CN110452279B - Triterpenoid and pharmaceutically acceptable salt thereof, and preparation method and application thereof - Google Patents

Triterpenoid and pharmaceutically acceptable salt thereof, and preparation method and application thereof Download PDF

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CN110452279B
CN110452279B CN201910394851.1A CN201910394851A CN110452279B CN 110452279 B CN110452279 B CN 110452279B CN 201910394851 A CN201910394851 A CN 201910394851A CN 110452279 B CN110452279 B CN 110452279B
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compound
pharmaceutically acceptable
methyl
pharmaceutical composition
butyryloxy
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白璐
蹇顺
凌勇根
张英帅
龚云
李春
廖端芳
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Qianjin Pharmaceutical Co ltd
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    • C07J—STEROIDS
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Abstract

The invention discloses triterpenoids, pharmaceutically acceptable salts thereof, a preparation method and application thereof. The structural formula of the compound is shown as a formula I, and the structure of the pharmaceutically acceptable salt is shown as a formula II; wherein M is an alkali metal ion, an alkaline earth metal ion or an ammonium group. The compound disclosed by the invention is novel in structure, originally derived from cherokee rose root, and separated from cherokee rose root extract, has a good antibacterial effect on escherichia coli, staphylococcus aureus or pseudomonas aeruginosa, also has a good anti-inflammatory effect, analgesic effect or antipyretic effect, and can be prepared into an antibacterial drug, an anti-inflammatory drug, an analgesic drug or an antipyretic drug for application; when salified, the resulting salified compound also has biological activity equivalent to or the same as that of the compound.
Figure DDA0002057835060000011

Description

Triterpenoid and pharmaceutically acceptable salt thereof, and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a triterpenoid and pharmaceutically acceptable salts thereof, and a preparation method and application thereof.
Background
The root of cherokee rose, named as cherokee rose Jiang and boneless pill, is the root of Rosa laevigata Michx, a dicotyledonous Rosaceae plant, and is originally recorded in Rihua Zi Bencao, which carries the root of Rosa laevigata, is flat and nontoxic. For instance, it is indicated for cun Bai Chong, checking diarrhea and bleeding, metrorrhagia and leukorrhagia. It is bitter, sour, astringent and mild in taste. It has effects in clearing away heat, promoting diuresis, removing toxic materials, relieving swelling, promoting blood circulation, stopping bleeding, and astringing. The cherokee rose root mainly contains triterpene, flavone and tannin components, can treat spermatorrhea, enuresis, dysentery and diarrhea, metrorrhagia and metrostaxis, uterine prolapse, hemorrhoids, scald and the like, and is a main medicament in clinical common traditional Chinese medicines and herbal tea such as Qianjin tablets, Jinji capsules, Guangdong herbal tea and the like for gynecology.
The cherokee rose fruit can be used as both medicine and food, has great development value in the aspect of health food and drink, is a good product for reinforcing yin and nourishing yin, but is forbidden to be taken by people with excess fire and pathogenic heat, and Ming, Mie Jong & Mie Yong (Ben Cao Jing Shu): it is contraindicated for diarrhea due to fire-heat sudden injection; it is contraindicated for urinary incontinence and loss of essence and qi due to yin deficiency with flaming of fire. In the aspect of medicine, the cherokee rose root is mainly used as a compound medicine, is widely applied to clinic, is mainly used for treating diseases such as prostatitis, gynecological diseases, spermatorrhea and enuresis, urinary system infection, burn and scald, and has larger dosage in marketable Chinese patent medicines such as Qianjin tablets, Sanjin tablets, Jinji capsules and tablets thereof.
The medicinal value of cherokee rose root is remarkable, but the active ingredient of cherokee rose root is still unclear so far, and the research on the medicinal effect of the monomer compound is less. In the application process of the traditional Chinese medicine extract, due to the complex components, the use amount is inevitably increased to achieve the treatment effect, on one hand, the cost of the medicine is easily increased, and on the other hand, the side effect is easily generated or enhanced due to the increase of the use amount. Therefore, there is a need for specific research on active compounds in cherokee rose root and providing a simple, convenient and safe chemical synthesis method, which can improve the therapeutic effect of the drug and reduce the cost of the drug.
Disclosure of Invention
The invention aims to provide triterpenoids and pharmaceutically acceptable salts thereof. The compound is originally derived from cherokee rose root and is separated from cherokee rose root extract, has good antibacterial action against escherichia coli, staphylococcus aureus or pseudomonas aeruginosa, also has good anti-inflammatory action and antipyretic action, and can be prepared into antibacterial drugs, anti-inflammatory drugs or antipyretic drugs for application.
The second purpose of the invention is to provide a pharmaceutical composition of the triterpenoid and/or the pharmaceutically acceptable salt thereof.
The third purpose of the invention is to provide an isolation method of the triterpenoid and the pharmaceutically acceptable salt thereof.
The fourth purpose of the invention is to provide the application of the triterpenoid and the pharmaceutically acceptable salt thereof.
The above object of the present invention is achieved by the following scheme:
triterpenoid and pharmaceutically acceptable salts thereof, wherein the structural formula of the triterpenoid is shown as a formula I, and the chemical name of the triterpenoid is as follows: 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-acid; the structure of the pharmaceutically acceptable salt is shown as a formula II:
Figure BDA0002057835040000021
wherein M is an alkali metal ion, an alkaline earth metal ion or an ammonium group; and n is 1 or 2.
The compound of formula I has the molecular formula C35H54O6The compound is a ursane pentacyclic triterpenoid compound, has good antibacterial action against escherichia coli, staphylococcus aureus or pseudomonas aeruginosa, also has good anti-inflammatory action, analgesic action or antipyretic action, and can be prepared into antibacterial drugs, anti-inflammatory drugs, analgesic drugs or antipyretic drugs for application.
Preferably, the alkali metal ions are potassium ions, sodium ions or lithium ions; the alkaline earth metal ions are calcium ions, magnesium ions or barium ions.
The invention also provides a pharmaceutical composition which contains the compound (2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid compound) separated from the cherokee rose root and shown in the formula I and/or a pharmaceutically acceptable salt shown in the formula II.
Preferably, the pharmaceutical composition contains the compound (2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid compound) isolated from the cherokee rose root and/or a pharmaceutically acceptable salt shown in formula II, and pharmaceutically acceptable auxiliary materials and/or carriers.
Preferably, the pharmaceutical composition comprises the compound (2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid compound) isolated from cherokee rose root as shown in formula I and/or a pharmaceutically acceptable salt thereof as shown in formula II, and other pharmaceutically acceptable ingredients.
Preferably, the pharmaceutical composition further comprises one or more of philippine flemingia root, zanthoxylum dissitum Hemsl, suberect spatholobus stem, leatherleaf mahonia stem, common andrographis herb, Chinese angelica and pilose asiabell root.
Preferably, the pharmaceutical composition further comprises one or more extracts of philippine flemingia root, zanthoxylum dissitum Hemsl, suberect spatholobus stem, leatherleaf mahonia stem, common andrographis herb, Chinese angelica and pilose asiabell root.
The extract is prepared by the extraction method described in any one or more of patent documents with patent publication numbers CN104529984B, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C and CN 1296073C.
Preferably, the pharmaceutical composition is in the form of tablets, capsules, powders, granules, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays, and other forms that can be realized by the prior art.
The compound of the formula I is originally derived from traditional Chinese medicinal material cherokee rose root, and is a new compound prepared from the cherokee rose root by solvent extraction, column chromatography separation, preparative liquid phase separation and purification, and the specific separation process comprises the following steps:
s1, reflux-extracting coarse powder of a cherokee rose root medicinal material by using ethanol, and concentrating to obtain dry paste; adding water into the dry extract of radix Rosae Laevigatae to obtain suspension, and sequentially extracting with petroleum ether, chloroform and n-butanol to obtain petroleum ether fraction, chloroform fraction and n-butanol fraction;
s2, performing primary separation on the chloroform part obtained in the step S1 by adopting a medium-pressure column, filling the chloroform part into the column by adopting a dry method, performing column chromatography separation by using the medium-pressure column, and performing gradient elution by using petroleum ether-ethyl acetate to obtain 4 different elution parts A1, A2, A3 and A4;
s3, combining the obtained A3 and A4 parts, separating by using a reverse phase column, and carrying out methanol-water gradient elution to obtain 5 parts, namely A3-4.1, A3-4.2, A3-4.3, A3-4.4 and A3-4.5;
s4, mixing A3-4.5 with methanol: eluting with 0.1% formic acid, collecting the peak at 70-75min, eluting with acetonitrile: and performing liquid-phase elution with 0.1% formic acid water to obtain the target compound.
Preferably, in the step S2, in the gradient elution process, the specific process is that petroleum ether-ethyl acetate is eluted at a volume ratio of 30:1 to obtain a position a 1; eluting at a volume ratio of 20:1 to obtain a site A2; eluting at a volume ratio of 10:1 and 5:1 to obtain a site A3; eluting at a ratio of 3:1 and 2:1 to obtain A4 part.
Preferably, in step S3, the methanol-water gradient elution process is: eluting with methanol-water at a volume ratio of 6:4 to obtain A3-4.1; eluting with an eluent at a volume ratio of 6.5: 3.5-6.8: 3.2 to obtain A3-4.2; eluting at a volume ratio of 7:3 to obtain A3-4.3; eluting with the solvent at a volume ratio of 7.5:2.5 to obtain A3-4.4; eluting at a volume ratio of 8:2 to obtain A3-4.5.
Preferably, in step S4, the target compound is obtained by eluting with methanol-0.1% formic acid water at 72:28 isocratic ratio for 72min, then replacing the eluent with acetonitrile-0.1% formic acid water at 55:45 isocratic ratio, and eluting for 62 min.
Preferably, in the step S1, the cherokee rose root crude powder is extracted for 3 times, and the volume fraction of ethanol adopted in each reflux for 1 hour is 50-100%; preferably 70%.
The application of the compound in preparing antibacterial drugs, anti-inflammatory drugs, analgesic drugs or pyrolytic drugs is also within the protection scope of the invention.
Preferably, the antibacterial drug is a drug against escherichia coli, staphylococcus aureus or pseudomonas aeruginosa.
Preferably, the medicament contains pharmaceutically acceptable adjuvants and/or carriers.
Preferably, the medicine also contains one or more of Flemingia philippinensis, radix zanthoxyli, caulis spatholobi, caulis Mahoniae, herba andrographitis, angelica sinensis and radix codonopsis.
Preferably, the medicine also contains one or more extracts of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, caulis Mahoniae, herba Andrographitis, radix Angelicae sinensis, and radix Codonopsis.
Preferably, the medicament is in the form of tablets, capsules, powders, granules, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays.
The compound of the invention can be separated from cherokee rose root and can also be artificially synthesized.
Compared with the prior art, the invention has the following beneficial effects:
the compound disclosed by the invention is novel in structure, originally derived from cherokee rose root, and separated from cherokee rose root extract, has a good antibacterial effect on escherichia coli, staphylococcus aureus or pseudomonas aeruginosa, also has a good anti-inflammatory effect, an analgesic effect and an antipyretic effect, and can be prepared into an antibacterial drug, an anti-inflammatory drug, an analgesic drug or an antipyretic drug for application; when salified, the resulting salified compound also has biological activity equivalent to or the same as that of the compound.
Drawings
FIG. 1 is a schematic diagram of the isolation scheme of compounds.
FIG. 2 is a (+) HR-ESI-MS spectrum of the compound.
FIG. 3 is a (-) HR-ESI-MS spectrum of compound.
FIG. 4 is a hydrogen spectrum of the compound.
FIG. 5 is a carbon spectrum of a compound.
Figure 6 is the DEPT 90 spectrum of the compound.
Figure 7 is the DEPT 135 spectrum of the compound.
FIG. 8 is the HSQC spectrum of the compound.
Figure 9 is an HMBC spectrum of a compound.
FIG. 10 is a drawing of a compound1H-1H COSY spectrum.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 extraction and isolation of the Compound 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-dien-28-oic acid
The Cherokee rose root is taken as a research object, and 70% ethanol extract of the Cherokee rose root is extracted and separated so as to clarify the specific activity cost and the biological activity of different components. Specifically, the adopted Rosa laevigata root medicinal material is provided by the limited company of the stephania japonica pharmaceutical industry, and the Rosa laevigata root medicinal material is identified as the root of Rosa laevigata michx.
The instrument to which the extraction and separation process is applied comprises: dionex U-3000 high performance liquid chromatograph; a WatersAcquirityUPLC/Xevo G2QTOF system; FW135 pulverizer; bruker Ascend 600M superconducting NMR; an Ultimate 3000 ultra-high performance liquid chromatograph and an LTQ Orbitray sources pro mass spectrometer; LC3000 preparative liquid chromatograph; qi step medium pressure preparative chromatography BUCHI
Figure BDA0002057835040000051
flash systems X-10 flash column systems.
The materials used include: conventional column chromatography silica gel, hydroxypropyl sephadex, RP-C18 column chromatography packing, high performance thin layer plate GF254, Thermo preparative chromatography column BDS HYPERSIL C18, Kromasil 1005C 18 semi-preparative chromatography column, deuterated reagent, chromatographic methanol and acetonitrile, and other reagents are analytical grade, chromogenic agent: vanillin-concentrated sulfuric acid color developer.
The specific extraction and separation process comprises:
1. reflux-extracting 10Kg of radix Rosae Laevigatae coarse powder with 70% ethanol for 3 times, each time for 1 hr, and concentrating under reduced pressure to obtain dry extract 1144.56 g; dispersing the above dried extract in water to obtain suspension, and sequentially extracting with petroleum ether, chloroform and n-butanol to obtain petroleum ether fraction (5.73g), chloroform fraction (90.9g), n-butanol fraction (504.67g) and water fraction (475.42 g).
2. Performing primary separation on the chloroform part obtained in the step 1 by adopting a step medium pressure column, namely taking the chloroform part (79.99g), performing dry column chromatography separation by using the medium pressure column, and performing gradient elution by using petroleum ether-ethyl acetate (30: 1-0: 1) to obtain 4 different elution parts (A1-A4); wherein the petroleum ether-ethyl acetate volume ratio is 30:1 to obtain the A1 part; eluting at a volume ratio of 20:1 to obtain a site A2; eluting at a volume ratio of 10:1 and 5:1 to obtain a site A3; eluting at a ratio of 3:1 and 2:1 to obtain A4 part.
3. Combining the obtained A3 and A4 parts, separating by using a reverse phase column, and carrying out methanol-water gradient elution to obtain 5 parts A3-4.1-A3-4.5; eluting with methanol-water at a volume ratio of 6:4 to obtain A3-4.1; eluting with an eluent at a volume ratio of 6.5: 3.5-6.8: 3.2 to obtain A3-4.2; eluting at a volume ratio of 7:3 to obtain A3-4.3; eluting with the solvent at a volume ratio of 7.5:2.5 to obtain A3-4.4; eluting at a volume ratio of 8:2 to obtain A3-4.5; and (3) eluting A3-4.5 with prepared liquid phase methanol-0.1% formic acid water at a volume ratio of 72:28 isocratic for 72min, replacing the eluent with acetonitrile-0.1% formic acid water at a volume ratio of 55:45 isocratic for 62min to obtain the compound 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid (tR is 62min, 10.3 mg).
Example 2
During the extraction process of the compound 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid, the coarse powder of the root of cherokee rose-hip medicinal material can be refluxed and extracted for 3 times by 50% ethanol, the reflux is carried out for 1 hour each time, and the concentration is carried out under reduced pressure to obtain dry paste, and then the extraction and separation processes are carried out according to the embodiment 1 to obtain the target compound.
Example 3
During the extraction process of the compound 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid, the coarse powder of the root of cherokee rose-hip medicinal material can be refluxed and extracted for 3 times by 100 percent ethanol, the reflux is carried out for 1 hour each time, and the concentration is carried out under reduced pressure to obtain dry paste, and then the extraction and separation processes are carried out according to the embodiment 1 to obtain the target compound.
EXAMPLE 4 structural characterization of the Compound of interest
The target compounds prepared in examples 1 to 3 are subjected to structural identification, and the structure of the target compounds is shown as a formula I:
Figure BDA0002057835040000061
the compound is white powder (methanol), sprayed with vanillin-concentrated sulfuric acid to show purple spot, and HR-ESI-MS to show excimer peak M/z593.3820[ M + Na ]]+(calcd.593.3818，C35H54O6Na)，m/z569.3856[M-H]-(calcd.569.3842，C35H53O6) Giving the formula C35H54O6The unsaturation degree was 9.1The H NMR high field region shows 8 methyl groups, δ H: 0.85(3H, s, H-26), 0.93(3H, d, J ═ 6.7Hz, H-30), 0.96(6H, d, J ═ 6.6Hz, H-4',5'), 1.13(3H, s, H-23), 1.36(3H, s, H-27), 1.19(3H, s, H-25) and 1.20(3H, s, H-29); 1 singlet δ H2.49(1H, s, H-18), two pairs of carbon-coupled protons δ H3.45 and δ H3.66(1H, d, J ═ 10.9, H-24), δ H4.63 and δ H4.66(1H, d, J ═ 13.5, H-1), and 2 alkene hydrogen signals δ H5.26(1H, d, J ═ 3.7, H-12) and δ H5.52(1H, s, H-3).13C NMR spectrum and DEPT spectrum showed 8 methyl groups, 10 methylene groups, 7Methine and 10 quaternary carbons, with δ c129.15, δ c140.60 being typical of ursane-type triterpenes with the alkene carbon signals at the 12, 13 positions, δ c182.42 being the carbonyl carbon signal at the 28 position, δ c63.52, δ c67.08 and δ c73.56 being the vicinal carbon signals. From the nuclear magnetic characteristics of the ursolic alkane type pentacyclic triterpenoid in the cherokee rose root summarized in the previous period, the deltac 73.56 is presumed to be a signal of the carbon substituted by the hydroxyl at the 19 th position. Comprehensive analysis1HNMR，13C NMR，1H-1HCOSY, HSQC and HMBC know that a 3-methyl butyryloxy fragment exists in the structure of the compound, and signals are delta C22.81(C-4',5'), delta C26.9(C-3'), delta C44.49 (C-2'), delta C174.56(C-1'), delta H0.96(H-4',5'), delta H2.10(H-3'), and delta H2.22(H-2 '). The above information suggests that the basic parent nucleus of the compound is ursane pentacyclic triterpene, and 19-position is substituted by hydroxyl, 28-position is substituted by carboxyl, and the structure also has 3-methyl butyryloxy substitution. In addition, the structure of the compound also contains 2 vicinal oxygen carbon signals delta C63.52(C-1), delta C67.08(C-24) and a pair of alkene carbon signals delta C136.51(C-3) and 152.42 (C-2).
Summarizing the ursane-type pentacyclic triterpene compounds in cherokee rose root, the 23-position and 24-position angular methyl groups are often substituted by hydroxyl, and the general rule is as follows: 23 th-position angle methyl (or-CH) of ursane type compound2OH) is more angular than the 24-position methyl (or-CH)2OH) is in low field and interacts with 23, 24-CH3Comparison, with 23-CH2When OH is substituted, 24-CH3A shift to high field of about 2.4ppm, typically 64-67 ppm and 14-20 ppm for C-23 and C-24 chemical shifts; and has 24-CH2When OH is substituted, 23-CH3The shift to high field is about 4.5ppm, and the C-23 and C-24 chemical shifts are typically 20-25 ppm and 62-67 ppm. According to this rule, it is assumed that the compound has a hydroxyl substitution at the 24-position, and the chemical shifts at the C-23 and C-24 positions are δ C24.53 and δ C67.08, respectively. On the HSQC spectrum, the delta 0C67.08 is related to delta 1H3.45 and delta 2H3.66 protons, on the HMBC spectrum, the delta 3H3.45 and the delta 4H3.66 are both related to delta 5C24.53(C-23), and the 24-position angle methyl group is proved to be changed into hydroxymethyl by hydroxylation. On the HMBC spectrum, Δ H0.93(H-30), Δ H1.20(H-29) and Δ H2.49(H-18) are all related to Δ C73.56(C-19), and the substitution at position 19 is also demonstrated. On the HMBC spectrum, δ H3.45(H-24), δ H3.66(H-24) and δ H1.13(H-23) are all related to carbon with chemical shift δ c136.51When δ C136.51 is a 3-position carbon signal, it is found that a double bond is present at the 2 and 3 positions and the chemical shift of C-2 is δ C152.42. The HMBC spectra also show that the chemical shifts are related to C-2(δ 2C152.42) and C-3(δ 3C136.51) for both the δ 0H4.63 and δ 1H4.66 protons, indicating that δ H4.63 and δ H4.66 are two hydrogen proton signals on the 1-position carbon. On HSQC, these two protons are correlated with the carbon signal at δ C63.52(C-1), indicating that C-1 is a vicinal oxygen carbon. Furthermore, on the HMBC spectrum, both hydrogen protons on the carbon at position 1(Δ H4.63 and Δ H4.66) are associated with a carbon having a chemical shift at δ C174.56(C-1'), indicating that 3-methylbutanoyloxy is attached at position 1. In addition, the spectrum of HMBC was clearly related to delta H1.19(H-25) and delta C152.42(C-2), indicating that the condensation of the A ring from a six-membered ring to a five-membered ring occurred.
Further comparison shows that the chemical shifts of C-5 (delta C64.29) and C-10 (delta C51.91) of the compound are lower than those of normal ursolic alkyl pentacyclic triterpene C-5 (delta C55) and C-10 (delta C37), and an HMBC spectrum shows that delta H5.52(H-3) is obviously related to delta C51.91(C-10), and the information also proves that the A ring is condensed into a five-membered ring. Compared with the known compound rosaMultic acid, the data are basically consistent except C-1, 2 and 3, so that the structure of the target compound is finally determined to be 2- [ (3'-methyl) -butyl acrylate ] methyl A (1) nor-19 alpha, 24-dihydrours-2, 12-dien-28-oic acid (1- (3' -methyl) -butyl acrylate rosaMultic acid).
The nuclear magnetic data of the target compound are subjected to full attribution according to one-dimensional, two-dimensional and related spectrums, and the results are shown in table 1.
Nuclear magnetic data for the compounds of table 1
Figure BDA0002057835040000091
(Note: delta in ppm, J in Hz: a: CD3OD，600MHz for 1H-NMR，150MHz for 13C-NMR：b：CDCl3，75MHz for 13C-NMR)
Example 22 preparation of- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid salt
1. Preparation of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid potassium or sodium salt: firstly, dissolving potassium hydroxide or sodium hydroxide in ethanol, then adding 2- [ (3'-methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid into the solution, heating and refluxing while stirring, cooling to room temperature after the refluxing is finished, then dropwise adding acetonitrile while stirring, carrying out suction filtration and drying to obtain a white solid, namely 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid potassium salt or sodium salt.
2. Preparation of ammonium salt of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid: dissolving 2- [ (3'-methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid in ethanol to prepare ethanol solution, then dropwise adding saturated ammonia water until the pH value of the solution is 9-11, dropwise adding acetonitrile while stirring, filtering, and drying to obtain white solid, namely 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid ammonium salt.
3. Preparation of calcium 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oate: dissolving 2- [ (3'-methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid in ethanol, then dropwise adding saturated ammonia water until the pH value of the solution is 9-13, dropwise adding a calcium ion-containing aqueous solution while stirring, continuously stirring, fully reacting, standing, precipitating, filtering, and drying to obtain a white solid, namely 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid calcium salt.
4. Preparation of magnesium salt of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid: preparation of calcium 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oate: dissolving 2- [ (3'-methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid in ethanol, then dropwise adding saturated ammonia water until the pH value of the solution is 9-13, dropwise adding a magnesium ion-containing aqueous solution while stirring, continuously stirring, fully reacting, standing, precipitating, filtering, and drying to obtain a white solid, namely the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid magnesium salt.
5. Preparation of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-carboxylic acid barium salt: preparation of calcium 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oate: dissolving 2- [ (3'-methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid in ethanol, then dropwise adding saturated ammonia water until the pH value of the solution is 9-13, dropwise adding an aqueous solution containing barium ions while stirring, continuously stirring, fully reacting, standing, precipitating, filtering and drying to obtain a white solid, namely 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid barium salt.
Example 3 biological Activity testing of Compounds
1. Bacteriostasis test
(1) Zone of inhibition test
The test strains are: staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus dysenteriae, Bacillus proteus, Bacillus typhi, Bacillus subtilis, and Candida albicans.
Preparing a solution to be detected: dissolving 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid compound in ethanol solution, adding water to prepare the solution with required concentration, wherein the volume ratio of ethanol in the prepared sample to be detected is lower than 2%. Streptomycin is used as a positive control drug.
The testing process of the zone of inhibition: respectively placing staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, dysentery bacillus, proteus, typhoid bacillus, bacillus subtilis and candida albicans into a broth culture medium test tube under the aseptic condition. And plugging a test tube plug after sterilizing the tube orifice, culturing in a 37 ℃ bacteria incubator for 24h, taking out, and storing in a refrigerator at 4 ℃ with the bacteria content of 5-10 CFU/mL. Boiling nutrient agar, dissolving and sterilizing, transferring to an ultraclean workbench, pouring the nutrient agar into a culture dish to be cooled, sucking 100 mu L of bacterial liquid by using an aseptic micro-sampler, pouring the bacterial liquid on a nutrient agar plate, uniformly coating, after the bacterial liquid is slightly dried, taking an aseptic circular paper sheet with the diameter of 6mm, respectively sucking to-be-detected sample or positive control drug solution with different concentrations, sucking 10 mu L of each sheet, and uniformly distributing on the surface of the nutrient agar plate. The diameter of the inhibition zone is observed and measured after the culture is carried out for 24 hours at 37 ℃ in a constant temperature and humidity incubator, the drug inhibition experiment of each concentration is repeated for 3 times, and the inhibition result is the average value of 3 experiments. The results are shown in Table 1.
TABLE 12- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid Compound for bacteriostatic results
Figure BDA0002057835040000111
(2) Determination of Minimum Inhibitory Concentration (MIC)
The tested concentrations of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-dien-28-oic acid, 0.5, 0.25, 0.125, 0.06 and 0.03mg/mL, were tested and the results are shown in table 2. The above test results show that 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-dien-28-oic acid exhibits a good bacteriostatic effect against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, but a weak bacteriostatic effect against Shigella dysenteriae, Proteus, Salmonella typhi and Candida albicans, and therefore only the MICs of the compounds against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were tested here.
TABLE 22- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid results for minimum inhibitory concentration
Figure BDA0002057835040000112
Figure BDA0002057835040000121
As can be seen from tables 1 and 2, the 2- [ (3'-methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of the present invention exhibits a certain degree of bacteriostatic action against staphylococcus aureus, pseudomonas aeruginosa and escherichia coli, and the diameter of the zone of inhibition increases with the increase in the concentration of the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound, wherein the bacteriostatic action against staphylococcus aureus, escherichia coli and pseudomonas aeruginosa is better.
2. Anti-inflammatory test
Indometacin tablet is used as positive control drug, the dosage is 6.5mg/kg, physiological sodium chloride solution is used as blank control, wherein the dosage of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid is 26mg/kg, 52mg/kg and 104 mg/kg.
Male mice were taken as 50 mice, 10 mice per group, and randomly divided into 5 groups. A clear transverse line is made on the upper end of the front side of the metatarsus of the left hind paw of the mouse, the volume of the paw of the mouse is measured by a volume measuring device and a drain valve, and the volumes are respectively measured for 3 times to obtain an average value. Then, the medicine is continuously administrated by stomach irrigation for 4 days according to the dose of 0.2mL/10g, 10% egg white (0.1 mL/plantar) prepared by a labor-saving sodium chloride solution is injected under the skin of the left hind foot and the plantar of the mouse after the last administration for 1 hour, and the swelling degree is the volume of the hind foot and the plantar of the proinflammatory foot. The results are shown in Table 3.
TABLE 32- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid Compounds for anti-inflammatory
Results
Figure BDA0002057835040000122
As is clear from table 3, the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound had an anti-inflammatory effect, and the anti-inflammatory effect was dose-dependent, and the effect of inhibiting the swelling of the mouse plantar foot was comparable to that of indomethacin tablets when the dose was 104 mg/kg.
3. Analgesic test
The amount of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid used was 26mg/kg, 52mg/kg and 104 mg/kg.
The test method comprises the following steps: the prophylactic administration is carried out for 5 days, mice in each group are fasted overnight before the test, 0.6% acetic acid (0.2 mL/mouse) is injected into the abdominal cavity after the corresponding drugs are infused into the stomach of each group for 1 hour on the test day, the times of writhing of the mice within 10min after the injection are recorded, the writhing reaction is positive by the repeated contraction of lumbar muscles, the torsion of the arch back and the hip and the extension of hind limbs of the mice, and the percent of writhing of the drugs inhibited is calculated according to the following formula:
inhibition (%) - (number of writhing in control group-number of writhing in administration group)/number of writhing in control group + 100%; wherein the writhing of the control group is a gavage physiological sodium chloride solution, and the Zaltoprofen (the administration dose is 80mg/kg) is used as a positive control.
The twist results are shown in table 4.
Table 4 results of mouse writhing times
Figure BDA0002057835040000131
As shown in Table 4, after different doses of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-oic acid are taken, the writhing frequency of the mice is obviously reduced, the inhibition rate is 36-80%, and the inhibition rate of the writhing of the mice is concentration-dependent; when the administration dose is 104mg/kg, the times of writhing of the mice are equivalent to that of zaltoprofen (the administration dose is 80mg/kg), which shows that the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid compound has better analgesic effect.
4. Fever reduction test
The amount of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid used was 26mg/kg, 52mg/kg and 104 mg/kg.
Measuring the body temperature 2 times every day 3d before the experiment, and selecting rats with the body temperature of 36.6-38.0 ℃ and the body temperature change of no more than 0.3 ℃ for preparing fever models. 60 healthy rats with the weight of 150-200 g are divided into 6 groups with half of male and female, the rats are subjected to continuous intragastric administration for 6 days, a 20% yeast powder suspension is injected subcutaneously on the 7 th day according to the dose of 1mL/100g, the anal temperature is measured after 1h, then the anal temperature is measured for 1 time every 1h, and when the 4 th hour, more than 80% of rat body temperature rises to more than 0.5 ℃, the molding is successful. Then, the body temperature was measured 1 time every 1 hour and 9 hours. The difference between the body temperature measured each time and the basal body temperature is delta ℃ to compare the antipyretic effect. The test results are shown in Table 5, using physiological sodium chloride solution as blank control group and aspirin (100mg/kg) as positive control drug.
TABLE 5 results of the mouse pyrolysis test
Figure BDA0002057835040000141
As can be seen from Table 5, the body temperature change of the mice was alleviated and concentration-dependent after administration of 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-dien-28-oic acid, and the thermolytic effect was even better than that of the positive control drug when administered at a dose of 104 mg/kg.
The experimental results show that the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 alpha, 24-dihydroxy-2, 12-diene-28-acid or pharmaceutically acceptable salt thereof has the effects of bacteriostasis, anti-inflammation, analgesia and antipyresis, and can be prepared into pharmaceutically common dosage forms for application or be compounded with other medicines to prepare pharmaceutical compositions for application.
EXAMPLE 4 preparation of different dosage forms
1. Preparation of tablets: the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I and the salt thereof with an alkali metal or ammonium were prepared as described in example 1, and excipients were added in a weight ratio of the compound or any of its salts to excipients of 1:10, and the mixture was granulated and tabletted.
2. Preparation of the powder: the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I, and its salt with an alkali metal or ammonium were prepared as described in example 1 and made into powders by conventional powder preparation methods.
3. Preparation of capsules or granules: the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I and the salt thereof with an alkali metal or ammonium were prepared according to the method of example 1, and excipients were added in a weight ratio of the compound or any one of the salts thereof to the excipients of 1:10 to prepare capsules or granules.
4. Preparation of injection: the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I and the salt thereof with alkali metal or ammonium were prepared as described in example 1, and the compound was used to prepare injection by conventional water for injection, fine filtration, encapsulation and sterilization.
EXAMPLE 5 pharmaceutical composition
A pharmaceutical composition comprises 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I as described in example 1, and powders and adjuvants of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, Mahonia, Andrographis paniculata, Angelica sinensis, and Codonopsis pilosula.
EXAMPLE 6 pharmaceutical composition
A pharmaceutical composition comprising the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I as described in example 1, and salts thereof with alkali metals, alkaline earth metals or ammonium, and powders and adjuvants of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, Mahonia, Andrographis, Angelica sinensis, Codonopsis pilosula.
EXAMPLE 7 pharmaceutical composition
A pharmaceutical composition comprises 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I as described in example 1, extract of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, Mahonia, Andrographis paniculata, Angelica sinensis, Codonopsis pilosula, and adjuvants. The extract is prepared by the extraction method described in any one or more of patent documents with patent publication numbers CN104529984B, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C and CN 1296073C.
EXAMPLE 8 pharmaceutical composition
A pharmaceutical composition comprising the 2- [ (3' -methyl) -butyryloxy ] demethyl-19 α, 24-dihydroxy-2, 12-diene-28-oic acid compound of formula I as described in example 1, and salts thereof with alkali metals, alkaline earth metals or ammonium, and extracts of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, Mahonia, Andrographis, Angelica sinensis, Codonopsis pilosula, and adjuvants. The extract is prepared by the extraction method described in any one or more of patent documents with patent publication numbers CN104529984B, CN1170549C, CN1158087C, CN1330335C, CN1296071C, CN1321631C, CN1296072C and CN 1296073C.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (17)

1. The triterpenoid compound and the pharmaceutically acceptable salt thereof are characterized in that the structural formula of the compound is shown as a formula I, and the structure of the pharmaceutically acceptable salt is shown as a formula II:
Figure FDA0003103986050000011
wherein M is an alkali metal ion, an alkaline earth metal ion or an ammonium group; and n is 1 or 2.
2. The triterpenoid compound and pharmaceutically acceptable salts thereof according to claim 1, wherein the alkali metal ion is potassium, sodium or lithium; the alkaline earth metal ions are calcium ions, magnesium ions or barium ions.
3. A pharmaceutical composition comprising the triterpenoid compound and/or pharmaceutically acceptable salt thereof according to claim 1 or 2.
4. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable excipient and/or carrier.
5. The pharmaceutical composition of claim 3, further comprising one or more of Philippine flemingia root, zanthoxylum dissitum Hemsl, Spatholobus suberectus Dunn, Mahonia dubia, Andrographis paniculata, Angelica sinensis and Codonopsis pilosula.
6. The pharmaceutical composition of claim 5, further comprising an extract of one or more of Flemingia philippinensis, Zanthoxylum nitidum, caulis Spatholobi, caulis Mahoniae, herba Andrographitis, radix Angelicae sinensis, and radix Codonopsis.
7. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is in the form of tablets, capsules, powders, granules, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays.
8. A process for the preparation of triterpenoids and pharmaceutically acceptable salts thereof according to claim 1 or 2, which comprises the steps of:
s1, reflux-extracting coarse powder of a cherokee rose root medicinal material by using ethanol, and concentrating to obtain dry paste; adding water into the dry extract of radix Rosae Laevigatae to obtain suspension, and sequentially extracting with petroleum ether, chloroform and n-butanol to obtain petroleum ether fraction, chloroform fraction and n-butanol fraction;
s2, performing primary separation on the chloroform part obtained in the step S1 by adopting a medium-pressure column, filling the chloroform part into the column by adopting a dry method, performing column chromatography separation by using the medium-pressure column, and performing gradient elution by using petroleum ether-ethyl acetate to obtain 4 different elution parts A1, A2, A3 and A4;
s3, combining the obtained A3 and A4 parts, separating by using a reverse phase column, and carrying out methanol-water gradient elution to obtain 5 parts, namely A3-4.1, A3-4.2, A3-4.3, A3-4.4 and A3-4.5;
s4, mixing A3-4.5 with methanol: eluting with 0.1% formic acid, collecting the peak at 70-75min, eluting with acetonitrile: and performing liquid-phase elution with 0.1% formic acid water to obtain the target compound.
9. The method according to claim 8, wherein the coarse powder of cherokee rose root is extracted 3 times in step S1, and the extraction is performed for 1 hour each time; the volume fraction of the ethanol is 50-100%.
10. The method according to claim 9, wherein the ethanol is used in a volume fraction of 70%.
11. Use of the triterpenoid and pharmaceutically acceptable salts thereof of claim 1 or 2 in the preparation of antibacterial, anti-inflammatory, analgesic or antipyretic medicaments.
12. The use of claim 11, wherein the antibacterial agent is an agent against escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, or candida albicans.
13. The use of claim 11, wherein the anti-inflammatory agent is an agent for the treatment of cervicitis, vaginitis, endometritis, pelvic inflammatory disease, or adnexitis.
14. The use according to claim 11, wherein the medicament comprises a pharmaceutically acceptable excipient and/or carrier.
15. The use of claim 11, wherein the medicament further comprises one or more of philippine flemingia root, zanthoxylum dissitum Hemsl, suberect spatholobus stem, leatherleaf mahonia stem, common andrographis herb, Chinese angelica, and pilose asiabell root.
16. The use of claim 11, wherein the medicament further comprises one or more extracts of philippine flemingia root, zanthoxylum dissitum Hemsl, suberect spatholobus stem, leatherleaf mahonia stem, andrographis paniculata, Chinese angelica, and dangshen.
17. The use according to claim 11, wherein the medicament is in the form of tablets, capsules, powders, granules, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays.
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