CN102621027A - Quantitative detection method for saponin in tea saponin - Google Patents
Quantitative detection method for saponin in tea saponin Download PDFInfo
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Abstract
The invention discloses a quantitative detection method for saponin in tea saponin. According to the quantitative detection method, 3.0g-3.5g of extract of tea seed or the tea saponin is taken, the extract is subjected to alkali and acid hydrolysis and then is dissolved in water, hydrolysate is poured into the water to allow precipitation of the extract so as to measure mass of sapogenin, and then the mass of the sapogenin is converted to mass fraction of the saponin. The quantitative detection method has the characteristics that equipment is simple, the cost is low, the operation is convenient and easy, the accuracy is high, and the direct detection can be preformed, thus being suitable for detection on tea seeds, crude extract of the tea saponin, and high-purity tea saponin. The quantitative detection method is particularly applicable to pioneering small and medium-sized enterprises which are lack of instruments and equipment and are equipped with personnel with low education degree.
Description
Technical field
The present invention relates to a kind of detection technique, relate in particular to the quantitative detecting method of saponin(e in a kind of Tea Saponin.
Background technology
Tea Saponin is claimed tea saponin, tea saponin again, by seven kinds of aglucons, four kinds of glycocides and two kinds of a kind of pentacyclic triterpene saponins that organic carboxyl acid is formed, is mainly derived from plant of theaceae (like tea, camellia, oil tea).Performances such as Tea Saponin has antibiotic, anti-oxidant, anti-high cholesterol high fat of blood, moistening foaming; Of many uses in industries such as medicine, building materials, aquatic products, agricultural chemicals, cosmetics, washing agent; As a kind of environmental type non-ionic surfactant, have a good application prospect.China is the world maximum country of oil tea output; The camellia oleifera lam area reaches 3,600,000 hectares at present; Account for more than 80% of national woody edible oil materials area; The remaining camellia seed meal in annual oil expression back has more than 50 ten thousand tons, and camellia seed meal contains Tea Saponin 10～20%, for producing Tea Saponin abundant raw material is provided.The domestic and international in recent years market demand to camellia seed meal and Tea Saponin demonstrates the rising tendency that is not had, and has promoted the fast development of camellia seed meal deep processing industry.
Meanwhile, the Tea Saponin product quality is more and more paid close attention to, and wherein saponin content has become the main price foundation of domestic and international trade.For satisfying the export market demand; State Administration for Quality Supervision and Inspection and Quarantine has issued " People's Republic of China's entry and exit quarantine quarantine industry standard: the mensuration of saponin content in the outlet Tea Saponin " (calling " SN/T 1852-2006 standard " in the following text) in 2006; But it is 30%～70% Tea Saponin product that the gravimetry that this standard adopted (chemical method) only is applicable to saponin content; Be not suitable for the detection of the Tea Saponin that extracts in the lower camellia seed meal of saponin content, and this method do not have the pressure effectiveness that detects sold inside the country product.Therefore, the detection by quantitative of home market Tea Saponin still lacks national standard at present, also lacks relevant industries standard and provincial standard.
The quantitative detecting method of saponin(e mainly contains in the Tea Saponin of past bibliographical information: gravimetry, vanillic aldehyde-concentrated sulphuric acid colourimetry, fluorophotometric method, fast differential spectrophotometry, thin layer chromatography scanning, HPLC method and liquid chromatography-flight time mass spectrum (LC/TOF-MS) combined instrument method etc., but the scope of application of these methods and accuracy do not seen more so far specialize in.And from cake of camellia oleifera seeds, extracting Tea Saponin, existing many in history researchs research reports announce with patented technology, as: " solvent extraction and selectively separating integration prepare the new technology of Tea Saponin " (open patent of invention number: 200710168737.4; 2007; Face flowing water, Luo Xubiao, Tai build east, Tu Xinman, Yu Bin, 2007), " a kind of method for distilling of Tea Saponin " (authorized patent of invention number: ZL99115525.4; Fang Xiamin; 2008), but related patent U.S. Patent No. generally only relates to the refining utilization of extraction, and is not suitable for camellia seed meal, Tea Saponin crude extract and high-purity tea saponin detection method.In recent years, it is very ripe from the tea dregs of rice, to extract Residual oil technology with normal butyl alcohol etc., form scale in many greases enterprise, but Shang Weijian has the report of Tea Saponin quantitative measurement.Simultaneously; When from camellia seed meal, extracting Tea Saponin; Because of containing many impurity such as grease, resin, protein, polysaccharide, tannic acid, make that the Tea Saponin product viscosity is big, content is not high, color and luster is pitch black, the content of saponin there are differences because of the difference of kind, planting environment and quantivative approach in the tea seed; Cause a lot of difficulties for the quantitative test of Tea Saponin, therefore do not see unified ripe detection method so far both at home and abroad as yet.And in Tea Saponin research and production run, quantitative measurement is extremely important, not only require detection method not only degree of accuracy high, simple, be convenient to operation, and applicable surface wants wide, especially will be applicable to the area and the enterprise of shortage checkout equipment.For this reason, the detection method research of the saponin content in the Tea Saponin that we have carried out extracting in the camellia seed meal is in the hope of providing scientific basis for working out the detection method standard.
Summary of the invention
The problems referred to above in view of existing Tea Saponin quantitative measurement existence; The object of the present invention is to provide the quantitative detecting method of saponin(e in a kind of Tea Saponin; This method not only degree of accuracy high, simple, be convenient to operation, and applicable surface wants wide, be particularly useful for lacking the area and the enterprise of checkout equipment.
The objective of the invention is to realize through following technical scheme: the quantitative detecting method of saponin(e in a kind of Tea Saponin, this method may further comprise the steps:
(1) take by weighing powdery camellia seed meal sample 3.0 ~ 3.5g, place Erlenmeyer flask, add 25 mL sodium hydrate methanol solutions, on connect serpentine condenser, put in the fuming cupboard 2 h that reflux in the boiling water bath, take out and be cooled to room temperature; Adding 80 mL methyl alcohol prepared after said sodium hydrate methanol solution was dissolved in 20 mL water by NaOH 10 g;
(2) using mass percent is that the solution of 20% hydrochloric acid solution regulating step 1 preparation approximates 7 to pH; Add 20 mL methyl alcohol; Slowly add 7 mL hydrochloric acid (ρ=1.18 g/mL) then, put 1 h that refluxes in the boiling water bath, remove condenser pipe; Put in the fuming cupboard, water-bath 30 min fling to about 30 mL of methyl alcohol to final volume;
(3) solution with step 2 preparation is poured in the 1000 mL beakers immediately, adds 500 mL distilled water mixings, with the distilled water washing Erlenmeyer flask that boils, incorporates in the beaker in addition, treats abundant cooling, post precipitation, filters;
(4) filter residue is washed till neutrality with distilled water, and oven dry places in the filtration paper cylinder, soak bottle graft with the Suo Shi extracting of 105 ± 2 ℃ of following constant weights and receive, with acetone do auxiliary agent in 75 ℃ of water-baths with Suo Shi extraction process extracting 2h, recovery acetone;
(5) bottle is soaked in the Suo Shi extracting and put 105 ℃ ± 2 ℃ dry 2h in the baking oven, take out, jump a queue, put into exsiccator and cool off weighing behind 0.5 h with bottle stopper; Repeat, until constant weight;
(6) blank test: do blank test in company with sample;
(7) saponin content (being massfraction) calculates by the following formula of SN/T 1852-2006 regulation:
In the formula,
wBe saponin content (%), m
1Be the quality (g) of sample, m
2Be the quality (g) that bottle is soaked in the Suo Shi extracting, m
3For bottle and extract quality (g), m are soaked in constant weight Hou Suoshi extracting
0Be blank residue quality (g), 1223.54 is the theoretical mean molecular weight of tea saponin, and 501 is the theoretical mean molecular weight of sapogenin.
The invention has the beneficial effects as follows:
(1) the invention provides the quantitative detecting method of saponin content in a kind of camellia seed meal, only be applicable to that than " SN/T 1852-2006 standard " saponin content is the detection by quantitative of the Tea Saponin product of 30%-70%, the present invention does not receive the restriction of saponin content.
(2) to have equipment simple in the present invention, and cost is low, easy and simple to handle, and repeated degree of accuracy is high, the characteristics that can directly measure.This method to lack instrument and equipment, foundation medium-sized and small enterprises that testing staff's educational level is lower are very suitable.
Description of drawings
Fig. 1 is the ultra-violet absorption spectrum (vanillic aldehyde concentrated sulphuric acid method) of camellia seed meal Tea Saponin standard items;
Fig. 2 is the typical curve (vanillic aldehyde-concentrated sulphuric acid development process) of oil tea saponin;
Fig. 3 is the spectrum of Sasanguasaponin standard items;
Fig. 4 is the typical curve (HPLC method) of Sasanguasaponin;
Fig. 5 is a Tea Saponin standard items methanol solution chromatogram;
Fig. 6 is camellia seed meal methanol solution chromatogram after methyl alcohol vibration extracting is handled;
Fig. 7 is camellia seed meal methanol solution chromatogram after the Suo Shi extracting is handled;
Fig. 8 is Tea Saponin crude extract and high-purity tea saponin sample methanol solution chromatogram.
Embodiment
The quantitative detecting method of saponin(e in the Tea Saponin of the present invention may further comprise the steps:
1, takes by weighing powdery camellia seed meal sample 3.0 ~ 3.5g, place Erlenmeyer flask, add 25 mL sodium hydrate methanol solutions (weighing sodium hydroxide 10 g; Be dissolved in the 20 mL water, add 80 mL methyl alcohol), on connect serpentine condenser; Put 2 h that reflux in the boiling water bath in the fuming cupboard, take out and be cooled to room temperature;
2, regulate above-mentioned pH value of solution with 20% hydrochloric acid solution and approximate 7, add 20 mL methyl alcohol, slowly add 7 mL hydrochloric acid (ρ=1.18 g/mL) then; Put 1 h that refluxes in the boiling water bath; Remove condenser pipe, put in the fuming cupboard, fling to about 30 mL of methyl alcohol to final volume among water-bath 30 min;
3, above-mentioned solution is poured in the 1000 mL beakers immediately, adds 500 mL distilled water mixings, with the distilled water washing Erlenmeyer flask that boils, incorporate in the beaker in addition, treat abundant cooling, post precipitation, filter;
4, filter residue is washed till neutrality with distilled water, and oven dry places in the filtration paper cylinder, soak bottle graft with the Suo Shi extracting of 105 ± 2 ℃ of following constant weights and receive, with acetone do auxiliary agent in 75 ℃ of water-baths with Suo Shi extraction process extracting 2h, recovery acetone;
5, bottle is soaked in the Suo Shi extracting and bottle stopper is put 105 ℃ ± 2 ℃ dry 2h in the baking oven, take out, jump a queue, put into weighing behind exsiccator cooling 0.5 h; Repeat, until constant weight;
6, blank test
Do blank test in company with sample.
7, saponin content (being massfraction) calculates by the following formula of SN/T 1852-2006 regulation:
In the formula,
wBe saponin content (%), m
1Be the quality (g) of sample, m
2Be the quality (g) that bottle is soaked in the Suo Shi extracting, m
3For bottle and extract quality (g), m are soaked in constant weight Hou Suoshi extracting
0Be blank residue quality (g), 1223.54 is the theoretical mean molecular weight of tea saponin, and 501 is the theoretical mean molecular weight of sapogenin.
Through following examples the present invention is done further detailed description, but the present invention is not limited by these contents.
Embodiment 1: the gravimetric method of camellia seed meal saponin(e, vanillic aldehyde-concentrated sulphuric acid method and HPLC standard measure detect
(1) gravimetric method detection by quantitative
Be ready to following reagent: (1) methyl alcohol (analyzing pure), hydrochloric acid (ρ=1.18 g/mL), NaOH (analyzing pure); Acetone (analyzing pure); 20% hydrochloric acid solution (measuring 504 mL hydrochloric acid, thin up to 1000 mL), sodium hydrate methanol solution (weighing sodium hydroxide 10 g; Be dissolved in the 20 mL water, add 80 mL methyl alcohol) mixing.Take by weighing powdery camellia seed meal sample 3.0 ~ 3.5g (being accurate to 0.0001 g), place Erlenmeyer flask, add 25 mL sodium hydrate methanol solutions, on connect serpentine condenser, put in the fuming cupboard boiling water bath 2 h that reflux, take out and be cooled to room temperature.Regulate above-mentioned solution to pH 7 with 20% hydrochloric acid solution, add methyl alcohol 20 mL, slowly add 20% hydrochloric acid then, put 1 h that refluxes in the boiling water bath, remove condenser pipe, put fuming cupboard and in water-bath, fling to methyl alcohol and put about 30 min.Solution is poured in the 1000 mL beakers immediately, adds mixing in the 500 mL distillation, with the distilled water washing Erlenmeyer flask that boils on a small quantity, washing is incorporated in the beaker, treats abundant cooling, post precipitation, filters in addition.Filter residue is washed till the oven dry of neutral back with distilled water, places in the filtration paper cylinder, soaks bottle graft with the Suo Shi extracting of 105 ℃ of following constant weights and receives, and with 75 ℃ of extracting 2 h of acetone, reclaims acetone.Receiving bottle and bottle stopper are put 105 ℃ of drying 2 h in the baking oven, take out, jump a queue, put into weighing behind exsiccator cooling 0.5 h; Put into 105 ℃ of dry 0.5 h of baking oven again, take out, jump a queue, put into exsiccator and cool off weighing behind 0.5 h.Do blank test in company with sample.Same sample need be done twice parallel experiment.The formula that saponin content (being massfraction) is pressed SN/T 1852-2006 regulation calculates.
In the formula:
wBe saponin(e massfraction/%,
m 1 Be sample quality/g,
m 2 Be receiving bottle quality/g,
m 3 Be receiving bottle after the constant weight and extract quality/g,
m 0 Be blank residue quality/g, 1223.54 is the theoretical average molecular mass of tea saponin, and 501 is the theoretical average molecular mass of sapogenin.
(2) vanillic aldehyde-concentrated sulphuric acid detection by quantitative
1. set up typical curve: accurately take by weighing vanillic aldehyde 0.8 ~ 1.0g and be dissolved in 10 mL absolute ethyl alcohols, be mixed with vanillic aldehyde solution.Get the concentrated sulphuric acid (analyzing pure) 77 mL, subsequent use with 23 mL deionized water mixings.Take by weighing Tea Saponin standard items 25.0 mg, be dissolved in the 50 mL volumetric flasks, constant volume, shake up, promptly get standard solution with 80% ethanolic solution.Get standard solution 0.5 mL, accurately add 80 g/L vanillic aldehyde solution, 0.5 mL, in frozen water, add 77% sulfuric acid solution, 4 mL; Shake up,, place ice-water bath cooling 10 min then in 60 ℃ of heating 15 min; Taking-up places under the room temperature; With reagent is blank, scans on ultraviolet-visible spectrophotometer with 1 cm cuvette, draws maximum absorption wavelength (Fig. 1).Get Tea Saponin standard solution 0.1,0.2,0.3,0.4,0.5,0.6 mL respectively; Place test tube with ground stopper, adding water, to be settled to liquor capacity be 1.0 mL, add the reagent reaction as stated above after; Under maximum absorption wavelength, measure absorbance; And with absorbance to concentration value drawing standard curve, set up regression equation: Y=38.766X-0.0333, R
2=0.9968 (Fig. 2).
2. camellia seed meal is measured: will wrap with filter paper through the powdery sample that grinds, and put 60 ℃ of baking ovens and be incubated 4 h, and dry to constant weight; Cross 60 mesh sieves; Accurately take by weighing 4.5 ~ 5.5g and put into apparatus,Soxhlet's, with 150 mL alcohol reflux, 4 h, control extraction rate 8 min that reflux.After the extract cooling, put into 500 mL volumetric flasks, with 80% ethanol constant volume.Get 1.0 mL and measure absorbance, be blended into corresponding regression equation, calculate the saponin(e massfraction according to typical curve.
(3)
Reversed-phased high performace liquid chromatographic (HPLC) detection by quantitative
Chromatographic column: Agilent Eclipse XDB-C18 (4.6 * 250 mm, 5 μ m); Moving phase: methyl alcohol; Flow velocity: 1 mL/min; The UV scanning result shows that the Tea Saponin standard specimen maximum absorption peak (Fig. 3) occurs at 280 nm places, detects wavelength: 280 nm; Column temperature: 30 ℃.Standard specimen is handled: accurately take by weighing Tea Saponin standard items 45.0 mg and place 50 mL volumetric flasks, the water constant volume is the Tea Saponin standard solution; Get 1 mL solution in sample bottle 1; Accurately pipette in 12.5 mL to the 25 mL volumetric flasks, add the water constant volume; Get 1 mL solution in sample bottle 2.Be prepared into sample bottle 3,4,5 according to the method described above.Solution in the sample thief bottle is analyzed with high performance liquid chromatograph through 0.45 μ m filtering with microporous membrane, and standard items are a simple spike (Fig. 3).Typical curve equation y=0.0028x+0.0199 (R
2=0.9999, Fig. 4).
Two kinds of sample-pretreating method effects are relatively: 1. methyl alcohol succusion: take by weighing camellia seed meal powder 1.5 ~ 2.5 g of drying to constant weight in 100 mL tool plug triangular flasks; Add methyl alcohol 50 mL; In reciprocating type oscillator, extract 24 h, extracting liquid filtering, filter residue is washed 2 times with 20 mL methyl alcohol; Merge extract in 500 mL volumetric flasks, use methanol constant volume.Fig. 6 is the HPLC figure of camellia seed meal sample methanol solution after the employing methyl alcohol oscillation treatment.2. soxhlet extraction method: will wrap with filter paper through the powdery sample that grinds, and put 60 ℃ of baking ovens and be incubated 4 h, and dry to constant weight; Cross 60 mesh sieves; Accurately take by weighing 4.5 ~ 5.5g and put into apparatus,Soxhlet's, extract 4 h, control extraction rate 8 min that reflux with 150 mL methanol eddies.After the extract cooling, put into 500 mL volumetric flasks, use methanol constant volume.Get 1.0 mL constant volume methanol solutions,, analyze with high performance liquid chromatograph respectively through 0.45 μ m filtering with microporous membrane.Fig. 7 is the HPLC figure of the camellia seed meal sample methanol solution of Suo Shi extracting after handling.The result shows that HPLC figure (Fig. 6, Fig. 7) difference of different pre-treating methods is very big, and is wherein more consistent with standard items with the sample methanol solution chromatogram (Fig. 7) of Suo Shi extraction process processing; And be not easy to separate with main peak with the impurity peaks of methyl alcohol oscillation treatment sample, impurity peaks fairly obvious (Fig. 6), the result is dried to be disturbed greatly to measuring.Experimental result shows and adopt the Suo Shi method of taking out to handle sample.
The gravimetric method of table 1 camellia seed meal saponin(e and vanillic aldehyde-concentrated sulphuric acid method and HPLC method effect comparison
* conspicuousness is added up
P>=0.05.
Measuring the result with vanillic aldehyde-concentrated sulphuric acid method and HPLC method is reference; The gravimetric determination result is carried out variance analysis; Result's (table 1) shows; The saponin(e massfraction result of gravimetric determination and vanillic aldehyde-concentrated sulphuric acid method and HPLC method are very approaching, and three's saponin(e massfraction is respectively 16.20%, 16.08% and 16.21%, statistics do not have significant difference (
P>=0.05).Show that improving the back gravimetric method can be used for measuring saponin(e massfraction in the camellia seed meal.
Embodiment 2: the gravimetric method of high-purity tea saponin and Tea Saponin crude extract, vanillic aldehyde-concentrated sulphuric acid method and HPLC standard measure detect
(1) gravimetric method detection by quantitative
Be ready to following reagent: (1) methyl alcohol (analyzing pure), hydrochloric acid (ρ=1.18 g/mL), NaOH (analyzing pure); Acetone (analyzing pure); 20% hydrochloric acid solution (measuring 504 mL hydrochloric acid, thin up to 1000 mL), sodium hydrate methanol solution (weighing sodium hydroxide 10 g; Be dissolved in the 20 mL water, add 80 mL methyl alcohol) mixing.Take by weighing high-purity tea saponin and Tea Saponin crude extract sample 3.0 ~ 3.5 g, place Erlenmeyer flask, add 25 m L sodium hydrate methanol solutions, on connect serpentine condenser, put in the fuming cupboard boiling water bath 2 h that reflux, take out and be cooled to room temperature.Using 20% hydrochloric acid solution to regulate above-mentioned solution to pH value is 7, adds methyl alcohol 20 mL.Slowly add hydrochloric acid then, put in the boiling water bath 1 h that refluxes, remove condenser pipe, putting fuming cupboard, in water-bath, to fling to methyl alcohol to final volume be about 30 mL.Solution is poured in the 1000 mL beakers immediately, adds mixing in the 500 mL distillation, with the distilled water washing Erlenmeyer flask that boils on a small quantity, washing is incorporated in the beaker, treats abundant cooling, post precipitation, filters in addition.Filter residue is washed till the oven dry of neutral back with distilled water, places in the filtration paper cylinder, soaks bottle graft with the Suo Shi extracting of constant weight (105 ± 2 ℃) and receives, and with acetone (75 ℃) extracting 2 h, reclaims acetone.Receiving bottle and bottle stopper are put (105 ± 2 ℃) dry 2 h in the baking oven, take out, jump a queue, put into weighing behind exsiccator cooling 0.5 h; Put into dry 0.5 h of baking oven (105 ± 2 ℃) again, take out, jump a queue, put into exsiccator and cool off weighing behind 0.5 h.The repetition above-mentioned steps is twice 2 mg that are no more than of poor quality to front and back, is constant weight.Do blank test in company with sample.
(2) vanillic aldehyde-concentrated sulphuric acid method is measured
1. typical curve is set up: accurately take by weighing vanillic aldehyde 0.8 g ~ 1.0 g and be dissolved in 10 mL absolute ethyl alcohols, be mixed with vanillic aldehyde solution.Get the concentrated sulphuric acid 77 mL, subsequent use with 23 mL deionized water mixings.Take by weighing Tea Saponin standard items 25.0 mg, be dissolved in the 50 mL volumetric flasks, constant volume, shake up, promptly get standard solution with 80% ethanolic solution.Get standard solution 0.5 mL, accurately add 80 g/L vanillic aldehyde solution, 0.5 mL, in frozen water, add 77% sulfuric acid solution, 4 mL; Shake up,, place ice-water bath cooling 10 min then in 60 ℃ of heating 15 min; Taking-up places under the room temperature; With reagent is blank, scans on ultraviolet-visible spectrophotometer with 1 cm cuvette, draws maximum absorption wavelength.After adding reagent reaction as stated above, mensuration absorbance under maximum absorption wavelength, the Tea Saponin standard items scan gained on ultraviolet-visible spectrophotometer maximum absorption wavelength 451 nm spectrum are seen Fig. 1.Get Tea Saponin standard solution 0.1,0.2,0.3,0.4,0.5,0.6 mL respectively; Place test tube with ground stopper; Adding water, to be settled to liquor capacity be 1.0 mL, add the reagent reaction as stated above after, mensuration absorbance under maximum absorption wavelength; And with absorbance to concentration value drawing standard curve, set up regression equation.And with absorbance to concentration value drawing standard curve, set up regression equation: Y=38.766X-0.0333, R
2=0.9968 (Fig. 2).
2. the mensuration of high-purity tea saponin and Tea Saponin crude extract: take by weighing respectively about sample 65.0 mg, be settled to 25 mL, get 1.0 mL and measure absorbance, be blended into corresponding regression equation, calculate the saponin(e massfraction according to typical curve with 80% ethanol.
(2) reversed-phased high performace liquid chromatographic (HPLC) is measured
1. sampling condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6 * 250 mm, 5 μ m); Moving phase: methyl alcohol; Flow velocity: 1 mL/min; The UV scanning result shows that the Tea Saponin standard specimen maximum absorption peak (Fig. 3) occurs at 280 nm places, detects wavelength: 280 nm; Column temperature: 30 ℃.Standard specimen is handled: accurately take by weighing Tea Saponin standard items 45.0 ~ 50.0 mg and place 50 mL volumetric flasks, the water constant volume is the Tea Saponin standard solution; Get 1 mL solution in sample bottle 1; Accurately pipette in 12.5 mL to the 25 mL volumetric flasks water constant volume; Get 1 mL solution in sample bottle 2.Be prepared into sample bottle 3,4,5 according to the method described above.Solution in the sample thief bottle is analyzed with high performance liquid chromatograph through 0.45 μ m filtering with microporous membrane.Because of Tea Saponin is the potpourri of multiple saponin monomer, each monomer peak all can show at chromatogram, and calculating the total saponins massfraction needs the peak face of each monomer is added up, again with the reference substance peak area ratio to after draw.Fig. 5 is the HPLC figure of Tea Saponin standard items methanol solution, and the typical curve regression equation is: y=0.0028x+0.0199, R
2=0.9999 (Fig. 4).
2. the mensuration of high-purity tea saponin and Tea Saponin crude extract: take by weighing sample 50 ~ 60 mg respectively; Put into 50 mL volumetric flasks, use methanol constant volume, respectively pipette 1.0 mL in sample bottle with transfer pipet; Through 0.45 μ m filtering with microporous membrane, analyze with high performance liquid chromatograph.Fig. 8 is the HPLC figure of Tea Saponin crude extract and high-purity tea saponin methanol solution, and the result shows that the HPLC figure of these two types of samples is all more consistent with standard items (Fig. 5).
The gravimetric method of table 2 high-purity tea saponin and Tea Saponin crude extract and vanillic aldehyde-concentrated sulphuric acid method and HPLC method effect comparison
* conspicuousness is added up
P>=0.05.
Measuring the result with vanillic aldehyde-concentrated sulphuric acid method and HPLC method is reference, carries out variance analysis relatively with the gravimetric determination result.Result's (table 2) shows, the saponin(e massfraction of the Tea Saponin crude extract that three kinds of methods are measured measure the result do not have significant difference (
P>=0.05); The high-purity oil tea saponin numerical value of three kinds of method mensuration is also more approaching.Therefore, gravimetric method is applicable to that the saponin(e massfraction is measured in Tea Saponin crude extract and the high-purity oil tea saponin.
Claims (1)
1. the quantitative detecting method of saponin(e in the Tea Saponin is characterized in that this method may further comprise the steps:
(1) take by weighing powdery camellia seed meal sample 3.0 ~ 3.5g, place Erlenmeyer flask, add 25 mL sodium hydrate methanol solutions, on connect serpentine condenser, put in the fuming cupboard 2 h that reflux in the boiling water bath, take out and be cooled to room temperature; Adding 80 mL methyl alcohol prepared after said sodium hydrate methanol solution was dissolved in 20 mL water by NaOH 10 g;
(2) using mass percent is that the solution of 20% hydrochloric acid solution regulating step 1 preparation approximates 7 to pH; Add 20 mL methyl alcohol; Slowly add 7 mL hydrochloric acid (ρ=1.18 g/mL) then, put 1 h that refluxes in the boiling water bath, remove condenser pipe; Put in the fuming cupboard, water-bath 30 min fling to about 30 mL of methyl alcohol to final volume;
(3) solution with step 2 preparation is poured in the 1000 mL beakers immediately, adds 500 mL distilled water mixings, with the distilled water washing Erlenmeyer flask that boils, incorporates in the beaker in addition, treats abundant cooling, post precipitation, filters;
(4) filter residue is washed till neutrality with distilled water, and oven dry places in the filtration paper cylinder, soak bottle graft with the Suo Shi extracting of 105 ± 2 ℃ of following constant weights and receive, with acetone do auxiliary agent in 75 ℃ of water-baths with Suo Shi extraction process extracting 2h, recovery acetone;
(5) bottle is soaked in the Suo Shi extracting and put 105 ℃ ± 2 ℃ dry 2h in the baking oven, take out, jump a queue, put into exsiccator and cool off weighing behind 0.5 h with bottle stopper; Repeat,, obtain constant weight Hou Suoshi extracting and soak bottle and extract quality until constant weight;
(6) blank test: do blank test in company with sample, obtain blank residue quality;
(7) saponin content (being massfraction) calculates by the following formula of SN/T 1852-2006 regulation:
In the formula,
wBe saponin content (%), m
1Be the quality (g) of sample, m
2Be the quality (g) that bottle is soaked in the Suo Shi extracting, m
3For bottle and extract quality (g), m are soaked in constant weight Hou Suoshi extracting
0Be blank residue quality (g), 1223.54 is the theoretical mean molecular weight of tea saponin, and 501 is the theoretical mean molecular weight of sapogenin.
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| CN106404685A (en) * | 2016-08-25 | 2017-02-15 | 海南椰岛酒业发展有限公司 | Method for determination of total saponin content in health wine |
| CN107687987A (en) * | 2017-10-18 | 2018-02-13 | 中国农业科学院特产研究所 | A kind of method using gravimetric detemination general ginsenoside |
| CN108982286A (en) * | 2018-08-28 | 2018-12-11 | 同济大学 | The separation and quantitative approach of different preservation state Soluble Organic Matters in mud shale |
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| CN106404685A (en) * | 2016-08-25 | 2017-02-15 | 海南椰岛酒业发展有限公司 | Method for determination of total saponin content in health wine |
| CN106404685B (en) * | 2016-08-25 | 2019-01-29 | 海南椰岛酒业发展有限公司 | The measuring method of total saponin content in a kind of health liquor |
| CN107687987A (en) * | 2017-10-18 | 2018-02-13 | 中国农业科学院特产研究所 | A kind of method using gravimetric detemination general ginsenoside |
| CN107687987B (en) * | 2017-10-18 | 2020-08-11 | 中国农业科学院特产研究所 | Method for determining total ginsenoside by gravimetric method |
| CN108982286A (en) * | 2018-08-28 | 2018-12-11 | 同济大学 | The separation and quantitative approach of different preservation state Soluble Organic Matters in mud shale |
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