Научная статья на тему 'Investigation of the interactions between human gut bacteria and herbal medicinal products in vitro using a combined lc-hrms- and 16S RNA sequencing approach'

Investigation of the interactions between human gut bacteria and herbal medicinal products in vitro using a combined lc-hrms- and 16S RNA sequencing approach Текст научной статьи по специальности «Биологические науки»

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Текст научной работы на тему «Investigation of the interactions between human gut bacteria and herbal medicinal products in vitro using a combined lc-hrms- and 16S RNA sequencing approach»


© Fernando Pereira Beserra1, Gabriela Lemos de Azevedo Maia2, Ariane Leite Rozza1, Claudia Helena Pellizzon1, Meilang Xue3, Christopher Jackson3

1 Department of Morphology, Institute of Biosciences, Sao Paulo State University, Botucatu, Sao Paulo, Brazil;

2 Department of Pharmacy, Federal University of Sao Francisco Valley, Petrolina, Pernambuco, Brazil;

3 Sutton Arthritis Research Laboratories, Kolling Institute of Medical Research, University of Sydney, Sydney, Australia

Keratinocytes are a major cell type of human skin and play a fundamental role in normal skin metabolism and cutaneous wound healing. Many natural products and medicinal plants have been used for study in vitro in the wound healing. For that reason, the aim of this study was to evaluate the effect of Lupeol (LU) on cell migration and wound healing using the scratching model as well as to determine its signaling mechanisms. Human primary epidermal keratinocytes were obtained from human neonatal foreskin in accordance to the guidelines of the local ethics committee. Cells were seeded into 24-well plates and cultured to subconfluence for in vitro migration and wound healing "scratch" assay treated with LU in different concentrations at 0.1, 1, 10 or 20 ^g/mL. Keratinocytes were lysed with lysis buffer, supplemented with protease inhibitor and phosphate inhibitor for western blot analysis. The primary antibodies used were: Akt, phospho-Akt, Erk, phospho-Erk, Tie-2, phospho-Tie-2, NF-kBp65 and MMP-2. Results were expressed as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed by one-way

analysis of variance (ANOVA) followed by Tukey test. P< 0.05 was considered significant. In the in vitro test performed to evaluate wound healing, LU accelerated wound closure at all concentrations used in the study, whereas in the cellular migration assay, only the two lowest concentrations (0.1 and 1 ^g/mL) stimulated cell migration significantly in relation to the control. LU also showed increase in the protein expression of Akt, phospho-Akt, Tie-2, phospho-Tie-2 and MMP-2 and a decrease in the NF-kB expression was observed. These results demonstrate a central role for LU in promoting cell migration through activating Akt and its phosphorylated form (p-Akt), Tie-2 and phospho-Tie-2, reduction of inflammation by inhibition of NF-kB, and increasing of MMP-2 expression. These regulatory properties implicate lupeol as having potential to promote re-epithelialization during wound healing and remains a novel and exciting avenue of investigation.

Financial support: CsF/CNPq: 202513/2015-71, Fapesp: 2014/23247-4 and SuttonLab


© Eva-Maria Pferschy-Wenzig13, Kaisa Koskinen2,3, Alexandra Rossmann2, Karin Ardjomand-Woelkart1, Christine Moissl-Eichinger23, Rudolf Bauer1,3

11nstitute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz; Graz, Austria;

2 Department of Internal Medicine, Medical University Graz; Graz, Austria;

3 BioTechMed, Graz, Austria

The microbial community colonizing the distal gut does not only have a strong impact on its human host's health, but also an enormous metabolic capacity. Compounds orally ingested via food or medicines that remain unabsorbed in the upper gastrointestinal tract are potentially degraded by gut microbes. This can lead to the formation of potentially bioavailable and bioactive metabolites. This aspect was often A platform should be established for studying the interaction between herbal medicinal product constituents and human gut bacteria. Several approved herbal medicinal products should be

tested, including willow bark extract, hawthorn leaves and flowers extract, and a fixed combination consisting of nine herbal extracts.

Extracts were incubated with 10% human fecal suspension under physiological conditions for 24 h. Samples were taken at 0.5 h, 4 h and 24 h. Changes in the composition of plant extracts were monitored by LC-HRMS analysis. Microbiome shifts were tracked by means of 16S RNA sequencing.

LC-HRMS analysis allowed to detect the microbial degradation of many compound classes occurring in

Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] vol. 15/2017/suppLement 1

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the studied extracts, including flavonoid glycosides and aglycones, procyanidins, salicyl alcohol derivatives, caffeic acid derivatives, and triterpene glycosides. Numerous newly formed compounds could be assigned to putative metabolites derived from these compound classes. Microbiome analyses allowed insights in microbial community changes and identification of some of the bacterial species that significantly increased or decreased upon incubation with the studied herbal extracts.

The combined LC-HRMS and next-generation sequencing approach allowed straightforward detection of potentially interesting microbiome shifts and metabolites resulting from the interaction between herbal constituents and gut microbes. In depth studies of the observed effects, including their verification in a higher number of different fecal samples from healthy and diseased donors, will follow.


© Picking D.1, Chambers B.2, Barker J.3, Shah I.4, Porter R.5, Delgoda R.1

1 Natural Products Institute, University of the West Indies, Kingston, Jamaica;

2 Tropical Medicine Research Institute, University of the West Indies, Kingston, Jamaica;

3 School of Life Sciences, Pharmacy & Chemistry, Kingston University, UK;

4 College of Science, United Arab Emirates University, Al-Ain United Arab Emirates;

5 Department of Chemistry, University of the West Indies, Kingston, Jamaica

Understanding the potential for adverse drug reactions (ADRs), resulting from plant-drug interactions, is a key aspect of medicinal plant safety. The aim of this study was to provide an in-depth assessment of standardised extracts of Hyptis verticillata Jacq. through its impact on the activities of key human cytochrome P450 (CYP) enzymes in vitro and antioxidant properties.

Crude extracts were prepared from fresh and dried aerial plant material (leaf and stem). The aqueous dried plant extract, was evaluated for its ability to inhibit the activities of human CYPs 1A1, 1A2, 1B1, 3A4 and 2D6, and characterised using high performance liquid chromatography (HPLC) and liquid chromatography -mass spectrometry (LC-MS/MS). Individual key phytochemicals identified were further assessed for their ability to inhibit the activity of the enzyme most impacted. All crude extracts were assessed for antioxidant properties, using a standard DPPH radical scavenging assay.

The dried plant aqueous extract demonstrated potent inhibition (IC50 values < 10 ig/ml) against the activities of CYPs 1A1 (7.6 ig/ml), 1A2 (1.9 ig/ml), 1B1 (9.4 ig/ml) and 3A4 (6.8 ig/ml) and its analysis confirmed the identity of seven phytochemicals, five lignans and two triterpenes. Screening these phytochemicals against the activity of CYP1A2 failed to identify a single agent responsible for the plant extract's potent bioactivity. Further analysis of other crude extracts demonstrated potent inhibition of

CYP1A2 activity for a dried plant ethanol extract (1.5 | g/ml), fresh plant ethanol extract (3.9 | g/ml), and moderate activity for a fresh plant aqueous extract (27.8 ig/ml). All four extracts demonstrated strong antioxidant activity (IC50 values<10 ig/ml), compared to the positive control, ascorbic acid (1.3 | g/ml), with the dried plant ethanol extract being the most potent (1.6 ig/ml).

Aqueous extracts, prepared following ethnomedical recipes for H. verticillata, demonstrated inhibition of key drug metabolising enzymes in vitro. Further analysis on the potential impact of this plant on key drug metabolizing enzymes in vivo appears warranted, given the extensive use of this plant in the Americas. Key phytochemical(s) responsible for the extracts' bioactivity could not be pinpointed, and led to one of two possible explanations: Firstly, the phytochemical(s) responsible has/have not yet been identified, or, secondly, the potent bioactivity is due to synergistic interactions amongst the many constituents of the whole plant extract being greater than the sum of individual phytochemical bioactivities tested. Four traditional preparations demonstrated strong antioxidant activity, which, together with the plant extracts potent inhibition of known carcinogen activators, CYPs 1A1 and 1B1, and previously reported anti-inflammatory and anticancer properties, warrants further research into the potential chemopreventive properties of this interesting plant.

Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy]

vol. 15/2Q17/suppLeMEnT 1

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