Compounds Isolated from Caryopteris terniflora, Preparation Method and Application Thereof

Abstract

This application relates to six clerodane diterpenoids isolated from Caryopteris terniflora Maxim. These compounds are obtained through methods such as ethanol extraction, elution, and chromatography. It has been confirmed by activity tests that the said compounds have applications in the preparation of estrogen biosynthesis inhibitors and proliferation inhibitors of MCF-7 breast cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical compounds, and particularly to compounds isolated from Caryopteris terniflora, and their preparation methods and applications. Background Art

[0002] Breast cancer seriously threatens the lives and health of women. Prevention, screening and treatment are important means to alleviate the current severe situation of breast cancer prevention and control. Among them, anti-breast cancer drugs with low toxicity and high efficiency are an effective guarantee for realizing breast cancer treatment and chronic disease management. [9] 。

[0003] Clinically, estrogen receptor (ER)-positive breast cancer is the main type, accounting for 60% of premenopausal breast cancer patients and 75% of postmenopausal breast cancer patients.

[10] Estrogen mainly refers to the steroid hormones secreted by the placenta and ovaries in the female body, which are converted from androgens by aromatase. Estrogen has a significant impact on the reproductive system, central nervous system, bone growth, body metabolism, etc.

[11] Excessively high local estrogen content in breast tissue is the main cause of breast cancer.

[12] 。

[0004] Aromatase (CYP19) is a member of the cytochrome P450 enzyme system, which can oxidize the substrates androstenedione and testosterone into estrone and estradiol respectively, and plays an important role in maintaining the homeostatic balance of estrogen in the body. Therefore, aromatase has become an ideal target for the treatment of breast cancer. At present, aromatase inhibitors (AIs) such as exemestane, letrozole, anastrozole, etc. have been widely used in the treatment of diseases such as breast cancer, endometrial cancer and polycystic ovary syndrome. [13-14] Existing studies have found that AIs can effectively inhibit the production of more than 90% of estrogen in postmenopausal women, and can significantly inhibit tumor progression in the comprehensive treatment of breast cancer, endometrial cancer and epithelial ovarian cancer. [15-16] Therefore, aromatase inhibitors have also received increasing attention in the treatment of breast cancer and have gradually become first-line drugs for the treatment of breast cancer. For example, exemestane can bind to the active site of aromatase, thereby reducing the estrogen level. [17-18] However, long-term use will lead to drug resistance, such as cross-resistance with non-steroidal AIs.

[19] Therefore, it is particularly important to discover new aromatase inhibitors with low toxicity, high efficiency, and low tolerance. The biological activity diversity, drug-likeness, and other characteristics of natural products, as well as their advantages such as definite curative effects and small toxic and side effects, have been widely recognized. Aromatase is an ideal target for treating breast cancer. Therefore, searching for unique and effective aromatase inhibitory active ingredients from natural products has become one of the effective ways to develop new anti-breast cancer drugs.

[0005] Caryopteris terniflora, also known as Liuyuehan, Lubianxue, Dafenghan Cao, Huagudan, etc., is a plant of the genus Caryopteris in the Verbenaceae family. It has the effects of relieving exterior cold, dispersing lung qi to relieve cough, and activating blood circulation to regulate menstruation. It is commonly used in folk medicine to treat cold headache, cough, chronic bronchitis, dysmenorrhea, etc.

[18] Although terpene components mainly composed of iridoids and diterpenes are the characteristic components and active components of Caryopteris plants [19-22] , there are limited reports on the terpene components in Caryopteris terniflora [23-26] , especially the application of this compound in the preparation of estrogen biosynthesis inhibitors and proliferation inhibitors of MCF-7 breast cancer cells has not been reported. Summary of the Invention

[0006] The object of the present invention is to provide compounds isolated from Caryopteris terniflora.

[0007] Another object of the present invention is to provide a preparation method of the compounds in Caryopteris terniflora.

[0008] Another object of the present invention is to provide the application of the provided compounds in the preparation of estrogen biosynthesis inhibitors and proliferation inhibitors of MCF-7 breast cancer cells.

[0009] The compounds isolated from Caryopteris terniflora provided by the present invention are 6 clerodane-type diterpenes: 16-oxo-14,15H-strictic acid (1), Jemesoniellide N (2), 15-oxo-14,16H-strictic acid (3), Strictic acid (4), Nor-strictic acid (5), Cleroinermin acid (6), and their structural formulas are:

[0010]

[0011] The preparation method of the compounds described in the present invention is as follows:

[0012] S1: Take 16 kg of dry crude powder of Caryopteris terniflora, and extract it with 5 - 35 L of 60 - 98% ethanol at room temperature for 1 - 6 times, each time for 2 - 12 days. Filter, combine the extracts, and recover ethanol under reduced pressure to obtain 1.725 kg of total extract. Disperse the total extract in 2 - 3 L of water to obtain a suspension of the extract and water. The suspension is successively extracted with petroleum ether, ethyl acetate, and n-butanol, and the solvents are recovered under reduced pressure to obtain the petroleum ether fraction P, the ethyl acetate fraction E, and the n-butanol fraction B;

[0013] S2: The petroleum ether fraction is subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate from 1:0 to 0:1. Detected by TLC, combine the same components to obtain 6 sub-components P1 - P6; Component P4 is subjected to silica gel column chromatography with petroleum ether / ethyl acetate 15:1 to obtain four sub-components P4A - P4D. P4A is recrystallized with petroleum ether - ethyl acetate (1:1) to obtain Compound 4;

[0014] S3: The ethyl acetate fraction E is subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate from 1:0 to 0:1. Detected by TLC, combine the same components to obtain 6 sub-components E1 - E6;

[0015] S4: Component E3 is subjected to gel column chromatography with dichloromethane / methanol 1:1. Detected by TLC, combine the same components to obtain 3 sub-components E3A - E3C; Among them, component E3B is subjected to silica gel column chromatography with petroleum ether / ethyl acetate 5:1. Detected by TLC, combine the same components to obtain 5 sub-components E3B1 - E3B5; Component E3B3 is subjected to gel column chromatography with dichloromethane / methanol 1:1 to obtain three sub-components E3B3A - E3B3C. Component E3B3B is subjected to silica gel column chromatography with dichloromethane / acetone 30:1 + formic acid (0.1%) to obtain Compound 1;

[0016] S5: E3C is subjected to gel column chromatography with dichloromethane / methanol 1:1. Detected by TLC, combine the same components to obtain 5 sub-components E3C1 - E3C5. Component E3C4 is subjected to silica gel column chromatography with petroleum ether / ethyl acetate 2:1. Detected by TLC, combine the same components to obtain Compound 5;

[0017] S6: Component E4 was subjected to gel column chromatography with dichloromethane / methanol 1:1. After TLC detection, the same components were combined to obtain 4 sub-components E4A - E4D. Component E4C was eluted with a dichloromethane / methanol 1:1 system in gel column chromatography. After TLC detection, the same components were combined to obtain 6 sub-components E4C1 - E4C6; Component E4C3 was subjected to gel column chromatography with dichloromethane / methanol 1:1. After TLC detection, the same components were combined to obtain 3 sub-components E4C3A - E4C3C. Among them, E4C3A was subjected to silica gel column chromatography with dichloromethane / ethyl acetate 10:1 to obtain Compound 3; Component E4C4 was subjected to silica gel column chromatography with petroleum ether / ethyl acetate 2:1. After TLC detection, the same components were combined to obtain 3 sub-components E4C4A - E4C4C; Component E4C4A was subjected to gel column chromatography with dichloromethane / methanol 1:1. After TLC detection, the same components were combined to obtain 5 sub-components E4C4A1 - E4C4A5; E4C4A2 was subjected to silica gel column chromatography with dichloromethane / acetone 35:1 to obtain Compound 2; Component E4C5 was subjected to silica gel column chromatography with petroleum ether / ethyl acetate 3:1. After TLC detection, the same components were combined to obtain 8 sub-components E4C5A - E4C5H. Among them, E4C5C was subjected to gel column chromatography with dichloromethane / methanol 1:1. After TLC detection, the same components were combined to obtain two sub-components E4C5C1 - E4C5C2. Component E4C5C1 was subjected to silica gel column chromatography with dichloromethane / ethyl acetate 20:1 to obtain Compound 6.

[0018] Preferably, in step S1, 10 - 30 L of 75 - 98% ethanol was used for extraction at room temperature for 1 - 5 times, 3 - 10 days each time.

[0019] More preferably, in step S1, 13 - 27 L of 80 - 95% ethanol was used for extraction at room temperature for 2 - 4 times, 4 - 9 days each time.

[0020] More preferably, in step S1, 20 L of 95% ethanol was used for extraction at room temperature for 3 times, 7 days each time.

[0021] In step S1, petroleum ether was used for extraction 2 - 5 times, 1 - 5 L each time, ethyl acetate was used for extraction 2 - 5 times, 1 - 5 L each time, and n-butanol was used for extraction 1 - 6 times, 1 - 5 L each time.

[0022] Preferably, in step S1, petroleum ether was used for extraction 3 - 5 times, 2 - 4 L each time, ethyl acetate was used for extraction 3 - 4 times, 2 - 4 L each time, and n-butanol was used for extraction 2 - 5 times, 2 - 4 L each time.

[0023] More preferably, in step S1, petroleum ether was used for extraction 4 times, 2 L each time, ethyl acetate was used for extraction 4 times, 2 L each time, and n-butanol was used for extraction 3 times, 2 L each time.

[0024] Use of the compound according to the present invention in the preparation of an estrogen biosynthesis inhibitor and a proliferation inhibitor of MCF-7 breast cancer cells.

[0025] The concentration of the compound according to the present invention in the preparation of an estrogen biosynthesis inhibitor is 20 μM; the concentration of the proliferation inhibitor of MCF-7 breast cancer cells is 20 μM.

[0026] The characteristics of the compound according to the present invention are respectively:

[0027] Compound 1: 16-oxo-14,15H-strictic acid 1 H NMR(600MHz,CDCl3)δ7.40(1H,d,J=2.0Hz,H-3),7.11(1H,d,J=1.3Hz,H-16),5.94(1H,dd,J=11.6,1.1Hz,H-2),5.43(1H,m,H-1),5.08(1H,s,H-19α),4.86(1H,s,H-19β),4.78(2H,d,J=1.7Hz,H-15),2.68(1H,d,J=14.0Hz,H-6α),2.31(1H,m,H-10α),2.21(2H,m,H-12),2.09(1H,td,J=13.7,2.0Hz,H-6β),1.81(1H,d,J=14.1Hz,H-10β),1.56(1H,t,J=13.5Hz,H-7α),1.48(2H,m,H-11),1.38(1H,dq,J=13.7,6.8Hz,H-8),0.84(1H,m,H-7β),0.77(3H,d,J=6.8Hz,H-17),0.72(3H,s,H-20); 13 C NMR(151MHz,CDCl3)δ174.57(C-14),171.43(C-18),144.98(C-4),144.04(C-3),143.97(C-16),136.54(C-5),135.43(C-13),127.88(C-1),127.88(C-2),118.42(C-19),70.54(C-15),38.24(C-9),36.18(C-10),35.95(C-8),35.46(C-11),34.12(C-6),29.48(C-7),20.72(C-12),18.89(C-20),14.16(C-17).

[0028] Compound 2: Jemesoniellide N 11H NMR (600 MHz, CD3OD) δ 7.19 (1H, d, H-3), 6.01 (1H, s, H-16), 5.96 (1H, d, J = 11.4 Hz, H-2), 5.88 (1H, s, H-14), 5.36 (1H, d, J = 11.9 Hz, H-1), 4.99 (1H, s, H-19α), 4.79 (1H, s, H-19β), 2.64 (1H, d, J = 13.4 Hz, H-6α), 2.45 (2H, t, J = 13.9 Hz, H-12), 2.36 (1H, t, J = 14.4 Hz, H-10β), 2.07 (1H, t, J = 13.6 Hz, H-6β), 1.78 (1H, d, J = 13.8 Hz, H-10α), 1.57 (2H, dd, J = 13.3, 1.4 Hz, H-7α / 8), 1.52–1.40 (2H, m, H-11), 0.85–0.81 (1H, m, H-7β), 0.80 (3H, s, H-17), 0.72 (3H, s, H-20); 13 13C NMR (151 MHz, CD3OD) δ 173.71 (C-13), 173.13 (C-15), 169.42 (C-18), 146.84 (C-4), 141.66 (C-5), 138.91 (C-3), 129.28 (C-2), 127.98 (C-1), 118.11 (C-14), 117.48 (C-19), 101.10 (C-16), 38.86 (C-9), 36.84 (C-8), 36.58 (C-10), 35.50 (C-11), 35.38 (C-6), 30.33 (C-7), 23.59 (C-12), 18.82 (C-20), 14.12 (C-17).

[0029] Compound 3: 15-oxo-14,16H-strictic acid 11H NMR (600 MHz, CD3OD) δ 7.21 (1H, dd, J = 2.1 Hz, H-3), 5.99 (1H, dd, J = 10.8 Hz, H-2), 5.90 (1H, dt, H-14), 5.44–5.34 (1H, m, H-1), 5.01 (1H, dd, H-19α), 4.89 (2H, d, J = 1.3 Hz, H-16), 4.81 (1H, s, br H-19β), 2.67 (1H, dd, J = 13.8, 1.5 Hz, H-6α), 2.52–2.44 (2H, m, H-12), 2.29 (1H, m, J = 13.3 Hz, H-10α), 2.09 (1H, td, J = 13.7, 2.3 Hz, H-6β), 1.81 (1H, ddd, H-10β), 1.68–1.56 (2H, m, H-7α / 11α), 1.55–1.43 (2H, m, H-8 / 11β), 0.88–0.83 (1H, m, H-7β), 0.82 (3H, d, J = 6.8 Hz, H-17), 0.75 (3H, s, H-20); 13 13C NMR (151 MHz, CD3OD) δ 177.12 (C-15), 175.24 (C-13), 175.24 (C-18), 146.82 (C-4), 141.61 (C-3), 129.30 (C-5), 127.93 (C-14), 118.09 (C-19), 115.03 (C-1), 115.03 (C-2), 75.00 (C-16), 38.87 (C-9), 36.79 (C-10), 36.56 (C-8), 35.91 (C-11), 34.93 (C-6), 30.32 (C-7), 24.41 (C-12), 18.83 (C-20), 14.13 (C-17).

[0030] Compound 4: Strictic acid 11H NMR (600 MHz, CDCl3) δ 7.41 (1H, s, H-3), 7.35 (1H, t, J = 1.5 Hz, H-16), 7.22 (1H, s, H-15), 6.28 (1H, s, H-14), 5.94 (1H, dd, J = 11.6, 1.1 Hz, H-2), 5.49 - 5.38 (1H, m, H-1), 5.10 (1H, t, J = 1.8 Hz, H-19α), 4.87 (1H, s, H-19β), 2.68 (1H, d, J = 13.8 Hz, H-6), 2.40 (2H, dd, J = 10.3, 6.9 Hz, H-12 / 12`), 2.23 (1H, t, J = 13.2 Hz, H-10), 2.10 (1H, td, J = 13.8, 2.3 Hz, H-6`), 1.84 (1H, d, J = 14.0 Hz, H-10`), 1.55 (2H, dt, J = 16.0, 10.2 Hz, H-11), 1.48 - 1.42 (1H, m, H-7α), 1.36 (1H, dt, J = 13.7, 6.9 Hz, H-8), 0.87 - 0.82 (1H, m, H-7β), 0.77 (3H, d, J = 6.8 Hz, H-17), 0.73 (3H, s, H-20); 13 13C NMR (151 MHz, CDCl3) δ 171.83 (C-18), 144.87 (C-4), 143.83 (C-1), 142.86 (C-15), 138.59 (C-16), 136.41 (C-5), 128.00 (C-2), 127.44 (C-3), 125.88 (C-13), 118.38 (C-19), 111.15 (C-14), 38.07 (C-6), 37.90 (C-9), 36.01 (C-8), 35.85 (C-10), 33.93 (C-12), 29.26 (C-7), 19.70 (C-11), 18.74 (C-20), 13.96 (C-17).

[0031] The structure of Compound 4 was also determined by single crystal diffraction.

[0032]

[0033] Compound 5: Nor-strictic acid 11H NMR (600 MHz, DMSO-d6) δ 12.29 (2H, s, H-13 / H-15), 7.09 (1H, m, H-3), 5.96 (1H, dd, J = 11.5, 1.1 Hz, H-2), 5.30 (1H, m, H-1), 5.00 (1H, m, H-16α), 4.75 (1H, d, J = 1.4 Hz, H-16β), 2.56 (1H, dd, J = 13.6, 1.4 Hz, H-12), 2.17 (2H, m, H-10), 2.09 (1H, dd, J = 16.2, 10.1 Hz, H-6α), 2.01 (1H, td, J = 13.5, 2.0 Hz, H-8), 1.63 (1H, d, J = 13.8 Hz, H-11α), 1.53 (1H, m, H-11β), 1.46 (1H, t, J = 12.9 Hz, H-7α), 1.34 (1H, m, H-7β), 1.29 (1H, dt, J = 16.0, 7.0 Hz, H-6β), 0.72 (3H, d, J = 6.8 Hz, H-14), 0.61 (3H, s, H-17); 13 13C NMR (151 MHz, DMSO-d6) δ 175.53 (C-13), 167.21 (C-15), 145.72 (C-4), 140.14 (C-1), 137.73 (C-5), 128.55 (C-2), 127.15 (C-3), 117.56 (C-16), 37.60 (C-9), 35.77 (C-8), 35.56 (C-10), 33.91 (C-11), 31.38 (C-12), 29.52 (C-6), 29.29 (C-7), 18.67 (C-17), 14.11 (C-14).

[0034] Compound 6: Cleroinermin acid 1 1H NMR (600 MHz, CDCl3) δ 6.85–6.79 (1H, m, H-3), 5.85 (1H, s, H-14), 4.74 (2H, s, H-16), 2.44 - 2.09 (6H, m, H-1 / 2 / 12), 1.70 - 1.64 (1H, m, H-11), 1.56 - 1.46 (4H, ddd, J = 17.7, 12.1, 5.0 Hz, H-6 / 7), 1.29 (1H, m, H-10), 1.26 (3H, m, H-19), 1.14 (1H, t, J = 12.4 Hz, H-8), 0.82 (3H, d, J = 5.7 Hz, H-17), 0.80 (3H, s, H-20); 1313C NMR (151 MHz, CDCl3) δ 173.97 (C-15), 171.41 (C-18), 170.73 (C-13), 141.29 (C-4), 139.85 (C-3), 115.18 (C-14), 73.06 (C-16), 46.73 (C-10), 38.80 (C-9), 37.60 (C-5), 36.34 (C-8), 35.72 (C-6), 35.44 (C-11), 27.41 (C-2), 27.11 (C-7), 22.26 (C-12), 20.50 (C-19), 18.19 (C-17), 17.49 (C-1), 15.92 (C-20).

[0035] Beneficial effects:

[0036] 1. For the compounds obtained by the preparation method of this application, the effects of the obtained clerodane diterpenoids on estrogen biosynthesis were evaluated using the KGN cell model. The results showed that at a safe concentration of 20 μM, both compounds 2 and 3 exhibited good inhibitory activity against estrogen biosynthesis, with inhibition rates of 49% and 44% respectively. At the same time, it was found that compounds 2 and 3 showed weak inhibitory effects on the proliferation of MCF-7 breast cancer cells, with inhibition rates of 26.3% and 20.9% respectively.

[0037] 2. Based on molecular docking analysis, it was found that both compounds 2 and 3 could bind well to the active pocket of aromatase: the carboxyl group at C-18 interacted with the heme-binding region of aromatase; the carboxyl group at C-18 and the lactone group at C-12 had hydrogen bond interactions with the key amino acid residues (such as Arg-115, Ala-306, Ala-307, Thr-310, and Met-374) of aromatase, and the affinity energies were -9.2 kcal / mol and -8.1 kcal / mol respectively. Description of the drawings

[0038] Figure 1 : Effects of clerodane diterpenoids in Caryopteris terniflora on estrogen biosynthesis

[0039] Figure 2 : Docking analysis of compounds 2 and 3 with aromatase

[0040] Figure 3 : Of compound 1 1 1H-NMR spectrum (600 MHz, CDCl3)

[0041] Figure 4 : Of compound 1 13 13C-NMR spectrum (151 MHz, CDCl3)

[0042] Figure 5 : 1H-NMR spectrum of Compound 2 1 (600 MHz, CD3OD)

[0043] Figure 6 : 1H-NMR spectrum of Compound 2 13 13C-NMR spectrum (151 MHz, CD3OD)

[0044] Figure 7 : 1H-NMR spectrum of Compound 3 1 (600 MHz, CDCl3)

[0045] Figure 8 : 1H-NMR spectrum of Compound 3 13 13C-NMR spectrum (151 MHz, CDCl3)

[0046] Figure 9 : 1H-NMR spectrum of Compound 4 (Y-20) 1 (600 MHz, CDCl3)

[0047] Figure 10 : 1H-NMR spectrum of Compound 4 13 13C-NMR spectrum (151 MHz, CDCl3)

[0048] Figure 11 : 1H-NMR spectrum of Compound 5 1 (600 MHz, DMSO-d6)

[0049] Figure 12 : 1H-NMR spectrum of Compound 5 13 13C-NMR spectrum (151 MHz, DMSO-d6)

[0050] Figure 13 : 1H-NMR spectrum of Compound 6 1 (600 MHz, CDCl3)

[0051] Figure 14 13C-NMR spectrum of Compound 6 13 (151 MHz, CDCl3) Detailed implementation manners

[0052] The technical solutions of the present invention will be further specifically described below through specific embodiments. Embodiment 1

[0053] The compounds isolated from Caryopteris terniflora Maxim. are 6 clerodane diterpenoids: 16-oxo-14,15H-strictic acid (1), Jemesoniellide N (2), 15-oxo-14,16H-strictic acid (3), Strictic acid (4), Nor-strictic acid (5), Cleroinermin acid (6), and their structural formulas are as follows:

[0054]

[0055] Example 2

[0056] Take 16 kg of dry crude powder of Caryopteris terniflora Maxim., and extract it with 20 L of 95% ethanol at room temperature for 3 times, 7 days each time. Filter, combine the 3 extraction solutions, and recover ethanol under reduced pressure to obtain 1.725 kg of total extract. The total extract is dispersed in 2.5 L of water to obtain a suspension of extract and water. The suspension is successively extracted with petroleum ether (4 times, 2 L each time), ethyl acetate (4 times, 2 L each time), and n-butanol (3 times, 2 L each time), and the solvents are recovered under reduced pressure to obtain the petroleum ether fraction (P, 320.0 g), ethyl acetate fraction (E, 219.0 g), and n-butanol fraction (B, 705.0 g).

[0057] The petroleum ether fraction is subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection is carried out, and the same components are combined to obtain 6 sub-fractions P1 - P6. Fraction P4 (112.3692 g) is subjected to silica gel column chromatography (petroleum ether / ethyl acetate 15:1) to obtain four sub-fractions P4A - P4D. P4A is recrystallized (petroleum ether - ethyl acetate 1:1) to obtain compound 4 (9.0 g).

[0058] The ethyl acetate fraction (E, 219.0 g) is subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection is carried out, and the same components are combined to obtain 6 sub-fractions E1 - E6.

[0059] Fraction E3 (14.0 g) is subjected to gel column chromatography (dichloromethane / methanol 1:1). TLC detection is carried out, and the same components are combined to obtain 3 sub-fractions E3A - E3C. Among them, fraction E3B (2.1288 g) is subjected to silica gel column chromatography (petroleum ether / ethyl acetate 5:1). TLC detection is carried out, and the same components are combined to obtain 5 sub-fractions E3B1 - E3B5. Fraction E3B3 is subjected to gel column chromatography (dichloromethane / methanol 1:1) to obtain three sub-fractions E3B3A - E3B3C. Fraction E3B3B is subjected to silica gel column chromatography (dichloromethane / acetone 30:1 + 0.1% formic acid) to obtain compound 1 (13.0 mg).

[0060] E3C was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 5 sub-components E3C1 - E3C5. Component E3C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), detected by TLC, and the same components were combined to obtain compound 5 (12.0 mg).

[0061] Component E4 (30.0 g) was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 4 sub-components E4A - E4D. Component E4C (13.1491 g) was eluted with a system of gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 6 sub-components E4C1 - E4C6. Component E4C3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C3A - E4C3C. Among them, E4C3A was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 10:1) to obtain compound 3 (18.0 mg); Component E4C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C4A - E4C4C. Component E4C4A was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 5 sub-components E4C4A1 - E4C4A5. E4C4A2 was subjected to silica gel column chromatography (dichloromethane / acetone 35:1) to obtain compound 2 (8.0 mg). Component E4C5 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 3:1), detected by TLC, and the same components were combined to obtain 8 sub-components E4C5A - E4C5H. Among them, E4C5C was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain two sub-components E4C5C1 - E4C5C2. Component E4C5C1 was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 20:1) to obtain compound 6 (11.0 mg).

[0062] Example 3

[0063] 16 kg of dry crude powder of Caryopteris terniflora was extracted twice with 10 L of 75% ethanol at room temperature for 3 days each time. After filtration, the three extraction solutions were combined, and ethanol was recovered under reduced pressure to obtain the total extract. The total extract was dispersed in 2 L of water to obtain a suspension of the extract and water. The suspension was successively extracted with petroleum ether (2 times, 2 L each time), ethyl acetate (2 times, 2 L each time), and n-butanol (2 times, 2 L each time), and the solvents were recovered under reduced pressure to obtain the petroleum ether fraction P, ethyl acetate fraction E, and n-butanol fraction B.

[0064] The petroleum ether fraction was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate (1:0 → 0:1). Detection by TLC was carried out, and the same components were combined to obtain 6 sub-fractions P1 - P6. Fraction P4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 15:1) to obtain four sub-fractions P4A - P4D. Compound 4 was obtained by recrystallization of P4A (using petroleum ether - ethyl acetate 1:1).

[0065] Fraction E of ethyl acetate was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate (1:0 → 0:1). Detection by TLC was carried out, and the same components were combined to obtain 6 sub-fractions E1 - E6.

[0066] Fraction E3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), and detection by TLC was carried out. The same components were combined to obtain 3 sub-fractions E3A - E3C. Among them, fraction E3B was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 5:1), and detection by TLC was carried out. The same components were combined to obtain 5 sub-fractions E3B1 - E3B5. Fraction E3B3 was subjected to gel column chromatography (dichloromethane / methanol 1:1) to obtain three sub-fractions E3B3A - E3B3C. Compound 1 was obtained after fraction E3B3B was subjected to silica gel column chromatography (dichloromethane / acetone 30:1 + 0.1% formic acid).

[0067] E3C was subjected to gel column chromatography (dichloromethane / methanol 1:1), and detection by TLC was carried out. The same components were combined to obtain 5 sub-fractions E3C1 - E3C5. Fraction E3C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), and detection by TLC was carried out. Compound 5 was obtained after combining the same components.

[0068] Component E4 was subjected to gel column chromatography (dichloromethane / methanol 1:1), and TLC detection was carried out. The same components were combined to obtain 4 sub-components E4A - E4D. Component E4C was eluted with a system of gel column chromatography (dichloromethane / methanol 1:1), and TLC detection was carried out. The same components were combined to obtain 6 sub-components E4C1 - E4C6. Component E4C3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), and TLC detection was carried out. The same components were combined to obtain 3 sub-components E4C3A - E4C3C. Among them, compound 3 was obtained from E4C3A by silica gel column chromatography (dichloromethane / ethyl acetate 10:1); Component E4C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), and TLC detection was carried out. The same components were combined to obtain 3 sub-components E4C4A - E4C4C. Component E4C4A was subjected to gel column chromatography (dichloromethane / methanol 1:1), and TLC detection was carried out. The same components were combined to obtain 5 sub-components E4C4A1 - E4C4A5. Compound 2 was obtained from E4C4A2 by silica gel column chromatography (dichloromethane / acetone 35:1). Component E4C5 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 3:1), and TLC detection was carried out. The same components were combined to obtain 8 sub-components E4C5A - E4C5H. Among them, component E4C5C was subjected to gel column chromatography (dichloromethane / methanol 1:1), and TLC detection was carried out. The same components were combined to obtain two sub-components E4C5C1 - E4C5C2. Compound 6 was obtained from E4C5C1 by silica gel column chromatography (dichloromethane / ethyl acetate 20:1).

[0069] Example 4

[0070] 16 kg of dry Crinopodium ternatum rough powder was extracted with 30 L of 98% ethanol at room temperature for 5 times, each time for 10 days. After filtration, the extraction solutions were combined, and ethanol was recovered under reduced pressure to obtain the total extract. The total extract was dispersed in 3 L of water to obtain a suspension of the extract and water. The suspension was successively extracted with petroleum ether (5 times, 5 L each time), ethyl acetate (5 times, 5 L each time), and n-butanol (3 times, 5 L each time). The solvents were recovered under reduced pressure to obtain the petroleum ether fraction P, the ethyl acetate fraction E, and the n-butanol fraction B.

[0071] The petroleum ether fraction was subjected to silica gel column chromatography and gradient elution was carried out with petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection was carried out, and the same components were combined to obtain 6 sub-components P1 - P6. Component P4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 15:1) to obtain four sub-components P4A - P4D. Compound 4 was obtained from P4A by recrystallization (petroleum ether - ethyl acetate in a ratio of 1:1).

[0072] The ethyl acetate fraction E was subjected to silica gel column chromatography and gradient elution was carried out with petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection was carried out, and the same components were combined to obtain 6 sub-components E1 - E6.

[0073] Component E3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 3 sub-components E3A - E3C. Among them, component E3B was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 5:1), detected by TLC, and the same components were combined to obtain 5 sub-components E3B1 - E3B5. Component E3B3 was subjected to gel column chromatography (dichloromethane / methanol 1:1) to obtain three sub-components E3B3A - E3B3C, and component E3B3B was subjected to silica gel column chromatography (dichloromethane / acetone 30:1 + 0.1% formic acid) to obtain compound 1.

[0074] E3C was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 5 sub-components E3C1 - E3C5. Component E3C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), detected by TLC, and the same components were combined to obtain compound 5.

[0075] Component E4 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 4 sub-components E4A - E4D. Component E4C was eluted with the system of gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 6 sub-components E4C1 - E4C6. Component E4C3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C3A - E4C3C. Among them, E4C3A was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 10:1) to obtain compound 3; Component E4C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C4A - E4C4C. Component E4C4A was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 5 sub-components E4C4A1 - E4C4A5. E4C4A2 was subjected to silica gel column chromatography (dichloromethane / acetone 35:1) to obtain compound 2. Component E4C5 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 3:1), detected by TLC, and the same components were combined to obtain 8 sub-components E4C5A - E4C5H. Among them, E4C5C was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain two sub-components E4C5C1 - E4C5C2. Component E4C5C1 was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 20:1) to obtain compound 6.

[0076] Example 5

[0077] Take 16 kg of dry crude powder of Caryopteris terniflora, and extract it with 27 L of 95% ethanol at room temperature for 4 times, each time for 9 days. Filter, combine the extracts, and recover ethanol under reduced pressure to obtain the total extract. The total extract is dispersed in 2.5 L of water to obtain a suspension of the extract and water. The suspension is successively extracted with petroleum ether (4 times, 4 L each time), ethyl acetate (4 times, 4 L each time), and n-butanol (2 times, 3 L each time), and the solvents are recovered under reduced pressure to obtain the petroleum ether fraction P, the ethyl acetate fraction E, and the n-butanol fraction B.

[0078] The petroleum ether fraction is subjected to silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection is carried out, and the same components are combined to obtain 6 sub-fractions P1 - P6. Fraction P4 is subjected to silica gel column chromatography (petroleum ether / ethyl acetate 15:1) to obtain four sub-fractions P4A - P4D. Compound 4 is obtained by recrystallization (petroleum ether - ethyl acetate in a ratio of 1:1) of P4A.

[0079] The ethyl acetate fraction E is subjected to silica gel column chromatography and gradient eluted with petroleum ether / ethyl acetate (1:0 → 0:1). TLC detection is carried out, and the same components are combined to obtain 6 sub-fractions E1 - E6.

[0080] Fraction E3 is subjected to gel column chromatography (dichloromethane / methanol 1:1). TLC detection is carried out, and the same components are combined to obtain 3 sub-fractions E3A - E3C. Among them, fraction E3B is subjected to silica gel column chromatography (petroleum ether / ethyl acetate 5:1). TLC detection is carried out, and the same components are combined to obtain 5 sub-fractions E3B1 - E3B5. Fraction E3B3 is subjected to gel column chromatography (dichloromethane / methanol 1:1) to obtain three sub-fractions E3B3A - E3B3C. Compound 1 is obtained after fraction E3B3B is subjected to silica gel column chromatography (dichloromethane / acetone 30:1 + 0.1% formic acid).

[0081] E3C is subjected to gel column chromatography (dichloromethane / methanol 1:1). TLC detection is carried out, and the same components are combined to obtain 5 sub-fractions E3C1 - E3C5. Fraction E3C4 is subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1). TLC detection is carried out, and the same components are combined to obtain compound 5.

[0082] Component E4 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 4 sub-components E4A - E4D. Component E4C was eluted with a system of gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 6 sub-components E4C1 - E4C6. Component E4C3 was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C3A - E4C3C. Among them, E4C3A was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 10:1) to obtain Compound 3; Component E4C4 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 2:1), detected by TLC, and the same components were combined to obtain 3 sub-components E4C4A - E4C4C. Component E4C4A was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain 5 sub-components E4C4A1 - E4C4A5. E4C4A2 was subjected to silica gel column chromatography (dichloromethane / acetone 35:1) to obtain Compound 2. Component E4C5 was subjected to silica gel column chromatography (petroleum ether / ethyl acetate 3:1), detected by TLC, and the same components were combined to obtain 8 sub-components E4C5A - E4C5H. Among them, E4C5C was subjected to gel column chromatography (dichloromethane / methanol 1:1), detected by TLC, and the same components were combined to obtain two sub-components E4C5C1 - E4C5C2. Component E4C5C1 was subjected to silica gel column chromatography (dichloromethane / ethyl acetate 20:1) to obtain Compound 6.

[0083] Example 6

[0084] Application of the compound in the preparation of an estrogen biosynthesis inhibitor and a proliferation inhibitor of MCF-7 breast cancer cells.

[0085] Example 7

[0086] The concentration of the compound in the preparation of an estrogen biosynthesis inhibitor is 20 μM; the concentration of the proliferation inhibitor of MCF-7 breast cancer cells is 20 μM.

[0087] Experimental Example

[0088] I. Operating Methods and Reagent Manufacturers

[0089] (1) Separating Fillers and Manufacturers

[0090] Silica gel: 200 - 300 mesh, Qingdao Ocean Chemical Co., Ltd.

[0091] Sephadex LH20: GE Healthcare Bio-Sciences AB, Sweden

[0092] Solvents: Commercially available analytical pure reagents.

[0093] (2) Activity

[0094] 1. Activity determination

[0095] A. Cell culture: KGN cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, 5% CO2 cell culture incubator. When the cells grew to the logarithmic phase, subculture was performed. Cells in good condition were selected for the experiment.

[0096] B. Cell proliferation experiment: A 96-well culture plate was taken, and KGN cells in the logarithmic growth phase were inoculated into each well (100 μL, density of 3×10 4 cells / mL). After that, they were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin for 24 h, and then different concentrations of the test samples were added. DMSO was used as the negative control. After culturing in a 5% CO2 incubator at 37°C for 24 h, 20 μL of MTT solution (5 mg / mL) was added to each well and cultured for another 4 h. After that, the supernatant was carefully aspirated, 150 μL of DMSO was added to each well, and the culture was placed in the incubator for 15 min to completely dissolve the purple crystals. The absorbance at a wavelength of 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0097] C. Estrogen content determination: A 24-well culture plate was taken, and KGN cells in the logarithmic growth phase were inoculated into each well (1 mL, density of 1×10 5 cells / mL). After that, they were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin for 24 h, and then DMEM / F12 medium containing 1% penicillin / streptomycin with the test sample (20 μM) was added. DMSO was used as the control, FSK was used as the positive control (50 μM), and FOR (50 μM) was used as the negative control. After culturing in a 5% CO2 incubator at 37°C for 24 h, 10 nM testosterone was added, and the cells were cultured in a 5% CO2 incubator at 37°C for 24 h. The supernatant was aspirated into a sterile ep tube as the test sample, and the total protein concentration was measured by lysing the cells for result correction.

[0098] Determination of estradiol content by enzyme-linked immunosorbent assay: Centrifuge the sample to be tested at 4000 rpm for 20 min at 4°C, and aspirate the supernatant for testing. Take out the enzyme plate strips, lyophilized standards, concentrated HRP enzyme conjugate (100×), standard & sample diluent, HRP enzyme conjugate diluent, concentrated washing solution, substrate solution, reaction termination solution, and sealing film, and equilibrate them at room temperature for 20 min. Centrifuge the lyophilized standards at 10000 rpm for 1 min at 4°C, add 1 mL of standard & sample diluent, let it stand for 10 min, and invert it several times up and down for gradient dilution; dilute the concentrated HRP enzyme conjugate with HRP enzyme conjugate diluent 15 min before use. Set the standard wells, blank wells, and sample wells respectively. Add 50 μL of gradient-diluted standards to the standard wells, add 50 μL of standard & sample diluent to the blank wells, add 50 μL of the sample to be tested to the sample wells, immediately add 50 μL of the diluted HRP enzyme conjugate diluent, heat it at a constant temperature of 37°C for 60 min, take it out, add 350 μL of the diluted washing solution to each well for washing, 5 times (spin dry the washing solution in the wells after each wash), after completion, add 90 μL of the substrate solution, cover it with a sealing film, and heat it at 37°C in the dark for 15 min. Preheat the microplate reader 15 min in advance, and measure the absorbance value at 450 nm absorbance.

[0099] Protein concentration determination: Rinse the cells 3 times with PBS, add 20 μL of RIRP lysis buffer, lyse them on ice for 30 min, aspirate the lysate, centrifuge it at 10000 rpm for 20 min at 4°C, aspirate the supernatant, and dilute the supernatant 20-fold with PBS. Prepare the standards by adding 21 μL of BSA standard solution to 189 μL of PBS for dilution. Prepare the BCA solution and Cu reagent in a ratio of 50:1. Take a 96-well plate, add 0, 2, 4, 6, 8, 12, 16, 20 μL of standards, make up the volume to 20 μL with PBS, add 20 μL of the sample solution to be tested, set 3 replicates, add 200 μL of BCA reagent in the order of adding samples, put it in an incubator at 37°C for 30 min, measure the absorbance value at 562 nm absorbance, and calculate the protein content.

[0100] 4. Reagents for activity determination and estrogen determination

[0101] DMEM / F12 complete medium: Procell

[0102] MTT kit, penicillin, streptomycin: Sevier

[0103] Human estradiol enzyme-linked immunosorbent assay kit: Elabscience

[0104] BCA protein assay kit, RIRP lysis buffer, protease inhibitor: Solarbio

[0105] II. Activity Evaluation

[0106] The applicant further evaluated the effect of the obtained clerodane diterpenoids on estrogen biosynthesis using the KGN cell model. The results showed that both 2 and 3 exhibited good inhibitory activities against estrogen biosynthesis at a safe concentration of 20 μM (Figure 1A), with inhibition rates of 49% and 44% respectively (Figure 1B). Meanwhile, it was found that compounds 2 and 3 showed weak inhibitory effects on the proliferation of MCF-7 breast cancer cells, with inhibition rates of 26.3% and 20.9% respectively.

[0107] Based on molecular docking analysis, it was found that both compounds 2 and 3 could bind well to the active pocket of aromatase: the carboxyl group at C-18 interacted with the heme-binding region of aromatase; there were hydrogen bond interactions between the carboxyl group at C-18 and the lactone group at C-12 and the key amino acid residues (such as Arg-115, Ala-306, Ala-307, Thr-310, and Met-374) of aromatase (Figure 2), and the affinity energies were -9.2 kcal / mol and -8.1 kcal / mol respectively.

[0108] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

[0109] References

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Claims

1. Use of a compound isolated from Caryopteris terniflora, characterized in that, The compound is a clerodane diterpene with the structural formula as follows: ； Use of the compound in the preparation of an estrogen biosynthesis inhibitor and a proliferation inhibitor of MCF-7 breast cancer cells.

2. Use of the compound according to claim 1, characterized in that, The concentration of the compound in the preparation of the estrogen biosynthesis inhibitor is 20 μM; the concentration of the proliferation inhibitor of MCF-7 breast cancer cells is 20 μM.

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