Isolation and identification of polyphenols from Marsilea quadrifolia with antioxidant properties in vitro and in vivo

Abstract Marsilea quadrifolia is an edible aquatic medicinal plant used as a traditional health food in Asia. Four new polyphenols including kaempferol 3-O-(2″-O-E-caffeoyl)-β-d-glucopyranoside (1), kaempferol 3-O-(3″-O-E-caffeoyl)-α-l-arabinopyranoside (3), 4-methy-3′-hydroxypsilotinin (4) and (±)-(E)-4b-methoxy-3b,5b-dihydroxyscirpusin A (18) together with 14 known ones (2, 5–17) were isolated from the ethanol extract of M. quadrifolia. Structures of the new compounds were elucidated by extensive spectroscopic analyses. In DPPH and oxygen radical absorbance capacity antioxidant assays, some compounds showed stronger antioxidant activities and quercetin (9) was the most potent antioxidant in both assays. In a restraint-induced oxidative stress model in mice, quercetin significantly attenuated the increase in plasma ALT and AST levels as well as liver MDA content of restrained mice. Liver SOD activity was also significantly increased by quercetin, indicating a significant in vivo antioxidant activity. As a rich source of polyphenols with strong antioxidant activities, M. quadrifolia may be developed to a product for relieving oxidative stress.


Introduction
Marsilea quadrifolia L, belonging to the family of Marsileaceae, is an edible aquatic fern plant widely distributed in tropical and temperate regions in Eurasia and America (Luo & Ikeda 2007). The plant is also known as water clover and consumed as health food in soup, vegetable and tea in some Asian countries (Bhadra et al. 2012;Fu 2012). In India, this herb is used in folk medicine for the treatments of snakebite, abscess, cough, bronchitis (Pepsi et al. 2012) and used as a vegetable for inducing sleep . In China, there are a number of patents concerning the use of M. quadrifolia in health food for the treatment of fever (Lin 2003), hepatitis and alcoholism (Liu & Zhao 2006a, 2006b. In Japan, this herb is used in skin care preparation (Hanano & Maeda 2004).
Modern scientific studies have revealed diverse pharmacological activities of M. quadrifolia. The water and ethanol extracts of the plant were effective in reducing the epileptic seizures induced by pentylenetetrazole in rats in both behavioural observation and electroencephalogram analysis (Sahu et al. 2012). Its methanol extract was found to inhibit acetylcholinesterase and butyrylcholinesterase activity with IC 50 values of 51.89 ± 0.24 and 109.43 ± 2.82 μg mL −1 , respectively (Bhadra et al. 2012). The ethanol extract could significantly improve the learning and memory of mice (Ashwini et al. 2012). In addition, the methanol extract showed significant antidiabetic and antioxidant activities (Zahan et al. 2011). In spite of the wide applications of this plant in folk medicine and health food and the pronounced pharmacological activities, chemical constituents of M. quadrifolia were rarely reported except for several phenolic acids (Venkataramaiah et al. 1981).
During the course of our ongoing search for antioxidants from health and medicinal materials, the ethanol extract of M. quadrifolia was found to show strong radical scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (IC₅₀ value of 13.9 ± 1.2 μg mL −1 ). In order to understand the active chemical components responsible for the biological effects, a phytochemical study was performed and four new polyphenols (1, 3, 4, 18) together with 14 known ones (2, 5-17) were isolated. We reported herein the isolation, structural elucidation and antioxidant activities of the isolated compounds.

In vitro assay
Antioxidant activities of the isolated compounds 1-11, 13, 15-18 (compounds 12 and 14 were not tested because of the limited quantities) from M. quadrifolia were evaluated by two in vitro methods, DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC) assay. The most active compound was further tested in an in vivo model. All compounds exhibited strong DPPH free radical scavenging activities except for 7, 8 and 13 (Figure 2(a)). The scavenging effects of 1, 3-6, 9, 15-18 were higher than those of the controls (ascorbic acid and butylated hydroxytoluene [BHT]). Among them, quercetin (9) showed the most potent scavenging activity. In ORAC assay (Figures 2(b) and S2), compound 9 also showed the strongest antioxidant activities (1.60-fold) followed by compound 6 (1.51-fold) and 17 (1.48-fold). Representative curves of fluorescence decay within 60 min induced by AAPH in the presence of five compounds at 4 μM and Trolox at 20 and 40 μM are shown in Figure 2(b). Both DPPH and ORAC assays revealed that some polyphenols from M. quadrifolia showed comparable or even stronger antioxidant activities than the positive controls, though the potency might vary between these two bioassays due to different experimental principles. In both assays, quercetin (9) showed the most potent antioxidant activity. Then compound 9 was further tested in a restraint-induced oxidative stress model in mice.

In vivo activity
Before induction of stress by physical restraint in restraining tubes for 20 h, mice were orally administered with different doses of quercetin (9) or distilled water daily for seven consecutive days. As shown in Table S4 and Figure 3, plasma ALT (125.31 ± 27.45 U/L) and AST (84.74 ± 17.62 U/L) levels significantly increased in restrained mice (p < 0.01 vs. normal control group). Quercetin treatment (50, 100, 150 mg/kg) could significantly attenuate the increase in plasma ALT and AST levels in stressed mice in dose-dependent manner. Liver SOD activity significantly decreased, while MDA level was significantly higher in the restraint model group when compared with the normal control group (SOD: 226.72 ± 56.02 vs. 323.90 ± 42.37 U/mgprot, p < 0.01; MDA: 6.50 ± 1.05 vs. 2.93 ± 0.73 nmol/mgprot, p < 0.01). Quercetin treatment at high dose (150 mg/kg) significantly increased SOD activity and in all three doses, it decreased hepatic MDA content in restrained mice. It was reported that M. quadrifolia possessed liver protective effect (Liu & Zhao 2006a, 2006b. Our results showed that quercetin could significantly alleviate the liver damages of the stress-loaded mice. Thus, quercetin could be considered as one of the active components responsible for the liver protection effect.

Conclusion
In summary, we have isolated 18 polyphenols, including four new compounds and 14 known ones. Though the potency varied among different bioassay methods, most compounds showed comparable or even stronger antioxidant activities than the positive controls, which might be due to the free phenolic hydroxyl groups in these polyphenols (Liu & Ng 2000). Among them, quercetin (9) showed the best antioxidant properties. In a restraintinduced oxidative stress model in mice, quercetin treatment could significantly decrease Figure 3. Effects of quercetin (9) on the activities of plasma ALT and AST, and MDA content and SOD activity in the liver of restraint-stressed mice. The results are represented as the mean ± SD of values in each group (n = 10). ALT -alanine aminotransferase, AST -aspartate aminotransferase, MDA -malondialdehyde, SOD -superoxide dismutase. **p ≤ 0.01 compared to the control group, # p ≤ 0.05 compared to the stressed model group, ## p ≤ 0.01 compared to the stressed model group.
plasma ALT and AST levels, increase SOD activity and decrease MDA content in the liver, indicating a significant in vivo antioxidant activity. As a rich source of polyphenols with strong antioxidant activities, M. quadrifolia might be developed to products for relieving oxidative stress.