Medicinal plants have been utilized since millennia by different civilizations, such as those from China, India, Egypt, Rome and Greece [1]. These plants are rich sources of food, drugs, cosmetics, dyes and essential oil [2, 3, 4]. They have been exploited for traditional as well as modern medicinal purposes since these plants and their constituents exhibit an array of beneficial ethno-pharmacological activities such as, spasmolytic, antiviral, sedative, hepatoprotective, anti-inflammatory, antiseptic, antihyperglycemic, and immuno-stimulating. In addition, the consumption of these plants or plant parts or their extracts are regarded effective and safe [5, 6]. Hence these plants are considered as future of medicine [7].
Most medicinally important plants contain an array of secondary metabolites produced in response to biotic as well as abiotic stresses mainly for protecting the plants [8]. These active constituents are responsible for various pharmacological bioactivities imparted by the medicinal plants [9]. However, these ethno-botanically valued plants are getting depleted day by day due to pressure of increasing population, urbanisation, and exploitation of herbal biodiversity. Additionally, the changes in environmental factors are also affecting the content of bioactive compounds in these plants [10]. In order to get adequate desirable secondary metabolites from these plants, biotechnological approaches such as plant tissue cultures provide a platform for sustainable production of these secondary metabolites, under controlled condition throughout the year to meet the increasing demand by pharmaceutical companies.Amongst these approaches, the application of plant cell suspension culture system has been reported to be an efficient method for the enhanced accumulation of several secondary metabolites like cosmetics, food and drugs [11]. However, the low yield of secondary metabolites as well as problems associated in scaling up of suspension cultures are the major challenges, thereby restricting accumulation of secondary metabolites in cell cultures [12]. Various strategies, such as elicitation and metabolic engineering, have been designed, optimized and applied to overcome these challenges. Moreover, several researchers reported the influence of various elicitors such as ultraviolet, and visible light, chitin, yeast extract (YE), salicylic acid (SA),and methyl jasmonate (MeJ) on the enhancement of biomass and accumulation of secondary metabolites in differentiated cells viz.hairy root, adventitious root, callus, suspension and cambial meristematic cells of various plants [11, 12, 13]. Ocimum tenuiflorum L. (Tulsi in Hindi and Holy Basil in English) is an aromatic annual herb belonging to the family Lamiaceae (tribe ocimeae). It is believed to have originated in north central India and now grows as native throughout the entire tropics of the eastern world. In Ayurveda, it is known as “The Queen of Herbs”, “The Incomparable One,” and “Mother Medicine of Nature”. The plant has been used as medicinal herb for curing various ailments for centuries [14]. Several studies reveal that it exhibit combination of actions including antimicrobial (antibacterial, antiviral, antifungal, antiprotozoal, antimalarial, anthelmintic), mosquito repellent, anti-diarrheal, anti-oxidant, anti-cataract, anti-inflammatory, hepato-protective, neuro-protective, cardio-protective, anti-diabetic, anti-hypercholesterolemia, anti-hypertensive, anti-carcinogenic, analgesic, anti-pyretic, anti-allergic, immunomodulatory, central nervous system depressant, memory enhancement, and anti-coagulant [15]. Some of the medicinally important bioactive metabolites reported in O. tenuiflorum are eugenol, linalool, oleanolic acid, linalyl, camphor, methyleugenol, citral, methyl chavicol, geraniol, methyl cinnamate, thymol, rosmarinic acid, safrol, taxol, ursolic acid etc. which impart an array of pharmacological potentials to the plant [9, 16, 17]. Amongst these metabolites, oleanolic acid (OA) is one of them in active constituents found in various parts of O. tenuiflorum.
OA (3β-hydroxy-olean-12-en-28-oic acid) is a pentacyclic triterpenoid compound having 30 carbon atoms in its backbone which is abundant in plants of the Oleaceae family such as olive [18]. OA are generally found in the form of free acid or aglycones of triterpenoid saponins in plants, the chemical structure is depicted in Fig. 1. It imparts anti-inflammatory, anti-hyperlipidemic, hepatoprotective and anticancer properties to the plants [19]. A recent study also demonstrated that OA can act as a potential inhibitor to Mpro protein having role in controlling viral replication during COVID-19 [20]. Since O. tenuiflorum is abundant and can be grown easily, it can be a cheaper source of OA and can be utilized for its extraction as an alternative source. Till now, several efforts have been applied for the establishment of cell, callus and suspension cultures of O. tenuiflorum for the accumulation of rosmarinic acid, eugenol and flavonoids [21, 22, 23]. Moreover, several researchers investigated the impact of important factorson augmenting biomass and accumulation of secondary metabolites like rosmarinic acid, phenolic compounds, alkaloids, terpenoids, phenylterpenoids) in suspension cultures of O. tenuiflorum through elicitation and culture optimization [24, 25, 26].
Enhanced OA accumulation in response to biotic elicitors in cell suspension cultures of Calendula officinalis has been reported [27]. However, till date there are no reports on influence of age of the culture for biomass production as well as accumulation of OA in suspension cultures of O. tenuiflorum in the presence of varied types and concentrations of elicitors with different exposure times. Thus, in the present study, we established the callus and suspension culture of O. tenuiflorum and investigated the effect of age of culture, concentration of elicitors and their exposure time on biomass and accumulation of OA.