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Phytochemical Analysis, Phenolic Content, Antioxidant, Antibacterial, Insecticidal and Cytotoxic Activites of Allium Reuterianum Boiss

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Phytochemical Analysis, Phenolic Content, Antioxidant, Antibacterial, Insecticidal and Cytotoxic Activites of Allium Reuterianum Boiss Indian Journal of Traditional Knowledge Vol 18 (2), April 2019, pp 290-298 Phytochemical analysis, phenolic content, antioxidant, antibacterial, insecticidal and cytotoxic activites of Allium reuterianum Boiss. extracts Çiğdem Aydin*,1,+ & Ramazan Mammadov1 1Department of Biology, Faculty of Science, Pamukkale University, Denizli, Turkey E-mail: [email protected] Received 04 December 2018; revised 06 February 2019 Dried bulbs and leaves of Allium reuterianum were extracted with different solvents and evaluated for antioxidant, antibacterial, insecticidal, in vitro cytotoxic activities. The obtained results indicated that the highest compound 3, 4-dihidroksi benzoic acid with 166.2 µg/g in extracts. The ethanolic extracts had the highest phenolic content and acetone extracts had the lowest fenolic content. In the β-carotene-linoleic acid test system, bulb methanol (ABM) and leaf methanol (ALM) extracts showed the highest antioxidant activity. The mean antioxidant activity of ABM and ALM were 75.76±1.22% and 73.42±1.03%, respectively. The highest ferric-reducing power of extract (ABM) was determined 12.75±0.010 trolox equivalent (mg/g). Methanolic extract (ALM) exhibited a dose-dependent scavenging of DPPH and ABTS radicals with IC50 values of 0.512±0.003 mg/mL and 0.378±0.002 mg/mL respectively. Very strong reduction of gram-positive Staphylococcus aureus growth were observed during incubation of bacteria in bulb extracts (MIC was 20±1.01 μg/mL). The brine shrimp lethality assay of bulb extract has showed good toxic to brine shrimp nauplii, with LC50 of 3.987 μg/mL. The bulb extract of A. reuterianum showed highest larvicidal activity against Cx. pipiens with value LC50 (6.4129 μg/mL). The results suggest that these plants could be used as a source of natural antioxidant and antibacterial agents. Keywords: Allium reuteriaum, Antibacterial, Antioxidant, Cytotoxicite, HPLC, Insecticidal IPC Code: Int. Cl.19: A01G 13/00, A61P 31/04, A61P 17/18, A61K 39/395, C07C 7/135, A01P 7/04 Extremely diverse and taxonomically difficult genus their precursors12,13. The many biological effects of Allium L. is one of the largest genus in Allium vegetables are mainly associated with Amaryllidaceae1-7. The genus Allium has more than organosulphur compounds. These compounds include 600 species all over the world, among them just a few four γ-glutamyl peptides: γ-l-glutamyl-S-allyl-l- species have been consumed so far as vegetables, cysteine (GSAC), γ-l-glutamyl-S-(trans-1-propenyl)-l- spices or ornamental plants Allium L. which a genus cysteine (GSPC), γ-l-glutamyl-S-methyl-l-cysteine the important of between geophyta is creates a group (GSMC) and γ-glutamyl phenylalanine (γGPA). In of natural antioxidants. Since ancient times, many addition, intermediate compounds in the biosynthesis Allium species, such as onion, garlic, leek and chives, of S-alk(en)yl-l-cysteine sulphoxides (ACSOs) from provided by NOPR View metadata, citation and similar papers at core.ac.uk CORE have been used as foods, spices and herbal remedies γ-glutamyl peptidesbrought to youconsist by of S-alk(en)yl-cysteines in widespread areas of the world, especially in the such as (+)-S-allyl-l-cysteine (SAC) and (+)-S-(trans- northern hemisphere8. Allium species have been used 1-propenyl)-lcysteine (SPC)14. for food and medicine for thousands of years, The species belonging to the Allium family have especially Allium sativum (garlic) and A. cepa been used for a long time as a remedy for the (onion), and recently interest in other species has been prevention and treatment of certain diseases15. A. 9-11 increasing . The Allium genus is one of the major reuterianum is a perennial bulbous plant, originally sources of polypphenolic compunds and the from south-west Turkey. There is no report on antioxidative activity of some Allium’ s species has antioxidant, antimicrobial and cytotoxic potential of been reported and has been mainly attributed to a A. reuterianum in the literature. Therefore, the aims of variety of organo-sulfurous compounds as wells as this study were to study phenolic acid constituent, total phenolic content and flavonoid content ——— antibacterial, antioxidant activity, insecticidal, *Corresponding author AYDIN & MAMMADOV: BIOLOGICAL ACTIVITIES OF A. REUTERIANUM 291 cytotoxic activites of A. reuterianum from Turkey. times and UV absorption spectra with those of pure Separation and quantitative determination of standards. Gallic acid, 3,4-dihydroxy, 4-dihydroxy, individual phenolic compounds was performed using chlorogenic acid, vanilic acid, caffeic acid, high-performance liquid chromatography (HPLC). p-coumaric acid, ferulic acid and cinnamic acid were used as standard. Peaks identified by comparing Materials and methods retention times and UV spectra with authentic Plant material standards. The amount of each phenolic compound Allium reuterianum Boiss (Family: Amaryllidaceae) was expressed as µg/g of the extract. species were collected in the spring 2015 from Kötekli locality, near Muğla province, in Turkey. The fresh Total phenolic content assay bulbs and leaves of the plants samples were cleaned The total phenolic content of extracts were and dried in the shadow for extraction. determined with Folin- Ciocalteau reagent, according 17 to the method of Slinkard and Singleton . Briefly, Plant extract preparations 0.75 mL of Folin–Ciocalteu reagent (1:9; Folin- Dried plant parts (bulbs and leaves) were Ciocalteu reagent: distilled water) and 100 mL of pulverized. Each ground sample was transferred into a sample (5 mg/mL) were put into a test tube. The beaker. Ethanol, methanol and acetone were added in mixture was allowed to stand at room temperature for the ratio of 1:10 and they were put in water bath at 5 min. 0.75 mL of 6% (w/v) Na2CO3 was added to the 55ºC for 6 h16. The extraction mixture was separated mixture and then mixed gently. The mixture was from the residue by filtration through Whatman No: 1 homogenized and allowed to stand at room filter paper. The plant residue was re-extracted twice temperature for 90 min. Total polyphenol content was with ethanol, methanol and acetone. After the determined using a spectrophotometer at 760 nm. The filtration two extracts were combined. The residual standard calibration (0.01–0.05 mg/mL) curve was solvent of methanol, ethanol and acetone extracts of plotted using gallic acid. The total phenolic content sample were removed under reduced pressure at 48- was expressed as Gallic Acid Equivalents (GAE) in 49°C using a rotary evaporator (rotavapor IKA VB mg/mL plant extract. 10, Germany). The water extract was lyophilized using a freeze dryer (Thermosavant Modulyo D, Total flavonoid content assay USA). Extracts were produced in duplicates and used Total flavonoid content was determined using the to assay the biological activity. Dowd method as adapted by Arvouet-Grand et al. 18 Plant extracts: Allium (A), Bulb-Methanol (ABM), (1994) . For each extract, 1 mL of methanolic -1 Bulb-Ethanol (ABE), Bulb-Acetone (ABA), Leaf- solution (100 μg mL ) was mixed with 1 mL of Methanol (ALM), Leaf-Ethanol (ALE), Leaf-Acetone aluminium trichloride (AlCl3) in methanol (2%). The (ALA). absorbance was read at 415 nm after 10 min against a blank sample consisting of a 1 mL of methanol and 1 Analysis of phenolic contents by HPLC mL of plant extract without AlCl3. The total flavonoid Phenolic compounds were evaluated by reversed- content was determined on a standard curve using phase High Performance Liquid Chromatography quercetin as a standard. The mean of three readings (RP-HPLC, Shimadzu Scientific Instruments). The was used and expressed as mg of quercetin conditions utilized were as follows: C-18 column equivalents (QE) per 100 mg of extract or fraction CTO-10ASVp, 4.6 mm × 250 mm, 5 μm; mobile (mgQE/g). phase was composed of solvent A (formic acid with 3% methanol) and solvent B (100% acetonitrile); In vitro antioxidant activity injection volume 20 μL, gradient elution from β-Carotene-linoleic acid assay 15-100% B; run time 45 min and flow rate was The antioxidant activity of the crude extracts was 1 mL/min. For analysis, the samples were dissolved in evaluated using the β-carotene-linoleic acid test methanol and 20 μL of this solution was injected into system with slight modifications19. 0.2 mg of β- the column. The chromatograms were examined at Carotene (Sigma-Aldrich) dissolved in 1 mL of 280 nm with a LC gradient detector. The phenolic chloroform was added to 20 μL of linoleic acid, and compounds were recognized by comparing retention 200 mg of Tween-20 emulsifier mixture. The mixture 292 INDIAN J TRADIT KNOWLE, APRIL 2019 was then evaporated at 40oC for 10 min by means of a (1986)21. The different concentrations (40-150 rotary evaporator to remove chloroform. After μg/mL) of extracts were mixed with 2.5 mL of evaporation of chloroform, 100 mL of distilled water phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of saturated with oxygen, 4.8 mL of this emulsion was potassium ferricyanide [K3Fe(CN)6] (1%). The placed into test tubes which had 0.2 mg of the sample mixture was incubated at 50◦ºC for 20 min. A portion and 0.2 units of the extract in them. For control, 0.2 (2.5 mL) of TCA (10%) was added to the mixture, mL of solvent (methanol, ethanol, acetone) was which was then centrifuged for 10 min at 3000 rpm placed in test tubes instead of the extract. As soon as (MSE Mistral 2000, UK). The supernatant of solution the emulsion was added into the test tubes, initial (2.5 mL) was mixed with 2.5 mL of distilled water 3+ 2+ absorbance was measured at 470 nm with a and 0.5 mL of FeCl3 (0.1%).
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