CN104337837A - Anti-lung cancer application of ruditapes philippinarum enzymatic hydrolysate - Google Patents
Anti-lung cancer application of ruditapes philippinarum enzymatic hydrolysate Download PDFInfo
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- CN104337837A CN104337837A CN201310312796.XA CN201310312796A CN104337837A CN 104337837 A CN104337837 A CN 104337837A CN 201310312796 A CN201310312796 A CN 201310312796A CN 104337837 A CN104337837 A CN 104337837A
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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
- A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/13—Preparation or pretreatment of starting material involving cleaning, e.g. washing or peeling
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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Abstract
The invention relates to an anti-lung cancer application of a ruditapes philippinarum enzymatic hydrolysate. Trypsin is adopted for enzymolysis of ruditapes philippinarum meat, and then the enzymatic hydrolysate having a molecular weight less than 3KDa is obtained by ultrafiltration. Influences of the enzymatic hydrolysate on activity of human lung cancer H1299 cells are detected by an MTT method, an HE staining method, an AO/EB staining method, and the like. Results show that: after the ruditapes philippinarum enzymatic hydrolysate having a molecular weight less than 3KDa acts on the H1299 cells, cell proliferation of the H1299 cells is obviously inhibited, and apoptosis morphological changes of the cells occur. Compared with the prior art, the ruditapes philippinarum enzymatic hydrolysate has obvious anti-lung cancer effects, a raw material of enzymolysis is wide in source, and a preparation process is simple in operation, high in efficiency and high in safety.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to the application of a kind of Ruditapes philippinarum zymolyte in anti-pulmonary carcinoma.
Background technology
Ruditapes philippinarum (Ruditapes philippinarum) belongs to Mollusca Bivalvia curtain clam order Veneridae, a kind of eurythermal small-sized bivalve shellfish, south is commonly called as colored clam, the large cultivated shellfish of China four is listed as with Concha Ostreae, Sinonovacula constricta, Carnis Arca inflata class, it is one of the main shellfish in offshore beach and shallow sea, its delicious flavour, nutritious, protein content is high.
Cancer, also known as malignant tumor, is a kind of by controlling the not normal and disease that causes of growth and proliferation of cell mechanism.Pulmonary carcinoma is the cancer occurred frequently of mankind nowadays, occupy first of cancer, the prevalence of nonsmall-cell lung cancer accounts for pulmonary carcinoma and always to fall ill 80% of number, the patient of about 2/3 has lost surgical engine meeting when making a definite diagnosis, Radiotherapy chemotherapy occupies critical role in Treatment for Non-small Cell Lung, but the toxic and side effects of Radiotherapy chemotherapy drastically influence therapeutic effect and quality of life.Very large progress has been had in recent years to the antitumor research of Ruditapes philippinarum extract, as (food industry science and technology such as Fan Xiuping, 2003) semifinished product of Ruditapes philippinarum glycosaminoglycans (CRG) is utilized to act on the HL-60 cell of In vitro culture, find that CRG has remarkable inhibitory action to HL-60 cell under 1.0mg/ml concentration, 59.8%, 67.7% and 96.2% can be reached respectively at 24h, 48h, 72h tumour inhibiting rate.(the Oceanologia et Limnologia Sinica such as Zhang Li, 2007) Ruditapes philippinarum meat is utilized to extract two kinds of Dan Baiduotang proteoglycan PG PG1 and PG2, wherein PG1 reaches 73.30% when 200ug/ml concentration to the growth inhibition ratio of human liver cancer cell SMMC7721, and does not affect growth and the function of Human normal hepatocyte HL-7002.From marine organisms, extract the focus that anti-tumor active substance is scientific circles' research, wherein anticancer peptide is particularly noticeable.Utilizing protease hydrolyzed to obtain biologically active peptide is that a kind of safety is high, the method that productive rate is high, Recent study finds that Ruditapes philippinarum zymolyte has multiple biological activity, as (Chinese Chinese medicine academic periodicals such as Yang Yongfang, 2011) utilize Ruditapes philippinarum enzymolysis oligopeptide to the scavenging action of hydroxy radical to examine or check the antioxidant activity of each zymolyte, found that the Scavenging action to hydroxyl free radical when zymolyte concentration is 2.5mg/ml, DPPH free radical scavenging activity and reducing power are all higher than Vit C.At present, about the cancer resistance of Ruditapes philippinarum zymolyte, the application especially in anti-pulmonary carcinoma yet there are no report, therefore has the space of research further.
Summary of the invention
Technical problem to be solved by this invention is the novelty teabag providing a kind of Philippine zymolyte for prior art, the application namely in anti-pulmonary carcinoma.
The present invention solves the problems of the technologies described above adopted technical scheme: the application of a kind of Ruditapes philippinarum zymolyte in anti-pulmonary carcinoma.
In pulmonary carcinoma, nonsmall-cell lung cancer accounts for 80% of pulmonary carcinoma sum, is modal pulmonary carcinoma, and as preferably, the pulmonary carcinoma in such scheme refers to nonsmall-cell lung cancer.
In such scheme, the molecular weight of Ruditapes philippinarum zymolyte is less than 3KDa, and it obtains by the following method:
1), after fresh Ruditapes philippinarum is cleaned, get meat, decontamination, homogenate smashed to pieces by using-system bruisher;
2) in step 1), add 1200u/g trypsin in gained homogenate, enzymolysis 24h, hydrolysis temperature is 37 DEG C, and enzymolysis pH is 8.0;
3) after enzyme digestion reaction terminates, 90 DEG C of enzyme denaturing 15min, then the centrifugal 15min of 8500r/min at 4 DEG C, gets supernatant;
4) adopt intercepting molecular weight to be the ultrafilter membrane of 3KDa, 20 DEG C of ultrafiltration 11h obtain enzymolysis solutions;
5) by after the enzymolysis solution rotary evaporation of step 4) gained, lyophilization is preserved, and is made into different concentration during experiment with F12 culture fluid.
For understanding essence of the present invention better, in the present invention, Ruditapes philippinarum zymolyte acts on the people pulmonary carcinoma H1299 cell strain of In vitro culture, H1299 cell after zymolyte effect is carried out respectively MTT detection, HE dyeing and AO/EB dyeing, wherein, mtt assay, also known as MTT colorimetry, is a kind of method detecting cell survival and growth; HE dyes, i.e. hematoxylin-eosin staining method, is the colouring method that a kind of ordinary optical microscope observes with identification of cell apoptosis and necrocytosis; AO/EB dyes, and namely the two fluorescence staining of acridine orange/ethidium bromide, is a kind of method detecting apoptotic cell form and quantity in cell culture.Result shows this Ruditapes philippinarum zymolyte can significantly suppress H1299 Growth of Cells, and with the increase of zymolyte reaction solution concentration and the prolongation of action time, suppression ratio obviously rises; The H1299 cell of inverted microscope and HE stain control group is Epithelial growth, form is basically identical, extensibility is good, after zymolyte effect, cellular morphology changes, and occurs cavity in kytoplasm, and nuclear chromatin limit is gathered, the morphological change of the apoptosis such as karyopycnosis, therefore Ruditapes philippinarum zymolyte significantly can suppress the propagation of H1299 cell and can induce H1299 apoptosis.
Compared with prior art, the invention has the advantages that:
1, in the present invention, Ruditapes philippinarum zymolyte suppresses H1299 cell proliferation, induces its apoptosis Be very effective.In the present invention after 30mg/ml zymolyte effect H1299 cell 48h, in culture vessel, the normal H1299 cell of form obviously reduces, and floating cells or floating thing obviously increase, and dead phenomenon appears Proliferation Ability and in cell.
2, in the present invention, zymolyte raw material sources are extensive, and preparation process is simple to operate, efficiency is high, safety is high.Ruditapes philippinarum is a kind of shellfish by nest RT-PCR common in offshore beach and shallow sea in China marine site, very common at places such as coastal food market, supermarkets, utilize tryptic specificity and high efficiency to prepare zymolyte simultaneously, such that the preparation of zymolyte is simple, efficient, safety.
Accompanying drawing explanation
Fig. 1 is MTT testing result schematic diagram in the embodiment of the present invention 2;
Fig. 2 is H1299 cellular morphology schematic diagram under the inverted microscope of matched group in the embodiment of the present invention 3;
Fig. 3 is H1299 cellular morphology schematic diagram under the inverted microscope of 10mg/ml group in the embodiment of the present invention 3;
Fig. 4 is H1299 cellular morphology schematic diagram under the inverted microscope of 20mg/ml group in the embodiment of the present invention 3;
Fig. 5 is H1299 cellular morphology schematic diagram under the inverted microscope of 30mg/ml group in the embodiment of the present invention 3;
Fig. 6 is H1299 cellular morphology schematic diagram after the HE dyeing of matched group in the embodiment of the present invention 3;
Fig. 7 is H1299 cellular morphology schematic diagram after the HE dyeing of 10mg/ml group in the embodiment of the present invention 3;
Fig. 8 is H1299 cellular morphology schematic diagram after the HE dyeing of 20mg/ml group in the embodiment of the present invention 3;
Fig. 9 is H1299 cellular morphology schematic diagram after the HE dyeing of 30mg/ml group in the embodiment of the present invention 3;
The rear H1299 cellular morphology schematic diagram of AO/EB dyeing of matched group in Figure 10 embodiment of the present invention 4;
The rear H1299 cellular morphology schematic diagram of AO/EB dyeing of 10mg/ml group in Figure 11 embodiment of the present invention 4;
The rear H1299 cellular morphology schematic diagram of AO/EB dyeing of 20mg/ml group in Figure 12 embodiment of the present invention 4;
The rear H1299 cellular morphology schematic diagram of AO/EB dyeing of 30mg/ml group in Figure 13 embodiment of the present invention 4.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of Ruditapes philippinarum zymolyte
Fresh Ruditapes philippinarum gets meat decontamination after cleaning, and smashs young for clam clean for process meat to pieces homogenate with tissue mashing machine.In homogenate, add the distilled water of 3 times of volumes, add 1200u/g trypsin and carry out enzymolysis, in 37 DEG C, pH value be 8.0 enzymolysis environment reaction 24h, sodium hydroxide and the pH value of hydrogen chloride to reactant liquor can be used to adjust.After enzyme digestion reaction terminates, 90 DEG C of enzyme denaturing 15min, make unreacted trypsin inactivation, and then by centrifugal for reactant liquor 15min, get supernatant, centrifugal condition is: 4 DEG C, 8500r/min.By gained supernatant ultrafiltration 11h at 20 DEG C, use during ultrafiltration and intercept the ultrafilter membrane that molecular weight is 3KDa.After ultrafiltration terminates, by ultrafiltrate rotary evaporation, preserve after lyophilization, obtained zymolyte F12 culture fluid is mixed with different concentration and carries out subsequent experimental.
Embodiment 2: cell growth inhibition assay
Adopt mtt assay to detect the impact of Ruditapes philippinarum zymolyte on H1299 cell proliferation, specific experiment method is as follows:
1) cultivation of H1299 cell: be inoculated in the culture bottle containing F12 culture fluid by H1299 single cell suspension, is placed in 37 DEG C, 5%CO
2incubator in cultivate, cell is monolayer adherence growth, when cell covers with 80%, chooses the good cell of growth conditions and adopts trypsinization to go down to posterity, with 1 × 10
4/ mL cell suspension inoculation in 96 orifice plates, every hole 200mL.
2) MTT detects: above-mentioned cell suspension abandons supernatant after cultivating 24h, carry out MTT detection, Setup Experiments matched group and dosing group, dosing component is 10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml5 group, and each dosage sets up 3 multiple holes separately, abandons culture fluid after cultivating 24h, 48h and 72h respectively, 1mg/ml MTT200mL is added in each hole, inhale after 4h and abandon MTT, add the DMSO of 150 μ L, fully mix.Finally survey absorbance A value at microplate reader 490nm place, calculate cell proliferation inhibition rate (IR), IR=[(control group A value-dosing group A value)/(control group A value-zeroing hole A value] × 100%, repeat experiment 3 times.
3) experimental result: as shown in Figure 1, after zymolyte acts on H1299 cell, cell presents obvious growth inhibited phenomenon, with increase (10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml) and the prolongation of action time (24h, 48h, 72h) of zymolyte concentration, proliferation inhibition rate obviously rises, compared with matched group, difference has statistical significance (p<0.05).
Embodiment 3:HE dyes
1) HE dyeing: put into through the coverslip steeping acid, autoclaving is crossed in 6 well culture plates, the H1299 single cell suspension concentration of cultivating in embodiment 2 step 1) is adjusted to 1 × 10
5be inoculated on the coverslip in culture plate after/mL, every hole adds 2ml single cell suspension, changes liquid after 24h.Establish matched group and dosing group in experiment, dosing component is 10mg/ml, 20mg/ml, 30mg/ml, observes and take pictures after cultivating 48h respectively under inverted microscope (× 200).Test the coverslip terminated in rear taking-up culture plate, fully clean rear use 95% ethanol with PBS and fix 25 minutes, finally carry out conventional H E dyeing, observe under optical microscope (× 400), take pictures with digital camera.
2) experimental result: under inverted microscope, morphologic observation result is as shown in Fig. 2 ~ Fig. 5: in matched group, H1299 cellular morphology is Epithelial as shown in Figure 2, full, stretches, cell boundary is clear; After 10mg/ml zymolyte acts on H1299 cell as shown in Figure 3, occur circular cell in culture fluid, float in culture fluid, part cell stretches out projection and mutually merges; H1299 cell as shown in Figure 4 after 20mg/ml effect, in culture fluid, circle and floating cells increase; As shown in Figure 5 after 30mg/ml effect, the normal H1299 cell of form obviously reduces, floating cells or floating thing more, there is Proliferation Ability and dead phenomenon in cell.
HE coloration result is as shown in figs. 6-9: the H1299 cell boundaries as shown in Figure 6 in matched group is clear, has obvious split coil method, full, and kernel number is many; More cavity is there is in H1299 cell cytoplasm as shown in Figure 7 in 10mg/ml group; Cellular morphology as shown in Figure 8 in 20mg/ml group is irregular, and cavity increases, the karyopycnosis of part cell; Cellular morphology as shown in Figure 9 in 30mg/ml group is irregular, and part cellular morphology diminishes rounded, and megalokaryocyte appears in the increase of part cell volume, and the kernel of most cells obviously reduces, nucleus generation pyknosis, and the apoptosis phenomenons such as Bian Ju occur chromatin.
Embodiment 4:AO/EB dyes
1) AO/EB dyeing: the method for cell inoculation dyes with HE, and take out the coverslip in culture plate after the cell culture 24h of matched group and dosing group, wash 3 times with PBS, 30min fixed by 95% ethanol.Getting final concentration is that the AO/EB mixed liquor of 20mg/L drips on microscope slide, and will have the coverslip of cell one side down, fluorescence microscope (× 200) is observed and taken pictures.
2) experimental result: in matched group, H1299 cellular morphology is substantially identical as shown in Figure 10, nuclear chromatin bright green is also in normal morphology; Viable apoptotic cell as shown in figure 11 in 10mg/ml group is more, and the pyknosis of nuclear chromatin bright green becomes lumps to be positioned at cell side, or in the ANOMALOUS VARIATIONS such as crescent; The non-viable apoptotic cell of 20mg/ml group is more as shown in figure 12, cell rounding, kernel decreased number, and chromatin pyknosis, in orange colour; 30mg/ml group middle and advanced stage apoptotic cell increases as shown in figure 13, cellular morphology is various, chromatin pyknosis is obvious, color burn is salmon pink, and visible part karyon forms fragment, or form semilune, cell membrane outwardly forms apoptotic body, the dead cell of non-apoptosis increases simultaneously, and nuclear chromatin salmon pink but structure is normal.
Note: the Ruditapes philippinarum used in embodiment 1 is purchased and food market, Zhoushan;
The people pulmonary carcinoma H1299 cell strain used in embodiment 2 ~ 4, purchased from Shanghai cell institute of the Chinese Academy of Sciences, is preserved by applicant place laboratory passage.
In each embodiment agents useful for same and key instrument information as follows:
Trypsin SIGMA company); F12 culture medium (GIBCO company) adds the hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio-engineering corporation) of 10%, includes dual anti-; MTT(SIGMA company); AO/EB(Hangzhou Hao Tian Bioisystech Co., Ltd); All the other reagent are analytical pure.
Organize blender (Shanghai is than bright company), CF16RXII High speed refrigerated centrifuge (HITACHI company of Hitachi), UV1600PC ultraviolet spectrophotometer (Shanghai Mei Puda company limited) at a high speed; Automatic fraction collector (BSZ-40-LCD) and Protein Detection instrument (HD-21-88) (Shanghai Qi Te Analytical Instrument Co., Ltd); MSC300 ultrafiltration cup (ultrafilter membrane: 3Kda, rub fast science equipment company limited in Shanghai); Forma3111CO
2incubator (Thermo company of the U.S.); Inverted phase contrast microscope and fluorescence microscope (OLYMPUS company); Super-clean bench (ZHJM-C12090, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.); Microplate reader (U.S. BIO-RAD); Microimaging CCD(promicroscan5898, USA).
Claims (4)
1. the application of Ruditapes philippinarum zymolyte in anti-pulmonary carcinoma.
2. the application of Ruditapes philippinarum zymolyte according to claim 1 in anti-pulmonary carcinoma, is characterized in that: described pulmonary carcinoma is nonsmall-cell lung cancer.
3. the application of Ruditapes philippinarum zymolyte according to claim 1 and 2 in anti-pulmonary carcinoma, is characterized in that: the molecular weight of described zymolyte is less than 3KDa.
4. the application of Ruditapes philippinarum zymolyte according to claim 3 in anti-pulmonary carcinoma, is characterized in that: described zymolyte obtains by the following method:
1), after fresh Ruditapes philippinarum is cleaned, get meat, decontamination, homogenate smashed to pieces by using-system bruisher;
2) in step 1), add 1200u/g trypsin in gained homogenate, enzymolysis 24h, hydrolysis temperature is 37 DEG C, and enzymolysis pH is 8.0;
3) after enzyme digestion reaction terminates, 90 DEG C of enzyme denaturing 15min, then the centrifugal 15min of 8500r/min at 4 DEG C, gets supernatant;
4) adopt intercepting molecular weight to be the ultrafilter membrane of 3KDa, 20 DEG C of ultrafiltration 11h obtain enzymolysis solutions;
5) by after the enzymolysis solution rotary evaporation of step 4) gained, lyophilization is preserved.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498015A (en) * | 2016-11-21 | 2017-03-15 | 浙江海洋大学 | A kind of preparation method and application of Ruditapes philippinarum blood pressure lowering peptide |
| CN106868084A (en) * | 2017-03-23 | 2017-06-20 | 福建省水产研究所 | The preparation method and application of Ruditapes philippinarum high activity natineoplaston |
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| KR20120079977A (en) * | 2011-01-06 | 2012-07-16 | 건국대학교 산학협력단 | Anticancer composition comprising enzymatic hydrolysates of ruditapes philippinarum |
-
2013
- 2013-07-23 CN CN201310312796.XA patent/CN104337837A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120079977A (en) * | 2011-01-06 | 2012-07-16 | 건국대학교 산학협력단 | Anticancer composition comprising enzymatic hydrolysates of ruditapes philippinarum |
Non-Patent Citations (2)
| Title |
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| 李莉等: ""菲律宾蛤仔酶解物抗肺癌H1299 细胞的活性研究", 《安徽农业科学》 * |
| 李莉等: "菲律宾蛤仔酶解物抗肺癌H1299 细胞的活性研究", 《安徽农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106498015A (en) * | 2016-11-21 | 2017-03-15 | 浙江海洋大学 | A kind of preparation method and application of Ruditapes philippinarum blood pressure lowering peptide |
| CN106868084A (en) * | 2017-03-23 | 2017-06-20 | 福建省水产研究所 | The preparation method and application of Ruditapes philippinarum high activity natineoplaston |
| CN106868084B (en) * | 2017-03-23 | 2020-10-09 | 福建省水产研究所 | Preparation method and application of Ruditapes philippinarum high-activity antitumor peptide |
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