Evaluation of antioxidants activity of some tree barks grown in Libya (Al Jabal Al Akhdar) Pinus halepensis Mill, Pistacia lentiscus L, Juniperus phoenicea L

The aim of the present study investigated anti-oxidant activity of various methanol-water extracts from tree barks grown in Libya (Al Jabal AL Akhdar) by four anti-oxidant assays FRAP. DPPH. H2O2 and Metal chelating activity. Additionally, these antioxidant activities were compared with BHA, BHT as reference antioxidants. Tree bark has a large, diverse class of compounds, many with antioxidant properties. This study showed FRAP inhibitory activity of the Pinus halepensis bark extracts 65.8%, while Juniperus phoenicea L 57.6%, and Pistacia lentiscus L bark extracts was 69.2% The DPPH radical scavenging in the bark extracts exerted an inhibition of 55. 8%, 52.3% ,47.5% for Pinus halepensis, Juniperus phoenicea and Pistacia lentiscus L respectively. While H2O2 activity shows variation, ranging from 67. 8 to 81. 3%. The Metal chelating activity of the barks extract was 59% in Pinus halepensis and 57% in Juniperus phoenicea L bark extracts furthermore Pistacia lentiscus L was 67.8% in addition the Metal chelating activity of BHT and BHA 92%, 94% respectively. The high antioxidant activity of bark was founded in Pistacia lentiscus L bark extracts.


Introduction
Al Jabal AL Akhdar region (Libya) has highest species diversity and having distinct environmental characteristics associated with evergreen forest along with the Mediterranean from the Atlas Mountains to the Levant, and it has an environment similar to other regions in Southern Europe such as Italy, the Greek islands and Turkey [1]. The number of plant species reach up of 1100 species from the total of plant species in Libya (2000 species) with about 75 species of plants that grow only in AL Jabal AL Akhdar and have been served for as basis of traditional medicinal systems for thousands of years [2]. Pinus halepensis is a steno-Mediterranean species distributed along the coasts and in the islands, it prefers warmer Calcareous areas like Libya, Italy, Algeria, Greece, Morocco, and Turkey, where it also succeeds in colonizing the less hospitable rocks because of its springiness Juniperus phoenicea L is a small tree that is native to the northern lands bordering the Mediterranean Sea from Portugal to Palestine. It is also native to North Africa found in Libya, Algeria, Morocco and Canary Islands [3]. This plant species is a conspicuous constituent of the vegetation of the Mediterranean basin, particularly in Al-Jabel Al-Akhdar region. It constitutes about 80% of the total number of the trees and evergreen shrubs that exist in Al-Jabal Al-Akhdar area [4] Pistacia lentiscus L. is an evergreen shrub of the family Anarcadiaceae. This dioecious species can reach 3 m in height and grows in many Mediterranean countries [5]. All trees have stems and branches composed of an internal wood tissue this wood is covered by bark with a percentage of 9 to 15% of the stem volume [6] Tree barks is morphologically diverse, which could be the result of specific ecological strategies. This diversity might also be the result of adaptation to environmental conditions, fire resistance, or insect attacks. This morphological diversity and its causes remain poorly understood [7]. The bark is very promising technological raw material and classic bio refinery object because of its unique bark's biomass chemical composition and possibility to get many different products with the added value of individual or group of compounds of the synergistic biological activity, raw materials to produce various materials [8]. Several types of plant materials, such as vegetables, fruits, seeds, hulls, wood, bark, roots and leaves have been examined as potential sources of antioxidant compounds. The crude extract of bark contains a wide variety of phenolic compounds. Studies on the investigation of its bark and characterization have been reported [9]. Polyphenols: Plants produce varying forms of polyphenols, a large, diverse class of compounds, many with antioxidant properties structurally; polyphenols all have one or more aromatic (phenolic) rings with different structural elements [10].

Sampling and processing
Tree barks samples (Pinus halepensis Mill, Pistacia lentiscus L, Juniperus phoenicea L) were collected from Aljabal Alakhdar province. The bark samples were dried at room temperature for two weeks, and grinded to fine powder. 40 -60 mesh and were stored in closed glass jars at room temperature until analysis.

Extraction of sample
Extracted the three samples in Soxhlet extractors according to (TAPPI T 204 om88) for 6 h using methanol-water mixture (65-35% v/v).Then the extract was filtered paper Whatman No. 1, The filtrates were frozen at -84 C in ultra low temperature freezer and lyophilized for analysıs of determınatıon antıoxıdantes actıvıty,The antioxidant capacities of plant extracts were assayed with four different assays including FRAP, Metal chelating, H2O2, and DPPH scavenging because the antioxidant capacity cannot be fully described by a single method. The experiments were carried out in triplicate all chemicals were purchased from Sigma (Sigma Aldrich GmbH. Sternheim), Merck (Darmstadt)

Ferric Reducing Antioxidant Power assay
FRAP (ferric reducing antioxidant power) process were carried out as described by Hamad AMA et al 2019 [11] with slight modifications. Solutions having 1, 0.5, 0.25 and 0.125 g/L concentrations were obtained from the extraction samples and a 0.5 mL sample was put in the test tubes for each concentration and 4.5 ml methanol was added to them. After that, 2.5ml of 1% K3 (FeCN) 6 was added. This mixture was heated at 50 centigrade for 20 minutes in a water bath, and 2.5ml of Trichloric acid (TCA) was added to it. Firstly, the UV absorption at 520 nm was measured at 0 min. When samples were centrifuged at 4500 rpm, the samples were shifted to another test tube and 2.5 ml alcohol and 0.1% FeCl3 were added to the tube, secondly the absorption was measured at 700 nm.

Radical Scavenging Activity assay
DPPH (radical scavenging activity) assay were carried out as described by Hamad AMA et al 2019 [11]. With slight modification solutions at 1, 0.5, 0.25 and 0.125 g/L concentrations. They were obtained from extraction samples. 2 ml of sample was taken in the test tubes for each concentration and 40 mM 2 ml DPPH solution was added to them. Centrifugation remained at 4500rpm for 10 minutes and UV absorbance was 520 nm.

Hydrogen peroxide scavenging assay
Hydrogen peroxide was determined according to Guder,et al 2012 [12]. 170 µl methanol-water extract was added to H2O2 solution, which was prepared using 40 mM phosphate solution according to the final volume, which was nearly 4 ml. After that, 230 nm absorption was determined by UV-VIS spectrophotometer.

Metal Chelating Activity assay
Metal chelating of ferrous ions by the extracts and standards was determined according to the procedure by Guder,et al 2012 [12] with slight modifications. Different extractive concentrations were prepared and 0.4ml extract was added to 50µl FeCl2 solution (2 mM). The reaction was initiated when 0.2 mL ferrozine (5 mM) was added and the mixture was shaken with force. After that, the solution was left at room temperature for 10 min. Finally, UV absorption was measured at 562 nm using a spectrophotometer. The ferric reducing antioxidant power (FRAP) method is important for the evaluation of antioxidants. FRAP was associated with antioxidant potential of extracts to confirm its level [13]. In this assay, the Fe 3+ / Fe 2+ transformation was investigated in the presence of samples and the absorbance values were measured at 700 nm. In addition, FRAP activity of the different bark extracts had significant variation, according to these results, we can say that especially Pistacia lentiscus L bark extracts have higher FRAP activity than the others.

Results and discussion
Moreover, the researchers to determine radical scavenging activity mostly use DPPH. The free radical scavenging activities of extracts depend on the ability of antioxidant compounds to lose hydrogen and the structural conformation of these components [14].
Furthermore, reactive oxygen species (ROS), i.e., hydrogen peroxide, singlet oxygen, hydroxyl and superoxide radicals, have crucial acts such as in energy production, phagocytosis, intercellular signal transfer, cell growth regulation and important biological compounds synthesis [15]. Although hydrogen peroxide is not very reactive, it can show toxic property to cell due to their transformation hydroxyl radical in the cells. By this way, H2O2 scavenging activity is very important for antioxidant defense in cell or food systems [12].
Iron and other transition metals such as copper, chromium, cobalt, vanadium, cadmium, arsenic, and nickel support oxidation owing to their catalyst properties on free radical reactions. These transition metals donate single electrons during redox reaction between them. When some compounds chelate metals, their pro-oxidant activity diminishes by reducing their redox potentials and stabilizing the oxidized form of the metal [16].
Bark extracts from other Pine species have been reported to possess potent antioxidant activity. Extracts of Pinus sylvestris, Pinus pinea, and Pinus massoniana barks showed cytotoxic activity against human cancer cell lines. In addition, pinus sylvestris and pinus massoniana bark extracts reduced the production of several inflammatory mediators , pinus pinea and Pinus densiflora bark extracts have been found very promising for diabetes treatment due to their inhibitory effects on glucose absorption and carbohydrate-hydrolysing enzymes, respectively. , all these data show that bark extracts from different pine species have remarkable biological effects, thus making pine bark a valuable raw material for the food and pharmaceutical industries [17].
The high phenolic content and the important antioxidant activities of P. lentiscus extract could be a useful source of natural products and may be increasingly important for human consumption, prevention of damages caused by oxygen free as well as for the agro-food, cosmetic and pharmaceutical industries. [21]. The results exhibited the highest inhibition activity of J. phoenicea against H2O2.
The Nano extract of J. phoenicea can be used effectively in the production of potential antioxidant, antiparasite antimicrobial and anticancer [22]. Pistacia lentiscus L bark extractives were 69.2%, furthermore that Butylated Hydroxyl Toluene (BHT) with an activity of 90% and Butylated Hydroxyl Anisole (BHA) 87%.
The bark sample extractives were analyzed with DPPH, a stable free radical. As DPPH picks up one electron in the presence of a free radical scavenger, the absorption decreases and the resulting discoloration is related to the number of electrons gained The DPPH radical scavenging (%) activity is shown in the Fig .2 Pinus halepensis bark extract exerted an inhibition of 66.8% and Juniperus phoenicea L bark extractives was 62.3%, and Pistacia lentiscus L bark extract 74.6 % while in addition that of BHT was 90% H2O2 antioxidant activity in Fig .3

Conclusion
According to the present results, the methanolic extracts of P. lentiscus bark have a considerable scavenging activity against the free radical DPPH; High ferric reducing power; Metal chelating and H2O2 scavenging activity this could be related to the high content of this plant on secondary metabolites. These extracts could therefore be a source of natural antioxidant molecules as an alternative to the use of synthetic antioxidants. Furthermore, these results support the large use of P. lentiscus in traditional medicine in Libya. For further studies, it is interesting to identify the chemical composition of the extracts and explore more biological activities of the plant.