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Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants
SAJB-02149; No of Pages 6 South African Journal of Botany xxx (2018) xxx–xxx
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Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants R. Attia a,b, C. Messaoud b, K. Arraki a, A. Zedet a, C. Demougeot a, M. Boussaïd b, C. Girard a,⁎ a
PEPITE EA4267, Univ. Bourgogne Franche-Comté, F-25000 Besançon, France Laboratory of Nanobiotechnology and Medicinal Plants, Department of Biology, National Institute of Applied Science and Technology (INSAT), University of Carthage, BP 676, 1080 Tunis Cedex, Tunisia
b
a r t i c l e
i n f o
Article history: Received 31 March 2018 Received in revised form 30 August 2018 Accepted 19 September 2018 Available online xxxx Edited by A Mocan Keywords: Arginase Naturally occurring inhibitors Tunisian medicinal plants Polyphenols
a b s t r a c t Arginase has been the focus of a particular interest since it has been shown that the increase of its activity is involved in various pathologies for which the inhibition of this enzyme could be a promising treatment. The several existing synthetic inhibitors of arginase are not suitable for a use in human therapeutics. Hence, interest has been initiated to explore natural resources, in particular medicinal plants for the search for new arginase inhibitors. Tunisia is famous for its great flora diversity, as well as a centuries-old tradition of medicinal plants used to treat many diseases including cardiovascular disorders. Therefore, seven Tunisian plants belonging to various families: Retama raetam (Forssk.) Webb & Berthel., Rhus tripartita (Ucria) Grande, Artemisia campestris L., Artemisia herba-alba Asso, Myrtus communis L., Crataegus azarolus L., Rubus ulmifolius Schott, were selected to be screened on arginase using an in vitro inhibition test. Fifty-one crude extracts obtained from different parts of these plants showed inhibition percentages ranging from 2.66 ± 7.57% to 90.00 ± 1.53% at 100 μg/mL. Five extracts were identified as the most active and their IC50 were determined, as well as their phenolic and flavonoid contents. Results showed that the methanolic extract from Crataegus azarolus stems is the most interesting crude material with the lowest IC50 (41.99 ± 1.53 μg/mL). This extract also shows the richest polyphenolic content (161.02 ± 1.24 μg GAE/mg CE). The analyses of total flavonoid content revealed that flavonoids represent only a small part of this polyphenolic content (22.32 ± 0.24 μg RE/mg CE) and cannot be considered as fully responsible for the observed activity. Further, bio-guided fractionations and structural elucidation will aim to identify secondary metabolites responsible for the bioactivity observed on arginase. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Worldwide traditional medicine has a long history of use in disease treatment (WHO, 2013). In particular, medicinal plants are widely used in many countries. As they constitute a well-known source of active metabolites, a lot of studies have been performed to isolate compounds responsible for their biological properties. Some of them led to the discovery of new drugs currently used in therapeutics (Newman and Cragg 2016). Among their numerous biological activities, natural compounds have the potential to inhibit enzymes (Rauf and Jehan, 2017). Arginase is a trimeric metalloenzyme that catalyzes hydrolysis of L-arginine to L-ornithine and urea and plays an important role in the ammonia detoxification in mammals. By substrate competition, arginase also plays a crucial role in the bioavailability of L-arginine for nitric oxide synthase (NOS). The result of this competition is the decrease of nitric oxide (NO) production and the increase of ⁎ Corresponding author at: PEPITE EA4267, Univ. Bourgogne Franche-Comté, 19 rue Ambroise Paré, F-25000 Besançon, France. E-mail address: [email protected] (C. Girard).
L-ornithine production. This latter is converted into polyamines or proline that can promote cell proliferation and collagen production, resulting in various health problems, in particular at the cardiovascular level (Pudlo et al. 2017). Over the last decades, studies on animal models and clinical studies have provided arguments in favor of arginase as a new promising target of inhibitors aiming to treat microbial (Martini et al. 2009) or parasitic infections (Heby et al. 2007), cancers (Pham et al. 2018) and inflammatory or cardiovascular diseases (Caldwell et al. 2015). Among the few synthetic arginase inhibitors commercially available, boronic acid derivatives (S-(2-boronoethyl)-L-cysteine (BEC), 2-(S)-amino-6-boronohexanoic acid (ABH) and Nω-hydroxynor-L-arginine (nor-NOHA) are the most potent, but their toxicity or their poor pharmacokinetic profile limits their potential therapeutic use in humans (Ivanenkov and Chufarova, 2014). Therefore the discovery of new structures of arginase inhibitors is needed and plants have proven their potential as a source of new arginase inhibitors (GirardThernier et al. 2015). Currently, polyphenols, that are well known to be antioxidant molecules and to display several biological properties, such as anti-inflammatory (Joseph et al. 2016; Chiavaroli et al. 2017) or multiple enzyme inhibition properties (Menghini et al., 2018),
https://doi.org/10.1016/j.sajb.2018.09.022 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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R. Attia et al. / South African Journal of Botany xxx (2018) xxx–xxx
constitute the most active natural plant metabolites with arginase inhibitory effects. Only very few other natural plant metabolites are reported for this activity (Minozzo et al. 2018). For this purpose and with the aim to valorize Tunisian flora, seven plants growing in Tunisia, Retama raetam (Forssk.) Webb & Berthel., Rhus tripartita (Ucria) Grande, Artemisia campestris L., Artemisia herba-alba Asso, Myrtus communis L., Crataegus azarolus L., Rubus ulmifolius Schott were first selected considering their biological activities (Table 1), some of which could be correlated with an effect on arginase. Then we evaluated the in vitro arginase inhibitory activity of 51 extracts prepared from different parts of these plants, as well as their phenolic and flavonoid contents, in order to assess whether their inhibitory activity might be supported, at least in part, by polyphenols and flavonoid compounds. 2. Materials and methods 2.1. Chemical agents All reagents were from Sigma–Aldrich (Saint-Quentin Fallavier, France), except for purified bovine liver arginase 1, which was purchased from MP Biomedicals (one unit (1 U) of bovine arginase corresponds to the amount of enzyme able to convert 1 μmol of L-arginine to urea and L-ornithine per minute at pH 7.5 and 37 °C). They were used without further purification. Methanol, dichloromethane, hexane, petroleum ether and dimethylsulfoxid were obtained from Carlo Erba Reagents (Val de Reuil, France) and VWR Chemicals (Fontenay-sousBois, France). Water was purified (resistivity N 18 mΩ/cm) using an ELGA water purification system (ELGA LabWater). 2.2. Plant materials Based on their biological activities previously determined in several studies, seven species belonging to different botanical families were considered (Table 1). Artemisia campestris (Ac-S/01–2016 and Ac-G/ 01–2016) was collected from the regions of Sousse (East-central
Tunisia, lower semi-arid, latitude 35°50′32″ N, longitude 10°31′46″ E, altitude 45 m) and Gafsa (Southwest of Tunisia, lower arid, latitude 34°35′59″ N, longitude 8°57′56″ E, altitude 380 m) in January 2016. Retama raetam (Rr/01-2017) and Rhus tripartita (Rt/01-2017) were collected from the Sousse region (East-central Tunisia, lower semiarid, latitude 35°50′32″ N, longitude 10°31′46″ E, altitude 45 m) in January 2017. Artemisia herba-alba (Aha/01-2016) was collected from Sidi Bouzid (Center of Tunisia, lower arid, latitude 35°17′37″ N, longitude 9°25′06″ E, altitude 390 m) and Myrtus communis (Mc/ 01-2016) from Korbous (Northeast of Tunisia, sub-humid, latitude 36°50′14″ N, longitude 10°35′0″ E, altitude 50 m) in January 2016. Finally Crataegus azarolus (Ca/02-2017) and Rubus ulmifolius (Ru/ 02-2017) were collected from the Morneg region (Northeast of Tunisia, upper semi-arid, latitude 36°39′57″ N, longitude 10°20′31″ E, altitude 130 m) in February 2017. The species were botanically identified by Professor Chokri Messaoud and voucher specimens of each plant were deposited at the Herbarium of the Faculty of Medical and Pharmaceutical Sciences of Besancon (France) for future reference. Before analyses, plant parts were air-dried at room temperature for 2 weeks. 2.3. Preparation of the extracts For each species, 15 g of the finely ground air-dried plant parts (stems, leaves, flowers, fruits, fruit stones or flower buds) were successively exhausted by maceration (room temperature) for 24 h in 750 mL hexane (H) or petroleum ether (PE), dichoromethane (D) and methanol (M). For each solvent, maceration was repeated seven times in order to exhaust the plant powder, and extractive solutions were filtered and concentrated to dryness under reduced pressure in a rotary evaporator. Fifty one final crude extracts (CE) were obtained. 2.4. Determination of total phenolic content Total phenol content was determined using the Folin–Ciocalteu method as described by Messaoud et al. (2012) with some
Table 1 Biological activities of the selected medicinal plants. Plant name
Family
Plant part used for the study
Myrtus communis L.
Myrtaceae
Leaves (Le), Fruits (Fr), Stems (St)
Retama raetam (Forssk.) Webb & Berthel. Rhus tripartita (Ucria) Grande
Rubus ulmifolius Schott Crataegus azarolus L.
Artemisia herba-alba Asso
Artemisia campestris L.
Biological activities
Hypotensive, antioxidant (Bouaziz et al. 2015), anti-hyperglycaemic (Elfellah et al. 1984), anti-inflammatory, antiseptic, treatment of candidiasis, lung disorders, treatment of stomach aches, hypoglycaemia, dysbiosis, cough, constipation and externally for wound healing (Aleksic and Knezevic 2014), Anti-genotoxic (Hayder et al. 2004) Fabaceae Flowers (Fl), Stems (St) Antihypertensive (Eddouks et al. 2008), antibacterial, antifungal, cytotoxic (Edziri et al. 2012), antidiabetic, laxative, diuretic (Maghrani et al. 2005a), vermifuge (Ziani et al. 2015), hypoglycemic (Maghrani et al. 2005b), antioxidant (Conforti et al. 2004), Anacardiaceae Leaves (Le), Stems (St), Anti-inflammatory, antioxidant (Mahjoub et al. 2010), anti-hepatotoxicity, anti-nephrotoxicity (Tlili et al. Fruits (Fr), Fruits 2016), anti-diarrheal (Ben Barka et al. 2016), antibacterial and antifungal (Abbassi and Hani 2012), stones (Fs) protective effect against gastric ulcer (Ben Barka et al. 2017), decrease of cardiotoxicity and oxidative stress (Shahat et al. 2016) Rosaceae Leaves (Le), Stems (St) Anti-Helicobacter pylori (Martini et al. 2009), antifungal (Sisti et al. 2008), antioxydant (Dall'Acqua et al. 2008), antipyretic (Ali et al. 2017), Rosaceae Leaves (Le), Stems (St), Antihypertensive (Haydari et al. 2017), antioxidant (Mustapha et al. 2015a), anti-inflammatory (Kallassy Buds (Bu) et al. 2017), cancer (Mustapha et al. 2016), antidiabetic (Henchiri and Zidi 2011), antihyperglycemic, antihyperlipidemic (Abu-Gharbieh and Gamil Shehab 2017), antihypercholesterolemic (Ben Jemaa et al. 2016). Asteraceae Whole plant (Wp) Hypotensive, diuretic (Zeggwagh et al. 2014), cancer (Khlifi et al. 2013), antidiabetic, cardiovascular diseases, antioxidant, antiradical, anti-spasmodic, neurological activities (Moufid and Eddouks 2012), antibacterial, antifungal (Bourgou et al. 2017), anti-inflammatory, analgesic, antipyretic, gastroprotective (Abdel Jaleel et al. 2016), cholinesterase inhibition (Orhan et al. 2010), hypoglycemic (Al Shamaony et al. 1994). Asteraceae Whole plant (Wp) Antihypertensive and vasorelaxant effects (Dib et al. 2017), anti-Leishmania (Aloui et al. 2016), anti-inflammatory, hepatoprotective, hypoglycemic, depurative, cholagogue, choleretic, depurative, digestive, antilithiasic, antimicrobial, anti-rheumatic, antivenin, obesity treatment and cholesterol decrease. (Al Snafi, 2015), antioxidant, antitumor (Akrout et al. 2011), gastroprotective (Corrêa-Ferreira et al. 2018).
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
R. Attia et al. / South African Journal of Botany xxx (2018) xxx–xxx
modifications. Briefly, 0.1 mL of a 1 mg/mL methanol solution of the extract was mixed with 0.5 mL of Folin–Ciocalteu's reagent (diluted 10 times with distilled water) and 0.4 mL of 7.5% sodium carbonate. After incubation at room temperature for 90 min, the absorbance of the resulting solution was measured at 765 nm. The total phenolic content was expressed as micrograms of gallic acid equivalent per milligram of crude extract (μg GAE/mg CE). 2.5. Determination of total flavonoid content Total flavonoid content was determined by using the colorimetric assay described by Messaoud et al. (2012) with some modifications. A volume of 0.5 mL of a 1 mg/mL methanolic extract solution was mixed with 0.5 mL of AlCl3 methanolic solution (2%) The mixture was allowed to stand for 30 min. The absorbance of the resulting solution was measured at 430 nm. Flavonoid content was expressed as micrograms of rutin equivalent per milligram of crude extract (μg RE/mg CE). 2.6. Arginase inhibition assay Urea produced by the hydrolysis of L-arginine by arginase [purified bovine liver arginase (b-ARGI)] was measured using the modified colorimetric method of Corraliza et al. (1994), as described below (Bordage et al. 2017). Briefly, in each well of a 96-well microplate the following solutions were added in this order: (1) buffer containing Tris–HCl (50 mM, pH 7.5) and 0.1% of bovine serum albumin (TBSA buffer) (10 μL), with or without (control) arginase (0.025 U/μL), (2) Tris–HCl solution (50 mM, pH 7.5) containing 10 mM MnCl2 as a cofactor (30 μL), a solution containing an extract (10 μL), (4) a solution of L-arginine (pH 9.7, 0.05 M) (20 μL). The microplate was incubated for 60 min in a 37 °C water bath after covering with a plastic sealing film. The addition of 120 μL of H2SO4/H3PO4/H2O (1:3:7) quenched the reaction. Thereafter, 10 μL of α-isonitrosopropiophenone (5% in absolute EtOH) was added, and the microplate was heated in an oven at 100 °C for 45 min after covering with an aluminum sealing film. The colored product being photosensitive, the microplate was kept in the dark until reading. After 5 min of centrifugation and cooling for another 10 min, the microplate was shaken for 2 min and the absorbance was read at 550 nm and 25 °C using a spectrophotometer (Synergy HT BioTeck). The level of arginase activity was expressed as relative to the “100% arginase activity”. The experiment was repeated three times with each microplate under similar experimental conditions. 2.7. Determination of percentages of arginase inhibition and IC50 values For each extract, a stock solution (21 mg/mL) was prepared in dimethylsulfoxid (DMSO) and stored at −26 °C. These stock solutions were extemporaneously and successively diluted in DMSO to afford the following concentrations: 21000, 6300, 2100, 630, 210, 63, 21, 6.3, and 2.1 μg/mL, corresponding to final concentrations in the wells of 3000, 900, 300, 90, 30, 9, 3, 0.9, 0.3 μg/mL, respectively. For the first preliminary screening, extracts were only tested at final concentrations of 10 and 100 μg/mL. Each solution was incubated with arginase for 1 h, as described above. The arginase inhibitory activity was calculated using the following formula: Inhibition of arginase activity (%) = 100 × [(Aaa – Ac1) – (Ae – Ac2)/(Aaa – Ac1)]; where, Aaa is the absorbance of 100% arginase activity, Ac1 is the absorbance of the control 1 (TBSA buffer instead of the enzyme), Ac2 is the absorbance of the control 2 (containing extract without arginase) and Ae is the absorbance of the reaction containing the extract and the enzyme. All tests were performed in triplicate. For the preliminary screening, results were expressed as percentage of inhibition at 100 and 10 μg/mL. IC50 for crude extracts showing inhibition percentage exceeding 70% at 100 μg/mL were calculated. IC50 were estimated by nonlinear sigmoidal curve-fitting using Prism® (GraphPad®
3
Software, version 5.0.3) after plotting the inhibition percentages versus sample concentrations on a semi logarithmic scale. 2.8. Data analysis All determinations were conducted in triplicates and results for each measured parameter were expressed as mean ± SEM. Variations of total phenol and flavonoid contents and the arginase inhibitory activity among extracts were determined by the analysis of variance (ANOVA) procedure (p b .05), followed by Duncan's multiple range test using the SAS program version 9. Correlations between the total phenol or flavonoid contents and the arginase inhibitory activity were determined with the PROC CORR procedure using SAS version 9. 3. Results 3.1. Phenolic and flavonoid contents The total phenolic and flavonoid contents of the most active extracts were measured and are given in Table 2. Significant variations (p b .05) were observed between the five plant extracts. The highest total phenol content (161.02 ± 1.25 μg GAE/mg CE) was detected in the methanolic extract from the stems of Crataegus azarolus and the highest flavonoid content (38.79 ± 0.34 μg RE/mg CE) was determined in the methanolic extract of buds of Crataegus azarolus. It can be noted that the total polyphenolic content of the dichloromethane extracts is consittuted by flavonoids. In contrast, flavonoids only represent a small part of the total phenolic content of methanolic extracts. 3.2. Effects of plant extracts on arginase activity The fifty one crude extracts obtained from the selected plants were screened for their arginase inhibitory activity. As shown in Fig. 1, the percentages of arginase inhibition varied according to the type of extract and the concentration used (10 or 100 μg/mL). Some crude extracts, such as petroleum ether extract from the whole plant of Artemisia herba-alba, hexane extract from the stems of Rubus ulmifolius, chloromethylenic extracts from the stems of Rhus tripartita and the flower buds of Crategus azarolus, had a concentration-independent inhibitory activity as their inhibition was almost the same at 10 and 100 μg/mL. However, most of the extracts showed concentration-dependent activity. Among them, this preliminary screening led us to identified five relevant crude fractions at the concentration of 100 μg/mL with inhibitory activities over 70%: two chloromethylenic extracts from stems of Retama raetam (71.66 ± 2.94%) and Crataegus azarolus (73.00 ± 1.18%) and the methanolic extracts from stems of Rhus tripartita (80.66 ± 3.69%), flower buds (80.00 ± 3.21%) and stems of Crataegus azarolus (90.00 ± 1.53%). Table 2 Crude extract (CE) per gram of dry matter, total phenolic compounds (TPC), and total flavonoids (TFC).
C. R. C. R. C.
azarolus tripartita azarolus raetam azarolus
Stems Stems Buds Stems Stems
M M M D D
CE (mg/g DM)
TPC (μg GAE/mg CE)
TFC (μg RE/mg CE)
104.1 140,8 188.6 7.4 9
161.02 ± 1.24a 127.56 ± 5.82b 79.35 ± 4.70c 38.35 ± 1.60d 25.96 ± 0.39e
22.32 ± 0.24c 9.82 ± 0.012d 38.79 ± 0.34a 32.79 ± 0.60b 23.41 ± 0.41c
CE: Crude Extract; D: Dichloromethane; M: methanol; DM: Dried Matter; TPC: Total Phenolic Content; GAE: Gallic Acid Equivalent; TFC: Total Flavonoid Content; RE: Rutin Equivalent. Values are given as mean ± SEM of three independent experiments performed in triplicate. Means followed by different letters are significantly different (p ˂ 0.05).
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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R. Attia et al. / South African Journal of Botany xxx (2018) xxx–xxx
Fig. 1. In vitro effect of the crude extracts on bovine liver arginase I.
The IC50 values for these extracts were also determined (Table 3). The analysis of variance (ANOVA) showed that the arginase inhibitory activity varied significantly among these relevant extracts (Table 3). The methanolic extract from Crataegus azarolus stems exhibited the best inhibitory activity (IC50 = 41.99 ± 5.77 μg/mL), whereas the methanolic extract from Crataegus azarolus flower buds was characterized by the lowest capacity to inhibit the activity of the arginase enzyme (IC50 = 96.69 ± 0.56 μg/mL). In our study, the variation of arginase inhibitory activity observed among samples reflects their phenolic composition and content differences. In fact, a significant correlation (r = −0.749; p = .0013 b 0.05) was observed between the total phenolic contents and the IC50 values of the arginase inhibitory activity. The flavonoid contents are inversely proportional to the arginase inhibitory activity (r = + 0.514; p = .0498). However, the arginase inhibitory activities determined for the investigated extracts remain lower than those observed with the pure synthetic reference inhibitors S-(2-boronethyl)-L-Cysteine (BEC) and Nωhydroxy-nor-Arginine (IC50 = 0.30 and 0.66 μg/mL, respectively) or the pure natural reference inhibitors chlorogenic acid and piceatannol IC50 = 3.75 and 2.95 μg/mL, respectively) (Bordage et al. 2017). 4. Discussion Arginase inhibition offers new therapeutic possibilities for the treatment of various diseases. Although potent synthetic arginase inhibitors Table 3 Arginase inhibitory activity and IC50 values of the five most active crude extracts. Plant
C. azarolus R. tripartita C. azarolus C. azarolus R. raetam
Plant part used
Extract type
Inhibition (%) 10 μg/mL
100 μg/mL
IC50 (μg/ml)
stems stems buds stems stems
M M M D D
22.00 ± 1.99b,c 33.00 ± 6.72a 25.66 ± 3.07a,b 15.33 ± 1.57b,c 14.00 ± 1.30c
90.00 ± 1.53a 80.66 ± 3.69b 80.00 ± 3.21b 73.00 ± 1.18c 71.66 ± 2.94c
41.99 ± 5.77d 71.48 ± 6.29c 96.69 ± 0.56a 85.84 ± 6.59b 84.91 ± 4.25b
D: Dichloromethane; M: Methanol. Values are given as mean ± SEM of three independent experiments performed in triplicate. Values followed by different letters are significantly different (p ˂ 0.05).
are available, none is currently useful for clinical application. Consequently there is a persistent need to identify new arginase inhibitors as potential drug candidates. Various plant extracts have been previously studied for their arginase inhibitory effect and the potential of natural plant-derived substances as arginase inhibitors has been highlighted over the last years. The majority of naturally occurring arginase inhibitors already described in the literature belongs to the class of polyphenols, such as chlorogenic acid, piceatannol, resveratrol, epicatechin, cyperusphenol B, carexinol A, scirpusin, viniferin and the flavonoids quercetin, (2S)-5,7-dihydroxy-8,2′-dimethoxyflavanone and (2S)-5,2′,5′-trihydroxy-7,8-dimethoxyflavanone (Girard-Thernier et al. 2015; Arraki et al. 2017; Minozzo et al. 2018). For this reason and in order to valorise Tunisian flora, we screened extracts from seven medicinal plants from the Tunisian traditional medicine for their arginase inhibitory potential. Plants belonging to different genera and families were selected regarding their biological activities related to arginase inhibition such as antihypertensive, antimicrobial or antiinflammatory properties (Table 1): Retama raetam, Rhus tripartita, Artemisia campestris, Artemisia herba-alba, Myrtus communis, Crataegus azarolus, Rubus ulmifolius. Five of the 51 prepared extracts from these plants showed an interesting inhibitory effect higher than 70% of at 100 μg/mL. Their IC50 values were found to be less than 100 μg/mL, with the best activity obtained with the methanolic stem extract from Crataegus azarolus (IC50 = 41,99 μg/mL) which constitutes an interesting value for an extract, if we consider the IC50 value of pure natural reference inhibitors; chlorogenic acid (3.75 μg/mL) or piceatannol (2.95 μg/mL) (Girard-Thernier et al. 2015). In order to establish a potential correlation between activity and polyphenolic content, we also determined the amount of phenols and flavonoids in these most active extracts. The results highlighted that methanolic extracts from Crataegus azarolus stems and from Rhus tripartita are also the richest in phenolic compounds, confirming the interest of this kind of secondary metabolites for arginase inhibition (Bordage et al. 2017; Minozzo et al. 2018). However, it also appears that flavonoid compounds constitute only a small part of the total phenolic content of these extracts and cannot consequently be considered as fully responsible for the observed activities. Other polyphenolic compounds are probably involved, which is not surprising regarding previous studies cited above (Girard-Thernier et al. 2015; Arraki et al. 2017). Crataegus azarolus, Retama Raetam and Rhus tripartita have not been extensively studied from a phytochemical point of view but a few
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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studies have reported that these plants contain polyphenols, sometimes associated with other classes of secondary metabolites (Abu-Gharbieh and Shehab, 2017; Edziri et al. 2012; Kassem et al. 2000; Mustapha et al. 2015a; Shahat et al. 2016). Retama raetam, for example, is characterized by its richness in alkaloids (El-Shazly et al. 1996; Abdel-Halim et al., 1992; Abdel-Halim, 1995; Schmid et al. 2006). For Rhus tripartita the majority of compounds described in the literature, so far, belongs to flavonoids (Alam et al. 2017; Mahjoub et al., 2005; Shahat et al. 2016). Finally, in Crataegus azarolus, triterpens are present in large quantities (Mustapha et al. 2015b). This latter plant showed the best activity on the in vitro arginase inhibition test. Considering that an antihypertensive effect of the fruit of Crataegus azarolus has been recently demonstrated by Haydari et al. (2017), further analysis on this plant should be considered with the aim to isolate and evaluate its active secondary metabolites on arginase as well as to correlate their arginase inhibitory effect with a potential vasorelaxant effect. Finally, considering that polyphenols represent only a fraction of the active crude extracts, it could be interesting to isolate and evaluate other various classes of secondary metabolites in order to highlight new natural structures of inhibitors. 5. Conclusion In conclusion, our study identified several extracts as a new source of arginase inhibitors, in particular the methanolic extract from the stems of Crataegus azarolus which is the richest in polyphenol content. These results encourage further studies of these active extracts in order to identify the bioactive molecules responsible for arginase inhibition. Funding sources This work was financially supported by the “PHC Utique” program of the French Ministries of Foreign Affairs and of Higher Education, Research and Innovation and the Tunisian Ministry of higher education and scientific research in the CMCU project number 18G0816. The authors would like to thank the University of Carthage, the National Institute of Applied Science and Technology, Campus France and PEPITE EA4267 for their financial support, and Dr. Lucie Bernard for the check of the English language. References Abbassi, F., Hani, K., 2012. In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine. Natural Product Research 26, 2215–2218. Abdel Jaleel, G.A.R., Ibrahim Abdallah, H.M., Sayed Gomaa, N.E.L., 2016. Pharmacological effects of ethanol extract of Egyptian Artemisia herba-alba in rats and mice. Asian Pacific Journal of Tropical Biomedicine 6, 44–49. Abdel-Halim, O.B., 1995. (−)-6α-Hydroxylupanine, a lupin alkaloid from Lygos raetam var. sarcocarpa. Phytochemistry 40, 1323–1325. Abdel-Halim, O.B., Sekine, T., Saito, K., Halim, A.F., Abdel Fattah, H., Murakoshi, I., 1992. (+)-12α-Hydroxylupanine, a lupin alkaloid from Lygos raetam. Phytochemistry, The International Journal of Plant Biochemistry 31, 3251–3253. Abu-Gharbieh, E., Shehab, N.G., 2017. Therapeutic potentials of Crataegus azarolus var. Euazarolus Maire leaves and its isolated compounds. BMC Complementary and Alternative Medicine 17, 218. Akrout, A., Alarcon Gonzalez, L., El Jani, H., Campra Madrid, P., 2011. Antioxidant and antitumor activities of Artemisia campestris and Thymelaea hirsuta from Southern Tunisia. Food and Chemical Toxicology 49, 342–347. Al Shamaony, L., Al Khazraji, S.M., Twaij, H.A., 1994. Hypoglycaemic effect of Artemisia herba-alba. II. Effect of a valuable extract on some blood parameters in diabetic animals. Journal of Ethnopharmacology 43, 167–171. Al Snafi, A.E., 2015. The pharmacological importance of Artemisia campestris. Asian Journal of Pharmaceutical Research 5, 88–92. Alam, P., Parvez, M.K., Arbab, A.H., Siddiqui, N.A., Al-Dosary, M.S., Al-Rehaily, A.J., Ahmed, S., Abul Kalam, M., Ahmad, M.S., 2017. Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita. Pharmaceutical Biology 55, 1450–1457. Aleksic, V., Knezevic, P., 2014. Antimicrobial and antioxidative activity of extracts and essential oils of Myrtus Communis L. microbiological research. Medicinal Extracts in Microbiology 169, 240–254.
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Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants R. Attia a,b, C. Messaoud b, K. Arraki a, A. Zedet a, C. Demougeot a, M. Boussaïd b, C. Girard a,⁎ a
PEPITE EA4267, Univ. Bourgogne Franche-Comté, F-25000 Besançon, France Laboratory of Nanobiotechnology and Medicinal Plants, Department of Biology, National Institute of Applied Science and Technology (INSAT), University of Carthage, BP 676, 1080 Tunis Cedex, Tunisia
b
a r t i c l e
i n f o
Article history: Received 31 March 2018 Received in revised form 30 August 2018 Accepted 19 September 2018 Available online xxxx Edited by A Mocan Keywords: Arginase Naturally occurring inhibitors Tunisian medicinal plants Polyphenols
a b s t r a c t Arginase has been the focus of a particular interest since it has been shown that the increase of its activity is involved in various pathologies for which the inhibition of this enzyme could be a promising treatment. The several existing synthetic inhibitors of arginase are not suitable for a use in human therapeutics. Hence, interest has been initiated to explore natural resources, in particular medicinal plants for the search for new arginase inhibitors. Tunisia is famous for its great flora diversity, as well as a centuries-old tradition of medicinal plants used to treat many diseases including cardiovascular disorders. Therefore, seven Tunisian plants belonging to various families: Retama raetam (Forssk.) Webb & Berthel., Rhus tripartita (Ucria) Grande, Artemisia campestris L., Artemisia herba-alba Asso, Myrtus communis L., Crataegus azarolus L., Rubus ulmifolius Schott, were selected to be screened on arginase using an in vitro inhibition test. Fifty-one crude extracts obtained from different parts of these plants showed inhibition percentages ranging from 2.66 ± 7.57% to 90.00 ± 1.53% at 100 μg/mL. Five extracts were identified as the most active and their IC50 were determined, as well as their phenolic and flavonoid contents. Results showed that the methanolic extract from Crataegus azarolus stems is the most interesting crude material with the lowest IC50 (41.99 ± 1.53 μg/mL). This extract also shows the richest polyphenolic content (161.02 ± 1.24 μg GAE/mg CE). The analyses of total flavonoid content revealed that flavonoids represent only a small part of this polyphenolic content (22.32 ± 0.24 μg RE/mg CE) and cannot be considered as fully responsible for the observed activity. Further, bio-guided fractionations and structural elucidation will aim to identify secondary metabolites responsible for the bioactivity observed on arginase. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Worldwide traditional medicine has a long history of use in disease treatment (WHO, 2013). In particular, medicinal plants are widely used in many countries. As they constitute a well-known source of active metabolites, a lot of studies have been performed to isolate compounds responsible for their biological properties. Some of them led to the discovery of new drugs currently used in therapeutics (Newman and Cragg 2016). Among their numerous biological activities, natural compounds have the potential to inhibit enzymes (Rauf and Jehan, 2017). Arginase is a trimeric metalloenzyme that catalyzes hydrolysis of L-arginine to L-ornithine and urea and plays an important role in the ammonia detoxification in mammals. By substrate competition, arginase also plays a crucial role in the bioavailability of L-arginine for nitric oxide synthase (NOS). The result of this competition is the decrease of nitric oxide (NO) production and the increase of ⁎ Corresponding author at: PEPITE EA4267, Univ. Bourgogne Franche-Comté, 19 rue Ambroise Paré, F-25000 Besançon, France. E-mail address: [email protected] (C. Girard).
L-ornithine production. This latter is converted into polyamines or proline that can promote cell proliferation and collagen production, resulting in various health problems, in particular at the cardiovascular level (Pudlo et al. 2017). Over the last decades, studies on animal models and clinical studies have provided arguments in favor of arginase as a new promising target of inhibitors aiming to treat microbial (Martini et al. 2009) or parasitic infections (Heby et al. 2007), cancers (Pham et al. 2018) and inflammatory or cardiovascular diseases (Caldwell et al. 2015). Among the few synthetic arginase inhibitors commercially available, boronic acid derivatives (S-(2-boronoethyl)-L-cysteine (BEC), 2-(S)-amino-6-boronohexanoic acid (ABH) and Nω-hydroxynor-L-arginine (nor-NOHA) are the most potent, but their toxicity or their poor pharmacokinetic profile limits their potential therapeutic use in humans (Ivanenkov and Chufarova, 2014). Therefore the discovery of new structures of arginase inhibitors is needed and plants have proven their potential as a source of new arginase inhibitors (GirardThernier et al. 2015). Currently, polyphenols, that are well known to be antioxidant molecules and to display several biological properties, such as anti-inflammatory (Joseph et al. 2016; Chiavaroli et al. 2017) or multiple enzyme inhibition properties (Menghini et al., 2018),
https://doi.org/10.1016/j.sajb.2018.09.022 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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constitute the most active natural plant metabolites with arginase inhibitory effects. Only very few other natural plant metabolites are reported for this activity (Minozzo et al. 2018). For this purpose and with the aim to valorize Tunisian flora, seven plants growing in Tunisia, Retama raetam (Forssk.) Webb & Berthel., Rhus tripartita (Ucria) Grande, Artemisia campestris L., Artemisia herba-alba Asso, Myrtus communis L., Crataegus azarolus L., Rubus ulmifolius Schott were first selected considering their biological activities (Table 1), some of which could be correlated with an effect on arginase. Then we evaluated the in vitro arginase inhibitory activity of 51 extracts prepared from different parts of these plants, as well as their phenolic and flavonoid contents, in order to assess whether their inhibitory activity might be supported, at least in part, by polyphenols and flavonoid compounds. 2. Materials and methods 2.1. Chemical agents All reagents were from Sigma–Aldrich (Saint-Quentin Fallavier, France), except for purified bovine liver arginase 1, which was purchased from MP Biomedicals (one unit (1 U) of bovine arginase corresponds to the amount of enzyme able to convert 1 μmol of L-arginine to urea and L-ornithine per minute at pH 7.5 and 37 °C). They were used without further purification. Methanol, dichloromethane, hexane, petroleum ether and dimethylsulfoxid were obtained from Carlo Erba Reagents (Val de Reuil, France) and VWR Chemicals (Fontenay-sousBois, France). Water was purified (resistivity N 18 mΩ/cm) using an ELGA water purification system (ELGA LabWater). 2.2. Plant materials Based on their biological activities previously determined in several studies, seven species belonging to different botanical families were considered (Table 1). Artemisia campestris (Ac-S/01–2016 and Ac-G/ 01–2016) was collected from the regions of Sousse (East-central
Tunisia, lower semi-arid, latitude 35°50′32″ N, longitude 10°31′46″ E, altitude 45 m) and Gafsa (Southwest of Tunisia, lower arid, latitude 34°35′59″ N, longitude 8°57′56″ E, altitude 380 m) in January 2016. Retama raetam (Rr/01-2017) and Rhus tripartita (Rt/01-2017) were collected from the Sousse region (East-central Tunisia, lower semiarid, latitude 35°50′32″ N, longitude 10°31′46″ E, altitude 45 m) in January 2017. Artemisia herba-alba (Aha/01-2016) was collected from Sidi Bouzid (Center of Tunisia, lower arid, latitude 35°17′37″ N, longitude 9°25′06″ E, altitude 390 m) and Myrtus communis (Mc/ 01-2016) from Korbous (Northeast of Tunisia, sub-humid, latitude 36°50′14″ N, longitude 10°35′0″ E, altitude 50 m) in January 2016. Finally Crataegus azarolus (Ca/02-2017) and Rubus ulmifolius (Ru/ 02-2017) were collected from the Morneg region (Northeast of Tunisia, upper semi-arid, latitude 36°39′57″ N, longitude 10°20′31″ E, altitude 130 m) in February 2017. The species were botanically identified by Professor Chokri Messaoud and voucher specimens of each plant were deposited at the Herbarium of the Faculty of Medical and Pharmaceutical Sciences of Besancon (France) for future reference. Before analyses, plant parts were air-dried at room temperature for 2 weeks. 2.3. Preparation of the extracts For each species, 15 g of the finely ground air-dried plant parts (stems, leaves, flowers, fruits, fruit stones or flower buds) were successively exhausted by maceration (room temperature) for 24 h in 750 mL hexane (H) or petroleum ether (PE), dichoromethane (D) and methanol (M). For each solvent, maceration was repeated seven times in order to exhaust the plant powder, and extractive solutions were filtered and concentrated to dryness under reduced pressure in a rotary evaporator. Fifty one final crude extracts (CE) were obtained. 2.4. Determination of total phenolic content Total phenol content was determined using the Folin–Ciocalteu method as described by Messaoud et al. (2012) with some
Table 1 Biological activities of the selected medicinal plants. Plant name
Family
Plant part used for the study
Myrtus communis L.
Myrtaceae
Leaves (Le), Fruits (Fr), Stems (St)
Retama raetam (Forssk.) Webb & Berthel. Rhus tripartita (Ucria) Grande
Rubus ulmifolius Schott Crataegus azarolus L.
Artemisia herba-alba Asso
Artemisia campestris L.
Biological activities
Hypotensive, antioxidant (Bouaziz et al. 2015), anti-hyperglycaemic (Elfellah et al. 1984), anti-inflammatory, antiseptic, treatment of candidiasis, lung disorders, treatment of stomach aches, hypoglycaemia, dysbiosis, cough, constipation and externally for wound healing (Aleksic and Knezevic 2014), Anti-genotoxic (Hayder et al. 2004) Fabaceae Flowers (Fl), Stems (St) Antihypertensive (Eddouks et al. 2008), antibacterial, antifungal, cytotoxic (Edziri et al. 2012), antidiabetic, laxative, diuretic (Maghrani et al. 2005a), vermifuge (Ziani et al. 2015), hypoglycemic (Maghrani et al. 2005b), antioxidant (Conforti et al. 2004), Anacardiaceae Leaves (Le), Stems (St), Anti-inflammatory, antioxidant (Mahjoub et al. 2010), anti-hepatotoxicity, anti-nephrotoxicity (Tlili et al. Fruits (Fr), Fruits 2016), anti-diarrheal (Ben Barka et al. 2016), antibacterial and antifungal (Abbassi and Hani 2012), stones (Fs) protective effect against gastric ulcer (Ben Barka et al. 2017), decrease of cardiotoxicity and oxidative stress (Shahat et al. 2016) Rosaceae Leaves (Le), Stems (St) Anti-Helicobacter pylori (Martini et al. 2009), antifungal (Sisti et al. 2008), antioxydant (Dall'Acqua et al. 2008), antipyretic (Ali et al. 2017), Rosaceae Leaves (Le), Stems (St), Antihypertensive (Haydari et al. 2017), antioxidant (Mustapha et al. 2015a), anti-inflammatory (Kallassy Buds (Bu) et al. 2017), cancer (Mustapha et al. 2016), antidiabetic (Henchiri and Zidi 2011), antihyperglycemic, antihyperlipidemic (Abu-Gharbieh and Gamil Shehab 2017), antihypercholesterolemic (Ben Jemaa et al. 2016). Asteraceae Whole plant (Wp) Hypotensive, diuretic (Zeggwagh et al. 2014), cancer (Khlifi et al. 2013), antidiabetic, cardiovascular diseases, antioxidant, antiradical, anti-spasmodic, neurological activities (Moufid and Eddouks 2012), antibacterial, antifungal (Bourgou et al. 2017), anti-inflammatory, analgesic, antipyretic, gastroprotective (Abdel Jaleel et al. 2016), cholinesterase inhibition (Orhan et al. 2010), hypoglycemic (Al Shamaony et al. 1994). Asteraceae Whole plant (Wp) Antihypertensive and vasorelaxant effects (Dib et al. 2017), anti-Leishmania (Aloui et al. 2016), anti-inflammatory, hepatoprotective, hypoglycemic, depurative, cholagogue, choleretic, depurative, digestive, antilithiasic, antimicrobial, anti-rheumatic, antivenin, obesity treatment and cholesterol decrease. (Al Snafi, 2015), antioxidant, antitumor (Akrout et al. 2011), gastroprotective (Corrêa-Ferreira et al. 2018).
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
R. Attia et al. / South African Journal of Botany xxx (2018) xxx–xxx
modifications. Briefly, 0.1 mL of a 1 mg/mL methanol solution of the extract was mixed with 0.5 mL of Folin–Ciocalteu's reagent (diluted 10 times with distilled water) and 0.4 mL of 7.5% sodium carbonate. After incubation at room temperature for 90 min, the absorbance of the resulting solution was measured at 765 nm. The total phenolic content was expressed as micrograms of gallic acid equivalent per milligram of crude extract (μg GAE/mg CE). 2.5. Determination of total flavonoid content Total flavonoid content was determined by using the colorimetric assay described by Messaoud et al. (2012) with some modifications. A volume of 0.5 mL of a 1 mg/mL methanolic extract solution was mixed with 0.5 mL of AlCl3 methanolic solution (2%) The mixture was allowed to stand for 30 min. The absorbance of the resulting solution was measured at 430 nm. Flavonoid content was expressed as micrograms of rutin equivalent per milligram of crude extract (μg RE/mg CE). 2.6. Arginase inhibition assay Urea produced by the hydrolysis of L-arginine by arginase [purified bovine liver arginase (b-ARGI)] was measured using the modified colorimetric method of Corraliza et al. (1994), as described below (Bordage et al. 2017). Briefly, in each well of a 96-well microplate the following solutions were added in this order: (1) buffer containing Tris–HCl (50 mM, pH 7.5) and 0.1% of bovine serum albumin (TBSA buffer) (10 μL), with or without (control) arginase (0.025 U/μL), (2) Tris–HCl solution (50 mM, pH 7.5) containing 10 mM MnCl2 as a cofactor (30 μL), a solution containing an extract (10 μL), (4) a solution of L-arginine (pH 9.7, 0.05 M) (20 μL). The microplate was incubated for 60 min in a 37 °C water bath after covering with a plastic sealing film. The addition of 120 μL of H2SO4/H3PO4/H2O (1:3:7) quenched the reaction. Thereafter, 10 μL of α-isonitrosopropiophenone (5% in absolute EtOH) was added, and the microplate was heated in an oven at 100 °C for 45 min after covering with an aluminum sealing film. The colored product being photosensitive, the microplate was kept in the dark until reading. After 5 min of centrifugation and cooling for another 10 min, the microplate was shaken for 2 min and the absorbance was read at 550 nm and 25 °C using a spectrophotometer (Synergy HT BioTeck). The level of arginase activity was expressed as relative to the “100% arginase activity”. The experiment was repeated three times with each microplate under similar experimental conditions. 2.7. Determination of percentages of arginase inhibition and IC50 values For each extract, a stock solution (21 mg/mL) was prepared in dimethylsulfoxid (DMSO) and stored at −26 °C. These stock solutions were extemporaneously and successively diluted in DMSO to afford the following concentrations: 21000, 6300, 2100, 630, 210, 63, 21, 6.3, and 2.1 μg/mL, corresponding to final concentrations in the wells of 3000, 900, 300, 90, 30, 9, 3, 0.9, 0.3 μg/mL, respectively. For the first preliminary screening, extracts were only tested at final concentrations of 10 and 100 μg/mL. Each solution was incubated with arginase for 1 h, as described above. The arginase inhibitory activity was calculated using the following formula: Inhibition of arginase activity (%) = 100 × [(Aaa – Ac1) – (Ae – Ac2)/(Aaa – Ac1)]; where, Aaa is the absorbance of 100% arginase activity, Ac1 is the absorbance of the control 1 (TBSA buffer instead of the enzyme), Ac2 is the absorbance of the control 2 (containing extract without arginase) and Ae is the absorbance of the reaction containing the extract and the enzyme. All tests were performed in triplicate. For the preliminary screening, results were expressed as percentage of inhibition at 100 and 10 μg/mL. IC50 for crude extracts showing inhibition percentage exceeding 70% at 100 μg/mL were calculated. IC50 were estimated by nonlinear sigmoidal curve-fitting using Prism® (GraphPad®
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Software, version 5.0.3) after plotting the inhibition percentages versus sample concentrations on a semi logarithmic scale. 2.8. Data analysis All determinations were conducted in triplicates and results for each measured parameter were expressed as mean ± SEM. Variations of total phenol and flavonoid contents and the arginase inhibitory activity among extracts were determined by the analysis of variance (ANOVA) procedure (p b .05), followed by Duncan's multiple range test using the SAS program version 9. Correlations between the total phenol or flavonoid contents and the arginase inhibitory activity were determined with the PROC CORR procedure using SAS version 9. 3. Results 3.1. Phenolic and flavonoid contents The total phenolic and flavonoid contents of the most active extracts were measured and are given in Table 2. Significant variations (p b .05) were observed between the five plant extracts. The highest total phenol content (161.02 ± 1.25 μg GAE/mg CE) was detected in the methanolic extract from the stems of Crataegus azarolus and the highest flavonoid content (38.79 ± 0.34 μg RE/mg CE) was determined in the methanolic extract of buds of Crataegus azarolus. It can be noted that the total polyphenolic content of the dichloromethane extracts is consittuted by flavonoids. In contrast, flavonoids only represent a small part of the total phenolic content of methanolic extracts. 3.2. Effects of plant extracts on arginase activity The fifty one crude extracts obtained from the selected plants were screened for their arginase inhibitory activity. As shown in Fig. 1, the percentages of arginase inhibition varied according to the type of extract and the concentration used (10 or 100 μg/mL). Some crude extracts, such as petroleum ether extract from the whole plant of Artemisia herba-alba, hexane extract from the stems of Rubus ulmifolius, chloromethylenic extracts from the stems of Rhus tripartita and the flower buds of Crategus azarolus, had a concentration-independent inhibitory activity as their inhibition was almost the same at 10 and 100 μg/mL. However, most of the extracts showed concentration-dependent activity. Among them, this preliminary screening led us to identified five relevant crude fractions at the concentration of 100 μg/mL with inhibitory activities over 70%: two chloromethylenic extracts from stems of Retama raetam (71.66 ± 2.94%) and Crataegus azarolus (73.00 ± 1.18%) and the methanolic extracts from stems of Rhus tripartita (80.66 ± 3.69%), flower buds (80.00 ± 3.21%) and stems of Crataegus azarolus (90.00 ± 1.53%). Table 2 Crude extract (CE) per gram of dry matter, total phenolic compounds (TPC), and total flavonoids (TFC).
C. R. C. R. C.
azarolus tripartita azarolus raetam azarolus
Stems Stems Buds Stems Stems
M M M D D
CE (mg/g DM)
TPC (μg GAE/mg CE)
TFC (μg RE/mg CE)
104.1 140,8 188.6 7.4 9
161.02 ± 1.24a 127.56 ± 5.82b 79.35 ± 4.70c 38.35 ± 1.60d 25.96 ± 0.39e
22.32 ± 0.24c 9.82 ± 0.012d 38.79 ± 0.34a 32.79 ± 0.60b 23.41 ± 0.41c
CE: Crude Extract; D: Dichloromethane; M: methanol; DM: Dried Matter; TPC: Total Phenolic Content; GAE: Gallic Acid Equivalent; TFC: Total Flavonoid Content; RE: Rutin Equivalent. Values are given as mean ± SEM of three independent experiments performed in triplicate. Means followed by different letters are significantly different (p ˂ 0.05).
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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R. Attia et al. / South African Journal of Botany xxx (2018) xxx–xxx
Fig. 1. In vitro effect of the crude extracts on bovine liver arginase I.
The IC50 values for these extracts were also determined (Table 3). The analysis of variance (ANOVA) showed that the arginase inhibitory activity varied significantly among these relevant extracts (Table 3). The methanolic extract from Crataegus azarolus stems exhibited the best inhibitory activity (IC50 = 41.99 ± 5.77 μg/mL), whereas the methanolic extract from Crataegus azarolus flower buds was characterized by the lowest capacity to inhibit the activity of the arginase enzyme (IC50 = 96.69 ± 0.56 μg/mL). In our study, the variation of arginase inhibitory activity observed among samples reflects their phenolic composition and content differences. In fact, a significant correlation (r = −0.749; p = .0013 b 0.05) was observed between the total phenolic contents and the IC50 values of the arginase inhibitory activity. The flavonoid contents are inversely proportional to the arginase inhibitory activity (r = + 0.514; p = .0498). However, the arginase inhibitory activities determined for the investigated extracts remain lower than those observed with the pure synthetic reference inhibitors S-(2-boronethyl)-L-Cysteine (BEC) and Nωhydroxy-nor-Arginine (IC50 = 0.30 and 0.66 μg/mL, respectively) or the pure natural reference inhibitors chlorogenic acid and piceatannol IC50 = 3.75 and 2.95 μg/mL, respectively) (Bordage et al. 2017). 4. Discussion Arginase inhibition offers new therapeutic possibilities for the treatment of various diseases. Although potent synthetic arginase inhibitors Table 3 Arginase inhibitory activity and IC50 values of the five most active crude extracts. Plant
C. azarolus R. tripartita C. azarolus C. azarolus R. raetam
Plant part used
Extract type
Inhibition (%) 10 μg/mL
100 μg/mL
IC50 (μg/ml)
stems stems buds stems stems
M M M D D
22.00 ± 1.99b,c 33.00 ± 6.72a 25.66 ± 3.07a,b 15.33 ± 1.57b,c 14.00 ± 1.30c
90.00 ± 1.53a 80.66 ± 3.69b 80.00 ± 3.21b 73.00 ± 1.18c 71.66 ± 2.94c
41.99 ± 5.77d 71.48 ± 6.29c 96.69 ± 0.56a 85.84 ± 6.59b 84.91 ± 4.25b
D: Dichloromethane; M: Methanol. Values are given as mean ± SEM of three independent experiments performed in triplicate. Values followed by different letters are significantly different (p ˂ 0.05).
are available, none is currently useful for clinical application. Consequently there is a persistent need to identify new arginase inhibitors as potential drug candidates. Various plant extracts have been previously studied for their arginase inhibitory effect and the potential of natural plant-derived substances as arginase inhibitors has been highlighted over the last years. The majority of naturally occurring arginase inhibitors already described in the literature belongs to the class of polyphenols, such as chlorogenic acid, piceatannol, resveratrol, epicatechin, cyperusphenol B, carexinol A, scirpusin, viniferin and the flavonoids quercetin, (2S)-5,7-dihydroxy-8,2′-dimethoxyflavanone and (2S)-5,2′,5′-trihydroxy-7,8-dimethoxyflavanone (Girard-Thernier et al. 2015; Arraki et al. 2017; Minozzo et al. 2018). For this reason and in order to valorise Tunisian flora, we screened extracts from seven medicinal plants from the Tunisian traditional medicine for their arginase inhibitory potential. Plants belonging to different genera and families were selected regarding their biological activities related to arginase inhibition such as antihypertensive, antimicrobial or antiinflammatory properties (Table 1): Retama raetam, Rhus tripartita, Artemisia campestris, Artemisia herba-alba, Myrtus communis, Crataegus azarolus, Rubus ulmifolius. Five of the 51 prepared extracts from these plants showed an interesting inhibitory effect higher than 70% of at 100 μg/mL. Their IC50 values were found to be less than 100 μg/mL, with the best activity obtained with the methanolic stem extract from Crataegus azarolus (IC50 = 41,99 μg/mL) which constitutes an interesting value for an extract, if we consider the IC50 value of pure natural reference inhibitors; chlorogenic acid (3.75 μg/mL) or piceatannol (2.95 μg/mL) (Girard-Thernier et al. 2015). In order to establish a potential correlation between activity and polyphenolic content, we also determined the amount of phenols and flavonoids in these most active extracts. The results highlighted that methanolic extracts from Crataegus azarolus stems and from Rhus tripartita are also the richest in phenolic compounds, confirming the interest of this kind of secondary metabolites for arginase inhibition (Bordage et al. 2017; Minozzo et al. 2018). However, it also appears that flavonoid compounds constitute only a small part of the total phenolic content of these extracts and cannot consequently be considered as fully responsible for the observed activities. Other polyphenolic compounds are probably involved, which is not surprising regarding previous studies cited above (Girard-Thernier et al. 2015; Arraki et al. 2017). Crataegus azarolus, Retama Raetam and Rhus tripartita have not been extensively studied from a phytochemical point of view but a few
Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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studies have reported that these plants contain polyphenols, sometimes associated with other classes of secondary metabolites (Abu-Gharbieh and Shehab, 2017; Edziri et al. 2012; Kassem et al. 2000; Mustapha et al. 2015a; Shahat et al. 2016). Retama raetam, for example, is characterized by its richness in alkaloids (El-Shazly et al. 1996; Abdel-Halim et al., 1992; Abdel-Halim, 1995; Schmid et al. 2006). For Rhus tripartita the majority of compounds described in the literature, so far, belongs to flavonoids (Alam et al. 2017; Mahjoub et al., 2005; Shahat et al. 2016). Finally, in Crataegus azarolus, triterpens are present in large quantities (Mustapha et al. 2015b). This latter plant showed the best activity on the in vitro arginase inhibition test. Considering that an antihypertensive effect of the fruit of Crataegus azarolus has been recently demonstrated by Haydari et al. (2017), further analysis on this plant should be considered with the aim to isolate and evaluate its active secondary metabolites on arginase as well as to correlate their arginase inhibitory effect with a potential vasorelaxant effect. Finally, considering that polyphenols represent only a fraction of the active crude extracts, it could be interesting to isolate and evaluate other various classes of secondary metabolites in order to highlight new natural structures of inhibitors. 5. Conclusion In conclusion, our study identified several extracts as a new source of arginase inhibitors, in particular the methanolic extract from the stems of Crataegus azarolus which is the richest in polyphenol content. These results encourage further studies of these active extracts in order to identify the bioactive molecules responsible for arginase inhibition. Funding sources This work was financially supported by the “PHC Utique” program of the French Ministries of Foreign Affairs and of Higher Education, Research and Innovation and the Tunisian Ministry of higher education and scientific research in the CMCU project number 18G0816. The authors would like to thank the University of Carthage, the National Institute of Applied Science and Technology, Campus France and PEPITE EA4267 for their financial support, and Dr. Lucie Bernard for the check of the English language. References Abbassi, F., Hani, K., 2012. In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine. Natural Product Research 26, 2215–2218. Abdel Jaleel, G.A.R., Ibrahim Abdallah, H.M., Sayed Gomaa, N.E.L., 2016. Pharmacological effects of ethanol extract of Egyptian Artemisia herba-alba in rats and mice. Asian Pacific Journal of Tropical Biomedicine 6, 44–49. Abdel-Halim, O.B., 1995. (−)-6α-Hydroxylupanine, a lupin alkaloid from Lygos raetam var. sarcocarpa. Phytochemistry 40, 1323–1325. Abdel-Halim, O.B., Sekine, T., Saito, K., Halim, A.F., Abdel Fattah, H., Murakoshi, I., 1992. (+)-12α-Hydroxylupanine, a lupin alkaloid from Lygos raetam. Phytochemistry, The International Journal of Plant Biochemistry 31, 3251–3253. Abu-Gharbieh, E., Shehab, N.G., 2017. Therapeutic potentials of Crataegus azarolus var. Euazarolus Maire leaves and its isolated compounds. BMC Complementary and Alternative Medicine 17, 218. Akrout, A., Alarcon Gonzalez, L., El Jani, H., Campra Madrid, P., 2011. Antioxidant and antitumor activities of Artemisia campestris and Thymelaea hirsuta from Southern Tunisia. Food and Chemical Toxicology 49, 342–347. Al Shamaony, L., Al Khazraji, S.M., Twaij, H.A., 1994. Hypoglycaemic effect of Artemisia herba-alba. II. Effect of a valuable extract on some blood parameters in diabetic animals. Journal of Ethnopharmacology 43, 167–171. Al Snafi, A.E., 2015. The pharmacological importance of Artemisia campestris. Asian Journal of Pharmaceutical Research 5, 88–92. Alam, P., Parvez, M.K., Arbab, A.H., Siddiqui, N.A., Al-Dosary, M.S., Al-Rehaily, A.J., Ahmed, S., Abul Kalam, M., Ahmad, M.S., 2017. Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita. Pharmaceutical Biology 55, 1450–1457. Aleksic, V., Knezevic, P., 2014. Antimicrobial and antioxidative activity of extracts and essential oils of Myrtus Communis L. microbiological research. Medicinal Extracts in Microbiology 169, 240–254.
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Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022
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Please cite this article as: Attia, R., et al., Phytochemical screening and arginase inhibitory activity of extracts from several Tunisian medicinal plants, South African Journal of Botany (2018), https://doi.org/10.1016/j.sajb.2018.09.022