High Performance Liquid Chromatography Dysosma the content of podophyllotoxin
【Abstract】 Objective To establish a high-performance liquid
chromatography of podophyllotoxin content in Dysosma method. Methods using high performance liquid chromatography column
was Diamonsiltm C18 column; mobile phase: methanol -0.1 mol /
L potassium dihydrogen phosphate solution (50:50); flow rate: 1.0 ml / min; detection wavelength: 290 nm . The results of podophyllotoxin in 0.047 69 ~ 0.476 9 mg / ml showed good
linear relationship (r = 0.999 6), the average recovery was 99.94%, RSD = 1.71% (n = 5). Conclusions The high-performance
liquid chromatography Dysosma in podophyllotoxin content, simple, accurate, specific and strong.
Key words high-performance liquid chromatography versipellis podophyllotoxin
Abstract: ObjectiveTo establish a method of determining Podophllotoxin in Dysosma versipellis by HPLC.MethodsHigh Performance Liquid Chromatography was performed Diamonsil C18. The chromatography conditions were as follows: methanol -
0.1mol / L monobasic potassium phosphate (50:50) as mobile phase, flow rate being 1.0ml/min and detecting wavelength was 290nm.ResultsA good linearity of Podophllotoxin was shown in range of 0.047 69 ~ 0.476 9 mg / ml (r = 0.999 6), the average recovery was 99.94% and RSD was 1.71% (n = 5). ConclusionThe method can supply evidence for the quality evaluation of Dysosma versipellis. It is simple and accurate.
Key words: HPLC; Dysosma versipellis; Podophllotoxin
Dysosma of Berberidaceae plant Dysosma Dysosma versipellis (Hance.) M. cheng (Podophyllum versipelle Hance.) Roots and rhizomes. A Qingrejiedu, phlegm Sanjie, expectorant swelling of the function. For the carbuncle swollen boils, M. scrofulaceum, sore throat, bruises, snake bites and so on. I had Dysosma traits, microstructure had conducted research . In this study, using high performance liquid chromatography Dysosma podophyllotoxin content in order to improve the quality of medicines Dysosma standards and provide the
scientific basis for clinical application.
An instrument and reagent
1.1 Instrument Diana UltiMateTM3000-type high-performance
liquid chromatography, including quaternary gradient pump, autosampler, column temperature box, UV detector, Chameleon (chromeleon) software, data processing system; AUW120D Shimadzu analytical balance (d = 0.1mg/0.01 mg); UP5200H Ultrasonic Cleaner (Panda Group Nanjing Electronic Measurement Co., Ltd.).
1.2 HPLC pure methanol reagent (Shanghai Chemical Reagent Factory Lu Du); potassium dihydrogen phosphate as the AR. Podophyllotoxin reference substance (content was determined with, to 93.5% of the total, batch number 111645-200301) from
Chinese medicines and biological products available; Dysosma
Dysosma versipellis (Hance.) M. Cheng (Podophyllum versipelle Hance.), From the South National University, Professor Wan Dingrong identification and provide.
2 Methods and Results
2.1 Chromatographic conditions Diamonsiltm C18 column
(250 mm × 4.6 mm, 5 μm); mobile phase was methanol -0.1 mol
/ L potassium dihydrogen phosphate (50:50); detection wavelength 290 nm; flow rate of 1.0 ml / min ; column temperature 30 ?; Sensitivity: 0.1AUFS; theoretical plate number calculated by podophyllotoxin peak of not less than 1500.
2.2 Preparation of reference substance solution, said precision obtained in the phosphorus pentoxide desiccator vacuum dried 12 h the amount of podophyllotoxin reference substance, add methanol produced contains about 0.5 mg per ml
of solution, shake, micro-filtration membrane filtration (0.45 μm), was podophyllotoxin reference substance solution.
2.3 Preparation of solution for the test materials were collected from roots and root powder Dysosma (over 3 screens),
each about 0.5 g, Precision said the take, set a Cypriot conical bottle, precision adding methanol 25 ml, Mesa, placed 12 h , saying that given the weight, ultrasound treatment 30 min, removed, put the cold, and then said that given the weight by using methanol to complement the weight loss, shaking, filtration, take added filtrate, microporous membrane
filter (0.45 μm), was octagonal lotus roots and root for the solution of test items.
2.4 octagonal lotus root and root HPLC spectra obtained respectively in roots and root Dysosma test items for the solution of 10 μl, injection of high-performance liquid
chromatography, by comparison of its HPLC diagram, from Figure 1 ~ 4 more can be seen that octagonal lotus root element of fundamental non-podophyllotoxin.
Figure 1 octagonal lotus rhizome HPLC diagram (omitted)
Figure 2 octagonal lotus root HPLC diagram (omitted)
Figure 3 podophyllotoxin reference substance HPLC diagram (omitted)
Figure 4 blank solvent HPLC diagram (omitted)
2.5 linear relationship between the precision study, said taking in the phosphorus pentoxide desiccator vacuum dried 12 h of podophyllotoxin reference substance 25.5 mg, set in 50 ml flask, using methanol dissolved and diluted to the scale, shake (0.51 × 93.5% = 0.476 9 mg / ml), precise volume of solution taken 1.0,2.0,3.0,4.0,5.0 ml, respectively, set 10 ml flask, add methanol was diluted to scale, shake, according to the above conditions for determination of chromatographic peak area of to peak area of the vertical axis, abscissa podophyllotoxin content, mapping the standard curve,
statistical calculation of the regression equation: Y = 0.011 08X +8.678 2 × 10-3, r = 0.999 6. The results showed that
injection of podophyllotoxin volume of 0.047 69 ~ 0.476 9 mg / ml within the framework of concentration and peak area showed
good linear relationship. Reposted elsewhere in the paper for free download http://
2.6 precision experimental precision drawing of podophyllotoxin reference substance solution 10 μl, according
to the above chromatographic conditions, continuous sampling five times, followed by the peak area were determined peak values, the results of podophyllotoxin reference substance RSD = 0.24%, that precision instrument a good degree.
2.7 stability test precision drawing Dysosma kept sealed at room temperature for the test product solution rhizome 10 μl, respectively, 0,2,5,8,10 h into the sample, measured peak
area and calculating the RSD = 0.84% (n = 3 ), results showed that the tested product to detect solution at 10 h had no effect on the outcome, good stability.
2.8 to reproduce the experiment to take samples of the same group of octagonal lotus roots, according to test products for the parallel solution of Preparation for the test solution was prepared by five copies, respectively, precision
drawing reference substance solution and test products for the solution of the 10 μl, were measured for the five test items in the Podophyllotoxin content, resulting RSD = 1.50%.
2.9 Determination of the sample taken three samples of roots Dysosma approved, according to preparation method for the solution of test product operations, respectively, precision drawing reference substance solution and test products for the solution of the 10 μl, injection of high-
performance liquid chromatography, using external standard
method to calculate podophyllotoxin content of the sample. The results in Table 1.
2.10 The average recovery experiments of known content to take samples of five copies of octagonal lotus roots, each about 0.25 g, precision, said determined podophyllotoxin
reference substances were added to 2.55 mg (2.55 × 93.5% =
2.3843 mg), according to the tested product Solution preparation method operation, according to the above-mentioned
chromatographic conditions were measured to calculate the
recovery rate, an average recovery of 99.94%, RSD = 1.71%.
Table 1 Dysosma Determination of podophyllotoxin results
Table 2 Dysosma recoveries of podophyllotoxin
determination results (omitted)
3.1 Dysosma podophyllotoxin content in the extraction method on the active ingredients of Dysosma for
podophyllotoxin have been reported in the literature Dysosma 80% methanol, ultrasonic extraction, the measured content of podophyllotoxin 0.94% , use of the text used in the
methanol measured ultrasonic extraction of podophyllotoxin content of 1.01%, The results show that podophyllotoxin Dysosma content may be related to extraction solvents and methods have a certain relationship, worthy of further study.
3.2 octagonal lotus root in the basic non-podophyllotoxin
by comparing the root and rhizome Dysosma HPLC diagram can be seen that the basic non-root Dysosma podophyllotoxin,
experimental results show that, mainly octagonal lotus roots with podophyllotoxin of anti-cancer anti-viral activity.
Law Dysosma determination of podophyllotoxin content in the sample pretreatment is simple and effective separation of good ingredients, and without interfering with other components of the quality control of medicinal Dysosma have a
certain reference value.
 Chen Li, CHEN Ji-Yan, An Zhibin, et al. Dysosma traits and microscopic identification [J]. Chinese herbal medicine, 2004,27 (8): 558.
 Yu Peizhong, YAO Li-Yun, Wang Liping. HPLC
determination of four kinds of podophyllotoxin Dysosma content [J]. Shanghai Medical University, 1998,25 (6): 452. Reposted elsewhere in the paper for free download http:/ /