CN106109668B - Inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp - Google Patents
Inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp Download PDFInfo
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- CN106109668B CN106109668B CN201610523840.5A CN201610523840A CN106109668B CN 106109668 B CN106109668 B CN 106109668B CN 201610523840 A CN201610523840 A CN 201610523840A CN 106109668 B CN106109668 B CN 106109668B
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- 241001629511 Litchi Species 0.000 title claims abstract description 62
- 230000000694 effects Effects 0.000 title claims abstract description 46
- 150000002989 phenols Chemical class 0.000 title claims abstract description 37
- 150000002632 lipids Chemical class 0.000 title claims abstract description 34
- 230000002776 aggregation Effects 0.000 title claims abstract description 25
- 238000004220 aggregation Methods 0.000 title claims abstract description 25
- 210000005229 liver cell Anatomy 0.000 title claims abstract description 22
- 238000000746 purification Methods 0.000 title claims abstract description 18
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 179
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 97
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 62
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 50
- 238000010828 elution Methods 0.000 claims description 50
- 229960000583 acetic acid Drugs 0.000 claims description 48
- 239000012362 glacial acetic acid Substances 0.000 claims description 47
- 235000013824 polyphenols Nutrition 0.000 claims description 37
- 239000000284 extract Substances 0.000 claims description 31
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 25
- 239000003480 eluent Substances 0.000 claims description 22
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 235000019441 ethanol Nutrition 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
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- 239000003463 adsorbent Substances 0.000 claims 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 abstract description 11
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 abstract description 11
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 abstract description 11
- 239000005642 Oleic acid Substances 0.000 abstract description 11
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 abstract description 11
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 abstract description 11
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 abstract description 11
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- 229930003935 flavonoid Natural products 0.000 abstract description 2
- 150000002215 flavonoids Chemical class 0.000 abstract description 2
- 235000017173 flavonoids Nutrition 0.000 abstract description 2
- 229920002414 procyanidin Polymers 0.000 abstract description 2
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- 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 26
- 230000003834 intracellular effect Effects 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 18
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 14
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- 238000007824 enzymatic assay Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
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- COTNUBDHGSIOTA-UHFFFAOYSA-N meoh methanol Chemical compound OC.OC COTNUBDHGSIOTA-UHFFFAOYSA-N 0.000 description 1
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- 230000000144 pharmacologic effect Effects 0.000 description 1
- 235000017807 phytochemicals Nutrition 0.000 description 1
- 229930000223 plant secondary metabolite Natural products 0.000 description 1
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- 239000012679 serum free medium Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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)
- A61K36/77—Sapindaceae (Soapberry family), e.g. lychee or soapberry
-
- 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
- A61K2236/30—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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses the purification process for inhibiting liver cell lipid aggregation main effect phenols component in a kind of litchi pulp, by the separation to 4 sections of different phenols components such as litchi pulp procyanidins, Flavonoid substances, the inhibiting effect for the liver cell inner lipid aggregation that this 4 sections of phenols components induce oleic acid is obtained.
Description
Technical field
The present invention relates to inhibit liver cell lipid aggregation main effect phenol in food processing field more particularly to a kind of litchi pulp
The purification process of constituents.
Background technique
Lichee is the main characteristics fruit of subtropical zone, deeply by state due to the bright-coloured beauty of its pericarp, gravy are fine and smooth fragrant and sweet
Inside and outside consumer's likes, is known as " treasure in fruit ".China is the main production country of lichee, cultivated area and the world yield Jun Ju
One.Guangdong Province accounts for 60% or more of national lichee total output as its yield of the main producing region of lichee.Due to lichee Time To Market collection
In, and fresh keeping time is short, marketing fresh market pressure is huge.How to carry out the intensive processing of lichee, alleviate marketing fresh market pressure, improves
The added value of lichee becomes a urgent problem to be solved.
Since ancient times, the various health-care effects of lichee are all recorded in many ancient medical books, Compendium of Material Medica is recorded: " often feeding
Scrofula is treated in lichee energy cerebrum tonifying body-building, and whet the appetite beneficial spleen；Drying energy tonifying primordial Qi, can be used as the tonic of puerpera and old weak person "；" the southern regions of the Yunnan Province
Book on Chinese herbal medicine " claim its " warm tonifying spleen essence, nourish liver blood ".However, so far, for material base with health role in lichee with
And its mechanism of action of performance bioactivity also lacks and clearly recognizes.Existing research shows in litchi pulp rich in Polyphenols
Substance, and find its mouse liver rouge that there is the damage of mouse liver caused by mitigating restraint stress, inhibit high lipid diet induction
The effects of fat denaturation, reduction blood lipid level.However, used is all the extract of litchi pulp polyphenol in the studies above,
Its polyphenol components complicated composition.Phenolic substances is a major class Secondary metabolites, and different compounds is because of its structure
Differential activities have apparent difference.Although the studies above has found that litchi pulp polyphenol has the biology of protect liver, reducing blood lipid etc.
Activity, wherein specific action component is but and indefinite.
In order to verify the effective active composition in certain raw materials, traditional Research Thinking is to utilize phytochemical method will
It gradually isolates and purifies the monomer prepared one by one, then is compared one by one to its activity, finds out wherein active strongest one
A or several ingredients regard as main active constituent in the raw material.However, recent research viewpoint thinks active substance of plant
Play pharmacological activity is frequently not the collective effect of certain single chemical component but one group of components group.Using the above method
The organic association and reciprocation between ingredient can be isolated, it is difficult to embody the collective effects features such as collaboration or the antagonism of its generation.And
And such research work trivial operations, research cost are high, in some instances it may even be possible to cannot achieve because of experiment condition limitation.If by litchi
Branch pulp is divided into different phenols component groups, is compared respectively to its activity and not only allows for that certain contents are lower or independent work
The synergistic function that may have with the weaker ingredient of activity to main active is determined closer to extract effect effect
Effective phenols component group of fruit, and working efficiency can be improved in separation compound more one by one.
Summary of the invention
In order to overcome the drawbacks of the prior art, of the invention to be to overcome the deficiencies of the prior art and provide a kind of simplification
The purification process for inhibiting liver cell lipid aggregation main effect phenols component in litchi pulp, solving compound, separation prepares lichee one by one
The loaded down with trivial details time-consuming of pulp polyphenol active constituent, and it is easy the problem of ignoring the synergistic effect between compound.
The purpose of the present invention is achieved by the following technical programs:
Inhibit the purification process of liver cell lipid aggregation main effect phenols component in a kind of litchi pulp, which is characterized in that packet
Include following steps:
S1, litchi pulp polyphenol extract is prepared；
S2, the litchi pulp polyphenol extract of step S1 is dissolved in DMSO to formation concentration 600mg/mL solution, then with steaming
Distilled water is diluted to final concentration of 1mg/mL；
S3, it is splined on C18After silica gel column chromatography, successively use concentration of volume percent for 1~2% acetonitrile water (v/v)
Gradient elution is carried out with combination liquid, collects flow point, and tested and analyzed with HPLC, the similar flow point of merging map composition, described group
Closing liquid includes the methanol aqueous solution that the concentration of volume percent containing 0.2% glacial acetic acid is 10~35%；
1%, 2% acetonitrile eluent and 10%, 15% methanol/0.2% glacial acetic acid water elution amalgamation liquid eluent are collected,
20% methanol/0.2% glacial acetic acid water elution is collected separately, collects 25% and 30% methanol/0.2% glacial acetic acid water elution and closes
And liquid, 35% methanol/0.2% glacial acetic acid water elution is collected separately；
S4, after the eluent collected in step S3 is successively diluted 1.5~2 times with distilled water respectively, it is loaded to HPD100
Type large pore resin absorption column first uses distilled water flushing, then meoh eluate is eluted and collected with 100% methanol, depressurizes back respectively
It is freeze-dried after receiving methanol, obtains four different litchi pulp phenols component groups of composition, F1, F2, F3 and F4；
S5, merge F2 and F3.
Wherein, 10%, 15% methanol/0.2% glacial acetic acid water elution refers to dense containing percent by volume in step S3
The methanol of degree 10% and the aqueous solution of 0.2% glacial acetic acid and methanol and 0.2% ice second containing concentration of volume percent 15%
The aqueous solution of acid is collected after eluting respectively；20% methanol/0.2% glacial acetic acid water elution refers to containing concentration of volume percent
It is collected after the elution of the aqueous solution of 20% methanol and 0.2% glacial acetic acid；25% and 30% methanol/0.2% glacial acetic acid water elution
Amalgamation liquid refers to aqueous solution with the methanol containing concentration of volume percent 25% and 0.2% glacial acetic acid and contains volume basis
The aqueous solution of the methanol of specific concentration 30% and 0.2% glacial acetic acid is collected after eluting respectively；35% methanol/0.2% glacial acetic acid washing
De- liquid refers to be collected after the aqueous solution of the methanol containing concentration of volume percent 35% and 0.2% glacial acetic acid elutes respectively.
Litchi pulp polyphenol extract is after AB-8 macroreticular resin preliminary purification, ingredient or more complicated, the inside
Some are effective in inhibiting liver cell lipid aggregation for phenols component, some grind without activity if carrying out separation one by one
Study carefully and very loaded down with trivial details, and some very possible ingredients do not work individually can but enhance the effect of other ingredients, if
Using the method separated one by one, the effect of these complementary compounds will be ignored, therefore, the effective component being finally recovered
Effect can not reach the coefficient effect of crude extract.Based on such a thinking, we want to invent a kind of simplification
The active substance of plant purification process that can isolate main active.
By silicagel column on litchi pulp polyphenol extract, using different types of compound because of the difference and silicon of its polarity etc.
The adsorption capacity of rubber column gel column is different, carries out stepwise elution using the eluant, eluent of opposed polarity and is isolated.In conjunction with liquid chromatographic detection and
Activity analysis, find Ingredients Active that 1%, 2% acetonitrile eluent and 10% methanol-water, 15% methanol-water elute compared with
Low, this part merges (F1)；20% water methanol eluent part (F2), there are also 25% and 30% water methanol eluent portions
Divide the activity of (F3) all relatively good, 35% water methanol eluent part (F4) activity is also poor.
The subsequent process handled using HPD100 macroporous absorbent resin is primarily to slough the moisture in eluent.Due to
Above-mentioned eluent moisture content is higher, if carrying out revolving and concentration, because phenolic substances is more sensitive to temperature etc.,
It will cause more degradation, while energy consumption is also very big.Therefore, by eluent on macroporous absorbent resin, in eluent
Polyphenol components can be combined with resin, and the combining phenolic substances of methanol is recycled to elute, and meoh eluate can be with
Faster revolving removal organic solvent obtains target separate substance in turn.Eluent is made of distilled water before macroreticular resin loading
1.5~2 times of dilutions, in order to the concentration of organic solvent in sample solution is reduced, because will affect when organic solvent content is high
The absorption of phenolic substances and macroreticular resin, causes phenolic substances to be lost.
During determining effective active component, it is contemplated that some ingredients individually still may be other without obvious activity
To the synergistic function of other ingredients, therefore after determining F2 and F3 activity preferably, also with further reference to each section thick
Composition in extract is cooperated, and whether activity changes after F4 is added in observation again on the basis of F2 and F3, and discovery does not have
Its activity is further increased, accordingly, it is determined that F2 and F3 is the main phenol that litchi pulp polyphenol inhibits liver cell lipid aggregation out
Constituents, so finally being merged to this two parts.
Further, specific step is as follows by step S1:
S11, prepare litchi pulp polyphenolic extract: fresh lichee pulp is immersed in 95% edible alcohol of 0~4 DEG C of pre-cooling
In, supernatant is collected after centrifugation in homogeneous, and ethyl alcohol is recovered under reduced pressure under the conditions of 45~55 DEG C, and remaining aqueous solution is litchi pulp
Polyphenolic extract；
This part is mainly the stability that the temperature rotated will affect polyphenol, and temperature is excessively high to be easy to cause polyphenol oxidase and drop
Solution
S12, by litchi pulp polyphenolic extract obtained in step S11 after macroporous resin adsorption, distill water elution
To remove soluble sugar and other small molecule compounds, then with 65~75% ethanol elutions, ethanol eluate is collected, in 45~55
It DEG C is concentrated under reduced pressure, obtains buff concentrate, be litchi pulp polyphenol extract after vacuum freeze drying.
This step is mainly the yield that macroporous resin type will affect purifying phenolic substances, preferably AB-8 but is not limited to
A kind of this resin of AB-8, several resins such as HPD826 have preferable separating effect；The concentration of revolving as above will affect polyphenol
Stability.
Preferably, the solid-liquid ratio of fresh lichee pulp and 95% edible alcohol is 1: (2~3) w/ when impregnating in step S11
v。
The dosage for controlling edible alcohol herein is mainly considered to save raw material, increases solid-liquid ratio, such as increase and arrive 1:4~5
Afterwards, polyphenol recovery rate can be improved slightly, but waste too many alcohol, therefore suitably limited solid-liquid ratio.
Wherein, the loading condition of step S3: loading flow velocity is 5~6BV/h, applied sample amount and C18Silica filler mass ratio is 1:
80~100, it is preferred to use 15cm × 6.0cm, 40 μm of C18Silica gel column chromatography.1~2% acetonitrile water of step S3,10% methanol/
0.2% glacial acetic acid water, 15% methanol/0.2% glacial acetic acid water, 20% methanol/0.2% glacial acetic acid water, 25% methanol/0.2% ice
The elution volume of acetic acid water is all 3.5~4BV, and 30% methanol/0.2% glacial acetic acid water elution volume is 14~16BV, 35%
Methanol/0.2% glacial acetic acid water elution volume is 5~6BV, and elution flow rate is 5~6BV/h.
The mass ratio of applied sample amount and silica filler will affect final result, applied sample amount too low consumption when, uneconomical, applied sample amount is too
It is high and be more than that silicagel column adsorbance will cause sample loss.Effluent volume will affect final result, and the too small elution of volume is endless
Entirely, volume causes greatly very much waste of solvent.
Eluting solvent type will affect as a result, the change of solvent type and concentration will affect its polarity, wash to will affect
The composition of phenols component in de- liquid.
Distilled water in step S4 for flushing is 2.5~3BV.The elution flow rate of step S4 is 5~6BV/h.
Resin type will affect final result, and different types of resin is different to different types of compound adsorption effect,
If resin type is improper, the phenomenon that adsorption rate is low or resolution factor is low, so that final yield be made to reduce.
Compared with prior art, the invention has the following beneficial effects:
The present invention is obtained by the separation to 4 sections of different phenols components such as litchi pulp procyanidins, Flavonoid substances
The inhibiting effect for obtaining the liver cell inner lipid aggregation that this 4 sections of phenols components induce oleic acid, can be used for preparing regulating lipid metabolism,
Inhibit reducing blood lipid, liver-protecting medicine or the functional food of hepatic steatosis.
Detailed description of the invention
Fig. 1 is that the HPLC of C18 silica gel post separation litchi pulp difference phenols component group is composed；
Fig. 2 is the timeliness and dose-effect relationship figure that litchi pulp polyphenol extract inhibits HepG2 intracellular triglyceride (TG)；
Fig. 3 is the timeliness and dose-effect relationship figure that litchi pulp polyphenol extract inhibits the intracellular total cholesterol of HepG2 (TC)；
Fig. 4 is the timeliness and dose-effect relationship figure that the F1 that purifying obtains inhibits HepG2 intracellular triglyceride (TG)；
Fig. 5 is the timeliness and dose-effect relationship figure that the F1 that purifying obtains inhibits the intracellular total cholesterol of HepG2 (TC)；
Fig. 6 is the timeliness and dose-effect relationship figure that the F2 that purifying obtains inhibits HepG2 intracellular triglyceride (TG)；
Fig. 7 is the timeliness and dose-effect relationship figure that the F2 that purifying obtains inhibits the intracellular total cholesterol of HepG2 (TC)；
Fig. 8 is the timeliness and dose-effect relationship figure that the F3 that purifying obtains inhibits HepG2 intracellular triglyceride (TG)；
Fig. 9 is the timeliness and dose-effect relationship figure that the F3 that purifying obtains inhibits the intracellular total cholesterol of HepG2 (TC)；
Figure 10 is the timeliness and dose-effect relationship figure that the F4 that purifying obtains inhibits HepG2 intracellular triglyceride (TG)；
Figure 11 is the timeliness and dose-effect relationship figure that the F4 that purifying obtains inhibits the intracellular total cholesterol of HepG2 (TC)；
Figure 12 is the timeliness and dose-effect relationship figure that compound F2+F3 inhibits HepG2 intracellular triglyceride (TG)；
Figure 13 is the timeliness and dose-effect relationship figure that compound F2+F3 inhibits the intracellular total cholesterol of HepG2 (TC)；
Figure 14 is the timeliness and dose-effect relationship figure that compound F2+F3+F4 inhibits HepG2 intracellular triglyceride (TG)；
Figure 15 is the timeliness and dose-effect relationship figure that compound F2+F3+F4 inhibits the intracellular total cholesterol of HepG2 (TC)；
Specific embodiment
The present invention is made combined with specific embodiments below and further being elaborated, but embodiment is not the present invention
Any type of restriction.
Embodiment 1
The purification process for inhibiting liver cell lipid aggregation main effect phenols component in a kind of litchi pulp, includes the following steps:
S1, litchi pulp polyphenol extract is prepared, included the following steps:
S11, prepare litchi pulp polyphenolic extract: fresh lichee pulp is immersed in 95% edible alcohol of 4 DEG C of pre-coolings,
Supernatant is collected after centrifugation in homogeneous, and ethyl alcohol is recovered under reduced pressure under the conditions of 45 DEG C, and remaining aqueous solution is that litchi pulp polyphenol slightly mentions
Liquid；
S12, by litchi pulp polyphenolic extract obtained in step S11 after AB-8 macroporous resin adsorption, distilled water
Elution is to remove soluble sugar and other small molecule compounds, then uses 70% ethanol elution, and collection ethanol eluate subtracts in 45 DEG C
Pressure concentration obtains buff concentrate, is litchi pulp polyphenol extract after vacuum freeze drying；
S2, the litchi pulp polyphenol extract of step S1 is dissolved in DMSO to formation concentration 600mg/mL solution, then with steaming
Distilled water is diluted to final concentration of 1mg/mL；
S3,15cm × 6.0cm, 40 μm of C are splined on18After silica gel column chromatography, successively use concentration of volume percent for 1~
The methanol that 2% acetonitrile water (v/v) and concentration of volume percent is 10~35%/0.2% glacial acetic acid aqueous solution carries out gradient and washes
It is de-, collect flow point, and tested and analyzed with HPLC, merge map and form similar flow point, collect 1%, 2% acetonitrile eluent and
10%, 20% methanol/0.2% glacial acetic acid water elution is collected separately in 15% water methanol eluent amalgamation liquid, collects 25% He
30% methanol/0.2% glacial acetic acid water elution amalgamation liquid, is collected separately 35% methanol/0.2% glacial acetic acid water elution；
S4, by the eluent collected in step S3 successively respectively with distilled water dilute 1.5 times after, be loaded to 15cm ×
6.0cm, 60 μm of HPD100 type large pore resin absorption column first use 2.5BV distilled water flushing, then are eluted and received with 100% methanol
Collect meoh eluate, elution flow rate 5BV/h is freeze-dried after methanol is recovered under reduced pressure respectively, obtains four different litchis of composition
Branch pulp phenols component group, F1, F2, F3 and F4；
S5, merge F2 and F3.
The solid-liquid ratio of fresh lichee pulp and ethanol solution is 1: 2w/v when impregnating in step S11.
Wherein, the loading condition of step S3: loading flow velocity is 5BV/h, applied sample amount and C18Silica filler mass ratio is 1:
100.1~2% acetonitrile water, 10% methanol/0.2% glacial acetic acid water, the 15% methanol/0.2% glacial acetic acid water, 20% first of step S3
Alcohol/0.2% glacial acetic acid water, 25% methanol/0.2% glacial acetic acid water elution volume are all 3.5BV, 30% methanol/0.2% ice second
The elution volume of sour water is 14BV, and 35% methanol/0.2% glacial acetic acid water elution volume is 5BV, and elution flow rate is 5BV/h.
It is specific as follows below by its activity for collecting component of experiment detection:
1, the yield and phenols content of each phenols component group of litchi pulp: the yield accounting of four components groups of gained is
1.11:2.15:1:1.26.Its total phenol and general flavone content such as following table.
The yield accounting and phenols content of 1 litchi pulp difference phenols component group of table
*: percentage contribution rate of the components group to litchi pulp polyphenol；The different letter expression significant differences of same row (p <
0.05)
2, it each phenols component group free phenol composition of litchi pulp and content analysis: weighs suitable each sample to be tested and is dissolved in
It is 2mgmL that concentration is configured in methanol-1Solution, component analysis is carried out after 0.22 μm of membrane filtration.HPLC-DAD detection
Condition: mobile phase A is the acetic acid of 0.4% (v/v), and Mobile phase B is acetonitrile, and gradient is 0-40min B 5%-25%, 40-
45min B 25%-35%, 45-50min B 35%-50%, it is rear to run equilibration time 5min；Chromatographic column is Zorbax SB-
C18 reverse-phase chromatographic column (4.6mm × 100mm, 5 μm, Agilent company)；Column temperature is 30 DEG C；Flow velocity is 1.0mL/min；Sample introduction
Volume is 20 μ L；Detection wavelength is full wavelength scanner, using 280nm as main detection wavelength.
As seen from Figure 1, F1 component is mainly separated to the ingredient of appearance before No. 1 peak in LPP, in this section of the peak 1-4
Component purification into F2 component, and highest No. 5 peaks of content concentrate on F3 sections, and the ingredient after No. 5 peaks then concentrates branch to exist
F4 sections.
3, the influence that litchi pulp polyphenol extract assembles HepG2 lipid within endothelial cells: growth conditions are good
HepG2 cell is with 5 × 105/ mL concentration is inoculated in 12 porocyte culture plates, and every hole 1mL is placed in 37 DEG C, 5% CO2 incubator
Middle culture is for 24 hours.It when cell fusion is to 70%-80%, inhales and abandons old culture solution, 1mL 1%BSA serum-free is added into each hole
Culture medium Nature enemy 16h replaces LPP containing various concentration or F1-F4 difference litchi pulp phenols component group and 400 μM of oleic acid
1%BSA serum free medium, be placed in incubator and cultivate for 24 hours.Measure intracellular triglyceride (TG) and total cholesterol (TC)
Content.
Content of triglyceride measurement: after the completion of HepG2 cell is through respective handling, discarding culture solution, washed 2 times with PBS,
Using triglycerides (histocyte) enzymic measuring reagent box of Beijing Puli's lema gene Technology Co., Ltd., it is added into every hole
130 μ L lysates, mechanical shaking extraction 10min take appropriate supernatant to be transferred in 0.5mL centrifuge tube, 2000rpm/min centrifugation
5min, separation supernatant measure protein content with BCA method；Meanwhile taking 10 μ L supernatants that 190 μ L enzyme working solutions are added, at room temperature
10min is reacted, measures absorbance value at microplate reader 550nm, standard curve is drawn with glycerol standard items and obtains regression equation, according to
Light absorption value after example reaction calculates TG concentration, with TG content in every mg protein concentration correction group of cells.With oleic acid model group
Make reference, calculates each concentration samples group TG relative amount.
Total cholesterol enzymatic assays: after the completion of HepG2 cell is through respective handling, discarding culture solution, is washed 2 times with PBS,
With total cholesterol (histocyte) enzymic measuring reagent box of Beijing Puli's lema gene Technology Co., Ltd., it is added into every hole
130 μ L lysates, mechanical shaking extraction 10min take appropriate supernatant to be transferred in 0.5 mL centrifuge tube, 2000rpm/min centrifugation
5min, separation supernatant measure protein content with BCA method；Take 10 μ L supernatants that 190 μ L total cholesterol levels measurement enzyme work is added
Make liquid, reacts 20min at room temperature, measure absorbance value at microplate reader 550nm, draw standard curve, root with cholesterol standards
TC concentration in sample is calculated according to light absorption value, with TC content in every mg protein concentration correction group of cells.Joined with oleic acid model group work
Than calculating each concentration samples group TC relative amount.
Fig. 2, the 3 HepG2 intracellular triglycerides that oleic acid is induced for various concentration litchi pulp polyphenol extract (LPP)
(TG), the inhibiting effect of total cholesterol (TC) aggregation.The LPP of various concentration all has certain inhibition to lipid within endothelial cells deposition
Effect, but no significant difference between the inhibitory effect of each concentration.
Fig. 4~11 are that tetra- phenols component groups of F1~F4 that purifying obtains make the inhibition that HepG2 lipid within endothelial cells are assembled
With.On the whole, in 4 phenols component groups, F2 components group inhibits the effect of HepG2 intracellular TG deposition best, when 5 μ g/mL
Intracellular TG content has dropped 26.9%, and it is 28% that when 10 μ g/mL, which reaches maximal percentage inhibition, the two no significant difference, but with
Concentration further increases, and inhibitory effect gradually weakens instead.F3 is only second to F2, concentration to the intracellular TG inhibiting effect deposited
Intracellular TG content decline 26.2% when for 10 μ g/mL, close to the maximal percentage inhibition of F2；Reach maximum suppression when 20 μ g/mL
Rate processed is 28.5%.The inhibitory effect of F4 is relatively weak, and it is 19.3% that maximal percentage inhibition is just reached in 20 μ g/mL concentration.And
The inhibitory effect of F1 is worst, and when 40 μ g/mL just has certain inhibiting effect, and intracellular TG content only declines 11%.4 phenols
The inhibiting rate trend of components group TC content intracellular to HepG2 is consistent with the result of TG, and inhibiting effect from being also to weak ordering by force
F2>F3>F4>F1.Show that F2 and F3 components group has apparent inhibiting effect to oleic acid inducing hepatocyte lipid aggregation.
The comparison diagram result of 2,3 and Fig. 4~11 is it can be found that equally under 5 μ g/mL activities, tetra- ingredients of F1~F4
Group will be lower than LPP to the inhibiting effect of the liver cell inner lipid aggregation of oleic acid induction, prompt aforementioned four components group independent role
Without method interpretation LPP to the inhibiting effect of liver cell lipid aggregation, there may be collaboration or summation actions between each components group.Into
And according to the yield of each phenols component group, it is multiple by the mass ratio of 2.15:1 with F3 based on active best components group F2
Match, is compound F2+F3；Further F2, F3 are compounded with F4 by the mass ratio of 2.15:1:1.26, are compound F2+F3+
F4.By compound obtained above, it is evaluated respectively by above-mentioned same method, the oleic acid induction intracellular TG and TC of HepG2 is contained
The inhibiting effect of amount.
Figure 12~15 are the inhibiting effect that two kinds of compounds induce oleic acid the aggregation of HepG2 lipid within endothelial cells.As seen from the figure,
F2+F3 compound is populated with certain inhibiting effect to lipid within endothelial cells in 1 μ g/mL of low concentration, the model with oleic acid processing
Group is compared, and intracellular TG and TC content declines about 15%, but further increasing with concentration, is assembled to lipid within endothelial cells
Inhibiting effect do not further enhance, gradually weaken instead, when concentration reaches 4 μ g/mL unrestraint act on, show
When low concentration there is certain synergistic effect in F2 and F3.The inhibiting effect that F2+F3+F4 compound assembles HepG2 lipid within endothelial cells
It is similar with the result of F2 and F3 compound, show not showing more obvious synergistic effect when F4 and F2 and F3 collective effect.Cause
This, F2 and F3 are the main effect phenols component that litchi pulp inhibits liver cell lipid aggregation.
Embodiment 2
The purification process for inhibiting liver cell lipid aggregation main effect phenols component in a kind of litchi pulp, includes the following steps:
S1, litchi pulp polyphenol extract is prepared, included the following steps:
S11, prepare litchi pulp polyphenolic extract: fresh lichee pulp is immersed in 95% edible alcohol of 0 DEG C of pre-cooling,
The solid-liquid ratio of fresh lichee pulp and 95% edible alcohol is 1: 3w/v, and supernatant is collected after centrifugation in homogeneous, under the conditions of 55 DEG C
Ethyl alcohol is recovered under reduced pressure, remaining aqueous solution is litchi pulp polyphenolic extract；
S12, by litchi pulp polyphenolic extract obtained in step S11 after AB-8 macroporous resin adsorption, distilled water
Elution is to remove soluble sugar and other small molecule compounds, then uses 65% ethanol elution, and collection ethanol eluate subtracts in 55 DEG C
Pressure concentration obtains buff concentrate, is litchi pulp polyphenol extract after vacuum freeze drying.
S2, the litchi pulp polyphenol extract of step S1 is dissolved in DMSO to formation concentration 600mg/mL solution, then with steaming
Distilled water is diluted to final concentration of 1mg/mL；
S3,15cm × 6.0cm, 40 μm of C are splined on18After silica gel column chromatography, successively use concentration of volume percent for 1~
The methanol that 2% acetonitrile water (v/v) and concentration of volume percent is 10~35%/0.2% glacial acetic acid aqueous solution carries out gradient and washes
It is de-, collect flow point, and tested and analyzed with HPLC, merge map and form similar flow point, collect 1%, 2% acetonitrile eluent and
10%, 20% methanol/0.2% glacial acetic acid water elution is collected separately in 15% water methanol eluent amalgamation liquid, collects 25% He
30% methanol/0.2% glacial acetic acid water elution amalgamation liquid, is collected separately 35% methanol/0.2% glacial acetic acid water elution；
S4, by the eluent collected in step S3 successively respectively with distilled water dilute 1.5 times after, be loaded to 15cm ×
6.0cm, 60 μm of HPD100 type large pore resin absorption column first use 3BV distilled water flushing, then are eluted and collected with 100% methanol
Meoh eluate, elution flow rate 6BV/h are freeze-dried after methanol is recovered under reduced pressure respectively, obtain four different lichee of composition
Pulp phenols component group, F1, F2, F3 and F4；
S5, merge F2 and F3.
Wherein, the loading condition of step S3: loading flow velocity is 6BV/h, applied sample amount and C18Silica filler mass ratio is 1: 80,
It is preferred that using 15cm × 6.0cm, 40 μm of C18Silica gel column chromatography.1~2% acetonitrile water, 10% methanol/0.2% ice of step S3
Acetic acid water, 15% methanol/0.2% glacial acetic acid water, 20% methanol/0.2% glacial acetic acid water, 25% methanol/0.2% glacial acetic acid water
Elution volume be all 4BV, 30% methanol/0.2% glacial acetic acid water elution volume is 16BV, 35% methanol/0.2% ice second
The elution volume of sour water is 6BV, and elution flow rate is 6BV/h.
Embodiment 3
Other than the ethyl alcohol percent by volume in S12 step for elution is 75%, other are the same as embodiment 1.
Claims (7)
1. inhibiting the purification process of liver cell lipid aggregation main effect phenols component in a kind of litchi pulp, which is characterized in that including
Following steps:
S1, litchi pulp polyphenol extract is prepared；
S2, the litchi pulp polyphenol extract of step S1 is dissolved in formation concentration 600mg/mL solution in DMSO, then uses distilled water
It is diluted to final concentration of 1mg/mL；
S3, it is splined on C18After silica gel column chromatography, acetonitrile water (v/v) and combination of the concentration of volume percent for 1 ~ 2% are successively used
Liquid carries out gradient elution, collects flow point, and tested and analyzed with HPLC, merges map and forms similar flow point, the combination liquid packet
Include the methanol aqueous solution that the concentration of volume percent containing 0.2% glacial acetic acid is 10 ~ 35%；Collect 1%, 2% acetonitrile eluent
With 10%, 15% methanol/0.2% glacial acetic acid water elution amalgamation liquid eluent, 20% methanol/0.2% glacial acetic acid washing is collected separately
De- liquid, collects 25% and 30% methanol/0.2% glacial acetic acid water elution amalgamation liquid, 35% methanol/0.2% glacial acetic acid is collected separately
Water elution；
S4, after the eluent collected in step S3 is successively diluted 1.5 ~ 2 times with distilled water respectively, it is big to be loaded to HPD100 type
Macroporous adsorbent resin column first uses distilled water flushing, then meoh eluate is eluted and collected with 100% methanol, and first is recovered under reduced pressure respectively
It is freeze-dried after alcohol, obtains four different litchi pulp phenols component groups of composition, F1, F2, F3 and F4；
S5, merge F2 and F3.
2. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 1,
It is characterized in that, specific step is as follows by step S1:
S11, prepare litchi pulp polyphenolic extract: fresh lichee pulp is immersed in 95% edible alcohol of 0 ~ 4 DEG C of pre-cooling,
Supernatant is collected after centrifugation in matter, and ethyl alcohol is recovered under reduced pressure under the conditions of 45 ~ 55 DEG C, and remaining aqueous solution is that litchi pulp polyphenol is thick
Extract；
S12, by litchi pulp polyphenolic extract obtained in step S11 after macroporous resin adsorption, distill water elution to remove
It removes soluble sugar and other small molecule compounds, then with 65 ~ 75% ethanol elutions, collects ethanol eluate, depressurized in 45 ~ 55 DEG C
Concentration obtains buff concentrate, is litchi pulp polyphenol extract after vacuum freeze drying.
3. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 2,
It is characterized in that, the solid-liquid ratio of fresh lichee pulp and ethanol solution is 1: (2 ~ 3) w/v when impregnating in step S11.
4. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 3,
Be characterized in that, the loading condition of step S3: loading flow velocity is 5 ~ 6BV/h, applied sample amount and C18Silica filler mass ratio is 1: (80 ~
100).
5. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 1,
Be characterized in that, 1~2% acetonitrile water of step S3,10% methanol/0.2% glacial acetic acid water, 15% methanol/0.2% glacial acetic acid water,
20% methanol/0.2% glacial acetic acid water, 25% methanol/0.2% glacial acetic acid water elution volume are all 3.5 ~ 4BV, 30% methanol/
The elution volume of 0.2% glacial acetic acid water is 14 ~ 16BV, and 35% methanol/0.2% glacial acetic acid water elution volume is 5 ~ 6BV, elution
Flow velocity is 5 ~ 6BV/h.
6. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 1,
It is characterized in that, the distilled water in step S4 for flushing is 2.5 ~ 3BV.
7. inhibit the purification process of liver cell lipid aggregation main effect phenols component in litchi pulp according to claim 1,
It is characterized in that, the elution flow rate of step S4 is 5 ~ 6BV/h.
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