CN103837618A - Method for controlling mass of semen cassiae polysaccharide by utilizing multi-dimensional fingerprint - Google Patents
Method for controlling mass of semen cassiae polysaccharide by utilizing multi-dimensional fingerprint Download PDFInfo
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Abstract
The invention belongs to the field of food and medicine analysis, and particularly relates to a method for controlling the mass of semen casiae polysaccharide by utilizing a multi-dimensional fingerprint. Semen cassiae is used as a raw amterial to establish a semen cassiae polysaccharide multi-dimensional fingerprint. The method comprises the steps of establishing the semen cassiae polysaccharide GC fingerprint, an HPLC (high performance liquid chromatography) fingerprint when the semen cassiae polysaccharide is completely hydrolyzed and an HPLC fingerprint when the semen cassiae polysaccharide is not completely hydrolyzed, and forming complete fingerprint characteristics of the semen cassiae polysaccharide collectively through the multi-dimensional fingerprints. The multi-dimensional fingerprint can be used for controlling the mass of the raw material. By adopting the method, more accurate and comprehensive mass control for the standard raw material can be realized.
Description
Technical field
The present invention relates to food, Pharmaceutical Analysis field, be specifically related to a kind of method of utilizing finger-print to control cassia seed polysaccharide quality.
Technical background
Cassia tora (Semen Cassiae) belongs to legume.Cassia seed is the dry mature seed of legume Cassia tora, the medicinal and edible plant that health ministry is promulgated.Begin to be loaded in " legendary god of farming's draft through ", cassia seed is listed in top grade, and its taste is sweet, bitter, is slightly cold, and enters liver, kidney channel, has step-down, lipopenicillinase, protects the liver, improving eyesight, ease constipation and the effect such as antibacterial.Have long use history at cassia seed among the people, or a drink of making tea, or after frying and other medicines compatibility (Yu Shouyang, Cui Hongbin. the progress [M] of National Health Foods. Beijing: People's Health Publisher, 2000.1).Cassia seed polysaccharide is one of principal ingredient having in cassia seed medical care effect, have free radical and the anti-oxidation function of removing (Liu Juan, Deng Zeyuan, Yu Huahong. the antioxidation [J] of Water-soluble Polysaccharide Isolated from Semen Cassia. Food Science, 2006,05:61-63; Guo Xiaoqiang, Yan Jun, Wu Xiaoyong, Xu Guang territory, Fan Chunhua, Gou little Jun. the purifying of Water-soluble Polysaccharide Isolated from Semen Cassia and antioxidation activity research [J]. Food Science, 2007,8:205-208).The cassia seed water extracting alcohol hypostasis or the water extract that are rich in polysaccharide have significant step-down (Liu Juxiu, Miao Rong, Di Junying, Hao Zhiqiang. the experimental study of cassia seed hypotensive effect. the Tianjin traditional Chinese medical science, 1990, 5:37-40), effect for reducing fat (Chen Weixing, Diao Guojun, Jiang Wenjuan, Liang Lian, the impact of cassia seed on high cholesterol disease mouse model. Chinese herbal medicine, 1991, 2:72-77) with adjusting immunity (Nan Jingyi, Wang Zhong, Shen Yuqin, Yang Zhengjuan etc. the experimental study of the impact of cassia seed on immune function of mice. Liaoning Journal of Traditional Chinese Medicine, 1989, the effect such as 5:43-44).
The quality of polysaccharide is the key point (R.Srivastava, D.K.Kulshreshtha, Bioactive polysaccharides from plants, Phytochemistry, 1989,28:2877-2883) that affects its effect activity.How with a kind of easy, fast, method is controlled the quality of cassia seed polysaccharide accurately, is a difficult problem urgently to be resolved hurrily.
Existing bibliographical information utilizes the quality of fingerprint pattern technology control polysaccharide, but the single finger-print of many employings, directly judge (CN102008515A according to the scope of retention time, a kind of construction method of lucidum spore powder finger-print and standard finger-print thereof), or aid in cluster analysis statistics means (CN102680607A, a kind of cocoa power detection of adulterations method based on finger-print) and carry out the quality judging of polysaccharide.Due to the polysaccharide for same cultivar origin, its higher structure has complicacy and monose constituent has relative stability, and the sensitivity of instrument detection method, therefore existing method has the shortcoming that is difficult to overcome, be difficult to judge whether unknown sample meets the finger-print of standard model, time and there is false positive results.The present invention adopts multi-Dimensional Fingerprint Chromatograms comprehensively to judge, aids in statistics means, synthesis result data, thus obtain reliable conclusion.
Summary of the invention
The present invention seeks to shortcoming and deficiency in order to overcome existing cassia seed polysaccharide Quality Control Technology, by the research of the multi-Dimensional Fingerprint Chromatograms construction method to cassia seed polysaccharide, provide a kind of method of utilizing multi-Dimensional Fingerprint Chromatograms effectively to control cassia seed polysaccharide quality.
For solving the existing problem that can not accurately determine polysaccharide contamination in cassia seed in prior art, the invention provides a kind of method of utilizing finger-print effectively to control cassia seed polysaccharide quality, it is characterized in that, described method comprises the steps:
The structure of described cassia seed polysaccharide GC standard finger-print is first by the aldoononitrile acetate of cassia seed polysaccharide standard items hydrolysate, adopts subsequently gc analysis need testing solution, finally obtains cassia seed polysaccharide GC finger-print;
The structure of the HPLC standard finger-print under described cassia seed polysaccharide complete hydrolysis is first cassia seed polysaccharide standard items to be carried out to complete hydrolysis, carry out subsequently 1-phenyl-3-methyl-5-pyrazolones ketone (the being PMP) derivative reaction of cassia seed polysaccharide standard items hydrolysate, carry out again the fingerprint analysis of high performance liquid chromatography, finally draw the HPLC standard finger-print under cassia seed polysaccharide complete hydrolysis according to the result obtaining;
The structure of the HPLC standard finger-print under described cassia seed polysaccharide is not exclusively hydrolyzed is first cassia seed polysaccharide standard items to be not exclusively hydrolyzed, carry out subsequently 1-phenyl-3-methyl-5-pyrazolones ketone (the being PMP) derivative reaction of cassia seed polysaccharide standard items hydrolysate, carry out again the fingerprint analysis of high performance liquid chromatography, finally draw the HPLC standard finger-print under the incomplete hydrolysis of cassia seed polysaccharide according to the result obtaining;
The structure of cassia seed polysaccharide GC standard finger-print of the present invention specifically comprises the steps:
(1) aldoononitrile acetate of cassia seed polysaccharide standard items hydrolysate: by the sample after trifluoroacetic acid hydrolysis evaporated under reduced pressure, add successively oxammonium hydrochloride and the 0.5ml anhydrous pyridine of 10mg, ultrasonic dissolution reacts 40min, after having reacted in 90 ℃ of water bath with thermostatic control oscillators, be cooled to room temperature, add the acetic anhydride of 0.6ml, ultrasonic mixing, reacts 40min in 90 ℃ of water bath with thermostatic control vibrations, obtain sugared nitrile acetyl derivatives, directly air inlet chromatography detects;
(2) adopt gc analysis need testing solution: chromatographic column is that (30m × 0.32mm × 0.25 μ m) for Agilent DB-5 capillary chromatographic column; The ratio of combustion gas, assisted gas and carrier gas is N2: H2: Air=40: 45: 400ml/min; Split ratio: 10: 1; Sample size: 2 μ L; Injector temperature: 220 ℃; Detector temperature: 260 ℃; Adopt temperature programme, its heating schedule is: 70 ℃ of initial temperatures, keep 8min; 30 ℃/min rises to 160 ℃, keeps 19min; 10 ℃/min rises to 170 ℃; Keep 29min; 10 ℃/min rises to 220 ℃, keeps 4min; Obtain with this understanding the chromatographic fingerprinting of sample polysaccharide, take No. 1 peak as reference, the relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates, finally obtains cassia seed polysaccharide GC finger-print.
By the gas chromatogram of polysaccharide hydrolysis product after aldoononitrile acetate in 10 batches of cassia seed samples compared, determine 7 common characteristic peaks, and chromatographic data is imported to finger-print special software and obtains the standard gas chromatograph finger-print of the cassia seed polysaccharide being formed by its common characteristic peak.Cassia seed polysaccharide GC standard finger-print of the present invention is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.042, RSD is 0.170%;
No. 3 average RT in peak are that 1.090, RSD is 0.511%;
No. 4 average RT in peak are that 1.199, RSD is 0.096%;
No. 5 average RT in peak are that 1.807, RSD is 0.559%;
No. 6 average RT in peak are that 1.841, RSD is 0.397%;
No. 7 average RT in peak are that 1.964, RSD is 0.505%.
Structure for the HPLC standard finger-print through complete hydrolysis or the incomplete cassia seed polysaccharide being hydrolyzed specifically comprises the steps:
(1) 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) the derivatization process of cassia seed polysaccharide hydrolysis solution: upper polysaccharide hydrolysis thing water is dissolved, then turn and hold to 10ml volumetric flask constant volume, good polysaccharide hydrolysis solution 0.2ml is placed in 5ml tool plug glass centrifuge tube accurately to measure constant volume, add successively 0.2ml, PMP methanol solution and the 0.2ml of 0.5mol/L, the NaOH solution of 0.3mol/L, ultrasonic mixing, 70 ℃ of water-bath 60min, be chilled to room temperature, add 0.2ml, the HCl solution neutralization of 0.3mol/L, in centrifuge tube, add chloroform to 2ml, high speed vortex 1min, extract remaining PMP, the centrifugal 5min of rotating speed of 4000rpm, remove lower floor's organic phase, retain supernatant, adopt 2mL chloroform re-extract 3 times, the last careful whole supernatants of sucking-off, thin up is settled to 5ml, cross the miillpore filter of 0.22 μ m, detect for HPLC, the PMP derivatization treatment method of mixed standard specimen product is identical with sample liquid, and the concentration of described mixed standard specimen product is 0.1mg/ml, (2) chromatographic condition: chromatographic column is C18 (250 × 4.60mm, 5 μ), mobile phase: 18% acetonitrile-82% phosphate buffer (0.05mol/mi, pH=6.74), flow rate of mobile phase is 0.8ml/min, column temperature is room temperature, detecting device is UV-detector, detection wavelength is 245nm, sample size: 20 μ L, take No. 1 peak (mannose) as reference, the relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates.
HPLC standard finger-print under cassia seed polysaccharide complete hydrolysis is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.607, RSD is 0.640%;
No. 3 average RT in peak are that 1.840, RSD is 1.243%;
No. 4 average RT in peak are that 2.093, RSD is 1.323%;
No. 5 average RT in peak are that 2.393, RSD is 1.839%;
No. 6 average RT in peak are that 2.500, RSD is 1.424%;
No. 7 average RT in peak are that 2.611, RSD is 1.752%.
HPLC standard finger-print under cassia seed polysaccharide is not exclusively hydrolyzed is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.058, RSD is 0.353%;
No. 3 average RT in peak are that 1.125, RSD is 0.506%;
No. 4 average RT in peak are that 1.503, RSD is 0.479%;
No. 5 average RT in peak are that 1.668, RSD is 0.841%;
No. 6 average RT in peak are that 2.015, RSD is 0.405%;
No. 7 average RT in peak are that 2.293, RSD is 0.528%;
No. 8 average RT in peak are that 2.365, RSD is 0.496%;
No. 9 average RT in peak are that 2.500, RSD is 0.545%.
The extraction of cassia seed polysaccharide involved in the present invention comprises the steps:
Take 1.0g and cross 40 object cassia seed samples, with sherwood oil (30-60 ℃) backflow degreasing 8h, by the 100ml that adds water of the cassia seed sample after degreaser drying, at 80 ℃ of water-baths magnetic agitation 3h, merging filtrate; Accurately take papain 0.01g, join in filtrate, be placed in 60 ℃ of water bath with thermostatic control oscillators, the 2h that slowly vibrates, is cooled to room temperature; In above-mentioned enzymolysis liquid, add the sevaga liquid of 50mL, quick oscillation 20min on constant temperature oscillator, the centrifugal 5min of rotating speed by mixing material with 4000rpm, after phase-splitting, get its supernatant, repeat again the process of above-mentioned " sevaga method is except albumen ", until upper and lower two alternate agalasisa white flocculent deposits, to eliminate the vegetable protein in extract; Then add the H of the massfraction 30% of 15% (V/V) toward extract
2o
2, be placed in 60 ℃ of water-baths and react 6h; Extract after decolouring is slowly splashed into absolute ethyl alcohol (by 4 times of its volume), be placed in rapid stirring on magnetic stirring apparatus simultaneously, room temperature leaves standstill 3 hours, and under 4000rpm rotating speed, centrifugal 10min gets precipitation; In gained precipitation, add 10ml acetone, ultrasonic 5min in ultrasonic cleaning machine, then with the centrifugal 5min of rotating speed of 4000rpm, repeat once; In residue after centrifugal, add 10ml absolute ether, supersound washing 5min, centrifugal, with the centrifugal 10min of rotating speed of 4000rpm, repeated washing once, dries up the residue of centrifugal rear gained with nitrogen, weighs, and sealing, in refrigerator, preserve, for subsequent use, make thick polysaccharide sample of the present invention.
Complete hydrolysis step involved in the present invention is: precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.5mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 3h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 50 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 4 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.The HPLC finger-print that the present invention builds under cassia seed polysaccharide GC finger-print and cassia seed polysaccharide complete hydrolysis is all the polysaccharide samples that adopt after this complete hydrolysis.
Incomplete hydrolysing step involved in the present invention is: precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.05mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 1h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 70 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 3 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.
In preceding method, the finger-print of the standard finger-print that obtains and unknown sample contrast, distinguish its similarities and differences.Total peak is carried out to area normalization, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates, then carry out principal component analysis (PCA).
The present invention has following advantage and effect with respect to prior art:
(1) the present invention sets up the fingerprint pattern technology standard of cassia seed polysaccharide, overall monitor material quality effectively, thereby the steady quality of guarantee product, even, controlled.
(2) the present invention adopts different determination methods, build three-dimensional finger-print java standard library, can monitor synergistically, complementally, all sidedly the quality of raw medicinal material, semi-manufacture, finished product, by the comparison of chromatographic fingerprint characteristic similarity, evaluate quality, stability and consistance, make up the deficiency of existing method of quality control or single finger-print.
(3), on basis of the present invention, can carry out the correlation research of finger-print information and effect activity, thereby illustrate the efficacy effect of intrinsic chemical composition.
Accompanying drawing explanation:
Cassia seed polysaccharide complete hydrolysis aldoononitrile acetate GC collection of illustrative plates in Fig. 1 embodiment 2;
Cassia seed polysaccharide complete hydrolysis PMP derivatization HPLC chromatogram in Fig. 2 embodiment 3;
In Fig. 3 embodiment 4, cassia seed polysaccharide is not exclusively hydrolyzed PMP derivatization HPLC chromatogram;
The principal component analysis (PCA) score two dimension scatter diagram of Fig. 4 cassia seed polysaccharide GC finger-print;
The principal component analysis (PCA) score two dimension scatter diagram of Fig. 5 cassia seed polysaccharide HPLC finger-print one;
The principal component analysis (PCA) score two dimension scatter diagram of Fig. 6 cassia seed polysaccharide HPLC finger-print two
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Take 1.0g and cross 40 object cassia seed samples, with sherwood oil (30-60 ℃) backflow degreasing 8h.By the 100ml that adds water of the cassia seed sample after degreaser drying, at 80 ℃ of water-baths magnetic agitation 3h, merging filtrate.Accurately take papain 0.01g, join in filtrate, be placed in 60 ℃ of water bath with thermostatic control oscillators, the 2h that slowly vibrates, is cooled to room temperature.In the sugared extract of above-mentioned enzymolysis, add the sevaga liquid of 50mL, quick oscillation 20min on constant temperature oscillator, the centrifugal 5min of rotating speed by mixing material with 4000rpm, after phase-splitting, get its supernatant, repeat again the process of above-mentioned " sevaga method is except albumen ", until upper and lower two alternate agalasisa white flocculent deposits, to eliminate the vegetable protein in extract.Then extract is added to the H of the massfraction 30% of 15% (V/V)
2o
2, be placed in 60 ℃ of water-baths and react 6h.Extract after decolouring is slowly splashed into absolute ethyl alcohol (by 4 times of its volume), be placed in rapid stirring on magnetic stirring apparatus simultaneously, room temperature leaves standstill 3 hours, and under 4000rpm rotating speed, centrifugal 10min gets precipitation.In gained precipitation, add 10ml acetone, ultrasonic 5min in ultrasonic cleaning machine, then with the centrifugal 5min of rotating speed of 4000rpm, repeat once.In residue after centrifugal, add 10ml absolute ether, supersound washing 5min, centrifugal, with the centrifugal 10min of rotating speed of 4000rpm, repeated washing once, dries up the residue of centrifugal rear gained with nitrogen, weighs, and sealing, preserves in refrigerator, for subsequent use.
The GC analytical approach of embodiment 2 cassia seed polysaccharide
10 batches of cassia seed raw materials are carried out to polysaccharide extraction (adopting the method for embodiment 1).
Cassia seed polysaccharide hydrolysis process: precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.5mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 3h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 50 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 4 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.
The aldoononitrile acetate of cassia seed polysaccharide sample water hydrolysis products; be the sample after trifluoroacetic acid hydrolysis evaporated under reduced pressure, add successively oxammonium hydrochloride and the 0.5ml anhydrous pyridine of 10mg, ultrasonic dissolution; in 90 ℃ of water bath with thermostatic control oscillators, react 40min; after having reacted, be cooled to room temperature, add the acetic anhydride of 0.6ml; ultrasonic mixing; in 90 ℃ of water bath with thermostatic control vibrations, react 40min, obtain sugared nitrile acetyl derivatives, directly air inlet chromatography detects.Adopt gc analysis need testing solution: chromatographic column is that (30m × 0.32mm × 0.25 μ m) for Agilent DB-5 capillary chromatographic column; The ratio of combustion gas, assisted gas and carrier gas is N
2: H
2: Air=40: 45: 400ml/min; Split ratio: 10: 1; Sample size: 2 μ L; Injector temperature: 220 ℃; Detector temperature: 260 ℃; Adopt temperature programme, its heating schedule is: 70 ℃ of initial temperatures, keep 8min; 30 ℃/min rises to 160 ℃, keeps 19min; 10 ℃/min rises to 170 ℃; Keep 29min; 10 ℃/min rises to 220 ℃, keeps 4min.Gather figure spectrum information at the 10th minute, obtain cassia seed polysaccharide complete hydrolysis aldoononitrile acetate GC chromatogram (Fig. 1).
The relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates.Obtain in cassia seed polysaccharide GC finger-print, total peak has 7, relative retention time RT (being reference take mannose peak), and deviation is less than 1%, and specific features is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.042, RSD is 0.170%;
No. 3 average RT in peak are that 1.090, RSD is 0.511%;
No. 4 average RT in peak are that 1.199, RSD is 0.096%;
No. 5 average RT in peak are that 1.807, RSD is 0.559%;
No. 6 average RT in peak are that 1.841, RSD is 0.397%;
No. 7 average RT in peak are that 1.964, RSD is 0.505%;
No. 1 peak, relative peak area 0.373～1.185%; No. 2 peaks, relative peak area 1.415～5.719%; No. 3 peaks, relative peak area 4.249～23.678%; No. 4 peaks, relative peak area 1.086～2.572%; No. 5 peaks, relative peak area 45.786～75.051%; No. 6 peaks, relative peak area 3.889～5.363%; No. 7 peaks, relative peak area 13.231～17.733%.
The HPLC analytical approach one of embodiment 3 cassia seed polysaccharide
Cassia seed polysaccharide extracts preparation: 10 batches of cassia seed raw materials are carried out to polysaccharide according to embodiment 1 and extract preparation.
Cassia seed polysaccharide complete hydrolysis process: 10 batches of cassia seed raw material polysaccharide are carried out to complete hydrolysis preparation according to embodiment 2.
1-phenyl-3-methyl-5-pyrazolones ketone (PMP) the derivatization process of cassia seed polysaccharide hydrolysis solution: upper polysaccharide hydrolysis thing water is dissolved, then turn and hold to 10ml volumetric flask constant volume, accurately measure 0.2ml polysaccharide hydrolysis solution and be placed in 5ml tool plug glass centrifuge tube, add successively 0.2ml, PMP methanol solution and the 0.2ml of 0.5mol/L, the NaOH solution of 0.3mol/L, ultrasonic mixing, 70 ℃ of water-bath 60min, be chilled to room temperature, add 0.2ml, the HCl solution neutralization of 0.3mol/L, in centrifuge tube, add chloroform to 2ml, high speed vortex 1min, extract remaining PMP, the centrifugal 5min of rotating speed of 4000rpm, remove lower floor's organic phase, retain supernatant, adopt 2mL chloroform re-extract 3 times, the last careful whole supernatants of sucking-off, thin up is settled to 5ml, cross the miillpore filter of 0.22 μ m, detect for HPLC.Chromatographic condition: chromatographic column is C18 (250 × 4.60mm, 5 μ); Mobile phase: 18% acetonitrile-82% phosphate buffer (0.05mol/ml; PH=6.74); Flow rate of mobile phase is 0.8ml/min; Column temperature is room temperature; Detecting device is UV-detector; Detection wavelength is 245nm; Sample size: 20 μ L.Gather figure spectrum information at the 19th minute, obtain cassia seed polysaccharide complete hydrolysis PMP derivatization method HPLC chromatogram (Fig. 2).
The relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates.Obtain in the HPLC finger-print of cassia seed polysaccharide complete hydrolysis, total peak has 7, relative retention time RT (being reference take mannose peak), and deviation is less than 1%, and specific features is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.607, RSD is 0.640%;
No. 3 average RT in peak are that 1.840, RSD is 1.243%;
No. 4 average RT in peak are that 2.093, RSD is 1.323%;
No. 5 average RT in peak are that 2.393, RSD is 1.839%;
No. 6 average RT in peak are that 2.500, RSD is 1.424%;
No. 7 average RT in peak are that 2.611, RSD is 1.752%.
No. 1 peak mannose, relative peak area 43.187～67.279%; No. 2 glucuronic acid peaks, relative peak area 1.394～2.469%; No. 3 amine-galactose peaks, relative peak area 1.882～2.748%; No. 4 glucose sugar peaks, relative peak area 2.483～10.125%; No. 5 galactose peaks, relative peak area 12.121～16.440%; No. 6 wood sugar peaks, relative peak area 4.436～26.411%; No. 7 arabinose peaks, relative peak area 1.742～6.544%.
The HPLC analytical approach two of embodiment 4 cassia seed polysaccharide
Cassia seed polysaccharide extracts preparation: 10 batches of cassia seed raw materials are carried out to polysaccharide according to embodiment 1 and extract preparation.
The incomplete hydrolytic process of cassia seed polysaccharide: precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.05mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 1h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 70 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 3 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.
1-phenyl-3-methyl-5-pyrazolones ketone (PMP) the derivatization process of the incomplete hydrolyzate of cassia seed polysaccharide: upper polysaccharide hydrolysis thing water is dissolved, then turn and hold to 10ml volumetric flask constant volume, accurately measure 0.2ml polysaccharide hydrolysis solution and be placed in 5ml tool plug glass centrifuge tube, add successively 0.2ml, PMP methanol solution and the 0.2ml of 0.5mol/L, the NaOH solution of 0.3mol/L, ultrasonic mixing, 70 ℃ of water-bath 60min, be chilled to room temperature, add 0.2ml, the HCl solution neutralization of 0.3mol/L, in centrifuge tube, add chloroform to 2ml, high speed vortex 1min, extract remaining PMP, the centrifugal 5min of rotating speed of 4000rpm, remove lower floor's organic phase, retain supernatant, adopt 2mL chloroform re-extract 3 times, the last careful whole supernatants of sucking-off, thin up is settled to 5ml, cross the miillpore filter of 0.22 μ m, detect for HPLC.Chromatographic condition: chromatographic column is Phenomenex Gemini C18 (250 × 4.60mm, 5 μ); Mobile phase: 18% acetonitrile-82% phosphate buffer (0.05mol/ml; PH=6.74); Flow rate of mobile phase is 0.8ml/min; Column temperature is room temperature; Detecting device is UV-detector; Detection wavelength is 245nm; Sample size: 20 μ L.Gathered figure spectrum information at the 19th minute, and obtain cassia seed polysaccharide and be not exclusively hydrolyzed PMP derivatization HPLC chromatogram (Fig. 3).
The relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates.Obtain in the HPLC finger-print of the incomplete hydrolysis of cassia seed polysaccharide, total peak has 7, relative retention time RT (take No. 1 peak as reference), and deviation is less than 1%, and specific features is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.058, RSD is 0.353%;
No. 3 average RT in peak are that 1.125, RSD is 0.506%;
No. 4 average RT in peak are that 1.503, RSD is 0.479%;
No. 5 average RT in peak are that 1.668, RSD is 0.841%;
No. 6 average RT in peak are that 2.015, RSD is 0.405%;
No. 7 average RT in peak are that 2.293, RSD is 0.528%;
No. 8 average RT in peak are that 2.365, RSD is 0.496%;
No. 9 average RT in peak are that 2.500, RSD is 0.545%.
No. 1 peak, relative peak area 24.225～58.653%; No. 2 peaks, relative peak area 1.438～6.213%; No. 3 peaks, relative peak area 0.251～1.621%; No. 4 peaks, relative peak area 1.642～5.476%; No. 5 peaks, relative peak area 1.650～5.818%; No. 6 peaks, relative peak area 2.324～3.652%; No. 7 peaks, relative peak area 11.580～16.885%; No. 8 peaks, relative peak area 9.009～33.197%; No. 9 peaks, relative peak area 4.830～11.443%.
The polysaccharide fingerprinting analysis of embodiment 5 place of production, Sichuan cassia seed samples (being called for short Sichuan cassia seed)
1. the preparation of polysaccharide: carry out according to embodiment 1.
2. the GC chromatogram of polysaccharide: according to embodiment 2, the polysaccharide of Sichuan cassia seed is carried out to complete hydrolysis, derivatization and GC instrumental analysis.
3. the HPLC chromatogram one of polysaccharide: according to embodiment 3, the polysaccharide of Sichuan cassia seed is carried out to complete hydrolysis, PMP derivatization and HPLC instrumental analysis.
4. the HPLC chromatogram two of polysaccharide: according to embodiment 4, by the polysaccharide of Sichuan cassia seed be not exclusively hydrolyzed, PMP derivatization and HPLC instrumental analysis.
The polysaccharide fingerprinting analysis of embodiment 6 place of production, Anhui cassia seed samples (being called for short Anhui cassia seed)
1. the preparation of polysaccharide: carry out according to embodiment 1.
2. the GC chromatogram of polysaccharide: according to embodiment 2, Anhui cassia seed polysaccharide is carried out to complete hydrolysis, derivatization and GC instrumental analysis.
3. the HPLC chromatogram one of polysaccharide: according to embodiment 3, Anhui cassia seed polysaccharide is carried out to complete hydrolysis, PMP derivatization and HPLC instrumental analysis.
4. the HPLC chromatogram two of polysaccharide: according to embodiment 4, by Anhui cassia seed polysaccharide be not exclusively hydrolyzed, PMP derivatization and HPLC instrumental analysis.
The principal component analysis (PCA) of embodiment 7 Sichuan cassia seeds and Anhui cassia seed sample and standard cassia seed polysaccharide fingerprinting data
The chromatogram obtaining from unknown sample and standard model proposes the chromatographic data at total peak, carries out principal component analysis (PCA).Principal component analysis (PCA) is a kind of method of mathematic(al) manipulation, and it changes into another one group of given correlated variables by linear transformation and organizes incoherent variable, and the order that these new variablees successively decrease successively according to variance is arranged.Select dimensionality reduction (data reduction) to carry out factor analysis herein, obtain two and be greater than 1 eigenwert, contribution rate of accumulative total is greater than 80%.Can obtain the corresponding coefficient of each index in major component by the data in major component loading matrix divided by the corresponding eigenwert sqrt of major component, the data after this coefficient and standardization are multiplied each other, just can obtain major component expression formula:
(1) principal component analysis (PCA) of Sichuan cassia seed, Anhui cassia seed and standard cassia seed polysaccharide GC finger-print data
Major component 1:F1=-0.381ZX
1-0.396ZX
2+ 0.284ZX
3+ 0.43ZX
4+ 0.424ZX
5-0.394ZX
6+ 0.311ZX
7
Major component 2:F1=0.43ZX
1+ 0.405ZX
2+ 0.399ZX
3+ 0.145ZX
4+ 0.221ZX
5+ 0.31ZX
6+ 0.571ZX
7
With major component 1 and 2 mappings, obtain the two-dimentional scatter diagram 4 of Sichuan cassia seed and the principal component analysis (PCA) of standard cassia seed polysaccharide.Can find out intuitively the distance between standard cassia seed sample from Fig. 4, part batch between distance differ far away.Under this analysis condition, Sichuan cassia seed and standard cassia seed are distinguished not obvious, and Anhui cassia seed can obviously be distinguished with other.
(2) principal component analysis (PCA) of Sichuan cassia seed, Anhui cassia seed and standard cassia seed polysaccharide HPLC finger-print one data
Major component 1:F1=-0.52ZX
1+ 0.365ZX
2+ 0.26ZX
3-0.399ZX
4+ 0.232ZX
5+ 0.225ZX
6+ 0.514ZX
7
Major component 2:F2=0.11ZX
1-0.256ZX
2+ 0.579ZX
3+ 0.245ZX
4+ 0.58ZX
5-0.422ZX
6+ 0.112ZX
7
With major component 1 and 2 mappings, obtain the two-dimentional scatter diagram 5 of Sichuan cassia seed and the principal component analysis (PCA) of standard cassia seed polysaccharide.Can find out intuitively that from Fig. 5 the distance difference between standard cassia seed sample is little, belong to a classification together.Under this analysis condition, Sichuan cassia seed and Anhui cassia seed and standard cassia seed are distinguished obviously, are respectively other two classifications.
(3) principal component analysis (PCA) of Sichuan cassia seed, Anhui cassia seed and standard cassia seed polysaccharide HPLC finger-print two data
Major component 1:F1=-0.185ZX
1+ 0.436ZX
2+ 0.398ZX
3+ 0.423ZX
4-0.309ZX
5-0.077ZX
6-0.039ZX
7+ 0.447ZX
8+ 0.369ZX
9
Major component 2:F2=0.536ZX
1-0.043ZX
2-0.104ZX
3+ 0.227ZX
4+ 0.273ZX
5+ 0.253ZX
6+ 0.618ZX
7+ 0.164ZX
8+ 0.319ZX
9
With major component 1 and 2 mappings, obtain the two-dimentional scatter diagram 6 of Sichuan cassia seed and the principal component analysis (PCA) of standard cassia seed polysaccharide.Can find out intuitively that from Fig. 6 distance difference between standard cassia seed sample is for belonging to a classification together.Under this analysis condition, Sichuan cassia seed and Anhui cassia seed and standard cassia seed are distinguished obviously, are respectively other two classifications.
From above result, by comparing with GC finger-print, the HPLC finger-print one and two of standard cassia seed polysaccharide, Sichuan cassia seed, Anhui cassia seed can be able to obvious differentiation, meet the quality control condition of cassia seed polysaccharide.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (9)
1. utilize finger-print effectively to control a method for cassia seed polysaccharide quality, it is characterized in that, described method comprises the steps:
Step 1, structure cassia seed standard items polysaccharide standard finger-print: selection standard cassia seed raw material, totally 10 batches, build respectively the HPLC standard finger-print under HPLC standard finger-print and the incomplete hydrolysis of cassia seed polysaccharide under cassia seed polysaccharide GC standard finger-print, cassia seed polysaccharide complete hydrolysis, build three-dimensional standard finger-print;
The structure of described cassia seed polysaccharide GC standard finger-print is first by the aldoononitrile acetate of cassia seed polysaccharide standard items hydrolysate, adopts subsequently gc analysis need testing solution, finally obtains cassia seed polysaccharide GC finger-print;
The structure of the HPLC standard finger-print under described cassia seed polysaccharide complete hydrolysis is first cassia seed polysaccharide standard items to be carried out to complete hydrolysis, carry out subsequently 1-phenyl-3-methyl-5-pyrazolones ketone (the being PMP) derivative reaction of cassia seed polysaccharide standard items hydrolysate, carry out again the fingerprint analysis of high performance liquid chromatography, finally draw the HPLC standard finger-print under cassia seed polysaccharide complete hydrolysis according to the result obtaining;
The structure of the HPLC standard finger-print under described cassia seed polysaccharide is not exclusively hydrolyzed is first cassia seed polysaccharide standard items to be not exclusively hydrolyzed, carry out subsequently 1-phenyl-3-methyl-5-pyrazolones ketone (the being PMP) derivative reaction of cassia seed polysaccharide standard items hydrolysate, carry out again the fingerprint analysis of high performance liquid chromatography, finally draw the HPLC standard finger-print under the incomplete hydrolysis of cassia seed polysaccharide according to the result obtaining;
Step 2, adopt definite method of cassia seed standard items polysaccharide standard finger-print in above-mentioned steps one, draw the not exclusively HPLC finger-print under hydrolysis of HPLC finger-print under cassia seed polysaccharide GC finger-print, the cassia seed polysaccharide complete hydrolysis of actual sample presentation cassia seed and cassia seed polysaccharide;
Step 3, by step 2 obtain result respectively with step 1 in three-dimensional standard finger-print carry out similarity evaluation, when similarity is all greater than more than 90%, show that the cassia seed polysaccharide in described actual sample presentation is qualified.
2. method according to claim 1, is characterized in that, the structure of described cassia seed polysaccharide GC standard finger-print specifically comprises the steps:
(1) aldoononitrile acetate of cassia seed polysaccharide standard items hydrolysate: by the sample after trifluoroacetic acid hydrolysis evaporated under reduced pressure, add successively oxammonium hydrochloride and the 0.5ml anhydrous pyridine of 10mg, ultrasonic dissolution reacts 40min, after having reacted in 90 ℃ of water bath with thermostatic control oscillators, be cooled to room temperature, add the acetic anhydride of 0.6ml, ultrasonic mixing, reacts 40min in 90 ℃ of water bath with thermostatic control vibrations, obtain sugared nitrile acetyl derivatives, directly air inlet chromatography detects;
(2) adopt gc analysis need testing solution: chromatographic column is that (30m × 0.32mm × 0.25 μ m) for Agilent DB-5 capillary chromatographic column; The ratio of combustion gas, assisted gas and carrier gas is N2: H2: Air=40: 45: 400ml/min; Split ratio: 10: 1; Sample size: 2 μ L; Injector temperature: 220 ℃; Detector temperature: 260 ℃; Adopt temperature programme, its heating schedule is: 70 ℃ of initial temperatures, keep 8min; 30 ℃/min rises to 160 ℃, keeps 19min; 10 ℃/min rises to 170 ℃; Keep 29min; 10 ℃/min rises to 220 ℃, keeps 4min; Obtain with this understanding the chromatographic fingerprinting of sample polysaccharide, take No. 1 peak (mannose) as reference, the relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates, finally obtains cassia seed polysaccharide GC standard finger-print.
3. according to the method for claim 2, it is characterized in that, described cassia seed polysaccharide GC standard finger-print is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.042, RSD is 0.170%;
No. 3 average RT in peak are that 1.090, RSD is 0.511%;
No. 4 average RT in peak are that 1.199, RSD is 0.096%;
No. 5 average RT in peak are that 1.807, RSD is 0.559%;
No. 6 average RT in peak are that 1.841, RSD is 0.397%;
No. 7 average RT in peak are that 1.964, RSD is 0.505%.
4. according to the method for claim 1, it is characterized in that, the structure of the HPLC standard finger-print of the described cassia seed polysaccharide through complete hydrolysis or incomplete hydrolysis specifically comprises the steps:
(1) 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) the derivatization process of cassia seed polysaccharide hydrolysis solution: polysaccharide hydrolysis thing water is dissolved, then turn and hold to 10ml volumetric flask constant volume, good polysaccharide hydrolysis solution 0.2ml is placed in 5ml tool plug glass centrifuge tube accurately to measure constant volume, add successively 0.2ml, PMP methanol solution and the 0.2ml of 0.5mol/L, the NaOH solution of 0.3mol/L, ultrasonic mixing, 70 ℃ of water-bath 60min, be chilled to room temperature, add 0.2ml, the HCl solution neutralization of 0.3mol/L, in centrifuge tube, add chloroform to 2ml, high speed vortex 1min, extract remaining PMP, the centrifugal 5min of rotating speed of 4000rpm, remove lower floor's organic phase, retain supernatant, adopt 2mL chloroform re-extract 3 times, the last careful whole supernatants of sucking-off, thin up is settled to 5ml, cross the miillpore filter of 0.22 μ m, detect for HPLC, the PMP derivatization treatment method of mixed standard specimen product is identical with sample liquid, and the concentration of described mixed standard specimen product is 0.1mg/ml, (2) chromatographic condition: chromatographic column is C18 (250 × 4.60mm, 5 μ), mobile phase: 18% acetonitrile-82% phosphate buffer (0.05mol/ml, pH=6.74), flow rate of mobile phase is 0.8ml/min, column temperature is room temperature, detecting device is UV-detector, detection wavelength is 245nm, sample size: 20 μ L, take No. 1 peak (mannose) as reference, the relative retention time at each peak in calculation sample, with the relative area at the each peak of areas of peak normalization method calculation sample collection of illustrative plates.
5. method according to claim 4, is characterized in that, the HPLC standard finger-print under cassia seed polysaccharide complete hydrolysis is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.607, RSD is 0.640%;
No. 3 average RT in peak are that 1.840, RSD is 1.243%;
No. 4 average RT in peak are that 2.093, RSD is 1.323%;
No. 5 average RT in peak are that 2.393, RSD is 1.839%;
No. 6 average RT in peak are that 2.500, RSD is 1.424%;
No. 7 average RT in peak are that 2.611, RSD is 1.752%.
6. method according to claim 4, is characterized in that, the HPLC standard finger-print under cassia seed polysaccharide is not exclusively hydrolyzed is as follows:
No. 1 average RT in peak is that 1, RSD is 0%;
No. 2 average RT in peak are that 1.058, RSD is 0.353%;
No. 3 average RT in peak are that 1.125, RSD is 0.506%;
No. 4 average RT in peak are that 1.503, RSD is 0.479%;
No. 5 average RT in peak are that 1.668, RSD is 0.841%;
No. 6 average RT in peak are that 2.015, RSD is 0.405%;
No. 7 average RT in peak are that 2.293, RSD is 0.528%;
No. 8 average RT in peak are that 2.365, RSD is 0.496%;
No. 9 average RT in peak are that 2.500, RSD is 0.545%.
7. method according to claim 1, is characterized in that, the extraction of described cassia seed polysaccharide comprises the steps:
Take 1.0g and cross 40 object cassia seed samples, with sherwood oil (30-60 ℃) backflow degreasing 8h, by the 100ml that adds water of the cassia seed sample after degreaser drying, at 80 ℃ of water-baths magnetic agitation 3h, merging filtrate; Accurately take papain 0.01g, join in filtrate, be placed in 60 ℃ of water bath with thermostatic control oscillators, the 2h that slowly vibrates, is cooled to room temperature; In above-mentioned enzymolysis liquid, add the sevaga liquid of 50mL, quick oscillation 20min on constant temperature oscillator, the centrifugal 5min of rotating speed by mixing material with 4000rpm, after phase-splitting, get its supernatant, repeat again the process of above-mentioned " sevaga method is except albumen ", until upper and lower two alternate agalasisa white flocculent deposits, to eliminate the vegetable protein in extract; Then add the H of the massfraction 30% of 15% (V/V) toward extract
2o
2, be placed in 60 ℃ of water-baths and react 6h; Extract after decolouring is slowly splashed into absolute ethyl alcohol (by 4 times of its volume), be placed in rapid stirring on magnetic stirring apparatus simultaneously, room temperature leaves standstill 3 hours, and under 4000rpm rotating speed, centrifugal 10min gets precipitation; In gained precipitation, add 10ml acetone, ultrasonic 5min in ultrasonic cleaning machine, then with the centrifugal 5min of rotating speed of 4000rpm, repeat once; In residue after centrifugal, add 10ml absolute ether, supersound washing 5min, centrifugal, with the centrifugal 10min of rotating speed of 4000rpm, repeated washing once, dries up the residue of centrifugal rear gained with nitrogen, weighs, and sealing, in refrigerator, preserve, for subsequent use, make thick polysaccharide sample of the present invention.
8. method according to claim 1, is characterized in that, described complete hydrolysis step is:
Precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.5mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 3h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 50 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 4 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.The HPLC finger-print that the present invention builds under cassia seed polysaccharide GC finger-print and cassia seed polysaccharide complete hydrolysis is all the polysaccharide samples that adopt after this complete hydrolysis.
9. method according to claim 1, is characterized in that, described incomplete hydrolysing step is:
Precision takes thick polysaccharide sample 10mg, be placed in 5mL peace and cut open bottle, add 2.0ml, the TFA solution of 0.05mol/L, seals with alcohol blast burner, be placed in 110 ℃ of hydrolysis 1h, after having reacted, sample is taken out, be cooled to room temperature, adopt methyl alcohol rinse to be transferred in the centrifuge tube of 5mL, the centrifugal 5min of 4000rpm; Get supernatant, turn molten to round-bottomed flask, 70 ℃ of decompression rotary evaporated to dryness, use methyl alcohol ultrasonic dissolution, and 50 ℃ are spin-dried for, and re-treatment 3 times to be to remove residual TFA, and the sample after evaporated under reduced pressure is for subsequent use.
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| CN113109489A (en) * | 2021-05-21 | 2021-07-13 | 夏永刚 | Method for simultaneously qualitatively and quantitatively analyzing aldose, ketose, sugar alcohol, sugar acid and aminosugar in traditional Chinese medicine polysaccharide |
| CN113281432A (en) * | 2021-05-19 | 2021-08-20 | 劲牌持正堂药业有限公司 | Method for identifying semen cassiae extract by using HPLC fingerprint |
| CN115406985A (en) * | 2022-08-17 | 2022-11-29 | 南京师范大学 | Method for characterizing polysaccharide structure information and method for identifying authenticity or quality of traditional Chinese medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113281432A (en) * | 2021-05-19 | 2021-08-20 | 劲牌持正堂药业有限公司 | Method for identifying semen cassiae extract by using HPLC fingerprint |
| CN113109489A (en) * | 2021-05-21 | 2021-07-13 | 夏永刚 | Method for simultaneously qualitatively and quantitatively analyzing aldose, ketose, sugar alcohol, sugar acid and aminosugar in traditional Chinese medicine polysaccharide |
| CN113109489B (en) * | 2021-05-21 | 2023-05-23 | 夏永刚 | Analysis method of traditional Chinese medicine polysaccharide aldose, ketose, sugar alcohol, uronic acid and amino sugar |
| CN115406985A (en) * | 2022-08-17 | 2022-11-29 | 南京师范大学 | Method for characterizing polysaccharide structure information and method for identifying authenticity or quality of traditional Chinese medicine |
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