In a normal cell, there is an appropriate balance between free radicals and antioxidants. However, this balance can be shifted towards free radicals when the production of reactive oxygen species is increased or when the antioxidants level is declined. This state is ‘oxidative stress’ and can lead to serious cell damage if stress is massive or prolonged [23,24]. Oxidative stress plays a major role in degenerative or pathological processes, such as diabetes mellitus, hypertension, cancers, heart failure, neurodegenerative disorders, etc [25] (Huang et al., 2005). Antioxidants from natural sources have attracted considerable attention from researchers and users on account of adverse toxicological reports of some synthetic antioxidants. In this context, medicinal plants are considered safer, cost-effective and cure the disease from its root cause [26]. Among these, medicinal plants are being viewed as an easily available and potent source of antioxidants as they contain a mixture of numerous different chemical compounds that may act individually or in synergy to cure disease and improve health [27]. In the majority of traditional systems like Ayurveda, African traditional medicine, Chinese system of medicine, etc., many diseases are better managed by herbs combination (Polyherbal) instead of single herb because of synergism and fewer side effects [28].

Polyherbal formulations magnify the therapeutic action and reduce the concentrations of single herbs, thereby reducing the adverse effects. In this study, 80% ethanolic extracts of a polyherbal formulation was evaluated. Phytochemical screening of the polyherbal extract revealed the presence of glycosides, alkaloids, flavonoids, steroids, phenolic compounds and saponins, suggesting its usage for various medicinal purposes in folk medicine. Most of the phytochemicals identified serve as natural antibiotics, which assist the body system in fighting microbial invasion and infections (Lillehoj et al., 2018). Flavonoids are known to have antioxidant effects and have shown to inhibit the initiation, promotion and progression of tumors [29]; this is done by either scavenging or quenching free radicals or by inhibiting enzymatic systems responsible for free radical generation [30]. Thus, as antioxidant present in this plant suggest its use as hepatoprotective, nephroprotective, antimicrobial, anti-inflammatory and anticarcinogenic [31,32]. Alkaloids may be responsible for its anti-malarial, analgesic properties and stomach disorder [33]. Many alkaloids such as morphine, codeine, sanguinarine etc are known for their psychotropic and stimulant, antihyperglycemic, antibacterial, antimalarial and anticancer activities [34,35]. While tannins have been reported to possess astringent properties, which hastens the healing of wounds [36]. 

Total phenol content of PHF was calculated as 25.24±3.56mg/ml (expressed as GAE % w/w), Similarly Total flavonoid content was  15.61±1.95mg/ml, (expressed as QE % w/w). The results obtained demonstrated that the combination of the herbal extracts increase significantly the amount of TPC and TFC. Williams et al., 2004 reported that Phenolic compounds and flavonoids are reported to have antioxidant and free-radical scavenging activity. They perform scavenging activity by stabilizing free radicals via their conjugated ring structures and hydroxyl groups (Amic et al., 2003). the high quantity of phenolic and flavonoid contents of PHF may contribute to its high antioxidant property. DPPH radical has been used extensively as a free radical to test the reductive ability of extracts or compounds as free radical scavengers or hydrogen donors and to evaluate the antioxidant activity of plant extracts and foods [37,38]. Antioxidants react with DPPH by providing electron or hydrogen atom, thus reducing it to 1,1-diphenyl-2-hydrazine (DPPH-H) or a substituted analogous hydrazine. The deep violet colour of DPPH at maximum absorption of 517 nm is changed to light yellow, colourless or bleached product, resulting in decrease in absorption [39,40]. The scavenging effect of plant extracts on DPPH has been shown to be related to the phenolic concentration of the extracts [38,41-43], which is believed to contribute to their electron transfer /hydrogen donating ability. It could therefore be suggested that PHF contains flavonoids with hydroxyl groups that could stabilize free radicals or scavenge their activities and that the method employed is reliable. The antioxidant activity of PHF for DPPH at 250µg/mL was significant when compared with ascorbic acid and tocophenol  as represented in Table 2.

This show that the scavenging activities of the crude extracts on DPPH was increased with increasing concentrations of the extract. Investigating the fifty percent inhibitory concentration (IC50) of the ethanolic extract of the polyherbal formulation was found to be 86.09±1.58 µg/mL at 250µg/mL compared with standard ascorbic acid and tocopherol 56.56±0.39 and 67.37±0.25µg/m. These results revealed the capability of the polyherbal formulation to quench the DPPH radical, which suggested that the extract may be a good antioxidant with high radical scavenging activity. This is in agreement with the study of Arun et al., 2020. they investigated the antioxidant activity of a polyherbal formulation containing three medicinal plants (Mormodica charantia, Andrographis paniculata and withania somnifera. It was reported in their study that the extract possessed free radical scavenging activity in a concentration dependant manner, individual extracts of the drugs show antioxidant potentials comparable to ascorbic acid but in combination their action got boosted dramatically. They reported an IC50 values of drug combination (MC:AP:WS) in DPPH (26.40± 0.56 µg/mL) and ABTS (28.91±0.34 µg/mL), which may be due to synergistic effect of polyherbal formulation with higher phenolic and flavonoid content.

ferric radical antioxidant assay depends on the capability of an antioxidant to reduce Fe3+ to Fe2+ in the presence of TPTZ, developing an intense blue Fe2+-TPTZ complex with an absorption maximum at 593 nm. The absorbance decrease is proportional to the antioxidant content (Hou et al., 2003). The tendency for ferric ions reducing activities of ethanolic extract at different concentrations is shown in Table 3. The IC50 value was found to be 66.95±0.03 µL/mg at 250 µg/mL compared with the  standard ascorbic acid (70.05±0.07) the result of the present study showed significantly ferric reducing property and thereby indicates the potential hydrogen-donating capacity of the extract.

Also in the current study, the hydroxyl radical scavenging activity of the ethanolic extract of herbal formulation showed maximum activity of 80.89 at 250 µg/mL, whereas Manitol at the same concentration exhibited 92.20±0.24µg/mL. Thus, the ethanolic extract shows a scavenging property of hydroxyl radicals Hydroxyl radicals are the major active oxygen species causing lipid peroxidation, damage to DNA and proteins (Spencer et al., 1994). According to Diplock 1997, the antioxidant action may be attributed to various mechanisms, some of which may be prevention of chain initiation, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging action. The reducing capacity of the compound/compounds in a mixture serve as a significant indicator of its potential antioxidant activity [44-49]. The reducing capacity of PHF was concentration dependent as presented in (Table 4). Phenolic compounds are powerful chain breaking antioxidants (Shahidi and Wanasundara 1992). The phytochemicals identified in the study may chelate the transition metals and inhibit the formation of free radicals at the initial level itself, Hence the various degree of inhibition of various oxidants can be seen in this aspect in the various scavenging models used in this study. It is reported that the crude extracts of plants are pharmacologically more active than their isolated active principles due to the synergistic effects of various phytochemicals present in the whole extract (Hamberger and Hastettman 1991). It has been reported that administration of C.sativus, A.sativum and C.longa together have shown increase in antigenotoxic effects against cyclophosphamide induced genotoxicity in mice against administration of individual agents separately, Premkumar et al 2004.

Similarly, PHF exhibited scavenging effect on nitric oxide radical (NO•) but concentration dependent. The antioxidant activity of PHF for Nitric oxide scavenging activity at 250µg/mL was significant when compared with the standard ascorbic acid, the IC50 of PHF was 68.13±0.16 and 72.134±.13 for the standard ascorbic acid as presented in Table 5. However, the PHF activity may be due to antioxidant principles in which they compete for oxygen to react with nitric oxide (Ilalenti et al. 1996), and thus inhibit the generation of nitrite. Similar mechanism was reported for the antioxidant action of Sphaeranthus indicus (Linn) by Shirwaiker et al., 2006.  

ABTS radical scavenging activity renders a redox functioned proton ion to unstable free radicals and performs a key role in stabilizing detrimental free radicals in the human body (Lee et al., 2015) [50-55]. The PHF powerfully scavenged ABTS radicals produced by the reaction between ABTS and ammonium persulfate, as shown in Table 6. The antiradical activity was found to increased in a dose-dependent manner. The extract showed an IC50 value of 65.54±0.55µg/mL while 65.32±0.33, 96.13±0.79, 67.49±.72 were reported for sodium ascorbate, Trolox and Galic acid respectively at 250 µg/ml. Hence, the ABTS radical scavenging activity of ethanol extract of polyherbal extract indicates its capability to scavenge free radicals, thus preventing lipid oxidation through a chain-breaking reaction [56-61].

Superoxide radical scavenging activity: Superoxide anion is the first reduction product of oxygen which is measured in terms of inhibition of generation of O2 (Kamalakkannan and Stanley 2003). Superoxide dismutase catalyses the dismutation of reactive superoxide anion to oxygen and hydrogen peroxide. The effect of PHF-5 in scavenging anions may be due to inhibition of generation of superoxide. Such an effect was reported for ethanolic extract of B. monnieri (Ghosh et al., 2007; Meena et al., 2012; Tripathi et al., 1996).