675 Rima Kumari and M.N.V. Prasad. / International Journal of Biological & Pharmaceutical Research. 2014; 5(8): 675-688.

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EFFECT OF UV-B PRETREATMENT ON ESSENTIAL OIL COMPONENTS, HEALTH SENSORY SECONDARY METABOLITES AND ANTIOXIDANT POTENTIAL OF COLEUS AROMATICUS Rima Kumari*1 and M.N.V. Prasad2 1

Departmental of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow-226025, India 2 Department of Plant Sciences, University of Hyderabad, Hyderabad-500047, India

ABSTRACT Effect of Ultraviolet-B radiation on secondary metabolism relates to mechanism involves in triggering of defense related gene expression, activation in enzyme activity, results to induced synthesis and/or accumulation of health sensory phyto-metabolites. In present study, we investigates on different level UV-B treatment response on comparative change in some medicinal bioactive metabolites, e.g. volatile oil, phenolic compounds, flavonoids, alkaloids, vitamins, saponin, tannins as well as at antioxidants level in a medicinally important aromatic plants Coleus aromaticus. In present experiment, plants were exposed to different level UV-B radiation exposure with an intensity of 3 J/s at low dose supplemental UV-B (UV1; 2h), high supplemental UV-B (UV2; 3h), and ambient level UV-B. A control group excluded UV-B radiation treatment. Various biochemical experiments conducted on field grown and growth chamber condition plants and analyzed the level of various phyto-constituents in Coleus leaf extracts i.e. total phenolics, alkaloids, total triterpenes, saponin, alkaloids, flavonoids and others. Plants grown under controlled growth chamber exposed with different dose UV-B treatment. In ambient UV-B condition (FC grown plants), total phenolic content was observed to be higher accumulated with respect to growth chamber plants. After UV-B treatment, various bioactive compounds were quantified and results ensures that UV-B supplementation induces the changes in concentration of volatile oil constituents, terpenes, alkaloids, ascorbic acid content at lower dose UV-B enhancement whereas at high intensity it may induces the inhibitory effects (hormesis effect). Key Words: Coleus aromaticus, UV-B, phyto-compounds, Antioxidants, Essential oil, Thymol. INTRODUCTION Medicinal plants are rich in nutritive compounds ―phytochemicals‖, which impart multiple health benefits. These compounds must be used as a nutritional supplement as well as natural antioxidants. Over the past few years, medicinal plants gained wide attention and serve as leading research for the development of novel pharmacological agents, due to its lesser side effects compared to allopathic medicine. Since many diseases have been related to the Corresponding Author Rima Kumari Email: [email protected]

occurrence of reactive oxygen species, plant derived antioxidants have gained importance for human health benefits in recent years. There has been an upsurge of interest in the therapeutic potential of various metabolites as antioxidants in reducing several stress related health problems. The potential value of antioxidants has prompted investigators to search plants rich in natural compounds with potent antioxidant activity. Epidemiological and in vitro studies on medicinal plants strongly supported this concept that plant secondary bioactive constituents is strong determinant of its antioxidant capacity, exerting protective effects against oxidative stress in biological systems (Lee et al., 2003). Polyphenols, flavonoids,

676 Rima Kumari and M.N.V. Prasad. / International Journal of Biological & Pharmaceutical Research. 2014; 5(8): 675-688.

terpenes and various plant extracts have been reported to be effective antioxidants, radical scavengers and inhibitors of lipid peroxidation (Liu and Ng, 2000). Volatile oils in medicinal plants have also been identified as the most important products of secondary metabolites, showing a broad range of applications in pharmaceutical fields including antioxidants, antiplatelets, anti-inflammatory, anti-tumour, anti-viral activities. Interest in the role of different phyto-compounds and antioxidants in concern to human health has promoted research in the field of pharmaceutical and food science to evaluate antioxidants rich natural products and to determine how their content and activity can be maintained or even improved through different agricultural practices and processing. The concentration of various phytoconstituents in medicinal plants in addition to be controlled by genetic processes, also being influenced by environmental factors i.e. light (quality, intensity and duration), temperature, irrigation, soil and nutrients amendment. Among various environmental factors, Ultraviolet-B (UV-B) light is reported to be one of the most important variables affecting phytochemical concentrations in plants. Based on the various previous literatures, UV-B radiation is proven beneficial in many cases to stimulate secondary metabolites (Zhang and Bjorn, 2009; Germ et al., 2010; Kumari and Prasad, 2013). For this reason, many researchers in the fields of plant biology and natural pharmaceutical chemistry have focused on the influence of UV-B radiation on different health sensory metabolites in number of medicinal plants (Hagen et al., 2007; Higashio et al., 2007; Kumari et al., 2009; Kumari and Agrawal, 2010). UV-B stimulates the level of metabolites such as phenolic contents, ascorbic acid, carotenoids etc. Mechanism involves can be related to the up-regulations of defense related secondary metabolism i.e., phenyl propanoids, isoprenoids pathway, results to the rise of phenolic compound concentrations (Gerhardt et al., 2008), alkaloids and terpenoid production (Johnson et al., 1999; Velikova, 2008). Though, there are many reports on the UV-B induced biosynthesis and accumulation of phenolics in epidermal leaf tissue but there is a rather limited understanding of the effect on other compounds, such as isoprenoid-derived terpenes, alkaloids, membrane components (sterols), saponin, tannin and others. These antioxidants act against possible stress effects of free radical-induced damage in humans. It should be noted that phytocompound extracts from fruits/vegetables or medicinal plants have strong antioxidant activities, and the foremost UV-B radiation plays a significant role on the concentration of various antioxidants. UV-B induced activation in antioxidants properties in medicinal may be proven to play a major health protective role (Kumari and Prasad, 2013). . Keeping the beneficial aspects of UV-B promoted secondary metabolites synthesis and antioxidants enzyme

activation , in present experiment, we investigate on the effect of different level UV-B treatments on various phytoconstituents i.e. phenolics, flavonoids, ascorbic acid, anthocyanin, total triterpenes, saponin, tanin as well as antioxidants assay in a medicinally important aromatic Coleus aromaticus. Coleus aromaticus is a good source of natural antioxidants. This plant is green perennial shrub that grows naturally in most tropical countries. In India, it is also popularly known as ―Indian Oregano. The leaves are highly aromatic, can be used to flavor foods & beverages. C. aromaticus is told as one of most important plant in medicinal herb garden, widely used in making native medicines in Ayurveda, Homeopathy and Unani. It is known to possess antimicrobial, antiepileptic, leishmanial, and antioxidant activities (Khare et al., 2011). This plant constitute various natural bioactive compounds; phenolics, terpenoids, alkaloids, saponin, tannins that may produces health beneficial effects by scavenging free radicals (Rout et al., 2012). This species produces essential oils, reported as an effective in vitro antimicrobial natural molecule against fungi and bacteria (Khare et al., 2011). Though, information on UV-B elicitor mediated changes on secondary plant metabolites on Coleus aromaticus is scanty. This study is quite interesting, informative and of innovative approach. In order to investigate on response of different UV-B treatment condition on medicinal bioactive compounds and antioxidants potential in chosen test plant C. aromaticus, we established the experiments under two different growth conditions i.e. in field condition and controlled growth chamber. We investigated the changes in different bioactive metabolites i.e. total phenolics, triterpenes, alkaloids, soluble protein, flavonoids, ascorbic acid, pigments analysis as well as antioxidants enzymes, total antioxidants assay under different level UV-B treatment. This study might therefore contribute to an assessment of a moderate stress inducing UV-B application on the dynamics of health promoting plant metabolites as well as enhancement in antioxidants status of medicinal plant extract. Information on different treatment conditions of UV-B light intensity on phytochemical production will help us in designing the most appropriate environment to obtain medicinal plants with enhanced phytochemical concentrations, which may be applicable for further commercialization and exploration of this novel technology on a pilot scale in pharmaceutical industries. MATERIAL AND METHODS Plant growth and propagation Coleus aromaticus is a succulent herb that grows in shallow soil habitats. For propagation, plants were collected from field bank maintained as stack plant in Botanical garden, University of Hyderabad, India. C. aromaticus have ability to propagate through stem cuttings and after establishment, adventitious roots, leaves and vegetative leaves were emerged out (Kumar and Prasad,

677 Rima Kumari and M.N.V. Prasad. / International Journal of Biological & Pharmaceutical Research. 2014; 5(8): 675-688.

2010). Growth conditions For experimentation, the present study was carried out in two growth conditions i) in natural environmental conditions (soil culture) and ii) in controlled growth chamber (in hydroponic medium). Flow chart diagram of experimental outline and UV-B treatment set up are represented in Figure. 1). Plant propagation in soil For propagation, equal sized (similar height and diameter) fresh healthy stem cutting of plants were potted in 5-L cement pots filled with mixture of red soil and sand in a green house condition. Optimum requirement of compost was mixed in soil for better plant growth. During experimental period, average daily mean temperature ranged between 24.6ºC and maximum to 32 ºC; the relative humidity ranged between 62-75%. At normal solar noon duration, the photosynthetic photon flux density ranged from 1200 -1500 µmol m-2 s-1 with an average sunshine light 7 h d-1. Plant propagation in hydroponic medium In Growth chamber (GC) condition, C. aromaticus stem cuttings were placed in 250 mL conical flask containing 100 ml of Hoagland’s solution (10% modified) (Hoagland’s and Arnon 1950). In the present study the culture medium was optimized for propagation and better ex vitro survival and growth. The Hoagland’s medium in hydroponic culture pots were changed twice in a week to provide fresh dose of nutrient supplement throughout the experiment duration. Stem cutting in hydroponic media under laboratory conditions were kept for two weeks for establishment and propagation and then exposed to UV-B supplementation. Experimental Design and UV-B treatment measurement Field grown plants received ambient level UV-B light and photosynthetic active radiation averaged 1500 μmol m-2 s-1 at midday. Growth chamber (GC) equipped with white fluorescent lamps as the main light source, and UV-B florescent lamps for exposing plant at different levels UV-B irradiation treatment. In GC, PPF was kept constant (300 μmol m−2 s−1 at the top canopy level; 16 h photoperiod) throughout the experimental period by controlling a dimmer switch. For each treatment, twelve replicate pots were maintained. In present experiment, different treatment conditions are as follow: ● UV1; (Low dose UV-B; PAR + UV-B for 2h treatment) ● UV2; (Low dose UV-B; PAR + UV-B for 3h treatment)  Ambient; (Solar light; optimum PAR+ UV-B)  Control; (PAR - UV-B excluded) The UV-B treatment system consists of three UVB (TL 20 W/12 RS SLV; Philips) fluorescent lamps. The

UV lamps were wrapped in pre-solarised (8 h) 0.13 mm thick cellulose diacetate film to avoid transmission of residual UV-C radiation (