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WO2017016355A1 - 一种中药提取物及其制备方法与应用 - Google Patents

一种中药提取物及其制备方法与应用 Download PDF

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Publication number
WO2017016355A1
WO2017016355A1 PCT/CN2016/087100 CN2016087100W WO2017016355A1 WO 2017016355 A1 WO2017016355 A1 WO 2017016355A1 CN 2016087100 W CN2016087100 W CN 2016087100W WO 2017016355 A1 WO2017016355 A1 WO 2017016355A1
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WIPO (PCT)
Prior art keywords
chinese medicine
traditional chinese
medicine extract
propolis
extract
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Ceased
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PCT/CN2016/087100
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English (en)
French (fr)
Inventor
唐健
邓文娟
刘光荣
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Infinitus China Co Ltd
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Infinitus China Co Ltd
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Priority to US15/561,178 priority Critical patent/US10675308B2/en
Priority to EP16829721.6A priority patent/EP3275451B1/en
Priority to JP2017556177A priority patent/JP6554181B2/ja
Publication of WO2017016355A1 publication Critical patent/WO2017016355A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • 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
    • A61K36/282—Artemisia, e.g. wormwood or sagebrush
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • A61K8/988—Honey; Royal jelly, Propolis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • 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
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction

Definitions

  • the invention relates to the field of traditional Chinese medicine, in particular to a traditional Chinese medicine extract and a preparation method and application thereof.
  • Calcineurin also known as protein phosphatase 2B (PP2B)
  • P2B protein phosphatase 2B
  • CaN acts by dephosphorylating the substrate.
  • Ca 2+ influx intracellular concentration of Ca 2+ increases, Ca 2+ binds to CaM and CaN, activates CaN, and activated CaN dephosphorylates substrate NF-AT into nucleus, causing IL- 2 and other expression of cytokines.
  • the CaN-NF-AT pathway plays a regulatory role in T cell activation.
  • Calcineurin inhibitors are currently the most potent immunosuppressive drugs in the clinic. For organ transplantation, control of graft rejection, treatment of autoimmune diseases (RA, CD, psoriasis), especially in recent years for the treatment of skin allergies, skin inflammation (such as eczema), and good treatment effect.
  • Calcineurin inhibitors (CNIs) as immunosuppressive agents, are divided into exogenous inhibitors and endogenous protein inhibitors. Exogenous inhibitors mainly include cyclosporine and tacrolimus, and endogenous protein inhibitors mainly include Cain and FKBP38.
  • Traditional Chinese medicine is a traditional medicine in China.
  • traditional Chinese medicine has a good effect in the treatment of skin inflammation such as eczema and autoimmune diseases.
  • Modern research has found that certain Chinese medicine monomer components can be straight It binds to CaN and inhibits its activity, such as quercetin, kaempferol, phenolic gossypol, etc.
  • some active ingredients of traditional Chinese medicine can effectively inhibit the activation of CaN-NF-AT pathway at the cellular level and inhibit Th1 and Th2 cells.
  • the expression of factors inhibits the activation of T cells, such as cordycepin, rhododendron, and fisetin.
  • Traditional Chinese medicine has the characteristics of wide source, low cost and low toxicity. Therefore, it should make full use of the resources of traditional Chinese medicine, and achieve the effect of treating skin inflammation no less than the efficacy of western medicine through the reasonable compound compatibility of traditional Chinese medicine, and use it for soothing and anti-resistance. Allergic cosmetics.
  • the technical problem to be solved by the present invention is to provide a traditional Chinese medicine extract and a preparation method and application thereof.
  • the traditional Chinese medicine extract provided by the invention can effectively block the CaN-NF-AT pathway, thereby functioning to treat skin inflammation or autoimmune diseases such as eczema.
  • the traditional Chinese medicine composition provided by the invention has a wide range of raw materials, simple preparation methods, and is suitable for large-scale production.
  • the traditional Chinese medicine extract provided by the invention is prepared from artemisia annua and propolis.
  • the mass ratio of Artemisia annua L. and propolis is from 3:5 to 5:3.
  • the mass ratio of Artemisia annua and propolis is 3:5.
  • the mass ratio of Artemisia annua and Propolis is 5:3.
  • Artemisia annua L. is the dry aerial part of the Artemisia annua L.
  • Artemisia annua contains various active substances such as artemisinin series compounds and flavonoids, which have the functions of clearing away heat and removing heat, removing steam and removing malaria.
  • artemisinin series compounds and flavonoids which have the functions of clearing away heat and removing heat, removing steam and removing malaria.
  • Propolis is a yellow-brown or dark-brown viscous substance secreted by the bee family to repair the hive, which can be used as a medicine. Its nature is flat, bitter, spicy, and slightly sweet. Recent studies have shown that propolis is rich in flavonoids, amino acids, vitamins and organic acids. It has the effect of moisturizing the skin, reducing inflammation and relieving pain, and can treat stomach ulcers, mouth ulcers, burns, skin cracks and radiation.
  • the present invention combines propolis and artemisia annua, and the extract obtained by the extraction can have a good effect of inhibiting the CaN-NF-AT pathway, and the effect thereof is remarkably superior to the effect of using artemisia annua or propolis alone. Moreover, experiments have shown that the extracts provided by the present invention have relatively low relative toxicity to cells.
  • the preparation method of the traditional Chinese medicine extract provided by the invention is: extracting the artemisia annua and propolis.
  • the extracted extract is a mixture of one or more of an alcohol, an alkane, chloroform, acetone, water or ethyl acetate.
  • the alcohol is a mixture of one or more of ethanol, methanol, glycerol, butylene glycol, propylene glycol, pentanediol, or hexanediol.
  • the alkane is methane, ethane or petroleum ether.
  • the extract is an aqueous ethanol solution.
  • the extract is an aqueous ethanol solution having an ethanol volume fraction of 95%.
  • the extraction method is a percolation method, a dipping method, a boiling method, a reflux extraction method, a continuous extraction method, a supercritical extraction method, or an ultrasonic extraction.
  • the extracted temperature is normal temperature, high temperature or intelligent temperature control;
  • the extraction pressure is vacuum, high pressure or normal pressure.
  • the method for preparing a traditional Chinese medicine extract comprises: extracting the artemisia annua and propolis with an aqueous solution of ethanol as an extracting agent by a percolation method, and concentrating the filtrate.
  • the volume fraction of ethanol in the aqueous ethanol solution is 95% extracted by percolation.
  • the mass of the aqueous ethanol solution is 20 to 30 times the sum of the masses of Artemisia annua and propolis.
  • the mass of the aqueous ethanol solution is 20, 25 or 30 times the sum of the mass of the artemisia annua and the propolis.
  • the time for extraction by percolation is from 40 h to 60 h.
  • the time of the percolation extraction is 48 h.
  • extraction is by percolation and concentration is concentrated by low temperature and reduced pressure.
  • it is extracted by percolation and concentrated to a level equal to the sum of the masses of Artemisia annua and propolis.
  • the preparation method of the traditional Chinese medicine extract provided by the present invention comprises: extracting the artemisia annua and propolis with methanol as an extracting agent by dip-drying method, and concentrating the filtrate.
  • the mass of methanol is 30 times the sum of the mass of Artemisia annua and propolis.
  • the dip extraction time is 48 h.
  • it is extracted by percolation and concentrated to a level equal to the sum of the masses of Artemisia annua and propolis.
  • the method for preparing a traditional Chinese medicine extract comprises: continuously extracting the artemisia annua and propolis with chloroform as an extractant, and concentrating the filtrate after concentration.
  • the extract is continuously refluxed and the mass of chloroform is 10 times the sum of the mass of Artemisia annua and propolis.
  • the continuous reflux extraction method takes 24 hours to extract.
  • the extraction is carried out with continuous reflux, and the concentration is concentrated until the mass of the filtrate is equal to the sum of the masses of Artemisia annua and propolis.
  • the method for preparing a traditional Chinese medicine extract comprises: refluxing the extract of Artemisia annua and propolis with ethyl acetate as an extracting agent, and concentrating the filtrate.
  • the extract is refluxed and the mass of ethyl acetate is 10 times the sum of the mass of Artemisia annua and propolis.
  • the reflux extraction method takes 36 hours to extract.
  • the extract is refluxed and concentrated until the mass of the filtrate is equal to the sum of the masses of Artemisia annua and propolis.
  • the method for preparing a traditional Chinese medicine extract comprises: extracting an extract of Artemisia annua and Propolis with a solution of butanediol or acetone as a solvent to obtain an extract.
  • the quality of the solvent is equal to the sum of the masses of Artemisia annua and propolis by ultrasonic extraction.
  • the ultrasonic extraction method extracts a temperature of 18 ° C to 50 ° C for a period of 1 h to 3 h.
  • the extract is sonicated to a mass equal to the sum of the mass of Artemisia annua and propolis by ultrasonic extraction.
  • the method for preparing a traditional Chinese medicine extract comprises: extracting the artemisia annua and propolis with petroleum ether as an extracting agent, and then concentrating the filtrate.
  • the quality of petroleum ether is 30 times the sum of the mass of Artemisia annua and propolis.
  • the extraction time by the impregnation extraction method is 48 h.
  • it is extracted by impregnation and concentrated until the mass of the filtrate is equal to the sum of the masses of Artemisia annua and propolis.
  • the method for preparing a traditional Chinese medicine extract provided by the present invention comprises: decocting the artemisia annua and propolis in water, and concentrating the filtrate.
  • the quality of the water used for decoction is 30 times the sum of the mass of Artemisia annua and propolis.
  • the boiling time is 48 h and the temperature is between 98 ° C and 105 ° C.
  • the concentration concentrated to the filtrate is equal to the sum of the mass of Artemisia annua and propolis.
  • the method for preparing a traditional Chinese medicine extract comprises: supercritical extraction of artemisia annua and propolis with ethanol as an entraining agent, and concentrating the filtrate.
  • the entrainer has a mass that is 30 times the sum of the mass of Artemisia annua and propolis.
  • the extraction pressure is 33 to 35 MPa
  • the extraction temperature is 50 ° C
  • the time is 48 hours
  • the CO 2 flow rate is 15 L/h to 25 L/h.
  • the filtrate is concentrated until the mass of the extract is equal to the sum of the mass of Artemisia annua and propolis.
  • the traditional Chinese medicine extract is prepared by the preparation method provided by the present invention.
  • the method is simple, easy to operate, does not require complicated instruments, and is suitable for mass production.
  • K562 cells are induced with PMA and A23187, and the NF-AT gene is activated as an experimental subject.
  • the traditional Chinese medicine extract provided by the invention is used as a test sample, and the results show that the traditional Chinese medicine extract provided by the invention can effectively inhibit the activation of the CaN-NF-AT pathway, and the inhibition rate can reach 69.43%, which is significantly better than using propolis alone or Artemisia annua. Since the traditional Chinese medicine composition provided by the present invention can significantly inhibit the CaN-NF-AT pathway, the extract can treat diseases targeting the CaN-NF-AT pathway.
  • the skin inflammation is eczema.
  • the autoimmune disease is RA, CD or psoriasis.
  • the invention provides a medicament for treating skin inflammation or autoimmune diseases, including the present The traditional Chinese medicine extract provided by the invention.
  • the dosage form of the medicament for treating skin inflammation or autoimmune diseases provided by the present invention is a cream.
  • the present invention also provides a soothing or anti-allergic cosmetic, comprising the traditional Chinese medicine extract provided by the present invention.
  • the invention provides an extract prepared from artemisia annua and propolis as a raw material, and the extract can have a good effect of inhibiting the CaN-NF-AT pathway, and the effect is remarkably better than the effect of using artemisia annua or propolis alone. Moreover, experiments have shown that the extracts provided by the present invention have relatively low relative toxicity to cells.
  • the invention provides a preparation method of the extract which is simple, easy to operate, does not require complicated instruments, and is suitable for mass production.
  • the invention provides a traditional Chinese medicine extract and a preparation method and application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application of the present invention may be modified or combined and modified to achieve and apply the present invention without departing from the scope of the present invention. Invention technology.
  • the instruments and reagents used in the present invention are all commercially available products, and are commercially available.
  • Samples to be tested All extracts were dissolved in 70% ethanol, diluted to 0.2 mg/ml working solution with deionized water, and 10 ⁇ l was added to the measuring tube during the test, and the final concentration of the extract to be tested was 0.2 mg/ml.
  • CaN enzyme activity assay Enzo life Scienceg's CaN activity assay kit was used according to the kit instructions. Take a small centrifuge tube, add 25 ⁇ l CaM working solution, 5 ⁇ L CaN working solution, 10 ⁇ l 50 ⁇ M test compound working solution, add 10 ⁇ l 0.5% DMSO aqueous solution to the control tube, mix, incubate at 30°C for 10 minutes, add 10 ⁇ L substrate to each tube. The solution was mixed and incubated at 30 ° C for 50-60 minutes. 100 ⁇ l of the color developer was added, mixed, and allowed to stand for 20-30 minutes. 135 ⁇ l per well was transferred to a 96-well plate, and the absorbance was measured at 620 nm.
  • K562 cells were stimulated with PMA (protein kinase CPKC activator) and A23187 (calcium channel activator) using the NFAT K562 Reporter Stable Cell Line, which was stably transfected with NFAT reporter gene from American affimatrix, and the transcription factor NFAT was detected.
  • the level of luciferase gene expression is driven by the level of activation of the calcineurin (CaN) pathway.
  • CaN calcineurin
  • PMA and A23187 can stimulate the expression of the reporter gene in a dose-dependent manner, and the difference in fluorescence value between the re-pore can be controlled within a reasonable range.
  • the above experiment basically established a suitable dose for determining the calcineurin pathway activator: PMA 10 ng/ml, A23187 0.5 ⁇ .
  • K562 cells stably transfected with the NFAT reporter gene were routinely subcultured in RPMI-1640 medium containing 10% fetal bovine serum.
  • the cells were grown in a healthy, logarithmic growth phase, and the cells were 2 ⁇ 10 5 /well.
  • the cells were inoculated into a 24-well cell culture plate, and the empty cells without the extract were left as a control group, and the wells without PMA and A23187 were left as a blank group.
  • the other wells were added to the extracts of Examples 1 to 3 and Comparative Examples 1 to 5, respectively, and incubated for 1 hour.
  • PMA (10 ng/ml) and A23187 (0.5 ⁇ M) (pathway stimulating agent) were added, and the cells were harvested for 18 hours.
  • the enzyme assay kit was operated according to the instructions, and the fluorescence intensity was measured at a value of 1 ⁇ m (reflecting the activation level of the intracellular response calcineurin pathway).
  • inhibition rate (%) W [1-(X-A) / (B-A)] ⁇ 100.
  • A is a blank l ⁇ m average value
  • B is a control group 1 ⁇ m average value
  • X is an average value of the extract 1 ⁇ m.
  • the present invention has a better inhibition of the CaN-NF-AT pathway and is capable of treating diseases targeting the CaN-NF-AT pathway, such as dermatitis (eczema), autoimmune diseases (RA, CD or psoriasis).
  • diseases targeting the CaN-NF-AT pathway such as dermatitis (eczema), autoimmune diseases (RA, CD or psoriasis).
  • the inhibitory rate of the extract prepared by the other embodiments of the present invention on the CaN-NF-AT pathway of K562 cells was similar to that of the extracts obtained in Examples 1 to 3, and all of them were significantly different from those of Comparative Examples 1 to 4.
  • the present invention adopts a pure natural Chinese medicine extract, overcomes the hidden troubles of the side effects of western medicine and hormonal medicine, and provides a material basis for anti-eczema and soothing cosmetics.

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Abstract

一种中药提取物及其制备方法与应用。该提取物的原料为青蒿和蜂胶，该提取物具有良好的抑制CaN-NF-AT通路的作用，对细胞的相对毒性较低。

Description

一种中药提取物及其制备方法与应用
本申请要求于2015年07月30日提交中国专利局、申请号为201510466544.1、发明名称为“一种中药提取物及其制备方法与应用”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
本发明涉及中药领域，尤其涉及一种中药提取物及其制备方法与应用。
背景技术
钙调磷酸酶(caleineurin，CaN)又称蛋白磷酸酶2B(PP2B)，属丝/苏氨酸蛋白磷酸酶家族成员，是迄今发现的唯一受Ca2+/钙调素(CaM)调节的丝/苏氨酸蛋白磷酸酶。CaN通过使底物脱磷酸发挥作用。在T细胞中，Ca2+内流、细胞内浓度Ca2+增加，Ca2+同CaM与CaN结合，激活CaN，活化的CaN使其底物NF-AT脱磷酸，入核，引起IL-2等一些列细胞因子的表达。CaN-NF-AT通路在T细胞活化中起着调节枢纽的作用。
钙调神经磷酸酶抑制剂目前是临床上最有效的免疫抑制剂药物。用于器官移植，控制移植物排斥反应、自身免疫性疾病的治疗(RA，CD，银屑病)、特别是其近年来用于皮肤过敏、皮肤炎症的治疗(如湿疹)，并取得良好治疗效果。钙调磷酸酶抑制剂(CNIs)作为一种免疫抑制剂，分为外源性抑制剂和内源性蛋白质抑制剂。外源性抑制剂主要有环孢素、他克莫司等，内源性蛋白质抑制剂主要有Cain、FKBP38等。目前，临床应用最多的是子囊霉素衍生物环孢素A、他克莫司和匹美克莫司，都具有相似的理化性质、作用机制和作用效果，但其毒副作用(如肾毒性，高血糖等)成为该类抑制剂应用的重要障碍。因此，以CaN-NF-AT通路为作用靶标寻找更加安全有效的抑制剂对研发新型皮肤炎症药物具有重要意义。
中药为我国的传统用药，在临床上，中药在湿疹等皮肤炎症和自身免疫性疾病的治疗中具有较好的效果。现代研究发现某些中药单体成分能直 接与CaN结合并抑制其活性，如槲皮素、山奈酚、酚醛棉酚等；同时，某些中药有效成分能在细胞水平有效抑制CaN-NF-AT通路的活化，抑制Th1和Th2型细胞因子的表达，抑制T细胞的活化，如虫草素、杜鹃素、非瑟酮等。中药具有来源广、成本低和毒性低的特点，因此，应当充分利用中药资源，通过中药的合理的复方配伍，达到不亚于西药疗效的治疗皮肤炎症的效果，并将其用于舒缓及抗过敏类化妆品中。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种中药提取物及其制备方法与应用。本发明提供的中药提取物能够有效阻断CaN-NF-AT通路，从而起到治疗湿疹等皮肤炎症或自身免疫性疾病的作用。并且，本发明提供的中药组合物的原料来源广泛，制备方法简单，适合大规模生产。
本发明提供的中药提取物由青蒿和蜂胶制得。
在本发明的实施例中，青蒿和蜂胶的质量比为3:5～5:3。
在一些实施例中，青蒿和蜂胶的质量比为3:5。
在一些实施例中，青蒿和蜂胶的质量比为5:3。
青蒿为菊科植物黄蒿Artemisia annua L.的干燥地上部分。青蒿含有青蒿素系列化合物、黄酮类等多种活性物质，具有清热解暑，除蒸，截疟的作用。用于暑邪发热，阴虚发热，夜热早凉，骨蒸劳热，疟疾寒热，湿热黄疸。
蜂胶是蜜蜂科动物修补蜂巢所分泌的黄褐色或黑褐色的粘性物质，可入药。其性平，味苦、辛、微甘。近年来研究表明，蜂胶中含有丰富的黄酮类、氨基酸、维生素和有机酸。有润肤生肌，消炎止痛的功效，可治疗胃溃疡、口腔溃疡、烧烫伤、皮肤裂痛，防辐射。
本发明将蜂胶和青蒿复配，经提取所得提取物能够具有良好的抑制CaN-NF-AT通路的作用，其效果显著优于单独使用青蒿或蜂胶的效果。并且，实验表明，本发明提供的提取物对细胞的相对毒性较低。
本发明提供的中药提取物的制备方法为：将青蒿和蜂胶经提取制得。
在本发明的实施例中，提取的提取液为醇类、烷烃、氯仿、丙酮、水或乙酸乙酯中的一种或两者以上的混合物。
在一些实施例中，醇类为乙醇、甲醇、甘油、丁二醇、丙二醇、戊二醇或己二醇中一种或两者以上的混合物。
在一些实施例中，烷烃为甲烷、乙烷或石油醚。
在一些实施例中，提取液为乙醇水溶液。
在一些实施例中，提取液为乙醇体积分数为95％的乙醇水溶液。
在本发明的实施例中，提取的方法为渗漉法、浸渍法、煎煮法、回流提取法、连续提取法、超临界萃取法或超声波提取。
在本发明的实施例中，提取的温度为常温、高温或智能温控；提取压力为真空、高压或常压。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以乙醇水溶液为提取剂经渗漉法提取后浓缩滤液。
在一些实施例中，以渗漉法提取，乙醇水溶液中乙醇的体积分数为95％。
在一些实施例中，以渗漉法提取，乙醇水溶液的质量为青蒿和蜂胶质量之和的20倍～30倍。
在一些实施例中，以渗漉法提取，乙醇水溶液的质量为青蒿和蜂胶质量之和的20倍、25倍或30倍。
在一些实施例中，渗漉法提取的时间为40h～60h。
在一些实施例中，渗漉法提取的时间为48h。
在一些实施例中，以渗漉法提取，浓缩采用低温减压浓缩。
在一些实施例中，以渗漉法提取，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以甲醇为提取剂经浸漉法提取后浓缩滤液。
在一些实施例中，以浸漉法提取，甲醇的质量为青蒿和蜂胶质量之和的30倍。
在一些实施例中，浸漉法提取的时间为48h。
在一些实施例中，以渗漉法提取，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以氯仿为提取剂连续回流提取后浓缩滤液。
在一些实施例中，以连续回流提取，氯仿的质量为青蒿和蜂胶质量之和的10倍。
在一些实施例中，连续回流提取法提取的时间为24h。
在一些实施例中，以连续回流提取，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以乙酸乙酯为提取剂回流提取后浓缩滤液。
在一些实施例中，以回流提取，乙酸乙酯的质量为青蒿和蜂胶质量之和的10倍。
在一些实施例中，回流提取法提取的时间为36h。
在一些实施例中，以回流提取，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以丁二醇或丙酮为溶剂经超声波提取后获得提取液。
在一些实施例中，以超声波提取，溶剂的质量与青蒿和蜂胶质量之和相等。
在一些实施例中，超声波提取法提取的温度为18℃～50℃，时间为1h～3h。
在一些实施例中，以超声波提取，提取液以溶剂定容至质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以石油醚为提取剂浸渍法提取后浓缩滤液。
在一些实施例中，以浸渍法提取，石油醚的质量为青蒿和蜂胶质量之和的30倍。
在一些实施例中，浸渍法提取法提取的时间为48h。
在一些实施例中，以浸渍法提取，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以水煎煮后浓缩滤液。
在一些实施例中，煎煮所用水的质量为青蒿和蜂胶质量之和的30倍。
在一些实施例中，煎煮的时间为48h，温度为98℃～105℃。
在一些实施例中，浓缩至滤液的质量与青蒿和蜂胶质量之和相等。
在本发明的实施例中，本发明提供的中药提取物的制备方法包括：将青蒿和蜂胶以乙醇为夹带剂超临界萃取后浓缩滤液。
在一些实施例中，夹带剂的质量为青蒿和蜂胶质量之和的30倍。
在一些实施例中，萃取的压力为33～35Mpa、萃取的温度为50℃，时间为48h，CO2流量为15L/h～25L/h。
在一些实施例中，滤液浓缩至提取液的质量与青蒿和蜂胶质量之和相等。
以本发明提供的制备方法制备中药提取物。方法简单、容易操作，不需要复杂的仪器，适用于大规模生产。
本发明提供的中药提取物在制备CaN-NF-AT通路阻断剂中的应用。
本发明以PMA和A23187对K562细胞进行诱导，激活其中NF-AT基因作为实验对象。以本发明提供的中药提取物作为受试样品，结果表明，本发明提供的中药提取物能够有效抑制CaN-NF-AT通路的激活，抑制率可达69.43％，显著优于单独使用蜂胶或青蒿。由于本发明提供的中药组合物能够显著抑制CaN-NF-AT通路，因此，该提取物能够治疗以CaN-NF-AT通路为靶标的疾病。
本发明提供的中药提取物在制备治疗皮肤炎症或自身免疫性疾病的药物中的应用。
作为优选，皮肤炎症为湿疹。
作为优选，自身免疫性疾病为RA、CD或银屑病。
本发明提供了一种治疗皮肤炎症或自身免疫性疾病的药物，包括本 发明提供的中药提取物。
本发明提供的治疗皮肤炎症或自身免疫性疾病的药物的剂型为乳膏。
本发明提供的中药提取物在制备舒缓或抗过敏的化妆品中的应用。
本发明还提供了一种舒缓或抗过敏的化妆品，包括本发明提供的中药提取物。
本发明提供了以青蒿和蜂胶为原料制得的提取物，该提取物能够具有良好的抑制CaN-NF-AT通路的作用，效果显著优于单独使用青蒿或蜂胶的效果。并且，实验表明，本发明提供的提取物对细胞的相对毒性较低。本发明提供提取物的制备方法简单，容易操作，不需要复杂的仪器，适用于大规模生产。
具体实施方式
本发明提供了一种中药提取物及其制备方法与应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的仪器、试剂皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：
将30g青蒿和50g蜂胶加入20倍质量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
实施例2：
将30g青蒿和50g蜂胶加入25倍质量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
实施例3：
将50g青蒿和30g蜂胶加入30倍质量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
实施例4
将50g青蒿和30g蜂胶的加入30倍质量的甲醇，浸漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
实施例5
将40g青蒿和40g蜂胶，常温下以10倍质量的氯仿连续回流提取(索氏提取)24h，收集滤液；低温减压浓缩至80g即得提取物。
实施例6
将35g青蒿和45g蜂胶，常温下以10倍量的乙酸乙酯回流提取36h，收集滤液；低温减压浓缩至80g即得提取物。
实施例7
将50g青蒿和30g蜂胶的加入1倍质量的丁二醇，常温下超声波提取2h，收集滤液，丁二醇定容至80g即得提取物。
实施例8
将50g青蒿和30g蜂胶的加入1倍质量的丙二酮，50℃超声波提取2h，收集滤液，丙二酮定容至80g即得提取物。
实施例9
将50g青蒿和30g蜂胶的加入30倍量的石油醚，常温下浸渍法提取48h，收集滤液，减压低温浓缩至80g即得提取物。
实施例10
将45g青蒿和35g蜂胶的加入30倍量的水，煎煮48h，收集滤液，减压低温浓缩至80g即得提取物。
实施例11
将30g青蒿和50g蜂胶进行超临界萃取，萃取压力33～35MPa，萃取温度50℃，萃取时间3.5h，CO2流量15～25L/h，乙醇夹带剂中乙醇的体积分数为15％，收集萃取液；低温减压浓缩至80g即得提取物。
对比例1：
将80g青蒿加入25倍量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
对比例2：
将80g蜂胶加入25倍量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
对比例3：
将60g青蒿和20g蜂胶的加入25倍量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
对比例4：
将20g青蒿和60g蜂胶的加入25倍量的95％乙醇，渗漉法提取48h，收集滤液；低温减压浓缩至80g即得提取物。
实施例12：
待测样品：所有提取物用70％的乙醇溶解，用去离子水稀释成0.2mg/ml的工作液，检测时取10μl加入测定管，待测提取物的终浓度为0.2mg/ml。
CaN酶活性检测：采用Enzo life Scienceg公司的CaN活性检测试剂盒，按试剂盒的说明书进行。取小离心管，加入25μl CaM工作液，5μL CaN工作液，10μl 50μM待测化合物工作液，对照管加入10μl 0.5％DMSO水溶液，混匀，30℃孵育10分钟，每管再加入10μL底物工作液，混匀，30℃孵育50-60分钟，加入100μl显色剂，混匀，静止20-30分钟，每孔取135μl转移到96孔板，620nm处测定吸光度。
刺激剂PMA和A23187最适浓度的确定：为了确定刺激CaN-NF-AT通路活化所需要的刺激剂PMA和A23187最适浓度，采用PMA浓度2.5ng/ml，5ng/ml，10ng/ml，20ng/ml，40ng/ml；A23187浓度0.125μΜ，0.25μΜ，0.5μΜ，1μΜ，2μΜ，每个浓度系列设4复孔，刺激18h后，检测各实验组荧光强度lμm值。
测定方法：采用从美国affimatrix公司购买了稳定转染NFAT报告基因的细胞系NFAT K562Reporter Stable Cell Line，以PMA(蛋白激酶CPKC激活剂)和A23187(钙通道激活剂)刺激K562细胞，检测转录因子NFAT驱动的荧光素酶基因表达水平，反应钙调磷酸酶(CaN)通路的活化水平。结果如表1：
表1 不同剂量的PMA和A23187对K562细胞NF-AT报告基因表达的影响
Figure PCTCN2016087100-appb-000001
由表1可知，PMA和A23187能够剂量依赖性地刺激报告基因的表达，复孔间的荧光度值差别可以控制在合理范围。上述实验基本建立了确定了钙调磷酸酶通路激活剂的合适剂量为PMA 10ng/ml,A23187 0.5μΜ。
稳定转染NFAT报告基因的K562细胞常规传代培养于含10％胎牛血清的RPMI-1640培养基中，实验采用生长健康、处于对数增长期的细胞，将上述细胞按2×105/孔接种于24孔细胞培养板，保留不加提取物的空为 对照组，保留不添加PMA和A23187的孔为空白组。其余各孔分别加入实施例1～3及对比例1～5提取物，孵育1小时，加入PMA(10ng/ml)和A23187(0.5μΜ)(通路刺激剂)，刺激18h，收集细胞，采用荧光素酶检测试剂盒按说明书操作，测定荧光强度lμm值(反映细胞内反应钙调磷酸酶通路活化水平)。
抑制率计算公式为：抑制率(％)W＝【1-(X-A)/(B-A)】×100。
其中，A为空白lμm平均值，B为对照组lμm平均值，X为提取物lμm平均值。结果见表2：
表2 各样品对K562细胞CaN-NF-AT通路的抑制率
  抑制率(％)
实施例1 66.58
实施例2 68.97
实施例3 69.43
对比例1 36.69
对比例2 42.37
对比例3 39.21
对比例4 45.46
由表2可知，青蒿和蜂胶总黄酮都具有较好的抑制CaN-NF-AT通路的作用。采用SPSS 19.0版统计软件进行分析：
实施例均值±标准差：68.32％±1.53％；
对比例均值±标准差：40.93％±3.81％。
通过单因素方差分析，实施例与对比例间具有显著的差别，具有统计学意义(P＜0.01)。
统计分析结果说明，将青蒿和蜂胶进行配伍后，效果显著优于单剂。因此，本发明组方具有较好的CaN-NF-AT通路的抑制作用，能够治疗以CaN-NF-AT通路为靶标的疾病，例如：皮炎(湿疹)、自身免疫性疾病(RA、CD或银屑病)。
本发明其他实施例制得的提取物对K562细胞CaN-NF-AT通路的抑制率与实施例1～3制得的提取物相似，皆与对比例1～4有显著性差异。
实施例13：
用常规的CCK8法检测实施例1～3和对比例1～5的提取物对细胞的毒性，方法为：
(1)在96孔板中配置100μL的K562细胞悬液(1×105/孔)，将96孔板在培养箱预培养24小时(37℃，5％CO2)；
(2)分别加入10μL实施例1～3和对比例1～5的提取物，留取6个孔作为空白对照；
(3)将96孔板在培养箱中孵育24小时；
(4)向每孔加入10μLCCK8溶液；
(5)将96孔板在培养箱内孵育4小时；
(6)用酶标仪测定在450nm处的吸光度。
细胞毒性的计算公式为：相对毒性(％)W＝【(A-X)/A】×100
其中：A为空白OD450平均值，X为提取物OD450平均值。结果如表3：
表3：本发明提取物对细胞的相对毒性
  相对毒性(％)
实施例1 2.9
实施例2 3.1
实施例3 3.3
对比例1 3.0
对比例2 3.2
对比例3 3.1
对比例4 3.2
结果显示，各待测样品对细胞的毒性之间不具有统计学差异。相对毒性都较小。可见，由于本发明采用的是纯天然的中药提取物，克服了西药、激素药较大副作用的隐患，为抗湿疹及舒缓类化妆品提供了物质基础。
本发明其他实施例制得的提取物对对细胞的相对毒性与实施例1～3或对比例1～4制得的提取物相似。
以上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. 一种中药提取物，其特征在于，由青蒿和蜂胶制得。
  2. 根据权利要求1所述的中药提取物，其特征在于，所述青蒿和蜂胶的质量比为3:5～5:3。
  3. 权利要求1或2所述的中药提取物的制备方法，其特征在于，包括：将青蒿和蜂胶经提取制得。
  4. 根据权利要求3所述的制备方法，其特征在于，所述提取的提取液为醇类、烷烃、氯仿、丙酮、水或乙酸乙酯中的一种或两者以上的混合物。
  5. 根据权利要求3所述的制备方法，其特征在于，所述提取的方法为渗漉法、浸渍法、煎煮法、回流提取法、连续回流提取法、超临界萃取法、超声波提取。
  6. 权利要求1或2所述的中药提取物或权利要求3～5任一项所述制备方法制备的中药提取物在制备CaN-NF-AT通路阻断剂中的应用。
  7. 权利要求1或2所述的中药提取物或权利要求3～5任一项所述制备方法制备的中药提取物在制备治疗皮肤炎症或自身免疫性疾病的药物中的应用。
  8. 一种治疗皮肤炎症或自身免疫性疾病的药物，其特征在于，包括权利要求1或2所述的中药提取物或权利要求3～5任一项所述制备方法制备的中药提取物。
  9. 权利要求1或2所述的中药提取物或权利要求3～5任一项所述制备方法制备的中药提取物在制备舒缓或抗过敏的化妆品中的应用。
  10. 一种舒缓或抗过敏的化妆品，其特征在于，包括权利要求1或2所述的中药提取物或权利要求3～5任一项所述制备方法制备的中药提取物。
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