Bitter Orange Peel Oil Biology Essay


Essential oil of Citrus aurantium volt-ampere. acrimonious orange was analyzed by GC-MS. Out of 8 constituents, 3 were identified from their atomization form. Among the identified components, limonene ( 98.173 % ) was found as a major constituent followed by ?-pinene ( 0.476 % ) and ?-pinene ( 0.

176 % ) .The antibacterial activity of the indispensable oil of C. aurantium was determined by paper phonograph record diffusion method, against Gram positive and Gram negative bacteriums ( Bacillus subtilis ATCC 6633, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa, Pseudomonas fluorescens, Staphylococcus aureus, Escherichia coli and Enterobacter species ) . Maximum zone of suppression was resulted against Gram positive bacteriums i-e Bacillus subtilis ATCC 6633 and Staphylococcus aureus while Gram negative strains i-e Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli were found immune to C. aurantium Peel oil.The Peel oil was found to hold extremist scavenging consequence on DPPH in the scope of 37.42-94.64 % with oil concentration 20-100 % when compared to BHT being strong antioxidant reagent.

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Cardinal words: Citrus aurantium, GC-MS, Limonene, antibacterial activity, DPPH.


Essential oils are the complex mixture of terpenic hydrocarbons and oxygenated derivates such as aldehydes, intoxicants, ketones, organic acids and esters1. These oils are widely used as flavorer and dissembling agents in many nutrient, cosmetics and pharmaceutical industries every bit good as in aromatherapy2. Essential oils have besides been shown to possess antibacterial, fungicidal, antiviral, insecticidal and antioxidant proreties3,4. Some oils have been used in malignant neoplastic disease treatment5.

The genus Citrus of household Rutaceae includes assorted species of oranges, Citrus reticulatas, calcium hydroxides, lemons and Citrus paradisi. Citrus fruits are chiefly possessed for juice go forthing Peels as a primary waste. These Peels are a possible beginning of indispensable oil6 and output oil in the scope of 0.5-3.0kg/tonne of fruit7. Citrus oil contains big sum of monoterpene hydrocarbon ( 70-95 % ) along with smaller sum of sesquiterpene hydrocarbons which are responsible for a characteristic flavour8,9.

The major usage of citrous fruit Peel oil is as a flavorer agent in confects, carbonated and non-carbonated drinks, bakeshop merchandises, ice-cream, bars and biscuits, confectionery and masticating gum10. The nutrient and pharmaceutical in Pakistan are extensively utilizing citrus oil and the present citrous fruit Peel oil is deserving Rs. 300 million11so there is a desperate demand to develop a executable technique for the production of indispensable oils from autochthonal resources.

27 species of Rutaceae exist in Pakistan 12. Out of these 27 species, 10 belong to Genus Citrus. Citrus aurantium volt-ampere. acrimonious orange is one of them.The indispensable oil of Citrus aurantium is aromatic, internally gastric and externally tonic13. It has been used to add olfactory property to drinks and spiritss and as an ingredient to give aroma to lather, detergents, cosmetics and perfumes14.The purpose of the present survey is to research the chemical composing of indispensable oil ofLocally available Citrus aurantium volt-ampere. acrimonious orange Peel oil every bit good as its anti microbic and antioxidant belongingss.


Extarction of oil

The fruit was washed, peeled off and Peels were cut into pieces. The cut pieces were subjected to hydro-distillation by utilizing rearward Dean Stark apparatus15. The steam distillation was removed and dried over anhydrous Na sulfate and stored at low temperature.

GC-MS analysis.

The analysis of the indispensable oil was carried out on GC-MS of Agilent Technologies, Model 6890N, runing in EI manner at 70 electron volts equipped with a split-splitless injector.

Helium used as a bearer gas at the flow rate of 1ml/min, while HP-5MS ( 30 m – 0.25 millimeter, 0.25 µm ) capillary column was used. The initial temperature was programmed at 50-140 & A ; deg ; C at the rate of 5 & A ; deg ; C/min and so 100-250 & A ; deg ; C at the rate of 3 & A ; deg ; C/min followed by a changeless temperature at 260 & A ; deg ; C for period of 20 proceedingss. Sample ( 2µL ) was injected to column programmed at 200 & A ; deg ; C and declarations of constituents were attained. The constituents were identified by their keeping clip and peak sweetening with standard samples in gas chromatographic manner and NIST library hunt from the derived atomization form of the assorted constituents of the oil.

Test beings

In vitro antimicrobic surveies were carried out on six bacterial strains ( Bacillus subtilis ATCC 6633, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli ) some of which were obtained from PCSIR research labs complex, Lahore and other from pathological research lab of a local infirmary.

The civilizations of bacteriums were maintained in their appropriate agar angles at 4 & A ; deg ; C throughout the survey and used as stock civilizations.

Antibacterial Assay

Paper disc diffusion method as reported by Bauer 16 was applied with little alteration to prove the antimicrobic activity of C. aurantium Peel indispensable oil. Discs of Whatman No.

1 filter paper holding a diameter of 6 millimeter wrapped in tin foil were oven dried at 65 & A ; deg ; C overnight for sterilisation.Normal strength food agar medium ( OXOID, England ) was prepared and autoclaved at 121±1 & A ; deg ; C for 15 proceedingss under 15psi for civilization growing through out the survey to measure the antibacterial activity of the oil under probe.For antibacterial check 24h old bacterial civilizations grown at 36±1 & A ; deg ; C were used. Cultures were diluted 10-1 in unfertile toller solution17 incorporating about 106CFU/mL in each instance. Twenty five micro-liters of these suspensions were inoculated to home bases incorporating unfertile alimentary agar medium utilizing a unfertile cotton swab in order to acquire a unvarying microbic growing on both control and trial home bases.Filter paper discs each impregnated with 10µl, 15µl and 20µl of oils were placed on pre-inoculated civilization media under sterile conditions individually and incubated at 36±1 & A ; deg ; C for 24h. The zone of suppression in each instance was measured as the diameter ( in millimetres ) of the clear zone around the phonograph record.

All experiments were performed in extra. Penicillin G and Streptomycin were used as positive controls. Inhibitory consequence of positive controls was tested for all micro-organisms used in this survey under the incubation conditions as mentioned above.

The working solution of control antibiotics were prepared in appropriate sums ( 0.01g/10mL ) so 25?L of each antibiotic solution was dropped on paper phonograph record which is so used during this survey.


Antiradical activity was evaluated by mensurating the scavenging activity of the examined C. aurantium oil on DPPH extremist. The DPPH check was performed as described by Epsin et al18.

The samples ( 100 µl each ) were assorted with 3 milliliters of DPPH solution. The optical density of the resulting solutions and the space ( with merely DPPH and no sample ) were recorded after an incubation clip of 30 proceedingss at room temperature against BHT as a positive control. For each sample, 3 replicates were recorded. The disappearing of DPPH was measured spectrophotometrically at 517 nanometer. The per centum of extremist scavenging activity was calculated utilizing the undermentioned equation ;DPPH scavenging consequence ( % ) = ( A0-A1 ) /A0 – 100Where A0 is the optical density of the control at 30 proceedingss and A1 is the optical density of the sample at 30 proceedingss.

Consequences and Discussion

The indispensable oil was extracted from Peels of C. aurantium volt-ampere. acrimonious oil by hydro-distillation.

The output of oil was 0.622 % . The output is somewhat less than every bit reported by Hifnaway et al19.GC-MS of the indispensable oil revealed the presence of 8 constituents. Out of which 3 have been identified from their atomization form by mass spectrometry utilizing MS library ( Table-1 ) .The indispensable oil of C.

aurantium was found to be constituted of monoterpene hydrocarbons merely. Among the components limonene ( 98.173 % ) ) was found as a main constituent followed by ?-pinene ( 0.476 % ) and ?-pinene ( 0.

176 % ) .The consequences are in conformance with old GC-MS surveies of C. aurantium Peel oil20 21 22 nevertheless the local assortment of C. aurantium showed a comparatively higher concentration of limonene i-e. 98.173 % .

It is used as a aroma stuff in aromatizing family merchandises and as a constituent of unreal indispensable oils. The other components of peel oil i-e ?-pinene and ?-pinene are used as a get downing stuff in aroma and flavour industry23.The information pertaining to the in vitro antimicrobic potency of C. aurantium indispensable oil against Gram positive and Gram negative bacteriums along with control antibiotic are presented in table-1.

These consequences showed that Bacillus subtilis exhibited maximal antimicrobic activity at 20?L concentration of oil with an suppression zone diameter ( IZD ) of 11±0.36mm. The IZD of Staphylococcus aureus was 12±0.28mm for phonograph record impregnated with 15µL of indispensable oil whereas with higher conc. of C.

aurantium Peel oil ( 20µL ) the IZD was besides increased to18±0.35mm. In present survey, the biological activity of C.

aurantium indispensable oil was besides evaluated against four G-ve bacteriums including Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae. The consequences indicated that all tested G-ve bacteriums were immune to C. aurantium indispensable oil as shown in table-1. Present findings indicted that is more effectual against Gram+ve bacterium which is in conformity with Kirbaslar et al20 who reported strong antimicrobic activity of citrous fruit Peel oil against tested Gram+ve bacteriums.

While sing the efficaciousness of the C. aurantium indispensable oil against Gram-ve bacteriums present consequences correlated with Quintero et. al21 who reported that Pseudomonas aeruginosa and E. coli were immune to C. aurantium Peel oil.

In instance of antibiotics Streptomycin exhibited antimicrobic activity against all tried micro-organisms as shown in table-1. Maximum zone of suppression observed in instance of Bacillus subtilis i.e 33.5±0.71mm whereas the minimal IZD was 18±0.71mm exhibited by Pseudomonas aeruginosa. Penicillin G was effectual against merely Bacillus subtilis and S.

aureus with an IZD of 15±0.71mm and 17.5±0.25mm severally. This probe supports the already documented fact Izzo et al.

, 1995 ; 24 25 26 27 that antimicrobic potency of the C. aurantium Peel oil is depending upon its constituents which may change with physiological development period, seasonal alterations in workss, extraction procedure and the sort of bacteriums used etc.Several natural compounds are known to slake free radicals28.

In the current survey ( Figure 1 ) indispensable Peel oil was able to cut down the stable extremist DPPH to yellow-colored DPPH-H making 94.64 % of DPPH scavenging consequence. The comparing of DPPH scavenging activity of C. aurantium Peel oil with good known antioxidant BHT showed that Peel oil has every bit strong antioxidant potency.


The consequences of the present survey recommend usage of C.

aurantium volt-ampere. acrimonious oil Peel oil in perfumery while its strong antimicrobic and antioxidant activities suggest it a possible replacement for man-made preservative in nutrient and medical specialties.


Writers are thankful to Dr. Zia-ur-Rehman, Senior Scientific Officer, PCSIR Labs Complex, Lahore for GC-MS analysis.Table-1: GS/MS analysis of indispensable oil of Citrus aurantium Peel oil.



% age

M/Z Values

4Limonene98.173M+ ( 136,31 ) ( 121,34 ) ( 107,29 ) ( 103,3 ) ( 93,85 ) ( 79,44 ) ( 68,100 ) ( 63,4 ) ( 53,25 )5?- pinene0.476M+ ( 136,8 ) ( 121,17 ) ( 105,13 ) ( 93,100 ) ( 80,6 ) ( 77,31 ) ( 67,6 ) ( 65,6 ) ( 55,6 ) ( 53,8 )6?-pinene1.008M+ ( 136,5 ) ( 121,8 ) ( 107,4 ) ( 93,100 ) ( 79,19 ) ( 65,7 ) ( 53,10 ) ( 50,3 )

Table-2. Assessment of antimicrobic activity of Citrus aurantium indispensable oil against six micro-organisms.

Test Micro-


G.staining/ Colony morphology

Antimicrobial activity of C. aurantium indispensable oil and some standard Antibiotics.

Zone of suppression ( millimeter )

Oil conc.10µL/DaOil conc.15µL/ DOil conc.20µL/ DStrepto-Mycin25µg/DPenicillin G25µg/DBacillus subtilis ATCC 6333G +ve rods/White, dry surface

11 ±0.3633.5±0.7115±0.71Staphylococcus aureus HIbG +ve rods/Light yellow, unit of ammunition, shiny

12±0.2818 ±0.3524±0.7017.5±0.25Salmonellatyphimurium HIG -ve rods/Transparent white,


Pseudomonas aeruginosa HIG -ve rods/ viridity, flat- spreaded


Klebsiella pneumoniae HIG -ve rods/ big grey- white, mucoid


Escherichia coli HIG -ve rods/Smooth, white


aPaper phonograph record ( 6mm diameter )bHospital isolated pathogen± Standard divergence-No suppression zone ( immune )

Figure1: Percentage antioxidant activity of indispensable oil of Citrus aurantium Peel oil in comparing with BHT as standard mention by DPHH check.


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