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Citation: Egypt. Acad. J. Biolog. Sci. (G.Microbiolog) Vol.13 (2) pp.1- 10 (2020) DOI: 10.21608/EAJBSG.2021.189671

Egypt. Acad. J. Biolog. Sci., 13(2):1-10(2021) Egyptian Academic Journal of Biological Sciences G. Microbiology

ISSN: 2090-0872 https://eajbsg.journals.ekb.eg/

Screening and Quantification of Bioactive Compounds and Antimicrobial Activities of Fresh Juice, Methanolic Peel and Pulp Extract of sinensis L. (Sweet ).

Reham M. Mostafa* and Heba S. Essawy Botany Department, Faculty of Science, Benha University, Benha, Qalyubiya 13518, Egypt *E.Mail: [email protected]

ARTICLE INFO ABSTRACT Article History In this work, phytochemical examination of fresh juice and Received: 4/5/2021 methanolic peel, pulp extract revealed the presence of diverse bioactive Accepted:19/7//2021 organic components. The antioxidant activity of Citrus sinensis L. (Sweet Available:20/7/2021 Orange) juice extract (JE) and crude methanolic extract of pulp and peel ______(MPPE) were tested. Two fungal strains and six bacterial strains (3 gramme Keywords: positive and 3 gramme negative) were tested. Citrus sinensis extracts Phytochemical show antibacterial action against both bacterial strains, according to the analysis, research, but there is no efficacy against fungal strains. MPPE has higher antimicrobial antibacterial activity than J.E, this may be because phenolic and flavonoid activities, Citrus compounds are found. sinensis. INTRODUCTION The family includes roughly 160 genera and 1700 species. Citrus was one of the Rutaceae family's most important economic genera, producing high levels of polyphenolic and vitamin C. (Mulero et al., 2012). Citrus fruits have a cell-protective effect because antioxidants such as vitamin C and flavonoids are present (Mokbel and Hashinaga 2006). The botanical name for the frequently cultivated sweet orange is Citrus sinensis. It belongs to the Citrus genus, which accounts for a considerable percentage of flowering (Jansz et al., 1994). The orange is the most well-known fruit in the world; it can be eaten raw or juiced (Topuz et al., 2005). The peel, pulp, seeds, and juice make up the fruit. In some places, in contrast to other fruits, C. sinensis can be used not just as a fruit but more as a medicinal plant (Blauer, 2003). Approximately thirty-four percent of the fruit can be used to make juice, resulting in approximately as a by-product, forty -four percent of the peels (Li et al., 2006). Citrus sinensis has a great number of secondary bioactive compounds, which play an important part in the plant's pharmacological activity. Many different sections of the plant, contain fatty acids, steroids, alkanes, and hydroxyamides (Rani et al., 2009), flavonoids, and other phytochemicals (Gattuso et al., 2007), peptides (Matsubara et al., 1991), carotenoids (Aschoff et al., 2015), and alimentary elements as sodium, potassium, calcium, and magnesium (Niu et al., 2009).

Citation: Egypt. Acad. J. Biolog. Sci. (G.Microbiolog) Vol.13 (2) pp.1- 10 (2020) DOI: 10.21608/EAJBSG.2021.189671

2 Reham M. Mostafa* and Heba S. Essawy

The peel and seeds are sensitive to freezer till use. The whole two studied microbial deterioration; they produced a high extracts included (1) juice extract (JE) and (2) amount of by-products that could pollute the crude methanolic extract of pulp and peel environment. Citrus by-products, which are (MPPE). high in dietary fibre and bioactive chemicals, Phytochemical Screening: can be utilized in nutrition as useful elements Alkaloids: in the development of efficient meals (Marin A-Wagner Test: The formation of reddish- et al., 2002). Peeling is used in a variety of brown precipitate appears when(1ml) of industrial sectors since it is a valuable Crude extracts are treated with Wagner’s resource of phenolic bioactive compounds reagent. that can be employed in the composition of B-Mayor’s test: When we mixed 2 ml foods or isolated as natural antioxidants to filtrate with 1 percent HCl and approximately keep certain foods from oxidising (Albishi et 6 drops of Mayor's reagents, we got a al.,2013). phenolic compounds present in a creamish or pale-yellow precipitate. high amount in Citrus peels and seeds, such as Terpenoid and Sterol Test (salkowski phenolic acids and flavonoids; while test): flavonoids are found in greater quantities in 20 mL of ethanol were added to peels than in seeds (Sawalha et al., 2009), Crude extracts and then boiled in a water bath. Citrus fruits are regarded to be the essential Subsequently, the filtration and vaporization sources of natural antioxidant, containing were done then 10 mL of diethyl ether was high amounts of bioactive compounds (Al- added into the residue and filtered once more. Juhaimi & Ghafoor, 2013). In addition, At room temperature, the filtrate was dried. volatile essential oils found in C. sinensis After that few drops of acetic acid were added peels are efficient at inhibiting bacterial and then a few drops of conc.H2so4 were growth and disinfecting wounds. Rios and added. The formation of blue or greenish-blue Recio (Rios and Recio, 2005). color indicated the existence of steroids and MATERIALS AND METHODS the presence of triterpenoids was revealed by Preparation of Extract: the production of reddish-brown color. Fresh fruits of Citrus sinensis were Saponins Test (Foam Test): The crude purchased from a local market for this study, extracts were mixed and shaked with distilled at Benha City, Egypt. Whole Citrus fruits water then heated to boiling point. The (300 g) were washed in the laboratory then formation of bubbles indicates that the surface sanitized with 70% alcohol, washed saponins are present. with sterile distilled water, peeled the Flavonoids Test (ammonia test): In a test fruit and juice extracted using the juice tube, 1-2 drops of 1% NH3 (ammonia) extractor into a separately sterilized container solution are added to the aqueous extract of and after that, it was filtered into a new sterile each sample. If flavonoids are present, they container to eliminate any remaining tissues produce a yellow color. and weight. (70 ml). It was peeled and cut into Phenolic Content Test: We added 5 mL smaller pieces then grinded with pulp. The distilled water to the crude extracts then added fresh peels with pulp (230 g) were soaked in few drops of 10% iron (III) chloride solution 300 ml methanol at room temp and shaking to the mixture When the color of the solution for 24 hours and filtrated. The filtrate was changed to blue or green, it meant the concentrated to dryness below vacuum phenolic content was positive. pressure using a rotary evaporator at 35 Tannins Test (ferric chloride test): Added 2 Celsius degrees. The plant residue that mL of distilled water to the Crude extracts and resulted was considered as a crude extract. It heated on a water bath, followed by adding of weighed (22.8 gm), was collected in glass 1-2 drops of diluted FeCl3. The presence of stoppered tubes, and stored at -10 °C in a deep Screening and Quantification of Bioactive Compounds and Antimicrobial Activities of Fresh Juice 3 tannins was shown by the formation of a dark tested is ABS, and the absorbance of the green tint tested sample is ATS. Test for amino acids (Ninhydrin test): 1-3 Antimicrobial Activity Test: drops of Ninhydrin reagent were added to 1 Antimicrobial activity tests were ml of the extract. The presence of amino acids conducted at Benha University's Faculty of is indicated by the occurrence of purple color. Sciences' Department of Botany and Quantification of Total Phenolic Microbiology. The tested samples were Compounds: - determined using a modified Kirby-Bauer To evaluate the concentration of total disc diffusion method (Bauer et al., 1966). phenolic compounds in dry extracts, the 100 l of the test bacteria/fungus were Folin-Ciocalteu technique (Singleton and cultivated in 10 ml of fresh media until they Rossi, 1965; Kähkönen et al., 1999) was reached roughly 108 cells/ml for bacteria and employed. 105 cells/ml for fungi (Pfaller, et al., 1988). Quantification of total flavonoids On plates, 100 µl of microbial suspension was compounds: spread corresponding to the broth in which Aluminum chloride colorimetric they were preserved. Inoculated fungi Plates assay (Zhishen et al., 1999; Zou et al., 2004) as Aspergillus flavus at 25 C for 48 hours; was used to determine the total flavonoid Inoculated bacterial plates; Gram (+) bacteria content. as Staphylococcus aureus, Bacillus subtilis; Antioxidant Activities Assays: Gram (-) bacteria as Escherichia According to (Pérez–Jiménez et al., coli, Pseudomonas aeuroginosa they were 2008), a DPPH radical scavenging assay was incubated at 35-37 °C for 24-48 hours and used. The DPPH radical scavenging activity yeast as Candida albicans incubated at 30 °C (percent RSA) of substances was estimated for 24-48 hours and, then the diameters of the using the equation from the absorbance at the inhibition zones were measured in start (0) and after a certain reaction time (T) millimeters (NCCLS, 1993). (1). The Type Strain of Microorganisms: (% RSA) = (ABS-ATS) / ABS x 100 Six bacterial strains (3 grams positive (1) and 3 grams negative (Table1), and two Where; The absorbance of the blank sample fungal strains were tested (Table 2). (DPPH) solution without the substance to be

Table 1: Bacterial species Name Gram reaction ATCC Bacillus cereus G + 14579 Staphylococcus aureus G + 6538 Streptococcus faecalis G + 25175 Escherichia coli G - 8739 Pseudomonas aeruginosa G - 9027 Salmonella typhimrium G - 14028

Table 2: Fungal species Name ATCC Candida parapsilosis 22019 Aspergillus flavus 9643

4 Reham M. Mostafa* and Heba S. Essawy

RESULTS AND DISCUSSION according to these findings. Citrus sinensis Phytochemical screening fruits contain carbohydrates, coumarin Extraction is a necessary step in the glycosides, flavonoids, volatile oils, organic analysis and utilization of the plant's bioactive acids, lipids fixed oils, and glycosides in compounds. Table (3) summarizes the varying proportions, according to Oikeh et al., phytochemical test findings for J.E. and 2013. Phytochemicals have a wide range of MPPE, which indicate positive results for health advantages, including anti- Terpenoids, Steroids, Alkaloids, Tannins, inflammatory, antibacterial, and anti-diabetic Phenolic, Protein Compounds, and properties (Ayoola et al., 2008; Oikeh et al., Flavonoids, but no results for Saponin. Citrus 2013). JE and MPPE are high in phytochemicals,

Table 3: Phytochemical analysis of JE and MPPE of Citrus sinensis

(+): Present (-):Absence

Quantification of Total Flavonoid and plants high in flavonoids, phenolics, and Phenolic Content: carotenoids have antioxidant action (Shoib Anti-inflammatory action, and Malik 2018). Because of its stability and antibacterial activity, enzyme inhibition simplicity, the DPPH Free Radical (Harborne and Baxter 1999), anti-allergic Scavenging Activity technique is commonly effect, and antioxidant activity are only a few utilized (Brand Williams et al., 1995). The of the therapeutic qualities of flavonoids results showed that when the concentration (Middleton and Chithan 1993). Total increased, the free radical scavenging activity flavonoid content was reported to be 0. 07 of both JE and MPPE increased (Fig. 1). This g/100g in JE and 0. 06 g/100g in MPPE, research backs up the findings of (Cillard and respectively. Follin Ciocalteu reagent method Cillard 1998), who found that the ethanolic using to calculate the content of total phenol, extract of C. sinensis Linn had anti- which was represented as g/100 g. Figure (1) inflammatory and antioxidant properties. The revealed the total phenolic content of JE and presence of key phytochemical components MPPE was 8.43 mg/g and 11.24 mg/g, such as phenolic acid, a flavonoid, was linked respectively. The peel of C. sinensis is high in to the antioxidant efficiency of C. sinensis vitamin C, fibre, and many nutrients, peel extract in the current study. The including phenolics and flavonoids. (Favela- antioxidant activity which belongs to the Hernández et al., 2016). phenolics and flavonoids content in plants is Antioxidant Activity: Because of their a critical aspect in the treatment and chemical structures and redox properties, prevention of disease. As a result, the Screening and Quantification of Bioactive Compounds and Antimicrobial Activities of Fresh Juice 5 therapeutic potential of a plant source may be action (Cai et al., 2004; Zheng and Wang, determined by its bioactive phytochemical 2001) components (Dai & Mumper, 2010). Rahman et al. (2011) supported our Antimicrobial Activities of Juice and findings, stating that the phytochemicals Methanolic Extracts of Citrus sinensis. responsible for plant antibacterial action are Many medications have been connected to the presence of tannins, discovered and designed using natural flavonoids, saponins, phenolic compounds, bioactive compounds derived from plants and essential oils. In contrast to JE, MPPE (Hafidh et al., 2011). Citrus sinensis JE and performed better in all bacterial strains, and MPPE were examined for antimicrobial this is an explanation that MPPE has a greater properties, and the results are displayed in phenolic content than JE (11.24 mg/g and Figures (2&3) and Table (1,2&4). Herbal 8.43 mg/g) respectively. The antibacterial medicine is regarded as a major source for the activity of plant phenolic aromatic discovery of novel antibiotic therapy for the compounds is diverse. Plants that deal with medication of bacterial infection-related microbial infestation produce these disorders. The antibacterial and antifungal chemicals. Their capability to form properties of C. sinensis JE and MPPE were complexes with extracellular and soluble investigated in this study. Citrus sinensis JE proteins, as well as bacterial cell walls, has and MPPE were tested against six bacterial been postulated as a possible explanation for strains (three Gram (+ve) and three Gram (- their action (Dhiman et al., 2012). These ve)) and two fungal strains. The findings of results are consistent with those of Ehigbai et this investigation revealed that the C. sinensis al. (2020), who discovered a significant extracted sample examined displayed content of tannin, flavonoids, and phenols in antibacterial properties versus Gram +ve and fresh C. sinensis peel extract. Gram-ve bacterial strains, but no possible Citrus sinensis peel extracts were antifungal activity. Table 4 and Figures 2&3 also shown to have antibacterial properties by demonstrate the maximum inhibitory El-Desouky et al., 2018; Baba et al., 2018). concentration (MIC) and lowest inhibitory Many biological activities of flavonoids have concentration (MBC) values. The occurrence been discovered, including antibacterial, of significant natural bioactive compounds antioxidant, and anti-inflammatory properties such as tannins, flavonoids, polyphenols, and (Gorniak et al., 2019). Tannins create alkaloids is linked to the antibacterial complexes with proline-rich proteins that stop activities of JE and MPPE in C. sinensis. In cells from making proteins. The synergistic many of the plants investigated, the main activity of alkaloids, flavonoids, tannins, and antioxidants are phenolic substances. saponins is linked to pathogen growth Components that support high antioxidant inhibition (Nwankwo et al., 2014).

6 Reham M. Mostafa* and Heba S. Essawy

Fig. 1: Total Flavonoids, Total Phenolic and Total antioxidant content of JE and MPPE of Citrus sinensis

Table 4: Antimicrobial activities of Citrus sinensis JE and MPPE against some bacterial and fungal strains tested by disc diffusion

Screening and Quantification of Bioactive Compounds and Antimicrobial Activities of Fresh Juice 7

Fig. 2: The agar diffusion method for the antimicrobial activity to Juice (1) and peel and pulp extract (2) against pathogenic bacteria (a) Streptococcus mutans (b) Pseudomona aeruginosa (c) Staphylococcus aureus, (d) Bacillus cereus (e) Salmonellatyphimrium (f) Escherichi coli.

A B

Fig. 3: The agar diffusion method for the antimicrobial activity to Juice (1) and peel and pulp extract (2) against pathogenic (a&b) Aspergillus flavus (b) Candida parapsilosis

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