1. Introduction

In the recent decades, there have been continuous increase in global demand for plant derived medicine and natural products, so there is a need to ensure abundance of various medicinal plants as well as to guarantee the quality of the herbal and natural products available to the public. Plants chemical constituents varies considerably depending on genetic and environmental factors, methods of cultivation, time of collection and post-harvest processing. This inherent variability in the chemistry may adversely affect the efficacy of medicinal plants. Therefore, it is imperative to ensure that the plants used for food, medicine and natural products are relatively of high quality that gives the desired efficacy (^{Kandeel, 1991}). To meet this requirement it is essential to establish qualitative and quantitative chemo-profiles of the samples. Production of medicinal plants is mainly under the circumstances of sustainable agricultural system. In this system, management of environmental parameters such as nutrient is very critical if the yield of medicinal plants would be maximized (^{Abd El-Wahab and Mohammed, 2008})

Plants nutritional requirements are obtained from atmosphere, soil, water and organic matter or by application of organic or inorganic fertilizers. Each plant species requires unique minimum nutrient level, below which the plant shows nutrient deficiency symptoms while excessive uptake could result into toxicity. Three most essential elements for plant growth (Nitrogen-N, Phosphorus-P and Potassium-K) has been applied in various proportions to enhance plant health and growth (^{Hellal et al., 2011}; ^{Oyedeji et al., 2014}). Also, Urea, which contains 46% nitrogen, is widely used in the agricultural industry as an animal feed additive and fertilizer. Proper application of urea would results in increased crop yield. Urea can be applied as a foliar spray on crops, such as potatoes, wheat, vegetables and soybeans due to its high water solubility. Foliage application of urea should contain no more than 0.25% biuret and the quantity of nitrogen applied at one time should not exceed 20 lb. of nitrogen per acre (^{Overdahl et al., 1991}; ^{Khalid, 2012})

Plant hormones (also known as phytohormones) are signal molecules produced within the plant, and occur in extremely low concentrations. These chemicals regulates cellular processes in targeted cells locally and in other locations in the plant and are the underlying factors of different plant mechanisms. They also regulates formation of flowers, stems, leaves, and ripening of fruit and controls senescence (^{Hellal et al., 2011}). Benzyl adenine (BA) is a first-generation synthetic cytokinin that elicits plant growth and development responses, setting blossoms and stimulating fruit richness. However, it is an inhibitor of respiratory kinase in plants and therefore increases post-harvest life of vegetables.

The use of organic matters in combination with inorganic fertilizers to optimize nutrient availability to plants is a difficult task as organic materials have variable and complex chemical nature. This requires the understanding and knowledge of the chemical composition, particularly the nutrient content and quality of organic materials and its interaction with inorganic nutrient sources. Studies have compared the yields from a given amount of inorganic fertilizer; an organic material; and their combinations (^{Khalid, 2012}; ^{Oyedeji et al., 2014}), the combinations of organic and inorganic nutrient sources have produced higher yields than solely organic or inorganic sources (^{Shah and Ahmad, 2006}).

Adhatoda zeylanica, is a shrub in the family Acanthaceae well-known to practitioners of Ayurvedic medicine in India. It is commonly called Vasaka (English), Arusa, Baka and Adusa, in Hindi with several other local names among different folks. The plant grows in several regions of India with high distribution in the lower Himalayan regions. The leaves of A. zeylanica have been used extensively in traditional medicine primarily for respiratory disorders. In addition, its utilization as astringent, diuretic and purgative herb is known in India and other countries for several years (^{Jain, 1981}).

Some decades back, the use of different parts of the plant for various diseases treatment have been documented (^{Gupta and Chopra, 1954}). In the recent time, ^{Rastogi (1990}) elucidated the extensive use of the plants parts to treat ailments. Furthermore, A. zeylanica has been used in modern and orthodox drug formulation particularly in treatment of bronchitis (^{Maikhuri and Gangwar, 1993}; ^{Kumar et al., 2005}). There are reports that some local folks used decoction of the leaves for antenatal and postnatal remedies and as an abortifacient (^{Nath et al., 1992}; ^{Jain et al., 1994}). The leaves extract contains pyrroloquinazoline alkaloids, chiefly vasicine and others such as vasicinone, anisotone, vasicoline, adhavasinone which exert effective pharmacological actions (^{Haq et al., 1967}; ^{Chowdhury and Bhattacharyya, 1985}).

Considering the potentials and utility of A. zeylanica, improvement in its cultivation and maintenance of quality to meet the industrial and local need is imperative. If its exploitation should be sustained, there is need to enhance the biomass and phytochemical yields of the plant. To this end, the present study aimed at evaluating foliar application of Kinetin and Benzyl adenine growth hormones as well artificial nutrients (urea) on yield and active compound vasicine composition of the plant.

Clones (stem cuttings with three nodes) of Adhatoda zeylanica collected from the Medicinal Garden of Zandu Foundation For Healthcare, Ambach, India were used for vegetative propagation of the plant. The field experiment was conducted at the C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, India from 2012 -2015. The experiment was laid-out in five (5) treatments with three replicates each in a completely randomized block design with 1 m x 1 m spacing in-between plants and rows. The first foliar treatment (T₁) was distilled water spray which also served as the control for the experiment. The second treatment (T₂) comprised 0.1% urea + 100 ppm kinetin; treatment three (T₃) was a mixture of 0.1 % urea + 100 ppm Benzyl adenine (BA). Treatment four (T₄) was a mixture of 0.2% urea + 100 ppm kinetin while treatment five (T₅) contained 0.2 % urea + 100 pm Benzyl adenine (BA). Hormones and nutrient combination treatments were sprayed on the plant leaves every four weeks. The plant were irrigated to field capacity every week, weeding and other cultural practices were carried out as when due.

The plants were evaluated for plant height, number of branches per plant, number of leaves per plant, fresh and dry leaf yield at maturity (36 months after planting)

Leaf extract preparation

Leaves were collected from the A. zeylanica plants treated with different hormones and nutrients for extract preparation at maturity in the months of January and July which spanned two different seasons (winter and monsoon). The harvested leaves were thoroughly washed, shade-dried and made into a coarse powder. Leaf extract was prepared using 2 g of powdered leaf with 50 ml of 95% ethanol under 80 KHz, 45 °C in ultrasonic extraction device for 30 min, repeated thrice. The extract was collected and filtered and the filtrate dried at 50 °C under reduced pressure on a rotary evaporator. The dried crude extract was dissolved in 100 ml mobile phases, then filtered through filter paper and a 0.45 mm membrane filter (Millipore). A commercial product “VASA Churna” which is a fine green powder from A. zeylanica leaves was used as a standard for comparison.

Qualitative determinations for the presence of bioactive compounds were carried out using standard analytical grade chemicals from Merck, Germany; Sigma-Aldrich, USA and Loba Chem. India. Saponins, carbohydrate, glycosides, alkaloids, phenolic compound, terpenoids, organic acid and tannin presence was determined by methods described by ^{Sofowora (1993}). Loss on drying was determined by placing 3 g of the prepared material in a dried and tarred flat dish, oven-dried at 100-105 °C in a desiccator over phosphorus pentoxide under atmospheric pressure and at room temperature. The loss in weight of the air-dried material (loss on drying) was gravimetrically determined. Other physicochemical parameters such as water solubility, alcohol solubility, acid insolubility ash, total ash and pH of the leaves from different treated A. zeylanica were determined by method of ^{Khandelwal (2008}).

The presences of various alkaloids were analyzed using Thin Layer Chromatography (TLC). The samples were spotted individually in TLC plate using toluene-methanol-dioxane-ammonia solvent system in ratio 1:1:2.5:0.5. Vasicine (Purity 99.95 %) 1 mg dissolved in 1 ml methanol was used as reference solution while Dragendorff’s reagent (R_f; 0.83) was used as standard. The procedure was performed as described by ^{Puratchimani and Jha (2004}). Vicisine, the major bioactive constituents of A. zeylanica leaves were analyzed using high-performance thin layer chromatography (HPTLC). The HPTLC analysis of the extracts was performed on CAMAG Linomat 5 Automatic “Linomat5 -180733 with 100 μL syringe (Hamilton); CAMAG glass twin trough chamber (20 × 10 × 4 cm) as the TLC Chamber and CAMAG TLC Scanner Desitometer linked to winCATS software. The TLC plates were silica gel 60 F254 (0.2 mm thickness) pre-coated (Merck, Germany).

A methanolic solution of Vasicine (1mg/ml) was prepared and further diluted to 100 μg/ml, the solution was applied on plates at 6 mm apart and 1 cm from edge of the plate by CAMAG Linomat 5 automatic sample applicator fitted with 100 μl Hamilton Syringe. Methanolic extracts of five different treated plants were applied to parallel track while the mobile phase; Toluene: Methanol : Dioxane : Ammonia ( 1 : 1 : 2.5 : 0.5 ) was poured into the second trough and the plates was left to equilibrate in the chamber for 20 min at 27 ° C. The plate was then moved to the second trough containing the mobile phase and developed to a distance of 90mm. The HPTLC analysis was conducted following the method described by ^{Fan et al. (2006}). After development, the plate was removed from the chamber, dried in current of hot air, and scanned at 343 nm, using a deuterium lamp coupled with Camag scanner III densitometer at scanning speed of 20 mm s^-1. The offset was 10%, and the sensitivity (SPAN) was optimized at 20 min.

The growth and yield data obtained from various treated A. zeylanica were analyzed using one-way analysis of variance (ANOVA) followed by Duncan Multiple Range Test (DMRT) at significant value of P ≤ 0.01. Peak height and area gotten from HPTLC analysis were integrated and the densitogram obtained by vasicine and extract characterization analyzed with winCATS’ software (version 3.17).

Plant height of A. zeylanica in response to different concentrations of the folia applied growth hormones and nutrient was highest (83.05 cm) in plants treated with 0.2% urea + 100 ppm benzyladenine (T5) which was not statistically different from effect produced by other treatments. However, foliar spray of distilled water (T1) produced least height among all the treatments (Table 1). In term of branches, T5 plants are the most branched with average of 27.45 branches per plant which was significantly higher that other treatments. Although least number of branches was found in T1 which also serves as control, the effect was not significantly different from those of T2 (0.1% urea + 100 ppm kinetin), T3 (0.1% urea + 100 ppm benzyladenine) and T4 (0.2% urea + 100 ppm kinetin). Number of leaf per plant was highest in T2, but statistically similar for T1, T4 and T5 with the least in T3. While T5 plants had the highest fresh weight, the least was recorded in T1, T2 plants had significantly highest dry weight as shown in Table 1.

The loss on drying weight of the treated A. zeylanicai varied with treatments, although, loss on drying of the samples collected for January and June were similar, however, the former had higher loss on drying than the later (Figure 1). The results of phytochemical screening of the extracts from treated A. zeylanica are presented in Table 2. The methods used showed carbohydrate, proteins, steriod, alkaloid, tannis and phenoloic compounds were present in the extracts which were also present in “VASA” used as the standard for comparison.

The results of the physicochemical properties of the treated plant samples collected in Jan and June is shown in Figures 2a-e. Highest loss on drying was recorded in T2 and the least in T1 which was also the control. Water solubility trend was similar to loss on drying, T2 had higest water solubility of 24.45 and 24.50 %w/w for the collections made in the months of January and June respectively and these values are higher than that of “VASA” (Figure 2b). Similarly, alcohol solubility was highest for T4 among the samples collected in January, while T1 (control) extract had highest alcohol solubilty among the samples collected in June and the two treament produced higher acohol solubility than the compared standard “VASA”. Treattment T4 produced highest total ash values for both samples collected in January and June, the least ash content (10.57 %w/w) was obtained by T5 which is also lower than the total ash of the standard comparison. The acid insoluble of the extract of the treated plants was in the order; T4>T1>T3>T2>T5 for January collections, whereas, the order was T1>T2>T3>T5>T4 for the June collection (Figure 2d). Extract obtained from T5 was the most alkaline with pH values of 7.45 and 7.42 for January and June samples respectively, in contrast, T2 produced the most acidic extract from the samples (Figure 2e)

The TLC of the active component Visicine was similar for the various hormonal and nutrient treatments applied to A. zeylanica. The January samples Rf values ranged from 0.84 to 0.82 (Table 3), with the least value obtained from T4, but the trend was different for the June samples, where T5 had Rf value of 0.84, T1 was 0.82 and other treatments had Rf value of 0.83 which is the same with the standard. The HPTLC chromatogram showed the spectral heights for the visicine obtained from the different treated A. zeylanica extracts (Figure 3). The peaks were between 33.80 - 63.60 and 13.50 - 46.40 AU for the Visicine present in the January and June samples respectively. Also, the percentage area ranged from 46.27 - 100% in January sample, the highest was from T3 while T5 had the least (Table 4). With respect to the standard, only T3 had less percentage area of visicine. Whereas T3 had highest (100%) percentage area amoung the June samples, T4 (53.51%) and T5 (46.27%) are below the percentage area of the used standard.

Establishment of the quality control profile of bioactive compounds in plant involved morphological and phytochemical evaluations by standard protocols for the isolation and characterization of the bioactive constituents. This of course is of great important in quality control and medicinal plants utilization, especially for those that could be used as pharmaceutical raw material or use directly by local folks. Therefore, quantitative determination of some phytochemicals and physicochemical parameters are useful for setting standard for crude extract application. Improved growth parameters obtained in this study as a result of foliar application of hormones and nutrients (Urea) is in tandem with ^{Abd El-Wahab and Mohammed (2008}), who reported better growth performance of Trachyspermum ammi on applications of some trace elements. Also, similar result was obtained by ^{Khalid (2012}) who worked on foliar application of nitrogen fertilizer on some members of Apiaceae. While maximum plant growth was obtained with higher combinations of urea and benzyladenine, leave formation was most favoured by less concentration of urea and kinetin foliar application. This implies that combine application of growth hormones and urea would increase growth and biological yield of plant which corroborated the findings of ^{Hellal et al. (2011}). This is because nutrient application promotes photosynthesis and transport assimilates of the carbohydrates to the storage organ.^{Kandeel (1991}) demonstrated that using trace elements as foliar application produced significant effect on plant height, fresh weight, dry weight, fruit yield and essential oil content of Petroselinum crispum. Furthermore, ^{Senthil Kumar et al. (2009}) revealed that Nitrogen application improves plant height, number of branches, fresh and dry weight of shoot, dry matter production, fresh herb yield and essential oil yield of Artemisia pallens.

Loss on drying of the samples collected in June were different from those collected in January, this may be due to higher moisture content of the later as a result of abundant rainfall and precipitation. Highest water solubility was obtained with 0.1% urea + 100 ppm kinetin, solubility for other treatments were similar to that of the standard control (VASA) while less for plant treated with distilled water. This suggests that treatment concentrations affects water solubility. In contrast, alcohol solubility of the extracts from the two seasons were similar even for the different treatments because solubility was not affected by season and nutrient availability or concentrations. Acid insoluble ash of the samples were less than the VASA, which mean acid insoluble ash was negatively affected by foliar hormonal and nutrient application to A. zeylanica. Nonetheless, since amount of water in a sample would affect the concertation of the hydrogen ions, the pH of the January samples was slightly higher than those of the June for the treatments due to less moisture content of the former.

The positive effects of foliar applications of growth hormones and urea on A. zeylanica may be due to the important physiological role of the hormones and nutrient in synthesis of the plant phytochemicals through the action of different enzymes activity and protein synthesis. Consequently, it reflected on an increase in quantity and quality of biochemical parameters of the plants. The obtained results are in accordance with those obtained by previous workers in this regard (^{Randhawa et al., 1996}; ^{Silva and Uchida, 2000}). In addition, it was demonstrated that Nitrogen base fertilizer caused a significant increase in the vegetative growth, essential oil, fixed oil, total carbohydrates, soluble sugars plants (^{Zheljazkov and Margina, 1996}; ^{Khalid, 2001}). Physicochemical constituent such as total ash provides information on the mineral composition and quality of the plant bio-constituents just as the extractive values help to determine the amount of active constituents which can be used as a major tool for obtaining a quality control profile of a medicinal plant (^{Sharma and Kumar, 2016}).

The treatments implication on the Rf values of the bioactive compound visicine revealed that foliar applications of growth hormones and nutrient treatments did not remarkably affect the quality of the bioactive compound. Although T3 produced same quality of vasicine as the market standard, other treatment are not too deviated in term of quality of the active substance. Thus, as the treatments increase growth and phytochemical yield, it may not significantly affect the quality of the chemical constituents. The quality of bioactive compound obtained in this study concurred with the study of ^{Sarkar et al. (2014}).

This study isolated vacisine from leaves of A. Zeylanica and characterized using HPTCL spectra. Although the alkaloid vasicine which is the main active constituent of the plant, however it may be easily oxidized in presence of light and moisture to its more stable form vasicinone which may account for more than 30% of crude extract (^{Chowdhuri et al., 1987}). The spectra of vacisine from HPTLC analysis in this study is similar to those obtained by ^{Suthar et al. (2009}). Thus the study revealed that foliar application of the growth hormones and urea though improves biochemical yield, it does not reduces the quality of bioactive compound as the present results agreed with the findings of ^{Srivastava et al. (2001}). Therefore, foliar application of growth hormones and nutrition in moderate concentrations could enhance growth, biochemical yield and active compound in A. zeylanica.

The present study evaluated effects of folia application of plant growth hormones and urea on growth parameters, biochemical yield and purity of the bioactive vacisine from the extract of A. zeylanica. The results showed that plant performance with respect to growth and phytochemical yield was enhanced by the treatment without compromising the quality of the major bioactive compound vacisine. In addition, it was revealed that phytochemical constituents of A. zeylanica collected in two different seasons differs slightly in phytochemical yield and physicochemical properties. The quality and characteristics of the bioactive compound vicisine from the treated plants are similar with the standard market product. The study concludes that foliar application of phytohormones and nutrient could be used to improve growth and biochemical yield of A. zeylanica. Thus providing an improved agricultural practices to increase cultivation and production of medicinal plant.