JPH0249730A - Method for extracting antitumor substance - Google Patents

Abstract

PURPOSE:To obtain an antitumor substance having stable and strong antitumor properties by extracting a crude drug by BAKU method with two kind of methods according to kind of the crude drug. CONSTITUTION:A crude drug selected from TENKISHI (semiaquilegiae redix), SOHGI, TETSUHAJU, SOUJISHI (Xanthii fructus), KUZETSUSOU, HOUBISOU (Dryopteris crassirhizomae rhizoma), YABUDOUTOU, TAKUSHITSU (root of Euphorbia Helioscopia L.), SUIYOUBAI, MOKKA (Chaenomelis fructus), SOUSHI, HOKOUEI (Taraxaci herba), KYUSEISHI (seed of Impatiens balsamica L.), BABOTSU (dried fruit body of Lasiosphaera nipponica Y.), RODORU (Euphorbiae pallasii radix), TOURIKON, RYOUKAOU, UDAIJU, NANSHOKUSHI, YOUTEIKON (root of Rumex japonicus Houtt.), KAGOSOU (Prunellae spica), HANPENREN (Lobeliae chinensis herba), MOKUFUYOU, SHUCHIN, HOKOKISHI, OUKASHA and BANRANKON (Baphicacanthis rhizoma et radix) is extracted with an acidic lower alcohol and shaken with n-butanol and them the butanol layer is collected to provide the active substance or a crude drug selected from JINKOTSUFU, KUBAKU (Dianthi herba), SEKISEN, NEKOBYOSOU, YOUTEIKON (root of Rumex japonicus Houtt.), KAGOSOU (Prunellae spica), HANPENREN (Lobeliae chinensis herba), MOKUFUYOU, SHOCHIN, HOKOKISHI, TOBANKI, SUIYOUBAI, MOKKA (Chaenomelis fructus), SOUSHI, HOROUEI (Taraxaci herba), KYUSEISHI (seed of Impatiens balsamica L.), BABOTSU (dried fruit body of Lasiosphaera nipponica Y.), SOUKASHA and OURANKON is extracted with a lower alcohol and extraction residue is extracted with hot water to provided the active substance.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for extracting antitumor substances from Chinese herbal medicines, and specifically relates to a method for extracting antitumor substances from Chinese herbal medicines, and specifically relates to a method for extracting antitumor substances from Chinese herbal medicines. Grass, wild grape wisteria, sawa lacquer, water yang plum, mokku, fusashi, kamagongei, acute child, mabo, kotozu, wisteria root, ryosaki, toriita tree, southern candle, false indigo root, skeletal style, konmugi , Iwami River, Cat Claw Grass, Yotei Root, Summer Dry Grass, Hanpen Lotus, Wood Furong, Stinky Camellia, Bamboo Shoot, Mubanki, Yellow Blue Root, and Soheguruma. A method for extracting antitumor substances from crude drugs, which involves converting the extract extracted with alcohol into an alkaline aqueous solution and collecting a butanol extract thereof, or collecting a hot water extract of the alcohol-extracted residue of the crude drug, and the extract. The present invention relates to a method of using the compound as an antitumor agent.

(Conventional technology) Each Chinese herbal medicine has its own unique pharmacological action, and more than 6,000 kinds of herbal medicines are listed in the Middle and Weak Encyclopedia (edited by Jiangsu New Medical Academy, Kamiyu People's Publishing Co., Ltd., 1977). There are more than 100 types of substances that are said to have tumor-causing effects. For about half of these, the substance of their antitumor activity has been elucidated.

However, the remaining half is said to have antitumor activity, but has not been studied in detail.

Although the active ingredients of some of them are unknown, they are thought to have relatively good signs of anticancer activity, and some are undergoing clinical trials or being combined with other herbal medicines. In Chinese medicine, only a single herbal medicine is rarely used, but a mixture of herbal medicines, or the combination thereof, is used, so the effect of the combined herbal medicine as a whole or its water-soluble parts cannot be determined. Therefore, it is unclear whether or not the herbal medicines that are said to have signs of anticancer activity contain antitumor active substances, and even if they do contain antitumor active substances, they cannot be efficiently It is unknown whether the material has been extracted.

(Problems to be Solved by the Present Invention) As mentioned above, the effects of Chinese herbal medicines are often judged based on the overall effect of the herbal medicines, partly because their active substances have not been confirmed. Herbal medicines that are thought to have antitumor properties do not necessarily contain antitumor substances. Therefore, it cannot be concluded that administration of a single herbal medicine will have an anticancer effect, and even if active substances are present, they are not necessarily efficiently extracted to the front side. The present invention relates to Chinese herbal medicines that are thought to have signs of tumor activity, and to determine whether the herbal medicines contain antitumor substances and, if so, to efficiently collect the active substances. The aim is to establish and use the substance as an antitumor agent containing the active substance.

(Means for solving the problem) As a result of the study in accordance with the above objectives, the herbal medicines that were recognized to contain antitumor substances were Tenkaiko, Jinkotsufu, Kumugi, Iwamiyo,
4 trees, iron-leafed trees, blue-eared grass, cat-claw grass, dog-tongued grass, thorn-tailed grass, wild grape wisteria, sawa lacquer, yotei roots, summer dried grass, grass-covered cars, half-side lotus, wood furong, fruit sticks, and hollyhock. These were the children, Kodoku, Fujitone, Mu Itanki, King Ryorai, Toriitashu, Nan Canshi, Huang Kuzi, Ita Aigen, Shui Yang Mei, Mokou, Sosashi, Fu Gongying, Kyukzi, and Ma Bo. These herbal medicines are considered based on the method of collecting their active substances, and are based on the following: Amansis, Takagi, Iron Leaf Tree, Blue-Eared Grass, Dog-tongue Grass, Feng-tail Grass, Wild Grape @ Wisteria, Sawa Lacquer, Water Yang Plum, Mokkuon,
Fusashi, Kamikoei, Kyuko, Mabo, Kotoku, Fujitone, Ryosakiou,
Toriboard tree, southern candle tree, (yotei root, summer dried grass, half-side lotus, Kifuyo,
The group of herbal roots (hereinafter referred to as herbal medicines A), the group of herbal roots (hereinafter referred to as herbal medicines A), the group of root roots, konmugi, iwami-yo, cat claw grass, yotei root, summer dried grass, hanpenren, Kifuyo, Jitsubo, Kamagoko,
杠板gui, (water yangmei, quince, fuzhi, puggongying, shuzi, mabo, saohesha) and huangjizi group (hereinafter these herbal medicines are referred to as herbal medicine B).
It is written as ) can be broadly classified into The crude drug A is obtained by extracting the crude drug with an acidic lower alcohol and distilling off the solvent to obtain an extracted extract. This extracted extract is dissolved in a dilute alkaline aqueous solution, shaken with n-butanol, the butanol layer is collected, and the butanol is distilled off to obtain the active substance. This active substance can be purified by conventional methods such as chromatography to obtain a pure product. The amount of acid added to the acidic lower alcohol solution to be used varies depending on the type of herbal medicine, but it is generally 1% or less, preferably 0.3 to 0.3%, based on the lower alcohol.
The lower alcohol is preferably methanol or ethanol, and is extracted under heating, but may be extracted at room temperature. Alkali concentration is 1% or less, preferably 0
．． It is about 1%.

The lower alcohol extraction residue of crude drug B is extracted with hot water, and the water is concentrated under reduced pressure to obtain an extracted extract. Generally, this extracted extract is used, but this extracted extract is further made into an acidic or alkaline solution and extracted with n-butanol,
Butanol extracts can also be used. The acid or alkali used in this case is a weak acid or a weak alkali.

The above-mentioned herbal medicines A and B groups include herbal medicines whose active substances can be obtained by any of the methods, such as yosei root, summer dried grass, hanpen lotus, wood furong, jitsubo, swamp root, water yang plum, quince, quince, It was also discovered that Pang Gong Ying, Chung Zi, and Ma Bo existed.

The substance thus obtained is stable and has strong antitumor properties as shown below, and can be easily made into tablets by conventional methods either alone or by mixing with other drugs, and can be used as an anticancer agent. Can be done.

(Function) The claimed substance obtained by extracting crude drugs with acidic alcohol or water, which is not possible with conventional techniques, is broadly classified into crude drug group A active substances and crude drug group 8 active substances, and each active substance is chemically Although isolation has not been confirmed in detail, based on the extraction method, it is estimated that the active substances of group A of crude drugs are nitrogen-containing compounds and alkaloids, and those of group B of crude drugs are phenolic compounds or glycosides.

The present invention will be specifically explained below with reference to Examples.

Shudo - 296g of wolf poison and 1.5 methanol! Add 5 ml of concentrated hydrochloric acid thereto, heat under reflux for 1 hour, and extract. This operation is repeated twice, the extracts are combined, and the solvent is distilled off under reduced pressure to obtain extract [1]. Yield 53.2g,
One gram of this was dissolved in 100 ml of 0.1% aqueous sodium hydroxide solution, extracted three times with an equivalent amount of n-butanol, and the solvent was distilled off under reduced pressure to obtain 165 ml of active material. (Wolf poison-1
).

Flame Lord - Under the same conditions as Example 1, 222 g of blue ear grass; 222 g of iron leaf tree
g; When 220g of Sawa lacquer is processed, the active substance, Soumiso-1
．． 11.7g, iron leaf tree-1,35,2g, sawa lacquer-1,3
Obtain 6.9 g.

Shaved - Add 1.52 g of methanol to 244 g of refined barley and heat under reflux for 1 hour for extraction. Separate the methanol and add 2 to the remaining medicine.
Add water from β and heat under reflux for 1 hour for extraction. The solvent was distilled off under reduced pressure to obtain 2.10.8 g of the active substance, refined barley.

■Soil - Using the same method as Example 3, 230g
g; Treated 222g of cat's claw grass to obtain active substance, Hikonefu-2
．． 6.2g; Iwami River-2,13g1 Cat Claw Grass-2,12,
4. Obtain g.

The antitumor effect of the substance obtained according to the present invention was determined by in vitro cell killing effect on cultured tumor cells and in animal experiments using experimental animal tumors.

The symbol -1 in the sample in the table indicates that this crude drug belongs to crude drug group A and was collected by the method in Example 1, and the symbol -2 indicates that this crude drug belongs to crude drug group B and was collected by the method in Example 3. It shows that it was done.

Example 5 Leukemia cells L-5178Y are subcultured in RPMI-1640 medium containing 10% live fetal serum with the active substance harvested according to the invention dissolved in saline.

1.0X105 pieces/m! ! After culturing in a 5% carbon dioxide incubator at 37°C for 48 hours, the living cells of the control group and the sample addition group were examined using trypan blue staining to determine the 50% inhibitory concentration (50% inhibitory concentration) for each active substance. I
Cs. ) was sought. The results are shown in Table 1.

Table 1 Tumoricidal cell effect on L-5178Y in vitro -1 Acute child -1 End of the tree -1 Yotei root -2 Summer dried grass -2 Half-sided lotus -2 Mokufuyo -2 Odor Camellia -2 Bamboo shoots -2 Yotei root -1 Summer dried grass -1 Half-sided lotus -1 Muffin root -1 Odor Tsubaki-1 Animal experiment Anti-tumor test using tumor P-388 Female BD
1.0 x 106 P-388 ascites cancer cells/mouse, which have been passaged once a week, were intraperitoneally transplanted into P I mice, and the active substance of the present invention was intraperitoneally administered from 24 hours after the transplant. was administered. The frequency of administration is once a day for 5 days.
The survival rate was calculated by comparing the survival days with that of the untreated group. The results are shown in Table-2.

Table 2 Anti-tumor effect on experimental animal tumor P-38,8 Iron leaf tree - 1200 μ/kg x 5 times 11.1% Blue ear grass -
1200〃X5// 18.1% Sawa lacquer-1200〃×
5 10.2% Poison -150 // X5 // 34.0%
10 〃 ×5 〃 16.
2% Equal pipe wind -2200// ×5〃15.0%
Wheat-2100// ×5 20.1% Iwamikawa-22 0 // X5// 11.3% Animal experiment llL
Antitumor test using Sarcoma 180 ascites-type Sarcoma 180 that has been passaged into female ICR mice.
．． 1 to 5 x 10' cells/mouse were implanted intraperitoneally in ICR mice, and the active substance of the present invention was intraperitoneally administered 24 hours after implantation. The frequency of administration was once a day for 5 days, and the survival rate was calculated by comparing the survival days with that of the untreated group. The results are shown in Table 3.

Table 3 Antitumor activity against experimental animal tumor Sarcoma 180 Sample Administration Fujitone - 1100 mg/kg Kiita Ki - 2190〃 Ryosaki - tioo〃 Toriitaju - 1100〃 Minami Toshi - 1100〃 Okutsuko - 2225〃 Itaikon -1100〃 Water yang plum -2100〃 Tree melon -2100〃 Zhuo sashimi-2100〃 Bamboo shoots-2150〃 Acute child-2100〃 Horse erection-2100〃 Quantity Life extension rate x 5 times 48.7% X5// 30.2% X5/ / 12.1% ×5 15.1% X5 // 13.3% X5 14.7% X 5 〃11.0% X5 // 15.3% X 5 // 13.5% X5 // 20.1% X 5 // 18.7% X 5 // 9.2% It became possible to manufacture the drug.

Claims (3)

[Claims] (1) Tenkazi, ■tree, iron leaf tree, blue-eared grass, dog tongue grass, thornweed grass, wild grape wisteria, sawa lacquer, water yang plum, mokku, ■thorn, kamagongei, acute child, horse erection, fox poison, Acidic lower alcohol of any one of the herbal medicines selected from Fujitone, Ryogeou, Toritaju, Nandanshi, Yotei root, summer dried grass, Hanben lotus, Kifuyo, stinky camellia, Kamako, Kusaheguruma, and Itaaikon. A method for extracting an antitumor substance, which comprises dissolving the extract in an alkaline aqueous solution and collecting a butanol extract thereof. (2) Ginkgo style, Kumugi, Iwami River, cat claw grass, yotei root, summer dry grass, half-side lotus, wood furong, stinky camellia, kaomako, kibanki, water yang plum, mokku, ■thorn, kamagongei, acute child, horse 1. A method for collecting an anti-tumor substance, which comprises extracting a hot water extract of the lower alcohol extraction residue of any one of the herbal medicines selected from the following. (3) A method of using the extract according to claim (1) and/or (2) as an antitumor agent.