CN114470139B

CN114470139B - Traditional Chinese medicine composition for treating liver cancer and preparation method thereof

Info

Publication number: CN114470139B
Application number: CN202210343852.5A
Authority: CN
Inventors: 韩卫东
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2023-06-30
Anticipated expiration: 2042-04-02
Also published as: CN114470139A

Abstract

The invention provides a traditional Chinese medicine composition for treating liver cancer, which comprises, by mass, 10-20 parts of rhizoma polygonati, 20-40 parts of astragalus, 10-20 parts of pagodatree flower, 10-20 parts of purslane, 10-20 parts of dandelion, 10-20 parts of curcuma longa, 10-20 parts of medlar and 10-20 parts of agaric. The traditional Chinese medicine composition integrates the functions of tonifying qi and blood, clearing heat and detoxicating, cooling blood and stopping bleeding, strengthening vital qi to eliminate pathogenic factors, inhibiting the growth of liver cancer cells and treating liver cancer, and provides a novel traditional Chinese medicine compound for treating liver cancer; the composition can be used together with cyclophosphamide, has good synergistic effect, and can effectively reduce the damage of immune system caused in the use process of cyclophosphamide; the application utilizes lactobacillus casei and lactobacillus bulgaricus to ferment for the first time to obtain the anti-liver cancer traditional Chinese medicine composition, can exert the compound treatment effect to the maximum extent, and provides a new method for preparing medicines.

Description

Traditional Chinese medicine composition for treating liver cancer and preparation method thereof

Technical Field

The application relates to the field of medicines, in particular to a traditional Chinese medicine composition for treating liver cancer and a preparation method thereof.

Background

Liver cancer is malignant tumor of liver, and the newly discovered liver cancer patients and the patients dying from liver cancer in China are about 11 ten thousand people each year, accounting for 45% of the world's liver cancer patients, and are one of three fatal cancers. And the liver cancer patients have no early symptoms or symptoms, develop rapidly and have high malignancy degree, and the liver cancer patients can usually live for three to six months on average after diagnosis. Therefore, the prevention and treatment of liver cancer is one of the tasks of medical and health industries of China. Cyclophosphamide is commonly used as an anti-tumor drug at present, but the cyclophosphamide is easy to cause immune system injury in the use process.

The traditional Chinese medicine is the magnificent of China. In long-term clinical practice of traditional Chinese medicine, abundant experience is accumulated for diagnosis and treatment of liver cancer, a large amount of traditional Chinese medicine prescription data is reserved, and the traditional Chinese medicine is currently used as an effective auxiliary method for treating liver tumor. However, at present, a traditional Chinese medicine compound which has obvious curative effect on liver tumor and can assist cyclophosphamide treatment is not available.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine compound which has obvious curative effect on liver tumors, can assist in cyclophosphamide treatment and reduce immune system injury caused by cyclophosphamide.

On the one hand, the application provides a traditional Chinese medicine composition for treating liver cancer, which comprises, by mass, 10-20 parts of rhizoma polygonati, 20-40 parts of astragalus membranaceus, 10-20 parts of sophora japonica, 10-20 parts of purslane, 10-20 parts of dandelion, 10-20 parts of curcuma zedoary, 10-20 parts of wolfberry fruit and 10-20 parts of agaric.

Further, the composition comprises 15 parts of rhizoma polygonati, 30 parts of astragalus, 15 parts of sophora flower, 15 parts of purslane, 15 parts of dandelion, 15 parts of curcuma longa, 15 parts of medlar and 15 parts of agaric.

Traditional Chinese medicine considers that the human body has vital qi deficiency and viscera disorder, so that pathogenic toxin enters the body to unbalance yin and yang and deficiency of both qi and blood, and causes pathological changes such as qi stagnation, blood stasis and the like, and tumors are formed for a long time. Therefore, the traditional Chinese medicine treatment method for treating tumors is mainly used for strengthening body resistance and eliminating evil. Strengthening body resistance has the methods of tonifying qi, enriching blood, nourishing yin, warming yang and the like; it has the functions of clearing away heat and toxic matter, eliminating blood stasis, softening hard mass, etc. The prescription has the functions of invigorating qi and blood, clearing heat and detoxicating, cooling blood and stopping bleeding, and strengthening body resistance and eliminating pathogenic factors.

Most of the single traditional Chinese medicines cannot effectively treat complex diseases, and if one medicine is taken for a long time, germs in a patient body can generate drug resistance; the compound combines a plurality of traditional Chinese medicines according to the compatibility principle, and can synergistically exert the curative effect.

In a preferred embodiment the compound composition is as follows:

15g of rhizoma polygonati, 30g of astragalus, 15g of sophora flower, 15g of purslane, 15g of dandelion, 15g of curcuma longa, 15g of medlar and 15g of agaric.

In another aspect, the present application further provides a method for preparing the above composition, where the method includes the following steps:

crushing rhizoma polygonati and astragalus, and extracting with an alcohol solvent to obtain a first extract and dregs;

step two, mixing the residues in the step one, the crushed agaric and the crushed medlar uniformly, and extracting by taking water as a solvent to obtain a second extract;

step three, taking pagodatree flower and curcuma zedoary, and carrying out ultrasonic extraction by taking ethyl acetate as a solvent to obtain a third extract;

step four, taking purslane and dandelion, and extracting with water as a solvent to obtain a fourth extract;

step five, combining the first extract, the second extract, the third extract and the fourth extract, uniformly mixing, and freeze-drying to obtain traditional Chinese medicine composition powder;

the method further comprises the step of fermenting the traditional Chinese medicine composition powder, wherein the inoculation ratio of the lactobacillus casei and lactobacillus bulgaricus which are fermented is 1: (1-9).

Further, in the first step, the mass ratio of the rhizoma polygonati to the astragalus to the alcohol solvent is 1:2: (24-36);

preferably, the alcohol solvent is ethanol;

more preferably, the ethanol concentration is 70% -80%;

preferably, the extraction temperature is 70-80 ℃ and the extraction time is 2-3 h.

Wherein, the yield of the saponin in the first extract can reach 10-14%.

Further, the mass ratio of the Chinese medicine residue, the agaric, the medlar and the water in the second step is 3:1:1: (75-125).

Preferably, the extraction temperature is 90-110 ℃ and the extraction time is 2-4 h.

Wherein, the yield of polysaccharide in the second extract can reach 40-60%.

Further, in the third step, the mass ratio of the pagodatree flower, the curcuma zedoary to the ethyl acetate is 1:1: (10-20);

preferably, the ultrasonic extraction is carried out with ultrasonic power of 140W-180W and ultrasonic time of 30 min-40 min.

Wherein, the extraction rate of the volatile oil in the third extract can reach 3-5%.

Further, in the fourth step, the mass ratio of the purslane to the dandelion to the water is 1:1: (30-50),

preferably, the extraction temperature is 50-60 ℃ and the extraction time is 50-80 min.

Wherein, the content of flavone in the fourth extract can reach 15 mg/g-25 mg/g.

Further, the inoculation is carried out with an inoculation amount of 1% -9% and a viable count of 2×10 ⁸ CFU～6×10 ⁸ CFU; preferably, the fermentation temperature is 35-40 ℃ and the fermentation is carried out for 3-5 days.

On the other hand, the application also provides application of the composition or the composition prepared by the preparation method in preparation of medicines for reducing immune system injury and/or medicines for treating liver cancer.

Further, the drug is used in combination with cyclophosphamide.

Rhizoma polygonati: replenishing qi, nourishing yin, strengthening spleen, moistening lung and tonifying spleen. Long Huang Qi is combined with Qi and Yin tonifying, and Qi tonifying and spleen tonifying are combined with Qi and Yin tonifying.

Radix astragali: spleen and middle-jiao strengthening, qi invigorating, yang ascending, diuresis and detumescence. Licorice root, radix Glycyrrhizae is combined in order to tonify qi, invigorate spleen and induce diuresis.

Flower of Chinese scholartree: cool blood and stop bleeding, clear liver and purge fire.

Purslane: clearing away heat and toxic materials, cooling blood and stopping bleeding. Can be used together with other heat-clearing and toxicity-removing medicines.

Dandelion: clearing away heat and toxic materials, relieving swelling, resolving hard mass, promoting urination, and treating stranguria. Huang Qi is usually combined to dispel swelling and remove stasis, clear liver and improve vision.

Rhizoma Wenyujin Concisa: break blood and promote qi circulation, promote menstruation and relieve menalgia. Long Huang Qi is combined with Qi tonifying and nutrient combining.

Wolfberry fruit: tonify kidney, nourish essence, nourish liver and improve vision.

Auricularia auricula-judae: invigorating qi, nourishing blood, moistening lung, stopping bleeding, lowering blood pressure, and resisting cancer.

The specific method comprises the following steps:

polygonatum sibiricum is neutral in nature and sweet in taste; the Chinese medicinal composition is used for restoring lung meridian, spleen meridian and kidney meridian; tonify middle energizer and qi, calm five viscera, benefit spleen and stomach, moisten heart and lung-Ben Cao from Xin (new materia Medica). Is a monarch drug.

Astragalus root has warm nature and flat taste; enter lung meridian and spleen meridian; it is the most important herb for tonifying qi, i.e., ben Cao Qi Zhen (true materia Medica). Is a monarch drug.

Dandelion is cold in nature, bitter in taste and sweet in flavor; enter liver meridian and stomach meridian; enters Yangming stomach, jueyin liver, cooling blood and relieving fever-Ben Cao Qizhen; for heat toxin, resolving food toxicity, detumescence and nuclear, furuncle, it is combined with fire-purging and pain-relieving herbs-like medical science. Is a ministerial drug.

Purslane is sour in nature and taste; enter liver meridian and large intestine meridian; yi Qi, clear summer-heat, widen middle energizer and descend Qi-Yuan nan Ben Cao (materia Medica of Yunnan), san Xue Ji (blood-dispelling and toxicity-removing) -Ben Cao from Xin (new materia Medica). Is a ministerial drug.

Wenyujin is warm in nature, bitter in taste and sweet; spleen meridian and liver meridian; for qi in blood, breaking blood and descending qi, dispelling wind and relieving swelling, it is used in Ben Cao from Xin (new materia Medica). Is an adjuvant drug.

The medlar is neutral and sweet; enter liver meridian and kidney meridian; yin nourishing, kidney tonifying, and essence replenishing, i.e. the theory of medicine property, moistening and nourishing, and combined with antipyresis, but is specially indicated for tonifying kidney, moistening lung, promoting fluid production, and qi-tonifying, i.e. the principal sketch is sparse, heavy and pure in taste, so it can nourish yin, and yin has yang, so it can tonify qi, i.e. the principal sketch is healthy. Is an adjuvant drug.

The agaric taste is sweet and neutral; the Chinese medicinal composition is used for treating lung meridian, spleen meridian, large intestine meridian and liver meridian; liwu zang (fructus Schisandrae chinensis) -Ben Cao (new materia Medica), bu Qi Tong Yi Huo (hunger resistance), huo Xue (blood circulation promoting) -Ji Huo Fang (food recipe for resolving food stagnation). Is an adjuvant drug.

Sophora flower is cold in nature and bitter in taste; enter liver meridian and large intestine meridian; is a key herb for cooling blood-Ben Cao is resolving primordium, clearing heat, cooling blood and stopping bleeding. Is an adjuvant drug.

The invention has the following beneficial effects:

1. the traditional Chinese medicine composition has the effects of tonifying qi and blood, clearing heat and detoxicating, cooling blood and stopping bleeding, strengthening vital qi to eliminate pathogenic factors, inhibiting the growth of liver cancer cells, and effectively treating liver cancer, and provides a novel traditional Chinese medicine compound for liver cancer and related diseases;

2. the traditional Chinese medicine composition can act cooperatively with cyclophosphamide, and can effectively reduce the damage of immune system caused by cyclophosphamide in the use process while treating liver cancer;

3. the traditional Chinese medicine composition has simple preparation process, and the anti-liver cancer traditional Chinese medicine composition is obtained by fermenting lactobacillus casei and lactobacillus bulgaricus for the first time, so that the compound treatment effect is exerted to the maximum extent, and a new method is provided for preparing medicines.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:

FIG. 1 is a graph showing the effect of treatment with different drug concentrations on liver cancer cell growth for 48 hours;

FIG. 2 is a graph showing the effect of treatment with different drug concentrations on liver cancer cell growth for 72 hours;

FIG. 3 is a graph showing morphology contrast of ascites tumor mice with normal mice;

FIG. 4 is a life extension rate plot;

FIG. 5 is a graph of spleen index of H22 ascites type mice;

FIG. 6 is a graph of thymus index of H22 ascites type mice;

FIG. 7 is a morphology of H22 ascites type mice spleen after dissection;

FIG. 8 is a diagram showing the thymus morphology of H22 ascites-type mice after dissection

FIG. 9 is a diagram of H22 solid mouse tumor weight statistics;

FIG. 10 is a diagram of H22 solid mouse tumor morphology;

FIG. 11 is a graph of spleen index of H22 entity type mice;

FIG. 12 is a spleen morphology of H22 entity type mice;

FIG. 13 is a chart of tumor histomorphometric observations (HE staining).

Detailed Description

In order to more clearly illustrate the general concepts of the present application, the following detailed description is made by way of example with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Wherein, rhizoma polygonati, astragalus root, pagodatree flower, purslane, dandelion, rhizoma curcumae longae, medlar and agaric are all provided by the Shandong colorful herbal medicine industry; lactobacillus casei, lactobacillus bulgaricus, is provided by the biotechnology of the Ningbo Tex Tuber Limited liability company; hep G2 liver cancer cells and H22 liver cancer cells are provided by Wuhan Shang En Biotechnology Co., ltd; SPF-grade KM mice were supplied by Peking Vitre Liwa laboratory animal technologies Co.

In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.

Hep G2 complete culture medium is MEM+10% FBS+1% P/S, and H22 mouse liver cancer cell complete culture medium is RPMI-1640+10% FBS+1% P/S.

Example 1 extraction method of Chinese medicinal composition

Test example 1

The formula is carried out according to the following raw materials and the dosage: 15g of rhizoma polygonati, 30g of astragalus, 15g of sophora flower, 15g of purslane, 15g of dandelion, 15g of curcuma longa, 15g of medlar and 15g of agaric.

The extraction method of the traditional Chinese medicine composition comprises the following steps:

(1) Extracting total saponins from rhizoma polygonati and astragalus:

mixing and crushing rhizoma polygonati and astragalus, and taking ethanol with the concentration of 70% -80% as a solvent, wherein the feed-liquid ratio is 1: (8-12), extracting at 70-80 ℃ for 2-3 h.

(2) Extracting rhizoma Polygonati, radix astragali residue, and polysaccharide in Auricularia and fructus Lycii:

mixing the residues of the rhizoma polygonati and the astragalus root which are extracted in the step (1) with the crushed agaric and the crushed matrimony vine, and taking water as a solvent, wherein the feed liquid ratio is 1: (15-25), extracting at 100 deg.c for 2-4 hr.

(3) Extracting volatile oil from flos Sophorae Immaturus and rhizoma Wenyujin Concisa with assistance of ultrasound:

mixing flos Sophorae Immaturus and rhizoma Wenyujin Concisa uniformly, pulverizing, and taking ethyl acetate as solvent, wherein the ratio of feed liquid is 1: (5-10), ultrasonic extraction is carried out under the conditions that the ultrasonic power is 140-180W and the ultrasonic time is 30-40 min.

(4) Extracting herba Portulacae and total flavonoids from herba Taraxaci:

evenly mixing purslane and dandelion, crushing, and taking water as a solvent, wherein the feed liquid ratio is 1: (15-25), extracting at 50-60 deg.c for 50-80 min.

(5) Mixing the extracts obtained in the steps (1) - (4), mixing uniformly, and freeze-drying to obtain the traditional Chinese medicine composition powder.

Test example 2

The difference from test example 1 is only that the formulation does not contain Polygonatum sibiricum.

Test example 3

The difference from test example 1 is only that the formulation does not contain astragalus root.

Test example 4

The only difference from test example 1 is that purslane is absent from the formulation.

Test example 5

The difference from test example 1 is only that the formulation does not contain dandelion.

Test example 6

The difference from test example 1 is only that the extraction was carried out by water decoction. The method comprises the following steps: decocting twice, wherein the first decoction-liquid ratio is 1:5, 60min, the second decoction liquid ratio is 1:3, decocting for 40min.

EXAMPLE 2 Mixed fermentation of Chinese medicinal composition

The powder of the traditional Chinese medicine composition obtained in the example 1 is further subjected to fermentation treatment, and the specific treatment steps are as follows:

the re-dissolution proportion of the traditional Chinese medicine composition freeze-dried powder by distilled water is 1: (10-20), lactobacillus casei and lactobacillus bulgaricus are used as fermentation strains, and inoculated into the solution for mixed fermentation. The inoculation ratio of lactobacillus casei to lactobacillus bulgaricus is 1: (1-9), the inoculation amount is 1-9%, and the viable count is 2X 10) ⁸ CFU～6×10 ⁸ CFU. Fermenting for 3-5 days at 35-40 ℃, and concentrating the fermentation liquor and the fermentation product into extract to obtain the compound extract.

Test example 7

The difference from test example 1 was only that lactobacillus plantarum and acetobacter were used for fermentation.

To investigate the changes in the composition components before and after fermentation, the polysaccharide content before and after fermentation was measured by a phenol-sulfuric acid method, the total flavone content before and after fermentation was measured by a sodium nitrite-aluminum nitrate chromogenic method, and the total saponin content before and after fermentation was measured by a vanillin-perchloric acid chromogenic method, and the measurement results are shown in Table 1.

TABLE 1 composition component Change before and after fermentation

As shown in Table 1, the polysaccharide content, the total flavone content and the total saponin content of the Chinese medicinal composition before and after fermentation were increased. Thus, the content of the active ingredients in the traditional Chinese medicine composition can be improved through the combined fermentation of lactobacillus casei and lactobacillus bulgaricus. In addition, experimental example 1 had higher polysaccharide, total flavone and total saponin contents than the other experiments. The composition after fermentation treatment can effectively inhibit the growth of liver cancer cells and improve the immunity.

Example 3 in vitro experiments: CCK-8 detection of inhibition effect of compound extract on Hep G2 liver cancer cells

The method for preparing the medicine-containing culture medium comprises the following steps: the compound extract obtained in example 2 was diluted to 5, 10, 25, 50, 100mg/mL with Hep G2 complete medium in a biosafety cabinet, and then sterilized by filtration with a sterile filter head of 0.25 μm to prepare a medicated medium.

The application adopts a CCK-8 colorimetric method to detect the influence of the drug on cell proliferation, and the specific method is as follows:

(1) The log phase cells were collected and 100. Mu.L (5X 10) was placed in a 96-well plate ⁴ Individual/ml). The plates were incubated at 37℃with 5% CO ₂ The incubator was pre-incubated for 24 hours.

(2) Cells adhere to the wall and the supernatant is discarded. Five experimental groups were set, 100. Mu.L of medium containing 5, 10, 25, 50 and 100mg/mL of drug was added respectively, and simultaneously, a negative control group (containing no drug) and zeroing holes (pure medium) were set, five duplicate holes were set for each group, and the plates were incubated in an incubator for 48h, 72h.

(3) To each well 10. Mu.L of CCK-8 solution was added and cultivation in the incubator was continued for 4 hours.

(4) OD at 450nm was measured with a microplate reader.

Inhibition (%) = (negative control OD-dosing group OD)/(negative control OD-zeroing well OD) ×100%

The survival of Hep G2 cells after 48h and 72h was observed, and the inhibition rate was calculated, and specific results are shown in tables 2 and 3.

Table 2: inhibiting result of compound extract on liver cancer cells after 48 hours

	Negative control	50(mg/ml)	100(mg/ml)	Zero setting hole
						1.2682	0.8437	0.0941	0.0881
	1.3624	1.0043	0.1264	0.0882
						1.4193	1.0284	0.1271	0.0886
	1.4213	1.0552	0.128	0.0908
						1.4734	1.0564	0.1342	0.0925
Average value of	1.401	1.0293	0.127167	0.0892
					Inhibition rate		28.3351％	97.1058％

As can be seen from Table 2 and FIG. 1, after 48 hours, the inhibition rate of the drug to liver cancer cells was 28.3351% when the drug concentration was 50 mg/ml; when the drug concentration is 100mg/ml, the inhibition rate of the drug to liver cancer cells is 97.1058%. Therefore, the drug concentration can effectively inhibit the growth of liver cancer cells at 48h no matter at 50mg/ml or 100 mg/ml.

Table 3: inhibiting result of compound extract on liver cancer cells after 72 hours

As can be seen from table 3 and fig. 2: when the drug concentration is 10mg/ml, the inhibition rate of the drug to liver cancer cells is 2.9976%; when the drug concentration is 25mg/ml, the inhibition rate of the drug to liver cancer cells is 21.5056%; when the drug concentration is 50mg/ml, the inhibition rate of the drug to liver cancer cells is 26.4162%; when the drug concentration is 100mg/ml, the inhibition rate of the drug to liver cancer cells is 96.4496%. Therefore, the drug concentration can be within the range of 10mg/ml to 100mg/ml, and can effectively inhibit the growth of liver cancer cells within 72 hours.

Example 4 in vivo experiment 1: anti-tumor effect of compound extract on H22 ascites type mice

SPF-grade KM mice are adopted in the experiments, H22 liver cancer cells are subjected to ascites passage of the KM mice and then injected into abdominal cavities of the mice for molding, and the injection is carried out the next day of molding.

Specifically, cells in log phase were collected for cell counting and diluted to 1X 10 with physiological saline in a biosafety cabinet ⁷ And each ml. Then, 0.2 ml/mouse was injected into the abdominal cavity, after which the change of the abdomen of the mouse was observed every day, and after 7 to 9 days, the abdomen of the mouse was significantly raised, as shown in fig. 3.

After the abdomen of the mice with ascites passage is obviously raised, taking out the ascites of the mice (the ascites contains a large amount of H22 liver cancer cells) in a sterile environment, counting cells and diluting to 1X 10 ⁷ And each ml. After which 0.2 ml/injection into the peritoneal cavity of the mice was performed.

The mice were randomly grouped the next day of modeling and started dosing for 17 consecutive days. Wherein the blank group is orally administered with the same volume of distilled water every day, the model group is orally administered with the same volume of distilled water every day, the cyclophosphamide group is orally administered with cyclophosphamide 20mg/kg every day, the compound low-dose group is orally administered with compound extract 17.5g/kg every day, the compound high-dose group is orally administered with compound extract 35g/kg every day, the combined low-dose group is cyclophosphamide 20mg/kg and compound extract 17.5g/kg, the combined high-dose group is cyclophosphamide 20mg/kg and compound extract 35g/kg, and the combined high-dose group is alternately administered every day.

After the last administration, half of mice are taken to detect the life extension rate, and the specific detection method is as follows: the death time of each mouse in each group is recorded, and the life extension rate of the mice to be dosed is calculated according to the following calculation formula:

life extension rate (%) = (average time to death of administration group mice-average time to death of model group mice)/average time to death of model group x 100%

The statistical results of the life extension rate of the mice are shown in Table 4.

TABLE 4 Life extension rate

Group of	Average survival days (d)	Life extension percentage (%)	Significance of the invention
				Model group	14.2±2.27
Cyclophosphamide group	17±4.12	19.72％	(P＞0.05)
				Compound high dosage	19.1±2.98	34.51％	(P＜0.01)

From the contents of table 4, fig. 4 was prepared, and it can be seen from table 4 and fig. 4 that the survival time of cyclophosphamide group was prolonged by 19.72% compared with the model group, and the result was not significant. Compared with cyclophosphamide, the survival time of the compound high-dose group is obviously prolonged by 34.51%, and compared with the model group, the survival time of the compound high-dose group is obviously prolonged. Proved by the results, the rhizoma polygonati compound extract has a certain treatment effect on the ascites tumor of mice.

Example 5 in vivo experiment 2: combined antitumor effect of compound extract and cyclophosphamide in H22 ascites type mice

After the last dose in example 4, the other half of the mice were fasted without water, and after 24h half of the mice were sacrificed and dissected.

Specifically, the thymus and spleen of the mouse are dissected, the thymus index and the spleen index of the mouse are calculated, and the calculation formula of the thymus index and the spleen index is as follows:

thymus index = thymus weight/mouse weight

Spleen index = spleen weight/mouse weight

TABLE 5 spleen and thymus index

Group of	Average spleen index (mg/g)	Average thymus index (mg/g)
			Blank group	2.22±0.48	1.85±0.36
Model group	0.69±0.12	0.05±0.016
			Cyclophosphamide group	0.89±0.18	0.18±0.015
Combination high dose group	1.13±0.7	0.25±0.01

From the contents of table 5, fig. 5 and 6 were prepared, and it can be seen from table 5 and fig. 5 to 8 that the spleen index and thymus index of the mice in the model group were significantly reduced compared with those in the blank group, thus proving that the molding of the mice was successful. After cyclophosphamide administration, spleen index and thymus index were elevated compared to model mice. Compared with the cyclophosphamide mice in the combined high-dose group, the organ index of the mice is obviously increased, which proves that the compound extract has a certain protection effect on the immune system damage caused by cyclophosphamide.

Example 6 in vivo experiment 3: antitumor effect of polygonatum sibiricum compound extract on H22 entity type mice

SPF-grade KM mice are adopted in the experiment, H22 liver cancer cells are subjected to ascites passage through the KM mice and then injected into the underarm of the right forelimb of the mice for subcutaneous molding, and the injection is carried out the next day of molding.

Cell culture and cell ascites passage procedure were the same as in example 4, and after the abdomen of the ascites passage mice was significantly raised, the mice were sterilizedTaking out ascites of mice (containing a large amount of H22 hepatoma cells) in the environment, performing cell count and diluting to 1×10 ⁷ And each ml. After which 0.2 ml/injection was made into the right forelimb underarm subcutaneous mould of the mice. The mice were randomly grouped the next day of modeling and started dosing for 21 consecutive days. Wherein, the blank group is orally administrated with the same volume of distilled water every day, the model group is orally administrated with the same volume of distilled water every day, the cyclophosphamide group is continuously and intraperitoneally administrated with the same volume of distilled water for 3 days, and then the same volume of distilled water is orally administrated for 3 days, so that the repetition is carried out, the compound low-dose group is orally administrated with 17.5g/kg of compound extractum every day, the compound high-dose group is orally administrated with 35g/kg of compound extractum every day, the combination low-dose group is intraperitoneally administrated with 30mg/kg of cyclophosphamide, 17.5g/kg of compound extractum, the 3-day cyclophosphamide 3-day compound extractum is alternately administrated, the combination high-dose group is intraperitoneally administrated with 30mg/kg of cyclophosphamide, 35g/kg of compound extractum, and the 3-day cyclophosphamide 3-day compound extractum is alternately administrated.

After the last dose, the mice were fasted without water, and after 24h, the mice were sacrificed and dissected.

The tumor of the mice is dissected, the tumor inhibition rate is calculated by weighing, the calculation formula of the tumor inhibition rate is as follows, and the statistical result is shown in table 6.

Tumor suppression rate (%) = (tumor weight of model group-tumor weight of administration group)/tumor weight of model group×100%

TABLE 6 tumor weight and tumor suppression Rate

As can be seen from Table 6 and FIGS. 9 to 10, the tumor inhibition rate of the compound high-dose group was 47.77%, and the tumor inhibition rate of the compound high-dose group was significantly reduced (P < 0.05) compared with the model group, and the tumor inhibition rate of the experimental group was 30% or more, which is significantly different from the model group. The sealwort compound extract has a certain treatment effect on the mice with solid tumors.

Example 7 in vivo experiment 4: combined antitumor effect of compound extract and cyclophosphamide in H22 solid mice

The mice in example 6 were dissected and spleens were obtained after the last administration, and the spleen index was calculated as shown in table 7.

Spleen index = spleen weight/mouse weight

TABLE 7 spleen index

Group of	Spleen index (mg/g)
		Blank group	2.68±0.44
Model group	3.39±1.05
		Cyclophosphamide group	3.49±1.1
Compound high-dose group	2.93±0.3
		Combination high dose group	2.89±0.75

As can be seen from Table 7 and FIGS. 11 to 12, the spleen index was increased after cyclophosphamide was applied, as compared with the model group. Compared with a model group, the compound high-dose compound extract has obviously reduced spleen index in combination with the high-dose group, and can be used for protecting the spleen injury of mice caused by tumors and cyclophosphamide application.

Example 8 Compound extract and cyclophosphamide histomorphometric detection of H22 solid mouse subcutaneous transplantation tumor

The fresh tumor pieces dissected in example 6 were fixed with 4% paraformaldehyde, sectioned after paraffin embedding, and examined for histopathological changes in each group under a microscope after Hematoxylin and Eosin (HE) staining. The arrangement, density, cell morphology, necrosis, presence or absence of vacuoles, etc. of each group of tumor cells were observed, and the results are shown in FIG. 13.

As can be seen from fig. 13, the results of HE staining under 200 x-fold mirror show that the tumor cells of the model group were closely arranged, whereas the tumor cells of the cyclophosphamide group and the compound high-dose group were loosely arranged. Compared with the model group, the cyclophosphamide group and the administration group can both see the tumor cell nucleus to shrink, obvious vacuole structures appear, and obvious necrosis areas exist. The compound extract of rhizoma polygonati has an inhibiting effect on the solid tumor of mice, and the compound extract can be synergistic with cyclophosphamide to inhibit the solid tumor of mice.

The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.

Claims

1. The traditional Chinese medicine composition for treating liver cancer is characterized by comprising 15 parts by weight of rhizoma polygonati, 30 parts by weight of astragalus, 15 parts by weight of sophora flower, 15 parts by weight of purslane, 15 parts by weight of dandelion, 15 parts by weight of curcuma zedoary, 15 parts by weight of medlar and 15 parts by weight of agaric;

the preparation method of the composition comprises the following steps:

(1) Mixing and crushing rhizoma polygonati and astragalus, and taking 70% -80% ethanol as a solvent, wherein the feed liquid ratio is 1: 8-12, extracting at 70-80 ℃ for 2-3 hours;

(2) Mixing the residues of the rhizoma polygonati and the astragalus which are extracted in the step (1) with crushed agaric and medlar, and taking water as a solvent, wherein the feed liquid ratio is 1: 15-25, extracting at 100 ℃ for 2-4 hours;

(3) Mixing flos Sophorae Immaturus and rhizoma Wenyujin Concisa uniformly, pulverizing, and mixing with ethyl acetate as solvent at a feed liquid ratio of 1: 5-10, performing ultrasonic extraction under the condition that the ultrasonic power is 140-180W and the ultrasonic time is 30-40 min;

(4) Evenly mixing purslane and dandelion, crushing, and taking water as a solvent, wherein the feed liquid ratio is 1: 15-25, extracting at 50-60 ℃ for 50-80 min;

(5) Mixing the extracts in the steps (1) - (4), uniformly mixing, and freeze-drying to obtain traditional Chinese medicine composition powder;

the method also comprises the steps of re-dissolving the traditional Chinese medicine composition powder with distilled water, wherein the re-dissolving proportion is 1: 10-20, inoculating lactobacillus casei and lactobacillus bulgaricus serving as fermentation strains into the solution for mixed fermentation; the inoculation ratio of lactobacillus casei to lactobacillus bulgaricus is 1: 1-9, the inoculation amount is 1-9%, and the viable count is 2×10 ⁸ CFU~6×10 ⁸ And (3) CFU, fermenting for 3-5 days at the temperature of 35-40 ℃, and concentrating the fermentation liquid and the fermentation product into extract to obtain the compound extract.

2. Use of a composition according to claim 1 for the manufacture of a medicament for reducing immune system damage and/or for treating liver cancer, wherein the medicament is in combination with cyclophosphamide.