CN105481770A - Preparation method for alkaloids by performing extraction on lindera glauca root and uses thereof - Google Patents
Preparation method for alkaloids by performing extraction on lindera glauca root and uses thereof Download PDFInfo
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Abstract
The invention discloses a preparation method for alkaloids by performing extraction on lindera glauca root and uses thereof. The preparation method comprises the following steps: 1) processing raw materials; 2) performing percolating extraction by using an acidic alcohol aqueous solution; 3) preparing a lindera-glauca-root total alkaloids extractive concentrate; 4) preparing an eluent; and 5)-8) preparing and purifying the compounds. By employing the acidic alcohol aqueous solution as the extraction solvent, the extraction solvent can be repeated recovered for utilization, and cost is saved. Percolating extraction is employed, the leachate can reach a relatively high concentration, and extraction efficiency can be improved. The method employs normal-temperature operation, does not need heating, the solvent usage amount is less, the filtration requirement is relatively low, the separation operation process is simplified, the extractive is less in impurities, extraction efficiency is high, and the solvent usage amount is less.
Description
Technical field
The invention belongs to extracted form natural plant separation technology field, be specifically related to one and extract alkaloidal preparation method and its usage from hickory root.
Background technology
The Lauraceae Lauraceae Lindera LinderaThunb. plant whole world about has 100 kinds, is distributed in Asia, temperature torrid areas, North America.There is 40 kind of 9 mutation 2 modification in China.Lindera is that in China's Lauraceae, kind is more, one of genus distributed more widely.Thymus mongolicus Linderaglauca (Siebold & Zucc.) Blume, is born in height above sea level less than about 900 meters hillside, border, roadside.On the south the Kunyu Mountain In Shandong Province of China, on the south Songxian, Henan, the provinces and regions such as on the south Yun County, Shaanxi and Gansu, Shanxi, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Guangdong, Guangxi, Hubei, Hunan, Sichuan are distributed more widely.Timber can make furniture; Leaf, pericarp can carry perfume oil; Seed kernel oil is containing lauric acid, and oil can make soap and lubricating oil; Root, branch, leaf, fruit medicinal; Leaf can stagnant, the detumescence of dispeling the wind of warming spleen and stomach for dispelling cold, relieving stagnant Qiization; Internal lesion caused by overexertion takes off power, water wets edema, four limbs are tingle, rheumatic arthritis, wound in radical cure; Fruit treats for stomachache.
More for Thymus mongolicus alkaloids component separating qualification work, but the alkaloid antitumor activity obtained is less for being separated in this plant, rarely seen Thymus mongolicus anti metastasis chemical composition to be reported, but to the further investigation of its activeconstituents, particularly to the inhibit activities research that different tumor cell line is bred, there is not yet report.
Therefore, select suitable extracting method to prepare Thymus mongolicus total alkaloids, and separation and purification research is carried out to the monomeric compound in total alkaloids, find the monomeric compound with anti-tumor activity, have significant to rational exploitation and utilization Thymus mongolicus resource.But, existingly mainly to have the following disadvantages for Thymus mongolicus total alkaloids extraction purification technical matters: (1) uses high density alcoholic solution as Extraction medium, and cost is higher, extracts complete not; And adopt and use water as Extraction medium, although economical, the efficiency of extracting effective components is low, and in Root of Greyblue Spicebush, alkaloids effective constituent is difficult to effective leaching.(2) traditional extraction process as decocted, backflow, impregnating, leaching process relates to heat treated or consuming time oversize, and extracting liquid filtering treatment step is many, and yield is also undesirable.In conjunction with above 2 points, the present invention adopts acid alcohol solution as Extraction solvent, repeatedly recycles extraction, can significantly improve extraction efficiency.And adopting seepage pressure effects, leach liquor can reach higher concentration, and leaching effect is better.This method normal-temperature operation does not need heating, and solvent load is few, and filtering requirement is lower, and separation operation process is simplified.And extract is impure less, extraction yield is high, use quantity of solvent few, saves solvent, reduces costs simultaneously.
The present invention obtains purity, alkaloid total extract that yield is higher.Consumption and the proportioning of organic solvent is continued to optimize in the selection of organic solvent.Further, the present invention adopts organic solvent extraction as preliminary purification means, have easy to operate, be easy to amplify and the advantage such as with low cost.
Summary of the invention
For problems of the prior art, the object of the invention is providing one to extract alkaloidal preparation method and its usage from hickory root.
The present invention is realized by the following technical programs:
Described one extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that comprising the following steps:
1) Feedstock treating: get after Root of Greyblue Spicebush dries in the shade and be 3-8% to water content, pulverize, cross 20-80 mesh sieve, obtain meal, for subsequent use;
2) acid alcohol solution solution seepage pressure effects: get the meal that step 1) is obtained, load in percolator, add the acid alcohol solution of the pH value 1-2 of 1-2 times amount, wherein the concentration of alcohol is 30%-80%, infiltrating loaded in diacolation bucket after 3-6 hour, carry out seepage pressure effects with the above-mentioned acid alcohol solution solution of 10-18 times amount, regulate flow velocity, collect percolate to the alkaloid lixiviate in Root of Greyblue Spicebush meal is clean with 10-20mL/min flow velocity; Adopt thin layer chromatography, aobvious brownish black spot under 254nm, bismuth potassium iodide solution shows safran, adds buck and regulates pH value to 9-10, then add organic solvent extraction 2-6 time, obtained extraction liquid in gained extracting solution; The volume ratio of described extracting solution and organic solvent is 1:1-3; Wherein chromatographic condition is for being carrier with GF254 fluorescence silica gel, and Preparative TLC chromatosheet, with chloroform: methyl alcohol=6:1 is developping agent, develops the color and observe under 254nm ultra-violet lamp;
3) by step 2) obtained extraction liquid, in bath temperature 40-55 DEG C, vacuum tightness is carry out concentrating under reduced pressure under the condition of 0.09-0.1Mpa, and the concentration being concentrated into concentrated solution is 0.5-1.0g/ml, obtained Root of Greyblue Spicebush total alkaloids extract concentrated solution;
4) the Root of Greyblue Spicebush total alkaloids extract concentrated solution that step 3) is obtained is crossed silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, collect elutriant, elutriant detects through thin-layer chromatography, merge same composition, obtain A-E totally 5 components, carry out anti-tumor activity tracking, B, component C anti-tumor activity are better;
5) to B component, through reversed-phase column chromatography, with the methanol-water gradient elution of 20-100%, elutriant detects through thin-layer chromatography, merges same composition, reclaims elutriant and obtains B1-B4 totally 4 components;
6) the B2 component that step 5) is obtained is crossed silicagel column, carry out purifying with chloroform-methanol, obtain brown color compound 1 and compound 2;
7) by the component C that step 4) is obtained, through reversed-phase column chromatography, with the methanol-water gradient elution of 30-100%, elutriant detects through thin-layer chromatography, and merge same composition, recycling design obtains C1-C8 totally 8 components;
8) the C4 component that step 7) is obtained is crossed gel column, purify with the methanol-water of 30-100%, obtain brown color compound 3;
Described compound 1 is N-methyllaurotetanine, and its structural formula is:
Described compound 2 is Litsoeine, and its structural formula is:
Described compound 3 is (+)-Boldine, and its structural formula is:
。
Described one extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in any one in the acetic acid of acid alcohol solution to be concentration be 0.5-2%, hydrochloric acid, sulfuric acid or tartrate.
Described one extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in buck be saturated sodium carbonate solution or the ammoniacal liquor of 15-25%.
Described one extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in organic solvent be in ethyl acetate, ether, acetone, propyl carbinol, chloroform or methylene dichloride one or more mixing.
Described one extracts alkaloidal preparation method from hickory root, to it is characterized in that in step 4) that the volume ratio of chloroform-methanol is followed successively by 10:1,8:1,6:1,4:1,2:1,0:1 and carries out gradient elution.
Described one extracts alkaloidal preparation method from hickory root, it is characterized in that methanol-water, the methanol-water of 40%, methanol-water, the methanol-water of 80%, the methyl alcohol gradient elution successively of 100% of 60% of using 20% in step 5) successively.
Described one extracts alkaloidal preparation method from hickory root, it is characterized in that the volume ratio of chloroform-methanol in step 6) is 8:1.
Described one extracts alkaloidal preparation method from hickory root, it is characterized in that the methanol-water wash-out first using 30% in step 7), then uses the methanol-water of 50%, the methanol-water wash-out of 70% successively, finally uses 100% methanol-eluted fractions; In step 8) existing 30% methanol-water purifying, then purify with the methanol-water of 50%, the methanol-water of 70%, finally purify with 100% methanol-water.
The application in anti-tumor medicinal preparation prepared by described a kind of alkaloid extracted from hickory root.
The present invention adopts low concentration alcohol-acid alcohol solution as Extraction solvent, repeatedly recycles extraction, can significantly improve extraction efficiency.And adopting seepage pressure effects, leach liquor can reach higher concentration, and leaching effect is better.This method normal-temperature operation does not need heating, and solvent load is few, and filtering requirement is lower, and separation operation process is simplified.And extract is impure less, extraction yield is high, use quantity of solvent few.
The present invention adopts organic solvent extraction to have the advantages such as extraction is convenient, simple, low cost, easily amplification as total alkaloids preliminary purification means.To the Root of Greyblue Spicebush total alkaloids extracted, active guiding is adopted to be separated, identify in Root of Greyblue Spicebush three alkaloids compositions with inhibition tumor cell proliferation activity, especially compound 1 has extremely strong suppression kinds of tumor cells proliferation activity, particularly suppress colon cancer cell HT-29, stomach cancer cell SGC-7901 cell-proliferation activity, under high dosage administrations, is even better than positive control medicine etoposide VP-16.This invention adopts conventional alkaloid extraction and separation method, and antitumor activity evaluation method, technical scheme is simple to operate, easily promotes.For the discovery of antitumor lead compound, the exploitation of anti-cancer agent has great importance.
Embodiment
By following specific embodiment, the invention will be further described.
Embodiment 1
1) Feedstock treating: get after Root of Greyblue Spicebush 600g dries in the shade and be 3-8% to water content, pulverize, cross 20-80 mesh sieve, obtain meal, for subsequent use; 2) acid alcohol solution solution seepage pressure effects: get the dry meal that step 1) is obtained, add the acid alcohol solution of the pH value 1-2 of 1.5 times amount, wherein the concentration of alcohol is 50%, infiltrating loaded in diacolation bucket after 3-6 hour, seepage pressure effects is carried out with the above-mentioned acid alcohol solution solution of 15 times amount, regulate flow velocity, collect percolate to the alkaloid lixiviate in Root of Greyblue Spicebush meal is clean with 10-20mL/min flow velocity; Adopt thin layer chromatography, aobvious brownish black spot under 254nm, bismuth potassium iodide solution shows safran, adds buck and regulates pH value to 9-10, then add organic solvent extraction 2-6 time, obtained extraction liquid in gained extracting solution; Wherein, the volume ratio of extracting solution and organic solvent is 1:2; Wherein chromatographic condition is for being carrier with GF254 fluorescence silica gel, and Preparative TLC chromatosheet, with chloroform: methyl alcohol=6:1 is developping agent, develops the color and observe under 254nm ultra-violet lamp; Acid alcohol solution to be concentration be 1% acetic acid, buck is saturated sodium carbonate solution, and machine solvent is ethyl acetate; 3) by step 2) obtained extraction liquid, in bath temperature 48 DEG C, vacuum tightness is carry out concentrating under reduced pressure under the condition of 0.09-0.1Mpa, and the concentration being concentrated into concentrated solution is 0.8g/ml, obtained Root of Greyblue Spicebush total alkaloids extract concentrated solution; 4) Root of Greyblue Spicebush total alkaloids extract concentrated solution is crossed silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, be specially, first carry out wash-out with chloroform-methanol volume ratio 10:1, being eluted to without becoming to distribute, changing chloroform-methanol volume ratio 8:1 and carrying out wash-out, being eluted to without becoming to distribute equally, in like manner, gradient elution is carried out by the ratio of 6:1,4:1,2:1,0:1 successively; Collect elutriant, elutriant detects through thin-layer chromatography, merges same composition, obtains A-E totally 5 components; 5) to B component, through reversed-phase column chromatography, with the methanol-water gradient elution of 20-100%, be specially, first carry out wash-out with the methanol-water of 20%, then carry out wash-out with the methanol-water of 40%, 60%, 80% successively, finally there is 100% methanol-eluted fractions, elutriant detects through thin-layer chromatography, merges same composition, reclaims elutriant and obtains B1-B4 totally 4 components; 6) the B2 component that step 5) is obtained is crossed silicagel column, carry out purifying with the chloroform-methanol that volume ratio is 8:1, obtain antitumor drug effect active substance: brown color compound 1 and compound 2; 7) by the component C that step 4) is obtained, through reversed-phase column chromatography, use the methanol-water gradient elution of 30%, 50%, 70%, 100% successively, elutriant detects through thin-layer chromatography, and merge same composition, recycling design obtains C1-C8 totally 8 components; 8) C4 component is crossed gel column, purify with the methanol-water of 30%, 50%, 70%, 100% successively, antitumor drug effect active substance must be obtained: brown color compound 3.
Embodiment 2
1) 1) Feedstock treating: get after Root of Greyblue Spicebush 600g dries in the shade and be 3-8% to water content, pulverize, cross 20-80 mesh sieve, obtain meal, for subsequent use; 2) acid alcohol solution solution seepage pressure effects: get the dry meal that step 1) is obtained, add the acid alcohol solution of the pH value 1-2 of 1 times amount, wherein the concentration of alcohol is 30%, infiltrating loaded in diacolation bucket after 3-6 hour, seepage pressure effects is carried out with the above-mentioned acid alcohol solution solution of 10 times amount, regulate flow velocity, collect percolate to the alkaloid lixiviate in Root of Greyblue Spicebush meal is clean with 10-20mL/min flow velocity; Adopt thin layer chromatography, aobvious brownish black spot under 254nm, bismuth potassium iodide solution shows safran, adds buck and regulates pH value to 9-10, then add organic solvent extraction 2-6 time, obtained extraction liquid in gained extracting solution; Wherein, the volume ratio of extracting solution and organic solvent is 1:1; Wherein chromatographic condition is for being carrier with GF254 fluorescence silica gel, and Preparative TLC chromatosheet, with chloroform: methyl alcohol=6:1 is developping agent, develops the color and observe under 254nm ultra-violet lamp; Acid alcohol solution to be concentration be 0.5% acetic acid, buck is the ammoniacal liquor of 20%, and machine solvent is chloroform; 3) by step 2) obtained extraction liquid, in bath temperature 40 DEG C, vacuum tightness is carry out concentrating under reduced pressure under the condition of 0.09-0.1Mpa, and the concentration being concentrated into concentrated solution is 0.5g/ml, obtained Root of Greyblue Spicebush total alkaloids extract concentrated solution; 4) Root of Greyblue Spicebush total alkaloids extract concentrated solution is crossed silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, be specially, first carry out wash-out with chloroform-methanol volume ratio 10:1, being eluted to without becoming to distribute, changing chloroform-methanol volume ratio 8:1 and carrying out wash-out, being eluted to without becoming to distribute equally, in like manner, gradient elution is carried out by the ratio of 6:1,4:1,2:1,0:1 successively; Collect elutriant, elutriant detects through thin-layer chromatography, merges same composition, obtains A-E totally 5 components; 5) to B component, through reversed-phase column chromatography, with the methanol-water gradient elution of 20-100%, be specially, first carry out wash-out with the methanol-water of 20%, then carry out wash-out with the methanol-water of 40%, 60%, 80% successively, finally there is 100% methanol-eluted fractions, elutriant detects through thin-layer chromatography, merges same composition, reclaims elutriant and obtains B1-B4 totally 4 components; 6) the B2 component that step 5) is obtained is crossed silicagel column, carry out purifying with the chloroform-methanol that volume ratio is 8:1, antitumor drug effect active substance must be obtained: brown color compound 1 and compound 2; 7) by the component C that step 4) is obtained, through reversed-phase column chromatography, use the methanol-water gradient elution of 30%, 50%, 70%, 100% successively, elutriant detects through thin-layer chromatography, and merge same composition, recycling design obtains C1-C8 totally 8 components; 8) C4 component is crossed gel column, purify with the methanol-water of 30%, 50%, 70%, 100% successively, antitumor drug effect active substance must be obtained: brown color compound 3.
Embodiment 3
1) Feedstock treating: get after Root of Greyblue Spicebush 600g dries in the shade and be 3-8% to water content, pulverize, cross 20-80 mesh sieve, obtain meal, for subsequent use; 2) acid alcohol solution solution seepage pressure effects: get the dry meal that step 1) is obtained, add the acid alcohol solution of the pH value 1-2 of 2 times amount, wherein the concentration of alcohol is 80%, infiltrating loaded in diacolation bucket after 3-6 hour, seepage pressure effects is carried out with the above-mentioned acid alcohol solution solution of 18 times amount, regulate flow velocity, collect percolate to the alkaloid lixiviate in Root of Greyblue Spicebush meal is clean with 10-20mL/min flow velocity; Adopt thin layer chromatography, aobvious brownish black spot under 254nm, bismuth potassium iodide solution shows safran, adds buck and regulates pH value to 9-10, then add organic solvent extraction 2-6 time, obtained extraction liquid in gained extracting solution; Wherein, the volume ratio of extracting solution and organic solvent is 1:3; Wherein chromatographic condition is for being carrier with GF254 fluorescence silica gel, and Preparative TLC chromatosheet, with chloroform: methyl alcohol=6:1 is developping agent, develops the color and observe under 254nm ultra-violet lamp; Acid alcohol solution to be concentration be 2% sulfuric acid, buck is the ammoniacal liquor of 25%, and machine solvent is methylene dichloride; 3) by step 2) obtained extraction liquid, in bath temperature 55 DEG C, vacuum tightness is carry out concentrating under reduced pressure under the condition of 0.09-0.1Mpa, and the concentration being concentrated into concentrated solution is 1.0g/ml, obtained Root of Greyblue Spicebush total alkaloids extract concentrated solution; 4) Root of Greyblue Spicebush total alkaloids extract concentrated solution is crossed silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, be specially, first carry out wash-out with chloroform-methanol volume ratio 10:1, being eluted to without becoming to distribute, changing chloroform-methanol volume ratio 8:1 and carrying out wash-out, being eluted to without becoming to distribute equally, in like manner, gradient elution is carried out by the ratio of 6:1,4:1,2:1,0:1 successively; Collect elutriant, elutriant detects through thin-layer chromatography, merges same composition, obtains A-E totally 5 components; 5) to B component, through reversed-phase column chromatography, with the methanol-water gradient elution of 20-100%, be specially, first carry out wash-out with the methanol-water of 20%, then carry out wash-out with the methanol-water of 40%, 60%, 80% successively, finally there is 100% methanol-eluted fractions, elutriant detects through thin-layer chromatography, merges same composition, reclaims elutriant and obtains B1-B4 totally 4 components; 6) the B2 component that step 5) is obtained is crossed silicagel column, carry out purifying with the chloroform-methanol that volume ratio is 8:1, antitumor drug effect active substance must be obtained: brown color compound 1 and compound 2; 7) by the component C that step 4) is obtained, through reversed-phase column chromatography, use the methanol-water gradient elution of 30%, 50%, 70%, 100% successively, elutriant detects through thin-layer chromatography, and merge same composition, recycling design obtains C1-C8 totally 8 components; 8) C4 component is crossed gel column, purify with the methanol-water of 30%, 50%, 70%, 100% successively, antitumor drug effect active substance must be obtained: brown color compound 3.
The qualification of the compound 1-3 in above-described embodiment:
Compound 1 yellow-brown solid: ESI-MSm/z342 ［ M+H ］
+ 1; H-NMR (CDCl
3, 400MHz)
: 2.54 (3H, s, N-CH
3), 2.56-3.23 (7H, m, H-4,5,6a, 7), 3.64 (3H, s, 1-OCH
3), 3.85 (3H, s, 2-OCH
3), 3.86 (3H, s, 10-OCH
3), 6.56 (1H, s, H-3), 6.77 (1H, s, H-8), 8.03 (1H, s, H-11); 13C-NMR (CDCl
3, 125MHz), 28.94 (C-4), 34.04 (C-7), 43.76 (N-CH
3), 53.28 (C-5), 55.85 (2-OCH
3), 56.01 (10-OCH
3), 60.19 (7-OCH
3), 62.59 (C-6a), 110.28 (C-3), 111.32 (C-11), 114.23 (C-8), 123.80 (C-11a), 126.74 (C-1a), 127.16 (C-1b), 128.74 (C-3a), 129.88 (C-7a), 144.27 (C-10), 145.14 (C-1), 145.58 (C-9), 152.09 (C-2).Above data and document Shoei-ShengLee, Yi-JeanLin, Chien-KuangChen, etal.QuaternaryAlkaloidsfromLitseacubebaandCryptocaryako nishii [J] .JournalofNaturalProduct, 1993, the data consistent of the N-methyll-aurotetanine of 56 (1): 1971-1976. reports, authenticating compound is N-methyllaurotetanine.
Compound 2 yellow-brown solid: ESI-MSm/z328 ［ M+H ］
+ 1; H-NMR (CD
3oD, 400MHz)
: 6.83 (1H, s, H-3), 3.26 (1H, dd, J=11.6,5.2, H-4a), 3.05 (1H, dd, J=11.6,5.2, H-4b), 3.72 (1H, dd, J=11.6,5.2, H-5a), 3.39 (1H, dd, J=12.8,4.4, H-5b), 4.15 (1H, dd, J=14.0,4.0, H-6a), 2.72 (1H, dd, J=11.6,5.2, H-5a), 2.98 (1H, dd, J=13.6,5.2, H-7a), 2.98 (1H, brt, J=14.0, H-7b), 6.76 (1H, s, H-8), 7.96 (1H, s, H-11), 3.90 (3H, s, 2-OCH
3), 3.84 (3H, s, 10-OCH
3), 3.66 (3H, s, 1-OCH
3); 13C-NMR (CDCl
3, 125MHz)
: 146.25 (C-1), 123.97 (C-1a), 127.44 (C-1b), 154.98 (C-2), 113.16 (C-3), 127.53 (C-3a), 26.26 (C-4), 42.45 (C-5), 54.30 (C-6a), 34.01 (C-7), 128.03 (C-7a), 115.83 (C-8), 147.67 (C-9), 148.10 (C-10), 111.81 (C-11), 121.77 (C-11a), 55.39 (2-OCH
3), 56.43 (10-OCH
3), 60.45 (1-OCH
3). above data and document Zhao lofty ideal, Zhao Yimin, Wang Kejun. the alkaloid component ［ J ］ in Twig and leaf of Narrowleaf Spicebush root. Acta Pharmaceutica Sinica, 2005,40(10): the data consistent of the Litsoeine (lauro-tetanine) of 931-934. report, authenticating compound is Litsoeine.
Compound 3 yellow-brown solid: ESI-MSm/z328 ［ M+H ］
+ 1; H-NMR (CDCl
3, 400MHz)
: 2.56 (3H, s, N-CH
3), 2.43-3.11 (7H, m, H-4,5,6a, 7), 3.57 (3H, s, 1-OCH
3), 3.87 (3H, s, 10-OCH
3), 6.55 (1H, s, H-3), 6.74 (1H, s, H-8), 7.98 (1H, s, H-11). above data and document Yuh-ChwenChang, Fang-RongChang, Yang-ChangWU.TheConstituentsofLinderaglauca [J] .JournaloftheChineseChemicalSociety, 2000, the data consistent of (+)-Boldine of 47 (2): 373-380. reports, authenticating compound is (+)-Boldine.
Determination of activity:
Utilize DMSO solvent, the mass concentration gradient of preparation compound 1-3 is 1,3 respectively, the test soln of 10,30,100 μ g/ml.Adopt mtt assay to study the inhibited proliferation of each compound to 5 kinds of human body tumour cells (HT-29, SGC-7901, SMMC-7721 and A549), find effective natural antitumor compound.Choose the cell of logarithmic phase, with 0.25g/100mL tryptic digestion adjustment concentration of cell suspension, with 5 × 10
3individual/hole is inoculated in 96 orifice plates, and perfect medium is supplied to 100uL in every hole.Separately establish the blank zeroing group not adding cell and only add substratum.After cell attachment, the compound adding different mass concentration intervenes tumor cell differentiation, every hole chemical feeding quantity 100 μ L, and each mass concentration arranges 3 multiple holes.Establish a control group simultaneously, namely add equivalent nutrient solution (compound quality concentration 0ug/mL).After dosing 72h, every hole adds MTT (5mg/mL) solution 20 μ L, and 37 DEG C are continued to hatch 4h.Supernatant liquor in centrifugal complete reject hole, every hole adds DMSO150 μ L, and vibration 10min, makes crystallisate fully dissolve.Microplate reader detects the absorbance A 492 at 492nm wavelength place.Calculate cell survival rate according to the following formula.
Adopt antitumor drug Etoposide to be positive control simultaneously, the results are shown in Table 1:
Table 1 compound 1 ~ 3, alkaloid general extractive are to the inhibited proliferation of 5 kinds of human body tumour cells
As can be seen from Table 1: from Root of Greyblue Spicebush, be separated the monomeric compound anti tumor activity in vitro effect obtained, be obviously better than total alkaloids extract.These 4 kinds of human body tumour cell propagation of monomeric compound 1 ~ 3 couple of HT-29, SGC-7901, SMMC-7721 and A549 all have stronger restraining effect.Especially compound 1 has extremely strong inhibition tumor cell proliferation activity, and particularly suppress colon cancer cell HT-29, stomach cancer cell SGC-7901 cell-proliferation activity, under high dosage administrations, is even better than positive control medicine etoposide VP-16.Therefore, the monomeric compound prepared by this invention is for the research and development of anti-cancer agent, and preparation has great importance.
Claims (9)
1. from Root of Greyblue Spicebush, extract an alkaloidal preparation method, it is characterized in that comprising the following steps:
1) Feedstock treating: get after Root of Greyblue Spicebush dries in the shade and be 3-8% to water content, pulverize, cross 20-80 mesh sieve, obtain meal, for subsequent use;
2) acid alcohol solution seepage pressure effects: get the meal that step 1) is obtained, load in percolator, add the acid alcohol solution of the pH value 1-2 of 1-2 times amount, wherein the concentration of alcohol is 30%-80%, infiltrating loaded in diacolation bucket after 3-6 hour, carry out seepage pressure effects with the above-mentioned acid alcohol solution solution of 10-18 times amount, regulate flow velocity, collect percolate to the alkaloid lixiviate in Root of Greyblue Spicebush meal is clean with 10-20mL/min flow velocity; Adopt thin layer chromatography, aobvious brownish black spot under ultraviolet lamp 254nm, bismuth potassium iodide solution shows safran, adds buck and regulates pH value to 9-10, then add organic solvent extraction 2-6 time, obtained extraction liquid in gained extracting solution; The volume ratio of described extracting solution and organic solvent is 1:1-3; Wherein chromatographic condition is: GF254 thin-layer chromatography chromatogram, with chloroform: methyl alcohol=6:1 is developping agent, and under 254nm ultra-violet lamp, colour developing is observed;
3) by step 2) obtained extraction liquid, in bath temperature 40-55 DEG C, vacuum tightness is carry out concentrating under reduced pressure under the condition of 0.09-0.1Mpa, and the concentration being concentrated into concentrated solution is 0.5-1.0g/ml, obtained Root of Greyblue Spicebush total alkaloids extract concentrated solution;
4) the Root of Greyblue Spicebush total alkaloids extract concentrated solution that step 3) is obtained is crossed silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, collect elutriant, elutriant detects through thin-layer chromatography, merges same composition, obtains A-E totally 5 components; Carry out anti-tumor activity tracking, B, component C anti-tumor activity are better;
5) to B component, through reversed-phase column chromatography, with the methanol-water gradient elution of 20-100%, elutriant detects through thin-layer chromatography, merges same composition, reclaims elutriant and obtains B1-B4 totally 4 components;
6) the B2 component that step 5) is obtained is crossed silicagel column, carry out purifying with chloroform-methanol, obtain yellow-brown solid compound 1 and compound 2;
7) by the component C that step 4) is obtained, through reversed-phase column chromatography, with the methanol-water gradient elution of 30-100%, elutriant detects through thin-layer chromatography, and merge same composition, recycling design obtains C1-C8 totally 8 components;
8) the C4 component that step 7) is obtained is crossed gel column, purify with the methanol-water of 30-100%, obtain yellow-brown solid compound 3;
Described compound 1 is N-methyllaurotetanine, and its structural formula is:
Described compound 2 is Litsoeine, and its structural formula is:
Described compound 3 is (+)-Boldine, and its structural formula is:
。
2. one according to claim 1 extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in any one in the acetic acid of acid alcohol solution to be concentration be 0.5-2%, hydrochloric acid, sulfuric acid or tartrate.
3. one according to claim 1 extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in buck be saturated sodium carbonate solution or the ammoniacal liquor of 15-25%.
4. one according to claim 1 extracts alkaloidal preparation method from Root of Greyblue Spicebush, it is characterized in that step 2) in organic solvent be in ethyl acetate, ether, acetone, propyl carbinol, chloroform or methylene dichloride one or more mixing.
5. one according to claim 1 extracts alkaloidal preparation method from hickory root, to it is characterized in that in step 4) that the volume ratio of chloroform-methanol is followed successively by 10:1,8:1,6:1,4:1,2:1,0:1 and carries out gradient elution.
6. one according to claim 1 extracts alkaloidal preparation method from hickory root, it is characterized in that methanol-water, the methanol-water of 40%, methanol-water, the methanol-water of 80%, the methyl alcohol gradient elution successively of 100% of 60% of using 20% in step 5) successively.
7. one according to claim 1 extracts alkaloidal preparation method from hickory root, it is characterized in that the volume ratio of chloroform-methanol in step 6) is 8:1.
8. one according to claim 1 extracts alkaloidal preparation method from hickory root, it is characterized in that the methanol-water wash-out first using 30% in step 7), then uses the methanol-water of 50%, the methanol-water wash-out of 70% successively, finally uses 100% methanol-eluted fractions; In step 8) existing 30% methanol-water purifying, then purify with the methanol-water of 50%, the methanol-water of 70%, finally purify with 100% methanol-water.
9. the application in anti-tumor medicinal preparation prepared by a kind of alkaloid extracted from hickory root as claimed in claim 1.
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| CN116982553B (en) * | 2023-06-29 | 2024-04-19 | 广西壮族自治区南宁良凤江国家森林公园 | Tissue culture method for cortex cinnamomi japonici |
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