CN102516213B - Sesquiterpene compound and preparation method and application thereof - Google Patents

Abstract

The invention discloses a sesquiterpene compound and a preparation method and application thereof. The molecular formula of the sesquiterpene compound is C15H18O4, and the preparation method of the sesquiterpene compound comprises the steps: adding a whole chloranthus henryi (Chlotanthushenryi Hemsl.) or tissue of the chloranthus henryi into an organic solvent to be leached to obtain a leaching solution, extracting the leaching solution by using ethyl acetate or chloroform, and concentrating, separating and purifying extraction liquid to obtain the sesquiterpene compound, wherein the organic solvent is one of or two of carbinol, ethanol, ethyl acetate and acetone. The sesquiterpene compound extracted and separated from the chloranthus henryi is provided with a novel structure, has good antioxidative activity, can be used for preparing antioxidant pharmaceuticals, antioxidant cosmetics and antioxidant health-care foods and has good developing prospect.

Description

A kind of sesquiterpene compound and its preparation method and application

technical field

The invention relates to the field of plant active ingredients, in particular to a sesquiterpene compound and its preparation method and application.

Background technique

Chloranthus henryi Hemsl. is a plant of the genus Chloranthus in the family Chloranthaceae. Chrysanthemum broadleaf is a perennial herb or subshrub, 40-65cm high (even as high as 100cm), rhizomes are thick, dark brown, with several slender brown fibrous roots; stems are erect, nodes are obviously enlarged, leaves are opposite, usually 4 The slices are born on the upper part of the stem, occasionally there are 6 slices (in whorls) broadly elliptic or obovate, 9-18cm long, 5-9cm wide, inflorescences spicate terminal and axillary, usually dichotomous or raceme branched, drupe Spherical; flowering and fruiting period from April to November, born in wetlands under hillsides and grass beside valleys. Distributed in Jiangsu, Anhui, Jiangxi, Fujian, Hunan, Hubei, Guangdong, Guangxi, Sichuan, Guizhou, Zhejiang, Shaanxi, Gansu, etc. ): 212-213.). Chrysanthemum broadleaves has the functions of expelling wind, dehumidification, promoting blood circulation, and removing blood stasis. It can be used to treat wind-cold-damp arthralgia, rheumatic numbness, pain, irregular menstruation, bruises, wind-cold cough, and carbuncle sores.

The phytochemical components of Chrysanthemum genus have been studied more, mainly sesquiterpene compounds, which have good medicinal prospects. As early as 1978, Masaaki Uchida et al. isolated two cycloeucalyptane-type sesquiterpene lactones from the grass coral Chloranthus glaber Makino, named chloranthalactone A and B (Uchida M, Kusano G, Kondo Y, Nozoe S, Takemoto T Two newsesquiterpenoids from Chloranthus glaber Makino. Heterocycles, 1978, 9(2): 139-44.).

In 1979 and 1981, Jun Kawabata et al. reported the isolation of two cycloeucalyptane-type sesquiterpenes, shizukanolide A and dehydro-shizukanolide (shizukanolide B), from Silverwort, among which dehydro-shizukanolide is chloranthalactone A reported by Masaaki Uchida. Activity test results show that dehydro-shizukanolide has moderate antifungal activity ((a) Kawabata J, Tahara S, Mizutani J, Furusaki A, Hashiba N, Matsumoto T. Shizukanolides, two sesquiterpenoids from Chloranthus japonicus (Chloranthaceae).Agric.Biol.Chem ., 1979, 43 (4): 885-887. (b) Kawabata J, Tahara S, Mizutani J. Isolation and structure elucidation of four sesquiterpenes from Chloranthus japonicus (Chloranthaceae). Agric. Bioc. Chem., 1981, 45 (6 ): 1447-1453.).

Masaaki Uchida et al. isolated five cycloeucalyptane-type sesquiterpenes from silvergrass produced in Japan, namely chloranthalactone AE. The absolute configurations of chloranthalactone A and B were deduced from the ORD curves determined by degradation. Chloranthalactone AC showed strong cytotoxic effect on mouse Lyphosarcima L-5178Y cells with _IC50 of 2.5, 1.0-2.5 and 20 μg/mL (Uchida M, Koike Y, Kusano G, Kondo Y, Nozoe S, Kabuto C , Takemoto T. Studies on the constituents of Chloranthus spp. III. Sixsesquiterpenes from Chloranthus japonicus. Chem. Pharm. Bull., 1980, 28(1): 92-102.).

Contents of the invention

The invention provides a sesquiterpene compound, which is a natural active substance extracted from Chrysanthemum broadleaf plant and has antioxidant activity.

A sesquiterpene compound, its structural formula is shown in formula (I):

The invention provides the preparation method of described sesquiterpene compound, comprises the following steps:

(1) adding the whole plant of Chloranthus henryi Hemsl. or its tissues into an organic solvent for extraction to obtain an extract;

Wherein, described organic solvent is one or both in methanol, ethanol, ethyl acetate and acetone;

(2) The extract is extracted with ethyl acetate or chloroform, and the extract is concentrated, separated and purified to obtain the sesquiterpene compound.

In step (1), the whole plant or tissue of Chrysanthemum broadleaf can be the whole plant or tissue of a naturally growing wild plant; it can also be the whole plant or tissue of a plant cultured through tissue culture, that is, it can be Tissue culture of Phalaenopsis phylloxera. The sesquiterpene compound of the present invention is extracted by using the tissue culture of Chrysanthemum broadleaf obtained by rapid propagation through tissue culture as a raw material, which has the advantages of short raw material culture period and stable source.

The tissue culture can be prepared by the following method: take the stem tip, budded stem segment or leaf of Chrysanthemum broadleaf as explants, inoculate them in induction medium after disinfection, and induce callus tissue and bud clusters ; then inoculated in subculture medium (MS+NAA 0.2, MS+6BA 0.02+NAA 0.5 or MS+6BA 2+NAA 0.2) for proliferation culture; after proliferation culture, transfer to rooting medium to induce rooting and obtain tissue culture things.

The said tissue can be the root, stem and leaf of wild or tissue-cultured Chrysanthemum broadleaf.

The whole plant of Chrysanthemum broadleaf or its tissues can be added directly or pulverized into an organic solvent for leaching, and the leaching after the raw material is pulverized is more conducive to the leaching of sesquiterpene compounds.

The weight ratio of the whole plant of Chrysanthemum broadleaf or its tissue to the organic solvent is preferably 1:1-1:8.

The extraction can be cold soaking or hot reflux extraction.

In step (2), the separation and purification can be as follows: the extract is subjected to silica gel column chromatography, and the obtained fraction is subjected to recrystallization, reversed-phase normal pressure chromatography or high performance liquid chromatography. Through multi-step separation and purification, sesquiterpene compounds with higher purity can be obtained.

Preferably, the silica gel column chromatography separation is as follows: using one or more mixtures of petroleum ether, n-hexane, ethyl acetate, alcohol and water as eluents, and performing gradient elution on the extract.

More preferably, the silica gel column chromatography is separated as follows: petroleum ether/ethyl acetate mixed solution and ethyl acetate in volume ratios of 9:1, 5:1, 1:1, 1:3, 1:9 Gradient elution is carried out on the extract; alternatively, the gradient elution is carried out on the extract with n-hexane/ethyl acetate mixed solution with a volume ratio of 9:1, 5:1, 1:1, 1:3, and 1:9 in sequence.

The present invention also provides the application of the sesquiterpene compound in the preparation of antioxidant drugs. The medicine takes the sesquiterpene compound of the present invention as the main active ingredient and is made by adding pharmaceutically acceptable auxiliary materials, and can be prepared according to the preparation method recorded in pharmacy. The preparations can be injections, drips, powder injections, granules, tablets, granules, powders, oral liquids, sugar-coated tablets, film-coated tablets, enteric-coated tablets, buccal preparations, granules , pills, ointments, pills, sprays, dropping pills, disintegrants, orally disintegrating tablets, pellets, etc.

The invention also provides the application of the sesquiterpene compound in the preparation of antioxidant cosmetics. The cosmetic is prepared by taking the sesquiterpene compound of the present invention as the main active ingredient and adding acceptable cosmetic auxiliary materials.

The invention also provides the application of the sesquiterpene compound in the preparation of antioxidant health food. The health food is prepared by taking the sesquiterpene compound of the invention as the main active ingredient and adding acceptable health food auxiliary materials.

The invention utilizes the difference in polarity of sesquiterpene to extract and separate a sesquiterpene compound with a novel structure from Chrysanthemum broadleaf. The method is simple to operate, has high extraction yield and high product purity, and is suitable for large-scale production.

Through the in vitro antioxidant test, it is shown that the sesquiterpene compound provided by the present invention has a good antioxidant effect, and its _IC50 value is 36 μ M, which can be used to prepare antioxidant drugs, antioxidant cosmetics or antioxidant health food, and has good development potential. prospect.

Detailed ways

Tissue culture and rapid propagation of embodiment 1 Chrysanthemum broadleaf

Take the leaves of Chloranthus henryi Hemsl. as explants, and carry out disinfection according to the following procedure: take materials→roughly wash with tap water→rinse with 5% aqueous solution of washing powder for 5min→rinse with tap water for 30min→scrub the surface with 75% ethanol→0.1% liter Sterilize in mercury solution for 5 minutes → sterilize in 2% sodium hypochlorite solution for 20 minutes → rinse with sterile water 4 to 6 times, then cut the explants into 0.5 to 1.0 cm long sections in a sterile workbench; inoculate them in the induction medium, The induced bud clusters were separated and cut into 2cm stem segments, transferred to a new medium with different hormone combinations (MS+6BA 2+NAA0.2) for subculture, and given all-day light after inoculation, room temperature 20 -28°C; after 3 days, the bases all began to swell, and after 1 week, the axillary buds began to germinate, and the sprouts grew rapidly; when the buds grew to 3-4 cm, cut the strong plants and transfer them to the rooting medium, and all the plants could be normal After one week of cultivation, roots are induced. After 10 days, each plant can grow 3 to 5 roots, and the root length is 4 to 5cm. After another week of cultivation, the plants are strong and the leaves are dark green. Then remove the paper cover and practice seedlings. .

The preparation of embodiment 2 sesquiterpene compounds

120mm)，依次以体积比9∶1、5∶1、1∶1、1∶3、1∶9的石油醚/乙酸乙酯混合液和乙酸乙酯进行梯度洗脱，每次5L；TLC检测馏分；再用甲醇重结晶。Take 5kg of Chrysanthemum broadleaves, soak in methanol for 2 weeks, suspend the soaking solution with 1L of distilled water after concentration, extract the aqueous suspension with 1L of ethyl acetate, and concentrate the ethyl acetate extract to obtain 110g of extract; use silica gel (100 mesh, 100g) mixed sample, carry out normal phase silica gel column chromatography separation (200-300 mesh, 1kg; silica gel column size L 500mm,

120mm), followed by a volume ratio of 9:1, 5:1, 1:1, 1:3, 1:9 petroleum ether/ethyl acetate mixture and ethyl acetate for gradient elution, each 5L; TLC detection Fraction; recrystallization from methanol.

The preparation of embodiment 3 sesquiterpene compounds

120mm)，依次以体积比9∶1、5∶1、1∶1、1∶3、1∶9的石油醚/乙酸乙酯混合液和乙酸乙酯进行梯度洗脱，每次5L；TLC检测馏分；再用甲醇重结晶。Get 5kg of Chrysanthemum broadleaves, reflux extraction with 5L ethanol, concentrate the extract, extract the aqueous suspension with 1L chloroform, concentrate the chloroform extract; mix the sample with silica gel (100 mesh, 100g), and carry out normal phase silica gel column chromatography separation ( 200-300 mesh, 1kg; silica gel column size L 500mm,

120mm), followed by a volume ratio of 9:1, 5:1, 1:1, 1:3, 1:9 petroleum ether/ethyl acetate mixture and ethyl acetate for gradient elution, each 5L; TLC detection Fraction; recrystallization from methanol.

The preparation of embodiment 4 sesquiterpene compounds

120mm)，依次以体积比9∶1、5∶1、1∶1、1∶3、1∶9的石油醚/乙酸乙酯混合液进行梯度洗脱，每次5L；TLC检测馏分；再用甲醇/氯仿(体积比3∶1)重结晶。Get 5kg of pulverized Chrysanthemum broadleaves, dry, reflux extraction with 5L ethyl acetate, the extract after separation is mixed with silica gel (100 mesh, 100g), and carry out normal phase silica gel column chromatography separation (200-300 mesh, 1kg ; Silica gel column size L 500mm,

120mm), followed by gradient elution with petroleum ether/ethyl acetate mixture with a volume ratio of 9:1, 5:1, 1:1, 1:3, 1:9, 5L each time; TLC detects the fraction; Methanol/chloroform (volume ratio 3:1) was used for recrystallization.

The preparation of embodiment 5 sesquiterpene compounds

120mm)，依次以体积比9∶1、5∶1、1∶1、1∶3、1∶9的正己烷/乙酸乙酯混合液进行梯度洗脱，每次5L；TLC检测馏分；再用丙酮/正己烷(体积比1∶1)重结晶。Get the Chrysanthemum broadleaf after 5kg pulverization, dry, reflux extraction with 5L acetone, the extract after separation mixes sample with silica gel (100 order, 100g), carries out normal phase silica gel column chromatography separation (200-300 order, 1kg; Silica gel Column size L 500mm,

120mm), followed by gradient elution with n-hexane/ethyl acetate mixture with a volume ratio of 9:1, 5:1, 1:1, 1:3, 1:9, 5L each time; TLC detects the fraction; Acetone/n-hexane (volume ratio 1:1) for recrystallization.

The preparation of embodiment 6 sesquiterpene compounds

120mm)，依次以体积比9∶1、5∶1、1∶1、1∶3、1∶9的正己烷/乙酸乙酯混合液进行梯度洗脱，每次5L；TLC检测馏分；再用丙酮/正己烷(体积比1∶1)重结晶。Take 5kg of crushed Chrysanthemum broadleaf, extract with 5L methanol/ethanol mixture, concentrate the extract, mix the sample with 100g diatomaceous earth, reflux with 5L ethyl acetate, and carry out normal phase silica gel column chromatography separation (200-300 mesh, 1kg; silica gel column size L 500mm,

120mm), followed by gradient elution with n-hexane/ethyl acetate mixed solution with a volume ratio of 9:1, 5:1, 1:1, 1:3, 1:9, 5L each time; TLC detects the fraction; Acetone/n-hexane (volume ratio 1:1) for recrystallization.

The preparation of embodiment 7 sesquiterpene compounds

120mm)；分段后用反相常压层析/高效液相色谱分离，洗脱剂为甲醇/水(1∶9-9∶1)，高效液相色谱的检测波长为210nm；用甲醇重结晶。Take 5kg of pulverized Chrysanthemum broadleaf, extract with 5L methanol/ethanol mixture, concentrate the extract, mix the sample with 100g diatomaceous earth, reflux with 5L ethyl acetate, and carry out normal phase silica gel column chromatography separation (200-300 mesh, 1kg; silica gel column size L 500mm,

120mm); Separation with reversed-phase atmospheric pressure chromatography/high performance liquid chromatography after segmentation, eluent is methanol/water (1: 9-9: 1), the detection wavelength of high performance liquid chromatography is 210nm; crystallization.

Among them, in Examples 2-7, silica gel for thin-layer chromatography and column chromatography is produced by Qingdao Ocean Chemical Factory; RP-18 or RP-8 column is used for reverse-phase silica gel; the boiling point of petroleum ether for chromatography is 60-90°C.

The structural identification of embodiment 8 sesquiterpene compounds

The purity of the prepared compound was identified by HPLC, and the structure of the sample with a purity greater than 98% was identified by mass spectrometry and nuclear magnetic resonance. The nuclear magnetic resonance was determined by Bruker AVANCE DRX-500NMR Sectrometer, and TMS was used as the internal standard; high-resolution mass spectrometry FTICRMS was used by Bruker Apex Spectrometer Determination; electrospray mass spectrometer ESI-MS was determined by Bruker Esquire 3000plus Spectrometer.

Table 1 shows the NMR analysis results of the compound. According to the structural analysis results, the substance is a sesquiterpene compound with a molecular formula of C ₁₅ H ₁₈ O ₄ , and the structure is as follows:

NMR data of table 1 sesquiterpene compounds

Example 9 Sesquiterpene Antioxidant Activity Analysis

Take a certain amount of sample, dissolve it in absolute ethanol, and make a solution with a concentration of 5mM; dilute it with absolute ethanol successively, and dilute it five times each time; take 96 empty plates, add 180 μl of DPPH ethanol solution (150 μM) to each well; 5 concentrations of sample solutions, 4 wells for each concentration, add 20 μl to each well; slightly shake the 96-well plate to mix evenly; place in an incubator and let stand at 37°C for 30 minutes; Measure the OD value of each hole; use the Bliss method to obtain the sample concentration when the DPPH clearance rate is 50%, that is, the half clearance experiment.

According to the results of the DPPH scavenging experiment, the IC ₅₀ of the compound is 36 μM, indicating that the compound has a good antioxidant effect.

The preparation of embodiment 10 containing sesquiterpene compound drop pill preparation

Take 0.5g of sesquiterpene compound and 10.5g of polyethylene glycol-6000, mix evenly, heat and melt, move the material into drip irrigation, drop the drug solution into liquid paraffin at 6-8°C, remove oil, and prepare drip Pills 300 capsules.

Example 11 Preparation of freeze-dried powder injection containing sesquiterpene compound

Take 0.5g of sesquiterpene compound, 4.5g of glucose, 0.9g of sodium thiosulfate and 1000ml of distilled water, mix the above components evenly, freeze-dry, and divide into 400 tubes to obtain the product.

Claims (4)

1. a sesquiterpenoid, its structural formula is as shown in formula I:

2. the preparation method of sesquiterpenoid as claimed in claim 1, is characterized in that, comprises the following steps:

(1) the whole strain of wide leaf Chloranthus spicatus or its tissue are added to lixiviate in organic solvent, obtain vat liquor;

Wherein, described organic solvent is one or both in methyl alcohol, ethanol, ethyl acetate and acetone;

(2) ethyl acetate or chloroform extraction for vat liquor, extraction liquid, after concentrated, separation and purification, obtains sesquiterpenoid;

Described separation and purification is: extraction liquid is carried out to the silica gel column chromatography separation, and the gained cut carries out the recrystallization separation again,

Described silica gel column chromatography is separated into: with petrol ether/ethyl acetate mixed solution and the ethyl acetate of volume ratio 9:1,5:1,1:1,1:3,1:9, extraction liquid is carried out to gradient elution successively; Perhaps, with the n-hexane/ethyl acetate mixed solution of volume ratio 9:1,5:1,1:1,1:3,1:9, extraction liquid is carried out to gradient elution successively.

3. preparation method according to claim 2, is characterized in that, in step (1), the weight ratio of the whole strain of described wide leaf Chloranthus spicatus or its tissue and organic solvent is 1:1-1:8.

4. the application of sesquiterpenoid as claimed in claim 1 in preparing anti-oxidation medicine, antioxidation cosmetic product or antioxidant functional food.