Benzophenone C- and O‑Glucosides from Cyclopia genistoides(Honeybush) Inhibit Mammalian α‑GlucosidaseTheresa Beelders,*,†,‡ D. Jacobus Brand,§ Dalene de Beer,‡ Christiaan J. Malherbe,‡

Sithandiwe E. Mazibuko,⊥ Christo J. F. Muller,⊥ and Elizabeth Joubert†,‡

†Department of Food Science, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa‡Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026,Stellenbosch 7599, South Africa§Central Analytical Facility (CAF), Nuclear Magnetic Resonance Unit, Private Bag X1, Matieland 7602, South Africa⊥Diabetes Discovery Platform, South African Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa

*S Supporting Information

ABSTRACT: An enriched fraction of an aqueous extract prepared from the aerial parts of Cyclopia genistoides Vent. yielded anew benzophenone di-C,O-glucoside, 3-C-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone (1), together with smallquantities of a known benzophenone C-glucoside, 3-C-β-D-glucopyranosylmaclurin (2). The isolated compounds showed α-glucosidase inhibitory activity against an enzyme mixture extracted from rat intestinal acetone powder. Compound 2 exhibitedsignificantly (p < 0.05) higher inhibitory activity (54%) than 1 (43%) at 200 μM. In vitro tests in several cell models showed that1 and its 3-C-monoglucosylated derivative (3-C-β-D-glucopyranosyliriflophenone) were marginally effective (p ≥ 0.05) inincreasing glucose uptake.

The global prevalence of diabetes is increasing at alarmingrates, leading to estimates of 439 million diabetics by

2030.1 In Africa, South Africa tips the scale with a nationalprevalence of over 7%.2 It is projected that diabetes will rank asthe ninth leading cause of death in low-income countries in thenext few decades.3 A sedentary lifestyle together with anunhealthy diet, high in refined carbohydrates accompanied by alow intake of fruits and vegetables, are considered contributingfactors. The search for natural and synthetic α-glucosidaseinhibitors4,5 that delay the breakdown and absorption ofcarbohydrates in the gut, thus mimicking the protective effect ofthe drug acarbose, but without its side-effects,6 is escalating.Much prominence is given to polyphenols, with promisingresults in vitro.4 Synergistic effects between acarbose andpolyphenols suggest benefits in terms of dose reduction of thedrug.7 Subclasses of phenolic compounds that show promise asα-glucosidase inhibitors include the polyhydroxyxanthones andpolyhydroxybenzophenones, together with their glycosylatedderivatives.8−11 Extracts of Cyclopia genistoides Vent. (honey-bush) are principally renowned for high levels of thetetrahydroxyxanthone C-glucoside mangiferin,12 a bioactivecompound13 with potent α-glucosidase inhibitory activity.9

Recent investigation of the phenolic constituents of C.genistoides revealed the presence of several benzophenone C-glucosides, including 3-C-β-D-glucopyranosyliriflophenone and3-C-β-D-glucopyranosylmaclurin.14−16 The major benzophe-none glycoside present in hot water extracts of C. genistoideshas been tentatively identified as an iriflophenone di-O,C-hexoside thus far.16 A compound with similar MS propertieshas also been observed in extracts of another Cyclopia species,C. subternata,17 albeit at lower concentrations. In the presentpaper the isolation and structural elucidation of thisbenzophenone glucoside, identified as 3-C-β-D-glucopyrano-syl-4-O-β-D-glucopyranosyliriflophenone (1), is described.During the isolation process, small quantities of the knownbenzophenone C-glucoside 3-C-β-D-glucopyranosylmaclurin(2) was also obtained. These compounds were isolated fromthe aerial parts of C. genistoides using solid-phase extraction(SPE) for extract enrichment, followed by purification bysemipreparative liquid chromatography (LC). The ability of 1and 2 to inhibit mammalian α-glucosidase was assessed and

Received: September 16, 2014

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compared to that of 3-C-β-D-glucopyranosyliriflophenone.Included in the α-glucosidase assay was the benzophenoneaglucone maclurin, with acarbose employed as positive control.To date, the α-glucosidase inhibitory activities of 1 and 2 as C-and O-glucosylated polyhydroxybenzophenones have not beenassessed. In vitro tests in several cell models were conducted toinvestigate the ability of 1 and 3-C-β-D-glucopyranosyliriflo-phenone to increase glucose uptake in vitro.

■ RESULTS AND DISCUSSIONCompound 1 was obtained as a white, amorphous powder. Itsmolecular formula was established as C25H30O15 from theanalyses of its HRESIMS and 13C NMR data. Initial LC-ESIMSMS analyses of this compound in a C. genistoides samplematrix16 had pointed to an iriflophenone di-O,C-hexoside. Theelucidation of the structure of 1 and, in particular, theidentification of the hexoside moieties and their point ofattachment to the aglycone, were done via 1D and 2D NMRexperiments. The relative configurations of the glycoside unitscan normally be determined by a 1D/2D NOESY NMRexperiment. However, NMR spectroscopic data for 1 provedinconclusive for unambiguous identification of the individualglycoside units, due to overlapping proton resonances. Sincethe NMR spectra of 1 closely resembled those of 3-C-β-D-glucopyranosyliriflophenone, it was essential to first selectivelyhydrolyze and identify the terminal O-linked sugar moiety,followed by comparison of the resulting iriflophenone C-hexoside to that of a commercial 3-C-β-D-glucopyranosyl-iriflophenone standard, also present in C. genistoides.18

The terminal O-linked hexoside moiety was identified as D-glucose by enzymatic hydrolysis and GC-MS analysis. Theresulting iriflophenone C-hexoside was purified, and its 1H and13C NMR data matched those of 3-C-β-D-glucopyranosyl-iriflophenone. Having identified the β-D-glucopyranosyl con-stituent units, the rest of the molecule and the point ofattachment of the O-β-D-glucopyranosyl unit were defined byadditional 1D NOESY, COSY, HSQC, and HMBC NMRexperiments.The aromatic region between 6.0 and 8.5 ppm of the 1H

NMR spectra showed two o-coupled doublets and a singlet, δ7.61 (2H, d, J = 8.6 Hz), 6.78 (2H, d, J = 8.6 Hz), 6.24 (1H, s),reminiscent of an AA′BB′ aromatic spin system with anuncoupled singlet on a separate aromatic ring and supported bythe presence of aromatic carbons in the 13C NMR spectra of 1.The 1H NMR spectra also presented the resonances of two β-coupled (∼8 Hz anomeric coupling constant) glucoside units inthe characteristic 2.5−5.0 ppm region.19 The presence of asingle carbon at δ 193.1 in addition to the 12 aromatic carbonsin the 13C NMR spectrum was reminiscent of a benzophenoneaglycone unit.19 The anomeric protons of the two glucopyr-

anosyl moieties at δ 4.73 (1H, d, J = 7.8 Hz) and 4.66 (1H, d, J= 9.8 Hz) were irradiated selectively utilizing a 1D NOESYexperiment to determine their position of attachment and otherproton resonances of the individual O- and C-linkedglucopyranosyl units. The anomeric proton of the glucopyr-anosyl residue at δ 4.73 (1H, d, J = 7.8 Hz, H-1‴) displayed astrong NOE association with the H-5 singlet at δ 6.24. Thisassigned the O-linked glucosyl unit to C-4 of thebenzophenone A-ring. A weak NOE association of H-1‴ withH-4″ (δ 3.24) of the neighboring glucosyl moiety indicated theclose proximity of the glucosyl entities.An NOE association was also observed between the three

axial H-1‴, H-3‴, and H-5‴ protons (Figure 1), affirming the

glucose configuration of the O-linked sugar unit. The nature ofthe C-linked sugar to the aglycone was confirmed bycomparison of the hydrolytic products to the NMR spectraof the commercial 3-C-β-D-glucopyranosyliriflophenone stand-ard.A COSY experiment confirmed the AA′BB′ aromatic spin

system of the B-ring and the connectivity of some of theprotons on the individual glucosyl moieties. This includes onlythe protons that are sufficiently resolved to be assigned by theCOSY experiment and did not overlap with the glucosyl protonresonances.The HSQC NMR experiment was used to assign all the

directly bonded protons (1JHC) to their respective carbons. Allthe carbons were resolved successfully by an HMBC experi-ment showing the long-range (2JHC,

3JHC) connectivity betweenprotons and carbons [up to four bonds (4JHC) in some cases].Those structurally significant HMBC correlations are shown inFigure 2, enabling the assignment of the unprotonated carbonsin the molecule. Compound 1 was thus identified as 3-C-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone.Compound 2 was obtained as a light yellow, amorphous

powder. Accurate measurement of the pseudomolecular ions inthe positive and negative HRESIMS data, in conjunction with13C NMR data, allowed a molecular formula of C19H20O11 to beassigned to 2. The structure of 2 was confirmed as 3-C-β-D-

Figure 1. Relevant NOE associations in 1.

Figure 2. Relevant long-range HMBC correlations in 1.

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glucopyranosylmaclurin by comparison of its observed andreported 1H NMR and 13C NMR spectroscopic data.14,20

Both benzophenone derivatives 1 and 2, together with thereference compounds, showed inhibitory activity againstmammalian α-glucosidase at various concentrations with allvalues significantly differing (p < 0.05) from all other values.The observed inhibitory activities were both concentration- andcompound-specific (Figure 3), showing a clear dose−response.

The activities of the benzophenones could thus be compared atequimolar concentration levels, which gave insight into possiblestructure−activity relations. Compound 2 was the most activeinhibitor, followed by 3-C-β-D-glucopyranosyliriflophenone and1, while maclurin showed the weakest inhibitory activity. Itappears that C-monoglucosides tend to be more effectiveinhibitors than their corresponding aglucones; that is, 2 issignificantly more active than maclurin (p < 0.05). This has alsobeen observed previously for 3-C-β-D-glucopyranosyl-iriflophenone and its aglucone, iriflophenone.9 Furthermore,the higher activity of 2 compared to that of 3-C-β-D-glucopyranosyliriflophenone was attributed to the additional3′-hydroxy group of 2. Previous studies on hydroxybenzophe-nones10 and hydroxyxanthones8 have shown that increases inthe number of phenolic hydroxy groups on the basic diphenylketone and dibenzo-γ-pyrone structures lead to significantincreases in α-glucosidase inhibitory activities. Moreover, forhydroxyflavones it has been shown that a 3′-hydroxy group inparticular leads to increased inhibitory activity.21 The presenceof an additional O-glucopyranosyl moiety at C-4 on thediphenyl ketone structure of 1 significantly (p < 0.05) loweredα-glucosidase inhibitory activity compared to 3-C-β-D-glucopyranosyliriflophenone. A similar decrease in activitydue to an additional C-glucopyranosyl moiety has also beenreported for 3-C-β-D-glucopyranosyl-5-C-β-D-glucopyranosyl-iriflophenone compared to 3-C-β-D-glucopyranosyl-iriflophenone.9

The effect of 1 and 3-C-β-D-glucopyranosyliriflophenone onin vitro glucose uptake in L6 myocytes demonstrated marginal,but not significant, increases in glucose uptake relative to thevehicle control at concentrations of 10 μM of 1 (ca. 22%increase; Figure 4A) and 100 μM of 3-C-β-D-glucopyranosyl-

iriflophenone (ca. 27% increase; Figure 4B). Similar marginaleffects on glucose uptake were observed in 3T3-L1 adipocytes(Figure 4C and D) and in C3A hepatocytes (data not shown).It was anticipated that the isolated benzophenone glucoside (1)and 3-C-β-D-glucopyranosyliriflophenone could increase invitro glucose uptake activity by activating the cellular energyregulator AMPK, as demonstrated for the latter compound22,23

in mature 3T3-L1 adipocytes and diabetic KK-Ay mice. Theassessed compounds were, however, less effective at increasingcellular glucose uptake than the reference pharmacologicalagent metformin, mechanistically known to be an activator ofAMPK.24

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. NMR spectra were recorded

on a Varian Unity Inova 600 NMR spectrometer with a 1H frequencyof 600 MHz and a 13C frequency of 150 MHz using a 5 mm inversedetection PFG probe. The chemical shift frequencies are indicated onthe δ scale. 1H and 13C NMR spectra were referenced to the residualDMSO-d6 peak at δ 2.5 and 39.5, respectively. The spectra in acetone-d6 have the residual acetone peaks referenced at δ 2.05 for 1H NMRand at δ 29.84 for the 13C NMR spectra. The spectra were recordedusing the standard VnmrJ instrument software and processed andexpanded further using the Mestrenova 9.0 software package.HRESIMS analyses were conducted on an Acquity UPLC system,fitted with a photodiode-array detector and coupled to a Synapt G2 Q-TOF equipped with an electrospray ionization source (Waters,Milford, MA, USA). UV spectra were recorded online. Chromato-graphic conditions and MS parameters were as described byBeelders.16 Semipreparative LC was performed on a LaChromHPLC system (Merck Hitachi, Hitachi High Technologies, Japan)comprising a quaternary pump, autosampler, variable-wavelengthdetector, and diode-array detector and fitted with a PhenomenexGemini C18 column (5 μm, 110 Å, 150 × 10 mm) (Phenomenex,Torrance, CA, USA). The column was protected by a guard column ofthe same stationary phase (10 × 10 mm) and a high-pressuresemipreparative in-line filter (IDEX Health & Science, Oak Harbor,WA, USA). Column temperature was maintained at 30 °C using anexternal HPLC column oven (LKB Bromma, Sweden). Gaschromatography was performed on an Agilent GC-MS instrument(Agilent 6890N GC and Agilent 5975 MSD, Agilent Technologies,Palo Alto, CA, USA) fitted with a 30 m Zebron ZB-SemiVolatilescolumn with 0.25 mm inner diameter and 0.25 μm film thickness. TheGC chromatograms and mass spectra were evaluated using AgilentMSD ChemStation (version D.02.00.237) software.

Plant Material. The leaves and fine stems from a selection ofCyclopia genistoides bushes (Overberg type) were harvested from acommercial plantation situated near Pearly Beach (Western Cape,South Africa; GPS coor. −34.702, 19.618). Plant material was driedwithout delay in a cross-flow drying tunnel at 40 °C for 16 h to amoisture content of less than 10% and pulverized using a Retsch rotarymill (1.4 mm sieve; Retsch GmbH, Haan, Germany).

Extraction and Isolation. Plant material (420 g) was extractedwith hot water (4.2 L, 93 °C) for 30 min using a ratio of 1:10 (w/v).The crude extract was filtered through Whatman #4 filter paper,frozen, and freeze-dried using a VirTis Advantage Plus freeze-drier (SPScientific, Warminster, PA, USA). The freeze-dried hot water extract(126.5 g) comprised 6.31% of 1 and 0.53% of 2.

Enrichment of the crude extract in terms of 1 and 2 was performedusing C18 solid-phase extraction (Discovery DSC-18; 10 g/60 mL;Sigma-Aldrich). The cartridge was conditioned sequentially withMeOH and deionized water (60 mL). A solution of freeze-driedextract (300 mg reconstituted in 50 mL of deionized water) wasapplied to the cartridge, followed by flushing with deionized water(100 mL). The target analytes were eluted with 5% aqueous MeOH(300 mL). This process was repeated 48 times with new cartridges(14.4 g of freeze-dried extract loaded), and the eluants were pooled,vacuum-evaporated, and freeze-dried, yielding ca. 1.2 g of enriched

Figure 3. Percentage activity of rat α-glucosidase challenged withvarious concentrations of 1, 3-C-β-D-glucopyranosyliriflophenone, 2,and maclurin.

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fraction. The enriched fraction comprised 56.5% of 1 and 4.8% of 2.The average recovery of target analytes after SPE was 75%.The enriched fraction was reconstituted in deionized water (ca. 6

mg/mL), filtered, and subjected to semipreparative LC (MeCN−2%HOAc (aq), 4:96, v/v) using a flow rate of 4.8 mL/min. Aliquots (400μL) of the reconstituted, enriched fraction were injected repeatedly,equaling 880 mg. The fractions containing 1 and 2 were collectedbased on retention times using a Gilson FC203B fraction collector(Gilson, Middleton, WI, USA) and pooled, and the organic solventwas evaporated under vacuum. The remaining aqueous solutions werefiltered, frozen, and freeze-dried, yielding 370 mg of 1 (purity >99% byHRESIMS; total yield of 74% from the enriched extract) and 30 mg of2 (purity 95% by HRESIMS; total yield of 71% from the enrichedextract).3-C -β -D -Glucopyranosyl -4-O -β -D-g lucopyranosy l -

iriflophenone (1): white, amorphous powder; UV λmax online 234,294 nm; 1H NMR 600 MHz (DMSO-d6, 298 K) δ 7.61 (2H, d, J = 8.6Hz, H-2′,6′), 6.78 (2H, d, J = 8.6 Hz, H-3′,5′), 6.24 (1H, s, H-5), 4.73(1H, d, J = 7.8 Hz, H-1‴), 4.66 (1H, d, J = 9.8 Hz, H-1″), 3.6 (3H, m,H-6″, 2 × H-6‴), 3.46 (2H, m, H-2″, H-6″), 3.25 (3H, m, H-3″, H-4″,H-5″), 3.18 (2H, m, H-3‴, H-5‴), 3.08 (1H, t, J = 9.2 Hz, H-4‴), 2.88(1H, t, J = 8.4 Hz, H-2″); 13C NMR 150 MHz (DMSO-d6, 298 K) δ193.1 (C, CO), 161.8 (C, C-1′), 157.7 (C, C-1), 155.2 (C, C-2),155.2 (C, C-4), 131.8 (CH, C-3′, 5′), 130.2 (CH, C-4′), 114.8 (CH,C-2′, 6′), 110.0 (C, C-6), 106.2 (C, C-3), 100.5 (CH, C-1‴), 94.8(CH, C-5), 81.0 (CH, C-3″), 78.0 (CH, C-5″), 77.1 (CH, C-5‴), 76.6(CH, C-3‴), 74.9 (CH, C-1″), 73.2 (CH, C-2‴), 72.3 (CH, C-2″),69.3 (CH, C-4″), 69.3 (CH, C-4‴), 60.5 (CH2, C-6‴), 60.1 (CH2, C-6″); HRESIMS m/z 569.1503 [M − H]− (calcd for C25H29O15,596.1506); ESIMS [m/z (%)] 569 (100) [M − H]−, 1139 (20) [2M −H]−; HRESIMS m/z 571.1661 [M + H]+ (calcd for C25H31O15,571.1663); ESIMS [m/z (%)] 1141 (10) [2M + H]+, 571 (60) [M +H]+, 409 (100) [M + H − 162]+, 391 (60) [M + H − 162 − H2O]

+,

373 (10) [M + H − 162 − 2 × H2O]+, 355 (10) [M + H − 162 − 3 ×

H2O]+, 313 (10) [M + H − 258]+, 289 (15) [M + H − 162 − 120]+.

Acid Hydrolysis of 1. Aliquots of a solution of 1 (105 mg in 70mL of 1.1 M HCl) were heated in 5 mL glass Reacti-vials at 60 °C in aStuart heating block (Bibby Scientific Limited, Stone, UK) for 24 h.The hydrolysis reaction was monitored using HPLC-DAD (86%degradation at t = 24 h). The hydrolyzed mixture was cooled to roomtemperature and adjusted to pH ∼4 using 1.1 M NaHCO3.

One half of this hydrolyzed mixture was vacuum-evaporated andsubjected to semipreparative LC (MeCN−2% HOAc (aq), 8:92, v/v)to obtain intact 1 and iriflophenone C-hexoside. The fractioncontaining iriflophenone C-hexoside was collected based on retentiontime, pooled, vacuum-evaporated, and freeze-dried, followed by NMRanalysis.

1H NMR and 13C NMR Data for 3-C-β-D-glucopyranosyl-iriflophenone (hydrolyzed product of 1): 1H NMR 600 MHz(acetone-d6, 298 K) δ 7.64 (2H, d, J = 8.6 Hz, H-2′, 6′), 6.85 (2H, d, J= 8.6 Hz, H-3′, 5′), 5.99 (1H, s, H-5), 4.92 (1H, d, J = 9.8 Hz, H-1″),3.85 (2H, d, J = 3.1 Hz, 2 × H-6″), 3.69 (H, t, J = 9.2 Hz, H-4″), 3.63(H, t, J = 9.2 Hz, H-2″), 3.57 (H, t, J = 8.9 Hz, H-3″), 3.49 (H, m, H-5″); 13C NMR 150 MHz (acetone-d6, 298 K) δ 197.8 (CO), 162.6(C-4′), 161.9 (C-4), 161.4 (C-2), 160.5 (C-6), 133.3 (C-1″), 132.5(C-2′, 6′), 115.3 (C-3′, 5′), 106.6 (C-1), 104.7 (C-3), 96.9 (C-5), 82.1(C-5″), 79.3 (C-3″), 76.6 (C-1″), 74.6 (C-4″), 70.6 (C-2″), 61.7 (C-6″).

Identification of the O-Linked Hexose Residue in theHydrolyzed Product. The other half of the hydrolyzed productwas partially evaporated under vacuum, and a small volume of theconcentrated liquid was removed for an enzyme robot assay. Thisassay was conducted on a Thermo Scientific Arena 20XT randomaccess chemistry analyzer (Thermo Fisher Scientific, Oy, Finland) withthe use of an Enytec fluid D-glucose no. 5140 enzyme reagent kit

Figure 4. 3H-2-DOG uptake in L6 myocytes exposed to (A) 1 and (B) 3-C-β-D-glucopyranosyliriflophenone and 3T3-L1 adipocytes exposed to (C)1 and (D) 3-C-β-D-glucopyranosyliriflophenone. Insulin (positive control) and metformin (reference drug control) were included at concentrationsof 1 μM. Mean activity is expressed relative to the vehicle control at 100% ± standard error of the means. Three independent experiments wereperformed each with three technical repeats. **p < 0.01; ***p < 0.001.

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(AEC-Amersham, Kayalami, South Africa) for the identification ofglucose according to the manufacturer’s instructions.The remaining sample volume was freeze-dried, derivatized with

bis(trimethylsilyl)trifluoroacetamide (BSTFA) using the proceduredescribed by Roessner,25 and subjected to GC-MS analysis. D-Glucoseand D-galactose standards (Sigma-Aldrich) were also derivatized priorto analysis. Sample volumes of 1 μL were injected with a split ratio of1:10. The injection temperature was 280 °C, the interface set to 280°C, and the ion source adjusted to 240 °C. The carrier gas was heliumset at a constant flow rate of 1.0 mL/min. The temperature programcomprised a 6 °C oven temperature ramp from 70 to 76 °C within 1min, followed by a 32 °C/min ramp to 300 °C, and a final 5 minheating at 300 °C. Mass spectra were recorded over an m/z scanningrange of 40 to 650. Electron energy was 70 eV and solvent delay 8 min.3-C-β-D-Glucopyranosylmaclurin (2): light yellow, amorphous

powder; UV λmax online 236, 290 (sh), 318 nm; 1H NMR 600 MHz(DMSO-d6, 298 K) δ 7.15 (1H, d, J = 1.93 Hz, H-2′), 7.06 (1H, dd, J= 1.93, 8.24 Hz, H-6′), 6.74 (1H, d, J = 8.24 Hz, H-5′), 5.94 (1H, s, H-5), 4.60 (1H, d, J = 9.77 Hz, H-1″), 3.61 (1H, d, 10.96 Hz, H-6a), 3.51(2H, m, H-2″, 6″), 3.20 (3H, H-3″, 4″, 5″); 13C NMR 150 MHz(DMSO-d6, 298 K) δ 194.6 (C, CO), 158.4 (C, C-4), 156.7 (C, C-6), 156.3 (C, C-2), 150.0 (C, C-4′), 144.6 (C, C-3′), 131.0 (C, C-1′),122.3 (CH, C-6′), 116.2 (CH, C-2′), 114.7 (CH, C-5′), 107.6 (C, C-3), 103.6 (C, C-1), 94.8 (CH, C-5), 81.1 (CH, C-5″), 78.3 (CH, C-3″), 74.8 (CH, C-1″), 72.1 (CH, C-4″), 69.6 (CH, C-2″), 60.4 (CH2,C-6″); HRESIMS m/z 423.0933 [M − H]− (calcd for C19H19O11,423.0927); ESIMS [m/z (%)] 1271 (5) [3M − H]−, 847 (70) [2M −H]−, 423 (100) [M − H]−, 303 (20) [M − H − 120]−; HRESIMS m/z 425.1075 [M + H]+ (calcd for C19H21O11, 425.1084); ESIMS [m/z(%)] 425 (80) [M + H]+, 407 (100) [M + H − H2O]

+, 389 (10) [M +H − 2 × H2O]

+, 371 (10) [M + H − 3 × H2O]+, 341 (10) [M + H −

84]+, 329 (15) [M + H − 96]+, 305 (20) [M + H − 120]+, 287 (5) [M+ H − 120 − H2O]

+.α-Glucosidase Inhibitory Activity. A method for the determi-

nation of α-glucosidase inhibitory effects26 was adapted for use on aBioTek SynergyHT microplate reader with dispenser (BioTekInstruments, Winooski, VT, USA). A mixture containing α-glucosidasewas extracted from rat intestinal acetone powder (Sigma-Aldrich) bysuspending ca. 350 mg of powder in 10 mL of cold KH2PO4 buffer(200 mM KH2PO4, pH 6.8 with KOH) followed by repeatedsonication on ice (sonication sequence repeated 12 times: 30 ssonication with 25% amplitude, 1 min rest) using a model VCX600high-intensity ultrasonic processor with a 3 mm stepped microtip(Sonics & Materials Inc., Newton, CT, USA). The crude mixture wascentrifuged at 10000g for 30 min at 4 °C in a Hettich Universal 320Rrefrigerated centrifuge (Hettich Holding GmbH & Co. oHG,Kirchlengern, Germany), and the supernatant was retrieved andfiltered using 0.45 μm pore size, 33 mm Millex HV PVDF filtermembranes (Merck Millipore). The supernatant was used as anenzyme mixture after dilution to the standardized concentration basedon activity testing.Activity determination of the enzyme mixture was performed daily

prior to each set of experiments, using the same procedure as for theinhibition assays, but with H2O as sample controls and varyingdilutions of the enzyme mixture. Fluorescence measurements wereused to determine the correct concentration for optimal enzymeactivity, ca. 15 to 20 mg/mL of the original powder estimated as anFL-value of 50 000 (λEX: 360 nm; λEM: 460 nm), 20 min after additionof the substrate.The inhibitory activities of 1, 2, 3-C-β-D-glucopyranosyl-

iriflophenone (Fluka, Sigma-Aldrich, St. Louis, MO, USA), andmaclurin (Sigma-Aldrich) were assessed at three concentration levelsranging between 50 and 400 μM. Acarbose (Sigma-Aldrich), a knowninhibitor of mammalian α-glucosidase, was used as positive control at65 μM. The following test procedure was employed: 20 μL of theassay control (H2O), positive control, or target analyte at selectedconcentration was added to 125 μL of a 200 mM KH2PO4 buffer (pH6.8) and 65 μL of the chosen enzyme dilution in 96-well black, flat-bottom microplates with a clear bottom (Greiner Bio-One GmbH,Rainbach im Muhlkreis, Austria). The mixture was preincubated at 37

°C for 15 min, followed by the addition of 40 μL of the substrate, 1.2mM 7-O-α-D-glucopyranosyl-4-methylumbelliferone, by dispenser.Fluorescence (λEX: 360 nm; λEM: 460 nm) was monitored over 30min, and the net fluorescence (net FL) and percent enzyme activitywere calculated using the following formulas:

= −Net FL Fluorescence Fluorescence30min 0min

= ×% Enzyme Activity 100Net FL

Net FLsample

assay control

Statistical analysis was performed with GraphPad Prism version 5.00for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com) using one-way ANOVA with Tukey’s multiplecomparison posthoc test to determine significant differences betweenvalues at the 95% confidence level (p < 0.05).

2-Deoxy-[3H]-D-glucose (3H-2-DOG) Uptake. To estimate invitro glucose uptake activity, cellular 3H-2-DOG uptake was assessedby liquid scintillation counting, using the method described byMazibuko.27 Briefly, L6 myoblasts (2.5 × 104 cells/mL) and 3T3-L1fibroblasts (2.0 × 104 cells/mL) were seeded into 24-well plates inDulbecco’s modified Eagle’s medium (DMEM) supplemented with10% fetal or normal calf serum, respectively. C3A hepatocytes wereseeded at 5.5 × 104 cells/mL in Eagle’s minimal essential medium(EMEM) supplemented with 10% fetal calf serum. All cells werecultured at 37 °C in humidified air with 5% CO2. Cell culture mediaDMEM, EMEM, and fetal and normal calf serum were obtained fromLonza (Walkersville, MD, USA). The L6 myoblasts and 3T3-L1fibroblasts were differentiated into myotubule-forming myocytes andadipocytes, respectively, while the C3A hepatocytes were used assemiconfluent cultures. For the 3H-2-DOG uptake experiments, cells(1 h for L6 myocytes, and 3 h for 3T3-L1 adipocytes and C3Ahepatocytes) were exposed to 1 and 3-C-β-D-glucopyranosyl-iriflophenone at concentrations ranging from 0.001 to 100 μM.Compounds were dissolved in DMSO and diluted with Krebs-Ringerbicarbonate HEPES buffer (KRBH) containing 8 mM glucose (finalDMSO concentrations <0.004%). For glucose uptake determination,cells were pulse-labeled for 15 min with 0.5 μCi/mL 3H-2-DOG(American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) inglucose- and serum-free KRBH containing 1 and 3-C-β-D-glucopyranosyliriflophenone at the relevant concentrations. Insulinand metformin (1,1-dimethylbiguanide hydrochloride) (Sigma-Al-drich) both at 1 μM were included as positive and drug referencecontrols, respectively. The amount of 3H-2-DOG taken up by cells wasmeasured using a liquid scintillation counter (2200CA Tricarb Series,Packard Instrument Company, Downers Grove, IL, USA), and theactivity calculated as fmol (3H-2-DOG)/mg protein. Statistical analysiswas performed with GraphPad Prism version 5.00 for Windows usingone-way ANOVA with Dunnett’s multiple comparison post hoc test todetermine significant differences between values at the 95% confidencelevel (p < 0.05).

■ ASSOCIATED CONTENT

*S Supporting InformationS1 (1H), S2 (13C), S3 (COSY), S4 (1D NOESY), S5 (HSQC),and S6 (HMBC) NMR spectra of 3-C-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone (1). S7: 1H NMR spectra for3-C-β-D-glucopyranosyliriflophenone (obtained by acid hydrol-ysis of 1), with an overlay of corresponding spectra for thecommercial reference standard. S8: The unreacted product of 1is also compared to the starting material of 1 to assess thestability of the sugar moiety during the acid hydrolysis reaction.This material is available free of charge via the Internet athttp://pubs.acs.org.

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■ AUTHOR INFORMATIONCorresponding Author*E-mail: beelderst1@arc.agric.za (T. Beelders). Phone: +27-21-809-3441. Fax: +27 21-809-3430.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work is based on the research supported in part by theSouth African Department of Science and Technology (DST/CON 0133/2012). Other financial support was receivedthrough a grant from the Economic Competitive SupportPackage for Agroprocessing to the ARC by the South AfricanGovernment. NRF grant holders (E.J. and C.J.M.) acknowledgethat opinions, findings, and conclusions or recommendationsexpressed in any publication generated by the NRF-supportedresearch (IFRR Grant 85277 and Thuthuka Grant 87849) arethose of the authors and that the NRF accepts no liabilitywhatsoever in this regard. An NRF-DST Professional Develop-ment Program Doctoral Scholarship to T.B. is acknowledged.Mr. F. Joubert (Koksrivier) is acknowledged for supplying plantmaterial. Ms. W. Kuhn (Water Analysis Division, CAF,Stellenbosch University) is thanked for the enzyme robotassay, and Mr. F. Hiten (Mass Spectrometry Unit, CAF,Stellenbosch University) for GC-MS analyses.

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