CN117503856A - 植物精油在制备治疗乙肝疾病药物或健康产品中的应用 - Google Patents
植物精油在制备治疗乙肝疾病药物或健康产品中的应用 Download PDFInfo
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- CN117503856A CN117503856A CN202311448760.4A CN202311448760A CN117503856A CN 117503856 A CN117503856 A CN 117503856A CN 202311448760 A CN202311448760 A CN 202311448760A CN 117503856 A CN117503856 A CN 117503856A
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- essential oil
- steam distillation
- volatile component
- plant
- hepatitis
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Abstract
本发明公布了植物精油在制备治疗乙肝疾病药物或健康产品中的应用,属于生物医药大健康技术领域。本发明植物精油在制备治疗乙肝疾病药物中的用途主要归功于植物精油在乙肝病毒体外细胞模型中显示显著的抗HBV活性。石菖蒲精油、莪术精油、紫苏精油、古巴香脂精油、有机胡萝卜籽精油、有机贞节树精油、乳香精油、灰毛莸精油、青花椒精油、艳山姜精油和黑胡椒精油对乙肝病毒显示显著抑制活性,对表面抗原(HBsAg)和乙肝病毒e抗原(HBeAg)的半数抑制浓度(IC50)分别在0.07~0.19mg/ml和0.10~0.22mg/ml之间。
Description
技术领域
本发明涉及植物精油的新用途,尤其涉及植物精油在制备治疗乙肝疾病药物或健康产品中的用途。
背景技术
乙型肝炎病毒(Hepatitis B virus,HBV)是一种噬肝性DNA病毒。HBV感染后会引发肝硬化、肝纤维化及原发性肝癌等疾病。全球感染乙肝病毒的人数约2.92亿,每年所导致的死亡人数超过80万,大多数死于肝硬化或肝细胞癌。尽管疫苗能有效预防HBV的感染,但是疫苗对已经感染HBV的患者无效。目前临床使用的治疗乙型肝炎的药物包括核苷类似物及干扰素两类,但存在耐药性和一系列不良反应。目前没有完全治疗乙肝的药物,感染后只能长期吃药来控制,患者用药面临着较大经济负担。目前临床上常用的治愈乙肝标准“功能性治愈”、“完全治愈”,“彻底根除”中血清乙肝病毒表面抗原(HBsAg)转阴都是必要的参考指标,说明HBsAg在HBV感染后治疗的监控过程中也发挥着重要作用。基于HBsAg筛选出具有抗HBV活性的植物精油,以实现功能性治愈,甚至完全治愈是非常重要的。
石菖蒲(Acorus tatarinowii,Acorus calamus)作为天南星科菖蒲属多年生草本植物,根茎常作药用。药典中记载石菖蒲具有开窍豁痰,醒神益智,化湿开胃等功效。用于神昏癫痫,健忘失眠,耳鸣耳聋,脘痞不饥,噤口下痢等病症。石菖蒲精油的成分较为复杂,α-细辛醚和β-细辛醚是其主要成分。石菖蒲精油具有镇静、催眠、抗惊厥、抗抑郁等生物活性。然而尚未有文献或专利报道石菖蒲精油具有显著抗HBV活性。
莪术(Curcuma zedoaroa)作为姜科姜黄属多年生草本植物,常以干燥根茎入药。药典中记载莪术归肝、脾经,味辛、苦,性温,具有行气破血,消积止痛等功效。用于瘴瘢痞块,瘀血经闭,胸痹心痛,食积胀痛等病症。莪术精油具有抗肿瘤、抗氧化、抗病毒等生物活性。然而尚未有文献或专利报道莪术精油具有抗HBV活性。
紫苏(Perilla frutescens)作为唇形科紫苏属一年生草本植物,紫苏叶和梗常做药用。药典中记载紫苏具有解表散寒,行气和胃等功效。用于风寒感冒,咳嗽呕恶,妊娠呕吐,鱼蟹中毒等病症。紫苏具有抗炎、抗菌、抗氧化、抗过敏、抗肿瘤等活性。然而尚未有文献或专利报道紫苏精油具有抗HBV活性。
古巴香脂(Copaifera officinalis)作为豆科古巴属古巴香脂树树脂。尚未有文献或专利报道古巴香脂精油具有抗HBV活性。
胡萝卜(Daucus carota)作为伞形科胡萝卜属两年生草本植物。胡萝卜籽为胡萝卜果实,胡萝卜籽精油具有抗菌活性。尚未有文献或专利报道胡萝卜籽精油具有抗HBV活性。
贞节树(Vitex agnus castus)又称穗花杜荆,作为马鞭草科杜荆属灌木。穗花杜荆具有抗菌、抗肿瘤的作用。尚未有文献或专利报道贞节树精油具有抗HBV活性。
乳香(Boswellia carteri)为橄榄科植物乳香树树皮渗出的树脂。药典中记载乳香具有活血定痛,消肿生肌等功效。用于胸痹心痛,胃脘疼痛,痛经经闭,产后瘀阻,瘢瘢腹痛,风湿痹痛,筋脉拘挛,跌打损伤,痈肿疮疡等病症。尚未有文献或专利报道乳香精油具有抗HBV活性。
胡椒(Piper nigrum)作为胡椒科胡椒属的多年生木质藤本植物。胡椒未成熟的果实干后,果皮黑色而皱缩,称黑胡椒。《唐本草》记载黑胡椒:“主下气,温中,去痰,除脏中风冷。”黑胡椒温补脾胃肾作用明显,有利尿功效,有助于消肿,主治痛风、猩红热、痢疾及斑疹伤寒等疾病。尚未有文献或专利报道黑胡椒精油具有抗HBV活性。
灰毛莸(Caryopteris forrestii)作为唇形科莸属的一种灌木。灰毛莸的根、叶主要用于疏风,止咳,祛湿,行气。在彝医药系统中,全株入药,用于治疗急、慢性胃炎,小儿疳积等疾病。尚未有文献或专利报道灰毛莸精油具有抗HBV活性。
青花椒(Zanthoxylum schinifolium)作为芸香科花椒属灌木。尚未有文献或专利报道青花椒精油具有抗HBV活性。
艳山姜(Alpinia zerumbet)作为姜科山姜属多年生草本植物。艳山姜精油是从艳山姜的干燥成熟果实中提取得来,具有广泛生物活性,包括抗炎、抗氧化、抗肿瘤以及防治心血管系统疾病等。尚未有文献或专利报道艳山姜精油具有抗HBV活性。
发明内容
本发明的目的在于针对现有技术的不足,提供植物精油在制备治疗乙肝疾病药物或健康产品中的用途。应用HepG2.2.15体外细胞模型研究了300多种植物精油抗HBV活性的效果,发现植物精油具有显著抗HBV活性,可以用于治疗/预防或缓解感染乙肝病毒疾病。
为实现上述目的,本发明采用以下技术方案:
本发明所述的用途是将植物精油作为制备治疗HBV的有效成分,还可以加入一种或多种药物或健康产品上可接受的辅料,并可将药物制成滴鼻剂、喷雾剂、贴剂、膜剂、片剂、凝胶剂、糊剂、漱口剂、丸剂、散剂、颗粒剂、胶囊剂、微囊片剂、内服液体制剂中的任何一种。
所述健康产品可以为化妆品、日用品、食品、保健品中的任何一种。
植物精油为石菖蒲精油、莪术精油、紫苏精油、古巴香脂精油、有机胡萝卜籽精油、有机贞节树精油、乳香精油、灰毛荻精油、青花椒精油、艳山姜精油或黑胡椒精油中的一种或多种;其中,所述石菖蒲精油为菖蒲属植物石菖蒲的地上部分和/或根茎通过水蒸气蒸馏法制备得到的挥发性成分;莪术精油为姜黄属多年生草本植物莪术的根茎通过水汽蒸馏法制备得到的挥发性成分;紫苏精油为紫苏属植物紫苏的地上部分通过水蒸气蒸馏法制备得到的挥发性成分;古巴香脂精油为古巴属古巴香脂树的树脂通过有机溶剂提取得到的挥发性成分;有机胡萝卜籽精油为胡萝卜属植物胡萝卜的种子通过水蒸气蒸馏法制备得到的挥发性成分;有机贞节树精油为杜荆属植物贞节树的果实通过水蒸气蒸馏法制备得到的挥发性成分;乳香精油为乳香属植物乳香的树脂通过水蒸气蒸馏法制备得到的挥发性成分;灰毛荻精油为荻属植物灰毛莸的叶通过水蒸气蒸馏法制备得到的挥发性成分;青花椒精油为花椒属植物青花椒的果实通过水蒸气蒸馏法制备得到的挥发性成分;艳山姜精油为山姜属植物艳山姜的果实跟果皮通过水蒸气蒸馏法制备得到的挥发性成分;黑胡椒精油为胡椒属植物黑胡椒的果实晒干通过水蒸气蒸馏法制备得到的挥发性成分。
在本发明之前尚未发现上述植物精油具有治疗HBV的作用;也尚未发现以上述植物精油为原料制备药物制剂或健康产品用于治疗乙肝疾病的报道。
本发明提供了植物精油的新用途,通过HepG2.2.15体外细胞模型进行筛选,首次发现石菖蒲精油、莪术精油、紫苏精油、古巴香脂精油、有机胡萝卜籽精油、有机贞节树精油、乳香精油、灰毛莸精油、青花椒精油、艳山姜精油和黑胡椒精油对乙肝病毒显示显著抑制活性。石菖蒲精油(jx-6)的HBsAg半数抑制浓度(IC50)为0.09±0.002mg/ml、HBeAg半数抑制浓度(IC50)为0.16±0.002mg/ml、半数毒性浓度(CC50)为0.22±0.02mg/ml;石菖蒲精油(zy-10)的HBsAg IC50为0.09±0.001mg/ml、HBeAg IC50为0.17±0.01mg/ml、CC50为0.27±0.04mg/ml;莪术精油(jx-4)的HBsAg IC50为0.12±0.009mg/ml、CC50为0.27±0.04mg/ml;紫苏SC006(样品)(jx-84)的HBsAg IC50为0.13±0.004mg/ml、CC50为0.18±0.02mg/ml;古巴香脂精油(zy-pl-2)的HBsAg IC50为0.18±0.001mg/ml、CC50为0.14±0.04mg/ml;有机胡萝卜籽精油(野生)(zy-pl-11)的HBsAg IC50为0.17±0.001mg/ml、CC50为0.14±0.04mg/ml;有机贞节树/贞洁树精油(zy-pl-12)的HBsAg IC50为0.18±0.01mg/ml、CC50为0.18±0.02mg/ml;乳香精油(印度)(zy-pl-99)的HBsAg IC50为0.19±0.001mg/ml、CC50为0.26±0.05mg/ml;灰毛莸(干燥)精油(zy-18)的HBsAg IC50为0.07±0.01mg/ml、HBeAg IC50为0.22±0.078mg/ml、CC50为0.15±0.01mg/ml;青花椒(新鲜)精油(zy-23)的HBsAg IC50为0.11±0.002mg/ml、CC50为0.16±0.02mg/ml;艳山姜(果实)(sx-77)的HBsAg IC50为0.09±0.02mg/ml、CC50为0.08±0.01mg/ml;艳山姜(全)(sx-79)的HBsAg IC50为0.07±0.002mg/ml、CC50为0.15±0.01mg/ml;黑胡椒(1080102014)精油(sx-110)的HBsAg IC50为0.15±0.02mg/ml、CC50为0.19±0.02mg/ml。
植物精油具有良好的脂溶性,代谢稳定,较长的半衰期或较小的副作用的特点,制备的药物可用于治疗乙肝疾病。
具体实施方式
本发明涉及的植物精油给药方式可为鼻腔给药、口腔给药、透皮给药、口服给药等途径。鼻腔给药的常见剂型包括滴鼻剂、喷雾剂、贴剂、膜剂等;口腔黏膜给药常用剂型包括片剂、贴剂、凝胶剂、糊剂、膜剂、喷雾剂、漱口剂、丸剂等;透皮给药常用剂型为贴剂;口服剂型包括片剂、散剂、颗粒剂、丸剂、内服液体制剂。
本发明所述可药用载体包括但不限于碳酸钙、磷酸钙、硫酸钙、蔗糖、葡萄糖、乳糖、果糖、木糖醇、山梨醇、淀粉、淀粉糊、纤维素衍生物、明胶、聚乙烯吡咯烷酮、氯化钠、糊精、硬脂酸、硬脂酸镁、硬脂酸钙、植物油、聚乙二醇、无菌磷酸盐缓冲盐水、盐水、林格氏溶液以及它们的组合。
此外,本发明中的植物精油还可以添加到食物和饮料中,用于口服施用,如配制成口香糖,以促进口服递送和吸收。本发明中的植物精油或其组合物还可以添加到化妆品、洗浴用品或大健康产品中,用于香薰、嗅吸、按摩、涂抹、泡浴使用。
本发明涉及的植物精油也可与其他药物活性成分联用,以制备其他的新药物组合物。
本发明的植物精油或其组合用以缓解乙肝疾病症状的证实性疗效和治疗相关活性,是通过细胞模型试验进行证实的。
上述本发明的植物精油或其组合的治疗作用、良好的药物代谢参数和通常无毒性使得本发明化合物成为用于治疗上述病症的理想药物。
下面将结合实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,应当理解为可以以各种形式实现本发明而不应该被所阐述的实施例限制。相反,提供这些实施例是为了更加透彻地理解本发明。实施例中方法如无特殊说明的采用常规方法,使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1
采集产自湖北省的石菖蒲(Acorus tatarinowii)的地上部分,阴凉干燥处晾干,将干燥的石菖蒲样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得石菖蒲精油(jx-6)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定石菖蒲精油(jx-6)中的主要成分为α-细辛醚(>50%)、γ-细辛醚(>17%)、顺式-甲基异丁香油酚(>2%)、4-烯丙基苯甲醚(>2%)、草蒿脑(>2%)。
实施例2
采集产自云南省的石菖蒲(Acortw tatarinowii)的根茎,阴凉干燥处晾干,将干燥的石菖蒲样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得石菖蒲精油(zy-10)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定石菖蒲精油中的成分。石菖蒲(zy-10)精油中的化合物按质量百分比为:α-细辛醚(>85%)、T-muurolol(>2%)、Aciphyllene(>3%)、β-细辛醚(>1%)。
实施例3
采集产自广西壮族自治区的莪术(Curcuma phaeocaulis)的根茎,阴凉干燥处晾干,将干燥的莪术样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得莪术精油(jx-4)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定莪术精油中的成分。莪术(jx-4)精油中的化合物按质量百分比为:樟脑(>15%)、trans-β-elemenone(>9%)、桉叶油醇(>6%)、异龙脑(>5%)、莪术烯(>5%)、β-榄香烯(>4%)、吉马酮(>3%)、乙酸龙脑酯(>3%)、苍术酮(>5%)、α-毕橙茄醇(>2%)。
实施例4
采集产自中国云南的紫苏(Perilla frutescens)的地上部分,阴凉干燥处晾干,将干燥的紫苏样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得紫苏精油(jx-84)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定紫苏精油中的成分。紫苏SC006(jx-84)精油中的化合物按质量百分比为:芳樟醇(>32%)、3-甲基-3-丁烯-1-醇(>21%)、正二十四烷(>10%)、正十七烷(>7%)、正十八烷(>7%)、2,2,2-三氟-1-(1H-咪唑-5-基)乙酮(>4%)、正二十烷(>4%)、β-石竹烯(>3%)、石竹素(>1%)。
实施例5
采集产自巴西的古巴香脂(Copaifera officinalis)树的树脂,阴凉干燥处晾干,将树脂样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。放入浸提设备中,加入乙醇浸提,控制浸提温度在2~80℃,浸提时间为1~48小时,浸提1~3次,然后将浸提液浓缩成浸膏,于常温常压和搅拌下向浸膏加入5wt%的碳酸氢钠溶液萃取1~5次,每次2小时,后静置10小时,收集沉淀层并用醚溶解;再分别用5wt%的碳酸氢钠溶液、1wt%的氢氧化钠溶液梯度洗脱,然后调pH,再用醚萃取1~3次,回收醚,得古巴香脂精油(zy-pl-2)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定古巴香脂精油中的成分。古巴香脂精油(zy-pl-2)中的化合物按质量百分比为:反式石竹烯(>55%)、germacrene D(>11%)、aciphyllene(>3%)、β-细辛醚(>1%)。
实施例6
采集产自加拿大的野生胡萝卜(Daucus carota)的种子,阴凉干燥处晾干,将干燥的胡萝卜籽样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得有机胡萝卜籽精油(野生)(zy-pl-11)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定有机胡萝卜籽精油中的成分。有机胡萝卜籽精油(野生)(zy-pl-11)中的化合物按质量百分比为:桧烯(>29%)、蒎烯(>13%)、(-)-4-萜品醇(>9%)、乙酸香叶酯(>4%)、邻-异丙基苯(>4%)、双戊烯(>4%)、β-蒎烯(>3%)、乙酸龙脑酯(>3%)月桂烯(>2%)、thujone(>1%)、γ-松油烯(>1%)、芳樟醇(>1%)、α-松油醇(>1%)、β-石竹烯(>1%)。
实施例7
采集产自波斯尼亚的贞节树(Vitex agnus castus)的果实,阴凉干燥处晾干,将干燥的样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得有机贞节树精油(zy-pl-12)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定有机贞节树精油中的成分。有机贞节树(zy-pl-12)精油中的化合物按质量百分比为:桧烯(>19%)、桉叶油醇(>19%)、(-)-α-蒎烯(>8%)、(E)-β-金合欢烯(>7%)、(±)-α-乙酸松油酯(>5%)、bicyclogermacrene(>2%)、(-)-萜品-4-醇(>2%)、γ-萜品烯(>2%)、月桂烯(>1%)、香树烯(>1%)、T-杜松醇(>1%)、松油烯(>1%)、α-松油醇(>1%)。
实施例8
采集产自印度的乳香(Boswellia carteri)的树脂,阴凉干燥处晾干,将干燥的样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得乳香精油(zy-pl-99)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定乳香精油中的成分。乳香(zy-pl-99)精油中的化合物按质量百分比为:侧柏酮(>57%)、桧烯(>5%)、4-烯丙基苯甲醚(>4%)、(+)-3-蒈烯(>4%)、柠檬烯(4%)、邻-异丙基苯(3%)、(-)-4-萜品醇(>2%)、α-水芹烯(>2%)、γ-松油烯(>2%)。
实施例9
采集产自云南省的灰毛莸(Caryopteris forrestii Diels)的叶,阴凉干燥处晾干,将干燥的灰毛莸样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得灰毛莸(干燥)精油(zy-18)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定灰毛莸(干燥)中的成分。灰毛莸(干燥)精油(zy-18)中的化合物按质量百分比为:石竹素(>9%)、4-异丙基甲苯(>7%)、(-)-4-萜品醇(>6%)、桃金娘烯醇(>6%)、3-辛炔(>3%)、Bicyclo[7.2.0]undecan-3-ol,11,11-dimethyl-4,8-bis(methylene)-(>3%)、(-)-反式一松香芹醇(>3%)、(-)-α-蒎烯(>3%)、2,4(10)-thujadiene(>3%)、2-辛炔(>3%)、5-Isopropylbicyclo<3.1.0>-hex-3-en-2-on(>3%)、2-(4-甲基苯基)丙-2-醇(>2%)、α-cadinol(>2%)、4-异丙基苯甲醇(>2%)、3-Furanpropanol(>2%)、cis-Sabinol(>2%)。
实施例10
采集产自云南省的青花椒(Zanthoxylum schinifolium)的果实,阴凉干燥处晾干,将干燥的青花椒果实样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得青花椒(新鲜)精油(zy-23)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定青花椒精油中的成分。青花椒(新鲜)精油(zy-23)中的化合物按质量百分比为:(+)-3-蒈烯(>92%)、7,7-二甲基-2-亚甲基-双环[2.2.1]庚烷(>1%)、氯仿(>3%)、柠檬烯(>0.5%)。
实施例11
采集自云南省的艳山姜(Alpinia zerumbet)的果实,阴凉干燥处晾干,将干燥的艳山姜样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得艳山姜精油(sx-77)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定艳山姜精油中的成分。艳山姜(sx-77)精油中的化合物按质量百分比为:(-)-β-蒎烯(>20%)、桧烯(>16%)、δ-杜松烯(>8%)、T-muurolol(>5%)、(+)-γ-cadinene(>5%)、α-cadinol(>4%)、(-)-α-蒎烯(>4%)、β-石竹烯(>3%)、α-muurolene(>2%)、α-律草烯(>2%)、2-epi-trans-β-caryophyllene(>1%)、cubebol(>1%)、β-榄香烯(>1%)、γ-muurolene(>1%)。
实施例12
采集自云南省的艳山姜(Alpinia zerumbet)的果实和果皮,阴凉干燥处晾干,将干燥的艳山姜样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得艳山姜精油(sx-79)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定艳山姜精油中的成分。艳山姜(sx-79)精油中的化合物按质量百分比为:β-侧柏烯(>18%)、(-)-β-蒎烯(>17%)、(-)-α-蒎烯(>6%)、(+)-δ杜松烯(>6%)、T-muurolol(>6%)、(-)-α-杜松醇(>4%)、β-石竹烯(>3%)、(±)-γ杜松烯(>3%)、4-异丙基甲苯(>1%)、石竹素(>1%)、γ-muurolene(>1%)。
实施例13
采集产自越南的黑胡椒(Piper nigrum)的果实,阴凉干燥处晾干,将干燥的黑胡椒样品用水冷式增速高效流水粉碎机粉碎,过药典标准筛3号筛。将样品放入圆底烧瓶,加入8倍量纯水,搅拌混匀,浸泡1h后采用水蒸气蒸馏法提取7小时,温度为60-70℃。收集蒸馏液,加入少量乙酸乙酯萃取3次,再加入无水硫酸钠除水,静置过夜,使乙酸乙酯挥发,分离无水硫酸钠后得黑胡椒精油(sx-110)。用气相色谱-质谱联用仪分析,并用Nist.14谱库联合检索,确定黑胡椒精油中的成分。黑胡椒(sx-110)精油中的化合物按质量百分比为:β-石竹烯(>25%)、α-蒎烯(>21%)、(+)-柠檬烯(>13%)、β-蒎烯(>8%)、α-水芹烯(>3%)、γ-榄香烯(>3%)、β-selinene(>2%)、β-榄香烯(>2%)、α-selinene(>2%)、月桂烯(>1%)。
实施例14
体外抗HBV作用的评价
材料与方法:使用稳定表达HBV的人肝母细胞瘤细胞系---HepG2.2.15。HepG2.2.15细胞系购自武汉大学细胞典藏中心,培养于含10%胎牛血清和380μg/ml G418的MEM培养基中。用MTT法检测细胞毒性,用商用ELISA试剂盒(上海科华生物工程股份有限公司)检测细胞培养上清液中的HBsAg、HBeAg水平。
试验过程:药物母液配置---精油用DMSO配置成100mg/ml的母液,初筛阶段临用前使用培养液进行稀释,作用浓度0.2mg/ml。复筛时挑选抗原抑制率>30%的精油进行复筛,加药处理前使用培养液对精油母液进行梯度稀释,作用梯度为0.4、0.2、0.1、0.05、0.05、0.0125mg/ml。
待细胞密度达90%时,将细胞用胰酶消化离心,接种48孔板。24h后加药处理(培养基不含G418),设置阳性对照组(PAC5)、溶剂对照组(含0.1%二甲基亚砜的培养液)、药物处理组,继续培养72h。处理结束时,收集上清用于抗原检测,每孔加入20μL 5mg/ml MTT溶液,再孵育4h。使用多功能酶标仪(INFINITE 200PRO)进行检测,对细胞毒性进行评估。用商用ELISA试剂盒(上海科华生物工程股份有限公司)检测细胞培养上清液中的HBsAg、HBeAg水平,检测步骤按说明书进行。
HBsAg检测,75μL上清液加到对应的孔板内(过高时需要稀释处理)37℃孵育1h。1h后每孔加入50μL酶结合物,敲打混匀,37℃孵育30min。手工洗板五次。加底物显色:显色液A和显色液B各50μL,37℃孵育30min。每孔加入50μL终止液,读板,OD450,OD630数值。
HBeAg检测,50μL上清液加到对应的孔板内(10μL上清加40μL PBS作5倍稀释)。每孔加50μL酶结合物,与样品敲打混匀。37℃孵育30min。手工洗板五次。加底物显色:显色液A和显色液B各50μL,37℃孵育15min。每孔加入50μL终止液,读板,OD450,OD630数值。
结果如表1所示,上述植物精油对HBsAg/HBeAg有较明显的抑制作用。
表1
上述实施例用来解释说明本发明,而不是对本发明进行限制,在本发明的精神和权利要求的保护范围内,对本发明做出的任何修改和改变,都落入本发明的保护范围。
Claims (3)
1.植物精油在制备治疗乙肝疾病药物或健康产品中的应用。
2.根据权利要求1所述的应用,其特征在于,植物精油为石菖蒲精油、莪术精油、紫苏精油、古巴香脂精油、有机胡萝卜籽精油、有机贞节树精油、乳香精油、灰毛荻精油、青花椒精油、艳山姜精油或黑胡椒精油中的一种或多种;其中,所述石菖蒲精油为菖蒲属植物石菖蒲的地上部分和/或根茎通过水蒸气蒸馏法制备得到的挥发性成分;莪术精油为姜黄属多年生草本植物莪术的根茎通过水汽蒸馏法制备得到的挥发性成分;紫苏精油为紫苏属植物紫苏的地上部分通过水蒸气蒸馏法制备得到的挥发性成分;古巴香脂精油为古巴属古巴香脂树的树脂通过有机溶剂提取得到的挥发性成分;有机胡萝卜籽精油为胡萝卜属植物胡萝卜的种子通过水蒸气蒸馏法制备得到的挥发性成分;有机贞节树精油为杜荆属植物贞节树的果实通过水蒸气蒸馏法制备得到的挥发性成分;乳香精油为乳香属植物乳香的树脂通过水蒸气蒸馏法制备得到的挥发性成分;灰毛荻精油为荻属植物灰毛莸的叶通过水蒸气蒸馏法制备得到的挥发性成分;青花椒精油为花椒属植物青花椒的果实通过水蒸气蒸馏法制备得到的挥发性成分;艳山姜精油为山姜属植物艳山姜的果实和/或果皮通过水蒸气蒸馏法制备得到的挥发性成分;黑胡椒精油为胡椒属植物黑胡椒的果实晒干通过水蒸气蒸馏法制备得到的挥发性成分。
3.根据权利要求1所述的应用,其特征在于,所述乙肝疾病药物为片剂、丸剂、胶囊剂、颗粒剂、微囊片剂、混悬剂、滴丸、口服液体、注射剂、气雾剂、栓剂、皮下给药剂型。
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