CN101444562A - Papaya anti-oxidant serum and preparation method thereof - Google Patents
Papaya anti-oxidant serum and preparation method thereof Download PDFInfo
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- CN101444562A CN101444562A CNA2008102372323A CN200810237232A CN101444562A CN 101444562 A CN101444562 A CN 101444562A CN A2008102372323 A CNA2008102372323 A CN A2008102372323A CN 200810237232 A CN200810237232 A CN 200810237232A CN 101444562 A CN101444562 A CN 101444562A
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Abstract
The invention discloses a papaya anti-oxidant serum, which comprises cyclodextrin inclusion compound of pawpaw natural antioxidant and carriers accepted in pharmacy. The papaya anti-oxidant serum consists of enzymolysis water extract and alcohol extract of the papaya, in which vitamin C and polyphenols are contained. Vitamin C contained in papaya anti-oxidant serum is no less than 2 milligram per gram, and the total polyphenols calculated in gallic acid is no less than 22 milligram. Therefore, the papaya anti-oxidant serum has the characteristics of high efficiency, durability and stable inoxidizability and easy storage and carrying, can be served as health food to eat, can used as cosmetics, animal food, food or medical mediate, and further can used for preparing antioxidation products; the invention also discloses a preparation method for the papaya anti-oxidant serum, which comprises seven steps of pulping, enzyme digestion, filtering, alcohol extraction, concentration, inclusion and pelletization, so as to be a system for efficiently, sufficiently and completely extracting papaya anti-oxidant, and the antioxidative activity can be effectively preserved.
Description
Technical field
The present invention relates to a kind of plant extract preparation, particularly a kind of papaya anti-oxidant serum also relates to the preparation method of this anti-oxidant serum.
Background technology
No matter antioxidant still is the external very strong free radical scavenging effect that all has in vivo, all has favorable effects at aspects such as antibacterial, antiviral, radioprotective, defying age, antitumor and anti-cardiovascular diseases.In recent years, flourish along with antioxidant, requirement to antioxidant is also more and more higher, various widely used synthetized oxidation preventive agents are because its genotoxic potential and carcinogenesis etc. are subjected to people's repulsion gradually, cause various countries scientific research personnel's great attention and from plant, seek antioxidant natural, efficient, low toxicity, become the inexorable trend of antioxidant development.From various plants, extract nearly hundred kinds of Natural antioxidants at present in succession, and proved conclusively it by external chemical assay and removed the oxygen-derived free radicals effect, as carotenoid, vitamin C, flavone etc., wherein most Natural antioxidants all are polyphenols.
But, previously the animal of the Natural antioxidant is intervened and crowd's clinical research result demonstration: the Natural antioxidant unification compound majority of purifying from plant has not been given play to the antioxidant activity and the relevant disease prophylactic-therapeutic effect of expection, directly edible former Study on plants result is inconsistent with early stage, trace it to its cause, may be relevant with the integrity that has destroyed former plant antioxidant system, thus be difficult to give play to the various active composition cooperative effect of former antioxidant system.Worldwide in recent years antioxidation dietary supplement is many to be that primary raw material is made with natural plant antioxidant crude extract, kept the dissimilar Natural antioxidants important more than 2 kinds as the Semen Vitis viniferae extract goods: flavonoid and proanthocyanidin, can remove HO and O simultaneously
2 -, fully suppressing lipid peroxidation, the effect of control oxidative damage relevant disease is better than traditional antioxidants and represents vitamin C.Therefore, natural plant antioxidant goods should keep the integrity of former plant antioxidant system as far as possible, make various oxidation-resistant active ingredients replenish mutually, coordinate, thereby make body have fairly perfect defense system and given play to its potential biological effect.
In addition, the antioxidant activity of natural plant antioxidant goods also is subjected to the influence of following several respects easily: in (1) preserving process after plant is gathered, because constantly carrying out of Repiration and enzymic catalytic reaction, its antioxidant system can form inevitably with content on variation, and the general trend that changes is to weaken oxidation resistance; (2) in the extraction and the course of processing, cellulose that plant contains and pectin may hinder the stripping of oxidation-resistant active ingredient, extraneous factors such as high temperature, ultraviolet, partial pressure of oxygen may cause the degraded or the volatilization of some oxidation-resistant active ingredient in the plant, the composition of antioxidant system and content are changed, reduce its antioxidant activity.Therefore, adopt advanced extraction process technology that the plant antioxidant system is carried out efficiently, fully, intactly extract, and prevent the degraded of oxidation-resistant active ingredient effectively or lose by post-production, to keeping the natural plant antioxidant activity, improving bioavailability and have crucial meaning.
Fructus Chaenomelis is the fruit of rosaceous plant chaenomeles lagenaria (Chaenomeles), mainly is divided into wrinkled papaya and Fructus Chaenomelis Chinesis two big classes, and wrinkled papaya mainly contains Fructus Chaenomelis, cloud Fructus Chaenomelis, hides Fructus Chaenomelis and Fructus Chaenomelis japonicae, and Fructus Chaenomelis Chinesis mainly contains mountain papaw.Fructus Chaenomelis is the integration of edible and medicinal herbs article, and it is not good to eat mouthfeel raw, makes the prepared slices of Chinese crude drugs more and uses, and has the effect of suppressing the hyperactive liver Shujin, stomach function regulating removing dampness.Modern component analysis shows that Fructus Chaenomelis contains rich dietary fiber and organic acid, especially contain a large amount of vitamin Cs, polyphenols (comprising chemical compounds such as flavonoid, tannin and phenolic acid) and superoxide dismutase (Superoxide dismutase, SOD).The inventor is through long-term a large amount of discovering, Fructus Chaenomelis extract has obvious antioxidation activity, but up to now, does not see the research report of relevant Fructus Chaenomelis Natural antioxidant goods both at home and abroad as yet.
Summary of the invention
In view of this, for overcoming the deficiencies in the prior art, one of purpose of the present invention is to provide a kind of papaya anti-oxidant serum, have efficient, lasting, stablize the antioxidative characteristics, and be easy to store, carry and use.
Two of purpose of the present invention is to provide a kind of method for preparing described papaya anti-oxidant serum, can efficiently, fully, intactly extract the Fructus Chaenomelis antioxidant system, and preserve its antioxidant activity effectively.
For achieving the above object, in a first aspect of the present invention, provide a kind of papaya anti-oxidant serum, comprise the cyclodextrin clathrate and the pharmaceutically acceptable carrier of the Fructus Chaenomelis Natural antioxidant; The described Fructus Chaenomelis Natural antioxidant is made up of enzymolysis water extract and the ethanol extract of Fructus Chaenomelis, comprises vitamin C and polyphenols; Every gram papaya anti-oxidant serum contains vitamin C and is no less than 2 milligrams, total polyphenols and is no less than 22 milligrams in gallic acid.
Further, described cyclodextrin is selected from alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, HP-or carboxymethyl-beta-cyclodextrin;
Further, described cyclodextrin is a gamma-cyclodextrin;
Further, described pharmaceutically acceptable carrier is selected from one or more in beta-schardinger dextrin-, dextrin and the starch;
Further, described pharmaceutically acceptable carrier is beta-schardinger dextrin-and dextrin.
In a second aspect of the present invention, a kind of method for preparing described papaya anti-oxidant serum is provided, may further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, be cut into small pieces after, be 1: 0.5～3 to add entry by the weight ratio of Fructus Chaenomelis and water, grinding makes the Fructus Chaenomelis pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and hydrolytic enzyme is 100: 0.2～10 adding hydrolytic enzyme, temperature be 30～50 ℃, pressure be-0.11～-carry out enzyme digestion reaction under the condition of 0.09Mpa, the time is 40～90 minutes, makes Fructus Chaenomelis enzymolysis pulp; The compound enzyme that described hydrolytic enzyme is selected from cellulase or is made up of pectase and cellulase;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be 1: 5～12 to add alcohols solvents by the w/v of filtering residue and alcohols solvent, reflux, extract, 1～2 time, each 1～2 hour, collect extracting solution, to filter, filtrate is the Fructus Chaenomelis ethanol extract; It is 50～100% methanol, ethanol or isopropyl alcohol that described alcohols solvent is selected from concentration expressed in percentage by volume;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/3～1/5 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 0.3～2 adding cyclodextrin, be-0.11 at pressure～-carry out inclusion reaction under the condition of 0.09Mpa, time is 4～6 hours, makes the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the pharmaceutically acceptable carrier of the step f gained Fructus Chaenomelis Natural antioxidant, be to push up spray fluidized bed granulation at 2～3: 1 by the weight ratio of cyclodextrin inclusion compound liquid and carrier, the gained granule is papaya anti-oxidant serum.
Further, hydrolytic enzyme is the compound enzyme of being made up of pectase and cellulase among the described step b, and the weight ratio of Fructus Chaenomelis pulp and this compound enzyme is 100: 2, the temperature of enzyme digestion reaction be 40 ℃, pressure for-0.09Mpa, time be 60 minutes;
Further, cyclodextrin is selected from alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, HP-or carboxymethyl-beta-cyclodextrin among the described step f;
Further, cyclodextrin is a gamma-cyclodextrin among the described step f, and the weight ratio of the Fructus Chaenomelis Natural antioxidant and gamma-cyclodextrin is 100: 0.5, the temperature of inclusion reaction be 20 ℃, pressure for-0.09Mpa, time be 4 hours;
Further, acceptable carrier is selected from one or more in beta-schardinger dextrin-, dextrin and the starch on the described step g Chinese materia medica, and the feed pump rotating speed of top spray fluidized bed granulation is that 4～8 rev/mins, fluidized bed body central temperature are that 45～65 ℃, air blast frequency are 3～7kHz.
Beneficial effect of the present invention is:
(1) the invention provides a kind of papaya anti-oxidant serum, have efficient, lasting, stablize the antioxidative characteristics, and be easy to store and carry, it is directly edible to can be used as anti-oxidation health food, also can be used as cosmetics, animal feed, food or pharmaceutical intermediate, further preparation has the product of anti-oxidation function.
(2) the invention provides a kind of method for preparing described papaya anti-oxidant serum, comprise slurrying, enzymolysis, filtration, alcohol extraction, concentrate, enclose, granulation totally 7 steps, can efficiently, fully, intactly extract Fructus Chaenomelis antioxidant system (main anti-oxidation active substance is vitamin C and polyphenols), and preserve its antioxidant activity effectively.
The cellulose of fresh Fructus Chaenomelis accounts for more than 4% of gross weight, adopts the conventional physical juice press method, and its crushing juice rate is very low, thereby causes the total amount of the oxidation-resistant active ingredient of stripping from cell not high, antioxidant system forms imperfect.The present invention adopts hydrolytic enzyme that the Fructus Chaenomelis pulp is carried out enzyme digestion reaction, can destroy the compact tissue cell wall that Fructus Chaenomelis is staggered to form, help in Cytoplasm and the vacuole oxidation-resistant active ingredient efficiently, fully, intactly stripping, extraction efficiency height, extracted amount are big, and reaction condition gentleness, energy consumption are low.Result of study shows that through enzymolysis processing, vitamin C and extraction of polyphenol substance total amount have improved 35.1% and 43.8% respectively in the Fructus Chaenomelis.In addition, the component analysis result of Fructus Chaenomelis extract shows, contains catechin, epicatechin, Quercetin, rutin, chlorogenic acid and procyanidin in the Fructus Chaenomelis extract.
Easy oxidation of antioxidant itself or degraded lose activity, the present invention adopts cyclodextrin inclusion technique, Fructus Chaenomelis natural anti-oxidation agent molecule bag is embedded in the void structure of cyclodextrin molecular and forms complex, can block contacting of the Fructus Chaenomelis Natural antioxidant and external environment effectively, prevent its oxidation, hydrolysis or volatilization, thereby increase antioxidative stabilizer, simultaneously, cyclodextrin inclusion compound also makes the Fructus Chaenomelis Natural antioxidant have slow-releasing, lastingly volatilization.The inventor has contrasted papaya anti-oxidant serum room temperature storage and fresh Fructus Chaenomelis keep in cold storage content and the activity change situation of anti-oxidation active substance in the process, the result shows, after storing nearly half a year, to retain rate higher for the content of anti-oxidation active substance and activity in the papaya anti-oxidant serum of the present invention, and vitamin C and total polyphenols content are respectively 2.2 times and 3.4 times of Fructus Chaenomelis fresh fruit; Vitamin C, total polyphenols and the SOD activity rate that retains exceeds Fructus Chaenomelis fresh fruit 10.7%, 8% and 2.3% respectively, points out papaya anti-oxidant serum of the present invention to have lastingly, stablize the antioxidative characteristics.The inventor also adopts gamma-cyclodextrin respectively the antioxidation unification compound that the Fructus Chaenomelis of having identified contains to be carried out enclose research, the result confirms, gamma-cyclodextrin all has clathration to vitamin C (furan enol form lactone structure), procyanidin (tannins polymer structure), chlorogenic acid (depside structure) and the rutin (flavonoid cellular construction) that contains in the Fructus Chaenomelis, and ascorbic enclose constant is 5.7 * 10
3, the enclose constant of polyphenols is 2.8 * 10
3～4.2 * 10
4Between, and increase with the molecular weight of polyphenols.The realization that cyclodextrin inclusion compound is modified helps the raising of processing stability, storage endurance and the bioavailability of follow-up granulation.In addition, in preparation method of the present invention, enzyme digestion reaction and inclusion reaction all are chosen under the condition of negative pressure and carry out, and the stability that this helps keeping the thermal sensitivity oxidation-resistant active ingredient prevents its degraded.
Spray granulation is that capsule core material is scattered in the capsule material solution, and by the thermal current atomizing, the solvent of pocket material is evaporated rapidly and makes the capsule material be set in the surface of capsule core material, forms microcapsule.The present invention combines spray granulation technology and fluid bed drying technology, adoption of innovation top spray fluidized bed granulation: pharmaceutically acceptable carrier (solid phase) rises from the bottom of material trough and is fluidized state, the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant sprays into fluid bed by the spray tip that is installed in diffusion (spraying) chamber, under the continuous stirring of dry air, hydrone in the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant forms fine membrane stage on solid phase carrier subparticle surface, gasification is discharged fast, thereby makes the cyclodextrin clathrate of the Fructus Chaenomelis Natural antioxidant be deposited on surface of solid phase carriers.Top spray fluid bed granulating process can guarantee that the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant carries out sufficient convection current with the dry air that rises under compressed-air actuated carrying, when mixing with solid phase carrier, cyclodextrin inclusion compound liquid at first runs into the thermal current preheating of lower temperature, higher levels of humidity, meeting the exsiccant hygropyrexia of leading develops to favourable process, thereby reduced normal " bed collapses " phenomenon that occurs in the spray granulation process, the problem of avoiding the effective ingredient total amount in the solid phase carrier absorption cyclodextrin inclusion compound liquid to reduce.In addition, top spray fluid bed granulating process also can improve " cocnentration factor " of oxidation-resistant active ingredient, shortens drying time, is easy to keep the stability of thermal sensitivity oxidation-resistant active ingredient.Discover, the every gram of papaya anti-oxidant serum that adopts top spray fluid bed granulating process to obtain contains vitamin C, total polyphenols and tannin content and is respectively 2.05 ± 0.09mg, 22.70 ± 1.57mg and 9.64 ± 0.65mg, has improved 219.8%, 344.2% and 348.4% before granulating.
The specific embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, will be described in detail the preferred embodiments of the present invention below.
The preparation of embodiment 1, papaya anti-oxidant serum
May further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, (1cm is cut into small pieces
3) after, be to add entry at 1: 0.5 by the weight ratio of Fructus Chaenomelis and water, grind, make pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and hydrolytic enzyme is the hydrolytic enzyme that adding in 100: 0.2 is made up of pectase and cellulase (weight ratio is 1: 1), under temperature to be 30 ℃, pressure for-0.11Mpa, mixing speed be 180 rev/mins condition, carry out enzyme digestion reaction, 90 minutes time, make Fructus Chaenomelis enzymolysis pulp;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be to add methanol at 1: 12 by the w/v of filtering residue and alcohols solvent, reflux, extract, 2 times, each 1 hour, collect extracting solution, to filter, filtrate is the Fructus Chaenomelis ethanol extract;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/5 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 0.3 adding o-cyclodextrin, under temperature to be 35 ℃, pressure for-0.09Mpa, mixing speed be 90 rev/mins condition, carry out inclusion reaction, time is 4 hours, makes the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the compound solid phase carrier of forming by beta-schardinger dextrin-and starch (weight ratio is 1: 3) of the step f gained Fructus Chaenomelis Natural antioxidant, weight ratio by cyclodextrin inclusion compound liquid and compound solid phase carrier is 2: 1, at the feed pump rotating speed is that 4 rev/mins, fluidized bed body central temperature are that 45 ℃, blower fan frequency are to push up the spray fluidized bed granulation under the condition of 3kHz, and the gained granule is papaya anti-oxidant serum.
The preparation of embodiment 2, papaya anti-oxidant serum
May further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, (1cm is cut into small pieces
3) after, be to add entry at 1: 1 by the weight ratio of Fructus Chaenomelis and water, grind, make pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and cellulase is 100: 6 adding cellulase, under temperature to be 40 ℃, pressure for-0.10Mpa, mixing speed be 220 rev/mins condition, carry out enzyme digestion reaction, 70 minutes time, make Fructus Chaenomelis enzymolysis pulp;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be that to add concentration expressed in percentage by volume at 1: 10 be 50% alcoholic solution by the w/v of filtering residue and alcohols solvent, reflux, extract, 2 times, each 1 hour, collect extracting solution, to filter, filtrate is the Fructus Chaenomelis ethanol extract;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/4 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 1 adding HP-, under temperature to be 30 ℃, pressure for-0.10Mpa, mixing speed be 120 rev/mins condition, carry out inclusion reaction, 5 hours time, make the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the compound solid phase carrier of forming by beta-schardinger dextrin-and dextrin (weight ratio is 1: 5) of the step f gained Fructus Chaenomelis Natural antioxidant, weight ratio by cyclodextrin inclusion compound liquid and compound solid phase carrier is 3: 1, at the feed pump rotating speed is that 5 rev/mins, fluidized bed body central temperature are that 55 ℃, blower fan frequency are to push up the spray fluidized bed granulation under the condition of 5kHz, and the gained granule is papaya anti-oxidant serum.
The preparation of embodiment 3, papaya anti-oxidant serum
May further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, (1cm is cut into small pieces
3) after, be to add entry at 1: 2 by the weight ratio of Fructus Chaenomelis and water, grind, make pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and hydrolytic enzyme is the hydrolytic enzyme that adding in 100: 2 is made up of pectase and cellulase (weight ratio is 1: 1), under temperature to be 40 ℃, pressure for-0.09Mpa, mixing speed be 200 rev/mins condition, carry out enzyme digestion reaction, 60 minutes time, make Fructus Chaenomelis enzymolysis pulp;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be that to add concentration expressed in percentage by volume at 1: 5 be 80% alcoholic solution by the w/v of filtering residue and alcohols solvent, reflux, extract, 1 time, the time is 2 hours, collects extracting solution, filters, filtrate is the Fructus Chaenomelis ethanol extract;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/3 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 0.5 adding gamma-cyclodextrins, under temperature to be 20 ℃, pressure for-0.09Mpa, mixing speed be 100 rev/mins condition, carry out inclusion reaction, 4 hours time, make the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the compound solid phase carrier of forming by beta-schardinger dextrin-and dextrin (weight ratio is 1: 3) of the step f gained Fructus Chaenomelis Natural antioxidant, weight ratio by cyclodextrin inclusion compound liquid and compound solid phase carrier is 2: 1, at the feed pump rotating speed is that 6 rev/mins, fluidized bed body central temperature are that 50 ℃, blower fan frequency are to push up the spray fluidized bed granulation under the condition of 4kHz, and the gained granule is papaya anti-oxidant serum.
The preparation of embodiment 4, papaya anti-oxidant serum
May further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, (1cm is cut into small pieces
3) after, be to add entry at 1: 3 by the weight ratio of Fructus Chaenomelis and water, grind, make pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and hydrolytic enzyme is the hydrolytic enzyme that adding in 100: 10 is made up of pectase and cellulase (weight ratio is 1: 1), under temperature to be 50 ℃, pressure for-0.09Mpa, mixing speed be 250 rev/mins condition, carry out enzyme digestion reaction, 40 minutes time, make Fructus Chaenomelis enzymolysis pulp;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be to add isopropyl alcohol at 1: 7 by the w/v of filtering residue and alcohols solvent, reflux, extract, 1 time, the time is 2 hours, collects extracting solution, filters, filtrate is the Fructus Chaenomelis ethanol extract;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/4 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 2 adding carboxymethyl-beta-cyclodextrins, under temperature to be 15 ℃, pressure for-0.11Mpa, mixing speed be 150 rev/mins condition, carry out inclusion reaction, time is 6 hours, makes the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the dextrin of the step f gained Fructus Chaenomelis Natural antioxidant, weight ratio by cyclodextrin inclusion compound liquid and dextrin is 2: 1, at the feed pump rotating speed is that 8 rev/mins, fluidized bed body central temperature are that 65 ℃, blower fan frequency are to push up the spray fluidized bed granulation under the condition of 7kHz, and the gained granule is papaya anti-oxidant serum.
The assay of the main anti-oxidation active substance of papaya anti-oxidant serum of the present invention
1, ascorbic assay
Method: adopt fluorescence method (national standard first method) to measure, 1. get the about 100mg of vitamin C reference substance, it is fixed accurately to claim, add Metaphosphoric acid-acetic acid solution and (take by weighing Metaphosphoric acid 15g, add glacial acetic acid 40mL and water 250mL, stir, placement is spent the night and is made dissolving, add water and be settled to 500mL, promptly) 100mL makes the solution that concentration is 1mg/mL, and precision is measured this solution 10mL, with the dilution of Metaphosphoric acid-acetic acid solution and be settled to 100mL, add activated active carbon 2g and carry out oxidation processes, filter, collect filtrate; Get each 5mL of filtrate, put respectively in 2 50mL volumetric flasks, wherein add mass percentage concentration in 1 volumetric flask and be 50% sodium acetate solution 5mL, water is settled to 50mL, precision is measured this solution 0.5,1.0,1.5,2.0mL, put respectively in the 10mL band plug scale test tube, be settled to 2mL with distilled water, as the standard solution of series concentration; Add boric acid-sodium acetate solution in another volumetric flask and (take by weighing boric acid 3g, add mass percentage concentration and be 50% sodium acetate solution 100mL and make dissolving, that is, face and use preceding preparation) 5mL, jolting 15 minutes, water is settled to 50mL, placed 2 hours for 4 ℃ in temperature, precision is measured this solution 0.5,1.0,1.5,2.0mL, puts respectively in the 10mL band plug scale test tube, be settled to 2mL with distilled water, as the standard blank solution of series concentration; 2. get the about 10g of papaya anti-oxidant serum of the present invention, the accurate title, decide, and adds Metaphosphoric acid-acetic acid solution dissolving and be settled to 100mL, adds activated active carbon 2g and carry out oxidation processes, filters, and collects filtrate; Get each 5mL of filtrate, put respectively in 2 50mL volumetric flasks, wherein add mass percentage concentration in 1 volumetric flask and be 50% sodium acetate solution 5mL, water is settled to 50mL, as sample solution; Add boric acid-sodium acetate solution 5mL in another volumetric flask, jolting 15 minutes, water is settled to 50mL again, places 2 hours for 4 ℃ in temperature, as sample blank solution; 3. get above-mentioned standard solution, the standard blank solution, sample solution and each 2mL of sample blank solution, put respectively in the 10mL band plug scale test tube, (take by weighing o-phenylenediamine 20mg in each pipe, adding o-phenylenediamine solution under the lucifuge condition rapidly, be diluted with water to 100mL before the Yu Linyong, i.e.) 5mL, jolting mixes, at room temperature reacted 35 minutes, use excitation wavelength 338nm, wavelength of transmitted light 420nm measures fluorescence intensity, Vitamin C content with standard solution is an abscissa, the corresponding actual fluorescence intensity difference of corresponding standard blank fluorescence intensity of solution (fluorescence intensity of standard solution with) is a vertical coordinate drawing standard curve, try to achieve regression equation, the actual fluorescence intensity of solution (difference of the fluorescence intensity of sample solution and sample blank fluorescence intensity of solution) checks in ascorbic content the sample from standard curve per sample.
The result: the every gram of papaya anti-oxidant serum of the present invention contains vitamin C and is not less than 2mg.
2, the assay of total polyphenols
Method: adopt the Folin-Ciocalteu method to measure, 1. get the about 30mg of gallic acid reference substance, the accurate title, decide, add dehydrated alcohol 100mL and make the solution that concentration is 0.3mg/mL, precision is measured this solution 0,0.1,0.2,0.4,0.6,0.8,1.0mL, put respectively in the 45mL band plug graduated centrifuge tube, be settled to 10mL with distilled water, add Folin-Ciocalteu reagent 0.5mL again, with mass percentage concentration is that 7.5% Carbon Dioxide sodium solution is settled to 20mL, left standstill 30 minutes, as the standard solution of series concentration; 2. get the about 0.5g of papaya anti-oxidant serum of the present invention, the accurate title, decide, put in the 45mL band plug graduated centrifuge tube, the adding distil water dissolving also is settled to 10mL, add Folin-Ciocalteu reagent 0.5mL again, with mass percentage concentration is that 7.5% Carbon Dioxide sodium solution is settled to 20mL, leaves standstill 30 minutes, as sample solution; 3. get above-mentioned standard solution and sample solution, measure absorbance respectively at wavelength 675nm place, gallic acid content with standard solution is that abscissa, corresponding absorbance are vertical coordinate drawing standard curve, try to achieve regression equation, solution absorbency checks in the content of total polyphenols the sample solution from standard curve per sample.
The result: the every gram of papaya anti-oxidant serum of the present invention contains total polyphenols and is not less than 22mg in gallic acid.
The antioxidant activity research of papaya anti-oxidant serum of the present invention
1, external DPPH (hexichol is for bitterness hydrazides free radical) removes experiment
Method: get the about 1g of papaya anti-oxidant serum of the present invention, the accurate title, decide, add water 50mL and make the solution that concentration is 20mg/mL, precision pipettes this solution 50,100,200,400 μ L, be that 50% alcoholic acid Tris-HCl buffer is settled to 2mL with containing concentration expressed in percentage by volume respectively, as the sample solution of series concentration; Each 100 μ L of sample solution that precision pipettes above-mentioned variable concentrations put in the 96 hole ELISA Plate, and each concentration is established 3 multiple holes, and it is the DPPH test solution 100 μ L of 0.4mg/mL that every hole adds concentration; Vibrated 30 seconds, 37 ℃ of insulation reaction of temperature are after 20 minutes, measure light absorption value (Asca) at wavelength 517nm place, measure the DPPH blank solution light absorption value (Amax) do not add the sample blank solution light absorption value (Acon) of DPPH test solution and not add sample solution (is 50% alcoholic acid Tris-HCl buffer 100 μ L replacement sample to contain concentration expressed in percentage by volume) simultaneously, calculate free radical scavenging activity according to the following equation: free radical scavenging activity (%)=[1-(Asca-Acon)]/Amax * 100%; The relation of the concentration of solution and clearance rate is calculated IC per sample then
50(IC
50For the DPPH clearance rate is 50% o'clock required sample size).
The result: the every gram of papaya anti-oxidant serum of the present invention is removed the DPPH of 945 ± 20 μ g.
2, antioxidant activity research in the body
(1) to the influence of serum glutathion peroxidase (GSH-PX) level and Total antioxidant capacity (T-AOC)
Method: with 40 ApoE gene defection type (ApoE
-/-) male 2 monthly age mices (strain C57BL/6J) are divided into 5 groups at random, are respectively blank group, sample low dose group, sample high dose group, super fine tea powder group, vitamin C group; Each is organized mice and divides cage to feed the drinking-water of freely ingesting in SPF level animal feeding room laminar-flow rack; With common nutrient fodder is normal feedstuff, and sample low dose group and high dose group are added mass percentage concentration respectively in normal feedstuff be 5% and 10% papaya anti-oxidant serum of the present invention; The super fine tea powder group is added mass percentage concentration in normal feedstuff be 3% ultrafine green tea powder (every g ultrafine green tea powder contains total polyphenols and counts 35mg with gallic acid); The vitamin C group is added mass percentage concentration in normal feedstuff be 0.02% vitamin C; The sample low dose group is identical with the total polyphenols addition of super fine tea powder group, and the sample high dose group is identical with the vitamin C addition of vitamin C group; The protein content of respectively organizing feedstuff with the casein adjustment is 18%, and the fat content of adjusting feedstuff with Adeps Sus domestica is 20%; Record mice food-intake and body weight change, fed for 16 weeks after, mice is plucked eyeball and gets blood, separation of serum is measured GSH-P
XContent and T-AOC.
The result: compare with the blank group, the sample low dose group makes ApoE respectively
-/-Mice serum GSH-P
XImproved 30.0% and 53.3% with T-AOC; The sample high dose group makes ApoE respectively
-/-Mice serum GSH-P
XImproved 47.1% and 126.7% with T-AOC, show that papaya anti-oxidant serum of the present invention has anti-oxidation efficacy in the good organism, can significantly improve the serum oxidation resistance, the generation that atherosclerosis (AS) is taken place the key risk factor OxLDL ELISA (ox-LDL) of development has inhibitory action potentially.Simultaneously, papaya anti-oxidant serum of the present invention and Folium Camelliae sinensis and vitamin C with clear and definite anti-oxidation efficacy are compared, the anti-oxidation efficacy of sample low dose group is close with the super fine tea powder group, be better than the vitamin C group, the anti-oxidation efficacy of sample high dose group is better than the super fine tea powder group, and littler than super fine tea powder to the appetite influence of mice, point out papaya anti-oxidant serum zest of the present invention little, safe and reliable.
(2) to the influence of (MDA) level of malonaldehyde in the body and SOD vigor
Method: (8～10 ages in week, body weight 18～20g) are divided into 6 groups at random with 60 Kunming kind male white mouses, every group 10,1 blank group, 1 model control group and 4 sample dose groups, 4 sample dose groups are irritated stomach respectively, and to give mass percentage concentration be 4%, 6%, 8% and 10% papaya anti-oxidant serum 0.1mL/kg/d of the present invention, and blank group and model control group all give the equivalent distilled water, after 30 days, except that the blank group, each group gives 5Gy
60The disposable total irradiation of Co gamma-rays, the irradiation back was put to death and is respectively organized mice on the 3rd day, plucked eyeball and got blood, and separation of serum is cut open the belly and is got brain, liver, heart and kidney, measures MDA content and SOD activity in serum and the tissue.
The result: interior MDA content of mice body and SOD activity all are higher than the blank group in the model control group, prompting modeling success; MDA content and SOD activity in each sample dose group in mice serum and the tissue significantly are lower than model control group (P＜0.05), show that papaya anti-oxidant serum of the present invention can effectively reduce MDA content and SOD activity in the mice body, and present the doses effect relation.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (10)
1, a kind of papaya anti-oxidant serum is characterized in that: the cyclodextrin clathrate and the pharmaceutically acceptable carrier that comprise the Fructus Chaenomelis Natural antioxidant; The described Fructus Chaenomelis Natural antioxidant is made up of enzymolysis water extract and the ethanol extract of Fructus Chaenomelis, comprises vitamin C and polyphenols; Every gram papaya anti-oxidant serum contains vitamin C and is no less than 2 milligrams, total polyphenols and is no less than 22 milligrams in gallic acid.
2, papaya anti-oxidant serum according to claim 1 is characterized in that: described cyclodextrin is selected from alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, HP-or carboxymethyl-beta-cyclodextrin.
3, papaya anti-oxidant serum according to claim 2 is characterized in that: described cyclodextrin is a gamma-cyclodextrin.
4, papaya anti-oxidant serum according to claim 1 is characterized in that: described pharmaceutically acceptable carrier is selected from one or more in beta-schardinger dextrin-, dextrin and the starch.
5, papaya anti-oxidant serum according to claim 4 is characterized in that: described pharmaceutically acceptable carrier is beta-schardinger dextrin-and dextrin.
6, a kind of method for preparing the described papaya anti-oxidant serum of claim 1 is characterized in that: may further comprise the steps:
A, slurrying: with fresh Fructus Chaenomelis clean, enucleation, be cut into small pieces after, be that 1:0.5～3 add entry by the weight ratio of Fructus Chaenomelis and water, grinding makes the Fructus Chaenomelis pulp;
B, enzymolysis: in step a gained Fructus Chaenomelis pulp, weight ratio by Fructus Chaenomelis pulp and hydrolytic enzyme is that 100:0.2～10 add hydrolytic enzyme, temperature be 30～50 ℃, pressure be-0.11～-carry out enzyme digestion reaction under the condition of 0.09Mpa, the time is 40～90 minutes, makes Fructus Chaenomelis enzymolysis pulp; The compound enzyme that described hydrolytic enzyme is selected from cellulase or is made up of pectase and cellulase;
C, filtration: step b gained Fructus Chaenomelis enzymolysis pulp 100 orders are filtered, collect filtrate and filtering residue respectively, filtrate is Fructus Chaenomelis enzymolysis water extract;
D, alcohol extraction: in step c gained filtering residue, be 1: 5～12 to add alcohols solvents by the w/v of filtering residue and alcohols solvent, reflux, extract, 1～2 time, each 1～2 hour, collect extracting solution, to filter, filtrate is the Fructus Chaenomelis ethanol extract; It is 50～100% methanol, ethanol or isopropyl alcohol that described alcohols solvent is selected from concentration expressed in percentage by volume;
E, concentrated: step c gained Fructus Chaenomelis enzymolysis water extract and steps d gained Fructus Chaenomelis ethanol extract are mixed, be concentrated into 1/3～1/5 of cumulative volume, promptly get the Fructus Chaenomelis Natural antioxidant;
F, enclose: in the Natural antioxidant of step e gained Fructus Chaenomelis, weight ratio by the Fructus Chaenomelis Natural antioxidant and cyclodextrin is 100: 0.3～2 adding cyclodextrin, be-0.11 at pressure～-carry out inclusion reaction under the condition of 0.09Mpa, time is 4～6 hours, makes the cyclodextrin inclusion compound liquid of the Fructus Chaenomelis Natural antioxidant;
G, granulation: with the cyclodextrin inclusion compound liquid and the pharmaceutically acceptable carrier of the step f gained Fructus Chaenomelis Natural antioxidant, be to push up spray fluidized bed granulation at 2～3: 1 by the weight ratio of cyclodextrin inclusion compound liquid and carrier, the gained granule is papaya anti-oxidant serum.
7, the method for preparing papaya anti-oxidant serum according to claim 6, it is characterized in that: the compound enzyme of hydrolytic enzyme for forming among the described step b by pectase and cellulase, the weight ratio of Fructus Chaenomelis pulp and this compound enzyme is 100: 2, the temperature of enzyme digestion reaction be 40 ℃, pressure for-0.09Mpa, time be 60 minutes.
8, the method for preparing papaya anti-oxidant serum according to claim 6, it is characterized in that: cyclodextrin is selected from alpha-cyclodextrin, beta-schardinger dextrin-, gamma-cyclodextrin, HP-or carboxymethyl-beta-cyclodextrin among the described step f.
9, the method for preparing papaya anti-oxidant serum according to claim 8, it is characterized in that: cyclodextrin is a gamma-cyclodextrin among the described step f, the weight ratio of the Fructus Chaenomelis Natural antioxidant and gamma-cyclodextrin is 100: 0.5, the temperature of inclusion reaction be 20 ℃, pressure for-0.09Mpa, time be 4 hours.
10, the method for preparing papaya anti-oxidant serum according to claim 6, it is characterized in that: acceptable carrier is selected from one or more in beta-schardinger dextrin-, dextrin and the starch on the described step g Chinese materia medica, and the feed pump rotating speed of top spray fluidized bed granulation is that 4～8 rev/mins, fluidized bed body central temperature are that 45～65 ℃, air blast frequency are 3～7kHz.
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| CN103380928A (en) * | 2013-06-11 | 2013-11-06 | 十堰渝川食品有限公司 | Shine skin papaya-chrysanthemum-wolfberry composite beverage and preparation method thereof |
| CN114306726A (en) * | 2021-12-17 | 2022-04-12 | 广西萌大夫生物技术有限公司 | Preparation method and use method of injectable bionic antioxidant hydrogel |
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| CN100372556C (en) * | 2005-12-29 | 2008-03-05 | 谢君 | Active component extraction method of plant polyphenol kind substance |
| CN100422205C (en) * | 2006-06-29 | 2008-10-01 | 宁蒗女儿珍生物工程有限公司 | Method for preparing oleanolic acid from wrinkled papaya |
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| CN103380928A (en) * | 2013-06-11 | 2013-11-06 | 十堰渝川食品有限公司 | Shine skin papaya-chrysanthemum-wolfberry composite beverage and preparation method thereof |
| CN103380928B (en) * | 2013-06-11 | 2015-03-18 | 十堰渝川食品有限公司 | Shine skin papaya-chrysanthemum-wolfberry composite beverage and preparation method thereof |
| CN114306726A (en) * | 2021-12-17 | 2022-04-12 | 广西萌大夫生物技术有限公司 | Preparation method and use method of injectable bionic antioxidant hydrogel |
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