Bioactivity-Guided Purification of Novel Herbal Antioxidant and Anti-NO Compounds from Euonymus laxiflorus Champ.

Euonymus laxiflorus Champ., a medicinal herb collected in Vietnam, has been reported to show several potent bioactivities, including anti-NO, enzyme inhibition, hypoglycemic and antidiabetic effects. Recently, the antioxidant activity of Euonymus laxiflorus Champ. trunk bark (ELCTB) has also been reported. However, the active antioxidant and anti-NO constituents existing in ELCTB have not been reported in the literature. The objective of this study was to purify the active antioxidants from ELCTB and investigate the anti-NO effect of the major constituents. Twenty-two phenolics isolated from ELCTB, including 12 compounds newly isolated in this study (1–12) and 10 constituents obtained from our previous work, were evaluated for their antioxidant activity. Of these, 12 compounds (4–6, 9, 13–15, 18–22) showed a potent antioxidant capacity (FRS50 = 7.8–58.11 µg/mL), in comparison to α-tocopherol (FRS50 = 23 µg/mL). In the anti-NO activity tests, Walterolactone A (1a) and B (1b) β-d-glucopyranoside (13) demonstrated the most effective and comparable activity to that of quercetin with max inhibition and IC50 values of 100%, 1.3 µg/mL, and 100%, 1.21 µg/mL, respectively. The crude extract and its major compounds showed no cytotoxicity on normal cells. Notably, three constituents (9, 11, and 12) were identified as new compounds, another three constituents, including 1, 7, and 8, were found to be new natural products, constituents 9 and 13 were determined to be new antioxidants, and compound 13 was reported to have novel potent anti-NO activity for the first time. The results of this study contribute to the enrichment of new natural products and compounds, as well as the novel biological activity of constituents isolated from Euonymus laxiflorus Champ. The current study also indicates ELCTB as a rich natural source of active phenolics. It is suggested that ELCTB could be developed as a health food with promising antioxidant and anti-NO effects, as well as other beneficial biological activities.


Introduction
Living organisms are negatively affected by free radicals in areas such as DNA, proteins, and lipid damage, and as such, they may cause various diseases [1]. These unexpected effects can be reduced and prevented by antioxidant compounds which can be synthesized or obtained from natural resources. However, synthetic antioxidants can cause carcinogenesis and liver damage [2]. Thus, the discovery of abundant and natural antioxidants is still needed. Antioxidants may be obtained from several natural sources, including higher plants [3][4][5], microbial fermentation [6,7], and marine products [8,9]. Of these, herbal antioxidants have been given much exploration in recent years [10].
Vietnam is a tropical country and is considered the sixteenth most biodiverse region in the world. It possesses around 10,000 herbal species, of which 4000 have been used as medicinal plants [11]. Therefore, there has been increased interest in the seeking and purification of bioactive constituents from Vietnamese herbs in recent years [4,5,10,[12][13][14][15][16][17]. Numerous herbs showing enzymic inhibitory, hypoglycemic, and antidiabetic effects have been investigated [4,5,[12][13][14][15][16][17]. The antioxidant and anticancer effects of the herbs collected in this biodiverse region have also been reported in several works [4,5,10]. However, only a few herbs possessing anti-NO properties have been reported, and there are also few studies working on the isolation and identification of active compounds (antioxidant and anti-NO constituents). Thus, the investigation of natural products processing beneficial properties from the herbs of this biodiverse area has been received with great attention.
Euonymus laxiflorus Champ.-A medicinal herb, is wildly distributed in Vietnam [12,17] and some other Asian countries, including China, Cambodia, India, and Myanmar [18]. This medicinal herbal species has been recognized as a rich source of bioactive natural products and has been extensively investigated to reclaim its potent antidiabetic properties via enzymic inhibition and animal tests [4,12,13]. These previous studies revealed that Euonymus laxiflorus Champ. shows potency for α-amylase inhibition [13], α-glucosidase inhibition [12], as well as a significant effect on the reduction of blood glucose in diabetic rats [4]. Recently, novel potent antidiabetic compounds have been isolated and identified from Euonymus laxiflorus trunk bark extract [14,17]. Of these, poly condensed tannin, a major antidiabetic compound, has demonstrated a significant effect on reducing plasma glucose in ICR mice [17]. Some constituents isolated from the leaves of this plant have shown anti-nitric oxide and antioxidant effects [19]. However, little data on the antioxidant properties of this herbal extract exists beyond the previous reports [4], and no compounds isolated from the trunk bark extract of this herb have been reported for the evaluation of antioxidant and anti-NO capacities.
As a part of the objective to indicate scientific proof for the development of Euonymus laxiflorus Champ. as potent functional food or drug, this study investigated the bioactivity of this herb to isolate and identify its chemical constituents. In the current study, the potent antioxidant capacity of Euonymus laxiflorus Champ. was reclaimed, and the isolation and identification of its active compounds, as well as an evaluation of its antioxidant and anti-NO activities, were also reported. The results of this study contributed to the enrichment of new natural products and novel compounds, as well as the new biological effects of the constituents isolated from Euonymus laxiflorus.

Reclamation of Euonymus laxiflorus Champ. Extracts as a Potential Source of Beneficial Bioactive Properties without Toxicity
The methanol extract of Euonymus laxiflorus Champ. trunk bark (ELCTB) was evaluated for its DPPH radical scavenging activity (antioxidant activity), and the results are illustrated in Figure 1 (%) and Table 1 (FRS50). ELCTB showed potent antioxidant activity with great max activity (%) and low FRS50 values of 97% and 20.2 µg/mL, respectively. The antioxidant activity of α-tocopherol, a commercial antioxidant compound, was also tested for comparison and showed approximately equal activity, with max activity (%) and low FRS50 values of 99% and 24.4 µg/mL, respectively. In the comparison, Euonymus laxiflorus Champ. also showed comparable or higher antioxidant activity than those of some recently-reported medicinal extracts collected in the central highlands of Vietnam (Table 1).

Molecules 2018
3 of 12 FRS50 values of 97% and 20.2 µ g/mL, respectively. The antioxidant activity of α-tocopherol, a commercial antioxidant compound, was also tested for comparison and showed approximately equal activity, with max activity (%) and low FRS50 values of 99% and 24.4 µ g/mL, respectively. In the comparison, Euonymus laxiflorus Champ. also showed comparable or higher antioxidant activity than those of some recently-reported medicinal extracts collected in the central highlands of Vietnam (Table 1).  The trunk bark of Euonymus laxiflorus Champ. has been reported to possess promising αglucosidase inhibitory and α-amylase inhibitory activities [12,13] and significant effects on the reduction of plasma glucose in normal and diabetic mice [4,17]. The anti-NO activity of the extract from the leaves of this herb was investigated by Liu et al. (2014) [19]. In addition, Euonymus laxiflorus has been proved to be non-toxic via animal models in previous reports [4]. In this study, ELCTB was found to show potential antioxidant activity, therefore an investigation was conducted to isolate and identify its active compounds, which were subsequently evaluated for their beneficial bioactivities.  The trunk bark of Euonymus laxiflorus Champ. has been reported to possess promising α-glucosidase inhibitory and α-amylase inhibitory activities [12,13] and significant effects on the reduction of plasma glucose in normal and diabetic mice [4,17]. The anti-NO activity of the extract from the leaves of this herb was investigated by Liu et al. (2014) [19]. In addition, Euonymus laxiflorus has been proved to be non-toxic via animal models in previous reports [4]. In this study, ELCTB was found to show potential antioxidant activity, therefore an investigation was conducted to isolate and identify its active compounds, which were subsequently evaluated for their beneficial bioactivities.

Selective Solvent Extraction for Max Antioxidant Productivity from Euonymus laxiflorus Champ. Extracts and Purification of Active Compounds
Five solvents, including water, butanol, ethanol, methanol, and ethyl acetate, were used to extract Euonymus laxiflorus Champ. trunk bark (ELCTB), and then all extracts were evaluated for antioxidant activity. As shown in Figure 2A, the ELCTB extracted by methanol demonstrated the highest activity due to its smallest FRS50 value of 20 µg/mL. Therefore, the methanol extract was chosen for the next step of purification.

No.
Isolated compounds DPPHFRS50 (µg/mL) Max (%) The purified compounds were tested for their antioxidant activity at concentrations in the range of 0.5-250 µg/mL. The results were presented as the means ± SD of multiple tests (n = 3). The means of max inhibition and FRS50 with different letters in the same column were significantly different based on Duncan's multiple range test at alpha = 0.01 (UD: unable to determine).

Anti-Inflammation and Cytotoxicity of Euonymus laxiflorus Champ. Extract and its Major Compounds
Nitric oxide (NO) has been suggested as a mediator of pro-inflammatory activity related to certain inflammatory disorders, including chronic hepatitis, pulmonary fibrosis, and rheumatoid arthritis. In this study, the crude extract (ELCTB) and its major identified compounds (1, 2, and 15) were evaluated for anti-NO activity with the use of LPS-stimulated RAW 264.7 cells. As shown in Figure 5a, Walterolactone A (1a) and B (1b) β-D-glucopyranoside (13) showed the most effective anti-inflammation properties and gallic acid (14) demonstrated acceptable activity with max inhibition and IC 50 values of 100%, 1.3 µg/mL and 56%, 145 µg/mL, respectively, while (+)-Catechin (19) and the crude extract possessed weak activity with a max inhibition of 33.7% and 6%, respectively. Quercetin, a commercial anti-NO compound, was tested for comparison and showed max inhibition and IC 50 values of 100%, 1.21 µg/mL, respectively. The results indicated that Walterolactone A (1a) and B (1b) β-D-glucopyranoside (13) demonstrated promising activity which was equal to that of the positive control. Notably, Walterolactone A (1a) and B (1b) β-D-glucopyranoside (13) was investigated for the efficacy of its anti-NO activity for the first time in the current study and was found to be a new effective anti-NO compound. In the previous study, a total of 11 constituents were isolated and identified from the stems and leaves of Euonymus laxiflorus. Of these purified compounds, laxifolone A (a triterpene) displayed significant anti-NO effects [29].
The cytotoxicity of Euonymus laxiflorus Champ. extract and its major compounds were also examined using the same cell model based on LPS-stimulated RAW 264.7 cells. The cytotoxicity of the crude extract and its major identified compounds were tested using a large concentration range of 20-160 µg/mL. The results (Figure 5b) showed that the major isolated compounds of ELCTB all slightly enhanced the growth of normal cells and that the ELCTB extract, as well as its major compounds (13,  14, and 19), had no toxicity to normal cells. In the previous study [3], The ELCTB extract was reported to show no toxic effects on a mouse model at a high dose of 300 mg/kg bw (body weight). The results of this study, as well as those of recently reported researches [3], confirmed that ELCTB was a natural and safe material with promising biomedical activities, including enzymic inhibition, hypoglycemic effects, and antidiabetic, antioxidant, and anti-NO activities.
Molecules 2018 9 of 12 effective anti-NO compound. In the previous study, a total of 11 constituents were isolated and identified from the stems and leaves of Euonymus laxiflorus. Of these purified compounds, laxifolone A (a triterpene) displayed significant anti-NO effects [29]. The cytotoxicity of Euonymus laxiflorus Champ. extract and its major compounds were also examined using the same cell model based on LPS-stimulated RAW 264.7 cells. The cytotoxicity of the crude extract and its major identified compounds were tested using a large concentration range of 20-160 µ g/mL. The results (Figure 5b) showed that the major isolated compounds of ELCTB all slightly enhanced the growth of normal cells and that the ELCTB extract, as well as its major compounds (13, 14, and 19), had no toxicity to normal cells. In the previous study [3], The ELCTB extract was reported to show no toxic effects on a mouse model at a high dose of 300 mg/kg bw (body weight). The results of this study, as well as those of recently reported researches [3], confirmed that ELCTB was a natural and safe material with promising biomedical activities, including enzymic inhibition, hypoglycemic effects, and antidiabetic, antioxidant, and anti-NO activities.

Materials
The ELCBT extract was obtained according to a previous study reported by Nguyen et al., 2017 [11] Reagents, other common chemicals, and solvents were available at the highest grade.

Biological Activity Assays
Free radical scavenging activity was determined through use of the DPPH radical scavenging activity assay described by Nguyen et al., 2018 [9] with minor modifications. One hundred and twenty microliters (120 µ L) of the samples (diluted with methanol at a concentration range of 0.5-250 μg/mL) were mixed with 30 µL of 0.75 mM DPPH in methanol in a 96-well plate and then kept in the dark for 30 min. The optical density of the mixtures at 517 nm (OD517nm) was measured [30]. The following formula (1) was used to calculate the antioxidant activity:

Materials
The ELCBT extract was obtained according to a previous study reported by Nguyen

Biological Activity Assays
Free radical scavenging activity was determined through use of the DPPH radical scavenging activity assay described by Nguyen et al., 2018 [9] with minor modifications. One hundred and twenty microliters (120 µL) of the samples (diluted with methanol at a concentration range of 0.5-250 µg/mL) were mixed with 30 µL of 0.75 mM DPPH in methanol in a 96-well plate and then kept in the dark for 30 min. The optical density of the mixtures at 517 nm (OD 517nm ) was measured [30]. The following formula (1) was used to calculate the antioxidant activity: where, A refers to the OD 517nm of the blank sample (no antioxidants/samples in the mixture solution) and B stands for the OD 517nm of the sample solution at 30 min of incubation. The concentration of an antioxidant compound that can reduce 50% of the purple color of the DPPH solution under the assay conditions was defined as the FRS50 value [9]. α-Tocopherol dissolved in MeOH was used as the positive control. An anti-inflammation assay was also performed according to the methods described in detail by Nguyen et al., 2018 [9]. The tests were all repeated in triplicate and the differences between the mean values of the antioxidant and anti-NO activity (p < 0.01) were analyzed using Statistical Analysis Software (SAS-9.4, provided by SAS Institute Taiwan Ltd., Minsheng East Road, Section 2, Taipei, Taiwan 149-8).

Conclusions
A total of 12 phenolics (1-12) were newly isolated and identified from Euonymus laxiflorus. Of these, three constituents (9), (11), and (12) were identified as new compounds, while (1), (7), and (8) were identified as new natural products. These 12 compounds and the other 10 compounds (13-22) obtained from our previous work were evaluated for their antioxidant activity. Notably, 12 compounds (4-6, 9, 13-15, 18-22) displayed potent antioxidant activity (FRS50 = 7.8-58.11 µg/mL) compared to α-tocopherol (FRS50 = 23 µg/mL). Among these, two constituents, (9) and (13), were determined to be new antioxidants. In the anti-NO activity tests, walterolactone A (1a) and B (1b) β-D-glucopyranoside (13) was found to have the most effective and comparable activity to that of quercetin, with max inhibition and IC 50 values of 100%, 1.3 µg/mL and 100%, 1.21 µg/mL, respectively, and this compound was reported to have anti-NO activity for the first time. The crude extract and its major compounds showed no cytotoxicity on normal cells. The results of the current study confirmed that ELCTB was a rich natural source of active phenolics that could be developed as a health food with promising antioxidant, anti-NO, and other beneficial biological activities. The results of this study also contributed to the enrichment of new natural products and the enrichment of novel compounds, as well as the novel biological activity of the constituents isolated from Euonymus laxiflorus.