Development of Chromatographic Methods for Phytochemical Standardization of Selected Herbal Raw Materials Used in Uganda

Abstract

Background: The quality assurance of herbal materials is challenging because of the wide variation in phytochemical composition. This variation occurs naturally due to genetic difference and also because of the different climatic and soil characteristics of the plant cultivation sites. Furthermore, compound variation can also be introduced by agronomic practices, as well as post-harvest handling and processing of the plant materials. This necessitates reliable standardization methods to demonstrate phytoequivalence of the plant materials and their products. Due to the multicomponent nature of plant drugs, analyzing all compounds in a sample is not only expensive but also not practical. To solve this challenge, the use of markers and fingerprinting comes in handy. This work was aimed at establishing markers, and developing chromatographic fingerprints for the quality control of commercially important herbal materials. It specifically aimed to answer the following questions: (i) What analytical markers are suitable for quality control of selected medicinal plant materials used in Uganda? (ii) What are the TLC and HPLC fingerprint characteristics for the medicinal plant materials? (iii) What are the optimum HPLC methods parameters for quantification of markers in the medicinal plant materials? (iv) Are herbal materials harvested from the different agroecological zones of Uganda phytoequivalent? Methods: This work employed the Herb Marker ranking system (HerbMaRS) to establish quality control markers for the most commercially used herbal materials in Uganda. Using a modified Herb MaRS, compounds were given scores from 0 to 8; where 8 indicated the most suitable chemical marker. Evidence of biological activity, was divided into three levels based on the number of symptoms of the disease condition a compound can treat or alleviate; (i) one symptom (1 point), two symptoms (2 points), 3 and more symptoms, with well elucidated mechanisms of action. The reported concentrations of the compounds were scored; concentration ≥5ppm (1 point), (concentration ≥50ppm, 2 points); then availability of analytical standards (1 point); and availability of an analytical method (1 point). HPLC methods were developed to quantify the identified markers in different batches of the plant materials. TLC and HPLC fingerprints were developed and, in conjunction with chemometric data analysis techniques, including similarity indices, Principal component analysis, hierarchical cluster analysis and analysis of variance, used to evaluate the phytoequivalence of different batches of the plant materials. The batches were defined as materials of the same plant harvested from different agroecological zones of Uganda. Results: Using the Herb MaRS ranking system, this study has identified suitable markers for the quality assurance of herbal materials and phytochemical standardization of six out of the sevenmost commonly used medicinal plants in commercial products in Uganda, namely, Eucalyptus globulus Labill, Aloe barbadensis (L.) Burm.f., Albizia coriaria Oliv., Mangifera indica L., Azadirachta indica A.Juss., Zingiber officinalis Roscoe. The suitable markers for each plant are as follows: aromadendrine, α-terpineol, globulol, and 1,8,- cineol (for Eucalyptus globulus Labill.); aloin, aloe emodin, acemannan (for Aloe barbadensis (L.) Burm.f.); lupeol, lupenone, betulinic acid, betulin, and catechin (for Albizia coriaria Oliv.); mangiferin, catechin, quercetin, and gallic acid (for Mangifera indica L.); azadirachtin, nimbin, nimbidin (for Azadirachta indica A.Juss.); and 6, 8, 10-gingerols, 6-shogaol (for Zingiber officinalis Roscoe). For W. ugandensis, the compounds with known biological activity scored poorly as chemical markers because they lack analytical standards and/or analytical methods. By comparing the TLC fingerprint images and calculating the jaccard indices of the bands, it was demonstrated that samples from different agroecological zones are phytoequivalent. While HPLC analysis of markers demonstrated marked variability among samples harvested from different agroecological zones., The chemometric analysis of HPLC fingerprints showed that samples of M. indica leaf materials and A. coriaria stembark materials were largely phytoequivalent. W. ugandensis stembark materials varied greatly. The possible sources of botanical reference materials were also highlighted: Kabarole, Pakwach and Ntungamo (M. indica leaves); Kabarole, Moroto, Mpigi (A. coriaria stembark); Kabarole, Kabale, Mpigi (W. ugandensis stembark). Recommendations: The National Drug Authority and manufacturers of herbal medicines should adopt the identified chemical markers for quality assurance and standardization of products containing Eucalyptus globulus Labill, Aloe barbadensis (L.) Burm.f., Albizia coriaria Oliv., Mangifera indica L., Azadirachta indica A.Juss., Zingiber officinalis Roscoe. The identified markers should be evaluated for suitability at the various stages of the production chain of the herbal medicines in Uganda i.e., from authentication and quality control of raw materials to evaluating reproducibility in efficacy, safety, and stability of finished products notified by the National Drug Authority. For Albizia coriaria, and Mangifera indica the developed HPLCquantitative method can be used to monitor the levels of markers in both the herbal materials and the finished herbal materials. The information about marker evaluation can be included in the future Ugandan medicinal plant monographs and or product data bases to guide their quality assurance. In addition, the developed chromatographic fingerprints should be used to control the quality of the Albizia coriaria, Mangifera indica and Warburgia ugandensis materials.