Bioactive Composition Analysis using HPLC-UV Profile and Evaluation of Antioxidant activities of different extracts from Aerial parts of Atractylis aristata batt.

 

Asma Abid¹*, Messaouda Dekmouche^1,2, Lazhar Bechki^1,2, Kamilia Bireche¹, Hakim Belkhalfa³, Abdeldjabbar Messaoudi¹, Mohamed Lakhder Belfar^1,2

¹Laboratoire de Valorisation et Promotion des Ressources Sahariennes (VPRS),

Université de Kasdi Merbah, Ouargla, Algérie.

²Département de Chimie, Université de Kasdi Merbah, Ouargla, Algérie.

³Centre de Recherche Scientifique et Technique en Analyse Physico-chimique (CRAPC), Ouargla, Algérie.

 

ABSTRACT:

Phytochemical compounds are known to be components of many plants and herbs; they have received great interest from the public and scientists due to their health benefits as antioxidants. The aim of the present study was to reveal the bioactive compounds and evaluation of antioxidant activity of the dichloromethane, ethyl acetate, n-butanol and residual water extracts of Atractylis aristata. The bioactive compounds analysis was investigated using HPLC-UV analysis obtained at 254nm and optimized with 16 standards, ABTS and DPPH methods were used for estimating the antioxidant capacity. Thirteen bioactive compounds were identified in the extracts by comparing the retention time. Major compounds detected in the extracts were Acetylsalicylic acid, Ascorbic acid, Gallic acid, Quercetin and Vanillin. All extracts give an antioxidant capacity varied with polarity of solvents. The residual water extract demonstrated a significant amount of total phenolics, flavonoids and condensed tannins (3.544±0.738mg of GAE/g DW, 3.104±0.6760mg of QE/g DW and 2.692±0.561mg of CE/g DW, respectively). In two methods tested to evaluate the antioxidant activity, ethyl acetate extract displayed the highest antioxidant capacity (IC₅₀ value: 0.097±0.003mg/ml in DPPH assay and IC₅₀ value: 0.077±0.003mg/ml in ABTS assay).

 

 

 

INTRODUCTION:

Atractylis aristata batt. (Ameskeki) is a North African dicotyledonous plant of the Asteraceae family which contains the largest number of species, with estimates of the total number up to 30,000. There are 1600-1700 genera distributed over many regions in the world¹. Atractylis is an herbaceous plant growing in sunlight areas in temperate and subtropical climates². This specie is used in traditional medicine in Hoggar Algeria, mainly for stomach diseases: colic, spasms, fever^3,4. Phenolic compounds are mainly represented in numerous health benefits, including antioxidant⁵, anti-inflammatory⁶,

 

antimicrobial⁷, anticoagulant⁸, antifungal⁹, analgesic¹⁰ antidiabetic¹¹, antiarthritic¹², anti-cancer¹³, antihyperlipidemic¹⁴ and cytotoxicity¹⁵ properties. Previous pharmacological studies have demonstrated that Atractylis bioactive compounds have anti-inflammatory, anti-pyretic and acute toxicity¹⁶, antioxidant¹⁷, antidiabetic¹⁸ and anticancer¹⁹ activities. Antioxidants have long been known for their ability to improve health and reduce the risk of cancer, hypertension, and heart disease^20,21. Plant-derived antioxidants are divided into different groups: phenolic compounds, vitamins, and carotenoids²². Phenolic compounds have an ideal chemical structure for free radical scavenging^23,24, hydroxyl groups are capable to donate an electron or a hydrogen atom to a free radical. DPPH and ABTS stable free radicals are frequently used to evaluate the antioxidant capacity to scavenge free radicals²⁵. In recent years herbs have been widely researched due to its numerous of therapeutically interests and effects, explained by their complex mixture of phytochemical compounds which known by their pharmacological activities and health benefits. The present study aimed to reveal the natural bioactive compounds among four different extracts, by using HPLC-UV, quantify of phenolic compounds by classical colorimetric technics and evaluate the antioxidant activity of each extract from aerial parts of Atractylis aristata by DPPH and ABTS assays, in order to compare and differentiate extracts as valuable sources of antioxidant compounds.

 

MATERIALS AND METHODS:

Chemical and reagents:

Folin-Ciocalteu, aluminum trichloride (AlCl₃), vanillin (C₈H₈O₃), concentrated (HCl), sulfuric acid (H₂SO₄), ascorbic acid (C₆H₈O₆), sodium carbonate (Na₂CO₃), Ethanol (C₂H₅OH), Potassium persulfate (K₂S₂O₈), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS), methanol (CH₃OH), dichloromethane (CH₂Cl₂), ethyl acetate (C₄H₈O₂), Butanol (C₄H₁₀O) petroleum ether, gallic acid (C₇H₆O₅), butylated hydroxytoluene (C₁₅H₂₄O).

 

Plant collection and preparation:

A. aristata aerial parts was collected from Tamanrasset (south of Algeria) during the winter of 2019, its scientific identification was performed on the bases of Ozenda²; Quezel and Santa²⁶ and Sahki and Sahki²⁷. The aerial parts of Atractylis aristata was dried in the dark and at room temperature for two weeks, the dried samples were powdered and used for solvent extraction; it was macerated with petroleum ether for 24 hours, then was extracted in methanol/water 8/2 (V: V) mixture for 48 hours once and 24 hours three times. After concentration at 45ºC, the filtrate recovered with distilled water and partitioned successively using petroleum ether, dichloromethane, ethyl acetate and n-butanol. The extracts were concentrated under reduced pressure.

 

HPLC-UV analysis of extracts:

A. aristata extracts were analysed using a Shimadzu NEXERA XR HPLC equipped with a variable wavelength UV detector (190–800nm) and an Ultra C18 column (5µm, 250 x 4.6mm). Mobile phases were 0.1% Acetic acid/ water (solvent A) and Acetonitrile (Solvent B) at a flow rate of 1ml/min with the detector set at 254 nm. The gradient system by pump (LC-20ADXR) was 0.01min (10% B), 55min (100% B). Using this methods, 10µl of extracts at concentration of 1mg/ml were filtrated with 0.45um filter and injected in Ultra C18 column.  

 

Antioxidant activity:

a) DPPH free radical assay:

The scavenging activities of A. aristata extracts were evaluated using the 1,1 diphenyl-2-picrylhydrazyl (DPPH free radical) method described by Brand-Williams²⁸. 3ml of 4 x 10-3% ethanolic DPPH free radical solution was added to 1ml of each concentration (10-100mg/ml) of sample solution. The absorbance of the preparations was measured at 517 after 30 minutes. The IC₅₀ (mg/ml) value was used to evaluate the antiradical activity, which is defined as the extract dose required to reduce the absorbance at 517nm by 50%. Increased antioxidant activity is indicated by a lower IC₅₀ value.

 

b) ABTS free radical assay:

The spectrophotometric activity of ABTS^•+ scavenging was calculated using the modified method of Re²⁹. The radical cation of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) is stable in its free form³⁰. It is formed by oxidation of ABTS (7mM) in the presence of potassium persulfate (K₂S₂O₈) (2.45mM) to give a coloured solution in green-blue then incubated in the dark at 12-16 hour. The mixture diluted by distilled water to obtain an absorbance of 0.7±0.005 at 734nm. 50 µl of extract was added to 950µl of ABTS radical cation solution and incubated at room temperature for 6 min. The antiradical activity was expressed as IC₅₀ (mg/mL), the positive control used is ascorbic acid.

 

Quantification of phenolic classes:

a) Total phenol quantification:

The Folin-Ciocalteu method was used to calculate total phenols (TPC) spectrophotometrically³¹, using gallic acid as standard. In the presence of Na₂CO₃ (20%) polyphenols reduce phosphomolybdic acid of Folin–Ciocalteu reagent and the mixture colour appearance of a dark blue colour. Maximum absorption of dark blue colour is between 725 and 765nm, which is proportional to the amount of polyphenols present in plant extracts. The results were expressed as mg gallic acid equivalent (GAE)/g of dry weight DW of plant.

 

b) Flavonoid quantification:

The TFC determined using spectrophotometric method³², it based on complex formation between phenolic compounds and aluminium trichloride (AlCl₃), using quercetin as standard. The complexes produced are yellow in the visible. 1.5ml of an of AlCl₃(2%) ethanolic solution is added to 1.5ml of the extract. The solutions are incubated in the dark for 30 minutes at room temperature. The absorbance reading of each solution was determined at 430nm. The amount of flavonoids present in plant extracts measured using calibration curve of prepared quercetin solution as standard and the results were expressed as mg quercetin equivalents (QE)/g of dry weight (DW) of plant.

 

c) Condensed Tannin quantification:

The CTC determined using spectrophotometric method³³, which catechin used as standard, 3 ml of ethanolic vanillin (4%) solution was added to 1.5 ml of concentrated HCl and 0.4 ml of extract. The solutions are kept at room temperature for 15 minutes in the dark. The amount of condensed tannin present in plant extracts measured using calibration curve of prepared catechin solution as standard and the results were expressed as mg catechin equivalents (CE)/g of dry weight (DW) of plant.

 

Statistical analysis:

Results are reported as the mean of three replicates (n = 3) performed by EXCEL 2016; it is based on calculating the mean and standard deviation ± SD for each studied parameter.

 

RESULTS AND DISCUSSION:

HPLC-UV fingerprinting analysis:

The chromatographic analysis of bioactive compounds of A. Aristata was performed using HPLC with absorbance detection at 254 nm. The HPLC profiles of extracts were presented in figure 2. The chromatogram of HPLC of sixteen reference standards was shown in figure 1. The phenolic composition in extracts and standards were well separated under the established HPLC conditions as shown in figure 1 and 2. Thirteen main bioactive compounds were recognized (table 1) by comparing the retention times and UV spectra of standards. The validation method of HPLC fingerprint analysis was performed based on the retention time.

 

Table 1. HPLC profile of bioactive composition of Atractylis aristata extracts

	Extracts
	Dichloromethane	Ethyl acetate	Butanol	Residual water
	RT	RT	RT	RT
Bioactive compound
Acetylsalicylic acid	17.662	17.654	17.673	17.682
Ascorbic acid	2.476	2.472	2.465	ND
Caffeic acid	10.976	10.859	ND	ND
Chlorogenic acid	ND	8.795	ND	ND
Gallic acid	3.557	3.765	3.949	ND
Para coumaric acid	ND	14.259	ND	ND
Caffeine	10.111	9.547	ND	ND
Catechin	ND	9.189	ND	ND
epicatechin	ND	ND	ND	ND
pyrogallol	ND	ND	ND	5.240
Quercetin	21.295	21.283	21.298	21.308
Rutin	13.289	13.304	ND	ND
Vitamin B1	1.767	1.774	ND	ND
Vitamin B6	ND	ND	ND	ND
Vitamin D3	ND	ND	ND	ND
vanillin	14.566	14.454	14.423	14.435

RT: Retention Time.                           ND: Not Detected.

 

Through comparing the HPLC chromatograms (figure 1 and 2), it was found that the fingerprint profiles of A. aristata was greatly similar from that of the standard compounds. In dichloromethane, ethyl acetate, n-butanol and residual water extracts, the bioactive compounds were always present are the acetylsalicylic acid, quercetin and vanillin. The ethyl acetate was very distinct from the others, which present 12 bioactive compounds. Other unknown peaks at retention time 17.188 min and 17.431 min, are present in ethyl acetate extract (figure 2B), and three unknown peaks at retention times 16.971 min, 17.192 min and 17.436 min are detected in dichloromethane extract (figure 2A), which not identified because there is no standard phenolic compound at the same retention time. These unknown peaks demonstrated the richness of the extracts by different bioactive compounds. 

 

Figure 1. Chromatogram of standard compounds

 

Figure 2. HPLC-UV chromatograms of different A. aristata extracts

 

Antioxidant activity:

The free radical scavenging activity of the different extracts was estimated using two free radical DPPH and ABTS tests based on the ability of antioxidant to scavenge DPPH and ABTS free radical, the results are summarized in (table 2). We have plotted the curves representing the variation in radical scavenging expressed in inhibition as a function of the concentration. The results are expressed by the IC₅₀ value, which is inversely proportional to the antioxidant activity, i.e., the smaller IC₅₀ value, has the higher antioxidant activity. 

 

All the extracts demonstrated high antiradical activity toward the stable DPPH and ABTS radicals. The IC₅₀ values of the different extracts of A. aristata vary between 0.097±0.003 and 1.256±0.013mg ∕ml against the free radical DPPH and vary between 0.094 ± 0.002 and 0.515 ± 0.017mg ∕ml against the free radical ABTS.

 

The highest antioxidant activity against the both free radical DPPH and ABTS was Ethyl acetate extract and the lowest was the residual water extract. The IC₅₀ values are classified in the same ascending order: Ethyl acetate extract < Butanol extract < Dichloromethane extract < Residual water extract.

 

High power of antioxidant activity was shown in the ethyl acetate extract, this extract showed also high number of bioactive compounds. All extracts show high antioxidant activity in DPPH and ABTS assays.  This could be explained by its different composition of active secondary metabolites, as well as their richness by naturally active phytochemical substances with hydrogen donating properties through their hydroxyl groups, which makes the H-abstraction reaction between antioxidant and radical easier, as well as their capacity to give electrons to act as an antioxidant. This lowers the rate of electron transfer between the antioxidant and oxygen, reducing the antioxidant's pro-oxidative potency. This study and various previous studies have reported that phenolic compounds exhibited noticeable antioxidant activities in vitro and in vivo ^34-36.

 

Quantification of phenolic classes:

a) Total phenolic content TPC:

The total phenolic content was determined by Folin-Ciocalteu method, the amount of TPC ranged from 0.900 ± 0.262 to 3.544 ± 0.738 mg of GAE/g dry weight (table 3). The results for the different extracts are classified in the following descending order: Residual water extract> Butanol extract > Dichloromethane extract > Ethyl acetate extract.

 

Table 2. Antioxidant activity results of A. aristata extracts

Extracts					Standards
Test	Dichloromethane	Ethyl acetate	n-Butanol	Residual water	BHT	Ascorbic acid
DPPH IC50 (mg/ml)	0.364 ± 0.117	0.097 ± 0.003	0.124 ± 0.005	1.256 ± 0.013	0.004 ± 0.0004	0.009 ± 0.0005
ABTS IC50 (mg/ml)	0.094 ± 0.002	0.077 ± 0.003	0.088 ± 0.002	0.515 ± 0.017	NT	0.057 ± 0.012

NT: Not Tested.

 

The results were consistent with those revealed in studies of other Atractylis plant species^37,38, they reported that TPC of Atractylis genus varied from 8.36 ± 0.06 GAE/ g DW to 17 mg GAE/g DW. Methodological difference may partially cause differences in the analytical values, as well as the difference between spices, source, and seasonality³⁹.

 

The determination of total polyphenols by the Folin-Ciocalteu test is a non-selective assay with respect to polyphenols, because it is implied that all the reducing molecules, such as reducing sugars or vitamin C⁴⁰. Therefore, the higher sugar compounds were in the higher polarity solvents, which were the residual water and butanol extracts may have influenced the TPC results.

 

b) Total flavonoids content TFC:

The flavonoids concentration of A. aristata extracts was determined by using method based on complex formation between phenolic compounds and aluminum trichloride (AlCl₃). The results showed that the concentrations values of the dichloromethane, Ethyl acetate, Butanol and Residual water extracts of Atractylis aristata aerial parts, are 0.380±0.060; 0.012±0.0050; 2.554±0.090 and 3.104±0.6760mg QE/g of dry weight material respectively (table 3).

 

The highest content of flavonoids was in the residual water fraction, it is of the order of 3.104±0.676mg QE/g, followed by the butanol fraction with a content of 2.554±0.090mg QE/g. These results could be justified by the investigation into flavonoids compounds from Atractylis genus⁴¹, that confirm well the presence of flavonoid glycosides, which explains the richness of residual water and butanol extracts on flavonoids content. According to our results, flavonoid contents from Atractylis aristata extracts are comparable to those of the similar genus Atractylis gummifera³⁷, Atractylis babelii³⁸. The results explained by the presence of similar type of flavonoid composition from Atractylis genus.

 

c) Condensed tannins content:

The results of concentration values of condensed tannins content of A. aristata extracts were varied from 0.396± 0.047 to 2.692±0.561mg CE/g of dry material. CTC for the different extracts is classified in the following descending order: Residual water extract > Butanol extract > Ethyl acetate extract >Dichloromethane extract.

 

The highest content of condensed tannins is observed in the residual water fraction, it is of the order of 2.692± 0.561mg CE/g DW, followed by the butanol fraction with a content of 1.856 0.765mg CE/g DW. Tannins are polar phenolic compounds soluble in water, which explains the richness of the butanol and residual water phases in condensed tannins. Our results of condensed tannin contents showed slightly higher amounts of dichloromethane extract (0.396±0.047 mg CE/ g DW), in comparison of the same extract from Atractylis babelii²⁹ (0.41mg CE/g DW) and showed slightly lower amounts from Atractylis gummifera³⁷ (1.7mg CE/g DW).

 

Atractylis aristata gave a moderate amount of phenolic compounds when compared to other South Sahara plant extracts collected in Tamanrasset from Asteraceae family, that have been collected during the flowering stage such as Varthemia sericea (TPC: 283.61 ± 0.58 µg GAE/mg; TFC: 54.48±1.29µg QE/mg)⁴², and Asteriscus graveolens (TPC: 107.16±3.01µg GAE / g extract; TFC: 114 ± 1.9µg QE/g extract; TCT: 21.18±8.09 µg CE/g extract)⁴³.

 

The differences of the results of plants in the similar genus explained by the hard environmental conditions such as hot temperatures, dryness, and short growing season may be led to varied accumulation of secondary metabolites in Saharan plants that help them cope with such conditions.  The amount of phenolics depends on several factors such as temperature, UV-light, nutrition available to the plant, and genetic factors⁴⁴.

 

The correlation between DPPH IC₅₀ and the phenolic content was R² = 0.604, with flavonoids was R²=0.388 and with condensed tannins was R² = 0.503 (table 4). This allows us to conclude that the capacity of DPPH radical is mainly due to 60.4% of the total phenolic content.

 

ABTS IC_{50 ,}total phenols content and condensed tannins have a high correlation value (R² = 0.702 and R² = 0.647 respectively), and a medium correlation with flavonoids (R² =0.481) Table 4. This allows us to conclude that the capacity of free radical ABTS is mainly due to the 70.2% of total phenol content, 48.1% of flavonoids, and 64.7 % of condensed tannins.

 

There are remarkable correlations between the two methods of antioxidant activity (DPPH and ABTS) R²=0.96 Table 4, these correlations attribute to the similar mechanism of action of antioxidant compounds in the assay followed.

 

Table 3. Total phenols, flavonoids and condensed tannins contents of dichloromethane, ethyl acetate, n-butanol and residual water extracts

	Extract
	Dichloromethane	Ethyl acetate	Butanol	Residual water
TPC (mg of GAE/g DW)	1.126 ± 0.135	0.900 ± 0.262	2.443 ± 1.007	3.544 ± 0.738
TFC (mg of QE/g DW)	0.380 ± 0.060	0.012 ± 0.0050	2.554 ± 0.090	3.104 ± 0.6760
CTC (mg of CE/g DW)	0.396 ± 0.047	0.683 ± 0.199	1.856 ± 0.765	2.692 ± 0.561

 

Microsoft Excel (2016) was used to calculate (R²) in order to confirm the correlation antioxidant activity-total phenols, antioxidant activity-flavonoids, and antioxidant activity-condensed tannins content. The results showed in (table 4).

 

Table 4. R² correlation between phenolic classes-antioxidant activity

	IC50 DPPH	IC50 ABTS
TPC	R2 =0.604	R2 = 0.702
TFC	R2=0.388	R2=0.481
CTC	R2= 0.503	R2= 0.647
IC50 ABTS	R2= 0.96	1

 

CONCLUSION:

Qualitative and quantitative analysis showed that the extracts have a different bioactive composition and different level of TPC, TFC and CTC, which may explain their differences of antioxidant activities. The results of the present study can propose that A. aristata extracts have high levels of the phenolic compounds and as a result higher antioxidant activity. Considering the importance of natural products and their high quality in medicine in today’s world, these extracts can be used as good source of antioxidants, which have relevance in the prevention of disease in which free radical are implicate.

 

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Received on 20.09.2021          Modified on 20.10.2021

Accepted on 08.11.2021         © RJPT All right reserved