This study was performed to evaluate the in vitro antimicrobial activity and the flavonoid content of Iraqi awsaj plant (Lycium barbarum L.) fruits. The fruits extracts contain important amounts of flavonoids. The results obtained in the antimicrobial tests revealed that flavonoid compound was more active than alcoholic extract both for Gram-positive and Gram-negative bacterial strains. The results suggest that these species are valuable sources of flavonoids with relevant antimicrobial activities

Alassadi, Iqbal J.

Alrubaie, Leaqaa A.

Sabah, Fatima S.

English

European Scientific Journal, ESJ

European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 268 ISOLATION OF FLAVONOID COMPOUND FROM IRAQI AWSAJ PLANT (LYCIUM BARBARUM L.) FRUITS AND THE STUDY OF ITS ANTIBACTERIAL ACTIVITY    Iqbal J. Alassadi Fatima S. Sabah Chemistry Department, College of Science, University of Basra, Iraq Leaqaa A. Alrubaie College of Pharmacy, University of Basra, Iraq   Abstract  This study was performed to evaluate the in vitro antimicrobial activity and the flavonoid content of Iraqi  awsaj plant  (Lycium barbarum L.) fruits. The fruits extracts contain important amounts of flavonoids. The results obtained in the antimicrobial tests revealed that flavonoid compound was more active than alcoholic extract both for Gram-positive and Gram-negative bacterial strains. The results suggest that these species are valuable sources of flavonoids with relevant antimicrobial activities.   Keywords: Awsaj, flavonoid, antimicrobial, herbal medicine  Introduction  Consequently, recent important epidemiological studies have concluded that certain natural foods and medicinal plants helps to prevent or hinder the development of different diseases (Dahech et al., 2013 and Vlase et al., 2013). Thus, the interest in developing natural nutritional antioxidants is increasing due to its well-documented impact on human health (Laurian et al., 2014).  The importance of plants belonging to the genus Lycium L. (Solanaceae) has increased rapidly in the last few years due to their traditional usages in Chinese herbal medicine. They are considered by most researchers as functional foods with a large variety of beneficial effects (Li et al., 2007 and  Qian et al., 2004). A sweet tonic decoction made from the fruits is used to lower blood pressure and blood cholesterol levels (Tahraoui et al.,  2007). However, it acts mainly on the liver and kidneys. The fruit is taken internally for the treatment of high blood pressure, diabetes, poor European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  269 eyesight, vertigo, lumbago, impotence, and menopausal complaints. The fruit is harvested when fully ripe and is dried for later use. The root bark is a bitter, cool, and is an antibacterial herb that controls coughs and lowers fevers, blood pressure, and blood cholesterol levels(Goji, 2014).    Materials and Methods Plant Material & Chemicals    The Awsaj plant (Lycium barbarum L.) fruits were purchased fresh from Abi –Algaseb and classified by the lush in the biology department of the College of Science at the University of Basra, Iraq .The fruits of the Awsaj plant were dried and turned to powder with the help of an electric grinder. All of the chemicals were purchased from Sigma- Aldrich Co. (St. Louis, MO, USA). Also, the solvents were obtained from E. Merck (Darmstadt, Germany). Furthermore, all of the reagents were prepared in deionized distilled water.  Preparation of Extracts Alcoholic Extract  20.000gm of Awsaj fruits powder soaked in 300ml (80% ethanol) was stirred using Magnetic stirrer for 24 hours, and was filtered through a filter paper (whatman No.541).The filtrate was placed in a Petri dish at room temperature until it dries up. Thus, the weight of the amorphous brown powder that was formed was 7.053gm.  Flavonoid Compound  25.000gm of Awsaj fruits powder was soaked in 300ml (80% ethanol) by a Magnetic stirrer for 24 hours, and the extract was filtered through a filter paper (whatman No.541). 2% aqueous lead acetate was then added until flocculent and brown precipitate was formed. Hence, the precipitate was separated by a filter paper (whatman No.532). Then, it was washed with water, methanol, and ethyl acetate consecutively.   The salt that was produced was converted to chloride by dissolving it in 30ml acetone and 10ml 2N HCl; also, it was filtered using a filter paper (whatman No. 540). The filtrate was placed in Petri dish at room temperature until it dries up. However, the weight of amorphous brown powder that was formed was 1.895 gm.  Preliminary Qualitative Test  Preliminary tests were carried out on the alcoholic extract and flavonoid compound as shown in table (1). (Harbone, 1984)   European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 270 Thin Layer Chromatography  Thin Layer Chromatography (TLC) were carried out on the alcoholic extract and flavonoid compound (Benzene: DMSO: Water: Acetic acid (4:0.5:0.2:1 )) .  Physical and Group Activity Test  The physical and group activity test is usually carried out on the flavonoid compound. Sepectroscopy  Ultra violet and visible spectra, UV-visible spectrum of the flavonoid compound using ethanol as the solvent, and the spectrum was recorded with a computerized thermos spectronic model LR 115161(England).  Infra-red spectrum FT-IR spectrum was recorded with FT-IR 8400SSHIMADZU–Japan.  The Determination of the Antimicrobial Activity of Alcoholic Extract and Flavonoid Compound Cultures  The Cultures of the bacteria (Escherichia coli Atcc25922 and Staphylococcus aureus ATCC25923) that were used was procured from the Department of Biology.  Cultures were maintained on the medium suggested by the respective laboratory, and sub-culturing was done fortnightly.   Antibacterial Activity of Flavonoid Compound and Alcoholic Extract  In this study, the experiments were performed to check the antibacterial properties of flavonoid compound and alcoholic extract.  The disc diffusion method was used to evaluate the antibacterial activity (Elgayyyar etal. 2001 and Andrews, 2001). Mueller Hinton agar was prepared in the plates as the media for the test microorganisms. Sterile filter paper discs (Whatman No. 1mm) were impregnated with 100 μl of each of the extracts (10mg/ml) and left to dry under the laminar flow cabinet overnight. The bacterial inoculum was spread evenly on the surface of the Mueller Hinton agar plates using a sterile glass L-form rod before the extract discs were positioned on the inoculated agar surface.  Each extract was assayed in triplicate. Thus, sterile distilled water served as a negative control. Furthermore, all the plates were incubated for 24 hr at 37°C. In addition, the antibacterial activity was interpreted from the size of the diameter of zone inhibition which was measured to the nearest millimeter (mm) as observed from the clear zones surrounding the discs.  The alcoholic extracts and flavonoid were dissolved in DMSO to a final concentration of 10mg/ml for disc diffusion assay. In addition, various European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  271 types of reference strains of gram positive and gram negative bacteria (Escherichia coli Atcc25922 and Staphylococcus aureus ATCC25923) were tested using plates of muller Hinton agar. The antimicrobial activity was defined as the clear zone of growth inhibition.  The minimum inhibition concentration of the alcoholic extract and flavonoid compound (MIC), was estimated against deferent types of reference strains of bacteria with deferent concentration of the alcoholic extract and flavonoid compound ranging from 1000 to 150µg/ml (Collee et al., 1996).    Results and Discussion      Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay (Andrews, 2001).  The alcoholic extract and flavonoid compound were isolated in a yield of 35.265% and 7.580% respectively from the dried fruits.  Table (1) indicates the preliminary phytochemical analysis for alcoholic extract and flavonoid compound. The results revealed that there were alkaloids, carbohydrate, glycosides, amino acid, and flavonoid in alcoholic extract. Thus, no saponin was present. The flavonoid compound contained only flavonoid compound. TLC procedure was applied for these compounds and the results are shown in table (2). Table (1): Preliminary qualitative test for petroleum ether and flavonoid extract Phytochemical Alcoholic extract Flavonoid compound Flavonoid test + + Carbohydrate test + - Alkaloids test + - Glycoside test + + Amino acid test + - Saponin test - - Absence = - ; Presence = +  Table (2): TLC for preliminary qualitative test for petroleum ether and flavonoid extract Reagent  Sample FeCl3+ K3Fe(CN)6 Ninhydrin Folin reagent Drangdroff   UV-Lump Vanilin  visible Alcoholic extract 0.23 0.48 0.40 0.3 0.23 0.06 0.23 0.23 0.23 Flavonoid compound 0.23 --- 0.23 --- 0.23 0.23 0.23  European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 272 Table (3): Activity test for flavonoid compound  Table (4): Physical test for flavonoid compound Test Result Physical Brown amorphous Melting point 176-1750C Solubility test Soluble in water ,ethanol, and DMSO   Fig (1, 2) shows the UV spectrum and IR- spectrum. The UV spectrum shows maximum absorption at 340nm due to π  →   π* transition which is the characteristic of unsaturated double bond. The visible spectrum also shows max absorption at λmax= 520 nm due to the n   →   π* transition, and due to the presence of pairs of electrons. Furthermore, it shows the most important absorption peaks of the functional groups which belonged to the IR- spectrum of isolated flavonoid compound. From IR- spectrum, we can conclude that the isolated compound has aromatic structure containing phenolic hydroxyl groups, ether, and carbonyl groups within its structure Table (5).  Fig. (1) shows the UV spectrum of flavonoid compound Phenols Double bond  alcohol Carboxylic  Acid Aldehyde& ketone amine             Test Sample --- + --- --- + --- Flavonoid Compound  00,20,40,60,811,2200 300 400 500 600 700Absorpance Wave Length   nm European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  273 Table (5) shows the full scan of IR spectrum of flavonoid compound    Fig. (2) shows IR for flavonoid compound   Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. However, flavonoids belong to the polyphenol family. Flavanoids can be visualized as two benzene rings which are joined together with a short three carbon chain. One of the carbons of the short chain is always connected to a carbon of one of the benzene rings, either directly or through an oxygen bridge. Thus, this forms a third middle ring, which can be five or six membered.  Furthermore, the flavonoids consist of 6 major subgroups: chalcone, flavone, flavonol, flavanone, anthocyanins, and isoflavonoids. Together with carotenes, flavanoids are also responsible for the coloring of fruits, vegetables, and herbs (Harborne, 1994 and Harborne et al., 2000).    The results of testing the Lycium barbarum L. extracts for antimicrobial activities for both Gram-positive and Gram-negative bacteria European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431 274 were summarized in Table (6). Results obtained in the present study relieved that Lycium barbarum L. flavonoid compound was found to be more active than alcoholic extract against both Gram-positive and Gram-negative bacterial strains. The MIC to flavonoid compound is 150µg/ml.  Generally, plant extracts are usually more active against Gram positive bacteria than Gram negative bacteria (Lin et al., 1999). The potent antibacterial activity of extracted flavonoid suggested that these extracts may have high total flavonoid content (Dijpa and Delmee, 2000). For phenols and phenolic compounds, an injury of membrane functions has been reported as a mechanism of action (Davidson etal., 1981, Sashidhar, 2002 and Abdul-Aziz , 2005).  The presence of alcoholic groups (-OH) in the structure of the flavonoid increase the activity of the plant extract to inhibit the microbial growth. So, the alcoholic compounds and their derivatives were considered as an antiseptic agents( Dey, and Harborne,1997).Thus, this agents are changing the cell protein nature and increasing the permeability of the cell membranes ( Feeny, 1998). Table (6) antimicrobial activities against both Gram-positive and Gram-negative bacteria for flavonoid test Bacterial strains Alcohol extract Flavonoid test S. aurens 0 15mm E.coli 0 12mm   Figure (3 ) :The antibacterial activity of flavonoid pigment(B) and alcoholic extract (A) against Staphylococcus aureus and Escherichia coli Bacteria.  European Scientific Journal August 2015 edition vol.11, No.24  ISSN: 1857 – 7881 (Print)  e - ISSN 1857- 7431  275 References: Dahech, I.; Farah, W.; Trigui, M.; Hssouna, A.B.; Belghith, H.; Belghith, K.S.; Abdallah, F.B. Antioxidant and antimicrobial activities of Lycium shawii fruits extract. Int. J. Biol. Macromol. 2013, 60, 328–333. Vlase, L.; Pârvu, M.; Pârvu, E.A.; Toiu, A. Chemical Constituents of Three Allium Species from Romania. Molecules 2013, 18, 114–127.  Laurian Vlase, Dan Cristian Vodnar, Cristina Bischin, Daniela Hanganu ,Ana-Maria Gheldiu , Radu Oprean , Radu Silaghi-Dumitrescu  and Gianina Crișan. Polyphenolic Content, Antioxidant and Antimicrobial Activitiesof Lycium barbarum L. and Lycium chinense Mill. Leaves Andrei Mocan Molecules 2014, 19, 10056-10073, Li, X.-M. Protective effect of Lycium barbarum polysaccharides on streptozocin-induced oxidative stress in rats. Int. J. Biol. Macromol. 2007, 40, 461–465.  Qian, J.-Y.; Liu, D.; Huang, A.-G. The efficiency of flavonoids in polar extracts of Lycium chinense Mill fruits as free radical scavenger. Food Chem. 2004, 87, 283–288.  Tahraoui A, El HJ, Israili ZH, Lyoussi B. Ethnopharmacological survey of plants used in the traditional treatment of hypertension and  diabetes in south eastern Morocco (Errachidia province). J. Ethnopharmacol. (2007), 110: 105-117. Plants For A Future (2014). Lycium barbarum Goji, Box Thorn, Matrimony vine PFAF Plant Database.html. Harbone, J. B. (1984). Phytochemical Methods: A Guide to Modern Technique of Plant Analysis. (2nd edn). Chapman and Hall, London, pp. 1, 19, 37 – 168.  Elgayyyar, M; Dranghon, F.A.; Golden, D. and Mount, J.R. (2001). Antimicrobial activity of essential oils from polanrs against selected pathologic and saprophytic microorganisms. J. Food Prot., 64(7): (1019-1024). Andrews, J.M. (2001). Standardized disc susceptibility testing methods. J. Antimicrob. Chemother., P.:(48-57). Harborne, J.B.(Ed.)(1994). The flavonoids, Advances in research Since, 1986. 1994, Chapman &Hall, London, Pp.(378382). Harborne, J.B. and Williams, Ch.A. (2000). Advances in flavonoid research sinc.1992. Phytochemistry, 2000, 55: (481504). Lin, J.; Opaka, A.R.; Geheeb-Keller, M.; Hutehings, A.D.; Terblanche , S.E. and Jager, A.K. (1999). Preliminary screening of some traditional zulu medicinal plants for anti-inflammatory and anti-microbial activities. J. Ethnopharmacol, 68:(267-274). Dijpa, C.D.;and Delmee, M.(2000). Antibacterial activity of bark extracts of Zyzygium jambos ( L.) Alston (Mytaceae).J. Ethnopharmacol, 71:( 307-31). 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Tables and figures contents  Table 1 Preliminary qualitative test for petroleum ether and flavonoid extract Table 2 TLC for preliminary qualitative test for petroleum ether and flavonoid extract Table 3 Activity test for flavonoid compound Table 4 Physical test for flavonoid compound  Table 5 shows the full scan of IR spectrum of flavonoid compound Table 6 antimicrobial activities against both Gram-positive and Gram-negative bacteria for flavonoid test Figure 1 the UV spectrum of flavonoid compound Figure 2 IR for flavonoid compound Figure 3 The antibacterial activity of flavonoid pigment(B) and alcoholic extract(A) against Staphylococcus aureus and Escherichia coli Bacteria      

ISOLATION OF FLAVONOID COMPOUND FROM IRAQI AWSAJ PLANT (LYCIUM BARBARUM L.) FRUITS AND THE STUDY OF ITS ANTIBACTERIAL ACTIVITY

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ISOLATION OF FLAVONOID COMPOUND FROM IRAQI AWSAJ PLANT (LYCIUM BARBARUM L.) FRUITS AND THE STUDY OF ITS ANTIBACTERIAL ACTIVITY

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