A hemanthanoid-type diterpenoid compound extracted and separated from compound golden cypress, and its preparation method and application

Abstract

The present invention discloses a cymene-type diterpenoid compound extracted and separated from the compound cymene, as well as its preparation method and application, relating to the technical field of natural plant extraction. The present invention subjects the compound cymene to a multi-stage separation process, including ethanol extraction, dilution with distilled water, extraction with petroleum ether and ethyl acetate, gradient elution with an organic solvent, and liquid chromatography preparation, to obtain a cymene-type diterpenoid compound I. Pharmaceutical activity experiments revealed that the cymene-type diterpenoid compound I exhibits significant anti-cervical cancer activity, demonstrating its promising application in the preparation of drugs for the prevention or treatment of cervical cancer. Furthermore, the compound preparation method is simple, feasible, and inexpensive.

Description

Technical Field

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

[0002] Cervicitis is a common gynecological disease, primarily caused by bacterial or viral infection. This inflammation can be acute or chronic. The former is characterized by increased vaginal discharge accompanied by a burning sensation, while the latter is characterized by vaginal itching and irregular vaginal bleeding. Cervicitis can induce gynecological inflammation, infertility, and cancer. Clinical studies have shown a close relationship between cervicitis and cervical cancer. Due to the effects of inflammation, the columnar epithelium of the cervical canal, under the long-term stimulation of chronic inflammation, develops atypical hyperplasia. If not promptly treated, it can lead to localized cancers such as cervical cancer.

[0003] Compound Jinbo Lotion, developed by Dr. Niu Xiangxin, a renowned Chinese medicine practitioner in Hainan Province, draws on his extensive clinical experience and is based on Hainan's geographical environment, climate, and the physical characteristics of the local population. It is composed of four Chinese herbs: Cnidium monnieri, Phellodendron amurense, Forsythia suspensa, and Stemona radix. It is used to treat cervical cancer caused by HPV infection. Cnidium monnieri is warm in nature, pungent and bitter in flavor, and serves as the main herb. Stemona radix is ​​sweet, bitter, and slightly warm in nature, and serves as the auxiliary herb. Phellodendron amurense is cold in nature, bitter in flavor, and has the effects of clearing heat and dampness, as well as treating sores, and serves as the adjuvant. Forsythia suspensa is slightly cold in nature, bitter in flavor, and has the effects of clearing heat and detoxifying, reducing swelling and dispersing nodules, and serves as the guiding herb. This recipe is one of the Liyao lotions developed based on traditional Chinese folk medicine. The chemical composition of these four herbs, Cnidium monnieri, Phellodendron amurense, Forsythia suspensa, and Stemona radix, has been extensively studied, primarily containing a variety of compounds, including flavonoids (glycosides), diterpenes, and alkaloids. Pharmacological studies have shown that many of these compounds possess promising biological activities, including anti-inflammatory, antibacterial, antioxidant, and anti-tumor properties, but the efficacy has been limited. Compound Golden Cypress contains a large number of biologically active compounds. The present invention aims to extract and separate the active ingredients from the compound Golden Cypress and study the active ingredients in order to obtain new structures of 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 hemipentane-type diterpenoid compound extracted and separated from compound golden cypress, and a preparation method and application thereof, 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 diterpenoid compound extracted and separated from compound golden cypress. The cymene-type diterpenoid compound has 21 carbon atoms in its structure and is substituted with a dicarbonyl group and a hydroxyl group at the C-7 position. The structural formula of the cymene-type diterpenoid compound is shown in Formula I:

[0007]

[0008] Preferably, the method for extracting and separating hemicyclane-type 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, with the elution gradient being a volume ratio of (100:1) to (1:100), and a total of 8 components, namely Fr.1 to Fr.8, were obtained according to the polarity;

[0012] (4) Fr.3 was first subjected to normal phase silica gel column chromatography using a dichloromethane-methanol mixed solvent as the eluent for gradient elution with a volume ratio of (10:1) to (1:1). 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 eluent for 3 to 6 column volumes, concentrated under reduced pressure, and then subjected to HPLC to obtain Compound I.

[0014] Preferably, in step (1), the extraction is performed more than three 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 three times each.

[0015] Preferably, 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 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 in 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, and 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 are obtained, 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, and 1:1, with each gradient elution lasting 2 to 5 column volumes;

[0019] Preferably, in step (5), the conditions for 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 with 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 tuberosum in equal mass ratios, add 3 to 5 times the mass 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.

[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 present invention subjects compound cyperus to multiple separation steps, including ethanol extraction, dilution with distilled water, extraction with petroleum ether and ethyl acetate, gradient elution with an organic solvent, and liquid chromatography preparation, to obtain a cyperane-type diterpenoid compound I. Pharmaceutical activity experiments revealed that the cyperane-type diterpenoid compound I exhibits significant anti-cervical cancer activity, demonstrating its promising application in the preparation of drugs for the prevention or treatment of cervical cancer. Furthermore, the compound preparation method is simple, feasible, and low-cost. Compound I exhibits significant anti-cervical cancer activity, with inhibition rates of 15.32%, 18.21%, and 25.95% against SiHa cells and 16.60%, 20.14%, and 27.50% against HeLa cells at concentrations of 0.025, 0.05, and 0.1 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0026] Figure 3 DEPT(135°) spectrum (MeOD-d4) of compound 1;

[0027] Figure 4 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 1;

[0030] Figure 7 is the NOESY spectrum (MeOD-d4) of compound 1;

[0031] Figure 8 is the HRESIMS spectrum of compound 1;

[0032] Figure 9 Comparison of the experimental ECD and theoretical ECD of compound 1. 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 examples of the present invention are all conventional methods.

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

[0036] Example 1 Preparation of hemanane-type diterpenoid compounds from compound golden cypress

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

[0038] (1) The dried compound golden cypress powder was crushed and extracted three times with 3 L of ethanol solution (75% by volume) heated to 40°C per kg of compound golden cypress powder, each extraction lasting 1 h. 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 three times with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain an extract; the volume of the organic solvent used in each extraction was 1.3 times the volume of water.

[0040] (3) The extract obtained in step (2) (about 22 g) was subjected 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, and 1:100. Three column volumes were collected for each gradient, and one fraction was obtained for each gradient. A total of eight fractions were obtained, and the eight fractions 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 the eluent for gradient elution. The elution gradient was 10:1, 8:1, 5:1, and 1:1, with each gradient elution lasting 3 column volumes. The fractions were concentrated under reduced pressure to obtain components Fr.3A-Fr.3D.

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

[0043] The preparation method of the compound golden cypress comprises the following steps: mixing the Chinese medicinal herbs Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosum in equal weight ratios, adding water three times the weight of the Chinese medicinal herbs and boiling for 20 minutes, filtering to obtain a filtrate, and concentrating the filtrate under reduced pressure to dryness to obtain the compound golden cypress.

[0044] Example 2 Preparation of hemipentane-type diterpenoid compounds from compound golden cypress

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

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

[0047] (2) After diluting 100 g of the crude extract with 400 mL of distilled water to prepare a suspension, the extract was extracted three times with petroleum ether and three times with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain an extract; the volume of the organic solvent used in each extraction was 1.2 times the volume of water.

[0048] (3) The extract obtained in step (2) (about 24 g) was subjected 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, and 1:100. Six column volumes were collected for each gradient, and one fraction was obtained for each gradient. A total of eight fractions were obtained, and the eight fractions 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 the eluent for gradient elution. The elution gradient was 10:1, 8:1, 5:1, and 1:1, with 5 column volumes for each gradient elution. 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 eluent for 6 column volumes, concentrated under reduced pressure, and then subjected to HPLC to obtain compound I; wherein the HPLC conditions were: chromatographic column H&EODS-A C 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O with a volume ratio of 30:70.

[0051] The preparation method of the compound golden cypress comprises the following steps: mixing the Chinese medicinal herbs Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosum in equal weight ratios, adding water 5 times the weight of the Chinese medicinal herbs and boiling for 30 minutes, filtering to obtain a filtrate, and concentrating the filtrate under reduced pressure to dryness to obtain the compound golden cypress.

[0052] Example 3 Structural Identification of Hemiflorin-Type Diterpenoids in Compound Golden Cypress

[0053] Using spectrum ( 1 H NMR, 13 The chemical structures of Compound 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 forsuspenoid-type diterpenoid containing two carbonyl groups and 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 1 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 Figure 9 shown.

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

[0066]

[0067]

[0068] After the above analysis, the structure of compound I was 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 Punosai 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 flasks (Corning);

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

[0075] 4.1.2 Test Method

[0076] Cell treatment: SiHa and HeLa cells in good condition in the logarithmic growth phase were obtained, digested with trypsin, and the cell density was adjusted to 2.5×10 5 Cells were plated at 200 nm and 200 nm, respectively, inoculated into six-well plates with 2 mL of cell suspension per well. The plates were cultured in a 37°C, 5% CO2 incubator for 24 h, then the culture medium was discarded and 0.025, 0.05, and 0.1 mg / mL concentrations of compound I were added, respectively, and placed in the incubator for 24 h.

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

[0078] 4.1.3 Test results

[0079] As shown in Table 2:

[0080] Table 2 Inhibitory effect 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 principles of the present invention should be included in the scope of protection 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 helianthane-type diterpenoid compound is shown in Formula I:

2. The method for extracting and separating hemane-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; The preparation method of the compound golden cypress is as follows: the Chinese medicines Cnidium monnieri, Forsythia suspensa, Phellodendron chinense and Stemona tuberosum are mixed in equal weight ratios, decocted with water 3 to 5 times the weight of the Chinese medicines for 20 to 30 minutes, filtered to obtain a filtrate, and concentrated and dried the filtrate under reduced pressure to obtain the compound golden cypress; (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. The elution gradient was a volume ratio of 100:1, 85:15, 70:30, 60:40, 50:50, 40:60, 20:80, and 1:

100. 3 to 6 column volumes were collected for each gradient, and one component was obtained for each gradient. A total of 8 components were obtained according to 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 the eluent for gradient elution with a volume ratio of 10:1 to 1:

1. 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 eluent for 3 to 6 column volumes, concentrated under reduced pressure, and then prepared by high performance liquid chromatography (HPLC) to obtain a hemi-petroleum diterpenoid compound.

3. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 2, characterized in that: In step (1), the extraction is performed more than three 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 three 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 hemerane-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 in 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 (4), the elution gradient of the dichloromethane-methanol mixed solvent is 10:1, 8:1, 5:1, and 1:1, with each gradient elution lasting 2 to 5 column volumes.

7. The method for extracting and separating hemerane-type diterpenoid compounds according to claim 2, characterized in that: In step (5), the HPLC conditions are: chromatographic column H&E ODS-AC 18 , the flow rate was 2 mL / min, and the mobile phase was CH3OH:H2O with a volume ratio of 30:

70.

8. 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 7 in the preparation of a drug for preventing or treating cervical cancer.

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