Callicarpa nudiflora leaf extract, labdane diterpenoid compound and preparation method and application of labdane diterpenoid compound

Abstract

The invention discloses a callicarpa nudiflora leaf extract, a labdane type diterpenoid compound as well as a preparation method and application of the labdane type diterpenoid compound, and a new 3, 4-split ring labdane type diterpenoid compound I containing a straight chain type monoterpenoid fragment is separated from the callicarpa nudiflora leaf extract. The compound I and the callicarpa nudiflora leaf extract both have the effects of resisting inflammation and inhibiting activity of alpha-glucosidase.

Description

Technical Field

[0001] The invention relates to a natural plant extract, in particular to a Callicarpa nudiflora leaf extract, a hesperanoid diterpenoid compound, and a preparation method and application thereof. Background Art

[0002] Callicarpa nudiflora Callicarpa nudiflora Hook. et Arn. ), also known as wind-driving firewood and red knotweed, is a member of the Verbenacase family ( Callicarpa ) plant. In my country, it is mainly distributed in coastal areas such as Guangdong, southern Guangxi, and Hainan Province. Callicarpa nudiflora is a typical representative of Hainan Li medicine. It was once a national protected Chinese medicine variety and the only new medicinal material variety added to the 2020 edition of the Chinese Pharmacopoeia. Callicarpa nudiflora is mainly used to dispel wind and promote blood circulation, detoxify and induce vomiting, and relieve inflammation and pain. It is often used clinically for various postoperative bleeding diseases in dermatology, gynecology, surgery, etc.

[0003] The research reports on the chemical components of Callicarpa nudiflora at home and abroad are mainly on leaves, including flavonoids (glycosides), triterpenes, diterpenes, phenylethanol (glycosides), etc. Some compounds show good biological activity. Zhao Xue-Rong (Nudifloids AN,structurally diverse3,4- seco -labdane diterpenoids from Callicarpa nudiflora with inflammatory inhibitory activity. Phytochemistry. 210:113665.) reported the anti-inflammatory effects of diterpenes in Callicarpa nudiflora; Chen Qian-Qian (Isolation and identification of 3,4- seco -labdane diterpenoids from Callicarpa nudiflora and investigation of their cytotoxicity against HepG2 cells. Phytochemistry.2023, 213:113773.) reported the anti-hepatocarcinogenic effects of diterpenes in Callicarpa nudiflora.

[0004] On June 30, 2020, the applicant applied for "A cleaved ring hemicyclane diterpenoid compound and its separation method and application" (application number 202010613626.5), and extracted new hemicyclane diterpenoid compounds A1-A3 with 3,4-ring cleavage and carbon reduction characteristics from Callicarpa nudiflora, and compound A1 has a C13-C17 cyclized new carbon skeleton feature. The compounds in this invention have certain anti-tumor activity, have obvious inhibitory effects on human liver cancer, gastric cancer or ovarian cancer cells, and can be used to prepare anti-tumor drugs.

[0005] The applicant also applied for "A kind of hemi-florinane diterpenoid compound and its separation method and application" (application number 202010615804.8) on June 30, 2020, and extracted and separated new skeleton hemi-florinane diterpenoid compounds A1 and A2 from the leaves of Callicarpa nudiflora. The two are a pair of diastereomers, which is the first discovery of such carbon skeleton structure. The drug activity experiment shows that the compound of the invention has obvious anti-inflammatory activity.

[0006] At present, few diterpenoid compounds with hypoglycemic effect have been found in Callicarpa nudiflora. The present invention aims to further separate and study the chemical components of Callicarpa nudiflora in order to obtain new diterpenoid compounds with anti-hyperglycemic effect. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a Callicarpa nudiflora leaf extract, a hemimerican diterpenoid compound and a preparation method and application thereof.

[0008] The technical solution to achieve the first object of the present invention is a hemiaran-type diterpenoid compound, the structural formula of which is as follows: (I).

[0009] The technical solution for achieving the second object of the present invention is the use of the compound I as described above in the preparation of anti-inflammatory, therapeutic and / or preventive drugs for diabetes.

[0010] The technical solution for achieving the third object of the present invention is a method for preparing compound I as described above, comprising the following steps: ① After the dried leaves of Callicarpa nudiflora are crushed, ultrasonic extraction is performed with dichloromethane for 2 to 4 times, the extracts are combined, and vacuum concentration is performed to obtain an extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora.

[0011] ② The extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora obtained in step ① is subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate solvent system is used for elution according to a gradient of a volume ratio of 100:0 to 0:100. The fraction of the petroleum ether-ethyl acetate solvent system of 60:40 (V / V) is concentrated by TLC detection to obtain a crude extract of the leaves of Callicarpa nudiflora.

[0012] ③ The crude extract of the leaves of Callicarpa nudiflora was separated by ODS column chromatography, the eluent was MeOH-H2O solvent, and the elution was performed according to the gradient of volume ratio of 5:95 to 100:0. After TLC detection, 8 components Fr.1-8 were obtained after combining the same components, and Fr.1-Fr.8 were combined to obtain the extract of the leaves of Callicarpa nudiflora.

[0013] ④ Fr.4 obtained in step ③ was first chromatographed on a normal phase silica gel column with a mixed system of dichloromethane and acetone as the eluent, eluted with a gradient of a volume ratio of 10:0 to 5:5 for 3 to 6 column volumes, and the fractions were collected and combined to obtain 7 components Fr.4a-Fr.4g.

[0014] ⑤ The Fr.4c obtained in step ④ was separated and prepared by high performance liquid chromatography HPLC, and the chromatographic column was Waters C 18 (9.4×250 mm, 4.6 μm), the mobile phase was MeCN:H2O=55:45, and compound I was separated.

[0015] In the above step ①, the amount of dichloromethane used is 1 to 1.2 L per kilogram of Callicarpa nudiflora leaf powder, and the ultrasonic extraction is performed for 2 to 3 hours each time.

[0016] Optionally, in the above step ②, fractions are collected every 3000 mL.

[0017] Optionally, in the above step ③, every 2000 mL fraction is collected.

[0018] The technical solution to achieve the fourth purpose of the present invention is a Callicarpa nudiflora leaf extract, which is obtained by extraction according to the following method: ① After the dried leaves of Callicarpa nudiflora are crushed, ultrasonic extraction is performed with dichloromethane for 2 to 4 times, the extracts are combined, and vacuum concentration is performed to obtain an extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora.

[0019] ② The extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora obtained in step ① is subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate solvent system is used for elution according to a gradient of a volume ratio of 100:0 to 0:100. The fraction of the petroleum ether-ethyl acetate solvent system of 60:40 (V / V) is concentrated by TLC detection to obtain a crude extract of the leaves of Callicarpa nudiflora.

[0020] ③ The crude extract of the leaves of Callicarpa nudiflora was separated by ODS column chromatography, the eluent was MeOH-H2O solvent, and the elution was performed according to the gradient of volume ratio of 5:95 to 100:0. After TLC detection, 8 components Fr.1-8 were obtained after combining the same components, and Fr.1-Fr.8 were combined to obtain the extract of the leaves of Callicarpa nudiflora.

[0021] In the above extraction step ①, the amount of dichloromethane used is 1 to 1.2 L per kilogram of Callicarpa nudiflora leaf powder, and the ultrasonic extraction is performed for 2 to 3 hours each time.

[0022] In the above extraction step ②, fractions were collected every 3000 mL; in the extraction step ③, fractions were collected every 2000 mL.

[0023] The technical solution for achieving the fifth object of the present invention is the use of the Callicarpa nudiflora leaf extract as described above in the preparation of anti-inflammatory, therapeutic and / or preventive diabetes drugs.

[0024] The present invention has positive effects: The invention uses the leaves of Callicarpa nudiflora as raw materials and dichloromethane as an extraction solvent to prepare a dichloromethane extraction part of the leaves of Callicarpa nudiflora, and the extraction part is eluted by a petroleum ether-ethyl acetate solvent and a methanol-water solvent, and then subjected to silica gel and ODS column chromatography to prepare a Callicarpa nudiflora extract. The Callicarpa nudiflora leaf extract has the effects of anti-inflammatory, anti-tumor and treatment of diabetes.

[0025] Furthermore, the present invention isolates a new 3,4-cleaved ring-containing hemiaran-type diterpenoid compound I from the leaf extract of Callicarpa nudiflora, which has anti-inflammatory and α-glucosidase inhibitory activity and has application prospects in the preparation of anti-inflammatory and diabetes drugs for treatment and / or prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For compound I 1 H-NMR spectrum (CD3OD- d 4).

[0027] Figure 2 For compound I 13 C-NMR spectrum (CD3OD- d 4).

[0028] Figure 3 DEPT (135°) spectrum of compound I (CD3OD- d 4).

[0029] Figure 4 For compound I 1 H- 1 H COSY spectrum (CD3OD- d 4).

[0030] Figure 5 is the HSQC spectrum of compound I (CD3OD- d 4).

[0031] Figure 6 is the HMBC spectrum of compound I (CD3OD-d 4).

[0032] Figure 7 NOESY spectrum of compound I (CD3OD- d 4).

[0033] Figure 8 is the HR-ESIMS spectrum of compound I. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0035] (Example 1, extracts and compounds) The structural formula of the hemipentane-type diterpenoid compound of this embodiment is as follows: (Ⅰ), abbreviated as Compound Ⅰ.

[0036] The compound I is isolated from the extract of the leaves of Callicarpa nudiflora.

[0037] The preparation method of the compound I comprises the following steps: ① After 5 kg of dried Callicarpa nudiflora leaves were crushed, 6 L of dichloromethane was added (the amount of dichloromethane per kilogram of Callicarpa nudiflora leaf powder was 1 to 1.2 L, 1.2 L in this embodiment), and ultrasonic extraction was performed at room temperature (10°C to 35°C) for 2 to 4 times (4 times in this embodiment), each time for 2 to 3 hours (2 hours in this embodiment), the extracts were combined, and the extracts were concentrated under reduced pressure to obtain an extract of the dichloromethane extraction part of Callicarpa nudiflora leaves.

[0038] ② The extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora obtained in step ① was subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate solvent system was used for elution according to an elution gradient of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. The fractions were collected every 3000 mL, and the fractions were concentrated. The fractions were detected by TLC, and the fractions of 60:40 (V / V) of the petroleum ether-ethyl acetate solvent system were concentrated to obtain a crude extract of Callicarpa nudiflora leaves.

[0039] ③ The crude extract of the leaves of Callicarpa nudiflora was separated by ODS column chromatography, the eluent was MeOH-H2O solvent, and the elution was carried out according to the gradient of 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 (V / V), and the fractions were collected every 2000 mL. The fractions were concentrated and detected by TLC. After combining the components with the same polarity, 8 components Fr.1-8 were obtained, and Fr.1-Fr.8 were combined to obtain the leaf extract of Callicarpa nudiflora.

[0040] ④ Fr.4 obtained in step ③ was first chromatographed on a normal phase silica gel column with a mixed system of dichloromethane and acetone as the eluent, and eluted with a gradient of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5 (V / V) for 3 to 6 (3 in this embodiment) column volumes. The fractions were collected according to their polarity, and 7 components Fr.4a-Fr.4g were obtained by combining the same components.

[0041] ⑤ The Fr.4c obtained in step ④ was separated and prepared by high performance liquid chromatography HPLC, and the chromatographic column was Waters C 18 (9.4×250 mm, 4.6 μm), the flow rate was 2 mL / min, the mobile phase was MeCN:H2O=55:45, and compound I was separated.

[0042] Using spectroscopy ( 1 H NMR, 13 C NMR, HSQC, HMBC) and MS and other modern structural identification techniques were used to determine the chemical structure of compound I.

[0043] Compound I 1 H-NMR spectrum (CD3OD- d 4) See Figure 1 ; Compound I 13 C-NMR spectrum (CD3OD- d 4) See Figure 2 DEPT(135°) spectrum of compound I (CD3OD- d 4) See Figure 3 ; Compound I 1 H- 1 H COSY spectrum (CD3OD- d 4) See Figure 4 HSQC spectrum of compound I (CD3OD- d 4) See Figure 5 ; HMBC spectrum of compound I (CD3OD- d 4) See Figure 6 ; NOESY spectrum of compound I (CD3OD- d4) See Figure 7 ; The HR-ESIMS spectrum of compound I is shown in Figure 8 .

[0044] The structural identification data are as follows: Compound I: Colorless oil, easily soluble in methanol or chloroform.

[0045] HR-ESIMS m / z 527.3345 [M + Na] + (calcd for C 30 H 48 O6Na,527.3343) to determine its molecular formula is C 30 H 48 O6.

[0046] according to 1 H, 13 Its structure was confirmed by C and 2D NMR data, and its skeleton type was a hemi-calendarane-type diterpene, which was named calnudiflopene A. 1 H and 13 C NMR data assignments are shown in Table 1, [400 MHz ( 1 H), 100 MHz ( 13 C), solvent: CD3OD- d 4].

[0047] Table 1 Compounds 1 H-NMR and 13 C-NMR data [ δ （ ppm), J (Hz)] Compound I was hydrolyzed under alkaline conditions according to the following scheme, and the parent compound (5 S ,9 S ,10 S ,12 S ,13 R )-3,4- seco -12,13-dihydroxy-4(18),8(17),14(15)-labdatrien-3-oicacid (II), and branched compound 8-hydroxy-2,6-dimethyl-2-octenoic acid (III); .

[0048] Compound II was dissolved in methanol, and the methanol was slowly evaporated to obtain the crystals shown below, with an absolute configuration of (5 S ,9 S ,10 S ,12 S ); The specific rotation of compound III is [ α ] +11.8 ( c 0.1, MeOH), and the absolute configuration was determined to be 6 by comparing with references. R .

[0049] The final structural formula of compound I is as follows: .

[0050] (Test Example 1, anti-inflammatory test) Experimental instruments and materials (1) Experimental instruments: full-wavelength multifunctional microplate reader, chemiluminescence detector (Analytik Jena, USA), electronic balance, high-temperature sterilizer, 96-well plate, pipette, pipette tips, and constant temperature incubator.

[0051] (2) Experimental Materials Cells: RAW264.7 macrophages.

[0052] Cell culture medium: DMEM (10% fetal bovine serum and 1% penicillin-streptomycin) culture medium.

[0053] Reagents: LPS (Sigma, USA); FBS (Gibco, USA); DMEM (Gibco, USA); NO kit (Shanghai Bio-Tech Co., Ltd.); MTT (Sigma, USA); dexamethasone (Sigma, USA).

[0054] (3) Experimental methods 1) MTT assay to determine cell viability The cytotoxic effect of the test samples on RAW264.7 cells was determined by MTT method. RAW 264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator with an air content of 95% and a CO2 content of 5% at 37°C.

[0055] RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 105 cells per well. After the cells were cultured at 37°C for 12 h, the cells were treated with test samples 1 to 12 for 24 h. Subsequently, 20 μL of MTT stock solution (5 μg / mL) was added to the wells. After incubation for 4 h, the supernatant was removed. Finally, the formazan crystals in each well were dissolved in DMSO (150 μL) and the absorbance was measured at a wavelength of 570 nm using a microplate reader (BD, Bioscience USA). These data are expressed as the average percentage of live cells compared to the respective controls.

[0056] 2) NO inhibition activity test The Griess method was used to test the release of nitric oxide (NO) in the supernatant of RAW264.7 cells induced by lipopolysaccharide (LPS), and dexamethasone (DEX) was used as a positive control.

[0057] RAW 264.7 cells were seeded in a 96-well plate at a density of 2 × 105 cells per well and cultured at 37°C for 12 h. The cells were then pretreated with the test samples and DEX (50 μg / mL) for 1 h. The cells were then stimulated with LPS (1 μg / mL) for 24 h. After 24 h, the accumulation of NO in the culture medium was determined. The cell supernatant (50 μL) was added to equal volumes of Griess reagent I and reagent II. The absorbance was measured at a wavelength of 540 nm using an enzyme reader.

[0058] The results of the anti-inflammatory test of hemi-penicillin-type diterpenoids I are as follows: name <![CDATA[IC 50 ( μ M) b ]]> Compound I 22.8 ± 0.52 Callicarpa nudiflora leaf extract 42.7 ± 0.33 Dexamethasone 39.62 ± 0.12 The experimental results show that the extract of the leaves of Callicarpa nudiflora has a certain anti-inflammatory effect, and the hemiarthane-type diterpenoid compound I isolated from the leaves of Callicarpa nudiflora has obvious anti-inflammatory effect.

[0059] (Test Example 2, α-glucosidase activity inhibition test) Experimental instruments and reagents: 96-well plate, electronic balance, pipette, constant temperature incubator, microplate reader (ELx800), constant temperature oscillator, acarbose, α -glucosidase, PNPG, anhydrous sodium carbonate, 0.1 mol / L pH 6.8 phosphate buffer (PBS).

[0060] Experimental methods: α-Glucosidase was diluted to 0.2 U / mL with 0.1 mol / L buffer (pH = 6.8) for use. The PNPG concentration was 2.5 mmol / L. Samples and acarbose were prepared with 0.1 mol / L buffer (pH = 6.8) to 1.0 mmol / L, 0.5 mmol / L, 0.25 mmol / L and 0.1 mmol / L for use.

[0061] This experiment was carried out in a 96-well culture plate, with a total reaction system of 200 µ L, add 50 µ L phosphate buffer (0.1 mol / L, pH = 6.8) and 10 µ L α -glucosidase (0.2 U / mL), followed by 20 µ L test sample, placed in a 37℃ water bath for 15 min, then added 20 µ The concentration of PNPG was 2.5 mmol / L, and after reacting at 37°C for 15 min, 100 µ The reaction was terminated by adding 2.5 mmol / L Na2CO3 solution, and the absorbance (A) value was measured at 405 nm after mixing evenly. Acarbose was used as a positive control.

[0062] The sample was measured three times in parallel and the average value was taken. α -Inhibition rate of glucosidase: Inhibition rate (%) = [1-(A 405nm Sample-A 405nm Blank) / A 405nm Control] × 100%.

[0063] (A 405nm Sample represents the A value of the sample, A 405nm Blank indicates the blank A value, A 405nm The control represents the A value excluding the interference of the sample).

[0064] The results of the α-glucosidase inhibition test are as follows: name <![CDATA[IC 50 ( μ M) b ]]> Compound I 175.3 ± 1.8 Callicarpa nudiflora leaf extract 250.8 ± 2.2 Acarbose 235.7 ± 2.4 The experimental results show that the leaf extract of Callicarpa nudiflora has the effect of inhibiting the activity of α-glucosidase; the hemiarthane-type diterpenoid compound I isolated from the leaves of Callicarpa nudiflora has a better effect of inhibiting the activity of α-glucosidase.

Claims

1. A hemipentane-type diterpenoid compound, the structural formula of which is as follows: （Ⅰ）。 2. Use of the compound I as claimed in claim 1 in the preparation of anti-inflammatory, therapeutic and / or preventive drugs for diabetes.

3. A method for preparing compound I as claimed in claim 1, characterized in that The following steps are involved: ① After the dried leaves of Callicarpa nudiflora were crushed, ultrasonic extraction was performed with dichloromethane for 2 to 4 times, the extracts were combined, and vacuum concentrated to obtain the extract of the dichloromethane extract of the leaves of Callicarpa nudiflora; ② The extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora obtained in step ① is subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate solvent system is used for elution according to a gradient of a volume ratio of 100:0 to 0:100, and the fraction of the petroleum ether-ethyl acetate solvent system of 60:40 is concentrated by TLC detection to obtain a crude extract of the leaves of Callicarpa nudiflora; ③ The crude extract of the leaves of Callicarpa nudiflora was separated by ODS column chromatography, the eluent was MeOH-H2O solvent, and the elution was performed according to the gradient of volume ratio of 5:95 to 100:0, and the same components were combined to obtain 8 components Fr.1-8, and Fr.1-Fr.8 were combined to obtain the extract of the leaves of Callicarpa nudiflora; ④ Fr.4 obtained in step ③ was first subjected to normal phase silica gel column chromatography, and the eluent was a mixed system of dichloromethane and acetone, and eluted according to a gradient of volume ratio of 10:0 to 5:5, eluted for 3 to 6 column volumes, and the fractions were collected and combined to obtain 7 components Fr.4a-Fr.4g; ⑤ The Fr.4c obtained in step ④ was separated and prepared by high performance liquid chromatography HPLC, and the chromatographic column was Waters C 18 , the mobile phase was MeCN:H2O=55:45, and compound I was separated.

4. The method for preparing compound I according to claim 3, characterized in that: In step ①, 1 to 1.2 L of dichloromethane is used per kilogram of Callicarpa nudiflora leaf powder, and ultrasonic extraction is performed for 2 to 3 hours each time.

5. The method for preparing compound I according to claim 3, characterized in that: In step ②, collect fractions every 3000 mL.

6. The method for preparing compound I according to claim 3, characterized in that: In step ③, collect fractions of 2000 mL each.

7. A Callicarpa nudiflora leaf extract, characterized in that Extracted as follows: ① After the dried leaves of Callicarpa nudiflora were crushed, ultrasonic extraction was performed with dichloromethane for 2 to 4 times, the extracts were combined, and vacuum concentrated to obtain the extract of the dichloromethane extract of the leaves of Callicarpa nudiflora; ② The extract of the dichloromethane extraction part of the leaves of Callicarpa nudiflora obtained in step ① is subjected to silica gel column chromatography, and a petroleum ether-ethyl acetate solvent system is used for elution according to a gradient of a volume ratio of 100:0 to 0:100, and the fraction of the petroleum ether-ethyl acetate solvent system of 60:40 is concentrated by TLC detection to obtain a crude extract of the leaves of Callicarpa nudiflora; ③ The crude extract of the leaves of Callicarpa nudiflora was separated by ODS column chromatography, the eluent was MeOH-H2O solvent, and the elution was performed according to the gradient of volume ratio of 5:95 to 100:

0. After TLC detection, 8 components Fr.1-8 were obtained after combining the same components, and Fr.1-Fr.8 were combined to obtain the extract of the leaves of Callicarpa nudiflora.

8. The Callicarpa nudiflora leaf extract according to claim 7, characterized in that: In the extraction step ①, the amount of dichloromethane used is 1 to 1.2 L per kilogram of Callicarpa nudiflora leaf powder, and the ultrasonic extraction is performed for 2 to 3 hours each time.

9. The Callicarpa nudiflora leaf extract according to claim 7, characterized in that: In extraction step ②, fractions were collected every 3000 mL; in extraction step ③, fractions were collected every 2000 mL.

10. Use of the Callicarpa nudiflora leaf extract as claimed in claim 7 in the preparation of anti-inflammatory, therapeutic and / or preventive drugs for diabetes.

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