Variability of phenolic compounds and antioxidant efficacy in needles extracts of Pinus nigra Arn

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Volume 53, Article 01

[Volume 53, Article 01]

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S. FKIRI*^,1,2

F. MEZNI¹

A. OUARGHI³

H. GHAZGHAZI¹

M. LARBI KHOUJA¹

A. KHALDI¹

Z. NASR¹

¹ National Research Institute of Rural Engineering, Water and Forestry, B.P 10, 2080 Ariana, Tunisia

² National institute of agronomy Tunis, BP 48, Tunis 1082, Tunisia

³ Superior Institute of Biotechnology Beja, Street Habib Bourguiba, Beja 9000, BP 382, Tunisia

Abstract – The phenolic compounds and antioxidant capacity of ethanol extracts, obtained from nineteen Pinus nigra provenances growing in Northwestern Tunisia, were invetigated. Phenols (Folin–Ciocalteu method), flavonoids (method based on the formation of a complex flavonoid–aluminium) and tannin (vanillin method) content were evaluated. The antioxidant capacity was determined using both phosphomolybdenum method and free radical scavenging activity (DPPH test). Total phenols ranged from 15.67 to 47.53 mg GAE/g of dry matter. The amount of flavonoids varies from 1.69 to 3.97 mg RE /g. Extracts showed an important antioxidant capacity ranging from 242.50 to 1206.87 GAE en mg/g for the total activity and from 36.08 to 99.05% for the free radical scavenging activity.

Keywords: Pinus nigra Arnold, variability, subspecies, phenolic compounds, antioxidant capacity.

 1. Introduction

Plants are a source of numerous free radical scavenging molecules and have high concentrations of various natural antioxidants, such as polyphenols, carotenoids and tocopherols which have a high antioxidant potential (Kahkonen et al. 1999; Zheng and Wang 2001; Kasinulainen and Holopainen 2002). It is knowing that natural antioxidants have been associated with the health benefits (Block et al. 1992; Parr and Bolwell 2000). Moreover, these compounds have been reported to have an essential role in plants defense, against herbivory and pathogens (Bravo 1998; Winks and Schimmer 1999; Blodgett et al. 2007).

Within conifers, Pine species are considered as a natural source of antioxidant compounds (Zulaica-Villagomez et al. 2005; Guri et al. 2006; Cretu et al. 2013). Almost pines were reported to have a high antioxidant capacity (Pinus halepensis, Pinus pinea, Pinus sylvestris, Pinus nigra…) (Laracine-Pittet and Lebreton 1988; Robles et al 2003; Yesil-Celiktas et al. 2009).

Pinus nigra Arnold is one of the most known medicinal plants in the Mediterranean countries (Tuzlaci and Erol 1999). This plant is known for its use in traditional medicine for respiratory diseases and for protection against both endoparasites and ectoparasites (Yeilada et al. 1995; Arı 2014; Grieve 1984; Fakir 2009; Gülçin 2003).

This species is discontinuously distributed around the northern Mediterranean from Southwest Europe to Asia Minor, extending to the Crimea and is also found in North Africa (Morocco and Algeria). The species is divided into six subspecies: P. nigra subsp. nigra (Host); P. nigra subsp. salzmannii (Dun.) P. nigra subsp. dalmatica (Vis.); P. nigra subsp. pallasiana (Lamb.); P. nigra subsp. laricio (Poir.) and P.nigra ssp mauretanica (Quézel and Médail 2003). This collective species is characterized by a high genetic, biochemical, phenotypic and morphological diversity (Arbez et al. 1974).

The aim of this study was to compare, for the first time, the total phenols, flavonoids, tannin content and the antioxidant capacity of needles collected from nineteen samples corresponding to different provenances from different regions which have been planted and grown in North West of Tunisia.

2. Materials and methods

2.1. Plant materiel

Needles of nineteen samples of Pinus nigra were collected in April 2015 from Souiniet arboretum in West-Northern Tunisia (8°48'E, 35°54'N, 492m). The nineteen samples correspond to different provenances from different regions which have been planted and grown in North West of Tunisia (Table 1) according to a comparative plantation assay since 1966.

2.2. Extract preparation

Needles of Pinus nigra were dried and then ground into a powder. 20 g of needle powder were mixed with 200 ml of solvent ethanol for 24 hours with continuous shaking. The filtrated plant extracts were dried to evaporation of all solvent. The residues were suspended in ethanol and used for the experiments. Needles extracts were conserved in obscurity at 4°C.

2.3. Extraction yield

Extraction yield was expressed as the percentage (%) of grams of extract per gram of dry needles.

2.4. Determination of total phenolic compounds

The total phenolic content of the extract was determined by the Folin–Ciocalteu method (Singleton and Rossi, 1965). 0.5 ml of needle extracts were mixed with 2.5 mL of Folin–Ciocalteu reagent (1:10) for 3 min, followed by the addition of 2 mL of sodium carbonate (7.5%). The mixture was allowed to stand for a further 30 min in the dark, and absorbance was measured at 765 nm. The total phenolic content was calculated from the calibration curve using 0, 0.03, 0.06, 0.12, 0.25 and 0.5 g/L solutions of gallic acid in water, and the results were expressed as mg of gallic acid equivalent per g dry weight (mg GAE/g).

2.5. Determination of flavonoids content

The total flavonoid content of crude extract was determined by the aluminium chloride colorimetric method (Quettier Deleu et al. 2000). 1 ml of diluted sample was mixed with 1 ml of 2% aluminum chloride methanolic solution. The mixture was allowed to stand for 15 min, and absorbance was measured at 430 nm.

The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg rutin equivalent per g dry weight (mg RE/g Ext).

2.6. Determination of condensed tannins

The method described by Broadhurst and Jones (1978) was used to determine the total condensed tannin content in needle extracts. 0.5 ml of the extract was mixed with 3 ml of vanillin (4% in methanol) and 1.5 ml of Hydrochloric acid. After incubation for 15 min at 20◦C in the dark, the absorbance was read at 500 nm. The condensed tannin content was calculated from a calibration curve prepared with a solution of catechin (30 ppm). The results were expressed in mg of catechin equivalent per g of dry weight of needles (mg CE/g).

2.7. Total antioxidant activity

The total antioxidant activity was evaluated according to the method described by Prieto et al. (1999). 0.5 mL of extracts was combined with 4.5 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). 0.5 mL of ethanol was used in case of blank. The tubes were placed in a boiling water bath at 95°C for 90 min. The samples were than cooled, and the absorbance was measured at 695 nm against blank. The antioxidant activity was expressed as equivalents of Gallic Acid (mg/g of dry matter).

2.8. Free radical scavenging activity

5 μl of the extracts was mixed with 5 ml of DPPH solution (0.004%, in ethanol). The reaction mixture was incubated for 30 min at room temperature and the absorbance was read at 517 nm against a blank (Brand-Williams et al. 1995).

The radical scavenging activity was calculated using the following formula:

2.9. Statistical analysis

The statistical analysis were done with the GLM procedure (General Linear Models) of the SAS (9.0) program. Correlations were performed by SPSS.20 program. Principal component analysis was evaluated with R (version 3.1.1) program.

3. Results

3.1. Extraction yield

Yields of extracts from needles of P. nigra are shown in table 2. Highest yield was reached by needles of Broug samples (31.29%). Bfr, Ala and Olet samples exhibited the lowest yield with 5.3%, 5.66% and 5.58%, respectively.

Statistical analysis showed that samples from nigra subspecies presented the most important yields while samples from pallasiana subsp. showed the lowest values.

3.2. Phenols, flavonoids and tannins contents

Results showed significant variations in the total phenols, flavonoids and tannin contents in P. nigra needles (p<0.0001).

The amount of total phenols ranged from 15.67 to 47.53 mg GAE/g of dry matter (Table 2).

The highest values was reached by needles of Marg sample and the lowest by Pug, Crim, Tent and Caz samples with respective values of 20.7,18.84, 17.08 and 15.67 mg GAE/g of dry matter. It has been demonstrated that total phenol content was higher in lariciosubsp. than the other subspecies.

The amount of flavonoids varies from 1.69 to 3.97 mg RE/g. The highest values were found in needles of Guil and Aspr samples (3.97 and 3.76 mg RE/g respectively). Needles of Bfr sample showed the lowest flavonoids content with 1.69 mg RE/g. Samples of salzmanni subsp. exhibited the most important flavonoids content.

Needles of Bfr, Purg and Guil samples were the richest in tannins. Its total contents were respectively 36.81; 27.73 and 32.61 mg CE/g. Samples belonging to nigra and laricio subspecies were the richest in tannins.

3.3. Antioxidant activity

Total antioxidant activity of needles extracts ranged from 242.5 to 1206.87 mg GAE/g (Table 3). The highest value was reached by Bfr, Ala and Crim samples with respective values of 1287.5, 1206.87 and 1179.37 mg GAE/g. Caz sample showed the lowest total antioxidant activity.

Using the free radical scavenging method, the highest percentage of inhibition was reached by needles of Kus and Marg samples (99.05 and 93.26%, respectively). Such as total antioxidant activity, Caz sample showed the less important free scavenging activity (36.08%).

Samples belonging to pallasiana subspecies showed the highest total antioxidant activity.

3.4. Correlation between antioxidant activity and total phenol, total flavonoids and tannin contents

The relationships between total phenols, flavonoids and tannins content and the antioxidant activity measured in P. nigra needles was determined using correlation analysis (Table 4).

There was a significant linear correlation between the antioxidant activity and total phenols, flavonoids and tannins contents in the case of salazmani, pallasiana and nigra subspecies. However, no significant relationship between antioxidant activity and total phenol, flavonoids and tannins was recorded for laricio subspecies.

 The strongest correlation was registered by pallasiana subspecies between antioxidant capacity (total antioxidant activity and DPPH inhibition) and total flavonoids (R² = 0.984).

3.5. Principal component analysis

The results of principal component analysis showed that total phenol content, total antioxidant activity and radical scavenging activity were the most significant variables for classification of the P. nigra extracts. These parameters were considerably loaded into the two major principal components (Dim1 and Dim 2) explaining more than 66% of the variance.

According to the analysis, three different groups were revealed (Figure 1). The first group contained Marg, Aspr and Barr2 samples which had the main concentrations of phenols and the strongest antioxidant activity. The second group contained only Caz sample which showed the lowest amount of phenols and the lowest antioxidant activity. The third group regrouped all the other samples studied.

4. Discussion

Extracts yields of black pine (P. nigra) needles of several provenances are more significant than those found in maritime pine (P. pinaster) varieties growing in the Souiniet site of Ain Drahem province (Maghrebiana variety (8.45%) and Renoui variety (5.77%)) (Fkiri al. 2018).

The amounts of total phenols in the Pinus nigra needles obtained in this study are more important than those found in other species of the same genus such as Pinus halepensis (25 mg GAE/g) (Laracine-Pittet and Lebreton, 1988), Pinus pinaster (17 mg GAE/g) (Alonso et al. 2002), Pinus roxburghii (10 mg GAE/g) and Pinus wallichiana (5 mg GAE/g) (Maimoona et al. 2011). Results showed that total phenol content was higher in laricio subsp. which is in accordance with levels reported in the literature (Cannac et al. 2007). Levels of total flavonoids were of the same importance as those found in other species, such as Pinus halepensis (Laracine-Pittet and Lebreton 1988) and Pinus sylvestris Robles et al (2003). In addition, needles of P. nigra showed a high amount of tannins when compared with P. pinaster needles (Alonso et al. 2002).

The analyses of variance showed that rates of total phenols, flavonoid and tannin contents varied considerably between the nineteen samples studied. In the literature, several factors were reported to explain the variability of these compounds. Numerous studies demonstrated that this diversity is related to the bioclimatic conditions especially rainfall, soil and temperature (Bhavana et al., 2013; Bencsik et al., 2011). Other authors explain it by a genetic variability.

In our case, significant difference found among provenances seem to support the hypothesis of high genetic variability of black pine. The divergence between the nineteen P. nigra samples, from different origins and growing under same bioclimatic and substrates conditions cannot result from local environmental factors. Arbez et al. (1974) highlighted a high degree of genotypic variability observed in P. nigra subspecies that was reflected in the biochemical variability of this plant (Fratianni et al. 2007).

Moreover, Boscaiu et al. (Boscaiu et al. 2010) showed that there is a positive correlation between the stress caused by environmental factors and the rate of phenolic compounds accumulated in the plants, signifying that these compounds appear to be good markers of stress in a lot of species. According to these results, we can suggest that samples of P. nigra which showed the highest amount of phenols and flavonoids were the less adapted to the bioclimatic conditions of the growing site and vice versa.

P. nigra needles exhibited an important antioxidant activity expressed as total antioxidant activity and DPPH free radical scavenging activity. It showed significant antioxidant activity when compared to alpha-tocopherol (Vijayakumar 2012).

The antioxidant capacity of Pinus nigra needles is more important than that found in other species such as P. roxburghii, P. wallchiana and P. gerardiana (Sharma et al. 2016). Many studies have revealed that there is an important relationship between antioxidant activity and phenolic and flavonoids compounds in plants (Cai et al. 2004; Shyi-Neng et al. 2014; Sharma et al. 2016). Needles of P. nigra showed a high amount of phenols and flavonoids. This can explain its important antioxidant capacity. On the other hand, antioxidant activity exhibited a high variability among the samples studied. This may be explained by the variability observed in antioxidant compounds, especially that a considerable correlation was revealed between the total phenols and the antioxidant capacity in the samples. Needles of Pinus nigra could be used as a source of natural antioxidants which is known by their positive effects on human health, due to their antioxidant properties, in particular protection from coronary heart disease and cancer (Catherine et al. 1997; Serafini et al. 1998; Knekt et al. 1996; Thomas 2000). It could be also used as a possible food supplement (Zupko et al. 2001) or in cosmetical and pharmaceutical industry (Lupo 2001).

5. Conclusion

This study showed the great presence of antioxidant compounds and confirmed high level of antioxidant activities in Pinus nigra needles. Significant differences were revealed between subspecies and provenances. Based on the results of our study, P. nigra needlesare a potent source of natural antioxidants applied in pharmaceutical and food industries. To highlight the properties of this plant, additional investigations concerning more biological activities require to be conducted.

 6. References

Alonso M, Rozados MJ, Vega JA, Perez-Gorostiaga P, Cuinas P, Fonturbel MT, Fernandez C (2002). Biochemical responses of Pinus pinaster trees to fire-induced trunk girdling and crown scorch: secondary metabolites and pigments as needle chemical indicators. J Chem Ecol 28: 687–700.

Arı S, Kargıoğlu M, Temel M, Konuk M (2014). Traditional Tar Production from the Anatolian Black Pine [Pinus nigra Arn. subsp. pallasiana (Lamb.) Holmboe var. pallasiana] and its usages in Afyonkarahisar, Central Western Turkey. J Ethnobiol Ethnomed 10: 10-29.

Arbez M, Bernard-Dagan C, Fillon C (1974). Variabilité intraspécifique des monoterpènes de Pinus nigra arn. bilan des premiers résultats. Ann Sci For 31: 57-70.

Bhavana GP, Chandrika R, Thara Saraswathi KJ (2013). Quantitative determination of secondary compounds in populations of Eryngium foetidum L. from India. Int J Curr Sci 9: 24-28.

Bencsik T, Horváth G, Papp N (2011).Variability of Total Flavonoid, Polyphenol and Tannin Contents in Some Lythrum salicaria Populations. Nat prod Commun 6: 1417 – 1420.

Block G, Patterson B, Subar A (1992). Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr Cancer 18: 1–29.

Blodgett JT, Eyles A, Bonello P (2007). Organ-dependent induction of systemic resistance and systemic susceptibility in Pinus nigra inoculated with Sphaeropsis sapinea and Diplodia scrobiculata. Tree Phys 27: 511–517.

Boscaiu M, Sánchez M, Bautista I, Donat P, Lidón A, Llinares J, Llul C, Mayoral O, Vicente O (2010). Phenolic Compounds as Stress Markers in Plants from Gypsum. Habitats Bull UASVM Hort 67: 1843-5394.

Bravo L (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 56: 317–333.

Brand-Williams W, Cuvelier ME, Berset C (1995). Use of a Free Radical Method to Evaluate Antioxidant Activity. Lebens Wiss Technol 28: 25-30.

Broadhurst RB, Jones WT (1978). Analysis of condensed tannins using acidified vanillin. J Sci Food Agr 29: 788-794.

Cai Y, Luob Q, Sunc M, Corke H (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 74: 2157–2184.

Cannac M, Pasqualini V, Greff S, Fernandez C, Ferrat L (2007). Characterization of Phenolic Compounds in Pinus laricio Needles and Their Responses to Prescribed Burnings. Molecules 12:1614-1622.

Catherine R E, Miller N, Paganga G (1997). Antioxidant properties of phenolic compounas. Tre Plant Sci 2: 153-159.

Cretu E, Karonen M, Salminen JP, Mircea C, Trifan A, Charalambous C, Constantinou AI, Miron A (2013). In Vitro Study on the Antioxidant Activity of a Polyphenol-Rich Extract from Pinus brutia Bark and Its Fractions. Journal of Medicinal Food 16: 984–991.

Fakir H, Korkmaz M, Güller B (2009). Medicinal Plant Diversity of Western Mediterrenean Region in Turkey. J Appl Biol Sci 3: 30-40.

Fkiri S, Mezni F, Ghazghazi H, Khouja ML, Khaldi A, Nasr Z Antioxidants (2018). Antioxydants and chlorophyll in Pinus pinaster Arn. Needles. Int J Food Sci Nutr 8: 176-17.Fratianni F, Tucci M, De Palma M, Pepe R, Nazzaro F (2007). Polyphenolic composition in different parts of some cultivars of globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori). Food Chem 104: 1282–1286.

Grieve M (1984). A Modern Herbal, London,United Kingdom: Penguin.

Gülçin İ, Büyükokuroǧlu ME, Oktay M, Küfrevioǧlu Öİ (2003). Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallasiana (Lamb.) Holmboe. J Ethnopharmacol 86: 51–58.

Guri A, Kefalas P, Roussis V (2006). Antioxidant potential of six pine species. Phytother Res 20: 263–266.

Kasinulainen P, Holopainen JK (2002). Concentrations of secondary compounds in Scots pine needles at different stages of decomposition Soi. Biol. Chem 34: 37–42.

Knekt P, Jarvinen R, Reunanen A, Maatela J (1996). Flavonoid intake and coronary mortality in Finland: a cohort study. Br Med J 312: 478-481.

Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS, Heinonen M (1999). Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem 47: 3954–3962.

Laracine-Pittet C, Lebreton P (1988). Flavonoid variability within Pinus sylvestrls. Phytochem 27: 2663-2666.

Lerski, RA, Collier BD, McKillop JH, Purnell GL, Pecking AP, Thomas FD, Harrison KA (1998). Palliation of pain associated with metastatic bone cancer using samarium-153 lexidronam: a double-blind placebo-controlled clinical trial. J Clin Oncol 16: 1574-81.

Lupo MP (2001). Antioxidants and vitamins in cosmetics. Clin Dermatol 19, 467–473.

Maimoona A, Naeem I, Saddiqe Z, Ali N, Ahmed G, Shah I (2011). Analysis of total flavonoids and phenolics in different fractions of bark and needle extracts of Pinus roxburghii and Pinus wallichiana. J Med Plants Res 5: 2724-2728.

Parr AJ, Bolwell GP (2000). Phenols in the plant and in man the potential for possible nutritional enhancement of the diet by modifying the phenols content or profile. J Sci Food Agric 80: 985–1012.

Prieto P, Pineda M, Anguilar M (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a Phosphomolybdenum Complex: Specific application to the determination of Vitamin E. Anal Biochem 269: 337-341.

Quettier-Deleu C, Gressier B, Vasseur J, Dine T, Brunet C, Luyckx M, Cazin M, Cazin JC, Bailleul F, Trotin F (2000). Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J Ethn 72 : 35-42.

Quézel P, Médail F (2003). Ecologie et biogéographie des forêts du bassin méditerranéen. France : Lavoisier.

Robles C, Greff S, Pasqualini V, Garzino S, Bousquet-Mélou A, Fernandez C, Korboulewsky N, Bonin G (2003). Phenols and Flavonoids in Aleppo Pine Needles as Bioindicators of Air Pollution. J Env Qual 32, 2265-2271.

Sharma A, Goyal R, Sharma L (2016). Potential biological efficacy of Pinus plant species against oxidative, inflammatory and microbial disorders. BMC Comp Alter Med 28: 16-35.

Serafini AN, Houston SJ, Resche I, Quick DP, Grund FM, Ell PJ, Bertrand A, Ahmann FR, Orihuela E, Reid RH, Shyi-Neng L, Ya-Siou H, Chi-Tang H (2014). Flavonoid compositions and antioxidant activity of calamondin extracts prepared using different solvents. J food drugs ann 22: 290–295.

Singleton VL, Rossi JA (1965). Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Eno Viti 16: 144-158.

Thomas M (2000). The role of free radicals and antioxidants. J Nutr 16: 7–8.

Tuzlaci E, Erol MK (1999). Part II: Egirdir (Isparta) Fitoterapia. Tur folk med plants 70: 593–610.

Vijayakumar S, Dhanapal R, Sarathchandran I, Kumar AS, Ratna JV (2012). Evaluation of antioxidant activity of Ammaniabaccifera (L.) Whole plant extract in rats Asian Pac. J Trop Biomed 2: 116-119.

Winks M, Schimmer O (1999). Modes of action of defensive secondary metabolites Function of Plant SMs and their exploitation in biotechnology. Ann Plant Rev. Academic Press, Sheffield.

Yesil-Celiktas O, Ganzera M, Akgun I, Sevimli C, Korkmaz KS, Bedir E (2009). Determination of polyphenolic constituents and biological activities of bark extracts from different Pinus species. Food Agr 89: 1339–1345.

Yeilada E, Honda G, Sezik E, Tabata M, Fujita T, Tanaka T, Takeda Y, Takaishi Y (1995). Traditional medicine in Turkey. V. Folk medicine in the inner Taurus Mountains. J Ethnophar 46: 133-152.

Zheng W, Wang SY (2001). Antioxidant activity and phenolic compounds in selected herbs. J Agr Food Chem 49: 5165 – 5170.

Zulaica-Villagomez H, Peterson DM, Herrin L, Young RA (2005). Antioxidant activity of different components of pine species. Int J Bio, Chem, Phys Tech Wood 59: 156–162.

Zupko I, Hohmann J, Redei D, Falkay G, Janicsak G, Mathe I (2001). Antioxidant activity of leaves of Salvia species in enzyme-dependent and enzyme-independent systems of lipid peroxidation and their phenolic constituents. Plant Med 67: 366–368.