CN103169752B - 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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- CN103169752B CN103169752B CN201110442493.0A CN201110442493A CN103169752B CN 103169752 B CN103169752 B CN 103169752B CN 201110442493 A CN201110442493 A CN 201110442493A CN 103169752 B CN103169752 B CN 103169752B
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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 the difficult diseases of serious threat human life, is to be only second to cardiopathic second killer.At present, clinical practice commonly use to the most first-selected chemotherapy of the drug treatment of malignant tumor, but, extremely serious side effect can be brought during chemotherapy.The some Chinese herbal medicine of prior art research report have distinctive feature in treatment difficult miscellaneous diseases, malignant tumor etc.Therefore, find from natural resources and screening antineoplastic active component, to develop the medicine of the treatment malignant tumor of effective low toxicity further, caused the extensive concern of pharmaceutical researchers.
Semen Momordicae is the dry mature seed of cucurbitaceous plant Semen Momordicae (Momordica cochinchinensis (Lour.) Spreng), main product in Guangxi, Sichuan, Hubei, Henan, Anhui, Zhejiang, Fujian, Guangdong, Guizhou, the ground such as Yunnan.Record according to " modern Chinese medicine voluminous dictionary ", Semen Momordicae nature and flavor are bitter, micro-sweet, poisonous, enter liver, spleen, stomach warp, tool mass dissipating and swelling eliminating, and counteracting toxic substances treats skin ulcer effect, is usually used in sore swollen toxin, acute mastitis, scrofula, anal fistula, chronic eczema, tinea alba.
Have research to be used in anti-tumor compound by Chinese crude drug Semen Momordicae, play synergism with other Chinese medicine ingredients, but Chinese medicine compound does not still reach country's promotion industrialization of Chinese medicine, internationalization and the requirement to Chinese medicine standard level.Relevant researcher is expected, screens antineoplastic active component to develop the medicine of the treatment malignant tumor of effective low toxicity further from natural Chinese medicine Semen Momordicae.
So far, there is not yet relevant its uniqueness of Semen Momordicae extract and the report of the anti-tumor activity proved by external pharmacological evaluation.
Summary of the invention
The object of the invention is the defect overcoming prior art existence, a kind of Semen Momordicae extract and preparation method thereof is provided.
A further object 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 Chinese crude drug Semen Momordicae, therefrom extract and obtain a kind of activity extract, prove that this extract has its growth of suppression to gastric carcinoma cells SGC7901, Human Prostate Cancer Cells PC-3 and mouse colonic cell CT-26 by external pharmacological evaluation, can be used as the medicine of active fraction preparation treatment people gastric cancer, human prostata cancer and colon cancer further, for Chinese crude drug Semen Momordicae provides new wide application prospect.
The invention provides the method preparing activity extract in Semen Momordicae, it comprises step:
1) sample preparation
Powder in Semen Momordicae, uses sodium acetate solution 20mM, and pH 5.0 4 DEG C of cold preservations in refrigerator are extracted and spent the night, and centrifugal rear filtrate lyophilizing obtains extract F;
2) sample separation and purification
Adopt semi-preparative liquid-phase chromatographic column, its filler is Octadecylsilane bonded phase silica gel, high-efficient liquid phase system adopts semi-preparative UV-detector and high-pressure pump, mobile phase adopts A:0.05% formic acid B:90% acetonitrile+10% water (containing 0.045% formic acid), gradient elution, according to going out peak situation and retention time collection, obtain effective site.
More specifically, in Semen Momordicae of the present invention, activity extract is prepared by following method:
Pulverized by Semen Momordicae Chinese crude drug and obtain powder in Semen Momordicae, get powder 100g in Semen Momordicae, extract with 500ml sodium acetate solution (20mM, pH 5.0) 4 DEG C of cold preservations in refrigerator and spend the night, centrifugal rear filtrate lyophilizing obtains extract, saves backup in-20 DEG C of refrigerators.After the Semen Momordicae extract lyophilized powder water dissolution that extraction is obtained, the a small amount of insoluble impurity of centrifugal filtration removing, further be separated (as shown in Figure 1) with chromatography, chromatographic condition is: chromatographic column: YMC-Pack ODS-A (150 × 10mm, 5 μm); 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), determined wavelength 254nm.
Type of elution: gradient elution (as shown in table 1), collects 26-40min fraction, merges after collecting liquid lyophilizing and saves backup in-20 DEG C of refrigerators.
Table 1 Semen Momordicae extract gradient elution timetable
The present invention obtains activity extract to extraction, carry out external pharmacological evaluation, pass through cell culture experiments, have detected Semen Momordicae extract in vitro 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, the propagation of Semen Momordicae extract remarkable anticancer by cell death inducing and retarding cell growth cycle, for the inhibitory action F3 > F > F1 > F2 of gastric carcinoma cells SGC7901, wherein F and F3 also has obvious inhibitory action (as Fig. 2 to other tumor cells, shown in table 2).
Table 2 Semen Momordicae extract F and F3 suppresses the IC of gastric carcinoma cells SGC7901, Human Prostate Cancer Cells PC-3 and mouse colonic cell CT-26 cell proliferation
50value.
Table 3 adopts PI dyeing and flow cytomery Semen Momordicae extract on the impact of gastric carcinoma cells SGC7901 growth cycle, and result shows 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 tool has the following advantages:
(1) preparation is simple, and employing merceration extraction method, not only facilitates and cost is low;
(2) extract components purity is high, carries out abstraction and purification obtain corresponding component with semi-preparative high performance liquid chromatography, and repeatability also and purity is high.
Accompanying drawing explanation
Fig. 1 Semen Momordicae extract chromatographic fractionation figure.
Fig. 2 Semen Momordicae extract (F, F1, F2, F3) effect that suppresses gastric carcinoma cells SGC7901 to breed, variable concentrations Semen Momordicae extract (F, F1, F2, F3) processing time is 72h, compared with blank group, * p < 0.05, #p < 0.01.
Fig. 3 adopts Hoechst 33258 to dye and the two dye of PI/Annexin V detects Semen Momordicae extract to the apoptotic effect of SGC7901.Variable concentrations Semen Momordicae extract treatment S GC7901 cell 48h, A.Hoechst 33258 dyes; The two dye of B.PI/Annexin V.
Detailed description of the invention
Embodiment 1. prepares Semen Momordicae extract
Semen Momordicae Chinese crude drug is pulverized and obtains powder in Semen Momordicae, get powder 100g in Semen Momordicae, with 500ml sodium acetate solution (20mM, pH 5.0) in refrigerator 4 DEG C of cold preservations extract and spend the night, centrifugal rear filtrate lyophilizing obtains extract (F), save backup in-20 DEG C of refrigerators, the Semen Momordicae extract lyophilized powder (F) extraction obtained is with after water dissolution, the a small amount of insoluble impurity of centrifugal filtration removing, further be separated (chromatogram as shown in Figure 1) with chromatography, collect 26-40min fraction, merge after collecting liquid lyophilizing and save backup in-20 DEG C of refrigerators.
Chromatographic condition is as follows:
Chromatographic column: YMC-Pack ODS-A (150 × 10mm, 5 μm)
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), determined 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 is tested
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 detection after 37 DEG C of cultivation 72h, calculate cell proliferation inhibition rate.Result shows, and along with dosage increases, Semen Momordicae extract significantly suppresses SGC7901 cell proliferation (as shown in Figure 2).
Embodiment 3. Semen Momordicae extract F and F3 is to the IC of different tumor cell
50value
Gastric carcinoma cells SGC7901, the Human Prostate Cancer Cells PC-3 of exponential phase and mouse colonic cell CT-26 are inoculated in 96 orifice plates respectively, 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 detection after 37 DEG C of cultivations 48h, 72h, 96h, calculate cell proliferation inhibition rate.Result shows, and along with dosage increases, Semen Momordicae extract significantly suppresses various cancer cell multiplication (as shown in table 2).
Embodiment 4. Semen Momordicae extract suppresses the effect of SGC7901 cell growth cycle
Be inoculated in 96 orifice plates by the SGC7901 cell of exponential phase, after adding Semen Momordicae extract process 48h, centrifugal collecting cell, PBS washes 2 times, and 70% ethanol adding pre-cooling fixedly spends the night.Centrifugal collecting cell, PBS washes 1 time, adds ethidium bromide (PI) lucifuge and hatches 30 minutes, with flow cytomery, result ModFit software analysis.Result show, along with dosage increase, Semen Momordicae extract by SGC7901 cell block in S phase (as shown in table 3).
The effect of embodiment 4. Semen Momordicae extract induction SGC7901 Growth of Cells apoptosis
Cell is inoculated in 24 orifice plates by SGC7901, and after adding Semen Momordicae extract process 48h, add the Hoechst33342 solution that final concentration is 5 μ g/ml, room temperature lucifuge places 5 minutes.Abandon culture medium, 4% formaldehyde fixes 10min, and suction is abandoned, and PBS washing 1-2 time, adds 1ml PBS.Observing apoptosis cell size, form under fluorescence microscope.In addition, collected by centrifugation SGC7901 cell, with pre-cooling PBS rinsing 1 time, adds Annexin V-FITC mix homogeneously, room temperature Incubation in dark 10min.PBS washes 1 time, adds appropriate PI, carries out apoptosis detection by quantitative with flow cytometer.Result shows, and Semen Momordicae extract has the obviously induction apoptotic effect of SGC7901 (as shown in Figure 3).
Claims (2)
1. the purposes of Semen Momordicae extract in preparation treatment entity tumor medicine;
Described Semen Momordicae extract is prepared by following method,
1) sample preparation
Powder in Semen Momordicae, uses sodium acetate solution 20mM, and pH5.0 4 DEG C of cold preservations in refrigerator are extracted and spent the night, and centrifugal rear filtrate lyophilizing obtains extract F;
2) sample separation and purification
Adopt semi-preparative liquid-phase chromatographic column, its filler is Octadecylsilane bonded phase silica gel, high-efficient liquid phase system adopts semi-preparative UV-detector and high-pressure pump, mobile phase adopts A:0.05% formic acid B:90% acetonitrile+10% water, wherein containing 0.045% formic acid, gradient elution, according to going out peak situation and retention time collection, obtains effective site;
Wherein, chromatographic condition is: chromatographic column: YMC-Pack ODS-A 150 × 10mm, 5 μm; Column temperature: room temperature; Mobile phase: A:0.05% formic acid B:90% acetonitrile+10% water, wherein containing 0.045% formic acid; Flow velocity: 2ml/min; Sample size: 1000 μ L; Detector: ultraviolet variable-wavelenght detector, determined wavelength 254nm;
Type of elution: gradient elution, collects 26-40min fraction, merges after collecting liquid lyophilizing for subsequent use in-20 DEG C of Refrigerator stores.
2., by the purposes of claim 1, it is characterized in that, described entity tumor is people's gastric cancer, human prostata cancer or colon cancer.
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| 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 |
| 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 |
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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (2)
| Title |
|---|
| 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);林志燕等;《Journal of Chinese Pharmaceutical Sciences》;20110715(第04期);397-403 * |
| 木鳖子及其活性成分的研究进展;王铭章等;《食品与药品》;20110910;第13卷(第09期);364-365 * |
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