CN117567424A - 一种链霉菌-芽孢杆菌共培养制备含硫吡喃萘醌类化合物的方法及其应用 - Google Patents
一种链霉菌-芽孢杆菌共培养制备含硫吡喃萘醌类化合物的方法及其应用 Download PDFInfo
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- CN117567424A CN117567424A CN202311548533.9A CN202311548533A CN117567424A CN 117567424 A CN117567424 A CN 117567424A CN 202311548533 A CN202311548533 A CN 202311548533A CN 117567424 A CN117567424 A CN 117567424A
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- sulfur
- compound
- frenolicin
- streptomyces
- crude extract
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Abstract
本发明阐述一种利用玫瑰石斛内生链霉菌Streptomyces sp.MG‑F‑1(CGMCCNO:28777)与蜡状芽孢杆菌Bacillus cereus共培养发酵，获得新型含硫吡喃萘醌类化合物的制备方法与应用。所述的含硫吡喃萘醌类化合物为从自然界首次分离得到。体外抗炎抑菌活性试验表明，本发明提供的含硫吡喃萘醌类化合物具有较好的抗炎抑菌活性。体内试验表明共培养发酵粗提物与石斛多糖组合物能够有效治疗金黄色葡萄球菌性小鼠乳腺炎。
Description
技术领域
本发明涉及一种链霉菌-芽孢杆菌共培养制备含硫吡喃萘醌类化合物的方法及其应用，属于医药技术领域。
背景技术
玫瑰石斛(Dendrobium crepidatum Lindl.et Paxt)为兰科石斛属多年生附生草本植物，主要分布于云南南部至西南部(勐海、勐腊、镇康、沧源)、贵州西南部(兴义、罗甸)等地，生于海拔1000-1800米的山地疏林中树干或山谷岩石上。境外分布于东南亚印度、尼泊尔、缅甸、越南等地，具有益胃生津，滋阴清热，润肺止咳等功效。现代研究表明，石斛等兰科植物具有与菌物形成典型共生关系的微生态系统特征，如内生菌对石斛具有促进其植物生长、增强抗性、诱导次生代谢产物合成等功能，是影响药材品质的重要因素。而内生菌在与宿主的协同作用下，能产生一系列特殊的次生代谢产物，它们可能是与宿主药材相同或类似甚至更强的活性物质。
通过影响、改造微生物的基因序列，激活内生菌“沉默”基因表达，进而改变微生物酶体系诱发新的酶催化反应，获得一系列结构新颖的类天然产物新骨架，可以丰富活性次生代谢产物的结构多样性。其中，改变培养条件被认为是最简单、最有效的策略，被称为“一株多化合物(OSMAC)”。包括改变培养基、改变培养条件、与其他菌株共培养、添加表观遗传修饰剂或生物合成前体。课题组前期从玫瑰石斛中分离得到了大量的内生放线菌，经过系列的化学和活性筛选，选择了玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)，并发现利用共培养方式，蜡状芽孢杆菌Bacillus cereus能够提高MG-F-1发酵产物中目标产物的结构多样性，最近我们发现一类具有抗炎抑菌活性的新含硫吡喃萘醌类化合物。
发明内容
发明目的：本发明的主要目的在于提供一类新型含硫吡喃萘醌类化合物Frenolicin H-L(I-V)及其制备方法和在抗炎抑菌方面的应用。
本发明是从玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus共培养发酵液中，通过分离纯化得到的一类新型含硫吡喃萘醌类化合物，命名为Frenolicin H-L(I-V)，其化学结构式如下：
本发明提出的Frenolicin H-L可以通过上述方法提取，经分离纯化得到，也可以是经化学合成的方法得到。
上述化合物的制备方法，通过以下步骤实现：
(1)发酵：取玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus各2mL，用100-200mL麦麸培养基(葡萄糖10.0g/L；玉米粉40.0g/L；麸质粉5.0g/L；麸皮10.0g/L；K2HPO4·3H2O 0.5g/L；CaCO3 2.0g/L；(NH4)2SO41.0g/L)，在26-30℃条件下共培养3-5天，得到的培养液取1mL，接种至100mL的麦麸培养基(共计10-20L)进行发酵，26-30℃条件下培养7-10天，得到发酵液。
(2)提取：将得到的发酵液离心，得到菌丝体和上清液。菌丝体直接采用甲醇超声提取3-5次，浓缩后得到粗体物A；上清液加入3％-6％的非离子型大孔树脂，搅拌4-8小时后过滤，弃去滤液，大孔树脂用甲醇洗脱至无色，回收甲醇得到粗提物B。合并粗提物A和B，经减压浓缩得到发酵液粗提物C。
(3)分离：将上述粗提物C分别采用MCI分离，10％，20％，40％，60％，80％，100％甲醇洗脱；得到12个流分(Fr.1-1to Fr.1-12)，浓缩得干燥样品，Fr.1-4采用凝胶柱层析进行分离得到4个流分(Fr.1-4-1to Fr.1-4-4)，Fr.1-4-4采用制备液相(30％MeOH)，即得化合物V(Frenolicin L)；Fr.1-5采用凝胶柱层析和制备液相(40％MeOH)，即得化合物III(Frenolicin J)；Fr.1-10采用凝胶柱层析和制备液相(60％MeOH)，即得化合物IV(Frenolicin K)；Fr.1-11采用凝胶柱层析和制备液相(80％MeOH)，即得化合物I(Frenolicin H)和化合物II(Frenolicin I)。
所述含硫吡喃萘醌类化合物Frenolicin H-L(I-V)在制备抗炎抑菌药物中的应用。
所述含硫吡喃萘醌类化合物Frenolicin H-L(I-V)，加入药物制剂辅料制成制剂。
所述含硫吡喃萘醌类化合物Frenolicin H-L(I-V)，制剂为粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、崩解剂、口崩剂和微丸中的一种。
一种治疗乳腺炎药物，将所述方法制备的发酵液粗提物C，放入真空冷冻干燥机中进行干燥，直至恒重，制成共培养发酵粗提物粉末，与石斛粗多糖组合制备成治疗乳腺炎药物，其中共培养发酵粗提物与石斛粗多糖两组分之间的重量比为5-0.2：1。
所述治疗乳腺炎药物，石斛粗多糖的制备方法：取鲜玫瑰石斛，加入适量95％乙醇鲜榨脱脂，过滤，挥干溶剂，滤渣加30倍量水榨汁，过滤，滤液加入乙醇，慢加快搅，至溶液中乙醇含量达到80％使多糖沉淀，静置过夜，沉淀离心，去上清液，沉淀放入-80℃冰箱冷冻6h，再放入真空冷冻干燥机中进行干燥，直至恒重，制成粉末，即得石斛粗多糖。
一种内生链霉菌，该内生链霉菌为玫瑰石斛内生链霉菌(Streptomyces sp.)MG-F-1保藏在中国微生物菌种保藏管理委员会普通微生物中心，其保藏号为CGMCC NO:28777，保藏时间为2023年10月26日，保藏地址为北京市朝阳区北辰西路1号院3号。
本发明的有益之处在于：
(1)提供一类从玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus共培养发酵液中提取的具有较好抗炎抑菌活性的新型含硫吡喃萘醌类化合物,制成相应制剂，可用于抗炎抑菌以及相关病症的药物治疗。
(2)提供一类能够有效治疗金黄色葡萄球菌性小鼠乳腺炎的组合物，组合物中两种有效成分表现为增效效果，比单个成分活性更为显著。同时组合物的两种成分化学结构差异很大，作用机理完全不同，不存在交互抗性，可延缓单独使用所产生的抗性问题。
附图说明
图1为化合物Frenolicin H-L(I-V)的结构式图；
图2为化合物I的1H NMR谱(DMSO-d6,500MHz)；
图3为化合物I的13C NMR谱(DMSO-d6,125MHz)；
图4为化合物I的高分辨质谱图；
图5为化合物II的1H NMR谱(DMSO-d6,500MHz)；
图6为化合物II的13C NMR谱(DMSO-d6,125MHz)；
图7为化合物II的高分辨质谱图；
图8为化合物III的1H NMR谱(DMSO-d6,500MHz)；
图9为化合物III的13C NMR谱(DMSO-d6,125MHz)；
图10为化合物III的高分辨质谱图；
图11为化合物IV的1H NMR谱(DMSO-d6,500MHz)；
图12为化合物IV的13C NMR谱(DMSO-d6,125MHz)；
图13为化合物IV的高分辨质谱图；
图14为化合物V的1H NMR谱(DMSO-d6,500MHz)；
图15为化合物V的13C NMR谱(DMSO-d6,125MHz)；
图16为化合物V的高分辨质谱图。
具体实施方式
下面将结合实施例进一步详细说明本发明的实质内容和有益效果，这些实施例仅用于说明本发明而非对本发明的限制。此外，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1：新型含硫吡喃萘醌类化合物Frenolicin H-L
取玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus菌液各2mL，用200mL麦麸培养基(葡萄糖10.0g/L；玉米粉40.0g/L；麸质粉5.0g/L；麸皮10.0g/L；K2HPO4·3H2O 0.5g/L；CaCO3 2.0g/L；(NH4)2SO4 1.0g/L)，在28℃条件下共培养4天。得到的培养液取1mL，接种至100mL的麦麸培养基(共10L)进行发酵，28℃培养7天，得到发酵液。将得到的发酵液离心，得到菌丝体和上清液。菌丝体直接采用甲醇超声提取3次，浓缩后得到粗体物A；上清液加入3％的非离子型大孔树脂，搅拌6小时后过滤，弃去滤液，大孔树脂用甲醇洗脱至无色，回收甲醇得到粗提物B；粗体物A和B合并，经旋转蒸发仪减压浓缩得到101.26g发酵液粗提物。
将上述粗提物采用MCI分离，10％，20％，40％，60％，80％，100％甲醇洗脱；得到12个流分(Fr.1-1to Fr.1-12)，浓缩得干燥样品，Fr.1-4采用凝胶柱层析进行分离得到4个流分(Fr.1-4-1to Fr.1-4-4)，Fr.1-4-4采用制备液相(30％ MeOH)，即得化合物V(Frenolicin L)；Fr.1-5采用凝胶柱层析和制备液相(40％ MeOH)，即得化合物III(Frenolicin J)；Fr.1-10采用凝胶柱层析和制备液相(60％ MeOH)，即得化合物IV(Frenolicin K)；Fr.1-11采用凝胶柱层析和制备液相(80％ MeOH)，即得化合物I(Frenolicin H)和化合物II(Frenolicin I)。
Frenolicin H-L的质谱数据为：Frenolicin H:(-)-HRESI-MS:m/z 425.0720[M-H]-(C19H22O7S2)。Frenolicin I:(-)-HRESI-MS:m/z 757.1602[M-H]-(C36H38O14S2)。Frenolicin J:(+)-HRESI-MS:m/z 576.2010[M+H]+(C27H33N3O9S)。Frenolicin K:(+)-HRESI-MS:m/z 727.2045[M+H]+(C36H38O14S)。Frenolicin L:(+)-HRESI-MS:m/z833.2665[M+H]+(C35H48N2O9S)。5个新化合物I-V的结构式见附图1，核磁数据见表1-3，核磁与质谱谱图见附图2-16。
表1.新化合物I-II的氢谱(500MHz)和碳谱(125MHz)数据(DMSO-d6)
表2.新化合物III-IV的氢谱(500MHz)和碳谱(125MHz)数据(DMSO-d6)
表3.新化合物V的氢谱(500MHz)和碳谱(125MHz)数据(DMSO-d6)
实施例2：新型含硫吡喃萘醌类化合物Frenolicin H-L
取玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus各菌液2mL，用100mL麦麸培养基(葡萄糖10.0g/L；玉米粉40.0g/L；麸质粉5.0g/L；麸皮10.0g/L；K2HPO4·3H2O 0.5g/L；CaCO3 2.0g/L；(NH4)2SO4 1.0g/L)，在28℃条件下共培养5天。得到的培养液取1mL，接种至100mL的麦麸培养基(共10L)进行发酵，28℃培养10天，得到发酵液。将得到的发酵液离心，得到菌丝体和上清液。菌丝体直接采用甲醇超声提取3次，浓缩后得到粗体物A；上清液加入3％的非离子型大孔树脂，搅拌6小时后过滤，弃去滤液，大孔树脂用甲醇洗脱至无色，回收甲醇得到粗提物B；粗体物A和B合并，经旋转蒸发仪减压浓缩得到95.77g发酵液粗提物。
将上述粗提物采用MCI分离，10％，20％，40％，60％，80％，100％甲醇洗脱；得到12个流分(Fr.1-1to Fr.1-12)，浓缩得干燥样品，Fr.1-4采用凝胶柱层析进行分离得到4个流分(Fr.1-4-1to Fr.1-4-4)，Fr.1-4-4采用制备液相(30％ MeOH)，即得化合物V(Frenolicin L)；Fr.1-5采用凝胶柱层析和制备液相(40％ MeOH)，即得化合物III(Frenolicin J)；Fr.1-10采用凝胶柱层析和制备液相(60％ MeOH)，即得化合物IV(Frenolicin K)；Fr.1-11采用凝胶柱层析和制备液相(80％ MeOH)，即得化合物I(Frenolicin H)和化合物II(Frenolicin I)。
实施例3：玫瑰石斛内生链霉菌与芽孢杆菌共培养发酵粗提物的制备
取玫瑰石斛内生链霉菌Streptomyces sp.MG-F-1(CGMCC NO:28777)与蜡状芽孢杆菌Bacillus cereus菌液各2mL，用200mL麦麸培养基(葡萄糖10.0g/L；玉米粉40.0g/L；麸质粉5.0g/L；麸皮10.0g/L；K2HPO4·3H2O 0.5g/L；CaCO3 2.0g/L；(NH4)2SO41.0g/L)，在28℃条件下共培养4天。得到的培养液取1mL，接种至100mL的麦麸培养基(共10L)进行发酵，28℃培养7天，得到发酵液。将得到的发酵液离心，得到菌丝体和上清液。菌丝体直接采用甲醇超声提取3次，浓缩后得到粗体物A；上清液加入3％的非离子型大孔树脂，搅拌6小时后过滤，弃去滤液，大孔树脂用甲醇洗脱至无色，回收甲醇得到粗提物B；粗体物A和B合并，经减压浓缩得到发酵液粗提物C，放入真空冷冻干燥机中进行干燥，直至恒重，制成粉末，即得共培养发酵粗提物96.77g。
实施例4：石斛粗多糖的制备：
取1000g鲜玫瑰石斛，加入适量的95％乙醇鲜榨脱脂，过滤，挥干溶剂，滤渣加30倍量水榨汁，过滤，滤液加入乙醇，慢加快搅，至溶液中乙醇含量达到80％使多糖沉淀，静置过夜，沉淀离心，去上清液，沉淀放入-80℃冰箱冷冻6h，再放入真空冷冻干燥机中进行干燥，直至恒重，制成粉末，即得石斛粗多糖30.65g。
实施例5：体外抗炎活性评价
取小鼠单核巨噬细胞白血病细胞RAW264.7，在添加10％胎牛血清的DMEM培养基培养。将实施例1方法制备的Frenolicin H-L(分别为化合物I、II、III、IV、V)配制成20，10，1，0.1，0.01μM的溶液，阳性对照采用地塞米松10μM。取对数生长期的RAW264.7细胞，以4×105cell/mL接种于96孔板(每孔200μL混悬液)，培育4h待细胞贴壁后，将上清液换为加药培养基，培育4h后，除空白对照组外各组加入LPS刺激(终浓度为5μg/mL)。置37℃，5％ CO2条件下培养20h，取50μL上清液加入到96孔板中，接着按照一氧化氮试剂盒的方法操作，于540nm处测定各孔吸光度，取平均值，计算NO释放抑制率(％)＝(模型组OD值-样品组OD值)/(模型组OD值-空白组OD值)×100％，利用Graphpad软件计算IC50值。结果见表4。
表4.新化合物I-V对LPS诱导RAW264.7产生NO的抑制率
| 化合物 | IC50,μM |
| I | 0.76 |
| II | 1.61 |
| III | 1.17 |
| IV | 0.92 |
| V | 0.22 |
结果显示：化合物I-V对LPS诱导RAW264.7产生NO均表现出较好的抑制活性，相对来说含单硫结构活性比双硫好。
实施例6：体外抑菌活性评价
测试抑菌活性的菌株为大肠杆菌B.subtilis(A186)、金黄色葡萄球菌S.aureus(ATCC29213)、大肠杆菌E.coli(ATCC 25922)、鲍曼不动杆菌A.baumannii(ATCC19606)和铜绿色假单胞菌P.aeruginosa(ATCC 27853)。将5种供试菌接种于LB肉汤培养基中，37℃孵育过夜。细菌隔夜培养计数是通过紫外分光光度计量化，稀释至106CFU/mL(OD600＝0.02)。将实施例1方法制备的化合物I-V用DMSO溶解，配成50mM储备液，4℃保存，使用时用LB培养液稀释成100μM。阳性对照组为氨苄青霉素Ampicillin(50mM)和多粘菌素B Polymyxin B(50mM)。通过二倍稀释法和96孔板对分离得到的化合物I-V进行抑菌活性测试，得到化合物I-V对指定菌的最小抑制浓度(MICs)。结果见表5。
表5化合物I-V的抗菌活性的MICs值(μM)
注：“-”表示抗菌活性大于100μM，“/”表示未测，*μg/mL
结果显示，化合物I和V对革兰氏阳性菌大肠杆菌和金黄色葡萄球菌表现出较好的抑菌活性，活性接近阳性对照氨苄青霉素。文献报道吡喃萘醌类(Pyranonaphthoquinones，PNQs)是II型聚酮类抗生素，主要来源于微生物。但在天然PNQs中，以S桥作为连接比较少见，目前被报道的只有(+)-BE-52440A、BE-52440B、frenolicin G、hypogeamicin A和naquihexcin A/B，而生物体中的低分子量硫醇在保护机体免疫受各种内源性和外源性应激等氧化还原过程中具有重要意义。故本发明制备的新型含硫吡喃萘醌类化合物作为抗炎抑菌药物以及其他相关病症药物具有一定的优势。
实施例7：共培养发酵粗提物与石斛粗多糖组合物的制备
取实施例3制备的玫瑰石斛内生链霉菌与蜡状芽孢杆菌共培养发酵粗提物、实施例4制备的石斛粗多糖，分别按照重量比5-0.2：1混合的制备组合物，具体方法为：将共培养发酵粗提物按照重量比5：1与石斛粗多糖组合制备成组合物1；按照重量比1：1与石斛粗多糖组合制备成组合物2；按照重量比1：5与石斛粗多糖组合制备成组合物3，进行下述实验。
实施例8：组合物对金黄色葡萄球菌性小鼠乳腺炎的影响
取金黄色葡萄球菌菌株接种于普通肉汤，镜检无杂菌者接种于血液琼脂斜面，培养24小时后，接种于营养琼脂平皿上，培养扩增后制备成细菌混悬液。采用平板计数法将细菌悬液调至1×104CFU/mL，4℃保存备用。取制备的金葡菌混悬液和雌性BALB/c小鼠，随机分组，对模型组及给药组小鼠第四对乳腺注射50uL金葡菌混悬液，对照组注射等量生理盐水，注射后对各给药组灌胃给药，剂量均为50mg/kg，模型组和对照组给予等量生理盐水，24小时后再次相同剂量给药，在饲养两天后，处死小鼠取乳腺组织，部分组织匀浆采用平板计数法测定乳腺组织中金葡菌菌落数，部分组织匀浆后，离心15min取上清液，采用ELISA试剂盒测定TNF-α含量。结果见表6。
表6不同比例组合物对小鼠乳房组织中金葡菌数量和TNF-α含量影响
| 处理 | 菌落数(CFU×105) | TNF-α(ng/ml) |
| 对照组 | - | 4.8 |
| 模型组 | 28.3 | 12.9 |
| 发酵粗提物组 | 18.5 | 7.3 |
| 石斛粗多糖组 | 21.7 | 9.7 |
| 组合物1(5:1) | 9.8 | 5.8 |
| 组合物2(1:1) | 12.6 | 7.9 |
| 组合物3(1:5) | 16.5 | 8.4 |
结果显示，本发明制备的发酵粗提物、石斛粗多糖和不同比例组合物，均能不同程度降低小鼠乳房组织中金黄色葡萄球菌数量和TNF-α含量，组合物活性优于单独给药，说明共培养发酵粗提物与石斛粗多糖有显著的协同增效作用。
以上实施方式只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人了解本发明内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所做的等效变化或修饰，都应涵盖在本发明的保护范围内。
Claims (8)
1.一种含硫吡喃萘醌类化合物，其结构式选自如下五种化合物中的任意一种，
2.一种根据权利要求1所述含硫吡喃萘醌类化合物的制备方法，其特征在于，该方法是利用玫瑰石斛内生链霉菌(Streptomyces sp.MG-F-1)CGMCC NO:28777与蜡状芽孢杆菌(Bacillus cereus)共培养发酵制备获得，具体包括如下步骤：
(1)取玫瑰石斛内生链霉菌(Streptomyces sp.)MG-F-1CGMCC NO:28777与蜡状芽孢杆菌(Bacillus cereus)菌液各2mL，用100-200mL麦麸培养基，培养基的配方为葡萄糖10.0g/L；玉米粉40.0g/L；麸质粉5.0g/L；麸皮10.0g/L；K2HPO4·3H2O 0.5g/L；CaCO3 2.0g/L；(NH4)2SO4 1.0g/L，在26-30℃条件下共培养3-5天；
(2)将步骤(1)得到的培养液取1mL，接种至100mL的麦麸培养基，共计10-20L，进行发酵，26-30℃条件下培养7-10天，得到发酵液；
(3)将步骤(2)得到发酵液离心，得到菌丝体和上清液；菌丝体直接采用甲醇超声提取3-5次，浓缩后得到粗提物A；上清液加入3％-6％的非离子型大孔树脂，搅拌4-8小时后过滤，弃去滤液，大孔树脂用甲醇洗脱至无色，回收甲醇得到粗提物B；合并粗提物A和B，经减压浓缩得到发酵液粗提物C；
(4)将步骤(3)得到的粗提物C采用MCI分离，10％，20％，40％，60％，80％，100％甲醇洗脱；得到12个流分(Fr.1-1to Fr.1-12)，浓缩得干燥样品，Fr.1-4采用凝胶柱层析进行分离得到4个流分(Fr.1-4-1to Fr.1-4-4)，Fr.1-4-4采用制备液相(30％MeOH)，即得化合物V(Frenolicin L)；Fr.1-5采用凝胶柱层析和制备液相(40％MeOH)，即得化合物III(Frenolicin J)；Fr.1-10采用凝胶柱层析和制备液相(60％MeOH)，即得化合物IV(Frenolicin K)；Fr.1-11采用凝胶柱层析和制备液相(80％MeOH)，即得化合物I(Frenolicin H)和化合物II(Frenolicin I)。
3.根据权利要求1所述含硫吡喃萘醌类化合物在制备抗炎抑菌药物中的应用。
4.根据权利要求3所述含硫吡喃萘醌类化合物在制备抗炎抑菌药物中的应用，其特征在于，所述药物是将所述含硫吡喃萘醌类化合物加入药物制剂辅料制成制剂。
5.根据权利要求4所述含硫吡喃萘醌类化合物在制备抗炎抑菌药物中的应用，其特征在于，所述制剂为粉针剂、颗粒剂、片剂、冲剂、散剂、口服液、糖衣剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、丸剂、膏剂、丹剂、喷雾剂、滴丸剂、崩解剂、口崩剂和微丸中的一种。
6.一种治疗乳腺炎药物，其特征在于，将权利要求2所述方法制备的发酵液粗提物C，放入真空冷冻干燥机中进行干燥，直至恒重，制成共培养发酵粗提物粉末，与石斛粗多糖组合制备成治疗乳腺炎药物，其中共培养发酵粗提物与石斛粗多糖两组分之间的重量比为5-0.2：1。
7.根据权利要求6所述治疗乳腺炎药物，其特征在于，石斛粗多糖的制备方法：取鲜玫瑰石斛，加入适量95％乙醇鲜榨脱脂，过滤，挥干溶剂，滤渣加30倍量水榨汁，过滤，滤液加入乙醇，慢加快搅，至溶液中乙醇含量达到80％使多糖沉淀，静置过夜，沉淀离心，去上清液，沉淀放入-80℃冰箱冷冻6h，再放入真空冷冻干燥机中进行干燥，直至恒重，制成粉末，即得石斛粗多糖。
8.一种内生链霉菌，其特征在于：该内生链霉菌为玫瑰石斛内生链霉菌(Streptomycessp.)MG-F-1，其保藏号为CGMCC NO:28777。
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| SUPONG, KHOMSAN等: "Frenolicins H and I from the caterpillar-associated Streptomyces sp. TBRC17107", NATURAL PRODUCT RESEARCH, 4 October 2023 (2023-10-04), pages 1 - 10 * |
| XIACHANG WANG等: "Frenolicins C−G, Pyranonaphthoquinones from Streptomyces sp. RM- 4-15", J. NAT. PROD., vol. 76, 14 August 2013 (2013-08-14), pages 1441, XP055483416, DOI: 10.1021/np400231r * |
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