Extracts and compounds with anti-diabetic complications and anti-cancer activity from Castanea mollissina Blume (Chinese chestnut)

Background Castanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima. Methods The kernels (CK), shells (CS) and involucres (CI) parts of C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay. Results CI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (5) and kaempferol-3-O-[2", 6"-di-O-(E)-p-coumaroyl]-β-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2–9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3–6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4–6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells. Conclusions Our results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of Castanea mollissima Blume. Electronic supplementary material The online version of this article (doi:10.1186/1472-6882-14-422) contains supplementary material, which is available to authorized users.

active agents, the anti-diabetic complications and anticancer activity of all fractions of alcohol extracts of kernels (CK), shells (CS) and involucres (CI) were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay in the present work, respectively.
Diabetes, as a complex metabolic disorder caused by insulin insufficiency and/or insulin dysfunction, is characterized by aberrant blood glucose and insulin levels [7]. Diabetic complications, including retinopathy, neuropathy, nephropathy, and arteriosclerosis are considered as risk factors for morbidity and death. Moreover, the diabetic patients are also susceptible to many diseases, including the cancer. For example, colorectal cancer which is the third leading cause of cancer-related death [8], is much easier happened in diabetic patient [9]. Many studies on chestnut seeds and other parts of this plant emphasised on the anti-oxidant property [2,10], while, the present work were to value their anti-diabetes complications and against the connecting cancer activity, also to find the part with much functional values.

Plant material
The kernel, shells and involucres parts of C. mollissima were collected respectively in September, 2005 in Qianxi County of Hebei province, and identified by Professor Sun Qishi (College of Traditional Chinese Medicine, Shenyang Pharmaceutical University). The voucher specimens were deposited at the Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education (No.ZB2005-026-028).

HR-AR assay
Aldose reductase activities of samples were assayed spectrophotometrically by determining the decrease in NADPH concentration at 340 nm in a UV-2201 Pharma Spec UV-vis spectrophotometer (Shimadzu, Japan). The reaction mixture contained 0.14 M phosphate buffer pH 6.2, 700 μL; 0.15 mM NADPH, 100 μL; 3 × 10 −2 units/mL AR, 100 μL; 10 mM D,L-glyceraldehyde, 3.3 mg/mL extracts or 1 mg/mL compounds in DMSO, 3 μL, in a total volume of 1 mL with a final concentration of 10 μg/mL for extracts and 3.3 μg/mL for compounds. The reference blank contained all of the above reagents, and buffer instead of AR, to correct for nonspecific reduction of NADPH. The control had only the sample solvent instead of the sample to correct for reduction of NADPH without inhibitor of AR (ARI). The reaction was initiated by the addition of substrate, and it was monitored spectrophotometrically for 3 min. All values were averages of three independent experiments [12]. The inhibition ratio of AR was calculated by following equation: Inhibit formation of AGEs assay The AGEs reaction solution (200 μL) was composed of 16 mg/mL bovine serum albumin, 50 μL; 0.1 M sodium phosphate buffer (pH 7.4), 96 μL; 144 mg/mL glucose, 50 μL; 0.5 mg/mL sample in DMSO, 4 μL with a final concentration of 10 μg/mL. The sample blank, control solvent and blank solvent contained all of the above reagents, except sample solvent with buffer instead of glucose in the sample blank, sample solvent instead of sample in the control solvent, and sample solvent instead of sample, buffer instead of glucose in the blank solvent. After incubating at 37°C for 7 days, the amounts of AGE products were determined by enzyme-linked immunosorbent assay (ELISA) [13]. Noncompetitive ELISA assays were performed at room temperature. Each well was incubated for 1 h with 0.1 mL of an AGE sample to be tested or its corresponding control sample in 50 mM carbonate buffer (pH 9.7) and washed three times with washing buffer (phosphate-buffered saline containing 0.05% Tween 20). Each well was then blocked for 1 h with 0.2 mL of 2.5% gelatin in 5 mM carbonate buffer (pH 9.7). Each well was washed three times with washing buffer and incubated for 1 h with 0.1 mL of anti-AGE antibody (50 ng/mL). Wells were then washed three times with washing buffer and incubated for 1 h with 0.1 mL of goat anti-mouse IgG, HRP conjugatesecondary antibody (Millipore, Merck) and then washed three times, followed by reaction with o-phenylenediamine dihydrochloride and hydrogen peroxide mixture. The reaction was terminated by adding 1 M sulfuric acid, and the absorbance at 492 nm was read on a micro-plate reader (MPR A4i II TOSOH). All values are averages of three independent experiments, each done in triplicate [14]. The percentage inhibition of AGE production was calculated by the following equation:

Cytotoxic assay
Human COLO 320 DM colon cancer cells (passage-10) were maintained in RPMI-1640 medium supplemented with 10% FBS and SMPC Gly at 37°C in an incubator. The cytotoxicity was determined by the MTT method. Briefly, exponentially growing cells, 100 μl, were attached at 5 × 10 4 cells/well, in 96-well plates, and the cellular viability was determined after 24 h, 48 h, and 72 h administration of the extractions (100 μg/mL) 100 μL. Cells were incubated with MTT tetrazolium salt for 1 h at 37°C, and the formation of formazan was measured by a microplate reader (MPR A4iIITOSOH). All values are averages of two independent experiments, each done in triplicate. The percentage inhibition of cell growth was calculated by the following equation: Where A0 is the absorbance of the control at 500 nm after incubations, and A1 is the absorbance in the presence of the samples. The study was performed in accordance with the Declaration of Helsinki.

Hydrolysis of compound 6
Compound 6 (1.0 mg) was dissolved in MeOH (9 mL) containing HCl (1 mL) and refluxed on a heated (80°C) water bath for 3 h. After cooling, the reaction mixture was concentrated and dried under reduced pressure, then analyzed by TLC using the solvent system CHCl 3 -MeOH-H 2 O (20:10:1) for identification of the complete reaction. The dried residues dissolved in EtOAc, and partitioned with water. The water solution was analyzed and subjected to HPLC with optical detector using a Kaseisorb LC NH 2 -60-5 column

Effects of fractions and compounds in HR-AR, AGEs and Cytotoxic assay
All crude materials and soluble fractions (EtOAc, n-BuOH and water part) of kernels (CK, CK-A, CK-B, CK- W), shells (CS, CS-A, CS-B, CS-W) and involucres (CI, CI-A, CI-B, CI-W) were evaluated on AR, AGEs and the growth inhibitory activity aginst human COLO 320 DM colon cancer cells. Among these fractions, the n-BuOH soluble fraction of involucres (CI-B) was found to be significantly more active than others (Figure 3), and its inhibitory rates were 88.6 ± 2.3% in AR, 77.0 ± 5.8% in AGEs, and 81.3 ± 3.7% in cytotoxic assay respectively. Effective compounds from CI-B were further studied. Six flavonoids, kaempferol (1) (6), and three polyphenolic acids, casuariin (7), casuarinin (8), and castalagin (9) were obtained. Structures of compounds (see Figure 1) were elucidated by the spectroscopic methods, including UV, ESI-MS, NMR experiments, and comparing their spectral data with reported in the references [3,16,17]. The inhibitory activities of compounds 1-9 were shown in Table 2.  13 C-NMR spectra data see Table 3.    Table 3.  Table 3.  13 C-NMR and 2D-NMR spectra data see Table 1.

Discussion
Flavonoids 1-5 and compound kaempferol-3-O-β-Dglucopyranoside were reported to have hypolipidemic, anti-oxidant, anti-inflammatory, analgesic, and antiaging activities [18]. The relationship between their structures and the efficiency was discussed. The presence of ortho-hydroxy group at B-ring, the double bond at C2-C3 for C-ring, and the presence of C 7 -OH are usually listed as important conditions for high AR inhibitory effects. Accordingly, the double bond of C2-C3, orthoor meta-hydroxy groups in B ring, or a glucose unit are associated with the enhanced cytotoxicity [19]. According to the data for compounds 1-6, the presence of p-coumaroyl groups would enhance the inhibitory activity in three assays, therein, flavonoids with more pcoumaroyl groups in structure are deemed to be the promising anti-diabetes complications and anti-cancer agents. Compounds 7, 8 and 9, as polyphenolic derivatives showed remarkable inhibitory effects in three bioactive systems, and the increasing number of galloyl groups in the structure could increase their activity. Some studies found that polyphenolic compounds possessed the potent anti-oxidant and anti-cancer activities to a greater or lesser extent [20], moreover, structure-activity relationships for their radical scavenging, anti-oxidant, anti-herbivore, and anti-herpetic activities also had been discussed [21][22][23]. Here, they were found having a healthy value against cancer or diabetic complications.
Based on our results, the anti-diabetes complications and anti-cancer activity of different parts of C. mollissina could be attributed to its containing amount of flavonoids and polyphenolic derivatives especially with more function groups in the structures. It is no doubt that as a food product the value for Ban li is not only for its nutrients but also for its functional values, including the value of its shells and involucres, which would be potential sources of phenolic compounds for using either as food additives or chemical medicines. We believe that systematic and thorough investigations on functions of Chinese chestnut are very necessary in future studies.