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Phenolic and flavonoid content of Elaeagnus angustifolia L. (leaf and flower)
Fereshte Saboonchian1, Rashid Jamei1*, Siavash Hosseini Sarghein1 1Department of Biology, Faculty of Science, Urmia University, Urmia, I. R. Iran
Article history: Abstract Received Sep:15, 2013 Received in revised form: Objectives: Leaves and flowers of Elaeagnus angustifolia contain Jul 11, 2013 phenolic and flavonoid compounds. These compounds have Accepted: Nov 24, 2013 antioxidant properties that protect cells from oxidative damage. Vol. 4, No. 4, Jul-Aug 2014, The aim of this study was to determine and analyze total phenolic 231-238. and flavonoid content of leaves and flowers in two E. angustifolia variants using different solvents (ethanol and methanol). * Corresponding Author: Materials and Methods: Ethanolic and methanolic extracts of the Tel: +989141464357 plant leaves and flowers were prepared. Experiments were carried Fax: +984412776707 out to measure their phenolic and flavonoid content using two [email protected] solvents. Data were analyzed using Instat-N software.
Keywords: Results: Results showed that the amount of phenolic and Elaeagnus flavonoid compounds in both ethanolic and methanolic extracts Angustifolia was higher in Fariman variant compared with Mashhad variant. Flavonoid Ethanolic and methanolic extracts of Fariman variant had the Phenolic compounds highest amount of phenolic compound (10.91±0.18, 10.28± 0.36 mg GAE/100gFW, respectively) and also the highest amounts of flavonoids (5.80±0.10, 3.36±0.05 mgQE/100gFW, respectively). Phenolic and flavonoids compounds were better extracted using methanol and ethanol solvent. Conclusions: In both varieties and solvents, the amount of phenolic and flavonoids compounds in leaves were higher than flowers. In addition, the phenolic and flavonoids compounds were
higher in Fariman compared to Mashhad variants
Please cite this paper as: Saboonchian F, Jamei R, Hosseini Sarghein S. Phenolic and flavonoid content of Elaeagnus angustifolia L. (leaf and flower). Avicenna J Phytomed, 2014; 4 (4): 231-238.
Introduction nitrate and thus slow or inhibit the In recent years, after proving the development of diseases (Rose et al., relationship between oxidative stress and 1982). In other words, the production of human diseases dietary supplements and free radicals in body cells and tissues is ingredients containing antioxidant associated with many diseases caused properties have attracted attention during aging (Sanchez-Moreno et al., (Pfannhauser et al., 2001). These 2003). Reports indicate reverse association compounds help to inhibit many of the between dietary antioxidant-rich foods and oxidative reactions caused by free radicals the incidence of human diseases (Olukemi such as superoxide, hydroxyl, and proxy et al., 2005).
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Thus, having a diet rich in antioxidant Thus, the phenol and flavonoid contents of compounds is important. The medicinal two variants of E. angustifolia found in value of plants depends on their Mashhad and Fariman were examined phytochemical constituents namely using two different solvents. alkaloids, tannins, flavonoids, and phenolic compounds which are the most important compounds (Shariff, 2001). E. angustifolia (Elaeagnus angustifolia Materials and Methods L.) is a European - Asian tree belonging to Plant materials the family Elaeagnaceae which grows in a The flowers and mature leaves of both wide range of environmental conditions variants were collected in April and (Shafroth, 1995). It is native to northern August, respectively, from two regions of Europe and Asia and is spread across the Mashhad and Fariman, Khorasan Razavi Himalayas. In English language, it is province, Iran. Then, they were transferred called Oleaster and has long elliptic leaves to the Herbarium of Faculty of Science, with sharp tips and short petioles (Zargari, Ferdowsi University of Mashhad to be 1990). Dried leaves and fruits of this identified. According to differences in species have astringent and antipyretic flowers and fruits and morphological effects. Its flower has been used in differences, the botanist of the mentioned flavoring some liqueurs. In addition, the herbarium, identified E. angustifolia from general idea is that it also has a febrifuge Mashhad and Fariman as two different effect. Iranians traditionally brewed its variants of the plant (Figure 1). fruit as a painkiller for patients with rheumatoid arthritis and its flowers were Extraction traditionally used to treat tetanus. The Thirty grams of the fresh herb of each fruits and flowers of this plant have also sample was extracted individually with been applied to treat nausea, vomiting, 100 ml of ethanol and methanol for 3 h on jaundice, asthma, and abdominal a magnetic stirrer. The yielded solutions distention (Zargari, 1990). Leaf extracts of were filtered through Whatman no.1 paper E. pungens have been proven to be a and refrigerated at 4 °C for the future tests useful herb in traditional Chinese medicine (kamkar et al., 2011). for the treatment of asthma and chronic bronchitis and are tussive and also bring Determination of total phenolic content up sputum (Ge, 2009). Total phenolic content of the extracts Antibacterial agents have been was determined using the Folin-Ciocalteau extracted from E. glabra bark (Nishino, reagent (Horwits, 1984; Jahanban et al. 1987). Moreover, ripe fruits of E. 2009). Folin-Ciocalteau reagent was philippinsis are used to treat amoebic diluted 10 times with distilled water. The dysentery (Perry, 1980). The most E. angustifolia extract solution (50 μL) important medicinal species of this genus was mixed with 1 ml diluted Folin- found in Iran is E. angustifolia (Elaegnus Ciocalteau reagent, 1 ml sodium tree) (Zargari, 1990). To supply body's bicarbonate solution (7.5%), and 2 ml necessary natural antioxidants, it is distilled water. The mixture was incubated recommended to use plants with high at room temperature for 15 min. The phenolic compounds (Frankel, 1999). absorbance of the solution was determined Since reactive oxygen species are involved at 730 nm using a spectrophotometer in the generation of most of the diseases, (Biowave, S2100, UK) and compared with investigation of phenolic compounds, in gallic acid equivalents (GAE) calibration order to collect and exploit them, is the curve. The total phenolic content was subject of attention. expressed as mg gallic acid equivalents of
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30 gram fresh E. angustifolia leaves and Table 1. Total phenolic content (mgGAE /100gFw) flowers. using ethanolic and methanolic extracts of two variety of E. angustifolia (for each group, n=4)
Determination of total flavonoid content Solvent Region Leaf Flower Flavonoid content was determined Mashhad 7.78±0.11 4.63±0.14 according to the method described by Ethanol Fariman 10.91±0.18 6.24±0.22 Bonvehi et al. (2001) with some Mashhad 8.64±0.18‡‡ 6.36±0.11‡‡‡ modifications. An appropriate dilution (50 Methanol μL)oftheextractwasmixedwith1mlof Fariman 10.28±0.36 5.86±0.13 2% AlCl in methanol solution (5% acetic 3 Statistical significance for the difference between acid in methanol). The mixture was the data of methanol vs. ethanol:‡‡:p<0.01,‡‡‡: allowed to react for 10 min and the p<0.001. absorbance was read at 430 nm against a blank sample without reactants. Quercetine Table 2. Total flavonoid content (mgQE/100gFw) was used as the standard for the calibration of two variety of E. angustifolia (for each group, curve. Total flavonoid content of the n=4) extracts was expressed as mg quercetin Solvent Region leaf Flower equivalents (QE) of 30 gram fresh E. Mashhad 4.81±0.12 1.43±0.05 angustifolia. Ethanol Fariman 5.80±0.10 2.35±0.04
Mashhad 3.34±0.0.05‡‡‡ 1.34±0.05 Statistical analysis Methanol The results are expressed as mean ± Fariman 3.36±0.0.05‡‡‡ 1.34±0.06‡‡‡
SEM. Differences were analyzed using Statistical significance for the difference between unpaired analysis of variance followed by the data of methanol vs. ethanol:‡‡‡:p<0.001. t-test. For all analyses, p-value < 0.05 was considered statistically significant. Data were analyzed using Instat-N software.
Results In this study, phenolic and flavonoids compounds from leaves and flowers in Fariman and Mashhad variants were measured using two solvents, ethanol and methanol (Tables 1 and 2). The phenolic compounds were more extracted by methanol (p<0.01 and p<0.001, Table 1) and flavonoids by ethanol (p<0.001, Table 2). The amount of phenolic compounds in the leaf of Fariman variant in both ethanolic (10.91±0.18 mgGAE/100gFW) and methanolic extract (10.28±0.36 mgGAE/100Gfw) was higher than Mashhadvariant’sleaf(p<0.001,Figure1 and p<0.01, Figure 2). Phenolic compounds of ethanolic extracts in Fariman variant’s flower were Figure 1. Flower, fruit and morphology of Fariman significantly higher than Mashhad variant of E. angustifolia (A, B, and C) and those variant’s(p<0.001,Figures1). of Mashhad variant (D, E, and F).
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12 *** Mashhad
7 Mashhad
10 Fariman *** 6
Fariman mgFW)
g FW) g 8 +++ 5
100
100 *** 6 4 +++
3 *** (mgGAE/ 4 Flavonoid Total Total Phenol content content Phenol Total 2 +++
2 content(mgQE/ 1
0 0 Leaf Flower Leaf Flower Organ Organ
Figure 2. The amount of total phenolic content Figure 4. The amount of total flavonoid content (mean ± SEM) in leaf and flower of E. angustifolia (mean±SEM) in leaf and flower of E. angustifolia of Fariman and Mashhad variants using ethanolic of Mashhad and Fariman variants using ethanol solvent. Statistical significance for the difference solvent. Statistical significance for the difference between the data of Fariman vs. Mashhad variants: between the data of Fariman vs. Mashhad: ***: ***: p<0.001. Statistical difference between the p<0.001. Statistical difference between the data of data of flower vs. leaf (using unpaired t-test, n=4). flower vs. leaf (using unpaired t-test, n=4): +++: +++: p<0.001. p<0.001.
4 Mashhad
12 Mashhad
g g 3.5 Fariman
** Fariman 100
10 3 mgFW)
8 100 2.5 +++ +++ * 2 6 FW) +++ +++
Total Flavonoid Flavonoid Total 1.5 4
1 content(mgQE/ 2 0.5
Total Phenol content (mgGAE/ content Phenol Total 0 0 Leaf Flower Leaf Flower Organ Organ
Figure 3. The amount of total phenolic content Figure 5. The amount of total flavonoid content (mean±SEM) in leaf and flower of E. angustifolia (mean±SEM) in leaf and flower of E. angustifolia of Fariman and Mashhad variants using methanol of Fariman and Mashhad variants using methanol solvent. Statistical significance for the difference solvent. Statistical difference between the data of between the data of Fariman vs. Mashhad variants: flower vs. leaf (using unpaired t-test, n=4): +++: *: p<0.05, **: p<0. 01. Statistical difference p<0.001. between the data of flower vs. leaf (using unpaired t-test, n=4): +++ : p<0.001.
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Flavonoid compounds of ethanolic extracts or electrons and scavenge free radicals in leaves (5.80±0.10 mgQE/100gFw) and (Sreedam et al., 2010). flowers (2.35±0.04 mgQE/100Gfw) of the In this study, the total phenol and Fariman variant showed significantly flavonoid content of two E. angustifolia higher value than the ethanolic extract of variants were compared. Results showed leaves (4.81±0.12 mgQE/100gFw) and that the amount of phenolic compounds flowers (1.43±0.05 mgQE/100gFw) in and flavonoids in both ethanolic and Mashhad variant (p<0.001 for both case, methanolic extracts were higher in Figure 3). However, no significant Fariman variant than Mashhad variant. difference was observed in flavonoid Many factors including climate, soil and content of methanolic extracts of the height are involved in levels of plant leaves and flowers between Fariman and secondary metabolites such as phenolic Mashhad variants (Figure 4). In both compounds. This difference might be variants and both solvents, phenolic associated with ecological conditions compounds and flavonoids of leaves were (Schwazt et al., 2009). In fact variations in higher than flowers (p<0.001 for all cases). developmental responses of E. angustifolia leaves to spatial heterogeneity was shown which could be related to its ecological Discussion strategies (Klich, 2000). In this study, two solvents, ethanol and Phenols are very important plant methanol, were used in order to extract constituents because of their radical phenolic and flavonoids compounds from scavenging ability resulting from their leaves and flowers of Fariman and hydroxyl groups (Hatano et al., 1989). The phenolic compounds may contribute Mashhad variants. We observed that directly to the antioxidative action (Duh et phenolic compounds were extracted better al., 1999). It is suggested that polyphenolic by methanol and flavonoids with ethanol compounds have inhibitory effects on solvent. Different solvent extractions mutagenesis and carcinogenesis in humans provide different types of compounds (Tanaka et al., 1998). Flavonoids and because of their variable chemical nature flavones are widely distributed secondary and sensitivity toward extraction or metabolites with antioxidant and hydrolysis methods (Jahanban Isfahlan et antiradical properties (Makari et al., 2008). al., 2010). Among different solvents Alexandru et al. determined the flavonoid commonly used for extraction of plant and total phenolic content of several herbs material for polar and non-polar including hyssop and yarrow flowers compounds, methanol and ethanol are the (Alexandru et al., 2007). most effective ones. Therefore, any The antioxidant activity of natural measurement with these alcoholic extracts sources is due to the active compounds can extract polar and non-polar present in the plants. The most natural compounds and thus leads to more phenol and flavonoid contents can be antioxidant properties (Harborne, 1998). In found in leaves, fruit, flower, and seeds of addition, ethanol is reported to be a better plants. Most of these compounds are solvent to extract phenolic compounds natural phenols or polyphenols, e.g., from persimmon compared with other tocopherols, flavonoids, and derivatives of solvents (In-Cheol et al., 2010). cinnamic acid, phosphatidic, and other Bang and colleagues (1999) showed organic acids. It is well known that the that using methanol for extraction shows antioxidant activity of plant extracts more total phenolic content compared with containing polyphenol components is due aqueous solvents and even the to their capacity to donate hydrogen atoms concentration of the used methanol can
AJP, Vol. 4, No. 4, Jul-Aug 2014 235 Saboonchian et al. affect the extraction of plant content. In anti-oxidative activities (Duh et al., 1999). other words, increasing the concentration Evidence suggests that increased intake of of methanol increases the amount of dietary antioxidants, fruits, or vegetables extracted phenol. Ethanolic and with anti-oxidative properties improves the methanolic solvents were used in several quality of life, delays the onset, or reduces studies previously, and the results showed the risk of degenerative diseases associated a different outcome of the two solvents with aging (Siriwardhana and Shahidi, (Mamashloo et al., 2012,) which support 2002). the results of the present study. Most The results of the present study showed phenolic compounds and flavonoids were that in both varieties and solvents, the found in the leaf extract. Due to amount of phenolic and flavonoids photosynthesis in leaves, flavonoid compounds in leaves were higher than biosynthetic pathway precursors (Shikimic flowers. In addition, the phenolic and acid) are more abundant in leaves than in flavonoids compounds were higher in other organs. This is also another reason Fariman compared to Mashhad variants. for the higher amount of flavonoids in leaves compared with flowers and other Conflict of Interest organs (Andersen and Markham, 2006). There is not any conflict of interest in Dokhani and colleagues (2005) also this study. reported the total amount of phenolic compounds in A. eriophora to be higher in leaf extract than flowers. Lowest flavonoid compound was observed in methanolic References flower extract of both variants. Ayaz et al. Alexandru V, Balan M, Gaspar A, in 1999 reported the presence of 4- Craciunescu O, Moldovan L. 2007. hydroxybenzoic acid, phenolic acid, and Studies on the antioxidant activity, acid caffeic in E. angustifolia fruit (Ayaz phenol and flavonoid contents of some et al., 1999). In another study significant selected Romanian medicinal plants amounts of flavonoid, terpenoid, and used for wound healing Biological cytosterol compounds were derived from Sciences. J Biosci, 296: 1831-1838. E. angustifolia leaves (Dembinska-Migas Andersen OM, Markham KR. 2006. and Gill, 1973). Flavonoids Chemistry, Biochemistry The content in polyphenolic compounds (flavones and polyphenolcarboxylic acids) and Applications. Pp.145-154,CRC in the soft extract of Elaeagnus Press, Tylor & Francis. angustifolia L. (oleaster) young branches, Ayaz FA, Kadioğlu A, Doğru A. 1999. using high-performance liquid Soluble suger composition of chromatography coupled with mass Elaeagnus angostiholio L. var. spectrometry (HPLC-MS) were also orientalis (L.) Kuntze (Russian olive) shown in two different types of extract fruits. Botany, 23: 349-354. (Bucur et al, 2009). Results of the present Bang IS, Park HY, Yuh CS, Kim AJ, YU study are in accordance with previous CY, Ghimire B, Lee HS, Park JG, studies in regard to the presence of Choung MG, Lim JD. 2007. phenolic and flavonoid compounds. Many Antioxidant Activities and Phenolic synthetic antioxidant compounds have Compounds Compisition of Extracts toxic effects or cause cancer which have from Mulberry (Morus alba L.) Fruit. turned attentions to the natural Korean J Med Crop Sci, 15: 120-127. antioxidants and phenolic compounds (as a Bonvehi JS, Torrento MS, Lorente EC. member of a family of natural 2001. Evaluation of polyphenolic and antioxidants) that participate directly in flavonoid compounds in honeybee-
AJP, Vol. 4, No. 4, Jul-Aug 2014 236 Phenolic and flavonoid content of Elaeagnus angustifolia L.
collected pollen produced in spain. J In-Cheol J, Eun-Kyung J, Myung-Suk B, Agricul Food Chem, 49: 1848-1853. Hyun-Jung L, Gyeong-Im J, Eunju P, Bucur L, lase L, Istudor V, Popescu A. Hyun-Gyun Y, Gwang-Hwan A, 2009. Hplc-ms analysis of the Seung-Cheol L. 2010. Antioxidant and polyphenols in two soft extracts of antigenotoxic activities of different Elaeagnus angustifolia l. note 2. soft parts of persimmon (Diospyros kaki cv. extract of young branches analysis. Fuyu) fruit. J Med Plants Res, 4: 155- Farmacia, 57: 736-742. 160. Dembinska-Migas W, Gill S. 1973. Jahanban Isfahlan A, Mahmoodzadeh A, Flavonoids in leaves of Elaeagnus Hassanzadeh A, Heidari R, Jamei R. angustifolia L. Pol J Pharmacol Pharm, 2009. Antioxidants and antiradicals in 25: 599-606. almond hull and shell (Amygdalus Dokhani SH, Cotrell T, Khajeddin J, Mazza communis L.) as a function of G. 2005. Analysis of Aroma and Phenolic genotype. Food Chem, 115, 529-533. Components of Selected Achillea Species. Jahanban Isfahlan A, Mahmoodzadeh A, Plant Foods Hum Nutr, 60: 55-62. Hassanzadeh A, Heidari R, Jamei R. Duh PD, Tu YY, Yen GC. 1999. 2010. Antioxidant and antiradical Antioxidant activity of water extract of activities of phenolic extracts from Harng Jyur (Chrysanthemum Iranian almond (Prunus amygdalus L.) morifolium Ramat). LWT - Food Sci hulls and shells. Turkish J Biol, 34: Technol, 32: 269-277. 165-173. Frankel EN. 1999. Recent advances in Kamkar A, Shariatifar N, Jamshidi AH, lipid oxidation. J Sci Food Agr, 54: Mohammadian M. 2011. Study of 495-511. Antioxidant Functional of the Water, Ge Y, Liu J, Su D. 2009. In vivo Methanol, and Ethanol Extracts of evaluation of the anti- asthmatic, Endemic Cuminum cyminum L. and antitussive and expectorant activities of Cardaria draba L. in the In-vitro extract and fraction from Elaeagnus Systems, pp 38, Ofoghe danesh. Pungens Leaf. J Ethnopharmacol, 126: Klich MG. 2000. Leaf variations in 538-542. Elaeagnus angustifolia related to Harborne JB. 1998. Phytochemical environmental heterogeneity. Environ methods. 3th ed, P.5-7, New York: Exp Bot, 44: 171-183. Chapman & Hall. Lu Y, Y Foo. 2000. Antioxidant activities of polyphenols from sage (Salvia Hatano T, Edamatsu R, Mori A, Fujita Y, officinalis) Food Chem, 75: 197-202. Yasuhara E. 1989. Effect of interaction Makari HK, N. Haraprasad HS, Patil, of tannins with co-existing substances. Ravikumar. 2008. In Vitro Antioxidant VI. Effects of tannins and related Activity of The Hexane And polyphenols on superoxide anion Methanolic Extracts Of Cordia radical and on DPPH radical. Chem Wallichii And Celastrus Paniculata. Pharm Bull (Tokyo), 37: 2016-2021. The Internet J. Aesthetic and Antiaging Horwitz W. 1984. Official methods of Medicine, 1: 1-10. analysis of the association of official Mamashloo S, Sadeghi Mahoonak A, analytical chemists. Washington, D. C. Ghorbani M, Alami M, Khomeiri M. 14th Ed. AOAC. 2012. The evaluation of antioxidant Huang MT, Ho CT, Tee CY. 1992. In properties and stability of phenolic Phenolic Compounds in Food and Their copmpounds from medlar (Mespilus Effects on Health ll. Antioxidants and germanica L.) fruit. J Food Sci Tech Cancer Prevention, PP 402, ACS: research and innovation, 1: 219-228. Washington.
AJP, Vol. 4, No. 4, Jul-Aug 2014 237 Saboonchian et al.
Nishino C, Enoki N, Tawata S, Mori A. Environmental Condition Affect the 1987. Antimicrobial activity of Color, Taste, Antioxidant Capacity of flavonoids against Staphylococ 11 Pomegranate Accessions, Fruits. epidermidis, a skin bacterium. Agric J Agric Food Chem, 57: 9197-9209. Biol Chem, 51: 139-143. Shafroth PB, Auble GT, Scott ML. 1995. Olukemi OA, Olukemi IO, Oluwatoyin Germination and establishment of SM, Austin AO, Mansurat LB, native plains cottonwood (Populus Olufunmilola TI. 2005. Antioxidant deltoids Marshall subsp. Monilifera) activity of Nigerian dietary species. and the exotic Russian-olive (Elaegnus Electron. J Environ Agric Food Chem, angustifolia L.). Conserv Biol, 1169- 4: 1086-1093. 1175. Perry LM. 1980. Medicinal Plants of the Shariff ZU. 2001. Modern herbal therapy East and Southeast Asia. MIT Press. for common ailmentsSpectrum Books London, 131. Ltd., Ibadan, Nigeria in Association Pfannhauser W, Fenwick GR, Khokhar S. with Safari Books Ltd. 1: 9-84, Nature 2001. Biologically active Pharamcy Series, UK. phytochemicals in food. PP. 448-452, Siriwardhana SSKW, Shahidi F. 2002. Cambridge: Royal Society of Antiradical activity of extracs of Chemistry. almond and its by-products. J Am Oil Sánchez-Moreno C, Cao G, Ou B, Prior Chem Soc, 79: 903-908. RL. 2003. Anthocyanin and Sreedam C D, Kaiser H, Israt Jahan B, 1 2 proanthocyanidin content in selected 3 4Shapna Sultana and 5Prof. Saiful white and red wines. Oxygen radical Islam MD.2010. In Vitro Antioxidant absorbance capacity comparison with Activity of Different Parts of the Plant nontraditional wines obtained from Diospyros discolor. Res J Agric Biol highbush blueberry. J Agric Food Sci, 6: 472-475. Chem, 51:4889-4896. Tanaka M, Kuei CW, Nagashima Y, Sanwal M, Trevithick GR. 1982. In vivo Taguchi T. 1998. Application of effects of vitamin E on cataractogenesis antioxidative maillrad reaction products in diabetic rats. Can J Ophtalmol, from histidine and glucose to sardine 17:61-66. products. Nippon Suisan Gakk, Schwartz E, Tzulker R, Glazer I, Bar-Ya, 54:1409-1414. akov I, Wiesman Z, Tripler E, Bar-Ilan Zargari A. 1990. Medicinal plants. 4: 275- I, Fromm H, Borochov- Neori H, 277.Tehran Univercity Press, Tehran. Holland D and Amir R. 2009.
AJP, Vol. 4, No. 4, Jul-Aug 2014 238