MATERIALS AND METHODS

Samples: Leaves of Moringa oleifera where collected randomly in three regions of Chad at july 2012, one sample in the vicinity of Faculty of Sciences (N’Djaména, central region of Chad), the second at Moundou (Southern) and the third one at Sarh (Southern east). In the former region (N’Djamena) the mean annual rainfall is 800 mm. Moundou and Sarh receive more rain, more than 1200 mm year^-1. In southern and south east regions, the Moringa trees are more vigorous than those of N’Djaména region probably due to the difference of rainfall per year.

The young green leaves were collected. One part is placed in plastic bags in coolers, covered with ice and transported to the “Laboratoire de Recherche sur les Substances naturelles” for the determination of vitamin C and pigments. A second part is air dried and 1kg of each are placed in polyethylene bags and transported to laboratory for other analysis.

Dry matter determination: Samples, 5 g of air dried Moringa leaves from N’Djamena (Ndj), Moundou (Mou) and Sarh (Sar) are weighted and placed in an drying oven at 110°C until there is no variation of mass. The dry sample is weighted and the dry matter calculated (AOAC, 1990).

Total protein: Total protein was determinate by the micro method of Kjeldahl through nitrogen content determination followed by its conversion by a factor of 6.25.

For amino acids determination, powder of dry leaves were hydrolysed, under nitrogen, in HCl vapour at 120°C for 24 h using a Pico-Tag work station (Waters). Along with 2-β-mercaptoethanol (4%) to preserve sulphur containing amino acids, 200 μL of 6N HCl were placed in the hydrolsysis tank. After hydrolysis, 10mmol of glucosamic acid per mg of sample were added as an internal standard. The samples were dried under vacuum in a Speedvac apparatus (Savant Instruments Inc., Farmingdale) and taken up with 0,05M lithium citrate buffer, pH 2.2. The samples were submitted to ion exchange chromatography in an automatic amino acids analyser (Beckman 3600). Amino acids were detected by the ninhydrin reaction, identified by their retention time and wavelength ratio and quantified by their absorption at 750 nm (440 nm for proline).

Total carbohydrates: Total carbohydrates were determinate by the phenol-sulphuric method (Saha and Brewer, 1994; Ausubel et al., 1995) after extraction with ethanol 80%.

Total lipids: Total lipids were determined by Soxlhet method using Soxtherm 2000 apparatus with n-hexane as solvent.

Crude fibbers: They were determinate by the insoluble method of Deymie et al. (1981) with a slight modification. In this method, sample of leaves powder is digested by formic acid 80% (V/V) in boiling water. The insoluble phase is recovered and submitted to NaOH digestion. The remaining insoluble phase was submitted to incineration. The crude fibbers are calculated as the difference of the mass between insoluble phase recovered after alkaline digestion and ash obtained after incineration.

Vitamin C: The total vitamin C content was estimated using the UV spectrometric method described by Khan et al. (2006). In this method, vitamin C is extracted with metaphosphoric-glacial acetic acid solution. The extracted vitamin C is converted to dehydroascorbic acid by a bromine solution. The dehydroascorbic acid reacts with 2, 4 dinitrophenylhydrazine in presence of H₂SO₄. The absorbance of the coloured solution is measured at 521 nm. A calibration curve is used to determine the vitamin C content.

Minerals determination: Potassium was determined using spectrometric method (AFNOR, 1982), Calcium, Magnesium, Copper, Iron and Sodium were determined using AOAC (2005). Manganese and Phosphorus were determined by the method approved by USEPA (1974). Zinc was determined using AOAC method (AOAC, 1990).

Pigments determination: Chlorophyll A, B and total carotenoid content of Moringa leaves were determinate using spectrometric method described by Dere et al. (1998). The samples are treated with methanol (96%). The absorbance of the supernatant obtained after centrifugation is measured at 470, 653 and 666 nm and the following formula are used to calculate the level of the pigments:

Statistical analysis: All essays were done in triplicate. The means and standard deviation are calculated using SPSS for windows 13. Student’s t-test was used for differences of mean performance of samples from the different regions implying significance at p<0.05.

RESULTS AND DISCUSSIONS

Proximate composition: Table 1 shows that all the 3 Moringa samples contain total protein average 31.5%. There is no significant difference of protein content of the 3 samples. These results are the same with those of some researchers but vary slightly from those of others. Moyo et al. (2011), in their nutritional characterization of Moringa leaves from Limpopo province of South Africa, found that samples contain 30% of protein. Oduro et al. (2008) found Moringa leaves from Kumasi (Ghana) contain average 27.5% of crude protein while Ayssiwede et al. (2011) found a crude protein content of 28.5 for Moringa leaves from Senegal. Joshi and Metha (2010) obtain less crude protein value of 23.32% for sun dried leaves of Moringa from Jaipur, India. Campaore et al. (2011) found a greatest protein content of 35.56% of Moringa leaves from Burkina Faso. In the same country, Yameogo et al. (2011) found smaller value of 27% of protein from samples at Ouagadougou. Suchada et al. (2010) found the crude protein content ranging from 19 to 27% from dried leaves of Moringa of eleven regions of Thailand. Mensah et al. (2012) in the study of nutritional value of Moringa leaves from central Nigeria found only 6.8% of total protein content.

Except the result of Mensah et al. (2012) all these research results show the protein content not less than 20%. The Moringa leaves constitute a good source of protein for poor people of less developed country which cannot find animal protein all days.

Table 1:	Chemical composition of Moringa oleifera leaves from the 3 regions (N’Djaména = Ndj, Moundou = Mou and Sarh = Sar)

The same table shows an average 13.5% of carbohydrates, 2.5% of lipids. Moyo et al. (2011) found a greater value (6.5%) for fat but they haven’t determinate carbohydrates. Oduro et al. (2008) found the same value (2.23%) for crude fat but much greater content of carbohydrates (43.88%). On Moringa news link, Mélanie Broin mentioned 40% for carbohydrates and 8% for crude fat of dry matter. Yameogo et al. (2011) found similar amont of carbohydrates ranging from 35.7% to 43.3% but a higher content of crude fat from 12.5 to 21.6% of dry matter. Suchada et al. (2010) found crude fat similar to the present results (average 2%) and they also reported other authors’ results which are not higher than 2%. Talreja (2011) obtain a similar among of crude fat (2.3%) and a higher content of carhydrates (38.2%) for dry matter. Mensah et al. (2012) found only 0.041% of carbohydrates and 0.5% of fat.

These results show that leaves of Moringa contain only a small quantity of carbohydrates and lipids and they cannot provide energy to human organism.

Using the coefficients of Merrill and Watt (1955) adopted the energy value of dry Moringa leaves is 206,75 Kcal per 100 g of dry matter. This result is lower than that found by Yameogo et al. (2011) for dry leaves from Burkina faso (average 339.1 Kacl).

In the culinary processing, oil is added and the sauce is eaten with dough of cereals flour which is carbohydrates rich which provide an additional energy to organism. The estimated dry matter, ash and crude fibber of this study are not significantly different of those of the authors mentioned above.

Amino acids profile: Table 2 presents the dry Moringa leaves amino acids profile. This table shows 17 of the 20 natural amino acids. Asparagine and glutamine have been converted to aspartic acid and glutamic acid while tryptophane has been destroyed by the hydrolysis. Moringa leaves also exhibit all essential amino acids (those with asterisk) making them suitable for infant and sick persons nutrition. The highest amino acid percent is that of glycine (average 9%) and the lowest those of cysteine and methionine (average 0.4%). These two amino acids are those which contain sulphur.

Table 2:	Amino acids profile of Moringa oleifera samples from the 3 regions (N’Djaména = Ndj, Moundou = Mou and Sarh = Sar)
*: Essential amino acids

Table 3:	Pigments and vitamin C content of Moringa oleifera of the 3 regions (N’Djaména = Ndj, Moundou = Mou and Sarh = Sar)

Moyo et al. (2011) have detected 19 of 20 natural amino acids using an amino acids detector after hydrolysis. They have also detected all the 10 essential amino acids like in this study. They found that the highest amino acid percent is that of alanine (3.03%) and the lowest that of cysteine, only as trace (0.01%). Talreja (2011), in a study of dry leaves from India, presents only the 10 essential amino acids. The highest content is that of leucine (1.95%), different to Moyo et al. (2011) and this study findings but the lowest is the same amino acid, methionine (0.35%). Moringanews website shows that the lowest amino acids in dry Moringa leaves are the sulphur amino acids with the same percent like these findings, 0.37% for methionine and 0.36% for cysteine. Although Mensah et al. (2012) found less total protein content, their study gave the same percents of almost all essential amino-acids.

All these results show that the greatest amino acid content of Moringa leaves is a neutral amino acid, glycine for this study, alanine or leucine for cited findings. The lowest amino acid percents are those of sulphur amino acids.

Pigments and vitamin C: All three samples of Moringa exhibit the same levels of chlophyll A, chlorophyll B, total carotenoid and vitamin C content (Table 3). No bibliographic data was found about pigments from Moringa oleifera. Pigments (Chlorophyll A, Chlorophyll B and carotenoids) are pigments which permit plant to realize photosynthesis, the processus that cause light energy to turn into chemical energy in organic compounds. Dere et al. (1998) have used three solvents (Diethy ether, methanol and acetone) for the determination of chlorophyll A, B and total carotenoid from four algae species. They found the highest content of chlorophyll A in Cladophora glomerata species but they found no significant difference in the chlorophyll B content with the three others species (Ulva rigita, Codium tomentosum and Cladosthephus verticilatus). The β-carotene, a carotenoid, is also known as provitamin A because it is converted to form vitamin A which protect human eyes.

There is also no significant difference of vitamin C content of the three location samples of Moringa. This mean value is average 250 mg/100 g of fresh matter. Vitamin C plays many functions in the human body including antioxidant agent, cofactor for enzymes. The daily requirement of vitamin C for adults range from 75 to 125 mg depending of activities. The value obtain in this study shows that only 50 g or less powder of Moringa is enough to achieve the required value.

Macro and micro-elements: Table 4 shows the minerals contents in Moringa leaves. Human body needs macro and micro elements for his development and metabolism. Calcium is the major constituent of bone and teeth and needed for the regulation of nerve and muscle function. Sodium and potassium are principal cations in extracellular fluids, they are involved in the regulation of plasma volume and acid-base balance. Phosphorus is a constituent of adenosine triphosphate, the energy compound of the body and is involved in many metabolism procedures.

Table 4:	Macro-elements (g/100 g DM) and micro-elements (ppm) in Moringa oleifera leaves from the 3 regions (N’Djaména = Ndj, Moundou = Mou and Sarh = Sar)

Table 5:	Mean content of minerals, daily requirement for adults and quantity needed day-1

Iron is a constituent of haemoglogin, the oxygen carrier in cellular respiration. Magnesium is a component in many metabolism systems. Copper is a constituent of enzymes and is necessary for the growth and bone formation (Soetan et al., 2010). There is no significant difference for the elemental composition of the samples from the three regions.

Table 5 shows the content of the 5 macro-elements and 4 micro-elements determinate, the daily requirement and the quantity a man will eat to achieve the quantity needed. As shown on Table 5, Moringa dry leaves are a good dietary source for only 2 macro-elements, calcium and magnesium. For the 3 others tested, potassium, sodium and phosphorus, they are poor source. Human need to eat more than 100 g to achieve the quantity needed of these minerals per day. For micro-elements, only the manganese and copper requirements per day can be achieve with less than 100 g of dry leaves.

CONCLUSION

There are no differences in chemical composition of Moringa oleifera leaves from the three regions of Chad. Moringa leaves are rich source of protein with well balance of essential aminoacids. They contain more vitamin C than lemon and orange which are considered the main source of this compound. Calcium, magnesium, copper and manganese could be provided to human health by the consumption of these leaves. They can thus constitute a good source of many nutrients for people of less developed country which cannot find meat sufficiently every day. They don’t contain enough lipids and carbohydrates but they are consumed with oil and dough prepared using cereals’ flour.