Structures and Antioxidant and Intestinal Disaccharidase Inhibitory

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Structures, Antioxidant and Intestinal Disaccharidase Inhibitory Activities of A-type Proanthocyanidins from Peanut Skin Huiwen Zhang, Rigui Ye, Yumei Yang, and Chaomei Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf402518k • Publication Date (Web): 26 Aug 2013 Downloaded from http://pubs.acs.org on August 31, 2013

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Journal of Agricultural and Food Chemistry

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Structures, Antioxidant and Intestinal Disaccharidase Inhibitory Activities of

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A-type Proanthocyanidins from Peanut Skin

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Huiwen Zhang,†,‡ Rigui Ye,† Yumei Yang,‡ and Chaomei Ma*,†

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School of Life Sciences, Inner Mongolian University, Huhhot, China ‡

Baotou Medical College, Baotou, Inner Mongolia, China

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Corresponding Author

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*Telephone: +86 471 4992435. Fax: +86 471 4992435. E-mail: [email protected]

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ABSTRACT: Nine compounds including a new A-type proanthocyanidin trimer,

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epicatechin-(2β→O→7,4β→8)-[catechin-(6→4β)]-epicatechin (8), and a known

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trimer,

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reported for peanut skin for the first time, were isolated and purified. Their structures

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were determined by spectroscopic methods and by degradation reactions with

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L-cysteine in acidic conditions. The DPPH radical scavenging activity and the

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inhibitory activity on maltase and sucrase of the isolated compounds were

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investigated. All compounds showed strong DPPH scavenging activities (EC50 < 20

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µg/ml). Compound 8 showed the strongest inhibitory activity on maltase with an

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IC50 value of 0.088 mg/ml, while compound 9 exhibited the strongest inhibition on

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sucrase with an IC50 value of 0.091 mg/ml.

epicatechin-(4β→8)-epicatechin-(2β→O→7,4β→8)-catechin

(9),

being

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Keywords: peanut skin, condensed tannin, proanthocyanidin trimer, inhibition on

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intestinal disaccharidases

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INTRODUCTION Peanut (Arachis hypogaea L. Fabaceae), the fourth oleaginous plant in the world, is

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known to be one of the most important economical crop owing to its wide distribution,

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nutritional characteristics and its great application in the food industry for centuries.

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China, being the largest producer and exporter of peanut, has a production of

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14,385,000 tons every year and almost 60% of the peanut is consumed as feedstock

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for oil, which is one of the main vegetal oils in Chinese daily life. However, over the

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years, the skin of peanut is generally neglected and considered as useless byproduct

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and, for the yearly generated peanut skin, only small part of it is used in traditional

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Chinese medicine (TCM) to treat illness such as chronic hemorrhage and

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bronchitis,etc.1 Early investigations have shown that the most important chemical

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constituents of peanut skin are proanthocyanidins, particularly the A-type, in which

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the subunits are connected by a 4→8 or 4→6 carbon bond and a 2→O→7 ether

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bond.2-3

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Proanthocyanidins in general have shown notable potentials of health benefits, e.g.,

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antioxidant4-5 and insulin action potentiation6 activities. Moreover, consumption of

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plant extract containing A-type proanthocyanidins has been shown to increase sugar

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tolerance in type II diabetes patients.7 Disaccharidase (or α-glucosidase) inhibitors

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could control postprandial hyperglycemia by delaying the absorption of intestinal

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carbohydrates as does the prototype clinically used anti-diabetic α-glucosidase

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inhibitor drug, acarbose.8

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In our further systematic search for proanthocyanidins, 9 compounds were isolated

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from the water-soluble fraction of peanut skin, including a novel trimer (compound 8)

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and another trimer (compound 9) which is being reported from peanut skin for the

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first time. Bioassays were carried out to test the antioxidant and intestinal

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disaccharidase inhibitory activities of the 9 compounds from peanut skin. This paper

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describes the isolation, structure elucidation, antioxidant activity and intestinal

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disaccharidase inhibitory activity of these proanthocyanidins to provide scientifically

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valuable information for better utilization of peanut skin.

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MATERIALS AND METHODS

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Materials and Apparatus. Solvents used for the extraction and isolation were of

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analytical grade. L-Cysteine hydrochloride was pursed from Sigm-Aldrich

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Corporation (St. Louis, MO, USA). High performance liquid chromatography (HPLC)

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grade solvents used for UPLC were purchased from Fisher scientific company (New

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Jersey, USA). The sources of other materials are: octadecylsilane (ODS, 38-63µm,

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wako Pure Chemcial Industries, td., Osaka, Japan), Sephadex LH-20 (GE Healthcare

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Bio-Sciences AB, Uppsala, Sweden) and high porous polymer from Mitsubishi

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Chemical Corporation (MCI CHP 20, Mitsubishi Chemical Corporation, Tokyo,

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Japan). TLC was performed on silica gel plates (GF254, 0.2mm, Rushan Taiyang

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Desiccant co., Itd, China). NMR spectra were obtained from a Bruker Avance III 500

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spectrometer with tetramethylsilane as internal standard. UPLC-DAD-ESI-MS

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experiments were carried out on an Agilent 1290 infinity UPLC-DAD system [Agilent

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Technologies Singapore (International) Pte. Ltd., Singapore] with an auto-sampler and

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a photo-diode array detector (DAD) coupled with an Agilent 6340 triple Quad MS.

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Fast atom bombardment mass spectrometry (FAB-MS) were measured with a Xevo

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G2 Q-TOF mass spectrometer (Waters). Circular dichroism (CD) was recorded with

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JASCO-J-18 spectro-polarimeter (Waters). UV and IR. Spectra were taken using a

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Shimadzu UV 240 spectrophotometer and a Shimadzu IR spectrophotometer,

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respectively.

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Plant Materials. Peanut skin used in this research was purchased from Weikang

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drug store, Huhhot, Inner Mongolia, China and identified by the authors to be the seed

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skin of Arachis hypogaea L. Fabaceae.

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Extraction and Purification. Peanut skin (2kg) was soaked with 70% acetone

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(32L) for 24h, and extracted with ultrasonication for 30 minutes. After filtration, the

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residue was extracted once again by repeating the above procedure. The solvent was

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evaporated under reduced pressure and the ensuing extract (276 g) fractionated by

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chromatography over Sephadex LH-20 with ethanol (7500 ml) to give 8 fractions

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(E1-E8). TLC and LC-MS analysis revealed that E2 contained proanthocyanidin

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monomers, while E3 and E5 contained dimeric and trimeric proanthocyanidins

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respectively. E2 was subjected to further chromatography on ODS, eluted with a

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0-100% methanol/water gradient to give compounds 1 (122 mg) and 2 (63 mg).

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Similarly E3 (27 g) was subjected to further chromatography on ODS, eluted with a

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0-100% methanol/water gradient. The 20%-30% gradient fraction contained

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polyphenols as indicated by TLC, and was further purified by repeated

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chromatography on MCI gel (CHP20/P120) and Sephadex LH-20 with

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methanol/water gradients to afford compounds 3-6. Compound 3 (780 mg) was

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recrystallized from water as colorless needles, while 4 (56 mg), 5 (25 mg) and 6 (68

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mg) were obtained as amorphous powders. Furthermore, E5 (20 g) was purified in the

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same way as described for E3 to obtain compounds 7 (145 mg), 8 (54 mg) and 9 (72

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mg) as amorphous powders.

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Compound 1-2: amorphous powder, MS (ESI) m/z 289 ([M-H]-, 100). Compound

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3-6: colorless needles (3) or amorphous powder (4-6), MS (ESI) m/z 575 ([M-H]-,

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100), NMR see supporting material. Compound 7-9: amorphous powder, MS (ESI)

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m/z 863 ([M-H]-, 100), NMR see supporting material.

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Degradation of Proanthocyanidin Trimers. L-cysteine-induced degradations

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of the trimeric proanthocyanidins were carried out according to the reported

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methods9,10 with modification. The trimer and cysteine•HCl (1:2) were dissolved in

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methanol in a centrifuge tube. The tube was tightly sealed and heated at 65 ºC for 24 h.

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The reaction mixture was filtered though a 0.22 µm microfilter and analyzed by

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UPLC-DAD-EIS-MS in comparison with standard 3 (procyanidins A1), and standards

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catechin-cystein and epicatechin-cystein which were recently prepared in our earlier

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work.11

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DPPH Radical Scavenging Assay. We examined the DPPH radical scavenging

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activity with the method described by Ma et al.12 The antioxidant activities were

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examined at four different concentration (2.5, 5, 10 and 20 µg/ml) in 96-well plates.

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10 µl of a compound solution (in DMSO) and 190 µl of DPPH solution

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(1,1-diphenyl-2- picrylhydrazyl radical in ethanol, 0.1 mM) were added to each well.

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For the color control, 10 µl of compound solution and 190 µl of ethanol were mixed in

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each well. In the control wells, 10 µL DMSO and 190 µL DPPH were added. After 20

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min at room temperature in the dark, the absorbance (A) was measured at 520 nm, and

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the activity was calculated as follow:

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Effect %=100×[Acontrol - (Acompound -Acolor)] /Acontrol

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IC50, the concentration of a sample that scavenged 50% of DPPH radical was obtained

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from a curve of effect % versus sample concentration.

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Determination of Inhibitory Activity against Maltase and Sucrase. To

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determine the maltase and sucrase inhibitory activities of samples, we prepared the

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stocking intestinal disaccharidases as reported before.11 The assay was carried out as

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following: 3 µl of sample (dissolved by DMSO), 7µl of disaccharidase [the stocking

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enzyme was diluted 10 times by phosphate buffer (pH 7)] and 20 µl maltose solution

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(2 mg/ml) were added in each well of a 96-well plate to test the inhibition activity on

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maltase. 3 µl DMSO was added instead of samples in the control wells. After 20

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minutes of incubation at 37 ºC, 10 µl of DMSO and the glucose detecting reagents of

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an assay kit from Nanjing Jiancheng Bio Company (Nan Jing, China) were added and

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the absorbances (A) were measured at 520 nm with a plate reader. The activity was

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calculated as follows:

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Inhibition %=100 × [(Acontrol-Asample)] ⁄Acontrol

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The inhibition activity on sucrase was tested in the same procedure as above,

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except that 20 µl of sucrose instead of maltose was used as the substrate and the

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concentration of the working enzyme solution was trice of that for the maltase assay.

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RESULTS AND DISCUSSION

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Proanthocyanidins in Peanut Skin. Peanut skin was extracted with 70 % (v/v)

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acetone and the extract was fractionated with Sephadex LH-20 column

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chromatogaphy. The fraction obtained from 95% ethanol eluted part was then

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subjected to repeated chromatography on ODS and MCI eluted with aqueous

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methanol. Compounds 1-9 were obtained and their structures were determined by

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interpretation of their spectral data and by L-cysteine-induced degradations (Figure 1).

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Compounds 1 and 2 were identified as catechin and epicatechin by comparison

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with authentic standards in UPLC-DAD-ESI-MS. Compounds 3~6 were identified as

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procyanidins A1, A2, epicatechin-(2β→O→7,4β→8)-ent-epicatechin, and

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epicatechin-(2β→O→7,4β→6)-catechin by comparison of their spectral data with

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those reported in literature.3

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Structural Elucidation of Compound 7. Compound 7 [circular dichroism (CD):

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∆ε272.6 -3.93, ∆ε231 +21.57] was obtained as a white amorphous powderIt showed a

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molecular formula of C45H36O18 from the negative FAB-MS (m/z [M-1]- 863.1823,

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which revealed that 7 was a trimeric proanthocyanidin. Degradation of 7 (Scheme 1)

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produced catechin and another compound with the poseudo-molecular weight of 694

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in negative ESIMS, suggesting it is composed by an A-type procyanidins and a

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catechin. By comparison of its 1H and 13C NMR spectral data with those preciously

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reported, 10-11 compound 7 was confirmed as cinnamtannin D-1.14

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Structural Elucidation of Compound 8. Compound 8 [UV (MeOH) λmax ( log

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ε): 280 (4.13) nm; IR (ν, KBr, cm-1): 3350, 1610, 1520, 1450, 1290, 1150, 1100;

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CD:∆ε273.8 -2.58, ∆ε239.4 +16.07] was obtained as a white amorphous powder. It

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showed a molecular formula of C45H36O18 from the negative HRFAB-MS (m/z

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[M-1]- 863.1823), which revealed that 8 was a trimeric proanthocyanidin. The

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presence of three flavanyl units was also indicated by 13C resonances at δ 100.88

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(C-2), 67.49 (C-3), 29.46 (C-4), 84.21 (C-2′), 68.02 (C-3′), 28.58 (C-4′), 77.21

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(C-2″), 67.49 (C-3″), 37.06 (C-4″) (Table 1), arising from the heterocyclic rings

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(rings C, I and F). The existence of an A-type proanthocyanidin unit was supported

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by the downfielded C-2 signal at δ 100.88 (Table 1) and an isolated AB coupling

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system at δ 4.20 (br.s, H-3) and 4.35 (br.s, H-4) (C-ring) in 1H NMR spectrum. The

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two flavan-3-ol units of the A-type entity in 8 were deduced to be linked through C4

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(C ring) and C-8′ (D ring) based on the long-range correlations of H-4 (C-ring) at δ

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4.35 with C-7′ at δ 150.1, C-8′ at δ 107.33, and C-9′ at δ 154.44 in the Heteronuclear

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multiple-bond correlation (HMBC) spectrum. The long-range correlations of H-4″

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(I-ring) at δ 4.63 with C-5′ at δ 149.29 , C-6′ at δ 120.08, and C-7′ at δ 150.1 in the

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HMBC indicated the C-4″→C-6′ linkage (Figure 2). The C-4″→C-6′ linkage was

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further confirmed by its complex NMR spectra arisen from conformational isomers

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due to the steric hindrance for the free rotation of the epicatechin unit.

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The terminal flavanol unit was determined to be 2,3-cis (I ring) from the singlet

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at δ 5.33 (1H, s, H-2″) and the corresponding carbon at δ 77.21(C-2″), 4 while the

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middle unit (the doubly-substituted terminal unit ) was deduced to have a 2,3-trans

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configuration (F ring) from the proton signal at δ 4.79 (1H, d, J=6.5, H-2′) and the

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corresponding carbon at δ 84.21 (C-2′) in 13C NMR.4

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Treatment of 8 with cysteine•HCl in methanol (Scheme 2) yielded compound 3,

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which was identified to be procyanidin A1 by direct comparison of its 1H and 13C

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NMR spectral data with those preciously reported.4 The cystein conjugate derived

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from the upper flavan-3-ol unit in the degradation of 8, was identified as

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epicatechin-cystein by direct comparison by UHPLC-MS with a standard compound

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which had been prepared and reported in our laboratory recently11 (Figure 3). Of the three trimeric proanthocyanidins (7-9) with the same molecular weight (864),

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the 1HNMR spectrum of 8 was observed to be relatively complicated compared to

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those of 7 and 9. This relative complication is alludable to the presence of conformers

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in 8, due to hindered free rotation across its 4″→6′ linkage, which the structures of 7

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and 9 conspicuously lack.

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The diagnostic high amplitude positive cotton effect at 239 nm wavelength of the

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CD spectrum of 8 informed the β-orientation assignment of the interflavonoid

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bonds.15 Consequently, the structure of 8 was determined to be epicatechin-

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(2β→O→7,4β→8)-[catechin-(6→4β)]-epicatechin. This is a branched A-type

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proanthocyanidin with C4-C6, C4-C8 linkages to the same A ring of a flavanyl unit.

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Structural Elucidation of Compound 9. Compound 9 (CD: ∆ε277.8 -4.67, ∆ε240

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+5.39, ∆ε228.2 +2.86) was obtained as a white amorphous powder. It showed a

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molecular formula of C45H36O18 from the negative FAB-MS (m/z [M-1]- 863.1823,

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which revealed that 9 was a trimeric proanthocyanidin. Treatment of 9 with

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cysteine•HCl in methanol yielded procyanidin A1 and epicatechin-cystein (Scheme 3,

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Figure 3), the same results as for the degradation of compound 8. The result indicated

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that compound 9 also composed of proanthocynidin A1 and epicatechin with

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epicatechin being its upper flavan-3-ol unit. It was thus deduced that compounds 8

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and 9 possess different linking positions between procyanidin A1 and epicatechin. The

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epicatechin was determined to be linked through C4→C8 with proanthocynidin A1,

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by direct comparison of the 1H and 13C NMR spectral data of 9 with references.16-17

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The high amplitude positive Cotton effect at a wavelength of 240 nm in the CD

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spectrum of 9 confirmed its β-orientated interflavonoid bond. Compound 9 was thus

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identified as epicatechin-(4β→8)-epicatechin-(2β→O→7,4β→8)-catechin, which is

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reported from peanut skin for the first time.

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Antioxidant activity. As shown in Table 2, all the isolated compounds, 1−9

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demonstrated strong antioxidant activities. They scavenged 50% of the DPPH radical

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at a concentration less than or near to10 µg/ml. The dimeric and trimeric

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proanthocyanidins showed radical scavenging activity with the potencies similar to

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those of the monomers, catechin and epicatechin, which are well known potent

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anti-oxidants.

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Inhibitory Activity Against Maltase and Sucrase. As shown in Table 3, five

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compounds showed maltase inhibitory activity (>50% inhibition), and four

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compounds showed sucrase inhibitory activity at concentrations of 1 mg/ml.

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Compound 8, epicatechin-(2β→O→7,4β→8)-[catechin-(6→4β)]-epicatechin, showed

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the strongest inhibition against maltase, with an IC50 value of 0.088 mg/ml. The

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lowest IC50 value (0.091 mg/ml) observed in the sucrase inhibition experiment was

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exhibited by 9, epicatechin-(4β→8)-epicatechin-(2β→O→7,4β→8)-catechin, making

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it the strongest sucrase inhibitor of all the samples. It can be deduced from the results

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that 8 and 9 have notable degrees of specificity for maltase and sucrase respectively.

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As the component units of 8 and 9 are essentially the same, the variation in their

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biological activities as by their observed differing enzyme specificities is alludable to

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their different unit-connectivity, impacting chirality (though of same configuration) at

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different positions. All the 3 trimeric proanthocyanidins showed inhibitory activities

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on maltase and sucrase. Their inhibitory activities were greater than those of their

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component dimeric proanthocyanidins. Further investigations are required to elucidate

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the detail structure-activity relationships of A-type proanthocyanidins on intestinal

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disaccharidases.

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Degradation of the trimeric proanthocyanidins was carried out with a mild reagent,

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L-cysteine, which also is relatively safe compared to the conventionally used thiolytic

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degradation reagent, benzyl mercaptan with a strong irritating smell. LC-MS analysis

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of the products of L-cysteine-induced degradation clearly indicated the component

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units and their linking order in the structures of pranthocyanidin polymers. Some

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L-cysteine conjugates of catechin/epicatechin and low-molecular-weight B-type

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proanthocyanidins have been reported to have antioxidant,10 glucosidase inhibitory,11

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and neuroprotective activities.18 However, no literature could be found for any A-type

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proanthocyanidin-cysteine conjugate. In the present research, we detected an A-type

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proanthocyanidin-cysteine conjugate for the first time from the degradation product of

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cinnamtannin D-1 (7). Preparation, purification and bioactivity evaluation of novel

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A-type proanthocyanidin-cysteine conjugates are planed to be carried out in our group

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in the near future.

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Proanthocyanidins are rich in hydrophobic aromatic rings and hydrophilic hydroxyl

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groups, which could interact with biological molecules, especially proteins. It is

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generally believed that the activity of proanthocyanidins becomes stronger as the

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number of flavanol units increases. The result of our study confirmed that

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proanthocyanidins have strong antioxidant activities, which could provide protection

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against oxidative stress and thereby prevent a variety of human diseases so

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associated.19-21

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In summary, we have isolated nine compounds, including one novel trimeric A-type

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proanthocyanidin (8), and a known trimeric A-type proanthocyanidin (9) that was

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reported for the first time for peanut skin. All these compounds showed antioxidant

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activity and the 2 trimeric A-type proanthocyanidins, 8 and 9 showed considerable

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inhibitory activities on the intestinal disaccharidases, maltase and sucrase.

268 269

ACKNOWLEDGMENTS

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We are grateful to the Government of Inner Mongolia for financial support and to Mr.

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Menghe (Inner Mongolian University, Huhhot, China) for measurement of NMR

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spectra. We thank Dr.. Lijun (School of Pharmaceutical Sciences, Peking University,

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Beijing, China) for performing the HRESI-MS.

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ASSOCIATED CONTENT

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Supporting information

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Additional figures. This material is available free of charge via the Internet at

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http://pubs.acs.org.

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Chemical components and antioxidant activity of the peels of commercial

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apple-shaped pear (fruit of pyrus pyrifolia cv. pingguoli). J. Food Sci. 2012, 10,

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proanthocyanidin trimer from Lindera aggregata (Sims) Kosterm. Chin. Chem. Lett.

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Daugherty, F. J.; Tempesta, M. S. Bioactive A-type proanthocyanidins from

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Cinnamomum cassia. J. Nat. Prod. 2011, 74, 1833−1841.

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proanthocyanidins. Part 6. Chiroptical studies. Part 95. Circular dichroism of

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procyanidins. J.Chem. Soc., Perkin Trans. I, 1979, 2375-2377.

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(16) Morimoto, S.; Nonaka, G.; Nishioka, I. Tannins and related compounds. LX.

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Isolation and characterization of proanthocyanidins with a doubly-linked unit from

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Vaccinium vitis-idaea L. Chem. Pharm. Bull. 1988, 36, 33−38.

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Antimicrobial activity of tannin components from Vaccinium vitis-idaea L. J. Pharma.

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Pharmacol. 2001, 53, 187−191.

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(18) Torres, J. L.; Lozano, C;Maher, P. Conjugation of catechins with cysteine

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generates antioxidant compounds with enhanced neuroprotective activity.

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Phytochemistry, 2005, 66, 2032−2037.

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(19) Shahidi, F. Natural antioxidants-and overview. In F. Shahidi (Ed.), Natural

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antioxidants: Chemistry, health effects, and application. Champaign, IL: The

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American Oil Chemists’ Society. 1997, pp. 1−10

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(20) Shahidi, F. Beneficial health effects and drawbacks of antinutrients and

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phytochemicals in foods. In F. Shahidi (Ed.), Antinutrients and phytochemicals in

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food (ACS symposium series 662). Washington, DC: American ChemicalSociety.

345

1997, pp. 1−9

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(21) Ren, Y.; Chen, X. Distribution, bioactivities and therapeutical potentials of

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pentagalloylglucopyranose. Curr. Bioact. Compd. 2007, 3, 81−89.

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Page 18 of 30

Figure Captions

350 351

Figure 1. Structures of compounds 1‒9 from peanut skin.

352

Scheme 1. Degradation of compound 7 with L-cysteine HCl. Figure 2. Key correlations observed in the HMBC and 1H-1H COSY spectra of 8. ) 1H-1H COSY (

353

HMBC (

)

354

Scheme 2. Degradation of compound 8 with L-cysteine HCl.

355

Scheme 3. Degradation of compound 9 with L-cysteine HCl.

356

Figure 3. Extracted ion chromatograms of the LC-MS of cysteine-induced

357

degradation of 7 (A), 8 (B), and 9 (C), in comparison with catechin (D), procyanidin

358

A1 (E) and epicatechin-cysteine (F).

359

Table 1. NMR spectral Data of Compound 8 in CD3OD.

360

Table 2. DPPH Radical Scavenging Activities of Proanthocyanidins from Peanut

361

Skin.

362

Table 3. The Maltase and Sucrase Inhibitory Activities of Compounds from Peanut

363

Skin.

364

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OH 13 8

HO

9

7

OH

14

12 11

O

15

2 16

OH 13 8

HO

O

9

7

OH OH

14

12 11

13

15

2

8

HO

16 10

5

7'

10

5

OH

OH

4

12'

13'

OH

10' 4'

2'

11'

3' 14'

OH

OH

16'

HO

15'

2

1

5'

O HO

6'

8' 9'

15

3

6

OH

4

2

O

OH

4

OH

16

3

6

O

9

7

10

5

OH

14

12 11

3

6

3 OH

OH 13

HO

8

7

O

9

13

15

8

HO

3

6 10

5

4

13'

12'

11'

HO

14'

15

4

8' 9'

5'

HO

O

10'

5'

2'

4'

OH

10'

7'

6'

OH 6'

11' 2'

O

OH

14'

4'

OH

OH

HO

8 7

O

9

11

2

13

OH

14

12

6 10

5

HO

15

OH

4

OH

O HO

13'

12'

HO

7'

11'

4'

8''

OH O

HO

13''

12''

11''

2''

16''

8'

OH O HO

13'

11''

12''

12'

11'

10' 4'

2'

OH

14'

16'

OH

9'

O

12'

2'

13' 14'

OH

3'

6' 5'

15''

10'

4'

HO

13''

12''

3''

14''

HO

8

16''

OH

10''

11'' 2''

OH

15'

7'' 6'' 5''

9''

O

15'

OHO 8''

OH

OH

OH

11' 16'

13''

3'

HO

8'

14''

OH 16''

5''

OH

2''

OH

4

3''

6' 5'

9'

4''

OH

7'

O

10'' 4''

10

OH HO 7'

9''

15''

7

365 366

4

HO O

15

3 5

8''

5''

2

6

7''

15

OH

6''

10'' 3''

14''

HO

11

2 3

10

5

7''

9''

15'

O

9

6

OH OH

2'

8

7

6'' 16

5' 10'

16'

HO

6'

OH

O

9

7

11 16

OH

14

12

9'

3' 14'

13

O

8'

OH

8

OH

14

12

16 3

OH

OH

6

5 13

15' 14'

13'

15'

4

16'

12'

OH

OH

16'

HO

15'

11'

3'

3'

OH

16'

13'

12'

10

7'

O

4'

9

11

2 3

5

9'

OH

10'

2'

7

O

6

O

8'

OH

3'

HO

OH

4

5'

9'

HO

10

5 6'

O

8

16

15

3

6

7'

8'

OH

11

2

HO

16

O

OH

O

9

7

16

OH

OH

14

12

14

12

11

2

OH

14

12

13

OH

4''

OH

15''

9

Figure 1. Structures of compounds 1-9 from peanut skin.

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367 OH

OH

OH

OH HO

HO

O OH

O

OH

O

L-cysteine HCl

OH OH

HO

O

O

OH

HO

HO

OH

HO

OH O

OH

HO O

OH

O

+

OH

OH

OH S OH H2N

OH

catechin COOH

HO OH

HO

7

368 369

Scheme 1. Degradation of compound 7 with L-cysteine HCl.

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370 OH OH OH HO

B

O

A

C 4

OH

G HO

3

O

OH

8'

F

H

E

H OH

D

O 9'

2"

4" 6'

H

HO

I O

10'

OH

H OH

OH

2'

OH

HO

Figure 2. Key correlations observed in the HMBC and 1H-1H COSY spectra of 8. 371

HMBC (

) 1H-1H COSY (

)

372

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373 OH

OH

OH OH

HO

OH

O

HO OH

HO O

OH

OH

OH O

HO

L-cysteine HCl

OH OH

HO

O

OH

OH O

OH

OH

S

HO H2N

OH HO

COOH

OH

procyanidins A1

374 375

O

+

OH

OH

HO

OH

O

epicatechin-cysteine

Scheme 2. Degradation of compound 8 with L-cysteine HCl.

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376 OH OH OH

HO

OH

OH OH

OH

HO

O OH

L-cysteine HCl

O

OH

O O

OH

HO OH

HO

OH

O OH

+

HO

HO HO

O

OH

OH

377

OH

O

OH

OH HO

OH

procyanidins A1

OH

S

H2N

COOH

epicatechin-cysteine

Scheme 3. Degradation of compound 9 with L-cysteine HCl.

378

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379 380

Figure 3. Extracted ion chromatograms of the LC-MS of cysteine-induced

381

degradation of 7 (A), 8 (B), and 9 (C), in comparison with catechin (D), procyanidin

382

A1 (E) and epicatechin-cysteine (F).

383 384 385

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Table 1. NMR spectral Data of Compound 8 in CD3OD Ring

No.

13

1

C

2 3 4

100.88 67.14 29.46

—— 4.20, br.s 4.35, br.s

A

5 6 7 8 9 10 11 12 13 14 15 16

156.54 96.05 157.82 98.35 156.54 104.07 131.86 116.14 146.09 146.94 116.01 120.19

D —— 6.10, br.s —— 5.96, br.s —— —— E —— 7.18, br.s —— —— 6.82, m 7.06, d (7.8)

B

C

H

Ring No . F 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′

13

1

Ring No.

13

1

84.21 67.49 28.58

4.79, d (6.5) 4.15, m 2.76, dd (16.1, 7.5) 2.50, dd (16.1, 7.5) —— —— —— —— —— —— —— 6.87, br.s —— —— 6.10, br.s 6.77, m

I

2″ 3″ 4″

77.21 73.34 37.06

5.33, br.s 4.09, m 4.63, br.s

G

5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ 14″ 15″ 16″

159.21 96.45 154.55 99.18 158.18 99.18 132.93 115.7 145.66 146.45 119.22 120.19

—— 6.04, br.s —— 5.93, br.s —— —— —— 6.94, br.s —— —— 6.78, m 6.74, m

C

149.29 109.35 150.1 107.33 154.44 103.93 130.43 115.26 145.91 146.85 120.59 120.08

H

H

C

H

388 389

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391 392

Table 2. DPPH Radical Scavenging Activities of Proanthocyanidins from Peanut Skin sample

393 394 395

Effect

EC50 (µg/ml)

%

20 (µg/ml)

10 (µg/ml)

5 (µg/ml)

2.5 (µg/ml)

1 2 3 4

83.01 94.52 94.02 86.91

61.69 74.33 63.14 58.93

37.42 42.07 29.93 28.80

31.88 34.59 26.28 20.00

8.21 6.12 8.55 9.71

5

79.43

47.29

30.06

15.28

11.36

6 7

93.39 93.33

67.29 54.21

47.10 26.79

38.61 19.43

5.68 9.74

8

84.21

53.01

37.61

34.02

8.61

89.37 85.66 48.49 20.56 9 Catechin (1), a known anti-oxidant was used as a positive control.

6.32

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396

Table 3. The Maltase and Sucrase Inhibitory Activities of Compounds from Peanut

397

Skin Inhibition% on IC50 on Inhibition% on IC50 on Sucrase Maltase Maltase Sucrase (mg/ml) at 1 mg/ml (mg/ml) at 1 mg/ml >1 1 44 45 >1 0.088 2 60 54 0.57 >1 3 >1 47 44 >1 4 >1 44 31 >1 >1 5 47 33 0.61 >1 6 67 42 0.78 0.66 7 62 55 0.088 0.41 8 83 61 0.54 0.091 9 79 64 0.0054 0.0074 Acarbose* nt nt Acarbose*, a known α–glucosidase inhibitor, was used as a positive control. nt: not tested, as this concentration is too high for the this very potent α–glucosidase inhibitor, acarbose. Sample

398 399 400 401

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Graphic Abstract

404 405 406 407 408 409

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