CN109966335B

CN109966335B - Anti-tumor composition

Info

Publication number: CN109966335B
Application number: CN201910301589.1A
Authority: CN
Inventors: 朱晓新; 王娅杰; 屠鹏飞; 杨庆; 李琦; 李玉洁; 翁小刚; 陈颖; 蔡维艳; 阚月一
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-04-16
Filing date: 2019-04-16
Publication date: 2021-11-19
Anticipated expiration: 2039-04-16
Also published as: CN109966335A

Abstract

The invention relates to an anti-tumor composition, which is prepared by processing semen momordicae and herba epimedii which are used as raw traditional Chinese medicine raw material medicines, wherein the semen momordicae and the herba epimedii are fed with raw traditional Chinese medicine according to the weight ratio of 1-10:1-10, preferably 1-5:1-5, more preferably 3:25,1:1,2:1 and 1:2, and most preferably 1: 1.

Description

Anti-tumor composition

The technical field is as follows:

the invention relates to a traditional Chinese medicine composition, in particular to a combination of cochinchina momordica seed and epimedium herb in combined application.

Background art:

the traditional Chinese medicine has wide application in the aspect of anti-tumor, in particular to a plurality of traditional Chinese medicine combinations with definite anti-tumor effect. Based on the prior art, the invention provides a novel traditional Chinese medicine composition with an anti-tumor effect.

Semen Momordicae (academic name: Momoradica cochinchinensis (Long.) Spreng.), separately named: semen Strychni, semen Panici Miliacei, and fructus Benincasae. Perennial herbaceous vine wood has the effects of reducing swelling, resolving masses and dispelling toxicity. Nature and taste: bitter, slightly sweet and cool; is toxic. Meridian tropism: it enters liver, spleen and stomach meridians. The functional indications are as follows: dissipate nodulation and resolve swelling, counteract toxic pathogen and cure sore. Can be used for treating pyocutaneous disease, pyogenic infection, acute mastitis, scrofula, anal fistula, tinea, and tinea. The usage and dosage are as follows: 0.9-1.2 g; proper dosage is applied externally, ground into powder and mixed with oil or vinegar to be applied to the affected part. Semen Momordicae contains momordic acid, gypsogenin, oleanolic acid, alpha-eleostearic acid, amino acids, and sterols. Modern pharmacological research shows that the momordica cochinchinensis seeds have certain effects on reducing blood sugar, resisting bacteria, resisting cancer, easing pain and the like as a poison attacking medicine. However, because of its toxicity, its clinical application is limited, and it is used as a few commonly used Chinese herbs, since ancient times, it is mostly used externally, and it is rarely used orally.

Epimedium brevicornum Maxim, a perennial herb, and the whole herb of Epimedium brevicornum is used for medicine. It can be used for treating sexual impotence, premature ejaculation, soreness of waist, skelalgia, numbness of limbs, hemiplegia, neurasthenia, amnesia, tinnitus, and blurred vision. Epimedium herb is pungent, sweet and warm in flavor and acts on the liver and kidney meridians. Is an essential herb for nourishing life gate, benefiting vital essence and qi, strengthening bones and muscles, tonifying kidney and strengthening yang, and is clinically used for treating impotence and impotence, spermatorrhea, premature ejaculation, urinary incontinence, infertility of women and other symptoms. Herba Epimedii contains icariine, volatile oil, ceryl alcohol, phytosterol, tannin, vitamin E, etc. Can excite sexual function, and promote semen secretion for animals. It also has blood pressure lowering (peripheral vasodilation inducing), blood glucose lowering, diuretic, antitussive, expectorant, and vitamin E-like effects. Pharmacological experimental research shows that epimedium can increase blood flow of heart and cerebral vessels, promote hemopoiesis, immunity and bone metabolism, and has the functions of resisting senility, resisting tumor, etc.

Herba Epimedii is used as a tonifying Chinese medicine, and has an anti-tumor effect by enhancing immunity in modern clinical practice.

The invention applies the principle that when the offensive traditional Chinese medicines are used for treating malignant tumors, deficiency-tonifying medicines, blood-activating and stasis-dissolving medicines, heat-clearing medicines and the like are often combined to improve the treatment effect, and for malignant tumors which are easy to consume qi and damage the body fluid, the application of treatment means such as chemotherapy, radiotherapy and the like can cause deficiency of qi and yin, and the offensive traditional Chinese medicines are combined with the deficiency-tonifying medicines to support healthy qi, tonify essence and tiny, defend evil and resist cancer. The compatibility of counteracting-toxic herbs and tonifying deficiency herbs meets the compatibility principle of counteracting pathogenic factors and protecting the middle energizer to treat healthy qi as proposed in jin Kui Yao L ü e.

Therefore, the invention combines the semen momordicae and the epimedium in the traditional Chinese medicine to create a combination therapy, combines the two medicines, and mutually cooperates to eliminate pathogenic factors and strengthen body resistance so as to resist tumors.

The invention applies the Chinese medicine semen momordicae and epimedium together, controls the dosage reasonably, keeps the effective components, pays attention to compatibility and attenuation, lightens the side effect, enhances the immune function of the organism and achieves better anti-tumor effect.

The invention content is as follows:

the invention provides a traditional Chinese medicine composition which is prepared by processing semen momordicae and herba epimedii which are used as traditional Chinese medicine raw materials.

The composition of the invention comprises semen momordicae and herba epimedii which are added with crude drugs in a weight ratio of 1-10:1-10, preferably 1-5:1-5, more preferably 3:25,1:1,2:1 and 1:2, and most preferably 1: 1.

The composition of the invention is prepared by simultaneously or respectively extracting and processing semen momordicae and epimedium to obtain traditional Chinese medicine active substances, and then preparing the active substances into a pharmaceutical preparation composition by the conventional pharmaceutical preparation technology.

The invention relates to a method for simultaneously or respectively extracting and processing momordica cochinchinensis and epimedium, which comprises the following steps:

pulverizing semen Momordicae, extracting with 95% ethanol and water respectively, heating and reflux-extracting the extractive solution for 2 times (2 hr each time), mixing extractive solutions, filtering with filter paper, and rotary-steaming the extractive solution.

Respectively extracting herba Epimedii with 95% ethanol and water, filtering the extractive solution, rotary steaming, freeze drying the water extract, and drying the ethanol extract in vacuum drying oven.

The invention respectively obtains four extracts of cochinchina momordica seed alcohol extract, cochinchina momordica seed water extract, epimedium alcohol extract and epimedium water extract by different extraction methods

The four extracts can be combined in different ways:

combining 1 semen Momordicae alcohol extract and herba Epimedii alcohol extract

Combination of 2 Water extraction of Momordica cochinchinensis seed and Water extraction of Epimedium

Combined with 3 alcohol extraction of cochinchina momordica seed and water extraction of epimedium

Combining 4 water extraction of semen momordicae and alcohol extraction of herba epimedii

The most preferable of the invention is the combination of 1 the momordicae semen alcohol extract and the epimedium alcohol extract, and certainly, the momordicae semen and the epimedium can be mixed and then extracted by ethanol to obtain a mixed extract.

The medicine extraction method of the invention can adopt the following method:

pulverizing semen Momordicae, sieving with No. 10 sieve, extracting with 95% ethanol and water respectively, heating and reflux-extracting the extractive solution for 2 times, each for 2 hr, mixing extractive solutions, filtering with filter paper, and rotary-steaming the extractive solution.

The epimedium crude product is directly extracted by adopting 95 percent ethanol and water respectively, and the extraction method is the same as that of the cochinchina momordica seed. After rotary evaporation, the aqueous extract is subjected to freeze drying treatment, and the alcohol extract is dried for 24 hours by a vacuum drying oven. Respectively obtaining four extracts of cochinchina momordica seed alcohol extract, cochinchina momordica seed water extract, epimedium alcohol extract and epimedium water extract by different extraction methods,

the invention takes the extract as the active ingredient of the traditional Chinese medicine, and the traditional Chinese medicine preparation composition is prepared after processing.

According to the traditional Chinese medicine preparation composition, a pharmaceutically acceptable carrier can be added according to the needs, wherein the weight percentage of the traditional Chinese medicine active ingredients in the preparation can be 0.1-99.9%, and the balance is the pharmaceutically acceptable carrier. The Chinese medicinal preparation composition can exist in a unit dosage form, and the unit dosage form refers to a unit of preparation, such as each tablet of a tablet, each capsule of a capsule, each bottle of oral liquid, each bag of granules and the like.

The Chinese medicinal preparation composition can be any medicinal dosage forms, and the dosage forms comprise: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal agents, granules, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches. The formulations of the present invention, preferably oral dosage forms, are: capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent. Most preferred are tablets.

The Chinese medicinal preparation composition of the present invention, which is a preparation for oral administration, may contain conventional excipients such as binders, fillers, diluents, tabletting agents, lubricants, disintegrants, coloring agents, flavoring agents and wetting agents, and the tablet may be coated if necessary.

Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.

The solid oral compositions can be prepared by conventional methods of mixing, filling, tabletting and the like. Repeated mixing can distribute the active throughout those compositions that use large amounts of filler. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerol, propylene glycol or ethyl alcohol; preservatives, for example p-hydroxybenzyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

For injections, liquid unit dosage forms are prepared containing the active substances of the invention and a sterile carrier. Depending on the carrier and concentration, the compound may be suspended or dissolved. Solutions are generally prepared by dissolving the active substance in a carrier, filter sterilising before filling it into a suitable vial or ampoule and then sealing. Adjuvants such as a local anaesthetic, preservatives and buffering agents may also be dissolved in the vehicle. To improve its stability, the composition can be frozen after filling into vials and the water removed under vacuum.

The traditional Chinese medicine preparation composition can be selectively added with a suitable pharmaceutically acceptable carrier when being prepared into a medicament, and the pharmaceutically acceptable carrier is selected from the following components: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, EDTA disodium, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and derivatives thereof, alginates, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid-based materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.

The preparation of the invention can be used for determining the usage amount according to the condition of a patient, and can be taken three times a day, 1-20 doses each time, such as: 1-20 bags or granules or tablets.

The beneficial effects of the present invention are further illustrated by experimental data as follows:

the invention adopts the momordica cochinchinensis seed and the epimedium herb of the embodiment 1 to extract the mixture by alcohol according to the proportion of 1:1 in the pharmacodynamic experiment.

Through optimization screening, the invention discovers that when the momordica cochinchinensis and the epimedium are used together in a ratio of 1:1, the momordica cochinchinensis and the epimedium have more obvious inhibition effect on tumor cells than the two medicines are used alone, and the two medicines have smaller influence on normal cells LO2 after being used together, so that the effect of attenuation and synergy are supposed to be realized when the two medicines are used together.

The experimental data are as follows:

experiment 1, MTT colorimetric method is used for researching the antitumor activity of momordica cochinchinensis and epimedium, so that an effective combination proportion and an antitumor spectrum are screened out. And different proportions of the drug were applied to normal hepatocytes LO 2. The effect of different ratios of drug on LO2 proliferation was observed by MTT method.

The data are as follows:

TABLE 1 inhibition rate of Momordica cochinchinensis seed and Epimedium brevicornum on A549 cell 72h by single decoction or combination

P <0.05 with statistical difference compared to placebo; ■ the p of the alcohol extract is less than 0.05 compared with the water extract, and the statistical difference is obtained; the ratio p <0.05 between single decoction and mixed decoction is statistically different; it is statistically different from Yu Yi Jian with p < 0.05.

TABLE 2 inhibition rate of Bel7402 cells for 72h by single decoction and combined decoction of semen Momordicae and herba Epimedii

TABLE 3 inhibition rate of SK-OV-3 cells by single decoction and combined decoction of semen Momordicae and herba Epimedii for 72h

TABLE 4 inhibition rate of single decoction and combined decoction of semen Momordicae and herba Epimedii on HT-29 cells for 72h

TABLE 5 inhibition rate of single decoction and combined decoction of semen Momordicae and herba Epimedii on MGC803 cells for 72h

The above table shows that the two drugs have certain proliferation inhibition effects on different human tumor cells, and by comparing IC50 of different groups, the proliferation inhibition rate of the cells is higher than that of the drugs when the momordica cochinchinensis and the epimedium are used in combination in a ratio of 3: 25. In order to further screen out the optimal proportion of the two drugs, the screening range of the proportion of the two drugs is expanded, and the drugs which are combined in different proportions are respectively applied to the A549 cells and the MGC803 cells with obvious action effects. The results are shown in Table 6.

TABLE 6 Epimeritol refers to the inhibition rate of two drugs on A549 and MGC803 cells for 72h in different combination ratios

Statistical differences from placebo p <0.05

As can be seen from table 6, the effect of inhibiting the proliferation of tumor cells was significant when the two drugs were used in a ratio of 3:25 to 1: 1. To further compare the effect of two different ratios on cells, the toxicity of momordicae semen and epimedium herb in different ratios on normal cells was observed by allowing momordicae semen and epimedium herb in a ratio of 3:25 to 1:1 to act on normal hepatocytes for 72h, and the results are shown in table 7.

TABLE 7 Epimeritol refers to the inhibition rate of two drugs on LO2 cells at different combination ratios

Table 7 shows that the alcohol extract of epimedium or the compatibility of cochinchina momordica seed and epimedium in different proportions has slight inhibition effect on normal liver cells. In contrast, when the medicines are combined in a ratio of 1:1, the influence on normal liver cells is weak, and the cells even have a negative growth trend. Thus, it was preliminarily presumed that the two drugs had a synergistic effect of attenuation when used in combination at a ratio of 1: 1.

Experiment 2, a Chou-Talalay combined index method is used for further verifying the synergistic and attenuated effects of the combined action of the two medicines on A549. Finally, the Combination Index (CIs) and the dose-reduction index (DRI) of the momordica cochinchinensis and the epimedium are calculated, and the combination ratio of the two medicines is 1: 1.

The results are shown in FIGS. 1 to 4.

Experiment 3. to observe the influence of the drugs on the cell proliferation capacity more intuitively, the plate clone formation capacity of the A549 cells acted by different concentrations after the two drugs are combined in a ratio of 1:1 is detected, and the statistical analysis is carried out by a factorial analysis method, so that the result shows that compared with the blank control group cells, the plate clone formation capacity of the drug-added group cells is obviously reduced, and the difference has statistical significance (P is less than 0.001) compared with the blank control group.

The results are shown in FIG. 5 and FIG. 6.

Experiment 4. in vivo constructing Lewis lung cancer model, verifying the anti-tumor effect of epimedium, the result is consistent with the in vitro experiment, and the two drugs can obviously inhibit the proliferation of Lewis subcutaneous transplantation tumor after being used together.

The results are shown in FIG. 7

TABLE 8 comparison of tumor mass and tumor inhibition ratio for different administration groups (n ═ 10)

TABLE 9 Effect of drugs on the mouse immune organs (n ═ 10)

Experiment 5, the expression levels of IFN-gamma and IL-4 in the serum of mice of each group are detected by ELISA, and the common action of the epimedium is considered to enhance the autoimmune function of Lewis lung cancer mice. The results are shown in FIG. 8:

description of the drawings:

FIG. 1 shows the relationship between the concentration and the inhibition ratio when different combination ratios act on A549

FIG. 2 shows the relationship between CI coefficient and inhibition ratio of A549 acted by different combination ratios

FIG. 3 shows the relationship between the inhibition rate and DRI value when two drugs act on A549 in a ratio of 3:25

FIG. 4 shows the relationship between the inhibition rate and DRI value when two drugs act on A549 at a ratio of 1:1

FIG. 5A 549 cell plate cloning capacity when two drugs are used together

FIG. 6 clone number comparison of A549 after the drugs with different concentrations act on them represents that the difference of the two drugs is significant (P <0.01)

FIG. 7 tumor volume changes in different groups (P >0.05)

FIG. 8 comparison of IFN-. gamma.and IL-4 levels in the middle dose group and the blank control group

FIG. 9. Process for extracting Epimedium cochinchinensis

The specific implementation mode is as follows:

the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1

Pulverizing semen Momordicae, sieving with No. 10 sieve, extracting with 95% ethanol respectively, heating and reflux extracting for 2 times (each for 2 hr), mixing extractive solutions, filtering with filter paper, and steaming to remove ethanol to obtain extract.

Extracting herba Epimedii with 95% ethanol under reflux for 2 times (2 hr each time), mixing extractive solutions, filtering with filter paper, and evaporating to remove ethanol to obtain extract.

Mixing the two extracts to obtain the medicinal active substance.

Example 2

Extracting semen Momordicae and herba Epimedii with 95% ethanol under reflux for 2 times (2 hr each time), mixing extractive solutions, filtering with filter paper, and steaming to remove ethanol to obtain mixed extract of the two drugs.

Example 3

Pulverizing semen Momordicae, sieving with No. 10 sieve, extracting with water, heating and reflux-extracting the extractive solution for 2 times (each for 2 hr), mixing extractive solutions, filtering with filter paper, and rotary-steaming the extractive solution.

The epimedium crude product is directly extracted by water, and the extraction method is the same as that of the cochinchina momordica seed. After rotary evaporation, the aqueous extract is subjected to freeze drying treatment, and the alcohol extract is dried for 24 hours by a vacuum drying oven.

Mixing the two extracts to obtain the medicinal active substance.

Example 4

Extracting semen Momordicae and herba Epimedii with water directly, heating and reflux-extracting the extractive solution for 2 times (2 hr each time), mixing extractive solutions, filtering with filter paper, and rotary steaming the extractive solution to obtain mixed extract of the two medicines.

Example 5

5g of the mixed extract obtained in the example 1 is uniformly mixed with 15g of starch, and povidone is added to be used as a binding agent, and the mixture is granulated and tabletted to obtain 100 tablets.

EXAMPLE 6 injection

Mixing 5g of the extract of example 2, 4.5g of glucose, 0.9g of sodium thiosulfate and 1ml of distilled water uniformly, filtering, sterilizing and packaging 100 pills.

Claims

1. A traditional Chinese medicine composition is prepared by processing semen momordicae and herba epimedii which are used as traditional Chinese medicine raw materials, wherein the semen momordicae and the herba epimedii are fed with crude drugs, the weight ratio of the semen momordicae to the herba epimedii is 1-5:1-5, four kinds of extracts of semen momordicae alcohol extraction, semen momordicae water extraction, herba epimedii alcohol extraction and herba epimedii water extraction are respectively obtained from the traditional Chinese medicine raw materials through different extraction methods, and the four kinds of extracts have different combinations as follows: the method comprises the steps of 1 cochinchina momordica seed alcohol extraction and epimedium alcohol extraction, 2 cochinchina momordica seed water extraction and epimedium water extraction, 3 cochinchina momordica seed alcohol extraction and epimedium water extraction, and 4 cochinchina momordica seed water extraction and epimedium alcohol extraction.

2. The Chinese medicinal composition according to claim 1, wherein the semen Momordicae and herba Epimedii are dosed in a weight ratio of 1:1,2:1 or 1: 2.

3. The Chinese medicinal composition according to claim 1, wherein the semen Momordicae and herba Epimedii are dosed at a weight ratio of 1: 1.

4. The Chinese medicinal composition according to claim 1, which is prepared by extracting semen Momordicae and herba Epimedii simultaneously or respectively to obtain Chinese medicinal active substances, and preparing the active substances into pharmaceutical preparation composition by conventional pharmaceutical preparation technology.

5. The composition of claim 1, wherein the composition is prepared by combining 1 the alcohol extraction of momordicae semen and the alcohol extraction of epimedium herb, or the mixed extract is prepared by mixing momordicae semen and epimedium herb and then extracting with ethanol.

6. The Chinese medicinal composition according to claim 1, wherein a pharmaceutically acceptable carrier is added as required, wherein the weight percentage of the Chinese medicinal active ingredients in the preparation is 0.1-99.9%, and the balance is the pharmaceutically acceptable carrier.