| Purpose:1. To establish measuring method of animal medicinal products fingerprint by using high-performance capillary electrophoresis (HPCE). 2. Apply HPCE fingerprints to establish quality evaluation model of animal medicinal products.Material and method:1. material:10 groups in different areas of Pheretima, Eupolyphaga seu steleophaga (Eupolyphaga sinensis Walker), Bombyx batryticatus, Scolopendra, Gecko, Agkistrodon, Zaocys medicinal products, Eupolyphaga sinensis Walker and Steleophaga plancyi(Boleny) control medicine.2. method2.1 Sample processing:The samples under test are all extracted by water or dilute alcohol, ultrasonic extraction.2.2 Reagents and instruments:American agilent 3D high-performance capillary electrophoresis instruments, DAD detector, agilent chem workstation; AS5150A ultrasonic cleaning;TDL-5-A centrifuge;AR2140 analytical balance. All the chemical reagents are for AR, purified water; the reagents are all through the membrane filter of 0.45μm.2.3 Electrophoresis conditions:Capillary diameter is respectively 50,75μm, effective length 40 ~ 65cm; Buffer system is boric acid– borax or boric acid, pH value between 8 and 10; voltage between 12 ~ 25KV; column temperature is 25℃; way for sample injection is pressure; detector is UV-DAD.2.4 The establishment of the each herbs HPCE fingerprint:Put HPCE fingerprints of samples into traditional Chinese medicine computer-aided similarity calculation software, fit out a control fingerprint, and then calculate the similarity of each sample compared with the control fingerprint. Results:1. With 30mmol/L borax buffer solution, pH 9.45, isolating voltage 15KV, column temperature is 25℃, the wavelength is 250nm, determine Pheretima medicinal product HPCE fingerprint, received 8 fingerprint peaks, retention time is respectively 3.985, 4.804, 5.259, 6.255, 6.641, 6.793, 7.537, 7.967, the similarity of every sample compared with its control fingerprint is respectively 0.846, 0.912, 0.719, 0.959, 0.859, 0.932, 0.874, 0.948, 0.949, 0.940, 0.849, 0.853, 0.899, 0.868, 0.928, 0.966, 0.821, 0.968, 0.868, 0.791, 0.825.2. With 20mmol/L borax buffer solution, pH 9.44, isolating voltage 13KV, column temperature is 25℃, the wavelength is 265nm, determine Eupolyphaga seu steleophaga medicinal product HPCE fingerprint, received 6 fingerprint peaks, retention time is respectively 4.336, 4.76, 5.293, 6.189, 8.107, 8.191, the similarity of every sample compared with its control fingerprint is respectively 0.962, 0.966, 0.997, 0.987, 0.963, 0.950, 0.958, 0.975, 0.982, 0.970.3. With 30mmol/L borax-0.2mol/L boricacid buffer solution, pH 8.6, isolating voltage 18KV, column temperature is 25℃, the wavelength is 245nm, determine Bombyx batryticatus medicinal product HPCE fingerprint, received 8 fingerprint peaks, retention time is respectively 6.663, 7.152, 7.305, 7.51, 8.054, 10.925, 12.751, 13.254, the similarity of every sample compared with its control fingerprint is respectively 0.997, 0.992, 0.996, 0.973, 0.992, 0.998, 0.996, 0.994, 0.959, 0.982.4. With 40mmol/L borax-0.2mol/L boricacid buffer solution, pH 8.9, isolating voltage 20KV, column temperature is 25℃, the wavelength is 256nm, determine Scolopendra medicinal product HPCE fingerprint, received 7 fingerprint peaks, retention time is respectively 6.014, 7.515, 8.93, 10.158, 12.232, 12.458, 14.808, the similarity of every sample compared with its control fingerprint is respectively 0.948, 0.981, 0.875, 0.957, 0.883, 0.949, 0.859, 0.956, 0.967, 0.849.5. With 10mmol/L borax-1% acetonitrile buffer solution, pH 9.18, isolating voltage 20 KV, column temperature is 25℃, the wavelength is 245nm, determine Gecko medicinal product HPCE fingerprint, received 7 fingerprint peaks, retention time is respectively 4.865, 6.004, 6.348, 7.064, 8.386, 8.692, 8.846, the similarity of every sample compared with its control fingerprint is respectively0.974, 0.987, 0.967, 0.963, 0.976, 0.990, 0.910, 0.961, 0.979, 0.526.6. With 20mmol/L borax-0.2mol/L boricacid buffer solution, pH 8.4, isolating voltage 19KV, column temperature is 25℃, the wavelength is 245nm, determine Agkistrodon medicinal product HPCE fingerprint, received 7 fingerprint peaks, retention time is respectively 5.908, 6.31, 6.461, 6.758, 9.063, 10.331, 11.174, the similarity of every sample compared with its control fingerprint is respectively 0.978, 0.914, 0.978, 0.967, 0.992, 0.982, 0.986, 0.988, 0.985, 0.974.7. With 80mmol/L borax-0.2mol/L boricacid buffer solution, pH 8.8, isolating voltage 18KV, column temperature is 25℃, the wavelength is 230nm, determine Zaocys medicinal product HPCE fingerprint, received 7 fingerprint peaks, retention time is respectively 7.806, 8.339, 8.872, 9.092, 9.228, 10.799, 17.803, the similarity of every sample compared with its control fingerprint is respectively 0.961, 0.865, 0.891, 0.987, 0.927, 0.942, 0.986, 0.923, 0.914, 0.982.Conclusion:1. In the established fingerprints of 7 species animal medicinal products, the similarity of each Eupolyphaga seu steleophaga, Bombyx batryticatus, Agkistrodon sample compared with their control fingerprints is all above 0.9. Therefore, we can consider the current market on the variety of Eupolyphaga seu steleophaga, Bombyx batryticatus, Agkistrodon medicinal products having uniformity; Each sample of Scolopendra, Gecko, Zaocys medicinal products compared with its fitting control fingerprint, several similarity is less than 0.9, except Gecko(0.526), the other samples'similarity were close to 0.9, it explained that Scolopendra, Zaocys medicinal products were in general accord; In the 21 samples of Pheretima medicinal products, 10 Pheretima aspergillum, 11 Hudilong, besides 1#,3# sample of Pheretima aspergillum, the other similarity were greater that 0.9 and the similarity between the samples is basically unify, while only 4 samples similarity of Hudilong were more than 0.9, 4 samples similarity were close to 0.9, 3 samples similarity were less than 0.9, it demonstrated that Hudilong is more complex in the variety of sources, and it was same as the actual situation; But whether the Pheretima aspergillum is better than Hudilong or not is still has to get a further study.2. The study found that the samples of animal medicinal products whose similarity were less than 0.9 have differences on the relative peak area of common peaks(like 10# similarity of Gecko is 0.526, its relative peak area is much higher than the average level), but their relative retention time is basically unify compared with the other samples. Therefore, HPCE law will not only be considered as the variety identification method of an animal medicine but also have become its means of quality evaluation.3. From analytical results of HPCE fingerprints of 7 species animal medicinal products, we can see that the animal medicines are most contained charged compounds of proteins, nucleosides and so on, while HPCE is such an analysis and effective method, so HPCE may be considered as the analytical method for fingerprints of animal medicinal products. |
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