3.1. Phytochemical screening
To detect the different classes of secondary metabolites present in T. articulata, we performed phytochemical tests on their organic and aqueous extracts. These tests have the advantage of revealing the chemical groups present in the plant material, which allows us to have a better appreciation of the proven and potential biological activities of the species in our study. The main chemical groups, identified by these tests (Table 1), were the polyphenols (flavonoids and tannins), osteoids, holosides, sterols, triterpenes, and mucilages.
For polyphenols, both gallic and catechic tannins were detected in T. articulata extracts. As for flavonoids, they exist in several forms, flavones, dihydroflavonols, flavonols, flavanonols, and anthocyanins. The characterization tests have shown the presence of leucoanthocyanins and free flavonoids and the absence of anthocyanins. The tests using the reagents of Mayer and Dragendorff allowed the demonstration of the richness of T. articulata extracts in alkaloids. The presence of this chemical family in T. articulata may increase the possibility that it exhibits important biological properties. Indeed, we can cite, for example, the alkaloids shown in N. latifolia gave it antimalarial and antimicrobial activity [14]. Also, the alkaloids (vincristine and vinblastine) extracted from the Madagascan periwinkle (Catharanthus roseus) are used in cancer treatments. However, some studies show that alkaloids, although therapeutically interesting, can constitute a significant risk of toxicity (Stegelmeier et al., 2016; [16]. Other tests of characterization have shown the presence of sterols and terpenes, oses, holosides, and mucilages while the carotenoids and Coumarins are absent. On the other hand, given that the decoction is the preferred method of preparation of traditional remedies, and based on the results of phytochemical tests, the remedies used by the populations seem to be rich in polyphenolic compounds, oses, holosides, and mucilages. These compounds with various activities are also known for their effects on the digestive system and pain relief [17]. This could help explain the fact that most plants have a use in traditional medicine to treat digestive disorders and abdominal pain. Also, despite the possible abundance of alkaloids in a plant, they could be found in low proportions in traditional remedies due to their insolubility in water. So, the risk of exposure to the toxic effects of alkaloids can be reduced. Polyphenolic compounds being the most represented in T. articulata and probably the most important in traditional remedies, we have advanced our research by performing assays of these polyphenolic compounds in the studied species.
Table 1
Phytochemical Screening of T. articulata
Chemical family | T.articulata |
Alcaloïds | Mayer | + |
Dragendorff | + |
Polyphenols | Tannins | Gallic tannins | - |
Catechic tannins | +++ |
Flavonoïds | Anthocyanes | - |
Reaction of Cyanidine | +++ |
Leucoanthocyanes | +++ |
free anthracenic derivates | - |
combinedanthracenicderivates | O-heterosides | - |
C-heterosides | - |
Sterols et triterpenes | +++ |
Mucilages | +++ |
Saponins | - |
Cardiotonic Heterosides | - |
Carotenoïds | - |
Oses and holosides | + |
Quinones | - |
+++ : Reaction strictly positive, + : reaction positive, -: reaction negative |
3.2. Yields of phenolic compounds
The solid-liquid extraction of the phenolic compounds from T. articulata, carried out by soxhlet using hydroalcoholic solutions: methanol/water and ethanol/water at 80% allowed obtaining two crude extracts for each sample, these are hydroethanolic extract (MeOH Ext.) and hydroethanolic extract (EtOH Ext.) respectively. The results are summarized in Table 2. The yield of hydromethanolic extract of T. articulata was stronger (38.36 ± 0.31%) than that observed in hydroethanolic one (17.23 ± 0.45%). These results are in agreement with those carried out by [18], who found that the effective solvent for extracting polyphenols, with the three used methods (maceration at room temperature, hot infusion and microwave), was methanol followed by ethanol and water. Moreover, the extraction of methanol gives a wide range of phenolic compounds, including anthocyanins, phenolic acids, catechins, flavanons, flavanols and procyanidins [19] [20]; Ross et al., 2009). For fractionation of crude extract (MeOH Ext.) of T. articulata, the results showed that the extract from the residual phase exhibited the highest yield (40.72 ± 0.81%) followed by BuOH Ext. (23.81 ± 0.09%), then AcEth Ext. (18.19 ± 0.11%). Indeed, the highest yield observed for ResPh Ext. allowed us to conclude that there are chemical compounds that are not soluble in either ethyl acetate or butanol. Differences in the extraction efficiency of various solvents have been attributed to their polarities [22]. So, the extraction yield depends on the polarity as well as the variety of intermolecular interactions or the selectivity of the solvents. It is difficult to compare the results with those in the literature, the yield is only relative and depends on the method and conditions under which the extraction was carried out.
Table 2
Yields of extracts by Soxhlet of the T.articulata
| EtOH Ext. | MeOH Ext. | AcEth Ext. | BuOH Ext. | Ph.Res Ext. |
T. articulata | 17.23 ± 0.45 | 38.36 ± 0.31 | 18.19 ± 0.11 | 23.81 ± 0.09 | 40.72 ± 0.81 |
3.3. Total Phenols, Flavonoids And Condensed Tannins Content
The contents of total phenols, total flavonoids and condensed tannins in extracts of T. articulata are expressed in mg equivalent of gallic acid, quercetin and catechin per gram of extract (mg EGA /g E) respectively (Table 3). AcEth Ext. recorded the highest content of total phenols, i.e. 654.69 ± 0.33mg EGA /g E, followed by BuOH Ext. (551.21 ± 0.41) mg EGA / g E, then MeOH Ext. (458.95 ± 0.19) mg EGA/ g E, and finally the ResPh Ext. and EtOH Ext. (144.52 ± 0.25) and 96.71 ± 0.08mg EGA / g E respectively. Compared to other studies, the aqueous extract of T. articulata was characterized by the presence of phenolic compounds with a content reaching 175.67 ± 10.21 µg EGA / mg MS [23]. The T. articulata hydroethanolic extract obtained by soxhlet from Tunisia reached a total phenol content (TPC) of about 38.1 ± 0.6 mg EGA/ g DM [24]. Likewise, the acetone extract of Tunisian T. articulata recorded a TPC of 32.47 mg EGA / g DM [25]. The EtOH Ext. and MeOH Ext. of T. articulata from Algeria get TPCs around 185 and 156 µg EGA / mg E respectively [26]. According to results of flavonoids dosage, the MeOH Ext. is displayed to be the richest in flavonoids among all tested extracts with a content (FC) of 20.31 ± 0.02 mg eq of quercetin EQ / g Extract, followed by EtOH Ext. (17.98 ± 0.13mg EQ/ g E) then AcEt Ext. (16.71 ± 0.03 mg EQ / g E). The FC of MeOH Ext. is lower than that observed in MeOH Ext. (37.14 𝜇g EQ/ mg MV) from T. articulata leaves collected in southern Morocco[27]. Also, [23] found that the aqueous extract of T. articulata, from the Ait Issi Ihahan region (Morocco), recorded a FC about 11.78 ± 0.30 𝜇g EQ / mgE which is greater than that obtained in our study (2.37 ± 0.21mg EQ / g of extract). Compared to our EtOH Ext., that from Algeria is richer in flavonoids (36.70 mg EQ/ g E) [28]. The T. articulata AcEt Ext. is completely free from condensed tannins. It can be inferred that ethyl acetate is not a suitable solvent for the extraction of condensed tannins.
Table 3
Polyphenol, flavonoids and condensed tannins of exctracts of T. articulata
Extracts | Polyphenols (mg EGA/gE) | Flavonoids (mgEQ/gE) | condensed tannins (mg EC/gE) |
EtOH Ext. | 96.71 ± 0.08 | 17.98 ± 0.13 | 12.09 ± 0.82 |
MeOH Ext. | 458.95 ± 0.19 | 20.31 ± 0.02 | 15.97 ± 0.74 |
AcEth Ext. | 654.69 ± 0.33 | 16.71 ± 0.03 | |
BuOH Ext. | 551.21 ± 0.41 | 8.75 ± 0.92 | 25.58 ± 0.14 |
Ph.Res Ext. | 144.52 ± 0.25 | 2.37 ± 0.21 | 27.72 ± 0.11 |
3.4 Identification of polyphenols in the MtOH Ext. from T. articulata by high-performance liquid chromatography coupled with mass spectrometry (HPLC / UV-ESI-MS)
Chromatographic profiles (Fig. 3) illustrate the peaks of phenolic compounds from T. articulata MeOH Ext. and standards and their retention times (RT) and relative abundances. Following the analytical and spectral data (chromatograms and mass spectra), we detected and identified seven compounds whose names and raw formulas are presented in Table 4. The MeOH Ext. chromatogram from T. articulata reveals the presence of the majority peaks which extend between 19.30 and 27.78 min. The analysis of the MS and the comparison with the results reported in the literature allowed determining the existence of molecules of the flavonoid type O-glycosides having ions: a pseudo-molecular ion [M + H] + and fragments [M-X + H]+ corresponding respectively to a loss of a hexose (X: 162) or of rhamnose (X: 146) or both at the same time rutinoside (X: 308). The most important compounds are Catechin, gallocatechin, Myrcetin-rhamnose, Quercetin-3-o-Rhamnoside and kampferol-deoxyhexose. These compounds were previously described in T. articulata extract, studied by[29] who identified nine compounds: (Epi) catechin dimer type B, (+) - catechin, (-) - Epi catechin, Myricetin-O-pentoxyl-O-hexoside, Myricetin-3-O rutinoside, Myricetin-3-O glucoside, Myricetin-3-O rhamnoside, Quercetin-3-O rhamnoside and Kaempferol-3- O rhamnoside. In addition, [30] have identified seven compounds which are: Catechine, Myricetin-hexose, Myricetin-rhamnoside, Quercetin-3-O-glucoside, Kaempferol-hexose, Quercetin-3-O-rhamnoside Kaempferol-deoxyhexose. Likewise, (+) - catechin and (-) - epicatechin have been found in extracts of T. articulata obtained by decoction (aqueous extract) and Soxhlet (ethyl acetate and methanolic extracts) [31]. Indeed, the phytochemical analysis of this extract revealed the presence of a certain number of polyphenolic compounds (flavonols, flavones, flavan). Moreover, Myricitrin is a flavonol known for its numerous pharmacological effects [32], it is used as a supplement in functional foods, cosmetics and drugs due to its strong antioxidant activity [33] [34], antinociceptive, anti-inflammatory [35] and antiallodynic activity [33].
Table 4
Identification by HPLC-MS of polyphenols in MtOH Ext. of T. articulata
N° | Name | Masse | Ion M/Z | Fragments | Area % |
1 | Catechin | 291 | 292 | 291 | 4.25 |
2 | B-type (Epi) catechin dimer | 587 | 588 | 578, 453, 288 | 9.35 |
3 | Gallocatechin | 306 | 307 | 306 | 6.89 |
4 | Myricetin-hexose | 480 | 481 | 480 | 4.67 |
5 | Myrcetin-rhamnose | 465 | 466 | 465,319 | 14.39 |
6 | Quercetin-3-o-Rhamnoside | 449 | 450 | 449,303 | 4.98 |
7 | kampferol-deoxyhexose | 433 | 434 | 433,287 | 6.27 |
3.5. Antioxidant activity
3.5.1. DPPH free radical scavenging test
The antiradical activity of T. articulata extracts and the reference standards (ascorbic acid and BHA) was carried out by the DPPH method. The results obtained are shown in Fig. 4. From obtained data, we see that MeOH Ext. and EtOH Ext. has inhibition percentages of about 93.94% and 91.99% respectively for a concentration of 472 µg / mL, these percentages stand out by being greater than the ascorbic acid and BHA. Indeed, AcEth Ext., BuOH Ext. and Res Ph Ext. have significant anti-free radicals on DPPH free radicals with inhibition percentages of 82.71% 82.71%, and 85.87% respectively while, those of ascorbic acid and BHA are of the order of 90.49% and 89.29% respectively for a concentration of 472µl / ml. According to the results about inhibitory concentrations of 50% (IC50) values (Table 5), the EtOH Ext. has moderate antioxidant power (IC50 = 70 ± 0.07 µg / ml) followed by MeOH Ext. (IC50 = 90,68 ± 0.32µg / ml), AcEth Ext. (156.36 ± 0.04µg / ml), BuOH Ext. (231.37 ± 0.14µg / ml) and finally Ph.Res Ext. (104.01 ± 0.05µg / ml). These values are much higher than those of ascorbic acid (31.44 ± 0.24µg / ml ) and BHA (46.25 ± 0.31µg / ml).
Table 5
The IC50 (µg/ml) of the reference standards and extracts of T. articulata
Extracts | Ascorbic. A | BHA | EtOH Ext. | MeOH Ext. | BuOH Ext. | AcEt Ext. | Ph.Res Ext. |
IC50 | 31.4 ± 0.24 | 46.25 ± 0.31 | 70 ± 0.07 | 90.68 ± 0.32 | 231.37 ± 0.14 | 156.36 ± 0.04 | 104.01 ± 0.05 |
3.5.2. Iron reduction test: FRAP (Ferric reducing antioxidant power)
The antioxidant activity of extracts of T. articulata was also evaluated using the FRAP method. The results (Fig. 5) show that the reducing power of the extracts is dose-dependent (concentration-dependent). At a concentration of 0.4 mg/ml, the reducing power of BuOH Ext. is much greater (OD = 1.50) compared to MeOH Ext., AcEth Ext. and Ph.Res Ext., but clearly lower than that of ascorbic acid (OD = 1.9), BHA (OD = 1.88) and BHT (OD = 1.81). The concentrations corresponding to the optical density of 0.5 are described in Table 6. the BuOH Ext. has a relatively interesting EC0.5 (100 ± 0.65 µg/ml), compared to other extracts MeOH Ext. (225.65 ± 0.14 µg /ml), AcEth Ext. (470 ± 1.52 µg/ml) and Ph.Res Ext. (980 ± 0.57 µg /ml). Furthermore, the positive controls of Ascorbic acid, BHA and BHT recorded EC0.5 values around 19 ± 0.35 µg /ml, 46.25 ± 0.78µg /ml and 75 ± 0.92 µg /ml respectively. These results agree with those reported by [23], who found that the aqueous extract from T. articulata gets IC50 = 27.38 ± 0.02 𝜇g / ml and 47.12 ± 0.15 𝜇g / ml by the DPPH and FRAP methods respectively. In addition, the study performed by [29] showed that the aqueous extract, ethyl acetate extract, butanolic extract from leaves of Algerian T. articulata, exhibit significant antiradical power with IC50 = 12.7; 4.5 and 8.7 µg /ml for aqueous extract, ethyl acetate extract, butanol extract respectively.
Table 6
The EC0.5 of extracts of T. articulata and reference standards
Extracts | EC0.5 (µg/ml) |
Ascorbic acid | 19 ± 0.35 |
BHA | 46,25 ± 0.78 |
BHT | 75 ± 0.92 |
EtOH Ext. | 104,12 ± 1.02 |
MeOH Ext. | 225,65 ± 0.14 |
AcEt Ext. | 470 ± 1.52 |
BuOH Ext. | 100 ± 0.65 |
Ph.Res Ext. | 980 ± 0.57 |
3.5.3. Correlation between the content of phenolic compounds and the antioxidant activities of the extracts
All the obtained results from the assays of phenolic compounds and antioxidant activities (DPPH and FRAP) indicate that the extracts that recorded significant antioxidant potential are very rich in polyphenols and flavonoids. So, there are proportional relationships between antioxidant activities and total polyphenols. To better characterize these possible relationships between the different variables, we calculated the correlation coefficients (Table 7). Depending on the results of the antioxidant activity evaluated by the two methods DPPH and FRAP, it appears that there is a weak positive correlation for the extracts of T. articulata, ie R2 = 0.23. This difference in antioxidant activity between both methods could be attributed to the difference in compounds of the extracts which intervene by different types of action mechanisms. The correlation established between the FC of the T. articulata extracts and the antioxidant power determined by the DPPH test is strongly positive (R2 = 0.82). Similarly, a weak positive correlation (R2 = 0.25) is observed between the reducing power and the FC. However, the correlation between the antioxidant power and the TPC is weakly negative, which shows the richness of these extracts by other phenolic compounds which do not have antioxidant potential. The antioxidant activity of MeOH Ext. may be due to its chemical profile rich in Catechin, Gallocatechin, Myrcetin-rhamnose, Quercetin-3-o-Rhamnoside, and kampferol-deoxyhexose. The identified phytochemicals have been reported to have excellent efficiency as antioxidants [36] [37] [38] [39] Myrcetin and quercetin, the main flavonols found in our sample, have already been cited as antioxidants by [40] [41]. It should be noted that the TPC still does not correlate with antioxidant activity. This may mean on the one hand that the antioxidant activity is mainly due to the quality of the phenolic compounds in the extracts and not to their amounts. On the other hand, the anti-free radical and reducing powers of the extracts found in this study may not relate to flavonoids and polyphenols, but other chemical groups with antioxidant activity.
Table 7
Linear correlation coefficients (R2) between the content of phenolic compounds and the antioxidant activity of the extracts of the plants studied
| TPT | TFT | ARP(DPPH) | 1/EC0.5(FRAP) |
T. articulata | TPT | 1 | -0.28 | -0.77 | -0.023 |
TFT | | 1 | 0.82 | 0.25 |
ARP(DPPH) | | | 1 | 0.23 |
1/EC0.5(FRAP) | | | | 1 |