LC-MS analysis , antioxidant and alpha-glucosidase inhibitory activities of Centaurea papposa extracts

This work aimed to ascertain the phenolic compounds and assess the antioxidant capacity and alpha-glucosidase inhibitory activity of Centaurea papposa extracts. Phenolic compounds were appraised using LC-MS technique. Moreover, antioxidant activity was investigated using DPPH, ABTS, CUPRAC and FRAP assays. In vitro alpha-glucosidase inhibitory effect was carried out. LC-MS analysis revealed the presence of 21 compounds among which 13 were phenolic acids, 6 flavonoids, 1 phenolic aldehyde and 1 benzopyrone. The ethyl acetate extract exhibited the highest activity in ferric reducing antioxidant power (FRAP) assay (IC50: 22.9 ± 2.8 µg/mL). Nevertheless the n-butanol extract was the most active in cupric reducing antioxidant capacity assay (IC50: 3.1 ± 0.1 µg/mL). A significant alpha-glucosidase inhibitory activity was displayed by dichloromethane extract (IC50: 227.6 ± 4.4 µg/mL). 
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Plant material
The aerial parts of the plant were collected in full bloom in Annaba (North East of Algeria) in September 2016. They were identified by Dr. Hamel Tarek, Department of Plant Biology and Environment, Badji Mokhtar University, Annaba, Algeria. A reference specimen was deposited in the herbarium of the laboratory under the reference code No.: ChifaDZUMCAPBC000039. The samples were dried in the shade at room temperature in a ventilated place and cut into small pieces.

Preparation of extracts
The air-dried powdered aerial parts of C. papposa (600 g) were successively macerated with dichloromethane (6 L × 3) and methanol (4L × 3) at room temperature for 24 hours. After concentration under reduced pressure, dichloromethane (10 g) and methanol (56 g) extracts were obtained. The methanol extract was dissolved in hot distilled water. The resulting solution was successively extracted by solvents with increasing polarity ethyl acetate and n-butanol evaporated under reduced pressure to obtain 2 g of ethyl acetate and 16 g of nbutanol extracts.
MS detection was performed using a Shimadzu brand. LCMS 8040 model tandem mass spectrometer equipped with an ESI source operating in negative ion mode. LC-ESI-MS/MS data were collected and shipped by LabSolutions Software (Shimadzu) software. Multiple reaction monitoring (MRM) was used to quantify it. The working conditions of the mass spectrometer were passed as interface temperature, 350°C; DL temperature, 250°C; temperature of the thermal block, 400°C; nebulization gas flow (nitrogen), 3L/min; and drying gas stream (nitrogen), 15 L/min.
A complete LC-MS/MS method was optimized and validated for the quantification of 37 phytochemical fingerprint compounds (17 flavonoids, 15 phenolic acids, 3 non-phenolic organic acids, 1 benzopyrene and 1 phenolic aldehyde) on the species studied. The performance characteristics of the method were determined using standard solutions, enriched and nonenriched samples. In this context, the developed method has been fully validated in terms of linearity, accuracy (recovery), interday and intraday precision (repeatability), detection and quantification limits (LOD/LOQ) and uncertainty relative standards (U% at 95% confidence level [k = 2]) ( Table I).

Determination of antioxidant activity DPPH radical scavenging assay
DPPH assays based on measurement of the scavenging capacity of antioxidants towards a stable free radical α, α-diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M=394.33). The odd electron of the nitrogen atom in DPPH was reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine (Kedare and Singh, 2011).
The antiradical activity of crude extracts obtained from the species C. papposa was evaluated by the free radical DPPH assay (Blois, 1958). A solution of 40 μL of the sample (extract or standard) at different dilutions was completed with 160 μL of the DPPH methanol solution (0.1 mM). DPPH reagent in methanol was used as a blank. After 31 min, the absorbance of each solution was detected at 517 nm using a microplate reader. Butyl hydroxyl toluene (BHT) and butyl hydroxyanisole (BHA) were used as positive controls. The percentage of radical scavenging activity was calculated as follows: Where, Acontrol is the absorbance of control reaction (containing all reagents except the test extract or standard), and Asample is the absorbance of the test extract or standard The result was expressed as IC50 value (µg/mL) corresponding of sample concentration that inhibits 50% DPPH of free radical.

ABTS radical scavenging assay
The ABTS antioxidant assay measures ABTS+• radical production induced by potassium persulfate.
The ABTS scavenging activity was determined according to the method described earlier (Re et al., 1999). ABTS ·+ was produced by the reaction between 7 mM of ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 hours before use. ABTS ·+ solution was then diluted with methanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. After the addition of 160 μL of diluted ABTS·+ to 40 μL of diffèrent concentrations of crude extracts and standards (BHT, BHA), the absorbance was measured at 734 nm after 10 min of the initial mixing using a 96well microplate reader. The scavenging activity of ABTS + radical was expressed as the inhibition percentage using the following equation: Where, Acontrol is control absorbance reaction (containing all reagents except the test extract or standard), and Asample is the absorbance of extract or standard

Reducing power assay (FRAP)
FRAP assay measures the change in absorbance at 593 nm which indicated with the appearance blue colored Fe 2+ -tripyridyltriazine compound from colorless oxidized Fe 3+ form by the action of electron donating antioxi-dants (Popova et al., 2014).
The FRAP of the crude extract was determined according to the method described earlier (Marco, 1968). In which, 10 μL of different concentrations of extracts were mixed with 40 μL of phosphate buffer (0.2 M, pH 6.6) and 50 μL of potassium ferricyanide (10 mg/mL). Then, the reaction mixture was incubated at 50°C for 20 min, after that the reaction mixture was acidified with 50 μL of trichloroacetic acid solution (10%), and 10 μL of ferric chloride solution (0.1%) was added to this solution. The absorbance was measured at 700 nm.

Alpha-glucosidase inhibitor activity
The α-glucosidase inhibitor activity was investigated using the method described elsewhere (Lordan et al., 2013). A volume of 50 μL of extract solution and 50 μL of p-nitrophenyl-a-D-glucopyranoside solution 5 mM (in phosphate buffer 100 mM, pH 6.9) were mixed and incubated at 37°C for 10 min. Then, 100 μL of alphaglucosidase (0.1 U/mL) was added and the absorbance was recorded at 405 nm after 0 and 30 min respectively. The pharmacological inhibitor, acarbose, was included as a positive control.
The activity of α-glucosidase was calculated as follows: %Activity = [(Absorbance of control -Absorbance of extract)/Absorbance of control] × 100

Statistical analysis
In this study, analyses were carried out in triplicate of each sample and each experiment was realized out in triplicate (n=3). The mean value and standard deviation were calculated from the data obtained. Data of bioassays were the subject of one-way analysis of variance (ANOVA) using the SPSS 18.0 software (SPSS Inc.) followed by Tukey's test. The level of significance was fixed at p<0.05.

Principle
The cupric ion reducing antioxidant capacity (CUPRAC assay) evaluates the capacity of antioxidants to reduce the Cu 2+ to Cu + in the presence of a chelating agent. It is efficient for glutathione and thiol-type antioxidants, for which the FRAP test is nonresponsive.
Step 2: Mixed well using vortex mixer.
Step 3: 40 μL of the sample solutions at different concentrations were added.
Step 4: Mixed well using vortex mixer.
Step 5: After 30 min, the absorbance was read at 450 nm using spectrophotometer.

Analysis of results
The results were given as 0.5 (μg/mL) which corresponding to concentration indicating 50% of absorbance intensity and results were compared with those of standards BHT and BHA.
On the other hand, the same previously cited phenolic compounds were detected in the n-butanol extract. Finally, coumarin showed the highest values (1075.4 μg/g extract) followed by ferulic acid, vanillic acid, apigenin and vanillin in dichloromethane extract.

Antioxidant activity
For DPPH test, maximum scavenging activity was found in n-butanol extract (IC50 value: 17.0 ± 0.9 µg/ mL), followed by ethyl acetate extract (IC50 value: 18.1 ± 0.6 µg/mL). Dichloromethane extract was inactive. In the ABTS method, the ethyl acetate extract exhibited the highest activity (IC50 value: 11.7 ± 0.2 µg/mL) among all extracts, followed by n-butanol (IC50 value: 22.6 ± 1.2 µg/mL). Results of CUPRAC of extracts were compared with those of BHA and BHT (Table II). Activity (absorbance) increased linearly with the increasing of extract amount. n-Butanol extract exhibited the highest activity, it was better than those of standards (A0.50 value: 3.1 ± 0.1 µg/mL). Ethyl acetate extract indicated the higher activity, it was better than that of BHT, but lower than the BHA (4.9 ± 0.0 µg/mL). Ethyl acetate extract exhibited higher ferric reducing power ability than the α-tocopherol (22.9 ± 2.8 µg/mL), which is adjoining to n-butanol extract (42.1 ± 2.9 µg/mL) but this activity was relatively lower than that of ascorbic acid and tannic acid (Table II).
A good correlation between total phenolic contents and antioxidant activity was demonstrated. Indeed, ethyl acetate and butanol which are richer with these compounds were generally significantly more actives. From literature, it has been well noted that medicinal plants with high amounts of phenols and flavonoids have potent antioxidant actions (da Silva et al., 2006;Ksouri et al., 2009;Falleh et al., 2011;Dehshiri et al., 2013).
On the other hand, the dichloromethane extract exhibits strong alpha-glucosidase inhibitory activity, the TLC profiling revealed with sulfuric vanillin that indicated the presence of terpenes (visible spots: blue, green, violet pink) might contribute to this activity (Ouattara et al., 2016).

Conclusion
A total of 21 compounds were identified of which the main constituents were flavonoids and phenolic acids. The ethyl acetate and n-butanol extracts of C. papposa have strong antioxidant properties in vitro. The dichloromethane extract exhibits strong alpha-glucosidase inhibitory activity, this result therefore clearly indicates the potential of this extract to manage hyperglycemia.