Phytochemical and Proximate Analysis of Some Medicinal Leaves

The proximate analysis and the phytochemical constituents were investigated in Magnifera, Morinda lucida, Parquetina nigrescens, Oscmium gratissimum, Chenopodium ambrosioides and Veronia amygaldalina using standard reference methods. The proximate analysis in % showed that Chenopodium ambrosioides had the highest amount of ash content of 17.30 and moisture content of 89.40 while Magnifera indica had the lowest amount of ash content 12.04 and moisture content 68.40. Veronia amygaldalina had the highest amount of protein 30.02 and carbohydrate 54.00 while Parquetina nigrescens had the least amount of protein 25.06 and carbohydrate 40.23. Parquetina nigrescens had the highest amount of crude fibre of 25.05 while Chenopodium ambrosioides had the highest amount of crude fat 18.22. The moisture content was found to be 89.40%, ash content 17.30, fat 18.22%, protein 30.02%, crude fibre 25.05% and carbohydrate 54.00%. The phytochemical constituents indicated flavonoid 28.58% alkaloid 25.84% and tannin 2.94% in Magnifera indica, Veronia amygaldalina and Morinda lucida respectively. The presence of high secondary metabolites in the leaves are good indication that if the plant is subjected to further research such as identification and characterization of plant, bioactive compounds with strong biological activities may be isolated and novel compounds may also be identified.


Introduction
Medicinal plants are plants which contain substances that could be used for therapeutic purposes or which are precursors for the synthesis of useful drugs [46]. The medicinal value of these plants lies in bioactive phytochemical constituents that produce definite physiological action on the human body [8]. Ocimum gratissimum which belongs to the family of Lamiaceae and found mostly in the tropical countries including: Nigeria, India, North and South America, Mexico and Brazil where it is popularly known as alfavaca-cravo, alfavacao, alfavaca [14]. The local names are Efinrin, Efirin aaja, Erumaba (Yoruba), Daidoyatagida (Hausa), Esewon (Edo-Akoko), Nehonwu, Nchanwu (Igbo) and Menthesauvage (French). It is traditionally used to relief pains and also used in the treatment of rheumatism, diarrhea, high fever, convulsions, diabetes, eczema, piles and as a repellant [15]. The decoction of the stem is inhaled for the treatment of catarrh and bronchitis [26].
Vernonia amygdalina is commonly called bitter leaf because of its bitter taste. It belongs to the member of Asteraceae family and a small ever-green shrub that grows all over Africa. It is reported to be a medicinal plant for diabetes and fever [16]. Bitter herbs are reportedly good for the body as they help tone the vital organs of the body like the kidney and liver. Ethnomedically, the leaves are consumed either as a vegetable (macerated leaves in soup) or aqueous extracts as tonics for the treatment of various illnesses [29].
The roots of V. amygdalina have been used for gingivitis and toothache due to its proven antimicrobial activity from the previous work being done on it [2]. In North America, of the 17 species of Vernonia all have the same effective properties as a blood purifier, uterus toner and helps also in the prevention of atherosclerosis [22,41]. Many herbalists and naturopathic doctors recommend aqueous extracts for their patients as treatment for anemia, nausea, diabetes, loss of appetite, dysentery and other gastro intestinal track problems. V. amygdalina extracts have also been reported to help suppress, delay, or kill cancerous cells [16].
Mangifera indica is commonly known as mango, they belong to the family of Anarcadiacae. It is widely used as a source of food, medicines and timber. In Nigeria, different parts of Mangifera indica (mango tree) are commonly used as herbal preparations in the treatment of tooth ache, gastrointestinal disorders, dysentery, diarrhea, gastrointestinal tract infections, respiratory and urinary tract infections [5]. It has been noted that infection of leaves and stems extract of Mangifera produce in dogs and rabbits hypotensive action [26].
Chenopodium ambrosioides Linn (Amaranthaceae), popularly known as "mastruz" and widely used in folk medicine, being considered one of the plant species with potential for use in the production chain and to generate products of interest to the Brazilian Unified Health [47]. It is an annual or perennial herbaceous plant with distinctive aroma, upright, that reaches up to 1.5 m high, with dark green and camphorated pubescent leaves. Several therapeutic activities are assigned to the species, such as antiinflammatory action, antifungal, antitumor, immunomodulatory, analgesic and antibacterial. The species is distinguished by its constitution rich in flavonoids, tannins and alkaloids reportedly said by Grassi [27]. Although there are studies that attribute to C. ambrosioides a possible toxic action, allegedly resulting from the presence of some monoterpenes ascaridole, constituent abundant in the specie [42].
The roots, stem, bark and leaves of Morinda lucida are widely used in tropical Africa due to their reputed therapeutic value in the treatment of antiinflamatory diseases and antiparasitic diseases [36], such as inflammation and malaria [49,13]. Phytochemical screening revealed that M. lucida contains various types of alkaloids-anthraquinones and anthraquinols [4]. In fact, Koumaglo associated the antiplasmodial activity of M. lucida stem bark and root to the presence of the anthraquinones digitolutein, rubiadin-1methylether and damnacanthal [38].
Parquetin nigrescens is widely used in traditional medicine as a medicinal herb. Usually small amount of Parquetin nigrescens are used as it is very toxic especially the latex, Many vital accidents have been recorded. A plant or leave decoction is taken as an enema to treat serious kidney problems, several constipation and to induce abortion. Sometimes fresh crushed leaves are taken as an emetic to treat severe constipation [31]. A plant or leave decoction or infusion, sometimes with part of other plant species, it is drunk to treat measles, intestinal worms, diarrheal, dysentery, diabetes, menstrual disorders and venereal diseases. In very small quantities, it is given to children to treat respiratory diseases [6]. A leave decoction with honey added is drunk to reduce fatigue, jaundice, stomach ulcer and anemia as a tonic. It is also taken to treat hypotension and to ease child birth. The body is washed with a leave decoction to treat general fatigue. The leaves are the common ingredient in medication to treat insanity [37].
The contribution of different species of plant parts to health status of human cannot be over emphasized. Various plants in Nigeria have served as source of, protein, fat and carbohydrates. They become important when their functions are considered in human body [3]. Most of these plants are used to cure some diseases [39]. The ability of these plants to cure human and animal diseases is as a result of the ethnopharmacological activities carried out by these plantcontaining bioactive constituents. So many medicinal plants have been used by traditional medicine practitioners in Nigeria for the treatment of different diseases. Among the various evidence revealing that medicinal and culinary herbs have some endemic species, a diet rich in fruits and vegetables and phytochemical which decrease the risk of cardiovascular diseases and some forms of cancer are of particular interest [34]. Many of these plants are underutilized. These medicinal leaves are underutilized for effective treatments of serious human ailments. This research work looks into the quantitative determination of the phytochemical and proximate constituent levels of these plants that makes them usable for curing some diseases.

Collection of Plant Materials
The fresh plants were collected from a local farm in Esa oke, Osun State, Nigeria. Identification and authentication of the plants was done by Prof. J. A. Ogunkunle at Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso, where the specimens was deposited in the herbarium.

Processing of Plant Materials
The fresh leaves of the following plants Morinda lucida, Parquetina nigrescens, Magnifera indica, Oscium gratissimum, Chenopodium ambrosioides and Veronia amygaldalina were air dried at 28°C for 30 days. They were grounded into fine powder using an electric blender and stored in a cool dry container until use.

Determination of Tannins
Five gram (5g) of each of the ground sample was weighed into conical flask in triplicates and 100ml 2M HCL was added. The content was boiled on a water bath for 30 minutes. The extract was cooled and filtered using Whatman No. 1 filter paper. The filtrate was taken up twice in 40ml each of diethylether. The ether extract was heated to dryness and weighed. The average of each sample was calculated and their percentage.

Determination of Flavonoids
Five gram (5g) of each sample was extracted with 50ml of 80% aqueous methanol in triplicate repeatedly at room temperature. The whole solution was filtered through Whatman filter No. 42 (125). The filtrate was then transferred into 200ml beaker and evaporated into dryness over a water bath, the weight of the material and the percentage quantify was calculated. The average of each sample was calculated and their percentages.

Determination of Alkaloids
Two and half gram (2.5g) of each sample was weighed into 250ml beaker; 200ml of 20% acetic acid ethanol was added and allowed to stand for 4hrs. This was then filtered and the extract was concentrated using a water bath to evaporate about a quarter of the original volume. Concentrated ammonia solution was added drop-wise to the precipitation and was completed. The entire solution was allowed to settle and the precipitation was collected by filtrate and weighed. The average of each sample was calculated and their percentage

Proximate Analysis
The proximate analysis of the samples for moisture, ash, fibre and fat were done by the method of AOAC (2005). The nitrogen was determined by micro-Kjeldahl method as described by Pearson (1976) the percentage Nitrogen was converted to crude protein by multiplying. All determinations were performed in triplicates.  [24]. It is however lower than the value reported for P. fistulosus (62.39) [28]. Carbohydrate constitutes a major class of naturally occurring organic compounds that are essential for the maintenance and sustenance of life in plants and animals and also provide raw materials for many industries [20]. The leave is a good source of carbohydrate when consumed because it meets the Recommended Dietary Allowance (RDA) values [23].

Results
The crude protein content (%) of V. amygaldalina, C.  29.00 and 25.06 respectively. These are higher than the protein content of Telfaria occidentalis (7.00) and Momordica balsania L. (11.29) [33], but however they are in the same range with Piper guineensis (29.78) and Talinum. triangulare (31.00) [7]. However, it compared favorably with the value reported for A. viridus (16.41) and S. oleraceae (23.74) [43]. The leaves are considered as a good source of protein because it gives us more than 12% of caloritic value from protein [44].  [43], and higher in Ipomea batatas with 11.10 [10]. They are however higher than that of A. sativum with 4.84 [28]. The ash content is a reflection of the amount of mineral elements present in the samples; therefore, the plants contained a good amount of minerals. The moisture content (in %) values for the leaves of C. ambrosioides were relatively high, therefore it would speed up the growth of microorganisms and life span of stored samples would be low. The moisture content of the leave is high when compared to that of Xylopia aethiopia (16.04) [1] and Acalypha hispida (11.91) [30].
The values of the crude fat (in %) for the leaves of C.  [28] and low when compare with P. thonningii 35.03 [21] but are higher than that of Gnetum africanum (4.60), M. ureans (4.00) and Parinari polyandra. The plants are good source of crude fibre when consumed because adequate intake of dietary fibre can lower the serum cholesterol level, heart disease, hypertension, constipation, diabetes and breast cancer [32].
The result of phytochemical analysis in percentages (table 2) shows that the six medicinal plants contained alkaloids, tannins and flavonoids while tannin was slightly present in M. indica and C. ambrosioides. Alkaloids has been found to have microbiocidal effect and the major anti-diarrheal effect is probably due to their effects on small intestine and antihypertensive antifungal, antiinflammatory, antifibrogenic effect [25]. However, the result of this work is similar to the findings of Awoyinka [12] who reported the presence of alkaloid in Cnidoscolus aconitifolius.
Some alkaloids are useful against HIV infection as well as intestinal infection associated with AIDS [40]. The presence of alkaloids in the six medicinal plants makes them recommendable for patient as alkaloids possess a significant pharmacological property.
Tannin is a non-toxic and can they generate physiological responses in animals that consume them [40]. Tannin can be toxic to filamentous fungi, yeast and bacterial. The presence of tannin in the medicinal plant suggests the ability of these plants to play key roles as antifungal antidiarrheal, antioxidant and antihemorrhoidal agent [11].
In this study, the leaves of P. nigrescens O. gratissimum, M. lucida, M. indica, V. amygaldalina, and C. ambrosioides contained flavonoid. It modifies the body's reaction to allergens, virus and caranogens. It has been reported to show anti-inflammatory, antifungi, antibacterial and antimicrobial activities based on the literature [18]. Akubugwo [9] reported the presence of flavonoids in A. hybridus. This perhaps justifies the already locally established function of the plant in the treatment and management of hypertension [48].

Conclusion and Recommendation
Plants have contributed immensely to the medical field. It has been the source of most drugs used for combating infections. The six plants used in this study were found to contain the important constituent needed to combat various kinds of infection in human. From the result of proximate analysis, it is quite interesting that V. amygaldalina has more protein content, carbohydrate and also the presence of high content of alkaloid, tannin and flavonoid from the phytochemical analysis done is an indication that if further research can be done on those samples, novel bioactive compounds can be derived from them after the identification, isolating the compounds and characterizing them using various spectroscopic techniques.