Labdane diterpenoid compound extracted and separated from compound cypress as well as preparation method and application of labdane diterpenoid compound

Abstract

The invention discloses labdane diterpenoid compounds extracted and separated from compound juniperus chinensis as well as a preparation method and application of the labdane diterpenoid compounds, and relates to the technical field of natural plant extraction. The labdane type diterpenoid compound I is obtained through the multi-stage separation steps of ethanol extraction, distilled water dilution, petroleum ether-ethyl acetate extraction, organic solvent gradient elution, liquid chromatography preparation and the like. Pharmaceutical activity experiments show that the labdane diterpenoid compound I has obvious anti-cervical cancer activity, which indicates that the labdane diterpenoid compound I has good application prospects in preparation of drugs for preventing or treating cervical cancer, and the preparation method of the compound is simple, feasible and low in cost.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant extraction, and in particular to a helianthane-type diterpenoid compound extracted and separated from compound golden cypress, and a preparation method and application thereof. Background Art

[0002] Cervicitis is a common gynecological disease, mainly caused by bacterial or viral infection. This inflammation can be acute or chronic. The former is mainly manifested by increased vaginal discharge accompanied by a burning sensation; the latter is mainly manifested by vaginal itching, irregular vaginal bleeding, etc. Cervicitis can induce gynecological inflammation, cause infertility and cause cancer. Clinical studies have shown that there is a close relationship between cervicitis and cervical cancer. Due to the influence of inflammation, the columnar epithelium of the cervical canal will undergo atypical hyperplasia under the long-term stimulation of chronic inflammation. If not treated in time, it will cause local cancers such as cervical cancer.

[0003] Compound Jinbo lotion is a prescription made by Dr. Niu Xiangxin, a famous Chinese medicine doctor in Hainan Province, based on his rich clinical experience, according to the geographical environment, climate and physical characteristics of Hainan locals. It is composed of four Chinese medicines: Cnidium monnieri, Phellodendron chinense, Forsythia suspensa and Stemona radix. It is used to treat cervical cancer caused by HPV infection. In the prescription, Cnidium monnieri is warm in nature, pungent and bitter in taste, and is the main medicine. Stemona radix is ​​sweet and bitter in nature, slightly warm, and is the ministerial medicine. Phellodendron chinense is cold in nature, bitter in taste, and has the effects of clearing heat and dampness, and treating sores. It is an adjuvant. Forsythia suspensa is slightly cold in nature, bitter in taste, and has the effects of clearing heat and detoxifying, reducing swelling and dispersing nodules. It is a guiding medicine. This prescription is one of the Li medicine lotions developed based on traditional Chinese ethnic medicine. There are many studies on the chemical components of the four Chinese medicines: Cnidium monnieri, Phellodendron chinense, Forsythia suspensa and Stemona radix. They are mainly flavonoids (glycosides), diterpenes, alkaloids and other types of compounds. Pharmacological studies have shown that many of these compounds have good biological activities, including anti-inflammatory, antibacterial, antioxidant, anti-tumor, etc., but the effects are not good. The compound golden cypress contains a large number of compounds with biological activity. The present invention aims to extract and separate effective ingredients from the compound golden cypress and study the effective ingredients, in order to obtain new structural compounds with significant anti-cervical cancer effects and give full play to the medicinal value of the compound golden cypress. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention proposes a hemerocalline-type diterpenoid compound extracted and separated from compound golden cypress and a preparation method and application thereof, so as to solve the problems raised by the above-mentioned background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A cymene-type diterpene compound extracted and separated from compound golden cypress, wherein the cymene-type diterpene compound has 21 carbon atoms in its structure, and has a dicarbonyl group and a C-7 hydroxyl group substitution, and the structural formula of the cymene-type diterpene compound is shown in Formula I:

[0007]

[0008] Preferably, the method for extracting and separating hemimeric diterpenoid compounds comprises the following steps:

[0009] (1) crushing the dried compound golden cypress, extracting the compound golden cypress powder with ethanol to obtain an extract, and concentrating under reduced pressure to obtain a crude extract;

[0010] (2) diluting the crude extract of step (1) with water to prepare a suspension, extracting with petroleum ether and ethyl acetate in sequence, combining the organic phases, and concentrating under reduced pressure to obtain an extract;

[0011] (3) The extract obtained in step (2) was first subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate mixed solvent was used as an eluent for gradient elution, and the elution gradient was (100:1) to (1:100) by volume, and a total of 8 components were obtained according to the polarity, namely Fr.1 to Fr.8;

[0012] (4) Fr.3 was first subjected to normal phase silica gel column chromatography, using a dichloromethane-methanol mixed solvent as an eluent for gradient elution, with the elution gradient being a volume ratio of (10:1) to (1:1), and the fractions were concentrated under reduced pressure to obtain components Fr.3A-Fr.3D;

[0013] (5) Fr.3A was subjected to Sephadex LH-20 gel column chromatography with MeOH as the eluent for 3 to 6 column volumes, concentrated under reduced pressure, and then subjected to high performance liquid chromatography (HPLC) to obtain Compound I.

[0014] Preferably, in step (1), the extraction times are 3 or more times, each extraction lasts 1 to 3 hours, and the extracts are combined; in step (2), petroleum ether and ethyl acetate are used for extraction for 3 or more times respectively.

[0015] Preferably, in step (1), the volume fraction of ethanol is 75% to 95%, and the amount used is 3 to 4 L of ethanol per kilogram of compound Jinbo powder.

[0016] Preferably, in step (2), the amount of water used is 200-300 mL per 100 g of crude extract, and the volume of the organic solvent used for each extraction is 1.2-1.3 times the volume of water.

[0017] Preferably, in step (3), the elution gradient of the petroleum ether-ethyl acetate mixed solvent is 100:1, 85:15, 70:30, 60:40, 50:50, 40:60, 20:80, 1:100, 3 to 6 column volumes are collected for each gradient, and one component is obtained for each gradient, for a total of 8 components, namely Fr.1 to Fr.8.

[0018] Preferably, in step (4), the elution gradient of the dichloromethane-methanol mixed solvent is 10:1, 8:1, 5:1, 1:1, and each gradient elution is 2 to 5 column volumes;

[0019] Preferably, in step (5), the conditions of high performance liquid chromatography are: chromatographic column H&E ODS-A C 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O in a volume ratio of 30:70.

[0020] Preferably, the preparation method of the compound golden cypress is to mix the Chinese medicines Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosa in equal mass ratios, add 3 to 5 times the mass of water of the Chinese medicines and boil for 20 to 30 minutes, filter to obtain the filtrate, and decompress and concentrate the filtrate to dryness to obtain the compound golden cypress.

[0021] Preferably, the cymene-type diterpenoid compounds described in the present application are used in the preparation of drugs for preventing or treating cervical cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention uses ethanol extraction, dilution with distilled water, petroleum ether-ethyl acetate extraction, organic solvent gradient elution and liquid chromatography preparation and other multi-stage separation steps to obtain a cymene-type diterpenoid compound I. Through drug activity experiments, it is found that the cymene-type diterpenoid compound I has obvious anti-cervical cancer activity, indicating that it has good application prospects in the preparation of drugs for preventing or treating cervical cancer, and the preparation method of the compound is simple, feasible and low-cost. Among them, compound I shows obvious anti-cervical cancer activity, and the inhibition rate of compound I on SiHa cells at concentrations of 0.025, 0.05 and 0.1 mg / mL is 15.32%, 18.21% and 25.95%, and the inhibition rate on HeLa cells is 16.60%, 20.14% and 27.50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For compound I 1 H-NMR spectrum (MeOD-d4);

[0025] Figure 2 For compound I 13 C-NMR spectrum (MeOD-d4);

[0026] Figure 3 DEPT (135°) spectrum (MeOD-d4) of compound I;

[0027] Figure 4 For compound I 1 H-1H COSY spectrum (MeOD-d4);

[0028] Figure 5 is the HSQC spectrum of compound 1 (MeOD-d4);

[0029] Figure 6 is the HMBC spectrum (MeOD-d4) of compound I;

[0030] Figure 7 is the NOESY spectrum (MeOD-d4) of compound I;

[0031] Figure 8 is the HRESIMS spectrum of compound I;

[0032] Fig. 9 Comparison of the experimental ECD and theoretical ECD of compound I. DETAILED DESCRIPTION

[0033] In order to better understand the technical content of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0034] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0036] Example 1 Preparation of hemiaran-type diterpenoid compounds in compound golden cypress

[0037] The method for separating the helianthane-type diterpenoid compounds in the compound golden cypress comprises the following steps:

[0038] (1) The dried compound golden cypress powder was crushed, and 3 L of ethanol solution (75% by volume) was heated to 40° C. and extracted three times per kg of the compound golden cypress powder, each time for 1 hour, and the extracts were combined and concentrated under reduced pressure to obtain a crude extract (about 280 g);

[0039] (2) After diluting 100 g of the crude extract with 300 mL of distilled water to prepare a suspension, the extract was extracted three times with petroleum ether and ethyl acetate respectively, and the organic phases were combined and concentrated under reduced pressure to obtain an extract; wherein the volume of the organic solvent used in each extraction was 1.3 times the volume of water;

[0040] (3) taking the extract (about 22 g) obtained in step (2), subjecting it to silica gel column chromatography, using a petroleum ether-ethyl acetate mixed solvent for gradient elution, the elution gradient was 100:1, 85:15, 70:30, 60:40, 50:50, 40:60, 20:80, 1:100, collecting 3 column volumes for each gradient, obtaining one component for each gradient, and obtaining a total of 8 components, which were labeled Fr.1 to Fr.8 in sequence;

[0041] (4) Fr.3 was first subjected to normal phase silica gel column chromatography, using a dichloromethane-methanol mixed solvent as an eluent for gradient elution, with an elution gradient of 10:1, 8:1, 5:1, and 1:1, with each gradient elution lasting 3 column volumes, and the fractions were concentrated under reduced pressure to obtain components Fr.3A-Fr.3D;

[0042] (5) Fr.3A was subjected to Sephadex LH-20 gel column chromatography with MeOH as the eluent for 3 column volumes, concentrated under reduced pressure, and then subjected to HPLC to obtain Compound I; wherein the conditions of HPLC were: chromatographic column H&EODS-AC 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O in a volume ratio of 30:70.

[0043] The preparation method of the compound golden cypress is as follows: mix the Chinese medicines Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosa in equal weight ratios, add water three times the weight of the Chinese medicines and boil for 20 minutes, filter to obtain a filtrate, and decompress, concentrate and dry the filtrate to obtain the compound golden cypress.

[0044] Example 2 Preparation of hemiaran-type diterpenoid compounds in compound golden cypress

[0045] The method for separating the helianthane-type diterpenoid compounds in the compound golden cypress comprises the following steps:

[0046] (1) The dried compound golden cypress powder was crushed, and 4 L of ethanol solution (volume fraction 95%) was heated to 50° C. and extracted three times per kg of the compound golden cypress powder, each time for 3 h, and the extracts were combined and concentrated under reduced pressure to obtain a crude extract (about 300 g);

[0047] (2) Taking 400 mL of distilled water per 100 g of the crude extract to dilute it into a suspension, extracting it with petroleum ether and ethyl acetate three times respectively, combining the organic phases, and concentrating them under reduced pressure to obtain an extract; wherein the volume of the organic solvent used in each extraction is 1.2 times the volume of water;

[0048] (3) taking the extract (about 24 g) obtained in step (2), subjecting it to silica gel column chromatography, using a petroleum ether-ethyl acetate mixed solvent for gradient elution, the elution gradient was 100:1, 85:15, 70:30, 60:40, 50:50, 40:60, 20:80, 1:100, collecting 6 column volumes for each gradient, obtaining one component for each gradient, and obtaining a total of 8 components, which were labeled Fr.1 to Fr.8 in sequence;

[0049] (4) Fr.3 was first subjected to normal phase silica gel column chromatography, using a dichloromethane-methanol mixed solvent as an eluent for gradient elution, with an elution gradient of 10:1, 8:1, 5:1, and 1:1, with each gradient elution lasting 5 column volumes, and the fractions were concentrated under reduced pressure to obtain components Fr.3A-Fr.3D;

[0050] (5) Fr.3A was subjected to Sephadex LH-20 gel column chromatography with MeOH as the eluent for 6 column volumes, concentrated under reduced pressure, and then subjected to HPLC to obtain Compound I; wherein the conditions of HPLC were: chromatographic column H&EODS-A C 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O in a volume ratio of 30:70.

[0051] The preparation method of the compound golden cypress is as follows: mix the Chinese medicines Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosa in equal weight ratios, add 5 times the weight of the Chinese medicines in water and boil for 30 minutes, filter to obtain a filtrate, and decompress, concentrate and dry the filtrate to obtain the compound golden cypress.

[0052] Example 3 Structural Identification of the Hemiptane-type Diterpenoids in the Compound Goldenrod

[0053] Using spectroscopy ( 1 H NMR, 13 The chemical structures of the compounds I obtained in Example 1 and Example 2 were determined using modern structural identification techniques such as C NMR, HSQC, HMBC, NOESY and MS.

[0054] The structural identification data are as follows:

[0055] Compound I: Colorless needle-shaped crystals, easily soluble in methanol. High-resolution mass spectrometry HR-ESI-MS m / z 387.2136 [M+Na] + (calcd for C 21 H 32 O5Na + ,387.2142) to determine its molecular formula is C 21 H 32 O5; according to 1 H, 13Its planar structure was determined by C and 2D NMR data, and its absolute configuration was determined by ECD calculation. The skeleton type was a hemipentane-type diterpene containing two carbonyl groups and substituted with a C-7 hydroxyl group. It was named forsuspenoid. 1 H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d4].

[0056] Compound I 1 H-NMR spectrum (MeOD-d4) Figure 1 As shown;

[0057] Compound I 13 C-NMR spectrum (MeOD-d4) Figure 2 As shown;

[0058] The DEPT (135°) spectrum (MeOD-d4) of compound I is as follows Figure 3 As shown;

[0059] Compound I 1 H- 1 H COSY spectrum (MeOD-d4) Figure 4 As shown;

[0060] The HSQC spectrum (MeOD-d4) of compound I is as follows Figure 5 As shown;

[0061] The HMBC spectrum (MeOD-d4) of compound I is as follows Figure 6 As shown;

[0062] The NOESY spectrum (MeOD-d4) of compound I is as follows Figure 7 As shown;

[0063] The HRESIMS spectrum of compound I is as follows: Figure 8 As shown;

[0064] Comparison of the experimental and theoretical ECD of compound I Fig. 9 shown.

[0065] Table 1 Compound I 1 H(400MHz) and 13 C(100MHz) data(MeOD-d4)

[0066]

[0067]

[0068] Through the above analysis, the structure of compound I is determined to be:

[0069]

[0070] Example 4 Pharmacological Activity Experiment

[0071] 4.1 Anti-cervical cancer activity experiment

[0072] 4.1.1 Experimental Materials Cell lines: Human cervical cancer HeLa cells and human cervical squamous carcinoma SiHa cells were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0073] Instruments: flow cytometer (cytoFLEX, Beckman coulter); clean bench (SW-CJ-1FC, Suzhou Group Antai Air Technology Co., Ltd.); CO2 constant temperature incubator (MCO-15AC, Sanyo Electric Co., Ltd., Japan); low-speed centrifuge (5702R, EPPENDORF); cell culture dishes and cell culture bottles (Corning);

[0074] Reagents: Compound I; cell inhibition detection kit (E-CK-A211, Wuhan Elerite Biotechnology Co., Ltd.); fetal bovine serum (FSP500, Ekosai Biotechnology (Taicang) Co., Ltd.); trypsin-EDTA (PB180225, Wuhan Pronosai Life Science Co., Ltd.); others were of analytical grade.

[0075] 4.1.2 Test Method

[0076] Cell treatment: SiHa and HeLa cells in logarithmic growth phase and in good condition were obtained. After trypsin digestion, the cell density was adjusted to 2.5×10 5 / mL, inoculated into a six-well plate, 2mL of cell suspension in each well, cultured in a 37°C, 5% CO2 incubator for 24h, then the culture medium was discarded, and samples of compound I at concentrations of 0.025, 0.05, and 0.1mg / mL were added, respectively, and placed in an incubator for treatment for 24h.

[0077] Detection: Digest the cells with 0.25% trypsin without EDTA, collect and centrifuge (1500rpm, 5min), remove the supernatant, resuspend in PBS buffer and rinse twice, add 500μL Binding Buffer solution to resuspend the cells again, then add 5μL Annexin VFITC and 5μL PI solution, mix well, and set the normal group cells without Annexin VFITC and PI solution. After reacting in the dark at room temperature for 5-15min, detect on the flow cytometer.

[0078] 4.1.3 Test results

[0079] As shown in Table 2:

[0080] Table 2 Inhibition rate of compound I on SiHa cells and HeLa cells

[0081]

[0082] As can be seen from Table 2, as the concentration of compound I increases, its inhibition rate on SiHa cells and HeLa cells also gradually increases. At concentrations of 0.025, 0.05, and 0.1 mg / mL, the inhibition rate of compound I on SiHa cells is 15.32%, 18.21%, and 25.95%, and the inhibition rate on HeLa cells is 16.60%, 20.14%, and 27.50%.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A hemipentane-type diterpenoid compound extracted and separated from compound golden cypress, characterized in that: The structural formula of the hemipentane-type diterpenoid compound is shown in Formula I:

2. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 1, characterized in that: The following steps are involved: (1) crushing the dried compound golden cypress, extracting the compound golden cypress powder with ethanol to obtain an extract, and concentrating under reduced pressure to obtain a crude extract; (2) diluting the crude extract of step (1) with water to prepare a suspension, extracting with petroleum ether and ethyl acetate in sequence, combining the organic phases, and concentrating under reduced pressure to obtain an extract; (3) The extract obtained in step (2) was first subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate mixed solvent was used as an eluent for gradient elution, and the elution gradient was (100:1) to (1:100) by volume, and a total of 8 components were obtained according to the polarity, namely Fr.1 to Fr.8; (4) Fr.3 was first subjected to normal phase silica gel column chromatography, using a dichloromethane-methanol mixed solvent as an eluent for gradient elution, with the elution gradient being a volume ratio of (10:1) to (1:1), and the fractions were concentrated under reduced pressure to obtain components Fr.3A-Fr.3D; (5) Fr.3A was subjected to Sephadex LH-20 gel column chromatography with MeOH as the eluent for 3 to 6 column volumes, concentrated under reduced pressure, and then subjected to high performance liquid chromatography (HPLC) to obtain Compound I.

3. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 2, characterized in that: In step (1), the extraction times are more than 3 times, each extraction lasts for 1 to 3 hours, and the extracts are combined; in step (2), petroleum ether and ethyl acetate are used for extraction more than 3 times each.

4. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 2, characterized in that: In step (1), the volume fraction of the ethanol is 75% to 95%, and the amount used is 3 to 4 L of ethanol per kilogram of compound golden cypress powder.

5. The method for extracting and separating cymene-type diterpenoid compounds according to claim 2, characterized in that: In step (2), the amount of water used is 200-300 mL per 100 g of crude extract, and the volume of the organic solvent used for each extraction is 1.2-1.3 times the volume of water.

6. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 2, characterized in that: In step (3), the elution gradient of the petroleum ether-ethyl acetate mixed solvent is 100:1, 85:15, 70:30, 60:40, 50:50, 40:60, 20:80, 1:100, and 3 to 6 column volumes are collected for each gradient. One component is obtained for each gradient, and a total of 8 components, namely Fr.1 to Fr.8, are obtained.

7. The method for extracting and separating the hemane-type diterpenoid compounds from the compound golden cypress according to claim 2, characterized in that: In step (4), the elution gradient of the dichloromethane-methanol mixed solvent is 10:1, 8:1, 5:1, 1:1, and each gradient elution is 2 to 5 column volumes.

8. The method for extracting and separating the hemane-type diterpenoid compounds from the compound golden cypress according to claim 2, characterized in that: In step (5), the conditions of high performance liquid chromatography are: chromatographic column H&E ODS-A C 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O in a volume ratio of 30:

70.

9. The method for extracting and separating cymene-type diterpenoid compounds according to claim 2, characterized in that: The preparation method of compound golden cypress is as follows: mix the Chinese medicines Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosa in equal weight ratios, add 3 to 5 times the weight of the Chinese medicines in water and boil for 20 to 30 minutes, filter to obtain the filtrate, and decompress and concentrate the filtrate to dryness to obtain the compound golden cypress.

10. Use of the cymene-type diterpenoid compound according to claim 1 or the cymene-type diterpenoid compound prepared by the extraction and separation method according to any one of claims 2 to 9 in preparing a drug for preventing or treating cervical cancer.

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