ANTIOXIDANT ACTIVITY AND TOTAL PHENOL CONTENT OF DIFFERENT PLANT PARTS OF LEBANESE ANNONA SQUAMOSA LINN

Objective: The aim of this work was to characterize the antioxidant properties and to evaluate the total phenol content of leaves, bark, pericarp, and pulp extracts of Lebanese Annona squamosa Linn. (A. squamosa),, as well as a total screening of secondary metabolites present in the various plant parts studied. Methods: Two solvent systems were used for extraction: ethanol 80 % and methanol 80 %. The antioxidant activity of different extracts was investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The Total Phenol Content (TPC) of the different plant parts are determined and compared via Folin-Ciocalteu method. The results were presented as the mean of three separate experiments and error bars were used to illustrate standard deviation. Results: The phenolic content was found to be highest in the A. squamosa leaves methanolic and ethanolic extracts (117.2 mg and 112.92 gallic acid extract/g, respectively). The results showed that A. squamosa leaves methanolic and ethanolic extracts display the highest antioxidant activities than the bark, pulp and pericarp extracts, with half-maximal inhibitory concentration values 13.61 and 15.97 μg. ml-1 respectively. Ethanol 80 % and methanol 80 % were found to be efficient for the extraction of phenolic compounds. Conclusion: Results of this study indicate the presence of promising compounds in Lebanese A. squamosa that are able to act as antioxidants and free radical scavengers.


INTRODUCTION
Free radicals are the inevitable by-products resulting from biological redox reactions. At high concentrations, they produce oxidative stress, a harmful process that can damage cell function and structures. They have been reported to inactivate enzymes and damage important cellular components causing tissue injury through covalent bond and lipid peroxidation [1]. Research in the medical field reports the implication of free radicals induced oxidative stress in the development of many diseases such as inflammatory diseases, atherosclerosis, aging, diabetes, and cancer [2][3][4].
The antioxidants are associated with the reduction of free radical generation. Thus, it may be beneficial to recover normal function and treat such diseases. Antioxidants are a crucial defense against free radical-induced damage and are critical in maintaining optimum health and wellbeing [5]. In recent years, there has been an increased interest in the therapeutic use of antioxidants in the treatment of diseases associated with oxidative stress. Antioxidantrich diet and natural antioxidant supplements as part of a healthy lifestyle are now being recognized to protect health from oxidative stress [6]. Plants and other organisms have evolved a wide range of mechanisms to surmount this problem with a wide variety of antioxidants for molecules and enzymes [7,8].
Annona squamosa Linn. (A. squamosa) is a small, well-branched tree of the family of Annonaceae. The plant is native to the tropical Americas and is cultivated mainly for its edible fruit commonly called "sugar apple". It was later taken to the Philippines and Asia via West Indies. A. squamosa grows at altitudes of 0 to 2,000 m and does well in hot, dry climates; at much lower altitudes than many of the other fruit bearers in its family [9,10]. In Lebanon, the A. squamosa crop is gaining in popularity and has been cultivated along the coastal zone at an altitude of 0-200 m above sea level [11]. All parts of A. squamosa tree are traditionally used to treat various diseases [12]. It is considered to be a good source of natural antioxidants for various diseases [13]. It has been reported to possess a wide variety of pharmacological activities [14][15][16][17][18]. 100 m from sea-level. The plant has been identified by Pr. Jean HABIB (Professor of Pharmacognosy at the Lebanese University) and confirmations were done through the Flora of the presidency of Madras, by Gamble J. S. 1921 [19]. The voucher specimens (No. 1803(No. -1804 are deposited at Pharmacognosy Departement, Faculty of Pharmacy of the Lebanese University ( fig. 1).

Fig. 1: A. squamosa: the appearance of leaves (A) and fruit (B)
A. squamosa leaves and bark were collected in the month of February and the beginning of March 2018 from Annona trees. The fruit was collected in the month of January 2018. Different plant parts, namely bark and leaves, were isolated from A. squamosa tree. Different fruit parts, namely pulp, and pericarp were isolated. The leaves were either used as fresh or shade dried on a well-ventilated rack for 3 w. The bark was also shade dried for two months. The dried samples were ground separately into fine powder and stored at room temperature for further use.

Preparation of plant extracts
Two solvents systems were used for the extraction: 80 % methanol (v/v in water) and 80 % ethanol (v/v in water). A. squamosa plant extracts were obtained by macerating 20 g finely cut fresh leaves/20 g pulverized dried leaves/20 g pulverized dried bark/96 g pericarp, or 96 g pulp (filtrated using filter paper) with frequent agitation for 24 h with 100 ml of the solvent system. The obtained extracts were then filtered twice through Whatman's filter paper No. 1 and the filtrates were concentrated under vacuum using a rotary evaporator at 40 . The condensed extracts were put in the refrigerator at-20 °C and then lyophilized to obtain a solid residue. The extracts were labelled and stored at room temperature in tightly closed glass containers for further analysis. The extraction yield (mg of dry extract per g of sample) was determined.

Phytochemical screening
Prepared plant extracts were analyzed for the presence or absence of chemical constituents like hydrolyzable and condensed tannins, flavonoids, and saponins. Alkaloids tests were performed directly on the plant material. The change of color or formation of precipitate was observed when the test reagent was added to the prepared sample for the phytochemical test. The result was recorded as present (+) or absent (-) depending on the outcome of the test. All the experiments were executed in triplicates.

Determination of total phenol content (TPC)
Total phenolic contents of A. squamosa parts were estimated using the Folin-Ciocalteu reagent based spectrophotometric assay [20]. Gallic acid is used as a standard for determining the phenol content of various extracts. For this purpose, 100 µl of different concentrations (0.5-0.88 g. ml -1 ) of the test extract solutions were combined with 0.5 ml of Folin-Ciocalteu reagent (2 N) and the mixture was incubated for 5 min in the dark. Then, 2 ml of sodium carbonate aqueous solution (7 % w/v) was added. Samples were incubated in darkness for 30 min at room temperature. The absorbance of the reaction mixture was measured at 760 nm against a blank using a UV-VIS spectrophotometer. Distilled water and 2 ml of Na2CO3 solution acted as a blank. A standard curve was plotted using different concentrations of gallic acid. The absorbance obtained was converted to gallic acid concentration using gallic acid standard curve. The total phenolic content was calculated as mg of gallic acid extract (GAE) per g of dry A. squamosa extract using the following formula (1): Where: -GAE = mg equivalent of GAE per gram of dry extract used.
-C is the concentration of gallic acid (mg. ml -1 ) calculated by the calibration curve regression equation.
-V is the volume of plant extract solution -m is the mass of a plant extract used to prepare the test extract solution used.
Further calculations were done to obtain the total phenol content in mg GAE per gram of initial plant and fruit part used.

Determination of antioxidant activity
The antioxidant activities of A. squamosa dried leaves, fresh leaves, dried bark, pulp, and pericarp of the fruit were assessed using DPPH assay as described by Kumawat et al. [21]. 1 ml of the methanolic extract solutions of different concentrations (0.001-0.26 mg. ml -1 ) was added to 1 ml of a methanolic solution of DPPH (81.14 µM) in a test tube, followed by vigorous stirring. After 30 min of incubation in the dark, the decrease in absorbance of each mixture (due to quenching of DPPH free radicals) was determined at 517 nm against a blank (methanol) using a UV-VIS spectrophotometer. The percentage scavenging activity of the DPPH radicals was calculated according to the following formula (2): Where: -Acontrol is the absorbance of the mixture of methanol and DPPH solution -Asample is the absorbance of the mixture of the sample extract/standard and DPPH solution.
Based on graphic values of the percentage of DPPH inhibition vs extract concentration, the half-maximal inhibitory concentration (IC50) (the concentration of the extract needed to inhibit the 50% of the DPPH) of each extract was estimated. The antioxidant activities of all the samples were compared to the antioxidant activity of ascorbic acid, i.e. ascorbic acid was used as a reference standard.

Statistical analysis
The experimental runs and the analyses were carried out in triplicate. The experimental results derived in the study were expressed as the mean±standard deviation (SD). Linear regression analysis was used to calculate the IC50 values. Data were analyzed through independent samples t-test using SPSS statistics 9.0 software, the same software was used to test IC50.

Extraction yields
The extraction yield depends on the nature of the solvent, the time, the temperature and the chemical nature of the sample used [22]. It is calculated as % w/w (weight of extract/weight of plant material).
In the present study, the obtained extraction yields for the two solvent systems used namely methanol 80 % and ethanol 80 % are listed in table 1. Although a number of factors have to be considered when selected the appropriate solvent, ethanol was investigated to test the possibility to replace methanol in order to reduce the toxicity. Knowing that same conditions of time and temperature were applied for the same plant part used during extraction, methanolic system was more effective in extraction compared to ethanol in the case of fresh leaves and dried bark. While ethanolic system was the suitable solvent for the extraction of pericarp. No significant difference is detected in the case of dried leaves and pulp (P ˂ 0.05).  The results of the phytochemical qualitative analysis are reported in . The screening test showed the presence of and alkaloids in all studied extracts of A. squamosa. Also, hydrolyzable tannins were absent in all studied extracts. Condensed tannins were absent in pulp extract, but were present in all other extracts and mostly abundant in fresh and dried leaves extra Dried bark and leaves were rich in alkaloids. ethanol is not more polar than methanol difference was revealed in terms of families of secondary metabolites extracted. In addition, our results are in go with the experimental data of Sabbah [24].  which interrupt chain oxidation reactions by donation of a hydrogen atom or chelating metals. So they act as reducing agents and antioxidants [25]. Dried bark and leaves were rich in alkaloids. Besides the fact that ethanol is not more polar than methanol, it is worth noting that no difference was revealed in terms of families of secondary metabolites extracted. In addition, our results are in good agreement with the experimental data of Sabbah et al. [23] and Mahawar et al. The conducted work showed no significant difference in the total phenol content between methanolic and ethanolic extract of squamosa dry leaves, dry bark, pulp and pericarp (P cases, ethanol can effectively substitute methanol in the extraction of polyphenols from these plant parts. However, extrac  To the best of our knowledge, there is no study available that has focused upon different parts of A. squamosa grown in Lebanon. On The highest amounts of total phenolic content were 117.2, 112.9, mg gallic acid extract/g plant part and they correspond dried leaves methanolic extract, dried leaves ethanolic extract and bark methanolic extract. The bark ethanolic extract as well as the methanolic and ethanolic extract of A.

Alkaloids (Buchardart reagent)
showed moderate amounts of TPC of 47.02, plant part respectively. The lowest amounts of TPC recorded were 7.7, 7.0, 1.1, 1.2 mg gallic acid extract/g plant part for methanolic, ethanolic extracts of pericarp The conducted work showed no significant difference in the total phenol content between methanolic and ethanolic extract of A.
dry leaves, dry bark, pulp and pericarp (P ˂ 0.05). In these cases, ethanol can effectively substitute methanol in the extraction of polyphenols from these plant parts. However, extraction solvents, methanol and ethanol, significantly affected squamosa fresh leaves phenolic compounds. be more efficient in the extraction of phenolic compounds from fresh leaves than ethanol (P ˃ 0.05).

Radical scavenging activity
DPPH radical scavenging activity assays of the 10 different extracts of A. squamosa plant parts extracted by both ethanol and methanol showed significant DPPH radical scavenging activity in a concentration-dependent manner. The results antioxidant activity increased with the concentration of the extracts. DPPH free radical scavenging assays were repeated in triplicate. The half-maximal inhibitory concentration (IC determined as the concentration of the t reduction in absorbance from that of the control and the mean was calculated ( fig. 3).

3: DPPH radical scavenging activity of different A. Squamosa extracts (Values are expressed as mean marked by the same letter are not significantly different (P ˂ 0.05))
The potent free radical scavenging activity was exhibited by the = 13.61 µg/ml), whereas the ethanolic extract exhibited relatively less free radical scavenging values of the fresh leaves ethanolic and methanolic extracts respectively. The IC50 values of the dried bark ethanol and methanol extracts were 55.77 µg/ml, values of the pericarp ethanol and µg/ml respectively. The poorest free radical scavenging activity was recorded for the ) and the ethanolic extract ) of pulp. However, ascorbic acid showed the highest DPPH radical scavenging activity (IC50 = 6.62 µg/ml). The conducted work proved no significant difference in scavenging activity A. squamosa dry and fresh ˂ 0.05). However, A. squamosa bark methanol extract exhibited significantly higher scavenging activity where IC50 ascorbic acid and IC concentrations of the ascorbic acid and the sample respectively. The higher the AEAC value, the greater is the antioxidant activity. showed the highest AEAC value of 0.233 mg AAE/g dry wt of extract. Polyphenolic compounds have been reported to have antioxidant activities due to the reactivity of the phenolic moiety, scavenging free radicals via electron donation or hydrogen donation [29]. Hence, the antioxidant activities of dried leaves come to confirm these rep significantly affected the amount of total A. fresh leaves phenolic compounds. Methanol was found to be more efficient in the extraction of phenolic compounds from fresh DPPH radical scavenging activity assays of the 10 different extracts plant parts extracted by both ethanol and methanol showed significant DPPH radical scavenging activity in a dependent manner. The results revealed that the antioxidant activity increased with the concentration of the extracts. DPPH free radical scavenging assays were repeated in triplicate. The maximal inhibitory concentration (IC50) values were determined as the concentration of the test mixture that gave 50% reduction in absorbance from that of the control and the mean was leaves and bark methanolic extracts and determined by values of 7.81 and 125.0 μg. ml -1 , respectively. This result is close to the one found in this work in the case of the leaves methanolic extract, but greater than that found in the present study for the bark methanolic extract [27].
The results were also expressed as ascorbic acid equivalent antioxidant capacity (AEAC) (table 4) i.e. mg ascorbic acid equivalents/mg dry wt, which was calculated as follows: [28] = X mg ascorbic acid equivalent mg dry wt (3) ascorbic acid and IC50 sample are the effective ascorbic acid and the sample respectively. The higher the AEAC value, the greater is the antioxidant activity.

AEAC (mg AAE/mg dry wt)
the phenolic moiety, scavenging free radicals via electron donation . Hence, the antioxidant activities of dried come to confirm these reports. IC50 and AEAC values obtained for methanolic dried leaves extract imply that the extract contains phytochemicals with antioxidant properties.

Correlation between TPC and antioxidant activity
Several studies have evaluated the relationship between the antioxidant activity of plant products and their phenolic content. It has been reported that phenolic compounds were the main antioxidant components, and their total contents were directly proportional to their antioxidant activity [30,31]. This is demonstrated in our study as far as A. squamosa dried leaves and bark extracts were concerned. These showed the highest TPC, the highest antioxidant activity as well as the highest AEAC. Regarding fresh leaves, they showed a TPC lower than dried leaves and comparable to dried bark. At the same time they showed a relatively higher antioxidant activity as well as higher ascorbic acid equivalent antioxidant capacity than dried bark. These results might be due to several reasons. The antioxidant activity of an extract is not only determined by the phenolics. Non-phenolic compounds could show more potent antioxidant activities. This is the concept of electron donor and acceptor which plays a very important role in the redox reaction so any functional group capable to donate an electron may act as a good antioxidant agent. For instance, the antioxidant activity of polysaccharides is shown to be related to their functional groups such as hydroxyl, amino, carbonyl, and carboxyl group [32]. Therefore, it is possible that fresh leaves contain nonphenolic antioxidants contributing to its antioxidant activity. Hence emerges the necessity of identification of various constituents of fresh leaves and bark through chromatographic techniques, for instance, and highlighting those responsible for antioxidant action.

CONCLUSION
In the present study, it is observed that the different parts of A. squamosa plant present a good contribution of phenolic compounds as well as high antioxidant activity. A. squamosa leaves showed a high total phenolic content and antioxidant activity compared to burk, pulp, and pericarp. Studies on A. squamosa anticancer and other biological properties are ongoing. However, outcomes encourages further investigations to identify the chemical constituents that are responsible for multiple biological activities.