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Abstract: In this experiment, we investigated the effects of crude Ephedrae herba, alkaloid extract of Ephedrae herba and 1-ephedrine, a major alkaloid component, on diabetic mice induced by streptozotocin (STZ). The alkaloid extract and 1-ephedrine showed suppression on the hyperglycemia. The suppression by Ephedrae herba of hyperglycemia may therefore be due to 1-ephedrine. Furthermore, we found that Ephedrae herba, alkaloid and 1-ephedrine promoted the regeneration of pancreas islets following atrophy induced by STZ. It is therefore suggested that Ephedrae herba may regenerate atrophied pancreatic islets, restore the secretion of insulin, and thus correct hyperglycemia.


Streptozotocin (STZ) is widely used experimentally to induce diabetes in animals (Zusman et al., 1985). The mechanism of diabetes induced by STZ is due to a change of the DNA chain in the pancreatic islets, a reduction in damage of the B cells (Okamoto, 1981).
The crude drug Ephedrae herba, obtained from the herb Ephedra sinica Stapf (Ephedraceae), has long been used for sudorific, antipyretic, antitussive and anti-inflammatory purposes in East-Asia. Diabetes is not included in the traditional applications of Ephedrae herba. However, we used an experimental model of mice with diabetes induced by STZ to investigate the effects of Mao-to, a Kampo formulation, and found that it could suppress the increase of blood glucose. Furthermore, Ephedrae herba, a major crude drug of Mao-to, protected the pancreas islet and improved the results of the glucose tolerance test (Kobayashi et al, 1998). Abundant alkaloid is found in Ephedrae herba, and the major component is ephedrine (Yamada et al., 1994).

In this study, we attempted to clarify the active components of Ephedrae herba, and used crude extract, alkaloid extract of Ephedrae herba and 1-ephedrine to investigate their effects on the diabetes induced in mice by STZ.
Materials and Methods
Animals
BALB/cA Jcl male mice were obtained from Clea Japan, Inc. at 8 weeks of age. They were kept in plastic cages with wood shavings, and maintained in an animal room, which was air-conditioned (22-24 [degrees]) and artificially illuminated, and provided with standard commercial pellets and tap water ad libitum.
Preparation of Samples
Ephedrae herba (Ephedra sinica Stapf) was obtained from Uchida Co. (Tokyo, Japan). 10 g of crude herb of Ephedrae herba was boiled with 600 ml of distilled water until the volume was reduced to 350 ml. The supernatant fluid was filtered and centrifuged. The yield was 15% from the original herb weight. Ephedrae herba was crushed into powder and extracted three times with ethanol; HCl was then added into the crude extracts and the resulting precipitate was collected. This was to provide 70 mg (0.7% yield) of the crude alkaloid extracts in the following experiment. Analytical grade of 1-ephedrine was purchased from Dainippon Pharmacy.
Induction of Diabetes
Mice were intraperitoneally injected with 200 mg/kg of streptozotocin (STZ, Sigma, USA) in 10 mM citrate buffer (pH 4.4) after 18 hours of fasting. Blood was then collected from the tail vein. Blood glucose levels were measured by the glucose dehydrogenase method on a portable glucometer (Yamanochi, Tokyo, Japan). On the 4th day after injection of STZ, mice showing 200-500 mg/dl blood glucose were divided into four groups. In the Ephedrae herba group, the herbal extract was diluted and given as drinking water consecutively for five weeks. The concentration of the extract was adjusted to 1.4 g/kg/day as original herb weight. In the alkaloid and 1-ephedrine groups, the samples were dissolved in tap water and given as drinking water. The concentration of the samples was adjusted to alkaloid fraction: 0.62 mg/kg body weight and 1-ephedrine: 0.62 mg/kg body weight, respectively. The samples administered for a mouse was 10-20 times the dose for human adults. The samples were administered orally consecutively from the 4th day after STZ injection for two weeks. Animals of the control group received only water. The numbers of mice in each group were as follows: control group 5, Ephedrae herba: 5, alkaloid fraction: 6 and 1-ephedrine: 6 mice.
Histological Analysis of the Pancreas
Two weeks after the administration, mice were killed by decapitation under light ether anesthesia and the pancreas was removed. Pancreatic tissue was fixed in 10% buffered formalin, and embedded in paraffin according to the conventional procedure. Paraffin sections were serially cut at 4[micro]m in thickness and stained with Hematoxylin. A part of the slide was refixed in Bouin solution and stained with Gomorri's aldehyde-fuchsin stain.
Statistics
Data were analyzed by Student's t-test, p<0.05 was considered statistically significant.
Results
Effects of Ephedrae herba and its Components on Blood Glucose
The mice showing hyperglycemia (200-500 mg/dl) were used to investigate the effects of Ephedrae herba extract, alkaloid extract and 1-ephedrine in the present experiment. The blood glucose was measured before administration and on the 3rd, 6th and 14th days after administration of samples. In the control group receiving only water, the blood glucose continued to be raised. The hyperglycemia was suppressed by all samples, but the period of this effect was different. The blood glucose in the Ephedrae herba extract group tended to decrease on the 3rd day, and significantly decrease by the 14th day, compared with the control group (p<0.05).>

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