Antiinflammatory Medicinal Plants from the Ugandan Greater Mpigi Region Act as Potent Inhibitors in the COX-2/PGH2 Pathway

Abstract

Our study investigates 16 medicinal plants via assessment of inhibition of proinflammatory enzymes such as cyclooxygenases (COX). The plants are used by traditional healers in the Greater Mpigi region in Uganda to treat inflammation and related disorders. We present results of diverse in vitro experiments performed with 76 different plant extracts, namely, (1) selective COX-2 and COX-1 inhibitor screening; (2) 15-LOX inhibition screening; (3) antibacterial resazurin assay against multidrug-resistant Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, and Escherichia coli K12; (4) DPPH assay for antioxidant activity; and (5) determination of the total phenolic content (TPC). Results showed a high correlation between traditional use and pharmacological activity, e.g., extracts of 15 out of the 16 plant species displayed significant selective COX-2 inhibition activity in the PGH₂ pathway. The most active COX-2 inhibitors (IC₅₀ < 20 µg/mL) were nine extracts from Leucas calostachys, Solanum aculeastrum, Sesamum calycinum subsp. angustifolium, Plectranthus hadiensis, Morella kandtiana, Zanthoxylum chalybeum, and Warburgia ugandensis. There was no counteractivity between COX-2 and 15-LOX inhibition in these nine extracts. The ethyl acetate extract of Leucas calostachys showed the lowest IC₅₀ value with 0.66 µg/mL (COX-2), as well as the most promising selectivity ratio with 0.1 (COX-2/COX-1). The TPCs and the EC₅₀ values for DPPH radical scavenging activity showed no correlation with COX-2 inhibitory activity. This led to the assumption that the mechanisms of action are most likely not based on scavenging of reactive oxygen species and antioxidant activities. The diethyl ether extract of Harungana madagascariensis stem bark displayed the highest growth inhibition activity against S. aureus (MIC value: 13 µg/mL), L. innocua (MIC value: 40 µg/mL), and L. monocytogenes (MIC value: 150 µg/mL). This study provides further evidence for the therapeutic use of the previously identified plants used medicinally in the Greater Mpigi region.

Keywords: Albizia coriaria; Cassine buchananii; Combretum molle; Erythrina abyssinica; Ficus saussureana; Harungana madagascariensis; Leucas calostachys; Microgramma lycopodioides; Morella kandtiana; Plectranthus hadiensis; Securidaca longipedunculata; Sesamum calycinum subsp. angustifolium; Solanum aculeastrum; Toddalia asiatica; Warburgia ugandensis; Zanthoxylum chalybeum; antibiotics; cyclooxygenase; ethnopharmacology; fever; inflammation; lipoxygenase; pain; traditional medicine.

Figure 1

Ethnopharmacological information, describing the traditional use of 16 medicinal plants from the Greater Mpigi region in Uganda (with emphasis on the treatment of cardinal signs of acute inflammation, cancer, and stomach and gastrointestinal (GI) tract infections). The histogram shows the relative frequencies of citation (RFCs) in %, a field assessment index that was calculated from an ethnobotanical survey of 39 traditional healers. The RFC describes the use of plants to treat a specific medical condition relative to the total number of interviewees in the study, assessing the significance of a plant species in the local traditional medicine system (y-axis). This ethnobotanical index can vary from 0% (no survey participant uses this plant in treatment of a specific medical condition) to 100% (all survey participants use this plant in treatment of a specific medical condition) [5].

Figure 2

Cyclooxygenase-1/2 pathway and its physiological functions; COX, cyclooxygenase; NSAIDs, nonsteroidal antiinflammatory drugs; IL-1, interleukin 1; IL-4, interleukin 4; TNF, tumor necrosis factor; PGH₂, prostaglandin H2; PGE₂, prostaglandin E₂; PGF_2α, prostaglandin F_2α; PGD₂, prostaglandin D₂; PGI₂, prostaglandin I₂; TX, thromboxane.

Figure 3

Overview of the 12/15-lipoxygenase (12/15-LOX) pathway, highlighting biosynthesis of the antiinflammatory mediator lipoxin via a 15-S-hydroxyeicosatetraenoic acid (15(S)-HpETE) precursor with relevance to the 15-LOX inhibition assay presented in this study [48].

Figure 4

Scheme describing the COX inhibition assay used for screening plant extracts for antiinflammatory activity.

Figure 5

Results of the 15-LOX inhibition extract library counterscreen at 10 μg/mL; positive control tested at 3.024 μg/mL and 0.3024 μg/mL.

Figure 6

Results of the in vitro investigation of antioxidant activity (free radical scavenging activity) and determination of TPC for assessment of potential mechanism of action of the COX-2/1 and 15-LOX inhibition activity; plant extracts identified in the initial library COX-2 screen for COX-2/COX-1 dose-response inhibition experiments are marked with *; n.a. = not available.

Figure 7

LOX-15 inhibition assay flow sheet depicting the experimental procedure.