NMR-based metabolomics as a quality control tool for herbal products

The full potential of the herbal market is mainly not realised due to the lack of knowledge of the chemical composition of most herbal products. The growth potential of the herbal medicine industry can only be achieved if the composition of herbal medicine is standardised to ensure proper quality control and accountability. Plant-based nuclear magnetic resonance metabolomics is one such way of ensuring quick and reliable quality control and metabolite profiling to ensure quality and reproducibility of herbal medicine. Nuclear magnetic resonance-based metabolomics is robust and relatively easy to use, thus ensuring that herbal medicine can be verified and quality controlled much quicker and more accurate than is currently the case. Although nuclear magnetic resonance is not as sensitive as other analytical techniques such as liquid chromatography and gas chromatography–mass spectroscopy, it is far more reproducible, non-destructive, covers a much wider dynamic range and sample preparation is simpler and quicker to use. Economical development of herbal medicine and the use of nuclear magnetic resonance-based metabolomics should go hand in hand for a better future for herbal medicine. In this review an introduction is given to herbal extracts as therapeutic agents and to the quality control aspects of herbal medicine by means of metabolomics. The experimental methodology for plant metabolomics which covers extraction, nuclear magnetic resonance analysis and multivariate data analysis is also discussed. Some examples are given on the possible applications of nuclear magnetic resonance-based metabolomics in the industry and finally the future of nuclear magnetic resonance-based metabolomics is discussed regarding advances in research and development.http://www.elsevier.com/locate/sajbhb2016Plant Scienc

An evaluation of the endophytic colonies present in pathogenic and non-pathogenic Vanguerieae using electron microscopy

Fadogia homblei, Pavetta harborii, Pavetta schumanniana, Vangueria pygmaea (=Pachystigma pygmaeum), Vangueria latifolia (=Pachystigma latifolium) and Vangueria thamnus (=Pachystigma thamnus) all induce one of the most important cardiotoxicoses of domestic ruminants in southern Africa, causing the sickness gousiekte. All the plants which cause gousiekte have previously been shown to contain bacterial endophytes. However, in this study other plants within the Vanguerieae tribe that have not been reported to cause gousiekte; namely Vangueria infausta, Vangueria macrocalyx and Vangueria madagascariensis, have now been shown to also contain endophytes within the inter-cellular spaces of the leaves. The disease gousiekte is difficult to characterise due to fluctuations in plant toxicity. The majority of reported cases of gousiekte poisoning are at the beginning of the growing season; and thus the plants are thought to be more toxic at this time. By using both transmission and scanning electron microscopy the endophytes within these Vanguerieae plants were compared visually. Using the plant reported most often for gousiekte poisoning, V. pygmaea, a basic seasonal comparison of the presence of endophytes was done. It was found that the bacterial endophyte colonies were most abundant during the spring season.The National Research Foundation of South Africa and Professor T. Coutinho.http:// www.elsevier.com/ locate/sajbam201

Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha-4,11-diene

AbstractArtemisinin is an antimalarial sesquiterpenoid isolated from the aerial parts of the plant Artemisia annua. CYP71AV1, a cytochrome P450 monooxygenase was identified in the artemisinin biosynthetic pathway. CYP71AV1 catalyzes three successive oxidation steps at the C12 position of amorpha-4,11-diene to produce artemisinic acid. In this study, we isolated putative CYP71AV1 orthologs in different species of Artemisia. Comparative functional analysis of CYP71AV1 and its putative orthologs, together with homology modeling, enabled us to identify an amino acid residue (Ser479) critical for the second oxidation reaction catalyzed by CYP71AV1. Our results clearly show that a comparative study of natural variants is useful to investigate the structure–function relationships of CYP71AV1

Identification of anti-HIV biomarkers of Helichrysum species by NMR-based metabolomic analysis

Several species of the Helichrysum genus have been used ethnobotanically to treat conditions that we today know have been caused by viral infections. Since HIV is a modern disease with no ethnobotanical history, we commenced with a study on the antiHIV activity of several Helichrysum species. Drug discovery of small molecules from natural resources that is based on the integration of chemical and biological activity by means of metabolomical analyses, enables faster and a more cost-effective path to identify active compounds without the need for a long process of bioassay-guided fractionation. This study used metabolomics to identify anti-HIV compounds as biomarkers from 57 Helichrysum species in a combined study of the chemical and biological data of two previous studies. In the OPLS-DA and hierarchical cluster analyses, anti-HIV activity data was included as a secondary observation, which assisted in the correlation of the phytochemical composition and biological activity of the samples. Clear grouping revealed similarity in chemical composition and bioactivity of the samples. Based on the biological activity of polar extracts, there was a distinct phytochemical difference between active and non-active groups of extracts. This NMR-based metabolomic investigation showed that the chlorogenic acids, compounds with cinnamoyl functional groups, and quinic acid were the most prominent compounds in the Helichrysum species with anti-HIV activity. This study further revealed that the chlorogenic acid type compounds and quinic acid are biomarkers for anti-HIV activity.The National Research Foundation of South Africa.https://www.frontiersin.org/journals/pharmacologydm2022Plant Production and Soil Scienc

Investigation of the anti-mycobacterial mechanism of action of 7-methyljuglone

Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis (MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified. The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of 7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods: Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of 0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.The National Research Foundation and the Medical Research Council, Pretoria, South Africa.http://www.SciRP.org/journal/ojrd)am2016ImmunologyPlant Scienc

Evaluation of four Cameroonian medicinal plants for anticancer, antigonorrheal and antireverse transcriptase activities

Methanol extracts from the leaves, bark and roots of four Cameroonian medicinal plants, Bersama engleriana, Cupressus lusitanica, Vitellaria paradoxa and Guibourtia tessmannii were tested for their in vitro cytotoxicity, antigonorrheal and antireverse transcriptase activities. The XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay, the dilution method and reverse transcriptase (RT) assay were used for the investigations. Preliminary phytochemical analysis of the extracts was also conducted using standard methods. Results showed that all extracts contained compounds belonging to the classes of phenols and terpenoids. They were also able to reduce in dose dependent manner, the proliferation of the cancer THP-1, DU145, HeLa, MCF-7, HepG2 and the normal Vero cells. IC50 values below 30 μg/ml were noted with extract from the three parts of B. engleriana on at least two of the five studied cancer cell lines, the lowest value of 5.9 μg/ml being obtained with sample from the bark. IC50 values below 30 μg/ml were also recorded with extracts from the leaves (on HeLa cells) and bark (on MCF-7) of G. tessmanii, and that from the bark of C. lusitanica on MCF-7. Extracts from B. engleriana and those from the bark of V. paradoxa gave the minimal inhibitory concentrations (MIC) values below 100 μg/ml on most of the 10 tested Nesseria gonorrhoeae strains. Extracts from B. engleriana also inhibited more than 80% the activity of the Human Immuno-deficiency Virus (HIV) enzyme. Finally, the results of the present study provide baseline information for the use of B. engleriana, C. lusitanica, G. tessmanii, V. paradoxa.http://www.elsevier.com/locate/etapnf201

Antibacterial constituents of three Cameroonian medicinal plants : Garcinia nobilis, Oricia suaveolens and Balsamocitrus camerunensis

BACKGROUND: Multidrug resistance is a worrying cause of treatment failure in bacterial infections. The search of bioactive constituents from medicinal plants against multidrug resistant (MDR) bacteria has significantly evolved in the two last decades. In the present study, twenty-two compounds (three terpenoids, eleven phenolics and eight alkaloids) isolated from three Cameroonian medicinal plants, namely Garcinia nobilis, Oricia suaveolens and Balsamocitrus camerunensis, as well as the crude extracts were tested for their antibacterial activities against Mycobacterium tuberculosis and Gram-negative bacteria amongst which were MDR active efflux pumps expressing phenotypes. METHODS: The microplate alamar blue assay (MABA) and the broth microdilution methods were used to determine the minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of the studied samples. RESULTS: The results of the MIC determinations indicate that, the best crude extract was that from G. nobilis (GNB), its inhibitory effects being noted against 12 of the 14 tested bacteria. The extract of GNB also exhibited better anti-tuberculosis (MIC of 128 μg/ml M. tuberculosis against ATCC 27294 strain) and antibacterial (MIC of 64 μg/ml against Escherichia coli ATCC10536) activities compared to the extracts of O. suaveolens and B. camerunensis. Interestingly, 4-prenyl-2-(3,7-dimethyl-2,6-octadienyl)-1,3,5,8-tetrahydroxyxanthone (2), isolated from the most active extract GNB, also showed the best activity amongst compounds, inhibiting the growth of all the fourteen tested microorganisms. The lowest MIC value obtained with compound 2 was 8 μg/ml against M. tuberculosis ATCC 27294 and M. tuberculosis clinical MTCS2 strains. Other compounds showed selective activities with 11 of the 14 tested bacteria being sensitive to the xanthone, morusignin I (5) and the alkaloid, kokusaginine (13). CONCLUSIONS: The results of the present investigation provide evidence that the crude extract from G. nobilis, O. suaveolens and B. camerunensis as well as some of their compounds, and mostly compound 2 (isolated from G. nobilis,) could be considered as interesting natural antibacterial products.International Foundation for Science (IFS-Grant F/4579-2 to VK), TWAS for financial support for ICCBSTWAS fellowship at the H.E.J. Research Institute of Chemistry, International Center of Chemical Sciences, University of Karachi, Pakistan on year 2010 (HF).http://www.biomedcentral.com/bmccomplementalternmedam201

Cytotoxicity of synthesized 1,4-naphthoquinone analogues on selected human cancer cell lines

In an effort to establish new candidates with enhanced anticancer activity of 5-hydroxy-7-methyl-1,4- naphthoquinone scaffold (7-methyljuglone) previously isolated from the root extract of Euclea natalensis, a series of 7-methyljuglone derivatives have been synthesized and assessed for cytotoxicity on selected human cancer lines. These compounds were screened in vitro for anticancer activity on MCF-7, HeLa, SNO and DU145 human cancer cell lines by MTT assay. Most of them exhibited significant toxicity on cancer cell lines with lower IC50 values. The most potent derivative (19) exhibited the toxicity on HeLa and DU145 cell lines with IC50 value of 5.3 and 6.8 lM followed by compound (5) with IC50 value of 10.1 and 9.3 lM, respectively. Structure–activity relationship reveals that the fluoro substituents at position C-8 while hydroxyl substituents at C-2 and C-5 positions played an important role in toxicity.University of Pretoria, South Africa and National Research Foundation (NRF), South Africa.http://www.elsevier.com/locate/bmc2015-09-30hb201

Fluoroacetate metabolism of Dichapetalum Cymosum

A fast and sensitive method was developed for the determination of fluoroacetate in Dichapetalum cymosum using high-performance liquid chromatography. The highest concentrations of fluoroacetate were found in the immature seeds, flowers and young leaves of the plant. The young leaves are more toxic than the older leaves, probably because the rate of fluoroacetate degradation is higher in old leaves than in younger leaves. Foliarly applied fluoroacetate is also more readily accumulated by the young than by the older leaves of D. cymosum. Fluoroacetate can be taken up by the roots of D. cymosum and be transported to the leaves. Whether this happens to a significant extent under natural conditions is unknown. It was, however, demonstrated in this study that aseptically grown D. cymosum seedlings, as well as an aseptic callus culture of the plant, is capable of producing fluoroacetate. A pseudomonad was isolated from D. cymosum and identified as Pseudomonas cepacia. It was established that an isolate of this bacterium could grow in fluoroacetate enriched solutions without any reduction in growth rate. This bacterium is capable of defluorinating fluoroacetate and also of liberating CO2 from fluoroacetate. It seems as though the synthesis of N-methyl alanine and the occurrence of N-methyl serine in D. cymosum, is a result of the symbiosis between the plant and its endophyte. An aseptic callus culture of D. cymoswa is capable of degrading fluoroacetate albeit at a much lower rate than the leaves of the plant. By contaminating the callus with P. cepacia, isolated from the plant, the rate of CO2 release from fluoroacetate was increased about five fold. A D. cymosum crude mitochondrial enzyme extract can release CoASH from fluoroacetyl-CoA at a rate of 29.5 nmoles/min/mg protein, indicating the presence in the extract of an enzyme which is probably fluoroacetyl-CoA hydrolase. This enzyme could not use acetyl-CoA as a substrate. The presence of the fluoroacetyl-CoA hydrolase-like enzyme in D. cymosum together with the ability of the plant and its endophyte to degrade the fluoroacetate, helps to explain why D. cymosum is not poisoned by the high fluoroacetate concentrations which occur in the plant at times.Thesis (PhD (Plant Physiology))--University of Pretoria, 1991.Plant SciencePhD (Plant Physiology)Unrestricte