Characterization of Secondary Metabolites from Purple Ipomoea batatas Leaves and Their Effects on Glucose Uptake

Ipomoea batatas has long been used in folk medicine for the treatment of hyperglycemia or as a food additive for the prevention of type 2 diabetes. However, neither the plant extract nor its active components have been evaluated systematically. In this work four crude extracts, including n-hexane- (IBH), 95% MeOH- (IBM), n-BuOH- (IBB), and H2O-soluble (IBW) fractions, were prepared by fractionation of a methanolic extract of purple I. batatas leaves. Twenty-four pure compounds 1–24 were then isolated by various chromatographic techniques and their structures identified from NMR and MS data. Glucose uptake assays in differentiated 3T3-L1 adipocytes and rat primary hepatocytes, as well as western blot analysis, were carried out to evaluate the antidiabetic activity of this species. The IBH crude fraction, with methyl decanoate (22) as a major and active compound, showed the greatest effect on glucose uptake, most likely via activation of Glut4 and regulation of the PI3K/AKT pathway. Quercetin 3-O-β-d-sophoroside (1), quercetin (3), benzyl β-d-glucoside (10), 4-hydroxy-3-methoxybenzaldehyde (12), and methyl decanoate (22) could be important components contributing to the antidiabetic effects. We conclude that purple I. batatas leaves have potential as an antidiabetic plant source and the active constituents 1, 3, 10, 12, and 22 are promising lead candidates for future investigation.


Introduction
Ipomoea batatas (L.) (Convolvulaceae), commonly known as sweet potato, is a well-known valuable medicinal food. While the roots and leaves of I. batatas play important roles as an energy source for humans and animals, they also have been used in traditional medicine for the treatment of various diseases [1]. Numerous pharmacological properties, including antidiabetic (caffeic acid derivatives, anthocyanosides, flavonoids, arabinogalactan-protein) [1][2][3], anti-oxidant (caffeic acid derivatives, anthocyanosides, coumarins) [1,4,5], anticancer (caffeic acid derivatives, anthocyanosides, coumarins) [1], antimicrobial (caffeic acid derivatives, triterpenes) [1], anticoagulant (coumarins) [1], and anti-inflammatory (resin glycosides) [6] activities, have been reported for this species. The green I. batatas leaves have long been used in folk medicine for the treatment of hyperglycemia or as a food additive for the prevention of type 2 diabetes [7][8][9], while the purple I. batatas leaves contain large quantities of rough fibers and in Taiwan they are usually discarded or fed to animals. A systematic investigation of the active antidiabetic compounds isolated from the latter variety has not been reported. Therefore, as part of our studies to identify promising antidiabetic drugs from natural products, the active components of purple sweet potato leaves were elucidated herein.

Glucose Uptake Efficacy of Four Crude Fractions Prepared from Purple I. batatas Leaves
A MeOH extract of the aerial parts of purple I. batatas was separated into n-hexane-(IBH), 95% MeOH-(IBM), n-BuOH-(IBB), and H 2 O-soluble (IBW) fractions by liquid-liquid partition chromatography (see Section 3.3. Extraction and Isolation). 3T3-L1 adipocyte and primary rat hepatocyte models were used to evaluate the glucose uptake efficacy of the above four fractions.
In MTT and trypan blue assays, IBM was cytotoxic to 3T3-L1 preadipocytes ( Figure 1A,B). In contrast, IBH, IBB, and IBW exhibited no significant cytotoxicity toward differentiated 3T3-L1 preadipocytes in the MTT assay ( Figure 1C), while IBB was cytotoxic to primary rat hepatocytes ( Figure 1D). Therefore, only IBH and IBW were tested for glucose uptake in 3T3-L1 adipocytes and rat hepatocytes. IBH showed a significant effect in both models (Figure 2A,B), while IBW affected glucose uptake activity only in the latter model. anthocyanosides, coumarins) [1,4,5], anticancer (caffeic acid derivatives, anthocyanosides, coumarins) [1], antimicrobial (caffeic acid derivatives, triterpenes) [1], anticoagulant (coumarins) [1], and anti-inflammatory (resin glycosides) [6] activities, have been reported for this species. The green I. batatas leaves have long been used in folk medicine for the treatment of hyperglycemia or as a food additive for the prevention of type 2 diabetes [7][8][9], while the purple I. batatas leaves contain large quantities of rough fibers and in Taiwan they are usually discarded or fed to animals. A systematic investigation of the active antidiabetic compounds isolated from the latter variety has not been reported. Therefore, as part of our studies to identify promising antidiabetic drugs from natural products, the active components of purple sweet potato leaves were elucidated herein.

Glucose Uptake Efficacy of Four Crude Fractions Prepared from Purple I. batatas Leaves
A MeOH extract of the aerial parts of purple I. batatas was separated into n-hexane-(IBH), 95% MeOH-(IBM), n-BuOH-(IBB), and H2O-soluble (IBW) fractions by liquid-liquid partition chromatography (see Section 3.3. Extraction and Isolation). 3T3-L1 adipocyte and primary rat hepatocyte models were used to evaluate the glucose uptake efficacy of the above four fractions.

Glucose Uptake Effect of the Components from IBH, IBM, IBB, and IBW
Adipocytes regulation of glucose uptake can decrease postprandial hyperglycemia. To determine the effect of the isolates from purple I. batatas leaves on glucose uptake and identify potential promising antidiabetic natural products leads, we evaluated selected isolated compounds in an insulin-stimulated glucose uptake assay using differentiated 3T3-L1 adipocytes and a fluorescent D-glucose analog (2-NBDG). Because the IBH fraction showed significant effects on glucose uptake (Figure 2), its three major components 22-24 (Table 1), purchased from commercial companies (see Section 3.1. General Procedures), were studied. Among these three compounds, only methyl decanoate (22) increased glucose uptake (27.5%) relative to the control ( Figure 5).

Glucose Uptake Effect of the Components from IBH, IBM, IBB, and IBW
Adipocytes regulation of glucose uptake can decrease postprandial hyperglycemia. To determine the effect of the isolates from purple I. batatas leaves on glucose uptake and identify potential promising antidiabetic natural products leads, we evaluated selected isolated compounds in an insulin-stimulated glucose uptake assay using differentiated 3T3-L1 adipocytes and a fluorescent D-glucose analog (2-NBDG). Because the IBH fraction showed significant effects on glucose uptake (Figure 2), its three major components 22-24 (Table 1), purchased from commercial companies (see Section 3.1. General Procedures), were studied. Among these three compounds, only methyl decanoate (22) increased glucose uptake (27.5%) relative to the control ( Figure 5). Differentiated 3T3-L1 adipocytes were treated with compound for 30 min, and then 2-NBDG uptake was measured. Compounds 4-5, 7-9, 13-17, and 20-21 were cytotoxic toward 3T3-L1 adipocytes and were not tested in this assay. ANOVA statistical analysis: *, p < 0.05; **, p < 0.01; ***, p < 0.005.

GC/MS Analysis of the n-Hexane-soluble Fraction (IBH)
(1) HS-SPME analysis: A 50/30-µm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Inc.) was used for aroma extraction. IBH samples was put into a 7 mL vial (Hole Cap PTFE/Silicone Septa) and sealed. The SPME fiber was exposed to each sample for 30 min at 25˝C, after which each sample was injected into a gas chromatograph injection unit [48]. The injector and detector temperatures were maintained at 250˝C and 300˝C, respectively. The oven temperature was held at 40˝C for 1 min and then raised to 150˝C at 5˝C/min and held for 1 min, finally raised to 200˝C at 10˝C/min and held for 11 min. The carrier gas (nitrogen) flow rate was 1 mL/min. Kovats indices were calculated for the separated components relative to a C 5 -C 25 n-alkane mixture [49]. Percentage composition was calculated using the peak area normalization measurements.

LC/MS Analysis of H 2 O-Soluble Fraction (IBW)
A UHPLC system (Ultimate 3000; Dionex, Germering, Germany) equipped with a C18 reversed-phase column (2.1ˆ150 mm, 3 µm, T3; Waters, Milford, MA, USA) was coupled with a hybrid Q-TOF mass spectrometer (maXis impact, Bruker Daltonics, Bremen, Germany) with an orthogonal electrospray ionization (ESI) source. The initial flow rate was 0.25 mL/min of 99% solvent A (0.1% formic acid) and 1% solvent B (MeCN with 0.1% formic acid). A volume of 2 µL of sample was injected. After injection, solvent B was maintained at 1% for 4 min, then increased to 45% during a span of 14 min, and finally to 99% over a period of 2 min after which this percentage composition was held for 2 min. After 0.5 min, solvent B was reduced back to 1% and held at this percentage for 2.5 min.
The mass spectrometer was operated in either positive or negative ion mode using the m/z range 50-1000 at 1 Hz (summation value of 9839). The capillary voltage of the ion source and negative was set at +4500 V for positive mode and´2500 V for negative mode, and the endplate offset was 500 V. The nebulizer gas flow was 1 bar and drying gas flow was 8 L/min. The drying temperature was set at 200˝C. Funnel 1 radiofrequency (RF) and Funnel 2 RF were both 200 Vpp. The hexapole RF was 30 Vpp and the low mass cutoff of quadrupole was 100 m/z. For the MS/MS settings, the eight most intense ions from each MS full scan spectrum were automatically selected as the precursor ion peaks for the following auto MS/MS experiments.

Acid Hydrolysis and Reversed-Phase HPLC Analysis of IBW
The IBW crude fraction (10.0 mg) was hydrolyzed in 1 M HCl/1,4-dioxane (1:1, 2.0 mL) at 90˝C for 3 h and then partitioned with CH 2 Cl 2 /H 2 O (1:1). The aqueous layer was neutralized with Amberlite IRA400. After drying, the residue was dissolved in pyridine (1.0 mL) containing L-cysteine methyl ester hydrochloride (10.0 mg) and heated at 60˝C for 1 h. A 10 µL solution of o-tosyl isothiocyanate in pyridine was added to the mixture, which was heated at 60˝C for 1 h. The final reaction mixture was directly analyzed by HPLC: Analytical HPLC was performed on a 250ˆ4.6 mm i.d. Cosmosil 5C18-AR II column at 35˝C with isocratic elution of 25% CH 3 CN in 50 mM H 3 PO 4 for 40 min at a flow rate 0.8 mL/min [50].

Cell Culture
The 3T3-L1 preadipocytes were cultured as previously described [51]. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) with high glucose containing 10% (v/v) FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in plates (10 5 cells/mL) at 37˝C in a humidified atmosphere of 10% CO 2 . The medium was changed every two days. Primary rat hepatocytes (HCs) were isolated and cultured as previously described [52]. Primary HCs were suspended in William's Medium E containing 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 0.86 µM insulin, 0.5 nM dexamethasone, and 10 mM HEPES and were plated on collagen-coated dishes (5ˆ10 5 cells/mL). Cells were cultured at 37˝C with 5% CO 2 for 3 h for attachment and were washed twice with PBS. The medium was then changed, and, the cells were used for experiments after overnight incubation.

Cell Viability and Proliferation
The cells were treated with different concentrations of crude fractions or pure compounds for the indicated time points. Cell viability was assayed using the Trypan blue exclusion method. The cells were stained with Trypan blue. The unstained (viable) and stained (dead) cells were counted separately using a hemocytometer under a microscope (Nikon TS100). The cell viability was calculated based on the ratio of viable cells to total cell population in each plate. Cell proliferation was assayed using MTT metabolic analysis. MTT was added to the cell medium, and after incubation at 37˝C for 4 h, the blue formazan reduction product was dissolved in isopropanol and measured on an ELISA reader at 570 nm [51].

Differentiation of 3T3-L1 Cells into Mature Adipocytes
3T3-L1 cells were cultured from preadipocytes and differentiated into adipocytes as previously described. Preadipocytes were grown in Dulbecco's modified Eagle's medium (DMEM) with high glucose containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in plates (10 5 cells/mL) at 37˝C in a humidified atmosphere of 10% CO 2 . The medium was changed every 2 days. Then, two days after confluence, the medium was replaced with DMEM (high glucose) containing 10% FBS and adipogenic agents (1.7 µM insulin, 0.25 µM DMX, and 0.5 mM IBMX); this day was designated day 0 and occurred after three days of culturing. The cells were then grown in DMEM containing 10% FBS, and the medium was changed every two days. Differentiated cells were used for experimentation on day 9 when the proportion of differentiated cells reached up to 90%. The differentiated cells were identified by Oil Red O staining and by gradient centrifugation with Percoll [53].

Western Blot Analysis
The cells were treated as indicated, detached, thoroughly washed with PBS, and then lysed in ice-cold lysis buffer. Following centrifugation at 13,000ˆg for 10 min at 4˝C, the supernatants (30 µg protein) were boiled with reducing sample buffer for 5 min, subjected to electrophoresis in SDS-polyacrylamide gels, and then transferred onto a PVDF membrane. The membrane was blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature and then washed with PBST. Proteins were detected by incubating the membrane overnight at 4˝C with antibodies against β-actin (Sigma-Aldrich), Glut4 (IF8), p-Glut4 (Ser 488) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt (pan), p-Akt (Ser 473), p-PI3K (Tyr458/Tyr199) (Cell Signaling Technology, Danvers, MA, USA). Next, the membrane was washed with PBST, and, finally, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for 1 h. An enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Arlington Heights, IL, USA or Millipore, Billerica, MA, USA) was used for protein detection.

Glucose Uptake Assay
The differentiated 3T3-L1 adipocytes or primary rat hepatocytes were cultured with low glucose DMEM for 6 h, and then PBS including 1% bovine serum albumin (BSA) was added. The crude extracts (0.1 mg/mL) and pure compounds (0.01 mg/mL) were added to the medium for the indicated time and then stimulated with 10´7 M insulin at 37˝C for another 30 min. A 10´4 M fluorescent D-glucose analog (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, 2-NBDG) was added to the former 3T3-L1 adipocytes for 30 min and then PBS was used to wash the cells three times. Some cell lysis buffers were used to obtain the cell extracts, which were homogenized with an ultrasonicator to assay the fluorescence with excitation and emission at 485 and 535 nm, respectively.

Conclusions
In conclusion, four crude fractions (IBH, IBM, IBB, and IBM) were prepared from purple I. batatas leaves and 21 compounds were isolated. The IBH crude extract, with methyl decanoate (22) as a major and active component, showed antihyperglycemic potential in vitro. In a glucose uptake assay, flavonoids 1 and 3, as well as benzene derivatives 10 and 12, were identified as promising lead compounds, especially quercetin (3). Overall, our data systematically demonstrated that purple sweet potato leaves are a potential antidiabetic plant source. We believe that further investigation is definitely merited.