WO2023040094A1 - 一种具有抑制肿瘤细胞活性作用的组合物及其应用 - Google Patents

一种具有抑制肿瘤细胞活性作用的组合物及其应用 Download PDF

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WO2023040094A1
WO2023040094A1 PCT/CN2021/138251 CN2021138251W WO2023040094A1 WO 2023040094 A1 WO2023040094 A1 WO 2023040094A1 CN 2021138251 W CN2021138251 W CN 2021138251W WO 2023040094 A1 WO2023040094 A1 WO 2023040094A1
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extract
composition
group
composition according
water
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何健文
江荣高
张丽杰
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海南鑫开源医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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)
    • 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)
    • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
    • 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
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • 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
    • 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
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether

Definitions

  • the invention belongs to the field of antitumor drugs, and in particular relates to a composition capable of inhibiting tumor cell activity and its application.
  • Human choriocarcinoma is a highly malignant trophoblastic tumor, and blood metastasis occurs in the early stage of its secondary disease. It is sensitive and resistant to chemotherapy drugs.
  • the current conventional chemotherapy methods are suitable for the treatment of high-risk, relapsed and drug-resistant cases. The effect is not good, and it is urgent to find new therapeutic drugs and open up new therapeutic targets.
  • the object of the present invention is to provide a composition with the effect of inhibiting tumor cell activity and its application, and the composition has the activity of inhibiting tumor cell.
  • the present invention provides a composition with the effect of inhibiting the activity of tumor cells, which comprises the following components: natural capsaicin, diosgenin, extract of Aquamania spp. Root wood extract.
  • the mass ratio of the natural capsaicin, diosgenin, Saccharomyces japonicus extract, iron chopsticks volatile oil extract, Brassica chinensis extract and single wood extract is 1:(0.9 ⁇ 1.1) :(0.47 ⁇ 0.52):(0.18 ⁇ 0.23):(0.48 ⁇ 0.53):(0.45 ⁇ 0.55).
  • the mass ratio of the natural capsaicin, diosgenin, Saccharomyces osmanthus extract, iron chopsticks volatile oil extract, eucalyptus bractiferae extract and single wood extract is 1:1:0.5:0.2 :0.5:0.5.
  • the natural capsaicin plays an anti-tumor effect in terms of inducing cycle arrest of tumor cells, inhibiting proliferation and invasion, and regulating autophagy and apoptosis.
  • the diosgenin inhibits tumor cell activity mainly by regulating signaling pathways to block tumor cell growth and migration, activating tumor suppressor gene expression to promote tumor cell apoptosis, and down-regulating tumor growth enzymes to cut off tumor cell proliferation.
  • the extract of Aquamania sativa is taken from the whole herb of the perennial bulbous plant Spider Orchid of the family Amaryllidaceae of Liliaceae, and its main component contains alkaloids; the total alkaloids of Shuimeniata alkaloids have significant cytotoxic and inhibitory effects on various cancer cells , have inhibitory effects on human melanoma, leukemia cells, pancreatic cancer cells, etc.
  • the volatile oil extract of Tiechoizi is taken from the root of the genus Tiechoizi in the family Ranunculaceae. Its anti-tumor activity is mainly concentrated in its volatile oil and amino acid components. Among them, the lipid chemical components in the volatile oil mainly exert anti-tumor activity by inhibiting the transport of ions .
  • Bractleaf extract is taken from the whole herb of the perennial herb of Compositae, and its active ingredients include sterol compounds, flavonoids, and amides.
  • the flavonoids in it promote the secretion of interferon by regulating signaling pathways, thereby inhibiting tumor growth.
  • the single wood extract is taken from the rhizome of the Apocynaceae Bermudagrass shrub, which contains a variety of chemical components such as alkaloids, lignin, terpenoids, etc. Studies have shown that the indole alkaloids in it have the effect on human oral epidermal cancer cells. Growth was significantly inhibited.
  • the existing form of the natural capsaicin is freeze-dried powder; the freeze-dried powder is made by the following method:
  • the solubilization component A includes one of PEG400, hydroxypropyl beta cyclodextrin, hydroxyethyl beta cyclodextrin, methyl beta cyclodextrin and sulfobutyl beta cyclodextrin sodium or Various.
  • the diosgenin exists in the form of freeze-dried powder;
  • the freeze-dried powder is prepared by the following method:
  • the insoluble diosgenin and the solubilizing component B are mixed, dissolved in water, and freeze-dried to obtain a freeze-dried powder.
  • the extract of Shuiguiban is prepared according to the following method:
  • Pulverize the whole plant of Saccharomyces japonicus use 95% ethanol as the extraction solvent, extract it by continuous reflux method, concentrate it into an extract, disperse it in water, extract it with ethyl acetate and n-butanol respectively, collect the two-phase solvent, spin dry and mix, Deshui ghost banana dry paste extract.
  • the iron chopsticks volatile oil extract is prepared according to the following method:
  • the rhizome of the iron chopsticks plant is crushed, and extracted by steam extraction for 9.5-10.5 hours to obtain the iron chopsticks volatile oil extract.
  • the extract of Zebrasia bractica is prepared according to the following method:
  • the whole herb of Bractia bractica was crushed, extracted with 80% ethanol as the extraction solvent, concentrated into an extract, dispersed in water, extracted with n-butanol, and spin-dried to obtain the extract of Bractia bractae.
  • the single wood extract is preferably prepared according to the following method:
  • the rhizome of a single woody plant is crushed, 95% ethanol is used as the extraction solvent, extracted by continuous reflux, concentrated into an extract, dispersed in water, extracted with petroleum ether and ethyl acetate respectively, the two-phase solvent is collected, spin-dried and mixed, Get single wood extract.
  • the present invention provides an application of the composition described in the above technical solution in the preparation of antitumor drugs.
  • the present invention provides a composition with the effect of inhibiting the activity of tumor cells, which comprises the following components: natural capsaicin, diosgenin, extract of Aquamania spp. Root wood extract.
  • the above compositions are all taken from natural medicinal plants.
  • the anti-tumor active ingredients extracted and isolated from natural plants have the characteristics of large number, diverse structure types, and remarkable drug effects. Diversified anti-tumor activities and multi-target effects Its characteristics can be regarded as an idea and potential strategy for the development of anticancer drugs.
  • This embodiment provides a composition with the effect of inhibiting the activity of tumor cells.
  • the active ingredients of the composition include the following proportions by weight: 1 part of natural capsaicin, 1 part of diosgenin, 0.5 parts of Shuigui banana extract, iron chopsticks 0.2 part of volatile oil extract, 0.5 part of eucalyptus extract, and 0.5 part of single tree extract.
  • This embodiment further provides the preparation method of the six active ingredients in the above composition.
  • Water ghost banana extract take the whole plant of the water ghost banana plant and crush it, use an appropriate amount of 95% ethanol as the extraction solvent, use the continuous reflux method to extract for 10 hours, and then concentrate it into a 10:1 extract by rotary evaporation, disperse the extract with an appropriate amount of water, and use Extract with ethyl acetate and n-butanol, collect the two-phase solvent, spin dry and mix to obtain the dry extract of Shuiguijiao paste.
  • Iron chopsticks volatile oil extract take the rhizome of the iron chopsticks plant and crush it, extract it by steam extraction for 10 hours, and obtain a yellow oily extract with special aroma.
  • Bractia chinensis extract Take the whole plant of Bractia chinensis and crush it, use an appropriate amount of 80% ethanol as the extraction solvent to extract for 10 hours, filter it through gauze, take the extract and spin-steam it into a 30:1 extract, and disperse the extract with appropriate amount of water , and then extracted with n-butanol, and the n-butanol phase was spin-dried to obtain the dry paste extract of Bractonia chinensis.
  • Single wood extract Take a single wood plant rhizome and crush it, use an appropriate amount of 95% ethanol as the extraction solvent, extract by continuous reflux for 10 hours, spin dry and concentrate to obtain a 10:1 extract, disperse the extract with an appropriate amount of water, and use petroleum ether respectively Extract with ethyl acetate, collect the two-phase solvent, spin dry and mix to obtain the extract of single root wood paste.
  • the 6 kinds of active components prepared above were respectively mixed in the weight ratio of 1 part: 1 part: 0.5 part: 0.2 part: 0.5 part: 0.5 part to obtain a composition.
  • composition of the present invention Take 1 part of the composition of the present invention, dissolve it in 500 parts of deionized water, and finally dilute it with 500 parts of RPMI-1640 culture solution to obtain the composition of the present invention that has the effect of inhibiting tumor cell activity.
  • Cell test 1 CCK8 method to detect the inhibitory effect of the composition on the proliferation of human choriocarcinoma cells
  • Culture of human choriocarcinoma cells inoculate human choriocarcinoma cells into RPMI-1640 medium containing 5% fetal bovine serum, incubate in 5% carbon dioxide, 37°C incubator, and passage once every 2 days.
  • Human choriocarcinoma cells in the logarithmic growth phase were digested with 0.25% trypsin, the cell suspension density was adjusted to 1.5 ⁇ 104 cells/mL with culture medium, and seeded into 96-well cell culture plates, and 100 ⁇ L of cell suspension was added to each well, Cultivate under the above incubator conditions until the cells grow into a single layer and adhere to the wall, discard the supernatant, wash with phosphate buffer twice, and obtain the in vitro culture of human choriocarcinoma cells.
  • the human choriocarcinoma cells cultured in vitro were divided into an experimental group, a negative control group, a positive control group and a blank group.
  • the experimental group was added the composition diluted with RPMI-1640 culture medium, 25 ⁇ L, 50 ⁇ L, 75 ⁇ L, and 100 ⁇ L were added respectively, and 6 replicate wells were set for each concentration.
  • the composition was replaced with 100 ⁇ L of 25 mg/mL fluorouracil injection, and the rest remained unchanged.
  • the blank group had no choriocarcinoma cells, and the rest remained unchanged.
  • the above four groups were incubated in a 5% carbon dioxide, 37°C incubator for 2 days, the supernatant was discarded, and washed twice with phosphate buffer.
  • Pre-test operation Add 20 ⁇ L of CCK-8 solution and 80 ⁇ L of RPMI-1640 culture solution to the above four groups, and incubate for 30 minutes.
  • the survival results of human choriocarcinoma cells in different groups are shown in the table below.
  • Table 1 The survival results of human choriocarcinoma cells in different groups by CCK8 method
  • the experimental results showed that the cell survival rates of 50 ⁇ l, 75 ⁇ l, and 100 ⁇ l in the experimental group were significantly lower than those of the negative control group, which was statistically significant compared with the negative control group (P ⁇ 0.05), and the cell survival rate values showed a trend that increased with the amount of the composition added. There is an obvious dose-effect relationship.
  • the human choriocarcinoma cells cultured in vitro were divided into an experimental group, a negative control group, a positive control group and a blank group.
  • the experimental group was added the composition diluted with RPMI-1640 culture medium, 25 ⁇ L, 50 ⁇ L, 75 ⁇ L, and 100 ⁇ L were added respectively, and 6 replicate wells were set for each concentration.
  • the composition was replaced with 100 ⁇ L of 25 mg/mL fluorouracil injection, and the rest remained unchanged.
  • the blank group had no choriocarcinoma cells, and the rest remained unchanged.
  • the above four groups were incubated in a 5% carbon dioxide, 37°C incubator for 2 days, the supernatant was discarded, and washed twice with phosphate buffer.
  • composition of the present invention dissolve and dilute to 100 parts with water for injection, filter through a 0.22 ⁇ m filter membrane, pack in ampoules, and sterilize by autoclaving at 115°C for 30 minutes to obtain 1-fold concentration, 5-fold concentration and 10-fold concentration of sterile composition dilutions.
  • SCID mice were selected to shave the back hair and disinfected, inject 0.3ml of JEG-3 cell suspension into the right back of the mouse with a sterile skin test needle, and then raise them in a clean room. Tumor-bearing mice were obtained. Twenty-five tumor-bearing mice were randomly divided into 5 groups, with 5 mice in each group. Administration by intraperitoneal injection:
  • mice in group A were injected with 500 ⁇ L sterile saline (negative control group).
  • mice in group B were injected with 500 ⁇ L of 1-fold concentration of the sterile composition dilution.
  • mice in group C were injected with 500 ⁇ L of a 5-fold concentration of sterile composition dilution.
  • mice in group D were injected with 500 ⁇ L of a 10-fold dilution of the sterile composition.
  • mice in group E were injected with 500 ⁇ L of 25 mg/mL fluorouracil injection (positive control group).
  • Example 1 the three active components prepared in "Example 1: Preparation of the composition" (example 1 is six kinds of compositions) in order by weight
  • the composition is obtained by mixing 1 part: 1 part: 0.5 part.
  • composition of this comparative example Take 1 part of the composition of this comparative example, dissolve it in 500 parts of deionized water, and finally dilute it with 500 parts of RPMI-1640 culture solution to obtain the composition with the effect of inhibiting tumor cell activity described in this comparative example.
  • Cell test 1 CCK8 method was used to detect the inhibitory effect of the composition on the proliferation of human choriocarcinoma cells.
  • the human choriocarcinoma cells cultured in vitro were divided into an experimental group, a negative control group, a positive control group and a blank group.
  • the experimental group 75 ⁇ l, 100 ⁇ l, 150 ⁇ l, and 200 ⁇ l of the composition diluted with RPMI-1640 culture medium were added, and 6 replicate wells were set for each concentration.
  • the composition was replaced with 100 ⁇ l of 25 mg/ml fluorouracil injection, and the rest remained unchanged.
  • the blank group had no choriocarcinoma cells, and the rest remained unchanged.
  • the above four groups were incubated in a 5% carbon dioxide, 37°C incubator for 2 days, the supernatant was discarded, and washed twice with phosphate buffer.
  • the experimental results showed that 200 ⁇ l of the experimental group had an effect on the survival rate of choriocarcinoma cells, and the statistical analysis showed that it was significantly different from the negative control group (P ⁇ 0.05).
  • the results of the MMT method were parallel to those of the CCK8 method.
  • Example 1 Composition Preparation
  • natural capsaicin natural capsaicin
  • volatile oil extract of iron chopsticks extract of Campanulaceae bractiferae
  • single root wood extract Example 1 is Six composition forms
  • composition of this comparative example Take 1 part of the composition of this comparative example, dissolve it in 500 parts of deionized water, and finally dilute it with 500 parts of RPMI-1640 culture solution to obtain the composition with the effect of inhibiting tumor cell activity described in this comparative example.
  • Cell test 1 CCK8 method was used to detect the inhibitory effect of the composition on the proliferation of human choriocarcinoma cells.
  • the human choriocarcinoma cells cultured in vitro were divided into an experimental group, a negative control group, a positive control group and a blank group.
  • 50 ⁇ l, 75 ⁇ l, 100 ⁇ l, and 150 ⁇ l of the composition diluted with RPMI-1640 culture medium were added, and 6 replicate wells were set for each concentration.
  • the composition was replaced with 100 ⁇ l of 25 mg/ml fluorouracil injection, and the rest remained unchanged.
  • the blank group had no choriocarcinoma cells, and the rest remained unchanged.
  • the above four groups were incubated in a 5% carbon dioxide, 37°C incubator for 2 days, the supernatant was discarded, and washed twice with phosphate buffer.
  • Experimental group 100 ⁇ l 83.71 ⁇ 5.75
  • Experimental group 150 ⁇ l 73.58 ⁇ 5.76 negative control group 104.67 ⁇ 3.75 positive control group 20.86 ⁇ 5.39 blank group 12.18 ⁇ 2.34
  • composition of the present invention dissolve and dilute to 100 parts with water for injection, filter through a 0.22 ⁇ m filter membrane, pack in ampoules, and sterilize by autoclaving at 115°C for 30 minutes to obtain 1-fold concentration, 5-fold concentration and 10-fold concentration of sterile composition dilutions.
  • mice The tumor-forming mice were divided into 5 groups, 5 mice in each group. Administration by intraperitoneal injection:
  • mice in group A were injected with 500 ⁇ L sterile saline (negative control group).
  • mice in group B were injected with 500 ⁇ L of 1-fold concentration of the sterile composition dilution.
  • mice in group C were injected with 500 ⁇ L of a 5-fold concentration of sterile composition dilution.
  • mice in group D were injected with 500 ⁇ L of a 10-fold dilution of the sterile composition.
  • mice in group E were injected with 500 ⁇ L of 25 mg/mL fluorouracil injection (positive control group).
  • the present invention provides a composition that has the effect of inhibiting the activity of tumor cells, including the following components: natural capsaicin, diosgenin, water ghost banana extract, iron chopsticks volatile oil extract, bract leaf wind Extract of chamomile and wood root.
  • the above compositions are all taken from natural medicinal plants.
  • the anti-tumor active ingredients extracted and isolated from natural plants have the characteristics of large number, diverse structure types, and remarkable drug effects. Diversified anti-tumor activities and multi-target effects Its characteristics can be regarded as an idea and potential strategy for the development of anticancer drugs.

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Abstract

一种具有抑制肿瘤细胞活性作用的组合物及其应用，组合物包括以下组分：天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物。上述组合物均取自天然药用植物，从天然植物中提取和分离出的抗肿瘤活性成分有数量众多、结构类型多样化、药效显著等特点。

Description

一种具有抑制肿瘤细胞活性作用的组合物及其应用
本申请要求于2021年09月18日提交中国专利局、申请号为202111109677.5、发明名称为“一种具有抑制肿瘤细胞活性作用的组合物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于抗肿瘤药物领域，尤其涉及一种具有抑制肿瘤细胞活性作用的组合物及其应用。
背景技术
人绒毛膜癌是一种高度恶性的滋养细胞肿瘤，其继发早期即发生血液转移，其对化疗药物的敏感性和耐药性，目前的常规化疗手段对于高危、复发、耐药病例的治疗效果不佳，迫切需要寻找新的治疗药物和开辟新的治疗靶点。
发明内容
有鉴于此，本发明的目的在于提供一种具有抑制肿瘤细胞活性作用的组合物及其应用，该组合物具有抑制肿瘤细胞活性。
本发明提供了一种具有抑制肿瘤细胞活性作用的组合物，包括以下组分：天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物。
在本发明中，所述天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物的质量比为1:(0.9～1.1):(0.47～0.52):(0.18～0.23):(0.48～0.53):(0.45～0.55)。
在本发明中，所述天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物的质量比为1:1:0.5:0.2:0.5:0.5。
在本发明中，所述天然辣椒素在肿瘤细胞的诱导周期阻滞、抑制增殖侵袭、调控自噬凋亡等方面起抗肿瘤作用。所述薯蓣皂苷元主要通过调控信号通路阻滞肿瘤细胞生长和迁移、激活抑癌基因表达促进肿瘤细胞凋亡、下调肿瘤生长酶切断肿瘤细胞增殖等机制发挥抑制肿瘤细胞活性的作用。所述水鬼蕉提取物取自百合目石蒜科多年生鳞茎草本植物蜘蛛兰的全草，其主要成分包含生物 碱；水鬼蕉生物总碱对多种癌细胞具有显著的细胞毒和抑制作用，对人黑色素瘤、白血病细胞、胰腺癌细胞等均有抑制作用。铁筷子挥发油提取物取自毛茛科铁筷子属铁筷子的根，其抗肿瘤活性主要集中在其挥发油和氨基酸类成分，其中挥发油中的脂类化学成分主要通过抑制离子的转运而发挥抗肿瘤活性。苞叶风毛菊提取物取自菊科风毛菊属多年生草本植物苞叶风毛菊全草，其活性成分包含甾醇类化合物、黄酮类化合物、酰胺类化合物等。其中的黄酮类化合物通过调控信号通路，促进干扰素分泌，从而抑制肿瘤生长。单根木提取物取自夹竹桃科狗牙花属灌木的根茎，含有生物碱、木质素、萜类化合物等多种化学成分，有研究表明其中的吲哚类生物碱对人类口腔表皮癌细胞生长有明显抑制作用。
在本发明中，所述天然辣椒素的存在形式为冻干粉；所述冻干粉由以下方法制得：
将难溶性天然辣椒素和增溶组分A混合，溶于水中，随后冷冻干燥，得到冻干粉；
所述增溶组分A包括PEG400、羟丙基倍他环糊精、羟乙基倍他环糊精、甲基倍他环糊精和磺丁基倍他环糊精钠中的一种或多种。
在本发明中，所述薯蓣皂苷元的存在形式为冻干粉；所述冻干粉由以下方法制得：
将难溶性薯蓣皂苷元和增溶组分B混合，溶于水中，冷冻干燥，得到冻干粉。
在本发明中，所述水鬼蕉提取物按照以下方法制得：
将水鬼蕉全草粉碎，采用95％乙醇作为提取溶剂，利用连续回流法提取，浓缩成浸膏，水分散后分别采用乙酸乙酯和正丁醇萃取，收集两相溶剂，旋干并混合，得水鬼蕉干膏提取物。
在本发明中，所述铁筷子挥发油提取物按照以下方法制得：
取铁筷子植物的根茎粉碎，利用水蒸气提取法提取9.5～10.5h，得到铁筷子挥发油提取物。
在本发明中，所述苞叶风毛菊提取物按照以下方法制得：
取苞叶风毛菊全草粉碎，采用80％乙醇作为提取溶剂浸提，浓缩成浸膏， 水分散后采用正丁醇萃取，旋干，得到苞叶风毛菊提取物。
在本发明中，所述单根木提取物优选按照以下方法制得：
取单根木植物的根茎粉碎，采用95％乙醇作为提取溶剂，利用连续回流法提取，浓缩成浸膏，水分散后分别采用石油醚和乙酸乙酯萃取，收集两相溶剂，旋干并混合，得到单根木提取物。
本发明提供了一种上述技术方案所述组合物在制备抗肿瘤药物中的应用。
本发明提供了一种具有抑制肿瘤细胞活性作用的组合物，包括以下组分：天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物。上述组合物均取自天然药用植物，从天然植物中提取和分离出的抗肿瘤活性成分有数量众多、结构类型多样化、药效显著等特点，多元化的抗肿瘤活性、多靶点作用的特点不失为一种抗肿瘤药物研发思路和潜在策略。
具体实施方式
为了进一步说明本发明，下面结合实施例对本发明提供的一种具有抑制肿瘤细胞活性作用的组合物及其应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
对比例
实施例1
组合物制备
本实施例提供一种具有抑制肿瘤细胞活性作用的组合物，组合物的活性成分包含以下重量份的比例：天然辣椒素1份、薯蓣皂苷元1份、水鬼蕉提取物0.5份、铁筷子挥发油提取物0.2份、苞叶风毛菊提取物0.5份、单根木提取物0.5份。
本实施例还进一步提供上述组合物中六种活性成分的制备方法。
难溶性天然辣椒素1份和19份磺丁基倍他环糊精钠，溶于适量水溶液中，随后冷冻干燥成粉末。
难溶性薯蓣皂苷元1份、39份普朗尼克F68，溶于适量水溶液中，随后冷冻干燥成粉末。
水鬼蕉提取物：取水鬼蕉植物全草粉碎，使用适量95％乙醇作为提取溶剂， 利用连续回流法提取10h，随后旋转蒸发浓缩成10:1浸膏，浸膏用适量水分散，分别用乙酸乙酯和正丁醇萃取，收集两相溶剂，旋干并混合，得水鬼蕉干膏提取物。
铁筷子挥发油提取物：取铁筷子植物的根茎粉碎，利用水蒸气提取法提取10h，得到具有特殊香气的黄色油状提取物。
苞叶风毛菊提取物：取苞叶风毛菊全草粉碎，使用适量80％乙醇作为提取溶剂浸提10h，纱布过滤后取提取液旋蒸成30:1的浸膏，浸膏用适量水分散，再用正丁醇萃取，取正丁醇相旋干，得苞叶风毛菊干膏提取物。
单根木提取物：取单根木植物的根茎粉碎，使用适量95％乙醇作为提取溶剂，利用连续回流法提取10h，旋干浓缩得10:1浸膏，浸膏用适量水分散，分别用石油醚和乙酸乙酯萃取，收集两相溶剂，旋干并混合，得单根木干膏提取物。
分别将以上制得的6种活性组分依次按重量比1份：1份：0.5份：0.2份：0.5份：0.5份混合，得到组合物。
细胞实验
取本发明所述的组合物1份，溶于500份去离子水，最后用500份RPMI-1640培养液稀释，即得本发明所述的具有抑制肿瘤细胞活性作用的组合物。
细胞试验1：CCK8法检测组合物对人绒毛膜癌细胞增殖的抑制作用
人绒癌细胞的培养：将人绒癌细胞接种至含5％胎牛血清的RPMI-1640培养基中，置于5％二氧化碳、37℃培养箱中孵化，约2天传代1次。用0.25％胰酶消化处于对数生长期的人绒癌细胞，用培养液调节细胞悬液密度至1.5×10 4个/mL并接种至96孔细胞培养板，每孔加入100μL细胞悬液,于上述孵箱条件下培养至细胞长出单层贴壁，吸弃上清液，用磷酸缓冲液冲洗2遍，得到人绒癌细胞体外培养物。
实验设计：将上述人绒癌细胞体外培养物分为实验组、阴性对照组、阳性对照组和空白组。实验组加入用RPMI-1640培养液稀释的组合物，分别加入25μL、50μL、75μL、100μL，每个浓度设置6个复孔。阴性对照组与实验组相比，不添加组合物，其余不变。阳性对照组与实验组相比，将组合物替换为25mg/mL氟尿嘧啶注射液100μL，其余不变。空白组与实验组相比，无人绒癌 细胞，其余不变。以上四组置于5％二氧化碳、37℃培养箱中孵化2天，吸弃上清液，用磷酸缓冲液冲洗2遍。
检测前操作：以上四组再分别加入20μL的CCK-8溶液和80μL的RPMI-1640培养液，培养30min。用酶标仪测定各孔在450nm的吸光度(OD)值，癌细胞的存活率(＝(OD实验-OD空白)/(OD对照-OD空白)×100)。不同组别人绒癌细胞存活结果见下表。
数据均采用SPSS(23.0版)统计软件进行分析，计量资料采用“均数±标准差(X±S)表示”，多组间均数比较采用单因素方差分析，两组间均数比较采用单样本t检验，以P＜0.05表示差异具有统计学意义。
表1 CCK8法中不同组别人绒癌细胞存活结果
组别 存活率％
实验组25μL 98.86±5.87
实验组50μL 68.78±3.64
实验组75μL 46.87±6.14
实验组100μL 28.97±4.73
阴性对照组 102.06±4.68
阳性对照组 23.57±6.47
空白组 8.28±0.78
实验结果显示，实验组50μl、75μl、100μl的细胞存活率明显低于阴性对照组，与阴性对照组相比具有统计学意义(P＜0.05)，且细胞存活率数值显示出随组合物添加量增大而逐渐变小的趋势，呈现明显的量效关系。
细胞实验2：MTT法检测组合物对人绒毛膜癌细胞增殖的抑制作用
人绒癌细胞的培养：同上述实验过程。
实验设计：将上述人绒癌细胞体外培养物分为实验组、阴性对照组、阳性对照组和空白组。实验组加入用RPMI-1640培养液稀释的组合物，分别加入25μL、50μL、75μL、100μL，每个浓度设置6个复孔。阴性对照组与实验组相比，不添加组合物，其余不变。阳性对照组与实验组相比，将组合物替换为25mg/mL氟尿嘧啶注射液100μL，其余不变。空白组与实验组相比，无人绒癌细胞，其余不变。以上四组置于5％二氧化碳、37℃培养箱中孵化2天，吸弃 上清液，用磷酸缓冲液冲洗2遍。
检测前操作：以上四组再分别加入20μL的MTT溶液和80μl的RPMI-1640培养液，培养4h，吸弃上清液。每组加入100μL二甲亚砜振摇使结晶物溶解。用酶标仪测定各孔在490nm的吸光度(OD)值，癌细胞的存活率(＝(OD实验-OD空白)/(OD对照-OD空白)×100)。不同组别人绒癌细胞存活结果见下表。
表2 MMT法中不同组别人绒癌细胞存活结果
组别 存活率％
实验组25μL 96.82±3.87
实验组50μL 72.71±4.72
实验组75μL 54.18±3.76
实验组100μL 35.28±4.17
阴性对照组 105.00±3.81
阳性对照组 29.79±4.27
空白组 5.98±2.47
结果显示，与阴性对照组相比，实验组50μl、75μl、100μl对绒癌细胞存活率有影响，差异具有统计学意义(P＜0.05)，表明经组合物处理的细胞存活率明显低于阴性对照组，且细胞存活率数值显示出随组合物添加量增大而逐渐变小的趋势，呈现明显的量效关系。
动物试验
分别取本发明所述的组合物1份、5份和10份，以注射用水溶解并稀释至100份，0.22μm滤膜过滤，分装于安瓿中，115℃热压灭菌30min，即得1倍浓度、5倍浓度和10倍浓度的无菌组合物稀释液。
选SCID小鼠剃净背部毛发并消毒，用无菌皮试针向小鼠右侧背部注射0.3mlJEG-3细胞悬液，随后饲养于洁净室内，待移植瘤潜伏期过后肿瘤体积生长达100mm 3后即得荷瘤小鼠。将25只荷瘤小鼠随机分为5组，每组5只。采用腹腔注射方式给药：
A组小鼠注射500μL无菌生理盐水(阴性对照组)。
B组小鼠注射500μL、1倍浓度的无菌组合物稀释液。
C组小鼠注射500μL、5倍浓度的无菌组合物稀释液。
D组小鼠注射500μL、10倍浓度的无菌组合物稀释液。
E组小鼠注射500μL、浓度为25mg/mL的氟尿嘧啶注射液(阳性对照组)。
给药后处理：第14天用游标卡尺测量小鼠皮下移植瘤的长径a和短径b，以公式V(mm)＝1/2*a*b 2计算移植瘤体积，随后计算各给药组移植瘤体积抑制率：＝(1-各组移植瘤平均增长体积/阴性对照组移植瘤平均增长体积)*100％。
表3组合物给药后各组移植瘤抑制率
组别 体积(mm 3) 体积抑制率％
A组(阴性对照组) 4359.35±158.98 —
B组 4087.14±89.73 6.24％
C组 3687.18±124.38 15.42％
D组 2368.94±137.24 45.66％
E组(阳性对照组) 469.58±48.25 89.23％
从实验结果可以看出，使用氟尿嘧啶注射液的给药方式对移植瘤抑制率最高；同时，统计结果显示，与阴性对照组A组比较，C组和D组具有明显差异(P＜0.05)，表明使用本发明组合物对移植瘤抑制率呈明显的量效关系。
对比例1
组合物制备：将“实施例1：组合物制备”中制备所得的天然辣椒素、薯蓣皂苷元、水鬼蕉提取物三种活性组分(实施例1为六种组合物形式)依次按重量比1份：1份：0.5份混合，得到组合物。
细胞实验
取本对比例的组合物1份，溶于500份去离子水，最后用500份RPMI-1640培养液稀释，即得本对比例所述的具有抑制肿瘤细胞活性作用的组合物。
细胞试验1：CCK8法检测组合物对人绒毛膜癌细胞增殖的抑制作用。
人绒癌细胞的培养：同实施例1。
实验设计：将上述人绒癌细胞体外培养物分为实验组、阴性对照组、阳性对照组和空白组。实验组加入用RPMI-1640培养液稀释的组合物，分别加入75μl、100μl、150μl、200μl，每个浓度设置6个复孔。阴性对照组与实验组相比，不添加组合物，其余不变。阳性对照组与实验组相比，将组合物替换为 25mg/ml氟尿嘧啶注射液100μl，其余不变。空白组与实验组相比，无人绒癌细胞，其余不变。以上四组置于5％二氧化碳、37℃培养箱中孵化2天，吸弃上清液，用磷酸缓冲液冲洗2遍。
检测前操作：同实施例1。不同组别人绒癌细胞存活结果见下表。
表4 CCK8法中不同组别人绒癌细胞存活结果
组别 存活率％
实验组75μl 96.15±2.23
实验组100μl 97.76±6.17
实验组150μl 86.27±4.37
实验组200μl 72.86±3.34
阴性对照组 103.79±5.73
阳性对照组 26.47±4.83
空白组 9.78±3.38
从实验结果可知，实验组中200ul组合物的加入量使绒癌细胞的存活率明显较其他加入量降低，且与阴性对照组相比差异具有统计学意义(P＜0.05)。本对比例1和实施例1比较，组合物的加入量提高到200μl才显示出对绒癌细胞明显的抑制作用，初步分析为对比例1的组合物只包含三种活性组分，而实施例1包含六种活性组分，六种活性组分的协同增效作用较三种活性组分强。
细胞实验2：MTT法检测组合物对人绒毛膜癌细胞增殖的抑制作用
人绒癌细胞的培养：同实施例1。
实验设计：同上述实验过程。
检测前操作：同上述实验过程。不同组别人绒癌细胞存活结果见下表。
表5 MMT法中不同组别人绒癌细胞存活结果
组别 存活率％
实验组75μl 102.38±3.34
实验组100μl 98.24±5.37
实验组150μl 92.34±3.67
实验组200μl 78.35±4.68
阴性对照组 103.69±6.27
阳性对照组 30.25±3.96
空白组 7.18±2.63
实验结果表明，实验组200μl对绒癌细胞存活率有影响，统计学分析显示其与阴性对照组差异显著(P＜0.05)，MMT法检测结果与CCK8法检测结果平行。
对比例2
组合物制备：将“实施例1：组合物制备”中制备所得的天然辣椒素、铁筷子挥发油提取物、苞叶风毛菊提取物、单根木提取物四种活性组分(实施例1为六种组合物形式)依次按重量比1份:0.2份:0.5份:0.5份混合，得到组合物。
细胞实验
取本对比例的组合物1份，溶于500份去离子水，最后用500份RPMI-1640培养液稀释，即得本对比例所述的具有抑制肿瘤细胞活性作用的组合物。
细胞试验1：CCK8法检测组合物对人绒毛膜癌细胞增殖的抑制作用。
人绒癌细胞的培养：同实施例1。
实验设计：将上述人绒癌细胞体外培养物分为实验组、阴性对照组、阳性对照组和空白组。实验组加入用RPMI-1640培养液稀释的组合物，分别加入50μl、75μl、100μl、150μl，每个浓度设置6个复孔。阴性对照组与实验组相比，不添加组合物，其余不变。阳性对照组与实验组相比，将组合物替换为25mg/ml氟尿嘧啶注射液100μl，其余不变。空白组与实验组相比，无人绒癌细胞，其余不变。以上四组置于5％二氧化碳、37℃培养箱中孵化2天，吸弃上清液，用磷酸缓冲液冲洗2遍。
检测前操作：同实施例1。不同组别人绒癌细胞存活结果见下表。
表6 CCK8法中不同组别人绒癌细胞存活结果
组别 存活率％
实验组50μl 98.81±3.83
实验组75μl 87.65±4.63
实验组100μl 83.71±5.75
实验组150μl 73.58±5.76
阴性对照组 104.67±3.75
阳性对照组 20.86±5.39
空白组 12.18±2.34
从实验结果可知，实验组中75μl、100μl和150μl组合物的加入量对绒癌细胞有明显的抑制作用，且与阴性对照组相比具有统计学差异(P＜0.05)。本对比例2和对比例1比较，组合物增加了一种活性成分，组合物的抑肿瘤活性作用增强。本对比例2和实施例1比较，组合物减少两种活性成分，但抑肿瘤活性作用依然明显。
动物试验
分别取本发明所述的组合物1份、5份和10份，以注射用水溶解并稀释至100份，0.22μm滤膜过滤，分装于安瓿中，115℃热压灭菌30min，即得1倍浓度、5倍浓度和10倍浓度的无菌组合物稀释液。
JEG-3人绒毛膜癌细胞小鼠移植瘤模型的建立：同实施例1。
将成瘤小鼠分为5组，每组5只。采用腹腔注射方式给药：
A组小鼠注射500μL无菌生理盐水(阴性对照组)。
B组小鼠注射500μL、1倍浓度的无菌组合物稀释液。
C组小鼠注射500μL、5倍浓度的无菌组合物稀释液。
D组小鼠注射500μL、10倍浓度的无菌组合物稀释液。
E组小鼠注射500μL、浓度为25mg/mL的氟尿嘧啶注射液(阳性对照组)。
给药后处理：同实施例1。
表7组合物给药后各组移植瘤抑制率
Figure PCTCN2021138251-appb-000001
Figure PCTCN2021138251-appb-000002
从实验结果可以看出，使用氟尿嘧啶注射液组别的抑肿瘤活性最强，本对比例D组亦有明显的抑肿瘤活性，与阴性对照组相比差异具有统计学意义(P＜0.05)，抑制率与实施例1相仿。说明本例的四种活性组分的组合物形式仍有一定的抑肿瘤活性，但较实施例1的六种活性组分的协同增效作用弱(P＜0.05)。
由以上实施例可知，本发明提供了一种具有抑制肿瘤细胞活性作用的组合物，包括以下组分：天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物。上述组合物均取自天然药用植物，从天然植物中提取和分离出的抗肿瘤活性成分有数量众多、结构类型多样化、药效显著等特点，多元化的抗肿瘤活性、多靶点作用的特点不失为一种抗肿瘤药物研发思路和潜在策略。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种具有抑制肿瘤细胞活性作用的组合物，包括以下组分：天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物。
  2. 根据权利要求1所述的组合物，其特征在于，所述天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物的质量比为1:(0.9～1.1):(0.47～0.52):(0.18～0.23):(0.48～0.53):(0.45～0.55)。
  3. 根据权利要求1所述的组合物，其特征在于，所述天然辣椒素、薯蓣皂苷元、水鬼蕉提取物、铁筷子挥发油提取物、苞叶风毛菊提取物和单根木提取物的质量比为1:1:0.5:0.2:0.5:0.5。
  4. 根据权利要求1所述的组合物，其特征在于，所述天然辣椒素的存在形式为冻干粉；所述冻干粉由以下方法制得：
    将难溶性天然辣椒素和增溶组分A混合，溶于水中，随后冷冻干燥，得到冻干粉；
    所述增溶组分A包括PEG400、羟丙基倍他环糊精、羟乙基倍他环糊精、甲基倍他环糊精和磺丁基倍他环糊精钠中的一种或多种。
  5. 根据权利要求1所述的组合物，其特征在于，所述薯蓣皂苷元的存在形式为冻干粉；所述冻干粉由以下方法制得：
    将难溶性薯蓣皂苷元和增溶组分B混合，溶于水中，冷冻干燥，得到冻干粉。
  6. 根据权利要求1所述的组合物，其特征在于，所述水鬼蕉提取物按照以下方法制得：
    将水鬼蕉全草粉碎，采用95％乙醇作为提取溶剂，利用连续回流法提取，浓缩成浸膏，水分散后分别采用乙酸乙酯和正丁醇萃取，收集两相溶剂，旋干并混合，得水鬼蕉干膏提取物。
  7. 根据权利要求1所述的组合物，其特征在于，所述铁筷子挥发油提取物按照以下方法制得：
    取铁筷子植物的根茎粉碎，利用水蒸气提取法提取9.5～10.5h，得到铁筷 子挥发油提取物。
  8. 根据权利要求1所述的组合物，其特征在于，所述苞叶风毛菊提取物按照以下方法制得：
    取苞叶风毛菊全草粉碎，采用80％乙醇作为提取溶剂浸提，浓缩成浸膏，水分散后采用正丁醇萃取，旋干，得到苞叶风毛菊提取物。
  9. 根据权利要求1所述的组合物，其特征在于，所述单根木提取物优选按照以下方法制得：
    取单根木植物的根茎粉碎，采用95％乙醇作为提取溶剂，利用连续回流法提取，浓缩成浸膏，水分散后分别采用石油醚和乙酸乙酯萃取，收集两相溶剂，旋干并混合，得到单根木提取物。
  10. 一种权利要求1～9任一项所述组合物在制备抗肿瘤药物中的应用。
PCT/CN2021/138251 2021-09-18 2021-12-15 一种具有抑制肿瘤细胞活性作用的组合物及其应用 WO2023040094A1 (zh)

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