ABSTRACT
The poor stability of the ligustilide (LIG) makes its quantitation in Angelica sinensis (AS) difficult. This study establishes a chemical conversion method for the determination of ligustilide content in AS and proposes a national pharmacopoeia standard. Mechanical agitation and sonication of a powdered AS extract in a methanol/cyprolamine mixture facilitated the stabilization and transformation of ligustilide. Using an external reference HPLC-DAD method, the cyclopropyl-ligustilide (LIGc) content in the mixture could be determined. The content of ligustilide was greater than 1.0% based on 144 AS specimens including 68 obtained from the originally planted areas of Qinghai and Gansu Province; 55 specimens were obtained from Minxian and Weiyuan County medicine markets, and 21 specimens for which the storage period reached or exceeded 1.5 years. According to the Hong Kong Chinese materis medica standards, the content of ligustilide in AS should not be lower than 0.6%. The developed method could also be applied to the quality control of other Chinese medicinal materials (such as Ligusticum chuanxiong) or Chinese patent medicines in which ligustilide is the main component.
ABSTRACT
Objective:To establish a rapid method to identify <italic>Levisticum officinale </italic>adulterated in<italic> Angelica sinensis</italic> by polymerase chain reaction -restriction fragment length polymophism（PCR-RFLP）. Method:By comparing sequences restriction sites in ribosomal DNA Internal Transcribed Spacer（ITS） of <italic>A. sinensis</italic> and <italic>L. officinale</italic>，the specific restriction site Fnu4HI of <italic>L. officinale</italic> was selected，and the primers for PCR-RFLP reaction were designed. Different <italic>A. sinensis</italic> and <italic>L. officinale</italic> were amplified by PCR. The conditions affecting the PCR-RFLP reaction，such as annealing temperature，primer concentration，cycle number and enzyme digestion reaction time，were optimized，and the accuracy of the method was investigated. The established PCR-RFLP identification method was used to investigate the applicability of <italic>L. officinale </italic>adulterated<italic> in A. sinensis</italic> with different aduleration ratios and different origins. Result:A PCR-RFLP method for identifying <italic>A. sinensis</italic> mixed with <italic>L. officinale</italic> was established. When the annealing temperature was 62 ℃ and the number of cycles was 30，when the <italic>L. officinale </italic>adulterated in<italic> A. sinensis</italic> could be digested by Fnu4H I restriction endonuclease after amplification with specific primers，and the two single DNA bands were detected between 100-500 bp，the <italic>A. sinensis</italic> were all negative. The minimum detection limit of this method for adulterated <italic>L. officinale</italic> in <italic>A. sinensis</italic> was 3%，which could be used for the detection of adulterated <italic>L. officinale</italic> in <italic>A. sinensis</italic>. Conclusion:The established PCR-RFLP identification method is sensitive and accurate in detecting whether there is <italic>L. officinale</italic> in <italic>A. sinensis</italic>，and it provides inspection reference and basis for the quality control of <italic>A. sinensis</italic>，with great significance to ensure the safety of its clinical medication.
ABSTRACT
Objective:Due to the limitation of traditional identification methods of Chinese medicinal materials， the study established a rapid method to identify Persicae Semen mixed with Armeniacae Semen Amarum by allele-specific polymerase chain reaction （PCR）. Method:By comparing the ribosomal DNA internal transcribed spacer （ITS） gene sequences of Persicae Semen and Armeniacae Semen Amarum， single nucleotide polymorphism （SNP） sites were searched and specific primers were designed. Different Persicae Semen and Armeniacae Semen Amarum samples were amplified by PCR， the effects of annealing temperature， primer concentration and cycle number on the PCR reaction system were optimized， and the specificity and detection limit of this method were investigated. In addition， the established PCR method was used to detect the samples of Persicae Semen mixed with different proportion of Armeniacae Semen Amarum from different sources and producing areas. Result:A specific PCR method for identifying Persicae Semen mixed with Armeniacae Semen Amarum was established. When the annealing temperature was 63 ℃ and the number of primer cycles was 30， only Armeniacae Semen Amarum could be amplified with 432 bp specific band， while Persicae Semen samples did not have this band. The minimum detection limit of this method for Armeniacae Semen Amarum was 0.2 ng， and the detection limit for Armeniacae Semen Amarum adulterated in Persicae Semen was 1%. Conclusion:The established allele-specific PCR method can accurately detect whether there is Armeniacae Semen Amarum in Persicae Semen， which can provide experimental basis for the quality control of Persicae Semen and guarantee the safety of its clinical use.
ABSTRACT
Objective To establish HPLC analysis methods and fingerprint of different proportions of Angelicae Pubescentis Radix in Angelicae Sinensis Radix; To use HPLC fingerprint method to identify the adulteration of Angelicae Pubescentis Radix in Angelicae Sinensis Radix. Methods Agilent TC-C18 column (5 μm, 4.6 mm × 250 mm) was adopted. Acetonitrile (A) - 0.05% phosphoric acid (B) was the mobile phase, and the gradient elution (0–20 min, 95%–75%B; 20–60 min, 75%–65%B; 60–80 min, 65%–5%B) was used; The flowrate was 1 mL/min; Detection wavelength was 254 nm; Column temperature was 30 ℃. The HPLC spectra of Angelicae Sinensis Radix, Angelicae Pubescentis Radix and adulterated samples were compared and analyzed by using TCM chromatogram fingerprint similarity evaluation system. Results Angelicae Sinensis Radix and Angelicae Pubescentis Radix showed good chromatographic peaks in chromatogram separation. 11 peaks in HPLC fingerprint could be used as the mark, including 7th peak for ferulic acid, 32th peak for osthole, and 34th peak for columbianadin. Conclusion This method is rapid, simple and feasible, can fully reflect the quality of Angelicae Sinensis Radix and clearly identify whether Angelicae Pubescentis Radix is adulterated in Angelicae Sinensis Radix.