CN104155371A - HPLC (High Performance Liquid Chromatography) fingerprint spectrum of Chinese herbal medicine compound preparation, namely fluid promoting pills, for treating diabetes and establishing method thereof - Google Patents
HPLC (High Performance Liquid Chromatography) fingerprint spectrum of Chinese herbal medicine compound preparation, namely fluid promoting pills, for treating diabetes and establishing method thereof Download PDFInfo
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Abstract
The invention discloses an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of a Chinese herbal medicine compound preparation, namely fluid promoting pills, for treating diabetes and an establishing method thereof. The establishing method comprises the following steps: (1) preparing a reference substance solution; (2) preparing a sample for testing; and (3) establishing and determining HPLC determination conditions. The establishing method is high in specificity, is simple and convenient to operate, is excellent in stability, precision and reproducibility, and can be used for comprehensively detecting multiple main effective ingredients in the fluid promoting pills, thereby constructing the scientific and reasonable fingerprint spectrum of the fluid promoting pills, and completely and accurately evaluating the safety, effectiveness, stability and quality uniformity of the fluid promoting pills.
Description
Technical field
The present invention relates to a kind of quality determining method of compound Chinese medicinal preparation diabetes pill for the treatment of diabetes, particularly relate to foundation and the HPLC finger-print thereof of diabetes pill HPLC finger-print.
Background technology
The compound Chinese medicinal preparation diabetes pill for the treatment of diabetes is the Chinese and Western medicine compound preparation of 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 composition, there is effect of nourishing kidney-YIN, nourishing generate fluid, be used for clinically treating the diabete (diabetes B) due to deficiency of both qi and yin, now record in the version Pharmacopoeia of the People's Republic of China in 2010.As the compound hypoglycemic medicine diabetes pill of Chinese and Western medicine, both brought into play the entirety conditioning of traditional Chinese medicine, prevent and treat the advantage of complication, overcome again chemical drugs long-term taking and easily produced the shortcoming of drug resistance and spinoff.Statistics so far, diabetes pill sales volume totally exceedes 5,000,000,000 yuan, occupies the 3rd, after only coming Acarbose and Diamicron at treatment diabetes medicine meta.
The Chinese prescription of diabetes pill derives from the side of quenching one's thirst of Yuan Dynasty well-known doctor Zhu Danxi and Yuquan of the well-known doctor Ye Tianshi of the Qing Dynasty is loose, 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 thereof (Yin Lihong etc., Heilungkiang medicine, 2010,23 (3): 371-372); In Chinese yam principal ingredient have polysaccharide, fatty acid etc. (Zhao Hong etc., today pharmacy, 2009,19 (3): 49-52); Corn stigma contains (Zhu Xu etc., Changchun University of Traditional Chinese Medicine's journal, 2009,25 (2): 183-184) such as flavones and glycoside, volatile ingredient, carbohydrates; In kadsura longepedunculata taking lignanoid and volatile oil as principal ingredient (grandson KUNG KUNG etc., ShanXi Chinese Medicine Academy journal, 2007,30 (4): 74-75); In root of Chinese trichosanthes, contain the composition such as triterpene and saponin(e and polysaccharide (Li Zhenhong etc., external medical autonomic drug fascicle, 2003,18 (1): 1-4).But at present its perfect square chemical composition is studied.In " pharmacopeia ", the quality control of diabetes pill is comprised the micro-discriminating of the Radix Astragali, kadsura longepedunculata, Chinese yam; The Qualitative Identification of thin-layered chromatography to Puerarin, Astragaloside IV, schizandrin A; The assay of high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) to 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 starts to be widely used in traditional Chinese medicine quality control field.This technology is in conjunction with the main effective constituent of extraction and separation technology, method of spectroscopy and the clear and definite Chinese medicine of high-efficient liquid phase chromatogram technology, determine the index components of finger-print key monitoring, both showed the global feature of Chinese medicine from macroscopic view, 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 HPLC finger-print thereof of the treatment Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print that can comprehensively reflect diabetes pill inherent quality.
A method for building up for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill HPLC finger-print, comprises 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 respectively methyl alcohol to dissolve 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 to 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, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.01%-0.1%, gradient elution;
Detect wavelength: 235-280nm.
In an embodiment, the gradient elution method described in step (3) is therein: elution program carries out with following volumetric concentration configuration:
0 minute time, the percent by volume of mobile phase A is 2%, Mobile phase B percent by volume is 98%;
10 minutes time, the percent by volume of mobile phase A is 5%, the percent by volume of Mobile phase B is 95%;
20 minutes time, the percent by volume of mobile phase A is 22%, the percent by volume of Mobile phase B is 78%;
25 minutes time, the percent by volume of mobile phase A is 25%, the percent by volume of Mobile phase B is 75%;
35 minutes time, the percent by volume of mobile phase A is 30%, the percent by volume of Mobile phase B is 70%;
60 minutes time, the percent by volume of mobile phase A is 50%, the percent by volume of Mobile phase B is 50%;
65 minutes time, the percent by volume of mobile phase A is 65%, the percent by volume of Mobile phase B is 35%;
70 minutes time, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%;
80 minutes time, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%.
In an embodiment, the chromatographic condition in step (3) also comprises therein:
Flow velocity: 0.8-1.2mL/min;
Sample size: 2-20 μ L.
In an embodiment, described in step (3), flow velocity is 1mL/min therein, and described sample size is 10 μ L.
In an embodiment, chromatographic column described in step (3) is Welch Materials XB-C therein
18post, specification is 4.6 × 260mm, 5 μ m, or Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μ m.。
In an embodiment, described in step (3), column temperature is 30 DEG C therein.
In an embodiment, Mobile phase B described in step (3) is that formic acid percent by volume is 0.02% formic acid-aqueous solution therein.
In the HPLC fingerprint chromatogram that above-mentioned diabetes pill HPLC fingerprint obtains, have 12 features to have peak, wherein No. 10 peaks be Dai with reference to peak, highest peak is No. 4 peaks, collection of illustrative plates total length is 78 minutes, specific as follows:
No. 1 peak, retention time is 13.837 minutes, 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, relative retention time is 0.2813; Peak area is 444.5910, and relative peak area is 0.2000;
No. 3 peak is 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 peak is 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, 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, 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 has identified by high performance liquid chromatography on the basis of the main chromatographic peak structure of diabetes pill, and a kind of construction method of the HPLC finger-print of pointing out diabetes pill multi-target ingredient is provided.Described HPLC fingerprint specificity is strong, easy and simple to handle, have excellent stability, precision and reappearance, can detect more all sidedly multiple main effective constituent in diabetes pill, thereby build scientific and reasonable diabetes pill HPLC finger-print, and then monitor exactly more comprehensively the quality of diabetes pill, complete, to evaluate exactly diabetes pill security, validity, stability and quality homogeneity.
Brief description of the drawings
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 of embodiment 1 '-hydroxyl Puerarin;
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of 3 of embodiment 1 '-hydroxyl Puerarin;
Fig. 4 is the DEPT figure and carbon-13 nmr spectra figure of 3 of embodiment 1 '-hydroxyl Puerarin;
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 the DEPT figure and carbon-13 nmr spectra figure of the Puerarin of embodiment 1;
Fig. 8 is the ESI-MS figure of 3 of embodiment 1 '-methoxy puerarin;
Fig. 9 is the hydrogen nuclear magnetic resonance spectrogram of 3 of embodiment 1 '-methoxy puerarin;
Figure 10 is the DEPT figure and carbon-13 nmr spectra figure of 3 of embodiment 1 '-methoxy puerarin;
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 the DEPT figure and 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 the DEPT figure and 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 the DEPT figure and 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 the DEPT figure and 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 the DEPT figure and 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 reference substance of embodiment 2 and the chromatogram of the main chromatographic peak contrast of diabetes pill test sample solution;
Figure 29 is the uv absorption match map of main chromatographic peak and its structure in the diabetes pill HPLC standard finger-print of embodiment 2;
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 source: Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd.;
Instrument: Bruker AV-400 type nuclear magnetic resonance analyser (Bruker company of the U.S.); Finnigan LCQ Advantage 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, water is ultrapure water, it is pure that all the other reagent are analysis.
Embodiment 1 treats separation, purifying and the Structural Identification of the compound Chinese medicinal preparation diabetes pill main chemical compositions of diabetes
To add mass percent concentration be 75% ethanol to medicinal powder before diabetes pill pill, and ultrasonic extraction twice adds 75% ethanol of 10 times of weight of pill prodrug powder for the first time, add for the second time 8 times of 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 to suitable quantity of water suspendible, be extracted with ethyl acetate, after recovery solvent, 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, use successively V (chloroform): V (methyl alcohol) 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, know and merge identical cut through thin-layer chromatography inspection, obtain 10 parts, be labeled as Fr.A～J.Fr.D separates out precipitation, and precipitation, through recrystallization repeatedly, obtains compound 10.Fr.C carries out silica gel (100-200 order) column chromatography, use successively V (sherwood oil): V (ethyl acetate) to carry out gradient elution for 100:0,95:5,9:1,85:15,8:2,7:3,5:5,0:100, know and merge identical cut through thin-layer chromatography inspection, obtain 8 parts, be labeled as Fr. I～VIII.Fr. I～VII is separated out respectively precipitation, and precipitation, through recrystallization repeatedly, obtains respectively compound 11～16.Wherein Fr. V is carried 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 through thin-layer chromatography inspection, obtains 8 parts, is labeled as Fr.1～8.Fr.1 is through twice Sephadex LH20 column chromatography, and preparation HPLC separation, obtains compound 17.Fr.2～5 are placed and are separated out respectively precipitation, and precipitation, through recrystallization repeatedly, obtains compound 18～21.
Fr.G and Fr.H carry out silica gel (200-300 order) column chromatography, use successively V (chloroform): V (methyl alcohol) to carry out gradient elution for 100:0,9:1,85:15,8:2,7:3,5:5,0:100, know and merge identical cut through thin-layer chromatography inspection, obtain 8 parts, be labeled as Fr.a～h.Fr.a, through twice Sephadex LH20 column chromatography, obtains respectively compound 5 and 7.Fr.e, Fr.f place and separate out respectively precipitation, and precipitation, through recrystallization repeatedly, obtains respectively compound 4 and 3.Fr.g separates 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;
Detect wavelength: 280nm;
Flow velocity: 5～8mL/min;
Sample size: 0.01～0.2mL.
Adopt the chemical composition containing in the method qualification diabetes pill of Wave Spectrum 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 to point out spectrum and the physicochemical data of compound 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 schemes 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 of 3 '-methoxy puerarin schemes 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 of daidzein-8-C-celery sugar (1 → 6) glucose schemes 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: colourless acicular crystal (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 of daidzin schemes 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: colourless acicular crystal (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 of Dai schemes 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 of Schisantherin C schemes 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 of schizandrin A schemes 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:
Methyl caffeate's structural formula 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 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 appropriate, accurately weighed, add methyl alcohol dissolving and be diluted to separately the solution that concentration is 0.01～0.1mg/mL, 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 10 of diabetes pills, porphyrize, accurately takes 0.2g fine powder and is placed in round-bottomed flask, add 95% ethanol 50mL, close plug, soaks 1 hour, add hot reflux 1 hour, take out, put to room temperature, filter, filtrate decompression is concentrated into dry, then dissolves with 50% methyl alcohol and is settled to 5mL volumetric flask, shake up, filter through 0.45 μ m miillpore filter, get filtrate, to obtain final product.
(3), high effective liquid chromatography for measuring
The test sample of step (2) is carried out to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μ m;
Column temperature: 30 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.02%, 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;
Detect wavelength: 280nm.
Adopt said method to detect diabetes pill, obtain chromatogram, adopt again the chromatographic condition of diabetes pill test sample to measure each part of reference substance solution, after mensuration completes, the retention time of contrast test sample solution and reference substance solution chromatographic peak, adopt diode array detector to detect online 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, the uv absorption match map of main chromatographic peak and its structure as shown in figure 29 for the chromatogram of the main chromatographic peak contrast of reference substance and diabetes pill test sample solution.
Adopt " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia committee 2,010 2.0 editions) simulation to generate diabetes pill HPLC finger-print the chromatogram of test sample.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 with respect to the relative retention time with reference to peak in table 3.
Retention time and the relative retention time at table 3 diabetes pill HPLC Fingerprints peak
Note: " S " is with reference to peak; "-" is structure the unknown.
Wherein, set up the minimum needed sample size of finger-print and be generally advisable above with 15 batches, 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 20 batches of diabetes pill finger-prints of gained, calculate each characteristic peak and relative retention time and relative peak area with reference to peak, testing result is in table 4,5.
The relative retention time testing result of 20 batches of diabetes pill HPLC finger-prints of table 4
Note: " S " is with reference to peak.
The relative peak area testing result of 20 batches of diabetes pill HPLC finger-prints of table 5
Note: " S " is with reference to peak.
By the mean value of each characteristic peak relative retention time and relative peak area in 20 batches of diabetes pill finger-prints, as the each characteristic peak relative retention time of diabetes pill HPLC standard finger-print and relative peak area, refer to table 6 respectively.
The HPLC standard finger-print data of table 6 diabetes pill
Note: " S " is with reference to peak.
By the finger-print stack of the HPLC of 20 batches of diabetes pills, utilize " similarity evaluation 2.0 editions " (state-promulgated pharmacopoeia committee 2,010 2.0 editions) software to analyze, adopt median method, time window 0.1, taking No. 10 peaks as with reference to 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 to " similarity evaluation 2.0 editions " software and analyze, adopt median method, time window 0.1, taking No. 10 peaks as with reference to peak, after Supplements Auto-matching, generate the HPLC standard finger-print of diabetes pill, determined 12 total peaks.The uv absorption behavior of 12 total peaks of contrast and each reference substance, determine the compound of 8 the total peak representatives that can chemical point out, built 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 decompression is concentrated into dry, then dissolves with 40% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μ m;
Column temperature: 20 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.01%, gradient elution, elution program is with embodiment 2:
Flow velocity: 0.8mL/min;
Sample size: 15 μ L;
Detect 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 decompression is concentrated into dry, then dissolves with 60% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μ m;
Column temperature: 30 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.01%, gradient elution, elution program is with embodiment 2;
Flow velocity: 1mL/min;
Sample size: 10 μ L;
Detect 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 decompression is concentrated into dry, then dissolves with 50% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μ m;
Column temperature: 50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.1%, gradient elution, elution program is with embodiment 2;
Flow velocity: 1.2mL/min;
Sample size: 20 μ L;
Detect 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 decompression is concentrated into dry, then dissolves with 50% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μ m;
Column temperature: 20 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.01%, gradient elution, elution program is with embodiment 2;
Flow velocity: 0.8mL/min;
Sample size: 10 μ L;
Detect 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 decompression is concentrated into dry, then dissolves with 50% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μ m;
Column temperature: 45 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.05%, gradient elution, elution program is with embodiment 2;
Flow velocity: 1mL/min;
Sample size: 20 μ L;
Detect 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 decompression is concentrated into dry, then dissolves with 50% methyl alcohol and is settled in 5mL volumetric flask, shakes up, and 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 to high effective liquid chromatography for measuring, and chromatographic condition is:
Chromatographic column: Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μ m;
Column temperature: 50 DEG C;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.1%, gradient elution, elution program is with embodiment 2;
Flow velocity: 1.2mL/min;
Sample size: 20 μ L;
Detect 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, step (2) according to diabetes pill test sample in embodiment 2 is prepared need testing solution, continuous sample introduction 6 times, and adopt the chromatographic condition of the step (3) in embodiment 2 to measure, measurement result is in table 7 and table 8.
Table 7 PO1310 diabetes pill HPLC finger-print precision is investigated result (relative retention time of main peaks)
Note: " S " is with reference to peak.
Table 8 PO1310 diabetes pill HPLC finger-print precision is investigated 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 committee 2,010 2.0 editions) similarity evaluation system-computed, result similarity all meets the technical requirement of finger-print, in table 9.
The similarity result of calculation (n=6) of table 9 instrument precision test
2, sample stability test
Get the diabetes pill that lot number is PO1310, prepare need testing solution by the step in embodiment 2 (2), respectively at 0,2,4,8,12,24h investigates stability, and adopt the chromatographic condition of the step (3) in embodiment 2 to measure, survey altogether 6 times, 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 committee 2,010 2.0 editions) similarity evaluation system-computed, result similarity all meets the technical requirement of finger-print, in table 12.
The similarity result of calculation 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 6 parts of need testing solutions of the parallel preparation of the step in embodiment 2 (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 is investigated result (relative retention time of main peaks)
Note: " S " is with reference to peak.
Table 14 PO1310 diabetes pill HPLC finger-print repeatability is investigated 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 committee 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 result of calculation (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, finger-print that can Accurate Determining said preparation.
Embodiment 10 treats the investigation of the compound Chinese medicinal preparation diabetes pill HPLC fingerprint similarity of diabetes
1, the acquisition of the mensuration of 20 batches of diabetes pills and 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 of embodiment 2 (2), adopt the chromatographic condition of step (3) respectively 20 batches of diabetes pills to be measured.
2, the investigation of diabetes pill HPLC fingerprint similarity
Taking the diabetes pill HPLC standard finger-print in embodiment 2 as reference, calculate the similarity of every batch of diabetes pill HPLC finger-print with " similarity evaluation 2.0 editions ".Result is shown in table 16.
20 batches of diabetes pill HPLC fingerprint similarities of table 16 are investigated result
As can be seen from Table 16, the similarity of the 20 batches of diabetes pill HPLC finger-prints and diabetes pill HPLC standard finger-print is all greater than 0.90, shows that the method for building up of described diabetes pill HPLC finger-print is reproducible, and similarity is high.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore 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 (8)
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 respectively methyl alcohol to dissolve 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 to 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, Mobile phase B is that formic acid percent by volume is formic acid-aqueous solution of 0.01%-0.1%, gradient elution;
Detect wavelength: 235-280nm.
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, is characterized in that, the gradient elution method described in step (3) is: elution program carries out with following volumetric concentration configuration:
0 minute time, the percent by volume of mobile phase A is 2%, Mobile phase B percent by volume is 98%;
10 minutes time, the percent by volume of mobile phase A is 5%, the percent by volume of Mobile phase B is 95%;
20 minutes time, the percent by volume of mobile phase A is 22%, the percent by volume of Mobile phase B is 78%;
25 minutes time, the percent by volume of mobile phase A is 25%, the percent by volume of Mobile phase B is 75%;
35 minutes time, the percent by volume of mobile phase A is 30%, the percent by volume of Mobile phase B is 70%;
60 minutes time, the percent by volume of mobile phase A is 50%, the percent by volume of Mobile phase B is 50%;
65 minutes time, the percent by volume of mobile phase A is 65%, the percent by volume of Mobile phase B is 35%;
70 minutes time, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%;
80 minutes time, the percent by volume of mobile phase A is 95%, the percent by volume of Mobile phase B is 5%.
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 1, 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.
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 3, is characterized in that, described in step (3), flow velocity is 1mL/min, and described sample size is 10 μ L.
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, is characterized in that, chromatographic column described in step (3) is Welch Materials XB-C
18post, specification is 4.6 × 260mm, 5 μ m, or Agilent ZORBAX SB-Aq post, specification is 4.6 × 250mm, 5 μ m.
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, described in step (3), column temperature is 30 DEG C.
7. 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) is that formic acid percent by volume is 0.02% formic acid-aqueous solution.
8. a HPLC finger-print for the treatment of Chinese medicine for treating diabetes compound preparation diabetes pill, is characterized in that, has 12 features to have peak in described HPLC finger-print, wherein No. 10 peaks are that Dai is with reference to peak, 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, 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, relative retention time is 0.2813; Peak area is 444.5910, and relative peak area is 0.2000;
No. 3 peak is 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 peak is 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, 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, 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.
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| 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 |
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| CN105232498A (en) * | 2015-09-30 | 2016-01-13 | 浙江大学 | Application of deoxyschizandrin to preparation of medicine for treating diabetes |
| CN105842353A (en) * | 2016-03-18 | 2016-08-10 | 江苏康缘药业股份有限公司 | Establishing method of fingerprint spectrum of honeysuckle-fructus forsythiae heat-clearing tablets and fingerprint spectrum |
| CN106370751A (en) * | 2016-08-30 | 2017-02-01 | 广州康臣药物研究有限公司 | Traditional Chinese medicine composition fingerprint construction method and a detection method thereof |
| CN107941967A (en) * | 2018-01-10 | 2018-04-20 | 广西壮族自治区食品药品检验所 | The fingerprint atlas detection method of Xiao Ke Lin piece and Xiaokelin capsule for diabetes |
| CN112076151A (en) * | 2020-08-28 | 2020-12-15 | 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 |
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Cited By (8)
| 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 |
| CN105842353A (en) * | 2016-03-18 | 2016-08-10 | 江苏康缘药业股份有限公司 | Establishing method of fingerprint spectrum of honeysuckle-fructus forsythiae heat-clearing tablets and fingerprint spectrum |
| 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 |
| CN106370751A (en) * | 2016-08-30 | 2017-02-01 | 广州康臣药物研究有限公司 | Traditional Chinese medicine composition fingerprint construction method and a detection method thereof |
| 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 |
| CN107941967A (en) * | 2018-01-10 | 2018-04-20 | 广西壮族自治区食品药品检验所 | The fingerprint atlas detection method of Xiao Ke Lin piece and Xiaokelin capsule for diabetes |
| 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 |
| CN112076151A (en) * | 2020-08-28 | 2020-12-15 | 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 |
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