Development of Tianma HPLC fingerprint and discriminant analysis / 中国中药杂志

Tianma(the tuber of Gastrodia eleta) is a widely used and pricy Chinese herb. Its counterfeits are often found in herbal markets, which are the plant materials with similar macroscopic characteristics of Tianma. Moreover, the prices of Winter Tianma(cultivated Tianma) and Spring Tianma(mostly wild Tianma) have significant difference. However, it is difficult to identify the true or false, good or bad quality of Tianma samples. Thus, a total of 48 Tianma samples with different characteristics(including Winter Tianma, Spring Tianma, slice, powder, etc.) and 9 plant species 10 samples of Tianma counterfeits were collected and analyzed by HPLC-DAD-MS techniques. After optimizing the procedure of sample preparation, chromatographic and mass-spectral conditions, the HPLC chromatograms of all those samples were collected and compared. The similarities and Fisher discriminant analysis were further conducted between the HPLC chromatograms of Tianma and counterfeit, Winter Tianma and Spring Tianma. The results showed the HPLC chromatograms of 48 Tianma samples were similar at the correlation coefficient more than 0.848(n=48). Their mean chromatogram was simulated and used as Tianma HPLC fingerprint. There were 11 common peaks on the HPLC chromatograms of Tianma, in which 6 main peaks were chosen as characteristic peaks and identified as gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C, parishin E, respectively by comparison of the retention time, UV and MS data with those of standard chemical compounds. All the six chemical compounds are bioactive in Tianma. However, the HPLC chromatograms of the 10 counterfeit samples were significantly different from Tianma fingerprint. The correlation coefficients between HPLC fingerprints of Tianma with the HPLC chromatograms of counterfeits were less than 0.042 and the characteristic peaks were not observed on the HPLC chromatograms of these counterfeit samples. It indicated the true or false Tianma can be identified by either the similarity or characteristic peaks on HPLC fingerprint. Comparing the Winter Tianma with Spring Tianma showed that the HPLC chromatograms of 15 winter Tianma samples and 11 spring Tianma samples were similar at the mean correlation coefficient of 0.908. But the intensity of the characteristic peaks were different between the two groups of Tianma samples, i.e. the intensity of gastrodin, paishin A and C in winter Tianma was lower than those in spring Tianma. The Winter Tianma and Spring Tianma could be discriminated by either the Fisher unstandardized discrimination function or Linear discriminant function, based on the peak areas of 11 common peaks on HPLC chromatograms as variate.

Development of FTIR fingerprint for identification of armand clematis stem (Chuanmutong) and related herbs / 中国中药杂志

Armand clematis stem (Clematidis Armandii Caulis, Chuanmutong) is a widely used Chinese herb to disinhibit urine and relieve stranguria. It is difficult to be identified owing to its various macroscopic feature and unknown characteristic compounds. Thus, total of 24 Chuanmutong samples and 7 related herbs including four manshurian aristolochia stem (Aristolochiae Manshuriensis Caulis, Guanmutong) and three akebia stem (Akebiae Caulis, Mutong) samples were collected and analyzed in the range of 4 000 - 400 cm⁻¹ by Fourier Transform Infrared (FTIR) and two-dimensional infrared correlation spectroscopy (2D-FTIR) techniques. The FTIR spectra of 24 Chuanmutong samples are consistent in the spectrum profiles, position and intensity of characteristic peaks. 20 of the 24 Chuanmutong samples were randomly selected as calibration samples to calculate and simulate mean spectrum. This mean spectrum is named as FTIR fingerprint of Chuanmutong with characteristic peaks at 3 412, 2 932, 1 739, 1 639, 1 509, 1 456, 1 426, 1 376, 1 332, 1 261, 1 159, 1 035, 897 ,609 cm⁻¹. Meanwhile, the limited level (Mean-3σ=0.992 6) to identify true or false Chuanmutong by correlation coefficient of FTIR spectra was calculated based on the 20 Chuanmutong calibration samples. Then, the rest 4 Chuanmutong, 4 Guanmutong and 3 Mutong samples were used as validation samples to evaluate the identification efficacy. The result shows that the FTIR spectra of 4 Chuanmutong validation samples were similar to the fingerprint. Their correlation coefficients of FTIR spectra were over the limited level and accepted as Chuanmutong. However, the spectra of Guanmutong and Mutong were significantly different from Chuanmutong fingerprint. The correlation coefficients of Guanmutong (0.902 1-0.940 4, n=4) and Mutong (0.954 9-0.978 9, n=3) FTIR spectra were less than the limited level and rejected from Chuanmutong. Furthermore, the number, position and intensity of auto-peaks on the 2D-FTIR were drastically different among the three herbs. It is concluded that the developed FTIR fingerprinting can be rapidly and accurately identify Chuanmutong and differentiate from related herbs.

Bioassay for enrich-blood bioactivity of Agelicae Sinensis Radix / 中国中药杂志

Danggui, Agelicae Sinensis Radix, is a widely used Chinese herb to enrich blood, but its quality cannot be effectively assessed by the known chemical markers such as ferulic acid, ligustilide, polysaccharides, etc. A new bioassay was therefore developed to quantify the Enrich-Blood Bioactivity (EBB) for the quality assessment of Danggui raw materials. Danggui sample was first extracted with ethanol and water, respectively. Then the ethanolic extract and water extract were mixed as a test sample to quantify the amount of EBB by mice experiment. The blood deficiency mode in mice was developed by intraperitoneal injecting cyclophospharmide and phenylhdrazine hydrochloride. The quantity of red blood cell was chosen as EBB marker. Cyclosporine A was chosen as a control substance. EBB in analytes was quantified by the amount reaction of parallel line analysis (3, 3') method. The results indicated that the reliability test for quantifying EBB was passed through and the measured value was valid. The analytes showed the significant EBB (P < 0.05). The correlation coefficient was 0.9984 (n=5) between the amount of cyclosporine A (0.035-0.56 g x kg(-1)) and the increased number of red blood cell. The relative standard deviation (RSY) on the amount of EBB was estimated to be 6.15% (n = 6) by six replicated tests, and the confidence limit rate was 26.68% (n = 6). Five Danggui samples, which were collected from different cultivation areas with various morphological characters, showed the variety of EBB in the range of 21.95-44.16 U x g(-1). It is concluded that the developed method is accurate to quantify the EBB of Danggui raw materials, and is therefore suitable to assess its quality.

Comparison on content of ligustilides in different danggui samples / 中国中药杂志

Bioactivity of Danggui is linked to the content of ligustilide, but the relationship between ligustilide with herb shape, cultivating areas and plant species is still unknown. The relationship was investigated by quantifying on the amounts of Z-ligustilide and E-ligustilide by HPLC-DAD-MS method, and then comparing the content of ligustilides (the sum of Z-ligustilide and E-ligustilide) among forty-four various "Danggui" samples containing thirty Chinese Danggui (CDG), six Japanese Danggui (JDG), four Korea Danggui (KDG) and four European Danggui (EDG). Results showed that the content of ligustilides in CDG samples (Angelica sinensis) was in the range of 5.63-24.53 mg x g(-1) with the mean of 11.02 mg x g(-1) (n = 30). Ligustilides amounts were varied among samples cultivated in different areas in China, i. e. 13.90 mg x g(-1) (n = 6) in Yannan, 12.51 mg x g(-1) (n = 6) in Sichuan and 10.04 mg x g(-1) (n = 13) in Gansu. It was also found that ligustilides content was related to the shape, color and fragrance of herb, e. g. the relative larger amount of ligustilides was in the small main root, long rootlet and perfumed sample. Further, ligustilides contents were estimated to be 1.00 mg x g(-1) (n = 6) in JDG samples (A. acutiloba and A. acutiloba var. sugiyamae) and 2.78 mg x g(-1) (n = 2) in EDG samples (lovage root, Levisticum officinale). However, ligustilides could not be detected in the four KDG samples (A. gigas) and two EDG samples (angelica root, A. archangelica). It has been concluded that ligustilide is significant variant among plant species, which may result in the variety of bioactivity and therapeutic effect.

Determination of ligustilide for quality assessment of Ligusticum chuanxiong / 中国中药杂志

<p><b>OBJECTIVE</b>To assay ligustilide content in the herb of Szechwan Lovage Rhizome (Chuanxiong, CX), which is the dried rhizome of Ligusticum chuanxiong in order to assess the quality.</p><p><b>METHOD</b>Ligustilide was quantitatively analyzed by high-performance liquid chromatography in 21 CX samples. An Alltima C18 column (4.6 mmx 150 mm, 5 microm) was used as the analytical column. The mobile phase consisted of water and acetonitrile (40:60). The flow rate was maintained at 1.0 mL x min(-1) with the column temperature at ambient conditions. The detection wavelength was set at 350 nm.</p><p><b>RESULT</b>The average content of Z-ligustilide in 21 CX samples was found to be 7.40 +/- 3.54 mg x g(-1)(x +/- s, n = 21). Therefore,the content of Z-ligustilide in CX should not be less than 0.66% (calculated on the dried basis).</p><p><b>CONCLUSION</b>The overall analytical procedure is rapid and accuracy which is considered suitable for the quantitative analysis of ligustilide in CX. The amount of ligustilide in CX samples collected from different cultivation areas was obviously different. However, a relatively higher content of ligustilide was generally found in the CX collected from its main cultivated areas.</p>