A sesquiterpenoid compound, and an extraction method and application thereof

Abstract

The application discloses a sesquiterpenoid compound and an extraction method and application thereof, belongs to the field of traditional Chinese medicine extraction, and particularly relates to a sesquiterpenoid compound separated from the rhizome of Curcuma kwangsiensis in Guangxi Zhuang Autonomous Region and having the structural formula shown in the description and an extraction method of the compound, and further provides the inhibition effect of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound on the production of NO in LPS-induced RAW264.7 cells, and the compound can be used for preparing anti-inflammatory drugs. The application further enriches the structural diversity of active substances in the rhizome of Curcuma kwangsiensis in Guangxi Zhuang Autonomous Region, lays a foundation for the subsequent related biological activity test of monomer compounds, provides an active lead compound for new drug development, and provides a theoretical basis for deep research and development of Curcuma kwangsiensis in Guangxi Zhuang Autonomous Region.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to a sesquiterpene compound separated from the rhizome of Curcuma zedoary in Guangxi Zhuang Autonomous Region, an extraction method of the sesquiterpene compound and application of the sesquiterpene compound in preparation of anti-inflammatory drugs. BACKGROUND

[0002] Curcuma kwangsiensis S.G.Lee et C.F.Ling is a perennial herb of Zingiberaceae, mainly distributed in Guangxi and Yunnan, China. It is also known as Gui E Zhi and Mao E Zhi. According to the Pharmacopoeia of the People's Republic of China (2020 edition, part one), it is one of the three main source plants of traditional Chinese medicine E Zhi. Its main functions are to remove blood stasis, promote blood circulation, and relieve pain (State Pharmacopoeia Commission of P.R.C. Pharmacopoeia of the People's Republic of China[M]. Beijing: China Medical Science Press, 2020). Sesquiterpenes, diarylheptanoids, and diterpenoids are the main chemical components of C. kwangsiensis. Modern pharmacology shows that curcumin isolated from C. kwangsiensis has significant activity in anti-tumor, especially for liver cancer, skin cancer, pancreatic cancer, prostate cancer, and ovarian cancer (Perrone D, Ardito F, Giannatempo G, et al. Biological and therapeutic activities, and anticancer properties of curcumin [J]. Experimental and Therapeutic Medicine, 2015, 10(5): 1615-1623.). Zeng J H, Dai P, Ren LY, et al. Apoptosis-induced anti-tumor effect of Curcuma kwangsiensis polysaccharides against human nasopharyngeal carcinoma cells [J]. Carbohydrate Polymers, 2012, 89(4): 1067-1072. found that C. kwangsiensis polysaccharides had a significant inhibitory effect on the proliferation of CNE-2 cells, and believed that the mechanism of action might be to weaken the expression of Bcl-2-mediated apoptosis induction and promote p53 expression. In addition, Liao HB et al. isolated salvialane-type sesquiterpenes from C. kwangsiensis, which had good anti-inflammatory activity, with an IC 501.85±0.44μM (Liao H B, Feng WY, Wang H S, et al. Sesquiterpenoid compounds from Curcuma kwangsiensis [J]. Chemistry & Biodiversity, 2019, 16(5):e1900123.) To maximize the medicinal value of Guangxi Zangzuang Autonomous Region Zedoary, the rhizomes of Guangxi Zangzuang Autonomous Region Zedoary were systematically studied, and new sesquiterpenoid compounds were extracted, and the structure of the compounds was confirmed by nuclear magnetic, infrared, mass spectrometry and other methods, and the anti-inflammatory activity was detected. SUMMARY

[0003] The primary object of the present application is to provide a sesquiterpenoid compound.

[0004] The second object of the present application is to provide a sesquiterpenoid compound extraction method.

[0005] The third object of the present application is to provide a pharmaceutical composition containing a sesquiterpenoid compound.

[0006] The fourth object of the present application is to provide a sesquiterpenoid compound or an isomer of the compound, a pharmaceutically acceptable salt of the compound or a pharmaceutical composition comprising the compound for use in the preparation of an anti-inflammatory drug.

[0007] The technical scheme of the present application is as follows:

[0008] A sesquiterpenoid compound is any one of the compounds shown in the following structural formula 1-3 or an isomer of the compound, a pharmaceutically acceptable salt of the compound.

[0009]

[0010] The pharmaceutically acceptable salt includes sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate.

[0011] The isomer includes optical isomer, cis-trans isomer, racemate and their mixture.

[0012] The present application also provides an extraction method of the sesquiterpenoid compound 1-3, comprising the following steps:

[0013] (1) The dried rhizomes of Guangxi Zangzuang Autonomous Region Zedoary are used as raw materials, 8-15 times the volume of 95% ethanol aqueous solution is added, reflux extraction is carried out 2-4 times, each time for 2-4 hours, the extract is obtained by combining, the solvent is recovered under reduced pressure, and the total extract is obtained after concentration;

[0014] (2) The total extract is dispersed in 5-10 times the mass of water, and extracted using ethyl acetate to obtain an ethyl acetate layer and a water layer;

[0015] (3) The ethyl acetate extraction concentrated liquid is separated by silica gel column chromatography, and eluted by gradient elution using petroleum ether-ethyl acetate with a volume ratio of 100:0-0:1 as the eluent, and the volume ratio of 100:0 of the collected fraction E1, the volume ratio of 50:1 of the fraction E2, the volume ratio of 20:1 of the fraction E3, the volume ratio of 2:1 of the fraction E4, and the volume ratio of 0:1 of the fraction E5;

[0016] (4) The fraction E3 is concentrated, and the concentrated liquid is separated by silica gel column chromatography, and further purified by gradient elution using petroleum ether-ethyl acetate with a volume ratio of 100:1-0:1 as the eluent to obtain compound 1-3.

[0017] The step (4) specifically comprises:

[0018] After the fraction E3 is concentrated, it is separated by silica gel column chromatography, and the fraction with a volume ratio of 15:1 of petroleum ether and ethyl acetate is collected, and is recorded as E32;

[0019] After the fraction E32 is concentrated, it is separated by MCI column chromatography, and the fractions eluted by 70% and 80% methanol water are collected, and are recorded as E321;

[0020] After the fraction E321 is concentrated, it is separated by ODS column chromatography, and is eluted by gradient elution using methanol-water with a volume ratio of 9:1, 7:3, 5:5, 3:7, 2:8, and 1:0 in sequence as the eluent, and the fraction with a volume ratio of 5:5 is collected, and is recorded as E3214;

[0021] After the fraction E3214 is concentrated, it is separated by silica gel column chromatography, and the fractions with a volume ratio of 20:1 and 8:1 of petroleum ether and acetone are collected, and are recorded as E32142 and E32144;

[0022] After the fraction E32142 is concentrated, it is separated by Sephadex LH-20 column chromatography (CH3OH) to obtain the fraction E321422;

[0023] The fraction E321422 is purified by preparative HPLC chromatography using methanol-water with a volume ratio of 60:40 as the mobile phase to obtain compound 1;

[0024] The fraction E32144 is purified by preparative HPLC chromatography using methanol-water with a volume ratio of 50:50 as the mobile phase to obtain compounds 2 and 3.

[0025] Extracts of the rhizomes of Curcuma zedoaria from Guangxi Zhuang Autonomous Region containing sesquiterpenes.

[0026] Use of Guangxi Zhuang Autonomous Region Curcuma Phaeocaulis Root Rhizome Extract in Preparation of Anti-inflammatory Drugs.

[0027] A pharmaceutical composition comprises one or more of the sesquiterpenes, isomers of the compound, pharmaceutically acceptable salts of the compound; and further comprises one or more of pharmaceutically acceptable carriers, excipients, diluents or a combination thereof. The pharmaceutical composition is administered orally or by injection, and the dosage forms include tablets, capsules, powders, syrups and injections.

[0028] The application also provides use of the sesquiterpenes, isomers of the compound, pharmaceutically acceptable salts of the compound or the pharmaceutical composition in preparation of anti-inflammatory drugs.

[0029] Advantages of the application:

[0030] The sesquiterpenes or isomers thereof, pharmaceutically acceptable salts thereof or the pharmaceutical composition comprising the compound of the application have inhibitory effect on liver cancer cells, and can be used in preparation of anti-inflammatory drugs. The application further enriches the structural diversity of active substances of Guangxi Zhuang Autonomous Region Curcuma Phaeocaulis Root Rhizome, and lays a foundation for subsequent biological activity tests of monomer compounds, provides active lead compounds for new drug development, and provides a theoretical basis for deep research and development of Guangxi Zhuang Autonomous Region Curcuma Phaeocaulis.

[0031] The sesquiterpenes of the application have inhibitory effect on NO production in LPS-induced RAW264.7 cells, and can be used in preparation of anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Inhibitory activity of compound 1-2 on NO production in LPS-induced RAW264.7. DETAILED DESCRIPTION

[0033] The technical solutions of the application will be further described below in combination with specific embodiments.

[0034] Example 1

[0035] The extraction method of the sesquiterpenes in Guangxi Zhuang Autonomous Region Curcuma Phaeocaulis comprises the following steps:

[0036] (1) 20.0 kg of dried root rhizome of Guangxi Zhuang Autonomous Region Curcuma Phaeocaulis is used as raw material, 10 times the mass of the raw material of volume concentration 95% ethanol aqueous solution is added, and reflux extraction is performed for 3 times, 3 hours each time, and the extraction liquid is obtained by combining, and the solvent is recovered under reduced pressure, and the total extract is obtained after concentration;

[0037] (2) The total extract was dispersed in 8 times the mass of water, and extracted with ethyl acetate to obtain an ethyl acetate layer (570.0 g) and an aqueous layer;

[0038] (3) The ethyl acetate extraction concentrated liquid was separated by silica gel column chromatography, and eluted with petroleum ether-ethyl acetate with a volume ratio of 100:0 to 0:1 as the eluent gradient, and the volume ratio of 100:0 of fraction E1 (55.0 g), the volume ratio of 50:1 of fraction E2 (175.0 g), the volume ratio of 20:1 of fraction E3 (129.1 g), the volume ratio of 2:1 of fraction E4 (99.6 g), and the volume ratio of 0:1 of fraction E5 (73.3 g) were collected;

[0039] (4) The fraction E3 was concentrated, and the concentrated liquid was separated by silica gel column chromatography, and eluted with petroleum ether-ethyl acetate with a volume ratio of 100:1 to 0:1 as the eluent gradient, and further purified to obtain compound 1-3.

[0040] The fraction E3 (129.1 g) was concentrated and separated by silica gel column chromatography, and the fraction with a volume ratio of 15:1 of petroleum ether and ethyl acetate was collected and recorded as E32 (30.8 g);

[0041] The fraction E32 was concentrated and separated by MCI column chromatography, and the fractions eluted with 70% and 80% methanol water were collected and recorded as E321 (26.2 g);

[0042] The fraction E321 was concentrated and separated by ODS column chromatography, and eluted with methanol-water with a volume ratio of 9:1, 7:3, 5:5, 3:7, 2:8, and 1:0 as the eluent gradient, and the fraction with a volume ratio of 5:5 was collected and recorded as E3214 (5.3 g);

[0043] The fraction E3214 was concentrated and separated by silica gel column chromatography, and the fractions with a volume ratio of 20:1 and 8:1 of petroleum ether and acetone were collected and recorded as E32142 (660.9 mg) and E32144 (1.0 g);

[0044] The fraction E32142 was concentrated and separated by Sephadex LH-20 column chromatography (CH3OH) to obtain the fraction E321422 (520.1 mg);

[0045] The fraction E321422 was purified by preparative HPLC chromatography with methanol-water with a volume ratio of 60:40 as the mobile phase to obtain compound 1 (2.9 mg);

[0046] The fraction E32144 was purified by preparative HPLC chromatography with methanol-water with a volume ratio of 50:50 as the mobile phase to obtain compound 2 (2.8 g) and 3 (1.1 mg).

[0047] The extracted and separated sesquiterpenes 1-3 were identified by structure, and the specific physical and chemical data were as follows:

[0048] Compound 1: colorless oil, which showed pink purple color after heating reaction with 1% vanillin-concentrated sulfuric acid. +27.0 (c = 0.10, CH3OH), HR-ESI-MS gave the quasi-molecular ion peak [M+Na] + m / z: 273.1480 (calcd. for C 15 H 22 O3Na + , 273.1467), the molecular formula was C 15 H 22 O3, 1 H-NMR (600MHz, CD3OD) and 13 C-NMR (150MHz, CD3OD) data are shown in Table 1.

[0049] Compound 2: colorless oil, which showed pink purple color after heating reaction with 1% vanillin-concentrated sulfuric acid. -16.1 (c = 0.10, CH3OH), HR-ESI-MS gave the quasi-molecular ion peak [M+Na] + m / z: 289.1411 (calcd. for C 15 H 22 O4Na + , 289.1416), the molecular formula was C 15 H 22 O4, 1 H-NMR (600MHz, CD3OD) and 13 C-NMR (150MHz, CD3OD) data are shown in Table 2.

[0050] Compound 3: colorless oil, which showed pink purple color after heating reaction with 1% vanillin-concentrated sulfuric acid. -26.8 (c = 0.10, CH3OH), HR-ESI-MS gave the quasi-molecular ion peak [M+Na] + m / z: 275.1618 (calcd. for C 15 H 24 O3Na + , 275.1623), the molecular formula was C 15 H 24 O3, 1 H-NMR (600MHz, CD3OD) and 13 C-NMR (150MHz, CD3OD) data are shown in Table 3.

[0051] Table 1 1 H-NMR (600MHz), 13 C-NMR (150MHz) data of compound 1 (CD3OD)

[0052]

[0053] Table 2 1 H-NMR (600MHz), 13 C-NMR (150MHz) data of compound 2 (CD3OD)

[0054]

[0055] Table 3 1 H-NMR (600MHz), 13 C-NMR (150MHz) data of compound 3 (CD3OD)

[0056]

[0057] The structures of the above compounds were identified by physicochemical data and modern spectroscopy methods (HRESIMS and NMR), combined with relevant data in the public literature, and it was determined that compounds 1-3 were all new compounds not reported in the literature, as shown below:

[0058]

[0059] Example 2

[0060] (1) Take the dried rhizome of Curcuma zedoary from Guangxi Zhuang Autonomous Region as raw material, add 8 times the volume of 95% ethanol aqueous solution by mass of the raw material, and reflux extract twice, each time for 2 hours. Combine the extract to obtain the extract, and recover the solvent under reduced pressure. Concentrate to obtain the total extract;

[0061] (2) Disperse the total extract into 5 times the mass of water, and extract using ethyl acetate to obtain an ethyl acetate layer and a water layer;

[0062] (3) The ethyl acetate extraction concentrated liquid is separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate with a volume ratio of 100:0 to 0:1 as eluent gradient, and the volume ratio of 100:0 of the fraction E1, the volume ratio of 50:1 of the fraction E2, the volume ratio of 20:1 of the fraction E3, the volume ratio of 2:1 of the fraction E4, and the volume ratio of 0:1 of the fraction E5 are collected;

[0063] (4) The concentrated fraction E3 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:1, to further purify compounds 1-3. The specific separation and purification process was the same as in Example 1.

[0064] Example 3

[0065] (1) Using the dried rhizome of Curcuma zedoaria from Guangxi Zhuang Autonomous Region as raw material, add 15 times the mass of the raw material to a 95% ethanol aqueous solution with a volume concentration of 95%, reflux extract 4 times, each extraction for 4 hours, combine the extracts, recover the solvent under reduced pressure, concentrate and obtain the total extract.

[0066] (2) Disperse the total extract in 10 times its weight of water and extract with ethyl acetate to obtain an ethyl acetate layer and an aqueous layer;

[0067] (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 100:0 to 0:1. The fractions E1 (100:0), E2 (50:1), E3 (20:1), E4 (2:1), and E5 (0:1) were collected.

[0068] (4) The concentrated fraction E3 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate at a volume ratio of 100:1 to 0:1, to further purify compounds 1-3. The specific separation and purification process was the same as in Example 1.

[0069] The effects of the extracted sesquiterpenoid compounds 1-2 on the anti-inflammatory activity of mouse macrophage RAW264.7 were studied in detail below:

[0070] 1. Principle: Excessive LPS induces the activation and expression of nitric oxide synthase (NOS) in macrophages, leading to the production of NO. NO then reacts with oxygen free radicals to rapidly form NO2. - NO2 formed - It can be quantitatively detected by Griess Reagent under acidic conditions. First, NO2 - The azo compound undergoes a diazotization reaction with sulfanilamide, followed by a coupling reaction with N-1-naphthylethylene-diamine dihydrochloride to generate a purple-red azo compound. Finally, the absorbance value is measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. By combining this with a standard curve, the NO content in the sample can be determined.

[0071] 2. Methods: RAW264.7 mouse mononuclear macrophages in the logarithmic growth phase were collected and their concentration adjusted to 3.5 × 10⁻⁶ cells.4 RAW264.7 cells / well were inoculated in 96-well plates, and 100 μL of cell suspension was added to each well. Control group (RAW264.7 cells, DMSO), model group (RAW264.7 cells, DMSO, 0.5 μg / mL of LPS), positive drug group (RAW264.7 cells, dexamethasone (20 μM), 0.5 μg / mL of LPS), and test drug group (RAW264.7 cells, each test compound (20 μM), 0.5 μg / mL of LPS) were set up in the experiment. After incubation in a 5% CO2, 37°C constant temperature incubator for 24 h, 40 μL of cell supernatant was taken and placed in an enzyme-labeled plate, and an equal volume of Griess reagent was added to measure the nitrite accumulation in the culture medium at 540 nm using an enzyme-labeled instrument. The results are shown in Table 1. Figure 1 As shown in Table 1, compound 1 has a slight inhibitory activity on NO release.

[0072] The above description of the embodiments is only used to help understand the method of the present application and its central idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection of the claims of the present application.

Claims

1. A method for extracting a sesquiterpene compound, characterized by, Comprise the following steps: (1) with Guangxi Zhuang Autonomous Region Curcuma zedoary dry rhizome as raw material, add 8~15 times the mass of ethanol aqueous solution of raw material, reflux extraction, combined to obtain the extract, recovery of solvent under reduced pressure, concentrated to obtain total extract; (2) the total extract is dispersed into 5~10 times the mass of water, using ethyl acetate extraction, obtain ethyl acetate layer and water layer; (3) ethyl acetate extraction concentrated liquid is separated by silica gel column chromatography, with volume ratio of 100:0~0:1 petroleum ether-ethyl acetate as eluent gradient elution, collect volume ratio 100:0 fraction E1, volume ratio 50:1 fraction E2, volume ratio 20:1 fraction E3, volume ratio 2:1 fraction E4, volume ratio 0:1 fraction E5; (4) concentrate fraction E3, the concentrated liquid is separated by silica gel column chromatography, with volume ratio of 100:1~0:1 petroleum ether-ethyl acetate as eluent gradient elution, purification to obtain compound 1; The compound 1 structural formula is: 。 2. The extraction method of the sesquiterpenoid compound according to claim 1, characterized by, The specific separation and purification process of fraction E3 in step (4) is: After fraction E3 is concentrated, it is separated by silica gel column chromatography, and the fraction with volume ratio of petroleum ether and ethyl acetate of 15:1 is collected, which is recorded as E32; After fraction E32 is concentrated, it is separated by MCI column chromatography, and the fraction eluted by 70% and 80% methanol water is collected, which is recorded as E321; After fraction E321 is concentrated, it is separated by ODS column chromatography, and the fraction with volume ratio of methanol and water of 5:5 is collected, which is recorded as E3214, by gradient elution with volume ratio of 9:1, 7:3, 5:5, 3:7, 2:8 and 1:0 methanol-water in turn; After fraction E3214 is concentrated, it is separated by silica gel column chromatography, and the fractions with volume ratio of petroleum ether and acetone of 20:1 and 8:1 are collected, which are recorded as E32142 and E32144; compound 1 is obtained after fraction E32142 is purified.

3. The method of claim 2, wherein the sesquiterpene compound is extracted from the plant of the genus Artemisia. The method for purifying fraction E32142 comprises: After fraction E32142 is concentrated, it is separated by Sephadex LH-20 column chromatography with CH3OH as eluent to obtain E321422 fraction; Fraction E321422 is purified by preparative HPLC chromatography with volume ratio of 60:40 methanol-water as mobile phase to obtain compound 1.

4. The extraction method of the sesquiterpenes according to claim 1, characterized in that, The obtained compound 1 is used for preparing anti-inflammatory drugs.

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