A method for separating halimane diterpenoid compounds and their anti-inflammatory application

Abstract

The present invention provides a method for separating a Halimane-type diterpenoid compound and its anti-inflammatory application, belonging to the technical field of traditional Chinese medicine separation. The present invention uses Hydrangea scutellariae as a raw material, adopts petroleum ether ultrasonic extraction to obtain a petroleum ether extract; then the petroleum ether extract is sequentially subjected to silica gel column chromatography, reverse phase column separation, gel column chromatography, and high performance liquid chromatography HPLC separation to obtain the target extract Halimane-type diterpenoid compound. The Halimane-type diterpenoid compound extracted from Hydrangea scutellariae is an aromatic and carbon-reduced Halimane-type diterpenoid compound. Pharmacological studies have shown that the Halimane-type diterpenoid compounds of formula (I) and (II) have good anti-inflammatory effects, IC 50 The values ​​were 23.13±0.66 and 25.94±1.03 μM, respectively.

Description

Technical Field

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

[0002] Diterpenes are terpenoid compounds containing four isoprene units, typically with a 20-carbon backbone. Many oxygenated diterpenoid derivatives, such as paclitaxel, andrographolide, tanshinone, ginkgolide, triptolide, yuanhuacine, and stevia, possess diverse biological activities, some of which have become important pharmaceuticals.

[0003] Chinese patent CN116063313A discloses a Halimane-type diterpene compound from honeycomb grass, and its preparation method and application. The Halimane-type diterpene compound from honeycomb grass obtained has a safe and effective effect in lowering blood sugar and can be well used in the preparation of hypoglycemic 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 synaptic growth of PC-12 nerve cells.

[0005] Inflammation is a fundamental pathological process that occurs when biological tissues are stimulated by certain damaging factors, such as trauma or infection. It is primarily a defensive response. Local manifestations of inflammation include redness, swelling, heat, pain, and functional impairment, and are also accompanied by systemic reactions such as fever and changes in peripheral white blood cell counts.

[0006] Hydrangea (Leucas ciliata Benth.), also known as hydrangea grass and wind-whispering grass, is a member of the Lamiaceae family. It is primarily found in subtropical regions, including Yunnan, Sichuan, Guangxi, and Hainan in my country. The entire plant can be used as medicine, benefiting from soothing the liver and promoting blood circulation, dispelling wind and improving eyesight, and detoxifying. It is primarily used to treat sores, swellings, rashes, dysentery, and other ailments.

[0007] Studies have found that Saposhnikovia divaricata contains a variety of chemical components, including flavonoids (glycosides), phenylpropanoids (glycosides), lignans, triterpenes, diterpenes, and phenolic acids, some of which have significant biological activity. Saposhnikovia divaricata is commonly used to treat blood stasis, amenorrhea, flank pain, and eye problems in children. However, few active compounds have been reported in Saposhnikovia divaricata.

[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 diterpenoid compound, which has good anti-inflammatory effect.

[0010] The halimane 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 aerial part of the hydrangea radix serrata is crushed and 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 radix serrata extracted with petroleum ether can also be sequentially extracted with ethyl acetate and ethanol to extract other active ingredients;

[0014] (2) The petroleum ether extract obtained in step (1) was subjected 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 7 components Fr.1-Fr.7;

[0015] (3) Fr.2 obtained from step (2) was separated by reverse phase column separation using a MeOH-H2O solvent system as an eluent for gradient elution, with each gradient elution lasting 3 to 6 column volumes. Fractions were then collected and combined to obtain nine fractions, Fr.2.1 to Fr.2.9.

[0016] (4) The combined Fr.2.3 fractions from step (3) were subjected to gel column chromatography using MeOH as the eluent, and 20 ml fractions were collected. After TLC analysis, 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, a 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 and ethyl acetate with a volume ratio 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-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.

[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, and the mobile phase was MeCN:H2O=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 relates to an application of the Halimanes diterpenoid compounds in the preparation of a drug 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 hydrangea salviae chinensis is an aromatized and carbon-reduced halimane-type diterpenoid compound. Pharmacological studies have shown that the halimane-type diterpenoid compounds of formula (I) and (II) have good anti-inflammatory effects, with IC50 values ​​of 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 1;

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

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

[0029] Figure 4 Compound I 1 H-NMR spectrum (MeOD-d4);

[0030] Figure 5 Compound I 13 C-NMR spectrum (MeOD-d4);

[0031] Figure 6DEPT(135°) spectrum (MeOD-d4) of compound 1;

[0032] Figure 7 is the HSQC spectrum of compound 1 (MeOD-d4);

[0033] Figure 8 Compound I 1 H- 1 H COSY spectrum (MeOD-d4);

[0034] Figure 9 is the HMBC spectrum (MeOD-d4) of compound 1;

[0035] Figure 10 is the NOESY spectrum (MeOD-d4) of compound 1;

[0036] Figure 11 is the HRESIMS spectrum of compound 1;

[0037] Figure 12 For compound II 1 H-NMR spectrum (MeOD-d4);

[0038] Figure 13 For compound II 13 C-NMR spectrum (MeOD-d4);

[0039] Figure 14 DEPT(135°) spectrum (MeOD-d4) of compound II;

[0040] Figure 15 is the HSQC spectrum of compound II (MeOD-d4);

[0041] Figure 16 For compound II 1 H- 1 H COSY spectrum (MeOD-d4);

[0042] Figure 17 is the HMBC spectrum (MeOD-d4) of compound II;

[0043] Figure 18 is the NOESY spectrum (MeOD-d4) of compound II;

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

[0045] The present invention will be further described below with reference to the embodiments.

[0046] Example 1

[0047] A method for separating a halimane diterpenoid compound,

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

[0049]

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

[0051] (1) 1.5 kg of the aerial part of hydrangea salsa was crushed and then ultrasonically extracted with 4.5 L of petroleum ether for 2 h. The extraction was repeated four times. The extracts from each extraction were combined and concentrated under reduced pressure to obtain the petroleum ether extract.

[0052] (2) The petroleum ether extract obtained in step (1) was subjected 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 with a volume ratio of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100;

[0054] (3) Fr.2 obtained from step (2) was separated by reverse phase silica gel column chromatography using MeOH-H2O as the eluent with an elution gradient of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. Fractions were collected after 5 column volumes of each gradient elution. After combining the same fractions, 9 fractions Fr.2.1 to Fr.2.9 were obtained in descending order of polarity.

[0055] (4) The combined Fr.2.3 fractions from step (3) were subjected to gel column chromatography (Sephadex LH-20) using MeOH as the eluent. 20 ml fractions were collected using a semi-automatic receiver. After TLC analysis, similar fractions were combined to obtain four fractions, Fr.2.3A to Fr.2.3D, with decreasing molecular weight.

[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, and the mobile phase was MeCN:H2O=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 The results of C-NMR (100 MHz) data (CD3OD) 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 O3Na + , 341.2093) to determine its molecular formula is C 20 H 30 O3; according to 1 H, 13 Its structure was confirmed 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 diterpenoid and named cilileucapenoid N. 1 H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d4].

[0063] The comparison results of the experimental ECD and theoretical ECD of compound I are shown in Figure 2. 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 forC 20 H 32 O4Na + , 359.2193) to determine its molecular formula is C20 H 32 O4; according to 1 H, 13 Its structure was confirmed 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. Its 1H and 13 C NMR data assignments are shown in Table 1. [400 MHz ( 1 H), 100MHz( 13 C), solvent: MeOD-d4].

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

[0066] Test example:

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

[0068] 1.1 Experimental Materials:

[0069] Cells: Mouse monocyte-macrophage Raw264.7 cells.

[0070] Cell culture medium: DMEM medium containing 10% fetal bovine serum (FBS), 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 in a 5% CO2 incubator. 5 / mL, 200μL / well were inoculated into 96-well plates, and blank control group, LPS-induced group, high-, medium-, and low-dose (50, 25, 12.5μM) groups of the test drug were set up respectively, and placed in a 37℃, 5% CO2 cell culture incubator to adhere for 24h.

[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 instructions, add 50 μL of reagent A and 50 μL of reagent B in sequence, and measure the OD value at 540 nm using a microplate reader.

[0075] The inhibitory activities of compounds I and II on LPS-induced NO release from RAW 264.7 cells are shown in Table 2 ( n=3) As shown in the results, 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 compounds I and II both showed good anti-inflammatory activity, IC 50 The values ​​were 23.13±0.66 and 25.94±1.03 μM, 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 Compound I 1 H-NMR spectrum (MeOD-d4); Figure 5 Compound I 13 C-NMR spectrum (MeOD-d4); Figure 6 DEPT(135°) spectrum (MeOD-d4) of compound 1; Figure 7 is the HSQC spectrum of compound 1 (MeOD-d4); Figure 8 Compound I 1 H- 1 H COSY spectrum (MeOD-d4); Figure 9 is the HMBC spectrum (MeOD-d4) of compound 1; Figure 10 is the NOESY spectrum (MeOD-d4) of compound 1; Figure 11 is the HRESIMS spectrum of compound 1; Figure 12 For compound II 1 H-NMR spectrum (MeOD-d4); Figure 13 For compound II 13 C-NMR spectrum (MeOD-d4); Figure 14 DEPT(135°) spectrum (MeOD-d4) of compound II; Figure 15 is the HSQC spectrum of compound II (MeOD-d4); Figure 16 For compound II 1 H- 1 H COSY spectrum (MeOD-d4); Figure 17 is the HMBC spectrum (MeOD-d4) of compound II; Figure 18 is the NOESY spectrum (MeOD-d4) of compound II; Figure 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

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

2. The method for separating the Halimane diterpenoid compound according to claim 1, wherein The following steps are involved: (1) The aerial parts of the hydrangea phyllostachys were crushed and ultrasonically extracted with petroleum ether for 2 to 4 times. The extracts were combined and concentrated under reduced pressure to obtain the petroleum ether extract. (2) The petroleum ether extract obtained in step (1) was subjected 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 7 components Fr.1-Fr.7; (3) Fr.2 obtained from step (2) was separated by reverse phase column separation using a MeOH-H2O solvent system as the eluent for gradient elution, with each gradient elution lasting 3 to 6 column volumes. The fractions were then collected and combined to obtain nine fractions, Fr.2.1 to Fr.2.

9. (4) The combined Fr.2.3 fractions from step (3) were subjected to gel column chromatography using MeOH as the eluent, and 20 ml fractions were collected. After TLC analysis, similar fractions were combined to obtain four fractions, Fr.2.3A-Fr.2.3D. (5) Separating the combined Fr.2.3C fractions from step (4) by high performance liquid chromatography (HPLC) to obtain the target extract, a Halimane-type diterpene compound; The mass volume ratio of the hydrangea salsa and petroleum ether in step (1) is 1-1.5 kg: 4-5 L; The gradient elution in step (2) is performed according to a gradient of petroleum ether and ethyl acetate with a volume ratio of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100; 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; The parameters of the high performance liquid chromatography in step (5) are as follows: the chromatographic column is H&E ODS-AC18, 10×250 mm, 5 μm, the flow rate is 2 mL / min, and the mobile phase is MeCN:H2O=60:

40.

3. The method for separating the Halimane diterpenoid compound according to claim 2, wherein: The gel in step (4) is Sephadex LH-20, and a semi-automatic receiver is used to collect one fraction per 20 ml.

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

5. Use of the Halimane diterpenoid compound according to claim 4 in the preparation of a medicament for preventing or treating inflammation, characterized in that: The inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis, enteritis or hepatitis.

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