Evaluation of Withania somnifera based supplement for immunomodulatory and antiviral properties against viral infection

Abstract

Background: Viral mediated diseases are continuously posing potent threat to human health. Nutraceuticals are being employed as novel therapeutics during viral outbreaks. MAM granules consist of Curcuma longa, Withania somnifera, and Piper nigrum, is one such patented Siddha nutraceutical supplement that has been proposed to be a therapeutic agent against viral diseases.

Objective: We characterised MAM for their phytochemical and physicochemical properties and evaluated its cytotoxicity via in vivo acute toxicity studies using Wistar rats and by cell-based MTT assays.

Materials and methods: The antiviral properties of the aqueous extract of MAM were investigated against SARS-CoV-2 and chikungunya virus (CHIKV). Further, using ABTS radical scavenging, SOD enzymatic assays and measurement of intracellular ROS, we investigated the antioxidant potential of MAM extract and its ingredients in RAW264.7 cells. Additionally, production of inflammatory mediators was evaluated via NO release, PGE2 production and release of pro-inflammatory cytokines (IL-1β and TNFα).

Results: The MAM granules and aqueous extracts demonstrated no significant toxicity and demonstrated potent antiviral activity during co-incubation assay with SARS-CoV-2 and CHIKV. Moreover, we observed potent antioxidant and anti-inflammatory activity of MAM extract in a dose dependent manner. Further investigations on the individual ingredients with respect to their antioxidant and anti-inflammatory activities showed that all ingredients contributed synergistically and Withania somnifera showed most potent anti-oxidant activity.

Conclusion: The overall in vitro, and in vivo analysis demonstrated that MAM granules were non-toxic and possessed potent antiviral activity. Additionally, observed significant anti-oxidant and anti-inflammatory properties of MAM suggested the modulation of innate immune response in the host validating its use as an effective nutraceutical during viral outbreaks.

Keywords: Antioxidant; Antiviral; Curcuma longa (Manjal); Inflammation; MAM granules; Nutraceutical; Piper nigrum (Milagu); SARS-CoV-2; Siddha; Withania somnifera (Amukkara).

Fig. 1

Microscopy analysis of MAM powder. (A–D) Parenchyma cells, Oleoresin cells, spiral vessels, and oil cells indicating Curcuma longa. (E–H) Perisperm cells, beaker-shaped stone cells, isodiametric stone cells, and starch grains indicating Piper nigrum. (I–L) Parenchyma cells, bordered pitted vessels, tracheid and starch grains indicating Withania somnifera.

Fig. 2

TLC photodocumentation of MAM granules. The TLC photos of MAM granules at UV 254, 366 nm and 520 nm after derivatization with vanillin-sulphuric acid showing presence of Curcuma longa, Piper nigrum and Withania somnifera respectively in the formulation.

Fig. 3

HPTLC Finger print profiling

Fig. 4

Antiviral activity of MAM aqueous extract against SARS-CoV-2 and CHIKV. (A) Shows cytotoxicity data of MAM aqueous extract treatment after 48 hours of incubation via MTT assay using Vero-E6 cells. Control represents untreated Vero-E6 cells. The bars represent mean ± SD. (B&C) demonstrates the antiviral activity of MAM aqueous extract during co-incubation and pre-incubation assay respectively with SARS-CoV-2 using Vero-E6 cells. VO represents untreated SARS-CoV-2 infected Vero-E6 cells and served as positive control to compare viral inhibition upon MAM treatment. The bars represent mean ± SD. (D–F) Shows antiviral activity for CHIKV using co-incubation, pre-treatment and post-treatment of MAM extract respectively using Vero cells. Control represents untreated CHIKV infected Vero cells. The bars represent mean ± SD.

Fig. 5

Investigation of cytotoxicity, anti-oxidant and anti-inflammatory properties of MAM aqueous extract. (A) Shows percentage cell survival of RAW 264.7 cells upon treatment of MAM via MTT assay. Control represents untreated RAW 264.7 cells. LPS represents RAW 264.7 cells treated with LPS. RAW 264.7 cells treated with 0.04, 0.4 and 4 mg/ml of MAM and 2 μg/ml of LPS showed no significant cytotoxicity. (B) demonstrate free radical scavenging capacity of MAM accessed via ABTS assay using RAW 264.7 cells. (C) Shows intracellular level of ROS induced by LPS and upon MAM treatment in RAW 264.7 cells. (D) Shows increase in intracellular SOD activity after MAM treatment in a dose dependant manner in RAW 264.7 cells. Untreated cells served as negative control and LPS treated cells as positive control. (E&F) shows effect on MAM treatment on the production of inflammatory mediators, NO measured as nitrite and PGE2 respectively using RAW 264.7 cells. Untreated cells served as negative control and LPS treated cells as positive control. (G&H) reflects the capability of MAM aqueous extract to counter the release of pro-inflammatory cytokines, TNF-alpha and IL-1 in RAW 264.7 cells. LPS treatment and untreated cells served as positive control and negative control respectively. The bars represent mean ± SD. One way ANOVA was used to calculate P-values. ∗P < 0.05, ∗∗P < 0.001 vs LPS treated Group.

Fig. 6

Investigation of anti-oxidant activity and cytotoxicity of MAM ingredients in RAW 264.7 cells. (A&B) demonstrates free radical and intracellular ROS scavenging capacity of each ingredient of MAM in a dose dependant manner assessed via ABTS and ROS assay respectively. Cl indicates Curcuma longa, Pn indicates Piper nigrum and Ws indicates Withania somnifera. (C) Shows increase in intracellular SOD activity after MAM treatment in RAW 264.7 cells. Untreated cells served as negative control and LPS treated cells as positive control. (D–G) Shows cytotoxicity of the three ingredients, Curcuma longa (Cl), Piper Nigrum (Pn) and Withania somnifera (Ws) respectively. The bars represent mean ± SD.