CN108452240B - Anti-tumor traditional Chinese medicine composition and application thereof - Google Patents
Anti-tumor traditional Chinese medicine composition and application thereof Download PDFInfo
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/902—Sparganiaceae (Bur-reed family)
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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)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/284—Atractylodes
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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)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
- A61K36/8884—Arisaema, e.g. Jack in the pulpit
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
- A61K36/8888—Pinellia
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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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
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- 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
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- 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
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- 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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
The invention relates to an anti-tumor traditional Chinese medicine composition and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 9-15 parts of rhizoma sparganii, 9-15 parts of curcuma zedoary, 15-20 parts of prepared pinellia ternate, 15-20 parts of prepared rhizoma arisaematis, 15-30 parts of astragalus membranaceus and 15-30 parts of bighead atractylodes rhizome. The traditional Chinese medicine composition has the effects of removing blood stasis and eliminating mass, reducing phlegm and softening hard masses, strengthening body resistance and invigorating spleen and qi and the like, animal experiments show that the traditional Chinese medicine composition can obviously inhibit the growth of the nude mouse MKN-45 gastric adenocarcinoma transplantable tumor, can reduce the expression of PCNA and EGFR in the nude mouse gastric cancer tissues, can change the tumor microenvironment of a walker-256 ascites tumor model rat, and can play a good role in treating tumors.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, in particular to an anti-tumor traditional Chinese medicine composition and application thereof.
Background
Malignant tumor is also called cancer, has become a common disease and a frequently encountered disease threatening human life, and is the first cause of death of urban population in China. According to the World Health Organization (WHO) report, the incidence of cancer worldwide is increasing at a rate of 3% per year, with as many as 600 million deaths per year due to cancer. Therefore, it is an urgent task for clinical oncologists to enhance the research on malignant tumor treatment, improve the treatment effects of various existing means, explore new treatment ways, improve the quality of life of cancer patients, reduce the recurrence rate and reduce the mortality rate.
Modern medical treatment methods are mainly surgical resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy and the like. The operation treatment is still the first choice for most of tumor treatments at present, but is only suitable for tumor patients with early and middle clinical stages, and only can adopt the palliative treatment means such as chemotherapy, radiotherapy and the like for the tumor patients with late-stage metastasis, and the treatment is characterized in that the tumor cells are killed and damaged and the normal tissue cells are damaged to a certain extent, so that the treatment of the patients can be stopped due to severe toxic and side effects; the immunization method opens up a new way for treating the tumor, but the curative effect and the indication are still searched.
The traditional Chinese medicine considers that malignant tumor is a systemic disease expressed in local, the generation and development of the malignant tumor are slow and gradual processes, and the malignant tumor is a local expression after the environment in a body is disordered. The occurrence of tumor is considered to be closely related to phlegm and stasis. As early as in the book of Shen nong Ben Cao Jing, it is recorded that the phlegm accumulation in the chest is phlegm accumulation in the chest, and it is considered that all the people with lumps in the upper, middle and lower parts are phlegm, and it is said that "cancer tumor is formed by stagnation of yin-yang healthy qi, which is caused by stasis of blood, turbidity, qi and blood in the five zang organs, and that" phlegm accumulation is caused by self-qi, phlegm with blood stasis and blood stasis, which is caused by the formation of the nestle sac. It indicates that the formation of tumor is due to the accumulation of phlegm and blood stasis. Aiming at the cause of phlegm and blood stasis, the adopted method for removing blood stasis, resolving phlegm and resolving masses is one of the traditional major methods for treating tumors. However, in traditional Chinese medicine, while eliminating local pathogenic factors, more attention is paid to mobilizing and improving the anticancer function of the human body, and supporting vital qi. Therefore, the traditional Chinese medicine has considerable knowledge on tumors for thousands of years, and not only careful clinical observation is carried out, but also a plurality of therapeutic medicines are accumulated. Therefore, the novel Chinese medicinal preparation with refined formula, exact curative effect, safety and effectiveness is searched and developed from the traditional Chinese medicine, and has important clinical and scientific values for preventing and treating tumors.
Chinese patent document CN200810079458.5, published japanese patent No. 2009.02.11, discloses a medicine for treating malignant tumor with dual functions of preventing and treating, which is composed of astragalus root, pilose asiabell root, honeysuckle flower, dandelion, bupleurum root, baikal skullcap root, root of herbaceous peony, irkutsk anemone rhizome, burred tuber, arisaema tuber, pinellia tuber, oldenlandia diffusa, notoginseng root, subprostrate sophora root, zedoary, and has the functions of inhibiting and killing the growth of various cancer cells. Chinese patent document CN201410430909.0, published japanese 2014.11.19, discloses a Chinese medicinal composition for treating malignant tumor, which is prepared from the following raw materials in parts by weight: 5-12 parts of trichosanthes root, 1-3 parts of rhizoma sparganii, 1-3 parts of curcuma zedoary, 3-5 parts of peach kernel, 3-5 parts of safflower, 1-2 parts of prepared nux vomica, 1-3 parts of eupolyphaga, 2-5 parts of scorpion, 1-3 parts of centipede, 3-5 parts of pinellia ternate, 1-2 parts of rhizoma arisaematis, 3-5 parts of frankincense, 3-8 parts of sappan wood, 5-8 parts of achyranthes bidentata, 5-8 parts of hawthorn, 8-12 parts of codonopsis pilosula and 10-20 parts of astragalus.
However, the theory of traditional Chinese medicine is profound, the compatibility of Chinese medicinal herbs is numerous, and the compatibility of the Chinese medicinal herbs is more delicate, so that the Chinese medical scholars can have unique experiences on the prescriptions and can re-combine the prescriptions to improve the curative effect. In addition, when applied to clinical application, the traditional Chinese medicine preparation also needs to take various factors such as difficulty and easiness in preparation, raw material cost and the like into consideration. Therefore, the traditional Chinese medicine preparation which has good curative effect, low cost and convenient preparation has wider market prospect.
Disclosure of Invention
The invention aims to provide an anti-tumor traditional Chinese medicine composition aiming at the defects in the prior art.
The invention further aims to provide application of the traditional Chinese medicine composition.
The other purpose of the invention is to provide a preparation method of the traditional Chinese medicine composition.
In order to achieve the first purpose, the invention adopts the technical scheme that:
an anti-tumor traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 9-15 parts of rhizoma sparganii, 9-15 parts of curcuma zedoary, 15-20 parts of prepared pinellia ternate, 15-20 parts of prepared rhizoma arisaematis, 15-30 parts of astragalus membranaceus and 15-30 parts of bighead atractylodes rhizome.
Preferably, the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 10-12 parts of rhizoma sparganii, 10-12 parts of curcuma zedoary, 16-18 parts of processed pinellia ternate, 16-18 parts of processed rhizoma arisaematis, 20-25 parts of astragalus membranaceus and 20-25 parts of bighead atractylodes rhizome.
More preferably, the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 10 parts of rhizoma sparganii, 10 parts of curcuma zedoary, 18 parts of processed pinellia ternate, 18 parts of processed rhizoma arisaematis, 20 parts of astragalus membranaceus and 20 parts of bighead atractylodes rhizome.
As a specific embodiment of the invention, the dosage form of the traditional Chinese medicine composition is mixture, granules, capsules or tablets.
Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6-10 times of distilled water, reflux-extracting for 4-8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into extraction container, adding distilled water 6-10 times of the total weight of the 3 medicinal materials, stirring, decocting for 2-3 times, each time adding water amount is 6-10 times of the total weight of the medicinal materials, each time adding water amount is 0.5-1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material with weight 6-10 times of the volatile oil, adding appropriate amount of distilled water, preparing into saturated aqueous solution at 40-60 deg.C, slowly adding anhydrous alcohol diluent of the volatile oil under stirring, stirring for 1-3 hr, placing in refrigerator at 2-8 deg.C for 12-24 hr, taking out, vacuum filtering, washing with petroleum ether at 30-60 deg.C for precipitation, drying, and weighing to obtain volatile oil inclusion compound.
In order to achieve the second object, the invention adopts the technical scheme that:
the application of the traditional Chinese medicine composition in preparing antitumor drugs.
In a preferred embodiment of the present invention, the tumor is gastric cancer, liver cancer, intestinal cancer, pancreatic cancer, gallbladder cancer or esophageal cancer.
In order to achieve the third object, the invention adopts the technical scheme that:
the preparation method of the traditional Chinese medicine composition comprises the following steps:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6-10 times of distilled water, reflux-extracting for 4-8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into extraction container, adding distilled water 6-10 times of the total weight of the 3 medicinal materials, stirring, decocting for 2-3 times, each time adding water amount is 6-10 times of the total weight of the medicinal materials, each time adding water amount is 0.5-1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material with weight 6-10 times of the volatile oil, adding appropriate amount of distilled water, preparing into saturated aqueous solution at 40-60 deg.C, slowly adding anhydrous alcohol diluent of the volatile oil under stirring, stirring for 1-3 hr, placing in refrigerator at 2-8 deg.C for 12-24 hr, taking out, vacuum filtering, washing with petroleum ether at 30-60 deg.C for precipitation, drying, and weighing to obtain volatile oil inclusion compound.
Preferably, the volatile oil inclusion material is selected from hydroxypropyl-beta-cyclodextrin and/or beta-cyclodextrin.
Herein, the processed pinellia tuber is a processed product of the pinellia tuber, and the preparation method comprises the following steps: taking purified pinellia ternate, separating the purified pinellia ternate into different sizes, soaking the purified pinellia ternate in water until no dry cores exist in the purified pinellia ternate, and taking out the purified pinellia ternate; and decocting proper amount of liquorice in water twice, mixing decoction, pouring into lime liquor prepared from proper amount of water, stirring uniformly, adding the soaked pinellia ternate, soaking, stirring for 1-2 times every day, keeping the pH value of the soaking solution above 12 until the section is yellow and uniform, taking out when the mouth tastes slightly numb tongue, cleaning, and drying in the shade or oven drying to obtain the traditional Chinese medicine composition, wherein 15kg of liquorice and 10kg of quicklime are used for each 100kg of purified pinellia ternate.
The processed rhizoma arisaematis is processed product of rhizoma arisaematis, and the preparation method comprises the following steps: soaking clean rhizoma arisaematis with water according to size respectively, changing water for 2-3 times every day, if foaming, adding Alumen (2 kg for every 100kg rhizoma arisaematis) after changing water, soaking for one day, changing water again, and taking out when the mouth is cut and the tongue is slightly numb. Adding appropriate amount of water into rhizoma Zingiberis recens slice and Alumen in a pan, boiling, adding rhizoma arisaematis, decocting until there is no dry heart, taking out, removing rhizoma Zingiberis recens slice, air drying until four to six turns dry, slicing, and drying, wherein each 100kg rhizoma arisaematis is 12.5kg rhizoma Zingiberis recens and Alumen respectively.
The invention has the advantages that:
1. the action mechanism of the traditional Chinese medicine composition is as follows: rhizoma Sparganii and Curcumae rhizoma are the monarch, rhizoma Sparganii is bitter and flat in taste, and is good at breaking qi in blood, and the strength of breaking blood is superior to breaking qi, which is recorded in KAI BAO BEO CAO (Kai Bao materia Medica) for treating senile nodules and abdominal mass; curcumae rhizoma, bitter, pungent and warm in flavor, has the effects of removing blood stasis and relieving swelling, and is the most important herb for treating accumulation of qi by modern physicians in the book Jing Ben Cao. The processed rhizoma pinelliae and the processed rhizoma arisaematis are ministerial, the processed rhizoma pinelliae is pungent and warm in flavor, has the effects of eliminating dampness and phlegm, and relieving distension and masses, and the processed rhizoma arisaematis is bitter, pungent and warm in flavor, has the effects of eliminating dampness and phlegm, and relieving swelling and dissipating masses. The astragalus root and the white atractylodes rhizome are used as adjuvant drugs, the astragalus root is sweet and slightly warm in taste, enters lung and spleen channels, tonifies qi, strengthens exterior, supports sores and promotes tissue regeneration; bighead atractylodes rhizome, bitter in taste, sweet and warm in flavor, enters spleen and stomach channels, invigorates qi and invigorates spleen, eliminates dampness and induces diuresis, and the pearl sac recorded in the patent of 'removing dampness and tonifying qi, regulating the middle warmer and tonifying yang' has the effect of tonifying qi and strengthening body resistance. The above medicines are used together to have the effects of removing blood stasis and eliminating mass, reducing phlegm and softening hard masses, strengthening body resistance, invigorating spleen and replenishing qi, and the like. Animal experiments show that the traditional Chinese medicine composition can obviously inhibit the growth of the MKN-45 gastric adenocarcinoma transplantation tumor of a nude mouse, can reduce the expression of PCNA and EGFR in gastric cancer tissues of the nude mouse, can change the tumor microenvironment of a walker-256 ascites tumor model rat, and can play a good role in treating tumors.
2. The traditional Chinese medicine composition has the advantages of fewer medicinal ingredients, convenient preparation, abundant and easily-obtained raw materials and low cost.
Detailed Description
The following provides a detailed description of specific embodiments of the present invention.
EXAMPLE 1 preparation of the Chinese medicinal composition of the present invention
The weight parts of the raw materials are as follows: 9 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 15 parts of processed pinellia tuber, 15 parts of processed rhizoma arisaematis, 15 parts of astragalus mongholicus and 15 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6 times of distilled water, extracting under reflux for 8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding distilled water 6 times the total weight of the 3 medicinal materials, stirring, decocting for 3 times, adding water 6 times the total weight of the medicinal materials each time, each time for 1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material hydroxypropyl-beta-cyclodextrin with the weight 6 times of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 60 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 1 hour, placing in a refrigerator at 8 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 2 preparation of the Chinese medicinal composition of the present invention
The weight parts of the raw materials are as follows: 9 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 20 parts of processed pinellia ternate, 20 parts of processed rhizoma arisaematis, 15 parts of astragalus membranaceus and 15 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 10 times of distilled water, extracting under reflux for 4 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 10 times of distilled water, stirring, decocting for 2 times, each time adding water amount 10 times of the total weight of the 3 medicinal materials, each time adding 0.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material beta-cyclodextrin which is 10 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 40 ℃, slowly adding absolute ethanol diluent of the volatile oil under continuous stirring, stirring for 3 hours, placing in a refrigerator at 2 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 3 preparation of the Chinese medicinal composition of the present invention
The weight parts of the raw materials are as follows: 9 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 15 parts of processed pinellia tuber, 15 parts of processed rhizoma arisaematis, 30 parts of astragalus mongholicus and 30 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 8 times of distilled water, extracting under reflux for 5 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 8 times of distilled water, stirring, decocting for 2 times, each time adding water amount is 8 times of the total weight of the 3 medicinal materials, each time adding 1 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion materials of hydroxypropyl-beta-cyclodextrin and beta-cyclodextrin which are 8 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 50 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 2 hours, placing in a refrigerator at 4 ℃ for 18 hours, taking out, filtering, washing and precipitating with petroleum ether at 50 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 4 preparation of the Chinese medicinal composition of the present Invention (IV)
The weight parts of the raw materials are as follows: 9 parts of rhizoma sparganii, 9 parts of curcuma zedoary, 20 parts of processed pinellia ternate, 20 parts of processed rhizoma arisaematis, 30 parts of astragalus membranaceus and 30 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6 times of distilled water, extracting under reflux for 8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding distilled water 6 times the total weight of the 3 medicinal materials, stirring, decocting for 3 times, adding water 6 times the total weight of the medicinal materials each time, each time for 1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material hydroxypropyl-beta-cyclodextrin with the weight 6 times of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 60 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 1 hour, placing in a refrigerator at 8 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 5 preparation of the Chinese medicinal composition of the present invention (V)
The weight parts of the raw materials are as follows: 15 parts of rhizoma sparganii, 15 parts of curcuma zedoary, 15 parts of processed pinellia ternate, 15 parts of processed rhizoma arisaematis, 15 parts of astragalus membranaceus and 15 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 10 times of distilled water, extracting under reflux for 4 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 10 times of distilled water, stirring, decocting for 2 times, each time adding water amount 10 times of the total weight of the 3 medicinal materials, each time adding 0.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material beta-cyclodextrin which is 10 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 40 ℃, slowly adding absolute ethanol diluent of the volatile oil under continuous stirring, stirring for 3 hours, placing in a refrigerator at 2 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 6 preparation of the Chinese medicinal composition of the present invention (VI)
The weight parts of the raw materials are as follows: 15 parts of rhizoma sparganii, 15 parts of curcuma zedoary, 20 parts of processed pinellia ternate, 20 parts of processed rhizoma arisaematis, 15 parts of astragalus membranaceus and 15 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 8 times of distilled water, extracting under reflux for 5 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 8 times of distilled water, stirring, decocting for 2 times, each time adding water amount is 8 times of the total weight of the 3 medicinal materials, each time adding 1 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion materials of hydroxypropyl-beta-cyclodextrin and beta-cyclodextrin which are 8 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 50 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 2 hours, placing in a refrigerator at 4 ℃ for 18 hours, taking out, filtering, washing and precipitating with petroleum ether at 50 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
EXAMPLE 7 preparation of the Chinese medicinal composition of the present invention (VII)
The weight parts of the raw materials are as follows: 15 parts of rhizoma sparganii, 15 parts of curcuma zedoary, 20 parts of processed pinellia ternate, 20 parts of processed rhizoma arisaematis, 30 parts of astragalus membranaceus and 30 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6 times of distilled water, extracting under reflux for 8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding distilled water 6 times the total weight of the 3 medicinal materials, stirring, decocting for 3 times, adding water 6 times the total weight of the medicinal materials each time, each time for 1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material hydroxypropyl-beta-cyclodextrin with the weight 6 times of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 60 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 1 hour, placing in a refrigerator at 8 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
EXAMPLE 8 preparation of the Chinese medicinal composition of the present invention (eight)
The weight parts of the raw materials are as follows: 15 parts of rhizoma sparganii, 15 parts of curcuma zedoary, 15 parts of processed pinellia tuber, 15 parts of processed rhizoma arisaematis, 30 parts of astragalus mongholicus and 30 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 10 times of distilled water, extracting under reflux for 4 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 10 times of distilled water, stirring, decocting for 2 times, each time adding water amount 10 times of the total weight of the 3 medicinal materials, each time adding 0.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material beta-cyclodextrin which is 10 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 40 ℃, slowly adding absolute ethanol diluent of the volatile oil under continuous stirring, stirring for 3 hours, placing in a refrigerator at 2 ℃ for 12 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 30 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 9 preparation of the Chinese medicinal composition of the present invention (nine)
The weight parts of the raw materials are as follows: 10 parts of rhizoma sparganii, 10 parts of curcuma zedoary, 18 parts of processed pinellia ternate, 18 parts of processed rhizoma arisaematis, 20 parts of astragalus membranaceus and 20 parts of bighead atractylodes rhizome. The preparation steps are as follows:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 8 times of distilled water, extracting under reflux for 5 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into an extraction container, adding 8 times of distilled water, stirring, decocting for 2 times, each time adding water amount is 8 times of the total weight of the 3 medicinal materials, each time adding 1 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion materials of hydroxypropyl-beta-cyclodextrin and beta-cyclodextrin which are 8 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 50 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 2 hours, placing in a refrigerator at 4 ℃ for 18 hours, taking out, filtering, washing and precipitating with petroleum ether at 50 ℃, drying, weighing, and obtaining the volatile oil inclusion compound;
c) adding conventional pharmaceutical adjuvants into the extract and volatile oil clathrate obtained in the above steps a) and b), and making into capsule, granule, tablet or mixture.
Example 10 Effect on human gastric carcinoma MKN-45 nude mouse transplant tumor size and expression of PCNA and EGFR
First, experiment method
1. The preparation of the medicine comprises the following steps:
the traditional Chinese medicine of the invention: the preparation steps are as follows: a) traditional Chinese medicine extraction: taking 100g of rhizoma sparganii, 100g of curcuma zedoary and 200g of bighead atractylodes rhizome, appropriately crushing the materials, putting the materials into an extraction container, adding distilled water with the weight being 8 times of that of 3 medicinal materials, performing reflux extraction for 5 hours by adopting a steam distillation method, and collecting volatile oil; adding 200g of crushed astragalus, 180g of processed rhizoma arisaematis and 180g of processed pinellia ternate into an extraction container, adding 8 times of distilled water of the total weight of the 3 medicinal materials, uniformly stirring, decocting for 2 times, wherein the water amount added each time is 8 times of the total weight of the medicinal materials, and each time lasts for 1 hour, combining decoction, concentrating, drying and weighing to obtain an extract; b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material beta-cyclodextrin which is 8 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 50 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 2 hours, placing in a refrigerator at 4 ℃ for 18 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 50 ℃, drying, weighing, and obtaining the volatile oil inclusion compound; c) adding beta-cyclodextrin into the extract and volatile oil inclusion compound obtained in the steps a) and b), uniformly mixing, drying, crushing into fine powder, adding a proper amount of ethanol, granulating, drying and preparing into 1000 g.
Comparing the traditional Chinese medicine I: the preparation steps are as follows: a) adding 100g of scorpion, 100g of centipede, 180g of prepared rhizoma arisaematis and 180g of prepared pinellia ternate into an extraction container, adding 8 times of distilled water of the total weight of the 4 medicinal materials, uniformly stirring, decocting for 2 times, adding water for each time, wherein the water amount is 8 times of the total weight of the medicinal materials, and adding water for 1 hour each time, combining decoction, concentrating, drying and weighing to obtain an extract; b) adding beta-cyclodextrin into the extract obtained in the step a), uniformly mixing, drying, crushing into fine powder, adding a proper amount of ethanol, granulating, and drying to prepare 1000 g.
Comparing the traditional Chinese medicine II: the preparation steps are as follows: a) traditional Chinese medicine extraction: taking 200g of bighead atractylodes rhizome, appropriately crushing the bighead atractylodes rhizome, putting the crushed bighead atractylodes rhizome into an extraction container, adding distilled water with the weight being 8 times of that of the medicinal materials, extracting the mixture for 5 hours by reflux by a steam distillation method, and collecting volatile oil; adding 100g of crushed scorpion, 100g of centipede, 200g of astragalus, 180g of prepared rhizoma arisaematis and 180g of prepared pinellia tuber into an extraction container, adding 8 times of distilled water of the total weight of the 5 medicinal materials, uniformly stirring, decocting for 2 times, adding 8 times of water of the total weight of the medicinal materials every time, 1 hour every time, combining decoction, concentrating, drying and weighing to obtain an extract; b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material beta-cyclodextrin which is 8 times of the weight of the volatile oil, adding a proper amount of distilled water, preparing a saturated aqueous solution at 50 ℃, slowly adding absolute ethyl alcohol diluent of the volatile oil under continuous stirring, stirring for 2 hours, placing in a refrigerator at 4 ℃ for 18 hours, taking out, carrying out suction filtration, washing and precipitating with petroleum ether at 50 ℃, drying, weighing, and obtaining the volatile oil inclusion compound; c) adding beta-cyclodextrin into the extract and volatile oil inclusion compound obtained in the steps a) and b), uniformly mixing, drying, crushing into fine powder, adding a proper amount of ethanol, granulating, drying and preparing into 1000 g.
Comparing the traditional Chinese medicines: the preparation steps are as follows: a) traditional Chinese medicine extraction: taking 150g of astragalus mongholicus, 150g of codonopsis pilosula, 150g of honeysuckle, 30g of dandelion, 30g of radix bupleuri, 30g of scutellaria baicalensis, 120g of radix paeoniae alba, 30g of irkutsk anemone rhizome, 90g of rhizoma sparganii, 90g of rhizoma arisaematis, 90g of pinellia ternate, 300g of oldenlandia diffusa, 30g of pseudo-ginseng, 90g of subprostrate sophora and 90g of curcuma zedoary, adding 8 times of distilled water of the total weight of the 15 medicinal materials, uniformly stirring, decocting for 2 times, adding water for each time, wherein the water amount is 8 times of the total weight of the medicinal materials, and each time lasts for 1 hour, combining decoction, concentrating; b) adding beta-cyclodextrin into the extract obtained in the step a), uniformly mixing, drying, crushing into fine powder, adding a proper amount of ethanol, granulating, and drying to prepare 1000 g.
Positive control drug: 5-Fluorouracil (Fluorouracil, 5-FU)10ml, 0.25 g.
2. Preparation of seeded cell suspension: aseptic operation, when the growth of the MKN-45 cells cultured in vitro is intensive, 0.05 percent of trypsin and 0.02 percent of ethylenediamine tetraacetic acid (EDTA) are digested for 1-2 min, digestion liquid is poured out, serum-free 1640 liquid is added for washing, centrifugation is carried out, the cells are prepared into inoculated cells by the serum-free 1640 liquid, and the concentration is 2 multiplied by 106And L. The homogeneous single cell suspension was observed by phase contrast microscopy.
3. Subcutaneous inoculation: aseptically handling, disinfecting skin, taking a l ml syringe, aspirating 0.4ml of cell suspension, and inoculating subcutaneously under the right forelimb root of nude mice. The nude mice are transplanted for passage under the skin, the nude mice about 14d after passage are taken, aseptic operation is carried out, tumor tissues are stripped from the skin of the armpit, fiber envelopes are removed, the nude mice are cut open, the tumor tissues which grow well and are light red and fish-shaped are selected, the tumor tissues are cut into small blocks of 1mm multiplied by 1mm, and the small blocks are placed in physiological saline for standby. The nude mice were weighed and anesthetized by intraperitoneal injection with 75mg/kg ketamine, and the prepared tumor tissue blocks were implanted by cutting about 1cm along the median line of the upper abdomen. The growth condition of the tumor is observed day by day after operation, and the change of the general activity, the nutrition state and the like of the mouse is observed by taking the accessible mass on the abdomen of the mouse as a tumor formation mark.
4. Grouping and administration of experimental animals: the inoculated BABL/c nude mice are randomly divided into 6 groups, namely a treatment group, a traditional Chinese medicine first control group, a traditional Chinese medicine second control group, a traditional Chinese medicine third control group, a chemotherapy group and a tumor-bearing control group, wherein each group comprises 15 mice, and the mice are bred in a laminar flow frame in cages under the condition of no Specific Pathogen (SPF). The administration was as follows: treatment groups: the pure water solution of the traditional Chinese medicine particles is drenched into each nude mouse the next day after inoculation, 9.2g of crude drug is given per kg of body mass for 1 time per day, and the stomach is drenched continuously for 6 weeks. Traditional Chinese medicine I control group: the following day after inoculation, each nude mouse is administered 9.2g/kg body mass of the crude drug per 1 time per day by drenching with a granule of pure water solution of the control traditional Chinese medicine for 6 weeks. And (3) a traditional Chinese medicine two control group: the control Chinese medicinal two-particle pure water solution is administered to each nude mouse on the next day after inoculation, 9.2g/kg body mass of the crude drug is administered, 1 time/d, and the administration is continued for 6 weeks. Three control groups of traditional Chinese medicines: the control Chinese medicinal three-particle pure water solution is administered to each nude mouse on the next day after inoculation, 9.2g/kg body mass of the crude drug is administered, 1 time/d, and the administration is continued for 6 weeks. Chemotherapy groups: diluting 5-FU with normal saline to 7.5mg/ml, and performing intraperitoneal injection at 60mg/kg for 1 time per week for 3 weeks. Tumor-bearing control group: the normal saline is infused into each nude mouse 1 time/d for 6 weeks on the day after inoculation.
5. Growth of transplanted tumor, mouse activity and nutrient status observation: after inoculation, mice were observed for activity, feeding and nutrition. After 6 weeks, the nude mice were sacrificed by pulling their necks, the tumor tissues were completely stripped from the axillary subcutaneous tissues, the fibrous envelopes were removed, and the tumor mass was weighed on an electronic balance. The tumor inhibition rate was calculated using the formula (%) ratio (average tumor mass after control treatment-average tumor mass after drug group treatment)/average tumor mass after control treatment × 100. After the observation, tumor tissues with the weighed mass were fixed with neutral formalin, embedded in paraffin, and sliced continuously at 5 μm.
6. Determination of PCNA and EGFR of transplanted tumor tissues: SP method, hematoxylin-eosin staining, paraffin section deparaffinization to water, microwave antigen retrieval. Dripping 1 drop of primary antibody (mouse anti-human PCNA and EGFR) into each section, standing overnight in a refrigerator at 4 ℃, washing for 3 times by PBS, dripping biotinylated secondary antibody, adding 1 drop of streptavidin-peroxidase solution into each section, developing for 10min by 3,3' -diamino and benzidine substrates, and sealing by xylene and neutral gum. The PCNA staining positive substance is brown yellow and granular, is positioned in the gastric cancer cell nucleus, more than 200 cells are randomly counted under an X200 oil microscope as an analysis base number, and the marked PCNA index, namely the percentage of the marked cells in a cell group, is counted. Most EGFR positive staining is located on cytoplasm and cell membrane, and each EGFR positive staining is located on nucleus and is a brown yellow particle which is respectively scored according to staining depth and the proportion of positive cells. (1) Dyeing depth: 0min, no coloring; 1 minute, light coloring; 2min, dark coloring. (2) Positive cell ratio: 0min, no coloring; 1 minute, coloring is less than or equal to 1/3; 2min, 1/3< coloring is less than or equal to 2/3; score 3, coloring > 2/3. A positive score was obtained by adding >3 of the two results.
Second, experimental results
1. Effect on growth of gastric adenocarcinoma MKN-45 in nude mice
In the tumor-bearing control group, the abdomen of the nude mouse starts to touch the nodular lumps about 13 days after the MKN-45 human gastric adenocarcinoma is inoculated under the skin of the nude mouse, and the masses gradually increase later; the tumor was touched later in each of the other groups than the control group. Nude mice were sacrificed after 6 weeks, tumor mass was weighed, and tumor inhibition rate was calculated, and the mass of transplanted tumors and the statistical results of tumor inhibition rate for each group are shown in table 1. The results show that the difference of the tumor mass after treatment of each administration group and the tumor-bearing control group has statistical significance (P is less than 0.05); compared with the traditional Chinese medicine one control group, the traditional Chinese medicine two control group and the traditional Chinese medicine three control group, the difference of the tumor quality of the treatment group has statistical significance (P <0.05), and compared with the chemotherapy group, the difference has no statistical significance (P > 0.05).
TABLE 1 quality and tumor inhibition rate of each group of transplanted tumors
2. PCNA, EGFR expression in tumor tissues
The statistical results of the positive expression rate of PCNA in each group of tumor tissues are shown in Table 2. The result shows that the PCNA positive expression rate of each administration group is obviously lower than that of a tumor-bearing control group (P is less than 0.05), which indicates that the cell proliferation activity is obviously reduced after the administration; the PCNA positive expression rate of the treatment group is respectively compared with a traditional Chinese medicine first control group, a traditional Chinese medicine second control group and a traditional Chinese medicine third control group, the difference has statistical significance (P <0.05), and compared with a chemotherapy group, the difference has no statistical significance (P > 0.05).
TABLE 2 Positive expression of PCNA in various tumor tissue groups
| Group of | n | Positive expression Rate (%) |
| Tumor-bearing control group | 15 | 67.94±18.66 |
| Treatment group | 14 | 10.74±2.59 |
| Control group of Chinese medicinal materials | 15 | 32.76±8.80 |
| Chinese medicine two control group | 13 | 29.44±6.26 |
| Three control groups of traditional Chinese medicines | 15 | 51.30±10.47 |
| 5-FU | 15 | 10.78±2.45 |
Statistical results of EGFR positivity in each group of tumor tissues are shown in table 2. The results show that the EGFR positive rate of each administration group is obviously lower than that of a tumor-bearing control group (P < 0.05); the EGFR positive rate of the treatment group is respectively compared with a traditional Chinese medicine first control group, a traditional Chinese medicine second control group and a traditional Chinese medicine third control group, the difference has statistical significance (P <0.05), and compared with a chemotherapy group, the difference has no statistical significance (P > 0.05).
TABLE 3 EGFR-positive expression in tumor tissues of various groups
| Group of | n | Positive for | Negative of | Positive rate (%) |
| Tumor-bearing control group | 15 | 13 | 2 | 86.67 |
| Treatment group | 14 | 4 | 10 | 28.57 |
| Control group of Chinese medicinal materials | 15 | 8 | 7 | 53.33 |
| Chinese medicine two control group | 13 | 7 | 6 | 53.85 |
| Three control groups of traditional Chinese medicines | 15 | 10 | 5 | 66.67 |
| 5-FU | 15 | 4 | 11 | 26.67 |
The results show that the traditional Chinese medicine composition can obviously inhibit the growth of the nude mouse MKN-45 gastric adenocarcinoma transplantation tumor and can down-regulate the PCNA and EGFR expression in the nude mouse gastric cancer tissues.
Example 11 inhibition of Walker-256 rat gastric neoplasia
First, experiment method
1. Rat stomach tumor implantation model establishment
Firstly establishing a rat walker-256 ascites tumor model, then extracting the abdominal dropsy by using a sterile syringe, inoculating the abdominal dropsy to the subcutaneous part of the right axilla of the rat, and growing solid tumors after 7 days. Tumor implantation in stomach: taking the subcutaneous solid tumor tissue, washing completely, cutting into tissue blocks with the size of about 0.5mm multiplied by 0.5mm, opening the abdomen after the Wistar rat is anesthetized, inoculating the tumor tissue blocks into the serosal layer of the stomach wall, and dripping OB biological glue for bonding and fixing. All rats were recorded daily for food and water intake, weighed every 3 days, and observed for changes in mental, activity, and fur gloss.
2. Experimental grouping and intervention
40 rats, 36 rats inoculated with tumor mass in stomach, the other 4 uninoculated rats were normal control group, Wistar rats implanted with tumor mass were randomly divided into three groups of 12 per group: treatment groups: the present invention was administered with a pure water-dissolved solution (equivalent to 55g/kg of crude drug in body mass) of the Chinese medicinal granules (prepared in the same manner as in example 10) immediately after molding, and the administration was continued for 21 days by intragastric administration daily. Traditional Chinese medicine control group: the control two-particle Chinese medicine (prepared by the same method as example 10) was administered with a pure water solution (equivalent to 55g/kg of crude drug) immediately after molding, and administered by intragastric administration daily for 21 days. Model control group: the normal saline is given about 3 mL/patient, and the medicine is given immediately the next day after the model is made, and the stomach is drenched every day for 21 days continuously. The normal control group was fed only on the normal diet daily. All rats were raised under equal SPF conditions.
3. Sampling of tissue specimens
After 21d, all rats are killed by pulling neck, the abdomen is opened, the conditions of macroscopic change in the abdominal cavity, ascites and organ metastasis are observed, and fresh tumor tissues of the stomach of each group (one stomach tissue of the rat without tumor implantation) are peeled and taken. The removed tissue blocks were formalin-fixed to prepare paraffin sections (5 μm thick) for immunohistochemical examination.
4. Immunohistochemical detection
The expression of CD34, alpha-Sma and FAP alpha protein in the gastric implantation tumor and gastric tissue specimen is detected by an immunohistochemical method. Fibroblasts of CD34(+) are mainly distributed in a mucous layer and a serous layer in the digestive tract, and CD34 is expressed on the fibroblast membrane; FAP alpha is selectively expressed in tumor-associated fibroblasts in malignant tumor stroma, and is mainly expressed in membrane and stroma; alpha-Sma is the most important marker for identifying tumor-associated fibroblasts, is mainly expressed on cell membranes, and is positive when appearing brown-yellow to brown-black. Randomly collecting 5 visual fields for each wafer, taking a picture by a microscope, collecting the integral optical density value of the positive staining part of the 5 visual fields/the positive area of the 5 visual fields by using Image J2x software, and calculating the average optical density value of the 5 visual fields as the expression quantity.
Second, experimental results
The statistical results of CD34, alpha-Sma, FAP alpha expression in rat gastric neoplasia and gastric tissue are shown in Table 4. The results show that the expression level of CD34 of the rats in other groups is obviously lower than that of the rats in the normal control group (P <0.05), and the expression levels of FAP alpha and alpha-SMA are obviously higher than that of the rats in the normal control group (P < 0.05); the expression level of CD34 of rats in the treatment group and the traditional Chinese medicine control group is obviously higher than that of rats in the model control group (P <0.05), and the expression levels of FAP alpha and alpha-SMA are obviously lower than that of rats in the model control group (P < 0.05); the expression level of CD34 in the treated rats is obviously higher than that in the traditional Chinese medicine control rats (P <0.05), and the expression levels of FAP alpha and alpha-SMA are obviously lower than that in the traditional Chinese medicine control rats (P < 0.05).
TABLE 4 expression of CD34, alpha-Sma, FAP alpha in gastric hybridomas and gastric tissues of various groups of rats
| Group of | Number of specimen | CD34 | α-Sma | FAPα |
| Normal control group | 4 | 809.52±11.68 | 647.85±20.46 | 620.73±10.29 |
| Model control group | 12 | 598.22±17.36 | 834.49±28.62 | 883.01±18.54 |
| Control group of traditional Chinese medicines | 12 | 680.15±22.47 | 753.16±19.53 | 771.19±20.66 |
| Treatment group | 12 | 741.40±27.39 | 691.40±11.75 | 704.85±23.87 |
The results show that the traditional Chinese medicine composition can change the tumor microenvironment, so that the tumor growth can be inhibited.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (9)
1. An anti-tumor traditional Chinese medicine composition is characterized by being prepared from the following raw material medicines in parts by weight: 9-15 parts of rhizoma sparganii, 9-15 parts of curcuma zedoary, 15-20 parts of prepared pinellia ternate, 15-20 parts of prepared rhizoma arisaematis, 15-30 parts of astragalus membranaceus and 15-30 parts of bighead atractylodes rhizome.
2. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw material medicines in parts by weight: 10-12 parts of rhizoma sparganii, 10-12 parts of curcuma zedoary, 16-18 parts of processed pinellia ternate, 16-18 parts of processed rhizoma arisaematis, 20-25 parts of astragalus membranaceus and 20-25 parts of bighead atractylodes rhizome.
3. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw material medicines in parts by weight: 10 parts of rhizoma sparganii, 10 parts of curcuma zedoary, 18 parts of processed pinellia ternate, 18 parts of processed rhizoma arisaematis, 20 parts of astragalus membranaceus and 20 parts of bighead atractylodes rhizome.
4. The Chinese medicinal composition according to any one of claims 1 to 3, wherein the Chinese medicinal composition is in the form of mixture, granule, capsule or tablet.
5. The Chinese medicinal composition according to any one of claims 1 to 3, wherein the preparation method of the Chinese medicinal composition comprises the following steps:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6-10 times of distilled water, reflux-extracting for 4-8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into extraction container, adding distilled water 6-10 times of the total weight of the 3 medicinal materials, stirring, decocting for 2-3 times, each time adding water amount is 6-10 times of the total weight of the medicinal materials, each time adding water amount is 0.5-1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material with weight 6-10 times of the volatile oil, adding appropriate amount of distilled water, preparing into saturated aqueous solution at 40-60 deg.C, slowly adding anhydrous alcohol diluent of the volatile oil under stirring, stirring for 1-3 hr, placing in refrigerator at 2-8 deg.C for 12-24 hr, taking out, vacuum filtering, washing with petroleum ether at 30-60 deg.C for precipitation, drying, and weighing to obtain volatile oil inclusion compound.
6. The use of the Chinese medicinal composition of any one of claims 1-3 in the preparation of an anti-tumor medicament.
7. The use of claim 6, wherein the tumor is gastric cancer, liver cancer, intestinal cancer, pancreatic cancer, gallbladder cancer or esophageal cancer.
8. A method for preparing the Chinese medicinal composition of any one of claims 1 to 3, which comprises the following steps:
a) traditional Chinese medicine extraction: pulverizing rhizoma Sparganii, Curcumae rhizoma and Atractylodis rhizoma, placing into an extraction container, adding 6-10 times of distilled water, reflux-extracting for 4-8 hr by steam distillation, and collecting volatile oil; adding pulverized radix astragali, rhizoma arisaematis preparata, and rhizoma Pinelliae preparata into extraction container, adding distilled water 6-10 times of the total weight of the 3 medicinal materials, stirring, decocting for 2-3 times, each time adding water amount is 6-10 times of the total weight of the medicinal materials, each time adding water amount is 0.5-1.5 hr, mixing decoctions, concentrating, drying, and weighing to obtain extract;
b) preparing a volatile oil clathrate compound: taking volatile oil inclusion material with weight 6-10 times of the volatile oil, adding appropriate amount of distilled water, preparing into saturated aqueous solution at 40-60 deg.C, slowly adding anhydrous alcohol diluent of the volatile oil under stirring, stirring for 1-3 hr, placing in refrigerator at 2-8 deg.C for 12-24 hr, taking out, vacuum filtering, washing with petroleum ether at 30-60 deg.C for precipitation, drying, and weighing to obtain volatile oil inclusion compound.
9. The method of claim 8, wherein the volatile oil inclusion material is selected from hydroxypropyl- β -cyclodextrin and/or β -cyclodextrin.
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