|Year : 2019 | Volume
| Issue : 6 | Page : 267-272
Contrasting actions of ginsenosides Rb1 and Rg1 on glucose tolerance in rats
Rungchai Chaunchaiyakul1, Naruemon Leelayuwat2, Jin-Fu Wu3, Chih-Yang Huang4, Chia-Hua Kuo5
1 Faculty of Sports Science and Technology, Mahidol University, Nakhonpathom, Thailand; Laboratory of Exercise Biochemistry, University of Taipei, Taipei, Taiwan
2 Exercise and Sport Sciences Development and Research Group, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
3 Laboratory of Regenerative Medicine in Sports Science, School of Physical Education & Sports Science, South China Normal University, Taichung, Taiwan
4 Graduate Institute of Basic Medicine, China Medical University, Taichung, Taiwan
5 Laboratory of Exercise Biochemistry, University of Taipei, Taipei, Taiwan
|Date of Submission||08-Sep-2019|
|Date of Acceptance||07-Oct-2019|
|Date of Web Publication||29-Nov-2019|
Prof. Chia-Hua Kuo
Laboratory of Exercise Biochemistry, University of Taipei, 101, Section 2, Jhong Cheng Road, Shihlin District, Taipei 111
Source of Support: This study was supported by grants from the Ministry of Science and Technology, Taiwan; Nuliv Sciences, CA, USA; and University of Taipei, Taipei, Taiwan., Conflict of Interest: This work was funded to develop a supplement for Nuliv Science, Taiwan, and the USA.
Ginsenoside profile of Panax ginseng is changing with season and cultivated soil. Yet, dose-response relationship of main ginsenosides on metabolic measures has not been documented in vivo. Here, we examined glucose and insulin responses after an oral glucose challenge (0.5 g/kg body weight) at various doses (0.01, 0.1, 1, and 10 mg/kg of body weight) under acute and chronic Rb1 and Rg1 supplemented conditions. The results show that Rb1 (0.01 and 0.1 mg/kg body weight) increased, whereas Rg1 (0.01 mg/kg body weight) decreased postprandial glucose levels compared with the Vehicle group (P < 0.05). This contrasting effect reduced as dose increased. Both Rb1 and Rg1 decreased the mitochondrial enzyme citrate synthase activity (P < 0.05) together with decreases in glycogen content in red gastrocnemius muscle and body temperature at low doses (P < 0.05), compared with the Vehicle group. These differences also diminished as dosage increases. For reliable ginseng research, dose standardization on Rg1 and Rb1 is essential based on their opposing action and peculiar dose-response relationship. Both major ginsenosides may influence dynamics of mitochondria turnover and alter muscle metabolism.
Keywords: Glucose, insulin, oral glucose tolerance test, Panax ginseng
|How to cite this article:|
Chaunchaiyakul R, Leelayuwat N, Wu JF, Huang CY, Kuo CH. Contrasting actions of ginsenosides Rb1 and Rg1 on glucose tolerance in rats. Chin J Physiol 2019;62:267-72
|How to cite this URL:|
Chaunchaiyakul R, Leelayuwat N, Wu JF, Huang CY, Kuo CH. Contrasting actions of ginsenosides Rb1 and Rg1 on glucose tolerance in rats. Chin J Physiol [serial online] 2019 [cited 2020 Oct 20];62:267-72. Available from: https://www.cjphysiology.org/text.asp?2019/62/6/267/272028
| Introduction|| |
Panax ginseng has been claimed to have illness curing property and used for several thousand years in humans. However, its efficacy on glucose metabolism is often inconsistent among different types of ginseng. Furthermore, a previous study has shown divergent outcomes in glucose tolerance using two batches of the same ginseng species cultivated from two seasons, which is associated with different ginsenoside profiles. These reports highlight a technical barrier in developing reliable ginseng-based nutraceuticals.
Severalin vivo studies indicate improved glucose tolerance and enhanced insulin sensitivity after short- and long-term intraperitoneal injections of Rb1 (10 mg/kg)., Reduced fat gains with increased energy expenditure with Rb1 injection were also observed in rats under a high-fat diet. With similar chemical structure to Rb1, Rg1 (50 mg/kg) has been found to decrease the hepatic glucose production in high-fat diet-fed mice and glucagon-challenged C57BL/6J mice. Improved insulin sensitivity in cultured adipocytes and myocytes by Rg1 incubation suggests its direct effect at the cellular level., Most of the previous studies showing enhanced glucose metabolism used intraperitoneal injection as a delivery method or conducted in cell culture, which is not practical in nutraceutical use.
Mixed ginsenosides from P. ginseng show a peculiar dose-response relationship in rats. Documentation regarding dose-response relationship of Rb1 and Rg1 on oral glucose tolerance test (OGTT) is presently scarce. Therefore, in the study, we examined the dose-response relationship aiming to answer the question of whether both ginseng components independently improve glucose tolerance in rats at doses ranging from 0.01 to 10 mg/kg body weight. Both mitochondria enzyme activity (citrate synthase) and glycogen of red and white gastrocnemius muscles were also measured to examine their action on muscle metabolism.
| Materials and Methods|| |
Sprague–Dawley rats (~120 g) purchased from Bio-LASCO Corporation (Taipei, Taiwan) were housed in the animal facility at University of Taipei on a 0800–2000 h light cycle and at a temperature of 23°C. They were housed in cages with standard laboratory chow (PMI Nutrition International, Brentwood, MO, USA) and tap water ad libitum. All animal experiment protocols were approved by the Ethics Committee of University of Taipei (Taipei, Taiwan), in accordance with the Guide for the Care and Use of Laboratory Animals and Taiwan's Animal Protection Law.
Ginsenosides Rb1 and Rg1
Rb1 and Rg1, provided by (NuLiv Science, Inc., Brea, CA, USA), were extracted from Asian ginseng according to method standardized by Tsu-Chung Chang in the Department of Biochemistry, National Defense Medical Center, Taiwan. Rb1 and Rg1 crystals were solubilized by a 58% alcohol solution for stock. Rb1 and Rg1 were delivered by oral intubation using gastric gavage after further dilution in a 1% saline solution. The same volume of 1% ethanol was given to the Vehicle group.
Experimental design and procedures
The first experiment was conducted to determine the acute effect of Rg1 supplementation on glucose and insulin responses after an OGTT (0.5 g/kg body weight). Rats were divided into three treatment groups: Rb1 (n = 16), Rg1 (n = 16), and Vehicle (n = 16). For the Rb1 and Rg1 groups, they were equally divided into four dose ranges: 0.01, 0.1, 1, and 10 mg/kg of body weight. The second experiment with the same design and daily dosage was conducted for 4 weeks to determine the effects of long-term supplementation on OGTT and insulin response. Blood samples were taken from a tail vein and analyzed for glucose and insulin. Red and white gastrocnemius muscles were collected 5 h after OGTT.
Rat temperature was measured at the right feet by an infrared body temperature thermometer (Brannan Thermometer and Instrument, Cumbria, CA, UK).
Oral glucose tolerance test
Glucose solution for OGTT was administered by oral intubation. Rats from all groups received 1 ml of a 50% (w/v) glucose solution, 12 h and 1 h before Rb1 or Rg1 supplementations to determine the acute effect of these ginsenosides on oral glucose tolerance. To determine the chronic effect, the same treatments were conduced daily for 4 weeks. OGTT was performed 12 h after the last supplementation of Rb1 or Rg1 under overnight fasted condition. Rat chow was continuously supplied ad libitum after OGTT until muscle tissue collection for glycogen and citrate synthase assays. During blood collection, rats were wrapped in a towel immediately postexercise, and a 0.6 ml blood sample was taken from a tail vein.
All rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg bw), 5 h after oral glucose intubation for OGTT before surgical collection. The red and white gastrocnemius muscles were excised and frozen immediately by liquid nitrogen, and then stored in liquid nitrogen tank until glycogen and citrate synthase analyses. Following muscle sampling, rats were euthanized by cardiac injection of pentobarbital sodium.
About 50 mg of red and white gastrocnemius muscles were weighed and dissolved in 1N KOH at 70°C for 30 min. The dissolved homogenate was neutralized by acetic acid and incubated overnight in acetate buffer (0.3 M sodium acetate, pH to 4.8) containing amyloglucosidase (Boehringer Mannheim, Indianapolis, IN, USA). The reaction mixture was neutralized with 1N NaOH. Samples were then analyzed by measuring glucosyl units by the Trinder reaction (Sigma, St. Louis, MO, USA) and normalized by muscle weight.
Glucose and insulin
Fasting blood samples collected from the tail were used for glucose and insulin analyses after a 12-h overnight fasting. Blood glucose level was measured in fresh by using Accu-Chek® performa system (Roche Diagnostics, Indiana, USA). Blood samples for insulin measurements were collected into EDTA-contained tube and centrifugation for 10 min (3000 rpm) at 4°C. Plasma insulin was measured using commercial enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Mercodia AB, Uppsala, Sweden). The optical density value was read at 450 nm using ELISA reader (Tecan GENios, A-5082, Austria).
Citrate synthase activity
Citrate synthase activity was assessed according to the method from Srere.
The analysis of variance was used to determine the statistical significance of mean among groups for all variables. Tukey post hoc test was used for comparing the difference between paired groups. The probability of Type I error is set at 5% or less for significance. All results are expressed as mean ± standard error.
| Results|| |
The main acute effects of Rb1 and Rg1 on fasting glucose were not observed [Figure 1]. However, under oral glucose challenged conditions, significant effects of Rb1 and Rg1 on glucose response were observed only when doses were low. In particular, Rb1 (0.01 mg/kg) showed moderately higher [Figure 1]a whereas Rg1 (0.01 mg/kg) showed moderately lower [Figure 1]b glucose response compared against the vehicle group. This opposing response of Rb1 and Rg1 diminished as dose increased. No significant differences in insulin between groups at all doses were observed [Figure 1]c and [Figure 1]d. Similarly, body temperature (foot) decreased by both Rb1 (0.1 mg/kg) and Rg1 (0.01 and 0.1 mg/kg) supplementation at low doses against the Vehicle group [Figure 2]a and [Figure 2]b. This effect was reversed as dosage increased. Following a 4-week ginsenoside treatments, increased glucose response during OGTT in the Rb1 group remained significant compared with the Vehicle group [Figure 3]a, but no significant Rg1 effect was detected [Figure 3]b. Both fasting insulin level and insulin response to OGTT were not different among all doses [Figure 3]c and [Figure 3]d. Glycogen content in red gastrocnemius muscle of the Rg1 group was moderately lower than that in the Vehicle group when dosage was low (0.1 mg/kg) [Figure 4]. Citrate synthase activity of red gastrocnemius muscles in the Rb1 and Rg1 groups was significantly lower than that of the Vehicle group at low dose (0.01 mg/kg) [Figure 5]a. No significant differences in citrate synthase of white gastrocnemius muscle among three groups were found [Figure 5]b.
|Figure 1: Oral glucose tolerance and insulin response after acute ginsenoside supplementations. Rb1 and Rg1 were orally delivered to rats 12 h and 1 h before oral glucose tolerance test. Low-dose Rb1 (0.01 and 0.1 mg/kg body weight) increased (a), whereas low-dose Rg1 (0.01 mg/kg body weight) decreased (b) glucose levels. This contrasting effect diminished as dose escalated. Insulin responses for Rb1 (c) and Rg1 (d) were not different among groups at all doses. *Significant difference against the Vehicle group, P < 0.05.|
Click here to view
|Figure 2: Body temperature changes after acute ginsenoside supplementations. Ginsenosides Rb1 and Rg1 were orally delivered to rats 12 h and 1 h before oral glucose tolerance test. Foot temperature decreased after both Rb1 (0.1 mg/kg body weight) (a) and Rg1 (0.01 and 0.1 mg/kg body weight) (b) supplementations at low doses but not high doses. *Significant difference against the Vehicle group, P < 0.05.|
Click here to view
|Figure 3: Oral glucose tolerance and insulin response after a 4-week daily ginsenoside supplementation. The oral glucose tolerance test was conducted 12 h after the last oral supplementations of ginsenosides Rb1 and Rg1. At low dose (0.1 mg/kg body weight), an increased glucose response in the Rb1 group was observed compared with the Vehicle group (a). Significant difference was not detected for Rg1 among all doses (b). Insulin responses for Rb1 (c) and Rg1 (d) were not different among groups at all doses. *Significant difference against the Vehicle group, P < 0.05.|
Click here to view
|Figure 4: Glycogen contents in red and white gastrocnemius muscles after a 4-week daily ginsenoside supplementation. A moderate glycogen-reducing effect supplementation in red gastrocnemius muscle was observed only under low dose (0.1 mg/kg) Rb1 and Rg1 supplementations (a), whereas no significant effect was observed in the white gastrocnemius muscle (b). *Significant difference against the Vehicle group, P < 0.05.|
Click here to view
|Figure 5: Citrate synthase activity in red and white gastrocnemius muscles after a 4-week daily ginsenoside supplementation. Significant effects of ginsenoside Rb1 and Rg1 on lowering citrate synthase activity (mitochondria marker) were detected in red gastrocnemius muscle (a) but not in white gastrocnemius muscle (b). *Significant difference against the Vehicle group, P < 0.05.|
Click here to view
| Discussion|| |
Rb1 and Rg1 are major steroidal components of ginseng, which have been claimed to influence glycemic control in vivo. In this study, we determined the acute and chronic effects of Rb1 and Rg1 supplementation on oral glucose tolerance in rats. The key findings of the study are: (1) An acute low-dose Rb1 supplementation moderately increased, whereas acute low-dose Rg1 supplementation decreased glucose levels under oral glucose challenged condition. However, this contrasting effect diminished as dose increased; (2) oral supplementations of Rb1 and Rg1 at low doses resulted in lower body temperature and decreased mitochondria enzyme citrate synthase activity and these effects diminished as dose increased, and (3) Rg1 at low dose decreased muscle glycogen content, suggesting increased energy reliance on glycogen consumptions. Intriguingly, this effect also diminished as dose increased.
Rg1 supplementation at low dosage appears to switch the energy reliance from fat to carbohydrate oxidation reflected by concurrent improvement in glucose tolerance and decreased glycogen storage. This is supported by the observation on small muscle glycogen depletion together with reduced mitochondria enzyme activity. Greater glycogen depletion of exercising human muscle has also been shown when Rg1 was supplemented 1 h before exercise. Furthermore, the dose-response patterns on OGTT are somewhat similar for both acute and chronic supplemented conditions. Therefore, it is likely that the observed effects of low dose Rb1 and Rg1 on glucose tolerance are acute in nature. The underlying mechanism to explain the opposing effect of Rb1 and Rg1 on oral glucose tolerance may, to some extent, associated with their effects on intestinal glucose absorption. Intestinal metabolite of Rb1 significantly stimulates the steady-state glucose transport rate by ~50%, whereas Rg1 inhibits the glucose transport by 70%.
It is not surprising that increasing doses in both Rb1 and Rg1 resulted in attenuation of metabolic actions in glucose response during OGTT, temperature, and citrate synthase activity. This is similar to a hormesis dose-response relationship reported previously using a collection of ginsenoside component, in which exercise-induced oxidative stress and decreased citrate synthase activity can be buffered by ginseng steroids only at low doses, and the observed response diminishes at higher doses. Hormesis refers to a biphasic dose-response to an intervention characterized by a stimulatory (or beneficial) action at low doses and an inhibitory (or toxic) action at high doses. A recent study has reported that ginsenoside Rg1 may exhibit a toxic effect to senescent cells. It remains unknown whether increasing the dosage of Rg1 could exert survival challenge to normal cells as well and therefore offset its benefit. Our data on Rg1 are consistent with a previous finding, in which no improvement in OGTT after oral Rg1 supplementation at extremely high dosage (50 mg/kg) was observed under normal dietary and high-fat diet conditions. In contrast to our studies, glucose-lowering effects of Rb1 on glucose tolerance under intraperitoneal injected condition at much higher doses have been reported., The discrepancy between the current study and previous reports suggests that delivering methods (injection and oral supplementation) and dosage of the ginsenosides could confound experimental observation in the area of ginseng research.
It remains unsettled whether decreased body temperature at low dosage is associated with decreased energy consumption due to improved energy efficiency by rapid clearance of aged mitochondria. It has been reported that American ginseng reduces body temperature, whereas Korean ginseng shows opposing action. This discrepancy may have been associated with different ginsenoside profiles of the ginseng. In the present study, a reduced body temperature with Rb1 or Rg1 supplementation under normal condition was observed under relatively low-dose condition. Similar result has also reported elsewhere under cytokine-injected condition. We found that this temperature-lowering effect diminished as doses of Rb1 or Rg1 increase. The attenuation of temperature-lowering effect at a high dosage may be due to increased energy expenditure. Rg1 has been shown as an activator of phagocytosis, which is expected to increase body temperature by accelerating cell turnoverin vivo within a short period and in high-fat diet-induced obese rats at high dose of 50 mg/kg.
The most important implication from the results of the study is that the dosage and ginsenoside profile of the P. ginseng can influence its metabolic efficacies in glucose tolerance, muscle glycogen, body temperature, and mitochondria enzyme activity. Furthermore, opposing properties between Rb1 and Rg1 from the same ginseng are not new. We must be aware that Rb1 and Rg1 components are changing with season and species type. According to our data, the observed hormesis dose-response suggests that the ginseng efficacy reports in the past are difficult to be compared unless dosage of Rb1 or Rg1 is standardized.
| Conclusion|| |
The contrasting effects of Rb1 and Rg1 at low dosage on glucose tolerance, as well as its peculiar dose-response relationship, suggest that the standardization of both ginsenoside components is essential to produce reliable ginseng-based nutraceuticals. Furthermore, both ginsenosides can significantly alter muscle glycogen metabolism and mitochondria enzyme activity in a hormesis dose-response fashion.
Financial support and sponsorship
This study was supported by grants from the Ministry of Science and Technology, Taiwan; Nuliv Sciences, CA, USA; and University of Taipei, Taipei, Taiwan.
Conflicts of interest
This work was funded to develop a supplement for Nuliv Science, Taiwan, and the USA.
| References|| |
Sievenpiper JL, Arnason JT, Leiter LA, Vuksan V. Decreasing, null and increasing effects of eight popular types of ginseng on acute postprandial glycemic indices in healthy humans: The role of ginsenosides. J Am Coll Nutr 2004;23:248-58.
Sievenpiper JL, Arnason JT, Leiter LA, Vuksan V. Variable effects of american ginseng: A batch of American ginseng (Panax quinquefolius L.) with a depressed ginsenoside profile does not affect postprandial glycemia. Eur J Clin Nutr 2003;57:243-8.
Shen L, Haas M, Wang DQ, May A, Lo CC, Obici S, et al.
Ginsenoside rb1 increases insulin sensitivity by activating AMP-activated protein kinase in male rats. Physiol Rep 2015;3. pii: e12543.
Xiong Y, Shen L, Liu KJ, Tso P, Xiong Y, Wang G, et al.
Antiobesity and antihyperglycemic effects of ginsenoside rb1 in rats. Diabetes 2010;59:2505-12.
Liu Q, Zhang FG, Zhang WS, Pan A, Yang YL, Liu JF, et al.
Ginsenoside rg1 inhibits glucagon-induced hepatic gluconeogenesis through akt-foxO1 interaction. Theranostics 2017;7:4001-12.
Chang TC, Huang SF, Yang TC, Chan FN, Lin HC, Chang WL. Effect of ginsenosides on glucose uptake in human Caco-2 cells is mediated through altered Na+
/glucose cotransporter 1 expression. J Agric Food Chem 2007;55:1993-8.
Lee HM, Lee OH, Kim KJ, Lee BY. Ginsenoside rg1 promotes glucose uptake through activated AMPK pathway in insulin-resistant muscle cells. Phytother Res 2012;26:1017-22.
Hsu MF, Yu SH, Korivi M, Jean WH, Lee SD, Huang CY, et al.
Hormetic property of ginseng steroids on anti-oxidant status against exercise challenge in rat skeletal muscle. Antioxidants (Basel) 2017;6. pii: E36.
Srere P. Citrate synthase. In: Methods in Enzymology. New York: Academic; 1969;13:3-5.
Hou CW, Lee SD, Kao CL, Cheng IS, Lin YN, Chuang SJ. Improved inflammatory balance of human skeletal muscle during exercise after supplementations of the ginseng-based steroid rg1. PLoS One 2015;10:e0116387.
Li SG, Yan MZ, Zhang D, Ye M, Deng JJ. Effects of ginsenoside rg1 on the senescence of vascular smooth muscle cells. Genet Mol Res 2016;15:gmr.15038409.
Park EY, Kim MH, Kim EH, Lee EK, Park IS, Yang DC, et al.
Efficacy comparison of korean ginseng and american ginseng on body temperature and metabolic parameters. Am J Chin Med 2014;42:173-87.
Kang M, Yoshimatsu H, Oohara A, Kurokawa M, Ogawa R, Sakata T. Ginsenoside rg1 modulates ingestive behavior and thermal response induced by interleukin-1 beta in rats. Physiol Behav 1995;57:393-6.
Fan ZH, Isobe K, Kiuchi K, Nakashima I. Enhancement of nitric oxide production from activated macrophages by a purified form of ginsenoside (Rg1). Am J Chin Med 1995;23:279-87.
Sengupta S, Toh SA, Sellers LA, Skepper JN, Koolwijk P, Leung HW, et al.
Modulating angiogenesis: The yin and the yang in ginseng. Circulation 2004;110:1219-25.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]