PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF DIFFERENT CITRUS FRUIT PEELS

PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF DIFFERENT CITRUS FRUIT PEELS

Swati Gupta, Adi Nath, Mahesh Kumar Gupta and Shanthy Sundaram *

Centres of Biotechnology, Department of Chemistry, University of Allahabad, Prayagraj, Uttar Pradesh, India.

ABSTRACT: Citrus fruit is a very rich source of Vitamin C, A, E, and flavonoids, alkaloids, and minerals. Citrus fruits are medicinal plants that present rutaceae family and include some fruits such as orange (Citrus sinensis), kinnow (Citrus reticulata), mosambi (Citrua ltmetta), grapefruit (Citrus maxima) lime, pomelo lemon. Antibacterial antiviral, antifungal activity of different solvent extracts (Acetone, ethanol, methanol, and distilled water) prepared by soxhlet extractor and aqueous solvent from three citrus fruit peel orange, kinnow, mosambi (Citrus sinensis, Citrus reticulata, and Citrus limetta respectively) were screened against four pathogenic bacteria staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi. The highest antibacterial activity was exhibited by the acetone peel and ethanol extract of Citrus reticulate peels. MICs were tested at concentrations ranging from 80-5.25 mg/mL as wells as their MBCs. The phytochemical analysis of the citrus peel extracts showed the presence of flavonoids, saponins, steroids, terpenoids, tannins and alkaloids.

Keywords: Phytochemical, Antibacterial, Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC)

INTRODUCTION: Citrus fruit is the most abundant fruit that grows worldwide and contains very rich amounts of phytochemicals and bioactive compound ¹. Citrus fruit is a medicinal plant belonging to the Rutaceae family. They are a rich source of Vitamin C, A, E alkaloids and flavonoids, and other minerals. Citrus plants originated from tropical, subtropical and East Asia and it is consumed all over world as an rich source of Vitamin C and other minerals and good source of vitamin A and contains a powerful natural antioxidant antiviral, antifungal and antibacterial activity that builds the strong body immune system.

The peel of different citrus fruit is most abundant source of phenolics content, flavonoids, flavones, flavanones content and many polyethoxylated flavones, which are very rare in other plants ². Citrus fruit peels are recognized as being a healthful source of bioactive compounds polyphenols, dietary fibre, essential phenolics, and ascorbic acid ³.

The phytochemicals are also known as secondary metabolites, which include many types of useful secondary metabolites such as alkaloids, flavonoids, tannins, saponin, Coumarins, carotenoids etc., which are derived from primary metabolites and are used as drugs ^{4, 1}. Phytochemical analyses of citrus fruit peels are very useful in the evaluation of some biologically active compounds in some vegetables and medicinal plants. Medically, the presence of phytochemicals explains the use of the secondary compound in a different area of medical, where used as a beneficial drug.

These secondary components of citrus fruit peels are extractable by various solvents that showed varied biological, biochemical and pharmacological actions in human beings. The citrus peels are a good and promising source of Vitamin C, minerals, fiber and different nutrients, and contain many rich amounts of phytochemical metabolites. Citrus plant can be used as a beneficial drug, food supplement in the food industry and Cosmetic industries and many different areas ^{5, 6}. Citrus peels enhance the antimicrobial activity of harmful bacteria, fungi, and viruses. Citrus fruits are rich sources of useful phytochemicals, such as vitamins A, C, and E, mineral elements, flavonoids, coumarins, limonoids, carotenoids, pectins and other compounds ⁷. These phytochemicals, consumed through fresh fruits or their derived products, have been suggested to have a wide variety of biological functions, including antioxidant, anti inflammatory, anti-mutagenicity, anti-carcinogenicity, and anti-aging ^{5, 8, 9, 10}.

MATERIALS AND METHODS:

Collection of the Plant Material: Plant material of citrus spp. fruit peels used in this study. Citrus spp. fruit were collected from the local fruit market of Allahabad from India then after, the peels were dried at room temperature (40 ºC) for 8 days. The Plant was authenticated by the scientist of the Botanical Survey of India (BSI), Prayagraj. After that 20 g of dried citrus peels were crushed by electric blender. The dried citrus peels were collected in an air-tight closed container.

Preparation of Solvent Extract:

Extract Prepared by Soxhlet Method: The dried and powdered of citrus peels extract (10 g) were dissolved in 100 ml of each different solvent and separated by soxhlet extractor for 6 hours at a room temperature ¹¹. The solvents acetone, ethanol, methanol and distilled water were used for the study. The extracts were filtered by Whatman filter paper no. 1 and then concentrated to dryness. After that, each different extract was stored in airtight container and kept in a cold place before use.

Aqueous Extraction: 15 g of the dried citrus peels powder soaked separately in 100 ml of distilled water at room temperature for 18 hours under shaking conditions at 120 rpm. After that extract was filtered by Whatman filter paper No 1 and extracts were stored to airtight container and kept in cold place for future use.

Phytochemical Analysis: The dried extracts of citrus fruit peels of the different extracts are a preliminary estimation of phytochemical ¹².

Alkaloids Test: 1 ml acidic and aqueous extract was mixed with few drops of Mayers reagent and add 0.1% HCl. A yellow color showed the presence of alkaloids.

Flavonoids Test: 1 ml of extract was mixed with conc HCl and adds magnesium Ribbon. The pink-red color showed the presence of flavonoids.

Saponins Test: Take 2 ml filtrate was added to 10 ml of sterile distilled water in a test tube. The test tube was stopped and shaken vigorously for about 30 seconds. It was then allowed to stand for half an hour. Honeycomb Froth indicated the presence of saponins.

Tannins Test: 1ml extract was mixed with 0.1% Ferric chloride. The brownish-green indicated the presence of tannins.

Steroids Test: 1 ml of extract was added to 2 ml acetic anhydride with add 1.5 ml sulfuric acid the violet to a blue colour indicating the presence of steroids.

Amino Acid Test: Take 3 ml of extract was mixed with 4 -5 drops of ninhydrin reagent. The purple color showed the presence of amino acids.

Reducing Sugar Test: 2 ml of extract add to 0.5 ml of both A and B Fehling's solution after that mixture was heated in a water bath for 30 min. The brick red colour indicates the presence of reducing sugar.

Terpenoids Test: 1 ml of extract was mixed with 0.5 ml sulfuric acid and adds 0.5 ml of chloroform. A reddish brown colour was formed to show the presence of terpenoids.

Cardiac Glycosides Test: 4 ml of each extract was mixed with glacial acetic acid and 1 ml of conc. sulphuric acid adds few drops of ferric chloride a brown color ring indicates the presence of cardiac glycosides.

Antraquinones Test: 2 ml of the extract was mixed with diluted conc. sulfuric acid and added 1 ml of dilute ammoniarose - pink color showed the presence of anthraquinones.

In-vitro Antimicrobial Screening: Antibacterial activity analysis by agar well diffusion method and disk diffusion method. The four bacteria of Gram-positive and Gram-negative (S. aureus, B. subtilis and E. coli, and S. typhi) were cultured in 24 hr, and fungi C. albicans, Aspergillus niger were prepared on Mueller-Hinton agar plates and Sabouraud dextrose agar (SDA). Where citrus extract C. sinensis C. reticulata C. limetta tested against two positive B. subtilis, S. aureus bacteria and two gram negative E. coli, S. typhi bacteria and fungi C. albicans, Aspergillus niger. These plates were incubated at 34 °C for 24 hours in an upright position, and the zone of inhibition formed around the wells was measured.

Minimum Inhibitory Concentration: The determination of minimum inhibitory concentration (MIC) by the lowest concentration of different citrus fruit extracts. Which essential to inhibit the multiplication of the organism is referred to as ^{13 14}. Here “Serial dilution technique” was followed using nutrient broth media. The four test bacteria grown at 34 °C in a nutrient broth medium for 24 hr. Here five different concentrations (80, 40, 20, 10, 5 mg/mL) of extracts are used for determination of MIC. 1 ml of nutrient broth medium was transferred to each of the test tubes. Next the test tubes were cotton plugged and sterilized in an autoclave for 20 minutes at 121 °C. After cooling, 1 ml of the sample solution was added to the 1st test tube and mixed well and then 1 ml of this content was shifted to the 2^nd test tube. The content of the 2^nd test tube was shaken well and then again, 1 ml of this mixture was moved to the third test tube. The entire process of serial dilution was carried up to the 5^th test tube. Finally, 1 ml of the sample was added to each of the test tubes and all the test tubes were incubated at 37 °C for 20 h.

Minimum Bactericidal Concentration: The determination of minimum bactericidal concentration (MBC) ¹⁵ for the study of each well in the MIC determination after that Inoculated test bacteria in Nutrient agar plates only use for as a control. The nutrient agar plates were incubated at 36 ºC for 18 h. After incubation, agar plates did not show any growth of organisms and were noted as a minimum bactericidal concentration.

Statistical Analysis: All the experiments were carried out in triplicate. One-way analysis of variance was applied to confirm the significance of data (p<0.05). Comparison with control and treatment’s means was carried out by using ANOVA by Dunnett’s multiple comparison tests and Duncan’s Multiple Range Test (DMRT) as per required. The principal component analysis was performed with PAST software (Version 2.17).

RESULTS AND DISCUSSION:

Extract of Citrus Fruit Peels: The extract of the citrus peel was carried out using different solvents such as ethanol, methanol, acetone and distilled water. Fig. 1, exhibited the comparison of yield of citrus peels extracts obtained from different solvents.

For acetone and distilled water extract, Citrus sinensis exhibited a good yield and the methanol extract showing the slowest percentage yield. Methanol and ethanol extract of citrus reticulate peel showed good yield. Acetone and distilled water extract of Citrus limetta showed good yield when compared to the methanol and ethanol extract. The aqueous, methanol, and Acetone extract exhibited a good yield of citrus fruit peels extract. So the variation of yield showed the solubility of extract in different solvents.

FIG. 1: YIELD (%) OF EXTRACT OF CITRUS FRUIT PEELS BY USING DIFFERENT SOLVENTS

Phytochemical Screening of Citrus Fruit Peels Extract: The phytochemical analysis of the various solvent extract showed the presence of secondary metabolites such as alkaloids, flavonoids, saponin, tannins, starch and terpenoids ^16-18.

Phytochemical Analysis of Different Solvent Extract of Citrus Fruit Peel:

TABLE 1: PHYTOCHEMICAL ANALYSIS

TABLE 2: QUANTITATE ANALYSIS (g/100 g.)

TABLE 3: DETERMINATION OF ANTIMICROBIAL ACTIVITY OF CITRUS FRUIT PEEL EXTRACT FROM DIFFERENT SOLVENT BY USING DISC DIFFUSION METHOD

Antimicrobial Activity of Citrus Fruit Peels: Citrus peel extracts showed a good antibacterial, antifungal, and antiviral activity against all different pathogenic bacteria. C. sinensis and C. reticulata peel extracts exhibited a good antibacterial and antifungal activity. Ethanol extract of Citrus sinensis showed a maximum zone of inhibition against bacteria  S. aureus (13mm), B. subtilis (18mm), S. typhi (11mm),  and showed antifungal activity in C. albicans (14mm), A. niger (16mm) acetone extract of Citrus reticulata exhibited maximum zone of inhibition against bacteria E. coli (25mm), S. typhi (18mm) followed by fungi A. niger (10mm) acetone and ethanol extract of citrus reticulate showed good activity against C. albicans (10mm), A. niger (16mm) and acetone extract of Citrus limetta exhibited zone of inhibition against bacteria E. coli (18mm) and fungi C. albicans (11mm) and ethanol extract showed A. niger (12mm) whereas the methanol and aqueous extract of Citrus sinensis, Citrus reticulate and Citrus limetta did not show such high antibacterial and antifungal activity ^19-21. In the case of Citrus limetta, Citrus sinensis, Citrus reticulata methanol and aqueous showed very less the same antibacterial activity and antifungal activity when compared to other solvents. So different extracts may have shown diverse antimicrobial agents that the bacterium was contained special metabolism to maintain its activity. Acetone and ethanol were showed a better solvent for the extraction of antibacterial and antifungal agents, which shown better higher yield of antibacterial and antifungal  activity relating to the high concentration of various phytochemicals, and so citrus peels showed high antibacterial and antifungal activity. This statement can be validated as acetone and ethanol has shown antibacterial and antifungal activity in Citrus sinensis and Citrus reticulate ^22-23. The determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of different solvent extracts of C. sinensis, C. reticulata, and C. limetta peels are shown in Table 4 and Table 5, respectively. The extracts showed significant activity. MIC activity detects by the nutrient broth dilution method, which showed excellent results. The broth method, carried out in a test tube, has the advantage of lower workloads for a larger number of replicates, and Lack of activity can thus only be proven by using large doses ^24-25.

TABLE 4: DETERMINATION OF MIC (MINIMUM INHIBITORY CONCENTRATION) OF DIFFERENT CITRUS PEEL EXTRACT AGAINST PATHOGENIC BACTERIA

Concentration- 40 mg/mL and (-); No Inhibition Zone

TABLE 5: DETERMINATION OF MBC (MINIMUM BACTERICIDAL CONCENTRATION) OF DIFFERENT CITRUS PEEL EXTRACT AGAINST PATHOGENIC BACTERIA

Principle Component Analysis: Protein, Moisture, Fiber, Calcium, Phosphorus, Energy, and carbo-hydrate contents were further analyzed via multi-variate analysis by principal component analysis (PCA) to determine principle component in each genus and their strains. PCA clearly resolved the data into two major components viz. MIC and MBC for all eight components in each three strains of Citrus. A maximum of 79.17%, 31.8%, 29.78% and 19.9% of the variance was recorded for Energy Calcium, phosphorus content, and carbohydrate content of these Citrus strains Fig. 2. PCA scores were determined the highest producing attributes of citrus strains ²⁶ of the Citrus limetta for efficient carbohydrate and protein accumulation. PCA was also used in multifactor analysis to easily analyze the probability area of productivity ²⁶.

FIG. 2: PRINCIPLE COMPONENT ANALYSES (PCA)

CONCLUSION: Re-use of peels of citrus waste which are very important innovative ways where produce the higher amount of beneficial, essential, a healthy new product in a very low cost, utilization of citrus fruit peels waste can be used in multiple areas such as food industry where the use of fruit waste peels as a supplement and can be use waste of peels in the cosmetic industry for make a beauty product in a very low price and citrus peels also use as drugs which are very effective against the pathogenic bacteria, fungi and virus apart from that citrus peels enhance the immunity of human being because it is a natural source of vitamin C. which protect human body to harm full disease. Citrus peels rich in nutrients which beneficial and required for human, animal, and plant nutrition. So it is used in the pharmaceutical. This work has identified the antibacterial, antiviral, antifungal, and antioxidant activity of citrus peels. Which act against the disease phytochemical and bioactive compounds in Citrus limetta, Citrus reticulate, and Citrus sinensis peels extracts obtained from different solvents. This study showed that dried fruit peels of different citrus fruit are rich sources of antioxidant compound and antibacterial activity, and the exploitation of these abundant and low-cost renewable resources could be anticipated for the pharmaceutical and food industry with opportunities of developing new ingredient for the formulation of functional food products and/ or pharmaceutical products.

ACKNOWLEDGEMENT: Swati Gupta is thankful to UGC New Delhi, India, for providing him UGC-D.Phil. research fellowship.

Author’s Contributions: Shanthy Sundaram and Swati Gupta designed the experiments; Swati Gupta performed all the experiments. Adi Nath and Mahesh Gupta helped Swati Gupta in manuscript drafting. Shanthy Sundaram reviewed the manuscript.

CONFLICTS OF INTEREST: The authors declare no conflict of interest.

REFERENCES:

1. Negi PS: Plant extracts for the control of bacterial growth: efficacy, stability and safety issues for food application. Int J Food Microbiol 2012; 156: 7-17.
2. Hayat K, Hussain S and Abbas S: Optimized microwave-assisted extraction of phenolic acids from citrus mandarin peels and evaluation of antioxidant activity in-vitro. Sep Purif Technol 2009; 70: 63-70.
3. Inga K, Malecka M, Szlachta M and Gliszczynska-Swiglo A: Effect of storage on the content of polyphenols, vitamin C and the antioxidant activity of orange juices. J Food Composition Anal 2007; 20: 313-22.
4. Wang Y, Chuang Y and Hsu H: The flavonoid, carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chem 2008; 106: 277-84.
5. Negi PS and Jayaprakasha GK: Antibacterial activity of grapefruit (Citrus paradisi) peel extracts. Eur Food Resour Technol 2001; 213: 484-87.
6. Moo-huchin VM, Moo-huchin MI and Estrada-león RJ: Antioxidant compounds, antioxidant activity and phenolic content in peel from three tropical fruits from yucatan, mexico. Food Chem 2014; 166: 17-22.
7. Goulas V and Mangaris GA: Exploring the phytochemical content and the antioxidant potential of citrus fruits grown in cyprus. Food Chem 2012; 131: 39-47.
8. Rajendran P, Nandakumar N and Rengarajan T: Antioxidants and human diseases. Clin Chim Acta 2014; 436: 332-47.
9. Ya-Jiao K, Xiang-Qun Z, Heng G, Yue M and Zhao-Hua C: Low field induced giant anisotropic magnetocaloric effect in DyFeO₃ single crystal. Chinese Phys B 2015; 24: 037501.
10. Zou Z, Xi W, Hu Y, Nie C and Zhou Z: Antioxidant activity of citrus fruits. Food Chem 2016; 196: 885-96.
11. Lin J, Opoku AR, Geheeb-keller M, Hutchings AD, Terblanche SE and van Staden J: Preliminary screening of some traditional zulu medicinal plants for anti-inflammatory and anti-microbial activities. J Ethnopharmacol 1999; 68: 267-74.
12. Sofowora A: Research on medicinal plants and traditional medicine in Africa. J Altern Complement Med 1996; 2: 365-72.
13. Andrews JM: Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001; 48: 5-16.
14. Rasooli I and Abyaneh MR: Inhibitory effects of thyme oils on growth and aflatoxin production by Aspergillus parasiticus. Food Control 2004; 15: 479-83.
15. Parvez GMM, Rana MM, Jahan EN and Mosaddik A: Alternation of antimicrobial potential of mango peel and pulp after formalin treatment against six bacteria. J Pharmacogn Phytochem 2016; 5: 158-16.
16. Mehmood B, Dar KK, Ali S, Awan UA, Nayyer AQ, Ghous T and Andleeb S: In-vitro assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis. Pakistan Journal of Pharmaceutical Sciences 2015; 28(1): 231-39.
17. Adham, AA: Phytochemical analysis and evaluation antibacterial activity of Citrus medica peel and juice growing in Kurdistan/Iraq. Journal of Applied Pharmaceutical Sciences 2015; 5(10): 136-41.
18. Wahyuono RA, Hesse J, Hipler UC, Elsner P and Böhm V: In-vitro lipophilic antioxidant capacity, antidiabetic and antibacterial activity of citrus fruits extracts from Aceh, Indonesia. Antioxidants 2017; 6(1): 11.
19. Fratianni F, Cozzolino A, De Feo V, Coppola R, Ombra MN and Nazzaro F: Polyphenols, antioxidant, antibacterial, and biofilm inhibitory activities of peel and pulp of Citrus medica, Citrus bergamia, and Citrus medica cv. Salò Cultivated in Southern Italy. Molecules 2019; 24(24): 4577.
20. Nata’ala MK, Dalhat MH, Omoye BS, Isah AA, Kabiru S, Bashiru I and Umar FA: Phytochemical Screening and Antibacterial Activity of Citrus sinensis (L.) Osbeck [Orange] and Citrus aurantifolia (Cristm.) Swingle [Lime] stem from bacteria associated with dental caries. Journal of Advances in Microbiology 2018; 1-9.
21. Oikeh EI, Oviasogie FE and Omoregie ES: Quantitative phytochemical analysis and antimicrobial activities of fresh and dry ethanol extracts of Citrus sinensis (L.) Osbeck (sweet Orange) peels. Clinical Phytoscience 2020; 6(1): 1-6.
22. Thakur N and Kumari S: Preliminary screening of phytochemicals and antimicrobial activity of Citrus pseudolimon. Advances in Traditional Medicine 2021; 1-11.
23. Khan NH, Qian CJ and Perveen N: Phytochemical screening, antimicrobial and antioxidant activity deter-mination of Citrus maxima Pharmacy & Pharmacology International Journal 2018; 6(4): 279-85.
24. Singh N, Jaiswal J, Tiwari P and Sharma B: Phytochemicals from Juice as Potential Antibacterial Agents. The Open Bioactive Compounds Journal 2020; 8(1).
25. Ekawati ER and Darmanto W: Lemon (Citrus limon) juice has antibacterial potential against diarrhea-causing pathogen. In IOP Conference Series: Earth and Environmental Science 2019; 217(1): 012023.
26. Nath A, Tiwari PK, Rai AK and Sundaram S: Evaluation of carbon capture in competent microalgal consortium for enhanced biomass, lipid, and carbohydrate production. 3 Biotech 2019; 9(11): 1-15.
    

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    How to cite this article:

    Gupta S, Nath A, Gupta MK and Sundaram S: Phytochemical analysis and antibacterial activity of different citrus fruit peels. Int J Pharm Sci & Res 2021; 12(11): 5820-26. doi: 10.13040/IJPSR.0975-8232.12(11).5820-26.

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