Unveiling the Hypoglycemic Potential of the Traditional Cuisine Jiao Hua Ji: The Role of Lotus Leaf Heteropolysaccharide

TL;DR

This study explores how a traditional Chinese dish, Jiao Hua Ji, may help lower blood sugar due to polysaccharides from lotus leaves.

Contribution

The study identifies and characterizes specific polysaccharides in lotus leaves and their hypoglycemic effects in Jiao Hua Ji.

Findings

Purified polysaccharides from lotus leaves inhibited α-amylase activity, correlating with molecular weight and glucose content.

Molecular docking showed polysaccharides bind to α-amylase with low energy, suggesting a mechanism for hypoglycemic effects.

Polysaccharides improved glucose utilization by 157.5% without causing cell toxicity.

Abstract

Lotus leaf provides unique nutritional properties to the traditional Chinese dish Jiao Hua Ji. However, its functional polysaccharides remain inadequately characterized. This study evaluates the physicochemical properties and hypoglycemic effects of lotus leaf polysaccharides in Jiao Hua Ji. Ultrasonic-assisted enzymatic extraction significantly improved the yield of polysaccharides to 10.35 ± 0.39%. The yield of the polysaccharides as well as uronic acid content demonstrated a strong correlation with the bioactivity. FTIR analysis confirmed the characteristic infrared spectral features associated with glucans. Four polysaccharides were purified and characterized as 719 kDa (Glc/Gal/Ara 98.91:0.44:0.65), 1010 kDa (Glc/Gal/Ara 98.43:1.18:0.39), 447 kDa (Glc/Gal/Ara 97.17:2.02:0.82), and 327 kDa (Glc/Gal/Ara 97.54:2.06:0.4). The purified polysaccharides exhibited enhanced inhibition of α-amylase, positively correlating with molecular weight and glucose content. Molecular docking studies revealed that the polysaccharide successfully occupies the hydrophobic pocket of α-amylase through hydrogen bonds, with a low binding energy of −6.548 kcal/mol. Notably, the purified polysaccharide significantly improved glucose utilization by 157.5% without cytotoxicity. This study may provide a foundational basis for the application of Jiao Hua Ji in hypoglycemic dietary intervention.

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1. Introduction

Dietary intervention is crucial for diabetes management [1]. Jiao Hua Ji, a traditional Chinese diet, incorporates lotus leaf, an aromatic herb that contributes distinctive nutritional and flavor qualities, highlighting the significance of traditional diets in contemporary health practices [2]. In ancient China, lotus leaf was orally administered to patients as an herbal remedy to relieve fever, combat dehydration, and treat sunstroke, demonstrating systemic effects in the human body that were ascribed to its marked antioxidant capacity and antifungal activity [3,4,5,6]. Furthermore, lotus leaf is frequently incorporated into health beverages aimed at weight loss and is utilized in traditional medicinal practices due to its beneficial properties [7]. As an emerging functional food ingredient, lotus leaf is increasingly employed to enhance both flavor and nutritional values, with previous studies highlighting its hypoglycemic effects [7,8,9]. These attributes underscore the hypoglycemic potential of Jiao Hua Ji in dietary interventions.

Polysaccharides are key active components in lotus leaf, exhibiting various functional activities such as anti-inflammatory and antioxidant properties [10,11,12]. Notably, these compositionally heterogeneous macromolecules play crucial roles in promoting hypoglycemic efficiency [7,9]. Lotus leaf polysaccharide has been shown to significantly enhance the lipid profile and antioxidant enzyme levels in gestational diabetes mellitus rats, positioning it as a promising functional food with potential blood glucose regulatory properties [13]. However, despite the documented hypoglycemic properties of lotus leaf polysaccharides, the impact of processing methods on its bioactivity remains unclear during the preparation of Jiao Hua Ji. This lack of clarity highlights the insufficient scientific theoretical support for its role in dietary intervention, thereby hindering the industrial application of Jiao Hua Ji as a hypoglycemic dietary option. Further investigation is necessary to elucidate the functional characteristics of lotus leaf polysaccharides in Jiao Hua Ji [14,15].

Traditional polysaccharide extraction methods, such as hot-water extraction, often lead to thermal degradation and loss of activity in lotus leaf polysaccharides. In contrast, low-temperature rapid extraction using ultrasound effectively preserves the polysaccharide structure, enhancing free radical scavenging activity. The cavitation effect of ultrasound disrupts cell walls, while the combination with enzymatic hydrolysis selectively hydrolyzes the pectin–cellulose network, facilitating polysaccharide dissolution [16,17]. Moreover, both methods operate in an aqueous phase without strong acids or bases, reducing the need for subsequent desalting and neutralization and promoting environmental sustainability. Thus, a “dual-assisted ultrasound-enzymatic” extraction approach enhances yield while obtaining structurally intact lotus leaf polysaccharides [14].

This research aims to elucidate the hypoglycemic mechanisms of lotus leaf heteropolysaccharides from Jiao Hua Ji. The yields, antioxidant activities, and amylase inhibitory activities of polysaccharides extracted by three methods were evaluated. Four distinct heteropolysaccharides were purified, and correlation analyses were conducted on their molecular weights, monosaccharide compositions, free radical scavenging activities, and hypoglycemic effects. Additionally, molecular docking studies were performed to investigate the glycemic regulation mechanisms at the molecular level. This study provides a theoretical foundation for the hypoglycemic effects of Jiao Hua Ji and its potential as a dietary intervention for diabetes.

2. Materials and Methods

2.1. Materials and Reagents

The Jiao Hua Ji lotus leaf was obtained from local farmers’ markets of Hangzhou (Zhejiang, China). DEAE- cellulose, α-amylase, soluble starch, the fetal bovine serum (FBS), dextran standards and cellulase were purchased from Hangzhou Zeheng-Bio Technology Co., Ltd. (Hangzhou, China). PMP (1-phenyl-3-methyl-5-pyrazolone) and standards of monosaccharides were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA). HepG2 cell line was kindly donated by Prof. L. R. Shen of Zhejiang University (Hangzhou, China). The other reagents were of analytical grade.

2.2. Extraction of the Polysaccharides

Jiao Hua Ji lotus leaf was dried and ground into 200 mesh powder, then mixed with distilled water at a 1:20 (m/v) ratio. The solution was treated with a JY92-IIN ultrasonic device (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at 400 W for 40 min. Ultrasonic-assisted enzymatic extraction was performed with cellulase at 50 °C and pH 4.5 for 1 h, followed by ultrasonic treatment. The extract was depigmented using AB-8 macroporous resin and concentrated at 50 °C. Dialysis through a 12 kDa filter removed small molecules. Precipitation with 95% ethanol at 4 °C overnight yielded crude polysaccharide [15]. The polysaccharide yield is calculated as follows:

$$[eqn]$$

where W1 (g) denotes the mass of the polysaccharide extract, while W2 (g) signifies the mass of the lotus leaf powder.

2.3. Purification

The crude polysaccharide (1 g) was redissolved in distilled water and filtered with a 0.22 μm membrane before being loaded onto a DEAE-cellulose column (55 cm × 3.5 cm). Distilled water and NaCl solutions (0.1, 0.5, and 0.9 mol/L) were used to elute the column at a flow rate of 1.0 mL/min, with fractions collected in 10 mL tubes. The fractions were combined according to the elution curve, and labeled HY-1, 2, 3, and 4, then lyophilized for further use [18].

2.4. FTIR

Using an infrared spectrometer (VERTEX 70, Bruker Corporation, Billerica, MA, USA), the FTIR spectrum of the polysaccharide (1 g/mL) dissolved in distilled water was recorded at a 4 cm^−1^ resolution with 32 scans, spanning the range of 4000 to 400 cm^−1^ in ATR (attenuated total reflection) mode.

2.5. Analysis of Polysaccharide and Uronic Acid Content

The total sugar and uronic acid contents were determined by phenol–sulfuric acid and carbazole–sulfuric acid methods, respectively, as described by Xiao et al. with modifications [19]. Briefly, total sugar was quantified using D-glucose standards (0.02–0.20 mg/mL) measured at 490 nm. Uronic acid was determined against D-glucuronic acid standards (0.02–0.20 mg/mL) following acid hydrolysis (80% (v/v) H_2_SO_4_, 80 °C, 1 h) and carbazole reaction, with absorbance at 530 nm. Both assays employed triplicate measurements with R^2^ > 0.99 for standard curves.

2.6. Monosaccharide Composition Analysis

Monosaccharide composition analysis was conducted using the PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization method with slight modifications [20]. Isolated polysaccharide fractions (10 mg) were dissolved in 10 mL of 4 mol/L trifluoroacetic acid and hydrolyzed at 115 °C for 6 h. After hydrolysis, 2 mL of methanol was added to remove residual trifluoroacetic acid. For PMP derivatization, 300 μL of the sample was mixed with 200 μL of 0.3 mol/L PMP in methanol, and 200 μL of 0.3 mol/L NaOH. The reaction was stopped with 200 μL of 0.3 mol/L HCl. Chloroform was used to remove PMP, and the aqueous layer underwent filtration through a 0.22 μm filter before analysis on a CarboPac PA-20 ion exchange column with 3.75 mmol/L NaOH at 0.5 mL/min using a pulsed amperometric detector.

2.7. Molecular Weight Analysis

The molecular weights (Mw) of the purified polysaccharide were analyzed using high-efficiency size-exclusion chromatography with a refractive index detector (RID). The eluent, 0.1 mol/L NaNO_3_, was injected at a volume of 20 μL and a flow rate of 0.5 mL/min at 25 °C, utilizing sequential ShodexOHpak SB-803 HQ, SB-804 HQ, and SB-805 HQ (3 × 8 mm × 300 mm). The polysaccharide solution in distilled water (15 mg/mL) was passed through a 0.22 μm filter prior to injection [21].

2.8. Radical-Scavenging Activity Analysis

The DPPH radical-scavenging activity was assessed using a methanolic DPPH solution following modified spectrophotometric methods [22]. The activity was calculated as below:

$$[eqn]$$

where OD1 and OD2 represent the absorbance of the control and sample, respectively.

Additionally, the ability to scavenge ABTS free radicals, the power to reduce ferric ions (FRAP), and the capacity to neutralize OH• radicals were determined following the instructions provided by the manufacturer (Nanjing Jiancheng, Nanjing, China).

2.9. Analysis of α-Amylase Inhibitory

The inhibitory activity of polysaccharides on α-amylase was assessed using a modified method based on Ofosu et al. [23]. Porcine pancreatic α-amylase (5 U/mg) was dissolved in sodium phosphate buffer (0.1 mol/L, pH 6.7) at 20 mg/mL. The reaction mixture, with a total volume of 100 μL, contained 20 μL of enzyme solution, 20 μL of sample (1 mg/mL), and 60 μL of soluble starch (2%, w/v). Following pre-incubation of enzyme and sample at 37 °C for 10 min to allow binding equilibrium, starch substrate was added and the mixture was vortexed and incubated at 37 °C for an additional 10 min. The reaction was monitored by measuring absorbance at 660 nm using a microplate reader. The inhibition percentage was calculated as follows:

$$[eqn]$$

where A_0_ is the absorbance of the initial reaction mixture containing enzyme, sample and buffer (without starch substrate), and A_t_ is the absorbance of the mixture containing enzyme and sample at different times. Acarbose (0.01–1.0 mg/mL) served as a positive control, and all measurements were performed in triplicate.

2.10. Glucose Uptake Analysis

Study of how glucose is absorbed in HepG2 cells with insulin resistance was conducted based on a modified protocol [18]. In 96-well plates, HepG2 cells at a density of 6000 cells per well were seeded with 100 μL DMEM and then incubated in a 5% CO_2_ humidified atmosphere at 37 °C for 12 h. After washing with PBS, high glucose DMEM medium containing 2% FBS and insulin (0.3 × 10^−6^ mol/L) was added for 36 h to induce insulin resistance. Following this, the insulin-resistant cells were washed with serum-free high glucose DMEM and treated with purified polysaccharide at 0.2 mg/mL. Medium instead of sample served as the normal control, while 3.5 mg/L metformin served as the positive control. After 24 h, glucose concentration in the culture medium was analyzed using a glucose test kit from Nanjing Jiancheng, China, following the instructions provided by the manufacturer.

2.11. Cell Viability Analysis

Cell viability of HepG2 cells treated with purified polysaccharide was assessed using the MTT assay. Following incubation, cells were cultured with 50 μL of 0.5 mg/mL MTT for 4 h. After removing supernatants, 150 μL of DMSO was added. Absorbance was recorded at 492 nm for each well using a microplate reader [24].

2.12. Molecular Docking

Molecular docking was conducted using blind docking with Autodock Vina 1.2.2 software [25]. Based on the structural characteristics of lotus leaf polysaccharides, fragments that retain essential functional groups and structural features were selected for molecular docking studies with α-amylase. The polysaccharide’s structures at the molecular level were sourced from PubChem Compound (https://pubchem.ncbi.nlm.nih.gov/), and the PDB file for α-amylase (PDB ID 1H7P; resolution 2.5 Å) was obtained from the PDB (http://www.rcsb.org/pdb/home/home.do). Protein and molecular files were transformed into PDBQT format, with water molecules removed and polar hydrogen atoms added. The grid box, centered on each protein domain, measured 30 Å × 30 Å × 30 Å with a grid point spacing of 0.05 nm.

2.13. Statistical Analysis

For the statistical analysis, SPSS version 18 (Chicago, IL, USA) was utilized. A significance threshold of p < 0.05 was applied, and data were presented as mean ± SD. Origin 2018 software was used for data visualization.

3. Results and Discussion

3.1. Lotus Leaf Polysaccharide Yield and Uronic Acid Content

Crude polysaccharides were extracted using ultrasonic (HE-U) and enzymatic (HE-E), yielding 7.67 ± 0.11% and 8.73 ± 0.25%, respectively, comparable to the previous study [7]. In contrast, ultrasonic-assisted enzymatic extraction (HE-UE) significantly enhanced the yield to 10.35 ± 0.39%, which was notably greater than that of the other methods (p < 0.05).

Uronic acid content is a key factor contributing to the hypoglycemic activity of polysaccharides. In this study, the uronic acid content of HY-UE (6.22 ± 0.13%) is significantly higher than that of HY-U (3.22 ± 0.07%) and HY-E (4.71 ± 0.11%) as indicated in Table 1. Higher uronic acid content indicates more negative charge sites. The carboxyl groups of uronic acid may bind to receptors in insulin-resistant cells, exerting blood glucose-lowering effects [26].

3.2. Radical-Scavenging Activity

Notably, at a concentration of 1 mg/mL, HY-UE exhibited significantly greater scavenging activity and reducing capacity compared to both HY-U and HY-E, as illustrated in Figure 1A,B. The polysaccharides prepared by three extraction methods exhibited notable scavenging abilities against DPPH, ABTS, and hydroxyl (•OH) free radicals, as well as demonstrated ferric reducing ability of antioxidants (FRAP). The effectiveness of these methods was ranked from highest to lowest as follows: HY-UE, HY-E, and HY-U.

Through Pearson correlation analysis, antioxidant activity (DPPH, ABTS, •OH, and FRAP) was significantly correlated with polysaccharide yield, with correlation coefficients of 0.94, 0.96, 0.95, and 0.96, respectively. Additionally, significant correlation was demonstrated between antioxidant activity and uronic acid content, with correlation coefficients of 0.97, 0.94, 0.94, and 0.97, respectively (Figure 2B).

3.3. FTIR Analysis of the Polysaccharides

The infrared spectra of the polysaccharides extracted via three methods exhibit similar peak patterns. A broad absorption peak around 3312 cm^−1^ indicates -OH stretching, reflecting hydrogen bonds. A weaker peak near 2933 cm^−1^ originates from saturated C-H stretching, likely from methyl or methylene groups in the monosaccharide units [27]. The peak at 1635 cm^−1^ suggests the presence of carboxylic acids or salts due to C=O stretching [28]. Additionally, the peak around 1427 cm^−1^ indicates glycosidic bonds and hydroxyl groups, while a strong peak at 1020 cm^−1^ signifies the stretching vibrations of C-O-C and bending vibrations of O-H, representing a typical infrared spectral feature of glucans, suggesting that lotus leaf polysaccharides have a glucan structure. The peak at 927 cm^−1^ corresponds to C-O stretching from β-D-pyranose, confirming the pyranose conformation of the polysaccharides [29]. However, a comprehensive elucidation of the detailed chemical structure, including glycosidic linkages and branching patterns, remains necessary.

3.4. Molecular Weight

Four polysaccharides, designated as HY-1, -2, -3, and -4, were purified from the HY-UE, yielding 0.06%, 0.54%, 0.32%, and 0.27%, respectively. The isolation profile is depicted in Figure 3. Additionally, the molecular weights (Mws) of HY-1, -2, -3, and -4 were estimated to be 719 kDa, 1010 kDa, 447 kDa, and 327 kDa, respectively (Table 1). The molecular weights of these purified polysaccharides are higher than those reported in previous studies, suggesting that the production process of Jiao Hua Ji may result in the differential molecular weight distribution of lotus leaf polysaccharides [7,15].

3.5. Monosaccharide Composition

Monosaccharide composition is crucial for fully understanding the structural of polysaccharides and its functional properties [30]. As shown in Figure 4B, HY-1, -2, -3, and -4 were primarily composed of glucose, galactose, and arabinose in the ratios of 98.91:0.44:0.65, 98.43:1.18:0.39, 97.17:2.02:0.82, and 97.54:2.06:0.4, respectively. In contrast, the monosaccharide composition of these polysaccharides differs from that reported by Hwang et al. [14], where the lotus leaf polysaccharide consisted of 29.8% galactose, 3.5% glucose, 21.2% arabinose, and 11.9% rhamnose. This variation may be attributed to multiple factors. In addition to the high-temperature preparation of Jia Hua Ji, batch variation, including genetic diversity, geographic origin, harvest timing, and post-harvest handling, can significantly influence monosaccharide composition. Future research should prioritize cultivar selection and the standardization of raw materials to enhance consistency and comparability [7,31].

3.6. Inhibition of α-Amylase Activity

Inhibition of α-amylase activity is the key indicator to evaluate the hypoglycemic activity of polysaccharides [32]. As shown in Figure 1C, inhibitory effects of the crude polysaccharides toward α-amylase were significantly lower than the positive control (p < 0.05). However, inhibitory effects of purified fractions obvious enhanced, and HY-2 (1 mg/mL) isolated from HY-UE exhibited stronger α-amylase inhibitory activity than the positive control acarbose, a well-known hypoglycemic constituent at 0.1 mg/mL (p < 0.01) (Figure 4A). Comparison with acarbose requires careful consideration of molecular weight differences and mechanism of action. While acarbose exhibits higher molar potency as a competitive active-site inhibitor, lotus leaf polysaccharides may achieve comparable functional effects through alternative mechanisms—including substrate sequestration, enzyme complexation, and viscosity-mediated diffusion limitation—at physiologically achievable concentrations. The in vitro inhibitory activity should be interpreted as indicative of biological potential rather than direct prediction of clinical efficacy [7,9,13].

Additionally, HY-2 has a higher Mw and has a stronger α-amylase inhibitory activity than HY-1, HY-3, and HY-4, which was similar to the previous studies that show the Mw can affect inhibitory activity of polysaccharides toward α-amylase [33]. Through Pearson correlation analysis, α-amylase inhibitory activity was significantly positively correlated with Mw and glucose proportion of the polysaccharide, with correlation coefficients of 0.96 and 0.86, respectively (Figure 5C) The inhibitory effects correlates with the observed glucose uptake enhancement in cellular assays, providing cross-validation between different experimental data.

3.7. Molecular Docking of the Polysaccharide into the Active Site of α-Amylase

AutodockVina 1.2.2 was utilized to examine the binding strengths and interaction patterns between the polysaccharide and α-amylase [25]. The docking results revealed that the polysaccharide interacts with α-amylase through visible hydrogen bonds and successfully occupies its hydrophobic pocket (Figure 4C). The low binding energy of −6.548 kcal/mol indicates a strong and stable binding affinity between the polysaccharide and the α-amylase target. However, the higher α-amylase inhibitory activity of the lotus leaf polysaccharide compared to acarbose, which has a binding energy of −7.18 kcal/mol, may be related to the difference in the number of hydrogen bonds [34].

3.8. Effects of the Polysaccharides on Glucose Uptake

To investigate the hypoglycemic activity, the effects of the purified polysaccharide on glucose uptake were analyzed in insulin-resistant HepG2 cells. The glucose uptake in untreated HepG2 cells was 1.65 mM (p < 0.01), confirming successful model establishment. Purified polysaccharides significantly increased glucose uptake in model cells (p < 0.01). Specifically, compared to the model group, glucose uptake in insulin-resistant cells treated with HY1, HY-2, HY-3, and HY-4 at a concentration of 0.2 mg/mL increased by 91.6%, 157.5%, 94.0%, and 91.8%, respectively. These findings are comparable to those of the positive control, metformin, and are consistent with prior research indicating that lotus leaf polysaccharides significantly enhance glucose uptake in a concentration-dependent manner in insulin-resistant HepG2 cells. This effect improves insulin sensitivity through the modulation of the IRS1/PI3K/Akt signaling pathway, highlighting their potential role in promoting glucose uptake [35]. However, the physiological relevance and signaling pathway analysis of these findings requires further confirmation through in vivo studies. Due to their high molecular weight, the polysaccharides may primarily rely on colonic microbial fermentation rather than direct absorption into the bloodstream [36]. The observed in vitro effects on HepG2 cells may not directly translate to in vivo efficacy, future studies should therefore focus on elucidating the metabolic fate of these polysaccharides, their interaction with gut microbiota, and the potential role of fermentation-derived metabolites in mediating hypoglycemic effects.

3.9. Effects of the Polysaccharides on Cells Viability

The MTT assay results indicated that purified polysaccharides at 0.2 mg/mL did not affect the viability of insulin-resistant HepG2 cells (Figure 5B), aligning with previous findings on the cytoprotective effects of lotus leaf [6]. Consequently, considering the impact on glucose uptake, these polysaccharides are capable of promoting glucose utilization and enhancing glucose metabolism in insulin-resistant cells without exhibiting cytotoxicity. This study further emphasizes the in vitro hypoglycemic potential of Jiao Hua Ji lotus leaf polysaccharides, which may warrant further investigation for potential dietary applications.

4. Conclusions

This study systematically compared the yield, antioxidant capacity, and infrared characteristics of lotus leaf polysaccharides derived from Jiao Hua Ji using various extraction methods. The yield of the polysaccharides and uronic acid content demonstrated a strong correlation with bioactivity. Four distinct heteropolysaccharides were purified, revealing significantly enhanced hypoglycemic activity positively correlated with glucose content and molecular weight. The polysaccharides exhibited typical glucan characteristics and effectively occupied the hydrophobic pocket of α-amylase through hydrogen bonding, leading to the inhibition of α-amylase activity and improved glucose uptake in insulin-resistant HepG2 cells without compromising cell viability. These findings underscore that lotus leaf polysaccharides, following comprehensive validation of their safety and efficacy, have the potential to serve as functional food ingredients in the management of diabetes.