Abstract

Phenolic compounds are highly solicited bioactive substances, present specifically in plants and playing a powerful antioxidant role. These secondary metabolites are endowed with several very interesting biological properties, which find applications in various fields, in particular in medicine, cosmetics and nutrition. Given the various virtues of these compounds, we were interested in determining by High Pressure Liquid Chromatography) HPLC their contents in the aqueous extract of roasted beans of Coffea canephora (EAGTCc) and particularly the contents of chlorogenic acid, caffeic acid and catechol. In addition, the antioxidant activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Total polyphenols and total flavonoids were also assayed spectrophotometrically. HPLC showed levels of 3.7 % catechol, 5.4 % chlorogenic acid and 2.8 % caffeic acid in 100 g of dry matter of EAGTCc. The concentration of EAGTCc responsible for 50 % inhibition (IC₅₀) of the DPPH radical is 75.54 ± 2.08 µg / mL. The contents of total polyphenols and total flavonoids are 81.67 mg EAG / g and 293.33 mg EQ / g. This antioxidant activity present in the EAGTCc is probably linked to the richness of the seeds of this plant in phenolic compounds. The results obtained are very interesting insofar as the use of natural compounds as antioxidants remains without side effects on the body and could replace the use of antioxidant molecules resulting from chemical synthesis.

1. Introduction

Plants synthesize secondary metabolites such as polyphenols, which are a large group of natural phytochemicals. These polyphenols are made up of aromatic ring(s) to which at least one hydroxyl group is attached. The main group of polyphenols are the Flavonoids and Phenolic acids and have been used as bioactive compounds in controlling human diet. They can be found in fruits, vegetables, herbs, cocoa, coffee, tea, and some other alcoholic beverages such as beer and wine. In addition to controlling human diet, Flavonoids have consistently gained a lot of attention from researchers due to their biological functions such as antioxidant, hepatoprotective, antibacterial, anti-inflammatory and anti-cancer activities. Furthermore, phenolic acids and flavonoids are important organic compounds and their consumption provide in vivo protection against free radical damage and reduces the risk of degenerative diseases associated with oxidative stress ^{1, 2, 3, 4, 5}. Previous research on the bean seeds of coffee trees indicate that they contain phenolic compounds and flavonoids ^{6, 7} and their consumption, especially in the Americans reveal some benefits, including overall mortality rates ^{8, 9}. The antioxidant activity of most fruits, vegetables and foods have been determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) ^{3, 10, 11}. This DPPH method could also be extended to determine the antioxidant activity in many plant extracts including the bean seeds of coffee trees ^{11, 12}. The coffee drink brewed in the coffee shops is a complex mixture of more than a thousand different chemical compounds, many of which are said to be biologically active ¹³. The coffee bean seeds contain many organic compounds such as flavonoids, chlorogenic acid and caffeic acid, etc, which have been used as antioxidant agents ^{6, 14}. In this study, our aim is to measure the anti-free radical activity of an aqueous extract of roasted bean seed of Coffea c. using the DPPH method.

2. Materials and Methods

Roasted and finely ground green bean seeds of a coffee plant (Coffea c.) was use in this research. The green coffee bean seeds were obtained from Ivory Coast in Africa.

Thirty grams (30 g) of roasted bean seeds of Coffea c. were ground into a powder and dissolved in 175.00 mL of hot distilled water. The resulting aqueouss solution was transferred into a Philips brand filter coffee maker and allowed to brew for 10 minutes. The filtrate obtained was dried in an oven (Binder, Tuttlingen, Germany) at a temperature of 60°C to a solid powder. 3 g of the dried powder was recovered from the aqueous extract of roasted bean seeds of Coffea c.; percent recovery was 10 %.

The determination of the total polyphenol content in our extract was done using the method proposed by method Vermerris and Nicholson ¹⁵. 0.10 mL of the sample was stirred continuously with 2.0 mL of a 2 % sodium carbonate solution. After 5 minutes, 0.10 mL of Folin-Ciocalteu reagent (0.2 N) was added dropwise to the resulting mixture and allowed to react for 30 minutes. The reading was obtained using a spectrophotometer at 700 nm against a blank. A calibration curve was produced under the same condition as the extract with gallic acid at different concentrations ranging from 100 to 1000 µg / mL. The analyzes was repeated three more times and the results expressed in milligrams equivalent of gallic acid per gram of dry extract (mg EAG / g E).

The colorimetric method reported by Ardestani and Yazdanparast ¹⁶ was used to determine the total flavonoids content in our sample. 500.00 µL of the extract was mixed with 2 mL of distilled water and 150 µL of 15 % sodium nitrite (NaNO₂) solution and incubated for six minutes. 150 µL of 10 % aluminum chloride (AlClᴣ) was added dropwise to the resulting solution. On the other hand, 2.00 mL of 4 % sodium hydroxide (NaOH) was added to the different test tubes and diluted to 5 mL with distilled water. After 15 minutes of continuous stirring, the absorbance of the solution was measured using a spectrophotometer set at 510 nm against a blank. A calibration curve was produced under the same condition as the sample with quercetin at different concentrations (100 to 1000 µg / mL). The process was repeated three more times and the results expressed in milligram quercetin equivalent per gram of dry extract (mg EQ / g E).

The extraction and determination of phenolic compound in our sample was done according the method proposed by Donovan et al. ¹⁷. 25.00 mL of the aqueous extract of the bean seeds of Coffea canephora was added to 75.00 mL of 80 % dilute methanol solution (v/v) and saturated with sodium chloride solution. After continuous stirring for 30 minutes, the resulting solution was filtered using a Watchman No.4 paper. The filtrate obtained was concentrated at 35 °C using a HEILDOLPH Laborata 4003 Control rotary evaporator (Schwabach, Germany). The resulting mixture was diluted with 100 mL of distilled water and the total phenolic compounds in the sample determined using an HPLC instrument (Shimadzu Corporation, Japan) consisting of a pump (Shimadzu LC-20A Liquid Chromatograph), a UV detector (Shimadzu SPD-20A UV Spectrophotometry detector). The retention times of the resulting spectra were compared the retention times of the reference molecules (chlorogenic acid, caffeic acid and catechol) and an internal standards was prepared from powders of the reference substances. The procedure was replicated and the concentration of the different constituents determined using the equation below:

where

CE = sample concentration

CT = Witness concentration

Aire T = sample peak surface

Aire E = sample peak area

2.2-diphenyl-1-picrylhydrazyl (DPPH) is an unstable free radical, which becomes stable by accepting a free hydrogen atom. The antioxidants activity of DPPH is determined by its ability to accept it a hydrogen atom. The method described by Mansouri et al. ¹⁸ was used to measure the anti-free radical activity by DPPH. In this research, 50.00 µL of different concentrations of the EAGTCc solution were added to 1,950.00 µL of a 0.025 mg/mL of DPPH solution. Similarly, a negative control was prepared in parallel, by adding 50.00 µL of methanol to 1,950.00 µL of a 0.025 mg/mL methanolic solution of DPPH. At the same time, a positive control was prepared from ascorbic acid at different concentrations ranging from 0.02 to 0.2 mg / mL. The above solutions were incubated for 10 minutes, their absorbances determined using a spectrophotometer set at 515 nm. The equation below was used to express our calculations as a percentage.

Where:

DPPH (%) = Percent reduction in DPPH

OD_control = Optical density of the negative control

OD_sample = Optical density of the sample.

The 50 % inhibition concentration (IC₅₀) is the concentration of the test sample necessary to reduce DPPH radical solution to 50 %. The IC₅₀ values were determined graphically by linear or logarithmic regressions of the plots of the percentage inhibition as a function of the various final concentrations of the extracts tested ¹⁹. This procedure was repeated three times.

Different methods were used to determine the number of ions present in our aqueous extracts (EAGTCc). The potassium ions (K⁺) were determined by flame spectrophotometry or flame test. The titanium yellow spectrophotometric method was used to determine the presence of magnesium ions (Mg²⁺). In addition, the complexometric method was to determine the presence of calcium ions (Ca²⁺). Finally, the phosphorus, zinc and copper ions were determined using the absorption spectrophotometry method ²⁰.

3. Results and Discussion

The total polyphenols and total flavonoids contents in the aqueous extracts our sample (EAGTCc) were determined to be 81.67 mg EAG / g and 293.33 mg EQ / g respectively (Table 1).

The total polyphenol content (81.67) determined in the aqueous extracts of roasted bean seeds of Coffea c. was seen to be higher than that reported from previous research done on aqueous extracts of roasted beans of coffee robusta (47.85 ± 0.077 mg EAG / g and 7 mg EAG / g to 42.37 mg EAG / g ^{21, 22}. The only research that has reported higher values of total polyphenol content (561.91) compared to our result was in 2017 by Khadra obtained from aqueous extracts of green robusta coffee beans ²³.

HPLC revealed levels of 3.7 % catechol, 5.4 % chlorogenic acid and 2.8 % caffeic acid in 100 g of dry matter of our sample (Figure 1 and Figure 2).

From Figure 1 above, the chlorogenic acid content decrease with increase in temperature. This might be due to roasting of the seed. During roasting, chlorogen acid is decomposed into different phenol derivatives, including caffeic acid, quinic acid, catechol, pyrogallol and hydroquinone ²⁴. Previous research showed that during roasting, about 95 % of n-chlorogenic acid was observed at 200°C ^{25, 26, 27}. This gives the content of n-chlorogenic acid in roasted coffee beans to be between 0.5 % and 7 %. Therefore, our chlorogenic acid content after roasting of 5.4 % agrees with that obtained from previous reports ²⁴. However, some reports show a higher chlorogenic acid content (13.00 % - 14.73 %) after roasting of green bean seeds than that obtained in our study ²⁸.

The concentration of EAGTCc responsible for 50 % inhibition (IC₅₀) of the DPPH radical solution in our sample was higher than that of quercetin (75.54 ± 2.08 µg / mL versus 4.45 ± 1.02 µg / mL) (Table 2).

The value of IC₅₀ correlated with its antioxidant power. A low IC₅₀ value would reflect a higher antioxidant effect ²⁹. The IC₅₀ value ofour extract (EAGTCc) was observed to be 75.54 ± 2.08 µg / mL. This IC₅₀ value is higher than that obtained with an aqueous extract of green robusta coffee bean seeds (51 μg / mL) ²³ indicating that our extract has a higher antioxidant effect compared an aqueous extract of green robusta coffee bean seeds. Therefore, the antioxidant activity contained in the coffee drink is linked, among other things, to the chlorogenic, ferulic, caffeic and n-coumaric acid contentit contains ^{30, 31}. Numerous studies have shown that polyphenols (mainly in the form of n-chlorogenic acid) and melanoidins have antioxidant activity and limit DNA degradation ^{32, 33}. This activity could be due to the scavenging effect of the radical and/or to the chelating effects of the metal ^{34, 35, 36}. Thus, the antioxidant activity of coffee is linked to its richness in phenolic compounds ^{37, 38}

Therefore, the aqueous extract of roasted bean seeds of Coffea c. (EAGTCc) contains 1,159.5 mg of potassium, 213.0 mg of magnesium, 106.2 mg of calcium, 100.5 mg of phosphorus, 31.0 mg of sodium, 3.3 mg of copper and 2.2 mg of zinc in 100 g of dry matter. These values correspond respectively to 1.16 % of potassium, 0.21 % of magnesium, 0.11 % of calcium, 0.10 % of phosphorus, 0.03 % of sodium, 0.003 % of copper and 0.002 % of zinc (Table 3).

The different electrolyte contents in our extract could be due to the method of preparation of the coffee drink and the composition of the coffee bean seed used. In addition, the different electrolyte contents in our extracts could be caused environmental conditions such as the soil texture, the climate, the agricultural practices and the storage of the green coffee bean seeds. This would affect the physiology of the seeds and the chemical composition of the coffees ^{26, 39}. The magnesium (0.212 % to 0.415 %) and calcium (0.106 % to 0.189 %) contents of our extract agrees to those in instant coffee made from a blend of robusta and arabica ^{40, 41}. However, the contents of potassium (3.35 % to 4.76 %), phosphorus (0.223 % to 0.410 %), copper (0.005 % to 0.026 %) and zinc (0.003 % to 0.015 %) contents in our extract (EAGTCc) are lower than those in coffee made from a blend of robusta and arabica.

4. Conclusion

In this study, we have successfully determine the organic content of an aqueous extract of roasted bean seeds of Coffea c. (EAGTCc). This extract was shown to contains a good content of total polyphenols and can be used as a good antioxidant. The IC₅₀ value (75.54 ± 2.08 µg / mL) was higher than that obtained with an aqueous extract of green robusta coffee bean seeds (51 μg / mL). In addition, the antioxidant activity of coffee could be attributed to its rich content in phenolic compounds different elements. Furthermore, these contents may vary due to environmental conditions. The antioxidant activity of coffee could be used to justify its use in preventing different diseases.

References