CN105055488A - Preparation method and application of loofah sponge antioxidant active extract - Google Patents
Preparation method and application of loofah sponge antioxidant active extract Download PDFInfo
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- CN105055488A CN105055488A CN201510455441.5A CN201510455441A CN105055488A CN 105055488 A CN105055488 A CN 105055488A CN 201510455441 A CN201510455441 A CN 201510455441A CN 105055488 A CN105055488 A CN 105055488A
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Abstract
The invention provides a preparation method and an application of loofah sponge antioxidant active extract, relating to a preparation method and an application of the loofah sponge extract, for solving the problems that in the existing preparation method of the loofah sponge antioxidant active extract, the residual organic solvent exists, the safety is poor, and the cost is high. The preparation method provided by the invention comprises the following steps: 1. taking loofah sponge medicinal material, soaking by 75% ethanol solution, carrying out ultrasonic extraction, respectively collecting solid-phase material A and liquid-phase material A, soaking the solid-phase material A by 75% ethanol solution, carrying out ultrasonic extraction, collecting liquid-phase material B, and mixing the liquid-phase material A and the liquid-phase material B, thus preparing leaching liquor; 2. carrying out vacuum concentration on the leaching liquor to obtain extract; and 3. dissolving the extract by adopting distilled water, coating a polyamide chromatographic column by the obtained solution, firstly, washing four column volumes by distilled water, then eluting four column volumes by adopting ethanol solution with the mass concentration being 30%, collecting the eluant, and carrying out vacuum concentration on the eluant till the eluant becomes dry, thus obtaining solid, namely, the loofah sponge antioxidant active extract. The preparation method is used for extracting antioxidant active substances.
Description
Technical field
The present invention relates to a kind of preparation method and its usage of Retinervus Luffae Fructus extract.
Background technology
Human body can produce a large amount of oxygen radical in the process of oxidative metabolism, free radical can destroy the structure of other molecule by cellular transport processes, cause cell damage, make human body respectively prevent the decline of cell function, aging, the over one hundred kind of diseases such as cancer, diabetes, cardiovascular disease, nephropathy, arteriosclerosis, ischemic heart desease, nervous system disease, disease of eye can be brought out.Research shows, the generation of body free radical and elimination can be kept to reach balance by supplementary antioxidant, thus prevent the damage of radical pair biological cells and tissues, be one of effective measures of preventing each disease, the antioxidant food of edible function is also a kind of not only convenient but also effectively supplements the method for antioxidant.
Retinervus Luffae Fructus, namely the vascular bundle of the dry mature fruit of cucurbitaceous plant Fructus Luffae (Luffacylindrical (L.) Rome), has the effects such as dredge the meridian passage, heat-clearing and toxic substances removing, inducing diuresis to remove edema.Retinervus Luffae Fructus contains the compositions such as sterol, triterpene, saponin, flavone, phenols, protein, aminoacid and organic acid.Retinervus Luffae Fructus is widely used in treatment pain in chest and hypochondrium, stomachache, lumbago, painful and swollen testis, cough due to lung-heat, women's amenorrhea, galactostasis, carbuncle hemorrhoid, the diseases such as metrorrhagia of having blood in stool clinically.Modern study shows, Retinervus Luffae Fructus has protective effect to the cardiac muscle in myocardial ischemia process.In addition, Retinervus Luffae Fructus also achieves good effect in blood fat reducing, the increase of suppression body weight, antiinflammatory, antalgic and sedative and antiallergic etc., but the research of its antioxidant activity almost has no report in the literature.Relevant to multiple pharmacological effect based on antioxidant activity, antioxidative active extractive that the invention provides Retinervus Luffae Fructus and preparation method thereof, this extract has the ability of stronger removing DPPH free radical, and FRAP test determination its there is stronger total antioxidant capacity, therefore, Retinervus Luffae Fructus antioxidant extract provided by the invention both as the antioxidant obtained from natural plants in field application such as food, also can be developed to new drug as the pharmacologically active extract of Retinervus Luffae Fructus and be applied to clinical in the future.
Summary of the invention
The present invention is that the preparation method that will solve existing Retinervus Luffae Fructus antioxidative active extractive has organic solvent residual, poor stability, and the problem that cost is high, a kind of preparation method and its usage of Retinervus Luffae Fructus antioxidative active extractive is provided.
The preparation method of Retinervus Luffae Fructus antioxidative active extractive of the present invention, comprises the steps:
One, Retinervus Luffae Fructus medical material is got, the mass percentage concentration adding 10 times of quality is the alcoholic solution of 75%, soaks 8 ~ 12h, ultrasonic 25 ~ 40min, filter, collect solid formation A and liquid phase thing A respectively, mass percentage concentration solid formation A being added 10 times of quality is soak 6 ~ 8h, ultrasonic 25 ~ 40min in the alcoholic solution of 75%, filter, collect liquid phase thing B, then liquid phase thing A and liquid phase thing B is mixed, obtain lixiviating solution;
Two, lixiviating solution concentrating under reduced pressure is obtained extractum;
Three, in polyamide chromatographic column on after extractum being dissolved with distilled water, first use distilled water wash 4 column volumes, be alcoholic solution eluting 4 column volumes of 30% again by mass concentration, collect eluent, then concentrating under reduced pressure eluent is to dry, and the solid obtained is Retinervus Luffae Fructus antioxidative active extractive.
Above-mentioned Retinervus Luffae Fructus antioxidative active extractive is preparing the purposes in anti-oxidation medicine and antioxidant food.
Beneficial effect of the present invention:
Only used water and ethanol in preparation method of the present invention, environmental protection, organic solvent-free remain, and safety is high.The inventive method adopts polyamide chromatographic column, and price is lower compared with sephadex chromatography etc., reduces the cost of method.Extracting method have employed ultrasonic extraction and cold-maceration combines, and avoids the destruction of heating and refluxing extraction to active component, improves extraction ratio, extracts yield >=5% of gained extractum, yield >=0.1% of antioxidative active extractive.It is high that polyamide column chromatography has bioaccumulation efficiency, and the advantage that can reuse, reduces cost, has feasibility.
Retinervus Luffae Fructus antioxidative active extractive prepared by the present invention, demonstrate this extract by measuring the test of its oxidation resistance to the test of DPPH free radical scavenging and FRAP method there is excellent antioxidant activity, can the anti-oxidation medicine that is suitable for Clinical practice be made with drug excipient or add in food and use as antioxidative food additive.Only used water and ethanol in preparation method of the present invention, environmental protection, organic solvent-free remain, and safety is high.Extracting method have employed ultrasonic extraction, has both avoided the destruction of heating and refluxing extraction to active component, has turn improved extraction ratio.It is high that polyamide column chromatography has bioaccumulation efficiency, and the advantage that can reuse, reduces cost, has feasibility.
Accompanying drawing explanation
Fig. 1 is FeSO
4solution standard curve.
Detailed description of the invention
Technical solution of the present invention is not limited to following cited detailed description of the invention, also comprises the combination in any between each detailed description of the invention.
Detailed description of the invention one: the preparation method of present embodiment Retinervus Luffae Fructus antioxidative active extractive, comprises the steps:
One, Retinervus Luffae Fructus medical material is got, the mass percentage concentration adding 10 times of quality is the alcoholic solution of 75%, soaks 8 ~ 12h, ultrasonic 25 ~ 40min, filter, collect solid formation A and liquid phase thing A respectively, mass percentage concentration solid formation A being added 10 times of quality is soak 6 ~ 8h, ultrasonic 25 ~ 40min in the alcoholic solution of 75%, filter, collect liquid phase thing B, then liquid phase thing A and liquid phase thing B is mixed, obtain lixiviating solution;
Two, lixiviating solution concentrating under reduced pressure is obtained extractum;
Three, in polyamide chromatographic column on after extractum being dissolved with distilled water, first use distilled water wash 4 column volumes, be alcoholic solution eluting 4 column volumes of 30% again by mass concentration, collect eluent, then concentrating under reduced pressure eluent is to dry, and the solid obtained is Retinervus Luffae Fructus antioxidative active extractive.
Only used water and ethanol in the preparation method of present embodiment, environmental protection, organic solvent-free remain, and safety is high.Extracting method have employed ultrasonic extraction and cold-maceration combines, and avoids the destruction of heating and refluxing extraction to active component, and improves extraction ratio.It is high that the polyamide column chromatography used in separation process has bioaccumulation efficiency, and the advantage that can reuse, reduces cost, is applicable to large-scale production.
Detailed description of the invention two: present embodiment and detailed description of the invention one unlike: the relative density of extractum described in step 2 is 1.10 ~ 1.14.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment and detailed description of the invention one or two unlike: in step 2, concentrating under reduced pressure obtains the temperature of extractum is 50 DEG C, namely at 50 DEG C, carries out concentrating under reduced pressure.Other is identical with detailed description of the invention one or two.
Detailed description of the invention four: one of present embodiment and detailed description of the invention one to three unlike: in step 3, extractum is 1:1 with the volume ratio of dissolving distilled water used.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention five: one of present embodiment and detailed description of the invention one to four unlike: in step 3, the flow velocity of eluting is 5mL/min.Other is identical with one of detailed description of the invention one to four.
Detailed description of the invention six: one of present embodiment and detailed description of the invention one to five unlike: being evaporated to dry temperature in step 3 is 60 DEG C, namely carries out being evaporated to dry at 60 DEG C.Other is identical with one of detailed description of the invention one to five.
Detailed description of the invention seven: one of present embodiment and detailed description of the invention one to six unlike: the column volume described in step 3 refers to bed volume, i.e. the volume of polyamide in post.Other is identical with one of detailed description of the invention one to six.
Detailed description of the invention eight: present embodiment Retinervus Luffae Fructus antioxidative active extractive is preparing the purposes in anti-oxidation medicine and antioxidant food.
Retinervus Luffae Fructus antioxidative active extractive prepared by present embodiment, demonstrate this extract by measuring the test of its oxidation resistance to the test of DPPH free radical scavenging and FRAP method there is excellent antioxidant activity, can the anti-oxidation medicine that is suitable for Clinical practice be made with drug excipient or add in food and use as antioxidative food additive.
By following verification experimental verification beneficial effect of the present invention:
The Retinervus Luffae Fructus of test 1, this test collection 10 batches of Different sources carries out extraction and isolation;
The preparation method of this test Retinervus Luffae Fructus antioxidative active extractive, comprises the steps:
One, 200g Retinervus Luffae Fructus medical material is got, the mass percentage concentration adding 10 times of quality is the alcoholic solution of 75%, soaks 12h, ultrasonic 30min, filter, collect solid formation A and liquid phase thing A respectively, mass percentage concentration solid formation A being added 10 times of quality is soak 8h in the alcoholic solution of 75%, ultrasonic 30min, filter, collect liquid phase thing B, then liquid phase thing A and liquid phase thing B is mixed, obtain lixiviating solution;
Two, lixiviating solution concentrating under reduced pressure is obtained extractum;
Three, in polyamide chromatographic column on after extractum being dissolved with distilled water, first use distilled water wash 4 column volumes, use 30% ethanol, 75% ethanol, each eluting of 95% ethanol 4 column volumes again, collect eluent respectively, then concentrating under reduced pressure 30% ethanol elution is to dry, obtain the solid of the drying of 30% ethanol elution, be Retinervus Luffae Fructus antioxidative active extractive, namely complete the preparation method of Retinervus Luffae Fructus antioxidative active extractive.
The extract parameter of the Retinervus Luffae Fructus of 10 batches of Different sources is as shown in table 1.
The extract parameter of the Retinervus Luffae Fructus of table 110 batch Different sources
In this experimental procedure three, the volume ratio of the distilled water that extractum is used with dissolving is 1:1; Elution flow rate described in this experimental procedure three is 5mL/min; Column volume described in this experimental procedure three refers to bed volume, i.e. the volume of polyamide in post; 75% ethanol elution in experimental procedure three and 95% ethanol elution are carried out respectively be evaporated to dry, obtain 75% ethanol elution thing and 95% ethanol elution thing.
Test 2, DPPH free radical scavenging method measure the antioxidant activity of above-mentioned three kinds of eluates:
Respectively the 95% ethanol elution thing of 10 batches, 75% ethanol elution thing and 30% ethanol elution thing are dissolved into the solution that concentration is 2mg/mL with the alcoholic solution that mass percentage is 95%, with the filtering with microporous membrane of 0.22 μm, the subsequent filtrate of gained is used for DPPH free radical scavenging method and measures antioxidant activity.Often group experiment in triplicate, according to suppression ratio (%)=[1-(A
1-A
2)/A
0] × 100% calculates; A
0for the contrast absorbance not containing extract; A
1for the absorbance containing extract; A
2that result of the test is as shown in table 2 not containing the absorbance of DPPH.As can be known from Table 2,30% ethanol elution thing and Retinervus Luffae Fructus antioxidative active extractive to the inhibitory action of DPPH free radical apparently higher than 95% ethanol elution thing and 75% ethanol elution thing, and between group, comparative result display three has significant difference (P<0.01), has stronger antioxidant activity.
The preparation method of the DPPH solution of this test: the DPPH powder taking 8mg, be that 95% ethanol is settled to 100mL with mass percentage, obtaining initial concentration is 2 × 10
-4the DPPH solution of mol/L, at being positioned over 4 DEG C, cold preservation is for subsequent use.
The suppression ratio (%) of the different eluate of table 210 batch Retinervus Luffae Fructus
Note: often group experiment repetition 3 times, result mean+SD represents
For comparing the oxidation resistance size of Retinervus Luffae Fructus antioxidative active extractive and vitamin C (Vc), to the EC of the antioxidant extract of above-mentioned 10 batches of Retinervus Luffae Fructus medical materials
50value measures.EC
50namely value removes the consumption of antioxidant needed for 50% original DPPH concentration, EC
50be worth less, sample Scavenging ability is stronger, and oxidation resistance is stronger.Concrete operations are as follows: pipette the 30% ethanol elution matter sample solution 0.80mL of 2.0mg/mL, 0.40mL, 0.20mL, the mixing of 0.10mL, 0.05mL and 2mLDPPH solution respectively, mend to 4mL with alcoholic solution again, in the dark react 30min after abundant mixing, measure absorbance (A in 517nm place
1), the sample solution of above-mentioned 5 concentration is mended to 4mL with alcoholic solution respectively simultaneously, survey mixed liquor light absorption value (A
2), and the light absorption value (A of 2mLDPPH and 2mL alcohol mixeding liquid
0), by formulae discovery suppression ratio.Then use GraphPadPrism software to carry out curve fitting and draw the EC of 10 batches of Retinervus Luffae Fructus antioxidant extracts
50value.Be measured in the same method the EC of Vc
50value does positive control, and result is as shown in table 3.The EC of Retinervus Luffae Fructus antioxidant extract
50between 0.068-0.181mg/mL, be about 1/8th of Vc, show stronger antioxidant activity.
The EC of table 310 batch Retinervus Luffae Fructus antioxidant extract
50value
| Sample number into spectrum | EC 50(mg/mL) | Sample number into spectrum | EC 50(mg/mL) |
| 1 | 0.094 | 7 | 0.181 |
| 2 | 0.152 | 8 | 0.153 |
| 3 | 0.079 | 9 | 0.078 |
| 4 | 0.090 | 10 | 0.093 |
| 5 | 0.091 | Vc | 0.009 |
| 6 | 0.068 |
Test 3, FRAP method measure the total antioxidant capacity of above-mentioned three kinds of eluates:
One, FeSO
4the drafting of standard curve
Accurately 0.1mmol/L is pipetted, the FeSO of 0.2mmol/L, 0.4mmol/L, 0.8mmol/L, 1.6mmol/L in reaction tube
4standard solution 0.2mL, more accurately add 6mLFRAP working solution (by 0.3mol/L acetate buffer, 10mmol/LTPTZ solution, 20mmol/LFeCl
3solution forms with the ratio of 10:1:1, now with the current), 0.6mL distilled water, mixing, reacts 10min under 37 DEG C of conditions, measures absorbance in 593nm place.With FeSO
4concentration is abscissa, and absorbance is vertical coordinate, carries out linear regression, and application Excel matching obtains FeSO
4standard curve, as shown in Figure 1.As seen from the figure, FeSO
4concentration has good linear relationship with absorbance within the scope of 0.1 ~ 1.6mmol/L.Regression equation Y=0.7734x-0.0044, R
2=0.9998
Two, FRAP method measures the total antioxidant capacity of above-mentioned three kinds of eluates
In reaction tube, accurately pipette the sample solution of 0.2mL0.5mg/mL eluate, more accurately add 6mLFRAP working solution, 0.6mL distilled water, mixing, reacts 10min under 37 DEG C of conditions, measures absorbance in 593nm place, and often group experiment in triplicate.The absorbance of three kinds of eluate samples of the 10 batches of Retinervus Luffae Fructus medical materials recorded is substituted into FeSO
4standard curve equation, obtains FRAP value as calculated, the results are shown in table 4.As can be seen from the table, the FRAP value often criticizing 30% ethanol elution thing of Retinervus Luffae Fructus is all greater than 75% ethanol elution thing and 95% ethanol elution thing, and statistics shows there is difference (P<0.01), illustrate that the antioxidative active extractive of 30% ethanol elution thing and Retinervus Luffae Fructus has stronger total antioxidant capacity.
The FRAP value of the different eluate of table 410 batch Retinervus Luffae Fructus
Claims (8)
1. a preparation method for Retinervus Luffae Fructus antioxidative active extractive, is characterized in that the method comprises the steps:
One, Retinervus Luffae Fructus medical material is got, the mass percentage concentration adding 10 times of quality is the alcoholic solution of 75%, soaks 8 ~ 12h, ultrasonic 25 ~ 40min, filter, collect solid formation A and liquid phase thing A respectively, mass percentage concentration solid formation A being added 10 times of quality is soak 6 ~ 8h, ultrasonic 25 ~ 40min in the alcoholic solution of 75%, filter, collect liquid phase thing B, then liquid phase thing A and liquid phase thing B is mixed, obtain lixiviating solution;
Two, lixiviating solution concentrating under reduced pressure is obtained extractum;
Three, in polyamide chromatographic column on after extractum being dissolved with distilled water, first use distilled water wash 4 column volumes, be alcoholic solution eluting 4 column volumes of 30% again by mass concentration, collect eluent, then concentrating under reduced pressure eluent is to dry, and the solid obtained is Retinervus Luffae Fructus antioxidative active extractive.
2. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that the relative density of extractum described in step 2 is 1.10 ~ 1.14.
3. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that the temperature that concentrating under reduced pressure in step 2 obtains extractum is 50 DEG C.
4. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that the volume ratio of the distilled water that extractum is used with dissolving in step 3 is 1:1.
5. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that the flow velocity of eluting in step 3 is 5mL/min.
6. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that being evaporated to dry temperature in step 3 is 60 DEG C.
7. the preparation method of a kind of Retinervus Luffae Fructus antioxidative active extractive according to claim 1, is characterized in that the column volume described in step 3 refers to bed volume, i.e. the volume of polyamide in post.
8. Retinervus Luffae Fructus antioxidative active extractive as claimed in claim 1 is preparing the purposes in anti-oxidation medicine and antioxidant food.
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Application publication date: 20151118 |