5 1 2 2 3 2 4 2* 100050 3. 210009 4. 236069 1 1. 100029 2. - 5 Lichrospher RP-C 18 4. 6 mm 250 mm 5 μm - 1. 0 ml min - 1 260 nm 25 5 68 5 HPLC doi 10. 11669 /cpj. 2015. 04. 006 R284 A 1001-2494 2015 04-0293 - 06 HPLC Characteristic Fingerprint and Chemical Pattern Recognition of Fermentation Mycelium Preparations ZHANG Ping 1 2 ZHENG Tian-jiao 2 3 ZHANG Wen-juan 2 JIAO Kun 4 WEI Feng 2* LIU Bin 1 1. Beijing University of Chinese Medicine Beijing 100029 China 2. National Institutes for Food and Drug Control Beijing 100050 China 3. China Pharmaceutical University Nanjing 210009 China 4. Fuyang Institute for Food and Drug Control Fuyang 236069 China ABSTRACT OBJECTIVE To establish a method of HPLC characteristic fingerprint annlysis for the quality control of five fermentation mycelium preparations. METHODS The HPLC analysis was carried out on a C 18 column 4. 6 mm 250 mm 5 μm by gradient elution with methanol-water as mobile phase at a folw rate of 1. 0 ml min - 1 the column temperature was set at 25 and the detection wavelength was set at 260 nm. The HPLC characteristic fingerprint of 68 batches of five fermentation mycelium preparations was developed and the components of adenosine uridine guanosine uracil and adenine were identified. Similarity analysisi and hierarchical cluster analysis HCA were applied to study the HPLC characteristic fingerprint and chemical pattern recognition. RE- SULTS The chemical pattern recognition analysis showed that 15 batches of Bailing capsules 18 batches of Jinshuibao capsules and 10 batches of Xinganbao capsules were clustered together respectively indicating that the preparation quality of single enterprise was consistent. The HPLC chromatograms of 15 batches of Zhiling capsules were different and the difference in samples of different enterprises was obvious. CONCLUSION This method is accurate and reliable and it can be used to control the quality of fermentation mycelium preparations. KEY WORDS fermentation mycelium preparation nucleoside HPLC characteristic chromatogram similarity analysis hierarchical cluster analysis Cordyceps sinensis BerK. Sacc. 80 10 2 1 3-4 1 Tel 010 67095432 * E-mail weifeng@ nifdc. org. cn 2015 2 50 4 Chin Pharm J 2015 February Vol. 50 No. 4 293
1 Tab. 1 Information of the fermentation mycelium preparations Name Source of standard Sample Strain Sample standard Batch Manufacturer Ningxinbao capsule The Ministry of health standards WS3-B-2120-96 YBZ03812003 Xinganbao capsule The Ministry of health standards WS3-B-2299-97 Zhiling capsule The Ministry of health standards WS3-B-2312-97 Bailing capsule Jinshuibao capsule China Pharmacopoeia 2010 edition Cs-C-Q80 China Pharmacopoeia 2010 edition Cs-4 Cordyceps mycelium powder Cephalosporium sinensis Chen. sp. nov WS-10001- HD-1280-2002 WS- 10 7 5391 B-0319-2002 / 10 1 Artificial Cordyceps mycelium Gliocladium roseum powder link Thom Cordyceps Mortierella powder Mortierella SP WS-10001- HD-1271-2002 15 4 Hirsutella sinensis Liu WS 3-181 Z-60-94 Z 15 1 Guo Yu-et Zeng Paecilomyces hepiali 18 1 WS 3 -C1-0001-95 Z chen 5 2. 1. 1 Waters 2695 HPLC Lichrospher RP-18 4. 6 mm 250 mm 5 μm B - A 0 ~ 13 min 0% B 13 ~ 30 min 0 15% B 30 ~ 40 min 15% 60% B 6-9 40 ~ 50 min 60% 95% B 1. 0 ml min - 1 25 260 nm 20 μl 1 2. 1. 2 70% 1 ml 1. 6 4. 8 24 24 24 μg 2. 1. 3 0. 25 g 1 70% 50 ml 1. 1 Waters 2695 Waters 2996 PDA 20 min 70% Empower KQ - 250DE 10 ml 10 ml METTLER TOLEDO AE240 2. 1. 4 1 1. 2 Milli-Q RSD 5 % RSD 1 % Sigma 98. 0% 1. 3 10 15 15 10 HPLC 18 RSD 5% 7 RSD 1. 5% 4 1 2 2. 1 294 Chin Pharm J 2015 February Vol. 50 No. 4 6 2. 1. 5 1 2. 1. 4 6 2. 1. 3 2. 1. 6 1 2. 1. 4 0 2 4 8 12 16 24 h 2015 2 50 4
HPLC RSD 5 % 5 S RSD 1. 0 % 24 1 h 2. 2 0. 19 0. 057 0. 43 0. 579 0. 65 0. 027 2. 2. 1 0. 78 0. 603 1. 00 1. 000 6 S 2004 0. 19 0. 093 0. 40 0. 089 0. 43 0. 681 0. 65 0. 095 0. 76 0. 657 1. 00 1. 000 5 6 6 S 6 6 0. 19 0. 105 0. 36 6 5 0. 045 0. 45 1. 187 0. 77 0. 687 0. 87 4 0. 059 1. 00 1. 000 6 S1 S2 S3 S4 S 1 0. 19 0. 195 0. 43 0. 711 0. 76 2. 2. 2 5 0. 638 0. 83 0. 038 0. 96 0. 094 1. 00 1 A - 5 HPLC B - C - D - E - F - Fig. 1 The HPLC characteristic chromatograms of the fermentation mycelium preparations and five reference substances A - HPLC characteristic chromatogram of five reference substances B - HPLC characteristic chromatogram of Bailing capsules C - HPLC characteristic chromatogram of Jinshuibao capsules D - HPLC characteristic chromatogram of Ningxinbao capsules E - HPLC characteristic chromatogram of Xinganbao capsules F - HPLC characteristic chromatogram of Zhiling capsules 2015 2 50 4 Chin Pharm J 2015 February Vol. 50 No. 4 295
1. 000 6 S chempattern 15 0. 19 0. 560 0. 41 0. 194 0. 44 0. 683 0. 67 0. 059 0. 76 0. 550 1. 00 1. 000 3 3 2. 3 5 2. 3. 1 11 3 2004 1 4 15 0. 97 ~ 15 0. 99 15 1 2 3 1 4 5 6 1 7 ~ 12 18 2 7 8 9 1 0. 92 ~ 0. 99 10 11 12 1 13 14 15 10 0. 94 ~ 0. 96 10 0. 97 ~ 0. 99 15 0. 87 ~ 0. 98 2 Fig. 2 The similarity analysis results of the fermentation mycelium 68 chempattern preparations 15 2 2. 3. 2 5 3 Fig. 3 The cluster analysis dendrogram of the fermentation mycelium 68 preparations 296 Chin Pharm J 2015 February Vol. 50 No. 4 2015 2 50 4
13 14 15 1 4 C 2. 4 15 0. 90 5A 18 0. 90 5B 10 0. 90 5C 10 0. 90 5D 15 2 0. 90 13 0. 90 5E 3. 2 3 3. 1 10-11 4 Fig. 4 The similarity distribution of Zhiling Capsules 5 A - B - C - D - E - Fig. 5 The HPLC characteristic chromatograms of the fermentation mycelium preparations A - HPLC characteristic chromatograms of 15 bathches of Bailing capsules B - HPLC characteristic chromatograms of 18 bathches of Jinshuibao capsules C - HPLC characteristic chromatograms of 10 bathches of Ningxinbao capsules D - HPLC characteristic chromatograms of 10 bathches of Xinganbao capsules E - HPLC characteristic chromatograms of 15 bathches of Zhiling capsule 2015 2 50 4 Chin Pharm J 2015 February Vol. 50 No. 4 297
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