Chemical Profile of Leaves and Roots of Miracle Fruit (Synsepalum dulcificum)

Proximate analysis, elemental analysis and phytochemical analysis were conducted on leaves and roots of S. dulcificum (miracle fruit). The results of proximate analysis of the leaves sample showed the following compositions: protein (6.62±0.02)%, crude lipid (12±2.00)%, crude fibre (17.5±0.50)%, moisture (40.30±1.53)%, ash (6.70±2.00)% and carbohydrate (57.60±0.01)%, while that of roots samples showed protein (5.6±0.07)%, crude lipid (7.5±1.4)%, crude fibre (20.30±1.53)%, moisture (29.2±1.06)%, ash (8.00±1.56)%, and carbohydrate (58.60±0.01)%. Meanwhile the phytochemical screening analysis of the leaves of S. dulcificum showed the following concentrations in the leaf smples; alkaloids (0.6±0.20)%, saponins (2.80±0.20)%, flavonoids (2.8±0.2)%, polyphenols (3.52±0.10)% cardiac glycosides (3.44±0.20)%. Similarly, the root samples exhibited the following concentrations: saponins (0.6±0.02)%, polyphenols (4.30±0.10)%, anthraquinones (0.02±0.12)% and cardiac glycosides (4.40±0.20)%. On the other hand, the mineral elements concentrations of the leaf samples were as follows; Ca(0.001±0.00) mg/100 g, Cr(0.0006±0.00) mg/100 g, Fe(0.0029±0.01) mg/100 g, Zn (0.0095±0.00) mg/100 g Cu (0.00082±0.01) mg/100 g while that of root samples were recorded thus; Ca(0.00134±0.01) mg/100 g, Cr(0.00073±0.01) mg/100 g, Original Research Article Osabor et al.; ACSJ, 12(1): 1-8, 2016; Article no.ACSJ.20456 2 Zn(0.0097±0.01) mg/100 g Fe(0.00025±0.01) mg/100 g and Cu (0.007±0.01) mg/100 g. Generally, the leaf samples of S. dulcificum were rich in carbohydrate and moisture while the roots were rich in carbohydrate, moisture and fibre. Ash, fat and protein also showed remarkable concentration. Essential mineral elements were present at required concentration thus making S. dulcificum an important source of phytomedicine in the study area.


INTRODUCTION
Right from the creation, plants and its products derived from parts of plants such as stem bark, leaves, fruits and seeds have been parts of phytomedicine, thus indicating that any part of a plant may contain useful active compounds [1]. Medicinal plants constitute the main source of raw pharmaceuticals and health care products [2]. Mandal [3] pointed out that extraction and characterization of several active phytocompounds from green plants have given birth to some high activity profile drugs. Edem and Dosunmu [4] also reported that plants are primary sources of medicines, food, shelters and other items used by human every day. While Beroumand and Deokule [5] identified fruits as sources of minerals, fibre and vitamins which also provide essential nutrients for the human health.
Thus, S. dulcificum has also been found as an important source of phytomedicine. S. dulcificum is commonly known as miracle fruit. It is known as Mkpatun by the Ibibio and Efik people of Akwa Ibom and Cross River States, Azimomo by the Benin people of Edo State and Igbayum by the Yoruba people of Western Nigeria. S. dulcificum, is a plant with a berry that when eaten, causes sour foods (such as lemons and limes) consumed to taste sweet [6]. The sweetening property is due to miraculin (a glycoprotein) which is used commercially as sugar substitute. Apart from miracle fruit, it is also commonly known as miracle berry. But S. dulcificum should not be confused with two other plants species Gymnema sylvestre and Thaumatococcus daniellii referred to as miracle fruit which also affect perception of taste.
S. dulcificum (sapotaceae) is an ever green shrub native to tropical West Africa, and the fruits, and red berries have the property of modifying sour taste into sweet taste remarkably. The active material of the berry is the glycoprotein, miraculin, which has no taste in itself [7]. The most outstanding property of the fruit is its effect on the taste buds of the tongue that causes every sour or acid foods eaten to taste very sweet. The testmodifying effect last up to two hours or more, causing acid food substances such as sour lime, lemon, grape juice and even vinegar to taste sweet [8].
Chen [7] pointed out that the sweetness intensity is reduced by 0.02% citric acid after 0.4% purified miraculin solution is held in the mouth is equivalent to that of about 0.3% sucrose. The fruits are small approximately 2-3 cm long ellipsoid berries that are bright red when riped and composed of a thin layer of edible pulp surrounding a single seed [9]. The leaves are thin, papery, leathering and evergreen. It is reported that African here used S. dulcificum to sweeten a sour gruel made from stale bread, soured palm win and pito, a sour alcoholic beverage made from fermented grain. It has also been used before eating a certain type of sour corn bread [10][11][12]. The present studies deals with the analysis of S. dulcificum obtained from Akwa Ibom State Nigeria with a view of obtaining it nutritional potentials and Therapeutic utility.

Sample Collection and Preparation
The fresh leaf and root samples of S. dulcificum were collected from Ibot village in Mkpat Enin Local Government Area of Akwa Ibom State. The samples were identified in the Department of Botany University of Calabar, stored in a polyethylene bag and dried at 160°C in an oven. After drying, both leaves and roots were blended to powder with a manual blender. The blended samples were put in a desiccator and labeled accordingly and stored in a desiccator.

Sample Extraction
Petroleum ether and distilled water were used to extract each of leaf and root samples. In each process, 20 g of the powdered samples were washed and packed into extraction thimble and fitted into soxhlet extractor. The samples were Soxhlet extracted for three (3) hours. The petroleum ether and water extracts obtained were put in the reagent bottles. Each bottle was distinctively labeled and kept in the laboratory for phytochemical screening.

Digestion of Sample
Five grams (5 g) of the powdered samples were accurately weighed into a conical flask. 25 ml of concentrated nitric acid was added to the sample. 5 ml of perchloric acid was then added to the sample. The conical flask with its content was gently heated (50-70°C) on a stuart hot plate until the colour changed from brown to colourless. The digest was made up to 100 ml with deionized distilled water. Appropriate dilutions were made for each element. All determinations were carried out in triplicates.

Phytochemical Screening
The phytochemical screening procedures carried out on the leaves and roots of S. dulcificium were adapted from the previous work on medicinal plant analysis [15,16]. Alkaloids, saponins, flavonoids, cardiac glycosides, polyphenols, phlobatannins, anthraquinones, anthranoids, tannins and reducing compounds were screened.

Mineral Element Analysis
The methods of AOAC [14] were used in these determinations. Standard solutions were prepared for each element. Sodium and potassium were determined by flame photometer (Gallen Kamp). Calcium, magnesium, iron, chromium, lead, mercury, nickel, cadmium, cobalt, manganese, zinc and copper, were determines using atomic absorption spectroscopy (AAS). Tables 1 and 2 Table 1 shows that the moisture content of the leaves and roots of S. dulcificum are (40.30±1.53%) and (29.20±1.06%) respectively. The percentages are slightly lower than the moisture content of Chrysophyllum africanum (66.67±0.02)% [4]. The moisture content of food is usually used as a meaure of stability and susceptibility to microbial contamination [19]. These compositions indicate that S. dulcificum can be stored for a long time without spoilage. The study on the ash content of the leaves and roots of S. dulcificum obtained were found to be (6.70±2.00%) and (8.00±1.56%) respectively. These compositions are higher when compared to (2.03%) reported by Edem [20] for Tetracarpidium conophorum. Again the ash content of S. dulcificum was found to be nearly the same with Alpinia allighas (6.40±0.15%) by Landan [21]. The results of the analysis for fibre contents as presented in Table 1 are (17.50±0.50%) and (20.30±1.53%) for leaves and roots respectively. Comparing these values to that reported for Chrysophyllum africanum fruits (4.45±0.02%) by Edem and Dosunmu [4]. S. dulcificum has a very high percentage of fibre. Emphasis has been placed on the importance of keeping fibre in takes low in the nutrition of infants and pre-school children [22].

RESULTS AND DISCUSSION
The crude lipid contents of the leaves and roots of S. dulcificum were found to be (12.0±2.00%) and (7.50±1.40%) respectively. The lipid content of the leaves of S. dulcificum is significantly higher than that reported for Chrysophyllum africanum (9.38±0.01) by Edem and Dosunmu [4]. Lipids are monosaturated and considered healthy when consumed on moderation. They are essentials because they provides body with maximum energy [23]. Similarly, the protein contents were found to be (6.62±0.02%) and (5.60±0.07%) respectively. Protein is an essential component of the diet needed for survival of animals and humans. Basic function is to supply adequate amount of required amino acids [24]. Protein deficiency causes growth retardation, muscle washing, abnormal swelling of the belly and collection of fluids in the body [25]. The daily protein intake for children and adult are 23-26 kg and 45-46 g respectively [26]. However, the carbohydrate contents were found to be (57.18±0.01 and 58.60±0.01%) for leaves and roots respectively. These values are higher than (53.20%) reported for Tetracarpidium conophorum fruit by Edem [19]. The result obtained in this study indicates that S. dulcificum is rich in carbohydrate. If carbohydrate is sufficient in food, it prevents the unnecessary usage of protein and allows it to be used for the body building processes.    The phytochemical screening of the leaves and roots of S. dulcificum show that cardiac glycosides were present in higher concentration in both petroleum ether and water extracts. Quantitatively, the levels of cardiac glycosides were found to (4.40±0.2%) and (4.20±0.2%) respectively. These concentrations are higher when compared to that of Cissus populnea Guill and Perr. Tannins showed a higher concentration in the petroleum ether extract of the leaves, but was however absent in water extract of both roots and leaves.
Phytochemical screening result of reducing compounds in the leaves and roots of S. dulcificum is given in Table 2. Reducing compounds were absent in both petroleum ether and water extracts of the leaf and root samples, even after boiling for 30 minutes in a water bath. The result indicated that leaves and roots of S. dulcificum are not rich in reducing compounds compared to the seeds, which is evidence of the taste modifying activity.
Phlobatannins were absent in both petroleum ether and water extracts of the leaf and root samples. Similarly, the screening for anthranoids showed the absent of anthranoids in both extracts (petroleum ether and water) of the leaf and root samples ( Table 2). Phytochemical screening analysis shows that the water extract of the root shows the presence of antraquinones (Table  2) while the quantification of anthraquinones revealed (0.02±0.12%) ( Table 3). It was however, absent in the leaves sample. Naturally occurring anthranol are easily oxidized by atmospheric oxygen to anthraquinones. Anthraceae are found as glycosides and aglycone [32]. Anthraquinones and related glycosides are thatatus, which increased smooth muscles tone of the large intestine [33].
The mineral elements analysis of S. dulcificum showed the composition of calcium to be (0.001±0.00 mg/100 g) and (0.00134±0.01 mg/100 g) for both leaves and roots respectively. These concentrations are very low when compared to that of calcium obtained from Aspilla Africana leaves (1.04±0.03) mg/l [34] and Achyranthes aspera Linn roots (849435) ppm [35]. About 200-400 g/day will provide the recommended daily calcium allowance of 360 mg-1200 mg for children and adults especially if combined with other food stuff rich in calcium. Calcium is required in the body for normal growth of bones and teeth [36]. Mineral element analysis result for magnesium is given in Table 4. Magnesium was not detected in both the leaves and root of S. dulcificum. The mineral levels of potassium in the leaves and roots of S. dulcificum is signifincantly low as potassium was not detected (Table 4). But this is not so with seed of the same plant the potassium in the seed is 569.500±2.820 mg/100 g [37]. Table 4 shows a significant composition of iron in both leaves and roots of S. dulcificum. The iron concentrations were found to be (0.0029±0.01 mg/100 g) and 0.0025±0.01 mg/100 g for the leaves and roots respectively. The obtained results are very low when compared to that reported for Securinegavirosa leaves (2.02%) [38]. The recommended intake of iron per day for children and adult is 10-18 mg/day [26]. Iron is required for the formation of blood and its deficiency causes anaemia [39]. Sodium was not detected in both leaves and roots of S. dulcificum as presented in Table 4. Meanwhile, chromium composition of the leaves and roots of S. dulcificum were found to be (0.0006±0.00 mg/100 g) and (0.00073±0.01 mg/100 g) respectively. For most plants species Cr at 10 mg/kg levels is considered to be toxic [40,41].
Zinc also recorded a low concentration in the leaves and roots of S. dulcificum which were found to be (0.0095±0.00 mg/100 g) and (0.0097±0.01 mg/100 g) respectively. The concentrations are very low when compared to that of the seed of the same plant reported to be (2.710±0.009 mg/100 g) [37]. The zinc present in the pancreas may aid in the storage of insulin, zinc in the plants could mean that plants can play essential roles in the management of diabetes, which result from insulin malfunction [42]. The results of the analysis for copper in the leaves and roots of S. dulcificum is presented in Table  4. The result obtained showed that leaf contains (0.00082±0.01 mg/100 g) while the roots contain (0.007±0.01 mg/100 g). Comparing the result to that reported for Averrhoa bilimbil (0.07±0.01) ppm [43], S. dulcificum is not a good sources of copper. Mineral elements composition of the leaves and roots of S. dulcificum for manganese is given in Table 4. Manganese was not detected in both leaves and roots. But Jeremiah [37] reported manganese content of the seeds to be (2.280±0.004 mg/100 g).
Similarly, lead, mercury, cadmium, cobalt and Nickel were not detected in the samples but Jeremiahs [37] reported that the seeds of S. dulcificum contain a Nickel concentration of (0.240±0.028 mg/100 g).

CONCLUSION
The leaves of S. dulcificum are rich in carbohydrate and moisture. Similarly the roots are rich in carbohydrate, moisture and fibre. Ash, fat and protein were appreciably present. Phytochemical screening of the extracts (petroleum ether and water) revealed that S. dulcificum is highly rich in cardiac glycosides and polyhenols when compared to other detected bioactive compounds. From the quantitive analysis of the phytochemicals, tannins, cardiac glycosides, polyphenols, flavonoids and saponins showed a high levels in the both leaves and roots. Elemental analysis revealed that all the detected mineral elements were present in a moderate concentration in both leaves and roots.