CN111635352B - Indole alkaloid in traditional Chinese medicine herba Rubi Corchorifolii, and extraction and purification method and application thereof - Google Patents

Indole alkaloid in traditional Chinese medicine herba Rubi Corchorifolii, and extraction and purification method and application thereof Download PDF

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CN111635352B
CN111635352B CN202010612484.0A CN202010612484A CN111635352B CN 111635352 B CN111635352 B CN 111635352B CN 202010612484 A CN202010612484 A CN 202010612484A CN 111635352 B CN111635352 B CN 111635352B
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indole
dichloromethane
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杨武德
郭正红
陶小艳
於祥
隋怡
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Guizhou University of Traditional Chinese Medicine
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Abstract

The invention relates to indole alkaloid in a traditional Chinese medicine of the blood basin grass and an extraction and purification method and application thereof, wherein the blood basin grass is researched for the initial detection of hemostatic activity, the separation of chemical components of hemostatic active parts, the activity screening of hemostatic material bases and the like, and through the researches, the hemostatic efficacy of the blood basin grass is preliminarily mastered, the chemical component composition of the hemostatic active parts of the blood basin grass in Guizhou province is analyzed, and the hemostatic material bases of the blood basin grass are determined; provides scientific basis for the development and utilization of the blood basin grass resource.

Description

Indole alkaloid in traditional Chinese medicine herba Rubi Corchorifolii, and extraction and purification method and application thereof
Technical Field
The invention relates to the field of pharmacy, in particular to indole alkaloid in traditional Chinese medicine rubus bidens and an extraction and purification method and application thereof.
Background
The hemostatic is a medicine for accelerating blood coagulation, reducing permeability of capillary vessels or promoting contraction of broken ends of blood vessels to stop bleeding; can be used for treating hemorrhage or hemorrhagic diseases caused by various reasons. The hemostatic has wide clinical application and wide application in life, and has important clinical significance and research value.
According to relevant statistics, the dosage of the hemostatic in the Chinese hospital basically keeps a relatively stable rising trend of medication level; if the western medicine is used for treatment, dangerous consequences caused by high coagulation can often occur, and the western medicine is forbidden or cautiously used under the condition of some inapplicable symptoms or postoperative sequelae, such as renal insufficiency or postoperative hematuria; the traditional hemostatic medicines can bring more adverse reactions, and the problems of hemostasis, thrombosis promotion, immunological rejection and the like cannot be solved. The combination of Chinese and Western medicine has become a new idea for researching and developing new hemostatics, and according to the four basic principles of safety, effectiveness, economy and appropriateness of reasonable medicine application, the traditional Chinese medicine dialectically treats and stops bleeding, and can act on three major systems of physiological hemostasis, blood coagulation and fibrinolysis. According to the intervention of the clinical scheme of dialectical treatment of the traditional Chinese medicine, the traditional Chinese medicine is supplemented with western medicines, and the application of the Chinese herbal medicines can also reduce the medical expense and lighten the economic burden of a patient, thereby embodying the superiority of the traditional Chinese medicine in treating diseases.
To date, some hemostatic active ingredients have been isolated and identified from plants while studying the hemostatic effects of traditional Chinese medicines. Reportedly, the quinone compound eriosema chinense element separated from the pyrola whole grass has the anti-bleeding effect and the retinoid K-like activity; isoverbascoside, a phenylpropanoid glycoside compound, is proved to have extremely strong effect of inhibiting oxidation hemolysis; wedelolactone and demethylwedelolactone separated from Hypericum plant also have good hemostatic activity; caffeic acid, one of the main hemostatic components of common cephalanoplos herb, can contract local blood vessels and inhibit fibrinolysis, thereby achieving the hemostatic effect. The action mechanism of other active ingredients is not clear, so the research on the hemostasis mechanism of the traditional Chinese medicine, the national medicine and the monomeric compound for hemostasis is increased, and new hemostatic medicines with good hemostasis effect, clear mechanism and wide clinical application prospect are searched; the monomer compound with hemostatic activity is formulated as the index of quality control of the hemostatic traditional Chinese medicine, and guarantees are provided for reasonable, safe and effective medication in clinic.
The Chinese herbaceous plants (with the scientific name of Salvia cavaliei var. simplicifolia Stib.) are also called as reversed (turning) red-back, and Guizhou copper kernel and most of areas are called Chinese herbaceous plants, are distributed in Guizhou, Hubei, Sichuan, Guangxi and other areas, and have abundant resources in the Guizhou areas; the Chinese basine herb is used as a medicine in folk, is mainly used for treating symptoms such as hematemesis, hemoptysis and the like, and mainly has the effects of cooling blood, detoxifying, dissipating blood stasis, stopping bleeding and the like; the hemostatic drug has wide treatment range and definite curative effect, and is a common hemostatic folk drug for the Guizhou Miao nationality, the Dong nationality and other minority nationalities. The method has abundant resources in each seedling region of Guizhou (including Guiyang city urban areas), and has sufficient resource foundation for reasonably developing and utilizing the sanguinea potted plant. In 1934-2015, reports of Wu Y-B and Pentron and the like are organized and systematically reviewed, and during this period, monomer components separated from the plants are nearly 900 or more, and the main types are diterpenes, triterpenes, phenolic acids, glycosides, alkaloids, flavonoids, lignans, fatty acids and other types of compounds. Literature research shows that the chemical composition research and other related research on the blood basin grass report a few and only how to group the blood basin grass[12]For the determination of the content of the total flavonoids in the blood basin grass, Zhao Li Min and the like are separated from an n-butanol part of a water extract of the blood basin grass to obtain two trace phenolic glycosides, namely isoquercitrin and milk vetch, Zhang Meng Ke separates 45 compounds from the water extract, Chen Qiong separates 17 compounds from the water extract, and Wang He Ying and the like separate 13 compounds from the water extract, but relevant research reports on the hemostasis aspect are not found.
With the increasingly deep modern research on traditional Chinese medicine and the continuous deepening of understanding on national traditional Chinese medicine, in order to more effectively control the quality of the medicinal materials and more effectively evaluate the medicinal materials, more reasonably utilize medicinal material resources and explore medicinal material resources and ensure more accurate, safe and effective clinical medication, the combination of modern clinical practice and the traditional Chinese medicine theory must be further strengthened, the hemostasis action mechanism and the hemostasis chemical components of the traditional Chinese medicine are researched and summarized by utilizing modern scientific means, and the enrichment and the perfection of the hemostasis components and the hemostasis theory of the national traditional Chinese medicine are realized, so that the theoretical basis of the traditional Chinese medicine can be broken through the theoretical experience of the national traditional Chinese medicine, the better inheritance and the development of the national traditional Chinese medicine is laid, and therefore, the hemostasis components, the medication mechanism and the medication performance of the national hemostatic medicinal herb of the Guizhou national hemostatic herb Xueyi Cai are researched, and the reasonable medicine is developed for the national herb Xueyi Cai, The utilization and resource evaluation have positive significance.
The climate belongs to the mountain type of subtropical plateau in the watershed zone at the east of the cloud plateau and the upstream of the two rivers of the Yangtze river and the Zhujiang river in Guizhou. Due to the special ecological environment and the complex and various natural conditions of Guizhou, the rare and special medicine resources are inoculated, and the difference of the chemical components of the medicine and the same plants is large due to the difference of the growth environment; the basic mechanism of the haemostasis by the sanguinaria japonica in the Guizhou national region is not clear, and in view of multiple purposes of the sanguinaria japonica in the Guizhou national region, the basic research report of the haemostasis is very few, and the effective components are not clear (haemostasis), so that the sanguinaria japonica is required to be subjected to deep systematic research, which is favorable for further mastering and evaluating the scientificity of the application of the sanguinaria japonica of the Guizhou national region.
In order to carry out relevant research on the hemostatic effect of the Guizhou folk medicinal herb, namely the sanguinea japonica, the invention carries out researches such as primary detection on hemostatic activity, separation of chemical components of hemostatic active parts, activity screening of hemostatic material bases and the like on the sanguinea japonica, preliminarily grasps the hemostatic efficacy of the sanguinea japonica, analyzes the chemical component composition of the hemostatic active parts of the Guizhou sanguinea japonica and determines the hemostatic material bases of the sanguinea japonica through the researches, can scientifically evaluate the folk medication basis of the sanguinea japonica on the basis, and simultaneously provides practical bases for the research and development of new hemostatic medicines and the quality evaluation standards of the sanguinea japonica; provides scientific basis for the development and utilization of the blood basin grass resource.
Disclosure of Invention
The invention aims to provide indole alkaloid in traditional Chinese medicine, namely, the hematuria bidentis;
the other purpose of the invention is to provide a method for extracting and purifying indole alkaloid in the traditional Chinese medicine, namely the hematuria bidentis;
the invention also aims to provide the application of indole alkaloid in the traditional Chinese medicine of the hematuria in the aspect of hemostatic medicines.
The indole alkaloid is indole-3-ethyl formate and 3-carboxyl indole.
The indole-3-carboxylic acid ethyl ester compound is characterized in that:
structural formula (xvi):
Figure GDA0003158323730000031
the molecular formula is as follows: c11H11NO2。
API-ES m/z：212[M+Na]+
The state is as follows: white powder (dichloromethane)
Silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red;
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in figure 1 and figure 2
Figure GDA0003158323730000032
The 3-carboxyindole compounds of the present invention are characterized as:
structural formula (xvi):
Figure GDA0003158323730000033
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]+。
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystals (methanol).
Silica gel thin layer identification: the dichloromethane-methanol with the ratio of 35:1 is developed to have fluorescence under 254nm, and the orange spots are shown after the dichloromethane-methanol is developed and baked by 10 percent concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4.
Figure GDA0003158323730000041
The method for extracting and purifying the indole alkaloid specifically comprises the following steps: crushing 7-12 kg of dried whole herb of rubus bidens into coarse powder, performing reflux extraction for 2-5 times by using 80-98% industrial ethanol, performing concentration under reduced pressure for 1-3 hours each time, and recovering a solvent to obtain 1300-2300 g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 4-8 times by using each polar solvent, respectively combining each extraction liquid, and recovering the solvent by using a rotary evaporator to respectively obtain 210-360 g of petroleum ether part extract, 180-320 g of chloroform part extract, 140-240 g of ethyl acetate part extract, 150-265 g of n-butanol part extract and water part;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 36-62 mg of Fr.6.6 by a silica gel column with chromatographic petroleum ether-dichloromethane according to the ratio of 3:1 to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 by a scraper to obtain 4.5-7.8 mg of indole-3-ethyl formate;
removing pigment from 7-13 g of Fr.9 by using a gel chromatographic column with dichloromethane-methanol in a ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 160-275 mg of Fr.9.5, carrying out column chromatography twice on a dichloromethane-methanol system in a ratio of 40:1 and 35:1, and scraping to obtain 3-carboxyindole 3.1-5.2 mg.
Preferably, the method for extracting and purifying indole alkaloids provided by the invention specifically comprises the following steps: crushing 8-11 kg of dried whole herb of the rubus bidens into coarse powder, performing reflux extraction for 2-4 times by using 85-97% industrial ethanol, performing concentration under reduced pressure for 1-3 hours each time, and recovering a solvent to obtain 1500-2100 g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 5-7 times by using each polar solvent, respectively combining each extraction liquid, and recovering the solvent by using a rotary evaporator to respectively obtain 230-340 g of petroleum ether extract, 200-300 g of chloroform extract, 160-220 g of ethyl acetate extract, 170-245 g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 36-62 mg of Fr.6.6 by a silica gel column with chromatographic petroleum ether-dichloromethane according to the ratio of 3:1 to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 by a scraper to obtain 5-7 mg of indole-3-ethyl formate;
removing pigment from 8-12 g of Fr.9 by using a gel chromatographic column through dichloromethane-methanol according to the ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 180-255 mg of Fr.9.5, carrying out column chromatography twice through a dichloromethane-methanol system according to the ratio of 40:1 and 35:1, and scraping to obtain 3-carboxyindole 3.5-4.8 mg.
Further preferably, the method for extracting and purifying indole alkaloids provided by the invention specifically comprises the following steps: pulverizing 9.3kg dried whole plant of herba Rubi Corchorifolii into coarse powder, extracting with 95% industrial ethanol under reflux for 3 times, each for 2 hr, concentrating under reduced pressure, and recovering solvent to obtain alcohol extract 1756 g; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 6 times by each polar solvent, respectively combining each extraction liquid, and recovering the solvent by a rotary evaporator to respectively obtain 286.6g of petroleum ether extract, 244.9g of chloroform extract, 188.6g of ethyl acetate extract, 205.1g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 48mg of Fr.6.6 with chromatographic petroleum ether-dichloromethane at a ratio of 3:1 through a silica gel column to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 with a scraper to obtain 6mg of indole-3-ethyl formate;
10g of Fr.9 is depigmented by a gel chromatographic column with dichloromethane-methanol in a ratio of 1:1, TLC tracking is carried out to obtain 5 components Fr.9.1-Fr.9.5, 212mg of Fr.9.5 is subjected to wet sample loading, and a dichloromethane-methanol system in a ratio of 40:1 and 35:1 is subjected to column chromatography twice and then is scraped to obtain 4mg of the compound 3-carboxyl indole.
The indole-3-ethyl formate and 3-carboxyl indole provided by the invention are applied to hemostatic drugs.
The application of the indole-3-ethyl formate and the 3-carboxyl indole in the aspect of hemostatic pharmaceutical preparations is provided.
The preparation is prepared into a pharmaceutically acceptable preparation by adding pharmaceutically acceptable auxiliary materials according to a conventional process, and the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation.
The solid preparation is granules, capsules, tablets, pills, powder and freeze-dried powder injection; the liquid preparation is injection preparation and oral liquid.
The pharmaceutically acceptable auxiliary materials are not limited, and can be one or more of common auxiliary materials in the field, such as a filling agent, a lubricating agent, a flavoring agent, a disintegrating agent, an antioxidant, a humectant, a surfactant and the like.
The principle of the invention is as follows: 2 indole alkaloids which are indole-3 ethyl formate and 3-carboxyl indole are respectively obtained by separating from a petroleum ether layer of a 95% ethanol extract of the blood basin grass by adopting an animal hemostatic activity guiding method, and the in-vitro plasma recalcification time of the indole alkaloids is determined by adopting a test tube method.
The problems in the prior art and the beneficial effects of the invention are as follows:
1. in the prior art, if the treatment is carried out by adopting pure western medicines, dangerous consequences caused by high coagulation state often appear, and the western medicines are forbidden or cautiously used under the condition of some inapplicable symptoms or postoperative sequelae, such as renal insufficiency or postoperative hematuria; the traditional hemostatic medicines can bring more adverse reactions, and the problems of hemostasis, thrombosis promotion, immunological rejection and the like cannot be solved.
The invention separates 2 indole alkaloids from the Chinese medicine of the hematuria which has the functions of cooling blood, removing toxicity, dissipating blood stasis and stopping bleeding, and pharmacodynamics experiments show that the invention has good hemostatic function and can be directly developed into hemostatic medicines or used as a lead compound to synthesize a series of medicines with good hemostatic activity.
2. Compared with the blank group, the plasma recalcification time of the indole-3-ethyl formate and the 3-carboxyl indole is obviously shortened, and the difference has statistical significance (P is less than 0.05); compared with the Yunnan white drug powder which is a positive drug, the plasma recalcification time of the indole-3-ethyl formate and the 3-carboxyl indole is shortened, and the difference of the plasma recalcification time in vitro has statistical significance (P is less than 0.05). Therefore, the invention not only solves the problems of hemostasis, but also solves the problems of thrombosis, immunological rejection and the like caused by the hemostasis process.
Drawings
FIG. 1 Process for preparing indole-3-carboxylic acid ethyl ester13C-NMR chart.
FIG. 2 Process for preparing indole-3-carboxylic acid ethyl ester1H-NMR chart.
FIG. 33 Process for preparing carboxyindoles13C-NMR chart.
FIG. 43 Process for preparing carboxyindoles1H-NMR chart.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
Example 1
Method for extracting and purifying indole alkaloids
Pulverizing 9.3kg dried whole plant of herba Rubi Seguinii into coarse powder, extracting with 95% industrial ethanol under reflux for 3 times, each for 2 hr, concentrating under reduced pressure, and recovering solvent to obtain 1756g ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 6 times by each polar solvent, respectively combining each extract, and recovering the solvent by a rotary evaporator to respectively obtain a petroleum ether part extract (286.6g), a chloroform part extract (244.9g), an ethyl acetate part extract (188.6g), an n-butanol part extract (205.1g) and a water part.
Dissolving the extractum at the petroleum ether part by adding a solvent, mixing the extractum with silica gel (100-200 meshes), applying a silica gel column by a dry method, performing gradient elution by silica gel column chromatography petroleum ether-ethyl acetate (20: 1-0: 1), tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components (Fr.1-12).
Fr.6 is subjected to pigment removal by a gel chromatographic column (dichloromethane-methanol 1:1) to obtain 6 components (Fr.6.1-Fr.6.6); fr.6.6(48mg) was eluted through silica gel column chromatography petroleum ether-dichloromethane (3:1) to give 3 fractions (Fr.6.6.1-Fr.6.6.3) and Fr.6.6.2 was scraped to give compound (indole-3-carboxylic acid ethyl ester) (6 mg).
After pigment removal from Fr.9 (about 10g) by gel chromatography (dichloromethane-methanol 1:1), TLC tracing gave 5 fractions (Fr.9.1 to Fr.9.5), and Fr.9.5(212mg) was subjected to wet-process loading, twice-column chromatography using a dichloromethane-methanol system (40:1, 35:1), and then scraped to give compound (3-carboxyindole) (4 mg).
Characterization of a chemical combination part:
compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000071
the molecular formula is as follows: c11H11NO2。
API-ES m/z：212[M+Na]+
The state is as follows: a white powder (dichloromethane),
silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in figure 1 and figure 2
Figure GDA0003158323730000072
Figure GDA0003158323730000081
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000082
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]+
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystal (methanol)
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4
Figure GDA0003158323730000083
Example 2
Method for extracting and purifying indole alkaloids
Pulverizing 7kg of dried whole herb of herba Rubi Corchorifolii into coarse powder, extracting with 8% industrial ethanol under reflux for 3 hr for 2 times, concentrating under reduced pressure, and recovering solvent to obtain 1300g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 8 times by each polar solvent, respectively combining each extract, and recovering the solvent by a rotary evaporator to respectively obtain 210g of petroleum ether extract, 180g of chloroform extract, 140g of ethyl acetate extract, 150g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; 36mg of Fr.6.6 is eluted by a silica gel column with 3:1 of chromatographic petroleum ether-dichloromethane to obtain 3 components of Fr.6.6.1-Fr.6.6.3, and Fr.6.6.2 is scraped to obtain 4.5mg of indole-3-ethyl formate;
removing pigment from 7g of Fr.9 by a gel chromatographic column by using dichloromethane-methanol at a ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 160mg of Fr.9.5, carrying out column chromatography twice by using a dichloromethane-methanol system at a ratio of 40:1 and 35:1, and scraping to obtain 3-carboxyl indole 3.1 mg.
Characterization of a chemical combination part:
compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000091
the molecular formula is as follows: c11H11NO2。
API-ES m/z：212[M+Na]+
The state is as follows: a white powder (dichloromethane),
silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600 MHz，CDCl3)、13C-NMR(150 MHz，CDCl3) The data are shown in figure 1 and figure 2
Figure GDA0003158323730000092
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000101
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]+
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystal (methanol)
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4
Figure GDA0003158323730000102
Example 3
Method for extracting and purifying indole alkaloids
Pulverizing 12kg of dried whole herb of herba Rubi Corchorifolii into coarse powder, reflux-extracting with 98% industrial ethanol for 2 times, each for 1 hr, concentrating under reduced pressure, and recovering solvent to obtain 2300g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 4 times by each polar solvent, respectively combining each extraction liquid, and recovering the solvent by a rotary evaporator to respectively obtain 360g of petroleum ether extract, 320g of chloroform extract, 240g of ethyl acetate extract, 265g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 62mg of Fr.6.6 with chromatographic petroleum ether-dichloromethane at a ratio of 3:1 through a silica gel column to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 with a scraper to obtain 7.8mg of indole-3-ethyl formate;
13g of Fr.9 is depigmented by a gel chromatographic column with dichloromethane-methanol in a ratio of 1:1, TLC tracking is carried out to obtain 5 components Fr.9.1-Fr.9.5, 275mg of Fr.9.5 is subjected to wet sample loading, and a dichloromethane-methanol system in a ratio of 40:1 and 35:1 is subjected to column chromatography twice and then scraped to obtain 5.2mg of the compound 3-carboxyl indole.
Characterization of a chemical combination part:
compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000111
the molecular formula is as follows: c11H11NO2。
API-ES m/z：212[M+Na]+
The state is as follows: a white powder (dichloromethane),
silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 1 and FIG. 2.
Figure GDA0003158323730000112
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000113
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]
Melting point (mp): 232-234 DEG C
The state is as follows: pale yellow needle crystal (methanol)
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4
Figure GDA0003158323730000121
Example 4
Pulverizing 8kg of dried whole herb of herba Rubi Corchorifolii into coarse powder, extracting with 85% industrial ethanol under reflux for 3 hr for 2 times, concentrating under reduced pressure, and recovering solvent to obtain 1500g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 5 times by each polar solvent, respectively combining each extraction liquid, and recovering the solvent by a rotary evaporator to respectively obtain 230g of petroleum ether extract, 200g of chloroform extract, 160g of ethyl acetate extract, 170g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; 36mg of Fr.6.6 is eluted by a silica gel column with 3:1 of chromatographic petroleum ether-dichloromethane to obtain 3 components of Fr.6.6.1-Fr.6.6.3, and Fr.6.6.2 is scraped to obtain 5mg of indole-3-ethyl formate;
removing pigment from 8g of Fr.9 by a gel chromatographic column by using dichloromethane-methanol at a ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, loading 180mg of Fr.9.5 by a wet method, performing column chromatography twice by using a dichloromethane-methanol system at a ratio of 40:1 and 35:1, and scraping to obtain 3-carboxyl indole 3.5 mg.
Characterization of a chemical combination part:
compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000131
the molecular formula is as follows: c11H11NO2
API-ES m/z：212[M+Na]+。
The state is as follows: white powder (dichloromethane).
Silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in figure 1 and figure 2
Figure GDA0003158323730000132
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000133
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]+。
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystals (methanol).
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4
Figure GDA0003158323730000141
Example 5
Pulverizing 11kg of dried whole herb of herba Rubi Corchorifolii into coarse powder, reflux-extracting with 97% industrial ethanol for 4 times, each for 1 hr, concentrating under reduced pressure, and recovering solvent to obtain 2100g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 7 times by each polar solvent, respectively combining each extraction liquid, and recovering the solvent by a rotary evaporator to respectively obtain 340g of petroleum ether part extract, 300g of chloroform part extract, 220g of ethyl acetate part extract, 245g of n-butanol part extract and water part;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 62mg of Fr.6.6 with chromatographic petroleum ether-dichloromethane at a ratio of 3:1 through a silica gel column to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 with a scraper to obtain 7mg of indole-3-ethyl formate;
removing pigment from 12g of Fr.9 by a gel chromatographic column by using dichloromethane-methanol at a ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 255mg of Fr.9.5, carrying out column chromatography twice by using a dichloromethane-methanol system at a ratio of 40:1 and 35:1, and scraping to obtain 4.8mg of the compound 3-carboxyl indole.
Characterization of a chemical combination part:
compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000142
the molecular formula is as follows: c11H11NO2
API-ES m/z：212[M+Na]+。
The state is as follows: white powder (dichloromethane).
Silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 1 and FIG. 2.
Figure GDA0003158323730000151
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000152
the molecular formula is as follows: C9H7NO2
EI-MS m/z：161[M]+。
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystals (methanol).
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4
Figure GDA0003158323730000153
Example 6
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as raw material, adding 1/9 dextrin, and granulating to obtain granule.
Example 7
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as raw material, adding 1/9 dextrin, mixing, and encapsulating to obtain capsule.
Example 8
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as raw material medicine, adding 1/10 dextrin, mixing, drying, and making into pill.
Example 9
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as raw material, adding 1/10 dextrin, granulating, tabletting, and making into tablet.
Example 10
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as a raw material medicine, adding 13 times of water for injection, stirring, filtering, and sterilizing to obtain the injection.
Example 11
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as a raw material medicine, adding 9 times of water for injection, stirring, filtering, and freeze-drying to obtain freeze-dried powder.
Example 12
Taking any one of indole-3-ethyl formate and 3-carboxyl indole as a raw material medicine, adding 13 times of purified water, uniformly mixing, filtering, and sterilizing to obtain the oral liquid.
In order to further verify the feasibility and effectiveness of the invention and screen out the best scheme, the inventor carries out a series of tests, specifically as follows:
the traditional hemostatic medicines can bring more adverse reactions, and the problems of hemostasis, thrombosis promotion, immunological rejection and the like cannot be solved. The combination of Chinese and Western medicine is a new way to develop new hemostatic medicine, and Chinese medicine can treat diseases through dialectical treatment to stop bleeding, and can act on three major systems of physiological hemostasis, blood coagulation and fibrinolysis. The invention separates 2 indole alkaloids from traditional Chinese medicine (Salvia calterii Levl. var. simplicifolia Stib.) with the functions of cooling blood, removing toxicity, dissipating blood stasis and stopping bleeding, and pharmacodynamics experiments show that the indole alkaloids have good hemostatic effect and can be directly developed into hemostatic drugs or used as lead compounds to synthesize a series of drugs with good hemostatic activity.
The principle is as follows: 2 indole alkaloids which are indole-3 ethyl formate and 3-carboxyl indole are respectively obtained by separating from a petroleum ether layer of a 95% ethanol extract of the blood basin grass by adopting an animal hemostatic activity guiding method, and the in-vitro plasma recalcification time of the indole alkaloids is determined by adopting a test tube method.
1. Experimental methods
1.1 extraction and separation of indole-3-carboxylic acid ethyl ester, 3-carboxyindole
The extraction and purification method comprises the following steps:
pulverizing 9.3kg dried whole plant of herba Rubi Seguinii into coarse powder, extracting with 95% industrial ethanol under reflux for 3 times, each for 2 hr, concentrating under reduced pressure, and recovering solvent to obtain 1756g ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 6 times by each polar solvent, respectively combining each extract, and recovering the solvent by a rotary evaporator to respectively obtain a petroleum ether part extract (286.6g), a chloroform part extract (244.9g), an ethyl acetate part extract (188.6g), an n-butanol part extract (205.1g) and a water part.
Dissolving the extractum at the petroleum ether part by adding a solvent, mixing the extractum with silica gel (100-200 meshes), applying a silica gel column by a dry method, performing gradient elution by silica gel column chromatography petroleum ether-ethyl acetate (20: 1-0: 1), tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components (Fr.1-12).
Fr.6 is subjected to pigment removal by a gel chromatographic column (dichloromethane-methanol 1:1) to obtain 6 components (Fr.6.1-Fr.6.6); fr.6.6(48mg) was eluted through silica gel column chromatography petroleum ether-dichloromethane (3:1) to give 3 fractions (Fr.6.6.1-Fr.6.6.3) and Fr.6.6.2 was scraped to give Compound 1 (indole-3-carboxylic acid ethyl ester) (6 mg).
After pigment removal from Fr.9 (about 10g) by gel chromatography (dichloromethane-methanol 1:1), TLC tracing gave 5 fractions (Fr.9.1 to Fr.9.5), and Fr.9.5(212mg) was subjected to wet loading, twice chromatography in a dichloromethane-methanol system (40:1, 35:1), and then scraped to give Compound 2 (3-carboxyindole) (4 mg).
1.2 characterization of the Compounds
Compound 1: indole-3-carboxylic acid ethyl ester
Structural formula (xvi):
Figure GDA0003158323730000171
the molecular formula is as follows: c11H11NO2。
API-ES m/z：212[M+Na]+。
The state is as follows: white powder (dichloromethane).
Silica gel thin layer identification: petroleum ether and ethyl acetate (8:1) are developed, fluorescence is generated under 254nm, orange spots are formed after coloration and baking are carried out by 10% concentrated sulfuric acid-ethanol, and the spots are continuously baked to be purple red.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 1 and FIG. 2.
Figure GDA0003158323730000181
Compound 2: 3-carboxyindoles
Structural formula (xvi):
Figure GDA0003158323730000182
the molecular formula is as follows: c9H7NO2
EI-MS m/z：161[M]+。
Melting point (mp): 232-234 ℃.
The state is as follows: pale yellow needle crystals (methanol).
Silica gel thin layer identification: the developed dichloromethane-methanol 35:1 has fluorescence under 254nm, and the orange spots appear after the development and baking of 10% concentrated sulfuric acid-ethanol.
1H-NMR(600MHz，CDCl3)、13C-NMR(150MHz，CDCl3) The data are shown in FIG. 3 and FIG. 4.
Figure GDA0003158323730000183
1.3 in vitro hemostatic Activity test
1.3.1 animal, Positive control
2-4kg of healthy rabbits are fed by green grass; yunnan Baiyao capsule (national standard Z53020799, Yunnan Baiyao group GmbH);
1.3.2 in vitro hemostasis test
1.3.2.1 preparation of sample solutions
Placing 5mg of indole-3 ethyl formate and 5mg of 3-carboxyl indole in a PE tube respectively, adding 1mL of deionized water to prepare a solution with the concentration of 5mg/mL, placing the solution in an ultrasonic cleaner for ultrasonic treatment for 30min, and filtering the solution by using a microporous filter to prepare a clear sample solution for later use.
1.3.2.2 Positive control drug with M/40CaCl2Preparation of the solution
Dissolving 0.05g of powder in Yunnan Baiyao capsule in 10mL of deionized water, and filtering with 0.45nm microporous membrane to obtain clear Yunnan Baiyao solution (5mg/mL) for use.
1.1100g of anhydrous CaCl2 solid is taken and dissolved in 400mL of deionized water to prepare M/40CaCl2 solution for later use.
1.3.2.3 preparation of anticoagulated plasma
After anesthesia of healthy rabbits by intraperitoneal injection of chloral hydrate (10 percent, 3.5mL/kg), blood is taken from the marginal veins of the rabbits by a blood taking needle and a negative pressure blood collection tube (sodium citrate 1:9) with an anticoagulant, the mixture is gently shaken and uniformly mixed, and then the mixture is centrifuged at the rotating speed of 3000r/min for 30min, and plasma is separated for later use.
1.3.2.4 Effect of the tube method on in vitro plasma recalcification time
Taking a clean glass test tube, and adding 0.1mL of the medicine and 0.1mL of M/40CaCl by using a pipette2And (3) shaking the solution uniformly, putting the solution on a test tube rack, putting the solution into a water bath kettle at 37 ℃, adding 0.1mL of anticoagulated plasma, starting timing, inclining the test tube every 10s for observation until white flocculent fibrin appears, and stopping timing, wherein the time is the plasma recalcification time. The positive control group medicine is Yunnan white drug powder solution, and the blank control group is deionized water. Each set of experiments was repeated 5 times.
1.3.3 results and conclusions
1.3.3.1 results of the experiment
Plasma recalcification time for the samples (N-5,
Figure GDA0003158323730000191
Figure GDA0003158323730000192
acomparison with blank group, P<0.05；bComparison with positive drugs, P<0.05
1.3.3.2 conclusion of the experiment
Compared with a blank group, the plasma recalcification time of the indole-3-ethyl formate and the 3-carboxyl indole is obviously shortened, and the difference has statistical significance (P is less than 0.05); compared with the Yunnan white drug powder which is a positive drug, the plasma recalcification time of the indole-3-ethyl formate and the 3-carboxyl indole is shortened, and the difference of the plasma recalcification time in vitro has statistical significance (P is less than 0.05).
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. A method for extracting and purifying indole alkaloids from Chinese medicinal herba Rubi, is characterized in that the indole alkaloids are indole-3-ethyl formate and 3-indolecarboxylic acid;
the method for extracting and purifying the indole alkaloid comprises the following steps: crushing 7-12 kg of dried whole herb of rubus bidens into coarse powder, performing reflux extraction for 2-5 times by using 80-98% industrial ethanol, performing concentration under reduced pressure for 1-3 hours each time, and recovering a solvent to obtain 1300-2300 g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 4-8 times by using each polar solvent, respectively combining each extraction liquid, and recovering the solvent by using a rotary evaporator to respectively obtain 210-360 g of petroleum ether part extract, 180-320 g of chloroform part extract, 140-240 g of ethyl acetate part extract, 150-265 g of n-butanol part extract and water part;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 36-62 mg of Fr.6.6 by a silica gel column with chromatographic petroleum ether-dichloromethane according to the ratio of 3:1 to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 by a scraper to obtain 4.5-7.8 mg of indole-3-ethyl formate;
removing pigment from 7-13 g of Fr.9 by using a gel chromatographic column with dichloromethane-methanol in a ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 160-275 mg of Fr.9.5, carrying out column chromatography twice on a dichloromethane-methanol system in a ratio of 40:1 and 35:1, and scraping to obtain 3.1-5.2 mg of the compound 3-indolecarboxylic acid.
2. The method for extracting and purifying indole alkaloids according to claim 1, wherein the method for extracting and purifying indole alkaloids specifically comprises: crushing 8-11 kg of dried whole herb of the rubus bidens into coarse powder, performing reflux extraction for 2-4 times by using 85-97% industrial ethanol, performing concentration under reduced pressure for 1-3 hours each time, and recovering a solvent to obtain 1500-2100 g of ethanol extract; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 5-7 times by using each polar solvent, respectively combining each extraction liquid, and recovering the solvent by using a rotary evaporator to respectively obtain 230-340 g of petroleum ether extract, 200-300 g of chloroform extract, 160-220 g of ethyl acetate extract, 170-245 g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 36-62 mg of Fr.6.6 by a silica gel column with chromatographic petroleum ether-dichloromethane according to the ratio of 3:1 to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 by a scraper to obtain 5-7 mg of indole-3-ethyl formate;
removing pigment from 8-12 g of Fr.9 by using a gel chromatographic column through dichloromethane-methanol according to the ratio of 1:1, tracking by TLC to obtain 5 components Fr.9.1-Fr.9.5, carrying out wet sample loading on 180-255 mg of Fr.9.5, carrying out column chromatography twice through a dichloromethane-methanol system according to the ratio of 40:1 and 35:1, and scraping to obtain 3.5-4.8 mg of the compound 3-indolecarboxylic acid.
3. The method for extracting and purifying indole alkaloids according to claim 2, wherein the method for extracting and purifying indole alkaloids specifically comprises: pulverizing 9.3kg dried whole plant of herba Rubi Corchorifolii into coarse powder, extracting with 95% industrial ethanol under reflux for 3 times, each for 2 hr, concentrating under reduced pressure, and recovering solvent to obtain alcohol extract 1756 g; the method comprises the following steps of 1:1, suspending in water, and sequentially performing 1:1, extracting for 6 times by each polar solvent, respectively combining each extraction liquid, and recovering the solvent by a rotary evaporator to respectively obtain 286.6g of petroleum ether extract, 244.9g of chloroform extract, 188.6g of ethyl acetate extract, 205.1g of n-butanol extract and water;
dissolving the petroleum ether part extract by adding a solvent, mixing the extract with silica gel, applying the mixture to a silica gel column by a dry method, performing gradient elution by the silica gel column with petroleum ether-ethyl acetate in a chromatography of 20: 1-0: 1, tracking by TLC, combining the same parts, and performing coarse segmentation to obtain 12 components Fr.1-12;
fr.6 is subjected to pigment removal by a gel chromatographic column by dichloromethane-methanol with the ratio of 1:1 to obtain 6 components Fr.6.1-Fr.6.6; eluting 48mg of Fr.6.6 with chromatographic petroleum ether-dichloromethane at a ratio of 3:1 through a silica gel column to obtain 3 components Fr.6.6.1-Fr.6.6.3, and scraping Fr.6.6.2 with a scraper to obtain 6mg of indole-3-ethyl formate;
10g of Fr.9 is depigmented by a gel chromatographic column with dichloromethane-methanol in a ratio of 1:1, TLC tracking is carried out to obtain 5 components Fr.9.1-Fr.9.5, 212mg of Fr.9.5 is subjected to wet sample loading, and a dichloromethane-methanol system in a ratio of 40:1 and 35:1 is subjected to column chromatography twice and then is scraped to obtain 4mg of the compound 3-indolecarboxylic acid.
4. The use of the indole alkaloid of claim 1, wherein said indole-3-carboxylic acid ethyl ester, 3-indolecarboxylic acid are used in the preparation of a hemostatic agent.
5. The use of indole alkaloids according to claim 4, wherein indole-3-carboxylic acid ethyl ester, 3-indolecarboxylic acid are used in the preparation of a hemostatic pharmaceutical formulation.
6. The use of indole alkaloids according to claim 5, wherein the preparation is prepared into pharmaceutically acceptable preparations by adding pharmaceutically acceptable adjuvants according to conventional process, and the pharmaceutically acceptable preparations are solid preparations or liquid preparations.
7. The use of indole alkaloids according to claim 6, wherein the solid formulation is a granule, a capsule, a tablet, a pill, a powder, a lyophilized powder for injection; the liquid preparation is injection preparation and oral liquid.
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