CN103169752A - Semen momordicae extractive as well as preparation method and use thereof - Google Patents
Semen momordicae extractive as well as preparation method and use thereof Download PDFInfo
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- CN103169752A CN103169752A CN2011104424930A CN201110442493A CN103169752A CN 103169752 A CN103169752 A CN 103169752A CN 2011104424930 A CN2011104424930 A CN 2011104424930A CN 201110442493 A CN201110442493 A CN 201110442493A CN 103169752 A CN103169752 A CN 103169752A
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Abstract
The invention belongs to the field of medical examination, and relates to a semen momordicae extractive as well as a preparation method and anti-tumour use thereof. The semen momordicae extractive having anti-tumour activity is prepared from corresponding components separated and purified by taking high performance liquid chromatography as the method after a sample to be measured and extracted is quenched. Found from in-vitro pharmacological experiments, the semen momordicae extractive is capable of obviously inhibiting proliferation of cancer cells by inducing cell apoptosis and retarding cell growth cycle, has an obvious inhibiting effect on human gastric carcinoma cells and other tumour cells, and can be used as the active component for preparing medicines for treating solid tumours, particularly medicines for treating human gastric carcinoma, human prostatic cancer or colon cancer. The preparation method disclosed by the invention is simple, convenient, low in cost and high in yield; and the extractive is high in component purity and good in reproducibility.
Description
Technical field
The invention belongs to field of medical examination, relate to the active substance in Chinese crude drug, be specifically related to a kind of Semen Momordicae extract and preparation method thereof and anti-tumor application.
Background technology
Report according to statistics, malignant tumor has become serious threat human life's difficult diseases, is to be only second to cardiopathic the second killer.At present, the most first-selected chemotherapy of the drug treatment to malignant tumor that clinical practice is commonly used, yet, can bring extremely serious side effect during chemotherapy.The some Chinese herbal medicine of prior art research report have distinctive feature at aspects such as treatment difficult miscellaneous diseases, malignant tumor.Therefore, seek from natural resources and screening antineoplastic active component, with the medicine of the treatment malignant tumor of the effective low toxicity of further exploitation, the person's that caused the medical research extensive concern.
Semen Momordicae is the dry mature seed of cucurbitaceous plant Semen Momordicae (Momordica cochinchinensis (Lour.) Spreng), main product in Guangxi, the ground such as Sichuan, Hubei, Henan, Anhui, Zhejiang, Fujian, Guangdong, Guizhou, Yunnan.According to " modern Chinese medicine voluminous dictionary " record, the Semen Momordicae nature and flavor are bitter, little sweet, poisonous, enter liver, spleen, stomach warp, the tool mass dissipating and swelling eliminating, and counteracting toxic substances is treated the skin ulcer effect, is usually used in sore swollen toxin, acute mastitis, scrofula, anal fistula, chronic eczema, tinea alba.
Have research that the Chinese crude drug Semen Momordicae is used in anti-tumor compound, play synergism with other Chinese medicine ingredients, but Chinese medicine compound does not reach still that country promotes industrialization of Chinese medicine, internationalization and to the requirement of Chinese medicine standard level.Relevant researcher expectation, screening antineoplastic active component is with the medicine of the treatment malignant tumor of the effective low toxicity of further exploitation from the natural Chinese medicine Semen Momordicae.
So far, there is not yet the report of anti-tumor activity relevant its uniqueness of Semen Momordicae extract and that experimental results show that by external pharmacology.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists, a kind of Semen Momordicae extract and preparation method thereof is provided.
Further purpose of the present invention is to provide the anti-tumor activity of this Semen Momordicae extract, especially to people's gastric cancer, human prostata cancer and colon cancer inhibitory action.
The present invention carries out the Chinese crude drug Semen Momordicae, therefrom extract and obtain a kind of activity extract, experimental results show that by external pharmacology this extract has to gastric carcinoma cells SGC7901, Human Prostate Cancer Cells PC-3 and mouse colonic cell CT-26 its growth of inhibition, further can be used as the medicine of active fraction preparation treatment people gastric cancer, human prostata cancer and colon cancer, for the Chinese crude drug Semen Momordicae provides new wide application prospect.
The invention provides the method for preparing activity extract in Semen Momordicae, it comprises step:
1) sample preparation
Powder in Semen Momordicae is used sodium acetate solution 20mM, and pH 5.0 4 ℃ of cold preservations in refrigerator are extracted and spent the night, and centrifugal rear filtrate lyophilizing gets extract F;
2) sample separation and purification
Adopt the liquid-phase chromatographic column of half preparation type, its filler is Octadecylsilane bonded phase silica gel, the high performance liquid chromatogram system adopts UV-detector and the high-pressure pump of half preparation type, mobile phase adopts A:0.05% formic acid B:90% acetonitrile+10% water (containing 0.045% formic acid), gradient elution, collect according to going out peak situation and retention time, get effective site.
More specifically, in Semen Momordicae of the present invention, activity extract prepares by following method:
The Semen Momordicae Chinese crude drug is pulverized obtained powder in Semen Momordicae, get powder 100g in Semen Momordicae, extract with 500ml sodium acetate solution (20mM, pH 5.0) 4 ℃ of cold preservations in refrigerator and spend the night, centrifugal rear filtrate lyophilizing gets extract, saves backup in-20 ℃ of refrigerators.The Semen Momordicae extract lyophilized powder that extraction is obtained with water dissolution after, a small amount of insoluble impurity is removed in centrifugal filtration, further separate (as shown in Figure 1) with chromatography, chromatographic condition is: chromatographic column: YMC-Pack ODS-A (150 * 10mm, 5 μ m); Column temperature: room temperature; Mobile phase: A:0.05% formic acid B:90% acetonitrile+10% water (containing 0.045% formic acid); Flow velocity: 2ml/min; Sample size: 1000 μ L; Detector: ultraviolet variable-wavelenght detector (VWD), detect wavelength 254nm.
Type of elution: gradient elution (as shown in table 1), collect the 26-40min fraction, merging saves backup in-20 ℃ of refrigerators after collecting the liquid lyophilizing.
Table 1 Semen Momordicae extract gradient elution timetable
the present invention obtains activity extract to extraction, carried out external pharmacological evaluation, pass through cell culture experiments, detected Semen Momordicae extract external to gastric carcinoma cells SGC7901, the effects anb Mechanism of Human Prostate Cancer Cells PC-3 and mouse colonic cell CT-26, experimental result shows, Semen Momordicae extract is by cell death inducing and block cell growth cycle and the propagation of remarkable anticancer, for the inhibitory action F3 of gastric carcinoma cells SGC7901＞F＞F1＞F2, wherein F and F3 also have obvious inhibitory action (as Fig. 2 to other tumor cells, shown in table 2).
Table 2 Semen Momordicae extract F and F3 suppress the IC of gastric carcinoma cells SGC7901, Human Prostate Cancer Cells PC-3 and mouse colonic cell CT-26 cell proliferation
50Value.
Table 3 is to adopt PI dyeing and flow cytometer to detect Semen Momordicae extract to the impact of gastric carcinoma cells SGC7901 growth cycle, and result has shown variable concentrations Semen Momordicae extract treatment S GC7901 cell 48h, wherein A.Control; B.0.1mg/L; C.0.2mg/L, D.0.4mg/L.
Table 2
Table 3
Semen Momordicae extract of the present invention can be used as the medicine of active fraction preparation treatment entity tumor, especially treats the medicine of people's gastric cancer, human prostata cancer or colon cancer.
Semen Momordicae extract of the present invention and preparation method thereof has following advantage:
(1) preparation is simple, adopts the merceration extraction method, and is not only convenient and cost is low;
(2) extract components purity is high, separates with purification obtaining corresponding component with semi-preparative high performance liquid chromatography, repeatability also and purity high.
Description of drawings
Fig. 1 Semen Momordicae extract chromatographic fractionation figure.
Fig. 2 Semen Momordicae extract (F, F1, F2, F3) suppresses the effect of gastric carcinoma cells SGC7901 propagation, and variable concentrations Semen Momordicae extract (F, F1, F2, the F3) processing time is 72h, compares * p＜0.05, #p＜0.01 with the blank group.
Fig. 3 adopts Hoechst 33258 dyeing and two the dying of PI/Annexin V to detect Semen Momordicae extract to the apoptotic effect of SGC7901.Variable concentrations Semen Momordicae extract treatment S GC7901 cell 48h, A.Hoechst 33258 dyeing; B.PI/Annexin V is two to be dyed.
The specific embodiment
Embodiment 1. preparation Semen Momordicae extracts
The Semen Momordicae Chinese crude drug is pulverized obtained powder in Semen Momordicae, get powder 100g in Semen Momordicae, with 500ml sodium acetate solution (20mM, pH 5.0) in refrigerator 4 ℃ of cold preservations extract and spend the night, centrifugal rear filtrate lyophilizing gets extract (F), save backup in-20 ℃ of refrigerators, the Semen Momordicae extract lyophilized powder (F) that extraction is obtained is with after water dissolution, a small amount of insoluble impurity is removed in centrifugal filtration, further separate (chromatogram as shown in Figure 1) with chromatography, collect the 26-40min fraction, merging saves backup in-20 ℃ of refrigerators after collecting the liquid lyophilizing.
Chromatographic condition is as follows:
Chromatographic column: YMC-Pack ODS-A (150 * 10mm, 5 μ m)
Column temperature: room temperature
Mobile phase: A:0.05% formic acid B:90% acetonitrile+10% water (containing 0.045% formic acid)
Flow velocity: 2ml/min
Sample size: 1000 μ L
Detector: ultraviolet variable-wavelenght detector (VWD), detect wavelength 254nm
Type of elution: gradient elution (table 1).
Table 1 Semen Momordicae extract gradient elution timetable
Embodiment 2. Semen Momordicae extract extracorporeal anti-tumor function tests
The SGC7901 cell of exponential phase is inoculated in 96 orifice plates, be divided into blank group, carboplatin matched group and Semen Momordicae extract processed group (0.1,0.2,0.4,0.6,0.8,1.0mg/ml), carry out MTT after 37 ℃ of cultivation 72h and detect, calculate cell proliferation inhibition rate.The result demonstration, along with dosage increases, Semen Momordicae extract significantly suppresses SGC7901 cell proliferation (as shown in Figure 2).
Embodiment 3. Semen Momordicae extract F and the F3 IC to different tumor cells
50Value
Gastric carcinoma cells SGC7901, Human Prostate Cancer Cells PC-3 and the mouse colonic cell CT-26 of exponential phase are inoculated in respectively in 96 orifice plates, be divided into blank group, carboplatin matched group and Semen Momordicae extract processed group (0.1,0.2,0.4,0.6,0.8,1.0mg/ml), cultivate for 37 ℃ and carry out MTT after 48h, 72h, 96h and detect, calculate cell proliferation inhibition rate.The result demonstration, along with dosage increases, Semen Momordicae extract significantly suppresses various cancer cell multiplications (as shown in table 2).
Embodiment 4. Semen Momordicae extracts suppress the effect of SGC7901 cell growth cycle
The SGC7901 cell of exponential phase is inoculated in 96 orifice plates, and after adding Semen Momordicae extract to process 48h, centrifugal collecting cell, PBS are washed 2 times, add 70% ethanol of pre-cooling fixedly to spend the night.Centrifugal collecting cell, PBS are washed 1 time, add ethidium bromide (PI) lucifuge to hatch 30 minutes, detect result ModFit software analysis with flow cytometer.Result shows, along with dosage increases, Semen Momordicae extract with the SGC7901 cell block in S phase (as shown in table 3).
Embodiment 4. Semen Momordicae extracts are induced the effect of SGC7901 Growth of Cells apoptosis
SGC7901 is inoculated in 24 orifice plates with cell, and after adding Semen Momordicae extract to process 48h, adding final concentration is the Hoechst33342 solution of 5 μ g/ml, and the room temperature lucifuge was placed 5 minutes.Abandon culture medium, 4% formaldehyde is 10min fixedly, and suction is abandoned, and PBS washing 1-2 time adds 1ml PBS.Observing apoptosis cell size, form under fluorescence microscope.In addition, centrifugal collection SGC7901 cell with pre-cooling PBS rinsing 1 time, adds Annexin V-FITC mix homogeneously, room temperature lucifuge incubation 10min.PBS washes 1 time, adds appropriate PI, carries out the apoptosis detection by quantitative with flow cytometer.The result demonstration, Semen Momordicae extract has obviously induces the apoptotic effect of SGC7901 (as shown in Figure 3).
Claims (4)
1. a Semen Momordicae extract, is characterized in that, this Semen Momordicae extract prepares by following method:
1) sample preparation
Powder in Semen Momordicae is used sodium acetate solution 20mM, and pH 5.0 4 ℃ of cold preservations in refrigerator are extracted and spent the night, and centrifugal rear filtrate lyophilizing gets extract F;
2) sample separation and purification
Adopt the liquid-phase chromatographic column of half preparation type, its filler is Octadecylsilane bonded phase silica gel, the high performance liquid chromatogram system adopts UV-detector and the high-pressure pump of half preparation type, mobile phase adopts A:0.05% formic acid B:90% acetonitrile+10% water, wherein contain 0.045% formic acid, gradient elution is collected according to going out peak situation and retention time, gets effective site.
2. by Semen Momordicae extract claimed in claim 1, it is characterized in that, in described Semen Momordicae extract preparation, chromatographic condition is: chromatographic column: YMC-Pack ODS-A 150 * 10mm, 5 μ m; Column temperature: room temperature; Mobile phase: A:0.05% formic acid B:90% acetonitrile+10% water wherein contains 0.045% formic acid; Flow velocity: 2ml/min; Sample size: 1000 μ L; Detector: the ultraviolet variable-wavelenght detector, detect wavelength 254nm;
Type of elution: gradient elution, collect the 26-40min fraction, standby in-20 ℃ of Refrigerator stores after the lyophilizing of merging collection liquid.
3. claim 1 Semen Momordicae extract is in the purposes for preparing in treating the entity tumor medicine.
4. by purposes claimed in claim 3, it is characterized in that, described entity tumor is people's gastric cancer, human prostata cancer or colon cancer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467258A (en) * | 2013-08-15 | 2013-12-25 | 华北制药集团新药研究开发有限责任公司 | Lignanoid compound, and preparation method and use thereof |
| CN104706729A (en) * | 2015-02-28 | 2015-06-17 | 西安交通大学 | Traditional Chinese medicine extract with antitumor effect as well as extraction method and application thereof |
| CN115097040A (en) * | 2022-06-24 | 2022-09-23 | 北京康仁堂药业有限公司 | UPLC characteristic map construction method and application of momordica cochinchinensis seeds |
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| CN1168278A (en) * | 1996-06-18 | 1997-12-24 | 上海地康营养保健食品开发有限公司 | Antineoplastic Chinese medicine prepn and its prepn process |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1168278A (en) * | 1996-06-18 | 1997-12-24 | 上海地康营养保健食品开发有限公司 | Antineoplastic Chinese medicine prepn and its prepn process |
Non-Patent Citations (2)
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| 林志燕等: "HPLC法测定胰蛋白酶抑制剂及其在中药木鳖子提取液研究中的应用(英文)", 《JOURNAL OF CHINESE PHARMACEUTICAL SCIENCES》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103467258A (en) * | 2013-08-15 | 2013-12-25 | 华北制药集团新药研究开发有限责任公司 | Lignanoid compound, and preparation method and use thereof |
| CN104706729A (en) * | 2015-02-28 | 2015-06-17 | 西安交通大学 | Traditional Chinese medicine extract with antitumor effect as well as extraction method and application thereof |
| CN104706729B (en) * | 2015-02-28 | 2019-04-16 | 西安交通大学 | A kind of Chinese medical extract and its extracting method and application with antitumor efficacy |
| CN115097040A (en) * | 2022-06-24 | 2022-09-23 | 北京康仁堂药业有限公司 | UPLC characteristic map construction method and application of momordica cochinchinensis seeds |
| CN115097040B (en) * | 2022-06-24 | 2023-06-02 | 北京康仁堂药业有限公司 | UPLC characteristic spectrum construction method and application of semen momordicae |
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