CN109806285A - One kind having active moringa oleifera leaf extractive of anti-trioxypurine and the preparation method and application thereof - Google Patents
One kind having active moringa oleifera leaf extractive of anti-trioxypurine and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses one kind to have active moringa oleifera leaf extractive of anti-trioxypurine and the preparation method and application thereof.It will pulverize and sieve after fresh leaf of Moringa microwave drying, using water-ethanol refluxing extraction, targetedly extract phenolic substances in leaf of Moringa, after centrifugation, alcohol extracting thing is concentrated under reduced pressure to obtain in supernatant；After residual residue drying, it is removed slag using alkaline process heat treatment, protease hydrolyzed, high speed centrifugation, supernatant ultrafiltration impurity elimination, secondary protease hydrolyzed, high speed centrifugation, supernatant reduced pressure, alcohol precipitation impurity elimination, secondary pressure concentration, targetedly extract polypeptides matter in leaf of Moringa, finally alcohol extracting thing is added in compounding, spray drying obtains a kind of with the active moringa oleifera leaf extractive of anti-trioxypurine.Extract aldehydes matter content > 10%, peptide matters content > 40% have good anti-trioxypurine effect in animal level.Extraction process of the present invention is simple, and entire process flow can reach food-grade requirement, can be applied to the fields such as drug, health care product and food.
Description
Technical field
The invention belongs to leaf of Moringa deep processings, high-valued field, are related to a kind of with the active leaf of Moringa extraction of anti-trioxypurine
Object and the preparation method and application thereof.
Background technique
Under normal purine diet state, non-empty stomach serum uric acid level male is higher than 420 μm of ol/L, Nv Xinggao twice on the same day
It is known as hyperuricemia in 360 μm of ol/L.Gout is a kind of metabolism made of developing on the biochemical basis of hyperuricemia
Property disease, hyperuricemia is the important biochemical basis of gout, and causes urarthritis, gouty nephropathy and tophus
The reason of deposition.In addition to this, hyperuricemia is related to hypertension, hyperlipidemia, obesity disease, coronary artery disease etc., is
The key risk factor of above-mentioned illness.With further increasing for living standards of the people, the disease incidence of hyperuricemia and gout
It is gradually increasing, age of onset shifts to an earlier date.Currently, China adult hyperuricemia disease incidence is 18%, gout disease incidence is about 2%,
And it is just increased sharply with 9.7% annual growth.Therefore, exploitation have anti-trioxypurine activity, the low health food of toxic side effect with
Drug has significant economic results in society.
Moringa (Moringa oleifera Lam.) is a kind of Moringaceae Moringa torrid zone fallen leaves xylophyta, in China
The ground such as Guangdong, Guangxi, Hainan, Sichuan and Yunnan large-scale plantation, has formd the raw material planting base of scale.Leaf of Moringa in
It is approved within 2012 new resource food, rich in nutrition content, protein content is 27% or so, and total sugar content is 15% left
In addition to this polyphenol, steroids are also contained in the right side, alkaloid isoreactivity ingredient has biggish potentiality to be exploited.In recent years research
It was found that leaf of Moringa have the effects that it is anti-inflammatory, anti-oxidant, antibacterial.
However, less about the relevant report with the active moringa oleifera leaf extractive of anti-trioxypurine.The present invention provides one kind
The preparation method of moringa oleifera leaf extractive, and its internal anti-trioxypurine activity is verified by animal model.
Summary of the invention
The purpose of the present invention is to provide one kind to have active moringa oleifera leaf extractive of anti-trioxypurine and preparation method thereof, obtains
With the active moringa oleifera leaf extractive of good anti-trioxypurine.The extract has good anti-trioxypurine function by zoopery verifying
Effect.Extraction process of the present invention is simple, entire process flow up to food-grade requirement, can be applied to drug, health care product and food
In equal fields.
Technical scheme is as follows.
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine will crush after fresh leaf of Moringa microwave drying
Sieving targetedly extracts phenolic substances in leaf of Moringa using water-ethanol refluxing extraction, and after centrifugation, supernatant is concentrated under reduced pressure
Obtain alcohol extracting thing；After residual residue drying, removed slag using alkaline process heat treatment, protease hydrolyzed, high speed centrifugation, supernatant ultrafiltration is gone
Miscellaneous, secondary protease hydrolyzed, high speed centrifugation, supernatant reduced pressure, alcohol precipitation impurity elimination, secondary pressure concentration, targetedly mention
Polypeptides matter in leaf of Moringa is taken, finally alcohol extracting thing is added in compounding, and spray drying obtains a kind of active peppery with anti-trioxypurine
The wooden leaf extract, extract aldehydes matter content > 10%, peptide matters content > 40% have good in animal level
Anti-trioxypurine effect.
Technical solution of the present invention comprises the following specific steps that:
(1) microwave drying: by fresh leaf of Moringa microwave drying, dry leaf of Moringa is obtained；
(2) it pulverizes and sieves: by dry leaf of Moringa grinding and sieving obtained by step (1), obtaining Moringa leaf dried powder；
(3) water-ethanol refluxing extraction: Moringa leaf dried powder obtained by step (2) is mixed with certain density ethanol water,
It at the uniform velocity stirs, heating and refluxing extraction, is centrifuged, supernatant is taken to obtain extracting solution 1, precipitating dries to obtain residue 1；
(4) it is concentrated under reduced pressure: extracting solution 1 being concentrated under reduced pressure, alcohol extract 2 is obtained；
(5) alkaline process is heat-treated: residue 1 being mixed with deionized water, carries out first time stirring, sodium hydroxide is added and adjusts
PH carries out second of heating stirring, obtains suspension 1；
(6) protease hydrolyzed: being added protease in suspension 1, and constant temperature stirring is digested, and enzyme deactivation obtains suspension 2；
(7) high speed centrifugation: after 2 high speed centrifugation of suspension, supernatant is taken to obtain extracting solution 3；
(8) ultrafiltration impurity elimination: ultrafiltration membrane ultra-filtration and separation extracting solution 3 is used, macromolecular trapped fluid is discarded, small molecule is taken to filter out
Liquid obtains extracting solution 4, and extracting solution 5 is concentrated under reduced pressure to obtain；
(9) secondary protease hydrolyzed: being added protease in extracting solution 5, and constant temperature stirring is digested, and enzyme deactivation must be suspended
Liquid 3；
(10) high speed centrifugation: after 3 high speed centrifugation of suspension, supernatant is taken to obtain extracting solution 6；
(11) it is concentrated under reduced pressure: extracting solution 6 being concentrated under reduced pressure, extracting solution 7 is obtained；
(12) alcohol precipitation impurity elimination: the dehydrated alcohol of pre-cooling being added into extracting solution 7, and after at the uniform velocity stirring, centrifugation takes supernatant to obtain
Extracting solution 8；
(13) secondary pressure is concentrated: extracting solution 8 being concentrated under reduced pressure, enzyme-extracting solution 9 is obtained；
(14) it compounds: enzyme-extracting solution 9 obtained by alcohol extract 2 obtained by step (4) and step (13) is mixed, heating is stirred
It mixes, obtains mixed extract 10；
(15) it is spray-dried: mixed extract 10 being spray-dried, moringa oleifera leaf extractive is obtained.
Further, in step (1), 1~3min of the microwave drying time, power is 400~600W.
Further, in step (2), the sieving was 40 meshes.
Further, in step (3), in the ethanol water concentration of alcohol be 60~80vol%, Moringa leaf dried powder with
The solid-liquid ratio of ethanol water is 1:6~10g/mL, and stirring rate is 120~180r/min, and reflux temperature is 80~90 DEG C, is returned
The stream time is 1~3h, and the centrifugation is that 4000~6000g is centrifuged 20~30min, and the drying temperature of residue is 50~60 DEG C.
Further, in step (4), the reduced pressure temperature is 50~60 DEG C, and solid content is 30 in concentrate
~40wt%.
Further, in step (5), the residue 1 and deionized water solid-liquid ratio are 1:8~1:16g/mL, are stirred for the first time
Mixing the time is 15~25min, and the pH value is 7.0~8.0, and stirring rate is 120~180r/min twice, second of stirring
Time is 20~40min, and second of whipping temp is 50~60 DEG C.
Further, in step (6), the additional amount of the protease is the 2~5% of mass of residue, and protease is alkalinity
Protease and compound protease, the 50~70% of the total enzyme amount of additional amount Zhan of alkali protease, the additional amount of compound protease accounts for
The 30~50% of total enzyme amount, the alkali protease is the protease that Novi believes model NS37071, and compound protease is Novi
The compound protease (Protamex) of letter company, the hydrolysis temperature are 52~58 DEG C, and enzymolysis time is 8~16h, stirring rate
For 120~180r/min, the enzyme deactivation is in 90~96 DEG C of enzyme deactivation 20-30min.
Further, step (7), in (10), the high speed centrifugation is that 6000~8000g is centrifuged 15~25min.
Further, in step (8), the ultrafiltration membrane molecular weight of the ultrafiltration removal of impurities is 10000Da, and ultrafiltration number is 4~6
Secondary, the extracting solution 4 is the ultrafiltration filter liquor of molecular weight < 10000Da, and the reduced pressure temperature is 50~60 DEG C, extracting solution
Solid content is 8~10wt% in 5.
Further, in step (9), the additional amount of the protease is the 3~6% of 5 solid content of extracting solution, albumen
Enzyme be pancreatin and compound protease, the 60~80% of the total enzyme amount of additional amount Zhan of pancreatin, the total enzyme of additional amount Zhan of compound protease
The 20~40% of amount, the pancreatin are the pancreatin of Nanning Pang Bo bio-engineering corporation, and compound protease is answering for Novozymes Company
Hop protein enzyme (Protamex).The hydrolysis temperature be 40~50 DEG C, enzymolysis time be 2~4h, stirring rate be 120~
180r/min, the enzyme deactivation are in 90~96 DEG C of enzyme deactivation 20-30min.
Further, step (11), in (13), the reduced pressure temperature is 50~60 DEG C, and solid content contains in concentrate
Amount is 30~40wt%.
Further, in step (12), the dehydrated alcohol of the pre-cooling is the dehydrated alcohol for being cooled to 4~8 DEG C in advance, anhydrous second
The additional amount of alcohol is that ethanol content in system is made to reach 10~20wt%, and the at the uniform velocity stirring is 120~180r/min at room temperature
Revolving speed stirs 3~5h, and the high speed centrifugation is that 6000~8000g is centrifuged 20~30min.
Further, in step (14), the stirring rate is 120~180r/min, and mixing time is 30~50min,
Temperature is 65~85 DEG C.
There is the active moringa oleifera leaf extractive of anti-trioxypurine by above method preparation the present invention provides a kind of.
The present invention also provides a kind of moringa oleifera leaf extractives to prepare the application in anti-trioxypurine health care product and drug.
The invention has the advantages that and effect:
(1) what preparation method of the invention was prepared a kind of mainly polyphenol containing leaf of Moringa and polypeptide has good anti-trioxypurine
The moringa oleifera leaf extractive of effect, extract aldehydes matter content > 10%, peptide matters content > 40%.
(2) entire preparation process flow of the invention is up to food-grade requirement.
(3) present invention gained moringa oleifera leaf extractive has good by the verifying of hyperuricemia rat animal experimental model
Anti-trioxypurine activity, can be effectively reduced the serum uric acid of hyperuricemia rat.
Specific embodiment
For a better understanding of the invention, below by the concrete operations for specifically illustrating zoopery and specific embodiment to this hair
Bright to be described further, embodiments of the present invention are not limited thereto.
Embodiment 1
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, specifically comprises the following steps:
(1) microwave drying: under microwave power 400W, by fresh leaf of Moringa microwave drying 1min, dry leaf of Moringa is obtained.
(2) it pulverizes and sieves: dry leaf of Moringa obtained by step (1) being smashed it through into 40 meshes, obtains Moringa leaf dried powder.
(3) water-ethanol refluxing extraction: the ethanol water for being 60vol% by Moringa leaf dried powder obtained by step (2) and concentration of alcohol
Solution is mixed by solid-liquid ratio 1:6g/mL, under 120r/min at the uniform velocity stirring condition, refluxing extraction 1h at 80 DEG C, 4000g centrifugation
20min, takes supernatant to obtain extracting solution A11, and 50 DEG C of drying precipitate to obtain residue B11.
(4) be concentrated under reduced pressure: it is 30wt% that extracting solution A11 is concentrated under reduced pressure into solid content at 50 DEG C, obtains alcohol extracting
Liquid A12.
(5) alkaline process is heat-treated: residue B11 being mixed with deionized water by solid-liquid ratio 1:8g/mL, 120r/min is at the uniform velocity stirred
15min is added sodium hydroxide adjusting pH value to 120r/min at 7.0,50 DEG C and at the uniform velocity stirs 20min, obtains suspension C11.
(6) protease hydrolyzed: being added protease in suspension C11, and additional amount is the 2% of residue B11 mass, Novi's letter
The 50% of the total enzyme amount of additional amount Zhan of the alkali protease NS37071 of company, the additional amount of the compound protease of Novozymes Company
The 50% of the total enzyme amount of Zhan, 120r/min is at the uniform velocity stirred at 52 DEG C, digests 8h, and 90 DEG C of enzyme deactivation 20min obtain suspension C12.
(7) high speed centrifugation: after suspension C12 is using 6000g centrifugation 15min, supernatant is taken to obtain extracting solution A13.
(8) ultrafiltration impurity elimination: using ultrafiltration membrane ultra-filtration and separation extracting solution A13, and ultrafiltration membrane molecular weight is 10000Da, ultrafiltration time
Number is 4 times, discards macromolecular trapped fluid, takes small molecule filter liquor, obtains extracting solution A14, and 50 DEG C of reduced pressures obtain solid content
For the extracting solution A15 of 8wt%.
(9) secondary protease hydrolyzed: being added protease in extracting solution A15, and additional amount is extracting solution A15 solid content
3%, the 60% of the total enzyme amount of additional amount Zhan of the pancreatin of Nanning Pang Bo bio-engineering corporation, the compound protease of Novozymes Company
The total enzyme amount of additional amount Zhan 40%, 120r/min is at the uniform velocity stirred at 40 DEG C, digests 2h, and 90 DEG C of enzyme deactivation 20min obtain suspension
C13。
(10) high speed centrifugation: after suspension C13 is using 6000g centrifugation 15min, supernatant is taken, extracting solution A16 is obtained.
(11) be concentrated under reduced pressure: it is 30wt% that extracting solution A16 is concentrated under reduced pressure into solid content at 50 DEG C, obtains extracting solution
A17。
(12) alcohol precipitation impurity elimination: the pre- dehydrated alcohol for being cooled to 4 DEG C is added into extracting solution A17, makes the ethanol content in system
Reach 10wt%, after 120r/min at the uniform velocity stirs 3h, 6000g is centrifuged 20min, and supernatant is taken to obtain extracting solution A18.
(13) secondary pressure is concentrated: it is 30wt% that extracting solution A18 is concentrated under reduced pressure into solid content at 50 DEG C, obtains enzyme
Solve extracting solution A19.
(14) it compounds: by enzyme-extracting solution A19 obtained by alcohol extract A12 obtained by step (4) and step (13) at 65 DEG C,
120r/min at the uniform velocity stirs 30min, obtains mixed extract A110.
(15) it is spray-dried: mixed extract A110 being spray-dried, moringa oleifera leaf extractive 1, phenolic compound are obtained
Content is 11.4%, and peptide matters content is 42.9%.
Embodiment 2
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, specifically comprises the following steps:
(1) microwave drying: under microwave power 500W, by fresh leaf of Moringa microwave drying 2min, dry leaf of Moringa is obtained.
(2) it pulverizes and sieves: dry leaf of Moringa obtained by step (1) being smashed it through into 40 meshes, obtains Moringa leaf dried powder.
(3) water-ethanol refluxing extraction: the ethanol water for being 70vol% by Moringa leaf dried powder obtained by step (2) and concentration of alcohol
Solution is mixed by solid-liquid ratio 1:8g/mL, under 150r/min at the uniform velocity stirring condition, refluxing extraction 2h at 85 DEG C, 5000g centrifugation
25min, takes supernatant to obtain extracting solution A21, and 55 DEG C of drying precipitate to obtain residue B21.
(4) be concentrated under reduced pressure: it is 35wt% that extracting solution A21 is concentrated under reduced pressure into solid content at 55 DEG C, obtains alcohol extracting
Liquid A22.
(5) alkaline process is heat-treated: residue B21 being mixed with deionized water by solid-liquid ratio 1:12g/mL, 150r/min is at the uniform velocity stirred
20min is mixed, sodium hydroxide adjusting pH value is added to 150r/min at 7.5,55 DEG C and at the uniform velocity stirs 30min, obtains suspension C21.
(6) protease hydrolyzed: being added protease in suspension C21, and additional amount is the 3.5% of residue B21 mass, Novi
The 60% of the total enzyme amount of additional amount Zhan of the alkali protease NS37071 of letter company, the addition of the compound protease of Novozymes Company
The 40% of the total enzyme amount of Zhan is measured, 150r/min is at the uniform velocity stirred at 55 DEG C, digests 12h, and 93 DEG C of enzyme deactivation 25min obtain suspension C22.
(7) high speed centrifugation: after suspension C22 is using 7000g centrifugation 20min, supernatant is taken to obtain extracting solution A23.
(8) ultrafiltration impurity elimination: using ultrafiltration membrane ultra-filtration and separation extracting solution A23, and ultrafiltration membrane molecular weight is 10000Da, ultrafiltration time
Number is 5 times, discards macromolecular trapped fluid, takes small molecule filter liquor, obtains extracting solution A24, and 55 DEG C of reduced pressures obtain solid content
For the extracting solution A25 of 9wt%.
(9) secondary protease hydrolyzed: being added protease in extracting solution A25, and additional amount is extracting solution A25 solid content
4.5%, the 70% of the total enzyme amount of additional amount Zhan of the pancreatin of Nanning Pang Bo bio-engineering corporation, the compound protein of Novozymes Company
The 30% of the total enzyme amount of additional amount Zhan of enzyme, 150r/min is at the uniform velocity stirred at 45 DEG C, digests 3h, and 93 DEG C of enzyme deactivation 25min obtain suspension
C23。
(10) high speed centrifugation: after suspension C23 is using 7000g centrifugation 20min, supernatant is taken, extracting solution A26 is obtained.
(11) be concentrated under reduced pressure: it is 35wt% that extracting solution A26 is concentrated under reduced pressure into solid content at 55 DEG C, obtains extracting solution
A27。
(12) alcohol precipitation impurity elimination: the pre- dehydrated alcohol for being cooled to 6 DEG C is added into extracting solution A27, makes the ethanol content in system
Reach 15wt%, after 150r/min at the uniform velocity stirs 4h, 7000g is centrifuged 25min, and supernatant is taken to obtain extracting solution A28.
(13) secondary pressure is concentrated: it is 35wt% that extracting solution A28 is concentrated under reduced pressure into solid content at 55 DEG C, obtains enzyme
Solve extracting solution A29.
(14) it compounds: by enzyme-extracting solution A29 obtained by alcohol extract A22 obtained by step (4) and step (13) at 75 DEG C,
150r/min at the uniform velocity stirs 40min, obtains mixed extract A210.
(15) it is spray-dried: mixed extract A210 being spray-dried, moringa oleifera leaf extractive 2, phenolic compound are obtained
Content is 10.7%, and peptide matters content is 45.3%.
Embodiment 3
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, specifically comprises the following steps:
(1) microwave drying: under microwave power 600W, by fresh leaf of Moringa microwave drying 3min, dry leaf of Moringa is obtained.
(2) it pulverizes and sieves: dry leaf of Moringa obtained by step (1) being smashed it through into 40 meshes, obtains Moringa leaf dried powder.
(3) water-ethanol refluxing extraction: the ethanol water for being 80vol% by Moringa leaf dried powder obtained by step (2) and concentration of alcohol
Solution is mixed by solid-liquid ratio 1:10g/mL, under 180r/min at the uniform velocity stirring condition, refluxing extraction 3h at 90 DEG C, 6000g centrifugation
30min, takes supernatant to obtain extracting solution A31, and 60 DEG C of drying precipitate to obtain residue B31.
(4) be concentrated under reduced pressure: it is 40wt% that extracting solution A31 is concentrated under reduced pressure into solid content at 60 DEG C, obtains alcohol extracting
Liquid A32.
(5) alkaline process is heat-treated: residue B31 being mixed with deionized water by solid-liquid ratio 1:16g/mL, 180r/min is at the uniform velocity stirred
25min is mixed, sodium hydroxide adjusting pH value is added to 180r/min at 8.0,60 DEG C and at the uniform velocity stirs 40min, obtains suspension C31.
(6) protease hydrolyzed: being added protease in suspension C31, and additional amount is the 5% of residue B31 mass, Novi's letter
The 70% of the total enzyme amount of additional amount Zhan of the alkali protease NS37071 of company, the additional amount of the compound protease of Novozymes Company
The 30% of the total enzyme amount of Zhan, 180r/min is at the uniform velocity stirred at 58 DEG C, digests 16h, and 96 DEG C of enzyme deactivation 30min obtain suspension C32.
(7) high speed centrifugation: after suspension C32 is using 8000g centrifugation 25min, supernatant is taken to obtain extracting solution A33.
(8) ultrafiltration impurity elimination: using ultrafiltration membrane ultra-filtration and separation extracting solution A33, and ultrafiltration membrane molecular weight is 10000Da, ultrafiltration time
Number is 6 times, discards macromolecular trapped fluid, takes small molecule filter liquor, obtains extracting solution A34, and 60 DEG C of reduced pressures obtain solid content
For the extracting solution A35 of 10wt%.
(9) secondary protease hydrolyzed: being added protease in extracting solution A35, and additional amount is extracting solution A35 solid content
6%, the 80% of the total enzyme amount of additional amount Zhan of the pancreatin of Nanning Pang Bo bio-engineering corporation, the compound protease of Novozymes Company
The total enzyme amount of additional amount Zhan 20%, 180r/min is at the uniform velocity stirred at 50 DEG C, digests 4h, and 96 DEG C of enzyme deactivation 30min obtain suspension
C33。
(10) high speed centrifugation: after suspension C33 is using 8000g centrifugation 25min, supernatant is taken, extracting solution A36 is obtained.
(11) be concentrated under reduced pressure: it is 40wt% that extracting solution A36 is concentrated under reduced pressure into solid content at 60 DEG C, obtains extracting solution
A37。
(12) alcohol precipitation impurity elimination: the pre- dehydrated alcohol for being cooled to 8 DEG C is added into extracting solution A37, makes the ethanol content in system
Reach 20wt%, after 180r/min at the uniform velocity stirs 5h, 8000g is centrifuged 30min, and supernatant is taken to obtain extracting solution A38.
(13) secondary pressure is concentrated: it is 40wt% that extracting solution A38 is concentrated under reduced pressure into solid content at 60 DEG C, obtains enzyme
Solve extracting solution A39.
(14) it compounds: by enzyme-extracting solution A39 obtained by alcohol extract A32 obtained by step (4) and step (13) at 85 DEG C,
180r/min at the uniform velocity stirs 50min, obtains mixed extract A310.
(15) it is spray-dried: mixed extract A310 being spray-dried, moringa oleifera leaf extractive 3, phenolic compound are obtained
Content is 10.3%, and peptide matters content is 44.1%.
Comparative example 4
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, specifically comprises the following steps:
(1) microwave drying: under microwave power 600W, by fresh leaf of Moringa microwave drying 3min, dry leaf of Moringa is obtained.
(2) it pulverizes and sieves: dry leaf of Moringa obtained by step (1) being smashed it through into 40 meshes, obtains Moringa leaf dried powder.
(3) water-ethanol refluxing extraction: the ethanol water for being 80vol% by Moringa leaf dried powder obtained by step (2) and concentration of alcohol
Solution is mixed by solid-liquid ratio 1:10g/mL, under 180r/min at the uniform velocity stirring condition, refluxing extraction 3h at 90 DEG C, 6000g centrifugation
30min takes supernatant to obtain extracting solution A41.
(4) be concentrated under reduced pressure: it is 40wt% that extracting solution A41 is concentrated under reduced pressure into solid content at 60 DEG C, obtains alcohol extracting
Liquid A42.
(5) it is spray-dried: mixed extract A42 being spray-dried, obtains moringa oleifera leaf extractive 4, phenolic compound contains
Amount is 23.8%.
Comparative example 5
A kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, specifically comprises the following steps:
(1) microwave drying: under microwave power 600W, by fresh leaf of Moringa microwave drying 3min, dry leaf of Moringa is obtained.
(2) it pulverizes and sieves: dry leaf of Moringa obtained by step (1) being smashed it through into 40 meshes, obtains Moringa leaf dried powder.
(3) alkaline process is heat-treated: Moringa leaf dried powder being mixed with deionized water by solid-liquid ratio 1:16g/mL, 180r/min is at the uniform velocity
25min is stirred, sodium hydroxide adjusting pH value is added to 180r/min at 8.0,60 DEG C and at the uniform velocity stirs 40min, obtains suspension C51.
(4) protease hydrolyzed: being added protease in suspension C51, and additional amount is the 5% of leaf of Moringa dry powder quality, promise
The 70% of the total enzyme amount of additional amount Zhan of the alkali protease NS37071 of Wei Xin company, the compound protease of Novozymes Company add
Enter the 30% of the total enzyme amount of amount Zhan, 180r/min is at the uniform velocity stirred at 58 DEG C, digests 16h, and 96 DEG C of enzyme deactivation 30min obtain suspension C52.
(5) high speed centrifugation: after suspension C52 is using 8000g centrifugation 25min, supernatant is taken to obtain extracting solution A53.
(6) ultrafiltration impurity elimination: using ultrafiltration membrane ultra-filtration and separation extracting solution A53, and ultrafiltration membrane molecular weight is 10000Da, ultrafiltration time
Number is 6 times, discards macromolecular trapped fluid, takes small molecule filter liquor, obtains extracting solution A54, and 60 DEG C of reduced pressures obtain solid content
For the extracting solution A55 of 10wt%.
(7) secondary protease hydrolyzed: being added protease in extracting solution A55, and additional amount is extracting solution A55 solid content
6%, the 80% of the total enzyme amount of additional amount Zhan of the pancreatin of Nanning Pang Bo bio-engineering corporation, the compound protease of Novozymes Company
The total enzyme amount of additional amount Zhan 20%, 180r/min is at the uniform velocity stirred at 50 DEG C, digests 4h, and 96 DEG C of enzyme deactivation 30min obtain suspension
C53。
(8) high speed centrifugation: after suspension C53 is using 8000g centrifugation 25min, supernatant is taken, extracting solution A56 is obtained.
(9) be concentrated under reduced pressure: it is 40wt% that extracting solution A56 is concentrated under reduced pressure into solid content at 60 DEG C, obtains extracting solution
A57。
(10) alcohol precipitation impurity elimination: the pre- dehydrated alcohol for being cooled to 8 DEG C is added into extracting solution A57, makes the ethanol content in system
Reach 20wt%, after 180r/min at the uniform velocity stirs 5h, 8000g is centrifuged 30min, and supernatant is taken to obtain extracting solution A58.
(11) secondary pressure is concentrated: it is 40wt% that extracting solution A58 is concentrated under reduced pressure into solid content at 60 DEG C, obtains enzyme
Solve extracting solution A59.
(12) it is spray-dried: mixed extract A59 being spray-dried, obtains moringa oleifera leaf extractive 5, phenolic compound contains
Amount is 2.9%, and peptide matters content is 37.4%.
The experimental study of hyperuricemia rat model:
(1) experimental animal: male SD rat, in 21d age, 100~110g is purchased from Guangdong Medical Lab Animal Center.
(2) animal feeding environment: 22 ± 2 DEG C of room temperature, relative humidity 50%~70%, each 12 hours of daily light dark,
Drinking water uses reverse osmosis ultraviolet sterilization water.
(3) after adaptive feeding 7 days, experimental group, normal group (12) zoopery: are randomly divided into.Experimental group is big
The daily stomach-filling Oteracil Potassium (2g/kg) of mouse, after 7 days, intraperitoneal injection 3% yellow Jackets (30mg/kg weight) is anaesthetized, after eye socket
Veniplex blood sampling, 4 DEG C, 3000g centrifugation 15min take upper serum measure uric acid content, normal rats stomach-filling isometric(al) it is molten
Matchmaker.110 μM of rat of uric acid content > is determined as modeling success.The successful rat of modeling is randomly divided into model control group,
Allopurinol group (25mg/kg), 1 group of moringa oleifera leaf extractive (500mg/kg), moringa oleifera leaf extractive 2 groups of (500mg/kg), Moringas
3 groups of leaf extract (500mg/kg), 4 groups of moringa oleifera leaf extractive (500mg/kg) and 5 groups of moringa oleifera leaf extractive (500mg/kg), often
Group 12, gastric infusion (administration volume 10mL/kg), rat model gives the distilled water of isometric(al).Continuous processing 30 days.Drug
Handle the 15th, 30 day, intraperitoneal injection 3% yellow Jackets (30mg/kg) anesthesia, retroorbital venous clump blood sampling, 4 DEG C, 3000g from
Heart 15min takes upper serum to measure uric acid content.In strict accordance with uric acid detection kit, (bioengineering is built up in Nanjing to serum uric acid
Research institute) illustrate to be operated and be measured.
Results of animal analysis
Influence of 1 moringa oleifera leaf extractive of table to hyperuricemia rat blood serum uric acid level
Different letters indicate there is significant difference p < 0.05 between contemporaneity groups, without aobvious between same letter expression group
Write sex differernce p > 0.05
As shown in Table 1, after modeling, the rat blood serum uric acid of model group, allopurinol group and 5 moringa oleifera leaf extractive groups
Level will be significantly higher than normal group (n=12, p < 0.05), and four groups of serum uric acid level there was no significant difference (n=12, p
> 0.05).When being administered 15 days and 30 days, the uric acid level of allopurinol group and 5 moringa oleifera leaf extractive groups is substantially less than
Model group (n=12, p < 0.05) illustrates that moringa oleifera leaf extractive can significantly reduce hyperuricemia rat blood serum uric acid level,
In, the anti-trioxypurine effect of moringa oleifera leaf extractive 1,2,3 in vivo is best.However, lacking in the preparation flow of moringa oleifera leaf extractive 4
The extraction of leaf of Moringa peptide and compound；The extraction of leaf of Moringa polyphenol and compound is lacked in the preparation flow of moringa oleifera leaf extractive 5；
The rat blood serum uric acid level that 4,5 groups of moringa oleifera leaf extractive is significantly higher than 1,2,3 groups of (n=12, p < of moringa oleifera leaf extractive
0.05)。
The embodiment of the present invention is as it can be seen that entire preparation process flow is simple, each link is up to food-grade requirement, preparation cost
It is low.
Above embodiments are only preferrred embodiment of the present invention, for explaining only the invention, are not intended to limit the present invention, this
Field technical staff should belong to guarantor of the invention without departing from change made under spirit of the invention, replacement, modification etc.
Protect range.
Claims (10)
1. a kind of preparation method with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that use microwave drying, flour
Alcohol extracting thing is concentrated under reduced pressure to obtain in sieving, water-ethanol refluxing extraction, centrifugation, supernatant；After residual residue drying, alkaline process heat is used
Processing, protease hydrolyzed, high speed centrifugation remove slag, supernatant ultrafiltration impurity elimination, secondary protease hydrolyzed, high speed centrifugation, supernatant subtract
Concentration, alcohol precipitation impurity elimination, secondary pressure is pressed to be concentrated, finally alcohol extracting thing is added in compounding, and spray drying obtains a kind of with anti-trioxypurine
Active moringa oleifera leaf extractive, extract aldehydes matter content > 10%, peptide matters content > 40%.
2. the preparation method according to claim 1 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that tool
Body the following steps are included:
(1) microwave drying: by fresh leaf of Moringa microwave drying, dry leaf of Moringa is obtained；
(2) it pulverizes and sieves: by dry leaf of Moringa grinding and sieving obtained by step (1), obtaining Moringa leaf dried powder；
(3) water-ethanol refluxing extraction: Moringa leaf dried powder obtained by step (2) is mixed with certain density ethanol water, at the uniform velocity
Stirring, heating and refluxing extraction, centrifugation take supernatant to obtain extracting solution 1, and precipitating dries to obtain residue 1；
(4) it is concentrated under reduced pressure: extracting solution 1 being concentrated under reduced pressure, alcohol extract 2 is obtained；
(5) alkaline process is heat-treated: residue 1 is mixed with deionized water, carries out first time stirring, sodium hydroxide is added and adjusts pH, into
Second of heating stirring of row, obtains suspension 1；
(6) protease hydrolyzed: being added protease in suspension 1, and constant temperature stirring is digested, and enzyme deactivation obtains suspension 2；
(7) high speed centrifugation: after 2 high speed centrifugation of suspension, supernatant is taken to obtain extracting solution 3；
(8) ultrafiltration impurity elimination: ultrafiltration membrane ultra-filtration and separation extracting solution 3 is used, macromolecular trapped fluid is discarded, takes small molecule filter liquor, obtain
Extracting solution 5 is concentrated under reduced pressure to obtain in extracting solution 4；
(9) secondary protease hydrolyzed: being added protease in extracting solution 5, and constant temperature stirring is digested, and enzyme deactivation obtains suspension 3；
(10) high speed centrifugation: after 3 high speed centrifugation of suspension, supernatant is taken to obtain extracting solution 6；
(11) it is concentrated under reduced pressure: extracting solution 6 being concentrated under reduced pressure, extracting solution 7 is obtained；
(12) alcohol precipitation impurity elimination: the dehydrated alcohol of pre-cooling being added into extracting solution 7, and after at the uniform velocity stirring, centrifugation takes supernatant that must extract
Liquid 8；
(13) secondary pressure is concentrated: extracting solution 8 being concentrated under reduced pressure, enzyme-extracting solution 9 is obtained；
(14) it compounds: enzyme-extracting solution 9 obtained by alcohol extract 2 obtained by step (4) and step (13) is mixed, heating stirring obtains
Mixed extract 10；
(15) it is spray-dried: mixed extract 10 being spray-dried, moringa oleifera leaf extractive is obtained.
3. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly in (1), the microwave drying time is 1 ~ 3min, and power is 400 ~ 600W；In step (2), the sieving was 40 meshes；
In step (3), concentration of alcohol is 60 ~ 80 vol %, the feed liquid of Moringa leaf dried powder and ethanol water in the ethanol water
Than for the g/mL of 1:6 ~ 10, stirring rate is 120 ~ 180 r/min, reflux temperature is 80 ~ 90 DEG C, and return time is 1 ~ 3h, described
Centrifugation is 4000 ~ 6000gIt is centrifuged 20 ~ 30min, the drying temperature of residue is 50 ~ 60 DEG C；In step (4), the reduced pressure temperature
Degree is 50 ~ 60 DEG C, and solid content is 30 ~ 40 wt% in concentrate.
4. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly in (5), the residue 1 is 1:8 ~ 1:16 g/mL with deionized water solid-liquid ratio, and first time mixing time is 15 ~ 25 min, institute
Stating pH value is 7.0 ~ 8.0, and stirring rate is 120 ~ 180 r/min twice, and second of mixing time is 20 ~ 40 min, second
Secondary whipping temp is 50 ~ 60 DEG C；The additional amount of step (6) described protease is the 2 ~ 5% of mass of residue, and protease is alkaline egg
White enzyme and compound protease, the 50 ~ 70% of the total enzyme amount of additional amount Zhan of alkali protease, the total enzyme of additional amount Zhan of compound protease
The 30 ~ 50% of amount, the alkali protease are the protease that Novi believes model NS37071, and compound protease is Novozymes Company
Compound protease (Protamex), the hydrolysis temperature be 52 ~ 58 DEG C, enzymolysis time be 8 ~ 16h, stirring rate be 120 ~
180 r/min, the enzyme deactivation are in 90 ~ 96 DEG C of enzyme deactivation 20-30 min.
5. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly (7), in (10), the high speed centrifugation is 6000 ~ 8000gIt is centrifuged 15 ~ 25 min；In step (8), the ultrafiltration removal of impurities surpasses
Filter membrane molecular weight is 10000Da, and ultrafiltration number is 4 ~ 6 times, and the extracting solution 4 is the ultrafiltration filter liquor of molecular weight < 10000Da,
The reduced pressure temperature is 50 ~ 60 DEG C, and solid content is 8 ~ 10 wt% in extracting solution 5.
6. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly the additional amount of (9) described protease is the 3 ~ 6% of 5 solid content of extracting solution, and protease is pancreatin and compound protease, pancreas
The 60 ~ 80% of the total enzyme amount of additional amount Zhan of enzyme, the 20 ~ 40% of the total enzyme amount of additional amount Zhan of compound protease, the pancreatin is Nanning
The pancreatin of Pang Bo bio-engineering corporation, compound protease are the compound protease Protamex of Novozymes Company, the enzymatic hydrolysis temperature
Degree is 40 ~ 50 DEG C, and enzymolysis time is 2 ~ 4 h, and stirring rate is 120 ~ 180 r/min, and the enzyme deactivation is in 90 ~ 96 DEG C of enzyme deactivations 20
~30 min。
7. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly (11), in (13), the reduced pressure temperature is 50 ~ 60 DEG C, and solid content is 30 ~ 40 wt% in concentrate；Step
(12) in, the dehydrated alcohol of the pre-cooling is the dehydrated alcohol for being cooled to 4 ~ 8 DEG C in advance, and the additional amount of dehydrated alcohol is to make second in system
Alcohol content reaches 10 ~ 20 wt%, and the at the uniform velocity stirring stirs 3 ~ 5h for 120 ~ 180 r/min revolving speeds at room temperature, the high speed from
The heart is 6000 ~ 8000g, it is centrifuged 20 ~ 30min.
8. the preparation method according to claim 2 with the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that step
Suddenly in (14), stirring rate is 120 ~ 180 r/min, and mixing time is 30 ~ 50 min, and temperature is 65 ~ 85 DEG C.
9. one kind has the active moringa oleifera leaf extractive of anti-trioxypurine, which is characterized in that by the described in any item systems of claim 1-8
Preparation Method is made.
10. moringa oleifera leaf extractive as claimed in claim 9 is preparing the application in anti-trioxypurine health care product and drug.
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| PCT/CN2019/114166 WO2020181784A1 (en) | 2019-03-13 | 2019-10-29 | Moringa oleifera leaf extract having uric acid lowering activity, preparation method therefor and application thereof |
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