WO2020010519A1 - 一种治疗肝癌和抑制肝癌术后复发的中药组合物 - Google Patents

一种治疗肝癌和抑制肝癌术后复发的中药组合物 Download PDF

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WO2020010519A1
WO2020010519A1 PCT/CN2018/095153 CN2018095153W WO2020010519A1 WO 2020010519 A1 WO2020010519 A1 WO 2020010519A1 CN 2018095153 W CN2018095153 W CN 2018095153W WO 2020010519 A1 WO2020010519 A1 WO 2020010519A1
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liver cancer
traditional chinese
chinese medicine
medicine composition
extract
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PCT/CN2018/095153
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English (en)
French (fr)
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林德良
郭平
康超
蒿长英
钟云
黄卉
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北京汉典制药有限公司
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Publication of WO2020010519A1 publication Critical patent/WO2020010519A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, and relates to a traditional Chinese medicine composition, in particular to a traditional Chinese medicine composition comprising Yexiazhu, Yunzhi, Salvia miltiorrhiza and comfrey. More specifically, the present invention relates to a traditional Chinese medicine composition for treating liver cancer and inhibiting recurrence of liver cancer after surgery, and a pharmaceutical combination of the traditional Chinese medicine composition and 5-Fu.
  • Liver cancer is a high-risk and extremely harmful malignant tumor. Because of the initial asymptomatic, high malignancy, rapid disease transition, short survival time, and high mortality rate, primary liver cancer is known as the "king of cancer.” An estimated 782,500 new cases of liver cancer and 745,500 deaths worldwide in 2012.
  • Radical surgery is the preferred treatment for liver cancer.
  • most patients due to the characteristics of hidden attack, long incubation period, high malignancy, rapid progress, strong invasiveness, easy metastasis, and poor prognosis, most patients have reached the middle and advanced stages of the disease when they are diagnosed, and many of them have lost the opportunity for surgery. According to statistics, only 20% of liver cancer patients can undergo surgery.
  • postoperative recurrence of liver cancer is also one of the main factors affecting the long-term efficacy of primary liver cancer. It is reported in the literature that the 5-year recurrence rate of primary liver cancer after radical resection is 60% -75%, and the recurrence rate of 1-year postoperative is as high as 51.4% -72% (Zhang Zhijian, Wu Mengchao, Clinical research progress in preventing recurrence of primary liver cancer , "Foreign Medical Surgery Volume", 1999, 26 (2): 129). The peak time of recurrence is 6 months after operation, and the peak time is 3 to 4 months after operation.
  • the average time of metastasis and recurrence is (198 ⁇ 29) d (Lu Xin, Zhao Haitao, Mao Yilei, etc., patients with hepatocellular carcinoma)
  • d Li Xin, Zhao Haitao, Mao Yilei, etc., patients with hepatocellular carcinoma
  • interventional embolization chemotherapy systemic chemotherapy, radiation therapy, radiofrequency ablation, and biological therapy are widely used in the treatment of liver cancer and inhibiting recurrence of liver cancer after surgery, the efficacy and prognosis are not ideal, and the cost is high. Big. Therefore, the drug treatment of liver cancer and the plan to suppress the recurrence of liver cancer after surgery are far from meeting the clinical needs.
  • Yunzhi, Gan and Ping is beneficial to nourishing yin, nourishing yin and relieving evil, reconciling yin and yang, and is a medicine for ministers.
  • Junyao detoxifies to dispel evil
  • Chenyao replenishes yin to remedy
  • Salvia miltiorrhiza, bitter and slightly cold enters the heart, liver meridian, clears the cooling blood, promotes blood circulation, and is used here to promote blood circulation, stasis, and relieve pain.
  • Lithospermum, bitter, salty, cold into the heart, liver meridian, cooling blood, promoting blood circulation, detoxification, used here, compatible with Ye Xiazhu, not only play the role of heat and cooling blood detoxification, and increase blood circulation, It is also a medicine.
  • Ziyedan is safe. Rats were administered gavage at 100, 50, and 25 times the human daily dose for 6 months. No toxic reactions related to Ziyedan and no cumulative toxicity were seen.
  • the invention provides a new option for the treatment of clinical liver cancer and clinically inhibiting the recurrence of liver cancer after surgery.
  • the invention provides a traditional Chinese medicine composition for treating liver cancer or inhibiting recurrence of liver cancer after surgery.
  • the traditional Chinese medicine composition is composed of the following parts by weight of medicinal materials: 5-20 parts of Yexiazhu, 2-8 parts of Yunzhi, and Danshen 1 -5 parts, 1-5 parts of comfrey.
  • the present invention provides a medicine for treating liver cancer or inhibiting recurrence of liver cancer after surgery.
  • the medicine comprises a combination of a traditional Chinese medicine composition and 5-Fu.
  • the traditional Chinese medicine composition is composed of the following parts by weight of medicinal materials: 5-20 parts of bead under the leaf, 2-8 parts of Yunzhi, 1-5 parts of Salvia miltiorrhiza, 1-5 parts of Sage.
  • the invention provides an application of a traditional Chinese medicine composition for preparing a medicament for treating liver cancer or inhibiting recurrence of liver cancer after surgery.
  • the traditional Chinese medicine composition is composed of the following parts by weight of medicinal materials: 5-20 parts of Yexiazhu, 2-8 of Yunzhi Servings, 1-5 servings of salvia miltiorrhiza, 1-5 servings of comfrey.
  • the present invention provides the application of a combination of a traditional Chinese medicine composition and 5-Fu in the preparation of a medicament for treating liver cancer or inhibiting recurrence of liver cancer after operation.
  • the traditional Chinese medicine composition is composed of the following parts by weight of medicinal materials: Ye Xiazhu 5 -20 servings, 2-8 servings of Yunzhi, 1-5 servings of Salvia miltiorrhiza, 1-5 servings of Sage.
  • the invention provides a method for treating liver cancer or inhibiting recurrence of liver cancer after surgery, which method comprises administering to a patient in need an effective amount of a traditional Chinese medicine composition composed of the following parts by weight of medicinal materials: 5-20 parts of Yexiazhu, Yun 2-8 servings of zhi, 1-5 servings of salvia miltiorrhiza, 1-5 servings of comfrey.
  • the present invention provides a method for treating liver cancer or inhibiting recurrence of liver cancer after surgery, the method comprising administering to a patient in need an effective amount of a traditional Chinese medicine composition consisting of the following parts by weight of medicinal materials: Ye Xiazhu 5-20 Servings, 2-8 servings of Yunzhi, 1-5 servings of Salvia miltiorrhiza, and 1-5 servings of Arnebia mirabilis, combined with 5-Fu.
  • the traditional Chinese medicine composition of the present invention is preferably composed of the following parts by weight of medicinal materials: 10 parts of leaf bead, 4 parts of Yunzhi, 1 part of Salvia miltiorrhiza, and 1 part of comfrey.
  • the traditional Chinese medicinal composition of the present invention is prepared by the following methods: the bead under the leaves is extracted with water, the macroporous adsorption resin on the obtained extract is adsorbed, and it is completely eluted with 30-75% ethanol; the eluates are combined and concentrated to obtain extract I; Extract the dregs after boiling down with ethanol to obtain an alcohol extract; extract Danwort together with ethanol to obtain an alcohol extract, and combine with the alcohol extract of Yexiazhu to recover the solvent to obtain extract II; Yunzhi is combined with the salvia of Salvia miltiorrhiza and comfrey, and extracted with water to obtain a water extract, and the solvent is recovered to obtain extract III; the extracts I, II, and III are mixed well.
  • the traditional Chinese medicine composition of the present invention may further include pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients include (1) diluents such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salts (such as: calcium sulfate, calcium hydrogen phosphate, medicine Use calcium carbonate, etc.), mannitol, etc., vegetable oil, polyethylene glycol, etc .; (2) binders, such as: distilled water, ethanol, starch pulp, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl fiber And ethyl cellulose, hypromellose, etc .; (3) disintegrating agents, such as: dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, and cross-linked carboxymethyl (4) Lubricants, such as: magnesium stearate, micronized silica
  • the Chinese medicinal composition of the present invention is a clinically acceptable preparation, including a preparation for parenteral administration and a preparation for parenteral administration;
  • the preparation for parenteral administration is selected from the group consisting of powder, tablet, granule, Capsules, pills, emulsions or suspensions;
  • the parenteral preparation is selected from the group consisting of injections, sprays, suppositories, infusions, patches or ointments.
  • mix extracts I, II, III granulate, dry, make capsules or granules, or tablet into tablets after granulation; if necessary, add in the granulation and molding process
  • the pharmaceutically acceptable excipient for example, mix extracts I, II, III, granulate, dry, make capsules or granules, or tablet into tablets after granulation; if necessary, add in the granulation and molding process
  • the pharmaceutically acceptable excipient for example, mix extracts I, II, III, granulate, dry, make capsules or granules, or tablet into tablets after granulation; if necessary, add in the granulation and molding process
  • the pharmaceutically acceptable excipient for example, mix extracts I, II, III, granulate, dry, make capsules or granules, or tablet into tablets after granulation; if necessary, add in the granulation and molding process
  • the pharmaceutically acceptable excipient for example, mix extracts I, II, III, granulate, dry, make capsules or
  • the Chinese medicinal composition of the present invention can be made into a capsule form, which is administered orally, a capsule of 0.5 g specification, 1 to 25 capsules at a time, 3 times a day, 24 weeks as a course of treatment.
  • the liver cancer is primary liver cancer or secondary liver cancer;
  • the primary liver cancer is hepatocellular carcinoma, hepatoblast carcinoma, bile duct cell carcinoma, intrahepatic gallbladder adenocarcinoma, and liver-biliary duct mixed cell carcinoma.
  • Hepatic sarcoma hepatic vascular endothelial sarcoma, hepatic epithelioid vascular endothelial cell tumor, or liver fibrous lamellar carcinoma.
  • the experimental methods in the following examples are conventional methods.
  • the amount of solvent (such as water, ethanol, etc.) used in the preparation of each test drug is a volume multiple based on the weight of the medicinal material.
  • Ziyedan capsules (hereinafter referred to as "ziyedan") are provided by Beijing Handian Pharmaceutical Co., Ltd., batch number: 13040102; Ziyedan capsules are prepared through the following prescription and process:
  • the leaves under the leaves are extracted with water.
  • the first time add 12 times the amount of water and cook for 1.5 hours, and the second time add the 10 times amount of water, and cook for 1 hour.
  • the decoction was combined, centrifuged, and the supernatant was adsorbed with H103 macroporous adsorption resin, which was completely eluted with 30 and 60% ethanol.
  • the amount was refluxed for 1 hour, and the salvia miltiorrhiza-purorium alcohol extract was combined with the extract from the leaves of bezoar, and the solvent was recovered to obtain extract II. Yunzhi was combined with the salvia salvia miltiorrhiza and purplishgrass slag, and decocted twice with water. 12 times the amount of water each time, 1.5 hours for the first time, and 1 hour for the second time, the water extract was obtained, and the solvent was recovered to obtain the extract III; the extracts I, II, and III were mixed uniformly, and pharmaceutically acceptable excipients were added. To 500g, granulated, dried, filled with capsules, 0.5g / capsule.
  • Yexiazhu extract (hereinafter referred to as "Yehxiazhu"), provided by Beijing Handian Pharmaceutical Co., Ltd .; prepared by the following method: extraction using traditional water decoction process, extracting Yexiazhu raw medicinal decoction twice, The two decoctions were combined, filtered, and the filtrate was concentrated to 1: 1 (the weight of the bead medicine under the leaf: the volume of the filtrate).
  • Fluorouracil (5-Fu) for injection 0.25g / 10ml per bottle, Tianjin Jinyao Amino Acid Co., Ltd.
  • Microplate reader (MULTLSKAN MK3), Hyclone, USA.
  • liver cancer cell line Human liver cancer cell SMMC-7721, purchased from Shanghai Cell Bank, Chinese Academy of Sciences.
  • Drug 1 (leaf under the leaf water extract through macroporous resin): Take 1100 g of raw medicine under the leaf, after recording and registration, add water to decoction twice, first add 12 times the amount of water, decoction for 1.5 hours, second time Add 10 times the amount of water and cook for 1 hour. The gauze was filtered after each decoction, and the filtrate was collected.
  • Drug 7 (mixed extract): According to the raw material composition of Ziye Dan (2100g of bead under the leaf, 800g of Yunzhi, 200g of Salvia miltiorrhiza, 200g of purple grass) and the yield of its drug 1-6, the amount shown in Table 1 The extracts prepared according to the methods described in the aforementioned Drugs 1-6 were separately taken and mixed to obtain Drug 7, a total of 259.28g.
  • Drug 8 (Ziye Dan preparation): take 2100g of Yexiazhu crude medicine, 800g of Yunzhi crude medicine, 200g of salvia crude medicine, 200g of purple herb medicine; extract the Yexiazhu twice with water, add 12 times the amount of water for the first time, and cook for 1.5 Hour, add 10 times the amount of water for the second time, and cook for 1 hour.
  • the H103 macroporous adsorption resin was adsorbed on the obtained extract, and was completely eluted with 30% and 60% ethanol. The eluates were combined and concentrated to obtain extract I.
  • the dregs after boiling under the leaf bead water were used for 12 times the amount of 95 % Ethanol extraction, to obtain the leaf under the bead alcohol extract; Dan participated in the joint extraction with 95% ethanol twice, the first addition of 10 times the amount of alcohol, reflux for 1.5 hours, the second addition of 6 times the amount of alcohol, reflux for 1 hour
  • the salvia miltiorrhiza- shikonin alcohol extract was combined with the extract from the leaves of bezoar, and the solvent was recovered to obtain extract II. Yunzhi was combined with the salvia of salvia miltiorrhiza and shikonin, and it was boiled twice, 12 times each time. Water, 1.5 hours for the first time, 1 hour for the second time, get the water extract, recover the solvent to obtain the extract III; mix the extracts I, II, III, dry, and add pharmaceutically acceptable excipients to 500g , Which is drug 8.
  • SMMC-7721 cells were seeded in a 25 cm 2 culture flask, and about 8 mL of RPIM-1640 culture solution (10% FBS, 100 U ⁇ mL -1 cyanogenin, 100 ⁇ g ⁇ mL -1 streptomycin sulfate) was added, and the temperature was 37 ° C and 5% CO. 2.
  • RPIM-1640 culture solution 10% FBS, 100 U ⁇ mL -1 cyanogenin, 100 ⁇ g ⁇ mL -1 streptomycin sulfate
  • a cell suspension of 1.5 ⁇ 10 4 mL -1 was seeded in a 96-well culture plate at 200 ⁇ L per well. The wells were adjusted to zero without cells and filled with PBS. After 24h, the above 16 working fluids were replaced, and 5 replicates were used for each work, and the culture was continued for 24h, 48h, or 72h. Add 20 ⁇ L of 5 mg ⁇ mL -1 MTT solution to each well. After incubating in the incubator for 4 hours, discard the incubation solution. Add 150 ⁇ L of DMSO to each well and shake and mix at room temperature (22 ° C) for 15 min. After the precipitate was completely dissolved, the optical density (A) was measured at 570 nm on a microplate reader. Calculate the inhibition rate according to the following formula:
  • Inhibition rate (%) (A blank hole- A measurement hole ) / (A blank hole- A zero adjustment hole ) * 100%.
  • Drugs 1-6 at low concentrations have basically no inhibitory or even promotion effect on the proliferation of human liver cancer cells SMMC-7721; especially drug 4 (Yunzhi water extract) shows a positive effect on the whole observation and culture time. Promotion of liver cancer cell growth.
  • High-concentration drug 4 (Yunzhi water extract) showed a promotion effect on the growth of liver cancer cells throughout the culture period of observation.
  • Drugs 7 and 8 at low concentrations showed a promotion effect on liver cancer cell proliferation at 24 hours of intervention, but with the extension of the effect time, 48 hours and 72 hours inhibited the proliferation of liver cancer cells to varying degrees.
  • the inhibitory effects of high concentrations of drugs 7 and 8 on the proliferation of liver cancer cells had a time-dependent effect, and after 72 hours of intervention, both showed a strong inhibitory effect on the growth of cancer cells.
  • the effect of drug 8 is stronger than that of drug 7.
  • the single-flavored leaf beads, Yunzhi, comfrey and Salvia miltiorrhiza have little or no inhibitory effect on the proliferation of human liver cancer SMMC-7721, and some have shown to promote the growth of cancer cells (such as water extract ).
  • cancer cells such as water extract
  • the combination of four Chinese herbs can inhibit the growth of liver cancer cells. It is shown that the anti-hepatocellular carcinoma effect of the traditional Chinese medicine composition of the present invention is not a simple addition of the effects of the individual medicinal herbs, but a new effect produced by the compatibility of the medicinal flavors.
  • the drug 8 prepared by a specific process has a stronger effect than the drug 7 obtained by simply mixing the extracts extracted from the four Chinese medicines. It is speculated that during the preparation of Drug 8, after multiple steps of pharmaceutical extraction process, the medicinal materials in the compound are reasonably compatible, and the various chemical components contained in them interact with each other, thereby providing the necessary material basis for its antitumor effect.
  • the preparation method of drug 8 is basically the same as the preparation process of Ziye Dan, so Ziye Dan is used in the following experiments for corresponding research.
  • ICR mice male, weighing about 18-22 g, 120, were purchased from Hua Fukang Biotechnology Co., Ltd., license number: SCXK (Jing) 2014-0004. After all animals were purchased, the animals' general physiological indicators, body weight, and diet were observed. Adapted feeding for 3 days. Feed on standard pellets and drink freely. Natural day and night light illumination, room temperature 18 ⁇ 26 °C, relative humidity 40% ⁇ 70%.
  • H22 liver cancer ascites tumor cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • Mouse liver cancer H22 cells were cultured in vitro in a 5% CO 2 , 37 ° C incubator with RPMI-1640 medium with 10% inactivated fetal bovine serum, and routinely passaged every 2-3 days. Cells were harvested during exponential growth and counted for tumor cell seeding.
  • Cell animal passage in vivo observe the general physiological indicators, body weight and feeding status of the animal. The animals are adaptively fed for 1 week. After observing that all the animals are normal, take 3-5 animals and inject 0.2ml in the left lower abdominal cavity at a density of 1 ⁇ 10 7 cells / ml. H22 tumor cells; normal breeding, observe the state of the animal, produce ascites after a few days, take 7-10 days, mice with full ascites in good animal condition take ascites, centrifuge at 1000 rpm, 3 minutes, wash with saline once, count tumor Cells were diluted and mixed.
  • Animal modeling Observe the general physiological indicators, body weight, and feeding status of the animals. The animals are adaptively fed for 1 week. After observing that everything is normal, the right neck and shoulder are subcutaneously injected with 0.1ml density of 2 ⁇ 10 7 cells / ml H22 tumor cells ( The upper ascites was diluted); the number of animals and cells in each group were recorded in the experimental design table.
  • Tumor volume Measure the tumor diameter twice a week or every other day with a vernier caliper.
  • Tumor weight After the administration, the tumor tissue was stripped and weighed, and the average tumor weight changes in each group were compared. The average tumor weight and tumor growth inhibition rate are used to evaluate the anti-cancer effect of the drug in vivo.
  • the calculation formula is:
  • Tumor growth inhibition rate (1-T / C) ⁇ 100%
  • T is the average tumor weight of the administration group
  • C is the average tumor weight of the control group.
  • BALB / C-nu nude mice male, 5 weeks, weighing about 18-20g, 115, purchased from Hua Fukang Biotechnology Co., Ltd., license number: SCXK (Jing) 2014-0004, quality certificate of experimental animals Number: 11401300035982.
  • SCXK Hua Fukang Biotechnology Co., Ltd.
  • SCXK Quality Certificate of experimental animals Number: 11401300035982.
  • HepG2 human liver cancer cells gifted by Beijing Wujiahe Institute of Molecular Medicine
  • mice 60kg for adults, 5 capsules orally three times a day for adults, 0.5g per capsule, 0.125g / kg, equivalent to the equivalent dose for mice (g / kg):
  • Human liver cancer HepG2 cells (derived from) were cultured in vitro, routinely cultured in 10% inactivated fetal bovine serum 1640 medium in a 5% CO 2 , 37 ° C incubator, and routinely passaged every 2-3 days, when the cells proliferated Cells were harvested during exponential growth phase and counted for tumor cell seeding.
  • Cell suspension preparation Take logarithmic growth stage HepG2 cells, trypsinize for 2-3 minutes, terminate digestion with 1640 medium containing 10% FBS, 1000 rpm, centrifuge for 4 min, wash 3 times with PBS, adjust the cell density to 2 ⁇ 10 7 cells / ml.
  • Animal modeling Observe the general physiological indicators, body weight, and feeding status of the animals. The animals were adaptively fed for 1 week. After observing that all the animals were normal, the neck and shoulders were injected subcutaneously with a density of 2 ⁇ 10 7 cells / ml HepG2 cells.
  • mice were subcutaneously inoculated with HepG2 cells. 10-15 days after inoculation, tumor-bearing nude mice with a diameter of less than 5 mm and more than 10 mm were removed, and the volume of solid tumors was measured. Tumor volume and body size were randomly divided into 9 groups with 8 animals in each group. See Table 7 for grouping.
  • Tumor volume Measure the tumor diameter with vernier caliper twice a week or every other day.
  • Tumor weight After the administration, the tumor tissue was stripped and weighed, and the average tumor weight changes in each group were compared. The average tumor weight and tumor growth inhibition rate are used to evaluate the anti-cancer effect of the drug in vivo.
  • the calculation formula is:
  • Tumor growth inhibition rate (1-T / C) ⁇ 100%
  • T is the average tumor weight of the administration group
  • C is the average tumor weight of the control group.
  • mice had no abnormal activity or diet before modeling. Before and after administration, there was no significant difference in body weight of each group (P> 0.05). The specific results are shown in Table 8.
  • ICR mice male, weighing about 18-22 g, 120, were purchased from Hua Fukang Biotechnology Co., Ltd., license number: SCXK (Jing) 2014-0004. After all animals were purchased, the animals' general physiological indicators, body weight, and diet were observed. Adapted feeding for 3 days. Feed on standard pellets and drink freely. Natural day and night light illumination, room temperature 18 ⁇ 26 °C, relative humidity 40% ⁇ 70%.
  • H22 liver cancer ascites tumor cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • Mouse liver cancer H22 cells were cultured in vitro in a 5% CO 2 , 37 ° C incubator with RPMI-1640 medium with 10% inactivated fetal bovine serum, and routinely passaged every 2-3 days. Cells were harvested during exponential growth and counted for tumor cell seeding.
  • Cell animal in vivo passage observe the general physiological indicators, body weight and feeding status of the animal. The animals are adaptively fed for 1 week. After observing that all the animals are normal, take 3-5 animals. Inject the 0.2ml density of 1 ⁇ 10 7 H22 tumor cells into the left lower abdominal cavity. ; Normal breeding, observe the state of the animal, produce ascites after a few days, take 7-10 days, ascites of mice that are in good condition and full of ascites, take ascites, centrifuge at 1000 rpm, 3 minutes, wash with normal saline once, count tumor cell dilution uniform.
  • Animal modeling Observe the general physiological indicators, body weight, and feeding of the animals. The animals are adaptively fed for 1 week. After observing that everything is normal, the right neck and shoulder are injected subcutaneously with a density of 2 ⁇ 10 7 H22 tumor cells / ml ( The upper ascites was diluted); the number of animals and cells in each group were recorded in the experimental design table.
  • Tumor volume Measure the tumor diameter with vernier caliper twice a week or every other day.
  • Tumor weight After the administration, the tumor tissue was stripped and weighed, and the average tumor weight changes in each group were compared. The average tumor weight and tumor growth inhibition rate are used to evaluate the anti-cancer effect of the drug in vivo.
  • the calculation formula is:
  • Tumor growth inhibition rate (1-T / C) ⁇ 100%
  • T is the average tumor weight of the administration group
  • C is the average tumor weight of the control group.
  • Ziyedan has obvious anti-tumor effect, and has a certain dose-effect relationship, which is superior to the Xiazhuzhu group and close to the anti-tumor effect of the 5-Fu group.
  • the results are shown in Table 12.
  • BALB / C-nu nude mice male, 5 weeks, 40, purchased from Hua Fukang Biotechnology Co., Ltd., license number: SCXK (Jing) 2014-0004, laboratory animal quality certification number: 11401300032435.
  • HepG2 human liver cancer cells gift from Institute of Tropical Medicine, Guangzhou University of Traditional Chinese Medicine
  • mice 60kg for adults, 5 capsules orally three times a day for adults, 0.5g per capsule, 0.125g / kg, equivalent to the equivalent dose for mice (g / kg):
  • Human liver cancer HepG2 cells (derived from) were cultured in vitro, routinely cultured in 10% inactivated fetal bovine serum 1640 medium in a 5% CO 2 , 37 ° C incubator, and routinely passaged every 2-3 days, when the cells proliferated Cells were harvested during exponential growth phase and counted for tumor cell seeding.
  • Cell suspension preparation Take logarithmic growth stage HepG2 cells, trypsinize for 2-3 minutes, terminate digestion with 1640 medium containing 10% FBS, 1000 rpm, centrifuge for 4 min, wash 3 times with PBS, adjust the cell density to 2 ⁇ 10 7 cells / ml.
  • Animal modeling Observe the general physiological indicators, body weight, and feeding status of the animals. The animals were adaptively fed for 1 week. After observing that all the animals were normal, the neck and shoulders were injected subcutaneously with a density of 2 ⁇ 10 7 cells / ml HepG2 cells.
  • HepG2 cells were inoculated subcutaneously into the neck and shoulders of 40 BALB / C-nu nude mice, and the group was randomly divided into groups according to body weight on the second day of inoculation. Dan low-dose group, 5-Fu group, model group, a total of 5 groups, 8 animals in each group, continuous administration for 7 weeks, the next day after the last dose, anesthesia and sacrificed and taken materials. See Table 13 for grouping.
  • Tumor volume Measure the tumor diameter with vernier caliper twice a week or every other day.
  • Tumor weight After the administration, the tumor tissue was stripped and weighed, and the average tumor weight changes in each group were compared. The average tumor weight and tumor growth inhibition rate are used to evaluate the anti-cancer effect of the drug in vivo.
  • the calculation formula is:
  • Tumor growth inhibition rate (1-T / C) ⁇ 100%
  • T is the average tumor weight of the administration group
  • C is the average tumor weight of the control group.
  • mice had no abnormal activity or diet before modeling. There was no significant difference in body weight between the groups before and after administration (P> 0.05). The specific results are shown in Table 14.
  • This product is a traditional Chinese medicine capsule, and the clinical route is oral.
  • the test uses the oral administration route, which is consistent with the clinical route.
  • mice were randomly divided into 2 groups according to body weight, 20 in each group, and 10 in male and female, one of which was the administration group and the other was the control group.
  • Mice in the administration group were intragastrically administered Ziyedan capsule powder suspension, 0.3ml / 10g, 3 times within 24 hours, 4 hours apart, and the total dose in one day was equivalent to 225 times the clinical dose.
  • the control group was given the same volume of distilled water for the same time as the administration group.
  • the animal's response (such as general performance, breathing, activity, and presence or absence of convulsions) was continuously observed, the time, duration and recovery of symptoms of poisoning were recorded, the number of animal deaths was recorded daily, and the animal weight was recorded every other day.
  • the dead animals were grossly dissected and the main organs were observed. Observation period is 7 days after administration. At the end of the 7-day observation period, gross dissection was performed on the surviving animals and the status of each major organ was observed.
  • mice in the Ziyedan administration group were generally in good condition, with shiny hair, normal posture, no abnormal secretions and abnormal breathing, and the body weight increased from 20.5 ⁇ 1.4g before the test to 24.5 ⁇ 2.7g, no animal death.
  • all mice were sacrificed by cervical dissection, and gross necropsy was performed.
  • all major organs and body cavities such as heart, liver, spleen, lung, kidney, stomach, intestine, thymus, adrenal gland, abdominal cavity, and thoracic cavity were not seen.
  • Pathological changes During maximal dosing, certain symptoms were observed in mice. The results showed that Ziyedan was administered to mice by intragastric administration at a dose of 225 times the clinical dose three times a day. No significant toxic reaction was observed in the animals and no animal death was caused.
  • the maximum tolerated dose of Ziyedan Capsule in mice by intragastric administration is equivalent to 225 times the clinical human dose.
  • Tested drugs Ziyedan capsule, Yexiazhu
  • Rats are adaptively bred for 2 weeks, kept in open rooms, air-conditioned, temperature controlled, indoor maximum temperature 27 °C, minimum temperature 24 °C, relative temperature 58% -70%, conventional ventilation, natural light source. Feed on solid feed, freely eat and drink regular water. Animals were housed in cages by sex, with 5 animals per cage, and mouse cages were cleaned once a day.
  • This product is a traditional Chinese medicine capsule, and the clinical route is oral.
  • the test uses the oral administration route, which is consistent with the clinical route.
  • Animals were randomly divided into 5 groups according to body weight, with 40 in each group and 20 in male and female.
  • Blank control group 40ml / kg
  • Ziyedan small dose group (equivalent to 25 times the adult daily dose)
  • Ziyedan medium dose group (equivalent to 50 times the adult daily dose)
  • Ziyedan high dose Group (equivalent to 100 times the daily dosage for adults)
  • the leaf bead group (451 g / kg).
  • the dosing cycle is 6 months, and some animals in each group (approximately 1/3 each of female and male) are sacrificed after 3 months of administration, and the remaining animals continue to be administered until 6 months or discontinued after 6 months It was executed at 14 days.
  • Animals in each group were dosed once a day in the morning and in the afternoon, with a second interval of 5-6 hours.
  • the dose volume was 40ml / kg body weight.
  • the morning dose time was 8: 00-10: 00, and the afternoon dose time was 2: 30-4: 00, dosing for 6 days per week, stopping once on Sunday.
  • WBC white blood cells
  • Pt platelets
  • NE neutrophils
  • LY lymphocytes
  • ALT serum alanine aminoconverting enzyme
  • AS albumin
  • Cr muscle liver
  • Pathological examination of the organs of all animals sacrificed at various stages including heart, liver, spleen, lung, kidney, adrenal, thymus, testis, epididymis, prostate, ovary, uterus, mesenteric lymph nodes, esophagus, stomach, jejunum, colon , Bladder, sternum (bone marrow), brain, pituitary, brain and other 20 tissues, fixed with 10% formaldehyde, paraffin-embedded sections, HE staining, for pathological examination.
  • the data of each group were divided into females and males, and the group average ⁇ standard deviation was calculated.
  • the t test was used to compare the differences between the drug administration group and the control group, and the measured blood cell counts and serum biochemical examination data were larger than the germline. Compare the normal range of the rats to determine whether the examinations are abnormal.
  • the pathological examination results of the sublobular bead group showed diffuse necrosis of hepatocytes around the central vein of the liver, some with monocyte infiltration and vacuole; renal tissue showed vacuolar degeneration of cortical renal tubular epithelial cells , With varying degrees of necrosis, shedding.
  • no abnormal results were found in the pathological examination of the bead group.
  • Ziye Dan was administered to rats at 100, 50, and 25 times the daily dose of human for 6 months. There were no drug-related toxic reactions or cumulative toxic reactions.
  • the lobular bead has a reversible toxic reaction in the liver and kidney.
  • the Chinese medicinal composition of the active ingredients of Xiazhuzhu, Yunzhi, Danshen, and Lithospermum according to the present invention is better in safety and efficacy than Xiazhuzhu, so it is more suitable for clinical treatment of liver cancer patients and inhibition of liver cancer. The patient relapsed after the operation.

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Abstract

一种用于治疗肝癌或抑制肝癌术后复发的中药组合物，该组合物由叶下珠5-20份、云芝2-8份、丹参1-5份、紫草1-5份组成；该中药组合物还能够与5-Fu组合制备药物，用于治疗肝癌或抑制肝癌术后复发。

Description

一种治疗肝癌和抑制肝癌术后复发的中药组合物 技术领域
本发明属于医药学领域，涉及一种中药组合物，具体涉及一种包含叶下珠、云芝、丹参和紫草的中药组合物。更具体地，本发明涉及一种用于治疗肝癌和抑制肝癌术后复发的中药组合物以及该中药组合物与5-Fu的药物组合。
背景技术
肝癌是高发的，危害极大的恶性肿瘤。因为初期无症状、恶性程度高、病情转变快、生存期短、病死率高，原发性肝癌被称为“癌中之王”。2012年全世界估计有782,500例新发的肝癌病例和745,500例死亡。
根治性手术是肝癌的首选治疗手段。但是因其起病隐袭、潜伏期长、高度恶性、进展快、侵袭性强、易转移、预后差等特征，大多数患者确诊时，已到疾病的中晚期，很多都丧失了手术机会。据统计，可以进行外科手术的肝癌病人仅为20％。
此外，肝癌术后复发也是影响原发性肝癌远期疗效的主要因素之一。文献报道，原发性肝癌根治术后5年复发率为60％-75％，其中术后1年复发率高达51.4％-72％(张智坚，吴孟超，预防原发性肝癌术后复发临床研究进展，《国外医学外科学分册》，1999，26(2)：129)。术后6个月是复发的高峰时间，其中又以术后3-4月为最高峰，平均转移复发时间为(198±29)d(卢欣，赵海涛，毛一雷等，肝细胞肝癌患者术后早期复发情况，《中国医学科学院学报》，2008，30(4)：415；吴孟超，原发性肝癌的诊断及治疗进展，《中国医学科学院学报》，2008，30(4)：363)。甚至还有文献报道，在原发性肝癌中大肝癌根治性切除术后5年复发率高达80％,小肝癌亦达60％左右(罗时敏，原发性肝癌术后复发机理及其影响因素[J]，《实用癌症杂志》，2001，16(1)：108-110)。与原发性肝癌相比，肝癌术后复发的恶性程度更大，进程更快；由于术后复发患者对化疗不如原发性肝癌患者敏感，造成治疗难度加大且易造成癌症转移。因此，肝癌术后复发对患者造成的影响比原发性肝癌更大。所以，如何控制肝癌术后复发，是提高生存率、改善预后的关键。
目前，虽然介入栓塞化疗、全身化疗、放射治疗、射频消融及生物治疗等多种治疗手段广泛应用于治疗肝癌和抑制肝癌术后复发，但是疗效和预后均不理想，且费用昂贵，毒副作用较大。因此，肝癌的药物治疗和抑制肝癌术后复发的方案远未达到满足的临床需求。
发明内容
为了克服现有技术的不足，寻找治疗肝癌和抑制肝癌术后复发的安全有效的中药制剂，发明人针对含有叶下珠、云芝、丹参和紫草的中药组合物进行了研究。
在该中药组合物中，叶下珠，甘苦、凉，入肝、肺经，具有平肝清热、利水解毒等功效，为君药。云芝，甘、平，有益气养阴、扶正祛邪、调和阴阳等功效，为臣药。君药解毒以祛邪，臣药补阴以扶正，君臣共奏祛邪扶正之功。丹参，苦、微寒，入心、 肝经，清营凉血，活血通经，此处用之活血化瘀、消肿止痛，是为佐使药。紫草，苦、咸、寒，入心、肝经，凉血、活血，解毒，此处用之，与叶下珠配伍，既发挥清热凉血解毒之效，又增活血化瘀之功，也是佐使药。
试验表明，分别将叶下珠、云芝、丹参和紫草四位药单独用于对肝癌细胞的增殖基本没有抑制作用，甚至还有促进作用；但是四味药配伍之后却能够显著抑制肝癌细胞的生长。动物试验也证明紫叶丹对负瘤小鼠的肝癌瘤块生长有显著的抑制，作用好于单用叶下珠；动物实验也证明紫叶丹对多个负瘤小鼠肿瘤复发模型有显著的肿瘤抑制，作用好于单用叶下珠；紫叶丹与化疗药物5-Fu联合后还有协同作用。且紫叶丹安全性好，以人日用剂量的100、50、25倍给大鼠灌胃6个月，未见与紫叶丹有关的毒性反应，亦未见蓄积性毒性反应。本发明为临床肝癌的治疗和临床上抑制肝癌术后复发提供了一种新的选择。
本发明提供一种用于治疗肝癌或抑制肝癌术后复发的中药组合物，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
进一步地，本发明提供一种用于治疗肝癌或抑制肝癌术后复发的药物，所述药物包含一种中药组合物与5-Fu的组合，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
本发明提供一种中药组合物在制备治疗肝癌或抑制肝癌术后复发的药物中的应用，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
进一步地，本发明提供一种中药组合物与5-Fu的组合在制备治疗肝癌或抑制肝癌术后复发的药物中的应用，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
本发明提供一种治疗肝癌或抑制肝癌术后复发的方法，所述方法包括向有需要的患者给予有效量的由如下重量份的药材组成的中药组合物：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
进一步地，本发明提供一种治疗肝癌或抑制肝癌术后复发的方法，所述方法包括向有需要的患者给予有效量的由如下重量份的药材组成的中药组合物：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份，联合给予5-Fu。
本发明的中药组合物优选由如下重量份的药材组成：叶下珠10份，云芝4份，丹参1份，紫草1份。
本发明的中药组合物通过如下方法制备：叶下珠用水提取，所得提取液上大孔吸附树脂吸附，用30-75％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用乙醇提取，得醇提取液；丹参与紫草共同用乙醇提取，得醇提液，与叶下珠的醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，水提取，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀。
本发明的中药组合物中还可包括药学上可以接受的辅料。所述药学上可以接受的辅料，包括(1)稀释剂，例如淀粉、糖粉、糊精、乳糖、预胶化淀粉、微晶纤维、无机钙盐(如：硫酸钙、磷酸氢钙、药用碳酸钙等)、甘露醇等、植物油、聚乙二醇等； (2)粘合剂，例如：蒸馏水、乙醇、淀粉浆、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素和乙基纤维素、羟丙甲纤维素等；(3)崩解剂，例如：干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠等；(4)润滑剂，例如：硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇类、月桂醇硫酸镁等；(5)矫味剂，如：蔗糖、甜菊素等；(6)溶剂，例如：水、酒精等。
本发明中药组合物为临床上可以接受的制剂，包括经胃肠道给药制剂和非经胃肠道给药制剂；所述经胃肠道给药制剂选自散剂、片剂、颗粒剂、胶囊剂、滴丸、乳剂或混悬剂；所述非经胃肠道给药制剂选自注射剂、喷雾剂、栓剂、灌注剂、贴剂或软膏剂。
例如，将浸膏I、II、III混匀，制粒，烘干，制成胶囊剂或颗粒剂，或制粒后压片制成片剂；如有必要，在制粒、成型过程中加入所述药学上可以接受的辅料。
本发明中药组合物可制成胶囊形式，口服施用，0.5g规格的胶囊，一次服用1～25粒，一日3次，24周为一疗程。
本发明中所述肝癌为原发性肝癌或继发性肝癌；所述原发性肝癌为肝细胞癌、肝胚细胞癌、胆管性细胞癌、肝内胆囊腺癌、肝-胆管混合细胞癌、肝肉瘤、肝血管内皮细胞肉瘤、肝上皮样血管内皮细胞瘤或肝纤维板层癌。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。如无特殊说明，各受试药物的制备过程中溶剂(如水、乙醇等)的用量都是以药材重量为基准的体积倍数。
下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
(1)受试药物
紫叶丹胶囊(以下简称“紫叶丹”)，由北京汉典制药有限公司提供，批号：13040102；紫叶丹胶囊通过如下处方、工艺制备：
处方：叶下珠2100g，云芝800g，丹参200g，紫草200g。
叶下珠用水提取，第一次加12倍量水，煎煮1.5小时，第二次加10倍量水，煎煮1小时。合并煎液，离心，上清液用H103大孔吸附树脂吸附，分别用30和60％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用12倍量95％乙醇提取，得叶下珠醇提取液；丹参与紫草共同用95％乙醇提取两次，第一次加醇10倍量，回流1.5小时，第二次加醇6倍量，回流1小时，得丹参-紫草醇提液，与叶下珠醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，加水煎煮两次，每次12倍量水，第一次1.5小时，第二次1小时，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀，加入药学上可以接受的辅料至500g，制粒，烘干，装胶囊，0.5g/胶囊。
叶下珠提取物(以下简称“叶下珠”)，由北京汉典制药有限公司提供；通过如下方法制备：使用传统水煎煮工艺提取，将叶下珠生药用煎煮法提取2次，合并两次煎液，过滤，滤液浓缩为1∶1(叶下珠生药重量：滤液体积)。
注射用氟尿嘧啶(5-Fu)，每瓶装0.25g/10ml，天津金耀氨基酸有限公司。
(2)主要试剂
RPMI-1640培养基，Hyclone公司，GIBCO公司；
胎牛血清，GIBCO公司
0.25％胰酶(含0.04％EDTA)，Hyclone公司；
4％多聚甲醛，Biotopped公司
(3)主要仪器
血球细胞计数板，上海化科实验器材有限公司；
奥特BDS200倒置生物显微镜，重庆奥特光学仪器有限责任公司；
超净工作台(YT-CJ-2ND)，北京亚泰科隆仪器技术有限公司；
二氧化碳培养箱(SANYO)，北京东迅天地医疗仪器有限公司；
低速离心机(SC-3616)，安徽中科中佳科学仪器有限公司；
电子天平(AL204)，METTLER TOLEDO；
电子天平(LQ-A10001)，瑞安市安特称重设备有限公司；
酶标仪，(MULTLSKAN MK3)，美国Hyclone公司。
实施例1叶下珠、云芝、紫草、丹参及其组合对肝癌细胞增殖的影响
1.1实验仪器材料：
(1)肝癌细胞株人肝癌细胞SMMC-7721，购自中科院上海细胞库。
(2)实验药物
对叶下珠、云芝、紫草、丹参分别进行如下处理，得到药物1-8：
药物1(叶下珠水提液过大孔树脂)：取叶下珠生药1100g，记录登记后，加水煎煮提取两次，第一次加12倍量水，煎煮1.5小时，第二次加10倍量水，煎煮1小时。每次煎煮后纱布过滤，收集滤液，则为叶下珠水提液，叶下珠药渣备用。该水提液上H103大孔吸附树脂柱，用纯水冲柱至洗脱液无色；依次用30％乙醇和60％乙醇洗脱完全，收集并合并洗脱液；测定出膏量为，浓缩至500ml，喷雾干燥，得喷干粉末10.5g，即为所述药物1；得率：10.5/1100*100＝0.95％。
药物2(叶下珠醇提)：上步水提后的叶下珠药渣用95％乙醇加热回流提取二次，每次95％乙醇的用量为12倍量，第一次1.5小时，第二次1小时，合并醇提液，浓缩至相对密度为1.18-1.20(50℃)，离心，收集上清液，放于4℃冰箱中静置过夜，过滤，喷雾干燥，得到干粉12.9g，即为所述药物2；得率：12.9/1100*100＝1.17％。
药物3(叶下珠水提)：取叶下珠生药500g，记录登记后，加水煎煮提取两次，第一次加12倍量水，煎煮1.5小时，第二次加10倍量水，煎煮1小时。每次煎煮后纱布过滤，收集滤液，则为叶下珠水提液，浓缩至300ml，喷雾干燥。得到干粉15.6g，即为所述药物3；得率：15.6/500*100＝3.12％。
药物4(云芝水提)：取云芝生药500g，记录登记后，用水煎煮两次，第一次煎煮1.5小时，第二次煎煮1小时，每次用12倍量水；纱布过滤，收集并合并滤液；浓缩至620ml，喷雾干燥，得到喷干粉37.15g，即为所述药物4，得率：37.15/500*100＝7.43％。
药物5(紫草和丹参醇提)：取紫草、丹参生药各500g(丹参粉碎机粉碎)，同放入提取容器内乙醇提取二次，第一次加入10倍量的95％乙醇，回流提取1.5h，第二次加入6倍量的95％乙醇，回流提取1h，趁热过滤，合并滤液，药渣备用；浓缩后离心计量浓缩液总体积为500ml；喷雾干燥，得到喷干粉57.80g，即为所述药物5；得率：57.80g/1000g*100＝5.78％。
药物6(紫草和丹参水提)：取上步紫草和丹参醇提后的药渣，用水煎煮两次，第一次煎煮1.5h，第二次煎煮1h，每次用12倍量水，趁热过滤，合并滤液；浓缩后离心计量浓缩液总体积为690ml，喷雾干燥，得到喷干粉166.72g，即为所述药物6，得率：166.72g/1000g*100＝16.67％。
药物7(混合提取物)：按照紫叶丹的原料组成处方(叶下珠2100g，云芝800g，丹参200g，紫草200g)及其药物1-6的得率，按照表1所示的量分别取按照上述药物1-6所述方法制备的提取物，混合，即为药物7，共259.28g。
表1药物7的配方组成
Figure PCTCN2018095153-appb-000001
药物8(紫叶丹制剂)：取叶下珠生药2100g、云芝生药800g、丹参生药200g、紫草生药200g；叶下珠用水提取两次，第一次加12倍量水，煎煮1.5小时，第二次加10倍量水，煎煮1小时。所得提取液上H103大孔吸附树脂吸附，分别用30％和60％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用12倍量95％乙醇提取，得叶下珠醇提取液；丹参与紫草共同用95％乙醇提取两次，第一次加醇10倍量，回流1.5小时，第二次加醇6倍量，回流1小时，得丹参-紫草醇提液，与叶下珠醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，加水煎煮两次，每次12倍量水，第一次1.5小时，第二次1小时，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀，烘干，加入药学上可以接受的辅料至500g，即为药物8。
1.2工作液配制
按各药材在处方中的比例以及各部位提取率换算得到药物1-8的取样量，如表2所示，分别按照如下方法配制成工作液I和工作液II：
按照表2所示称取药物，加RPIM-1640培养液(含10％FBS、100U·mL -1青雾素、100μg·mL -1硫酸链霉素)至10mL，4000rpm离心20min，无菌过滤，即为母液。临用时，母液用RPIM-1640培养液稀释200倍，即为工作液I，母液用RPIM-1640培养液稀释100倍，即为工作液II。共计16个工作液。
表2药物1-8的工作液配制
  药物1 药物2 药物3 药物4 药物5 药物6 药物7 药物8
称取 0.0040g 0.0049g 0.0131g 0.0119g 0.0046g 0.0133g 0.0519g 0.1000g
工作液I(μg/mL) 20 25 66 60 23 67 260 500
工作液II(μg/mL) 40 50 132 120 46 134 520 1000
1.3实验方法：
细胞培养
SMMC-7721细胞接种于25cm 2培养瓶，加入约8mLRPIM-1640培养液(10％FBS、100U·mL -1青雾素、100μg·mL -1硫酸链霉素)，于37℃、5％CO 2、饱和湿度的培养箱中静置培养，每48h换液。约3～4d细胞长成致密单层，铺满培养瓶底，进行传代：弃去原培养液，用PBS液冲洗两遍，每瓶加入1mL的胰酶消化，倒置相差显微镜下观察细胞，约1～2min细胞收缩成圆形，弃去消化液，加培养液，吹打培养瓶底，使细胞脱落，制成细胞悬液。
药物干预及检测
1.5×10 4mL -1的细胞悬液，以每孔200μL接种于96孔培养板，调零孔不加细胞，周围PBS填充。24h后换用上述16个工作液，每个工作5复孔，继续培养24h、48h或72h。每孔加入5mg·mL -1的MTT溶液20μL，培养箱内孵育4h后，弃去孵育液，每孔加入150μL DMSO，室温(22℃)振荡混匀15min。待沉淀完全溶解后，酶标仪上570nm测定光密度(A)。按照如下公式计算抑制率：
抑制率(％)＝(A 空白孔-A 测量孔)/(A 空白孔-A 调零孔)*100％。
1.4实验结果
各工作液处理24h、48h和72h时人肝癌细胞SMMC-7721的抑制率，见表3。
表3药物1-8对人肝癌细胞SMMC-7721增殖的影响(x±SD)(n＝5)
Figure PCTCN2018095153-appb-000002
表3的数据示出：
1)低浓度的药物1-6，对人肝癌细胞SMMC-7721的增殖基本没有抑制作用，甚至有促进作用；尤其是药物4(云芝水提)，在整个观察培养时间内均表现出对肝癌细胞生长的促进作用。
2)高浓度的药物1-3(均为叶下珠的不同提取物)和5长时间干预下(72h)，对肝癌细胞仅表现出非常微弱的抑制作用。
3)高浓度的药物4(云芝水提)，在整个观察培养时间内均表现出对肝癌细胞生长的促进作用。
4)低浓度的药物7和8，虽然在干预24h时显示对肝癌细胞增殖的促进作用，但是随着作用时间的延长，48h和72h均不同程度的抑制肝癌细胞增殖。高浓度的药物7和8对肝癌细胞增殖的抑制作用具有时间依赖效应，干预72h时，两者均表现出较强的抑制癌细胞生长的作用。其中药物8的作用强于药物7。
1.5实验结论
单味叶下珠、云芝、紫草和丹参对人肝癌SMMC-7721的增殖基本没有抑制作用，或者仅有很弱的作用，有的反而表现出促进癌细胞生长(如云芝水提物)。令人意想不到的是，四味中药合用后能够抑制肝癌细胞的生长。说明本发明的中药组合物抗肝癌的作用不是各单味药材作用简单的加和，而是药味之间配伍后产生的新作用。
更让人意想不到的是，通过特定工艺制备的药物8，作用强于四味中药分别提取得到的提取物简单混合而成的药物7。推测在药物8制备过程中，经过多步药学提取工艺，复方中各药材配伍合理，且所含有的多种化学成分之间相互作用，进而为其抗肿瘤作用提供了必要的物质基础。
药物8的制备方法与紫叶丹的制剂工艺基本相同，因此下面的实验中都采用紫叶丹进行相应的研究。
实施例2紫叶丹对H22荷瘤小鼠的治疗作用
2.1实验仪器材料：
(1)实验动物
ICR小鼠，雄性，重约18-22g，120只，购买于华阜康生物科技股份有限公司，许可证号：SCXK(京)2014-0004。所有动物购入后，观察动物的一般生理指标、体重和进食情况。适应性喂养3天。标准颗粒饲料饲养，自由饮水。自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。
(2)肿瘤细胞株
H22肝癌腹水瘤细胞，购自中科院上海细胞库。
(3)实验药物
紫叶丹胶囊。
成人剂量:成人60kg，成人口服一日3次，一次5粒，每粒装0.5g，即0.125g/kg，按等效剂量换算小鼠用药(提取物)剂量(g/kg):
0.125g/kg×9.01＝1.126g/kg
注射用5-Fu
2.2实验方法：
小鼠肝癌H22细胞体外培养，在5％CO 2，37℃恒温箱内，用10％灭活胎牛血清的RPMI-1640培养基进行常规培养，每2-3d例行传代，当细胞增殖呈指数生长期时收取细胞并计数进行肿瘤细胞接种。
细胞动物体内传代：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1周，观察动物一切正常后，取3-5只，左下腹腔注射0.2ml密度为1×10 7个/ml H22肿瘤细胞；正常饲养，观察动物状态，几天后产生腹水，取7-10天，动物状态良好的腹水饱满的小鼠取腹水，离心1000转、3分钟，生理盐水洗1次，计数肿瘤细胞稀释混匀。
动物造模：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1周，观察动物一切正常后，右颈肩部皮下注射0.1ml密度为2×10 7个/ml H22肿瘤细胞(上方腹水取得稀释)；每组的动物数和细胞数记录在实验设计表中。
接种后第5天，根据肿瘤体积随机分组，分组情况见下表4。
表4实施例2分组及给药表
Figure PCTCN2018095153-appb-000003
一般状态观察：观察动物精神状态，反应性，毛发色泽，饮食及排泄状况。动物呈现恶化和不好状态和动物不能获得充足的食物和水(失能)用CO 2使其安乐死亡。
抑瘤作用：
(1)体重：每天称量体重，观察各组平均体重变化；
(2)肿瘤体积：每周两次或隔天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5×ab 2，a、b分别表示肿瘤的长径和短径。
(3)肿瘤体重：给药结束后，剥离瘤组织，称重，比较各组平均瘤重大小变化。以平均瘤重及肿瘤生长抑制率来评价药物的体内抗癌效果，计算公式为：
肿瘤生长抑制率＝(1-T/C)×100％，
式中T为给药组平均瘤重，C为对照组平均瘤重。
数据处理：两组间比较，使用独立样本t检验。三个或以上的团体之间的比较，使用单向ANOVA。如果计算F-统计(治疗方差误差方差的比率)，多重比较程序将被应用，方差分析。治疗之间的潜在协同效应，将双向方差分析。将分析所有数据采用SPSS17.0，P<0.05被认为有统计学意义。
2.3实验结果：
(1)体重：
造模前小鼠活动、饮食、毛色均无异常。给药前后各组的体重均无显著性差异(P＞0.05)，结果见表5。
表5用药前后体重的变化
Figure PCTCN2018095153-appb-000004
Figure PCTCN2018095153-appb-000005
与正常组比较， *P＜0.05， **P＜0.01；与模型组比较， ΔP＜0.05， ΔΔP＜0.01
(2)对H22移植瘤的抑制作用：
紫叶丹单独使用及与5-Fu联合使用均具有不同程度的抑瘤作用，且与化疗药物具有协同增效作用，结果见表6。
表6紫叶丹对H22肝癌肿瘤的抑制作用
Figure PCTCN2018095153-appb-000006
Figure PCTCN2018095153-appb-000007
与模型组比较， *P＜0.05， **P＜0.01
2.4结论：
(1)紫叶丹对H22肝癌荷瘤小鼠体重无影响；
(2)与模型组比较，紫叶丹单独使用，高中低三个剂量组对H22肝癌荷瘤小鼠均具有显著的抑瘤作用(P＜0.05)；
(3)与模型组比较，紫叶丹与5-Fu联合使用对H22肝癌荷瘤小鼠均具有显著及非常显著的抑瘤作用(P＜0.05，P＜0.01)，且呈剂量依赖关系。提示紫叶丹与化疗药物具有协同增效作用。
实施例3紫叶丹对HepG2荷瘤小鼠的治疗作用
3.1实验仪器材料：
(1)实验动物
BALB/C-nu裸鼠，雄性，5周，重约18-20g，115只，购买于华阜康生物科技股份有限公司，许可证号：SCXK(京)2014-0004，实验动物质量合格证号：11401300035982。于北京市口腔医院动物室适应性喂养一周，采用标准颗粒饲料饲养，自由饮水，自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。
(2)肿瘤细胞株
HepG2人肝癌细胞，北京五加和分子医学研究所有限公司惠赠
(3)实验药物
紫叶丹胶囊。
成人剂量：成人60kg,成人口服一日3次，一次5粒，每粒装0.5g，即0.125g/kg，按等效剂量换算小鼠用药剂量(g/kg)：
0.125g/kg×9.01＝1.126g/kg
3.2实验方法：
人肝癌HepG2细胞(来源于)体外培养，在5％CO 2，37℃恒温箱内，用10％灭活胎牛血清的1640培养基进行常规培养，每2-3d例行传代，当细胞增殖呈指数生长期时收取细胞并计数进行肿瘤细胞接种。
细胞悬液制备：取对数生长期肝癌HepG2细胞，胰酶消化2-3min，用含10％FBS的1640培养液终止消化，1000转/分，离心4min，PBS洗3次，调整细胞密度为2×10 7个/ml。
动物造模：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1周，观察动物一切正常后，颈肩部皮下注射0.1ml密度为2×10 7个/ml肝癌HepG2细胞。
将115只Balb/C-nu裸鼠，皮下接种HepG2细胞，接种10-15天后，剔除直径在5mm以下与10mm以上的荷瘤裸鼠，测量实体瘤体积，将72只荷瘤裸鼠根据实体瘤体积体大小随机区组，共9组，每组8只。分组情况见表7。
表7实施例3分组及给药表
Figure PCTCN2018095153-appb-000008
3.3观察项目及评价指标
3.3.1一般状态观察
观察动物精神状态，反应性，饮食及排泄状况，有无皮疹，肿瘤是否破溃。动物呈现恶化和不好状态和动物不能获得充足的食物和水(失能)用CO 2使其安乐死亡。
3.3.2抑瘤作用
(1)体重：每天称量体重，观察各组平均体重变化；
(2)肿瘤体积：每周两次或隔天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5×ab 2，a、b分别表示肿瘤的长径和短径。
(3)肿瘤体重：给药结束后，剥离瘤组织，称重，比较各组平均瘤重大小变化。以平均瘤重及肿瘤生长抑制率来评价药物的体内抗癌效果，计算公式为：
肿瘤生长抑制率＝(1-T/C)×100％，
式中T为给药组平均瘤重，C为对照组平均瘤重。
3.4数据处理
两组间比较，使用独立样本T检验，三个或以上的团体之间的比较，使用单向ANOVA检验；若方差不齐，使用非参数秩和检验。所有数据采用SPSS19.0进行分析，P<0.05被认为有统计学意义。
3.5实验结果：
(1)体重：
造模前BALB/C-nu裸鼠的活动、饮食均无异常。给药前后，各组的体重均无显著性差异(P＞0.05)。具体结果见表8。
表8用药前后体重的变化
Figure PCTCN2018095153-appb-000009
Figure PCTCN2018095153-appb-000010
与模型组比较， *P＜0.05， **P＜0.01。
(2)对HepG2移植瘤的抑制作用：
紫叶丹单独使用及与化疗药联用，对人源肝癌小鼠具有不同程度的抑瘤作用，且与化疗药联用发挥增效作用。具体结果见表9。
表9紫叶丹对HepG2肝癌肿瘤的抑制作用
Figure PCTCN2018095153-appb-000011
Figure PCTCN2018095153-appb-000012
Figure PCTCN2018095153-appb-000013
与模型组组比较， *P＜0.05， **P＜0.01。
3.6结论：
(1)紫叶丹对HepG2肝癌荷瘤小鼠体重无影响；
(2)与模型组比较，叶下珠单独使用，对HepG2肝癌荷瘤小鼠没有抑瘤作用(P＞0.05)；
(3)与模型组比较，紫叶丹单独使用，高中低三个剂量组对HepG2肝癌荷瘤小鼠均具有显著的抑瘤作用(P＜0.05)；且作用好于叶下珠；
(4)与模型组比较，紫叶丹三个剂量组与5-Fu联合使用对HepG2肝癌荷瘤小鼠均具有显著及非常显著的抑瘤作用(P＜0.05，P＜0.01)，且呈剂量依赖关系。提示紫叶丹与化疗药物具有协同增效作用。
实施例4紫叶丹对H22荷瘤小鼠肿瘤复发的抑制作用
4.1实验仪器材料：
(1)实验动物
ICR小鼠，雄性，重约18-22g，120只，购买于华阜康生物科技股份有限公司，许可证号：SCXK(京)2014-0004。所有动物购入后，观察动物的一般生理指标、体重和进食情况。适应性喂养3天。标准颗粒饲料饲养，自由饮水。自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。
(2)肿瘤细胞株
H22肝癌腹水瘤细胞，购自中科院上海细胞库。
(3)实验药物
紫叶丹胶囊
成人剂量:成人60kg，成人口服一日3次，一次5粒，每粒装0.5g，即0.125g/kg，按等效剂量换算小鼠用药(提取物)剂量(g/kg)：
0.125g/kg×9.01＝1.126g/kg
叶下珠提取物
注射用5-Fu
4.2实验方法：
小鼠肝癌H22细胞体外培养，在5％CO 2，37℃恒温箱内，用10％灭活胎牛血清的RPMI-1640培养基进行常规培养，每2-3d例行传代，当细胞增殖呈指数生长期时收取细胞并计数进行肿瘤细胞接种。
细胞动物体内传代：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1 周，观察动物一切正常后，取3-5只，左下腹腔注射0.2ml密度为1×10 7H22肿瘤细胞；正常饲养，观察动物状态，几天后产生腹水，取7-10天，动物状态良好的腹水饱满的小鼠取腹水，离心1000转、3分钟，生理盐水洗1次，计数肿瘤细胞稀释混匀。
动物造模：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1周，观察动物一切正常后，右颈肩部皮下注射0.1ml密度为2×10 7H22个/ml肿瘤细胞(上方腹水取得稀释)；每组的动物数和细胞数记录在实验设计表中。
接种后第二天，称重，根据体重大小随机分组(随机区组设计)，每组10只，分组情况见表10。
表10实施例4分组及给药表
Figure PCTCN2018095153-appb-000014
4.3观察项目及评价指标
4.3.1一般状态观察
观察动物精神状态，反应性，毛发色泽，饮食及排泄状况。动物呈现恶化和不好状态和动物不能获得充足的食物和水(失能)用CO 2使其安乐死亡。
4.3.2抑瘤作用
(1)体重：每天称量体重，观察各组平均体重变化；
(2)肿瘤体积：每周两次或隔天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5×ab 2，a、b分别表示肿瘤的长径和短径。
(3)肿瘤体重：给药结束后，剥离瘤组织，称重，比较各组平均瘤重大小变化。以平均瘤重及肿瘤生长抑制率来评价药物的体内抗癌效果，计算公式为：
肿瘤生长抑制率＝(1-T/C)×100％，
式中T为给药组平均瘤重，C为对照组平均瘤重。
4.4数据处理
两组间比较，使用独立样本t检验。三个或以上的团体之间的比较，使用单向ANOVA。如果计算F-统计(治疗方差误差方差的比率)，多重比较程序将被应用，方差分析。治疗之间的潜在协同效应，将双向方差分析。将分析所有数据采用SPSS17.0，P<0.05被认为有统计学意义。
4.5实验结果：
(1)体重：
造模前小鼠活动、饮食、毛色均无异常。给药前后各组的体重均无显著性差异(P ＞0.05)，结果见表11。
表11用药前后体重的变化
Figure PCTCN2018095153-appb-000015
Figure PCTCN2018095153-appb-000016
与模型组组比较， *P＜0.05， **P＜0.01
(2)对H22移植瘤的抑制作用：
紫叶丹具有明显的抑瘤作用，且呈一定的量效关系，优于叶下珠组，并与5-Fu组的抗肿瘤效果接近，其结果见表12。
表12紫叶丹对H22肝癌肿瘤的抑制作用
Figure PCTCN2018095153-appb-000017
Figure PCTCN2018095153-appb-000018
与模型组组比较， *P＜0.05， **P＜0.01
4.6结论：
(1)紫叶丹对H22肝癌小鼠肿瘤复发模型体重无影响；
(2)与模型组比较，紫叶丹高中低三个剂量组对H22肝癌小鼠肿瘤复发模型均具有显著或非常显著的抑瘤作用(P＜0.05，P＜0.01)，且呈量效依赖关系；
(3)紫叶丹高剂量组对H22肝癌小鼠肿瘤复发模型的抑瘤作用与5-Fu相当。
(4)与模型组比较，叶下珠单用对H22肝癌小鼠肿瘤复发模型未表现出显著的抑瘤作用(P＞0.05)。该结果提示紫叶丹对肝癌复发的治疗作用好于叶下珠。
实施例5紫叶丹对HepG2荷瘤小鼠肿瘤复发的抑制作用
5.1实验仪器材料：
(1)实验动物
BALB/C-nu裸鼠，雄性，5周，40只，购买于华阜康生物科技股份有限公司，许可证号：SCXK(京)2014-0004，实验动物质量合格证号：11401300032435。于北京市口 腔医院动物室适应性喂养25天，采用标准颗粒饲料饲养，自由饮水，自然昼夜光线照明，室温18～26℃，相对湿度40％～70％。
(2)肿瘤细胞株
HepG2人肝癌细胞，广州中医药大学热带医学研究所惠赠
(3)实验药物
紫叶丹胶囊。
成人剂量：成人60kg,成人口服一日3次，一次5粒，每粒装0.5g，即0.125g/kg，按等效剂量换算小鼠用药剂量(g/kg)：
0.125g/kg×9.01＝1.126g/kg
注射用氟尿嘧啶(5-Fu)
5.2实验方法：
人肝癌HepG2细胞(来源于)体外培养，在5％CO 2，37℃恒温箱内，用10％灭活胎牛血清的1640培养基进行常规培养，每2-3d例行传代，当细胞增殖呈指数生长期时收取细胞并计数进行肿瘤细胞接种。
细胞悬液制备：取对数生长期肝癌HepG2细胞，胰酶消化2-3min，用含10％FBS的1640培养液终止消化，1000转/分，离心4min，PBS洗3次，调整细胞密度为2×10 7个/ml。
动物造模：观察动物的一般生理指标、体重和进食情况，动物适应性喂养1周，观察动物一切正常后，颈肩部皮下注射0.1ml密度为2×10 7个/ml肝癌HepG2细胞。
将HepG2细胞接种于40只BALB/C-nu裸鼠颈肩背部皮下，与接种第二天按体重大小随机区组分组，分为紫叶丹高剂量组、紫叶丹中剂量组、紫叶丹低剂量组、5-Fu组、模型组，共5组，每组8只，连续给药7周，于末次给药后第二天，麻醉处死并取材。分组情况见下表13。
表13实施例5分组及给药表
Figure PCTCN2018095153-appb-000019
5.3观察项目及评价指标
5.3.1一般状态观察
观察动物精神状态，反应性，饮食及排泄状况，有无皮疹，肿瘤是否破溃。动物呈现恶化和不好状态和动物不能获得充足的食物和水(失能)用CO 2使其安乐死亡。
5.3.2抑瘤作用
(1)体重：每天称量体重，观察各组平均体重变化；
(2)肿瘤体积：每周两次或隔天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为： V＝0.5×ab 2，a、b分别表示肿瘤的长径和短径。
(3)肿瘤体重：给药结束后，剥离瘤组织，称重，比较各组平均瘤重大小变化。以平均瘤重及肿瘤生长抑制率来评价药物的体内抗癌效果，计算公式为：
肿瘤生长抑制率＝(1-T/C)×100％，
式中T为给药组平均瘤重，C为对照组平均瘤重。
5.4数据处理
两组间比较，使用独立样本T检验，三个或以上的团体之间的比较，使用单向ANOVA检验；若方差不齐，使用非参数秩和检验。所有数据采用SPSS19.0进行分析，P<0.05被认为有统计学意义。
5.5实验结果：
(1)体重：
造模前BALB/C-nu裸鼠的活动、饮食均无异常。给药前后各组的体重均无显著性差异(P＞0.05)。具体结果见表14。
表14用药前后体重的变化
Figure PCTCN2018095153-appb-000020
Figure PCTCN2018095153-appb-000021
与模型组组比较， *P＜0.05， **P＜0.01。
(2)对HepG2荷瘤小鼠肿瘤复发的抑制作用：
紫叶丹可有效抑制人源肝癌细胞在小鼠体内的生长，提示其对肝癌的复发转移有抑制作用。其结果见表15。
表15紫叶丹对HepG2荷瘤小鼠肿瘤复发的抑制作用
Figure PCTCN2018095153-appb-000022
Figure PCTCN2018095153-appb-000023
与模型组组比较， *P＜0.05， **P＜0.01。
5.6结论：
(1)紫叶丹对HepG2肝癌小鼠肿瘤复发模型体重无影响；
(2)与模型组比较，紫叶丹高中低三个剂量组对HepG2肝癌小鼠复发模型均具有显著或非常显著的抑瘤作用(P＜0.05，P＜0.01)；且呈一定量效关系；
(3)紫叶丹低组对HepG2肝癌小鼠复发模型的抑制作用与5-Fu相当，紫叶丹高中剂量的作用强于5-Fu。提示紫叶丹对肝癌复发的治疗作用好于5-Fu。
实施例6紫叶丹动物急性毒性(最大耐受量)试验
6.1实验材料：
(1)实验动物
昆明小鼠，雌雄各半，20±2g，40只，由西安交通大学实验动物中心提供，合格证：陕医动09-003号。
(2)实验药物
受试药物：紫叶丹胶囊
空白对照：蒸馏水
6.2实验方法：
(1)给药途径选择说明
本品为中药胶囊剂，临床用药途径为口服，本试验采取灌胃给药途径，与临床用药途径一致。
(2)实验分组及给药剂量
将动物按体重随机分成2组，每组20只，雌雄各10只，其中一组为给药组，另一组为对照组。给药组小鼠按体重灌胃给予紫叶丹胶囊药粉水悬液，0.3ml/10g，24小时内给药3次，间隔4小时，一日内总剂量相当于临床剂量的225倍。对照组给予同体积的蒸馏水，给药时间同给药组。
(3)观察指标和时间
给药后连续观察动物的反应情况(如一般表现、呼吸、活动及有无惊厥等)，中毒症状发生的时间、持续时间、恢复情况，逐日记录动物死亡数，隔日一次记录动物体重。死亡动物进行大体解剖，观察各主要脏器情况。观察时间为给药后7天期间。7天观察期结束后，对存活的动物进行大体解剖，观察各主要脏器情况。
6.3实验结果：
在给药后7天观察期内，紫叶丹给药组小鼠一般状况良好，毛发光泽，神态活动正常，未见异常分泌物及异常呼吸，体重由试验前的20.5±1.4g，增至24.5±2.7g，未见动物死亡。7天后将全部小鼠颈椎脱颈处死，行肉眼尸检，结果所有小鼠心、肝、脾、肺、肾、胃、肠、胸腺、肾上腺、腹腔、胸腔等主要脏器及体腔等均未见病理改变。最大限度给药期间，对小鼠出现的某症状进行了观察。结果表明，紫叶丹225倍临床剂量一日内分三次给小鼠灌胃给药，动物未见明显的毒性反应，也未引起动物死亡。
6.4结论：
紫叶丹胶囊药粉小鼠灌胃给药的最大耐受量相当于临床人用量的225倍。
实施例7紫叶丹大鼠长期毒性试验
7.1实验材料：
(1)实验动物
SD大鼠，雌雄各半，10-112g，由西安医科大学实验动物中心提供，合格证：陕医动证字08-005号。
(2)实验药物
受试药物：紫叶丹胶囊、叶下珠
空白对照：蒸馏水
(3)动物饲养条件：大鼠适应性饲养2周，饲养于开放房间中，空调控温，室内最高温度27℃，最低温度24℃，相对温度58％-70％，常规通风，自然光源。固体饲料饲养，自由摄食和饮用常水。动物按性别分笼饲养，每笼5只，每天清洁鼠笼1次。
7.2实验方法：
(1)给药途径选择说明
本品为中药胶囊剂，临床用药途径为口服，本试验采取灌胃给药途径，与临床用药途径一致。
(2)实验分组及给药剂量
将动物按体重随机分成5组，每组40只，雌雄各20只。分别为空白对照组(40ml/kg)、紫叶丹小剂量组(相当于成人日用量的25倍)、紫叶丹中剂量组(相当于成人日用量的50倍)、紫叶丹大剂量组(相当于成人日用量的100倍)、叶下珠组(451g/kg)。给药周期为6个月,其中各组部分动物(雌、雄各约1/3)于给药3个月后处死，其余动物继续给药至6个月或给药6个月后停药14天时处死。各组动物均每日上、下午各给药1次，二次间隔5-6小时，给药体积为40ml/kg体重，上午给药时间为8:00-10:00，下午给药时间为2:30-4:00，每周给药6天，周日停药一次。
(3)观察指标和时间
A、一般表现
逐日观察动物的神态、毛发、行为、活动、饮食、粪便、分泌物等情况。
B、血液学指标
分别于给药3个月和6个月，停药2周时，各组动物取血，用全自动血球计数仪测定各组动物的白细胞(WBC)、血小板(Pt)、中性细胞(NE)和淋巴细胞(LY)。
C、血液生化学指标
分别于给药3个月和6个月，停药2周时，各组动物取血，分离血清，用全自动生化分析仪测定血清丙氨酸氨基转换酶(ALT)、天门冬氨酸氨基转换酶(AST)、白蛋白(ALB)、肌肝(Cr)。
D、组织病理学检查
对各期处死的所有动物的脏器进行病理学检查，包括心、肝、脾、肺、肾、肾上腺、胸腺、睾丸、附睾、前列腺、卵巢、子宫、肠系膜淋巴结、食道、胃、空肠、结肠、膀胱、胸骨(骨髓)、脑、垂体、大脑等20个组织，用10％的甲醛固定，石蜡包埋切片，HE染色，作病理检查。
7.3统计分析
各组数据均分雌性和雄性分别计算组平均值±标准差，采用t检验比较各组给药组与对照组之间的差异，并且将测定的血细胞计数和血清生化检查数据与该种系大鼠的正常 值范围比较，确定各项检查是否出现异常。
7.4实验结果：
(1)对大鼠一般状况的影响
给药期期间，叶下珠组神态萎靡，毛发无光泽，行为活动倦怠，停药2周后上述症状消失。其余各组大鼠未见异常。
(2)血液学检查
于给药3个月和6个月，停药2周时，各组动物取血，进行血液常规检查。结果表明，叶下珠组给药6个月时，雌性和雄性大鼠的WBC、NE％明显升高，LY％、Pt明显降低(P﹤0.05)，停药2周时恢复正常。紫叶丹组给药3个月、6个月以及停药2周，对血液学指标均无明显影响(P﹥0.05)。结果见表16。
表16紫叶丹对大鼠血象的影响
Figure PCTCN2018095153-appb-000024
Figure PCTCN2018095153-appb-000025
注：与对照组比较， *P﹤0.05
(3)血液生化检查
于给药3个月和6个月，停药2周时，各组动物取血，分离血清，测定各项生化指标。结果表明，叶下珠组给药6个月时，雌性和雄性大鼠的ALT、AST、Cr明显升高，雌性ALB明显降低(P﹤0.05)，停药2周时恢复正常。紫叶丹各剂量组在各时期的各项生化指标均无显著性差异(P﹥0.05)。结果见表17。
表17紫叶丹对大鼠血液生化的影响
Figure PCTCN2018095153-appb-000026
Figure PCTCN2018095153-appb-000027
注：与对照组比较， *P﹤0.05
(4)组织病理学检查
于试验3个月、6个月和停药2周后进行的病理检查结果显示，对照组和紫叶丹各剂量组，除个别动物中肺、肾等组织有散在性非特异炎症外，其余各给药剂量组的各种组织包括心、肝、脾、肺、肾、肾上腺、胸腺、睾丸、附睾、前列腺、卵巢、子宫、肠系膜淋巴结、食道、胃、空肠、结肠、膀胱、胸骨(骨髓)、脑、垂体、大脑等的细胞结构和形态均未见异常病理变化。给药6个月时，叶下珠组病理检查结果显示，肝脏中央静脉周围肝细胞弥漫性坏死，部分伴有单核细胞浸润及空泡化；肾脏组织可见皮质部肾小管上皮细胞空泡变性，伴不同程度的坏死，脱落。给药3个月和给药6个月时，叶下珠组病理检查结果未见异常。
7.5结论：
紫叶丹以人日用剂量的100、50、25倍给大鼠灌胃6个月，未见与药有关的毒性反应，亦未见蓄积性毒性反应。而叶下珠在肝脏和肾脏出现了可逆性的毒性反应。
上述实验提示叶下珠、云芝、丹参和紫草合用，能够降低叶下珠的毒性。肝癌的治疗是一个长期的过程，患者在生存期内需要长时间服药；肝癌术后复发患者其生理机能由于疾病的影响已比较脆弱，治疗时应选择对肝肾功能影响小、更安全的药物。本发明的叶下珠、云芝、丹参和紫草构成活性原料的中药组合物，相较于叶下珠，安全性更好、疗效更确切，因此更适于临床上治疗肝癌患者及抑制肝癌术后患者复发。

Claims (21)

  1. 一种用于治疗肝癌或抑制肝癌术后复发的中药组合物，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
  2. 一种用于治疗肝癌或抑制肝癌术后复发的药物，所述药物包含一种中药组合物与5-Fu的组合，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
  3. 根据权利要求1或2所述的中药组合物或药物，其特征在于，所述中药组合物由如下重量份的药材组成：叶下珠10份，云芝4份，丹参1份，紫草1份。
  4. 根据权利要求1至3中任一项所述的中药组合物或药物，其特征在于，所述中药组合物通过如下方法制备：
    叶下珠用水提取，所得提取液上大孔吸附树脂吸附，用30-75％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用乙醇提取，得醇提取液；丹参与紫草共同用乙醇提取，得醇提液，与叶下珠的醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，水提取，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀。
  5. 根据权利要求1至4中任一项所述的中药组合物或药物，其特征在于，所述中药组合物中还包括药学上可以接受的辅料。
  6. 根据权利要求1至5中任一项所述的中药组合物或药物，其特征在于，所述中药组合物为临床上可以接受的制剂，包括经胃肠道给药制剂和非经胃肠道给药制剂；所述经胃肠道给药制剂选自散剂、片剂、颗粒剂、胶囊剂、滴丸、乳剂或混悬剂；所述非经胃肠道给药制剂选自注射剂、喷雾剂、栓剂、灌注剂、贴剂或软膏剂。
  7. 根据权利要求1至6中任一项所述的中药组合物或药物，其特征在于，所述肝癌为原发性肝癌或继发性肝癌；所述原发性肝癌为肝细胞癌、肝胚细胞癌、胆管性细胞癌、肝内胆囊腺癌、肝-胆管混合细胞癌、肝肉瘤、肝血管内皮细胞肉瘤、肝上皮样血管内皮细胞瘤或肝纤维板层癌。
  8. 一种中药组合物在制备治疗肝癌或抑制肝癌术后复发的药物中的应用，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
  9. 一种中药组合物与5-Fu的组合在制备治疗肝癌或抑制肝癌术后复发的药物中的应用，所述中药组合物由如下重量份的药材组成：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
  10. 根据权利要求8或9所述的应用，其特征在于，所述中药组合物由如下重量份的药材组成：叶下珠10份，云芝4份，丹参1份，紫草1份。
  11. 根据权利要求8至10中任一项所述的应用，其特征在于，所述中药组合物通过如下方法制备：
    叶下珠用水提取，所得提取液上大孔吸附树脂吸附，用30-75％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用乙醇提取，得醇提取液；丹参 与紫草共同用乙醇提取，得醇提液，与叶下珠的醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，水提取，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀。
  12. 根据权利要求8至11中任一项所述的应用，其特征在于，所述中药组合物中还包括药学上可以接受的辅料。
  13. 根据权利要求8至12中任一项所述的应用，其特征在于，所述中药组合物为临床上可以接受的制剂，包括经胃肠道给药制剂和非经胃肠道给药制剂；所述经胃肠道给药制剂选自散剂、片剂、颗粒剂、胶囊剂、滴丸、乳剂或混悬剂；所述非经胃肠道给药制剂选自注射剂、喷雾剂、栓剂、灌注剂、贴剂或软膏剂。
  14. 根据权利要求8至13中任一项所述的应用，其特征在于，所述肝癌为原发性肝癌或继发性肝癌；所述原发性肝癌为肝细胞癌、肝胚细胞癌、胆管性细胞癌、肝内胆囊腺癌、肝-胆管混合细胞癌、肝肉瘤、肝血管内皮细胞肉瘤、肝上皮样血管内皮细胞瘤或肝纤维板层癌。
  15. 一种治疗肝癌或抑制肝癌术后复发的方法，所述方法包括向有需要的患者给予有效量的由如下重量份的药材组成的中药组合物：叶下珠5-20份，云芝2-8份，丹参1-5份，紫草1-5份。
  16. 根据权利要求15的方法，其特征在于，联合给予5-Fu。
  17. 根据权利要求15或16所述的方法，其特征在于，所述中药组合物由如下重量份的药材组成：叶下珠10份，云芝4份，丹参1份，紫草1份。
  18. 根据权利要求15-17中任一项所述的方法，其特征在于，所述中药组合物通过如下方法制备：
    叶下珠用水提取，所得提取液上大孔吸附树脂吸附，用30-75％的乙醇洗脱完全，合并洗脱液，浓缩得浸膏I；叶下珠水煎煮后的药渣用乙醇提取，得醇提取液；丹参与紫草共同用乙醇提取，得醇提液，与叶下珠的醇提液合并，回收溶剂，得浸膏II；云芝与丹参、紫草的药渣合并，水提取，得水提取液，回收溶剂，得浸膏III；将浸膏I、II、III混匀。
  19. 根据权利要求15至18中任一项所述的方法，其特征在于，所述中药组合物中还包括药学上可以接受的辅料。
  20. 根据权利要求15至19中任一项所述的方法，其特征在于，所述中药组合物为临床上可以接受的制剂，包括经胃肠道给药制剂和非经胃肠道给药制剂；所述经胃肠道给药制剂选自散剂、片剂、颗粒剂、胶囊剂、滴丸、乳剂或混悬剂；所述非经胃肠道给药制剂选自注射剂、喷雾剂、栓剂、灌注剂、贴剂或软膏剂。
  21. 根据权利要求15至20中任一项所述的方法，其特征在于，所述肝癌为原发性肝癌或继发性肝癌；所述原发性肝癌为肝细胞癌、肝胚细胞癌、胆管性细胞癌、肝内胆囊腺癌、肝-胆管混合细胞癌、肝肉瘤、肝血管内皮细胞肉瘤、肝上皮样血管内皮细胞瘤或肝纤维板层癌。
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