Abstract
BackgroundDysregulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) form the basis of immune-mediated inflammatory diseases. Vitex trifolia L. is a medicinal plant growing in countries such as China, India, Australia and Singapore. Its dried ripe fruits are documented in Traditional Chinese Medicine to treat ailments like rhinitis and dizziness. Its leaves are used traditionally to treat inflammation-related conditions like rheumatic pain.ObjectiveThis study aimed to investigate the effects of V. trifolia leaf extracts prepared by different extraction methods (Soxhlet, ultrasonication, and maceration) in various solvents on cytokine production in human U937 macrophages, and identify phytoconstituents from the most active leaf extract.MethodsFresh leaves of V. trifolia were extracted using Soxhlet, ultrasonication, and maceration in hexane, dichloromethane, methanol, ethanol or water. Each extract was evaluated for its effects on TNF-α and IL-1β cytokine production by enzyme-linked immunosorbent assay in lipopolysaccharide-stimulated human U937 macrophages. The most active extract was analyzed and further purified by different chemical and spectroscopic techniques.ResultsAmongst 14 different leaf extracts investigated, extracts prepared by ultrasonication in dichloromethane and maceration in ethanol were most active in inhibiting TNF-α and IL-1β production in human U937 macrophages. Further purification led to the isolation of artemetin, casticin, vitexilactone and maslinic acid, and their effects on TNF-α and IL-1β production were evaluated. We report for the first time that artemetin suppressed TNF-α and IL-1β production. Gas chromatography-mass spectrometry analyses revealed the presence of eight other compounds. To the best of our knowledge, this is the first report of butylated hydroxytoluene, 2,4-di-tert-butylphenol, campesterol and maslinic acid in V. trifolia leaf extracts.ConclusionsIn conclusion, leaf extracts of V. trifolia obtained using different solvents and extraction methods were successfully investigated for their effects on cytokine production in human U937 macrophages. The findings provide scientific evidence for the traditional use of V. trifolia leaves (a sustainable resource) and highlight the importance of conservation of medicinal plants as resources for drug discovery. Our results together with others suggest further investigation on V. trifolia and constituents to develop novel treatment strategies in immune-mediated inflammatory conditions is warranted.
Highlights
Dysregulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin1β (IL-1β) form the basis of immune-mediated inflammatory diseases
Amongst 14 different leaf extracts investigated, extracts prepared by ultrasonication in dichloromethane and maceration in ethanol were most active in inhibiting TNF-α and IL-1β production in human U937 macrophages
Further purification led to the isolation of artemetin, casticin, vitexilactone and maslinic acid, and their effects on TNF-α and IL-1β production were evaluated
Summary
Fresh leaves of V. trifolia were extracted using Soxhlet, ultrasonication, and maceration in hexane, dichloromethane, methanol, ethanol or water. Reagents Analytical grade solvents (acetone, dichloromethane, ethanol, ethyl acetate, hexane and methanol) and HPLC-grade methanol and acetonitrile were purchased from Tedia (Fairfield, USA). Chemical standards for artemetin, casticin and vitexilactone were from ChemFaces (China), while αamyrin, β-amyrin, butylated hydroxytoluene (BHT), campesterol, 2,4-Di-tert-butylphenol, maslinic acid, phytol, βsitosterol, and stigmasterol were from Sigma-Aldrich (USA). Plant source and preparation of plant extracts Fresh, healthy and mature leaves of V. trifolia were harvested from Singapore (Leeward Pacific Pte. Ltd) for extraction. A voucher specimen of V. trifolia (VT-0101) was deposited at the Department of Pharmacy Herbarium in National University of Singapore. Air dried and blended using a dry grinder, and extracted using Soxhlet, ultrasonication or maceration in hexane, dichloromethane, 70% v/v methanol, 70% v/v ethanol and water. The extracts were concentrated under vacuum and stored at 25 °C
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