Antifungal activity and toxicity assessment of traditional medicinal plants commonly used in the treatment of respiratory diseases in South Africa

Abstract

Cryptococcus neoformans, Cryprococcus gattii, Aspergillus fumigatus and Candida albicans cause fungal respiratory coinfections in Human immunodeficiency virus (HIV) and Coronavirus disease 2019 (COVID-19) patients. Medicinal plants have been reported to have antifungal activities and other pharmacological properties against these fungal species. Defender is the indigenous herbal medicinal supplement prepared from medicinal plants, including Zingiber officinale, Salvia rosmarinus, Petroselium crispum, Allium sativum, Capsicum annuum, and Cannabis sativa, which have been used traditionally to treat various respiratory conditions and diseases. Felicia and Searsia species have been used traditionally to treat respiratory diseases and conditions. The aim of the study was to assess the antifungal activity and toxicity of traditional medicinal plants commonly used in the treatment of respiratory diseases in South Africa. This study also determined other pharmacological activities of these medicinal plants. Qualitative phytochemical analysis included tests for flavonoids, anthraquinones, tannins, steroids, and terpenoids. Quantitative phytochemical analysis included tests for total flavonoid and total phenolic contents and liquid chromatography coupled with mass spectrometry (LCMS). Total flavonoid and phenolic contents were tested using the Folin-Ciocalteu reagent and the aluminium chloride colorimetric method and were measured spectrophotometrically. The antioxidant activity was analysed using the diphenyl picrylhydrazyl (DPPH) radical scavenging method. The anti-inflammatory activities of the medical plant extract and Defender were determined by measuring their ability to inhibit nitric oxide production in RAW 264.7 macrophages activated by Lipopolysaccharide (LPS). Cytotoxicity of plant extracts and Defender was performed using the Hoechst 33342/Propidium iodide (PI) dual staining method and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Antifungal activity of plant extracts and Defender was determined against Cryptococcus neoformans, Cryptococcus gattii, Candida albicans and Aspergillus fumigatus using the serial dilution assay. The potential inhibition of plant extracts on human P450 3A4 was determined using a Vivid® CYP450 Screening Kit. Plant extracts and Defender were tested against Cryptococcus gattii and Cryptococcus neoformans in the absence and presence (100-800 μg/ml) of exogenous ergosterol, and their Minimum Inhibitory Concentrations (MICs) were determined after 24 h of incubation at 35°C. Phytochemical analysis revealed the presence of flavonoids, tannins, anthraquinones, and steroids in the aqueous and methanol extracts of Searsia erosa and Felicia filifolia, and steroids and flavonoids in Defender. The highest phenolic contents were found in Searsia erosa methanol extract (426±0.1 mg GAE/g sample) and Defender (236±0.0 mg GAE/g sample), while Felicia filifolia aqueous extract had a phenolic amount of 127±0.0 mg GAE/g sample. The highest flavonoid amounts were observed in Searsia erosa methanol extract (257±3.3 mg QE/g extract) and Defender (86±1.4 mg QE/g extract), and Felicia filifolia methanol extract had a flavonoid content of 83±1.1 mg QE/g extract. LCMS analysis of Felicia filifolia methanol extract revealed the presence of Acerosin, Aphidicolin, Dictamnoside B, Lamiide, and Prostaglandin E2, 5,7,4'-Trihydroxy-3,6,8,3',5'-pentamethoxyflavone, Ranupenin 3-rutinoside, Rutin, Scandoside, and Loniphenyruviridoside D. LCMS analysis of Searsia erosa confirmed the presence of phytochemicals, including chlorogenic acid, caffeoyl quinic acid, 3,4-Dicaffeoyl quinic acid, flavonoid-7-O-glycoside, pentacarboxylic acids, quinic acids and derivatives, saccharolipids, and unspecified terpene glycosides. The antioxidant activities of Defender (74%), Searsia erosa methanol extract (74%), and Felicia filifolia aqueous extract (68%) were higher than those of the positive controls, trolox (64%) and ascorbic acid (53%), at a concentration of 250 μg/mL. The Felicia filifolia methanol extract at concentrations of 50 and 200 μg/ml, the Searsia erosa methanol extract at 7.5 μg/mL, the Searsia erosa aqueous extracts at 50 and 200 μg/mL, and Defender at 500 μg/mL demonstrated potential anti-inflammatory activities. Felicia filifolia methanol extract exhibited the most significant cytotoxicity at a minimum concentration (MIC) of 62.5 μg/mL on Vero cells. Searsia erosa aqueous extract (MIC=1 μg/mL), Defender (MIC=1, 10, 50 μg/mL), and Felicia filifolia methanol extract (MIC=1 μg/mL) were effective against C. neoformans, C. gattii, C. albicans, and A. fumigatus. Searsia erosa methanol extract (MIC=1 μg/mL) only inhibited the growth of C. albicans and A. fumigatus. MICs of all tested extracts and Defender increased in the presence of ergosterol, and the increase was concentration dependent. Results of the CYP3A4 activity assay revealed a concentration-dependent decrease in substrate production for tested extracts. Searsia erosa and Felicia filifolia methanol extracts are potential anti-inflammatory, antioxidant, and cytotoxic (anticancer) agents. They are also potential antifungal treatments against C. albicans, C. neoformans, C. gattii, and A. fumigatus. Defender is a potential antioxidant, anti-inflammatory agent, and antifungal treatment against C. albicans, A. fumigatus, C. neoformans, and C. gattii. Searsia erosa and Felicia filifolia are possible inhibitors of CYP3A4, and this finding has dose implications. Tested plant extracts and Defender are possible antifungal treatments for infections caused by C. neoformans and C. gattii, working by binding to ergosterol. Therefore, these medicinal plants and Defender can be used to treat fungal respiratory infections caused by C. albicans, C. neoformans, C. gattii, and A. fumigatus in COVID-19 and HIV patients.