Antioxidant activity, carotenoids, chlorophylls and mineral composition from leaves of Pallenis spinosa: An Algerian medicinal plant

Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd DE GRUYTER Journal of Complementary and Integrative Medicine. 2019; 20170081 Hanane Amrani-Allalou1/ Lila Boulekbache-Makhlouf2/ Paula Mapelli-Brahm3/ Sabrina Sait1,4 / Gian Carlo Tenore5/ Akila Benmeziane1/ Nabil Kadri1,4 / Khodir Madani6,7 / Antonio Jesús Meléndez Martínez3 Antioxidant activity, carotenoids, chlorophylls and mineral composition from leaves of Pallenis spinosa: an Algerian medicinal plant 1Biologie Physico-Chimique, Laboratoire de Biomathématiques, Biophysique, Biochimie, et Scientométrie, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algérie, E-mail: [email protected], [email protected], [email protected], [email protected] 2Food sciences, Université A.Mira de Bejaia, Bejaia 06000, Algeria, E-mail: [email protected] 3Food Colour & Quality Lab., Dpt. of Nutrition & Food Science, Universidad de Sevilla, Seville, Spain, E-mail: [email protected], [email protected] 4Département de Biologie, Faculté des sciences de la Nature et de la Vie et Sciences de la Terre, Université de Bouira, Bouira 10000, Algérie, E-mail: [email protected], [email protected] 5Department of Pharmacy, University of Naples “Federico II”Via Domenico Montesano, Via Domenico Montesano, 49 Napoli, Campania 80131, Italy, E-mail: giancarlo.[email protected] 6Laboratoire de Biomathématiques, Biophysique, Biochimie, et Scientométrie, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Bejaia 06000, Algérie, E-mail: [email protected] 7Centre de recherche en technologie agro-alimentaire, Route de tergua-ouzemour, 06000, Bejaia, Algérie, E-mail: [email protected] Abstract: Background: Plant and medicinal herbs are important sources of bioactive compounds and minerals that can play a role in preventing various diseases and they are considered a factor indispensable for the proper functioning of the human body. Methods: We investigated the content of carotenoids and chlorophylls of leaves from Pallenis spinosa (P. spinosa), as well as their antioxidant activity and mineral composition then, we optimized the solvent extraction for the recovery of total carotenoids and chlorophylls using spectrophotometric method. Finally, we tested the antioxidant activity of the optimized extract by three assays (DPPH, ABTS and FRAP) and we determined the mineral composition by Emission Spectrometer Induced Couple Plasma (ICP). Results: Carotenoid (CART), chlorophylls (CHLa + b), chlorophyll a (CHLA), chlorophyll b (CHLB) contents were about 36.337 ± 0.312; 347.769 ± 6.326; 224.286 ± 5.601; 123.483 ± 1.339 mg/100 g dw, respectively. We revealed an interesting antioxidant capacity by the tested extract (DPPH: 127.522 ± 1.406 mmol ET/Kgdw, ABTS: 104.827 ± 1.222 mmol ET/Kgdw and FRAP 71.89 ± 0.495 ± 0.994 mmol ET/Kgdw). Carotenoids and chlorophylls content correlate positively with the antioxidant activity of P. spinosa leaves extract (r=0.646–0.986). Eight minerals have been detected (Mg, Ca, P, Fe, Mn, Zn, Cu and Cr), Mg and Ca being the predominant ones (6479.32 ± 48.33 and 3851.88 ± 130.63 mg/Kg, respectively). Conclusions: These results have shown that P. spinosa leaves are a good source of carotenoids and chlorophylls with a potent antioxidant potential with high amount of minerals. Keywords: antioxidant activity, carotenoids, chlorophylls, extraction, mineral composition, Pallenis spinosa DOI: 10.1515/jcim-2017-0081 Received: June 5, 2017; Accepted: April 13, 2019 Introduction Phytochemicals have been receiving an increased attention for their important health benefits, providing a potential protection against several bimolecular damages, among these compounds we can cite carotenoids and chlorophylls [1]. Lila Boulekbache-Makhlouf is the corresponding author. © 2019 Walter de Gruyter GmbH, Berlin/Boston. 1 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Amrani-Allalou et al. DE GRUYTER Carotenoids are natural pigments which participate in the physiological processes such as respiration, photosynthesis and regulation of growth and development [2]. Carotenoids are also occurred in leafy vegetables with green color where chlorophylls cover their color (lettuce, pepper, spinach, etc.). Carotenoids are beneficial to humans, because some of them show vitamin A and antioxidant activities. The consumption of carotenoids is inversely related to the risk of developing certain diseases [3], such as coronary heart dysfunction, cancers and ophthalmological diseases such as macular degeneration and cataract [4, 5]. They contribute in the immune response by enhancing macrophage, cytotoxic T cell and natural killer cell tumoricidal capacities, as well as increase the production of certain interleukins [6]. Carotenoids have been reported for the effect on intracellular signaling cascades, thereby influencing gene expression and protein translation. By blocking the translocation of nuclear factor κB to the nucleus, carotenoids are able to interact with the nuclear factor κB pathway and thus inhibit the downstream production of inflammatory cytokines [6– 9]. They contribute also to the process of vision, and are considered as a good supplement for the development of body cells [10]. Due to their essential roles in photosynthesis, chlorophylls are the most photosynthetic pigments present in algae, bacteria and higher plants. These photochemical compounds have been reported to possess benefits to human health such as antimutagenic effect, antigeno-toxic properties and potent antioxidant capacity by scavenging the free radicals and preventing the lipid oxidation [3, 6, 11–13], they have been reported to their anti-inflammatory and wound healing properties [13, 14]. Chlorophylline (chlorophyll derivatives) is responsible for the increasing immune defense by its action on immune cells [13]. Some studies have reported its beneficial effect against cancer [3, 15] by forming complexes with carcinogenic chemical molecules tobacco [15] and heterocyclic amines from cooked meat [16]. Trace elements have also an importance on health and their role in traditional medicines have been reported in many published works [17–19]. They are essential components of proteins (hemoprotein, hemoglobin and hormones) and vitamins; in low dose they play an important role in the biochemical and essential enzymatic functions. They contribute also to the structure of bones and teeth [17–19] and are associated with heart function, muscle contraction, nerve conduction and to the homeostasis [18]. The large family of Asteraceae (=Compositae) contains 25,000–30,000 species belonging to over 1,000 genera [20]. Many species have been used as sources of rubber, medicines, edible oils, vegetables and pesticides, and some of them are popular ornamental plants. P. spinosa (L.) Cass (Buphthalmum spinosum L.) belongs to the family of Asteraceae, and the tribe of Inuleae [20]. The genus Pallenis is distributed in southern Europe, northern Africa, the Canary Islands, the Middle East and in the Mediterranean region, especially in the desert, and coastal habitats [21]. Limited studies have been conducted on the selected health effects of the proposed plant, and to the best of our knowledge no study has been carried out on its pigments (carotenoids and chlorophylls) and mineral composition. The main objectives of this paper were: (1) the optimization of the solvent extraction of CRTS and CHLS; (2) evaluation of the antioxidant activity of the optimized extract and (3) determination of the mineral composition from leaves of P. spinosa. Materials and methods Plant material P. spinosa plant was collected at flowering stage in May 2015, from Chabet-el-Ameur in Boumerdès, located in the East of Algiers (36 °38′00″N, 3 °42′00″E). Leaves were separated manually, washed with distilled water and air dried at the shade for 20 days; then they were grounded into a fine powder using an electrical grinder (IKA model-A11, Staufen, Baden-Württemberg, Germany). The obtained powder was passed through a standard sieve (125 µm) and only the fraction with particle size <125 µm was collected and stored at room temperature (26 °C) in an airtight polythene bag before ready for analyses. Chemicals and standards The extraction solvents were of analytical grade. Other chemicals were purchased from Sigma. 2 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd DE GRUYTER Amrani-Allalou et al. Extraction of pigments The extraction of pigments has been done according to the method recommended by Lichtenthaler [22]. Different solvents were tested (Acetone 100%, aqueous acetone 80%, diethyl ether 100%, ethanol 95%, MeOH 90% and MeOH 100%). The obtained extracts were stored under dry nitrogen at −20 °C using a µ-24 bioreactor (applikon Biotechnology, Netherlands). Spectrophotometric determination of total CART and CHLS content The quantitative determination of CARS and CHLS in leaves extracts of P. spinosa has been conducted by the methodology of Lichtenthaler [23]. The results are then expressed in mg/100 g of extract. Antioxidant activity In attempt to have a full profile of antioxidant capacity of chlorophylls extract from P. spinosa leaves, we have used methods based on two different modes of action: (1) hydrogen transfer (HAT) in term of 1,1-diphenyl-2picrylhydrazyl (DPPH) and 2,2�-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activities and (2) single electron transfer (SET) as determined by ferric reducing antioxidant power (FRAP) assay. Scavenging ability of DPPH radical DPPH test was carried out according to the modified method of Jiménez-Escrig et al. [24]. About 3.9 mL of DPPH solution in MeOH was added to 100 µL of different concentrations of sample extract (1, 3, 7 and 13 mg/mL), and then the tubes were shaken gently by vortex and put in dark place for 10 min. The absorbance of the mixture was recorded by UV-spectrophotometer at 580 nm. The antioxidant activity of the extract was calculated by using the following formula: %Inhibition = [(Astandard −Aextract)/Astandard)] × 100. where A extract was the absorbance of samples and Astandard the absorbance of MeOHic DPPH solution. The percentage of DPPH remaining (% DPPHrem) was calculated using the equation below: %DPPHrem =[DPPH]t/[DPPH]0×100 where [DPPH]0is the concentration of DPPH without the sample extract and [DPPH]tat is the concentration of DPPH after the reaction. Scavenging ability of ABTS radical The antioxidant activity by ABTS assay was measured by the method of Re et al. [25], as described below. After the preparation of the radical (ABTS•+) (7 mM ABTS stock solution and 2.45 mM potassium persulfate), the mixture was incubated for 16 h. After that, the solution was diluted with ethanol to an absorbance of 0.700 ± 0.050 at 734 nm. One milliliter of ABTS• + solution was added to 100 µL of the tested sample and vortexed thoroughly. The reactive mixture was left in the dark at room temperature for 2.5 min and the absorbance was recorded at 734 nm. Ferric reducing antioxidant power (FRAP assay) Antioxidant power was tested by FRAP assay according to Thaipong et al. [26] method. Three stock solutions were previously prepared: 300 mM acetate buffer (pH 3.6); 10 mM of 2,4,6-tris(2pyridyl)-s-triazine (TPTZ) solution in 40 mMHCl; and 20 mM FeCl3solution in distilled water. A fresh solution was prepared by mixing 3 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Amrani-Allalou et al. DE GRUYTER 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3 solution. In a reaction tube, 150 µL of extract was allowed to react with 2850 µL of the FRAP working solution for 4 min at room temperature in the dark. The reading of the absorbance was done at 593 nm. For all tests, Trolox was used as a standard, and the results were expressed as micromole Trolox equivalent antioxidant capacity per Kg of dry weight (μmol TEAC/Kg DW). Mineral salts analysis Mineral composition of leaves of the studied plant has been determined by Emission Spectrometer Induced Couple Plasma (ICP) (HORIBA JobinYvon, model LAST 2, France). The reagents used for the digestion are concentrated HNO3(Plasma Pure quality, Brand: SCP SCIENCE) and concentrated H2O2(Suprapure quality, Brand: MERCK). Once treatment is complete, the sample made up to mark with ultrapure quality water. Statistical analysis All the experiments were conducted in triplicate and the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed by analysis of variance with ANOVA and Tukey’s test in the JMP 10 software from SAS Institute Inc. (Cary, NC, USA). Correlation between CHLS and CART content and antioxidant activity was performed by XLSTAT-pro7.5. Differences were considered to be significant at p<0.05. Results and discussion Optimization of solvent extraction Seven different extraction solvents, acetone 100%, aqueous acetone 80%, diethyl ether, ethanol 95%, MeOH 90% and MeOH 100%, were used to select the best solvent for pigments extraction from P. spinosa leaves. The results were presented in Table 1, it was clearly shown that the type of solvent significantly influenced (p<0.05) the CARS and CHLS yields from leaves of P. spinosa. Table 1: Influence of solvent on extraction efficiency of carotenoids and chlorophylls from P. spinosa leaves. Photosynthetic pigments (mg/100 g dw) CARS (µg/100 g dw) CHLA CHLB (mg/100 g dw) CHLA + CHLB D Ether 21.819 ± 0.000c77.279 ± 0.00c24.854 ± 0.000d102.132 ± 0.000d Hexane 10.666 ± 0.330d32.786 ± 3.057d2.554 ± 0.964e35.340 ± 2.162e EtOH 95% 35.062 ± 0.612b226.271 ± 0.601a123.439 ± 0.871b349.710 ± 0.327a Ace 100% 394.43 ± 0.611a198.956 ± 1.773b101.855 ± 0.857c300.811 ± 2.620c Ace 80% 363.37 ± 0.312a224.286 ± 5.601a123.483 ± 1.339b347.769 ± 6.326a MeOH 100% 22.512 ± 0.730c226.183 ± 5.602a131.179 ± 1.458ab 357.362 ± 7.047a MeOH 90% 24.894 ± 2.856c194.815 ± 3.538b133.624 ± 7.879a328.439 ± 5.780b Results are expressed as mg/100 mg dw. Values are mean of three determinations; values marked by the same letter are not significantly different (p>0.05). The effect of extraction solvents on total carotenoids yield Compared with other solvents, acetone (80–100%) is the best one for the extraction of CARS from leaves of P. spinosa (Table 1). There is no significant difference (p>0.05) between the extraction yield obtained by pure acetone and that obtained by acetone 80% (/100 g dw). These are in agreement with those reported by Warkoyo and Saati [27] and Nobre et al. [28] on their study on seaweed pigment and Haematococcus pluvialis. Furthermore, Denery et al. [29] have also reported that acetone is the best solvent for extracting carotenoids from of Haematococcus pluvialis and Dunaliella salina. Amin et al. [10] have reported that the carotenoid content was 4 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd DE GRUYTER Amrani-Allalou et al. found highest in all acetone extracts of three plants (Triticuma estivum,Spinaciaoleracea,Chromolaena odorata). Moreover, Suzuki and Shioi [30] have extracted more carotenoids by acetone (80%) than by hot water from major teas. Thus, the effectiveness of extraction of a substance is determined by the properties of both solute and solvent [27]. Indeed, aqueous acetone is the best extraction solvent as it extracts most photosynthetic pigments [31, 32]. The effect of extraction solvents on chlorophylls yield The selection of the solvent to promote the extraction is a very important issue since it determines the degree of affinity to the chemical composition of the substances to be extracted. Apart from the dissolution ability towards the compounds to be extracted and quantified, the solvent also plays an important role in cell lysis. It is clear that chlorophyll has been detected in all the solvents, with no significant difference (p>0.05) between ethanol 95%, MeOH 100% and acetone 80% (349.71 ± 0.33; 357.36 ± 7.04; 347.77 ± 6.33 mg/100 gdw, respectively). Aqueous acetone is the most widely used solvent because it allows better stability for CHLA compared to methanol. Johan et al. [33, 34] (please remove the refernce number 34) have reported that acetone results in higher CHLA concentration. Indeed, acetone gave the highest extraction efficiency for total pigments in the green algae [29] and in red seaweed [27]. Furthermore, chlorophyll content was found highest in acetone extract of Triticuma estivum,Spinaciaocelera,Eupatorium odoratum [10]. The effectiveness of extraction of a substance is determined by the match between the natural properties of the solute with a similar solvent that dissolves like them because of their polarity [27]. Based on these data, aqueous acetone 80% was selected as the best solvent for the extraction of pigments from leaves of the studied plant. Carotenoids content of the leaves of the studied plant (36.337 mg/100 g of dw) is higher than that of some plants which are known by their nutritional and medicinal importance such as B. oleracea (27.8 mg/100 g of dw), Amaranthus sp. (24.6 mg/100g of dw), A. polygonoides (20.6 mg/100 g of dw), P. crispum (18.4 mg/100 g of dw), B. diffusa (2.80 mg/100 g of dw), B.chinensis (1.50 mg/100 g of dw), C. annuum (3.79 mg/100 g of dw), I. pes-tigridis (4.31 mg/100 g of dw), L. sativa (2.87 mg/100 g of dw), P. oleracae (4.20 mg/100 g of dw), T. indica (4.77 mg/100 g of dw) [32]. Compared with some fruits and vegetables known to be rich in carotenoids (carrot: 10.432 mg/100 g fresh weight, spinach: 10.75 mg/100 g fresh weight, respectively), P. spinosa had a relatively high concentration of these compounds, which corresponds to half (5.65 mg/100 g fresh weight) of the latter, but it remains higher than that of apricots (3.69 mg/100 g) [34]. As for chlorophylls (CHLa + b), the content of the leaves of P. spinosa (347.769 ± 6.326 mg/100 g dw), is higher than that reported for some selected leafy vegetables (cv. Anivip: 328.05 mg/100 g dw, cv. Monivip: 200.44 mg/100 g dw, Dandelion: 248.25 mg/100 g dw, Garden rocket: 359.62 mg/100 g dw and Wild rocket: 303.23 mg/100 g dw) [35]. Antioxidant activity The antioxidant activity of pigments extract (CRTS and CHLS) from P. spinosa was evaluated by three tests. As it is shown in Figure 1, the highest activity was obtained with DPPH test (127.522 ± 1.406 mmol ET/Kgdw) followed by that of ABTS method (104.827 ± 1.222 mmol ET/Kgdw), and the lowest one was obtained by FRAP assay (71.89 ± 0.495 ± 0.994 mmol ET/Kgdw). 5 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd Amrani-Allalou et al. DE GRUYTER Figure 1: Antioxidant activity of pigments extracts from P. spinosa leaves. Results are expressed as means ± standard deviation. Values are mean of three determinations; values marked by the same letter are not significantly different (p>0.05). In Table 2 are presented the matrices of linear correlation between pigments contents (CRTS, CHLS, CHLa and CHLb) and antioxidant activity of the extract from leaves of the studied plant. Table 2: Correlation matrix of the variables for the extract of P. Spinosa. CART CHL a CHL b CHLS DPPH ABTS FRAP CART 1 CHL a 0.766* 1 CHL b 0.596 0.965** 1 CHLS 0.715* 0.996** 0.984** 1 DPPH 0.646 0.981** 0.973** 0.986** 1 ABTS 0.932** 0.801** 0.707* 0.776* 0.675* 1 FRAP 0.771* 1.000** 0.966** 0.996** 0.978** 0.812** 1 * p value less than 0.05, ** p value less than 0.01. Except for the carotenoids (r=0.646), a strong correlation has been found (r=0.981; 0.973; 0.986) between CHLa, CHLb, CHLS and the DPPH assay. Concerning the ABTS test, a very high correlation (r=0.932) has been found with carotenoids content and high correlation (r=with chlorophyll contents [0.801; 0.707; 0.776] for CHLa, CHLb and CHLS, respectively). A strong correlation (r=1; 0.966; 0.996) has been shown between CHLa, CHLb and CHLS, respectively, and FRAP assay, and high coefficient has been found with CART. Our results are in agreement with those reported by Mitićet al. [36, 37], in their work on the effect of food preparation technique on antioxidant activity and pigment content in some vegetables; indeed, the highest correlation was found with FRAP (0.99) assay and the lowest one was shown by DPPH and ABTS (0.68) tests. Carotenoid and chlorophyll pigments have long been considered to be antioxidants [38] and CHLa and its oxidized derivatives had a higher antioxidant activity than other carotenoids [36]. Porphyrin seems to be an essential chemical structure for the antioxidant activity of chlorophyll, which reduced the free radicals such as DPPH; based on electron donation of antioxidants [39]. No works have been made on the antioxidant activity of the pigments of P. spinosa, the only studies reported in the literature were on their phenolic extracts. Mineral salts analysis From the obtained results (Table 3), we can distinguish three groups of minerals depending on whether the concentrations were greater than 1.000 mg/kg (major nutrient), between 100 and 1.000 mg/kg (minor nutrient) or less than 100 mg/kg (trace element). Comparing the minerals to each other, magnesium, calcium, phosphorus are the major elements and, iron, manganese are minor nutrients, while zinc, copper and chromium are found in traces. Calcium plays a very important role in the functioning of our body, especially in the formation of bones with the large contribution of phosphorus [17, 18]. Concerning magnesium, it is a cofactor of enzyme systems which are involved in the biochemical reactions in the body and play an important role in the intestinal absorption [40]. Adequate iron in a diet is very critical for decreasing the incidence of anemia [41], it play a vital role in controlling various functions such as cellular respiration and the formation of the hemoglobin in red blood cells [18]. Manganese has been considered essential in human diets [17], it is found both as a structural component of some enzymes and as an activator of others [41]. Copper is a component of several enzymes (cuproenzymes) its deficiency increased incidence of several anomalies (infections, osteoporosis, hypochromic, microcytic anemia, neutropenia and bone changes) [42]. Zinc is an essential metal for the normal functioning of various enzyme systems (metalloproteinase and enzymes complexes). Indeed, this mineral plays several functions (structural, signaling, and regulatory) and its deficiency, particularly in children, can lead to loss of appetite, growth retardation, weakness and even stagnation of sexual growth. Regarding the chromium, its biological function is associated to that of insulin [18], it is an essential nutrient required for normal carbohydrate metabolism [19]. Table 3: Mineral contents of P. spinosa leaves. Mineral salts Concentration, mg/Kg of dw Mg 6479.32 ± 48.33a 6 Automatically generated rough PDF by ProofCheck from River Valley Technologies Ltd DE GRUYTER Amrani-Allalou et al. Ca 3851.88 ± 130.63b P 1642.6 ± 15.37c Fe 829.35 ± 10.47d Mn 155.97 ± 1.05e Zn 41.86 ± 0.57f Cu 9.53 ± 0.15g Cr 2.77 ± 0.06h Values are mean of three determinations; values marked by the same letter are not significantly different (p>0.05). Comparing our results with those of three vegetable green leaves, Dandelion, Ayoyo and Baobab [43], mineral contents are much higher in the leaves of the studied plant, with the exception for zinc and copper which are higher in Dandelion plant, from the family of Asteraceae (Compositae). Indeed, iron content of P. spinosa is much higher than that of Baobab leaves which is reported by its richness on this element. Also and with the exception for copper element, iron, zinc and manganese contents are much higher in P. spinosa leaves than in Syzygium caryophylatum and Syzygium densiflorum plants [41]. Conclusion Different solvents were used for the optimization of the extraction of pigments from P. Spinosa plant, and acetone 80% was selected as the best one. The pigment extract showed a good antioxidant activity, which correlate positively with the content of all pigments. Through this work, we can conclude that P. spinosa is a good source of chlorophylls and carotenoids and consequently it can be considered an excellent source of antioxidants. Leaves of the studied plant are also a good sources of minerals with highest contents of Ca, Mg and P. As this is the first report on pigments, their antioxidant activity and mineral elements from P. spinosa plant, we can conclude that this work is a good contribution to the improvement of knowledge and data on its chemical composition and biological activity [44–63]. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. References [1] Barroso MF, Ramalhosa MJ, Alves RC, Dias A, Soares CM, Oliva-Teles MT, et al. Total antioxidant capacity of plant infusions: assessment using electrochemical DNA-based biosensor and spectrophotometric methods. Food Control 2016;68:153–61. 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