Preparation method and application of curzerene in zedoary turmeric

Abstract

This invention relates to the field of pharmaceutical technology, and particularly to the preparation method and application of diphenylheptane in Curcuma zedoaria. The preparation method of diphenylheptane includes the following steps: (1) crushing Curcuma zedoaria rhizomes, extracting with ethyl acetate, concentrating the extract to obtain a residual extract, separating the extract by silica gel column chromatography with a 100-200 mesh, and eluting with a gradient of a petroleum ether-ethyl acetate mixed solution with a volume ratio of 1:0-0:1 to obtain six components Fr.I to VI; (2) separating the Fr.III component by silica gel column chromatography, and using a volume ratio of 1:0-0:1 to obtain six components Fr.I to VI; (2) A gradient elution with a petroleum ether-ethyl acetate mixed solution with a product ratio of 6:1 to 0:1 was used to obtain five components Fr.Ⅲ1 to Fr.Ⅲ5; (3) Fr.Ⅲ3 was separated by medium-pressure reversed-phase silica gel column chromatography with a gradient elution of MeOH-H2O solution at a volume concentration gradient of 20-100% to obtain nine sub-components Fr.Ⅲ3a to Fr.Ⅲ3i; (4) The Fr.Ⅲ3f sub-component was separated by semi-preparative high-performance liquid chromatography to obtain the diphenylheptane. The diphenylheptane has good antitumor activity and can be used in the preparation of antitumor pharmaceutical products.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the preparation method and application of diphenylheptane in Curcuma zedoaria. Background Technology

[0002] Curcuma zedoaria ( Curcuma zedoaria(chiristm.)Rosc Curcuma zedoaria (also known as white turmeric, turmeric root, or turmeric genus) is a plant belonging to the ginger family (Zingiberaceae). It is a perennial monocotyledonous herb, and its rhizome is used medicinally. It is widely distributed in my country, cultivated in Sichuan, Yunnan, Guangxi, Zhejiang, and other regions. Traditional Chinese medicine believes that Curcuma zedoaria is warm in nature, pungent and bitter in taste, and has the effects of promoting qi circulation, breaking up blood stasis, relieving muscle tension, and alleviating pain. It can be used to treat muscle pain caused by pathogenic factors, regulate food stagnation, treat amenorrhea, dysmenorrhea, and injuries from falls and blows.

[0003] Cancer is a major challenge in the medical field, with its incidence rate increasing year by year, seriously threatening human health and life. The active components in Curcuma zedoaria volatile oil, such as curcumin and β-elemene, can inhibit the occurrence and development of tumors by affecting multiple stages, including tumor cell growth, proliferation, apoptosis, and metastasis. Modern pharmacological and research studies have shown that Curcuma zedoaria possesses anti-tumor properties; curcumol, isolated from it, exhibits significant cytotoxicity to cancer cells and has been widely used clinically as an anti-cancer drug. To explore other anti-tumor active ingredients from this plant, we conducted phytochemical research on Curcuma zedoaria, native to Lingshan County, Guangxi Zhuang Autonomous Region, and isolated a new diphenylheptane compound. This compound exhibits good anti-tumor activity. Currently, there are no reports on the isolation and extraction of this diphenylheptane compound from Curcuma zedoaria and its anti-tumor activity. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying diphenylheptane in Curcuma zedoaria, addressing the aforementioned problems.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The structural formula of diphenylheptane in Curcuma zedoaria of this invention is as follows:

[0007] .

[0008] The present invention also provides a method for preparing diphenylheptane from the above-mentioned Curcuma zedoaria, comprising the following steps:

[0009] (1) The rhizome of Curcuma zedoaria was crushed and extracted with ethyl acetate. The extract was concentrated to obtain a residual extract. The extract was separated by silica gel column chromatography with a 100-200 mesh and eluted with a gradient of petroleum ether-ethyl acetate mixed solution with a volume ratio of 1:0-0:1 to obtain six components Fr.I ~ VI.

[0010] (2) The Fr.Ⅲ components were separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate mixed solution with a volume ratio of 6:1-0:1 to obtain 5 components Fr.Ⅲ1 ~ Fr.Ⅲ5;

[0011] (3) Fr.Ⅲ3 was separated by medium-pressure reversed-phase silica gel column chromatography and eluted with a gradient of MeOH-H2O solution with a volume concentration gradient of 20~100% to obtain 9 sub-fractions Fr.Ⅲ3a ~ Fr.Ⅲ3i;

[0012] (4) The Fr.Ⅲ3f subfraction was separated by semi-preparative high performance liquid chromatography to obtain the diphenylheptane.

[0013] In this invention, preferably, in step (1), the extraction is carried out at room temperature, and the extraction is performed 3 times, with each extraction lasting 3 days.

[0014] In this invention, preferably, in step (1), the method for concentrating the extract is vacuum concentration.

[0015] In this invention, preferably, in step (1), the gradient elution is performed by sequentially eluting with a petroleum ether-ethyl acetate mixed solution with volume ratios of 1:0, 9:1, 8:2, 2:1, 1:1, and 0:1.

[0016] In this invention, preferably, in step (2), the gradient elution is performed by sequentially eluting with a petroleum ether-ethyl acetate mixed solution with volume ratios of 6:1, 4:1, 2:1, 1:1, and 0:1.

[0017] In this invention, preferably, the flow rate of gradient elution in step (3) is 20 mL / min.

[0018] In this invention, preferably, in step (4), the separation conditions of semi-preparative high performance liquid chromatography are as follows: separation is performed using CH3CN:H2O eluent at a volume ratio of 35:65.

[0019] The diphenylheptane extracted by this invention has antitumor activity and can be used in the preparation of antitumor pharmaceutical products.

[0020] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0021] 1. This invention is the first to isolate a new diphenylheptane compound, Diphenylheptane A, from Curcuma zedoaria, which is of positive significance for the further in-depth research and utilization of Curcuma zedoaria.

[0022] 2. The turmeric diphenylheptane A isolated in this invention has good antitumor activity, and exhibits good antitumor activity against HepG2 cells and A549 cells at IC50 levels. 50 The values ​​were 70.34 ± 11.38 μM and 41.17 ± 1.51 μM, respectively, and the toxicity was almost non-toxic to normal human liver cells, making it suitable for use in the preparation of anti-tumor pharmaceutical products.

[0023] 3. The separation method of the present invention is reasonable, simple to operate, safe, suitable for industrial production, and has industrialization significance. Attached Figure Description

[0024] Figure 1 This is the structural diagram of compound 1.

[0025] Figure 2 It is key to compound 1 1 H– 1 H COSY ( ) and HMBC ( ) spectrum.

[0026] Figure 3 It is the C-3 adjacent proton in the (S)-MTPA ester and (R)-MTPA ester of compound 1. 1 H-NMR chemical shift difference. Detailed Implementation

[0027] To more clearly illustrate the present invention, the following specific embodiments will be used to further explain the invention.

[0028] The structural formula of diphenylheptane in the turmeric of this invention is as follows: Figure 1 As shown

[0029] The compound is a yellow oily substance. a ]26.0 D-2.80 ( c 0.1, MeOH); UV (MeOH) λmax(log ɛ): 201 (4.24), 223 (4.26), 279(3.66) nm; IR(KBr) ν max 3392, 2934, 2858,1703, 1613, 1514, 1446, 1379, 1266, 1026, 827 cm -1 According to ESI-MS: m / z 365 [M + Na] + The molecular formula of the compound was determined to be C643C ... 21 H 26O4, with an unsaturation degree of 9; the ultraviolet spectrum shows three maximum absorption peaks at wavelengths of 201 nm, 223 nm, and 279 nm; the infrared spectrum shows that the compound contains hydroxyl groups (3392 cm⁻¹). -1 ), carbonyl (1703 cm) -1 ) and benzene ring (1514 cm) -1 ).

[0030] The method for extracting the above-mentioned compound diphenylheptane from Curcuma zedoaria includes the following steps:

[0031] (1) The rhizome of Curcuma zedoaria was crushed and extracted with ethyl acetate. The extract was concentrated to obtain a residual extract. The extract was separated by silica gel column chromatography with a 100-200 mesh and eluted with a gradient of petroleum ether-ethyl acetate mixed solution to obtain six components Fr.I ~VI.

[0032] Among them, Curcuma zedoaria (50 kg) was collected from Lingshan County, Guangxi, and identified as a Zingiberaceae plant by Associate Professor Huang Deqing of the School of Pharmacy, Guilin Medical College. Curcuma zedoaria (chiristm.) Rosc The rhizomes were extracted at room temperature, three times, for three days each time. The extract was concentrated by vacuum concentration. The elution conditions for silica gel column chromatography were a gradient elution with a petroleum ether-ethyl acetate mixture at volume ratios of 1:0, 9:1, 8:2, 2:1, 1:1, and 0:1, to obtain six components Fr.I ~ VI.

[0033] (2) The Fr.Ⅲ components were separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate mixture to obtain five components Fr.Ⅲ1 ~ Fr.Ⅲ5;

[0034] The elution conditions for silica gel column chromatography were as follows: a petroleum ether-ethyl acetate mixture was used for gradient elution at volume ratios of 6:1, 4:1, 2:1, 1:1, and 0:1 to obtain five components, Fr.Ⅲ1 to Fr.Ⅲ5.

[0035] (3) Fr.Ⅲ3 was separated by medium-pressure reversed-phase silica gel column chromatography and eluted with a gradient of MeOH-H2O solution with a volume concentration gradient of 20~100% to obtain 9 sub-fractions Fr.Ⅲ3a ~ Fr.Ⅲ3i;

[0036] The gradient elution flow rate was 20 mL / min.

[0037] (4) The Fr.Ⅲ3f subfraction was separated by semi-preparative high performance liquid chromatography to obtain the diphenylheptane;

[0038] The separation conditions for the semi-preparative high performance liquid chromatography method are as follows: using CH3CN: H2O as the eluent at a volume ratio of 35:65.

[0039] The instruments and reagents used in the embodiments of this invention are as follows: optical rotation is expressed digitally. J Measurements were taken using an ASCO P-1020 spectrometer; IR measurements were performed on a PerkinElmer Spectrum Two FT-IR spectrometer with KBr compression; UV measurements were taken using a Shimadzu UV2401PC UV spectrometer; HRESIMS spectra were determined using liquid chromatography-mass spectrometry (Agilent 6200 series TOF, Agilent Technologies, USA); 1D and 2D NMR measurements were performed on a Bruker DRX-400 MHz NMR spectrometer; an Agilent preparative HPLC system (Agilent LC1260 Infinity, Agilent Technologies, USA); a CO2 incubator and microplate reader (Thermo Scientific, USA); silica gel (100-200 mesh, Qingdao Ocean Chemical Plant, China); pre-coated silica gel GF254 for thin-layer chromatography (Qingdao Ocean Chemical Plant, China); reversed-phase packing material (RP-18, YMC, Tokyo, Japan); reversed-phase column (Zorbax SB-C18, 5 μm, 9.4 × 250 mm, Agilent Technologies, USA). Petroleum ether, ethyl acetate, methanol (AR, Guangdong Xilong Technology Co., Ltd.); DMEM high-glucose culture medium (Gibco, USA); penicillin-streptomycin solution, 0.25% trypsin, MTT powder, dimethyl sulfoxide, paclitaxel (Beijing Solarbio Technology Co., Ltd.); fetal bovine serum (FBS) (Gibco, USA); PBS powder (Beijing Regen Biotechnology Co., Ltd.); cisplatin (GlpBio, USA). Specific product names are not intended to limit the scope of protection of this invention.

[0040] I. Preparation Examples Example

[0041] (1) The rhizomes of Curcuma zedoaria were crushed and extracted with ethyl acetate at room temperature. The extraction was repeated three times, with each extraction lasting three days. The extract was concentrated under vacuum to obtain a residual extract. The extract was separated by silica gel column chromatography using a 100-200 mesh filter, eluting sequentially with a petroleum ether-ethyl acetate mixture at volume ratios of 1:0, 9:1, 8:2, 2:1, 1:1, and 0:1. Six components, Fr.I ~ VI, were obtained.

[0042] (2) The Fr.Ⅲ components were separated by silica gel column chromatography and eluted sequentially with a petroleum ether-ethyl acetate mixture with volume ratios of 6:1, 4:1, 2:1, 1:1 and 0:1 to obtain five components Fr.Ⅲ1 ~ Fr.Ⅲ5;

[0043] (3) Fr.Ⅲ3 was separated by medium-pressure reversed-phase silica gel column chromatography, and eluted with MeOH-H2O solution gradient. The volume concentration gradient of MeOH-H2O solution was 20~100%, and the flow rate was 20 mL / min, to obtain 9 sub-fractions Fr.Ⅲ3a ~ Fr.Ⅲ3i;

[0044] (4) The Fr.Ⅲ3f subfraction was separated by semi-preparative high performance liquid chromatography. The separation conditions were: CH3CN: H2O eluent at a volume ratio of 35:65 to obtain compound 1, diphenylheptane.

[0045] Compound 1 is a yellow oily substance, [ a ]26.0 D-2.80 ( c 0.1, MeOH); UV (MeOH) λmax (logɛ): 201 (4.24), 223 (4.26), 279(3.66) nm; IR(KBr) ν max 3392, 2934, 2858, 1703,1613, 1514, 1446, 1379, 1266, 1026, 827 cm -1 According to ESI-MS: m / z 365 [M + Na] + The molecular formula of the compound was determined to be C643C ... 21 H 26 O4, with an unsaturation degree of 9; the ultraviolet spectrum shows three maximum absorption peaks at wavelengths of 201 nm, 223 nm, and 279 nm; the infrared spectrum shows that this compound contains hydroxyl groups (3392 cm⁻¹). -1 ), carbonyl (1703 cm) -1 ) and benzene ring (1514 cm) -1 ).

[0046] 1H of compound 1 and 13 The C NMR data are shown in Table 1 below.

[0047] Table 1. Compound 1 1 H and 13 C NMR data ( d in ppm, J (in Hz).

[0048]

[0049] Compound 1 in 1 H-NMR data (Table 1) indicate that the compound has one methyl proton signal. d H2.08 (s, 3H,H-4′-OAc)], 6 methylene proton signals [ d H 2.55 (t, J = 7.6 Hz, 2H, H-7), 1.91 (td, J =13.4, 12.0, 6.4 Hz, 2H, H-2), 1.69-1.49 (m, 4H, H-5, H-6), 1.38 (m, 2H, H-4)], 1 methylene proton signal [ d H 5.13 (t, J = 6.4 Hz, 1H, H-3). 13 C-NMR data showed one ester group carbon signal ( d C 170.5), 12 aromatic carbon signals ( d C 116.0, 116.0, 116.1, 116.1, 129.7, 129.7, 129.7, 129.7, 132.3, 133.0, 156.8, 156.9), 1 oxygen-carbon signal ( d C 73.6), with 6 methylene groups remaining. d C 24.9, 31.0, 31.8, 34.1, 34.9, 36.4) and 1 methyl carbon signal ( d C 20.9). NMR data of compound 1 indicate that it is a diphenylheptane compound with two para-substituted benzene rings.

[0050] exist 1 H- 1 H COSY spectrum ( Figure 2 Correlation signals can be observed in H-1 / H-2 / H-3 / H-4 / H-5 / H-6 / H-7, and H-1( d H 2.67 m, 2H) and C-1′( d C 132.3), C-2′( d C 129.7), C-6′ d C 129.7) has relevant signals, H-7 ( d H 2.55 t, J= 7.6 Hz, 2H) and C-1′′( d C 133.0), C-2′′( d C 129.7), C-6′′( d C There are relevant signals at 129.7). d H The methyl hydrogen signal at 2.08 (s, 3H, H-4′-OAc) and d C The carbon at 170.5 is correlated, which indicates the planar structure of compound 1.

[0051] The absolute configuration of the compound was determined by a modified Mosher method, in which the hydroxyl group at C-3 reacted with Mosher reagents (R)-(-)-MTPA-Cl and (S)-MTPA-Cl, respectively, under pyridine conditions and in a dry environment to generate (R)-MTPA esters (1-a) and (S)-MTPA esters (1-b). [The following text appears to be incomplete and requires further context: "Collect its..."] 1 The 1H NMR data are as follows:

[0052] (S)-MTPA ester (1-a): Yellow oily substance; 1 H NMR (600 MHz, Pyridine- d 5) d 5.066765(t, J = 7.7 Hz, 1H, H-3), 2.69457-2.603194 (m, 2H, H-1), 2.519606 (t, J = 7.7Hz, 2H, H-7), 1.986664-1.718473 (m, 2H, H-2), 1.645679-1.544150 (m, 2H, H-6), 1.546065-1.440705 (m, 1H, H-5), 1.313321 (m, 2H, H-4).

[0053] (R)-MTPA ester (1-b): Yellow oily substance. 1 H NMR (600 MHz, Pyridine- d 5) d 5.065871(t, J = 7.7 Hz, 1H, H-3), 2.723950 – 2.603064 (m, 2H, H-1), 2.519881 (t, J=7.7 Hz, 2H, H-7), 1.853407 (m, 2H, H-2), 1.634294 – 1.520124 (m, 2H, H-6), 1.563777 – 1.419385 (m, 2H, H-5), 1.312694 (m, 2H, H-4).

[0054] Analyze the proton chemical shift difference around the chiral center C-3 in MTPA esters, such as Figure 3 Its configuration was determined to be R, with the following structural formula, and it was named Diphenylheptane A.

[0055]

[0056] II. Antitumor Activity Test

[0057] The cytotoxicity of compound 1 against two human cancer cells, HepG2 and A549, and normal human hepatocytes, LO2, was determined using the MTT assay. The specific experimental steps are as follows:

[0058] Cells in good growth condition were digested with 0.25% trypsin. After complete digestion, the reaction was terminated with prepared DMEM complete medium. After centrifugation and discarding the supernatant, the cells were resuspended in DMEM complete medium and seeded into 96-well plates (6 × 10⁶ cells per well). 3 After incubating in a 5% CO2, 37 ℃ incubator for 12 h, test compounds were added at concentrations of 5, 10, 20, 40, and 80 μM. Cisplatin and paclitaxel were used as positive controls. Each concentration was tested in quadruplicates. After incubation for 48 h, 20 μL of 5 mg / mL MTT solution was added to each well. After incubation in a 5% CO2, 37 ℃ incubator in the dark for 4 h, 150 μL of DMSO was added to each well. The OD value was measured at 490 nm using a microplate reader.

[0059] The experiment was repeated at least three times. The inhibitory rate of different concentrations of the compound on cells was calculated using the following formula, and the IC50 was also calculated. 50 The values ​​and results are shown in Table 2:

[0060] Cell inhibition rate = [1 - (OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%

[0061] Table 2. Inhibition rates of the compounds on HepG2, A549, and LO2 cells after 48 hours of treatment (x ± s, n = 3)

[0062]

[0063] Note: [a] Data are presented as x ± s and are from three parallel trials. The drug concentrations were 5, 10, 20, 40, and 80 μM.

[0064] As shown in Table 2, compound 1, diphenylheptane, prepared in this invention exhibits cytotoxic effects on both HepG2 and A549 cells, demonstrating good antitumor activity. Although the antitumor activity of compound 1 is lower than that of existing cisplatin and paclitaxel, cisplatin and paclitaxel are highly toxic to normal human liver cells, while compound 1, diphenylheptane, is almost non-toxic to normal human liver cells, and thus has certain application prospects in the development of antitumor pharmaceutical products.

[0065] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. Diphenylheptane in Curcuma zedoaria, characterized in that, The structural formula is as follows: 。 2. The method for preparing diphenylheptane according to claim 1, characterized in that, Includes the following steps: (1) The rhizome of Curcuma zedoaria was crushed and extracted with ethyl acetate. The extract was concentrated to obtain a residual extract. The extract was separated by silica gel column chromatography with a 100-200 mesh and eluted with a gradient of petroleum ether-ethyl acetate mixed solution with a volume ratio of 1:0-0:1 to obtain six components Fr.I ~ VI. (2) The Fr.Ⅲ components were separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate mixed solution with a volume ratio of 6:1-0:1 to obtain 5 components Fr.Ⅲ1 ~ Fr.Ⅲ5; (3) Fr.Ⅲ3 was separated by medium-pressure reversed-phase silica gel column chromatography and eluted with a gradient of MeOH-H2O solution with a volume concentration gradient of 20~100% to obtain 9 sub-fractions Fr.Ⅲ3a ~ Fr.Ⅲ3i; (4) The Fr.Ⅲ3f subfraction was separated by semi-preparative high performance liquid chromatography to obtain the diphenylheptane; In step (4), the separation conditions for semi-preparative high performance liquid chromatography are as follows: separation is performed using CH3CN: H2O eluent at a volume ratio of 35:

65.

3. The preparation method according to claim 2, characterized in that, In step (1), the extraction is carried out at room temperature, and the extraction is performed 3 times, with each extraction lasting 3 days.

4. The preparation method according to claim 2, characterized in that, In step (1), the extraction solution is concentrated by vacuum concentration.

5. The preparation method according to claim 2, characterized in that, In step (1), the gradient elution is performed by sequentially eluting with a petroleum ether-ethyl acetate mixed solution with volume ratios of 1:0, 9:1, 8:2, 2:1, 1:1, and 0:

1.

6. The preparation method according to claim 2, characterized in that, In step (2), the gradient elution is performed by sequentially eluting with a petroleum ether-ethyl acetate mixed solution with volume ratios of 6:1, 4:1, 2:1, 1:1, and 0:

1.

7. The preparation method according to claim 2, characterized in that, In step (3), the gradient elution flow rate is 20 mL / min.

8. The use of diphenylheptane in Curcuma zedoaria as described in claim 1 in the preparation of pharmaceutical products against HepG2 cells or A549 cells.

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