CN113730466A - Preparation method and application of blackberry extract - Google Patents
Preparation method and application of blackberry extract Download PDFInfo
- Publication number
- CN113730466A CN113730466A CN202111072889.0A CN202111072889A CN113730466A CN 113730466 A CN113730466 A CN 113730466A CN 202111072889 A CN202111072889 A CN 202111072889A CN 113730466 A CN113730466 A CN 113730466A
- Authority
- CN
- China
- Prior art keywords
- blackberry
- extract
- freeze
- extraction
- ultrasonic
- 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
- 239000000284 extract Substances 0.000 title claims abstract description 133
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 162
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 74
- 238000004108 freeze drying Methods 0.000 claims abstract description 31
- 206010067125 Liver injury Diseases 0.000 claims abstract description 23
- 231100000753 hepatic injury Toxicity 0.000 claims abstract description 23
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 20
- 238000001035 drying Methods 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 208000010706 fatty liver disease Diseases 0.000 claims abstract description 4
- 235000020510 functional beverage Nutrition 0.000 claims abstract description 4
- 208000006454 hepatitis Diseases 0.000 claims abstract description 3
- 231100000283 hepatitis Toxicity 0.000 claims abstract description 3
- 244000078534 Vaccinium myrtillus Species 0.000 claims abstract 26
- 238000000605 extraction Methods 0.000 claims description 141
- 150000004636 anthocyanins Chemical class 0.000 claims description 117
- 229930002877 anthocyanin Natural products 0.000 claims description 116
- 235000010208 anthocyanin Nutrition 0.000 claims description 116
- 239000004410 anthocyanin Substances 0.000 claims description 116
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 60
- 239000007788 liquid Substances 0.000 claims description 49
- 239000000843 powder Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 42
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 30
- 235000019253 formic acid Nutrition 0.000 claims description 30
- 235000013399 edible fruits Nutrition 0.000 claims description 27
- 238000005303 weighing Methods 0.000 claims description 23
- 238000000967 suction filtration Methods 0.000 claims description 19
- 238000010992 reflux Methods 0.000 claims description 16
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 14
- 230000014759 maintenance of location Effects 0.000 claims description 13
- 239000003480 eluent Substances 0.000 claims description 10
- 238000007654 immersion Methods 0.000 claims description 7
- 239000006187 pill Substances 0.000 claims description 7
- 210000004185 liver Anatomy 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 239000004952 Polyamide Substances 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 208000019425 cirrhosis of liver Diseases 0.000 claims description 4
- 239000008187 granular material Substances 0.000 claims description 4
- 238000002386 leaching Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229920002647 polyamide Polymers 0.000 claims description 4
- 239000006188 syrup Substances 0.000 claims description 4
- 235000020357 syrup Nutrition 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 3
- 239000003826 tablet Substances 0.000 claims description 3
- 208000007082 Alcoholic Fatty Liver Diseases 0.000 claims description 2
- 206010003445 Ascites Diseases 0.000 claims description 2
- 206010016262 Fatty liver alcoholic Diseases 0.000 claims description 2
- 208000026594 alcoholic fatty liver disease Diseases 0.000 claims description 2
- 238000007598 dipping method Methods 0.000 claims description 2
- 238000005470 impregnation Methods 0.000 claims description 2
- 235000013402 health food Nutrition 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 235000021029 blackberry Nutrition 0.000 abstract description 176
- 241000699670 Mus sp. Species 0.000 abstract description 9
- 235000013305 food Nutrition 0.000 abstract description 8
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 6
- 230000006378 damage Effects 0.000 abstract description 6
- 210000002966 serum Anatomy 0.000 abstract description 5
- 206010019668 Hepatic fibrosis Diseases 0.000 abstract description 4
- 208000004930 Fatty Liver Diseases 0.000 abstract description 2
- 206010019708 Hepatic steatosis Diseases 0.000 abstract description 2
- 231100000240 steatosis hepatitis Toxicity 0.000 abstract description 2
- 206010019692 hepatic necrosis Diseases 0.000 abstract 1
- 231100000149 liver necrosis Toxicity 0.000 abstract 1
- 210000005228 liver tissue Anatomy 0.000 abstract 1
- 229920006122 polyamide resin Polymers 0.000 abstract 1
- 241001092459 Rubus Species 0.000 description 172
- 239000000243 solution Substances 0.000 description 60
- 230000000694 effects Effects 0.000 description 28
- 238000006731 degradation reaction Methods 0.000 description 25
- 230000015556 catabolic process Effects 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 21
- 230000004044 response Effects 0.000 description 19
- 238000012360 testing method Methods 0.000 description 16
- 238000005457 optimization Methods 0.000 description 14
- 239000002904 solvent Substances 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 239000000463 material Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000002137 ultrasound extraction Methods 0.000 description 10
- 230000004913 activation Effects 0.000 description 9
- 230000001154 acute effect Effects 0.000 description 8
- 230000001684 chronic effect Effects 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- 238000002604 ultrasonography Methods 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000002354 daily effect Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 208000007848 Alcoholism Diseases 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000002535 acidifier Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 208000019423 liver disease Diseases 0.000 description 4
- JMZOMFYRADAWOG-UHFFFAOYSA-N methyl 7-methoxy-4-(7-methoxy-5-methoxycarbonyl-1,3-benzodioxol-4-yl)-1,3-benzodioxole-5-carboxylate Chemical compound COC(=O)C1=CC(OC)=C2OCOC2=C1C1=C2OCOC2=C(OC)C=C1C(=O)OC JMZOMFYRADAWOG-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 201000007930 alcohol dependence Diseases 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012803 optimization experiment Methods 0.000 description 3
- 230000004792 oxidative damage Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000005211 surface analysis Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- ORTBMTXABUAMJS-CRTITRSXSA-N (4s,5r)-2-[2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromenylium-3-yl]oxyoxane-3,4,5-triol;chloride Chemical compound [Cl-].OC1[C@@H](O)[C@H](O)COC1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 ORTBMTXABUAMJS-CRTITRSXSA-N 0.000 description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 241000415078 Anemone hepatica Species 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- KUCVMQMKRICXJC-LMVHDODDSA-O Cyanidin 3-O-alpha-L-arabinopyranoside Natural products O([C@@H]1[C@H](O)[C@H](O)[C@@H](O)CO1)c1c(-c2cc(O)c(O)cc2)[o+]c2c(c(O)cc(O)c2)c1 KUCVMQMKRICXJC-LMVHDODDSA-O 0.000 description 2
- KUCVMQMKRICXJC-FBVAEJEDSA-O Cyanidin 3-arabinoside Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@H](O)CO1)c1c(-c2cc(O)c(O)cc2)[o+]c2c(c(O)cc(O)c2)c1 KUCVMQMKRICXJC-FBVAEJEDSA-O 0.000 description 2
- ORTBMTXABUAMJS-VGEDXCMYSA-N Cyanidin 3-arabinoside Chemical compound [Cl-].O[C@H]1[C@H](O)[C@H](O)CO[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 ORTBMTXABUAMJS-VGEDXCMYSA-N 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 235000017848 Rubus fruticosus Nutrition 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 206010001584 alcohol abuse Diseases 0.000 description 2
- 208000025746 alcohol use disease Diseases 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- YTMNONATNXDQJF-UBNZBFALSA-N chrysanthemin Chemical compound [Cl-].O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 YTMNONATNXDQJF-UBNZBFALSA-N 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- ROQLTZUOXIQBDO-UHFFFAOYSA-O cyanidin 3-(6''-malonylglucoside) Chemical compound OC1C(O)C(O)C(COC(=O)CC(O)=O)OC1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 ROQLTZUOXIQBDO-UHFFFAOYSA-O 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 238000010562 histological examination Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000012933 kinetic analysis Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000000199 molecular distillation Methods 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 238000003305 oral gavage Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229960002275 pentobarbital sodium Drugs 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000004537 pulping Methods 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 240000007651 Rubus glaucus Species 0.000 description 1
- 235000011034 Rubus glaucus Nutrition 0.000 description 1
- 235000009122 Rubus idaeus Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002075 anti-alcohol Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 239000006084 composite stabilizer Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000021022 fresh fruits Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- -1 ultrasonic time Substances 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Alternative & Traditional Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Preparation Of Fruits And Vegetables (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to a preparation method and application of a blackberry extract, wherein the blackberry extract is prepared by freeze-drying fresh blackberries, extracting with methanol, and then concentrating and drying or concentrating and drying after purifying by polyamide resin. The effective dose of the extract is 25-100mg/kg/d, the extract can reduce the harm of alcoholic liver injury, and remarkably reduce the levels of AST, ALT, SOD and MDA in mouse serum, meanwhile, the blackberry extract can remarkably improve liver injury such as alcohol-induced liver tissue hepatitis, liver necrosis, fatty liver, hepatic fibrosis and the like of mice, and the blackberry extract can be well applied to medicines, functional beverages and health-care foods for resisting alcoholic liver injury.
Description
Technical Field
The invention relates to the field of medicines and foods, in particular to a preparation method and application of a blackberry extract.
Background
Alcohol abuse and dependence are important public health problems worldwide. With the continuous improvement of the living standard of people, alcohol consumption people are increased year by year in China, and the damage to nerves and a digestive system caused by alcoholism is increased day by day. The liver is a main organ in the alcohol metabolism process, and long-term drinking can cause Alcoholic liver injury (ALD), such as fatty liver, Alcoholic hepatitis, hepatic fibrosis and liver cirrhosis, and simultaneously can increase the incidence of liver cancer. According to the report of the World Health Organization (WHO), 20 hundred million people in the world drink alcohol, wherein over 750 million people have the phenomenon of alcohol abuse, and excessive drinking is a main cause of liver diseases. Alcoholic liver disease is the second largest type of liver disease in China after infectious liver disease, and the number of patients is increasing at an alarming rate in a state of being younger. Liver disease has become the fifth leading fatal disease worldwide following heart disease and stroke. In recent years, natural products with the advantages of low toxic and side effects, multiple ways and multiple targets are concerned, various food-borne natural products are reported to have the effect of protecting alcoholic liver injury, and the important significance is achieved in finding a medicine, a functional beverage or a health-care food which has definite curative effect and strong activity and is used for resisting alcoholic liver injury.
Blackberry (Blackberry) is a rosaceous rubus plant, named raspberry, and native north america, and is a new fruit which has rapid world development in recent years and integrates nutrition and health care. The fresh fruits are soft and juicy, have unique flavor and rich nutritive value; the blackberry has very high medicinal value, has the effects of regulating metabolic function, delaying senility, eliminating fatigue, improving immunity and the like, particularly has the effects of reducing cholesterol content, preventing and treating heart diseases and resisting cancers (reducing toxic and side effects caused by chemotherapy), and is known as purple life fruit.
Application number CN201711145154.X, invention name is a preparation method and application of novel blackberry powder, and discloses a preparation method: cleaning, namely cleaning the harvested blackberries by using a cleaning agent; crushing and pulping: primarily pressing the cleaned blackberries, then spraying high-pressure steam, rapidly cooling the blackberries to below 5 ℃, and pulping to obtain a first blackberry stock solution; mixing and stirring: adding a composite stabilizer into the blackberry stock solution, uniformly mixing, and then crushing by using a high-speed crusher to obtain a second blackberry stock solution; biological fermentation: fermenting the second blackberry stock solution with yeast to obtain a third blackberry stock solution; and (3) sterile treatment: performing high-temperature instantaneous sterilization, and then cooling to 30-40 ℃ to obtain a fourth blackberry stock solution; and (3) freeze-drying treatment: freeze-drying and pulverizing the fourth blackberry stock solution with a vacuum freeze-drying machine, and sieving with a 100-mesh sieve to obtain novel blackberry powder; also discloses application of the blackberry powder food.
Technical problem with application No. cn201711145154. x: 1) the blackberry stock solution is extracted for four times, so that the operation is complex, the cost is high, and the industrial production is not easy to realize; 2) the biological fermentation easily damages blackberry cells and influences the original components and quality of the blackberry; the effective components of the blackberry are easy to damage by adopting high-temperature instantaneous sterilization; 4) only the application of the blackberry powder food is mentioned, and the application of the blackberry extract in resisting alcoholic liver injury is not mentioned.
The application number CN201510433616.2 is the name of a preparation method of a blackberry extract, and discloses a preparation method of a blackberry extract, wherein the blackberry cleaned is placed in a homogenizer, and an ethanol solution with the weight 5-10 times of that of the blackberry is added for homogenizing and extraction; placing the filtrate after homogenate extraction in an ultrasonic device for ultrasonic extraction; standing the filtrate after ultrasonic extraction at 2-8 ℃, and centrifuging; and (3) performing molecular distillation on the centrifuged filtrate, and separating under the conditions that the feeding speed is 100-200 g/h, the film scraping speed is 300-500 r/min, the distillation temperature is 40-50 ℃, and the distillation pressure is 100-200 Pa to obtain the blackberry extract. The extraction method is a pure physical method, the homogenate extraction method realizes the crushing and extraction of the materials in one step, the ultrasonic extraction method can go deep into the inside of the cells of the materials, and the extraction rate of effective components is greatly improved; can be carried out under mild conditions, thereby avoiding the damage of effective components.
The technical problem of application No. CN201510433616.2 is: 1) directly placing the blackberries into a homogenizer, adding 5-10 times of ethanol solution for extraction, and removing impurities without peracid, so that the phenomena of more impurities, incomplete extraction, low extract yield and the like are easily caused; 2) the preparation of the extract by molecular distillation is costly.
Application number CN201510018395.2, the name of the invention is a blackberry extract and application thereof in preparing liver cell oxidative damage inhibitor, and discloses a blackberry extract, which comprises the following components by mass percent: 58.3 to 72.5 percent of cyanidin-3-glucoside; cyanidin-3-xyloside 11.6% -15.8%; cyanidin-3-oxalyl glucoside 9.8% -14.6%; 3.1 to 6.4 percent of cyanidin-3-malonyl glucoside; 2.7 to 4.1 percent of delphinidin-3-xylose and 0.3 to 0.8 percent of cyanidin-3-arabinoside. The extraction steps are as follows: and (2) carrying out enzymolysis on the fresh blackberry fruits by pepsin and trypsin in sequence, carrying out centrifugal separation to obtain a supernatant, filtering the supernatant by an ultrafiltration membrane, intercepting components with the molecular weight of more than 10kDa, and carrying out vacuum freeze drying on the filtrate to obtain the blackberry extract. The invention also discloses application of the blackberry extract in preparation of an inhibitor for oxidative damage of liver cells.
The technical problem of application No. CN201510018395.2 is: 1) the extraction method is complex, pepsin, trypsin, ultrafiltration membrane filtration and the like are required for extraction, and the cost is high; 2) the extract comprises the following main components: cyanidin-3-glucoside, cyanidin-3-xyloside, cyanidin-3-oxalylglucoside, cyanidin-3-malonylglucoside, delphinidin-3-xyloside and cyanidin-3-arabinoside, with no mention of blackberry anthocyanins in the extract; 3) the extract is mentioned for application in an inhibitor of acrylamide-induced oxidative damage of liver cells, but is not directed to application in an anti-alcoholic liver injury medicament.
Aiming at the problems, aiming at solving the problem of liver injury caused by alcoholism in the current alcohol consumer group, the invention group combines the technical problems existing in the blackberry extraction, and obtains the preparation method of the blackberry extract by a large amount of experimental researches on the influence of an acidifier, an extractant, ultrasonic time, methanol volume fraction, ultrasonic temperature, ultrasonic time and ultrasonic power on the extraction rate of the blackberry anthocyanin in the extraction.
Disclosure of Invention
The invention aims to provide a preparation method of a blackberry extract.
The invention also aims to provide application of the blackberry extract in preparing a medicament for treating alcoholic liver injury.
The preparation method of the blackberry extract comprises the following steps:
(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 45-52h at the freeze drying parameter of-50 ℃, the vacuum degree of 60Pa and the tray temperature of 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing a proper amount of freeze-dried powder, adding 60-80% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:4-10g/mL, extracting for 3 times by adopting any one of an ultrasonic method, a reflux method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.1-1.5 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
It is preferable that
The preparation method of the blackberry extract comprises the following steps:
(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing a proper amount of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 3 times by adopting any one of a reflux method, an ultrasonic method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
The ultrasonic method, the reflux method and the dipping method in the step 2) of the invention; wherein the ultrasonic method: the temperature is 40 ℃, the power is 300W, and the time is 90 min; the reflux method comprises the following steps: extracting at 35 deg.C for 90 min; the impregnation method comprises the following steps: the leaching time is 3 h/time.
The step 3) of the invention can also be:
taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 5-10, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extract complex solution by polyamide chromatography with 30-60 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting eluent until the eluent is colorless, concentrating under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.
The blackberry extract disclosed by the invention is applied to the preparation of medicines for treating alcoholic liver injury.
The blackberry extract is applied to the preparation of medicines for treating alcoholic fatty liver, hepatic fibrosis, liver cirrhosis, hepatic ascites and hepatitis.
The medicine can be added with pharmaceutically acceptable auxiliary materials to be prepared into granules, tablets, capsules, oral liquid, syrup, mixture, pills or powder.
The total effective dose of the blackberry extract in resisting alcoholic liver injury is 25-100 mg/kg/d.
The blackberry extract contains blackberry anthocyanin as main ingredient.
The blackberry extract disclosed by the invention is applied to the preparation of health-care food and functional beverage thereof.
The invention has the beneficial effects that:
1. the blackberry extract obtained by the preparation method can effectively reduce the harm of alcoholic liver injury, and compared with a model group, the blackberry extract can remarkably reduce liver index of a mouse and the levels of AST, ALT, SOD and MDA in serum, and H & E staining shows that the blackberry extract can improve cell morphological changes such as tissue vacuolization, edema, inflammation, cell necrosis and the like caused by the alcoholic liver injury of the mouse; the red oil O staining shows that the blackberry extract can improve fat deposition caused by alcoholic liver injury of mice and reduce the area of fat drop halo; the sirius red staining shows that the blackberry extract can improve the extension of collagen fiber in alcoholic liver injury and weaken the degree of hepatic fibrosis caused by alcohol.
2. The parameters of the preparation method of the invention pass through the establishment and analysis experiments of a response surface model, and the Design-Expert 8.0 software is adopted to carry out variance analysis on a regression model to obtain a multivariate quadratic equation model of anthocyanin extraction rate to methanol volume fraction (A), feed-liquid ratio (B), ultrasonic time (C), ultrasonic power (D) and ultrasonic temperature (E), and finally obtain the blackberry anthocyanin extraction rate and factor variables: y (anthocyanin extraction ratio) is 2775.40+46.8A +17.08B +0.16C +0.27D +12.64CE +0.16DE +1356.10a2+668.75B2+597.06C2+772.99D2+1341.82E2, and it can be seen from the analysis of variance table 3 that the significance level P of the model is less than 0.0001, indicating that the regression variance model is significant. In addition, kinetic analysis of blackberry extract degradation was also performed.
3. According to the three-dimensional graph and the contour map of the response surface of the blackberry anthocyanin content of each factor in the experimental result, the trend that the response surface is open downwards and the extraction rate of anthocyanin increases and then decreases along with the change of each factor is shown, and the model has a stable point with the maximum value. Finally, according to the F value and the P value, the regression model can be well fitted with the measured value, so that the model can predict and analyze the extraction process result of the blackberry anthocyanin. According to the steep degree of the response surface, the influence of various factors on the extraction amount of the blackberry anthocyanin is shown in the following sequence: ultrasonic temperature (E) > methanol volume fraction (A) > material-liquid ratio (B) > ultrasonic power (C) > ultrasonic time (D). According to the obtained model, the optimal technological parameters for extracting the blackberry anthocyanin can be predicted to be as follows: 70 percent of methanol acidified by formic acid, the ratio of material to liquid is 1:8, the ultrasonic time is 90min, the ultrasonic temperature is 40 ℃, the ultrasonic power is 300W, and the extraction rate is about 52.00 percent.
4. According to the invention, through determination of the optimal conditions and verification of a regression model, the optimal extraction scheme of the result system model optimization is that the ultrasonic power is 299.81W, the ultrasonic temperature is 37.69 ℃, the methanol volume fraction is 70.99%, the material-liquid ratio is 1:9.58, the ultrasonic time is 89.90min, and the extraction efficiency model value is 52.2467%.
5. According to the invention, through the degradation kinetics analysis of the blackberry anthocyanin, the result shows that the degradation of the blackberry anthocyanin accords with the first-order reaction of the kinetics, and the degradation constant K is increased along with the rise of the temperature and the pH value; t1/2 is reduced along with the increase of temperature and pH value, the activation energy is reduced along with the increase of pH value, the thermal stability is highest when the pH value is 2, which indicates that the degradation energy of the blackberry anthocyanin is the highest when the pH value is approximate to 2, otherwise, the degradation energy is the most stable and the blackberry anthocyanin is not easy to decompose. The experimental result provides a certain experimental basis and theoretical basis for the extraction and the prevention of decomposition of the blackberry anthocyanin in the processes of production, transportation, storage, use and the like in the industries of food, medicine, cosmetics and the like.
Description of the drawings:
FIG. 1 influence of the extraction method on anthocyanin extraction;
FIG. 2 effect of extraction solvent on anthocyanin extraction yield;
FIG. 3 effect of acidulant on anthocyanin extraction;
FIG. 4 effect of methanol volume fraction on anthocyanin extraction;
FIG. 5 effect of feed-liquid ratio on anthocyanin extraction rate;
FIG. 6 the effect of ultrasound power on anthocyanin extraction rate;
FIG. 7 effect of ultrasound time on anthocyanin extraction rate;
FIG. 8 effect of ultrasound temperature on anthocyanin extraction;
FIG. 9 is a three-dimensional graph of blackberry anthocyanin optimization using response surface optimization;
FIG. 10 is a graph of response surface optimization for blackberry anthocyanin contour;
fig. 11 thermal degradation of blackberry anthocyanins under different pH, temperature and heating time treatments (in the figure: a: pH 1, B: pH 2, C: pH 3, D: pH 4);
FIG. 12 histopathological observations H & E staining to evaluate the effect of blackberry extract on chronic (A), acute (B) alcoholic liver injury (x 400 fold) (left to right once: blank group, model group, low dose group, medium dose group, high dose group, positive drug group);
FIG. 13 histopathology observations Red oil O staining to evaluate the effect of blackberry anthocyanin on chronic (A), acute (B) alcoholic liver injury (x 400 times) (left to right once: blank group, model group, low dose group, medium dose group, high dose group, positive drug group);
FIG. 14 histological observations of sirius red stain to evaluate the effect of blackberry anthocyanin on chronic (A), acute (B) alcoholic liver injury (x 100 fold) (from left to right once: blank, model, low, medium, high, positive).
The technical solution of the present invention will be further specifically described below by way of specific examples.
Example 1 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 2 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 45 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 60% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:4g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.1 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 3 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 52h at the freeze drying parameter of-50 ℃, the vacuum degree of 60Pa and the tray temperature of 20 ℃, and crushing to 30 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 80% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:10g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.5 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 4 preparation of blackberry extract
(1) Putting 1000g of blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 5 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, performing reflux extraction at 35 ℃ for 90min, repeatedly extracting for 3 times, performing suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 6 preparation of blackberry extract
(1) Putting 1000g of blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, performing reflux extraction at 35 ℃ for 90min, repeatedly extracting for 3 times, performing suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 7 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, performing immersion extraction for 3 h/time, repeatedly extracting for 3 times, filtering and combining the extracting solutions, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 8 preparation of blackberry extract
(1) Putting 1000g of blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, performing immersion extraction for 3 h/time, repeatedly extracting for 3 times, filtering and combining the extracting solutions, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
Example 9 preparation of blackberry extract
(1) Putting 1000g of fresh blackberry fruits into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 8, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extractum complex solution by polyamide chromatography with 40 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting the eluent until the eluent is colorless, concentrating the eluent under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.
Example 10 preparation of blackberry extract
(1) Putting 1000g of blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ and the freeze drying parameter of-50 ℃ and the vacuum degree of 60Pa, and crushing to 20 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing 3.00g of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 90min by an ultrasonic method at 40 ℃ and 300W for 3 times, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 8, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extractum complex solution by polyamide chromatography with 40 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting the eluent until the eluent is colorless, concentrating the eluent under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.
Example 11 blackberry extract is added with 5% microcrystalline cellulose and 20% sucrose to obtain granules.
Example 12 blackberry extract is added with 20% starch syrup and 0.2% dextrin, granulated, tabletted, and made into tablets.
Example 13 the blackberry extract is mixed with 5% of pregelatinized starch, 5% of microcrystalline cellulose and 3% of talcum powder, and the mixture is encapsulated to obtain capsules.
Example 14 blackberry extract was taken, 15 times of purified water and 0.02% propylene glycol were added, mixed well, filtered, and sterilized to obtain oral liquid.
Example 15 blackberry extract was taken, 5 times of purified water and 45% of sucrose and 0.03% of p-hydroxybenzoic acid were added, mixed well, and sterilized to obtain syrup.
Example 16 blackberry extract is added with 5% refined honey and 5% water and mixed well to make into pill.
Example 17 blackberry extract was added with 5% ethyl cellulose and 20% starch to obtain granules.
Example 18 blackberry extract was added with sodium benzoate 0.03% and 10% water to obtain a mixture.
In order to further verify the feasibility and the effectiveness of the invention, the inventor carries out a series of tests, which are as follows:
screening of blackberry extract extraction method
1 apparatus, reagent and Material
1.1 instruments and reagents
1.2 materials
Experimental crude (fresh blackberry): nanjing Youyou fruit vitamin blackberry base, batch number 20200823
2 screening by extraction method
2.1 Freeze-drying of fresh blackberry
Taking a proper amount of fresh blackberry fruits, putting the fresh blackberry fruits into a tray of a freeze dryer, uniformly spreading the fresh blackberry fruits, setting freeze drying parameters to be-50 ℃, vacuum degree to be 60Pa and tray temperature to be 20 ℃. Freeze drying for 48 hr, taking out, freezing at-20 deg.C for storage, and pulverizing into powder.
2.2 optimization of the extraction method
3.00g of blackberry freeze-dried fruit powder is accurately weighed, 70% methanol (containing 0.1% formic acid) is respectively added as extraction solution according to the material-liquid ratio of 1:10g/mL, the extraction is respectively carried out by adopting a reflux method, an ultrasonic method and an immersion method, the ultrasonic and reflux temperature is 35 ℃, the time is 90min, the ultrasonic power is 300W, the extraction time is 3 h/time, the extraction is carried out for 3 times, the extraction solutions are filtered and combined, concentrated and freeze-dried, the anthocyanin extraction rate is calculated, and the experiment is carried out for 3 times in parallel.
As a result: the effect of the extraction method on anthocyanin extraction yield is shown in FIG. 1. As can be seen from FIG. 1, the extraction of the blackberry anthocyanins is carried out by three methods of ultrasound, extraction and reflux, and the extraction rates are different. The extraction rate of the anthocyanin obtained by reflux extraction is the lowest, probably because the local temperature is too high in the reflux extraction process, so that the structure of the anthocyanin is damaged, and meanwhile, the reflux extraction is easy to cause more impurities to be dissolved out. The extraction rates of ultrasound and leaching are not very different, but ultrasound is selected as the optimal extraction method in consideration of the fact that leaching takes longer.
2.3 optimization of extraction solvent
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, respectively adding 70% methanol solution, 75% ethanol solution, 75% acetone solution and ultrapure water as extracting agents according to the material-liquid ratio of 1:10g/mL, respectively, extracting for 3 times at the ultrasonic temperature of 35 ℃, ultrasonic time of 90min and ultrasonic power of 300W, carrying out suction filtration and combination on extracting solutions, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of solvent on anthocyanin extraction is shown in FIG. 2. As can be seen from fig. 2, when 75% acetone, 70% methanol, 75% ethanol and ultrapure water were used as extractants to extract the blackberry anthocyanins, the extraction rates were significantly different (P < 0.05). The extraction rate of anthocyanin is highest when 70% methanol is used as an extracting agent, and 75% ethanol, 75% acetone and ultrapure water are used as the reason that the polarity of anthocyanin is higher and the solubility of anthocyanin in acetone is lower, while in the ultrapure water, besides anthocyanin components, a large amount of polysaccharide is extracted, so that the extraction rate of anthocyanin is reduced, and the safety problem of a reagent is considered, so 70% methanol is selected as an extraction solvent.
2.4 optimization of acidulants
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, respectively adding 0.1% of acetic acid, hydrochloric acid, phosphoric acid and formic acid as acidifying agents into 70% of methanol according to the material-liquid ratio of 1:10g/mL, respectively, extracting for 3 times at the ultrasonic temperature of 35 ℃, the ultrasonic time of 90min and the ultrasonic power of 300W, carrying out suction filtration and combination on extract, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of acidulant on anthocyanin extraction is shown in FIG. 3. As can be seen from fig. 3, formic acid was selected as the acidulant since it extracted anthocyanin with the highest extraction rate, followed by acetic acid, citric acid, hydrochloric acid and phosphoric acid.
2.5 blackberry anthocyanin extraction Single factor investigation experiment
Weighing 3.00g of blackberry freeze-dried fruit powder, taking acidic methanol as an extractant, carrying out ultrasonic-assisted extraction, and selecting methanol volume fraction, ultrasonic power, ultrasonic time, ultrasonic temperature and material-liquid ratio as independent variables to carry out single-factor tests respectively.
2.5.1 optimization of methanol volume fraction
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, adding 35%, 55%, 70% and 95% methanol (containing 0.1% formic acid) as extraction solvents according to the material-liquid ratio of 1:10g/mL, respectively, performing ultrasonic extraction at the power of 300W, wherein the ultrasonic temperature is 35 ℃, the ultrasonic time is 90min, extracting for 3 times, performing suction filtration and combination on extract, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and performing parallel experiments for 3 times.
As a result: the effect of methanol volume fraction on anthocyanin extraction is shown in FIG. 4. As can be seen from fig. 4, the extraction yield of anthocyanin was the highest with 70% methanol, followed by 35% methanol, 55% methanol and 95% methanol. This is probably due to the fact that under low concentration methanol, a large amount of the more polar impurities are extracted together, reducing the purification of anthocyanins, whereas at high concentration 95% methanol, the polarity is less, resulting in a large amount of the more polar anthocyanins not being completely extracted, and therefore a suitable methanol concentration is 70% methanol.
2.5.2 optimization of the feed-to-liquid ratio
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, taking 70% methanol (containing 0.1% formic acid) as an extraction solvent, carrying out ultrasonic extraction at the power of 300W and the ultrasonic temperature of 35 ℃, the ultrasonic time of 90min and extracting for 3 times according to the material-liquid ratio of 1:2, 1:4, 1:8 and 1:16g/mL, carrying out suction filtration and combination on the extracting solution, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of the feed liquid ratio on the anthocyanin extraction rate is shown in FIG. 5. As can be seen from FIG. 5, the feed-to-liquid ratio is in the range of 1.4 to 1:8, the extraction rate of anthocyanin increases with the increase of the feed-to-liquid ratio, the highest extraction rate of anthocyanin is obtained when the feed-to-liquid ratio is 1:8, and the extraction rate is reduced when the feed-to-liquid ratio is higher than 1: 8. Probably because the ratio of the material to the liquid is too small, the solvent cannot effectively infiltrate the sample and the anthocyanin cannot be completely and effectively extracted, so that the extraction rate of the anthocyanin is lower; the increase of the material-liquid ratio can increase the concentration difference of solid-liquid phases, improve the mass transfer driving force and facilitate the extraction of anthocyanin, and when the material-liquid ratio is too large, a large amount of alcohol-soluble impurities in a sample are adsorbed, the temperature rise speed of the solution is slow, so that anthocyanin cannot be completely leached. It is also possible that when the ultrasonic wave propagates in the solution, the acoustic energy rapidly attenuates with the increase of the propagation distance, and thus the total volume of the solution in the system increases with the increase of the solid-to-liquid ratio, and at a distance farther from the acoustic source, the ultrasonic energy decreases, the cavitation ability is impaired, and the elution of anthocyanin is not facilitated. Therefore, the ratio of the materials to the liquid is 1:8, which is a proper ratio.
2.5.3 optimization of ultrasonic Power
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, carrying out ultrasonic extraction at the ultrasonic temperature of 35 ℃ for 90min under the conditions of 100, 200, 300 and 400W according to the material-liquid ratio of 1:10g/mL and the extraction solvent of 70% methanol (containing 0.1% formic acid), extracting for 3 times, carrying out suction filtration and combination on the extracting solution, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of ultrasonic power on anthocyanin extraction yield is shown in FIG. 6. As can be seen from FIG. 6, in the range of ultrasonic power of 200-300W, the extraction rate of anthocyanin increases with the increase of power, and when the ultrasonic power is higher than 300W, the extraction rate decreases with the increase of power. This is probably because the ultrasonic wave propagates in the medium to generate cavitation, the broken object is broken instantaneously by the great pressure generated in the cavitation, and the vibration action generated by the ultrasonic wave enhances the diffusion and dissolution of the broken object. The power is increased, and cavitation and mechanical action are carried out to increase the movement frequency and speed of the substance molecules, thereby increasing the penetrating power of the solvent and improving the dissolution of anthocyanin. The power is more than 300W, and the pigment content is reduced. The possible reasons are that the ultrasonic frequency is too high, the medium temperature is too high in the action process, so that anthocyanin is degraded, and the extraction amount is reduced. Therefore, 300W of ultrasonic power was selected as the optimum power.
2.5.4 optimization of ultrasound time
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, carrying out ultrasonic extraction at the power of 300W by using 70% methanol (containing 0.1% formic acid) as an extraction solvent according to the material-liquid ratio of 1:10g/mL for 3 times at the ultrasonic temperature of 35 ℃ for 30, 60, 90 and 120min respectively, carrying out suction filtration and combination on extract, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of sonication time on anthocyanin extraction yield is shown in FIG. 7. As can be seen from fig. 7, the anthocyanin extraction rate increased with the increase of the ultrasonic time within 30-90min, and decreased when the ultrasonic time exceeded 90min, probably because the total anthocyanins gradually dissolved in the methanol extract with the increase of the ultrasonic time, and the yield gradually increased. When the ultrasonic time exceeds 90min, on one hand, the solvent is evaporated, and on the other hand, the total anthocyanin structure can be damaged, decomposed, degraded or oxidized into other substances, so that the extraction rate is reduced[30,31]。
2.5.5 optimization of ultrasonic temperature
Accurately weighing 4 parts of 3.00g blackberry freeze-dried fruit powder, carrying out ultrasonic extraction at the ultrasonic temperature of 20, 40, 60 and 80 ℃ respectively and the ultrasonic time of 90min according to the material-liquid ratio of 1:10g/mL and the extraction solvent of 70% methanol (containing 0.1% formic acid) under the condition of 300W, extracting for 3 times, carrying out suction filtration and combination on the extracting solution, concentrating, freeze-drying, calculating the extraction rate of anthocyanin, and carrying out parallel experiments for 3 times.
As a result: the effect of ultrasound temperature on anthocyanin extraction yield is shown in FIG. 8. As is clear from fig. 8, in the range of 20 to 40 ℃, the extraction rate of anthocyanins increases with increasing temperature, and after exceeding 40 ℃, the extraction rate of anthocyanins decreases with increasing temperature, which is probably because the structure of anthocyanins is broken under high temperature conditions because anthocyanins are heat-sensitive components, the glycosidic bond is easily broken, and the dissolution of anthocyanins is reduced due to volatilization of the extraction solvent, and the extraction rate of anthocyanins decreases. Therefore, 40 ℃ was chosen as the sonication temperature for the experiment.
2.6 design of response surface optimization experiment
And according to the experimental results of the single-factor investigation, adopting a Box-Behnken model of Design Expert 8.0 software to carry out experimental Design and response surface analysis. And (3) synthesizing the single-factor test result, designing a five-factor three-level response surface test by using the ultrasonic power, the ultrasonic temperature, the ultrasonic time, the material-liquid ratio and the methanol volume fraction, and analyzing by using the anthocyanin extraction rate as a response value to obtain the optimal extraction process condition. The results are shown in tables 1 and 2.
TABLE 1 response surface analysis factors and levels
2.6.1 creation and analysis of response surface model
And (3) carrying out variance analysis on the regression model by adopting Design-Expert 8.0 software to obtain the anthocyanin extraction rate to methanol volume fraction (A), the feed-liquid ratio (B), the ultrasonic time (C), the ultrasonic power (D) and the ultrasonic temperature (E), and finally obtaining a multivariate quadratic equation model of the blackberry anthocyanin extraction rate and the factor variables: y (anthocyanin extraction ratio) is 2775.40+46.82A +17.08B +0.16C +0.27D +12.64CE +0.16DE +1356.10a2+668.75B2+597.06C2+772.99D2+1341.82E2, and it can be seen from the analysis of variance table 3 that the significance level P of the model is less than 0.0001, indicating that the regression variance model is significant.
According to the experimental result, the three-dimensional graph and the contour graph of the response surface of each factor relative to the content of the anthocyanin in the blackberry are shown in fig. 9 and fig. 10, and the trend that the extraction rate of the anthocyanin increases and then decreases along with the change of each factor when the response surface is opened downwards is seen from the graph, which shows that the model has a stable point with the maximum value. Finally, according to the F value and the P value, the regression model can be well fitted with the measured value, so that the model can predict and analyze the extraction process result of the blackberry anthocyanin. According to the steep degree of the response surface, the influence of various factors on the extraction amount of the blackberry anthocyanin is shown in the following sequence: ultrasonic temperature (E) > methanol volume fraction (A) > material-liquid ratio (B) > ultrasonic power (C) > ultrasonic time (D). According to the obtained model, the optimal technological parameters for extracting the blackberry anthocyanin can be predicted to be as follows: 70 percent of methanol acidified by formic acid, the ratio of material to liquid is 1:8, the ultrasonic time is 90min, the ultrasonic temperature is 40 ℃, the ultrasonic power is 300W, and the extraction rate is about 52.00 percent.
TABLE 2 Box-Behnken test design and response surface analysis results
TABLE 3 regression model analysis of variance
2.6.2 determination of optimal conditions and validation of regression models
The optimal extraction scheme of the system model optimization is that the ultrasonic power is 299.81W, the ultrasonic temperature is 37.69 ℃, the methanol volume fraction is 70.99%, the material-liquid ratio is 1:9.58, the ultrasonic time is 89.90min, and the extraction efficiency model value is 52.2467%.
2.7 kinetic analysis of thermal degradation of blackberry anthocyanins
Preparing deionized water with pH values of 1, 2, 3 and 4 for later use, putting 4 parts of 1.00g of blackberry lyophilized fruit powder into a 100mL volumetric flask, fixing the volume with the deionized water to a constant volume to prepare a solution of 1.0mg/mL, sucking a proper amount of the solution, respectively putting the solution into a constant-temperature water bath kettle at 25 ℃, 55 ℃, 75 ℃ and 95 ℃, taking 1mL every 1 hour, rapidly cooling with cold water, keeping in the dark for later test, and taking 5 times in total. The mean value is calculated by parallel measurement and three times. The results are shown in FIG. 11.
Calculating a first order kinetic reaction rate constant (K) according to the anthocyanin residual rate by the following formula:
ln(Ct/C0)＝-Kt
wherein C0 is the initial concentration of heated blackberry anthocyanin, Ct is the concentration of heated blackberry anthocyanin, and t is the heat treatment time (h).
The half-life of degradation of blackberry anthocyanins (t1/2) was calculated using the formula:
t1/2＝-ln0.5/K
the thermal degradation activation energy [ Ea (kJ/mol) ] of blackberry anthocyanin was calculated according to the following formula:
lnk＝lnK0-Ea/RT
wherein K is degradation rate constant (min-1), R is gas constant (8.314X 10-3 kJ/mol. K), K0Is the frequency constant (min-1) and T is the temperature (K).
2.7.1 Effect of pH and temperature on anthocyanin stability of blackberries
As can be seen from fig. 11, the degradation rate of anthocyanins increases with increasing temperature under the same pH condition, indicating that it is advantageous for the stability of anthocyanins under low temperature conditions, and anthocyanins are unstable and are easily degraded or converted into other substances under high temperature conditions. In addition, as the pH value is increased, the degradation rate K value is increased, which shows that the anthocyanin is easy to degrade under the high pH environment and is stable under the low pH environment.
2.6.2 blackberry anthocyanin degradation kinetics resolution
The mass concentrations of blackberry anthocyanins 1h after water bath at 25, 55, 75 and 95 ℃ are respectively taken as initial values, the absorbance (the mass concentration is in direct proportion to the absorbance A) is measured, and the negative value-ln (Ct/C0) of the logarithm of the retention rate of the mass concentration of the anthocyanins is plotted against the heating time t, as shown in the above figure 11. Linear regression is respectively carried out to obtain a regression equation and a slope, the-ln (Ct/C0) and the t are obviously in a linear relation, and the formula ln (Ct/C0) is-Kt, so that the thermal degradation of the blackberry anthocyanin accords with a first-order reaction model, and the reaction order is a first-order reaction.
As can be seen from the formula-ln (Ct/C0) — Kt and fig. 11, the slope of the straight line obtained by plotting the negative value of the logarithm of the storage percentage of blackberry anthocyanin concentration — ln (Ct/C0) against the heating time t is the thermal degradation reaction constant K at that temperature. The activation energy Ea can be obtained from the slope and intercept of a straight line by performing linear regression on 1/T with lnK according to the K value at the same temperature and obtaining the activation energy Ea from the formula lnK0-Ea/RT, wherein the slope of the straight line is-Ea/R.
The changes of thermal degradation rate constant (K), half-life period (t1/2) and activation energy (Ea) of blackberry anthocyanin under different temperature and pH value conditions are shown in Table 4. As can be seen from the data in Table 4, different temperatures and pH values have significant effects on the thermal degradation rate constant (k) and half-life (t1/2) of blackberry anthocyanin. The K value increases with the temperature and the pH value; t1/2 decreases with increasing temperature and pH; the activation energy represents an energy barrier which the substance needs to cross when the substance is converted, the higher the energy barrier, the higher the stability of the substance, and analysis of combined test data shows that the activation energy of anthocyanin decreases with the increase of pH value, which indicates that the anthocyanin has the highest thermal stability at pH 2. These results indicate that at higher temperatures, blackberry anthocyanins are very susceptible to degradation, and at lower temperatures and low pH values, it is beneficial to maintain their stability.
TABLE 4 influence of different temperatures and pH values on thermal degradation rate constant (k), half-life (t1/2) and activation energy (Ea) of blackberry anthocyanins
3 conclusion
The method researches the influence of an acidifying agent, an extracting agent, ultrasonic time, methanol volume fraction, ultrasonic temperature, ultrasonic time and ultrasonic power on the extraction rate of the blackberry anthocyanin, determines that formic acid is the acidifying agent, adopts ultrasonic-assisted extraction, takes methanol as the extracting agent, and optimizes the influence of the ultrasonic time, the ultrasonic temperature, the methanol volume fraction, the ultrasonic power and the ultrasonic time on the extraction rate of the blackberry anthocyanin by a response surface according to the following optimal process parameters: 70 percent of methanol acidified by formic acid, the ratio of material to liquid is 1:8, the ultrasonic time is 90min, the ultrasonic temperature is 40 ℃, the ultrasonic power is 300W, and the extraction rate is about 52.00 percent. The response surface optimization experiment result shows that the significant influence of all factors on the response value is that the ultrasonic temperature (E) is greater than the methanol volume fraction (A) and is greater than the feed-liquid ratio (B) and the ultrasonic power is greater than the ultrasonic time (D), and the optimization experiment result shows that the model has certain practical value on the extraction process optimization of the blackberry anthocyanin. Thermal degradation kinetic experiments show that the degradation of blackberry anthocyanin accords with the first-order reaction of kinetics, and the degradation constant K is increased along with the rise of temperature and pH value; t1/2 is reduced along with the increase of temperature and pH value, the activation energy is reduced along with the increase of pH value, the thermal stability is highest when the pH value is 2, which indicates that the degradation energy of the blackberry anthocyanin is the highest when the pH value is approximate to 2, otherwise, the degradation energy is the most stable and the blackberry anthocyanin is not easy to decompose. The experimental result shows that the blackberry anthocyanin has been produced, transported, stored, used and the like in the industries of food, medicine, cosmetics and the like
The extraction and the decomposition prevention in the process provide certain experimental basis and theoretical basis.
Second, research on pharmacodynamics experiment of extract
1 materials and methods
1.1 sample: blackberry extract.
1.2 Experimental animals: kunming mouse, male, 18-22 g, each group of 10.
2, experimental method:
2.1 dose and grouping
The experiment is divided into three dose groups of blank group (acute and chronic), model group (acute and chronic), blackberry extract low (25mg/kg/d), medium (50mg/kg/d) and high (100mg/kg/d) (acute and chronic) and bifendate dripping pill positive drug group (acute and chronic).
2.2 administration of test samples
Weighing appropriate amount of test sample, grinding, dissolving in 0.5% sodium carboxymethylcellulose solution, mixing well, administering test sample via oral gavage, and recording daily feed intake or water intake of each animal. Administration was continued for 30 days.
2.3 Experimental procedures
2.3.1 acute alcoholic liver injury test
Daily mice were given test samples by oral gavage, with no intervention in the blank and model groups. The mice in the administration group are subjected to intragastric administration with blackberry extract and bifendate dripping pills with corresponding doses every day, and after 4 hours of the last administration, the mice in the model group and the administration group are subjected to intragastric administration with 50% ethanol (12mL/kg BW) at one time. After fasting and water prohibition for 16h, carrying out intraperitoneal injection of 60mg/kg BW pentobarbital sodium solution for anesthesia, picking eyeballs and taking blood, detecting AST, ALT, SOD and MDA in the serum, fixing the left liver leaf of a mouse by 4% paraformaldehyde, taking a cross section from the middle part of the left liver leaf, carrying out flaking, dyeing, and carrying out detection and pathological histological examination of various indexes. During the experiment, mice were weighed daily to adjust the test sample dose in time.
2.3.2 Chronic Alcoholic liver injury test
Daily mice were gavaged orally with test samples, blank groups were not intervened, model groups were left undisturbed for thousands of 15 days, and 15 days before the end of the experiment were gavaged orally with 30% ethanol (10mL/kg BW) daily. The mice in the administration group are fed with the blackberry extract and the bifendate dripping pill with corresponding doses by intragastric administration every day, and 30% ethanol is fed by intragastric administration after the blackberry extract and the bifendate dripping pill with corresponding doses are fed by intragastric administration every day for 4 hours 15 days before the experiment is finished. After fasting for 4 hours, after anesthesia by intraperitoneal injection of 60mg/kg BW pentobarbital sodium solution, the eyeballs are picked up and blood is taken out, AST, ALT, SOD and MDA in the serum are detected, the left lobe of the liver of a mouse is fixed by 4% paraformaldehyde, the cross section of the middle part of the left lobe of the liver is taken out, and the section is sliced, dyed, and the detection of each index and the pathological histological examination are carried out. During the experiment, mice were weighed daily to adjust the test sample dose in time.
2.3.3 results, see table 5.
TABLE 5 effects of blackberry extracts on mouse body weight, liver index and serum indices
Values are the mean±standard deviation of 10determinations.Compared with control:***,p<0.001.Compared with model:###,p<0.001.
2.4 microscopic examination
The whole tissue section is continuously observed by a microscope with corresponding magnification, pathological changes such as hepatocyte degeneration, hepatocyte necrotic fat deposition, collagen fiber extension and the like are mainly observed, and records are given at the same time. The results are shown in FIGS. 12, 13 and 14.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A preparation method of a blackberry extract is characterized by comprising the following steps:
(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 45-52h under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing a proper amount of freeze-dried powder, adding 60-80% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:4-10g/mL, extracting for 3 times by adopting any one of an ultrasonic method, a reflux method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.1-1.5 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
2. The method of claim 1, comprising the steps of:
(1) putting fresh blackberry fruits or blackberry leaves into a tray of a freeze dryer, drying for 48 hours at the temperature of 20 ℃ under the conditions that the freeze drying parameters are-50 ℃, the vacuum degree is 60Pa and the tray temperature is 20 ℃, and crushing to 20-30 meshes to obtain freeze-dried powder for later use;
(2) precisely weighing a proper amount of freeze-dried powder, adding 70% methanol solution containing 0.1% formic acid as an extraction solution according to the material-liquid ratio of 1:8g/mL, extracting for 3 times by adopting any one of a reflux method, an ultrasonic method and an immersion method, carrying out suction filtration and combination on the extraction solution, and concentrating to obtain an extract with the relative density of 1.3 for later use;
(3) and (4) taking the extract, and performing vacuum freeze drying to obtain the blackberry extract.
3. The method according to any one of claims 1 or 2, wherein the ultrasonic method, the reflux method, the dipping method in step 2); wherein the ultrasonic method: the temperature is 40 ℃, the power is 300W, and the time is 90 min; the reflux method comprises the following steps: extracting at 35 deg.C for 90 min; the impregnation method comprises the following steps: the leaching time is 3 h/time.
4. The method according to any one of claims 1 or 2, wherein step 3) further comprises: taking the extract, and redissolving by using 10% ethanol containing 0.1% formic acid, wherein the redissolution ratio is 1: and 5-10, completely dissolving the blackberry extract by ultrasonic treatment, purifying the extract complex solution by polyamide chromatography with 30-60 meshes, eluting by 10 percent of 10 retention volumes, 30 percent ethanol with 20 retention volumes and 50 percent ethanol with 15 retention volumes, collecting 30 percent and 50 percent ethanol elution fractions, collecting eluent until the eluent is colorless, concentrating under reduced pressure at 40 ℃, and freeze-drying to obtain the blackberry extract.
5. The blackberry extract prepared by the preparation method according to any one of claims 1 or 2, and is characterized in that the blackberry extract is applied to the preparation of the drug for treating alcoholic liver injury.
6. The blackberry extract of claim 5, wherein the blackberry extract is used for preparing the drug for treating alcoholic fatty liver, liver fibrosis, liver cirrhosis, liver ascites and hepatitis.
7. The blackberry extract according to claim 5, wherein the blackberry extract can be made into granules, tablets, capsules, oral liquids, syrups, mixtures, pills or powders with pharmaceutically acceptable excipients.
8. The extract as claimed in claim 5, wherein the total effective dose of the blackberry extract in resisting alcoholic liver injury is 25-100 mg/kg/d.
9. The blackberry extract of claim 5, wherein the blackberry extract is used in the preparation of health food and functional beverage thereof.
10. The blackberry extract of claim 5, wherein the blackberry extract has a major component of blackberry anthocyanin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111072889.0A CN113730466A (en) | 2021-09-14 | 2021-09-14 | Preparation method and application of blackberry extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111072889.0A CN113730466A (en) | 2021-09-14 | 2021-09-14 | Preparation method and application of blackberry extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113730466A true CN113730466A (en) | 2021-12-03 |
Family
ID=78738613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111072889.0A Pending CN113730466A (en) | 2021-09-14 | 2021-09-14 | Preparation method and application of blackberry extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113730466A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116686922A (en) * | 2023-05-31 | 2023-09-05 | 江苏悠维有机食品有限公司 | Composition containing blackberry berries as well as preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104606306A (en) * | 2015-01-14 | 2015-05-13 | 浙江大学 | Blackberry extract and application of blackberry extract in preparation of liver cell oxidative damage inhibitor |
| CN106811371A (en) * | 2017-03-29 | 2017-06-09 | 贵州穗蓝酒业科技有限公司 | A kind of blackberry, blueberry health liquor and preparation method thereof |
| CN110292596A (en) * | 2019-08-12 | 2019-10-01 | 沈阳药科大学 | Black raspberry anthocyanin is preparing the application in anti-liver injury medicament or health food |
| CN111067052A (en) * | 2019-12-31 | 2020-04-28 | 贵州师范大学 | Blackberry refined powder preparation method and product |
-
2021
- 2021-09-14 CN CN202111072889.0A patent/CN113730466A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104606306A (en) * | 2015-01-14 | 2015-05-13 | 浙江大学 | Blackberry extract and application of blackberry extract in preparation of liver cell oxidative damage inhibitor |
| CN106811371A (en) * | 2017-03-29 | 2017-06-09 | 贵州穗蓝酒业科技有限公司 | A kind of blackberry, blueberry health liquor and preparation method thereof |
| CN110292596A (en) * | 2019-08-12 | 2019-10-01 | 沈阳药科大学 | Black raspberry anthocyanin is preparing the application in anti-liver injury medicament or health food |
| CN111067052A (en) * | 2019-12-31 | 2020-04-28 | 贵州师范大学 | Blackberry refined powder preparation method and product |
Non-Patent Citations (3)
| Title |
|---|
| 刘红锦等: "大孔树脂对黑莓花色苷的吸附与解吸特性", 《江苏农业学报》 * |
| 王兴鹏等: "《医师考核培训规范教程 消化内科分册》", 30 April 2016 * |
| 赵慧芳等: "黑莓果实中花色苷的提取和测定方法研究", 《食品工业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116686922A (en) * | 2023-05-31 | 2023-09-05 | 江苏悠维有机食品有限公司 | Composition containing blackberry berries as well as preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103230473B (en) | Lycium ruthenicum Murr. effective extract, extracting method and extract are applied | |
| CN110574927B (en) | Anti-saccharification composition and preparation method thereof | |
| CN1931270A (en) | Sobering up and liver protecting Chinese medicine prepn and its prepn process | |
| CN107412721B (en) | Blood sugar-reducing bitter gourd polypeptide compound capsule and preparation method thereof | |
| CN111905084B (en) | Traditional Chinese medicine extract and preparation process and application thereof | |
| CN112656707A (en) | Extraction method of centella asiatica polysaccharide extract, centella asiatica polysaccharide extract and application thereof | |
| CN108379305A (en) | Application of the Phellinus water extract in anti-uric acid | |
| CN109601795A (en) | Using penthorum chinense pursh as composition alcohol-decomposing beverage of primary raw material and preparation method thereof | |
| CN104435034B (en) | A kind of arasaponin and preparation method thereof | |
| CN101401829A (en) | Wild Jinchai liveness extract, preparation and uses thereof | |
| CN102908375B (en) | Method for simultaneously extracting and separating saponin enrichment matters and polysaccharide enrichment matters from ginseng rootlets | |
| CN111437310A (en) | Preparation method of high-activity oldenlandia diffusa total flavone and application of high-activity oldenlandia diffusa total flavone in liver peroxidation injury | |
| CN114699468A (en) | Preparation method of vine tea extract | |
| CN113730466A (en) | Preparation method and application of blackberry extract | |
| CN106619782A (en) | Herba rhodiolae extract and healthy product containing herba rhodiolae extract | |
| CN102940650B (en) | Propolis ethanol extract for dispeling the effects of alcohol, preparation method of propolis ethanol extract and application of propolis ethanol extract for producing enteric-coated tablets | |
| CN109232757B (en) | Walnut leaf polysaccharide extract and application thereof | |
| CN101333204B (en) | Method for extracting dihydroquercetin form larch | |
| CN113694104B (en) | Traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function, preparation method and application thereof | |
| CN103054906B (en) | Propolis ethanol extract for alleviating hangover and preparation method thereof, and application of propolis ethanol extract in producing buccal tablets | |
| CN109602759A (en) | The purposes of kusamaki broad-leaved podocarpus seed and receptacle polysaccharide | |
| JP2006290876A (en) | Anti-diabetic agent | |
| CN101757059B (en) | Method for extracting alpha-glucosidase activity inhibitor from plants | |
| CN104926958A (en) | Method for extracting herba lycopi polysaccharide from herba lycopi and application of herba lycopi polysaccharide | |
| CN105920051B (en) | Flammulina velutipes extract and preparation method and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211203 |
|
| RJ01 | Rejection of invention patent application after publication |