MATERIALS AND METHODS

1. Quality Control and Standardization Technology of Medicinal and Dietary Non-Timber Forest Products Based on Tannin Analysis Silber Moris L., MD, PhD, Davitt, B.B., Freed, J., PhD Washington State University • INTRODUCTION • American forests, once seen primarily as stands of timber, are now prized as a rich source of wide range of non-timber forest products (NTFP) with hundreds of medicinal plants among them whose uses have only begun to be fully realized. • The state of scientific knowledge of medicinal and dietary botanicals determines their commercial and noncommercial use, sustainable management, social, economic, and biological benefits, as well as potential negative consequences of their harvesting on their own population and the forest ecosystems. • Expansion of botanicals markets and tenure issues raises ecological concerns that address more sophisticated policy and management for not only ensuring their ongoing use but also protecting the future of the forests. • Success or failure of sustainable management of medicinal and dietary products resource based communities strongly depends on how the industry implements new technologies providing for high quality and standards. • Currently, quality control and standardization (QCS) of medicinal plants and plant-derived dietary supplements is restricted to large pharmaceutical companies and is not designed for in-the-field use. However, these technologies are notorious for complexity (HPLC purification and isolation of singular ingredients with specific pharmacological activity), inconsistency and high cost. • New simple and consistent QCS technologies are needed which could be readily applied at in-the-field conditions during the whole farming cycle, growing, harvesting, processing, and marketing. Such implementation will add value to botanical commodities and augment their competitiveness at local, national, and international markets • KEYWORDS: Non timber forest products (NTFP), medicinal and dietary botanicals, quality control and standardization (QCS),singular biomarkers over tannin specific content (SBM/TSC) • HYPOTHESIS • Tannins are the most ubiquitous metabolites in plants. Tannins are distinct among other secondary metabolites due to their structural diversity, uniquely high affinity for precipitating proteins, from the multiple perspectives of health protection and promotion because of the pharmacological utility, as well as for their influence on ecological interactions between the plant and its environment. • We hypothesize that the therapeutic and dietary effects of whole NTFP on mammalian organism arise primarily from the in orchestra combination of thousands of secondary metabolites, rather than a restricted singular biochemical compound. In these effects important role is played by tannins, as they affect directly the body’s first line defense system (anti-nutritional effect). • Tannins are easy to detect for in-the-field bio prospecting screening. We suggest that QCS of medicinal and dietary plants by specific tannin content (STC) is more relevant from the biological, pharmacological, and nutritional viewpoints, compared to an HPLC purified singular biomarker. • OBJECTIVES • To implement into NTFP resource-based industry a new, simple, rapid, accurate, and low cost analytical technology for tannin measurement in multiple plant samples (screening). This will assist to: i) adding quality value to medicinal and dietary herbal commodities, ii) augment their market competitiveness, and iii) guarantee sustainable management. RATIONALE We possess a newly patented technology for simple, rapid, accurate, and low cost tannin measurement in multiple plant samples (screening). The technology employs tannin’s high precipitation affinity for proteins at low acidic pH. Chemical design and manufacture of a highly sensitive and specific alkaline CBB-BSA reagent for binding tannins is the core of our new technology. Its chemical stability and analytical consistency allows to modify the technology for in-the field use in the form of a hand-held portable Kit with a digital read out device – TANALYZER. MATERIALS AND METHODS Three botanicals were used in this study, i.e. St. John’s Wort (Hypericum perforatum), Echinacea angustifolia, and Cranberry (Vaccinium macrocarpon). Tannins were extracted from lyophilyzed samples (0.4 g) with 50% aqueous MeOH. A newly patented alkaline CBB-BSA technology for tannin measurement was carried out in a modified protein-precipitation protocol (Silber and Davitt, 2000) to identify the biological activity of tannins within the samples of medicinal botanicals: In this analytical protocol the coomassie brilliant blue dye (CBB) combines hydrophobically with bovine serum albumin (BSA) protein under alkaline conditions to yield a stable indicator compound which will easily bind to and precipitate out hydrolysable and condensed tannins at relatively neutral acidity (pH 4-6). Absorbance of the supernatant and the redissolved precipitate were read at 602 nm. The resulting curve plots along with a tannic acid (TA) standard were scanned to locate the interception points. Total tannin content was reported as mg of tannic acid equivalents (TAE)/g sample dry basis. • RESULTS • The new alkaline CBB-BSA method was used in this study to determine specific tannin content (STC) in various botanicals. The method demonstrated high sensitivity, accuracy , and consistency. The inter-species variation in STC was higher than 100%, while the intra-sample variation was always lower than 5%. • Each of the three botanicals analyzed by this method was characterized by its specific tannin content. The STC was the highest in St. John’s Wort, about three times less in Cranberry (Vaccinium macrocarpon), and the lowest (eleven times less) in Echinecae angustifolia. This data is compatible with our earlier analyses of the STC in Hypericum perforatum and Echinaceaangustifolia and purpurea from other locations of the Pacific NW. Here, we first characterize the botanicals by the ratio of the singular biomarker over tannin specific content (BM/STC), in order to scientifically predict the expected biological activity and pharmacological effect of the whole botanical commodities. • CONCLUSION • The long-term goal of this study is to create a database of tannin content and BM/STC ratios for a wide variety of medicinal and dietary plants grown in various locations of the PNW, under different environments, and differently harvested and processed. REFERENCES Silber, ML, Davitt, BB, Khairutdinov, RF, and Hurst, JK, Anal. Biochem., 263, 46-50, 1998. Silber, ML, Davitt, BB, Prep. Biochem. Biotechnol.,30 (3), 209-229, 2000.