1. Introduction
Exploration of the therapeutic potential of plants indicates the presence of antimicrobial principles. Renewed interest in plant antimicrobials has emerged in the last 20 years, possibly due to growing drug resistance of human pathogens, over and above the undesirable side effects of synthetic antibiotics [
1]. Many bacteria, such as
Staphylococcus aureus or
enterococci are resistant to antibiotics such as methicillin or vancomycin. The development of potential multidrug resistance in pathogens is a key motivator for finding novel molecules or groups of compounds that can be used in pharmaceuticals without the toxic effects of synthetic chemical compounds [
2]. Ultimately, research should be focused in order to discover as much potentially appealing data as possible, together with negative and positive interactions with general antibiotics and so forth. Such findings could further improve the use of medicinally important plants, their extracts, or other natural products, in any form: alone or in combination with antibiotics. The researchers developed their interest in biologically potent compounds which are isolated from different plant species meant for the removal of pathogenic microorganisms due to resistance built into microorganisms against antibiotics [
3].
In plants, harsh environmental conditions, for example salt constraint, cause improved production and accumulation of the reactive oxygen species (ROS), initiating cellular damage, severe metabolic disorders, and senescence pathways [
4]. In living beings, different ROS are able to form in diverse methods. ROS have been involved in over 100 ailments, counting heart disease, malaria, stroke, acquired immunodeficiency syndrome, arteriosclerosis, cancer, and diabetes [
5]. Plants are well-known for their capability to withstand unfavorable environments and quench toxic ROS, as they are equipped by means of powerful antioxidant systems that involve both non enzymatic and enzymatic components [
6]. Antioxidant compounds are able to delay oxidation of lipids and prevent oxidation of other molecules through inhibition of initiation and proliferation of oxidative chain reaction. Therefore, they can prevent and repair cellular damage triggered by oxygen species [
7]. All these compounds are considered as hydrogen donors, chelating agents of prooxidants, reducing agents, free radical scavengers, and singlet oxygen quenchers [
8]. In recent times, there is a huge interest in measurement as well as exploitation of plant antioxidants for scientific research and industrial uses (dietary, cosmetic, and pharmaceutical). This is primarily as a result of strong biological activity, surpassing those of synthetic antioxidant components which are active as carcinogens and considered to be its promoters [
9]. Thus, a need exists for powerful, economic, natural, and safe antioxidants in the replacement of synthetic antioxidants [
10].
The plant kingdom is considered to be the best source of medication for a variety of pains and ailments. For this reason, medicinally important plants have played key roles in maintenance of health worldwide. Higher plants and their natural products are a vital source of effective therapeutic agents. Thus, countless research groups are presently involved in the screening of plants for their diverse biological activities.
Otostegia limbata (syn.
Ballota limbata) belongs to Lamiaceae (Labiatae) family and genus Otostegia possesses twenty species distributed in the Mediterranean region. Only three species of genus Otostegia, i.e.,
O. persica,
O. aucheri, and
O. limbata are reported from Pakistan.
Otostegia limbata (Benth) Boiss is a valuable bioactive plant extensively distributed in hilly regions of Khyber Pakhtoonkhawa (KPK) and Punjab provinces of Pakistan. Locally it is known as Spina ghazai, Koi booi, Chota kanda, Bui, Chittie bootie or Chitta jand. It is a bushy, slender, branched, pubescent, spiny, and small shrub up to almost 2 ft tall while its flowering period is April–June. Its stem is woody, branched, erect, spiny, with gray and whitish bark. Small, dentate, oblanceolate in shape, not entirely divided leaves with short petiole and spiny bracts. Flowers of the plant are long, pale yellow to orange throated, prominently bilabiate with straight upper-lip and spread lower-lip and present in axillary clusters [
11].
O. limbata is well known for many traditional medicines intended for several purposes. Juice of the plant is effective for the treatment of wounds, as an ophthalmic medication, and a valuable product to treat bleeding gum problems in children. Crushed fresh leaves with low amounts of water in the form of an extract are used locally to treat different types of eye infections [
12]. Antimicrobial agents are extremely vital in reducing the large-scale burden of infectious maladies. People indigenous to the area in which it grows have been consuming numerous plant species as conventional remedies for many years, however there has been a paucity of information regarding in vivo and in vitro efficacy. Yet, there are inadequately detailed or thorough investigations into the potential role of the plant as an antimicrobial and therapeutic entity for MDR bacteria and pathogenic fungi [
13]. Considering extensive potentiality of the plant as an antimicrobial drug source, this analysis was aimed to examine its in vitro antioxidant, antibacterial, and antifungal activity against the most common contagious pathogens. To this end, the main objectives of this study were (i) to estimate antioxidant activity using various tests, (ii) and to assess antimicrobial capacity against different human pathogenic microbial (bacterial and fungal) strains.
4. Discussions
Results revealed a decrease in value of absorbance with increase in concentration of MEP and AA extract. The decline in value of absorbance of DPPH radical is due to antioxidants, as a result of the reaction between radical progressed and antioxidant molecules, results in radicals scavenging by donation of hydrogen. 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a free radical assay for scavenging an unpaired electron which delocalized over the whole molecule. In this method the change in color was observed from the violet color of the DPPH solution to the yellow colored diphenylpicryl hydrazine product. This is due to addition of the plant sample in a concentration dependent manner which works to determine antioxidant potential. Absorption of DPPH being proportionate to radical concentration being scavenged. Antioxidants respond with free radicals through the mechanism of electron transfer, the antioxidant gives an electron to free radicals and becomes a radical cation. Ionization potential of antioxidant in this mechanism is the significant energetic factor for assessing antioxidant activity. This process has been used widely to predict antioxidant behaviors despite the relatively less time needed for scrutiny. The observed trend is coherent with previous reports in which an inverse relationship was observed between concentration of sample and absorbance value [
18]. The radical scavenging potential determined in this assay in terms of inhibition percentage rather gives more comparable results than absorbance [
19]. MEP possess a fine ability to scavenge the free radicals, with percentage scavenging ranging from 37.89–63.50% as tested at several concentrations. The 250 µg/mL of AA showed highest observed percentage scavenging i.e., 84.31% while MEP was capable of scavenging free radicals at 63.50% (
Figure 1). The results showed the dependence of scavenging potential on the concentration of sample, and a direct relation was observed, so antioxidant potential is directly interrelated with sample concentration. Several other investigations support these result that inhibitory aptitude of all plants strongly interlinked with its concentration [
20].
The results obtained through PMA facilitate the validation of a direct relationship between absorbance and sample concentration. The phosphomolybdate assay is based on reduction of Mo (VI)-Mo (V) by MEP at acidic pH and successive formation of green phosphate/Mo (V) complex [
21]. The sample absorbance signifies the reducing ability and higher value of absorbance is an indication of strong reducing potential, hence it clarifies the dose dependence relationship. It is worth noting that obtained absorbance in the case of MEP was found to be comparable with that of AA with a minor difference explaining the significant level of scavenging capability. The reducing aptitude of the plant in comparison with AA may serve as a significant reflector of antioxidant potential. The obtained outcomes supported in RPA are in confirmation of a direct relationship between sample concentration and absorbance value. RPA is a convenient and fast screening to evaluate reductive capability of MEP. The mechanism of RPA is the capacity of MEP to donate an electron and transformation capability of Fe
3+ to Fe
2+, in terms of rising absorbance which increase with concentration [
22]. The observed absorbance value reveals the reducing potential of that sample and this may serve as a considerable indicator of its antioxidant activity.
Its antioxidant potential is primarily due to the presence of phytochemical constituent such as bioactive anthocyanin, flavonoids, phenols, and isoflavones [
23]. Flavonoid compounds and phenolic components are renowned for their competence and ability to behave as antioxidants. These compounds oxidize free radicals of sample extracts and show standard activity towards less reactive and comparatively more stable radicals. For this reason, flavonoids are considered responsible for stabilizing the ROS through reaction with reactive components of these free radicals. Positive correlation among total polyphenol contents along with antioxidant capability and the considerable amount of TPC and TFC reported in the literature highlight its antioxidant potential. Total phenolic content varied in the range of 489–1273 mg, GAE/100 g and total flavonoid content in the range of 198–3018 mg, QE/100 g [
24]. These active phenolic and flavonoid content present in the plant highlights the reason for their reducing ability which is a significant reflector of antioxidant capacity. Hence, the antioxidant capability of the plant may possibly be due to the existence of phytochemicals responsible for the activity.
The susceptibility of the selected
staphylococcus bacterial strains are rational and coherent with several previously reported outcomes of studies [
25]. The
Staphylococcus epidermidis showed strong susceptibility against
Aquilaria crassna leaf extract, revealing its considerable antibacterial potential [
26]. These findings support the previous outcomes that Lamiaceae family is a very competent contributor to combat skin infection problems caused by highly resistant
S. epidermidis strains. Synergistic action of plant extracts with essential oil present in members of Lamiaceae family in very significant amounts might be the reason of significant potency against bacterial strains [
27]. Hence
Otostegia limbata may possibly be a remarkable potential source for obstructing particularly problematic skin infections caused by
S. epidermidis; consequently, further exploration of some of their active compounds should be investigated in the near future.
Regarding the least inhibition showed by various strains of bacteria, the analogous discovery was reported [
26] of the susceptibility of various bacterial strains. Various isolates were examined and all isolates were either not or less sensitive to eight antibiotics tested and resistant towards at least one antibiotic commercial drug. Parallel to this report, many other findings also explain the lesser susceptibility and higher resistance of
E. coli to several tested medicinal plants and many commercial antibiotic drugs [
28]. The efficiency of other medicinal plants was also analyzed against these sensitive bacterial strains in various reports which reveal consistent the results with the current investigations [
28,
29]. Therefore, the above mentioned susceptible two bacterial species were the most, sensitive thus the documented literature is coherent with our current findings. The formerly investigated reports are strongly coherent to the current observation that gram-positive strains are relatively more susceptible, since gram-negative species have an additional outer membrane similar to a covering which guards their cell wall, while others lack this extra cover [
30].
Alternaria spp. was the most susceptible tested fungal strain in various previously reported studies. Thus, effectiveness of several medicinal plants in previously reported literature against Alternaria spp. reveals consistency of these findings.
Fluconazole was strongly active against
Aspergillus terreus and also sensitive for MEP with observed linear growth for this strain in test tube at 14.16 mm and 32.0 mm, respectively, while linear growth observed against negative control tube was 100 mm. Calculated percentage inhibition of MEP against
A. terreus was found to be 68% and Fluconazole was 77.33% active against
A. terreus (
Table 4).
Casuarina equisetifolia was reported as an active medicinal plant with percentage inhibition ranged from 51.78% to 85.80% against
A. terreus, thus previous reports are in accordance with recent findings (Lagnika et al., 2014). In accordance with current outcomes,
O. limbata could be a potent source for the control of several fungal infections caused by
A. terreus and
Alternaria spp. For this reason, it is recommended that the aptitude of this plant to restrain the fungal strains and bioactive compounds responsible for the antifungal potential be explored further.