JPS6038376B2 - Cell activator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Gynostemma pentaphyllum makino, a perennial vine of the Cucurbitaceae family.
Concerning a Cell-Active Pharmaceutical Composition Containing as an Active Ingredient a Saponin Component Existing in the Whole Plant of Jiaogulan (Makino ntaphyllum MAKINO) The leaves of Jiaogulan have a sweet taste and have been used as a kind of sweetener in the folk industry.

This invention was made based on the new finding that the sabonin component in Jiaogulan has cellular effects on animals including humans and is useful as a stimulant. The saponin component of the present invention can be produced by extracting, separating, and purifying the whole plant of Jiaogulan, or by culturing the tissue of a section of the whole Jiaogulan plant, and then extracting, separating, and purifying it.

In this invention, the term "saponin component" refers to a mixture substantially consisting only of saponins obtained by these methods. Saponin components can be obtained from the whole Jiaogulan plant by the following method, for example.

The whole Jiaogulan plant or its dried product is extracted using water, lower aliphatic alcohols, or water-containing lower aliphatic alcohols, and the extract is concentrated to obtain an extract.This extract is extracted using ordinary fat-soluble organic solvents. is used to defatte and remove most of the chlorophyll. Next, this defatted extract is dissolved in water-saturated n-butanol, water is added to the solution, shaken well, and then allowed to settle. The upper n-butanol layer is separated and sugars and pigments are removed together with water. This n-butanol layer is evaporated to dryness, the residue is dissolved in a lower aliphatic alcohol, and the substance that precipitates when injected into a large amount of ether or benzene is taken out and dried in an oven to produce a product. The substance thus obtained contains substantially only saponin components and can be used as is as an active ingredient in the present invention. The overall properties of the saponin component of this invention are as follows:
It is a yellowish-white or odorless powder with a slightly bitter taste. It is easily soluble in methanol and diluted methanol, soluble in water and ethanol, and does not dissolve in benzene, chloroform, ether, hexane, and petroleum ether.

2 A 1% aqueous solution is neutral.

3 Infrared absorption spectrum・R maX(KBr)Cm-1:3370,1
650.1070, 10404 Nuclear magnetic resonance spectrum NMR (heavy pyridine) 6ppm: 4.0 (broad).
1.6 (Broad), 1.2 (Broad), 0.9 (Broad) 5 When this product is added to water and shaken, it generates persistent small bubbles.

6 Lieberman reaction and Zarkowski reaction are positive. 7 Sugars such as glucose, rhamnose, and xylose are obtained from the water-soluble part of the acid hydrolyzate, and panaxadiol (C38 ratio 203, melting point: 2060) and 26-hydroxypanaxadiol are obtained.

Based on these results, the saponin component of the present invention is considered to be a dammaran saponin. 8. Thin layer chromatography When this product is subjected to thin layer chromatography under the following conditions, a reddish-purple saponin spot as shown in Figure 1 appears.

Plate: Kieselgel, hexavalent 2I (manufactured by Merck) Developing solvent: Chloroform-methanol-water (65:35:1
0) Lower layer development distance: 10 meters Detection: 1% ceric sulfate-10% sulfuric acid solution was heated at 105q○ for 5 minutes after dawn.

. This saponin component can be separated and purified into each component saponin by silica gel column chromatography or high performance liquid chromatography, etc. to obtain each component saponin, but from a pharmaceutical standpoint, it is used after separating into each component saponin. It is more preferable to use them as a mixture.

Urao when this saponin component was administered intraperitoneally to mice
has an o/X9 of 755, and its toxicity is extremely low. Furthermore, when administered to humans, no side effects are observed. The composition of the present invention may be an internal preparation for oral administration, an injection for parenteral administration, or an external preparation, and comprises a saponin component and a solid or liquid excipient.

Most commonly, oral forms are preferred.

The dosage forms of oral preparations usually include several tablets, tablets, emulsions, capsules, tea tablets, and liquid preparations (including liquid extracts, syrups, etc.). Specific examples of excipients for oral preparations include powders, and other excipients for oral powders include lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, and dry water. Aluminum oxide, magnesium stearate, sodium bicarbonate, dried yeast, etc., and for topical powders, zinc oxide, talc, starch, kaolin, boric acid powder, zinc stearate, magnesium stearate, magnesium carbonate, precipitated calcium carbonate. , bismuth precipitate, potassium aluminum sulfate powder, etc.

Excipients in liquid formulations include water, glycerin, propylene glycol, simple syrup, ethanol, fatty oils, ethylene glycol, polyethylene glycol, sorbitol, and the like. Also, examples of liquid excipients for injections include sterile distilled water.

The dosage forms of external preparations include suppositories, ointments, liquids, powders for external use, syrups, atomizers, sieves, and emulsions.

The solid or liquid excipients used herein are those known in the art, and in the case of cartilage agents, fat,
Fatty oil, lanolin, petrolatum, glycerin, beeswax,
Hydrophobic bases or hydrophilic bases (emulsion bases, water-droppable bases and suspension bases) made by combining wax, paraffin, liquid paraffin, resins, higher alcohols, plastics, glycols, water, surfactants, etc. (including cloudy bases) are used as excipients. The above formulations may be made by methods known in the art, but are preferably formulated to contain the saponin component of this invention as required for a single dose. Furthermore, among these preparations, one that is convenient and appropriate is selected and used depending on the purpose.

The sabonin component of this invention is effective as a fine hoe activator (anti-aging, fatigue recovery agent, etc.).
It can be effective by administering it in three doses.

Administration may be either oral or parenteral. In the case of oral administration, it is carried out in the form of the above-mentioned internal medicine, and in the case of parenteral administration, it is injected into the body in the form of an injection, or absorbed directly under the armpit or mucous membrane in the form of a suppository or hydrophilic ointment. In the case of injections containing 5 to 50 saponin components per dose, suppositories or ointments, it is desirable to administer the required amount once at a concentration of 0.1 to 1% by weight. Next, a pharmacological test example will be described in which the sabonin component of the present invention has a medicinal effect as an activator (anti-aging, fatigue recovery agent).

Note that the "saponin component" used below means the saponin component of Jiaogulan obtained by the method of the above production example.

1 Cell Activation Lifespan Extension Test Example It is widely known that cells are the basic unit of biological life.

Currently, dividing cells, which are the basic units of these cells, are taken out from the tissue, dwarfed twice in a particularly suitable medium, for example, MEM medium supplemented with 10% calf serum, allowed to divide and proliferate, and then passed on to examine the nature and nature of their growth decline. It is now possible to do so.
The progress of this subculture is divided into three stages: the first stage, which is the primary doubling stage, is followed by the 0th stage, which grows at a constant rate.
No matter how you try to adjust the conditions, the rate of proliferation gradually declines and you reach the m-th stage, where you are unable to subculture and die. Phase 0 is the longest period, during which the cell's own protein synthesis function increases, DNA increases, and division actively occurs at a constant rate.
At the m-th stage, ribosome activity is observed in the cells themselves, the protein synthesis function decreases, passage becomes impossible, the cells die, and the lifespan of the cells ends. When the same medium is used, the number of passages at which the cells become unpassageable, which can be called the lifespan of the cells, is almost determined depending on the cell. For example, in the case of normal diploid fibroblasts (WI-1) isolated from the lungs of a human fetus, the cell division time in phase 0 (proliferation phase) is It repeats division at a rate of 35±2 hours and survives, but it weakens and dies at the m-th stage, becoming unable to be subcultured at the 51st generation. In addition, when human Wilner syndrome skin cells are used, they become unsubcultured and die during subculture in Matsuda. At this time, if a saponin component at a concentration of 200 muta/heart is added to the leased land at the beginning of the D period, cell activity on the first day will be prolonged and the number of rope changes will increase. That is, by adding a concentration of 200 mta/', it is possible to subculture up to 27 generations. 7%
It was confirmed that the lifespan of the product was extended. In addition, saponin components of 200
By adding Muta/No', it is possible to subculture up to 5 needles (15.7%)
It was confirmed that the lifespan of the product was extended. Next, an experimental example will be described. Experiment 1 Trypsin treatment of human Wirner syndrome skin tissue (
Use 0.05% trypsin aqueous solution to make a cell suspension, and add this to MEM culture solution* (10% calf serum added).
Niuetsugi 3 doubles with 0 (seeding ratio 1:4).

In 2 to 3 days, the cells will cover the entire glass surface.

This is treated with trypsin again to obtain a cell suspension, and the contents of one culture bottle are divided into two bottles and poured into MEM culture solution (added with 10% calf blood) for subculture. When subculturing was repeated regularly twice a week using the same procedure, no cell proliferation was observed due to the subculture of Matsushiro, making subculture impossible (control). On the other hand, saponin components were added to the culture solution from the 7th generation of subculturing to a concentration of 200 muta/ml, and the same procedure was applied to the culture solution in subsequent subcultures. If the subculturing was continued, the lifespan of the cells was significantly extended and the subculture was extended, as described below, compared to the cells that were not supplemented, which were unable to be subcultured at the female stage. Experimental Example 2 Normal diploid chick cells (W) isolated from human fetal lung tissue
I-1) as a cell suspension in the same manner as in Experimental Example 1.
Multiply the seam allowance into the M culture solution* (added with 10% calf blood).

When the same procedure was repeated twice a week regularly, no cell proliferation was observed after 51 generations of subculture, making it impossible to culture the chicks (control). On the other hand, saponin components were added to the culture solution from the 21st generation of subculture to a concentration of 200r/kite, and the same procedure was applied to the culture solution for subsequent subcultures. If you continue feeding, 5
Capable of doubling up to needles, increasing cell lifespan by 15.7%
I was able to extend it. Note* MEM culture solution (Minimum Essenti
alMediyama for Suspension) L-arginine hydrochloride 126. Monoo/SoL Cystine
24.0L-Glutamine
292.3L-Histidine Hydrochloride
41.9L-isoleucine 52.5
L-leucine 52.5L-lysine hydrochloride 731L-methionine
14.9L-Phenylalanine
deep red. OL-Threonine 47.6L
-Tryptophan 10.2L-Tyrosine
362L-Varin
469L-choline hydrochloride 1
．． 0D-Ca bentothenaate 1.0 Aoi acid
1U2 Swimming Fatigue Method '1' Test Method One group of 20 DR mice (male) weighing 15 to 20 days were fasted, and after 1 hour of water deprivation, Jiaogulan saponin components 50/K9 and 200 O/K9 were added to water. After combing and administering orally, 3% of the body weight was added to the ridge after 1 hour.
The durability time (seconds) was determined by swimming in water at zero temperature.

The same amount of water was orally administered to the control group. The fatigue limit was defined as when the mouse lowered its head more than 1 sand beneath the water surface.
■ Test Results As mentioned above, it is clear that the saponin component has the function of significantly prolonging swimming time and has an anti-fatigue effect.

The above pharmacological experiments confirm that the saporin component of the present invention has an activating effect on the cells themselves, prevents aging, extends lifespan, and has anti-fatigue effects.

,

[Brief explanation of the drawing]

FIG. 1 is a chromatogram obtained when the Jiaogulan saponin component of the present invention was subjected to thin layer chromatography under the following conditions. Carrier: Kieselgel F2 (manufactured by Merck & Co.) Solvent:
Chloroform/methanol/water (65:35:10 lower layer) Next to development: Other development Color: 1% ceric sulfate - 10% sulfuric acid goodwill 1
Heated at 05°C for 5 minutes.Each saporin red-purple star color (Kono) ■: Dark color ○: Normal, 0: Light color. Figure 1

Claims (1)

[Claims] 1. Gynostemma pentaphyllum
A cell activator containing the saponin component of Makino) as an active substance.