Physicochemical Properties of Essential oils of Ginger ( Zingiber officinale ) , African Nutmeg ( Monodora myristica ) and Ashanti Black Pepper ( Piper guineense ) 1

This study was carried out to evaluate the physicochemical characteristics of Ginger (Zingiber officinale), African nutmeg (Monodora myristica) and Ashanti Black pepper (Piper guineense) essential oils. Dried plant materials were obtained from the Oba market in Edo State, Nigeria. All samples were ground into a fine powder and stored in an airtight plastic container at room temperature (28±2oC) until when used. The essential oils were extracted using steam distillation and their physicochemical properties analyzed using standard procedures. The results showed that color varied from yellow for Z. officinale to colorless for M. myristica and P. guineense. The highest yield (3.55%), acid value (6.73 mg KOH/g), saponification value (185.13 mg KOH/g), free fatty acid value (3.37%) and iodine value (69.49 gI2/100 g) were recorded for M. myristica, the highest specific gravity value (0.88) was for P. guineense and M. myristica, the highest peroxide value (2.67) was for P. guineense while the highest refractive index value (1.49) was for both P. guineense and Z. officinale. There were significant differences (p<0.05) in all physicochemical parameters except for the refractive index and specific gravity of the essential oils. The result indicates that the essential oils of the selected plants can be utilized as edible oils and suitable for nutritional purposes. The oils are stable and would not easily undergo rancidity, thus they can be used as a good component of food additives.


INTRODUCTION
Spices are aromatic vegetative substances used for seasoning of food and from which no portion of any volatile oil or flavouring principles have been removed and are free from artificial colouring matters, adulterants and impurities (Farrel, 1990). They are Generally Recognized As Safe'' (GRAS) when used at concentrations normally found in foods. In Nigeria commonly used spices include ginger, garlic, onions, nutmeg, clove, Ashanti pepper etc. They can be used whole, pulverized or as extractives such as essentials oils or oleoresins. Most spices contain carbohydrate, protein and small amount of minerals. Only a small fraction of the dry matter which contribute to flavoring for example; tannins, resins, pigments, volatile, essential and fixed oils occur in traces (Cowan, 1999). Essential oils are volatile and aromatic produces or mixtures of produce which are formed in cytoplasm occurring as tiny droplets amongst cells of plants (Toure and Xiaoming, 2007). The plant from which they are gotten gives them a characteristic taste and odor (British Pharmacopoeia Commission, 2013;Burdock, 2005). This disparity in composition may be as a result of a variety of plants (Sarac and Ugur, 2008), geographical locations (Mechergui et al., 2010;Sarac and Ugur, 2008), harvesting seasons (Hussain et al., 2008;Figueiredo et al., 2008;Taiz and Zeiger, 2010), drying methods (Di Cesare et al., 2003) and extraction methods (Karakaya et al., 2011;Burt, 2004). Quality, freshness and distinctiveness of the oil are main concerns that affect the value of essential oils (Shahidi and Zhong, 2005). Due to the nutritional values of oils and importance for industrial purposes, the use and demand of oils have increased resulting in their exploration from plants (Adolf et al., 2018). Thus, the importance of physicochemical characteristics of these oils is of utmost importance in our daily life's. The quality of essential oils is ultimately affected by the physicochemical properties. These properties are used in regulating their consumption as well as determining their market value (Parthiban et al., 2011;Bamboye and Adejumo, 2010). Despite numerous studies carried out on these spices, there is a dearth of literature on the physicochemical characteristics of plant essential oil. This study is intended to uncover the nature and importance associated with the physicochemical characterization of the essential oils to ascertain their suitable application in the food industry.

Collection of materials and preparation of samples:
The dried plant materials of Ginger (Zingiber officinale), African nutmeg (Monodora myristica), Ashanti Black pepper (Piper guineense) were obtained Oba market in Edo State, Nigeria. They were then taxonomically identified and authenticated in the Department of Plant Biology and Biotechnology, University of Benin, Benin City Edo State. All plant materials were pulverized into fine powder. The powder was stored at room temperature (28±2 o C) in an air tight container until it was used.
Extraction of the essential oils: Three thousand gram of the ground dried powder of each plant material was measured into distillation flask fitted with condensers. Steam was provided to the flask through a steam generator at constant flow. The essential oil which vaporizes with the steam was condensed into a collecting separating funnel. The oil was separated by gravity and dried over anhydrous sodium sulphate, filtered and stored at 4°C in a refrigerator until analyzed (Hussain et al., 2008).

Physicochemical analysis of essential oils:
The extracted essential oils were analysed for yield, refractive index, relative density (specific gravity), saponification value, iodine value, acid value, peroxide value and free fatty acid value of the essential oils were determined according to the methods below.

Determination of yield:
This is the quantity of essential oil extracted from the plant materials. The yield of the oil (hydrophobic fraction) was recorded as volume: weight ratio (Ayoola et al., 2008;Gundidza et al., 2009): Weight of extract recovered weight of dried plant material × 100 Color determination: Color of the oils was determined by physical observation in day light and under ultraviolet radiation of 254 and 366 nm by means of an ultra violet chamber (Bamgboye and Adejumo, 2010).

Determination of refractive index:
Refractometer was used to determine the refractive index of the extracted oil. One drop of the oil was dropped on the cell compartments of the instruments. The necessary adjustment was made and the result recorded when the lower part became darker (AOAC, 1990).
Determination of specific gravity: The density bottle was cleaned, dried, weighed and filled with distilled water, then immersed in a bath at 20°C until the water inside reaches 20°C. The outside of the bottle was wiped and weighed. The bottle was emptied, dried and filled with the oil at 20°C. It was later kept in a bath, wiped and weighed again (AOAC, 1990):

Determination of saponification value:
The Saponification Value (SV) is a measure of the amount of alkali necessary to saponify all the triglycerides present in the sample. Two grams of the oil was weighed into a 250 cm 3 conical flask containing 25 cm 3 of alcoholic potassium hydroxide solution. A reflux condenser was attached to the flask and heated on a boiling water bath for 1 h with occasional shaking. One cm 3 of phenolphthalein indicator was added and titrated while hot with standard hydrochloric acid (0.5 M) until end point is colourless (AOAC, 1990). Blank determination was carried out: where, a = Volume of 0.5M HCL used in oil titration b = Volume of 0.5M HCL used in Blank titration

Determination of iodine value:
The iodine value of oil is a measure of the saturation of fatty acids. Iodine value determination is used to determine the amount of unsaturation in fatty acids. Iodine value is the weight of iodine absorbed by 100 parts by weight of the sample. It was determined using Wij's method. Zero-point five gram of the sample was weighed into a dry glass stoppered flask of about 250 cm 3 capacity, 10 cm 3 of carbon tetrachloride was added and swirled until it dissolved. Twenty cm 3 of Wij's solution was added to it, the solution was mixed and it was allowed to stand in the dark for 30 min with occasional shaking. Fifteen cm 3 potassium iodide and 100 cm 3 of water were added to the mixture. The whole solution was mixed and titrated with the standard sodium thiosulphate using freshly prepared starch as indicator (AOAC, 1990). Blank determination was carried out. Calculation: Iodine value = Determination of free fatty acid: Twenty-five cm 3 diethylether and 25 cm 3 ethanol were mixed inside a 250 cm 3 capacity of conical flask and warmed on hot plate for 5 min. One g of oil was dissolved in the solvent-mixture and also warmed on hot plate for 5 min and removed. Two drops of phenolphthalein indicator was added and titrated with standardized 0.1M NaOH until a faint colour appeared. The process was repeated for three consecutive titre value (AOAC, 1990): Free fatty acid = ]^?_ IJEH> X Statistical analysis: All data obtained in this study were represented as mean±Standard Error (S.E.) of triplicates values. The data were then subjected to a One-way analysis of variance laid in a completely randomized design. Duncan Multiple Range Test at 95% confidence level was used to separate significant.

RESULTS AND DISCUSSION
The physico-chemical parameters of the essential oil's samples are presented in Table 1.
Colour: Physical observation of the essential oils for colour showed that M. myristica and P. guineense were colourless while Z. officinale was pale yellow. These results are in accordance with studies by Udoh et al. (2004) for M. myristica, Nandi et al. (2013) for Z. officinale but differed from Uzeh and Oguntosin (2013) who recorded yellow for P. guinneense.

Yield:
The highest yield of 3.55% was recorded for M. myristica while the lowest 1.17% was recorded for Z. officinale. Significant differences (p<0.05) were recorded in the yield between samples. The essential oil yield for the samples was low. M. myristica had the highest yield compared to Z. officinale and P. guineense. Ifesan et al. (2010) recorded a yield that is about 13 times the yield obtained in this study for Z. officinale, although the yield was higher than 0.47 and 0.50% reported by Talla et al. (2013) and Nguefack et al., (2004). The yield of M. myristica and P. guineense were also low compared to 2.16 and 37.82% reported by Owokotomo and Ekundayo (2012) and Ogbonna et al. (2015) respectively. Generally, the percentage yield of the oils was higher than the essential oils of Eucalyptus globulus (0.2%) and Eucalyptus robusta (0.13%) as reported by Boukhatem et al. (2014). The yield of oil obtained in this study varied with other researchers, this may be due to the application of different methodologies. According to international standards, a product essential oil is deemed commercially acceptable when its concentration is higher than 0.4%, (Hanus et al., 2006;Marquard and kroth, 2002). This study showed that the essential oils yield from these plants were higher than 0.4% as such can be regarded as commercially acceptable.  Specific gravity: The specific gravity was 0.87 for Z. officinale, 0.88 for M. myristica and 0.88 for P. guineense respectively. There were no significant differences (p>0.05) recorded between samples. The ratio of the density of a respective substance to the density of water at 4°C is known as specific gravity (Bamboye and Adejumo, 2010). It is used to assess the purity of the oils (Ogbonna et al., 2015). The specific gravity of all the oils were <1, this implies water is heavier than the oils. The specific gravity of the oils obtained in this study were lower than 1.05 for Z. officinale, 0.96 for M myristica and 0.91 for P. guineense reported by Ifesan et al. (2010), Adolf et al. (2018) and Ogbonna et al. (2015) respectively. However, they were within the specific gravity values range of 0.82-0.92 reported by Akubugwo and Ugbogu (2007).

Refractive index:
The results of the refractive index recorded for the essential oils samples ranged between 1.48 for M. myristica and 1.49 for Z. officinale and P. guineense. There were no significant differences (p>0.05) between samples. The results of the refractive index recorded of the essential oils for the samples were slightly higher than the value of 1.46 obtained for B. sapida oil (Akintayo et al., 2002) but comparable with the values for most drying oils whose refractive index are between 1.48 and 1.49 (Oluba et al., 2008). When oils have high number of carbon-atoms in their fatty acids, it indicates the refractive index is high (Adolf et al., 2018). As the double bond increases, the refractive index also increases leading to high degree of unsaturation (Adolf et al., 2018).

Acid value:
The highest acid value of 6.73 mg KOH/g was recorded in M. Myristica while the lowest 5.01 mg KOH/g was recorded in Z. officinale. There were significant differences (p<0.05) in acid values recorded among samples. The acid value is a measure of the amount of Free Fatty Acids (FFA) present in the oil (Popoola and Yangomodou, 2006). It is often an ideal measure of the breakdown of triacylglycerols into free fatty acids, which reflects on the essential property or quality of many lipids. Generally, it points to the degree of edibility of the oil (Amira et al., 2014). This has an inverse relationship with the free fatty acid content (Bachheti et al., 2012). These values were found in the permissible limits of 10 mg KOH/g of oil and found suitable for dietary purposes (Essein and Amadi, 2009).

Free fatty acid:
The highest free fatty acid values 3.37% was recorded in M. Myristica while the lowest 9.56% was recorded in Z. officinale. Results showed significant differences (p<0.05) recorded between samples. The Free fatty acid is the percentage weight of a specified fatty acid (expressed in oleic acid). According to Oladiji et al. (2010) andOluba et al. (2008, the lower the free fatty acid value, the better the quality of the oil. When there is an increase in the quantity of FFA in a sample of oil or fat, it shows that hydrolysis of tryglycerides has occurred (Akubugwo et al., 2008). The free fatty acids values were greater than one so this indicates the edibility of the oils (Amira et al., 2014). Also, Free Fatty Acids (FFA) values were higher than the minimum limit of 2.0% set for highgrade oils by Codex Alimenarius Commission (1993). The oils of these samples can therefore be used as edible oils and are suitable for nutritional purposes.
Perioxide value: Perioxide values ranged between 2.33 meq/kg for M. myristica and 2.67 meq/kg for P. guineense. There were significant differences (p<0.05) recorded between samples. Peroxide Value (PV) is used to measure the extent to which rancidity reactions have occurred during storage. It could be used to show the quality and stability of fats and oils (Stoilova et al., 2007;Gulfraz et al., 2006). The oils in this study recorded low peroxide values compared to the maximum acceptable value of 10 meq/kg specified by Standard Organization of Nigeria (SON) (2000) and the Codex Alimentarius Commission (2001). Consequently, the oils are thus stable and would not undergo rancidity easily.
Saponification value: Saponification values obtained were 173.21 mg KOH/g for Z. officinale, 185.13 mg KOH/g for M. myristica and 171.25 mg KOH/g for P. guineense respectively. Significant differences (p<0.05) were recorded between samples. The saponification value is a measure of the proportion of low molecular weight triacylglycerols in oil (Oladiji et al., 2010). The high values of saponification in this study indicates high mean molecular weight of fatty acids or high number of ester bonds (Zahir et al., 2014). Furthermore, saponification value indicates the average molecular mass of fatty acid which is inversely proportional to the chain length of fatty acid in fats and oils (Oladiji et al., 2010). This signifies that; a longer average fatty acid chain length, results in a smaller saponification value (Mansor et al., 2012). Therefore, P. guineense has fatty acid with the longest chain length followed by Z. officinale and M. myristica being the shortest. As such, the high saponification values recorded for these oil samples in this study, are indications of their possible use in the industry (Aolf et al., 2018;Ardabili et al., 2011;Amoo et al., 2004).

Iodine value:
The highest iodine value 69.49 gI2/100 g was recorded in M. myristica while the lowest 39.35 gI2/100 g was recorded in Z. officinale. There were significant differences (p<0.05) recorded between samples. The iodine value is a marker for the extent of unsaturation of the oil (Shad et al., 2012). It influences the stability of oils to oxidation and also determines the quality of the overall unsaturation of the fat (Asuquo et al., 2012;Bello and Makanju, 2011). The iodine value of M. myristica indicated the greatest degree of unsaturation therefore has the highest ability to remain in liquid form at room temperature than other oils. The values in this study are comparable to (64.9 g I2/100 g) reported by Adolf et al. (2018) for M. myristica, but higher than (17.1 and 13.66 g I2/100 g) reported by Aletor (2014) and Ogbonna et al. (2015) for Z. officinale and P. guineense respectively. However, the iodine values for the oils were lower than those recorded for vegetable oils like peanut (86-107 gI2/100 g) cottonseed (100-123 gI2/100 g), sesame (104-120 gI2/100 g), soyabean oil (124-139 gI2/100 g) as well as sunflower oil (118-141 gI2/100 g) (Aremu et al., 2006). The fact that iodine values less than 115 gI2/100 g are regarded as non-drying oils and those above 130 gI2/100 g are considered as drying oils (Ogbonna et al., 2015). These oils can be categorized as non-drying oils. Higher iodine values in oils are desirable nutritionally. Nevertheless, higher iodine value indicates a lesser stability of the oils and greater vulnerability to undergo oxidation and production of free radicals. Oils with low iodine values have been reportedly used as a component of food additives because they are less liable to oxidative rancidity and it is effective in controlling microbial growth (Stoilova et al., 2007).

CONCLUSION
The result of this study suggests that the essential oil of the selected plants can be utilized as edible oils and suitable for nutritional purposes, stable and would not easily undergo rancidity as such can be used as a good component of food additives.