PHYSICOCHEMICAL PROPERTIES AND INHIBITORY EFFECTS OF ESSENTIAL OILS FROM SELECTED LOCAL SPICES

Spices are rich in essential oils and are known to possess antiviral, antibacterial, antifungal and insecticidal properties. This study evaluated the inhibitory effect and physicochemical properties of essential oil from some selected spices. Cinnamon, ginger and garlic were pulverized and extracted using the soxhlet method with n-Hexane. The extracted oils were subjected to physicochemical and microbial analysis. Results showed that ginger gave the highest oil yield (7.01%) in comparison with cinnamon and garlic: 2.75 and 1.33 % oil yields at respective 10.62, 14.29and 11.16 % moisture contents. Iodine value, acid value and free fatty acid value was significantly (p<0.05) higher in cinnamon oil (506.15, 20.66 mgKOH/g and 10.33 mgKOH/g respectively) compared to the respective 348.65, 15.41 mgKOH/g and 7.71 mgKOH/g for ginger and 128.15, 17.90 mgKOH/g and 8.95 mgKOH/g for garlic. Saponification value was significantly (p<0.05) higher in ginger (226.15mgKOH/g) than in cinnamon (214.75 mgKOH/g) and garlic (198.54 mgKOH/g). A significantly high (p<0.05) peroxide value was observed in garlic with 247.12 milli eq/Kg, and 234.15 and 247.12 milli eq/Kg for cinnamon and ginger respectively. Cinnamon oil was observed to be the most potent against the tested microorganisms, showing maximum inhibition zones of 0.9 cm for E . coli , 1.1 cm for P . aeruginosa and 2.4 cm for each of K. pneumonia , Serratia spp. and S. aureus. Result from the study revealed that the extracted oils are not fit for consumption, highly susceptible to rancidity and oxidation but are of good potential use in paint and soap making.


INTRODUCTION
Essential oils (EOs) are plant-derived volatiles with a hydrophobic character. They have been defined as intricate mixtures of volatile, odoriferous, lipophilic and liquid substances (Krisch, 2011;Simõeset al., 2004). Essential oils, being products of secondary plants' metabolism,play important roles in plant protection such as antiviral, antibacterial, antifungal, insecticidal properties, also against herbivore attack. Majority of them are used as seasonings and medicines (Simões, 2004;Chouhanet al., 2017).According to Bakkaliet al.(2008),of about 3,000 essentials oils which are currently known,approximately 300 of them are commercially important in the food, sanitation, pharmaceutical, agronomic, cosmetics, and perfume industries.
Variousadditives are used to extend the storage period of foods and to prevent the growth of microorganisms (Özcanet al.,2005).In spite of that, there is an immerse interest in the use of alternatives, mainly natural products, such asplants and spices (Marino et al.,2001;Huntanen, 1980). Spices are rich in essential oils and extracts, with established antimicrobial activity. Due to the recognition of the antimicrobial propertiesfor the past years (Hammer et al.,1999),these materials can be used to suppressor delay the emergence of microorganisms.Most herbs and spices exhibit antimicrobial activity due to the fractions of their essential oils.Several EOs from medicinal and aromatic plants have been known since ancient years to possess biological activity, particularlyantifungal, antibacterial and antioxidant properties (Barattaet al.,1998;Cosentinoet al.,1999;Bounatirouet al.,2007).Essential oils can be obtained through fermentation, expression, effleurage or extraction but according to Burt (2004), the most generally used method for mass production is the steam method.
Basically, the antimicrobial activity of plant oils and their extracts have become the basis of various applications,including raw and processed food preservation, pharmaceuticals,medicine and natural therapies (Hammer et al.,1999) The aim and objectiveof this study was to determine the inhibitory effect of essential oils gotten from three (3) different spices against some food spoilage microorganisms and to ascertain the physicochemical properties of the essential oils with a hope to further the study for food preservation and storage.

MATERIALS AND METHODS
All reagents used for this research work were of analytical grade unless otherwise stated. This research work was carried out at the Chemistry/Biochemistry and Microbiology laboratories of the Nigerian Stored Products Research Institute, Ilorin. Kwara state.

Sample Collection and Preparation
Cinnamon, ginger and garlic were purchased from Yoruba Road market in Ilorin. Ginger and garlic samples were peeled, washed and sliced to small pieces, Cinnamon bark was cleaned and broken to pieces, the samples were then oven dried at 50 o C to a constant weight. The samples were ONYEGBULA 1 *, AKUDO FRANCILIA, LAWAL 1 , ISRAEL OLUWASANMI, FASHANU 1 , TITILOPE ABOSEDE, SALAWU 1 , RUKAYAT ADENIKE, ADEBAYO 1 ODUNAYO BENEDICTA, AYANDA 1 , IFEDAPO SOLOMON pulverised with the aid of hammer mill. The milled samples were placed in neat and well labeled air tight plastic bottles and kept for further use.

Determination of moisture content
The moisture content determination was carried out adopting the AOAC methods. A weighed portion (5g) of the milled samples were dried in an oven to a constant weight at 105 0 C for 5 hours.

Oil Extraction and Oil Yield Determination (%)
The oil content of each sample wasdeterminedseparately bycomplete extraction using the Soxhlet extractor (Konte, USA).50g of milledsamples were placed intoa porous thimble in a soxhlet extractor, using n-hexane (150mL) as the extracting solvent for 6 hours. The oil was obtainedafter the solvent was removed under reduced temperature andpressure and refluxing at 70 o C to remove the excess solventfrom the extracted oils (Abdulhamid et al., 2014). The oils were placed in bijou bottles and kept for further use.
The % oil yield was determined by placing the extracted oils over water bath for 30min at 70 o C to ensure complete evaporation of solvent and weight of the oil was recorded and expressed as oil contentas described by AOAC (2000). The oil yield (%) was calculated as thus:

Physicochemical Analyses
The physicochemical analyses of the oils were determined using standard methods as reported in each of the parameters. The parameters analysed were refractive index, saponification value, acid value, free fatty acid, peroxide value and iodine value and the methods of analysis used are as follows:

Saponification value
As described by Abdulhamidet al. (2014), 2g of each oil sample was added to a flask with 30 mL of ethanolic KOH and was then attached to a reflux condenser for 30minutes to ensure that the sample was fully dissolved. Aftersample was cooled, 1ml of phenolphthalein was added andtitrated against 0.5 M HCl until a pink colour appeared which indicatedthe end point.A blank was also carried out which contains all reagents but no oil and was titrated against 0.5M HCl. The saponification value was calculated as thus: Where V0 = Volume of the solution used for blank test V1 = Volume of the solution used for sample N = Actual Normality of the HCl used M = Mass of the sample.

Iodine Value (IV)
The Iodine Value of the oils were determined using a mathematical relationship betweenrefractive index and iodine value has been describedby Perkins (1995) and reported by Amos-Tautuaet al. (2013)as η/D 30 Refractive index (RI) = 1.45765+0.0001164 IV (Iodine value). The relationship was adjusted and used to calculate theiodine value of oils since the refractive indices are is knownas follows: Iodine Value (gI2/100g)= Acid value/Free Fatty Acid 25ml ofneutral ethanol was heated to boiling and added to 1g of each oilextract to dissolve in a conical flask. The heating was stopped and the solution was titratedwith 0.1MNaOH solution using phenolphthalein as indicator (Amos-Tautuaet al., 2013).
The free fatty acid of the oil can be determined from the expression of: Free Fatty Acid (% as Oleic acid) =

Peroxide Value
The peroxide value of the extracted oils were determined using the standard method described by Akpanet al. (2006). Exactly 1.0g of KI and 20ml of solvent mixture (glacialacetic acid: chloroform, 2:1 v/v) were added to 1.0g of the oilsample and the mixture was boiled for one minute. The hotsolution was poured into a flask containing 20ml of 5% KIO3solution. Few drops of starch solution were added to themixture and the latter was titrated with 0.025M Na2S2O3solution. A blank was also carried out and titrated with 0.025M Na2S2O3.

Microorganisms used and standardization of the inoculums
The experiment was undertaken using a modification of Sofyet al. (2017), basically the agar well diffusion assay in a duplicate setup.

Extract Description
Three plant extracts presented as Garlic, Ginger and Cinnamon were evaluated for antimicrobial efficacy against five clinical/food bacterial isolates.

Culture Media Used
Nutrient Agar: 28g of the commercial medium (Lab M Limited, Lancashire) was dissolved in 1000mL of distilled water, homogenized and sterilized at 121°C for 15mins in the autoclave. Molten medium was allowed to cool before pouring. Mueller Hinton Broth: 21g of the product (HiMedia, India) was weighed into 1000ml distilled water; the mixture was homogenized on a laboratory hot plate with repeated swirling before sterilization in the autoclave at 121°C for 15mins. Mueller Hinton Agar: 28g of the commercial medium (Oxoid, UK) was dissolved in 1000ml distilled water; with frequent agitation, the solution was homogenized on the magnetic hot plate and autoclaved at 121°C for 15mins. Upon cooling 40 -45°C, the molten medium was dispensed into sterile petridishes on a uniform laboratory bench to achieve uniform depths in the agar plates.

Test microorganisms and Inoculums Preparation
The identified bacterial species: Escherichia coli, Klebsiellapneumoniae, Pseudomonas aeruginosa, Serratia sp. and Staphylococcus aureus were obtained from the University of Ilorin Teaching Hospital on nutrient agar slants. From the overnight bacteria cultures, 2 -4 colonies respectively was discretely suspended in 15ml Mueller Hinton Broth and incubated at 37°C overnight. Each test tube containing 5ml bacteria suspension had turbidity adjusted to 0.5 MacFarland standard of 1.5 x 10 8 cfu/ml.

Sensitivity Test
Solidified Mueller Hinton Agar plates were welled with a 0.8cm sterile stainless steel cork borer; the bored agar plates were seeded with 3ml respective bacteria inoculums, swabbed over the entire agar surface using sterile swab sticks.
Using a micropipette, 500μL of each extract was dispensed into the wells occupying the centre of each agar plates; covered with the Petri dish lid, plates were incubated in the incubator at 37°C for 18 -24 hours. Control plates lack the incorporated extracts.
Observed zones of inhibition were accurately measured using a laboratory meter rule and recorded appropriately.

Statistical Analysis
All analysis was carried out in three replicates, except stated otherwise. Data was subjected to analysis of variance (ANOVA) and tested for significance difference among spices by New Duncan's Multiple Range F-Test (DMRT) at (p<0.05) using SPSS software package version 20.0.0 (IBM Statistics).

Physicochemical Properties
The physicochemical properties of oils extracted from the selected spices are presented in Table 1. As shown below, cinnamon, ginger and garlic were extracted at 14.29%, 10.62% and 11.16% moisture contents with the resulting 2.75%, 7.01% and 1.33% yields respectively. In comparison with some conventional oil seed cropslike soybean, mustard andcotton with yields in the range of 17.0 -21.0%, 24.0-40.0% and 15.0 -24.0% respectively, (Pritchard, 1991) yields recorded in this study was found to be relatively low. This has revealed that the selected spices cannot be regarded as oil crops.
Therefractive index is a physical parameter that reveals the level of purity of oils and also explains the degree of the deflection that occurs when a beam of light passes from one transparent medium to the other (Pearson, 1976). The results of the refractive index of the essential oilsmeasured were 1.52, 1.50 and 1.47 for cinnamon, gingerand garlic respectively. The valuegotten for the essential oils is evidence that the samples might be of long carbon chain with high degree of unsaturation. It's also an indication that the oilsare pure in order of decreasing values.
The iodine value of oil gives an indication of the degree of unsaturation of the oil. The higher the iodine value of an oil, the higher the degree of unsaturation (Carbon to carbon double bonds) of the oil, which is also a good advantage in the production of soap (Akinhanmiet al., 2008). The greater the iodine value, the higher the susceptibility of the oil to oxidation (Amos-Tautua, and Onigbinde, 2013). The Iodine values of the essential oils were found to be 506.15, 348.65 and 128.15 for cinnamon, ginger and garlic respectively. In this context, cinnamon oil has the highest iodine value, hence the most unsaturated of the oils, followed by ginger while garlic has the lowest iodine value and least susceptible to oxidation.
The acid values for cinnamon, ginger and garlic oils were found to be 20.66, 15.41 and 17.90 mgKOH/g respectively. These values are relatively higher than that of olive oil 17mgKOH/g as reported by (Warraet al., 2012). Acid value indicates the amount of free fatty acids present in oils and the higher the acid value, the lower the storability and the higher the risk of the oil undergoing rancidity. Essential oils contain several volatile aroma compounds, mostly as free fatty acids. (Abdulhamidet al., 2014). The free fatty acids present in the extracted oils as indicated below were found to be high, with cinnamon being significantly (p<0.05) higher than garlic which was in turn significantly (p<0.05) higher than ginger.
The high values recorded for acid value suggests the oils not useful for skin care products (Coenon, 1976). Also, the free fatty acid values were found to be higher than 2.33% recorded by (Amos-Tautuaet al 2013) for groundnut oil and much lower than 32.95% for corn oil. For oils to be considered edible, it should fall within the allowable limit of 1.0-3.0% for free fatty acid (Paul and Mittal, 1997). The free fatty acid of the oils under study suggests the oils are not suitable for consumption, highly susceptible to rancidity and have a low storability.  (Mabrouk, 2005). This justifies that the oils used in this study would be of good use in the production of soap.
The peroxide values of cinnamon, ginger and garlic oils are as shown in Table 1. It was discovered that the peroxide values are relatively high with a significant difference between the samples. The high values contribute to increase in rancidity rate (Epka, 1996). This fact suggests that the oils understudy are highly susceptible to rancidity at room temperature.

Antimicrobial Properties
The antibacterial efficacy of the respective extracts (Cinnamon, Garlic and Ginger) was tested against five bacteria species: Escherichia coli, Klebsiellapneumoniae, Pseudomonas aeruginosa, Serratia sp. and Staphylococcus aureus( Table 2 below). Generally, the zones of inhibition diameter ranged 0.4 -2.5cm as revealed in Table 3  The results obtained for antibacterial effect of this study utilizing n-Hexane as the extractant, presents higher efficacy against Pseudomonas aeruginosa than ethanolic extract of garlic as reported by Akintobiet al., 2013. The resistance of Escherichia coli to ethanolic extract of garlic as presented by the authors was subjected in the bacteria's susceptibility to n-Hexane extract in this study. The resistance of Pseudomonas aeruginosa was also reported byAbdulzahra and Mohammed (2014) to aqueous extracts of garlic and ginger; this study presents similar resistance to the n-Hexane extracts of garlic and ginger but susceptibility to cinnamon extract, although relatively lower than the garlic-ginger ethanolic extract as reported by the authors. Thus, in the qualitative yield of an extractant for antibacterial potency, n-Hexane is presented.From the results obtained, the Cinnamon extract has the highest antibacterial potency against the test bacterial species.

CONCLUSION
The result of this study has shown that the selected essential oil are not suitable for consumption due to their high acid value and free fatty acid values but of good use in the paint, soap making and pharmaceutical companies/industries with cinnamon oil being the most important due to its highest degree of unsaturation compared to others. It can also be concluded thatthe selected essential oils have inhibitory effects on microorganism with cinnamon oil being the most