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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 44  |  Page : 545-549  

Hypoglycemic effects of a standardized extract of salvia miltiorrhiza roots in rats


1 Neuroscience Institute, National Research Council of Italy, Section of Cagliari, Monserrato (CA); Cagliari Pharmacological Research S.R.L., Cagliari (CA), Italy
2 Neuroscience Institute, National Research Council of Italy, Section of Cagliari, Monserrato (CA), Italy
3 Indena S.p.A, Milan (MI), Italy

Date of Web Publication31-Dec-2015

Correspondence Address:
Giancarlo Colombo
Neuroscience Institute, National Research Council of Italy, Section of Cagliari, Monserrato (CA)
Italy
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.172959

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   Abstract 

Background and Aims: Salvia miltiorrhiza Bunge (Labiatae) is a Chinese medicinal plant, the dried roots of which (known as Dan-Shen) have been used for hundreds of years in the treatment of a series of ailments, including hyperglycemia. This study was designed to evaluate the hypoglycemic effect of a new, standardized extract of S. miltiorrhiza. Materials and Methods: S. miltiorrhiza extract (containing 21% total tanshinones and 3.7% tanshinone IIA) was administered acutely and intragastrically at the doses of 0, 50, 100, and 200 mg/kg to male, healthy, fasted Wistar rats 60 min before the intragastric infusion of a bolus of starch (3 g/kg; a semi-naturalistic experimental condition) (Experiment 1) or glucose (2 g/kg) (Experiment 2). Results: In both experiments, treatment with S. miltiorrhiza extract produced a dose-related decrease in glycemia, evidenced in terms of reduction of peak value and/or area under the curve of the time-course of glycemia. The effect of S. miltiorrhiza extract occurred at doses devoid of any behavioral toxicity in rats. Conclusion: The results of this study suggest that the hypoglycemic effect of S. miltiorrhiza extract was likely secondary to an action on carbohydrate metabolism. These results are consistent with several preclinical and clinical data and add further support to the hypothesis that S. miltiorrhiza extracts may act as effective anti-hyperglycemic remedies.

Keywords: Rats, standardized extract of S. miltiorrhiza roots, starch- and glucose-induced rise in glycemia, type-2 diabetes


How to cite this article:
Carai MA, Colombo G, Loi B, Zaru A, Riva A, Cabri W, Morazzoni P. Hypoglycemic effects of a standardized extract of salvia miltiorrhiza roots in rats . Phcog Mag 2015;11, Suppl S4:545-9

How to cite this URL:
Carai MA, Colombo G, Loi B, Zaru A, Riva A, Cabri W, Morazzoni P. Hypoglycemic effects of a standardized extract of salvia miltiorrhiza roots in rats . Phcog Mag [serial online] 2015 [cited 2019 Nov 12];11, Suppl S4:545-9. Available from: http://www.phcog.com/text.asp?2015/11/44/545/172959

Summary

  • Acute treatment with Salvia miltiorrhiza extract reduced the glycemia rise induced by a bolus of starch in rats
  • Acute treatment with Salvia miltiorrhiza extract reduced the glycemia rise induced by a bolus of glucose in rats
  • This effect relates to both glycemia peak value and area under the curve of the time-course of glycemia.





   Introduction Top


Recent research and re-evaluation of knowledge deriving from traditional medicines suggest the potential effectiveness of herbal remedies in the treatment of diabetes and its complications; a series of herbal extracts have been described to decrease hyperglycemia and prevent diabetes-induced ailments in animal models, clinical surveys, and anecdotal reports. [1],[2]

Salvia miltiorrhiza Bunge (Labiatae) is a medicinal plant, the dried roots of which (known as Dan-Shen) are officially listed in the Chinese Pharmacopoeia; these roots have been used for hundreds of years in Chinese folk medicine in the treatment of a series of pathological conditions, including cardiovascular disease, hematological abnormalities, hyperlipidemia, hepatitis, hemorrhage, menstrual disorders, miscarriage, edema, and insomnia. [3],[4],[5],[6] More recently, the potential for the treatment of alcohol use disorders has been proposed. [7],[8] In addition, several preclinical and clinical Chinese studies have reported the efficacy of S. miltiorrhiza extracts in decreasing hyperglycemia as well as its traditional use for treating diabetes mellitus and its complications. [1],[2],[9]

This study was aimed at providing an additional contribution to the anti-diabetes potential of S. miltiorrhiza extracts; to this end, the present study evaluated the capacity of a new, standardized extract of S. miltiorrhiza roots to lower the rise in glycemia produced by administration of a bolus of starch (Experiment 1) and glucose (Experiment 2) in healthy, adult rats.


   Materials And Methods Top


The experimental procedures employed in the present study were in accordance with the Italian Law on "Protection of animals used for scientific reasons."

Animals

Healthy, adult male Wistar rats (Charles River Laboratories, Calco, Italy), weighing approximately 250 g at the start of each experiment, were used. In both experiments, starting from the age of 60 days, rats were individually housed in standard plastic cages with wood chip bedding. The animal facility was under an inverted 12:12 h light/dark cycle (lights on at 9:00 pm), at a constant temperature of 22 ± 2°C and relative humidity of approximately 60%. Standard rat chow (Mucedola, Settimo Milanese, Italy) and tap water were available ad libitum in the homecage, except as noted below. Rats were extensively habituated to handling and intragastric (i.g.) infusion. Independent groups of rats were used in each experiment. In both experiments, rats were food-deprived for 12 h before the test.

Extract preparation

S. miltiorrhiza roots were collected in 2005 in the Eastern part of China and identified using the genus treatment found in Flora of China [10] and Chinese Pharmacopoeia. [5] A voucher specimen is kept at Indena S.p.A., Milan, Italy, under registry no: 137127.

Roots of S. miltiorrhiza were dried and then extracted with acetone. After concentration under vacuum, the residue was re-dissolved in alcohol and the solution defatted with n-hexane. The aqueous layer was concentrated and extracted with ethyl acetate. The final extract (named IDN5655) was obtained after removal of ethyl acetate by alcohol and drying under vacuum at 50°C for approximately 48 h. The S. miltiorrhiza extract used in the present study contained 21% total tanshinones and 3.7% tanshinone IIA.

Experimental procedures

Experiment 1: Effect of Salvia miltiorrhiza extract on starch-induced rise in glycemia

On the test day (occurring approximately 15 days after the start of the single-housing period), rats were divided into 4 groups of n = 12, matched for body weight, and treated acutely with 0, 50, 100, and 200 mg/kg S. miltiorrhiza extract. S. miltiorrhiza extract (code: IDN5655) was dissolved in a 1:1 mixture of polysorbate 80 plus polyethylene glycol 600. The solution was kept under continuous agitation, at room temperature and in a dark container, for at least 12 consecutive h before administration. S. miltiorrhiza extract was administered i.g. (infusion volume: 4 mL/kg) 60 min before lights off. Doses of S. miltiorrhiza extract were chosen on the basis of preliminary data (this laboratory, unpublished results).

At lights off, rats were treated i.g. with 3 g/kg corn starch (Unilever, Milan, Italy; suspended in 4.5 mL/kg distilled water). Glycemia was determined 0, 30, 60, 120, and 240 min after starch administration. At each recording time, a small (0.05 mL) blood sample was collected from the tip of the tail of each rat and analyzed enzymatically by means of GL5 Analox (Analox Ltd, London, UK). Glycemia was expressed in mg/dl.

Experiment 2: Effect of Salvia miltiorrhiza extract on glucose-induced rise in glycemia

On the test day (occurring approximately 15 d after the start of the single-housing period), rats were divided into four groups of n = 7-8, matched for body weight, and treated acutely with 0, 50, 100, and 200 mg/kg S. miltiorrhiza extract. S. miltiorrhiza extract was dissolved as described above and administered i.g. (infusion volume: 4 mL/kg) 60 min before lights off.

At lights off, rats were treated i.g. with 2 g/kg glucose (ACEF, Fiorenzuola d'Arda, Italy; dissolved in 3.5 mL/kg distilled water). Glycemia was determined 0, 30, 60, and 90 min after glucose administration. At each recording time, a small (0.05 mL) blood sample was collected and analyzed as described above.

Statistical analysis

In both experiments, data on the effect of treatment with S. miltiorrhiza extract on glycemia over time were analyzed by 2-way (treatment; time) ANOVA with repeated measures on the factor "time," followed by the least significant difference (LSD) test for post hoc comparisons. Data on the effect of treatment with S. miltiorrhiza extract on the area under the curve of the time-course of glycemia were expressed as mg/dL and mg·min/dL, respectively, and analyzed by 1-way ANOVA, followed by the LSD test for post hoc comparisons.


   Results Top


Experiment 1: Effect of Salvia miltiorrhiza extract on starch-induced rise in glycemia

In vehicle-treated rats, glycemia peaked up to approximately 130 mg/dL at the 30- and 60-min recording times and then decreased progressively [Figure 1], top panel]. ANOVA revealed a significant effect of treatment with S. miltiorrhiza extract (F (3,44) =4.60, P < 0.01) and a highly significant effect of time (F (3,132) =115.47, P < 0.0001), but not a significant interaction (F (9,132) =1.35, P > 0.05), on glycemia over the 240-min recording period. Post hoc analysis revealed that glycemia was significantly reduced, compared to vehicle, by (a) 100 mg/kg S. miltiorrhiza extract at the 60-min recording time, and (b) 200 mg/kg S. miltiorrhiza extract at the 30-, 60-, and 120-min recording times [Figure 1], top panel].
Figure 1: Effect of the acute, intragastric administration of different doses of a standardized extract of Salvia miltiorrhiza on time-course of glycemia (top panel) and area under the curve of the time-course of glycemia (bottom panel) in Wistar rats treated intragastric with a bolus of starch (3 g/kg) at time 0. Each point or bar is the mean ± standard error mean of n = 12 rats. *P < 0.05, **P < 0.005, and ***P < 0.001 in comparison to vehicle-treated rat group (least significant difference test)

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Treatment with S. miltiorrhiza extract also reduced the area under the curve of the time-course of glycemia (F (3,44) =3.88, P < 0.05) [Figure 1], bottom panel]. In the rat group treated with 200 mg/kg S. miltiorrhiza extract, this reduction (in comparison to vehicle-treated rat group) averaged approximately 25% and achieved statistical significance at post hoc test [Figure 1], bottom panel].

Experiment 2: Effect of Salvia miltiorrhiza extract on glucose-induced rise in glycemia

In vehicle-treated rats, glycemia reached an average value of approximately 130 mg/dL at the 30- and 60-min recording times, and decreased markedly at the subsequent 90-min recording time [Figure 2], top panel]. ANOVA revealed a significant effect of treatment with S. miltiorrhiza extract (F (3,27) =4.67, P < 0.01), a highly significant effect of time (F (2,54) = 48.94, P < 0.0001), and a significant interaction (F (6,54) = 2.44, P < 0.05), on glycemia over the 90-min recording period. Post hoc analysis revealed that glycemia was significantly reduced, compared to vehicle, by (a) all three doses of S. miltiorrhiza extract at the 60-min recording time, and (b) 50 and 200 mg/kg S. miltiorrhiza extract at the 90-min recording time [Figure 2], top panel].
Figure 2: Effect of the acute, intragastric administration of different doses of a standardized extract of Salvia miltiorrhiza on time-course of glycemia (top panel) and area under the curve of the time-course of glycemia (bottom panel) in Wistar rats treated intragastric with a bolus of glucose (2 g/kg) at time 0. Each point or bar is the mean ± standard error mean of n = 7-8 rats. *P < 0.5, **P < 0.005, and ***P < 0.0005 in comparison to vehicle-treated rat group (least significant difference test)

Click here to view


Treatment with S. miltiorrhiza extract also reduced the area under the curve of the time-course of glycemia (F (3,27) =5.88, P < 0.005) [Figure 2], bottom panel]. Compared to vehicle-treated rat group, magnitude of the reducing effect of treatment with 50, 100, and 200 mg/kg S. miltiorrhiza extract on the area under the curve of the time-course of glycemia averaged approximately 30%, 30%, and 45%, respectively; this reducing effect reached statistical significance at post hoc analysis at each dose of S. miltiorrhiza extract [Figure 2], bottom panel].


   Discussion Top


The results of this study indicate that acute administration of a standardized extract of roots from the Chinese medicinal plant, S. miltiorrhiza, effectively reduced the rise in glycemia produced in healthy, fasted rats by administration of a bolus of starch (Experiment 1) or glucose (Experiment 2). This effect of S. miltiorrhiza extract (a) tended to be dose-related, as evidenced more clearly by the sets of data on the curve of the time-course of glycemia, and (b) occurred at doses known to be devoid of behavioral toxicity in rats. [7],[8]

Data from Experiment 1 demonstrate the hypoglycemic properties of S. miltiorrhiza extract in a semi-naturalistic experimental condition, represented by the infusion of a bolus of starch. However, these data may not fully clarify the issue of efficacy of S. miltiorrhiza extract on glucose metabolism, as the active ingredient(s) of the extract may have acted in the lumen of the small intestine, inhibiting hydrolysis of complex starches or oligosaccharides, thus resulting in poor absorption of monosaccharides and a modest rise in glycemia. However, data from Experiment 2 demonstrate that the hypoglycemic effect of S. miltiorrhiza extract was likely secondary to an action on carbohydrate metabolism, as administration of the extract inhibited the rise in glycemia induced by infusion of a bolus of pure glucose (the final product of dietary carbohydrate catabolism) to an extent comparable to that observed following infusion of the starch bolus (Experiment 1).

The results of the present study are in close agreement with recent lines of experimental evidence demonstrating that chronic treatment with (a) a polyphenolic fraction of S. miltiorrhiza decreased fasting glycemia in rats made diabetic by treatment with streptozotocin (experimental model of hyperglycemia and diabetes) [11] and (b) the active ingredient, tanshinone IIA, reduced the rise in glycemia produced in fasted mice by acute injection of a glucose bolus. [12] However, divergent data have also been collected, depicting the inability of S. miltiorrhiza extracts to affect glycemia in rats; [13],[14] the mode of extract preparation, content of active ingredient(s), and tested dose-ranges of S. miltiorrhiza extracts may be fundamental in justifying these discrepancies. Preclinical data showing the anti-hyperglycemic effects of S. miltiorrhiza extracts [11],[12],[ present study] are however consistent with findings reported elsewhere, in traditional Chinese medicine, on the hypoglycemic effects of S. miltiorrhiza extracts or preparations given to humans affected by type-2 diabetes. [1],[2],[9]

The present study did not specifically address the biochemical mechanism(s) of action and active ingredient(s) responsible for the hypoglycemic effect of the tested S. miltiorrhiza extract. However, data present in literature may be of help in advancing several hypotheses. S. miltiorrhiza extracts - as well as their active lipophilic constituents, tanshinone IIA, tanshinone I, cryptotanshinone, and 15,16-dihydrotanshinone (DHTH) - have been found to exert insulin-sensitizing effects, enhancing phosphorylation, and downstream signaling at the insulin receptor. [15] This potentiation of insulin activity is likely responsible for the observed increase in glucose uptake exerted by DHTH. [16] Additionally, DHTH has been found to inhibit gluconeogenesis. [16] Finally, a polyphenolic fraction from S. miltiorrhiza decreased blood insulin in diabetic rats. [11] Similar data were collected in mice after chronic treatment with tanshinone IIA. [12]

Notably, the beneficial effects of S. miltiorrhiza extracts apparently extend to the complications of diabetes; recent studies have indeed demonstrated the ability of S. miltiorrhiza extracts to decrease (a) urinary protein excretion (experimental model of diabetes-induced nephropathy) [13] and (b) oxidative stress in the eye and aorta (key event of pathogenesis of diabetes complications) [14] in streptozotocin-treated rats. When considering treatment for type-2 diabetes and associated and/or promoting disorders such as hyperlipidemia and overweight, it should be taken into account that S. miltiorrhiza extracts and tanshinone IIA have been reported to regulate lipid metabolism, (a) decreasing plasma levels of total cholesterol, low-density lipoprotein cholesterol, and triglycerides in rats and mice, [11],[12],[17] (b) increasing high-density lipoprotein cholesterol in rats and mice, [12],[17] and (c) reducing adipose mass and body weight in mice. [12]


   Conclusion Top


These results indicate an interesting and promising anti-diabetic profile for S. miltiorrhiza extracts, including the extract tested in this study. To this end, it may be of interest to note how several clinical trials in China have reported a good safety profile and small number of side effects when using products containing S. miltiorrhiza. [6]

Acknowledgments

The authors are grateful to Mrs. Carla Acciaro for animal breeding and care, and Ms. Anne Farmer for language editing of the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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Li WL, Zheng HC, Bukuru J, De Kimpe N. Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. J Ethnopharmacol 2004;92:1-21.  Back to cited text no. 1
    
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Xie W, Du L. Diabetes is an inflammatory disease: Evidence from traditional Chinese medicines. Diabetes Obes Metab 2011;13:289-301.  Back to cited text no. 2
    
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Tang W, Eisenbrand G, editors. Salvia miltiorrhiza Bge. In: Chinese Drugs of Plant Origin - Chemistry, Pharmacology and Use in Traditional and Modern Medicine. Berlin: Springer-Verlag; 1992. p. 891-902.  Back to cited text no. 3
    
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Huang KC, Tan S. Salvia miltiorrhiza. In: Huang KC, editor. The Pharmacology of Chinese Herbs. 2 nd ed. Boca Raton: CRC Press; 1993. p. 90-3.  Back to cited text no. 4
    
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State Pharmacopoeia Commission of the People's Republic of China. Pharmacopoeia of the People's Republic of China, English edition. Beijing: Chemical Industry Press; 2000.  Back to cited text no. 5
    
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Zhou L, Zuo Z, Chow MS. Danshen: An overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol 2005;45:1345-59.  Back to cited text no. 6
    
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Colombo G, Serra S, Vacca G, Orrù A, Maccioni P, Morazzoni P, et al. Identification of miltirone as active ingredient of Salvia miltiorrhiza responsible for the reducing effect of root extracts on alcohol intake in rats. Alcohol Clin Exp Res 2006;30:754-62.  Back to cited text no. 7
    
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Maccioni P, Vargiolu D, Falchi M, Morazzoni P, Riva A, Cabri W, et al. Reducing effect of the Chinese medicinal herb, Salvia miltiorrhiza, on alcohol self-administration in Sardinian alcohol-preferring rats. Alcohol 2014;48:587-93.  Back to cited text no. 8
    
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Huang M, Xie Y, Chen L, Chu K, Wu S, Lu J, et al. Antidiabetic effect of the total polyphenolic acids fraction from Salvia miltiorrhiza Bunge in diabetic rats. Phytother Res 2012;26:944-8.  Back to cited text no. 9
    
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Gong Z, Huang C, Sheng X, Zhang Y, Li Q, Wang MW, et al. The role of tanshinone IIA in the treatment of obesity through peroxisome proliferator-activated receptor gamma antagonism. Endocrinology 2009;150:104-13.  Back to cited text no. 10
    
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Lee SH, Kim YS, Lee SJ, Lee BC. The protective effect of Salvia miltiorrhiza in an animal model of early experimentally induced diabetic nephropathy. J Ethnopharmacol 2011;137:1409-14.  Back to cited text no. 11
    
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Yue KK, Lee KW, Chan KK, Leung KS, Leung AW, Cheng CH. Danshen prevents the occurrence of oxidative stress in the eye and aorta of diabetic rats without affecting the hyperglycemic state. J Ethnopharmacol 2006;106:136-41.  Back to cited text no. 12
    
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Jung SH, Seol HJ, Jeon SJ, Son KH, Lee JR. Insulin-sensitizing activities of tanshinones, diterpene compounds of the root of Salvia miltiorrhiza Bunge. Phytomedicine 2009;16:327-35.  Back to cited text no. 13
    
14.
Fan Z. A survey of treatment of diabetic complications with Chinese drugs. J Tradit Chin Med 2005;25:153-9.  Back to cited text no. 14
    
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Xi-wen L, Hedge IC. Lamiaceae (Labiatae). In: Zheng-yi W, Raven PH, editors. Flora of China. St. Louis: Science Press and Missouri Botanical Garden; 1994. p. 195-222.  Back to cited text no. 15
    
16.
Liu Q, Zhang Y, Lin Z, Shen H, Chen L, Hu L, et al. Danshen extract 15,16-dihydrotanshinone I functions as a potential modulator against metabolic syndrome through multi-target pathways. J Steroid Biochem Mol Biol 2010;120:155-63.  Back to cited text no. 16
    
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Ji W, Gong BQ. Hypolipidemic activity and mechanism of purified herbal extract of Salvia miltiorrhiza in hyperlipidemic rats. J Ethnopharmacol 2008;119:291-8.  Back to cited text no. 17
    

 
   Authors Top


Mauro A.M. Carai, is CEO of Cagliari Pharmacological Research, Cagliari, Italy. He is a pharmacologist with primary research interest on natural remedies for eating disorders, obesity, diabetes, metabolic syndrome, and alcohol dependence.








Giancarlo Colombo, is a researcher at the CNR Neuroscience Institute, Section of Cagliari, Italy. He is a behavioral pharmacologist with research interest on natural remedies for eating disorders, obesity, diabetes, metabolic syndrome, and alcohol dependence.








Barbara Loi, is currently a PhD student at the University of Hertfordshire, UK. During her research training at the CNR Neuroscience Institute, Section of Cagliari, Italy, she was involved in studies on the control of alcohol and food intake and glycemia by medicinal plants.








Alessandro Zaru, is currently a PhD student at the University of Cagliari, Italy. During his research training at the CNR Neuroscience Institute, Section of Cagliari, Italy, he was involved in studies on the control of alcohol and food intake and glycemia by medicinal plants.








Antonella Riva, is a Senior Research Scientist deeply involved in the biological and clinical characterizations of botanicals. She is the Head of Preclinical and Clinical Development at Indena S.p.A., Milan, Italy.








Walter Cabri, is former R&D Director in Indena S.p.A., Milan Italy. The actual position is VP Innovation and Development in Fresenius-kabi, Italy/ India. His expertise are in all areas of preclinical development in the pharma and nutraceutical business. He has published more than 180 publications and patents.








Paolo Morazzoni, is Scientific Director, Indena S.p.A., Milan, Italy. He is involved in Research and Development of new active botanical products for pharmaceutical and nutraceuticals fields.


    Figures

  [Figure 1], [Figure 2]


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