CN108904540A - Extraction, purification process and the application of alkaloid in a kind of eupolyphoge sinensis - Google Patents
Extraction, purification process and the application of alkaloid in a kind of eupolyphoge sinensis Download PDFInfo
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- CN108904540A CN108904540A CN201810791612.5A CN201810791612A CN108904540A CN 108904540 A CN108904540 A CN 108904540A CN 201810791612 A CN201810791612 A CN 201810791612A CN 108904540 A CN108904540 A CN 108904540A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C249/00—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C249/16—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton of hydrazones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
- C07C2603/18—Fluorenes; Hydrogenated fluorenes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention belongs to extraction, purification process and the applications of alkaloid in Chemistry for Chinese Traditional Medicine extractive technique field more particularly to a kind of eupolyphoge sinensis.The present invention provides a kind of from eupolyphoge sinensis extracts the extracting method of alkaloid, the present invention also provides a kind of purification process of resulting alkaloid of said extracted method, a kind of application of the purifying biological alkali obtained the present invention also provides alkaloid that said extracted method obtains or above-mentioned purification process in antibacterial, the purifying biological alkali obtained the present invention also provides a kind of alkaloid that said extracted method obtains or above-mentioned purification process are inhibiting the application in Growth of Gastric.Method for extraction and purification provided by the invention, it is separable to obtain GBA18；It can be obtained through measuring, products therefrom has good effect in inhibition gastric cancer and antibacterial aspect, meanwhile, median lethal dose and minimum lethal dose belong to the safe handling range that Chinese Pharmacopoeia is recommended；It solves in the prior art, effective component is indefinite in eupolyphoge sinensis, seriously limits the technological deficiency of its application.
Description
Technical field
The invention belongs to the extractions of alkaloid, purifying side in Chemistry for Chinese Traditional Medicine extractive technique field more particularly to a kind of eupolyphoge sinensis
Method and application.
Background technique
Eupolyphoge sinensis (Eupolyphagesinensis), also known as ground beetle, Eupolyphoge sinensis etc. are subordinate to Insecta (Hexapeda) Blattaria
(Blattaria) Corydiidae (Corydiidae) insect.Protein, carbohydrate, organic acid, steroidal, life are mainly contained in eupolyphoge sinensis body
The organic matters such as alkaloids, amino acid, grease, terpene lactone, cumarin, phenols and various trace elements, in the application field of Chinese medicine, tool
It is with a long history.
Modern biochemical molecular and pharmacological testing prove：Eupolyphoge sinensis has antineoplastic action.The existing lot of documents report in the country
It points out that eupolyphoge sinensis has anti-mutation, antineoplastic action, and has begun for clinical treatment.Research points out, eupolyphoge sinensis fibrinolytic
Protein component has the function of inhibition human microvascular endothelial cell (mvec) proliferation, and eupolyphoge sinensis water decoction can obviously inhibit epidermal growth factor to swash
The proliferation of epithelial cell in tumour layer living；Eupolyphoge sinensis can inhibit the breathing of liver cancer, stomach cancer cell, and leukaemic can be inhibited white thin
Born of the same parents etc., while also with the ability of anti-frameshift mutation；Explanation：Eupolyphoge sinensis has the function of good inhibition tumour, has clinic
The prospect of application value.
Currently, the research of effective component is simultaneously indefinite in eupolyphoge sinensis, it can not confirm and really play Essential Action in eupolyphoge sinensis
Substance, seriously limit application of the eupolyphoge sinensis in clinic.
Therefore, extraction, purification process and the application for developing alkaloid in a kind of eupolyphoge sinensis, for solving in the prior art,
Effective component is indefinite in eupolyphoge sinensis, seriously limits the technological deficiency of its application, it is urgently to be resolved to become those skilled in the art
Problem.
Summary of the invention
In view of this, the present invention provides extraction, purification process and the application of alkaloid in a kind of eupolyphoge sinensis, it is existing for solving
Have in technology, effective component is indefinite in eupolyphoge sinensis, seriously limits the technological deficiency of its application.
The present invention provides a kind of from eupolyphoge sinensis extracts the extracting method of alkaloid, and the extracting method is：
Step 1: merging extracting solution twice after eupolyphoge sinensis alcohol extracting twice, obtaining the first product；
Step 2: diluted hydrochloric acid dissolution is used after first product is dry for the first time, and it is dry through chloroform and second, it obtains
Alkaloid total alkali.
Preferably, in step 1, the method for the alcohol extracting is：The ethyl alcohol that concentration is 95% is after 70 DEG C of extraction 8h, filtering,
Filter residue uses methanol that concentration is 70% in 70 DEG C of extractions 8h again, merging extracting solution twice.
Preferably, the extracting method further includes：Pre-treatment, the pre-treatment are carried out before step 1；
The method of the pre-treatment is：After eupolyphoge sinensis is dry, grind into powder.
Preferably, in step 2, dry method is to be dried under reduced pressure for the first time, and second of dry method is that decompression is dry
It is dry；
In step 2, the concentration of the dilute hydrochloric acid is 0.5%.
The present invention also provides a kind of purifying sides including the resulting alkaloid of extracting method described in any of the above one
Method, the purification process are：
PH to 8.0 is adjusted after S1, alkaloid dissolution, is dried under reduced pressure after extracting, obtains the first intermediate product；
S2, the first intermediate product silica gel column chromatography, eluent are chloroform and methyl alcohol mixed liquor, wash for the first time
De-, gained eluent is dried under reduced pressure, and obtains the second intermediate product；
S3, the second intermediate product silica gel column chromatography, eluent be chloroform, methanol and ethyl acetate mixtures, into
Second of elution of row, gained eluent are dried under reduced pressure, and obtain third intermediate product；
S4, the third product are chromatographed with Sephadex LH-20 column, and eluent is methanol and water mixed liquid, carry out third
Secondary elution, gained eluent are dried under reduced pressure, and obtain the 4th intermediate product；
S5, the 4th intermediate product column chromatography, eluent are methanol and water mixed liquid, collect that peak value is highest washes
De- liquid, is dried under reduced pressure to obtain purifying biological alkali.
Preferably, in S2, in terms of parts by volume, the volume ratio of chloroform and methanol is 9:1；
In S3, in terms of parts by volume, chloroform, methanol, ethyl acetate volume ratio be 9:1:9；
In S4, in terms of parts by volume, the volume ratio of first alcohol and water is 3:5；
In S5, in terms of parts by volume, the volume ratio of chloroform and methanol is 9:1.
The present invention also provides a kind of alkaloid obtained including extracting method described in any of the above one or to take up an official post
Application of the obtained purifying biological alkali of purification process described in meaning one in antibacterial.
Preferably, the antibacterial strain includes：Gram-E. coli, Gram positive S grape ball
Bacterium, bacillus subtilis and saccharomyces cerevisiae.
The present invention also provides a kind of alkaloid obtained including extracting method described in any of the above one or to take up an official post
The purifying biological alkali that purification process described in meaning one obtains is inhibiting the application in Growth of Gastric.
Preferably, the stomach cancer cell include MKN28, MGC803, SGC7901, AGS, MKN45, KATOIII and
BGC823。
The extracting method of alkaloid is extracted in conclusion the present invention provides a kind of from eupolyphoge sinensis, the present invention also provides
A kind of purification process of the resulting alkaloid of said extracted method, the present invention also provides a kind of lifes that said extracted method obtains
Application of the purifying biological alkali that alkaloids or above-mentioned purification process obtain in antibacterial, the present invention also provides a kind of said extracted sides
The purifying biological alkali that the alkaloid or above-mentioned purification process that method obtains obtain is inhibiting the application in Growth of Gastric.The present invention
The method for extraction and purification of offer, it is separable obtain 9- (2', 2'- dimethyl propylene acylhydrazone) bis- chloro- 2,7- of -3,6- it is bis--[2- (diethyl
Amine)-ethyoxyl] fluorenes；It can be obtained through measuring, products therefrom has good effect in inhibition gastric cancer and antibacterial aspect, meanwhile,
Median lethal dose and minimum lethal dose belong to the safe handling range of Chinese Pharmacopoeia recommendation.In a kind of eupolyphoge sinensis provided by the invention
Extraction, purification process and the application of alkaloid, solve in the prior art, and effective component is indefinite in eupolyphoge sinensis, seriously limits it
The technological deficiency of application.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is to purify the chemical structural formula of resulting GBA18 in embodiment 2；
Fig. 2 be embodiment 3 in, GBA18's1H-NMR spectrum；
Fig. 3 be embodiment 3 in, GBA18's13C-NMR spectrogram；
Fig. 4 is in embodiment 6, and GBA18 inhibits the result schematic diagram of stomach cancer cell BGC823 related gene expression.
Specific embodiment
The present invention provides extraction, purification process and the applications of alkaloid in a kind of eupolyphoge sinensis, for solving in the prior art,
Effective component is indefinite in eupolyphoge sinensis, seriously limits the technological deficiency of its application.
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
In order to which the present invention is described in more detail, alkaloid in a kind of eupolyphoge sinensis provided by the invention is mentioned below with reference to embodiment
It takes, purification process and application, is specifically described.
Embodiment 1
The present embodiment is the extracting method of total alkaloid in eupolyphoge sinensis.
Fresh ground beetle is clean wash with distilled water, and 45 DEG C of drying, grind into powder, 4 DEG C of cryo-conservations are for future use.
Take 20g eupolyphoge sinensis powder with 95% ethyl alcohol 200ml (material ratio 1:10), under the conditions of 70 DEG C, Soxhlet extraction 8 hours,
Filtering；Filtered filter residue uses 70% methanol 200ml (material ratio 1 again:10), under the conditions of 70 DEG C, Soxhlet extraction 8 hours, mistake
Filter merges extracting solution twice.
After extracting solution is dried under reduced pressure, with 0.5% dilute hydrochloric acid 50ml dissolve, then use 150ml chloroform, extracting solution decompression does
It is dry, obtain that there is antibacterial and anticancer activity eupolyphoge sinensis total alkaloid component 1.124g, extracting yield is 5.62%.
Embodiment 2
The present embodiment is that eupolyphoge sinensis total alkaloid is purified, obtains 9- (2', 2'- dimethyl propylene acylhydrazone) -3,6- bis- chloro- 2,7-
The specific embodiment of double-[2- (diethylamine)-ethyoxyl] fluorenes (GBA18).
(1), 1 gained eupolyphoge sinensis total alkaloid 5g of embodiment is dissolved in 0.5% dilute hydrochloric acid of 50ml, is arrived with weak aqua ammonia tune pH value
8.0；Then, with 150ml chloroform, extracting solution is dried under reduced pressure to obtain solid extract 89mg.
(2), the silica gel 500g for taking 200-300 mesh mixes wet process with chloroform after activation and is packed into chromatographic column (3cm*50cm).Step
Suddenly wet process is loaded after (1) obtained solid extract 89mg is dissolved in chloroform, chloroform/methanol (9:1) it elutes, eluent is dried under reduced pressure
Obtain solid extract 50mg.
(3), the silica gel 500g for taking 200-300 mesh mixes wet process with chloroform after activation and is packed into chromatographic column (3cm*50cm).Step
Suddenly wet process is loaded after (2) obtained solid extract 50mg is dissolved in chloroform, chloroform/methanol/ethyl acetate (9:1:9) it elutes, washes
De- liquid is dried under reduced pressure to obtain solid extract 23mg.
(4), step (1), (2), (3) is repeated several times, merges the eupolyphoge sinensis alkaloid for extracting and obtaining, for future use.
(5), step (4) obtained solid extract 50mg is chromatographed with Sephadex LH-20 column, reversed solvent methanol/water
(3:5) it elutes, eluent is dried under reduced pressure to obtain solid extract 41mg.
(6), step (5) obtained solid extract 41mg is with preparing chromatography, methanol/water (9:1) it elutes, fraction collection
Each fraction, it is main with antibacterial and anticancer activity acylhydrazone alkaloid list that peak value highest elution fraction is dried under reduced pressure to obtain eupolyphoge sinensis
Body (GBA18) 34mg.
Embodiment 3
The present embodiment is the specific embodiment of GBA18 carbon spectrum and hydrogen spectrum analysis.3.11H-NMR analysis
Herein further referring to table 1 and Fig. 2.
Table 1
3.213C-NMR analysis
Herein further referring to table 2 and Fig. 3.
Table 2
Embodiment 4
The present embodiment is the specific embodiment that GBA18 inhibits stomach cancer cell in vitro.
The HEPES (pH7.5) that GBA18 is dissolved in 50mM is configured to the mother liquor of 1mg/ml, and it is spare to filter sterilizing.Utilize MTT
Method detects the activity that GBA18 inhibits Growth of Gastric, wherein gastric carcinoma cell lines include：MKN28,MGC803,SGC7901,
AGS、MKN45、KATOIII、BGC823。
Using HEPES buffer solution as negative control, 5-FU is as positive control.0.22 μm of sterilised membrane filter mistake of each component
After filtering out bacterium, saved backup in -20 DEG C.
Mtt assay detection GBA18 inhibits stomach cancer cell in vitro, and steps are as follows：
(1) each cell strain of RPMI-1604 culture solution culture is to logarithmic growth phase.
(2) logarithmic phase cell inoculation is in 96 orifice plates, cell density about 105A/hole, culture is for 24 hours.
(3) GBA18 of various concentration (1-50 μ g/ml) is added in every hole, and every group sets 6 in parallel.
(4) HEPES makees negative control, and 5-FU makees positive control, continues culture for 24 hours.
(5) remove culture solution, the culture solution of 150 μ lMTT is added in every hole, continues to cultivate 3h.
(6) remove culture solution in hole, 150 μ lDMSO are added in every hole.
(7) 10min is shaken, formazan crystallization dissolution Nei hole is made.
(8) microplate reader detects 490nm light absorption value.
(9) cell survival rate and inhibiting rate are calculated.
Wherein, cell survival rate=experimental group absorbance value/control group absorbance value x100%, inhibiting rate=(control group
Absorbance value-experimental group absorbance value)/control group absorbance value x100%
GBA18 is to the IC in various stomach cancer cell body outer suppressioning experiments50Please refer to table 3.
Table 3
Embodiment 5
The present embodiment is GBA18 and the specific embodiment of GRPR affinity detection.
The gene of human cloning hGRPR receptor, and be connected on pCD2 plasmid vector, it utilizes
LIPOFECTamine3000 transfects gastric carcinoma cell lines BGC823；72 hours after transfection, with positive gram of G418 screening of 800 μ g/ml
It is grand.
GRP is obtained using Ez-NHS-PEG4-Biotin label GRPbiotin；The BGC823 cell culture transfected is in 96 holes
Plate, removes culture solution, and 200 GRPs of the μ l containing 100000cpm are added in each holebiotinWith the PBS of the GBA18 of various concentration, 25 DEG C of trainings
60min is supported, supernatant is removed in centrifugation.
In each reacting hole, the 50 μ l of Streptavidin of the HRP label of diluted fresh is added.37 DEG C of incubation 60min, are washed
It washs；The 100 μ l of tmb substrate solution of Extemporaneous is added in each reacting hole, 37 DEG C of dark places are incubated for 30min, 50 μ of 2M sulfuric acid is added
L terminates reaction；It is returned to zero with blank well, 450nm wavelength sequentially measures the absorbance (OD value) in each hole.
Using BW2256U89 and PD176252 as positive control, PRELIMINARY RESULTS shows that GBA18 is able to suppress GRPbiotin
It is incorporated into BGC823 cell, and there is concentration dependent, it is provable herein herein further referring to Fig. 4：GBA18 and GRPR
Compatibility with higher.
Embodiment 6
The present embodiment is the specific embodiment that GBA18 inhibits the detection of stomach cancer cell BGC823 related gene expression.
Current research data shows that multiple signal paths of GRPR and tumour all have certain relationship；Wherein, compare
More data shows that the isogenic expression of EGFR, c-fos and c-jun can be by under using after GRPR antagonist inhibition GRPR access
It adjusts.
After handling gastric carcinoma cell lines BGC823 using the GBA18 of 20 μ g/ml, three above is detected using Westernblot
Gene, acquired results please refer to table 4.As can be drawn from Table 4, above three gene protein level have it is different degrees of under
It adjusts.
Table 4
Embodiment 7
The present embodiment is the specific embodiment of GBA18 bacteriostatic experiment.
Escherichia coli, staphylococcus aureus, bacillus subtilis with LB liquid medium in 37 DEG C of overnight incubations, it is sterile
Distilled water dilutes bacterium solution to 104-105CFU/ml.Saccharomyces cerevisiae is cultivated with PDB is based on 30 DEG C of overnight incubations, sterile distilled water dilution
Bacterium solution is to 104-105CFU/ml.GBA18 is added in 10mlLB solid medium (or PDA), make its final concentration of 10,20,40,
60,80,100,150,200,300,400,500 μ g/ml is poured into glass culture dish before solidification, while being made with ampicillin
For control group.
20 μ l bacterial suspensions are added on the solid medium prepared and smear uniformly, 37 DEG C (or 30 DEG C) cultures 24
Hour, bacterium and yeast growth situation are observed, the minimum sample concentration generated without any bacterium colony is minimum inhibitory concentration MIC
(μg/ml).Acquired results please refer to table 5.
Table 5
The extracting method of alkaloid is extracted in conclusion the present invention provides a kind of from eupolyphoge sinensis, the present invention also provides
A kind of purification process of the resulting alkaloid of said extracted method, the present invention also provides a kind of lifes that said extracted method obtains
Application of the purifying biological alkali that alkaloids or above-mentioned purification process obtain in antibacterial, the present invention also provides a kind of said extracted sides
The purifying biological alkali that the alkaloid or above-mentioned purification process that method obtains obtain is inhibiting the application in Growth of Gastric.The present invention
The method for extraction and purification of offer, it is separable obtain 9- (2', 2'- dimethyl propylene acylhydrazone) bis- chloro- 2,7- of -3,6- it is bis--[2- (diethyl
Amine)-ethyoxyl] fluorenes；It can be obtained through measuring, products therefrom has good effect in inhibition gastric cancer and antibacterial aspect, meanwhile,
Median lethal dose and minimum lethal dose belong to the safe handling range of Chinese Pharmacopoeia recommendation.In a kind of eupolyphoge sinensis provided by the invention
Extraction, purification process and the application of alkaloid, solve in the prior art, and effective component is indefinite in eupolyphoge sinensis, seriously limits it
The technological deficiency of application.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of extracting method for extracting alkaloid from eupolyphoge sinensis, which is characterized in that the extracting method is：
Step 1: merging extracting solution twice after eupolyphoge sinensis alcohol extracting twice, obtaining the first product；
Step 2: diluted hydrochloric acid dissolution is used after first product is dry for the first time, and it is dry through chloroform and second, it obtains biological
Alkali total alkali.
2. extracting method according to claim 1, which is characterized in that in step 1, the method for the alcohol extracting is：Concentration is
95% ethyl alcohol is after 70 DEG C of extraction 8h, filtering, and filter residue uses methanol that concentration is 70% to merge and mention twice in 70 DEG C of extractions 8h again
Take liquid.
3. extracting method according to claim 1, which is characterized in that the extracting method further includes：Pre-treatment, before described
Processing is carried out before step 1；
The method of the pre-treatment is：After eupolyphoge sinensis is dry, grind into powder.
4. extracting method according to claim 1, which is characterized in that in step 2, dry method is decompression for the first time
Dry, second of dry method is to be dried under reduced pressure；
In step 2, the concentration of the dilute hydrochloric acid is 0.5%.
5. a kind of purification process including the resulting alkaloid of extracting method described in Claims 1-4 any one, feature
It is, the purification process is：
PH to 8.0 is adjusted after S1, alkaloid dissolution, is dried under reduced pressure after extracting, obtains the first intermediate product；
S2, the first intermediate product silica gel column chromatography, eluent are chloroform and methyl alcohol mixed liquor, carry out first time elution,
Gained eluent is dried under reduced pressure, and obtains the second intermediate product；
S3, the second intermediate product silica gel column chromatography, eluent are chloroform, methanol and ethyl acetate mixtures, carry out the
Secondary elution, gained eluent are dried under reduced pressure, and obtain third intermediate product；
S4, the third product are chromatographed with Sephadex LH-20 column, and eluent is methanol and water mixed liquid, are carried out third time and are washed
De-, gained eluent is dried under reduced pressure, and obtains the 4th intermediate product；
S5, the 4th intermediate product column chromatography, eluent are methanol and water mixed liquid, collect the highest elution of peak value
Liquid is dried under reduced pressure to obtain purifying biological alkali.
6. purification process according to claim 5, which is characterized in that in S2, in terms of parts by volume, the volume of chloroform and methanol
Than being 9:1；
In S3, in terms of parts by volume, chloroform, methanol, ethyl acetate volume ratio be 9:1:9；
In S4, in terms of parts by volume, the volume ratio of first alcohol and water is 3:5；
In S5, in terms of parts by volume, the volume ratio of chloroform and methanol is 9:1.
7. a kind of alkaloid obtained including extracting method described in Claims 1-4 any one or claim 5 to 6 times
Application of the obtained purifying biological alkali of purification process described in meaning one in antibacterial.
8. application according to claim 7, which is characterized in that the antibacterial strain includes：Gram-negative large intestine bar
Bacterium, Gram positive staphylococcus aureus, bacillus subtilis and saccharomyces cerevisiae.
9. a kind of alkaloid obtained including extracting method described in Claims 1-4 any one or claim 5 to 6 times
The purifying biological alkali that purification process described in meaning one obtains is inhibiting the application in Growth of Gastric.
10. application according to claim 9, which is characterized in that the stomach cancer cell include MKN28, MGC803,
SGC7901, AGS, MKN45, KATOIII and BGC823.
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