CN102914508A - Method for measuring content of polysaccharide in camellia seeds - Google Patents
Method for measuring content of polysaccharide in camellia seeds Download PDFInfo
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- CN102914508A CN102914508A CN2012104016616A CN201210401661A CN102914508A CN 102914508 A CN102914508 A CN 102914508A CN 2012104016616 A CN2012104016616 A CN 2012104016616A CN 201210401661 A CN201210401661 A CN 201210401661A CN 102914508 A CN102914508 A CN 102914508A
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Abstract
The invention provides a method for measuring the content of polysaccharide in camellia seeds. The method comprises the following steps of: firstly, sequentially extracting camellia seeds by using a low-polarity solvent and alcohol, removing liposoluble substances and soluble glycoside substances from the camellia seeds; extracting polysaccharide from the camellia seeds by using water; and finally measuring the content of the polysaccharide in the camellia seeds by adopting a sulfuric acid-phenol method. The method provided by the invention has the advantages of strong maneuverability, accuracy in measuring results and high reproducibility, can be carried out in common conventional routine test laboratories, and provides an effective guarantee for development, production and quality control of polysaccharide products.
Description
Technical field
The present invention relates to a kind of measurement of the polysaccharide content method, particularly measurement of the polysaccharide content method in a kind of tea seed.
Background technology
Oil tea is commonly called as herb mixtures tea, builds tea, spends tea in vain, becomes one of outstanding edible vegetable oil because can squeeze unsaturated fatty acid content from its seed up to 93% tea oil.Simultaneously, studies show that, also contain polysaccharide, Tea Saponin, glycosides displayed and caffeine etc. in the tea seed and have bioactive effective constituent, very high research and using value are arranged.
The plant water-soluble polysaccharide is isolated natural constituent from natural products; have diversified biologically active and pharmacological action; mainly comprise: strengthen immune, antitumor, hypotensive, reducing blood lipid, hypoglycemic, anti-ageing, strengthen marrow hemopoiesis function, radioresistance, to the protective effect of the main organs such as liver kidney etc., be widely used in fields such as health care, disease treatments.Therefore, the content of the water-soluble polysaccharide in the plant is commonly used to as one of index of estimating some medicinal plant quality, the mensuration of its content can provide for the explo iting and researching of natural biological resource effective reference data, therefore how to judge quantitatively that the content of tea polysaccharide is that people pay close attention in the tea seed.
The mensuration of camellia seed polysaccharide content there is no national standard at present, and the assay method of relevant camellia seed polysaccharide content rarely has report, and the mensuration of plant water-soluble polysaccharide all is to continue to use the assay method of thick polysaccharide.Sulfuric acid-phynol method is the classical way of measuring polyoses content, its principle is: water extracts the polysaccharide component in the plant, under effect of sulfuric acid, be hydrolyzed into monose, and dehydration generates the alditol derivant rapidly, then be condensed into colored compound with phenol, measure its polyoses content with spectrophotometric method in suitable wavelength.In the camellia seed polysaccharide assay, camellia seed polysaccharide is converted into monose under concentrated sulphuric acid effect, and then can measure contents of monosaccharides.Yet owing to also containing other various active compositions such as tea oil, Tea Saponin, flavone compound in the tea seed, therefore strengthened the difficulty of effective more much higher sugar of dna purity from tea seed, and affected the degree of accuracy of determination of polysaccharide.And the quantitative measurement of polyoses content becomes key technical problem in polyose exploitation, production and the quality control in the tea seed, therefore in view of existing methodical deficiency, find a kind of how can the Accurate Measurement tea seed in the method for polyoses content become present problem demanding prompt solution.
Summary of the invention
The objective of the invention is for the existing deficiency of Methods in Determination of Polysaccaride Content in the present tea seed, provide a kind of accurate quantification to measure the method for polyoses content in the tea seed, that the method has advantages of is workable, measurement result is accurate, reappearance is high, can measure at general Routine Test Lab, and provides reliable reference frame for the Accurate Measurement of the polyoses content of other natural productss.
Purpose of the present invention is achieved through the following technical solutions:
Measurement of the polysaccharide content method in a kind of tea seed may further comprise the steps:
(1) takes by weighing the tea seed to be measured that oven dry grinds, with low polar solvent described tea seed is extracted, get tea seed I and low polar solvent extract;
(2) be that 70～85% ethanolic solution extracts the tea seed I with the quality percentage composition, get tea seed II and ethanolic solution extract;
(3) water extracts the tea seed II, gets aqueous extract, with gained aqueous extract constant volume, as polysaccharide solution to be measured;
(4) adopt sulfuric acid-phynol method to measure the content of polysaccharide in the above-mentioned solution to be measured, concrete steps are as follows: draw the polysaccharide sample solution, use distilled water diluting, get polysaccharide solution to be measured, draw 2.0ml polysaccharide solution to be measured in test tube, add 5%～10% phenol reagent 1.0ml, shake up, drip rapidly concentrated sulphuric acid 5.0ml, shake up rapidly, after leaving standstill, place boiling water bath to heat, rapidly cooling is done blank with 2ml distilled water in addition after taking out, measure absorbance at the 490nm place by ultraviolet spectrophotometry, draw the quality of polysaccharide in the liquid to be measured according to typical curve.
The purpose of step of the present invention (1) is to remove liposoluble constituent in the tea seed, such as tea oil, the purpose of step (2) is to remove solubility glucoside material in the tea seed, such as oil-tea camellia seed saponin, the compounds such as glycosides displayed, because some solubility glucoside components also can be hydrolyzed generation monose in the tea seed under effect of sulfuric acid, when therefore adopting classic method to measure polyoses content, it is quantitative that these monose that are hydrolyzed also can be treated as polysaccharide, therefore the polyoses content of measuring is far above actual value, thereby affect the accuracy of quantitative measurement, so the present invention adopts the method for extraction to remove first liposoluble substance and solubility glucoside material in the tea seed, can not only guarantee the effectively more much higher sugar of dna purity, and avoid the impact of these materials on the determination of polysaccharide degree of accuracy.
Preferably, the extracting method in step (1), step (2) and the step (3) is the method for Soxhlet extraction or extracted many times.
Preferably, described low polar solvent is one or more mixing in sherwood oil, n-pentane, normal hexane, the cyclohexane.
Preferably, in step (1), step (2) and step (3), extraction time is 2～6h.
Preferably, the extraction temperature in the step (1) is 60～80 ℃, and the extraction temperature in the step (2) is 80～100 ℃, and the extraction temperature in the step (3) is 100～120 ℃.
Preferably, the constant volume of aqueous extract is 100～250ml in the step (3).
Preferably, the uptake of polysaccharide sample solution is 0.1～15ml in the step (4).
Preferably, placing the heat time heating time of boiling water bath in the step (4) is 12～18min.
Preferably, in step (4), described polysaccharide sample solution is 10～1000 with the volume multiple of distilled water diluting.
The present invention compared with prior art has the following advantages:
Method of the present invention, have advantages of workable, measurement result is accurate, reappearance is high, can measure at general Routine Test Lab, for the Accurate Measurement of the polyoses content of other natural productss provides reliable reference frame.In addition, the present invention provides effective assurance for camellia seed polysaccharide product development, production and quality control.
Embodiment
Soxhlet used in the present invention is extracted or the method for extracted many times is all carried out according to the experimental implementation step of this area routine.
The below will be clearly and completely described the technical scheme in the embodiment of the invention, and obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
Embodiment 1
Measurement of the polysaccharide content method in a kind of tea seed may further comprise the steps:
(1) accurate weighing tea seed sample 5g, wrap with double-deck filter paper, place apparatus,Soxhlet's, add the 80ml sherwood oil at round-bottomed flask, add hot reflux in temperature is 70 ℃ oil bath pan, extracting 4 hours gets tea seed I and petroleum ether extract, keep the tea seed I, discard petroleum ether extract;
(2) add 70% ethanol 80ml in the round-bottomed flask that above-mentioned tea seed I is housed, in temperature is 90 ℃ oil bath pan, add hot reflux, extracting 4 hours, get tea seed II and ethanol extract, discard ethanol extract, described tea seed II is taken out, naturally dry in the lucifuge ventilation;
(3) dried tea seed II in the step (2) is wrapped with double-deck filter paper, place apparatus,Soxhlet's, add 80ml distilled water at round-bottomed flask, in being 110 ℃ oil bath pan, temperature adds hot reflux, extracting 4 hours, move into after cooling in the 250ml volumetric flask, to 250ml, obtain the polysaccharide sample solution with distilled water diluting.
(4) adopt sulfuric acid-phynol method to measure the content of polysaccharide in the above-mentioned solution to be measured:
4.1 the drafting of typical curve:
4.1.1 the preparation of phenol solution
Take by weighing 10g phenol, add and be transferred in the brown bottle after water 150ml fully dissolves, place refrigerator for subsequent use.
4.1.2 the preparation of glucose storing solution
Accurately take by weighing 105 ℃ of standard glucose 100mg that are dried to constant weight, add suitable quantity of water and make it dissolving, place the 100ml volumetric flask, add water and be settled to 100ml, it is for subsequent use that even mixing is placed on refrigerator.
4.1.3 glucose uses the preparation of liquid
Accurately draw glucose storing solution 10ml, place the 100ml volumetric flask, be diluted with water to 100ml, the glucose that obtains 0.1mg/ml uses liquid.It is for subsequent use that even mixing is placed on refrigerator.
4.1.4 the drafting of typical curve:
Accurately draw glucose and use liquid 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml 0.8ml places respectively the 10ml test tube, in every test tube, add distilled water to 2.0ml, in every test tube, add the phenol reagent 1.0ml that configures respectively again, shake up, drip rapidly concentrated sulphuric acid 5.0ml, shake up, place 5min, place boiling water bath to heat 15min, take out, be cooled to rapidly room temperature with cold water; Repeat aforesaid operations with 2.0ml distilled water in addition, do blank.In the 1cm cuvette, measure the absorption photometric value at 490nm wavelength place by ultraviolet spectrophotometry, as shown in table 1.
Table 1 concentration of glucose is in the absorption photometric value at 490nm wavelength place
| Concentration of glucose (mg/ml) | The absorption photometric value at 490nm wavelength place |
| 0.005 | 0.17 |
| 0.010 | 0.25 |
| 0.015 | 0.33 |
| 0.020 | 0.41 |
| 0.025 | 0.49 |
| 0.030 | 0.57 |
| 0.035 | 0.65 |
| 0.040 | 0.73 |
Take glucose content as horizontal ordinate, absorbance is ordinate, and the drawing standard curve gets regression equation: A=0.0887+16.293*C, R=0.9931.
4.2 mensuration polyoses content
Draw polysaccharide sample solution 10ml, with 100 times of distilled water dilutings, get polysaccharide solution to be measured.Draw 2.0ml polysaccharide solution to be measured in test tube, add again the phenol reagent that 1ml configures, shake up, drip rapidly concentrated sulphuric acid 5.0ml, shake up rapidly, place 5min, place boiling water bath to heat 15min, take out, be cooled to rapidly room temperature with cold water, do blank with 2ml distilled water in addition, measuring the absorption photometric value by ultraviolet spectrophotometry at the 490nm place in the 1cm cuvette is 0.51, and the mass concentration that draws polysaccharide in the liquid to be measured according to typical curve is 0.026mg/ml.
4.3 the result calculates, the Mass Calculation of polysaccharide goes out to treat the content of polysaccharide in the test sample in the liquid to be measured, and computing formula is as follows:
X=(C*N
1*V
1)/(m
1*1000)*100%
In the formula, the content of polysaccharide in the test sample is treated in the X representative;
C represents the mass concentration of polysaccharide in the liquid to be measured, the mg/ml of unit;
N
1When polyoses content is measured in representative to the extension rate of sample solution;
V
1The volume of representative sample solution, unit/ml;
m
1Represent the tea seed sample and get sampling quality, the g of unit.
The content that calculates polysaccharide is 1.3%.Wherein polyoses content is in glucosan.
The above; only for the better embodiment of the present invention, but protection scope of the present invention do not limit to therewith, anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.
Claims (9)
1. measurement of the polysaccharide content method in the tea seed is characterized in that, may further comprise the steps:
(1) takes by weighing the tea seed to be measured that oven dry grinds, with low polar solvent described tea seed is extracted, get tea seed I and low polar solvent extract;
(2) be that 70～85% ethanolic solution extracts the tea seed I with the quality percentage composition, get tea seed II and ethanolic solution extract;
(3) water extracts the tea seed II, gets aqueous extract, with gained aqueous extract constant volume, as the polysaccharide sample solution;
(4) adopt sulfuric acid-phynol method to measure the content of polysaccharide in the above-mentioned solution to be measured, concrete steps are as follows: draw the polysaccharide sample solution, use distilled water diluting, get polysaccharide solution to be measured, draw 2ml polysaccharide solution to be measured in test tube, the phenol reagent 1.0ml that adds 5～10 % by weight, shake up, drip rapidly concentrated sulphuric acid 5.0ml, shake up rapidly, after leaving standstill, place boiling water bath to heat, rapidly cooling is done blank with 2ml distilled water in addition after taking out, measure absorbance at the 490nm place by ultraviolet spectrophotometry, draw the quality of polysaccharide in the liquid to be measured according to typical curve.
2. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, the extracting method in step (1), step (2) and the step (3) is the method for Soxhlet extraction or extracted many times.
3. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, described low polar solvent is one or more mixing in sherwood oil, n-pentane, normal hexane, the cyclohexane.
4. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, in step (1), step (2) and step (3), extraction time is 2～6h.
5. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, the extraction temperature in the step (1) is 60～80 ℃, and the extraction temperature in the step (2) is 80～100 ℃, and the extraction temperature in the step (3) is 100～120 ℃.
6. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, the constant volume of aqueous extract is 100～250ml in the step (3).
7. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, the uptake of polysaccharide sample solution is 0.1～15ml in the step (4).
8. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, be 12～18min the heat time heating time that places boiling water bath in the step (4).
9. measurement of the polysaccharide content method in the tea seed as claimed in claim 1 is characterized in that, in step (4), described polysaccharide sample solution is 10～1000 with the volume multiple of distilled water diluting.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728263A (en) * | 2013-12-31 | 2014-04-16 | 无限极（中国）有限公司 | Method for quantitatively detecting polysaccharide of semen cassiae |
| CN103743869A (en) * | 2014-01-08 | 2014-04-23 | 中国农业科学院作物科学研究所 | Determination method of substances for improving blood sugar level reduction effect of small red beans |
| CN112304886A (en) * | 2020-10-30 | 2021-02-02 | 温州瑞雪农业开发有限公司 | Rapid identification method for squeezing and leaching camellia seed oil |
| CN113426155A (en) * | 2021-06-04 | 2021-09-24 | 北京理工大学 | Comprehensive utilization process of coffee grounds |
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2012
- 2012-10-19 CN CN2012104016616A patent/CN102914508A/en active Pending
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| 孙锋等: "山药粗多糖的提取工艺", 《食品与生物技术学报》 * |
| 杜清等: "明党参多糖提取工艺研究", 《现代中药研究与实践》 * |
| 王元凤等: "从油茶饼粕中提取油茶籽多糖的差皂素的工艺研究", 《食品工业》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728263A (en) * | 2013-12-31 | 2014-04-16 | 无限极（中国）有限公司 | Method for quantitatively detecting polysaccharide of semen cassiae |
| CN103728263B (en) * | 2013-12-31 | 2016-03-02 | 无限极（中国）有限公司 | A kind of cassia seeds polysaccharides quantitative detecting method |
| CN103743869A (en) * | 2014-01-08 | 2014-04-23 | 中国农业科学院作物科学研究所 | Determination method of substances for improving blood sugar level reduction effect of small red beans |
| CN103743869B (en) * | 2014-01-08 | 2015-08-12 | 吉林睿德生物科技有限公司 | A kind of defining method improving the material of red bean blood sugar decreasing effect |
| CN112304886A (en) * | 2020-10-30 | 2021-02-02 | 温州瑞雪农业开发有限公司 | Rapid identification method for squeezing and leaching camellia seed oil |
| CN112304886B (en) * | 2020-10-30 | 2022-05-20 | 温州瑞雪农业开发有限公司 | Rapid identification method for squeezed and extracted camellia seed oil |
| CN113426155A (en) * | 2021-06-04 | 2021-09-24 | 北京理工大学 | Comprehensive utilization process of coffee grounds |
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Application publication date: 20130206 |