International Journal of Natural Products Research

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1 Available online at International Journal of Natural Products Research Universal Research Publications. All rights reserved ISSN: Original Article SCREENING OF PHYTOCHEMICALS IN CITRUS LIMONUM PEEL EXTRACT TO EVALUATE ITS ANTIMICROBIAL POTENTIAL SHIKHA RANA 1, DR SAVITA DIXIT* 1. RESEARCH SCHOLAR, MAULANA AZAD NATIONAL INSTITUTE OF TECHNOLOGY, BHOPAL (Corresponding Author) *PROFESSOR, HOD, DEPARTMENT OF CHEMISTRY, MAULANA AZAD NATIONAL INSTITUTE OF TECHNOLOGY, BHOPAL Received 1 July 217; accepted 1 August 217 Abstract Citrus (lemon) is one of the most commercial fruit crops grown all over the world. It is one of the largest plant species and is further consisting of 4 species which are found around the world. The present study was carried out for phytochemical and antimicrobial activity analysis in the hydroalcoholic lemon peel extracts. The phytochemical analysis and analysis of bioactive compounds are done, the lemon peel extracts are found to have carbohydrates, alkaloids, flavonoids, steroids, tannic acids and phenolic compounds. The antioxidant activity, DPPH free radical scavenging activity, nitric oxide radical scavenging activity was analyzed. The antimicrobial activity is studied by the disc diffusion method. Thus, from the present study it is concluded that lemon peel extracts are rich source of phytochemicals, bioactive compounds such as antioxidants and antimicrobial activity so can be used for various pharmaceutical and therapeutic purposes so as to increase the immunity of a body against microorganisms causing diseases. 216 Universal Research Publications. All rights reserved Key words:- lemon peel, antioxidant activity, DPPH free radical scavenging, nitric oxide radical scavenging, antimicrobial activity. 1. Introduction An antimicrobial can be defined as a substance that kills or inhibits the growth of microbes such as bacteria, fungi, protozoa or viruses. The substances which are produced by the microorganisms which kills or prevents the growth of other microorganisms is known as antibiotics. In general, these are used against bacteria. Similarly, antivirals are used against viral infections and antifungals are used in treatment against fungal infections. But proper use of these drugs is advised always as if not used accordingly then these may be life threatening [1]. Various antibiotics derived from microorganisms are presently employed for the treatment of various human diseases but these must be controlled and focus must be towards the development of new drugs which can be done either synthetically or naturally. India is a rich source of medicinal plants. These medicinal plants have found their description even in Ayurveda. Phytochemicals are found in plants and are non-nutritive in nature but have certain disease preventing properties. They offer protection against pathogens and are not required by human beings for perpetuating life [2]. Phytochemicals work in many ways such as antioxidants which protect the cells from damage. These can stimulate certain enzymes and can reduce the risk of various ailments. These can also act as antimicrobial and hormonal stimulant. These also provide a unique platform for anatomical diversity and biological functionality which is imperative for drug discovery. Citrus (lemon) is one of the most commercial fruit crops grown all over the world. It is one of the largest plant species and is further consisting of 4 species which are found around the world [3]. The major consumption of the fruit is through juices as it is beneficial for prevention of diseases. The fruit juices contain Vitamin C (ascorbic acid) [4,5]. These fruits contain phenolics, flavonoids, vitamins, and essential oils which are responsible for protection 7

2 Figure 1: Citrus Lemon Peel against various diseases which are related to antioxidative, antitumor and antimicrobial activities [6]. Citrus fruits are mainly used by juice processing industries while the peels are generally wasted. Since the juice yield of citrus is less half of the fruit weight, very large amounts of by product wastes, such as peels are formed every year [7]. Peel waste are highly perishable and seasonal. There is always an increased attention in bringing useful products from waste materials and citrus wastes are no exceptions [8]. The citrus peels are rich in nutrients and contain many phytochemicals, they can be efficiently used as drugs or as food supplements too. Researchers focuses on natural components as of the increasing antibiotic resistance pathogens. Citrus (lemon) peel can be tested for antibacterial properties and can utilised in various food industries which generates large peel wastes as food preservatives. 1.1 Plant profile Botanical name: Citrus Limon L. 1.2 Taxonomical classification Kingdom: Plantae Order: Sapindales Family: Rutaceae Genus: Citrus Species: C.Limon 2. Materials and Methods 2.1 Collection of Sample: Fresh lemon fruits were bought from the Bittan Market, Bhopal, Madhya Pradesh, India. The fruits were washed and then disinfected. The juice was removed from the fruit thoroughly and the peel was carefully removed. Then the lemon peels were grinded in the grinder (figure1,2). These peels were then shade dried for 14 days. Then these were taken for extraction process. 2.2 Preparation of Extract: Organic solvents are often used for the extraction of citrus compounds. There are two main operations to extract the compounds from the citrus matrix. One is simple maceration, with solvents extracting the compounds by diffusion from the citrus matrix where methanol is a frequently used solvent. The second one is centrifugation of the juices, eliminating aqueous phases [9]. The Soxhlet extraction has widely been used for extracting valuable bioactive compounds from various natural sources. It is Figure 2: Grinded Citrus Limon Peel used for the comparison of new extraction alternatives. The small amount of the dried powdered material is placed in the thimble. The thimble is placed in distillation flask which contains methanol and water as solvent (7:3). After reaching to an overflow level, the solution of the thimble-holder is aspirated by a siphon. Siphon unloads the solution back into the distillation flask. This solution carries extracted solutes into the bulk liquid. Solute is remained in the distillation flask and solvent passes back to the solid bed of plant. The process runs repeatedly until the extraction is completed (Figure 3). The above method is used in accordance to [1]. Figure 3: Extraction Process Initial weight of Lemon peels = 39 grams Weight after drying = grams Solvent system used = Hydro-alcoholic system (2 ml) Percentage yield = 3.62% 8

3 Table 1: Tannic and Phenolic compounds results Reagent Observation 1. 5% FeCl 3solution Solution will appear deep blue black colour. 2. Lead acetate solution (1%). Solution will form white ppt. 3. Gelatin solution (1%)Solution Solution will form white ppt. 4. Acetic acid solution. Solution will appear red colour 5. Dilute iodine solution. Solution will appear red colour 6. Dilute potassium permagnate solution Solution will decolourizes its colour. 2.3 Phytochemical Analysis: - The crude methanolic extracts of lemon peel were tested for the presence of Carbohydrates, Steroids, Proteins, Alkaloids, Tannic Acid and Phenolic compounds, Sulphates, Chlorides, Flavonoids. The qualitative results are expressed as (+) for the presence and ( ) for the absence of phytochemicals. a) Molisch test This test is performed to identify carbohydrates. In this 2.3 ml of the extract was taken and 4 drops of naphthol in alcohol is added. This is then shaked well and conc. H2SO4 is then poured from the sides of the test tube [11]. b) Test for Steroids This test is known as the Salkowiski test. In this 2 ml of chloroform and 2 ml of conc. H2SO4 are added to 2ml of the extract. This is then shaken well [11]. c) Test for Flavanoids Take 2 ml of test solution then add Small quantity of reduced lead acetate solution (1%) [11]. d) Test for Sulphates With lead acetate reagent gives white ppt. will form that soluble in NaOH solution [11]. e) Test for Chloride Take 5-7 ml. filterate plant extract solution and add 3-5 ml. lead acetate solution [11]. f) Millions Test 2ml. solution and Million Reagent will mix properly [12]. g) Maeyer s Test Take 2-3 ml. filtrate extract solution with few drops mayer reagent [13]. h) Wagner s test 2-3 ml. filtrate extract solution with few drops wagners reagent [14]. i) Tannic acid and Phenolic tests Take 2-3ml. of aqueous or alcoholic extract add few drops for following reagent [15] j) Test for saponins Foam test The extract and powder were mixed vigorously with water [16]. 2.4 Analysis of Bioactive Compounds Total Flavonoid content:- Aluminium chloride complex forming assay was used to determine the total flavonoid content of the extracts [17]. Quercetin was used as standard and flavonoid content was determined as quercetin equivalent. A clean test tube was taken and.5 ml of the sample (Extract) is added, containing 1.25 ml of distilled water. Then.75 ml of 5 % sodium nitrite solution is added and allowed to stand for 5 min..15 ml of 1 % aluminium chloride is added, after 6 min.5 ml of 1. M sodium hydroxide was added and the mixture were diluted with another.275 ml of distilled water. The absorbance of the mixture at 51 nm was measured on UV spectrophotometer immediately. The flavonoid content was expressed as mg querecitin equivalents /g sample Total phenolic content:- The total phenolic content of the methanolic extract of lemon peel was determined by using Folin- Ciocalteu method [18]. 1. ml of sample was mixed with 1. ml of Folin and Ciocalteu's phenol reagent. After 3 min, 1. ml of saturated Na2CO3 (~35 %) was added to 2 3 the mixture and made up to 1 ml by adding distilled water. The reaction was kept in the dark for 9 min, observed under UV-Vis spectrophotometer at 76 nm absorbance. Gallic acid was used as a standard with varied concentration from 2 ppm to 1 ppm. A calibration curve was constructed with different concentrations of tannic acid (.2-.1 mm) as standard. The results were expressed as mg of catechol equivalents/g of extract DPPH Free Radical Scavenging Activity:- The free radical scavenging activities of the extracts were determined by using 2, 2- Diphenyl- 1-picrylhydrazyl (DPPH) free radical scavenging method [19]. DPPH in oxidized form gives a deep violet color in methanol. An antioxidant compound donates the electron to DPPH thus causing its reduction and in reduced from its color changes from deep violet to yellow. The free-radical scavenging activities were tested by their ability to bleach the stable radical DPPH. The antioxidant activity using the DPPH (1, 1-diphenyl-2-Picrylhydrazyl) assay was assessed by this method. The sample extracts and standards (BHA and ascorbic acid) were prepared at various concentrations (2-1 ppm) and mixed with ethanolic solution of DPPH with a concentration of.4 mg/ml. After stand for 2 min in the dark, the mixtures were measured at 517 nm against ethanol as blank using UV-Vis Spectrophotometer. The percentage inhibition of DPPH by extracts was calculated by using following formula Scavenging Activity (%) = (Abs Control Abs Sample) Abs Control X Nitric oxide radical scavenging activity:- The procedure is based on the principle that, sodium nitroprusside in aqueous solution at physiological ph spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide complete with 9

4 oxygen, leading to reduced production of nitrite ions. Plant extract was dissolved in distilled water for this quantification. Sodium Nitroprusside (5 mm) in standard phosphate buffer saline (.25m, ph 7.4) was incubated with different concentration (1-4μg/ml) of methanol extract and tubes were incubated at 29oC for 3 hours. Control experiment without the test compounds but with equivalent amount of buffer was conducted in an identical manner. After 3 hours incubated samples were diluted with 1 ml of Griess reagents. The absorbance of the colour developed during diazotization of Nitrite with sulphanilamide and its subsequent coupling with Napthylethylenediamine hydro-chloride was observed at 55nm on spectrophotometer. Same procedure was done with ascorbic acid which was standard in comparison to methanol extract. Calculated the % inhibition by formula and plot graph in compared to standard. Nitric Oxide (%) = (Acontrol - Atest) X Acontrol Acontrol Where, Acontrol = Absorbance of control reaction Atest = Absorbance in the presence of the samples of extract Total Antioxidant Activity: - Determination of total antioxidant capacity was evaluated by the Phosphomolybdenum method [2]..3 ml of extract and sub-fraction in ethanol, ascorbic acid used as standard (5to 2g/ml) and blank (ethanol) were combined with 3 ml of reagent mixture separately and incubated at 95 C for 9 min. After cooling to room temperature, the absorbance of each sample was measured at 695 nm against the blank. Ascorbic acid was used as standard and the total antioxidant capacity is expressed as equivalents of ascorbic acid or Gallic acid. The antioxidant activity is expressed as the number of equivalents of ascorbic acid and was calculated by the following equation: A= (c V) ( ) m Where, A = total content of antioxidant compounds, mg/g plant extract, in Ascorbic Acid Equivalent, c = the concentration of Ascorbic acid established from the calibration curve, mg/ml, V = the volume of extract (ml), and m = the weight of crude plant extract (g) Antimicrobial Activity:- Here agar disk diffusion method is used to test the antimicrobial activity. This method was developed in 194 [21]. Many accepted and approved standards are published by the Clinical and Laboratory Standard Institute (CLSI) yet this method is the most efficient for testing various microorganisms using specific culture media, incubation condition and interpretive criteria of inhibition zones [22,23]. In this well-known procedure, agar plates are inoculated with a standardized inoculum of the test microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test compound at a desired concentration, are placed on the agar surface. The Petri dishes are incubated under suitable conditions. Generally, antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism and then the diameters of inhibition growth zones are measured [24]. Based on the various literature review following 2 gram positive and 2 gram negative bacteria are tested by agar diffusion method. 1. Bacillus subtilis 2. S. auerus 3. P. aeruginosa 4. E. coli Method: Grow over night culture in liquid broth. Maintain O. D at 66 nm up to (1 x 1 6). Spread the culture on NAM agar medium. Place the 6 mm whattman filter paper on disc of different conc of plant extract 75 mg/ml dilution (25,5,75,1 %). Incubate at 37⁰C for 8 hr. Measure the zone of inhibition in mm by zonal scale (Hi-Media) [25]. 3. Results Phytochemicals, also referred to as phytonutrients, are found in fruits, vegetables, whole grains, legumes, beans, herbs, spices, nuts, and seeds and are classified according to their chemical structures and functional properties. The terminology used to describe phytochemicals can be confusing. Phytochemicals include compounds such as salicylates, phytosterols, saponins, glucosinolates, polyphenols, protease inhibitors, monoterpenes, phytoestrogens, sulphides, terpenes, lectins, and many more. Naturally occurring compounds, known as phytochemicals (phyto means plant in Greek) are thought to be largely responsible for the protective health benefits of these plantbased foods and beverages, beyond those conferred by their vitamin and mineral contents. These phytochemicals, which are part of a large and varied group of chemical compounds, also are responsible for the colour, flavour, and odour of plant foods [26]. In the hydroalcoholic extracts of citrus limon peel following phytochemicals were found (Table2) The table 2 shows that the methanolic extract of citrus limon peel shows positive results for carbohydrates, steroids, alkaloids, tannic acid and phenolic compounds, chlorides and flavonoids whereas proteins and sulphates were absent. These results explain that citrus limon peel is a rich source of phytochemicals. Generally, phytochemicals are known to confer certain health benefits such as anti inflammatory, antimicrobial, antihypertensive, and antidiabetic effects [27,28]. 3.1 Total Flavonoid content: Flavonoids are reported to possess many useful properties such as anti-inflammatory activity, antimicrobial activity, enzyme inhibition [29,3] anti-allergic activity, antioxidant activity [31] etc. The present study showed that flavonoid content in the lemon peel is found to be as shown in figure 4. The absorbance is found to show an increasing trend 1

5 Table 2: Phytochemical Results S.N. Phytochemical Name of Test Result 1. Carbohydrates Molisch test + 2. Steroids Salkowiski test + 3. Proteins Millions Test - 4. Alkaloids Maeyer s test + 5. Alkaloids Wagner s Test + 6. Tannic Acid and Phenolic compounds Tannic Acid and Phenolic Compounds test + 7. Sulphates Sulphate Test - 8. Chlorides Chloride Test + 9. Flavanoids Flavanoid Test + Table 3: Showing DPPH Assay of lemon peel extract S No Concentration (µg) Standard Ascorbic Acid Lemon Peel Extract Table 4: NO Scavenging activity of lemon peel extract S No Concentration (µg/ml) Standard Ascorbic Acid Lemon Peel Extract Table 5: Showing total antioxidant activity of lemon peel extract S No Concentration (µg/ml) Standard Ascorbic Acid Lemon Peel Extract Table 6: Zone of inhibition of microrganisms S No Micro organism 25mg/ml 5mg/ml 75mg/ml 1mg/ml 1. B Subtilis S. Aureus P.Aeruginosa E.Coli with an increase in the concentration. The TFP of extracts were determined from regression equations for the calibration curve of Quercetin (y =.2441x +.697, R2 =.995). The total flavonoid content in lemon peel extract was found to be 75.3 mg/g. 3.2 Total phenolic content: Total phenolic content in the extract is determined by Folin-Ciocalteu (F-C) method using gallic acid as the standard. The absorbance values obtained at different concentrations of gallic acid was used for the construction of calibration curve (Fig 5). F-C method is based on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybedic phosphotungstic acid complexes to form blue coloured complexes, (PMoW11O4)-4 that are determined spectro - -photometrically at 76 nm. Total phenolic content of the extracts was calculated from the regression equation of calibration curve (Y =.145x +.211; R2 =.9932) and expressed as mg gallic acid equivalents (GE) per gra of sample in dry weight (mg/g). The total phenolic content in the lemon peel extract is found to be mg/g which is much higher as shown by [32]. 3.3 Antioxidant Activity Plants rich in secondary metabolites, including phenolics, flavonoids and carotenoids, have antioxidant activity due to their redox properties and chemical structures [33]. The 11

6 Absorbance methanolic extract of citrus limonum peel had strong antioxidant activity against all the free radicals investigated. 3.4 DPPH Free Radical Scavenging Activity This method is largely used because of its simplicity and stability [34,35]. It is based on the decoloration reaction between nitrogen electron (from DPPH+) and hydrogen atom of the hydroxyl group (from the antioxidant) [36]. In the lemon peel extract the absorbance obtained is shown in table 3 and figure Nitric oxide radical scavenging activity: NO is an important bioregulatory molecule that has several physiological effects, including control of blood pressure, neural signal transduction, platelet function, as well as antimicrobial and antitumor activities. Low concentrations of NO are sufficient, in most cases, to affect these beneficial functions. But during infections and inflammations the formation of NO gets elevated and can lead to undesired deleterious effects [37]. An excess generation of NO is deleterious to human health thus making it important to screen the extracts for their capacity to scavenge nitric oxide [38]. Presently the results of NO radical scavenging activity show that the results for the citrus limonum peel extracts are higher as compared than the standards. Thus, confirming that the % scavenging is higher as shown by table 4 and figure 7 in the lemon peel extracts. The present results show that citrus peel extract have potentially high antioxidant activity. 3.6 Total Antioxidant Activity: - This method is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of green phosphate/mo (V) complex at acid ρh. Total antioxidant capacity of the phosphomolybdenum model evaluates both water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity) [39]. The results are shown in table 5 and figure 8. The results show that lemon peel extract has very high value as compared to the standard and contain as much quantity of antioxidants compounds as equivalents of ascorbic acid to effectively reduce the oxidant in the reaction matrix. 3.7 Antimicrobial Activity: Results of antimicrobial activity of lemon peel extracts against Gram positive and negative isolates by the agar well diffusion method were shown on Table 6. The microbial susceptibility was collectively summarized in Figure 9. The result of antimicrobial susceptibility assay showed promising evidence for the antimicrobial effects of lemon fruit peels against bacterial pathogens. Methanolic extract of lemon peels showed that S. aureus shows maximum zone of inhibition of 22mm followed by B subtilis, E.Coli and P. aeruginosa. 4. Conclusions Finding healing powers in plants is an ancient idea. These are as old as prehistoric times. Clinical microbiologists are researching on the antimicrobial plant extracts. It is known that due to the presence of various phytochemicals such as alkaloids, flavonoids, glycosides, saponins, steroids, anthraquinones, phenols, resins, fatty acids and gums present in the plant extracts are responsible for the antibacterial properties. The total phenolic content values show promising figures which confirms that lemon peel is an enormous source of antimicrobial activity. Further presence of various other phytochemicals such as steroids, flavonoids, alkaloids, tannic acid and phenolic compounds assist in confirming the strong antimicrobial activity of lemon peel extracts. The antimicrobial studies show that lemon peel extracts are effective against both gram positive and gram negative bacteria. Many studies show that analysis of various plant extracts and phenolic content can inhibit bacterial growth and also, they have the ability to scavenge radicals (antioxidant activity). Thus there is a correlation between the antioxidant activity and antibacterial activities [4]. The antioxidant activity tested under DPPH radical scavenging activity, NO scavenging 2.5 TFC - Lemon y =.2441x R² = Concentration Figure 4: Graph Showing TFC of lemon peel extract 12

7 % Scavenging Absorbance.8 TPC - Lemon y =.145x R² = Concentration Figure 5: Graph Showing TPC of lemon peel extract DPPH ASSAY - LEMON y = -.337x R² = y =.1265x -.73 R² = Standard ascorbic acid Lemon.1µg Linear (Standard ascorbic acid).2 µg.3 µg Linear (Lemon).4 µg.5 µg Figure 6: Graph Showing DPPH Assay of lemon peel extract Nitrous Oxide Scavenging Activity µg/ml 4 µg/ml 6 µg/ml 8 µg/ml 1 µg/ml Standard Ascorbic Acid Lemon Figure 7: Graph Showing NO scavenging activity of lemon peel extract 13

8 % Inhibition 25 Total Antioxidant µg/ml 4 µg/ml 6 µg/ml 8 µg/ml 1 µg/ml Standard Ascorbic Acid Lemon Figure 8: Graph Showing total antioxidant activity of lemon peel extract Antimicrobial activity Zone of Inhibition (mm) mg/ml 5 mg/ml 75 mg/ml 1 mg/ml Bacillus subtilis S.auerus P.aeruginosa E.coli Figure 9: Graph showing antimicrobial activity activity and total antioxidant activity shows that lemon peel characterization and biological activities of Citrus have promising antioxidant activity. Thus, confirming that aurantium bloom. Molecules 17: [PubMed] the lemon peel has strong antimicrobial and antioxidant 4. Boudries H., Madani K., Touati N., Souagui S., properties. Even though the lemon peel shows extremely Medouni S., and Chibane M Pulp antioxidant powerful results in vivo studies must be carried out to activities, mineral contents and juice nutritional validate these results. properties of Algerian Clementine Cultivars and REFERENCES: Mandarin. Afr. J. Biotechnol. 11: Pandey Amit et.al Evaluation of antimicrobial activity 5. Rekha C., Poornima G., Manasa M., Abhipsa V., and phytochemical analysis of Citrus limon. JPBMS, Pavithra Devi J., Vijay Kumar H. T., et al. 211, 13 (17). 212.Ascorbic acid, total phenol content and 2. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. antioxidant activity of fresh juices of four ripe and 34th ed.: Nirali Prakashan; 26. unripe citrus fruits. Chem. Sci. Trans. 1: Karimi E., Oskoueian E., Hendra R., Oskoueian 6. Aruoma O. I., Landes B., Ramful Baboolall D., A., and Jaafar H. Z. E Phenolic compounds 14

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