
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00291-8
10.1016/j.ultsonch.2024.107043
107043
Original Research Article
Simultaneous extraction of oil, protein and polysaccharide from Idesia polycarpa Maxim cake meal using ultrasound combined with three phase partitioning
Shi Xin a
Zhang Qiuqiu a
Yang Jintao a
Huang Renshuai a
Ge Yonghui ab
Wang Jinhua ab
Chen Guangjing gyugjchen@gyu.edu.cn
gjchen1989@126.com
ab⁎
a College of Food Science and Engineering, Guiyang University, Guiyang, Guizhou 550005, PR China
b Engineering Technology Research Center for Processing and Comprehensive Utilization of Idesia polycarpa, National Forestry and Grassland Administration of the People′s Republic of China, Guiyang, Guizhou 550005, PR China
⁎ Corresponding author at: College of Food Science and Engineering, Guiyang University, 103 Jianlongdong Road, Nanming District, Guiyang, Guizhou 550005, PR China (G. Chen). gyugjchen@gyu.edu.cngjchen1989@126.com
24 8 2024
11 2024
24 8 2024
110 10704324 7 2024
19 8 2024
23 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Highlights

• Utilized an efficient ultrasound-assisted three-phase partitioning (UTPP) method.

• Simultaneously extracted oil, protein, and polysaccharides from IPM cake meal.

• Achieved high recovery rates for oil, protein, and polysaccharides.

• Identified oil rich in linoleic acid and protein high in Glu, Asp, and Ser.

• Demonstrated strong inhibitory effects of polysaccharides on α-glucosidase and glycation processes.

This study explored the potential of ultrasonic-assisted three-phase partitioning (UTPP) to simultaneously extract lipids, proteins, and polysaccharides from Idesia polycarpa Maxim (IPM) cake meal, a significant byproduct of oil extraction. The impact of variables such as inorganic salt type, solid–liquid ratio, salt concentration, pH, ultrasonic time, temperature, and volume of dimethyl carbonate was examined. Based on the single-factor tests and response surface methodology (RSM), optimal conditions were identified as 30 % ammonium citrate, a 1:26 solid–liquid ratio, pH 3, 31 min of ultrasonic time, 30 °C temperature, and 15 mL of dimethyl carbonate. These conditions achieved extraction rates of 8.10 % for lipids, 5.03 % for proteins, and 10.03 % for polysaccharides, with recovery rates of 91.62 %, 83.08 %, and 93.95 % respectively. Chemical analysis showed the lipid fraction rich in linoleic acid, and the protein fraction high in glutamic acid, aspartate, and serine. The polysaccharide fraction, mainly RG-I pectin with a molecular weight of 226.58 kDa, exhibited strong thermal stability and inhibitory effects on α-glucosidase and glycation, suggesting potential for functional food and dietary supplement applications. This highlights UTPP as a sustainable method for effectively utilizing valuable compounds from IPM cake meal, outperforming traditional extraction techniques.

Keywords

Idesia polycarpa Maxim cake meal
Ultrasound-assisted three phase partitioning
Response surface methodology
Polysaccharide
Hypoglycemic activity
Anti-glycosylation activity
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pmc1 Introduction

Idesia polycarpa Maxim (IPM), commonly known as the oil grape, is a deciduous tree belonging to the Idesia genus of the Flacourtiaceae family [1]. Known for its adaptability, rapid growth, and ease of cultivation [2], IPM boasts well-developed lateral roots that support its fast growth and production of oil-rich fruits [3]. Recent medical research highlights the nutritional and health benefits of IPM oil, including its association with lower mortality rates from malignant tumors, reduced hypertension prevalence, and fewer genetic diseases [4]. Furthermore, IPM fruit extracts exhibit antioxidant, anti-lipogenic, and apoptosis-inducing properties [5], and the fruits are nutrient-dense, containing tryptophan, protein, linoleic acid, beta-sitosterol, and tocopherol [6]. IPM is a high-yielding perennial oil plant, producing 3000–4500 kg/ha of fruit over a lifespan of 70–100 years, with oil content ranging from 21.27 ± 0.83 % to 39.47 ± 0.32 % [4]. Conventional methods for extracting edible oils from plants typically involve organic solvent extraction using hexane or mechanical squeezing. However, these traditional methods are costly and environmentally harmful due to hexane pollution. Additionally, the nutrient-rich defatted meal cake, a by-product of the extraction process, is often discarded as waste despite containing valuable compounds like (−)-idesolide and (−)-idescarpin, which have potential applications in treating obesity, metabolic syndromes, and skin conditions [2]. Residual organic solvents from conventional extraction methods further limit the practical use of IPM meal cake. There is an urgent need for environmentally friendly and cost-effective oil extraction methods to replace traditional techniques. Currently, the substantial quantity of cake meal produced during extraction is underutilized, with only a small portion used as compost [7]. To address environmental concerns and resource wastage, it is crucial to explore recycling opportunities for these by-products. Our preliminary experiments indicate that IPM meal cake is rich in polysaccharides, protein, and residual oil. However, comprehensive research on the polysaccharides, proteins, and oils within IPM cake meal, the primary by-product of IPM fruit processing, is still lacking. Addressing these challenges can better utilize IPM fruit processing by-products, reduce environmental impact, and unlock new opportunities for sustainable development and health benefits.

IPM's oil, rich in linoleic acid (over 60 % of its total fatty acid content), is a valuable source of edible oils. The peel and seeds of IPM fruit represent 62.3 % and 37.6 % of the fruit's weight, respectively, with oil contents of 37.22 % and 24.29 % [4]. The fatty acid composition and bioactive compounds like tocopherols, phytosterols, and polyphenols in vegetable oils are critical determinants of their quality, nutritional value, and associated health benefits, including reduced cholesterol levels and lower cardiovascular disease risks [8]. Proteins, primarily sourced from plants like grains, legumes, and nuts, play essential roles in tissue formation, physiological regulation, and various biochemical reactions [9]. Research into proteins is vital for addressing major health issues such as cancer, kidney disease, inflammation, and hypertension [10]. Polysaccharides, natural macromolecules known for their biodegradability, non-toxicity, and renewable nature, exhibit antioxidative, anti-tumor, and immune-regulatory activities [9]. Understanding their structures is crucial for exploring their biological activities [11]. There is increasing interest in identifying natural α-glucosidase inhibitors from plant sources due to their hypoglycemic effects, intricately linked to their chemical composition, molecular weight, monosaccharide composition, glycosidic bond configurations, and structural characteristics [12], [13]. Additionally, plant polysaccharides are being explored as natural inhibitors of advanced glycation end-products (AGEs), important for managing age-related diseases such as diabetes and cardiovascular disorders [14], [15]. This exploration of IPM oil, proteins, and polysaccharides highlights their significant potential in health, nutrition, and disease prevention, warranting further research and application in these fields.

Three-phase partitioning (TPP) is a multifunctional method for extracting, purifying, and concentrating macromolecules. It employs various principles such as salting out, isoionic precipitation, co-solvents, osmolytic effects, and kosmotropic precipitation [16]. This technique involves an organic solvent, typically butanol, and a significant amount of salt, such as ammonium sulfate, in a crude extract or suspension. Following stirring and centrifugation, the mixture divides into three distinct layers: the top organic phase (low molecular weight pigments, lipids, and nonpolar compounds), the middle-precipitated phase (proteins), and the bottom aqueous phase (polar components like polysaccharides) [17]. Historically, TPP predominantly used butanol, but its industrial use is restricted due to its volatility and low flash point. Dimethyl carbonate stands out as an excellent alternative, offering benefits such as recyclability, low toxicity, and minimal skin irritation, making it ideal for safer TPP applications [18]. Recent advancements in ultrasonic technology have revolutionized the extraction of biological macromolecules. Ultrasonic waves generate cavitation bubbles that burst, creating intense shockwaves and mechanical shear forces, enhancing mass transfer between the solvent and solute [19]. This method drastically cuts down extraction times and boosts efficiency. Ultrasonic-assisted extraction can achieve higher yields of polysaccharides in less time than traditional methods due to reduced mass transfer resistance [20]. Ultrasound not only speeds up the extraction process but can also alter the biological activities of extracted compounds depending on the operational conditions [21]. The integration of dimethyl carbonate in ultrasonic TPP is a significant advancement, allowing effective extraction from various biological sources such as pumpkin seeds [19], custard apple seed oil [22], Corbicula fluminea [20], and fresh passion fruit peel [23], enhancing the recovery of oils, proteins, and polysaccharides. Despite these advancements, research on the simultaneous extraction of oil, protein, and pectin from IPM cake meal using ultrasonic three-phase partitioning (UTPP) with green dimethyl carbonate as the solvent remains limited. Additionally, the biological activity of the extracted polysaccharides has not yet been investigated. Addressing these gaps could yield substantial industrial and environmental benefits, creating a robust framework for sustainable extraction of valuable biological compounds from IPM cake meal.

Therefore, this study explores the use of ultrasound in conjunction with TPP for the simultaneous extraction of oils (IPMO), proteins (IPMP), and pectin polysaccharides (IPMPP) from IPM cake meal. A triaqueous system was created using dimethyl carbonate (DMC) and an inorganic salt, with optimal extraction conditions determined through single-factor experiments and response surface methodology (RSM). The processed IPM cake meal was analyzed for fatty acid profiles of the oil, amino acid compositions of the proteins, and the chemical and structural characteristics of the polysaccharides. Additionally, the bioactivities of these polysaccharides were evaluated using in vitro hypoglycemic and anti-glycosylation assays, providing critical data for the potential commercial application and value enhancement of IPM cake meal. The experimental approach is detailed in Fig. 1.Fig. 1 The schematic of the experimental design.

2 Materials and methods

2.1 Materials and chemicals

The fresh Idesia polycarpa fruit was collected from Jiubao Qiang Ethnic Township, Pingwu County, Mianyang, Sichuan Province, on November 4, 2022, at coordinates 32°20′54.18′′N, 104°23′47.50′′E, with an elevation of 1584 m. The fruit was dried using a commercial microwave vacuum dryer (WBZ-10PLC, Guiyang Xinqi Microwave Industry Co., Ltd., China) through a staged process at a vacuum level of 0.07 MPa and varying microwave powers: 2 kW for 50 min, 1 kW for 20 min, 0.5 kW for 10 min, and 0.3 kW for 10 min. Oil was extracted using a screw press (CZR309, Dehaiwei Industrial Equipment Co., LTD., Guangzhou, China) at 110 °C, and the cake meal was stored at −20 °C for subsequent analysis. The cake meal composition includes 5.52 % protein, 8.85 % oil, and 10.67 % polysaccharides.

Hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate, methanol phosphate, ammonium sulfate, sodium citrate, ammonium citrate, dimethyl carbonate, acarbose, and p-nitrophenyl-α-D-glucopyranoside (pPNG) were acquired from Shanghai Maclin Biochemical Technology Co., Ltd. Coomassie Brilliant Blue G250 was purchased from Biofrxx, Germany. The pullulan polysaccharide calibration kit was procured from Agilent Co., USA. α-Glucosidase was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). L-fucose (Fuc), L-rhamnose (Rha), L-arabinose (Ara), D-mannose (Man), D-galactose (Gal), D-glucose (Glc), D-xylose (Xyl), D-galacturonic acid (GalA), and D-glucuronic acid (GlcA) were obtained from Tokyo Chemical Industry Co., Ltd. (Japan).

2.2 Extraction of IPMO, IPMP, IPMPP with the UTPP system

Initially, an inorganic salt solution (10–50 % w/v) was prepared with a pH adjusted between 2 and 9 using 1 M NaOH or HCl. IPM cake meal was then combined with 20 mL of this solution, maintaining a material-to-liquid ratio of 1:15 to 1:40 (m/v). After thorough mixing, 5–25 mL of dimethyl carbonate was added, and the mixture underwent ultrasound (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ltd., China) treatment for 10–50 min at 500 W. The mixture was then incubated in a vibration incubator at 20–45 °C for 30 min, followed by centrifugation at 4000 rpm for 15 min, resulting in a three-phase solution. The upper phase containing dimethyl carbonate and IPM oil was collected, concentrated under reduced pressure using a rotary evaporator, and stored at −20 °C. The middle layer, composed of precipitated proteins, was dissolved in 0.05 M Tris-HCl buffer (pH 8.0) and refrigerated at 4 °C for over 12 h. Subsequently, it was dialyzed against deionized water within a molecular weight range of 8000–14000 Da, then freeze-dried and reserved at −20 °C for subsequent research. The lower phase, containing inorganic salts, polysaccharides, and residual meal powder, was adjusted to pH 6.5 using 1 M NaOH. The polysaccharides were concentrated, dialyzed for 48 h (8000–14000 Da), and precipitated by gradually adding four times the volume of anhydrous ethanol under stirring, followed by overnight settling at 4 °C. After centrifugation at 4000 rpm for 10 min and washing twice with anhydrous ethanol, the polysaccharides were redissolved in deionized water at 50 °C, and centrifuged again to remove insoluble impurities. The final polysaccharides extract, obtained via UTPP, was vacuum freeze-dried and stored at −20 °C to prevent degradation from temperature fluctuations, thereby preserving the integrity of the samples for further analysis.

The upper solvent layer was subjected to vacuum concentration to recuperate the dimethyl carbonate. The extraction of IPMO (YIPMO) was determined using the provided formula:(1) YIPMO(%)=M1M×100

where M1 represents the weight of the extracted IPMO (g) and M represents the weight of the initial IPM cake meal sample (g).

The protein content in the IPM cake meal samples was determined using Coomassie Brilliant Blue colorimetry, with the absorbance measured at 595 nm. The extraction yield of IPMP (YIPMP) was then determined using the equation:(2) YIPMP%=CL×VLM×100

where CL represents the concentration of IPMP within the intermediate phase measured in mg/mL, VL indicates the volume of this phase (mL), M denotes the mass of the original IPM cake meal sample (mg).

The concentration of IPMPP was assessed using the phenol–sulfuric acid method, where glucose served as the reference standard. The extraction yield of IPMPP (YIPMPP) was determined as follows:(3) YIPMPP%=CL×VLM×100

where CL represents the concentration of IPMPP in the lower aqueous phase expressed in mg/mL, VL denotes the volume of this phase in mL, and M represents the weight of the IPM cake meal sample in mg.

2.3 Optimization via the response surface methodology test

To optimize the extraction conditions for IPMP and IPMPP, response surface methodology (RSM) was employed using a Box-Behnken design (BBD). This design was configured with four factors—ammonium citrate concentration (X1), solid–liquid ratio (X2), pH (X3), and ultrasound time (X4), each at three levels, identified as the most influential based on preliminary single-factor experiments. The extraction rate was targeted as the dependent variable. The experimental setup, as detailed in Table 1, Table 2, was composed of 24 factorial points and 5 axial points to evaluate method repeatability. The appropriate ranges for each parameter were established from the initial single-factor experiments. A total of 29 randomized experiments were conducted, each being replicated three times, as indicated in Table 2. The data from these experiments were analyzed using a quadratic polynomial model:(4) Y=k0+k1X1+k2X2+k3X3+k4X4+k5X1X2+k6X1X3+k7X1X4+k8X2X3+k9X3X4+k10X12+k11X22+k12X32+k13X42

Table 1 Experimental factors and levels of the Box-Behnken experimental design.

Factors	Units	Symbols	Level of factors	
−1	0	1	
Ammonium citrate concentration	%	X1	20	30	40	
Solid-liquid ratio		X2	20	25	30	
pH		X3	2	3	4	
Ultrasound time	min	X4	20	30	40	

Table 2 The Box–Behnken design and results for the yield of YIPMPP and YIPMP.

Run	Independent variable	YIPMPP (%)	YIPMP (%)	
X1	X2	X3	X4	Actual values	Predicted values	Actual values	Predicted values	
1	1	0	−1	0	8.19	8.24	4.19	4.30	
2	−1	0	0	1	8.28	8.38	4.60	4.73	
3	0	−1	0	−1	7.36	7.59	3.87	3.85	
4	0	−1	1	0	6.62	6.40	3.63	3.23	
5	1	1	0	0	8.24	8.35	4.27	4.28	
6	0	0	0	0	9.63	9.82	4.86	5.31	
7	0	0	0	0	9.91	9.82	5.40	5.31	
8	1	0	1	0	6.9	6.76	3.40	3.52	
9	−1	0	−1	0	7.1	7.35	3.62	3.63	
10	0	1	−1	0	7.69	7.87	3.68	3.94	
11	1	0	0	−1	8.17	8.03	4.74	4.48	
12	0	0	−1	1	8.63	8.34	4.69	4.32	
13	0	−1	0	1	7.02	7.29	3.33	3.50	
14	−1	−1	0	0	7.64	7.46	3.69	3.68	
15	−1	0	0	−1	8.41	8.30	4.05	4.08	
16	1	0	0	1	7.98	8.05	4.06	3.89	
17	−1	1	0	0	8.18	8.05	4.56	4.37	
18	0	−1	−1	0	7.56	7.41	3.50	3.57	
19	0	0	0	0	9.76	9.82	5.40	5.31	
20	0	1	1	0	8.21	8.32	4.70	4.50	
21	0	1	0	1	8.94	8.83	4.54	4.69	
22	−1	0	1	0	8.19	8.26	4.60	4.63	
23	0	0	−1	−1	8.17	8.12	4.03	3.94	
24	0	0	1	1	7.9	7.87	4.00	4.08	
25	0	0	1	−1	7.79	8.01	4.03	4.40	
26	0	0	0	0	9.96	9.82	5.40	5.31	
27	0	1	0	−1	8.59	8.44	4.33	4.29	
28	0	0	0	0	9.84	9.82	5.50	5.31	
29	1	−1	0	0	6.51	6.56	3.15	3.34	

The predicted response (Y), representing YIPMPP and YIPMP, was calculated using a quadratic polynomial model. In this mode, k0 is identified as the intercept term, k1, k2, k3, and k4 are the linear effect terms, k5, k6, k7, k8, and k9 are the interaction effects, and k10, k11, k12 and k13 are the squared effects. The fitness of the model was confirmed by the regression coefficient R2, and its statistical significance was evaluated using the F-test. The significance of the regression was assessed with the t-test. An increase in the absolute F-value and a decrease in the P-value were used as indicators of greater significance of the variables.

2.4 Fatty acid profiles of IPMO

The assessment of the fatty acid composition was conducted using gas chromatography, following the methodology outlined by Li et al. [4]. Fatty acid methyl esters were prepared by the addition of 1 mL of n-hexane to 30 mg of IPMO, followed by 200 μL of 2 mol/L sodium methoxide. The mixture was briefly warmed in a 50 °C bath, followed by the addition of 200 μL of 2 mol/L HCl. Then, 1 μL from the upper layer was injected into a gas chromatograph (Agilent 8890A, USA) equipped with a flame ionization detector (FID) and an SH-Rt2560 capillary column (60 m × 0.25 mm × 0.20 μm). The heating program commenced at an initial temperature of 130 ℃, held for 5 min, followed by a temperature increase at a rate of 4 ℃/min until reaching 240 ℃, where it was maintained for 20 min.

2.5 Amino acid composition of IPMP

An appropriate amount of the sample was placed in a digestion tube, to which 3 drops of phenol were added, followed by the addition of 6 mol/L hydrochloric acid to fill up to 5 mL. The tube was then injected with nitrogen, sealed, and hydrolyzed at 100 °C for 48 h. After suitable dilution, derivatization was performed using the phenyl isothiocyanate (PITC) method. To the 100 μL sample solution, 50 μL of triethylamine-acetonitrile solution (1 mol/L) and 50 μL of PITC acetonitrile solution (0.1 mol/L) were added, mixed, and left to react for 2 h at room temperature. Following the reaction, 200 μL of n-hexane was added, thoroughly mixed, and allowed to stand for 10 min. The lower solution was then collected for analysis using a Thermo Q-Exactive high-resolution mass spectrometer system. The system utilized a Shimazu InsertSustain C18 column (4.6 × 250 mm). The elution procedure was carried out as follows: initial conditions of 10 % A to 10 % B for 0–1 min; 10 % A to 25 % B from 1–1.1 min; 25 % A to 30 % B from 1.1–7 min; maintained at 30 % A to 30 % B from 7–10 min; gradual increase from 30 % A to 33 % B from 10–15 min; sharp increase from 33 % A to 90 % B from 15–15.1 min; held at 90 % A to 90 % B from 15.1-20 min; a return to 90 % A to 10 % B from 20-20.1 min; and finally 10 % A to 10 % B from 20.1–25 min. Mass spectrometry conditions included an ionized transmission tube temperature of 400 ℃, an evaporation temperature of 500n ℃, sheath gas pressure at 75 arb, auxiliary gas pressure at 20 arb, a spray voltage of 3.5 kV, and monitoring of target ions.

2.6 Structural characterization of IPMPP

2.6.1 Chemical composition analysis

Neutral sugar, protein, and uronic acid contents were assessed using the phenol–sulfuric acid method [24], Coomassie brilliant blue method [25], and m-hydroxyphenyl method [24], respectively.

2.6.2 Monosaccharide composition and molecular weight distribution determination

The detection of the monosaccharide composition begins by placing 5 mg of the IPMPP sample into a 10 mL amber bottle. Then, 4 mL of trifluoroacetic acid (2 M) was added, the bottle was sealed using an alcohol blowtorch, and the mixture was hydrolyzed at 105 °C for 6 h. Subsequently, the trifluoroacetic acid was removed by steam distillation under reduced pressure. For chromatographic analysis, the sample was filtered through a 0.22 μm nylon filter membrane into a 50 mL bottle. The analysis was conducted using an ion chromatography system (Dionex ICS 6000, Thermo Fisher, USA) equipped with a Dionex CarboPac PA20 column (3 × 150 mm). The detector was set to electrochemical mode, and the column temperature was maintained at 30 °C. The flow rate was maintained at 0.5 mL/min, and a sample size of 25 μL was injected. The mobile phase gradient elution was performed in three stages: From 0 to 16.05 min: 5 % NaOH (20 mM) and 95 % ultra-pure water; From 16.05 to 30.05 min: 20 % NaOH (20 mM), 25 % CH3COONa (500 mM), and 55 % ultra-pure water; From 30.05 to 50 min: 80 % NaOH (20 mM) and 20 % ultra-pure water. Monosaccharide standards, ranging in concentrations from 1 to 10 ppm, were prepared and the chromatographic analysis was carried out using the same ion chromatography method. The monosaccharide composition and molar percentage of the polysaccharide samples were determined based on the retention times and peak areas of the standard products.

The molecular weight distribution of IPMPP was determined through high-performance gel permeation chromatography (HPGPC), using dextran standards that ranged from 6,300 Da to 739,000 Da, as described in our previous study [26]. The HPGPC analysis was conducted on an Agilent 1260GPC system, which included a refractive index detector (1260 RID, Agilent, USA) and a TSK-Gel GMPWXL column (7.8 × 300 mm, Tosoh Biosep, Japan), maintained at 35 °C. For this analysis, 0.02 M potassium dihydrogen phosphate was used as the mobile phase. An injection volume of 25 μL was utilized, with the column flow rate set at 0.5 mL/min. The calibration curve was constructed, represented by the equation y = − 0.4501x + 11.86, with an R2 of 0.9927.

2.6.3 Zeta potential and particle size distribution analysis

The zeta potential and particle size distribution of the IPMPP in a distilled water solution (2 mg/mL) were examined using dynamic light scattering (DLS). The measurements were conducted at 25 °C using a Malvern Zetasizer, NS-90Z (Malvern, UK).

2.6.4 Fourier transform infrared spectroscopy (FTIR) and degree of methyl-esterification (DM) analysis

The molecular structures of IPMPP were analyzed using FTIR with a Spectrum Two instrument from PerkinElmer Co., USA. A dried sample was finely ground with KBr (1:100, w/w), pressed into a tablet, and examined over a spectral range of 4000–400 cm−1 at a resolution of 4 cm−1. The DM of IPMPP was determined using FTIR. The areas under the peaks corresponding to methyl-esterified groups at 1735 cm−1 (A1735) and free carboxyl groups at 1628 cm−1 (A1628) were measured. The DM was calculated as follows [27]:(5) DM(%)=A1735A1735+A1628×100

2.6.5 Congo red test

The triple-helical structure of IPMPP was investigated using a Congo red test, conducted according to the method previously described in [25]. Initially, a 2 mL aliquot of a 2.5 mg/mL IPMPP solution was mixed with an equal volume of an 80 μmol/L Congo red solution. To adjust the NaOH concentrations from 0.0 to 0.5 mol/L, various volumes (0–4 mL) of 1 mol/L NaOH were added. A control mixture without IPMPP was also prepared. After equilibrating for 10 min, the maximum wavelength (λmax) was recorded using a Thermo Multiskan SkyHigh spectrophotometer in the 200–600 nm range.

2.6.6 X-ray diffraction (XRD) analysis

XRD analyses of dehydrated samples were conducted using a D8 Advance diffractometer (Bruker, Germany). The scanning speed was set to 10°/min, and an incident current of 40 mA was applied, covering a 2θ range of 5–80°.

2.6.7 Thermal stability property

The thermal properties of IPMPP were analyzed using a DSC-4000 apparatus (PerkinElmer, USA). Samples of IPMPP, each weighing 4 mg, were analyzed in a dynamic nitrogen atmosphere. The temperature was increased at a rate of 10 °C/min, covering a range from 45 to 445 °C.

2.6.8 Scanning electron microscopy (SEM)

The surface and microstructure of IPMPP were observed using a SIGMA 300 SEM (ZEISS, Germany). Samples were mounted on an aluminum holder, gold-sputtered, and imaged at magnifications of 100×, 5,000×, and 10,000 × .

2.7 Determination of in vitro hypoglycemic activity of IPMPP

2.7.1 α-Glucosidase inhibitory activity assay

The α-glucosidase inhibitory potential of IPMPP was assessed according to our established method [14] method with adaptations. IPMPP at concentrations of 0.5–6.0 mg/mL, α-glucosidase (0.5 U/mL), and pPNG (5 mM) were prepared in phosphate-buffered saline (100 mM, pH 6.9). Controls included mixtures without IPMPP or enzyme. Acarbose served as the positive control. In the reaction vials, 100 μL of IPMPP and 100 μL of α-glucosidase were combined and incubated at 37 °C for 10 min. Following the initial steps, 100 μL of pPNG was introduced, and the mixture was further incubated at 37 °C for 20 min. The reaction was terminated by adding 1 mL of sodium carbonate (1 M). Absorbance at 405 nm was subsequently measured using a microplate reader (Model 1500, Thermo Multiskan SkyHigh, USA). To calculate the α-glucosidase inhibitory rate of IPMPP, the following approach was used:(6) Inhibition rate (\%) =1-Asample-Acontrol-1Acontrol-2×100

where Asample was the absorbance of the sample reaction solution, Acontrol-1 was the absorbance of the solution without α-glucosidase, and Acontrol-2 was the absorbance of the solution without the sample.

2.7.2 Determination of inhibitory kinetics of IPMPP

The kinetic properties of α-glucosidase (0.5 U) using various concentrations of IPMPP (1, 3, and 6 mg/mL) and the pPNG substrate (1–4 mM) were assessed. Using Lineweaver–Burk plots and linear regression analyses, the inhibition pattern and mechanism of IPMPP on α-glucosidase was examined. These plots facilitated the derivation of the Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax) according to the Michaelis–Menten equation (Eq. (7)). We determined the dissociation constant for the enzyme-inhibitor complex (Ki) as the absolute value where the slope of the Lineweaver-Burk plot intersects the X-axis, corresponding to the curve fit of inhibitor concentration. Additionally, the dissociation constant for the enzyme-inhibitor-substrate complex (Kis) was identified from the absolute value where the Y-axis intercept of the Lineweaver-Burk plot and the X-axis intercept of the inhibitor concentration fit curve intersect [26].(7) 1V=1Vmax+KmVmax·1S

2.7.3 Fluorescence quenching

The interaction between IPMPP and α-glucosidase was investigated using an F-320 FL fluorescence spectrophotometer (Tianjin Gangdong Technology Co., China). Each sample, containing 1.5 mL mixed with α-glucosidase solution (0.75 U/mL), was incubated at 30 °C for 10 min. Fluorescence data were recorded from 300 to 500 nm, with an excitation wavelength of 280 nm. Slit widths were set at 5 nm for both excitation and emission, and a scanning rate of 1200 nm/min was employed. The Stern-Volmer equations were applied to analyze the mechanism of fluorescence quenching and to determine the binding constant and binding site of IPMPP on α-glucosidase. The equations are as follows [26], [28]:(8) FF0=1+kqτ0Q=1+KSVQ

(9) lgF0-FF=lgKa+nlgQ

In the analysis, F0 and F represented the fluorescence intensities before and after the interaction of α-glucosidase with IPMPP, respectively. The concentration of IPMPP was denoted by [Q], while τ0 signified the average lifetime of the fluorescent substance without the quencher, approximately 10-8 s. The Stern-Volmer constant, KSV, was derived via linear regression of F0/F against [Q]. Ka represented the binding constant, and n indicated the number of binding sites. The linear relationship of lg [(F0 − F)/F] with lg [Q] revealed n as the slope and lgKa as the intercept.

2.8 Analysis of non-enzymatic glycation inhibition activity of IPMPP

2.8.1 Non-enzymatic glycation of bovine serum albumin (BSA)

The effect of IPMPP on BSA glycation was investigated using the BSA-fructose model, employing a reported method with slight modifications [29]. BSA (20 mg/mL) was reacted with fructose (90.1 mg/mL) in a PBS solution (0.1 mM, pH 7.4, 0.02 % NaN3), with either IPMPP (1.0–6.0 mg/mL) or AG (1.0–6.0 mg/mL) added. The mixture was kept at 50 °C for 24 h, after which the resulting solution was utilized for further assays.

2.8.2 Measurement of fructosamine concentration

Fructosamine levels were assessed using the nitroblue tetrazolium (NBT) reductive assay, with absorbance measurements taken at 530 nm. The BSA-fructose-water and BSA-fructose-polysaccharide solution reaction systems were incubated at 50 °C for 24 h, respectively. After incubation, 400 μL of the reaction mixture, 320 μL of ultrapure water, and 1.6 mL of NBT (0.3 mM, dissolved in 100 mM sodium carbonate buffer, pH 10.35) were mixed and incubated at 25 °C for 15 min. AG served as the positive control [30]. The fructosamine inhibition rate (IR, %) was determined using the following formula:(10) IR(%)=A0-AA0×100

where A0 and A denoted the absorbance of the BSA-fructose model without and with IPMPP, respectively.

2.8.3 Measurement of α-dicarbonyl compounds

α-Dicarbonyl compounds were detected via the Girard-T assay. The procedure involved mixing 500 μL of the sample solution with 40 μL of ultrapure water, 50 μL of 500 mM Girard-T reagent, and 400 μL of 500 mM sodium formate (pH 2.9). The mixture was incubated at 25 °C for 1 h. After incubation, the absorbance was measured at 290 nm. AG was employed as the positive control. The inhibition rate (IR, %) of α-dicarbonyl compounds was calculated using a specific equation:(11) IR(%)=A0-AA0×100

where A0 and A denoted the absorbance of the BSA-fructose model without and with IPMPP, respectively.

2.8.4 Measurement of fluorescent AGEs

For fluorescent AGEs, the glycated solution (60 μL) was mixed with 2 mL of PBS (0.2 M, pH 7.4). Its fluorescence intensity was measured using a spectro-fluorophotometer (Model F-320, Tianjin Gangdong Technology Co., China) at an excitation wavelength of 370 nm and an emission wavelength of 440 nm [31]. The fluorescent AGEs inhibition rate was determined using the following formula:(12) IR(%)=F0-FF0×100

where F0 and F represented the fluorescence intensity of the glycated solution without and with IPMPP, respectively.

2.9 Statistical analysis

The data are presented as mean ± SD from a minimum of three independent experiments per sample. Regression analysis and graphical optimization were conducted using the trial version 12 of “Design Expert” software (Stat-Ease, Minneapolis). Experimental results underwent one-way analysis of variance (ANOVA) via SPSS software (Version 23.0, SPSS Inc., Chicago, IL), and figures were generated using Origin 2021 software (Origin Lab Corp., MA, USA).

3 Results and discussion

3.1 Effects of single factors on the extraction yield of IPMP and IPMPP

3.1.1 Effects of inorganic salt type

Within the TPP system, (NH4)2SO4 is preferred due to its compatibility with proteins and high solubility. Citric acid is also frequently used for its cost-effectiveness in extracting pectin polysaccharides [19]. The addition of salt triggers osmotic shock, which increases cellular permeability and enhances protein solubility in the mixture. The effects of inorganic salt types on the extraction rate of protein and polysaccharide were studied under the conditions of solid–liquid ratio of 1:15 (m/v), inorganic salt concentration of 20 %, pH 6.0, dimethyl carbonate volume of 15 mL, ultrasonic time of 20 min and temperature of 30 °C. As shown in Fig. 2A, the yields of IPMP (3.90 ± 0.10 %) and IPMPP (6.73 ± 0.20 %) were significantly higher when ammonium citrate was used as the solute, compared to ammonium sulfate and sodium citrate. Therefore, ammonium citrate was selected as the inorganic salt for subsequent experiments.Fig. 2 (A) The effects of inorganic salt types on the extraction rate of IPMP and IPMPP; (B) The effects of ammonium citrate concentration on the extraction rate of IPMP and IPMPP; (C) The effects of solid–liquid ratio on the extraction rate of IPMP and IPMPP; (D) The effects of pH on the extraction rate of IPMP and IPMPP; (E) The effects of ultrasonic time on the extraction rate of IPMP and IPMPP; (F) The effects of temperature on the extraction rate of IPMP and IPMPP; (G) The effects of DMC volume on the extraction rate of IPMP and IPMPP.

3.1.2 Effects of ammonium citrate concentration

The effects of ammonium citrate concentration (10–50 %) on the extraction rate of protein and polysaccharide were studied under the conditions of solid–liquid ratio of 1:15 (m/v), pH 6.0, dimethyl carbonate volume of 15 mL, ultrasonic time of 20 min and temperature of 30 °C. As illustrated in Fig. 2B, the yield of IPMPP initially increased and then declined as the concentration of ammonium citrate rose from 10 g/100 mL to 50 g/100 mL. This trend is attributed to an intensified salting-out effect with increasing ammonium citrate concentration. An optimal concentration of ammonium citrate promotes protein flocculation, thereby enhancing the release of proteins bound to lipids [19]. At a 30 % concentration, the extraction rates for IPMPP and IPMP peaked at 7.63 % and 4.10 %, respectively. Beyond this concentration, the extraction rates for both IPMPP and IPMP gradually decreased, likely due to the increased binding of salt ions with water molecules. This binding reduces the hydrogen bond network between the polysaccharides and water, adversely affecting the polysaccharide extraction process [32]. Consequently, 30 % ammonium citrate was selected as the central point for subsequent response surface experiment.

3.1.3 Effects of solid–liquid ratio

In the initial stage of the UTPP process, a specified concentration of ammonium citrate is added to the cake meal, causing the cells to absorb water and increase the mixture's viscosity. If the volume of the inorganic salt solution is too small, the resulting high viscosity negatively impacts the extraction rate [22]. Conversely, excessive addition of the inorganic salt solution escalates process costs due to increased salt requirements and a larger volume of the extraction mixture. Therefore, optimizing the solid–liquid ratio is critical. The effects of solid–liquid ratio 1:15 to 1:40 (m/v) on the extraction rate of protein and polysaccharide were studied under the conditions of ammonium citrate concentration 30 %, pH 6.0, dimethyl carbonate volume 15 mL, ultrasonic time 20 min, and temperature 30 °C. As demonstrated in Fig. 2C, a 1:25 ratio was identified as optimal, yielding extraction rates of 7.51 % for IPMP and 3.91 % for IPMPP. Adjusting the solid–liquid ratio from 1:15 to 1:25 raised the IPMPP yield from 5.98 % to 7.51 %. However, increasing the ratio further from 1:30 to 1:45 reduced the yield from 7.03 % to 5.79 %, with IPMP exhibiting a similar trend. This decrease is likely due to the reduced viscosity at higher solid–liquid ratios, which may impede the efficient extraction of IPMPP and IPMP. Consequently, a solid–liquid ratio of 1:25 was chosen as the central point for the subsequent response surface experiments.

3.1.4 Effects of pH

The separation efficiency of UTPP is highly dependent on pH alterations, which influence the ionization of amino acids in proteins and, consequently, the properties of macromolecules. As a result, the distribution of proteins and polysaccharides within the UTPP system varies with pH changes [19]. The effects of pH (2.0–9.0) on the extraction rate of protein and polysaccharide were studied under the conditions of ammonium citrate concentration of 30 %, solid–liquid ratio of 1:15 (m/v), dimethyl carbonate volume of 15 mL, ultrasonic time of 20 min, temperature of 30 °C, constant. Within the pH range of 2.0 to 3.0, the extraction rate of IPMPP gradually increased. However, beyond pH 3.0, the rate began to decline. Notably, at pH 3.0 and 4.0, the extraction rate of IPMP peaked at 3.73 % and 3.71 %, respectively, with no significant difference observed between these values (Fig. 2D). Adjusting the pH influences the surface charge of ionized proteins, thereby impacting the extraction efficiency of both polysaccharides and proteins [16]. This study found that the UTPP system was particularly effective under acidic conditions (pH 3.0) for extracting and separating IPMPP from IPM cake meal. At this pH, IPMPP achieved a maximum yield of 8.33 %. Previous studies suggested that under acidic conditions, polysaccharides exhibit the lowest solubility due to a net charge of zero, which facilitates maximal protein precipitation. This phenomenon aids in the separation of oil and protein in the cake meal and enhances the solubility of oil in dimethyl carbonate [33]. As the pH increases, the separation efficiency of glycoproteins bound to polysaccharides is affected, thereby influencing IPMPP extraction. Consequently, the optimal IPMPP yield was recorded at pH 3.0, marking significant changes in its extraction rate (p < 0.05). Moreover, the effects of pH 3.0 and pH 4.0 on IPMP extraction showed no significant variations (p > 0.05). Given the impact of pH on both IPMP and IPMPP yields, pH 3.0 was selected as the central point for further response surface experiments.

3.1.5 Effects of ultrasound time

Ultrasonic time is a pivotal factor in UTPP, impacting both yield and production costs. The effects of ultrasonic time (10–50 min) on the extraction rate of protein and polysaccharide were studied under the conditions of ammonium citrate concentration of 30 %, solid–liquid ratio of 1:15 (m/v), pH 6.0, dimethyl carbonate volume of 15 mL, temperature of 30 °C, constant. Extending the extraction time from 10 to 30 min led to an increase in the extraction rate of IPMPP from 6.54 % to 8.81 %, as illustrated in Fig. 2E, with IPMP showing a similar increase from 3.77 % to 4.89 %. Further extension beyond 30 min did yield significant descent in the extraction rates of IPMPP and IPMP. UTPP is likely to enhance the purity of polysaccharides, as ultrasound helps to break down or disperse polysaccharides in the aqueous phase, thereby enhancing mass transfer during UTPP separation [20]. This facilitates the comprehensive extraction of IPMPP and IPMP. Based on these results, 30 min was determined to be the optimal ultrasonic time for subsequent response surface experiments.

3.1.6 Effects of temperature

The effects of temperature (20–45 °C) on the extraction rate of protein and polysaccharide were studied under the conditions of ammonium citrate concentration of 30 %, solid–liquid ratio of 1:15 (v/v), pH 6.0, dimethyl carbonate volume of 15 mL and ultrasonic time of 20 min. Between 20 ℃ and 30 ℃, the extraction rates for IPMPP (up to 7.55 %) and IPMP (up to 2.63 %) increased significantly with temperature (p < 0.05) (Fig. 2F). However, as the temperature continued to rise from 30 ℃ to 45 ℃, the extraction rates decreased. In the UTPP system, higher temperatures are thought to facilitate the enrichment of IPMPP in the lower phase. This effect may be due to increased temperatures promoting the exposure of hydroxyl groups on macromolecules, leading to enhanced formation of hydrogen bonds or linkages, rendering IPMPP more hydrophilic and thus more concentrated in the lower phase [34]. However, excessively high extraction temperatures can affect protein solubility, potentially causing the re-dissolution of the precipitated phase into the aqueous phase, thereby reducing protein yield. Additionally, higher temperatures negatively impact the cavitation effect in the UTPP process, decreasing the extraction yield [35]. Based on these results, it is determined that 30 °C is the temperature for subsequent experiments.

3.1.7 Effects of dimethyl carbonate (DMC) volume

One advantage of using dimethyl carbonate (DMC) is that it does not cause salting out, even with excessive use, allowing proteins to transfer from the aqueous phase to the intermediate phase. Additionally, DMC's partial solubility in water (up to 13 %) aids in concentrating proteins in the intermediate phase. As a green solvent, DMC enhances the environmental friendliness of the process. Increasing the volume of DMC promotes the flotation of precipitated proteins above the lower water phase, facilitating the separation of proteins from lipids and polysaccharides [18]. The effects of DMC volume (5–25 mL) on the extraction rate of protein and polysaccharide were studied under the conditions of ammonium citrate concentration of 30 %, solid–liquid ratio of 1:15 (m/v), pH 6.0, temperature of 30 °C and ultrasonic time of 20 min. As shown in Fig. 2G, the highest extraction rates for IPMP (4.16 %) and IPMPP (7.31 %) were observed when the volume of DMC was 15 mL. However, increasing the DMC volume to 25 mL led to decreased extraction rates for both IPMP and IPMPP. This decrease is likely due to protein denaturation caused by excessive organic solvent, which hinders the proteins' ability to settle into the intermediate phase. Based on these results, the DMC volume of 15 mL was determined as the volume of subsequent experiments.

3.2 Optimization of IPMPP conditions by BBD

3.2.1 Statistical analysis and model fitting

The optimization of the extraction procedure for IPMP and IPMPP was conducted using RSM. A BBD was implemented, involving four independent factors—ammonium citrate concentration (X1), solid–liquid ratio (X2), pH (X3), and ultrasound time (X4)—over 29 experimental runs to optimize the extraction of IPMP and IPMPP. The values of these variables and their corresponding predicted extraction yields for IPMP and IPMPP are detailed in Table 2. The predictive model was validated through multiple regression analysis of the experimental data. The extraction yields for IPMP and IPMPP (Y) are represented by the following second-order polynomial equation:YIPMP=5.31-0.1099X1+0.4096X2-0.0551X3+0.0149X4+0.0614X1X2-0.4436X1X3-0.3074X1X4+0.2211X2X3+0.1852X2X4-0.1725X3X4-0.5935X12-0.8026X22-0.6999X32-0.4250X42

YIPMPP=9.82-0.1508X1+0.5950X2-0.1442X3+0.0217X4+0.2975X1X2-0.5950X1X3-0.0150X1X4+0.3650X2X3+0.1725X2X4-0.0875X3X4-1.03X12-1.18X22-1.14X32-0.5996X42

A comprehensive summary of the analysis of variance (ANOVA) and model adequacy is detailed in Table 3. The models demonstrated significant predictive power, with F-values of 36.88 and 10.08 and p-values < 0.0001, indicating a significant nonlinear relationship between the four factors and the response values. The R-squared values for IPMPP and IPMP were high at 0.9736 and 0.9098, respectively, showing that the models explained the majority of the variance. The adjusted R-squared values, 0.9472 for IPMPP and 0.8196 for IPMP, indicated a strong fit. Additionally, Fig. S1(A1–A2) illustrates a robust correlation between observed and predicted values. The lack of fit F-values was 3.61 for IPMPP and 0.9297 for IPMP, with corresponding p-values of 0.4422 and 0.1137, suggesting that random noise did not significantly impact the model predictions. The coefficients of variation (C.V.) were 2.69 % and 6.55 %, indicating good reproducibility. Empirical testing further validated the regression model's accuracy, affirming the effectiveness of the response surface methodology in predicting and analyzing IPMPP yield. The residual normal probability plot (Fig. S1(B1–B2)) demonstrated normally distributed residuals along a straight line, underscoring the model's precision. Biochemical residuals, shown in Fig. S1(C1–C2), were randomly distributed between −2 and + 2 compared to predicted values, confirming their adherence to a normal distribution. Additionally, the perturbation diagram (Fig. S1(D1–D2)) highlighted the solid–liquid ratio as exerting the most significant impact on IPMPP and IPMP yields, aligning with ANOVA findings and substantiating the model's high accuracy and reliability for extracting polysaccharide and protein from IPM cake meal using UTPP.Table 3 ANOVA for response surface quadratic model for the yield of IPMP and IPMPP.

Source	IPMP					IPMPP					
SS	DF	MS	F-value	P-value	SS	DF	MS	F-value	P-value	
Model	11.03	14	0.7877	10.08	< 0.0001	25.08	14	1.79	36.88	< 0.0001	
X1	0.1450	1	0.1450	1.86	0.1947	0.2730	1	0.2730	5.62	0.0326	
X2	2.01	1	2.01	25.78	0.0002	4.25	1	4.25	87.46	< 0.0001	
X3	0.0364	1	0.0364	0.4664	0.5058	0.2494	1	0.2494	5.13	0.0398	
X4	0.0027	1	0.0027	0.0340	0.8563	0.0056	1	0.0056	0.1160	0.7385	
X1X2	0.0151	1	0.0151	0.1929	0.6672	0.3540	1	0.3540	7.29	0.0173	
X1X3	0.7871	1	0.7871	10.08	0.0068	1.42	1	1.42	29.15	< 0.0001	
X1X4	0.3780	1	0.3780	4.84	0.0451	0.0009	1	0.0009	0.0185	0.8937	
X2X3	0.1955	1	0.1955	2.50	0.1360	0.5329	1	0.5329	10.97	0.0051	
X2X4	0.1372	1	0.1372	1.76	0.2064	0.1190	1	0.1190	2.45	0.1398	
X3X4	0.1190	1	0.1190	1.52	0.2374	0.0306	1	0.0306	0.6305	0.4404	
X12	2.28	1	2.28	30.32	< 0.0001	6.89	1	6.89	141.90	< 0.0001	
X22	4.18	1	4.18	55.42	< 0.0001	9.10	1	9.10	187.38	< 0.0001	
X32	3.18	1	3.18	43.38	< 0.0001	8.37	1	8.37	172.27	< 0.0001	
X42	1.17	1	1.17	15.71	0.0017	2.33	1	2.33	48.01	< 0.0001	
Residual	1.09	14	0.0781			0.6801	14	0.0486			
Lack of Fit	0.8312	10	0.0831	0.9297	0.4422	0.6123	10	0.0612	3.61	0.1137	
Pure Error	0.2625	4	0.0656			0.0678	4	0.0169			
Cor Total	12.12	28				25.76	28				
R2	0.9098	0.9736	
Adjusted R2	0.8196	0.9472	
Predicted R2	0.5712	0.8590	
C.V. %	6.55	2.69	

The significance of each coefficient within the models was determined by p-values from an F-test, with p < 0.05 indicating statistical significance. A smaller p-value signifies a more significant contribution of the variable. As presented in Table 3, the terms X1, X2, X3, X1X2, X1X3, X2X3, X12, X22 X32and X42 were significant for the YIPMPP model (p < 0.05), while the terms X2, X1X3, X1X4, X12, X22, X32and X42 were significant for the YIPMP model (p < 0.05). Analysis of the F values derived from linear coefficients reveals that the impact of experimental factors on the yields of IPMPP and IPMP consistently follows the same order: solid–liquid ratio > ammonium citrate concentration > pH>ultrasonic time.

3.2.2 Optimization of the extraction parameters of the UTPP method

Interactions between variables and the response were elucidated through 3D response surface and 2D contour diagrams. The 2D contour map's shape—elliptical contours indicating stronger interactions and circular contours suggesting weaker ones—reflects the intensity of variable interactions. The 3D response surface diagrams showed steeper surfaces where variables exerted more significant impacts on the response values. As shown in Figs. S2 and S3, interactions between ammonium citrate concentration (X1) and solid–liquid ratio (X2), as well as ammonium citrate concentration and pH (X3), exhibited quadratic effects on the yield, in line with the results detailed in Table 3. Specifically, increases in ammonium citrate concentration from 20 mg/mL to 30 mg/mL and solid–liquid ratios from 1:20 to 1:25 significantly enhanced IPMPP and IPMP yields. Similarly, elevating the ammonium citrate concentration from 20 mg/mL to 30 mg/mL and pH from 2 to 3 also improved yields when other parameters remained constant. These findings affirm that response surface methodology is a robust tool for optimizing extraction conditions, offering precise and reliable predictions through the established regression model.

The optimized extraction conditions for IPMPP and IPMP, determined through a mathematical model established by response surface methodology, were initially set at a ammonium citrate concentration of 29.84 %, a solid–liquid ratio of 1:26.24, a pH of 2.98, and an ultrasonic time of 30.55 min for IPMPP. For IPMP, the conditions were slightly different, with an ammonium citrate concentration of 29.43 %, a solid–liquid ratio of 1:26.45, a pH of 3.08, and an ultrasonic time of 30.85 min. To enhance practical applicability, these conditions were rounded to an ammonium citrate concentration of 30 %, a solid–liquid ratio of 1:26, a pH of 3, and an ultrasonic time of 31 min. Under the optimal conditions, the predicted values were 4.91 % for IPMP and 9.90 ± 0.31 % for IPMPP. Validation experiments conducted under these adjusted optimal conditions yielded extraction rates of 10.03 ± 0.31 % for IPMPP and 5.03 ± 0.10 % for IPMP, aligning closely with the model’s predictions. These results confirm the feasibility of using the regression model derived from the experimental data to accurately predict and analyze the yields of IPMPP and IPMP, demonstrating its effectiveness and practicality. These conditions achieved extraction rates of 8.10 % for lipids, 5.03 % for proteins, and 10.03 % for polysaccharides, with recovery rates of 91.62 %, 83.08 %, and 93.95 % respectively. These results demonstrate that UTPP can effectively recover the majority of oil, protein, and polysaccharide components from IPM cake meal, underscoring the practicality and reliability of this extraction method.

3.3 Analysis of the fatty acid composition of IPMO

Fig. 3A presents the GC chromatogram illustrating the fatty acid composition of IPMO extracted via UTPP. As depicted in Fig. 3B, linoleic acid is the predominant fatty acid, constituting 84.57 % of the total fatty acids and serving as the primary unsaturated fatty acid. This high concentration of linoleic acid contributes significantly to lowering blood cholesterol, which is vital for preventing atherosclerosis, and serves as a precursor for essential signaling molecules that regulate skin health and inflammation [36]. The major saturated fatty acids identified are palmitic acid (5.69 %) and palmitoleic acid (PA, 4.37 %). Additionally, oleic acid, stearic acid, and erucic acid were detected at concentrations of 3.53 %, 1.37 %, and 0.47 %, respectively. These results align with prior studies, which consistently identify linoleic acid as the dominant fatty acid in IPM fruit oil, typically comprising over 60 % [4], [37]. Overall, the findings indicate that the UTPP extraction technique has a minimal impact on the fatty acid composition of IPMO.Fig. 3 Comprehensive Analyses of IPMO and IPMP: (A) GC chromatogram displaying the fatty acid mixture standard alongside IPMO, with identified peaks for palmitic acid (1), palmitoleic acid (2), stearic acid (3), oleic acid (4), linoleic acid (5), and erucic acid (6), and the solvent peak at 10.25 min. (B) Fatty acid composition profile of IPMO, illustrating the distribution of various fatty acids. (C) Amino acid composition of IPMP, detailing the types and concentrations of amino acids present. (D) HPAEC chromatography of standard monosaccharides compared with IPMPP, showcasing the monosaccharide components.

3.4 Analysis of the amino acid composition of IPMP

The quality of protein is often gauged by its essential amino acid content. The protein derived from IPM cake meal, as detailed in Fig. 3C, comprises 17 types of amino acids, confirming its high nutritional quality. IPMP meets the requirements for all essential amino acids necessary for human nutrition, except for Try. Notable essential amino acids in IPMP include Thr, Val, Met, Leu, Ile, Phe, and Lys, as illustrated in Fig. 3C. Amino acids can be categorized based on the polarity of their side chains into hydrophobic and hydrophilic groups. Hydrophobic amino acids in IPMP include Gly, Pro, Ala, Val, Met, lLeu, Ile, and Phe. The hydrophilic group includes Asp, Glu, His, Ser, Arg, Thr, Tyr, Cys, and Lys. Among these, Glu (24.9 %), Asp (12.2 %), and Ser (9.9 %) are present in substantial amounts, indicative of a typical amino acid profile for plant seed proteins. This profile aligns with findings from simultaneous extraction of oil, protein, and polysaccharides from Oiltea residue seed cake using three-phase partitioning, which also highlighted proteins rich in Glu and Asp [35]. Therefore, the amino acid profile of IPMP extracted using UTPP is well-suited as an excellent supplement for cereal and grain-based diets, enhancing their nutritional value.

3.5 Analysis of the physicochemical characteristics of IPMPP

3.5.1 Analysis of the chemical composition

Table 4 details the primary chemical components of IPMPP, with neutral sugars constituting 56.17 % of its composition. IPMPP contains 39.77 % uronic acid, characterizing it as a typical acidic heteropolysaccharide. Uronic acid, an important component in hypoglycemic drugs and moisturizing cosmetics, shows a high concentration likely enhanced by ultrasonic waves facilitating its release into the extraction solvent through cavitation processes [25]. Additionally, IPMPP exhibited a relatively high protein content of 4.48 %, suggesting the presence of polysaccharide-protein complexes within the extract. This residual protein is probably due to its conjugation with IPMPP, as free proteins are extracted using the UTPP method.Table 4 Chemical composition, monosaccharide composition, molecular weight distribution, particle size and Zeta potential distribution of IPMPP.

Chemical composition (%, g/g)	IPMPP	
Neutral sugar	56.17 ± 2.11	
Protein	4.48 ± 0.18	
Uronic acid	39.77 ± 2.03	
Monosaccharide composition (molar%)	
Fuc	4.02 ± 0.26	
Ara	14.01 ± 0.57	
Rha	7.50 ± 0.16	
Gal	12.97 ± 0.25	
Glc	13.91 ± 0.01	
Xyl	6.59 ± 0.08	
Man	4.37 ± 0.44	
GalA	30.04 ± 0.50	
GlcA	6.60 ± 0.20	
HG	22.54 ± 0.35	
RG-I	41.97 ± 0.64	
Rha/GalA	0.25 ± 0.01	
(Ara + Gal)/Rha	3.60 ± 0.03	
Molecular weight distribution	
Mw (kDa)	226.58 ± 8.03	
Mn (kDa)	89.79 ± 2.33	
Mw/Mn	2.52 ± 0.02	
DM (%)	36.64 ± 0.63	
Particle size and zeta potential distribution	
Particle size (nm)	1331.67 ± 22.50	
PDI	0.34 ± 0.03	
ζ potential (mV)	−35.13 ± 1.40	

Values are presented as the mean ± SD (standard deviation) based on triplicate determinations. The structural characteristics of IPMPP were determined by analyzing its monosaccharide composition. HG %= (GalA − Rha) mol %, RG-I %= (2 Rha + Gal + Ara) mol %, Rha/GalA: the contribution of RG-I region to the entire pectin, (Ara + Gal)/Rha: the degree of RG-I branching.

3.5.2 Monosaccharide composition

Fig. 3D illustrates the monosaccharide composition and content of IPMPP. As detailed in Table 4, the composition includes Fuc, Ara, Rha, Gal, Glc, Xyl, Man, GalA, and GlcA with respective molar percentages of 4.02 %, 14.01 %, 7.50 %, 12.97 %, 13.91 %, 6.59 %, 4.37 %, 30.04 %, and 6.60 %. The predominant presence of GalA, Ara, Gal, and Glc classifies IPMPP primarily as a pectic polysaccharide. A higher content of GalA would have better antioxidant activity, while the presence of Xyl and Ara is conducive to their hypoglycemic effect [13]. The 'smooth' region of pectin of contains the homogalacturonan (HG) domain, composed of partially methylated and acetylated galacturonic acid. Conversely, the 'hairy' region comprises more intricate structures such as rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II), and xylogalacturonan (XGA), characterized by their branched chains [27]. IPMPP, with an RG-I content exceeding 41.97 %, is notable for its substantial Ara or Gal side chains, demonstrating that UTPP is an effective method for isolating RG-I enriched pectin from IPM cake meal. The Rha/GalA ratio, indicative of the balance between HG and RG-I domains, typically ranges from 0.05 to 1 for RG-I pectin. IPMPP fell within this range with a ratio of 0.25 [38]. The (Ara + Gal)/Rha ratio, reflecting the branching level in the RG-I domain, suggests that higher values indicate longer side chains linked by rhamnose. Previous studies have reported (Ara + Gal)/Rha ratios ranging from 1.89 to 29.83. IPMPP's ratio of 3.60 signified a relative high degree of branching within its RG-I domain, underscoring its potential as a functional dietary supplement [39].

3.5.3 Molecular weight distribution

The molecular weight distribution of IPMPP was analyzed using HPGPC. The results, including the weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity index (Mw/Mn ratio), are detailed in Table 4. As illustrated in Fig. 4A, the HPGPC distribution curve for IPMPP featured a prominent peak, underscoring its homogeneity. Specifically, the Mw of IPMPP is 226.58 kDa, Mn is 89.79 kDa, and the Mw/Mn ratio is 2.52. Comparatively, the molecular weight of IPMPP aligns with that of sunflower seed meal polysaccharide, which is 236,741 Da, but remains lower than that of pectic polysaccharides derived from Arabica coffee husks, noted to be approximately 1.04 × 106 Da [40], [41]. The polydispersity index, a measure of molar mass distribution where higher values indicate a broader distribution [42], for IPMPP is 2.52, suggesting a relatively narrow molecular weight distribution. These observations confirmed that IPMPP comprises highly homogeneous polysaccharides with a concentrated molecular weight distribution. The findings substantiate the efficacy of UTPP as a method for extracting highly homogeneous polysaccharides from IPM cake meal, emphasizing its practical application in industry.Fig. 4 Comprehensive characterization of IPMPP: (A) HPGPC molecular weight distribution curve; (B) FT-IR spectrum; (C) Particle size distribution; (D) Triple-helix conformation; (E) XRD spectrum; (F) DSC curve.

3.5.4 FT-IR spectrum

The FT-IR spectrum of IPMPP is presented in Fig. 4B. A strong, wide absorption peaks at 3400 cm−1 was caused by O–H stretching vibrations [38]. Additionally, the absorption peak around 2935 cm−1 corresponds to the C-H stretch of the CH2 group. Peaks at 1735 cm−1 for the esterified carboxyl group (−COOR) and 1628 cm−1 for the free carboxyl group (–COO-) reveal the degree of esterification of the pectin [43]. The FT-IR method also enables the determination of the degree of esterification (DE) by analyzing the areas of these peaks [27]. The DM of IPMPP is noted as 36.64 ± 0.63 %, classifying it as a low methoxylated pectin, suitable for use in low-sugar or sugar-free products [44]. Furthermore, the absorption peak near 1419 cm−1 is associated with the bending vibrations of O-H or CH2 groups. Peaks at 1247 cm−1, 1155 cm−1, 1015 cm−1, and 780 cm−1 further substantiate the presence of β-type or α-(1 → 6) glucosidic bonds in IPMPP [45]. Overall, the FT-IR analysis of IPMPP provides a comprehensive depiction of its structural characteristics, exhibiting typical pectin spectral peaks and offering essential insights into its chemical structure.

3.5.5 Particle sizes and zeta potentials

As depicted in Fig. 4C, IPMPP displayed a single peak indicative of a relatively uniform particle size distribution. The dispersion coefficient, or polydispersity index (PDI), is recorded at 0.34, reflecting substantial uniformity in particle size distribution, as detailed in Table 4. The hydrodynamic radius of the molecules in solution, representing the particle size, is measured at 1331.67 nm for IPMPP. The particle size of a polymer is influenced by its molecular weight and the extent of molecular chain extension, which in turn affects the aggregation state of the molecule [46]. IPMPP carries a negative charge, evidenced by its ζ-potential value exceeding |±30| mV. This characteristic is instrumental in preventing large-scale aggregation of the particles. Smaller polysaccharide particle sizes, associated with higher absolute zeta potentials, enhance both the solubility of the polysaccharide and the stability of the solution [25]. This property is especially crucial for applications where dispersion stability is paramount.

3.5.6 Complex formation with Congo red of IPMPP

Congo red test is essential for investigating the sugar chain conformation of polysaccharides in aqueous solutions, particularly their ability to form complexes with polysaccharides that exhibit a three-strand helical structure. This structural feature is crucially linked to the bioactivity of polysaccharides [47]. When polysaccharides complex with Congo red, the maximum absorption wavelength (λmax) in the visible spectrum typically undergoes a redshift towards longer wavelengths. However, within certain NaOH concentrations, this λmax will gradually decrease, indicating changes in polysaccharide conformation [25], [48]. For IPMPP, complexation with Congo red led to observable redshifts in the λmax as the concentration of NaOH solution increased. Notably, at an NaOH concentration of 0.05 mol/L, the λmax peaked at 496.33 nm, as shown in Fig. 4D. These findings indicate that IPMPP maintains triple-helix structures at lower alkaline concentrations, underscoring its potential bioactive conformation. Such structural integrity at specific pH levels highlights IPMPP's relevance in biological contexts, suggesting its utility in applications where molecular conformation impacts functional efficacy.

3.5.7 XRD analysis

XRD is an expedient analytical technique employed to distinguish between crystalline and amorphous materials. Crystalline substances are characterized by narrow diffraction peaks, whereas amorphous materials display broader peaks, indicative of their disordered structures [11]. By analyzing the crystal structure of polysaccharides, insights into properties such as expansibility, flexibility, tensile strength, and solubility can be derived [49]. The molecular crystal structure of IPMPP was examined using XRD, as shown in Fig. 4E. The XRD pattern of IPMPP revealed a single broad dispersion peak at a diffraction angle of 22.55°, with an absence of significant sharp peaks across the diffraction range from 5° to 90°. This pattern suggests that IPMPP lacks a crystalline structure and is predominantly amorphous. The absence of crystallinity in IPMPP is indicative of its molecular disarray, which may influence its functional properties in industrial and biomedical applications.

3.5.8 Thermal characteristic

Differential Scanning Calorimetry (DSC) was employed to evaluate the thermodynamic properties of IPMPP, particularly focusing on the thermal transformations occurring during heating. Typically, DSC heat maps of pectin samples display distinct endothermic and exothermic peaks, where higher melting temperatures (Tm) and enthalpies of melting (ΔHm) are indicative of strong water retention capabilities in pectin [43]. In the endothermic phase, as illustrated in Fig. 4F, IPMPP exhibited a Tm at 125.96 ℃ and a ΔHm at 122.01 J/g, suggesting relatively poor water retention [43]. Conversely, during the exothermic phase, IPMPP displayed a single exothermic peak, which aligns with the singular peak observed in molecular weight assessments. The onset temperature of this exothermic phase (To) registered at 330.46 ℃, the glass transition temperature (Tg) at 357.72 ℃, and the enthalpy of the glass transition (ΔHg) at −12.71 J/g. Sharper and more defined exothermic peaks indicate a narrower melting range, implying a concentrated molecular weight distribution and an orderly molecular arrangement within the sample [42]. These results indicate that IPMPP is well-suited for applications that involve thermal processing, demonstrating its ability to withstand high temperatures.

3.5.9 Scanning electron microscopy (SEM)

The surface microstructure of IPMPP was meticulously analyzed using SEM at magnifications ranging from 100 × to 10,000×, as depicted in Fig. 5. At a magnification of 100×, IPMPP presented a lamellar structure characterized by a curly surface and some debris, indicative of its complex physical makeup. Upon closer examination at 5,000 × magnification, the fragments appeared small, consistent with the size characteristics typically associated with pectin; additionally, the surface exhibited varying degrees of protrusions, suggesting structural diversity. At an even higher magnification of 10,000×, the surface was observed to be uneven, and some particles exhibited a rod-like structure. This observation correlates with the established size and molecular weight distribution of IPMPP, confirming the heterogeneous nature of this polysaccharide.Fig. 5 SEM images of IPMPP at magnifications of 100×, 5000× and 10 000×.

3.6 In vitro hypoglycemic activity of IPMPP

3.6.1 Inhibition effect on α-glucosidase

α-Glucosidase inhibitors play a critical role in diabetes management by slowing the conversion of carbohydrates to glucose in the small intestine, thereby reducing postprandial hyperglycemia, enhancing insulin sensitivity, and preventing diabetes and its related complications [50]. As depicted in Fig. 6A, the inhibitory effect of IPMPP on α-glucosidase activity was observed to increase with the concentration, achieving an inhibition rate of 68.82 % at a concentration of 6.0 mg/mL. This indicates that the inhibitory effect is concentration-dependent. The half-maximal inhibitory concentration (IC50) of IPMPP was determined to be 1.40 mg/mL, which is significantly higher than the 0.11 mg/mL for acarbose. Although less potent than acarbose, IPMPP still holds potential as an antidiabetic agent, offering the advantage of fewer side effects and multiple health benefits. The efficacy of polysaccharides like IPMPP as α-glucosidase inhibitors is influenced by several factors, including molecular weight, uronic acid content, and monosaccharide composition [51]. Notably, polysaccharides rich in arabinose and galacturonic acid have been demonstrated to exhibit significant α-glucosidase inhibitory activity [12], [47]. Thus, the pronounced α-glucosidase inhibitory activity of IPMPP could be attributed to its high levels of arabinose and galacturonic acid, underscoring its potential therapeutic value in diabetes management.Fig. 6 (A) Inhibitory effect of IPMPP on α-glucosidase activity; (B) Lineweaver–Burk plots of the reaction of α-glucosidase in the presence of IPMPP; (C) Fluorescence spectra of α-glucosidase in the presence of various concentrations of IPMPP; (C1) Stern–Volmer plot of α-glucosidase with different IPMPP concentrations; (C2) Double logarithm regression plot of log [(F0 − F)/F] against log [Q] for α-glucosidase with different IPMPP concentrations; (D) Fructosamine inhibition by IPMPP; (E) α-Dicarbonyl compound inhibition by IPMPP; (F) AGEs inhibition by IPMPP; (G) Fluorescence spectra of AGEs in the presence of increasing concentrations of IPMPP and AG.

3.6.2 Inhibition kinetics analysis

The inhibition type of IPMPP on α-glucosidase was investigated using Lineweaver-Burk double reciprocal plots. With the substrate pPNG concentrations ranging from 1 to 4 mM, IPMPP at 1, 3, and 6 mg/mL was used to assess changes in the enzyme reaction rate. As illustrated in Fig. 6B, an increase in IPMPP concentration resulted in a corresponding increase in the slope of the curve. The double reciprocal plots displayed a clear linear relationship, with the fitting curves intersecting in the third quadrant of the coordinate system. This intersection pattern indicates that IPMPP exhibits mixed-type inhibition of α-glucosidase [14]. According to the Lineweaver-Burk kinetic model detailed in Table 5, in the absence of IPMPP, the Km for α-glucosidase is 16.629 mM, and the Vmax is 0.481 (ΔA405/min). When IPMPP was introduced at concentrations of 1, 3, and 6 mg/mL, the Km value decreased to 5.156 mM, and the Vmax dropped to 0.0583 (ΔA405/min), respectively. Additionally, the Ki for IPMPP was determined to be 3.595 mg/mL, and the Kis is 0.792 mg/mL. Typically, lower Ki and Kis values suggest a tighter binding between the inhibitor-enzyme and inhibitor-enzyme-substrate complexes, reflecting stronger inhibition [14], [50]. These findings suggest that IPMPP inhibits α-glucosidase activity by forming a polysaccharide-enzyme-substrate complex, which significantly impacts the enzymatic reaction process. This mechanism underlines the potential of IPMPP as an effective natural inhibitor in diabetes management by modulating enzyme activity.Table 5 Kinetic parameters of α-glucosidase inhibition in the presence of IPMPP and the half maximal inhibitory concentration (IC50 value) of IPMPP for non-enzymatic glycation inhibition activity.

ɑ-Glucosidase inhibition	
IPMPP (mg/mL)	0	1	3	6	
Inhibition type	Mixed competitive	
Km (mM)	16.629	9.873	5.811	5.156	
Vmax (ΔA405/min)	0.481	0.245	0.0972	0.0583	
Ki (mg/mL)	3.595	
Kis (mg/mL)	0.792	
Kq (M−1·S-1)	1.034 × 1013	
Ksv (M−1)	1.034 × 105	
Ka (M−1)	8.123 × 104	
n	0.986	
	IC50 (mg/mL)	
Fructosamine inhibition	α-Dicarbonyl compounds inhibition	AGEs inhibition.	
AG (mg/mL)	2.45 ± 0.10	3.81 ± 0.06	0.98 ± 0.01	
IPMPP (mg/mL)	4.26 ± 0.06	7.29 ± 0.54	2.44 ± 0.02	

3.6.3 The effect of IPMPP on fluorescent characteristics of α-glucosidase

Fluorescence quenching, which involves the reduction in the fluorescence quantum yield of a fluorophore, can occur through various mechanisms, including excited state reactions, energy transfer, molecular rearrangement, ground state complex formation, and collision quenching [52]. Proteins such as α-glucosidase, which contain aromatic amino acids like tyrosine, phenylalanine, and tryptophan, exhibit strong fluorescence. Tryptophan is particularly significant as the primary contributor to fluorescence emission. When α-glucosidase interacts with inhibitors or experiences changes in its microenvironment, its fluorescence intensity generally decreases [14], [50]. As depicted in Fig. 6C, the fluorescence intensity of α-glucosidase decreased with increasing concentrations of IPMPP, indicating that IPMPP effectively reduced the fluorescence intensity of α-glucosidase. The quenching effect became more pronounced with higher concentrations of IPMPP. Furthermore, after treatment with IPMPP, the maximum fluorescence emission wavelength of α-glucosidase shifted from 334 nm to 335.4 nm under an excitation of 300 nm. This shift suggests an interaction between IPMPP and α-glucosidase that alters the polarity around the tyrosine and tryptophan residues, transitioning the microenvironment from hydrophilic to hydrophobic. Such alterations can lead to the unfolding of fluorescent amino acid residues and the subsequent quenching of the fluorophores, indicative of significant structural changes in the enzyme [14], [50].

The fluorescence quenching mechanisms of enzyme inhibitors and enzymes are typically classified into static quenching, dynamic quenching, or a combination of both [52]. To determine the binding mechanism between IPMPP and α-glucosidase, the Stern-Volmer equation was applied to the fluorescence quenching data. The resulting Stern-Volmer plot, depicted in Fig. 6C1, shows a linear relationship between α-glucosidase and IPMPP, with a high regression coefficient (R2 = 0.9828). The molecular weight of IPMPP is 226.58 kDa, yielding a Kq of 1.034 × 1013 M−1s−1 and a KSV of 1.034 × 105 M−1, as documented in Table 5. Given that the Kq value substantially exceeded the typical maximum for dynamic fluorescence quenching rate constants (2.0 × 1010 M−1s−1), this suggests that the quenching mechanism of α-glucosidase by IPMPP predominantly involves static quenching. This form of quenching likely results from the formation of non-fluorescent complexes between the enzyme and inhibitor, which either do not emit light or exhibit reduced luminous efficiency due to the fluorophore's interaction with the quencher [47], [50]. Further analysis using the plot of lg[(F0-F)/F] against lg[Q], shown in Fig. 6C2, allowed the determination of Ka for α-glucosidase inhibition by IPMPP to be 8.123 × 104 M−1 (Table 5). This Ka value indicates a strong binding affinity between the inhibitor and enzyme. Additionally, the binding stoichiometry (n) value of approximately 0.986, close to 1, suggests that that α-glucosidase has one binding site for IPMPP, confirming a specific interaction [53]. The n values of IPMPP were consistent with those reported for Lentinus edodes mycelia polysaccharide, ranging from 1.01 to 1.14 [54]. These results underscore IPMPP’s potential as a novel α-glucosidase inhibitor for use in functional foods and dietary supplements, showcasing its capability to regulate postprandial blood glucose levels effectively.

3.7 In vitro non-enzymatic glycation inhibition activity of IPMPP

Chronic diabetes frequently leads to the development of non-enzymatic AGEs, which are implicated in various diabetic complications such as microangiopathy, nephropathy, and atherosclerosis [55]. The glycation process initiates with the formation of a Schiff base, a reversible intermediate that occurs when amino groups in proteins react with carbonyl groups from reducing sugars. This Schiff base then undergoes the Amadori rearrangement, leading to more stable products that participate in the Maillard reaction. This sequence of reactions ultimately culminates in the formation of harmful AGEs through processes like dehydration, oxidation, and further molecular rearrangement [56]. AGEs contribute to the exacerbation of diabetic complications by interacting with specific receptors known as RAGEs. This interaction promotes the production of reactive oxygen species (ROS) and triggers inflammatory responses, thereby amplifying oxidative stress and activating pro-inflammatory mediators, which exacerbate the progression of the disease [57]. Consequently, targeting and inhibiting the formation of AGEs represents a promising strategy to mitigate the complications associated with diabetes. This study evaluates the efficacy of IPMPP in inhibiting non-enzymatic protein glycation, employing a BSA-fructose model with aminoguanidine (AG) as the positive control. The glycation process is analyzed through its early, intermediate, and late stages, assessing IPMPP's potential to interrupt these critical phases and its effectiveness compared to established inhibitors.

During the early stage of glycation, Schiff bases and Amadori products are formed, which react with nitroblue tetrazolium (NBT) to yield a colored product detectable at a wavelength of 530 nm [30]. Fructosamine, a marker of early glycosylation products, is employed to measure the extent of this reaction. As illustrated in Fig. 6D, IPMPP exhibited a significant inhibitory effect on fructosamine formation. With increasing concentrations of IPMPP, the inhibition rate of fructosamine progressively increased, reaching 63.77 ± 0.59 % at a concentration of 6 mg/mL. The IC50 values for AG and IPMPP were determined to be 2.45 ± 0.10 mg/mL and 4.26 ± 0.06 mg/mL, respectively, as documented in Table 5. Although IPMPP is less potent than AG, its substantial inhibitory effect demonstrate its potential as an effective agent for managing early glycation processes. This suggests that IPMPP could be considered as an alternative or complementary therapeutic approach in diabetes care, particularly for mitigating the formation of early glycation products.

Studies have shown that dicarbonyl compounds significantly contribute to the rapid cross-linking of proteins, leading to the formation of stable AGEs; in fact, nearly 50 % of AGEs are derived from these compounds [58]. During the intermediate phase of glycation, fructosamine breaks down into α-dicarbonyl compounds such as methylglyoxal, glyoxal, and 3-deoxyglucosone, which act as markers for intermediate glycosylation products [30], [56]. The inhibitory effect of IPMPP on the formation of α-dicarbonyl compounds was observed to increase with its concentration, as depicted in Fig. 6E. At a concentration of 6 mg/mL, IPMPP achieved an inhibition rate of 41.21 ± 1.37 %. The IC50 for AG and IPMPP were 3.81 ± 0.06 mg/mL and 7.29 ± 0.54 mg/mL, respectively (Table 5). Although IPMPP’s effectiveness was lower compared to AG, its ability to inhibit the formation of harmful intermediates underscores its potential utility in managing glycation processes. The ability of IPMPP to inhibit the formation of dicarbonyl compounds was consistent with that observed in Actinidia arguta polysaccharides, which are rich in galacturonic acid [59]. This suggests that IPMPP could serve as a beneficial agent in interventions aimed at mitigating the progression of diseases associated with AGEs.

In the final stage of glycation, the formation of AGEs was assessed. AGEs are oxidizing compounds that form both in vivo and in vitro, resulting from reactions between proteins and reducing sugars. The fluorescence intensity of AGEs serves as a quantitative measure of advanced glycosylation, reflecting the extent of non-enzymatic glycosylation systems and the inhibitory activity of samples [56]. Previous research has indicated that the molar concentration of monosaccharides, particularly the high content of galacturonic acid units in pectic polysaccharides, can effectively inhibit AGE formation [44]. Various concentrations of IPMPP were introduced into a BSA-glycosylation system to evaluate its concentration-dependent effects on AGE inhibition. As depicted in Fig. 6F, IPMPP significantly curtailed the formation of AGEs, with inhibition rates increasing alongside IPMPP concentration. At 6 mg/mL of IPMPP, the inhibition rate peaked at 81.13 ± 0.11 %. The IC50 values for AG and IPMPP were 0.98 ± 0.01 mg/mL and 2.44 ± 0.02 mg/mL, respectively, indicating that IPMPP was effective at scavenging AGEs and blocking the glycosylation reaction (Table 5). The inhibitory ability of IPMPP in the final stage was significantly stronger than in the early and middle stages, primarily because it may block the cross-linking of proteins. This action helps prevent the formation of advanced glycation end-products (AGEs) in the final stage. [60]. Further analysis, presented in Fig. 6G, showed a dose-dependent decrease in fluorescence intensity when IPMPP and AG were used, with AG exhibiting a stronger quenching effect. A noticeable blue shift in the maximum absorption peak indicated an increase in polarity around the fluorophores due to interactions with IPMPP, suggesting a protective mechanism against AGE formation. The antioxidant properties of pectic polysaccharides, particularly those rich in GalA, effectively inhibit AGE formation by reducing the release of reactive oxygen species. Notably, the inhibition capability of IPMPP in the late stage was significantly more potent than in the early and intermediate stages, primarily inhibiting protein cross-linking and preventing the formation of late-stage AGEs [30]. This effect is likely due to the high content of galacturonic acid in IPMPP. This observation aligns with prior research, which indicated that blackberry polysaccharides, particularly those abundant in galacturonic acid, exhibited strong inhibitory effects on the formation of AGEs [29]. These findings highlight the potential of IPMPP as a promising alternative therapeutic agent for managing conditions associated with glycosylation.

4 Conclusion

T-butanol, traditionally used in three-phase partitioning (TPP), poses environmental concerns. In response, a sustainable ultrasound-assisted three-phase partitioning (UTPP) system utilizing dimethyl carbonate was developed to extract and separate oil, protein, and polysaccharides simultaneously from IPM cake meal. Optimization of various parameters, including inorganic salt type, solid–liquid ratio, salt concentration, pH, ultrasonic duration, temperature, and volume of dimethyl carbonate, was achieved through single-factor experiments and response surface methodology. The most favorable conditions were determined to be a 30 % ammonium citrate concentration, a 1:26 solid–liquid ratio, pH 3, ultrasonic time of 31 min, temperature of 30 °C, and a 15 mL volume of dimethyl carbonate. These conditions led to optimal extraction rates of 10.03 ± 0.31 % for IPMPP, 5.03 ± 0.10 % for IPMP, and 8.10 ± 0.75 % for IPMO, with high recovery rates (91.62 % for oil, 83.08 % for protein, and 93.95 % for polysaccharides). Chemical analysis of IPMO identified linoleic acid as the predominant unsaturated fatty acid. IPMP was characterized by a typical plant seed protein profile with glutamic acid, aspartate, and serine being the most abundant amino acids. IPMPP, consisting mainly of RG-I pectin with a molecular weight of 226.58 kDa, demonstrated a triple helix structure, robust thermal stability, and significant inhibitory activities against α-glucosidase and non-enzymatic glycation. Additionally, IPMPP was found to inhibit α-glucosidase through a mixed-type mechanism, predominantly quenching the enzyme's fluorescence through static interaction and binding at a single site. These findings highlight IPMPP's potential as a novel α-glucosidase inhibitor and anti-glycation agent, suitable for inclusion in functional foods and dietary supplements. Overall, this research underlines the effectiveness of the UTPP method as a novel and environmentally friendly alternative for the extraction and utilization of valuable compounds from IPM cake meal, improving the conventional TPP approach.

CRediT authorship contribution statement

Xin Shi: Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Qiuqiu Zhang: Writing – original draft, Data curation. Jintao Yang: Formal analysis. Renshuai Huang: Writing – review & editing. Yonghui Ge: Writing – review & editing. Jinhua Wang: Writing – review & editing. Guangjing Chen: Writing – review & editing, Visualization, Supervision, Project administration, Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Acknowledgements

This work was supported by the Academic New Seedling Cultivation and Free Exploration Innovation Special Project of the Guizhou Provincial 10.13039/100019242 Department of Science and Technology (2023) and Guizhou Forestry and Grassland Development Co., Ltd. Special Project (GZ-LFGS-HZ-135-011 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107043.
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