Article
Heavy metals content, phytochemical composition, antimicrobial and insecticidal evaluation of Elaeagnus angustifolia
Toxicology and Industrial Health 2016, Vol. 32(1) 154–161 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713498459 tih.sagepub.com
Shahid Ullah Khan1, Arif-ullah Khan2,3, Azhar-ul-Haq Ali Shah1, Syed Majid Shah2, Sajid Hussain2, Mohammad Ayaz2 and Sultan Ayaz4 Abstract Elaeagnus angustifolia was analyzed for determination of metals, phytoconstituents, bactericidal, fungicidal and insecticidal effects and to explore its chemical and biological potential. The root, branches, leaves, stem bark and root bark parts of E. angustifolia were found to contain iron, lead, copper, cadmium, zinc, chromium, nickel and cobalt in different concentrations. Crude extract of Elaeagnus angustifolia (Ea.Cr) was tested positive for the presence of alkaloids, flavonoids, saponins and tannins. Ea.Cr and its fractions, n-hexane (Ea.Hex), ethyl acetate (Ea.EtAc) and aqueous (Ea.Aq) showed bactericidal activity against Escherichia coli and Staphylococcus aureus, while against Pseudomonas aeruginosa only Ea.Hex and Ea.EtAc were effective. When tested for antifungal effect, Ea.Cr exhibited fungicidal action against Aspergillus fumagatus, Ea.EtAc and Ea.Aq against Aspergillus flavis and Ea.EtAc against Aspergillus niger. Ea.Hex was active against all three fungal strains. The chloroform fraction (Ea.CHCl3) was found inactive against the used microbes. Ea.Cr, Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq caused mortality of Tribolium castaneum and Ephestia cautella insects observed after 24 and 48 h of treatment. These data indicate that E. angustifolia exhibits different heavy metals and compound groups. Methanolic extract of E. angustifolia and its various fractions possess antibacterial, antifungal and insecticidal activities, which elucidate medicinal application of the plant. Keywords Elaeagnus angustifolia, metals and phytochemistry profile, germicide, insecticide
Introduction Medicinal plants are nature’s gift to human beings, making for a disease-free and healthy life. Since antiquity, plants have been used for prevention and treatment of various ailments. There has been a great resurgence in the use of herbal remedies especially in the last decade. World Health Organization (WHO) estimates that approximately 80% of the world population relies on medicinal plants for their primary health care (Farnsworth et al., 1985). Elaeagnus angustifolia L. (Russian olive), which belongs to the family Elaeagnaceae, is a Eurasian tree native to western and central Asia, from southern Russia and Kazakhstan to Turkey and Iran. It is now also widely established in North America. In traditional medical system, E. angustifolia is used for the treatment of asthma, catarrh, diarrhoea,
fever, flatulence, jaundice, nausea, tetanus, urinary diseases and vomiting (Asadiar et al., 2012; Chopra et al., 1986). Recently, it has been reported to possess 1 Department of Chemistry, Kohat University of Science and Technology, Kohat, Pakistan 2 Department of Pharmacy, Kohat University of Science and Technology, Kohat, Pakistan 3 Department of Pharmacology, Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan 4 Department of Zoology, Kohat University of Science and Technology, Kohat, Pakistan
Corresponding author: Arif-ullah Khan, Department of Pharmacology, Riphah Institute of Pharmacology, Riphah International University, 7th Avenue, Sector G-7/4, Islamabad 44000, Pakistan. Email:
[email protected];
[email protected]
Khan et al.
an analgesic effect (Karimi et al., 2010). In the present research, we evaluated the chemical (heavy metals and phytoconstituents) and pharmacological (antibacterial, antifungal and insecticidal) properties of E. angustifolia.
Materials and methods Plant material, extraction and fractionation The whole plant of E. angustifolia was collected in March 2010 from Bannu district, Khyber Pakhtunkhwa (KPK), Pakistan, and identified by a taxonomist, Abdur Rahman (Department of Botany, Government Post Graduate College, Bannu, KPK, Pakistan). The shade dried plant (6 kg) was crushed into a coarse powder. Plant material was macerated in methanol with occasional shaking at room temperature for 30 days and then filtered using Whatman filter paper (Williamson et al., 1998). The filtrate was concentrated at 40 C using a rotary evaporator to obtain the crude extract of E. angustifolia (Ea.Cr), yielding approximately 3.0%. Activity-guided fractionation was carried out using solvents of increasing polarity. Ea.Cr was suspended in distilled water and sequentially partitioned with n-hexane, chloroform (CHCl3) and ethyl acetate (EtAc) to get E. angustifolia n-hexane fraction (Ea.Hex), CHCl3 fraction (Ea.CHCl3) and ethyl acetate fraction (Ea.EtAc), respectively. The remaining lower layer was collected and evaporated to obtain the aqueous fraction (Ea.Aq).
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spectrophotometer (model 1100; Perkin Elmer, Waltham, Massachusetts, USA).
Chemical constituents’ identification Preliminary phytochemical analysis was carried out for the presence of alkaloids, anthraquinones, flavonoids, glycosides, saponins, sterols, tannins and terpenoids according to reported qualitative methods (Meriga et al., 2012; Rajan et al., 2011) with some modifications. Briefly, alkaloids were detected by Dragendorff’s reagent resulting in orange–red precipitate. Presence of saponins was detected based on the appearance of froth upon vigorous shaking of diluted samples. For the detection of sterols and terpenoids, plant material was treated with petroleum ether and subsequently extracted with CHCl3. The gradual appearance of green to pink (for sterols) and reddish brown colours (for terpenoids) was then noted after treatment of CHCl3 layer with acetic anhydride and concentrated hydrochloric acid in succession. Plant material was detected as positive for flavonoids when it gave yellow colour with aluminium chloride reagent and for tannins when green or black colour was produced with aqueous ferric chloride. In case of glycosides detection, the extracts were hydrolyzed with hydrochloric acid and neutralized with sodium hydroxide solution. A few drops of Fehling’s solution were added. Red precipitate indicates the presence of glycosides. Finally, for detecting anthraquinones, the extract was dissolved in 1% hydrochloric acid, then in benzene and finally in ammonium hydroxide showing pink, violet or red colour.
Metals detection The chemicals of analytical grade used for sample preparation were 65% nitric acid and 30% hydrogen peroxide. The different portion of the plant was washed with water. The samples were dried at 80 C for 24 h in an oven. After drying, each sample was converted into finely powdered form with the help of an electrical grinder. Each sample of 2 g was taken in crucible and was ignited for 6 h at 550 C in a muffle furnace. The ash formed was digested in concentrated nitric acid (5 ml) and evaporated on a hot plate. Thereafter, little amount of distilled water was added in the digested residue, filtered and volume was made up to 25–30 ml using distilled water (Khan et al., 2007, 2008). The resulting solution of each sample was analyzed for quantitative analysis of heavy metals (iron (Fe), lead (Pb), copper (Cu), cadmium (Cd), zinc (Zn), chromium (Cr), nickel (Ni) and cobalt (Co)) using atomic absorption
Bactericidal activity Susceptibility of the selected bacteria to the plant samples was determined using well assay method (Mufti et al., 2012; Prabuseenivasan et al., 2006). Briefly, nutrient agar media was prepared and was inoculated with the test organisms. Holes were made equidistantly on the media using sterile cork borer. The plant extract was dissolved in dimethyl sulphoxide (DMSO) as concentration of 5 g/5 ml and 100 ml solution was added to holes. The inoculated agar plates were incubated at 37 C for 24 h and diameter of inhibitory zone around the hole was measured.
Fungicidal activity Nutrient broth base powder was used for culturing fungal strains. The media was prepared according to
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the manufacturer’s specifications and was transferred to sterile flasks at 65 C under laminar flow hood (Khan et al., 2011). These flasks were inoculated with the test fungi and were incubated at 25 C for primary growth. For antifungal assay Mueller Hinton agar was prepared according to the manufacturer’s specifications by dissolving and autoclaving the specified quantity of the dry powder in sufficient quantity of distilled water and was transferred to sterile test tubes (10 ml). To each test tube, 1 ml solution of the test sample was added and inoculated with different fungal species. Test tubes were incubated at 25 C for 8 days and inhibition of fungal growth was observed (Hussain et al., 2010; Ismail et al., 2012). The minimum inhibitory concentration (MIC) values were obtained.
Insecticidal assay Insecticidal activity of plant extracts was tested on adult insects of Tribolium castaneum and Ephestia cautella. Different concentrations of plant samples (0.15 and 0. 25 mg/ml) were prepared in 10% DMSO and added to sterile labelled petridishes containing about 100 ml of distilled water. In total, 20 insects were placed in each of petridish containing the extract. After adding the insects, petridishes were kept in growth room (Roman, 2004). The insecticidal effect of various extracts was determined by counting the number of dead insects after 24 and 48 h. The experiments were run in triplicate, and the results were expressed as the percentage of insect mortality.
Results Metals analysis The concentrations of various metals in different parts of E. angustifolia, that is, root, branches, leaves, stem bark and root bark, respectively were: Cu: 0.283 + 0.001, 1.058 + 0.0026, 0.072 + 0.0009, 0.147 + 0.0001 and 1.497 + 0.0032; Ni: 0.015 + 0.008, 0.012 + 0.0007, 0.022 + 0.0014, 0.016 + 0.001 and 0.010 + 0.0012; Fe: 0.009 + 0.0011, 0.693 + 0.0004, 1.042 + 0.0024, 0.790 + 0.0019 and 0.816 + 0.2281; Cr: 0.000 + 0.0013, 0.003 + 0.0009, 0.000 + 0.0005, 0.005 + 0.0001 and 0.002 + 0.0011; Pb: 0.005 + 0.0019, 0.004 + 0.0007, 0.006 + 0.0007, 0.002 + 0.0007 and 0.030 + 0.0014; Cd: 0.005 + 0.0002, 0.006 + 0.0005, 0.009 + 0.0004, 0.016 + 0.0007 and 0.008 + 0.0003, Zn: 0.638 + 0.0864, 0.510 + 0.0043, 0.780 + 0.0011, 0.376 +
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0.0017 and 0.457 + 0.0024; Co: 0.199 + 0.0020, 0.556 + 0.0006, 0.008 + 0.0034, 0.006 + 0.0012 and 0.009 + 0.0003 ppm, as presented in Table 1.
Phytochemical screening Ea.Cr was found to contain alkaloids, flavonoids, saponins and tannins, while tested negative for the presence of anthraquinones, glycosides, steroids and terpenoids (Table 2).
Antibacterial effect The plant crude extract and its resultant fractions showed bactericidal action against different pathogenic bacteria, measured as diameter of inhibitory zone. Ea.Cr, Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq, respectively, scored inhibitory zone of 20 + 0.78, 17 + 0.70, 0.0 + 0.0, 15 + 0.68 and 25 + 0.73 mm against Escherichia coli; 15 + 0.58, 17 + 0.62, 0.0 + 0.0, 25 + 0.67 and 20 + 0.50 mm against Staphylococcus aureus; 0 + 0.0, 18 + 0.95, 0.0 + 0.0, 25 + 1.0 and 0.0 + 0.0 mm against Pseudomonas aeruginosa (Figure 1).
Antifungal action Fungicidal effect of E. angustifolia samples was determined against three types of fungal species. The respective MICs values of Ea.Cr, Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq were against Aspergillus fumagatus 2.5 + 0.25, 3.5 + 0.1, 0, 0 and 0 mg/ml, Aspergillus flavis 0, 4.5 + 0.25, 0, 2.5 + 0.2 and 3.5 + 0.50 mg/ml, Aspergillus niger 0, 3.5 + 0.75, 0, 2.5 + 0.25 and 0 mg/ml as presented in Table 3.
Insecticidal activity When tested against T. castaneum and E. cautella insects, Ea.Cr, Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq after 24 h of treatment caused 58 + 0.0, 34 + 0.25, 62 + 0.75, 63 + 0.0 and 55 + 0.75%, 69 + 0.25, 41 + 0.5, 55 + 0.25, 62 + 0.0 and 61 + 0.5%, while after 48 h produced 75 + 0.50, 49 + 0.0, 85 + 0.75, 79 + 0.5 and 69 + 0.25%, 92 + 0.5, 62 + 0.5, 71 + 0.5, 91 + 0.25 and 77 + 0.25% mortality, respectively (Table 4).
Discussion Effects of heavy metals on human health and their interaction with essential trace elements may produce serious consequences. WHO recommends that
0.283 + 1.058 + 0.072 + 0.147 + 1.497 +
0.0010 0.0026 0.0009 0.0001 0.0032
Cu
0.015 0.012 0.022 0.016 0.010
+ 0.0080 + 0.0007 + 0.0014 + 0.0010 + 0.0012
Ni 0.009 0.693 1.042 0.790 0.816
+ 0.0011 + 0.0004 + 0.0024 + 0.0019 + 0.2281
Fe 0.000 0.003 0.000 0.005 0.002 + 0.0013 + 0.0009 + 0.0005 + 0.0001 + 0.0011
Cr
Fe: iron; Pb: lead; Cu: copper; Cd: cadmium; Zn: zinc; Cr: chromium; Ni: nickel; Co: cobalt.
Root Branches Leaves Stem bark Root bark
Part
Table 1. The metal contents in different parts of Elaeagnus angustifolia (in parts per million).
0.005 + 0.004 + 0.006 + 0.002 + 0.030 +
Pb 0.0019 0.0007 0.0007 0.0007 0.0014
0.005 + 0.006 + 0.009 + 0.016 + 0.008 +
0.0002 0.0005 0.0004 .0007 0.0003
Cd 0.638 0.510 0.780 0.376 0.457
+ 0.0864 + 0.0043 + 0.0011 + 0.0017 + 0.0024
Zn
0.199 + 0.556 + 0.008 + 0.006 + 0.009 +
0.0020 0.0006 0.0034 0.0012 0.0003
Co
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Table 2. Phytochemical constituents of Ea.Cr. Phytochemical class Alkaloids Flavonoids Saponins Tannins Anthraquinones Glycosides Steroids Terpenoids
Result þ þ þ þ
Ea.Cr: crude extract of Elaeagnus angustifolia. þ: Present; : absent.
medicinal plants, which form the materials for the finished products, may be checked for the presence of heavy metals, pesticide, bacterial and fungal contamination. According to WHO, the permissible limit of Cr, Pb, Zn, Cu, Ni and Co in medicinal plants is 1.5, 10, 50, 10, 1.5 and 0.2 ppm, respectively (Khuda et al., 2012). In the present study, we observed that various parts of E. angustifolia, such as root, branches, leaves, stem bark and root bark contains the aforementioned metals in lower concentrations than the permissibility. Fe and Cd were found in concentration range of 0.009–1.042 and 0.005–0.016 ppm, respectively. When tested against the pathogenic bacteria E. coli, S. aureus and P. aeruginosa, E. angustifolia samples displayed bactericidal effect, at their extent. These bacteria cause infections of skin, soft tissues, ear, respiratory and urinary tracts (Brooks et al., 2010; Pommerville, 2011). Aqueous fraction was found to be the most effective in inhibiting the E. coli, while the EtAc fraction showed the highest affectivity to eradicate the growth of S. aureus and P. aeruginosa. The CHCl3 fraction was found inactive against all the tested bacterial strains. The crude extract and aqueous fraction showed no effect against P. aeruginosa. We also reported antifungal potential of the E. angustifolia extract and its various fractions against three fungal species: A. fumagatus, A. flavis and A. niger with different potencies. These fungi cause major infections in liver, lungs, mouth, blood and skin (Pelczar et al., 2006; Willey et al., 2008). The n-hexane fraction was found the most potent against all types of microbes, followed by the plant crude extract in case of A. fumagatus, aqueous and EtAc fractions against A. flavis and EtAc fraction in case of A. niger. The CHCl3 fraction was found devoid of any effect against all the tested fungal strains. Nowadays, researchers have focused to increase the food production due to increased
Figure 1. Antibacterial effect of Ea.Cr and its resultant fractions: Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq against (a) E.coli, (b) S.aureus and (c) P.aeruginosa, measured as DIZ. Ea.Cr: crude extract of Elaeagnus angustifolia; Ea.Hex: nhexane; Ea.CHCl3: chloroform; Ea.EtAc: ethyl acetate; Ea.Aq: aqueous; E.coli: Escherichia coli; S.aureus: Staphylococcus aureus; P.aeruginosa: Pseudomonas aeruginosa; DIZ: diameter of inhibitory zone.
population of the world. Unfortunately, economically important crops are lost due to insects and plant diseases caused by microorganisms, which is a major
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Table 3. Antifungal action of Ea.Cr and its resultant fractions: Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq against different fungal species. MICs (mg/ml) Fungi Aspergillus fumagatus Aspergillus flavis Aspergillus niger
Ea.Cr
Ea.Hex
Ea.CHCl3
Ea.EtAc
Ea.Aq
2.5 + 0.25 NA NA
3.5 + 0.1 4.5 + 0.25 3.5 + 0.75
NA NA NA
NA 2.5 + 0.20 2.5 + 0.25
NA 3.5 + 0.50 NA
Ea.Cr: crude extract of Elaeagnus angustifolia; Ea.Hex: n-hexane; Ea.CHCl3: chloroform; Ea.EtAc: ethyl acetate; Ea.Aq: aqueous; MICs: minimum inhibitory concentrations; NA: not active.
Table 4. Insecticidal activity of Ea.Cr and its resultant fractions: Ea.Hex, Ea.CHCl3, Ea.EtAc and Ea.Aq against adult Tribolium castaneum and Ephestia cautella insects. Plant sample Ea.Cr Ea.Hex Ea.CHCl3 Ea.EtAc Ea.Aq
After 24 h Tribolium castaneum mortality (%) 58 34 62 63 55
+ 0.0 + 0.25 + 0.75 + 0.0 + 0.75
After 24 h Ephestia cautella mortality (%) 69 41 55 62 61
+ 0.25 + 0.5 + 0.25 + 0.0 + 0.5
After 48 h Tribolium castaneum mortality (%) 75 + 49 + 85 + 79 + 69 +
0.50 0.0 0.75 0.5 0.25
After 48 h Ephestia cautella mortality (%) 92 + 62 + 71 + 91 + 77 +
0.5 0.5 0.5 0.25 0.25
Ea.Cr: crude extract of Elaeagnus angustifolia; Ea.Hex: n-hexane; Ea.CHCl3: chloroform; Ea.EtAc: ethyl acetate; Ea.Aq: aqueous.
problem. Insect pests are responsible for the extensive loss of food grains and their products in tropical and semi-tropical environment. Stored grain insect pests have been damaging food grains in granaries and store houses, accounting for 10–40% loss worldwide. Insect pests cause quantitative and qualitative damage to grains. Quantitative damage includes weight loss of grain due to direct feeding of insects, while loss of nutritional and aesthetic value of grains is the qualitative damage. Despite application of improved storage structures and traditional control techniques, 70–90% of food grain is still stored not more than 6 months to 1 year at farmers level (Peeyush et al., 2011). Hence, there is great need to use safe insecticide or repellents to save food grains from damage. Synthetic insecticides are effective, but their wide spread use has resulted in the development of insect resistant, high cost and toxic residue on grain, so there is a dire need to develop natural insecticidal agents, which are inexpensive, safe and environment friendly, pest specific, locally available and have low pesticide resistance. The use of plant extracts in insects control is an alternative pest control method for decreasing the undesirable effects of some pesticidal compounds on environment, wild life, livestock and nontarget insect species (Ciccia et al., 2000). About 1500 species of plants have been reported to possess insecticidal value.
The most promising natural grain protectants were generally observed in the plant families of Annonaceae, Asteraceae, Canellaceae, Labiatae, Meliaceae, and Rutaceae. The botanical insecticides that have primarily been used and are commercially available include ryania, rotenone, pyrethrin, nicotine, azadirachtin and sabadilla (Rajashekar et al., 2012). T. castaneum (Coleoptera: Tenebrionidae), red flour beetle is a common insect pest of food processing facilities, such as mills, processing plants, warehouses and retail stores (Campbell, 2012). It has long association with human stored food and has been found in association with wide range of commodities including grain, flour, peas, beans, nuts, dried fruits and spices (Pugazhvendan et al., 2012). E. cautella (Lepidoptera: Pyralidae) is a small stored pest insect, which infests wide range of plant material including grain and cereal products (Arbogast and Shahpar, 2005). In the present research, we observed that E. angustifolia extract and its resultant fractions, after 24 and 48 h of treatment, caused significant mortality of adult T. castaneum and E. cautella insects. The insecticidal effect increased with increment of treatment duration (after 48 h), as expected. In case of after 24 h pretreatment results, EtAc fraction was found to be the most effective against T. castaneum, while crude extract on E. cautella. After 48 h of treatment, CHCl3 fraction was the
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most effective against T. castaneum, while the plant crude extract again on E. cautella. Preliminary phytochemical analysis of E. angustifolia extract showed the presence of alkaloids, flavonoids, saponins and tannins. The observed antibacterial, antifungal and insecticidal activities of the plant could be due to alkaloids and flavonoids as such compounds are known to possess the aforementioned effects (Acree and Haller, 1950; El-Sayed et al., 1998; Li et al., 2004; Orhan et al., 2010; Zhang et al., 2012). However, the role of other constituents cannot be ignored.
Conclusions This study reveals that E. angustifolia contain Fe, Pb, Cu, Cd, Zn, Cr, Ni, Co, alkaloids, flavonoids, saponins and tannins. Ea.Cr and its various fractions showed bactericidal, fungicidal and insecticidal actions, and therefore considered as a valuable source with potential therapeutic applications in remedy of microbialoriginated disorders. It is also used to control insects in stored products to minimize pest infestation. Further investigations are going on to identify active components of the plant, accounting for observed effects. Acknowledgements The authors are grateful to Kohat University of Science and Technology, Kohat, Pakistan, for supporting this research.
Conflict of interest The authors declared no conflicts of interest.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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