CN103040917B - Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides - Google Patents
Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides Download PDFInfo
- Publication number
- CN103040917B CN103040917B CN201210574017.9A CN201210574017A CN103040917B CN 103040917 B CN103040917 B CN 103040917B CN 201210574017 A CN201210574017 A CN 201210574017A CN 103040917 B CN103040917 B CN 103040917B
- Authority
- CN
- China
- Prior art keywords
- bean taro
- polysaccharide
- ethanol
- bean
- reflux
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously and relates to the technical field of plant extraction. The method comprises the following steps: (1) harvesting raw materials, cleaning and drying; (2) reflux-extracting with ethanol, and filtering to obtain a supernatant and filter residues; (3) defatting the supernatant with petroleum ether, extracting with n-butanol, performing adsorption and elution, concentrating, and drying to obtain powdery Apios americana flower saponins; (4) removing the solvent from the filter residues, reflux-extracting with water, filtering, collecting and mixing the filtrates; and (5) decolorizing the filtrate, performing deproteinization, concentrating, precipitating with alcohols, dialyzing and concentrating, and drying to obtain Apios americana flower polysaccharides. The method has the advantages of simple process, high yield and low cost. Besides, the total saponins and polysaccharides of Apios americana flowers are prepared simultaneously, have high purity and strong activity, and can be widely used in pharmaceuticals or functional foods with antioxidant and blood sugar regulating effects.
Description
Technical field
The present invention relates to technical field of plant extraction, be specifically related to a kind of from greatly more prepare method of saponin and polysaccharide and uses thereof bean taro simultaneously.
Background technology
Large more bean taro (
apios americanamedikus) belong to perennial leguminous plant, claim again Potato bean, American ground nut, be wound around liana, 5-7 sheet lobule one compound leaf, purple little Hua, the crown of papilionaceous flower, originate in US West, underground tuber, as asexual propagation or edible, is a kind of natural high protein, high calcium food.Introduced and develop by Japanese government the sixties owing to thering is its high nutrition and health care effect, and be widely used in various food or food ingredient, become the widely known health food of Japan, mainly to modern way of life disease (as hypertension, diabetes, obesity, osteoporosis etc.) the tool effect that has clear improvement, and there is anticancer and HIV (human immunodeficiency virus)-resistant activity composition.At present, greatly more bean taro is successfully introduced a fine variety to China, all has cultivation and utilize in area, Hangzhou, Zhejiang province, Ningbo area, Wenzhou Area, Jinhua Region.But find no large more bean taro saponin and the extracting method of polysaccharide and the relevant report of purposes at present.In addition, greatly more bean taro, at present substantially in discarded state, fails to develop, and has caused the waste of resource.
On other plant, there is at present the report of the technique that saponin and polysaccharide extract simultaneously, as Chinese patent (application number: 201110029129.1, application publication number CN102178720A) a kind of method of simultaneously preparing total soap battalion and polysaccharide from Herb Gynostemmae Pentaphylli is disclosed, but there is the deficiency of the aspects such as purity is low.
Summary of the invention
The object of this invention is to provide that a kind of cost is low, raw material comprehensive utilization, and when environmental pollution is little, prepare the method for bean taro saponin and polysaccharide, and provide the large more bean taro saponin prepared by the method and polysaccharide to there is the application in medicine or the functional food of antioxidation, blood glucose regulation effect in preparation.
Technical scheme of the present invention is as follows:
The large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that according to the following steps:
1) pluck large more bean taro, clean, dry, pulverize, cross 40 mesh sieves, obtain greatly more bean taro pollen end;
2) bean taro pollen end greatly more, according to solid-liquid ratio 1:10-1:20, adding concentration is 70%-90% ethanol, 50-80 DEG C of reflux, extract, 1-3 time, each time 1-3 hour, filtration, obtains supernatant and filtering residue;
3) supernatant, volatilizes the rear water dissolution of using, defat with petroleum ether, and n-butanol extraction, gets n-butyl alcohol part after absorption with macroporous adsorbent resin eluting, concentrated, the dry bean taro saponin powder that obtains;
4) filtering residue, volatilizes solvent, and with its weight 10-20 distilled water doubly, in 70-90 DEG C of reflux, extract, 1-3 time, each time 2-3 hour, filters, and collects merging filtered solution;
5) filtered solution decolouring, deproteinization, concentrated, add ethanol, hold over night is carried out precipitate with ethanol, and collecting precipitation, after dialysis, use DEAE-cellulose chromatography, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M-0.5M NaCl eluent, dialysis, concentrated, dry, obtain bean taro polysaccharide.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that step 2) described solid-liquid ratio is 1:15-1:20, is preferably 1:20; Concentration of alcohol is 80-90%, preferably 90%; Reflux, extract, temperature is 60-70 DEG C, preferably 70 DEG C; Reflux, extract, 2 times, each time 2 h.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that the macroporous adsorbent resin described in step 3) is D101 or AB-8, and the eluent that eluting adopts is that mass concentration is the ethanol of 60%-90%.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that the discoloration method described in step 5) is activated carbon decolorizing or hydrogen peroxide decolouring; Described method for removing protein is sevage method, trichloroacetic acid method, sodium chloride method or Calcium Chloride Method; Described precipitate with ethanol is 75-95% for adding ethanol to make alcohol precipitation concentration; Described dialysis adopts 3KD dialyzer.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that the drying means described in step 1), step 3) and step 5) is lyophilization or vacuum drying.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that described eluent is the ethanol of mass concentration 75%.
The described large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that alcohol precipitation concentration is 90-95%.
Said method has been prepared large more bean taro flower total saponine and polysaccharide simultaneously, and the large more bean taro saponin and the polysaccharide that prepare can reach higher extraction ratio and purity, and wherein greatly more bean taro saponin output can reach 2-3.5%, and purity is 90-97%; Large more bean taro polysaccharide yield can reach 0.5-2%, and purity is 94-98%.
The large more bean taro flower total saponine preparing can be used as medicine or the function food additive of preparing antioxidant activity, large more bean taro polysaccharide can be used as medicine or the function food additive of preparation adjusting blood glucose, if large more bean taro polysaccharide is for the preparation of medicines such as treatment diabetes.The active component that above-mentioned preparation method is extracted is as removing free radical, regulating the medicines such as blood glucose, blood fat and functional food ingredient to use, good effect, applied range, safety non-toxic, large more bean taro active component, as the green treasure-house of a class natural drug, the function of health food factor, dietary supplement, if can be developed, can be exploitation antioxidant, regulate blood sugar disorders that new way is provided, also provide technical support to the development regional economy of China and the people's health.
In sum, the present invention has advantages of that step is simple, yield is high, cost is low, and the present invention more prepares two class materials bean taro from large simultaneously, raw material availability is high, and prepared large more bean taro flower total saponine and purity of polysaccharide are high, active strong, of many uses, not only can be used alone and can also be used in conjunction with other natural drugs.
Detailed description of the invention
Now in conjunction with embodiments of the invention, the invention will be further described.
embodiment 1:
Large more bean taro flower total saponine extraction process is optimized:
Bean taro has been spent the clear assorted postlyophilization of stalk, cryopreservation; Before extracting, measure the actual moisture in style with fast tester for water content, sample saponin content and antioxygenic property are all amounted to into colored butt and are calculated.Technological process: bean taro pollen end → reflux, extract, → filtered while hot → washery slag → standardize solution → analytical test under certain condition.
The assay method of total saponin content is as follows:
Precision takes ursolic acid reference substance 2.5mg, adds dehydrated alcohol and is settled to 10mL, obtains 250 μ g/mL ursolic acid titers.The accurate reference substance solution 0,100,150 of drawing, 200,250,300,350,400 μ l are placed in respectively 5mL volumetric flask, volatilize solvent, 5% vanillin-glacial acetic acid, the 200 μ l that add respectively new preparation, perchloric acid 800 μ l, shake up, put 60 DEG C of water-bath 15min, taking-up is placed in frozen water cooling, and precision adds 4ml glacial acetic acid, be settled to scale, mix, after 20min, measure trap A at 548nm wavelength place.Taking trap (A) as vertical coordinate, concentration (C) is abscissa, and drawing standard curve obtains standard curve regression equation: Y=0.0458X-0.0002(R
2=0.9998).The mensuration of sample saponin content is measured trap by above-mentioned standard curve formulating method, and calculates the total saponin content of liquid to be measured according to standard curve.
The different extracting parameters settings of large more bean taro flower total saponine and analysis result are in table 1.
As shown in Table 1, each factor optimum process condition of optimization is: extracting temperature is 70 DEG C, and solid-liquid ratio is 1: 20, concentration of alcohol 90%, and extraction time is 2.0h.Adopt the condition of this optimization to extract bean taro flower total saponine, recording the content that total saponins accounts for butt is 5.55%.
embodiment 2:
Large more bean taro polysaccharide extracting process optimization:
Bean taro has been spent the clear assorted postlyophilization of stalk, cryopreservation; Before extracting, measure the actual moisture in style with fast tester for water content, sample polysaccharide yield is amounted to into colored butt and is calculated.Technological process: bean taro pollen end → 90% alcohol reflux → filtered while hot → filtering residue volatilizes solvent → distilled water reflux, extract, → filtered while hot → supernatant concentration, and to certain volume → 4, DEG C ethanol precipitation → 5000r/min is centrifugal → precipitate is dissolved in water → dialyses → obtain crude polysaccharides analytical test.
The computational methods of polysaccharide yield are as follows:
Weigh/greatly more bean taro dry basis × 100% of polysaccharide yield (%)=crude polysaccharides
The different extracting parameters settings of large more bean taro polysaccharide and analysis result are in table 2.
As shown in Table 2, each factor optimum process condition of optimization is: extracting temperature is 80 DEG C, and solid-liquid ratio is 1: 20, and extraction time is 100min, and the concentration of alcohol of precipitate with ethanol is 90%.Adopt the condition of this optimization to extract bean taro polysaccharide, recording polysaccharide yield is 4.91%.
embodiment 3:
Large more bean taro flower total saponine, polysaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and drains, and lyophilization, pulverizes, and crosses 40 mesh sieves;
(2) in 10g bean taro pollen end, adding 200mL concentration is 90% alcoholic solution, reflux, extract, 3 times in 70 DEG C of water-baths, and each time 2 h, sucking filtration while hot, with 90% ethanol washery slag, obtains supernatant and filtering residue;
(3) supernatant, after volatilizing with Rotary Evaporators, water is settled to 100mL.After equal-volume defat with petroleum ether, with isopyknic water-saturated n-butanol extraction 3 times, get n-butyl alcohol part, Rotary Evaporators is concentrated, after vacuum drying, with water dissolution, the upper macroporous adsorbent resin AB-8(fast science equipment company limited that rubs in Shanghai produces, lower same), adopt 75% ethanol to carry out eluting, collect eluent, Rotary Evaporators is concentrated, lyophilization, obtains bean taro saponin powder;
(4) filtering residue, vacuum drying volatilizes solvent, with the distilled water of 20 times of its weight, reflux, extract, 3 times in 80 DEG C of water-baths, each time 2 h, filtered while hot, water washery slag, filtered solution activated carbon decolorizing, after sevage method deproteinization, Rotary Evaporators is concentrated, add ethanol to concentration of alcohol 90%, be placed on hold over night in 4 DEG C of refrigerators and carry out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, (GE-GE produces upper DEAE-52 post, lower same), use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M and 0.5M NaCl eluant solution liquid Rotary Evaporators concentrated, the dialysis of 3KD dialyzer, Rotary Evaporators is concentrated, lyophilization, obtain bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 3.5%, purity 96.96 %; Bean taro polysaccharide yield 2%, purity is 97.53%.
embodiment 4:
Large more bean taro flower total saponine, polysaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and drains, and vacuum drying, pulverizes, and crosses 40 mesh sieves;
(2) in 10g bean taro pollen end, adding 100mL concentration is 80% alcoholic solution, reflux, extract, 2 times in 50 DEG C of water-baths, and each time 2 h, sucking filtration while hot, with 80% ethanol washery slag, obtains supernatant and filtering residue;
(3) supernatant, after volatilizing with Rotary Evaporators, water is settled to 100mL.After equal-volume defat with petroleum ether, with isopyknic water-saturated n-butanol extraction 3 times, get n-butyl alcohol part, Rotary Evaporators is concentrated, after vacuum drying, with water dissolution, upper macroporous adsorbent resin AB-8, adopts 60% ethanol to carry out eluting, collect eluent, Rotary Evaporators is concentrated, and lyophilization, obtains bean taro saponin powder;
(4) filtering residue, vacuum drying volatilizes solvent, with the distilled water of 10 times of its weight, reflux, extract, 1 time in 70 DEG C of water-baths, each 3 hours time, filtered while hot, water washery slag, filtered solution activated carbon decolorizing, after trichloroacetic acid method deproteinization, Rotary Evaporators is concentrated, add ethanol to concentration of alcohol 75%, be placed on hold over night in 4 DEG C of refrigerators and carry out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M and 0.5M NaCl eluant solution liquid Rotary Evaporators concentrated, the dialysis of 3KD dialyzer, Rotary Evaporators is concentrated, lyophilization, obtain bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 2.98%, purity 94.17 %; Bean taro polysaccharide yield 1.54%, purity is 96.79%.
embodiment 5:
Large more bean taro flower total saponine, polysaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and drains, and lyophilization, pulverizes, and crosses 40 mesh sieves;
(2) in 10g bean taro pollen end, adding 100mL concentration is 70% alcoholic solution, reflux, extract, 1 time in 80 DEG C of water-baths, and 3 hours time, sucking filtration while hot, with 70% ethanol washery slag, obtains supernatant and filtering residue;
(3) supernatant, after volatilizing with Rotary Evaporators, water is settled to 100mL.After equal-volume defat with petroleum ether, with isopyknic water-saturated n-butanol extraction 3 times, get n-butyl alcohol part, Rotary Evaporators is concentrated, after vacuum drying, with water dissolution, the upper macroporous adsorbent resin D-101(fast science equipment company limited that rubs in Shanghai produces, lower same), adopt 90% ethanol to carry out eluting, collect eluent, Rotary Evaporators is concentrated, lyophilization, obtains bean taro saponin powder;
(4) filtering residue, vacuum drying volatilizes solvent, with the distilled water of 20 times of its weight, reflux, extract, 2 times in 90 DEG C of water-baths, each 1 hour time, filtered while hot, water washery slag, the decolouring of filtered solution hydrogen peroxide, after sodium chloride method deproteinization, Rotary Evaporators is concentrated, add ethanol to concentration of alcohol 80%, be placed on hold over night in 4 DEG C of refrigerators and carry out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M and 0.5M NaCl eluant solution liquid Rotary Evaporators concentrated, the dialysis of 3KD dialyzer, Rotary Evaporators is concentrated, lyophilization, obtain bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 3.12%, purity 95.52 %; Bean taro polysaccharide yield 1.82%, purity is 97.14%.
embodiment 6:
Large more bean taro flower total saponine, polysaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and drains, and vacuum drying, pulverizes, and crosses 40 mesh sieves;
(2) in 10g bean taro pollen end, adding 300mL concentration is 80% alcoholic solution, reflux, extract, 1 time in 80 DEG C of water-baths, and each 3 hours time, sucking filtration while hot, with 80% ethanol washery slag, obtains supernatant and filtering residue;
(3) supernatant, after volatilizing with Rotary Evaporators, water is settled to 100mL.After equal-volume defat with petroleum ether, with isopyknic water-saturated n-butanol extraction 3 times, get n-butyl alcohol part, Rotary Evaporators is concentrated, after vacuum drying, with water dissolution, upper macroporous adsorbent resin D-101, adopts 90% ethanol to carry out eluting, collect eluent, Rotary Evaporators is concentrated, and lyophilization, obtains bean taro saponin powder;
(4) filtering residue, vacuum drying volatilizes solvent, with the distilled water of 10 times of its weight, reflux, extract, 2 times in 90 DEG C of water-baths, each 3 hours time, filtered while hot, water washery slag, the decolouring of filtered solution hydrogen peroxide, after Calcium Chloride Method deproteinization, Rotary Evaporators is concentrated, add ethanol to concentration of alcohol 95%, be placed on hold over night in 4 DEG C of refrigerators and carry out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M and 0.5M NaCl eluant solution liquid Rotary Evaporators concentrated, does 3KD(also have other model?) dialyzer dialysis, Rotary Evaporators is concentrated, lyophilization, obtain bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 2.47%, purity 93.64 %; Bean taro polysaccharide yield 1.95%, purity is 97.28%.
embodiment 7:
Large more bean taro flower total saponine, polysaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and drains, and lyophilization, pulverizes, and crosses 40 mesh sieves;
(2) in 10g bean taro pollen end, adding 200mL concentration is 90% alcoholic solution, reflux, extract, 2 times in 80 DEG C of water-baths, and each time 2 h, sucking filtration while hot, with 90% ethanol washery slag, obtains supernatant and filtering residue;
(3) supernatant, after volatilizing with Rotary Evaporators, water is settled to 100mL.After equal-volume defat with petroleum ether, with isopyknic water-saturated n-butanol extraction 3 times, get n-butyl alcohol part, Rotary Evaporators is concentrated, after vacuum drying, with water dissolution, upper macroporous adsorbent resin AB-8, adopts 75% ethanol to carry out eluting, collect eluent, Rotary Evaporators is concentrated, and lyophilization, obtains bean taro saponin powder;
(4) filtering residue, vacuum drying volatilizes solvent, with the distilled water of 20 times of its weight, reflux, extract, 2 times in 90 DEG C of water-baths, each 3 hours time, filtered while hot, water washery slag, filtered solution activated carbon decolorizing, after sevage method deproteinization, Rotary Evaporators is concentrated, add ethanol to concentration of alcohol 95%, be placed on hold over night in 4 DEG C of refrigerators and carry out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M and 0.5M NaCl eluant solution liquid Rotary Evaporators concentrated, the dialysis of 3KD dialyzer, Rotary Evaporators is concentrated, lyophilization, obtain bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 3.26 %, purity 96.53 %; Bean taro polysaccharide yield 1.96%, purity is 97.33%.
example 8:a kind of large more bean taro flower total saponine, holosaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and dry, pulverize, and crosses 40 mesh sieves;
(2) will in bean taro pollen end, add 70% alcoholic solution of 10 times of quality multiples, 70 DEG C of reflux, extract, 3 times, each time 2 h, filters, and obtains supernatant and filtering residue;
(3) supernatant, defat with petroleum ether, after equal-volume n-butanol extraction 3 times, gets n-butyl alcohol part, concentrated, and upper macroporous adsorbent resin AB-8 adopts 60% ethanol to carry out eluting, obtains eluent, and concentrating under reduced pressure is dry, obtains bean taro saponin powder;
(4) filtering residue, volatilizes solvent, and with the distilled water of 20 times of its weight, 80 DEG C of reflux, extract, 3 times, each time 2 h, filters.Concentrated after filtered solution activated carbon decolorizing, sevage method deproteinization, add ethanol to concentration of alcohol 90%, hold over night is carried out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collects 0.3M and 0.5M NaCl eluant solution liquid concentrated, the dialysis of 3KD dialyzer, concentrated, lyophilization, obtains bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 2.6%, purity 95.43 %; Bean taro polysaccharide yield 1.9%, purity is 97.38%.
example 9:a kind of large more bean taro flower total saponine, holosaccharide preparation method simultaneously, comprise the steps:
The bean taro of (1) 6, plucking July, cleans, and dry, pulverize, and crosses 40 mesh sieves;
(2) will in bean taro pollen end, add 80% alcoholic solution of 15 times of quality multiples, 70 DEG C of reflux, extract, 3 times, each time 2 h, filters, and obtains supernatant and filtering residue;
(3) supernatant, defat with petroleum ether, after equal-volume n-butanol extraction 3 times, gets n-butyl alcohol part, concentrated, and upper macroporous adsorbent resin AB-8 adopts 85% ethanol to carry out eluting, obtains eluent, and concentrating under reduced pressure is dry, obtains bean taro saponin powder;
(4) filtering residue, volatilizes solvent, and with the distilled water of 20 times of its weight, 80 DEG C of reflux, extract, 3 times, each time 2 h, filters.Concentrated after filtered solution activated carbon decolorizing, sevage method deproteinization, add ethanol to concentration of alcohol 90%, hold over night is carried out precipitate with ethanol, collecting precipitation thing, after the dialysis of 3KD dialyzer, upper DEAE-52 post, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collects 0.3M and 0.5M NaCl eluant solution liquid concentrated, the dialysis of 3KD dialyzer, concentrated, lyophilization, obtains bean taro holosaccharide.
Wherein bean taro flower total saponine productive rate 3.1%, purity 96.66 %; Bean taro polysaccharide yield 2%, purity is 97.53%.
embodiment 10:
The bean taro saponin antioxidation in vitro performance evaluation preparing with above-described embodiment 3:
(1) scavenging action of bean taro saponin to DPPH free radical:
Bean taro saponin is mixed with the liquid to be measured (0.1,0.2,0.4,0.8,1.6,3.2mg/mL) of different solubility with ethanol.3.9 mL DPPH solution (0.101mmol) and 0.1ml are treated to the mixing of test sample solution shakes up, at room temperature react 1h, under 517nm wavelength, measure absorbance A.Taking methanol as blank reagent, spectrophotometer is returned to zero, 3.9 mL DPPH and 0.1ml alcohol mixeding liquid are blank, record absorbance A
0.Suppression ratio (%)=(A
0– A)/A
0× 100.Taking liquid solubility to be measured as abscissa (X), suppression ratio is the relation curve that vertical coordinate (Y) obtains testing sample removing DPPH free radical.According to equation, what calculate DPPH free radical scavenging activity partly suppresses solubility (IC
50).
(2) bean taro saponin is to ABTS
+the scavenging action of free radical:
The persulfate aqueous solution of getting 176 ul concentration and be 140 mM/L joins in the ABTS solution (distilled water diluting) that 10 mL solubility are 7 mM/L and mixes, and lucifuge reaction 12-16h, obtains ABTS working solution.
Bean taro saponin is mixed with the liquid to be measured (0.1,0.2,0.4,0.8,1.6,3.2mg/mL) of different solubility with ethanol.
Before measuring, add ethanol by ABTS
+solution dilution to light absorption value is 0.700 ± 0.02(734nm) under.Taking ethanol as blank reagent, spectrophotometer is returned to zero.By the ABTS of 3.9 mL
+solution and 0.1 mL treat that test sample solution mixes and shake up, and at room temperature react 6min, under 734nm wavelength, measuring light absorption value is A.The ABTS of 3.9 mL
+solution and 0.1 m L ethanol are blank, record light absorption value A
0.Suppression ratio (%)=(A
0– A)/A
0× 100.Taking liquid solubility to be measured as abscissa (X), suppression ratio is the relation curve that vertical coordinate (Y) obtains testing sample removing DPPH free radical.According to equation, what calculate DPPH free radical scavenging activity partly suppresses solubility (IC
50).
(3) scavenging action of bean taro saponin to OH free radical:
Utilize at Sirius single hose chemiluminescence detector and adopt FeSO
4-luminol-H
2o
2system detects sample and removes OH free radical ability.
The phosphate buffered solution (PBS, pH=7.8, and the EDTA that contains 0.1 mmol/L) of preparation 50mmol/L.After NaOH melting luminol with a small amount of 1mmol/L, then be configured to the luminol solution that concentration is 0.2mmol/L with PBS.Water is configured to 1.5% hydrogen peroxide solution and the copperas solution of 0.6mmol/L.Bean taro saponin is mixed with the liquid to be measured (0.1,0.2,0.4,0.8,1.6,3.2mg/mL) of different solubility with ethanol.
The sample solution of first hand sampling 50 μ L in the luminous tube of luminous detection instrument, the copperas solution of the luminol solution of 600 μ L and 50 μ L, add 50 μ L hydrogen peroxide solutions to start reaction with automatic sampler again, record luminous value every 2s, acquisition time is 120s.Luminous value automatically carries out integration to luminosity curve by software that instrument is with and obtains.Sample is to O
2 -the calculating that the clearance rate of free radical is calculated by following formula, clearance rate %=[(
cL blank-
cL sample)/
cL blank-
cL background] × 100.
cL blankfor blank luminosity curve integral area,
cL samplefor the integral area of the luminosity curve of sample sets,
cL backgroundluminosity curve integral area when not adding pyrogallol.(blank replace to ethanol for sample; Background is that hydrogen peroxide replaces to water).Taking liquid solubility to be measured as abscissa (X), suppression ratio is the relation curve that vertical coordinate (Y) obtains testing sample removing DPPH free radical.According to equation, what calculate DPPH free radical scavenging activity partly suppresses solubility (IC
50).
(4) bean taro saponin is to O
2 -the scavenging action of free radical:
Utilizing at Sirius single hose chemiluminescence detector adopts pyrogallol-Luminol to detect sample removing O
2 -free radical ability.
Use NaCO
3and NaHCO
3be made into the phosphate buffered solution (CBS, pH=10.2, containing the EDTA of 0.1mmol/L) of 50mmol/L.After NaOH melting luminol with a small amount of 1mmol/L, then be configured to the luminol solution that concentration is 0.5mmol/L with CBS.It is 3.125 × 10 that water is mixed with concentration
-5the pyrogallol solution of mmol/L.Bean taro saponin is mixed with the liquid to be measured (0.1,0.2,0.4,0.8,1.6,3.2mg/mL) of different solubility with ethanol.
The sample solution of first hand sampling 50 μ L in light pipe on the offensive in the luminous tube of luminous detection instrument, the CBS solution of the luminol solution of 200 μ L and 150 μ L, add 250 μ L pyrogallol solution to start reaction with automatic sampler again, record luminous value every 2s, acquisition time is 120s, and luminous value automatically carries out integration to luminosity curve by software that instrument is with and obtains.Sample is to O
2 -the calculating that the clearance rate of free radical is calculated by following formula, clearance rate %=[(
cL blank-
cL sample)/
cL blank-
cL background] × 100.
cL blankfor blank luminosity curve integral area,
cL samplefor the integral area of the luminosity curve of sample sets,
cL backgroundluminosity curve integral area when not adding pyrogallol.(blank replace to ethanol for sample; Background is that pyrogallol replaces to water).Taking liquid solubility to be measured as abscissa (X), suppression ratio is the relation curve that vertical coordinate (Y) obtains testing sample removing DPPH free radical.According to equation, what calculate DPPH free radical scavenging activity partly suppresses solubility (IC
50).
Bean taro removed saponin free radical ability is as in table 3, table 4.
According to table 3, table 4 is known, and bean taro saponin is to DPPH, ABTS, OH, O
2 -four kinds of free radicals all have very strong removing ability, and are all concentration dependence.Wherein to O
2 -the removing ability of free radical is higher than ascorbic acid Vc.
embodiment 11:the bean taro polysaccharide preparing with above-described embodiment 3 is to tissue of experimental diabetic mice hypoglycemic activity:
Male mouse of kunming, 20g ± 2g, age in 6-7 week, 80, adaptability is fed fasting after 3 days and be can't help water 24 hours, gets at random 10 as normal control, the normal saline of injection equivalent, all the other 70 lumbar injection alloxan (220mg/kgbw), within 4 hours, pneumoretroperitoneum is injected 20% glucose solution (0.2 mL/kgbw).Fasting 3-5 hour after 3 days, docking is got blood and is surveyed blood glucose, and blood glucose value >11.1 mmol/L is hyperglycemia model success mice, for experiment.
50 groups of diabetic mices, be divided at random 5 groups by body weight and blood glucose homeostatic principle, 10 every group, be divided into diabetes model matched group, positive drug matched group (glibenclamide 5 mg/kgbwd), administration group (100,200,400 mg/kgbwd), separately establishes Normal group.Each group gavage gives relative medicine, and wherein diabetes model matched group and Normal group give isopyknic normal saline, successive administration 7 days, and fasting after last administration, measures fasting glucose concentration.
As seen from table, compared with Normal group, the blood glucose in diabetic mice value of alloxan induction significantly raises, but no significant difference between group shows modeling success.After bean taro polysaccharide treatment, mouse blood sugar value obviously declines, there were significant differences compared with diabetes model control group mice blood glucose value P < 0.01).The bean taro polysaccharide of high dose has the hypoglycemic effect close to glibenclamide.The hypoglycemic activity of bean taro polysaccharide presents certain dose-effect relationship.
embodiment 12:the bean taro saponin preparing with above-described embodiment 1, the experiment of the chmice acute Oral toxicity of polysaccharide:
Laboratory animal: select 40 of the health of body weight 20g ± 2g, ripe ICR mices, male and female half and half.
Dosage and medication: establish 20.0 g/kg body weight dosage groups by mtd test method, take the bean taro saponin 40g that example 1 prepares, be mixed with 40mL suspension with 1%CMC as solvent.Each 10 of male and female.The 16h that stops eating before mouse stomach, by 20mL/kg body weight gavage capacity secondary gavage, interval 6h.
Dosage and medication: establish 20.0 g/kg body weight dosage groups by mtd test method, take the bean taro polysaccharide 40g that example 1 prepares, be mixed with 40mL suspension with 1%CMC as solvent.Each 10 of male and female.The 16h that stops eating before mouse stomach, by 20mL/kg body weight gavage capacity secondary gavage, interval 6h.
After administration, observe general status, the poisoning and death condition of mice, observe two weeks time limits, the mice starting weight and heavy eventually of weighing.
Duration of test, each mice is showed no manifest symptom, also without dead.Bean taro polysaccharide is all greater than 20.0g/kg body weight to male and female its mouse oral maximum tolerated dose, belongs to nontoxic level.
Claims (10)
1. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously, is characterized in that according to the following steps:
1) pluck large more bean taro, clean, dry, pulverize, cross 40 mesh sieves, obtain greatly more bean taro pollen end;
2) bean taro pollen end greatly more, according to solid-liquid ratio 1:10-1:20, adding concentration is 70%-90% ethanol, 50-80 DEG C of reflux, extract, 1-3 time, each time 1-3 hour, filtration, obtains supernatant and filtering residue;
3) supernatant, volatilizes the rear water dissolution of using, defat with petroleum ether, and n-butanol extraction, gets n-butyl alcohol part after absorption with macroporous adsorbent resin eluting, concentrated, the dry bean taro saponin powder that obtains;
4) filtering residue, volatilizes solvent, and with its weight 10-20 distilled water doubly, in 70-90 DEG C of reflux, extract, 1-3 time, each time 2-3 hour, filters, and collects merging filtered solution;
5) filtered solution decolouring, deproteinization, concentrated, add ethanol, hold over night is carried out precipitate with ethanol, and collecting precipitation, after dialysis, use DEAE-cellulose chromatography, use respectively distilled water, 0.1M, 0.3M, 0.5M NaCl eluant solution liquid, collect 0.3M-0.5M NaCl eluent, dialysis, concentrated, dry, obtain bean taro polysaccharide.
2. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 1, is characterized in that step 2) described solid-liquid ratio is 1:15-1:20; Concentration of alcohol is 80-90%; Reflux, extract, temperature is 60-70 DEG C; Reflux, extract, 2 times, each time 2 h.
3. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 1, is characterized in that the macroporous adsorbent resin described in step 3) is D101 or AB-8, and the eluent that eluting adopts is that mass concentration is the ethanol of 60%-90%.
4. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 1, is characterized in that the discoloration method described in step 5) is activated carbon decolorizing or hydrogen peroxide decolouring; Described method for removing protein is sevage method, trichloroacetic acid method, sodium chloride method or Calcium Chloride Method; Described precipitate with ethanol is 75-95% for adding ethanol to make alcohol precipitation concentration; Described dialysis adopts 3KD dialyzer.
5. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 1, is characterized in that the drying means described in step 1), step 3) and step 5) is lyophilization or vacuum drying.
6. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 3, is characterized in that described eluent is the ethanol of mass concentration 75%.
7. according to the large method that more bean taro flower total saponine, polysaccharide are prepared described in claim 1 or 4 simultaneously, it is characterized in that alcohol precipitation concentration is 90-95%.
8. the large more bean taro flower total saponine preparing according to method described in any one claim in claim 1-7 is as the application of medicine or function food additive of preparing antioxidant activity.
9. the large more bean taro flower total saponine preparing according to method described in any one claim in claim 1-7 is being removed DPPH free radical, ABTS
+free radical, OH free radical, O
2 -application on free radical.
10. the large method that more bean taro flower total saponine, polysaccharide are prepared simultaneously according to claim 2, is characterized in that described solid-liquid ratio is 1:20; Concentration of alcohol is 90%; Reflux, extract, temperature is 70 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210574017.9A CN103040917B (en) | 2012-12-26 | 2012-12-26 | Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210574017.9A CN103040917B (en) | 2012-12-26 | 2012-12-26 | Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103040917A CN103040917A (en) | 2013-04-17 |
| CN103040917B true CN103040917B (en) | 2014-10-08 |
Family
ID=48053928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210574017.9A Expired - Fee Related CN103040917B (en) | 2012-12-26 | 2012-12-26 | Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103040917B (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844720B (en) * | 2015-06-19 | 2016-11-09 | 贵州师范学院 | A kind of separating and extracting process of hundred tail gracilis polysaccharides |
| CN107353358A (en) * | 2017-08-28 | 2017-11-17 | 浙江大学 | Beans taro polysaccharide and preparation method thereof |
| CN107582607B (en) * | 2017-08-28 | 2020-11-24 | 浙江大学 | Application of alocasia esculenta flower ethanol extract in reducing liver cell lipid deposition |
| CN107510698B (en) * | 2017-08-28 | 2021-03-02 | 浙江大学 | Antioxidation application of taro polysaccharide |
| CN107582606B (en) * | 2017-08-28 | 2020-11-24 | 浙江大学 | Preparation method of alocasia esculenta flower ethanol extract and antioxidation application thereof |
| CN107582562B (en) * | 2017-08-28 | 2020-11-24 | 浙江大学 | Application of taro polysaccharide in reducing liver cell lipid deposition |
| CN107793492A (en) * | 2017-11-17 | 2018-03-13 | 浙江海洋大学 | A kind of extracting method of fructus cannabis polysaccharide |
| CN107823245B (en) * | 2017-11-22 | 2021-03-02 | 浙江大学 | Application of water extract of soybean taro leaves in reducing lipid deposition of liver cells |
| CN107812032B (en) * | 2017-11-22 | 2021-01-26 | 浙江大学 | Application of soybean taro leaf ethanol extract in reducing liver cell lipid deposition |
| CN107823246B (en) * | 2017-11-22 | 2021-03-02 | 浙江大学 | Preparation method of ethanol extract of taro tuber and antioxidant application thereof |
| CN107823244B (en) * | 2017-11-22 | 2021-03-02 | 浙江大学 | Preparation method of soybean taro leaf ethanol extract and antioxidation application thereof |
| CN107812031B (en) * | 2017-11-22 | 2021-01-26 | 浙江大学 | Preparation method of water extract of Doudou taro leaves and antioxidation application thereof |
| CN108478593A (en) * | 2018-04-28 | 2018-09-04 | 浙江大学 | The hypoglycemic purposes of beans taro stem tuber polysaccharide |
| CN108403752A (en) * | 2018-04-28 | 2018-08-17 | 浙江大学 | The hypoglycemic purposes of beans taro ethanol extract |
| CN109662236A (en) * | 2018-12-12 | 2019-04-23 | 漳州市吾梓贸易有限公司 | A kind of food additives of the more saponin(es of selenium-rich polysaccharide, preparation method and applications |
| CN114163541A (en) * | 2021-03-19 | 2022-03-11 | 贵州省生物研究所 | Green separation and purification method and application of blueberry polysaccharide |
| CN113940427A (en) * | 2021-11-01 | 2022-01-18 | 大连海洋大学 | Preparation process of asterias amurensis Lutken saponin with antioxidant activity |
| CN113797220A (en) * | 2021-11-03 | 2021-12-17 | 王砚桐 | Preparation method and application of mouse injection medicine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5219344B2 (en) * | 2006-06-16 | 2013-06-26 | 三省製薬株式会社 | Topical skin preparation |
| CN102443072A (en) * | 2010-10-12 | 2012-05-09 | 沈阳药科大学 | Method for simultaneously extracting polysaccharides and saponins from astragalus |
| KR101227171B1 (en) * | 2010-10-13 | 2013-01-28 | 전남과학대학 산학협력단 | Cosmetic composition using Apios and manufacturing method thereof |
| CN102178720A (en) * | 2011-01-24 | 2011-09-14 | 南京泽朗医药科技有限公司 | Method for simultaneously preparing total saponin and polysaccharide from fiveleaf gynostemma herb |
-
2012
- 2012-12-26 CN CN201210574017.9A patent/CN103040917B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103040917A (en) | 2013-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103040917B (en) | Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides | |
| CN102600219B (en) | Total flavone extract of abelmoschus manihot and preparing method of total flavone extract | |
| CN100464764C (en) | Composition of white mulberry leaf blood-sugar-reducing effective components and preparing method | |
| CA3149142C (en) | Plant extraction method | |
| CN1931270B (en) | Sobering up and liver protecting Chinese medicine preparation and its preparation process | |
| CN109833377B (en) | Camellia oleifera Abel extract and preparation method and application thereof | |
| CA2979576A1 (en) | Pharmaceutical composition containing silibinin | |
| CN103156869A (en) | Sanggenone C and sanggenone D extracted from morus plants and new medicine application of composition | |
| CN111249338A (en) | Cistanche deserticola extract and industrial preparation method and application thereof | |
| CN102726479A (en) | Mulberry leaf flour with hyperglycemic effect and preparation method thereof | |
| CN101816723B (en) | Sobering up and liver protecting Chinese medicinal preparation and preparation method thereof | |
| CN105267257A (en) | Preparation method of ginkgo leaf extract | |
| JP2017518381A (en) | Medicinal use of anti-tumor for rutile pentacyclic triterpene saponins compounds | |
| CN1840067A (en) | Chewing tablet of asparagus and preparation process thereof | |
| CN1434053A (en) | Rhizoma anemarrhenae extract and preparation process and use thereof | |
| CN102228513B (en) | Medicinal composition for treating diabetes or diabetic complications and preparation method thereof | |
| CN107857826A (en) | A kind of isolation and purification method of hypoglycemic banana flowers polysaccharide | |
| CN101912425A (en) | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof | |
| CN102302615B (en) | Effective site group of daphne giraldii nitsche leaf, preparation method, medicinal composition and application thereof | |
| CN102058822B (en) | Pharmaceutical composition for strengthening stomach and promoting digestion | |
| CN104945532B (en) | The preparation method of Gynura divaricata polysaccharide | |
| CN108743795A (en) | A kind of prevention diabetic nephropathy towards medicament extract and its preparation method and application | |
| CN101559166B (en) | Ophiopogon japonicus extract, preparation method and hypoglycemic application thereof | |
| CN103054038A (en) | Preparation method of bamboo leaf flavonoid extract for reducing blood sugar activity | |
| CN107050161A (en) | A kind of extraction of one side pin total alkaloid and isolation and purification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141008 Termination date: 20161226 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |