Separation method of Halimane diterpenoid compound and anti-inflammatory application of Halimane diterpenoid compound

Abstract

The invention provides a separation method of Halimane diterpenoid compounds and anti-inflammatory application of the Halimane diterpenoid compounds, and belongs to the technical field of traditional Chinese medicine separation. The method comprises the following steps of: performing ultrasonic extraction by using petroleum ether to obtain petroleum ether part extract by using the leucas davidii as a raw material; and sequentially carrying out silica gel column chromatography, reversed-phase column separation, gel column chromatography and high performance liquid chromatography (HPLC) separation on the petroleum ether part extract to obtain the target extract Halimane type diterpenoid compound. According to the present invention, the Halimane type diterpenoid compound extracted from the Saposhnikovia divaricata is the aromatized carbon reduction Halimane type diterpenoid compound, and the pharmacological research results show that the Halimane type diterpenoid compound represented by the formula (I) and the Halimane type diterpenoid compound represented by the formula (II) have good anti-inflammatory effects, and the IC50 values of the Halimane type diterpenoid compound represented by the formula (I) and the Halimane type diterpenoid compound represented by the formula (II) are respectively 23.13 + / -0.66 [mu] M and 25.94 + / -1.03 [mu] M;

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine separation, and in particular to a method for separating a halimane diterpene compound and an anti-inflammatory application thereof. Background Art

[0002] Diterpenes are terpene compounds containing four isoprene units, and their skeletons are usually 20 carbon atoms. Many oxygen-containing diterpene derivatives have various biological activities, such as paclitaxel, andrographolide, tanshinone, ginkgolide, triptolide, yuanhualide and stevia, and some of them have become important drugs.

[0003] Chinese patent CN116063313A discloses a Halimane-type diterpene compound in honeycomb grass and its preparation method and application. The Halimane-type diterpene compound in honeycomb grass obtained has a safe and effective effect in lowering blood sugar and can be well used in the preparation of blood sugar-lowering drugs.

[0004] Chinese patent CN112094174B discloses guanane-type diterpene compounds and applications thereof, and specifically discloses that guanane-type diterpene compounds have significant neurotrophic activity and can significantly promote the synaptic growth of PC-12 nerve cells.

[0005] Inflammation is a basic pathological process that occurs when biological tissues are stimulated by certain damaging factors such as trauma, infection, etc., and is mainly a defensive response. The local manifestations of inflammation are redness, swelling, heat, pain and dysfunction, and are also accompanied by systemic reactions such as fever and changes in peripheral white blood cell counts.

[0006] Leucas ciliata Benth. is a plant of the genus Leucas ciliata in the family Lamiaceae. It is also known as hydrangea grass and wind-blowing grass. It is mainly distributed in subtropical areas, including Yunnan, Sichuan, Guangxi and Hainan in my country. The whole plant can be used as medicine, which has the effects of soothing the liver and promoting blood circulation, dispelling wind and improving eyesight, and detoxifying. It is mainly used to treat diseases such as sores, swellings, rashes, and dysentery.

[0007] Studies have found that hydrangea radix windii mainly contains flavonoids (glycosides), phenylpropanoids (glycosides), lignans, triterpenes, diterpenes, phenolic acids and other chemical components, some of which have good biological activity. Hydrangea radix windii is often used for women's blood stasis, amenorrhea, rib pain, and children's eye. However, there are few active compounds found in hydrangea radix windii reported so far.

[0008] The current problem to be solved is to further separate and study the chemical components of hydrangea serrata to obtain new diterpenoid compounds with anti-inflammatory effects. Summary of the invention

[0009] In view of this, the present invention provides a Halimane diterpene compound, which has a good anti-inflammatory effect.

[0010] The Halimanes diterpenoid compounds of the present invention include Compound I and Compound II, and the specific structures are:

[0011]

[0012] The method for separating the halimane diterpenoid compounds of the present invention comprises the following steps:

[0013] (1) The aboveground part of the hydrangea sessilifolia is crushed and then ultrasonically extracted with petroleum ether for 2 to 4 times, the extracts are combined, and the extracts are concentrated under reduced pressure to obtain a petroleum ether extract; the hydrangea sessilifolia extracted with petroleum ether can also be sequentially extracted with ethyl acetate and ethanol to extract other effective components;

[0014] (2) subjecting the petroleum ether extract obtained in step (1) to silica gel column chromatography, using a petroleum ether-ethyl acetate solvent system for gradient elution, collecting fractions of 500 mL each, concentrating each fraction, and combining fractions containing the same components after TLC detection to obtain seven components Fr.1-Fr.7;

[0015] (3) Fr.2 combined in step (2) was separated by reverse phase column with MeOH-H 2 O solvent system was used as the eluent for gradient elution, each gradient elution lasted 3 to 6 column volumes, and then the fractions were collected and the same components were combined to obtain 9 components Fr.2.1-Fr.2.9;

[0016] (4) The combined Fr.2.3 fractions in step (3) were subjected to gel column chromatography, using MeOH as the eluent, collecting fractions of 20 ml each, and analyzed by TLC thin layer chromatography. Similar fractions were combined to obtain four fractions Fr.2.3A-Fr.2.3D;

[0017] (5) The combined Fr.2.3C components in step (4) are separated by high performance liquid chromatography (HPLC) to obtain the target extract, the Halimane-type diterpene compound.

[0018] Preferably, the mass volume ratio of the hydrangea salsa and petroleum ether in step (1) is 1-1.5 kg: 4-5 L.

[0019] Preferably, the gradient elution in step (2) is performed according to a gradient of petroleum ether to ethyl acetate volume ratios of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100.

[0020] Preferably, the elution gradient of the gradient elution in step (3) is MeOH-H 2 The volume ratio of O is 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10.

[0021] Preferably, the parameters of the high performance liquid chromatography in step (5) are: the chromatographic column is H&E ODS-A C 18 , 10×250mm, 5μm, flow rate was 2mL / min, mobile phase was MeCN:H 2 O=60:40.

[0022] Preferably, the gel in step (4) is Sephadex LH-20, and a semi-automatic receiver is used to collect one fraction per 20 ml.

[0023] The invention discloses an application of the Halimanes diterpenoid compounds in the preparation of drugs for preventing or treating inflammation; the inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, enteritis or hepatitis.

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

[0025] The Halimane diterpenoid compound extracted from the hydrangea salviae chinensis of the present invention is an aromatized and carbon-reduced Halimane diterpenoid compound. Pharmacological studies have shown that the Halimane diterpenoid compounds of formula (I) and (II) have good anti-inflammatory effects, and the IC50 values ​​are 23.13±0.66 and 25.94±1.03 μM, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the comparison result between the experimental ECD and the theoretical ECD of compound I;

[0027] Figure 2 is the comparison result between the experimental ECD and the theoretical ECD of compound II;

[0028] Figure 3 The effects of compounds I and II on NO in LPS-induced RAW264.7 cells;

[0029] Figure 4 For compound I 1 H-NMR spectrum (MeOD-d 4 );

[0030] Figure 5 For compound I 13 C-NMR spectrum (MeOD-d 4 );

[0031] Figure 6 DEPT(135°) spectrum of compound I (MeOD-d 4 );

[0032] Figure 7 is the HSQC spectrum of compound I (MeOD-d 4 );

[0033] Figure 8 For compound I 1 H- 1 H COSY spectrum (MeOD-d 4 );

[0034] Fig. 9 is the HMBC spectrum of compound I (MeOD-d 4 );

[0035] Fig.10 NOESY spectrum of compound I (MeOD-d 4 );

[0036] Fig.11 is the HRESIMS spectrum of compound I;

[0037] Fig.12 For compound II 1 H-NMR spectrum (MeOD-d 4 );

[0038] Fig.13 For compound II 13 C-NMR spectrum (MeOD-d 4 );

[0039] Fig.14 DEPT(135°) spectrum of compound II (MeOD-d 4 );

[0040] Fig.15 is the HSQC spectrum of compound II (MeOD-d 4 );

[0041] Fig.16 For compound II 1 H- 1 H COSY spectrum (MeOD-d 4 );

[0042] Fig.17 is the HMBC spectrum of compound II (MeOD-d 4 );

[0043] Fig.18 NOESY spectrum of compound II (MeOD-d4 );

[0044] Fig.19 This is the HRESIMS spectrum of compound II. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with the embodiments.

[0046] Example 1

[0047] A method for separating halimane diterpenoid compounds,

[0048] The Halimanes diterpenoid compounds include Compound I and Compound II:

[0049]

[0050] The method for separating the Halimane diterpenoid compounds comprises the following steps:

[0051] (1) After pulverizing 1.5 kg of the aboveground part of hydrangea serrata, ultrasonically immerse and extract with 4.5 L of petroleum ether for 2 h, immerse for 4 times in succession, combine the extracts from each time, and concentrate under reduced pressure to obtain the petroleum ether extraction part;

[0052] (2) subjecting the petroleum ether extract obtained in step (1) to silica gel column chromatography, using a petroleum ether-ethyl acetate solvent system for gradient elution, collecting fractions of 500 mL each, concentrating each fraction, and combining fractions containing the same components after TLC detection to obtain seven components Fr.1-Fr.7 in increasing polarity;

[0053] The gradient elution is performed according to a gradient of petroleum ether and ethyl acetate in a volume ratio of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100;

[0054] (3) Fr.2 combined in step (2) was separated by reverse phase silica gel column chromatography, and the eluent was MeOH-H 2 O solvent, elution gradient was 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, after each gradient elution for 5 column volumes, the fractions were collected, and after combining the same components, 9 components Fr.2.1-Fr.2.9 with decreasing polarity were obtained;

[0055] (4) The combined Fr.2.3 fractions in step (3) were subjected to gel column chromatography (model: Sephadex LH-20) with MeOH as the eluent and 20 ml fractions were collected by a semi-automatic receiver. After TLC thin layer analysis, similar fractions were combined to obtain four fractions Fr.2.3A-Fr.2.3D with decreasing molecular weights;

[0056] (5) The combined Fr.2.3C fractions from step (4) were separated by high performance liquid chromatography (HPLC) to obtain target extracts of Halimane-type diterpenoid compounds I (4.5 g) and II (5.2 g);

[0057] The parameters of the HPLC are as follows: the chromatographic column is H&E ODS-A C 18 , 10×250mm, 5μm, flow rate was 2mL / min, mobile phase was MeCN:H 2 O=60:40.

[0058] The structures of compound I and compound II prepared in Example 1 were identified to obtain 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data (CD 3 The results of OD are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] The structural identification results of compound I are as follows: Compound I is a colorless oil, easily soluble in methanol. High resolution mass spectrometry HR-ESI-MS m / z 341.2088[M+Na] + (calcd for C 20 H 30 O 3 Na + , 341.2093) to determine its molecular formula is C 20 H 30 O 3 ;according to 1 H, 13 Its structure was determined by C and 2D NMR data, and its absolute configuration was determined to be (4S, 6R, 8R, 9R, 13S) by ECD calculation and testing. Compound I was identified as a ring-aromatic reduced carbon Halimane-type diterpene and was named cilileucapenoid N. 1 H and 13C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d 4 ].

[0063] The comparison results of the experimental ECD and theoretical ECD of compound I are shown in Figure 1 .

[0064] The structural identification results of compound II are as follows: Compound II is a colorless oil, easily soluble in methanol. High resolution mass spectrometry HR-ESI-MS m / z 359.2178[M+Na] + (calcd for C 20 H 32 O 4 Na + , 359.2193) to determine its molecular formula is C 20 H 32 O 4 ;according to 1 H, 13 Its structure was determined by C and 2D NMR data, and its absolute configuration was determined by ECD calculation and testing. It is a reduced-carbon Halimane-type diterpene and was named cilileucapenoid M. 1 H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d 4 ].

[0065] The comparison results of the experimental ECD and theoretical ECD of compound II are shown in Figure 2 .

[0066] Test example:

[0067] The in vitro anti-inflammatory activities of compounds I and II were evaluated.

[0068] 1.1 Experimental Materials:

[0069] Cells: Mouse mononuclear macrophage Raw264.7.

[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] 1.2 Experimental methods:

[0073] (1) Induction: Raw264.7 cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO 2 The cells were cultured in a conventional incubator. 5 / mL, 200 μL / well were inoculated in 96-well plates, and blank control group, LPS-induced group, high, medium and low (50, 25, 12.5 μM) dose groups of the tested drugs were set up respectively. 2 The cells were allowed to adhere to the culture medium for 24 h.

[0074] (2) 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 ELISA reader to measure the OD value at 540 nm.

[0075] The inhibitory activity of compounds I and II on LPS-induced NO release from RAW 264.7 cells is shown in Table 2 ( n=3), it can be seen from the results that compounds I and II showed good anti-inflammatory effects at 25μM and 12.5μM, IC 50 23.13 and 25.94 μM respectively.

[0076] Table 2

[0077] Compound <![CDATA[IC 50 (μM)]]> <![CDATA[CC 50 (μM)]]> I 23.13±0.66 ＞100 II 25.94±1.03 ＞100 Dexamethasone 67.13±0.48 ＞100

[0078] Effects of compounds I and II on LPS-induced NO in RAW264.7 cells Figure 3 . Figure 3 middle: ## P<0.001vs.Con, *** P<0.001 vs. LPS, ** P<0.05vs.LPS.n=3.

[0079] From Table 2 and Figure 3 It can be seen that both compounds I and II showed good anti-inflammatory activity, IC 50 The values ​​were 23.13±0.66 and 25.94±1.03μM, respectively, which were stronger than the positive control drug dexamethasone (IC 50 =67.13±0.48 μM).

[0080] Compound (I) and compound (II) were tested, and the results were as follows Figures 4 to 19 .in Figure 4 For compound I 1 H-NMR spectrum (MeOD-d 4 ); Figure 5 For compound I 13 C-NMR spectrum (MeOD-d4 ); Figure 6 DEPT(135°) spectrum of compound I (MeOD-d 4 ); Figure 7 is the HSQC spectrum of compound I (MeOD-d 4 ); Figure 8 For compound I 1 H- 1 H COSY spectrum (MeOD-d 4 ); Fig. 9 is the HMBC spectrum of compound I (MeOD-d 4 ); Fig.10 NOESY spectrum of compound I (MeOD-d 4 ); Fig.11 is the HRESIMS spectrum of compound I; Fig.12 For compound II 1 H-NMR spectrum (MeOD-d 4 ); Fig.13 For compound II 13 C-NMR spectrum (MeOD-d 4 ); Fig.14 DEPT(135°) spectrum of compound II (MeOD-d 4 ); Fig.15 is the HSQC spectrum of compound II (MeOD-d 4 ); Fig.16 For compound II 1 H- 1 H COSY spectrum (MeOD-d 4 ); Fig.17 is the HMBC spectrum of compound II (MeOD-d 4 ); Fig.18 NOESY spectrum of compound II (MeOD-d 4 ); Fig.19 This is the HRESIMS spectrum of compound II.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A halimane diterpene compound, characterized in that: Including compound I and compound II, the specific structure is:

2. The method for separating the halimane diterpenoid compounds according to claim 1, characterized in that: The following steps are involved: (1) The aboveground part of hydrangea serrata was crushed and ultrasonically extracted with petroleum ether for 2 to 4 times, the extracts were combined, and the extracts were concentrated under reduced pressure to obtain a petroleum ether extract; (2) subjecting the petroleum ether extract obtained in step (1) to silica gel column chromatography, using a petroleum ether-ethyl acetate solvent system for gradient elution, collecting fractions of 500 mL each, concentrating each fraction, and combining fractions containing the same components after TLC detection to obtain seven components Fr.1-Fr.7; (3) Fr.2 combined in step (2) was separated by reverse phase column, and gradient elution was performed using MeOH-H2O solvent system as eluent, each gradient elution lasted 3 to 6 column volumes, and then the fractions were collected and the same components were combined to obtain 9 components Fr.2.1-Fr.2.9; (4) The combined Fr.2.3 fractions in step (3) were subjected to gel column chromatography, using MeOH as the eluent, collecting fractions of 20 ml each, and analyzed by TLC thin layer chromatography. Similar fractions were combined to obtain four fractions Fr.2.3A-Fr.2.3D; (5) The combined Fr.2.3C components in step (4) are separated by high performance liquid chromatography (HPLC) to obtain the target extract, the Halimane-type diterpene compound.

3. The method for separating the Halimane diterpenoid compounds according to claim 2, characterized in that: The mass volume ratio of the hydrangea salsa and petroleum ether in step (1) is 1-1.5 kg: 4-5 L.

4. The method for separating the Halimanes diterpenoid compounds according to claim 2, characterized in that: The gradient elution in step (2) is performed according to a gradient of petroleum ether to ethyl acetate volume ratios of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:

100.

5. The method for separating the Halimanes diterpenoid compounds according to claim 2, characterized in that: The elution gradient of the gradient elution in step (3) is MeOH-H2O with a volume ratio of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10, respectively.

6. The method for separating the Halimanes diterpenoid compounds according to claim 2, characterized in that: The parameters of the high performance liquid chromatography in step (5) are as follows: the chromatographic column is H&E ODS-A C 18 , 10×250mm, 5μm, flow rate was 2mL / min, and the mobile phase was MeCN:H2O=60:

40.

7. The method for separating the Halimanes diterpenoid compounds according to claim 2, characterized in that: The gel in step (4) is Sephadex LH-20, and a semi-automatic receiver is used to collect one fraction per 20 ml.

8. Use of the Halimanes diterpenoid compound according to claim 1 in the preparation of a medicament for preventing or treating inflammation.

9. Use of the Halimanes diterpenoid compound according to claim 8 in the preparation of a drug for preventing or treating inflammation, characterized in that: The inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, enteritis or hepatitis.

Images