Preparation and lipid-lowering application of a tetranor-eudesmane sesquiterpene

Abstract

The present application relates to a preparation of a tetra-cinerin sesquiterpene and its application in preparing lipid-lowering products, and belongs to the field of traditional Chinese medicine and natural medicine pharmacy. The lipid-lowering drug with the tetra-cinerin sesquiterpene compound as an active ingredient. The present application expands the medicinal value of the cinerinsesquiterpene compound.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and natural medicine pharmaceutical manufacturing, specifically relating to a drug combination with a tetradecane sesquiterpene compound as the active ingredient, and its application in lipid-lowering agents. Background Technology

[0002] Eucalyptane sesquiterpenes are abundant in nature, especially in plants of the Asteraceae family. However, eucalyptane sesquiterpenes are rarely found in nature. Literature reports that eucalyptane sesquiterpenes possess cytotoxic, antibacterial, antifungal, anti-inflammatory, and insect repellent activities (Wu Q.-X., et al.). Natural Product Reports ,2006, 23(5): 699-734); The natural products with lipid-lowering activity currently discovered are mainly phenolic components and saponins (Singh SP, et al.). European Journal of Medicinal Chemistry , 2017,140: 331-348; Nie L., et al. Current Topics in Medicinal Chemistry , 2016, 16(23), 2605-2624; Bhutani KK, et al. Natural Product Communications , 2007,2(3), 331-348; Mahdavi A., et al. Biofactors ,2020, 46(6), 906-926). To date, no reports have been found on the lipid-lowering activity of eucalyptane sesquiterpenoids. Summary of the Invention

[0003] The present invention aims to provide a tetradecane sesquiterpene compound, pharmaceutical compositions thereof as active ingredients, methods for their preparation, and their use in the preparation of lipid-lowering agents.

[0004] The above-mentioned objective of the present invention is achieved through the following technical solution: The following structure shows tetra-noreucan sesquiterpene compound 1. .

[0005] The preparation method of compound 1 involves taking *Anacardiaceae* species (*Anacardia* genus)... Dobinea The underground parts or whole plant of the plant were directly cold-soaked or hot-refluxed with organic solvents such as chloroform, ethyl acetate, acetone, methanol, ethanol or water, or first cold-soaked or refluxed with the above organic solvents or water and then extracted with ethyl acetate to obtain the total extract. The total extract was subjected to repeated column chromatography to obtain compound 1.

[0006] The method for preparing compound 1 of the present invention more specifically uses: A: The underground parts or whole plant of the genus *Nyctaginata* were extracted with acetone, methanol, ethanol, or water by cold soaking or hot reflux to obtain the total extract. The extract was then extracted with ethyl acetate to obtain the ethyl acetate extract. Compound 1 was obtained by repeated column chromatography.

[0007] B: The crude powder of the underground parts or whole plant of the genus *Nyctaginata* was obtained by direct cold maceration or hot reflux extraction with organic solvents (such as chloroform, methanol, ethanol, acetone, dichloromethane, etc.). The total extract was then subjected to repeated column chromatography to obtain compound 1.

[0008] More specifically, the preparation method of compound 1 involves air-drying the underground parts or whole plant of *Hemiberlesia javanica*, pulverizing them to 30 mesh, extracting them three times with 95% ethanol at room temperature for 24 hours each time, combining the extracts, concentrating the extract under reduced pressure to obtain a paste, suspending it in an appropriate amount of water, and then partitioning it several times with ethyl acetate to obtain an ethyl acetate extract. The extract is dissolved in an appropriate amount of acetone, mixed with silica gel at 80-100 mesh, and then subjected to column chromatography at 200-300 mesh for fractional separation. Gradient elution is performed using 1:0-0:1 chloroform / acetone or 1:0-0:1 chloroform / methanol to obtain eight main fractions. The 1:0 chloro / acetone fraction, the 9:1 chloro / acetone fraction, and the 8:2 chloro / acetone fraction are then subjected to silica gel column chromatography at a 25:1-1:1 ratio. Compound 1 was obtained by gradient elution with petroleum ether / acetone followed by repeated silica gel, RP-18 and Sephadex LH-20 column chromatography.

[0009] Lipid-lowering agent, containing compound 1 and conventional excipients.

[0010] A pharmaceutical composition comprising a therapeutically effective amount of compound 1 and a pharmaceutically acceptable carrier.

[0011] Application of compound 1 in the preparation of lipid-lowering agents.

[0012] The application of compound 1 in the preparation of drugs for treating hyperlipidemia and obesity.

[0013] .

[0014] The preparation method of compound 1 involves taking *Anacardiaceae* species (*Anacardia* genus)... Dobinea The underground parts or whole plant of the plant were directly cold-soaked or hot-refluxed with organic solvents such as chloroform, ethyl acetate, acetone, methanol, ethanol or water, or first cold-soaked or refluxed with the above organic solvents or water and then extracted with ethyl acetate to obtain the total extract. The total extract was subjected to repeated column chromatography to obtain compound 1.

[0015] The method for preparing compound 1 of the present invention more specifically uses: A: The underground parts or whole plant of the genus *Nyctaginata* were extracted with acetone, methanol, ethanol, or water by cold soaking or hot reflux to obtain the total extract. The extract was then extracted with ethyl acetate to obtain the ethyl acetate extract. Compound 1 was obtained by repeated column chromatography.

[0016] B: The crude powder of the underground parts or whole plant of the genus *Nyctaginata* was obtained by direct cold maceration or hot reflux extraction with organic solvents (such as chloroform, methanol, ethanol, acetone, dichloromethane, etc.). The total extract was then subjected to repeated column chromatography to obtain compound 1.

[0017] More specifically, the preparation method of compound 1 involves air-drying the underground parts or whole plant of *Hemiberlesia javanica*, pulverizing them to 30 mesh, extracting them three times with 95% ethanol at room temperature for 24 hours each time, combining the extracts, concentrating the extract under reduced pressure to obtain a paste, suspending it in an appropriate amount of water, and then partitioning it several times with ethyl acetate to obtain an ethyl acetate extract. The extract is dissolved in an appropriate amount of acetone, mixed with silica gel at 80-100 mesh, and then subjected to column chromatography at 200-300 mesh for fractional separation. Gradient elution is performed using 1:0-0:1 chloroform / acetone or 1:0-0:1 chloroform / methanol to obtain eight main fractions. The 1:0 chloro / acetone fraction, the 9:1 chloro / acetone fraction, and the 8:2 chloro / acetone fraction are then subjected to silica gel column chromatography at a 25:1-1:1 ratio. Compound 1 was obtained by gradient elution with petroleum ether / acetone followed by repeated silica gel, RP-18 and Sephadex LH-20 column chromatography.

[0018] Lipid-lowering agent, containing compound 1 and conventional excipients.

[0019] A pharmaceutical composition comprising a therapeutically effective amount of compound 1 and a pharmaceutically acceptable carrier.

[0020] Application of compound 1 in the preparation of lipid-lowering agents.

[0021] The application of compound 1 in the preparation of drugs for treating hyperlipidemia and obesity.

[0022] The present invention relates to a pharmaceutical composition for hyperlipidemia and obesity, comprising compound 1 and a pharmaceutically acceptable carrier.

[0023] The pharmaceutically acceptable carrier described in the pharmaceutical compositions of this invention refers to a pharmaceutical carrier conventional in the pharmaceutical field. The compounds of this invention can be administered in combination to patients requiring this treatment via oral, nasal inhalation, rectal, or parenteral administration. For oral administration, they can be formulated as conventional solid dosage forms such as tablets, powders, granules, capsules, etc., or as liquid dosage forms such as oil suspensions, syrups, elixirs, etc.; for parenteral administration, they can be formulated as solutions for injection, etc. Preferred forms are tablets, capsules, and injections.

[0024] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient can be mixed with one or more carriers and then formulated into the desired dosage form.

[0025] The pharmaceutical composition of the present invention preferably contains 0.1%-99.5% by weight of an active ingredient, and most preferably contains 0.5%-95% by weight of an active ingredient.

[0026] The dosage of the compound of the present invention can vary depending on the route of administration, the patient's age, weight, the type and severity of the disease being treated, etc., and the daily dose can be 0.01-10 mg / kg body weight, preferably 0.1-5 mg / kg body weight. It can be administered once or multiple times.

[0027] The compounds of this invention exhibit good lipid-lowering activity.

[0028] This invention screened compound 1 for lipid-lowering activity, and this class of compounds showed good lipid-lowering activity. In the application of lipid-lowering activity, compound 1 is applied to a substrate or group of compounds in an amount ranging from 1 to 1000 µM, preferably from 10 to 200 µM, optionally in combination with a carrier and / or a medium. Detailed Implementation

[0029] The following embodiments of the present invention will further illustrate the substantive content of the present invention, enabling those skilled in the art to gain a more comprehensive understanding of the present invention, but without limiting the present invention in any way. Example 1:

[0030] Extraction, separation and purification of compound 1 of the present invention: The underground parts (9.6 kg) of *Gastrodia elata*, a plant of the *Gastrodia* genus, were air-dried, pulverized to 30 mesh, and extracted three times with 80% ethanol at room temperature, 50 L each time for 24 h. The extracts were combined and concentrated under reduced pressure to obtain an extract (1.8 kg). This extract was dissolved in acetone and adsorbed onto a suitable amount of 80-100 mesh silica gel. Then, it was subjected to column chromatography with 3.5 kg of 200-300 mesh silica gel for fractional separation. A gradient elution with chloroform / acetone (1:0-0:1) yielded eight main fractions. The 1:0 chloroform fraction, the 9:1 chloro / acetone fraction, and the 8:2 chloro / acetone fraction were subjected to silica gel column chromatography with a gradient elution of 25:1-1:2 petroleum ether / acetone, yielding 11 fractions. The fifth eluted fraction was subjected to repeated silica gel, RP-18, and Sephadex LH-20 column chromatography to obtain compound 1. Example 2:

[0031] Physical and spectroscopic data of compound 1 of this invention: Compound 1: a colorless oily substance.1 H-NMR (400 MHz, acetone- d 6) δ : 1.73 (1H, m, H-1a), 1.55 (1H, m, H-1b), 1.88 (1H, m, H-2a), 1.63 (1H, m, H-2b), 3.95 (1H, dd, J =10.9, 5.2 Hz, H-3), 2.39 (1H, t-like, J = 13.0 Hz, H-5), 2.38 (1H, t-like, J = 13.0 Hz, H-6a), 2.16 (1H, t-like, J = 13.0 Hz, H-6b), 2.12 (1H, d, J = 16.6Hz, H-9a), 2.00 (1H, d, J = 16.6 Hz, H-9b), 0.97 (3H, s, Me-14), 1.41 (3H, s, Me-15), 1.98 (3H, s, 4-OAc); 13 C-NMR (100 MHz, acetone- d 6) δ : 36.4 (t, C-1),29.6 (t, C-2), 76.1 (d, C-3), 90.1 (s, C-4), 51.4 (d, C-5), 37.5 (t, C-6),215.6 (s, C-7), 57.5 (t, C-9), 41.0 (s, C-10), 19.6 (q, C-14), 14.5 (q, C-15), 172.4 (s, 4-OAc), 22.3 (q, 4-OAc). HR-ESI-MS m / z 263.1255 [M+Na] + (calcdfor C 13 H 20 O4Na, 2633.1254). Example 3:

[0032] Detection of the lipid-lowering activity of the compounds of this invention: HepG2 cells were fed at a concentration of 1.5 × 10⁻⁶. 4Cells were seeded per well in 96-well plates and cultured at 37 °C with 5% CO2. When cell abundance reached 90%, the old culture medium was replaced with fresh culture medium containing 0.6 mM oleic acid and 0.3 mM sodium palmitate (oleic acid and sodium palmitate were bound with 40% bovine serum albumin). After 24 h of culture, 20 μL of the compound solution was added to each well of the test group, and 20 μL of the corresponding solvent was added to each well of the blank group (undeveloped cells) and the model group. After another 24 h of culture, the culture medium was carefully removed from the wells, and the cells were washed once with phosphate-buffered saline (PBS). 60 µL of 10% formaldehyde solution was added to each well. After 30 min of fixation, the fixative was removed, the cells were washed once with PBS, and 60 µL of 0.4 mg / mL Oil Red O working solution was added to each well for staining. After 60 min, the staining solution was removed, the cells were rinsed three times with PBS, and the intracellular lipid droplet staining was observed under an inverted microscope. Finally, add 200 μL of isopropanol to each well, dissolve thoroughly, and then measure the OD value of each group at 510 nm using a microplate reader. The lipid reduction rate is calculated using the following formula: Lipid reduction rate % = [1 − (OD value of test group − OD value of blank group) / (OD value of model group − OD value of blank group)] × 100.

[0033] Activity data are shown in Table 1.

[0034] Table 1. Lipid-lowering activity data of compound 1 (mean ± √3) s , n = 4)

[0035] Example 4: Tablets: Compound 1 obtained in Examples 1 and 2, 10 mg; lactose, 180 mg; starch, 55 mg; magnesium stearate, 5 mg. Preparation method: Mix the compound, lactose and starch, wet them evenly with propylene glycol, sieve and dry the wetted mixture, sieve it again, add magnesium stearate, and then compress the mixture into tablets, each weighing 250 mg and containing 10 mg of the compound. Example 5:

[0036] Ampoule preparation: 1-2 mg of the compounds obtained in Examples 1 and 2; Preparation method: Compound 1 obtained in Examples 1 and 2 was dissolved in 3 mL of propylene glycol, the resulting solution was filtered, and then filled into ampoules under sterile conditions. Example 6:

[0037] Capsules: Compound 1 obtained in Examples 1 and 2, 10 mg; lactose, 187 mg; magnesium stearate, 3 mg; Preparation method: Mix the compound with the excipients, sieve, mix evenly, and fill the resulting mixture into hard gelatin capsules, each capsule containing 200 mg of active ingredient and 10 mg of active ingredient.

Claims

1. Tetranor-cadinenic sesquiterpene compounds of the following structural formula, 。 2. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier.

3. A lipid-lowering agent comprising the compound of claim 1 and conventional adjuvants.

4. Use of the compound of claim 1 for the preparation of a lipid-lowering agent.

5. Use of the compound of claim 1 for the preparation of a medicament for the treatment of hyperlipidemia and obesity.

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