CN114699441A

CN114699441A - Application of traditional Chinese medicine composition containing phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum in preparation of medicine for inhibiting postoperative recurrence of liver cancer

Info

Publication number: CN114699441A
Application number: CN202210265247.0A
Authority: CN
Inventors: 林德良; 郭平; 康超; 蒿长英; 钟云; 黄卉
Original assignee: Beijing Handian Pharmaceutical Co ltd
Current assignee: Beijing Handian Pharmaceutical Co ltd
Priority date: 2017-02-22
Filing date: 2017-02-22
Publication date: 2022-07-05
Also published as: CN108452008A

Abstract

The invention relates to an application of a traditional Chinese medicine composition containing phyllanthus urinaria, coriolus versicolor, salvia miltiorrhiza and lithospermum in preparing a medicine for inhibiting postoperative recurrence of liver cancer, wherein the traditional Chinese medicine composition comprises the following medicinal materials in parts by weight: 10 parts of phyllanthus urinaria, 4 parts of coriolus versicolor, 1 part of salvia miltiorrhiza and 1 part of lithospermum. The traditional Chinese medicine composition has a remarkable inhibiting effect on multiple liver cancer recurrence models, and has good treatment effect and safety.

Description

Application of traditional Chinese medicine composition containing phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum in preparation of medicine for inhibiting postoperative recurrence of liver cancer

Technical Field

The invention belongs to the field of medicine and pharmacology, and particularly relates to a new medical application of a traditional Chinese medicine composition containing phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum. More specifically, the medical application refers to the application of the traditional Chinese medicine composition in preparing a medicine for inhibiting postoperative recurrence of liver cancer.

Background

The Primary liver malignant tumor originates from the epithelium or mesenchymal tissue of the liver, the former is called Primary Hepatic Cancer (PHC), which is a highly-developed malignant tumor with great harm in China; the latter is called sarcoma, and is less common than primary liver cancer.

The primary liver cancer accounts for 95% of hepatocellular carcinoma, so the daily primary liver cancer is exclusively referred to as hepatocellular carcinoma. The primary liver cancer is called "king of cancer" because of its initial asymptomatic stage, high degree of malignancy, rapid disease transition, short survival time and high mortality. 782,500 newly-discovered liver cancer cases and 745,500 deaths are estimated worldwide in 2012, and only China accounts for about 50% of the total number of cases and deaths, so that the liver cancer cases and the deaths are serious diseases seriously threatening the health and the life of people in China.

Radical surgery is the first choice for treating liver cancer. With the progress of diagnosis technology and the popularization of health education, the early diagnosis rate of liver cancer is continuously improved, and more patients have the chance of operation. However, postoperative recurrence of liver cancer has become one of the main factors affecting the long-term efficacy of primary liver cancer. The literature reports that the 5-year recurrence rate after primary radical hepatoma surgery is 60-75%, wherein the 1-year recurrence rate after surgery is as high as 51.4-72% (Zhang Zhijian, Wu Meng super, clinical research progress for preventing postoperative recurrence of primary hepatoma, division of foreign medicine and surgery, 1999, 26 (2): 129). The peak time of recurrence is 6 months after operation, the peak time is 3-4 months after operation, the average recurrence time of metastasis is (198 +/-29) d (Luxin, Zhao Hao, Maoyei and the like), the early recurrence condition of the liver cell liver cancer patient after operation is reported in Chinese academy of medicine, 2008, 30 (4): 415, Wumeng, the diagnosis and treatment progress of the primary liver cancer, and 2008, 30 (4): 363. Even in the case of primary liver cancer, it has been reported in the literature that the recurrence rate of 5 years after radical resection of large liver cancer is as high as 80%, and that of small liver cancer is about 60% (Roshimin, recurrence mechanism and its influencing factor [ J ] after primary liver cancer surgery, J.M. Kokai, 2001, 16 (1): 108-.

Compared with primary liver cancer, the postoperative recurrence of liver cancer has higher malignancy degree and faster process; because patients with postoperative recurrence are less sensitive to chemotherapy than patients with primary liver cancer, treatment difficulty is increased, and cancer metastasis is easily caused, postoperative recurrence of liver cancer has a greater effect on patients than primary liver cancer. Therefore, how to control the postoperative recurrence of liver cancer is the key to improve survival rate and prognosis. Therefore, the research and the development of the medicine for preventing and treating the postoperative liver cancer have important significance.

At present, various treatment means such as interventional embolization chemotherapy, systemic chemotherapy, radiotherapy, radiofrequency ablation, biological treatment and the like are widely applied to postoperative recurrence of liver cancer, but the treatment effect and prognosis are not ideal, the cost is high, and the toxic and side effects are large. Therefore, prevention and treatment of postoperative recurrence of liver cancer is a far from being met clinical need.

Traditional Chinese medicine has a long history, and forms a recognition and treatment rule for liver cancer in the treatment practice of doctors of all ages. According to the traditional Chinese medicine, liver cancer belongs to the syndrome of origin deficiency and secondary excess. The deficiency of the essential qi means deficiency of qi and blood, and the deficiency of the healthy qi means excess of pathogenic qi, blood stasis and fire toxin. In the occurrence and development of liver cancer, qi stagnation, phlegm and fluid retention and blood stasis are often mixed and fixed and difficult to separate, and dampness and cold and heat cementation formed on the basis of the deficiency are important pathogenic factors causing the liver cancer. In combination with the above, most scholars think that the cause of the disease is not related to improper diet, emotional disorder, affection of six excesses and pathogenic factors, and weak constitution due to long-term illness. The basic pathogenesis of the liver-qi deficiency type liver-qi stagnation type blood stagnation type liver-qi stagnation type lung-qi stagnation type liver-qi stagnation type lung-qi stagnation type liver-qi stagnation type lung cancer.

According to the above traditional Chinese medicine mechanism, the inventor has studied a traditional Chinese medicine composition containing phyllanthus urinaria, coriolus versicolor, salvia miltiorrhiza and lithospermum. More specifically, the inventor researches the Ziye Dan capsule, and the active ingredients of the Ziye Dan capsule comprise phyllanthus urinaria, coriolus versicolor, Salvia miltiorrhiza and lithospermum. The monarch drug phyllanthus urinaria is sweet and bitter and cool, enters liver and lung channels, and has the effects of calming liver, clearing heat, promoting diuresis, removing toxicity and the like. The coriolus versicolor is sweet and mild, has the effects of benefiting qi and nourishing yin, strengthening body resistance and eliminating evil, harmonizing yin and yang and the like, and is used as a ministerial drug. Monarch drugs detoxify to eliminate pathogens, ministerial drugs tonify yin to strengthen body resistance, and monarch drugs and ministerial drugs play a role of eliminating pathogens and strengthening body resistance together. Salvia miltiorrhiza is bitter and slightly cold. Enter heart and liver meridians, clear nutrient and cool blood, activate blood and dredge meridians. The medicine has the functions of promoting blood circulation to disperse blood clots, eliminating swelling and relieving pain, and is used as assistant and guiding medicine. Shi Zi Cao is bitter, salty and cold, entering heart and liver meridians. Cool blood, activate blood and remove toxicity. It is used together with Ye Xie Zhu to play the actions of clearing heat, cooling blood, removing toxicity, promoting blood circulation, removing blood stasis, and also as an adjuvant drug. So far, no report that the Ziye Dan can inhibit postoperative recurrence of liver cancer is available.

The inventor finds in research that although the monarch drug phyllanthus urinaria is commonly used for treating hepatitis B in the prior art, reversible toxic reactions of the liver and the kidney appear in animal long-term toxicity tests. While searching for a drug suitable for inhibiting postoperative recurrence of liver cancer, those skilled in the art would like to search for a safe and effective Chinese medicinal preparation, and thus, for patients with postoperative recurrence of liver cancer, the liver function of the patients is impaired, and toxicity such as phyllanthus is obviously disadvantageous.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a new medical application of a traditional Chinese medicine composition containing phyllanthus urinaria, coriolus versicolor, salvia miltiorrhiza and lithospermum for inhibiting postoperative recurrence of liver cancer. More specifically, the invention provides an application of the minium for preparing a medicine for inhibiting postoperative recurrence of liver cancer. Animal experiments show that the minium has obvious tumor inhibition effect on a plurality of tumor recurrence models of tumor-bearing mice, and the effect is better than that of single-use phyllanthus urinaria; and the safety is good, the 100, 50 and 25 times of daily dosage of human is used for intragastric administration for 6 months to rats, and the purple-leaved salvia does not have drug-related toxic reaction and also does not have accumulative toxic reaction. The invention provides a new choice for clinically inhibiting postoperative recurrence of the liver cancer.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the application of a traditional Chinese medicine composition comprising phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum in preparing a medicine for inhibiting postoperative recurrence of liver cancer, wherein the traditional Chinese medicine composition comprises the following medicinal materials in parts by weight: 5-20 parts of phyllanthus urinaria, 2-8 parts of coriolus versicolor, 1-5 parts of salvia miltiorrhiza and 1-5 parts of lithospermum.

Preferably, the liver cancer comprises primary liver cancer and secondary liver cancer.

The primary liver cancer comprises hepatocellular carcinoma, hepatoblastcarcinoma, cholangiocarcinoma, intrahepatic gallbladder adenocarcinoma, hepatobiliary mixed cell carcinoma, hepatosarcoma, hepatoangiomyosarcoma, hepatoepithelioid hemangiocytoma and liver fibrolamellar carcinoma.

Preferably, the traditional Chinese medicine composition comprising common leafflower herb, coriolus versicolor, red sage root and lithospermum comprises the following medicinal materials in parts by weight: 10 parts of phyllanthus urinaria, 4 parts of coriolus versicolor, 1 part of salvia miltiorrhiza and 1 part of lithospermum.

Preferably, the traditional Chinese medicine composition comprises phyllanthus urinaria, coriolus versicolor, salvia miltiorrhiza and lithospermum and also comprises pharmaceutically acceptable auxiliary materials.

The pharmaceutically acceptable adjuvants include (1) diluent such as starch, sugar powder, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salt (such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, etc.), mannitol, etc., vegetable oil, polyethylene glycol, etc.; (2) binders such as distilled water, ethanol, starch slurry, sodium carboxymethylcellulose, hydroxypropyl cellulose, methyl and ethyl cellulose, hypromellose, and the like; (3) disintegrants, for example: dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, and the like; (4) lubricants, for example: magnesium stearate, silica gel micropowder, pulvis Talci, hydrogenated vegetable oil, polyethylene glycols, magnesium lauryl sulfate, etc.; (5) flavoring agents, such as: sucrose, steviosin, etc.; (6) solvents, for example: water, alcohol, and the like.

Preferably, the traditional Chinese medicine composition comprising phyllanthus urinaria, coriolus versicolor, salvia miltiorrhiza and lithospermum is a clinically acceptable preparation, and comprises a gastrointestinal tract administration preparation and a non-gastrointestinal tract administration preparation.

Preferably, the preparation for gastrointestinal tract administration is selected from powder, tablets, granules, capsules, dripping pills, emulsion or suspension; the parenteral preparation is selected from injection, spray, suppository, perfusion, patch or ointment.

Preferably, the traditional Chinese medicine composition comprising phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum is the Chinese sage herb, the raw medicines comprise 10 parts of phyllanthus urinaria, 4 parts of corious versicolor, 1 part of salvia miltiorrhiza and 1 part of lithospermum, and the traditional Chinese medicine composition is prepared by the following method:

extracting cacumen Securinegae Suffruticosae with water, adsorbing the obtained extractive solution with macroporous adsorbent resin, eluting with 30-75% ethanol, mixing eluates, and concentrating to obtain extract I; extracting the residue after decocting cacumen Securinegae Suffruticosae with ethanol to obtain ethanol extractive solution; extracting Saviae Miltiorrhizae radix and radix Arnebiae with ethanol to obtain ethanol extractive solution, mixing with the ethanol extractive solution of cacumen Securinegae Suffruticosae, and recovering solvent to obtain extract II; mixing the residue of Coriolus versicolor, Saviae Miltiorrhizae radix and radix Arnebiae, extracting with water to obtain water extract, and recovering solvent to obtain extract III; mixing the extracts I, II and III, granulating, oven drying, and making into capsule or granule, or granulating and tabletting to make into tablet; if necessary, the pharmaceutically acceptable auxiliary materials are added in the granulating and forming processes.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein:

(1) test drug

Ziyedan capsules (hereinafter referred to as "ziyedan") are provided by beijing handian pharmaceutical limited, lot number: 13040102; the preparation method comprises the following steps:

prescription: 2000g of phyllanthus urinaria, 800g of coriolus versicolor, 200g of salvia miltiorrhiza and 200g of lithospermum

Extracting Phyllanthus urinaria L with water, adding 12 times of water for the first time, decocting for 1.5 hr, adding 10 times of water for the second time, and decocting for 1 hr. Mixing decoctions, centrifuging, adsorbing supernatant with H103 macroporous adsorbent resin, eluting with 30% and 60% ethanol respectively, mixing eluates, and concentrating to obtain extract I; extracting the residue after decocting cacumen Securinegae Suffruticosae with 12 times of 95% ethanol to obtain cacumen Securinegae Suffruticosae ethanol extract; extracting Saviae Miltiorrhizae radix and radix Arnebiae with 95% ethanol twice, adding 10 times of ethanol for the first time, refluxing for 1.5 hr, adding 6 times of ethanol for the second time, refluxing for 1 hr to obtain Saviae Miltiorrhizae radix-radix Arnebiae ethanol extractive solution, mixing with cacumen Securinegae Suffruticosae ethanol extractive solution, and recovering solvent to obtain extract II; mixing the residue of Coriolus versicolor, Saviae Miltiorrhizae radix and radix Arnebiae, decocting with 12 times of water twice, 1.5 hr for the first time and 1 hr for the second time to obtain water extractive solution, and recovering solvent to obtain extract III; mixing the extracts I, II and III, adding pharmaceutically acceptable adjuvants to 500g, granulating, oven drying, and making into capsule (0.5 g/capsule).

Phyllanthus urinaria extract (hereinafter referred to as "Phyllanthus urinaria") is provided by Beijing Hanclassical drug manufacturing Co., Ltd; the preparation method comprises the following steps:

extracting with conventional water decoction method, extracting cacumen Securinegae Suffruticosae for 2 times, mixing decoctions, filtering, and concentrating the filtrate to 1: 1 (weight of cacumen Securinegae Suffruticosae: volume of filtrate).

Fluorouracil for injection (5-Fu), 0.25g/10ml per vial, Tianjin Jinyao amino acids Co., Ltd.

(2) Primary reagent

RPMI-1640 medium, Hyclone, GIBCO;

fetal bovine serum, GIBCO Co

0.25% pancreatin (containing 0.04% EDTA), Hyclone;

4% Paraformaldehyde, Biotopped

(3) Main instrument

A blood cell counter, Shanghai chemical laboratory instruments Co., Ltd.;

ott BDS200 inverted biomicroscope, Chongqing ott optical instruments, llc;

ultra clean bench (YT-CJ-2ND), Beijing Adita Crohn instruments & Technical Co., Ltd;

carbon dioxide incubator (SANYO), north kyoto express medical instruments ltd;

low speed centrifuge (SC-3616), Anhui Zhongkezhongjia scientific instruments, Inc.;

electronic balance (AL204), METTLER TOLEDO;

electronic balance (LQ-A10001), Ant weighing apparatus, Inc., of Reian;

microplate reader (MULTILSKAN MK3), Hyclone, USA.

Example 1 Effect of Phyllanthus urinaria, Coriolus versicolor, Lithospermum erythrorhizon, Salvia miltiorrhiza and combinations thereof on proliferation of hepatoma carcinoma cells

1.1 materials of laboratory instruments:

(1) hepatoma cell line human hepatoma cells SMMC-7721, purchased from Shanghai cell Bank of Chinese academy of sciences.

(2) Experimental drugs

Respectively processing Phyllanthus urinaria, Coriolus versicolor, radix Arnebiae, and Saviae Miltiorrhizae radix as follows to obtain medicines 1-8:

medicine 1 (aqueous extract of phyllanthus urinaria L. goes through macroporous resin): 1100g of Phyllanthus urinaria L crude drug is taken, recorded and registered, and then decocted and extracted twice by adding water, wherein 12 times of water is added for the first time and decocted for 1.5 hours, and 10 times of water is added for the second time and decocted for 1 hour. Filtering with gauze after each decoction, and collecting filtrate to obtain water extractive solution of cacumen Securinegae Suffruticosae and medicinal residues of cacumen Securinegae Suffruticosae. Loading the water extract on H103 macroporous adsorbent resin column, and washing the column with pure water until the eluate is colorless; completely eluting with 30% ethanol and 60% ethanol in sequence, collecting and mixing eluates; the measured amount of the ointment is concentrated to 500ml, and spray-dried to obtain 10.5g of spray-dried powder, namely the medicine 1; the yield is as follows: 10.5/1100 × 100 ═ 0.95%.

Medicine 2 (cacumen Securinegae Suffruticosae alcoholic extract): heating and refluxing the phyllanthus urinaria dregs subjected to water extraction in the previous step with 95% ethanol twice, wherein the dosage of the 95% ethanol is 12 times of that of the medicine residues, the first time is 1.5 hours, the second time is 1 hour, merging ethanol extract, concentrating until the relative density is 1.18-1.20(50 ℃), centrifuging, collecting supernatant, placing the supernatant in a refrigerator at 4 ℃ for standing overnight, filtering, and performing spray drying to obtain 12.9g of dry powder, namely the medicine 2; the yield is as follows: 12.9/1100 × 100 ═ 1.17%.

Drug 3 (underleaf pearl aqueous extract): 500g of crude drugs of Phyllanthus urinaria L is taken, and after registration, the crude drugs are decocted and extracted twice by adding water, wherein 12 times of water is added for the first time and decocted for 1.5 hours, and 10 times of water is added for the second time and decocted for 1 hour. Decocting each time, filtering with gauze, collecting filtrate to obtain water extractive solution of cacumen Securinegae Suffruticosae, concentrating to 300ml, and spray drying. 15.6g of dry powder is obtained, namely the medicine 3; the yield is as follows: 15.6/500 × 100 ═ 3.12%.

Medicine 4 (aqueous extract of coriolus versicolor): taking 500g crude coriolus versicolor, recording and registering, and decocting with water twice, wherein the first time of decoction is 1.5 hours, the second time of decoction is 1 hour, and 12 times of water is used for each time; filtering with gauze, collecting and combining the filtrates; concentrating to 620ml, and spray drying to obtain spray dried powder 37.15g, namely the medicine 4, wherein the yield is as follows: 37.15/500 × 100 ═ 7.43%.

Medicine 5 (alkanna tinctoria and salvia miltiorrhiza extract): taking 500g of crude drugs of lithospermum and salvia miltiorrhiza respectively (the crude drugs are crushed by a salvia miltiorrhiza crusher), putting the crude drugs into an extraction container for ethanol extraction twice, adding 10L of 95% ethanol 10 times of the amount of the crude drugs for the first time, carrying out reflux extraction for 1.5h, adding 6L of 95% ethanol 6 times of the amount of the crude drugs for the second time, carrying out reflux extraction for 1h, filtering while hot, combining filtrates, and keeping dregs for later use; after concentration, the total volume of the concentrated solution is measured by centrifugation to be 500 ml; spray drying to obtain spray dried powder 57.80g, namely the medicine 5; the yield is as follows: 57.80g/1000g 100 ═ 5.78%.

Medicine 6 (water extraction of lithospermum and salvia miltiorrhiza): decocting the residue with water twice (1.5 hr for the first time and 1 hr for the second time, each time with 12 times of water), filtering while hot, and mixing filtrates; after concentration, the total volume of the concentrated solution is measured by centrifugation to be 690ml, spray drying is carried out to obtain 166.72g of spray dried powder, namely the medicine 6, and the yield is as follows: 166.72g/1000g 100 ═ 16.67%.

Drug 7 (mixed extract): according to the raw material composition formula of the Ziye Dan (2100 g of Phyllanthus urinaria L., 800g of Coriolus versicolor, 200g of Salvia miltiorrhiza Bunge, 200g of Lithospermum erythrorhizon Sieb. et Zucc.) and the yield of the medicines 1-6, respectively taking the extracts prepared by the methods of the medicines 1-6 according to the amount shown in the table 1, mixing to obtain the medicine 7, wherein the total amount is 259.28 g.

TABLE 1 formulation composition of drug 7

Medicine 8 (ziye dan preparation): taking 2100g of Phyllanthus urinaria L crude drug, 800g of Coriolus versicolor crude drug, 200g of Salvia miltiorrhiza crude drug and 200g of lithospermum; extracting Phyllanthus urinaria L with water twice, adding 12 times of water for the first time, decocting for 1.5 hr, adding 10 times of water for the second time, and decocting for 1 hr. Adsorbing the obtained extractive solution with H103 macroporous adsorbent resin, eluting with 30% and 60% ethanol respectively, mixing eluates, and concentrating to obtain extract I; extracting the residue after decocting cacumen Securinegae Suffruticosae with 12 times of 95% ethanol to obtain cacumen Securinegae Suffruticosae ethanol extract; extracting Saviae Miltiorrhizae radix and radix Arnebiae with 95% ethanol twice, adding 10 times of ethanol for the first time, refluxing for 1.5 hr, adding 6 times of ethanol for the second time, refluxing for 1 hr to obtain Saviae Miltiorrhizae radix-radix Arnebiae ethanol extractive solution, mixing with cacumen Securinegae Suffruticosae ethanol extractive solution, and recovering solvent to obtain extract II; mixing the residue of Coriolus versicolor, Saviae Miltiorrhizae radix and radix Arnebiae, decocting with 12 times of water twice, 1.5 hr for the first time and 1 hr for the second time to obtain water extractive solution, and recovering solvent to obtain extract III; and (3) uniformly mixing the extractum I, II and III, drying, and adding pharmaceutically acceptable auxiliary materials to 500g to obtain the medicine 8.

1.2 preparation of working solution

The sampling amounts of the medicines 1 to 8 are obtained by conversion according to the proportion of the medicinal materials in the prescription and the extraction rate of each part, and as shown in table 2, the working solution I and the working solution II are prepared respectively according to the following methods:

the drugs were weighed as shown in Table 2, and RPIM-1640 culture medium (containing 10% FBS and 100U. mL) was added^-1Qinghazu, 100 mug. mL^-1Streptomycin sulfate) to 10mL, centrifuging at 4000rpm for 20min, and performing sterile filtration to obtain a mother solution. When the mother liquor is used, the mother liquor is diluted by 200 times by using the RPIM-1640 culture solution to obtain working solution I, and the mother liquor is diluted by 100 times by using the RPIM-1640 culture solution to obtain working solution II. A total of 16 working fluids.

TABLE 2 working solution preparation of drugs 1-8

	Medicine 1	Medicine 2	Medicine 3	Medicine 4	Medicine 5	Medicine 6	Medicine 7	Medicament 8
									Weighing	0.0040g	0.0049g	0.0131g	0.0119g	0.0046g	0.0133g	0.0519g	0.1000g
Working solution I (mu g/mL)	20	25	66	60	23	67	260	500
									Working solution II (mug/mL)	40	50	132	120	46	134	520	1000

1.3 Experimental methods:

cell culture

SMMC-7721 cells were seeded at 25cm²The culture flask was supplemented with about 8mL of culture medium (10% FBS, 100U. mL) LRPIM-1640^-1Cyanoxin, 100. mu.g.mL^-1Streptomycin sulfate) at 37 deg.C, 5% CO₂And carrying out static culture in an incubator with saturated humidity, and changing the culture solution every 48 hours. Growing the cells into a compact monolayer in about 3-4 days, paving the compact monolayer on the bottom of a culture bottle, and carrying out passage: discarding the original culture solution, washing twice with PBS, adding 1mL pancreatin per bottle for digestion, observing cells under an inverted phase-contrast microscope, shrinking the cells into a circle in about 1-2 min, discarding the digestive juice, adding the culture solution, blowing the bottom of the culture bottle to make the cells fall off, and preparing the cell suspension.

Pharmaceutical intervention and detection

1.5×10⁴mL^-1The cell suspension of (4) was inoculated in a 96-well plate at 200. mu.L per well, and the wells were zeroed without cells and filled with PBS. After 24h, the 16 working solutions are used, 5 wells are worked for each work, and the culture is continued for 24h, 48h or 72 h. 5 mg. about.mL of the solution was added to each well^-1After incubation for 4h in an incubator, the incubation solution was discarded, 150. mu.L of DMSO was added to each well, and the mixture was shaken and mixed at room temperature (22 ℃) for 15 min. After the precipitate was completely dissolved, the optical density (A) was measured at 570nm on a microplate reader. The inhibition rate was calculated according to the following formula:

inhibition ratio (%) (A)_{Blank hole}-A_{Measuring hole})/(A_{Blank hole}-A_{Zero setting hole})*100％

1.4 results of the experiment

The inhibition rate of human liver cancer cell SMMC-7721 in each working solution treatment for 24h, 48h and 72h is shown in Table 3.

The data in table 3 shows:

1) 1-6 of low-concentration medicine basically has no inhibition effect or even promotion effect on the proliferation of human liver cancer cells SMMC-7721; especially, the medicine 4 (water extract of Coriolus versicolor) shows the promotion effect on the growth of liver cancer cells in the whole observation and culture time.

2) High-concentration drugs 1-3 (all different extracts of phyllanthus urinaria) and 5 show only very weak inhibition effect on liver cancer cells under long-time intervention (72 h).

3) The high concentration medicine 4 (water extract of Coriolus versicolor) shows the promotion effect on the growth of hepatoma carcinoma cells in the whole observation and culture time.

4) The low-concentration drugs 7 and 8 show the effect of promoting the proliferation of the liver cancer cells at the time of intervention for 24 hours, but inhibit the proliferation of the liver cancer cells to different degrees in 48 hours and 72 hours along with the prolonging of the action time. The inhibition effect of the high-concentration medicaments 7 and 8 on the liver cancer cell proliferation has a time-dependent effect, and the two medicaments show stronger inhibition effect on the cancer cell growth when the intervention is carried out for 72 hours. Wherein drug 8 is more potent than drug 7.

TABLE 3 Effect of drugs 1-8 on proliferation of human hepatoma cells SMMC-7721 (x + -SD) (n ═ 5)

1.5 conclusion of the experiment

The Phyllanthus urinaria, Coriolus versicolor, Lithospermum erythrorhizon and Salvia miltiorrhiza have no inhibitory effect or only weak effect on proliferation of human liver cancer SMMC-7721, and some of them show promotion of cancer cell growth. Unexpectedly, the four traditional Chinese medicines can inhibit the growth of liver cancer cells after being used together. The anti-liver cancer effect of the traditional Chinese medicine composition is not the simple addition of the effects of the single medicinal materials, but the new effect generated after the compatibility of the medicinal materials.

Moreover, the medicine 8 prepared by a specific process is the medicine 7 formed by simply mixing extracts obtained by respectively extracting four traditional Chinese medicines, and supposing that in the preparation process of the medicine 8, through a multi-step pharmaceutical extraction process, all medicinal materials in the compound are reasonably compatible and various contained chemical components interact with each other, so that a necessary material basis is provided for the anti-tumor effect of the compound.

The preparation method of the medicament 8 is basically the same as the preparation process of the red sage root, so the red sage root is adopted in the following experiments for carrying out corresponding research.

Example 2Inhibition effect of ziye Dan on tumor recurrence of H22 tumor-bearing mice

2.1 materials of laboratory instruments:

(1) laboratory animal

ICR mice, male, weighing about 18-22g, 120, purchased from wakaukang biotechnology, inc, license number: SCXK (Jing) 2014-. After purchase of all animals, the animals were observed for general physiological indices, body weight and feeding. Adaptive feeding for 3 days. Standard pellet feed, free water. Natural day and night light illumination is carried out, the room temperature is 18-26 ℃, and the relative humidity is 40-70%.

(2) Tumor cell strain

H22 hepatoma ascites tumor cells, purchased from Shanghai cell Bank of Chinese academy of sciences.

(3) Experimental drugs

Ziye Dan capsule

Adult dose is 60kg for adult, and the adult takes orally 5 granules 3 times a day, each granule contains 0.5g, namely 0.125g/kg, and the mouse drug (extract) dose (g/kg) is converted according to equivalent dose:

0.125g/kg×9.01＝1.126g/kg

phyllanthus urinaria extract

5-Fu for injection

2.2 Experimental methods:

mouse liver cancer H22 cell in vitro culture in 5% CO₂In a 37 ℃ incubator, conventional culture is carried out in 10% inactivated fetal bovine serum RPMI-1640 medium, and the cells are harvested and counted for tumor cell inoculation every 2-3d routine passage when the cell proliferation shows exponential growth phase.

In vivo passage of cell animals: watch withObserving general physiological index, weight and feeding condition of animal, adaptively feeding for 1 week, observing all animal is normal, collecting 3-5 animals, injecting into abdominal cavity at left side with 0.2ml density of 1 × 10⁷H22 tumor cells; normally feeding, observing the state of the animal, generating ascites after a few days, taking ascites from a mouse with good ascites and plump animal state for 7-10 days, centrifuging for 1000 turns and 3 minutes, washing with physiological saline for 1 time, counting tumor cells, diluting and uniformly mixing.

Animal molding: observing general physiological index, weight and eating condition of animal, adaptively feeding animal for 1 week, and injecting 0.1ml subcutaneous injection with density of 2 × 10 into right neck and shoulder after observing animal all normal⁷H22 tumor cells/ml (diluted in ascites fluid above); the number of animals and cells per group are recorded in the experimental design table.

The day after inoculation, the animals were weighed and randomly grouped according to body weight (randomized block design) into 10 animals per group, as shown in Table 4.

Table 4 example 2 grouping and dosing table

2.3 Observation items and evaluation indexes

2.3.1 general State Observation

Animal mental state, reactivity, hair color, diet and excretion were observed. Animals presented with deteriorated and bad condition and animals were unable to obtain sufficient food and water (incapacitated) and were euthanized with CO 2.

2.3.2 tumor suppressor action

(1) Weight: weighing the body weight every day, and observing the average body weight change of each group;

(2) tumor volume: tumor diameters were measured twice weekly or every other day with a vernier caliper. The formula for tumor volume is: v0.5 Xab²And a and b represent the major and minor diameters of the tumor, respectively.

(3) Tumor weight: after the administration, tumor tissues were dissected, weighed, and the mean tumor size changes of each group were compared. The in vivo anticancer effect of the medicine is evaluated by the average tumor weight and the tumor growth inhibition rate, and the calculation formula is as follows:

the tumor growth inhibition rate is (1-T/C) multiplied by 100 percent,

wherein T is the average tumor weight of the administration group, and C is the average tumor weight of the control group.

2.4 data processing

Comparisons between groups were made using independent sample t-tests. Comparisons between three or more groups, one-way ANOVA was used. If F-statistics (the ratio of treatment variance error variance) are calculated, a multiple comparison procedure will be applied, analysis of variance. Potential synergy between treatments, two-way anova was analyzed. All data will be analyzed using SPSS17.0, with P <0.05 considered statistically significant.

2.5 Experimental results:

(1) weight:

the mouse had no abnormal activity, diet and hair color before molding. There was no significant difference in body weight average (P > 0.05) between groups before and after administration, and the results are shown in Table 5.

TABLE 5 weight changes before and after drug administration

In comparison with the set of models,^*P＜0.05，^**P＜0.01

(2) inhibition of H22 transplantable tumors:

the Ziye Dan has obvious tumor inhibiting effect, is in certain dose-effect relationship, is superior to the Phyllanthus urinaria group, and has the anti-tumor effect similar to that of the 5-Fu group, and the result is shown in Table 6.

TABLE 6 inhibitory effect of ZIYEDAN on H22 hepatocarcinoma tumor

In comparison with the set of models,^*P＜0.05，^**P＜0.01

2.6 conclusion:

(1) the minium preparatium has no influence on the weight of a tumor recurrence model of an H22 liver cancer mouse;

(2) compared with a model group, the three dose groups of the high, middle and low of the red sage root have obvious or very obvious tumor inhibition effects (P is less than 0.05, P is less than 0.01) on a tumor recurrence model of an H22 liver cancer mouse, and are in dose-effect dependence;

(3) the tumor inhibition effect of the high-dose group of the ziyedan on the tumor recurrence model of the H22 liver cancer mouse is equivalent to 5-Fu.

(4) Compared with the model group, the phyllanthus urinaria single application does not show obvious tumor inhibition effect (P is more than 0.05) on a tumor recurrence model of an H22 liver cancer mouse. The results suggest that the therapeutic effect of ziye dan on liver cancer recurrence is better than that of phyllanthus urinaria.

Example 3Inhibition effect of ziye Dan on tumor recurrence of HepG2 tumor-bearing mice

3.1 materials of laboratory instruments:

(1) laboratory animal

BALB/C-nu nude mice, male, 5 weeks, 40, purchased from Wafukang Biotechnology GmbH, license number: SCXK (jing) 2014-: 11401300032435. the animal is adaptively fed in an oral hospital in Beijing for 25 days, and is fed by adopting standard pellet feed, drinking water freely, illuminating by natural day and night light, the room temperature is 18-26 ℃, and the relative humidity is 40-70%.

(2) Tumor cell strain

HepG2 human hepatoma cell, a gift from the institute of tropical medicine of Guangzhou university of traditional Chinese medicine

(3) Experimental drugs

Ziye Dan capsule.

Adult dose: 60kg for adult, 5 granules for adult orally taking 3 times a day, each granule containing 0.5g, namely 0.125g/kg, and the mouse dosage (g/kg) converted according to equivalent dose:

0.125g/kg×9.01＝1.126g/kg

fluorouracil for injection (5-Fu)

3.2 Experimental methods:

human liver cancer HepG2 cells (from) cultured in vitro in 5% CO₂In a 37 ℃ incubator, conventional culture is carried out by using a 1640 culture medium of 10% inactivated fetal bovine serum, and the cells are harvested and counted for tumor cell inoculation every 2-3d routine passage when the cell proliferation shows an exponential growth phase.

Preparing a cell suspension: collecting liver cancer HepG2 cells in logarithmic growth phase, digesting with pancreatin for 2-3min, stopping digestion with 1640 culture solution containing 10% FBS, centrifuging at 1000 rpm for 4min, washing with PBS for 3 times, and adjusting cell density to 2 × 10⁷One per ml.

Animal molding: observing general physiological index, weight and eating condition of animal, adaptively feeding animal for 1 week, and injecting 0.1ml subcutaneously into neck and shoulder with density of 2 × 10⁷Liver cancer HepG2 cells per ml.

The HepG2 cells are inoculated under the skin of the neck, shoulder and back of 40 BALB/C-nu nude mice, and are grouped with random blocks according to the body weight on the next day of inoculation, and the groups are divided into a high-dose group of the minium, a medium-dose group of the minium, a low-dose group of the minium, a 5-Fu group and a model group, 5 groups are included, 8 cells in each group are continuously administrated for 7 weeks, and on the next day after the last administration, the minium is killed and the materials are obtained by anesthesia. The grouping is shown in table 7 below.

Table 7 example 3 components and dosing schedule

3.3 Observation items and evaluation indexes

3.3.1 general State Observation

The animal was observed for mental state, reactivity, diet and excretion status, rash and tumor ulceration. Animals presented with deteriorated and bad condition and animals were unable to obtain sufficient food and water (incapacity) with CO₂So that the patient is happy and dies.

3.3.2 tumor suppressor action

the tumor growth inhibition rate is (1-T/C) multiplied by 100 percent,

3.4 data processing

Comparisons between groups, using independent sample T-test, comparisons between three or more groups, using one-way ANOVA test; if the variance is not uniform, non-parametric rank-sum test is used. All data were analyzed using SPSS19.0 and P <0.05 was considered statistically significant.

3.5 Experimental results:

(1) weight:

no abnormality exists in activity and diet of BALB/C-nu nude mice before molding. There was no significant difference in body weight average (P > 0.05) between groups before and after administration. The specific results are shown in Table 8.

TABLE 8 weight change before and after drug administration

(2) Inhibition of tumor recurrence in HepG2 tumor-bearing mice:

the red sage root can effectively inhibit the growth of human liver cancer cells in a mouse body, and has an inhibiting effect on the recurrence and metastasis of liver cancer. The results are shown in Table 9.

TABLE 9 inhibitory Effect of ZIYEDAN on tumor recurrence in HepG2 tumor-bearing mice

In comparison with the set of models,^*P＜0.05，^**P＜0.01。

3.6 conclusion:

(1) the minium preparatium has no influence on the weight of a tumor recurrence model of a HepG2 liver cancer mouse;

(2) compared with a model group, the three high, medium and low dose groups of the paeonia lactiflora have obvious or very obvious tumor inhibition effect on a HepG2 liver cancer mouse relapse model (P is less than 0.05, and P is less than 0.01); and has a quantitative effect relationship;

(3) the inhibition effect of the low group of the ziye Dan on a HepG2 liver cancer mouse recurrence model is equivalent to 5-Fu, and the effect of the high and medium dose of the ziye Dan is stronger than 5-Fu. The treatment effect of the red sage root on the recurrence of the liver cancer is better than that of 5-Fu.

Example 4Acute toxicity (maximum tolerated dose) test for Ziye Dan animals

4.1 Experimental materials:

(1) laboratory animal

Half of Kunming mice, 20 +/-2 g of male and female, 40 mice, provided by the Experimental animal center of Sian university of transportation, and the qualification certificate: shanxi medical science No. 09-003.

(2) Experimental drugs

The tested drugs are: ziye Dan capsule

Blank control: distilled water

4.2 Experimental methods:

(1) instructions for selection of route of administration

The product is a Chinese medicinal capsule, the clinical administration route is oral administration, and the test adopts the intragastric administration route which is consistent with the clinical administration route.

(2) Experimental groups and dosages

Animals were randomly divided by body weight into 2 groups of 20 animals each and 10 animals each, one of which was a drug-administered group and the other was a control group. The mice in the administration group are subjected to intragastric administration according to the body weight, 0.3ml/10g of the Ziye Dan capsule, namely the powder and the water are administered for 3 times within 24 hours at intervals of 4 hours, and the total dose in one day is 225 times of the clinical dose. The control group was given the same volume of distilled water for the same administration time as the administration group.

(3) Observation of indicators and times

After administration, the animals were continuously observed for their response (e.g., general manifestations, respiration, activity, presence or absence of convulsions), duration of toxic symptoms, recovery, daily mortality, and every other day body weight. The dead animals were roughly dissected and observed for the condition of each major organ. The observation time was 7 days after the administration. After the 7-day observation period, the surviving animals were roughly dissected and the conditions of the major organs were observed.

4.3 Experimental results:

within an observation period of 7 days after the administration, the mice of the ziyedan administration group have good general condition, glossy hair, normal mental activities, no abnormal secretion and abnormal respiration, the body weight is increased from 20.5 +/-1.4 g before the test to 24.5 +/-2.7 g, and no death of the animals is seen. After 7 days, all mice were killed by cervical spondylolysis and necropsy, and as a result, no pathological changes were observed in the heart, liver, spleen, lung, kidney, stomach, intestine, thymus, adrenal gland, abdominal cavity, thoracic cavity and other major organs and body cavities of all mice. During the maximal dosing period, mice were observed for certain symptoms. The result shows that the 225 times of clinical dose of the purple-leaf lead is administrated to the mice by intragastric administration for three times every day, and the animals have no obvious toxic reaction and do not cause death.

4.4 conclusion:

the maximum tolerance of the mice with the capsule powder of the Ziye Dan is 225 times of the clinical human dosage.

Example 5Long-term toxicity test of ziye Dan rat

5.1 Experimental materials:

(1) laboratory animal

SD rats, hermaphrodite halves, 10-112g, provided by the experimental animals center of the western medicine university, certification: shanxi medical certificate 08-005.

(2) Experimental drugs

The tested drugs are: ziye Dan capsule and Yexiazhu

Blank control: distilled water

(3) Animal feeding conditions: the rats are bred for 2 weeks adaptively, bred in an open room, air-conditioned to control the temperature, controlled at the highest temperature of 27 ℃ and the lowest temperature of 24 ℃ in the room, and controlled at the relative temperature of 58-70 percent by conventional ventilation and natural light source. Feeding with solid feed, and freely taking food and drinking normal water. Animals were housed in cages of 5 animals per sex, and the cages were cleaned 1 time per day.

5.2 Experimental methods:

(1) instructions for selection of route of administration

(2) Experimental groups and administration dose

Animals were randomly divided into 5 groups of 40 animals each, 20 animals each. Respectively, blank control group (40ml/kg), small dose group (equivalent to 25 times of daily dosage of adults), medium dose group (equivalent to 50 times of daily dosage of adults), large dose group (equivalent to 100 times of daily dosage of adults) and phyllanthus urinaria (451 g/kg). The dosing period was 6 months, with a subset of animals (approximately 1/3 for female and male) in each group sacrificed 3 months after dosing, and the remaining animals sacrificed by continued dosing for 6 months or 14 days after 6 months. The animals in each group are respectively administrated 1 time in the morning and afternoon, the time interval is 5-6 hours, the administration volume is 40ml/kg body weight, and the administration time in the morning is 8: 00-10: 00, dosing time in the afternoon was 2: 30-4: 00, 6 days per week, once every week.

(3) Observation of indicators and times

A. General expression of

The animal's expression, hair, behavior, activity, diet, feces, secretions and the like were observed day by day.

B. Index of hematology

At 3 months and 6 months, respectively, and 2 weeks after discontinuation of the administration, blood was taken from each group of animals, and White Blood Cells (WBC), platelets (Pt), Neutrophils (NE), and Lymphocytes (LY) were measured using a fully automatic blood cell counter.

C. Biochemical index of blood

Respectively taking blood from each group of animals at 3 months and 6 months of administration and 2 weeks of withdrawal, separating serum, and measuring alanine amino transferase (ALT), aspartate amino transferase (AST), Albumin (ALB) and myoliver (Cr) in serum with full-automatic biochemical analyzer.

D. Histopathological examination

Pathological examination was performed on organs of all animals sacrificed at each stage, including 20 tissues of heart, liver, spleen, lung, kidney, adrenal gland, thymus, testis, epididymis, prostate, ovary, uterus, mesenteric lymph node, esophagus, stomach, jejunum, colon, bladder, sternum (bone marrow), brain, pituitary, brain, etc., fixed with 10% formaldehyde, paraffin-embedded sections, HE-stained, and pathological examination was performed.

5.3 statistical analysis

The mean value of each group was calculated as. + -. standard deviation from the mean value of the group calculated for female and male groups, the differences between the administered groups and the control group were compared using the t-test, and the measured blood cell count and serum biochemical test data were compared with the range of normal values for the germ line rats to determine whether each test was abnormal.

5.4 Experimental results:

(1) effect on general conditions in rats

During the administration period, the cacumen Securinegae Suffruticosae has listlessness, lackluster hair, listlessness, and disappearance of above symptoms after stopping administration for 2 weeks. The rest of the rats in each group are not abnormal.

(2) Hematology examination

At 3 months and 6 months of administration, and 2 weeks after discontinuation, blood was taken from each group of animals and subjected to routine blood tests. The results show that when the phyllanthus urinaria group is administrated for 6 months, the WBC and NE% of female and male rats are obviously increased, LY% and Pt are obviously reduced (P < 0.05), and the rats are recovered to be normal when the drug is stopped for 2 weeks. The administration time of the Ziye Dan group is 3 months, 6 months and 2 weeks after stopping administration, and no obvious influence is caused on hematology index (P > 0.05). The results are shown in Table 10.

TABLE 10 influence of ZIYEDAN on hemogram in rats

Note: compared with the control group, the compound of the formula,^*P﹤0.05

(3) biochemical examination of blood

At 3 months and 6 months after stopping administration for 2 weeks, blood is taken from each group of animals, serum is separated, and various biochemical indexes are measured. The results show that ALT, AST and Cr of female and male rats are obviously increased when the phyllanthus urinaria group is administrated for 6 months, ALB of the female is obviously reduced (P < 0.05), and the female recovers to be normal when the medicine is stopped for 2 weeks. The biochemical indexes of various dosage groups of the red sage root have no significant difference (P > 0.05) in various periods. The results are shown in Table 11.

TABLE 11 Biochemical Effect of ZIYEDAN on rat blood

Note: compared with the control group, the compound of the formula,^*P﹤0.05

(4) histopathological examination

Pathological examination results carried out 3 months, 6 months and 2 weeks after drug withdrawal show that the cell structures and morphologies of various tissues including heart, liver, spleen, lung, kidney, adrenal gland, thymus, testis, epididymis, prostate, ovary, uterus, mesenteric lymph node, esophagus, stomach, jejunum, colon, bladder, sternum (bone marrow), brain, pituitary and brain of each dose group of the control group and the ziyedan have no abnormal pathological changes except for the sporadic non-specific inflammation of the tissues including lung, kidney and the like in individual animals. After 6 months of administration, the results of pathological examination of phyllanthus group showed that liver cells around the central vein of liver undergo diffuse necrosis, and part of the liver cells are accompanied by monocyte infiltration and vacuolization; the kidney tissue can be seen in the vacuolar degeneration of the cortical tubular epithelial cells with different degrees of necrosis and shedding. The pathological examination result of the phyllanthus urinaria group is not abnormal when the medicine is administered for 3 months and 6 months.

5.5 conclusion:

the Ziye Dan is administrated to rat by 100, 50 and 25 times of daily dosage for 6 months, and has no drug-related toxic reaction and no accumulative toxic reaction. While the phyllanthus urinaria presents reversible toxic reactions in the liver and kidney.

The above test results suggest that the combination of Phyllanthus urinaria, Coriolus versicolor, Salvia miltiorrhiza and Lithospermum erythrorhizon can reduce toxicity of Phyllanthus urinaria. The treatment of liver cancer is a long-term process, the physiological functions of patients who relapse after liver cancer operation are fragile due to the influence of diseases, and medicines which have small influence on the functions of liver and kidney and are safer should be selected during treatment. Compared with phyllanthus urinaria, the traditional Chinese medicine composition with active raw materials consisting of phyllanthus urinaria, corious versicolor, salvia miltiorrhiza and lithospermum has better safety and more definite curative effect, thereby being more suitable for clinically inhibiting patients with postoperative recurrence of liver cancer.

Claims

1. The application of a traditional Chinese medicine composition comprising common leafflower herb, coriolus versicolor, salvia miltiorrhiza and lithospermum in preparing a medicine for inhibiting postoperative recurrence of liver cancer is disclosed, wherein the traditional Chinese medicine composition is prepared from the following medicinal materials in parts by weight: 10 parts of phyllanthus urinaria, 4 parts of coriolus versicolor, 1 part of salvia miltiorrhiza and 1 part of lithospermum; the preparation method comprises the following steps:

extracting cacumen Securinegae Suffruticosae with water, adsorbing the obtained extractive solution with macroporous adsorbent resin, eluting with 30-75% ethanol, mixing eluates, and concentrating to obtain extract I; extracting the residue after decocting cacumen Securinegae Suffruticosae with ethanol to obtain ethanol extractive solution; extracting Saviae Miltiorrhizae radix and radix Arnebiae with ethanol to obtain ethanol extractive solution, mixing with the ethanol extractive solution of cacumen Securinegae Suffruticosae, and recovering solvent to obtain extract II; mixing the residue of Coriolus versicolor, Saviae Miltiorrhizae radix and radix Arnebiae, extracting with water to obtain water extract, and recovering solvent to obtain extract III; mixing the extracts I, II and III, granulating, oven drying, and making into capsule or granule, or granulating and tabletting to make tablet.

2. The use of claim 1, wherein the liver cancer comprises primary and secondary liver cancer.

3. The use of claim 2, wherein the primary liver cancer comprises hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, intrahepatic cholecystadenocarcinoma, hepato-cholangiocarcinoma, hepatosarcoma, hepatoendotheliocytoma, hepatoepithelioid endotheliocytoma, and hepatic fibrolamellar carcinoma.

4. The use according to claim 1, wherein the necessary adjuvants are added during the granulation and shaping process.

5. The use of claim 1, wherein the traditional Chinese medicine composition comprising Phyllanthus urinaria L, Coriolus versicolor, Salvia miltiorrhiza Bunge and Lithospermum erythrorhizon is Bordetella purpurea Bunge.