CN114276405A

CN114276405A - Pentacyclic triterpenoid, preparation method and application thereof

Info

Publication number: CN114276405A
Application number: CN202111474636.6A
Authority: CN
Inventors: 杨洲; 房强强; 卢宗元; 叶晓仪; 钱勇; 谢天培
Original assignee: Shanghai Standard Technology Co ltd
Current assignee: Shanghai Standard Technology Co ltd
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2022-04-05
Anticipated expiration: 2041-12-06
Also published as: CN114276405B

Abstract

The application relates to the field of biomedicine, and particularly discloses a pentacyclic triterpenoid compound, and a preparation method and application thereof. The molecular formula of the pentacyclic triterpenoid is C₂₉H₄₂O₆Molecular weight of 486.65, structural formula:

(ii) a The preparation method comprises the following steps: s1, extracting: extracting dry pterocephalus hookeri with ethanol to obtain an extract; s2, extraction: extracting the extract by using an extraction solvent, and performing spin drying to obtain an extract; s3, purification: sequentially subjecting the extract to macroporous resin column, silica gel column I, silica gel column II, MCI column, and silica gelAnd performing column III and gel column chromatography, collecting eluent, and spin-drying to obtain the final product. The pentacyclic triterpenoid has hypoglycemic activity and anti-inflammatory activity, and has no influence on cell survival at the concentration of less than 20 mu mol/L; by adopting the preparation method, 9mg of the pentacyclic triterpenoid can be extracted from every 20kg of dried pterocephalus hookeri medicinal materials, and the purity is more than or equal to 98%.

Description

Pentacyclic triterpenoid, preparation method and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to a pentacyclic triterpenoid, and a preparation method and application thereof.

Background

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, which occurs chronically, leading to chronic damage and dysfunction of various tissues, particularly the eye, kidneys, heart, blood vessels, nerves. Diabetes has become the third leading "health killer" that causes human death following cardiovascular disease, malignant tumors.

At present, western medicines are mainly used for controlling symptoms aiming at diabetes, wherein acarbose is a very widely applied hypoglycemic medicine. Acarbose is an alpha-glucosidase inhibitor, and can reduce the concentration of fasting blood glucose and glycated hemoglobin after long-term administration. However, acarbose is decomposed in the small intestine, and is slowly absorbed, the retention time is prolonged, and the fermentation of intestinal bacteria increases gas production, which may cause abdominal distension, abdominal pain, diarrhea, and other discomfort symptoms. China has a medical book Huangdi's classic on traditional Chinese medicines two thousand years ago, the history of treating diseases by the traditional Chinese medicines is long, abundant experiences are accumulated, the traditional Chinese medicines have the characteristics of mild and lasting action, small toxic and side effects, suitability for long-term administration of patients and the like, have irreplaceable comprehensive advantages of other medicines and have attracted more and more attention in the medical field. Therefore, the natural active substance with hypoglycemic activity is searched from the traditional Chinese medicine, and the development prospect is quite broad.

The Pterocephalus hookeri (C.B. Clarke) hoeck is a dried whole herb of Pterocarpus hookeri (C.B. Clarke) belonging to Dipsacaceae, and has a long history of administration. The pterocephalus hookeri has cold nature and bitter taste, has the effects of detoxifying and removing plague, clearing heat and stopping dysentery, dispelling wind and freeing Bi, and is mainly used for treating diseases such as plague epidemic, cold and fever, dysentery, arthritis and the like. Researches show that the main active ingredients of the pterocarpus includes triterpenes and glycosides thereof, iridoid, flavone and the like. However, most of the pharmaceutical studies on pterocephalus hookeri currently focus on the research on the anti-rheumatoid arthritis and anti-tumor aspects of pterocephalus hookeri, and no research report on the hypoglycemic activity and related active substances of pterocephalus hookeri is available.

Disclosure of Invention

In order to further explore the pharmaceutical properties of pterocephalus hookeri, particularly the hypoglycemic activity and determine the corresponding natural active substances, the application provides a pentacyclic triterpenoid compound, a preparation method and application thereof.

In a first aspect, the present application provides a pentacyclic triterpenoid, which adopts the following technical scheme:

a pentacyclic triterpenoid has a molecular formula of C₂₉H₄₂O₆Molecular weight is 486.65, and the structure formula is shown as formula I:

preferably, the pentacyclic triterpenoid is extracted from pterocephalus hookeri.

The inventor adopts an ethanol extraction mode to extract the traditional Chinese medicine of pterocephalus hookeri, and then obtains the active substance of pterocephalus hookeri through extraction and purification steps. Through MS,¹H NMR、¹³C NMR、DEPT、¹H-¹H COSY、¹H- ¹After H NOESY and HMBC analysis, the molecular formula of the active substance is determined to be C₂₉H₄₂O₆Molecular weight is 486.65, and the structural formula is shown in the formula I.

Further, cytotoxicity studies were conducted on the compound of formula I using RAW 264.7 cells as a test sample, and the results showed that the compound of formula I had no effect on cell survival when administered at a concentration of 20. mu. mol/L or less. After that, the compound of formula I was studied about the inhibitory activity of alpha-glucosidase and acarbose was used as a positive control, and the results showed that the alpha-glucosidase was inhibited by 50.37% at 500. mu. mol/L, whereas the compound of formula I reached 60.31% at 10. mu. mol/L. It can be seen that, in the case of a compound of formula I with an acarbose concentration of only 2%, the α -glucosidase inhibition rate of the compound of formula I of the present application exceeds the α -glucosidase inhibition rate of acarbose, which is 119.7% of the α -glucosidase inhibition rate of acarbose. The compound shown in the formula I has very excellent hypoglycemic activity. In addition, the anti-inflammatory activity of the compound shown in the formula I is researched by using RAW 264.7 cells as a test sample, and the result shows that the compound shown in the formula I can obviously reduce the content of nitric oxide and the content of an inflammatory factor IL-6 in a culture solution, and the compound shown in the formula I also has very excellent anti-inflammatory activity.

In a second aspect, the present application provides a preparation method of the pentacyclic triterpenoid, which adopts the following technical scheme: the preparation method of the pentacyclic triterpenoid comprises the following steps:

s1, extracting: extracting dry pterocephalus hookeri medicinal materials serving as raw materials in an ethanol leaching mode to obtain an extract;

s2, extraction: sequentially using an extraction solvent I and an extraction solvent II to extract the extract obtained in the step S1, and performing spin drying to obtain an extract;

s3, purification: performing column chromatography on the extract obtained in the step S2 by using a macroporous resin column, a silica gel column I, a silica gel column II, an MCI column, a silica gel column III and a gel column in sequence, collecting eluent, and performing spin drying to obtain the extract;

in step S2, the extraction solvent i is any one of chloroform, dichloromethane, and ethyl acetate; the extraction solvent II is any one of isopropanol, methanol and n-butanol;

in step S3, the silica gel column I, the silica gel column II and the silica gel column III are all forward silica gel columns.

When the compound of the formula I is extracted, an ethanol water solution is used as an extraction solvent, various primula sikkmensis maxim active substances are extracted from the extractive solution, then the extractive solution is extracted by using solvents such as dichloromethane, chloroform and ethyl acetate with low polarity, the extractive solution is kept still for layering, solvents such as isopropanol, methanol and n-butyl alcohol with high polarity are used again for extraction and separation, the extract is obtained after spin drying, finally, a proper elution solvent is selected to sequentially carry out column chromatography on the extract for multiple times, and a target product is gradually separated from the extract, so that the compound of the formula I is obtained. By adopting the steps, 9-10 mg of the compound shown in the formula I can be separated and extracted from every 20kg of dried pterocephalus hookeri Chinese herbal medicine, and the purity is more than or equal to 98%.

Preferably, the specific steps of S1 are:

crushing a dried pterocephalus hookeri medicinal material, adding an extraction solvent with the mass of 8-20 times that of the pterocephalus hookeri medicinal material, controlling the temperature at 60-75 ℃, leaching for 2-3 hours, filtering, collecting filtrate, repeatedly leaching filter residues for 1-3 times at the same temperature and leaching time, filtering, combining the filtrates, and concentrating the obtained filtrate to obtain an extract;

the extraction solvent is an ethanol water solution with the mass percentage concentration of 70-95%.

More preferably, in step S1, the extraction solvent is an aqueous ethanol solution with a mass percentage concentration of 80%, the extraction temperature is controlled at 70 ℃, and the extraction is performed 3 times, each time for 2 hours.

According to the technical scheme, the effective active substances in the pterocephalus hookeri medicinal material are fully extracted and obtained by selecting the type, concentration and usage amount of the extraction solvent and controlling the temperature, times and time of leaching, so that a foundation is laid for the subsequent separation of the compound shown in the formula I. In the process of realizing the application, the inventor determines the optimal condition of leaching, namely leaching for 3 times at 70 ℃ and leaching for 2 hours each time through a single-factor test.

Preferably, the specific steps of S2 are:

extracting the extract for 3-5 times by using an extraction solvent I with the volume being 1-3 times of that of the extract, standing for layering, combining organic phases, extracting for 3-5 times by using an extraction solvent II with the volume being 1-3 times of that of the extract, standing for layering, combining the organic phases, and spin-drying to obtain the extract.

The extraction solvent I and the extraction solvent II are sequentially used for extracting the extract, and each extraction is carried out in a multi-extraction mode, so that the extraction effect on effective substances is improved.

Preferably, the specific steps of S3 are:

s3-1, purifying the extract by using a macroporous resin column, eluting by using a solvent I, namely water, in a volume ratio of an elution solvent I to 9: 1-49: 1, wherein the flow rate of the eluent passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain eluent I;

s3-2, purifying the eluent I by using a silica gel column I, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate being 1:1, wherein the flow rate of passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent II;

s3-3, purifying the eluent II by using a silica gel column II, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate being 5:1, wherein the flow rate of passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent III;

s3-4, purifying the eluent III by using an MCI column, eluting by using a solvent with an elution solvent II and water in a volume ratio of 2: 3-1: 1, collecting the eluent in sections at a flow rate of 50-70 mL/min, performing MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent IV;

s3-5, purifying the eluent IV by using a silica gel column III, eluting by using a solvent with a volume ratio of dichloromethane to methanol being 200:1, wherein the flow rate of the passing column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent V;

s3-6, purifying the eluent V by using a gel column, eluting by using a solvent III and water in a volume ratio of 3: 2-9: 1, collecting the eluent in sections at a column flow rate of 0.8-1.2L/min, carrying out MS analysis on the eluent, combining the eluents containing the target product to obtain an eluent VI, and carrying out spin drying to obtain the compound I;

in step S3-1, the elution solvent I is any one of methanol, ethanol and acetonitrile;

in step S3-4, the elution solvent II is any one of methanol, ethanol and acetonitrile;

in step S3-6, the elution solvent iii is any one of methanol, ethanol, and acetonitrile.

In the purification step, the extract is subjected to macroporous resin column chromatography, the eluates are collected in sections, then the eluates are subjected to MS analysis, the eluates containing the target product (m/z 486.65 +/-1) are combined, then the eluates are subjected to column chromatography by using a silica gel column I, a silica gel column II, an MCI column, a silica gel column III and a gel column in sequence, the eluates are collected in sections, the eluates are also subjected to MS analysis, finally obtained eluates containing the target product are combined and dried in a spinning mode, and the compound shown in the formula I is obtained. By adopting the method, the compound shown in the formula I is successfully separated from a plurality of active substances of pterocephalus hookeri, and the purity of the obtained compound shown in the formula I is higher and can reach more than 98 percent.

More preferably, in step S3-1, elution is performed using a solvent in which the volume ratio of elution solvent i to water is 19:1, and elution solvent i is ethanol.

More preferably, in step S3-4, the elution is performed using a solvent in which the volume ratio of the elution solvent ii to water is 1:1, and the elution solvent ii is methanol.

More preferably, in step S3-6, elution is performed using a solvent in which the volume ratio of elution solvent iii to water is 7:3, and elution solvent iii is methanol.

In a third aspect, the application provides the use of the pentacyclic triterpenoid in the preparation of hypoglycemic drugs and anti-inflammatory drugs.

In a fourth aspect, the present application provides a pharmaceutical composition, which adopts the following technical scheme:

a pharmaceutical composition comprises the pentacyclic triterpenoid and one or more pharmaceutically acceptable carriers.

The pharmaceutically acceptable carrier may be selected according to different needs, and may include diluents, such as mannitol, sorbitol, glucose, dextran, fructose, microcrystalline cellulose, lactose, pregelatinized starch, dextrin, calcium phosphate, sucrose, water, polyethylene glycol, propylene glycol, glycerol, cyclodextrin and its derivatives; binders such as polyvinylpyrrolidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, guar gum, xanthan gum, and the like; lubricants such as magnesium stearate, stearic acid, talc powder, sodium stearyl fumarate, sodium lauryl sulfate, and the like; disintegrating agents such as sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, crospovidone, croscarmellose sodium, sodium cross-linked carboxymethyl starch, pregelatinized starch, and the like; surfactants such as sodium lauryl sulfate, polysorbate-80, and the like may also be included; may also include pH adjusting agents such as phosphate buffer, citric acid, sodium citrate, acetate buffer, dilute hydrochloric acid, sodium carbonate, sodium hydroxide, etc.; preservatives such as sodium benzoate, potassium sorbate, methylparaben, propylparaben, and the like; stabilizers such as calcium sodium edetate, sodium sulfite, vitamin C, etc.; optionally taste modifier such as maltitol, fructose, sucrose, saccharin sodium, orange essence, strawberry essence, etc.; in addition, other conventional carriers which are pharmaceutically acceptable may also be included.

Preferably, the dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form.

The dosage form of the pharmaceutical composition can be selected conventionally according to different needs, and can be any pharmaceutically acceptable dosage form, including liquid dosage forms, such as aromatic water, solution, injection, mixture, lotion, liniment, etc.; also included are solid dosage forms, such as powders, pills, tablets, capsules, granules, powders, and the like; also included are gaseous dosage forms, such as aerosols, sprays, and the like; also included are semisolid dosage forms such as ointments, gels, suppositories, pastes, and the like.

In summary, the present application has the following beneficial effects:

1. the pentacyclic triterpenoid has good hypoglycemic activity, and when the concentration is 10 mu mol/L, the inhibition rate of the pentacyclic triterpenoid on alpha-glucosidase can reach 60.31 percent, so that the pentacyclic triterpenoid can be used for preparing hypoglycemic drugs;

2. the pentacyclic triterpenoid is extracted from natural Chinese medicinal material pterocephalus hookeri, is a natural active substance, has small toxic action on human bodies, and has no influence on the survival of RAW 264.7 cells when the concentration of the pentacyclic triterpenoid is below 20 mu mol/L as shown by a cytotoxicity test result;

3. the pentacyclic triterpenoid compound has good anti-inflammatory activity, and anti-inflammatory activity test results with RAW 264.7 cells as test samples show that the compound can obviously reduce the content of nitric oxide and the content of inflammatory factors IL-6 in a culture solution;

4. according to the preparation method, 9-10 mg of the pentacyclic triterpenoid can be extracted from 20kg of dried pterocephalus hookeri medicinal materials, and the purity is more than or equal to 98%;

5. the pentacyclic triterpenoid can be prepared into a pharmaceutical composition with one or more pharmaceutically acceptable carriers, and the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form according to the use requirement.

Drawings

FIG. 1 is a MS-detection scheme of a compound of formula I in example 1 of the present application;

FIG. 2 shows the compounds of formula I in example 1 of the present application¹H NMR chart;

FIG. 3 shows a scheme for the preparation of the compounds of formula I in example 1 of the present application¹³C NMR check chart;

FIG. 4 shows DEPT and¹³c NMR contrast chart;

FIG. 5 shows a scheme for the preparation of the compounds of formula I in example 1 of the present application¹H-¹H COSY spectrogram;

FIG. 6 shows a formula in example 1 of the present applicationOf the compounds of formula I¹H-¹H NOESY spectrum;

FIG. 7 shows a spectrum of HMBC (one) of the compound of formula I of example 1 herein;

FIG. 8 shows a spectrum of HMBC for the compound of formula I of example 1 of the present application (II);

FIG. 9 is a schematic representation of the carbon positions of the compound of formula I in example 1 of the present application;

FIG. 10 is a bar graph of the concentration of drug administered versus cell viability in the performance assays of the present application;

FIG. 11 is a bar graph of the concentration of the drug administered versus the amount of nitric oxide present in the performance assays of the present application;

FIG. 12 is a bar graph of the relationship between the concentration of the drug administered and the level of the inflammatory factor IL-6 in the performance assays of the present application.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples.

The raw materials used in the examples of the present application are commercially available, except for the following specific descriptions:

example (b): pentacyclic triterpenoid

Example 1

A pentacyclic triterpene compound with molecular formula of C₂₉H₄₂O₆Molecular weight is 486.65, and the structural formula is shown in formula I:

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method comprises the following steps:

s1, extracting:

pulverizing 20kg of dried pterocephalus hookeri, adding 200L of 80% ethanol water solution, controlling the temperature at 70 ℃, leaching for 2h, filtering, collecting filtrate, repeatedly leaching filter residue for 2 times at the same temperature and leaching time, filtering, mixing filtrates, and concentrating the obtained filtrate at 60 ℃ to 100L by rotary drying to obtain extract;

s2, extraction:

extracting the extract obtained in the step S1 with 300L ethyl acetate for 3 times, standing for layering, combining organic phases, extracting with 300L n-butanol for 3 times, standing for layering, combining organic phases, and spin-drying at 60 deg.C until no solvent remains to obtain extract;

s3, purification:

s3-1, purifying the extract by using a macroporous resin column, wherein the macroporous resin column is a macroporous resin column D101 which is collected from the daceae, eluting by using a solvent with the volume ratio of ethanol to water being 19:1, the flow rate of the eluent passing through the column being 60mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing a target product (m/z 486.65 +/-1) to obtain an eluent I;

s3-2, purifying the eluent I by using a silica gel column I, wherein the silica gel column I is a forward silica gel column and is collected from Qingdao Yonghai, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate being 1:1, the flow rate of passing through the column is 60mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing a target product (m/z 486.65 +/-1) to obtain an eluent II;

s3-3, purifying the eluent II by using a silica gel column II, wherein the silica gel column II is a forward silica gel column and is collected from Qingdao Yonghai, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate being 5:1, the flow rate of passing through the column is 60mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing a target product (m/z 486.65 +/-1) to obtain an eluent III;

s3-4, purifying the eluent III by using an MCI (methanol-ethanol) column, wherein the MCI column is an MCI column CHP20P which is collected from sigma, eluting by using a solvent with the volume ratio of methanol to water being 1:1, the flow rate of the solvent passing through the column being 60mL/min, collecting the eluent in sections, carrying out MS (mass spectrometry) analysis on the eluent, and combining the eluents containing target products (m/z 486.65 +/-1) to obtain an eluent IV;

s3-5, purifying the eluent IV by using a silica gel column III, wherein the silica gel column III is a forward silica gel column and is collected from Qingdao Yonghai, eluting by using a solvent with the volume ratio of dichloromethane to methanol being 200:1, the flow rate of passing through the column is 60mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing a target product (m/z 486.65 +/-1) to obtain an eluent V;

s3-6, purifying the eluent V by using a gel column, wherein the gel column is a gel column LH-20 which is collected from GE Healthcare, eluting by using a solvent with the volume ratio of methanol to water being 7:3, the flow rate of passing through the column is 1L/min, collecting the eluent in sections, carrying out MS analysis on the eluent, combining the eluents containing a target product (m/z 486.65 +/-1) to obtain an eluent VI, and spin-drying at the temperature of 60 ℃ until no solvent remains, thus obtaining the target product.

Combining the profile data of FIGS. 1-8, and combining FIG. 9 with the following Table (NMR data, CDCL)₃) Carrying out structural analysis on the pentacyclic triterpenoid.

And (3) structural analysis result:

the pentacyclic triterpenoid is in a colorless needle crystal shape, and the low field region of a hydrogen spectrum of the pentacyclic triterpenoid shows 3 continuous oxygen methine hydrogen signals of 4.42(1H, dd, J ═ 12.0, 7.8Hz), 3.34(1H, dd, J ═ 3.6, 1.8Hz) and 3.02(1H, d, J ═ 3.6 Hz); the high field region shows 3 unimodal methyl signals 1.20(3H, s), 1.47(3H, s) and 1.04(3H, s); 3 bimodal methyl signals 1.60(3H, d, J ═ 6.0Hz), 1.16(3H, d, J ═ 6.6Hz) and 0.98(3H, d, J ═ 6.0 Hz). A carbon spectrum showing 29 carbon signals, including a characteristic carbonyl carbon signal (δ C213.2); a characteristic ester carbonyl carbon signal (δ C179.0); four characteristic oxygen-linked sp3 hybridized carbon signals (δ C88.8, 71.8, 56.1 and 54.4); the rest of carbonThe signals are all displayed in the high field area. The data are analyzed comprehensively to show that the compound is the carbon-reduced triterpenoid. In that¹H-¹The connection of the A ring can be determined by combining H-1 and H-2 in an H COSY spectrum, H-4 and H-5 in an H-4 and H-23 in an HMBC spectrum and H-2/4 and C-3 in an HMBC spectrum and H-24 and C-5/10 in an HMBC spectrum; in that¹H-¹The connection of the B ring can be determined by the correlation of H-5 and H-6, H-6 and H-7 in the H COSY spectrum and the correlation of H-24 and C-9/10/5, and H-25 and C-9/8/7 in the HMBC spectrum; in that¹H-¹The connection of the C ring can be determined by combining H-9 with H-11 and H-11 with H-12 in an H COSY spectrum and combining H-25 with C-9/8/14, H-26 with C-8/13/14 and H-12 with C-13 in an HMBC spectrum; in that¹H-¹The connection of the D ring can be determined by combining H-15 and H-16 in an H COSY spectrum and H-26 and C-13/14/15 and H-18/16 and C-17/27 in an HMBC spectrum; in that¹H-¹The connection of the E ring can be determined by combining the correlation between H-18/22 and C-17 in the HMBC spectrum, wherein H-18 and H-19 in the H COSY spectrum, H-19 and H-20 in the H-19 spectrum, H-20 and H-21 in the H-21 spectrum, H-22 in the H-19 spectrum and H-28 in the H-20 spectrum and H-29 in the HMBC spectrum. It can be seen that this compound is very similar to the known compound 11 α,12 α -epoxy-3 β,6 β -dihydroxy-24-norurs-2-oxo- (28 → 13) -olide, with the exception of the difference in the A ring, indicating that this compound has the same structure as the B, C, D, E ring in this known compound. The relative configuration of the A ring of the compound is determined by correlating H-2 with H-4, and H-2 with H-24, respectively, in the NOESY spectrum, indicating that these hydrogens are on the same side, so that the structure of the compound is as shown in formula I above.

According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.5mg, and the purity is 98.9%.

Example 2

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S1 except that the extraction solvent is 70% ethanol water solution by mass percentage, and the other steps are the same as those in the embodiment 1.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.3mg, and the purity is 98.4%.

Example 3

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S1 except that the extraction solvent is 95% ethanol water solution by mass percentage, and the other steps are the same as those in the embodiment 1.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.2mg, and the purity is 98.5%.

Example 4

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that of the extraction method in the step S1 in the embodiment 1 except that the extraction temperature is 60 ℃, and the total extraction is carried out for 4 times.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.3mg, and the purity is 98.3%.

Example 5

Pentacyclic triterpenoid compound and preparation method thereofMolecular formula C₂₉H₄₂O₆Molecular weight is 486.65, and the structural formula is shown in formula I:

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that of the extraction method in the step S1 in the embodiment 1 except that the extraction temperature is 75 ℃, and the extraction is carried out for 2 times.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.4mg, and the purity is 98.6%.

Example 6

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S3-1 except that a solvent with the volume ratio of methanol to water being 9:1 is used for elution in the step S3-1, and the other condition steps are the same as the steps in the example 1.

Example 7

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S3-1 except that a solvent with the volume ratio of acetonitrile to water being 49:1 is used for elution in the step S3-1, and the other conditions and steps are the same.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.4mg, and the purity is 98.3%.

Example 8

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S3-4 of the example 1 except that a solvent with the volume ratio of ethanol to water to 2 to 3 is used for elution, and the other conditions are the same.

Example 9

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S3-6 of the example 1 except that a solvent with the volume ratio of ethanol to water being 3:2 is used for elution, and the other condition steps are the same.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.1mg, and the purity is 98.3%.

Example 10

Pentacyclic triterpenoid compoundCompound of formula C₂₉H₄₂O₆Molecular weight is 486.65, and the structural formula is shown in formula I:

the pentacyclic triterpenoid is extracted from pterocephalus hookeri, and the extraction method is the same as that in the step S3-6 of the example 1 except that the solvent with the volume ratio of acetonitrile to water being 9:1 is used for elution, and the other conditions are the same.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.2mg, and the purity is 98.2%.

Example 11

s1, extracting:

pulverizing 20kg of dried pterocephalus hookeri, adding 160L of 80% ethanol water solution, controlling the temperature at 70 ℃, leaching for 2h, filtering, collecting filtrate, repeatedly leaching filter residue for 2 times at the same temperature and leaching time, filtering, mixing filtrates, and concentrating the obtained filtrate at 60 ℃ to 100L by rotary drying to obtain extract;

s2, extraction:

extracting the extract obtained in the step S1 with 200L ethyl acetate for 5 times, standing for layering, combining organic phases, extracting with 200L n-butanol for 5 times, standing for layering, combining organic phases, and spin-drying at 60 deg.C until no solvent remains to obtain extract;

s3, purification:

the purification procedure of this example was the same as that of example 1 except that the column flow rate in S3-1 to S3-5 was 50mL/min and the column flow rate in S3-6 was 0.8L/min.

The spectra of the compound obtained in this example are consistent with those of example 1. According to detection, in the embodiment, the mass of the obtained pentacyclic triterpenoid is 9.3mg, and the purity is 98.5%.

Example 12

s1, extracting:

pulverizing 20kg of dried pterocephalus hookeri, adding 400L of 80% ethanol water solution, controlling the temperature at 70 ℃, leaching for 2h, filtering, collecting filtrate, repeatedly leaching filter residue for 2 times at the same temperature and leaching time, filtering, mixing filtrates, and concentrating the obtained filtrate at 60 ℃ to 100L by rotary drying to obtain extract;

s2, extraction:

extracting the extract obtained in the step S1 with 100L ethyl acetate for 4 times, standing for layering, combining organic phases, extracting with 100L n-butanol for 4 times, standing for layering, combining organic phases, and spin-drying at 60 deg.C until no solvent remains to obtain extract;

s3, purification:

the purification procedure of this example was the same as that of example 1 except that the column flow rate in S3-1 to S3-5 was 70mL/min and the column flow rate in S3-6 was 1.2L/min.

Performance test

The pentacyclic triterpenoid obtained in example 1 was used as a test sample to test the cytotoxicity, alpha-glucosidase inhibitory activity and anti-inflammatory activity of the compound.

Test one: cytotoxicity assays

Inoculating RAW 264.7 cells in a dish, observing the growth state of the cells, blowing down the cells by PBS when the fusion degree is 70%, counting and inoculating 96-well plates, and adjusting the cell density in the plates to be 5 multiplied by 10⁴cells/mL, 100. mu.L cell suspension, cultured overnight for adherence. The test is divided into a blank group (DMSO with the same volume), a blank control group (the administration concentration is 0) and an administration group (the administration concentrations are respectively 5 mu mol/L, 10 mu mol/L, 20 mu mol/L, 40 mu mol/L and 80 mu mol/L), each group is provided with 3 multiple holes, after the culture is continued for 48 hours, the supernatant is sucked by an aspirator, 100 mu L/hole CCK-8 reagent with the working concentration of 1mg/mL is added, the culture medium is discarded after the culture is placed in an incubator for 3.5 hours, 100 mu L/hole DMSO is added, and the absorbance value of 490nm wavelength is detected after the incubation is carried out on a shaking table for 30 minutes.

Cell survival (%) × (administration absorbance-blank absorbance)/(blank absorbance-blank absorbance) × 100%.

The results are shown in fig. 10, in which the administration concentration in the blank control group or the administration group was plotted as the abscissa and the cell survival rate was plotted as the ordinate.

As can be seen from FIG. 10, pentacyclic triterpenoids have no effect on cell survival (P > 0.05) when administered at concentrations below 20. mu. mol/L, compared to the blank control group.

And (2) test II: alpha-glucosidase inhibitory activity assay substrate preparation: 0.3766g of PNPG was weighed out accurately, dissolved in 10mL of phosphate buffer (0.1mol/L, pH 6.8), and then diluted to 50mL to obtain 25mmol/L of PNPG substrate.

Preparing an alpha-glucosidase solution: lyophilized enzyme powder (enzyme activity 14u/mg) was dissolved in phosphate buffer (0.1mol/L, pH 6.8) to prepare a 2u/mL α -glucosidase solution.

Preparing an acarbose solution: acarbose was dissolved in a phosphate buffer (0.1mol/L, pH 6.8) to prepare a 500 μmol/L acarbose solution.

Preparing a test sample solution: the test sample (pentacyclic triterpenoid obtained in example 1) was dissolved in phosphoric acid buffer (0.1mol/L, pH 6.8) to prepare a test sample solution of 10 μmol/L.

The assay was divided into blank, background, negative control, positive control and sample groups, each reaction was loaded in a 96-well plate according to the doses in the table below, with 3 duplicate wells per group. Adding PBS solution, inhibitor solution and PNPG substrate in sequence, mixing well, and preserving heat in 37 ℃ water bath for 10 min. Wherein the inhibitor solution of the positive control group is acarbose solution; the inhibitor solution of the sample set is the test sample solution. After the heat preservation is finished, adding alpha-glucosidase solution in water bath at 37 ℃ according to the dosage in the following table, mixing uniformly, reacting in water bath at 37 ℃ for 20min, and adding 150 mu L of 0.2mol/L Na₂CO₃The reaction was stopped with the solution.

Since PNPG is hydrolyzed by alpha-glucosidase to produce glucose and PNP, PNP has maximum absorption at 405 nm. Therefore, the inhibition rate of α -glucosidase in each test group can be calculated by measuring the absorbance at 405nm in each test group. The assay was performed with the aid of an alpha-glucosidase activity detection kit (purchased from Beijing boxed Ltd.).

The inhibition rate (%) [1- (sample group or positive control absorbance-sample blank absorbance)/(background group absorbance-blank absorbance) ] × 100%.

The inhibition rates of the positive control group and the sample group are shown in the following table.

Group of	Inhibition ratio (％)
		Positive control group	50.37
Sample set	60.31

As can be seen from the data in the table, the inhibition rate of acarbose on alpha-glucosidase at the concentration of 500. mu. mol/L is 50.37%, while the inhibition rate of the test sample on alpha-glucosidase at the concentration of 10. mu. mol/L reaches 60.31%. Through further analysis, the inhibition rate of the test sample can reach 119.7% of the inhibition rate of acarbose under the condition that the concentration of the test sample is only 2% of the acarbose concentration, so that the pentacyclic triterpenoid compound has good blood sugar reducing activity and can be applied to preparation of blood sugar reducing medicines.

And (3) test III: test for anti-inflammatory Activity

Inoculating RAW 264.7 cells in a dish, observing the growth state of the cells, blowing down the cells by PBS when the fusion degree is 70%, counting and inoculating 96-well plates, and adjusting the cell density in the plates to be 5 multiplied by 10⁴cells/mL, 100. mu.L cell suspension, cultured overnight for adherence. After overnight adherence, the medium was discarded and returned to room temperature (25 ℃) at 2.5 mL/well. The test was divided into a model control group (equivalent 1mg/mL of lipopolysaccharide), a blank group (administration concentration of 0) and an administration group (administration concentrations of 5. mu. mol/L, 10. mu. mol/L, 15. mu. mol/L, and 20. mu. mol/L, respectively), and 3 duplicate wells were provided for each group. After further culturing for 48h, the content of nitric oxide and inflammatory factor IL-6 in the cell culture fluid of each well was determined using a nitric oxide assay kit (purchased from Biyun), and a mouse IL-6ELISA assay kit (purchased from Biyun).

The results are shown in fig. 11, in which the administration concentration in the model control group, blank group or administration group is plotted as the abscissa and the nitric oxide content is plotted as the ordinate, respectively. The results are shown in FIG. 12, in which the administration concentration in the model control group, blank group or administration group is plotted as the abscissa and the content of the inflammatory factor IL-6 is plotted as the ordinate.

As can be seen from FIG. 11, the amount of nitric oxide in the cell culture medium of the model control group was compared with that of the blank groupThe content is obviously increased (^###P is less than 0.001); the content of nitrogen monoxide in the cell culture solution was reduced in the administration group at an administration concentration of 5. mu. mol/L as compared with that in the model control group: (^**P < 0.01), the reduction of the content of nitric oxide in the cell culture solution of the administration group at the administration concentration of 10. mu. mol/L, 15. mu. mol/L, or 20. mu. mol/L is more remarkable: (^**P is less than 0.001), and the content of nitric oxide has obvious reduction trend along with the increase of the dosage, and the two show a certain dose-effect relationship.

As can be seen from FIG. 12, the IL-6 content in the model control group was significantly increased compared to that in the blank group (^###P is less than 0.001); IL-6 expression was significantly reduced in the cell culture fluid of the administered group at an administration concentration of 5. mu. mol/L, as compared with that of the model control group (^**P < 0.01), the IL-6 content in the cell culture fluid of the administration group with the administration concentration of 10 mu mol/L, 15 mu mol/L and 20 mu mol/L is also obviously reduced (^**P is less than 0.001), and the content of IL-6 is reduced obviously with the increase of the dosage, and the two show a more obvious dose-effect relationship. Therefore, the pentacyclic triterpenoid has good anti-inflammatory activity and can be applied to preparation of anti-inflammatory drugs.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. A pentacyclic triterpenoid is characterized in that the molecular formula of the pentacyclic triterpenoid is C₂₉H₄₂O₆Molecular weight is 486.65, and the structural formula is shown in formula I:

formula I.

2. The pentacyclic triterpenoid of claim 1, extracted from pterocephalus hookeri.

3. A method for preparing pentacyclic triterpenoid of claim 1 or 2, which comprises the following steps:

4. The preparation method of pentacyclic triterpenoid as claimed in claim 3, wherein the specific step of S1 is as follows:

5. The preparation method of pentacyclic triterpenoid as claimed in claim 4, wherein the extraction solvent is 80% ethanol water solution by mass, the extraction temperature is controlled at 70 ℃, and the extraction is performed for 3 times, each time for 2 h.

6. The preparation method of pentacyclic triterpenoid as claimed in claim 3, wherein the specific step of S2 is as follows:

7. The preparation method of pentacyclic triterpenoid as claimed in claim 3, wherein the specific step of S3 is as follows:

s3-1, purifying the extract by using a macroporous resin column, eluting by using a solvent I and water =9: 1-49: 1 in a volume ratio, wherein the flow rate of the eluent passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain eluent I;

s3-2, purifying the eluent I by using a silica gel column I, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate =1:1, and the flow rate of passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent II;

s3-3, purifying the eluent II by using a silica gel column II, eluting by using a solvent with the volume ratio of petroleum ether to ethyl acetate =5:1, and the flow rate of passing through the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent III;

s3-4, purifying the eluent III by using an MCI column, eluting by using a solvent with a volume ratio of an elution solvent II to water =2: 3-1: 1, wherein the flow rate of the eluent in the column is 50-70 mL/min, collecting the eluent in sections, carrying out MS analysis on the eluent, and combining the eluents containing the target product to obtain an eluent IV;

s3-5, purifying the eluent IV by using a silica gel column III, eluting by using a solvent with the volume ratio of dichloromethane to methanol =200:1, collecting the eluent in sections at the flow rate of 50-70 mL/min, carrying out MS analysis on the eluent, and combining the eluates containing the target product to obtain an eluent V;

s3-6, purifying the eluent V by using a gel column, eluting by using a solvent III, namely water =3: 2-9: 1 according to a volume ratio, wherein the flow rate of the eluent is 0.8-1.2L/min, collecting the eluent in sections, carrying out MS analysis on the eluent, combining the eluents containing the target product to obtain an eluent VI, and carrying out spin drying to obtain the final product;

8. The method for preparing pentacyclic triterpenoid compounds according to claim 7, wherein in step S3-1, elution is performed by using a solvent with a volume ratio of elution solvent I to water =19 to 1, wherein the elution solvent I is ethanol.

9. The method for preparing pentacyclic triterpenoid compounds according to claim 7, wherein in the step S3-4, a solvent with a volume ratio of elution solvent II to water =1 to 1 is used for elution, and the elution solvent II is methanol.

10. The method for preparing pentacyclic triterpenoid compounds according to claim 7, wherein in the step S3-6, elution is performed by using a solvent with a volume ratio of elution solvent III to water =7 to 3, wherein the elution solvent III is methanol.