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Physicochemical characteristics and biological activities of polysaccharides from the leaves of different loquat (Eriobotrya japonica) cultivars
International Journal of Biological Macromolecules 135 (2019) 274–281
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical characteristics and biological activities of polysaccharides from the leaves of different loquat (Eriobotrya japonica) cultivars Yuan Fu a,1, Qin Yuan a,1, Shang Lin a, Wen Liu a, Gang Du c, Li Zhao a, Qing Zhang a, De-Rong Lin a, Yun-Tao Liu a, Wen Qin a,1, De-Qiang Li b,⁎, Ding-Tao Wu a,⁎ a b c
Institute of Food Processing and Safety, College of Food Science, Sichuan Agricultural University, Ya'an 625014, Sichuan, China Department of Pharmacy, the Second Hospital of Hebei Medical University, Shijiazhuang, China Sichuan Provincial Institute for Food and Drug Control, Chengdu, Sichuan, China
a r t i c l e
i n f o
Article history: Received 5 April 2019 Received in revised form 30 April 2019 Accepted 21 May 2019 Available online 22 May 2019 Keywords: Loquat Eriobotrya japonica Polysaccharides Weight-average molecular weights Binding properties Enzyme inhibition
a b s t r a c t In order to explore polysaccharides from loquat leaves (LLPs) as functional food ingredients for prevention and treatment of obesity and type 2 diabetes, the physicochemical characteristics, in vitro binding properties, and inhibitory effects on α-amylase and α-glucosidase of polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’, ‘Chuannong8’, ‘Yuanbao’, and ‘Dawuxing’, were investigated and compared. Results showed that the contents of total uronic acids and degree of esterification in LLPs ranged from (27.04 ± 1.76)% to (41.46 ± 1.91)%, and from (20.54 ± 0.11)% to (26.90 ± 0.10)%, respectively. The weight-average molecular weights of LLPs ranged from 4.307 × 106 Da to 5.101 × 106 Da, and the major constituent monosaccharides of LLPs were rhamnose, galacturonic acid, arabinose, and galactose. Furthermore, LLPs exerted strong in vitro fat binding, cholesterol binding, and bile acid binding capacities, as well as remarkable inhibitory effects on α-amylase and αglucosidase, which might be attributed to their molecular weights, molecular weight distributions, and degree of esterification. Results are helpful for better understanding of chemical structures and bioactivities of polysaccharides from loquat leaves, and LLPs had good potential to be explored as functional food ingredients for prevention and treatment of obesity and type 2 diabetes. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Overweight and obesity are the fifth leading risk of global deaths [1]. Obesity has been proven to be a major risk factor for developing other diseases, such as type 2 diabetes, hyperlipidemia, and cardiovascular diseases [2]. Indeed, 44% of the diabetes burden is attributable to overweight and obesity [1]. According to the World Health Organization, overweight and obesity as well as their related diseases can be reduced by limiting energy intake from total fats and sugars. The reducing of dietary fat absorption from the intestine seems to be an effective approach to prevent obesity [3,4]. Furthermore, the inhibition of target enzymes (α-amylase and α-glucosidase) for type 2 diabetes is one of the main strategies to counteract metabolic alterations related to hyperglycemia and type 2 diabetes [3,5,6]. Nowadays, many synthetic drugs have been widely used for the treatment of obesity and type 2 diabetes [7,8]. However, due to the side effects of some synthetic drugs [7,8], there is an increasing interest in seeking for non-toxic, safe, and ⁎ Corresponding authors. E-mail addresses: [email protected] (D.-Q. Li), [email protected] (D.-T. Wu). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.05.157 0141-8130/© 2019 Elsevier B.V. All rights reserved.
effective natural products for prevention and treatment of obesity and type 2 diabetes. A variety of natural polysaccharides have attracted great interest due to their anti-obesity and anti-diabetic activities. At present, lots of different polysaccharides with excellent anti-obesity and anti-diabetic activities have been isolated from natural resources [6,9–11]. Therefore, natural polysaccharides have great potential to be further explored as medicines and functional food ingredients for prevention and treatment of obesity and type 2 diabetes. Eriobotrya japonica (Thunb.) Lindl., also known as ‘loquat’, belongs to the Rosaceae family [12]. It is a subtropical evergreen fruit tree originating in southeastern China, and is now commercially cultivated in Japan, Korea, India, Spain, Italy, and many other countries [13]. The leaf of loquat is a very popular tea material, and an important food and medicine dual-purpose plant in China, Japan, and Korea [12–14]. The leaves of loquat have been traditionally used for the treatment of pain, cough, asthma, chronic bronchitis, diabetes, inflammation, and cancer [13]. Pharmacological studies have shown that the extracts of loquat leaves possess multiple bioactivities [13], such as anti-diabetic [15,16], antiobesity [16,17], anti-inflammatory [12,14], anti-oxidant [18], and hepatoprotective effects [19]. Phenolics and terpenoids in ethanol or methanol extracts have been identified as the major contributors toward the
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anti-diabetic and anti-obesity effects of loquat leaves [13,16,17,20]. However, to the best of our knowledge, chemical structures and bioactivities of polysaccharides, which abundant exist in loquat leaf tea (water decoction) [21], have seldom been investigated. Therefore, the investigation of physicochemical characteristics and bioactivities of polysaccharides from loquat leaves is necessary, which is beneficial to well understand the chemical characters and bioactivities of loquat leaf tea. In this study, in order to well understand the chemical characters and bioactivities of polysaccharides from loquat leaves, and to explore loquat leaf polysaccharides as functional food ingredients for prevention of obesity and type 2 diabetes, the physicochemical characteristics, in vitro binding properties, and inhibitory activities on α-amylase and α-glucosidase of polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’, ‘Chuannong8’, ‘Yuanbao’, and ‘Dawuxing’, were investigated and compared. 2. Material and methods 2.1. Material and chemicals The leaves of four widely cultivated loquat cultivars in Sichuan Province, including E. japonica cv. Dawuxing, E. japonica cv. Chuannong8, E. japonica cv. Yuanbao, and Eriobotrya japonica cv. Baiyu, were collected in Chengdu, Sichuan Province, China. Samples were dried at 45 °C for 48 h, and pulverized. The powder of each sample was passed through a 60 mesh sieve, and stored at −20 °C for further analysis. Trifluoroacetic acid, sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, cholesterol, oleic acid, carboxymethyl cellulose, soluble starch, α-amylase (1000 U/mg), αglucosidase (10 U/mg), acarbose, rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), 1-phenyl-3-methyl-5-pyrazolone (PMP), m-hydroxydiphenyl, and 4-nitrophenyl β-D-glucopyranoside (pNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Heat stable α-amylase and a free cholesterol assay kit were purchased from Solarbio (Beijing, China). All other reagents and chemicals used were of analytical grade. 2.2. Preparation of polysaccharides from loquat leaves The hot water extraction was performed according to our previously reported method with minor modifications [22]. In brief, 10.0 g of samples were firstly refluxed twice with 100 mL of 80% (v/v) ethanol at 80 °C for 2 h to remove most of the small molecules. Subsequently, polysaccharides from the leaves of loquat (LLPs) were extracted twice with 200 mL of deionized water at 95 °C for 2 h. After centrifugation at 4000 ×g for 15 min (Heraeus Multifuge X3R Centrifuge, Thermo Fisher scientific, Waltham, MA, USA), the supernatants were combined, and concentrated to 1/3 of the original volume by a rotary evaporator (RE52AA, YaRong Company, Shanghai, China) under a vacuum at 60 °C. Then, the starch in the supernatant was removed by heat stable αamylase (1 U/mL). Furthermore, the supernatants were precipitated with three volumes of 95% (v/v) ethanol overnight at 4 °C. After centrifugation at 4000 ×g for 20 min, the precipitation was washed with 70% (v/v) ethanol and re-dissolved in deionized water. Afterwards, the crude polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’ (LLP-B), ‘Chuannong8’ (LLP-C), ‘Yuanbao’ (LLP-Y), and ‘Dawuxing’ (LLP-D), were freeze dried and stored at −20 °C for further analysis. 2.3. Characterization of LLPs from loquat leaves 2.3.1. Chemical composition analysis The content of total polysaccharides, content of uronic acids, and content of proteins in LLPs from the leaves of different loquat cultivars
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were analyzed by the phenol sulfuric acid method, the mhydroxydiphenyl method, and the Bradford's method [3], respectively. Briefly, in order to reduce the interference of different monosaccharides on the response of the phenol-sulfuric acid method, a mixture standard was prepared by 40% of Rha, 30% of GalA, and 30% of Ara according to the constituent monosaccharides in LLPs determined by a highperformance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA). Furthermore, the content of total uronic acids in LLPs was determined with GalA as a standard. The content of proteins in LLPs was determined with bovine serum albumin as a standard.
2.3.2. Determination of weight-average molecular weights The absolute weight-average molecular weights (Mw) and polydispersities (Mw/Mn) of LLP-B, LLP-C, LLP-Y, and LLP-D were measured by high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (HPSECMALLS-RID, Wyatt Technology Co., Santa Barbara, CA, USA) according to our previously reported method with minor modifications [23]. Briefly, HPSEC-MALLS-RID measurements were carried out on a multi angle laser light scattering detector (DAWN HELEOS, Wyatt Technology Co., Santa Barbara, CA, USA) with an Agilent 1260 series LC system (Agilent Technologies, Palo Alto, CA, USA). TSKgel GMPWXL (300 mm × 7.8 mm, i.d.) was used at 30 °C. An Optilab rEX refractometer (DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA) was simultaneously connected. The mobile phase was 0.9% NaCl aqueous solution at a flow rate of 0.5 mL/min. The sample concentration was about 1.0 mg/mL. An injection volume of 100 μL was used. The dn/dc value of LLPs was selected as 0.15 mL/g according to a previous study [24]. The Astra software (version 6.1.2, Wyatt Technology Co., Santa Barbara, CA, USA) was utilized for data acquisition and analysis. The Mw of LLPs was calculated by the Zimm method of static light scattering based on the basic light scattering equation.
2.3.3. Determination of constituent monosaccharides Constituent monosaccharides of LLP-B, LLP-C, LLP-Y, and LLP-D were determined by high performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) analysis according to our previously reported method with minor modifications [3]. Briefly, 4.0 mg of each sample was hydrolyzed with 2.0 M trifluoroacetic acid (2.0 mL) at 95 °C for 10 h. After hydrolysis, the hydrolysates were mixed with 1 mL of methanol, and evaporated to dryness by a rotary evaporator under a vacuum. Furthermore, the same amount of methanol was added again, and dried by the same method as above. Subsequently, the dried hydrolysates were dissolved in 1 mL of deionized water for subsequent derivatization. 50 μL of hydrolysates were mixed with 50 μL of 0.6 M sodium hydroxide and 100 μL of 0.5 M 1-phenyl-3-methyl-5pyrazolone (PMP) methanol solution. The mixture was incubated at 70 °C for 100 min in the dark, and shaken continuously. Then, 85 μL of 0.3 M hydrochloric acid solution was used to neutralize the mixture, and the mixture was diluted to 1 mL with deionized water. Furthermore, 1 mL of chloroform was added. After vigorous shaking and layering, the organic phase was discarded. Finally, the solution was passed through a 0.22 μm organic syringe filter for HPLC analysis. Meanwhile, the monosaccharide standard solution, containing Rha, Man, GlcA, GalA, Glc, Gal, Xyl, and Ara, was performed as described above. The PMP derivatives were analyzed by an Agilent 1260 series LC system (Agilent Technologies, Palo Alto, CA, USA) with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, i.d. 5 μm, Agilent Technologies Inc., CA, USA). A 20 μL of PMP derivatives was injected into the HPLC system at the operation temperature of 30 °C, and eluted with a mixture of 0.1 M phosphate buffer solution (pH = 6.7) and acetonitrile (83: 17, v/v) at a flow rate of 1.0 mL/min. The wavelength of diode array detector was set at 245 nm.
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2.3.4. Fourier transform infrared spectroscopy analysis The Fourier transform infrared (FT-IR) analysis of LLP-B, LLP-C, LLPY, and LLP-D was performed according to a previously reported method with minor modifications [3]. Briefly, each sample (1.0 mg) was mixed with 80 mg of dried KBr, and pressed into disk for the analysis. The IR spectra were recorded in the frequency range of 4000–500 cm−1 with a Nicolet iS 10 FT-IR (ThermoFisher scientific, Waltham, MA, USA). Furthermore, the esterification degree (DE) of LLP-B, LLP-C, LLP-Y, and LLPD was determined from FT-IR spectra according to previously reported methods [3,22]. The determination of DE was based on the band areas at 1700–1750 cm−1 (esterified uronic acids) and 1600–1630 cm−1 (free uronic acids). Afterwards, the DE was calculated by the equation as follows, DE ð%Þ ¼
A1735 100 A1735 þ A1624
2.4. In vitro binding properties of LLPs from loquat leaves The in vitro fat binding, cholesterol binding, and bile acid binding capacities of LLP-B, LLP-C, LLP-Y, and LLP-D were measured according to previously reported methods with minor modifications [2,3]. In brief, in order to determine the fat binding capacity of LLPs, 20 mg of each sample was dissolved in 2.0 mL of deionized water. Then 1 mL of peanut oil was added, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (10,000 ×g for 20 min), the supernatant was carefully removed. The bound oil of LLPs was extracted with petroleum ether for four times. Carboxymethyl cellulose was used as a positive control, and the deionized water was used as a substrate blank. The fat binding capacity of LLPs was expressed as gram of binding fat per gram of LLPs (g/g). Furthermore, in order to determine the cholesterol binding capacity, 20 mL of cholesterol micellar solution were prepared by sonication at 480 W for 1 h, composed of 10 mM sodium taurocholate, 10 mM cholesterol, 5 mM oleic acid, 132 mM sodium chloride, and 15 mM sodium phosphate buffer (pH 7.4). Then, 5 mg of each sample was added into 3 mL of micellar solution, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of cholesterol. The content of cholesterol in the supernatant was measured by the free cholesterol kit. In brief, 10 μL of supernatant were mixed with 190 μL of free cholesterol working solution. After incubation at 40 °C for 5 min, the absorbance was measured at 500 nm. Carboxymethyl cellulose was used as a positive control, and deionized water was used as a substrate blank control. The cholesterol binding capacity of LLPs was expressed as milligrams of binding cholesterol per gram of LLPs (mg/g). Finally, in order to determine the bile acid binding capacity, the bile acid mixture was prepared with sodium cholate, sodium deoxycholate, sodium glycocholate, and sodium taurocholate with proportions as 35%, 35%, 15%, and 15% (w/w) in 50 mM phosphate buffer (pH 6.9), respectively. Each sample (10 mg) was digested with 1 mL of 0.01 M HCl in a shaking water bath at 37 °C for 1 h, which
simulated gastric digestion. Then, to each sample, 4 mL of 1.4 μM bile acid mixture and 5 mL of porcine pancreatin (10 U/mL) were added, and incubate at 37 °C for 1 h. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of unbound bile acid. The content of bile acid was measured by furfural-sulfuric acid method. The cholestyramine and carboxymethyl cellulose were used as positive and negative controls, respectively. The bile acid binding capacity of LLPs was expressed as a percent of blank control (%). 2.5. In vitro α-amylase and α-glucosidase inhibition of LLPs from loquat leaves Both α-glucosidase and α-amylase inhibitory effects of LLP-B, LLP-C, LLP-Y, and LLP-D were measured according to our previously reported method with slight modifications [3,5]. In brief, for the determination of α-amylase inhibitory effects of LLPs, 100 μL of each sample at different concentrations (ranged from 100 μg/mL to 1200 μg/mL) was mixed with 100 μL of α-amylase solution (30 U/mL, dissolved in 0.1 M, pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Then, 200 μL of soluble starch (0.5%, w/v) was added into the mixture, and incubated at 37 °C for 10 min. Subsequently, 100 μL of mixture was incubated with 400 μL of 3,5dinitrosalicylic acid reagent (DNS) at a boiling water bath for 5 min. Finally, the absorbance of the mixture was measured at 540 nm. Acarbose standard was used as a positive control. α-Amylase inhibitory effect was measured at five different concentrations, and a logarithmic regression curve was established to calculate IC50 values (μg/mL). The α-amylase inhibitory activity was calculated as follows, α−Amylase inhibition ð%Þ ¼
Asample −Acontrol Þ 100% 1− Ablank −Acontrol
where Asample is the absorbance of the mixture of sample, starch solution, α-amylase, and DNS reagent; Acontrol is the absorbance of the mixture of phosphate buffer, starch solution, and DNS reagent; Ablank is the absorbance of the mixture of phosphate buffer, starch solution, αamylase, and DNS reagent. Furthermore, for the determination of α-glucosidase inhibitory effect, 200 μL of each sample at different concentrations (ranged from 100 μg/mL to 600 μg/mL) was mixed with 40 μL of α-glucosidase solution (0.5 U/mL, dissolved in 0.1 M pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Subsequently, 100 μL of PNPG (4 mM, dissolved in 0.1 M pH 6.8 phosphate buffer) was added into the mixture, and incubated at 37 °C for 10 min. Finally, the absorbance of the mixture was measured at 405 nm, and acarbose standard was used as a positive control. α-Glucosidase effect was measured at five different concentrations, and a logarithmic regression curve was established to calculate IC50 values (μg/mL). The α-glucosidase inhibitory activity was calculated as follows, α−Glucosidase inhibition ð%Þ ¼
Asample −Acontrol Þ 100% 1− Ablank −Acontrol
Table 1 Chemical composition, molecular weight (Mw), and polydispersity (Mw/Mn) of polysaccharides from the leaves of different loquat cultivars (LLPs).
Extraction yield (%) Total polysaccharides (%) Total uronic acids (%) Protein content (%) Degree of esterification (%) Mw × 106 (Da) Mw/Mn
LLP-B
LLP-C
LLP-Y
LLP-D
3.62 ± 0.20c 81.80 ± 1.53a 32.35 ± 1.45b 3.02 ± 0.32ab 20.54 ± 0.11d 5.101 (±2.9%)a 1.553 (±2.8%)
3.95 ± 0.12b 83.07 ± 0.90a 41.46 ± 1.91a 3.18 ± 0.16a 21.02 ± 0.34c 4.786 (±2.1%)b 2.233 (±2.5%)
3.94 ± 0.16b 82.67 ± 1.87a 27.04 ± 1.76c 2.89 ± 0.17ab 21.65 ± 0.17b 4.606 (±3.2%)b 1.675 (±3.2%)
5.29 ± 0.14a 82.05 ± 0.42a 32.68 ± 0.54b 2.72 ± 0.14b 26.90 ± 0.10a 4.307 (±2.5%)c 1.742 (±3.2%)
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). Values represent mean ± standard deviation, and superscripts a–d differ significantly (p b 0.05) among LLPs; statistical significances were carried out by ANOVA and Duncan's test.
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where Asample is the absorbance of the mixture of sample, PNPG, and αglucosidase; Acontrol is the absorbance of the mixture of phosphate buffer and PNPG; Ablank is the absorbance of the mixture of phosphate buffer, α-glucosidase, and PNPG.
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2.6. Statistical analysis All experiments were conducted in triplicate, and data were expressed in means ± standard deviations. Statistical analysis was
Fig. 1. High performance size exclusion chromatograms (left) and high performance liquid chromatography profiles (right) of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar); PMP, 1-phenyl-3-methyl-5-pyrazolone; Rha, rhamnose; GalA, galacturonic acid; Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Glc, glucose; Xyl, xylose; Man, mannose.
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performed using Origin 9.0 software (OriginLab Corporation, Northampton, MA, USA). Statistical significances were carried out by oneway analysis of variance (ANOVA), taking a level of p b 0.05 as significant to Duncan's multiple range test. 3. Results and discussions 3.1. Physicochemical characteristics of LLPs from loquat leaves 3.1.1. Chemical composition of LLPs The extraction yields and chemical compositions of LLPs from the leaves of different loquat cultivars are summarized in Table 1. As shown in Table 1, the extraction yields of LLP-B, LLP-C, LLP-Y, and LLPD ranged from (3.62 ± 0.20)% to (5.29 ± 0.14)%, which were similar with previous studies [21]. Indeed, the extraction yield of LLP-D was significantly (p b 0.05) higher than that of LLP-B, LLP-C, and LLP-Y. The results indicated that the genotype of loquat cultivars affected their contents of polysaccharides, which were similar with other studies that different cultivars of hulless barley affected their contents of βglucans [25]. Moreover, the total polysaccharide contents of LLP-B, LLP-C, LLP-Y, and LLP-D were similar, which were determined to be (81.80 ± 1.53)%, (83.07 ± 0.90)%, (82.67 ± 1.87)% and (82.05 ± 0.42)%, respectively. A few proteins were detected in LLP-B, LLP-C, LLP-Y, and LLP-D, which ranged from (2.72 ± 0.14)% to (3.18 ± 0.16)%. Furthermore, the contents of total uronic acids in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be (32.35 ± 1.45)%, (41.46 ± 1.91)%, (27.04 ± 1.76)%, and (32.68 ± 0.54)%. The relatively high uronic acids contents in LLPs suggested that pectin-like acidic polysaccharides existed in loquat leaves [3,22,26]. 3.1.2. Weight-average molecular weights and constituent monosaccharides of LLPs It is considered that bioactivities of natural polysaccharides are closely correlated to their molecular weights and constituent monosaccharides [27]. In addition, analysis of molecular weights and constituent monosaccharides is necessary for structural characterization of natural polysaccharides [27]. Therefore, the weight-average molecular weights and constituent monosaccharides of LLPs from the leaves of different loquat cultivars were investigated and compared. Briefly, Fig. 1 showed that the HPSEC-MALLS-RID chromatograms of LLP-B, LLP-C, LLP-Y, and LLP-D were similar. The polysaccharide fraction 1 in LLPs exhibited strong laser light scattering signal, and there was no laser light scattering signal of the solvent peak (ranged from 20 min to 22 min). Therefore, the Mw of polysaccharide fraction 1 in LLPs were summarized in Table 1. As shown in Table 1, the Mw of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 4.307 × 106 Da to 5.101 × 106 Da. Results showed that the Mw of LLP-B was higher than that of LLP-C, LLP-Y, and LLP-D. Furthermore, the Mw of LLPs from the loquat leaf tea were relatively higher than that of polysaccharides from other commonly consumed tea material, such as chrysanthemum tea [22], wolfberry tea [27], bitter gourd tea [28], and green tea [29]. The high molecular weights of natural polysaccharides may contribute to their relatively high anti-obesity and anti-diabetic effects in vitro [2,3,30,31]. The polydispersities of polysaccharide fraction 1 in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be 1.553, 2.233, 1.675, and 1.742, respectively. Results showed that LLPC possessed a relatively wide molecular weight distribution, which was in accordance with the HPSEC chromatogram. Furthermore, Fig. 1 also showed that the HPLC-UV profiles of LLPB, LLP-C, LLP-Y, and LLP-D were similar. Results showed that the constituent monosaccharides of LLPs were measured as Rha, GalA, Ara, Gal, GlcA, Glc, Xyl, and Man. The molar ratios of Rha, GalA, Ara, Gal, GlcA, Glc, Xyl, and Man in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be about 1.00:0.91:0.87:0.54:0.14:0.11:0.06:0.03, 1.00:1.30:1.14:0.47:0.50:0.30:0.09:0.04, 1.00:0.64:0.66:0.40:0.06:0.04: 0.05:0.10, and 1.00:0.89:0.88:0.44:0.09:0.02:0.06:0.02, respectively (Table 2). Results showed that Rha, GalA, Ara, and Gal were the
Table 2 Molar ratios of constituent monosaccharides of polysaccharides from the leaves of different loquat cultivars (LLPs). Monosaccharides and molar ratios
LLP-B LLP-C LLP-Y LLP-D
Rha
GalA
Ara
Gal
GlcA
Glc
Xyl
Man
1.00 1.00 1.00 1.00
0.91 1.30 0.64 0.89
0.87 1.14 0.66 0.88
0.54 0.47 0.40 0.44
0.14 0.50 0.06 0.09
0.11 0.30 0.04 0.02
0.06 0.09 0.05 0.06
0.03 0.04 0.10 0.02
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar); Rha, rhamnose; GalA, galacturonic acid; Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Glc, glucose; Xyl, xylose; Man, mannose.
dominant monosaccharides in LLPs, which further confirmed that LLPs from loquat leaves were pectic-polysaccharides. According to the constituent monosaccharides in LLPs, results suggested that rhamnogalacturonan I (RG I), homogalacturonan (HG), and arabinogalactan (AG II) might exist in LLPs from loquat leaves [3,22,32]. 3.1.3. FT-IR spectra and esterification degree of LLPs The FT-IR spectra were used for determination of the structural features of LLPs from the leaves of different loquat cultivars. As shown in Fig. 2, the FT-IR spectra of LLP-B, LLP-C, LLP-Y, and LLP-D were similar, which indicated that LLPs extracted from different loquat cultivars had similar structures. Briefly, the intense and broad bands around 3200 cm−1 and 3600 cm−1 are the characteristic bands of hydroxyl groups [22]. Bands in the region of 3000–2800 cm−1 are assigned to C\\H absorption that includes CH, CH2, and CH3 stretching vibrations [3]. The absorption peak at approximately 1735.00 cm−1 is assigned to the stretching vibration of the esterified carboxylic groups (\\COOR) [22]. Furthermore, the intense band appeared at 1624.73 cm−1 is the C_ O asymmetric stretching of COO−, suggesting the LLPs are all acidic polysaccharides [22,26]. The band at 1420.02 cm−1 is attributed to bending vibration of C\\H or O\\H [22]. Moreover, the absorption peak at 1247.49 cm−1 is attributed to the asymmetric C\\O\\C stretching vibration, suggesting the presence of \\OCH3 [3]. And the peaks in 800–1200 cm−1 called fingerprint region are not significantly different, indicating the structures of LLPs from different loquat cultivars are quite similar [33]. Typical protein band at 1651 cm−1 and 1555 cm−1 were not detected, which indicated the low amount of protein in LLPs. Furthermore, the degree of esterification (DE) of LLPs from the leaves of different loquat cultivars was also investigated by FT-IR spectroscopy analysis. The highest DE was observed in LLP-D (26.90%),
Fig. 2. FT-IR spectra of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar).
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followed by lower DE in LLP-Y (21.65%) and LLP-C (21.024%), and the lowest DE in LLP-B (20.54%). Previous studies have indicated that the low DE might contribute to the relatively high antioxidant activity of natural polysaccharides [22,26], and the high DE might contribute to the relative high inhibitory effects on digestive enzymes [34]. 3.2. In vitro binding properties of LLPs The over absorption of fat, cholesterol, and bile acid can lead some obesity issues, which are associated with cardiovascular disease, diabetes, and cancer [2,3]. The reducing or inhibition of dietary fat absorption from the intestine seems to be an effective approach to prevent obesity. It is recognized that fat binding, cholesterol binding, bile acid binding capacities of natural polysaccharides with high molecular weights are involved in bioactivities, such as hypolipidemic effect and hypocholesterolemic effect [35]. Therefore, in order to explore loquat leaf polysaccharides as functional food ingredients for prevention of obesity, the in vitro binding properties, including fat binding, cholesterol binding, bile acid binding capacities, of LLPs from the leaves of different loquat cultivars were estimated and compared. In vitro fat, cholesterol, and bile acid binding capacities of LLPs from loquat leaves are summarized in Table 3. Results showed that the fat binding, cholesterol binding, and bile acid binding capacities of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 5.09 ± 0.03 g/g to 8.18 ± 0.08 g/g, from 19.23 ± 0.16 mg/g to 22.63 ± 0.42 mg/g, and from (32.05 ± 0.67)% to (59.18 ± 0.58)%, respectively. The highest fat binding, cholesterol binding, and bile acid binding capacities were found in LLP-C among all tested LLPs. Furthermore, the fat binding and bile acid binding capacities of LLPs were significantly higher than that of β-glucans from barley and oat [2,35,36], which are commonly consumed to reduce the risk of obesity [37]. The relatively high binding capacities of LLPs from loquat leaves might be attributed to their high molecular weights, wide molecular weight distributions, and high degree of esterification. Previous studies have also revealed that the binding properties of natural polysaccharides are influenced by their molecular weights [3,35]. Furthermore, compared with the positive control (carboxymethyl cellulose), all LLPs exerted significantly higher fat and cholesterol binding capacities. Indeed, the bile acid binding capacities of LLP-B and LLP-C were also significantly higher than that of cholestyramine (a positive control). Results suggested that polysaccharides especially LLP-C, from loquat leaves had great potential to be explored as functional food ingredients for prevention of obesity and hypercholesterolemia.
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Therefore, in this study, in order to explore loquat leaf polysaccharides as functional food ingredients for prevention of type 2 diabetes, the in vitro inhibitory effects of LLPs against target enzymes (α-amylase and α-glucosidase) for type 2 diabetes were investigated. As shown in Fig. 3, results showed that LLPs exerted remarkable inhibitory effects on α-amylase and α-glucosidase. Briefly, the IC50 values of α-amylase inhibition of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 253.51 ± 1.41 μg/mL to 751.33 ± 5.66 μg/mL. The strongest α-amylase inhibition effect was determined in LLP-C among all tested LLPs. Compared with the positive control (acarbose, IC50 = 36.85 ± 2.66 μg/mL), LLPs exhibited moderate inhibitory effects on α-amylase. However, the α-amylase inhibition effects of LLPs from loquat leaves were higher than that of some pectic-polysaccharides extracted from Momordica charantia [28,40], Symphytum officinale [38], etc., and thereby LLPs could be explored as a promising natural α-amylase inhibitor. Furthermore, the IC50 values of α-glucosidase inhibition of LLP-B, LLP-C, LLP-Y, and LLPD ranged from 276.67 ± 1.53 μg/mL to 402.33 ± 4.16 μg/mL. The strongest α-glucosidase inhibition effect was also determined in LLP-C among all tested LLPs. Indeed, LLPs extracted from loquat leaves exerted much stronger inhibitory effects on α-glucosidase than that of the positive control (Acarbose, IC50 = 393.76 ± 3.57 μg/mL). Furthermore, the α-glucosidase inhibition effects of LLPs from loquat leaves were also higher than that of some reported pectic-polysaccharides [28,38,41].
3.3. In vitro α-amylase and α-glucosidase inhibitory effects of LLPs Inhibition of α-glucosidase and α-amylase is one of the main strategies to counteract metabolic alterations related to type 2 diabetes [33]. It is recognized that pectic-polysaccharides from edible and medicinal plants exhibit remarkable in vitro anti-diabetic effects [3,28,38,39].
Table 3 The fat binding, cholesterol binding, and bile acid binding capacities of polysaccharides from the leaves of different loquat cultivars (LLPs). Fat binding (g/g) LLP-B LLP-C LLP-Y LLP-D Positive control
6.67 ± 0.03b 8.18 ± 0.08a 6.34 ± 0.03c 5.09 ± 0.03d 1.06 ± 0.13e
Cholesterol binding (mg/g)
Bile acid binding (%)
21.17 ± 0.27b 22.63 ± 0.42a 20.18 ± 0.15c 19.23 ± 0.16d 13.85 ± 0.34e
50.06 ± 0.03b 59.18 ± 0.58a 32.05 ± 0.67d 32.97 ± 0.19d 36.46 ± 0.21c
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). Values represent mean ± standard deviation, and superscripts a–e differ significantly (p b 0.05) among LLPs; statistical significances were carried out by ANOVA, followed by Duncan's test.
Fig. 3. In vitro inhibitory activities on α-amylase (A) and α-glucosidase (B) of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). The error bars are standard deviations; Significant (p b 0.05) differences are shown by data bearing different letters (a–d); statistical significances were carried out by ANOVA and Duncan's test.
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Generally, the inhibitory effects of pectic-polysaccharides on digestive enzymes are considered as a non-competitive interaction, and it is estimated that the reaction of enzymes and substrate would be inhibited by surroundings [34]. The remarkable inhibitory effects of LLPs extracted from loquat leaves on α-amylase and α-glucosidase might be attributed to their high molecular weights, high contents of uronic acids, and high degree of esterification [3,28,34]. Finally, results suggested that LLPs, especially LLP-C, had potential to be explored further as functional food ingredients for prevention and treatment of type 2 diabetes. 4. Conclusions In this study, the physicochemical characteristics, in vitro fat binding, cholesterol binding, and bile acid binding capacities, and inhibitory effects on α-amylase and α-glucosidase of LLPs extracted from the leaves of different loquat cultivars were systematically investigated and compared. Results showed that the weight-average molecular weights, constituent monosaccharides, and FT-IR spectra of LLPs were similar. Indeed, LLPs extracted from loquat leaves exhibited strong in vitro fat binding, cholesterol binding, and bile acid binding capacities, as well as remarkable inhibitory effects on α-amylase and α-glucosidase. Results are helpful for the better understanding of the chemical structures and bioactivities of LLPs extracted from loquat leaves, and LLPs could be further explored as functional food ingredients for prevention and treatment of obesity and type 2 diabetes. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Scientific Research Foundation of Sichuan Agricultural University (grant number 03120321), the Scientific Research Fund Project of Department of Science and Technology of Sichuan Province (grant number 2018JY0149), and the National Natural Science Foundation of China (grant number 81603066). References [1] A. Podsedek, I. Majewska, M. Redzynia, D. Sosnowska, M. Koziolkiewicz, In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits, J. Agric. Food Chem. 62 (2014) 4610–4617. [2] H. Guo, S. Lin, M. Lu, J.D.B. Gong, L. Wang, Q. Zhang, D.R. Lin, W. Qin, D.T. Wu, Characterization, in vitro binding properties, and inhibitory activity on pancreatic lipase of beta-glucans from different Qingke (Tibetan hulless barley) cultivars, Int. J. Biol. Macromol. 120 (2018) 2517–2522. [3] Q. Yuan, S. Lin, Y. Fu, X.R. Nie, W. Liu, Y. Su, Q.H. Han, L. Zhao, Q. Zhang, D.R. Lin, W. Qin, D.T. Wu, Effects of extraction methods on the physicochemical characteristics and biological activities of polysaccharides from okra (Abelmoschus esculentus), Int. J. Biol. Macromol. 127 (2019) 178–186. [4] Q. Jin, H.H. Yu, X.Q. Wang, K.C. Li, P.C. Li, Effect of the molecular weight of watersoluble chitosan on its fat-/cholesterol-binding capacities and inhibitory activities to pancreatic lipase, PeerJ 5 (2017) 3279. [5] H.Y. Li, Q. Yuan, Y.L. Yang, Q.H. Han, J.L. He, L. Zhao, Q. Zhang, S.X. Liu, D.R. Lin, D.T. Wu, W. Qin, Phenolic profiles, antioxidant capacities, and inhibitory effects on digestive enzymes of different kiwifruits, Molecules 23 (2018) 2957. [6] J.J. Wu, S.S. Shi, H.J. Wang, S.C. Wang, Mechanisms underlying the effect of polysaccharides in the treatment of type 2 diabetes: a review, Carbohydr. Polym. 144 (2016) 474–494. [7] N.N. Sun, T.Y. Wu, C.F. Chau, Natural dietary and herbal products in anti-obesity treatment, Molecules 21 (2016) 1351. [8] X. Xu, B. Shan, C.H. Liao, J.H. Xie, P.W. Wen, J.Y. Shi, Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice, Int. J. Biol. Macromol. 81 (2015) 538–543. [9] R.D. Devaraj, C.K. Reddy, B.J. Xu, Health-promoting effects of konjac glucomannan and its practical applications: a critical review, Int. J. Biol. Macromol. 126 (2019) 273–281. [10] Y. Zheng, L. Bai, Y. Zhou, R. Tong, M. Zeng, X. Li, J. Shi, Polysaccharides from Chinese herbal medicine for anti-diabetes recent advances, Int. J. Biol. Macromol. 121 (2019) 1240–1253. [11] P.C. Wang, S. Zhao, B.Y. Yang, Q.H. Wang, H.X. Kuang, Anti-diabetic polysaccharides from natural sources: a review, Carbohydr. Polym. 148 (2016) 86–97.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical characteristics and biological activities of polysaccharides from the leaves of different loquat (Eriobotrya japonica) cultivars Yuan Fu a,1, Qin Yuan a,1, Shang Lin a, Wen Liu a, Gang Du c, Li Zhao a, Qing Zhang a, De-Rong Lin a, Yun-Tao Liu a, Wen Qin a,1, De-Qiang Li b,⁎, Ding-Tao Wu a,⁎ a b c
Institute of Food Processing and Safety, College of Food Science, Sichuan Agricultural University, Ya'an 625014, Sichuan, China Department of Pharmacy, the Second Hospital of Hebei Medical University, Shijiazhuang, China Sichuan Provincial Institute for Food and Drug Control, Chengdu, Sichuan, China
a r t i c l e
i n f o
Article history: Received 5 April 2019 Received in revised form 30 April 2019 Accepted 21 May 2019 Available online 22 May 2019 Keywords: Loquat Eriobotrya japonica Polysaccharides Weight-average molecular weights Binding properties Enzyme inhibition
a b s t r a c t In order to explore polysaccharides from loquat leaves (LLPs) as functional food ingredients for prevention and treatment of obesity and type 2 diabetes, the physicochemical characteristics, in vitro binding properties, and inhibitory effects on α-amylase and α-glucosidase of polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’, ‘Chuannong8’, ‘Yuanbao’, and ‘Dawuxing’, were investigated and compared. Results showed that the contents of total uronic acids and degree of esterification in LLPs ranged from (27.04 ± 1.76)% to (41.46 ± 1.91)%, and from (20.54 ± 0.11)% to (26.90 ± 0.10)%, respectively. The weight-average molecular weights of LLPs ranged from 4.307 × 106 Da to 5.101 × 106 Da, and the major constituent monosaccharides of LLPs were rhamnose, galacturonic acid, arabinose, and galactose. Furthermore, LLPs exerted strong in vitro fat binding, cholesterol binding, and bile acid binding capacities, as well as remarkable inhibitory effects on α-amylase and αglucosidase, which might be attributed to their molecular weights, molecular weight distributions, and degree of esterification. Results are helpful for better understanding of chemical structures and bioactivities of polysaccharides from loquat leaves, and LLPs had good potential to be explored as functional food ingredients for prevention and treatment of obesity and type 2 diabetes. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Overweight and obesity are the fifth leading risk of global deaths [1]. Obesity has been proven to be a major risk factor for developing other diseases, such as type 2 diabetes, hyperlipidemia, and cardiovascular diseases [2]. Indeed, 44% of the diabetes burden is attributable to overweight and obesity [1]. According to the World Health Organization, overweight and obesity as well as their related diseases can be reduced by limiting energy intake from total fats and sugars. The reducing of dietary fat absorption from the intestine seems to be an effective approach to prevent obesity [3,4]. Furthermore, the inhibition of target enzymes (α-amylase and α-glucosidase) for type 2 diabetes is one of the main strategies to counteract metabolic alterations related to hyperglycemia and type 2 diabetes [3,5,6]. Nowadays, many synthetic drugs have been widely used for the treatment of obesity and type 2 diabetes [7,8]. However, due to the side effects of some synthetic drugs [7,8], there is an increasing interest in seeking for non-toxic, safe, and ⁎ Corresponding authors. E-mail addresses: [email protected] (D.-Q. Li), [email protected] (D.-T. Wu). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.05.157 0141-8130/© 2019 Elsevier B.V. All rights reserved.
effective natural products for prevention and treatment of obesity and type 2 diabetes. A variety of natural polysaccharides have attracted great interest due to their anti-obesity and anti-diabetic activities. At present, lots of different polysaccharides with excellent anti-obesity and anti-diabetic activities have been isolated from natural resources [6,9–11]. Therefore, natural polysaccharides have great potential to be further explored as medicines and functional food ingredients for prevention and treatment of obesity and type 2 diabetes. Eriobotrya japonica (Thunb.) Lindl., also known as ‘loquat’, belongs to the Rosaceae family [12]. It is a subtropical evergreen fruit tree originating in southeastern China, and is now commercially cultivated in Japan, Korea, India, Spain, Italy, and many other countries [13]. The leaf of loquat is a very popular tea material, and an important food and medicine dual-purpose plant in China, Japan, and Korea [12–14]. The leaves of loquat have been traditionally used for the treatment of pain, cough, asthma, chronic bronchitis, diabetes, inflammation, and cancer [13]. Pharmacological studies have shown that the extracts of loquat leaves possess multiple bioactivities [13], such as anti-diabetic [15,16], antiobesity [16,17], anti-inflammatory [12,14], anti-oxidant [18], and hepatoprotective effects [19]. Phenolics and terpenoids in ethanol or methanol extracts have been identified as the major contributors toward the
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anti-diabetic and anti-obesity effects of loquat leaves [13,16,17,20]. However, to the best of our knowledge, chemical structures and bioactivities of polysaccharides, which abundant exist in loquat leaf tea (water decoction) [21], have seldom been investigated. Therefore, the investigation of physicochemical characteristics and bioactivities of polysaccharides from loquat leaves is necessary, which is beneficial to well understand the chemical characters and bioactivities of loquat leaf tea. In this study, in order to well understand the chemical characters and bioactivities of polysaccharides from loquat leaves, and to explore loquat leaf polysaccharides as functional food ingredients for prevention of obesity and type 2 diabetes, the physicochemical characteristics, in vitro binding properties, and inhibitory activities on α-amylase and α-glucosidase of polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’, ‘Chuannong8’, ‘Yuanbao’, and ‘Dawuxing’, were investigated and compared. 2. Material and methods 2.1. Material and chemicals The leaves of four widely cultivated loquat cultivars in Sichuan Province, including E. japonica cv. Dawuxing, E. japonica cv. Chuannong8, E. japonica cv. Yuanbao, and Eriobotrya japonica cv. Baiyu, were collected in Chengdu, Sichuan Province, China. Samples were dried at 45 °C for 48 h, and pulverized. The powder of each sample was passed through a 60 mesh sieve, and stored at −20 °C for further analysis. Trifluoroacetic acid, sodium cholate, sodium deoxycholate, sodium glycocholate, sodium taurocholate, cholesterol, oleic acid, carboxymethyl cellulose, soluble starch, α-amylase (1000 U/mg), αglucosidase (10 U/mg), acarbose, rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), 1-phenyl-3-methyl-5-pyrazolone (PMP), m-hydroxydiphenyl, and 4-nitrophenyl β-D-glucopyranoside (pNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Heat stable α-amylase and a free cholesterol assay kit were purchased from Solarbio (Beijing, China). All other reagents and chemicals used were of analytical grade. 2.2. Preparation of polysaccharides from loquat leaves The hot water extraction was performed according to our previously reported method with minor modifications [22]. In brief, 10.0 g of samples were firstly refluxed twice with 100 mL of 80% (v/v) ethanol at 80 °C for 2 h to remove most of the small molecules. Subsequently, polysaccharides from the leaves of loquat (LLPs) were extracted twice with 200 mL of deionized water at 95 °C for 2 h. After centrifugation at 4000 ×g for 15 min (Heraeus Multifuge X3R Centrifuge, Thermo Fisher scientific, Waltham, MA, USA), the supernatants were combined, and concentrated to 1/3 of the original volume by a rotary evaporator (RE52AA, YaRong Company, Shanghai, China) under a vacuum at 60 °C. Then, the starch in the supernatant was removed by heat stable αamylase (1 U/mL). Furthermore, the supernatants were precipitated with three volumes of 95% (v/v) ethanol overnight at 4 °C. After centrifugation at 4000 ×g for 20 min, the precipitation was washed with 70% (v/v) ethanol and re-dissolved in deionized water. Afterwards, the crude polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’ (LLP-B), ‘Chuannong8’ (LLP-C), ‘Yuanbao’ (LLP-Y), and ‘Dawuxing’ (LLP-D), were freeze dried and stored at −20 °C for further analysis. 2.3. Characterization of LLPs from loquat leaves 2.3.1. Chemical composition analysis The content of total polysaccharides, content of uronic acids, and content of proteins in LLPs from the leaves of different loquat cultivars
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were analyzed by the phenol sulfuric acid method, the mhydroxydiphenyl method, and the Bradford's method [3], respectively. Briefly, in order to reduce the interference of different monosaccharides on the response of the phenol-sulfuric acid method, a mixture standard was prepared by 40% of Rha, 30% of GalA, and 30% of Ara according to the constituent monosaccharides in LLPs determined by a highperformance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA). Furthermore, the content of total uronic acids in LLPs was determined with GalA as a standard. The content of proteins in LLPs was determined with bovine serum albumin as a standard.
2.3.2. Determination of weight-average molecular weights The absolute weight-average molecular weights (Mw) and polydispersities (Mw/Mn) of LLP-B, LLP-C, LLP-Y, and LLP-D were measured by high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (HPSECMALLS-RID, Wyatt Technology Co., Santa Barbara, CA, USA) according to our previously reported method with minor modifications [23]. Briefly, HPSEC-MALLS-RID measurements were carried out on a multi angle laser light scattering detector (DAWN HELEOS, Wyatt Technology Co., Santa Barbara, CA, USA) with an Agilent 1260 series LC system (Agilent Technologies, Palo Alto, CA, USA). TSKgel GMPWXL (300 mm × 7.8 mm, i.d.) was used at 30 °C. An Optilab rEX refractometer (DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA) was simultaneously connected. The mobile phase was 0.9% NaCl aqueous solution at a flow rate of 0.5 mL/min. The sample concentration was about 1.0 mg/mL. An injection volume of 100 μL was used. The dn/dc value of LLPs was selected as 0.15 mL/g according to a previous study [24]. The Astra software (version 6.1.2, Wyatt Technology Co., Santa Barbara, CA, USA) was utilized for data acquisition and analysis. The Mw of LLPs was calculated by the Zimm method of static light scattering based on the basic light scattering equation.
2.3.3. Determination of constituent monosaccharides Constituent monosaccharides of LLP-B, LLP-C, LLP-Y, and LLP-D were determined by high performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) analysis according to our previously reported method with minor modifications [3]. Briefly, 4.0 mg of each sample was hydrolyzed with 2.0 M trifluoroacetic acid (2.0 mL) at 95 °C for 10 h. After hydrolysis, the hydrolysates were mixed with 1 mL of methanol, and evaporated to dryness by a rotary evaporator under a vacuum. Furthermore, the same amount of methanol was added again, and dried by the same method as above. Subsequently, the dried hydrolysates were dissolved in 1 mL of deionized water for subsequent derivatization. 50 μL of hydrolysates were mixed with 50 μL of 0.6 M sodium hydroxide and 100 μL of 0.5 M 1-phenyl-3-methyl-5pyrazolone (PMP) methanol solution. The mixture was incubated at 70 °C for 100 min in the dark, and shaken continuously. Then, 85 μL of 0.3 M hydrochloric acid solution was used to neutralize the mixture, and the mixture was diluted to 1 mL with deionized water. Furthermore, 1 mL of chloroform was added. After vigorous shaking and layering, the organic phase was discarded. Finally, the solution was passed through a 0.22 μm organic syringe filter for HPLC analysis. Meanwhile, the monosaccharide standard solution, containing Rha, Man, GlcA, GalA, Glc, Gal, Xyl, and Ara, was performed as described above. The PMP derivatives were analyzed by an Agilent 1260 series LC system (Agilent Technologies, Palo Alto, CA, USA) with a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, i.d. 5 μm, Agilent Technologies Inc., CA, USA). A 20 μL of PMP derivatives was injected into the HPLC system at the operation temperature of 30 °C, and eluted with a mixture of 0.1 M phosphate buffer solution (pH = 6.7) and acetonitrile (83: 17, v/v) at a flow rate of 1.0 mL/min. The wavelength of diode array detector was set at 245 nm.
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2.3.4. Fourier transform infrared spectroscopy analysis The Fourier transform infrared (FT-IR) analysis of LLP-B, LLP-C, LLPY, and LLP-D was performed according to a previously reported method with minor modifications [3]. Briefly, each sample (1.0 mg) was mixed with 80 mg of dried KBr, and pressed into disk for the analysis. The IR spectra were recorded in the frequency range of 4000–500 cm−1 with a Nicolet iS 10 FT-IR (ThermoFisher scientific, Waltham, MA, USA). Furthermore, the esterification degree (DE) of LLP-B, LLP-C, LLP-Y, and LLPD was determined from FT-IR spectra according to previously reported methods [3,22]. The determination of DE was based on the band areas at 1700–1750 cm−1 (esterified uronic acids) and 1600–1630 cm−1 (free uronic acids). Afterwards, the DE was calculated by the equation as follows, DE ð%Þ ¼
A1735 100 A1735 þ A1624
2.4. In vitro binding properties of LLPs from loquat leaves The in vitro fat binding, cholesterol binding, and bile acid binding capacities of LLP-B, LLP-C, LLP-Y, and LLP-D were measured according to previously reported methods with minor modifications [2,3]. In brief, in order to determine the fat binding capacity of LLPs, 20 mg of each sample was dissolved in 2.0 mL of deionized water. Then 1 mL of peanut oil was added, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (10,000 ×g for 20 min), the supernatant was carefully removed. The bound oil of LLPs was extracted with petroleum ether for four times. Carboxymethyl cellulose was used as a positive control, and the deionized water was used as a substrate blank. The fat binding capacity of LLPs was expressed as gram of binding fat per gram of LLPs (g/g). Furthermore, in order to determine the cholesterol binding capacity, 20 mL of cholesterol micellar solution were prepared by sonication at 480 W for 1 h, composed of 10 mM sodium taurocholate, 10 mM cholesterol, 5 mM oleic acid, 132 mM sodium chloride, and 15 mM sodium phosphate buffer (pH 7.4). Then, 5 mg of each sample was added into 3 mL of micellar solution, and the mixture was incubated at 37 °C for 2 h with continuous shaking. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of cholesterol. The content of cholesterol in the supernatant was measured by the free cholesterol kit. In brief, 10 μL of supernatant were mixed with 190 μL of free cholesterol working solution. After incubation at 40 °C for 5 min, the absorbance was measured at 500 nm. Carboxymethyl cellulose was used as a positive control, and deionized water was used as a substrate blank control. The cholesterol binding capacity of LLPs was expressed as milligrams of binding cholesterol per gram of LLPs (mg/g). Finally, in order to determine the bile acid binding capacity, the bile acid mixture was prepared with sodium cholate, sodium deoxycholate, sodium glycocholate, and sodium taurocholate with proportions as 35%, 35%, 15%, and 15% (w/w) in 50 mM phosphate buffer (pH 6.9), respectively. Each sample (10 mg) was digested with 1 mL of 0.01 M HCl in a shaking water bath at 37 °C for 1 h, which
simulated gastric digestion. Then, to each sample, 4 mL of 1.4 μM bile acid mixture and 5 mL of porcine pancreatin (10 U/mL) were added, and incubate at 37 °C for 1 h. After centrifugation (13,000 ×g, 1 h), the supernatant was collected for the determination of unbound bile acid. The content of bile acid was measured by furfural-sulfuric acid method. The cholestyramine and carboxymethyl cellulose were used as positive and negative controls, respectively. The bile acid binding capacity of LLPs was expressed as a percent of blank control (%). 2.5. In vitro α-amylase and α-glucosidase inhibition of LLPs from loquat leaves Both α-glucosidase and α-amylase inhibitory effects of LLP-B, LLP-C, LLP-Y, and LLP-D were measured according to our previously reported method with slight modifications [3,5]. In brief, for the determination of α-amylase inhibitory effects of LLPs, 100 μL of each sample at different concentrations (ranged from 100 μg/mL to 1200 μg/mL) was mixed with 100 μL of α-amylase solution (30 U/mL, dissolved in 0.1 M, pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Then, 200 μL of soluble starch (0.5%, w/v) was added into the mixture, and incubated at 37 °C for 10 min. Subsequently, 100 μL of mixture was incubated with 400 μL of 3,5dinitrosalicylic acid reagent (DNS) at a boiling water bath for 5 min. Finally, the absorbance of the mixture was measured at 540 nm. Acarbose standard was used as a positive control. α-Amylase inhibitory effect was measured at five different concentrations, and a logarithmic regression curve was established to calculate IC50 values (μg/mL). The α-amylase inhibitory activity was calculated as follows, α−Amylase inhibition ð%Þ ¼
Asample −Acontrol Þ 100% 1− Ablank −Acontrol
where Asample is the absorbance of the mixture of sample, starch solution, α-amylase, and DNS reagent; Acontrol is the absorbance of the mixture of phosphate buffer, starch solution, and DNS reagent; Ablank is the absorbance of the mixture of phosphate buffer, starch solution, αamylase, and DNS reagent. Furthermore, for the determination of α-glucosidase inhibitory effect, 200 μL of each sample at different concentrations (ranged from 100 μg/mL to 600 μg/mL) was mixed with 40 μL of α-glucosidase solution (0.5 U/mL, dissolved in 0.1 M pH 6.8 phosphate buffer), and incubated at 37 °C for 30 min with continuous shaking. Subsequently, 100 μL of PNPG (4 mM, dissolved in 0.1 M pH 6.8 phosphate buffer) was added into the mixture, and incubated at 37 °C for 10 min. Finally, the absorbance of the mixture was measured at 405 nm, and acarbose standard was used as a positive control. α-Glucosidase effect was measured at five different concentrations, and a logarithmic regression curve was established to calculate IC50 values (μg/mL). The α-glucosidase inhibitory activity was calculated as follows, α−Glucosidase inhibition ð%Þ ¼
Asample −Acontrol Þ 100% 1− Ablank −Acontrol
Table 1 Chemical composition, molecular weight (Mw), and polydispersity (Mw/Mn) of polysaccharides from the leaves of different loquat cultivars (LLPs).
Extraction yield (%) Total polysaccharides (%) Total uronic acids (%) Protein content (%) Degree of esterification (%) Mw × 106 (Da) Mw/Mn
LLP-B
LLP-C
LLP-Y
LLP-D
3.62 ± 0.20c 81.80 ± 1.53a 32.35 ± 1.45b 3.02 ± 0.32ab 20.54 ± 0.11d 5.101 (±2.9%)a 1.553 (±2.8%)
3.95 ± 0.12b 83.07 ± 0.90a 41.46 ± 1.91a 3.18 ± 0.16a 21.02 ± 0.34c 4.786 (±2.1%)b 2.233 (±2.5%)
3.94 ± 0.16b 82.67 ± 1.87a 27.04 ± 1.76c 2.89 ± 0.17ab 21.65 ± 0.17b 4.606 (±3.2%)b 1.675 (±3.2%)
5.29 ± 0.14a 82.05 ± 0.42a 32.68 ± 0.54b 2.72 ± 0.14b 26.90 ± 0.10a 4.307 (±2.5%)c 1.742 (±3.2%)
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). Values represent mean ± standard deviation, and superscripts a–d differ significantly (p b 0.05) among LLPs; statistical significances were carried out by ANOVA and Duncan's test.
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where Asample is the absorbance of the mixture of sample, PNPG, and αglucosidase; Acontrol is the absorbance of the mixture of phosphate buffer and PNPG; Ablank is the absorbance of the mixture of phosphate buffer, α-glucosidase, and PNPG.
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2.6. Statistical analysis All experiments were conducted in triplicate, and data were expressed in means ± standard deviations. Statistical analysis was
Fig. 1. High performance size exclusion chromatograms (left) and high performance liquid chromatography profiles (right) of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar); PMP, 1-phenyl-3-methyl-5-pyrazolone; Rha, rhamnose; GalA, galacturonic acid; Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Glc, glucose; Xyl, xylose; Man, mannose.
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performed using Origin 9.0 software (OriginLab Corporation, Northampton, MA, USA). Statistical significances were carried out by oneway analysis of variance (ANOVA), taking a level of p b 0.05 as significant to Duncan's multiple range test. 3. Results and discussions 3.1. Physicochemical characteristics of LLPs from loquat leaves 3.1.1. Chemical composition of LLPs The extraction yields and chemical compositions of LLPs from the leaves of different loquat cultivars are summarized in Table 1. As shown in Table 1, the extraction yields of LLP-B, LLP-C, LLP-Y, and LLPD ranged from (3.62 ± 0.20)% to (5.29 ± 0.14)%, which were similar with previous studies [21]. Indeed, the extraction yield of LLP-D was significantly (p b 0.05) higher than that of LLP-B, LLP-C, and LLP-Y. The results indicated that the genotype of loquat cultivars affected their contents of polysaccharides, which were similar with other studies that different cultivars of hulless barley affected their contents of βglucans [25]. Moreover, the total polysaccharide contents of LLP-B, LLP-C, LLP-Y, and LLP-D were similar, which were determined to be (81.80 ± 1.53)%, (83.07 ± 0.90)%, (82.67 ± 1.87)% and (82.05 ± 0.42)%, respectively. A few proteins were detected in LLP-B, LLP-C, LLP-Y, and LLP-D, which ranged from (2.72 ± 0.14)% to (3.18 ± 0.16)%. Furthermore, the contents of total uronic acids in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be (32.35 ± 1.45)%, (41.46 ± 1.91)%, (27.04 ± 1.76)%, and (32.68 ± 0.54)%. The relatively high uronic acids contents in LLPs suggested that pectin-like acidic polysaccharides existed in loquat leaves [3,22,26]. 3.1.2. Weight-average molecular weights and constituent monosaccharides of LLPs It is considered that bioactivities of natural polysaccharides are closely correlated to their molecular weights and constituent monosaccharides [27]. In addition, analysis of molecular weights and constituent monosaccharides is necessary for structural characterization of natural polysaccharides [27]. Therefore, the weight-average molecular weights and constituent monosaccharides of LLPs from the leaves of different loquat cultivars were investigated and compared. Briefly, Fig. 1 showed that the HPSEC-MALLS-RID chromatograms of LLP-B, LLP-C, LLP-Y, and LLP-D were similar. The polysaccharide fraction 1 in LLPs exhibited strong laser light scattering signal, and there was no laser light scattering signal of the solvent peak (ranged from 20 min to 22 min). Therefore, the Mw of polysaccharide fraction 1 in LLPs were summarized in Table 1. As shown in Table 1, the Mw of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 4.307 × 106 Da to 5.101 × 106 Da. Results showed that the Mw of LLP-B was higher than that of LLP-C, LLP-Y, and LLP-D. Furthermore, the Mw of LLPs from the loquat leaf tea were relatively higher than that of polysaccharides from other commonly consumed tea material, such as chrysanthemum tea [22], wolfberry tea [27], bitter gourd tea [28], and green tea [29]. The high molecular weights of natural polysaccharides may contribute to their relatively high anti-obesity and anti-diabetic effects in vitro [2,3,30,31]. The polydispersities of polysaccharide fraction 1 in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be 1.553, 2.233, 1.675, and 1.742, respectively. Results showed that LLPC possessed a relatively wide molecular weight distribution, which was in accordance with the HPSEC chromatogram. Furthermore, Fig. 1 also showed that the HPLC-UV profiles of LLPB, LLP-C, LLP-Y, and LLP-D were similar. Results showed that the constituent monosaccharides of LLPs were measured as Rha, GalA, Ara, Gal, GlcA, Glc, Xyl, and Man. The molar ratios of Rha, GalA, Ara, Gal, GlcA, Glc, Xyl, and Man in LLP-B, LLP-C, LLP-Y, and LLP-D were determined to be about 1.00:0.91:0.87:0.54:0.14:0.11:0.06:0.03, 1.00:1.30:1.14:0.47:0.50:0.30:0.09:0.04, 1.00:0.64:0.66:0.40:0.06:0.04: 0.05:0.10, and 1.00:0.89:0.88:0.44:0.09:0.02:0.06:0.02, respectively (Table 2). Results showed that Rha, GalA, Ara, and Gal were the
Table 2 Molar ratios of constituent monosaccharides of polysaccharides from the leaves of different loquat cultivars (LLPs). Monosaccharides and molar ratios
LLP-B LLP-C LLP-Y LLP-D
Rha
GalA
Ara
Gal
GlcA
Glc
Xyl
Man
1.00 1.00 1.00 1.00
0.91 1.30 0.64 0.89
0.87 1.14 0.66 0.88
0.54 0.47 0.40 0.44
0.14 0.50 0.06 0.09
0.11 0.30 0.04 0.02
0.06 0.09 0.05 0.06
0.03 0.04 0.10 0.02
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar); Rha, rhamnose; GalA, galacturonic acid; Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Glc, glucose; Xyl, xylose; Man, mannose.
dominant monosaccharides in LLPs, which further confirmed that LLPs from loquat leaves were pectic-polysaccharides. According to the constituent monosaccharides in LLPs, results suggested that rhamnogalacturonan I (RG I), homogalacturonan (HG), and arabinogalactan (AG II) might exist in LLPs from loquat leaves [3,22,32]. 3.1.3. FT-IR spectra and esterification degree of LLPs The FT-IR spectra were used for determination of the structural features of LLPs from the leaves of different loquat cultivars. As shown in Fig. 2, the FT-IR spectra of LLP-B, LLP-C, LLP-Y, and LLP-D were similar, which indicated that LLPs extracted from different loquat cultivars had similar structures. Briefly, the intense and broad bands around 3200 cm−1 and 3600 cm−1 are the characteristic bands of hydroxyl groups [22]. Bands in the region of 3000–2800 cm−1 are assigned to C\\H absorption that includes CH, CH2, and CH3 stretching vibrations [3]. The absorption peak at approximately 1735.00 cm−1 is assigned to the stretching vibration of the esterified carboxylic groups (\\COOR) [22]. Furthermore, the intense band appeared at 1624.73 cm−1 is the C_ O asymmetric stretching of COO−, suggesting the LLPs are all acidic polysaccharides [22,26]. The band at 1420.02 cm−1 is attributed to bending vibration of C\\H or O\\H [22]. Moreover, the absorption peak at 1247.49 cm−1 is attributed to the asymmetric C\\O\\C stretching vibration, suggesting the presence of \\OCH3 [3]. And the peaks in 800–1200 cm−1 called fingerprint region are not significantly different, indicating the structures of LLPs from different loquat cultivars are quite similar [33]. Typical protein band at 1651 cm−1 and 1555 cm−1 were not detected, which indicated the low amount of protein in LLPs. Furthermore, the degree of esterification (DE) of LLPs from the leaves of different loquat cultivars was also investigated by FT-IR spectroscopy analysis. The highest DE was observed in LLP-D (26.90%),
Fig. 2. FT-IR spectra of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar).
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followed by lower DE in LLP-Y (21.65%) and LLP-C (21.024%), and the lowest DE in LLP-B (20.54%). Previous studies have indicated that the low DE might contribute to the relatively high antioxidant activity of natural polysaccharides [22,26], and the high DE might contribute to the relative high inhibitory effects on digestive enzymes [34]. 3.2. In vitro binding properties of LLPs The over absorption of fat, cholesterol, and bile acid can lead some obesity issues, which are associated with cardiovascular disease, diabetes, and cancer [2,3]. The reducing or inhibition of dietary fat absorption from the intestine seems to be an effective approach to prevent obesity. It is recognized that fat binding, cholesterol binding, bile acid binding capacities of natural polysaccharides with high molecular weights are involved in bioactivities, such as hypolipidemic effect and hypocholesterolemic effect [35]. Therefore, in order to explore loquat leaf polysaccharides as functional food ingredients for prevention of obesity, the in vitro binding properties, including fat binding, cholesterol binding, bile acid binding capacities, of LLPs from the leaves of different loquat cultivars were estimated and compared. In vitro fat, cholesterol, and bile acid binding capacities of LLPs from loquat leaves are summarized in Table 3. Results showed that the fat binding, cholesterol binding, and bile acid binding capacities of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 5.09 ± 0.03 g/g to 8.18 ± 0.08 g/g, from 19.23 ± 0.16 mg/g to 22.63 ± 0.42 mg/g, and from (32.05 ± 0.67)% to (59.18 ± 0.58)%, respectively. The highest fat binding, cholesterol binding, and bile acid binding capacities were found in LLP-C among all tested LLPs. Furthermore, the fat binding and bile acid binding capacities of LLPs were significantly higher than that of β-glucans from barley and oat [2,35,36], which are commonly consumed to reduce the risk of obesity [37]. The relatively high binding capacities of LLPs from loquat leaves might be attributed to their high molecular weights, wide molecular weight distributions, and high degree of esterification. Previous studies have also revealed that the binding properties of natural polysaccharides are influenced by their molecular weights [3,35]. Furthermore, compared with the positive control (carboxymethyl cellulose), all LLPs exerted significantly higher fat and cholesterol binding capacities. Indeed, the bile acid binding capacities of LLP-B and LLP-C were also significantly higher than that of cholestyramine (a positive control). Results suggested that polysaccharides especially LLP-C, from loquat leaves had great potential to be explored as functional food ingredients for prevention of obesity and hypercholesterolemia.
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Therefore, in this study, in order to explore loquat leaf polysaccharides as functional food ingredients for prevention of type 2 diabetes, the in vitro inhibitory effects of LLPs against target enzymes (α-amylase and α-glucosidase) for type 2 diabetes were investigated. As shown in Fig. 3, results showed that LLPs exerted remarkable inhibitory effects on α-amylase and α-glucosidase. Briefly, the IC50 values of α-amylase inhibition of LLP-B, LLP-C, LLP-Y, and LLP-D ranged from 253.51 ± 1.41 μg/mL to 751.33 ± 5.66 μg/mL. The strongest α-amylase inhibition effect was determined in LLP-C among all tested LLPs. Compared with the positive control (acarbose, IC50 = 36.85 ± 2.66 μg/mL), LLPs exhibited moderate inhibitory effects on α-amylase. However, the α-amylase inhibition effects of LLPs from loquat leaves were higher than that of some pectic-polysaccharides extracted from Momordica charantia [28,40], Symphytum officinale [38], etc., and thereby LLPs could be explored as a promising natural α-amylase inhibitor. Furthermore, the IC50 values of α-glucosidase inhibition of LLP-B, LLP-C, LLP-Y, and LLPD ranged from 276.67 ± 1.53 μg/mL to 402.33 ± 4.16 μg/mL. The strongest α-glucosidase inhibition effect was also determined in LLP-C among all tested LLPs. Indeed, LLPs extracted from loquat leaves exerted much stronger inhibitory effects on α-glucosidase than that of the positive control (Acarbose, IC50 = 393.76 ± 3.57 μg/mL). Furthermore, the α-glucosidase inhibition effects of LLPs from loquat leaves were also higher than that of some reported pectic-polysaccharides [28,38,41].
3.3. In vitro α-amylase and α-glucosidase inhibitory effects of LLPs Inhibition of α-glucosidase and α-amylase is one of the main strategies to counteract metabolic alterations related to type 2 diabetes [33]. It is recognized that pectic-polysaccharides from edible and medicinal plants exhibit remarkable in vitro anti-diabetic effects [3,28,38,39].
Table 3 The fat binding, cholesterol binding, and bile acid binding capacities of polysaccharides from the leaves of different loquat cultivars (LLPs). Fat binding (g/g) LLP-B LLP-C LLP-Y LLP-D Positive control
6.67 ± 0.03b 8.18 ± 0.08a 6.34 ± 0.03c 5.09 ± 0.03d 1.06 ± 0.13e
Cholesterol binding (mg/g)
Bile acid binding (%)
21.17 ± 0.27b 22.63 ± 0.42a 20.18 ± 0.15c 19.23 ± 0.16d 13.85 ± 0.34e
50.06 ± 0.03b 59.18 ± 0.58a 32.05 ± 0.67d 32.97 ± 0.19d 36.46 ± 0.21c
LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). Values represent mean ± standard deviation, and superscripts a–e differ significantly (p b 0.05) among LLPs; statistical significances were carried out by ANOVA, followed by Duncan's test.
Fig. 3. In vitro inhibitory activities on α-amylase (A) and α-glucosidase (B) of LLP-B, LLP-C, LLP-Y, and LLP-D from the leaves of different loquat cultivars. LLP-B, polysaccharides from loquat leaves (Baiyu cultivar); LLP-C, polysaccharides from loquat leaves (Chuannong8 cultivar); LLP-Y, polysaccharides from loquat leaves (Yuanbao cultivar); LLP-D, polysaccharides from loquat leaves (Dawuxing cultivar). The error bars are standard deviations; Significant (p b 0.05) differences are shown by data bearing different letters (a–d); statistical significances were carried out by ANOVA and Duncan's test.
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Generally, the inhibitory effects of pectic-polysaccharides on digestive enzymes are considered as a non-competitive interaction, and it is estimated that the reaction of enzymes and substrate would be inhibited by surroundings [34]. The remarkable inhibitory effects of LLPs extracted from loquat leaves on α-amylase and α-glucosidase might be attributed to their high molecular weights, high contents of uronic acids, and high degree of esterification [3,28,34]. Finally, results suggested that LLPs, especially LLP-C, had potential to be explored further as functional food ingredients for prevention and treatment of type 2 diabetes. 4. Conclusions In this study, the physicochemical characteristics, in vitro fat binding, cholesterol binding, and bile acid binding capacities, and inhibitory effects on α-amylase and α-glucosidase of LLPs extracted from the leaves of different loquat cultivars were systematically investigated and compared. Results showed that the weight-average molecular weights, constituent monosaccharides, and FT-IR spectra of LLPs were similar. Indeed, LLPs extracted from loquat leaves exhibited strong in vitro fat binding, cholesterol binding, and bile acid binding capacities, as well as remarkable inhibitory effects on α-amylase and α-glucosidase. Results are helpful for the better understanding of the chemical structures and bioactivities of LLPs extracted from loquat leaves, and LLPs could be further explored as functional food ingredients for prevention and treatment of obesity and type 2 diabetes. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the Scientific Research Foundation of Sichuan Agricultural University (grant number 03120321), the Scientific Research Fund Project of Department of Science and Technology of Sichuan Province (grant number 2018JY0149), and the National Natural Science Foundation of China (grant number 81603066). References [1] A. Podsedek, I. Majewska, M. Redzynia, D. Sosnowska, M. Koziolkiewicz, In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits, J. Agric. Food Chem. 62 (2014) 4610–4617. [2] H. Guo, S. Lin, M. Lu, J.D.B. Gong, L. Wang, Q. Zhang, D.R. Lin, W. Qin, D.T. Wu, Characterization, in vitro binding properties, and inhibitory activity on pancreatic lipase of beta-glucans from different Qingke (Tibetan hulless barley) cultivars, Int. J. Biol. Macromol. 120 (2018) 2517–2522. [3] Q. Yuan, S. Lin, Y. Fu, X.R. Nie, W. Liu, Y. Su, Q.H. Han, L. Zhao, Q. Zhang, D.R. Lin, W. Qin, D.T. Wu, Effects of extraction methods on the physicochemical characteristics and biological activities of polysaccharides from okra (Abelmoschus esculentus), Int. J. Biol. Macromol. 127 (2019) 178–186. [4] Q. Jin, H.H. Yu, X.Q. Wang, K.C. Li, P.C. Li, Effect of the molecular weight of watersoluble chitosan on its fat-/cholesterol-binding capacities and inhibitory activities to pancreatic lipase, PeerJ 5 (2017) 3279. [5] H.Y. Li, Q. Yuan, Y.L. Yang, Q.H. Han, J.L. He, L. Zhao, Q. Zhang, S.X. Liu, D.R. Lin, D.T. Wu, W. Qin, Phenolic profiles, antioxidant capacities, and inhibitory effects on digestive enzymes of different kiwifruits, Molecules 23 (2018) 2957. [6] J.J. Wu, S.S. Shi, H.J. Wang, S.C. Wang, Mechanisms underlying the effect of polysaccharides in the treatment of type 2 diabetes: a review, Carbohydr. Polym. 144 (2016) 474–494. [7] N.N. Sun, T.Y. Wu, C.F. Chau, Natural dietary and herbal products in anti-obesity treatment, Molecules 21 (2016) 1351. [8] X. Xu, B. Shan, C.H. Liao, J.H. Xie, P.W. Wen, J.Y. Shi, Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice, Int. J. Biol. Macromol. 81 (2015) 538–543. [9] R.D. Devaraj, C.K. Reddy, B.J. Xu, Health-promoting effects of konjac glucomannan and its practical applications: a critical review, Int. J. Biol. Macromol. 126 (2019) 273–281. [10] Y. Zheng, L. Bai, Y. Zhou, R. Tong, M. Zeng, X. Li, J. Shi, Polysaccharides from Chinese herbal medicine for anti-diabetes recent advances, Int. J. Biol. Macromol. 121 (2019) 1240–1253. [11] P.C. Wang, S. Zhao, B.Y. Yang, Q.H. Wang, H.X. Kuang, Anti-diabetic polysaccharides from natural sources: a review, Carbohydr. Polym. 148 (2016) 86–97.
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