WO2014075563A1 - 一种具有抗菌作用的中药复方总黄酮及其制备方法 - Google Patents

一种具有抗菌作用的中药复方总黄酮及其制备方法 Download PDF

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WO2014075563A1
WO2014075563A1 PCT/CN2013/086390 CN2013086390W WO2014075563A1 WO 2014075563 A1 WO2014075563 A1 WO 2014075563A1 CN 2013086390 W CN2013086390 W CN 2013086390W WO 2014075563 A1 WO2014075563 A1 WO 2014075563A1
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chinese medicine
traditional chinese
medicine compound
total flavonoids
extract
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PCT/CN2013/086390
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English (en)
French (fr)
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赵亚
孙懿
卢玲巧
陈园园
祝乐
董亚丽
杨俊�
魏少敏
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上海家化联合股份有限公司
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Publication of WO2014075563A1 publication Critical patent/WO2014075563A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones

Definitions

  • the invention relates to the field of natural medicinal chemistry, household products, disinfecting articles and cosmetics, and particularly relates to a traditional Chinese medicine compound total flavonol for preventing Staphylococcus aureus, Escherichia coli and Candida albicans and a preparation method thereof.
  • Background technique
  • the invention provides a new method for extracting total flavonoids from traditional Chinese medicine compound, and the total flavonoids extracted from the traditional Chinese medicine compound according to the method can inhibit and kill various pathogenic bacteria.
  • the traditional Chinese medicine compound total flavonoid is an extract of a natural plant and is therefore harmless to the human body.
  • the traditional Chinese medicine compound total flavonoid extract prepared by the method of the invention can also be added as a functional additive to household products, disinfecting articles or cosmetics. Summary of the invention
  • the present invention provides a total flavonoid of traditional Chinese medicine having anti-S. aureus, Escherichia coli and Candida albicans.
  • the compound total flavonoid extract can also be used as an antibacterial ingredient in household products, disinfecting articles, and cosmetics.
  • the invention provides a method for extracting total flavonoids from traditional Chinese medicine, the method comprising the following steps: a) taking the following proportion of Chinese medicinal materials and extracting with polar solvent to obtain crude extract, raw oyster shell 20-60 weight Share
  • the polar solvent in step a) above is selected from the group consisting of: methanol, ethanol, isopropanol, acetone, ethyl acetate or polyol.
  • the extraction method of step a) above may be reflux extraction, impregnation or percolation.
  • the macroporous resin in step b) above is a non-polar to moderately polar adsorbent resin.
  • the macroporous resin may be a macroporous resin of the type D 101, DM130 or DM301.
  • the invention also relates to a traditional Chinese medicine compound flavonoid prepared according to the method of the invention.
  • the invention also relates to the use of the total flavonoids of the traditional Chinese medicine compound against Staphylococcus aureus, Escherichia coli and Candida albicans.
  • the traditional Chinese medicine compound total flavonoid is an extract prepared according to the extraction method of the present invention.
  • the invention also relates to the use of the total flavonoids of the traditional Chinese medicine compound as an additive with the action against Staphylococcus aureus, Escherichia coli and Candida albicans in household products, disinfecting articles or cosmetics, wherein the total flavonoids of the traditional Chinese medicine compound are prepared by the extraction method of the invention Extract.
  • the invention further relates to a household product, a disinfecting article or a cosmetic comprising a total flavonoid of traditional Chinese medicine prepared according to the extraction method of the invention and an acceptable adjuvant.
  • the extraction solvent may be a medium or high polarity organic solvent, such as different concentrations of ethanol, different concentrations of methanol, acetone, etc.; the extraction method may be reflux extraction, impregnation, percolation, and the like.
  • the macroporous resin may be a non-polar to medium-polar adsorption resin such as a D101 type, a DM130 type, a DM301 type macroporous resin, or the like. In the above-mentioned traditional Chinese medicine compound, the raw oyster shell is wide and chest, and the stagnation and elimination are accumulated.
  • the total flavonoids can be extracted by using the Chinese medicinal materials of the raw oyster shell, the green skin, the Bupleurum, the Magnolia officinalis and the Astragalus membranaceus according to the present invention, and the compound obtained by the extraction according to the ratio is not mentioned.
  • the extract has a bactericidal or bacteriostatic effect.
  • the present invention first proposes a method for extracting compound total flavonoids by a two-step method using macroporous resin.
  • the total flavonoids extracted from the traditional Chinese medicine compound proved to have a broad-spectrum bactericidal effect, and can significantly kill Staphylococcus aureus, Escherichia coli and Candida albicans, and the effect is significantly better than the conventional method extract.
  • the total flavonoid content was also determined to show that the total flavonoid content in the extract prepared according to the extraction method of the present invention was greatly enriched by the mass ratio of the Chinese medicinal material according to the present invention.
  • the invention provides a two-step method for extracting total flavonoids from traditional Chinese medicine compound.
  • the method firstly extracts the crude extract of the traditional Chinese medicine compound by the solvent extraction method, and the second step utilizes the adsorption of the macroporous resin to obtain the total flavonoids of the traditional Chinese medicine compound of the invention.
  • the present invention finds for the first time that the traditional Chinese flavonoids extracted according to the above two-step method have an antibacterial effect. Therefore, the total flavonoids of the traditional Chinese medicine compound can also be added as an efficacy additive to household products, disinfecting articles or cosmetics.
  • the literature does not involve the antibacterial effect of the total flavonoids of the traditional Chinese medicine compound, nor does it mention it as a functional additive in household products, disinfecting products or cosmetics. Therefore, the present invention is the first to propose that the total flavonoids of the traditional Chinese medicine compound have an antibacterial effect and can be added as a functional additive to household products, disinfecting articles or cosmetics.
  • the total flavonoids of the traditional Chinese medicine compound can be used at a concentration of 5 mg/ml or more. High, 6 mg/ml or higher, 7 mg/ml or higher, 8 mg/ml or higher, 9 mg/ml or higher, 10 mg/ml or higher, 12 mg/ml or higher, 16 mg/ml or higher, 18 mg/ml or higher.
  • the total flavonoids of the traditional Chinese medicine compound may be used at a concentration of 5 to 150 mg/ml, 6 to 150 mg/ml, ⁇ -150 mg/ml, 8-150 mg/ml, 9-150 mg/ml, 10-150 mg/ml, 12-150 mg/ml, 16-150 mg/ml, 18-150 mg/ml.
  • the total flavonoids of the traditional Chinese medicine compound may be used at a concentration of 5-100 mg/ml, 6-100 mg/ml, ⁇ -100 mg/ml, 8-100 mg/ml, 9-100 mg/ml, 10-100 mg/ml, 12-100 mg/ml, 16-100 mg/ml, 18-100 mg/ml.
  • the total flavonoids of the traditional Chinese medicine compound can be used at a concentration of 5 - 75 mg / ml, 6 - 75 mg / ml, 7 - 75 mg / ml, 8- 75 mg / ml, 9 - 75 mg / ml, 10-75 mg/ml, 12-75 mg/ml, 16-75 mg/ml, 18-75 mg/ml.
  • the total flavonoids of the traditional Chinese medicine compound can be used at a concentration of 5 to 50 mg/ml, 6 to 50 mg/ml, ⁇ -50 mg/ml, 8-50 mg/ml, 9 to 50 mg/ml, 10- 50 mg/ml, 12- 50 mg/ml, 16-50 mg/ml, 18- 50 mg/ml.
  • the total flavonoids of the traditional Chinese medicine compound can be added to various household products, anti-drug products or cosmetics in a proportion of 0.001 to 50% by weight.
  • the total flavonoids of the traditional Chinese medicine compound may be used in an amount of 0.001 to 20% by weight.
  • the total flavonoids of the traditional Chinese medicine compound may be used in an amount of 0.01 to 1% by weight.
  • the raw medicinal materials and quality for extracting the total flavonoids of the compound are as follows:
  • the crude extract of traditional Chinese medicine compound is reconstituted with water to a concentration of 0.25g/ml of crude drug, which is the sample solution.
  • the macroporous resin is D101 type (Tianjin Haiguang Chemical Co., Ltd.), and the loading flow rate is 1 column volume/hour (BV/h). . It was washed with water at a flow rate of 2 BV/h until it was substantially colorless. The active ingredient was eluted at a flow rate of 2 BV/h and a total of 6 BV of 80% ethanol, and the ethanol was recovered and concentrated to obtain a solid powder, which was a total flavonoid powder of the traditional Chinese medicine compound.
  • Example 2 Example 2:
  • the raw medicinal materials and quality for extracting the total flavonoids of the compound are as follows:
  • the crude extract of Chinese herbal compound is reconstituted with water to a concentration of 0.25g/ml of crude drug, which is the sample solution.
  • the macroporous resin is DM130 (Tianjin Haiguang Chemical Co., Ltd.), and the loading flow rate is 1 column volume/hour (BV/h). . Wash with water at a flow rate of 2 BV/h until it is substantially colorless.
  • the active ingredient was eluted at a flow rate of 2 BV/h and a total of 6 BV of 80% ethanol, and the ethanol was recovered and concentrated to obtain a solid powder, which was a total flavonoid powder of the traditional Chinese medicine compound.
  • Example 3 Example 3:
  • the raw medicinal materials and quality for extracting the total flavonoids of the compound are as follows:
  • the crude extract of traditional Chinese medicine compound is reconstituted with water to a concentration of 0.25g/ml of crude drug, which is the sample solution.
  • the macroporous resin is D301 type (Tianjin Haiguang Chemical Co., Ltd.), and the loading flow rate is 1 column volume/hour (BV/h). . It was washed with water at a flow rate of 2 BV/h until it was substantially colorless. The active ingredient was eluted at a flow rate of 2 BV/h and a total of 6 BV of 80% ethanol, and the ethanol was recovered and concentrated to obtain a solid powder, which was a total flavonoid powder of the traditional Chinese medicine compound.
  • Example 4 Minimum inhibitory concentration - minimum bactericidal concentration (MIC-MBC) experiment
  • Conventional Method Extract Extracted by conventional methods in the art, the specific steps are as follows. According to the ratio of the first embodiment, take the appropriate amount of the pure Chinese herbal medicine group, mix well, put it into the extractor, add 15 times the amount of water to cook for 1 hour; collect the extract, the dregs and then boil with 10 times the amount of water 1 Hour; combine the two extracts, filter, and concentrate to obtain the conventional method extract.
  • the traditional Chinese medicine compound total flavonoid of the present invention is obtained by the process of Example 1.
  • Preparation of sample solution Take the sample solution as the stock solution, and mix the appropriate amount of the original solution with double broth medium and sterile deionized water to make 5ml containing 50%, 25%, 12.5%, 6.2 respectively. %, 3.1% were subjected to a single broth medium of the sample solution. At the same time, 2.5 ml of the solvent used in the sample was mixed with the double broth medium as a solvent control; sterile deionized water was used as a negative control.
  • test bacteria The bacterial and fungal suspensions were adjusted to 106 CFU/ml and 105 CFU/ml, respectively, and a 50 ⁇ l suspension was pipetted into each test tube such as the prepared sample solution, while the single meat was The culture medium test tube was not added with a suspension as a blank control. After shaking and mixing, the tubes were incubated at 30 ° C and 160 rpm for about 18 hours. After the negative control (sterile deionization) tube was cloudy, the tubes were taken out.
  • MIC result discrimination Visually observe whether each sample tube is clear or turbid, when the sterile deionized water control tube has bacteria growth (turbidity), the blank control tube has no bacterial growth (clarification), and the highest dilution of the test group for aseptic growth
  • concentration of the sample solution is the MIC of the sample against the test bacteria:
  • the sample at this concentration has no bacteriostatic effect; +++ indicates that the colony growth on the plate covers 1/2 medium surface, and the sample at this concentration is considered to have no bacteriostatic effect; ++ indicates that the colony growth on the plate covers 1/4 On the surface of the medium, it is considered that the sample at this concentration has a certain antibacterial effect; + indicates that there is sporadic colony growth, and the sample at this concentration is considered to have a certain antibacterial effect; one indicates the aseptic growth, and the sample at the concentration is considered to have Significant antibacterial and bactericidal effect.
  • Table 1 shows the comparison of the bactericidal effects of the conventional method extract and the total flavonoids of the traditional Chinese medicine compound.
  • Table 2 shows the MBC of the conventional method extract and the total flavonoids of the Chinese herbal compound.
  • the total flavonoid content was determined by ultraviolet spectrophotometry. This method uses rutin as the standard and methanol as the solvent. Weigh a certain amount of extract powder and dissolve it in the appropriate amount of methanol as the test sample solution. The color was developed by aluminum trichloride, and the absorbance was measured at 395 nm. The blank UV absorption of the sample is subtracted from the blank solution of the equal concentration. Calculate total flavonoids based on standard curve and dilution factor
  • Total flavonoid content (total flavonoids / extract solids mass) X 100%
  • Table 3 shows the comparison of flavonoids content of the conventional method extracts and total flavonoids of traditional Chinese medicine compound.
  • the total flavonoid content in the total flavonoids of the traditional Chinese medicine compound extracted by the extraction method of the present invention is as high as 14.9%, while the conventional method extract is only 0.30%. It can be seen that the total flavonoids of the traditional Chinese medicine compound obtained by the extraction method of the present invention is 49.7 times that of the conventional method, and the flavonoid content is greatly enriched.

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Abstract

本发明公开了一种中药复方总黄酮的制备方法，用该方法制备得到的中药复方总黄酮具有抗菌作用。本发明也公开了中药复方总黄酮的抗菌用途。所述中药复方总黄酮还可作为具有抗菌剂添加到家用产品、消毒用品或化妆品中。

Description

一种具有抗菌作用的中药复方总黄酮及其制备方法 技术领域
本发明涉及天然药物化学、 家用产品、 消毒用品、 化妆品领域， 具体 涉及一种用于抗金黄色葡萄球菌、 大肠杆菌、 白色念珠菌的中药复方总黄 酮及其制备方法。 背景技术
目前国内外多数抗菌添加剂以合成化合物为主， 随着天然、安全与环保要 求的提出， 天然植物添加剂越来越为人们所青睐。 但天然抗菌添加剂的开发较 少，不能满足消费者要求，特别是中草药复方抗菌添加剂的开发，则少之又少。
本发明提出一种提取中药复方总黄酮的新方法，根据该方法提取得到的中 药复方总黄酮能够抑制、 杀灭多种致病菌。 所述中药复方总黄酮是一种天然植 物的提取物， 因而对人体无害。 本发明方法制备得到的中药复方总黄酮提取物 还可作为功能添加剂添加到家用产品、 消毒用品或化妆品中。 发明内容
本发明目的在于提供一种具有广谱抗菌作用的复方提取物。 具体说， 本发明提供了一种具有抗金黄色葡萄球菌、 大肠杆菌和白色念珠菌作用的 中药复方总黄酮。 所述复方总黄酮提取物也可作为抗菌成分加入到家用产 品、 消毒用品、 化妆品中使用。
本发明提供了一种中药复方总黄酮的提取方法， 所述方法包括以下步骤： a) 取以下配比用量的中药材， 用极性溶剂提取， 得到粗提物， 生枳壳 20-60重量份
青皮 15-40重量份
柴胡 10〜35重量份
厚朴 10〜 30重量份 黄芩 5〜25重量份
b) 将所述粗提物复溶， 大孔树脂吸附， 洗脱， 浓缩干燥后得到中药复 方总黄酮。
在一些实施方式中， 上述步骤 a)中的极性溶剂选自： 甲醇、 乙醇、 异 丙醇、 丙酮、 乙酸乙酯或多元醇。 在一些实施方式中， 上述步骤 a)的提取方 法可以是回流提取、 浸渍或渗漉。
在其他实施方式中， 上述步骤 b)中的大孔树脂是非极性到中等极性的 吸附树脂。 例如， 大孔树脂可以是 D 101型、 DM130型或 DM301型大孔树 脂。
本发明还涉及按照本发明方法制备得到的中药复方总黄酮。
本发明还涉及中药复方总黄酮在抗金黄色葡萄球菌、 大肠杆菌、 白色念 珠菌的中的用途。在一些实施方式中， 所述中药复方总黄酮是按照本发明提取 方法制备得到的提取物。
本发明还涉及中药复方总黄酮作为具有抗金黄色葡萄球菌、 大肠杆菌、 白色念珠菌作用的添加剂在家用产品、消毒用品或化妆品中的用途，所述中药 复方总黄酮是本发明提取方法制备得到的提取物。
本发明还涉及一种家用产品、消毒用品或化妆品， 其包含按照本发明提取 方法制备得到的中药复方总黄酮以及可接受的辅料。
制取本发明复方总黄酮的一般工艺流程如下：
取生枳壳 20〜60重量份， 青皮 15〜40重量份， 柴胡 10〜35重量份， 厚朴
10〜 30重量份， 黄芩 5〜25重量份， 充分混合， 放置于提取器中。 加入适量的 溶剂， 采用溶剂提取法进行提取。待提取结束后， 回收溶剂得到粗提物。然后， 将粗提物用水复溶至一定浓度，大孔树脂吸附，水洗除杂， 乙醇洗脱有效成分。 回收乙醇， 浓缩干燥得固体粉末， 即为中药复方总黄酮。 再根据需要将固体粉 末溶解于所需溶剂中， 即可得一定浓度的中药复方总黄酮溶液。
提取溶剂可以是中、 高极性的有机溶剂， 如不同浓度的乙醇、 不同浓度的 甲醇， 丙酮等； 提取方法可以是回流提取、 浸渍、 渗漉等方法。 大孔树脂可以 是非极性到中等极性吸附树脂， 如 D101型、 DM130型、 DM301型大孔树脂 等。 上述中药复方中， 生枳壳理气宽胸、 行滞消积， 《药性论》 ： "枳壳主肠 风痔疾， 心腹气结…… "， 《日华子》： "枳壳治反胃， 霍乱泻痢， 消食…… "； 青皮疏肝破气，消积化滞， 《本草纲目》： "青皮治胸膈气逆、肋痛…… "， 《新 增治疔汇要》 ： "青皮解疔毒" ； 柴胡解表退热， 疏肝解郁， 升举阳气， 《本 经》 ： "柴胡主心腹， 去肠胃结气， 饮食积聚， 寒热邪气…… " ； 厚朴行气导 滞，燥湿， 降逆平喘， 《本经》： "主中风伤寒，头痛，寒热惊悸… 去三虫"， 《别录》 ： "疗霍乱及腹痛胀满…… "； 黄芩清热泻火， 燥湿解毒， 《本经》 ： "黄芩主诸热黄疸， 肠擗， 泄痢…… "，《别录》 ： "疗痰热， 胃中热， 小腹绞 痛， 消谷， 利小肠" 。
文献中均未提及可以采用本发明所述的生枳壳、 青皮、柴胡、 厚朴和黄芩 质量配比的中药材来提取总黄酮， 也未提及按照所述配比提取得到的复方提取 物具有杀菌或抑菌作用。本发明首次提出了利用大孔树脂的两步法提取复方总 黄酮的方法。 该方法提取得到的中药复方总黄酮经证实具有广谱杀菌作用， 能 够显著杀灭金黄色葡萄球菌、 大肠杆菌和白色念珠菌， 且效果显著优于常规方 法提取物。 总黄酮含量也测定表明， 采用本发明所述的中药材质量配比， 根据 本发明的提取方法制备得到的提取物中总黄酮含量大大富集。 具体实施方式
本发明提供了一种两步法提取中药复方总黄酮的方法。该方法首先采用溶 剂提取法提取得到中药复方粗提物， 第二步利用大孔树脂吸附， 得到本发明的 中药复方总黄酮。
本发明首次发现了根据上述两步法提取得到的中药复方总黄酮具有抗菌 作用。 因此， 所述中药复方总黄酮也可作为功效添加剂加入到家用产品、 消毒 用品或化妆品中。
文献中均未涉及中药复方总黄酮具有抗菌作用，也未提及将其作为功能添 加剂加入家用产品、 消毒用品或化妆品中。 因此， 本发明首次提出中药复方 总黄酮具有抗菌作用， 可以作为功能添加剂加入家用产品、 消毒用品或化妆 品中。
在一些实施方式中， 中药复方总黄酮的使用浓度可以是 5 毫克 /毫升或更 高， 6 毫克 /毫升或更高， 7 毫克 /毫升或更高， 8 毫克 /毫升或更高， 9 毫克 / 毫升或更高， 10 毫克 /毫升或更高， 12 毫克 /毫升或更高， 16 毫克 /毫升或更 高， 18 毫克 /毫升或更高。 在一些实施方式中， 中药复方总黄酮的使用浓度可 以是 5- 150 毫克 /毫升， 6- 150 毫克 /毫升， Ί- 150 毫克 /毫升， 8- 150 毫克 /毫 升， 9- 150 毫克 /毫升， 10- 150 毫克 /毫升， 12- 150 毫克 /毫升， 16- 150 毫克 / 毫升， 18- 150 毫克 /毫升。 在一些实施方式中， 中药复方总黄酮的使用浓度可 以是 5- 100 毫克 /毫升， 6- 100 毫克 /毫升， Ί- 100 毫克 /毫升， 8- 100 毫克 /毫 升， 9- 100 毫克 /毫升， 10- 100 毫克 /毫升， 12- 100 毫克 /毫升， 16- 100 毫克 / 毫升， 18- 100 毫克 /毫升。 在一些实施方式中， 中药复方总黄酮的使用浓度可 以是 5- 75 毫克 /毫升， 6- 75 毫克 /毫升， 7- 75 毫克 /毫升， 8- 75 毫克 /毫升， 9- 75 毫克 /毫升， 10- 75 毫克 /毫升， 12- 75 毫克 /毫升， 16- 75 毫克 /毫升， 18- 75 毫克 /毫升。在一些实施方式中，中药复方总黄酮的使用浓度可以是 5- 50 毫 克 /毫升， 6- 50 毫克 /毫升， Ί- 50 毫克 /毫升， 8- 50 毫克 /毫升， 9- 50 毫克 /毫 升， 10- 50 毫克 /毫升， 12- 50 毫克 /毫升， 16- 50 毫克 /毫升， 18- 50 毫克 /毫 升。
可以将中药复方总黄酮按照 0.001-50重量％的比例加到各种家用产品、消 毒用品或化妆品中。 例如， 在消毒用品中， 中药复方总黄酮的用量可以是 0.001-20重量％。 在化妆品中， 中药复方总黄酮的用量可以是 0.01-1重量％。
下面结合具体的实施例进一步阐述本发明。 但是， 应该明白， 这些实 施例仅用于说明本发明而不构成对本发明范围的限制。 下列实施例中未注 明具体条件的试验方法， 通常按照常规条件， 或按照制造厂商所建议的条 件。 除非另有说明， 所有的百分比和份数按重量计。 下面各实施例中使用 的中药材 （包括生枳壳、 青皮、 柴胡、 厚朴和黄芩） 均购于上海华宇药业。 实施例 1 :
用于提取复方总黄酮的生药材及质量配比如下：
生枳壳 35
青皮 20
柴胡 18 黄芩 12
在制取时， 工艺流程如下：
取配比用量下药材纯正的中草药组合适量， 混合均匀， 放入提取器中， 加入 15倍量 60%乙醇回流 1小时； 收集提取液， 药渣再用 10倍量 60%乙 醇回流提取一次； 合并两次提取液， 过滤、 回收乙醇至无醇味， 浓缩干燥 得中药复方粗提物。
中药复方粗提物加水复溶至生药浓度 0.25g/ml， 为上样液， 大孔树脂 为 D101 型 （天津市海光化工有限公司） ， 上样流速为 1 柱体积 /小时 (BV/h) 。 以 2BV/h流速水洗， 至基本无色。 以 2BV/h流速、 共 6BV的 80%乙醇洗脱有效成分， 回收乙醇， 浓缩干燥得固体粉末， 为中药复方总 黄酮粉末。 实施例 2:
用于提取复方总黄酮的生药材及质量配比如下：
生枳壳 40
青皮 30
柴胡 15
厚朴 10
黄芩 5
在制取时， 工艺流程如下：
取配比用量下药材纯正的中草药组合适量， 混合均匀， 放入提取器中， 加入 20倍量甲醇浸渍 24小时； 收集提取液， 药渣再用 15倍量甲醇浸渍 24 小时； 合并两次提取液， 过滤、 回收甲醇， 浓缩干燥得中药复方粗提物。
中药复方粗提物加水复溶至生药浓度 0.25g/ml， 为上样液， 大孔树脂 为 DM130 型 （天津市海光化工有限公司） ， 上样流速为 1 柱体积 /小时 (BV/h) 。 以 2BV/h流速水洗， 至基本无色。 以 2BV/h流速、 共 6BV的 80%乙醇洗脱有效成分， 回收乙醇， 浓缩干燥得固体粉末， 为中药复方总 黄酮粉末。 实施例 3:
用于提取复方总黄酮的生药材及质量配比如下:
生枳壳 25
青皮 25
柴胡 20
厚朴 15
黄芩 15
在制取时， 工艺流程如下：
取配比用量下药材纯正的中草药组合适量， 混合均匀， 放入提取器中， 加入 40倍量丙酮渗漉提取， 提取液过滤， 回收丙酮， 浓缩干燥得中药复方 粗提物。
中药复方粗提物加水复溶至生药浓度 0.25g/ml， 为上样液， 大孔树脂 为 D301 型 （天津市海光化工有限公司） ， 上样流速为 1 柱体积 /小时 (BV/h) 。 以 2BV/h流速水洗， 至基本无色。 以 2BV/h流速、 共 6BV的 80%乙醇洗脱有效成分， 回收乙醇， 浓缩干燥得固体粉末， 为中药复方总 黄酮粉末。 实施例 4: 最小抑菌浓度-最小杀菌浓度 （MIC-MBC) 实验
1. 样品制备
常规方法提取物： 采用本领域常规方法提取， 具体步骤如下。 按实施 例 1 的配比， 取药材纯正的中草药组合适量， 混合均匀， 放入提取器中， 加入 15倍量水煎煮 1小时； 收集提取液， 药渣再用 10倍量水煎煮 1小时； 合并两次提取液， 过滤、 浓缩干燥得常规方法提取物。
本发明的中药复方总黄酮： 按实施例 1的工艺获得。
取常规方法提取物 6g于 40ml的无菌水， 搅拌溶解完全即得质量浓度 为 150mg/ml的常规方法提取物受试原液。取中药复方总黄酮粉末 6g于 40ml 的 60%乙醇，搅拌溶解完全即得质量浓度为 150mg/ml的中药复方总黄酮受 试原液； 同时以 60%乙醇为溶剂对照。 2.实验方法
受试样液的制备： 以提供的样品液为原液， 分别取适量原液与双倍肉 汤培养基及无菌去离子水混匀， 分别制成 5ml含 50%、 25%、 12.5%、 6.2%、 3.1%受试样液的单倍肉汤培养基。 同时以样品所使用的溶剂 2.5ml 与双倍 肉汤培养基混匀， 为溶剂对照； 以无菌去离子水作阴性对照。
试验菌接种：分别将细菌、真菌菌悬液调整至 106CFU/ml、 105 CFU/ml, 以移液器吸取 50μ1菌悬液至已制备好的受试样液等各试管中， 同时单倍肉 汤培养基试管中未加菌悬液为空白对照。 各试管振荡混匀后， 30°C 160rpm 振荡培养 18h左右， 待阴性对照试 （无菌去离子） 管呈浑浊生长后， 取出 各试管。
MIC结果判别： 肉眼观察各样品试管是否澄清或浑浊， 当无菌去离子 水对照管有菌生长 （混浊） ， 空白对照管无细菌生长 （澄清） ， 试验组无 菌生长的最高稀释度所对应的样液浓度为该样品对受试菌的 MIC：。
MBC结果判别： 取 MIC实验后的样液 lml， 分别浇注细菌 /真菌培养 基培养后， 观察平皿上的菌落总数， 以 ++++表明平板上菌落生长覆盖整个 培养基表面， 无法计数， 认为该浓度下的样品无抑菌效果； +++表明平板上 菌落生长覆盖 1/2个培养基表面， 认为该浓度下的样品无抑菌效果； ++表 明平板上菌落生长覆盖 1/4个培养基表面，认为该浓度下的样品具有一定的 抑菌功效； +表示有零星菌落生长， 认为该浓度下的样品具有一定的抑菌功 效； 一表示无菌落生长， 认为该浓度下的样品具有显著的抑菌杀菌功效。
3. 实验结果
表 1显示了常规方法提取物和中药复方总黄酮的杀菌效果比较。 表 1
金黄色葡萄 白色念珠 样品名称 作用浓度 大肠杆菌
球菌
50%
150mg/ml的常规方法 25%
12.5%
提取物 6.2%
3.1%
50%
25%
150mg/ml的中药复方
12.5%
总黄酮
6.2%
3.1% +++ +++ +
50%
25%
溶剂对照 （60%乙醇） 12.5%
6.2%
3.1% 表 2显示了常规方法提取物和中药复方总黄酮的 MBC：。
表 2
样品名称 大肠杆菌 MBC 金黄色葡萄球菌 白色念珠菌
( mg/ml) MBC ( mg/ml) MBC ( mg/ml) 常规方法提取物 >75.0 75.0 >75.0 中药复方总黄酮 9.38 9.38 9.38 结果表明， 中药复方总黄酮具有广谱抗金黄色葡萄球菌、 大肠杆菌、 白色念珠菌的作用， 在 9.38mg/ml 的浓度下仍能杀灭这三种细菌。 而传统 提取得到的常规方法提取物仅对金黄色葡萄球菌有一定的杀菌作用， MBC 为 75.0mg/ml， 但对大肠杆菌、 白色念珠菌无效。 可见， 按照上述中药配比 及特定的工艺提取得到的中药复方总黄酮具有显著的抗菌作用， 效果大大 优于传统提取物。 实施例 5: 总黄酮含量比较
1. 实验方法
总黄酮含量的测定采用紫外分光光度法， 本方法以芦丁为标准品， 甲 醇为溶剂， 称取一定量的提取物粉末溶于适量甲醇作为受试样品溶液， 通 过三氯化铝显色， 395nm下检测吸光度。 并以等浓度样品溶液为空白 扣除样品空白紫外吸收。 根据标准曲线以及稀释倍数计算出总黄酮质
总黄酮含量 = (总黄酮质量 /提取物固体质量） X 100%
2. 实验结果
表 3显示了常规方法提取物、 中药复方总黄酮的黄酮含量比较。
表 3
样品名称 总黄酮含量
( %)
常规方法提取物 0.30
中药复方总黄酮 14.9
实验表明， 通过本发明提取方法提取得到的中药复方总黄酮中的总黄 酮含量高达 14.9%， 而常规方法提取物只有 0.30%。 比较可知， 本发明提取 方法得到的中药复方总黄酮是常规方法提取物的 49.7倍， 黄酮含量得以大 大富集。

Claims

1. 一种中药复方总黄酮的提取方法， 所述方法包括以下步骤：
a) 取以下配比用量的中药材， 用极性溶剂提取， 得到粗提物， 生枳壳 20-60重量份
青皮 15-40重量份
柴胡 10〜35重量份
厚朴 10〜 30重量份
黄芩 5-25重量份
b) 将所述粗提物复溶， 大孔树脂吸附， 洗脱， 浓缩干燥后得到中药复 方总黄酮。
2. 如权利要求 1所述的方法， 其特征在于， 所述步骤 a)中的极性溶剂 选自： 甲醇、 乙醇、 异丙醇、 丙酮、 乙酸乙酯或多元醇。
3. 如权利要求 1所述的方法， 其特征在于， 所述步骤 a)的提取方法可以 是回流提取、 浸渍或渗漉。
4. 如权利要求 1所述的方法， 其特征在于， 所述步骤 b)中的大孔树脂 是非极性到中等极性的吸附树脂。
5. 如权利要求 4所述的方法， 其特征在于， 所述大孔树脂选自： D101 型、 DM130型或 DM301型大孔树脂。
6. 按照权利要求 1-5中任一项所述方法制备得到的中药复方总黄酮。
7. 中药复方总黄酮在抗金黄色葡萄球菌、 大肠杆菌、 白色念珠菌的中 的用途。
8. 如权利要求 7所述的用途， 其中， 所述中药复方总黄酮是按照权利要 求 1-5中任一项所述方法制备的提取物。
9. 中药复方总黄酮作为具有抗金黄色葡萄球菌、大肠杆菌、 白色念珠菌 作用的添加剂在家用产品、 消毒用品或化妆品中的用途， 所述中药复方总黄酮 是按照权利要求 1-5中任一项所述方法制备的提取物。
10. 一种家用产品、 消毒用品或化妆品， 其包含如权利要求 1-5中任一项 所述方法制备得到的中药复方总黄酮以及可接受的辅料。
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