CN105585574A

CN105585574A - Diterpene compound and preparation method thereof

Info

Publication number: CN105585574A
Application number: CN201511004812.4A
Authority: CN
Inventors: 吴芊葭
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-12-28
Filing date: 2015-12-28
Publication date: 2016-05-18

Abstract

The invention discloses a diterpene compound and a preparation method thereof, belongs to the field of novel compounds and particularly relates to a novel diterpene compound separated from Daphne genkwa sieb et zucc, a preparation method and a medical application of the compound. The compound is reported for the first time, can be obtained from the Daphne genkwa sieb et zucc through extraction, separation and purification, and is high in purity. In-vitro tests prove that the diterpene compound can inhibit proliferation of various types of breast cancer cells, shows stronger in-vitro cytotoxicity, has an in-vitro breast cancer resisting effect and can be further researched and developed into a breast cancer treatment drug.

Description

A kind of diterpene compound and preparation method thereof

Technical field

The invention belongs to noval chemical compound field, be specifically related to separate a kind of new diterpene compound obtaining and preparation method thereof from lilac daphne.

Background technology

Lilac daphne claims again purple lilac daphne, southern lilac daphne, headache flower, medicine water shield etc., begins to be loaded in Shennong's Herbal, classifies low-grades as, is traditional drastically purgating water drug. Compendium of Material Medica is listed poisonous weeds class in, by " Wu Pu Bencao " meaning lilac daphne " February raw leafiness, thicken black, Hua Youzi, red, Bai Zhe, March is honest most, Ye Naisheng. " " shortcut has it everywhere to Han Bao Noboru " another name for Sichuan Province book on Chinese herbal medicine " meaning, height of seedling two or three chis. Leaf is like Cynanchum glaucescens and willow leaf, and root Beijing opera is like mulberry root, and February in the first month of the lunar year, flower was sent out, and purple green look, receives when Ye Weisheng. " see in conjunction with " Bencao Tujing " " Anhui Chuzhou's lilac daphne " and " Jinzhou, Sichuan lilac daphne " accompanying drawing, medicinal lilac daphne kind is consistent at all times, and its former plant is Daphnegenkwasieb.Etzucc. undoubtedly. " Chinese pharmacopoeia " 2010 editions recorded its dry flower that is Thymelaeceae (Thymelaeaceae) Daphne (Daphne) plant lilac daphne (Daphnegenkwasiebetzucc). Xie Zongwan had once carried out the textual criticism of this agrostology to lilac daphne, and having confirmed the lilac daphne of recording in successive dynasties book on Chinese herbal medicine should be the dry flower of purple lilac daphne Daphnegenkwasieb.Etzucc. This plant is the clinical kind of commonly using in national most of area, its wild province such as Henan, Shandong, Jiangsu, Anhui, Sichuan of mainly originating in, and Henan Province is mainly distributed in Xinyang, Deng Di mountain area, Nanyang.

Up to the present, from the flower of lilac daphne, bud, leaf, branch, root, extract and isolate compound and have kind more than 70, wherein contain the Multiple components such as flavones ingredient, diterpene ortho-ester composition, Coumarins composition, chlorogenic acid composition, lignan component, phenolic glycoside class.

Lilac daphne is traditional drastically purgating water drug, poisonous, function removing water retention by purgation, and removing toxic substances desinsection, modern study shows that it has following pharmacological action: 1. diuresis; 2. Antitussive and Expectorant Effect; 3. desinsection, anti parasitic effect; 4. induced labor effect; 5. anti-inflammatory, antitumor action; 6. immunoregulation effect.

Summary of the invention

The object of the present invention is to provide a kind of a kind of new diterpene-kind compound obtaining that separates from lilac daphne;

Another object of the present invention is to provide the preparation method of this noval chemical compound;

Above-mentioned purpose of the present invention is to be achieved by technical scheme below:

A kind of diterpene compound, it is the compound (I) with following structural formula,

As preferably, it is from dry lilac daphne, is separated and is obtained by following extraction step:

A, dry lilac daphne is pulverized, alcohol heat reflux extracts, concentrated, uses successively benzinum, ethyl acetate and water saturated extracting n-butyl alcohol, obtains respectively petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;

Acetic acid ethyl ester extract macroreticular resin removal of impurities in b, step a, uses 10% ethanol and 75% ethanol elution successively, collects 75% ethanol eluate, and reduced pressure concentration obtains 75% ethanol elution thing medicinal extract;

In c, step b, 75% ethanol elution medicinal extract separates by purification on normal-phase silica gel, obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 80:1,40:1,20:1,10:1 and 1:1;

In d, step c, component 3 use purification on normal-phase silica gel further separate, and obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 25:1,20:1 and 10:1;

In e, steps d, the reverse phase silica gel of component 2 use octadecylsilane bondings separates, and the methanol aqueous solution isocratic elution that is 70% by concentration expressed in percentage by volume, collects 9-13 column volume eluent, and eluent reduced pressure concentration obtains pure diterpene compound.

A preparation method for above-mentioned diterpene compound, comprises the steps:

In e, steps d, the reverse phase silica gel of component 2 use octadecylsilane bondings separates, and the methanol aqueous solution isocratic elution that is 70% by concentration expressed in percentage by volume, collects 9-13 column volume eluent, and eluent reduced pressure concentration obtains pure compound (I).

As the preparation method's of above-mentioned diterpene compound further preferred version, wherein:

It is to adopt that alcohol heat reflux in step a extracts; 75% alcohol heat reflux extracts, and merges extract.

In step a, concentrated terminal is to be concentrated into without alcohol taste.

Described macroreticular resin is AB-8 type macroporous absorbent resin.

Beneficial effect of the present invention: the compounds of this invention can be used as medicine, has medical value. When as medicine, can directly use, or use with the form of pharmaceutical composition. While use with the form of pharmaceutical composition, this pharmaceutical composition contains the diterpene compound of the present invention (I) for the treatment of effective dose, and all the other are pharmaceutically suitable carrier and/or the excipient of acceptable on materia medica, nontoxic to humans and animals and inertia. Pharmaceutically suitable carrier or excipient can be that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent. The compounds of this invention is made to the form that pharmaceutical composition can per weight dose to be used. Diterpene compound of the present invention (I) effect is pharmaceutically: diterpene compound of the present invention (I) can significantly be induced Apoptosis of Breast Cancer, performance antitumor action. Specifically, chemical compounds I can make three kinds of Cells Proliferation of Human Breast Cancer activity decreaseds, significantly suppresses its propagation, demonstrates stronger vitro cytotoxicity, has In Vitro Anti breast cancer effect.

During as clinical medicine, medicine of the present invention can be applied to the patient who needs treatment by form oral or injection. When oral, can be made into tablet, sustained release tablets, controlled release tablet, capsule, dripping pill, micropill, supensoid agent, emulsion, powder or granule, oral liquid etc.; While being used for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.

Brief description of the drawings

Fig. 1 is compound (I) structural formula;

Fig. 2 is the impact of various dose compound (I) on Cells Proliferation of Human Breast Cancer vigor;

Fig. 3 is the impact of various dose compound (I) on Apoptosis of Breast Cancer.

Detailed description of the invention

Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit protection domain of the present invention with this. Although the present invention is explained in detail with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify or be equal to replacement technical scheme of the present invention, and do not depart from essence and the scope of technical solution of the present invention.

Embodiment 1: compound (I) separates preparation and structural identification

Medicinal material and reagent source: ethanol, benzinum, ethyl acetate, n-butanol, carrene are pure for analyzing, purchased from Shanghai Ling Feng chemical reagent Co., Ltd, methyl alcohol, analyze pure, purchased from Jiangsu Han Bang chemical reagent Co., Ltd. Lilac daphne is purchased from Hui nationality's Chinese Medicinal Materials Markets, Fujian, the place of production.

Preparation method: (a) lilac daphne (8kg) is crushed to mung bean size, with 75% alcohol heat reflux extraction (25L × 3 time), merge extract, be concentrated into without alcohol taste (6L), use successively benzinum (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated n-butanol (6L × 3 time) extraction, concentrated, obtain respectively petroleum ether extract, acetic acid ethyl ester extract (315g) and n-butyl alcohol extract; (b) in step (a) acetic acid ethyl ester extract by dissolved in purified water to 2L, medical absorbent cotton filters, filtrate separates with AB-8 type macroreticular resin (1.5kg), use successively 10% ethanol (10L) and 75% (12L) ethanol elution, collect 75% ethanol eluate, reduced pressure concentration obtains 75% ethanol elution thing medicinal extract (155g); (c) in step (b), 75% ethanol elution medicinal extract separates by 200-300 order purification on normal-phase silica gel, obtains 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 80:1 (10 column volumes), 40:1 (8 column volumes), 20:1 (8 column volumes), 10:1 (8 column volumes) and 1:1 (5 column volumes); (d) in step (c), component 3 (36g) further separates by 200-300 order purification on normal-phase silica gel, obtains 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 25:1 (8 column volumes), 20:1 (8 column volumes) and 10:1 (5 column volumes); (e) in step (d), component 2 (12g) separates with the reverse phase silica gel ODS-C18 of octadecylsilane bonding, the methanol aqueous solution isocratic elution that is 70% by concentration expressed in percentage by volume, collect 9-13 column volume eluent, eluent reduced pressure concentration obtains pure compound (I) (25mg).

Structural identification: HR-ESIMS shows [M+Na]⁺For m/z401.1612, can obtain molecular formula in conjunction with nuclear-magnetism feature is C₂₀H₂₆O₇, degree of unsaturation is 8. Proton nmr spectra data δ_H(ppm,DMSO-d₆, 500MHz): H-1 (1.95, m), H-1 (2.07, m), H-2 (1.63, m), H-2 (2.86, m), H-3 (4.63, m), H-4 (1.98, br, s), H-6 (4.61, t, J=8.9), H-7 (2.26, dd, J=14.4, 8.4), H-7 (1.80, dd, J=14.4, 9.6), H-10 (2.24, m), H-11 (2.20, dd, J=14.4, 8.4), H-11 (2.33, dd, J=14.4, 6.0), H-12 (5.29, dd, J=8.4, 6.0), H-14 (6.28, br, s), H-15 (7.38, br, s), H-16 (7.35, br, s), H-17 (1.21, s), H-19 (1.37, s), H-20 (5.25, s), carbon-13 nmr spectra data δ_C(ppm,DMSO-d₆，125Hz)：18.2(CH₂，1-C)，25.0(CH₂，2-C)，72.1(CH，3-C)，54.9(CH，4-C)，39.1(C，5-C)，84.6(CH，6-C)，41.8(CH₂，7-C)，73.2(C，8-C)，56.0(C，9-C)，42.7(CH，10-C)，40.0(CH₂，11-C)，70.6(CH，12-C)，128.9(C，13-C)，108.2(CH，14-C)，144.0(CH，15-C)，138.5(CH，16-C)，26.9(CH₃，17-C)，177.8(C，18-C)，29.6(CH₃, 19-C), 105.8 (CH, 20-C); Carbon atom mark is referring to Fig. 1. IR spectrum shows that this compound contains hydroxyl and lactone groups (3444cm^-1And 1770cm^-1). NMR spectrum data show that this compound contains a typical β-monosubstituted furan nucleus [δ H6.28 (br, s, H-14), 7.38 (br, s, H-15), and 7.35 (br, s, H-16); δ C128.9 (C-13), 108.2 (C-14), 144.0 (C-15), and 138.5 (C-16)), an ester carbonyl group [δ C177.8 (C-18)], three containing oxygen methine [δ C72.1 (C-3), 84.6 (C-6), and 70.6 (C-12)], connect oxygen quaternary carbon [δ C73.2 (C-8)] and two methyl [δ H1.21 (3H, s, H₃-17) and 1.37 (3H, s, H₃-19)]. According to its molecular formula and NMR data, this compound identification is high containing oxygen diterpene-kind compound.¹H-¹The bright C-1 of HCOSY stave and C-4, C-6 and C-7, C-11 and C-12 and C-14 and C-15 have correlation. In HMBC spectrum, H₃-19 and C-4, C-5, C-6, and C-10 and H₃-17 and C-7, the correlation of C-8 and C-9 shows that compound is for containing the oxygen containing hexa-atomic two ring diterpenes of 5,8-dimethyl and 3,6,8-tri-. In HMBC spectrum, the correlation of H-6 and C-18 and H-3 and C-18, shows to exist a gamma lactone ring. In addition according to the correlation of H-12 and C-14 and C-16 in HMBC spectrum, deducibility furan nucleus is positioned on C-12 position. H in NOESY spectrum₃-19 and H-4, H-6 and H-10 correlation show, they are α configuration. H-16 and H₃-17 correlation shows CH₃-17 is beta comfiguration. In addition H,₃-17 show that with the correlation of H-20 H-20, H-11 and H-7 are beta comfiguration. This this compound structure as shown in Figure 1.

Embodiment 2: compound (I) pharmacological action test

One, material and instrument

DMEM/F12 is high, and sugared nutrient solution is purchased from Beijing Hyclone company. Modified form RPMI-l640 culture medium is purchased from Beijing Hyclone company. Standard hyclone is purchased from Tianjin TBD company. CCK-8 cell viability detects reagent and is purchased from the colleague of Amada Co., Ltd. chemistry institute. AnnexinV-FITC/PI apoptosis detection kit is purchased from Shanghai Yan Bin Chemical Industry Science Co., Ltd. JC-1 mitochondrial membrane potential probe is purchased from the green skies, Jiangsu biotechnology research institute. Dimethyl alum (DMSO) and PBS (PBS) are purchased from Sigma company product.

-70 DEG C of low temperature refrigerator (FormaScientificInc. companies, the U.S.), electronic balance (Shimadzu AEL-200 type, Japan), superclean bench (SW-CJ-IC type, Suzhou), constant temperature oscillator (THZ mono-C type, domestic), disinfection with high pressure steam pot (YX-400B type, domestic), CO₂Cell culture incubator (FormaSeries II, Thermalelectroncorp., the U.S.), thermostat water bath (BHW2-I type, domestic), inverted phase contrast microscope (CK40 type, Olympus, Japan), microscope (BX51 type, Olympus, Japan), micro-digit collecting system (DP71 type, Olympus, Japan), multi-functional ELIASA (InfiniteM200 type, TECAN, Switzerland), desk-top room temperature supercentrifuge (TGL-16G type, Beijing), desk-top low-temperature and high-speed centrifuge (2K15 type, sigma company, the U.S.), BL60 electronic balance (Beijing Sai Duolisi instrument system Co., Ltd), the large capacity centrifuge of LD4-40 (the vertical centrifuge of system in Beijing Jing Co., Ltd), 2K15 high temperature low speed refrigerated centrifuge (sigma company), UV765 ultraviolet specrophotometer (Shanghai Precision Scientific Apparatus Co., Ltd), laser confocal scanning microscope (MRC-1024 type, Bio-Rad, Britain).

Two, test method

1, cell line and cultivation

Select MCF-7 Breast Cancer Cell (estrogen receptor positive ER⁺, the negative HERZ/neu in human epidermal growth factor acceptor-2^-), breast cancer MDA-MB-231 cell (ER^-，HERZ/neu^-) and breast cancer MDA-MB-453 (ER^-，HERZ/neu⁺) cell line, three kinds of cell lines all come from Chinese Sciences Academy Biochemistry And Cell Biology Institute. MCF-7 cell and MDA-MB-231 cell are incubated in the high sugared nutrient solution of DMEM/F12 containing 10% standard hyclone, and MDA-MB-453 cell is incubated at containing in the RPMI-1640 nutrient solution of 10% standard hyclone, and above cell is all in 37 DEG C, 5%CO₂Under condition, cellar culture goes down to posterity. Three kinds of breast cancer cells are attached cell, going down to posterity of attached cell: in the time of cell attachment growth 2～3d to 80% fusion, discard old nutrient solution, with the 0.1mol/LPBS preparation that adds appropriate 0.25% pancreatin (pH7.4) in the backward bottle of appropriate 0.1mol/LPBS (pH7.4) rinsing cell 2 times, filtration sterilization. Digestion, is advisable just to cover Tissue Culture Flask bottom, and light Microscopic observation, treats cytoplasm retraction, after space between cells increases, adds immediately fresh nutrient solution to stop digestion, and with suction pipe piping and druming repeatedly gently, makes to form cell suspension at the bottom of cell detachment bottle; Adjust cell concentration, draw appropriate cell suspension and add in new blake bottle, add 6mL fresh culture, micro-Microscopic observation, is positioned over cell culture incubator.

2, cell proliferation vigor detects

Utilize the impact of CCK-8 method detection compound (I) on three kinds of Cells Proliferation of Human Breast Cancer vigor. CCK-8 kit (CellCountingKit-8) is the kit by the detection cell proliferation of Japanese colleague's chemistry institute exploitation, in CCK-8 reagent, contain WST-8[2-(2-methoxyl group-4-nitrobenzophenone)-3-(4-nitrobenzophenone)-5-(2, 4-disulfonic acid benzene)-2H-tetrazolium list sodium salt], it is reduced to the yellow formazan product with high water soluble by the dehydrogenase in cell mitochondrial under the effect of electron carrier 1-methoxyl group-5-toluphenazine dimethyl suflfate (1-MethoxyPMS), the quantity that generates formazan thing is directly proportional to the quantity of living cells. measure its absorbance value with ELIASA at 450nm wavelength place, can indirectly reflect living cells quantity.

Concrete operations are: breast cancer cell MCF-7, the MDA-MB-231 of exponential phase, MDA-MB-453 routine are gone down to posterity, and cell count, with 2.5 × 10⁵/ mL cell concentration is inoculated in 96 porocyte culture plates, every hole 200 μ L, and overnight incubation, treats cell attachment and well-grown. Experiment divides 5 groups, make 6 parallel holes for every group, add respectively the compound (I) of different amounts, make that its final concentration is respectively 200,150,100,50mg/mL, control group adds equivalent distilled water, cultivate 24h, discard nutrient solution, wash 3 times with PBS, add the every hole 200 μ L of fresh medium, then every hole adds 10 μ LCCK-8, cultivate 4h harvesting, detect 450nm place OD value [D (450)] with ELIASA, with the blank zeroing of distilled water, calculate the proliferation inhibition rate of variable concentrations compound (I) effect.

Cell proliferation inhibition rate=[contrast D (450) one experimental group D (450)]/contrast D (450) × 100%

3, apoptotic detection

Utilize AnnexinV/PI pair to dye the impact of flow cytometry technology for detection compound (I) on breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 apoptosis. Experiment is carried out in strict accordance with AnnexinV apoptosis detection kit operating instruction. Concrete operations are: breast cancer cell MCF-7, the MDA-MB-231 of exponential phase, MDA-MB-453 routine are gone down to posterity, and cell count, with 10⁵/ mL cell concentration is inoculated in 6 porocyte culture plates, every hole 5mL, and overnight incubation, treats cell attachment and well-grown. Experiment divides 5 groups, makes 3 parallel holes for every group, adds respectively the compound (I) of various dose, make that its final concentration is respectively 150,100,50mg/mL, control group adds equivalent distilled water, cultivates 24h, discards nutrient solution, wash cell 3 times with the PBS (pH7.2) of 4 DEG C of precoolings, conventional trypsinization, makes single cell suspension, centrifugal, with the binding buffer solution Eddy diffusion cell of 250 μ L dilutions, and to make its concentration be 1 × 10⁶/ mL, gets the cell suspension of 100 μ L in 5mL streaming pipe, adds 5 μ LAnnexinV/FITC solution, leave standstill 5min, add the PI solution 10 μ L of 20 μ g/mL, after mixing, place 10min in room temperature lucifuge, in reaction tube, add 400 μ LPBS, utilize machine testing on flow cytometer respectively to organize fluorescence situation. Cell divides respectively to 4 phases according to AnnexinV and PI dyeing situation, AnnexinV and the PI equal negative cells that dyes is normal survivaling cell, PI stained positive, AnnexinV dyeing feminine gender are mechanical damage or ruptured cell, AnnexinV and PI dyeing are all positive is necrosis or non-viable apoptotic cell, and AnnexinV stained positive, PI dyeing feminine gender are judged to be viable apoptotic cell.

4, the detection of mitochondrial membrane potential in anoxic

Utilize mitochondrial membrane potential in anoxic molecular probe JC-1 and the impact of laser confocal scanning microscope detection compound (I) on breast cancer cell MBA-MD-453 mitochondrial membrane potential. Concrete operations are: the breast cancer cell MDA-MB-453 routine of exponential phase is gone down to posterity, and cell count, with 10⁵/ mL cell concentration is inoculated in 6 porocyte culture plates, every hole 5mL, overnight incubation, treat that cell attachment and well-grown experiment divide 5 groups, make 3 parallel holes for every group, add respectively the compound (I) of various dose, make that its final concentration is respectively 150,100,50mg/mL, control group adds equivalent distilled water, cultivates 24h, discard nutrient solution, wash cell 3 times with the PBS (pH7.2) of 4 DEG C of precoolings, add 1.0mLPBS, add the JC-l of 10 μ L2.5mmol/L, making its final concentration is 25 μ mol/L, at 37 DEG C, 5%CO₂In incubator, lucifuge is hatched 20min, and with PBS washing 3 times, laser confocal scanning microscope detects to be analyzed.

5, the statistical analysis of data

All data are all with average scholar standard deviationRepresent, adopt SPSS12.0 statistical software, experimental result is carried out to one-way analysis of variance, P < 0.05 is thought and is had significant difference between the two.

Three, result and conclusion

1, cell proliferation vigor testing result

Utilize CCK-8 method to detect variable concentrations compound (I) and act on the impact on three kinds of Cells Proliferation of Human Breast Cancer vigor, result shows, compound (I) effect can make three kinds of Cells Proliferation of Human Breast Cancer activity all reduce, and the increase reduction degree with activity increases, demonstrate dose-effect relationship, 100, 150 all there is significant difference (P < 0.05 compared with control group with 200mg/mL group, P < 0.01), compound (I) effect can significantly suppress breast cancer cell MCF-7, MDA-MB-231, the propagation of MDA-MB-453, demonstrate stronger vitro cytotoxicity, show that compound (I) has stronger In Vitro Anti breast cancer effect. the results are shown in Figure 2 and table 1.

The inhibiting rate (%) of table 1 various dose compound (I) to Cells Proliferation of Human Breast Cancer

	50mg/mL	100mg/mL	150mg/mL	200mg/mL
					MDA-MB-453 cell	14.80	29.80	33.10	44.30
MDA-MB-231 cell	11.40	24.00	30.60	37.50
					MCF-7 cell	10.30	20.20	26.20	35.10

2, apoptotic testing result

Utilize AnnexinV/PI pair to dye the impact of flow cytometry technology for detection compound (I) on breast cancer cell MCF-7, MDA-MB-231, MDA-MB-453 apoptosis, apoptosis analysis result shows, compound (I) effect 24h can make the apoptosis rate of three kinds of breast cancer cells all raise, demonstrate short apoptosis effect, and there is dose-effect relationship, 100 have significant difference (P < 0.05) with 150mg/mL group compared with control group shows that compound (I) can significantly induce Apoptosis of Breast Cancer, performance antitumor action. The results are shown in Figure 3.

Sum up, this research, by chemical compounds I anti-breast cancer effect research, finds that chemical compounds I can make three kinds of Cells Proliferation of Human Breast Cancer activity decreaseds, significantly suppresses its propagation, demonstrates stronger vitro cytotoxicity, has In Vitro Anti breast cancer effect.

Embodiment 3

The preparation of tablet: first make compound (I) by embodiment 1 method, and the salt that utilizes organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid example hydrochloric acid or sulfuric acid or phosphoric acid, the ratio that is 1:9 in itself and excipient weight ratio adds excipient, pelletizing press sheet.

Embodiment 4

The preparation of oral liquid: first make compound (I) by embodiment 1 method, and the salt that utilizes organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making is made oral liquid routinely.

Embodiment 5

The preparation of capsule or granule: first make compound (I) by embodiment 1 method, and the salt that utilizes organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid example hydrochloric acid or sulfuric acid or phosphoric acid, the ratio that is 1:9 in itself and excipient weight ratio adds excipient, makes capsule or granule.

Embodiment 6

The preparation of parenteral solution: first make compound (I) by embodiment 1 method, and the salt that utilizes organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject routinely water, essence filter, parenteral solution is made in embedding sterilizing.

Embodiment 7

The preparation of aseptic powder injection: first make compound (I) by embodiment 1 method, and the salt that utilizes organic acid to make as tartaric acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, with aseptic suction funnel filtration, aseptic essence filter, is sub-packed in ampoule again, and after frozen drying, aseptic sealing by fusing obtains powder-injection.

Claims

1. a diterpene compound, is characterized in that: it is the compound (I) with following structural formula,

2. diterpene compound according to claim 1, is characterized in that: it is from dry lilac daphne, by carrying as followsGet step and separate acquisition:

A, dry lilac daphne is pulverized, alcohol heat reflux extracts, concentrated, uses successively benzinum, ethyl acetate and just water saturatedButanols extraction, obtains respectively petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;

Acetic acid ethyl ester extract macroreticular resin removal of impurities in b, step a, uses 10% ethanol and 75% ethanol elution successively, collects 75%Ethanol eluate, reduced pressure concentration obtains 75% ethanol elution thing medicinal extract;

In c, step b, 75% ethanol elution medicinal extract separates by purification on normal-phase silica gel, is 80:1,40:1,20:1,10:1 successively by volume ratioObtain 5 components with the methylene chloride-methanol gradient elution of 1:1;

In d, step c, component 3 use purification on normal-phase silica gel further separate, and are the dichloromethane of 25:1,20:1 and 10:1 successively by volume ratioAlkane-methyl alcohol gradient elution obtains 3 components;

In e, steps d, the reverse phase silica gel of component 2 use octadecylsilane bondings separates, the methyl alcohol that is 70% by concentration expressed in percentage by volumeAqueous solution isocratic elution, collects 9-13 column volume eluent, and eluent reduced pressure concentration obtains pure diterpene compound.

3. the preparation method of diterpene compound claimed in claim 1, is characterized in that: comprise the steps:

In e, steps d, the reverse phase silica gel of component 2 use octadecylsilane bondings separates, the methyl alcohol that is 70% by concentration expressed in percentage by volumeAqueous solution isocratic elution, collects 9-13 column volume eluent, and eluent reduced pressure concentration obtains pure compound (I).

4. the preparation method of diterpene compound according to claim 3, is characterized in that:

5. the preparation method of diterpene compound according to claim 3, is characterized in that:

6. the preparation method of diterpene compound according to claim 3, is characterized in that:

Described macroreticular resin is AB-8 type macroporous absorbent resin.