2 Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China;
3 Research Institute for Science and Technology, Tokyo University of Science, Chiba 278-8510, Japan;
4 World Agroforestry Centre(ICRAF), Nelson Marlbolough Institute of Technology(NMIT), Nelson 7010, New Zealand;
5 School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China;
6 Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, China
Recently, there has been increasing attention to the natural extracts as a rich source of bioactive compounds with fewer side-effects [1-2], such as ginkgo biloba extract, green tea extract, licorice extract, stevia extract [3-6], which could be used in treatment of cerebral dysfunction disturbance, hyperlipidemia, diabetes, etc [7-10]. These extracts were obtained from the leaves, roots, rhizomes and the other parts of plants [3-6], however, many researchers generally ignored the development of waste parts like nutshell, which tended to cause the resource waste. The V. paradoxa nutshell waste separated from shea kernel during industrial production usually brought environmental pollution. Therefore, we made full pre-literature research on V. paradoxa nutshell for resolving environmental pollution and resource utilization for treating diabetes.
The shea tree (Vitellaria paradoxa C. F. Gaertner) belongs to Sapotaceae family and is indigenous to the savanna belt from 16° W to 34° E longitude and 1° N to 15° N altitude . As a medicinal and edible plant, the fruit of V. paradoxa is edible and nutritious, and the leave, root and stem bark have been traditionally used in the treatment of inflammation, diarrhea, helminthes, sunburn, dermatitis and rashes for local people [12-13]. Modern pharmacological studies have shown that V. paradoxa extract displayed anti-inflammatory, antioxidant, melanogenesis-inhibitory and anthelmintic activities. Phytochemical investigations have discovered that the presence of flavonoids, phenolic acids, terpenoids and glycosides in V. paradoxa [12, 14]. Especially, shea butter extracted from shea kernel was widely used in cosmetics industry as product formulations and effective components [14-15].
Diabetes mellitus, a serious metabolic disease, is closely related to oxidative stress characterized by an excessive increase of oxidizing species like reactive oxygen species (ROS), and α-glucsidase is usually used to reduce the postprandial plasma glucose for patients with type 2 diabetes . Moreover, effective a-glucosidase inhibitors from herbal plant are gaining more and more attention due to their high effectivities and low toxicities in recent years. Therefore, evaluation of antioxidant activity is an important part of the screening for potential anti-diabetic resources. The pre-experiment was introduced by thin layer chromatography (TLC) method to determine that many phenolic constituents were found in V. paradoxa nutshell methanol extracts, which might be used as natural antidiabetic herbal source.
Taking the above into account, high performance liquid chromatography-diode array detector-quadrupole-time of flight (HPLC-DAD-Q-TOF-MS) and phytochemical methods were applied to characterize the main ingredients in V. paradoxa nutshell samples. Four characteristic constituents were quantified and chemometric analysis including similarity analysis (SA), hierarchical cluster analysis (HCA) and principal components analysis (PCA) was applied in comparison of the origin on HPLC fingerprints. Twenty-eight V. paradoxa nutshells from seven sub-Saharan countries were determined for their yield percentage, total phenolic content (TPC), antioxidant and α-glucosidase inhibitory activities. In addition, all fractions and isolated compounds were evaluated for their antioxidant and α-glucosidase inhibitory activities, and further research was carried out for molecular docking of four characteristic constituents to investigate α-glucosidase inhibitory action mechanism. Our study would help to resolve V. paradoxa nutshell waste environmental pollution problem and provide a potential herbal resource for treating diabetes.Materials and Methods Chemicals, reagents, and plant materials
Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-car-boxylicacid), 1, 1-diphenyl-2-picrylhydrazul (DPPH), 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2, 4, 6-tripyridyls-triazine (TPTZ), α-glucosidase powder, p-nitrophenyl-α-D-glucopyranoside (pNPG) and arbutin were purchased from Yuanye Bio Technology (Shanghai, China). Water was purified by means of a Millipore Milli Q-Plus system (Millipore, Bedford). Folin-Ciocalteu reagent, ferric chloride (FeCl3), acarbose, protocatechuic acid and quercetin (purity ≥ 98%) were obtained from Aladdin (Shanghai, China). 3, 5, 7-Trihydroxycoumarin and (2R, 3R)-(+)-taxifolin were isolated from V. paradoxa nutshell methanol extract in our laboratory and the purity of the standards were over 98% determined by HPLC method. All other reagents and solvents used were of analytical grade, which were purchased from Nanjing Chemical Reagent Corp (Nanjing, China).
The V. paradoxa nutshells were obtained from the nut samples collected from twenty-eight sites of seven sub-Saharan countries in Africa, i.e., Côte d'Ivoire (sample no. S1), Ghana (S2), Nigeria (S3-S13), Cameroon (S14-S16), Chad (S17-S24), Sudan (S25-S26), and Uganda (S27-S28) in 2006 (Table 1 and Fig. 1) by one of the authors (E. T. M.) on behalf of the World Agroforestry Centre .
Each accurately weighed powder of V. paradoxa nutshell (1.0 g), which had been passed through a 40 mesh screen, was refluxed (60 ℃) for 3 h in 10 mL of 100% methanol (MeOH). The extract was then centrifuged at 15 000 r·min-1 for 5 min with a HR/T16M centrifuge (Herexi Instrument & Equipment Co., Ltd., Hunan, China). The supernatant solution was evaporated with a rotary vacuum evaporator under reduced pressure at 50 ℃ and the yield of the extract was determined gravimetrically by dividing the extracted material by the dried weight of V. paradoxa nutshell. The sample solution was filtered through 0.45 μm membrane before injected for HPLC analysis.HPLC-UV and HPLC-DAD-Q-TOF-MS analysis
HPLC analysis was carried out using a L-2000 HPLC instrument (Hitachi, Tokyo, Japan) equipped with a ultraviolet (UV) detector, a quaternary pump, loop injection system, and a column oven. Chromatographic separation was performed on a reversed-phase column (CAPCELL PAK C18, 4.6 mm × 150 mm, 5 μm). To achieve a better chromatographic separation of the chromatograms of twenty-eight V. paradoxa nutshell samples, various HPLC parameters were optimized. We compared different mobile phase (MeOH-H2O and MeCN-H2O) with different additive (HCOOH and CH3COOH) in different concentrations (0.02%, 0.05%, 0.1% and 0.2%). Besides, different wavelength (220, 228, 236, 244, 252, 260, 268, 272, 280, 320 and 360 nm), column temperature (30, 35 and 40 ℃) and flow rate (0.8, 0.9 and 1.0 mL·min-1) were also compared.
Qualitative analysis in the V. paradoxa nutshell extract was performed by means of high-performance liquid chromatography with quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a diode array detector (DAD) and electrospray interface (ESI). Major operating parameters were as follows: negative ion mode, nebulizer pressure of 40 psi, scan spectra from m/z 50 to 1000, drying gas temperature of 325 ℃, drying gas (N2) flow rate 8.0 L·min-1, skimmer of 65 V, fragmentor voltage of 100 V, capillary voltage of 3500 V and collision energy 25 eV. Data were processed by Agilent Mass Hunter Workstation Data Acquisition Software Version B.04.00 (Agilent Technologies, Santa Clara, CA).Validation of methodology
The intraday and interday precisions test were determined by analyzing six injections of the same sample solution on the same day and on three consecutive days, respectively. The repeatability was assessed by analysing six independently prepared sample solutions, respectively. The sample stability test was appraised by examining a single sample solution at room temperature for 0, 2, 4, 8, 12 and 24 h, respectively.
Reference standards were accurately weighed and dissolved separately in MeOH for reservation. And then they were diluted to obtain a series of mixed standard stock solutions with different concentrations. The assay linearity was determined by analyzing six appropriate concentrations of the mixed standard solutions. The standard curves were obtained by plotting peak area against nominal concentration of these four compounds. The limits of detection (LOD) and quantitation (LOQ) of each compound were determined at S/N (signal-noise ratio) = 3 and 10, respectively. The recovery test was conducted to assess the accuracy, spiking known amounts of mixed standard solution (about 50% of the content) with known concentration of V. paradoxa nutshell sample, and the mean recoveries were calculated for six replicate determinations. All sample solutions stored at room temperature during this period.Determination of total phenolic content
The TPC of twenty-eight V. paradoxa nutshell samples was determined by the Folin-Ciocalteau method with slight modifications . 100 μL sample solution was put into flask and then 1 mL Foline Ciocalteau's reagent was mixed. After 5 min, 2 mL (29%, W/V) sodium carbonate was added. After 1 h of incubation, the absorbance was measured (725 nm) at room temperature. The TPC of each sample was expressed as mg of gallic acid equivalents per g dry-extract weight (mg GAEs/g DW).Measurement of antioxidant capacity
The antioxidant activity of samples was measured using different chemical-based assays including free radicals-scavenging activity (DPPH and ABTS assays) and ferric reducing antioxidant power (FRAP assay). For DPPH assay, result was expressed as IC50 values estimated by a nonlinear regression algorithm and for ABTS and FRAP assays, the antioxidant capacity was expressed as mmol of trolox equivalents (TEs) per gram (mmol TEs/g).DPPH assay
DPPH assay was performed according to previous report with slight modifications . Briefly, a 50 μL aliquot of the samples was added to 3 mL of 60 μmol·L-1 DPPH (dissolved in MeOH), and mixed solution was incubated for 30 min in the dark at room temperature and measured at 517 nm.ABTS assay
ABTS assay was conducted according to previous report with slight modifications . Briefly, ABTS radical cation (ABTS+) solution was prepared by reacting 2.45 mmol·L-1 potassium persulfate with ABTS stock solution in the dark at room temperature for 14 h, and then the absorbance of the resulting ABTS+ solution was adjusted to 0.70 ± 0.02 at 734 nm diluted with ethanol. 10 μL of samples or trolox standard solution was added to 200 μL of diluted ABTS+ solution and the absorbance was recorded at 734 nm after 30 min incubation in the dark at room temperature.FRAP assay
The FRAP assay was performed according to previous report with slight modifications . Briefly, the FRAP working solution was prepared from sodium acetate buffer solution (300 mmol·L-1), TPTZ solution (10 mmol·L-1) and FeCl3 solution (20 mmol·L-1) at a ratio of 10 : 1 : 1, respectively, and kept in the dark at room temperature before use. 5 μL of samples or trolox standard solution was added to 180 μL of FRAP reagent and kept for 30 min in the dark at room temperature and the absorbance was recorded at 593 nm after 30 min incubation in the dark at room temperature.Determination of α-glucosidase inhibitory activity
The α-glucosidase inhibitory activity of samples was determined according to previous report with slight modifications . Briefly, 20 μL of samples, 50 μL of 0.1 mol·L-1 phosphate buffer (pH 6.9) and 10 μL the enzyme solution (1 U·mL-1) were plated into 96-well plates and incubated at 37 ℃ for 20 min. Then, 20 μL pNPG solution (5 mmol·L-1) was added. After incubation at 37 ℃ for 30 min, the absorbance was recorded at 405 nm. Result was expressed as IC50 values estimated by a nonlinear regression algorithm.Molecular docking
To explore the probable binding of four characteristic compounds (protocatechuic acid, 3, 5, 7-trihydroxycoumarin, (2R, 3R)-(+)-taxifolin and quercetin) with α-glucosidase, docking study was carried out. Homology modeling of α-glucosidase was conducted by using the crystal structure of S. cerevisiae isomaltase (PDB code: 3A4A) as the template , which was downloaded from the Protein Data Bank (PDB) website (http://www.rcsb.org). Result was expressed in terms of free energy (kcal/mol) of ligand-protein binding.Statistical analysis
All results were expressed as mean ± SD (n = 3). The relative retention time (RRT) and relative peak area (RPA) were used as a normalised data for fingerprint analysis, and the similarity analysis was performed using the software "Similarity Evaluation System for chromatographic fingerprint of TCMs" (Version 2004 A, Chinese Pharmacopoeia Committee). The statistical analyses for hierarchical cluster analysis and principal component analysis were performed with SPSS software (SPSS 19.0, SPSS, Chicago, IL, USA). Significant differences (P < 0.05) between means were determined by a one-way analysis of variance (one-way ANOVA) and correlations between variables were computed using Pearson's correlation values. The molecular docking method was performed using the AutoDock (4.2) tools.Results and Discussion Optimization of chromatographic conditions and method validation
In order to achieve satisfactory separations among the chromatographic peaks of test solutions, the optimized HPLC parameters were as follows: the mobile phase consisted of MeCN (A) and H2O (with 0.2% CH3COOH) (B) using a gradient elution program (0-10 min, 10%-23% A; 10-17 min, 23%-25% A; 17-30 min, 25%-35% A). In addition, the flow rate was 1.0 mL·min-1 and the injection volume was 5 μL. The column temperature was maintained at 30 ℃ and the detection of peaks was recorded at 252 nm.
According to the operation method, precision, repeatability and stability of V. paradoxa nutshell samples were evaluated by calculating the RSD of retention time (tR) and of the common peak areas (PAs). The RSD of tR and PAs of precisions varied from 0.07% to 0.87% and 0.64% to 2.09%, respectively, whereas RSD of the repeatability test was within the range 0.07%-0.78% (tR) and 0.60%-2.48% (PAs), respectively. What's more, stability test yielded RSD between 0.10% and 0.66% (tR) and 0.58% and 2.42% (PAs), respectively. All results suggested that the HPLC method for fingerprint analysis of V. paradoxa nutshell was valid and applicable.
The proposed HPLC method was also fully validated of the four characteristic compounds in this study (Table 1 in Supplementary material). The RSD values of intra-and inter-day precision, repeatability and stability of the four compounds were all lower than 3%, and the calibration curves of these four compounds demonstrated good linearity (r2 ≥ 0.999 5) within the test ranges. The LODs and LOQs of were in the range of 0.06-1.56 and 0.24-3.91 μg·mL-1, respectively, which indicated the high detection sensitivity of the analysis method. All the mean recoveries were between 96.22% and 102.75% with RSD less than 3%. These results revealed that the developed method was validated and satisfactory for quantitative analysis of V. paradoxa nutshell samples.Identification of chemical constituents Identification of constituents by HPLC-DAD-Q-TOF-MS
The negative and positive ion modes were both adopted for MS analysis. Our results indicated that chemical constituents of V. paradoxa nutshell showed more obvious response in the negative mode than positive mode. Therefore, the negative ion mode was finally adopted, and the typical total ion chromatogram (TIC) was illustrated in Fig. 2A.
A total of twenty-eight constituents were identified by comparing tR, [M-H]- ions and fragmentations with literature [22-36]. These compounds included fourteen flavonoids (10, 13, 15-19, 22, 23, 25, 26, 28, 29, 33), nine phenolic acids (4-9, 11, 12, 21), two lignins (27, 30) and three organic acids (1-3), among which, twenty-seven compounds (1-3, 5-13, 14-19, 21-23, 25-28, 30, 33) were reported for the first time in V. paradoxa extract. Compounds were numbered in their elution order (Table 2).
V. paradoxa nutshell (S15) was extracted with 100% MeOH to get the extract, which was suspended in H2O and then partitioned with PE, CH2Cl2, EtOAc and n-BuOH, respectively. Separation of CH2Cl2 and EtOAc fractions by repeated column chromatography on silica gel, ODS and RP-HPLC resulted in the isolation of twenty-one compounds (VP-1-VP-21), which were identified as (+)-pinoresinol (VP-1), (+)-medioresinol (VP-2), (+)-syringaresinol (VP-3), 5, 7, 3', 5'-tetrahy-droxyflavanone (VP-4), (2R, 3R)-(+)-taxifolin (VP-5), quercetin (VP-6), kaempferol (VP-7), luteolin (VP-8), 5, 7-dihydroxyl coumarin (VP-9), 3, 5, 7-trihydroxyl coumarin (VP-10), p-hydroxybenzoic acid (VP-11), protocatechuic acid (VP-12), salicylic acid (VP-13), p-hydroxybenzaldehyde (VP-14), protocatechuic acid methyl ester (VP-15), p-methoxybenzoic acid (VP-16), p-methoxyphenylacetic acid (VP-17), vanillic acid (VP-18), 2-hydroxy-5-(2-metho-xyethyl) benzoic acid (VP-19) coniferaldehyde (VP-20) and ferulic acid (VP-21) by comparing their MS and NMR spectroscopic data and optical rotation with literature. Among which, nineteen compounds were reported for the first time in V. paradoxa extract except compounds VP-6 and VP-12. Their extraction, separation, structures, spectral data and reference literature were shown in the Supplementary material.Quantitative application
The established quantification method was subsequently applied to measure amounts of four characteristic compounds, including protocatechuic acid (6), 3, 5, 7-trihydroxycoumarin (14), (2R, 3R)-(+)-taxifolin (16) and quercetin (29) (Fig. 2B and 2C) in twenty-eight samples. The contents of four characteristic compounds differed between twenty-eight samples (Table 3), and the total concentration of the four compounds was from 29.29 (S19) to 139.31 mg·g-1 (S10). Compound 16 was the most predominant component and showed high concentrations (27.64-132.04 mg·g-1, mean 67.16 mg·g-1) in all MeOH extracts. Compound 29 was the second most abundant in all V. paradoxa nutshells and its content was within 0.49-11.56 mg·g-1 (mean 2.08 mg·g-1), while compounds 6 and 14 were found the lower contents in all samples (mean 0.46 and 0.80 mg·g-1, respectively). Meanwhile, there was significant difference (P < 0.01) in contents of four characteristic compounds between twenty-eight samples, which might cause their difference in bioactivities. Therefore, we would discuss the correlation between them in the following section.
Chromatographic fingerprint analysis is an effective and comprehensive technique for quality assessment of herbal medicines. As a statistical tool, chemometrics could overview complex data from the chromatographic profiles . Therefore, HPLC fingerprints combined with powerful chemometrics would be an important tool for systemic quality assessment of herbal medicine.Similarity analysis
The chromatographic fingerprints for V. paradoxa nutshells (S1-S28) were estalished and reference (R) was produced by the median of twenty-eight chromatograms. Twenty peaks (1 + 2, 3, 6, 8, 10, 14, 16, 17, 18 + 19, 20 + 21, 22, 24, 25, 29, 31 + 32, 34) existing in all chromatograms of twenty-eight V. paradoxa nutshell samples were assigned as "common peaks". The correlation coefficients of all sample fingerprints were calculated and shown in Table 3. The results showed that V. paradoxa nutshell samples from Chad (S18) and Nigeriahad (S10) relatively low similarities (0.586 and 0.818) and the others were all more than 0.9, which indicated that quality of the samples S10 and S18 was different from others from other origins in the contents of common peaks.Hierarchical cluster analysis
For HCA analysis, calculation of degree of association is expressed as distance and the smallest distance between two groups indicates how highest the degree of association was . The sample clusters were shown in a dendrogram (Fig. 3A) using the relative areas of common eight characteristic peaks as clustering variable. The results demonstrated that the twenty-eight samples could be categorized into three main clusters (Cluster Ⅰ, Cluster Ⅱ and Cluster Ⅲ). The cluster Ⅰ consisted of S18, cluster Ⅱ consisted of S10, and the other samples, S1-S9, S11-S17, and S19-S28 were belonging to cluster Ⅲ, which was in accordance with the result of HPLC fingerprints similarity above.
The aim of PCA is to reduce the dimensionality of multivariate data while preserving data from a higher to a lower dimensional space (called principal components) . The full 28 × 20 (sample size × number of variables) data matrix was formed for PCA analysis. Three principal components (PC1 = 46.74%, PC2 = 20.85%, PC3 = 8.46%) were obtained when performing PCA using the complete data set, jointly accounting for 76.04% of the total variance, which were considered significant . PCA 3D score plot of V. paradoxa nutshells from different origins was shown in Fig. 3B. From the scatter points, the twenty-eight V. paradoxa nutshell samples could be classified into three groups. Group Ⅰ was formed by S18, group Ⅱ by S10, and group Ⅲ was marked by the rest of the samples, S1-S9, S11-S17, and S19-S28.
In summary, the results of SA, HCA and PCA were consistent and demonstrated an unignorable quality variation in the contents of common peaks between groups Ⅰ, Ⅱ and Ⅲ, which indicated that the geographical origin, including longitude, altitude and elevation, were the major effects on quality difference of V. paradoxa nutshells.Yields and total phenolic content
The yield percentage of twenty-eight sample extracts ranged from 3.10% to 6.92% (mean 5.04%) (Table 1). What's more, all samples contained high TPC (mean 190.08 mg GAE/g DW), and the sample from S10 had the highest TPC (358.78 mg GAE/g DW) and the lowest TPC was the sample S22 which was only 111.79 mg GAE/g DW. Besides, there was significant difference (P < 0.01) in yields and TPC between twenty-eight samples, which indicated that the geographical origin, including longitude, altitude and elevation, might be the major effects on quality difference of V. paradoxa nutshells.Bioactivity evaluation Antioxidant and α-glucosidase inhibitory activities of MeOH extracts
For the herbal medicine, different contents of chemical constituents might lead to significant differences in bioactivities. As shown in Table 4, all twenty-eight samples exhibited moderate-higher antioxidant capacity (DPPH: IC50 6.16-15.17 μg·mL-1, ABTS: 2.31-5.85 mmol TEs/g, and FRAP: 1.15-4.81 mmol TEs/g, respectively) compared with reference trolox (DPPH IC50 6.43 μg·mL-1). Among these, S10 exhibited the most potent antioxidant capacity (DPPH: IC50 6.16 μg·mL-1, ABTS: 5.85 mmol TEs/g, and FRAP: 4.81 mmol TEs/g, respectively). It was observed the contents of phenols played a very important role in the antioxidative capacity of V. paradoxa nutshells. Besides, diabetes mellitus is closely related to oxidative stress and α-glucsidase is usually used to reduce the postprandial plasma glucose for patients with type Ⅱ diabetes . Therefore, α-glucosidase activity of V. paradoxa nutshell MeOH extracts was determined for screening potential anti-diabetic resources furtherly.
All extracts showed strong α-glucosidase inhibitory activity with IC50 values ranging from 8.76 to 30.68 μg·mL-1, which were significantly higher than the reference acarbose (IC50 119.71 μg·mL-1). Meanwhile, S10 exhibited the highest inhibitory activity (IC50 8.76 μg·mL-1). Considering phenolic constituents as an important compound group that contributed to the bioactivities of V. paradoxa nutshell, bioactive components of V. paradoxa nutshell MeOH extract were isolated and purified by bioassay-guided method and main characteristic compounds were quantified. The correlation was also discussed between TPC and bioactivities in the following section.Antioxidant and α-glucosidase inhibitory activities of fractions and isolated compounds
Due to the good bioactivities of CH2Cl2 and AcOEt fractions, the isolated compounds VP-1-VP-21 were made for further antioxidant and α-glucosidase inhibitory activities evaluation and the results were summarized in Table 5. The CH2Cl2 and AcOEt fractions showed obvious antioxidant and α-glucosidase inhibitory activities (for CH2Cl2 fraction DPPH: IC50 10.48 μg·mL-1, ABTS: 4.19 mmol TEs/g, FRAP: 3.56 mmol TEs/g and α-glucosidase: IC50 9.03 μg·mL-1, respectively; for AcOEt fraction DPPH: IC50 9.10 μg·mL-1, ABTS: 5.62 mmol TEs/g, FRAP: 3.71 mmol TEs/g and α-glucosidase: IC50 4.81 μg·mL-1, respectively), and the n-BuOH and H2O fractions exhibited moderate antioxidant activity (for n-BuOH fraction DPPH: IC50 39.44 μg·mL-1, ABTS: 0.99 mmol TEs/g, FRAP: 0.63 mmol TEs/g and α-glucosidase: IC50 32.40 μg·mL-1, respectively; for H2O fraction DPPH: IC50 65.39 μg·mL-1, ABTS: 0.87 mmol TEs/g, FRAP: 0.37 mmol TEs/g and α-glucosidase: IC50 12.12 μg·mL-1, respectively) compared with reference trolox (DPPH IC50 6.43 μg·mL-1) and acarbose (IC50 119.71 μg·mL-1).
Among the isolated compounds tested (Table 6), compounds VP-1-8, VP-12, VP-15 and VP-20 exhibited moderate-higher DPPH radical-scavenging activity (IC50 5.55-87.35 μmol·L-1), and compounds VP-1, VP-4-8, VP-12, VP-15 and VP-20 showed potent radical-scavenging activity in the range of 1.02-4.29 μmol·L-1 Trolox/μmol·L-1 by ABTS analysis. Meanwhile, compounds VP-1 and VP-4-8 displayed ferric reducing power (1.09-3.53 μmol·L-1 Trolox/μmol·L-1) by FRAP test. Besides, compounds VP-2-10 exhibited strong α-glucosidase inhibitory activity (IC50 7.70-87.17 μmol·L-1). It was noteworthy that (2R, 3R)-(+)-taxifolin (DPPH: IC50 17.48 μmol·L-1, ABTS: 1.72 μmol·L-1 Trolox/μmol·L-1, FRAP: 1.43 μmol·L-1 Trolox/μmol·L-1 and α-glucosidase: IC50 26.79 μmol·L-1, respectively) and quercetin (DPPH: IC50 12.78 μmol·L-1, ABTS: 1.62 μmol·L-1 Trolox/μmol·L-1, FRAP: 2.46 μmol·L-1 Trolox/μmol·L-1 and α-glucosidase: IC50 20.87 μmol·L-1, respectively) showed more potent antioxidant and α-glucosidase inhibitory activities compared with reference trolox (IC50 25.69 μmol·L-1) and acarbose (IC50 185.42 μmol·L-1). Protocatechuic acid exhibited lower antioxidant and α-glucosidase inhibitory effects (DPPH: IC50 21.67 μmol·L-1, ABTS: 1.02 μmol·L-1 Trolox/μmol·L-1 and FRAP: 0.83 μmol·L-1 Trolox/mol·L-1, respectively) than (2R, 3R)-(+)-taxifolin and quercetin. There was minor antioxidant activity (DPPH, ABTS and FRAP) of 3, 5, 7-trihy-droxycoumarin observed, yet it exhibited higher α-glucosidase inhibitory activity (IC50 43.28 μmol·L-1) than acarbose. The results revealed that four characteristic components with high contents could be the main active constituents in V. paradoxa nutshell extracts.
Pearson correlation analysis was carried out to reflect the correlation between chemical component contents and bioactivities of V. paradoxa nutshell extracts, and the most significant correlation between chemical component contents and bioactivities how highest the contribution degree of association was.
The correlation between the antioxidant (DPPH, ABTS and FRAP) and α-glucosidase inhibitory activities and TPC was evaluated (Table 7), and the results showed significant negative or positive correlation (r = -0.937, 0.872, 0.918 and -0.696 at the level of 0.01) between TPC and DPPH, TPC and ABTS, TPC and FRAP and TPC and α-glucosidase inhibitory activity, respectively. For pure compounds, (2R, 3R)-(+)-taxifolin and 3, 5, 7-trihydroxycoumarin significantly correlated with DPPH, ABTS and FRAP at P < 0.01 or 0.05 level (r = -0.854 and -0.386), while protocatechuic acid and quercetin showed no significant correlation with DPPH, ABTS and FRAP (P > 0.05 level). Only (2R, 3R)-(+)-taxifolin correlated with α-glucosidase inhibitory activity significantly at P < 0.01 level (r = -0.817). 3, 5, 7-Trihydroxycoumarin also contributed to the α-glucosidase inhibitory activity significantly. It was noteworthy that protocatechuic acid and quercetin with higher contents and better bioactivities didn't significantly relate to antioxidant or α-glucosidase inhibitory activities, we inferred that the factor of high level of (2R, 3R)-(+)-taxifolin contributed to this case, certainly with the synergistic reaction of compounds. Moreover, antioxidant activity (DPPH, ABTS and FRAP) and α-glucosidase inhibitory activity also were significantly correlated with each other (r = 0.731, -0.606, and -0.766 at the level of 0.01), which supported the close connection between them.
Due to the high contents and good α-glucosidase inhibitory activity of four characteristic compounds, they could be the major active constituents responsible for the majority of medical effects of V. paradoxa nutshell. Thus, their binding mode to the allosteric site of S. cerevisiae isomaltase was investigated by a docking simulation, which was conducted to better understand the the mechanism of α-glucosidase inhibition of V. paradoxa nutshell resources and the interaction between the compound and the protein. The molecular docking results were shown in Fig. 3, which included best docking pose with the lowest docking energy (A), docked pose showing interacting residues (B) and binding mode (C). From the docking results, it could be obtained that the selected docked energies for the protocatechuic acid, 3, 5, 7-trihydroxycoumarin, (2R, 3R)-(+)-taxifolin and quercetin were -5.13, -6.32, -6.55 and -7.00 kcal/mol, respectively. This result displayed that the quercetin and (2R, 3R)-(+)-taxifolin molecules had larger binding affinities towards a-glucosidase of S. cerevisiae isomaltase, supporting for their α-glucosidase inhibitory activity, which reflected the inhibition mainly depended on the binding modes and binding affinities of the molecules to the enzyme . Besides, it could be seen that the binding site between the α-glucosidase and four molecules was stabilized by hydrogen bond, and protocatechuic acid docked to α-glucosidase was stabilized by four hydrogen bonds to Leu471, Pro467 and Lys466, which for 3, 5, 7-trihydroxycoumarin was stabilized by one hydrogen bonds to Lys156. The (2R, 3R)-(+)-taxifolin-α-glucosidase interactions was stabilized by four hydrogen bonds to Pro488, Gly564, Lys568 and Tyr566, which for quercetin was stabilized by two hydrogen bonds to Asp69. Namely, the four characteristic compounds inhibited α-glucosidase by blocking the interaction between four ligands and the receptor at the binding site, and the high binding affinity was mainly ascribed to the hydrogen bonding [41-42]. Thus, the docking study method employed was adequate and effective, and the α-glucosidase inhibitory activity could be a result of a synergistic effect between different phenolic constituents in V. paradoxa nutshell.Conclusions
In conclusion, it is the first time that we carried out research on the V. paradoxa nutshell waste and this study revealed all V. paradoxa nutshell samples from seven sub-Saharan countries could be used as cheap natural antidiabetic herbal source, specially rich in (2R, 3R)-(+)-taxifolin. Future research would deeply probe into the hypoglycemic mechanism of the target component and further in vivo experiments are also needed to evaluate. Besides, the synergy effect between different chemical constituents in V. paradoxa nutshell samples remained to be studied, which would establish foundation for prospect of resource utilization.
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