JPS63316729A - Tonic - Google Patents

Abstract

PURPOSE:To obtain a tonic containing saponin of Gynostemma pentaphyllum as an active ingredient. CONSTITUTION:The whole dried plant of Gynostemma pentaphyllum is extracted with water under heating, the extracted solution is concentrated and dried to give a water extracted essence. The essence is extracted with a methanol under heating to remove insoluble materials, the extracted solution is concentrated, then dissolved in water, mixed with an ether and well shaken. The lower water layer is collected, well shaken with water-saturated n-butanol, the upper n-butanol layer is collected and concentrated to give crude saponin of Gynostemma pentaphyllum. Then the crude saponin of Gynostemma pentaphyllum is dissolved in a small amount of methanol and an ether is added to the solution to precipitate saponin of Gynostemma pentaphyllum. A daily dose is 1-500mg, preferably 10-100mg saponin of Gynostemma pentaphyllum as an active ingredient per weight in the case of oral administration and 0.25-125mg, preferably 2.5-25mg in the case of parenteral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the Gynostemsa pe
The present invention relates to a tonic containing saponins present in Ntaphyllum Makino as an active ingredient.

The present inventors have conducted extensive research on the physiological activities of Jiaogulan, and found that some people who take Jiaogulan aqueous extract often complain of increased virility, and upon investigating the cause, found that They discovered that saponin from Jiaogulan can produce this, and based on this new knowledge, they completed the present invention.

That is, the present invention provides a tonic containing Jiaogulan saponin as an active ingredient.

Jiaogulan belongs to the Cucurbitaceae family and is a perennial vine that grows naturally in the mountains and fields of East Asia. Moreover, this plant is easy to cultivate, and the plant that is the raw material for the tonic of the present invention is extremely easy to obtain. Jiaogulan saponin, which is an active ingredient of the tonic of the present invention, can be obtained from Jiaogulan by the following method, for example. Water is added to the dried whole plant of Jiaogulan, extracted by heating, and the extract is concentrated to dryness to obtain a water extract. Add methanol to this water extract,
Extract by heating, remove insoluble matter, and layer the extract to obtain a methanol extract. Dissolve this methanol extract in water, add ether to it, shake well, separate the lower aqueous layer, add water-saturated n-butanol to it, shake well, and remove the upper one-butanol layer. It is separated and concentrated to produce Jiaogulan crude saponin. Next, this crude Jiaogulan saponin is dissolved in as little methanol as possible and poured into ether to precipitate Jiaogulan saponin.

Specific examples are as follows.

Production example l 250g of dried Jiaogulan whole plant, 10 times the amount of distilled water,
That is, add 2.5Q of distilled water and boil for 120 minutes. Cooling, filter the extract, and reduce the filtrate to dryness under reduced pressure for 6 hours.
80 g of reddish brown powder was obtained. Next, 10 times the amount of methanol, that is, 800 liters of methanol, is added to this reddish-brown powder and boiled for 20 minutes. After cooling and filtering the extract, the filtrate was concentrated to dryness under reduced pressure to obtain brown powder 309. Next, add 200 zQ of distilled water to this brown powder, and after completely dissolving the powder, transfer it to a separatory funnel, and then add 200 zQ of distilled water.
After adding 212 and shaking well, separate the lower aqueous layer. This aqueous layer was extracted with ether twice in the same manner, and then water-saturated n-butanol 200x (
After adding 7 and shaking well, separate the upper n-butanol layer. The lower aqueous layer was further extracted with n-butanol in the same manner three times, and all the n-butanol extracts were combined and concentrated to dryness under reduced pressure to form a light brown powder! 2g was obtained.

Next, 85mC of methanol was added to this light brown powder, and once the powder was completely dissolved, it was poured into 6Q ether, the precipitate was filtered through a glass filter (G4), and the yellowish white powder of Jiaogulan saponin 9° 6g was obtained.

The physical properties of Jiaogulan saponin obtained as described above are as follows.

Properties of Jiaogulan saponin (+) Appearance, smell, taste It is a yellowish white powder, odorless and slightly bitter.

(2) Solubility Soluble in water, methanol, ethanol, insoluble in benzene, chloroform, ether, n-hexane, petroleum ether (3) pH of 1% aqueous solution is 6.1.

(4) IR (KBr) 3360.1630.1070,1040cJl-'(
5) Qualitative reactions Lieberman reaction, Sarcolasky reaction, and vanillin/sulfuric acid reaction are positive. Furthermore, when an 81% O 81% aqueous solution of quartz is vigorously shaken, persistent fine bubbles are produced.

(6) Thin layer chromatography A solution of crystal 50R9 dissolved in methanol 2xQ 1OμQ
When performing thin layer chromatography under the following conditions using as a sample solution, a thin layer chromatogram as shown in FIG. 1 is obtained.

Plate: Kieselgel 60F254 (Merck) Developing solvent: Chloroform-methanol-water mixture (65:
Detection: 1% ceric sulfate-10% sulfuric acid solution is sprayed and then heated at 105° C. for 5 minutes.

(7) High performance liquid chromatography Solution 10 in which main island 75 shoulder 9 was dissolved in methanol 2j112
When high performance liquid chromatography is performed using μg as a sample liquid under the following conditions, a high performance liquid chromatogram as shown in FIG. 2 is obtained.

Equipment: High performance liquid chromatograph L (-6A (with system controller 5CL-A) Column: L 1ch
rosorb RP-18 (manufactured by Merck & Co.) Inner diameter 4 m
m, length 25cx stainless steel column Column temperature = 30°C Mobile phase: Water-acetonitrile mixture (65.35) Flow rate: 0 min (0.5xL/min) - 15 min (0.5a+ff/
minutes) = 30 minutes (1,0x12/minute) → 45 minutes (2,4W
! /min) Detector: Ultraviolet spectrophotometer 5PD-6A (Takashi)
Measurement wavelength 202nm Sensitivity 0.16Aufs Data processing device: Chromatopack C-43A (Island 11t
) Chart bee 15 mm/min Next, the tonic effect of Jiaogulan saponin will be explained using experimental examples.

Experimental example! The animals used in the test were male and female ICR mice, and they were used for the test after 10 weeks of compliance. To observe sexual behavior, 10 female mice and 1 male mouse were allowed to live together for 10 minutes once a day, and the number of copulations was measured using a monitor TV. In order to keep female mice in constant estrus, estradiol benzoate was subcutaneously injected at 10μ9 per gram of body weight once a day from 4 days before the start of the experiment until the end of the experiment, and progesterone was injected once per gram of body weight 2 days before the start of the experiment. Ix9 was injected subcutaneously. Male mice were left untreated for the first 8 days, and the number of copulations was measured by allowing them to cohabit with female mice administered with the hormone once a day. Select male mice that were not recognized and prepare 3 groups of 8 mice per group: a control group and test groups A and B. From then on, the control group received 10x water once a day.
For test group A, an 8% aqueous solution of the reddish brown powder obtained by the water extraction procedure of Production Example 1 was added once a day to 10 mC/9 body weight (1 OOx9/ as Jiaogulan saponin).
kg body weight), test group B received 1% aqueous solution of Jiaogulan saponin obtained in Production Example 1 at a rate of 10 x ff/kg body weight once a day.
9 body weight (100 x 9/ as Jiaogulan saponin)
kg body weight), and sexual behavior was observed for 8 days in the same manner as before administration. In addition, the Student' - test or the χ" test was used for statistical processing of the experimental results. As a result, as shown in Table 1, the number of copulations in test group A and test group B during the test substance administration period and The number of mated animals increased significantly compared to the control group, and the test substances, the reddish-brown powder of Production Example 1 and Jiaogulan saponin, were found to have a strong tonic effect.The reddish-brown powder administered to test group A was found to have a strong tonic effect. First, the content of Jiaogulan saponin was determined by the method for quantifying Jiaogulan saponin devised by the present inventors (Public Patent Publication No. 6O-219566), and based on this quantitative result, administration group B was determined.
The same amount of Jiaogulan saponin was administered as that contained in the reddish-brown powder administered to administration group A, but there was no significant difference in the number of copulations or the total number of mated animals between the two groups. It became clear that the tonic effect was mainly due to Jiaogulan saponin.

Next, the toxicity of Jiaogulan saponin will be explained.

Experimental Example 2 The animals used in the test were ICR male mice, 8 mice per group. 4, weighing 18 to 229, and feeding them solid feed (MP, manufactured by Oriental Yeast Industry) and water ad libitum in a constant temperature and humidity room (23 ± i ° C, 55 ± 5%). They were preliminarily bred for a week, and those with good growth were selected for testing. After the test animals had been fasted for 16 hours, the test substance was orally administered or intraperitoneally injected. For oral administration, the test substance should be dissolved in water and
In the case of intraperitoneal injection, it was dissolved in physiological saline and administered at a dose of 10*(2/ky body weight).

Based on the mortality rate 168 hours after administration, the 50% lethal dose was calculated using the Van der Waerden method [Yoshio Ban, Pharmaceutical Research Methods, pp. 101-102, Asakura Shoten (1)
970)]. As a result, the 50% lethal dose of Jiaogulan saponin was 9 body weight at 712 sites/9 body weight (95% confidence limit: 655 to 775 H/9 body weight) when administered intraperitoneally to mice. 4000 H/kg for oral administration
No deaths were observed with administration of body weight, and LD! . measurement was not possible.

As is clear from the above toxicity tests, the toxicity of Jiaogulan saponin is extremely low and can be used as a pharmaceutical.

Considering the practical data and the results of acute toxicity tests regarding the tonic effect of Jiaogulan saponin, the dosage of Jiaogulan saponin according to the present invention may vary depending on the age, sex, weight, symptoms, etc., or it can be administered orally. 1 to 500*g per kg of body weight per day, preferably 10-
For parenteral administration, a range of 0.25 to 125 g, preferably 2.5 to 25 x g per kg of body weight per day is advantageous. In addition, it is preferable to administer the daily dose in several doses. In addition, depending on the symptoms, it is also possible to administer doses exceeding this upper limit.

Jiaogulan saponin can be used as a tonic as it is, but by adding excipients, adjuvants, etc. used in normal preparations, it can be prepared into powders, granules, tablets, capsules, and injections according to the usual method of preparation. It can be used in preparations such as drugs.

Next, the present invention will be specifically explained with reference to formulation examples, but the present invention is not limited thereto.

Formulation Example 1 9 parts of Jiaogulan saponin obtained in Production Example 1 were added to 5 parts by weight of the powder.
Mix evenly with 5 parts of lactose to form a powder.

Formulation Example 2 Granules 5 parts by weight of Jiaogulan saponin obtained in Production Example 1 were added to 9
Mix with 3 parts of lactose and granulate using 2 parts of hydroxypropylcellulose as binder in a conventional manner.

Formulation Example 3 Manufacturing example of 5 parts by weight of tablets! 9 Jiaogulan saponins obtained from
After mixing with 1 part of lactose and granulating in a conventional manner using 2 parts of hydroxypropylcellulose as a binder, 1 part of talc and 1 part of magnesium stearate are added and compression molded to obtain tablets.

Formulation Example 4 Production example of 5 parts by weight of capsules! 9 Jiaogulan saponins obtained from
Mix with 3 parts of lactose, granulate using 2 parts of hydroxypropyl cellulose as a binder in a conventional manner and fill into hard gelatin capsules.

Formulation Example 5 Injection 1 part by weight of Jiaogulan saponin obtained in Production Example 1 was added to 9
After heating and dissolving in 9 parts of physiological saline, the solution is sterilized to prepare an injection.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a thin layer chromatogram of Jiaogulan saponin according to the present invention. In the figure, * represents a dark red-purple spot, O represents a red-purple spot, and (:', * represent a light red-purple spot. FIG. 2 is a graph showing a high-performance liquid chromatogram of Jiaogulan saponin according to the present invention.

Claims (1)

[Claims] A tonic containing Jiaogulan saponin as an active ingredient