Proximate Characterisation and Physicochemical Properties of Raw and Boiled Milk Bush (Thevetia Peruviana) Seed

This study examined the powdered sample of the raw and boiled seeds of thevetia Peruviana, the seeds were analysed for proximate composition, mineral content, while the oil extracted from the seeds were evaluated for physicochemical properties using standard methods. The results of the study showed that the proximate composition (%) of the raw and boiled samples respectively are moisture (2.00 and 2.89), ash (3.33 and 2.96), crude protein (30.10 and 29.6), crude fibre (4.79 and 5.21), crude fat ( 58.3 and 59.20), carbohydrate ( 1.80 and 0.30), and energy (2524.5 and 2521.1) KJ/g. While the physiochemical properties of the extracted oil are acid value (1.71 and 1.41) mg/g, peroxide value (3.85 and 11.51) mg/g, iodine value (83.89 and 76.20) mgKOH/g, saponification value (224.26 and 193.78)mg/g, unsaponification value (2.04 and 1.59)mg/g, density (0.89 and 0.88) g/dm, specific gravity (0.91 and 0.93), refractive index at 32C (1.46 and 1.47) and viscosity (28.21 and 30.59) pal/sec. The mineral contents of both raw and boiled samples are in the order of P > K > Na > mg > Fe > Ca > Zn > Mn. Cd, Pb, Ca and Cr were detected in raw and boiled samples except Ca that was detected in raw sample. Generally, the high protein and fat contents show that the samples can be used in food and feed formulation while the saponification values, iodine values and the presence of some valuable minerals indicate that they can be used for other industrial purpose.


Introduction
Seeds have nutritive and caloric values which make them necessary in diet. (Odeomelam, 2005). Africa, indeed the most tropical and sub-tropical, countries are blessed with numerous seeds and nuts, many of which are yet to be fully used due to insufficient information on their chemical, biological and industrial importance. One of such seed-bearing plants is thevetia peruviana commonly known as lucky-nut or milk-bush. Thevetia peruviana belongs to Apocynaceae family; Thevetia Genus; Yoruba name is called Olomi-Ojo. The plant is commonly known as yellow oleander, lucky-nut, be-still tree and milk bush. (Ibiyemi et al, 2002).
Thevetia peruviana is used medicinally throughout the tropics in spite of its toxicity. In Côte d'Ivoire and Benin the leaf sap is used as eye drops and nose drops to cure violent headaches; the seeds may be used as a purgative. The seed oil is applied externally in India to treat skin infections. In southern Africa and Cameroon the seeds are used as an arrow or ordeal poison. Other reports state the use of the seeds as an abortifacient. The seeds act as a contact poison; mashed with a soap solution they are used as an insecticide. In Ghana and Uganda the wood is used to make tool handles and building poles (Vandana D. and Varsha Z. 2014). It is also used as fuel. The fruit pulp is sometimes eaten. Thevetia peruviana is widely planted as an ornamental in gardens, and also as a hedge. In cooler climates it can be grown in tubs in the glasshouse and outdoors in summer.
It is a plant toxin insecticide for termites. Thevetia peruviana inhibited spermatogenesis in rats, indicating the possibility of developing a herbal male contraceptive. (Gupta R. et al, 2011).
The aim of this work is to evaluate the effect of boiling on the chemical constituents and physicochemical properties of the extracted oil. The research is expected to provide information on the usefulness of chemical constituents of the seed and serve as a resource in advancement of medicine and industrial applications.

Materials and Methods Preparation
The milk bush seed (Thevetia peruviana) also called olomiojo in Yoruba used in this study was obtained from Lafe Area in Akure Ondo State Nigeria. The fruits were air dried, cracked to remove the seed; some of the seeds were boiled while the remaining were left unboiled (raw). Both the raw and boiled dried seeds were ground to powder, packaged in airtight sample plastics and stored in the refrigerator at 4 ºC prior to laboratory analysis.

Chemical Reagents
All reagents/chemical were of analytical grades and they were obtained at the Department of Chemistry, Federal University of Technology Akure (FUTA). (d) Crude Protein Determination: Crude protein determination was carried out, using the micro kjeldahl method. About 1.0 g of the sample was weighed and transferred into the microkjeldahl flask. One tablet of catalyst and 10ml of concentrated H2SO4 were added to the sample inside the flask. The flask with its content was heated on a heating mantle inside a fume cupboard for 3 hrs until the black solution turned colourless. The clear solution was diluted with distilled water and made up to 100 ml. This was followed by distillation. 10ml of resulting solution from the digest was measured and transferred into a distillation apparatus. Then, 25 ml of 40% NaOH was added to the digested sample solution in order to make it alkaline. The cloudy nature of the sample solution after the addition of the 40%NaOH was in excess. 25 ml of 2 % boric acid was pipetted into a receiving conical flask; to which 2 drops of mixed indicator was added to produce a pink colour solution. The distillation was carried out with all the joints tightened, making sure the end of delivery tube dipping below the boric acid solution. As the distillation proceeds, the pink colour solution of the receiver turned light green, indicating the presence of NH3. Distillation was continued until the distillate was about 50 ml after which the delivery end of the condenser was rinsed with distilled water into the receiving flask. The third step was the titration stage where the received ammonia in the boric acid was titrated against standard 0.01HCl. A colour change of this solution from light green back to pink due to the presence of the mixed indicator that was earlier introduced in the distillation stage indicated the end point. The titre value was noted and recorded. The final step was to estimate the % Nitrogen in the sample and hence the crude protein by multiplying that value by general factor; 6.25 (e) Crude fibre determination: 200 ml of freshly prepared 1.25% H2SO4 was added to 5 g of sample, which had been defatted by extraction with n-hexane, and brought to boiling quickly. Boiling was continued for 30 mins after which the mixture was filtered. The residue was washed free of acid with plenty of warm water. The residue was then transferred quantitatively into a digestion flask; 200 ml of 1.25 % NaOH was added and boiled for 30 mins. The mixture was then filtered and the residue washed free of alkali with warm water. The residue was then washed thrice with methylated spirit, thrice with petroleum ether and was allowed to properly drain. The residue was then transferred to a dried, weighed silica dish and dried to a constant weight at 105 ºC. The organic matter of the residue was burnt off by igniting for 30 mins in a muffle furnace at 600 ºC. The loss in weight on ignition was reported as crude fibre.

(f) Determination of carbohydrate
The total carbohydrate present in a sample is determined by difference i.e. % carbohydrate= 100% -(% moisture + % ash + % crude fibre + % crude protein + % crude fat). (A.O.A.C, 1990).  (i) Determination of refractive Index: The refractive index was determined with a refractometer. The prism of the refractometer was wiped clean with a tissue paper and moistened with acetone. A drop of the oil sample was placed on the prism surface and clamped. Viewing through the telescope, the control knob was adjusted so that the path of view coincided with the dark part as indicated in the cross wire. The refractive index was read directly from the calibrated scale as observed through the telescope and recorded.

Mineral content determination:
The ashed content of the sample was dissolved in 20 ml 10%HCl solution, heated and filtered. The filtrate was then made up to 50 ml with distilled water. The solution obtained was used to determine the mineral content. Sodium and potassium contents were determined using JENWAY Flame Photometer, the vanadomolybdate method was used for phosphorus determination while the other minerals were determined using Atomic Absorption Spectrophotometer (AAS). Results are the means of triplicate determinations ± standard deviation. Table 1 shows the values of proximate composition (%) of the samples for raw and boiled seed of Thevetia peruviana. The moisture content for raw and boiled seeds are 2.00±0.00 and 2.89±0.19 respectively. The moisture content of boiled sample is significantly higher than that of the raw sample indicating that the seed can be stored without growing mould or any other organisms for many days. These results are lower than that of 4.66± 0.28 reported for Hura crepitans seed by Jokotagba and Amoo (2012). The ash content for raw and boiled seeds are 3.33±0.58 and 2.96±0.01 respectively. The ash content for boiled sample is lower than that of the raw, which may be due to leaching of nutrients in the boiling water or chemical interaction during boiling. This indicates that the inorganic residue of the seed is very low compared to the organic matter which had been burnt off before evaluating the ash content. These results are lower than 3.55± 0.10% reported for Hura crepitans seed by Muhammed et al, (2013). It was also discovered that the protein content (30.10±0.00) of the raw seed was significantly higher than that of the boiled seed (29.6±0.00), which could be as a result of denaturation of the protein by heat during boiling process. These values are close to the protein values reported for some other melons like three varieties of Lageneria siceraria with protein contents (27.71, 32.70 and 34.64) Ogundele and Oshodi (2010). The higher crude fibre in boiled sample may be due to the dissolution effect of boiling on the sample. The crude fibre is an index for digestivity of food sample. These results are higher than 1.14 % reported for Trichosanthes cucumerinia seed by Adeniyi and Amoo (2013). The fat content of the seeds showed that the oil seed contained high level of fat in boiled (59.20±0.00) than in raw (58.30±0.00). The results obtained are higher than 51.33 % reported for Trichosanthes cucumerina seeds by Adeniyi and Amoo (2013); it is also quite higher than 43.5 % and 47.7 % reported by Amoo and Owoeye (2002) for snake gourd seeds. The carbohydrate by difference are given as raw    Adeniyi and Amoo (2013). Peroxide value unit (3.85±0.00) for raw is lower than that of boiled (11.51±0.00). Oils with peroxide values ranging from 20.00 to 40.00 mg/g are considered rancid (Cock and Rede, 1986). The peroxide values obtained are below this range. The peroxide value is usually used as an indicator of deterioration of fats or oil (Asiedu, 1989).

Results and Discussion
The saponification value unit (244.26±0.00 mg/g) of the raw oil is higher than that of the boiled (193.76±0.00), High saponification value make the oil useful in the production of liquid soaps and shampoos (Ibiyemi et al, 2002) these implies that saponification of the oil seed of thevetia peruviana will be useful in industrial applications.  Table 3 shows the mineral content (mg/l) in the oil of Thevetia peruviana seed oil. The results showed that the seed contained both macro and micro minerals. Phosphorus is the most abundant with 11,088.09 in the raw sample. The implication of this is that Phosphorus in the form of adenosine (ADP) is important for production of energy in the cells (Sharma, 2006). Next in abundance to phosphorus is potassium, with a raw value 22.80 and boiled value of 17.60. Sodium ranks next to potassium in the sample with a raw value of 3.50 and boiled value of 3.10. Evidence has showed that the boiled sample contain the lowest sodium concentration. The result also indicates that magnesium is the next to sodium in the sample with boiled sample of 2.70 which exceeded that of the raw sample value at 2.00. Magnesium is a mineral that is essential to a variety of cellular metabolic reactions and sometimes has the ability to replace calcium in the body. (Encyclopaedia Britannica, 2008). Processing of the sample by boiling causes an appreciable increase in magnesium content. The result further showed that the raw mineral composition value of Iron content is 0.63 against the boiled value which was 0.60. This implies that processing of the sample by boiling does not cause any appreciable increase in the content. The concentration of zinc for both samples is the same with (0.26). Sharma (2006) has reported that zinc is a crucial nutrient for immune function, healing nervous system, brain function, blood sugar, balanced reproduction and optimal health. Manganese content of the raw sample is 0.17 while that of the boiled sample is 0.15. The manganese in the raw is higher than the manganese in boiled sample. The raw sample only contains copper with 0.06 while the boiled sample did not detect the concentration of copper. Other minerals such as cadmium, lead and chromium were not detected while the rest minerals were present in trace amounts. The values for sodium, calcium and potassium are lower than those obtained for Hura crepitans seeds by Muhammed et al, (2013). The magnesium and iron are lower than those obtained for Hura crepitans seeds as reported by Muhammed et al, (2013). Thus, the seed is a good source of phosphorus and potassium.

Conclusion
This research work has given insight into proximate composition, mineral content and physicochemical parameters of the seed of thevetia peruviana. This shows that thevetia peruviana seed contained both macro and micro mineral elements which are essential for normal functioning of the body. The result had shown that boiled thevetia peruviana seed has little reduction in ash, protein, carbohydrate and increase in fat content. This was due to the effect of boiling on the seeds which is normal for industrial application and in curing of disesases. In conclusion, the high protein and fat contents show that the samples can be used in food and feed formulation while the saponification values, iodine values and the presence of some valuable minerals indicate that they can be used for other industrial purpose.