CN109021129B - Method for extracting black tiger fruit polysaccharide - Google Patents
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Abstract
The invention discloses an extraction method of black tiger fruit polysaccharide, which comprises the following steps: step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder; step two, taking the fruit powder and the ethanol water solution, refluxing and stirring, filtering to obtain filter residue, and repeating the steps for three times; step three, mixing filter residues with distilled water, refluxing, stirring, concentrating, adding absolute ethyl alcohol, refrigerating, storing, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and drying to obtain crude polysaccharide; and step four, deproteinizing the crude polysaccharide, and performing vacuum freeze drying to obtain the black tiger fruit polysaccharide. The method has the advantages of simplicity, feasibility, high extraction rate of the black tiger fruit polysaccharide and good antioxidant activity.
Description
Technical Field
The invention relates to the technical field of plant extraction. More specifically, the invention relates to a method for extracting black tiger fruit polysaccharide.
Background
The black tiger is a evergreen woody vine wild plant of the schisandra of Magnoliaceae, the black tiger fruit is a polymerized fruit, which is also called as the rice ball, the Bufna and the like, and is a rare folk Chinese herbal medicine, and the medicinal parts comprise roots, stems and fruits. In modern medical research, pharmacological experiments such as Liu Xie Jun prove that the root of the black tiger has a certain sedative and anti-inflammatory effects, studies such as Ci-seal etc. find that the root of the black tiger has an antibacterial effect on pneumococcus and studies such as Goh MJ etc. show that the extract of the black tiger can inhibit melanin synthesis, studies such as Queenmo etc. find that the extract of the black tiger has the effects of regulating alcoholic fatty liver and protecting liver, etc., studies such as Pengmy et al find that pulp contains rich amino acids and trace elements, studies on the extract of pericarp such as Sunja etc. show that the extract of the black tiger has antioxidant activity, but studies on the extraction method and antioxidant activity of the fruit polysaccharide of the black tiger are rarely reported at present.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide an extraction method of the black tiger fruit polysaccharide, which is simple and feasible, and has high extraction rate and good antioxidant activity.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for extracting black tiger fruit polysaccharide, comprising the steps of:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
secondly, taking the fruit powder and an ethanol water solution according to the mass ratio of 1: 2.8-3.2, refluxing and stirring for 1-3 hours, filtering to obtain filter residues, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 20-30 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 3.5-4.5 h, concentrating the mixed aqueous solution to 5-6 parts by mass, adding 18-22 parts by mass of absolute ethyl alcohol, carrying out cold storage at 0-4 ℃ for 12-16 h, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
Preferably, the drying temperature in the first step is 40-50 ℃, and the mesh number of the sieved mesh is 40.
Preferably, the stirring in the second step adopts an electric stirring paddle, and the rotating speed of the electric stirring paddle is 300-600 r/min.
Preferably, the mixed aqueous solution is concentrated in the third step by ultrafiltration with an ultrafiltration membrane with the molecular weight cutoff of 12000Da, and the rotation speed of centrifugation is 8000 r/min.
Preferably, the deproteinization treatment in the fourth step is carried out by the following specific method: mixing the crude polysaccharide, distilled water and a Sevag reagent according to the mass ratio of 1:20: 9.5-10.5, oscillating, standing, centrifuging to remove lower-layer precipitates, repeatedly centrifuging for 6-8 times at the centrifugal speed of 4000r/min, dialyzing upper-layer solution, wherein the cut-off molecular weight of a dialysis bag is 3500Da, boiling the dialysis bag with boiling water for 20min, pouring the upper-layer solution into the dialysis bag with one end fastened, pouring 2/3 of the upper-layer solution with the volume not exceeding that of the dialysis bag, fastening the other end of the dialysis bag with a thread rope, placing the dialysis bag into a sealable water tank, dialyzing for 48h with flowing distilled water, and carrying out vacuum freeze-drying on the upper-layer solution after dialysis.
Preferably, the volume ratio of chloroform to n-butanol in the Sevag reagent in the fourth step is 3-4: 1.
Preferably, the mixed aqueous solution in the third step further comprises: 0.1 parts by mass of acetic acid.
Preferably, the mixed aqueous solution in the third step further comprises: 1 part by mass of hydrochloric acid having a mass fraction of 0.3%.
The invention at least comprises the following beneficial effects: the extraction method is simple and feasible, the using amounts of ethanol and Sevag reagents are reduced, the extraction rate of the black tiger fruit polysaccharide is high, the antioxidant activity is good, and the dissolution rate of the polysaccharide can be improved by respectively adding 0.1 part by mass of acetic acid and 1 part by mass of hydrochloric acid with the mass fraction of 0.3% into the mixed aqueous solution in the third step.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
secondly, taking fruit powder and 92% ethanol water solution according to the mass ratio of 1:2.8, refluxing and stirring for 1h, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 20 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 3.5 hours, concentrating the mixed aqueous solution to 5 parts by mass, adding 18 parts by mass of absolute ethyl alcohol, carrying out cold storage at 0 ℃ for 12 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
< example 2>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
secondly, taking the fruit powder and 98% ethanol water solution according to the mass ratio of 1:3.2, refluxing and stirring for 2 hours, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 30 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 4.5 hours, concentrating the mixed aqueous solution to 6 parts by mass, adding 22 parts by mass of absolute ethyl alcohol, carrying out cold storage at 4 ℃ for 16 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
< example 3>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
step two, taking the fruit powder and 95% ethanol water solution according to the mass ratio of 1:3, refluxing and stirring for 1.5h, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 25 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 4 hours, concentrating the mixed aqueous solution to 5.5 parts by mass, adding 20 parts by mass of absolute ethyl alcohol, carrying out cold storage at 2 ℃ for 14 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
< example 4>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
step two, taking the fruit powder and 90% ethanol water solution according to the mass ratio of 1:3, refluxing and stirring for 1.5h, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 25 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 4 hours, concentrating the mixed aqueous solution to 5.5 parts by mass, adding 20 parts by mass of absolute ethyl alcohol, carrying out cold storage at 2 ℃ for 14 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
In the first step, the drying temperature is 40-50 ℃, and the mesh number of the sieved mesh is 40.
And stirring in the second step adopts an electric stirring paddle, and the rotating speed of the electric stirring paddle is 450 r/min.
And the mixed aqueous solution is concentrated in the third step, and ultrafiltration concentration is carried out by adopting an ultrafiltration membrane with the molecular weight cutoff of 12000Da, and the centrifugal rotating speed is 8000 r/min.
The specific method for deproteinizing in the fourth step comprises the following steps: mixing the crude polysaccharide, distilled water and Sevag reagent according to the mass ratio of 1:20:10, oscillating, standing, centrifuging to remove lower-layer precipitates, repeatedly centrifuging for 7 times at the centrifugal speed of 4000r/min, dialyzing upper-layer solution, wherein the cut-off molecular weight of a dialysis bag is 3500Da, boiling the dialysis bag in boiling water for 20min, pouring the upper-layer solution into the dialysis bag with one end fastened, pouring 2/3 of the upper-layer solution with the volume not more than that of the dialysis bag, fastening the other end of the dialysis bag by a thread rope, placing the dialysis bag in a sealable water tank, dialyzing for 48h by flowing distilled water, and carrying out vacuum freeze drying on the upper-layer solution after dialysis.
The volume ratio of chloroform to n-butanol in the Sevag reagent in the fourth step is 4: 1.
< example 5>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
step two, taking the fruit powder and 90% ethanol water solution according to the mass ratio of 1:3, refluxing and stirring for 1.5h, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 25 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 4 hours, concentrating the mixed aqueous solution to 5.5 parts by mass, adding 20 parts by mass of absolute ethyl alcohol, carrying out cold storage at 2 ℃ for 14 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
In the first step, the drying temperature is 40-50 ℃, and the mesh number of the sieved mesh is 40.
And stirring in the second step adopts an electric stirring paddle, and the rotating speed of the electric stirring paddle is 450 r/min.
And the mixed aqueous solution is concentrated in the third step, and ultrafiltration concentration is carried out by adopting an ultrafiltration membrane with the molecular weight cutoff of 12000Da, and the centrifugal rotating speed is 8000 r/min.
The specific method for deproteinizing in the fourth step comprises the following steps: mixing the crude polysaccharide, distilled water and Sevag reagent according to the mass ratio of 1:20:10, oscillating, standing, centrifuging to remove lower-layer precipitates, repeatedly centrifuging for 7 times at the centrifugal speed of 4000r/min, dialyzing upper-layer solution, wherein the cut-off molecular weight of a dialysis bag is 3500Da, boiling the dialysis bag in boiling water for 20min, pouring the upper-layer solution into the dialysis bag with one end fastened, pouring 2/3 of the upper-layer solution with the volume not more than that of the dialysis bag, fastening the other end of the dialysis bag by a thread rope, placing the dialysis bag in a sealable water tank, dialyzing for 48h by flowing distilled water, and carrying out vacuum freeze drying on the upper-layer solution after dialysis.
In the step four, the volume ratio of chloroform to n-butyl alcohol in the Sevag reagent is 3-4: 1.
The mixed aqueous solution in the third step also comprises: 0.3 parts by mass of acetic acid.
< example 6>
A method for extracting black tiger fruit polysaccharide comprises the following steps:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
step two, taking the fruit powder and 90% ethanol water solution according to the mass ratio of 1:3, refluxing and stirring for 1.5h, filtering to obtain filter residue, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 25 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 4 hours, concentrating the mixed aqueous solution to 5.5 parts by mass, adding 20 parts by mass of absolute ethyl alcohol, carrying out cold storage at 2 ℃ for 14 hours, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, taking the crude polysaccharide to carry out deproteinization treatment to obtain the black tiger fruit polysaccharide.
In the first step, the drying temperature is 40-50 ℃, and the mesh number of the sieved mesh is 40.
And stirring in the second step adopts an electric stirring paddle, and the rotating speed of the electric stirring paddle is 450 r/min.
And the mixed aqueous solution is concentrated in the third step, and ultrafiltration concentration is carried out by adopting an ultrafiltration membrane with the molecular weight cutoff of 12000Da, and the centrifugal rotating speed is 8000 r/min.
The specific method for deproteinizing in the fourth step comprises the following steps: mixing the crude polysaccharide, distilled water and Sevag reagent according to the mass ratio of 1:20:10, oscillating, standing, centrifuging to remove lower-layer precipitates, repeatedly centrifuging for 7 times at the centrifugal speed of 4000r/min, dialyzing upper-layer solution, wherein the cut-off molecular weight of a dialysis bag is 3500Da, boiling the dialysis bag in boiling water for 20min, pouring the upper-layer solution into the dialysis bag with one end fastened, pouring 2/3 of the upper-layer solution with the volume not more than that of the dialysis bag, fastening the other end of the dialysis bag by a thread rope, placing the dialysis bag in a sealable water tank, dialyzing for 48h by flowing distilled water, and carrying out vacuum freeze drying on the upper-layer solution after dialysis.
The volume ratio of chloroform to n-butanol in the Sevag reagent in the fourth step is 4: 1.
The mixed aqueous solution in the third step also comprises: 1 part by mass of hydrochloric acid having a mass fraction of 0.3%.
< comparative example 1>
The extraction method of the black tiger fruit polysaccharide is the same as that in example 3, except that in the second step, fruit powder and 95% ethanol water solution are taken according to the mass ratio of 1:5, and 25 parts by mass of absolute ethyl alcohol is added in the third step.
< comparative example 2>
The extraction method of the black tiger fruit polysaccharide is the same as that in example 4, except that the volume ratio of chloroform to n-butanol in the Sevag reagent in the fourth step is 5: 1.
< evaluation of Tiger fruit polysaccharide >
Taking the extraction rate of the black tiger fruit polysaccharide, the total sugar content of the extracted black tiger fruit polysaccharide, the uronic acid content of the extracted black tiger fruit polysaccharide and the antioxidant capacity of the extracted black tiger fruit polysaccharide as evaluation objects, statistically recording the extraction rate, the total sugar content, the uronic acid content and the antioxidant capacity of the black tiger fruit polysaccharide in the embodiments 1-6 and the comparative examples 1-2 of the invention, wherein the calculation method of the extraction rate is 100% of the total sugar content of the black tiger fruit polysaccharide extracted per 100g of black tiger fruit powder, the phenol-sulfuric acid method is used for measuring the total sugar content of the black tiger fruit polysaccharide, the sulfuric acid-carbazole method is used for measuring the uronic acid content, and the FRAP method is used for detecting the total antioxidant capacity of the black tiger fruit polysaccharide.
The extraction rate% | Total sugar content% | Uronic acid content% | Total antioxidant capacity mg/mL | |
Example 1 | 1.83 | 46.11±0.76 | 72.62±1.80 | 0.068 |
Example 2 | 1.80 | 46.76±1.02 | 72.95±2.35 | 0.072 |
Example 3 | 1.82 | 46.56±1.27 | 73.28±1.78 | 0.070 |
Example 4 | 1.97 | 49.09±2.24 | 79.80±1.98 | 0.080 |
Example 5 | 2.30 | 52.01±1.86 | 83.56±1.65 | 0.092 |
Example 6 | 2.50 | 53.12±1.09 | 84.35±2.65 | 0.102 |
Comparative example 1 | 1.69 | 45.25±2.05 | 71.00±2.18 | 0.060 |
Comparative example 2 | 1.70 | 45.48±1.68 | 71.35±2.52 | 0.061 |
Comparing the fruit powder and 95% ethanol water solution in the mass ratio of 1:5 in the second step of comparative examples 1-2, adding 25 parts by mass of absolute ethanol in the third step, and processing the chloroform-n-butanol in the Sevag reagent in the fourth step in the volume ratio of 5:1 with examples 1-3, the results show that the use amounts of ethanol and Sevag reagent are reduced, the extraction rate, total sugar content, uronic acid content and total antioxidant capacity of the extracted Kadsura coccinea fruit polysaccharide can be improved, the extraction rate, total sugar content, uronic acid content and total antioxidant capacity of the Kadsura coccinea fruit polysaccharide extracted in example 5 are obviously improved compared with example 4, the results show that 0.1 part by mass of acetic acid is added into the mixed water solution in the third step, the extraction rate, total sugar content, uronic acid content and total antioxidant capacity of the Kadsura coccinea fruit polysaccharide extracted in example 6 are obviously improved compared with example 4, the results show that the hydrochloric acid with the mass fraction of 0.3% added in 1 part by mass in the mixed aqueous solution in the third step has a positive effect on the extraction of the black tiger fruit polysaccharide.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (7)
1. The extraction method of the black tiger fruit polysaccharide is characterized by comprising the following steps of:
step one, cleaning, removing seeds, drying, crushing and sieving black tiger fruits in sequence to obtain fruit powder;
secondly, taking fruit powder and an ethanol water solution according to the mass ratio of 1: 2.8-3.2, refluxing and stirring for 1-2 hours, filtering to obtain filter residues, and repeating the steps for three times;
step three, taking 1 part by mass of filter residue and 20-30 parts by mass of distilled water to form a mixed aqueous solution, carrying out reflux stirring for 3.5-4.5 h, concentrating the mixed aqueous solution to 5-6 parts by mass, adding 18-22 parts by mass of absolute ethyl alcohol, carrying out cold storage at 0-4 ℃ for 12-16 h, centrifuging, washing the lower-layer precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and airing to obtain crude polysaccharide;
and step four, mixing the crude polysaccharide, distilled water and Sevag reagent according to the mass ratio of 1:20: 9.5-10.5, oscillating, standing, centrifuging to remove lower-layer precipitates, repeatedly centrifuging for 6-8 times at the centrifugal rotation speed of 4000r/min, dialyzing upper-layer solution, wherein the cut-off molecular weight of a dialysis bag is 3500Da, boiling the dialysis bag with boiling water for 20min, pouring the upper-layer solution into the dialysis bag with one end fastened, pouring 2/3 of the upper-layer solution with the volume not exceeding that of the dialysis bag, fastening the other end of the dialysis bag with a thread rope, placing the dialysis bag into a sealable water tank, dialyzing for 48h with flowing distilled water, and carrying out vacuum freeze drying on the upper-layer solution after dialysis to obtain the black cutworm fruit polysaccharide.
2. The method for extracting black cutworm fruit polysaccharide as claimed in claim 1, wherein the drying temperature in the first step is 40-50 ℃, and the mesh number of the sieve is 40.
3. The method for extracting black cutworm fruit polysaccharide as claimed in claim 1, wherein the stirring in the second step is performed by using an electric stirring paddle, and the rotation speed of the electric stirring paddle is 300-600 r/min.
4. The method for extracting black cutworm fruit polysaccharide as claimed in claim 1, wherein the concentration of the mixed aqueous solution in the third step is performed by ultrafiltration concentration using an ultrafiltration membrane with a molecular weight cut-off of 12000Da, and the rotation speed of the centrifugation is 8000 r/min.
5. The method for extracting black cutworm fruit polysaccharide as claimed in claim 1, wherein the volume ratio of chloroform to n-butanol in the Sevag reagent in the fourth step is 3-4: 1.
6. The method for extracting Tiger fruit polysaccharide of claim 1, wherein the mixed aqueous solution in step three further comprises: 0.1 parts by mass of acetic acid.
7. The method for extracting Tiger fruit polysaccharide of claim 1, wherein the mixed aqueous solution in step three further comprises: 1 part by mass of hydrochloric acid having a mass fraction of 0.3%.
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