CN1733130A - Process for extracting litchi polyphenol from litchi - Google Patents
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Abstract
The invention discloses a process for extracting litchi polyphenol from litchi through dissolvent abstraction, concentrating, separation and purification, and drying, wherein water or low carbon alcohols such as methanol, ethanol, propanol, isopropanol, butanol and amylic alcohol are used as the dissolvent.
Description
Technical field
The present invention relates to a kind of preparation method of Fructus Litchi extract, specifically, relate to a kind of method of from litchi rind or Semen Litchi, extracting lichee polyphenol.
Background technology
Fructus Litchi belongs to Sapindaceae aithullium fruit, originates in Fujian Yue Ersheng and Taiwan.According to the modern medicine study assay determination, Fructus Litchi nutrition is very abundant, contain rich in protein, fat, nicotinic acid, citric acid, pectin, calcium, phosphorus, ferrum etc., contain glucose in the litchi pulp up to 66%, also contain fructose, sucrose and abundant vitamin C, vitamin B, provitamin A and folic acid, malic acid and a large amount of free amino acid etc.The medical value of Fructus Litchi is also higher.China is among the people to be considered as tonic to Fructus Litchi, and the traditional Chinese medical science is used as Fructus Litchi and cures the disease.Li Shizhen (1518-1593 A.D.) is put down in writing in Compendium of Material Medica: " Fructus Litchi can quench the thirst, beneficial people's color, logical god, Fructus Alpiniae Oxyphyllae, strong gas ", can control " top hernia common fault, married woman's pricking caused by the stagnation of QI and blood ".Among the people sincerely believe in Fructus Litchi in also as dried longan, can blood yiqi, be the tonic of old children, gynecological.The leaf of Fructus Litchi, flower, nuclear, shell all can be used as medicine.Suitably eat a little Fructus Litchi, can protect brain and enhancing fitness, appetizing strengthening the spleen, life lengthening.
Polyphenol component in the plant has antitumaous effect and has obtained proof, and for example, quercetin in the gossypol in catechin monomers ECG in the green tea and EGCG, the Semen Gossypii, the chlorogenic acid in the bark of willow, robur and the Oak Tree and datiscetin etc. all belong to Polyphenols.Germany scientist has had further understanding to the mechanism of polyphenol inhibition growth of tumour cell a few days ago, and finds all to comprise in many plants this natural anti-cancer material.Research group has been chosen leukaemia, lung tumors cell and breast cancer cell as object of study in experiment, found that, even have only the polyphenol of trace also can suppress enzyme in the tumor cell, and these enzymes are essential to the generation of triggering tumor cell growth " signal " molecule.In the plant amedica therapy, polyphenol is considered to prevent and suppress the material of tumor.
Scientist thinks: plant polyphenol has multiple pharmacological effect, mainly is:
1. anticancer, antioxidation (free radical) effect.
Arteriosclerosis, prevent and treat cardiovascular disease such as coronary heart disease and apoplexy.
3. cartilage protection effect and sclerotin potentiation.
4. hair regrowth and black hair effect.
5. protect retina, anti-visual deterioration.
6. antibiotic, antivirus action.
According to Japanese scholar research, the contained glycocide composition of Semen Litchi has multiple physiological activity, as anticancer, anticollagenase and blood sugar reducing function or the like.
Originally be considered as by the people is that the litchi rind of refuse and Semen Litchi will be expected to exploitation from now on and become the novel healthy food raw material always.
Through patent retrieval, also do not find singlely at present with litchi rind or Semen Litchi, be the correlational study report that primary raw material extracts lichee polyphenol perhaps with litchi rind or Semen Litchi.
Summary of the invention
The technical issues that need to address of the present invention just are to overcome that not have a kind of in the prior art be the defective that raw material extracts the method for lichee polyphenol with litchi rind or Semen Litchi singlely, a kind of method of extracting lichee polyphenol from Fructus Litchi is provided, it be a kind of be raw material with litchi rind or Semen Litchi, through extraction solvent extract, concentrate, the method for separation and purification, dry extraction litchi rind polyphenol.The lichee polyphenol weight content is more than 70% in the extract.The lichee polyphenol content height that the inventive method is produced, avoided active component loss, improved production technology, shortened the production cycle.
For addressing the above problem, the present invention adopts following technical scheme:
A kind of method of from Fructus Litchi, extracting lichee polyphenol of the present invention, it be a kind of be raw material with litchi rind and/or Semen Litchi, through the method that extraction solvent extracts, concentrates, lichee polyphenol is extracted in separation and purification, the lichee polyphenol weight content is more than 70% in the extract.
The inventive method can be pulverized litchi rind earlier before extraction, and litchi rind helps the abundant stripping of various compositions after crushed.
Extracting method of the present invention can be heating extraction or ultrasonic extraction or microwave extraction.
Extraction solvent is water or low-carbon alcohols during heating extraction of the present invention, and extraction time is 1～3 time, and the solvent consumption is 3～15 times of amounts of raw material weight; Temperature is 40～100 ℃, and extraction time is solvent boiling back 1～3 hour.Wherein said low-carbon alcohols is methanol, ethanol, propanol, isopropyl alcohol or butanols, and the weight concentration of described low-carbon alcohols is 1%～95%.
Need to reclaim low-carbon alcohols after litchi rind of the present invention or the Semen Litchi alcohol extraction and handle, temperature is controlled at below 60 ℃, and vacuum degree control is more than 0.06Mpa, and the medicinal liquid and the raw material weight ratio that reclaim low-carbon alcohols should be controlled between 1: 2～5.
Ultrasonic extraction condition of the present invention is operating frequency 15～80kHz, time 5～120min.
Microwave extraction condition of the present invention is operating frequency 900～2500MHz, time 5～120min.
Separation and purification of the present invention is a column chromatographic isolation and purification.
Column chromatographic isolation and purification of the present invention is:
1) extracting solution is through concentrating (vacuum concentration, temperature is controlled at below 60 degrees centigrade, more than the vacuum 0.08MPa, be concentrated into the 1/4-1/2 of original extracting liquid volume), reclaim low-carbon alcohols, centrifugal (horizontal centrifuge: 4800r/min), directly enter macroporous resin column, flow speed control is about 0.5～1.5BV/hr;
2) it is limpid to effluent to wash post with deionized water, about 2h;
3) wash post to eluent clarification, transparent with low-carbon alcohols, flow velocity is slightly slower than the speed of absorption;
4) collect effusive clarification from macroporous resin column, transparent, the eluent of alcohol flavor slightly, about 1～3 times of collecting eluent and be approximately amount of resin;
5) pure water elution liquid is through concentrating (vacuum concentration, temperature is controlled at below 60 degrees centigrade, and vacuum 0.06-0.1MPa is concentrated into about 10～15 mother-in-law U.S. degree), dry (spray drying: 160～200 ℃ of intake air temperatures, 80～110 ℃ of air outlet temperature) gets the litchi rind polyphenol.
Wherein said low-carbon alcohols is methanol, ethanol, propanol, isopropyl alcohol, butanols or amylalcohol, and the concentration of low concentration alcohol-water solution is minimum to be volumetric concentration 30%, and the concentration of high concentration alcohol-water solution is up to volumetric concentration 95%; Wherein said macroporous resin is for being the material of skeleton with the polystyrene, and as the DIAION brand, model is: D101, D130, AB-8 or HP-20.
The lichee polyphenol content height that the inventive method is produced, its weight content is more than 70%, avoided active component loss, improved production technology, shortened the production cycle.
Antioxygenic property index TEAC and ORAC show that lichee polyphenol has extremely strong antioxidant activity in the lichee polyphenol in the extract of the inventive method preparation, can effectively remove the peroxidating material.Resulting lichee polyphenol has the characteristic that is evenly distributed from the oligomer to the high polymer.
The test of lichee polyphenol antioxygenic property index TEAC (Trolox Equivalent AntioxidantCapacity) and ORAC (Oxygen Radical Absorbance Capacity) antioxidant activity:
The antioxygenic property of ORAC test in the serum
Reagent is prepared:
1.225mM phosphate buffer (pH 7.0): with 39mL 0.45M NaH
2PO
4With 61mL0.45M Na
2HPO
4Mix, regulate pH to 7.0, be diluted to 200mL (room temperature preservation) with deionized water;
2.60nM R-phycoerythrin (PE): with 10mL deionized water dissolving 19.80 μ L R-phycoerythrin (shaking up before the use);
3.18mM AAPH: with 10mL deionized water dissolving 488.1mg AAPH (shaking up before the use)
Method:
The pre-treatment of serum
1. with perchloric acid and the 60 μ L serum mixing in the test tube of 1.5-mL of 20 μ L;
2. then, add 300 μ L n-butanols, waited for for ten seconds;
With test tube in 12,000rpm centrifugal ten minutes;
4. the last supernatant liquid that pipettes 50L is in the test tube of another 1.5-mL;
5. dry up with purity nitrogen;
6. add 2mL 225mM phosphate buffer (pH 7.0)
The ORAC test
1. the serum that 2mL is pretreated and the PE of 2mL mixing in brown test tube;
2.37 ℃ hatched in advance 5 minutes;
3. begin fluorescence analysis (exciting light 540nm by adding 2mLAAPH reagent down at 37 degrees centigrade, radiant light 565nm), after partial reaction solution being transferred to a small test tube in per 3 to 5 minutes, test fluorescence intensity (, stopping test) if fluorescence intensity is reduced to below 20% of original value
Attention:
1.ORAC want lucifuge in the process of the test, close the laboratory fluorescent lamp;
2.R-phycoerythrin will in time use after handling;
3.AAPH after the dissolving, must use in ten minutes, because the solution instability;
4.ORAC brown test tube is washed with 1N HCl in the experiment back, removes fluorescin.
The antioxygenic property of TEAC test in the serum
Reagent is prepared
(1) 1.25mM trolox solution (standard)
In 10ml 5mM PBS (pH 7.4) lining dissolving 3.13mg Trolox ,-20 ℃ of preservations
(2) 450mM hydrogen peroxide (culture medium)
With 0.5ml 30% hydrogen peroxide (H
2O
2) and 9.3ml 5mM PBS mixing, 4 ℃ of preservations (are diluted to 450 μ M H with 5mM PBS during use
2O
2Solution)
(3) 5mM ABTS (DAP) solution (chromogen)
27.43mg ABTS is dissolved among the 10ml 5mM PBS 4 ℃ of brown test tubes keep in Dark Place (being diluted to 500 μ M ABTS solution with 5mM PBS during use)
(4) 70 μ M Met Mb solution (chromogen)
1) purification Met Mb
68mg myoglobin (Mb) is dissolved in 10ml 5mM PBS (400 μ M Mb)
With 2.44mg of K3[Fe (CN)
6] be dissolved in 10ml 5mM PBS (740 μ MK3[Fe (CN)
6])
Get 10ml 400 μ M Mb and 10ml 740 μ M K3[Fe (CN)
6] after the mixing, place under the room temperature, activate 15 minutes
With the Sephadex G15-120 post (2.5 φ x 40cm) of Met Mb solution by crossing with 5mM PBS balance
Calculate the elution yield with following formula:
2) calculate purity and concentration
Purity with following little EQUILIBRIUM CALCULATION FOR PROCESS each several part:
[Met?Mb]＝146A490-108A560+2.1A580
[Ferryl?Mb]＝-62A490+242A560-123A580
[MbO2]＝2.8A490-127A560+153A580
(value that deducts the 700nm place is revised background absorption)
When total protein haemachrome＞95% is collected Met Mb part, with the concentration in the top formula calculating eluate.
3) preparation 70 μ M Met Mb solution
With the Met Mb solution behind the 5mM PBS dilution purification, 4 ℃ of preservations
Experimental technique:
(1) 36 μ l 70 μ M Met Mb are in the sample tube of 1.5-ml;
(2) in test tube, add 300 μ l, 500 μ M ABTS and 487 μ l 5mM PBS
(3) add 10 μ l serum or 1.25mM trolox solution
(4) slowly behind the mixing, test tube is stored 5 minutes in 0 ℃
(5) add 167 μ l, 450 μ M H then
2O
2, mixing placed for ten seconds
(6) in (20-25 ℃) accurate control time under the room temperature, reacted 5 minutes
(7) use ultraviolet spectrophotometer in the 734nm specimen
Trolox?equivalent?antioxidant?capacity(TEAC)
(1) calculates the absorptance of blood serum sample with reference to per unit 1.25mM trolox solution
(2) calculate the value of TEAC in the blood serum sample (mM) with reference to 1.00mM trolox
Description of drawings
Fig. 1 is lichee polyphenol characteristic fingerprint pattern in the extract of the present invention.
Wherein: Figure 1A is reversed-phase HPLC (1.0mg/ml)
Figure 1B is along phase HPLC (1.0mg/ml).
The specific embodiment
Embodiment 1
1) pre-treatment: the Semen Litchi that will choose, reject behind the impurity is pulverized;
2) extract: with raw material and deionized water by 1: 3 mixed extraction pot of packing into, be heated to the boiling back and kept temperature 2 hours, collect and extract feed liquid for the first time, extract the deionized water that adds 3 times of amounts more for the second time, ebuillition of heated insulation 2 hours is collected and is extracted feed liquid for the second time.
3) concentrating under reduced pressure: extracting solution imported carry out concentrating under reduced pressure in the concentrator, temperature is controlled at below 60 ℃, and vacuum degree control is more than 0.06MPa.
4) centrifugal: the extracting solution after will concentrating is centrifugal by centrifuge.
5) concentrate, the extracting solution after centrifugal directly advances macroporous resin column;
6) it is limpid to effluent to wash post with water;
7) it is tasteless to effluent to wash post with ethanol;
8) effluent of collection from macroporous resin column;
9) eluent carries out concentrating under reduced pressure, is concentrated into 10 mother-in-law U.S. degree;
10) spray drying, sterilization packaging
Embodiment 2
1) pre-treatment: litchi rind is pulverized.
2) extract: qualified raw material is put in the clean extraction pot, add 5 times of amount 65% (v/v) ethanol, reflux, extract, 3h for the first time, from solvent boiling timing, 80 ℃ of control temperature, insulation then extrude medicinal liquid, the alcohol reflux 2h that adds for the second time 3 times of amount 65% (v/v) again, merging filtrate;
3) reclaim ethanol: will carry out concentrating under reduced pressure in the medicinal liquid suction concentration tank that extract, reclaim ethanol, temperature be controlled at below 60 ℃, and vacuum degree control is more than 0.06Mpa.After being recycled to nothing alcohol flavor, medicinal liquid is pressed into fluid reservoir;
4) add water, leave standstill, cooling: reclaim alcoholic acid medicinal liquid and raw material ratio and should be controlled at about 1: 4, then add water inadequately, leave standstill, cooling;
5) centrifugal: refrigerative medicinal liquid is centrifugal with centrifuge, and medicinal liquid should be no any precipitate;
6) post absorption: the medicinal liquid after centrifugal is advanced the D101 resin column;
7) washing: after absorption finishes, wash, wash about about 2h with deionized water.
8) eluting: carry out eluting with 70% (v/v) ethanol, collect slightly alcohol flavor, and the eluent clarification, transparent, the collection eluent is about 3 times of amount of resin approximately;
9) concentrate: eluent carries out concentrating under reduced pressure, and temperature is controlled at below 60 ℃, and vacuum should be controlled at more than the 0.06MPa, is concentrated into about 15 mother-in-law U.S. degree.
10) spray drying, sterilization packaging.
Embodiment 3
1) pre-treatment: will choose, reject litchi rind behind the impurity and Semen Litchi and mix and pulverize;
2) extract: with qualified raw material and weight concentration be 70% ethanol by 1: 10 mixed, carry out ultrasonic extraction, extraction conditions is operating frequency 60kHz, time 90min extracts 2 times;
3) reclaim propanol: will carry out concentrating under reduced pressure in the medicinal liquid suction concentration tank that extract, reclaim propanol, temperature be controlled at below 60 ℃, and vacuum degree control is more than 0.06Mpa.After being recycled to nothing alcohol flavor, medicinal liquid is pressed into fluid reservoir;
4) add water, leave standstill, cooling: the medicinal liquid and the raw material ratio that reclaim propanol should be controlled at about 1: 3, then add water inadequately, leave standstill cooling;
5) centrifugal: refrigerative medicinal liquid is centrifugal with centrifuge, and medicinal liquid should be no any precipitate;
6) post absorption: the medicinal liquid after centrifugal is advanced the D130 resin column;
7) washing: after absorption finishes, wash, wash about about 2h with deionized water.
8) eluting: carry out eluting with 80% (v/v) propanol, collect slightly alcohol flavor, and the eluent clarification, transparent, the collection eluent is about 3 times of amount of resin approximately;
9) concentrate: eluent carries out concentrating under reduced pressure, and temperature is controlled at below 60 ℃, and vacuum should be controlled at more than the 0.06MPa, is concentrated into about 13 mother-in-law U.S. degree.
10) spray drying, sterilization packaging.
Claims (11)
1, a kind of method of from Fructus Litchi, extracting lichee polyphenol, it is characterized in that it be a kind of be raw material with litchi rind and/or Semen Litchi, through the method that extraction solvent extracts, concentrates, lichee polyphenol is extracted in separation and purification, the lichee polyphenol weight content is more than 70% in the extract.
2, extract the method for lichee polyphenol according to claim 1 from Fructus Litchi, it is characterized in that: described extracting method is heating extraction or ultrasonic extraction or microwave extraction.
3, as extract the method for lichee polyphenol as described in the claim 2 from Fructus Litchi, it is characterized in that: extraction solvent is water or low-carbon alcohols during heating extraction, and extraction time is 1～3 time, and the solvent consumption is 3～15 times of amounts of raw material weight; Temperature is 40～100 ℃, and extraction time is solvent boiling back 1～3 hour.
4, as extract the method for lichee polyphenol as described in the claim 3 from Fructus Litchi, it is characterized in that: described low-carbon alcohols is methanol, ethanol, propanol, isopropyl alcohol or butanols.
5, as from Fructus Litchi, extracting the method for lichee polyphenol as described in claim 3 or 4, it is characterized in that: need to reclaim low-carbon alcohols after the alcohol extraction and handle, temperature is controlled at below 60 ℃, and vacuum degree control is more than 0.06Mpa, and the medicinal liquid that reclaims low-carbon alcohols is 1: 2～5 with the raw material weight ratio.
6, as extract the method for lichee polyphenol as described in the claim 2 from Fructus Litchi, it is characterized in that: described ultrasonic extraction condition is that operating frequency is 15～80kHz, time 5～120min.
7, as extract the method for lichee polyphenol as described in the claim 2 from Fructus Litchi, it is characterized in that: described microwave extraction condition is that operating frequency is 900～2500MHz, and the time is 5～120min.
8, as extract the method for lichee polyphenol as described in claim 1-7 arbitrary from Fructus Litchi, it is characterized in that: described separation and purification is a column chromatographic isolation and purification.
9, as extract the method for lichee polyphenol as described in the claim 8 from Fructus Litchi, it is characterized in that: described column chromatographic isolation and purification step is:
The A extracting solution through concentrate, reclaim low-carbon alcohols, centrifugal after, directly enter macroporous resin column, flow velocity is 0.5～1.5BV/hr; Described simmer down to vacuum concentration, thickening temperature are controlled at below 60 ℃, and vacuum is more than the 0.08MPa, is concentrated into the 1/4-1/2 of original extracting liquid volume; Centrifugal employing horizontal centrifuge, rotating speed are 4800r/min;
It is limpid to effluent that B washes post with deionized water, and the time is 2h;
C washes post to eluent clarification, transparent with low-carbon alcohols, and flow velocity is slightly slower than the speed of absorption;
D collects effusive clarification from macroporous resin column, transparent, the eluent of alcohol flavor slightly, about 1～3 times of collecting eluent and be amount of resin;
E alcohol water elution liquid is concentrated, dry, described simmer down to vacuum concentration, and temperature is controlled at below 60 ℃, and vacuum 0.06-0.1MPa is concentrated into 10～15 mother-in-law U.S. degree; Drying is a spray drying: 160～200 ℃ of intake air temperatures, 80～110 ℃ of air outlet temperature.
10, as from Fructus Litchi, extracting the method for lichee polyphenol as described in the claim 9, it is characterized in that: described low-carbon alcohols is methanol, ethanol, propanol, isopropyl alcohol, butanols or amylalcohol, the concentration of low concentration alcohol-water solution is minimum to be volumetric concentration 30%, and the concentration of high concentration alcohol-water solution is up to volumetric concentration 95%; Described macroporous resin is for being the material of skeleton with the polystyrene, and described macroporous resin model is: D101, D130, AB-8 or HP-20.
11, a kind of arbitrary described preparation method preparation by claim 1-10 the lichee polyphenol extract, it is characterized in that: antioxygenic property index TEAC and ORAC show that lichee polyphenol has extremely strong antioxidant activity in the lichee polyphenol in the extract, can effectively remove the peroxidating material.
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2005
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