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The plants belonging to Berberis species are extensively used in various Ayurvedic, homeopathic and ethno-medicines as raw material or as an ingredient. Presence of various major and minor secondary metabolites and their amount plays very crucial role in the standardization and quality control of herbal medicines. Therefore, more objective and definitive methods are necessary for knowing about the chemical variability amongst the genuine and commonly available species of the same plant.In the present study an attempt has been made to critically analyse the variability among the popular Berberis species on the basis of conventional single marker based approach and subsequently using the more advanced method of fingerprinting with principle component analysis and similarity analysis. A comparative analysis was done among the three commonly used analytical techniques TLC, HPLC and ATR-FTIR to select the most appropriate method. Based on the results of optimization of various parameters, the final protocol for sample preparation of Berberis species was optimized on the basis of statistical Duncan’s ranking method based on total extractive value (%) and /or berberine content (%).
Fingerprints of different Berberis species to authentic the raw materials were developed. A comparative analysis was done among the three fingerprinting methods and HPLC fingerprinting with PCA was selected as the best method due to its capability to describe the variability and similarity amongst different Berberis sample in terms of parts, species and locations. The variability among the most common Berberis species on the basis of conventional approach and subsequently using the more advanced method of HPLC fingerprinting with principle component analysis was studied.
In the present study the overall average extractive value of stem, root and leaves samples were found to be 8.53%, 13.50% and 28.05% respectively using mixture of methanol and water (3:1) as solvent. The maximum extractive value was found in leaves followed by the root and stem. A study on correlationof altitude on extractive value of different Berberis plant parts reveals that extractive value of root and stem samples have a weak correlation(R2= 0.31 and R2=0.27 respectively) to altitude. The plants of low altitude have more extractive value in comparison to higher altitude. No such correlation could be observed for the leaves samples (R2=0.006). The maximum average berberine content (%)was obtained in root followed by the stem and leaves corresponding to 1.6%, 0.29%, and 0.098% respectively using HPLC analysis method.
The maximum berberine content was obtained from the Champawat root samples (2.72%). A comparative analysis for the berberine content (%) in stem revealed maximum content in B. lycium (0.46%) whereas minimum berberine content was estimated in B. umbellate (0.02%). In case of root maximum berberine content was estimated in B. asiatica (2.13%) and minimum content in B. chitria (0.58%). It was found that the effect of location dominates over the species and variation in berberine content was less for species if they were collected from the same location. The PCA score plotted with root extract to study interspecies chemical variabiltiy showed distinct clusters for B. aristata and other Berberis species under present study. From the PCA plot, it appears more appropriate to support the fact that chemical variability in the Berberis species is dependent upon geographical factors.
To evaluate the inter species variability; the mean HPLC fingerprints of B. asiatica, B. chitria, B. lycium, B. tinctoria and B. nepalnesis were generated and compared with mean chromatogram of B. aristata. Similarity index was calculated on the basis of Pearson correlation coefficient. B. lycium and B. asiatica are almost similar species (R2=0.95) whereas; B. chitria stands alone, as have low similarity index (R2 value between 0.33-0.22) with either of the species. The present study which has been carried out on the basis of comprehensive HPLC fingerprint revealed that, B. chitria could not be used as the substitute species of B. aristata as reported earlier. Even, B. lycium and B. asiatica are moderately similar to B. aristata. B. tinctoria; the distinctive species of South India has strong similarity to B. lycium. It was evident from the results of interspecies similarity evaluation that B. lycium and B. asiatica has strong correlation and could be considered as substitute species of each other. Ability of similarity analysis to identify the unknown species samples was demonstrated experimentally.
The HPLC fingerprints were also developed for Berberis plant market raw material and formulations. It was demonstrated by similarity analysis and multivariate PCA analysis that the methodologies developed in this thesis successfully demonstrated that HPLC fingerprinting with chemometric methods can successfully differentiate between various Berberis species plants and raw materials. The results of similarity analysis revealed that although Delhi is closely placed withWestern Himalaya region but most of its samples show weak correlation with B. aristata directing towards the use of substitute or adulterant species under the name of B. aristata. The sample collected from Bangalore has a weak correlation with B. tinctoria (R2=0.375) which is supposed be the popular Berberis species distributed in South India. Sample of Delhi and Calcutta showed moderate correlation with B. nepalnesis (R2=0.655, 0.613 and 0.648). The PCA score plot could group raw materials on the basis of location and type of material respectively. The ability of HPLC fingerprinting and PCA to classify different formulations on the basis of their pharmacological applications, variation in amount of the phytoconstituents, comparing the similar type of herbal drugs from different manufacturers and verification of the label claims and utilization of HPLC fingerprinting method combined with PCA for the assessment of batch to batch consistency or variability was also demonstrated successfully.
Hence, it could be concluded that the combination of HPLC fingerprinting and chemometric techniques can be a suitable tool for the authentication and identification of raw material and herbal drugs containing B. aristata even if the number and / or concentration of chemically characteristic constituents are not very similar in different samples of herbal medicines.
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