Comparative survey of three processes used for the extraction of total phenol content and total flavonoid content of Anacardium occidentale L . and the assessment of its antioxidant activity

1 Department of Chemistry. Faculty of Sciences and Techniques University Adam Barka of Abeche (UNABA), Chad. 2 Department of Chemistry. Faculty of Sciences. University of Ngaoundere (UN), Cameroun. 3 Department of Applied Chemistry. National Advanced School Of Agro-Industrial Sciences, University of Ngaoundere (UN) Cameroon. 4 Laboratory of Research on the Natural Substances, University of N’Djamena (UNDJ), Chad.


INTRODUCTION
Researchers have done studies on how to arrange and measure chlorophylls as well as its effect on plants.These studies also show that plants are bioactive compounds.They explain what causes inhibition of root growth, as well as reduced water absorption; what causes the morphological, biochemical and physiological changes in parts of different plants.Recent studies show that medicinal plants are an important source of new chemical substances with potential materials (Kravkaz et al., 2018).
Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects.Cashew (Anacardium occidentale) is a tree in the family of the flowering plant Anacardiaceae.The family contains 73 genera and about 600 species.Anacardium contains eight species, native to tropical America, of which the cashew is by far the most important economically.It is a multipurpose tree that grows up to 8 to 10 m height.It has a thick and tortuous trunk with branches winding that they frequently reach the ground (John et al., 2017).The cashew tree produces many resources and products.The cashew nut has an international appeal and market value as food.Even the shell oil around the nut is used medicinally and has industrial applications in plastics and resin industries for its phenolic content.The pseudo-fruit, a large pulpy and juicy part, has a fine sweet flavor and is commonly referred to as the "cashew fruit" or the "cashew apple" (John et al., 2017).In Chad, it is also used to treat diabetes, weakness, muscular debility, urinary disorders, asthma, eczema, psoriasis, scrofula, dyspepsia, genital problems, bronchitis, cough, intestinal colic, venereal diseases, as well as impotence, and syphilis-related skin disorders.In addition to being delicious, cashew fruit is a rich source of vitamins, minerals, and other essential nutrients.Several clinical studies have shown that anacardic acid, a component of cashew, with the highest concentration in the nutshells curbs the darkening effect of aging by inhibiting tyrosinase activity, and that it is toxic to certain cancer cells.Moreover, anacardic acid causes allergies and dermatitis (Jothi et al., 2013;Honoré et al., 2017).
Phytochemical studies revealed that the stem barks of the plant contain tannins and flavonoids.Resorcinolic acid, ascorbic acid, carotenoids, vitamin C and phenolic compounds were also identified in the apples and stem barks (Ajileye et al., 2015).Edy et al. (2007) quantified total flavonoids from the methanol/water extracts of the apples.From the ethanoic extract, phenolics were the main components.Glucosyl quercetin, derivative of amentoflavone and a tetramere of proanthocyanidin were also identified (André et al., 2012).
Due to the fact that A. occidentale is very useful, as shown earlier, there is a need to find out more about the potentiality of this plant as an antioxidant agent.The objective of the present work was to point out the best process of extraction of the phenolic and flavonoid compounds and to determine the anti-oxidant activity of extracts with a perspective of doing further research on new active molecules.

Plant
Samples of stem barks, leaves and nuts of cashew were collected from Southwestern part of Chad (Pala Region of Mayo-Kebbi) in April 2014.The species were identified and voucher specimen was deposited at the National Institute of Research for Development (IRED), Farcha, N'djamena, Chad (n° 1344).
The air-dried materials were ground and then pulverized.Obtained powders were kept in closed containers until needed.

General experimental procedure
Column chromatography (CC) was performed on silica gel 60 (70-230 mesh, Merck), monitored by analytical thin layer chromatography (TLC) on silica gel precoated plates F-254 Merck (20 × 20 cm).Spots were visualized under UV light (254 and 365 nm), sprayed with 20% sulfuric acid then heated at 105°C for 15 min.The melting point of the new compound was recorded in an open capillary using Stuart melting point apparatus (SMP-3) and is uncorrected.ESIMS spectra (ionization voltage 3 kV) were registered on a Q-TOF Ultima spectrometer (Waters).A spectrometer Bruker Avance AV-500 (125 and 500 MHz) was used for NMR spectra.CDCl3 or DMSO-d6 was used as solvents and TMS as internal standard.Ciprofloxacin (Hollande) and ketoconazole (European Pharmacopoeia (EP) Reference Standard, Sigma-Aldrich), were used as reference antibiotics whereas hiazolyl blue tetrazolium blue (MTT) from Sigma Aldrich was used as bacterial growth indicator for antimicrobial assay.α-Glucosidase from Saccharomyces cerevisiae recombinant G0660-750UN, 4nitrophenyl α-D-glucopyranoside ≥ 99% N13771G, acarbose (positive control) and sodium dihydrogen phosphate were purchased from Sigma Aldrich, (Germany).100% dimethylsulfoxide (DMSO) was purchased from LAB-SCAN (Нailand).Solvents used were of analytical grade and water was distilled.

Extraction procedure
Different raw samples were separately submitted to three processes of extraction, using methanol as solvent: mechanical agitation, Soxhlet apparatus and microwave assisted.In order to recognize the best process used for the extraction of total phenolic and flavonoid compounds, optimization study and multi-response analysis were carried out.For each extract, the yield was determined.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License were shaken and left to stand for a few minutes at room temperature to allow the reagent to react completely with the oxidizable substances or phenolates.0.4 ml of Na2CO3 (20% in water) was added to destroy the residual reagent.Absorbances were measured at 760 nm, using a spectrophotometer (spectrophotometer UV Rayleigh Vis-723N) after incubation for 20 min at 40°C against distilled water as a blank.Total phenolic contents of the samples determined from the calibration curve equation (y = 0.1236x, R 2 = 0.9831) were expressed in mg gallic acid equivalents (GAE)/100 g of dry material.All measurements were performed in three replications.

Determination of total flavonoids contents
Total flavonoid contents of the extracts were evaluated using UV spectroscopic method.A volume of methanolic solution of aluminium chloride (0.5 ml, 2% w/v) was mixed with the methanolic solution of the extract (0.5 ml, 0.1 mg/ml).After 10 min, absorbances were measured at 415 nm.Results, determined from the calibration curve equation (y = 0,009x, R 2 = 0,9963) were calculated as follows and expressed in mg of quercetin equivalents (QE)/100 g dry material (DM) (Anyin et al., 2016): , Where, F is the content of total flavonoids (mg QE/100 g DM); Aext is the absorbance of the extract; Aq is the absorbance of quercetin; Cext is the concentration of plant extract (10 mg/ml).All measurements were performed in three replications.

Determination of anti-oxidant activity
The potential antioxidant activity of the extracts was assessed on the basis of the scavenging activity of stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical according to Ali et al. (2015).For the control, 3.8 mL of 100 mM DPPH was added to 0.2 mL of MeOH extract.The step was repeated by replacing extract with BHT (5 mgmL -1 ).All samples were incubated for 30 min and following incubation, absorbance was read at 517 nm.The percentage of inhibition of the sample against DPPH radicals was calculated from the following equation.

Yield of extraction
Microwave assisted extraction gave the best yield, followed by Soxhlet and mechanical agitation.These results explained that, in microwave, the sample comes quickly in contact with solvent and there is more transfer matter toward the solvent.Yield extractions are shown in Table 1.

Exploratory qualitative analysis of the extracts
The tests achieved on the plant materials reveal that methanol extract was richer in phenol, flavonoid, tannins and less rich in saponins, followed by ethyl acetate.The methanol extract with ethyl acetate was only rich in phenol but less rich in flavonoids and terpenoid.while,the extract with hexane was poor in these compounds.This could be due to the difference of polarity between these solvents.These results corroborate the works of Honoré et al. (2017).

Quantitative analysis
The results of the quantitative analyses by spectrophotometer visible UV of the different extracts are shown in Table 3.The method used is the one of Chen et al. ( 2011).In the survey, the absorbance of a set of concentrations of gallic acid and quercetin led to the curves of standardization with the equations and coefficients of correlation, respectively: , for gallic acid and , for quercetin These results reflect the rich content of the plant in phenolic compounds as flavonoids and phenols.Table 2 shows the total phenol content and total flavonoid content.These total phenol content and total flavonoid content got are better than the result obtained by Honoré et al. ( 2017).

Anti-oxidant activity of the extracts
In terms of antioxidant activities, it can be observed that the stem barks had the best anti-oxidant activity, followed by the leaf extracts.The coefficient of determination (R 2 ) of the predicted anti-oxidant activity was 0.983.The predicted high values were found by microwave that IC 50 of the methanolic extracts of the stem bark was not better than BHT.The antioxidant activity of the extracts was expressed in IC 50 that defines the efficient concentration of the substratum that causes 50% loss of the activity of the radical DPPH.This IC 50 is shown in Table 3. Results of IC 50 determined from the calibration curve equation (Y = 0.123x, R 2 = 0.983) of standardization of BTH for DPPH test were obtained and expressed in mg/mL.Smaller IC 50 value had important activity and more value was considered as a poor antioxidant activity.
The results obtained earlier for the percentage of inhibition of the extracts are as shown in Figures 1, 2 and  3.The curves of the percentage of inhibition were function of the concentration from different processes of extraction.It was noticed that the extracts of the stem barks inhibit the DPPH better that the other extracts, followed by the extracts of the leaves.As for the extracts of the cashew nuts, the percentages of inhibition were weak in relation to the other extracts.These results corroborated with those of John et al. (2017).

Yield
The yield (output) of these different methanol extracts is calculated and reported in Table 4.The microwave assisted extraction had a better output, followed by the  Soxhlet extraction, and then the mechanical agitation.

Total phenol and total flavonoid content
Whatever the technique of extraction used, the flavonoids and phenols are in the same quantity in the extracts.The strong content in phenol observed in the microwave extracts could be due to the fact that this system of extraction is similar to that used in cooking device (Tsatsop et al., 2016).As for the Soxhlet technical and mechanical agitation, the contents were nearly the same.Figures 4 and 5 show the total flavonoids and total phenols contents.

Antioxidant activity of the extracts
The microwave extraction technique of extracts gave compounds that inhibit the DPPH better after the BTH than the techniques by Soxhlet and mechanical agitation that nearly had the same percentages of inhibition.Figure 6 shows the percentages of inhibition.

Isolation of compounds
A portion of 50 g of methanol extract of the leaves obtained by Soxhlet extraction was subjected to column chromatography of silica gel using a gradient of Hex-EtOAc (1:0 to 0:1) and EtOAc-MeOH (1:0 to 0:1) with increasing polarity.The main column chromatography led to the isolation of two known compounds: Quercetin-3-Oα-D-glucopyranoside (4 mg, 1) and Kaempferol-3-O-β-Dglucopyranoside (5 mg, 2) (Figure 7).To the best of our knowledge, this is the first report of these two compounds in A. occidentale.

Conclusion
The extraction of the different organs obtained by different processes makes us to conclude that microwave is the best extraction process used for the extraction of the total phenol content and total flavonoid content and the assessment of the anti-oxidant activity of this extract is high, while Soxhlet and mechanical agitation give almost the same results.These two compounds isolated reveal the presence of flavonoids in the plant.

Inhibition
Four experiments were carried out at each experimental design point and the mean values were stated as observed responses.Experimental runs were randomized to minimize the effects of unexpected variability in the observed responses.Comparison of means was performed by one-way analysis of variance (ANOVA) followed by Duncan's test.Statistical analyses (p<0.05) were performed using Statgraphics centurion software (Version XVI.I).

Figure 1 .
Figure 1.Curves of the percentage of inhibition of methanol extracts gotten by mechanical agitation.

Figure 2 .
Figure 2. Curves of the percentage of inhibition of methanol extracts gotten by Soxhlet.

Figure 3 .
Figure 3. Curves of the percentage of inhibition of methanol extracts gotten by microwave.

Figure 4 .Figure 5 .
Figure 4. Content in total phenols of the extracts by different methods.MO, Microwave; Sox, Soxhlet; AM, mechanical agitation; (A) MeOH extract of the steam barks; (B) MeOH extract of the leaves; (C) MeOH extract of the cashew nuts.

Figure 6 .
Figure 6.Percentage of inhibition from different processes of extraction.(A) MeOH excerpt of the steam barks; (B) MeOH extract of the leaves; (C) MeOH extract of the cashew nuts.

Table 1 .
Yield of extraction.

Table 2 .
Content in total phenols and in total flavonoids.The values carrying as exponents the same letters in the columns are not meaningfully different (p < 0.05) in accordance with the test of multiple comparison of Duncan. b

Table 3 .
Concentration of IC50 inhibition of the different extracts.