CN102659595A - Method for extraction and separation of rosmarinic acid, apigenin and luteolin in elsholtzia splendens - Google Patents
Method for extraction and separation of rosmarinic acid, apigenin and luteolin in elsholtzia splendens Download PDFInfo
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Abstract
The invention discloses a method for extraction and separation of rosmarinic acid, apigenin and luteolin in elsholtzia splendens. The method comprises the steps of: (1) ethanol immersion extraction: performing air drying, smash, immersion extraction and extraction on branches and leaves of elsholtzia splendens harvested in the full-bloom stage to obtain a brownish black extractum; (2) performing macroporous resin coarse separation and concentration on the brownish black extractum to obtain a yellowish-brown extractum; (3) performing polyamide column separation on the obtained yellowish-brown extractum to obtain an extractum A, an extractum B and an extractum C; and (4) performing silica-gel column chromatography purification and refinement on the extractum A, the extractum B and the extractum C to obtain the apigenin, the apigenin and the rosmarinic acid correspondingly. Highly purified rosmarinic acid, apigenin and luteolin can be obtained by adopting the method provided in the invention.
Description
Technical field
The biologic recycling that the invention belongs to natural product utilizes the field, relates to the method for rosmarinic acid, apigenin and luteolin in the special plant elsholtzia splendens of extraction separation environment.
Background technology
From plant, extract antitumor activity component, be the popular domain of natural product chemistry research always.The effective constituent kind of natural antitumor medicine is various, is mainly alkaloids, herbal polysaccharide class, Polyphenols, protein-based, quinones, terpene etc.; The plant polyphenol constituents has antitumor action and has obtained proof.It is reported; Choose leukemia cell, lung tumors cell and breast cancer cell as research object; Even find to have only the polyphenol of trace also can suppress the enzyme in the tumour cell, and these enzymes are that essential (the Deco scholar finds that polyphenol suppresses tumour cell mechanism, " Chinese patent medicine " to the generation of triggering tumor cell growth " signal " molecule; 2005,27 (4): 455).Plant polyphenol is considered to prevent and suppress in the plant amedica therapy composition of tumour.Plain type (phenylpropanoids) the caffeinic antineoplastic invasion of natural polyphenol analog derivative phenylpropyl alcohol is relevant with its strongly inhibited effect to matrix metalloproteinase-9 (MMP 9) with transfer activity.The black coffee acid phenethyl ester (CAPE) that is obtained by the propolis ethanol extract is the potent inhibitor (IC of MMP 9
50Value is 1.0～2.0nmol/L).Phenyl propanoid derivative rosmarinic acid (rosmarinic acid), Lithospermic acid A (lithospermic acid) have strongly inhibited effect, its IC to matrix metalloproteinase-2 (MMP 2)
50Value is respectively 27.2,10.2 μ mol/L.Vegetable flavonoid (Flavonoids) is signaling molecule and the midbody or the metabolite of Polyphenols in the plant materials, and is more to exist in the plants such as Labiatae, rank of nobility fiber crops section, Gesneriaceae, scrophulariaceae, composite family.Discover that wherein quite a few flavones has significant physiology and pharmacologically active; Like oxidation-resistance; Improve organism endocrine, remove free radical activity etc., can be used to study the molecular mechanism of cell signaling, the exploitation flavones is in the function of aspects such as anticancer, antitumor and cardiovascular protection.Because acute, the subacute toxicity of flavones has determined its wide development potentiality in medicine well below Theelin,dihydro-.
Elsholtzia splendens (Elsholtzia splendens) is a Labiatae elscholtiza platymiscium, perennial herb, ground such as distribution Liaoning, Hebei, Shandong, Henan, Anhui, Jiangsu, Zhejiang, Jiangxi, Hubei, Sichuan, Guizhou, Yunnan, Shaanxi, Gansu.The application among the people of elsholtzia splendens is comparatively extensive, and effect is like elscholtiza.Confirmed that the plain type coffic acid of antineoplastic component phenylpropyl alcohol is one type of main active ingredient in the elsholtzia splendens plant, coffic acid content is 0.1305% of elsholtzia splendens plant flowers leaf dry weight; There is plain type of coffic acid metabolite of the phenylpropyl alcohol relevant in the elsholtzia splendens plant materials with copper.The method of from the elsholtzia splendens plant, extracting the higher coffic acid composition of productive rate is disclosed in the patent of invention (ZL201110136277.3: extract the caffeinic method of antitumor composition in the elsholtzia splendens).Experiment finds to exist in the elsholtzia splendens the plain analog derivative rosmarinic acid of phenylpropyl alcohol, a kind of water-soluble polyphenolic compound.Rosmarinic acid is the natural active matter with multiple function, has pharmacotoxicological effect widely such as anti-oxidant, anti-inflammatory, antibiotic, immunomodulatory.Simultaneously; Patent of invention (ZL201010151950.6: disclose the medical active of extractive of general flavone in vasodilation in the elsholtzia splendens the application of the elsholtzia splendens extractive of general flavone of copper polluted soil in preparation vasodilation medicine), obviously be superior to Flos Chrysanthemi extractive of general flavone, Ginkgo total flavones extract and soybean extractive of general flavone.Discover at present two kinds of important biological flavone composition apigenin and luteolins are arranged in the elsholtzia splendens.Apigenin has the carcinogenic activity that suppresses carcinogenic substance, as the antiviral of treatment HIV and other virus infection; Be used to treat various inflammation; It also is inhibitor; Have calmness simultaneously, calm the nerves, hypotensive effect.Compare with other Flavonoid substances (Quercetin, kaempferia galamga flavones), apigenin has characteristics such as low toxicity, no mutagenicity.Luteolin is very representative natural flavone, distributes wider in vegitabilia; Luteolin has cough-relieving clinically, eliminate the phlegm and effect such as anti-inflammatory, has antibiotic, antiviral, cardiovascular protection effect in vivo, spasmolysis, anticancer, press down enzyme effect, inhibitor, diuresis choleretic effect and effects such as reduce fat and SUV.Therefore; Extract the plain analog derivative rosmarinic acid of phenylpropyl alcohol, bioflavonoid apigenin and luteolin in the special plant elsholtzia splendens of development environment effectively; The antitumor efficiently natural drug of preparation plant-sourced low toxicity is the effective way of its high added value recycling.
Up to now, the domestic method of still not having report about while extraction separation rosmarinic acid, apigenin and luteolin from the special plant elsholtzia splendens of environment.
Summary of the invention
The technical problem that the present invention will solve provides the method for rosmarinic acid, apigenin and luteolin in a kind of extraction separation elsholtzia splendens, adopts method of the present invention can obtain highly purified rosmarinic acid, apigenin and luteolin.
In order to solve the problems of the technologies described above, the present invention provides the method for rosmarinic acid, apigenin and luteolin in a kind of extraction separation elsholtzia splendens, may further comprise the steps successively:
(1), ethanol lixiviate: the elsholtzia splendens spray and the air-dry back of blade of full-bloom stage results are pulverized, and with the ethanolic soln lixiviate of volumetric concentration >=95%, the extracting solution of gained gets CE behind vacuum concentration; CE is used water dissolution, get lysate; In lysate, add petroleum ether extraction, the volume ratio of said sherwood oil and lysate is 1: 0.9～1.1, abandons petroleum ether layer; In the water layer of gained, add ETHYLE ACETATE and extract repeatedly, do not have color until the ethyl acetate extraction layer; In said each when extraction,, the consumption of ETHYLE ACETATE was 0.9～1.1 times of lysate volume; All ethyl acetate extraction layers are merged the back concentrate (being conventional concentrating under reduced pressure), get brownish black medicinal extract;
(2), macroporous resin roughing out: the brownish black medicinal extract of gained is processed the aqueous solution; Be splined on D101 macroporous resin and fully absorption, water and volumetric concentration are that 10%, 20%, 30%, 40% ethanolic soln carries out wash-out with 1.4～1.6BV/h gradient and separates successively; Collected volume concentration is the corresponding elutriant of 30% and 40% ethanolic soln, concentrates and obtains yellowish brown medicinal extract;
The amount ratio of said brownish black medicinal extract and D101 macroporous resin is: 1g brownish black medicinal extract/10～15mlD101 macroporous resin;
The brownish black medicinal extract that contains 15～25g in said every 100ml aqueous solution;
The consumption that said water, volumetric concentration are respectively 10%, 20%, 30%, 40% ethanolic soln is respectively: 0.8～1.2BV, 1.8～2.2BV, 4.8～5.2BV, 5.8～6.2BV, 3.8～4.2BV;
(3), polyamide column separates: the yellowish brown medicinal extract of gained is used dissolve with methanol, get solution, the amount ratio of said yellowish brown medicinal extract and methyl alcohol is 1g/2～5ml; By yellowish brown medicinal extract and Silon is 1: 1～2 mass ratio, in said solution, adds Silon, and the suspension liquid that obtains is evaporated to dried under 20～30 ℃, appearance thing on the powdery;
In the ratio of the mass ratio 1: 30～50 of yellowish brown medicinal extract and polymeric amide, get successively the polyamide solution after ethanol and water treatment and pour in the chromatography column, and with the water in the methylene dichloride displacement polyamide solution;
Appearance thing on the powdery evenly is splined on the polyamide column top, with CH
2Cl
2: CH
3OH: HCOOH=(60: 1: 3), (50: 1: 2.5), (40: 1: 2); (30: 1: 1.5); The volume by volume concentration gradient of (20: 1: the 1) gradient elution that pressurizes, (generally speaking, the corresponding every gradient of the yellowish brown medicinal extract of per 4～6g is used elutriant 150～400ml); Analyze with TLC, with CH
2Cl
2: CH
3OH: HCOOH=20: be developping agent at 2: 1, through the FeCl of mass concentration 3%
3Ethanolic soln colour developing indication according to colour developing spot distribution situation, is collected the similar elutriant of composition; Concentrating under reduced pressure respectively, 25～50 ℃ heat up gradually to revolve and steam to doing, thereby obtain extractum A, medicinal extract B, medicinal extract C respectively; Wherein,
Extractum A corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=60: 1: 3;
Medicinal extract B corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=50: 1: 2.5 and CH
2Cl
2: CH
3OH: HCOOH=40: 1: 2;
Medicinal extract C corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=30: 1: 1.5 CH
2Cl
2: CH
3OH: HCOOH=20: 1: 1;
(4), purification by silica gel column chromatography is refining:
1., silica gel (for example being 200 orders) is press-fited post with adding behind ETHYLE ACETATE or the petroleum ether dissolution, the mass volume ratio of silica gel and ETHYLE ACETATE or sherwood oil is 1g/4～8ml; The silica gel of said supporting extractum A is used acetic acid ethyl dissolution, and the silica gel of supporting medicinal extract B and supporting medicinal extract C is all used petroleum ether dissolution (that is, the silica gel for chromatography of extractum A is used acetic acid ethyl dissolution, and the chromatographic silica gel of medicinal extract B and medicinal extract C is used petroleum ether dissolution);
2., the extractum A with above-mentioned gained, medicinal extract B and medicinal extract C, carry out following operation respectively: mix appearance with adding silica gel behind the dissolve with methanol, the weight ratio of silica gel and extractum A, medicinal extract B and medicinal extract C is 2: 1～4: 1; Revolve after doing with gained 3 kinds and mix appearance and be contained in the capital end respectively, carry out purification by silica gel column chromatography respectively after the last appearance and make with extra care; Thereby corresponding respectively apigenin, apigenin and the rosmarinic acid of obtaining;
The weight ratio that 2. gross weight of the silica gel that said step is 1. used and step mix appearance use silica gel is 20～30: 1.
Improvement as the method for rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens of the present invention: in the step (4) with on the extractum A of gained the appearance; With ETHYLE ACETATE: methyl alcohol: the mixed solution of the volume ratio of formic acid=20: 3: 1 is an eluent system; Collect and respectively manage effluent; And carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, mass concentration 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, and can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, with R
f=0.5 (Rf value of spot on silica-gel plate), and be each pipe merging of single spot; Repeated post (promptly " waste liquid " being gone up the appearance wash-out again) until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain the apigenin of yellow powder shape, and purity is 100%.Generally speaking, the eluent system of extractum A adapted 400～600ml of every 1g.
Further improvements in methods as rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens of the present invention: the medicinal extract B with gained in the step (4) goes up appearance; With sherwood oil: ETHYLE ACETATE: the volume ratio of formic acid=10: 10: 1 is an eluent system; Collect and respectively manage effluent; And carry out TLC point plate at any time and follow the tracks of, with the AlCl of apparent blue-fluorescence, mass concentration 1% under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, and can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, with R
f=0.65 (Rf value of spot on silica-gel plate), and be each pipe merging of single spot; Repeated post (promptly " waste liquid " being gone up the appearance wash-out again) until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain the luteolin of pale yellow powder shape, and purity is 100%.Generally speaking, the eluent system of medicinal extract B adapted 400～600ml of every 1g.
Further improvements in methods as rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens of the present invention: the medicinal extract C with gained in the step (4) goes up appearance; With sherwood oil: ETHYLE ACETATE: formic acid=(20: 10: 1); (15: 10: 1), the volume ratio of (10: 10: 1) is carried out gradient elution for the expansion system, collects and respectively manages effluent; And carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and is as the criterion with smoked three indexs of silica-gel plate inclusion-free of iodine, can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, and with sherwood oil: ETHYLE ACETATE: formic acid=20: 10: 1 is developping agent, with R
f=0.55 (Rf value of spot on silica-gel plate), and be each pipe merging of single spot; Repeated post until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain faint yellow crystalloid rosmarinic acid, and purity is 98.3%.Generally speaking, the eluent of every kind of gradient of the medicinal extract C adapted of every 1g is 150～250ml.
In above-mentioned steps 2) the invention process of " macroporous resin roughing out " in, above-mentioned collection step is specific as follows: collect effluent and respectively the effluent in every pipe is carried out TLC and analyze (developping agent CH
2Cl
2: CH
3OH: HCOOH=20: 4: 1 volume ratio), through the AlCl of mass concentration 1%
3Ethanolic soln shows glassy yellow, 3%FeCl
3Ethanolic soln shows greyish-green, and the effluent composition of observing behind volumetric concentration 30% and 40% ethanol elution changes, thereby volume merges above-mentioned 30% and 40% ethanol stream fluid (that is, volumetric concentration is the elutriant of 30% and 40% ethanolic soln correspondence); Concentrate and obtain yellowish brown medicinal extract.
In " the polyamide column separation " of step 3) of the present invention, the polymeric amide chromatography post of handling well is pressurizeed with air pump, make it tightr, the water surface is not less than the height of polymeric amide bed.Wait to be forced into after the polymeric amide height remains unchanged,, the water in the polymeric amide all is replaced into methylene dichloride with the flow velocity of 1BV/h with the methylene dichloride of 3～5BV.
In " the ethanol lixiviate " of step of the present invention (1), with the ethanolic soln lixiviate (ultrasonic frequency in 30～50KHz was handled 0.8～1.2 hour) of volumetric concentration >=95%, the extracting solution suction filtration of gained must be filtrated; The residue of suction filtration gained is repeated said process 1～3 time (promptly with residue alternative dry powder, repeating above-mentioned lixiviate).The whole filtratings that merge above-mentioned lixiviate gained; Through 45 ℃ of vacuum concentration, get CE again.The for example available ultrapure water of CE dissolves.
The present invention adopts ethanol lixiviate, macroporous resin roughing out, polyamide column separation, purification by silica gel column chromatography process for purification; From the floral leaf dry powder of the special plant elsholtzia splendens of environment; Obtain rosmarinic acid, apigenin and luteolin product simultaneously, purity all is higher than 98.3%.
The present invention utilizes elsholtzia splendens to extract rosmarinic acid, apigenin and the luteolin of gained, all can be used to prepare antitumor drug.The resulting rosmarinic acid of the present invention, apigenin and luteolin; All show certain anti-tumor activity; Wherein the anti-tumor activity of luteolin is best; Inhibiting rate to lung adenocarcinoma cell A549, epidermal carcinoma cell A431, human breast carcinoma Bcap37 reaches 71.94%, 37.41%, 46.78% respectively, has many targets activity.
In the present invention, adopt MTT colorimetric method for determining target compound (rosmarinic acid, luteolin, apigenin) active to the inhibition of tumour cell.MTT is a kind of dyestuff that can accept Wasserstoffatoms, chemistry 3-(4,5-dimethylthiazole-2)-2 by name, 5-phenylbenzene tetrazole bromine salt.The MTT colourimetry is a kind of method that detects cell survival and growth; Its principle is that exogenous MTT can be reduced to water-fast bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and in cell, deposit by succinodehydrogenase in the viable cell plastosome, and dead cell does not have this phenomenon.DMSO 99.8MIN. (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation, under the 570nm wavelength, measures its light absorption value with enzyme-linked immunosorbent assay instrument, can reflect its viable cell quantity indirectly.The present invention is made into certain concentration with test-compound (rosmarinic acid, luteolin, apigenin); After incubating 72 hours altogether with human cancer cell strain (human breast carcinoma Bcap37, lung adenocarcinoma cell A549, epidermal carcinoma cell A431), measure its inhibiting rate to JEG-3.
Measuring method is following: (1) is incubated at the RPMI-1640 substratum that contains 10% NBCS with tumour cell (human breast carcinoma Bcap37, lung adenocarcinoma cell A549, epidermal carcinoma cell A431), puts 37 ℃, 5%CO
2Routine cultivations of going down to posterity divides bottle to increase behind 2～3d in the incubator, and the cell in the vegetative period of taking the logarithm experimentizes.(2) the human breast carcinoma Bcap37 that takes the logarithm respectively vegetative period, lung adenocarcinoma cell A549, epidermal carcinoma cell A431; 0.05% trysinization; Be diluted to the single cell suspension of 50000/ml with the RPMI-1640 substratum that contains 10% NBCS; It is inoculated in 96 orifice plates, and every hole 100 μ l establish 3 multiple holes for every group.(3) dosing (adding target compound) behind the cultivation 24h.Adding consistency is set at the DMSO solution of 50 μ mol/L, establishes the ZD1939 positive control simultaneously, establishes DMSO simultaneously and makes solvent control, and each concentration is established 3 multiple holes.37 ℃, 5%CO2 incubator are hatched, and behind the dosing 72h, every hole adds the RPMI-1640 substratum 100 μ l that contain MTT solution, after in cell culture incubator, continuing to cultivate 3h, produce bluish voilet crystallization first a ceremonial jade-ladle, used in libation.(4) centrifugal 6min carefully blots the supernatant in 96 orifice plates, and every hole adds DMSO at least 100 μ l; The shaking table 5min at least that slightly vibrates; Make the dissolving of first a ceremonial jade-ladle, used in libation, measure every hole absorbancy (OD value), reference wavelength 620nm in 562nm or 570nm place with enzyme-linked immunosorbent assay instrument.Get 3 multiple hole OD values, average, calculate cell inhibitory rate.
Remarkable advantage of the present invention and effect:
The method of extraction separation rosmarinic acid, apigenin and luteolin in the elsholtzia splendens plant of the present invention; Be the natural phant phenols; Combine with supersound extraction through the room temperature lixiviate; Again with sherwood oil, the extraction of the ETHYLE ACETATE degree of depth, macroporous resin roughing out, polyamide column chromatography-silica gel column chromatography separating for several times, the product purity that obtains height.The rosmarinic acid that is extracted among the present invention, apigenin and luteolin have better antitumor activity, and its effect obviously is superior to coffic acid.And luteolin is the antitumor natural drug of many targets.
In sum, the method for extraction separation rosmarinic acid, apigenin and luteolin in the elsholtzia splendens provided by the invention has realistic meaning to the development and use of active skull cap components in the special plant elsholtzia splendens of environment.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is rosmarinic acid in the elsholtzia splendens, apigenin and luteolin extraction separation schema;
Fig. 2 is the infrared spectrogram of rosmarinic acid of the present invention, luteolin and apigenin;
Fig. 3 is the HPLC-ESI-MS spectrogram of rosmarinic acid of the present invention, luteolin and apigenin.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Rosmarinic acid, apigenin and luteolin extraction separation flow process are as shown in Figure 1 in the elsholtzia splendens.
In embodiment 1, the elsholtzia splendens of the present invention the solvent extraction of floral leaf dry powder with separate
(1), alcohol extracting:
The elsholtzia splendens spray of full-bloom stage results is worn into dry powder (water ratio is lower than 0.5%, weight ratio) with blade after air-dry, cross 100 purposes and sieve.Get above-mentioned 500g dry powder, place the plastic tank of 10L, add 95% (V/V) ethanol 5L, stir and soak 3 days to the complete imbibition of material, the ultrasonic frequency of 40KHz was handled 1 hour, and suction filtration must be filtrated.The residue of suction filtration gained is repeated said process 2 times (promptly with residue alternative dry powder, repeating above-mentioned lixiviate).The filtrating that merges 3 lixiviate gained; Again through 45 ℃ of vacuum concentration, the medicinal extract 1. (that is CE) of 126.1g; Productive rate 25.22%.
1. the medicinal extract of above-mentioned 126.1g dissolved with being settled to the 750ml ultrapure water, with petroleum ether extraction 3 times (consumption of each sherwood oil is 750ml).Abandon petroleum ether layer; 3 times water layer is merged.With ethyl acetate extraction 3 times (consumption of each ETHYLE ACETATE is 750ml), this moment, the ethyl acetate extraction layer did not have color with the water layer that merges the back gained; 3 times ethyl acetate extraction layers are merged, and 35 ℃ of rotary evaporations reclaim ETHYLE ACETATE, and the medicinal extract that obtains the 19.755g brownish black is (that is, brownish black medicinal extract) 2., productive rate 3.951%.
(2), the roughing out of macroporous resin:
Prepare D101 type macroporous adsorbent resin 250ml; With a large amount of distilled water flushings; Remove and be loaded on chromatography column behind the buoyant broken resin particle (among the 3cm * 50cm); Water with in the ethanol of 95% (volumetric concentration) the displacement resin column spends the night with above-mentioned alcohol immersion in chromatography column, and the ethanol of using 95% (volumetric concentration) does not again have muddiness with flow velocity drip washing to effluent and the water of 1.5BV/h by 1: 5 mixed.Again the ethanol in the above-mentioned D101 type macroporous adsorbent resin is rinsed with a large amount of zero(ppm) water, do not have alcohol to effluent and distinguish the flavor of.
2. the medicinal extract of above-mentioned 19.755g added ultrapure water process the 100ml aqueous solution, be added in the top of the D101 type macroporous resin chromatography column of above-mentioned wash clean; The above-mentioned aqueous solution is fully absorbed.
With 10% (V/V) ethanol of the 1BV water of (BV refers to the resin bed volume), 2BV, 20% (V/V) ethanol of 5BV, 30% (V/V) ethanol of 6BV, 40% (V/V) ethanol of 4BV D101 type macroporous resin is carried out gradient elution respectively, flow rate control is at 1.5BV/h.The about 50ml of every pipe collects and respectively manages elutriant.Respectively the elutriant in every pipe is carried out TLC point plate (polymeric amide thin-layer chromatography, developping agent CH
2Cl
2: CH
3OH: HCOOH=20: 4: 1 volume ratio), through mass concentration 1%AlCl
3Ethanolic soln shows glassy yellow, mass concentration 3%FeCl
3Ethanolic soln shows greyish-green, observe 30% with 40% ethanol elution after effluent in composition similar, merge the above-mentioned 30% and 40% ethanol stream fluid that contains, the medicinal extract that obtains the 5.213g yellowish brown after concentrating is (that is, yellowish brown medicinal extract) 3., productive rate 1.042%; Other elutriants that do not meet above-mentioned standard are done the waste liquid processing.
(3), polyamide column separates again:
Get 100 purpose polymeric amide 200g; 95% (volumetric concentration) alcohol immersion with 300ml is spent the night, and reflux 2h pours in the beaker and leaves standstill; Treat that the polymeric amide post precipitation outwells upper strata ethanol and swimmer; Adding chromatography column behind 95% (volumetric concentration) dissolve with ethanol with 300ml (among the 3cm * 50cm), and adds ultrapure water and replaces ethanol repeatedly, do not have the alcohol flavor to effluent.The polymeric amide chromatography post of handling well is pressurizeed with air pump, make it tightr, the water surface is not less than the height of polymeric amide bed.Wait to be forced into after the polymeric amide height remains unchanged,, the water in the polymeric amide all is replaced into methylene dichloride with the flow velocity of 1BV/h with the methylene dichloride liquid of 4BV.
3. 5.213g medicinal extract used the 15ml dissolve with methanol, and add the 8g Silon, the suspension liquid that obtains is evaporated to dried under 20～30 ℃, obtains reddish-brown powder 15g.
The reddish-brown powder is splined on the top of polyamide column, with CH
2Cl
2: CH
3OH: HCOOH (volume ratio)=(60: 1: 3), (50: 1: 2.5), (40: 1: 2), (30: 1: 1.5), the volume by volume concentration gradient of (20: 1: the 1) gradient elution that pressurizes, every gradient is used elutriant 300ml, and every 50ml collects 1 pipe.Use the polymeric amide tlc analysis, with CH
2Cl
2: CH
3OH: HCOOH=20: 2: 1 volume ratio is a developping agent, the FeCl of mass concentration 3%
3The ethanolic soln colour developing is indication, according to the distribution situation of colour developing spot, merges the similar elutriant of composition.
(that is, the corresponding volume concentration gradient is CH to collect the 1st～6 pipe
2Cl
2: CH
3OH: HCOOH=60: 1: 3), to get extractum A be 0.782g to concentrating under reduced pressure;
(that is, the corresponding volume concentration gradient is CH to collect the 7th～18 pipe
2Cl
2: CH
3OH: HCOOH=50: 1: 2.5 and CH
2Cl
2: CH
3OH: HCOOH=40: 1: 2), to get medicinal extract B be 0.927g to concentrating under reduced pressure;
(that is, the corresponding volume concentration gradient is CH to collect the 19th～30 pipe
2Cl
2: CH
3OH: HCOOH=30: 1: 1.5CH
2Cl
2: CH
3OH: HCOOH=20: 1: 1), to get medicinal extract C be 0.821g to concentrating under reduced pressure.
The further refining purifying of silicagel column of crossing of above-mentioned extractum A, medicinal extract B and medicinal extract C.
The thin-layer silicon glue purification of apigenin, luteolin, rosmarinic acid is refining in embodiment 2, the elsholtzia splendens of the present invention
(1) the thin-layer silicon glue purification of apigenin is refining.
The 200 order silica gel of 50g are used the 250ml acetic acid ethyl dissolution, stir the bubble that degass, add and press-fit post.
The 0.782g extractum A of gained among the embodiment 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With ETHYLE ACETATE: methyl alcohol: the volume ratio of formic acid=20: 3: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 400ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.5 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 30th～36 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain yellow powder (compound a---apigenin), about 0.033g, productive rate 0.0066%.Purity is 100%.
(2) the thin-layer silicon glue purification of luteolin is refining.
The 200 order silica gel of 65g are used the 350ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.927g medicinal extract B of gained among the embodiment 1 is used the 5ml dissolve with methanol, and add the silica gel mixed sample of 3g, revolve and it is contained in the capital end after doing.With sherwood oil: ETHYLE ACETATE: the volume ratio of formic acid=10: 10: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 500ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.65 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 40th～47 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain pale yellow powder (compound b---luteolin), about 0.062g, productive rate 0.0124%.Purity is 100%.
(3) the thin-layer silicon glue purification of rosmarinic acid is refining.
The 200 order silica gel of 50g are used the 250ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.821g medicinal extract C of gained among the embodiment 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With medicinal extract C with sherwood oil: ETHYLE ACETATE: formic acid=(20: 10: 1), (15: 10: 1), the volume ratio of (10: 10: 1) is carried out gradient elution for the expansion system, every gradient eluent 200ml collects and respectively manages effluent, every 10ml collects 1 pipe; And with sherwood oil: ETHYLE ACETATE: formic acid=20: 10: 1 is developping agent, carries out TLC point plate at any time and follows the tracks of.AlCl with apparent blue-fluorescence, 1% (mass concentration) under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.55 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 45th～56 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain faint yellow crystallization (compound c---rosmarinic acid, about 0.154g, productive rate 0.0308%.Purity 98.3%.
The chemical structure of apigenin, luteolin, rosmarinic acid is identified in embodiment 3, the elsholtzia splendens of the present invention
With the faint yellow compound that obtains among the embodiment 2 (compound a, b, c) with the dissolving of the ultrapure water of 20ml ,-40 ℃ freezing 2 hours, vacuum freezing (80 ℃) drying obtains product.Dissolve for DMSO with deuterium respectively, measure proton nmr spectra, carbon spectrum, and measured ir spectra, be used to identify structure with pressed disc method.Data are following:
The proton nmr spectra of apigenin (purity 100%), carbon are composed as shown in Figure 2, and characteristic peak belongs to as follows:
1H-NMR (500MHz, DMSO-d
6) δ (ppm): 12.975 (s, 1H), 10.605 (s, 1H), 7.936 (d, J=8.0Hz, 2H), 6.938 (d, J=8.3Hz, 2H), 6.785 (s, 1H), 6.487 (s, 1H), 6.199 (s, 1H).
13C-NMR(125MHz，DMSO-d
6)δ(ppm)：182.208，164.562，164.179，161.914，161.547，157.773，128.927，121.653，116.369，116.369，104.126，103.37，99.26，94.464，94.391。HPLC-ESI/MS:m/z 270.8 [M+H]
+, molecular weight is 270.
The proton nmr spectra of luteolin (purity 100%), carbon are composed as shown in Figure 3, and characteristic peak belongs to as follows:
1H NMR (500MHz, DMSO-d
6): δ 12.98 (s, 1H), 10.05 (br), 7.42 (s, 1H), 7.41 (d, J=8.5Hz, 1H), 6.89 (d, J=8.5Hz, 1H), 6.67 (s, 1H), 6.44 (s, 1H), 6.19 (s, 1H).
13C?NMR(125MHz，DMSO-d
6)：δ182.1，164.7，164.4，162.0，157.8，150.2，146.2，122.0，119.4，116.5，113.8，104.2，103.3，99.3，94.3。HPLC-ESI/MS:m/z 286.8 [M+H]
+, molecular weight is 286.
The characteristic peak of the proton nmr spectra of rosmarinic acid (purity 98.3%), carbon spectrum belongs to as follows:
1H-NMR (500MHz, DMSO-d
6): 7.47 (d, J=16Hz), 7.07 (d, J=2Hz), 7.01 (dd, J=8Hz, 2Hz); 6.78 (d, J=8Hz), 6.69 (d, J=2Hz), 6.65 (d, J=8Hz), 6.54 (dd; J=8Hz, 2Hz), 6.25 (d, J=16Hz), 5.04 (dd, J=8.5Hz, 4Hz); 3.00 (dd, J=14.5Hz, 4Hz), 2.91 (dd, J=14.5Hz, 8.5Hz).
13C-NMR(125MHz，DMSO-d
6)：δ171.4，166.4，149.1，146.3，146.1，145.4，144.5，127.9，125.9，122.0，120.5，117.2，116.3，115.9，115.4，113.8，73.4，36.6。Principal character peak in the ir spectra (KBr compressing tablet): phenolic hydroxyl group (stretching vibration (3367cm OH)
-1), phenolic hydroxyl group-OH flexural vibration (1353cm
-1), phenolic hydroxyl group C-OH stretching vibration (1220cm
-1), the ester group on the phenyl ring-C=O stretching vibration (1692cm
-1), ester group-C=C stretching vibration (1605cm
-1), phenyl ring=C-H stretching vibration (3239cm
-1), phenyl ring C=C stretching vibration (1524cm
-1) and phenyl ring=C-H out-of-plane deformation vibration (813cm
-1), Ar-C=C=C-H out-of-plane deformation vibration (980-960cm
-1), the characteristic peak (1284cm of aromatic acid
-1, 2600cm
-1, 950cm
-1).HPLC-ESI/MS (: m/z 383.1 [M+23]
+, 359 [M-1]
-, molecular weight is 360, molecular formula is C
18H
16O
8
Embodiment 4: rosmarinic acid in the elsholtzia splendens of the present invention, luteolin, apigenin are active to the inhibition of human breast cancer cell Bcap37, lung adenocarcinoma cell A549 and epidermal carcinoma cell A431
Adopt mtt assay to measure target compound (rosmarinic acid, luteolin, apigenin) human breast cancer cell Bcap37, lung adenocarcinoma cell A549 and epidermal carcinoma cell A431 are suppressed active.Test-compound (rosmarinic acid, luteolin, apigenin) is made into certain concentration, incubate 72 hours altogether with human cancer cell strain (human breast carcinoma Bcap37, lung adenocarcinoma cell A549, epidermal carcinoma cell A431) after, measure its inhibiting rate to JEG-3.Measuring method is following:
(1) tumour cell (human breast carcinoma Bcap37, lung adenocarcinoma cell A549, epidermal carcinoma cell A431) is incubated at the RPMI-1640 substratum that contains 10% NBCS, puts 37 ℃, 5%CO
2Routine cultivations of going down to posterity divides bottle to increase behind 2～3d in the incubator, and the cell in the vegetative period of taking the logarithm experimentizes.(2) the human breast carcinoma Bcap37 that takes the logarithm respectively vegetative period, lung adenocarcinoma cell A549, epidermal carcinoma cell A431; 0.05% trysinization; Be diluted to the single cell suspension of 50000/ml with the RPMI-1640 substratum that contains 10% NBCS; It is inoculated in 96 orifice plates, and every hole 100 μ l establish 3 multiple holes for every group.(3) dosing (adding target compound) behind the cultivation 24h.Adding consistency is set at the DMSO solution of 50 μ mol/L, establishes the ZD1939 positive control simultaneously, establishes DMSO simultaneously and makes solvent control, and each concentration is established 3 multiple holes.37 ℃, 5%CO2 incubator are hatched, and behind the dosing 72h, every hole adds the RPMI-1640 substratum 100 μ l that contain MTT solution, after in cell culture incubator, continuing to cultivate 3h, produce bluish voilet crystallization first a ceremonial jade-ladle, used in libation.(4) centrifugal 6min carefully blots the supernatant in 96 orifice plates, and every hole adds DMSO at least 100 μ l; The shaking table 5min at least that slightly vibrates; Make the dissolving of first a ceremonial jade-ladle, used in libation, measure every hole absorbancy (OD value), reference wavelength 620nm in 562nm or 570nm place with enzyme-linked immunosorbent assay instrument.Get 3 multiple hole OD values, average, calculate cell inhibitory rate.
Inhibiting rate (IR%)=(1-TOD/COD) * 100%; Wherein, TOD: administration group OD average; COD: solvent control group OD average.Measure the active substance such as coffic acid, rosmarinic acid, apigenin, apigenin glucosides and the luteolin that extract in the elsholtzia splendens, under 50 μ mol/l concentration to the inhibiting rate (like table 1) of tumor cell line (lung adenocarcinoma cell A549, epidermal carcinoma cell A431, human breast carcinoma Bcap37).
Active compound is to the cytotoxic activity (representing with inhibiting rate) of tumor cell line under the table 1 50 μ mol concentration
Annotate: it is active that NA representes not detect inhibition
Can know by table 1; Under 50 μ mol/L concentration; The anti-tumor activity of luteolin is best; Inhibiting rate to lung adenocarcinoma cell A549, epidermal carcinoma cell A431, human breast carcinoma Bcap37 reaches 71.94%, 37.41%, 46.78% respectively, has many targets activity, can be used for preparing anti-tumor medicine.Apigenin reaches 77.07%, 5.63%, 24.3% respectively to the inhibiting rate of lung adenocarcinoma cell A549, epidermal carcinoma cell A431, human breast carcinoma Bcap37; Wherein best in measuring the result to the inhibition effect of lung adenocarcinoma cell A549; Suitable with finished medicines ZD1939 (inhibiting rate 80%) effect, have good DEVELOPMENT PROSPECT.Apigenin glucosides and rosmarinic acid have certain inhibition effect to specific tumour cell, obviously are better than coffic acid.
The comparative example 1: " polyamide column separates again " of embodiment 1 step 3) made into following content:
Get 100 purpose polymeric amide 200g, washing, (method of 3cm * 50cm) is with embodiment 1 for the chromatography column of packing into.
Macroporous resin among the embodiment 1 is separated the 5.213g medicinal extract that obtains 3. use the 15ml dissolve with methanol, and add the 8g Silon, the suspension liquid that obtains is evaporated to dried under 20～30 ℃, directly obtains reddish-brown powder 15g.The reddish-brown powder is splined on the top of polyamide column, with CH
2Cl
2: CH
3OH: HCOOH (volume ratio)=(70: 1: 3), (60: 1: 2.5), (50: 1: 2), (40: 1: 1.5), the concentration gradient of (30: 1: the 1) gradient elution that pressurizes, every gradient is used elutriant 400ml, and every 50ml collects 1 pipe.Use the polymeric amide tlc analysis, with CH
2Cl
2: CH
3OH: HCOOH=20: 2: 1 volume ratio is a developping agent, 3%FeCl
3The ethanolic soln colour developing is indication, according to the distribution situation of colour developing spot, merges the similar elutriant of composition.
Collect the 1st～8 pipe, it is 0.750g that concentrating under reduced pressure gets extractum A;
Collect the 9th～27 pipe, it is 0.901g that concentrating under reduced pressure gets medicinal extract B;
Collect the 28th～40 pipe, it is 0.795g that concentrating under reduced pressure gets medicinal extract C.
The further refining purifying of silicagel column of crossing of above-mentioned extractum A, medicinal extract B and medicinal extract C.
The comparative example 2: the thin-layer silicon glue purification of apigenin, luteolin and rosmarinic acid is refining in the elsholtzia splendens
(1) the thin-layer silicon glue purification of apigenin is refining.
The 200 order silica gel of 50g are used the 250ml acetic acid ethyl dissolution, stir the bubble that degass, add and press-fit post.The 0.750g extractum A of gained among the comparative example 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With ETHYLE ACETATE: methyl alcohol: the volume ratio of formic acid=15: 3: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 350ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.5 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 29th～33 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain yellow powder (compound a---apigenin), about 0.029g, productive rate 0.0058%.
(2) the thin-layer silicon glue purification of luteolin is refining.
The 200 order silica gel of 65g are used the 350ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.901g medicinal extract B of gained among the comparative example 1 is used the 5ml dissolve with methanol, and add the silica gel mixed sample of 3g, revolve and it is contained in the capital end after doing.With sherwood oil: ETHYLE ACETATE: the volume ratio of formic acid=10: 15: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 430ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.65 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 37th～41 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain pale yellow powder (compound b---luteolin), about 0.0536g, productive rate 0.0107%.
(3) the thin-layer silicon glue purification of rosmarinic acid is refining.
The 200 order silica gel of 50g are used the 250ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.795g medicinal extract C of gained among the comparative example 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With medicinal extract C with sherwood oil: ETHYLE ACETATE: formic acid=(15-10-5): 10: 1 volume ratio is that elutriant carries out gradient elution, and every gradient eluent 180ml collects and respectively manages effluent, and every 10ml collects 1 pipe; And with sherwood oil: ETHYLE ACETATE: formic acid=20: 10: 1 is developping agent, carries out TLC point plate at any time and follows the tracks of.With apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.55 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 46th～52 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain faint yellow crystallization (compound c---rosmarinic acid, about 0.143g, productive rate 0.0286%.
The comparative example 3: the thin-layer silicon glue purification of apigenin, luteolin and rosmarinic acid is refining in the elsholtzia splendens
(1) the thin-layer silicon glue purification of apigenin is refining
The 200 order silica gel of 50g are used the 250ml acetic acid ethyl dissolution, stir the bubble that degass, add and press-fit post.The 0.782g extractum A of gained among the embodiment 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With ETHYLE ACETATE: methyl alcohol: the volume ratio of formic acid=25: 3: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 500ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.5 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 39th～48 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain yellow powder (compound a---apigenin), about 0.027g, productive rate 0.0054%.
(2) the thin-layer silicon glue purification of luteolin is refining
The 200 order silica gel of 65g are used the 350ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.927g medicinal extract B of gained among the embodiment 1 is used the 5ml dissolve with methanol, and add the silica gel mixed sample of 3g, revolve and it is contained in the capital end after doing.With sherwood oil: ETHYLE ACETATE: the volume ratio of formic acid=15: 12: 1 is the expansion system post wash-out that pressurizeed; Need elutriant 600ml altogether; Collect and respectively manage effluent (every 10ml collects 1 pipe), and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.65 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 43rd～57 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain pale yellow powder (compound b---luteolin), about 0.060g, productive rate 0.012%.
(3) the thin-layer silicon glue purification of rosmarinic acid is refining.
The 200 order silica gel of 50g are used the 250ml petroleum ether dissolution, stir the bubble that degass, add and press-fit post.The 0.821g medicinal extract C of gained among the embodiment 1 is used the 4ml dissolve with methanol, and add the silica gel mixed sample of 2g, revolve and it is contained in the capital end after doing.With medicinal extract C with sherwood oil: ETHYLE ACETATE: formic acid=(25-20-15): 10: 1 volume ratio is that elutriant carries out gradient elution, and every gradient eluent 250ml collects and respectively manages effluent, and every 10ml collects 1 pipe; And with sherwood oil: ETHYLE ACETATE: formic acid=20: 10: 1 is developping agent, carries out TLC point plate at any time and follows the tracks of.With apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, with R
f=0.55 (Rf value of spot on silica-gel plate), and be that each pipe of single spot merges (being about to the 55th～70 pipe merges), 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain faint yellow crystallization (compound c---rosmarinic acid, about 0.150g, productive rate 0.03%.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1. the method for rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens is characterized in that may further comprise the steps successively:
(1), ethanol lixiviate: the elsholtzia splendens spray and the air-dry back of blade of full-bloom stage results are pulverized, and with the ethanolic soln lixiviate of volumetric concentration >=95%, the extracting solution of gained gets CE behind vacuum concentration; CE is used water dissolution, get lysate; In lysate, add petroleum ether extraction, the volume ratio of said sherwood oil and lysate is 1: 0.9～1.1, abandons petroleum ether layer; In the water layer of gained, add ETHYLE ACETATE and extract repeatedly, do not have color until the ethyl acetate extraction layer; In said each when extraction,, the consumption of ETHYLE ACETATE was 0.9～1.1 times of lysate volume; All ethyl acetate extraction layers are merged the back concentrate, get brownish black medicinal extract;
(2), macroporous resin roughing out: the brownish black medicinal extract of gained is processed the aqueous solution; Be splined on D101 macroporous resin and fully absorption, water and volumetric concentration are that 10%, 20%, 30%, 40% ethanolic soln carries out wash-out with 1.4～1.6BV/h gradient and separates successively; Collected volume concentration is the corresponding elutriant of 30% and 40% ethanolic soln, concentrates and obtains yellowish brown medicinal extract;
The amount ratio of said brownish black medicinal extract and D101 macroporous resin is: 1g brownish black medicinal extract/10～15mlD101 macroporous resin;
The brownish black medicinal extract that contains 15～25g in said every 100ml aqueous solution;
The consumption that said water, volumetric concentration are respectively 10%, 20%, 30%, 40% ethanolic soln is respectively: 0.8～1.2BV, 1.8～2.2BV, 4.8～5.2BV, 5.8～6.2BV, 3.8～4.2BV;
(3), polyamide column separates: the yellowish brown medicinal extract of gained is used dissolve with methanol, get solution, the amount ratio of said yellowish brown medicinal extract and methyl alcohol is 1g/2～5ml; By yellowish brown medicinal extract and Silon is 1: 1～2 mass ratio, in said solution, adds Silon, and the suspension liquid that obtains is evaporated to dried under 20～30 ℃, appearance thing on the powdery;
In the ratio of the mass ratio 1: 30～50 of yellowish brown medicinal extract and polymeric amide, get successively the polyamide solution after ethanol and water treatment and pour in the chromatography column, and with the water in the methylene dichloride displacement polyamide solution;
Appearance thing on the powdery evenly is splined on the polyamide column top, with CH
2Cl
2: CH
3OH: HCOOH=(60: 1: 3), (50: 1: 2.5), (40: 1: 2), (30: 1: 1.5), the volume by volume concentration gradient of (20: 1: the 1) gradient elution that pressurizes is analyzed with TLC, with CH
2Cl
2: CH
3OH: HCOOH=20: be developping agent at 2: 1, through the FeCl of mass concentration 3%
3Ethanolic soln colour developing indication according to colour developing spot distribution situation, is collected the similar elutriant of composition; Concentrating under reduced pressure respectively, 25～50 ℃ heat up gradually to revolve and steam to doing, thereby obtain extractum A, medicinal extract B, medicinal extract C respectively; Wherein,
Extractum A corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=60: 1: 3;
Medicinal extract B corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=50: 1: 2.5 and CH
2Cl
2: CH
3OH: HCOOH=40: 1: 2;
Medicinal extract C corresponding concentration gradient is CH
2Cl
2: CH
3OH: HCOOH=30: 1: 1.5 CH
2Cl
2: CH
3OH: HCOOH=20: 1: 1;
(4), purification by silica gel column chromatography is refining:
1., silica gel added after with ETHYLE ACETATE or petroleum ether dissolution press-fit post, the mass volume ratio of silica gel and ETHYLE ACETATE or sherwood oil is 1g/4～8ml; The silica gel of said supporting extractum A is used acetic acid ethyl dissolution, and the silica gel of supporting medicinal extract B and supporting medicinal extract C is all used petroleum ether dissolution;
2., the extractum A with above-mentioned gained, medicinal extract B and medicinal extract C, carry out following operation respectively: mix appearance with adding silica gel behind the dissolve with methanol, the weight ratio of silica gel and extractum A, medicinal extract B and medicinal extract C is 2: 1～4: 1; Revolve after doing with gained 3 kinds and mix appearance and be contained in the capital end respectively, carry out purification by silica gel column chromatography respectively after the last appearance and make with extra care; Thereby corresponding respectively apigenin, apigenin and the rosmarinic acid of obtaining;
The weight ratio that 2. gross weight of the silica gel that said step is 1. used and step mix appearance use silica gel is 20～30: 1.
2. the method for rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens according to claim 1; It is characterized in that in the said step (4): with appearance on the extractum A of gained; With ETHYLE ACETATE: methyl alcohol: the mixed solution of the volume ratio of formic acid=20: 3: 1 is an eluent system; Collect and respectively manage effluent, and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, mass concentration 1%AlCl under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, and can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, with R
f=0.5, and be each pipe merging of single spot; Repeated post until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain the apigenin of yellow powder shape.
3. the method for rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens according to claim 1 and 2; It is characterized in that in the said step (4): the medicinal extract B of gained is gone up appearance; With sherwood oil: ETHYLE ACETATE: the volume ratio of formic acid=10: 10: 1 is an eluent system; Collect and respectively manage effluent, and carry out TLC point plate at any time and follow the tracks of, with the AlCl of apparent blue-fluorescence, mass concentration 1% under the uv lamp
3Solution shows glassy yellow and smoked three indexs of silica-gel plate inclusion-free of iodine are as the criterion, and can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, with R
f=0.65, and be each pipe merging of single spot; Repeated post until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain the luteolin of pale yellow powder shape.
4. the method for rosmarinic acid, apigenin and luteolin in the extraction separation elsholtzia splendens according to claim 3; It is characterized in that in the said step (4): the medicinal extract C of gained is gone up appearance, with sherwood oil: ETHYLE ACETATE: formic acid=(20: 10: 1), (15: 10: 1); The volume ratio of (10: 10: 1) is carried out gradient elution for the expansion system; Collect and respectively manage effluent, and carry out TLC point plate at any time and follow the tracks of, with apparent blue-fluorescence, 1%AlCl under the uv lamp
3Solution shows glassy yellow and is as the criterion with smoked three indexs of silica-gel plate inclusion-free of iodine, can not satisfy the effluent of these 3 standards simultaneously and do the waste liquid processing, and with sherwood oil: ETHYLE ACETATE: formic acid=20: 10: 1 is developping agent, with R
f=0.55, and be each pipe merging of single spot; Repeated post until the driftlessness compound; 25～50 ℃ heat up gradually to revolve and steam to doing, and obtain faint yellow crystalloid rosmarinic acid.
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| CN103342689A (en) * | 2013-06-18 | 2013-10-09 | 聊城大学 | Method for separation and purification of luteolin, apigenin and diosmetin in trichosanthes peel |
| CN105218605A (en) * | 2015-09-14 | 2016-01-06 | 浙江大学 | The method of Nepeta glycosides, Homoplantaginin and apigenin glucosides in extraction and isolation elsholtzia splendens |
| CN105954386A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | ASE extraction method for rosmarinic acid in perilla seed |
| CN116999484A (en) * | 2023-10-07 | 2023-11-07 | 云南英格生物技术有限公司 | Application of perilla seed oil meal extract in preparation of 5 alpha-reductase inhibitor |
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| CN109985087B (en) * | 2017-12-29 | 2023-04-07 | 百岳特生物技术（上海）有限公司 | Application of herba violae and citronella extract in treating lung tissue injury and inhibiting pulmonary fibrosis |
| TWI698246B (en) * | 2017-12-29 | 2020-07-11 | 大江生醫股份有限公司 | Use of elsholtzia ciliata hylander extracts for treating lung tissue damage and inhibiting pulmonary fibrosis |
| CN112794872B (en) * | 2021-04-02 | 2022-07-15 | 湖南德诺贝莱健康产业有限公司 | Method for extracting luteolin-3' -glucuronide from rosemary |
| CN113480507B (en) * | 2021-07-16 | 2022-09-27 | 湖南朗林生物资源股份有限公司 | Method for simultaneously extracting apigenin and luteolin from chamomile |
-
2012
- 2012-02-17 CN CN2012100359123A patent/CN102659595A/en active Pending
-
2013
- 2013-01-31 CN CN201310042896.5A patent/CN103130644B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103342689A (en) * | 2013-06-18 | 2013-10-09 | 聊城大学 | Method for separation and purification of luteolin, apigenin and diosmetin in trichosanthes peel |
| CN105218605A (en) * | 2015-09-14 | 2016-01-06 | 浙江大学 | The method of Nepeta glycosides, Homoplantaginin and apigenin glucosides in extraction and isolation elsholtzia splendens |
| CN105218605B (en) * | 2015-09-14 | 2018-05-08 | 浙江大学 | The method of Nepeta glycosides, Homoplantaginin and apiolin glucosides in extraction separation elsholtzia splendens |
| CN105954386A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | ASE extraction method for rosmarinic acid in perilla seed |
| CN116999484A (en) * | 2023-10-07 | 2023-11-07 | 云南英格生物技术有限公司 | Application of perilla seed oil meal extract in preparation of 5 alpha-reductase inhibitor |
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| Publication number | Publication date |
|---|---|
| CN103130644A (en) | 2013-06-05 |
| CN103130644B (en) | 2014-10-22 |
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