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Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur. a
a
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Khawla Kerbab , Tarak Mekhelfi , Lahcene Zaiter , Samir a
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Benayache , Fadila Benayache , Patrizia Picerno , Teresa b
b
b
b
Mencherini , Francesca Sansone , Rita P. Aquino & Luca Rastrelli a
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Unité de recherche Valorisation des Ressources Naturelles, Molécules Bioactives et Analyses Physicochimiques et Biologiques (VARENBIOMOL), Université Constantine 1, Route de Aïn El Bey, Constantine, Algérie b
Dipartimento di Farmacia, Università degli Studi di Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy Published online: 25 Nov 2014.
To cite this article: Khawla Kerbab, Tarak Mekhelfi, Lahcene Zaiter, Samir Benayache, Fadila Benayache, Patrizia Picerno, Teresa Mencherini, Francesca Sansone, Rita P. Aquino & Luca Rastrelli (2014): Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.979422 To link to this article: http://dx.doi.org/10.1080/14786419.2014.979422
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.979422
SHORT COMMUNICATION Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur.
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Khawla Kerbaba, Tarak Mekhelfia, Lahcene Zaitera, Samir Benayachea, Fadila Benayachea, Patrizia Picernob, Teresa Mencherinib*, Francesca Sansoneb, Rita P. Aquinob and Luca Rastrellib a
Unite´ de recherche Valorisation des Ressources Naturelles, Mole´cules Bioactives et Analyses Physicochimiques et Biologiques (VARENBIOMOL), Universite´ Constantine 1, Route de Aı¨n El Bey, Constantine, Alge´rie; bDipartimento di Farmacia, Universita` degli Studi di Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy (Received 21 July 2014; final version received 12 October 2014)
Thymelaea microphylla Coss. et Dur. (Thymelaeaceae) is a rare medicinal plant endemic to Algeria. In order to continue our studies on this species, herein we report the isolation and characterisation of 20 compounds from a hydroalcoholic extract (EtOH – H2O 7:3) of the aerial parts. They include monoterpene glucosides (1 – 3), phenolic acid derivatives (4, 8 and 9), phenylpropanoid glucosides (5 and 6), flavonoids (7, 10 and 11), a benzyl alcohol glucoside (12), ionol glucosides (13 – 16), lignans (17 – 19) and a bis-coumarin (20). All the structures were elucidated by spectroscopic methods including 1D and 2D NMR experiments, as well as ESI-MS analysis. Moreover, the extract of T. microphylla showed a significant and concentration-dependent free radical-scavenging activity in vitro, correlated to the presence of phenolic and chlorogenic acid derivatives (8, 9 and 4). Keywords: Thymelaea microphylla; polyphenols; lignans; ionol glucosides; monoterpenes; bis-coumarin; free radical-scavenging activity
1. Introduction Thymelaea is a genus (Thymelaeaceae family) comprising about 30 species of evergreen shrubs under the flowering plant family Thymelaeaceae. Thymelaea microphylla Coss. et Dur. is a rare medicinal species endemic to Algeria (Galicia-Herbada 2006). The leaves decoction is used in folk medicine to treat skin diseases and abdominal pain (Boukef 1986). Another species of
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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K. Kerbab et al.
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Thymelaea genus (Thymelaea hirsuta Endl.) is traditionally used for its several and welldocumented antiseptic, anti-inflammatory, antimelanogenesis and antihypertensive properties (Miyamae et al. 2009; Akrout et al. 2011; Trigui et al. 2013; Amari et al. 2014). Furthermore, the antioxidant properties of its aerial parts have recently been investigated (Amari et al. 2014). On the contrary, only few data dealing with the antibacterial and antioxidant activity of the extracts of T. microphylla are available (Djeridane et al. 2011; Ladjel et al. 2011). In order to continue our studies on this species (Mekhelfi et al. 2014), herein we report the isolation and characterisation of 20 compounds from a hydroalcoholic extract (EtOH –H2O 7:3) of the aerial parts. Moreover, the in vitro free radical-scavenging activity of the extract, major fractions and isolated phenolic compounds has also been investigated. 2. Results and discussion Aerial parts of T. microphylla were collected in April 2010 from M’Sila, east of Algeria. The plant material was identified by Professors Nadra Khalfallah (Constantine 1 University) and Mohamed Kaabache (Setif 1 University). A voucher specimen has been kept at the Herbarium of the VARENBIOMOL research unit, University of Constantine 1. Air-dried leaves and flowers of T. microphylla were macerated at room temperature with EtOH – H2O (7:3, v/v). After filtration, the filtrate was concentrated and dissolved in H2O. The resulting solution was successively extracted with chloroform, ethyl acetate and n-butanol. A part of the n-BuOH extract was chromatographed on a silica gel column followed by RP-HPLC to yield 20 known compounds (Figure 1). The chemical structures of the isolated compounds were identified by their NMR and MS data in comparison with those reported in the literature. They include three monoterpene
Figure 1. Structures of compounds 1 – 20 isolated from T. microphylla.
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glucosides, (3S,6R)-cis-linalool-3,7-oxide b-D -glucopyranoside (1) (Jiang et al. 2001), 3,7dimethyl-1-octene-3,6,7-triol-6-O-b-D -glucopyranoside (2) (Manns 1995; Yamamoto et al. 2008) and betulabuside B (3) (Kim et al. 2008; Park et al. 2010), three phenolic acid derivatives, chlorogenic acid butyl ester (4) (Osawa et al. 2001), protocatechuic acid (8) (Juliao L de S et al. 2010) and ethyl gallate (9) (Picerno et al. 2011), two phenylpropanoid glucosides, coniferin (5) (Huang et al. 2004) and syringin (6) (Kiem et al. 2003) and three flavonoids, yuankanin (7) (Ayers et al. 2008), kaempferol 3-O-{300 ,600 -di-O-[(E)-p-coumaroyl]-b-D -glucopyranoside} (10) and stenopalustroside A (11) (Liu et al. 1999). Moreover, a benzyl alcohol glucoside (12) (Seigler et al. 2002), four ionol glucosides, (E)-4-[300 -(b-D -glucopyranosyloxy)butylidene]3,5,5-trimethyl-2-cyclohexen-1-one (13) (Khan et al. 2003), 3-oxo-a-ionol-9-O-b-D -glucopyranoside (14) (De Tommasi et al. 1992; Sueyoshi et al. 2006), blumenol C-glucoside (15) (Matsunami et al. 2010) and vomifoliol-9-O-b-D -glucopyranoside (16) (De Tommasi et al. 1992; Sueyoshi et al. 2006), three lignans, phyllyrin (17) (Masataka & Masao 1991; Kwak et al. 2009), syringaresinol-4-O-b-D -glucopyranoside (18) (Gohari et al. 2011; Jung et al. 2004) and lariciresinol-400 -O-b-D -glucopyranoside (19) (Tanahashi et al. 1995; Shoeb et al. 2004) and a biscoumarin giraldoid A (20) (Li et al. 2005) were found. Several isolated compounds (1 –4 and 9 – 17) are new for the family Thymelaeaceae, and compounds 5 –8 and 18– 20 are isolated for the first time from the genus Thymelaea. The free radical-scavenging activity of the hydroalcoholic extract, fractions (I –VII) and phenolic compounds (4– 11 and 20) has been evaluated by the DPPH test (Herna´ndez et al. 2014). The extract showed a significant and concentration-dependent free radical-scavenging activity (EC50 ¼ 180.8 mg/mL). In comparison with the whole extract, fraction VI was more potent in the same test, suggesting that it contained a higher concentration of the active free radical-scavenging compounds. The activity of fraction VI (EC50 ¼ 11.2 mg/mL) was comparable to that of the well-known antioxidant, a-tocopherol (EC50 10.1 mg/mL), used as positive control. Instead, fractions III and V exhibited a moderate free radical-scavenging activity (EC50 ¼ 79.3 and 52.0 mg/mL, respectively), and fractions I, II, IV and VII resulted in less activity with EC50 values ranging from 115.0 to 157.0 mg/mL (Table 1).
Table 1. Free radical-scavenging activity (DPPH test) of T. microphylla extract, fractions and compounds. Extract, fractions and compounds Extract Fr. I Fr. II Fr. III Fr. IV Fr. V Fr. VI Fr. VII 4 5 6 8 9 10 11 20 a-Tocopherolb a b
EC50 ^ standard deviation (data from three experiments in triplicate). Positive control of the DPPH test.
DPPH test EC50 (mg/mL)a 180.8 ^ 3.0 115.0 ^ 3.4 117.6 ^ 2.9 79.3 ^ 2.5 157.0 ^ 3.6 52.0 ^ 2.1 11.2 ^ 0.5 150.8 ^ 1.8 16.3 ^ 1.1 111.6 ^ 1.9 107.0 ^ 1.5 5.4 ^ 0.1 1.9 ^ 0.1 119.7 ^ 3.8 109.1 ^ 2.9 76.7 ^ 2.2 10.1 ^ 1.3
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As is well known, phenolics are powerful antioxidant plant components. Their activity is mainly due to the redox properties, which can play an important role in neutralising free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Rice-Evans et al. 1995). In order to identify the compounds responsible for the observed activity of T. microphylla fractions, the free radical-scavenging activity of the main isolated phenolic compounds (4 –6, 8– 11 and 20) was evaluated. The strong activity of fraction VI seems to be correlated to the presence of protocatechuic acid (8) and ethyl gallate (9), which exhibited high potency in scavenging DPPH (EC50 ¼ 5.4 and 1.9 mg/mL, respectively) than the positive control, in agreement with the literature data (Kalaivani et al. 2011; Akihisa et al. 2013). Chlorogenic acid butyl ester (4), isolated from fraction V, was less active (EC50 ¼ 16.3 mg/mL) than the previous phenol compounds. Finally, the other tested compounds (5, 6, 10, 11 and 20) showed a weak free radical-scavenging activity in vitro.
3. Conclusions The extensive phytochemical investigation of a polar extract from the aerial parts of T. microphylla led to the isolation and characterisation of 20 compounds of various classes of phytoconstituents. Several isolated compounds (1 – 4 and 9 –17) are new for the family Thymelaeaceae, or found for the first time in the genus Thymelaea (5– 8 and 18 –20). Moreover, the whole extract showed an interesting free radical-scavenging activity in vitro correlated to the content of phenolic and chlorogenic acid derivatives (8, 9 and 4). In conclusion, this study provides a scientific background for the use of T. microphylla as an antioxidant medicinal plant in Algeria.
Supplementary material Experimental details relating to this paper are available online.
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur. a
a
a
Khawla Kerbab , Tarak Mekhelfi , Lahcene Zaiter , Samir a
a
b
Benayache , Fadila Benayache , Patrizia Picerno , Teresa b
b
b
b
Mencherini , Francesca Sansone , Rita P. Aquino & Luca Rastrelli a
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Unité de recherche Valorisation des Ressources Naturelles, Molécules Bioactives et Analyses Physicochimiques et Biologiques (VARENBIOMOL), Université Constantine 1, Route de Aïn El Bey, Constantine, Algérie b
Dipartimento di Farmacia, Università degli Studi di Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy Published online: 25 Nov 2014.
To cite this article: Khawla Kerbab, Tarak Mekhelfi, Lahcene Zaiter, Samir Benayache, Fadila Benayache, Patrizia Picerno, Teresa Mencherini, Francesca Sansone, Rita P. Aquino & Luca Rastrelli (2014): Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.979422 To link to this article: http://dx.doi.org/10.1080/14786419.2014.979422
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.979422
SHORT COMMUNICATION Chemical composition and antioxidant activity of a polar extract of Thymelaea microphylla Coss. et Dur.
Downloaded by [University of Saskatchewan Library] at 01:57 17 March 2015
Khawla Kerbaba, Tarak Mekhelfia, Lahcene Zaitera, Samir Benayachea, Fadila Benayachea, Patrizia Picernob, Teresa Mencherinib*, Francesca Sansoneb, Rita P. Aquinob and Luca Rastrellib a
Unite´ de recherche Valorisation des Ressources Naturelles, Mole´cules Bioactives et Analyses Physicochimiques et Biologiques (VARENBIOMOL), Universite´ Constantine 1, Route de Aı¨n El Bey, Constantine, Alge´rie; bDipartimento di Farmacia, Universita` degli Studi di Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy (Received 21 July 2014; final version received 12 October 2014)
Thymelaea microphylla Coss. et Dur. (Thymelaeaceae) is a rare medicinal plant endemic to Algeria. In order to continue our studies on this species, herein we report the isolation and characterisation of 20 compounds from a hydroalcoholic extract (EtOH – H2O 7:3) of the aerial parts. They include monoterpene glucosides (1 – 3), phenolic acid derivatives (4, 8 and 9), phenylpropanoid glucosides (5 and 6), flavonoids (7, 10 and 11), a benzyl alcohol glucoside (12), ionol glucosides (13 – 16), lignans (17 – 19) and a bis-coumarin (20). All the structures were elucidated by spectroscopic methods including 1D and 2D NMR experiments, as well as ESI-MS analysis. Moreover, the extract of T. microphylla showed a significant and concentration-dependent free radical-scavenging activity in vitro, correlated to the presence of phenolic and chlorogenic acid derivatives (8, 9 and 4). Keywords: Thymelaea microphylla; polyphenols; lignans; ionol glucosides; monoterpenes; bis-coumarin; free radical-scavenging activity
1. Introduction Thymelaea is a genus (Thymelaeaceae family) comprising about 30 species of evergreen shrubs under the flowering plant family Thymelaeaceae. Thymelaea microphylla Coss. et Dur. is a rare medicinal species endemic to Algeria (Galicia-Herbada 2006). The leaves decoction is used in folk medicine to treat skin diseases and abdominal pain (Boukef 1986). Another species of
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
2
K. Kerbab et al.
Downloaded by [University of Saskatchewan Library] at 01:57 17 March 2015
Thymelaea genus (Thymelaea hirsuta Endl.) is traditionally used for its several and welldocumented antiseptic, anti-inflammatory, antimelanogenesis and antihypertensive properties (Miyamae et al. 2009; Akrout et al. 2011; Trigui et al. 2013; Amari et al. 2014). Furthermore, the antioxidant properties of its aerial parts have recently been investigated (Amari et al. 2014). On the contrary, only few data dealing with the antibacterial and antioxidant activity of the extracts of T. microphylla are available (Djeridane et al. 2011; Ladjel et al. 2011). In order to continue our studies on this species (Mekhelfi et al. 2014), herein we report the isolation and characterisation of 20 compounds from a hydroalcoholic extract (EtOH –H2O 7:3) of the aerial parts. Moreover, the in vitro free radical-scavenging activity of the extract, major fractions and isolated phenolic compounds has also been investigated. 2. Results and discussion Aerial parts of T. microphylla were collected in April 2010 from M’Sila, east of Algeria. The plant material was identified by Professors Nadra Khalfallah (Constantine 1 University) and Mohamed Kaabache (Setif 1 University). A voucher specimen has been kept at the Herbarium of the VARENBIOMOL research unit, University of Constantine 1. Air-dried leaves and flowers of T. microphylla were macerated at room temperature with EtOH – H2O (7:3, v/v). After filtration, the filtrate was concentrated and dissolved in H2O. The resulting solution was successively extracted with chloroform, ethyl acetate and n-butanol. A part of the n-BuOH extract was chromatographed on a silica gel column followed by RP-HPLC to yield 20 known compounds (Figure 1). The chemical structures of the isolated compounds were identified by their NMR and MS data in comparison with those reported in the literature. They include three monoterpene
Figure 1. Structures of compounds 1 – 20 isolated from T. microphylla.
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glucosides, (3S,6R)-cis-linalool-3,7-oxide b-D -glucopyranoside (1) (Jiang et al. 2001), 3,7dimethyl-1-octene-3,6,7-triol-6-O-b-D -glucopyranoside (2) (Manns 1995; Yamamoto et al. 2008) and betulabuside B (3) (Kim et al. 2008; Park et al. 2010), three phenolic acid derivatives, chlorogenic acid butyl ester (4) (Osawa et al. 2001), protocatechuic acid (8) (Juliao L de S et al. 2010) and ethyl gallate (9) (Picerno et al. 2011), two phenylpropanoid glucosides, coniferin (5) (Huang et al. 2004) and syringin (6) (Kiem et al. 2003) and three flavonoids, yuankanin (7) (Ayers et al. 2008), kaempferol 3-O-{300 ,600 -di-O-[(E)-p-coumaroyl]-b-D -glucopyranoside} (10) and stenopalustroside A (11) (Liu et al. 1999). Moreover, a benzyl alcohol glucoside (12) (Seigler et al. 2002), four ionol glucosides, (E)-4-[300 -(b-D -glucopyranosyloxy)butylidene]3,5,5-trimethyl-2-cyclohexen-1-one (13) (Khan et al. 2003), 3-oxo-a-ionol-9-O-b-D -glucopyranoside (14) (De Tommasi et al. 1992; Sueyoshi et al. 2006), blumenol C-glucoside (15) (Matsunami et al. 2010) and vomifoliol-9-O-b-D -glucopyranoside (16) (De Tommasi et al. 1992; Sueyoshi et al. 2006), three lignans, phyllyrin (17) (Masataka & Masao 1991; Kwak et al. 2009), syringaresinol-4-O-b-D -glucopyranoside (18) (Gohari et al. 2011; Jung et al. 2004) and lariciresinol-400 -O-b-D -glucopyranoside (19) (Tanahashi et al. 1995; Shoeb et al. 2004) and a biscoumarin giraldoid A (20) (Li et al. 2005) were found. Several isolated compounds (1 –4 and 9 – 17) are new for the family Thymelaeaceae, and compounds 5 –8 and 18– 20 are isolated for the first time from the genus Thymelaea. The free radical-scavenging activity of the hydroalcoholic extract, fractions (I –VII) and phenolic compounds (4– 11 and 20) has been evaluated by the DPPH test (Herna´ndez et al. 2014). The extract showed a significant and concentration-dependent free radical-scavenging activity (EC50 ¼ 180.8 mg/mL). In comparison with the whole extract, fraction VI was more potent in the same test, suggesting that it contained a higher concentration of the active free radical-scavenging compounds. The activity of fraction VI (EC50 ¼ 11.2 mg/mL) was comparable to that of the well-known antioxidant, a-tocopherol (EC50 10.1 mg/mL), used as positive control. Instead, fractions III and V exhibited a moderate free radical-scavenging activity (EC50 ¼ 79.3 and 52.0 mg/mL, respectively), and fractions I, II, IV and VII resulted in less activity with EC50 values ranging from 115.0 to 157.0 mg/mL (Table 1).
Table 1. Free radical-scavenging activity (DPPH test) of T. microphylla extract, fractions and compounds. Extract, fractions and compounds Extract Fr. I Fr. II Fr. III Fr. IV Fr. V Fr. VI Fr. VII 4 5 6 8 9 10 11 20 a-Tocopherolb a b
EC50 ^ standard deviation (data from three experiments in triplicate). Positive control of the DPPH test.
DPPH test EC50 (mg/mL)a 180.8 ^ 3.0 115.0 ^ 3.4 117.6 ^ 2.9 79.3 ^ 2.5 157.0 ^ 3.6 52.0 ^ 2.1 11.2 ^ 0.5 150.8 ^ 1.8 16.3 ^ 1.1 111.6 ^ 1.9 107.0 ^ 1.5 5.4 ^ 0.1 1.9 ^ 0.1 119.7 ^ 3.8 109.1 ^ 2.9 76.7 ^ 2.2 10.1 ^ 1.3
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K. Kerbab et al.
As is well known, phenolics are powerful antioxidant plant components. Their activity is mainly due to the redox properties, which can play an important role in neutralising free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Rice-Evans et al. 1995). In order to identify the compounds responsible for the observed activity of T. microphylla fractions, the free radical-scavenging activity of the main isolated phenolic compounds (4 –6, 8– 11 and 20) was evaluated. The strong activity of fraction VI seems to be correlated to the presence of protocatechuic acid (8) and ethyl gallate (9), which exhibited high potency in scavenging DPPH (EC50 ¼ 5.4 and 1.9 mg/mL, respectively) than the positive control, in agreement with the literature data (Kalaivani et al. 2011; Akihisa et al. 2013). Chlorogenic acid butyl ester (4), isolated from fraction V, was less active (EC50 ¼ 16.3 mg/mL) than the previous phenol compounds. Finally, the other tested compounds (5, 6, 10, 11 and 20) showed a weak free radical-scavenging activity in vitro.
3. Conclusions The extensive phytochemical investigation of a polar extract from the aerial parts of T. microphylla led to the isolation and characterisation of 20 compounds of various classes of phytoconstituents. Several isolated compounds (1 – 4 and 9 –17) are new for the family Thymelaeaceae, or found for the first time in the genus Thymelaea (5– 8 and 18 –20). Moreover, the whole extract showed an interesting free radical-scavenging activity in vitro correlated to the content of phenolic and chlorogenic acid derivatives (8, 9 and 4). In conclusion, this study provides a scientific background for the use of T. microphylla as an antioxidant medicinal plant in Algeria.
Supplementary material Experimental details relating to this paper are available online.
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