WO2020147275A1 - 一种抗肿瘤中药组合物及其挥发油提取物 - Google Patents

一种抗肿瘤中药组合物及其挥发油提取物 Download PDF

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WO2020147275A1
WO2020147275A1 PCT/CN2019/095410 CN2019095410W WO2020147275A1 WO 2020147275 A1 WO2020147275 A1 WO 2020147275A1 CN 2019095410 W CN2019095410 W CN 2019095410W WO 2020147275 A1 WO2020147275 A1 WO 2020147275A1
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chinese medicine
traditional chinese
volatile oil
oil extract
parts
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PCT/CN2019/095410
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French (fr)
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金家骅
丁礼琴
曹无介
朱晓萍
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上海中华药业有限公司
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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)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
    • 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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/285—Aucklandia
    • 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)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • 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)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • 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)
    • A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9064—Amomum, e.g. round cardamom
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction

Definitions

  • the invention relates to an application of a traditional Chinese medicine composition and a volatile oil extract thereof in an anti-tumor drug that inhibits overexpression of ras proto-oncogene mutations, and belongs to the field of anti-tumor drugs.
  • KRAS codon 12 mutations occur in 83% of pancreatic cancers and 85% of cholangiocarcinomas.
  • cancers such as lung cancer, stomach cancer, breast cancer, bladder cancer and melanoma are closely related to mutations in the ras gene (Yang Qiwei et al., 2018).
  • Abnormal Ras-MAPK signal transduction pathway is involved in the process of liver cancer.
  • Anti-tumor therapy targeting the Ras signaling pathway has also become a research hotspot in tumor therapy. Therefore, ras has become a recognized target for screening related malignant cancer drugs (Liu Xuemei et al., 2008).
  • the Ras signaling pathway is highly conserved from C. elegans to humans. In C.
  • the let-60 gene encodes a conserved RAS protein, which has 83% homology compared with the human RAS protein. If the ras gene is mutated, it will cause the nematode to produce multiple vaginas in Caenorhabditis elegans, and in humans, it will cause the malignant proliferation of cells and cause tumors (Li Rui et al., 2016).
  • the model organism Caenorhabditis elegans (Caenorhabditis elegans) Ras/MAPK signaling pathway over-activated mutants are widely used as tumor models to screen for anti-tumor drugs that inhibit the over-activation of the Ras pathway (Hara and Han, 1995; Wang Fang, 2005) ; Liu Xuemei et al., 2008).
  • the present invention uses Caenorhabditis elegans as a screening tool to screen down-regulated Ras/MAPK signal pathway over-activation of anti-tumor drug candidates.
  • Longhu Ren Dan was originally derived from the ancient Chinese recipe "Zhuge Xingjun San”. After several improvements, the existing prescription was obtained.
  • Longhurendan is mainly composed of menthol, borneol, cloves, amomum, star anise, cinnamon, pepper, woody, dried ginger, catechu, and licorice. It can invigorate the body, dispel heat and turbidity, and stop vomiting. It is mainly used for heat stroke, dizziness, nausea and vomiting, diarrhea, motion sickness, and seasickness. So far, no literature has reported that Longhu Ren Dan has anti-tumor activity.
  • the present invention is improved on the basis of Longhuren Dan, and discovered a traditional Chinese medicine composition and its volatile oil extract which can inhibit the excessive activation of ras gene.
  • the traditional Chinese medicine composition and its volatile oil extract can be used in the treatment of ras protocarcinoma. Application in tumors with excessive gene activation. Compared with Longhu Rendan, the traditional Chinese medicine composition and its volatile oil extract can be applied in treating tumors with excessive activation of ras proto-oncogene.
  • the traditional Chinese medicine composition and its volatile oil extract provided by the present invention can be mixed with any pharmaceutically acceptable auxiliary material to prepare various dosage forms, and are used in the treatment of tumors with excessive activation of ras proto-oncogene.
  • the purpose of the present invention is to provide a traditional Chinese medicine composition consisting of cloves, Amomum villosum, star anise, cinnamon, white pepper, wood incense, and dried ginger, which is extracted by steam distillation to obtain the crude volatile oil extract of the traditional Chinese medicine composition. Then it is extracted with an organic solvent, and the obtained organic phase is extracted and evaporated to dry the solvent, dehydrated and dried to obtain the volatile oil extract of the traditional Chinese medicine.
  • the present invention provides the application of the traditional Chinese medicine composition and its volatile oil extract in inhibiting the overexpression of ras proto-oncogene.
  • the specific tumors are the treatment of liver cancer, pancreatic cancer, lung cancer and colon cancer.
  • the present invention is implemented through the following technical solutions.
  • the purpose of the present invention is to provide a traditional Chinese medicine composition made of the following raw materials in parts by weight: 5 parts of cloves, 5 parts of Amomum villosum, 3 parts of star anise, 8 parts of cinnamon, 3 parts of white pepper, wood 3 parts fragrant, 5 parts dried ginger.
  • Another object of the present invention is to provide a traditional Chinese medicine volatile oil extract, which consists of 5 parts of clove, 5 parts of Amomum villosum, 3 parts of star anise, 8 parts of cinnamon, 3 parts of white pepper, 3 parts of woody 5 parts of ginger, the preparation method is as follows: take the above proportions of cloves, amomum, star anise, cinnamon, white pepper, woody, dry ginger, add distilled water, and extract by steam distillation to obtain crude volatile oil extract. The crude volatile oil extract is extracted with an organic solvent, and the obtained organic phase is extracted to evaporate the solvent, dehydrated and dried to obtain the volatile oil extract of traditional Chinese medicine.
  • the symbolic chemical components of the volatile oil extract of traditional Chinese medicine contain eugenol 42.43-59.19%, eugenol acetate 13.05-15.28%, anethole 10.02-13.11%, caryophyllene 2.07-2.88%, and bornyl acetate 1.3-1.71% .
  • the density of the volatile oil extract of the traditional Chinese medicine is 1.0-1.1 g/mL.
  • Another object of the present invention is to provide the application of the traditional Chinese medicine composition and its volatile oil extract in the preparation of anti-tumor drugs.
  • the tumor is caused by overexpression of the ras proto-oncogene mutation.
  • the tumors specifically involved are liver cancer, pancreatic cancer, lung cancer and colon cancer.
  • Ras signaling pathway is highly conserved from C. elegans to humans, in C. elegans, let-60 gene encodes a conserved RAS protein, which has 83% homology compared with human RAS protein. If ras is mutated, it will cause the nematode's multiple vaginal phenotype in Caenorhabditis elegans, and in humans it will cause the malignant proliferation of cells, leading to the occurrence of tumors. Therefore, the model organism Caenorhabditis elegans (Caenorhabditis elegans) Ras/MAPK signaling pathway over-activated mutants are widely used as tumor models to screen anti-tumor drugs that inhibit the over-activation of the Ras pathway.
  • the present invention uses the Caenorhabditis elegans mutant as a model for evaluating drugs that inhibit the excessive activation of the ras proto-oncogene pathway, and concludes that the traditional Chinese medicine composition and the volatile oil extract of traditional Chinese medicine can significantly down-regulate the overexpression of ras proto-oncogene It has the conclusion that it can inhibit the excessive activation of the ras proto-oncogene pathway, and the traditional Chinese medicine composition and the volatile oil extract of traditional Chinese medicine only act on tumor cells caused by the excessive activation of ras caused by let-60 mutation, while the lin-15 deletion mutation causes The wild-type ras does not work on tumor cells caused by excessive activation.
  • the traditional Chinese medicine composition and the volatile oil extract of traditional Chinese medicine can be used in the preparation of medicines for treating tumors overexpressing ras proto-oncogene.
  • the traditional Chinese medicine composition and the traditional Chinese medicine volatile oil extract of the present invention can specifically down-regulate the overexpression caused by the mutation of ras proto-oncogene, and can be used in the preparation of drugs for treating malignant tumors caused by the overexpression of the ras proto-oncogene mutation, and specifically relates to
  • the tumors are liver cancer, pancreatic cancer, lung cancer and colon cancer.
  • the therapeutic effect of the traditional Chinese medicine composition and traditional Chinese medicine volatile oil extract on ras gene overexpression tumors does not depend on extensive cytotoxicity, but specifically down-regulates the overexpression of ras proto-oncogene; the traditional Chinese medicine composition and traditional Chinese medicine volatile oil extract are It only works on tumor cells caused by overactivation of ras caused by let-60 mutation, but does not work on tumor cells caused by overactivation of wild-type ras caused by lin-15 deletion mutation, that is, the traditional Chinese medicine composition and traditional Chinese medicine volatile oil extract It only acts on cancer cells, not normal cells, does not cause teratogenicity, and is safe to use.
  • Raw materials cloves, amomum, star anise, cinnamon, white pepper, woody, dried ginger
  • the above-mentioned raw material medicine is crushed, 10 times the amount of water is added, and steam distillation is used to extract for 3 hours to obtain a crude Chinese medicine volatile oil extract.
  • the crude Chinese medicine volatile oil extract is extracted twice with ether, the organic phases obtained from the extraction are combined, and then the ether is evaporated to obtain a Chinese medicine volatile oil extract, which is a light yellow oily substance.
  • Volatile oil composition analysis GC-MS analysis of the chemical composition of the volatile oil extract, 42.43-59.19% eugenol, 11.85-15.28% eugenol acetate, 10.02-13.11% anethole, 2.07-2.88% caryophyllene, 1.3-1.71% bornyl acetate.
  • Example 2 The effect of volatile oil extract of Chinese medicine on Caenorhabditis elegans MT2124 (let-60 mutation)
  • MT2124 is a Caenorhabditis elegans that is over-activated by let-60/ras. This strain of nematodes has a multiple vaginal phenotype, which is equivalent to a refractory tumor suffering from human ras proto-oncogene mutations.
  • the test drug is used to treat C. elegans MT2124. If the test drug can inhibit the production of the multi-vulva phenotype of the tested nematode and promote them to develop into a wild type, it means that the drug acts on the Ras pathway and down-regulates the excessive activation of this pathway. Has anti-tumor activity. The higher the proportion of wild-type nematodes in the tested nematode population, the better the effect of the tested drug.
  • Caenorhabditis elegans MT2124 genotype: let-60(n1046sd, gf) IV, purchased from CGC; Escherichia coli OP50, uracil leaking mutant strain, used as food for Caenorhabditis elegans, purchased from CGC.
  • 1M citric acid buffer 2g citric acid, 29.35g potassium citrate, 100mL distilled water, shake to dissolve, sterilize at 121°C for 20min;
  • 1M magnesium sulfate solution 24.647g MgSO 4 ⁇ 7H 2 O, 100 mL of distilled water, shake to dissolve, and sterilize at 121°C for 20 min;
  • cholesterol solution Cholesterol 0.05g, absolute ethanol 10mL, shake to dissolve, store at 4°C for later use;
  • S solution medium 100mL of S basic solution, 1mL of 1M citrate buffer solution, 1mL of Trace solution, 0.3mL of 1M magnesium sulfate solution, 0.3mL of 1M calcium chloride solution, and 0.1mL of 5mg/mL cholesterol solution. Mix well.
  • M9 buffer 2.486g of sodium chloride, 2.982g of disodium hydrogen phosphate, 1.496g of potassium dihydrogen phosphate, 0.06g of magnesium sulfate, 500mL of distilled water, shake to dissolve, sterilize at 121°C for 20 minutes;
  • Cultivation of nematodes The nematodes are attached to a solid NGM plate coated with E. coli OP50, and then cultured in an incubator at 20°C. When the nematodes grow to adults and there are eggs and larvae on the plate, the nematodes are synchronized ⁇ .
  • Nematode synchronization Wash the nematodes on the plate with M9 buffer, and suck the washing liquid into the centrifuge tube; centrifuge at 4000 rpm for 3 minutes to remove the supernatant, then add M9 buffer, centrifuge, and repeat several times, the purpose is to wash the E.
  • coli Clean add lysate (final concentration: 3.2% NaClO and 1M NaOH); shake on a vortexer for 7 minutes to rupture the nematodes and obtain eggs; centrifuge at 4000 rpm for 3 minutes, remove the supernatant, and collect the precipitate to obtain a large number of nematode eggs; M9 Rinse twice with buffer solution, add 1mL M9 buffer solution, and incubate in an incubator at 20°C for 48 hours.
  • Liquid medicine preparation Dilute the volatile oil extract of traditional Chinese medicine to the following concentration:
  • Drug effect Dilute the synchronized nematodes to about 80/20 ⁇ L, add 20 ⁇ L of the above-mentioned nematode solution, 120 ⁇ L of S solution, 10mg/mL E. coli OP50 20 ⁇ L, 5mg/mL to each well in a 96-well plate Cholesterol 20 ⁇ L, medicinal solution 20 ⁇ L, each concentration set five parallel.
  • the blank control group no liquid was added, and the liquid was replaced with an equal volume of S liquid.
  • the volatile oil extract of traditional Chinese medicine significantly increases the wild-type ratio of C. elegans MT2124, and is in a concentration-dependent relationship, indicating that the volatile oil extract of traditional Chinese medicine acts on the Ras pathway and significantly down-regulates the excessive activation of the Ras/MAPK pathway. Effect, the mutant becomes wild type. Therefore, the volatile oil extract of the traditional Chinese medicine can act on the Ras pathway, down-regulate the excessive activation of the pathway, has anti-tumor activity, and can be used in the preparation of drugs for the treatment of tumors overexpressing ras proto-oncogene.
  • Example 3 The effect of volatile oil extracts of traditional Chinese medicine on Caenorhabditis elegans MT8189 (lin-15 mutation)
  • Lin-15 is located in the upstream of ras and is the suppressor gene of ras. After lin-15 deletion mutation, the inhibitory effect on ras disappears, and the nematode shows a polyvulva phenotype. MT8189 (lin-15) nematode contains wild-type let60/ras copy. When the volatile oil extract of Chinese medicine of the present invention can significantly increase the wild-type ratio of C. elegans MT2124 (let-60), it cannot inhibit MT8189 (lin-15).
  • the volatile oil extract of traditional Chinese medicine of the present invention cannot inhibit the polypusal phenotype of lin-15 (n765).
  • the experimental results of Example 2 show that the volatile oil extract of traditional Chinese medicine of the present invention can reverse its downstream let60
  • the multiple vaginal phenotype caused by the excessive activation of the /ras proto-oncogene that is, the volatile oil extract of traditional Chinese medicine of the present invention cannot inhibit the multiple vaginal phenotype of let60/ras upstream gene l in-15 (n765). This is because the in-15(n765) nematode contains a copy of wild-type let60/ras.
  • the volatile oil extract of traditional Chinese medicine of the present invention only works on tumors that are over-activated by let60/ras caused by let-60 mutation.
  • the let60/ras over-activated tumor caused by the deletion mutation is not sensitive, that is, the volatile oil extract of Chinese medicine of the present invention only acts on the tumor cells caused by the over-activation of ras caused by the let-60 mutation, but not on normal cells.
  • Example 4 The effect of volatile oil extracts of traditional Chinese medicine on Caenorhabditis elegans CB1275 (lin-1 deletion mutation)
  • Lin-1 is a gene downstream of ras. It is a nuclear transcription factor that can be phosphorylated by MAPK. It is a negative regulator of vulva development and can regulate the signaling pathway of vulva development downstream of let-60. Lin-1 and lin-31 form a complex, lin-1 encodes a member of the ETS family, and lin-31 encodes a winged spiral transcription factor. When the ras signal is not activated, lin-1 interacts with lin-31 to form a complex. The complex combines with the priming part of the ovipositor function genes to prevent their expression.
  • the complex cannot be formed, the complex cannot be formed, and it cannot be combined with the priming part of the ovipositor functional gene, and the ovipositor gene is expressed, and the false vulva develops to form a polyvulva phenotype (Ferguson, 1989 ).
  • lin-1 is a gene downstream of ras, which is a negative regulator of vulva development.
  • ras a negative regulator of vulva development.
  • ovipositor gene expression nematodes show multiple vulva phenotypes, and volatile oil extracts of traditional Chinese medicine cannot inhibit lin-1.
  • the multiple vaginal phenotype indicates that the drug action site is upstream of lin-1 instead of lin-1, that is, the drug down-regulates the overactivation of ras instead of directly inhibiting the development of vulva cells.
  • Caenorhabditis elegans Genotype lin-1(e1275) IV is derived from CGC (Caenorhabditis Genetics Center), purchased from CGC; Escherichia coli OP50, uracil leaking mutant strain, as food for Caenorhabditis elegans, purchased From CGC.
  • Example 2 show that the drug inhibits the multi-vulva phenotype of nematodes, but this result only suggests that the drug significantly down-regulates the excessive activation of let-60/ras at the site of let-60 or its downstream, and can inhibit MT2124 (let-60).
  • the drug with multiple vulva phenotype of mutation also treats Caenorhabditis elegans CB1275. If the multiple vulva phenotype cannot be inhibited, the drug action site is upstream of lin-1. In addition, because lin-1 is a nuclear transcription factor, it interacts with lin-31 to form a complex that regulates the development of vulva cells.
  • the drug has a significant inhibitory effect on the multiple vulva phenotype of the gene inactivation mutation, it means that the drug can directly inhibit the development of vulva precursor cells and prevent them from forming a multiple vulva phenotype, which can inhibit the overexpression of ras proto-oncogene Of anti-tumor.
  • the multiple vaginal phenotype of the CB1275 strain of nematodes was not affected by the volatile oil extracts of traditional Chinese medicine, which indicates that the drug acting on the Ras signaling pathway can inhibit the multiple vaginal phenotype formed by the excessive activation of ras caused by let-60 mutation.
  • the inability to inhibit the multiple vaginal phenotype formed by the deletion mutation of lin-1 indicates that the volatile oil extract of traditional Chinese medicine of the present invention acts on ras but not on lin-1, which proves that the drug down-regulates the excessive activation of ras instead of directly inhibiting cell proliferation and differentiation.
  • the effect that is, the effect of the volatile oil extract of traditional Chinese medicine of the present invention is dependent on the mechanism of the Ras pathway, rather than extensive cytotoxicity.
  • Example 5 The effect of volatile oil extracts of traditional Chinese medicine and single medicine on Caenorhabditis elegans MT2124 (let-60 mutation)
  • the dosage of each single medicine is consistent with that of the volatile oil extract of traditional Chinese medicine.
  • the anti-tumor activity of the volatile oil of the traditional Chinese medicine extract prepared by the preparation method of the present invention is significantly higher than that of the volatile oil of the single medicine.
  • Example 5 The effect of volatile oil extract of traditional Chinese medicine and eugenol on Caenorhabditis elegans MT2124 (let-60 mutation)
  • the anti-tumor activity of the volatile oil extract of Chinese medicine disclosed in the present invention is significantly higher than that of eugenol.
  • the invention achieves the technical effect of synergy and synergy with the traditional Chinese medicine volatile oil extract obtained through reasonable proportioning and extraction treatment.
  • the present invention relates to the application of the traditional Chinese medicine composition and the volatile oil extract thereof in the treatment of tumors overexpressing ras proto-oncogene, and the tumors specifically involved are liver cancer, pancreatic cancer, lung cancer and colon cancer.
  • the therapeutic effect of the traditional Chinese medicine composition and traditional Chinese medicine volatile oil extract on tumors with overexpression of ras gene does not depend on extensive cytotoxicity, but specifically down-regulates the overexpression of ras proto-oncogene; the traditional Chinese medicine composition and traditional Chinese medicine volatile oil extract only It works on tumor cells caused by excessive activation of ras caused by let-60 mutation, but does not work on tumor cells caused by excessive activation of wild-type ras caused by lin-15 deletion mutation, that is, the traditional Chinese medicine composition and the volatile oil extract of traditional Chinese medicine of the present invention It only acts on cancer cells, not normal cells, is not teratogenic, and is safe to use.

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Abstract

一种中药组合物及其挥发油提取物在制备抑制ras原癌基因突变后过表达肿瘤的药物中的应用。该中药组合物由丁香、砂仁、八角茴香、肉桂、白胡椒、木香、干姜组成。该中药组合物及其挥发油提取物可显著抑制let‑60突变导致的ras原癌基因过表达的秀丽隐杆线虫的肿瘤样多阴门表型，使其转变为具有真阴门的野生型表型。上述结果表明该中药组合物及其挥发油提取物具备治疗ras基因过表达的肿瘤的作用，可以在制备抑制ras原癌基因突变后过表达的抗肿瘤药物中应用。

Description

一种抗肿瘤中药组合物及其挥发油提取物 技术领域
本发明涉及一种中药组合物及其挥发油提取物在抑制ras原癌基因突变后过表达的抗肿瘤药物中的应用，属于抗肿瘤药物领域。
背景技术
目前，恶性肿瘤已经成为人类的头号杀手，严重的危害着人类的健康，世界卫生组织的资料表明，每年全世界肿瘤的发病为1000万人，死亡约700万人，我国癌症每年的新发病例为200万，因癌症死亡的约为140万，我国居民死亡5人中，即有1人死于癌症(王明政，2017)。
约30％的人类肿瘤是由于ras原癌基因突变后导致RAS蛋白过度激活造成的。在30％的人类肿瘤中存在ras基因的突变和RAS蛋白的过度激活，其中约90％的胰腺癌，50％左右的结肠癌和20％左右的急性白血病存在ras基因突变(Hara and Han,1995)。在40％的遗传性非息肉病性结直肠癌中检测到KRAS突变，49％的常规滤泡性甲状腺癌有RAS突变，48％的Hurthle细胞甲状腺肿瘤有RAS突变。83％的胰腺癌和85％胆管癌中发生KRAS密码子12突变。除此以外，肺癌、胃癌、乳腺癌、膀胱癌以及黑色素瘤等癌症都与ras基因的突变密切相关(杨麒巍等，2018)。Ras-MAPK信号转导通路异常参与了肝癌的进程。以Ras信号通路为靶标的抗肿瘤治疗也成为肿瘤治疗学的研究热点，因此，ras已成为公认的筛选相关恶性癌药物靶点(刘雪梅等,2008)。Ras信号通路从线虫到人高度保守，在秀丽隐杆线虫中let-60基因编码保守的RAS蛋白，它与人类的RAS蛋白相比具有83％的同源性。如果ras基因突变，在秀丽隐杆线虫中会引起线虫产生多阴门，在人类中则是引起细胞的恶性增殖，导致肿瘤的发生(李蕊等，2016)。因此，模式生物秀丽隐杆线虫(Caenorhabditis elegans)Ras/MAPK信号通路过度激活型突变体被作为肿瘤模型广泛应用于筛选抑制Ras通路过度激活的抗肿瘤药物(Hara and Han,1995；王昉,2005；刘雪梅等,2008)。本发明采用秀丽隐杆线虫为筛选工具，进行下调Ras/MAPK信号通路过度激活抗肿瘤药物候选物的筛选。
传统的化学药物治疗和手术、放疗等结合，虽成功提高了多种恶性肿瘤的治疗率，但由于较强的副作用以及耐药性，使得寻找新的治疗肿瘤的研究刻不容缓(张秀云，2012)。我国的中药在治疗癌症中具有多方位、多靶点、不易产生耐药等优势，符合肿瘤多因素、多环节致病的机理，其抗肿瘤的有效部位、有效成分及作用机理研究已成为目前国内外研究的热点，并在临床肿瘤治疗方面取得了一定疗效，且毒性较低，为肿瘤预防和治疗开辟了一条新路，近年来中药及其有效成分抗肿瘤研究工作取得了一系列的成果，有的成果已经转化为临床上广泛使用的抗肿瘤药物(尹龙等，2006；张秀云，2012；朱元章等，2017)。
龙虎人丹最早源自于中国古有的成方“诸葛行军散”，几经改进，得到现有的处方。龙虎人丹主要由薄荷脑、冰片、丁香、砂仁、八角茴香、肉桂、胡椒、木香、干姜、儿茶、甘草组成。可以开窍醒神，祛暑化浊，和中止呕，主要用于中暑头晕，恶心呕吐，腹泻及晕车，晕船。迄今为止，没有文献报道过龙虎人丹具有抗肿瘤活性。
而本发明在龙虎人丹的基础上进行了改进，发现了一种具有抑制ras基因过度激 活的中药组合物及其挥发油提取物，该中药组合物及其挥发油提取物可以在治疗ras原癌基因过度激活的肿瘤中应用。相较于龙虎人丹，该中药组合物及其挥发油提取物可以在治疗ras原癌基因过度激活的肿瘤中应用。
本发明提供的中药组合物及其挥发油提取物可以与任何一种药学上可以接受的辅料混合制成各种剂型，在治疗ras原癌基因过度激活的肿瘤中应用。
参考文献
王明政，我国恶性肿瘤预防与控制策略探索[J].中医药管理杂志，2017,25(06):8-9
刘雪梅，邓平等，基于Ras信号转导途径的药物靶标研究进展[J].药学进展，2008,32(11):481-485
王昉(2005).利用线虫作为模式生物筛选能够抑制癌基因ras活性的药物.复旦大学博士后出站报告.
Hara M，Han M et al.Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.[J].Developmental Biology，1995,92(4):3333-3337
张秀云等，抗肿瘤中药有效部位及化学成分研究进展[J].科技视界，2012,7(3):177-178
尹龙，徐亮等，抗肿瘤中药及其有效成分的作用研究现状[J].动物医学进展，2006,27(1):39-43
朱元章，张贵彪等，中药挥发油抗肿瘤机制研究进展[J].中国实验方剂学杂志，16(8):227-234
杨麒巍,隋玉杰,杜珍武,张桂珍.RAS基因家族在实体瘤中突变率的研究进展[J].中国体视学与图像分析,2018(03):303-310.
李蕊,宋威江,张少鹏,姚树坤.Ras-MAPK通路在肝癌发病机制中的作用[J].中日友好医院学报,2016,30(01):47-49+54.
发明内容
本发明的目的是提供一种中药组合物由丁香、砂仁、八角茴香、肉桂、白胡椒、木香、干姜组成，采用水蒸气蒸馏法提取，得到该中药组合物的挥发油提取物粗品，再用有机溶剂萃取，萃取所得的有机相挥干溶剂，脱水干燥，得到该中药挥发油提取物。本发明是提供该中药组合物及其挥发油提取物在抑制ras原癌基因过表达中的应用，具体的肿瘤为治疗肝癌、胰腺癌、肺癌和结肠癌。
本发明通过下述技术方案予以实施。
本发明的目的是提供一种中药组合物，所述中药组合物由以下重量份的原料制成：丁香5份、砂仁5份、八角茴香3份、肉桂8份、白胡椒3份、木香3份、干姜5份。
本发明的另一个目的是提供一种中药挥发油提取物，该中药挥发油提取物由丁香5份、砂仁5份、八角茴香3份、肉桂8份、白胡椒3份、木香3份、干姜5份制成，制备方法如下：取上述比例的丁香、砂仁、八角茴香、肉桂、白胡椒、木香、干姜，加入蒸馏水，采用水蒸气蒸馏法提取，得到挥发油提取物粗品，所述挥发油提物粗品再用有机溶剂萃取，萃取所得的有机 相挥干溶剂，脱水干燥，得到中药挥发油提取物。
该中药挥发油提取物的标志性化学组分含有丁香酚42.43-59.19％，乙酸丁香酚酯13.05-15.28％，茴香脑10.02-13.11％，石竹烯2.07-2.88％，乙酸龙脑酯1.3-1.71％。
该中药挥发油提取物的密度为1.0～1.1g/mL。
本发明的另一个目的是提供所述中药组合物及其中药挥发油提取物在制备抗肿瘤药物中的应用。
所述的肿瘤是由ras原癌基因突变后过表达导致的。
具体涉及的肿瘤为肝癌、胰腺癌、肺癌和结肠癌。
由于Ras信号通路从线虫到人高度保守，在秀丽隐杆线虫中，let-60基因编码保守的RAS蛋白，它与人类的RAS蛋白相比具有83％的同源性。如果ras突变，在秀丽隐杆线虫中会引起线虫的多阴门表型，在人类中则是引起细胞的恶性增殖，导致肿瘤的发生。因此，模式生物秀丽隐杆线虫(Caenorhabditis elegans)Ras/MAPK信号通路过度激活型突变体被作为肿瘤模型广泛应用于筛选抑制Ras通路过度激活的抗肿瘤药物。
因此，本发明采用了秀丽隐杆线虫突变体作为评估抑制ras原癌基因通路过度激活药物的模型，并得出了该中药组合物和中药挥发油提取物对ras原癌基因过表达有明显的下调作用、具有抑制ras原癌基因通路过度激活的结论，并且该中药组合物和中药挥发油提取物仅仅对let-60突变导致的ras过度激活引起的肿瘤细胞起作用，而对lin-15缺失突变导致的野生型ras过度激活引起的肿瘤细胞不起作用，应用安全，即它仅作用于癌细胞，而不会作用于正常细胞，可显著下调ras原癌基因过表达，并不导致基因突变，不致畸。因此，该中药组合物和中药挥发油提取物可以在制备治疗ras原癌基因过表达肿瘤的药物中应用。
有益效果：
本发明的中药组合物和中药挥发油提取物可以特异性下调ras原癌基因突变后所致的过表达，可在制备治疗由ras原癌基因突变后过表达引起的恶性肿瘤药物中应用，具体涉及的肿瘤为肝癌、胰腺癌、肺癌和结肠癌。该中药组合物和中药挥发油提取物对ras基因过表达肿瘤的治疗作用不是依赖于广泛的细胞毒作用，而是特异性下调ras原癌基因的过表达；该中药组合物和中药挥发油提取物对仅对let-60突变导致的ras过度激活引起的肿瘤细胞起作用，而对lin-15缺失突变导致的野生型ras过度激活引起的肿瘤细胞不起作用，即该中药组合物和中药挥发油提取物对仅作用于癌细胞，不作用于正常细胞，不致畸，应用安全。
具体实施方式
以下提供具体实施例来说明本发明，但本发明权利要求书和说明书的保护范围并不限于这些实施例。
实施例一中药挥发油提取物的制备
原料药：丁香、砂仁、八角茴香、肉桂、白胡椒、木香、干姜
丁香7.5g、砂仁7.5g、八角茴香4.5g、肉桂12g、白胡椒4,5g、木香4.5g、干姜7.5g。
将上述原料药粉碎，加10倍量水，水蒸汽蒸馏法提取3小时，得到中药挥发油提取物粗品。
中药挥发油提取物粗品用乙醚萃取两次，萃取得到的有机相合并，然后挥干乙醚，得到中药挥发油提取物，所得的中药挥发油提取物为淡黄色油状物质。
挥发油成分分析：用GC-MS分析挥发油提取物的化学成分，42.43-59.19％的丁香酚，11.85-15.28％的乙酸丁香酚酯，10.02-13.11％的茴香脑，2.07-2.88％的石竹烯，1.3-1.71％的乙酸龙脑酯。
表1中药挥发油提取物GC-MS结果
Figure PCTCN2019095410-appb-000001
实施例二中药挥发油提取物对秀丽隐杆线虫MT2124(let-60突变)的作用
ras过度激活在线虫中会引起线虫产生多阴门，在人类中则是引起细胞的恶性增殖，如果受试药物能够抑制受试线虫多阴门表型的产生，促使它们发育成为野生型，则说明药物作用于Ras通路，下调了该通路的过度激活，具有抗肿瘤活性。MT2124是let-60/ras过度激活的秀丽隐杆线虫，该株系线虫为多阴门表型，相当于罹患了人类ras原癌基因突变的难治性肿瘤。采用受试药物处理秀丽隐杆线虫MT2124，如果受试药物能够抑制受试线虫多阴门表型的产生，促使它们发育成为野生型，则说明药物作用于Ras通路，下调了该通路的过度激活，具有抗肿瘤活性。野生型线虫在受试线虫群体中所占的比例越高，表明受试药物作用效果越好。
实验材料：秀丽隐杆线虫MT2124，基因型：let-60(n1046sd,gf)IV，购自CGC；大肠杆菌OP50，尿嘧啶渗漏突变株，作为秀丽隐杆线虫的食物，购自CGC。
S基础液：氯化钠2.925g，磷酸氢二钾0.5g，磷酸二氢钾3g，蒸馏水500mL，震摇使溶解，于121℃灭菌20min；
1M柠檬酸缓冲液：柠檬酸2g，柠檬酸钾29.35g，蒸馏水100mL，震摇使溶解，于121℃灭菌20min；
Trace液：EDTA0.186g，FeSO 4·7H 2O 0.06g，MnCl 2·4H 2O 0.02g，CuSO 4·5H 2O0.0025g，ZnSO 4·7H 2O 0.029g，蒸馏水100mL，震摇使溶解，于121℃灭菌20min；
1M硫酸镁溶液：MgSO 4·7H 2O 24.647g，蒸馏水100mL，震摇使溶解，于121℃灭菌 20min；
1M氯化钙溶液：CaCl 2·2H 2O14.702g，蒸馏水100mL，震摇使溶解，于121℃灭菌20min；
5mg/mL胆固醇溶液：胆固醇0.05g，无水乙醇10mL，震摇使溶解，置4℃保存备用；
S液培养基：S基础液100mL，1M柠檬酸缓冲液1mL，Trace液1mL，1M硫酸镁溶液0.3mL，1M氯化钙溶液0.3mL，5mg/mL胆固醇溶液0.1mL，混合均匀即可。
M9缓冲液：氯化钠2.486g，磷酸氢二钠2.982g，磷酸二氢钾1.496g，硫酸镁0.06g，蒸馏水500mL，震摇使溶解，于121℃灭菌20min；
具体实验步骤：
线虫的培养：将线虫接在涂有大肠杆菌OP50的固体NGM板上，然后将放在20℃的培养箱中培养，当线虫长到成虫，并且板子上有卵和幼体线虫的时候进行线虫同步化。
线虫同步化：用M9缓冲液将平板上的线虫冲下来，将冲洗液吸入离心管；4000rpm离心3min，去除上清，然后在加入M9缓冲液，离心，重复几次，目的是将大肠杆菌洗干净；加入裂解液(终浓度：3.2％NaClO和1M NaOH)；在涡旋仪上震荡7min，使线虫破裂，得到卵；4000rpm离心3min，去除上清，收集沉淀获得大量的线虫卵；M9缓冲液冲洗两次，加入1mL M9缓冲液，置于20℃培养箱中孵化48小时。
药液配制：将中药挥发油提取物稀释成下列浓度：
L：12.5μg/mL；M：25μg/mL；H：50μg/mL；HH：100μg/mL
药物作用：将同步化好的线虫稀释到80条/20μL左右，在96孔板中，每个孔中加入上述线虫液20μL，S液120μL，10mg/mL的大肠杆菌OP50 20μL，5mg/mL的胆固醇20μL，药液20μL，每个浓度设置五个平行。空白对照组不加药液，将药液换成等体积的S液。20℃恒温培养至线虫成熟，在显微镜下检测线虫的野生型比例(即只有一个真阴门的线虫比例)。与空白对照组相比，野生型的比例越高，说明药物抑制ras激活的作用更强。结果如下表所示：
表2中药挥发油提取物对秀丽隐杆线虫MT2124(let-60突变)野生型比例的影响
Figure PCTCN2019095410-appb-000002
*为与空白组比较P＜0.05
从表2可以看出，中药挥发油提取物使秀丽隐杆线虫MT2124的野生型比例显著增加，并呈浓度依赖关系，说明中药挥发油提取物作用于Ras通路，对Ras/MAPK通路过度激活有明显下调作用，使突变体变为野生型。因此，本中药挥发油提取物能够作用于Ras通路，下 调了该通路的过度激活，具有抗肿瘤活性，可以在制备治疗ras原癌基因过表达肿瘤的药物中应用。
实施例三中药挥发油提取物对秀丽隐杆线虫MT8189(lin-15突变)的作用
lin-15位于ras的上游，是ras的抑制基因，lin-15缺失突变后，对ras的抑制作用消失，线虫表现出多阴门表型。MT8189(lin-15)线虫中包含的是野生型let60/ras拷贝，当本发明中药挥发油提取物能够使秀丽隐杆线虫MT2124(let-60)的野生型比例明显增加，而不能抑制MT8189(lin-15)线虫的多阴门表型时，说明本发明中药挥发油提取物仅对let-60突变导致的ras过度激活引起的肿瘤细胞起作用，而对lin-15缺失突变导致的野生型ras过度激活引起的肿瘤细胞不起作用。因此，证明本发明中药挥发油提取物仅仅作用于let-60突变导致的ras过度激活引起的肿瘤细胞，而不作用于正常的细胞。
实验材料：秀丽隐杆线虫MT8189，多阴门表型，基因型:l in-15(n765ts)X，购自CGC；大肠杆菌OP50，尿嘧啶渗漏突变株，作为秀丽隐杆线虫的食物，购自CGC。
具体实验步骤：同实施例二。
表3中药挥发油提取物对秀丽隐杆线虫MT8189(lin-15缺失突变)的作用
Figure PCTCN2019095410-appb-000003
如表3所示，本发明中药挥发油提取物不能抑制lin-15(n765)的多阴门表型，根据遗传上位的原则，实施例二实验结果显示本发明中药挥发油提取物可逆转其下游的let60/ras原癌基因过度激活导致的多阴门表型，即本发明中药挥发油提取物不能抑制let60/ras上游基因l in-15(n765)的多阴门表型。这是由于l in-15(n765)线虫中包含的是野生型let60/ras拷贝，本发明中药挥发油提取物仅仅对let-60突变导致的let60/ras过度激活的肿瘤起作用，对lin-15缺失突变导致的let60/ras过度激活的肿瘤不敏感，即本发明中药挥发油提取物仅仅作用于let-60突变导致的ras过度激活引起的肿瘤细胞，而不作用于正常的细胞。
实施例四中药挥发油提取物对秀丽隐杆线虫CB1275(lin-1缺失突变)的作用
lin-1是ras下游的一个基因，是可被MAPK磷酸化的核转录因子，是阴门发育的负调节因子，可以调节let-60下游的阴门发育的信号通路。lin-1与lin-31组成复合体，lin-1编码ETS家族成员，而lin-31编码带翼螺旋转录因，ras信号未激活时，lin-1与lin-31相互作用组成复合体，这个复合体与产卵器功能基因的起动部分结合而阻止它们的表达。lin-1缺失突变后，复合体就无法形成，复合体不能形成，无法与产卵器功能基因的起动部分结合，产卵器基因表达，进而产生假阴门发育形成多阴门表型(Ferguson,1989)。
即lin-1是ras下游的一个基因，是阴门发育的负调节因子，lin-1缺失突变后，产卵器基因表达，线虫表现出多阴门表型，中药挥发油提取物不能抑制lin-1的多阴门表型，说明药物作用位点在lin-1上游，而不是lin-1，即药物下调了ras的过度激活而不是直接抑制了阴门细胞的发育起作用。
实验材料：秀丽隐杆线虫基因型：lin-1(e1275)IV来源于CGC(Caenorhabditis Genetics Center)，购自CGC；大肠杆菌OP50，尿嘧啶渗漏突变株，作为秀丽隐杆线虫的食物，购自CGC。
具体实验步骤：同实施例二
实施例二的结果表明药物抑制了线虫的多阴门表型，但这一结果仅提示药物显著下调let-60/ras过度激活的位点在于let-60或其下游，能够抑制MT2124(let-60突变)的多阴门表型的药物，同样处理秀丽隐杆线虫CB1275，如果不能抑制其多阴门表型，说明药物作用位点在lin-1上游。此外，由于lin-1为核转录因子，与lin-31相互作用组成复合体，调节阴门细胞发育的过程。若药物对该基因失活突变的多阴门表型具显著的抑制作用，则说明药物可直接抑制阴门前体细胞的发育，阻止其形成多阴门表型，即可以抑制ras原癌基因过表达的抗肿瘤。
表4中药挥发油提取物对秀丽隐杆线虫CB1275(lin-1缺失突变)的作用
Figure PCTCN2019095410-appb-000004
如表4所示，CB1275株系线虫的多阴门表型未受到中药挥发油提取物的影响，这表明药物作用于Ras信号通路可抑制let-60突变导致的ras过度激活形成的多阴门表型，不能抑制lin-1缺失突变形成的多阴门表型，说明本发明中药挥发油提取物作用于ras而不作用于lin-1，证明药物下调了ras的过度激活而不是直接抑制了细胞的增殖分化起作用的，即本发明中药挥发油提取物起作用是Ras通路机制依赖地，而非广泛的细胞毒作用。
实施例五中药挥发油提取物及单味药对秀丽隐杆线虫MT2124(let-60突变)的作用
实验材料：同实施例二。
各单味药剂量与中药挥发油提取物保持生药量一致。
表5各单味药与中药挥发油提取物复方生药量
Figure PCTCN2019095410-appb-000005
具体实验步骤：同实施例二。
表6中药挥发油提取物与丁香挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000006
表7中药挥发油提取物与砂仁挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000007
表8中药挥发油提取物与八角茴香挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000008
表9中药挥发油提取物与肉桂挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000009
Figure PCTCN2019095410-appb-000010
表10中药挥发油提取物与白胡椒挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000011
表11中药挥发油提取物与木香挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000012
表12中药挥发油提取物与干姜挥发油对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000013
如表6-12所示，本发明所述制备方法制备得到的中药提取物挥发油的抗肿瘤活性均明显高于单味药挥发油的抗肿瘤活性。
实施例五中药挥发油提取物与丁香酚对秀丽隐杆线虫MT2124(let-60突变)的作用
检索现有技术发现，丁香酚具有抗肿瘤的活性，因此，本实施例就中药挥发油提取物与丁香酚在抑制ras原癌基因过度激活的肿瘤中的作用进行了比较，实验结果如下：
表13中药挥发油提取物与丁香酚对秀丽隐杆线虫MT2124(let-60突变)的作用比较
Figure PCTCN2019095410-appb-000014
如表13所示，虽然丁香酚也表现出抑制ras原癌基因过表达的效果，但是本发明公开的中药挥发油提取物的抗肿瘤活性显著高于丁香酚的抗肿瘤活性，由此可见，本发明经过合理配比和提取处理获得的中药挥发油提取物实现了协同增效的技术效果。
因此，本发明涉及中药组合物及其挥发油提取物在治疗ras原癌基因过表达肿瘤的应用，具体涉及的肿瘤为肝癌、胰腺癌、肺癌和结肠癌。该中药组合物和中药挥发油提取物对ras基因过表达肿瘤的治疗作用不是依赖于广泛的细胞毒作用，而是特异性下调ras原癌基因的过表达；该中药组合物和中药挥发油提取物仅对let-60突变导致的ras过度激活 引起的肿瘤细胞起作用，而对lin-15缺失突变导致的野生型ras过度激活引起的肿瘤细胞不起作用，即本发明中药组合物和中药挥发油提取物仅作用于癌细胞，不作用于正常细胞，不致畸，应用安全。

Claims (10)

  1. 一种中药组合物，其特征在于，所述中药组合物由以下重量份的原料制成：丁香5份、砂仁5份、八角茴香3份、肉桂8份、白胡椒3份、木香3份、干姜5份。
  2. 一种中药挥发油提取物，其特征在于，所述中药挥发油提取物由丁香5份、砂仁5份、八角茴香3份、肉桂8份、白胡椒3份、木香3份、干姜5份制成，制备方法如下：取上述比例的丁香、砂仁、八角茴香、肉桂、白胡椒、木香、干姜，加入蒸馏水，采用水蒸气蒸馏法提取，得到挥发油提取物粗品，所述挥发油提物粗品再用有机溶剂萃取，萃取所得的有机相挥干溶剂，脱水干燥，得到中药挥发油提取物。
  3. 根据权利要求2所述的中药挥发油提取物，其特征在于，所述中药挥发油提取物的标志性化学组分含有丁香酚42.43-59.19％，乙酸丁香酚酯13.05-15.28％，茴香脑10.02-13.11％，石竹烯2.07-2.88％，乙酸龙脑酯1.3-1.71％。
  4. 根据权利要求3所述的一种中药挥发油提取物，其特征在于，所述中药挥发油提取物的密度为1.0～1.1g/mL。
  5. 根据权利要求1所述的中药组合物在制备抗肿瘤药物中的应用。
  6. 根据权利要求5所述的中药组合物在制备抗肿瘤药物中的应用，其特征在于，所述的肿瘤是由ras原癌基因突变后过表达导致的。
  7. 根据权利要求6所述的中药组合物在制备抗肿瘤药物中的应用，其特征在于，所述的肿瘤为肝癌、胰腺癌、肺癌和结肠癌。
  8. 根据权利要求2所述的中药挥发油提取物在制备抗肿瘤药物中的应用。
  9. 根据权利要求8所述的中药挥发油提取物在制备抗肿瘤药物中的应用，其特征在于，所述的肿瘤是由ras原癌基因突变后过表达导致的。
  10. 根据权利要求9所述的中药挥发油提取物在制备抗肿瘤药物中的应用，其特征在于，所述的肿瘤为肝癌、胰腺癌、肺癌和结肠癌。
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