Rhodiosin and herbacetin in Rhodiola rosea preparations: additional markers for quality control?

Context: Rhodiola rosea L. (Crassulaceae) is well-known to contain flavonoids such as the herbacetin derivative rhodiosin. However, flavonoids are not typically used in quality control. Objective: This study analyses two flavonoids of R. rosea rhizomes and roots for their potential as analytical markers. Materials and methods: Two constituents were isolated from ethanolic extracts via HPLC, identified via NMR and quantified via RP-HPLC. Presence and content variation was investigated according to extraction (solvent and repetitions), drying (temperature and duration) and sample origin (homogenously cultivated plants of different provenance, commercial samples). Results: Rhodiosin was identified as a main flavonoid, accompanied by 10-fold lower concentrated herbacetin. Both compounds were best extracted with 70-90% ethanol, but were also detectable in more aqueous extracts. Different drying conditions had no effect on the flavonoid content. These two flavonoids were consistently identified in rhizome and root extracts of over 100 R. rosea samples. Rhizomes tend to contain less flavonoids, with average ratios of rosavins to flavonoids of 1.4 (rhizomes) and 0.4 (roots). Provenance differences were detected in the range (rhodiosin plus herbacetin) of 760-6300 µg/mL extract corresponding to a maximum of approximately 0.5-4.2% (w/w) in the dry drug. Conclusions: For the first time, two main flavonoids present in R. rosea were quantified systematically. Rhodiosin and herbacetin can be detected simultaneously to phenylpropenoids or salidroside in authentic samples, influenced by the plant part examined and the plant origin. Rhodiosin and herbacetin may serve as additional marker to guarantee a consistent content of R. rosea products.

Quality Control of Traditional Cannabis Tinctures: Pattern, Markers, and Stability.

Traditional tinctures of Cannabis sativa L. became obsolete before elucidation of the main cannabinoids and routine quality testing for medicines. In view of increasing medicinal use of cannabinoids and associated safety concerns, tinctures from a Δ9-tetrahydrocannabinol (THC)-type chemovar were studied. High-performance liquid chromatography with diode-array detection (HPLC/DAD) was used to determine THC, Δ9-tetrahydrocannabinolic acid A (THCA), cannabinol (CBN), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannflavin A/B, and total phenolics. Derived group and ratio markers describe absolute and relative profiles when varying plant part (flos, folium), extraction solvent (EtOH percentage), storage conditions ('shelf' or 'fridge' up to 15 months), and pasteurization (2 h 70 °C, 20 min 80 °C). Tinctures from female flowering tops contained ten-fold more cannabinoids than tinctures from leaves; tinctures (80%-90% EtOH) contained ten-fold more cannabinoids than tinctures (40% EtOH). The analysis of CBGA + CBG, the main co-cannabinoids aside from THCA + THC, appears more relevant than CBDA + CBD. The decarboxylation of THCA to THC-the main change during storage of freshly prepared tinctures-is after 15 months in the 'fridge' comparable to 3 months on the 'shelf'. Minimally increased CBN totals did not correlate to diminished totals of THCA and THC (up to 15% after 3 months 'shelf', 45% after 15 months 'fridge'). Instead, total cannabinoids or acidic/neutral cannabinoid ratios are better stability markers. Moderate changes after pasteurization and partial losses below 10% for total cannabinoids after 9 months 'fridge' indicate possibilities for a reasonable shelf life. Yet storage and use of non-stabilized tinctures remain critical without authorized specification and stability data because a consistent cannabinoid content is not guaranteed.

¹H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification.

The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ∆9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of (1)H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide together with two new validated HPLC/DAD methods used for identification and extract profiling based on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, cannabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid acids in non-heated extracts suggest their consideration for total values in chemotype distinction and specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile. Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2-4 lead cannabinoids are considered essential. The suitability of analytical methods and the range of compound groups summarized in group and ratio markers are discussed regarding plant classification and pharmaceutical specification.

The use of community herbal monographs to facilitate registrations and authorisations of herbal medicinal products in the European Union 2004-2012.

The provisions for the simplified registration of traditional herbal medicinal products in the European Union were introduced by Directive 2004/24/EC amending Directive 2001/83/EC (Chapter 2a) in 2004. Since implementation in the European member states until December 2012 a total of 1015 registrations (traditional use) and 514 authorisations (well-established use) have been granted for products containing substances/ preparations from about 200 different herbal drugs. The overall number of received applications with more than one third still under assessment suggests a further increase for the next years. This review summarises the main features of registered and authorised herbal medicinal products in the EU and evaluates available data against provisions of Directive 2004/24/EC and European standards established by the Committee on Herbal Medicinal Products at the European Medicines Agency. The supportive function of Community herbal monographs is described as regards availability and their use in national procedures, which is complemented by an analysis of specific future challenges from experiences made with the implementation of Directive 2004/24/EC so far.

Effect of provenance, plant part and processing on extract profiles from cultivated European Rhodiola rosea L. for medicinal use.

The demand for plant material of Rhodiola rosea L. (Crassulaceae) for medicinal use has increased recently, amid concerns about its quality and sustainability. We have analysed the content of phenylpropanoids (total rosavins) and salidroside in liquid extracts from 3-year old cultivated plants of European origin, and mapped the influence of plant part (rhizome versus root), genotype, drying, cutting, and extraction solvent to chemical composition. Rhizomes contained 1.5-4 times more salidroside (0.3-0.4% dry wt) and total rosavins (1.2-3.0%) than roots. The qualitative decisive phenylpropanoid content in the extracts was most influenced by plant part, solvent, and genotype, while drying temperature and cutting conditions were of less importance. We have shown that R. rosea from different boreal European provenances can be grown under temperate conditions and identified factors to obtain consistent high quality extracts provided that authentic germplasm is used and distinguished between rhizome, roots and their mixtures.

Effects of 20-hydroxyecdysone, Leuzea carthamoides extracts, dexamethasone and their combinations on the NF-κB activation in HeLa cells.

OBJECTIVES: The plant steroid 20-hydroxyecdysterone (20E) and 20E-containing extracts from Leuzea carthamoides (Willd.) DC are sold with claims of anabolic and immunomodulatory effects. Yet their effect on the activation of nuclear factor kappa B (NF-κB), a key player in immune response and cell fate, and their influence on the NF-κB-inhibiting activity of steroidal anti-inflammatory drugs is still unknown. METHODS: The ability of 20E, Leuzea extracts and selected steroidal/non-steroidal anti-inflammatory drugs to influence the activation of NF-κB was explored using, as the experimental model, human cervical cancer HeLa-IL-6 cells stably transfected with an IL-6-bound reporter gene. Effects on cell viability and proliferation were monitored (MTT assay). HPLC-DAD was used to establish links between chemical patterns of Leuzea extracts and their bioactivities. KEY FINDINGS: 20E inhibited NF-κB activation (IC50 31.8 µm) but was less active than other plant metabolites (xanthohumol 3.8 µm, withaferin A 1.4 µm). Leuzea extracts with high content in 20E had a fair activating effect, but in contrast, some extracts with low 20E content significantly inhibited NF-κB activation at IC50s ranging from 3.5 to 6.2 µg/ml. Combination tests confirmed that 20E does not explain the NF-κB modulation achieved by Leuzea extracts. The extracts but not 20E itself showed a significant modulation of the NF-κB inhibitory effect of dexamethasone. CONCLUSIONS: 20E is unlikely a major player in the NF-κB inhibitory effects displayed by some Leuzea extracts in vitro. If confirmed in vivo, caution should prevail towards marketed Leuzea extracts that are non-standardised or standardised on 20E only, since different starting materials and extracts may even cause opposite effects. More importantly, our results indicate the interaction potential of Leuzea with steroidal anti-inflammatory drugs.

Variability of total flavonoids in Crataegus--factor evaluation for the monitored production of industrial starting material.

European Hawthorn species (Crataegus spp.) are traditionally used for their demonstrated cardioprotective benefits. Most products are based on the compendial leaf and flower drug (LFD), which is standardised on the total flavonoid content (TF). In order to reduce variability associated with the wild plant origin and to ease product standardisation, we set out to develop a sustainable source of high quality raw material. Firstly, the LFD of wild trees in Germany was screened in terms of TF content (spectrophotometric analysis, calculated as hyperoside) according to the current pharmacopoeia requirements. Secondly, eleven high value provenances were selected, propagated, cultivated and the grafted clones were reanalysed. Thirdly, major environmental and sourcing influences were assessed: the year of harvest, the growing location, exposure to sunlight, the harvest time and the portion of leaf, flower and wood within the LFD. We found the TF in LFD of 150 wild grown Crataegus ranged between 0.28% and 1.92% (average 1.15%). Pure single styled Crataegus monogyna and hybrid trees represented the major portion (57%) of all screened trees. The hybrids with mainly two-styled flowers (pure Crataegus laevigata and hybrids) showed slightly higher TF values. The selected clones proved to maintain a high TF profile in cultivation, although superiority was attenuated when not only O-glycosides, but also C-glycosides were covered by the assay. Environmental influence surpassed in part the genetic variation of the selection in this study. In conclusion, cultivated high performance trees under central European conditions produced LFD exceeding the Pharmacopoeia standards. Careful monitoring of production--particularly in terms of the harvest time and actual LFD location and composition--has decisive impact to guarantee consistent high TF values.