1. Introduction
Plant tissues contain several substances with phenolic functions and, the most common are flavonoids, tannins and phenolic acids [1] . In the large group of compounds called tannins, a variety of chemical structures are possible, and the number of plant species containing these compounds is immense [2] [3] . Hydrolysable tannins consisting of simple phenolic acids such as gallic acid esterified with polyols, usually glucose, and condensed tannins are polymers of flavonoid units [4] . Several basic human foods, including fruits, beverages, and some seeds contain condensed and hydrolysable tannins [4] .
Mucuna is seasonal legume, and is cultivated for the good quality of its fodder, its seeds, its ability to fix atmospheric nitrogen at ground level. It is a creeping and climbing plant that adapts to the temperature of 15˚C to 35˚C [5] .
Mucuna leaves are given as a supplement to cows to increase the quality and quantity of milk and to other livestock, especially ruminants. The seeds can be combined with animal feed after processing [5] .
However, it is known that the seeds of Mucuna species us contain anti-nutritional factors, including tannins, which can present an intoxication danger in the case of direct feeding of ruminants [6] . However, these seeds are crushed to be used as a complementary food for draught cattle, cattle, or sheep for fattening [6] .
This study aimed to determine the tannin content in untreated and treated seeds of the four Mucuna varieties.
2. Material and Methods
2.1. Plant Material
The study focuses on the untreated seeds of the four Mucuna varieties and the seeds are treated according to four protocols. The species studied are the seeds of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada harvested in Burkina—Faso. To better identify the part of the seed containing more tannins, the different parts, namely the epicarp, the starch and the crushed seed set, of each of the four untreated species were studied. The tannin determination for the treated species was done on the whole seed set.
2.2. Seed Treatment
The different seed processing methods [7] for four Mucuna species are as follows:
§ Mucuna seeds are boiled directly for 2 hours;
§ Mucuna seeds are soaked for 12 hours in water followed by boiling for 2 hours;
§ Mucuna seeds are boiled in the presence of KOH (0.5%) for 2 hours;
§ Mucuna seeds were soaked in water for 12 hours followed by scalding in the presence of KOH (0.5%) for 2 hours.
2.3. Extraction of Phenolic Compounds
Different species of the seed were finely ground in a mortar. Then, 5 g of powder of each species were weighed and put into 30 mL of acetone-water system (80: 20, v/v) and kept in the refrigerator at 4˚C for three days [8] . The extracts were filtered and the filtrates kept in the refrigerator at 4˚C for subsequent tannin determination.
2.4. Determination of Tannins
The method adopted to determine the tannins of the different Mucuna species is the reference method used to quantify the tannins of sorghum [9] .
Ferric ammonium citrate (III) and ammonia were added to part of the acetone phase and the absorbance of the resulting solution was measured spectrophotometrically at 525 nm.
The determination of the tannin content is done using a calibration curve obtained from gallic acid as reference.
2.5. Identification Tests of Tannins (FeCl3)
2.5 g of powder of the plant material were added into 50 mL of boiling water and left to infuse for 15 min before filtering. A few drops of FeCl3 are added to the infused, precipitation with green, brown or blue coloration shows:
§ The presence of gallic tannins if the color is dark blue;
§ The presence of catechic tannins if the color is green brown [10] .
2.6. Determination of Total Tannins
After extraction of phenolic compounds by chloroform, then comes the processing of the total tannin determination.
1 mL of the acetone phase was measured with a pipette and introduced into a test tube N˚ 1. 5 mL of distilled water and 1 mL of ammonia were added and the whole was stirred for a few seconds with a vortex mixer.
A second sample of 1 mL of the acetone phase is taken in a test tube N˚ 2 by adding 5 mL of distilled water and 1 mL of the iron (III) ammonium citrate solution while stirring for a few seconds with a vortex mixer.
The two solutions from tubes N˚ 1 and N˚ 2 are transferred to the measuring vessels (1 cm) for measurement of absorbances with a spectrophotometer (UV-1700: UV-Visible spectrophotometer—SHIMADZU) at 525 nm against water, 10 min after operations 1 and 2. The result is based on differing absorbances.
The establishment of the calibration curve was done as follows:
The gallic acid solution was pipetted into 6 test tubes 0, 1, 2, 3, 4, and 5 mL, respectively, and then each test tube was supplemented with acetone such that 10 mL of solution was obtained.
1 mL of each of these 6 solutions was pipetted into 6 other test tubes and successively supplemented with 5 mL of distilled water, 1 mL of the iron (III) ammonium citrate solution. The whole was shaken for a few seconds with a vortex mixer.
The curve was plotted with the absorbance values on the ordinates and the corresponding concentrations on the abscissa.
The resulting solutions are transferred to the measuring cessels and the absorbances are measured 10 minutes later at 525 nm against water.
2.7. Statistical Analysis
The statistical study was carried out by Excel software at the 5% probability threshold. All experiments were performed in triplicate. The comparison of means was performed by analysis of variance (ANOVA). Results are expressed as mean ± standard deviation. Values of p < 0.05 are considered statistically significant [11] .
3. Results
3.1. Qualitative Analysis
The results of the phytochemical screening on the tannins of the Mucuna different species seeds are shown in Table 1.
3.2. Quantitative Analysis
A calibration curve was plotted with gallic acid at different concentrations. The tannin content was determined from Equation (1):
(1)
With the correlation coefficient R2 = 0.9985.
3.3. Dosage of Untreated Seeds
The results of tannin determination in different parts of untreated seeds of the four Mucuna species are shown in Table 2.
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Table 1. Results of tannin identification tests four species of Mucuna seeds.
++: intense coloring and +: less intense coloring.
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Table 2. Estimated values of tannins in the different parts of untreated Mucuna seeds.
Superscripted values with the same letters in the lines were not significantly different (p < 0.05) according to Duncan’s multiple comparison test.
3.4. Dosage of Treated Seeds
The tannin content values in the treated seeds of the different Mucuna species are recorded in Table 3.
3.5. Tannin Reduction Rate
The percentages of tannin reduction in the treated seeds of the four Mucuna varieties are recorded in Table 4.
4. Discussion
4.1. Qualitative Analysis
Intense staining was observed in the tubes containing extracts of Mucuna deerigiana and Mucuna nagaland seeds. This implies a strong presence of tannins in the seeds of these Mucuna species. The tubes containing the extracts of Mucuna rajada and Mucuna cochinchinensis seeds are less colored indicating a light presence of tannins in the seeds of these two species.
The action of iron (III) chloride on the aqueous extract showed that all four seed varieties contain tannins (Table 1). This corroborates to the work of Agbafor and Nwachukwu [12] and later Murthy et al. [13] .
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Table 3. Estimated values of tannins in Mucuna seeds after different treatments.
Superscripted values with the same letters in the columns were not significantly different (p < 0.05) according to Duncan’s multiple comparison test. †A: seeds scalded directly for 2 hours; †B: seeds soaked for 12 hours in water followed by scalding for 2 hours; †C: seeds boiled in the presence of 0.5% KOH for 2 hours; †D: seeds soaked in water for 12 hours followed by scalding in the presence of KOH (0.5%) for 2 hours.
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Table 4. Rate of tannin reduction in Mucuna seeds according to the treatments performed.
4.2. Determination of Untreated Seeds
The test results of the different parts of the untreated seeds (Table 2) revealed, on the one hand, that the extract of the starch contains more tannins than the extract of the epicarp. On the other hand, for the crushed seed set, the results show that the Mucuna deerigiana seed extract is richer in tannins (0.370 g GAE/100g DM). It is followed by the seed extracts of Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada with the respective values of 0.336 g GAE/100g DM, 0.266 g GAE/100g DM, and 0.203 g GAE/100g DM thus confirming the results of the identification test. These values are similar to those found by Murthy et al. [13] which was 0.306 g GAE/100g DM for Mucuna cochinchinensis (white seeds) but lower than the value of 0.577 g GAE/100g DM found for Mucuna cochinchinensis from black seeds [13] .
Tannins are much more concentrated in starches than in epicarps. If they were more concentrated in the epicarp, the epicarp would be removed and the starch left for feeding. Since the starch constitutes the largest part of the seed, hence the need for the treatment processing.
4.3. Determination of Treated Seeds
Determination results of extracts from treated seeds of the four Mucuna varieties showed an effective decrease in tannins (Table 3).
All seed treatment methods resulted in a decrease of tannin content. However, the method of soaking the seeds in water for 12 hours and boiling in the presence of KOH (0.5%) for 2 hours gave the best result.
4.4. Tannin Reduction Rates
The percentages of tannin content reduction in the treated seeds of different Mucuna species are recorded in Table 4. The results revealed that the method of direct boiling of seeds for 2 hours decreased the tannin content by 24.86% in Mucuna deerigiana seed extract, 42.85% in Mucuna nagaland seed extract, 18.42% in Mucuna cochinchinensis seed extract and 3.94% in Mucuna rajada seed extract. This decrease in tannins from the different seeds of four species of Mucuna can be explained by the increase in the temperature of the water which broke some tannin-protein bonds allowing the polar solvent to extract the tannins from these seeds.
In addition, the method of soaking the seeds for 12 hours in water followed by boiling for 2 hours reduced the tannin content in the extracts of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada by 27.02%, 45.53%, 32.27% and 5.91% respectively. It is noted that this method reduced more tannins in the seeds of the different Mucuna species than the previous one. The high rate of tannin reduction can be explained by the duration of the soaking of the seeds.
On the other hand, the method of treating seeds by boiling in the presence of KOH (0.5%) for 2 hours resulted in low tannin reduction rates. It resulted in 8.37% tannin reduction in Mucuna deerigiana seed extract, 31.25% in Mucuna nagaland seed extract, 1.50% in Mucuna cochinchinensis seed extract and 3.94% in Mucuna rajada seed extract. This low reduction rate can be explained by the formation of potassium salts with some tannins that are trapped.
Finally, the method of soaking seeds in water for 12 hours, then boiling in the presence of KOH (0.5%) for 2 hours gave for the extracts of the seeds of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada, the percentages of reduction of tannins respectively 47.29% , 48.21% , 66.16% and 15.76%. These results, compared to those obtained by the first treatment method which seems to be the best, indicate a clear increase in the rate of tannin reduction for the seeds of these different Mucuna. However, this method of soaking seeds in water for 12 hours and then boiling in the presence of KOH (0.5%) for 2 hours resulted in a high reduction of tannins in the seeds of all four species of Mucuna. This high reduction rate can be explained by the soaking time, which has already extracted some seeds that can form salts.
5. Conclusions
The seed extracts from the different Mucuna species were subjected to qualitative and quantitative analysis. Phytochemical screening revealed excessive presence of tannins in the seeds of Mucuna deerigiana and Mucuna nagaland and slight presence of tannins in the seeds of Mucuna cochinchinensis and Mucuna rajada.
The results of the tannin analysis showed that the crushed seeds as a whole contain more tannins. The tannin content in the seed extract of Mucuna deerigiana is high. It is followed by the seed extracts of Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada respectively. These results also show that tannins are much more concentrated in the seed starches of the four Mucuna species than the epicarps.
The different seed treatment methods were effective in reducing the tannin content in the seeds of the different Mucuna species. However, the method of soaking the seeds for 12 hours and then boiling them in the presence of 0.5% KOH for 2 hours was the most effective of the four methods.