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Journal of Taibah University Medical Sciences (2018) -(-), 1e5

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Taibah University
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Journal of Taibah University Medical Sciences 59
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www.sciencedirect.com 62
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Original Article 65
1 66
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3 Chemical composition and in-vitro antioxidant and antimicrobial 68
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5 activity of the essential oil of Citrus aurantifolia L. leaves grown in 70
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7 eastern Oman 71
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9 Q11 Maha S. Al-Aamri, B. Pharm a, Nour M. Al-Abousi, B. Pharm a, 74
10 Sausan S. Al-Jabri, B. Pharm a, Tanveer Alam, Ph.D b and Shah A. Khan, Ph.D a, * 75
11 76
12 a
Department of Pharmacy, Oman Medical College, Muscat, Oman 77
Q1
13 b
Oman’s Medicinal Plants & Marine Natural Products, University of Nizwa, Oman 78
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Received 3 August 2017; revised 12 December 2017; accepted 19 December 2017; Available online - - - 80
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20 ‫ﺍﻟﻤﻠﺨﺺ‬ Abstract
85
21 ‫ ﻭﻧﺸﺎﻁ ﻣﻀﺎﺩﺍﺕ‬،‫ ﻳﺪﺭﺱ ﻫﺬﻩ ﺍﻟﺒﺤﺚ ﻣﻀﺎﺩﺍﺕ ﺍﻷﻛﺴﺪﺓ‬:‫ﺍﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ‬ 86
Objectives: This study investigated the chemical compo-
22 ‫ﺍﻟﻤﻴﻜﺮﻭﺑﺎﺕ ﻭﺍﻟﺘﺮﻛﻴﺐ ﺍﻟﻜﻴﻤﻴﺎﺋﻲ ﻟﻠﺰﻳﺖ ﺍﻷﺳﺎﺳﻲ ﺍﻟﺬﻱ ﺗﻢ ﻋﺰﻟﻪ ﻣﻦ ﺍﻷﺟﺰﺍﺀ‬ 87
23 sition and the antioxidant and antimicrobial activities of
.‫ﺍﻟﻬﻮﺍﺋﻴﺔ ﻟﺤﻤﺾ ﺃﻭﺭﺍﻧﺘﻴﻔﻮﻟﻴﺎ‬ 88
24 the essential oil isolated from the aerial parts of Citrus
89
25 ‫ ﺗﻢ ﺟﻤﻊ ﺍﻷﻭﺭﺍﻕ ﺍﻟﻄﺎﺯﺟﺔ ﻟﺤﻤﺾ ﺃﻭﺭﺍﻧﺘﻴﻔﻮﻟﻴﺎ ﻣﻦ ﻣﺰﺍﺭﻉ ﻣﺤﻠﻴﺔ‬:‫ﻃﺮﻕ ﺍﻟﺒﺤﺚ‬ aurantifolia L.
90
26 ،‫ ﻳﻮﻟﻴﻮ‬-‫ﻓﻲ ﻣﺪﻳﻨﺔ ﺻﻮﺭ )ﺍﻟﻤﻨﻄﻘﺔ ﺍﻟﺸﺮﻗﻴﺔ( ﻟﺴﻠﻄﻨﺔ ﻋﻤﺎﻥ ﺧﻼﻝ ﺍﻟﻔﺘﺮﺓ ﻣﻦ ﻳﻮﻧﻴﻮ‬
91
27 ‫ ﻭﺍﺳﺘﺨﺪﻡ ﺍﻟﻐﺎﺯ‬.‫ ﺍﺳﺘﺨﺪﻣﺖ ﻃﺮﻳﻘﺔ ﺍﻟﺘﻘﻄﻴﺮ ﺍﻟﻤﺎﺋﻲ ﻟﻌﺰﻝ ﺍﻟﺰﻳﺖ ﺍﻷﺳﺎﺳﻲ‬.٢٠١٥ Methods: Fresh Citrus aurantifolia L. leaves were 92
28 .‫ ﻗﻴﺎﺱ ﺍﻟﻄﻴﻒ ﺍﻟﻜﺘﻠﻲ ﻟﻠﺘﻌﺮﻑ ﻭﻟﺘﺤﺪﻳﺪ ﻛﻤﻴﺔ ﺍﻟﻤﻜﻮﻧﺎﺕ ﺍﻟﻜﻴﻤﺎﺋﻴﺔ ﻓﻲ ﺍﻟﺰﻳﺖ‬-‫ﺍﻟﻠﻮﻧﻲ‬ collected from farms in Sur city, located in the Al-Sharqia 93
29 ‫ﺍﺳﺘﺨﺪﻣﺖ ﻃﺮﻳﻘﺔ ﺍﻟﺠﺮﻑ ﺍﻟﺤﺮﺓ ﻟﻠﺠﺬﻭﺭ ﻓﻲ – ﺍﻟﻤﺨﺘﺒﺮ ﻟﺘﺤﺪﻳﺪ ﻧﺸﺎﻁ ﻣﻀﺎﺩﺍﺕ‬ (Eastern) region of the Sultanate of Oman, during June 94
30 ‫ ﺑﻴﻨﻤﺎ ﺗﻢ ﺗﻘﻴﻴﻢ ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ‬،‫ﺍﻷﻛﺴﺪﺓ ﻟﻠﺰﻳﺖ ﺍﻟﺬﻱ ﺗﻢ ﻋﺰﻟﻪ ﻣﻦ ﺃﻭﺭﺍﻕ ﺍﻟﻠﻴﻤﻮﻥ‬ eJuly of 2015. The essential oil was isolated using 95
31 ‫ﻟﻠﺠﺮﺍﺛﻴﻢ ﺿﺪ ﺍﻟﺒﻜﺘﻴﺮﻳﺎ ﺇﻳﺠﺎﺑﻴﺔ ﺍﻟﻐﺮﺍﻡ ﻭﺍﻟﺒﻜﺘﻴﺮﻳﺎ ﺳﻠﺒﻴﺔ ﺍﻟﻐﺮﺍﻡ ﺑﻄﺮﻳﻘﺔ ﺍﻧﺘﺸﺎﺭ‬ hydrodistillation. Gas chromatographyemass spectrom- 96
32 .‫ﺍﻟﻘﺮﺹ‬ etry (GCeMS) was used to identify and quantify the 97
33 chemical constituents of the oil. An in-vitro 1,1-diphenyl- 98
34 -‫ ﺗﻢ ﺗﺤﺪﻳﺪ ﻣﺎ ﻣﺠﻤﻮﻋﺔ ﺛﻼﺛﺔ ﻭﺛﻼﺛﻴﻦ ﻣﺮﻛﺒﺎ ﻛﻴﻤﻴﺎﺋﻴﺎ ﻭﺗﻢ ﺍﻟﻌﺜﻮﺭ ﻋﻠﻰ ﺩ‬:‫ﺍﻟﻨﺘﺎﺋﺞ‬
2-picrylhydrazyl (DPPH) free radical scavenging method Q3 99
35 ‫ ﺗﻢ ﺗﺤﺪﻳﺪ ﻣﻜﻮﻧﺎﺕ ﺭﺋﻴﺴﺔ ﺃﺧﺮﻯ‬.‫( ﻟﻴﻜﻮﻥ ﺍﻟﻤﻜﻮﻥ ﺍﻷﺳﺎﺳﻲ‬٪٦٣.٣٥) ‫ﻟﻴﻤﻮﻧﻴﻦ‬
was used to determine the antioxidant activity of the 100
36 ‫ ﻛﻤﺎ ﺃﻇﻬﺮ ﺍﻟﺰﻳﺖ ﺍﻷﺳﺎﺳﻲ ﻟﺤﻤﺾ ﺃﻭﺭﺍﻧﺘﻴﻔﻮﻟﻴﺎ ﻧﺸﺎﻃﺎ ﻣﻤﺘﺎﺯﺍ‬.‫ﺑﻨﺴﺐ ﻣﺨﺘﻠﻔﺔ‬
isolated oil from the lime leaves while a disc diffusion 101
37 ‫ﻣﻀﺎﺩﺍ ﻟﻠﺠﺮﺍﺛﻴﻢ ﺿﺪ ﺍﻟﻤﻜﻮﺭﺍﺕ ﺍﻟﻌﻨﻘﻮﺩﻳﺔ ﺍﻟﺬﻫﺒﻴﺔ ﻭﻧﺸﺎﻁ ﻣﺘﻮﺳﻄﺎ ﺿﺪ ﺍﻟﺴﻼﻻﺕ‬
method was used to evaluate the antibacterial activity 102
38 ‫ ﻭﺃﻇﻬﺮ ﺍﻟﺰﻳﺖ ﻛﺬﻟﻚ ﻧﺸﺎﻃﺎ ﻭﺍﻋﺪﺍ ﻟﻤﻀﺎﺩﺍﺕ ﺍﻷﻛﺴﺪﺓ ﻓﻲ‬.‫ﺍﻹﺷﺮﻳﻜﻴﺔ ﺍﻟﻘﻮﻟﻮﻧﻴﺔ‬
against Gram-negative and Gram positive bacteria. 103
39 .‫ﺍﻟﻤﺨﺘﺒﺮ ﻭﻟﻜﻨﻪ ﺃﻇﻬﺮ ﻧﺸﺎﻃﺎ ﻣﻀﺎﺩﺍ ﻟﻠﺠﺮﺍﺛﻴﻢ ﻣﺘﻮﺳﻄﺎ‬ 104
40 ‫ ﻳﺘﻤﻴﺰ ﺍﻟﺰﻳﺖ ﺍﻷﺳﺎﺳﻲ ﻷﻭﺭﺍﻕ ﺍﻟﻠﻴﻤﻮﻥ ﺍﻟﻌﻤﺎﻧﻲ ﺑﻤﺤﺘﻮﻯ ﻋﺎﻟﻲ ﻣﻦ‬:‫ﺍﻻﺳﺘﻨﺘﺎﺟﺎﺕ‬ 105
Results: Thirty-three chemical compounds were identi-
41 .‫ ﻭﺍﻟﺼﻨﺎﻋﺎﺕ ﺍﻟﺪﻭﺍﺋﻴﺔ ﻭﺍﻟﻌﻄﻮﺭ‬،‫ ﻟﻴﻤﻮﻧﻴﻦ ﺍﻟﺬﻱ ﻳﺠﻌﻠﻪ ﻣﻔﻴﺪﺍ ﻓﻲ ﺍﻟﻐﺬﺍﺀ‬-‫ﺩ‬ 106
fied, with D-limonene (63.35%) forming the major
42 107
constituent. Other prominent constituents include 3,7-
43 ‫ ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻟﻠﺠﺮﺍﺛﻴﻢ؛ ﺣﻤﺾ ﺃﻭﺭﺍﻧﺘﻴﻔﻮﻟﻴﺎ؛ ﺍﻟﺰﻳﺖ‬:‫ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺣﻴﺔ‬ 108
dimethyl-2,6-octadien-1-ol (7.07%), geraniol (6.23%),
44 ‫ ﻟﻴﻤﻮﻧﻴﻦ‬-‫ﺍﻷﺳﺎﺳﻲ؛ ﺩ‬ 109
45 E-citral (4.35%), Z-citral (3.29%), and b-ocimene
110
46 (2.25%). The essential oil of Citrus aurantifolia L. leaves
* Corresponding address: Department of Pharmacy, Oman 111
Q2 showed excellent antibacterial activity against Staphylo-
47 112
Medical College, Muscat, Oman. coccus aureus and moderate activity against pathogenic
48 113
E-mail: shahalamkhan@yahoo.com, sakhan@omc.edu.om (S.
49 Escherichia coli strains. The oil exhibited promising invi- 114
A. Khan)
50 tro antioxidant activity (IC50 value ¼ 21.57 mg/mL) but 115
Peer review under responsibility of Taibah University.
51 showed moderate antibacterial activities. 116
52 117
53 Conclusions: The essential oil from Omani lime leaves is 118
54 Production and hosting by Elsevier characterized by a high D-limonene content, making 119
1658-3612 Ó 2018 Taibah University.
Production and hosting by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.jtumed.2017.12.002
Please cite this article in press as: Al-Aamri MS, et al., Chemical composition and in-vitro antioxidant and antimicrobial activity of the essential oil of Citrus
aurantifolia L. leaves grown in eastern Oman, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/j.jtumed.2017.12.002
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2 M.S. Al-Aamri et al.

1 it useful for food, pharmaceutical, and perfumery leaves by GCeMS and (2) investigate the antioxidant and 66
2 industries. antibacterial activity of the isolated oil. 67
3 68
4 Keywords: Antibacterial activity; Citrus aurantifolia; D-limo- Materials and Methods 69
5 nene; Essential oil; GCeMS 70
6 71
7 Ó 2018 Taibah University.
Chemicals and test microorganisms 72
8 Production and hosting by Elsevier Ltd. This is an open 73
9 access article under the CC BY-NC-ND license (http:// Chemicals and reagents were obtained from a local sup- 74
10 creativecommons.org/licenses/by-nc-nd/4.0/). plier. A medium-size glass Clevenger apparatus made by Q4 75
11 BorosilÒ, India, was used to isolate the essential oil. To 76
12 evaluate the antibacterial activity, two pathogenic bacterial 77
13 strains (Escherichia coli-ATCC 8739, Gram-negative and 78
Introduction
14 Staphylococcus aureus-ATCC 29213, Gram-positive) were 79
15 obtained from the Department of Natural Sciences, Oman 80
16 Citrus aurantifolia L., the Swingle or Omani lime (Family: 81
Medical College, Sultanate of Oman.
17 Rutaceae), is a major citrus crop in the Sultanate of Oman in 82
18 terms of cultivation, production, and consumption.1 This 83
Collection of lime leaves
19 edible and medicinal plant is native to Southeast Asia and 84
20 was widely cultivated in the tropics and subtropics before 85
21 its introduction to the African and European continents Fresh C. aurantifolia L. leaves grown in Sur city, which is 86
22 through Oman. It is a perennial, flowering, evergreen tree, located in the Al-Sharqia region of the Sultanate of Oman, 87
23 which is 3e5 m in height. Its stem is unusually slender and were collected from farms from June to July of 2015. The 88
24 branched, with sharp thorns and spines. Leaves are leaves were identified by a subject expert from Oman Medical 89
25 alternately arranged and elliptical to oval in shape with College, and a voucher specimen (PHAR425/2015/4) was 90
26 rounded teeth on their perimeters; they usually measure 4e deposited in the Department of Pharmacy, Oman Medical 91
27 6 cm in length by 2.5e4.5 cm in width. The tree’s flowers College, Oman. 92
28 are white in color and have a strong aroma. The lime fruits 93
29
are round in shape (3e5 cm in diameter), and green to Isolation of essential oils by hydro-distillation method 94
30 95
yellow in color with a thin skin; they are juicy, aromatic,
31 96
and acidic in nature.2 The fruit is known as lime in English C. aurantifolia leaves (150 g) were washed under running
32 water to remove dust and insects and then cut into small pieces 97
and lomi or limah in Arabic. The Omani lime variety
33 98
34
resembles the Indian, Mexican, or Floridian key lime.3 to increase their surface area. The material was transferred to a
99
Citrus aurantifolia L. is a very popular and valued citrus 1-L round bottom flask and covered with a sufficient quantity
35 100
species in the Gulf region due to its nutritional qualities, of water. Hydro-distillation in a Clevenger apparatus for 6 h Q5
36 101
distinct flavor, and health benefits. Various parts of the plant yielded a strongly aromatic light green volatile oil.3 The oil was
37 102
38 are used in traditional medicine to treat cataracts, colds, sore separated from the aqueous layer, collected in plastic sample 103
39 throats, fevers, chest pains, earaches, headaches, stomach tubes, dried over anhydrous sodium sulfate, and stored in 104
40 ailments, and edema, and it is considered an antiseptic, the dark at 4  C until further use. The yield of the isolated 105
41 anthelmintic, mosquito repellent, anti-scurvy, astringent, essential oil was calculated based on the weight of the fresh 106
42 digestive, and appetite stimulant, among others.4 Lime juice leaves. Q6 107
43 and its essential oil are also commonly used in the food, drug, 108
44 and cosmetic industries because of their medicinal properties Gas chromatographyemass spectrometry analysis 109
45 and fragrance. The traditional and pharmacological uses of 110
46 Citrus aurantifolia L. plants are attributed to the presence A small portion of the essential oil was diluted in diethyl Q7 111
47 of secondary plant metabolites including flavonoids, ether to determine the chemical composition using a gas 112
48 coumarins, and terpenoids.5e7 chromatographyemass spectrometry (GCeMS) instrument 113
49 Biologists have recently become increasingly interested in equipped with an auto sampler, including a Perkin Elmer 114
50 the useful biological activities of essential oils, especially their Clarus 600 GC system fitted with a Rtx-5MS capillary col- 115
51 116
broad antimicrobial abilities against a wide range of patho- umn (30 m  0.25 mm i.d.  0.25 mm film thickness;
52 117
genic microbes.8 This antimicrobial activity is primarily due to maximum temperature ¼ 350  C) which was coupled to a
53 118
their complex chemical composition, including substances Perkin Elmer Clarus 600C MS at SQU. Ultra-high purity
54 119
belonging to a broad range of chemical classes including helium (99.9999%) was used as a carrier gas at a constant
55 120
56
terpenes, aldehydes, alcohols, esters, phenols, ethers, and flow of 1.0 mL/min. The injection, transfer line, and ion 121
57 ketones.9,10 Thus, understanding the chemical constitution source temperatures were 280, 260, and 260  C, respectively. 122
58 of volatile natural essential oils could prove a viable The ionizing energy was 70 eV. The electron multiplier (EM) 123
59 approach to identify and develop novel antimicrobial agents voltage was obtained using auto-tune. All data were ob- 124
60 to overcome the problem of antimicrobial drug resistance.11 tained by collecting a full-scan mass spectrum within the 125
61 An extensive literature review revealed that very little is range of 40e550 amu. The injected sample volume was 1 mL 126
62 known about the chemical composition and antimicrobial with a split ratio of 100:1. The oven temperature was pro- 127
63 activity of the essential oil of C. aurantifolia L. leaves grown grammed to begin at 60  C and heat at a ramp rate of 3  C/ 128
64 in eastern Oman. Hence, the goals of this study were to (1) min to a final temperature of 280  C, which was held for 129
65 analyze the composition of the essential oil of C. aurantifolia 2 min. Unknown compounds were identified by comparing 130

Please cite this article in press as: Al-Aamri MS, et al., Chemical composition and in-vitro antioxidant and antimicrobial activity of the essential oil of Citrus
aurantifolia L. leaves grown in eastern Oman, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/j.jtumed.2017.12.002
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GCeMS analysis and biological evaluation of essential oil from Omani lime leaves 3

1 the experimental spectra with those in mass spectrum li- et al. detected a total of 59 chemical constituents with 66
2 braries (NIST 2011 v.2.2 and Wiley, 9th edition). D-limonene, pinene, and sabinene as the major components, 67
3 followed by citronellal, geranial, linalool, and neral.14 Lawal 68
4 Identification of volatile constituents of essential oil et al. analyzed the essential oil of C. aurantifolia grown in the 69
5 Lagos state of Nigeria and determined that D-limonene 70
6 (45%) and geranial (38%) form the chief constituents in 71
Volatile constituents were identified based on their
7 72
retention time relative to n-alkanes (C6eC24), with corre- the oil.15
8 73
9 sponding literature data in conjunction with that available in 74
10 mass spectrometry libraries (NIST 2011 v.2.3 and Wiley, 9th 75
Antioxidant activity
11 edition). 76
12 77
In vitro anti-oxidant activity A number of studies have recommended the use of essential
13 78
oils in the food and drug industries as natural antioxidants
14 79
because of the combination of their promising antioxidant
15 The in vitro free radical scavenging activity of the essential 80
16
activities and relatively safe toxicological profiles.16 In 2000, 81
Q8 oil of lime leaves was determined with DPPH using a slightly
Choi et al. tested the antioxidant activity of 31 essential oils
17 modified adaptation of a previously reported method.12 82
from citrus fruits and found them to be similar or better
18 Briefly, 50 mL solutions of essential oil in ethyl acetate at 83
antioxidants than Trolox.17 Prompted by these and other
19 various concentrations (5e50 mg/mL) were measured using 84
20 studies, a simple and reliable in vitro assay method using 85
a micropipette and added to 2.95 mL of a DPPH/ethyl
21 DPPH free radicals was used to investigate the antioxidant 86
acetate solution (0.01 mM) in a test tube. After 30 min in
22 potential of the essential oil prepared by lime leaves from the 87
Q9 the dark at room temperature, the absorbance (At) of the
23 Al-Sharqia region. The results of the antioxidant assay, 88
reaction mixture was measured at 517 nm on a UVeVis
24 which are presented in Table 2, indicate that concentrations of 89
spectrophotometer (UV Analyst-CT 8200). Ethyl acetate
25 5e50 mg/mL, the oil has a comparable antioxidant activity 90
26
was used as a blank while DPPH solution was used as the 91
27 control (Ab). The % inhibition of DPPH radical was calcu- 92
28 lated using the formula [(Ab e At/Ab)  100]. The IC50 93
Table 1: Chemical composition of essential oil isolated from Al-
29 value was also calculated using the plot of percentage inhi- 94
bition versus sample concentration. Sharqia lime leaves.
30 Q10 95
31 S. No. Compound name RT KI % 96
32 Evaluation of antibacterial activity (min) composition 97
33 1. a-Pinene 5.152 924.24 1.7485 98
34 The antibacterial activity of the lime leaves essential oil 2. Sabinene 6.063 974.36 0.2596 99
35 was evaluated against Gram-positive and negative patho- 3. b-Pinene 6.16 911.26 0.3195 100
36 genic bacteria, S. aureus and E. coli, respectively. The anti- 4. D-Limonene 7.558 1026.03 63.3539 101
37 5. Trans-b-Ocimene 7.774 1039.02 0.4397 102
bacterial activity was determined using the disc diffusion
38 6. b-Ocimene 8.078 1028.74 2.2450 103
method with standard Mueller Hinton agar (MHA) media.13
39 7. b-Thujene 8.403 1060.86 0.0625 104
Sterile filter paper discs (6 mm in diameter) were impregnated 8. Isoterpinolene 9.302 1092.08 0.0503
40 105
with 5 and 10 mL of pure extracted essential oil and then 9. Linalool 9.692 1090.2 1.6491
41 106
placed on inoculated petri plates. The plates were then 10. UI 10.386 1127.01 0.0476
42 107
incubated at 37  C for 24 h before measuring the diameter 11. Limonene oxide 10.7 1136.90 0.0563
43 108
44
of the zone of inhibition (clear zone) around the disc. The 12. Trans-Limonene oxide 10.841 1141.35 0.0749
109
antimicrobial activity of the test samples was compared 13. Citronellal 11.307 105.06 0.8985
45 110
46 with that of the positive control, Ampicillin (25 mg/disc). 14. Terpinen-4-ol 12.174 1183.4 0.0562
111
All experiments were performed in triplicate. 15. Cis-Verbenol 12.271 1186.46 0.0885
47 16. L-a-Terpineol 12.64 1222.44 0.2821 112
48 17. 3,7-dimethyl-2, 13.821 1234.81 7.07054 113
49 Results and discussion 114
6-Octadien-1-ol
50 18. Z-Citral 14.157 1236.01 3.2880 115
51 Yield and chemical composition of Omani lime leaf essential 19. Geraniol 14.666 1261.05 6.2331 116
52 oil 20. E-Citral 15.11 1267.35 4.3481 117
53 21. UI 17.83 1361.21 0.0439 118
54 22. Neryl acetate 18.046 1368.14 0.6930 119
The slightly green essential oil was obtained in a 0.53% v/
55 23. Geranyl acetate 18.653 1387.59 1.8138 120
56
w (0.8 mL) yield (calculated from the fresh weight of the 24. b-Elemene 18.935 1396.63 0.1800 121
57 leaves). The results of the GCeMS analysis, including the 25. Trans-Caryophyllene 19.78 1407.75 1.6035 122
58 content and composition of the lime essential oil, are pre- 26. Humulene 20.82 1458.94 0.2702 123
59 sented in Table 1. A total of 30 chemical substances out of 33 27. ()-Germacrene D 21.644 1486.22 0.0965 124
60 were identified (90.9%). D-limonene (Figure 1) formed the 28. UI 22.424 1512.72 0.1776 125
61 major component (63.35%), followed by 3,7-dimethyl-2,6- 29. a-Springene 23.41 1547.20 0.0958 126
62 octadien-1-ol (7.07%), geraniol (6.23%), E-citral (4.35%), 30. g-Elemene 23.865 1563.11 0.2437 127
63 Z-citral (3.29%) and b-ocimene (2.25%). These results 31. Caryophyllene oxide 24.613 1565.53 0.4910
128
32. Spathulenol 26.01 1639.92 0.2303
64 confirm that the Omani lime leaves belong to the limonene 129
33. a-Bisabolol 27.506 1694.72 0.0777
65 chemotype. In a similar study of various lime species, Lota 130

Please cite this article in press as: Al-Aamri MS, et al., Chemical composition and in-vitro antioxidant and antimicrobial activity of the essential oil of Citrus
aurantifolia L. leaves grown in eastern Oman, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/j.jtumed.2017.12.002
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4 M.S. Al-Aamri et al.

1 66
2
Table 3: Antibacterial activity of essential oil isolated from 67
3 lime leaves. 68
4 Essential Conc/disc Inhibition zone (mm)a 69
5 oil/Standard against microbes 70
6 S. aureus E. coli 71
7 72
8 Al- Sharqia region 5 mL 5.8  1.5 1.7  0.2 73
9 10 mL 7.9  1.2 3.1  0.6 74
10 Ampicillin 25 mg 19.3  1.2 19.0  0.0 75
11 a
Values are mean  SD; n ¼ 3. 76
12 77
13 78
14 79
dependent activity with more pronounced effects against
15 80
S. aureus (5.8e7.9 mm) than in E. coli (1.7e3.1 mm). How-
16 81
ever, its antibacterial activity is much weaker than that of the
17 82
positive control, ampicillin.
18 83
19
Citrus oils are generally recognized as safe (GRAS) and 84
20 therefore, lime essential oil can be used as a natural flavoring 85
21 agent or food additive for its aroma as well as its antioxidant 86
22 and antibacterial activities. D-limonene, the major constituent 87
23 of lime oil, is an effective gastroprotective agent22; therefore, 88
24 lime oil may be used in combination with anti-inflammatory 89
25 agents to overcome their gastrotoxicity. Lime essential oil 90
Figure 1: Chemical structure of D-limonene.
26 can also be explored as an alternative to conventional therapy 91
27 for some common ailments. 92
28 93
29 Table 2: In- vitro anti-oxidant activities of lime essential oil and 94
Conclusions
30 ascorbic acid by the DPPH method. 95
31 Concentration (mg/mL) % inhibition of DPPH A GCeMS analysis of lime leaf essential oils detected 33
96
32 97
33 Ascorbic acid Lime oil volatile chemical compounds, three of which remain un- 98
34 5 42.18  3.46 27.9  1.20 identified (9.1%). D-limonene was found to be the majority 99
35 10 70.06  2.09 63.23  0.27 constituent, confirming the limonene chemotype of the Al- 100
36 25 92.89  0.81 85.93  1.99 Sharqia lime variety. The lime leaves oil demonstrated 101
37 50 93.83  0.19 87.54  0.36 concentration-dependent inhibition of DPPH radicals with 102
38 IC50 value 13.68 21.87 an IC50 value of 21.87 mg/mL. Its in-vitro free radical scav- 103
39 Values are mean  SD, n ¼ 3. enging activity was nearly comparable to that of ascorbic 104
40 acid at a concentration of 50 mg/mL. On the other hand, the 105
41 oil had moderate anti-bacterial activities. Further, more 106
42 detailed studies are recommended to explore the potential of 107
(27.9e87.54%) to that of a reference compound, ascorbic acid
43 C. aurantifolia L. leaves essential oil as a food preservative 108
44
(42.18e93.83%). The free radical scavenging activities of the 109
and a source of natural antioxidants.
45 test and reference compounds increased as concentrations 110
46 increased; however, ascorbic acid (IC50 ¼ 13.68) was found 111
to be approximately 1.5 times as potent as the essential oil Conflicts of interest statement
47 112
48 based on the IC50 value. 113
49 The authors have no conflict of interest to declare. 114
50 Antibacterial activity 115
51 Authors’ contribution 116
52 117
Essential oils have been well known to exert antimicrobial
53 118
activity and are potential candidates for the development of SAK designed the experiment, analyzed the results and
54 119
antimicrobial agents from alternative sources.18,19 Because of edited the manuscript; TA performed the antioxidant activ-
55 120
their lipophilic nature, essential oils can interact with and alter ity and wrote the manuscript; MSA, NMA, and SSA per-
56 121
the permeability of the cell membrane in microorganisms, formed the experiment, collected the data, and analyzed the
57 122
58 eventually leading to the microorganism’s death.20 The results (all three authors contributed equally). All authors 123
59 antimicrobial spectrum of a volatile oil invariably depends read and approved the final manuscript. 124
60 upon its chemical composition. In the present study, D- 125
61 limonene was found to be the major chemical constituent of Acknowledgements 126
62 the citrus oil, contributing to its sharp aroma and 127
63 antibacterial actions.21 The results of the antibacterial The authors wish to thank Dean Dr. Yaseen M. Al 128
64 activity analysis (reported as the diameter of the zone of Lawatia and Dr. Jayasekhar P., Pharmacy Head of Oman 129
65 inhibition, Table 3) indicate that the essential oil exerts dose- Medical College for providing necessary research facilities. 130

Please cite this article in press as: Al-Aamri MS, et al., Chemical composition and in-vitro antioxidant and antimicrobial activity of the essential oil of Citrus
aurantifolia L. leaves grown in eastern Oman, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/j.jtumed.2017.12.002
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GCeMS analysis and biological evaluation of essential oil from Omani lime leaves 5

1 We also acknowledge the help of Mr. Ahmed Kashif in 12. Al-Owaisi M, Al-Hadiwi N, Khan SA. GC-MS analysis, 49
2 carrying out the antimicrobial activity experiments. determination of total phenolics, flavonoid content and free 50
3 radical scavenging activities of various crude extracts of Mor- 51
4 Appendix A. Supplementary data inga peregrina (Forssk.) Fiori leaves. Asian Pac J Trop Biomed 52
5 2014; 4(12): 964e970. 53
6 13. Al-Abbasy DW, Pathare N, Khan SA, Al-Sabahi JN. Chemical 54
7
Supplementary data related to this article can be found at composition and antibacterial activity of essential oil isolated 55
8 https://doi.org/10.1016/j.jtumed.2017.12.002. from Omani basil (Ocimum basilicum Linn). Asian Pac J Trop 56
9 Dis 2015; 5(8): 645e649. 57
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Please cite this article in press as: Al-Aamri MS, et al., Chemical composition and in-vitro antioxidant and antimicrobial activity of the essential oil of Citrus
aurantifolia L. leaves grown in eastern Oman, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/j.jtumed.2017.12.002