CN111303237B - Polyhydroxy-substituted oleanane pentacyclic triterpenoid, preparation method thereof and application thereof in preparing antifungal medicines - Google Patents
Polyhydroxy-substituted oleanane pentacyclic triterpenoid, preparation method thereof and application thereof in preparing antifungal medicines Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Abstract
The invention discloses a polyhydroxy-substituted oleanane pentacyclic triterpenoid compound, a preparation method thereof and application thereof in preparing antifungal medicaments. The 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid is extracted and separated from the raspberry, and the compound is subjected to a fungus inhibitory activity experiment, and the test result shows that the compound has obvious fungus inhibitory activity, can be used for preparing antifungal medicines or serving as a lead compound of the medicines, and has potential clinical application value.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a polyhydroxy-substituted oleanane pentacyclic triterpenoid compound, a preparation method thereof and application thereof in preparing antifungal medicines.
Background
Modern medicine divides fungal infections into two main categories, superficial fungal infections and deep fungal infections. Superficial fungal infections refer to infections of the keratin tissues of the skin (including the stratum corneum, nail plate, hair, etc.); deep fungal infections are also called Invasive Fungal Infections (IFI), which refer to diseases in which fungi invade the body, grow and multiply in tissues, organs or blood, and cause inflammatory reactions and tissue damage. In recent years, the incidence of invasive fungal infections has increased dramatically due to the widespread use of hormones and antibiotic drugs, increasing the frequency of bone marrow, organ transplantation and the use of antineoplastic drugs (Brown et al, Science 2012, 647-648; Brown et al, Sci. Transl. Med.2012, 165rv13). It is estimated that approximately 150 to 200 million deaths per year are caused by invasive fungal infections. Invasive fungal infections have become a leading cause of death in immunocompromised patients, with candida infections, especially candida albicans, always being the first and still having a mortality rate of up to 40% after treatment. Thus, fungal infections have become a clinically urgent problem (Ostrosky-Zeichner et al. Nat. Rev. Drug Discov.2010, 719-727; Odds et al. trends Microbiol.2003, 272-279). Although a series of antifungal drugs are available on the market since the 50 s in the 20 th century, the antifungal drugs face various problems such as limited product types (Ostrosky-Zeichhner et al Nat. Rev. drug Discov.2010,719-727) and large toxic and side effects (Ostrosky-Zeichner et al, Clin. Infect. Dis.2003, 415-425). At present, the development of antifungal drugs is slow, and the development of antifungal drugs is lack of substantial progress in recent decades, so that the clinical choice of drugs for treating invasive fungal infection is still very limited. Fluconazole (fluconazole) is the antifungal drug which is the most widely used clinically at present, but the drug promotes the rapid development of fungal drug resistance in the process of repeated treatment and long-term administration, and causes cross drug resistance, so that the existing drug has no policy for more and more drug-resistant fungal restraints. The limited reserves of antifungal drugs highlight the urgent need to develop a new generation of antifungal (especially drug-resistant fungal) drugs.
Natural products are considered as an important source of novel chemical entities with unique pharmacological activity and have long been considered as a valuable source of drug design (Hattum et al, J.Am.Chem.Soc.2014, 11853-11859; Harvey et al, Nat.Rev.drug Discov.2015, 111-119; Koch et al, Proc.Natl.Acad.Sci.USA 2005, 17272-17277; Eschenbrenner-Lux et al, Angew. Chem.Int.Ed.2014, 2134-2137; Tiago et al, Nature m.2016,531-541), and especially in the development of anti-cancer and anti-infective drugs (Newman and Cragg, J.Nat.Prod.629-661; New Nature m.2016,531-541, 2000). Therefore, the method has important research value in searching novel antifungal medicines from natural products. Chinese medicine resources are abundant in China, and part of Chinese medicines have a long history of being used for treating fungal infection, so that screening antifungal medicines from Chinese medicines and chemical components thereof and researching the action mechanism of the antifungal medicines become important directions for researching and developing the antifungal medicines at present. The traditional Chinese medicine raspberry is the dried fruit of Rubus chingii (Rubus) of Rubus of Rosaceae (Rosaceae), is a common Chinese medicine collected in Chinese pharmacopoeia, and has the effects of tonifying kidney, securing essence, reducing urination, nourishing liver and improving eyesight (pharmacopoeia of the people's republic of China, first part 2015, 382). The polyhydroxy oleanane-type triterpene compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid obtained by first separation in the invention is proved to have remarkable anti-candida albicans SC5314 activity by a plurality of in vitro pharmacological tests. The compound is found to have the activity for the first time and can be applied to the preparation of antifungal medicines.
Disclosure of Invention
The invention aims to solve the technical problems and provides a polyhydroxy-substituted oleanane pentacyclic triterpenoid compound, a preparation method thereof and application thereof in preparing antifungal medicines.
The invention is realized by the following technical scheme:
a polyhydroxy-substituted oleanane-type pentacyclic triterpenoid is 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid, and has a structural formula as follows:
a preparation method of polyhydroxy-substituted oleanane pentacyclic triterpenoid comprises the following steps:
a. drying and pulverizing Rubi fructus fruit;
b. extracting with 75% ethanol at normal temperature for 3-5 times, each time for 12-24 hours;
c. mixing extractive solutions, concentrating under reduced pressure to remove methanol to obtain extract;
d. dispersing the extract with water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol of equal volume for 2-3 times respectively to obtain four components of petroleum ether, ethyl acetate, n-butanol and water;
e. and d, gradient eluting the ethyl acetate part obtained in the step d by using a silica gel column and petroleum ether-ethyl acetate, gradient eluting the ethyl acetate part by using a MCI column and methanol-water, and further purifying the ethyl acetate part by using gel column chromatography and semi-preparative HPLC to obtain the compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid.
In the embodiment, in the step e, the elution ratio of the petroleum ether to the ethyl acetate is 30:1-0:1, v/v; the elution ratio of methanol to water is 50:50-70: 30-85: 15-100:0, v/v.
In the present embodiment, 2 α,3 α, 23-trihydroxyoleanane-12-en-28-oic acid can be isolated and purified from plants by the above-mentioned method; can also be obtained by chemical synthesis methods well known to those skilled in the art.
An application of polyhydroxy-substituted oleanane pentacyclic triterpene compounds in preparing antifungal medicines is provided.
In this embodiment, the polyhydroxy-substituted oleanane-type pentacyclic triterpenoid is 2 α,3 α, 23-trihydroxyoleanane-12-en-28-oic acid.
In this particular embodiment, the antifungal agent is an anti-candida albicans SC5314 agent.
In this embodiment, the compounds can be used alone or in combination, or can be combined with pharmaceutically acceptable carriers or excipients to make oral or non-oral dosage forms according to conventional methods.
An antifungal medicine, which takes 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid as an active component.
In the specific embodiment, the antifungal medicine contains pharmaceutically acceptable auxiliary materials and is prepared into a pharmaceutically acceptable dosage form.
In the embodiment, the dosage form of the antifungal drug comprises tablets, capsules and injections.
The invention has the beneficial effects that:
the polyhydroxy pentacyclic triterpenoid compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid has obvious fungal inhibition activity and good application prospect on diseases caused by high-incidence fungal infection of modern people.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions of the present invention are further described below with reference to examples.
Mass spectra (ESI-MS) were obtained from an Agilent 1100LC/MSD type mass spectrometer using an ESI ion source to obtain both positive and negative ion modes; NMR data were obtained from a Bruker AvanceII400 NMR spectrometer or an Avance II 600 NMR spectrometer, chemical shifts being referenced to the non-deuterated residual solvent peak and expressed in delta (ppm); thin layer chromatography plates (TLC) used for the analysis were purchased from Tintamiu silica gel development, Inc., and developed using UV (. lamda.: 254nm,365nm) and sulfuric acid-vanillin solution; column chromatography was carried out using mainly MCI microporous resin CHP20P (Japan, Mitsubishi Chemical Industries,75-150 μm), silica gel (200-300mesh, Ji-Yi-Da silicasia Chemical Ltd, Qingdao P.R.China) and Sephadex LH-20 (Sweenen, GE Healthcare BioSciences AB); analytical and semi-preparative liquid phase apparatus Waters e2695 equipped with Waters 2998PDA and Waters 2424ELSD dual detectors, and Waters Sunfire column (5 μm, 250X 10 mm); analytical pure solvents used in the experiments, such as petroleum ether, acetone, ethyl acetate, methanol and the like, were purchased from Shanghai Tantan chemical Co., Ltd, chromatographic grade methanol and acetonitrile were purchased from Shanghai Hai Kangke high purity solvent Co., Ltd, fluconazole was purchased from Peui pharmaceutical Co., Ltd (fluconazole injection, 2mg/mL, production lot: B359505), and Rubi fructus was purchased from Shanghai Hai hong Qiao Chinese medicinal decoction pieces Co., Ltd.
Example 1: preparation of triterpene compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid
Drying and pulverizing 10kg Rubi fructus (Rubuschingii), and extracting with 75% ethanol (10L) for 5 times each for 24 hr at room temperature. The combined extract was concentrated under reduced pressure to remove methanol to obtain 2.1kg of extract (semi-dry). And (3) dispersing the extract with 2L of water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol which are equal in volume for three times respectively to obtain four components of the petroleum ether, the ethyl acetate, the n-butanol and the water. The total amount of 125.3g of ethyl acetate fraction was eluted through a silica gel column with a petroleum ether-ethyl acetate gradient (30:1 → 0:1, v/v) and combined into 12 fractions Fr.1-Fr.12 according to TLC color development. The component Fr.8(6.7g) passes through an MCI column and is eluted by methanol-water (50:50-70: 30-85: 15-100:0, v/v) in a gradient way to obtain 8 subcomponents Fr.8A-Fr.8H. Subfraction Fr.8F (217mg) by gel (Sephadex LH-20, MeOH) column chromatography and semi-preparative HPLC (MeOH-H)2O74:26, v/v) to obtain the compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid (4.3mg, t)R＝20.6min)。
The physicochemical data for the compounds are as follows:
2 α,3 α, 23-trihydroxyoleanane-12-en-28-oic acid: white amorphous powder [ alpha ]]D 26: +48.5(c 0.3,MeOH).1H NMR(in C5D5N,400MHz):δ0.86(3H,s,Me-24),0.89 (3H,s,Me-25),0.97(3H,s,Me-26),1.00(3H,s,Me-30),1.03(3H,s,Me-30), 1.19(3H,s,Me-27),3.28(1H,dd,J＝13.7,4.6Hz,H-18),3.76(1H,d,J＝10.4 Hz,H-23),3.93(1H,d,J＝10.4Hz,H-23),4.16(1H,br s,H-3),4.30(1H,br dd, J＝10.6Hz,H-2),5.46(1H,br s,H-12)；13C NMR(in C5D5N,150MHz):δ42.2 (C-1),66.2(C-2),78.9(C-3),41.8(C-4),43.5(C-5),18.2(C-6),33.1(C-7),39.8 (C-8),48.0(C-9),38.4(C-10),23.6(C-11),122.4(C-12),144.8(C-13),42.5 (C-14),28.1(C-15),23.7(C-16),46.6(C-17),41.8(C-18),46.3(C-19),30.8 (C-20),34.1(C-21),32.8(C-22),71.2(C-23),17.7(C-24),16.9(C-25),17.5 (C-26),26.1(C-27),180.2(C-28),33.1(C-29),23.8(C-30).ESIMS m/z487[M- H]-.
Example 2: determination of fungal inhibitory Activity of 2 alpha, 3 alpha, 23-Trihydroxyl Oleanopan-12-ene-28-oic acid
The experimental method comprises the following steps: candida albicans SC5314 is provided by professor William A.Fonzi of Georgetown University microorganisms and immunology, USA. A small amount of Candida albicans SC5314 was picked from the SDA medium stored at 4 ℃ and inoculated into 1mL YEPD culture medium, and after shaking culture at 37 ℃ and 200rpm, the fungus was activated twice, and then was in the late exponential phase of growth. Adding the bacterial liquid into YEPD culture solution, activating again by the above method, counting with a blood cell counting plate after 16 hr, adjusting bacterial liquid concentration to 1 × 10 with RPMI 1640 culture solution3～5×103one/mL. Taking a sterile 96-well plate, and adding 100 mu L of RPMI 1640 liquid culture medium into each row of No. 1 wells as a blank control; adding 100 mu L of freshly prepared bacterial liquid into each hole 3-12; no. 2 well is 193.6. mu.L of inoculum and the sample is 6.4. mu.L of 2mg/mL solution. Dilution was performed in duplicate for wells 2-11 to give final drug concentrations of 64, 32, 16, 8, 4,2, 1, 0.5, 0.25, 0.125. mu.g/mL for each well. No. 12 wells contained no drug, and 100. mu.L of inoculum was added as a positive growth control. After culturing Candida albicans in an incubator at 30 ℃ for 24 or 48 hours, the OD value of each well was measured at 630nm using an enzyme-labeled analyzer. The OD value of the positive control hole is controlled to be about 0.2, and compared with the positive control hole, the MIC is the concentration of the drug in the hole with the lowest concentration and the OD value of the drug is reduced by more than 80 percent80(drug concentration at which fungal growth was 80% inhibited). MIC of drug80When the value exceeds the measurement range, the statistics is carried out according to the following method: MIC80Values above the maximum concentration of 64. mu.g/mL, measured as "> 64. mu.g/mL"; MIC80When the concentration is the lowest concentration or below, the values are calculated to be less than or equal to 0.125 mu g/mL without distinction "
All the above experiments were performed in parallel 2 to 3 times when MIC80Values can be accurately repeated or accepted only at one concentration difference, and higher concentrations are taken as MICs80A value; when MIC80If the values differ by more than two concentrations, the experiment needs to be repeated until the above requirements are met. The results are shown in Table 1.
TABLE 1 drug vs. Candida albicans SC5314 MIC80Value of
As can be seen from Table 1, the MIC of positive control fluconazole against Candida albicans SC5314800.125 mu g/mL, MIC of compound 2 alpha, 3 alpha, 23-trihydroxyoleanane-12-ene-28-acid to Candida albicans SC531480Is 32 mu g/mL, and has potential clinical application value.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (6)
1. A preparation method of polyhydroxy-substituted oleanane pentacyclic triterpenoid is characterized in that: the method comprises the following steps:
a. drying and pulverizing Rubi fructus fruit;
b. extracting with 75% ethanol at normal temperature for 3-5 times, each time for 12-24 hours;
c. mixing extractive solutions, concentrating under reduced pressure to remove methanol to obtain extract;
d. dispersing the extract with water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol of equal volume for 2-3 times respectively to obtain four components of petroleum ether, ethyl acetate, n-butanol and water;
e. gradient eluting the ethyl acetate part obtained in the step d by a silica gel column by using petroleum ether-ethyl acetate, wherein the elution ratio of the petroleum ether to the ethyl acetate is 30:1-0:1, and v/v; performing gradient elution with methanol-water through MCI column at methanol-water elution ratio of 50:50-70: 30-85: 15-100:0, v/v; finally, further purifying by gel column chromatography and semi-preparative HPLC to obtain the compound 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid, the structural formula is as follows:
2. the application of polyhydroxy-substituted oleanane pentacyclic triterpene compounds in preparing antifungal medicaments is characterized in that: the polyhydroxy-substituted oleanane pentacyclic triterpenoid is 2 alpha, 3 alpha, 23-trihydroxyoleanane-12-alkene-28-acid; the antifungal medicine is an anti-candida albicans SC5314 medicine.
3. The use of the polyhydroxy-substituted oleanane pentacyclic triterpenoid in the preparation of antifungal drugs according to claim 2, is characterized in that: the compound is used alone or together, or combined with pharmaceutically acceptable carriers or excipients, and prepared into oral or non-oral dosage forms according to a conventional method.
4. An antifungal drug, which is characterized in that: the antifungal medicine takes 2 alpha, 3 alpha, 23-trihydroxy oleanane-12-alkene-28-acid as an active component, and the fungus is candida albicans SC 5314.
5. The antifungal agent of claim 4, wherein: the antifungal medicine contains pharmaceutically acceptable auxiliary materials and is prepared into pharmaceutically acceptable dosage forms.
6. The antifungal agent of claim 5, wherein: the antifungal medicine is in the dosage form of tablet, capsule or injection.
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Citations (1)
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| CN101830964A (en) * | 2010-05-21 | 2010-09-15 | 中国人民解放军第二军医大学 | Triterpenoid compounds with anti-inflammatory activities |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101830964A (en) * | 2010-05-21 | 2010-09-15 | 中国人民解放军第二军医大学 | Triterpenoid compounds with anti-inflammatory activities |
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| Anti-inflammatory Triterpenes from the Leaves of Rosa laevigata;Na Zeng 等;《Journal of Natural Products》;20110308;第74卷(第4期);732-738 * |
| 粗叶悬钩子三萜类化合物的分离鉴定;甘露 等;《中国中药杂志》;19980630;第23卷(第6期);361-363 * |
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