CN104155371B - The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof - Google Patents
The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof Download PDFInfo
- Publication number
- CN104155371B CN104155371B CN201310179700.7A CN201310179700A CN104155371B CN 104155371 B CN104155371 B CN 104155371B CN 201310179700 A CN201310179700 A CN 201310179700A CN 104155371 B CN104155371 B CN 104155371B
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- volume
- percent
- diabetes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of method for building up and HPLC finger-print thereof for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print, described method for building up comprises the following steps: the preparation of (1), reference substance solution; (2), the preparation of test sample; (3), the foundation of high effective liquid chromatography for measuring condition and mensuration.The method for building up specificity of above-mentioned diabetes pill HPLC fingerprint chromatogram is strong, easy and simple to handle, have excellent stability, precision and reappearance, multiple principle active component in diabetes pill can be detected more all sidedly, thus build scientific and reasonable diabetes pill HPLC finger-print, evaluate the security of diabetes pill, validity, stability and mass uniformity complete and accurate.
Description
Technical field
The present invention relates to a kind of quality determining method for the treatment of the compound Chinese medicinal preparation diabetes pill of diabetes, particularly relate to foundation and the HPLC finger-print thereof of diabetes pill HPLC finger-print.
Background technology
The Chinese and Western medicine compound preparation that the compound Chinese medicinal preparation diabetes pill for the treatment of diabetes is Chinese herbal medicine astragalus, glutinous rehmannia, the root of kudzu vine, Chinese yam, corn stigma, kadsura longepedunculata, root of Chinese trichosanthes and Western medicine glibenclamide form, there is effect of nourishing kidney-YIN, nourishing generate fluid, be used for clinically treating the diabete (diabetes B) caused by deficiency of both qi and yin, now record in the version Pharmacopoeia of the People's Republic of China in 2010.As the hypoglycemic medicine diabetes pill of Chinese and Western medicine compound, both played the entirety conditioning of traditional Chinese medicine, and prevented and treated the advantage of complication, overcome again the shortcoming that chemical drugs long-term taking easily produces drug resistance and spinoff.Add up so far, diabetes pill sales volume is accumulative more than 5,000,000,000 yuan, occupies the 3rd, after only coming Acarbose and Diamicron at treatment diabetes medicine meta.
Fall apart in Yuquan of Diabetes decoction and Qing Dynasty well-known doctor YE Tian shi that the Chinese prescription of diabetes pill derives from Yuan Dynasty well-known doctor ZHU Dan-xi, the complex chemical composition of its contained seven taste Chinese medicines, wherein the principal ingredient of the Radix Astragali is flavones and saponins compound (Wen Yanmei, Chinese patent drug, 2006,28 (6): 879-893); The principal ingredient of glutinous rehmannia is iridoid glycoside (Zeng Yan etc., Chinese patent drug, 2006,28 (4): 609-611); The principal ingredient of the root of kudzu vine comprises isoflavones, triterpene and saponins compound (Yin Lihong etc., Heilungkiang medicine, 2010,23 (3): 371-372) thereof; In Chinese yam principal ingredient have polysaccharide, fatty acid etc. (Zhao Hong etc., today pharmacy, 2009,19 (3): 49-52); Corn stigma contains flavones and glycoside, volatile ingredient, carbohydrate etc. (Zhu Xu etc., Changchun University of Traditional Chinese Medicine's journal, 2009,25 (2): 183-184); In kadsura longepedunculata with lignanoid and volatile oil for principal ingredient (grandson KUNG KUNG etc., ShanXi Chinese Medicine Academy journal, 2007,30 (4): 74-75); Then containing triterpene and the composition such as saponin(e and polysaccharide (Li Zhenhong etc., external Medicinal Plants medicine fascicle, 2003,18 (1): 1-4) thereof in root of Chinese trichosanthes.But at present its perfect square chemical composition is studied.In " pharmacopeia ", the quality control of diabetes pill is comprised to the microscopical characters of the Radix Astragali, kadsura longepedunculata, Chinese yam; Thin-layered chromatography is to the Qualitative Identification of Puerarin, Astragaloside IV, schizandrin A; High performance liquid chromatography (High Performance Liquid Chromatography, HPLC) is to the assay of Puerarin and glibenclamide.Due to these methods can not system, intactly reflect the inherent quality of diabetes pill therefore in the quality control of diabetes pill, to there is certain limitation.
Along with day by day showing especially of high-efficient liquid phase chromatogram technology advantage, HPLC fingerprint spectrum method, particularly multi-target ingredient HPLC fingerprint pattern technology start to be widely used in traditional Chinese medicine quality control field.The principle active component of the clear and definite Chinese medicine of this combine with technique extraction and separation technology, method of spectroscopy and high-efficient liquid phase chromatogram technology, determine the index components of finger-print key monitoring, both from the global feature macroscopically showing Chinese medicine, monitor its multiple index components from microcosmic again, make the quality standard of Chinese medicine preparation more perfect.
Summary of the invention
Based on this, be necessary to provide a kind of method for building up and the HPLC finger-print thereof that can reflect the treatment Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print of diabetes pill inherent quality comprehensively.
Treat a method for building up for Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print, comprise the following steps:
(1), the preparation of reference substance solution
Precision takes 3 '-hydroxyl Puerarin, Puerarin, 3 '-methoxy puerarin, daidzein-8-C-celery sugar (1 → 6) glucose, daidzin, Dai, Schisantherin C, schizandrin A, add methyl alcohol dissolving respectively and be respectively diluted to the solution that concentration is 0.01 ~ 0.1mg/mL, filter, to obtain final product;
(2), the preparation of test sample
Accurately take diabetes pill fine powder, add 85-95% ethanol, add hot reflux, be chilled to room temperature, filter, concentrated, then dissolve and constant volume with 40-60% methyl alcohol, shake up, filter, to obtain final product;
(3), high effective liquid chromatography for measuring
The test sample of step (2) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: octadecylsilane chemically bonded silica post;
Column temperature: 20-50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, the formic acid-aqueous solution of Mobile phase B to be formic acid percent by volume be 0.01%-0.1%, gradient elution;
Determined wavelength: 235-280nm.
Wherein in an embodiment, the gradient elution method described in step (3) is: elution program carries out with following volumetric concentration configuration:
When 0 minute, the percent by volume of mobile phase A is 2%, Mobile phase B percent by volume is 98%;
When 10 minutes, the percent by volume of mobile phase A is 5%, the percent by volume of Mobile phase B is 95%;
When 20 minutes, the percent by volume of mobile phase A is 22%, the percent by volume of Mobile phase B is 78%;
When 25 minutes, the percent by volume of mobile phase A is 25%, the percent by volume of Mobile phase B is 75%;
When 35 minutes, the percent by volume of mobile phase A is 30%, the percent by volume of Mobile phase B is 70%;
When 60 minutes, the percent by volume of mobile phase A is 50%, the percent by volume of Mobile phase B is 50%;
When 65 minutes, the percent by volume of mobile phase A is 65%, the percent by volume of Mobile phase B is 35%;
When 70 minutes, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%;
When 80 minutes, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%.
Wherein in an embodiment, the chromatographic condition in step (3) also comprises:
Flow velocity: 0.8-1.2mL/min;
Sample size: 2-20 μ L.
Wherein in an embodiment, described in step (3), flow velocity is 1mL/min, and described sample size is 10 μ L.
Wherein in an embodiment, chromatographic column described in step (3) is Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm, or Agilent ZORBAX SB-Aq post, and specification is 4.6 × 250mm, 5 μm.。
Wherein in an embodiment, described in step (3), column temperature is 30 DEG C.
Wherein in an embodiment, Mobile phase B described in step (3) is formic acid percent by volume is 0.02% formic acid-aqueous solution.
Have 12 features to have peak in the HPLC fingerprint chromatogram that above-mentioned diabetes pill HPLC fingerprint obtains, wherein No. 10 peaks are Dai reference peak, and highest peak is No. 4 peaks, and collection of illustrative plates total length is 78 minutes, specific as follows:
No. 1 peak, retention time is 13.837 minutes, and relative retention time is 0.2184; Peak area is 581.0910, and relative peak area is 0.2566;
No. 2 peaks, retention time is 17.823 minutes, and relative retention time is 0.2813; Peak area is 444.5910, and relative peak area is 0.2000;
No. 3 peaks are 3 '-hydroxyl Puerarin, and retention time is 29.488 minutes, and relative retention time is 0.4654; Peak area is 986.5500, and relative peak area is 0.4252;
No. 4 peaks are Puerarin, and retention time is 35.372 minutes, and relative retention time is 0.5583; Peak area is 5942.3160, and relative peak area is 2.5649;
No. 5 peaks are 3 '-methoxy puerarin, and retention time is 36.998 minutes, and relative retention time is 0.5839; Peak area is 1474.1900, and relative peak area is 0.6362;
No. 6 peaks are daidzein-8-C-celery sugar (1 → 6) glucose, and retention time is 39.204 minutes, and relative retention time is 0.6187; Peak area is 842.2190, and relative peak area is 0.3641;
No. 7 peaks are daidzin, and retention time is 40.906 minutes, and relative retention time is 0.6456; Peak area is 1090.1780, and relative peak area is 0.4715;
No. 8 peaks, retention time is 54.680 minutes, and relative retention time is 0.8630; Peak area is 301.5630, and relative peak area is 0.1309;
No. 9 peaks, retention time is 57.767 minutes, and relative retention time is 0.9117; Peak area is 1085.8930, and relative peak area is 0.4613;
No. 10 peaks are Dai, and retention time is 63.362 minutes, and relative retention time is 1.0000; Peak area is 2340.8880, and relative peak area is 1.0000;
No. 11 peaks are Schisantherin C, and retention time is 73.174 minutes, and relative retention time is 1.1549; Peak area is 273.2360, and relative peak area is 0.1188;
No. 12 peaks are schizandrin A, and retention time is 74.116 minutes, and relative retention time is 1.1697, and peak area is 161.8080, and relative peak area is 0.0704.
The present invention is carrying out cycle chemistry composition Study to diabetes pill perfect square, and is identified by high performance liquid chromatography on the basis of the main chromatographic peak structure of diabetes pill, provides a kind of construction method pointing out the HPLC finger-print of diabetes pill multi-target ingredient.Described HPLC fingerprint specificity is strong, easy and simple to handle, have excellent stability, precision and reappearance, multiple principle active component in diabetes pill can be detected more all sidedly, thus build scientific and reasonable diabetes pill HPLC finger-print, and then monitor the quality of diabetes pill more comprehensively exactly, complete, to evaluate diabetes pill exactly security, validity, stability and mass uniformity.
Accompanying drawing explanation
Fig. 1 is the separation process figure of diabetes pill main chemical compositions in embodiment 1;
Fig. 2 is the ESI-MS figure of 3 '-hydroxyl Puerarin of embodiment 1;
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of 3 '-hydroxyl Puerarin of embodiment 1;
Fig. 4 is DEPT figure and the carbon-13 nmr spectra figure of 3 '-hydroxyl Puerarin of embodiment 1;
Fig. 5 is the ESI-MS figure of the Puerarin of embodiment 1;
Fig. 6 is the hydrogen nuclear magnetic resonance spectrogram of the Puerarin of embodiment 1;
Fig. 7 is DEPT figure and the carbon-13 nmr spectra figure of the Puerarin of embodiment 1;
Fig. 8 is the ESI-MS figure of 3 '-methoxy puerarin of embodiment 1;
Fig. 9 is the hydrogen nuclear magnetic resonance spectrogram of 3 '-methoxy puerarin of embodiment 1;
Figure 10 is DEPT figure and the carbon-13 nmr spectra figure of 3 '-methoxy puerarin of embodiment 1;
Figure 11 is the ESI-MS figure of daidzein-8-C-celery sugar (1 → 6) glucose of embodiment 1;
Figure 12 is the hydrogen nuclear magnetic resonance spectrogram of daidzein-8-C-celery sugar (1 → 6) glucose of embodiment 1;
Figure 13 is DEPT figure and the carbon-13 nmr spectra figure of daidzein-8-C-celery sugar (1 → 6) glucose of embodiment 1;
Figure 14 is the ESI-MS figure of the daidzin of embodiment 1;
Figure 15 is the hydrogen nuclear magnetic resonance spectrogram of the daidzin of embodiment 1;
Figure 16 is DEPT figure and the carbon-13 nmr spectra figure of the daidzin of embodiment 1;
Figure 17 is the ESI-MS figure of the Dai of embodiment 1;
Figure 18 is the hydrogen nuclear magnetic resonance spectrogram of the Dai of embodiment 1;
Figure 19 is DEPT figure and the carbon-13 nmr spectra figure of the Dai of embodiment 1;
Figure 20 is the ESI-MS figure of the Schisantherin C of embodiment 1;
Figure 21 is the hydrogen nuclear magnetic resonance spectrogram of the Schisantherin C of embodiment 1;
Figure 22 is DEPT figure and the carbon-13 nmr spectra figure of the Schisantherin C of embodiment 1;
Figure 23 is the ESI-MS figure of the schizandrin A of embodiment 1;
Figure 24 is the hydrogen nuclear magnetic resonance spectrogram of the schizandrin A of embodiment 1;
Figure 25 is DEPT figure and the carbon-13 nmr spectra figure of the schizandrin A of embodiment 1;
Figure 26 is the HPLC chromatogram of 20 batches of diabetes pills of embodiment 2;
Figure 27 is the diabetes pill HPLC standard finger-print of embodiment 2;
Figure 28 is the chromatogram that the reference substance of embodiment 2 and the main chromatographic peak of diabetes pill test sample solution contrast;
Figure 29 is that in the diabetes pill HPLC standard finger-print of embodiment 2, main chromatographic peak mates figure with the uv absorption of its structure;
Figure 30 is the diabetes pill HPLC finger-print that can chemical point out of embodiment 2.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated.
Test sample is originated: Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.;
Instrument: Bruker AV-400 type nuclear magnetic resonance analyser (Bruker company of the U.S.); Finnigan LCQAdvantage MAX type mass spectrometer (Thermo Finnigan company); Agilent1200 analytic type high performance liquid chromatograph (Agilent company of the U.S.); Agilent1260 preparative high performance liquid chromatography instrument (Agilent company of the U.S.);
Reagent used in chromatographic determination: methyl alcohol is chromatographically pure, and water is ultrapure water, it is pure that all the other reagent are analysis.
Embodiment 1 treats the separation of the compound Chinese medicinal preparation diabetes pill main chemical compositions of diabetes, purifying and Structural Identification
Before diabetes pill pill, medicinal powder adds the ethanol that mass percent concentration is 75%, ultrasonic extraction twice, adds 75% ethanol of pill prodrug powder 10 times of weight for the first time, second time adds 8 times amount 75% ethanol, each ultrasonic extraction 30min, gets supernatant, is concentrated into density and is about 1.10g/cm
3, obtain diabetes pill alcohol extract.Diabetes pill alcohol extract is added suitable quantity of water suspendible, is extracted with ethyl acetate, after recycling design, obtain acetic acid ethyl ester extract 400g.
As shown in Figure 1, get diabetes pill alcohol extracting acetic acid ethyl ester extract 400g, carry out silica gel (100-200 order) column chromatography, V (chloroform): V (methyl alcohol) is used to carry out gradient elution for 100:0,97:3,95:5,9:1,85:15,8:2,7:3,6:4,5:5,0:100 successively, know through thin-layer chromatography inspection and merge identical cut, obtain 10 parts, be labeled as Fr.A ~ J.Fr.D separates out precipitation, precipitates through recrystallization repeatedly, obtains compound 10.Fr.C carries out silica gel (100-200 order) column chromatography, V (sherwood oil): V (ethyl acetate) is used to carry out gradient elution for 100:0,95:5,9:1,85:15,8:2,7:3,5:5,0:100 successively, know through thin-layer chromatography inspection and merge identical cut, obtain 8 parts, be labeled as Fr. I ~ VIII.Fr. I ~ VII separate out precipitation respectively, precipitate through recrystallization repeatedly, obtain compound 11 ~ 16 respectively.Wherein Fr. V carries out reversed-phase bonded silica column chromatography, carries out gradient elution with methanol-water (10% methyl alcohol ~ 90% methyl alcohol), knows and merges identical cut, obtain 8 parts, be labeled as Fr.1 ~ 8 through thin-layer chromatography inspection.Fr.1 is through twice Sephadex LH20 column chromatography, and preparation HPLC is separated, and obtains compound 17.Fr.2 ~ 5 are placed and are separated out precipitation respectively, precipitate through recrystallization repeatedly, obtain compound 18 ~ 21.
Fr.G and Fr.H carries out silica gel (200-300 order) column chromatography, V (chloroform): V (methyl alcohol) is used to carry out gradient elution for 100:0,9:1,85:15,8:2,7:3,5:5,0:100 successively, know through thin-layer chromatography inspection and merge identical cut, obtain 8 parts, be labeled as Fr.a ~ h.Fr.a, through twice Sephadex LH20 column chromatography, obtains compound 5 and 7 respectively.Fr.e, Fr.f place and separate out precipitation respectively, precipitate through recrystallization repeatedly, obtain compound 4 and 3 respectively.Fr.g is separated through preparation HPLC, obtains compound 6 and 22.
The chromatographic condition of described preparation HPLC is as follows:
Chromatographic column: octadecylsilane chemically bonded silica post;
Mobile phase: 20 ~ 45%(v/v) methanol aqueous solution;
Column temperature: 20 ~ 40 DEG C;
Determined wavelength: 280nm;
Flow velocity: 5 ~ 8mL/min;
Sample size: 0.01 ~ 0.2mL.
The chemical composition contained in the method for Wave Spectrum qualification diabetes pill is adopted to be respectively 3 '-hydroxyl Puerarin 3, Puerarin 4,3 '-methoxy puerarin 5, daidzein-8-C-celery sugar (1 → 6) glucose 6, daidzin 7, Dai 10, Schisantherin C 11, schizandrin A 12, formoononetin, 3 '-methoxyl Dai, calycosin, Puerarin xyloside, Astragaloside IV, astragaloside II, Heptadecanoic acide, 5-hydroxyl maltol, DHAP and methyl caffeate; Wherein, finger-print can chemistry point out the spectrum of compound and physicochemical data as follows:
3 '-hydroxyl Puerarin 3: white powder (methyl alcohol), fusing point: 216 ~ 217 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：433[M+H]
+。ESI-MS figure as shown in Figure 2.
1h-NMR (DMSO-d
6, 300MHz) and δ: 10.38 (1H, s, 7-OH), 9.53 (1H, s, 4'-OH) 8.97 (1H, s, 3'-OH), 8.29 (1H, s, H-2), (7.94 1H, d, J=8.8Hz, H-5), (6.99 1H, d, J=8.8Hz, H-6), (7.03 1H, d, J=1.9Hz, H-2'), (6.76 1H, d, J=8.1Hz, H-5'), 6.81 (1H, dd, J=8.1Hz, 1.9Hz, H-6'), 4.81 (1H, d, J=9.8Hz, H-1 "), 3.17 ~ 4.99 (m, each protons on sugar).The hydrogen nuclear magnetic resonance spectrogram of 3 '-hydroxyl Puerarin as shown in Figure 3.
13C-NMR(DMSO-d
6，75MHz)δ：174.9(C-4)，161.1(C-7)，155.9(C-9)，152.6(C-2)，1445.3(C-4')，1444.8(C-3')，126.3(C-5)，123.3(C-3)，123.0(C-1')，119.8(C-6')，116.7(C-10)，116.7(C-5')，115.4(C-6)，115.4(C-2')，112.7(C-8)，81.8(C-5″)，78.8(C-3″)，73.5(C-1″)，70.5(C-2″)，70.8(C-4″)，61.4(C-6″)。The carbon-13 nmr spectra figure of 3 '-hydroxyl Puerarin as shown in Figure 4.
Puerarin 4: white cluster crystal (methyl alcohol), fusing point: 186 ~ 187 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：417[M+H]
+。The ESI-MS of Puerarin as shown in Figure 5.
1H-NMR(DMSO-d
6，300MHz)δ：9.53(1H，s，4'-OH)，8.34(1H，s，H-2)，7.94(1H，d，J=8.8Hz，H-5)，7.39(2H，d，J=8.6Hz，H-2'，H-6')，6.98(1H，d，J=8.8Hz，H-6)，6.80(2H，d，J=8.6Hz，H-3'，H-5')，4.81(1H，d，J=9.5Hz，H-1″)，4.52～4.98(4H，m)，3.16～4.01(6H，m)。The hydrogen nuclear magnetic resonance spectrogram of Puerarin as shown in Figure 6.
13C-NMR(DMSO-d
6，75MHz)δ：174.9(C-4)，161.1(C-7)，157.1(C-9)，157.1(C-4')，152.7(C-2)，130.0(C-2')，130.0(C-6')，126.2(C-5)，123.1(C-1')，122.5(C-3)，116.9(C-10)，115.5(C-6)，115.0(C-3')，115.0(C-5')，112.7(C-8)，81.9(C-5″)，78.7(C-3″)，73.4(C-1″)，70.7(C-4″)，70.5(C-2″)，61.6(C-6″)。The carbon-13 nmr spectra figure of Puerarin as shown in Figure 7.
3 '-methoxy puerarin 5: white powder (methyl alcohol), fusing point: 215 ~ 216 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：447[M+H]
+。The ESI-MS figure of 3 '-methoxy puerarin as shown in Figure 8.
1h-NMR (DMSO-d
6, 300MHz) and δ: 9.09 (1H, s, 4'-OH), 8.40 (1H, s, H-2), 7.96 (1H, d, J=8.8Hz, H-5), 7.17 (1H, d, J=1.9Hz, H-2'), 7.04 (1H, dd, J=8.2,1.9Hz, H-6'), 6.99 (1H, d, J=8.8Hz, H-6), 6.81 (1H, d, J=8.2Hz, H-5'), 4.82 (1H, d, J=9.8Hz, H-1 "), 3.80 (3H, s, OCH
3), 3.17 ~ 4.99 (m, each protons on sugar).The hydrogen nuclear magnetic resonance spectrogram of 3 '-methoxy puerarin as shown in Figure 9.
13C-NMR(DMSO-d
6，75MHz)δ：174.9(C-4)，161.2(C-7)，156.4(C-9)，153.0(C-2)，147.2(C-3')，146.4(C-4')，126.3(C-5)，123.1(C-1')，123.0(C-6')，115.7(C-6)，115.2(C-10)，115.2(C-5')，113.1(C-2')，112.7(C-8)，81.9(C-5″)，78.8(C-3″)，73.5(C-1″)，70.8(C-2″)，70.5(C-4″)，61.3(C-6″)，55.7(3'-OCH
3)。The carbon-13 nmr spectra figure of 3 '-methoxy puerarin as shown in Figure 10.
Daidzein-8-C-celery sugar (1 → 6) glucose 6: white powder (methyl alcohol), fusing point: 189 ~ 190 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：549[M+H]
+。The ESI-MS figure of daidzein-8-C-celery sugar (1 → 6) glucose as shown in figure 11.
1h-NMR (DMSO-d
6, 300MHz) and δ: 9.53 (1H, s, 4'-OH), 8.32 (1H, s, H-2), 7.93 (1H, d, J=8.8Hz, H-5), 7.40 (2H, d, J=8.6Hz, H-2', H-6'), 6.98 (1H, d, J=8.8Hz, H-6), 6.80 (2H, d, J=8.6Hz, H-3' and H-5'), 5.07 (1H, d, J=9.5Hz, H-1 "); 4.78 (1H, d, J=3.2Hz; H-1 " '), 3.17 ~ 4.07 (m, each protons on sugar).The hydrogen nuclear magnetic resonance spectrogram of daidzein-8-C-celery sugar (1 → 6) glucose as shown in figure 12.
13C-NMR(DMSO-d
6，75MHz)δ：174.9(C-4)，161.4(C-7)，157.2(C-9)，157.2(C-4')，152.6(C-2)，130.1(C-2')，130.1(C-6')，126.3(C-5)，123.1(C-3)，122.6(C-1')，116.7(C-10)，115.0(C-6)，115.0(C-3')，115.0(C-5')，112.5(C-8)，109.1(C-1″′)，80.1(C-5″)，78.8(C-3″′)，78.8(C-3″)，73.4(C-1″)，73.2(C-4″′)，70.7(C-2″)，70.6(C-4″)，68.4(C-6″)，63.0(C-5″′)。The carbon-13 nmr spectra figure of daidzein-8-C-celery sugar (1 → 6) glucose as shown in figure 13.
Daidzin 7: colorless needle crystals (methyl alcohol), fusing point: 234 ~ 235 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：439[M+Na]
+。The ESI-MS figure of daidzin as shown in figure 14.
1h-NMR (DMSO-d
6, 300MHz) and δ: 9.53 (1H, s, 4'-OH), 8.35 (1H, s, H-2), 8.01 (1H, d, J=8.9Hz, H-5), 7.37 (2H, d, J=8.5Hz, H-2', H-6'), (7.19 1H, d, J=2.10Hz, H-8), 7.10 (1H, dd, J=8.9,2.1Hz, H-6), 6.78 (2H, d, J=8.5Hz, H-3' and H-5'), 5.06 (1H, d, J=5.0Hz, H-1 "), 4.58 ~ 5.43 (4H; m), 3.12 ~ 3.70 (6H, m).The hydrogen nuclear magnetic resonance spectrogram of daidzin as shown in figure 15.
13C-NMR(DMSO-d
6，75MHz)δ：174.3(C-4)，160.9(C-7)，156.8(C-9)，156.6(C-4')，152.9(C-2)，129.6(C-6')，129.6(C-2')，126.5(C-5)，123.2(C-3)，121.8(C-1')，118.0(C-10)，115.1(C-6)，114.5(C-5')，114.5(C-3')，102.9(C-8)，99.5(C-1″)，76.7(C-5″)，75.9(C-3″)，72.7(C-2″)，69.2(C-4″)，60.2(C-6″)。The carbon-13 nmr spectra figure of daidzin as shown in figure 16.
Dai 10: colorless needle crystals (methyl alcohol), fusing point: 314 ~ 315 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：277[M+Na]
+。The ESI-MS figure of Dai as shown in figure 17.
1h-NMR (DMSO-d
6, 300MHz) and δ: 10.79 (1H, br s, 7-OH), 9.53 (1H, br s, 4'-OH), 8.28 (1H, s, H-2), 7.96 (1H, d, J=8.8Hz, H-5), 7.37 (2H, d, J=8.8Hz, H-2', H-6'), 6.93 (1H, dd, J=8.8,2.20Hz, H-6), 6.85 (1H, d, J=2.2Hz, H-8), 6.80 (2H, d, J=8.8Hz, H-3' and H-5').The hydrogen nuclear magnetic resonance spectrogram of Dai as shown in figure 18.
13C-NMR(DMSO-d
6，75MHz)δ：174.7(C-4)，162.5(C-7)，157.4(C-4')，157.2(C-9)，152.0(C-2)，130.1(C-2')，130.1(C-6')，127.3(C-5)，123.5(C-1')，122.6(C-3)，116.6(C-10)，115.1(C-6)，115.0(C-3')，115.0(C-5')，102.1(C-8)。The carbon-13 nmr spectra figure of Dai as shown in figure 19.
Schisantherin C 11: white powder (methyl alcohol), fusing point: 112 ~ 113 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：559[M+Na]
+。The ESI-MS figure of Schisantherin C as shown in figure 20.
1H-NMR(Pyridine-d
5，400MHz)δ：7.27-7.78(-COPh)，6.84(1H，s，H-4)，6.41(1H，s，H-11)，5.90(1H，s，H-6)，5.86(2H，s，-OCH
2O-)，3.86，3.68，3.65，3.49(3H，s，OCH
3×4)，3.10(1H，dd，J=13.7，9.6Hz，H-9α)，2.36(1H，d，J=13.7Hz，H-9β)，2.26(1H，m，H-8)，1.57(3H，s，7-CH
3)，1.39(3H，d，J=7.1Hz，8-CH
3)。The hydrogen nuclear magnetic resonance spectrogram of Schisantherin C as shown in figure 21.
13C-NMR(Pyridine-d
5，100MHz)δ：165.6(C=O)，152.8(C-1)，152.6(C-3)，149.8(C-12)，142.6(C-2)，141.5(C-14)，137.1(C-10)，135.1(C-13)，133.8(C-4')，133.6(C-5)，130.9(C-2')，130.4(C-6')，130.4(C-1')，128.9(C-3')，128.9(C-5')，123.3(C-15)，122.8(C-16)，113.2(C-4)，103.4(C-11)，101.5(-OCH
2O-)，86.5(C-6)，72.9(C-7)，61.2(2-OCH
3)，61.1(1-OCH
3)，59.1(14-OCH
3)，56.2(3-OCH
3)，44.1(C-8)，37.5(C-9)，29.7(C-18)，20.0(C-17)。The carbon-13 nmr spectra figure of Schisantherin C as shown in figure 22.
Schizandrin A 12: white powder (methyl alcohol), fusing point: 113 ~ 114 DEG C, its chemical structural formula is:
Spectroscopic data is as follows:
ESI-MS m/z：417[M+H]
+。The ESI-MS figure of schizandrin A as shown in figure 23.
1h-NMR (CDCl
3, 300MHz) and δ: 6.51 (2H, d, J=2.18Hz, H-4 and H-11), 3.56 (6H, s), 3.85 (3H, s), 3.86 (6H, s), 3.87 (3H, s), 2.53 (2H, m, H-6), 2.26 (1H, dd, J=13.0,9.5Hz, H-9 α), 2.03 (1H, d, J=13.0Hz, H-9 β), 1.88 (1H, m, H-8), 1.77 (1H, m, H-7), 0.96 (3H, d, J=7.1Hz, 7-CH
3), 0.71 (3H, d, J=7.1Hz, 8-CH
3).The hydrogen nuclear magnetic resonance spectrogram of schizandrin A as shown in figure 24.
13C-NMR(CDCl
3，75MHz)δ：153.0(C-2)，151.8(C-1)，151.7(C-13)，151.6(C-14)，140.3(C-3)，139.9(C-16)，139.3(C-12)，134.1(C-15)，123.5(C-10)，122.5(C-5)，110.6(C-11)，107.3(C-4)，61.1(OCH
3)，61.1(OCH
3)，60.7(OCH
3)，60.7(OCH
3)，56.1(OCH
3)，56.1(OCH
3)，41.0(C-7)，39.3(C-9)，35.8(C-6)，33.9(C-8)，22.0(C-18)，12.9(C-17)。The carbon-13 nmr spectra figure of schizandrin A as shown in figure 25.
The structural formula of formoononetin is:
The structural formula of 3 '-methoxyl Dai is:
The structural formula of calycosin is:
The structural formula of Puerarin xyloside is:
The structural formula of Astragaloside IV is:
The structural formula of astragaloside II is:
The structural formula of Heptadecanoic acide is: CH
3(CH
2)
13cH
2cH
2cOOH
The structural formula of 5-hydroxyl maltol is:
The structural formula of DHAP is:
The structural formula of methyl caffeate is:
Embodiment 2 treats method for building up and the HPLC finger-print thereof of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of reference substance solution
Get 3 '-hydroxyl Puerarin 3 respectively, Puerarin 4,3 '-methoxy puerarin 5, daidzein-8-C-celery sugar (1 → 6) glucose 6, daidzin 7, Dai 10, Schisantherin C 11, schizandrin A 12 are appropriate, accurately weighed, add methyl alcohol dissolving and be diluted to the solution that concentration is 0.01 ~ 0.1mg/mL separately, filter with miillpore filter, get filtrate, to obtain final product.
Reference substance purity is as shown in table 1:
Table 1 reference substance purity
Note: " S " is with reference to peak.
(2), the preparation of test sample
Get diabetes pill 10, porphyrize, accurately take 0.2g fine powder and be placed in round-bottomed flask, add 95% ethanol 50mL, close plug, soak 1 hour, add hot reflux 1 hour, take out, put to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 50% methyl alcohol and is settled to 5mL volumetric flask, shake up, filter through 0.45 μm of miillpore filter, get filtrate, to obtain final product.
(3), high effective liquid chromatography for measuring
The test sample of step (2) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm;
Column temperature: 30 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.02% formic acid-aqueous solution, gradient elution, elution program is shown in Table 2:
The elution program of table 2 diabetes pill HPLC
Flow velocity: 1mL/min;
Sample size: 10 μ L;
Determined wavelength: 280nm.
Said method is adopted to detect diabetes pill, obtain chromatogram, the chromatographic condition of diabetes pill test sample is adopted to measure each part reference substance solution again, after mensuration completes, the retention time of contrast test sample solution and reference substance solution chromatographic peak, adopt diode array detector to carry out on-line checkingi in 190-400nm ultraviolet region, the uv absorption behavior of contrast test sample and each reference substance simultaneously.As shown in figure 28, in diabetes pill test sample chromatogram, main chromatographic peak mates with the uv absorption of its structure and schemes as shown in figure 29 the chromatogram that reference substance and the main chromatographic peak of diabetes pill test sample solution contrast.
Adopted by the chromatogram of test sample " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia entrusts 2,010 2.0 editions) to simulate and generate diabetes pill HPLC finger-print.Gained test sample HPLC finger-print at least comprises 11 characteristic peaks and 1 with reference to peak (S), according to reference substance and test sample chromatogram retention time and the contrast of UV spectrum, can point out the main chromatographic peak in diabetes pill HPLC finger-print.The retention time of each characteristic peak and relative to the relative retention time with reference to peak in table 3.
The retention time at table 3 diabetes pill HPLC Fingerprints peak and relative retention time
Note: " S " is with reference to peak; "-" is structure the unknown.
Wherein, the minimum required sample size setting up finger-print is generally advisable with more than 15 batches, and the diabetes pill sample that the present invention detects is 20 batches.
20 batches of diabetes pills are produced by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd., and lot number is respectively NO1479, NO2020, PO2111, PO1310, NO1324, NO1431, NO2258, NO1554, PO1136, PO1242, PO1195, PY0003, MO0082, LO3597, PO1216, PO1246, PO2145, PO2013, PO1510, NY0011.
Prepare test sample solution according to step (2), adopt the chromatographic condition of step (3) to measure, the finger-print of 20 batches of diabetes pills of gained as shown in figure 26.
Analyze the detection data of gained 20 batches of diabetes pill finger-prints, calculate relative retention time and the relative peak area at each characteristic peak and reference peak, testing result is in table 4,5.
The relative retention time testing result of table 4 20 batches of diabetes pill HPLC finger-prints
Note: " S " is with reference to peak.
The relative peak area testing result of table 5 20 batches of diabetes pill HPLC finger-prints
Note: " S " is with reference to peak.
By the mean value of characteristic peak relative retention time each in 20 batches of diabetes pill finger-prints and relative peak area, respectively as each characteristic peak relative retention time of diabetes pill HPLC standard finger-print and relative peak area, refer to table 6.
The HPLC standard finger-print data of table 6 diabetes pill
Note: " S " is with reference to peak.
The finger-print of the HPLC of 20 batches of diabetes pills is superposed, " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia entrusts 2,010 2.0 editions) software is utilized to analyze, adopt median method, time window 0.1, with No. 10 peaks for reference peak, after Supplements Auto-matching, generate the HPLC standard finger-print of diabetes pill.The HPLC standard finger-print of described diabetes pill as shown in figure 27.
The standard HPLC finger-print that described employing similarity evaluation software simulation generates diabetes pill is that conventional method operates: the AIA data file of 20 batches of diabetes pill HPLC finger-prints is imported " similarity evaluation 2.0 editions " software and analyzes, adopt median method, time window 0.1, with No. 10 peaks for reference peak, after Supplements Auto-matching, generate the HPLC standard finger-print of diabetes pill, determine 12 total peaks.The uv absorption behavior of contrast 12 total peaks and each reference substance, determine 8 compounds representated by total peak that can chemical point out, namely construct the diabetes pill HPLC finger-print that can chemical point out, the described diabetes pill HPLC finger-print that can chemical point out as shown in figure 30.
Embodiment 3 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 85% ethanol 50mL, add hot reflux 30min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 40% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm;
Column temperature: 20 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.01% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2:
Flow velocity: 0.8mL/min;
Sample size: 15 μ L;
Determined wavelength: 225nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 4 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 95% ethanol 50mL, add hot reflux 60min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 60% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm;
Column temperature: 30 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.01% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2;
Flow velocity: 1mL/min;
Sample size: 10 μ L;
Determined wavelength: 254nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 5 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 90% ethanol 50mL, add hot reflux 90min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 50% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm;
Column temperature: 50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.1% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2;
Flow velocity: 1.2mL/min;
Sample size: 20 μ L;
Determined wavelength: 280nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 6 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 95% ethanol 50mL, add hot reflux 30min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 50% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μm;
Column temperature: 20 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.01% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2;
Flow velocity: 0.8mL/min;
Sample size: 10 μ L;
Determined wavelength: 235nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 7 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 95% ethanol 50mL, add hot reflux 60min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 50% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μm;
Column temperature: 45 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.05% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2;
Flow velocity: 1mL/min;
Sample size: 20 μ L;
Determined wavelength: 365nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 8 treats the assay method of the compound Chinese medicinal preparation diabetes pill HPLC finger-print of diabetes
(1), the preparation of test sample
Accurately take diabetes pill fine powder 0.2g, add 95% ethanol 50mL, add hot reflux 90min, be chilled to room temperature, filter, filtrate reduced in volume, to dry, then dissolve with 50% methyl alcohol and is settled in 5mL volumetric flask, shaking up, through filtering with microporous membrane, to obtain final product;
(2), high effective liquid chromatography for measuring
The test sample of step (1) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μm;
Column temperature: 50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B to be formic acid percent by volume be 0.1% formic acid-aqueous solution, gradient elution, elution program is with embodiment 2;
Flow velocity: 1.2mL/min;
Sample size: 20 μ L;
Determined wavelength: 280nm.
Adopt said method to detect diabetes pill, obtain chromatogram, other steps are with embodiment 2.
Embodiment 9 treats the checking of the compound Chinese medicinal preparation diabetes pill HPLC fingerprint of diabetes
1, instrument precision test
Get the diabetes pill that lot number is PO1310, need testing solution is prepared according to the step (2) of diabetes pill test sample in embodiment 2, continuous sample introduction 6 times, and adopt the chromatographic condition of the step in embodiment 2 (3) to measure, measurement result is in table 7 and table 8.
Table 7 PO1310 diabetes pill HPLC finger-print precision investigates result (relative retention time of main peaks)
Note: " S " is with reference to peak.
Table 8 PO1310 diabetes pill HPLC finger-print precision investigates result (relative peak area of main peaks)
Note: " S " is with reference to peak.
Result shows, the retention time at each total peak and the peak area of main peaks basically identical (RSD < 3%) in need testing solution, using the finger-print of the 1st sample introduction gained as reference, by the similarity of 5 sample introduction gained finger-prints after " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia entrusts 2,010 2.0 editions) similarity evaluation system-computed, result similarity all meets the technical requirement of finger-print, in table 9.
The Similarity Measure result (n=6) of table 9 instrument precision test
2, sample stability test
Get the diabetes pill that lot number is PO1310, need testing solution is prepared by the step (2) in embodiment 2, respectively at 0,2,4,8,12,24h investigates stability, and adopt the chromatographic condition of the step in embodiment 2 (3) to measure, survey 6 times altogether, measurement result is in table 10,11.
Table 10 PO1310 diabetes pill HPLC finger-print study on the stability result (relative retention time of main peaks)
Note: " S " is with reference to peak.
Table 11 PO1310 diabetes pill HPLC finger-print study on the stability result (relative peak area of main peaks)
Note: " S " is with reference to peak.
Result shows, the retention time at each total peak and the peak area of main peaks basically identical (RSD < 3%) in need testing solution, using the finger-print of the 1st sample introduction gained as reference, by the similarity of 5 sample introduction gained finger-prints after " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia entrusts 2,010 2.0 editions) similarity evaluation system-computed, result similarity all meets the technical requirement of finger-print, in table 12.
The Similarity Measure result of table 12 sample stability test
Above result shows, need testing solution is surveyed stable in 24h.
3, method replica test
Get the diabetes pill that lot number is PO1310, according to the parallel preparation of step (2) the 6 parts of need testing solutions in embodiment 2, adopt the chromatographic condition of the step (3) in embodiment 2 to measure, measurement result is in table 13,14.
Table 13 PO1310 diabetes pill HPLC finger-print repeatability investigates result (relative retention time of main peaks)
Note: " S " is with reference to peak.
Table 14 PO1310 diabetes pill HPLC finger-print repeatability investigates result ((relative peak area of main peaks)
Note: " S " is with reference to peak.
Result shows, the retention time at each total peak and the peak area of main peaks basically identical (RSD < 3%) in need testing solution, using the finger-print of the 1st sample introduction gained as reference, by the similarity of 5 sample introduction gained finger-prints after " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia entrusts 2,010 2.0 editions) similarity evaluation system-computed, result similarity all meets the technical requirement of finger-print, and measurement result is in table 15.
The Similarity Measure result (n=6) of table 15 method replica test
Above methodological study result shows, sets up the HPLC finger-print of diabetes pill by this method, and sample stability, instrument precision, method repeatability are all better, can the finger-print of Accurate Determining said preparation.
Embodiment 10 treats the investigation of the compound Chinese medicinal preparation diabetes pill HPLC fingerprint similarity of diabetes
1, the mensuration of 20 batches of diabetes pills and the acquisition of HPLC standard finger-print
20 batches of diabetes pills are produced by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd., and lot number is respectively NO1479, NO2020, PO2111, PO1310, NO1324, NO1431, NO2258, NO1554, PO1136, PO1242, PO1195, PY0003, MO0082, LO3597, PO1216, PO1246, PO2145, PO2013, PO1510, NY0011.
Prepare need testing solution according to the step (2) of embodiment 2, adopt the chromatographic condition of step (3) to measure 20 batches of diabetes pills respectively.
2, the investigation of diabetes pill HPLC fingerprint similarity
With the diabetes pill HPLC standard finger-print in embodiment 2 for reference, calculate the similarity often criticizing diabetes pill HPLC finger-print with " similarity evaluation 2.0 editions ".Result is shown in table 16.
Table 16 20 batches of diabetes pill HPLC fingerprint similarities investigate result
As can be seen from Table 16, the similarity of 20 batches of diabetes pill HPLC finger-prints and diabetes pill HPLC standard finger-print is all greater than 0.90, and show that the method for building up of described diabetes pill HPLC finger-print is reproducible, similarity is high.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. treat the method for building up of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print for one kind, described diabetes pill comprises Chinese medicament kudzu-vine root, glutinous rehmannia, corn stigma, root of Chinese trichosanthes, the Radix Astragali, kadsura longepedunculata, Chinese yam, chemical drugs glibenclamide, it is characterized in that, comprise the following steps:
(1), the preparation of reference substance solution
Precision takes 3 '-hydroxyl Puerarin, Puerarin, 3 '-methoxy puerarin, daidzein-8-C-celery sugar (1 → 6) glucose, daidzin, Dai, Schisantherin C, schizandrin A, add methyl alcohol dissolving respectively and be respectively diluted to the solution that concentration is 0.01 ~ 0.1mg/mL, filter, to obtain final product;
(2), the preparation of test sample
Accurately take diabetes pill fine powder, add 85-95% ethanol, add hot reflux, be chilled to room temperature, filter, concentrated, then dissolve and constant volume with 40-60% methyl alcohol, shake up, filter, to obtain final product;
(3), high effective liquid chromatography for measuring
The test sample of step (2) is carried out high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: octadecylsilane chemically bonded silica post;
Column temperature: 20-50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, the formic acid-aqueous solution of Mobile phase B to be formic acid percent by volume be 0.01%-0.1%, gradient elution;
Determined wavelength: 235-280nm;
Elution program carries out with following volumetric concentration configuration:
When 0 minute, the percent by volume of mobile phase A is 2%, Mobile phase B percent by volume is 98%;
When 10 minutes, the percent by volume of mobile phase A is 5%, the percent by volume of Mobile phase B is 95%;
When 20 minutes, the percent by volume of mobile phase A is 22%, the percent by volume of Mobile phase B is 78%;
When 25 minutes, the percent by volume of mobile phase A is 25%, the percent by volume of Mobile phase B is 75%;
When 35 minutes, the percent by volume of mobile phase A is 30%, the percent by volume of Mobile phase B is 70%;
When 60 minutes, the percent by volume of mobile phase A is 50%, the percent by volume of Mobile phase B is 50%;
When 65 minutes, the percent by volume of mobile phase A is 65%, the percent by volume of Mobile phase B is 35%;
When 70 minutes, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%;
When 80 minutes, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%.
2. the method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print according to claim 1, it is characterized in that, the chromatographic condition in step (3) also comprises:
Flow velocity: 0.8-1.2mL/min;
Sample size: 2-20 μ L.
3. the method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print according to claim 2, it is characterized in that, described in step (3), flow velocity is 1mL/min, and described sample size is 10 μ L.
4. the method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print according to claim 1, it is characterized in that, chromatographic column described in step (3) is Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μm, or Agilent ZORBAX SB-Aq post, and specification is 4.6 × 250mm, 5 μm.
5. the method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print according to claim 1, it is characterized in that, described in step (3), column temperature is 30 DEG C.
6. the method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print according to claim 1, is characterized in that, Mobile phase B described in step (3) to be formic acid percent by volume be 0.02% formic acid-aqueous solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310179700.7A CN104155371B (en) | 2013-05-15 | 2013-05-15 | The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310179700.7A CN104155371B (en) | 2013-05-15 | 2013-05-15 | The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104155371A CN104155371A (en) | 2014-11-19 |
| CN104155371B true CN104155371B (en) | 2015-09-30 |
Family
ID=51880927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310179700.7A Active CN104155371B (en) | 2013-05-15 | 2013-05-15 | The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104155371B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105232498A (en) * | 2015-09-30 | 2016-01-13 | 浙江大学 | Application of deoxyschizandrin to preparation of medicine for treating diabetes |
| CN105842353B (en) * | 2016-03-18 | 2018-08-28 | 江苏康缘药业股份有限公司 | The method for building up and its finger-print of Lonicera and Forsythia heat clearing tablet finger-print |
| CN106370751B (en) * | 2016-08-30 | 2018-09-14 | 广州康臣药物研究有限公司 | The fingerprint map construction method and detection method of Chinese medicine composition |
| CN107941967B (en) * | 2018-01-10 | 2020-08-04 | 广西壮族自治区食品药品检验所 | Fingerprint detection method of xiaokeling tablets and xiaokeling capsules |
| CN112076151B (en) * | 2020-08-28 | 2023-04-18 | 乐泰药业有限公司 | A Chinese medicinal oral liquid for treating diabetes due to deficiency of both qi and yin, and its preparation method and quality control method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588037A (en) * | 2004-08-30 | 2005-03-02 | 广州中一药业有限公司 | <<XIAOKEWAN>> medicine quality control method for treating diabetes |
| CN101301425A (en) * | 2008-05-19 | 2008-11-12 | 河南省新四方制药有限公司 | Formulation for treating diabetes and promoting coronary circulation and quality control method |
-
2013
- 2013-05-15 CN CN201310179700.7A patent/CN104155371B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588037A (en) * | 2004-08-30 | 2005-03-02 | 广州中一药业有限公司 | <<XIAOKEWAN>> medicine quality control method for treating diabetes |
| CN101301425A (en) * | 2008-05-19 | 2008-11-12 | 河南省新四方制药有限公司 | Formulation for treating diabetes and promoting coronary circulation and quality control method |
Non-Patent Citations (3)
| Title |
|---|
| 中药天然药物活性成分的研究方法;姚新生;《药学服务与研究》;20031231;第3卷(第4期);205-209 * |
| 消渴丸HPLC特征图谱研究;孙一蕊等;《药物分析杂志》;20141231;第34卷(第4期);539-546 * |
| 高效液相色谱法测定消渴胶囊中葛根素的含量;王举涛等;《食品与药品》;20071231;第9卷(第03A期);17-19 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104155371A (en) | 2014-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104155371B (en) | The method for building up of the compound Chinese medicinal preparation diabetes pill HPLC finger-print for the treatment of diabetes and HPLC finger-print thereof | |
| CN102749348B (en) | Method for identifying active components in medicinal plant | |
| CN102091132B (en) | Method for detecting esculin, aesculetin, fraxin and fraxetin in cortex fraxini or extract thereof | |
| CN101851261B (en) | Polygonum perfoliatum medicinal material, method for preparing reference substance of active constituents in preparation thereof as well as content determination method | |
| CN105911154A (en) | Method for determination of chlorogenic acid, galuteolin and total flavone content of honeysuckle | |
| CN103969350B (en) | A kind of method for building up of antiphlogistic Chunwang capsule HPLC finger printing | |
| CN107677745A (en) | The quality determining method of Desmodium styracifolium extractive of general flavone | |
| CN110346462A (en) | Leaf of Moringa UPLC fingerprint and its UPLC finger-print | |
| CN101091749A (en) | Medicinal material of polygonum capilalum, extractive, and quality control method | |
| He et al. | Differentiation of Cuscuta chinensis and Cuscuta australis by HPLC-DAD-MS analysis and HPLC-UV quantitation | |
| CN109239220B (en) | A kind of quality determining method of Yupingfeng Granules | |
| CN104678004B (en) | A kind of method of quality control of Ge Shan blood-fat-lowering granule | |
| CN109856270A (en) | A method of with 7 index components in hplc simultaneous determination canopy powder granule | |
| CN103969355B (en) | A kind of discrimination method of the finger printing of Milkvetch Root | |
| CN102879516A (en) | Method for identifying Buyang Huanwu soup and measuring content of Buyang Huanwu soup | |
| CN104292203A (en) | Isocoumarin compound and preparation method and application thereof | |
| CN110346463A (en) | A kind of method for building up of Flos Ilicis Asprellae root herb HPLC-ELSD finger-print | |
| CN104758365A (en) | Method for extracting and preparing blood-sugar-reducing substance--secoiridoid glucoside compounds from seeds of Fraxinus rhynchophylla Hance | |
| CN110687219B (en) | Detection method of suhuang cough-relieving capsule fingerprint and application thereof | |
| CN114636779A (en) | Method for constructing sanhua decoction reference sample freeze-dried powder fingerprint spectrum and fingerprint spectrum thereof | |
| CN103926344B (en) | The detection method of selfheal finger-print | |
| CN109557233B (en) | Method for determining content of multi-index components in white paeony root extracting solution | |
| CN111956734A (en) | Optimized extraction method of yankang buccal tablets | |
| CN103399097B (en) | Method for establishing HPLC (High Performance Liquid Chromatography) fingerprint chromatogram of fructus perillae antioxidant active extract and standard fingerprint chromatogram and applications thereof | |
| CN105372370A (en) | Method for detecting 10-deacetylbaccatin III in Chinese yew branches and leaves |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |