Antimicrobial Activity and Phytochemical
Analysis of Citrus Fruit Peels -Utilization of
Fruit Waste
1K. Ashok kumar, 2M. Narayani, 3A. Subanthini and 4M. Jayakumar
1
Department of Biotechnology, 4
Department of Chemical Engineering, Arulmigu Meenakshi Amman College of Engineering,
Vadamavandal (Near Kanchipuram), T.V. Malai District – 604 410, Tamilnadu, India. E-Mail: ashokkumar_kh5@yahoo.com, jaimchem@gmail.com
2
Department of Chemical Engineering,
National Institute of Technology (NIT), Karnataka, India. E-Mail: narayani@ymail.com
3
Department of Biotechnology,
Bannari Amman Institute of Technology, Sathyamangalam – 638 401, Tamilnadu, India. E-Mail: subanthini@gmail.com
Abstract:
Antibacterial activity of five different solvent extracts(ethyl acetate, acetone, ethanol, petroleum ether and water) prepared by soxhlet extractor from two citrus fruit peel (Citrus sinensis and Citrus limon) were screened against five pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli , Klebsiella pneumonia and Salmonella typhi. The highest antibacterial potentiality was exhibited by the acetone peel extract of Citrus sinensis followed by the ethyl acetate peel extract of Citrus limon. The peel extract of Citrus sinensis and Citrus limon can be considered to be as equally potent as the antibiotics, such as metacillin and penicillin. MICs were tested at concentrations ranging from 50-6.25 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: Antibacterial- Soxhlet extractor-Minimum Inhibitory Concentration (MIC) Minimum bactericidal concentration (MBC) – Phytochemicals
1. Introduction
some pathogens [Wilson and Droby (2000); Friedman et al. (2002);Soković et al (2007)]. The food industry has tended to reduce the use of chemical preservatives in their products due to increasing pressure of consumers or legal authorities, to either completely remove or to adopt more natural alternatives for the maintenance or extension of product shelf life [Nychas (1995)].
The peel of Citrus fruit is a rich source of flavanones and many polymethoxylated flavones, which are very rare in other plants [Ahmad et al. (2006)].The antimicrobial abilities of essential oils, among which citrus oils, are also shown to be a particularly interesting field for applications within the food and cosmetic industries [Caccioni et al., (1998)]. It has also been used as an anti-diabetic (Hamendra and Anand (2007)]), antimicrobial [Caccioni et al. (1998)], antifungal [Stange Jr et al. 1993], hypotensive agent [Kumamoto et al. (1986)], antioxidant [Proteggente et al. (2003)]; [Kanaze et al. (2008)], carminative, insect repellent, antibacterial, larvicidal, antiviral, uricosuric, anti-yeast, antihepatotoxic and antimutagenic agent [Han (1998)].
This study was aimed to focus on waste minimization in fruit juice processing industry. The combined efforts of waste minimization during the production process and recovery of valuable product substantially reduce the amount of waste, as well as boost the environmental profile of fruit juice processing industry. This study investigates the antibacterial activity and the fundamental scientific basis for the use of peels of citrus fruits by determining the chemical constituents as well as quantifying the yield percentage of crude phytochemicals.
2. Materials and Methods 2.1. Plant materials
The plants used in this study were Citrus limon (common name: Lemon) and Citrus sinensis (common name: Sweet orange).The peels were collected from the local fruit juice shops. After collection, the peels were shade dried at room temperature (32 - 35ºC) to constant weight over a period of 5 days. 15 g of each of the plant parts were coarsely powdered using a mortar and pestle and were further reduced to powder using an electric blender. The powder was transferred into closed containers.
2.2. Preparation of extracts 2.2.1. Soxhlet extraction:
The dried and powdered plant materials (15 g) were extracted successively with 200 ml of each solvent separately by using soxhlet extractor for 5 h at a temperature not exceeding the boiling point of the Solvent [Lin et al., (1999)]. The solvents used for the study were Ethyl acetate, Petroleum ether Acetone, Ethanol and Water. The extracts were filtered and then concentrated to dryness. Yield of the extract obtained was calculated as follows:
%) = ℎ ℎ )×
Each extract were transferred to glass vials and kept at 4º C before use. The extracts were dissolved in 25% aqueous dimethyl sulfoxide (DMSO) to produce a stock solution of 100 mg ml-1.
2.2.2. Aqueous Extraction
The method of Dupont et al. (2005) was adopted for extraction with little modification. Briefly, 15g of the powdered plant were soaked separately in 200 ml of distilled water at ambient temperature for 24 hour under shaking condition at 130 rpm. The extract was then filtered using Whatman filter paper No 1 .Each extracts were transferred to glass vials and kept at 4 ºC before use.
2.3. In vitro testing of extracts for antimicrobial activity:
2.3.1. Antimicrobial assay:
Overnight cultures of the Gram positive strains S. aureus, B. subtilis and the Gram negative strains E. coli, K. pneumoniae and S. typhi were prepared on nutrient agar plates. All bacterial isolates were suspended in saline to a turbidity equivalent to 0.5 McFarland (1.5 x 108 CFU/ml) and 0.1% standardized inoculum suspension was swabbed uniformly in Mueller Hinton Agar (MHA, pH 7.3 ± 0.1, Difco) obtained from HiMedia, Mumbai, India. Sterile HiMedia paper disc (6mm) were soaked in 20 µl of the extract diluted in 25% DMSO and dried at 37°C overnight. The loaded disc was placed on the surface of medium, the compound was allowed to diffuse and the plates were kept for incubation at 37°C for 24 hrs. Antibiotic discs containing Penicillin, Methicillin and Gentamycin (5-30µg) were used as controls.
2.3.2. Determination of Minimum Inhibitory Concentration (MIC) of the extracts on the test organisms
A sterile 16 well plate was labeled. 100 μL of nutrient broth was added to all the wells. A volume of 100 μL of test material in 10% (v/v) DMSO or sterile water (usually a stock concentration of 100 mg/mL was pipetted into the first row of the plate. Serial dilutions were performed using a micropipette to obtain dilutions: 50mg/ml, 25mg/ml, 12.5mg/ml, and finally 6.25mg/ml. Tips were discarded after use such that each well had 100 μL of the test material in serially descending concentrations. Finally, 10 μL of bacterial suspension (1-2 × 108 cfu/mL) was added to each well .Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Negative controls were set up as follows: Nutrient broth only; Nutrient broth and sterile plant extract; and finally positive control containing Nutrient broth, and a test organism. The plates were prepared in duplicate, and placed in an incubator set at 37 °C for 18–24 h. To each well 10 μL of resazurin indicator solution was added and incubated for another 6-7 h. The color change was then assessed visually. Any color changes from purple to pink or colorless were recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of two values was calculated and that was the MIC for the test material and bacterial strain.
2.3.3. Determination of minimum bactericidal concentration (MBC)
To determine the MBC, for each set of well in the MIC determination(before the addition of resazurin dye), a loopful of broth was collected from those plates well ,which did not show any visible sign of growth and inoculated on sterile nutrient agar by streaking. Nutrient agar plates were streaked with the test organisms only to serve as control. The plates were then incubated at 37ºC for 24 h. After incubation the concentration at which no visible growth was seen was noted as the minimum bactericidal concentration.
2.4. Preliminary phytochemical analysis (Qualitative analysis):
The powered plant parts as well as the extracts were subjected to preliminary phytochemical screening following the methodology of Sofowora (1994), Harborne (1998) and Kokate (2001).
2.4.1. Test for alkoloids: 2 ml filtrate was mixed with 1% HCl and about 6 drops of Mayor’s reagents. A Creamish or pale yellow precipitate indicated the presence of respective alkaloids.
2.4.2. Test for flavonoids: 2 ml filtrate was added to conc. HCl and magnesium ribbon. Pink-tomato red color indicated the presence of flavonoids.
2.4.3. Test for amino acids: 1 ml of the extract was treated with few drops of Ninhydrin reagent. Appearance of purple color shows the presence of amino acids.
2.4.4. Test for tannins: 1 ml of the extract was treated with few drops of 0.1% ferric chloride and observed for brownish green or a blue-black coloration.
2.4.5. Test for phlobatannins: Deposition of a red precipitate when an aqueous extract of each plant sample was boiled with 1% aqueous hydrochloric acid was taken as evidence for the presence of phlobatanins.
2.4.6. Test for anthraquinones (Borntrager’s test): 1 ml of the extract solution was hydrolyzed with diluted Conc. H2SO4 extracted with benzene. 1 ml of dilute ammonia was added to it. Rose pink coloration suggested the positive response for anthraquinones.
2.4.7. Test for saponins: Froth test for saponins was used. 1g of the sample was weighed into a conical flask in which 10ml of sterile distilled water was added and boiled for 5 min. The mixture was filtered and 2.5ml of the filtrate was added to 10ml of sterile distilled water in a test tube. The test tube was stopped and shaken vigorously for about 30 second. It was then allowed to stand for half an hour. Honeycomb froth indicated the presence of saponins.
2.4.8. Test for steroids: 2 ml of acetic anhydride was added to 0.5 g ethanolic extract of each sample with 2 ml H2SO4. The color changed from violet to blue or green in some samples indicating the presence of steroids. 2.4.9. Test for phytosterol: The extract was refluxed with solution of alcoholic potassium hydroxide till complete saponification takes place. The mixture was diluted and extracted with ether. The ether layer was evaporated and the residue was tested for the presence of
2.4.11. Test for terpenoids (Salkowski test):5 ml of each extract was mixed in 2 ml of chloroform, and concentrated H2SO4 (3 ml) was carefully added to form a layer. A reddish brown coloration of the inter face was formed to show positive results for the presence of terpenoids.
2.4.12. Test for cardiac glycosides (Keller-Killani test): 5 ml of each extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 ml of concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.
2.4.13. Test for Chalcones: 2 ml of Ammonium hydroxide was added to 0.5 g ethanolic extract of each sample. Appearance of reddish color showed the presence of chalcones.
3. Results and discussion:
The soxhlet extract of the citrus peel using different solvents yielded different results in each of the experiment conducted in the this study. There existed, a difference in the percentage yield of the extract obtained between various solvents. Figure 1, shows the comparison of the percentage yield of extracts obtained from different solvent with respect to various sources. For Citrus limon extract, the ethyl acetate extract showed the highest yield of about 18% followed by the ethanol extract and the acetone extract showing the least percentage yield. Citrus sinensis peel extract showed highest yield when acetone was used as a solvent with a yield percentage of about 17% followed by ethyl acetate (12%). The aqueous extract of both the citrus peel showed a moderate percentage yield. This variation in yield between ranges of solvents explains that solubility of different plant compound in different solvent.
Citrus peel extracts showed a significant antibacterial activity against all the test organisms. Citrus sinensis peel extracts showed a very good antimicrobial activity when compared to Citrus limon .Acetone extract of Citrus sinensis showed a maximum zone of inhibition against E.coli (16mm) followed by S. typhi (15mm), B.subtilis and K.pnemonia (14mm) and S.aureus (13mm) whereas the ethanol and aqueous extract of Citrus sinensis did not show such high antibacterial activity. This antibacterial activity may be indicative of the presence of metabolic toxins or broad spectrum antibiotic compounds. In case of Citrus limon, ethyl acetate and acetone showed more or less the same antibacterial activity. Aqueous extracts showed very less antibacterial activity when compared to other solvents. Petroleum ether did not show any significant effect against all the tested strains except E.coli. This shows that petroleum ether has the capability of extracting one such antibacterial agent which may be very toxic to E.coli. Thus it indicates that different extracts may have diverse antibacterial agent that has different modes of action or the bacteria may have a special metabolism to overcome or adapt its activity. Ethyl acetate proves to be a good solvent for the extraction of antibacterial agents from both the sources as it has shown better yield as well as antibacterial activity relating that higher yield means high concentration of single or variety of phytochemicals and therefore high antibacterial activity. This statement can be validated as acetone has shown highest yield as well antibacterial activity in Citrus sinensis.
Ekwenye and Edeha (2010) reported antibacterial activity of the ethanol and aqueous extract of Citrus sinensis leaf against the test organisms taken in their study (E.coli: aqueous extract -7mm, ethanol extract -3mm; K.pneumoniae: 3mm for both the extracts; S.aureus: aqueous extract -1mm, ethanol extract-2mm). Comparing their results with our study with the same test organism and solvent shows that Citrus sinensis peel extract(E.coli :aqueous extract -9mm, ethanol extract -8mm; K.pneumoniae : aqueous extract -7mm, ethanol extract - 8mm; S.aureus: aqueous extract -9mm, ethanol extract-7mm) has high degree of antibacterial activity as compared to the leaf extract. However, this difference may be because of the difference in the phytochemical composition in various part of the plant or may be also due to the extraction method used and/or environmental factors or difference in the genotypes of the citrus plant used. The citrus peel extract exhibited similar or higher antibacterial activity as that of the standard antibiotics used in the study. However, gentamycin showed higher activity relatively.
The zone of inhibition for Citrus limon and Citrus sinensis peel oil against E. coli was 14mm and 13mm respectively; S. aureus was 14mm and 12mm respectively; K. pneumonia was 13mm and 11mm respectively [F. Gülay kirbaşlar et al (2009)]. Amandeep et al (2009) reported that Citrus sinensis peel oil showed zone of inhibition of 13mm and 17mm against E.coli and B. subtilis respectively.
use of small volumes of the test substance and growth medium [Soković et al. (2007)].Lack of activity can thus only be proven by using large doses (Farnsworth, 1993).Alternatively, if the active principle is present in large quantities, there could be other constituents exerting antagonistic effects or negating the positive effects of the bioactive agents [Jager et al.,(1996)].
The difference in the antibacterial activity with the same source when extracted with different solvent has proven that not all phytochemicals that are responsible for antibacterial activity are soluble in a single solvent. Hence solvents of different polarity should be employed as discussed in this study (polar: water, acetone, ethanol; non-polar: ethyl acetate, petroleum ether). Sequential or successive solvent extraction is as good option for better solubility of many of the phytochemcials but it is always necessary to know the phytochemicals extracted by each individual solvent so as to avoid the inclusion of unnecessary solvents for extraction process as well as to understand the role of each solvent in the extraction of a individual or class of phytochemicals. With no antibacterial activity, extracts may be active against other bacterial species which were not tested [Shale et al., (1999)].
The medicinal value of these plants lies in bioactive phytochemical constituents that produce definite physiological action on the human body [Akinmoladun et al., (2007)]. The preliminary phytochemcial investigation revealed the presence of various constituents of citrus peels. The results are shown in the table. Different solvent showed different class of phytochemicals .they showed the presence of flavanods, saponins etc. antraquiones were completely absent in both the citrus peels. These constituents could account for the antibacterial activity but it is difficult to correlate their action to a specific phyochemical.
The presence of phenol further indicated that Citrus limon and Citrus sinensis peels could act as anti-inflammatory, anti clotting, antioxidant, immune enhancers and hormone modulators. Citrus lemon and Citrus sinensis peels have high quantity of saponin which has hemolytic activity and cholesterol binding properties .Therefore, in addition to their use as drugs, citrus peels can be used as a food preservative or even as food supplement as many literature says that they are highly nutritive.
Figure 1 Percentage yield of citrus peel extracts
0 2 4 6 8 10 12 14 16 18 20
C. limon C.sineses
Y
ie
ld
(%
)(w/
w)
Various extracts
Ac
EtOAc
EtOH
Pet ether
Table 1. Zone of inhibition (mm) of Citrus limon peel and citrus sinensis peel extract against test bacteria on Mueller-Hinton agar medium
using disc diffusion method
Bacteria
Zone of inhibition(mm)
Citrus peel extracts* Standard antibiotics
Acetone Petroleum ether
Ethanol Ethyl acetate
Aqueous Pen Met Gen
C.l C.s C.l C.s C.l C.s C.l C.s C.l C.s
E. coli 12 16 13 10 11 8 11 9 10 9 7 7 29
S.aureus 10 13 7 - 9 7 10 11 - 9 6 6 18
S. typhi 10 15 - 7 9 8 10 9 7 - - - 24
B. subtilis 9 14 7 - 9 7 10 10 9 9 8 9 20
K.Pneumonia 12 14 8 9 10 8 12 11 - 7 - - 25
*disc concentration 100 mg/ml C.l- Citrus limon; C.s- Citrus sinensis
- indicates < 5mm
Table 2. MIC values of Citrus limon and Citrus sinensis peel extract for different bacterial strains
Bacteria
Citrus lemon peel extract (mg/ml)
Citrus sinensis peel extract (mg/ml)
Acetone Petroleum
ether Ethanol Eth yl acet ate
Aqueou s
Acetone Petroleum ether
Ethano l
Ethyl acetate
Aqueo us
E.coli 12.5 6.25 25 50 50 6.25 25 25 50 50
S.aureus 50 50 50 50 - 25 - 50 12.5 50
S. typhi 50 - 50 25 50 6.25 50 50 50 50
B. subtlis 50 50 25 50 50 6.25 - 50 50 50
K.pneumonia 25 50 25 25 - 6.25 50 50 25 50
Table 3. MBC values of Citrus limon and Citrus sinensis peel extract for different bacterial strains
Bacteria
Citrus lemon peel extract (mg/ml)
Citrus sinensis peel extract (mg/ml)
Aceto
ne Petroleum ether
Ethano l
Ethyl acetate
Aqueo us
Aceton
e Petroleum
ether
Ethano l
Ethyl
acetate Aqueous
E.coli 50 12.5 50 - - 12.5 50 50 - -
S.aureus 50 - - - - 50 - - 25 -
S. typhi - - - 50 - 25 - - - - B. subtlis - - 50 - - 25 - - - -
Table 4. Phytochemical analysis of Citrus limon and Citrus sinensis peel extracts
+ indicates presence, - indicates absence
C. lim- Citrus limon , C.sin- Citrus sinensis
5. Conclusions
Recycling of fruit waste is one of the most important means of utilizing it in a number of innovative ways yielding new products and meeting the requirements of essential products required in human, animal and plant nutrition as well as in the pharmaceutical industry. This work has identified the antibacterial activity against the test organisms and phytochemical constituents in Citrus limon and Citrus sinensis peels extracts obtained from different solvents. However, further evaluation performed with the pure compounds is required for the definite conclusion of the bioactive compounds contributing to the antimicrobial activity, although the nature and number ofactive components involved in each extract are not clear, however they are promising. This finding can form the basis for further studies to prepare an optimize preparation of the herbal extract.
Acknowledgement
The authors sincerely thank the authorities of Arulmigu Meenakshi Amman College of Engineering for the successful completion of the work.
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