CN117256457A - Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots - Google Patents
Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots Download PDFInfo
- Publication number
- CN117256457A CN117256457A CN202310987861.2A CN202310987861A CN117256457A CN 117256457 A CN117256457 A CN 117256457A CN 202310987861 A CN202310987861 A CN 202310987861A CN 117256457 A CN117256457 A CN 117256457A
- Authority
- CN
- China
- Prior art keywords
- rosewood
- cedrene
- cedrol
- culture
- roots
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- DYLPEFGBWGEFBB-OSFYFWSMSA-N (+)-β-cedrene Chemical compound C1[C@]23[C@H](C)CC[C@H]3C(C)(C)[C@@H]1C(=C)CC2 DYLPEFGBWGEFBB-OSFYFWSMSA-N 0.000 title claims abstract description 120
- 235000009984 Pterocarpus indicus Nutrition 0.000 title claims abstract description 85
- DYLPEFGBWGEFBB-UHFFFAOYSA-N beta-Cedren Natural products C1C23C(C)CCC3C(C)(C)C1C(=C)CC2 DYLPEFGBWGEFBB-UHFFFAOYSA-N 0.000 title claims abstract description 60
- SVURIXNDRWRAFU-OGMFBOKVSA-N cedrol Chemical compound C1[C@]23[C@H](C)CC[C@H]3C(C)(C)[C@@H]1[C@@](O)(C)CC2 SVURIXNDRWRAFU-OGMFBOKVSA-N 0.000 title claims abstract description 49
- 229940026455 cedrol Drugs 0.000 title claims abstract description 49
- SVURIXNDRWRAFU-UHFFFAOYSA-N juniperanol Natural products C1C23C(C)CCC3C(C)(C)C1C(O)(C)CC2 SVURIXNDRWRAFU-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 229930004725 sesquiterpene Natural products 0.000 title claims abstract description 29
- 150000004354 sesquiterpene derivatives Chemical class 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 24
- 241000533793 Tipuana tipu Species 0.000 title claims abstract 16
- 239000000443 aerosol Substances 0.000 claims abstract description 32
- 241000219195 Arabidopsis thaliana Species 0.000 claims abstract description 24
- 230000012010 growth Effects 0.000 claims abstract description 24
- 230000006698 induction Effects 0.000 claims abstract description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 15
- 239000003814 drug Substances 0.000 claims abstract description 15
- 201000005202 lung cancer Diseases 0.000 claims abstract description 15
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 15
- 230000001737 promoting effect Effects 0.000 claims abstract description 9
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 7
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 54
- 235000015097 nutrients Nutrition 0.000 claims description 50
- 230000000694 effects Effects 0.000 claims description 34
- 238000011282 treatment Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- UDPGUMQDCGORJQ-UHFFFAOYSA-N (2-chloroethyl)phosphonic acid Chemical compound OP(O)(=O)CCCl UDPGUMQDCGORJQ-UHFFFAOYSA-N 0.000 claims description 14
- 239000005976 Ethephon Substances 0.000 claims description 14
- 238000002386 leaching Methods 0.000 claims description 12
- 230000001965 increasing effect Effects 0.000 claims description 10
- 238000005520 cutting process Methods 0.000 claims description 8
- 238000001819 mass spectrum Methods 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 238000007598 dipping method Methods 0.000 claims description 6
- 235000016709 nutrition Nutrition 0.000 claims description 6
- 230000035764 nutrition Effects 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000010606 normalization Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- PCROEXHGMUJCDB-UHFFFAOYSA-N cedrol Natural products CC1CCC2C(C)(C)C3CC(C)(O)CC12C3 PCROEXHGMUJCDB-UHFFFAOYSA-N 0.000 abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 244000086363 Pterocarpus indicus Species 0.000 description 69
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 21
- 230000035882 stress Effects 0.000 description 18
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 17
- 241000196324 Embryophyta Species 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 14
- 239000007789 gas Substances 0.000 description 14
- 239000005556 hormone Substances 0.000 description 10
- 229940088597 hormone Drugs 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 9
- 238000011161 development Methods 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 241000218691 Cupressaceae Species 0.000 description 8
- 241000522195 Dalbergia Species 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 8
- 239000002023 wood Substances 0.000 description 8
- 235000017060 Arachis glabrata Nutrition 0.000 description 6
- 244000105624 Arachis hypogaea Species 0.000 description 6
- 235000010777 Arachis hypogaea Nutrition 0.000 description 6
- 235000018262 Arachis monticola Nutrition 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 235000020232 peanut Nutrition 0.000 description 6
- 229910052698 phosphorus Inorganic materials 0.000 description 6
- 239000011574 phosphorus Substances 0.000 description 6
- 241000219194 Arabidopsis Species 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- CXENHBSYCFFKJS-UHFFFAOYSA-N (3E,6E)-3,7,11-Trimethyl-1,3,6,10-dodecatetraene Natural products CC(C)=CCCC(C)=CCC=C(C)C=C CXENHBSYCFFKJS-UHFFFAOYSA-N 0.000 description 3
- JSNRRGGBADWTMC-UHFFFAOYSA-N (6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene Chemical compound CC(C)=CCCC(C)=CCCC(=C)C=C JSNRRGGBADWTMC-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 150000001336 alkenes Chemical class 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000003337 fertilizer Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000002786 root growth Effects 0.000 description 3
- 229930000044 secondary metabolite Natural products 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000000341 volatile oil Substances 0.000 description 3
- RGZSQWQPBWRIAQ-LSDHHAIUSA-N (+)-alpha-Bisabolol Chemical compound CC(C)=CCC[C@@](C)(O)[C@@H]1CCC(C)=CC1 RGZSQWQPBWRIAQ-LSDHHAIUSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- OSVXSBDYLRYLIG-UHFFFAOYSA-N dioxidochlorine(.) Chemical compound O=Cl=O OSVXSBDYLRYLIG-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000026267 regulation of growth Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- FQTLCLSUCSAZDY-UHFFFAOYSA-N (+) E(S) nerolidol Natural products CC(C)=CCCC(C)=CCCC(C)(O)C=C FQTLCLSUCSAZDY-UHFFFAOYSA-N 0.000 description 1
- 229960003164 (+)- alpha-bisabolol Drugs 0.000 description 1
- RGZSQWQPBWRIAQ-CABCVRRESA-N (+)-alpha-Bisabolol Natural products CC(C)=CCC[C@](C)(O)[C@H]1CCC(C)=CC1 RGZSQWQPBWRIAQ-CABCVRRESA-N 0.000 description 1
- MIHSPYMZXYONJN-RMKNXTFCSA-N (2e)-2,4-dimethylhepta-2,6-dienal Chemical compound C=CCC(C)\C=C(/C)C=O MIHSPYMZXYONJN-RMKNXTFCSA-N 0.000 description 1
- FQTLCLSUCSAZDY-SDNWHVSQSA-N (6E)-nerolidol Chemical compound CC(C)=CCC\C(C)=C\CCC(C)(O)C=C FQTLCLSUCSAZDY-SDNWHVSQSA-N 0.000 description 1
- FQTLCLSUCSAZDY-SZGZABIGSA-N (E)-Nerolidol Natural products CC(C)=CCC\C(C)=C/CC[C@@](C)(O)C=C FQTLCLSUCSAZDY-SZGZABIGSA-N 0.000 description 1
- JSNRRGGBADWTMC-QINSGFPZSA-N (E)-beta-Farnesene Natural products CC(C)=CCC\C(C)=C/CCC(=C)C=C JSNRRGGBADWTMC-QINSGFPZSA-N 0.000 description 1
- CXENHBSYCFFKJS-OXYODPPFSA-N (Z,E)-alpha-farnesene Chemical compound CC(C)=CCC\C(C)=C\C\C=C(\C)C=C CXENHBSYCFFKJS-OXYODPPFSA-N 0.000 description 1
- XBGUIVFBMBVUEG-UHFFFAOYSA-N 1-methyl-4-(1,5-dimethyl-4-hexenylidene)-1-cyclohexene Chemical compound CC(C)=CCCC(C)=C1CCC(C)=CC1 XBGUIVFBMBVUEG-UHFFFAOYSA-N 0.000 description 1
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- JXBSHSBNOVLGHF-UHFFFAOYSA-N 10-cis-Dihydrofarnesen Natural products CC=C(C)CCC=C(C)CCC=C(C)C JXBSHSBNOVLGHF-UHFFFAOYSA-N 0.000 description 1
- 125000006022 2-methyl-2-propenyl group Chemical group 0.000 description 1
- 101100438273 Arabidopsis thaliana CAN1 gene Proteins 0.000 description 1
- 239000004155 Chlorine dioxide Substances 0.000 description 1
- 240000007311 Commiphora myrrha Species 0.000 description 1
- 235000006965 Commiphora myrrha Nutrition 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000657528 Dalbergia odorifera Species 0.000 description 1
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 235000007265 Myrrhis odorata Nutrition 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- YHBUQBJHSRGZNF-HNNXBMFYSA-N alpha-bisabolene Natural products CC(C)=CCC=C(C)[C@@H]1CCC(C)=CC1 YHBUQBJHSRGZNF-HNNXBMFYSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229930003493 bisabolene Natural products 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019398 chlorine dioxide Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000035613 defoliation Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000011419 induction treatment Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
- 238000010150 least significant difference test Methods 0.000 description 1
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 description 1
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 description 1
- 235000009498 luteolin Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- -1 sigma) Chemical compound 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- ZLWGOLLBNDIBMM-UHFFFAOYSA-N trans-nerolidol Natural products CC(C)C(=C)C(O)CCC=C(/C)CCC=C(C)C ZLWGOLLBNDIBMM-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- 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
- A61K31/01—Hydrocarbons
- A61K31/015—Hydrocarbons carbocyclic
-
- 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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/005—Processes comprising at least two steps in series
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/93—Spiro compounds
- C07C2603/95—Spiro compounds containing "not free" spiro atoms
- C07C2603/96—Spiro compounds containing "not free" spiro atoms containing at least one ring with less than six members
- C07C2603/97—Spiro compounds containing "not free" spiro atoms containing at least one ring with less than six members containing five-membered rings
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Dentistry (AREA)
- Botany (AREA)
- Forests & Forestry (AREA)
- Agronomy & Crop Science (AREA)
- Ecology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood gas roots, which comprises the following steps: (S1) establishing a rosewood aerosol culture system; (S2) stress induction of rosewood gas roots to generate sesquiterpenes; (S3) extracting rosewood gas root sesquiterpenes by using ethyl acetate, and identifying by using GC-MS. The invention also discloses application of the beta-cedrene or the cedrol in preparing a reagent for promoting the growth of arabidopsis thaliana, and application of the beta-cedrene or the cedrol in preparing a medicament for inhibiting lung cancer cells.
Description
Technical Field
The invention belongs to the technical field of rosewood, and particularly relates to a method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots.
Background
Lignum Dalbergiae Odoriferae (Dalbergia odorifera T. Chen) is also called Hainan yellow pear, and is rare rosewood with medicinal and economic values. At present, wild resources of the dalbergia wood are scarce, the dalbergia wood is slow, the growing of the seedling planting industry can only primarily solve the problem of the depletion of the dalbergia wood resources, and the development of downstream industries such as medicines, spices, literaries and the like is not sufficiently promoted. Therefore, shortening the cultivation period of the dalbergia wood, and digging new medicinal parts and medicinal components is very important for the deep development of dalbergia wood resources.
Modern agricultural technology and biotechnology are introduced into the traditional Chinese medicine planting industry, so that the method is an effective way for traditional resource protection and new resource development, and a good material system can be provided for subsequent basic research. The aeroponic cultivation is a modern agricultural cultivation technology and has the advantages of being beneficial to the growth and development of root organs and short in cultivation period. At present, the aerosol cultivation industry in China is mostly concentrated on vegetable planting, and few reports are reported on the cultivation of woody medicinal plants, so that aerosol cultivation is cited to the cultivation of rosewood roots, and the method is a beneficial attempt for the development of new resources of the rosewood. In addition, the secondary metabolite in the plant root has various biological activities, is a great way for developing new traditional Chinese medicines, is used as a basic plant of traditional Chinese medicine dalbergia wood, is worth focusing on the composition, activity and synthesis mechanism of the secondary metabolite in the dalbergia wood seedling root, and provides a certain reference for understanding the drug effect components of the dalbergia wood and expanding the medicinal parts.
At present, the researches on the chemical components of the rosewood are concentrated on heartwood, and the related researches show that the rosewood is rich in volatile oil and flavonoid components, the main component of the volatile oil is trans-nerolidol, and the volatile oil also contains (E) -beta-farnesene, (3Z, 6E) -alpha-farnesene, myrrh alkene, huang Tansu, bisabolene, alpha-farnesene, dihydro-3- (2-methyl-2-propenyl) -2,5-furandione7- (2, 6-dimethyl-hepta-1, 5-diene) -3,8,8-trimethyl-bicyclo [4.2.0] oct-2-en luteolin, 2, 4-dimethyl-2, 6-heptadienal, (+ -) -alpha-bisabolol and other compounds. Of note, little research is currently done on the chemical composition of the roots of rosewood seedlings, which is to be further supplemented.
Therefore, the invention aims at taking the aerial root of the rosewood seedlings as an object, discussing the application prospect of aerial fog cultivation in the aspect of rosewood resource development, and providing reference information for knowing the medicinal components of the rosewood.
Disclosure of Invention
The invention aims to provide a method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots.
The invention also aims to provide the application of the beta-cedrene or the cedrol in preparing an agent for promoting the growth of arabidopsis thaliana and the application of the beta-cedrene or the cedrol in preparing a medicament for inhibiting lung cancer cells.
The first object of the present invention can be achieved by the following technical means: a method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots, comprising the following steps:
(S1) selecting annual rosewood soil culture seedlings as transplanting seedlings, cutting off all lateral roots and part of main roots, cleaning after disinfection, dipping the roots with IBA+NAA, transferring into an aerosol culture system for culture, wherein pure water rooting induction culture is adopted in the early stage, and nutrient solution is used in the later stage for nutrient culture;
(S2) adding ethephon solution into the aeroponic nutrient solution, and subjecting the aeroponic seedlings subjected to nutrition culture to stress treatment;
(S3) selecting side roots of the rosewood aerosol seedlings subjected to stress treatment, freeze-drying, grinding into powder, adding ethyl acetate, performing vortex leaching for a plurality of times, centrifuging to obtain supernatant, and concentrating to obtain a sample to be tested;
(S4) detecting the sample to be detected by adopting GC-MS, and identifying to obtain the volatile substances containing beta-cedrene and cedrol.
In the above method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots:
optionally, the disinfection in the step (S1) adopts KMnO with the mass percentage of 0.08 to 0.12 percent 4 And (5) sterilizing the solution for 55-65 s.
More preferably, in step (S1) the disinfection is performed using KMnO in an amount of 0.10% by mass 4 The solution was sterilized for 60s.
Optionally, in the step (S1), IBA+NAA with the concentration of 0.8-1.2 g/L is used for dipping roots for 8-12S, wherein the mass ratio of IBA to NAA is 1:1.
more preferably, in the step (S1), IBA+NAA with the concentration of 1.0g/L is used for dipping roots for 10 seconds, wherein the mass ratio of IBA to NAA is 1:1.
optionally, transferring the culture medium into an aerosol culture system for culturing for 28-32 days in the step (S1), wherein pure water rooting induction culture is adopted for the first 6-8 days, and nutrient solution is adopted for the rest days in the later period for nutrition culture.
More preferably, in the step (S1), the culture is carried out by transferring into an aerial fog culture system for 30 days, wherein pure water rooting induction culture is adopted in the first 7 days, and nutrient solution is adopted in the later 23 days for nutrient culture.
Optionally, the nutrient solution in the step (S1) is a Huaduo 15-grade nutrient solution, and the concentration of the nutrient solution is 0.4-0.6 g/L.
More preferably, the nutrient solution in the step (S1) is a Huaduo No. 15 nutrient solution, and the concentration of the nutrient solution is 0.5g/L.
Optionally, in the step (S2), the final concentration of the ethephon solution added into the aeroponic nutrient solution is 4-6wt%, and the time for stress treatment of the aeroponic seedlings after nutrient culture is 13-16 days.
More preferably, in the step (S2), the final concentration of the ethephon solution added into the aeroponic nutrient solution is 5wt%, and the time for stress treatment of the aeroponic seedlings after nutrient culture is 13-16 days.
The experimental result shows that 5wt% of ethephon can induce the accumulation of volatile components in rosewood gas roots.
Optionally, in the step (S2), the usage amount relationship between the side root of the rosewood aerosol seedling and the ethyl acetate is as follows: 1g: 4-6 mL, the leaching temperature is 3-5 ℃, the leaching time is 22-26 h each time, and the leaching times are 2-4 times.
More preferably, in the step (S2), the usage amount relationship between the side root of the rosewood aerosol seedling and the ethyl acetate is: 1g:5mL, the leaching temperature is 4 ℃, each leaching time is 24 hours, and the leaching times are 3 times.
Optionally, the GC-MS detection conditions in step (S3) are: the column HP-5ms,30 m.times.0.25 mm.times.0.25. Mu.m; the initial temperature is 35 ℃, the temperature is kept for 5min, the temperature is increased to 300 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample injection amount is 1 mu L; the temperature of the sample inlet is 250 ℃; the temperature of the quadrupole rods is 150 ℃; the ion source temperature is 230 ℃; ionization mode EI; electron energy 70eV; the carrier gas is He, the purity is 99.99 percent, and the flow rate is 1mL/min; the mass scanning range is 35-450 AMU; delaying the solvent for 3min, searching and comparing the obtained spectrum and mass spectrum data with a Willy and NIST standard mass spectrum library, qualifying each component, and calculating the relative content of each component by an area normalization method.
As a preferred embodiment of the invention, the invention establishes a complete rosewood aerosol culture system through screening culture conditions: selecting annual lignum Dalbergiae Odoriferae soil culture seedling as transplanting seedling, cutting off all lateral root and part of main root, and using 0.1% KMnO 4 The solution is sterilized for 1min, washed clean by pure water, then dipped with IBA+NAA (equal mass ratio) of 1.0g/L for 10s, and transferred into an aerosol culture system for culture, wherein the culture period is 30 days, the first 7 days are pure water rooting induction culture, and the last 23 days are nutrient culture with a flower bud of 0.5g/L and a flower bud of 15 #. The culture system can collect enough high-quality rosewood aerosol roots in a short culture period, and is an ideal culture system.
Wherein the aerosol culture system can be a conventional aerosol culture system disclosed in the prior art, preferably an aerosol culture system invented earlier by the inventor of the present application, with the application number of CN202120640461.0 and the like.
The second object of the present invention can be achieved by the following technical means: use of beta-cedrene or cedrol in the preparation of a medicament having the effect of promoting the growth of arabidopsis thaliana.
Alternatively, the concentration of beta-cedrene and cedrol is lower than 5. Mu. Mol/L, more preferably 5. Mu. Mol/L.
The experiment proves that the growth promotion activity of the beta-cedrene and the cedrol is proved, and the result shows that the beta-cedrene and the cedrol with the concentration of 5 mu mol/L can promote the growth of arabidopsis thaliana, and the method is particularly characterized in that the total biomass is increased, the number of lateral roots is increased, and the high-concentration beta-cedrene does not inhibit the growth of arabidopsis thaliana, but plays a promoting role, and can be used as a growth regulator.
The invention further provides application of beta-cedrene or cedrol in preparing medicines with an inhibitory effect on lung cancer cells.
Optionally, the concentration of beta-cedrene and cedrol is greater than 200. Mu. Mol/L, more preferably 400-600. Mu. Mol/L.
In the aspect of anti-tumor, the invention selects lung cancer strains H1299 and A549 for examination, and experimental results show that high-concentration (more than 200 mu mol/L, more preferably 400-600 mu mol/L) beta-cedrene and cedrol have obvious inhibition effect on lung cancer cells, and the inhibition effect increases with the increase of the administration time.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, a rosewood aerosol culture system is established through screening of hormone induction conditions and nutrient solution culture conditions;
(2) The invention also discusses the feasibility of stress induction of the rosewood gas root to generate volatile components, and the result shows that 5wt% of ethephon can obviously promote the accumulation of sesquiterpene components in the rosewood gas root;
(3) The invention further verifies the biological activity of the beta-cedrene and the cedrol, and the experimental result shows that the low-concentration beta-cedrene and the cedrol have the effect of promoting the growth of arabidopsis thaliana, and the proliferation of lung cancer cells can be inhibited by high-dose administration;
(4) Therefore, the invention obtains a large amount of high-quality aerial roots in a short culture period by establishing an aerial fog culture system of the rosewood, carries out stress treatment on the aerial roots of the rosewood, primarily explores the influence of ethephon on volatile components of the aerial roots, and verifies the biological activity of sesquiterpenes beta-cedrene and cedarwood alcohol serving as main volatile components.
Drawings
The invention is further illustrated by the following figures.
FIG. 1 is a pre-treatment of rosewood aerosol cultivation transplanting in example 1;
FIG. 2 shows rooting of rosewood under different hormone induction in example 1;
FIG. 3 shows rooting of rosewood in different kinds of nutrient solutions in example 1;
FIG. 4 shows rooting conditions of lignum Dalbergiae Odoriferae in example 1 under different concentrations of nutrient solutions;
FIG. 5 is a rooting profile of side roots of rosewood seedlings at 10, 13, 16, 19 and 25 days using 5wt% ethephon stress in example 1;
FIG. 6 is a graph showing total ion superposition for each sample in example 1;
FIG. 7 is a graph showing the time variation of total sesquiterpenes content in rosewood aerial roots in example 1;
FIG. 8 is a graph showing the effect of β -cedrene on the growth of Arabidopsis thaliana in example 2, wherein (A) is a graph showing the root-promoting effect of β -cedrene, (B) is a graph showing the main root length statistics of Arabidopsis thaliana, (C) is a graph showing the number of side roots of Arabidopsis thaliana, and (D) is a graph showing the total biomass statistics of Arabidopsis thaliana;
FIG. 9 is a graph showing the effect of cypress alcohol on the growth of Arabidopsis thaliana in example 2, wherein (A) is a graph showing the effect of beta-cypress alkene on root promotion, (B) is a graph showing the main root length statistics of Arabidopsis thaliana, (C) is a graph showing the number statistics of side roots of Arabidopsis thaliana, and (D) is a graph showing the total biomass statistics of Arabidopsis thaliana;
FIG. 10 shows the anti-lung cancer activity of β -cedrene and cypress wood alcohol in example 2, wherein (A) shows the activity of β -cedrene in the group of H1299 cells, (B) shows the activity of β -cedrene in the group of A549 cells, (C) shows the activity of cypress wood alcohol in the group of H1299 cells, and (D) shows the activity of cypress wood alcohol in the group of A549 cells.
Detailed Description
The raw materials or reagents used hereinafter, unless otherwise indicated, are commercially available or are obtained by conventional methods.
Example 1
The following materials and equipment are preferred but not limited to:
the annual seedlings of rosewood are purchased from Guangzhou forest seedling fields.
Water mist cultivation machine see application number: CN202120640461.0.
Potassium permanganate, indolebutyric acid (IBA, sigma), naphthylacetic acid (NAA, sigma), flower 1 (flower brand store with many-in-the-language), flower 10 (flower brand store with many-in-the-language), flower 15 (spring gardening).
1. Aerosol box and pretreatment of seedlings before transplanting
The chlorine dioxide disinfectant is used for immersing and disinfecting the field planting cup and the field planting cotton, spraying and disinfecting the tank body of the aerosol tank and the circulating system, and then cleaning the tank body by pure water so as to ensure that the interior of the aerosol tank is in a sterile state as much as possible. Good selective growth stateCutting lignum Dalbergiae Odoriferae seedling, adjusting plant height, soaking in clear water, cleaning the root system of the seedling, cutting off excessive lateral root and partial main root, and treating with 0.1% KMnO 4 The solution was sterilized for 1min for subsequent experimental treatment.
The aerosol culture transplanting pretreatment of lignum Dalbergiae Odoriferae is shown in figure 1.
2. Induction of rosewood gas root
The experiment designs 3 hormone types (IBA, NAA, IBA +NAA and other mass proportion compounding), 9 induction treatment groups are 3 concentration gradients (0.5 g/L, 1g/L and 1.5 g/L), and 1 blank control group is used as an induction scheme of aerial roots.
Specifically, hormone root dipping treatment is carried out on the sterilized rosewood seedlings according to experimental groups, the treatment time is 10s, then the rosewood seedlings are transferred into an aerosol culture system for rooting culture, and the rooting rate (/%) and the rooting number (/ strip) are counted after 20 days.
The rooting condition of rosewood under different hormone induction is shown in figure 2, in each hormone treatment group, the induction effect of an IBA+NAA equal proportion compound group is the best, the IBA group is inferior, the NAA group is slightly inferior, the optimal induction scheme is 1.0g/L IBA+NAA, and the rooting number of single aerial fog seedlings is 87.5 (75.3-96.3) pieces, as shown in table 1.
TABLE 1 hormone-induced rooting statistics (M (P25-P75))
3. Nutrient solution condition screening for rosewood gas rooting
The screening of nutrient solution conditions mainly relates to the consideration of nutrient element proportion and nutrient solution concentration. Firstly, in order to explore the influence of nutrient element proportion on growth of rosewood gas roots, a rosewood gas spray seedling is cultivated for 30 days by taking a rosewood general fertilizer No. 1, a rosewood high nitrogen fertilizer No. 10 and a rosewood high phosphorus fertilizer No. 15 as a research object, wherein pure water cultivation is carried out for the first 7 days, different types of nutrient solution cultivation is carried out for the last 23 days, and the rooting condition of the rosewood under the different types of nutrient solution cultivation is shown in a figure 3.
The rooting condition of lignum Dalbergiae Odoriferae under culture of nutrient solutions with different concentrations is shown in figure 4, and the investigation index comprises underground part including total root length and root fresh weight. The experiment carries out concentration screening on the nutrient solution with the optimal growth promoting effect, three concentrations of 0.5g/L, 1.0g/L and 1.5g/L are designed, the culture period is prolonged to 40 days, the nutrient solution with low concentration (0.1 g/L) is uniformly added for culture in the first 20 days, the nutrient solution with designed concentration is added for culture in the last 20 days, and the investigation indexes comprise total root length (/ m) and fresh weight (/ g).
For the statistics of root number, total root length and fresh weight, the experiment selects a biological statistical method of single plant repetition.
The results show that the culture effect of the peanut and peanut number 15 group is obviously better than that of the peanut and peanut number 10 group and the peanut and peanut number 1 group, the fresh weight of the individual plant aerial root is 0.88 (0.71-1.10) g, and the total root length is 3.08 (2.27-5.67) m (Table 2).
TABLE 2 influence of nutrient solution types on aerial root growth (M (P25-P75))
| Number of examples | Fresh weight/g | Total root length/m | |
| Flower as many as 15 | 10 | 0.88(0.71～1.10) | 3.08(2.27～5.67) |
| Flower as many as 10 | 10 | 0.09(0.03～0.25) | 0.35(0.22～0.78) |
| Huaduoduo 1 | 10 | 0.19(0.03～0.44) | 0.69(0.20～1.09) |
| x 2 | 18.880 a | 18.614 a | |
| P | 0.000 | 0.000 |
The concentration of the nutrient solution is very important for the growth of the aerial fog seedlings, and taking phosphorus as an example, the phosphorus with too high concentration can generate toxicity to plants and inhibit the growth of the plants. In order to obtain a better culture effect, the concentration of the rosewood branches with 15 numbers is screened, the M (P25-P75) values of fresh weight and total root length are examined, and experimental results show that the fresh weight of rosewood aerial roots is 1.84 (1.36-2.56) g, the total root length is 6.41 (5.60-8.58) M under the culture condition of 0.5g/L, and the culture effect is obviously higher than that of other culture groups, so that the rosewood aerial roots are suitable for culture concentration. (Table 3).
TABLE 3 influence of nutrient solution concentration on aerial root growth (M (P25-P75))
| Number of examples | Fresh weight/g | Total root length/m | |
| 0.5g/L | 10 | 1.84(1.36～2.56) | 6.41(5.60～8.58) |
| 1.0g/L | 10 | 1.17(0.77～1.73) | 4.07(3.38～4.95) |
| 1.5g/L | 10 | 0.31(0.08～1.61) | 1.399(0.70～7.09) |
| x 2 | 8.841a | 7.946a | |
| P | 0.012 | 0.019 |
The aeroponic cultivation is a novel soilless cultivation technology, and the key links comprise the selection of transplanted seedlings, rooting induction, nutrition cultivation and the like. In the aerosol cultivation of the rosewood, annual rosewood seedlings are experimentally selected as transplanting materials, and part of main roots are reserved so as to shorten the occurrence time of the aerial roots.
In the aspect of root growth induction, experiments mainly evaluate the induction effect of IBA, NAA, IBA +NAA equal-proportion compounding, and the optimal induction scheme is IBA+NAA of 1.0 g/L.
It is worth noting that in the three induction schemes, the induction effect of NAA treatment groups is weaker, NAA is an important hormone for inducing callus to generate adventitious roots, observation is carried out on the generation process of rosewood gas roots, experiments show that the rosewood gas roots originate from the center pillar of the main root and break through the cortex to grow into the aerosol environment, and no callus participates in the process, so that the generation mode of the rosewood gas roots is presumed to be a main reason for weakening the NAA induction effect, the rooting mode of cutting slips should be fully considered when the induction hormone is selected, and if the roots are used as cutting slips, independent application of NAA or proper reduction of the consumption of NAA in the compound hormone should be avoided.
In addition, in the nutrition culture stage, the influence of different element proportions and different nutrient solution concentrations on the growth of rosewood aerial roots is mainly evaluated, and experimental results show that 0.5g/L of the rosewood multi-15-number (high-phosphorus) water-soluble fertilizer has a good culture effect. The phosphorus element is an important element participating in plant energy metabolism and nucleic acid metabolism, can promote early development of plants, and because the requirement of the plants for the phosphorus element can be changed along with the change of growth state, the dynamic adjustment of the concentration of the nutrient solution also becomes the direction of optimizing the subsequent rosewood culture conditions. It is worth noting that observation of the long-period (40 days) cultivation process of the rosewood gas root shows different degrees of bacterial contamination and aging along with the extension of the cultivation time, which is very unfavorable for the development of the subsequent experiment, so that the final cultivation period is shortened to 30 days.
In combination, the experiment is carried out by cultureThe selection of the cultivation conditions establishes a complete rosewood aerosol cultivation system, and specifically comprises the following steps of selecting annual rosewood soil cultivation seedlings as transplanting seedlings, cutting off all lateral roots and part of main roots, and using 0.1% KMnO 4 The solution is sterilized for 1min, washed clean by pure water, then dipped with IBA+NAA of 1.0g/L for 10s, and transferred into an aerosol culture system for culture, wherein the culture period is 30 days, the first 7 days are pure water rooting induction culture, and the second 23 days are nutrient culture with 0.5g/L of Huaduo 15-numbered nutrient solution. The culture system can collect enough high-quality rosewood aerosol roots in a short culture period, and is an ideal culture system.
4. Stress treatment of rosewood gas roots
Adding 40wt% ethephon solution into the aeroponic nutrient solution, adjusting the concentration to 5wt%, then applying the nutrient solution to aeroponic seedlings which have grown for 30 days for stress treatment, replacing the nutrient solution every 3 days for ensuring the ethephon concentration, sampling and chemically detecting on the 10 th, 13 th, 16 th, 19 th and 25 th days of stress, wherein each group of stress samples are mixed samples of lateral roots of 3 seedlings, and rooting conditions are shown in figure 5.
5. Sample preparation
Sampling: selecting 3 rosewood aerosol seedlings subjected to stress treatment, cleaning lateral roots, sucking water by using filter paper, rapidly cutting down, placing in a freezing tube, quick-freezing by liquid nitrogen for 10min, and placing in a-80 ℃ refrigerator for subsequent detection.
Sample preparation: grinding the low-temperature frozen sample into powder by liquid nitrogen, weighing 1.00g of powder in a 10mL centrifuge tube, adding 5mL of ethyl acetate, leaching at 4 ℃ for 24 hours, and swirling for 3 times in order to ensure the extraction efficiency. After the extraction is finished, centrifuging and sucking the supernatant, concentrating to 1mL by a nitrogen blower to obtain a sample to be tested, and repeating the experiment three times.
6. GC-MS detection conditions
Chromatographic column HP-5ms (30 m. Times.0.25 mm. Times.0.25 μm); the initial temperature is 35 ℃, the temperature is kept for 5min, the temperature is increased to 300 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample injection amount is 1 mu L; the temperature of the sample inlet is 250 ℃; the temperature of the quadrupole rods is 150 ℃; the ion source temperature is 230 ℃; ionization mode EI; electron energy 70eV; carrier gas He (99.99%) flow rate 1mL/min; the mass scanning range is 35-450 AMU; the solvent was delayed for 3min. And searching and comparing the obtained map and mass spectrum data by using a Willy standard mass spectrum library and a NIST standard mass spectrum library, qualifying each component, and calculating the relative content of each component by using an area normalization method.
GC-MS detection results show that ethephon induces accumulation of sesquiterpenes in the rosewood aerial root, and the total sesquiterpenes content in the rosewood aerial root tends to be increased and then decreased with the increase of stress time (figures 6-7). Wherein the structural formulas of the (+) -beta-cedrene and the cedrol are as follows:
comparison of sesquiterpene content at different stress times: the sesquiterpene components with the relative content of more than 0.5% in each group of samples are subjected to summarized analysis, 17 sesquiterpene substances including beta-cedrene and cedrol are experimentally screened, and the sesquiterpene substances induced by the 16-day stress treatment group are the most abundant and highest in content and are stressed for 13 days.
TABLE 4 comparison of rosewood aerial root sesquiterpene Components
The invention adopts 5wt% ethephon to carry out stress treatment and volatile component detection on the rosewood gas root so as to provide reference for the subsequent deep development of the rosewood gas root resource. Experimental results show that the ethephon stress promotes the aging of the rosewood aerosol seedlings, particularly comprises defoliation and root yellowing, and the total sesquiterpene content in the rosewood aerosol roots is changed in a trend of rising and then falling along with the increase of the stress time.
Example 2
Of the 17 sesquiterpenes obtained in example 1, two of the components β -cedrene and cedrol having a good application prospect were screened for efficacy experiments, wherein β -cedrene and cedrol can be obtained by subjecting the supernatant obtained in example 1 to column chromatography, eluting with an eluent, subjecting the sub-fractions to column chromatography, and separating after chromatography, wherein since the amounts of β -cedrene and cedrol obtained in example 1 are small, the following experiments were performed using β -cedrene and cedrol standards instead of β -cedrene and cedrol obtained in example 1:
wherein: seeds of wild arabidopsis thaliana (Arabidopsis thaliana (l.) Heynh) were donated to the university of chinese medicine He Rui teacher subject group; the H1299 and A549 cell lines were given to the university of Guangzhou traditional Chinese medicine, university Liu Yongjiang teacher subject group, beta-cedrene standard (Posty technology), cedrol standard (Michelin).
Data statistics and analysis: experimental data were initially collated using Excel 2019 and Shapiro-Wilk normal distribution test using SPSS26.0 software followed by one-way analysis of variance for data meeting normal distribution and variance alignment, multiple comparisons using LSD test, all results expressed as mean ± standard deviation. Independent samples were tested for Kruskal-Wallis H Test (Kruskal-Wallis H Test) against the comparison group which did not meet the normal distribution, and the results were expressed as M (P25 to P75). In addition, statistical graphs were drawn with GraphPad Prism 8.
2.1 pharmaceutical Co-cultivation of Arabidopsis thaliana
The effect of beta-cedrene and cedrol on the growth of arabidopsis was evaluated by drug co-cultivation. Firstly, adding beta-cedrene into a sterilized 1/2MS culture medium to prepare a dosing plate with the final concentration of 0 mu mol/L, 5 mu mol/L, 10 mu mol/L, 15 mu mol/L, 20 mu mol/L, 25 mu mol/L, 100 mu mol/L and 300 mu mol/L, soaking and sterilizing Arabidopsis seeds with 2% NaClO for 15min, washing the seeds with sterile water for 6 times to ensure no NaClO residues, finally inoculating the treated seeds onto the dosing plate, vernalizing at 4 ℃ for two days, and culturing in an illumination incubator for 15 days. After the end of the culture, the main root length (/ cm), the lateral root number (/ strip) and the total biomass (/ mg) of the Arabidopsis seedlings in each treatment group were counted. The experiment selects a biological statistical method of single plant repetition, namely, 10 most representative samples are screened in each treatment group for index statistics. The operation of cypress alcohol is the same as that of beta-cypress alkene.
2.2 Effect of beta-cedrene and cedrol on Arabidopsis growth
In the beta-cedrene treated group, the administration concentrations of 5 mu mol/L, 10 mu mol/L, 15 mu mol/L, 100 mu mol/L and 300 mu mol/L significantly promoted the occurrence of lateral roots of Arabidopsis thaliana, and increased the total biomass of the plants, wherein the optimal concentration was 5 mu mol/L, the lateral roots of Arabidopsis thaliana could reach 39.50 (27.50-51) pieces, and the total biomass could reach 14.90 (12.05-24.63) mg (FIG. 8).
In the cypress alcohol treatment group, only 5 mu mol/L of the drug concentration plays a role in promoting the growth of the arabidopsis thaliana, but the effect is not obvious, at the moment, the number of the lateral roots of the arabidopsis thaliana is 19.5 (8.75-27.25), the total biomass is 11.5 (6.70-18.33) mg, and when the cypress alcohol concentration is more than 15 mu mol/L, the growth of the main roots of the arabidopsis thaliana can be obviously inhibited; above 50. Mu. Mol/L, the occurrence of Arabidopsis lateral roots and plant total biomass were significantly inhibited (FIG. 9).
2.3 evaluation of anti-Lung cancer Activity by MTT method
The influence of beta-cedrene and cedrol on proliferation of cell lines H1299 and A549 is evaluated by adopting an MTT method, and further the anti-lung cancer activity of the two medicines is explored, wherein the experiments are divided into a blank group, a control group and a dosing group, and each group is treated 3 times, and the specific experimental operation is as follows:
two cells in logarithmic growth phase were resuspended by digestion and the suspension concentration was adjusted to 8X 10 by cell count 4 The resulting cell suspension was then plated in 96-well plates at 100. Mu.L per well, and after 24h of incubation the crude medium was changed to drug-containing medium at a concentration of 600. Mu. Mol/L, 400. Mu. Mol/L, 200. Mu. Mol/L, 100. Mu. Mol/L, 50. Mu. Mol/L, 25. Mu. Mol/L, 12.5. Mu. Mol/L, 6.25. Mu. Mol/L, 0. Mu. Mol/L for further co-incubation with co-incubation periods of 24h and 48h. After the culture period was completed, 10. Mu. LMTT solution was added to each culture well and placed in an incubator to react for 4 hours, and the concentration of MTT solution was 5mg/mL. After the reaction was completed, the medium was discarded, 100. Mu.L of DMSO was added to each culture well, and the absorbance at 490nm was dissolved with shaking and measured, followed by calculation of cell viability: cell viability = (dosing group-blank)/(control group-blank) ×100%, and each dosing was calculated using SPSSHalf Inhibition Concentration (IC) 48h after group administration 50 )。
2.4 anti-lung cancer Activity of beta-cedrene and cedrol
MTT assay results show that beta-cedrene and cypress wood alcohol with more than 200 mu mol/L, especially 400-600 mu mol/L, can significantly inhibit proliferation of lung cancer cells H1299 and A549, and the inhibition effect can be enhanced by prolonging the administration time (figure 10). The calculation result of the half inhibition concentration shows that the half inhibition concentration of the beta-cedrene on H1299 and A549 is 1043.644 mu mol/L and 461.749 mu mol/L respectively; the half inhibition concentrations of the cedrol on H1299 and A549 are 147.463 mu mol/L and 202.516 mu mol/L respectively, compared with the cedrol, the anti-lung cancer activity is better.
Therefore, the biological activity of the beta-cedrene, a key secondary metabolite of the roots of the rosewood seedlings, and the related compound cedarwood alcohol is discussed in the embodiment, and the biological activity mainly comprises two parts of evaluation of growth regulation capacity and evaluation of anti-tumor capacity. First, in terms of growth regulation, the experiment verifies the growth promoting activity of beta-cedrene and cedrol, and the result shows that beta-cedrene and cedrol with the concentration of less than 5 mu mol/L can promote the growth of arabidopsis thaliana, and the method is characterized by increasing the total biomass and increasing the number of lateral roots. The high concentration of beta-cedrene does not inhibit the growth of arabidopsis thaliana, but rather plays a promoting role. Secondly, in the aspect of anti-tumor, lung cancer strains H1299 and A549 are selected for investigation, and experimental results show that high-concentration beta-cedrene and cedrol have obvious inhibition effect on lung cancer cells, and the inhibition effect is increased along with the increase of the administration time.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (10)
1. A method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots, characterized by comprising the following steps:
(S1) selecting annual rosewood soil culture seedlings as transplanting seedlings, cutting off all lateral roots and part of main roots, cleaning after disinfection, dipping the roots with IBA+NAA, transferring into an aerosol culture system for culture, wherein pure water rooting induction culture is adopted in the early stage, and nutrient solution is used in the later stage for nutrient culture;
(S2) adding ethephon solution into the aeroponic nutrient solution, and subjecting the aeroponic seedlings subjected to nutrition culture to stress treatment;
(S3) selecting side roots of the rosewood aerosol seedlings subjected to stress treatment, freeze-drying, grinding into powder, adding ethyl acetate, performing vortex leaching for a plurality of times, centrifuging to obtain supernatant, and concentrating to obtain a sample to be tested;
(S4) detecting the sample to be detected by adopting GC-MS, and identifying to obtain the volatile substances containing beta-cedrene and cedrol.
2. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: in the step (S1), KMnO with the mass percent of 0.08-0.12% is adopted for disinfection 4 And (5) sterilizing the solution for 55-65 s.
3. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: in the step (S1), IBA+NAA with the concentration of 0.8-1.2 g/L is used for dipping roots for 8-12S, wherein the mass part ratio of IBA to NAA is 1:1.
4. the method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: and (S1) transferring the culture medium into an aerial fog culture system for culturing for 28-32 days, wherein pure water rooting induction culture is adopted for the first 6-8 days, and nutrient solution is adopted for the rest days in the later period for nutrition culture.
5. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: the nutrient solution in the step (S1) is a Huaduo 15-grade nutrient solution, and the concentration of the nutrient solution is 0.4-0.6 g/L.
6. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: in the step (S2), the final concentration of the ethephon solution added into the aeroponic nutrient solution is 4-6wt%, and the stress treatment time of the aeroponic seedlings after nutrient culture is 13-16 days.
7. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: the dosage relationship between the side roots of the rosewood aerosol seedling and the ethyl acetate in the step (S2) is as follows: 1g: 4-6 mL, the leaching temperature is 3-5 ℃, the leaching time is 22-26 h each time, and the leaching times are 2-4 times.
8. The method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots according to claim 1, characterized in that: the GC-MS detection conditions in the step (S3) are as follows: the column HP-5ms,30 m.times.0.25 mm.times.0.25. Mu.m; the initial temperature is 35 ℃, the temperature is kept for 5min, the temperature is increased to 300 ℃ at 8 ℃/min, and the temperature is kept for 5min; the sample injection amount is 1 mu L; the temperature of the sample inlet is 250 ℃; the temperature of the quadrupole rods is 150 ℃; the ion source temperature is 230 ℃; ionization mode EI; electron energy 70eV; the carrier gas is He, the purity is 99.99 percent, and the flow rate is 1mL/min; the mass scanning range is 35-450 AMU; delaying the solvent for 3min, searching and comparing the obtained spectrum and mass spectrum data with a Willy and NIST standard mass spectrum library, qualifying each component, and calculating the relative content of each component by an area normalization method.
9. Use of beta-cedrene or cedrol in the preparation of a medicament having the effect of promoting the growth of arabidopsis thaliana.
10. The use of beta-cedrene or cedrol in the manufacture of a medicament having an inhibitory effect on lung cancer cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310987861.2A CN117256457A (en) | 2023-08-08 | 2023-08-08 | Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310987861.2A CN117256457A (en) | 2023-08-08 | 2023-08-08 | Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117256457A true CN117256457A (en) | 2023-12-22 |
Family
ID=89203431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310987861.2A Pending CN117256457A (en) | 2023-08-08 | 2023-08-08 | Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117256457A (en) |
-
2023
- 2023-08-08 CN CN202310987861.2A patent/CN117256457A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Malabadi et al. | Micropropagation of Dendrobium nobile from shoot tip sections | |
| Di Giacinto et al. | Vineyard management system affects soil microbiological properties | |
| CN102599005A (en) | Method for producing seed source of Cordyceps militaris with high-yield cordycepin by radiation breeding | |
| CN104938184A (en) | Method for cultivating pakchoi which is highly rich in vitamin C | |
| Samadimatin et al. | Effect of ethanol and humic acid foliar spraying on morphological traits, photosynthetic pigments and quality and quantity of essential oil content of Dracocephalum moldavica L. | |
| Chongkid et al. | Effects of vanadium on rice growth and vanadium accumulation in rice tissues | |
| CN111052997B (en) | Preparation and application method of biological stimulin for improving strawberry continuous cropping obstacle resistance | |
| CN110352850B (en) | Method for breeding high-yield, high-quality and stress-resistant excellent new species of common andrographis herb | |
| CN117256457A (en) | Method for obtaining sesquiterpenes containing beta-cedrene and cedrol from rosewood aerial roots | |
| KR101830058B1 (en) | Micrococcus luteus PNL10-2 and uses thereof | |
| CN112314626B (en) | Application of biological stimulin for improving activity of continuous cropping salvia miltiorrhiza seedlings | |
| Lara et al. | Microalga improve the growth, yield, and contents of sugar, amino acid, and protein of tomato | |
| Intanon et al. | Field evaluation of meadowfoam (Limnanthes alba) seed meal for weed management | |
| Yıldırım et al. | Effects of different doses of plant growth regulators on some characteristics of summer snowflakes (Leucojum aestivum L.) | |
| CN108192628A (en) | A kind of carried titanium dioxide even ploughs the preparation method of soil-repairing agent | |
| CN114478122A (en) | Novel functional fertilizer synergist and application | |
| CN103931452A (en) | Filbert high-yield cultivation method | |
| Hussein et al. | Study the effect of dry bread yeast Saccharomyces cerevisiae, in some qualities of vegetative growth and nutritional status of the Lens culinaris | |
| CN106862265B (en) | Method for reducing cadmium absorption and accumulation of rape by using thick-leaf ophiopogon japonicus | |
| CN117016549B (en) | Application of nucleoside compound and composition thereof in promoting plant growth | |
| CN109953042A (en) | A kind of inhibitor that acacia confusa is grown based on grignard evergreen chinquapin litter leaching liquor | |
| Szabó et al. | Improved rooting by different plant growth regulator treatments on Prunus mahaleb L. cuttings | |
| CN114703070B (en) | Extraction method and application of endophytic fungi of genus Celastrus and secondary metabolites thereof | |
| Bekusarova et al. | On the effect of PABA on germination | |
| CN115669468B (en) | Use of light treatment to increase the yield of secondary metabolites in plants of the genus nepeta |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |