WO2024000845A1 - 广东凉茶中药组合物在制备抗冠状病毒药物中的应用 - Google Patents

广东凉茶中药组合物在制备抗冠状病毒药物中的应用 Download PDF

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WO2024000845A1
WO2024000845A1 PCT/CN2022/120081 CN2022120081W WO2024000845A1 WO 2024000845 A1 WO2024000845 A1 WO 2024000845A1 CN 2022120081 W CN2022120081 W CN 2022120081W WO 2024000845 A1 WO2024000845 A1 WO 2024000845A1
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herbal tea
chinese medicine
traditional chinese
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PCT/CN2022/120081
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French (fr)
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黄晓丹
伍柏坚
谢君
郑荣波
杨子峰
彭绍忠
罗燕玉
徐凡
符皖
马钦海
朱志红
李振羽
严嘉豪
方广宏
陈伟平
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广州王老吉药业股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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
    • A61K36/11—Pteridophyta or Filicophyta (ferns)
    • A61K36/12—Filicopsida or Pteridopsida
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • 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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/738—Rosa (rose)
    • 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)
    • A61K36/85—Verbenaceae (Verbena family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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 or 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
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of medicine, and specifically to the application of a Guangdong herbal tea traditional Chinese medicine composition in the preparation of anti-coronavirus drugs.
  • Herbal tea is a type of health-care tea and medicinal tea. It was originally made from tea leaves. In order to enhance the heat-clearing and saliva-promoting effect of herbal tea, some Chinese herbal medicines were gradually added during the drinking process. Now, herbal teas are basically made of some Chinese herbal medicines. Since then, the concept of herbal tea has been continuously extended. As long as it can clear away heat and detoxify, remove dampness and relieve summer heat, refresh the mind, produce body fluids and quench thirst, it is defined as herbal tea.
  • Herbal tea is an oral decoction made from a variety of herbs in Guangdong, Fujian, and even Guangxi and Yunnan in southeastern China today to prevent and treat febrile diseases or damp-heat diseases.
  • This kind of decoction has cold properties and can clear away heat and detoxify.
  • To treat gastrointestinal damp-heat symptoms such as sores in the mouth and tongue, sore throat, red eyes and headache, dizziness and tinnitus, swollen boils, dry stools, bitter mouth and yellow urine, diarrhea, abdominal pain, tiredness after eating less, etc., Guangdong Commonly known as the person who is on fire. Drinking it by those who are preventing diseases can remove dampness and promote fluid production, clear fire and improve eyesight, dissipate stagnation and reduce swelling, and has the medicinal effect of regulating the body.
  • herbal tea has developed from traditional decoction to modern granules, tea bags, finished tea, and then to beverages.
  • the Guangdong herbal tea granules of Guangzhou Wonglaoji Pharmaceutical Co., Ltd. are composed of ten medicinal herbs, including plum blossoms, sesame seeds, five-finger mandarin oranges, light bamboo leaves, wood butterflies, slag leaves, fotan mother, golden sand vine, Desmodium guava, and golden cherry root. Its main functions are: clearing away heat and relieving summer heat, removing dampness and promoting fluid production. It is used for colds in four seasons, fever and sore throat, stagnant damp-heat, dry mouth and yellow urine. There are currently no reports of Guangdong herbal tea granules being used for antiviral treatment.
  • This application provides the application of a Guangdong herbal tea traditional Chinese medicine composition in the preparation of anti-coronavirus drugs.
  • coronavirus belongs to the genus Coronavirus in the family Coronaviridae of the order Nidovirales in terms of systematic classification.
  • Viruses of the genus Coronavirus are enveloped (envelope) RNA viruses with a linear single-stranded positive-sense genome. They are a large group of viruses that exist widely in nature. The diameter of the virus is about 80-120nm. The 5' end of the genome has a methylated cap-like structure and the 3' end has a poly(A) tail. The full genome length is about 27-32kb. It is the largest genome among currently known RNA viruses.
  • coronaviruses that can infect humans, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and the 2019 novel coronavirus (COVID-19).
  • the Guangdong herbal tea traditional Chinese medicine composition of the present application that is, Guangdong herbal tea granules, is composed of ten medicinal materials, including plum blossoms, mountain sesame, five-finger mandarin oranges, light bamboo leaves, wood butterflies, slag leaves, fotan mother, Golden sand vine, Desmodium candida, and golden cherry root.
  • the Guangdong herbal tea traditional Chinese medicine composition of the present application consists of the following components by weight: 205 to 462 parts by weight of Prunus grandiflorum, 28 to 65 parts by weight of Sesame chinensis, 39 to 89 parts by weight of Five-finger Mandarin orange, and 12-27 parts by weight of Light Bamboo Leaves. , 0.5 to 1.5 parts by weight of wood butterfly, 12 to 27 parts by weight of slag leaves, 30 to 69 parts by weight of charcoal mother, 69 to 156 parts by weight of golden sand vine, 18 to 41 parts by weight of Desmodium grandiflorum, and 70 to 159 parts by weight of golden cherry root.
  • the Guangdong herbal tea traditional Chinese medicine composition of the present application consists of the following components by weight: 277 to 342 parts by weight of Prunus grandiflora, 38 to 48 parts by weight of mountain sesame, 53-66 parts by weight of Five-finger Mandarin orange, and 16-20 parts by weight of pale bamboo leaves. , 0.9 to 1.2 parts by weight of wood butterfly, 16 to 20 parts by weight of slag leaves, 41 to 52 parts by weight of charcoal mother, 93 to 116 parts by weight of golden sand vine, 24 to 30 parts by weight of Desmodium grandiflorum, and 95 to 118 parts by weight of golden cherry root.
  • the Guangdong herbal tea Chinese medicine composition of the present application consists of the following components by weight: 308 parts by weight of Prunus japonica, 43 parts by weight of Sesame japonica, 59 parts by weight of Five-finger Mandarin, 18 parts by weight of Pale bamboo Leaves, and 1 part by weight of Wood Butterfly, 18 parts by weight of Buza leaves, 46 parts by weight of Foshan mother, 104 parts by weight of Golden sand vine, 27 parts by weight of Desmodium grandiflorum, and 106 parts by weight of Golden cherry root.
  • the anti-coronavirus drug described in this application is composed of Guangdong herbal tea traditional Chinese medicine composition and pharmaceutically acceptable excipients.
  • the Guangdong herbal tea traditional Chinese medicine composition described in this application can be prepared according to the following method:
  • the coronavirus described in this application is a human coronavirus.
  • the coronavirus described in this application is HCoV-229E.
  • Figure 3 The efficacy of Guangdong herbal tea granules on inflammation of human coronavirus HCoV-229E: compared with the virus control group, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001; compared with the positive control group, #p ⁇ 0.05 , ##p ⁇ 0.01, ###p ⁇ 0.001
  • Example 1 Preparation method of the traditional Chinese medicine composition of the present application
  • the anti-human coronavirus (HCoV-229E) effect of the Guangdong herbal tea traditional Chinese medicine composition of the present application is evaluated at the cellular level.
  • Test drug The Guangdong herbal tea granule extract in this application is provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd. (batch number 1911606). The drug name and concentration are shown in Table 3.
  • the positive control drug Remdesivir (GS-5734) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.
  • Table 1 List of commonly used reagents and consumables
  • Inhibition rate (average OD value of the cell control group - average OD value of the experimental group)/(average OD value of the cell control group - average OD value of the blank control group) ⁇ 100%.
  • the Reed-Meunch method was used to calculate the half toxic concentration (TC 50 ) of the drug.
  • Inhibition rate (average OD value of the experimental group - average OD value of the virus control group) / (average OD value of the cell control group - average OD value of the virus control group) ⁇ 100%.
  • the drug concentration that can inhibit 50% or more of virus-induced cytopathic effects (CPE) is regarded as the effective concentration, and the half inhibitory concentration (IC 50 ) is calculated using the Reed-Muench method or GraphPad Prism8.0.
  • Huh-7 cells were inoculated into 6-well plates and divided into cell control group, virus control group, positive control group and experimental group. After the cells grow to a monolayer, discard the culture medium and wash once with PBS.
  • the virus control group, positive control group and experimental group are diluted with 2% serum medium and inoculated with 100 TCID 50 human coronavirus (HCoV-229E). Normal The culture medium containing 2% serum was added to the group and adsorbed at 37°C for 2 hours.
  • mRNA is reverse transcribed into cDNA; configure the reverse transcription reaction system as follows:
  • Reverse transcription reaction conditions are as follows:
  • the reaction system is as follows:
  • primer design software Primer Premier 5.0 to design primers, and submit the designed designs to Life Company for synthesis.
  • the primer sequences are as follows:
  • cytokine mRNA was detected by RT-qPCR, and the calculation results were analyzed and calculated using GraphPad Prism 8.0, as shown in Table 8 and Figure 3.

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Abstract

一种广东凉茶中药组合物在制备抗冠状病毒的药物中的应用。该广东凉茶中药组合物由岗梅、山芝麻、五指柑、淡竹叶、木蝴蝶、布渣叶、火炭母、金沙藤、广金钱草、金樱根制成。实验结果表明，该广东凉茶中药组合物在体外对人冠状病毒(HCoV-229E)感染 Huh-7 细胞致细胞病变具有抑制作用，能抑制人冠状病毒(HCoV-229E)诱导的炎症因子IL-6、IL-8、MCP-1和TNF-α的mRNA过度表达，具剂量依赖关系。

Description

广东凉茶中药组合物在制备抗冠状病毒药物中的应用 技术领域
本发明涉及医药领域，具体而言是涉及一种广东凉茶中药组合物在制备抗冠状病毒药物中的应用。
背景技术
凉茶，是保健茶、药茶的一种，最早是以茶叶为原料，为了增强凉茶的清热生津的作用，在饮用的过程中逐渐加入了一些中草药，到了现在，凉茶基本上都由一些中草药配伍而成，凉茶的概念也不断延伸，只要能起到清热解毒、祛湿消暑、提神醒脑、生津止渴等功效的产品，都被定义为凉茶。
凉茶是今日中国东南方广东、福建乃至西南面的广西及云南地区，用多种草药煎煮而成以防治温病或湿热类疾患的口服汤剂。此类汤剂药性偏寒，能清热解毒。对治诸如口舌生疮、咽喉肿痛、目赤头痛、头晕耳鸣、疔疮肿毒、大便干结、口苦尿黄、兼有泄泻、腹痛、食少体倦等胃肠湿热诸症状，粤俗谓之上火者。防病者饮用能去湿生津、清火明目、散结消肿，有调理身体的药效。
随着现代制药技术的应用，凉茶从传统的汤剂，发展到现代的颗粒剂、袋泡茶，成品茶剂，再发展成饮料。如：王老吉颗粒、夏桑菊颗粒、板蓝根颗粒、源吉林甘和茶、白云山口炎清饮料、王老吉饮料、夏桑菊饮料等。
广州王老吉药业股份有限公司的广东凉茶颗粒，是由岗梅、山芝麻、五指柑、淡竹叶、木蝴蝶、布渣叶、火炭母、金沙藤、广金钱草、金樱根十味药材组成的，其功能主治为：清热解暑，去湿生津。用于四时感冒，发热喉痛，湿热积滞，口干尿黄。目前尚未有广东凉茶颗粒用于抗病毒治疗的报道。
发明内容
本申请提供一种广东凉茶中药组合物在制备抗冠状病毒的药物中的应用。
其中，冠状病毒，在系统分类上属套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。冠状病毒属的病毒是具囊膜(envelope)、基因组为线性单股正链的RNA病毒，是自然界广泛存在的一大类病毒。病毒直径约80～120nm，基因组5′端具有甲基化的帽状结构，3′端具有poly(A)尾，基因组全长约27-32kb，是目前已知RNA病毒中基因组最大的病毒。目前已知7种可以感染人的冠状病毒，分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV以及2019新型冠状病毒(COVID-19)。
本申请的广东凉茶中药组合物，即广东凉茶颗粒，由岗梅、山芝麻、五指柑、淡竹叶、木蝴蝶、布渣叶、火炭母、金沙藤、广金钱草、金樱根十味药材组成。
具体的，本申请的广东凉茶中药组合物由以下重量份的成分组成：岗梅205～462重量份，山芝麻28～65重量份，五指柑39～89重量份，淡竹叶12～27重量份，木蝴蝶0.5～1.5重量份，布渣叶12～27重量份，火炭母30～69重量份，金沙藤69～156重量份，广金钱草18～41重量份，金樱根70～159重量份。
优选的，本申请的广东凉茶中药组合物由以下重量份的成分组成：岗梅277～342重量份，山芝麻38～48重量份，五指柑53～66重量份，淡竹叶16～20重量份，木蝴蝶0.9～1.2重量份，布渣叶16～20重量份，火炭母41～52重量份，金沙藤93～116重量份，广金钱草24～30重量份，金樱根95～118重量份。
最优选的，本申请的广东凉茶中药组合物由以下重量份的成分组成：岗梅308重量份，山芝麻43重量份，五指柑59重量份，淡竹叶18重量份，木蝴蝶1重量份，布渣叶18重量份，火炭母46重量份，金沙藤104重量份，广金钱草27重量份，金樱根106重量份。
本申请所述的抗冠状病毒的药物是由广东凉茶中药组合物和药学上可接受的辅料组成。
本申请所述的广东凉茶中药组合物可以按照以下方法进行制备：
按配比称取全部十味药材，加水煎煮二次，每次半小时，滤过，合并滤液，浓缩至相对密度在45℃时1.03，以转速为15000转/分超高速离心分离至澄清，取分离液浓缩成稠膏，即得。
本申请所述的冠状病毒为人冠状病毒。
优选的，本申请所述的冠状病毒为HCoV-229E。
附图说明
图1广东凉茶颗粒对细胞毒性检测结果
图2广东凉茶颗粒对人冠状病毒(HCoV-229E)药效检测结果
图3广东凉茶颗粒对人冠状病毒HCoV-229E炎症药效：与病毒对照组比较，*p＜0.05，**p＜0.01，***p＜0.001；与阳性对照组比较，#p＜0.05，##p＜0.01，###p＜0.001
具体实施方式
以下通过实施例进一步阐述本发明药物的制备方法。
实施例1：本申请中药组合物的制备方法
岗梅308g，山芝麻43g，五指柑59g，淡竹叶18g，木蝴蝶1g，布渣叶18g，火炭母46g，金沙藤104g，广金钱草27g，金樱根106g。
以上十味药材，加水煎煮二次，每次半小时，滤过，合并滤液，浓缩至相对密度为1.03(45℃)，以转速为15000转/分超高速离心分离至澄清，取分离液浓缩成稠膏。取稠膏，加适量蔗糖，制颗粒，干燥，制成240g即得。
实施例2：本申请中药组合物的制备方法
岗梅308g，山芝麻43g，五指柑59g，淡竹叶18g，木蝴蝶1g，布渣叶18g，火炭母46g，金沙藤104g，广金钱草27g，金樱根106g。
以上十味药材，加水煎煮二次，每次半小时，滤过，合并滤液，浓缩至相对密度为1.03(45℃)，以转速为15000转/分超高速离心分离至澄清，取分离液浓缩成稠膏。取稠膏，加适量淀粉，制颗粒，干燥，制成24g即得。
实验例1本申请广东凉茶中药组合物对人冠状病毒(HCoV-229E)体外筛选实验
一、实验目的
在细胞水平评价本申请广东凉茶中药组合物抗人冠状病毒(HCoV-229E)的效果。
二、实验材料：
1.受试药物：本申请广东凉茶颗粒浸膏，由广州王老吉药业股份有限公司提供(批号1911606)，药物名称及浓度见表3。阳性对照药物瑞德西韦(GS-5734)购于上海吉至生化科技有限公司。
2.细胞：Huh-7细胞，由广州呼吸健康研究院呼吸疾病国家重点实验室病毒室保存。
3.病毒：人冠状病毒HCoV-229E，滴度为TCID 50＝10 -5/100μL,由广州呼吸健康研究院呼吸疾病国家重点实验室病毒室-80℃保存；使用病毒滴度为100TCID 50。
4.试剂及耗材
表1：常用试剂及耗材列表
Figure PCTCN2022120081-appb-000001
Figure PCTCN2022120081-appb-000002
5.主要仪器
表2：主要仪器列表
Figure PCTCN2022120081-appb-000003
三、实验方法：
(一)细胞毒性试验(MTT法)
(1)受试药物：
表3：药物名称、实验浓度和分组
Figure PCTCN2022120081-appb-000004
(2)无菌96孔培养板，每孔加入100μL浓度为2×10 5cells/mL Huh-7细胞，37℃5％CO 2培养24小时；
(3)96孔板单层细胞用PBS洗涤1次，每孔加入100μL 2倍梯度稀释的药物。空白对照组 和细胞对照组每孔加入等体积培养液，37℃培养4日；
(4)每孔加入浓度为5mɡ/mL的MTT溶液20μL，继续孵育4小时。弃上清，每孔加入100μL DMSO，低速振荡5分钟，使结晶物充分溶解；
(5)选择490nm波长，在酶联免疫仪上测定吸光值，计算抑制率。抑制率＝(细胞对照组平均OD值－实验组平均OD值)/(细胞对照组平均OD值－空白对照组平均OD值)×100％。Reed-Meunch法计算药物半数毒性浓度(TC 50)。
(6)实验条件：以上实验操作均在BSL-2实验室内完成。
(二)受试药物抗病毒实验
(1)受试药物：
表4：药物名称、实验浓度和分组
Figure PCTCN2022120081-appb-000005
(2)无菌96孔培养板,每孔加入100μL浓度为2×10 5cells/mL Huh-7细胞，37℃5％CO 2培养24小时；
(3)培养板实验组和病毒对照组加入2％血清培养基稀释的100TCID 50病毒液100μL/孔，细胞对照组加入等体积2％血清培养基，37、5％CO 2培养箱吸附2h。
(4)2h后，弃去96孔培养板中细胞培养液；受试药物使用2％血清培养基稀释成表4中的各个浓度，每个浓度3个复孔，100μL/孔加入上述药液；
(5)细胞对照组和病毒对照组加入等体积2％血清培养基；
(6)细胞37℃5％CO 2孵箱孵育3-4天；
(7)每孔加入浓度为5mɡ/mL的MTT溶液20μL，继续孵育4小时。弃上清，每孔加入100μL DMSO，低速振荡5分钟，使结晶物充分融解；
(8)选择490nm波长，在酶联免疫仪上测定吸光值，计算抑制率。抑制率＝(实验组平均OD值－病毒对照组平均OD值)/(细胞对照组平均OD值－病毒对照组平均OD值)×100％。能够抑制50％及以上病毒引起的细胞病变(CPE)的药物浓度视为有效浓度，采用Reed-Muench法或GraphPad Prism8.0计算半数抑制浓度(IC 50)。
(9)实验条件：以上实验操作均在BSL-2实验室内完成。
(三)RT-qPCR检测炎症因子
(1)受试药物：
表5：药物名称、实验浓度和分组
Figure PCTCN2022120081-appb-000006
(2)Huh-7细胞接种至6孔板中，分为细胞对照组、病毒对照组、阳性对照组和实验组。细胞长至单层后，弃去培养液，用PBS洗一次，病毒对照组、阳性对照组和实验组使用含2％血清培养基稀释并接种100TCID 50的人冠状病毒(HCoV-229E)，正常组加入含2％血清培养基，37℃吸附2小时。
(3)弃去病毒液，实验组加入含不同浓度药物的2％血清培养基2mL，阳性对照组加入含5μM瑞德西韦的2％血清培养基2mL，正常组和病毒组加入等体积含2％血清培养基。37℃作用48小时后，弃上清，PBS洗细胞一次，加入TRIzol试剂裂解细胞并转移至1.5mL EP管。
(4)RNA的提取
向细胞中加入氯仿200μL，震荡15s，在室温孵育5min，4℃、10000rpm离心10min；离心后，取上清至新的1.5mL EP管中，加入异丙醇500μL，室温孵育10min，4℃、10000rpm离心10min；离心弃上清后加入75％乙醇1000μL混匀，4℃、10000rpm离心10min；弃上清，室温中放置5min，加入适量无RNA酶的ddH 2O溶解RNA，检测RNA浓度并进行逆转录cDNA。
(5)样本RNA的逆转录
mRNA逆转录成cDNA；配置逆转录反应体系如下：
Figure PCTCN2022120081-appb-000007
逆转录反应条件如下：
Figure PCTCN2022120081-appb-000008
Figure PCTCN2022120081-appb-000009
(6)实时荧光定量PCR检测
反应体系如下：
Figure PCTCN2022120081-appb-000010
PCR反应条件如下：
Figure PCTCN2022120081-appb-000011
(7)引物的设计和合成
利用引物设计软件Primer Premier 5.0进行引物设计，将设计好的提交给life公司合成，引物序列如下：
Figure PCTCN2022120081-appb-000012
Figure PCTCN2022120081-appb-000013
(8)实验条件：以上实验操作均在BSL-2实验室内完成。
(四)抗病毒药效结果判定标准：能够抑制50％及以上病毒引起的细胞病变(CPE)的药物浓度视为有效浓度,，以选择指数
Figure PCTCN2022120081-appb-000014
判定药效标准，SI>1表示有效，选择指数越大，抗病毒效果越好。
四、实验结果
(一)药物毒性
MTT实验结果如表6及图1所示。
表6：细胞毒性试验(MTT法)结果
Figure PCTCN2022120081-appb-000015
实验结果表明，在Huh-7细胞中，广东凉茶颗粒半数毒性浓度(TC 50)为3206μɡ生药/mL(图1)。
(二)抗病毒药效
通过MTT法测试吸光值并记录实验结果，采用Reed-Muench法或GraphPad Prism 8.0计算半数抑制浓度(IC 50)。实验结果如表7及图2所示。
表7：广东凉茶颗粒抗病毒实验结果
Figure PCTCN2022120081-appb-000016
广东凉茶颗粒在此次实验的治疗模式中IC 50为266.00μɡ/mL(图2)，SI＝12.05。
(二)炎症细胞因子的结果
通过RT-qPCR检测细胞因子mRNA的表达，采用GraphPad Prism 8.0分析计算结果如表8及图3所示。
表8：各实验组对细胞因子mRNA的表达的影响
Figure PCTCN2022120081-appb-000017
五、结论
上述实验结果表明广东凉茶颗粒在体外对人冠状病毒(HCoV-229E)感染Huh-7细胞致细胞病变具有抑制作用(选择指数SI为12.05)；体外能抑制人冠状病毒(HCoV-229E)诱导炎症因子IL-6、IL-8、MCP-1和TNF-α的mRNA过度表达，具剂量依赖关系。

Claims (9)

  1. 一种广东凉茶中药组合物在制备抗冠状病毒的药物中的应用。
  2. 如权利要求1所述的应用，其中广东凉茶中药组合物由以下重量份的成分组成：岗梅205～462重量份，山芝麻28～65重量份，五指柑39～89重量份，淡竹叶12～27重量份，木蝴蝶0.5～1.5重量份，布渣叶12～27重量份，火炭母30～69重量份，金沙藤69～156重量份，广金钱草18～41重量份，金樱根70～159重量份。
  3. 如权利要求2所述的应用，其中广东凉茶中药组合物由以下重量份的成分组成：岗梅277～342重量份，山芝麻38～48重量份，五指柑53～66重量份，淡竹叶16～20重量份，木蝴蝶0.9～1.2重量份，布渣叶16～20重量份，火炭母41～52重量份，金沙藤93～116重量份，广金钱草24～30重量份，金樱根95～118重量份。
  4. 如权利要求3所述的应用，其中广东凉茶中药组合物由以下重量份的成分组成：岗梅308重量份，山芝麻43重量份，五指柑59重量份，淡竹叶18重量份，木蝴蝶1重量份，布渣叶18重量份，火炭母46重量份，金沙藤104重量份，广金钱草27重量份，金樱根106重量份。
  5. 如权利要求1-4任一项所述的应用，其中所述药物是由中药组合物和药学上可接受的辅料组成。
  6. 如权利要求1-4任一项所述的应用，其中所述广东凉茶中药组合物由以下方法制备：
    按配比称取全部十味药材，加水煎煮二次，每次半小时，滤过，合并滤液，浓缩至相对密度在45℃时1.03，以转速为15000转/分超高速离心分离至澄清，取分离液浓缩成稠膏，即得。
  7. 如权利要求1所述的应用，其中所述冠状病毒为人冠状病毒。
  8. 如权利要求7所述的应用，其中所述冠状病毒为HCoV-229E。
  9. 如权利要求1所述的应用，其中所述抗冠状病毒，包括治疗冠状病毒引起的炎症。
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Publication number Priority date Publication date Assignee Title
CN107744040A (zh) * 2017-12-05 2018-03-02 广东康奇力药业股份有限公司 一种凉茶颗粒及制备方法
CN113101310A (zh) * 2021-03-26 2021-07-13 香港科技大学 预防或/和治疗冠状病毒感染的药物及其制备方法
CN115025184A (zh) * 2022-07-01 2022-09-09 广州王老吉药业股份有限公司 广东凉茶中药组合物在制备抗冠状病毒药物中的应用

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Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107744040A (zh) * 2017-12-05 2018-03-02 广东康奇力药业股份有限公司 一种凉茶颗粒及制备方法
CN113101310A (zh) * 2021-03-26 2021-07-13 香港科技大学 预防或/和治疗冠状病毒感染的药物及其制备方法
CN115025184A (zh) * 2022-07-01 2022-09-09 广州王老吉药业股份有限公司 广东凉茶中药组合物在制备抗冠状病毒药物中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Chinese Traditional Patent Medicine of Drug Specifications Promulgated by the Ministry of Public Health, volume 19", 31 December 1998, article CHINESE PHARMACOPOEIA COMMISSION: "Guangdong Herbal Tea Granules", pages: 27 - 28, XP009552379 *
JUN ZHANG: "10 keywords that make Wong Lo Kat popular", CHINA BRAND, 28 February 2007 (2007-02-28), pages 76 - 79, XP093124103 *
RONGRONG HE, YAO XINSHENG: "The"Xiehuo"Effect of Guangdong Herbal Tea and Its Compositio", WORLD SCIENCE AND TECHNOLOGY(MODERNIZATION OF TRADITIONAL CHINESE MEDICINE AND MATERIA MEDICA), vol. 11, no. 6, 20 December 2009 (2009-12-20), pages 834 - 839, XP093124102 *

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