LYOPHILIZED AQUEOUS EXTRACT OF PINUS HALEPENSIS (MILL.) RESIN: CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIDERMATOPHYTIC ACTIVITIES

In traditional Algerian medicine, Pinus halepensis (Mill.) resin is used as antimicrobial, anti-inflammatory, analgesic, wound healer, for the treatment of respiratory and urinary diseases. In the present study, a lyophilized aqueous extract (LAE) of Pinus halepensis (Mill.) resin is subjected to chemical composition analysis by gas chromatography coupled to mass spectrometry (GC/MS), antioxidant activity was evaluated in vitro using 2,2-diphenyl-b,picrylhydrazyl (DPPH) and β-carotene bleaching assays. LAE of Pinus halepensis (Mill.) resin was also tested for its potential activity against four dermatophytic fungi: Trichophyton rubrum, Trichophyton equinum, Trichophyton mentagrophytes, Trichophyton tonsurans. Results showed that LAE of Pinus halepensis (Mill.) resin contained 64.57% of aromatic compounds. This extract contains also succinic acid, sugars and alcohol sugars, alkaloids and other compounds that represent respectively 0.58%, 6.45%, 0.87% and 2.05%. The obtained extract exhibited satisfying antioxidant activity with an inhibition percentage of 93.76±0.41 at a concentration of 300 µg/mL and IC50 of 83.64µg/mL by the DPPH method. The tested extract showed also a good antioxidant activity using β-carotene test. At resin concentration of 100 µg/mL antifungal tests showed, inhibition rates of 58.9%, 58.44%, 41.36% and 34.43% against T. tonsurans, T. equinum, T. rubrum and T. mentagrophytes respectively. Upon the obtained results, this extract can be used as external treatments for wounds to prevent oxidation and fungal infections.


INTRODUCTION
Plants have been used as an important source of medicine since ancient times and their products are being used for different purposes such as medicine, food, health care, agriculture, agrochemicals, pharmaceutical, etc. The medicinal properties of different plant species have contributed to the origin and evolution of many traditional herbal therapies (Malik et al., 2012). The importance of medicinal plants lies in their biological active principles, which are the real healers in the process of medication (Kumar, 2009). The Aleppo pine or Pinus halepensisis (Mill.) is a tree species of primary importance in the Mediterranean by the area it occupies and the role it plays in the economy of countries in this region (Nahal, 1962). The resins are considered to play a role in the defence of the coniferous trees against insects and microbial pathogens (Trapp and Croteau, 2001). Natural coniferous resins extracts are raw materials for various products in the industry and have been used as traditional medicines as a homemade salve for skin wounds and infections (Sipponen, 2013). Natural resins have a very wide range of applications, mainly used in industries coatings (varnishes, lacquers, paints, sealants, cements, adhesives...), printing (printing inks...), stationery (paper sizing), polymers (adhesives, rubber, various materials). Gums and many resins are also the sources of varied products for the food industry, perfumery, cosmetics, pharmacy and medicine (Delmond, 2002;Ghanmi et al., 2007). Several medical traditionally uses of P. halepensis (Mill.) were recorded in Algeria according to regions and diseases. Different parts of pine were used in decoction; infusion, pine resin powder mixed with honey or olive oil (Meddour and Meddour-Sahar, 2015). Fruits infusion for the treatment of arterial hypertension (Sarri et al., 2012), buds, leaves and resin decoction and poultice for the treatment of respiratory and urinary tract disorders, antiseptic and stimulant of adrenalglands (Benderradji et al., 2014), fruits infusion and powder for haemorrhoids, tuberculosis, ulcer treatments and pulmonary conditions (Sarri et al., 2015). Seeds decoction for the treatment of gastric and intestinal ulcers, respiratory infections, prostate infection, sterility, toothache (Bouasla and Bouasla, 2017), stomachache and kidney inflammation (Senouci et al., 2019). Roots decoction for the treatment of respiratory disorders (Chohra and Ferchichi, 2019). The resin was used against rheumatism (Rebbas et al., 2012), to treat asthma (Meddour and Meddour-Sahar, 2015), as antifungal (Chermat and Gharzouli, 2015) and to treat mainly burns, wounds and skin inflammation, in flu and cough cases (Meddour and Meddour-Sahar, 2015). Leaves, resin infusion and decoction for the treatment of respiratory ailments (bronchitis, pneumonia and colds), urinary problems, parasitosis and wounds (Miara et al., 2019;Bendif et al., 2020). Several studies reported that the most important fungal species implied in dermatophytic diseases in Algeria belonged to Tricophyton genus. They were predominant in the Northeast, 66.68% including Trichophyton rubrum and Trichophyton tonsurans (Hammadi et al., 2007). Trichophyton rubrum was the predominant pathogen of feet and the inguinal folds in Sétif (Ilham and Touabti, 2013), Trichophyton mentagrophytes primarily responsible for the inflammatory disease in Batna (Chelgham et al., 2012). While in 2016, Ennaghra et al. found that the occurrence of trichophytic was 72% among this percentage there were 24% T. rubrum and 16% T. mentagrophytes. In the last decade most research done on pine in Algeria were focused on the organic solvent extraction of essential oils from aerial parts of the tree, spines In traditional Algerian medicine, Pinus halepensis (Mill.) resin is used as antimicrobial, anti-inflammatory, analgesic, wound healer, for the treatment of respiratory and urinary diseases. In the present study, a lyophilized aqueous extract (LAE) of Pinus halepensis (Mill.) resin is subjected to chemical composition analysis by gas chromatography coupled to mass spectrometry (GC/MS), antioxidant activity was evaluated in vitro using 2,2-diphenyl-b,picrylhydrazyl (DPPH) and β-carotene bleaching assays. LAE of Pinus halepensis (Mill.) resin was also tested for its potential activity against four dermatophytic fungi: Trichophyton rubrum, Trichophyton equinum, Trichophyton mentagrophytes, Trichophyton tonsurans. Results showed that LAE of Pinus halepensis (Mill.) resin contained 64.57% of aromatic compounds. This extract contains also succinic acid, sugars and alcohol sugars, alkaloids and other compounds that represent respectively 0.58%, 6.45%, 0.87% and 2.05%. The obtained extract exhibited satisfying antioxidant activity with an inhibition percentage of 93.76±0.41 at a concentration of 300 µg/mL and IC50 of 83.64µg/mL by the DPPH method. The tested extract showed also a good antioxidant activity using β-carotene test. At resin concentration of 100 µg/mL antifungal tests showed, inhibition rates of 58.9%, 58.44%, 41.36% and 34.43% against T. tonsurans, T. equinum, T. rubrum and T. mentagrophytes respectively. Upon the obtained results, this extract can be used as external treatments for wounds to prevent oxidation and fungal infections. On the bases of the available literature about the extracts and the ethnobotanical use of Pinus halepensis (Mill.) and the biological activities focused on antioxidant and antifungal especially phytopathogenic fungi (El Omari et al., 2021), the extract and the use of pine resin aqueous extract seem to be more close to the folk use of pine. Thus the aim of this work, which is in our knowledge the first study done in Algeria, was to determine the chemical composition as well as the antioxidant and antifungal activities of a lyophilized aqueous extract of Pinus halepensis (Mill.) resin collected in Sétif region (Northeast of Algeria).

Preparation of lyophilized aqueous extract (LAE) of Pinus halepensis (Mill.) resin
Resin samples are collected in the north of Sétif region (in the Northeast of Algeria). A sample of 1.9 g of air-dried resin is ground into a fine powder in a blender and mixed with 90 mL boiling water by a magnetic stirrer for 4h. The aqueous extract was then filtered over cheesecloth and Whatman paper No. 1, respectively. The filtrates were frozen at -20°C in ultra-low temperature freezer and lyophilized in a dry freezer machine (Cryorivoire) under 260-mTorr pressure at-81°C (Gülçin et al., 2007).

Chemical composition of LAE of Pinus halepensis (Mill.) resin by GC / MS
The identification of the components of LAE of P. halepensis resin was performed based on the gas chromatography coupled to mass spectrometry (GC / MS) as described by  (5) mg of pine resin samples of LAE were mixed with 50μl of dry pyridine and 75μL N,O-bis (trimethylsilyl) trifluoracetamide, heated at 80°C for 20 min and analyzed by GC-MS. This technique is performed using a Hewlett Packard Gas Chromatograph 6890 Series II Plus linked to Hewlett Packard 6972 mass spectrometer system equipped with capillary column HP5-MS (30 m long, 0.25 mm id and 0.5 μm film thicknesses). The column temperature is programmed from 100 to 325°C at a rate of 5°C/min. The carrier gas is helium with a flow rate set at 20 mL/min. The injection mode is Split (Split ratio 50:1, injector temperature 280°C). The identification of the compounds present in pine resin LAE samples is accomplished using computer searches on commercial libraries. In some cases, when identical spectra have not been found, only the structural type of the corresponding component was proposed based on its mass-spectral fragmentation. If available, reference compounds were co-chromatographed to confirm GC retention times. Components identification is verified by search in NIST Data Base using CAS numbers (http://webbook.nist.gov/chemistry/).

Radical scavenging by DPPH method
Antioxidant activity of pine resin LAE is evaluated according to the DPPH (1,1diphényl-di-picrylhydrazyl) method proposed by Chen et al. (2004) and Que et al. (2006). Two (2) mL of LAE of P. halepensis (Mill.) resin solution (2mg/mL) and different concentrations of BHT (butylated hydroxytoluene) in distilled water are mixed with 2 mL of 0.1 mM DPPH solution in methanol. Control is prepared with the same method but the tested substance is substituted by distilled water. After 30 min in dark, DPPH discoloration is measured at 517 nm with a spectrophotometer (thermo-Sientific: HEʎIOS°490) using distilled water as blank. All tests are performed in triplicate. Extracts scavenger activity in % is calculated according to the following formula: scavenger activity in %= [(Ac -Ae)/Ac] x 100 where: Ac= (Absorbance of control), Ae= (Absorbance of tested extract). The graph of the inhibition percentage variation as a function of tested extract concentrations allows determining the IC50, concentration corresponding to 50% inhibition of DPPH radicals.This value is compared to that recorded for the reference compound (BHT).

β-carotene bleaching test
The antioxidant capacity is determined by measuring inhibition of volatile organic compounds production and the formation of conjugated dienehydroperoxides arising from linoleic acid oxidation which results in the discoloration of β-carotene (Krishnaiah et al., 2011). β-carotene bleaching test is realized according to the method described by Sun and Ho (2005). In a flask, 1ml of β-Carotene solution (1 mg/mL of pure chloroform) is added to 25 µL of linoleic acid and 200 mg of the Tween 40 emulsifier mixture. After evaporation of the chloroform by rotary evaporator (Rotamantle) at 42°C, 100 mL of oxygensaturated distilled water are added with vigorous shaking until emulsification.
Next, 10 ml of this mixture are transferred into test tubes containing 2mL of distilled water (control), 2 mL of BHT solution (positive control, standard antioxidant) at 2mg/mL of methanol or 2 mL of different concentrations of the tested extract; all tests are performed in triplicate. As soon as the emulsion is added to each tube, the zero time point absorbance is measured at 490 nm by a spectrophotometer (thermo-Sientific: HEʎIOS°490), blank contains all emulsion components but devoid of β -carotene. After incubating emulsion for 2 h at 50°C, absorbance readings are taken at regular intervals (15min) until 2h (decolorized β-carotene).

Antifungal activity of resin LAE of P.halepensis (Mill.) resin
The LAE of Pinus halepensis (Mill.) resin was tested in plate based poisoned bait assays against four dermatophytic fungi Trichophyton rubrum, Trichophyton equinum, Trichophyton mentagrophytes and Trichophyton tonsurans obtained from the laboratory of Dr. Jane Nicklin, School of Biological and Chemical Sciences, Birkbeck College, University of London, UK. The dermatophytic fungi are cultivated on Sabouraud dextrose agar (Oxoid LTD, Basing Stoke, Hampshire, England). Resin LAE dissolved in methanol is added to the medium at the concentrations of 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 100µg/mL. The medium containing the appropriate concentration of MeOH without pine resin extract is used as control. Plates are incubated at 25°C for 9 days. The fungal growth inhibition rates are calculated according to the equation: Inhibition %=[(Cd-Td)/Cd)]x100 where Cd: control colony diameter, Td: test colony diameter. Results converted to prohibit values (Finney, 1971), are plotted against the log2 of the dilution factor in order to determine the dilution required to inhibit germination by 50%.

Statistical analysis
Results are expressed as mean ± SD. Graphs were realized by Graph Pad Prism 8.4.2 Statistical software (Graph Pad Software, USA). Statistical significance of the difference was assessed by a two-way analysis of variance. Differences were considered significant at p<0.05.

Chemical composition of resin LAE of P.halepensis (Mill.)
Analysis of the chemical composition of the LAE of pine resin by GC/MS resulted in the identification of 53 compounds.The most representative results according to the NIST database (Table 1), are aromatic compounds such as cinnamic and benzoic acids derivatives that represent, quantitatively the most important fraction. Among phenolic compounds, cafeic acid area is 35.52%. Methyl ester of salicylic acid, 3,5-Bis (1,1-dimethylethyl) catechol, vanillin, [1,1'-biphenyl]-4,4'-diol, 3,3'-dimethoxy-, isoferulic acid, coumarin 3-(1,1dimethylallyl)-7-hydroxy-6-methoxy-), p-salicylic acid (4-hydroxybenzoic acid), protocatechuic acid and even thymol are respectively present at percentages of 5.53, 5.09, 3.84, 3.05, 2.73, 1.80, 1.78, 0.97 and 0.63. Besides, the analysis reveals the presence of a variety of sugars representing 6.45%. This aqueous extract also contains succinic and phosphoric acids, D-verbenone and alkaloids as tetra hydroharmane. Total percentage area of identified compounds, referring to the NIST database, is 74.52%. Natural resins are complex mixtures of several classes of compounds, the main ones as described by Delmond (2002) are in addition to essential oils and polysachrides, acid constituents, aliphatic as succinic acid, aromatic as benzoic and cinnamic acids and phenolic acids as salicylic, p-coumaric and ferulic acids combined as esters to resin alcohols (resinols and resinotannols) and phenolic compounds as urushiol. Natural resins contain also neutral constituents, essentially triterpenic in the majority (Ghanmi et al., 2007), resin alcohols, free or as esters and are generally phenolic. The compounds present in greater quantities in the Pinus genus are mono or sesquiterpenes (Ghanmi et al., 2007;Simard, 2007; Abi-Ayad et al., 2011), their concentrations vary with species. Several diterpenes were isolated from different parts of pines (Simard, 2007). Earlier Karepova et al. (1983) reported that the aqueous extract of the woody verdure of Pinus sylvestris contained aliphatic mono-, di-and tricarboxylic acids and benzene carboxylic acids. Among aromatic acids, they found benzoic acid in the largest amounts. These results are consistent with the obtained results, Pinus halepensis (Mill.) resin LAE also contained aliphatic acids such as succinic acid and a significant amount of aromatic compounds such as benzoic and cinnamic acids derivatives greatly represented by cafeic acid. Sugars are present at 6.45%; these sugars derived probably from the hydrolysis of polysaccharides following sample boiling. This is reinforced by data of Sipponen (2013), who found constantly coumaric acid (hydroxycinnamic acid), a group of resin acids (hydroxyabietic and abietic acids) and a group of lignans specially pinoresinolin natural coniferous resins and other terpenic wood extracts.  3,3, 3

DPPH test
The test results of the free radicals scavenging effect from the LAE of pine resin and BHT (Fig. 1) (2010) reported that the ability of extracts to scavenge H2O2 is strongly associated with total phenolics, proanthocyanidins and cinnamic acids. Similarly, the ability of extracts to scavenge O2 − was strongly correlated with total phenolics whereas the extracts ability to scavenge the OH radical was strongly correlated with the cinnamic acids content. These results suggest that overall antioxidant effect towards the H2O2 and O2 − could be due to the synergistic influence of different phenolic classes present in the extract. According to Bougandoura and Bendimerad (2013), the inhibition percentage of aqueous and methanolic extracts of Satureja calamintha was greater than 90% at concentrations of 4.624 and 5.0 mg/mL respectively. They also suggested that the power reducing of Satureja calamintha species is probably due to the presence of hydroxyl group in the phenolic compounds that can serve as an electron donor. Aqueous extracts are then good antioxidants, indeed LAE of P. halepensis (Mill.) resin shows inhibition of 92.23±0.13% at a concentration of only 200 µg/mL.

β-carotene bleaching test
Based on the results of β-carotene bleaching assay of pine resin LAE (Fig. 2), the OD recorded at T=0min is higher than those recorded for other time intervals. The decrease in OD occured between 0 and 15 minutes and then became slower for the resin and BHT. The decrease in absorbance is however very marked for the aqueous and methanolic controls devoid of resin extract following the disappearance of the β-carotene, the oxidation of linoleic acid is at a maximum after 60 min. BHT appeared the most potent antioxidant; the LAE of P. halepensis resin also showed a remarkable and significant antioxidant effect by inhibiting β-carotene oxidation.  Pycnogenol containing organic acids-cafeic, cinnamic, fumaric, gallic, vanillic, ferulic, and protacatechuic, extracted from French Maritime pine tree bark, (Simpson et al., 2019), provided photoprotection, reduce hyperpigmentation of human skin and improveed skin barrier function and extracellular matrix homeostasis (Grether-Beck et al., 2016). Several research studies have demonstrated the antioxidant potential of derivatives of the components of Pinus genus as proanthocyanidin, cinnamic acid and its derivatives. The tested LAE pine resin extract contained a relatively large fraction consisting of cinnamic acid derivatives which are more active antioxidants than those of benzoic acid derivatives (Eom et al., 2012). According to Ku et al. (2007), hot water extracts from Pinus densiflora, P. radiata and P. rigida indicated greater antioxidant activity owing to high proanthocyanidin content. The same authors also suggest that P. radiata bark can be considered as a natural resource for biological and pharmaceutical applications why not the LAE resin; which was also obtained by hot water extraction. The present results showed also the presence of cafeic, vanillic, isoferulic and protocatechuic acids in P. halepensis resin LAE obtained by hot water that can be used as a potent radical scavenger and consequently as potent antioxidant.   Fig. 3). When probits were plotted against Log2 concentration of resin LAE samples, the relation was linear and the concentration required for 50% inhibition is 77 µg/mL for T. tonsurans and 78 µg/mL for T. Rubrum and T. equinum. T. tonsurans was the most sensitive to the extract and T. mentagrophytes was the most resistant one. The antifungal activity of the pine resin aqueous extract may be related to its chemical composition. The pine resin aqueous extract contains some compound which has antifungal activity such as caffeic acid ( found that resin purified from Norway spruce (Piceaabies) in salve mixture had antifungal activity against trichophytic fungi. These authors confirm that the salve with a resin concentration of 20% or more caused a significant fungicidal effect against T. rubrum, T. mentagrophytes and T. tonsurans. Increasingly, plant aqueous extracts are effective as significant and remarkable antifungal. Schmourlo et al. (2005) reported that lyophilized aqueous extracts of Xanthosomas agittifolium, according to popular use, inhibited the growth of T. rubrum strains isolated from clinical cases by an inhibition zone diameter of 18 mm with a lowest MIC of 100 ng/mL against T. rubrum. Shams Ghahfarokhi et al. (2004) reported that AOE (aqueous onion extract), at different concentrations, inhibited significantly the growth of both T. rubrum and T. mentagrophytes in a dose-and time-dependent manner compared with the controls. It was found that the AOE had fungistatic effect from 0.78% to 3.12 (v/v) and fungicidal properties at the range of 6.25to 50% (v/v) for both fungi at all culture periods.

CONCLUSION
The results of this study showed that Pinus halepensis (Mill.) resin lyophilized aqueous extract has considerable anti-oxidant and antifungal activities in vitro. Also, it contains important amounts of phenolic compounds and can be used as natural source of antifungal and antioxidants valuable in food supplement. It is hoped that efforts will continue to discover natural active compounds derived from plants with potent therapeutic action devoid of toxicity. We should then maintain our efforts in protecting and valorizing our natural patrimony, as well as scientific research on P. halepensis (Mill.) resin from chemical analysis, biological, toxicological and pharmacological investigations to therapeutic aspects.