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CN111329866B - Application of pentacyclic triterpenoid in preparation of anti-migraine medicine - Google Patents

Application of pentacyclic triterpenoid in preparation of anti-migraine medicine Download PDF

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CN111329866B
CN111329866B CN202010282388.4A CN202010282388A CN111329866B CN 111329866 B CN111329866 B CN 111329866B CN 202010282388 A CN202010282388 A CN 202010282388A CN 111329866 B CN111329866 B CN 111329866B
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黄相中
于海波
李艳红
田凯
蒋孟圆
李育晓
李红锐
李育逵
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Abstract

The invention relates to application of a pentacyclic triterpenoid in preparing an anti-migraine medicament. Specifically, the invention relates to application of a pentacyclic triterpenoid compound shown as a formula I in treating migraine:
Figure DDA0002447184070000011
in formula I, R = H, OH. The pentacyclic triterpenoid can be a naturally-occurring organic compound or a synthetic organic compound, has strong anti-migraine activity, and is used as an active ingredient to be prepared into an anti-migraine medicine or a pharmaceutical composition together with a pharmaceutically acceptable carrier or auxiliary material.

Description

Application of pentacyclic triterpenoid in preparation of anti-migraine drugs
Technical Field
The invention relates to application of a triterpenoid in preparing an anti-migraine medicine, in particular to application of a triterpenoid which can be separated from centella plants (such as centella asiatica) in the Umbelliferae in preparing an anti-migraine medicine.
Background
Migraine is the most common type of headache in clinic, generally without organic lesions, and clinically with paroxysmal moderate-severe and pulsatile headache as the main manifestations, mainly pain on one side or two sides of the head. Migraine is often genetically related and is more likely to develop in people with poor physical fitness, poor sports, and mental work. Migraine mainly includes three major classes of typical migraine, common migraine and cluster migraine. Typical migraine headache patients usually have periodic attacks, and are common to women. Before onset, most patients can have blurred vision, flash vision, pseudoscopic vision, blind spots, eye swelling and emotional instability, almost all patients are afraid of light, and one-sided headache can occur after a few minutes, and the majority of patients mainly take the parts of the head, the temples, the peripheries of the eye sockets, the temples and the like. Pain typically peaks in 1-2 hours, lasting 4-6 hours or more than ten hours, and can last for days in severe cases. Common migraine accounts for 80% of migraine patients, is common, has no obvious aura symptoms before onset, and has the symptoms of mental disorder, fatigue, yawning, inappetence, general malaise and the like before onset in some patients. The cluster migraine attack has no aura symptom, and the time of each attack is about the same. During the attack, the eye socket is swollen, lacrimation, conjunctival congestion, nasal obstruction, sweating and other symptoms occur. In addition, migraine includes neuropsychiatric migraine, abdominal migraine, familial hemiplegic migraine, and the like. The prevalence of migraine is 15% to 18% in women and 6% -12% in men. The incidence of migraine in age samples 25 to 64 years old is 8.1 people per thousand per year.
Currently, the drugs commonly used for treating migraine mainly comprise ibuprofen, mexicarpipe, nimodipine, aspirin, ergotamine preparation, sumatriptan, propranolol, amitriptyline, clonidine, flunarizine, sodium valproate and other chemical drugs. Although the medicine has better therapeutic effect on migraine, the medicine also shows serious adverse reaction in clinical application, and causes harm to organisms when being applied for many times or for a long time, and the medicine is highly regarded in recent years. The adverse reactions mainly comprise fatigue, nausea, lethargy, weakness, dry mouth, emesis, dyspepsia, gastrointestinal reaction, colitis, mental disorder, blood system damage, liver and kidney damage, etc., and serious adverse events comprise myocardial infarction, arrhythmia and apoplexy. At present, researchers at home and abroad pay great attention to exploring and searching anti-migraine medicaments from natural medicaments, which have good curative effects and small adverse reactions. The anti-migraine active ingredients of the botanical drugs such as the dahurian angelica root, the divaricate saposhnikovia root, the Szechuan lovage rhizome, the tetrandra root, the dolichos root and the like are researched, and a group of compounds with excellent activity, including alkaloids, diterpenes, triterpenes, coumarins, steroids, volatile oil and the like, are obtained. Although more active compounds were found, the efficacy was less strong. At present, there are also anti-migraine drugs or pharmaceutical compositions prepared from various medicinal plants, but the therapeutic effect is not ideal.
Disclosure of Invention
Technical problem to be solved
The invention aims to solve the technical problem that the compound in the prior art has poor treatment effect on migraine.
(II) technical scheme
In order to solve the technical problems, the invention provides an application of a pentacyclic triterpenoid in treating migraine, a medicine or a medicine composition for treating the migraine and a preparation method thereof.
Specifically, the present invention provides:
(1) The application of the pentacyclic triterpenoid in preparing the anti-migraine medicine,
Figure BDA0002447184050000021
Figure BDA0002447184050000031
in formula I, R = H, OH.
(2) The use according to (1), wherein the pentacyclic triterpenoid is one or two of compounds shown in formulas II-III:
formula II:
Figure BDA0002447184050000032
formula III:
Figure BDA0002447184050000033
(3) The use according to (2), wherein one or two of the compounds represented by the formulae II to III are present in and used in the form of an extract obtained by extracting centella with a solvent; wherein the solvent is 70-100 vol% ethanol/water, 70-100 vol% methanol/water, or 50-80 vol% acetone/water, wherein the centella plant is centella asiatica.
(4) The use according to (1) to (3), wherein the pentacyclic triterpenoid is used as an active ingredient and is prepared into a pharmaceutical composition together with a pharmaceutically acceptable carrier or auxiliary material.
(5) The use according to (1) - (4), wherein the migraine is a typical migraine, a common migraine or a cluster migraine.
(6) The use according to any one of (1) to (5), wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills, and injections.
(7) The use according to any one of (1) to (5), wherein the pharmaceutical composition is selected from a sustained release preparation or a controlled release preparation.
(8) The use according to any one of (1) to (7), wherein the pharmaceutically acceptable carrier or adjuvant comprises oral preparation adjuvant, parenteral administration or external administration adjuvant, and the administration route can be oral administration, injection, external topical administration and the like; the administration dosage form can be liquid dosage form, solid dosage form, the liquid dosage form can be syrup, injection solution, non-aqueous solution, suspension or emulsion, the solid dosage form can be tablet, lozenge, capsule, dripping pill, granule, powder, cream, solution, suppository, dispersible powder such as lyophilized powder for injection, aerosol, etc.; the used auxiliary materials comprise: lactose, calcium carbonate, calcium phosphate, sodium phosphate, starch, cyclodextrin, sucrose, mannitol, microcrystalline cellulose sodium, calcium sulfate, water, ethanol, propanol, glycerol, propylene glycol, isopropanol, syrup, honey, glucose, gelatin syrup, sodium carboxymethylcellulose, potassium phosphate, dried starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecyl sulfonate, methyl cellulose, glyceryl tristearate, cocoa butter, hydrogenated oil, quaternary ammonium salt, talc, triethylamine magnesium stearate, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin.
In addition, the present invention also provides:
(9) The use of centella asiatica extract in the preparation of anti-migraine agent is to extract centella asiatica with solvent to obtain extract, and use the extract; wherein the solvent is 70 to 100 volume percent of ethanol/water, 70 to 100 volume percent of methanol/water, or 50 to 80 volume percent of acetone/water.
(10) The use according to (9), wherein the migraine is a typical migraine, a common migraine or a cluster migraine.
(III) advantageous effects
The technical scheme of the invention has the following advantages:
1. the compound of the formula I can obviously reduce the times of head scratching of rats, reduce the content of NO in serum, lower the content of Calcitonin Gene Related Peptide (CGRP) in brain tissues of rats and increase the content of 5 hydroxytryptamine (5-HT) and beta endorphin (beta-EP) in the brain tissues of rats in a nitroglycerin migraine headache causing experiment, thereby prompting that the compound has good anti-migraine activity.
2. The centella plants, especially the centella asiatica, which can be adopted in the preparation method of the compound shown in the formula I are widely distributed in China, the resources are rich, and the raw material source is simple; in addition, the compound of the formula I has high content in centella plants, especially centella, and is easy to obtain.
3. The preparation method of the compound of the formula I can adopt a conventional column chromatography preparation method, the preparation operation flow of the compound is simple, the purity of the obtained compound is high, and the subsequent industrial production is easy to realize.
4. The invention discovers that asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-alkene-28-ursolic acid in the compound shown in the formula I have better treatment effect on migraine for the first time. The compounds can be used as anti-migraine active ingredients or lead compounds, and have good application prospects.
Drawings
FIG. 1 is a schematic diagram of the activity tracking and isolation scheme of 2 anti-migraine triterpenoids;
FIG. 2 shows the NMR spectrum of asiatic acid in the present invention ( 1 H NMR)；
FIG. 3 shows the NMR spectrum of asiatic acid in the present invention (C) 13 C NMR)；
FIG. 4 is a DEPT spectrum of asiatic acid in the present invention;
FIG. 5 is a mass spectrum (ESI-MS) of asiatic acid in the present invention;
FIG. 6 shows NMR spectra of 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid in the present invention: ( 1 H NMR)；
FIG. 7 is the NMR spectrum of 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid in the present invention: ( 13 C NMR)；
FIG. 8 is a DEPT spectrum of 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid in accordance with the present invention;
FIG. 9 is a mass spectrum (ESI-MS) of 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid in accordance with the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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 some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The compound of formula I in the present invention may be a naturally occurring compound or a synthetic compound.
The anti-migraine ingredients of the present invention include one or both of the compounds represented by formula I:
Figure BDA0002447184050000061
in formula I, R = H, OH.
The anti-migraine component can also be an extract obtained by extracting centella plants by using a solvent; wherein the solvent is 70-100 vol% ethanol/water, 70-100 vol% methanol/water, or 50-80 vol% acetone/water.
Centella asiatica is dry whole plant of Centella asiatica (l.) Urban (Centella asiatica) of Centella of Umbelliferae, belonging to perennial creeping herbaceous plants, produced in China, malaysia, indonesia, srilanca, vietnam, etc. The method is mainly distributed in China such as east China, south China, china and the southwest China. Centella asiatica is bitter and pungent in taste and cold in nature; the medicine is mainly used for clearing heat, promoting diuresis, reducing swelling and relieving pain and the like, and has certain curative effects on fever, enteritis, swelling and pain in throat, dysentery, damp-heat jaundice, edema, stranguria, hematuria, epistaxis, dysmenorrheal, metrorrhagia, scrofula, furuncle pyogenic infections, traumatic injury and gall, traumatic hemorrhage, snake and insect bite and the like (Jiamei, shiwei, xilijia, and the like. Research progress of centella asiatica. Modern Chinese medicine 2016,18 (2): 49-50.). Centella asiatica is widely applied in various forms, and is also widely eaten in the traditional application besides being used as a medicine, for example, centella asiatica is added into tea by dry leaves in China and is mostly used as herbal tea (Shiwei, liuyong, xilijia, and the like. Tea drinking and health. Chinese modern traditional medicine: 2014,16 (1): 1-3.), and fresh leaves can be used as vegetables or fruit juice in some countries in the east-south Asia (Wanggui, yuanqig, zhao friendship. European and American medicinal plant (III). Chinese wild plant resource: 2003,22 (6): 96-97.). Centella asiatica mainly contains triterpenes, glycosides, polyacetylenes, volatile oil, flavonoids, alkaloids and other compounds (Pengjinyu, junxiantong, zhangun, and the like). Centella asiatica's chemical components and quality control research progress, chinese folk medicine 2011 (14): 233-238.). Among them, asiaticoside, which is the triterpenes, has been most studied and is considered to be the main active ingredient of Centella asiatica that exerts related effects (Siddiqui B.S., aslam H., ali S.T., et al. Chemical associations of Centella asiatica.J.Asian Nat. Prod. Res.2007,9 (4): 407-414.). Pharmacological research shows that the plant has activities in various aspects such as anti-tumor, anti-depression, immunity enhancement, antibiosis and anti-inflammation, skin injury repair, nerve protection and the like.
The invention discovers that the solvent (such as 95 volume percent ethanol/water) extract of centella plants (such as centella asiatica) has better anti-migraine activity, and chemical composition research is carried out on the extract under the guidance of a biological activity test, so as to obtain a triterpenoid active ingredient with better anti-migraine activity.
The triterpenoid is mainly from centella plants, and comprises (but is not limited to): asiatic acid (asiatic acid), 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid (2 α,3 β,6 β,23 α -tetrahydroxyurs-12-en-28-oic acid), and the like.
The structural formula of asiatic acid (2 alpha, 3 beta, 23 alpha-trihydroxy-12-ene-28-ursolic acid) (aspartic acid;2 alpha, 3 beta, 23 alpha-trihydroxyurs-12-en-28-oic acid) is as follows:
Figure BDA0002447184050000071
the structural formula of 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-ene-28-ursolic acid (2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxyurs-12-en-28-oic acid) is as follows:
Figure BDA0002447184050000081
the compound of the formula I has an obvious inhibition effect on rat migraine induced by nitroglycerin. Preferably, the asiatic acid in said compound of formula I has the strongest inhibitory effect on nitroglycerin-induced migraine in rats. The application of the compound shown in the formula I in preparing an anti-migraine medicine or a medicine composition is characterized in that the anti-migraine medicine or the medicine composition is used for treating migraine.
The invention adopts the existing nitroglycerin migraine headache model recognized at home and abroad to carry out the anti-migraine activity test on the compound shown in the formula I. Experimental results show that asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid can obviously reduce the times of rat head scratching, reduce the content of NO in serum, reduce the content of CGRP in rat brain tissues and increase the content of 5-HT and beta-EP in rat brain tissues, and the asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid have good anti-migraine activity, wherein the anti-migraine effect of asiatic acid is strong.
The present invention also provides a process for the preparation of a compound of formula I as described above, which process comprises preparing said compound from a plant of the genus centella, such as centella asiatica. Preferably, centella asiatica and 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid are prepared from the roots, stems, leaves, fruits of centella asiatica, a plant of the genus centella. Preferably, the method has the following steps:
(1) Drying herba Centellae, pulverizing, extracting with solvent, and mixing extractive solutions;
(2) Concentrating the extracting solution in the step (1) under reduced pressure to obtain extract;
(3) Suspending the extract in the step (2) in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and evaporating the solvent with a rotary evaporator to obtain petroleum ether, ethyl acetate and n-butanol extracts respectively;
(4) The ethyl acetate extract in (3) was separated by silica gel column chromatography, and was gradient-eluted with chloroform-methanol (volume ratio 30: 1-0: 1) to obtain 10 fractions (fr.1-10).
(5) Subjecting Fr.3 in (4) to silica gel column chromatography, eluting with chloroform-methanol (volume ratio 30. Fr.3-3 was purified by Sephadex LH-20 column chromatography (methanol) and then eluted by silica gel column chromatography with chloroform-methanol (volume ratio 25; subjecting Fr.4 in (4) to silica gel column chromatography, and performing gradient elution by using chloroform-methanol at a volume ratio of 20. Fr.4-2 was purified by Sephadex LH-20 column chromatography (methanol), and then eluted with chloroform-methanol (volume ratio 15.
In the step (1), the solvent can be ethanol/water with volume percentage of 70-100%, or methanol/water with volume percentage of 70-100%, or acetone/water with volume percentage of 50-80%, the dosage of the solvent is 6-10 times of the weight of the centella asiatica, the reflux extraction time is 2 hours each time, the reflux extraction is repeated for 3 times, and the filtrate is combined to obtain the extract solution of the centella medicinal material.
The invention also provides the use of a compound of formula I in the manufacture of an anti-migraine agent or pharmaceutical composition: the compound of the formula I is used as an active ingredient and is prepared into an anti-migraine medicament or a medicament composition together with a pharmaceutically acceptable carrier or auxiliary material.
The anti-migraine drug or the pharmaceutical composition can be administered in a unit dosage form, and the administration route can be intestinal tract or parenteral tract, such as oral administration, muscle administration, nasal cavity administration, oral mucosa administration, skin administration, transdermal administration, subcutaneous administration, intradermal administration, peritoneal administration, rectal administration, intravenous administration, intramuscular administration, epidural administration, intraocular administration, intracranial administration, vaginal administration and the like;
the administration route of the anti-migraine drug or the pharmaceutical composition of the present invention may be injection administration. The injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, intrathecal injection, peritoneal injection, etc.
The administration dosage form can be liquid dosage form or solid dosage form. The solution properties of the liquid dosage form can be true solutions, colloids, microparticles, emulsions, and suspensions. The liquid dosage form can be syrup, injection solution, non-aqueous solution, suspension or emulsion; solid dosage forms such as tablet, lozenge, capsule, dripping pill, granule, powder, cream, solution, suppository, dispersible powder such as lyophilized powder for injection, aerosol, etc.
The anti-migraine drug or the pharmaceutical composition can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.
The pharmaceutically acceptable carrier or auxiliary material comprises an oral preparation auxiliary material and an auxiliary material for parenteral administration or external administration. The adjuvants include excipient such as lactose, calcium carbonate, calcium phosphate, and sodium phosphate; diluents and absorbents such as starch, cyclodextrin, lactose, sucrose, mannitol, microcrystalline cellulose sodium, calcium sulfate, and the like; wetting agents and binders such as water, ethanol, propanol, glycerol, propylene glycol, isopropanol, syrup, honey, glucose, gelatin syrup, sodium carboxymethylcellulose, potassium phosphate, etc.; disintegrating agents such as dry starch, agar powder, calcium carbonate, sodium bicarbonate, sodium dodecylsulfate, methylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants such as talc, triethylamine magnesium stearate, silica, corn starch, stearate, boric acid, liquid paraffin, and the like. The tablets may be further prepared into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets, in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide sustained action over a longer period.
In order to better understand the present invention, the following further explains or illustrates the present invention by specific examples, but these examples should not be construed as limiting the scope of the present invention.
Example 1
Centella asiatica extract anti-migraine activity test
Material sources are as follows: centella asiatica belonging to the genus Centella is collected from Yunnan Kunming and identified as Centella asiatica (L.) Urban by professor Yang Qing Song of national medical institute of Yunnan national university. The specimens are stored in the specimen museum of national institute of medicine and medicine of Yunnan national university.
Preparation of centella asiatica extract: crushing the whole dried centella to obtain centella broken blocks; then reflux-extracting the fragments of herba Centellae with 95 vol% ethanol/water for 3 times, each for 2 hr to obtain extractive solution; filtering the herba Centellae extractive solutions respectively, and concentrating under reduced pressure with a rotary evaporator to obtain extract.
The anti-migraine activity of centella asiatica different solvent extracts was tested using a nitroglycerin migraine headache rat model (see scientific literature: zhang Xiao-Fan, zhang Wen-Jun, dong Cui-Lan et al. Analgesia effect of baicalein against NTG-induced migaine in rates [ J ]. Biomed. Pharmacother, 2017, 90. 60 male healthy SD rats with the weight of 180-220 g are randomly divided into 6 groups of 10 rats. The groups are as follows: blank control group (distilled water), negative control group (distilled water), positive control group (ibuprofen tablet, 80 mg/kg), and herba Centellae high, medium, and low dose groups (2.0 g/kg,0.5g/kg,0.13 g/kg), and continuously administering for 7d. After 60min of the last administration, except for the blank group, each group was subcutaneously injected with nitroglycerin 10mg/kg in the right shoulder for 30min as a time period, and the number of times of head bending of the rats in 6 time periods after molding was observed and recorded. Experimental results show that the centella asiatica extract has obvious anti-migraine effect in high and medium dose groups. The results are shown in Table 1.
TABLE 1 Effect of 95% ethanol extract of centella asiatica on migraine headaches
Figure BDA0002447184050000111
n＝10
Note: ## comparison with blank control, P<0.01， # Comparison with blank control, P<0.05； ** Comparison with negative control group, P<0.01， * Comparison with negative control group, P<0.05。
Example 2
Further tests with centella asiatica
Example 1 was repeated using 95% methanol/water, 70% ethanol/water and 80% acetone/water, respectively, by volume as extraction solvents. The experimental results show that 95% methanol extract, 70% ethanol extract and 80% acetone/water extract of centella asiatica obtained by using 95% methanol/water, 70% ethanol/water and 80% acetone/water as extraction solvents respectively also have significant anti-migraine effect on rats, so that the anti-migraine active ingredient of centella asiatica can also be obtained by using ethanol/water, methanol/water or acetone/water as extraction solvents with different concentrations. The results are shown in Table 2.
TABLE 2 influence of different solvent extracts of centella asiatica on the number of times of head-bending in migraine rats
Figure BDA0002447184050000121
n＝10
Note: ## comparison with blank control, P<0.01， # Comparison with blank control, P<0.05； ** Comparison with negative control group, P<0.01， * Comparison with negative control group, P<0.05。
Example 3
Isolation and identification of anti-migraine active compounds from the whole plant of centella asiatica
(1) Drying 10Kg of whole plant of centella asiatica, crushing into granules with the diameter of 0.1cm, obtaining centella asiatica powder, carrying out reflux extraction on the centella asiatica powder for 4 times at the temperature of 70-74 ℃ by 60Kg of ethanol with the concentration of 95% each time, and combining ethanol extract for later use, wherein the mixture is 2 hours each time;
(2) Filtering the ethanol extract obtained in the step (1) by using 80-120 micron filter paper, and performing reduced pressure concentration by using a rotary evaporator at the temperature of 50 ℃ until the specific gravity is 1.2 to obtain 1219g of extract for later use;
(3) Suspending 1219g of the extract obtained in (2) in 4500ml of water, sequentially extracting with 4500ml of petroleum ether, 4500ml of ethyl acetate and 4500ml of n-butanol, extracting each solvent for 5 times, and evaporating the solvents by using a rotary evaporator to obtain a petroleum ether extract (218 g), an ethyl acetate extract (255 g) and an n-butanol extract (471 g), respectively;
(4) The ethyl acetate extract obtained in (3) was subjected to 100-200 mesh silica gel column chromatography and eluted with a chloroform-methanol gradient in a volume ratio of 30. Fr.3 (33.0 g) was subjected to silica gel column chromatography of 200 to 300 meshes, and eluted with a chloroform-methanol gradient having a volume ratio of 30. Fr.3-3 (9.0 g) was purified by Sephadex LH-20 column chromatography (methanol) and then eluted through 200-300 mesh silica gel column chromatography with chloroform-methanol (volume ratio 25; subjecting Fr.4 (17.0 g) to 200-300-mesh silica gel column chromatography, and eluting with a chloroform-methanol gradient in a volume ratio of 20. Fr.4-2 (7.0 g) was purified by Sephadex LH-20 column chromatography (methanol) and then eluted through 200-300 mesh silica gel column chromatography with chloroform-methanol (volume ratio 15: 1) to give 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid (2) (3.6 g). The separation and identification process of the anti-migraine active ingredient in the whole herb of centella asiatica is shown in figure 1.
Nuclear magnetic resonance spectrum for chemical structure of compound of the present invention ( 1 H NMR， 13 And (4) identifying the spectrograms of C NMR, DEPT, COSY, HSQC, HMBC), ESI-MS (anion mode), IR and the like. Spectroscopic data of compounds 1 and 2 were analyzed and identified as asiatic acid (asiatic acid) and 2 α,3 β,6 β,23 α -tetrahydroxy-12-ene-28-ursolic acid (2 α,3 β,6 β, 23-tetrahydroxy-12-en-28-oic acid), respectively, according to the relevant literature (He w.n., dai j.g., ye m., wu l.j., guo d.a. Microbial transformation of asiatic acid by Alternaria longipes.j.asian nat. Prod. Res.2008,10 (3): 760-764; liu acid, zhao, yu qing. Chemical composition of centella asiatica. Modern chinese traditional medicine 2010,12 (9): 7-9).
Physicochemical data for compound 1: the compound (asiatic acid) is white amorphous powder (chloroform-methanol), has a melting point of 326-328 deg.C, and ESI-MS (negative ions) m/z:487[ m-H ], [] - Molecular formula C 30 H 48 O 5 。 1 H NMR(400MHz，C 5 D 5 N)δH:0.92(3H，d，J＝6.2Hz，H-29)，0.96(3H，d，J＝6.4Hz，H-30)，1.07(3H，s，H-24)，1.07(3H，s，H-26)，1.07(3H，s，H-27)，1.14(3H，s，H-25)，2.61(1H，d，J＝11.2Hz，H-18)，3.73(1H，d，J＝13.0Hz，H-23a)，4.24(1H，overlap，H-23b)，4.24(1H，overlap，H-2)，4.24(1H，overlap，H-3)，5.47(1H，t，J＝3.4Hz，H-12)； 13 C NMR(100MHz，C 5 D 5 N)δC:48.4(C-1)，69.4(C-2)，78.7(C-3)，44.1(C-4)，48.6(C-5)，19.0(C-6)，33.7(C-7)，40.5(C-8)，48.4(C-9)，38.8(C-10)，24.2(C-11)，126.1(C-12)，139.8(C-13)，43.0(C-14)，29.1(C-15)，25.4(C-16)，48.5(C-17)，54.0(C-18)，39.9(C-19)，39.9(C-20)，31.5(C-21)，37.9(C-22)，67.0(C-23)，14.9(C-24)，18.0(C-25)，18.0(C-26)，24.4(C-27)，180.4(C-28)，18.0(C-29)，21.8(C-30).
Physicochemical data for compound 2: the compound (2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-ene-28-ursolic acid) is white amorphous powder (chloroform-methanol), the melting point is 253-255 ℃, ESI-MS (negative ions) m/z is 503[ M-H ], [] - Molecular formula C 30 H 48 O 6 。 1 H NMR(400MHz，C 5 D 5 N)δH:0.93(3H，d，J＝6.1Hz，H-30)，0.99(3H，d，J＝6.3Hz，H-29)，1.17(3H，s，H-27)，1.65(3H，s，H-26)，1.75(3H，s，H-24)，1.78(3H，s，H-25)，2.66(1H，d，J＝11.3Hz，H-18)，4.06(1H，d，J＝10.4Hz，H-23a)，4.26(1H，d，J＝9.4Hz，H-3)，4.42(2H，m，H-23b，H-2)，5.11(1H，brs，H-6)，5.56(1H，brs，H-12)； 13 C NMR(100MHz，C 5 D 5 N)δC:50.8(C-1)，69.6(C-2)，78.8(C-3)，45.0(C-4)，49.1(C-5)，68.0(C-6)，41.8(C-7)，40.0(C-8)，49.1(C-9)，38.6(C-10)，24.4(C-11)，126.5(C-12)，139.2(C-13)，43.6(C-14)，29.1(C-15)，25.4(C-16)，48.6(C-17)，54.1(C-18)，40.0(C-19)，39.9(C-20)，31.6(C-21)，38.0(C-22)，66.6(C-23)，16.5(C-24)，19.6(C-25)，19.4(C-26)，24.5(C-27)，180.4(C-28)，18.0(C-29)，21.9(C-30).
Example 4
Anti-migraine activity assay of the Compounds of formula I
The anti-migraine activity of asiatic acid and 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid was tested using a nitroglycerin migraine headache rat model. Male SD rats with the weight of 180-220 g are measured according to the experimental requirement and are randomly grouped into 10 rats each group. The groups are as follows: blank control group (distilled water), negative control group (distilled water), sample group (asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid), and positive control group (ibuprofen tablet dosage is respectively 80 mg/kg), and continuous administration is carried out for 7d. After 60min of the last administration, nitroglycerin was subcutaneously injected into the right shoulder of each group at 10mg/kg, except for the blank group to which physiological saline was administered. After the injection of nitroglycerin, the number of times of head bending of the rat in 6 time periods after the molding was observed for 30min as one time period and recorded. Injecting nitroglycerin into rat subcutaneously for 4h, anesthetizing with 10% chloral hydrate, collecting blood, separating plasma, and collecting supernatantTaking brain by cutting head, separating brain stem on ice box, preparing brain stem tissue homogenate, centrifuging at 4 deg.C for 15min (3500 r/min), collecting supernatant, and storing. Detecting the content of NO in the blood plasma of the rat and detecting the content of 5-HT, CGRP and beta-EP in the brain tissue of the rat by using an enzyme-linked immunosorbent assay (ELISA). The measured data is averaged + -SD
Figure BDA0002447184050000152
Showing that the statistical processing is carried out by prism6 statistical software. The results are shown in tables 3, 4 and 5. Experimental results show that asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-ene-28-ursolic acid can obviously reduce the times of rat head scratching, reduce the content of NO in serum, reduce the content of CGRP in brain tissues of rats and increase the content of 5-HT and beta-EP in brain tissues of rats, and the asiatic acid and the 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-ene-28-ursolic acid have good anti-migraine activity and can be used as anti-migraine components or lead compounds.
TABLE 3 Effect of Compounds of formula I on the number of head movements in migraine rats
Figure BDA0002447184050000151
Figure BDA0002447184050000161
n＝10
Note: ## comparison with blank control, P<0.01， # Comparison with blank control, P<0.05； ** Comparison with negative control group, P<0.01， * Comparison with negative control group, P<0.05。
TABLE 4 Effect of Compounds of formula I on NO content in serum of migraine rats
Figure BDA0002447184050000162
n＝10
Note: ## comparison with blank control, P<0.01， # Comparison with blank control, P<0.05； ** Comparison with negative control group, P<0.01， * Comparison with negative control group, P<0.05。
TABLE 5 Effect of Compounds of formula I on 5-HT, CGRP and beta-EP content in migraine rat brain tissue
Figure BDA0002447184050000171
n＝10
Note: ## comparison with blank control, P<0.01， # Comparison with blank control, P<0.05； ** Comparison with negative control group, P<0.01， * Comparison with negative control group, P<0.05。
Example 5
Asiatic acid and 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid obtained according to the method described in example 3 were prepared, and tablets were prepared by conventional preparation methods, with the addition of conventional excipients, respectively.
Or mixing asiatic acid obtained by the method described in example 3 with 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid at any ratio, adding common adjuvants, and making into tablet by conventional method.
Example 6
The asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid obtained by the method of example 3 are respectively added with common injection auxiliary materials and prepared into injection by the conventional preparation process.
Or mixing asiatic acid obtained by the method of example 3 with 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid at any ratio, adding common adjuvants for injection, and making into injection by conventional preparation method.
Example 7
The asiatic acid and 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid obtained in example 3 were prepared, and the capsules were prepared by the conventional preparation method, with the addition of common adjuvants for capsules.
Or mixing asiatic acid obtained by the method described in example 3 with 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid at any ratio, adding common adjuvants for capsule, and making into capsule by conventional method.
Example 8
The asiatic acid and 2 alpha, 3 beta, 6 beta, 23 alpha-tetrahydroxy-12-en-28-ursolic acid obtained by the method of example 3 are respectively added with common auxiliary materials of the cataplasm to prepare the cataplasm by a conventional preparation process.
Or mixing asiatic acid obtained by the method of example 3 with 2 α,3 β,6 β,23 α -tetrahydroxy-12-en-28-ursolic acid at any ratio, adding conventional adjuvants, and making into cataplasma by conventional method.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (7)

1. The application of the pentacyclic triterpenoid in preparing the anti-migraine medicine,
Figure QLYQS_1
in formula I, R = H or OH.
2. The use according to claim 1, wherein the pentacyclic triterpenoid is one or two of the compounds shown in formula II-III:
formula II:
Figure QLYQS_2
formula III:
Figure QLYQS_3
3. the use according to claim 2, wherein one or both of the compounds of formulae II-III are present in and used in the form of an extract obtained by extracting centella with a solvent; wherein the solvent is 70-100 vol% ethanol/water, 70-100 vol% methanol/water, or 50-80 vol% acetone/water, wherein the centella plant is centella asiatica.
4. The use according to any one of claims 1-3, wherein the pentacyclic triterpenoid is used as an active ingredient to prepare a pharmaceutical composition together with a pharmaceutically acceptable carrier or adjuvant.
5. Use according to any one of claims 1 to 3, wherein the migraine is a typical migraine, a common migraine or a cluster migraine.
6. The use according to claim 4, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills, injections.
7. The use according to claim 4, wherein said pharmaceutical composition is selected from a sustained release formulation or a controlled release formulation.
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