CN105115925A - Method for determining total alkaloid content in black nightshade - Google Patents
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Abstract
The invention discloses a method for determining total alkaloid content in black nightshade with an ultraviolet spectrophotometer by adopting an acid dye colorimetric method. The method is characterized in that by adopting black nightshade alkali as a reference substance, a reference substance solution is prepared by using chloroform; ultrasonic extraction of total alkaloid in black nightshade is carried out by adopting an acid-alcohol mixed solvent to prepare a test solution; and chromogenic conditions of the acid dye colorimetric method are as follows: pH is equal to 3.6, 3.5 mL of a buffer solution and 2.0 mL of a bromocresol green solution are used, extraction is carried out three times with 10 mL of chloroform, and standing is kept for 30 min. The detection wavelength is 243 nm, and content determination is carried out according to the absorbance. The total alkaloid of the black nightshade is in a linear relationship within a range of 5-30 mu g/mL, the average value of recovery rate is 99.7%, and RSD is equal to 1.4% (n is equal to 6). The method for determining the total alkaloid content in black nightshade disclosed by the invention is simple and convenient to operate, low in cost, high in recovery rate and good in reproducibility, and can be used for determining the total alkaloid content in the black nightshade.
Description
Technical field
The invention belongs to traditional Chinese medicine quality control field, relate to a kind of content assaying method of total Steroidal Alkaloids from Solanum nigrum L.
Background technology
Black nightshade is the dry aerial parts of plant of Solanaceae black nightshade Solanumnigrum, cold in nature, and bitter is micro-sweet, slightly poisonous, returns lung, stomach two warp, has effect of clearing heat and detoxicating, dispersing swelling and dissipating binds, anti-inflammatory diuresis.The pharmacological component of black nightshade is mainly alkaloids, comprises solanine, solamargine, solasonine, the bright alkali of Australia eggplant, solancarpine etc.The minority bibliographical information content assaying method of black nightshade medicinal material and preparation, all with solasonine, solamargine for reference substance, adopt high performance liquid chromatography or measure with mass spectrometry.The assay of the method to monomeric compound is sensitive and accurate, but require high for operative technique during Determination of Total Alkaloid, comparatively loaded down with trivial details, instrument maintains and cost of use higher, reagent cost expends comparatively large, and the selection of mobile phase when HPLC measures content simultaneously, the phosphoric acid solution etc. that adds still have certain influence to peak shape, retention time etc.Solasonine in the black nightshade medicinal material of different regions different parts, solamargine content difference are larger, the multiple steroid alkaloid of modern pharmacological research display black nightshade all has good antitumor activity in vivo and in vitro, therefore more reasonable as medicinal material Testing index using the content of black nightshade total alkaloids.This experiment measures the method for total Steroidal Alkaloids from Solanum nigrum L content with ultraviolet spectrophotometer by acid-dye colorimetry, easy and simple to handle, with low cost, precision is good, the recovery is high, favorable reproducibility, can be used as the index of black nightshade evaluation of medical materials' quality, to overcome the deficiencies in the prior art.
Summary of the invention
In order to solve the aforementioned problems in the prior, the invention provides a kind of black nightshade total alkaloid content detection method, easy and simple to handle, with low cost, precision is good, the recovery is high, favorable reproducibility, can be used as the index of black nightshade evaluation of medical materials' quality.
1, measure a method for total Steroidal Alkaloids from Solanum nigrum L content, it is characterized in that comprising the following steps:
(1) preparation of reference substance solution: precision takes the solanine reference substance 5.00mg being dried to constant weight, dissolve with chloroform, ultrasonic 20min, is transferred to 50mL volumetric flask, adds chloroform and is diluted to scale, shakes up and obtain the reference substance storing solution of 0.1mg/mL;
(2) making of typical curve: accurate reference substance storing solution of drawing dilutes the standard solution being made into gradient in 5ml measuring bottle with chloroform, accurate 2ml standard solution of drawing is placed in separating funnel, add acidic buffer solution and bromcresol green dye solution develops the color, add chloroform extraction again, divide and get chloroform layer processed, get supernatant according to ultraviolet spectrophotometry, measure absorbance in 243nm place; Blank is done with identical reagent and step process with chloroform; Take solanine content as horizontal ordinate, with its absorbance for ordinate, obtain black nightshade Determination of Total Alkaloid typical curve;
(3) preparation of need testing solution: precision takes the black nightshade medicinal powder 2.0g crossing 14 mesh sieves, be placed in conical flask, add 2mol/L hydrochloric acid and 90% alcohol mixed solvent 30mL that volume ratio is 1: 4, ultrasonic extraction 35min at 40 DEG C, extract decompression recycling ethanol is to the medicinal extract without alcohol taste, medicinal extract 10% dissolving with hydrochloric acid, ultrasonic 20min hydrotropy, suction filtration removes precipitation, is settled to 10mL with 10% hydrochloric acid, regulates pH to 10-11 with 10%NaOH, add chloroform extraction 3 times, collect chloroform solution, be settled to 25ml, obtain need testing solution;
(4) assay of sample solution: accurate absorption need testing solution 2.0mL, described in step (2), add acidic buffer solution and bromcresol green dye solution develops the color, add chloroform extraction again, divide and get chloroform layer processed, get supernatant according to ultraviolet spectrophotometry, measure absorbance in 243nm place, substitute into typical curve, calculate the content of black nightshade total alkaloids.
Further, step (2) and (4) middle acidic buffer solution are the NaAc_HAc buffer solution of pH=3.6, and addition is 3.5ml.
Step (2) and (4) middle bromcresol green dye solution, addition is 2ml.
In step (2) and (4) chloroform extraction and processed require be, three extractions are divided with 10ml chloroform, 4mL, 3mL, 3mL respectively, after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer to filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, point get supernatant.
The present invention prepares reference substance solution with solanine, black nightshade medicinal material is prepared into need testing solution, in acidic buffer solution, bromcresol green dyestuff and alkaloid is utilized to be combined into colored complex and with chloroform extraction, ultraviolet spectrophotometer is adopted to measure the absorbance log of this colored solutions at a given wavelength, in order to calculate alkaloidal content.
For making those skilled in the art better understand the present invention, the applicant has carried out series of experiment research, to prove effect of the present invention:
1 reagent
Black nightshade medicine materical crude slice, purchased from Zhejiang Jia Xin Pharmaceuticals Ltd, through differentiating to meet version Chinese Pharmacopoeia () in 2010.Solanine reference substance, purchased from Shaanxi Ci Yuan Bioisystech Co., Ltd, lot number 20131223.Bromcresol green, chloroform, ethanol, acetic acid, sodium acetate, anhydrous sodium sulfate etc., purchased from Jiaxing City Yong Hong Chemical Co., Ltd., be analysis pure.
2 instruments
UV2201 type ultraviolet-visible pectrophotometer (Japanese Shimadzu), BS224S type electronic analytical balance (Beijing Sai Duolisi), BGZ246 type digital display Constant Temp. Oven (the rich news in Shanghai), LH08A type continuous dosing comminutor (the evident sincere machinery in Xinchang), RE5299 type Rotary Evaporators (Shanghai is sub-flourish), HH4 type digital display electric-heated thermostatic water bath (China of Changzhou state), GM0.5B type diaphragm-type vacuum pump (Tian Jinjin rises), LX920S1 type electric suction apparatus (this Man Feng of Shanghai), KQ300DE type digital display temperature-control ultrasonic cleaning instrument (Kunshan Shu Mei).
The preparation of 3 solution
3.1 buffer solution first prepare acetum and the sodium acetate solution of 0.2mol/L.A liquid: measure acetic acid 11.55mL, adding distil water is diluted to 1000mL, stored refrigerated.B liquid: take 16.4g sodium acetate or 27.22g water-containing acetic acid sodium, adding distil water dissolves, and dilution is settled to 1000mL, stored refrigerated.The damping fluid of different pH value is prepared again respectively: A liquid xmL+B liquid ymL, is diluted with water to 100mL, corrects finally by pH meter according to following table.
3.2 dye solution 0.05% bromcresol green solution: precision takes 0.1g, is dissolved in 0.05mol/L sodium hydroxide solution 2.8mL, is diluted with water to 200mL, shake up and get final product.
3.3 reference substance solution precisions take the solanine reference substance 5.00mg being dried to constant weight, and dissolve with chloroform, ultrasonic 20min, is transferred to 50mL volumetric flask, adds chloroform and is diluted to scale, shakes up and obtain the reference substance stock solution of 0.1mg/mL.
3.4 need testing solution black nightshade medicine materical crude slice 60 DEG C dry, pulverize 14 mesh sieves.Precision takes black nightshade medicinal powder 2.0g, is placed in conical flask, adds 2mol/L hydrochloric acid: the mixed solvent 30mL of 90% ethanol (volume ratio 1: 4), ultrasonic extraction 35min at 40 DEG C, extract decompression recycling ethanol to without the medicinal extract of alcohol taste, medicinal extract 10% dissolving with hydrochloric acid, ultrasonic 20min hydrotropy, suction filtration removes precipitation, is settled to 10mL with 10% hydrochloric acid, regulates pH to 10-11 with 10%NaOH, add chloroform extraction 3 times, collect chloroform solution, be settled to 25ml, obtain need testing solution.
The selection of 4 mensuration wavelength
Accurate absorption solanine reference substance solution and each 2mL of black nightshade need testing solution, be placed in separating funnel respectively, the accurate NaAc_HAc buffer solution 3.5mL adding pH3.6 respectively, and bromcresol green dyestuff 2mL, add chloroform 10mL again and divide 3 extractions (4,3,3mL), after the violent jolting 5min of close plug, leave standstill 30min, divide and get chloroform layer, filter with dry quantitative filter paper, after adding (having added 0.25g anhydrous sodium sulfate) in dry tool plug Erlenmeyer flask standing 30min, get supernatant and scan respectively within the scope of 200-700nm at ultraviolet spectrophotometer.Another precision draws chloroform 2mL, and be placed in the same operation of separating funnel, gained chloroform solution is blank liquid.Result shows, solanine reference substance solution and black nightshade need testing solution all have maximum absorption band at 243nm place, and blank liquid at this place without absorption peak, therefore select 243nm for measure wavelength.
The foundation of 5 acid-dye colorimetries
The investigation precision of 5.1 pH of buffer draw solanine reference substance solution 2.0mL totally 5 parts in separating funnel, add the Acetic acid-sodium acetate damping fluid that pH value is 3.6,4.0,4.4,4.8,5.2 respectively, all the other operations are the same, and gained measures liquid and measures absorbance respectively at 243nm place.When result shows that pH of cushioning fluid is 3.6, measuring liquid has maximum absorbance.The accurate need testing solution 2.0mL that draws operates with method, and result is surveyed consistent with reference substance.
The investigation precision of 5.2 damping fluid consumptions draw solanine reference substance solution 2.0mL totally 5 parts in separating funnel, add the damping fluid 1.0,1.5,2.0,2.5,3.0mL of pH value 3.6 respectively, all the other operations are the same, and gained measures liquid respectively at 243nm place mensuration absorbance.When result shows that damping fluid consumption is 3.5mL, absorbance is maximum.The accurate need testing solution 2.0mL that draws operates with method, and result is surveyed consistent with reference substance.
The investigation precision of 5.3 acid dyes consumptions draw solanine reference substance solution 2.0mL totally 5 parts in separating funnel, add the damping fluid 3.5mL of pH3.6 respectively, add bromcresol green dye solution 1.0 respectively, 1.5,2.0,2.5,3.0mL, all the other operations are the same, and gained measures liquid and measures absorbance respectively at 243nm place.When result shows that acid dyes consumption is 2mL, absorbance is maximum.The accurate need testing solution 2.0mL that draws operates with method, and result is surveyed consistent with reference substance.
The investigation precision of 5.4 color stabilities draw black nightshade reference substance solution 2.0mL totally 10 parts in separating funnel, according to embodiment 2 the 1st method operation, respectively after colour developing 10,20,30,40,50,60,70,80,90,100min time, measure absorbance log.Result to show that after colour developing after 30min, absorbance log is without significant change.
The methodological study of 6 Wan withAcid-dye Colorimetry total Steroidal Alkaloids from Solanum nigrum L content
The investigation precision of 6.1 precision draws solanine reference substance solution 2.0mL, and by 4 lower method time-and-motion studies, METHOD FOR CONTINUOUS DETERMINATION 6 times, result records the RSD=0.49% of absorption value, shows that instrument precision is good.
The investigation precision of 6.2 stability draws solanine reference substance solution 2.0mL, by 4 lower method time-and-motion studies, surveys 1 absorption value every 30min, surveys 5 times altogether, and absorption value RSD=0.8% in result display 2h, interpret sample liquid is good at 2h internal stability.
The investigation precision of 6.3 linear relationships draws reference substance solution 0.5,1.0,1.5,2.0,2.5,3.0mL is made into the standard solution of gradient in 5ml measuring bottle with chloroform dilution, the accurate 2ml that draws is placed in separating funnel respectively, by 4 lower method operations, measures liquid and measures absorbance at 243nm.Take solanine content as horizontal ordinate, absorbance is ordinate, drawing standard curve, and calculating regression equation is Y=0.0129X-0.0087, R
2=0.9960, the range of linearity is 5-30 μ g/mL.See Fig. 1.
6.4 replica test precisions take the black nightshade dried powder 5.0g of same lot number, and by 4 parallel preparations of lower method, 6 parts of need testing solutions, each precision gets 2.0mL, according to embodiment 2 the 1st method time-and-motion study absorbance, and result display RSD=1.53%.
6.5 average recovery experiments are accurate draws each 6 parts of the solanine need testing solution 2.0mL having measured content, add the solanine reference substance solution 2.0mL of concentration known, by 4 lower method time-and-motion study absorbances, according to regression equation, calculate solanine content, and be calculated as follows the recovery.Result display average recovery rate is 99.7%, RSD=1.4%.
Average recovery (%)=(measured amount-test sample content)/reference substance content × 100%
Table 1 recovery test result (n=6)
Wan withAcid-dye Colorimetry total Steroidal Alkaloids from Solanum nigrum L content of the present invention is that alkaline drug (B) can be combined into kation (BH with hydrogen ion according in suitable medium
+), and some acid dyess, as bromcresol green, bromthymol blue etc., negative ion (In can be dissociated into
-).Above-mentioned kation BH
+with negative ion In
-quantitatively be combined into colored complex ion pair (BH
+in
-), can be quantitative by Solvent Extract methods, measure the right absorbance log of this solution coloured ion at a given wavelength, namely can calculate the content of alkaline drug.The method is used for the assay of black nightshade total alkaloids, the interference of the impurity such as albumen, tannin in the precipitation method can be avoided, or the complex process of sample liquid and inefficiency before measuring in the endpoint error brought of difficulty and HPLC method in diphasic titration, be applicable to the express-analysis of a large amount of sample, be easy to promote the use of.
Determine that bromcresol green and alkaloid form the optimal pH of colored complex ten points important.When pH is low, dyestuff can not fully dissociate, and during pH height, organic base can not form ionic species, all fully can not extract ion pair, affect measurement result.Be mixed into chloroform layer if any moisture during extraction, producing muddiness affects colorimetric, affects measurement result.This experiment not only uses dry Filter paper filtering, adopts anhydrous sodium sulfate to dewater simultaneously, gets rid of the interference of moisture to greatest extent.
The present invention adopts in test chloroform extraction ion pair, finds by the result discreteness of 10mL chloroform 1 extraction large, and not exclusively, therefore adopt 3 extractions, be respectively 4,3,3mL, experimental result is more satisfactory in extraction.
The present invention is to measuring the determined wavelength of black nightshade total alkaloid content, the pH value of damping fluid and consumption, and the experiment conditions such as the consumption of acid dyes are optimized screening.Determine that best condition determination is: with the acid dyes of bromcresol green solution for colour developing, consumption 2.0mL, be that the Acetic acid-sodium acetate solution of 3.6 is for damping fluid with pH value, consumption 3.5mL, 3 extractions are divided with chloroform 10mL, leave standstill 30min, dewater with dry Filter paper filtering and 0.25g anhydrous sodium sulfate, measure under 243nm wavelength.The present invention applies this assay method and compares three kinds of extracting method to the extraction effect of black nightshade total alkaloids, and the black nightshade total alkaloids massfraction mean value that result display ultrasonic extraction extracts is 11.433, is better than refluxing extraction and homogenate extraction.The method set up herein is easy and simple to handle, reliable, favorable reproducibility, can be used for the assay of black nightshade total alkaloids.Compared with other detection methods, beneficial effect of the present invention is as follows:
(1) the method is used for the assay of black nightshade total alkaloids, the interference of the impurity such as albumen, tannin in the precipitation method can be avoided, or the complex process of sample liquid and inefficiency before measuring in the endpoint error brought of difficulty and HPLC method in diphasic titration, be applicable to the express-analysis of a large amount of sample, be easy to promote the use of.
(2) the method is to the pH value of determined wavelength, damping fluid and consumption, and the conditions such as the consumption of acid dyes are optimized screening, and adopts dry Filter paper filtering and anhydrous sodium sulfate to get rid of moisture interference.The method set up is reliably easy and simple to handle, with low cost, and precision is good, the recovery is high, favorable reproducibility, can be used for the assay of black nightshade total alkaloids.
Accompanying drawing explanation
Fig. 1 is black nightshade alkaloid canonical plotting.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but the present invention is not limited only to described embodiment.
Embodiment 1 adopts the present invention to the assay of black nightshade medicinal material through ultrasonic extract total alkaloids
Black nightshade medicine materical crude slice 60 DEG C dry, pulverize 14 mesh sieves.Precision takes Black Nightshade Herb 10.0g, be placed in conical flask, add 2mol/L hydrochloric acid: the mixed solvent 150mL of 90% ethanol (volume ratio 1: 4), ultrasonic extraction 35min at 40 DEG C, extracts 2 times, and extract decompression recycling ethanol is to the medicinal extract without alcohol taste, medicinal extract 10% dissolving with hydrochloric acid, ultrasonic 20min hydrotropy, suction filtration removes precipitation, is settled to 50mL with 10% hydrochloric acid.Therefrom accurate absorption 10ml, regulates pH to 10-11 with 10%NaOH, adds chloroform extraction 3 times, collect chloroform solution, be settled to 25ml, obtain need testing solution.
Precision takes the solanine reference substance 5.00mg being dried to constant weight, and dissolve with chloroform, ultrasonic 20min, is transferred to 50mL volumetric flask, adds chloroform and is diluted to scale, shakes up and obtain the reference substance solution of 0.1mg/mL.Accurate absorption reference substance solution 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mL is made into the standard solution of gradient in 5ml measuring bottle with chloroform dilution, the accurate 2ml that draws is placed in separating funnel respectively, precision adds the NaAc_HAc buffer solution 3.5mL of pH3.6, and bromcresol green dye solution 2mL, add chloroform 10mL again and divide three extractions (4, 3, 3mL), after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer to filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, get supernatant ultraviolet spectrophotometer and measure absorbance in 243nm place., measure liquid and measure absorbance at 243nm.Take solanine content as horizontal ordinate, absorbance is ordinate, drawing standard curve, and calculating regression equation is Y=0.0129X-0.0087, R2=0.9960, and the range of linearity is 5-30 μ g/mL.
Accurate absorption need testing solution 2.0mL, precision adds the NaAc_HAc buffer solution 3.5mL of pH3.6, and bromcresol green dye solution 2mL, then add chloroform 10mL and divide three extractions (4,3,3mL), after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer and filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, get supernatant ultraviolet spectrophotometer in 243nm place mensuration absorbance.Absorbance according to the reference substance solution of gradient concentration calculates typical curve.Again according to the absorbance of need testing solution, substitute into the content that typical curve calculates black nightshade total alkaloids.Test in triplicate parallel, the massfraction that black nightshade medicinal material obtains total alkaloids through ultrasonic extraction process is 11.433 ± 5.85mg/g.
Embodiment 2 adopts the present invention to the assay of black nightshade medicinal material refluxing extraction total alkaloids
Black nightshade medicine materical crude slice 60 DEG C dry, pulverize 14 mesh sieves.Precision takes Black Nightshade Herb 10.0g, be placed in round-bottomed flask, add 2mol/L hydrochloric acid: the mixed solvent 150mL of 90% ethanol (1: 4), covered and enclosed soaks 8h, 80 DEG C of heating water bath refluxing extraction 2h, extract 2 times, merge extract, extract decompression recycling ethanol is to the medicinal extract without alcohol taste, medicinal extract 10% dissolving with hydrochloric acid, ultrasonic 20min hydrotropy, suction filtration removes precipitation, is settled to 50mL volumetric flask with 10% hydrochloric acid.Accurate absorption acid liquid 10mL, regulate pH to 10-11 with 10%NaOH, be transferred to separating funnel, add chloroform extraction 3 times, collect chloroform solution, be placed in 25mL volumetric flask, chloroform constant volume, obtains need testing solution.
Precision takes the solanine reference substance 5.00mg being dried to constant weight, and dissolve with chloroform, ultrasonic 20min, is transferred to 50mL volumetric flask, adds chloroform and is diluted to scale, shakes up and obtain the reference substance solution of 0.1mg/mL.Accurate absorption reference substance solution 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mL is made into the standard solution of gradient in 5ml measuring bottle with chloroform dilution, the accurate 2ml that draws is placed in separating funnel respectively, precision adds the NaAc_HAc buffer solution 3.5mL of pH3.6, and bromcresol green dye solution 2mL, add chloroform 10mL again and divide three extractions (4, 3, 3mL), after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer to filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, get supernatant ultraviolet spectrophotometer and measure absorbance in 243nm place., measure liquid and measure absorbance at 243nm.Take solanine content as horizontal ordinate, absorbance is ordinate, drawing standard curve, and calculating regression equation is Y=0.0129X-0.0087, R2=0.9960, and the range of linearity is 5-30 μ g/mL.See Fig. 1.
Accurate absorption need testing solution 2.0mL, precision adds the NaAc_HAc buffer solution 3.5mL of pH3.6, and bromcresol green dye solution 2mL, then add chloroform 10mL and divide three extractions (4,3,3mL), after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer and filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, get supernatant ultraviolet spectrophotometer in 243nm place mensuration absorbance.Absorbance according to the reference substance solution of gradient concentration calculates typical curve.Again according to the absorbance of need testing solution, substitute into the content that typical curve calculates black nightshade total alkaloids.Test in triplicate parallel, the massfraction that black nightshade medicinal material obtains total alkaloids through Effect of Alcohol Extracting Procedureto Total is 3.342 ± 3.21mg/g.
Claims (4)
1. measure a method for total Steroidal Alkaloids from Solanum nigrum L content, it is characterized in that comprising the following steps:
(1) preparation of reference substance solution: precision takes the solanine reference substance 5.00mg being dried to constant weight, dissolve with chloroform, ultrasonic 20min, is transferred to 50mL volumetric flask, adds chloroform and is diluted to scale, shakes up and obtain the reference substance storing solution of 0.1mg/mL;
(2) making of typical curve: accurate reference substance storing solution of drawing dilutes the standard solution being made into gradient in 5ml measuring bottle with chloroform, accurate 2ml standard solution of drawing is placed in separating funnel, add acidic buffer solution and bromcresol green dye solution develops the color, add chloroform extraction again, divide and get chloroform layer processed, get supernatant according to ultraviolet spectrophotometry, measure absorbance in 243nm place; Blank is done with identical reagent and step process with chloroform; Take solanine content as horizontal ordinate, with its absorbance for ordinate, obtain black nightshade Determination of Total Alkaloid typical curve;
(3) preparation of need testing solution: precision takes the black nightshade medicinal powder 2.0g crossing 14 mesh sieves, be placed in conical flask, add 2mol/L hydrochloric acid and 90% alcohol mixed solvent 30mL that volume ratio is 1: 4, ultrasonic extraction 35min at 40 DEG C, extract decompression recycling ethanol is to the medicinal extract without alcohol taste, medicinal extract 10% dissolving with hydrochloric acid, ultrasonic 20min hydrotropy, suction filtration removes precipitation, is settled to 10mL with 10% hydrochloric acid, regulates pH to 10-11 with 10%NaOH, add chloroform extraction 3 times, collect chloroform solution, be settled to 25ml, obtain need testing solution;
(4) assay of sample solution: accurate absorption need testing solution 2.0mL, described in step (2), add acidic buffer solution and bromcresol green dye solution develops the color, add chloroform extraction again, divide and get chloroform layer processed, get supernatant according to ultraviolet spectrophotometry, measure absorbance in 243nm place, substitute into typical curve, calculate the content of black nightshade total alkaloids.
2. method according to claim 1, is characterized in that, step (2) and (4) middle acidic buffer solution are the NaAc_HAc buffer solution of pH=3.6, and addition is 3.5ml.
3. method according to claim 1, is characterized in that, step (2) and (4) middle bromcresol green dye solution, addition is 2ml.
4. according to claim 1 containing method, it is characterized in that, in step (2) and (4) chloroform extraction and processed require be, dividing three extractions with 10ml chloroform, is 4mL, 3mL, 3mL respectively, after the violent jolting 5min of close plug, leave standstill 30min, get the dry quantitative filter paper of chloroform layer to filter, add dry tool plug and added in the Erlenmeyer flask of 0.25g anhydrous sodium sulfate and leave standstill after 30min, point get supernatant.
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