One of the uses of
The aim of the study was to investigate the chemical composition of
Fresh, mature leaves were harvested from the Herbal Garden, Forestry Research Institute of Nigeria, Jericho Hill, Ibadan, Nigeria. The plant samples were identified at Forest Herbarium, Ibadan, Nigeria, sorted to eliminate extraneous matter and then air-dried on a cabinet dryer for five days at ambient temperature and oven-dried at 40°C for five minutes. The dried leaves were milled to powder using a medium-sized milling machine and stored in an air-tight container until needed for analysis.
The proximate composition, phytochemical screening and mineral analysis were determined using standard analytic methods. Minerals such as iron, zinc, calcium, manganese and magnesium were determined by the atomic absorption spectrophotometer; sodium and potassium by the flame photometer and phosphorus by the spectrophotometer.
The proximate analysis results revealed that the samples contained carbohydrate (40.46%), crude protein (16.15%), crude fibre (19.71%), crude fat (2.91%), ash (9.77%) and moisture (11.03%), while the phytochemical screening confirmed the presence of cardiac glycosides, saponins, tannins, anthraquinones and flavonoids. The mineral compositions (mg/g) decreased in the order Ca (6.0) > K (5.0) > Mg (2.2) > P (1.5) > Na (0.2) > Fe = Zn (0.6) > Cu = Mn (0.03).
These results conceivably indicate that
Medicinal plants are the source of many important drugs of the modern world. Many indigenous medicinal plants are also used as spices in food (Okwu
Fresh and matured leaves of
The powdered samples of
Moisture was determined by oven drying, where 2 g of well-mixed sample was accurately weighed in a clean and dry crucible (W1). The crucible was placed into an oven at 105°C for 12 h until a constant weight was achieved. Then, the crucible was placed in a desiccator for 30 min to cool. After cooling, it was weighed again (W2). The percentage of moisture was calculated in
where W1 (initial weight of crucible + sample) and W2 (final weight of crucible + sample)
Empty crucibles were cleaned and then heated in a muffle furnace at 600°C for 1 h. The crucibles were cooled in a desiccator and then reweighed as (W1). Sample (1 g) was weighed into crucible (W2); the crucible was then placed in muffle furnace at 550°C for 24 h. The appearance of grey white ash indicated complete oxidation of all organic matter in the sample. After ashing, the crucible was cooled and weighed (W3). Percentage of ash was calculated in
W1 – W3 = Difference in weight of ash
Protein in the sample was determined by the Kjeldahl method where powdered sample (1 g) was taken into a digestion flask containing 10 mL of concentrated H2SO4 and 8 g of digestion mixture (K2SO4, CuSO4 [8: 1]). The flask was swirled for 2 h in order to mix the contents thoroughly and then placed on a heater to start digestion till the mixture became clear. The digest was cooled and transferred to a 100 mL volumetric flask and volume was made up to mark by the addition of distilled water. Distillation of the digest was performed in Markam Still Distillation Apparatus for at least 10 min. The digest (10 mL) was introduced into the distillation tube and then 10 mL of 0.5 N NaOH was gradually added in the same way. The NH3 produced was collected as NH4OH in a conical flask containing 20 mL of 4% boric acid solution with few drops of modified methyl red indicator. During distillation, a yellowish colour appeared because of the NH4OH. The distillate was then titrated against 0.1 N HCl standard solution until the solution turned pink. A blank was also run through all steps as above. The percentage of crude protein content of the sample was calculated using
where
Crude fat was determined by ether extract method using Soxhlet apparatus where powdered sample (1 g) was wrapped in filter paper, placed in a fat-free thimble and then added to the extraction tube. The weighed, cleaned and dried receiving beaker was filled with petroleum ether and fitted into the apparatus and then water and heater were turned on to start the extraction. After 6 rounds of siphoning, ether was allowed to evaporate and the beaker was disconnected before the last siphoning. The extract was then transferred into a clean glass dish where the ether was washed and evaporated on the water bath. The dish was then placed in an oven at 105°C for 2 h and cooled in a desiccator. The crude fat was determined using
This was done on moisture-free, ether-extracted powdered sample where 0.2 g of the sample was weighed (W0) and transferred to a porous crucible with the crucible then placed into a Dosi-Fibre Unit with the valve kept in the ‘OFF’ position. After that, 150 mL of preheated H2SO4 solution was added and some drops of acetone (foam suppresser) were added to each column. Then, the cooling circuit was opened and the heating elements turned on (power at 90%). When it started boiling, the power was reduced to 30% and left for 30 min. Valves were opened for drainage of acid and rinsed with distilled water thrice to completely ensure the removal of acid from the sample. The same procedure was used for alkali digestion by using KOH instead of H2SO4. The sample was dried in an oven at 105°C for 1 h until a constant weight was attained. Then, the sample was allowed to cool in a desiccator and weighed (W1). The sample crucibles were ashed in a muffle furnace at 550°C for 4 h. After ashing, samples were removed from the furnace and cooled in a desiccator and weighed (W2). Calculation was done by using
Carbohydrate was calculated by difference after analysis of all the other items using the formula: NFE = 100 - (% moisture + % crude protein + % crude fat + % crude fibre + % ash).
Phytochemical screenings were carried out on the powdered sample using standard procedures to confirm the presence of constituents (alkaloids, anthraquinones, flavonoids, saponins, tannins, steroids, cardiac glycosides and total phenolic compounds) as described by Harborne (
Test for saponins: Powdered sample (1 g) was boiled with 10 mL of distilled water in a bottle bath for 10 min. The mixture was filtered while hot and allowed to cool. The following tests were then carried out:
Demonstration of frothing: 2.5 mL of filtrate was diluted to 10 mL with distilled water and shaken vigorously for 2 min (frothing indicated the presence of saponins in the filtrate).
Demonstration of emulsifying properties: Two drops of olive oil were added to the solution obtained from diluting 2.5 mL of filtrate to 10 mL with distilled water (as above), then shaken vigorously for a few minutes. The formation of persistent foam was evidence of the presence of saponins.
Test for alkaloids: Sample (1 g) was stirred in 10 mL of concentrated HCl on a steam bath followed by filtration. Filtrate (1 mL) was mixed with two drops of Wagner’s reagent, then two drops of Dragendorff’s reagent were added to another 1 mL of the filtrate, and the mixtures were then observed for turbidity.
Test for tannins: Powdered sample (1 g) was boiled with 20 mL of distilled water in a water bath and was filtered while hot. Cooled filtrate (1 mL) was distilled to 5 mL with distilled water and two to three drops of 10% ferric chloride were added and observed for any formation of precipitates and any colour change. A bluish-black or brownish-green precipitate indicated the presence of tannins.
Test for flavonoids: Sample (1 g) was boiled with 10 mL of ethanol, and two drops of ferric chloride were added to 5 mL of the extract. The mixture was observed for a dusty green colouration as positive result.
Test for free anthraquinones: Sample (0.5 g) was shaken with 5 mL of chloroform for 10 min, filtered and 5 mL of 10% ammonium solution was added to the filtrate. The mixture was shaken and the presence of a pink, red or violent colour in the ammonia phase indicated the presence of free anthraquinones.
Test for cardiac glycoside: Sample (1 g) was extracted with 10 mL of 80% ethanol for 5 min on a water bath. The extract was filtered and diluted with equal volume of distilled water and two drops of lead acetate solution were added, shaken and filtered after standing for few minutes. The filtrate was then extracted with aliquots of chloroform, and the extract was dissolved in 2 mL of glacial acetic acid containing one drop of FeCl3 solution in a clean test tube. Concentrated H2SO4 acid (2 mL) was then poured down the side of the tube so as to form a layer below the acetic acid. The formation of a reddish-brown or brown ring at the interface and a green colour in the acetic layer was taken for a positive result.
Test for total phenolics: Sample (1 g) was soaked in 25 mL of 2% of HCl for 1 h and then filtered through a 10 cm Whatman No. 1 filter paper. Plant extract (5 mL) was mixed with 1 mL of 0.30% ammonium thiocyanate solution and three drops of ferric chloride solution. A brownish-yellow colour indicated the presence of phenols.
Mineral contents of
Note: Dilution factor for phosphorus is 2500, for magnesium 10 000 and for other minerals, including calcium, iron, potassium, sodium, manganese and chromium, is 100.
Determination of sodium (Na) and potassium (K) was done by flame photometry. Standard solutions of 20, 40, 60, 80 and 100 mg/mL were used both for Na and K. The calculations for the total mineral intake involve the same procedure as given in atomic absorption spectrophotometry above. Phosphorus (P) in the sample was determined by spectrophotometry by mixing 12 g of ammonium molybdate with 250 mL of distilled water in a beaker (solution A). Antimony potassium tartrate 0.2 g was taken and dissolved in 500 mL H2SO4 solution in a volumetric flask. Enough distilled water was added to make the solution up to 1000 mL (solution B). The two solutions were mixed in a 2000 mL volumetric flask to get the mix reagent. The volume of the mix reagent was increased to 2000 mL by adding distilled water. Ascorbic acid (0.7 g) was mixed with 140 mL of the mix reagent in a beaker and left until dissolved to make the colour reagent. Wet digested sample (1 g) was taken in a plastic bottle labelled properly and 4 mL of distilled water was added to make a diluted volume of 5 mL. Colour reagent (5 mL) was added to this volume and the total volume of this mixture (final solution) was increased to 25 mL. The dilution factor of this solution was 2500. After some time, the colour of this final solution turned blue. Sample from the final blue solution was taken in a cuvette and read using a spectrophotometer. The readings of phosphorus were recorded in ppm. The calculations for the total mineral intake involve the same procedure as given in atomic absorption spectrophotemtry.
Data were analysed using the descriptive statistical analyses where means and standard deviation (SD) were obtained using Microsoft Excel 2013 version. Results were expressed as mean ± SD. A value of
The analyses of proximate composition of
Proximate composition of
Parameters | Quantity (%) |
---|---|
11.04 ± 0.01 | |
16.08 ± 0.17 | |
19.72 ± 0.03 | |
2.92 ± 0.02 | |
9.77 ± 0.01 | |
40.48 ± 0.11 |
Values are expressed as mean ± how many SD in triplicates.
The result of phytochemical screening as shown in
Qualitative analysis of phytochemical composition of
Components | Tests for phytochemical components | Observation | Inference |
---|---|---|---|
1 g of sample + 10 mL of HCl + heat + filter + 2 drops of Dragendorff’s reagents. | The resulting solution was not turbid. | Absent | |
0.5 g of sample + 5 mL of chloroform + filter + 5 mL of 10% NH4 solution + shaken for 10 min. | No pink, violet, red solution was observed. | Absent | |
1 g of sample + 10 mL of 80% ethanol + heat + filter + 2 drops of lead acetate solution + filter + 2 mL of chloroform + 2 drops of glacial acetic acid + FeCl3 solution + 2 mL conc H2SO4. | A brown ring at the interface and green colour in the acetic layer were observed. | Present | |
1 g of sample + 10 mL of ethanol + heat + filter + 2 drops of FeCl3 solution. | A dusty green colour was observed. | Present | |
1 g of sample + 25 mL of 2% HCl + filter + 5 mL of extract + 1 mL of 0.3% NH4 thiocyanate solution + 3 drops of FeCl3 solution. | Brownish-yellow colouration was observed. | Present | |
1 g of sample + 10 mL of distilled H2O + heat + filter + 10 mL of distilled H2O in 2.5 mL filtrate + 2 drops of olive oil + vigorous shaking for 2 min. | Persistent foam was observed. | Present | |
1 g of sample + 20 mL of distilled H2O + heat + filter + 5 mL of distilled H2O + 2 drops of 10% FeCl3 solution. | A brownish green precipitate indicated presence of tannins. | Present |
Results for the mineral composition of
Mineral composition of
Elements | Quantity (mg/100g) |
---|---|
613 ± 0.012 | |
207 ± 0.012 | |
150 ± 0.001 | |
513 ± 0.012 | |
21 ± 0.001 | |
3 ± 0.001 | |
61 ± 0.001 | |
3 ± 0.001 | |
61 ± 0.001 |
Values are expressed as mean ± SD in triplicates.
The proximate analysis shows that moisture content (11.04% ± 0.01%) found in
The presence of phytochemicals such as saponins, cardiac glycosides, flavonoids, phenols and tannins in the leaves of
The mineral content in the
This study revealed the presence of phytochemical constituents in the leaves of
The authors thank the head and the entire staff of Bio-Medicinal Research Centre, Forestry Research Institute of Nigeria, P.M.B 5054, Jericho Hill, Ibadan, Nigeria, for their immense contributions to achieving this scholarly research work.
The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.
M.B.O. designed the study, coordinated plant material storage, carried out all the experiments and drafted the manuscript. I.O.L. was responsible for the collection of plant materials and identification, supervised the laboratory experiments and made substantial contributions to revise the manuscript critically. A.A.O. performed data analysis and edited the manuscript. All authors read and approved the final manuscript.