阅读说明：本技术 芭蕉根两个新化合物分离纯化方法及用途 (Method for separating and purifying two new compounds of Japanese banana root and application thereof ) 是由 李勇军 聂雨杉 马雪 李月婷 黄勇 刘亭 杨畅 刘春花 潘洁 于 2020-06-22 设计创作，主要内容包括：本发明公开了一种芭蕉根两个新化合物分离纯化方法及用途,新化合物的化学名4-Phenyl-benzo[de]isoquinoline-1,3-dione(1),4-(4-Hy droxy-phenyl)-benzo[de]isoquinoline-1,3-dione(2)结构式为：<Image he="329" wi="700" file="DDA0002549556810000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明采用高分辨质谱HR-ESI-MS、<Sup>1</Sup>H NMR、<Sup>13</Sup>C NMR、二维HMBC、HMQC核磁谱对这两个新化合物进行结构鉴定,推导出两个新化合物的分子式及分子结构。经体外细胞活性研究表明,从芭蕉根中分离得到的这两个新化合物对人肝癌细胞、人肺癌细胞及人乳腺癌细胞具有较好的抑制活性,可将该化合物开发制备成防治相关肿瘤的药物。(The invention discloses a method for separating and purifying two new compounds of Japanese banana root and application thereof, wherein the chemical name of the new compounds is 4-Phenyl-benzol [ de ]]isoquinoline‑1,3‑dione(1),4‑(4‑Hy droxy‑phenyl)‑benzo[de]The isoquinoline-1,3-dione (2) has the structural formula: the invention adopts high resolution mass spectrum HR-ESI-MS, 1 H NMR、 13 And carrying out structural identification on the two new compounds by using the nuclear magnetic spectrum of C NMR, two-dimensional HMBC and HMQC, and deducing molecular formulas and molecular structures of the two new compounds. In vitro cell activity research shows that the two new compounds separated from the roots of Japanese banana have good inhibitory activity on human liver cancer cells, human lung cancer cells and human breast cancer cells, and the compounds can be developed and prepared into medicaments for preventing and treating related tumors.)

1. Two new compounds of Japanese banana root have the structures shown in formula I:

4-Phenyl-benzoic [ de ] isoquinoline-1,3-dione (1),4- (4-Hydroxy-Phenyl) -benzoic [ de ] isoquinoline-1,3-dione (2), having the following structural formula:

2. a method for separating and purifying two novel compounds of plantain root according to claim 1, comprising the steps of:

step 1) taking a fresh or dried rhizome part from the roots of the Japanese bananas, drying and crushing the fresh or dried rhizome part, performing reflux extraction for three times by 70% ethanol for 1.5 hours/time, respectively adding 7 times, 7 times and 5 times of ethanol, combining the three extracting solutions, and performing reduced pressure concentration to obtain a total extract;

step 2) mixing the total extract obtained in the step 1), loading the sample on macroporous adsorption resin, eluting with water, 50% ethanol and 95% ethanol in sequence, and concentrating under reduced pressure to obtain three parts of extracts which are respectively a water section, a 50% ethanol section and a 95% ethanol section;

step 3) mixing the 50% ethanol section extract obtained in the step 2) with silica gel, loading the mixture into a column chromatography, eluting, and obtaining a plurality of components according to different polarities by combining a TLC detection method;

and 4) passing one component obtained in the step 3) through a chromatographic column, eluting to obtain two sub-components, and crystallizing and recrystallizing one sub-component to obtain the new compound 1.

And 5) passing one component in the step 3) through a normal phase silica gel column, eluting with dichloromethane/ethyl acetate, passing the concentrated solution through repeated Sephadex LH-20 and Toyopearl HW-40C, eluting, concentrating the eluent containing the target compound, finally passing the concentrated solution through a Toyopearl HW-40F gel column, and eluting with pure methanol to obtain the new compound 2.

3. The method for separating and purifying two novel compounds of Japanese banana root according to claim 2, wherein: the macroporous adsorption resin in the step 2) is D101 macroporous adsorption resin.

4. The method for separating and purifying two novel compounds of Japanese banana root according to claim 2, wherein: the column chromatography in the step 3) is silica gel column chromatography.

5. The method for separating and purifying two novel compounds of Japanese banana root according to claim 2, wherein: the eluent in the step 3) is dichloromethane-methanol, 15:1 → 1: 1.

6. The method for separating and purifying two novel compounds of Japanese banana root according to claim 2, wherein: the chromatographic column in the step 4) is Toyopearl HW-40C.

7. The method for separating and purifying two novel compounds of Japanese banana root according to claim 2, wherein: eluting in step 4) and step 5) with chloroform/methanol at a ratio of 1: 1.

8. The use of two novel compounds of plantain root isolated and purified according to claim 2 in the preparation of anti-tumor medicaments.

Technical Field

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a novel compound extracted, separated and identified from a plantain root medicinal material and an extraction and separation method thereof.

Background

The root of Japanese banana is fresh or dried rhizome of Japanese banana of Musaceae, is a perennial herb with short stem and most seeds, which can be harvested all year round and is planted in farmhouse courtyard. Japanese banana is recorded in Han Shu, also called Ba, named banana in Shi Ji, named as Lu Tian and Fan Xian in Ju Fang Men, and named as Xiang Hui, Ganlu Tree, big leaf Japanese banana and Japanese banana head in Classification herb medicine property. The plantain roots are mainly used for treating diseases such as jaundice, diabetes, heat toxin in the daytime, and the like, have the effects of diuresis and clearing heat and removing toxicity, can also be used for clinically treating diseases such as erysipelas, wind-heat headache, bloody stranguria, skin swelling and pain, and the like, and are contained in bone-recovering capsules and swelling and pain-relieving compound preparations, and the effects of dredging channels and collaterals, and relieving swelling and pain are mainly achieved. The banana is eradicated and has the medicinal function, and is often used as a food material for folk eating.

Modern research shows that: the banana root contains many chemical components, mainly flavone, saponin, volatile oil, coumarin, alkaloid and anthraquinone, and has pharmacological activities of resisting inflammation, reducing blood sugar, relieving pain, inhibiting alpha-glucosidase and inhibiting bacteria. Modern research identifies that the banana root contains 16 amino acids, wherein 7 of the amino acids are essential amino acids for human body, namely methionine, lysine, threonine, phenylalanine, leucine, isoleucine and valine, which are important for human health.

At present, more plantain roots are identified by methods such as microscopic identification, ultraviolet spectrum identification, Raman spectrum identification, fingerprint spectrum identification and the like, but the seedling medicine plantain roots have various and complex chemical components, the pharmacological action mechanism is not clear, the research is shallow, and the specific chemical components of the plantain roots are reported. Because the material basis is not clear enough, most of the medicines containing the plantain roots used clinically at present are only compound or coarse preparations, the development and utilization of the effective components of the plantain roots are lacked, the quality control level is low, and the clinical curative effect is difficult to ensure. Therefore, the research on the single chemical components of the banana root is enlarged, and the effective components are determined, so that the method has important significance.

Disclosure of Invention

The invention aims to provide a method for separating and purifying two new compounds of plantain roots and application thereof.

The invention provides two new compounds separated from Japanese banana root, the chemical names of the components of the new compounds are respectively: 4-Phenyl-benzoic [ de ] isoquinoline-1,3-dione (1),4- (4-Hydroxy-Phenyl) -benzoic [ de ] isoquinoline-1,3-dione (2), and the chemical structure formula is as follows:

the invention also provides a separation and purification method of the compound, which comprises the following steps:

1) taking a fresh or dried rhizome part from the roots of the Japanese bananas, drying and crushing the rhizome part, performing reflux extraction for three times for 1.5 hours per time by using 70% ethanol, adding 7 times, 7 times and 5 times of ethanol respectively, combining the three extracting solutions, and performing reduced pressure concentration to obtain a total extract;

2) mixing the total extract obtained in the step 1), loading the sample on D101 macroporous adsorption resin, eluting with water, 50% ethanol and 95% ethanol in sequence, and concentrating under reduced pressure to obtain three parts of extract which are respectively a water section, a 50% ethanol section and a 95% ethanol section;

3) mixing the 50% ethanol extract obtained in step 2) with silica gel, subjecting to silica gel column chromatography, eluting with dichloromethane-methanol (15:1 → 1:1), and detecting by TLC to obtain several components (Fr.1-Fr.10) according to different polarities;

4) passing the component Fr.8 in the step 3) through Toyopearl HW-40C, eluting with chloroform/methanol (1:1) to obtain two sub-components, and crystallizing and recrystallizing one of the sub-components to obtain the new compound 1.

5) Passing a certain component in the step 3) through a normal phase silica gel column, eluting with dichloromethane/ethyl acetate, passing the concentrated solution through repeated Sephadex LH-20 and Toyopearl HW-40C, wherein the eluent is chloroform/methanol (1:1), concentrating the eluent containing the target compound, finally passing the concentrated solution through a Toyopearl HW-40F gel column, and eluting with pure methanol to obtain a new compound 2.

The novel compound extracted from the roots of the plantain has the advantages that the novel compound is firstly subjected to silica gel column chromatography, a target compound is tracked by a TLC method, similar components are combined, low-content components are enriched, and finally, the sample loss is reduced by repeated gel column chromatography, the gel filler can be repeatedly used, the cost is lower, the operation is simpler, and the controllability is good. The two new compounds can be applied to the preparation of antitumor drugs.

Drawings

FIG. 1 is a HR-ESI-MS diagram of novel Compound 1 of the present invention;

FIG. 2 is a 1H-NMR spectrum of novel compound 1 of the present invention;

FIG. 3 is a 13C-NMR spectrum of novel compound 1 of the present invention;

FIG. 4 is a chart of the NMR HMQC spectrum of novel Compound 1 of the present invention;

FIG. 5 is a chart of the NMR spectrum of HMBC of the novel compound 1;

FIG. 6 is a HR-ESI-MS plot of novel Compound 2 of the present invention;

FIG. 7 is a 1H-NMR spectrum chart of the novel compound 2 of the present invention;

FIG. 8 is a 13C-NMR spectrum of novel compound 2 of the present invention;

FIG. 9 is a chart of the NMR HMQC spectrum of novel Compound 2 of the present invention;

FIG. 10 is a nuclear magnetic resonance HMBC spectrum of the novel compound 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.