J. Chem Soc. , Vol. 43, No. 2, pp 141 - 150 [2018]

CHEMICAL COMPOSITION AND ANTI-MICROBIAL ACTIVITY OF THE VOLATILE OILS OF AVERRHOA CARAMBOLA L. (STAR FRUIT) GROWN IN NIGERIA

*G. I. Ndukwe and J. O. Okhiku Department of Chemistry, Rivers State University, Nkpolu-Oroworukwo, Port Harcourt, Rivers State, Nigeria. *Corresponding author: [email protected]

Received 22 September 2017; accepted 18 December 2017, published online 25 March 2018

Abstract The chemical composition and antimicrobial activity of the essential oils extracted from the leaf and fruit of Averrhoa carambola L. (family Oxalidaceae) grown in Nigeria were evaluated. These extractions were carried out via hydrodistillation using a Clevenger apparatus. Essential oils from the leaf and fruit were analyzed using gas chromatography-mass spectrometry. The essential oil of the fruit revealed the presence of seventeen components, whose major compounds were estragole (62.312 %), trans-α- bisabolene (12.209 %) and pinene (8.246 %). Seven components from the essential oil of the leaf were identified; the major compounds were estragole (66.847 %), bis(2-ethylhexyl) phthalate (11.964 %) and α-ocimene (9.699 %). The volatile oil from the fruit showed antimicrobial activity against the test organisms (, , and Staphylococcus aureus) with minimum inhibitory concentrations ranging from 10 to 20 μg/ml; while that of the leaf inhibited the growth of only Staphylococcus aureus with minimum inhibitory concentration of 20 μg/ml. These results show that the , Averrhoa carambola, is a good antimicrobial agent.

Keywords: Averrhoa carambola, volatile oil, hydrodistilation, antimicrobial activity, estragole, pinene, α-ocimene, star fruit

Introduction leaves, for treating boils, colds, gastroenteritis, Plant extracts and volatile oils have been used postpartum edema, and traumatic injury [2]. for many purposes for centuries. The Phytochemical analysis of Averrhoa carambola antimicrobial activity of plant oils and extracts fruit indicated the presence of saponins, has been the basis of its several applications in alkaloids, flavonoids, [3], antioxidants food preservation, processing, pharmaceuticals, such as polyphenoloxidase, proanthocyanidins, alternative medicine and natural therapies [1]. epicatechin and vitamin C [4], O-glycosyl Averrhoa carambola L. (Oxalidaceae) popularly flavonoid components such as quercetin-3-O-β- known as Star fruit is found in America, Brazil, d-glucoside and rutin [5]. Other compounds Australia, South-East Asia and some parts of isolated from Averrhoa carambola include; β- Africa including Southern Nigeria. The sitosterol, lupeol, anthraquinone, cyanidin-3-0- carambola tree is slow-growing, short-trunked β-d-glycoside, β-amirin and C-glycoside with a much-branched, bushy, broad, rounded flavones [6-8]. crown and reaches 6-9 m in height. The edible Averrhoa carambola has been reported to fruits have thin, waxy, orange-yellow skin and exhibit several pharmacological activities which juicy, crisp, yellow flesh when fully ripe. Slices include: antioxidant [9], anti-inflammatory [10], of the fruit cut in cross-section have the form of antimicrobial and antifungal [11, 12], anti-ulcer a star [2]. In traditional medicine, the fruit is [13] and hypoglycaemic [14]. It also has used for treating ailments such as cough, food electrophysiological effects [15], amongst other poisoning, sore throat and malaria; the root, for properties. treating arthralgia (joint pain), chronic headache, Averrhoa carambola, though not widely and epistaxis (bleeding from the nose); the distributed in Nigeria, has shown in traditional

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medicine that the fruit, root and leaves can be thickness with 5 % phenyl methyl silicone as the used in treating several aliments [2]. Its versatile stationary phase. The carrier gas was Helium use in traditional medicine necessitated this (1.2 ml min-1) and the injector temperature was research. This study was aimed at investigating kept at 250 °C. The oven temperature was the chemical composition and antimicrobial programmed at 50 °C (1min) to 180 °C at 10 activity of the essential oils of the fruit and leaf °Cmin-1 (0min) and then 230 °C for 5 °Cmin-1 of Averrhoa carambola. (0min) to 330 °C for 10 °Cmin-1 (2min). The mass spectra were acquired at 70 eV within a Materials and Methods mass range of 38-550 Da with a scan time of Collection and preparation of plant material 0.73 scan s-1 and ion source temperature Averrhoa carambola fruits and leaves were maintained at 230 °C. obtained from a farm at Umuebule 4, Oyigbo Town in Oyigbo Local Government Area of Analysis of essential oil Rivers State, Nigeria on the 28th of June 2017. GC-MS was used to analyze the extracted The plant was identified by Professor (Mrs.) B. essential oils of Averrhoa carambola fruit and Green, a Taxonomist of the Department of Plant leaf. The components of the oil were identified Science and Biotechnology, Rivers State based on the comparison of their retention time University, Nigeria. The fruits and leaves were and mass spectra with those of standards and handpicked, washed to remove debris and National Institute of Standards and Technology cleaned. The fresh fruits (2 kg) and fresh leaves (NIST) Standard Reference Database 69 of the (1.5 kg) were separately chopped in to smaller GC-MS system. sizes using a kitchen knife to allow for easy placement into the round bottom flask and to Bioassay of Averrhoa carambola Essential increase surface area. Oils Preparation of Averrhoa carambola leaf and Essential oil extraction fruit essential oils Extraction was done via hydrodistillation using a Essential oil (0.1g) of both leaf and fruit of Clevenger apparatus [16-18] on the 29th of June Averrhoa carambola were separately dissolved 2017. The chopped fruits and leaves were in 10 ml dimethylsulphoxide (DMSO) and 10 ml respectively introduced into the round bottom 0.5 % Tween 80 [19] to produce 100 µg/ml flask fitted with a Clevenger apparatus, and was standard stock solutions for both leaf and fruit heated for about 1hour 30 minutes at a oils used for the disc diffusion assay. temperature of 60 °C to extract the volatile organic compounds present in the fruit and leaf. Test microorganisms The distillates were collected into airtight glass Antimicrobial and antifungal activities of the vials and stored at 4 °C under refrigeration until volatile oils were evaluated against two Gram- analysis. The following formula was used to positive cocci and two Gram-negative bacilli determine the essential oil yield: bacteria and one fungus by the disc diffusion Essential oil yield (%) = W1/W2 x 100 % method. The microorganisms used were Where: W1 = net weight of oil (grams) Staphylococcus aureus, Escherichia coli, W2 = total weight of the plant sample , Bacillus subtilis and (grams) Aspergillus niger. All the microorganisms used were clinical isolates obtained from Department Instrumentation of Medical Laboratory Science, Rivers State The instrument used for the analysis is gas University, Nkpolu-Oroworokwo, Port Harcourt, chromatography-mass spectrometry (GC-MS) Nigeria. with Agilent 7890A model GC system coupled with 5975C VL MSD. The GC equipment was Antimicrobial analysis fitted with HP 5MS capillary column, length The disc diffusion method [20, 21] was used to 30m; internal diameter 0.30 mm; 0.25 µm film assess the antibacterial activity as well as the

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Minimum Inhibitory Concentration (MIC) of the The yield of volatile oil from Averrhoa volatile oil from Averrhoa carambola leaf and carambola fruit was 1.06 % (w/w). From the fruit. Nutrient Agar plates were prepared and a GC-MS analysis, 17 compounds were identified 24hour old culture of the bacterial suspension (Table 1). The data and spectrum (Fig. 1) (equivalent to 0.5 McFarland suspensions) of interpretations were done using the National test microorganisms were inoculated by the Institute of Standards and Technology (NIST) spread plate method. Sterilized filter paper discs Standard Reference Database 69 of the GC-MS approximately 5 mm in diameter were soaked system. The result showed the oil to be with the already prepared essential oil stock composed of estragole (1-methoxy-4-(prop-2- solutions and placed on the prepared agar plates. en-1-yl)benzene) (62.312 %) (3), trans-α- Each disc was pressed down to ensure complete bisabolene (4-[(1E)-1,5-dimethyl-1,4-hexadien- contact with the agar surface and distributed 1-yl]-1-methyl-cyclohexene) (12.209 %) (12), evenly so that they were no closer than 24 mm pinene ((1R)-2,6,6-Trimethylbicyclo[3.1.1]hept- from each other, centre to centre. The agar plates 2-ene) (8.246 %) (1), ocimene (3,7-dimethyl- were incubated at 37 °C for 18 to 24 hours for 1,3,6-Octatriene) (4.095 %) (2) and other minor bacteria and 25 °C for 5 to 6 days for the fungus; compounds. Among constituents identified, after which each plate was examined, and the terpenoids (63.335 %) were the most abundant zones of inhibition were measured including the constituents (Table 2). diameter of the disc. The zones of inhibitions With respect to Averrhoa carambola fruit observed for the essential oils were within the essential oil composition, there have been some range of 0 mm – 14 mm. While those of the investigations providing informative data which standard control (Ciprofloxacin) were within 0 vary from the result of our study. Butyl acetate, mm – 35 mm. DMSO and 0.5 % Tween 80 were ethyl decanoate and hexadecanoic acid were used as negative controls. They showed no shown to be the most abundant components of activity. Three other concentrations (50 µg/ml, Averrhoa carambola fruit essential oil from 20 µg/ml and 10 µg/ml) were prepared from the Cuba [22]. Higher amounts of esters stock solutions and used for the disc diffusion (predominantly methyl anthranilate) were analysis. The least concentrations that inhibited reported for Averrhoa carambola fruit essential the growth of test organisms were taken as the oil from United States of America [23]. Frohlich minimum inhibitory concentration (MIC) of the and Schreier [24] indicated that (E)-hex-2-enal essential oil. (2.4 mg/kg) and methyl benzoate (1.9 mg/kg) were the major aroma compounds of Averrhoa Results and Discussion carambola fruit. These variations in the essential Chemical composition of the volatile oil from oil constituents may be attributed to difference Averrhoa carambola fruit in geographical locations [25].

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A b u n d a n c e

TIC: STAR FRUIT 1.D\data.ms

9 5 0 0 0 0 0

9 0 0 0 0 0 0 1 1 .3 8 9 8 5 0 0 0 0 0

8 0 0 0 0 0 0

7 5 0 0 0 0 0

7 0 0 0 0 0 0

6 5 0 0 0 0 0

6 0 0 0 0 0 0 7 .3 7 6 7 .3 1 0 5 5 0 0 0 0 0 7 .2 24 6

5 0 0 0 0 0 0

4 5 0 0 0 0 0 9 .9 8 4 4 0 0 0 0 0 0

3 5 0 0 0 0 0

3 0 0 0 0 0 0 9 .7 5 5 2 5 0 0 0 0 0

2 0 0 0 0 0 0 5 .5 0 9 1 0 .1 9 4 5 .6 2 8 1 5 0 0 0 0 0 2 3 .6 4 1 1 0 .2 4 1 1 1 .8 1 7 1 0 0 0 0 0 0 7 7 .5 .8 7 9 3 1 1 0 .5 38 81 1 0 .8 9 3 2 1 .0 2 3 2 4 .0 2 4 5 0 0 0 0 0

5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 T im e --> Fig 1: A Chromatogram showing the respective peaks and retention time (Rt) of the various components of the volatile oil from Averrhoa carambola fruit.

Table 1: Chemical Composition of the Volatile Oil from Averrhoa carambola Fruit S/N Compound Rt % Composition 1 Pinene ((1R)-2,6,6-Trimethylbicyclo [3.1.1] hept-2-ene) (1) 5.509 8.246 2 α-Ocimene (3,7-dimethyl-1,3,7-Octatriene) (2) 5.628 4.095 3 Estragole (1-methoxy-4-(prop-2-en-1-yl)benzene) (3) 7.226 62.312 4 Citral B (cis-3,7-dimethyl-2,6-Octadienal) Neral (4) 7.573 0.396 5 Citral A (trans-3,7-dimethyl-2,6-Octadienal) Geranial 7.893 0.647 6 β-Caryophyllene ([1R-(1R,4Z,9S)]-4,11,11 -trimethyl-8-methylene- 9.753 1.979 Bicyclo [7.2.0] undec-4-ene) (5) 7 Trans- α-Bergamotene (2,6-dimethyl-6-(4-methyl-3-pentenyl)- 9.982 3.378 Bicyclo [3.1.1]hept-2-ene) (6) 8 Humulene (2,6,6,9-tetramethyl-1,4-8-cycloundecatriene) (7) 10.194 0.973 9 (E)-β-Farnesene (7,11-dimethyl-3-methylene-1,6,10-dodecatriene) 10.239 0.872 (8) 10 Germacerene D ((S,1Z,6Z)-8-isopropyl-1-methyl-5- 10.538 0.512 methylenecyclodeca-1,6-diene) (9) 11 β-Sesquiphellandrene ([S-(R*,S*)]-3-(1,5-dimethyl-4-enyl)-6- 10.581 0.617 methylene-cyclohexene) (10) 12 β-Bisabolene ((S)-1-Methyl-4-(6-methylhepta-1,5-diene-2-yl) 10.892 0.306 cyclohexa-1-ene) (11) 13 trans-α-Bisabolene (Cyclohexene, 4-[(1E)-1,5-dimethyl-1,4- 11.389 12.209 hexadien-1-yl]-1-methyl-) (12) 14 Caryophyllene oxide (4,12,12-trimethyl-9-methylene-5-oxatricyclo 11.817 0.639 [8.2.0.] dodecane) (13) 15 2,6,11-trimethyl- Dodecane (14) 21.023 0.575 16 Pentacos-1-ene (15) 23.641 1.537 17 Pentacosane (16) 24.024 0.708

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Table 2: Major Classes of Compound in the Volatile Oil from Averrhoa carambola Fruit S/N Class of Compounds No. of Compounds % Concentration 1 Monoterpenes 2 4.095 2 Sesquiterpenes 8 21.485 3 Aliphatic hydrocarbon 3 2.82 4 Terpenoids 3 63.335

Chemical composition of the volatile oil from bisabolene (4.784 %) (11), pentadecanal (3.237 Averrhoa carambola leaf %) (20), (R)-1-methyl-4-(6-methylhept-5-en-2- The yield of volatile oil from Averrhoa yl) cyclohexa-1,4-diene (1.767 %) (18), and carambola leaf was 0.87 % (w/w). The GC-MS 1,2,4-trimethylbenzene (1.702 %) (17). Among analysis identified seven compounds (Table 3). constituents identified, terpenoid (66.847 %) The data and spectrum (Fig. 3) interpretations was the most abundant constituent (Table 4). were carried out using National Institute of The leaf and fruit essential oils contain the same Standards and Technology (NIST) Standard major compound (estragole), but considerable Reference Database 69 of the GC/MS system. variations could be discerned from the leaf and The result showed the oil to be composed of fruit volatile oils composition; presumably estragole (1-methoxy-4-(prop-2-en-1- because the essential oils are from different parts yl)benzene) (66.847 %) (3), bis(2- of the plant. To the best our knowledge, there is ethylhexyl)phthalate (11.964 %) (21), 3,7- no reported data on the essential oil of Averrhoa dimethyl-1,3,7-octatriene (9.699 %) (2), cis-α- carambola leaf.

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A b u n d a n c e

TIC: STAR FRUIT LEAVE 1.D\data.ms

2 9 0 0 0 0 0 6 . 8 0 8 2 8 0 0 0 0 0 2 7 0 0 0 0 0 2 6 0 0 0 0 0 2 5 0 0 0 0 0 2 4 0 0 0 0 0 2 3 0 0 0 0 0 2 2 0 0 0 0 0 2 1 0 0 0 0 0 2 0 0 0 0 0 0 1 9 0 0 0 0 0 1 8 0 0 0 0 0 1 7 0 0 0 0 0 1 6 0 0 0 0 0 1 5 0 0 0 0 0 1 4 0 0 0 0 0 1 3 0 0 0 0 0 1 2 0 0 0 0 0 1 1 0 0 0 0 0 2 4 . 7 0 9 1 0 0 0 0 0 0 9 0 0 0 0 0 8 0 0 0 0 0 5 . 2 8 2 7 0 0 0 0 0 6 0 0 0 0 0 5 0 0 0 0 0 1 1 . 3 2 2 4 0 0 0 0 0 3 0 0 0 0 0 3 . 6 7 1 9 . 9 2 9 1 3 . 3 7 5 2 0 0 0 0 0 1 0 0 0 0 0

5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 T im e - -> Fig 3: A Chromatogram showing the respective peaks and retention time (RT) of the various components of the volatile oil from Averrhoa carambola Leaf

Table 3: Chemical Composition of the Volatile Oil from Averrhoa carambola Leaf S/N Compound Rt % Composition 1 1,2,4-trimethyl-Benzene (17) 3.671 1.702 2 α-Ocimene (3,7-dimethyl-1,3,7-Octatriene) (2) 5.282 9.699 3 Estragole (1-methoxy-4-(prop-2-en-1-yl)benzene) (3) 6.808 66.847 4 β-Curcumenene ((R)-1-Methyl-4-(6-methylhept-5-en-2-yl) cyclohexa- 9.929 1.767 1,4-diene) (18) 5 cis-α-Bisabolene (19) 11.322 4.784 6 Pentadecanal (20) 13.375 3.237 7 Bis(2-ethylhexyl) phthalate (21) 24.709 11.964

Table 4: Major Classes of Compound in the Volatile Oil from Averrhoa carambola Leaf S/N Class of Compounds No. of Compounds % Concentration 1 Monoterpenes 1 9.699 2 Sesquiterpenes 1 4.784 3 Aliphatic hydrocarbon 1 1.767 4 Aromatic Hydrocarbon 1 1.702 5 Ester 1 11.964 6 Aldehyde 1 3.237 7 Terpenoid 1 66.847

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Antimicrobial activity and Gram-negative bacteria. This could be In-vitro antimicrobial activities of essential oils attributed to the ineffectiveness of the emulsifier of the fruit and leaf of Averrhoa carambola (Tween 80) in enhancing the diffusion of the oils showed a range of growth inhibition pattern through the agar medium. DMSO emulsified oils against pathogenic microorganisms (Table 5 and exhibited good activity by inhibiting the growth 6 respectively), these results tend to support its of three out of five test organisms; it current use in alternative medicine to treat successfully inhibited the growth of both Gram- infectious diseases [1, 2]. In this study, the positive and Gram-negative bacteria but showed result showed that 100 µg/ml Averrhoa no activity against the fungus. carambola (fruit and leaf) essential oils inhibited MIC values of 10 μg/ml - 20 μg/ml suggest that the growth of three pathogenic bacteria, the essential oils of Averrhoa carambola in although showed no activity for the pathogenic higher concentrations will outperform the fungus employed in the research. These standard drug (25mg Cipro-Floxacin) used. activities were obvious on Gram-positive However, essential oils of both leaf and fruit bacteria when compared with Gram-negative showed no fungicidal activity. bacteria. The presence of some compounds identified in The fruit essential oil showed a remarkable both the Averrhoa carambola leaf and fruit activity when tested against Gram-positive and essential oils may have played a major role in Gram-negative bacteria. It inhibited the growth the antimicrobial activity observed in this work of three pathogenic bacteria (two Gram-positive and as previously reported [11, 12]. Compounds and one Gram-negative), however, the leaf like estragole (1-methoxy-4-(prop-2-en-1- essential oil showed activity against only one yl)benzene) which showed to be the most Gram-positive bacteria. predominant volatile chemical component has Tween 80 emulsified oils of both Averrhoa been reported to exhibit antimicrobial activities carambola fruit and leaf showed very poor [26-28]. activity when tested against the Gram-positive

Table 5: Antibacterial and Antifungal Activity of Volatile Oil from Averrhoa carambola Fruit using Disc Diffusion Method Micro-organisms Zone of Inhibition (mm) MIC 100 µg/ml of 100 µg/ml of Cipro- Fungusol (µg/ml) volatile oil in volatile oil in Floxacin (60 mg) DMSO 0.5% Tween 80 (25 mg) Escherichia coli 10.75 + 0.75 0 35.50+0.50 0 20.00

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Pseudomonas 0 0 32.50 + 0.50 0 0 aeruginosa Bacillus subtilis 13.50 + 0.50 11.30+ 0.30 30.00 + 0.00 0 10.00 Staphylococcus 12.00+ 0.50 0 0 0 10.00 aureus Aspergillus niger 0 0 0 0 0 *MIC- Minimum inhibitory concentration, 0= no activity. Zone of Inhibition = Mean + SD

Table 6: Antibacterial and Antifungal Activity of Volatile Oil from Averrhoa carambola Leaf Using Disc Diffusion Method Micro-organisms Zone of Inhibition (mm) MIC 100 µg/ml 100 µg/ml Cipro- Fungusol (µg/ml) volatile oil in volatile oil in Floxacin (60 mg) DMSO 0.5% Tween 80 (25 mg) Escherichia coli 0 0 35.50+0.50 0 0 Pseudomonas 0 0 32.50 + 0.50 0 0 aeruginosa Bacillus subtilis 0 0 30.00 + 0.00 0 0 Staphylococcus aureus 10.50 +0.60 0 0 0 20.00 Aspergillus niger 0 0 0 0 0 *MIC- Minimum inhibitory concentration, 0 = no activity, Zone of Inhibition = Mean + SD

Conclusion of the Department of Plant Science and Essential oils from the fruit and leaf of Averrhoa Biotechnology, Rivers State University, for the carambola L. have good potentials as identification of the plant. antimicrobial agents and can also be used in treating infectious diseases caused by Gram- References positive organisms. Irrespective of the solvent 1. J. E. F. Reynolds. (1998). Martindale: The used in the emulsification of the essential oil and Extra Pharmacopeia. London: Royal very low concentrations used, the essential oil of Pharmaceutical Society of Great Britain, 31: the fruit of Averrhoa carambola L. showed good 592-601. antibacterial activity. This work reports that the 2. C. K. Sung, T. Kimura, P. P. H. But and J. essential oils from both fruit and leaf of X. Guo. (1998). International Collation of Averrhoa carambola L. grown in Southern Traditional and Folk Medicine: Northeast Nigeria comprised majorly of estragole (1- Asia, Singapore: World Scientific methoxy-4-(prop-2-en-1-yl)benzene). Publishing Company: 75-76. 3. S. Thomas, D. A. Patil, A. G. Patil and N. Chandra. (2008). Pharmacognostic Acknowledgements evaluation and physiochemical analysis of The authors are grateful to Rivers State Averrhoa carambola L. fruit, Journal of University and University of Lagos for their Herbal Medicine and Toxicology, 2(2): 51- laboratory assistance; and Prof. (Mrs.) B. Green 54.

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