JPH0469633B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention is directed to Solaunm.
lyratum Thunb (Hakuei, Hakue Wisteria) as an active ingredient. [Prior Art and its Problems (Background of the Invention)] Bulbul japonica is a climbing perennial herb belonging to the Solanaceae family that grows wild in the mountains and roadsides of Japan, India, mainland China, etc., and has been used as a folk medicine to treat fever and reduce fever. diuretic,
Although it is known to have effects such as tonicity, stomach health, and analgesic, there is a general tendency to avoid using it because it is said to be toxic, and its effects, ingredients, toxicity, etc. have not yet been investigated. It wasn't done enough. In view of this current situation, the inventors of the present invention conducted a detailed study on the physiological effects of the ingredients contained in Bulbul stingray, and found that
The present inventors have discovered that an extract of P. elegans with water has an antitumor effect and, surprisingly, is not particularly toxic, leading to the completion of the present invention. [Structure and Effects of the Invention] The gist of the present invention, which was made against the background of the above circumstances, is to construct an antitumor agent containing a red-spotted bulb extract as an active ingredient.
In the present specification, the term "Libernator stingray extract" refers to an extract obtained by extracting Red-breasted bulbul with water, a water-soluble organic solvent, or a mixture of water and a water-soluble organic solvent. The extract used as an active ingredient in the present invention is, for example, fresh, dried, or semi-dried leaves, stems, foliage, and whole plants of the bulbul extract used in water or a water-soluble organic solvent (e.g., methanol, ethanol, etc.). After filtration, the extract obtained by leaching at room temperature or heating with a mixture of these alcohols or other alcohols) or a mixture thereof is dried by a conventional drying method such as spray drying, freeze drying, or concentration drying. can be obtained.
Note that it is also possible to use other known water-soluble organic solvents. The extract obtained in this way has an excellent suppressive effect on various tumors. For example, as described below
It shows an excellent suppressive effect on Sarcoma180, and also shows a specific suppressive effect on JTC-26 (human cervical cancer-derived cells). It also has extremely low toxicity. Oral administration or parenteral administration such as subcutaneous injection is adopted for the bulbul extract. For oral administration, the extract can be used as is, but
Add excipients, binders, lubricants, adjuvants, etc. used in ordinary formulations and follow the usual method for manufacturing formulations.
It is administered in the form of powders, granules, tablets, capsules, syrups, etc., and parenteral administration includes injections such as aqueous solutions, suspensions, oily or aqueous emulsions, and aqueous liquids such as sterile water and physiological saline. It is administered in the form of an injection prepared by dissolving or suspending it in a medium.
The extract may be further purified and used by conventional methods such as dialysis and various chromatography methods. Although the dosage varies depending on symptoms, age, and dosage form, it is usually desirable for adults to be about 1 g per day for oral administration, and about 60 mg per day for parenteral administration. [Examples] The present invention will be further explained below with reference to Examples and Test Examples, but the present invention is not limited thereto. Example 1 60kg of dried bulbul leaves and 1600g of water
was added and extracted at 100°C for 1 hour using an extract extractor, and after filtering insoluble matter, the resulting extract was concentrated under reduced pressure at 40°C and further spray-dried to obtain 4 kg of yellowish brown dry extract powder. The yield was about 6.7%. Example 2 45 kg of dried whole plant of Bulbul spp. at 100℃
Extract with hot water for 1 hour and filter the insoluble matter.
A soft extract (fluid extract) 38 with a concentration of 12% was obtained by vacuum low temperature concentration at 40°C. The amount of dry extract powder per ml of extract was 0.28 g/ml. Formulation Example 1 (Tablet) Approximately 2 kg of dry extract powder produced in Example 1 above
, about 4 kg of lactose, about 4 kg of magnesium stearate
This mixture was compressed using a single-shot tablet machine to produce tablets with a diameter of 9 mm and a weight of approximately 330 mg.
Take 3 to 4 tablets at a time, 3 times a day, depending on your symptoms. Formulation Example 2 (Capsule) 166 mg of the dried extract powder prepared in Example 1 was filled into hard capsules. This capsule is taken 1 to 2 capsules at a time, three times a day, depending on the symptoms. Test example 1 Inhibitory effect on Sarcoma180 using mice (sample preparation) Extract 700 g of air-dried stems of the sagebrush with warm water at 60°C, and partition the extract between the solvents of water and n-butanol to form an aqueous layer. and butanol (BuOH)
The aqueous layer fraction is further divided into Amberlite.
By column chromatography using XAD-2 (trade name), methanol (MeOH) eluate,
It was fractionated into an aqueous eluate. (Sample Administration and Effect) The activity against Sarcoma180 was investigated using the test solution obtained from the above fractionation. In other words, aqueous layer, BuOH layer,
Fractions of water eluate and MeOH eluate were collected, respectively.
When administered once a day for a total of 10 times at a rate of 2000, 3000, 3000, and 1000 mg/Kg body weight, proliferation was suppressed compared to the control group (no administration group) as shown in the table. In particular, the BuOH layer showed the strongest suppressive effect when the tumor weight was approximately one-third that of the control group.

【table】

[Table] Test example 2 JTC-26 using mice (human cervical cancer-derived cells)
Inhibitory effect on (preparation of sample) Bulbul stingray was extracted as shown in FIG. 1 to obtain a sample. i.e. 1.1 stems of air-dried bulbul
Repeat the extraction of Kg with warm water at 60℃ 3 times and extract the extract.
The mixture was concentrated under reduced pressure at a temperature below 60°C to obtain 133 g of water extract.
After treating this with 400 ml of ethyl acetate (AcOEt), 124 g of the residue was suspended in 3 parts of water and extracted by shaking with BuOH3. On the other hand, the stems after hot water extraction were heated with MeOH2.
The extraction was repeated twice, and the extract was concentrated under reduced pressure.
38 g of MeOH extract was obtained. In addition, water extract,
When the AcOEt soluble part, BuOH layer, aqueous layer, and MeOH extract were searched using thin layer chromatography, the chromatogram shown in Figure 2 was obtained. It was found that mainly sugar was transferred. The BuOH layer was then concentrated under reduced pressure to obtain a residue 51
g and Sephadex LH-20 as shown in Figure 1.
Column chromatography using (product name),
Repeated silica gel column chromatography, recrystallization, etc., steroid saponin SL-0(2), SL
−(1) and SL−(3) respectively as crystals, 7.9,
1.6, 3.4g were obtained. The yields based on dry raw materials were 0.72, 0.15, and 0.31%, respectively. Further, from the MeOH extract, 3.9 g (0.36% based on the dry raw material) of SL-(1) was obtained by repeating silica gel column chromatography. (Sample administration and effect) Steroid saponin SL-0 obtained by the above method
(2), SL-(1), and SL-(3) were examined for their effects on JTC-26 (human cervical cancer-derived cells).
MEM JTC-26 to 1×10 ⁵ pieces/ml
Eagles90%, Feta-1Calf Serum (Product name:
The cells were placed in a 10% medium (manufactured by Microbiological), and the compound was further adjusted to each concentration and injected. Five samples were prepared from each sample, and the non-additive sample was used as a control group and incubated at 37°C in a _CO2 stream for 144 hours.The number of viable cells was counted and the average value was compared with the control group to inhibit proliferation. The rate was measured. The results are shown in the table. From this result, SL−(1) and
It was found that SL-(3) had a strong inhibitory effect.
SL-(3) shows no activity at low concentrations, but at 8.0μ
When the concentration is higher than g/ml, the inhibition rapidly reaches 100%. On the other hand, furostanol glycoside SL
-0(2) showed no activity at all. As can be seen from the above results of investigating the inhibitory effect on JTC-26, spirostanol glycoside showed activity, but furostanol glycoside showed no activity at all.

【table】

[Table] Acute toxicity test Example 1 on mice (BALB/c, Anw Crj)
Acute toxicity tests were conducted by administering orally or intraperitoneally the red-spotted bulb extract obtained in 2. In the case of oral administration, the Psyllium japonica extract obtained in Example 2 was administered to groups of 10 8-week-old male mice at doses of 5.6 g/Kg body weight and 11.2 g/Kg body weight. LD ₅₀ after 72 hours is 11.2
g/Kg or more. In the case of intraperitoneal administration, the extract concentration of the brown-spotted bulbul extract obtained in Example 1 was 0.38 g/Kg, 0.95 g/Kg, 1.9 g/Kg, or 3.8 g/Kg.
The suspension was suspended in physiological saline at a concentration of 7.6 g/Kg, and administered to a group of 10 4-week-old male mice at a volume of 10 ml/Kg. LD ₅₀ after 72 hours was 2.05 g/Kg. The LD ₅₀ of the hot water soluble fraction of dried leaves of Kumazasa is 10 g/Kg or more when administered orally and 2.20 g/Kg when administered intraperitoneally.
(Published in the Japan Pharmacological Journal by Mr. Shibata et al. of Hoshi Pharmaceutical University in 1975: Pharmacological research on bear bamboo).
LD ₅₀ is also about the same level. In addition, acute toxicity tests were conducted using 4-week-old and 8-week-old male and female mice (BALB/c, AnwCrj), 6-week-old female rats (Crj, Wistar), and 4-week-old female guinea pigs (CrJ, Hartley). (In the case of administration for 8 weeks, the internal intake per animal body weight is approximately 38g/Kg in terms of dry extract powder for mice, approximately 12.2g/Kg for rats, 14.8g/Kg for guinea pigs, and 14.8g/Kg for guinea pigs. That is 55g/Kg for mice and 55g/Kg for rats.
(16.7 g/Kg, 24.4 g/Kg for guinea pigs), but no significant changes due to the difference in period were observed in the 8-week and 13-week observations, and the general condition was normal, and body weight changes and food intake were also compared. No significant difference was observed compared to the groups. Furthermore, no gender-related changes were observed in histopathological findings, protein metabolism, etc., and no significant differences were observed in blood cell counts, etc. compared to the control group. Furthermore, histopathological observations of major organs etc. were normal in mice, rats, and guinea pigs, and no significant abnormal conditions were observed overall. From the above tests, it is clear that the toxicity of the Red-spotted Bulbul extract is extremely low. Incidentally, in the above-mentioned examples, dried lily pads are used, but fresh or semi-dried ones can also be used.

[Brief explanation of drawings]

FIG. 1 is a diagram of the fractionation process of a test sample of the present invention, and FIG. 2 is a thin layer chromatogram of a red-spotted bulb extract.

Claims (1)

[Claims] 1. An anti-tumor agent containing bulbul extract as an active ingredient.