Elemental analysis
Results presented in Table 1 showed the levels of ETEs (iron, zinc and calcium) concentration in FMB extract and standardised extract, respectively. From the result, it is ascertained that ETEs were successfully replenished into the standardised extract.
Table 1
The content of ETEs analyzed by ICP-OES (n = 3)
Name of ETEs | ETEs in FMB extract (PPM) | ETEs in standardised extract (PPM) |
Zinc | 212.07 | 1.61 |
Iron | 32.51 | 3.75 |
Immunomodulatory Activity
Pinocytic assay
Both the test samples (standardized extract and FMB extract) treatment enhanced the pinocytic rate in a dose-dependent manner (Fig. 1). Figure 1 clearly shows that at initial concentration pinocytic activity was more pronounced in standardized extract alone but at higher doses i.e. 1000µg/ml FMB extract showed more processing results. FMB extract almost shows equivalent effects as standard septalin. We cultured mouse peritoneal macrophages in RPMI medium with or without supplementation of external stimuli and, after 48 hours of incubation, used neutral red, which is readily taken up by macrophages to measure the pinocytic activity. After the incubation of macrophages with neutral red, macrophages were lysed to measure the concentration of neutral red and determine the extent of pinocytic activity by the macrophages.
Splenocyte proliferation observed by MTT assay
The splenocyte was isolated and cultured in the RPMI medium for 72 hours with or without the test drug. Thereafter, the cell viability was measured using an MTT assay. Data obtained revealed that cell viability was significantly increased in the case of the test drug compared to the untreated cells. Interestingly, FMB extract showed higher cell viability. Here five different concentrations (65-1000 µg/ml) of the formulations were tested for splenocyte proliferation assay, and it was found that there was a dose-dependent increase in cell proliferation (Fig. 2).
Spleen is the body's largest immune organ, capable of producing a large number of lymphocytes. Splenocyte proliferation is a crucial event in the activation cascade of both cellular and humoral immune responses [13]. Our in vitro studies showed comparable spleen proliferation with the previously reported study [14].
The significance of Zinc in the growth and development of all living things has long been acknowledged as that an essential trace element. In recent years, it has been discovered that zinc homeostasis is crucial for the innate immune system, particularly for sustaining the functionality of macrophages. In another study, it is reported that zinc is one of the most important transition metals in the biological system. It is said to have an impact on more than 100 enzymes related to the nervous system, metabolism, stress management, immunity, and DNA transcription. It can function as an antioxidant, vitamin, and pro-oxidant when ingested in moderation. It is vital for the expression of essential antioxidant molecules like superoxide dismutase and metallothionein that maintain redox equilibrium in living things [15].
Iron homeostasis is essential for the health of several cellular processes, including DNA synthesis, electron transfer, oxygen transport, and storage. A previous study reported that iron deficiency in macrophages of individuals with hereditary hemochromatosis may actually increase their resistance to a number of intracellular bacterial pathogens [16]. The results obtained from the immumodulation assay affirmed that FMB extract possesses strong immunomodulatory efficacy as compared to standardized extract. It might occur due to present of the high content of iron and zinc. Possibly, ETEs synergize the action of bioactive metabolites present in the extract.
Cytotoxicity
To assess, the nontoxic concentrations of standardized extract and FMB extract, respectively for cytoprotective studies, we carried out an MTT-based cytotoxicity assay in HepG2 cells. The obtained results of standardized extract and FMB extract were presented in Figs. 3A and B. The standardized extract showed more significant toxicity as compared to FMB extract and was found safe up to 250 µg/ml. Kong et al described that the cytoprotective effect of the plant extracts is highly dependent on their concentration, bioavailability, and together with complex interaction among the phytochemicals [17].
Effect Of Standardized Extract And Fmb Extract On The Expression Of Tnf-α In Cell Line
The level of TNF-α was assessed in experimental groups against high glucose induce stress. After treatment with the formulations, the level of TNF-α was demised (Fig. 4). Standardized extract and FMB extract showed an anti-inflammatory response in a dose-dependent manner. FMB extract showed comparatively better activity as compared to standardized extract alone. Among various inflammatory cytokines, TNF-α contributes its decisive role in inducing tissue-specific inflammation which may weaken the defence system of the body. Previous literature cited that several anti-inflammatory molecules have been found to ameliorate immune reactions underlying many pathogeneses, via directly regulating production/function/activation of the prime components of the immune system and limiting tissue damage [18].
Cited literature revealed that supplementation of zinc in the elderly showed that the incidence of infections was decreased by approximately 66 percent in the zinc group. Zinc supplementation also decreased oxidative stress biomarkers. Zinc is involved in the modulation of the proinflammatory response by targeting Nuclear Factor Kappa B, a transcription factor that is the master regulator of proinflammatory responses [19]. Growth retardation, testicular failure, rough skin, low appetite, mental lassitude, delayed wound healing, T cell-mediated immunological dysfunction, and neurosensory abnormalities are only a few of the clinical symptoms that can occur even with a modest zinc deficit [20].
Iron and its homeostasis are directly related to the inflammatory response. The chronic disease or inflammation that results from the adaptation to iron deficiency, which imparts resistance to infection and intensifies the inflammatory condition, is likely the relationship that is most obvious [21]. Moreover, iron homeostasis is necessary for proper immune cell proliferation and maturation, particularly lymphocytes, associated with the generation of a specific response to infection [22].
According to reports, ETEs (Iron and Zinc) have a significant role in maintaining the immune defence system and have strong antioxidant capacity. They also boost the anti-inflammatory cytokine levels in the body's inflammatory response [6].