Phytochemistry 117 (2015) 340–350
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Natural lignans from Arctium lappa as antiaging agents in Caenorhabditis elegans Shan Su, Michael Wink ⇑ Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
a r t i c l e
i n f o
Article history: Received 27 January 2015 Received in revised form 15 June 2015 Accepted 22 June 2015
Keywords: Arctium lappa Lignans Caenorhabditis elegans Antiaging agent Free radical scavenger DAF-16
a b s t r a c t Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites, which have a wide range of bioactivities. Yet, their antiaging potential has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The antioxidant and antiaging properties of the isolated lignans were studied using Caenorhabditis elegans as a relevant animal model. All lignans at concentrations of 10 and 100 lM significantly extended the mean life span of C. elegans. The strongest effect was observed with matairesinol, which at a concentration of 100 lM extended the life span of worms by 25%. Additionally, we observed that five lignans are strong free radical-scavengers in vitro and in vivo and all lignans can improve survival of C. elegans under oxidative stress. Furthermore, the lignans can induce the nuclear translocation of the transcription factor DAF-16 and up-regulate its expression, suggesting that a possible underlying mechanism of the observed longevity-promoting activity of lignans depends on DAF-16 mediated signaling pathway. All lignans up-regulated the expression of jnk-1, indicating that lignans may promote the C. elegans longevity and stress resistance through a JNK-1-DAF-16 cascade. Our study reports new antiaging activities of lignans, which might be candidates for developing antiaging agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aging is an inevitable biological process, which can be defined as a progressive decline in physiological capacities accompanied by an increased vulnerability to environmental challenges and aging-related diseases (Holliday, 2004). Although the exact biological and cellular mechanisms of the aging process are not well understood, a large body of evidence indicates that free radical-induced oxidative damage of cellular components plays a key role in aging and aging-related diseases (Beckman and Ames, 1998; Cadenas and Davies, 2000; Varadarajan et al., 2000). Production of free radicals is an unavoidable process in the course of cellular Abbreviations: DCF, 20 ,70 -dichlorofluorescein; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EGCG, epigallocatechin gallate; FOXO, Forkhead box O; GFP, green fluorescent protein; H2DCF, 2,7-dichlorodihydrofluorescein; HPLC, high performance liquid chromatography; IGF-1, insulin/insulin-like growth factor; IIS, insulin/insulin-like growth factor signaling; MS, mass spectrometry; NGM, nematode growth medium; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; ROS, reactive oxygen species; TCM, Traditional Chinese Medicine. ⇑ Corresponding author. E-mail address: [email protected] (M. Wink). http://dx.doi.org/10.1016/j.phytochem.2015.06.021 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
metabolism. For example, reactive oxygen species (ROS) are wellknown byproducts of ATP production via mitochondrial respiration (Getoff, 2007). Cells need a certain level of ROS and their concentrations are usually fine-tuned in vivo, However, overproduction and overaccumulation of ROS can lead to DNA damage (mutations), lipid peroxidation and protein oxidation, which are commonly implicated in aging and aging-related diseases, such as cancer, neurodegenerative and circulation disorders (Bergamini et al., 2004). As such, the scavenging of detrimental free radicals by antioxidants may alleviate oxidative damage of cells, therefore promoting longevity and preventing aging-related disorders. Medicinal plants provide a high diversity of natural products, which can be exploited for potential antiaging agents. Previous studies have successfully identified several natural antioxidants that have promising antiaging capacities, such as resveratrol (Chen et al., 2013b), epigallocatechin gallate (Abbas and Wink, 2009) and quercetin (Kampkotter et al., 2008). Arctium lappa, commonly known as burdock, is an important medicinal plant in China (TCM) and Europe that has been used to treat sore throat, skin infections and to alleviate rheumatic pain and fever for thousands
S. Su, M. Wink / Phytochemistry 117 (2015) 340–350
of years (Van Wyk and Wink, 2004). Nowadays it continues to serve as a valuable source for secondary metabolites that can be explored for new biological and pharmacological applications. Particularly, A. lappa is a rich source of bioactive lignans. At least 24 lignans have been isolated and identified from its seed and fruit extracts (Umehara et al., 1993). Lignans comprise a large group of natural compounds that mostly consists of two phenylpropanoid moieties connected via C8-C80 at their side chain or by additional ether, lactone, or carbon bonds (Laurence and Davin, 2003). A large body of research has focused on the pharmacological and biological properties of natural lignans in A. lappa. Emerging evidence has shown that lignans have anticancer, anti-inflammatory, antidiabetic, antimicrobial and antiviral properties. For example, arctigenin, one of the major bioactive lignans in A. lappa, has strong antiproliferative and apoptotic effects against different cancer cell lines (Ryu et al., 1995). Besides, arctigenin and arctiin have potent anti-inflammatory effects via inhibiting lipopolysaccharide-induced nitric oxide (NO) production and the release of pro-inflammatory cytokines in murine macrophages (Kou et al., 2011; Lee et al., 2011). However, a possible antiaging potential of the bioactive lignans from A. lappa has not been reported. The nematode Caenorhabditis elegans is an important model organism to study aging and oxidative stress-related diseases, because it has a rapid reproduction rate and a short life span, and most importantly, the major signaling pathways that regulate longevity and stress resistance in mammals are well conserved in C. elegans (Pinero Gonzalez et al., 2009). The evolutionary conserved insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway is one of the well-understood longevity-regulating pathways in animals. In this pathway, the Forkhead box O (FOXO) transcription factors are key players, which are under control of IGF-1 receptors. In C. elegans, similar to other systems, activation of DAF2, the worm homolog of IGF-1 receptor, recruits and activates the phosphoinositide 3-kinase/protein kinase B signaling cascade, which in turn results in phosphorylation of DAF-16, the single FOXO transcription factor in the worms. Being phosphorylated, DAF-16 is prevented from its nuclear translocation and the induction of gene expression of downstream longevity-promoting genes (Accili and Arden, 2004). On the contrary, inactivation of IIS pathway by using genetic and pharmacological approaches has been shown to significantly increase the life span of C. elegans and elevate the worm resistance to different stressors (Accili and Arden, 2004). Besides the IIS pathway, the Jun-N-terminal kinase-1 (JNK-1) signaling pathway is another important upstream pathway that can regulate the DAF-16 nuclear localization by alternating the phosphorylation status of DAF-16 (Oh et al., 2005). Once in the nucleus, several proteins cooperate with DAF-16 to modulate downstream gene transcription. Among these co-regulators, the heat-shock-factor-1 (HSF-1) acts together with DAF-16 to activate expression of specific genes, such as genes encoding small heatshock proteins, which enhance stress resistance and longevity in C. elegans (Hsu et al., 2003). Additionally, the p38 mitogen-activated protein kinase (PMK-1), a parallel pathway to DAF-16, also contributes to longevity of C. elegans (Troemel et al., 2006). In this study, six natural lignans were isolated and identified from the seed extracts of A. lappa, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F. We individually investigated their antiaging potential using C. elegans. We report here for the first time that the major lignans from A. lappa can significantly extend the life span of C. elegans under normal conditions and promote the survival of the worms under oxidative stress. The possible mechanism of longevity-promoting effects of the lignans on C. elegans has also been investigated. This study reports novel antiaging capabilities of natural lignans isolated from A. lappa and suggests a novel bioactivity for this medicinally important plant.
341
2. Results 2.1. The isolated lignans from A. lappa extended the mean life span of wild type C. elegans We have successfully isolated six lignans from the extract of A. lappa seeds, namely as arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F (Fig. 1). Their structures were identified by 1 H-NMR, 13C-NMR, EI-MS and ESI-MS analyses (Supplementary data). To study whether the isolated lignans have potential antiaging effects, we investigated their potential to extend life span of the wild type C. elegans under normal conditions. As shown in Table 1 and Fig. 2, all lignans significantly increased the mean life span of the N2 worms in a concentration-dependent manner compared with the control group. Among these lignans, matairesinol exerted the strongest life span-prolonging activity. At 10 lM and 100 lM concentrations, matairesinol significantly prolonged the mean life span of the worms by 14.0% and 25.0%, respectively (P < 0.001). At a moderate concentration of 100 lM, arctigenin, arctiin and (iso)lappaol A, lappaol C, lappaol F increased the mean life span of the worms by 13.7%, 15.3%, 11.2%, 11.5% and 12.5%, respectively (P < 0.05) (Table 1). We observed that the bacteria mass on top of the agar plates remained the same when we performed the lifespan experiments. Additionally, the minimal inhibitory concentration (MIC) values of all lignans are higher than 2000 lM, both after treatment of 24 h and 48 h at 20 °C. However the possibility that the living Escherichia coli may have an effect on the lifespan of C. elegans was not completely ruled out. 2.2. Antioxidant activities of the isolated lignans in vitro and in vivo Because a large body of research has clearly shown that the longevity-promoting capabilities of many natural products are related to their free radical-scavenging activities (Harrington and Harley, 1988), we were interested to know whether these natural lignans are also active in scavenging free radicals. For this purpose, we examined their free radical-scavenging activities by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay, in which quenching DPPH free radical is used as an indicator for the free radical-scavenging capabilities of the lignans (Eklund et al., 2005). As a result, five lignans (except arctiin) were found to have strong activities in scavenging DPPH free radicals with low IC50 values (Fig. 5A). The data indicate that all the lignans except arctiin are powerful in vitro free radical scavengers. To assess whether the isolated lignans exhibit in vivo antioxidant activities, we measured endogenous ROS levels in the lignans-treated worms by using H2DCF-DA as an indicator for intracellular ROS. Pre-treatment with arctigenin, matairesinol, arctiin, (iso)lappaol A and lappaol F (100 lM each) significantly attenuated ROS levels in the worms by 19.20% (P < 0.0001), 21.33% (P = 0.0093), 18.58% (P = 0.0261), 11.03% (P = 0.0038) and 13.97% (P = 0.0047), respectively. Additionally, arctigenin, arctiin, (iso)lappaol A and lappaol C of 10 lM markedly decreased ROS level in the worms by 12.53% (P = 0.0217), 14.35% (P = 0.0040), 12.13% (P = 0.0114) and 16.71% (P = 0.0026), respectively (Fig. 5B). However the highest concentration of lignans (200 lM) failed to further decrease the ROS level in the worms, possibly due to their toxicity to the worms. These results indicate that the isolated lignans at moderate concentrations have in vivo antioxidant activities by decreasing intracellular ROS level in C. elegans. 2.3. Lignans increase the resistance of nematodes to oxidative stress To test whether the antioxidant lignans protect the worms against oxidative stress, we further assessed the effects of the
342
S. Su, M. Wink / Phytochemistry 117 (2015) 340–350
Fig. 1. Chemical structures of the lignans isolated from A. lappa.
lignans on the survival of the worms that were exposed to oxidative stress. 2-Day-old wild type worms were pre-treated with different concentrations of lignans (50 lM, 100 lM and 200 lM), followed by the exposure to 80 lM freshly prepared juglone, which is a strong oxidant that can produce lethal oxidative stress. After the exposure to juglone for 24 h, only 29.50 ± 2.63% of the untreated worms were alive. In contrast, the pretreatments with lignans significantly increased the survival rate of the worms. Notably, treatments with 100 lM arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F caused a survival of the worms to 67.00 ± 5.13% (P = 0.0009), 62.33 ± 7.53% (P = 0.0055), 43.00 ± 1.53% (P = 0.0102), 64.33 ± 6.01% (P = 0.0020), 45.67 ± 4.48% (P = 0.0211), 63.33 ± 6.77% (P = 0.0034), respectively (Fig. 6). Apparently, the treatments with 200 lM lignans did not further increase the survival rate of the worms under oxidative stress, possibly due to their own toxicity. Because optimal survival and extended life span were recorded in worms treated with lignans at 100 lM, this concentration was selected as the working concentration in further experiments. Our results clearly indicated that the lignans strongly protect C. elegans from oxidative stress. 2.4. Stress-induced expression of HSP-16.2/GFP was suppressed in the transgenic C. elegans pre-treated with lignans The small heat shock protein hsp-16.2 is known as a sensitive stress sensor in C. elegans, in which its expression can be dramatically induced by thermal or oxidative stressors. To further evaluate the protective activities of the lignans against oxidative stress, we analyzed the effects of the lignans on the expression of hsp-16.2 (coupled to GFP) in a transgenic worm strain TJ375. The expression of hsp-16.2 can be quantitatively measured by the GFP fluorescence in the head of the worms. Under the exposure to the pro-oxidant juglone, a strong GFP fluorescence was clearly visible, indicating a high expression of hsp-16.2. However, hsp-16.2 expression under juglone-induced oxidative stress was markedly
suppressed in worms pre-treated with 100 lM lignans (Fig. 7) as well as with 200 lM EGCG as a positive control. By measuring the fluorescence intensity, we found that treatments with arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, lappaol F (100 lM each) significantly attenuated the juglone-induced expression of hsp-16-2/GFP by 31.67% (P = 0.0205), 45.09% (P = 0.0027), 36.94% (P = 0.0003), 34.22% (P = 0.0109), 26.39% (P = 0.0173), 19.51% (P = 0.0180), respectively. This result suggests that the lignans are apparently taken up and exhibit in vivo antioxidant activities to reduce the burden of oxidative stress in the worms. 2.5. Life span extension by lignans involves the daf-16-related signaling pathway To explore the possible mechanism by which lignans can extend life span in C. elegans, we first tested their effects on mev-1 mutant worms, which are short-lived due to a high endogenous oxidative flux (Ishii et al., 1998). We found that none of the lignans can extend the life span of this mutant worms (Table 1 and Fig. 4), indicating that endogenous signaling pathways rather than the antioxidative activity alone are required to promote longevity. We then focused on the insulin/insulin-like growth factor-1 (IGF1) signaling (IIS) pathway, in which the translocation of the FOXO transcription factor DAF-16 into the nucleus is believed to modulate stress response and longevity of the worms (Accili and Arden, 2004). Due to the important role of DAF-16 in this pathway, we assessed the effects of the lignans on the life span of a mutant GR 1307 strain, which lacks of DAF-16 (daf-16(mgDf50)). As a result, all lignans failed to extend the life span of this mutant (Table 1 and Fig. 3). Taken together with the results on the wild-type worms, our data strongly suggest that DAF-16 is indispensable for the life span-extending effects of the lignans. In addition, we examined the effect of lignans on the subcellular localization of DAF-16 in daf16::GFP transgenic worms (TJ356), in which GFP is integrated with
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S. Su, M. Wink / Phytochemistry 117 (2015) 340–350 Table 1 Effect of lignans on the life span of different strains of C. elegans. Strain
Treatment (lM)
Mean life span (day) ± SE
N2
Control
11.36 ± 0.34
N2
Arctigenin (10)
12.12 ± 0.35
6.7
0.0121
5
N2
Arctigenin (100)
12.93 ± 0.40
13.7
0.0121
2
N2
Matairesinol (10)
12.95 ± 0.42
14.0
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Natural lignans from Arctium lappa as antiaging agents in Caenorhabditis elegans Shan Su, Michael Wink ⇑ Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
a r t i c l e
i n f o
Article history: Received 27 January 2015 Received in revised form 15 June 2015 Accepted 22 June 2015
Keywords: Arctium lappa Lignans Caenorhabditis elegans Antiaging agent Free radical scavenger DAF-16
a b s t r a c t Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites, which have a wide range of bioactivities. Yet, their antiaging potential has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The antioxidant and antiaging properties of the isolated lignans were studied using Caenorhabditis elegans as a relevant animal model. All lignans at concentrations of 10 and 100 lM significantly extended the mean life span of C. elegans. The strongest effect was observed with matairesinol, which at a concentration of 100 lM extended the life span of worms by 25%. Additionally, we observed that five lignans are strong free radical-scavengers in vitro and in vivo and all lignans can improve survival of C. elegans under oxidative stress. Furthermore, the lignans can induce the nuclear translocation of the transcription factor DAF-16 and up-regulate its expression, suggesting that a possible underlying mechanism of the observed longevity-promoting activity of lignans depends on DAF-16 mediated signaling pathway. All lignans up-regulated the expression of jnk-1, indicating that lignans may promote the C. elegans longevity and stress resistance through a JNK-1-DAF-16 cascade. Our study reports new antiaging activities of lignans, which might be candidates for developing antiaging agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aging is an inevitable biological process, which can be defined as a progressive decline in physiological capacities accompanied by an increased vulnerability to environmental challenges and aging-related diseases (Holliday, 2004). Although the exact biological and cellular mechanisms of the aging process are not well understood, a large body of evidence indicates that free radical-induced oxidative damage of cellular components plays a key role in aging and aging-related diseases (Beckman and Ames, 1998; Cadenas and Davies, 2000; Varadarajan et al., 2000). Production of free radicals is an unavoidable process in the course of cellular Abbreviations: DCF, 20 ,70 -dichlorofluorescein; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EGCG, epigallocatechin gallate; FOXO, Forkhead box O; GFP, green fluorescent protein; H2DCF, 2,7-dichlorodihydrofluorescein; HPLC, high performance liquid chromatography; IGF-1, insulin/insulin-like growth factor; IIS, insulin/insulin-like growth factor signaling; MS, mass spectrometry; NGM, nematode growth medium; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; ROS, reactive oxygen species; TCM, Traditional Chinese Medicine. ⇑ Corresponding author. E-mail address: [email protected] (M. Wink). http://dx.doi.org/10.1016/j.phytochem.2015.06.021 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
metabolism. For example, reactive oxygen species (ROS) are wellknown byproducts of ATP production via mitochondrial respiration (Getoff, 2007). Cells need a certain level of ROS and their concentrations are usually fine-tuned in vivo, However, overproduction and overaccumulation of ROS can lead to DNA damage (mutations), lipid peroxidation and protein oxidation, which are commonly implicated in aging and aging-related diseases, such as cancer, neurodegenerative and circulation disorders (Bergamini et al., 2004). As such, the scavenging of detrimental free radicals by antioxidants may alleviate oxidative damage of cells, therefore promoting longevity and preventing aging-related disorders. Medicinal plants provide a high diversity of natural products, which can be exploited for potential antiaging agents. Previous studies have successfully identified several natural antioxidants that have promising antiaging capacities, such as resveratrol (Chen et al., 2013b), epigallocatechin gallate (Abbas and Wink, 2009) and quercetin (Kampkotter et al., 2008). Arctium lappa, commonly known as burdock, is an important medicinal plant in China (TCM) and Europe that has been used to treat sore throat, skin infections and to alleviate rheumatic pain and fever for thousands
S. Su, M. Wink / Phytochemistry 117 (2015) 340–350
of years (Van Wyk and Wink, 2004). Nowadays it continues to serve as a valuable source for secondary metabolites that can be explored for new biological and pharmacological applications. Particularly, A. lappa is a rich source of bioactive lignans. At least 24 lignans have been isolated and identified from its seed and fruit extracts (Umehara et al., 1993). Lignans comprise a large group of natural compounds that mostly consists of two phenylpropanoid moieties connected via C8-C80 at their side chain or by additional ether, lactone, or carbon bonds (Laurence and Davin, 2003). A large body of research has focused on the pharmacological and biological properties of natural lignans in A. lappa. Emerging evidence has shown that lignans have anticancer, anti-inflammatory, antidiabetic, antimicrobial and antiviral properties. For example, arctigenin, one of the major bioactive lignans in A. lappa, has strong antiproliferative and apoptotic effects against different cancer cell lines (Ryu et al., 1995). Besides, arctigenin and arctiin have potent anti-inflammatory effects via inhibiting lipopolysaccharide-induced nitric oxide (NO) production and the release of pro-inflammatory cytokines in murine macrophages (Kou et al., 2011; Lee et al., 2011). However, a possible antiaging potential of the bioactive lignans from A. lappa has not been reported. The nematode Caenorhabditis elegans is an important model organism to study aging and oxidative stress-related diseases, because it has a rapid reproduction rate and a short life span, and most importantly, the major signaling pathways that regulate longevity and stress resistance in mammals are well conserved in C. elegans (Pinero Gonzalez et al., 2009). The evolutionary conserved insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway is one of the well-understood longevity-regulating pathways in animals. In this pathway, the Forkhead box O (FOXO) transcription factors are key players, which are under control of IGF-1 receptors. In C. elegans, similar to other systems, activation of DAF2, the worm homolog of IGF-1 receptor, recruits and activates the phosphoinositide 3-kinase/protein kinase B signaling cascade, which in turn results in phosphorylation of DAF-16, the single FOXO transcription factor in the worms. Being phosphorylated, DAF-16 is prevented from its nuclear translocation and the induction of gene expression of downstream longevity-promoting genes (Accili and Arden, 2004). On the contrary, inactivation of IIS pathway by using genetic and pharmacological approaches has been shown to significantly increase the life span of C. elegans and elevate the worm resistance to different stressors (Accili and Arden, 2004). Besides the IIS pathway, the Jun-N-terminal kinase-1 (JNK-1) signaling pathway is another important upstream pathway that can regulate the DAF-16 nuclear localization by alternating the phosphorylation status of DAF-16 (Oh et al., 2005). Once in the nucleus, several proteins cooperate with DAF-16 to modulate downstream gene transcription. Among these co-regulators, the heat-shock-factor-1 (HSF-1) acts together with DAF-16 to activate expression of specific genes, such as genes encoding small heatshock proteins, which enhance stress resistance and longevity in C. elegans (Hsu et al., 2003). Additionally, the p38 mitogen-activated protein kinase (PMK-1), a parallel pathway to DAF-16, also contributes to longevity of C. elegans (Troemel et al., 2006). In this study, six natural lignans were isolated and identified from the seed extracts of A. lappa, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F. We individually investigated their antiaging potential using C. elegans. We report here for the first time that the major lignans from A. lappa can significantly extend the life span of C. elegans under normal conditions and promote the survival of the worms under oxidative stress. The possible mechanism of longevity-promoting effects of the lignans on C. elegans has also been investigated. This study reports novel antiaging capabilities of natural lignans isolated from A. lappa and suggests a novel bioactivity for this medicinally important plant.
341
2. Results 2.1. The isolated lignans from A. lappa extended the mean life span of wild type C. elegans We have successfully isolated six lignans from the extract of A. lappa seeds, namely as arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F (Fig. 1). Their structures were identified by 1 H-NMR, 13C-NMR, EI-MS and ESI-MS analyses (Supplementary data). To study whether the isolated lignans have potential antiaging effects, we investigated their potential to extend life span of the wild type C. elegans under normal conditions. As shown in Table 1 and Fig. 2, all lignans significantly increased the mean life span of the N2 worms in a concentration-dependent manner compared with the control group. Among these lignans, matairesinol exerted the strongest life span-prolonging activity. At 10 lM and 100 lM concentrations, matairesinol significantly prolonged the mean life span of the worms by 14.0% and 25.0%, respectively (P < 0.001). At a moderate concentration of 100 lM, arctigenin, arctiin and (iso)lappaol A, lappaol C, lappaol F increased the mean life span of the worms by 13.7%, 15.3%, 11.2%, 11.5% and 12.5%, respectively (P < 0.05) (Table 1). We observed that the bacteria mass on top of the agar plates remained the same when we performed the lifespan experiments. Additionally, the minimal inhibitory concentration (MIC) values of all lignans are higher than 2000 lM, both after treatment of 24 h and 48 h at 20 °C. However the possibility that the living Escherichia coli may have an effect on the lifespan of C. elegans was not completely ruled out. 2.2. Antioxidant activities of the isolated lignans in vitro and in vivo Because a large body of research has clearly shown that the longevity-promoting capabilities of many natural products are related to their free radical-scavenging activities (Harrington and Harley, 1988), we were interested to know whether these natural lignans are also active in scavenging free radicals. For this purpose, we examined their free radical-scavenging activities by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay, in which quenching DPPH free radical is used as an indicator for the free radical-scavenging capabilities of the lignans (Eklund et al., 2005). As a result, five lignans (except arctiin) were found to have strong activities in scavenging DPPH free radicals with low IC50 values (Fig. 5A). The data indicate that all the lignans except arctiin are powerful in vitro free radical scavengers. To assess whether the isolated lignans exhibit in vivo antioxidant activities, we measured endogenous ROS levels in the lignans-treated worms by using H2DCF-DA as an indicator for intracellular ROS. Pre-treatment with arctigenin, matairesinol, arctiin, (iso)lappaol A and lappaol F (100 lM each) significantly attenuated ROS levels in the worms by 19.20% (P < 0.0001), 21.33% (P = 0.0093), 18.58% (P = 0.0261), 11.03% (P = 0.0038) and 13.97% (P = 0.0047), respectively. Additionally, arctigenin, arctiin, (iso)lappaol A and lappaol C of 10 lM markedly decreased ROS level in the worms by 12.53% (P = 0.0217), 14.35% (P = 0.0040), 12.13% (P = 0.0114) and 16.71% (P = 0.0026), respectively (Fig. 5B). However the highest concentration of lignans (200 lM) failed to further decrease the ROS level in the worms, possibly due to their toxicity to the worms. These results indicate that the isolated lignans at moderate concentrations have in vivo antioxidant activities by decreasing intracellular ROS level in C. elegans. 2.3. Lignans increase the resistance of nematodes to oxidative stress To test whether the antioxidant lignans protect the worms against oxidative stress, we further assessed the effects of the
342
S. Su, M. Wink / Phytochemistry 117 (2015) 340–350
Fig. 1. Chemical structures of the lignans isolated from A. lappa.
lignans on the survival of the worms that were exposed to oxidative stress. 2-Day-old wild type worms were pre-treated with different concentrations of lignans (50 lM, 100 lM and 200 lM), followed by the exposure to 80 lM freshly prepared juglone, which is a strong oxidant that can produce lethal oxidative stress. After the exposure to juglone for 24 h, only 29.50 ± 2.63% of the untreated worms were alive. In contrast, the pretreatments with lignans significantly increased the survival rate of the worms. Notably, treatments with 100 lM arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C and lappaol F caused a survival of the worms to 67.00 ± 5.13% (P = 0.0009), 62.33 ± 7.53% (P = 0.0055), 43.00 ± 1.53% (P = 0.0102), 64.33 ± 6.01% (P = 0.0020), 45.67 ± 4.48% (P = 0.0211), 63.33 ± 6.77% (P = 0.0034), respectively (Fig. 6). Apparently, the treatments with 200 lM lignans did not further increase the survival rate of the worms under oxidative stress, possibly due to their own toxicity. Because optimal survival and extended life span were recorded in worms treated with lignans at 100 lM, this concentration was selected as the working concentration in further experiments. Our results clearly indicated that the lignans strongly protect C. elegans from oxidative stress. 2.4. Stress-induced expression of HSP-16.2/GFP was suppressed in the transgenic C. elegans pre-treated with lignans The small heat shock protein hsp-16.2 is known as a sensitive stress sensor in C. elegans, in which its expression can be dramatically induced by thermal or oxidative stressors. To further evaluate the protective activities of the lignans against oxidative stress, we analyzed the effects of the lignans on the expression of hsp-16.2 (coupled to GFP) in a transgenic worm strain TJ375. The expression of hsp-16.2 can be quantitatively measured by the GFP fluorescence in the head of the worms. Under the exposure to the pro-oxidant juglone, a strong GFP fluorescence was clearly visible, indicating a high expression of hsp-16.2. However, hsp-16.2 expression under juglone-induced oxidative stress was markedly
suppressed in worms pre-treated with 100 lM lignans (Fig. 7) as well as with 200 lM EGCG as a positive control. By measuring the fluorescence intensity, we found that treatments with arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, lappaol F (100 lM each) significantly attenuated the juglone-induced expression of hsp-16-2/GFP by 31.67% (P = 0.0205), 45.09% (P = 0.0027), 36.94% (P = 0.0003), 34.22% (P = 0.0109), 26.39% (P = 0.0173), 19.51% (P = 0.0180), respectively. This result suggests that the lignans are apparently taken up and exhibit in vivo antioxidant activities to reduce the burden of oxidative stress in the worms. 2.5. Life span extension by lignans involves the daf-16-related signaling pathway To explore the possible mechanism by which lignans can extend life span in C. elegans, we first tested their effects on mev-1 mutant worms, which are short-lived due to a high endogenous oxidative flux (Ishii et al., 1998). We found that none of the lignans can extend the life span of this mutant worms (Table 1 and Fig. 4), indicating that endogenous signaling pathways rather than the antioxidative activity alone are required to promote longevity. We then focused on the insulin/insulin-like growth factor-1 (IGF1) signaling (IIS) pathway, in which the translocation of the FOXO transcription factor DAF-16 into the nucleus is believed to modulate stress response and longevity of the worms (Accili and Arden, 2004). Due to the important role of DAF-16 in this pathway, we assessed the effects of the lignans on the life span of a mutant GR 1307 strain, which lacks of DAF-16 (daf-16(mgDf50)). As a result, all lignans failed to extend the life span of this mutant (Table 1 and Fig. 3). Taken together with the results on the wild-type worms, our data strongly suggest that DAF-16 is indispensable for the life span-extending effects of the lignans. In addition, we examined the effect of lignans on the subcellular localization of DAF-16 in daf16::GFP transgenic worms (TJ356), in which GFP is integrated with
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S. Su, M. Wink / Phytochemistry 117 (2015) 340–350 Table 1 Effect of lignans on the life span of different strains of C. elegans. Strain
Treatment (lM)
Mean life span (day) ± SE
N2
Control
11.36 ± 0.34
N2
Arctigenin (10)
12.12 ± 0.35
6.7
0.0121
5
N2
Arctigenin (100)
12.93 ± 0.40
13.7
0.0121
2
N2
Matairesinol (10)
12.95 ± 0.42
14.0
