CN106497860A - Culture medium and the preparation method of Garcinia mangostana cell secondary metabolitess - Google Patents
Culture medium and the preparation method of Garcinia mangostana cell secondary metabolitess Download PDFInfo
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- CN106497860A CN106497860A CN201610963845.XA CN201610963845A CN106497860A CN 106497860 A CN106497860 A CN 106497860A CN 201610963845 A CN201610963845 A CN 201610963845A CN 106497860 A CN106497860 A CN 106497860A
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- garcinia mangostana
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- mangostana
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- 240000006053 Garcinia mangostana Species 0.000 title claims abstract description 103
- 235000017048 Garcinia mangostana Nutrition 0.000 title claims abstract description 103
- 239000001963 growth medium Substances 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000007640 basal medium Substances 0.000 claims abstract description 6
- 239000002537 cosmetic Substances 0.000 claims abstract description 6
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 25
- 230000000694 effects Effects 0.000 claims description 14
- 108010059892 Cellulase Proteins 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 229940106157 cellulase Drugs 0.000 claims description 13
- 229940088598 enzyme Drugs 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 239000012531 culture fluid Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- JNELGWHKGNBSMD-UHFFFAOYSA-N xanthone powder Natural products C1=CC=C2C(=O)C3=CC=CC=C3OC2=C1 JNELGWHKGNBSMD-UHFFFAOYSA-N 0.000 abstract description 27
- 239000008176 lyophilized powder Substances 0.000 abstract description 26
- -1 xanthone compound Chemical class 0.000 abstract description 10
- 238000004113 cell culture Methods 0.000 abstract description 9
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 5
- 229930000044 secondary metabolite Natural products 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 72
- 230000001954 sterilising effect Effects 0.000 description 30
- 238000001914 filtration Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 229930192334 Auxin Natural products 0.000 description 10
- 239000002363 auxin Substances 0.000 description 10
- 230000032823 cell division Effects 0.000 description 10
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 210000003491 skin Anatomy 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 238000004114 suspension culture Methods 0.000 description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 239000005708 Sodium hypochlorite Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000003035 anti-peroxidant effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 210000002249 digestive system Anatomy 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000004540 process dynamic Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 4
- 150000007964 xanthones Chemical class 0.000 description 4
- QTDMGAWIBXJNRR-UHFFFAOYSA-N Mangostin Natural products CC(=CCc1c(O)cc2Oc3cc(C)c(O)c(CC=C(C)C)c3C(=O)c2c1O)C QTDMGAWIBXJNRR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- GNRIZKKCNOBBMO-UHFFFAOYSA-N alpha-mangostin Chemical compound OC1=C(CC=C(C)C)C(O)=C2C(=O)C3=C(CC=C(C)C)C(OC)=C(O)C=C3OC2=C1 GNRIZKKCNOBBMO-UHFFFAOYSA-N 0.000 description 3
- ZVFQDLCERPGZMO-UHFFFAOYSA-N alpha-mangostin Natural products OC1=C(CC=C(C)C)C(O)=C2C(=O)C3=C(CC=C(C)C)C(OC)=C(O)C=C3CC2=C1 ZVFQDLCERPGZMO-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 150000002576 ketones Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000546193 Clusiaceae Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000593508 Garcinia Species 0.000 description 2
- 235000000885 Garcinia xanthochymus Nutrition 0.000 description 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- VEZXFTKZUMARDU-UHFFFAOYSA-N gamma-mangostin Chemical compound OC1=C(O)C(CC=C(C)C)=C2C(=O)C3=C(O)C(CC=C(C)C)=C(O)C=C3OC2=C1 VEZXFTKZUMARDU-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000973497 Siphonognathus argyrophanes Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- JLDSOYXADOWAKB-UHFFFAOYSA-N aluminium nitrate Chemical class [Al+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O JLDSOYXADOWAKB-UHFFFAOYSA-N 0.000 description 1
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Natural products C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- ACRGTLGOYYTGNX-UHFFFAOYSA-N gamma-mangostin Natural products OC1=C(O)C=C2C(=O)C3=C(O)C(CC=C(C)C)=C(O)C=C3OC2=C1 ACRGTLGOYYTGNX-UHFFFAOYSA-N 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
- C07D311/86—Oxygen atoms, e.g. xanthones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
Abstract
The present invention relates to cell field, the more particularly to preparation method of culture medium and Garcinia mangostana cell secondary metabolitess.The culture medium includes 2,4 D, 6 BA and basal medium.Secondary metabolitess in the Garcinia mangostana cell culture obtained by the culture medium contain total xanthone compound, and its secondary metabolites is collected carries out lyophilizing preservation.Have anti-oxidation efficacy using total xanthone compound, reach the purpose of defying age, the lyophilized powder can be applied in cosmetics.
Description
Technical field
The present invention relates to cell field, the more particularly to preparation method of culture medium and Garcinia mangostana cell secondary metabolitess.
Background technology
Garcinia mangostana, also known as Cortex Garciniae, mangosteen, garcinia mangostana, is evergreen Qiao of Guttiferae (Guttiferae) Garcinia (Garcinia)
The fruit of wooden Garcinia mangostana.The pharmacological action of Garcinia mangostana becomes study hotspot in recent years.Garcinia mangostana shell contains multiple xanthone derivatives,
Mainly include α-mangostin, γ-mangostin etc..These xanthones show multiple biological activities, such as antiinflammatory, anti-swollen
Tumor, antioxidation and there is antibacterial activity to various bacteria.On biochemistry, xanthone is considered as that unique nature is looked for
To replaced by various phenols, methoxy base class and isoprene by tricyclic aromatic, so as to produce a series of family of derivants
Race.At present, research worker has been identified and isolated from about 200 kinds of xanthones, and wherein 40 kinds are found in mangosteen fruit
's.Many important xanthones such as α-Fructus Garciniae oblongifoliae ketone, β-Fructus Garciniae oblongifoliae ketone, γ-Fructus Garciniae oblongifoliae ketone.
The xanthone compound of Garcinia mangostana is with chemical leaching test or conbined enzymolysis at present.It is primarily present and asks as follows
Topic：1. chemical synthesis process complex process.2. safety is low, and used in production process, a large amount of organic solvents, easily remain in into
In product.3. the season of growth of Garcinia mangostana is limited to, the yield of finished product is affected.4. cell mass propgation has the work(of concentration working substance
Effect.
Content of the invention
In view of this, the present invention provides a kind of culture medium and Garcinia mangostana cell secondary metabolitess total xanthone compound
Preparation method.The present invention (is synthesized in Garcinia mangostana cell cultivation process and is discharged using the secondary metabolitess in Garcinia mangostana cell culture
Have the general name of bioactive substance into culture fluid) contain total xanthone compound, its secondary metabolites is collected to be carried out
Lyophilizing is preserved.Have anti-oxidation efficacy using total xanthone compound, reach the purpose of defying age, the lyophilized powder can be used
To in cosmetics.
In order to realize that foregoing invention purpose, the present invention provide technical scheme below：
The invention provides a kind of culture medium, including 2,4-D, 6-BA and basal medium；
Concentration of the 2,4-D in the culture medium is 0.2～2.0mg/L；The 6-BA is in the culture medium
Concentration is 0.5～3.0mg/L.
In some specific embodiments of the present invention, the culture medium also includes glucose.
The present invention some specific embodiments in, concentration of the glucose in the culture medium be 1.0～
5.0g/L.In some specific embodiments of the present invention, basal medium described in the culture medium is MS culture medium.
Present invention also offers the culture medium prepares the application in secondary metabolitess in culture Garcinia mangostana cell.
In some specific embodiments of the present invention, the Garcinia mangostana cell secondary metabolitess are total xanthone chemical combination
Thing.
Present invention also offers a kind of preparation method of Garcinia mangostana cell secondary metabolitess, it is characterised in that including following step
Suddenly：
Step 1：Obtain Garcinia mangostana cell；
Step 2：Take step 1 acquisition Garcinia mangostana cell with the present invention provide culture medium mix after tentatively cultivate, then with
Amplification culture after the culture medium mixing that invention is provided, collects culture fluid.
In some specific embodiments of the present invention, the preparation side of Garcinia mangostana cell described in the preparation method step 1
Method digest after peel of Carcinia mangostana L. is mixed with basal medium, cellulase and pectase for taking, and filters, collects filtrate.
In some specific embodiments of the present invention, the addition of cellulase described in the preparation method is described
The 0.01～0.10% of peel of Carcinia mangostana L. quality, the addition of the pectase be the peel of Carcinia mangostana L. quality 0.01～
0.10%.
In some specific embodiments of the present invention, described in the preparation method, the enzyme activity of cellulase is
30000U/mg, the enzyme activity of the pectase is 50000U/mg.
In some specific embodiments of the present invention, the volume ratio 1 of cellulase and pectase:1.
In some specific embodiments of the present invention, the time of the enzymolysis is 3 hours.
In some specific embodiments of the present invention, the condition of the preliminary culture is：Take Garcinia mangostana cell addition I to go out
Garcinia mangostana cell strain is configured to bacterium culture medium the Garcinia mangostana Cell sap of 1-5 × 105/ml density, takes the addition of 100ml Garcinia mangostana Cell sap
In the aseptic conical flasks of 250ml, on rotary shaker, with 100-200rpm/min, 25-26 DEG C, cultivate 14 days.
The formula (in 1L MS culture medium) of I culture medium
| Title | Content | Title | Content |
| 2,4-D | 0.2～1.0mg/L | 6-BA | 1.5～3.0mg/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division.
In some specific embodiments of the present invention, the condition of the amplification culture is：After preliminary culture, 250g
Centrifugation 10min removes supernatant, and the Garcinia mangostana for adding No. II sterilising medium that Garcinia mangostana cell is configured to 1-3x106/ml density is thin
Cytosol is placed in fermentation tank carries out suspension culture, 130-140rpm/min, 25-26 DEG C, is passed through air filtering, and speed is 0.01-
0.05m3/ s, cultivates 14 days.
The formula (in 1L MS culture medium) of No. II sterilising medium
| Title | Content | Title | Content |
| 2,4-D | 1.0～2.0mg/L | 6-BA | 0.5～1.5mg/L |
| Glucose | 1.0～5.0g/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division.
In some specific embodiments of the present invention, after step 2 collects culture fluid, also include filtering, concentrate, collect
Degerming after concentrated solution, the step of lyophilizing.
In some specific embodiments of the present invention, the lyophilizing is that vacuum is 50～200Pa in -20～-35 DEG C
Under conditions of, lyophilizing 24～36 hours.
Present invention also offers Garcinia mangostana cell secondary metabolitess obtained in the preparation method.
In some specific embodiments of the present invention, the Garcinia mangostana cell secondary metabolitess are total xanthone chemical combination
Thing.
Present invention also offers the Garcinia mangostana cell secondary metabolitess prepare the medicine of anti peroxidation of lipid, food and/
Or the application in cosmetics.
Present invention also offers the Garcinia mangostana cell secondary metabolitess are preparing medicine, food and/or the cosmetic of defying age
Application in product.
The invention provides a kind of culture medium, including 2,4-D, 6-BA and basal medium.By the culture medium and originally
Secondary metabolitess in Garcinia mangostana cell culture obtained in the method that invention is provided (synthesize in Garcinia mangostana cell cultivation process and discharge
Have the general name of bioactive substance into culture fluid) contain total xanthone compound, its secondary metabolites is collected to be carried out
Lyophilizing is preserved.Have anti-oxidation efficacy using total xanthone compound, reach the purpose of defying age, the lyophilized powder can be used
To in cosmetics.
Specific embodiment
The invention discloses the preparation method of a kind of culture medium and Garcinia mangostana cell secondary metabolitess, those skilled in the art can
To use for reference present disclosure, technological parameter realization is suitably modified.Specifically, all similar replacements and change are to this
It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the present invention and application are
It is described by preferred embodiment, related personnel substantially can be in without departing from present invention, spirit and scope to herein
Described methods and applications are modified or suitably change and combine, and realize and apply the technology of the present invention.
The invention provides a kind of preparation method of Garcinia mangostana cell secondary metabolitess, comprises the steps：
Obtain Garcinia mangostana cell strain：
Taking the fresh Garcinia mangostana of 1kg carries out cleaning and sterilizing, is immersed in 20 minutes in 10L 15%-20% sodium hypochlorite.Remove time chlorine
Acid sodium solution, aseptically, is cleaned using 2L sterile deionized water every time, totally three times, is gently stirred in each cleaning process
Dynamic.
Under sterile conditions, remove the peel of Carcinia mangostana L. after sterilization, with sterilizing after shears shred, every chip size≤
1mm2.
Peel of Carcinia mangostana L. fragment is transferred in the beaker of sterile clean, 10ml MS (Murashige is added per 1.0g fragments
And Skoog) culture medium, 0.01-0.10% cellulase and 0.01-0.10% pectases, digest 3 hours.Preferably, fiber
Plain enzyme and pectase volume ratio are 1:1.
Digestive system is filtered with 200 mesh aseptic mesh screen, remove impurity, obtain Garcinia mangostana cell strain.
Garcinia mangostana cell mass propgation：
Add I sterilising mediums that Garcinia mangostana cell strain is configured to 1～5 × 105The Garcinia mangostana Cell sap of/ml density, takes
100ml Garcinia mangostana Cell sap is added in the aseptic conical flasks of 250ml, on rotary shaker, with 100-200rpm/min, 25-26
DEG C, cultivate 14 days.
After preliminary culture, 250g centrifugation 10min remove supernatant, add No. II sterilising medium to match somebody with somebody Garcinia mangostana cell
Make 1-3 × 106The Garcinia mangostana Cell sap of/ml density is placed in fermentation tank carries out suspension culture, 130-140rpm/min, 25-26
DEG C, air filtering was passed through, speed is 0.01-0.05m3/ s, cultivates 14 days.
The formula (in 1L MS culture medium) of I culture medium：
| Title | Content | Title | Content |
| 2,4-D | 0.2-1.0mg/L | 6-BA | 1.5-3.0mg/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The formula (in 1L MS culture medium) of No. II sterilising medium：
| Title | Content | Title | Content |
| 2,4-D | 1.0-2.0mg/L | 6-BA | 0.5-1.5mg/L |
| Glucose | 1.0-5.0g/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The preparation of Garcinia mangostana cell secondary metabolitess lyophilized powder：
Garcinia mangostana cell culture is collected, which is filtered with 0.45 μM of filter membrane, concentrated, the concentration supernatant for obtaining is used
0.22 μM of filtration sterilization.
Carry out frozen dried after degerming, freeze temperature is -20～-35 DEG C, vacuum is 50～200Pa, freeze-drying time is
24～36 hours, that is, obtain the secondary metabolitess lyophilized powder of Garcinia mangostana cell.Lyophilized powder is distributed into 1ml/.
The present invention extracts total xanthone compound in Garcinia mangostana to total oxygen for extracting using culture plant cell method
Miscellaneous anthracene ketone compounds carry out frozen dried, it is easy to preserve and apply, increase stability.
Beneficial effect includes：
1. extraction process is stable, and effective ingredient concentration is big, and can preserve its activity.
2. safe, not residual organic solvent.
3. the season of growth for being not only restricted to Garcinia mangostana affects.
Raw materials used and reagent in the preparation method of the culture medium of present invention offer and Garcinia mangostana cell secondary metabolitess
Buied by market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
Obtain Garcinia mangostana cell strain：
Taking the fresh Garcinia mangostana of 1kg carries out cleaning and sterilizing, is immersed in 20 minutes in 10L 15%-20% sodium hypochlorite.Remove time chlorine
Acid sodium solution, aseptically, is cleaned using 2L sterile deionized water every time, totally three times, is gently stirred in each cleaning process
Dynamic.
Under sterile conditions, remove the peel of Carcinia mangostana L. after sterilization, with sterilizing after shears shred, every chip size≤
1mm2.
Peel of Carcinia mangostana L. fragment is transferred in the beaker of sterile clean, 10ml MS (Murashige is added per 1.0g fragments
And Skoog) culture medium, 0.01% cellulase and 0.05% pectase, digest 3 hours.The enzyme activity of cellulase is
30000U/mg, the enzyme activity of pectase is 50000U/mg.
Digestive system is filtered with 200 mesh aseptic mesh screen, remove impurity, obtain Garcinia mangostana cell strain.
Garcinia mangostana cell mass propgation：
Add I sterilising mediums that Garcinia mangostana cell strain is configured to 1～5 × 105The Garcinia mangostana Cell sap of/ml density, takes
100ml Garcinia mangostana Cell sap is added in the aseptic conical flasks of 250ml, on rotary shaker, with 100-200rpm/min, 25-26
DEG C, cultivate 14 days.
After preliminary culture, 250g centrifugation 10min remove supernatant, add No. II sterilising medium to match somebody with somebody Garcinia mangostana cell
Make 1-3 × 106The Garcinia mangostana Cell sap of/ml density is placed in fermentation tank carries out suspension culture, 130-140rpm/min, 25-26
DEG C, air filtering was passed through, speed is 0.01-0.05m3/ s, cultivates 14 days.
The formula (in 1L MS culture medium) of 1 I culture medium of table
| Title | Content | Title | Content |
| 2,4-D | 0.2mg/L | 6-BA | 3.0mg/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The formula (in 1L MS culture medium) of 2 No. II sterilising mediums of table
| Title | Content | Title | Content |
| 2,4-D | 1.0mg/L | 6-BA | 1.5mg/L |
| Glucose | 3.0g/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The preparation of Garcinia mangostana cell secondary metabolitess lyophilized powder：
Garcinia mangostana cell culture is collected, which is filtered with 0.45 μM of filter membrane, concentrated, the concentration supernatant for obtaining is used
0.22 μM of filtration sterilization.
Frozen dried is carried out after degerming, freeze temperature is -28 DEG C, vacuum is 50Pa, and freeze-drying time is 30 hours, obtains final product
Secondary metabolitess lyophilized powder to Garcinia mangostana cell.Lyophilized powder is distributed into 1ml/.
Embodiment 2
Obtain Garcinia mangostana cell strain：
Taking the fresh Garcinia mangostana of 1kg carries out cleaning and sterilizing, is immersed in 20 minutes in 10L 15%-20% sodium hypochlorite.Remove time chlorine
Acid sodium solution, aseptically, is cleaned using 2L sterile deionized water every time, totally three times, is gently stirred in each cleaning process
Dynamic.
Under sterile conditions, remove the peel of Carcinia mangostana L. after sterilization, with sterilizing after shears shred, every chip size≤
1mm2.
Peel of Carcinia mangostana L. fragment is transferred in the beaker of sterile clean, 10ml MS (Murashige is added per 1.0g fragments
And Skoog) culture medium, 0.10% cellulase and 0.01% pectase, digest 3 hours.The enzyme activity of cellulase is
30000U/mg, the enzyme activity of pectase is 50000U/mg.
Digestive system is filtered with 200 mesh aseptic mesh screen, remove impurity, obtain Garcinia mangostana cell strain.
Garcinia mangostana cell mass propgation：
Add I sterilising mediums that Garcinia mangostana cell strain is configured to 1～5 × 105The Garcinia mangostana Cell sap of/ml density, takes
100ml Garcinia mangostana Cell sap is added in the aseptic conical flasks of 250ml, on rotary shaker, with 100-200rpm/min, 25-26
DEG C, cultivate 14 days.
After preliminary culture, 250g centrifugation 10min remove supernatant, add No. II sterilising medium to match somebody with somebody Garcinia mangostana cell
Make 1-3 × 106The Garcinia mangostana Cell sap of/ml density is placed in fermentation tank carries out suspension culture, 130-140rpm/min, 25-26
DEG C, air filtering was passed through, speed is 0.01-0.05m3/ s, cultivates 14 days.
The formula (in 1L MS culture medium) of 3 I culture medium of table
| Title | Content | Title | Content |
| 2,4-D | 1.0mg/L | 6-BA | 1.5mg/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The formula (in 1L MS culture medium) of 4 No. II sterilising mediums of table
| Title | Content | Title | Content |
| 2,4-D | 1.5mg/L | 6-BA | 1.0mg/L |
| Glucose | 1.0g/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The preparation of Garcinia mangostana cell secondary metabolitess lyophilized powder：
Garcinia mangostana cell culture is collected, which is filtered with 0.45 μM of filter membrane, concentrated, the concentration supernatant for obtaining is used
0.22 μM of filtration sterilization.
Frozen dried is carried out after degerming, freeze temperature is -35 DEG C, vacuum is 200Pa, and freeze-drying time is 36 hours, i.e.,
Obtain the secondary metabolitess lyophilized powder of Garcinia mangostana cell.Lyophilized powder is distributed into 1ml/.
Embodiment 3
Obtain Garcinia mangostana cell strain：
Taking the fresh Garcinia mangostana of 1kg carries out cleaning and sterilizing, is immersed in 20 minutes in 10L 15%-20% sodium hypochlorite.Remove time chlorine
Acid sodium solution, aseptically, is cleaned using 2L sterile deionized water every time, totally three times, is gently stirred in each cleaning process
Dynamic.
Under sterile conditions, remove the peel of Carcinia mangostana L. after sterilization, with sterilizing after shears shred, every chip size≤
1mm2.
Peel of Carcinia mangostana L. fragment is transferred in the beaker of sterile clean, 10ml MS (Murashige is added per 1.0g fragments
And Skoog) culture medium, 0.05% cellulase and 0.10% pectase, digest 3 hours.The enzyme activity of cellulase is
30000U/mg, the enzyme activity of pectase is 50000U/mg.
Digestive system is filtered with 200 mesh aseptic mesh screen, remove impurity, obtain Garcinia mangostana cell strain.
Garcinia mangostana cell mass propgation：
Add I sterilising mediums that Garcinia mangostana cell strain is configured to 1～5 × 105The Garcinia mangostana Cell sap of/ml density, takes
100ml Garcinia mangostana Cell sap is added in the aseptic conical flasks of 250ml, on rotary shaker, with 100-200rpm/min, 25-26
DEG C, cultivate 14 days.
After preliminary culture, 250g centrifugation 10min remove supernatant, add No. II sterilising medium to match somebody with somebody Garcinia mangostana cell
Make 1-3 × 106The Garcinia mangostana Cell sap of/ml density is placed in fermentation tank carries out suspension culture, 130-140rpm/min, 25-26
DEG C, air filtering was passed through, speed is 0.01-0.05m3/ s, cultivates 14 days.
The formula (in 1L MS culture medium) of 5 I culture medium of table
| Title | Content | Title | Content |
| 2,4-D | 0.6mg/L | 6-BA | 2.2mg/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The formula (in 1L MS culture medium) of 6 No. II sterilising mediums of table
| Title | Content | Title | Content |
| 2,4-D | 2.0mg/L | 6-BA | 0.5mg/L |
| Glucose | 5.0g/L |
Note：2,4-D is auxin, and 6-BA is the basic element of cell division
The preparation of Garcinia mangostana cell secondary metabolitess lyophilized powder：
Garcinia mangostana cell culture is collected, which is filtered with 0.45 μM of filter membrane, concentrated, the concentration supernatant for obtaining is used
0.22 μM of filtration sterilization.
Frozen dried is carried out after degerming, freeze temperature is -20 DEG C, vacuum is 120Pa, and freeze-drying time is 24 hours, i.e.,
Obtain the secondary metabolitess lyophilized powder of Garcinia mangostana cell.Lyophilized powder is distributed into 1ml/.
The measure of total xanthone content in 4 secondary metabolitess lyophilized powder of embodiment
The preparation of standard curve：
Precision measures 0,5,10,15,20,25,30 μ l of α-mangostin reference substance solution, total xanthone concentration point
Not Wei 0,10,15,20,25,30 μ g/ml, be respectively placed in 10ml tool plug test tubes, plus methanol is to 1.0ml, shakes up, and with methanol is
Blank.Respectively plus 0.25ml5% sodium nitrite solutions, shake up, room temperature places 9min.Add 10% aluminum nitrates of 0.35ml molten
Liquid, shakes up, and room temperature places 6min.3.0ml4% sodium hydroxide solutions are added, plus methanol supplies volume to 5ml, shakes up, room temperature
15min is placed, mensuration absorbance at 370nm wavelength makees standard curve with the amount of α-mangostin to absorbance.
The extraction of total xanthone and measure：
1～sample sets of sample sets 3：The lyophilized powder prepared using 1～embodiment of embodiment 3 is dissolved into liquid.
Sample powder 0.25g is taken, accurately weighed, it is placed in tool plug test tube, adds 5ml citric acid-sodium citrate buffer solutions
With the enzyme of certain mass fraction, water-bath certain time under uniform temperature and pH, filtration, enzyme liquid is discarded, then is added in medicinal residues
Liquid-solid ratio is 90% ethanol of 1: 17 (g/ml), after supersound extraction 74min, filters, and is transferred in 10ml measuring bottles, plus ethanol is extremely
Scale, shakes up, filtration, takes subsequent filtrate as need testing solution.Reference substance solution and need testing solution each 20 μ l are drawn respectively, with
Shi Xianse, mensuration absorbance.The results are shown in Table 7.
Table 7
| Group | Total xanthone content (μ g/ml) |
| Sample sets 1 (prepared by embodiment 1) | 31.2 |
| Sample sets 2 (prepared by embodiment 2) | 30.7 |
| Sample sets 3 (prepared by embodiment 3) | 30.4 |
Conclusion：The culture plant cell method of this patent can successfully extract abundant total xanthone material, and not
With the content between batch without significant difference (P<0.05), it was demonstrated that method is stable.
Impact of the 5 Garcinia mangostana secondary metabolitess lyophilized powder of embodiment to MDA growing amounts in the homogenate of rat skin histology
After free radical is formed, its metabolite malonaldehyde (MDA) content substantially increases, and MDA is as lively as a cricket cross-linking agent, it
Can make dermis that macromolecules cross-linking occurs, increase corium fabric distortion thick disorderly, make the skin day by day aging in appearance of people,
The height of MDA contents can characterize anti peroxidation of lipid (the i.e. anti-MDA is generated) activity of material indirectly, and MDA contents are less, show thing
The anti peroxidation of lipid ability of matter is stronger；Conversely, then anti peroxidation of lipid ability is weaker.
40 rat are divided into 4 groups, 10 per group, its back are gone to by hair, exposed area about 6cm.,
The Garcinia mangostana cell secondary metabolitess lyophilized powder prepared with a batch of embodiment 1 is taken, will be matched with which before smearing
2ml lyases are poured in lyophilized powder completely, fully mix, 2ml mixture is all equably applied to the exposed back of mice, per
Smear once within two days.
First group, put to death after the Garcinia mangostana cell secondary metabolitess lyophilized powder 5 times for continuously smearing the preparation of embodiment 1, take back
Skin histology is homogenized, and is made as 10% homogenate.Second group, continuously smear the Garcinia mangostana cell secondary metabolitess of the preparation of embodiment 1
Put to death after lyophilized powder 10 times, take back skin tissues homogenate, be made as 10% homogenate.3rd group, continuously smear embodiment 1 and make
Put to death after standby Garcinia mangostana cell secondary metabolitess lyophilized powder 15 times, take back skin tissues homogenate, be made as 10% homogenate.The
Four groups of Garcinia mangostana cell secondary metabolitess lyophilized powders for not smearing the preparation of embodiment 1, put to death after 30 days, take back skin tissues even
Slurry, is made as 10% homogenate.
Standard pipe take 0.2ml10mol/ml standard substance, standard blank tube take 0.2ml dehydrated alcohol, determine pipe take 0.2ml survey
Test agent, then three pipes addition 0.2ml reagents one, after mixing, add 3ml reagents two and 1ml reagents three.
Swirl mixing device is mixed, and test tube mouth tightened with antistaling film, pierces an aperture, 95 DEG C of water-baths 40 minutes, is flowed after taking-up
Water cooling, is centrifuged 10 minutes by then 3500 revs/min.Supernatant is taken, at 532mm, 1cm optical paths, distilled water return to zero, and colorimetric surveys each pipe
Absorbance.The results are shown in Table 8.
Table 8
Note：* data are shown between matched group there were significant differences (P<0.05)
Conclusion：Prove that the total xanthone that this patent is extracted has obvious antioxidation (P<0.05), with use
The increase of natural law, effect are obviously improved.
Garcinia mangostana cell secondary metabolitess lyophilized powder prepared by Example 2, embodiment 3 carries out above-mentioned experiment, experimental result
The experimental result of the Garcinia mangostana cell secondary metabolitess lyophilized powder prepared with embodiment 1 is close, with which without significant difference (P ＞
0.05).
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of culture medium, it is characterised in that including 2,4-D, 6-BA and basal medium；
Concentration of the 2,4-D in the culture medium is 0.2～2.0mg/L；Concentration of the 6-BA in the culture medium
For 0.5～3.0mg/L.
2. culture medium according to claim 1, it is characterised in that also include glucose.
3. culture medium according to claim 2, it is characterised in that concentration of the glucose in the culture medium is
1.0～5.0g/L.
4. the culture medium according to any one of claims 1 to 3 prepares the application in secondary metabolitess in culture Garcinia mangostana cell.
5. a kind of preparation method of Garcinia mangostana cell secondary metabolitess, it is characterised in that comprise the steps：
Step 1：Obtain Garcinia mangostana cell；
Step 2：Take step 1 acquisition Garcinia mangostana cell mix with culture medium as claimed in claim 1 after tentatively cultivate, then with such as
Amplification culture after culture medium mixing described in claim 2 or claim 3, collects culture fluid.
6. preparation method according to claim 5, it is characterised in that the preparation method of Garcinia mangostana cell described in step 1 is
Take, filter, collect filtrate.
7. preparation method according to claim 6, it is characterised in that the addition of the cellulase is the Carcinia mangostana L.
The 0.01～0.10% of cortex amount, the addition of the pectase are the 0.01～0.10% of the peel of Carcinia mangostana L. quality；
The enzyme activity of the cellulase is 30000U/mg, and the enzyme activity of the pectase is 50000U/mg.
8. Garcinia mangostana cell secondary metabolitess obtained in the preparation method according to any one of claim 5 to 7.
9. Garcinia mangostana cell secondary metabolitess according to claim 8 are preparing medicine, food and/or the cosmetics of defying age
In application.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104221859A (en) * | 2014-09-01 | 2014-12-24 | 中国热带农业科学院海口实验站 | Rapid propagation and cultivation method of mangosteen |
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2016
- 2016-11-04 CN CN201610963845.XA patent/CN106497860A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104221859A (en) * | 2014-09-01 | 2014-12-24 | 中国热带农业科学院海口实验站 | Rapid propagation and cultivation method of mangosteen |
Non-Patent Citations (3)
| Title |
|---|
| TRAN ET AL: "Application of Tissue Culture Techniques in Woody Species Conservation, Improvement and Development in Vietnam: Mangosteen (Garcinia mangostana L.) via Embryogenesis Culture", 《PROC. IIND IS ON BIOTECH. OF TROP & SUBTROP. SPECIES》 * |
| 傅术琳等: "木立芦荟愈伤组织的诱导及快速繁殖", 《植物生理学通讯》 * |
| 李干龙: "山竹果皮化学成分及其抗菌机理研究", 《万方数据库》 * |
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