A guaiacol-alkane sesquiterpene compound, and a preparation method and application thereof

Abstract

The application discloses a guaiacol alkane sesquiterpene compound and a preparation method and application thereof, various chromatographic technologies such as normal phase silica gel column chromatography and reverse phase silica gel column chromatography and high performance liquid chromatography are comprehensively used to separate and purify an ethyl acetate extraction part of an ethanol extract of Ligularia fischeri, the structure of the compound is identified according to physical and chemical properties and spectral data of the compound, and a guaiacol alkane sesquiterpene compound C 15 H 24 O3, and MTT experiments show that the effect of delaying iTEC cell aging of the compound 1 is best when the concentration is 1.25 μ g / mL and the action time is 72 h. The application enriches the diversity of chemical components of the Ligularia fischeri and lays a foundation for research and development of a new type of anti-aging drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a guaiacolane-type sesquiterpene compound, its preparation method, and its application. Background Technology

[0002] Ligustrum lucidum Ligularia fischeri (Ledeb.) Turcz. is a perennial herb belonging to the genus *Ligustrum* in the family Asteraceae. It is warm in nature and has a pungent and bitter taste. Its root and rhizome are called "Shan Zi Wan" and it is widely distributed in Northeast China, Shaanxi, Hubei, and other regions. *Ligustrum lucidum* is a plant used both as food and medicine, with its medicinal history dating back to the Song Dynasty's *Illustrated Materia Medica*. It is commonly used in folk medicine to treat chronic bronchitis, whooping cough, and injuries from falls and blows. Furthermore, the tender stems and leaves of *Ligustrum lucidum* are an important seasonal wild vegetable in Northeast Asia, especially in the Korean culinary culture, where it has a long history of consumption. The main chemical components of *Ligustrum lucidum* include sesquiterpenes, diterpenes, and flavonoids, with sesquiterpenes and flavonoids being the main active ingredients. Modern pharmacological studies have shown that *Ligustrum lucidum* exhibits a variety of pharmacological activities, encompassing anti-inflammatory, antioxidant, antitumor, and hepatoprotective effects.

[0003] Guaiacone sesquiterpenes are a wide range of natural products with important biological activities. They contain a typical [5,7] fused ring structural unit and are mainly distributed in plants of the Asteraceae and Zingiberaceae families, as well as some marine organisms. Guaiacone sesquiterpenes have attracted much attention due to their diverse biological activities, especially showing great potential in the pharmaceutical field. For example, guaiacone sesquiterpenes extracted from *Cephalotaxus fortunei* have the potential to be used as adjuvant therapy for esophageal cancer, gastric cancer, and lung cancer. *Elliptisides AD*, found in *Cephalotaxus fortunei*, has anti-inflammatory activity and can inhibit the production of the inflammatory mediator nitric oxide (NO). Guaiacone compounds isolated from *Dapoxetus daphne* have a significant protective effect against oxidative damage to nerve cells, providing new ideas for the treatment of neurodegenerative diseases such as Alzheimer's disease. Some guaiacone sesquiterpenes also have antibacterial and antioxidant effects.

[0004] Chinese patent CN110194754A discloses a fat-soluble extract of Ligustrum lucidum leaves, its preparation method, and its uses. The extract is obtained from dried and pulverized flowers and stems, yielding an ethyl acetate solution. Active monomeric components, including styracil lactone A, styracil aldehyde, and luteolin, are isolated from this solution. This extract inhibits the proliferation of tumor cells SH-SY5Y and MCF-7, and also has a certain DPPH free radical scavenging effect. It provides pharmaceutical or health product uses for anti-oxidation, anti-aging, improving immunity, and cancer prevention. It can also be used as a natural antioxidant. Furthermore, it provides medicinal and health product uses for obesity and obesity-related diseases such as hyperlipidemia, hypertension, diabetes, fatty liver, coronary heart disease, and sleep apnea syndrome, as well as related diseases.

[0005] Chinese patent CN101759675B discloses a method for preparing sesquiterpenes with anticancer and antibacterial activities from *Ligustrum lucidum*. The method involves extracting and refining *Ligustrum lucidum* to obtain an ethyl acetate extract, followed by silica gel column chromatography and gradient elution with a mixed solution to separate sesquiterpene compound II. This II is then reacted with dilute hydrochloric acid upon heating to obtain sesquiterpene compound I. This sesquiterpene exhibits inhibitory activity against the growth of oral epithelial cancer cells and significantly inhibits the growth of *Staphylococcus aureus* and beta-hemolytic streptococci. This invention's preparation method has a high extraction conversion rate, low cost, simple operation, and high product purity, making it suitable for use as a standard and in drug development.

[0006] However, there are currently few studies on the isolation and identification of sesquiterpenes contained in the roots of Ligustrum lucidum, and the application of guaiacolane-type sesquiterpenes is also still in its early stages, especially in terms of their anti-aging effects, which have great research potential. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a guaiacol-type sesquiterpene compound, its preparation method, and its application. A novel guaiacol-type sesquiterpene compound was obtained by directional isolation from the ethyl acetate extract of the roots of Ligustrum lucidum. Cell experiments revealed that this novel guaiacol-type sesquiterpene compound can delay the aging of iTEC cells and can be used to research novel anti-aging drugs.

[0008] To achieve the above objective, the present invention provides a guaiacolane-type sesquiterpene compound, wherein the guaiacolane-type sesquiterpene compound is C 15 H 24 O3 has an unsaturation degree of 4.

[0009] Preferably, the C 15 H 24O3 is ligulafischenolide A and / or ligulafischenolide B, wherein the chemical structural formula of ligulafischenolide A is as follows: The chemical structural formula of ligulafischenolide B is as follows: .

[0010] This invention also provides a method for preparing guaiacolane-type sesquiterpenoid compounds, comprising the following steps:

[0011] (1) Take dried roots of Ligustrum lucidum, extract with ethanol, combine the extracts, concentrate under reduced pressure and then suspend in water; (2) The suspension was extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the ethyl acetate extract; (3) The ethyl acetate extract was separated and purified by normal phase silica gel column chromatography, and gradient elution was performed using dichloromethane-ethyl acetate as the eluent to obtain the fraction; (4) Combine the fractions Fr.54-66, separate and purify them by reversed-phase silica gel column chromatography, and perform gradient elution with methanol-water as the eluent to obtain the subfraction; (5) Subfraction Frr.19 was separated and purified by semi-preparative HPLC to obtain guaiacol-type sesquiterpenoid compounds.

[0012] Preferably, the ethanol in step (1) has a mass fraction of 92%, the amount used is 10 times that of Ligusticum striatum, and the number of cold extractions is 4.

[0013] Preferably, the extraction of petroleum ether, ethyl acetate and n-butanol in step (2) is performed 6 times.

[0014] Preferably, the mesh size of the normal-phase silica gel column chromatography in step (3) is 200-300 mesh; the gradient of dichloromethane-ethyl acetate is... V 二氯甲烷 : V 乙酸乙酯 =100∶0, 50∶1, 30∶1, 20∶1, 10∶1, 5∶1, 3∶1, 2∶1, 1∶1, 1∶2.

[0015] Preferably, the methanol-water gradient in step (4) is: V 甲醇 : V 水 =30∶70, 50∶50, 70∶30, 90∶10, 100∶0.

[0016] Preferably, the mobile phase of the semi-preparative HPLC in step (5) is... V 甲醇 :V 水 =42∶58, flow rate is 3 mL / min.

[0017] This invention also provides an application of guaiacolane-type sesquiterpenoids in delaying cell aging.

[0018] Preferably, the cells are iTEC cells.

[0019] The beneficial effects of this invention are as follows: This invention utilizes a combination of chromatographic methods, including normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, and high-performance liquid chromatography, to study the ethyl acetate extract of the 92% ethanol extract of *Ligustrum lucidum* root. Two novel guaiacolane-type sesquiterpenoid compounds, ligulafischenolide A and ligulafischenolide B, were isolated from this extract. Anti-aging activity evaluation experiments showed that ligulafischenolide A, at a concentration of 1.25 μg / mL, exhibited the best effect in delaying the senescence of iTECs cells after 72 h of administration. Attached Figure Description

[0020] Figure 1 The figures show the structural formulas of the compounds in this invention, where 1 represents compound 1 and 2 represents compound 2.

[0021] Figure 2 The HPLC chromatograms are for fractions Fr.54 to 66 in Example 1.

[0022] Figure 3 The HPLC chromatogram of subfraction Frir.19 in Example 1 is shown.

[0023] Figure 4 The image shows the HR-ESI-MS spectrum of compound 1 in Example 2.

[0024] Figure 5 For compound 1 in Example 2 1 H NMR spectrum.

[0025] Figure 6 For compound 1 in Example 2 13 C10 NMR and DEPT 135 spectra.

[0026] Figure 7 The image shows the HSQC spectrum of compound 1 in Example 2.

[0027] Figure 8 For compound 1 in Example 2 1 H 1 H COSY spectrum.

[0028] Figure 9 The image shows the HMBC spectrum of compound 1 in Example 2.

[0029] Figure 10 The NOESY spectrum of compound 1 in Example 2 is shown.

[0030] Figure 11 For compound 1 in Example 2 1 H 1 H COSY and HMBC are key related signals.

[0031] Figure 12 This is the key NOESY correlation signal for compound 1 in Example 2.

[0032] Figure 13 The X-ray crystal structure of compound 1 in Example 2 is shown.

[0033] Figure 14 The image shows the HR-ESI-MS spectrum of compound 2 in Example 3.

[0034] Figure 15 Compound 2 in Example 3 1 H NMR spectrum.

[0035] Figure 16 Compound 2 in Example 3 13 C10 NMR and DEPT 135 spectra.

[0036] Figure 17 The image shows the HSQC spectrum of compound 2 in Example 3.

[0037] Figure 18 Compound 2 in Example 3 1 H 1 H COSY spectrum.

[0038] Figure 19 The image shows the HMBC spectrum of compound 2 in Example 3.

[0039] Figure 20 The NOESY spectrum of compound 2 in Example 3 is shown.

[0040] Figure 21 Compound 2 in Example 3 1 H 1 H COSY and HMBC are key related signals.

[0041] Figure 22 This is the key NOESY correlation signal for compound 2 in Example 3.

[0042] Figure 23 The effect of compounds 1 and 2 in Example 4 on the activity of aging iTEC cells (initial screening).

[0043] Figure 24 The effect of compound 1 in Example 4 on the activity of aging iTEC cells (rescreening). Detailed Implementation

[0044] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0045] Instruments and materials: Bruker AV 400 NMR spectrometer (Bruker AG, Switzerland); Xevo-G2Q-Tof high-resolution mass spectrometer (Waters Corporation, USA); Lab-1B-50E BIOCOOL vacuum freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd.); LC-2050 high-performance liquid chromatograph (Ruihe Technology Co., Ltd.); LC-16 high-performance liquid chromatograph (Shimadzu Corporation, Japan) with a semi-preparative column (Daisogel-10). m-100 Å 250 mm × 10 mm, Ruihe Technology Co., Ltd.); Analytical column (Cosmosil 5C18-MS-II, 250 mm × 4.6 mm, Nacalai Tesque, Japan); Antonpaar MCP 5100 high-precision polarimeter (Antonpaar, Austria); Vortex-Genie 2 vortex mixer (Orran Technologies, USA); Stat Fax-2100 ELISA reader (Awareness, USA); 2406-2 CO2 incubator (Shellab, USA); 5702 benchtop low-speed centrifuge (Eppendorf, Germany); 200-300 mesh normal-phase column chromatography silica gel (Qingdao Ocean Chemical Co., Ltd.); reversed-phase column chromatography silica gel (YMC, Japan); chromatographic grade methanol (Tianjin Tiandi Chemical Co., Ltd.); analytical grade anhydrous ethanol (Sinopharm Group); analytical grade methanol, analytical grade ethyl acetate, analytical grade dichloromethane, analytical grade petroleum ether, analytical grade n-butanol (Tianjin Kemei Chemical Reagent Co., Ltd.); newborn fetal bovine serum (Gibco, USA); DMEM culture medium (Hyclone, USA); bovine Bauer's counting chamber (Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.); MTT (Lanjieke Technology Co., Ltd.); doxorubicin (DOX) (Shanghai Shangyao Xinya Pharmaceutical Co., Ltd.); dimethyl sulfoxide (DMSO) (Amresco, USA). The root of *Ligustrum lucidum* was collected in Yichang City, Hubei Province in August 2023 and identified as *Ligustrum lucidum* by Professor Wang Yubing of the School of Biology and Pharmaceutical Sciences, Three Gorges University. Ligularia fischeri The rhizome of (Ledeb.) Turcz., specimen number LF202301, is deposited at the Hubei Provincial Key Laboratory of Natural Product Research and Utilization, Three Gorges University.

[0046] Example 1: Preparation of Compounds (1) Take 2.1 kg of Ligustrum lucidum root that has been naturally dried at room temperature and crush it. Add 10 times the mass of 92% ethanol and soak it in cold for 48 h before extraction. The extraction is repeated 4 times. (2) Combine all extracts and concentrate under reduced pressure to 300 mL to obtain a concentrated solution; add 200 mL of purified water to the concentrated solution to disperse and obtain a suspension; (3) Take the suspension and extract it 6 times each with 1 L petroleum ether, 1 L ethyl acetate and 1 L n-butanol. Combine the extracts and concentrate them under reduced pressure until they are dry to obtain 28.1 g of ethyl acetate extract. (4) The ethyl acetate extract was separated and purified by normal-phase silica gel column chromatography (200-300 mesh) using a gradient elution with dichloromethane-ethyl acetate as the eluent, yielding 87 fractions. The elution gradient of dichloromethane-ethyl acetate was as follows:V 二氯甲烷 : V 乙酸乙酯 =100∶0, 50∶1, 30∶1, 20∶1, 10∶1, 5∶1, 3∶1, 2∶1, 1∶1, 1∶2; (5) HPLC analysis of 87 fractions showed that the peak compositions (number of peaks and retention times) of fractions Fr.54–66 were basically the same, with only differences in the attribution of the main peak. Figure 2 (This indicates that components with the same composition can be combined.) (6) Combine Fr. 54–66 (4.1 g), and separate and purify by reversed-phase silica gel column chromatography with a methanol:water gradient elution to obtain 114 subfractions (Frr. 1–114); wherein the methanol:water elution gradient is as follows: V 甲醇 : V 水 =30∶70, 50∶50, 70∶30, 90∶10, 100∶0; (7) Subfractions Frr.1–114 were analyzed by HPLC. Based on the principle of preparation feasibility, Frr.19 (140.0 mg) was selected. Figure 3 The separation and purification were performed using a semi-preparative HPLC method, wherein the eluent was [missing information]. V 甲醇 : V 水 =42∶58, the eluent flow rate was 3 mL / min, yielding compound 1 (5.4 mg, t R =54.8 min) and compound 2 (12.5 mg, t R =59.6 min) Example 2: Structural Identification of Compound 1 Compound 1: Colorless crystals; l max =250 nm; -46.0° ( c 0.10, CH3OH); HR-ESI-MS m / z :253.1813 [M+H] + (Theoretical value is C) 15 H 24 O3H + (253.1799) Figure 4 Its molecular formula is determined to be C. 15 H 24 O3, the calculated degree of unsaturation is 4.

[0047] 13C NMR (400 MHz, DMSO- d 6) The spectrum is combined with the DEPT135 spectrum ( Figure 6 It can be seen that this compound contains 4 methyl groups, 3 methylene groups, 4 methine groups, and 4 quaternary carbons. Combined with... 1 H NMR ( Figure 5 ), 13 C10 NMR and HSQC spectra ( Figure 7 It can be seen that this compound contains one carbonyl carbon signal. d C 207.5), 2 alkenyl carbon signals ( d C 134.0, d C 177.6), a quaternary carbon signal with oxygen ( d C 71.1), a single oxygen-bound methine signal [ d H 3.56 (1H, s), d C 70.4], three methine signals linked to aliphatic hydrogen protons [ d H 1.65~1.70 (1H, m), d C 41.4; d H 2.71~2.75 (1H, m), d C 43.9; d H 1.29~1.36 (1H, m), d C 47.5], three methylene signals linked to aliphatic hydrogen protons [ d H 2.18 (1H, t, J = 11.6 Hz), 2.91(1H, d, J = 11.6 Hz), d C 29.4; d H 1.58~1.64 (1H, m), d H 1.70~1.76 (1H, m), d C 34.8; d H 1.97(1H, dd, J= 18.2, 3.2 Hz), 2.56 (1H, dd, J = 18.2, 6.4 Hz), d C 42.2], 4 methyl signals [ d H 1.56 (3H, s), d C 7.4; d H 1.08 (3H, d, J = 4.8 Hz), d C 18.5; d H 1.09 (3H, s), 25.4; d H 1.11(3H, s), d C 27.7], 2 active hydrogen signals ( d H 4.36 (1H, s); d H 4.60 (1H, s)]. A search of the microspectral database revealed that the NMR data of this compound was similar to those of torololone, a known compound reported in the literature (Fu HW, Zhang L, Yi T, et al. . Guaiane type sesquiterpenes and other constituents from Daucus carrot L. [J]. Biochemical Systematics and Ecology , 2010, 38(03): 309-312.), the difference lies in the transfer of a hydroxyl group from the C-8 position to the C-9 position in compound torololone. Compound 1 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 1.

[0048] 1 H 1 H COSY spectrum ( Figure 8 In ), H-1 ( d H 2.71~2.75) and H-2 ( d H 1.97, d H 2.56) / H-10 ( d HA significant correlation exists between H-9 and H-9 (1.65~1.70). d H 3.56) and H-8 ( d H 1.58~1.64, d H 1.70~1.76) / H-10 ( d H A significant correlation exists between H-7 and H-7 (1.65~1.70). d H 1.29~1.36) and H-6 ( d H 2.18, d H 2.91) / H-8 ( d H 1.58~1.64, d H A significant correlation exists between HMBC and 1.70–1.76. Figure 9 In ), H-2 ( d H 1.97, d H 2.56) and C-1 ( d C 43.9) / C-3( d C 207.5) / C-5 ( d C 177.6) / C-10 ( d C 41.4) shows a clear correlation; H-6 ( d H 2.18, d H 2.91) and C-1 ( d C 43.9) / C-4 ( d C 134.0) / C-5 ( d C 177.6) / C-7 ( d C 47.5) / C-8 ( d C 34.8) / C-11 ( d C 71.1) shows a clear correlation; H-7 ( d H 1.29~1.36) and C-5 ( d C177.6) is correlated; H3-12 ( d H 1.11) and C-11 ( d C 71.1) / C-7 ( d C 47.5) shows a significant correlation; H3-13 ( d H 1.09) and C-11 ( d C 71.1) / C-7 ( d C 47.5) showed a significant correlation; H3-14 ( d H 1.08) and C-1 ( d C 43.9) / C-9 ( d C 70.4) / C-10 ( d C 41.4) shows a clear correlation; H3-15 ( d H 1.56) and C-3 ( d C 207.5) / C-4 ( d C 134.0) / C-5 ( d C There is a clear correlation between 177.6) and 177.6). 1 H 1 HCOSY and HMBC key signals such as Figure 11 As shown.

[0049] NOESY spectrum ( Figure 10 In this context, H-1b / H-6b, H-2a / H-10a, H-6a / H-7a, H-8a / H-9a, and H-9a / H-10a exhibit NOESY correlations, while H-1b / H-6a, H-1b / H-9a, H-2b / H-10a, H-6b / H-7a, and H-8b / H-9a do not exhibit NOESY correlations. Figure 12 Literature reports that H-1b is generally classified as... β Based on the configuration, it is inferred that H-2a, H-6a, H-7a, H-8a, H-9a, and H-10a are located in... α The H-2b, H-6b, H-8b, and 9-OH groups are located at these positions. β The absolute configuration of the compound was determined by X-ray single-crystal diffraction. Figure 13A Scifinder search revealed no relevant reports; compound 1 is a new compound, named ligulafischenolide A.

[0050] Table 1. Compound 1 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data (DMSO- d 6)

[0051] Note: 'a' represents the location α 'b' represents the position. β Bit.

[0052] Example 3: Structural Identification of Compound 2 Compound 2: a colorless oily substance; l max = 247 nm; -36.0° ( c 0.10, CH3OH); HR-ESI-MS m / z 253.1808 [M+H] + (Theoretical value is C) 15 H 24 O3H + (253.1799) Figure 14 Its molecular formula is determined to be C. 15 H 24 O3, the calculated degree of unsaturation is 4.

[0053] 13 C NMR (400 MHz, DMSO-) d 6) Spectrum combined with DEPT 135 spectrum ( Figure 16 It can be seen that this compound contains 4 methyl groups, 3 methylene groups, 4 methine groups, and 4 quaternary carbons. Combined with... 1 H NMR ( Figure 15 ), 13 C10 NMR and HSQC spectra ( Figure 17 It can be seen that this compound contains one carbonyl carbon signal. d C 206.3), 2 alkenyl carbon signals ( d C 140.9, d C 179.2), 1 oxygen-linked quaternary carbon signal ( d C 71.1), a single oxygen-bound methine signal [ dH 3.34~3.38 (1H, m), d C 70.7], three methine signals linked to aliphatic hydrogen protons [ d H 2.79~2.85 (1H, m), d C 33.1; d H 2.69~2.72 (1H, m), d C 37.1; d H 1.26(1H, t, J = 11.4 Hz), d C 48.0], three methylene signals linked to aliphatic hydrogen protons [ d H 2.10 (1H, t, J = 14.8Hz), 2.67 (1H, d, J = 14.8 Hz), d C 30.5; d H 1.57 (1H, dd, J = 24.4, 13.2 Hz), d H 1.94(1H, d, J = 13.2 Hz), d C 35.3; d H 1.86 (1H, d, J = 18.4 Hz), 2.50~2.51 (1H, m), d C 42.3], 4 methyl signals [ d H 0.90 (3H, d, J = 7.2 Hz), d C 10.5; d H 1.10 (3H, d, J = 6.0 Hz), d C 19.1; d H 1.11 (3H, s), 26.3; d H1.11 (3H, s), d C 27.0], 2 active hydrogen signals [ d H 4.33 (1H, s); d H 4.61 (1H, s)]. A search of the microspectral database revealed that the NMR data of this compound was similar to those of the known compound pancherione reported in the literature (Miyazawa M, Sugawara A. Biotransformation of (2)-guaiol byeurotium rubrum as a biocatalyst [J]. Natural Product Research , 2006, 20(08):731-734.). Compound 2 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 2.

[0054] 1 H 1 In the H COSY spectrum ( Figure 18 H-3 d H 1.86, d H 2.50~2.51) and H-4 ( d H A significant correlation exists between H-7 and H-7 (2.69~2.72). d H 1.26) and H-8 ( d H 1.57, d H 1.94) / H-6 ( d H 2.10, d H 2.67) shows a significant correlation; H-9 ( d H 3.34~3.38) and H-8 ( d H 1.57, d H 1.94) / H-10 ( d H A significant correlation exists between H-10 and H-10 (2.79~2.85). d H 2.79~2.85) and H-14 ( d HA significant correlation exists between 0.90 and HMBC spectra. Figure 19 H-3 d H 1.86, d H 2.50~2.51) and C-2 ( d C 206.3) / C-4 ( d C 37.1) / C-5 ( d C 179.2) / C-15 ( d C 19.1) shows a clear correlation; H-7 ( d H 1.26) and C-5 ( d C 179.2) / C-6 ( d C 30.5) / C-8 ( d C 35.3) / C-9 ( d C 70.7) / C-11 ( d C 71.1) shows a clear correlation; H-10 ( d H 2.79~2.85) and C-1 ( d C 140.9) / C-2 ( d C 206.3) / C-5 ( d C 179.2) / C-8 ( d C 35.3) / C-9( d C 70.7) / C-14 ( d C A significant correlation exists between 10.5 and H3-12. d H 1.11) and C-7 ( d C 48.0) / C-11 ( d C 71.1) shows a significant correlation; H3-13 ( d H 1.11) and C-7 ( d C 48.0) / C-11 ( d C71.1) shows a clear correlation; H3-15 ( d H 1.10) and C-3 ( d C 42.3) / C-4 ( d C 37.1) / C-5 ( d C There is a clear correlation between 179.2) and 279.2). 1 H 1 H COSY and HMBC key signals such as Figure 21 As shown.

[0055] The relative configuration of compound 2 was determined by NOESY spectroscopy ( Figure 20 To determine this, literature reports that H-10a is all positioned as... α Configuration and observation of NOESY spectra: NOESY correlations are observed in H-3a / H-4a, H-6a / H-7a, H-7a / H-8a, H-7a / H-9a, and H-9a / H-10a, while no NOESY correlations are observed in H-6b / H-4a, H-6b / H-7a, and H-7a / H-8b. Figure 22 Therefore, it is inferred that H-3a, H-6a, H-8a, H-4a, H-7a, H-9a, and H-10a are located in... α The H-3b, H-6b, H-8b, and 9-OH groups are located at these positions. β No relevant reports were found through a SciFinder search. Compound 2 is a new compound, named ligulafischenolide B.

[0056] Table 2. 1H NMR (400 MHz) and 13C NMR (100 MHz) data of compound 2 (DMSO- d 6)

[0057] Note: 'a' represents the location α 'b' represents the position. β Bit.

[0058] Example 4 Activity Evaluation Initial screening for anti-aging activity (1) Plating: Collect iTEC cells in the logarithmic growth phase, resuspend them in DMEM complete medium (containing 10% fetal bovine serum), mix the cells well, adjust the cell concentration, and add 100 μL of the mixture to each well of a flat-bottomed 96-well plate. µ L single-cell suspension, with 4000 cells per well (with PBS added around the perimeter).

[0059] (2) DOX-induced senescence: After iTEC cells adhered (approximately 4–6 h), 100 mg of DOX was added to each well. µ The prepared DOX (without adding the outer ring) was cultured in a cell culture incubator at 37 ℃ and saturated humidity for 36 h. DOX preparation: final concentration 150 ng / mL.

[0060] (3) Addition of reagents: Dissolve compound 1 and compound 2 in DMSO to obtain a stock solution of 20 mg / mL. Add the solution to four replicates. Discard 100 mg / mL of the solution in the plate. m After adding liquid L, add 100 mg of the drug to each well. m L, the specific concentration is given in the corresponding plating scheme (see Table 3).

[0061] (4) Cell viability measurement: After culturing for 24 h with the drug, discard all the liquid in the well plate and add 100 g of blank medium mixed with MTT. m L (MTT:empty plate = 1:10). After culturing for 4 hours, discard the liquid in the well plate again, and add 100-150 mg DMSO to each well depending on the amount of crystals. m L, measure on the instrument. It is necessary to ensure that the amount of DMSO in each well is completely consistent.

[0062] (5) Experimental Results: The average OD values ​​of the experimental group, negative group, and blank group were taken to calculate the cell viability. The calculation formula was: Cell viability = (OD experiment - OD blank) / (OD control - OD blank) × 100%. GraphPadPrism software was used to analyze the experimental data. One-way ANOVA was used to perform statistical analysis on the comparison of data among multiple groups. All data were analyzed using... ±SD means, P A value < 0.05 indicates that the difference is statistically significant. represent P <0.05, represent P <0.01, represent P <0.001, (This represents p < 0.0001).

[0063] Anti-aging activity rescreening Based on the initial screening results, some compounds were further screened using more refined concentration gradients and multi-time dosing.

[0064] (1) The steps of plating and DOX-induced aging are the same as above.

[0065] (2) Addition of reagents: Dissolve the test compound 1 in DMSO to obtain a stock solution of 20 mg / mL, and discard 100 mg / mL of the solution in the well plate. m After adding liquid L, add 100 mg of the drug to each well. m L, incubate for 24 h.

[0066] (3) Semi-quantitative fluid replacement: Perform a semi-quantitative fluid replacement every 24 hours (applicable to 48 h and 72 h administration), discarding 100 μL of liquid in each well and adding 100 μL of drug back to each well. m L, the specific concentration is shown in the corresponding plating scheme (see Table 4), and the culture continues.

[0067] (4) Cell viability measurement: After culturing for 24 h, 48 h, and 72 h with the drug, discard all the liquid in the well plate and add 100 g of blank medium mixed with MTT. m L (MTT: empty culture = 1:10); after culturing for 4 hours, discard the liquid in the well plate again, and add 100-150 mg DMSO to each well depending on the amount of crystals. m L, measure on the instrument. It is necessary to ensure that the amount of DMSO in each well is completely consistent.

[0068] (5) Experimental results: The processing steps are the same as above.

[0069] Table 3 Anti-aging activity plating scheme (initial screening)

[0070] Note: Columns 2-6 show the plating scheme for compound 1, and columns 7-11 show the plating scheme for compound 2. Each concentration was repeated four times.

[0071] Table 4 Anti-aging activity plating scheme (secondary screening)

[0072] Initial screening results are as follows Figure 23 As shown: Compound 1 at 0.4, 4, and 40 m Compound 1 at a concentration of g / mL can delay DOX-induced senescence in iTEC cells; compound 2 has no effect on delaying senescence in iTEC cells. Therefore, compound 1 should be screened with more refined concentration gradients and multiple time-dependent administration.

[0073] The results of the second screening are as follows Figure 24 As shown: After 48 h of treatment with compound 1, the concentrations of 1.25 and 2.5... m g / mL P <0.05) and 20 m g / mL P <0.01) The cell viability of the treatment group was better than that of the blank control group; after 72 h of treatment, 1.25 mg / mL P <0.0001), 2.5 and 5 m g / mL P <0.001) and 20 m g / mL P Cell viability in the <0.01) treatment group was superior to that in the blank control group, with 1.25% showing the highest activity. m At a concentration of 1.25 g / mL, senescent iTEC cells showed the best activity. In summary, compound 1, after 72 h of treatment at a concentration of 1.25 g / mL, showed optimal activity. m The optimal concentration (g / mL) is the most effective at delaying iTEC cell senescence.

[0074] Both compounds 1 and 2 are guaiacolane-type sesquiterpenes, differing in their structure: the carbonyl group in compound 1 is located at C-3, with a double bond between C-4 and C-5; while in compound 2, the carbonyl group is located at C-2, with a double bond between C-1 and C-5. The anti-aging activity exhibited by compound 1 may be related to its unique carbonyl / double bond substitution pattern.

Claims

1. A guaiacolane-type sesquiterpene compound, characterized in that: The guaiacolane-type sesquiterpene compound is C 15 H 24 O3 has an unsaturation degree of 4.

2. The guaiacolane-type sesquiterpene compound according to claim 1, characterized in that: The C 15 H 24 O3 is ligulafischenolide A and / or ligulafischenolide B, wherein the chemical structural formula of ligulafischenolide A is as follows: ; The chemical structural formula of ligulafischenolide B is as follows: .

3. A method for preparing a guaiacolane-type sesquiterpene compound as described in any one of claims 1-2, characterized in that: Includes the following steps: (1) Take dried roots of Ligustrum lucidum, extract with ethanol, combine the extracts, concentrate under reduced pressure and then suspend in water; (2) The suspension was extracted sequentially with petroleum ether, ethyl acetate and n-butanol to obtain the ethyl acetate extract; (3) The ethyl acetate extract was separated and purified by normal phase silica gel column chromatography, and gradient elution was performed using dichloromethane-ethyl acetate as the eluent to obtain the fraction; (4) Combine the fractions Fr.54-66, separate and purify them by reversed-phase silica gel column chromatography, and perform gradient elution with methanol-water as the eluent to obtain the subfraction; (5) Subfraction Frr.19 was separated and purified by semi-preparative HPLC to obtain guaiacol-type sesquiterpenoid compounds.

4. The preparation method according to claim 3, characterized in that: The ethanol used in step (1) has a mass fraction of 92%, and the amount used is 10 times that of Ligusticum striatum. The cold extraction is performed 4 times.

5. The preparation method according to claim 3, characterized in that: The extraction of petroleum ether, ethyl acetate and n-butanol in step (2) is performed 6 times.

6. The preparation method according to claim 3, characterized in that: The mesh size of the normal silica gel column chromatography in step (3) is 200-300 mesh; the gradient of dichloromethane-ethyl acetate is... V 二氯甲烷 : V 乙酸乙酯 =100∶0, 50∶1, 30∶1, 20∶1, 10∶1, 5∶1, 3∶1, 2∶1, 1∶1, 1∶2.

7. The preparation method according to claim 3, characterized in that: The methanol-water gradient in step (4) is as follows: V 甲醇 : V 水 =30∶70, 50∶50, 70∶30, 90∶10, 100∶0.

8. The preparation method according to claim 3, characterized in that: The mobile phase of the semi-preparative HPLC in step (5) is V 甲醇 : V 水 =42∶58, flow rate is 3 mL / min.

9. The use of a guaiacolane-type sesquiterpene compound as described in any one of claims 1-2 in delaying cell aging.

10. The application according to claim 9, characterized in that: The cells in question are iTEC cells.

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