Phytochemical Analysis, Antioxidant Potential and Antibacterial Activities of Different Anatomical Parts of Hypericum cordifolium Choisy.

The genus Hypericum comprises a large number of species. The flower, leaf, stem, and root of the Hypericum species are widely used in traditional medicine in different cultures. Many Hypericum species have been well investigated phytochemically and pharmacologically. However, only a few reports are available on the H. cordifolium native to Nepal. The present study aims to evaluate the phytochemical composition of different extracts, qualitative analysis of methanol extract of the flower and leaf using thin-layer chromatography (TLC), and the antioxidant properties of components by the TLC-DPPH. assay. The phenolic and flavonoid contents were estimated in different extracts of the leaf and stem, and their antioxidant and antibacterial activities were evaluated. In the phytochemical screening, phenolics and flavonoids were present in ethyl acetate, methanol, and 50% aq methanol extracts of both the leaf and stem. In TLC analysis, the methanol extract of flowers showed the presence of 11 compounds and the leaf extract showed the presence of 8 compounds. Both extracts contained chlorogenic acid and mangiferin. Hyperoside and quercetin were present only in the flower extract. In the TLC-DPPH. assay, almost all of the flower extracts and 5 compounds of the leaf extract showed radical scavenging potential. Estimation of phenolics and flavonoids showed that all the leaf extracts showed higher amounts of phenolics and flavonoids than stem extracts. Among leaf extracts, greater amounts of phenolics were detected in 50% aqueous methanol extract (261.25 ± 1.66 GAE/g extract) and greater amounts of flavonoids were detected in methanol extract (232.60 ± 10.52 CE/g extract). Among stem extracts, greater amounts of flavonoids were detected in the methanol extract (155.12 ± 4.30 CE/g extract). In the DPPH radical scavenging assay, the methanol extract of the leaf showed IC50 60.85 ± 2.67 µg/ml and 50% aq. methanol extract of the leaf showed IC50 63.09 ± 2.98 µg/ml. The methanol extract of the stem showed IC50 89.39 ± 3.23 µg/ml, whereas ethyl acetate and 50% aq. methanol extract showed IC50 > 100 µg/ml. In the antibacterial assay, the methanol extract of the leaf showed the inhibition zone of 12-13 mm and the stem extract showed the inhibition zone of 7-11 mm against S. aureus, E. coli, and S. sonnei, whereas both extracts were inactive against S. typhi. The findings of this study support the traditional use of this plant in Nepal for the treatment of diseases associated with bacterial infections. The present study revealed that the underutilized anatomical parts of H. cordifolium could be the source of various bioactive phytochemicals like other Hypericum species.

Pretreatment of Melanoma Cells with Aqueous Ethanol Extract from Madhuca longifolia Bark Strongly Potentiates the Activity of a Low Dose of Dacarbazine.

Madhuca longifolia is an evergreen tree distributed in India, Nepal, and Sri Lanka. This tree is commonly known as Mahua and is used in traditional medicine. It was demonstrated that ethanol extract from the bark of M. longifolia possessed potent cytotoxic activity towards two melanoma cell lines, in contrast to aqueous extract that exhibited no activity. Apart from being selectively cytotoxic to cancer cells (with no activity towards non-cancerous fibroblasts), the studied extract induced apoptosis and increased reactive oxygen species generation in melanoma cells. Additionally, the use of the extract together with dacarbazine (both in non-toxic concentrations) resulted in the enhancement of their anticancer activity. Moreover, the pretreatment of melanoma cells with M. longifolia extract potentiated the activity of a low dose of dacarbazine to an even higher extent. It was concluded that ethanol extract of M. longifolia sensitized human melanoma cells to chemotherapeutic drugs. It can therefore be interesting as a promising source of compounds for prospective combination therapy.

Bassia longifolia (= Madhuca longifolia): Isolation of flavan-3-ols and their contribution to the antibacterial and antidiabetic activity in vitro.

Bassia longifoliaKOENIG (= Madhuca longifolia (L.) is an evergreen tree that is widely distributed throughout Nepal, India, and Sri Lanka. The bark has various traditional uses: as a paste in the treatment of cuts and wounds or internally as a decoction that is given to diabetic patients. Chemical-analytical and pharmacological investigations regarding the bark are not sufficiently available. We focused on the isolation of flavan-3-ols from the methanolic extract and their contribution to the described traditional uses in wound healing and diabetes treatment. Therefore, an antibacterial assay and an α-glucosidase assay were performed. The isolation process was performed by a combination of Sephadex®-, MCI®-Gel-, and RP-18 chromatography. The structures of the isolated compounds were elucidated by 1H- and 13C-NMR-spectroscopy including COSY, ROESY, HSQC, and HMBC methods. Optical characterization was performed by polarimetry and circular dichroism. Two monomeric, seven dimeric, six trimeric, and one tetrameric flavan-3-ols were found including one dimer and three trimers with rare epiafzelechin units. Two compounds were isolated for the first time. A fraction containing higher oligomeric and polymeric proanthocyanidins (PAs) was examined by 13C NMR spectroscopy and revealed an average degree of polymerization of 8-9. PA with cis-configurated subunits predominated at 90 % and the presence of further monohydroxylated flavan-3-ols was revealed. Minimal inhibitory concentrations (MICs) were investigated by the serial microdilution broth assay with Staphylococcus aureus. The bacterial suspension was inoculated on agar plates for determining the MICs. The α-glucosidase assay was performed in 96 well plates with α-glucosidase from Bacillus stearothermophilus. For the detection of enzyme inhibition, p-nitrophenyl-α-d-glucopyranoside was used as a substrate and after incubation absorbance was measured at 405 nm. Antibacterial effects were only found for fractions enriched with PAs or containing higher oligomeric and polymeric flavan-3-ols. All tested substances showed high α-glucosidase inhibition. Whereby 4ß→8 conjugated dimers and the monomers showed the lowest inhibition, procyanidin (PC) B5 as 4ß→6 conjugated and cinnamtannin A2 as tetrameric flavan-3-ol showed the highest. PAs with epiafzelechin units are rarely found in nature but their reoccurring appearance in B. longifolia could be characteristic of this plant. For its traditional uses, the antibacterial activity of the PA-enriched fractions could contribute to the wound healing process when applied to the injured skin. Moreover, all tested substances and fractions showed α-glucosidase inhibition, which could also explain the use of a decoction in the treatment of diabetes. In conclusion, pharmacological investigations could provide scientific evidence for traditional uses of B. longifolia.

Influence of traditionally used Nepalese plants on wound healing and immunological properties using primary human cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The improvement of wound healing has always been an important issue for both ethnopharmacological and modern medical research. In this study, we used state-of-the-art methods to investigate extracts of plants used traditionally in Nepal for more than 1000 years to treat inflammatory injuries. AIM OF THE STUDY: We focused on the potential of the plant extracts to ameliorate wound healing and to influence immune modulatory properties. MATERIALS AND METHODS: Nine Nepalese plant extracts in three different solvents (methanol, ethyl acetate, petroleum ether) were immunologically characterised. Water-soluble tetrazolium (WST-1) assays and scratch assays were performed to determine their impact on viability and wound healing capacity of human keratinocytes and fibroblasts. Effects on proliferation, viability and function of physiologically relevant anti-CD3 and anti-CD28 stimulated primary human T lymphocytes were assessed using carboxyfluorescein succinimidyl ester (CFSE), annexin V/propidium iodide staining assays and flow cytometry-based surface receptor characterisation. The secretion level of interleukin-2 (IL-2) was analysed with the ELISA technique. Dendritic cells were generated out of peripheral blood mononuclear cells (PBMC) by CD14+ magnetic bead selection. Flow cytometry-based surface receptor characterisation and ELISA-based technique were used to evaluate the DC activation state and the interleukin-8 (IL-8) secretion level. RESULTS: We demonstrate that an ethyl acetate extract of Bassia longifolia and of Gmelina arborea have anti-inflammatory capacities, indicated by reduced proliferation, inhibition of IL-2 secretion and degranulation capacity of activated human T cells, when compared with adequate concentrations of synthetic positive drug controls. Furthermore, Gmelina arborea improved the wound healing of keratinocytes and fibroblasts and has tendency to increase the secretion of IL-8 by human primary dendritic cells. CONCLUSION: With this preliminary screening, we offer a scientific basis for the immunomodulatory properties of the two Nepalese medicinal plants Bassia longifolia and Gmelina arborea. However, further detailed studies regarding the responsible compounds are necessary.