Phenylalkyl Glycosides from the Flowers of Brugmansia arborea L. and Their Radical Scavenging Effect and Protective Effect on Pancreatic Islets Damaged by Alloxan in Zebrafish (Danio rerio) Larvae

The study aimed to investigate the antioxidant and antidiabetic activity of Brugmansia arborea L. flower extracts, solvent fractions, and isolated compounds. B. arborea L flowers were extracted with aqueous methanol, and concentrated extract was successively partitioned into EtOAc, n-BuOH, and H2O fractions. Repeated silica gel and octadecyl silica gel column chromatographies for EtOAc and n-BuOH fractions led to the isolation of a new phenylalkyl glycoside (6), along with five known ones. Several spectroscopic data led to the structure determination of one new phenylalky glycoside as brugmansioside C (named) (6) and five known ones as benzyl-O-β-D-glucopyranoside (1), benzyl-O-β-D-glucosyl-(1→6)-β-D-glucopyranoside (2), 2-phenylethyl-O-β-D-glucopyranoside (3), 2-phenylethyl-O-β-D-glucosyl-(1→6)-β-D-glucopyranoside (4), and 3-phenylpropyl-O-β-D-glucopyranoside (5). The five known ones (1–5) were isolated from B. arborea flowers for the first time in this study. The extract, solvent fractions, and all isolated compounds showed radical scavenging activities using ABTS radical, and EtOAc fraction showed the highest scavenging capacity, whereas compounds 2, 4, and 6 did not display the capacity to use the DPPH radical. The extract, solvent fractions, and all isolated compounds showed a protective effect on pancreatic islets damaged by alloxan treatment in zebrafish larvae. The pancreatic islet size treated with EtOAc, n-BuOH fractions, and all compounds significantly increased by 64.0%, 69.4%, 82.0%, 89.8%, 80.0%, 97.8%, 103.1%, and 99.6%, respectively, compared to the alloxan-induced group. These results indicate that B. arborea flowers and their isolated compounds are useful as potential antioxidant and antidiabetic agents.


Introduction
Diabetes mellitus (DM), a metabolic disease, is characterized by an abnormally high level of serum glucose caused by insulin deficiency or resistance.DM is classified as type 1 and type 2 according to etiology and characteristics [1][2][3].Type 1 DM (insulin-dependent) is closely related to autoimmunity.Patients with type 1 DM show insulin deficiency due to the destruction of pancreatic β-cells.Type 2 DM (non-insulin-dependent diabetes mellitus) is related to hyperglycemia and insulin resistance [4].DM can lead to serious microvascular complications that result in retinopathy and nephropathy [5].In the search for antidiabetic drugs, the zebrafish model has been widely used because of its physiological and genetic similarities to mammalian systems [6] as well as its size, handy conservation in laboratories, abundant offspring, transparent embryos, and obedience to genetic and chemical screens [7].Zebrafish can be treated with alloxan to damage the pancreatic islets, resulting in variations in islet size and glucose absorption.
Brugmansia arborea L. (Solanaceae), an evergreen shrub distributed in America, Africa, Australia, and Asia [8], is also called 'angel's trumpet' because the flowers resemble the long trumpet envisioned of angels.The plant reaches up to 3-11 m in height.The ovate leaves are coarsely toothed, and the flowers are strongly fragrant.B. arborea has been commonly used for ornamental purposes [8].In ancient South American Indian culture, B. arborea was used as a hallucinogenic drug for rituals, medical supplies, and poison.In particular, B. arborea was force-fed to the wives, children, and slaves of Native American rulers who died so that all could be buried together.This plant has also been used for its analgesic, antirheumatic, vulnerary, decongestant, and antispasmodic activities [8][9][10].Anti-cholinergic activity of B. arborea has also been reported [11,12], with the principal compounds revealed to be tropane alkaloids.Despite the several practical uses and reported pharmacological activities of B. arborea, the constituents of B. arborea flowers are not well described.Accordingly, authors carried out a study to identify the active principals of B. arborea flowers.
We isolated and identified one new phenolic glycoside and five known ones.And the extract, solvent fractions, and identified phenolic glycosides were evaluated for the radical scavenging activities using the DPPH and ABTS radicals and the protective effect on pancreatic islets damaged by alloxan treatment in zebrafish larvae.The description of the purification, structure determination, and antioxidant and antidiabetic potential of phenolic glycosides was included in this paper.Since the extract, fractions, and isolated compounds found in this study have diverse variations in their structural characteristics and potent antioxidant and antidiabetic activities, their successive study will lead to the development of safe and effective functional materials against metabolic disease.

Chemical Structure Elucidation
The flowers of B. arborea were extracted in aqueous methanol (MeOH) (80%), and the concentrated extracts were partitioned into EtOAc, n-BuOH, and water fractions, successively.Among the fractions, both organic fractions yielded one new phenylbutyl glycoside along with five phenylalkyl glycosides.

Radical Scavenging Capacity
The antioxidant capacities of extract, solvent fractions, and phenylalkyl glycosides 1-6 of B. arborea flowers by the ABTS and DPPH assays are shown in Table 1.The experimental information with a description of the measurements of radical scavenging assay

Radical Scavenging Capacity
The antioxidant capacities of extract, solvent fractions, and phenylalkyl glycosides 1-6 of B. arborea flowers by the ABTS and DPPH assays are shown in Table 1.The experimental information with a description of the measurements of radical scavenging assay was described in a previous study [17].EtOAc fraction (BAFE) showed the highest antioxidant capacities in both ABTS and DPPH assays.It was thought that the ethyl acetate fraction mainly contained compounds that contribute more to antioxidant capacities.The isolated compounds 1-6 showed ABTS radical scavenging capacities in order 1 > 3 > 2 ≥ 5 > 4 > 6.
The DPPH radical scavenging activity was similar to ABTS radical scavenging activity (Table 1).BAFE showed the highest capacity, whereas compounds 2, 4, and 6 did not show DPPH scavenging capacity.Because the DPPH assay (80% methanol) measures the radical scavenging ability in the non-polar solvent system, compounds 2, 4, and 6, including two sugars in the structure, showed lower DPPH radical scavenging activity than ABTS [19,20].Monoglycoside compounds 1 and 3 exhibited higher activity than the diglycoside compounds 2 and 4, respectively.513.2 ± 12.9 a 130.9 ± 3.7 a 1 VCE stands for vitamin C equivalent. 2 DW stands for dry weight. 3Data are presented as the mean ± standard deviations (n = 3). 4Means with different superscripts in the same column are significantly different by Tukey-Kramer's HSD (p < 0.05). 5Extract from Brugmansia arborea L. flowers. 6n-BuOH fraction from Brugmansia arborea L. flowers. 7EtOAc fraction from Brugmansia arborea L. flowers. 8No determined.

Protective Effects on Pancreatic Islets in Zebrafish Treated by Alloxan
Extracts, solvent fractions (EtOAc, n-BuOH), and isolated phenylalkyl glucosides 1-6 from B. arborea flowers were evaluated for protective activity against the pancreatic islets of zebrafish larvae damaged by alloxan.The larvae treated with alloxan were used to model type 1 diabetes due to their physiological similarities to mammals [21,22].Alloxan is a diabetogenic chemical that has been reported to decrease β-cell mass in pancreatic islets [23].To assess pancreatic islets treated with alloxan, the size changes of the pancreatic islets and fluorescence intensities of the NBDG-stained pancreatic islets under a fluorescence microscope were analyzed.When the zebrafish larvae were exposed to alloxan, pancreatic islet size decreased significantly by 51.8% (p = 0.0003) compared to the normal group (Figure 2a).Zebrafish larvae treated with glimepiride, a positive control, showed a pancreatic islet increase of 89.5% (p = 0.0047) compared to the alloxan group.The pancreatic islet sizes in the groups treated with EtOAc (BAFE) and n-BuOH (BAFB) fractions significantly increased up to 64.0 and 69.4% (p 0.0091 and 0.0065) compared with alloxan treatment (Figure 2).All of the phenylalkyl glucosides from B. arborea flowers also resulted in increases in pancreatic islet size.Compounds 1-6 (BAP 1-6) increased the injured pancreatic islets up to 82.0, 89.8, 80.0, 97.8, 103.1, and 99.6% (p 0.0011, 0.0037, 0.0012, 0.0002, 0.0011, 0.0011, and 0.0011), respectively, compared with alloxan treatment (Figure 2).All phenylalkyl glucosides isolated from B. arborea flowers in this study increased the sizes of pancreatic islets damaged by alloxan treatment in zebrafish larvae with high levels of significance.In particular, phenylpropyl glucoside 5 and the new phenylbutyl glucoside 6 displayed recovery effects greater than glimepiride.These diverse rates also showed structure-activity relationships.Monoglycoside compounds 1 and 3 exhibited lower activity than diglycoside compounds 2 and 4, respectively.And, compared to compounds 1, 3, and 5, the propyl group attached to phenyl was more effective than the methyl and ethyl groups.
structure-activity relationships.Monoglycoside compounds 1 and 3 exhibited lower activity than diglycoside compounds 2 and 4, respectively.And, compared to compounds 1, 3, and 5, the propyl group attached to phenyl was more effective than the methyl and ethyl groups.

Action of Diazoxide (DZ) on Alloxan-Induced Pancreatic Islets in Zebrafish
The metabolism of glucose in pancreatic cells is the key step in glucose-stimulated insulin secretion [24].To study the involvement of the pancreatic β-Cell KATP channel stimulation activity, diazoxide (DZ), a KATP channel opener, was used.The size of the pancreatic islets in the DZ-treated normal group was significantly smaller (43.4%, p = 0.0052) relative to the normal group without DZ treatment.Furthermore, the alloxan (AX) group showed no significant difference compared to the DZ-treated group.Pancreatic islet size in the 10 µg/mL glimepiride (GLM), AX, and DZ co-treatment groups was significantly lower (48.9%,p = 0.0202) compared to the 10 µg/mL GLM and AX co-treated groups without DZ.Groups co-treated with compounds 1, 2, 4, or 6 and AX were not significantly

Action of Diazoxide (DZ) on Alloxan-Induced Pancreatic Islets in Zebrafish
The metabolism of glucose in pancreatic cells is the key step in glucose-stimulated insulin secretion [24].To study the involvement of the pancreatic β-Cell KATP channel stimulation activity, diazoxide (DZ), a K ATP channel opener, was used.The size of the pancreatic islets in the DZ-treated normal group was significantly smaller (43.4%, p = 0.0052) relative to the normal group without DZ treatment.Furthermore, the alloxan (AX) group showed no significant difference compared to the DZ-treated group.Pancreatic islet size in the 10 µg/mL glimepiride (GLM), AX, and DZ co-treatment groups was significantly lower (48.9%,p = 0.0202) compared to the 10 µg/mL GLM and AX co-treated groups without DZ.Groups co-treated with compounds 1, 2, 4, or 6 and AX were not significantly different after treatment with DZ, indicating no relationship with K ATP channels.Compounds 3 and 5, in addition to AX, yielded significantly smaller pancreatic islet sizes after treatment with DZ (3: 57.6%, p = 0.0358; 5: 69.3%, p = 0.0358) compared to the compound+AX groups (Figure 3).These results suggest that compounds 3 and 5 might stimulate insulin secretion by Ca 2+ influx via the closure of the K ATP channels in β-cells.
different after treatment with DZ, indicating no relationship with KATP channels.Compounds 3 and 5, in addition to AX, yielded significantly smaller pancreatic islet sizes after treatment with DZ (3: 57.6%, p = 0.0358; 5: 69.3%, p = 0.0358) compared to the com-pound+AX groups (Figure 3).These results suggest that compounds 3 and 5 might stimulate insulin secretion by Ca 2+ influx via the closure of the KATP channels in β-cells.

Plant Materials
The dried flowers of B. arborea L. were supplied by Herb Island, Pocheon, Korea, in June 2014 and were identified by Professor Dae-Keun Kim, Woosuk University, Jeonju, Korea.A voucher specimen (KHU2014-0623) is reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Plant Materials
The dried flowers of B. arborea L. were supplied by Herb Island, Pocheon, Korea, in June 2014 and were identified by Professor Dae-Keun Kim, Woosuk University, Jeonju, Korea.A voucher specimen (KHU2014-0623) is reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Free Radical Scavenging Activity
The materials, equipment, and methods used for the free radical scavenging assay of extract, solvent fractions, and compounds 1-6 from B. arborea flowers were described in a previous study [17].

Chemicals and Animals
The chemical materials and animal preparation used for the antidiabetic activity are described in a previous study [25].

Ethics Statement
All zebrafish experimental procedures were carried out in accordance with standard zebrafish protocols and were approved by the Animal Care and Use Committee of Kyung Hee University [KHUASP(SE)-15-10].

Evaluation of Recovery Efficacy on Pancreatic Islet Damaged by Alloxan in Zebrafish
The materials, equipment, and methods used for evaluation of recovery efficacy of extract (BAF), solvent fractions (BAFE and BAFB), and compounds 1-6 from B. arborea flowers on pancreatic islet damaged by alloxan in zebrafish are described in a previous study [25].

Action of Diazoxide on Alloxan-Induced Diabetic Zebrafish
The materials, equipment, and methods used for the action of diazoxide on alloxaninduced diabetic zebrafish are described in a previous study [25].

Conclusions
This study endeavored to find new active compounds of B. arborea flowers.Six phenylalkyl glucosides, including one new phenylbutyl diglucoside, were isolated through repeated column chromatography using SiO 2 , ODS, and Sephadex LH-20 resins and iden-

Figure 1 .
Figure 1.Chemical structures of phenylalkyl glucosides 1-6 isolated from the flowers of Brugmansia arborea.Glc: β-D-glucopyranosyl; the gHMBC key correlations are represented by single-headed arrows from H to C.

Figure 1 .
Figure 1.Chemical structures of phenylalkyl glucosides 1-6 isolated from the flowers of Brugmansia arborea.Glc: β-D-glucopyranosyl; the gHMBC key correlations are represented by single-headed arrows from H to C.

Table 1 .
Radical scavenging capacity of extract, solvent fractions, and isolated compounds from Brugmansia arborea L. flowers using ABTS and DPPH radicals.