Ficus carica polysaccharide with anti-aging effect, efficient extraction method and application thereof

Abstract

The application discloses a ficus carica polysaccharide with an anti-aging effect and an efficient extraction method and application, adopts an ultrasonic-assisted low eutectic solvent extraction technology, efficiently extracts ficus carica polysaccharide FCPs from ficus carica, successfully obtains the ficus carica polysaccharide FCPs by optimizing extraction conditions including a liquid-solid ratio, ultrasonic treatment time, water content and temperature, and the yield is as high as 62.26+ / -0.16%, which is higher than that of an existing reported extraction method. The FCPs are composed of two components with molecular weights of 10118 Da and 532 Da, can effectively scavenge oxygen free radicals, and can significantly prolong the lifespan of Caenorhabditis elegans, and provide a scientific basis for the application of the ficus carica polysaccharide in the anti-aging field.

Description

Technical Field

[0001] This invention relates to an extraction method, and more particularly to a fig polysaccharide with anti-aging effects, its efficient extraction method, and its application. Background Technology

[0002] Fig is an important plant used for both food and medicine, and polysaccharides are one of its main active ingredients. Studies have shown that fig polysaccharides possess anti-diabetic, immunomodulatory, and anti-tumor activities. Currently, the main extraction techniques for fig polysaccharides include traditional hot water extraction (HWE, yield 10.45%), enzyme-assisted extraction (EAE, highest yield 34.13%), ultrasonic-assisted extraction (UAE, yield 2.81-11.78%), microwave-assisted extraction (MAE, yield 4.65-27.6%), and supercritical CO2 extraction (scCO2E, yield 17.31%). Subcritical water extraction (scWE, 150℃), while showing high extraction efficiency (yield 56.48%), suffers from bottlenecks in industrial application, such as high equipment requirements and high energy consumption. In summary, current fig polysaccharide extraction methods generally cannot simultaneously achieve the advantages of high yield and low cost. Summary of the Invention

[0003] To address the shortcomings of the aforementioned technologies, this invention provides a fig polysaccharide with anti-aging effects, an efficient extraction method, and its applications.

[0004] To solve the above technical problems, the technical solution adopted in this invention is: a highly efficient extraction method for fig polysaccharides with anti-aging effects, comprising the following steps: Step 1, Raw material pretreatment: Prepare fresh figs, and pretreat the figs by washing and crushing them; Step 2: Prepare the eutectic solvent DESs, which consists of hydrogen bond donor HBD and hydrogen bond acceptor HBA; Step 3: Mix the fig powder obtained in Step 1 with the eutectic solvent DESs prepared in Step 2, and extract it under ultrasonic assistance. Design a single-factor experiment to determine the optimal extraction conditions. The factors of the extraction conditions include liquid-solid ratio, ultrasonic time, water content and extraction temperature. Step 4, Polysaccharide preparation: After selecting the most suitable eutectic solvent and optimal extraction conditions, use ultrasound-assisted eutectic solvent to efficiently extract fig polysaccharides; Step 5: The fig polysaccharide extract obtained in Step 4 is subjected to alcohol precipitation, purification and drying to obtain the fig polysaccharide product, named FCPs.

[0005] Preferably, in step 1, the specific method of crushing is as follows: the washed figs are dried to constant weight, ground into powder, and then sieved through a 100-mesh sieve to obtain uniform fig powder.

[0006] Preferably, in step 2, the eutectic solvent DESs includes: The following are molar ratios: choline chloride / urea (1:2), choline chloride / lactic acid (1:1), choline chloride / oxalic acid (1:1), glycerol / glycine / water (3:1:1), glycerol / threonine / water (3:1:1), acetamide / lactic acid (1:1), choline chloride / ethylene glycol (1:2), choline chloride / 1,3-butanediol (1:2), choline chloride / sorbitol (1:2), and choline chloride / 1,3-butanediol / sorbitol (1:1:1).

[0007] Preferably, in step 3, the optimal eutectic solvent for fig polysaccharide extraction is choline chloride / 1,3-butanediol with a molar ratio of 1:2.

[0008] Preferably, in step 3, the optimal conditions for extracting fig polysaccharides are: a liquid-to-solid ratio of eutectic solvent to fig powder of 10-50:1 mL / g, an ultrasonic time of 20-60 min, a water content of eutectic solvent of 0-20%, and an extraction temperature of 30-70℃.

[0009] Preferably, in step 5, the specific alcohol precipitation, purification and drying process after extraction is as follows: After the fig polysaccharide is extracted, centrifuge to remove the filter residue, precipitate with four times the volume of ethanol, leave overnight, wash the precipitate with ethanol, air dry naturally, redissolve in deionized water, purify using a D101 macroporous resin column, concentrate by rotary evaporation, and finally freeze dry to obtain the fig polysaccharide product.

[0010] A fig polysaccharide with anti-aging properties was obtained by the above-mentioned efficient extraction method for fig polysaccharides with anti-aging properties.

[0011] Preferably, the fig polysaccharide FCPs consist of two components with molecular weights of 10,118 Da and 532 Da, and their monosaccharide composition includes mannose, rhamnose, glucose, galactose, xylose, arabinose and galacturonic acid, with a molar ratio of 1.00:0.51:1.45:1.23:0.14:1.20:2.02.

[0012] An application of fig polysaccharides with anti-aging properties: Fig polysaccharide FCPs are used as texture modifiers, thickeners, gelling agents and emulsifiers in the food and biomedical industries.

[0013] An application of fig polysaccharides with anti-aging effects: Fig polysaccharide FCPs are used in anti-aging and antioxidant products.

[0014] This invention discloses a fig polysaccharide with anti-aging effects and its efficient extraction method. This efficient extraction method innovatively introduces eutectic solvent (DESs)-assisted ultrasonic extraction technology for fig polysaccharide extraction. Through the specific binding of hydrogen bond donors and acceptors, a eutectic system is formed. Its unique physicochemical properties effectively disrupt the plant cell wall structure, significantly improving polysaccharide dissolution efficiency, while also offering advantages such as environmental friendliness and mild operation. The fig polysaccharides (FCPs) extracted using this efficient method were characterized using modern analytical techniques, and their anti-aging activity was studied. The results showed that the fig polysaccharides (FCPs) possess antioxidant and life-extending effects, providing a scientific basis for the application of fig polysaccharides in the field of anti-aging. Attached Figure Description

[0015] Figure 1 The yields of FCPs under different conditions were: (a) different solvents; (b) ultrasonic time; (c) water content; (d) liquid-to-solid ratio; and (e) extraction temperature.

[0016] Figure 2 Molecular weight and monosaccharide composition of FCPs: (a) High-pressure gel permeation chromatography (HPGPC) elution curve; (b) High-performance liquid chromatography (HPLC) chromatogram of monosaccharide composition.

[0017] Figure 3 Physical properties of FCPs: (a) Thermogravimetric (TG) analysis diagram; (b) Differential thermogravimetric (DTG) analysis diagram; (c) Steady-state shear flow curve; (d) Dynamic rheological curve.

[0018] Figure 4 The in vitro antioxidant activity of FCPs: the scavenging rate of DPPH by FCPs.

[0019] Figure 5 To extend the lifespan of nematodes using FCPs: (a) Survival curves of nematodes treated with different doses of FCP; (b) Average lifespan of nematodes treated with different doses of FCPs; (c) Representative images of lipofuscin in nematodes synchronized to the adult stage and treated with astaxanthin for 6 days; (d) Relative fluorescence intensity of lipofuscin.

[0020] Figure 6 To reduce ROS levels in nematodes for FCPs. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0022] First, this invention discloses a highly efficient extraction method for fig polysaccharides with anti-aging effects. It is a new technology that uses ultrasound-assisted eutectic solvent to efficiently extract figs. By optimizing the extraction conditions, including liquid-to-solid ratio, ultrasonic treatment time, water content, and temperature, fig polysaccharides (FCPs) were successfully obtained with a yield as high as 62.26±0.16%, which is higher than the extraction methods reported in the prior art.

[0023] The above-mentioned efficient extraction method for fig polysaccharides with anti-aging effects specifically includes the following extraction steps: Step 1, Raw material pretreatment: Prepare fresh figs, and pretreat the figs by washing and crushing them. Crushing refers to processing the washed figs into powder to obtain particles of suitable size, which facilitates solvent penetration and polysaccharide release in the subsequent extraction process. The specific methods for crushing and processing include: drying the washed figs to a constant weight, grinding them into powder, and then sieving them through a 100-mesh sieve to obtain uniform fig powder.

[0024] Step 2, Solvent Selection and Design: This invention prepared 10 eutectic solvents (DESs), which consist of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), as shown in Table 1. The synthesis conditions for the 10 eutectic solvents are as follows: Table 1. Synthesis conditions of 10 DESs

[0025] Based on the composition of the target eutectic solvent, hydrogen bond acceptors and hydrogen bond donors are precisely weighed and mixed according to the molar ratio. The mixture is then heated to a specified temperature. During heating, HBA and HBD gradually melt through hydrogen bonding. After melting for a specified time, a DES stable at room temperature is obtained. For example, choline chloride / urea DES is obtained by mixing choline chloride and urea in a 1:2 molar ratio and melting at 80°C for 2.0 h.

[0026] After obtaining 10 DESs, fig polysaccharides were extracted at 60℃ for 30 min. The content of fig polysaccharides in the extract was determined by the phenol-sulfuric acid method. The DES with the highest extraction rate was identified as choline chloride / 1,3-butanediol (ChCl / But). This solvent has good permeability to fig cell walls and effective dissolution ability for polysaccharides. The highest yield of fig polysaccharides was obtained by using this solvent.

[0027] Step 3: Optimization of extraction conditions: Fig powder and eutectic solvent were mixed and fig polysaccharides were extracted under ultrasonic assistance. Single-factor experiments were designed to investigate the effects of four key parameters, namely liquid-solid ratio, ultrasonic time, water content and extraction temperature, on the yield of fig polysaccharides, and the optimal extraction conditions were determined.

[0028] Ultimately, the optimal extraction conditions were determined to be: liquid-to-solid ratio 10-50:1 mL / g, ultrasonic time 20-60 min, water content 0-20%, and extraction temperature 30-70℃.

[0029] Step 4: Polysaccharide preparation: After selecting the most suitable eutectic solvent and optimal extraction conditions, fig polysaccharides are extracted efficiently using ultrasound-assisted eutectic solvent extraction. After extraction, the fig polysaccharide products are obtained by alcohol precipitation, purification and drying, and named FCPs.

[0030] The specific alcohol precipitation, purification and drying process is as follows: After the fig polysaccharide is extracted, the filter residue is removed by centrifugation, and the product is precipitated with four times the volume of ethanol overnight. The precipitate is repeatedly washed with ethanol, air-dried naturally, reconstituted with deionized water, purified by D101 macroporous resin column, concentrated by rotary evaporation, and finally freeze-dried to obtain the fig polysaccharide product.

[0031] Step 5: Evaluation of the physicochemical properties and activity of polysaccharides: The molecular weight, monosaccharide composition, thermogravimetric analysis and rheological analysis of the extracted FCPs are performed, and their life-extending effect is evaluated to ensure the extraction effect and application value of polysaccharides.

[0032] The yield of fig polysaccharides obtained from the above extraction method is as high as 62.26±0.16%, which is much higher than that of hot water extraction (yield of 10.45%), enzyme-assisted extraction (yield of 34.13%), and subcritical water extraction (yield of 56.48%) reported in the literature.

[0033] Analysis revealed that fig polysaccharides (FCPs) are mainly composed of two components with molecular weights of 10,118 Da and 532 Da. Their monosaccharide composition includes mannose, rhamnose, glucose, galactose, xylose, arabinose, and galacturonic acid, with a molar ratio of 1.00:0.51:1.45:1.23:0.14:1.20:2.02.

[0034] Furthermore, thermal stability assessments have confirmed that the FCPs extracted in this invention can be used as texture modifiers, thickeners, gelling agents, and emulsifiers in the food and biomedical industries.

[0035] Furthermore, through in vitro antioxidant activity and lifespan studies of Caenorhabditis elegans, it was determined that the FCPs extracted in this invention can be applied to anti-aging and antioxidant products.

[0036] In summary, this invention forms a eutectic system through the specific binding of hydrogen bond donors and hydrogen bond acceptors, and extracts fig polysaccharides under ultrasonic assistance. Its unique physicochemical properties can effectively disrupt the plant cell wall structure, significantly improve the polysaccharide dissolution efficiency, and solve the bottlenecks in industrial applications such as high requirements and high energy consumption of subcritical water extraction equipment. It also has the advantages of being environmentally friendly and having a mild operating environment.

[0037] It is known that aging is a complex biological process in which the physiological functions of an organism gradually decline with age, characterized by the accumulation of cell damage, decreased tissue function, and increased susceptibility to disease. Polysaccharides, as an important class of biomolecules, show broad application prospects in the field of anti-aging due to their significant antioxidant activity and low toxicity. This invention uses ultrasound-assisted low-eutectic solvent (UAE-DES) technology to extract polysaccharide components from figs. The physicochemical properties and physical characteristics of these components are characterized using modern analytical techniques, and their anti-aging activity is studied. The results show that the fig polysaccharides (FCPs) extracted in this invention have good potential for application as texture modifiers, thickeners, gelling agents, and emulsifiers in anti-aging, antioxidant products, and the food and biomedical industries.

[0038] The following section, in conjunction with specific experimental verification processes, further explains the fig polysaccharide with anti-aging effects disclosed in this invention, its efficient extraction method, and its applications.

[0039] I. High-Yield Extraction Method for Fig Polysaccharides

[0040] (1) Synthesis of DESs, screening of optimal solvents and optimization of extraction conditions

[0041] Fresh figs were washed, dried to constant weight, ground, and filtered through a 100-mesh sieve to obtain a uniform powder. After removing fat-soluble impurities from the sample with 95% ethanol, different DESs (specific synthesis conditions for DESs are shown in Table 1) were added, and extraction was performed at 60℃ for 30 min. Figure 1 As shown in a, the extraction rate using choline chloride / 1,3-butanediol (ChCl / 1,3-But) (34.96±0.49%) was significantly higher than that using the traditional solvent water (H2O) (5.74±0.05%).

[0042] Single-factor investigations were conducted on the extraction conditions of UAE-DES (liquid-to-solid ratio, ultrasonic time, water content, and temperature). Figure 1 As shown in b, the conditions for obtaining the optimal yield (20:1 mL / g, 50 min, 10%, 50°C) were determined, and the extraction rate of crude polysaccharide was 62.26 ± 0.16%, as shown in Table 2.

[0043] The yield of polysaccharides was calculated as follows: Y = (C × V) / W × 100%. Where C is the concentration of polysaccharides in the sample solution (mg / mL), V is the volume of the sample solution (mL), and W is the mass of fig powder (mg).

[0044] (2) Purification and physicochemical properties of FCPs

[0045] Fig polysaccharide extract was obtained using the extraction conditions described in (1). Four times the volume of ethanol was added to the extract to precipitate the polysaccharides. The precipitate was then repeatedly washed with ethanol, allowed to air dry, reconstituted with deionized water, and dialyzed against the deionized water using a semi-permeable membrane bag with a molecular weight cutoff of 3500 Da. The dialysate was then mixed, concentrated, separated using D101 macroporous resin, and finally freeze-dried. The polysaccharide obtained by this method was named FCPs.

[0046] As shown in Table 2, the contents of total sugar, reducing sugar, and protein in FCPs were 98.69±2.42%, 0.04±0.01%, and 0.01±0.01%, respectively. The uronic acid content in FCPs was 21.1±0.95. The molecular weights of the two polysaccharide components in FCPs were 10118 Da and 532 Da, respectively. FCPs were composed of mannose, rhamnose, glucose, galactose, xylose, arabinose, and galacturonic acid, with a molar ratio of 1.00:0.51:1.45:1.23:0.14:1.20:2.02. (See Table 2 for details.) Figure 2 b.

[0047] Table 2 Physicochemical properties of FCPs Yield 62.26±0.16% Total sugar content 98.69±2.42% reducing sugar content 0.04±0.01% Protein content 0.01±0.01% Polyphenol content Nd Glucuronic acid content 21.1±0.95 molecular weight 10118Da, 532Da Monosaccharide composition (molar ratio) Mannose 1.00 Rhamnose 0.51 glucose 1.45 Galactose 1.23 Xylose 0.14 Arabic sugar 1.20 Galacturonic acid 2.02

[0048] (3) The thermal stability of polysaccharides is an important basis for evaluating their application potential and processing conditions. Thermogravimetric analysis (TG) and derivative thermogravimetric analysis (DTG) were used to characterize the thermal behavior of FCPs. With increasing temperature, the weight of FCPs exhibited a three-step degradation pattern ( Figure 3 a and Figure 3 b). The initial mass loss (30–210°C) is primarily due to the evaporation of free and bound water. Rapid and maximum mass loss is observed in the second stage (210–320°C), attributed to the breaking of carbon chains and hydrogen bonds. Slower mass loss subsequently occurs in the third stage (320–600°C), further confirming that FCPs are relatively stable components.

[0049] The viscosity of FCP decreases significantly with increasing shear rate, which is a typical characteristic of pseudoplastic fluids exhibiting shear-thinning flow behavior. Figure 3 c). Furthermore, the viscosity of FCPs remains almost constant at high shear rates, indicating that they are close to Newtonian fluids. Figure 3Figure d shows the dependence of the storage modulus G' and loss modulus G" of FCP solutions on angular frequency. The excellent rheological properties of FCPs support their potential applications as texture modifiers, thickeners, gelling agents and emulsifiers in the food and biomedical industries.

[0050] II. FCPs have anti-aging activity

[0051] (1) In vitro antioxidant activity of FCPs

[0052] The in vitro antioxidant activity of different concentrations of FCPs was evaluated using the DPPH free radical scavenging assay. The results showed a significant dose-dependent relationship between FCP concentration and DPPH free radical scavenging rate. When the FCP concentration increased from 0 mg / mL to 1.25 mg / mL, the scavenging rate significantly increased from 1.08% to 86.83%. Figure 4 This indicates that FCPs have a strong free radical scavenging ability.

[0053] (2) FCPs reduce reactive oxygen species and prolong lifespan in Caenorhabditis elegans models.

[0054] *Caenorhabditis elegans* is a commonly used model organism for aging studies. Feeding nematodes with different concentrations of FCPs (50, 100, 200, and 400 ng / mL) resulted in an increase in their average lifespan of 8.15%, 15.18%, 16.01%, and 7.30%, respectively. Figure 5 a, Figure 5 b). The average lifespan of nematodes reaches its maximum when the concentration of FCPs is 200 ng / mL. Lipofuscin, a marker of aging, is deposited in senescent tissues and forms autofluorescence; its level is negatively correlated with healthy lifespan. Figure 5 As shown in c, treatment with 100 ng / ml and 200 ng / ml FCPs significantly reduced lipofuscin levels by 22.91% and 24.12%, respectively (p<0.05).

[0055] Aging is accompanied by increased production of reactive oxygen species (ROS), and oxidative stress accelerates the aging process. Using the ROS probe dichlorofluorescein (DCFH) to stain nematodes, it was found that FCP treatment reduced ROS levels in nematodes, such as... Figure 6 As shown.

[0056] These results indicate that FCPs have the effect of delaying aging and prolonging lifespan in nematode models.

[0057] The above embodiments are not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the technical solution of the present invention are also within the protection scope of the present invention.

Claims

1. A highly efficient extraction method for fig polysaccharides with anti-aging effects, characterized in that: The extraction method includes the following steps: Step 1, Raw material pretreatment: Prepare fresh figs, and pretreat the figs by washing and crushing them; Step 2: Prepare the eutectic solvent DESs, which consists of hydrogen bond donor HBD and hydrogen bond acceptor HBA; The eutectic solvent DESs is selected from: acetamide / lactic acid with a molar ratio of 1:1, choline chloride / 1,3-butanediol with a molar ratio of 1:2, and choline chloride / 1,3-butanediol / sorbitol with a molar ratio of 1:1:

1. Step 3: Mix the fig powder obtained in Step 1 with the eutectic solvent DESs prepared in Step 2, and extract it under ultrasonic assistance. Design a single-factor experiment to determine the optimal extraction conditions. The factors of the extraction conditions include liquid-solid ratio, ultrasonic time, water content and extraction temperature. The optimal extraction conditions are: a liquid-to-solid ratio of eutectic solvent to fig powder of 10-50:1 mL / g, ultrasonic time of 20-60 min, water content of eutectic solvent of 0-20%, and extraction temperature of 30-70℃. Step 4, Polysaccharide preparation: After selecting the optimal extraction conditions, fig polysaccharides are extracted efficiently using an ultrasonic-assisted eutectic solvent. Step 5: The fig polysaccharide extract obtained in Step 4 is subjected to alcohol precipitation, purification and drying to obtain the fig polysaccharide product, named FCPs.

2. The efficient extraction method for fig polysaccharides with anti-aging effects according to claim 1, characterized in that: In step 1, the specific methods for crushing are as follows: after washing, the figs are dried to a constant weight, ground into powder, and then sieved through a 100-mesh sieve to obtain uniform fig powder.

3. The efficient extraction method for fig polysaccharides with anti-aging effects according to claim 1, characterized in that: In step 3, the eutectic solvent used for fig polysaccharide extraction is choline chloride / 1,3-butanediol with a molar ratio of 1:

2.

4. The efficient extraction method for fig polysaccharides with anti-aging effects according to claim 1, characterized in that: In step 5, the specific alcohol precipitation, purification and drying process after extraction is as follows: After the fig polysaccharide is extracted, centrifuge to remove the filter residue, precipitate with four times the volume of ethanol, leave overnight, wash the precipitate with ethanol, air dry naturally, redissolve in deionized water, purify using a D101 macroporous resin column, concentrate by rotary evaporation, and finally freeze dry to obtain the fig polysaccharide product.

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