African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5239

Full Length Research Paper

Antimicrobial and antioxidant activities of Citrus sinensis var. late Valencia fruits at various stages of development

Francis Adu*
  • Francis Adu*
  • Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
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John Antwi Apenteng
  • John Antwi Apenteng
  • Department of Pharmaceutical Sciences, Central University College, Accra, Ghana.
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Noble Kuntworbe
  • Noble Kuntworbe
  • Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
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William Gariba Akanwariwiak
  • William Gariba Akanwariwiak
  • Department of Theoretical and Applied Biology, College of Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
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Theresa Appiah
  • Theresa Appiah
  • Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
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David Ntinagyei Mintah
  • David Ntinagyei Mintah
  • Department of Pharmaceutical Sciences, Central University College, Accra, Ghana.
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  •  Received: 18 August 2016
  •  Published: 21 January 2016

 ABSTRACT

Fruit juice extracts of Citrus sinensis var. late Valencia at different stages of development (3, 6, 10 and 12 months and fallen senescent fruits) were investigated for antimicrobial and antioxidant activities. Antimicrobial activity was determined using a modified Kirby-Bauer agar diffusion method and minimum inhibitory concentrations (MIC) were determined by the micro broth dilution method against strains of Bacillus subtilis NCTC 10073, Candida albicans ATCC 10231, Escherichia coli ATCC 25922, Proteus vulgaris NCTC 4175, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923. Antioxidant activity was evaluated by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging method using N-propyl gallate as standard antioxidant. IC50 values were then determined. Results revealed that the fruit juice extracts demonstrated broad spectrum antibacterial as well as antifungal activity with MIC values ranging from 8.00 to 20.00%, 16.00 to 28.00% , 24.00 to 32.00%, 28.00 to 40.00% and 32.00 to 44.00% v/v for 3, 6, 10, 12 months fruits as well as fallen fruits senescence, respectively. The antimicrobial activity was observed to decrease with increasing age of the fruits. The fruit juice extracts also demonstrated antioxidant activity with IC50 values of 0.4424, 0.6841, 7.357, 12.65 and 41.65% v/v for 3, 6, 10, 12 months and fallen fruits senescence, respectively. The antioxidant activity was also observed to decrease with increasing fruit age. 
 
Key words: Citrus sinensis, free radical, antimicrobial, antioxidant, various stages of development.
 


 INTRODUCTION

The problem of bacterial resistance to antibiotics has necessitated  the  need  for  a  continual  search  for  new antimicrobial compounds (Sibanda and Okoh, 2002). The search for new antibiotics is  usually  difficult  considering the number and nature of mechanisms and factors associated with resistance (Stewart and Costerton, 2001). Medicinal plants continue to provide new and important lead compounds against various pharmacological targets including cancer, HIV/AIDS, Alzheimer's disease, malaria and pain (Balunas and Kinghorn, 2005).
 
Citrus fruits contain many nutritional, therapeutic and pharmaceutical qualities which include antioxidant, anti-tumour and antimicrobial properties (Cotelle et al., 1996). The benefits of citrus fruits is from their wide content of bioactive compounds such as ascorbic acid, flavonoids, phenolic compounds and pectins which are important for human nutrition (Cheruvanky, 2004; Fernandez-Lopez et al., 2005; Jayaprakasha et al., 2008; Ebrahimzadeh et al., 2008). Studies have shown that phytonutrients such as citrus flavanones, polyphenols, anthocyanins and hydroxycinnamic acids are beneficial to human health. Studies have also shown that the fruit, which contains hesperidin, possess anti-inflammatory and some anti-hypertensive properties (Stavric, 1993; Elangovan et al., 1994).
 
The above constituents of citrus fruits give protection against cancer-causing free radicals, boost immune system function and reduce the risk of death associated with cardiovascular diseases (Harats et al., 1998; Rauf et al., 2014). Research conducted by Rauf et al, (2014) indicates that Citrus sinensis exhibits potent antioxidant potentials. Oranges also contain the polyphenol gallic acid which exerts anti-allergic, antihistamine, anti-inflammatory and anti-carcinogenic properties (Elangovan et al., 1994).
 
Citrus fruit juices, peels as well as essential oils are known to possess antimicrobial properties and hence are incorporated into some topical formulations for the management of infections (Pandey et al., 2011; Al-Ani et al., 2010). Studies conducted by Bocco et al. (1998), have however shown that the vitamin C content of the citrus fruits, tends to decrease as the fruit matures. The acid content has also been known to decrease as fruits mature (Sinclair and Ramsey, 1994). It is therefore important to know if fruit maturity and its resultant reduction in acidity will have any effect on the antioxidant and antimicrobial activity of the citrus fruits. This study therefore seeks to investigate the antimicrobial and antioxidant activity of C. sinensis (Late Valencia variety) at its various developmental stages.


 MATERIALS AND METHODS

Sample collection
 
C. sinensis (Late Valencia variety) was identified and the fruits at various stages of development (3, 6, 10, 12 months and fallen senescent fruits) were collected from different localities (Kwame Nkrumah University of Science and Technology (KNUST) Faculty of Agriculture,   Horticulture   Department,   Citrus   Farmland;    Crops Research Institute, Citrus Division, Kwadaso and Fumesua) in the Ashanti region of Ghana. The collected C. sinensis samples were authenticated by Mr. Paul Yaw Agyei, Senior Lecturer and Head of the Pomology Section, Horticulture Department, KNUST.
 
Preparation of fruit juice extracts
 
The Valencia orange fruits at the various stages were each washed thoroughly with running tap water and rinsed with distilled water and then blotted dry using absorbent cotton wool. The rind was removed, and then sliced into pieces. The rind were removed, and then sliced into pieces. The seeds were then removed. They were then blended to form a suspension. The suspension was filtered using a filter paper. A volume of 100 mL fruit juice was obtained for each stage of fruit development. Fruit juices obtained were kept refrigerated at 4°C and used within 48 h.
 
Test microorganisms
 
The test organisms employed in the determination of the antimicrobial activity of the C. sinensis fruit juice extracts were, Gram-positive bacteria (Bacillus subtilis NCTC 10073 and Staphylococcus aureus ATCC 25923), Gram-negative bacteria (Escherichia coli ATCC 25922, Proteus vulgaris NCTC 4175 and Pseudomonas aeruginosa ATCC 27853) and Candida albicans ATCC 1023.
 
Determination of antimicrobial activity
 
The antimicrobial activities of C. sinensis fruit juice extracts were determined using a modified Kirby-Bauer agar well diffusion method as described by Adu et al. (2014). Petri dishes containing 20 mL Muller-Hinton Agar (Sigma-Aldrich, St Louis, MO, USA), were poured and allowed to set. Overnight cultures of the test organisms grown at 37°C in Muller-Hinton Broth (Sigma-Aldrich, St Louis, MO, USA) and diluted to 0.5 McFarland standards with normal saline were used for the tests. Aliquots (10 μL) of the bacterial culture was spread over the surface of the agar and allowed to dry for 10 min. Five wells were made in the agar using a 5 mm cork-borer. Four of the wells were filled with 100 μL of the various concentrations (12.5, 25, 50 and 100%v/v) of juice. The fifth well was filled with either tetracycline (10 μg/mL) or ketoconazole (10 μg/mL) as control for bacteria and C. albicans, respectively.
 
The plates were allowed to stay on the bench for an hour to allow effective diffusion of the extract and then incubated at 37°C for 24 h. The procedure was repeated for the various fruit juice extracts. The antibacterial activity against each test organism was quantified by determining the mean diameter of zone of growth inhibition after 24 h incubation.’ The experiments were done in replicates to ensure consistency.
 
Determination of minimum inhibitory concentration (MIC)
 
The minimum inhibitory concentrations of the fruit juice extracts were determined by the micro broth dilution method using 96 well micro-titre plates as described by Wiegand et al. (2008). The micro-titre plates were filled with 100 µL of double strength nutrient broth. Different concentrations of each juice extracts (3, 6, 10, 12 months and fallen senescent fruits) were prepared and tested against the micro-organisms. The micro-titre plates were then incubated at 37°C for 24 h. The MIC was detected as the lowest concentration of extract that inhibited microbial growth. This was indicated by the absence of purple colouration upon the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) to the micro-titre tubes after the 24 h incubation period.
 
Determination of antioxidant activity
 
The antioxidant activity was performed using the 2,2-diphenyl-1-picryl hydrazyl (DPPH) assay method as described by Annan et al. (2009). A 40 μg/mL solution of DPPH was prepared in methanol and stored away from light. Serial dilutions of the juice extract were made in methanol to obtain concentrations of 2.5, 5, 25, 50% v/v. A volume of 1 mL of each of the four concentrations of the extract was added to 3 mL of 40 μg/mL DPPH. Methanol containing 40 μg/mL of DPPH was used as blank. The tubes were incubated in the dark at 25°C for 30 min and their absorbance read at 517 nm on a Thermo Spectronic UV spectrophotometer. The free radical scavenging activity was observed by bleaching of the colour of DPPH solution from violet to light yellow. The procedure was repeated for N-propyl gallate solutions (1.0, 3.0, 10.0 and 30 µg/mL) as reference standard. All the tests were done in replicates. The IC50 values were then obtained for the various juice extracts.
 
Statistical analysis and data evaluation
 
Results were analysed and plotted using Graph Pad prism version 5 for windows (GraphPad software, San Diego, CA, USA).
 
IC50 values were obtained from graph pad prism by a non-linear relation of log concentration against percentage free radical scavenging activity. Zones of microbial growth inhibition were obtained by subtracting the cork borer diameter (5 mm) from the diameter of the zone after 24 h incubation. 


 RESULTS

Antimicrobial activity  
 
The fruit juice extracts of C. sinensis at the various stages of development exhibited antimicrobial activity against all test organisms used (Table 1). The minimum inhibitory concentrations (MIC) against the various test organisms are shown in Table 2. The 3 months fruit juice extracts demonstrated potent antimicrobial activity, while the fallen fruit senescence demonstrated the least. The antimicrobial activity of C. sinensis fruit juice extracts was observed to decrease as the fruits aged.
 
Antioxidant activity
 
The IC50 indicates the concentration of an agent which scavenges 50% of free radicals, thus the lower the IC50, the more potent the antioxidant activity. The results obtained clearly indicate that the antioxidant activity of the fruit juices decreased as the fruits aged. Table 3 and Figure 1 indicate the scavenging activities of the fruit juice extracts at different concentrations.
 
 
 
 
 
 


 DISCUSSION

The study shows that C. sinensis fruit juice at all stages of development has antimicrobial activity. The fruit juice extracts exhibited  broad  spectrum  antimicrobial  activity against Gram positive bacteria, Gram negative bacteria and fungi. Other researchers have found that bioactive compounds such as flavones, flavonoids and flavonols found in citrus fruits are known to exert antimicrobial activity and are usually synthesized by citrus plants in response to microbial infection (Dixon et al., 1983).
 
The antimicrobial activity was also observed to decrease with increasing age of the fruits. S. aureus was consistently the most sensitive organism showing the least MICs at all the stages of development except at 6 months old where P. vulgaris exhibited the smallest MIC. P. aeruginosa was the most resistant organism for all stages of development.
 
The minimum inhibitory concentrations (MIC) of the various fruit juice extracts ranged from 8.00 to 20.00% v/v for the 3 months, 16.00 to 28.00% v/v for the 6 months, 24.00 to 32.00% v/v for the 10 months, 28.00 to 40.00% v/v for the 12 months old and 32.00 to 44.00% v/v for the fallen senescent fruit juice extract. This indicates that all the extracts especially in their pure state (100% v/v) were active against the test microorganisms. The 3 months old fruits recorded the lowest MIC for all the test organisms, followed by the 6, 10, 12 months old and fallen senescent fruits. The results obtained show that whatever may be responsible for the antimicrobial activity showed up in high concentrations in young fruits but breakdown or are converted to other substances as the fruit ages. According to a study carried out by Sinclair and Ramsey (1994), this effect is probably due to the accumulation of acids in young orange fruits, which decreases as the fruits mature. Studies conducted by Rasmussen (1964), has shown that total citric acid content per fruit in Valencia oranges decline by at least two-thirds as the fruit became thoroughly mature due to the accumulation of water which causes a dilution effect resulting in an increase in the fruit size. High acidity may result in low pH which is inhibitory to many organisms, therefore, as the fruits mature and the acidity declines, inhibitory activity against organisms also decline.
 
DPPH free radical scavenging activity is one of the methods commonly employed to determine antioxidant activity. The IC50, which is a measure of antioxidant potency is the concentration required to scavenge 50% of available free radicals. From Figure 1 and Table 3, it is evident that the amount of the DPPH scavenged by the 3 months old fruit extracts was high with an IC50 value of 0.4424% v/v. This activity decreased consistently as the fruit aged making the senescent fruits produce the weakest antioxidant activity with a high IC50 value of 41.650% v/v. The results show that whatever phyto-constituents were responsible for the antioxidant activity, either declined in concentration due to metabolism into other inactive compounds increased amounts of water with age or changed to inactive conformations as the fruits aged.
 
The presence of antioxidant phytochemicals in the Valencia orange juice’s extracts (flavonoids,quercetin and isoflavones) as well as ascorbic acid (vitamin C) are possibly responsible for the free radical scavenging activities exhibited by the extracts (Rauf et al., 2014). The above findings could be attributed to the reduction of vitamin C levels as well as other bioactive constituents as fruits age. According to Bocco et al. (1998), bioactive constituents such as flavonoids and flavones in citrus fruits tend to decrease as the fruits age. The reduction in antioxidant activity as the fruits aged could possibly be attributed to a reduction in vitamin C levels. Studies have also revealed that, lemons which contain high percentages of acid concentrations as compared to other members of the citrus family, demonstrated high free radical scavenging activities (Spada et al., 2008; Sinclair and Ramsey, 1994). This therefore implies that a possible reduction in acid content as the fruits aged due to the accumulation of water  (Rasmussen,  1964),  could have resulted in both a reduction in antimicrobial and antioxidant activity.
 
The results of this study may hold true for other medicinal plants. Where the fruits are the plant parts to be used, the stage of development at which it is employed should be a critical point. Studies should be carried out to determine at which stage the fruit will be best for the condition being managed and the same time being least toxic since if the fruits has some level of toxicity it may also vary with the stage of development.


 CONCLUSION

Late Valencia orange fruit juice at different stages of development exhibit antioxidant and broad spectrum antimicrobial  activities.  Both  activities  decrease  as  the fruits age with the 3 and 6 months old fruits showing higher antimicrobial and antioxidant activities than the 10 and 12 months old and the fallen senescent fruits. 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The authors acknowledge the technical staff of the Department of Pharmaceutics (Microbiology Section), KNUST, for their technical support.



 REFERENCES

Adu F, Sam GH, Agyare C, Apenteng JA, Boamah VE, Annan K, Ntinagyei DM (2014). The Effects of the Methanol Extracts of Myristica fragrans (Myristicaceae) Fruits and Leaves on the Antibacterial Activities of Ciprofloxacin, Tetracycline, Erythromycin and Amoxicillin. Afr. J. Microbiol. Res. 8(19):1982-1986.
Crossref

 

Al-Ani WN, Al-Haliem SM, Tawfik NO (2010). Evaluation of antibacterial activity of citrus juice. An in-vitro study. Al-Rafidain Dent. J. 10(2):376-382.

 
 

Annan K, Gbedema S, Adu F (2009). Antibacterial and radical scavenging activity of fatty acids from Paullinia pinnata L. Pharmacogn. Mag. 4(Suppl):119-123.

 
 

Balunas JM, Kinghorn DA (2005). Drug discovery from medicinal plants. Life Sci. 78(5):431-441.
Crossref

 
 

Bocco A, Cuvelier ME, Richard H, Berset C (1998). Antioxidant activity and phenolic composition of citrus peel and seed extracts. J. Agric. Food. Chem. 46:2123-2129.
Crossref

 
 

Cheruvanky H (2004). Method for treating hepercholesterolemia, hyperlipidemia and artherosclerosis. United. Stat. Path. 6(4):733-799.

 
 

Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet J C, Gaydou EM (1996). Antioxidant properties of hydroxylflavones. Free. Radic. Biol. Med. 20(1):35-43.
Crossref

 
 

Dixon RA, Dey PM, Lamb CJ (1983). Phytoalexins: Enzymology and molecular biology. Adv. Enzymol. Relat. Areas. Mol. Biol. 55:1-69.
Crossref

 
 

Ebrahimzadeh MA, Hosseinimehr SJ, Hamidinia A, Jafari M (2008). Antioxidant and free radical scavenging activity of Feijoa sallowiana fruits peel and leaves. Pharmacology online 1:7-14.

 
 

Elangovan V, Sekar N, Govindasamy S (1994). Chemo-protective potential of dietary bioflavonoids against 20-methylcholanthrene- induced tumorigenesis. Cancer Lett. 87:107-113.
Crossref

 
 

Fernandez-Lopez J, Zhi N, Aleson-Carbonell L, Perez-Alvarez JA, Kuri V (2005). Antioxidant and antibacterial activities of natural extracts: application in beef meatballs. Meat Sci. 69:371-380.
Crossref

 
 

Harats D, Chevion S, Nahir M, Norman Y, Sagee O, Berry B (1998). Citrus fruit supplementation reduces lipoprotein oxidation in young men ingesting a diet high in saturated fat: presumptive evidence for an interaction between vitamins C and E in vivo. Am. J. Clin. Nutr. 67:240-245.

 
 

Jayaprakasha GK, Girennavar B, Patil BS (2008). Radical scavenging activities of Rio Red grapefruits and Sour orange fruit extracts in different in vitro model systems. Bioresour. Technol. 99(10):4484-4494.
Crossref

 
 

Pandey A, Kaushik A, Tiwari K (2011). Evaluation of antimicrobial activity and phytochemical analysis of Citrus limon. J. Pharm. Biomed. Sci. 13(17):1-5.

 
 

Rasmussen GK (1964). Seasonal changes in the organic content of Valencia orange fruit in Florida. J. Am. Soc. Hortic. Sci. 84:181-187.

 
 

Rauf A, Uddin G, Ali J (2014). Phytochemical analysis and radical scavenging profile of juices of Citrus sinensis, Citrus anrantifolia and Citrus limonum. Org. Med. Chem. Lett. 4:5.
Crossref

 
 

Sibanda T, Okoh AI (2002). The challenges of overcoming antibiotic resistance: Plant extracts as potential sources of antimicrobial and resistance modifying agents. Afr. J. Biotechnol. 6(25):2886-2896.

 
 

Sinclair WB, Ramsey RC (1994). Changes in the organic acid content of Valencia oranges during development. Bot. Gaz. 106:140-148.
Crossref

 
 

Spada PDS, De Souza GN, Bortolini GV, Henriques JAP, Salvador M (2008). Antioxidant, and antimutagenic activity of frozen fruits. J. Med. Food. 11:144-151.
Crossref

 
 

Stavric B (1993). Antimutagens and anticarcinogens in foods. Food Chem. Toxicol. 32:79-90.
Crossref

 
 

Stewart PS, Costerton JW (2001). Antibiotic resistance in bacterial biofilms. Lancet 358(9276):135-138.
Crossref

 
 

Wiegand I, Hilpert K, Hancock RW (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration of antimicrobial substances. Nat. Protoc. 3:163-175.
Crossref

 

 




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