CN104232718B - The preparation method and application of dendrobium candidum antineoplastic polypeptide - Google Patents
The preparation method and application of dendrobium candidum antineoplastic polypeptide Download PDFInfo
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Abstract
The present invention provides the preparation method and application of dendrobium candidum antineoplastic polypeptide, and its method is：Dendrobium officinale powder is added into cushioning liquid, ammonium sulphate precipitation, dialysis freeze-drying obtains protein powder；It is redissolved, is made into dendrobium candidum protein liquid, is separately added into 3 kinds of protease, i.e. alkali protease Alcalase 2.4L, alkali protease 37017 and trypsase, digests, obtain three kinds of enzymolysis liquids；Gel chromatography column chromatography is carried out, water elution, collection obtains 9 polypeptide fractions, i.e. dendrobium candidum antineoplastic polypeptide.Products obtained therefrom A3 is respectively to liver cancer cells HepG 2, stomach cancer cell SGC 7901 and breast cancer cell MCF 7 503nhibiting concentration IC50：169.62 µg/mL、155.20 µg/mL、107.53 µg/mL.The dendrobium candidum polypeptide that the present invention is obtained is conducive to the exploitation of functional food and field of medicaments.
Description
Technical field
The invention belongs to biological technical field, and in particular to the preparation method and application of dendrobium candidum antineoplastic polypeptide.
Background technology
Dendrobium candidum (Dendrobium officinale) is herbaceos perennial, also known as ribbed hedyotis herb, and it dries stem warp
Cross processing and can be made into maple bucket, the successive dynasties, doctor write such as《Taoist Scriptures》、《Sheng Nong's herbal classic》、《A Supplement to the Compendium of Materia Medica》Deng on the books to it,
It is described as first of Chinese nine big celestial grass.Version in 2012《Pharmacopoeia of People's Republic of China》Described in dendrobium candidum there is beneficial stomach life
Tianjin, effect of nourishing Yin and clearing heat, available for hectic fever due to yin labor heat, deficiency of stomach-Yin is fire excess from yin deficiency, the symptom such as dry polydipsia.In recent years, separate
Technology and biotechnology are fast-developing, and researcher is able to carry out detailed grind to the chemical composition and bioactivity of dendrobium candidum
Study carefully, it is found that dendrobium candidum composition is various, and possess multiple biological activities, not only immunity of organisms can be strengthened, also with reinforcing stomach reg fluid
With antitumor, hypoglycemic effect.
Biologically active peptide (Bioactive peptide) is that small to two amino acid being made up of 20 natural amino acids are big
To hundreds of amino acid and with special biological function polypeptide compounds.Although peptide is made up of amino acid, still
With the function not available for many single amino acids.Biologically active peptide can be obtained by aminosal, its bioactivity
Also it is released in hydrolytic process.With going deep into for research, the multiple biological activities of biologically active peptide are substantial amounts of
Research confirms that be often mentioned activity has hypoglycemic activity, bacteriostatic activity, active anticancer, antiviral activity, immunoregulatory activity
Deng.It is expected that biologically active peptide will screen the natural resources treasure-house of medicine as the mankind in the near future.
In the last few years, natural polypeptides and artificial synthetic polypeptide with antitumor activity were widely paid close attention to.It is antitumor
Bioactive peptide molecule amount is small, penetration capacity is strong, immunogenicity is low, and is readily synthesized.At present, a variety of antineoplastic polypeptide class medicines on
City enters clinical research, and not only activity is high for polypeptide drug, toxic side effect is small, can also increase tumour cell to other treatments
The susceptibility of method.Therefore the research of anticancer active peptide has important value for the clinical treatment of tumour.At present both at home and abroad to iron
Isolating and purifying for skin stem of noble dendrobium antineoplastic polypeptide not yet has been reported that with applying.
The content of the invention
To expand dendrobium candidum in the application of food and biomedical sector, its added value, mesh of the invention are greatly improved
The preparation method and application for being to provide dendrobium candidum antineoplastic polypeptide.
To realize the object of the invention, adopt the following technical scheme that：
The preparation method of dendrobium candidum antineoplastic polypeptide, the preparation method is：
（1）Dendrobium officinale powder is added into soak extraction albumen in cushioning liquid PBS, ammonium sulphate precipitation, dialysis freezing is dry
It is dry, obtain protein powder；
（2）Take step（1）Gained protein powder is dissolved in distilled water, is made into dendrobium candidum protein liquid, is separately added into 3
Protease, i.e. alkali protease Alcalase 2.4L, alkali protease 37017 and trypsase are planted, is entered under controlled conditions
Row enzymolysis, obtains three kinds of enzymolysis liquids；
（3）Take step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 posts
Chromatography, water elution is collected under Detection wavelength 280nm and obtains 9 polypeptide fractions, i.e. dendrobium candidum antineoplastic polypeptide.
In the above method, comprise the following steps that：
(1) 20-200 g dendrobium candidum powder is weighed, 12-36 h are soaked with the PBS that 1-4 L are added after liquid nitrogen grinding, then
Fall the PBS extract solutions that residue obtains dendrobium candidum with filtered through gauze；Ammonium sulfate is added in PBS extract solutions to saturation degree 10%-
90%, overnight, supernatant is removed after being centrifuged under the conditions of rotating speed is 5000-10000 r/min, albumen medicinal extract is obtained, it is molten with PBS
The bag filter for loading molecular cut off scope 5000-10000 after albumen medicinal extract is solved, is dialysed three days, then freezes and obtains protein
Powder；
(2) 1-5g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 1-10%,
It is separately added into alkali protease Alcalase 2.4L, alkali protease 37017 and trypsase to be hydrolyzed, alkali protease
Alcalase 2.4L enzymatic hydrolysis condition is：Concentration (E/S) of the enzyme in dendrobium candidum protein liquid is 2-10%, temperature 30-60
DEG C, pH=6-9, the reaction time is 6-12 h；The enzymatic hydrolysis condition of trypsase is：Concentration of the enzyme in dendrobium candidum protein liquid
(E/S) it is 2-12%, 25-55 DEG C of temperature, pH=6-10, the reaction time is 6-12 h；The enzymatic hydrolysis condition of alkali protease 37017
For：Concentration (E/S) of the enzyme in dendrobium candidum protein liquid be 2-12%, 20-65 DEG C of temperature, pH=7-10, the reaction time is 6-13
h；Adjust the pH value of reaction system during this with 0.05-0.5 mol/L NaOH and HCL in real time, control ph pH ±
Within 0.05, after hydrolysis, go out enzyme 10-30 min in 90-100 DEG C of water-bath, is cooled to after room temperature in 5000-10000 r/
Supernatant is taken after centrifuging 5-30 min under min, three kinds of enzymolysis liquids are obtained；
(3) to step（2）It is pure that three kinds of obtained enzymolysis liquids carry out gel chromatography sephadex Sephadex G-25 separation
Change, actual conditions is：Column volume is 80-200 mL, and applied sample amount is 1-5mL, wherein the enzymolysis polypeptide dry powder of mg containing 200-600, stream
Dynamic is mutually water, and flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280
nm；Alkali protease Alcalase 2.4L enzymolysis products are collected into 3 peaks A1, A2 and A3 under Detection wavelength 280nm;Pancreas egg
White enzyme enzymolysis product is collected into 3 peaks Y1, Y2 and Y3 under Detection wavelength 280nm;The enzymolysis product of alkali protease 37017 exists
3 peak S1 are collected under Detection wavelength 280nm, so, three kinds of enzymolysis products are through Sephedax G-25 column chromatographies by S2 and S3.
After 9 polypeptide fractions, i.e. dendrobium candidum antineoplastic polypeptide are obtained.
3rd, the preparation method of the dendrobium candidum antineoplastic polypeptide according to claim 1-2, it is characterised in that the iron
The specific of skin stem of noble dendrobium antineoplastic polypeptide component has the peptide masses content to be：A1 is that 39.6%, A2 is that 47.2%, A3 is that 99.2%, Y1 is
33.7%, Y2 are that 54.0%, Y3 is that 58.2%, S1 is that 33.8%, S2 is that 40.6%, S3 is 86.4%.
In the above method, A3 contains 10 peptides, MALDI-TOF-TOF matter in the dendrobium candidum antineoplastic polypeptide component
Analysis of spectrum identifies that the sequence of wherein 3 peptides is respectively:RHPFDGPLLPPGD, KPEEVGGAGDRWTC and
RCGVNAFLPKSYLVHFGWKLLFHFD.
In the above method, the dendrobium candidum antineoplastic polypeptide component has antitumor activity；The component A3 is to liver cancer
Cell HepG-2, stomach cancer cell SGC-7901 and breast cancer cell MCF-7 503nhibiting concentration IC50 is respectively：169.62 µg/
mL、155.20 µg/mL、107.53 µg/mL。
The dendrobium candidum antineoplastic polypeptide can be applied to functional food or antitumor medical preparation.
Compared with prior art, the present invention has the following technical effect that and advantage：
9 polypeptide fractions of dendrobium candidum that the present invention is obtained, be with content of peptides：A1 is that 39.6%, A2 is 47.2%, A3
It is that 33.7%, Y2 is that 54.0%, Y3 is that 58.2%, S1 is that 33.8%, S2 is that 40.6%, S3 is 86.4% for 99.2%, Y1.MALDI-
TOF-TOF mass spectral analyses show, containing 10 peptides in A3, wherein, the sequence of 3 peptides is identified respectively as:
RHPFDGPLLPPGD, KPEEVGGAGDRWTC and RCGVNAFLPKSYLVHFGWKLLFHFD. more importantly, through antitumor
Active (mtt assay) detection, 9 components breed all inhibited to stomach cancer cell SGC-7901, and especially, A3 is to liver
Cancer cell HepG-2 and breast cancer cell MCF-7 also have inhibitory action.A3 is to HepG-2 cell, stomach cancer cell SGC-7901
503nhibiting concentration IC50 with breast cancer cell MCF-7 is respectively：169.62 µg/mL、155.20 µg/mL、107.53 µg/
mL.The dendrobium candidum polypeptide that thus present invention is obtained is conducive to the exploitation of functional food and field of medicaments.
Brief description of the drawings
Fig. 1 is the gel chromatography sephadex of the neutral and alkali protease A lcalase 2.4L enzymolysis products of embodiment 1
SephadexG-25 column chromatography elution curves；
Fig. 2 is the gel chromatography sephadex SephadexG-25 column chromatographies of trypsin digestion product in embodiment 1
Elution curve；
Fig. 3 is the gel chromatography sephadex SephadexG-25 of the enzymolysis product of 1 neutral and alkali protease 3 of embodiment 7017
Column chromatography elution curve；
Fig. 4 is inhibition of 9 polypeptide fractions of dendrobium candidum prepared by embodiment 1 to stomach cancer cell SGC-7901
Figure.
Fig. 5 is the first mass spectrometric figure of the dendrobium candidum polypeptide A 3 prepared by embodiment 1.
The second order mses figure of charge-mass ratio m/z=1417.840 in dendrobium candidum polypeptide As 3 of the Fig. 6 prepared by embodiment 1.
The second order mses figure of charge-mass ratio m/z=1504.81 in dendrobium candidum polypeptide As 3 of the Fig. 7 prepared by embodiment 1.
The second order mses figure of charge-mass ratio m/z=2994.743 in dendrobium candidum polypeptide As 3 of the Fig. 8 prepared by embodiment 1.
Embodiment
Specific implementation below in conjunction with accompanying drawing and example to the present invention is described further, but the implementation and protection of the present invention
Scope not limited to this.
Embodiment 1
The preparation method of dendrobium candidum antineoplastic polypeptide, step is as follows：
(1) 100 g dendrobium candidum powder are weighed, 20 h are soaked with the PBS that 4 L are added after liquid nitrogen grinding, then use gauze mistake
Filter the PBS extract solutions that residue obtains dendrobium candidum.The ammonium sulfate of addition 70% in PBS extract solutions, overnight, centrifugation (7000
R/min supernatant) is removed afterwards, obtains albumen medicinal extract.With loading the saturating of molecular cut off scope 6000 after PBS soluble protein medicinal extract
Bag is analysed, is dialysed three days, then freezes and obtains protein powder；
(2) 2g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 3%, respectively
Add alkali protease Alcalase 2.4L (Novozymes), alkali protease 37017 (Novozymes) and trypsase
(BioBasic Unit) is hydrolyzed, and alkali protease Alcalase 2.4L enzymatic hydrolysis condition is：Enzyme-to-substrate concentration ratio (E/
S it is) 3%, 45 DEG C of temperature, pH=7, the reaction time is 8 h.The enzymatic hydrolysis condition of trypsase is：Enzyme-to-substrate concentration ratio (E/S)
For 6%, 35 DEG C of temperature, pH=8, the reaction time is 10 h.The enzymatic hydrolysis condition of alkali protease 37017 is：Enzyme-to-substrate concentration
It is 8% than (E/S), 50 DEG C of temperature, pH=9, the reaction time is 12 h.In experimentation in real time with 0.08 mol/L NaOH with
HCL adjusts the pH value of reaction system, and control ph is within pH ± 0.05.After hydrolysis, go out enzyme 15 in 95 DEG C of water-bath
Min, is cooled to after room temperature after centrifuging 15 min under 9000 r/min and takes supernatant, obtains three kinds of enzymolysis liquids.
(3) step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 separation
Purify, actual conditions is：Column volume is 100 mL, and applied sample amount is 3mL (containing 400 mg enzymolysis polypeptides dry powder), and mobile phase is water,
Flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280 nm.Alkalescence
Protease A lcalase 2.4L enzymolysis products are collected into 3 peak A1, A2 and A3 (Fig. 1) under Detection wavelength 280nm;Pancreas egg
White enzyme enzymolysis product is collected into 3 peak Y1, Y2 and Y3 (Fig. 2) under Detection wavelength 280nm;Alkali protease 37017 is digested
Product is collected into 3 peak S1, S2 and S3 (Fig. 3) so under Detection wavelength 280nm, and three kinds of enzymolysis products are through Sephedax
It is obtained 9 polypeptide fractions A1, A2, A3, Y1, Y2, Y3, S1, S2, S3 after G-25 column chromatographies, content of peptides is followed successively by 39.6%,
47.2%、99.2%、33.7%、54.0%、58.2%、33.8%、40.6%、86.4%.Mtt assay detects this nine components to stomach cancer cell
SGC-7901, which increases, has inhibitory action (Fig. 4) especially, and polypeptide A 3 is to HepG-2 cell, stomach cancer cell SGC-7901
There is certain inhibitory action (table 1) with breast cancer cell MCF-7,503nhibiting concentration IC50 is respectively：169.62 µg/mL、
155.20 μ g/mL, 107.53 μ g/mL. MALDI-TOF-TOF-MS mass spectral analyses show, 10 polypeptides are mainly contained in A3
(Fig. 5), wherein charge-mass ratio account for further two grades of 61.5%. altogether for 1417.840,1504.81 and 2994.743 three peptides
Mass spectrum (Fig. 6-8) identifies that the amino acid sequence of this 3 peptides is respectively:RHPFDGPLLPPGD, KPEEVGGAGDRWTC and
RCGVNAFLPKSYLVHFGWKLLFHFD.
Inhibition (± s, the n=5) (%) of the 3 pairs of three kinds of cancer cells of polypeptide A of table 1
| Concentration (μ g/mL) | Negative control | 50 | 100 | 200 | 300 | 400 | 500 |
| HepG-2 | 0 | 35.04±0.011 | 38.87±0.003 | 46.11±0.002 | 58.04±0.004 | 64.00±0.016 | 73.38±0.006 |
| SGC-7901 | 0 | 26.09±0.006 | 42.28±0.003 | 53.35±0.007 | 61.24±0.024 | 72.31±0.010 | 78.91±0.005 |
| MCF-7 | 0 | 36.10±0.003 | 48.88±0.012 | 59.53±0.011 | 65.28±0.009 | 77.22±0.019 | 86.80±0.006 |
Embodiment 2
The preparation method of dendrobium candidum antineoplastic polypeptide, step is as follows：
(1) 120 g dendrobium candidum powder are weighed, 30 h are soaked with the PBS that 2 L are added after liquid nitrogen grinding, then use gauze mistake
Filter the PBS extract solutions that residue obtains dendrobium candidum.The ammonium sulfate of addition 60% in PBS extract solutions, overnight, centrifugation (9000
R/min supernatant) is removed afterwards, obtains albumen medicinal extract.With loading the saturating of molecular cut off scope 5000 after PBS soluble protein medicinal extract
Bag is analysed, is dialysed three days, then freezes and obtains protein powder；
(2) 3g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 8%, respectively
Add Alcalase 2.4L (Novozymes), alkali protease 37017 (Novozymes) and trypsase (BioBasic
Unit the enzymatic hydrolysis condition that alkali protease Alcalase 2.4L) are hydrolyzed is：Enzyme-to-substrate concentration ratio (E/S) is 2%, temperature
40 DEG C of degree, pH=9, the reaction time is 10 h.The enzymatic hydrolysis condition of trypsase is：Enzyme-to-substrate concentration ratio (E/S) is 7%, temperature
40 DEG C of degree, pH=7, the reaction time is 9 h.The enzymatic hydrolysis condition of alkali protease 37017 is：Enzyme-to-substrate concentration ratio (E/S)
For 5%, 35 DEG C of temperature, pH=8, the reaction time is 11 h.Adjusted in real time with 0.1 mol/L NaOH and HCL in experimentation
The pH value of reaction system, control ph is within pH ± 0.05.After hydrolysis, go out the min of enzyme 10 in 100 DEG C of water-bath, cooling
Supernatant is taken after centrifuging 25 min under 6000 r/min after to room temperature, three kinds of enzymolysis liquids are obtained.
(3) step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 separation
Purify, actual conditions is：Column volume is 120 mL, and applied sample amount is 4mL (containing 300 mg enzymolysis polypeptides dry powder), and mobile phase is water,
Flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280 nm.Alkalescence
Protease A lcalase 2.4L enzymolysis products are collected into 3 peaks A1, A2 and A3 under Detection wavelength 280nm;Trypsase enzyme
Solution product is collected into 3 peaks Y1, Y2 and Y3 under Detection wavelength 280nm;The enzymolysis product of alkali protease 37017 is in detection ripple
3 peak S1 are collected under long 280nm, so, three kinds of enzymolysis products there are after Sephedax G-25 column chromatographies by S2 and S3.
To 9 polypeptide fractions.Detection method is with result with reference to embodiment 1.
Embodiment 3
The preparation method of dendrobium candidum antineoplastic polypeptide, step is as follows：
(1) 80 g dendrobium candidum powder are weighed, 24 h are soaked with the PBS that 1 L is added after liquid nitrogen grinding, then use filtered through gauze
Fall the PBS extract solutions that residue obtains dendrobium candidum.The ammonium sulfate of addition 70% in PBS extract solutions, overnight, centrifuges (7000 r/
Min supernatant) is removed afterwards, obtains albumen medicinal extract.With the dialysis for loading molecular cut off scope 7000 after PBS soluble protein medicinal extract
Bag, dialyses three days, then freezes and obtains protein powder；
(2) 4g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 9%, respectively
Add Alcalase 2.4L (Novozymes), alkali protease 37017 (Novozymes) and trypsase (BioBasic
Unit the enzymatic hydrolysis condition that alkali protease Alcalase 2.4L) are hydrolyzed is：Enzyme-to-substrate concentration ratio (E/S) is 7%, temperature
50 DEG C of degree, pH=6, the reaction time is 11 h.The enzymatic hydrolysis condition of trypsase is：Enzyme-to-substrate concentration ratio (E/S) is 6%, temperature
50 DEG C of degree, pH=10, the reaction time is 12 h.The enzymatic hydrolysis condition of alkali protease 37017 is：Enzyme-to-substrate concentration ratio (E/
S it is) 4%, 60 DEG C of temperature, pH=9, the reaction time is 12 h.Adjusted in real time with 0.3 mol/L NaOH and HCL in experimentation
The pH value of reaction system is saved, control ph is within pH ± 0.05.After hydrolysis, go out the min of enzyme 30 in 90 DEG C of water-bath, cooling
Supernatant is taken after centrifuging 10 min under 6000 r/min after to room temperature, three kinds of enzymolysis liquids are obtained.
(3) step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 separation
Purify, actual conditions is：Column volume is 150 mL, and applied sample amount is 5mL (containing 600 mg enzymolysis polypeptides dry powder), and mobile phase is water,
Flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280 nm.Alkalescence
Protease A lcalase 2.4L enzymolysis products are collected into 3 peaks A1, A2 and A3 under Detection wavelength 280nm;Trypsase enzyme
Solution product is collected into 3 peaks Y1, Y2 and Y3 under Detection wavelength 280nm;The enzymolysis product of alkali protease 37017 is in detection ripple
3 peak S1 are collected under long 280nm, so, three kinds of enzymolysis products there are after Sephedax G-25 column chromatographies by S2 and S3.
To 9 polypeptide fractions.Detection method is with result with reference to embodiment 1.
Embodiment 4
The preparation method of dendrobium candidum antineoplastic polypeptide, step is as follows：
(1) 200 g dendrobium candidum powder are weighed, 30 h are soaked with the PBS that 3 L are added after liquid nitrogen grinding, then use gauze mistake
Filter the PBS extract solutions that residue obtains dendrobium candidum.The ammonium sulfate of addition 90% in PBS extract solutions, overnight, centrifugation (8000
R/min supernatant) is removed afterwards, obtains albumen medicinal extract.With loading the saturating of molecular cut off scope 8000 after PBS soluble protein medicinal extract
Bag is analysed, is dialysed three days, then freezes and obtains protein powder；
(2) 4g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 10%, respectively
Add Alcalase 2.4L (Novozymes), alkali protease 37017 (Novozymes) and trypsase (BioBasic
Unit the enzymatic hydrolysis condition that alkali protease Alcalase 2.4L) are hydrolyzed is：Enzyme-to-substrate concentration ratio (E/S) is 3%, temperature
60 DEG C of degree, pH=7, the reaction time is 6 h.The enzymatic hydrolysis condition of trypsase is：Enzyme-to-substrate concentration ratio (E/S) is 10%, temperature
55 DEG C of degree, pH=6, the reaction time is 7 h.The enzymatic hydrolysis condition of alkali protease 37017 is：Enzyme-to-substrate concentration ratio (E/S)
For 6%, 55 DEG C of temperature, pH=7, the reaction time is 9 h.Adjusted in real time with 0.15 mol/L NaOH and HCL in experimentation
The pH value of reaction system, control ph is within pH ± 0.05.After hydrolysis, go out the min of enzyme 25 in 95 DEG C of water-bath, is cooled to
Supernatant is taken after centrifuging 30 min under 5000 r/min after room temperature, three kinds of enzymolysis liquids are obtained.
(3) step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 separation
Purify, actual conditions is：Column volume is 200 mL, and applied sample amount is 3mL (containing 200 mg enzymolysis polypeptides dry powder), and mobile phase is water,
Flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280 nm.Alkalescence
Protease A lcalase 2.4L enzymolysis products are collected into 3 peaks A1, A2 and A3 under Detection wavelength 280nm;Trypsase enzyme
Solution product is collected into 3 peaks Y1, Y2 and Y3 under Detection wavelength 280nm;The enzymolysis product of alkali protease 37017 is in detection ripple
3 peak S1 are collected under long 280nm, so, three kinds of enzymolysis products there are after Sephedax G-25 column chromatographies by S2 and S3.
To 9 polypeptide fractions.Detection method is with result with reference to embodiment 1.
Claims (4)
1. the preparation method of dendrobium candidum antineoplastic polypeptide, it is characterised in that the preparation method is：
（1）Dendrobium officinale powder is added into soak extraction albumen in cushioning liquid PBS, ammonium sulphate precipitation, dialysis freeze-drying is obtained
To protein powder；
（2）Take step（1）Gained protein powder is dissolved in distilled water, is made into dendrobium candidum protein liquid, is separately added into 3 hatching eggs
White enzyme, i.e. alkali protease Alcalase 2.4L, alkali protease 37017 and trypsase, carry out enzyme under controlled conditions
Solution, obtains three kinds of enzymolysis liquids；
（3）Take step（2）Three kinds of obtained enzymolysis liquids, then carry out gel chromatography sephadex Sephadex G-25 column chromatographies,
Water elution, collects under Detection wavelength 280nm and obtains 9 polypeptide fractions, i.e. dendrobium candidum antineoplastic polypeptide；
The preparation method is comprised the following steps that：
(1) 20-200 g dendrobium candidum powder is weighed, 12-36 h are soaked with the PBS that 1-4 L are added after liquid nitrogen grinding, then use yarn
Cloth filters out the PBS extract solutions that residue obtains dendrobium candidum；Ammonium sulfate is added in PBS extract solutions to saturation degree 10%-90%, mistake
At night, supernatant is removed after being centrifuged under the conditions of rotating speed is 5000-10000 r/min, albumen medicinal extract is obtained, uses PBS soluble proteins
Load molecular cut off scope 5000-10000 bag filter after medicinal extract, dialyse three days, then freeze and obtain protein powder；
(2) 1-5g protein powders are weighed, distilled water is made into the dendrobium candidum protein liquid that mass percent concentration is 1-10%, respectively
Add alkali protease Alcalase 2.4L, alkali protease 37017 and trypsase to be hydrolyzed, alkali protease
Alcalase 2.4L enzymatic hydrolysis condition is：Concentration (E/S) of the enzyme in dendrobium candidum protein liquid is 2-10%, temperature 30-60
DEG C, pH=6-9, the reaction time is 6-12 h；The enzymatic hydrolysis condition of trypsase is：Concentration of the enzyme in dendrobium candidum protein liquid
(E/S) it is 2-12%, 25-55 DEG C of temperature, pH=6-10, the reaction time is 6-12 h；The enzymatic hydrolysis condition of alkali protease 37017
For：Concentration (E/S) of the enzyme in dendrobium candidum protein liquid be 2-12%, 20-65 DEG C of temperature, pH=7-10, the reaction time is 6-13
h；Adjust the pH value of reaction system during this with 0.05-0.5 mol/L NaOH and HCL in real time, control ph pH ±
Within 0.05, after hydrolysis, go out enzyme 10-30 min in 90-100 DEG C of water-bath, is cooled to after room temperature in 5000-10000 r/
Supernatant is taken after centrifuging 5-30 min under min, three kinds of enzymolysis liquids are obtained；
(3) to step（2）Three kinds of obtained enzymolysis liquids carry out gel chromatography sephadex Sephadex G-25 and isolated and purified,
Actual conditions is：Column volume is 80-200 mL, and applied sample amount is 1-5mL, wherein the enzymolysis polypeptide dry powder of mg containing 200-600, flowing
Xiang Weishui, flow velocity is 0.5 mL/min, and every 6 min collects one and managed, and 100 are collected altogether and is managed, and Detection wavelength is 215 nm and 280
nm；Alkali protease Alcalase 2.4L enzymolysis products are collected into 3 peaks A1, A2 and A3 under Detection wavelength 280nm;Pancreas egg
White enzyme enzymolysis product is collected into 3 peaks Y1, Y2 and Y3 under Detection wavelength 280nm;The enzymolysis product of alkali protease 37017 exists
3 peaks S1, S2 and S3 are collected under Detection wavelength 280nm, so, three kinds of enzymolysis products are through Sephedax G-25 column chromatographies
After 9 polypeptide fractions, i.e. dendrobium candidum antineoplastic polypeptide are obtained.
2. the preparation method of dendrobium candidum antineoplastic polypeptide according to claim 1, it is characterised in that the dendrobium candidum
A3 contains 10 peptides in antineoplastic polypeptide component, and MALDI-TOF-TOF mass spectral analyses identify that the sequence of wherein 3 peptides is respectively:
RHPFDGPLLPPGD, KPEEVGGAGDRWTC and RCGVNAFLPKSYLVHFGWKLLFHFD.
3. the preparation method of dendrobium candidum antineoplastic polypeptide according to claim 1, it is characterised in that the dendrobium candidum
Antineoplastic polypeptide component has antitumor activity；The component A3 is to HepG-2 cell, stomach cancer cell SGC-7901 and breast
Adenocarcinoma cell MCF-7 503nhibiting concentration IC50 is respectively：169.62 µg/mL、155.20 µg/mL、107.53 µg/mL.
4. the dendrobium candidum antineoplastic polypeptide that the preparation method described in any one of claim 1-3 is prepared is applied to function
Food or antitumor medical preparation；It is described antitumor including suppressing one or more of liver cancer, stomach cancer or breast cancer.
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| CN105713944A (en) * | 2016-03-30 | 2016-06-29 | 蔡庭守 | Small molecular peptide composition of Dendrobium officinale Kimura et Migo as well as extraction method and application of small molecular peptide composition |
| CN105861604A (en) * | 2016-04-07 | 2016-08-17 | 金寨金栗源生物技术有限公司 | Method for preparing dendrobium officinale peptide |
| CN107446978A (en) * | 2017-08-15 | 2017-12-08 | 陈玫辛 | A kind of stem of noble dendrobium small-molecular peptides and preparation method thereof |
| CN108314707B (en) * | 2018-02-26 | 2019-09-03 | 天津科技大学 | Anti-tumor activity peptide and its preparation method and application |
| CN110274972A (en) * | 2019-06-21 | 2019-09-24 | 海南大学 | A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution |
| CN112662722B (en) * | 2020-12-30 | 2024-01-23 | 江苏耐雀生物工程技术有限公司 | Dendrobium nobile polypeptide extract with antiseptic effect and preparation method and application thereof |
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