CN103739586A

CN103739586A - Method for extracting diterpenoid compounds from Blumea aromatic DC.

Info

Publication number: CN103739586A
Application number: CN201310588947.4A
Authority: CN
Inventors: 宋志军; 陈广钜; 姚彩云; 杜芳黎; 李汉浠; 陈路; 缪剑华; 蓝鸣生
Original assignee: Guangxi Botanical Garden of Medicinal Plants
Current assignee: Guangxi Botanical Garden of Medicinal Plants
Priority date: 2013-11-22
Filing date: 2013-11-22
Publication date: 2014-04-23

Abstract

The invention relates to a method for extracting diterpenoid compounds from Blumea aromatic DC., belongs to the technical field of medicines, and discloses a method for preparing diterpenoid compounds by extracting the diterpenoid compounds from the aerial part of Blumea aromatic DC.. The method is characterized in that crude medicinal powder is extracted through high-concentration lower alcohol (like methanol and ethanol, same below), the obtained extract liquid is concentrated to obtain a concentrate, the concentrate is dispersed by a small amount of water to obtain a dispersion, the dispersion is extracted by petroleum ether to remove low-polarity components (like chlorophyll), the obtained petroleum ether extraction liquid is extracted by ethyl acetate, the solvent of the obtained ethyl acetate extraction liquid is recovered, the solvent recovered substance is dissolved by a proper amount of the lower alcohol, the obtained solution sequentially goes through an MCI resin, polyamide and a SephadexLH-20 gel chromatograph column, and is eluted by the lower alcohol, a specific color section is collected, the lower alcohol solution low temperature crystallization and recrystallization of the specific color section are carried out to obtain diterpenoid compound crystals or amorphous powder. The compounds have latent medicinal values. The method has the advantages of good repetitiveness, stable content and high purity of the extracted compounds, simple technology, and low cost, and is of great significance to popularize and apply the Blumea aromatic DC..

Description

A kind of method of extracting diterpene-kind compound from strong medicine mountain breeze

Technical field

The present invention relates to Natural Medicine Chemistry field, be specifically related to a kind of method of extracting diterpene-kind compound from strong medicine mountain breeze.

Background technology

Mountain breeze is composite family Herba Blumeae Balsamiferae platymiscium fragrance virtue Herba Blumeae Balsamiferae Blumea aromatica DC., is Zhuang nationality in Guangxi medicinal herbs most in use, medicinal over-ground part, pungent, the micro-hardship of taste, warm in nature, gas perfume (or spice), have dispel rheumatism, swelling and pain relieving, clearing and activating the channels and collaterals effect.Mountain breeze is the main raw material of the rheumatism series product such as the famous Chinese patent medicine in Guangxi ' Huatuo's wind pain is precious ', ' effective rheumatism medicine medicinal liquor '.

Although mountain breeze in Guangxi utilization among the people extensively and have longer history, to the chemical research of its system, carry out very less.Only have at present inventor team this medicinal material ethyl acetate extract to be carried out to the pertinent literature of chemical constitution study, also only have a small amount of clinical and pharmacological action report, as: the externally used medicinal liquor being made into is dispelled rheumatism, the toothpaste anti-inflammation hemostasia of making etc.And the present invention finds a series of labdane diterpenes compounds that a large amount of skeletons are identical first from Herba Blumeae Balsamiferae platymiscium.

Diterpene-kind compound distributes very wide in vegitabilia, many diterpene containing oxygen derivatives have good biological activity, as rographolide, Triptolide, bilobalide etc., some has been clinical conventional medicine, particularly, thering is obvious effect aspect antitumor and anti-immune disease, there is good DEVELOPMENT PROSPECT.Labdane type diterpenoids is the parent nucleus that forms take the perhydronaphthalene cyclization diterpene as basic framework, in vegitabilia, Ladanum alkane exists in a large number, in Coleus forskohlii Briq. sheath stamen florigen (forskolin), Herba Andrographis in rographolide all belong to Ladanum alkane diterpene structure.That rographolide has is antibacterial, the effect of anti-inflammatory, in recent years finds that there is again the activity of cytodifferentiation induced activity and anti-toxic liver injury, has the application of wide model in medicine.Forskolin (forskolin) is used for the treatment of cardiovascular disorder clinically, glaucoma, bronchial asthma etc., it can also regulate various tissues by activated adenyl cyclase simultaneously, it is a kind of activator of other medicines, only in plant Coleus forskohlii Briq., find at home at present, and that diterpene-kind compound in mountain breeze has skeleton structure is completely identical with fosikelin, content is also high, mountain breeze is easier to establishing in large scale, therefore the diterpene-kind compound in mountain breeze has very large pharmaceutical prod potentiality to be exploited, and the present invention extracts the labdane diterpenes compound of preparation from mountain breeze, also may be the effective constituent of strong medicine mountain breeze.

Summary of the invention

Mountain breeze (Blumea aromatica DC) medicinal material, after suitably pulverizing, with 70% to 100% lower alcohol (methyl alcohol, ethanol, lower same), in static state or dynamically, room temperature or heating (comprise dipping, ultrasonic, diacolation, reflux, microwave etc.) extract 3 times, extracting solution decompression or air distillation are reclaimed alcoholic solvent to 1.10～1.50/30 ℃ of relative densities, adding 0.2 to 5 times of volume water mixes, with equal-volume petroleum ether extraction three times, then mother liquor changes and is extracted with ethyl acetate three times, the concentrated organic solvent of removing of ethyl acetate extraction part decompression or atmospheric evaporation, obtain medicinal extract.Appropriate lower alcohol (methyl alcohol for medicinal extract, ethanol, down together) after dissolving, through MCI macroporous resin column chromatography, with 50%, 85%, 95% ethanol wash-out successively, collect concentrated 85% alcohol elution, then with polyamide column chromatography, remove the sour phenolic constituents such as tannin and flavones, again through sephadex LH-20 dextrane gel column chromatography, with lower alcohol wash-out, under TLC or HPLC guidance, collect special color section eluting solvent, elutriant reclaims alcoholic solvent and obtains amber fluid extract, with moisture lower alcohol dissolution process generation crystallization, obtain crystallization or unformed Powdered diterpene compound sterling with moisture lower alcohol recrystallization.Recrystallization mother liquor can be used for next batch and recycles.

Accompanying drawing explanation

Fig. 1 is compound 1 (aromaticasin B) extraction process general flow chart

Fig. 2 is compound 1 (aromaticasin B) ¹h-NMR spectrogram (CDCl ₃)

Fig. 3 is compound 1 (aromaticasin B) ¹³c-NMR spectrogram (CDCl ₃)

Fig. 4 is that the single crystal diffraction of compound 1 (aromaticasin B) records relative configuration figure

Concrete embodiment 1

The extraction of Aromaticasin B separates

Mountain breeze (Blumea aromatica DC) medicinal material coarse powder 500g extracts 3 times by 85% alcohol immersion, each 6 times of amount (m/v, g/ml), soak 5 days, extracting solution suction filtration merges, filtrate recycling ethanol is 1.25/30 ℃ to relative density, add 2 times of volume water and stir evenly rear equal-volume petroleum ether extraction 3 times of using, after extraction liquid reclaims solvent, residue discards; Equal-volume ethyl acetate extraction 3 times for mother liquor after extraction, acetic acid ethyl acetate extract reclaims solvent and obtains medicinal extract, the a small amount of dissolve with methanol of medicinal extract, through MCI post with 85% ethanol elution, after reclaiming solvent, gained medicinal extract is through sephadex-LHX20 dextrane gel column chromatography, after methanol-eluted fractions, under instructing, TLC collects aromaticasin B high-content elute soln, reclaim methyl alcohol, obtain amber fluid extract, use ethanolic soln heating for dissolving, let cool crystallization, 80% ethyl alcohol recrystallization, obtains whitening compound 1 (aromaticasin B) crystallization.

The Structural Identification of Aromaticasin B

Compound 1 is white cube crystallization.By ESI-MS, determine that its its molecular formula is: C ₂₀h ₃₄o ₃, through two dimensional NMR technology such as HSQC, HMBC, determined compound two dimensional structure, relative configuration warp ¹h- ¹it is by name that H COSY and X-ray single crystal diffraction have obtained confirmation (accompanying drawing 4) chemistry: (4R, 5R, 8R, 9R, 10S, 12R, 13S)-2 β, 18-dihydroxyl-14-alkene-tear pool ether (or its enantiomorph), English name is decided to be: Aromaticasin B, Chinese name is decided to be: mountain breeze ether second (seeing Aromaticasin B chemical structural formula).

Compound 1 nuclear magnetic data is as follows:

¹H?NMR(600MHz，in?CDCl ₃)δ(ppm)：

1.36(1H，m，H-1a)，1.20(1H，m，H-1b)，1.60(1H，overlapped，H-2a)，1.46(1H，m，H-2b)，0.83(1H，td，J＝12.6，3.0，H-3a)，1.52(1H，m，H-3b)，1.60(1H，overlapped)，1.76(1H，td，J=13.2，3.0，H-6a)，1.60(1H，overlapped，H-6b)，1.71(1H，td，J=12.6，3.0，H-7a)，1.39(1H，td，J=13.2，4.2，H-7b)，1.25(1H，m，H-9)，1.51(1H，m，H-11a)，1.24(1H，overlapped，H-11b)，4.01(1H，t，J＝3.0，H-12)，6.00(1H，dd，J=11.4，18.6，H-14)，4.91(1H，d，J=11.4，H-15a)，4.90(1H，d，J＝18.6，H-15b)，1.13(3H，s，H-16)，1.18(3H，d，J=0.6，H-17)，0.67(3H，s，H-18)，3.35，3.04(2H，d，J=10.8，H-19)，0.72(3H，s，H-20).

¹³C?NMR(151MHz，in?CDCl ₃)δ(ppm)：

146.1(C-14)，109.6(C-15)，75.2(C-8)，75.1(C-13)，70.9(C-19)，68.1(C-12)，48.8(C-11)，41.4(C-7)，37.5(C-3)，36.6(C-4)，35.3(C-10)，34.3(C-1)，25.9(C-16)，23.3(C-17)，22.6(C-6)，18.7(C-11)，16.8(C-2)，16.1(C-18)，15.2(C-20).

Claims

1. from the over-ground part of Zhuang herbal medicine mountain breeze (Blumeaaromatica DC.), extract the method for preparing diterpene (falling diterpene) compounds, it is characterized in that medicinal material coarse powder passes through high density lower alcohol (as methyl alcohol, ethanol, lower same) extract, after extracting solution is concentrated, use a small amount of water-dispersion, through petroleum ether extraction, remove low polarity component (as chlorophyll etc.), be extracted with ethyl acetate again, after acetic acid ethyl acetate extract recovery solvent is concentrated, appropriate lower alcohol dissolves, successively through MCI resin, SephadexLH-20 gel chromatographic columns, with low-alcohol solution wash-out, collect special color section, recovery solvent is concentrated, obtain fluid extract, the aqueous solution of lower alcohol is processed crystallization, recrystallization can obtain diterpene-kind compound crystallization or unformed powder.

2. the chemical two dimensional structure formula of diterpene-kind compound claimed in claim 1 (I) meets following skeleton:

3. its substituting group of diterpene-kind compound claimed in claim 2 can be as follows:

R ₁:-CH ₂oH ,-CH ₂oCH ₃or-COOCH ₃,-COOH

R ₂: R ₃, R ₆, R ₇: can be-OH ,-OCH ₃,-OCOCH ₃,=O

R ₅：-OH、-OCH ₃、-OCOCH ₃

R4：-CH=CH ₂、-CHOHCH ₃、-CHOHCH ₂OH

4. compound claimed in claim 2 can be also that R4 sloughs, and epoxy bond fracture and form a series of contain 18 carbon atoms diterpenes derivative falls.

Compound claimed in claim 2 can be also dehydration and be formed on two keys of 1,2,3,11 or 12.

5. the preferred following compound 1 of diterpene-kind compound claimed in claim 2 (I), its molecular formula is: C ₂₀h ₃₄o ₃, chemistry is by name: (4R, 5R, 8R, 9R, 10S, 12R, 13S)-2 β, and 18-dihydroxyl-14-alkene-tear pool ether (or its enantiomorph), English name is decided to be: Aromaticasin B, Chinese name is decided to be: mountain breeze ether second.

6. the extraction and separation process of diterpene-kind compound claimed in claim 1 is:

6.1 starting material are the over-ground part of Herba Blumeae Balsamiferae platymiscium mountain breeze (Blumea aromatica DC.), and its meal adopts high density lower alcohols (methyl alcohol, ethanol, lower same) to extract;

6.2 extracting solutions reclaim after alcoholic solvent, residual solution adds 0.2 to 5 times of volume that water is equivalent to residual solution, fully suspendible, suspension is used respectively sherwood oil, ethyl acetate fully extracts, collect ethyl acetate part, recovery is removed organic solvent and is made ethyl acetate extract, medicinal extract dissolves with a small amount of lower alcohol, MCI removes pigment, polymeric amide removes after the sour phenolic constituents such as flavones, upper dextrane gel column chromatography, lower alcohol wash-out, at thin-layer chromatography, under the guidance of high performance liquid chromatography, collect particular elutriated part, reclaim alcoholic solvent, obtain amber total diterpene fluid extract, the aqueous solution with lower alcohol is processed crystallization, recrystallization obtains the crystallization of diterpene-kind compound monomer or solid diterpene mixture,

6.3 explanations about said extracted separating technology:

1) described in 6.1, the concentration of lower alcohols is 70% to 100%;

2) described in 6.1, extracting method can be: the several different methods such as backflow, dipping, diacolation, ultrasonic, microwave extraction;

3) described in 6.2, reclaim after alcoholic solvent, residual solution relative density is 1.1～1.50/30 ℃;

4) the preferred sephadex-LHX20 of dextrane gel for chromatography described in 6.2.

7. described in claim 1, diterpene-kind compound can be used as reference substance for medicinal material mountain breeze and formulation products quality control thereof, also has potential pharmaceutical value.