Corrigendum: An in vitro study to elucidate the effects of Product Nkabinde on immune response in peripheral blood mononuclear cells of healthy donors.

[This corrects the article DOI: 10.3389/fphar.2024.1308913.].

An in vitro study to elucidate the effects of product Nkabinde on immune response in peripheral blood mononuclear cells of healthy donors.

Introduction: A significant number of the South African population still rely on traditional medicines (TM) as their primary healthcare due to their belief in their holistic healing and immune-boosting properties. However, little to no scientific data is available on the effects of most TM products on cytokine and cellular biomarkers of the immune response. Here, we evaluated the impact of traditional medicine [Product Nkabinde (PN)] in inducing cellular and cytokine biomarkers of inflammation in peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers. Methods: PN was supplied by a local Traditional Health Practitioner (THP). The IC50 (half maximum concentration) of the standardized extract on isolated PBMCs was established using the cell viability assay over 24 h of incubation. Luminex and flow cytometry assays were used to measure cytokine and cellular levels in PBMCs stimulated with PN and/or PHA over 24, 48, and 72 h, respectively. Results: The IC50 concentration of PN in treated PBMCs was established at 325.3 µg/mL. In the cellular activation assay, the percentages of CD38-HLA-DR + on total CD4+ T cells were significantly increased in PBMCs stimulated with PN compared to unstimulated controls after 24 h (p = 0.008). PN significantly induced the production of anti-inflammatory IL-10 (p = 0.041); proinflammatory cytokines IL-1α (p = 0.003), TNF-α (p < 0.0001); and chemokine MIP-1ß (p = 0.046) compared to the unstimulated control after 24 h. At 48 h incubation, the production of proinflammatory cytokines IL-1α (p = 0.034) and TNF-α (p = 0.011) were significantly induced following treatment with PN. Conclusion: We conclude that the PN possesses in vitro immunomodulatory properties that may influence immune and inflammatory responses. More studies using PN are needed to further understand key parameters mediating induction, expression, and regulation of the immune response in the context of pathogen-associated infections.

HPLC-Based Purification and Isolation of Potent Anti-HIV and Latency Reversing Daphnane Diterpenes from the Medicinal Plant Gnidia sericocephala (Thymelaeaceae).

Despite the success of combination antiretroviral therapy (cART), HIV persists in low- and middle-income countries (LMIC) due to emerging drug resistance and insufficient drug accessibility. Furthermore, cART does not target latently-infected CD4+ T cells, which represent a major barrier to HIV eradication. The "shock and kill" therapeutic approach aims to reactivate provirus expression in latently-infected cells in the presence of cART and target virus-expressing cells for elimination. An attractive therapeutic prototype in LMICs would therefore be capable of simultaneously inhibiting viral replication and inducing latency reversal. Here we report that Gnidia sericocephala, which is used by traditional health practitioners in South Africa for HIV/AIDS management to supplement cART, contains at least four daphnane-type compounds (yuanhuacine A (1), yuanhuacine as part of a mixture (2), yuanhuajine (3), and gniditrin (4)) that inhibit viral replication and/or reverse HIV latency. For example, 1 and 2 inhibit HIV replication in peripheral blood mononuclear cells (PBMC) by >80% at 0.08 µg/mL, while 1 further inhibits a subtype C virus in PBMC with a half-maximal effective concentration (EC50) of 0.03 µM without cytotoxicity. Both 1 and 2 also reverse HIV latency in vitro consistent with protein kinase C activation but at 16.7-fold lower concentrations than the control prostratin. Both 1 and 2 also reverse latency in primary CD4+ T cells from cART-suppressed donors with HIV similar to prostratin but at 6.7-fold lower concentrations. These results highlight G. sericocephala and components 1 and 2 as anti-HIV agents for improving cART efficacy and supporting HIV cure efforts in resource-limited regions.

In vitro dual activity of Aloe marlothii roots and its chemical constituents against Plasmodium falciparum asexual and sexual stage parasites.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe marlothii A.Berger (Xanthorrhoeaceae) is indigenous to southern African countries where its aqueous preparations are used in traditional medicine to treat several ailments including hypertension, respiratory infections, venereal diseases, chest pain, sore throat and malaria. AIM OF THE STUDY: The aims of this study were as follows: (i) isolate and identify the antiplasmodial active compounds in A. marlothii roots. As the water extract was previously inactive, the dichloromethane:methanol (DCM:MeOH) (1:1) was used, (ii) examine the activity of the isolated compounds against Plasmodium falciparum asexual blood stage (ABS) parasites as well as for transmission-blocking activity against gametocytes and gametes, and (iii) to use in silico tools to predict the target(s) of the active molecules. MATERIALS AND METHODS: The crude DCM:MeOH (1:1) extract of A. marlothii roots was fractionated on a reverse phase C8 column, using a positive pressure solid-phase extraction (ppSPE) workstation to produce seven fractions. The resulting fractions and the crude DCM:MeOH extract were tested in vitro against P. falciparum (NF54) ABS parasites using the malaria SYBR Green I based-fluorescence assay. Flash silica chromatography and mass-directed preparative high-performance liquid chromatography were utilised to isolate the active compounds. The isolated compounds were evaluated in vitro against P. falciparum asexual (NF54 and K1 strains) and sexual (gametocytes and gametes) stage parasites. Molecular docking was then used for the in silico prediction of targets for the isolated active compounds in P. falciparum. RESULTS: The crude extract and two SPE fractions displayed good antiplasmodial activity with >97% and 100% inhibition of ABS parasites proliferation at 10 and 20 µg/mL, respectively. Following UPLC-MS analysis of these active fractions, a targeted purification resulted in the isolation of six compounds identified as aloesaponol I (1), aloesaponarin I (2), aloesaponol IV (3), ß-sorigenin-1-O-methylether (4), emodin (5), and chrysophanol (6). Aloesaponarin I (2) was the most bioactive, compared to other isolated constituents, against P. falciparum ABS parasites exhibiting equipotency against the drug-sensitive (NF54) (IC50 = 1.54 µg/mL (5 µM)) and multidrug-resistant (K1) (IC50 = 1.58 µg/mL (5 µM)) strains. Aloesaponol IV (3) showed pronounced activity against late-stage (>90% stage IV/V) gametocytes (IC50 = 6.53 µg/mL (22.6 µM)) demonstrating a 3-fold selective potency towards these sexual stages compared to asexual forms of the parasite (IC50 = 19.77 ± 6.835 µg/mL (68 µM)). Transmission-blocking potential of aloesaponol IV (3) was validated by in vitro inhibition of exflagellation of male gametes (94% inhibition at 20 µg/mL). In silico studies identified ß-hematin and DNA topoisomerase II as potential biological targets of compounds 2 and 3, respectively. CONCLUSION: The findings from our study substantiate the traditional use of A. marlothii to treat malaria. To our knowledge, this study has provided the first report on the isolation and identification of antiplasmodial compounds from A. marlothii roots. Furthermore, our study has provided the first report on the transmission-blocking potential of one of the compounds from the genus Aloe, motivating for the investigation of other species within this genus for their potential P. falciparum transmission-blocking activity.

Use of hyphenated analytical techniques to identify the bioactive constituents of Gunnera perpensa L., a South African medicinal plant, which potently inhibit SARS-CoV-2 spike glycoprotein-host ACE2 binding.

SARS-CoV-2, the causative agent of COVID-19, continues to cause global morbidity and mortality despite the increasing availability of vaccines. Alongside vaccines, antivirals are urgently needed to combat SARS-CoV-2 infection and spread, particularly in resource-limited regions which lack access to existing therapeutics. Small molecules isolated from medicinal plants may be able to block cellular entry by SARS-CoV-2 by antagonising the interaction of the viral spike glycoprotein receptor-binding domain (RBD) with the host angiotensin-converting enzyme II (ACE2) receptor. As the medicinal plant Gunnera perpensa L. is being used by some South African traditional healers for SARS-CoV-2/COVID-19 management, we hypothesised that it may contain chemical constituents that inhibit the RBD-ACE2 interaction. Using a previously described AlphaScreen-based protein interaction assay, we show here that the DCM:MeOH extract of G. perpensa readily disrupts RBD (USA-WA1/2020)-ACE2 interactions with a half-maximal inhibition concentration (IC50) of < 0.001 µg/mL, compared to an IC50 of 0.025 µg/mL for the control neutralising antibody REGN10987. Employing hyphenated analytical techniques like UPLC-IMS-HRMS (method developed and validated as per the International Conference on Harmonization guidelines), we identified two ellagitannins, punicalin (2.12% w/w) and punicalagin (1.51% w/w), as plant constituents in the DCM:MeOH extract of G. perpensa which antagonised RBD-ACE2 binding with respective IC50s of 9 and 29 nM. This good potency makes both compounds promising leads for development of future entry-based SARS-CoV-2 antivirals. The results also highlight the advantages of combining reverse pharmacology (based on medicinal plant use) with hyphenated analytical techniques to expedite identification of urgently needed antivirals.

Lobostemon trigonus (Thunb.) H. Buek, a medicinal plant from South Africa as a potential natural microbicide against HIV-1.

ETHNOPHARMACOLOGICAL RELEVANCE: There have been different methods proposed to prevent the sexual transmission of HIV-1 and many of them have centered on the use of anti-retrovirals as microbicides. Given that a large section of the African population still relies on herbal medicine, Lobostemon trigonus (L. trigonus), a traditionally used medicinal plant in South Africa to treat HIV-1 was further investigated for its potential as a natural microbicide to prevent the sexual transmission of HIV-1. METHODS: The aerial parts of L. trigonus were oven-dried at 80 °C, ground, extracted with boiling water for 30 min and then filtered. The aqueous extract produced was then bioassayed using different HIV-1 inhibition assays. The active components were purified and chemically profiled using ultra-performance liquid chromatography/quadrupole time-of flight mass spectrometry (UPLC-qTOF-MS). The mechanism of HIV-1 inhibition was determined by fusion arrest assay and time of addition assay. Molecular modelling and molecular dynamic simulations, using Schrödinger, were used to better understand the molecule's mechanism of entry inhibition by evaluating their docking affinity and stability against the gp120 of HIV-1. RESULTS: The aqueous extract of this plant had a broad spectrum of activity against different subtypes of the virus; neutralizing subtype A, B and C in the TZM-bl cells, with IC50 values ranging from 0.10 to 7.21 µg/mL. The extract was also inhibitory to the virus induced cytopathic effects in CEM-SS cells with an EC50 of 8.9 µg/mL. In addition, it inhibited infection in peripheral blood mononuclear cells (PBMC) and macrophages with IC50 values of 0.97 and 4.4 µg/mL, respectively. In the presence of vaginal and seminal simulants, and in human semen it retained its inhibitory activity albeit with a decrease in efficiency, by about 3-fold. Studies of the mode of action suggested that the extract blocked HIV-1 attachment to target cells. No toxicity was observed when the Lactobacilli strains, L. acidophilus, L. jensenii, and L. crispatus that populate the female genital tract were cultured in the presence of L. trigonus extract. UPLC-qTOF-MS analyses of the purified fraction of the extract, confirmed the presence of six compounds of which four were identified as rosmarinic acid, salvianolic acids B and C and lithospermic acid. The additional molecular dynamic simulations provided further insight into the entry inhibitory characteristics of salvianolic acid B against the HIV-1 gp120, with a stable pose being found within the CD4 binding site. CONCLUSION: The data suggests that the inhibitory effect of L. trigonus may be due to the presence of organic acids which are known to possess anti-HIV-1 properties. The molecules salvianolic acids B and C have been identified for the first time in L. trigonus species. Our study also showed that the L. trigonus extract blocked HIV-1 attachment to target cells, and that it has a broad spectrum of activity against different subtypes of the virus; thus, justifying further investigation as a HIV-1 microbicide.