CN104034823B - The detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material - Google Patents
The detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material Download PDFInfo
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Abstract
The invention provides the detection method of indoles alkaloid ingredients fingerprint in a kind of dried venom of toads medicinal material, comprising key step is: the preparation of need testing solution and reference substance solution, mensuration, and after the liquid chromatogram obtaining need testing solution and reference substance solution compares, according to relative retention time, determine 2 kinds of indoles alkaloid compositions in need testing solution, according to chromatographic peak area, adopt the content of 2 kinds of indoles alkaloid compositions in external standard method determination need testing solution.The present invention can carry out effective qualitative and quantitative analysis to 2 kinds of main indoles alkaloid compositions in the dried venom of toads simultaneously, method is easy, feasible, accurate, precision, the repeatability of assay method, to have good stability, can effectively control dried venom of toads quality, and provide reference for the clinical application of the dried venom of toads.
Description
Technical field
The present invention relates to traditional Chinese medicine ingredients technical field of analysis and detection, be specifically related to the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material.
Background technology
2010 editions " Chinese Pharmacopoeia (one) " records the medicinal material that the dried venom of toads obtains after dry processing as the ear rear gland of Bufonidae animal bufo gargarizans Cantor BufobufogargarizansCantor or Bufo melanostictus BufomelanostictusSchneider and the white slurries of skin gland secretion.Its proterties is oblate lumps or sheet, and lumps person's matter is hard, not frangibility, and section is sepia, cutin shape, micro-glossy; Sheet person's matter is crisp, frangible, section rufous, translucent.Dried venom of toads taste is just sweet then lasting spicy sense, and what powder was smelt sneezes.Have removing toxic substances, pain relieving, the effect of inducing resuscitation of having one's ideas straightened out, cure mainly ulcer furunculosis, abscess of throat, heatstroke coma, the diseases such as stomachache vomiting and diarrhoea, have very good curative effect.
Complex chemical composition in the dried venom of toads, principal ingredient has bufanolide dienes, cardenolides alkene toadpoison class, indoles bases, sterols and adrenaline, polysaccharide, protein, amino acid and organic acid etc.Wherein, indoles alkali components, also referred to as indoles alkaloid composition, is a very important class chemical composition in the dried venom of toads, has extraordinary raising immunity of organisms, and have certain anti-tumor capacity.At present, indoles alkali components contained in the dried venom of toads mainly contains serotonin and serotonin derivant, and wherein serotonin and bufotenine are as two kinds of important composition compositions in indoles alkaloid contained in the dried venom of toads, are starved of and carry out qualitative and quantitative analysis.
At present, for indoles alkaloid composition in the dried venom of toads, " Chinese Pharmacopoeia " is differentiated by means of only paradime thylaminobenzaldehyde development process, and prior art does not record quantivative approach and the correlated condition thereof of Analyze 5-HT and bufotenine simultaneously.Therefore, be necessary the pre-treatment and the testing conditions that adopt optimization, set up indoles alkaloid composition in the dried venom of toads: serotonin and bufotenine assay method, the method compensate for the shortcoming of the specificity deficiency that alkaloids composition relies on chromogenic reaction to differentiate, more comprehensively, effectively can control the quality of the dried venom of toads.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material, adopt the pre-treatment of optimal conditions and detection method to 2 kinds of indoles alkaloid compositions: while serotonin and bufotenine, to carry out qualitative and quantitative analysis, and then the inherent quality of reflection dried venom of toads medicinal material, for dried venom of toads medicinal material judges that the true and false, evaluation quality, investigation stability and consistance provide foundation.
For achieving the above object, first the present invention provides the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material, comprises the following steps:
Preferably, in described dried venom of toads medicinal material, indoles alkaloid composition is serotonin and bufotenine.
1) preparation of need testing solution: dried venom of toads fine powder is added ethanol water, after ultrasonic extraction, cools, shakes up; Centrifugal, Aspirate supernatant heating evaporate to dryness, adds dissolution with solvents and constant volume, filtering membrane, gets subsequent filtrate as need testing solution;
Preferably, described ethanol water is 50% ethanol water.Concrete, described 50% ethanol water to be percent by volume be 50% ethanol.
Further, 50% ethanol water added is the 100-800 times amount of dried venom of toads fine powder.Preferably, 50% ethanol water added described in is 200 times amount of dried venom of toads fine powder.
Concrete, described in 50% ethanol water that adds be that the 100-800 times amount of dried venom of toads fine powder refers to, add 50% ethanol water 10-80ml in the dried venom of toads fine powder of every 0.1g.Preferably, when the weight of described dried venom of toads fine powder is 0.1g, the addition of described 50% ethanol water is 20ml.
Preferably, after described dried venom of toads fine powder adds ethanol water, precise weighing is needed.
Preferably, after described ultrasonic extraction, cooling, need weigh again, and supply weightlessness with 50% ethanol water.
Preferably, described ultrasonic extraction time is 20-60min.Preferably, described ultrasonic extraction time is 20min.
Preferably, described centrifugation time is 20min; Described centrifugal rotational speed is 4000rpm.
Preferably, described Aspirate supernatant heating evaporate to dryness refers to: the supernatant of certain volume precision drawn is placed in evaporating dish, puts into water bath with thermostatic control heating, steams near dry.Further, the volume of described Aspirate supernatant is 3ml.
Preferably, described dissolving and constant volume time the solvent that uses be 20% ethanol water.Concrete, described 20% ethanol water to be percent by volume be 20% ethanol.Further, the constant volume adding 20% ethanol water described in is 10ml.
Preferably, described filter membrane is 0.45 μm of filter membrane.
Described subsequent filtrate refers to: during filtering membrane, and the solution after first filtrate is subsequent filtrate.
2) preparation of reference substance solution: serotonin and bufotenine reference substance are added dissolution with solvents and constant volume, shakes up, obtains mixing reference substance solution;
Preferably, in described reference substance solution No. CAS of serotonin be 50-67-9; No. CAS of bufotenine is 487-93-4.
Preferably, in described reference substance solution, the content range of each composition is: serotonin 120.0-1800.0 μ g/ml, bufotenine 14.4-216.0 μ g/ml.
Preferably, in described reference substance solution, the content range of each composition is: serotonin 120.0 μ g/ml, bufotenine 14.4 μ g/ml.
Preferably, described dissolving and constant volume time the solvent that uses be 20% ethanol water.Concrete, described 20% ethanol water to be percent by volume be 20% ethanol.
3) qualitative detection: adopt high performance liquid chromatography to measure need testing solution and reference substance solution respectively, by the liquid chromatogram of the need testing solution of acquisition, compare with the liquid chromatogram of reference substance solution, according to relative retention time, determine 2 kinds of indoles alkaloid compositions in need testing solution;
4) quantitatively detect: adopt high performance liquid chromatography to measure need testing solution and reference substance solution respectively, according to chromatographic peak area, adopt the content of 2 kinds of indoles alkaloid compositions in external standard method determination need testing solution.
Preferably, the chromatographic condition of described high performance liquid chromatography is: chromatographic column: AlltimaC
18(250mm × 4.6mm, 5 μm) chromatographic column; Column temperature: 25-35 DEG C, preferably 25 DEG C; Determined wavelength: 275nm; Flow velocity: 0.7-0.8ml/min, preferred 0.7ml/min; Sample size: 10 μ l; Mobile phase: acetonitrile-0.5% potassium dihydrogen phosphate buffer solution (phosphoric acid adjusts buffer solution pH to 3.2), wherein, A phase is acetonitrile, and B phase is 0.5% potassium dihydrogen phosphate buffer solution; Analysis time: 25min; Isocratic elution.
Further, described isocratic elution at 0-25min, with mobile phase A phase: B phase volume ratio is that 5:95 carries out wash-out.
Further, described 0.5% potassium dihydrogen phosphate buffer solution to be percent by weight be 0.5% potassium dihydrogen phosphate aqueous buffer solution.
Preferably, described external standard method refers to: the step B pipetting a series of different volumes respectively) described mixing reference substance solution, adopt the analysis of high performance liquid chromatograph sample introduction, obtain the linear relationship of indoles alkaloid 2 kinds of main active content and peak area in mixing reference substance solution, with each active component chromatographic peak area its corresponding content corresponding, draw corresponding standard working curve, calculate the regression equation of each standard working curve.High performance liquid chromatograph is adopted to detect need testing solution again, by the chromatographic peak area of 2 kinds of indoles alkaloid compositions in the need testing solution of acquisition, substitute into respectively in the regression equation of described each standard working curve, the content of corresponding indoles alkaloid composition can be obtained.
Preferably, the volume that pipettes of described mixing reference substance solution is 1 μ l, 2 μ l, 4 μ l, 8 μ l, 10 μ l, 15 μ l.
As mentioned above, the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material of the present invention, larger for indoles alkaloid polarity, in general chromatographic column almost without reserve and the feature of degree of separation difference, through investigating the optimization of chromatographic column screening and the condition such as flow phase system, sample preparation, analysis, adopt the pre-treatment of Optimal reaction conditions and high efficient liquid phase analysis method to establish in the dried venom of toads while 2 kinds of indoles alkaloid compositions, simply, quantitative and qualitative detection method accurately.Wherein, the present invention adopts mobile phase: acetonitrile--0.5% potassium dihydrogen phosphate buffer solution (phosphoric acid adjusts buffer solution pH to 3.2), without the need to the gradient elution program of complexity, under simple isocratic condition, separated compound just can be made to keep good degree of separation and peak shape, carry out Simultaneous Determination.The present invention passes through the quantitative test to indoles alkaloid 2 kinds of compositions, the coefficient R of the regression equation of 2 kinds of compositions in quantitative analysis results
2all be greater than 0.9997, the recovery is all greater than 99%, RSD and is all less than 2.0%, precision, the repeatability of assay method, has good stability.Qualitative and quantitative analysis method of the present invention is synchronous, easy, feasible, accurate, can effectively control dried venom of toads quality, and provides reference for the clinical application of the dried venom of toads.
Accompanying drawing explanation
Fig. 1 is shown as Different Extraction Method of the present invention and compares liquid chromatogram 1A, 1B, 1C
Fig. 2 is shown as different buffer salinity of the present invention and compares liquid chromatogram 2A, 2B, 2C
Fig. 3 is shown as different pH of cushioning fluid of the present invention and compares liquid chromatogram 3A, 3B, 3C
Fig. 4 is shown as different chromatographic column of the present invention and compares liquid chromatogram 4A, 4B, 4C
Fig. 5 is shown as different in flow rate of the present invention, column temperature compares liquid chromatogram 5A, 5B, 5C
Fig. 6 is shown as the reference substance liquid chromatogram of 2 kinds of compositions in indoles alkaloid of the present invention, wherein, and I:5-hydroxytryptamine, II: bufotenine
Fig. 7 is shown as the biased sample liquid chromatogram of 2 kinds of compositions in indoles alkaloid of the present invention, wherein, and I:5-hydroxytryptamine, II: bufotenine
Fig. 8 is shown as regression equation and the range of linearity schematic diagram of serotonin in indoles alkaloid of the present invention
Fig. 9 is shown as regression equation and the range of linearity schematic diagram of bufotenine in indoles alkaloid of the present invention
Embodiment
Set forth the present invention further below in conjunction with specific embodiment, should be understood that these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.
The material that following examples use, reagent and instrument are as follows:
1, material, reagent
The dried venom of toads (Huayu Pharmaceutical Co., Ltd, Shanghai); Ethanol (analyzing pure, Shanghai Hao Shen chemical reagent company limited); Phosphoric acid (chromatographic grade, TEDIA company); Potassium dihydrogen phosphate (top grade is pure, Shanghai Ling Feng chemical reagent company limited); Acetonitrile (chromatographically pure, TEDIA company); Serotonin, bufotenine (HPLC purity >=98%, lot number 20130630, Shanghai Kelike Biomedical Technology Co., Ltd.);
2, instrument
Tool plug conical flask, transfer pipet and associated glass vessel (Shanghai He Qi Glass Co., Ltd.) thereof; 50ml evaporating dish with round bottom (Shanghai Xi Bo reaches glass apparatus company limited); HSS type electric-heated thermostatic water bath (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); Filter membrane (0.45 μm, Shanghai ANPEL Scientific Instrument Co., Ltd.); TDL-40 type desk centrifuge (Anting Scientific Instrument Factory, Shanghai); Chromatographic column: NucleosilC
18(German MACHEREY-NAGEL company), AllitmaC
18(250mm × 4.6mm, 5 μm) (GRACE company of the U.S.), WatersSymmetryC
18(water generation Science and Technology Ltd. of the U.S.); SB-5200 ultrasonic extraction instrument (NingBo XinZhi Biology Science Co., Ltd); Agilent1260 liquid chromatograph (Anjelen Sci. & Tech. Inc of the U.S.).
The mensuration of 2 kinds of indoles alkaloid compositions in embodiment 1 dried venom of toads medicinal material
1 experimental section
1.1 sample pre-treatments
The preparation of need testing solution: the dried venom of toads fine powder of accurately weighed 0.1g, precision adds 50% ethanol water, weighed weight, ultrasonic extraction, after cooling, more weighed weight, supply weightlessness with 50% ethanol water, shake up.Adopt centrifuge, centrifugation time is 20min, centrifugal rotational speed is 4000rpm.The supernatant of the accurate 3ml of absorption is placed in evaporating dish heating, steams near dry again.Add 20% ethanol water dissolve and be settled to 10ml, cross 0.45 μm of filter membrane, get subsequent filtrate as need testing solution.
The preparation of reference substance solution: to take serotonin and bufotenine reference substance appropriate for precision respectively, adds 20% ethanol water and dissolve and constant volume, shake up, obtain mixing reference substance solution; Wherein, the content range of each composition is: serotonin 120.0-1800.0 μ g/ml, bufotenine 14.4-216.0 μ g/ml.
1.2 chromatographic condition
The chromatographic condition of high performance liquid chromatography is: chromatographic column: AlltimaC
18(250mm × 4.6mm, 5 μm) chromatographic column; Column temperature: 25-35 DEG C, preferably 25 DEG C; Determined wavelength: 275nm; Flow velocity: 0.7-0.8ml/min, preferred 0.7ml/min; Sample size: 10 μ l; Mobile phase: acetonitrile-0.5% potassium dihydrogen phosphate buffer solution (phosphoric acid adjusts buffer solution pH to 3.2), wherein, A phase is acetonitrile, and B phase is 0.5% potassium dihydrogen phosphate buffer solution; Analysis time: 25min; Isocratic elution.
Isocratic elution at 0-25min, with mobile phase A phase: B phase volume ratio is that 5:95 carries out wash-out.
1.3 measure
Adopt external standard method, volume aspirated is the mixing reference substance solution of 1 μ l, 2 μ l, 4 μ l, 8 μ l, 10 μ l, 15 μ l respectively, adopts the analysis of high performance liquid chromatograph sample introduction, drawing standard curve.Volume aspirated is the need testing solution of 10 μ l again, adopts the analysis of high performance liquid chromatograph sample introduction.By the liquid chromatogram of the need testing solution of acquisition, compare with the liquid chromatogram of reference substance solution, according to relative retention time, determine 2 kinds of indoles alkaloid compositions in need testing solution; According to chromatographic peak area, calculate the content obtaining 2 kinds of indoles alkaloid compositions in need testing solution.
2 results and discussion
The optimization of 2.1 Pretreatments
2.1.1 the selection of extracting method
Compare hot reflux, ultrasonic and cold soaking extraction method respectively, wherein, adopt circumfluence distillation method to extract 1 hour; Adopt the ultrasonic 20min of ultrasonic extracting method; Cold soaking extraction method cold soaking is adopted to spend the night extraction.Dried venom of toads medicinal material adopts identical HPLC testing conditions measurement result to see Fig. 1, table 1 under Different Extraction Method.
Table 1 three kinds of extracting method affect result to the chromatographic peak area of 2 kinds of compositions
| Extracting method | Serotonin (A) | Bufotenine (A) |
| Cold soaking | 1735 | 176 |
| Hot reflux | 1798 | 185 |
| Ultrasonic | 1781 | 178 |
From Fig. 1, table 1, under the sufficient condition of extraction, the peak area no significant difference (RSD<3.0%) of two kinds of compositions in the chromatic graph spectrum that three kinds of extracting method obtain, owing to all there being powder agglomates phenomenon in various degree in the solution after cold-maceration and heat reflow method extraction, after ultrasonic extraction, dried venom of toads powder is uniformly dispersed in the solution, and easy and simple to handle, rapid, therefore selective extraction mode is ultrasonic extraction.
2.1.2 the selection of Extraction solvent
Adopt identical pre-treatment and high-performance liquid chromatogram determination method, compare the extraction effect of Extraction solvent to indoles alkaloid respectively, Extraction solvent concrete outcome is in table 2.Shown by the peak area comparative result of 2 assay index compositions, 80% alcohol extract efficiency is minimum, 50% alcohol extract is most effective, simultaneously 20% ethanol, 30% ethanol extract are more muddy, cause in subsequent operation and cross 0.45 μm of filter membrane difficulty, therefore select 50% ethanol to be Extraction solvent.
Table 2 different solvents affects result to the chromatographic peak area of 2 kinds of compositions
| Extraction solvent | Serotonin (A) | Bufotenine (A) |
| 20% ethanol | 1554 | 152 |
| 30% ethanol | 1577 | 157 |
| 50% ethanol | 1781 | 178 |
| 50% methyl alcohol | 1695 | 167 |
| 70% ethanol | 1572 | 157 |
| 80% ethanol | 964 | 86 |
2.1.3 the selection of ultrasonic time and solvent volume
Adopt identical pre-treatment and high-performance liquid chromatogram determination method, compare different ultrasonic extraction time, Extraction solvent volume to the impact of extraction efficiency, with the peak area of 2 assay index compositions for index compares, concrete measurement result is in table 3,4.Result shows, extraction time, solvent volume do not make significant difference to extraction efficiency thing, and ultrasonic 20min can extract completely; More muddy after 100 times of solvent extraction liquid are centrifugal, be unfavorable for subsequent operation, 200 times of solvents can extract completely, and therefore selective solvent volume is 200 times.
The different ultrasonic time of table 3 affects result to the chromatographic peak area of 2 kinds of compositions
| Ultrasonic time | Serotonin (A) | Bufotenine (A) |
| 20min | 1716 | 170 |
| 30min | 1681 | 168 |
| 45min | 1734 | 172 |
| 60min | 1745 | 173 |
Table 4 different solvents volume affects result to the chromatographic peak area of 2 kinds of compositions
| Solvent volume | Serotonin (A) | Bufotenine (A) |
| 100 times | 846 | 69 |
| 200 times | 885 | 72 |
| 400 times | 860 | 71 |
| 800 times | 868 | 71 |
From above-mentioned experimental result, in dried venom of toads medicinal material, the optimum condition of the test sample pre-treatment of indoles alkaloid composition is: get a certain amount of dried venom of toads fine powder accurately weighed, put in tool plug conical flask, precision adds 50% ethanol of 200 times amount, weighed weight, ultrasonic extraction 20min, let cool, weighed weight again, supplies weightlessness with 50% ethanol, shakes up; Centrifugal 20min (4000rpm) gets 3ml supernatant heating evaporate to dryness, adds 20% ethanol and dissolves and be settled to 10ml, cross 0.45 μm of filter membrane, get subsequent filtrate, to obtain final product.
The optimization of 2.2 chromatographic conditions
2.2.1 the selection of buffer concentration
Adopt identical pre-treatment and high-performance liquid chromatogram determination method, compare the impact of potassium dihydrogen phosphate buffer solution on separating effect of variable concentrations respectively, concrete outcome is shown in Fig. 2.As shown in Figure 2, when potassium dihydrogen phosphate buffer solution concentration is 40mM (i.e. mmol), chromatographic peak broadening improves, and 60mM and 40mM compares, and difference is little, considers, should select the buffer salt of low concentration from protection chromatographic column angle; Recording " dried venom of toads content assaying method " due to " Chinese Pharmacopoeia " uses mobile phase for acetonitrile-0.5% potassium dihydrogen phosphate, with 40mM potassium dihydrogen phosphate concentration comparable, therefore potassium dihydrogen phosphate buffer solution concentration is selected to be 0.5%, namely add 5.0g potassium dihydrogen phosphate in 1L water, namely percent by weight is 0.5% potassium dihydrogen phosphate buffer solution.
2.2.2 the selection of pH of cushioning fluid
Adopt identical pre-treatment and high-performance liquid chromatogram determination method, the potassium dihydrogen phosphate buffer solution of more different pH is on the impact of separating effect respectively, and concrete outcome is shown in Fig. 3.As shown in Figure 3, when adjusting pH=3.2, chromatographic peak broadening improves, and adjusts pH=2.8 compared with 3.2, and difference is little, and therefore selection phosphoric acid tune buffer solution pH is 0.5% potassium dihydrogen phosphate buffer solution of 3.2.
2.2.3 the selection of mobile phase and condition of gradient elution thereof
By the above-mentioned selection to concentration in buffer solution and pH value, the flow phase system adopted in the present invention: acetonitrile-0.5% potassium dihydrogen phosphate buffer solution (phosphoric acid adjusts pH=3.2), and separated compound can be made under specific isocratic condition to keep good degree of separation and peak shape, can a sample preparation single injected sampling analysis, efficient qualitative and quantitative analysis can be carried out to indoles alkaloid composition.
2.2.4 the selection of chromatographic column
Adopting identical pre-treatment and high-performance liquid chromatogram determination method, in order to improve the tailing problem of chromatographic peak further, comparing with NucleosilC respectively
18, AllitmaC
18, WatersC
18three sections of chromatographic columns are on the impact of indoles alkaloid component separating effect, and concrete outcome is shown in Fig. 4.Result shows, AlltimaC
18chromatographic column can be improved chromatogram preferably and broaden and hangover situation, therefore selects AlltimaC
18chromatographic column is analyzed.
2.2.5 the selection of flow velocity, column temperature
Adopt identical pre-treatment and high-performance liquid chromatogram determination method, compare respectively different column temperature 25-35 DEG C, flow velocity 0.7-0.8ml/min on the impact of compound separation effect, concrete outcome is shown in Fig. 5.As shown in Figure 5, find to be chosen as 0.7ml/min when flow velocity, when column temperature is chosen as 25 DEG C, the chromatographic peak of composition to be determined can effectively be separated, and the degree of separation of 2 chromatographic peaks is all greater than 1.5, and the consumes least to chromatographic column, optimized separating effect can be obtained.
From above-mentioned experimental result, the optimization chromatographic condition measuring 2 kinds of indoles alkaloid component contents is: AlltimaC
18(250mm × 4.6mm, 5 μm) chromatographic column; Mobile phase is acetonitrile-0.5% potassium dihydrogen phosphate buffer solution (phosphoric acid adjusts buffer solution pH to 3.2); Isocratic elution; Column temperature 25 DEG C; Determined wavelength 275nm; Flow velocity 0.7ml/min; Sample size: 10 μ l; Analysis time 25min.
The precision of 2.3 methods, repeatability, stability
2.3.1 precision
The same need testing solution of accurate absorption, under the above-mentioned 2.2 HPLC optimal conditions confirmed, continuous sample introduction analyzes 6 times, and measure peak area, precision concrete outcome is in table 5.Result shows, the chromatographic peak area RSD of serotonin, bufotenine is less than 0.64%, illustrates that the method precision is good.
Table 52 kind of indoles alkaloid composition precision investigates result
| Project | Serotonin (A) | Bufotenine (A) |
| 1 | 1450.7 | 146.8 |
| 2 | 1452.1 | 146.8 |
| 3 | 1456.4 | 147.0 |
| 4 | 1452.3 | 146.6 |
| 5 | 1452.5 | 146.2 |
| 6 | 1454.0 | 144.5 |
| Mean value | 1453.0 | 146.3 |
| RSD(％) | 0.14 | 0.64 |
2.3.2 repeated
Get with a collection of dried venom of toads medicinal powder 6 parts, by above-mentioned 2.1 need testing solution pre-treatment optimal conditions operations, under above-mentioned 2.2 chromatographic conditions, continuous sample introduction analyzes 6 times, and measure peak area, repeated concrete outcome is in table 6.Result shows, the peak area RSD of serotonin, bufotenine is less than 2.16%, illustrates that the method repeatability is good.
Table 62 kind of indoles alkaloid composition repeatability investigates result
| Sample weighting amount (g) | Serotonin (A) | Bufotenine (A) |
| 0.1002 | 1484.0 | 148.8 |
| 0.1003 | 1471.2 | 148.0 |
| 0.1005 | 1476.6 | 148.4 |
| 0.1003 | 1490.1 | 153.6 |
| 0.1002 | 1452.3 | 145.7 |
| 0.1003 | 1453.7 | 144.3 |
| Mean value | 1471.3 | 148.1 |
| RSD(％) | 1.06 | 2.16 |
2.3.3 stability
Accurate draw same need testing solution, according to the optimal conditions that above-mentioned 2.1 and 2.2 confirm, respectively at 0,2,4,8,12,24h sample introduction analyzes, measure peak area, concrete outcome is in table 7.Study on the stability result shows, the relative peak area RSD of serotonin, bufotenine is less than 0.18%, illustrates that the method is good at 24h internal stability.
Table 72 kind of indoles alkaloid ingredient stability investigates result
| Time | Serotonin (A) | Bufotenine (A) |
| 0h | 1471.4 | 147.3 |
| 2h | 1466.2 | 147.4 |
| 4h | 1464.9 | 147.3 |
| 8h | 1467.5 | 147.6 |
| 12h | 1470.3 | 147.4 |
| 24h | 1470.1 | 147.0 |
| Mean value | 1468.4 | 147.3 |
| RSD(％) | 0.18 | 0.13 |
The linear relationship of 2.42 kinds of indoles alkaloid component content assay methods
It is appropriate that precision takes reference substance solution, add 20% ethanol dissolve and be settled to scale, be prepared into the mixing reference substance solution containing different quality concentration components, wherein, in mixing reference substance solution, each component content is: serotonin 120.0 μ g/ml, bufotenine 14.4 μ g/ml.Accurate absorption mixes reference substance solution 1 μ l, 2 μ l, 4 μ l, 8 μ l, 10 μ l, 15 μ l sample introductions respectively, measures and calculates the standard regressive method, related coefficient and the range of linearity that obtain each composition.Concrete outcome is shown in Fig. 8-9, table 8.
The regression equation of table 82 kind of indoles alkaloid composition and the range of linearity
| Compound | Typical curve | R 2 | The range of linearity (μ g) |
| Serotonin | y＝1928.1770x-8.5860 | 1.0000 | 0.12～1.8 |
| Bufotenine | y＝1586.9509x-5.5306 | 0.9997 | 0.0144～0.216 |
Known according to Fig. 8-9, table 8, standard regressive method take chromatographic peak area as ordinate (Y), compound quality is horizontal ordinate (X), simultaneously, 2 kinds of compositions are all in good linear relationship in range of liner, and the related coefficient of its standard regressive method is all greater than 0.9997.
The recovery of 2.52 kinds of indoles alkaloid component content assay methods
The dried venom of toads sample that precision takes 6 parts of concentration known is appropriate, and accurately add the serotonin of certain content, bufotenine reference substance respectively, the optimal conditions process by 2.1 and 2.2 also measures, and the results are shown in Table 9.As shown in Table 9, the average recovery rate of the content assaying method of 2 kinds of compositions is all greater than 99%, RSD and is all less than 2.0%, and the recovery of its measurement result is good.
Table 92 kind of indoles alkaloid composition average recovery result
The mensuration of 2 kinds of indoles alkaloid component contents in 2.6 actual samples
By the optimization to need testing solution pre-treatment and chromatographic condition, and methodological study, we establish the assay method of 2 kinds of indoles alkaloid compositions in dried venom of toads medicinal material.Get 10 batches of dried venom of toads medicinal materials and prepare sample by the optimal conditions of test sample pre-treatment in above-mentioned 2.1, optimize chromatographic condition by HPLC in above-mentioned 2.2 and measure, measure the content of 2 kinds of indoles alkaloid compositions in 10 batches of dried venom of toads samples.The comparison chromatogram of dried venom of toads medicinal material test sample and 2 mixing reference substance compositions is shown in Fig. 6-7.From Fig. 6-7, according to relative retention time, determine 2 kinds of indoles alkaloid compositions in need testing solution; And adopt external standard method, by the chromatographic peak area of 2 kinds of indoles alkaloid compositions in reference substance solution and need testing solution, calculate the content obtaining 2 kinds of indoles alkaloid compositions in need testing solution.Concrete outcome is in table 10.
The comparision contents of 2 kinds of indoles alkaloid compositions in table 1010 batch dried venom of toads sample
As shown in Table 10,2 kinds of indoles alkaloid component content significant differences in dried venom of toads medicinal material, wherein, according to mensuration, the content range of serotonin is 3.55% ~ 5.08%; The content range of bufotenine is 0.32% ~ 0.39%.Further, in 10 batches of dried venom of toads medicinal materials in 2 kinds of indoles alkaloid compositions, the content mean value of serotonin is 4.476%; The content mean value of bufotenine is 0.364%, all drops within the scope of practical measurement, realistic mensuration situation.
In sum, the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material of the present invention, effective qualitative and quantitative analysis can be carried out to 2 kinds of main indoles alkaloid compositions in the dried venom of toads simultaneously, method is easy, feasible, accurate, can effectively control dried venom of toads quality, and provide reference for the clinical application of the dried venom of toads.So the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (5)
1. the detection method of indoles alkaloid composition in dried venom of toads medicinal material, comprises the following steps:
1) preparation of need testing solution: dried venom of toads fine powder is added ethanol water, after ultrasonic extraction, cools, shakes up; Centrifugal, Aspirate supernatant heating evaporate to dryness, adds dissolution with solvents and constant volume, filtering membrane, gets subsequent filtrate as need testing solution;
The described ethanol water added is 50% ethanol water, is the 100-800 times amount of dried venom of toads fine powder; Described ultrasonic extraction time is 20-60min;
2) preparation of reference substance solution: serotonin and bufotenine reference substance are added dissolution with solvents and constant volume, shakes up, obtains mixing reference substance solution;
Described step 1) or 2) in, described dissolving and constant volume time the solvent that uses be 20% ethanol water;
3) qualitative detection: adopt high performance liquid chromatography to measure need testing solution and reference substance solution respectively, by the liquid chromatogram of the need testing solution of acquisition, compare with the liquid chromatogram of reference substance solution, according to relative retention time, determine 2 kinds of indoles alkaloid compositions in need testing solution;
4) quantitatively detect: adopt high performance liquid chromatography to measure need testing solution and reference substance solution respectively, according to chromatographic peak area, adopt the content of 2 kinds of indoles alkaloid compositions in external standard method determination need testing solution;
The chromatographic condition of described high performance liquid chromatography is: chromatographic column: AlltimaC
18chromatographic column; Column temperature: 25-35 DEG C; Determined wavelength: 275nm; Flow velocity: 0.7-0.8ml/min; Sample size: 10 μ l; Mobile phase: acetonitrile-0.5% potassium dihydrogen phosphate buffer solution, phosphoric acid adjust buffer solution pH to 3.2, and wherein, A phase is acetonitrile, and B phase is 0.5% potassium dihydrogen phosphate aqueous solution; Analysis time: 25min; Isocratic elution;
Described isocratic elution at 0-25min, with mobile phase A phase: B phase volume ratio is that 5:95 carries out wash-out.
2. the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material according to claim 1, is characterized in that, described step 1) in, described in 50% ethanol that adds be 200 times amount of dried venom of toads fine powder; Described ultrasonic extraction time is 20min.
3. the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material according to claim 1, is characterized in that, described step 1) in, described centrifugation time is 20min; Described centrifugal rotational speed is 4000rpm.
4. the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material according to claim 1, is characterized in that, described step 1) in, after described dried venom of toads fine powder adds ethanol water, need precise weighing; After described ultrasonic extraction, cooling, need weigh again, and supply weightlessness with 50% ethanol water.
5. the detection method of indoles alkaloid composition in a kind of dried venom of toads medicinal material according to claim 1, it is characterized in that, described step 2) in, in described reference substance solution, the content range of each composition is: serotonin 120.0-1800.0 μ g/ml, bufotenine 14.4-216.0 μ g/ml.
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