Flavonoid 8-O-Glucuronides from the Aerial Parts of Malva verticillata and Their Recovery Effects on Alloxan-Induced Pancreatic Islets in Zebrafish

Malva verticillata (Cluster mallow), a leafy vegetable that has been popular in East Asia for a long time, has also been used in herbal teas and medicines. The aqueous fraction of the aerial parts of Malva verticillata, exhibiting a very high quantity of flavonoids compared to the EtOAc and n-BuOH fractions, exhibited significant recovery effects on pancreatic islets damaged by alloxan in zebrafish larvae. Thus, the bioactive components responsible for this anti-diabetic activity were investigated. A new flavonoid glucuronide (1) and five known flavonoids were isolated from the aqueous fraction. Based on several spectroscopic methods, compound 1 was identified to be nortangeretin-8-O-β-d-glucuronide, and was named malvaflavone A. The A-ring of compound 1 had a 5,6,7,8-tetrahydroxy moiety, which rarely occurs in plant systems. Also 8-O-glucuronide attached to the flavonoid moiety was rarely occurred in plant system. Compounds 1, 3, 4, and 6 significantly improved the pancreatic islet size in zebrafish at 0.1 μM, and compounds 1 and 6 were found to block β-cell K+ channels in experiments with diazoxide. In ABTS, ORAC, and SOD assays, compounds 1–5 exhibited high anti-oxidant activities compared with quercetin and BHA (positive controls), indicating that the 8-O-glucuronide attached to the flavonoid moiety is a key structure for the expression of anti-oxidant activity. This is the first report of the isolation of compounds 1–6 from M. verticillata as well evaluated for anti-diabetic and anti-oxidant ativities.


Introduction
Diabetes is characterized by variations in the metabolism of carbohydrates, proteins and fats.It results from insulin secretion disorders or reduced insulin sensitivity to blood sugar levels [1].In diabetic patients and animal models, the pancreatic islet (PI) size and β-cell number are smaller than normal [2].Protection or recovery of the PI constitution could be an efficient pharmacological approach for the treatment of diabetes.The most important route of insulin secretion is the release of insulin from islet cells, a process which is regulated by K ATP channels and voltage-gated Ca 2+ channels [3,4].K ATP channels are closed during insulin secretion, but are open when insulin release is inhibited by β-cell metabolism.
Epidemiological studies have consistently shown that regular consumption of foods such as vegetables is strongly related to a lower risk of developing chronic diseases such as diabetes [5].
Recently, consumers increasingly believe that foods contribute directly to their health [6].Today, foods are intended not only to satisfy hunger and provide necessary nutrients, but also to prevent nutrition-related diseases and improve physical and mental well-being [7].It is generally accepted that a food can have health-promoting properties that go beyond its traditional nutritional value.Bioactive compounds (Coms) are defined as components of food that influence physiological or cellular activity in the animals or humans that consume them.Recently, much attention has been given to bioactive food components that may be beneficial for the prevention of chronic diseases such as diabetes [8].A considerable amount of evidence has indicated that increased oxidative damage contributes to the development of chronic diseases such as diabetes, and several epidemiologic studies have demonstrated that the consumption of polyphenol-rich vegetables is associated with a reduced risk of oxidative stress-related diseases [9,10].Hence, there is a need to search for anti-diabetic food components that prevent chronic diseases and also promote health.
Malva verticillata (Cluster mallow), a leafy vegetable that has been popular in East Asia for a long time, has also been used in herbal teas and medicines [11].Its seeds in particular have been used in traditional Chinese medicinal formulas as diuretic, laxative, and galactopoietic materials [12].However, reports on the chemical (Chem) constituents and biological activities of the aerial parts of M. verticillata were very insufficient.In our preliminary study, the aqueous fraction (Fr) of the aerial parts of M. verticillata, exhibiting very high quantity of flavonoids comparing to the ethyl acetate (EtOAc) and n-butanol (n-BuOH) Frs [13,14].The TLC experiment exhibited the major components of the aqueous Fr of M. verticillata to be oligosaccharides and flavonoids.Some literature has reported that oligosaccharides have significant anti-diabetic activity, showing a hypoglycaemic effect [15], increasing β-cell function and insulin levels [16], inhibiting α-glucosidase activity [17], and promoting peripheral tissue utilization of glucose [18].However, it is relatively difficult to prepare the oligosaccharide Frs.Flavonoids are a group of naturally occurring polyphenolic Coms present in various fruits and vegetables, and are constituents of a number of effective traditional remedies [19].To date, a considerable number of flavonoids have been revealed to have anti-oxidant and therapeutic effects in chronic diseases in human populations [20][21][22].Some studies have indicated that dietary flavonoids reduce the risk of type 2 diabetes [23][24][25].
Therefore, in this study, flavonoids with anti-diabetic activity were isolated and identified from the aqueous Fr of the aerial parts of M. verticillata.Six flavonoids were isolated, identified, and investigated for their recovery effects on alloxan (AX)-induced PI damage in zebrafish.The recovery effects of certain Coms were found to be derived from the closing of K ATP channels in PIs.The anti-oxidant potential of these flavonoids was measured with DPPH, ABTS, ORAC radical scavenging (RS), and superoxide dismutase (SOD) assays.

Results and Discussion
Repeated column chromatography for the aqueous Frs of the aerial parts of M. verticillata resulted in the isolation of one new flavone glucuronide (1) and five known Coms (2-6) (Figure 1).
To evaluate the toxicity of the EtOAc, n-BuOH, and aqueous Frs on zebrafish embryos, the survival rate related to EtOAc, n-BuOH, and aqueous Frs was investigated in zebrafish and LC 50 value was calculated.Zebrafish were treated with EtOAc, n-BuOH, and aqueous Frs at ten different concentrations.The LC 50 values of EtOAc, n-BuOH, and aqueous Frs were 91.5, 270.9 and 401.1 µg/mL, respectively (Figure 2).The isolated Coms 1-6 were then evaluated for their recovery effects on AX-induced PI damage in zebrafish larvae.AX is a well-known diabetogenic agent that causes a reduction of insulin release through a β-cell mass decrease.In addition, AX has already been proven in a zebrafish model in previous studies.The PI size (µm 2 ) average in normal zebrafish was 1720 ± 300 µm 2 ; furthermore, in AX-and glimepiride (GLM)-treated zebrafish, the average was 950 ± 200 µm 2 and 1680 ± 220 µm 2 , respectively.PI size was 40.2% lower (p < 0.0001) in the AX treatment group than in the normal group (Figure 3).According to this model, after the exposure to AX, an enhanced recovery from that damage could be observed after treating the zebrafish with some Coms or extracts, the GLM is used as a positive control, since it is a well-known antidiabetic drug, and like the AX, has also been proven in the zebrafish model [30,31].GLM was used as a positive control, as it promotes insulin secretion by closing the K ATP channel [32].Additionally, glucose uptake was assessed in the zebrafish model with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), which is a fluorescent dye derived from glucose, modified with an amino group at the C-2 position [33].In diabetes studies, 2-NBDG is widely used to measure the ability of cells to absorb glucose [34].Both the PI size and fluorescence intensity were significantly greater in the GLM-treated group than in the AX-treated group.When Coms 1-6 were added to AX-treated zebrafish larvae, Coms 1, 3, 4, and 6 significantly increased the size of the injured PIs, by 49.4% (p = 0.0072), 46.8% (p = 0.0028), 62.4% (p < 0.0001), and 54.9% (p = 0.0088), respectively, compared with AX alone (Figure 3D,E).Some reports have mentioned that anti-diabetes activity was influenced in compliance with the number of hydroxyl groups in an aromatic moiety.Because Coms 1, 3, and 4 included one more hydroxyl group in the structure than Com 2, the former three showed higher activity than the latter.Additionally, Com 5 has two sugar moieties in the structure, which induces the decease of the anti-diabetes activity [35,36].Furthermore, flavonoids with 7,8-dihydroxy moiety, such as Com 6, have been reported to improve blood insulin concentration, to lower blood glucose level, and to increase insulin sensitivity in tissues such as liver, fat, and muscles [37].
As shown in Figure 3B,D, the aqueous Fr showed higher recovery effect on the AX-induced pancreatic islets in zebrafish than those of each components.The preliminary TLC experiment indicated the aqueous Fr contained high amount of oligosaccharides, in addition to flavonoids.The oligosaccharides have been reported to have significant anti-diabetic activity in many previous works [15][16][17][18].Due to their effects on PI size, Coms 1, 3, 4, and 6 were investigated for their ability to alter insulin secretion by modulating K ATP channels.Diazoxide (DZ) was used as a K ATP channel opener, and pancreatic β-cell K ATP channel stimulation activity was measured.The size of the PIs was significantly smaller (23.5%, p = 0.0105) in the DZ-treated normal group than in the normal group without DZ.The PI size in the AX group did not change significantly after the zebrafish larvae were treated with 25 µM DZ.The PI sizes in AX-induced zebrafish larvae treated with 0.1 µM GLM were significantly reduced following cotreatment with 25 µM DZ (41.1%, p = 0.0001).Similarly, in AX-induced zebrafish larvae treated with Coms 1 and 6, the PI sizes were significantly reduced following cotreatment with 25 µM DZ (32.2%, p = 0.0027 and 29.7%, p = 0.0182, respectively).However, in AX-induced zebrafish larvae treated with Coms 3 and 4, the PI sizes were almost the same after cotreatment with 25 µM DZ, indicating that Coms 3 and 4 had no relationship with K ATP channels (Figure 4).Thus, Coms 1, 3, 4, and 6 (0.1 µM) exerted significant recovery effects on the size of PIs damaged by AX in zebrafish, and Coms 1 and 6 were confirmed to influence K ATP channels in DZ experiments.According to this study, the natural Coms of M. verticillata may exert their action by closing the K ATP channels, which is demonstrated by the inhibition of the effect when cotreated with DZ, a well-known K ATP channel opener, resulting in an increased insulin secretion and a consequently increased glucose uptake.In the DPPH RS assay, Coms 3-5 exhibited activity comparing to positive controls (quercetin and BHA).And Coms 1-6 displayed significant activity, comparing to positive controls in the ABTS RS, oxygen radical absorbance capacity (ORAC), and superoxide dismutase (SOD) assays (Table 1).Coms 1-5 were revealed to have high anti-oxidant activities in the ABTS, ORAC, and SOD assays, indicating that the 8-O-glucuronic acid moiety is a key structure for the expression of anti-oxidant activity.In particular, because Coms 1 and 3 exhibited very high anti-oxidant activity, the 1,2,3-trihydroxy benzene moiety in the flavonoid A-ring and the 1,2-dihydroxy benzene moiety in the flavonoid B-ring appear to contribute to anti-oxidant activity.The glucuronic acid moieties in Coms 1-5 may have reduced their solubility in 70% MeOH, which was used as a solvent for the DPPH assay.Therefore, the DPPH RS activities of Coms 1-5 were not as high as expected.Despite low solubility of Com 3 in 70% MeOH, B-ring of Com 3 has the 1,2-dihydroxy (catechol) moiety, which is well known to exhibit high radical scavenging activity [38,39].Com 3 showed the highest anti-oxidant activity; on the contrary, Com 6 showed the lowest activity in most assays.As mentioned above, Com 3 has the 1,2-dihydroxy (catechol) moiety.Additionally, among six flavonoid glycosides, Coms 1-5 have the glucuronic acid in the structure, but Com 6 has glucoside.In this study, a new flavonoid glucuronide (1) and five known flavonoids (2-6) were first isolated from the aqueous Fr of the aerial parts of M. verticillata and identified.The 5,6,7,8-tetrahydroxy benzene ring in Com 1 was of particular interest, as it rarely occurs in plant systems.Coms 1, 3, 4, and 6 (0.1 µM) were revealed to restore the PI mass in zebrafish damaged by AX treatment, and Coms 1 and 6 were found to block β-cell K + channels in experiments with DZ.Additionally, Coms 1-5 exhibited significant anti-oxidant activity in ABTS, ORAC, and SOD assays, indicating that the 8-O-glucuronide attached to the flavonoid moiety is a key structure for the expression of anti-oxidant activity.Coms 1 and 3 exhibited especially high RS activities in ABTS and ORAC assays, indicating that the 1,2,3-trihydroxy benzene moiety in the flavonoid A-ring and the 1,2-dihydroxy benzene moiety in the flavonoid B-ring are closely related to anti-oxidant activity (Table 2).
In conclusion, Coms 1-6 were isolated from M. verticillata, and their anti-diabetic and antioxidant activities were evaluated for the first time.These results suggest that steady intake of M. verticillata could have anti-diabetic and antioxidant effects.

Materials
The aerial parts of M. verticillata were purchased from a commercial farm in Namyangju city, Korea in April 2016.The voucher specimen (KHU20160419) is deposited at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Reagents and Instrumentation
The silica gel (SiO 2 ) and octadecyl SiO 2 (ODS) resins used for column chromatography (CC) were Kiesel gel 60 (Merck, Darmstadt, Germany) and Lichroprep RP-18 (40-60 µm, Merck, Darmstadt, Germany), respectively.Sephadex LH-20 was purchased from Amersham Biosciences (Uppsala, Sweden).Thin layer chromatography (TLC) analysis was carried out with Kiesel gel 60 F 254 and RP-18 F 254S (Merck) TLC plates, and the Coms were detected with a Spectroline Model ENF-240 C/F UV lamp (Spectronics Corporation, Westbury, NY, USA) and a 10% H 2 SO 4 solution.Nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz FT-NMR spectrometer (Varian Inova AS-400, Palo Alto, CA, USA).Deuterium solvents were purchased from Merck Co. Ltd and Sigma Aldrich Co. Ltd (St. Louis, MO, USA).IR spectra were obtained on a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England).Quadrupole time-of-flight tandem mass spectrometry (ESI-QTof-MS) spectra were recorded on a Vion IMS QTof Mass Spectrometer (Waters Corporation, Milford, MA, USA).Melting points (Mp) were obtained with a Fisher-John's Melting Point Apparatus (Fisher Scientific, Miami, FL, USA) with a microscope, and the values obtained were uncorrected.Optical rotation was measured on a JASCO P-1010 digital polarimeter (Jasco, Tokyo, Japan).AX and sea salts were purchased from Sigma Chem Co. (St.Louis, MO, USA).GLM was obtained from Cayman Chem Co. (Ann Arbor, MI, USA).2-NBDG was purchased from Invitrogen (Life Technologies, Grand Island, NY, USA).DZ was purchased Santa Cruz Biotechnology (Dallas, TX, USA).Fluorescence microscopy was performed on an Olympus 1X70 microscope (Tokyo, Japan).For image analysis, Focus Lite (Focus Co., Daejeon, Korea) was used.

Zebrafish Care and Maintenance
Adult zebrafish were maintained in a zebrafish system S type (1500[W] × 400[D] × 2050[H] mm) (Daejeon, Korea) and a 14 h light/ 10 h dark cycle at 28.5 • C. Two pairs of adult zebrafish were placed in a spawning box overnight to obtain zebrafish larvae.The zebrafish spawned during a 30 min period of light.Zebrafish embryos were then collected at 3 h post-fertilization for incubation, and were maintained in a 0.03% sea salt solution for a 14/10 h, light/dark photocycle in an incubator at 28.5 • C. The fish were cared for in accordance with standard zebrafish protocols approved by the Animal Care and Use Committee of Kyung Hee University (KHUASP[SE]-15-10).

Evaluation of the Toxicity in Zebrafish Embryo
Twenty zebrafish embryos were used treatment for toxic test.Exposure of fish embryos was performed for 96 h as outlined in the OECD TG 236 for the Fish Embryo Acute Toxicity (FET) Test [40].31 treatments were used: normal, EtOAc, n-BuOH, and aqueous Frs at the concentration 25, 50, 75, 100, 150, 200, 300, 400, 500 and 600 µg/mL, respectively.The zebrafish were observed under the microscope after 96 h treatment and dead zebrafish were recorded.LC 50 values were calculated by non-linear regression using GraphPad Prism version 5.01 software.

Evaluation of Recovery Efficacy for AX-Induced PI Damage in Zebrafish Larvae
The zebrafish larvae were divided into the normal group, AX-induced group (control group), and AX-induced groups treated with the different Frs and Coms 1-6.Wild-type zebrafish larvae (5 dpf) were placed into 24-well plates.The larvae were exposed to 600 µM AX for 3 h to induce PI damage.The AX-induced larvae were treated with 10 µg/mL Frs or 0.1 µM Coms for 12 h so that the recovery efficacy of the Frs and Coms could be determined.Then, the larvae were stained for 30 min with 40 µM 2-NBDG and rinsed with a 0.03% sea salt solution for 20 min.After the staining, the PIs were confirmed by fluorescence microscope and analyzed with Focus Lite software.

Action of DZ on AX-induced Diabetic Zebrafish
Wild-type zebrafish larvae (5 dpf) were placed in 24-well plates (10 zebrafish per well).The larvae were divided into the following 14 groups: normal, normal treated with DZ or AX, and AX-induced diabetic treated with DZ, GLM, GLM + DZ, Com 1, Com 1 + DZ, Com 3, Com 3 + DZ, Com 4, Com 4 + DZ, Com 6, and Com 6 + DZ.The Coms were applied at 0.1 µM each, and DZ was applied at 25 µM.The zebrafish larvae were treated with 600 µM AX for 3 h, after which the solution was rinsed with a 0.03% sea salt solution.The AX-induced zebrafish larvae were treated with the respective Coms and/or treatments for 12 h.Following treatment, the zebrafish larvae were stained with 40 µM 2-NBDG for 30 min and rinsed with a 0.03% sea salt solution for 20 min.After the staining, PI images were captured by fluorescence microscopy and analyzed with Focus Lite software.

Statistical Analysis
Statistical analysis was performed with GraphPad Prism (version 5).Data are expressed as the mean ± standard error of the mean (SEM) for three replicates.Significance was determined with repeated one-way ANOVA followed by Tukey's test.The probability level for statistical significance was p < 0.05.

DPPH RS Activity
According to the method of Brand-Williams [41], DPPH RS activity was measured.First, 100 µM DPPH• was dissolved in 70% aqueous methanol.Then, Coms 1-6 (0.1 mL) were added to 2.9 mL of the methanolic DPPH• solution.The mixture was shaken vigorously and allowed to stand at 23 • C in the dark for 30 min.The decrease in absorbance of the resulting solution was monitored at 517 nm for 30 min.The control consisted of 0.1 mL of 70% aqueous methanol and 2.9 mL of DPPH• solution.Positive controls (the natural anti-oxidant quercetin and the synthetic anti-oxidant BHA [butylated hydroxyl anisole]) were also subjected to the same procedure for comparison.The results were confirmed in triplicate analyses, and the DPPH RS activity was calculated with the following equation: RS activity (%) = (A517 control -A517 sample )/A517 control × 100 The EC50 was determined as the concentration required to obtain a 50% RS effect.

ABTS RS Activity
The ABTS RS assay was carried out according to a published protocol [42].A radical initiator, 1.0 mM AAPH, was added to 2.5 mM ABTS in phosphate-buffered saline (PBS; pH 7.4; 0.1 M K 2 HPO 4 /KH 2 PO 4 buffer; 150 mM NaCl).The mixed solution was heated in a water bath at 68 • C. The resulting blue-green ABTS radical solution was adjusted to an absorbance of 0.650 ± 0.020 at 734 nm with additional PBS.Then, 20 µL of the sample was added to 980 µL of the ABTS radical solution.The mixture was incubated in a 37 • C water bath under restricted light for 10 min.A control (20 µL of 70% methanol and 980 µL of the ABTS radical solution) was run with each series of samples.Positive controls (the natural anti-oxidant quercetin and the synthetic anti-oxidant BHA) were also subjected to the same procedure for comparison.The reduction of absorbance at 734 nm was measured 10 min later.The ABTS radical, exhibiting a characteristic blue-green color in its odd-electron state, loses color when its unpaired electron is paired with an electron from an anti-oxidant.The results were confirmed in triplicate analyses, and the ABTS RS activity was calculated with the following equation: RS activity (%) = (A734 control -A734 sample )/A734 control × 100 The EC50 was determined as the concentration required to obtain a 50% RS effect.

Oxygen Radical Absorbance Capacity (ORAC) Assay
The ABTS RS assay was carried out according to a published protocol [43].Appropriately diluted samples and the standards with 150 µL of 81.6 nM fluorescein solution were added to a 96-well plate and incubated at 37 • C for 10 min with 3 min of shaking.25 µL of 153 mM AAPH solution was added and fluorescence was then detected every minute for 90 min using a microplate reader (Infinite M200,
a EC 50 is a concentration required to obtain a 50% antioxidant effect.b Each result was expressed as mean ± SD (µM or µmol TE/ µmol) in triplicate studies.
a All proton and carbon positions were assigned by HSQC and HMBC experiments.b This signals were overlapped.