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Diabetes mellitus is a chronic disease of metabolic disorder (Pallavi et al., 2015) which is treated by either insulin or oral anti-diabetic drugs. Among the oral anti-diabetic drugs, alpha-glucosidase inhibitor retards the liberation of d-glucose from dietary complex carbohydrates and delays glucose absorption, resulting in reduced postprandial plasma glucose level and suppression of postprandial hyperglycemia. Plants such as Camellia sinensis (Hara and Honda, 1992), Ipomoea batatas (Matsui et al., 1996), berry (McDougall and Stewart, 2005), Syzygium zeylanicum, Cleistocalyx operculatus, Horsfieldia amygdalina and Careya arborea (Truong et al., 2007) show alpha-glucosidase inhibitor activity.

The genus Centaurea L. (Asteraceae, Carduae) is characterized by biosynthesis of sesquiterpene lactones (Medjroubi et al., 1998) and flavonoids (Akkal et al., 2003). They have long been used in traditional medicine to treat fever, diabetes and hemorrhoid and peptid ulcer (Honda et al., 1996; Kargıoğlu et al., 2008). Centaurea papposa is a species belonging to the Centaurea section, subsection Acrolophus (Cass.) DC (Hilpold et al., 2011), increasing wild in Algeria and Tunisia.

The chemical profile of the endemic species C. papposa of Algeria, has not been reported before. For this reason, this plant was chosen to explore its chemical compounds and quantities of the phenolic compounds by LC-MS/MS and to evaluate the α-glucosidase inhibitory activities and antioxidants in vitro by different methods.

Materials and Methods

For LC-MS/MS analysis: The analytical standards of quinic acid, malic acid, fumaric acid, gallic acid, protocatechic acid, pyrocatechol, chlorogenic acid, 4-OH-benzoic acid, vanillic acid, caffeic acid, syringic acid, vanillin, salicylic acid, p-coumaric acid, rutin, ferulic acid, sinapic acid, hesperidin, isoquercitrin, rosmarinic acid, nicotiflorin, α-coumaric acid, rhoifolin, quercitrin, apigetrin, coumarin, myricetin, fisetin, cinnamic acid, liquiritigenin, quercetin, luteolin, naringenin, apigenin, hesperetin, kaempferol and chrysin were purchased from Sigma-Aldrich, Italy. HPLC-grade acetonitrile, ammonium formate and formic acid were purchased from Sigma-Aldrich, Italy.

For extracts and biological activities: Quercetin, potassium persulfate, ferrous chloride, dichloromethane, ethyl acetate, n-butanol, α-tocopherol, acarbose and ethylenediamine tetraacetic acid (EDTA) were obtained from E. Merck, Germany. Folin–Ciocalteu’s reagent (FCR), neocuproine, butylated hydroxytoluene (BHT), DPPH dye, sodium carbonate, aluminum chloride, phosphate buffer, ammonium acetate buffer, potassium ferricyanide, butyl hydroxyanisole (BHA), methanol and ethanol were obtained from Sigma Chemical Co., Germany. 2,20-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), p-nitrophenyl-a-D-glucopyranoside solution, alpha-glucosidase, ascorbic acid, tannic acid were obtained from Fluka Chemie, Germany.

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.

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 n-butanol extracts.

The LC-MS analyses of phenolic compounds were per-formed using a Nexera model Shimadzu UHPLC coupled to a tandem MS instrument. The liquid chromatography was equipped with LC30AD binary pumps, CTO-10ASvp column oven, DGU-20A3R degasser and SIL-30AC autosampler. The chromatographic separation was performed on an RP-C18 Inertsil ODS-4 (100 mm × 2.1 mm, 2 μm) analytical column. Reversed-phase ultrahigh performance liquid chromatography was optimized to achieve optimum separation for 37 phytochemical compounds and to overcome the suppression effects. The column temperature was fixed at 35°C. The elution gradient consisted of eluent A (water, 10 mM ammonium formate and 0.1% formic acid) and eluent B (acetonitrile). The following gradient elution program was applied: 5-20% B (0-10 min), 20% B (10-22 min), 20-50% B (22-36 min), 95% B (36-40 min), 5% B (40-50 min). The solvent flow rate was maintained at 0.25 mL/min and the injection volume was settled as 4 μL.

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 non-enriched 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).

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 anti-oxidants 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, A_controlis the absorbance of control reaction (containing all reagents except the test extract or standard), and A_sample 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.

Box 1: Cupric reducing antioxidant capacity

Principle

The cupric ion reducing antioxidant capacity (CUPRAC assay) evaluates the capacity of antioxidants to reduce the Cu2+ 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.

Requirements

Ammonium acetate buffer; Cu (II) solution; Extract; Neocuproine; UV-Vis spectrophotometer

Procedure

Step 1: Ammonium acetate buffer (60 μL; 1 M, pH 7.0) solution, neocuproine solution (50 μL; 7.5 mM) and Cu (II) solution (50 μL, 10 mM) were added to a test tube.

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.

References

Apak et al., 2004; Rubio et al., 2016

The ABTS anti-oxidant 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 96-well microplate reader. The scavenging activity of ABTS⁺ radical was expressed as the inhibition percentage using the following equation:

Where, A_control is control absorbance reaction (containing all reagents except the test extract or standard), and A_sample is the absorbance of extract or standard

FRAP assay measures the change in absorbance at 593 nm which indicated with the appearance blue colored Fe²⁺-tripyridyltriazine compound from colorless oxidized Fe³⁺ form by the action of electron donating antioxidants (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.

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 alpha-glucosidase (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.

%Activity = [(Absorbance of control — Absorbance of extract)/Absorbance of control] × 100

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.

Results

Following the LC-MS results, phenolic acid contents in C. papposa extracts were higher than their of flavonoid contents (Table I). The LC-MS analysis of ethyl acetate extract revealed the presence of quinic acid, malic acid, gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, syringic acid, apigetrin, apigenin and coumarin showed the highest concentrations (200579.8, 1436.7, 2807.1, 3221.9, 4 539.34, 1834.9, 1658.7, 2521.8, 1608.0 μg/g extract) respectively. While 4-OH-benzoic acid, caffeic acid, salicylic acid, p-coumaric acid, ferulic acid, rosmarinic acid, hesperidin, isoquercitrin, rhoifolin and quercitrin were found with the lowest values.

No.	Analyte	Retention time	Amount (μg/g extract)
			Dichloromethane extract	Ethyl acetate extract	n-Butanol extract
1	Quinic acid	1.1	NI	200579.5	162905
2	Malic acid	1.2	NI	1 436.7	1 201.4
3	Fumaric acid	1.5	NI	NI	NI
4	Gallic acid	3	NI	2 807.1	2 599.3
5	Protocatechuic acid	4.9	NI	3 221.9	2 640.1
6	Pyrocatechol	6.5	NI	NI	NI
7	Chlorogenic acid	7.1	7.6	4539.3	4188
8	4-OH-Benzoic acid	7.4	NI	827.8	704.1
9	Vanillic acid	8.6	183.4	1834.9	1539
10	Caffeic acid	8.8	1.6	650.9	594.7
11	Syringic acid	9	NI	145.4	120.8
12	Vanillin	10.9	54.8	NI	NI
13	Salicylic acid	11.2	17.4	197.4	194
14	p-Coumaric acid	11.5	9.9	471.01	416.2
15	Rutin	12.6	NI	NI	NI
16	Ferulic acid	12.6	214.8	654.2	546.3
17	Sinapinic acid	12.7	NI	NI	NI
18	Hesperidin	12.7	NI	61.8	67.7
19	Isoquercitrin	13.4	NI	19	33
20	Rosmarinic acid	14.5	NI	31.4	29.1
21	Nicotiflorin	14.7	NI	NI	NI
22	o-Coumaric acid	15.5	NI	NI	NI
23	Rhoifolin	16.1	NI	283.6	268
24	Quercitrin	16.4	NI	783.5	661.4
25	Apigetrin	16.6	NI	1658.7	1567.7
26	Coumarin	17.4	1075.4	2521.8	2430.5
27	Myricetin	18.7	NI	NI	NI
28	Fisetin	19.3	NI	NI	NI
29	Cinnamic acid	25.6	NI	NI	NI
30	Liquiritigenin	25.6	NI	NI	NI
31	Quercetin	28.2	NI	NI	NI
32	Luteolin	28.3	NI	NI	NI
33	Naringenin	30.7	NI	NI	NI
34	Apigenin	31.4	146.1	1608	1506.3
35	Hesperetin	31.8	NI	NI	NI
36	Kaempferol	31.9	NI	NI	NI
37	Chrysin	36.7	NI	NI	NI
Total detected products			9	20	20
NI : Non-identifier

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.

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).

Extract	DPPH IC₅₀ (μg/mL)	ABTS IC₅₀ (μg/mL)	CUPRAC A0.50 (μg/mL)	Reducing power A0.50 (μg/mL)
Dichloromethane	NA	>100	>100	> 200
Ethyl acetate	18.1 ± 0.6	11.7 ± 0.2	4.9 ± 0.0	22.9 ± 2.8
n-Butanol	17.0 ± 0.9	22.6 ± 1.2	3.1 ± 0.1	42.1 ± 2.9
Butyl hydroxyl toluene	6.8 ± 0.5	1.6 ± 0.0	9.6 ± 0.9	NT
Butyl hydroxyl anisole	6.8 ± 0.5	1.0 ± 0.0	3.6 ± 0.2	NT
Ascorbic acid (4 μg/μL)	NT	NT	NT	6.8 ± 1.2
Tannic acid (4 μg/μL)	NT	NT	NT	5.4 ± 0.9
α-Tocopherol (4 μg/μL)	NT	NT	NT	34.9 ± 2.4
Values were expressed with means ± SD of three parallel measurements, (p<0.05). SD: Standard deviation, C. azarolus: Crataegus azarolus, ABTS: 2,2′‑azino‑bis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid, IC₅₀: Half maximal inhibitory concentration; nt: not tested, na: not absorbance

Dichloromethane extract showed a greater inhibition activity compared to acarbose (IC₅₀ value: 227.6 ± 4.4 µg/mL). Ethyl acetate extract (IC₅₀ value: 791.9 ± 1.8 µg/mL) exhibited weak inhibitory activity against alpha-glucosidase. The n-butanol extract, however, was inactive (Table III).

Discussion

Extracts (4 μg/μL)	IC₅₀ (μg/mL)
Dichloromethane	227.6 ± 4.4
Ethyl acetate	791.9 ± 1.8
n-Butanol	NA
Acarbose	275.4 ± 1.6

Biological potential and total phenolic contents of C. papposa are reported for the first time in this work. The LC-MS/MS analysis of C. papposa extracts revealed the presence of 21 phenolic compounds. The protocatechuic acid, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, coumarin, salicylic acid, vanillic acid and apigenin presented the highest values. Previous experiments on Centaurea have shown the presence of major phenolic compounds, quercetin, quercetin-3-β-D-glucoside and protocatechuic from C. amaena and C. aksoyi (Albayrak et al., 2017), chlorogenic, caffeic, ferulic, and p-coumaric acids, isoquercitrin, and coumarin from C. cyanus (Escher et al., 2018), β-sitosterol 3-glucoside, protocatechuic acid, scopoletin, chlorogenic acid, centaurein, kaempferol-3-glucoside, jacein, arctiin, quercetin-3-glucoside and janerin from C. isaurica (Flamini et al., 2004). Sixteen compounds including protocatechuic acid, hexoside and ferulic acid were determined in the methanol extract of C. baseri using LC/MS (Kösen et al., 2016).

The ethyl acetate and n-butanol extract of C. papposa have the best antioxidant effect, it was more potential than other Centaurea species investigated in earlier studies from C. pulchella (Zengin et al., 2010), C. calcitrapa subsp. calcitrapa, C. ptosimopappa, C. spicata (Erol-Dayi et al., 2011), C. kurdika, C. rigida, C. amanicola, C. cheiroolopha and C. ptosimopappoides (Aktumsek et al., 2013).

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 antioxidat 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.

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| Research | Article |

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

Abstract

Introduction

Materials and Methods

Box 1: Cupric reducing antioxidant capacity

Results

Discussion

Conclusion

Conflict of Interest

Acknowledgements

References