Batches Of Plant Material Research Articles

Determination of Toxic Elements in Botanical Dietary Supplement Ingredient Reference Materials.

The National Institute of Standards and Technology (NIST) has produced over 40 botanical dietary supplement Standard Reference Materials (SRMs) and reference materials (RMs) with values assigned for chemical markers and/or active compounds. Although environmental accumulation or inadvertent introduction of toxic elements (arsenic, cadmium, lead, and mercury) is a potential source of exposure in botanical dietary supplement products, the majority of the dietary supplement SRMs/RMs do not have values assigned for the four major toxic elements. To determine As, Cd, Pb, and Hg content in the current inventory of NIST botanical dietary supplement SRMs/RMs. Fifteen SRMs/RMs suites of plant part, extract, and finished products (i.e., solid oral dosage form [SODF]) were analyzed for As, Cd, Pb, and Hg using nitric acid microwave-assisted digestion followed by quantification using inductively coupled plasma-mass spectrometry. Results for control samples were in good agreement with certified values indicating that the analyses of 38 individual botanical SRMs/RMs were in control. Characterization of linked plant/extract SRMs/RMs derived from the same source materials demonstrated that while extraction processes can often yield extracts with lower toxic element content for Hg or As, it is also possible for mass fraction levels to remain unchanged or even to increase after extraction. The results fill significant knowledge gaps in toxic element content ranges for SRMs/RMs where no NIST assigned values existed, in particular for Hg content and for extract and SODF matrixes. With comprehensive toxic element content now available, researchers can better select appropriate dietary supplement SRMs/RMs for use as controls in the analysis of dietary supplement ingredients and products. Results for As, Cd, Pb, and Hg are reported for 38 dietary supplement SRMs/RMs including six suites of plant, extract, and SODF and nine pairs of plant and extract from the same batch of plant material.

Membrane selection and optimisation of tangential flow ultrafiltration of Cyclopia genistoides extract for benzophenone and xanthone enrichment

Ultrafiltration of Cyclopia genistoides extract was optimised to increase its benzophenone and xanthone content as quantified using HPLC-DAD. Regenerated cellulose (RC) and polyethersulphone membranes with molecular weight cut-offs of 10 and 30 kDa were evaluated in terms of compound enrichment, permeate flux and permeate yield, using dead-end ultrafiltration. Compound enrichment was subsequently optimised using the 10 kDa RC membrane and tangential flow ultrafiltration (TFU). The effect of extract composition on compound enrichment, due to natural variation in the source material, was assessed using extracts from different batches of plant material (n = 11). Transmembrane pressure and feed flow rate affected (p < 0.05) process efficiency (mean permeate flux, compound enrichment and membrane fouling). TFU achieved ≥20% enrichment of the target compounds, proving its suitability for preparation of a nutraceutical extract of C. genistoides.

Metabolic profiling of rhizomes of native populations of the strictly endemic Croatian species Iris adriatica

Iris adriatica Trinajstić ex Mitić (Iridaceae L.) is a strictly endemic taxon from Croatia. It is a rhizomatous dwarf plant from the I. pumila complex with a distribution area limited to the Croatian part of the Mediterranean area, mainly central Dalmatia. The genus Iris is known for its richness in isoflavonoids which also play a significant role in chemotaxonomy and biological activity. Hence, in the current study, different plant batches of I. adriatica collected in early spring of 2016 were analysed for their phytochemical profiles and qualitatively compared. UHPLC-PDA-ESI-MS analyses of methanolic rhizome extracts were performed. Altogether, 36 compounds, representing isoflavonoids (including 6,7-methylendioxy derivatives), benzophenones and xanthones were found as aglycones or in glycosidically bound form to be the main constituent groups of I. adriatica rhizomes. Qualitative results were identical between different batches of plant material from collection sites in central Dalmatia, they differed only in quantity. For some phenolic compounds of I. adriatica, chemotaxonomic relevance was detected.

Simultaneous optimisation of extraction of xanthone and benzophenone α-glucosidase inhibitors from Cyclopia genistoides and identification of superior genotypes for propagation

Extraction of xanthones and benzophenones from Cyclopia genistoides was optimised in terms of solvent composition (ethanol concentration) and extraction temperature, using a central composite design, response surface methodology and multi-response desirability profiling. Compounds of interest were the xanthones, mangiferin and isomangiferin, and the benzophenones, 3-β-d-glucopyranosyl-4-β-d-glucopyranosyloxyiriflophenone and 3-β-d-glucopyranosyliriflophenone, present in high levels in C. genistoides. Optimum extraction conditions (40% ethanol; 70°C; 10:1 solvent:solid ratio; 30min) applied to different batches of plant material (n=10), delivered mean extract, xanthone and benzophenone yields of 37.8, 4.12 and 1.99g/100g plant material, respectively, with a mean α-glucosidase inhibitory activity of 39.5% at 100μg/mL. Similar α-glucosidase inhibitory activity was demonstrated for mangiferin and isomangiferin, indicating that the position of the sugar moiety on the dibenzo-γ-pirone structure did not affect activity. Superior C. genistoides genotypes in terms of xanthone and benzophenone content were identified.

Optimisation and validation of high-temperature oxidation of Cyclopia intermedia (honeybush) – From laboratory to factory

Inconsistent and poor quality honeybush herbal tea, produced from Cyclopia intermedia, required that comprehensive sensory profiling of this Cyclopia species be undertaken to identify not only its characteristic sensory attributes, but also to identify negative attributes, including taints responsible for poor quality. This was achieved by descriptive sensory analysis of infusions, prepared at “cup-of-tea” strength of a large sample set sourced from processors and retail outlets. The aroma attributes, “fynbos-floral”, “fynbos-sweet” and “woody”, and to a lesser extent, “fruity-sweet” and “apricot jam”, were the most prominent. The presence of taints such as “smokey” and “wet fur/farm animals” at relative high intensities in some samples indicated poor processing practices. The presence of “green grass” and “dusty” aroma notes is most likely attributable to under- and over-fermentation, respectively. Fermentation is the high-temperature oxidation step essential for the development of the characteristic sensory attributes of traditional honeybush tea. High turbidity levels of some infusions further confirmed sub-optimal processing of plant material. The effects of fermentation temperature (70, 80 and 90°C) and time (12, 16, 24, 36, 48, and 60h) on the sensory characteristics of C. intermedia infusions were thus investigated on laboratory-scale to establish optimum conditions. Different fermentation temperatures produced teas with slightly different sensory profiles, with infusions of plant material fermented at 70°C predominantly floral, at 80°C predominantly fruity and at 90°C overall most characteristic of C. intermedia. Fermentation at 90°C for 24h or 36h proved effective to increase the major positive aroma attributes to prominent levels, while decreasing the negative aroma attributes to negligible levels. These conditions were thereafter validated on factory-scale. Fermentation performed concurrently on the same batches of plant material on laboratory- and factory-scale delivered more or less the same product in terms of aroma profile and “cup-of-tea” strength, with the latter assessed according to the soluble solids content, colour and turbidity of the infusion. For optimum quality, inherent batch-to-batch variation in plant material may require careful monitoring of aroma development during fermentation between 24 and 36h. Application of the optimum fermentation temperature-time combination by industry will contribute towards improved and consistent product quality.

Antitrypanosomal isoflavan quinones from Abrus precatorius

Human African trypanosomiasis is a neglected tropical disease in sub Saharan Africa that is fatal if left untreated. In a search for new natural products with antitrypanosomal activity, we recently identified abruquinones B and I from Abrus precatorius as potent in vitro trypanocidal compounds with high selectivity indices. To obtain sufficient compound for in vivo efficacy tests in mice, a second batch of plant material was re-collected and extracted. However, the chemical profiles of the two batches differed, and additional abruquinones were isolated and identified by HR-ESI-MS, and 1D and 2D NMR (1H, 13C, COSY, HMBC, HSQC, and NOESY) spectroscopy. Abruquinones J (1), K (2), and L (3) were new, while abruquinones A (4) and D (5) were known from the first batch of plant material. The absolute configuration of compounds 1 to 3 was determined by comparison of electronic circular dichroism (ECD) spectra with calculated ECD data. Compounds 2 to 5 showed high in vitro activity against T. b. rhodesiense (IC50 of 0.01, 0.02, 0.02 and 0.01μM, respectively), and remarkable SIs of 508, 374, 1379, and 668, respectively.

Antiprotozoal isoflavan quinones from Abrus precatorius

A library of 309 extracts from selected South African plants was screened in vitro against a panel of protozoan parasites. A CH2Cl2/MeOH (1:1) extract of the whole plant of Abrus precatorius L. ssp. africanus Verdc. (Fabaceae) inhibited Plasmodium falciparum (97.8%), Trypanosoma brucei rhodesiense (100%), and Leishmania donovani (75.5%) when tested at 4.8 mg/mL. Active constituents in two different batches of plant material were tracked by HPLC-based activity profiling, and isolated by normal phase flash chromatography and RP-HPLC. Structures and relative configuration of compounds were established by NMR (1H, 13C, COSY, HMBC, HSQC, NOE difference). The absolute configuration was determined by comparison of electronic circular dichroism (ECD) spectra with calculated ECD data. Ten compounds (Fig. 1) were obtained and identified as isoflavan quinones and hydroquinones, among them five new natural products. Abruquinone I (1) and abruquinone B (8) showed strong in vitro activity against T. brucei rhodesiense (IC50s of 0.30µM ± 0.1 and 0.16µM ± 0.1, respectively). Selectivity indices (SI) as calculated from cytotoxicity data in L-6 cells were 78.3 and 61.3. These SI qualify 1 and 8 as good candidates for assessment of in vivo activity. In contrast, compound 2 was not selective, even though very active (IC50 0.88µM ± 0.1; SI 4.5) [1]. Given that 1 and 8 possess a tenfold higher SI than cynaropicrin, the only plant derived compound with demonstrated in vivo activity against T. b. rhodesiense [2], 1 and 8 are of considerable interest.

Combined Analysis by GC (RI), GC-MS and 13C NMR of the Supercritical Fluid Extract of Abies alba Twigs

Two samples (leaves and twigs) of Abies alba Miller from Corsica were extracted using supercritical CO2 and their chemical compositions were compared with those of the essential oils obtained from the same batch of plant material. In total 45 components were identified using combined analysis by GC (RI), GC-MS and 13C NMR. It was observed that the contents of monoterpenes (mainly represented by limonene, alpha-pinene and camphene) were significantly lower in the supercritical fluid extract (SFE) than in the essential oil (EO). Conversely, the proportions of sesquiterpenes were much higher in CO2 extracts than in essential oils (around 30% vs 4%). Cis-abienol, a diterpene alcohol, was identified only in SFE, and the proportions of this constituent (7.5% and 17.3%) were determined using quantitative 13C NMR since it was under estimated using the standard conditions of GC.

Passiflora incarnata L. (Passionflower) extracts elicit GABA currents in hippocampal neurons in vitro, and show anxiogenic and anticonvulsant effects in vivo, varying with extraction method

Potential mechanisms of Passiflora incarnata extracts and the effect of extraction methods on ingredients and biological effects were explored. Using the same batch of plant material, total flavonoid yields as measured by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) increased substantially with hot versus cold extraction methods. Whole Passiflora extract induced prominent, dose-dependent direct GABA(A) currents in hippocampal slices, but the expected modulation of synaptic GABA(A) currents was not seen. GABA was found to be a prominent ingredient of Passiflora extract, and GABA currents were absent when amino acids were removed from the extract. Five different extracts, prepared from a single batch of Passiflora incarnata, were administered to CF-1 mice for 1 week in their drinking water prior to evaluation of their behavioral effects. Anticonvulsant effects against PTZ-induced seizures were seen in mice that received 2 of the 5 Passiflora extracts. Instead of the anxiolytic effects described by others, anxiogenic effects in the elevated plus maze were seen in mice receiving any of the 5 Passiflora extracts.

Screening of medicinal plants from Reunion Island for antimalarial and cytotoxic activity

Aim of the study Nine plants from Reunion Island, selected using ethnopharmacology and chemotaxonomy, were investigated for their potential antimalarial value. Materials and methods Thirty-eight extracts were prepared by maceration using CH 2Cl 2 and MeOH, and were tested for in vitro activity against the 3D7 and W2 strain of Plasmodium falciparum. The most active extracts were then tested for in vitro cytotoxicity on human WI-38 fibroblasts to determine the selectivity index. Those extracts were also investigated in vivo against Plasmodium berghei infected mice. Results Most active of the extracts tested were the dichloromethane leaves extracts of Nuxia verticillata Lam. (Buddlejaceae), Psiadia arguta Voigt. (Asteraceae), Lantana camara L. (Verbenaceae), the methanol extracts from Aphloia theiformis (Vahl) Benn. (Aphloiaceae) bark, and Terminalia bentzoe L. (Combretaceae) leaves displaying in vitro IC 50 values ranging from 5.7 to 14.1 μg/ml. Extracts from Psiadia, Aphloia at 200 mg/(kg day) and Teminalia at 50 mg/(kg day) also exhibited significant ( p < 0.0005) parasite inhibition in mice: 75.5%, 65.6% and 83.5%, respectively. Conclusion Two plants showed interesting antimalarial activity with good selectivity: Aphloia theiformis and Terminalia bentzoe. Nuxia verticillata still needs to be tested in vivo, with a new batch of plant material.

Isolation and characterization of 3-methoxy-2(5H)-furanone as the principal nephrotoxin from Narthecium ossifragum (L.) Huds.

The principal substance in Narthecium ossifragum (L.) Huds, responsible for the nephrotoxic effects on cattle, moose, goats and other ruminants has been isolated and identified by X-ray crystallography as 3-methoxy-2(5H)-furanone. The Fourier-transform infra-red, 1H and 13C nuclear magnetic resonance, and mass spectra are also given. The concentration in four different batches of plant material varied from 113 to 344 microg g(-1) (wet weight). Extracts of N. ossifragum and fractions derived from them, including purified 3-methoxy-2(5H)-furanone, were each dosed intraruminally, to young goats. 3-Methoxy-2(5H)-furanone of 99.9% purity (15 mg kg(-1) live weight) caused increased concentration of creatinine in serum within 2-3 days, typical of kidney damage caused by N. ossifragum, while toxic effect was obtained down to 4 mg kg(-1) live weight with less purified material (> or = 95%). Toxic effect was also obtained with synthesized 3-methoxy-2(5H)-furanone (30 mg kg(-1) live weight). The isomer 4-methoxy-2(5H)-furanone, detected in some of the batches of the plant material, was not toxic when dosed at 60 mg kg(-1) live weight.