Labdane-type diterpenoid compound in leucas davidii as well as separation method and application of labdane-type diterpenoid compound

Abstract

The invention belongs to the technical field of phytochemistry, and discloses labdane diterpenoid compounds in leucas davidii as well as a separation method and application of the labdane diterpenoid compounds. According to the invention, compounds I and II with novel skeleton structures are obtained through multi-stage separation steps of ethanol extraction, distilled water dispersion, polar solvent extraction, organic solvent gradient elution, liquid chromatography preparation and the like. Drug activity experiments find that labdane diterpenoid compounds I and II separated from Saposhnikovia divaricata have obvious anti-inflammatory activity, which indicates that the labdane diterpenoid compounds I and II have good application prospects in preparation of drugs for preventing or treating inflammation. The method is simple, feasible and low in cost.

Description

Technical Field

[0001] The invention relates to the technical field of plant chemistry, and in particular to a hemantanol-type diterpenoid compound in hydrangea salviae raphani and a separation method and application thereof. Background Art

[0002] Leucas ciliata Benth., also known as hydrangea grass and wind-blowing grass, is a herbaceous plant of the genus Leucas ciliata of the Lamiaceae family. It is mainly distributed in subtropical areas and in Guangxi, Sichuan, Guizhou, Yunnan and other places in my country. The whole plant can be used as medicine, which has the effects of soothing the liver and activating blood circulation, dispelling wind and improving eyesight, and detoxifying. It is mainly used to treat women's blood stasis, amenorrhea, flank pain, children's nystagmus, amblyopia, carbuncle, waxberry toxicity, scabies, rashes, etc.

[0003] There are few reports on the chemical composition of hydrangea salviae radix, which are mainly flavonoids (glycosides), diterpenes, phenolic acids 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, α-glucosidase inhibition, etc., providing precursor compound screening for new drug development. Some hemipentane-type diterpenoid compounds have anti-inflammatory effects, but the effect is poor. Hydrangea salviae radix contains a large number of compounds with life activity. The present invention aims to further separate and study the chemical components of hydrangea salviae radix, in order to obtain new structures of hemipentane-type diterpenoid compounds with significant anti-inflammatory effects, and give full play to the medicinal value of hydrangea salviae radix. Summary of the invention

[0004] In view of this, the present invention proposes a hemiaran-type diterpenoid compound in hydrangea salviae chinensis, which has a significant anti-inflammatory effect, and provides a method for separating and applying the target compound.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A hemiaran-type diterpenoid compound from hydrangea radix has a carbon skeleton of 19 carbon atoms and contains a tetrahydrofuran spiro ring, which are compound I and compound II, respectively, and their structures are:

[0007]

[0008] The present invention also provides a method for separating compounds I and II, comprising the following steps:

[0009] (1) grinding the dried stems and leaves of hydrangea scutellariae, extracting the hydrangea scutellariae powder by heating it at 50-60° C. with an ethanol solution to obtain an extract, and concentrating it under reduced pressure to obtain a crude extract;

[0010] (2) diluting the crude extract 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 by ethyl acetate extraction in step (2) was first subjected to silica gel column chromatography, and gradient elution was performed using a dichloromethane-methanol mixed solvent as an eluent, with an elution gradient of (10:1) to (1:10), and a total of 7 components, namely Fr.1 to Fr.7, were obtained according to the polarity;

[0012] (4) Fr.2 was first subjected to normal phase silica gel column chromatography, using a petroleum ether-acetone mixed solvent as an eluent for gradient elution, with an elution gradient of (10:1) to (1:1), and the fractions were concentrated under reduced pressure to obtain components Fr.2A-FR.2D;

[0013] (5) Fr.2A 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 compounds I and II, respectively.

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

[0015] Preferably, in step (1), the volume fraction of the ethanol solution is 75% to 95%, and the amount used is 2 to 3 L of ethanol solution per kilogram of hydrangea scoparia powder.

[0016] Preferably, the amount of water used in step (2) is 300-400 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 dichloromethane-methanol mixed solvent is 20:1, 10:1, 8:1, 5:1, 1:1, 1:5, 1:10, 3 column volumes are collected for each gradient, and one component is obtained for each gradient, for a total of 7 components, namely Fr.1 to Fr.7.

[0018] Preferably, in step (4), the elution gradient of the petroleum ether-acetone 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 for high performance liquid chromatography are: chromatographic column Waters C 18 , the flow rate was 2 mL / min, and the mobile phase was MeCN:H2O in a volume ratio of 58:42.

[0020] Preferably, the hydrangea siler is collected from Bawangling, Hainan.

[0021] The present invention confirms through pharmacological activity experiments that the obtained compounds I and II have good anti-inflammatory effects, indicating that compounds I and II have good application prospects in the preparation of drugs for preventing or treating inflammation.

[0022] The present invention also provides application of the hemimeric diterpenoid compounds in the hydrangea salviae radix in preparing medicines for preventing or treating inflammation.

[0023] Preferably, the inflammation includes systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, enteritis and hepatitis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention extracts and separates novel cymene-type diterpenoid compounds I and II with a carbon skeleton of 19 carbon atoms and containing a tetrahydrofuran spiro ring from hydrangea salviae radix, and the two are a pair of diastereomers. Drug activity experiments show that both compounds have obvious anti-inflammatory effects. The compounds I and II of the present invention are obtained from hydrangea salviae radix extract by a multi-stage separation and extraction method such as solvent extraction, distilled water dissolution and dispersion, polar solvent extraction, solvent gradient elution and liquid chromatography separation, and novel cymene-type diterpenoid compounds with a tetrahydrofuran spiro ring characteristic are obtained from hydrangea salviae radix extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For compound I 1 H-NMR spectrum (MeOD-d4).

[0027] Figure 2 For compound I 13 C-NMR spectrum (MeOD-d4).

[0028] Figure 3 DEPT (135°) spectrum of compound I (MeOD-d4).

[0029] Figure 4 is the HSQC spectrum of compound 1 (MeOD-d4).

[0030] Figure 5 For compound I 1 H- 1 H COSY spectrum (MeOD-d4).

[0031] Figure 6 It is the HMBC spectrum of compound I (MeOD-d4).

[0032] Figure 7 It is the NOESY spectrum of compound I (MeOD-d4).

[0033] Figure 8is the HRESIMS spectrum of compound I.

[0034] Fig. 9 For compound II 1 H-NMR spectrum (MeOD-d4).

[0035] Fig.10 For compound II 13 C-NMR spectrum (MeOD-d4).

[0036] Fig.11 DEPT (135°) spectrum (MeOD-d4) of compound II.

[0037] Fig.12 This is the HSQC spectrum (MeOD-d4) of compound II.

[0038] Fig.13 For compound II 1 H- 1 H COSY spectrum (MeOD-d4).

[0039] Fig.14 This is the HMBC spectrum (MeOD-d4) of compound II.

[0040] Fig.15 This is the NOESY spectrum (MeOD-d4) of compound II.

[0041] Fig.16 This is the HRESIMS spectrum of compound II.

[0042] Fig.17 This is the X-ray single crystal diffraction ORTEP map of compound I (Cu Kα).

[0043] Fig.18 Comparison of the experimental ECD and theoretical ECD of compound I.

[0044] Fig.19 Comparison of theoretical and experimental ECD of compounds I and II.

[0045] Fig. 20 The effects of compounds I and II on the production of NO in LPS-induced RAW264.7 cells. DETAILED DESCRIPTION

[0046] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0047] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0048] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0049] The experimental material of the present invention, the hydrangea sessilifolia, was collected from the hydrangea sessilifolia in Bawangling, Hainan.

[0050] Example 1 Preparation of hemimeric diterpenoid compounds from Saposhnikovia divaricata

[0051] The method for separating hemiaran-type diterpenoid compounds from hydrangea radix siler comprises the following steps:

[0052] (1) The dried stems and leaves of Hydrangea serrata were crushed, and 2 L of ethanol solution (75% by volume) was used for extraction three times at 50-60° C. per kg of Hydrangea serrata powder, each extraction lasting 3 h. The extracts were combined and concentrated under reduced pressure to obtain a crude extract (about 300 g);

[0053] (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; the volume of the organic solvent used for each extraction was 1.2 times the volume of water;

[0054] (3) taking the extract (about 37 g) obtained by ethyl acetate extraction in step (2), and performing gradient elution on a silica gel column using a dichloromethane-methanol mixed solvent as an eluent, the elution gradient being 20:1, 10:1, 8:1, 5:1, 1:1, 1:5, 1:10, collecting 3 column volumes for each gradient, obtaining one component for each gradient, and obtaining a total of 7 components, which were labeled Fr.1 to Fr.7 in sequence;

[0055] (4) Fr.2 was first subjected to normal phase silica gel column chromatography, using a petroleum ether-acetone 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.2A-FR.2D;

[0056] (5) Fr.2A 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 compounds I and II; the conditions for HPLC were: chromatographic column Waters C 18 , the flow rate was 2 mL / min, and the mobile phase was MeCN:H2O in a volume ratio of 58:42.

[0057] Example 2 Structural Identification of Hemiptane-Type Diterpenoids in Saposhnikovia divaricata

[0058] Using spectroscopy ( 1 H NMR, 13The chemical structures of compounds I and II obtained in Example 1 were determined by modern structural identification techniques such as C NMR, HSQC, HMBC, NOESY) and MS.

[0059] The structural identification data are as follows:

[0060] Compound I: Colorless needle-shaped crystals, easily soluble in methanol. High-resolution mass spectrometry HR-ESI-MS m / z 387.2143 [M+Na] + (calcd for C 21 H 32 O5Na + ,387.2147) to determine its molecular formula is C 21 H 32 O5; according to 1 H, 13 C and 2D NMR data and single crystal diffraction ( Fig.17 ) to determine its structure, and the absolute configuration was determined by ECD calculation ( Fig.18 ), the skeleton type is a semi-floral diterpene containing a tetrahydrofuran ring, which is named cilileucapenoid A. 1 H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d4].

[0061] Compound II: Colorless oil, easily soluble in methanol. High resolution mass spectrometry HR-ESI-MS m / z 387.2143 [M+Na] + (calcd for C 21 H 32 O5Na + ,387.2147) to determine its molecular formula is C 21 H 32 O5; according to 1 H, 13 Its structure was determined by C and 2D NMR data, and its absolute configuration was determined by ECD calculations ( Fig.19 ), the skeleton type is consistent with I, which is a semi-floral type diterpene, and is named cilileucapenoid B. 1 H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d4].

[0062] Table 1 Compounds I and II 1 H(400MHz) and13 C(100MHz) data(MeOD-d4)

[0063]

[0064]

[0065] After the above analysis, the structures of compounds I and II are determined as follows:

[0066]

[0067] Example 3 Pharmacological Activity Experiment

[0068] Experimental Materials:

[0069] Cells: Mouse mononuclear macrophage Raw 264.7.

[0070] Cell culture medium: DMEM medium containing 10% fetal bovine serum (FBS) and lipopolysaccharide (LPS) carbohydrate.

[0071] NO detection kit: APPLYGEN, catalog number: E1030.

[0072] Experimental methods:

[0073] Induction: Raw 264.7 cells were routinely cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. 5 / mL, 200μL / well were inoculated in 96-well plates, and blank control group, LPS-induced group, high-, medium-, and low-dose (25, 12.5, and 6.25μM) groups of the test drug were set up respectively, and placed in a 37°C, 5% CO2 cell culture incubator for 24 hours.

[0074] Detection: Pipette 50 μL of supernatant as the test solution into a 96-well plate. According to the detection method in the kit manual, add 50 μL of reagent A and 50 μL of reagent B in sequence, and use an enzyme reader to measure the OD value at 540 nm.

[0075] Table 2 Inhibitory activity of compounds I and II on NO release from RAW 264.7 cells induced by LPS ( n=3)

[0076]

[0077] The test results are shown in Table 2 and Fig. 20 ( ## P<0.001vs.Con, *** P<0.001vs.LPS, **P<0.05 vs. LPS. n=3), compounds I and II showed significant anti-inflammatory effects, IC 50 The values ​​were 9.13±0.84 and 19.63±1.16μM, respectively, which were more active than the positive control drug dexamethasone (IC 50 =67.13±0.48 μM).

[0078] 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 hemiaran-type diterpenoid compound in hydrangea radix siler, characterized in that: The carbon skeleton of the compound has 19 carbon atoms and contains a tetrahydrofuran spiro ring. The structural formula of the compound is shown in Formula I or II:

2. The method for separating hemantanol-type diterpenoid compounds from the herb of claim 1, characterized in that: The following steps are involved: (1) grinding the dried stems and leaves of hydrangea scutellariae, extracting the hydrangea scutellariae powder by heating it at 50-60° C. with an ethanol solution to obtain an extract, and concentrating it 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 by ethyl acetate extraction in step (2) was first subjected to silica gel column chromatography, and gradient elution was performed using a dichloromethane-methanol mixed solvent as an eluent, with an elution gradient of (10:1) to (1:10), and a total of 7 components, namely Fr.1 to Fr.7, were obtained according to the polarity; (4) Fr.2 was first subjected to normal phase silica gel column chromatography, using a petroleum ether-acetone mixed solvent as an eluent for gradient elution, with an elution gradient of (10:1) to (1:1), and the fractions were concentrated under reduced pressure to obtain components Fr.2A-FR.2D; (5) Fr.2A 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 compounds I and II, respectively.

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

4. The method for separating hemimeric diterpenoid compounds from Saposhnikovia divaricata according to claim 2, characterized in that: In step (1), the volume fraction of the ethanol solution is 75% to 95%, and the amount used is 2 to 3 L of ethanol solution per kilogram of hydrangea scoparia powder.

5. The method for separating hemimeric diterpenoid compounds from Saposhnikovia divaricata according to claim 2, characterized in that: In step (2), the amount of water used is 300-400 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 separating hemimeric diterpenoid compounds from Saposhnikovia divaricata according to claim 2, characterized in that: In step (3), the elution gradient of the dichloromethane-methanol mixed solvent is 20:1, 10:1, 8:1, 5:1, 1:1, 1:5, 1:10, 3 column volumes are collected for each gradient, and one component is obtained for each gradient, for a total of 7 components, namely Fr.1 to Fr.

7.

7. The method for separating hemimeric diterpenoid compounds from Saposhnikovia divaricata according to claim 2, characterized in that: In step (4), the elution gradient of the petroleum ether-acetone mixed solvent is 10:1, 8:1, 5:1, 1:1, and each gradient elution is 2 to 5 column volumes; in step (5), the conditions of the high performance liquid chromatography are: chromatographic column Waters C 18 , the flow rate was 2 mL / min, and the mobile phase was MeCN:H2O in a volume ratio of 58:

42.

8. The method for separating hemantanol-type diterpenoid compounds from Saposhnikovia divaricata according to claim 2, characterized in that: The hydrangea siler is collected from Bawangling, Hainan.

9. Use of the hemane-type diterpenoid compounds in the hydrangea salviae radix as claimed in claim 1 in the preparation of a medicament for preventing or treating inflammation.

10. The use according to claim 9, characterized in that: The inflammation includes systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, enteritis and hepatitis.

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