CN105481770B - A kind of preparation method and its usage extracting alkaloid from greyblue spicebush root - Google Patents
A kind of preparation method and its usage extracting alkaloid from greyblue spicebush root Download PDFInfo
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Abstract
The invention discloses a kind of from greyblue spicebush root extracts the preparation method and its usage of alkaloid, includes the following steps：1）Feedstock processing；2）Acid alcohol solution seepage pressure effects；3）The preparation of greyblue spicebush root total alkaloid extract concentrate；4）The preparation of eluent；5）‑8）The preparation and purifying of compound.The present invention, as Extraction solvent, recycles extraction repeatedly using acid alcohol solution, cost-effective.And seepage pressure effects are used, leachate can reach higher concentration, can improve extraction efficiency.This method normal-temperature operation is not required to heat, and solvent dosage is few, and filtering requirement is relatively low, and separation operation process is made to simplify.And extract is impure less, recovery rate is high, few using quantity of solvent.
Description
Technical field
The invention belongs to extracted form natural plant separation technology fields, and in particular to one kind extracting alkaloid from greyblue spicebush root
Preparation method and its usage.
Background technology
Lauraceae Lauraceae LinderasLindera ThunbThe plant whole world is distributed in Asia there are about 100 kinds
Continent, North America temperature torrid areas.There is 40 kind of 92 modification of mutation in China.Lindera is that type is more in China's Lauraceae, distributed more widely
One of category.Lindera glaucaLindera glauca(Siebold Zucc.) Blume is born in 900 meters or so of height above sea level to go down the hill
Slope, border, roadside.On the south the Kunyu Mountain In Shandong Province in China, on the south Songxian, Henan, on the south the Yun County of Shaanxi and Gansu, Shanxi, river
The provinces and regions such as Soviet Union, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Guangdong, Guangxi, Hubei, Hunan, Sichuan are distributed more widely.Timber can be made
Furniture；Leaf, pericarp can carry aromatic oil；Seed kernel oil contains lauric acid, and oil can make soap and lubricating oil；Root, branch, leaf, fruit are medicinal；Ye Ke
Warming spleen and stomach for dispelling cold, relieving stagnant Qiization are stagnant, wind-dispelling is subsided a swelling；Effect a radical cure tingle internal lesion caused by overexertion power off, the wet edema of water, four limbs, rheumatic arthritis, bruise damage
Wound；Fruit treats for stomachache.
It is more for lindera glauca composition of alkaloids separation appraisal, but for biology isolated in the plant
Alkali antitumor activity is less, rarely seen to be reported lindera glauca anti-tumor metastasis chemical composition, but to its active constituent
Further investigation, especially to different tumor cell lines proliferation inhibitory activity research, there is not yet report.
Therefore, suitable extracting method is selected to prepare lindera glauca total alkaloid, and to the monomeric compound in total alkaloid
It carries out isolating and purifying research, finds monomeric compound with anti-tumor activity, have to rational exploitation and utilization lindera glauca resource
It is significant.But existing it is primarily present following shortcoming for lindera glauca total alkaloid extraction purification technical matters：
（1）Using high concentration alcoholic solution as Extraction medium, cost is higher, and extraction is not complete enough；And Extraction medium is made using water, although
Economy, but the efficiency of effective component extracting is low, and alkaloids active ingredient is difficult to effectively leach in greyblue spicebush root.（2）Tradition carries
Take method as decocted, flowing back, impregnating, extraction process is related to heat treatment or time-consuming too long, and at extracting solution filtering
Reason step is more, and yield is also undesirable.In conjunction with above 2 points, the present invention, as Extraction solvent, is returned repeatedly using acid alcohol solution
It receives using extraction, extraction efficiency can be significantly improved.And seepage pressure effects are used, leachate can reach higher concentration, leaching effect
Preferably.This method normal-temperature operation is not required to heat, and solvent dosage is few, and filtering requirement is relatively low, and separation operation process is made to simplify.And extract
It is impure less, recovery rate it is high, few using quantity of solvent, while saving solvent, reduce cost.
The present invention is to obtain the higher alkaloid total extract of purity, yield.It is continued to optimize in terms of the selection of organic solvent
The dosage and proportioning of organic solvent.Also, the present invention is used as preliminary purification means using organic solvent extraction, has operation side
Just the advantages that, being easy to amplify and is of low cost.
Invention content
For problems of the prior art, present invention aims at providing one kind extraction biology from greyblue spicebush root
The preparation method and its usage of alkali.
The present invention is realized by the following technical programs：
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that include the following steps：
1）Feedstock processing：It takes greyblue spicebush root to dry in the shade to water content after 3-8%, to crush, crosses 20-80 mesh sieve, obtain coarse powder, it is standby
With；
2）Acid alcohol solution solution seepage pressure effects：Take step 1）Coarse powder obtained, is fitted into percolator, adds 1-2 times to measure
PH value 1-2 acid alcohol solution, the wherein a concentration of 30%-80% of alcohol, infiltration 3-6 hour after be fitted into diacolation bucket, use
It measures above-mentioned acid alcohol solution solution for 10-18 times and carries out seepage pressure effects, adjust flow velocity, diacolation is collected with 10-20mL/min flow velocitys
Until alkaloid extraction totally in extracting solution to greyblue spicebush root coarse powder；Using thin layer chromatography, dark brown color spot is shown at 254nm
Point, bismuth potassium iodide solution show crocus, and buck is added in gained extracting solution and adjusts pH value to 9-10, organic solvent extraction is then added
It takes 2-6 times, extract liquor is made；The volume ratio of the extracting solution and organic solvent is 1:1-3；Wherein chromatographic condition is to use GF254
Fluorescence silica gel is carrier, prepares chromatographic sheet, with chloroform：Methanol=6:1 is solvent, develops the color and sees under 254nm ultraviolet lamps
It examines；
3）By step 2）Extract liquor obtained, in 40-55 DEG C of bath temperature, under conditions of vacuum degree is 0.09-0.1Mpa
It is concentrated under reduced pressure, is concentrated into a concentration of 0.5-1.0g/ml of concentrate, greyblue spicebush root total alkaloid extract concentration is made
Liquid；
4）By step 3）Greyblue spicebush root total alkaloid extract concentrate obtained crosses silica gel column chromatography, uses chloroform-methanol
Volume ratio 10:1-0:1 gradient elution collects eluent, and eluent detects through thin-layer chromatography, merges same composition, obtains A-E totally 5
A component carries out antitumor activity tracking, and B, component C antitumor activity are preferable；
5）To B component, inverted column chromatography, with the methanol-water gradient elution of 20-100%, eluent is examined through thin-layer chromatography
It surveys, merges same composition, recycling eluent obtains B1-B4 totally 4 components；
6）By step 5）B2 components obtained cross silicagel column, are purified with chloroform-methanol, obtain 1 He of brown color compound
Compound 2；
7）By step 4）Component C obtained, inverted column chromatography, with the methanol-water gradient elution of 30-100%, eluent
It is detected through thin-layer chromatography, merges same composition, recycling design obtains C1-C8 totally 8 components；
8）By step 7）C4 components obtained cross gel column, are purified with the methanol-water of 30-100%, obtain brown color compound
3；
The compound 1 is N- methyllaurotetanines, and structural formula is：
The compound 2 is laurotetanine, and structural formula is：
The compound 3 is（+）- Boldine, structural formula are：
。
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 2）Middle acidity alcohol water
Solution is any one of acetic acid, hydrochloric acid, sulfuric acid or the tartaric acid of a concentration of 0.5-2%.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 2）Middle buck is full
The sodium carbonate liquor of sum or the ammonium hydroxide of 15-25%.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 2）Middle organic solvent
It is mixed for one or more of ethyl acetate, ether, acetone, n-butanol, chloroform or dichloromethane.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 4）Middle chloroform-first
The volume ratio of alcohol is followed successively by 10:1、8:1、6:1、4:1、2:1、0:1 carries out gradient elution.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 5）In use successively
20% methanol-water, 40% methanol-water, 60% methanol-water, 80% methanol-water, 100% methanol gradient elution successively.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 6）Middle chloroform-first
The volume ratio of alcohol is 8:1.
A kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that step 7）It is middle first with 30%
Methanol-water elution, then successively use 50% methanol-water, 70% methanol-water elution, finally with 100% methanol elution；Step
8）In existing 30% methanol-water purifying, then purified with the methanol-water of 50% methanol-water, 70%, finally with 100% methanol-
Water purifies.
A kind of application from the alkaloid extracted in greyblue spicebush root in preparing anti-tumor medicinal preparation.
The present invention, as Extraction solvent, is recycled extraction, can significantly carried repeatedly using low concentration alcohol-acidity alcohol solution
High extracting efficiency.And seepage pressure effects are used, leachate can reach higher concentration, and leaching effect is preferable.This method normal-temperature operation is not
It needs to heat, solvent dosage is few, and filtering requirement is relatively low, and separation operation process is made to simplify.And extract is impure less, recovery rate is high,
It is few using quantity of solvent.
The present invention using organic solvent extraction as total alkaloid preliminary purification means have extract it is convenient, simple, low at
Originally the advantages that, easily amplifying.To the greyblue spicebush root total alkaloid of extraction, it is oriented to and is detached using activity, identified in greyblue spicebush root and have
There are three composition of alkaloids for inhibiting proliferative activity o f tumor, especially compound 1 that there is extremely strong inhibition kinds of tumors
Cell-proliferation activity especially inhibits colon cancer cell HT-29, stomach cancer cell SGC-7901 cell-proliferation activities, in high dose
Under administrations, even better than positive control medicine etoposide VP-16.The invention extracts separation side using conventional alkaloid
Method, antitumor activity evaluation method, technical solution is easy to operate, is easy to promote.Discovery for antitumor lead compound resists
The exploitation of tumour new drug has great importance.
Specific implementation mode
By following specific examples, the invention will be further described.
Embodiment 1
1）Feedstock processing：It takes greyblue spicebush root 600g to dry in the shade to water content after 3-8%, to crush, crosses 20-80 mesh sieve, obtain slightly
Powder, it is spare；2）Acid alcohol solution solution seepage pressure effects：Take step 1）Dry coarse powder obtained adds the pH value 1-2 of 1.5 times of amounts
Acid alcohol solution, wherein a concentration of the 50% of alcohol, infiltration is fitted into after 3-6 hour in diacolation bucket, and above-mentioned acidity are measured with 15 times
Alcohol solution solution carries out seepage pressure effects, adjusts flow velocity, and percolate is collected to lindera glauca rough with 10-20mL/min flow velocitys
Until alkaloid extraction totally in powder；Using thin layer chromatography, brownish black spot is shown at 254nm, bismuth potassium iodide solution is aobvious
Buck is added in gained extracting solution and adjusts pH value to 9-10, organic solvent is then added and extracts 2-6 time, is made and extracts for crocus
Liquid；Wherein, the volume ratio of extracting solution and organic solvent is 1:2；It is carrier that wherein chromatographic condition, which is with GF254 fluorescence silica gels, system
Standby chromatographic sheet, with chloroform：Methanol=6:1 is solvent, and develop the color observation under 254nm ultraviolet lamps；Acid alcohol solution is
A concentration of 1% acetic acid, buck are the sodium carbonate liquor of saturation, and solvent is ethyl acetate；3）By step 2）Extraction obtained
Liquid, in 48 DEG C of bath temperature, vacuum degree is concentrated under reduced pressure under conditions of being 0.09-0.1Mpa, is concentrated into the concentration of concentrate
For 0.8g/ml, greyblue spicebush root total alkaloid extract concentrate is made；4）By greyblue spicebush root total alkaloid extract concentrate mistake
Silica gel column chromatography, with chloroform-methanol volume ratio 10:1-0:1 gradient elution, specifically, first using chloroform-methanol volume ratio 10:1 into
Row elution, is eluted to without at distributing, changes chloroform-methanol volume ratio 8：1 is eluted, and is equally eluted to without at distributing, together
Reason, successively with 6:1、4:1、2:1、0:1 ratio carries out gradient elution；Eluent is collected, eluent is detected through thin-layer chromatography, is closed
And same composition, obtain A-E totally 5 components；5）To B component, inverted column chromatography, with the methanol-water gradient elution of 20-100%,
Specifically, first being eluted with 20% methanol-water, is then eluted with 40%, 60%, 80% methanol-water, finally had successively
100% methanol elutes, and eluent is detected through thin-layer chromatography, merges same composition, and recycling eluent obtains B1-B4 totally 4 components；6）
By step 5）B2 components obtained cross silicagel column, are 8 with volume ratio:1 chloroform-methanol is purified, and is obtained antitumor drug effect and is lived
Property substance：Brown color compound 1 and compound 2；7）By step 4）Component C obtained, inverted column chromatography, successively with 30%,
50%, 70%, 100% methanol-water gradient elution, eluent are detected through thin-layer chromatography, merge same composition, and recycling design obtains C1-
C8 totally 8 components；8）C4 components are crossed into gel column, are purified successively with 30%, 50%, 70%, 100% methanol-water, obtain antitumor
Drug activity substance：Brown color compound 3.
Embodiment 2
1）1）Feedstock processing：It takes greyblue spicebush root 600g to dry in the shade to water content after 3-8%, to crush, crosses 20-80 mesh sieve, obtain slightly
Powder, it is spare；2）Acid alcohol solution solution seepage pressure effects：Take step 1）Dry coarse powder obtained adds the pH value 1-2's of 1 times of amount
Acid alcohol solution, wherein a concentration of the 30% of alcohol, infiltration are fitted into after 3-6 hours in diacolation bucket, with the above-mentioned acid alcohol of 10 times of amounts
Aqueous solution carries out seepage pressure effects, adjusts flow velocity, and percolate is collected to greyblue spicebush root coarse powder with 10-20mL/min flow velocitys
In alkaloid extraction it is clean until；Using thin layer chromatography, brownish black spot is shown at 254nm, bismuth potassium iodide solution shows tangerine
Buck is added in gained extracting solution and adjusts pH value to 9-10, organic solvent is then added and extracts 2-6 time for yellow, obtained extract liquor；
Wherein, the volume ratio of extracting solution and organic solvent is 1:1；It is carrier that wherein chromatographic condition, which is with GF254 fluorescence silica gels, is prepared thin
Layer chromatography plate, with chloroform：Methanol=6:1 is solvent, and develop the color observation under 254nm ultraviolet lamps；Acid alcohol solution is concentration
For 0.5% acetic acid, the ammonium hydroxide that buck is 20%, solvent is chloroform；3）By step 2）Extract liquor obtained, in bath temperature 40
DEG C, vacuum degree is concentrated under reduced pressure under conditions of being 0.09-0.1Mpa, is concentrated into a concentration of 0.5g/ml of concentrate, and mountain is made
Pepper root total alkaloid extract concentrate；4）Greyblue spicebush root total alkaloid extract concentrate is crossed into silica gel column chromatography, uses chlorine
Imitation-carbinol volume ratio 10:1-0:1 gradient elution, specifically, first using chloroform-methanol volume ratio 10:1 is eluted, and nothing is eluted to
At distributing, chloroform-methanol volume ratio 8 is changed：1 is eluted, and is equally eluted to without at distributing, similarly, successively with 6:1、4:
1、2:1、0:1 ratio carries out gradient elution；Eluent is collected, eluent is detected through thin-layer chromatography, is merged same composition, is obtained A-
E totally 5 components；5）To B component, inverted column chromatography, with the methanol-water gradient elution of 20-100%, specifically, first with 20%
Methanol-water is eluted, and is then eluted successively with 40%, 60%, 80% methanol-water, is finally had the elution of 100% methanol, is washed
De- liquid is detected through thin-layer chromatography, merges same composition, and recycling eluent obtains B1-B4 totally 4 components；6）By step 5）B2 obtained
Component crosses silicagel column, is 8 with volume ratio:1 chloroform-methanol is purified, and antitumor drug effect active material is obtained to obtain：Brown color
Close object 1 and compound 2；7）By step 4）Component C obtained, inverted column chromatography, successively with 30%, 50%, 70%, 100% first
Alcohol-water gradient elution, eluent are detected through thin-layer chromatography, merge same composition, and recycling design obtains C1-C8 totally 8 components；8）It will
C4 components cross gel column, are purified successively with 30%, 50%, 70%, 100% methanol-water, obtain to obtain antitumor drug effect active material：Palm fibre
Yellow compound 3.
Embodiment 3
1）Feedstock processing：It takes greyblue spicebush root 600g to dry in the shade to water content after 3-8%, to crush, crosses 20-80 mesh sieve, obtain slightly
Powder, it is spare；2）Acid alcohol solution solution seepage pressure effects：Take step 1）Dry coarse powder obtained adds the pH value 1-2's of 2 times of amounts
Acid alcohol solution, wherein a concentration of the 80% of alcohol, infiltration are fitted into after 3-6 hours in diacolation bucket, with the above-mentioned acid alcohol of 18 times of amounts
Aqueous solution carries out seepage pressure effects, adjusts flow velocity, and percolate is collected to greyblue spicebush root coarse powder with 10-20mL/min flow velocitys
In alkaloid extraction it is clean until；Using thin layer chromatography, brownish black spot is shown at 254nm, bismuth potassium iodide solution shows tangerine
Buck is added in gained extracting solution and adjusts pH value to 9-10, organic solvent is then added and extracts 2-6 time for yellow, obtained extract liquor；
Wherein, the volume ratio of extracting solution and organic solvent is 1:3；It is carrier that wherein chromatographic condition, which is with GF254 fluorescence silica gels, is prepared thin
Layer chromatography plate, with chloroform：Methanol=6:1 is solvent, and develop the color observation under 254nm ultraviolet lamps；Acid alcohol solution is concentration
For 2% sulfuric acid, the ammonium hydroxide that buck is 25%, solvent is dichloromethane；3）By step 2）Extract liquor obtained, in bath temperature
55 DEG C, vacuum degree is concentrated under reduced pressure under conditions of being 0.09-0.1Mpa, is concentrated into a concentration of 1.0g/ml of concentrate, is made
Greyblue spicebush root total alkaloid extract concentrate；4）Greyblue spicebush root total alkaloid extract concentrate is crossed into silica gel column chromatography, is used
Chloroform-methanol volume ratio 10:1-0:1 gradient elution, specifically, first using chloroform-methanol volume ratio 10:1 is eluted, and is eluted to
Without at distributing, chloroform-methanol volume ratio 8 is changed：1 is eluted, and is equally eluted to without at distributing, similarly, successively with 6:1、
4:1、2:1、0:1 ratio carries out gradient elution；Eluent is collected, eluent is detected through thin-layer chromatography, is merged same composition, is obtained
A-E totally 5 components；5）To B component, inverted column chromatography, with the methanol-water gradient elution of 20-100%, specifically, first with 20%
Methanol-water eluted, then eluted successively with 40%, 60%, 80% methanol-water, finally have 100% methanol elution,
Eluent is detected through thin-layer chromatography, merges same composition, and recycling eluent obtains B1-B4 totally 4 components；6）By step 5）It is obtained
B2 components cross silicagel column, are 8 with volume ratio:1 chloroform-methanol is purified, and antitumor drug effect active material is obtained to obtain：Brown color
Compound 1 and compound 2；7）By step 4）Component C obtained, inverted column chromatography, successively with 30%, 50%, 70%, 100%
Methanol-water gradient elution, eluent are detected through thin-layer chromatography, merge same composition, and recycling design obtains C1-C8 totally 8 components；8）
C4 components are crossed into gel column, are purified successively with 30%, 50%, 70%, 100% methanol-water, antitumor drug effect active material is obtained to obtain：
Brown color compound 3.
The identification of compound 1-3 in above-described embodiment：
1 yellow-brown solid of compound：ESI-MS m/z 342 ［ M+H ］+1; H-NMR ( CDCl3,400 MHz
): d:2. 54 ( 3H, s, N-CH3 ) , 2.56-3.23 ( 7H, m, H-4, 5, 6a, 7) ,3. 64 ( 3H,s,
1-OCH3 ),3. 85( 3H, s, 2-OCH3 ), 3. 86( 3H ,s, 10-OCH3 ), 6. 56( 1H, s, H-3 ),
6. 77 ( 1H, s, H-8), 8. 03 ( 1H, s, H-11); 13C-NMR ( CDCl3, 125 MHz ), 28. 94
( C-4 ), 34. 04( C-7 ), 43. 76 ( N-CH3 ), 53. 28( C-5 ), 55. 85(2-OCH3 ), 56.
01 (10-OCH3 ), 60.19( 7-OCH3 ), 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).Data above and document Shoei-Sheng Lee, Yi-Jean Lin, Chien-Kuang
Chen, et al. Quaternary Alkaloids from Litsea cubeba and Cryptocarya konishii
[J]. Journal of Natural Product, 1993, 56(1):N-the methyll-of 1971-1976. reports
The data of aurotetanine are consistent, and authenticating compound is N- methyllaurotetanines.
2 yellow-brown solid of compound：ESI-MS m/z 328 ［ M+H ］+1; H-NMR ( CD3OD, 400
MHz ) d: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,
br t, J = 14.0, H-7b ), 6. 76 ( 1H, s, H-8 ), 7. 96 ( 1H, s, H-11), 3. 90(
3H,s,2-OCH3 ), 3. 84( 3H, s, 10-OCH3 ) , 3. 66 ( 3H, s, 1-OCH3 ); 13C-NMR (
CDCl3, 125 MHz ) d: 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-OCH3 ) ,56. 43 ( 10-OCH3 ) ,
60.45 ( 1-OCH3 ) data above and document Zhao's lofty ideal, Zhao Yimin, the alkaloid in Wang Ke armies narrowleaf spicebush branchlet leaf or root roots
Ingredient ［ J ］ Acta Pharmaceutica Sinicas, 2005,40（10）：The data one of the laurotetanine (lauro-tetanine) of 931-934. reports
It causes, authenticating compound is laurotetanine.
3 yellow-brown solid of compound：ESI-MS m/z 328 ［ M+H ］+1; H-NMR ( CDCl3, 400
MHz ) d: 2. 56 ( 3H, s, N-CH3 ), 2.43-3.11 ( 7H, m, H-4, 5, 6a, 7 ), 3. 57 (
3H, s, 1-OCH3 ), 3. 87( 3H, s, 10-OCH3 ), 6. 55( 1H, s, H-3 ), 6. 74 ( 1H, s,
), H-8 7. 98 (1H, s, H-11) data above and document Yuh-Chwen Chang, Fang-Rong Chang,
Yang-Chang WU. The Constituents of Lindera glauca [J]. Journal of the Chinese
Chemical Society, 2000, 47(2):The data of (+)-Boldine of 373-380. reports are consistent, identificationization
Conjunction object is (+)-Boldine.
Determination of activity：
Using DMSO solvents, the mass concentration gradient for preparing compound 1-3 respectively is the survey of 1,3,10,30,100 μ g/ml
Try solution.Each 5 kinds of human body tumour cells (HT-29, SGC-7901, SMMC-7721 and A549) of compound pair are studied using mtt assay
Inhibited proliferation, find effective natural antitumor compound.The cell for choosing exponential phase, uses 0.25g/100mL
Trypsin digestion adjusts concentration of cell suspension, with 5 × 103A/hole is inoculated in 96 orifice plates, and complete medium is supplied extremely per hole
100 u L.Another set is not added with the blank zeroing group that cell only adds culture medium.After cell is adherent, the change of different quality concentration is added
It closes object and intervenes tumor cell differentiation, per 100 μ L of hole dosage, 3 multiple holes are arranged in each mass concentration.A control group is set simultaneously,
I.e. plus equivalent culture solution (compound quality concentration 0ug/mL).After dosing 72h, 20 μ L of MTT (5mg/mL) solution are added per hole,
37 DEG C are continued to be incubated 4h.Supernatant in complete reject hole is centrifuged, 150 μ L of DMSO are added per hole, 10 min is vibrated, makes crystal
Fully dissolving.Microplate reader detects the absorbance A 492 at 492nm wavelength.Cell survival rate is calculated according to the following formula.
It uses antitumor drug Etoposide for positive control simultaneously, the results are shown in Table 1：
The inhibited proliferation of 5 kinds of 1 compound 1 ~ 3 of table, alkaloid total extract pair human body tumour cells
As can be seen from Table 1：The isolated monomeric compound anti tumor activity in vitro effect from greyblue spicebush root, hence it is evident that
It is better than total alkaloid extract.This 4 kinds of human bodies of monomeric compound 1 ~ 3 couple of HT-29, SGC-7901, SMMC-7721 and A549 are swollen
Tumor cell proliferation all has stronger inhibiting effect.Especially compound 1 has extremely strong inhibition proliferative activity o f tumor, special
It is not to inhibit colon cancer cell HT-29, stomach cancer cell SGC-7901 cell-proliferation activities, under high dose administrations, even
Better than positive control medicine etoposide VP-16.Therefore, the monomeric compound being prepared by the invention is for antineoplastic new
The research and development of medicine, preparation have great importance.
Claims (4)
1. a kind of preparation method for extracting alkaloid from greyblue spicebush root, it is characterised in that include the following steps：
1）Feedstock processing：It takes greyblue spicebush root to dry in the shade to water content after 3-8%, to crush, crosses 20-80 mesh sieve, obtain coarse powder, it is spare；
2）Acid alcohol solution seepage pressure effects：Take step 1）Coarse powder obtained, is fitted into percolator, adds the 1-2 times of pH value 1-2 measured
Acid alcohol solution, the wherein a concentration of 30%-80% of alcohol, infiltration 3-6 hours after be fitted into diacolation bucket, measured with 10-18 times
Above-mentioned acidity alcohol solution carries out seepage pressure effects, adjusts flow velocity, and percolate is collected to lindera glauca with 10-20mL/min flow velocitys
Until alkaloid extraction totally in rough powder；Using thin layer chromatography, brownish black spot, iodate are shown at ultraviolet lamp 254nm
Bismuth potassium solution shows crocus, and buck is added in gained extracting solution and adjusts pH value to 9-10, organic solvent is then added and extracts 2-6
It is secondary, extract liquor is made；The volume ratio of the extracting solution and organic solvent is 1:1-3；Wherein chromatographic condition is：GF254 thin layers
Chromatography is analysed, with chloroform：Methanol=6:1 is solvent, and develop the color observation under 254nm ultraviolet lamps；The acidity alcohol solution is dense
Degree is any one of acetic acid, hydrochloric acid, sulfuric acid or the tartaric acid of 0.5-2%；Buck is the sodium carbonate liquor or 15-25% of saturation
Ammonium hydroxide；
3）By step 2）Extract liquor obtained, in 40-55 DEG C of bath temperature, vacuum degree carries out under conditions of being 0.09-0.1Mpa
It is concentrated under reduced pressure, is concentrated into a concentration of 0.5-1.0g/ml of concentrate, greyblue spicebush root total alkaloid extract concentrate is made；
4）By step 3）Greyblue spicebush root total alkaloid extract concentrate obtained crosses silica gel column chromatography, with the body of chloroform-methanol
Product ratio is followed successively by 10:1、8:1、6:1、4:1、2:1、0:1 carries out gradient elution, collects eluent, and eluent is examined through thin-layer chromatography
It surveys, merges same composition, obtain A-E totally 5 components；Antitumor activity tracking is carried out, B, component C antitumor activity are preferable；
5）To B component, inverted column chromatography, with the methanol-water gradient elution of 20-100%, eluent is detected through thin-layer chromatography, is closed
And same composition, recycling eluent obtain B1-B4 totally 4 components；
6）By step 5）B2 components obtained cross silicagel column, are purified with chloroform-methanol, obtain 1 He of yellow-brown solid compound
Compound 2；The volume ratio of chloroform-methanol is 8:1；
7）By step 4）Component C obtained, inverted column chromatography, with the methanol-water gradient elution of 30-100%, eluent is through thin
Layer chromatography detects, and merges same composition, and recycling design obtains C1-C8 totally 8 components；
8）By step 7）C4 components obtained cross gel column, are purified with the methanol-water of 30-100%, obtain yellow-brown solid compound
3；
The compound 1 is N- methyllaurotetanines, and structural formula is：
The compound 2 is laurotetanine, and structural formula is：
The compound 3 is（+）- Boldine, structural formula are：
。
2. a kind of preparation method for extracting alkaloid from greyblue spicebush root according to claim 1, it is characterised in that step
2）Middle organic solvent is the mixing of one or more of ethyl acetate, ether, acetone, n-butanol, chloroform or dichloromethane；
3. a kind of preparation method for extracting alkaloid from greyblue spicebush root according to claim 1, it is characterised in that step
5）In successively with 20% methanol-water, 40% methanol-water, 60% methanol-water, 80% methanol-water, 100% methanol successively
Gradient elution.
4. a kind of preparation method for extracting alkaloid from greyblue spicebush root according to claim 1, it is characterised in that step
7）It is middle first to be eluted with 30% methanol-water, it is then eluted successively with the methanol-water of 50% methanol-water, 70%, finally with 100% first
Alcohol elutes；Step 8）It is middle first to be purified with 30% methanol-water, then purified with the methanol-water of 50% methanol-water, 70%, finally
Purified with 100% methanol-water.
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| CN104829529A (en) * | 2015-05-15 | 2015-08-12 | 海南师范大学 | Artabotrys pilosus total alkaloid extract and application thereof |
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