Alpha-glucosidase inhibition from a Chinese medical herb (Ramulus mori) in normal and diabetic rats and mice.

Summary

Alpha-glucosidase inhibitors are oral antidiabetic drugs. A traditional Chinese medical herb, Sangzhi (Ramulus mori), appears to have properties similar to those of a-glucosidase inhibitors. The effects of an aqueous extract of Shangzhi (SZ) were studied in normal and alloxan diabetic rats and mice, and these results compared with those for acarbose, an [alpha]-glucosidase inhibitor. In our grade-dose studies, SZ was found to lower and prolong the zenith of blood glucose concentration (ZBG) after sucrose or starch loading and stabilize blood glucose levels in fasting normal and alloxan diabetic mice. After 2 weeks of SZ administration with high-calorie chow or a normal diet, the fasting and non-fasting blood glucose concentrations in alloxan diabetic mice and rats were decreased. In alloxan rats, the blood fructosamine concentration was lowered. Results for acarbose and SZ were similar. These indicate that SZ has [alpha]-glucosidase inhibitory effects.

Key words: aqueous extract of Sangzhi (SZ), Morus alba L., [alpha]-glucosidase inhibition, oral carbohydrate tolerance test, blood fructosamine concentration

* Introduction

Since the early 1990s, a new class of antidiabetic drugs, [alpha]-glucosidase inhibitors, has been known as an approach for treating diabetes, and was introduced with the marketing of acarbose (made by Bayer Germany AG). Alpha-glucosidase inhibitors delay the digestion of oligosaccharide and disaccharide to monosaccharide by inhibiting [alpha]-glucosidases on the small intestinal brush-border, and reduce the rate of glucose absorption. As a result, they decrease the postprandial rise in blood glucose concentration (Bischoff, 1995; Hanefeld et al., 1991; Lam et al., 1998). This more stable blood glucose concentration is important for diabetic patients, because it prevents hyperglycemia and the complications associated with diabetes. Therefore, the [alpha]-glucosidase inhibitor acarbose is a first-line drug for treating type-2 diabetes mellitus that is insufficiently controlled through diet alone (Hanefeld et al., 1991).

After screening hundreds of traditional Chinese medicines, the aqueous extract of Sangzhi (SZ) was found to potently inhibit [alpha]-glucosidase activity in the small intestine of rats and mice. The effects of SZ were studied in normal and alloxan diabetic rats and mice, and these results compared with those for acarbose, an [alpha]-glucosidase inhibitor. The animals were divided into five groups: untreated control; a group given acarbose; and three groups given SZ at 1.25, 2.50, and 5.00 g/kg body wt., respectively. After sucrose and starch loading, SZ was shown to significantly reduce the rise of blood glucose and to lower and prolong the zenith of blood glucose concentration (ZBG). During 10 to 15 days of repeated SZ administration and high-calorie chow, lower fasting and non-fasting blood glucose concentrations were found in animals administered SZ and acarbose than in control animals. Blood fructosamine concentrations were also significantly lower in these animals. All of the results show that SZ has eff ects similar to an [alpha]-glucosidase inhibitor.

Materials and Methods

Preparation of SZ

Ramulus mori (Sangzhi), the branches of Morus alba L., was purchased from the Tong Ren Tang Drugstore in Beijing, China, and identified by the Department of Botany in our institute under the voucher-No. of Morus alba L. sp. is 27-10-1. It was cut into small pieces and boiled in water (1:12, w/v) for 2 hours, twice. The solution was cooled and filtered. The filtrates were combined, concentrated under reduce pressure at 50-60[degrees]C. and spray-dried.

Animals

Male Kunming mice weighing 22 to 28 g each and male Wistar rats weighing 220 to 240 g each were purchased from the Experimental Animal Center, Chinese Academy of Medical Sciences, Beijing. They were cared for humanely in accordance with the standards for laboratory animals established by the Peoples Republic of China (GB 14923-94, GB 14922-94, and GB/T 14925-94).

The animals were rendered diabetic by a single injection of alloxan (mice 100 mg/kg body wt., rats 50 mg/kg body wt.) intravenously into a tail vein, and confirmed by hyperglycemie (>11.1 mM). There was no significant deviation in blood glucose level among the animals at the beginning of each experiment.

Assay of oral carbohydrate tolerance

Blood glucose concentration was determined using the glucose oxidase method with a Spectrophotometer S 500 (SECOMAM, France).

Oral sucrose tolerance test: The animals were restricted from food overnight and given sucrose at 4 g/kg body wt. orally at 9:00 am, with or without SZ. Blood samples were collected 0, 30, 60, and 120 minutes after sucrose loading to measure blood glucose (BG) concentrations. Blood glucose concentration-time curves were plotted, and the zenith blood glucose concentration (ZBG) and the area under the curve (AUC) were determined. The formula for AUC calculation follows:

AUC (mmol/1.hr) = ([BG.sub.0]+[BG.sub.30]) X 0.5 / 2 +([BG.sub.30]+[BG.sub.60]) X 0.5 / 2 + ([BG.sub.120]+[BG.sub.60]) X 1 / 2

([BG.sub.0], [BG.sub.30] [BG.sub.60], and [BG.sub.120] represent blood glucose concentrations at 0, 30, 60, and 120 minutes after loading).

Oral starch tolerance test: These tests and measurements were carried out in the same way as the oral sucrose tolerance tests, but using starch at 3 g/kg body wt. instead of sucrose.

Oral glucose tolerance test: The animals were restricted from food overnight, and given glucose at 2 g/kg body wt. orally with or without SZ. Blood glucose concentrations were measured 0, 30, 60 and 120 minutes after glucose administration.

Effects of SZ on blood glucose concentration in diabetic mice and rats

Fructosamine concentration was measured using a Fructosamine Determining Kit purchased from the Beijing Zhong Sheng Biological Project High-technology Company (China) with a 752Z UV/VIS spectrophotometer (BOIF, Beijing, China).

The high-calorie chow was prepared with 50% sucrose and 50% normal foodstuff for rodents. According to the animals' body weight and the amount of their feed intake, the quantities of SZ in the feed were 0.86 g per 100 g feed, for diabetic mice, and 1.36 or 2.72 g per 100 g feed, for diabetic rats.

The diabetic mice were given either a normal diet or high-calorie chow, with or without SZ. On the experimental day the animals were restricted from eating for 3 hours (from 8:00 am to 11:00am). Fasting blood glucose concentration (FBG) was measured on the 11th day. Non-fasting blood glucose concentration (Non-FBG), which means postprandial blood glucose concentration, was measured on the 14th day.

The diabetic rats were given a high-calorie chow with or without SZ. Non-FBG and FBG were measured separately on the 12th and 15th days. Fructosamine concentrations were measured on the 15th day.

Statistical analysis

The data conform to the assumptions of analysis of variance, using the computer application Stata; p < 0.05 was considered significant. Values were expressed as mean [+ or -] SD.

Results

Effects of SZ on oral sucrose tolerance in normal and alloxan diabetic mice

The results of the oral sucrose (4 g/kg body wt.) tolerance test showed that SZ significantly reduced the increase of blood glucose level after sucrose loading, especially at 30 minutes (Figure 1). SZ shifted the ZBG from 30 minutes to 60 minutes and decreased the ZBG and AUG significantly (Figure 1 and Table 1). These data suggested that BG did not increase severely in the oral sucrose tolerance test with SZ administration in healthy mice. All of the effects correlated with the SZ doses. These effects were similar to those for acarbose.

The oral sucrose tolerance test was repeated in alloxan diabetic mice. The results were similar to those for normal mice (Figure 2 and Table 1). SZ treatment smoothed the BG curves and decreased the ZBG and the AUG significantly.

Effect of SZ on oral starch tolerance in normal and diabetic mice

The results of the oral starch tolerance test in normal and diabetic mice showed that SZ inhibited the BO increase and made the BG curve flat after starch (3 g/kg body wt.) was given (Figures 3 and 4). SZ decreased and prolonged the ZBG, and reduced the AUC significandy (Table 2, Figures 3 and 4). These data correlated with the SZ dose, and were similar to those for acarbose.

Effects of SZ on oral glucose tolerance in mice

The animals were averaged into 4 groups (n = 10): Con, SZ 1.25, SZ 2.5, and Acar and given glucose at 2 g/kg body wt. with water, SZ at 1.25 and 2.50 g/kg body wt., and acarbose at 10 mg/kg body wt., respectively. Among the 4 groups, BG reached the peak at 30 minutes, then returned to normal at 120 minutes. ZBG values of Con, SZ 1.25, SZ 2.5, and Acar were 15.89 [+ or -] 3.67, 14.22 [+ or -] 2.27, 13.33 [+ or -] 2.76, and 15.57 [+ or -] 2.47 mmol/l. None showed statistically significant difference (p < 0.05). The AUC values of Con, SZ 1.25, SZ 2.5, and Acar groups were 22.72 [+ or -] 4.22, 20.86 [+ or -] 2.23, 21.25 [+ or -] 3.20, and 21.80 [+ or -] 3.34 mmol/l, respectively. They did not differ significantly (p > 0.05). Blood glucose level did not differ significantly at any time among the 4 groups. These results demonstrate that neither SZ nor acarbose affect glucose absorption in the small intestine.

Effects of SZ on blood glucose concentration in diabetic mice

After the administration of SZ mixed in normal or high-calorie chow for the appointed days to alloxan diabetic mice, blood glucose concentration was determined at 8.30 am as non-fasting blood glucose concentration and at 10:30 am, with 2 hours' food restriction, as fasting blood glucose. SZ was shown to significantly decrease non-fasting blood glucose concentration in both SZ and H-SZ mice on the 13th day after the diet was given. The fasting blood glucose concentration was decreased only in H-SZ animals on the 11th day (Table 3).

Effects of SZ on blood glucose concentration in diabetic rats

Three groups of alloxan diabetic rats were given high-calorie chow with or without SZ. According to the amount of SZ mixed in the chow, the food intake and the body weight, the average quantities of SZ were eaten by Con, SZ1, and SZ2 groups were approximately 0, 1.25 and 2.50 g/kg body wt. Meanwhile, ten normal rats given normal diet were used as control (N-Con). Non-fasting blood glucose concentrations were determined on the 12th day, fasting blood glucose concentrations and fructosamine concentrations on the 15th day after the diet and SZ were given. Fructosamine concentrations, fasting and non-fasting blood glucose concentrations were increased significantly in alloxan diabetic rats compared with N-Con, and with SZ administration all of the parameters were decreased significantly (Table 4 and Figure 5).

Discussion

Sangzhi (Ramulus mori) is the branch of Morus alba L., family Moraceae (Committee of the Peoples Republic of China Pharmacopeia, 1995). According to the theories of traditional Chinese medicine, it is slightly bitter in taste, mild in nature, and attributive to the liver meridian. In traditional Chinese medicine, its pharmacological actions are to expel wind, dredge the meridians, and ease joint pain (Ou, 1992). Our experimental results show that Sangzhi aqueous extract has the effects of an [alpha]-glucosidase inhibitor (Shen et al., 1998).

Alpha-Glucosidases are a series of enzymes located on the intestinal brush-border. The most important carbohydrates in food, such as starch and sucrose, are hydrolyzed to monosaccharide, such as glucose and fructose, by an [alpha]-glucosidase, and then absorbed into the blood, thereby increasing BG value. Usually, these processes take place in the upper portion of the small intestine and greatly increase BG concentration, especially in diabetic patients. Alpha-glucosidase inhibitors can prolong the processes along the entire intestine, lengthen the duration of carbohydrate absorption, and flatten the blood glucose concentrations over time curve (Bischoff, 1993). Because the [alpha]-glucosidase inhibitor acarbose prevents an abnormally high rise in postprandial BG concentrations, it is a first-line drug in treatment of type-2 diabetes that is not controlled through diet alone (Hanefeld et al., 1991). In our experiments, SZ reduced the increases in blood glucose concentrations and decreased the ZBG and AUC afte r sucrose or starch loading in normal and alloxan diabetic mice. However, in normal mice, SZ did not affect glucose absorption in the small intestine. It was demonstrated that SZ delayed the rapid digestion of starch and sucrose, and reduced the postprandial zenith of BG concentration. These effects are similar to those for the [alpha]-glucosidase inhibitor, acarbose.

Persistent hyperglycemia, the common characteristic of diabetes, can cause most diabetic complications. In all patients, treatment should aim to lower blood glucose to or near-normal levels (American Diabetes-Association, 1998). We found that after SZ administration, fasting and non-fasting blood glucose concentrations were decreased in alloxan diabetic mice and rats. In addition, in alloxan rats fed high-calorie diets for 2 weeks, blood fructosamine concentrations were decreased, indicating that SZ had decreased the mean blood glucose levels throughout the previous 2 weeks.

SZ affected the absorption of starch and sucrose, improved both fasting and non-fasting hyperglycemia, and gained the overall glycemic control as measured by the blood fructosamine concentrations. SZ is a kind of [alpha]-glucosidase inhibitor extracted from natural products. It is possible that SZ will be used as a very effective antidiabetic drug for improving blood glucose control and preventing diabetic complications.

Investigations are in progress to identify the active principles responsible for the [alpha]-glucosidase inhibitory effect of the Ramulus Mori-extract.

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Table 1

Effect of SZ on blood glucose concentration after surcrose loading in
normal mice and alloxan diabetic mice.

Group                     ZBG (mmol/l)        p        AUC (mmol/l)

Normal mice

Con                     8.30 [+ or -] 0.97   1.000   9.55 [+ or -] 0.56
SZ 1.25                 6.29 [+ or -] 0.49  <0.001   8.79 [+ or -] 0.27
SZ 2.50                 6.22 [+ or -] 0.27  <0.001   8.34 [+ or -] 0.68
SZ 5.00                 5.95 [+ or -] 0.40  <0.001   8.47 [+ or -] 0.57
Acar                    5.64 [+ or -] 0.65  <0.001   7.52 [+ or -] 1.29

Alloxan diabetic mice

Con                    22.08 [+ or -] 2.96   1.000  26.95 [+ or -] 1.90
SZ 1.25                15.59 [+ or -] 1.40  <0.001  22.29 [+ or -] 1.13
SZ 2.50                13.84 [+ or -] 3.36  <0.001  21.79 [+ or -] 1.48
Acar                   15.43 [+ or -] 1.69  <0.001  21.35 [+ or -] 1.61

Group                  p

Normal mice

Con                    1.000
SZ 1.25                0.017
SZ 2.50                0.006
SZ 5.00                0.007
Acar                   0.003

Alloxan diabetic mice

Con                    1.000
SZ 1.25                <0.001
SZ 2.50                <0.001
Acar                   <0.001

ZBG indicates the maximum blood glucose concentration. AUC, the area
under the blood glucose concentration curve, was calculated using the
formula described in the text. The data were presented as mean [+ or
-]AD. p, compared with Control. n = 10.
Table 2

Effect of SZ on blood glucose concentration after starch loading in
normal mice and alloxan diabetic mice (mean [+ or -] SD, n = 10).

Group                     ZBG (mmol/l)      p           AUC (mmol/l)

Normal mice

Con                    10.43 [+ or -] 1.24  1.000    10.92 [+ or -] 0.86
SZ 1.25                 8.26 [+ or -] 1.22  0.006    10.75 [+ or -] 0.90
SZ 2.50                 7.36 [+ or -] 1.31  0.001     9.70 [+ or -] 1.06
SZ 5.00                 6.69 [+ or -] 0.62  < 0.001   9.67 [+ or -] 0.69
Acar                    6.77 [+ or -] 0.24  < 0.001   9.11 [+ or -] 0.87

Alloxan diabetic mice

Con                    22.51 [+ or -] 1.87  1.000    24.74 [+ or -] 1.77
SZ 1.25                16.43 [+ or -] 2.48  < 0.001  20.48 [+ or -] 2.51
SZ 2.50                15.14 [+ or -] 1.39  < 0.001  20.15 [+ or -] 1.23
Acar                   13.59 [+ or -] 2.23  < 0.001  18.59 [+ or -] 2.16

Group                  p

Normal mice

Con                    1.000
SZ 1.25                0.720
SZ 2.50                0.034
SZ 5.00                0.010
Acar                   0.002

Alloxan diabetic mice

Con                    1.000
SZ 1.25                < 0.001
SZ 2.50                < 0.001
Acar                   < 0.001
Table 3

Effect of SZ on blood glucose concentrations in diabetic mice

Group  Blood glucose concent ration (mmol/l)

       Fasting              p         non-fasting          p

Con    21.64 [+ or -] 3.13  1.000     33.90 [+ or -] 4.62  1.000
SZ     18.82 [+ or -] 4.91  0.150     27.59 [+ or -] 4.31  0.007
H-Con  20.93 [+ or -] 3.47  0.639     27.91 [+ or -] 4.98  0.012
H-SZ   13.42 [+ or -] 4.31  < 0.001   21.26 [+ or -] 8.02  < 0.001

Four groups of alloxan diabetic mice were given different diets: Con was
normal diet; SZ, normal diet with SZ 0.6 g/100g feed; H-Con,
high-calorie chow; H-SZ, high-calorie chow with SZ 0.86 g/100 g feed.
The fasting blood glucose concentrations were measured on 11th day after
administration of high-calorie chow and SZ, and non-fasting on the 13th
day. The data were presented as mean [+ or -] SD. n = 10.
Table 4

Effect of SZ on blood glucose concentration in diabetic rats

Groups   Blood glucose concentration (mmol/l)

         Fasting              p        non-fasting          p

N-Con     3.38 [+ or -] 0.47  < 0.001   5.45 [+ or -] 0.21  < 0.001
Con      17.95 [+ or -] 4.49  1.000    18.90 [+ or -] 2.91  1.000
SZ 1.25  10.77 [+ or -] 6.54  0.017    11.88 [+ or -] 5.58  0.005
ST 2.5    7.97 [+ or -] 3.07  < 0.001   8.35 [+ or -] 5.35  < 0.001

There groups of alloxan diabetic rats were given high-calorie chow, with
or without SZ. According to the amounts of SZ mixed in the chow, the
animals' food intake and their body weight, the average quantities of SZ
ate by Con, SZ 1.25, and SZ 2.5 groups were approximately 0, 1.25 and
2.50 g/kg body wt. Meanwhile, ten normal rats ate normal diet as control
(N-Con). The fasting and non-fasting blood glucose concentrations were
measured on the 15th and 12th days, respectively, after the
administration of high-calorie chow and SZ. The data were presented as
mean [+ or -] SD. n = 10.

Acknowledgements

We thank Professor Lilian Zhu and Ms. Renyun Wang for providing the extract, Professor Peirang Cao for his skilled support in drug identification and Professor Linmao Ma for his help with statistics.

References

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Shen, Z., Xie, M., Ye, F. Study and exploitation of [alpha]-glucosidase inhibitor from traditional Chinese medicine. Abstracts of Chinese Academy of Medical Sciences and Peking Union Medical College Scientific Annual (1998, Beijing), p. 147, Peking Medical University and Peking Union Medical College, Beijing, 1998.

Address

Z. Shen, Department of Pharmacology, Institute of Materia Medica, Peking Union Medical University, 1 Xian Nong Tan Street, Beijing 100050, China

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