CN111518070A - Rotavirus-resistant compound in cassia wingnut, preparation method and application thereof - Google Patents
Rotavirus-resistant compound in cassia wingnut, preparation method and application thereof Download PDFInfo
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Abstract
Description
技术领域technical field
本发明属于植物化学领域,具体涉及一种翅荚决明中抗轮状病毒的化合物、制备方法及其应用。The invention belongs to the field of phytochemistry, and in particular relates to a compound against rotavirus in Cassia podae, a preparation method and an application thereof.
背景技术Background technique
翅荚决明(Cassia alata L. )为豆科决明属下的一个种。原产美洲热带地区,广布于全世界热带地区,在我国分布于广东和云南南部地区。该植物花色艳丽,有较高的观赏价值,常用作园林绿化树种。同时,它也是重要的药用植物,具有杀真菌、抗炎症、抗病毒的作用,可以用来治疗皮肤病,是香皂、洗发液、洗液的常用原料之一。它的种子含有的皂角苷可以作为驱除肠道寄生虫的驱虫剂;其叶水煎液常被用来治疗高血压、胃病、发烧、哮喘、毒蛇咬伤、性病等。从天然植物中寻找高效低毒的抗病毒活性分子也是当前天然产物化学的研究热点,临床上,多种药用植物资源广泛用于治疗各种病毒性感染疾病,如板蓝根、忍冬、甘草、苦参、大黄、菊花等。云南天然药物资源极为丰富,且与民族多样性相互交融,多种特色药物在长期民间用药中被证实具有抗病毒功效,因此,从特色药用植物资源中发现病毒抑制剂前景非常广阔。 Cassia alata L. is a species of the genus Cassia in the family Leguminosae. Native to tropical America, widely distributed in tropical regions of the world, in my country in Guangdong and southern Yunnan. The plant is colorful and has high ornamental value, and is often used as a landscaping tree species. At the same time, it is also an important medicinal plant with fungicidal, anti-inflammatory and antiviral properties, and can be used to treat skin diseases. It is one of the common raw materials for soaps, shampoos, and lotions. The saponins contained in its seeds can be used as anthelmintics to expel intestinal parasites; its leaf decoction is often used to treat high blood pressure, stomach problems, fever, asthma, snake bites, venereal diseases, etc. Finding high-efficiency and low-toxic antiviral active molecules from natural plants is also a research hotspot in natural product chemistry. Ginseng, rhubarb, chrysanthemum, etc. Yunnan is extremely rich in natural medicinal resources, and blends with ethnic diversity. Many characteristic medicines have been proven to have antiviral effects in long-term folk medicine. Therefore, the discovery of virus inhibitors from characteristic medicinal plant resources has a very broad prospect.
综上,为了充分利用云南的丰富的翅荚决明资源,寻找高效低毒的抗病毒活性分子,本发明对翅荚决明的化学成分进行研究,并从中分离到一个新骨架类型的化合物,该化合物为色酮和异香豆素的聚合体,具有明显的抗轮状病毒活性。To sum up, in order to make full use of the abundant resources of Cassia japonica in Yunnan, and to find antiviral active molecules with high efficiency and low toxicity, the present invention studies the chemical constituents of Cassia japonica, and isolates a new skeleton type compound from it, The compound is a polymer of chromone and isocoumarin, and has obvious anti-rotavirus activity.
发明内容SUMMARY OF THE INVENTION
本发明的第一目的是提供一种翅荚决明中抗轮状病毒的化合物,本发明的第二目的是提供一种抗轮状病毒的化合物的制备方法,本发明的第三目的是提供抗轮状病毒的化合物在制备抗轮状病毒药物中的应用。The first object of the present invention is to provide a compound against rotavirus in Cassia podae, the second object of the present invention is to provide a preparation method of the compound against rotavirus, and the third object of the present invention is to provide Application of anti-rotavirus compound in preparation of anti-rotavirus medicine.
本发明的第一目的是这样实现的,一种翅荚决明中抗轮状病毒的化合物,所述化合物为色酮和异香豆素的聚合体,命名为翅荚决明素B,英文名为:alatain B, 分子式为C24H20O8。The first object of the present invention is achieved in this way, a compound against rotavirus in Cassia serrata, the compound is a polymer of chromone and isocoumarin, named as Cassia spp. B, English name It is: alatain B, the molecular formula is C 24 H 20 O 8 .
结构式如下:The structure is as follows:
其中,本化合物的a片段为色酮,b片段为异香豆素。Wherein, the a fragment of this compound is chromone, and the b fragment is isocoumarin.
本发明的第二目的是这样实现的,所述抗轮状病毒病毒的化合物的制备方法,包括样品提取、硅胶柱层析、高效液相色谱分离及凝胶柱层析,具体包括以下步骤:The second object of the present invention is achieved in this way. The preparation method of the compound against rotavirus includes sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically includes the following steps:
A、样品提取:以翅荚决明枝叶为原料,将其晒干、粉碎至30~50目,用第一溶剂浸泡提取2~4次,再将提取液合并、过滤并浓缩得到枝叶提取物浸膏;A. Extraction of samples: take the branches and leaves of Cassia japonica as raw materials, dry them in the sun, pulverize them to 30-50 mesh, soak and extract with the first solvent for 2-4 times, then combine the extracts, filter and concentrate to obtain branch and leaf extracts extract;
B、硅胶柱层析:将所述浸膏上硅胶柱层析,以氯仿和丙酮体积比依次为10:0、9:1、8:2、7:3、6:4和5:5的一系列氯仿-丙酮溶液进行梯度洗脱,20:1的氯仿-丙酮洗脱液进行梯度洗脱,经TLC监测后,收集各梯度的梯度洗脱液并浓缩合并;B, silica gel column chromatography: silica gel column chromatography is applied to the extract, with the volume ratio of chloroform and acetone being 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 successively A series of chloroform-acetone solutions were subjected to gradient elution, and a 20:1 chloroform-acetone eluent was subjected to gradient elution. After monitoring by TLC, the gradient eluents of each gradient were collected, concentrated and combined;
C、高压液相色谱分离:将B步骤中9:1的氯仿-丙酮洗脱浓缩得到的样品经高效液相色谱分离纯化,得目标物粗品;C, high pressure liquid chromatography separation: the sample obtained by 9:1 chloroform-acetone elution concentration in step B is separated and purified by high pressure liquid chromatography to obtain the crude product of the target;
D、凝胶柱纯化:将所述目标物粗品用甲醇溶解,以纯甲醇为流动相,用凝胶柱层析进一步分离纯化得目标物纯品;D. Gel column purification: the crude product of the target product is dissolved in methanol, and pure methanol is used as the mobile phase, and further separation and purification is performed by gel column chromatography to obtain the pure product of the target product;
其中,所述高效液相色谱分离纯化条件如下:以体积浓度为70~80%的甲醇水溶液为流动相,流速15~25 mL/min,以21.2 × 250 mm,5 μm的ZorbaxSB-C18反相制备柱为固定相,紫外检测器检测波长为340~365 nm,每次进样0.5 ~ 1.0 mL,收集30 ~ 40 min的色谱峰,多次累加后蒸干得目标物粗品。Wherein, the high-performance liquid chromatography separation and purification conditions are as follows: take the methanol aqueous solution with a volume concentration of 70 to 80% as the mobile phase, the flow rate is 15 to 25 mL/min, and the ZorbaxSB-C 18 of 21.2 × 250 mm and 5 μm is used as the mobile phase. The reversed-phase preparative column is a stationary phase, and the detection wavelength of the UV detector is 340-365 nm, and each injection is 0.5-1.0 mL. The chromatographic peaks of 30-40 min are collected, and the crude product of the target substance is obtained after multiple accumulation and evaporation.
本发明的第三目的是这样实现的,所述抗轮状病毒的化合物在制备抗轮状病毒药物中的应用,所述抗轮状病毒药物包括所述化合物即翅荚决明素B。The third object of the present invention is achieved by the use of the anti-rotavirus compound in the preparation of an anti-rotavirus drug, the anti-rotavirus drug comprising the compound, viridian B.
所述抗轮状病毒药物包括翅荚决明素B和/或翅荚决明素B药学上可接受的载体或赋形剂。The anti-rotavirus drug includes cassia B and/or cassia B pharmaceutically acceptable carrier or excipient.
本发明的有益效果为:The beneficial effects of the present invention are:
1、本发明以翅荚决明的枝叶为原料,提取出一种结构新颖的具有抗病毒活性的化合物,其具有较强的抗轮状病毒活性,抗轮状病毒的实验表明,所述化合物的TC50值为208.2 µg/mL、IC50值为7.62 µg/mL,治疗指数TI为27.3;其治疗指数远高于对照病毒唑的治疗指数20.8;另外,本化合物毒性低,抗病毒活性好,可为抗轮状病毒药物研发提供高效、低毒的先导性化合物。1. The present invention uses the branches and leaves of Cassia spp. as raw materials to extract a compound with novel structure and antiviral activity, which has strong anti-rotavirus activity. Anti-rotavirus experiments show that the compound Its TC 50 value was 208.2 µg/mL, IC 50 value was 7.62 µg/mL, and the therapeutic index TI was 27.3; its therapeutic index was much higher than the control ribavirin's therapeutic index of 20.8; in addition, the compound had low toxicity and good antiviral activity , which can provide high-efficiency and low-toxicity lead compounds for the development of anti-rotavirus drugs.
2、本化合物的制备方法简单,另外,翅荚决明在我国南方发布广泛,生长迅速、生物产量达,原料来源广泛、成本低;而且枝叶再生能力非常强,可持续为本发明所述的活性化合物的分离制备提供原料,因此本化合物可以进行可持续的大规模生产与制备。2. The preparation method of this compound is simple, and in addition, Cassia spp. is widely distributed in southern my country, with rapid growth, high biological yield, wide source of raw materials, and low cost; and the regeneration ability of branches and leaves is very strong, and it is sustainable as described in the present invention. The isolated preparation of the active compound provides the raw material so that the present compound can be produced and prepared on a sustainable scale.
3、本发明所述化合物为色酮和异香豆素的聚合体,其中化合物的a片段为色酮,b片段为异香豆素;该类型化合物的抗病毒活性在公开文献中从未见报道,因此,本化合物抗轮状病毒活性的分析为抗病毒药物筛选提供了新的化合物骨架类型,为抗病毒药物及机制的研究发展提供了一种新的思路。3. The compound of the present invention is a polymer of chromone and isocoumarin, wherein the a fragment of the compound is chromone, and the b fragment is isocoumarin; the antiviral activity of this type of compound has never been reported in the open literature, Therefore, the analysis of the anti-rotavirus activity of this compound provides a new compound skeleton type for antiviral drug screening, and provides a new idea for the research and development of antiviral drugs and mechanisms.
附图说明Description of drawings
图1本发明化合物的核磁共振碳谱(13C NMR)。Fig. 1 Carbon nuclear magnetic resonance spectrum ( 13 C NMR) of the compounds of the present invention.
图2为本发明化合物的核磁共振氢谱(1H NMR)。Figure 2 is the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the compound of the present invention.
图3本发明化合物的关键HMBC相关图。Figure 3 Key HMBC correlograms of compounds of the present invention.
具体实施方式Detailed ways
下面结合实施例对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。本发明所采用的原料和设备若非特指,均可从市场购得或是本领域常用的,实施例中的方法,如无特别说明,均为本领域的常规方法。The present invention is further described below in conjunction with the examples, but the present invention is not limited in any way, and any transformation or replacement made based on the teachings of the present invention belongs to the protection scope of the present invention. Unless otherwise specified, the raw materials and equipment used in the present invention can be purchased from the market or are commonly used in the art. The methods in the examples, unless otherwise specified, are all conventional methods in the art.
本发明一种翅荚决明中抗轮状病毒的化合物,结构式如下:A compound of the present invention against rotavirus in Cassia podae, the structural formula is as follows:
本发明抗轮状病毒的化合物的制备方法,包括样品提取、硅胶柱层析、高效液相色谱分离及凝胶柱层析,具体包括以下步骤:The preparation method of the anti-rotavirus compound of the present invention includes sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically includes the following steps:
A、制备翅荚决明枝叶提取物浸膏:以翅荚决明枝叶为原料,将其晒干、粉碎至30~50目,用第一溶剂浸泡提取2~4次,再将提取液合并、过滤并浓缩得到枝叶提取物浸膏;A. Preparation of Cassia serrata branch and leaf extract extract: take the branches and leaves of Cassia serrata as raw material, dry and pulverize it to 30~50 meshes, soak and extract with the first solvent for 2~4 times, and then combine the extracts , filter and concentrate to obtain branch and leaf extract extract;
B、硅胶柱层析:将所述浸膏上硅胶柱层析,以氯仿和丙酮体积比依次为10:0、9:1、8:2、7:3、6:4和5:5的一系列氯仿-丙酮溶液进行梯度洗脱,20:1的氯仿-丙酮洗脱液进行梯度洗脱,经TLC监测后,收集各梯度的梯度洗脱液并浓缩合并;B, silica gel column chromatography: silica gel column chromatography is applied to the extract, with the volume ratio of chloroform and acetone being 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 successively A series of chloroform-acetone solutions were subjected to gradient elution, and a 20:1 chloroform-acetone eluent was subjected to gradient elution. After monitoring by TLC, the gradient eluents of each gradient were collected, concentrated and combined;
C、高压液相色谱分离:将B步骤中9:1的氯仿-丙酮洗脱浓缩得到的样品经高效液相色谱分离纯化,得目标物粗品;C, high pressure liquid chromatography separation: the sample obtained by 9:1 chloroform-acetone elution concentration in step B is separated and purified by high pressure liquid chromatography to obtain the crude product of the target;
D、凝胶柱纯化:将所述目标物粗品用甲醇溶解,以纯甲醇为流动相,用凝胶柱层析进一步分离纯化得目标物纯品;D. Gel column purification: the crude product of the target product is dissolved in methanol, and pure methanol is used as the mobile phase, and further separation and purification is performed by gel column chromatography to obtain the pure product of the target product;
其中,所述高效液相色谱分离纯化条件如下:以体积浓度为70~80%的甲醇水溶液为流动相,流速15~25 mL/min,以21.2 × 250 mm,5 μm的Zorbax SB-C18反相制备柱为固定相,紫外检测器检测波长为340~365 nm,每次进样0.5 ~ 1.0 mL,收集30 ~ 40 min的色谱峰,多次累加后蒸干得目标物粗品。Wherein, the high-performance liquid chromatography separation and purification conditions are as follows: take the methanol aqueous solution with a volume concentration of 70 to 80% as the mobile phase, the flow rate is 15 to 25 mL/min, and the Zorbax SB-C of 21.2 × 250 mm and 5 μm is used as the mobile phase. 18 The reversed-phase preparative column is the stationary phase, and the detection wavelength of the UV detector is 340-365 nm. Each injection is 0.5-1.0 mL, and the chromatographic peaks of 30-40 min are collected, and the crude product of the target substance is obtained by evaporation after multiple accumulation.
所述有机溶剂是60 %~100 %的乙醇、甲醇或丙酮的水溶液。The organic solvent is an aqueous solution of 60%-100% ethanol, methanol or acetone.
所述步骤A中,每次用1.5~4倍样品质量份的溶剂进行提取,提取时间为12 h~72h。In the step A, 1.5 to 4 times the sample mass parts of the solvent is used for extraction each time, and the extraction time is 12 h to 72 h.
所述步骤B中,所述浸膏在经硅胶柱层析前,用1~3倍浸膏重量份的丙酮、乙醇或甲醇溶解,再用1~1.6倍浸膏重量份的60~120目硅胶拌样后上样。In the step B, the extract is dissolved with acetone, ethanol or methanol of 1 to 3 times the weight of the extract, and then used 1 to 1.6 times the weight of the extract of 60 to 120 mesh before being subjected to silica gel column chromatography. The samples were loaded after the silica gel was mixed.
所述步骤B中,装柱硅胶为160~300目,所用硅胶的重量为2~5倍量浸膏重量。In the step B, the column packing silica gel is 160-300 mesh, and the weight of the silica gel used is 2-5 times the weight of the extract.
本发明抗轮状病毒的化合物在制备抗轮状病毒药物中的应用。The application of the anti-rotavirus compound of the present invention in the preparation of anti-rotavirus medicine.
所述抗轮状病毒药物包括所述化合物。The anti-rotavirus drug includes the compound.
所述抗轮状病毒药物包括所述化合物和/或所述化合物药学上可接受的载体或赋形剂。The anti-rotavirus drug comprises the compound and/or a pharmaceutically acceptable carrier or excipient for the compound.
以下结合实施例对本发明作进一步说明。The present invention will be further described below in conjunction with the examples.
实施例1Example 1
本实施例中翅荚决明枝叶采于云南德宏。In this example, the branches and leaves of Cassia serrata were collected in Dehong, Yunnan.
将翅荚决明枝叶晒干、粉碎到30目,取样3.0 kg以70%的丙酮水溶液提取3次,每次提取24h,提取液合并,过滤,减压浓缩得浸膏150 g。The branches and leaves of Cassia serrata were sun-dried and crushed to 30 mesh, and 3.0 kg was sampled and extracted with 70% acetone aqueous solution for 3 times, each extraction was 24 hours, the extracts were combined, filtered, and concentrated under reduced pressure to obtain 150 g of extract.
将浸膏用180g的甲醇溶解,然后加入80目粗硅胶200 g拌样,用160目硅胶1.0 kg装柱,拌样后上柱;用体积比依次为10:0、9:1、8:2、7:3、6:4和5:5的一系列氯仿-丙酮溶液进行梯度洗脱,收集其中用体积比为9:1的氯仿-丙酮溶液洗脱得到的洗脱液并浓缩得16.8克浓缩物。浓缩物以78%的甲醇水溶液为流动相,Zorbax SB-C18 (21.2 × 250 mm, 5 μm)制备柱为固定相,流动相流速为20 mL/min,紫外检测器检测波长为357 nm,每次进样1.0mL,收集停留时间为36.8 min的色谱峰,多次累加后蒸干,即得本发明翅荚决明素B粗品;将所述翅荚决明素B粗品用纯甲醇溶解,以纯甲醇为流动相,用Sephadex LH-20 凝胶柱层析纯化即得翅荚决明素B纯品。Dissolve the extract with 180 g of methanol, then add 200 g of 80-mesh thick silica gel to mix the sample, pack the column with 1.0 kg of 160-mesh silica gel, and load the column after mixing the sample; the volume ratios are 10:0, 9:1, and 8:1. A series of chloroform-acetone solutions of 2, 7:3, 6:4 and 5:5 were subjected to gradient elution, and the eluent obtained by elution with a 9:1 volume ratio of chloroform-acetone solution was collected and concentrated to obtain 16.8 gram concentrate. The concentrate used 78% methanol aqueous solution as the mobile phase, Zorbax SB-C 18 (21.2 × 250 mm, 5 μm ) preparative column as the stationary phase, the mobile phase flow rate was 20 mL/min, and the detection wavelength of the UV detector was 357 nm. , inject 1.0 mL each time, collect chromatographic peaks with a residence time of 36.8 min, and evaporate to dryness after accumulating for many times to obtain the crude cassia B of the present invention; the crude cassia B is treated with pure methanol Dissolve, use pure methanol as mobile phase, and purify by Sephadex LH-20 gel column chromatography to obtain pure cassia B.
实施例2Example 2
本实施例中翅荚决明枝叶采于云南西双版纳。In this example, the branches and leaves of Cassia serrata were collected in Xishuangbanna, Yunnan.
将翅荚决明枝叶晒干粉碎至50目,取样3.5 kg,以95%的乙醇提取3次,每次提取48h,提取液合并,过滤,减压浓缩得浸膏140 g。浸膏用280g甲醇溶解后,用120目粗硅胶150g拌样,用200目硅胶0.9 kg 装柱,拌样后上柱;再用体积比依次为10:0、9:1、8:2、7:3、6:4和5:5的一系列氯仿-丙酮溶液进行梯度洗脱,收集其中用体积比为9:1的氯仿-丙酮溶液洗脱得到的洗脱液并浓缩得26.4克浓缩物。以70%的甲醇水溶液为流动相,Zorbax SB-C18 (21.2 × 250 mm, 5 μm)制备柱为固定相,流动相流速为25ml/min,紫外检测器检测波长为357 nm,每次进样1.0 mL,收集停留时间为36.8 min的色谱峰,多次累加后蒸干,即得翅荚决明素B粗品。将翅荚决明素B粗品用甲醇溶液溶解,再以甲醇溶液为流动相,用SephadexLH-20 凝胶柱层析分离,即得翅荚决明素B纯品。The branches and leaves of Cassia serrata were sun-dried and crushed to 50 mesh, 3.5 kg were sampled, extracted with 95% ethanol for 3 times, each extraction was 48 hours, the extracts were combined, filtered, and concentrated under reduced pressure to obtain 140 g of extract. After the extract was dissolved in 280g methanol, the sample was mixed with 150g of 120-mesh coarse silica gel, and the column was packed with 0.9 kg of 200-mesh silica gel. A series of chloroform-acetone solutions of 7:3, 6:4 and 5:5 were subjected to gradient elution, and the eluent obtained by elution with a 9:1 volume ratio of chloroform-acetone solution was collected and concentrated to obtain 26.4 g of concentrated thing. A 70% methanol aqueous solution was used as the mobile phase, a Zorbax SB-C 18 (21.2 × 250 mm, 5 μm ) preparative column was used as the stationary phase, the flow rate of the mobile phase was 25 ml/min, and the detection wavelength of the UV detector was 357 nm. Inject 1.0 mL of sample, collect the chromatographic peaks with a residence time of 36.8 min, accumulate for many times and evaporate to dryness to obtain the crude product of Cassia B. The crude cassia B is dissolved in methanol solution, and then the methanol solution is used as the mobile phase to separate by SephadexLH-20 gel column chromatography to obtain the pure cassia B.
试验例1Test Example 1
本化合物为黄色胶状化合物,通过MS、HRMS、 1H NMR、1H和13C NMR 、HMBC及DEPT 等光谱技术对实施例1-2分离得到的抗轮状病毒病毒的化合物进行结构鉴定,具体数据及分析如下:This compound is a yellow colloidal compound. The structure of the anti-rotavirus compound isolated in Example 1-2 was identified by spectroscopic techniques such as MS, HRMS, 1H NMR, 1H and 13C NMR, HMBC and DEPT. The specific data and analyse as below:
化合物波谱数据:紫外光谱(溶剂为甲醇) 210 (4.26)、258 (3.68)、292(3.72)、357 (3.47) nm。红外光谱(溴化钾压片) 3418、2943、1722、1657、1606、1548、1432、1359、1146、1050、978、826 cm–1。ESIMS峰m/z 459 [M+Na]+;HRESIMS m/z 459.1050[M+Na]+ (计算值459.1056,C24H20O8Na);1H和13C NMR谱(图1和图2),数据见表1。Compound spectral data: UV spectrum (solvent is methanol) 210 (4.26), 258 (3.68), 292 (3.72), 357 (3.47) nm. Infrared Spectroscopy (Potassium Bromide Tablets) 3418, 2943, 1722, 1657, 1606, 1548, 1432, 1359, 1146, 1050, 978, 826 cm –1 . ESIMS peak m/z 459 [M+Na] + ; HRESIMS m/z 459.1050 [ M +Na] + (calcd 459.1056 for C24H20O8Na ); 1 H and 13 C NMR spectra (Fig. 1 and Fig. 1 2), see Table 1 for the data.
本化合物紫外光谱、红外光谱显示有羟基、羰基和芳环的特征吸收。高分辨质谱(HRESIMS) 给出准分子离子峰m/z 459.1050 [M+Na]+ (计算值459.1056)。结合1H和13 CNMR谱给出一个分子式C24H20O8,不饱和度为15。The ultraviolet spectrum and infrared spectrum of this compound show the characteristic absorption of hydroxyl, carbonyl and aromatic ring. High resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak m/z 459.1050 [M+Na] + (calcd. 459.1056). Combining the 1 H and 13 C NMR spectra gave a molecular formula of C 24 H 20 O 8 with an unsaturation of 15.
翅荚决明素A的1H NMR谱 (图1) 显示了一些特征信号,包括1个甲基,1个甲氧基、三个亚甲基,两个双键单峰信号、一个1,2,3,5-四取代苯环信号和一个1,2,3,4-四取代苯环信号。其13C和DEPT NMR谱 (图2) 显示了24个碳信号包括了2个甲基碳 (包括一个含氧甲基),3个亚甲基(包括1个含氧碳信号),6个烯碳次甲基乙基13个季碳原子信号(包括了3个羰基和5个含氧碳信号)。其中,3个羰基和 18个烯碳占据了12个不饱和度,提示化合物为1个四环体系的化合物。综上所述,初步可以判断翅荚决明素A为一个高度芳构化的异分子二聚体(见图3),它主要由一个C13色酮母核 (1a片段)和一个二环芳烃片段构成 (1b片段),其平面结构通过1D和2D核磁共振综合解析得到确定:在片段1a的色酮母核 (环A和B)通过特征1H和13C信号得到确定。该片段通过H-3 (δ H 6.28, s)和C-2/C-4/C-10,H-6 (δ H 6.56, d,J = 1.8 Hz)与C-8/C-10以及H-8 (δ H 6.67, d, J = 1.8 Hz)与C-6/C-10的HMBC相关得到确定。除此之外,一个羰基片段 (C-11 至 C-13的三碳单元)和一个羟基 (7-OH) 分别连接在色酮片段的C-5 和C-7位,该推论通过H2-11 (δ H 4.15, s) 与 C-6/C-10,以及 7-OH (δ H10.70, s) 与 C-6/C-7/C-8 得到证实。这些特征与已知化合物 5-acetonyl-7-hydroxy-2-methylchromone 非常类似,不同之处仅仅是原本A环中的C-14甲基变成了1a中的亚甲基,该推测通过H-3与C-14,以及H2-14和C-2、C-3 、C-9 (4 J CH)得到证实,推测1a片段通过C-14与1b片段连接起来。The 1 H NMR spectrum of Cassia A (Figure 1) showed some characteristic signals, including 1 methyl group, 1 methoxy group, three methylene groups, two double bond singlet signals, one 1, 2,3,5-tetrasubstituted benzene ring signal and one 1,2,3,4-tetrasubstituted benzene ring signal. Its 13 C and DEPT NMR spectra (Fig. 2) showed 24 carbon signals including 2 methyl carbons (including an oxygenated methyl group), 3 methylene groups (including an oxygenated carbon signal), 6 13 quaternary carbon atom signals (including 3 carbonyl and 5 oxygen-containing carbon signals) of alkenylcarbmethineethyl. Among them, 3 carbonyl groups and 18 alkene carbons occupy 12 unsaturations, suggesting that the compound is a compound of a tetracyclic system. In summary, it can be preliminarily judged that cassiacin A is a highly aromatized heterodimer (see Figure 3), which is mainly composed of a C 13 chromone nucleus (fragment 1a) and a bicyclic ring The aromatic hydrocarbon fragment constitutes (fragment 1b), and its planar structure was determined by comprehensive 1D and 2D NMR analysis: the chromone nucleus (rings A and B) in fragment 1a was determined by the characteristic 1 H and 13 C signals. The fragment passes through H-3 ( δ H 6.28, s) and C-2/C-4/C-10, H-6 ( δ H 6.56, d, J = 1.8 Hz) with C-8/C-10 and The HMBC correlation of H-8 ( δ H 6.67, d, J = 1.8 Hz) with C-6/C-10 was determined. In addition, a carbonyl fragment (three carbon units from C-11 to C-13) and a hydroxyl group (7-OH) are attached at the C-5 and C-7 positions of the chromone fragment, respectively, which is inferred by H 2 -11 ( δ H 4.15, s) with C-6/C-10, and 7-OH ( δ H 10.70, s) with C-6/C-7/C-8 were confirmed. These features are very similar to the known compound 5-acetonyl-7-hydroxy-2-methylchromone, except that the original C-14 methyl group in the A ring has been changed to a methylene group in 1a, which is presumed to be caused by H- 3 was confirmed with C-14, as well as H2-14 and C- 2 , C-3, C-9 (4JCH ) , and it was speculated that the 1a fragment was linked to the 1b fragment through C-14.
余下的11个碳信号(包括1个甲氧基、1个含氧的亚甲基、3个烯碳次甲基和6个季碳信号)归属为1个含有1个甲氧基和一个羟甲基的C9二环结构(1b片段)。进一步通过关键的HMBC信号H-4'与C-1'(4 J CH)、C-3'、C-4a'、C-5'、C-8a'和 C-9'相关,H-7'与C-5'、C-6'、C-8'和C-8a'相关,H-8'与 C-4a'、C-6'、C-7'和 C-8a'相关确定该C9母核为一个异香豆素骨架。除此之外,取代基1个甲氧基和一个羟甲基通过相应的HMBC确定分别连接在C-8' 和 C-3'位置上。最终,片段1a和1b通过关键的H2-14 与 C-4a'/C-5'/C-6' HMBC相关证实其通过C-14和C-5'连接起来,至此,化合物翅荚决明素B结构得以确定,其英文名为alatain B。The remaining 11 carbon signals (including 1 methoxy group, 1 oxygen-containing methylene group, 3 alkenyl carbonmethine groups, and 6 quaternary carbon signals) were assigned to 1 containing 1 methoxy group and one hydroxyl group. C9 bicyclic structure of methyl (fragment 1b). Further related to C-1' ( 4JCH ), C -3', C-4a', C-5', C-8a' and C-9' through the key HMBC signal H-4', H -7 ' is related to C-5', C-6', C-8' and C-8a', and H-8' is related to C-4a', C-6', C-7' and C-8a' to determine the The C9 parent nucleus is an isocoumarin skeleton. In addition, the
试验例2 本发明化合物的抗轮状病毒活性检测Test Example 2 Detection of anti-rotavirus activity of the compounds of the present invention
方法:取实施例1~2制备的化合物作为样品进行抗轮状病毒活性试验,采用体外细胞测试法,即样品与病毒同时作用于MA104细胞后,通过Alarmablue法检测样品对病毒感染致细胞死亡的保护作用,从而测定样品对轮状病毒的活性作用。Methods: The compounds prepared in Examples 1-2 were taken as samples for anti-rotavirus activity test, and the in vitro cell test method was used, that is, after the samples and the virus acted on MA104 cells at the same time, the alarmablue method was used to detect the effect of the samples on cell death caused by virus infection. protection, to determine the activity of the sample against rotavirus.
(a) 药物的细胞毒性检测(a) Cytotoxicity assay of drugs
MA104细胞在 96 孔细胞培养板中培养形成单层后,加入不同浓度的样品液,继续培养3天后,更换含Alamarblue的培养液,继续培养2~3小时后检测其530/590nm处的荧光值,从而检测样品对 MA104 细胞的毒性,并计算半数细胞毒浓度(TC50)。MA104 cells were cultured in a 96-well cell culture plate to form a monolayer, and sample solutions of different concentrations were added. After culturing for 3 days, the culture solution containing Alamarblue was replaced. After culturing for 2 to 3 hours, the fluorescence values at 530/590 nm were detected. , so as to detect the toxicity of the sample to MA104 cells, and calculate the half cytotoxic concentration (TC 50 ).
(b) 药物抗轮状病毒作用检测(b) Detection of drug anti-rotavirus effect
MA104细胞在 96 孔细胞培养板中培养形成单层后,100TCID50的病毒液和不超过20%细胞毒性的梯度浓度药物溶液同时加到MA104细胞上,继续培养4-6天后,更换含Alamarblue的培养液继续培养2~3小时后检测其530/590nm处的荧光值,并计算半数抑制浓度(IC50)。After MA104 cells were cultured in a 96-well cell culture plate to form a monolayer, 100 TCID50 of virus solution and a gradient concentration of drug solution with no more than 20% cytotoxicity were added to MA104 cells at the same time. After continuing to culture for 4-6 days, the culture containing Alamarblue was replaced After incubation for 2-3 hours, the fluorescence value at 530/590nm was detected, and the median inhibitory concentration (IC 50 ) was calculated.
(c) 根基TC50/ IC50计算化合物的治疗指数(c) Calculation of the therapeutic index of the compound based on TC50 / IC50
结果表明,本发明化合物的TC50值为208.2 µg/mL、IC50值为7.62 µg/mL,治疗指数TI为28.8;其治疗指数超过对照病毒唑(TC50值258.4、IC50值9.5)的治疗指数27.3,表明本化合物具有很好的抗轮状病毒活性。The results showed that the TC 50 value of the compound of the present invention was 208.2 μg/mL, the IC 50 value was 7.62 μ g /mL, and the therapeutic index TI was 28.8 ; ), the therapeutic index was 27.3, indicating that the compound had good anti-rotavirus activity.
综上所述,本发明的化合物在制备抗轮状病毒药物中有良好的应用前景;其结构简单,活性较好,可作为抗轮状病毒药物研发的先导性化合物用于抗轮状病毒药物制剂的研发。To sum up, the compound of the present invention has a good application prospect in the preparation of anti-rotavirus drugs; its structure is simple and the activity is good, and it can be used as a leading compound for the development of anti-rotavirus drugs for anti-rotavirus drugs Development of formulations.
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012080685A1 (en) * | 2010-12-17 | 2012-06-21 | Teane Cosmetiques | Extract of cassia alata against stretchmarks |
JP2014074019A (en) * | 2012-09-11 | 2014-04-24 | Daiichi Sankyo Healthcare Co Ltd | Antirotavirus agent |
CN105348192A (en) * | 2015-12-16 | 2016-02-24 | 云南民族大学 | Antiviral-activity isoquinoline alkaloid compound in Cassia alata L. and preparation method of antiviral-activity isoquinoline alkaloid compound |
CN105399656A (en) * | 2015-12-21 | 2016-03-16 | 云南民族大学 | Isobenzazole alkaloid compound, and preparation method and applications thereof |
CN105481817A (en) * | 2015-11-17 | 2016-04-13 | 云南民族大学 | Isocoumarin compound, and preparation method and application thereof |
CN105884588A (en) * | 2016-04-19 | 2016-08-24 | 秦瑞欣 | Norsesquiterpenoid compounds as well as preparation method and application thereof |
CN106008219A (en) * | 2016-05-20 | 2016-10-12 | 云南中烟工业有限责任公司 | Sesquiterpenoid compound, preparation method of sesquiterpenoid compound and application of sesquiterpenoid compound to preparation of anti-rotavirus medicines |
FR3046353A1 (en) * | 2015-12-31 | 2017-07-07 | Laboratoires Teane | EXTRACT OF CASSIA ALATA AGAINST DRY SKIN WITH ATOPIC TREND |
-
2020
- 2020-06-08 CN CN202010513773.5A patent/CN111518070B/en not_active Expired - Fee Related
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012080685A1 (en) * | 2010-12-17 | 2012-06-21 | Teane Cosmetiques | Extract of cassia alata against stretchmarks |
JP2014074019A (en) * | 2012-09-11 | 2014-04-24 | Daiichi Sankyo Healthcare Co Ltd | Antirotavirus agent |
CN105481817A (en) * | 2015-11-17 | 2016-04-13 | 云南民族大学 | Isocoumarin compound, and preparation method and application thereof |
CN105348192A (en) * | 2015-12-16 | 2016-02-24 | 云南民族大学 | Antiviral-activity isoquinoline alkaloid compound in Cassia alata L. and preparation method of antiviral-activity isoquinoline alkaloid compound |
CN105399656A (en) * | 2015-12-21 | 2016-03-16 | 云南民族大学 | Isobenzazole alkaloid compound, and preparation method and applications thereof |
FR3046353A1 (en) * | 2015-12-31 | 2017-07-07 | Laboratoires Teane | EXTRACT OF CASSIA ALATA AGAINST DRY SKIN WITH ATOPIC TREND |
CN105884588A (en) * | 2016-04-19 | 2016-08-24 | 秦瑞欣 | Norsesquiterpenoid compounds as well as preparation method and application thereof |
CN106008219A (en) * | 2016-05-20 | 2016-10-12 | 云南中烟工业有限责任公司 | Sesquiterpenoid compound, preparation method of sesquiterpenoid compound and application of sesquiterpenoid compound to preparation of anti-rotavirus medicines |
Non-Patent Citations (6)
Title |
---|
HANG-YING MA,ET.: ""ANTHRAQUINONES FROM THE BARKS OF Cassia alata AND THEIR ANTI-TOBACCO MOSAIC VIRUS ACTIVITY"", 《CHEMISTRY OF NATURAL COMPOUNDS》 * |
XIN LIU,ET.: ""TWO NEW FLAVONES FROM THE BARKS OF Cassia alata AND THEIR BIOACTIVITY"", 《CHEMISTRY OF NATURAL COMPOUNDS》 * |
YAN YANG,ET.: ""TWO NEW ALKALOIDS FROM THE SEEDS OF Cassia alata AND THEIR ANTI-TOBACCO MOSAIC VIRUS ACTIVITY"", 《CHEMISTRY OF NATURAL COMPOUNDS》 * |
周玲等: ""傣药翅荚决明树皮中1 个新呋喃-2-甲酸类化合物"", 《中国中药杂志》 * |
周玲等: ""傣药翅荚决明树皮中一个新异喹啉生物碱及其细胞毒活性"", 《天然产物研究与开发》 * |
马航赢等: ""傣药翅荚决明细枝中一个新的2-芳基苯并呋喃类化合物及细胞毒活性"", 《中草药》 * |
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