Antioxidant, Xanthine Oxidase, α-Amylase and α-Glucosidase Inhibitory Activities of Bioactive Compounds from Rumex crispus L. Root

The root of Rumex crispus L. has been shown to possess anti-gout and anti-diabetic properties, but the compounds responsible for these pharmaceutical effects have not yet been reported. In this study, we aimed to isolate and purify active components from the root of R. crispus, and to evaluate their anti-radical, anti-gout and anti-diabetic capacities. From the ethyl acetate (EtOAc) extract, two compounds, chrysophanol (1) and physcion (2), were isolated by column chromatography with an elution of hexane and EtOAc at a 9:1 ratio. Their structures were identified by spectrometric techniques including gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), X-ray diffraction analyses and nuclear magnetic resonance (NMR). The results of bioassays indicated that (1) showed stronger activities than (2). For antioxidant activity, (1) and (2) exhibited remarkable DPPH radical scavenging capacity (IC50 = 9.8 and 12.1 µg/mL), which was about two times stronger than BHT (IC50 = 19.4 µg/mL). The anti-gout property of (1) and (2) were comparable to the positive control allopurinol, these compounds exerted strong inhibition against the activity of xanthine oxidase (IC50 = 36.4 and 45.0 µg/mL, respectively). In the anti-diabetic assay, (1) and (2) displayed considerable inhibitory ability on α-glucosidase, their IC50 values (IC50 = 20.1 and 18.9 µg/mL, respectively) were higher than that of standard acarbose (IC50 = 143.4 µg/mL). Findings of this study highlight that (1) and (2) may be promising agents to treat gout and diabetes, which may greatly contribute to the medicinal properties of Rumex crispus root.


Introduction
Rumex crispus L. is known as the curly dock or yellow dock in most of Europe, North Africa, Turkey, Northern Iran, Central and East Asia, and North America [1]. The roots of this plant have been used in traditional medicine as a tonic, laxative, and for hemostasis medication. The fruits (seeds) are used for the treatment of dysentery. The young leaves of the plant are eaten as servings of mixed greens and soups. The plant has been utilized for treating the relevant disease, dermatology contaminations, better understanding of the value of medicinal plants, as well as contribute to the database for the production of gout and diabetes medications. Biological properties of the isolated compounds were also examined for their efficacy and safety in antioxidant activity and inhibition of xanthine oxidase, α-amylase and α-glucosidase enzyme by in vitro methods. The obtained compounds were identified by gas chromatography-mass spectrometry (GC-MS), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), nuclear magnetic resonance (NMR), and X-ray diffraction.

Structure Elucidation of Isolated Fractions
Separation of bioactive compounds from Rumex crispus root (RCR) was conducted following the procedure illustrated in Figure 1. This study aims to isolate compounds from R. crispus root that can scavenge free radicals and inhibit xanthine oxidase, α-amylase and α-glucosidase. The results of the study will contribute to a better understanding of the value of medicinal plants, as well as contribute to the database for the production of gout and diabetes medications. Biological properties of the isolated compounds were also examined for their efficacy and safety in antioxidant activity and inhibition of xanthine oxidase, α-amylase and α-glucosidase enzyme by in vitro methods. The obtained compounds were identified by gas chromatography-mass spectrometry (GC-MS), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), nuclear magnetic resonance (NMR), and X-ray diffraction.

Structure Elucidation of Isolated Fractions
Separation of bioactive compounds from Rumex crispus root (RCR) was conducted following the procedure illustrated in Figure 1. The isolated metabolites were characterized and identified by GC-MS, ESI-MS, APCI-MS, 1 Hand 13 C-NMR and X-ray analyses (Supplementary Figures S1-S21 and Tables S1-S3).
On the basis of GC-MS data (Table 1), isolated fractions were identified. The structure and formula of compounds were further confirmed by ESI-MS. Two fractions, C2 and C3, were elucidated by 1 H NMR and 13   The isolated metabolites were characterized and identified by GC-MS, ESI-MS, APCI-MS, 1 H-and 13 C-NMR and X-ray analyses (Supplementary Figures S1-S21 and Tables S1-S3).
On the basis of GC-MS data (Table 1), isolated fractions were identified. The structure and formula of compounds were further confirmed by ESI-MS. Two fractions, C2 and C3, were elucidated by 1 H NMR and 13 C NMR. Chemical structures of the identified compounds are illustrated in Figures 2 and 3.   11.99 (1H, s, OH-1). The 13 C NMR of 1 displayed 15 carbon signals including a methyl, two ketone groups, five methines, seven quaternary carbons (two of which are bonded to O atoms) ( Table 2). Assignments of all 1 H and 13 C NMR signals were accomplished by interpretation of HSQC and HMBC spectra. The melting point of compound was 196 °C. The above spectral data were diagnostic of an anthraquinone having two hydroxyl groups at C-1, C-8 position, and a methyl group. Moreover, methyl group and two hydroxyl group positions were determined by the HMBC spectral correlation between the proton of methyl group H-11 (δH 2.46) with C-2 (δC 124.4)/C-4 (δC 121.4); OH-1 (δH 11.99)    11.99 (1H, s, OH-1). The 13 C NMR of 1 displayed 15 carbon signals including a methyl, two ketone groups, five methines, seven quaternary carbons (two of which are bonded to O atoms) ( Table 2). Assignments of all 1 H and 13 C NMR signals were accomplished by interpretation of HSQC and HMBC spectra. The melting point of compound was 196 °C. The above spectral data were diagnostic of an anthraquinone having two hydroxyl groups at C-1, C-8 position, and a methyl group. Moreover, methyl group and two hydroxyl group positions were determined by the HMBC spectral correlation between the proton of methyl group H-11 (δH 2.46) with C-2 (δC 124.4)/C-4 (δC 121.4); OH-1 (δH 11.99)   11.99 (1H, s, OH-1). The 13 C NMR of 1 displayed 15 carbon signals including a methyl, two ketone groups, five methines, seven quaternary carbons (two of which are bonded to O atoms) ( Table 2). Assignments of all 1 H and 13 C NMR signals were accomplished by interpretation of HSQC and HMBC spectra. The melting point of compound was 196 • C. The above spectral data were diagnostic of an anthraquinone having two hydroxyl groups at C-1, C-8 position, and a methyl group. Moreover, methyl group and two hydroxyl group positions were determined by the HMBC spectral correlation between the proton of methyl group H-11 (δ H 2.46) with C-2 (δ C 124.4)/C-4 (δ C 121.4); OH-1 (δ H 11.99) with C-2 (δ C 124.4)/C-9a (δ C 113.8) and OH-8 (δ H 12.1) with C-7 (δ C 124.6)/C-8a (δ C 115.9). By means of its spectrometric analysis and comparison with previously published data [27], compound 1 was determined as chrysophanol or 1,8-dihydroxy-3-methylanthraquinone.

NMR Structural Elucidation
Compound 2 was obtained as orange-yellow needles. The 1 H and 13 C NMR spectra of 2 closely resembled those of 1, except for the appearance of one methoxy group (δ H 3.93, (3H, s), δ C 56.0) and the chemical shift of C-6 ( Table 2). This suggested that 2 also has an anthraquinone of 1 and 2 was the result of replacing the aromatic ring proton at C-6 in 1 by a methoxy group. This was confirmed by comparison of the NMR data of 2 with those of a known anthraquinone in Reference [28]. Thus 2 was determined to be 1,8-dihydroxy-3-methoxy-6-methylanthraquinone or physcion.

Quantitative Analysis of Fraxetin from Rumex Crispus Root
The content of two pure compounds chrysophanol and physcion in RCR is presented in Table 3. The quantity of these compounds was determined by the instrumentation used in this study.

Antioxidant Activities of the Isolated Fractions
Antioxidant activities of the isolated fractions were determined using three assays namely DPPH, ABTS free radical scavenging, and reducing power. BHT was used as a standard for all methods. The results are summarized in Table 4. Table 4 summarizes the antioxidant activities of the EtOAc R. crispus root extract. For the DPPH scavenging assay, the antioxidant activity of C2 was the highest as its IC 50 value was the lowest (10.0 µg/mL), followed by C3 (IC 50 = 12.0 µg/mL). Statistically, the DPPH radical scavenging properties of C2 and C3 were stronger than BHT (IC 50 = 19.2 µg/mL). The fraction C1 (IC 50 = 35.0 µg/mL) exhibited intermediate antioxidant capacities. For ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)), C2 exhibited the maximum scavenging activity with IC 50 value of 34.3 µg/mL, followed by C3 and BHT (IC 50 = 44.8 and 46.9 µg/mL). The lowest antioxidant property was fraction C1 (IC 50 = 194.7 µg/mL). For the reducing power ability, C2 displayed the maximum reducing power with IC 50 value of 312.6 µg/mL. Generally, the antioxidant properties of the pure compounds C2 and C3 were higher than standard BHT.

Discussion
In this study, two compounds were isolated and identified from the EtOAc extract of R. crispus root, namely chrysophanol (1) (fraction C2) and physcion (2) (fraction C3). They are biologically active compounds belonging to the flavonoid group. The radical scavenging activity of the isolated fractions was examined using DPPH, which is a frequently used method in natural product antioxidant evaluation [27], and ABTS assays. The results of this present study serve as an additional scientific finding with regards to the radical scavenging activity of the EtOAc extract of R. crispus root. The IC 50 values of (1) (10.0 ± 0.3 µg/mL) and (2) (12.0 ± 0.2 µg/mL) are comparable to the value reported by a previous study on the acetone extract of the R. crispus root with IC 50 of 14.0 µg/mL [28]. Moreover, a study reported that methanol extract of the fruits of R. crispus exhibited antioxidant activity with IC 50 value of 3.7 µg/mL [29]. These findings strengthen the antioxidant potential of the roots of R. crispus over its fruits. Furthermore, this results shows that the antioxidant activities of the pure compounds (1) and (2) are higher than the standard BHT ( Table 4). The results also show a strong correlation between the concentration of the pure compound (Table 3) and its antioxidant activity. Compound (1) (32.50 ± 0.11 µg/g DW) exhibits a stronger radical scavenging activity as seen in both DPPH and ABTS assays (IC 50 = 10.0 ± 0.3 and 34.3 ± 0.7 µg/mL, respectively) than (2) (25.04 ± 0.08 µg/g DW, 12.0 ± 0.2 and 44.8 ± 0.8 µg/mL, respectively). This result is in agreement with the findings of Elzaawely et al. [30] who reported the correlation between the phenolic content and DPPH assay of the EtOAc extract of the aerial parts of R. japonicus. The same correlation is observed between the concentration of the pure compound and its reducing power, in that the higher the concentration, the stronger its reducing power.
Xanthine oxidase (XO) plays a role in gout formation since it catalyzes the oxidation of xanthine to uric acid. Compounds that inhibit the activity of XO, therefore, can be used to treat gout [31,32]. Dietary flavonoids have been reported to possess inhibitory activity against free radicals and xanthine oxidase. The antioxidant property is mainly characterized by flavonoid contents that can effortlessly release hydrogen donors to naturalize free radicals. Therefore, flavonoids could be a promising remedy for human gout and ischemia by decreasing both uric acid and superoxide concentrations in human tissues [13]. According to Mohamed Isa et al. [15], Plumeria rubra contains a high amount of flavonoids and could be used as a new alternative to allopurinol with increased therapeutic activity and fewer side effects. The XO inhibitory activity in vitro assay of methanol extract of Plumeria rubra flowers possesses the highest inhibition effects at IC 50 = 23.91 µg/mL. Baicalein, baicalin and wogonin, isolated from Scutellaria rivularis, have been reported to exhibit a strong xanthine oxidase inhibition as evaluated by modified xanthine oxidase inhibition methods. The results showed that the order was baicalein > wogonin > baicalin, IC 50 = 3.12, 157.38 and 215.19 µM, respectively [33]. From this present study, (1) (IC 50 = 143.3 µM) exhibited a higher inhibitory effect on XO than wogonin and baicalin, whereas (2) (IC 50 = 158.5 µM) inhibited XO stronger than baicalin and showed comparable inhibitory activity as wogonin. The XO inhibition activity of (1) and (2) is directly related to their concentration as extracted from the root of R. crispus (Table 3). Allopurinol showed a much lower IC 50 value of 20.5 ± 0.5 µg/mL, which is why allopurinol is the preferred treatment for gout.
Both α-amylase (AA) (responsible for starch digestion) and α-glucosidase (AG) (produces glucose in the final step of the digestive process of carbohydrates) are related to type 2 diabetes since these enzymes are responsible for postprandial blood glucose levels [34]. AA and AG inhibitors are therefore widely used in the treatment of patients with type 2 diabetes, which is related to elevated postprandial blood glucose levels. Results of this present study showed consistent inhibition activities of (1) and (2) in both AA and AG in that (2) exhibited a stronger inhibition than (1) ( Table 5). However, in terms of AGI, (1) and (2) turned out to be much better inhibitors compared to acarbose (Table 5). Both terpenoids and flavonoids might play crucial roles in α-amylase and α-glucosidase inhibition [35,36]. The pure compounds (1) and (2) registered a stronger anti-diabetic activity than the mixture C3. Moreover, the synergic effect of the two pure compounds, C3 exerted stronger inhibition against α-glucosidase than acarbose (IC 50 = 143.8 µg/mL). Although this study to isolate and purify active components from the root of R. crispus, as well as test their biological activities, was successful, in vivo tests should be considered in order to ascertain the anti-gout and anti-diabetic properties of this prospective plant.

Materials
Rumex crispus root (RCR) was collected (34 • 23 28.3 N 132 • 43 06.0" E) in October 2017. The specimen with voucher number RCR-M2017 was deposited at the Plant Physiology and Biochemistry Laboratory (IDEC, Hiroshima University, Japan). The samples were cleaned by 1% NaOCl. After blotting with tissues, samples were dried at 45 • C in the oven for one week then ground to a fine powder [37].

Extraction of Rumex Crispus Root
The RCR powder (1.2 kg) was soaked in MeOH (2 L) to produce 23.8 g of MeOH crude extract. The crude extract was suspended in water (300 mL), then fractionated in hexane and EtOAc to obtain 6.6, 5.8, and 9.7 g water, hexane and EtOAc extracts, respectively.

Isolation of Pure Compounds
The hexane extract was dried at room temperature, then was purified by MeOH to obtain fraction C1 (200 mg).

Identification and Quantification of Isolated Compounds
Identification and quantification of isolated compounds was by GC-MS following previous study [40,41]. Therefore, the GC-MS system was equipped with a DB-5MS column (30 m × 0.25 mm internal diameter × 0.25 µm in thickness (Agilent Technologies, J & W Scientific Products, Folsom, CA, USA). The carrier gas was Helium and the split ratio was 5:1. GC oven temperatures operated with the initial temperature of 50 • C without hold time, followed by an increase of 10 • C/min up to a final temperature of 300 • C and holding time of 20 min. The injector and detector temperature were programmed at 300 • C and 320 • C, respectively. The MS ranged from 29 to 800 amu. JEOL's GC-MS Mass Center System Version 2.65a was used to control the GC-MS system and process the data peak.
ESI-MS analysis was conducted on negative/positive ion mode [42]. APCI-MS analysis was implemented using a mass spectrometer with an electrospray ion source [43].
X-ray diffraction measurement was conducted with a Bruker APEX-II ULTRA diffractometer (Bruker Corporation, Billerica, MA, USA). The crystals were irradiated using Mo-Kα radiation at −100 • C. Cell parameters and intensities for the reflection were estimated using the APEX2 crystallographic software package, in which data reduction and absorption corrections were carried out with SAINT and SADABS, respectively. The structures were solved and refined with the aid of the program SHELXL. The refined structures of crystals were reported as a crystallographic information file (CIF) [44].

Xanthine Oxidase Inhibition (XOD) Activity
The inhibitory effect on xanthine oxidase (XO) of isolated compounds (C1-C3) was measured spectrophotometrically according to the method reported previously [10] with minor modifications.

α-Amylase Inhibition (AAI) Assay
The inhibitory effect of C1-C3 on α-amylase was assessed by a starch-iodine method [34] with modification as follows: in each well of a microplate (U-shape, Greiner Bio-one, NC, USA), 20 µL of each isolated compound was pre-incubated with 20 µL of 1 U/mL α-amylase solution (from Aspergillus oryzae, Sigma-Aldrich, St. Louis, MO, USA) at 37 • C for 10 min. The reaction was initiated by pipetting 30 µL of soluble starch (0.5% in deionized water). After 6 min of incubation at 37 • C, an aliquot of 20 µL of hydrochloric acid (1 M) was added to stop reaction, followed by 120 µL of 0.25 mM iodine solution. The absorbance at 565 nm was read by a microplate reader (Multiskan TM Microplate Spectrophotometer, Thermo Fisher Scientific, Osaka, Japan). The inhibitory activity of C1-C3 on α-amylase was performed as the inhibition percentage calculated using the formula: where A is the absorbance of the compound, B is the absorbance of reaction without enzyme, C is the absorbance of the negative control. A commercial diabetes inhibitor acarbose was used as a positive reference. Dilutions of test samples and dissolutions of enzyme used 20 mM sodium phosphate buffer (pH 6.9 comprising of 6 mM sodium chloride). α-Amylase solution and soluble starch solution were prepared and used on the day of the experiment. The IC 50 value was calculated to exhibit 50% inhibitory activity of C1-C3 against α-amylase.

α-Glucosidase Inhibition (AGI) Assay
The anti-α-glucosidase activity of C1-C3 was evaluated using a method described previously [35] with some modifications. In brief, an amount of 20 µL methanolic stock solution of isolated compounds was pre-mixed with an equal volume of 0.1 M potassium phosphate buffer (pH 7) and 40 µL of α-glucosidase (from Saccharomyces cerevisiae, Sigma-Aldrich, St. Louis, MO, USA) enzyme solution (0.5 U/mL in 0.1 M potassium phosphate buffer, pH 7). After 6 min incubation at 25 • C, a 20 µL aliquot of 5 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) substrate (in 0.1 M potassium phosphate buffer, pH 7) was added to each reaction and the mixture was incubated for another 8 min. The reaction was terminated by adding 100 µL of 0.2 M Na 2 CO 3 , and absorbance was recorded at 405 nm. The inhibition percentage was calculated by the following equation: where A sample is absorbance of isolated compound, A control is the abosrbance of positive controls (acarbose or quercetin).

Statistical Analysis
All obtained data were analyzed by Minitab Software (version 16.0, copyright 2015, Minitab Inc., State College, PA, USA). One-way analysis of variance (ANOVA) and Tukey's post hoc test were used to identify the significant difference among mean values with p < 0.05. All trials were designed randomly in triplicate.

Conclusions
This study documented that the root of Rumex crispus possessed potent antioxidant, xanthine oxidase, α-amylase and α-glucosidase inhibitory activity in in vitro assays. More specifically, the compounds isolated from the EtOAc extracts emerged as a promising source of natural antioxidants, xanthine oxidase, α-amylase, and α-glucosidase inhibitors. In vivo tests should be conducted to affirm the bioactivity of the isolated compounds from the root of Rumex crispus for the development of food additives and supplements to reduce the risks type 2 diabetes and gout. The isolation of novel constituents, as well as investigations on the potent pharmaceutical properties of the root of Rumex crispus need to be considered.