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Effect of Sanguis draxonis (a Chinese traditional herb) on the formation of insulin resistance in rats
Diabetes Research and Clinical Practice 68 (2005) 3–11 www.elsevier.com/locate/diabres
Effect of Sanguis draxonis (a Chinese traditional herb) on the formation of insulin resistance in rats Zhenqing Hou, Zhenxi Zhang*, Hong Wu Institute of Biomedical Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China Received 12 June 2003; received in revised form 8 January 2004; accepted 2 August 2004 Available online 12 October 2004
Abstract Sanguis draxonis (SD) is a Chinese traditional herb that is prescribed for the handling of diabetic disorders. In this study, the effects of an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively, on the formation of insulin resistance were investigated in vivo in two models of insulin-resistant rats, HFD rats (high-fat diet-induced insulinresistant rats) and IILI rats (induced by the intraperitoneal injections of long-acting insulin at dosage of 0.5 U kg1 three times daily). The insulin resistance was indicated using the loss of tolbutamide-induced hypoglycemic activity. After the oral administration of SD (300 and 500 mg kg1 once a day for 7 days) to HFD rats, both plasma glucose and insulin concentration were decreased significantly, while the hypoglycemic activity of tolbutamide (10 mg kg1, i.p.) was significantly enhanced as compared with that of the vehicle-treatment (0.9% saline solution used as vehicle to disperse SD, w/v). Moreover, the formation of insulin resistance in IILI rats had been improved significantly with SD treatment (100, 300, 500 mg kg1 once a day for 14 days), but the influence of SD treatment on both plasma glucose and insulin concentration was not observed. For STZ-induced diabetic rats, the action of SD (300 and 500 mg kg1 once a day for 14 days) showed more effective on an increase of response to the exogenous short-acting porcine insulin than that of the metformin administrated orally at dosage of 320 mg kg1 three times daily. The present studies suggest that an oral administration of SD can increase insulin sensitivity and improve the development of insulin resistance in rats. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Insulin resistance; Sensitivity; Diabetic rats; Sanguis draxonis; Chinese herb
1. Introduction Before the advent of insulin and other synthesis medicine for diabetes, the herbal medication was the * Corresponding author. Tel.: +86 29 82668774; fax: +86 29 82668664. E-mail address: [email protected] (Z. Zhang).
mainstay in anti-diabetic therapies [1]. Nowadays, insulin and other synthesis medicine take the place of herbal medication gradually in foreign countries due to their remarkable therapeutic effects. However, the complications in macrovascular, retinal and neuropathic functions are still associated in the patients receiving insulin injection or the synthesis medicine [2]. Also, the insulin resistance is another serious problem,
0168-8227/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2004.08.011
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which is a common consequence of overweight and a cause of impaired glucose tolerance in type II diabetes [3]. In clinical practice, the insulin resistance was usually observed in association with hypertension [4], impaired glucose tolerance, hyperinsulinaemia [5], and oxidant stress [6]. Treatment of diabetes with insulin and oral drugs failed to prevent these complications in many patients, therefore the use of alternative medicine, especially the consumption of botanicals has been increasing, mostly because of the supposedly less frequent side effects and multiple effects when compared with modern western medicine [7,8]. Sanguis draxonis (SD) is a traditional Chinese herb also called Dragon’s Blood or Resina draconis. Its commercial product is known as Longxuejie1 capsule, which is processed from the resinous extract of Dracaena cochinchinensis (Lour.) S.C. Chen (Agavaceae) in China. It comprises more than 12 kinds of methanol soluble compounds [9] identified as [(1) 26-O-b-D-glucopyranosyl-furostan-5,25 (27)diene-1b,3b,22b,26-tetrahydroxy-1-O-a-L- arabinpyranoside; (2) 3,4-dihydroxy-allylbenzene-4-O-b-Dglucopyranoside; (3) 7-hydroxy-3-(r-hydroxyphenyl)-chroman; (4) 7,40 -dihydroxy-30 -methoxyflavan; (5) 3,4-dihydroxy-yallylbenzene; (6) resveratrol; (7) 7,4-dihy-droxy-flavanone; (8) di-(r-hydroxyphenyl)methane; (9) acanthoside B; (10) r-hydroxybenzoic acid; (11) hydroquinone and (12) protocatechualdehyde]. In the past 1500 years, SD was widely used in China as a famous drug for promoting blood circulation. It was generally received and recognized by herbal practitioners and passed on from generation to generation due to its miracle effects in the treatment of blood disorders [10,11]. A recent study indicated that SD had no obvious effects in decreasing the plasma sugar level in normal fasted rats but could decrease the plasma sugar level and improve the glucose-resistance in glucoseinduced or adrenaline-induced hyperglycemic rats and alloxan-induced fasted diabetic rats, and also increased insulin secretion in diabetic rats. The study also reported SD could also decrease the plasma lipid level in alloxan-induced diabetic rats [12]. These findings raised us a question whether SD could improve the insulin resistance and increase insulin sensitivity in rats. In order to develop agents without side effect for good handling of diabetic, SD was investigated in present study.
2. Materials and methods Longxuejie1 capsule, a purely natural botanic drug containing SD powder, was from Kunming arboretum pharmaceutical plant (Yunnan, China). STZ, tolbutamide and metformin were obtained from Sigma (St. Louis, U.S.A.). The short-acting porcine insulin was from Xuzhou biochemical plant (Xuzhou. China). The long-acting human insulin (Protamine zinc insulin) was supplied by Shanghai biochemical plant (Shanghai, China). 2.1. Animals Wistar male rats weighing 230–280 g, 12–13 weeks old were provided by the animal service of experimental center of Fourth Military Medical University of China. The STZ-induced diabetic rats, used as the insulin-dependent diabetes mellitus model (IDDM), were prepared by an injection of streptozotocin (65 mg kg1 i.v.) in a citrate buffer at pH 4.5. The blood glucose levels of diabetic rats were in the range of 14.6–17.4 mmol 11, 2 weeks after streptozotocin treatment. One model of insulin-resistance male Wistar rats (HFD rats), used as no insulindependent diabetes mellitus model (NIDDM), was induced after 16 week of a high-fat diet (see Table 1). At that time, a well established and permanent insulin resistance animal model with hyperglycemia, hyperinsulinaemia and hypertriglyceridemia, was present. Table 1 Composition of experimental diets Ingredient Casein Corn starch Soybean oil Beef tallow Mineral mixa Vitamin mixa Cellulose Choline bitartarate L-Methionine Fat Carbohydrate Protein
Control diet 1 (g/100 g diet) 17.0 62.5 8.0 – 3.5 1.0 7.5 0.2 0.3 (% of calories) 18.5 64 17.5
Fat-rich diet 22.9 41.4 1.87 18.0 9.2 0.92 5.0 0.39 0.35 45 35 20
a Provided by the animal service of experiment center of Fourth Military Medical University.
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Another model of insulin-resistance rats (IILI rats) was induced in male Wistar by intraperitoneal (i.p.) injections of long-acting human insulin (0.5 U kg1 three times daily for 14 days) [13]. All animals were housed in individual cages in a room controlled for temperature (23 2) 8C, humidity (45 5)% and light (08:00–20:00 h), and were maintained on a laboratory diet (see Table 1) except HFD rats, the food intake and body weight of all rats were measured everyday and water ad libitum. 2.2. Manufacture and standardization of SD formulations SD powder inside of Longxuejie1 capsules was made from the Chinese Dragon’s Blood powder, resin of Dracaena cochinchinensis which is a tropical medicinal plant, by means of following purifying step: an amount of crude SD dissolved in a certain volume of ethanol and filtered to remove the insoluble substance, SD ethanol solution was rotoevaporated under vacuum to get rid of ethanol. Finally, the refined SD was pulverized and sieved using a nest of standard sieve (200 mm). The size of less than 200 mm of SD powder was selected for Longxuejie1 capsules. Each capsule contains 300 mg SD presented in the form of brown–red powder with slight flavour and mild taste. The effective and active ingredients of Longxuejie1 capsules provided by the drug usage direction include five aromatic compounds (also called phytoalexins) and a steroid saponin with the following chemical names and structural formulas: ethyl-p-hydroxy benzoate; 7,40 -dihydroxyflavan; 7-hydroxy-40 -methoxyflavan; 7,40 -dihydroxyflavone; loureirin A; neoruscogenin-1-O-a-L-rhamnopyranosyl- (l-2)-O-a-L-arabinopyranoside. Different standard amounts (2, 6 and 10 g) of SD powder inside of Longxuejie1 capsules were suspended, respectively, in 100 ml of 0.9% (w/v) saline solution with 2 min of ultrasonification for uniformly dispersion. The final suspensions contained 20, 60 and 100 mg ml1 of SD, respectively. Five milliliter of three different concentrations of SD formulations, representing 100, 300 and 500 mg, respectively, were given to rats per kilogram by a stomach tube and the same weight-adjusted volume of vehicle was given to rats to make sure the whole amount of SD was delivered into stomach. The same weight-adjusted
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volume of vehicle (0.9% saline solution, w/v) was given to rats as control in each experiment. In addition, the suspension of SD must be agitated to make sure the suspensions remain evenly distributed during the period of dosing to the animals. 2.3. Effect of SD on insulin resistance in IILI rats Four groups of eight Wistar rats each were used to receive an i.p. injection of long-action human insulin to induce insulin-resistance. At the same time, three groups of rats received an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively. The fourth group of rats received the same weight-adjusted volume of vehicle. Blood samples from the femoral vein were drawn before and after the treatment. After treatment as above for 14 days. the development of insulin resistance was identified using the loss of tolbutamide-induced hypoglycemic activity according to the previous report [7,14]. In brief, these Wistar rats received an i.p. injection of 10 mg kg1 tolbutamide at 5 h later of the treatment with SD or vehicle. Effects on both plasma glucose and insulin concentration were determined using blood samples collected from femoral vein of rats at 1 h after tolbutamide injection. Results, the hypoglycemic activity of tolbutamide, were calculated as the percentage decrease of the initial value according to the following formula: ðCi Ct Þ 100% Ci Where Ci is the initial plasma glucose concentration (the blood sample was taken before treatment during the fasting state) and Ct is the plasma glucose concentration after treatment with tolbutamide or another agent. 2.4. Effect of SD on insulin resistance in HFD rats Three groups of eight HFD rats each were used to receive an oral administration of SD at dosages of 100, 300 and 500 mg kg1 once a day, respectively. One group of eight HFD rats received the same weightadjusted volume of vehicle. After treatment for 7 days, blood samples from the femoral vein were drawn for the measurement of plasma glucose and insulin concentration, the insulin resistance was indicated
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by the hypoglycemic response to tolbutamide (10 mg kg1, i.p.) as described above.
samples were immediately separated by centrifugation at 3000 rpm for 20 min at 4 8C, and the plasma was stored at 70 8C until analysis.
2.5. Effect of SD on insulin sensitivity in STZ-induced diabetic rats 2.7. Statistical analysis Five groups of eight STZ-induced diabetic rats each were used to investigate the response to the exogenous insulin. In the beginning of experiment, each group of STZ-induced diabetic rats received an injection of long-acting human insulin (1 U kg1 once a day) to normalize the insulin sensitivity [7]. Three days later, three groups of STZ-induced diabetic rats received an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively. The fourth group of rats received the same weight-adjusted volume of vehicle as control. The last group of rats received an oral administration of metformin (320 mg kg1 thrice a day) [7,15] as positive control. After 14 days of treatment, blood samples from the femoral vein were drawn for the measurement of both plasma glucose and insulin concentration. After that, all rats were prepared to challenge with the exogenous insulin. The intravenous insulin challenge tests were carried out by giving the short-acting porcine insulin (0.1, 0.5, 1.0, 1.5, and 2.0 U kg1) into these rats, and each animal was given all the doses of insulin one after another at 2-hourly intervals. Blood samples were drawn at 30 min following the intravenous insulin challenge test for the measurement of the plasma glucose concentration. The difference in response to the exogenous insulin was compared among the five groups of animals. 2.6. Determination of plasma glucose and insulin concentration Plasma glucose concentration was determined by the glucose-oxidase method (Glucose GOD-PAD kit, Beijing Ruikang Biochemical Reagent Industry, Beijing, China). Plasma insulin was assayed with a radioimmunological assay kit (Insulin RIA kit, Northern biotechnology institute, Beijing, China). The values obtained were immunoreactive insulin including insulin and proinsulin. The sample from an individual was analysed in triplicate at the same time. Intraassay and interassay coefficients of variation for insulin RIA were 7.6 and 8.9%, respectively. Blood
All data were expressed as mean S.D. for the number (n) of animals in the group. Repeated measures analysis of variation (ANOVA) was used to analyze the changes in plasma glucose and other parameters. The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. The obtained p value of 0.05 or less was considered statistically significant.
3. Results 3.1. Effect of SD on IILI rats As shown in Table 2, the initial plasma glucose concentration of control rats was 5.4 0.3 mmol l1 (n = 8), after an injection of long-acting human insulin at 0.5 U kg1 into peritoneal cavity of rats was repeated three times daily for 14 days, the significant increase (p < 0.05) of plasma glucose concentration (7.9 0.4 mmol l1, n = 8) was observed. However, both plasma glucose and insulin concentration in IILI rats receiving SD treatment in combination during the induction of insulin resistance showed no significant variation (p > 0.05) compared with their initial value, but it was significantly different (p < 0.01) from the values of vehicle-treated animals. In addition, after the induction of insulin-resistance for 14 days, the hypoglycemic activity of tolbutamide (10 mg kg1, i.p.) in IILI rats with SD treatment in combination was much higher (p < 0.01) than those with the vehicle-treatment. And, at 1 h after the tolbutamide treatment, the concentration of plasma insulin in IILI rats (including the control animals) was significantly higher (p < 0.01) than the value of the tolbutamide treatment before. But the pattern of plasma insulin increase among four groups of rats showed no significant difference (data not shown). Furthermore, the food intake and changes of body weight of SD treated-rats were not significantly different from that of the control group of animals.
Food intake and change of body weight was also included. Before: before SD or insulin treatment. After: after 14 days of SD or insulin treatment. 0.9% (w/v) of saline solution was used as vehicles to suspend powder. The hypoglycemic response to tolbutamide was defined using the loss of tolbutamide (10 mg kg1 i.p.)-induced hypoglycemic activity and calculated as the percentage decrease of the initial blood glucose concentration, n = 8, Mean S.D. a p < 0.01 vs. control (the same weight-adjusted volume of vehicle). ** p < 0.01 vs. the initial value.
11 2 93 82 10 4
Gain After
286 7 277 8 278 6 283 9 275 6 268 4 270 7 273 5
Before
3.1 2.3 14.5 2.9a 2l.4 3.3a 24.4 3.8a 25.6 3.6 26.4 5.4 24.8 4.3 27.2 3.9
43 2 45 3 47 3 49 2 After Before
96.3 5.6 99.2 4.1 92.4 5.4 96.8 4.8 7.9 0.4** 6.2 0.5a 5.7 0.3a 5.1 0.2a
After Before After Before
0.5 100 300 500 Insulin Insulin + SD
5.4 0.3 5.4 0.2 5.9 0.4 5.6 0.3
120.6 6.2** 101.3 6.8a 99.2 4.9a 104.2 5.7a
Body weight (g) Food intake (g/day) (control diet) Hypoglycemic activity of tolbutamide (%) Plasma insulin (pmol l1) glucose Blood (mmol l1) Dosage Wister rats
Table 2 Changes of the plasma glucose and insulin concentration and the hypoglycemic response to tolbutamide (10 mg kg1 i.p.) in Wister rats that received an r.p. injection of long-acting human insulin at dosage of 0.5 U kg1 three times a day in combination with an oral administration of Sanguis draxonis (SD) at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively
Z. Hou et al. / Diabetes Research and Clinical Practice 68 (2005) 3–11
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3.2. Effect of SD on insulin resistance in HFD rats As shown in Table 3, after 7 days of SD treatment, both plasma glucose and insulin concentration were not significantly but slightly decreased in the group of HFD rats receiving 100 mg kg1 of SD treatment. However, for the groups of HFD rats with SD treatment at dosages of 300 and 500 mg kg1, both plasma glucose and insulin concentration were significantly decreased compared with the value of vehicle-treatment (p < 0.01). Furthermore, the hypoglycemic activity of tolbutamide in HFD rats treated with SD at dosages of 300 and 500 mg kg1 showed significantly higher than those with the vehicle treatment. In addition, no difference on the amount of food intake and changes in body weight was observed between HFD rats with SD treatment and those with the vehicle-treatment. And the changing pattern of plasma insulin concentration in HFD rats before and after tolbutamide treatment showed no significant difference (data not shown). 3.3. Effect of SD on the insulin sensitivity in STZ-induced diabetic rats According to the results shown in Table 4, both plasma glucose and insulin concentration of STZinduced diabetic rats receiving SD at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively, were not significantly different from the values of vehicle-treated rats. However, the hypoglycemic activity of short-acting porcine insulin at dosage of 0.1–2.0 U kg1 in these STZ-induced diabetic rats with SD treatment once a day for 14 days were higher than those with the vehicle treatment (see Fig. 1). The hypoglycemic activity of exogenous insulin was more prominent (p < 0.01 versus control) at dosage of insulin up to 1.0 U kg1 for rats receiving SD at dosage of 300 mg kg1, or at dosage of insulin up to 0.5 U kg1 for rats receiving SD at dosage of 500 mg kg1 (p < 0.05 versus control). For the group of rats receiving SD at dosage of 100 mg kg1, the result of exogenous insulin was not significantly different from the value of control. In addition, the food intake and the body weight of STZ rats were not greatly influenced by SD treatment compared with the control (data no shown).
52 61 52 73
Food intake and change of body weight was also included. Before: before SD treatment. After: after 7 days of SD treatment. 0.9% (w/v) of saline solution was used as vehicles to suspend powder. The hypoglycemic response to tolbutamide was defined using the loss of tolbutamide (10 mg kg1 i.p.)-induced hypoglycemic activity and calculated as the percentage decrease of the initial blood glucose concentration according to the text, n = 8, Mean S.D. a p < 0.05. b p < 0.01 vs. control (the same weight-adjusted volume of vehicle). * p < 0.05. ** p < 0.01 vs. the initial value.
After
339 6 348 7 341 6 346 8 334 8 342 7 336 6 339 9
Before
46 4 43 6 46 4 47 5 10.8 3.3 14.1 2.9 19.4 3.3b** 21.9 3.8b**
After Before
11.6 3.6 12.4 3.4 12.8 2.3 13.2 3.9 130.2 7.9 117.1 8.4a* 101.6 7.2b** 96.9 7.6b** 8.9 0.4 7.5 0.5 5.7 0.3b* 5.1 0.2b** 8.4 0.3 8.2 0.5 7.9 0.4 8.6 0.3 – 100 300 500 Control SD
After Before After Before
Dosage (mg kg1) HFD rats
137.6 8.6 135.8 7.7 136.4 8.2 138.7 7.9
Plasma insulin (pmol l1) Blood glucose (mmol l1)
Hypoglycemic activity of tolbutamide (%)
Food intake (g/day) (control diet)
Body weight (g)
Gain
Z. Hou et al. / Diabetes Research and Clinical Practice 68 (2005) 3–11 Table 3 Changes of the plasma glucose and insulin concentration and the hypoglycemic response to tolbutamide (10 mg kg1 i.p) in high-fat diet (HFD) rats with Sanguis draxonis (SD) treatment for 7 days
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After the metformin was administrated orally at dosage of 320 mg kg1 three times daily for 14 days, the hypoglycemic activity of short-acting insulin in STZ-diabetic rats was similar to the vehicle-treated group rats at dosages of insulin less than 1.0 U kg1 (Fig. 1), but the hypoglycemic activity of exogenous insulin was significantly prominent at dosage of insulin up to 1.5 U kg1 (p < 0.05).
4. Discussion The insulin-resistant animal model induced by repeatedly injection of long action human insulin was used to identify the influence of SD on the formation of insulin resistance. It had been documented that tolbutamide-induced hypoglycemia occurs through the stimulation of endogenous insulin release [14], so, the loss of tolbutamide-induced hypoglycemic activity can be interpreted as the development of insulin resistance. In normal Walter rats, before the inducement of insulin resistance, the hypoglycemic activity induced by tolbutamide at 10 mg kg1 (i.p.) was about 25.6 3.6%, but it was only 3.1 3.2% in IILI rats that received three times daily injection of long-acting insulin for 14 days. This denoted that an insulin resistance had been developed in these rats. However, the hypoglycemic activities of tolbutamide were still high and no significant increase of both plasma glucose and insulin concentration was obtained in rats with SD treatment in combination during the induction of insulin-resistance. These results suggest that the development of insulin resistance can be improved in IILI rats with SD treatment. Since overconsumption of dietary fat is a major contribution factor to human obesity and obesitylinked NIDDM. HFD rats were used as another NIDDM animal model to verify the effect of SD on the formation of insulin resistance. After 7 days of treatment with SD, both blood glucose and insulin concentration in HFD rats were decreased significantly compared with control. On the other hand, the hypoglycemic activity of tolbutamide was also significantly higher in HFD rats with SD treatment than those with the vehicle treatment. Thus, the improvement of insulin resistance in HFD rats with SD treatment can be considered.
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Table 4 Plasma glucose and insulin concentration in STZ-induced rats receiving Sanguis draxonis (SD) and Metformin (positive control) treatment for 14 days
STZ-induced rats Vehicle (control) SD (mg kg1 oid)
Metformin (mg kg1 tid)
Dosage
Plasma glucose (mmol l1)
Plasma insulin (pmol l1)
– 100 300 500 320
16.9 2.9 15.5 3.6 14.7 2.8 13.9 3.2 14.4 3.8
45.8 4.2 47.2 3.9 49.4 4.6 52.1 4.2 50.8 4.7
oid: one time daily, tid: three times daily. Value represent means S.D. n = 8. Control (the same weight-adjusted volume of vehicle, 0.9% (w/v) of saline solution).
The pattern of plasma insulin increase before and after tolbutamide treatment among the four groups of IILI rats or HFD rats showed no significant difference, which suggests that the stimulation of endogenous insulin release in rats with boltutamide treatment was not influenced significantly by different dosages of SD treatment and inducement of insulin-resistance with IILI or HFD. So, the high hypoglycemic activity of tolbutamide in both IILI rats and HFD rats with SD treatment was attributed not to the increase of plasma insulin concentration, but to the increase of insulin sensitivity. To clarify the increase of insulin sensitivity with SD treatment, STZ-diabetic rats were used to investigate the response to exogenous insulin.
The first method developed to evaluate insulin sensitivity in vivo was the intravenous insulin challenge test, which was based on the change of plasma glucose level after an injection of regular insulin [16]. Then, the intravenous insulin challenge test was performed in STZ-diabetic rats. The advantage of using STZ-diabetic rats in this study was the negligible endogenous insulin. The hypoglycemic action was directly due to the activity of exogenous insulin in STZ-diabetic rats. The obtained result can be used to indicate insulin sensitivity. It was found in this study that SD at dosages of 100, 300, and 500 mg kg1 once a day for 14 days enhanced the hypoglycemic action of exogenous insulin in STZ-
Fig. 1. Hypoglycemic response to exogenous short-acting porcine insulin in STZ-induced diabetic rats after receiving an oral administration of Sanguis draxonis (SD) at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively. STZ-induced diabetic rats receiving an oral administration of metformin (320 mg kg1 thrice a day) was used as positive control. STZ-induced diabetic rats receiving the same weightadjusted volume of vehicle (0.9% saline solution, w/v) were taken as control, n = 8. Mean S.D. a: p < 0.05, b: p < 0.01 vs. control.
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diabetic rats, and the effective dose of SD should be more than 300 mg kg1 in STZ-diabetic rats. Results also indicated that both plasma glucose and insulin concentration showed no significant variation before and after SD treatment in STZ-diabetic rats, which was in good agreement with application direction of SD drug (Longxuejie1 capsules) suitable for the type II diabetic patients. But it was not consistent with previous report [12] that SD could decrease the plasma sugar level in alloxan-induced fasted diabetic-rats and increase insulin secretion in alloxan-induced diabetic rats significantly. This discrepancy may be attributed in part to the differences between the different druginduced diabetic animal models or other unknown reasons. In addition, the effect of metformin was used as positive control in the present study. Metformin had been shown to improve the insulin sensitivity in NIDDM subjects by activating post-receptor insulin signaling pathways [17]. And an increase of the response to exogenous insulin was also observed in STZ-diabetic rats that received metformin at an effective dose as described previously [18]. In this study, the action of SD at dosages of 300 and 500 mg kg1 was more effective than that of the metformin at dosage of 320 mg kg1 three times daily. Because SD failed to modify the plasma insulin significantly in STZ-diabetic rats in our study, the increase of exogenous insulin action with SD treatment seems to be associated with an improvement of insulin sensitivity. Nevertheless, since the increase of secretion of endogenous insulin with SD treatment could not be completely ruled out [12], enhancement of insulin action brought by SD treatment might produce a synergistic effect on the hypoglycemic action of insulin in STZ-diabetic rats. Resistance to the action of insulin can result from a variety of causes, including defects in the receptor binding and at the post-receptor level [19,20]. The mechanism of SD to improve the insulin resistance in rats might be complex and probably multifactorial due to its mixture of many kinds of compounds. It is now recognized that SD also has beneficial effects on platelet aggregation, thrombus formation and myocardial ischemia [21]. Moreover, SD was found to possess strong antioxidant activity [22]. Although the chemical constituents and other pharmacological properties of SD have been analyzed [9,10] and the effective and active ingredients of Longxuejie1
capsules are also identified, the active ingredients for the improvement of insulin resistance and insulin sensitivity are still unclear. Otherwise, drug synergistic action is still the primary issue for Chinese traditional herbs. Therefore, it is possible that our reported effects about SD could result from many kinds of ingredients among SD components. Further studies are required to clarify the detail molecular mechanisms associated to these SD effects. And Lots of work remains to be achieved before Longxuejie1 capsules can be seen as an effective drug for the improvement of insulin resistance in clinical practice. In conclusion, the most expressing findings of this study were that an oral administration of SD can (1) improve the formation of insulin resistance in HFD rats (2) improve the development of insulin resistance in IILI rats, and (3) increase exogenous insulin sensitivity in STZ-induced diabetic rats. From these findings, SD is expected to be useful both as monotherapy in type II diabetic patients and in combination with insulin in type I diabetic patients.
Acknowledgement This work was supported by the National Nature Science Foundation of China (60178034) and the Doctorate Foundation of Xi’an Jiaotong University (DFXJTU 2001-2).
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[15] J. Rouru, R. Huupponen, U. Pesonen, M. Koulu, Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats, Life Sci. 50 (1992) 1813–1820. [16] H.N. Ginsberg, Investigation of insulin sensitivity in treated subjects with ketosis-prone diabetes mellitus, Diabetes 26 (1977) 278–283. [17] C.J. Bailey, Metformin-an update, Gen. Pharmacol. 24 (1993) 1299–1309. [18] G. Kanigur-Sultuybek, M. Guven, I. Onaran, V. Genani, H. Hatemi, The effect of metformin on insulin receptors and lipid peroxidation in alloxan and streptozotocin induced diabetes, J. Basic Clin. Physiol. Pharmacol. 6 (1995) 271–280. [19] B.A. Nelson, K.A. Robinson, M.G. Buse, High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes, Diabetes 49 (2000) 981–991. [20] A. Dale, P. Odile, K. Jason, et al. Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver, Nature 409 (2001) 729–733. [21] J.J. Ma, Y. Song, M. Jia, C.L. Li, Effect of total flavone in Sanguis Draconis on platelet aggregation, thrombus formation and myocardial ischemia, Chin. Tradit. Herbal Drugs 33 (2002) 1008–1010 (in Chinese). [22] X.C. Weng, G.P. Ren, S. Duan, et al. Screening of natural antioxidants from Chinese medicines, herbs and spices, Journal of the Chinese Cereals and Oils Association 13 (1998) 46– 48 (in Chinese).
Effect of Sanguis draxonis (a Chinese traditional herb) on the formation of insulin resistance in rats Zhenqing Hou, Zhenxi Zhang*, Hong Wu Institute of Biomedical Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China Received 12 June 2003; received in revised form 8 January 2004; accepted 2 August 2004 Available online 12 October 2004
Abstract Sanguis draxonis (SD) is a Chinese traditional herb that is prescribed for the handling of diabetic disorders. In this study, the effects of an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively, on the formation of insulin resistance were investigated in vivo in two models of insulin-resistant rats, HFD rats (high-fat diet-induced insulinresistant rats) and IILI rats (induced by the intraperitoneal injections of long-acting insulin at dosage of 0.5 U kg1 three times daily). The insulin resistance was indicated using the loss of tolbutamide-induced hypoglycemic activity. After the oral administration of SD (300 and 500 mg kg1 once a day for 7 days) to HFD rats, both plasma glucose and insulin concentration were decreased significantly, while the hypoglycemic activity of tolbutamide (10 mg kg1, i.p.) was significantly enhanced as compared with that of the vehicle-treatment (0.9% saline solution used as vehicle to disperse SD, w/v). Moreover, the formation of insulin resistance in IILI rats had been improved significantly with SD treatment (100, 300, 500 mg kg1 once a day for 14 days), but the influence of SD treatment on both plasma glucose and insulin concentration was not observed. For STZ-induced diabetic rats, the action of SD (300 and 500 mg kg1 once a day for 14 days) showed more effective on an increase of response to the exogenous short-acting porcine insulin than that of the metformin administrated orally at dosage of 320 mg kg1 three times daily. The present studies suggest that an oral administration of SD can increase insulin sensitivity and improve the development of insulin resistance in rats. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Insulin resistance; Sensitivity; Diabetic rats; Sanguis draxonis; Chinese herb
1. Introduction Before the advent of insulin and other synthesis medicine for diabetes, the herbal medication was the * Corresponding author. Tel.: +86 29 82668774; fax: +86 29 82668664. E-mail address: [email protected] (Z. Zhang).
mainstay in anti-diabetic therapies [1]. Nowadays, insulin and other synthesis medicine take the place of herbal medication gradually in foreign countries due to their remarkable therapeutic effects. However, the complications in macrovascular, retinal and neuropathic functions are still associated in the patients receiving insulin injection or the synthesis medicine [2]. Also, the insulin resistance is another serious problem,
0168-8227/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2004.08.011
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which is a common consequence of overweight and a cause of impaired glucose tolerance in type II diabetes [3]. In clinical practice, the insulin resistance was usually observed in association with hypertension [4], impaired glucose tolerance, hyperinsulinaemia [5], and oxidant stress [6]. Treatment of diabetes with insulin and oral drugs failed to prevent these complications in many patients, therefore the use of alternative medicine, especially the consumption of botanicals has been increasing, mostly because of the supposedly less frequent side effects and multiple effects when compared with modern western medicine [7,8]. Sanguis draxonis (SD) is a traditional Chinese herb also called Dragon’s Blood or Resina draconis. Its commercial product is known as Longxuejie1 capsule, which is processed from the resinous extract of Dracaena cochinchinensis (Lour.) S.C. Chen (Agavaceae) in China. It comprises more than 12 kinds of methanol soluble compounds [9] identified as [(1) 26-O-b-D-glucopyranosyl-furostan-5,25 (27)diene-1b,3b,22b,26-tetrahydroxy-1-O-a-L- arabinpyranoside; (2) 3,4-dihydroxy-allylbenzene-4-O-b-Dglucopyranoside; (3) 7-hydroxy-3-(r-hydroxyphenyl)-chroman; (4) 7,40 -dihydroxy-30 -methoxyflavan; (5) 3,4-dihydroxy-yallylbenzene; (6) resveratrol; (7) 7,4-dihy-droxy-flavanone; (8) di-(r-hydroxyphenyl)methane; (9) acanthoside B; (10) r-hydroxybenzoic acid; (11) hydroquinone and (12) protocatechualdehyde]. In the past 1500 years, SD was widely used in China as a famous drug for promoting blood circulation. It was generally received and recognized by herbal practitioners and passed on from generation to generation due to its miracle effects in the treatment of blood disorders [10,11]. A recent study indicated that SD had no obvious effects in decreasing the plasma sugar level in normal fasted rats but could decrease the plasma sugar level and improve the glucose-resistance in glucoseinduced or adrenaline-induced hyperglycemic rats and alloxan-induced fasted diabetic rats, and also increased insulin secretion in diabetic rats. The study also reported SD could also decrease the plasma lipid level in alloxan-induced diabetic rats [12]. These findings raised us a question whether SD could improve the insulin resistance and increase insulin sensitivity in rats. In order to develop agents without side effect for good handling of diabetic, SD was investigated in present study.
2. Materials and methods Longxuejie1 capsule, a purely natural botanic drug containing SD powder, was from Kunming arboretum pharmaceutical plant (Yunnan, China). STZ, tolbutamide and metformin were obtained from Sigma (St. Louis, U.S.A.). The short-acting porcine insulin was from Xuzhou biochemical plant (Xuzhou. China). The long-acting human insulin (Protamine zinc insulin) was supplied by Shanghai biochemical plant (Shanghai, China). 2.1. Animals Wistar male rats weighing 230–280 g, 12–13 weeks old were provided by the animal service of experimental center of Fourth Military Medical University of China. The STZ-induced diabetic rats, used as the insulin-dependent diabetes mellitus model (IDDM), were prepared by an injection of streptozotocin (65 mg kg1 i.v.) in a citrate buffer at pH 4.5. The blood glucose levels of diabetic rats were in the range of 14.6–17.4 mmol 11, 2 weeks after streptozotocin treatment. One model of insulin-resistance male Wistar rats (HFD rats), used as no insulindependent diabetes mellitus model (NIDDM), was induced after 16 week of a high-fat diet (see Table 1). At that time, a well established and permanent insulin resistance animal model with hyperglycemia, hyperinsulinaemia and hypertriglyceridemia, was present. Table 1 Composition of experimental diets Ingredient Casein Corn starch Soybean oil Beef tallow Mineral mixa Vitamin mixa Cellulose Choline bitartarate L-Methionine Fat Carbohydrate Protein
Control diet 1 (g/100 g diet) 17.0 62.5 8.0 – 3.5 1.0 7.5 0.2 0.3 (% of calories) 18.5 64 17.5
Fat-rich diet 22.9 41.4 1.87 18.0 9.2 0.92 5.0 0.39 0.35 45 35 20
a Provided by the animal service of experiment center of Fourth Military Medical University.
Z. Hou et al. / Diabetes Research and Clinical Practice 68 (2005) 3–11
Another model of insulin-resistance rats (IILI rats) was induced in male Wistar by intraperitoneal (i.p.) injections of long-acting human insulin (0.5 U kg1 three times daily for 14 days) [13]. All animals were housed in individual cages in a room controlled for temperature (23 2) 8C, humidity (45 5)% and light (08:00–20:00 h), and were maintained on a laboratory diet (see Table 1) except HFD rats, the food intake and body weight of all rats were measured everyday and water ad libitum. 2.2. Manufacture and standardization of SD formulations SD powder inside of Longxuejie1 capsules was made from the Chinese Dragon’s Blood powder, resin of Dracaena cochinchinensis which is a tropical medicinal plant, by means of following purifying step: an amount of crude SD dissolved in a certain volume of ethanol and filtered to remove the insoluble substance, SD ethanol solution was rotoevaporated under vacuum to get rid of ethanol. Finally, the refined SD was pulverized and sieved using a nest of standard sieve (200 mm). The size of less than 200 mm of SD powder was selected for Longxuejie1 capsules. Each capsule contains 300 mg SD presented in the form of brown–red powder with slight flavour and mild taste. The effective and active ingredients of Longxuejie1 capsules provided by the drug usage direction include five aromatic compounds (also called phytoalexins) and a steroid saponin with the following chemical names and structural formulas: ethyl-p-hydroxy benzoate; 7,40 -dihydroxyflavan; 7-hydroxy-40 -methoxyflavan; 7,40 -dihydroxyflavone; loureirin A; neoruscogenin-1-O-a-L-rhamnopyranosyl- (l-2)-O-a-L-arabinopyranoside. Different standard amounts (2, 6 and 10 g) of SD powder inside of Longxuejie1 capsules were suspended, respectively, in 100 ml of 0.9% (w/v) saline solution with 2 min of ultrasonification for uniformly dispersion. The final suspensions contained 20, 60 and 100 mg ml1 of SD, respectively. Five milliliter of three different concentrations of SD formulations, representing 100, 300 and 500 mg, respectively, were given to rats per kilogram by a stomach tube and the same weight-adjusted volume of vehicle was given to rats to make sure the whole amount of SD was delivered into stomach. The same weight-adjusted
5
volume of vehicle (0.9% saline solution, w/v) was given to rats as control in each experiment. In addition, the suspension of SD must be agitated to make sure the suspensions remain evenly distributed during the period of dosing to the animals. 2.3. Effect of SD on insulin resistance in IILI rats Four groups of eight Wistar rats each were used to receive an i.p. injection of long-action human insulin to induce insulin-resistance. At the same time, three groups of rats received an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively. The fourth group of rats received the same weight-adjusted volume of vehicle. Blood samples from the femoral vein were drawn before and after the treatment. After treatment as above for 14 days. the development of insulin resistance was identified using the loss of tolbutamide-induced hypoglycemic activity according to the previous report [7,14]. In brief, these Wistar rats received an i.p. injection of 10 mg kg1 tolbutamide at 5 h later of the treatment with SD or vehicle. Effects on both plasma glucose and insulin concentration were determined using blood samples collected from femoral vein of rats at 1 h after tolbutamide injection. Results, the hypoglycemic activity of tolbutamide, were calculated as the percentage decrease of the initial value according to the following formula: ðCi Ct Þ 100% Ci Where Ci is the initial plasma glucose concentration (the blood sample was taken before treatment during the fasting state) and Ct is the plasma glucose concentration after treatment with tolbutamide or another agent. 2.4. Effect of SD on insulin resistance in HFD rats Three groups of eight HFD rats each were used to receive an oral administration of SD at dosages of 100, 300 and 500 mg kg1 once a day, respectively. One group of eight HFD rats received the same weightadjusted volume of vehicle. After treatment for 7 days, blood samples from the femoral vein were drawn for the measurement of plasma glucose and insulin concentration, the insulin resistance was indicated
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by the hypoglycemic response to tolbutamide (10 mg kg1, i.p.) as described above.
samples were immediately separated by centrifugation at 3000 rpm for 20 min at 4 8C, and the plasma was stored at 70 8C until analysis.
2.5. Effect of SD on insulin sensitivity in STZ-induced diabetic rats 2.7. Statistical analysis Five groups of eight STZ-induced diabetic rats each were used to investigate the response to the exogenous insulin. In the beginning of experiment, each group of STZ-induced diabetic rats received an injection of long-acting human insulin (1 U kg1 once a day) to normalize the insulin sensitivity [7]. Three days later, three groups of STZ-induced diabetic rats received an oral administration of SD at dosages of 100, 300, and 500 mg kg1 once a day, respectively. The fourth group of rats received the same weight-adjusted volume of vehicle as control. The last group of rats received an oral administration of metformin (320 mg kg1 thrice a day) [7,15] as positive control. After 14 days of treatment, blood samples from the femoral vein were drawn for the measurement of both plasma glucose and insulin concentration. After that, all rats were prepared to challenge with the exogenous insulin. The intravenous insulin challenge tests were carried out by giving the short-acting porcine insulin (0.1, 0.5, 1.0, 1.5, and 2.0 U kg1) into these rats, and each animal was given all the doses of insulin one after another at 2-hourly intervals. Blood samples were drawn at 30 min following the intravenous insulin challenge test for the measurement of the plasma glucose concentration. The difference in response to the exogenous insulin was compared among the five groups of animals. 2.6. Determination of plasma glucose and insulin concentration Plasma glucose concentration was determined by the glucose-oxidase method (Glucose GOD-PAD kit, Beijing Ruikang Biochemical Reagent Industry, Beijing, China). Plasma insulin was assayed with a radioimmunological assay kit (Insulin RIA kit, Northern biotechnology institute, Beijing, China). The values obtained were immunoreactive insulin including insulin and proinsulin. The sample from an individual was analysed in triplicate at the same time. Intraassay and interassay coefficients of variation for insulin RIA were 7.6 and 8.9%, respectively. Blood
All data were expressed as mean S.D. for the number (n) of animals in the group. Repeated measures analysis of variation (ANOVA) was used to analyze the changes in plasma glucose and other parameters. The Dunnett range post-hoc comparisons were used to determine the source of significant differences where appropriate. The obtained p value of 0.05 or less was considered statistically significant.
3. Results 3.1. Effect of SD on IILI rats As shown in Table 2, the initial plasma glucose concentration of control rats was 5.4 0.3 mmol l1 (n = 8), after an injection of long-acting human insulin at 0.5 U kg1 into peritoneal cavity of rats was repeated three times daily for 14 days, the significant increase (p < 0.05) of plasma glucose concentration (7.9 0.4 mmol l1, n = 8) was observed. However, both plasma glucose and insulin concentration in IILI rats receiving SD treatment in combination during the induction of insulin resistance showed no significant variation (p > 0.05) compared with their initial value, but it was significantly different (p < 0.01) from the values of vehicle-treated animals. In addition, after the induction of insulin-resistance for 14 days, the hypoglycemic activity of tolbutamide (10 mg kg1, i.p.) in IILI rats with SD treatment in combination was much higher (p < 0.01) than those with the vehicle-treatment. And, at 1 h after the tolbutamide treatment, the concentration of plasma insulin in IILI rats (including the control animals) was significantly higher (p < 0.01) than the value of the tolbutamide treatment before. But the pattern of plasma insulin increase among four groups of rats showed no significant difference (data not shown). Furthermore, the food intake and changes of body weight of SD treated-rats were not significantly different from that of the control group of animals.
Food intake and change of body weight was also included. Before: before SD or insulin treatment. After: after 14 days of SD or insulin treatment. 0.9% (w/v) of saline solution was used as vehicles to suspend powder. The hypoglycemic response to tolbutamide was defined using the loss of tolbutamide (10 mg kg1 i.p.)-induced hypoglycemic activity and calculated as the percentage decrease of the initial blood glucose concentration, n = 8, Mean S.D. a p < 0.01 vs. control (the same weight-adjusted volume of vehicle). ** p < 0.01 vs. the initial value.
11 2 93 82 10 4
Gain After
286 7 277 8 278 6 283 9 275 6 268 4 270 7 273 5
Before
3.1 2.3 14.5 2.9a 2l.4 3.3a 24.4 3.8a 25.6 3.6 26.4 5.4 24.8 4.3 27.2 3.9
43 2 45 3 47 3 49 2 After Before
96.3 5.6 99.2 4.1 92.4 5.4 96.8 4.8 7.9 0.4** 6.2 0.5a 5.7 0.3a 5.1 0.2a
After Before After Before
0.5 100 300 500 Insulin Insulin + SD
5.4 0.3 5.4 0.2 5.9 0.4 5.6 0.3
120.6 6.2** 101.3 6.8a 99.2 4.9a 104.2 5.7a
Body weight (g) Food intake (g/day) (control diet) Hypoglycemic activity of tolbutamide (%) Plasma insulin (pmol l1) glucose Blood (mmol l1) Dosage Wister rats
Table 2 Changes of the plasma glucose and insulin concentration and the hypoglycemic response to tolbutamide (10 mg kg1 i.p.) in Wister rats that received an r.p. injection of long-acting human insulin at dosage of 0.5 U kg1 three times a day in combination with an oral administration of Sanguis draxonis (SD) at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively
Z. Hou et al. / Diabetes Research and Clinical Practice 68 (2005) 3–11
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3.2. Effect of SD on insulin resistance in HFD rats As shown in Table 3, after 7 days of SD treatment, both plasma glucose and insulin concentration were not significantly but slightly decreased in the group of HFD rats receiving 100 mg kg1 of SD treatment. However, for the groups of HFD rats with SD treatment at dosages of 300 and 500 mg kg1, both plasma glucose and insulin concentration were significantly decreased compared with the value of vehicle-treatment (p < 0.01). Furthermore, the hypoglycemic activity of tolbutamide in HFD rats treated with SD at dosages of 300 and 500 mg kg1 showed significantly higher than those with the vehicle treatment. In addition, no difference on the amount of food intake and changes in body weight was observed between HFD rats with SD treatment and those with the vehicle-treatment. And the changing pattern of plasma insulin concentration in HFD rats before and after tolbutamide treatment showed no significant difference (data not shown). 3.3. Effect of SD on the insulin sensitivity in STZ-induced diabetic rats According to the results shown in Table 4, both plasma glucose and insulin concentration of STZinduced diabetic rats receiving SD at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively, were not significantly different from the values of vehicle-treated rats. However, the hypoglycemic activity of short-acting porcine insulin at dosage of 0.1–2.0 U kg1 in these STZ-induced diabetic rats with SD treatment once a day for 14 days were higher than those with the vehicle treatment (see Fig. 1). The hypoglycemic activity of exogenous insulin was more prominent (p < 0.01 versus control) at dosage of insulin up to 1.0 U kg1 for rats receiving SD at dosage of 300 mg kg1, or at dosage of insulin up to 0.5 U kg1 for rats receiving SD at dosage of 500 mg kg1 (p < 0.05 versus control). For the group of rats receiving SD at dosage of 100 mg kg1, the result of exogenous insulin was not significantly different from the value of control. In addition, the food intake and the body weight of STZ rats were not greatly influenced by SD treatment compared with the control (data no shown).
52 61 52 73
Food intake and change of body weight was also included. Before: before SD treatment. After: after 7 days of SD treatment. 0.9% (w/v) of saline solution was used as vehicles to suspend powder. The hypoglycemic response to tolbutamide was defined using the loss of tolbutamide (10 mg kg1 i.p.)-induced hypoglycemic activity and calculated as the percentage decrease of the initial blood glucose concentration according to the text, n = 8, Mean S.D. a p < 0.05. b p < 0.01 vs. control (the same weight-adjusted volume of vehicle). * p < 0.05. ** p < 0.01 vs. the initial value.
After
339 6 348 7 341 6 346 8 334 8 342 7 336 6 339 9
Before
46 4 43 6 46 4 47 5 10.8 3.3 14.1 2.9 19.4 3.3b** 21.9 3.8b**
After Before
11.6 3.6 12.4 3.4 12.8 2.3 13.2 3.9 130.2 7.9 117.1 8.4a* 101.6 7.2b** 96.9 7.6b** 8.9 0.4 7.5 0.5 5.7 0.3b* 5.1 0.2b** 8.4 0.3 8.2 0.5 7.9 0.4 8.6 0.3 – 100 300 500 Control SD
After Before After Before
Dosage (mg kg1) HFD rats
137.6 8.6 135.8 7.7 136.4 8.2 138.7 7.9
Plasma insulin (pmol l1) Blood glucose (mmol l1)
Hypoglycemic activity of tolbutamide (%)
Food intake (g/day) (control diet)
Body weight (g)
Gain
Z. Hou et al. / Diabetes Research and Clinical Practice 68 (2005) 3–11 Table 3 Changes of the plasma glucose and insulin concentration and the hypoglycemic response to tolbutamide (10 mg kg1 i.p) in high-fat diet (HFD) rats with Sanguis draxonis (SD) treatment for 7 days
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After the metformin was administrated orally at dosage of 320 mg kg1 three times daily for 14 days, the hypoglycemic activity of short-acting insulin in STZ-diabetic rats was similar to the vehicle-treated group rats at dosages of insulin less than 1.0 U kg1 (Fig. 1), but the hypoglycemic activity of exogenous insulin was significantly prominent at dosage of insulin up to 1.5 U kg1 (p < 0.05).
4. Discussion The insulin-resistant animal model induced by repeatedly injection of long action human insulin was used to identify the influence of SD on the formation of insulin resistance. It had been documented that tolbutamide-induced hypoglycemia occurs through the stimulation of endogenous insulin release [14], so, the loss of tolbutamide-induced hypoglycemic activity can be interpreted as the development of insulin resistance. In normal Walter rats, before the inducement of insulin resistance, the hypoglycemic activity induced by tolbutamide at 10 mg kg1 (i.p.) was about 25.6 3.6%, but it was only 3.1 3.2% in IILI rats that received three times daily injection of long-acting insulin for 14 days. This denoted that an insulin resistance had been developed in these rats. However, the hypoglycemic activities of tolbutamide were still high and no significant increase of both plasma glucose and insulin concentration was obtained in rats with SD treatment in combination during the induction of insulin-resistance. These results suggest that the development of insulin resistance can be improved in IILI rats with SD treatment. Since overconsumption of dietary fat is a major contribution factor to human obesity and obesitylinked NIDDM. HFD rats were used as another NIDDM animal model to verify the effect of SD on the formation of insulin resistance. After 7 days of treatment with SD, both blood glucose and insulin concentration in HFD rats were decreased significantly compared with control. On the other hand, the hypoglycemic activity of tolbutamide was also significantly higher in HFD rats with SD treatment than those with the vehicle treatment. Thus, the improvement of insulin resistance in HFD rats with SD treatment can be considered.
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Table 4 Plasma glucose and insulin concentration in STZ-induced rats receiving Sanguis draxonis (SD) and Metformin (positive control) treatment for 14 days
STZ-induced rats Vehicle (control) SD (mg kg1 oid)
Metformin (mg kg1 tid)
Dosage
Plasma glucose (mmol l1)
Plasma insulin (pmol l1)
– 100 300 500 320
16.9 2.9 15.5 3.6 14.7 2.8 13.9 3.2 14.4 3.8
45.8 4.2 47.2 3.9 49.4 4.6 52.1 4.2 50.8 4.7
oid: one time daily, tid: three times daily. Value represent means S.D. n = 8. Control (the same weight-adjusted volume of vehicle, 0.9% (w/v) of saline solution).
The pattern of plasma insulin increase before and after tolbutamide treatment among the four groups of IILI rats or HFD rats showed no significant difference, which suggests that the stimulation of endogenous insulin release in rats with boltutamide treatment was not influenced significantly by different dosages of SD treatment and inducement of insulin-resistance with IILI or HFD. So, the high hypoglycemic activity of tolbutamide in both IILI rats and HFD rats with SD treatment was attributed not to the increase of plasma insulin concentration, but to the increase of insulin sensitivity. To clarify the increase of insulin sensitivity with SD treatment, STZ-diabetic rats were used to investigate the response to exogenous insulin.
The first method developed to evaluate insulin sensitivity in vivo was the intravenous insulin challenge test, which was based on the change of plasma glucose level after an injection of regular insulin [16]. Then, the intravenous insulin challenge test was performed in STZ-diabetic rats. The advantage of using STZ-diabetic rats in this study was the negligible endogenous insulin. The hypoglycemic action was directly due to the activity of exogenous insulin in STZ-diabetic rats. The obtained result can be used to indicate insulin sensitivity. It was found in this study that SD at dosages of 100, 300, and 500 mg kg1 once a day for 14 days enhanced the hypoglycemic action of exogenous insulin in STZ-
Fig. 1. Hypoglycemic response to exogenous short-acting porcine insulin in STZ-induced diabetic rats after receiving an oral administration of Sanguis draxonis (SD) at dosages of 100, 300, and 500 mg kg1 once a day for 14 days, respectively. STZ-induced diabetic rats receiving an oral administration of metformin (320 mg kg1 thrice a day) was used as positive control. STZ-induced diabetic rats receiving the same weightadjusted volume of vehicle (0.9% saline solution, w/v) were taken as control, n = 8. Mean S.D. a: p < 0.05, b: p < 0.01 vs. control.
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diabetic rats, and the effective dose of SD should be more than 300 mg kg1 in STZ-diabetic rats. Results also indicated that both plasma glucose and insulin concentration showed no significant variation before and after SD treatment in STZ-diabetic rats, which was in good agreement with application direction of SD drug (Longxuejie1 capsules) suitable for the type II diabetic patients. But it was not consistent with previous report [12] that SD could decrease the plasma sugar level in alloxan-induced fasted diabetic-rats and increase insulin secretion in alloxan-induced diabetic rats significantly. This discrepancy may be attributed in part to the differences between the different druginduced diabetic animal models or other unknown reasons. In addition, the effect of metformin was used as positive control in the present study. Metformin had been shown to improve the insulin sensitivity in NIDDM subjects by activating post-receptor insulin signaling pathways [17]. And an increase of the response to exogenous insulin was also observed in STZ-diabetic rats that received metformin at an effective dose as described previously [18]. In this study, the action of SD at dosages of 300 and 500 mg kg1 was more effective than that of the metformin at dosage of 320 mg kg1 three times daily. Because SD failed to modify the plasma insulin significantly in STZ-diabetic rats in our study, the increase of exogenous insulin action with SD treatment seems to be associated with an improvement of insulin sensitivity. Nevertheless, since the increase of secretion of endogenous insulin with SD treatment could not be completely ruled out [12], enhancement of insulin action brought by SD treatment might produce a synergistic effect on the hypoglycemic action of insulin in STZ-diabetic rats. Resistance to the action of insulin can result from a variety of causes, including defects in the receptor binding and at the post-receptor level [19,20]. The mechanism of SD to improve the insulin resistance in rats might be complex and probably multifactorial due to its mixture of many kinds of compounds. It is now recognized that SD also has beneficial effects on platelet aggregation, thrombus formation and myocardial ischemia [21]. Moreover, SD was found to possess strong antioxidant activity [22]. Although the chemical constituents and other pharmacological properties of SD have been analyzed [9,10] and the effective and active ingredients of Longxuejie1
capsules are also identified, the active ingredients for the improvement of insulin resistance and insulin sensitivity are still unclear. Otherwise, drug synergistic action is still the primary issue for Chinese traditional herbs. Therefore, it is possible that our reported effects about SD could result from many kinds of ingredients among SD components. Further studies are required to clarify the detail molecular mechanisms associated to these SD effects. And Lots of work remains to be achieved before Longxuejie1 capsules can be seen as an effective drug for the improvement of insulin resistance in clinical practice. In conclusion, the most expressing findings of this study were that an oral administration of SD can (1) improve the formation of insulin resistance in HFD rats (2) improve the development of insulin resistance in IILI rats, and (3) increase exogenous insulin sensitivity in STZ-induced diabetic rats. From these findings, SD is expected to be useful both as monotherapy in type II diabetic patients and in combination with insulin in type I diabetic patients.
Acknowledgement This work was supported by the National Nature Science Foundation of China (60178034) and the Doctorate Foundation of Xi’an Jiaotong University (DFXJTU 2001-2).
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