An Insight into Antioxidant and Antimicrobial Activities of Ethnotherapeutically Important Trans Himalayan Medicinal Plants: A Review

The high altitude of the Himalayan cold desert represents a valuable habitat of natural resources. The extreme climatic condition manifested by intense mutagenic UV-radiation, physiological drought, desiccation and strong winds, makes the survival of plants really difficult. As a consequence of this atmospheric stressor, the plants produce unique metabolites which play a preventive role in intrinsic mechanism of sustenance. Many plant species of this region have been investigated in search of novel antioxidants and antimicrobials. Plants synthesize several antioxidants that aid in antioxidant defense system, thereby protecting plants against damage caused by active ROS. These compounds include chlorophyll derivatives, alkaloids, essential oils, phytosterols, phenolics and polyphenolics. Some of the antioxidants that have been isolated from plants include curcumin, quercetin, ascorbic acid, resveratrol amongst many other compounds. Additionally, the emergence of resistance to multiple antimicrobial agents has become a major threat to public health. Hence, fresh efforts towards new drug identification and development are greatly needed. Plants have long been used in traditional Indian medicine for numerous therapeutic Review Article Avasthi et al.; JPRI, 33(36A): 195-212, 2021; Article no.JPRI.70139 196 benefits and low toxicity. Considering the growing interest in quest for search of plant based antimicrobials and antioxidants; an effort has been carried to systematically record the antioxidants and antimicrobial potential of plants of Himalayan region.


INTRODUCTION
The northern part of India, especially Himalayan terrain is a hot spot of medicinal plants. In particular, Indian Trans-Himalayan mountain range supports low vegetation cover due to harsh climatic conditions along with short growing season. More than 8000 species of angiosperms, 44 species of gymnosperms and 600 species of pteridophytes have been reported from the Indian Himalayas, and of these, 1748 species are known for their therapeutic potential [1]. The high altitudes of the Himalayan cold desert represent a valuable habitat of natural resources. The extreme climatic condition manifested by intense mutagenic UV-radiation, physiological drought, desiccation and strong winds, makes the survival of plants really difficult. As a consequence of this atmospheric stressor, the plants produce unique metabolites which play a preventive role in intrinsic mechanism of sustenance [2]. However, high altitude medicinal plants (HAMP) are amongst the least well studied organisms of terrestrial ecosystems. This is largely due to misconceptions about poor biodiversity, small number of individuals per species, reduced surface inaccessibility, and relative simplicity of these habitats. These plants are capable of growing successfully at altitudes surpassing 5000 m mainly because of specialized physiological processes that include synthesis of special lipids which alter the flexibility and water permeability of cell membranes, anti-freeze carbohydrates and other unique secondary metabolites [3,4]. These conditions induce the biosynthesis of UV protection and free radical scavenging compounds in these plants.
From this Trans Himalayan region, 414 species of higher plants covering 56 families and 202 genera have been recorded. Almost a quarter of the recorded species (102 species) are reported to be used in traditional system of medicine whereas over 80 species are largely associated with cultivated fields and human habitation. These also include 49 species of crop plants like barley and buckwheat [4]. The local medical practitioners of this region, Amchis, use local herbs either as extract or sometimes in combination with salts or minerals and these concoctions are well acclaimed for their

Fig. 1. Map of Trans Himalayan region of India
medicinal values for many disorders like memory loss, osteoporosis, age related disorders, AIDS and cancer [5]. This traditional knowledge is not only useful for local health care, but also for the conservation and sustainable use of medicinal plants. This knowledge has been slowly diminishing due to the changing socio-economic circumstances of the region, but there has been a renewal and a revitalization of this ancient and time-tested tradition of healing in recent times. Considering the growing interest in quest for search of plant based antimicrobials and antioxidants; an effort has been carried to systematically record the antioxidants and antimicrobial potential of plants of this Trans Himalayan region.

METHODOLOGY
A systematic web search analysis and review was conducted on research literature pertaining to medicinal plants from Trans Himalayan region of India with reported biological activities. The search engines used for retrieving published data (from 2000 to 2020) include universally recognized databases, specifically, Scopus, PubMed, ScienceDirect, and Google Scholar. The search strategy was to retrieve and download published literature dealing with medicinal plants and compounds having antibacterial and antioxidant activities. Specific keywords such as "Trans Himalyan", "antioxidant", "antimicrobial" and "in vitro or in vivo activity". Studies reporting in vitro/in vivo efficacy of medicinal plants were included in this review.

PLANTS REPORTED WITH ANTIOXIDANT ACTIVITY
It is well established that plants have an innate ability to synthesize wide variety of nonenzymatic antioxidants which are capable of mitigating reactive oxygen species (ROS) induced oxidative damage. These antioxidants can delay or prevent the oxidation of various intracellular oxidizable substrates even at significantly lower concentrations than that of the substrate 20 . It is believed that two-thirds of the available plants on this earth have at least some medicinal importance, and majority of them possess significant antioxidant potential [6]. The interest in the exogenous plant derived antioxidants was first aroused by the discovery and subsequent isolation of Vitamin C (ascorbic acid) from plants. Since then, the antioxidant potential of plants has received a boundless attention because increased oxidative stress has been recognised as a major causative factor in the development and progression of many lifethreatening disorders, including neurodegenerative and cardiovascular diseases [7]. This antioxidant activity is mostly credited to the presence of phenolic acids, phenolic diterpenes, flavonoids and volatile oils as secondary metabolites in plants. Some plant pigments like anthocyanin also possess antioxidant activity [8]. For each of these metabolites, a distinct mechanism of action is followed which may include decreasing localized oxygen concentration in the tissues, preventing chain initiation by scavenging radicals, disintegrating lipid peroxides to peroxyl and alkoxyl radicals; decomposing peroxides by altering them to non-radical products or by terminating the free radical propagation to prevent sustained hydrogen abstraction [9].
The most effective antioxidants are those which can interrupt free radical chain reactions, which starts under various stress conditions. They usually contain phenolic or aromatic rings and can donate hydrogen atom to the free radicals formed during oxidation (Fig. 2). The freshly formed phenolic radical intermediates are stabilized by resonance within the aromatic ring [10]. Phenols (ArOH) donate H to scavenge the reactive free radical. They in turn gets converted into phenoxyl radical (ArO·). The phenoxyl radical is stabilised by resonance.
Phenolic compounds are also capable of suppressing lipid peroxidation by recycling other endogenous antioxidant Vitamin E (αtocopherol). They can also bind to pro-oxidant metals, such as iron or copper and hence prevent the formation of free radicals [11]. They are also reported to induce synthesis of antioxidant proteins including antioxidant enzymes like SOD and CAT [12].
Similarly, flavonoids can also donate hydrogen and in turn forms stable flavonoid semiquinone radicals, which may later be scavenged by intracellular antioxidant glutathione (GSH, reduced state). The ratio of intracellular reduced glutathione to oxidized glutathione is often used measure of cellular oxidative stress. Glutathione acts as an electro donor to reduce the disulfide bonds formed within cytoplasmic proteins to cysteines. In the process, glutathione is converted to its oxidized form, glutathione disulfide (GSSG, oxidized state) [13] as depicted in Fig. 3. Once oxidized, the GSSG can be reduced back to GSH by another enzyme GR (glutathione reductase) that uses NADPH as an electron donor.
A flavonoid compound donates H to scavenge free radicals and gets converted to a stable flavonoid free radical. This radical is scavenged by intracellular non-enzymatic antioxidant glutathione (GSH).
Since flavonoids contain multiple hydroxyl groups, they are considered more effective antioxidants. The presence of the ortho-3, 4dihydroxy moiety further enhances the antioxidant activity of these secondary metabolites [14]. Additionally, flavones and some flavanones can favourably bind to prooxidant metals thus rendering them ineffective [15]. Apart from number and position of OH groups, it is the ability to partition between the lipid and hydrophilic phase also plays an important role in the bioactivity of these compounds. Compounds that are capable of correctly orienting at the interphase of oil droplet in emulsion can act as a better inhibitor of ROS induced lipid peroxidation [16].

PLANTS REPORTED WITH ANTIMICROBIAL ACTIVITY
Beside antioxidant potential plant secondary metabolites also exhibit multiple pharmacological activities including anti-inflammatory, antimicrobial, anti-cancer etc. Also, they play significant role in self-defence and ecology [17]. Bacterial species also have genetic ability to acquire and subsequently transfer resistance to subsequent generations against currently available antibiotics/antibacterial agents, thus, becoming multi-resistant to the commercially available medications [18]. Hence, as a substitute to commercially used antimicrobial drugs, bioprospecting of plants as a source for new and safe antimicrobials is now being explored globally. Antimicrobial properties in plants are also credited to the presence of bioactive secondary metabolites. Plant derived antimicrobial peptides are also a part of plant defense systems and these are analogous to human antimicrobial peptides in their structure and function [19]. The mode of action of these natural plant derived antimicrobials include disintegration of cytoplasmic membrane, destabilization of the mitochondrial proton motive force (PMF), disruption of electron flow and ATP based active transport and coagulation of the cellular content [20].
Here, we present a few important classes of plant derived secondary metabolites with wellestablished antimicrobial potential for example quinones, alkaloids, flavonoids, couramins, essential oils, tannins, lignans, glucosinolates and thionins.
Quinones are a class of compounds which possess fully conjugated cyclic dione structure, example benzoquinones. They are capable of binding to the bacterial cell wall and also inhibit key bacterial enzymes [21]. Lawsonia inermis, commonly known as henna, also contains quinones exhibiting antibacterial activity against Pseudomonas aeruginosa [22]. Quinone rich extract of Hypericum perforatum, has demonstrated general antimicrobial properties and has also been found to be active against methicillin-resistant and methicillinsensitive Staphylococcus [23].
Another group of plant metabolites are alkaloids which contain a basic nitrogen atom. This group also comprises of some other related compounds which have neutral or weakly acidic properties. Their occurrence is common in angiosperms. The pharmacological importance of alkaloids was first recognized with the isolation of morphine from Papaver somniferum. Since then, many medicinally important alkaloids have been reported viz., berberine, caffeine, quinine, codeine, strychnine, ephedrine, emetine and narcotine.
Many plant species like Berberis spp., Cortex phellodendri and Rhizoma coptidis have shown significant antimicrobial activity due to the presence of high concentrations of alkaloids in their extracts [24]. However, these compounds are not frequently used in folk medicine due to their associated toxic effects at higher concentrations.
Chemical structure of flavonoids can be abbreviated as C6-C3-C6 containing two phenyl rings and one heterocyclic ring. Flavonoids are widely distributed in plant kingdom [8]. They are also abundant in many plant products like honey, fruits, seeds, vegetables, wines and tea. Beside antioxidant activity, a number of flavonoids also possess antimicrobial, antiviral, antiallergic and anti-inflammatory properties [25]. Few flavonoids have shown significant antifungal activity against Botrytis cinerea and Aspergillus flavus [26]. Flavonoids from Galium fissurense, Viscum album ssp. album and Cirsium hypoleucum have been reported to have antibacterial activity against extended-spectrum β-lactamase, producing multidrug-resistant bacteria Klebsiella pneumoniae [27]. These compounds are able to bind to bacterial cell wall causing membrane disruption of the pathogenic organism [21].
Coumarins are phenolic secondary metabolites of benzopyrone chemical class, containing fused benzene and an alpha pyrone ring [28]. Extract of Ferulago campestris containing pyranocoumarins possess antibacterial activity against many Gram-positive and Gram-negative clinical isolates [29]. Similarly, coumarins from Angelica lucida L. have been found to be active against Streptococcus mutans and S. viridians, causative agents of oral cavity and dental infections [30].
Essential oils are present in almost all plants and are the largest group of plant derived secondary metabolites. This class constitutes mainly of a number fatty acids/esters and lower homologues of terpenoids. These oils often possess sweet aroma. Five carbon isoprene unit is the building block of all terpenes. When a terpene molecule contains an additional functional group they are called terpenoids. In general, terpenoids are reported to be active against bacteria, fungi, viruses, and protozoa. Some widely prevalent terpenoids with antimicrobial properties include menthol, citral, camphor, salvinorin A and cannabinoids. They are generally amphipathic and hence are able to cause membrane disruption in the target organisms [21].
Tannins are a class of polyphenolic compounds containing large number of -OH and other functional groups like -COOH to form strong complexes with various macromolecules. The tannin compounds are also widely distributed in many plant species e.g., red wine and green tea, where they play a role in defense against microorganisms and pests. The tannin from Sorghum species had been active against S. aureus, Salmonella typhimurium, A. niger, A. flavus and Saccharomyces cerevisae [31].
Lignans are a group of dimeric-phenylpropanoids formed by fusion of two cinnamyl alcohol/cinnamic acids via the β-carbon of the aliphatic chain. Some plant derived lignans also possess antimicrobial activity e.g., lignans from Pseudo larixkaempferi were found to be active against Candida albican and S. aureus [32].
Glucosinolates present in many species of Brassicaceae family are sulphur and nitrogen containing secondary metabolites. They are abundant in broccoli, mustard and Brussels sprouts and have antifungal, antimicrobial, anticancer, antioxidant and anti-inflammatory activity [33].
Thionins are a group of small proteins found solely in higher plants. They have disulphide bonds and are positively charged. The positive charge of these thionins (antimicrobial peptides) can bind to negatively charged membrane constituents like phospholipids, teichoic acid and lipopolysacharide, and can disrupt membrane structure and result in death of the bacterial cell. For example fabatin which has been extracted from the fava beans contains 47 peptide residues that have shown antimicrobial activity against P. aeruginosa and E. coli [34].

ADAPTATIONS OF PLANTS IN NATURAL HABITAT OF HIMALAYAS
The plants of Himalayas are extremely specialized group that have metabolic and propagative adaptations suited for maximizing their activity under adverse harsh climatic conditions [35]. They exhibit many ecological, morphological and physiological adaptations which help them to offset the impact of severe climate prevailing in this area. They have developed a very deep and extensive root system which can absorb water even from great depth, and can also endure strong winds, snow blizzards and thus help in preventing damage due to prevalent subzero temperature during winter season. Another survival mechanism for these plants is the underground modified stem or rhizome and bulbs which help in the survival of the perennial herbs in harsh winters. The major physiological adaptation in these plants, however, is their resistance to frost, either through inhibition or reduction in the ice crystal formation. Due to short growing season, these plants have to complete their entire life cycle starting from seedling growth to sprouting of leaves and flowers, fruiting and dispersal of seeds in a span of few months. This is promoted through reproduction being carried out both by sexual as well as vegetative methods [36].

RESULTS AND DISCUSSION
The plants of Ladakh Himalaya are exposed to severe environmental stress which manifests in the production of high content of secondary metabolites. This has also been established that antioxidant potential of these plants is comparable and even greater than established commercially available antioxidant extracts like green tea and Indian gooseberry.  [74].
Some of the commercially important compounds having antioxidant activity include quercetin, ascorbic acid (Vitamin C) and tocopherol (Vitamin E). These compounds showed similar IC 50 9.479, 15.62, 11.23 µg/ ml respectively, measured by DPPH assay [75][76][77]. In addition to high radical scavenging activities, few extracts also induced antioxidant defence systems by enhancing GST and SOD levels e.g., Podophyllum hexandrum. Hence these plants can reduce the free radicals induced oxidative damage through multi-functional approaches.

CONCLUSION
The plants of Ladakh, India are integral part of tribal diet, and are associated with many health protection benefits too. The literature search clearly showed that many plants of this region showed potential antioxidant and antimicrobial activities against human pathogenic bacteria in vitro. In addition, most of the antimicrobial and antioxidant active plants species are non-toxic. The results of this study suggest that extract of these species could be used as natural antioxidant to reduce free radical mediated disorders and may also be a source of active molecule against disease causing pathogens. Several antioxidant and antimicrobial compounds could be obtained from these plants resources. Therefore, further works of isolation and characterization of antioxidant and antimicrobial compounds merits from these plants resources.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.