads to loss of body weight. In the present study, administration of TCMGARs (250 and 500 mg/kg body weight) helped the diabetic rats in restoration of body weight significantly. Changes in blood glucose levels in diabetic rats which were administered with TCMGARs extract and Korean ginseng extract are shown in Fig. 4. Blood glucose levels increased significantly in the STZ-control group of rats compared to the control group of normal rats. The groups of rats administered with TCMGARs extract and field cultivated Korean ginseng extract showed significant reduction in blood glucose levels after one week of sample administration when compared to STZ-control groups. Blood glucose levels decreased more significantly with the
Fig. 4. Effect of tissue cultured mountain ginseng adventitious root (TCMAGR) extract on the blood glucose levels in STZ-induced diabetic rats after four weeks of treatment. G, normal control; G2, STZ control, G3, diabetic group fed with 125 mg/kg of TCMGARs extract; G4, diabetic group fed with 250 mg/kg of TCMGARs extract; G5, diabetic group fed with 500 mg/kg of TCMGARs extract; G6, diabetic group fed with 250 mg/kg field cultivated Korean ginseng extract. *Po0.05 vs normal control, Po0.05 vs STZ control.
increased duration of the feeding period, and 2, 3 and 4 weeks after sample administration, the 250 and 500 mg/kg TCMGARs treated groups showed a more significant reduction in blood glucose levels when compared to STZ-control group. The present experimental results are consistent with previous reports demonstrating anti-glycemic effects of Korean ginseng and American ginseng (Liu et al., 2009; Xie et al., 2009), and fermented red ginseng (Kim et al., 2010). We used field cultivated Korean ginseng extract treatments as positive control to compare the effect of TCMGARs extract on STZ-induced diabetic rats. It was observed that TCMGARs extract treatments especially 250 and 500 mg/kg treatments were far superior to Korean ginseng treatments, this positive effect might be due to ginsenoside concentration and composition of TCMGARs (Table 1). Following the TCMGARs extract administration, the total plasma cholesterol concentration was 25.473.2 mg/dL in the control group of normal rats when compared with 44.376.8 mg/dL in diabetic control group. The plasma cholesterol concentrations were significantly low in the TCMGARs extract treated group of individuals especially with 250 and 500 mg/kg treatment groups
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
(Table 2). Yao et al. (2008) reported that the increase in total cholesterol levels in diabetes induced rats was due to inability of diabetic individuals to metabolize carbohydrates as an energy source and the use of available free fatty acids in the system for the energy synthesis which leads to cholesterol accumulation. The observation on accumulation of total cholesterol accumulation in diabetic rats is in accordance with reported results of Kim et al. (2010). There was also a significant increase in triglyceride concentration in diabetes induced rats when compared to the control group of normal rats (Table 2). Whereas, there was a significant decrease in the triglyceride levels in diabetic rats which were fed with the extracts of TCMGARs as well as the extract of field cultivated Korean ginseng (Table 2). These results are in agreement with the reports by Kim et al. (2010) that ginsenosides stimulate the improved lipid metabolism which is manifested in diabetic individuals. The present data of in vivo experiments on anti-glycemic effects of TCMGARs are in accordance with the various other experimental reports that ginseng improves glucose homeostasis. It was observed that the effects of TCMGARs are more dramatic when administered for a longer duration with elevated dose levels. TCMGARs contained higher amounts of diol-type of saponins such as ginsenoside Rb1, Rb2, Rc, Rd, Rg3 and Rh2 (Table 1) and these levels are optimal when compared to the ginsenoside amounts which were reported in white ginseng or red ginseng. The richness of ginsenosides in TCMGARs is due to the methyl jasmonate elicitation strategy applied during the in vitro production of ginseng. The present studies reveal that Korean ginseng is efficient in lowering the glucose and lipid levels, this might be due to richness of ginsenoside Re content when compared to TCMGARs (Table 2). Recently, several authors have reported that ginsenoside Re also lowers blood glucose and lipid levels (Attele et al., 2002; Quan et al., 2012). The above results prove the efficacy of TCMGARs. Earlier we have reported biosafety of TCMGARs based on mutagenicity and toxicological evaluations (Sivakumar et al., 2006) and TCMGARs are reported to possess biophenols (Sivakumar and Paek, 2005), enzymatic and non-enzymatic antioxidants (Ali et al., 2006). Therefore, TCMGARs and their products can be used as dietary supplements.
Acknowledgments This study was supported by a Grant of Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (Grant no. A103017). Dr. H.N. Murthy is thankful to the Ministry of Education, Science and Technology, Republic of Korea for the award of Brain Pool Fellowship (131S-4-3-0523); this study was also supported by the Ministry of Science, ICT and Planning (MSIP). Authors are thankful to Prof. Yeravinthalimath, Department of English, Karnatak University, India for language editing of the manuscript.
921
References Ali, M.B., Yu, K.W., Hahn, E.J., Paek, K.Y., 2006. Methyl jasmonate and salicylic acid elicitation induces ginsenoside accumulation, enzymatic and non-enzymatic antioxidant in suspension culture of Panax ginseng roots in bioreactors. Plant Cell Rep. 25, 613–620. Attele, A.S., Wu, J.A., Yuan, C.S., 1999. Ginseng pharmacology, multiple constituents and multiple actions. Biochem Pharmacol. 58, 1685–1693. Attele, A.S., Zhou, Y.P., Xie, J.T., Wu, J.A., Zhang, L., Dey, L., Pugh, W., Rue, P.A., Polonsky, K.S., Yuan, C.S., 2002. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component. Diabetes 51, 1851–1858. Bae, E.A., Han, M.J., Kim, E.J., Kim, D.H., 2004. Transformation of ginseng saponins to ginseonoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch. Pharm Res. 27, 61–67. Gilman, A.G., Rall, T.W., Nies, A.S., Tayer, P. (Eds.), 1990. Goodman and Gilman's the Pharmacological Basis of Therapeutics, eighth ed. Pergamon Press, New York. George, P., Ludvik, B., 2000. Lipids and diabetes. J. Clin. Basic Cardiol. 3, 159–162. Ivorra, M.D., Paya, M., Villar, A., 1989. A review of natural products and plants as potential antidiabetic drugs. J. Ethnopharmacol. 27, 243–275. Jia, W., Gao, W., Tang, L., 2003. Antidiabetic herbal drugs officially approved in China. Phytother. Res. 17, 1127–1134. Kim, H.J., Chae, I.G., Lee, S.G., Jeong, H.J., Lee, E.J., Lee, I.S., 2010. Effects of fermented red ginseng on hyperglycemia in streptozotocin-induced diabetic rats. J. Ginseng Res. 34, 104–112. Kim, Y.S., Hahn, E.J., Murthy, H.N., Paek, K.Y., 2004. Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnol. Lett. 26, 1619–1622. Lai, D.M., Tu, Y.K., Liu, I.M., Chen, P.F., Cheng, J.T., 2006. Mediation of beta-endorphin by ginsenoside Rh2 to lower plasma glucose in streptozotocin-induced diabetic rats. Planta Med. 72, 9–13. Liu, Z., Wang, L.J., Li, X., Hu, J.N., Chen, Y., Ruan, C.C., Sun, G.Z., 2009. Hyperglycemic effects of malonyl-ginsenosides extracted from roots of Panax ginseng on streptozotocin-induced diabetic mice. Phytother. Res. 23, 1426–1430. Paek, K.Y., Murthy, H.N., Hahn, E.J., 2009. Large scale culture of ginseng adventitious roots for production of ginsenosides. Adv. Biochem. Eng./Biotechnol. 113, 151–176. Park, H.R., Cho, J.S., 2006. Effect of natural medicinal plants extracts on organ weight and blood glucose challenge in sterptozotocin-induced diabetic rats. J. Nat. Sci. 25, 43–53. Park, J.D., Rhee, D.K., Lee, Y.H., 2005. Biological activities and chemistry of saponins form Panax ginseng C.A. Meyer. Phytochem. Rev. 4, 159–175. Quan, H., Yan, H, Jung, M.S., Ko, S.K., Park, Y.G., Chung, S.H., 2012. Ginsenoside Re lowers blood glucose and lipid levels via activation of AMP-activated protein kinase in HepG2 cells and high-fat diet fed mice. Int. J. Mol. Med. 29, 73–80. Sivakumar, G., Yu, K.W., Lee, J.S., Kang, J.K., Lee, H.L., Kim, W.J., Paek, K.Y., 2006. Tissue cultured mountain ginseng adventitious roots™: safety and toxicology evaluation. Eng. Life Sci. 6, 372–383. Sivakumar, G., Yu, K.W., Paek, K.Y., 2005. Production of biomass and ginsenosides form adventitious roots of Panax ginseng in bioreactor cultures. Eng. Life Sci. 5, 333–342. Sivakumar, G., Paek, K.Y., 2005. Methyl jasmonate induce enhanced production of soluble biophenols in Panax ginseng advnetitious roots from commercial scale bioreactors. Chem. Nat. Compd. 41, 669–673. Suda, K., Marakami, K., Jun, Murata, Hasegawa, H., Saiki, I.L., 2000. An intestinal bacterial metabolite (M1) of ginseng protopanaxadiol saponins inhibits tumorinduced neovascualization. J. Tradit. Med. 17, 144–150. Trinh, H.T., Han, S.J., Kim, S.W., Lee, Y.C., Kim, D.H., 2007. Bifidus fermentation increases hypolipidemic and hypoglycemic effects of red ginseng. J. Microbiol. Biotechnol. 17, 1127–1133. Xie, J.T., Wang, C.Z., Li, X.L., Ni, M., Fishbein, A., Yuan, C.S., 2009. Anti-diabetic effect of American ginseng and Scutellaria baicalensis using ob/ob mice model. Fitoterapia 80, 306–311. Yao, H.T., Huang, S.Y., Chiang, M.T., 2008. A comparative study on hyperglycemic and hypocholesterolemic effects of high and low molecular weight chitoson in streptozotocin-induced diabetic rats. Food Chem. Toxicol. 46, 1525–1534. Yu, K.W., Gao, W., Hahn, E.J., Paek, K.Y., 2002. Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer. Biochem. Eng. J. 11, 211–215.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Efficacy of ginseng adventitious root extract on hyperglycemia in streptozotocin-induced diabetic rats Hosakatte Niranjana Murthy a,b,n, Vijayalaxmi S. Dandin b, Eun Jung Lee c, Kee Yoeup Paek a,c,n a
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Botany, Karnatak University, Dharwad 580003, India c Cheongsol Biotech Co. Ltd., Industry Academic Cooperation Foundation Agribusiness Incubator Center, 205, Chungbuk National University, Cheongju 361-763, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 December 2013 Received in revised form 23 March 2014 Accepted 28 March 2014 Available online 5 April 2014
Ethnopharmacological relevance: Ginseng has various bioactive effects on human health including its potential activity of improving the glucose homeostasis and insulin sensitivity. Materials and methods: Tissue culture raised mountain ginseng adventitious root (TCMGARs) extract enriched with ginsenosides was used as experimental material. Streptozotocin-induced diabetic ‘Sprague Dawley’ male rats were used as experimental systems and were fed with Tissue culture raised mountain ginseng adventitious root extract. Field cultivated Korean ginseng root extract fed rats were used as positive control and several indices such as body weight, blood glucose level and other serological indicators were tested. Results: Chemical profile showed TCMGARs were rich in varied ginsenosides especially Rb1, Rb2, Rc, Rd, Rg3, and Rh2 when compared to field cultivated Korean ginseng. TCMGARs extract at dosage levels of 250 and 500 mg/kg body weight significantly lowered the blood glucose, total cholesterol and triglyceride content in streptozotocin-induced diabetic rats. Conclusion: The data of in vivo experiments on anti-glycemic effects of TCMGARs proves their efficacy and also their use as dietary supplement. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antidiabetic Blood glucose Dietary supplement Ginsenosides
1. Introduction Diabetes mellitus is a metabolic disease resulting due to the reduced insulin secretion by pancreas or due to a low biological activity of the insulin secreted and is classified as insulindependent (type 1) and insulin independent type (type 2) (Geroge and Ludvik, 2000). Panax ginseng C.A. Meyer (Korean ginseng) is traditionally used for treating hyperglycemia including diabetes mellitus (Ivorra et al., 1989; Attele et al., 1999) and it is recognized officially as one of the herbal drug ingredients in China for treating diabetes mellitus (Jia et al., 2003). Ginseng is available in commercial market in two forms namely red ginseng and white ginseng. The roots of Panax ginseng is steamed and dried to prepare red ginseng, while the peeled roots dried without steaming are designated as white ginseng. The major components of
n Corresponding authors at: Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea. Tel: þ82 43 266 3245; fax: þ 82 43 266 3246. E-mail addresses: [email protected] (H.N. Murthy), [email protected] (K.Y. Paek).
http://dx.doi.org/10.1016/j.jep.2014.03.062 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
ginseng are triterpenoidal dammarane glycosides (saponins) called ginsenosides. More than 30 kinds of ginsenosides have been reported and they have been named as ‘Rx’ based on their mobility on thin layer chromatographic plates, with polarity decreasing from index ‘a’ to ‘h’. They differ from one another by the type of sugar moieties, their number and the site of attachment (Park et al., 2005). Ginsenosides are classified into three groups by their structure i.e., Rb group (protopanaxadiols including Rb1, Rb2, Rc and Rd, etc.), the Rg group (protopanaxatriols including Rg1, Re, Rf, and Rg2, etc.) and the Ro group (Oleanolic acid; Park et al., 2005). It was reported that diol-type ginsenosides such as Rb1, Rb2, Rc, Rd, Rg3 and Rh2 are having anti-diabetic activities (Suda et al., 2000) and ginsenoside Rh2 increases insulin secretion in streptozotocin-induced diabetic rats to decrease the blood glucose concentration (Lai et al., 2006). Recently, microbial fermentation methods have been introduced and red ginseng powder was fermented using micro-organisms such as lactic acid bacteria to transform ginsenosides such as Rb1, Rb2, RC and Rd into readily absorbable forms (Bae et al., 2004; Trinh et al., 2007; Kim et al., 2010) and these fermented red ginseng powders and extracts were
918
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
also made available in the market. Simultaneously, a group of scientists have induced adventitious roots from 100 year old mountain ginseng and called them as ‘tissue cultured mountain ginseng adventitious roots’ (TCMGARs), and also adopted methyl jasmonate elicitation strategy to overproduce pharmacologically useful ginsenosides (Paek et al., 2009). TCMGARs were reported to be rich in diol-group of ginsenosides (Sivakumar et al., 2005) and were proved as ‘biosafe’ through toxicological evaluations (Sivakumar et al., 2006). The aims of the study were to investigate the efficacy of TCMGARs, the evaluation of antidiabetic effect of TCMGARs following the administration of TCMGARs extracts in streptozotocin (STZ) induced diabetic rats and the examination of body weight, blood glucose level and other serological indicators. Chemical profiling of ginsenosides present in TCMGARs as well as field cultivated Korean ginseng was also carried out before their administration for antidiabetic evaluations.
2. Materials and methods 2.1. Material Tissue cultured mountain ginseng adventitious roots (TCMGARs) and freshly harvested field cultivated ginseng roots (positive control) were dried (forced air drying at 50 1C), powdered and soaked in 50% aqueous ethanol for 10 days at 25 1C and filtered. The solution evaporated in vacuo gave a semi-gelatinous extract and yielded the crude ginsenosides of 128.51 mg/g and 100.08 mg/g from ginseng adventitious roots and cultivated ginseng roots respectively. 2.2. Determination of ginsenoside content The chemical profiling of ginsenosides in these two materials was carried out by high pressure liquid chromatography (HPLC) at the Korean Food Research Institute, Sungnam-Si, Republic of Korea (File no. AO2012-06-26-200). Extraction and analysis of ginsenosides were carried out using the protocol by Yu et al. (2002). The ginsenoside fractions were analyzed using HPLC system (Shimadzu, Kyoto) consisting of 10AT pump, 10AXL autosampler, SPDM10A photodiode array detector, and CTO-10A column oven, 5 μM Lichrosorb column (250 4.6 mm2) (Altech, Deerfild, IL), and a C18 guard column, at 40 1C. The eluted peaks were detected at 203 nm and quantified against external standards of ginsenosides, Rf, Rb2, Rd, (Karl Roth, Germany), Re, Rg1, Rg2, Rh1, Rh2, Rb1, Rb3, Rc and Rg2 (Wako, Osaka, Japan). The mobile phase was a gradient elution of water (A) and acetonitrile (B), commencing with 20% B, rising to 22% B after 20 min then to 46% after 45 min and 55% B after 50 min. 2.3. Experimental animals Experiments were carried out in Biotoxtech Laboratory, South Korea on Sprague Dawley male rats. All the experimental animals were supplemented with a standard diet and were maintained in a room kept under environmentally controlled conditions of 2471 1C and 12:12 h light/dark cycles. The animals had free access to water and a standard diet [a normal laboratory commercial stock diet containing 16% protein, 56% carbohydrate and 8% fat (w/w)]. 2.4. Oral glucose tolerance test Blood glucose levels of normal rats kept under fasting for at least 12 h were determined. Then the rats were orally administered TCMGARs (125, 250 and 500 mg/kg body weight) or field cultivated Korean ginseng extract (250 mg/kg body weight) dissolved in distilled water. For the control group, an equal amount of normal saline was
given. Subsequently, oral dose of 40% glucose was administered to all the experimental group of rats at 1 g/kg rate and later blood samples were collected at 30, 60 and 120 min post-glucose administration to record the variation in blood glucose levels. 2.5. Induction of diabetes in experimental rats In the treatment studies, animals were divided into six groups of seven animals in each (G). All animals were of seven weeks old and the average body weight was 305 g. Group G1 consisted of normal rats and which served as a control and the remaining five groups consisted of the rats induced with diabetes. The G2 was streptozotocin control (diabetes control) and G3, G4 and G5 groups were included with diabetic rats which were fed with diet containing tissue cultured mountain ginseng extract at a dose of 125, 250 and 500 mg/kg body weight respectively. Group G6 had diabetic rats which were fed with a diet containing field cultivated Korean ginseng extract of 250 mg/kg body weight. All rats were kept on observation for four weeks and further used for biochemical analysis. For the induction of diabetes, rats that had undergone one week adjustment period were kept under fasting for a minimum of 12 h, and the intra-peritoneal injection of streptozotocin (STZ) diluted in 0.01 M citrate buffer was administered at 50 mg/kg of body weight. In the normal control group, the same concentration of normal saline (without streptozotocin) was used for intra peritoneal injection. Blood collected from the veins of tail region was used to confirm the induction of diabetes, and the rats with fasting blood glucose levels of about 200 mg/dL were used for further experiments. 2.6. Analysis of body weight, blood glucose, and other biochemical tests Increase in body weight was measured every week at the same time in a study period of four weeks. Alterations in blood glucose levels during the feeding period were measured at weekly intervals using a blood glucose monitoring system (ACCU-CHEK Sensor; Roche Diagnostics GmbH, Mannheim, Germany), and blood was collected from the veins of tail region of the rats which were kept on fasting for over 12 h. The total triglyceride level and cholesterol contents were measured using a triglyceride measuring kit and a total cholesterol content measuring kit respectively (Asan Pharmaceuticals, Whasung, Korea). 2.7. Statistical analysis The results were statistically analyzed by using analysis of variance (ANOVA) followed by a Student's t-test and all the values are expressed as mean with standard errors.
3. Results and discussion 3.1. Ginsenoside content in TCMGARs and Korean ginseng The details of ginsenosides present in TCMGARs and field cultivated Korean ginseng are presented in Fig. 1 and Table 1. The TCMGARs possessed higher amounts of Rb1, Rb2, Rc, Rd, Re, Rg1and Rf ginsenosides when compared to field cultivated Korean ginseng, additionally, ginsenosides such as Rb3, Rg2, Rg3, Rh1 and Rh2 which were lacking in the extracts of field cultivated Korean ginseng were abundant in the TCMGARs. The higher accumulation of ginsenosides and other novel ginsenosides is obvious because of the influence of methyl jasmonate elicitation during the cultivation of TCMGARs but it was not observed with field cultivated Korean ginseng (Paek et al., 2009). It is reported that methyl
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
919
Fig. 1. HPLC chromatogram of field cultivated ginseng (above) and tissue cultured mountain ginseng adventitious roots (below).
Table 1 Ginsenoside contents of tissue cultured mountain ginseng adventitious roots in comparison with cultivated Korean ginseng.a Ginsenoside
Rb1 Rb2 Rb3 Rc Rd Re Rg1 Rg2 Rg3 Rh1 Rh2 Rf
Content (mg/g dry weight) TCMGARs
Korean ginseng
4.1 2.3 0.9 2.0 4.2 0.2 0.19 0.38 11.2 0.49 3.8 1.64
0.05 0.06 – 0.03 0.06 0.24 0.17 – – – – 0.03
a Analysis was carried out by the Korea Food Research Institute, Sungnam-si, Republic of Korea.
jasmonate stimulates the ginsenoside biosynthetic pathway thus enhancing the accumulation of ginsenosides up to 5–7 fold in TCMGARs (Yu et al., 2002; Kim et al., 2004). In ginseng, the dioltype of ginsenosides such as Rb1, Rb2, Rc, Rd, Rg3 and Rh2 exhibit anti-diabetic activity (Suda et al., 2000), and the ginsenoside Rh2 increases the insulin secretion in STZ-induced diabetic rats to reduce the blood glucose level (Lai et al., 2006). As TCMGARs possess higher concentrations of Rb1, Rb2, Rc, Rd, Rg3 and Rh2 ginsenosides, we had expected a greater impact of TCMGARs extracts in combating STZ-induced hyperglycemic activity in the present studies. Efforts were also made in the past for the enhancement in accumulation of diol-type of ginsenosides in red ginseng by the fermentation technology i.e., fermentation of red ginseng powder with intestinal bacteria such as Lactobacillus fermentum and also improved accumulation of Rg3 and Rh2 type of ginsenosides has been reported (Kim et al., 2010). 3.2. Oral glucose tolerance test in experimental rats The oral glucose tolerance tests in the rats administered with TCMGARs extracts showed a gradual decrease in the glucose levels
Fig. 2. Oral glucose tolerance test: effect of tissue cultured mountain ginseng adventitious root extract (TCMAGR) and field cultivated Korean ginseng (KG) extract feeding. In the oral glucose tolerance test, glucose levels were estimated followed by the oral administration of 1 g/kg D-(þ )-glucose at indicated time points. Control group (Control), TCMGAR 125, TCMGAR 250, TCMGAR 500, KG 250. Mean 7standard error (n¼ 7).
with the increase in time duration as well as with an increase in the dosage levels of TCMGARs extract (Fig. 2). The blood glucose level was highest at 30 min after glucose administration, thereafter showed a decrease in blood glucose levels. The groups fed with TCMGARs extract as well as Korean ginseng showed a significant reduction in the blood glucose levels when compared to the group that was administered with glucose alone and the one without sample administration. Relatively, the groups fed with 250 and 500 mg/kg TCMGARs extract demonstrated a greater reduction in blood glucose level than that of groups fed with 125 mg/kg. At 120 min post-glucose administration, the blood glucose levels were reduced to almost the level before fasting, regardless of the sample concentration used. These experimental results clearly demonstrate the effects of TCMGARs extracts as well as Korean ginseng extracts in the improvement of glucose metabolism.
920
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
Table 2 Effects of tissue cultured mountain ginseng adventitious root (TCMGR) extracts on total cholesterol and triglyceride contents of plasma in streptozotocin-induced rats for 4 weeks. Group
Dose (mg/kg)
G1
0
G2 STZ treated
STZ control
G3 STZþTCMGARa
125
G4 STZþ TCMGAR G5 STZþ TCMGAR G6 STZþ Korean ginseng
250
500
250
Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N Mean SEM N
Total cholesterol (mg/dL)
Triglyceride (mg/dL)
25.4 3.2 7 44.3n 6.8 7 39.7 2.9 7 32.4† 7.5 7 31.4† 3.1 7 39.9 7.4 7
43.3 4.8 7 60.5n 5.4 7 28.1† 4.8 7 33.2† 4.8 7 23.5† 3.8 7 29.5† 3.3 7
Fig. 3. Effect of tissue cultured mountain ginseng adventitious root (TCMAGR) extract on body weight in STZ-induced diabetic rats after four weeks of treatment. G, normal control; G2, STZ control, G3, diabetic group fed with 125 mg/kg of TCMGARs extract; G4, diabetic group fed with 250 mg/kg of TCMGARs extract; G5, diabetic group fed with 500 mg/kg of TCMGARs extract; G6, diabetic group fed with 250 mg/kg field cultivated Korean ginseng extract. *Po 0.05 vs normal control, P o 0.05 vs STZ control.
N ¼Number of animals. Values are expressed as mean7 S.E. a n
†
TCMGAR—Tissue cultured mountain ginseng adventitious root extract. Po 0.05 vs normal control. Po 0.05 vs STZ control.
3.3. Anti-diabetic property of TCMGARs STZ treatment of rats leads to selective destruction of the insulin secreting β-cells of the pancreas resulting in diabetes mellitus (Gilman et al., 1990). The symptoms of diabetes mellitus are polydipsia, polyphagia, polyuria and the most significant noticeable morphological change is decrease in body weight (Kim et al., 2010). All these symptoms were observed with STZ treated rats in the present studies. Changes in the body weight of rats with STZ-induced diabetes and after feeding for four weeks with TCMGARs as well as field cultivated Korean ginseng are shown in Table 2. Consistent increase in body weight over a time period was observed with the control group of normal rats. There was a significant decrease in body weight in STZ-induced group of rats (Fig. 3). The body weight of the TCMGARs extractadministered group was decreased when compared to the normal group, but it was significantly higher than the body weight of STZ control groups especially with G4 and G5 (i.e., the groups of rats administered with 250 and 500 mg/kg of TCMGARs). However, there was no improvement in the body weight of STZ-induced diabetic rats administered with 125 mg/kg TCMGARs extract and 250 mg/kg Korean ginseng extract. It was reported that the inefficient energy production during glucose metabolism affects the growth and development in diabetic individuals (Park and Cho, 2006), which leads to loss of body weight. In the present study, administration of TCMGARs (250 and 500 mg/kg body weight) helped the diabetic rats in restoration of body weight significantly. Changes in blood glucose levels in diabetic rats which were administered with TCMGARs extract and Korean ginseng extract are shown in Fig. 4. Blood glucose levels increased significantly in the STZ-control group of rats compared to the control group of normal rats. The groups of rats administered with TCMGARs extract and field cultivated Korean ginseng extract showed significant reduction in blood glucose levels after one week of sample administration when compared to STZ-control groups. Blood glucose levels decreased more significantly with the
Fig. 4. Effect of tissue cultured mountain ginseng adventitious root (TCMAGR) extract on the blood glucose levels in STZ-induced diabetic rats after four weeks of treatment. G, normal control; G2, STZ control, G3, diabetic group fed with 125 mg/kg of TCMGARs extract; G4, diabetic group fed with 250 mg/kg of TCMGARs extract; G5, diabetic group fed with 500 mg/kg of TCMGARs extract; G6, diabetic group fed with 250 mg/kg field cultivated Korean ginseng extract. *Po0.05 vs normal control, Po0.05 vs STZ control.
increased duration of the feeding period, and 2, 3 and 4 weeks after sample administration, the 250 and 500 mg/kg TCMGARs treated groups showed a more significant reduction in blood glucose levels when compared to STZ-control group. The present experimental results are consistent with previous reports demonstrating anti-glycemic effects of Korean ginseng and American ginseng (Liu et al., 2009; Xie et al., 2009), and fermented red ginseng (Kim et al., 2010). We used field cultivated Korean ginseng extract treatments as positive control to compare the effect of TCMGARs extract on STZ-induced diabetic rats. It was observed that TCMGARs extract treatments especially 250 and 500 mg/kg treatments were far superior to Korean ginseng treatments, this positive effect might be due to ginsenoside concentration and composition of TCMGARs (Table 1). Following the TCMGARs extract administration, the total plasma cholesterol concentration was 25.473.2 mg/dL in the control group of normal rats when compared with 44.376.8 mg/dL in diabetic control group. The plasma cholesterol concentrations were significantly low in the TCMGARs extract treated group of individuals especially with 250 and 500 mg/kg treatment groups
H.N. Murthy et al. / Journal of Ethnopharmacology 153 (2014) 917–921
(Table 2). Yao et al. (2008) reported that the increase in total cholesterol levels in diabetes induced rats was due to inability of diabetic individuals to metabolize carbohydrates as an energy source and the use of available free fatty acids in the system for the energy synthesis which leads to cholesterol accumulation. The observation on accumulation of total cholesterol accumulation in diabetic rats is in accordance with reported results of Kim et al. (2010). There was also a significant increase in triglyceride concentration in diabetes induced rats when compared to the control group of normal rats (Table 2). Whereas, there was a significant decrease in the triglyceride levels in diabetic rats which were fed with the extracts of TCMGARs as well as the extract of field cultivated Korean ginseng (Table 2). These results are in agreement with the reports by Kim et al. (2010) that ginsenosides stimulate the improved lipid metabolism which is manifested in diabetic individuals. The present data of in vivo experiments on anti-glycemic effects of TCMGARs are in accordance with the various other experimental reports that ginseng improves glucose homeostasis. It was observed that the effects of TCMGARs are more dramatic when administered for a longer duration with elevated dose levels. TCMGARs contained higher amounts of diol-type of saponins such as ginsenoside Rb1, Rb2, Rc, Rd, Rg3 and Rh2 (Table 1) and these levels are optimal when compared to the ginsenoside amounts which were reported in white ginseng or red ginseng. The richness of ginsenosides in TCMGARs is due to the methyl jasmonate elicitation strategy applied during the in vitro production of ginseng. The present studies reveal that Korean ginseng is efficient in lowering the glucose and lipid levels, this might be due to richness of ginsenoside Re content when compared to TCMGARs (Table 2). Recently, several authors have reported that ginsenoside Re also lowers blood glucose and lipid levels (Attele et al., 2002; Quan et al., 2012). The above results prove the efficacy of TCMGARs. Earlier we have reported biosafety of TCMGARs based on mutagenicity and toxicological evaluations (Sivakumar et al., 2006) and TCMGARs are reported to possess biophenols (Sivakumar and Paek, 2005), enzymatic and non-enzymatic antioxidants (Ali et al., 2006). Therefore, TCMGARs and their products can be used as dietary supplements.
Acknowledgments This study was supported by a Grant of Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (Grant no. A103017). Dr. H.N. Murthy is thankful to the Ministry of Education, Science and Technology, Republic of Korea for the award of Brain Pool Fellowship (131S-4-3-0523); this study was also supported by the Ministry of Science, ICT and Planning (MSIP). Authors are thankful to Prof. Yeravinthalimath, Department of English, Karnatak University, India for language editing of the manuscript.
921
References Ali, M.B., Yu, K.W., Hahn, E.J., Paek, K.Y., 2006. Methyl jasmonate and salicylic acid elicitation induces ginsenoside accumulation, enzymatic and non-enzymatic antioxidant in suspension culture of Panax ginseng roots in bioreactors. Plant Cell Rep. 25, 613–620. Attele, A.S., Wu, J.A., Yuan, C.S., 1999. Ginseng pharmacology, multiple constituents and multiple actions. Biochem Pharmacol. 58, 1685–1693. Attele, A.S., Zhou, Y.P., Xie, J.T., Wu, J.A., Zhang, L., Dey, L., Pugh, W., Rue, P.A., Polonsky, K.S., Yuan, C.S., 2002. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component. Diabetes 51, 1851–1858. Bae, E.A., Han, M.J., Kim, E.J., Kim, D.H., 2004. Transformation of ginseng saponins to ginseonoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch. Pharm Res. 27, 61–67. Gilman, A.G., Rall, T.W., Nies, A.S., Tayer, P. (Eds.), 1990. Goodman and Gilman's the Pharmacological Basis of Therapeutics, eighth ed. Pergamon Press, New York. George, P., Ludvik, B., 2000. Lipids and diabetes. J. Clin. Basic Cardiol. 3, 159–162. Ivorra, M.D., Paya, M., Villar, A., 1989. A review of natural products and plants as potential antidiabetic drugs. J. Ethnopharmacol. 27, 243–275. Jia, W., Gao, W., Tang, L., 2003. Antidiabetic herbal drugs officially approved in China. Phytother. Res. 17, 1127–1134. Kim, H.J., Chae, I.G., Lee, S.G., Jeong, H.J., Lee, E.J., Lee, I.S., 2010. Effects of fermented red ginseng on hyperglycemia in streptozotocin-induced diabetic rats. J. Ginseng Res. 34, 104–112. Kim, Y.S., Hahn, E.J., Murthy, H.N., Paek, K.Y., 2004. Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnol. Lett. 26, 1619–1622. Lai, D.M., Tu, Y.K., Liu, I.M., Chen, P.F., Cheng, J.T., 2006. Mediation of beta-endorphin by ginsenoside Rh2 to lower plasma glucose in streptozotocin-induced diabetic rats. Planta Med. 72, 9–13. Liu, Z., Wang, L.J., Li, X., Hu, J.N., Chen, Y., Ruan, C.C., Sun, G.Z., 2009. Hyperglycemic effects of malonyl-ginsenosides extracted from roots of Panax ginseng on streptozotocin-induced diabetic mice. Phytother. Res. 23, 1426–1430. Paek, K.Y., Murthy, H.N., Hahn, E.J., 2009. Large scale culture of ginseng adventitious roots for production of ginsenosides. Adv. Biochem. Eng./Biotechnol. 113, 151–176. Park, H.R., Cho, J.S., 2006. Effect of natural medicinal plants extracts on organ weight and blood glucose challenge in sterptozotocin-induced diabetic rats. J. Nat. Sci. 25, 43–53. Park, J.D., Rhee, D.K., Lee, Y.H., 2005. Biological activities and chemistry of saponins form Panax ginseng C.A. Meyer. Phytochem. Rev. 4, 159–175. Quan, H., Yan, H, Jung, M.S., Ko, S.K., Park, Y.G., Chung, S.H., 2012. Ginsenoside Re lowers blood glucose and lipid levels via activation of AMP-activated protein kinase in HepG2 cells and high-fat diet fed mice. Int. J. Mol. Med. 29, 73–80. Sivakumar, G., Yu, K.W., Lee, J.S., Kang, J.K., Lee, H.L., Kim, W.J., Paek, K.Y., 2006. Tissue cultured mountain ginseng adventitious roots™: safety and toxicology evaluation. Eng. Life Sci. 6, 372–383. Sivakumar, G., Yu, K.W., Paek, K.Y., 2005. Production of biomass and ginsenosides form adventitious roots of Panax ginseng in bioreactor cultures. Eng. Life Sci. 5, 333–342. Sivakumar, G., Paek, K.Y., 2005. Methyl jasmonate induce enhanced production of soluble biophenols in Panax ginseng advnetitious roots from commercial scale bioreactors. Chem. Nat. Compd. 41, 669–673. Suda, K., Marakami, K., Jun, Murata, Hasegawa, H., Saiki, I.L., 2000. An intestinal bacterial metabolite (M1) of ginseng protopanaxadiol saponins inhibits tumorinduced neovascualization. J. Tradit. Med. 17, 144–150. Trinh, H.T., Han, S.J., Kim, S.W., Lee, Y.C., Kim, D.H., 2007. Bifidus fermentation increases hypolipidemic and hypoglycemic effects of red ginseng. J. Microbiol. Biotechnol. 17, 1127–1133. Xie, J.T., Wang, C.Z., Li, X.L., Ni, M., Fishbein, A., Yuan, C.S., 2009. Anti-diabetic effect of American ginseng and Scutellaria baicalensis using ob/ob mice model. Fitoterapia 80, 306–311. Yao, H.T., Huang, S.Y., Chiang, M.T., 2008. A comparative study on hyperglycemic and hypocholesterolemic effects of high and low molecular weight chitoson in streptozotocin-induced diabetic rats. Food Chem. Toxicol. 46, 1525–1534. Yu, K.W., Gao, W., Hahn, E.J., Paek, K.Y., 2002. Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer. Biochem. Eng. J. 11, 211–215.
