Phytochemicals Screening, Phenolic Estimation and Evaluation for Anti-Oxidant, Anti-Inflammatory and Anti-Microbial Activities of Sequentially Soxhlet Extracted Coconut Testa

Background: In many coconut industries, the outer layer of thin brown skin of coconut kernel known as testa is peeled out as a byproduct. Despite the testa is rich in fat and plenty of polyphenolic compounds, it has been underu-tilized either as animal feed, serving as raw materials for bio-diesel production or discarded directly. Anticipating coconut testa (CT) as a natural source of multiple phyto-chemicals, its exploitation for the pharmacological activity or utilization as value added product is required which may reduce the disposal costs as well. Methods: Secondary metabolites from CT were extracted sequentially with different organic solvents based on polarity in the soxhlet apparatus followed by extraction with sterilized water. The crude dried extracts thus prepared were evaluated for qualitative screening of phytochemicals and quantitative estimation of total phenols, flavonoids and tannin content. Moreover, the antioxidant, anti-inflammatory and anti-microbial activities were also investigated. Results: Phytochemicals screening revealed the presence of polyphenolic compounds in methanolic fraction including phenols (822.60 ± 16.36 mg/g), flavonoids (103.30 ± 9.78 mg/g) and tannin (663.50 ± 19.26 mg/g), whereas non-phenolic compounds were present in other fractions. While methanolic fraction showed invariably the highest anti-oxidant activity in multiple assay methods, non-phenolic compounds in aqueous and chloroform fractions exhibited high anti-inflammatory activity. Antimicrobial activity was observed by both phenolic and non-phenolic compounds. Conclusion: The findings of the study reveal that CT is a rich source of various polyphenolic and non-phenolic natural antioxidants, anti-inflammatory and antimicrobial compounds. These findings are promising and form the basis to identify the number of active components and their characterization.


Introduction
Coconut (Cocos nucifera L.) belongs to the family Arecaceae [Palmae] and the subfamily Cocoideae is an important monocotyledon plant widely grown in the tropic and sub-tropics [1]. Further, certain religious and traditional values of coconut in most parts of India and other South East Asian countries have fared the production and productivity of coconut in these countries much better than many other countries. The coconut palm is sometimes referred to as 'great nut of India' owing to the use of all of its parts in some way or the other in coconut-growing areas. Apart from the use of coconut water, the natural sterile liquid from the young immature coconuts as a beverage; the kernel is used as the source of food and oil. Further, the wastes such as shell or coir are also used as raw materials for many industries for the production of shell-activated carbon or fibers for car seats and other household materials [2] [3] [4]. With the increasing demand for coconut oil, preparation of desiccated coconut powder or other products such as coconut cream and milk powder, the thin brown skin of coconut kernel known as testa is peeled out and processed in many coconut industries and the byproduct testa is usually underutilized. Of the more than 93 countries growing coconut Worldwide, India ranks 3 rd by producing about 12,000 -14,000 metric tons coconut annually, which contributes to about 19.3% of total coconut production of the World [5]. In an estimate, it has observed that the coconut testa (CT) constitutes about 18% of the total dry weight of kernel [6] and that about 23.7 thousand tons testa are produced in Hainan Island annually [4]. The worldwide production of this waste is even more and requires alternative ways for its re-utilization as valuable products. Several studies have been conducted to assess the bio-active potential or nutritional value of coconut testa.
Studies have shown that the testa is rich in fat [7] and contains plenty of polyphenolic compounds with a potent antioxidant capacity [8] [9]. Polyphenolic compounds ranging from simple phenolic acids to polyphenols such as flavonoids, tannins, anthocyanins, etc. are important plant derived phytochemicals which have anti-oxidant and anti-ageing property and have been shown to improve physical fitness or degenerative diseases like cancer, diabetes, hypertension and cardiovascular problems [10] [11] [12] [13]. However, the use of testa is largely limited either as an animal feed [14], serving as raw material for bio-diesel production [15] or discarded directly despite being a rich source of polyphenol compounds. The fact that plant secondary metabolites including polyphenolic compounds being diverse, different classes of compounds are soluble only in specific solvent system [16] and the extraction yields depends on methods adopted, nature  [19]. A previous study examining for identification of suitable solvent system for maximum extractability from the coconut testa has revealed significant differences in total phenolic content (TPC) and total flavonoid content (TFC) with antioxidant potential in various solvent systems studied [9]. However, to date, only ethanolic extract of defatted coconut testa has been analyzed for the qualitative and quantitative determination of phytochemicals [20]. Moreover, the ethanolic extract of testa has been found to possess anti-hyperglycaemic activity [8], exhibit inhibition effect on the oxidation of human low-density lipoprotein [21] or evaluated for cosmeceutical potentials [22] [14]. Further, the ethanolic extract of coconut testa has been shown to contain more natural anti-oxidants compared to kernel oil [20]. The oil extracts of testa have also been proved to be protective against oxidative damage induced by reactive oxygen species [23] [21]. Despite the fact of testa as a natural source of multiple phyto-chemicals, its exploitation for potential human health benefits is yet to be achieved. Utilization of coconut testa as value added product may have the economic and environmental impacts of reducing the disposal costs.
Therefore, in the present study secondary metabolites from coconut testa (CT) were extracted sequentially using organic solvents [Petrolium benzene, chloroform, ethyl acetate and methanol] and water based on polarity in soxhlet apparatus and evaluated for qualitative screening of phytochemicals. Besides, with the aim at exploring the potential benefit, the different solvent extracts of coconut testa were evaluated for quantitative estimation of total phenols, flavonoids and tannin content. Moreover, the antioxidant activity, anti-inflammatory activity and anti-microbial activity were also investigated.

Collection of Sample
Healthy coconuts were collected in sterile plastic bags from the different locality of Baripada, Mayurbhanj, Odisha and processed to peel off the thin, brown outer covering of kernel called the testa with the help of a scraper. These peeled off testa were then allowed to shed dry for 10 -15 days followed by pulverization into coarse powder using the blender.

Phytochemical Screening
Preliminary qualitative screening for the presence of various phytochemicals was carried out using the protocols of Sheel et al. [24], Nanna et al. [25], Mishra et al. [26], and Sharma et al. [27].

Estimation for Total Phenolic Content
The total phenolic content in various solvent fractions of CT was determined based on Ebrahimzadeh et al. [28], with partial modification.

Estimation for Total Flavonoid Content
The total flavonoid content was determined by the Aluminum chloride colorimetric method described by Islam et al. [29], with minor modification as detailed below. Quercetin was used instead of gallic acid as standard. Dilution of plant extracts and quercetin were carried out as mentioned previously in this paper for total phenol estimation. To 0.

Estimation for Total Tannin Content [TTC]
The total tannin content in each of the solvent fraction of CT was determined
Briefly, the reaction was initiated by adding 0.3 ml of various concentrations RT. The percentage of inhibition was calculated using Equation (1). Ascorbic acid, gallic acid, tannic acid, quercetin and BHT were used as positive controls.

Phosphomolybdate Assay
Phosphomolybdate assay was carried out by the method proposed by Prieto et al. [33].

Metal Chelating Assay
The metal chelating assay was carried out by the method reported in Dinis et al., [34]. In this method, to 1ml of various concentrations [312.5 -10,000 μg/ml in two fold dilution] of solvent extract, 0.5 ml of 2 M ferrous chloride and 2 ml of 5 mM ferrozine solution were added. The solution was mixed thoroughly and incubated in dark at room temperature for 10 minutes. The control was run in the same process except that distilled water replaced solvent extract. The absorbance was read at 562 nm against distilled water as a blank. EDTA was used as a standard metal chelator. Percent of inhibition was calculated using Equation (1).

Reducing Power Assay
Antioxidant capacities of different solvent fractions of CT were measured in reducing power assay according to a method reported by Oyaizu, [35]. To 1 ml of each of the solvent extracts of various concentrations [62 -1000 μg/ml in two fold dilution] in 10% DMSO, 2.5 ml of sodium phosphate buffer [PH 6.6] and 2.5 ml of potassium ferricyanide [1% w/v] were added and mixed well. After incubation for 20 minutes at 50˚C, 2.5 ml of trichloroacetic acid was added and the whole mixture was centrifuged at 3000 rpm for 10 minutes. To 2.5 ml of the supernatant, 2.5 ml of distilled water and 0.5 ml of ferric chloride [0.1% w/v in distilled water] solution were added and the absorbance was measured at 700 nm against the blank. Blank for each solvent extract was run using the same procedure but replacing the extracts with an equal volume of solvent [10% DMSO or water]. Quercetin and ascorbic acid were used as positive controls 2.7.6. CUPRAC Assay CUPRAC assay was carried out according to the method proposed by Karaman et al. [36].

Antimicrobial Activity
The test pathogens such as two bacterial strains, Escherichia coli [ were carried out in aseptic condition in a laminar air hood. Following this, the plates were incubated at 37˚C for 24 hours and the zone of inhibition was measured in mm.

In Vitro Anti-Inflammatory Activity
The anti-inflammatory activity was assessed by membrane stabilization of heat induced or hypotonic induced haemolysis of human RBCs as per the method described in Shinde et al. [38] and inhibition of heat induced albumin denaturation following the protocol of Mizushima et al. [39] with some minor modifications.

Preparation of HRBC Suspension
About 3 ml of blood from a healthy volunteer was freshly collected in a heparinized vial and centrifuged at 1000 rpm for about 2 minutes at RT.
where OD 1 = Absorbance of test sample unheated, OD 2 = absorbance of test sample heated and OD 3 = absorbance of control sample heated.

Hypotonic Induced Haemolysis
In hypotonic induced haemolysis of HRBC also, 11 number of centrifuge tubes of 15 ml capacity were taken and named as described for heat induced haemolysis. However, in one set labeled as 1 -5, 2 ml of different concentrations of the solvent extract [0.062 -1 mg/ml] were taken while in another set, 2 ml of ibuprofen of corresponding concentration [0.062 -1 mg/ml] were taken. 2 ml of normal saline was taken in a centrifuge tube labeled as "0". In all these eleven centrifuge tubes, 2 ml of hypotonic solution [50 mM NaCl in 10 mM sodium phosphate buffer] was added followed by the addition of 0.5 ml of 10% HRBC solution. After 10 minutes of incubation at RT, these were centrifuged at 1000 rpm for 10 minutes and the absorbance of the supernatant was recorded against the blank [normal saline] at 540 nm. The percentage inhibition of haemolysis was calculated as per Equation (1).

Heat Induced Albumin Denaturation
The inhibition potential of various solvent fractions of CT to heat induced albumin denaturation was carried out as per Mizushima et al., [39] with modification and compared to the known anti-inflammatory drug ibuprofen.

Statistical Analysis
Data generated from quantitative assays were expressed as mean values with standard deviation. The percentage yield of the different solvent extract of CT was determined as % yield = W1/W2 × 100, where W1 is the weight of dry ex-

In Vitro Antioxidant Activity
The antioxidant activities of various solvent extracts of coconut testa were investigated by in vitro scavenging ability to free radicals such as DPPH and ABTS, reduc-  [25], Mishra et al. [26], and Sharma et al. [27]. "+" indicates presence and "−" indicates absence. The increase in + sign indicates the intensity. stronger antioxidant activity (Table 2). In CUPRAC and reducing power assays, the anti-oxidant activities increased with absorption value at respective wavelengths.

DPPH˙ Scavenging Activity
The results showed that chloroform [EC 50 Table 2). The ethyl acetate fraction had the least activity.

Metal Chelating Assay
Assessment of various solvent extracts of coconut testa for their chelating ability of ferrous ions revealed that only methanolic and aqueous fractions had the power   Figure 1(a) and Figure 1  Food and Nutrition Sciences range of 125 -500 μg/ml, this activity was comparable at 1000 μg/ml. However, in CUPRAC assay the methanolic fraction exhibited high-reduction activity compared to the control, quercetion and ascorbic acid at all range of studied concentration. Besides, the ethyl acetate fraction also showed high anti-oxidant activity than quercetion and ascorbic acid at a concentration beyond 500 μg/ml.

Anti-Microbial Assay
The screening results for antimicrobial activity of different solvent extracts against two bacterial strains; one gram positive such as Staphylococcus aureus, and one gram negative such as Escherichia coli, and one fungal strain Candida albicans are presented in Table 3. The results indicated that petroleum benzene, chloroform and methanolic fractions of coconut testa had antibacterial and antifungal activity against all tested pathogens with a maximum zone of inhibition obtained for the methanolic fraction. However, while aqueous extract showed antibacterial activity against S. aureus only with a maximum zone of inhibition [17.33 mm] which is highest compared to all other solvent extracts, ethyl acetate fraction did not exhibit any antimicrobial activity against the test pathogens at a concentration of 10 mg/ml.

Anti-Inflammatory Assay
Investigation for anti-inflammatory activity of different solvent fractions of CT by membrane stabilization of heat and hypotonic solution induced human red blood cell haemolysis and inhibition of heat induced albumin denaturation assay revealed that aqueous fraction had the highest anti-inflammatory activity comparable to that of the known anti-inflammatory drug ibuprofen followed by the CH fraction and methanolic fraction ( Table 4). The PB and EA fractions had the least activity. The heat induced HRBC assay for the EA extract could not be carried out due to lack of the extract as the percentage yield of EA fraction was very very less.

Discussion
The present investigation shows that CT contains a diverse group of phytochemicals such as alkaloids, glycosides, saponin, sterol, phenols, flavonids, tan- viously. CT is anticipated as a potential source of polyphenolic compounds as evidenced by a significant increase in the total phenol content of coconut milk obtained from the grating with testa [41]. Besides, increased phenolic contents in coconut oil extracted from the kernel with testa had been observed compared to white coconut kernel alone [23] and the oil extracted from coconut testa was rich in phenolic compounds [7]. In a recent study, it has been observed that the extractability of phenolics and flavonoid compounds from CT were found to be affected largely by the polarity and acidification of the solvents used in extraction and that the maximum yield of total phenolic compounds [167 mg GAE/g] was obtained with 80% acidified acetone [9]. Several other studies also evidence for aqueous acetone as the efficient solvent system in extracting phenolic com- in the present study we documented the total phenolic content of CT to be as high as 822.6 mg GAE/g in the methanolic fraction. Previous sequential extraction studies with soxhlet also reveal high phenolic content in the methanolic fraction from various parts of Moringa oleifera [46] and of Caesalpinia digyna root [47]. The fact that polyphenolic compounds in plants are mostly found in conjugated forms with glycosides, proteins and sugars which are soluble in the specific solvent system [16] [48] [49]. The present finding of enhanced phenolic content in the methanolic fraction during sequential extraction with different solvents of varying polarity could be explained by the sequential elimination of solvent soluble non-polar compounds and interfering unwanted non-phenolic substances such as wax, fats, terpenes, etc. This is partially evidenced by the removal of almost all glycosides, sterol, saponin, resin, fats and oil before extraction with methanol ( Table 1) [39]. Therefore, stabilizing the lysosomal membrane or preventing the release of lysosomal enzymes along with inhibition of protein denaturation have been considered as a measure of anti-inflammatory activity for plant extracts or drugs.
Owing to the structural analogy of human red blood cell [HRBC] with lysosomal membrane [59] [60] [61], the in vitro anti-inflammatory activity of various solvent extracts of CT was determined by percentage inhibition of hypotonicity or heat induced HRBC membrane lysis. Besides, the ability of different solvent extracts to inhibit heat induced albumin denaturation was studied. Our results showed that the aqueous fraction had the comparable anti-inflammatory activity with the known drug ibuprofen followed by the CH fraction and that both of these fractions derived from CT do not contain any polyphenolic compounds.
We observed some anti-inflammatory activity of methanolic fraction containing polyphenolic compounds. However, these activities were less than that of aqueous and CH fraction. Although anti-inflammatory activities are usually attributed to the polyphenolic compounds present in the plant extracts [4] [62] [63] [64], there are limited studies [65] [66] including ours which witness non-polyphenolics as potent anti-inflammatory compounds. In the absence of any mechanistic study, the resistance to cell lysis of these CT derived compounds could be due to alteration of the surface charges of the cells preventing or promoting physical interaction with different agents involved in the haemolysis of red blood cells [67], expansion of membrane or shrinkage of the cell or by aureus. Earlier studies also suggest gram-negative bacteria to be generally more resistant to various drugs and antibiotics than gram-positive bacteria [73] and these differences in susceptibility by the aqueous fraction in the present study could be explained by the differences in cell wall composition or drug resistant genes on plasmids they inherited with. However, fractions of coconut testa obtained with organic solvents [PB, CH and methanol] had antibacterial and antifungal activity against all tested pathogens with the maximum zone of inhibition obtained for methanolic fraction. In most of the cases, organic extracts have been shown to exhibit greater antimicrobial activity compared to aqueous extract [74], although this is not universal. This suggests that relatively hydrophobic nature of the compounds in CT had the ability to kill both gram positive and gram negative bacteria along with fungal pathogens.

Conclusion
Overall, our study reveals that CT is a rich source of various polyphenolic and non-phenolic natural antioxidants, anti-inflammatory and antimicrobial compounds. Although the number of active components and their chemical and physical nature in each extract are unclear, the findings of the study are promising and can form the basis to exploit the CT for therapeutic benefits. Further research is warranted to characterize the individual component of the active principle.