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Distribution of Bioactive Compounds and Antioxidant Capacity of Different Parts of Justicia adhatoda L. (Acanthaceae)

Justicia adhatoda L is a well-known medicinal plant used in traditional systems of medicine globally. Different parts of J. adhatoda have been used for treatment of various ailments, mainly for the respiratory tract-based ailments. The present study was conducted to quantify the total antioxidant capacity (TAC), total phenolic content (TPC) and total flavonoid content (TFC) of different parts (mature leaves, immature leaves, flowers, bracts, soft stems, bark of mature stem and roots) of J. adhatoda using ferric reducing antioxidant power (FRAP) assay, modified Folin-Ciocalteu method and colorimetric method respectively. The results revealed that all tested parts of J. adhatoda contained marked amounts of TAC, TPC and TFC. Among tested parts, immature leaves showed a significantly higher TAC (19.28 ± 1.96 mg/TE g DW) followed by flowers (16.97 ± 0.99 mg/TE g DW) and bracts (14.19 ± 1.85 mg/TE g DW). Significantly the highest TPC (11.33 ± 0.14 mg/GAE g DW) and TFC (16.66 ± 3.06 mg/RE g DW) were observed in flowers followed by bracts and immature leaves. Moreover, there were positive correlations of TAC with TPC (R2 = 0.5411) and TFC (R2 = 0.5209). According to the results, it can be concluded that immature leaves, flowers and bracts of J. adhatoda contain marked amounts of bioactive compounds and hence could be effectively used for pharmaceutical industries.


Introduction
Justica adathoda L. is a therapeutically important medicinal plant belonging to the family Acanthaceae. This plant is native to Asia and distributed throughout Sri Lanka, India, Pakistan and Myanmar. J, adathoda is commonly known in English as Malabar nut, Pawatta in Sinhala, Adadodai in Tamil and Vasaka in Sanskrit [1]. This is an evergreen shrub with average height of three meters. It has opposite and ascending branches. The leaves are broad, leathery and entirely lanceolate in shape with opposite formation. The stem is soft while the flowers are dense, terminal spikes with large, attractive white petals, streaked with purple on the lower lip. Different parts of J. adathoda have multiple uses in traditional Unani and Ayurvedic medicines [1,2].
The main phytochemical group present in J. adathoda is alkaloids. The prominent compounds present in J. adhatoda are vasicine, vasicinone, deoxyvasicine, vasicol, adhatodinine, and vasicinol which are present in different parts of the plant. In addition, vitamin C, saponins, flavonoids, steroids, and fatty acids are also present [2,3] Due to the presence of an array of therapeutically active phytochemicals in different parts of J. adathoda, has been extensively used in traditional and Ayurveda systems of medicine to treat respiratory disorders such as cold cough, whooping cough, chronic bronchitis and asthma [1]. The powdered herb is boiled with sesame oil, is used to heal ear infections and arrest bleeding. Boiled leaves are used to treat rheumatic pain, and to relieve the pain of urinary tract infections [4] It was discovered that the main component vasicine present in J. adathoda shows oxytocin action helping for speeding up the delivery during child birth [1]. Moreover, this herb is important due to its anti-asthmatic and bronchodilator activity, wound healing activity, anti-ulcer activity, antibacterial activity, anti-allergy activity and anti-tubercular activity [4]. Apart from that medicinal uses, the tender leaves and flowers of J. adathoda are used as a vegetable in India and Nepal [5] In addition, leaf aqueous extract of J. adhatoda and O. tenuiflorum exhibited antioxidative effects and impede hyperlipidemia [6].
Generation of reactive oxygen species (ROS) and other free radicals which are normally produced during the metabolism of the body cells are able to neutralized by natural antioxidant systems. But imbalance of free radicals resulted to oxidative stress which related to cardiovascular disease, cancer and other chronic diseases. Reducing ability of plant extracts are directly related to the phytochemical content of the extract [7]. Though J. adathoda is widely used in traditional systems of medicine, information on its bioactive compounds and antioxidant capacity present in different parts of the plant are limited. Therefore, current study was aimed to determine the bioactive compounds and antioxidant capacity of different parts of J. adathoda.

Sample Collection
Mature leaves, immature leaves (bud with two leaves), flowers, bracts, soft stems, bark of mature stem, and roots of J. adathoda plant were collected from Kegalle district in Sri Lanka and brought to the laboratory.

Sample Preparation
The fresh leaves of mature and immature stages, flowers, bracts, soft stems, bark of mature stem and roots were thoroughly washed under running water. Then they were cut into small pieces and air dried at room temperature (28 ± 2°C) for three days.

Extraction of Phytochemicals
All air-dried samples were powdered using a coffee grinder and sieved with 0.25 mm mesh. Powdered sample (0.1 g) was mixed with 5mL of 80% methanol and vortexed for 15 min. Then it was placed in a water bath at 60°C for 40 min and vortex procedure was repeated in 10 min intervals. After centrifugation at 4,000 rpm for 5 minutes to remove the solid fraction, the supernatant was decanted into a 15 mL centrifuge tube and the pellet was re-extracted with 5 mL of 80% methanol. Supernatants were pooled and stored at -20°C until further usage.

Determination of Total Antioxidant Capacity (TAC)
Total antioxidant capacity was determined using ferric ion reducing antioxidant power (FRAP) assay described by Benzie and Strain [8], with slight modifications. Briefly, 100 µL of methanolic extracts of samples were mixed with 900 µL of freshly prepared FRAP (Mixing 25 mL of 300 mM Sodium acetate buffer, 2.5 mL of 10 mM TPTZ solution and 2.5 mL of 20 mM ferric chloride solution) reagent of pH 3.6 and incubated for 4 min. Absorbance was measured at 593 nm using spectrophotometer. TAC was calculated using the standard trolox curve and expressed as milligrams of trolox equivalents (TE) per g of DW.

Determination of Total Phenolic Content (TPC)
Total phenolic content of all samples was determined using a modified Folin-Ciocalteu method [9]. Briefly, 4 mL of distilled water and 0.5 mL of extract were added into a test tube. Then the same amounts of (0.5 mL) 0.2 N Folin-Ciocalteu reagent was added and allowed to react for 3 min. Then 1 mL of a saturated sodium carbonate solution was mixed and incubated in a water bath for 2 hrs. at 30°C. The absorbance was measured at 760 nm using a spectrophotometer. TPC was calculated using the standard gallic acid curve and expressed as milligrams of gallic acid equivalents (GAE) per g of DW.

Determination of Total Flavonoid Content (TFC)
Total flavonoid content was determined by a colorimetric method as described by Liu et al. [10] with slight modifications. Briefly, a volume of 0.5 mLof plant extract was added to centrifuge tube containing 3.5 mL of distilled water. Then the solution was mixed with 0.3 mL of 5% NaNO 2. After 6 min 0.3 mL of 10% Al (NO 3 ) 3 .6H 2 O solution was added and allowed to stand another 6 min. Then, 2 mL of 2 M NaOH was added. The mixture was diluted with 1.4 mL of distilled water and absorbance was measured immediately using a spectrophotometer at 510 nm wavelength. TFC was calculated using the standard rutin curve and expressed as milligrams of rutin equivalents (RE) per g DW.

Statistical Analysis
To verify the statistical significance of all parameters, mean values ± SD were calculated. Statistical comparison of mean values was performed by General Linear Model (GLM) of ANOVA followed by Tukey Multiple Range Test using SAS statistical software (Version 9.4).
Moreover, order of decrease of TAC of different extracts of J. adathoda was as follows; immature leaves > flowers > bracts > bark of mature stem > roots > soft stem > mature leaves. From the results obtained, it is clear that the higher TAC is concentrated around the bud region of J. adathoda and decreased with the maturity. This phenomenon agreed with Izzreen and Fadzelly [11], and Panawala et al [12] , which suggested that antioxidant values are high in immature leaves and decreased with the increment of maturity stage of tea and allspice respectively.. This may be due to the physiological changes of leaf with age and the unique chemical compounds transportation within the plant [13].  Table 1. TPC and TFC contents of different parts were ranged from 1.94 ± 0.12 to 11.33 ± 0.14 mg/GAE g DW and 3.54. ± 0.46 to 16.66 a ± 3.06 respectively. Significantly the higher TPC was observed in flowers (11.33 ± 0.14 mg/GAE g DW) followed by bracts (8.46 ± 0.66 mg/GAE g DW) and immature leaves (7.4 ± 0.32 mg/GAE g DW). The significantly higher TFC was recorded from flowers (16.66 ± 3.06 mg/RE g DW) followed by immature leaves, bracts, mature leaves, bark and roots respectively. However, there was no significant difference observed among the results in immature leaves, flowers and bracts parts whereas, least TFC was recorded in soft stem parts (3.54 ± 0.46 mg/RE g DW).
Both TPC and TFC were highly reported in flowers followed by the immature bud region of the plant. Results obtained from this study agreed with the findings of Saha et al. [14], who observed the higher content of phenolics in Saraca asoca flowers among different parts of the plant.