CN114539192B - 松香烷型二萜化合物及其制备方法和应用 - Google Patents
松香烷型二萜化合物及其制备方法和应用 Download PDFInfo
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- CN114539192B CN114539192B CN202011293368.3A CN202011293368A CN114539192B CN 114539192 B CN114539192 B CN 114539192B CN 202011293368 A CN202011293368 A CN 202011293368A CN 114539192 B CN114539192 B CN 114539192B
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- diterpenoid compound
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- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 title claims description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
- A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/60—Salicylic acid; Derivatives thereof
- A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
- A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
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- A61P19/00—Drugs for skeletal disorders
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
本发明公开了一种松香烷型二萜化合物及其制备方法和应用,该松香烷型二萜化合物如式I所示,其能够有效抑制RAW264.7细胞和BV2细胞中TNF‑α、IL‑1β和/或NO的含量,具有显著的抗炎活性,可用于制备预防、治疗和/或缓解炎症疾病的药物。
Description
技术领域
本发明涉及松香烷型二萜化合物及其制备方法和应用。
背景技术
炎症指的是具有血管系统的活体组织对于损伤因子所引起的防御反应。炎症过程的中心环节是血管反应,主要表现为红,肿,热,痛和功能障碍。炎症与多种疾病的发病机制有关,包括心脑血管疾病,糖尿病,类风湿关节炎等。
香青兰(Dracocephalum moldavica L.)为唇形科青兰属植物,又名巴迪然吉布亚,其味辛,苦,性凉。具有清肺解表,凉肝止血,通路开窍,止痛解毒的作用。松香烷型二萜类成分则首次从香青兰中分离得到,该类化合物的生物活性主要分为两类,一类是在植物体内作为防御性成分,可以起到防止昆虫取食,抗植物病原菌等作用;另外一类是作为药用活性成分,有抗溃疡、抗肿瘤、抗疟疾、抗菌、抗病毒活性等。
发明内容
本发明所要解决的技术问题是提供一种松香烷型二萜化合物及其制备方法和应用。本发明的松香烷型二萜化合物具有抗炎活性。
本发明通过下述技术方案来解决上述的技术问题。
本发明提供了一种如式I所示的松香烷型二萜化合物或其药学上可接受的盐,
本发明提供了一种如式I所示的松香烷型二萜化合物的制备方法,其包括如下步骤:从香青兰(Dracocephalum moldavica L.)中提取分离得到所述如式I所示的松香烷型二萜化合物。
本发明中,较佳地,从香青兰提取物中分离得到所述如式I所示的松香烷型二萜化合物。
本发明中,所述从香青兰提取物中分离的过程可以包括如下步骤:将香青兰提取物依次经硅胶柱层析、凝胶柱层析、反相硅胶ODS-C18层析和HPLC分离获得所述如式I所示的松香烷型二萜化合物。
本发明中,所述香青兰提取物进行硅胶柱层析时,较佳地收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=100:1时Rf值=0.60的组分A。所述硅胶柱层析优选以石油醚-丙酮为洗脱剂,更优选梯度洗脱,较佳地所述梯度洗脱中洗脱剂石油醚-丙酮的体积比依次为100:1、50:1、20:1、10:1、5:1、2:1和1:1。
本发明中,所述组分A进行凝胶柱层析时,较佳地收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=50:1时Rf=0.53~0.54的组分B。所述凝胶柱层析中的固定相优选羟丙基葡聚糖凝胶;所述凝胶柱层析优选以二氯甲烷-甲醇为洗脱剂,较佳地所述洗脱剂二氯甲烷-甲醇的体积比为1:1。
本发明中,所述组分B进行反向硅胶ODS-C18层析时,较佳地收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=50:1时Rf=0.53~0.54的组分C。所述反向硅胶ODS-C18层析优选以甲醇-水为洗脱剂,更优选梯度洗脱,较佳地所述洗脱剂甲醇-水的体积比依次为60%、80%和100%。
本发明中,所述组分C经HPLC制备得到tR=13.5min的如式I所示的松香烷型二萜化合物,其中HPLC制备的固定相为C18键合硅胶;所述tR值是指在如下测定条件下测得的tR值:色谱柱为XBridge BEH C18,5μm,10×250mm;进样量为200μL;柱温为25℃;流动相为40%乙腈水溶液;检测波长为210nm;流速15mL/min;所述HPLC制备优选包括如下条件:色谱柱为XBridge BEH C18,流动相优选为乙腈水溶液,更优选为40%乙腈水溶液,所述百分数是指体积百分数。
本发明中,所述香青兰提取物的制备可以包括如下步骤:在溶剂中,对香青兰(Dracocephalum moldavica L.)在溶剂回流温度下提取。
在某一实施方案中,所述溶剂可为本领域常规,例如醇类溶剂。所述醇类溶剂优选为乙醇,更优选为70%的乙醇水溶液,所述的百分数是指体积百分数。
在某一实施方案中,所述溶剂和所述香青兰的体积质量比可为本领域常规,优选为(8~15):1,例如10:1。
在某一实施方案中,所述香青兰的提取的次数优选不小于1次,例如2次,每次优选进行1h。
在某一实施方案中,所述香青兰的提取还可以包括后处理:将香青兰的提取液过滤、干燥、混悬和萃取。例如,经所述干燥过程得到香青兰干浸膏后,将所述香青兰干浸膏混悬于水中,用有机溶剂进行萃取。所述香青兰干浸膏和水的质量比优选为1:(1.2~3),更优选为1:2。所述萃取较佳地依次以石油醚、二氯甲烷、乙酸乙酯、正丁醇进行萃取,收集得到乙酸乙酯萃取物,即为所述香青兰提取物。
本发明提供了一种所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐在制备用于预防、治疗和/或缓解炎症疾病药物中的应用。
本发明还提供了一种药物组合物,其包含:
(i)所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐;和
(ii)至少一种药用辅料。
本发明还提供了一种药物组合物,其包含:
(i)所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐;
(ii)非甾体抗炎成分;和
(iii)至少一种药用辅料。
所述的非甾体抗炎成分为不含有甾体结构的抗炎成分,优选吲哚美辛、阿司匹林和槲皮素中的一种或多种。所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐和非甾体抗炎成分的浓度比优选为(10~30):1。所述的药用辅料的选择因施用途径和作用特点而异,通常是填充剂、稀释剂、粘合剂、润湿剂、崩解剂、润滑剂、乳化剂或助悬剂。
本发明还提供了一种如上所述的药物组合物在预防、治疗和/或缓解炎症疾病中的应用。
本发明还提供了一种体外、体内或离体条件下抑制细胞分泌TNF-α、IL-1β和/或NO的方法,其包括如下步骤:将所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐与细胞进行接触。
在某一实施方案中,所述细胞可为本领域常规,优选为RAW264.7细胞和BV2细胞。
本发明的药物组合物可根据公开的内容使用本领域技术人员已知的任何方法来制备。例如,常规混合、溶解、造粒、乳化、磨细、包封、包埋或冻干工艺。
本发明所述的药物组合物的施用方式没有特别限制,代表性的施用方式包括(但并不限于):口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。固体口服制剂的实例包括但不限于颗粒剂、片剂和胶囊剂。
除非另有规定,本文使用的所有技术术语和科学术语具有要求保护主题所属领域的标准含义。倘若对于某术语存在多个定义,则以本文定义为准。
本发明“炎症疾病”指的是具有血管的活体组织对损伤因子所发生的防御反应,通常表现为红、肿、热、痛和功能障碍。所述的炎症疾病例如类风湿性关节炎、咽喉炎、中耳炎、胃炎或牙周炎。在炎症过程中,一方面损伤因子直接或间接造成组织细胞的破坏,另一方面通过炎症反应和渗出反应,以稀释、杀伤和包围损伤因子,同时通过实质和间质细胞的再生使受损的组织得以修复和愈合,因此可以说炎症是损伤和抗损伤的统一过程。
在不违背本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:本发明新发现的如式I所示的松香烷型二萜化合物或其药学上可接受的盐能够有效地抑制RAW264.7细胞和BV2细胞中TNF-α、IL-1β和/或NO的含量,表明其具有显著的抗炎活性。
附图说明
图1显示了实施例1制得的如式I所示的松香烷型二萜化合物的DEPT135°谱(CDCl3,150MHz)。
图2显示了实施例1制得的如式I所示的松香烷型二萜化合物的1H-1HCOSY谱(CDCl3,600MHz)。
图3显示了实施例1制得的如式I所示的松香烷型二萜化合物的HSQC谱(CDCl3,600MHz)。
图4显示了实施例1制得的如式I所示的松香烷型二萜化合物的HMBC谱(CDCl3,600MHz)。
图5显示了实施例1制得的如式I所示的松香烷型二萜化合物的NOESY谱(CDCl3,600MHz)。
图6显示了实施例1制得的如式I所示的松香烷型二萜化合物的NOESY谱(CDCl3,600MHz)局部放大图。
图7显示了实施例1制得的如式I所示的松香烷型二萜化合物的实测ECD图和其绝对构型分别为3R,5R,10S,16S和3S,5S,10R,16R的计算ECD图(JASCO Corp.,J-810.所用溶剂:色谱级乙腈;测试浓度:0.02mg/mL)。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1如式I所示的松香烷型二萜化合物的制备
1)提取:取香青兰药材加70%乙醇回流提取,70%乙醇用量为药材量的10(v/m)倍,提取2次,每次1小时,将提取液过滤后合并,干燥,得香青兰干浸膏。浸膏混悬于2倍量水中,以石油醚、二氯甲烷、乙酸乙酯、正丁醇依次萃取,得到乙酸乙酯萃取物。
2)柱层析:将得到的乙酸乙酯萃取物,进行硅胶柱层析,分别用石油醚:丙酮溶液(100:1→50:1→20:1→10:1→5:1→2:1→1:1)梯度洗脱,每个梯度所用混合溶剂量为3倍柱体积(v/v)(即溶剂使用体积为硅胶装柱体积的3倍),按每个柱体积依次收集,共得到21个组份。收集组分17(即洗脱剂的用量为第17倍柱体积时收集到的洗脱液,也即混合溶剂(石油醚:丙酮溶液=2:1)的量为2倍柱体积时收集到的洗脱液),经硅胶薄层层析检测展开剂为二氯甲烷:甲醇=100:1时Rf值=0.60;减压浓缩后经凝胶柱(羟丙基葡聚糖凝胶LH-20(Sephadex LH-20),GE Healthcare Bio-Sciences AB,Uppsala,Sweden)层析,采用二氯甲烷:甲醇为1:1进行洗脱,采用10mL试管依次收集,并经硅胶薄层板鉴别,根据Rf值的不同,合并试管,得到4个组分,收集Rf=0.53~0.54(硅胶薄层层析检测,展开剂为二氯甲烷:甲醇=50:1)的组分17-3;浓缩干燥后经反相硅胶ODS-C18(ODS-A-HG,YMC Co.,Ltd.,Japan)层析,分别用甲醇/水(60%→80%→100%)梯度洗脱,每个梯度所用混合溶剂量为3倍柱体积,按每个柱体积依次收集,共得到9个组份,收集组分17-3-5(即洗脱剂的用量为第5倍柱体积时收集到的洗脱液,也即混合溶剂(甲醇/水=80%)的量为2倍柱体积时收集到的洗脱液),经硅胶薄层层析检测展开剂为二氯甲烷:甲醇=50:1时Rf=0.53~0.54,浓缩干燥后得到组分I;
3)HPLC制备:将上述组分I用HPLC制备(色谱柱型号:XBridge BEH C18,5μm,10×250mm;进样量:200μL;柱温:25℃;固定相为C18键合硅胶;流动相为40%乙腈水溶液;检测波长210nm;流速15mL/min;制备液相为LC3050N型高效液相色谱仪),得到如式I所示的松香烷型二萜化合物:12,16–环氧–3,11,14–三羟基–17(15→16),18(4→3)–移–4(19),8,11,13–松香烷四烯–7–酮(tR=13.5min)。
实施例2如式I所示的松香烷型二萜化合物的结构鉴定
ESI-MS m/z 343.1542[M-H]-,确定化合物分子量为344。
1H-NMR(CDCl3,600MHz)谱中显示3个甲基氢原子的吸收峰:δH 1.50(3H,d,J=6Hz,Me-17),δH 1.45(3H,s,Me-18),δH 1.15(3H,s,Me-20);4个亚甲基上的氢原子:δH 1.87(1H,m,H-1α),δH 3.09(1H,m,H-1β),δH 1.79(1H,m,H-2α),δH 1.71(1H,m,H-2β),δH 2.69(1H,t,J=14.4Hz,H-6α),δH 2.43(1H,dd,J=3.0,16.2Hz,H-6β),δH 3.36(1H,dd,J=9.0,15.6Hz,H-15α),δH 2.84(1H,dd,J=7.2,15.0Hz,H-15β);1个次甲基上的氢原子:δH 3.31(1H,dt,H-5);1个连氧次甲基上的氢原子δH 5.12(1H,m,H-16);2个末端双键上氢原子:δH 5.18(1H,d,J=1.2Hz,H-19α),δH 4.71(1H,d,J=1.8Hz,H-19α)。并在HSQC相关的帮助下完成氢谱的归属。
13C-NMR谱(CDCl3,150MHz)显示分子中含有20个碳原子,结合DEPT谱可知分子中含有3个甲基,5个亚甲基,2个次甲基,10个季碳。碳谱中显示含有3个甲基碳原子,并结合HSQC图谱发现Me-18(δH 1.45,3H,s)和δC 28.0,Me-17(δH 1.50,3H,d,J=6Hz)和δC 22.2,Me-20(δH 1.15,3H,s)和δC 14.6有相关,进一步对碳谱数据归属如下:δC 22.2(C-17),δC 28.0(C-18),δC 14.6(C-20);结合HSQC图谱发现H-1和δC 31.0,H-2和δC 36.9,H-6和δC 37.5,H-15和δC 34.5,H-19和δC 108.1有相关,进一步对5个亚甲基碳原子归属如下:δC 31.0(C-1),δC 36.9(C-2),δC 37.5(C-6),δC 34.5(C-15)和1个双键碳原子δC 108.1(C-19);2个次甲基碳原子δC 42.2(C-5),δC 83.5(C-16);10个季碳原子包括1个羰基碳原子δC 203.8(C-7);6个苯环碳原子δC111.1(C-8),δC 137.7(C-9),δC 132.0(C-11),δC 156.0(C-12、14),δC110.6(C-13);一个双键碳原子δC 152.0(C-4)和δC 71.2(C-3),δC 41.0(C-10),所以该化合物具有松香烷型二萜骨架。且该化合物的核磁数据与化合物caryopterisoid C相似,除了C-5和C-6的不饱和双键被饱和。并结合HMBC和1H-1H COSY谱图发现H-16和H-15、H-17;H-5和H-6、H-19;H-1和H-2有相关;H-1和C-2,C-3,C-10;H-2和C-1,C-3,C-10;H-6和C-5,C-7;H-15和C-13,C-14,C-16,C-17有相关,所以确定化合物平面结构。
由松香烷型二萜化合物的生源途径分析,C-10与C-16的构型均为S。由NOESY图谱发现,H-5和H-1α,H-20和H-1β相关,H-18和H-2β,H-20和H-2β相关;由ECD图谱发现如式I所示松香烷型二萜化合物的绝对构型为3R,5R,10S,16S,对应的计算ECD曲线在195nm、220nm、300nm左右呈现正cotton效应,在270nm处呈现负cotton效应;如式I所示松香烷型二萜化合物的绝对构型为3S,5S,10R,16R,对应的ECD曲线在195nm、220nm、300nm左右呈现负cotton效应,在270nm处呈现正cotton效应;由NOESY图谱结合ECD图谱可确定C-3,C-5的构型为R,R。故该如式I所示的松香烷型二萜化合物的立体结构如下:
实施例3如式I所示的松香烷型二萜化合物的抗炎活性
1.仪器
酶标仪(BioTek,EPOCH);CO2培养箱,(Thermo 3111);可调式移液器(Eppendorff);显微镜(奥林巴斯CX23);生物安全柜(Heal Force,HFsafe-1200LC)。
2.材料
细胞培养皿:Coning,430167;96孔细胞培养板:Corning,3599;75cm2细胞培养瓶:Corning,43063;DMEM培养基(Gibco,C11995500CP),RPMI1640培养基(Gibco,C11875500BT);胎牛血清(FBS,Gibco,2110875CP);抗菌-抗真菌剂(Antibiotic-Antimycotic,Lifetechnologies,15240-112);磷酸缓冲盐溶液(PBS),pH7.4(Gibco,10010-500BT);胰酶-EDTA(Trypsin-EDTA,0.25%)(Gibco,25200-056);牛血清白蛋白(Lifetechnologies,15561012);小鼠TNFαELISA试剂盒,MultiSciences,货号EK282/3;小鼠IL-1βELISA试剂盒,MultiSciences,货号EK201B/3;脂多糖(LPS,碧云天,S1732);总一氧化氮检测试剂盒(南京建成,A013-2)。
3.方法
1)细胞常规培养在10%FBS的DMEM培养基,将细胞按照20000/100μL/孔,接种于96孔细胞培养板,血清浓度为10%;
2)第二天,按照实验设置加入培养基配置的药物及阴性对照,作用细胞2小时;将LPS用10%FBS的DMEM配置成10μg/mL,按照10μL/孔加入96孔细胞培养板,继续培养8小时;
3)按照试剂盒要求,使用多功能酶标仪,测吸光值,进行各指标浓度检测,计算各指标浓度,计算不同浓度药物对LPS刺激细胞产生各指标的抑制率,从而得出不同化合物的IC50。
4)待测样品和吲哚美辛精确称样,实验前使用检测缓冲液稀释到工作浓度。
4.计算
5.实验结果
5.1初筛
表1.如式I所示的松香烷型二萜化合物对RAW264.7细胞和BV2细胞中TNF-α,IL-1β,NO抑制率
5.2IC50值测定
表2.如式I所示的松香烷型二萜化合物、caryopterisoid C[a]和吲哚美辛在RAW264.7细胞TNF-α,IL-1β,NO抑制作用的IC50表
[a]引用文献“J.Nat.Prod.2018,81,1508-1516”表6中化合物35的数据结果;“--”表示未测试该数据。
表3.如式I所示的松香烷型二萜化合物和吲哚美辛在BV2细胞TNF-α,IL-1β,NO抑制作用的IC50表
结果如表1-表3所示,结果表明,本发明的如式I所示的松香烷型二萜化合物对TNF-α,IL-1β,NO均有较强的抑制活性,其IC50值在RAW264.7细胞和BV2细胞中均小于5μM,表明如式I所示的松香烷型二萜化合物对炎症有显著的抑制作用。因此,本发明的如式I所示的松香烷型二萜化合物可为炎症疾病的预防、治疗和/或缓解提供一种新途径,同时可以开发为抗炎药物。
对比实施例1松香烷型二萜类化合物a-d的抗炎活性
化合物a-d的抗炎活性测试所需仪器、材料以及操作方法均同实施例3。
实验结果:
表5.化合物a-d对RAW264.7细胞中TNF-α,IL-1β,NO抑制率
表6.化合物a-d对BV2细胞中TNF-α,IL-1β,NO抑制率
结果如表5-表6所示,结果表明,当化合物C4位不存在末端双键时,单体化合物对LPS诱导的RAW264.7细胞和BV2细胞中TNF-α,IL-1β,NO抑制能力较弱,其抗炎活性较弱或没有抗炎活性。
对比实施例2松香烷型二萜类化合物e-g的抗炎活性
化合物e-g的抗炎活性测试所需仪器、材料以及操作方法均同实施例3。
实验结果:
表7.化合物e-g对RAW264.7细胞中TNF-α,IL-1β,NO抑制率
表8.化合物e-g对BV2细胞中TNF-α,IL-1β,NO抑制率
结果如表7-表8所示,结果表明,当松香烷型二萜类化合物中的五元环开环时(如化合物e,f,g),单体化合物对LPS诱导的RAW264.7细胞和BV2细胞中TNF-α,IL-1β,NO抑制能力明显减弱,基本没有抗炎活性。
Claims (17)
1.一种如式I所示的松香烷型二萜化合物或其药学上可接受的盐,
2.一种如权利要求1所述的如式I所示的松香烷型二萜化合物的制备方法,包括如下步骤:从香青兰(Dracocephalum moldavica L.)中提取分离得到所述如式I所示的松香烷型二萜化合物。
3.如权利要求2所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,从香青兰提取物中分离得到所述如式I所示的松香烷型二萜化合物;
所述从香青兰提取物中分离的过程包括如下步骤:将香青兰提取物依次经硅胶柱层析、凝胶柱层析、反相硅胶ODS-C18层析和HPLC分离获得所述如式I所示的松香烷型二萜化合物。
4.如权利要求3所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,
所述香青兰提取物进行硅胶柱层析时,收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=100:1时Rf值=0.60的组分A;
所述组分A进行凝胶柱层析时,收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=50:1时Rf=0.53~0.54的组分B;所述凝胶柱层析中的固定相为羟丙基葡聚糖凝胶;
所述组分B进行反向硅胶ODS-C18层析时,收集硅胶薄层层析中展开剂为二氯甲烷:甲醇=50:1时Rf=0.53~0.54的组分C;
所述组分C经HPLC制备得到tR=13.5min的如式I所示的松香烷型二萜化合物,其中HPLC制备的固定相为C18键合硅胶;所述tR值是指在如下测定条件下测得的tR值:色谱柱为XBridge BEH C18,5μm,10×250mm;进样量为200μL;柱温为25℃;流动相为40%乙腈水溶液;检测波长为210nm;流速15mL/min。
5.如权利要求4所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,所述香青兰提取物进行硅胶柱层析时,所述硅胶柱层析以石油醚-丙酮为洗脱剂;
所述组分A进行凝胶柱层析时,所述凝胶柱层析以二氯甲烷-甲醇为洗脱剂;
所述组分B进行反向硅胶ODS-C18层析时,所述反向硅胶ODS-C18层析以甲醇-水为洗脱剂;
当所述组分C经HPLC制备得到tR=13.5min的如式I所示的松香烷型二萜化合物时,所述HPLC制备包括如下条件:色谱柱为XBridge BEH C18,流动相为乙腈水溶液。
6.如权利要求5所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,所述香青兰提取物进行硅胶柱层析时,所述的硅胶柱层析的洗脱方式为梯度洗脱;
所述组分A进行凝胶柱层析时,所述洗脱剂二氯甲烷-甲醇的体积比为1:1;
所述组分B进行反向硅胶ODS-C18层析时,所述的反向硅胶ODS-C18层析的洗脱方式为梯度洗脱;
当所述组分C经HPLC制备得到tR=13.5min的如式I所示的松香烷型二萜化合物时,所述HPLC制备条件为40%乙腈水溶液,所述百分数是指体积百分数。
7.如权利要求6所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,所述香青兰提取物进行硅胶柱层析时,所述梯度洗脱中洗脱剂石油醚-丙酮的体积比依次为100:1、50:1、20:1、10:1、5:1、2:1和1:1;
所述组分B进行反向硅胶ODS-C18层析时,所述梯度洗脱中洗脱剂甲醇-水的体积比依次为60%、80%和100%。
8.如权利要求3所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,
所述香青兰提取物的制备包括如下步骤:在溶剂中,对香青兰(Dracocephalummoldavica L.)在溶剂回流温度下提取;
所述溶剂为醇类溶剂;
和/或,所述溶剂和所述香青兰的体积质量比为(8~15):1;
和/或,所述香青兰的提取次数不小于1次;
和/或,所述香青兰的提取还包括后处理,所述后处理包括如下步骤:将香青兰的提取液过滤、干燥、混悬和萃取;所述萃取依次以石油醚、二氯甲烷、乙酸乙酯、正丁醇进行萃取,收集得到乙酸乙酯萃取物,即为所述香青兰提取物。
9.如权利要求8所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,所述醇类溶剂为乙醇;
和/或,所述溶剂和所述香青兰的体积质量比为1:1;
和/或,所述香青兰的提取次数为2次。
10.如权利要求9所述的如式I所示的松香烷型二萜化合物的制备方法,其特征在于,所述醇类溶剂为70%的乙醇水溶液,所述百分数是指体积百分数。
11.一种药物组合物,其包含:
(i)如权利要求1所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐;和
(ii)至少一种药用辅料。
12.一种药物组合物,其包含:
(i)如权利要求1所述如式I所示的松香烷型二萜化合物或其药学上可接受的盐;
(ii)非甾体抗炎成分;和
(iii)至少一种药用辅料;
所述的非甾体抗炎成分为不含有甾体结构的抗炎成分。
13.如权利要求12所述的一种药物组合物,其特征在于,所述的组合物中非甾体抗炎成分为吲哚美辛、阿司匹林和槲皮素中的一种或多种。
14.如权利要求11-13中任一项所述的药物组合物,其特征在于,用于预防、治疗和/或缓解炎症疾病。
15.如权利要求14所述的药物组合物,其特征在于,用于预防、治疗和/或缓解炎症疾病为类风湿性关节炎、咽喉炎、中耳炎、胃炎或牙周炎。
16.一种如权利要求1所述的如式I所示的松香烷型二萜化合物或其药学上可接受的盐在制备用于预防、治疗和/或缓解炎症疾病药物中的应用。
17.一种如权利要求1所述的如式I所示的松香烷型二萜化合物或其药学上可接受的盐在制备用于预防、治疗和/或缓解类风湿性关节炎、咽喉炎、中耳炎、胃炎或牙周炎药物中的应用。
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