An in vitro investigation of the immunomodulatory effects of a traditional polyherbal traditional medicine product

Abstract

In South Africa, human immunodeficiency virus (HIV) remains one of the major public health concerns. HIV is a highly infectious lentivirus which causes a progressive degeneration of the immune system. When not treated, it leads to the development of acquired immunodeficiency syndrome (AIDS). AIDS is an immunosuppressive disease that results in opportunistic infections that can lead to death. Antiretroviral therapy (ART) is the most common and widely used treatment. Despite the availability of ART, HIV prevalence is on the rise. This has led to a surge in traditional herbal preparations that are purported as immune boosters for treatment in HIV patients. It is known that many patients switch from using ART to traditional herbal medicine due to side effects associated with ART. In South Africa and on the African continent, traditional medicine is not used only as an alternative and supplementary medicine, but also as a primary source of health care due to its ease of accessibility, affordability and also as it forms part of traditional and cultural practices. Safety and efficacy of many traditional medicines is not yet established and the pharmacological properties of the latter are based on anecdotal evidence with no scientific evidence. Prijap Health is a herbal traditional medicine preparation that is sold as an immune booster for immunocompromised patients. Prijap Health traditional herbal medicine is comprised of the following plants: Acorus calamus, Aloe arborescens, Artemisia afra, Drimia robusta, Elephantorrhiza elephantina, Erythrina lysistemon, Persea americana, Senecio serratuloides and Xysmalobium undulatum. It is claimed to have anti-viral, antioxidant, anti-inflammatory, blood cleansing and appetising properties. It is currently traded in select stores across South Africa. The aim of this study was to investigate the immune modulating properties of Prijap Health herbal concoction and the individual plant species that comprise the concoction. Water was used as a solvent to prepare plant extracts mimicking the traditional healer’s preparation technique. Phytochemical analysis was carried out using thin layer chromatography (TLC) and ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). Cytotoxicity of the extracts was evaluated by the sulphorhodamine B (SRB) protein staining assay using differentiated human acute leukaemia monocytic (THP-1) and human histiocytic lymphoma (U937) cell lines after incubation for 72 h. The free-radical scavenging activity of the extracts was evaluated against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals to determine the antioxidant potential. Evaluation of prostaglandin E2 concentration in phorbol-12-myristate 13-acetate (PMA)-differentiated and lipopolysaccharide (LPS)-activated THP-1 and U937 was conducted using enzyme-linked immunosorbent assay (ELISA). Determination of Th1/Th2/Th17 (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-ᵞ and IL-17A) and human inflammatory (IL-8, IL-1β, IL-6, IL-10, TNF-α and IL-12p70) cytokines concentrations was done using BD cytometric bead array (CBA) kits. The TLC revealed the presence of flavonoids in the extracts of A. afra, E. elephantina, P. americana and S. serratuloides. Sterols were detected in the extracts of A. arborescens, E. elephantina, P. americana, X. undulatum. Saponins were present in the extracts of A. afra, A. calamus, D. robusta, E. elephantina and P. americana. Alkaloids were not detected in any of the extracts. Phytochemical markers were identified in the plant extracts using UPLC-MS. These include uzarin (X. undulatum), β-asarone (A. calamus), catechin (P. americana), (-)-epicatechin (E. elephantina), rutin (A. afra), D-saccharic acid (D. robusta), 3-caffeoylquinic acid (S. serratuloides) and aloesin (A. arborescens). From the SRB assay, it was evident that the hot water extracts of A. afra, A. arborescens, A. calamus, E. elephantina, E. lysistemon, P. americana and S. serratuloides had no cytotoxic effects in both cell lines at the highest concentration tested (IC50>100 µg/mL). The THP-1 cells were found to be more sensitive and indicated lower cell viability than the U937 cells. The D. robusta, X. undulatum extracts and Prijap Health traditional herbal medicine displayed a potential for toxicity in the THP-1 cell line with IC50 = 39.29±1.76, 76.05±1.21 and 74.52±1.31 µg/mL, respectively. The DPPH and ABTS radical scavenging activity was most potent for the E. elephantina extract (DPPH scavenging EC50= 6.98±1.04 µg/mL; ABTS scavenging EC50= 2.45±1.05 µg/mL) and was found to be comparable to that of Trolox, the positive control (DPPH EC50 = 7.39±1.04 µg/mL; ABTS EC50 = 1.79±1.04 µg/mL). Prijap Health traditional herbal medicine showed greater antioxidant activity than all the individual plant extracts with the exception of E. elephantina. Curcumin (positive control), Prijap, D. robusta and X. undulatum extracts decreased the extracellular and intracellular PGE2 concentration in THP-1 cells, but not in U937 cells. Cytokine concentration determination by flow cytometry revealed that curcumin (positive control), A. calamus, Prijap Health traditional herbal medicine, and E. elephantina significantly (p<0.05) reduced IL-1β concentration. Curcumin, A. calamus and Prijap significantly (p<0.05) reduced TNF-α concentration. E. elephantina, A. arborescens and P. americana increased IL-10 concentration whereas curcumin, A. afra and Prijap significantly (p<0.05) reduced IFN-ᵞ concentration in both THP-1 and U937 cells. These findings suggest that the above-mentioned extracts and Prijap Health traditional herbal medicine have the potential to be used as anti-inflammatory agents. In conclusion, the results of the present study provide evidence of the antioxidant, anti-inflammatory, immune modulatory potential as well as safety of use of Prijap Health traditional herbal medicine. With this said, E. elephantina as a single plant showed to contain better activity than Prijap Health traditional herbal medicine concoction. Further studies in drug-herb interaction and antiviral properties should be conducted to investigate all the pharmacological properties of Prijap Health traditional herbal medicine.