A type of C 21 Steroidal saponins, their preparation methods, and applications

Abstract

The present invention belongs to the technical field of natural compound preparation, and specifically relates to a C 21 Steroidal saponin and its preparation method and application. 21 Steroidal saponin is Δ 5 ‑Pregnene‑3 β ,20( S )-diol-3- O ‑[2‑ O ‑Acetyl‑ β ‑D‑Digitinose‑(1→4)‑ β ‑D‑Canadian hemp glycosides‑20‑ O ‑[ β ‑D‑glucose‑(1→2)‑ β The present invention is the first to separate and obtain C-D-digitalin from Periploca odorata cortex. 21 Steroidal saponin; the component and its derivatives can be used as precursors for the synthesis of other compounds, as well as reference components for quality control of related products, raw materials for new drug development and pharmacological activity research, etc.; the separation method provided by the present invention is simple and rapid, and can repeatedly and continuously obtain the target compound; the purity of the compound separated by the method is high; in vitro anti-inflammatory activity screening found that it has certain anti-inflammatory activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural compound preparation, and specifically relates to a C 21 Steroidal saponin and its preparation method and application. Background Art

[0002] Periplocae Cortex, also known as Northern Acanthopanax and Fragrant Acanthopanax, is a plant of the Asclepiadaceae family. Periploca sepium Bge.) The dried root bark of Perennial Herb has a long history of medicinal use and is a traditional Chinese medicinal material. The preface to Shennong's Herbal Classic says: "The inferior medicinal properties are mainly offensive, with strong toxicity that damages the balance. It cannot be taken regularly and should be stopped when the disease is cured." According to research, the "inferior" in the above statement refers to the toxic Perennial Herb. Periploca odorata is widely distributed and is mostly found in North China, East China and other regions. Modern pharmacological studies have shown that Perennial Herb has multiple activities such as anti-tumor, cardiotonic, immunomodulatory, and anti-inflammatory. Its chemical components include steroids, triterpenes, phenylpropanoids, simple aromatics, long-chain aliphatics, oligosaccharides and other components.

[0003] C 21 Steroids are a class of steroidal derivatives with a parent nucleus containing 21 carbon atoms. They are derivatives of pregnane or its isomers. They have anti-tumor, anti-inflammatory, analgesic, anti-fertility, anti-depression, immunomodulatory and other pharmacological activities and have very important medicinal value. At present, most of the chemical components isolated from Perennial Cortex are known and have low structural novelty; there are relatively few studies on the chemical components and medicinal effects of Perennial Cortex, and the bioavailability is low. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a C 21 Steroidal saponin Δ 5 -Pregnene-3 β ,20( S )-diol-3- O -[2- O -Acetyl- β -D-Digitinose-(1→4)- β -D-Canadian hemp glycoside]-20- O -[ β -D-Glucose-(1→2)- β -D-digitalin glycoside], which is isolated from the 70% ethanol extract of Periplocoside Root and named periplocoside G1. The lipopolysaccharide-induced RAW264.7 cell release of inflammatory factors model was used to study the component and found that it has anti-inflammatory activity.

[0005] The present invention also provides the above-mentioned C 21A method for preparing steroidal saponins.

[0006] Another object of the present invention is to provide the above-mentioned C 21 Application of steroidal saponins.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] The present invention provides a C 21 Steroidal saponin, the structural formula is shown in Formula 1:

[0009] Formula 1: ;

[0010] Where cym: β -D-pyranose; dig: β -D-Digitalinose; glu: β -D-pyranose.

[0011] The present invention also provides the above-mentioned C 21 The preparation method of steroidal saponin comprises the following steps:

[0012] (1) crushing the Cortex Periplocae, adding a solvent, heating and refluxing for extraction, combining the extracts, and recovering under reduced pressure to obtain an ethanol extract;

[0013] (2) dispersing the ethanol extract with water, extracting with ether, ethyl acetate and n-butanol in sequence, and concentrating under reduced pressure to obtain ether extract phase, ethyl acetate extract phase and n-butanol extract phase respectively;

[0014] (3) The n-butanol extract was separated by an MCI resin column and gradient eluted to obtain 7 fractions: fraction 1 to fraction 7;

[0015] (4) fraction 3 was subjected to gradient elution through a silica gel column, and the same components were detected by TLC and combined to obtain 22 fractions, Fr. A to V;

[0016] (5) Fraction Fr. O was subjected to gradient elution on a silica gel column to obtain three fractions, Fr. O1 to O3. Fraction Fr. O2 was crystallized to obtain the target compound C. 21 Steroidal saponins.

[0017] Preferably, in step (1), the heating reflux extraction is performed by heating 70% ethanol to 70-90° C. and reflux extraction for 3 times, each time for 1 hour.

[0018] Preferably, in step (3), the gradient elution uses a 40% to 100% methanol aqueous solution; the gradient elution program is: 40%, 50%, 60%, 70%, 80%, 90%, 100%.

[0019] Preferably, in step (4), the fraction 3 is the 60% methanol aqueous solution elution portion in step (3).

[0020] Preferably, in step (4), the gradient elution adopts a dichloromethane-methanol system of 100:1 to 1:1; the gradient of the dichloromethane-methanol gradient elution is: 100:1, 99:1, 98:2, 97:3, 96:4, 95:5, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1, v / v.

[0021] Preferably, in step (5), the fraction Fr. O is the elution portion in step (4) with a volume ratio of dichloromethane to methanol of 97:3.

[0022] Preferably, in step (5), the gradient elution adopts a dichloromethane-methanol system of 100:1~13:1; the gradient of the dichloromethane-methanol gradient elution is: 100:1, 50:1, 30:1, 15:1, 13:1, v / v.

[0023] Preferably, in step (5), the fraction Fr. O2 is an elution portion having a volume ratio of dichloromethane to methanol of 30:1.

[0024] The present invention also provides the above-mentioned C 21 Application of steroidal saponins in the preparation of anti-inflammatory drugs.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention is the first to separate and obtain a new structure of C from Perilla frutescens cortex. 21 Steroidal saponin compounds; the compounds are non-toxic to cells within the concentration range, and their salts, derivatives, etc. can be used as precursors for the synthesis of other compounds, as quality control reference ingredients for related products (such as Chinese medicinal materials, Chinese medicine formula granules, Chinese patent medicines, health foods, cosmetics, etc.), and as raw materials for new drug development and pharmacological activity research;

[0027] (2) The separation method provided by the present invention is simple and rapid, and can repeatedly and continuously obtain the target compound; the compound separated by this method has a high purity; and the new compound was screened for in vitro anti-inflammatory activity, and it was found that the compound has certain anti-inflammatory activity;

[0028] (3) The compounds separated by the present invention enrich the number of chemical components in the Cortex Periplocae, and the research on its anti-inflammatory activity provides a certain reference basis for the research on the mechanism of action of the Cortex Periplocae medicinal material, as well as the development and utilization of its resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1is the key HMBC (→) of compound 1 and 1 H- 1 H COSY(━) correlation diagram;

[0030] Figure 2 The inhibition of NO release by compound 1 ( n = 3, DiDOX: positive control; C: blank control; LPS: LPS was added);

[0031] Figure 3 For compound 1 1 H-NMR spectrum;

[0032] Figure 4 For compound 1 13 C-NMR spectrum;

[0033] Figure 5 DEPT135 spectrum of compound 1;

[0034] Figure 6 is the HMBC spectrum of compound 1;

[0035] Figure 7 is the HSQC spectrum of compound 1;

[0036] Figure 8 For compound 1 1 H- 1 H COSY spectrum;

[0037] Fig. 9 is the NOESY spectrum of compound 1;

[0038] Fig.10 HR-ESI-MS (m / z) of compound 1. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. Unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available.

[0043] Example 1

[0044] 1. Instruments and Materials

[0045] Bruker Avance Ⅲ600 superconducting supershielded Fourier transform nuclear magnetic resonance spectrometer (Bruker, Germany); METTLER TOLEDO XSE205 electronic balance (Mettler Toledo, Switzerland); Buchi R210 rotary evaporator (Buch, Switzerland); HPLC-IT-TOF-MS liquid spectrometer (Shimadzu, Japan); Bio Tek EPOCH2NS microplate reader (Porton, USA).

[0046] Ether, ethyl acetate, n-butanol, methanol, ethanol, petroleum ether, acetone, dichloromethane and other reagents were of analytical grade (Sinopharm Chemical Reagent Co., Ltd.); MCI GEL CHP20 / P120 resin (37-75 μm, Mitsubishi Chemical Corporation, Japan); column chromatography silica gel (200-300 mesh, Qingdao Bay Fine Chemical Co., Ltd.); Sephadex LH-20 hydroxypropyl dextran gel (Beijing Solebao Technology Co., Ltd.); normal phase silica gel thin layer plate (Yantai Institute of Chemical Industry); MTT (Beijing Dingguo Changsheng Biotechnology Co., Ltd.); DMEM medium (Gibco, USA); fetal bovine serum (Shanghai Dathill Biotechnology Co., Ltd.); dimethyl sulfoxide (DMSO, LIFE SCIENCE); lipopolysaccharide [LPS, Sigma-Aldrich (Shanghai) Trading Co., Ltd.]; p-aminobenzenesulfonamide and pancreatin (Beijing Dingguo Changsheng Biotechnology Co., Ltd.); 3,4-dihydroxybenzohydroxamic acid (DiDOX, MedChem, USA) Express Company, batch number: HY-19387 / CS-5292).

[0047] The origin of Perilla frutescens is Shaanxi Province, and it is identified as Perilla frutescens of the Apocynaceae family. P. sepium The dried root bark of Bge. was stored in Shandong Institute of Food and Drug Control with the code number SDIFDC-G-PSZ01.

[0048] 2 Methods

[0049] 2.1 Extraction and separation

[0050] (1) 4 kg of Periploca odorata Cortex was crushed and refluxed with 70% ethanol heated to 80°C for 3 times, each time for 1 h. The extracts were combined and recovered under reduced pressure to obtain an ethanol extract concentrate of about 600 g.

[0051] (2) The obtained ethanol extract is dispersed in water, extracted with ether, ethyl acetate and n-butanol in sequence, and concentrated under reduced pressure to obtain ether extract phase, ethyl acetate extract phase and n-butanol extract phase respectively;

[0052] (3) The n-butanol extract was separated by an MCI resin column and gradient eluted with a methanol-water solution (volume fraction 40% to 100%) (gradient: 40%, 50%, 60%, 70%, 80%, 90%, 100%) to obtain 7 fractions in total;

[0053] (4) Fraction 3 (the eluted portion with 60% methanol aqueous solution) was subjected to silica gel column chromatography and gradient eluted using a dichloromethane-methanol system (100:1 to 1:1) (gradient: 100:1, 99:1, 98:2, 97:3, 96:4, 95:5, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1, v / v). The same components were detected by TLC and combined to obtain a total of 22 fractions, Fr. A to V.

[0054] (5) Fraction Fr. O (the elution portion with a dichloromethane-methanol volume ratio of 97:3) was subjected to gradient elution (gradient: 100:1, 50:1, 30:1, 15:1, 13:1, v / v) on a silica gel column (dichloromethane-methanol system 100:1-13:1) to obtain three fractions, Fr.O1-O3. Fraction Fr. O2 (the elution portion with a dichloromethane-methanol volume ratio of 30:1) was crystallized to obtain compound 1 (4.6 mg).

[0055] 2.2 Anti-inflammatory activity studies

[0056] 2.2.1 RAW264.7 cell culture

[0057] RAW264.7 cells were added with DMEM high-glucose culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin double antibodies, and placed in an incubator with a concentration of 5% CO2, a humidity of 95%, and a constant temperature of 37°C. The culture medium was replaced and subcultured regularly, and the cells were cultured to the logarithmic phase for use.

[0058] 2.2.2 MTT assay to detect the effects of compounds on RAW264.7 cell viability

[0059] RAW264.7 cells in the logarithmic phase were seeded in 96-well plates (8 × 10 4The blank control group cells were not added with drugs, the positive control group cells were added with positive drug DiDOX, and the experimental group cells were added with solutions of different concentrations of compounds. After culturing the cells for 24 h, 20 μL of 2 mg mL -1 MTT solution was added and cultured for 2-3 h. The supernatant in the culture medium was discarded and 100 μL DMSO was added. The cells were gently shaken for about 10 min in a dark place until the crystals dissolved. The optical density (OD) value of each well was detected at a wavelength of 570 nm using a full-wavelength microplate reader to calculate the cell survival rate.

[0060] 2.2.3 Detection of NO release in RAW264.7 cells by Griess method

[0061] RAW264.7 cells in the logarithmic phase were seeded in 96-well plates (1×10 4 The cells were randomly divided into three groups: blank control group (0.1 mL blank culture medium without LPS), model group (0.1 mL 1 μmol·L -1 LPS), experimental group (0.1 mL 1.0 µg mL -1 LPS+100, 50, 25, 12.5, 6.25, 3.125 μg·mL -1 different compounds), positive drug group (add 0.1 mL 1.0 µg mL -1 LPS+50.0, 100.0 µg·mL -1 DiDOX). Continue to culture for 24 h, take 100 μL of the supernatant, add an equal amount of Griess reagent, mix well, let stand at room temperature for 10 min, set the wavelength to 570 nm, measure the absorbance on an enzyme reader, and substitute it into the standard curve ( A = 0.0064 C + 0.0024, R² = 0.9994, where A is the absorbance, C The amount of NO released was calculated.

[0062] 3. Results Analysis

[0063] 3.1 Structure identification

[0064] Compound 1 is a white amorphous powder. α ]25 D = -127.273 ( c0.50, MeOH); UV λMeOH max (nm): 207 (2.04), 278 (0.121); IR νKBr max (cm −1 ): 3445, 2936, 1743, 1636, 1447, 1373, 1312, 1236, 1066, 564, 549, 521. HR-ESI-MS ( m / z )( Fig.10 ): 1031.5427 [M+COOH] - The theoretical value is 1031.5432, with a deviation of 0.5 ppm. The inferred molecular formula is C 50 H 82 O 19 , the unsaturation is 10.

[0065] Both Libermann-Burchard and Molish reactions were positive, suggesting that it was a steroidal compound. 1 H-NMR spectrum ( Figure 3 )and 13 C-NMR spectrum ( Figure 4 ), combined with HSQC spectra ( Figure 7 ), δ C 12.9 and δ H 0.70 (3H, s), δ C 19.9 and δ H 1.00(3H, s), δ C 23.0 and δ H 1.33 (3H, d, J = 6.0 Hz) are C 21 The methyl groups at C-18, C-19 and C-21 of pregnane are typical features; δ C 141.8 (C-5), 122.9 (C-6) and δ H 5.37 (1H, br d, J = 5.4 Hz, H-6) shows that there is a Δ 5 Double bonds; δ C 79.3 and δ H 3.49 (1H, m), δ C 83.6 and δH 3.58 (1H, m) are C-3 and C-20 connected to oxygen methyl groups. Therefore, the core of this component is double oxidized Δ 5 -Pregnene. 1 The H-NMR spectrum shows the terminal proton signals of four sugars in the low field region δ H 4.85 (1H, br d, J = 9.0 Hz), 4.49 (1H, d, J = 8.4 Hz), 4.38 (1H,d, J = 7.8 Hz), 4.66 (1H, d, J = 7.8 Hz), combined with 13 The signals in the sugar region of the C-NMR spectrum suggest that there are four sugar moieties in the structure; 1 The coupling constants of H-NMR spectrum show that the terminal protons are β Configuration( 3 J H1, H2 >7 Hz) [7] ; 1 H-NMR chemical shift values δ H Around 3.40, 3 methoxy signals are displayed δ H 3.43 (3H, s), 3.42 (3H, s), 3.37 (3H, s), in the HMBC spectrum ( Figure 6 ) and the sugar region carbon signal δ C 85.8 (C-3′′′), 78.1 (C-3′), 82.8 (C-3′′), indicating that there are three methoxy groups in the structure of the sugar. 1 H-NMR spectrum and 13 C-NMR spectrum also showed that there is an acetyl group in the structure [ δ C 172.9, 21.1, δ H 2.05 (3H, s)].

[0066] 13 C-NMR carbon spectrum showed 50 carbon signals, combined with DEPT135 ( Figure 5 ) and HSQC ( Figure 7 ) spectrum analysis, the component structure has 10 methyl groups (including 3 methoxy groups), 10 methylene groups (including 1 sp 3 Hybridized oxymethylene, 9 sp 3hybridized methylene), 26 methines (including 1 sp 2 Hybridized methine, 4 sp 3 Hybridized dioxymethylene, 17 sp 3 Hybridized oxymethyl group, 4 sp 3 hybridized methine), 4 quaternary carbons (including 1 carbonyl quaternary carbon, 1 sp 2 Hybridized quaternary carbon, 2 sp 3 Hybridized quaternary carbon). Combined with the literature comparison, the compound should be based on Δ 5 -Pregnene is the mother nucleus, and its structure contains steroidal glycoside components including 1 acetyl group, 1 glucose (glu), 1 cymose (cym), and 2 digitalis sugars (dig).

[0067] From the HMBC spectrum, we can see that the cym terminal proton δ H 4.85 (1H, br d, J = 9.0 Hz) and the steroid nucleus C-3 ( δ C 79.3) There is a long-range correlation, so it is judged that cym is connected to the C-3 position of the steroid nucleus; dig is the terminal proton δ H 4.49 (1H, d, J = 7.8 Hz) and cym's C-4′ ( δ 84.6) has long-range correlation, so it is judged that its connection mode is dig C-1′′→cymC-4′; H-2′′ signal δ H 5.06 (t, J = 8.4 Hz) has a long-range correlation with the carbonyl carbon of the acetyl group, indicating that the acetyl group is connected to the C-2′′ of dig. In the HMBC spectrum, another dig terminal proton signal δ H 4.38 (1H, d, J = 7.8 Hz) and the steroidal nucleus C-20 ( δ C 83.6) is remotely related, so the sugar group is attached to the C-20 position of the steroid nucleus; the glu terminal proton signal δ 4.66 (1H, d, J = 7.8 Hz) and the C-2′′′ of the above dig ( δ C 76.0) has long-range correlation, so its connection mode is glu C-1′′′′→dig C-2′′′.

[0068] Comprehensive analysis Figure 1, 1 H-NMR( Figure 3 )and 13 C-NMR( Figure 4 )、HSQC( Figure 7 )、HMBC( Figure 6 )、 1 H- 1 H COSY( Figure 8 ) and NOESY spectra ( Fig. 9 ), compound 1 was identified as Δ 5 -Pregnene-3 β ,20( S )-diol-3- O -[2- O -Acetyl- β -D-Digitinose-(1→4)- β -D-Canadian hemp glycoside]-20- O -[ β -D-Glucose-(1→2)- β -D-digitonoside]. According to literature search, this compound has not been reported before and is a new compound named periplocoside G1 (Δ 5 -Pregnene-3 β ,20( S )-diol-3- O -[2- O -Acetyl- β -D-Digitinose-(1→4)- β -D-Canadian hemp glycoside]-20- O -[ β -D-Glucose-(1→2)- β -D-digitalin glycoside]), the structural formula is:

[0069]

[0070] Formula 1

[0071] In formula 1: cym: β -D-pyranose; dig: β -D-Digitalinose; glu: β -D-pyranose.

[0072] Combined with one-dimensional and two-dimensional NMR spectra, the component 1 H-NMR and 13 The C-NMR data were assigned and are listed in Table 1.

[0073] Table 1 Compound 1 1 H-NMR and 13C-NMR data (600 / 150 MHz, CD3OD)

[0074]

[0075]

[0076]

[0077] 3.2 Anti-inflammatory activity studies

[0078] 3.2.1 Effects on RAW264.7 cell viability

[0079] According to the method in 2.2.2, the concentration of compound 1 was 3.125~100 μmol·L -1 , the cell viability was above 80%, with no significant difference, so it had no toxic effect on cells within this concentration range.

[0080] 3.2.2 Determination of NO content in LPS-stimulated RAW264.7 cells

[0081] The results are shown in the following table. Figure 2 Compound 1 has certain anti-inflammatory activity and shows significant dose-dependency. -1 The effect of positive drug DiDOX 100 μmol·L -1 quite.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A C 21 Steroidal saponin, characterized in that The structural formula is shown in Formula 1: Formula 1: ; Where cym: β -D-pyranose; dig: β -D-Digitalinose; glu: β -D-pyranose.

2. A C as claimed in claim 1 21 The method for preparing steroidal saponin is characterized in that: The following steps are involved: (1) crushing the Cortex Periplocae, adding a solvent, heating and refluxing for extraction, combining the extracts, and recovering under reduced pressure to obtain an ethanol extract; (2) dispersing the ethanol extract with water, extracting with ether, ethyl acetate and n-butanol in sequence, and concentrating under reduced pressure to obtain ether extract phase, ethyl acetate extract phase and n-butanol extract phase respectively; (3) The n-butanol extract was separated by an MCI resin column and gradient eluted to obtain 7 fractions: fraction 1 to fraction 7; (4) fraction 3 was subjected to gradient elution through a silica gel column chromatography column, and the same components were detected by TLC and combined to obtain 22 fractions, Fr. A to V; (5) Fraction Fr. O was subjected to gradient elution on a silica gel column to obtain three fractions, Fr. O1 to O3. Fraction Fr. O2 was crystallized to obtain the target compound C. 21 Steroidal saponins; In step (1), the heating reflux extraction is performed by heating 70% ethanol to 70-90°C and reflux extraction for 3 times, each time for 1 hour; In step (3), the gradient elution uses a 40% to 100% methanol aqueous solution; the gradient elution program is: 40%, 50%, 60%, 70%, 80%, 90%, 100%; In step (4), the fraction 3 is the elution portion of the 60% methanol aqueous solution in step (3); In step (4), the gradient elution adopts a dichloromethane-methanol system of 100:1 to 1:1; the gradient of the dichloromethane-methanol gradient elution is: 100:1, 99:1, 98:2, 97:3, 96:4, 95:5, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1, v / v; In step (5), the fraction Fr. O is the eluted portion of the dichloromethane-methanol volume ratio of 97:3 in step (4); In step (5), the gradient elution adopts a dichloromethane-methanol system of 100:1-13:1; the gradient of the dichloromethane-methanol gradient elution is: 100:1, 50:1, 30:1, 15:1, 13:1, v / v; In step (5), the fraction Fr. O2 is the elution portion having a volume ratio of dichloromethane to methanol of 30:

1.

3. A C as claimed in claim 1 21 Application of steroidal saponins in the preparation of anti-inflammatory drugs.

Images