2.2.1 Pretreatment of raw materials

According to reference (Hui, & Gao, 2022), the raw material of Herba Patriniae was powdered and passed through a 60-mesh sieve. 200 g of Herba Patriniae powder was taken accurately, added with 3 times the amount of 80 % ethanol, and refluxed to degrease for 3 times (2 h/time). The supernatant was removed and the residues were collected and placed in the ventilation cabinet dry standby.

2.2.2 Hot water extraction (HW)

Taken the above treated Patriniae powder, added 20 times its volume of distilled water to boil extraction for 3 times (2 h/time), then filtered by multi-layer gauze, collected the filtrate, centrifuged at 3000 rpm for 5 min, and the supernatant was concentrated under reduced pressure to 1/10 of the original volume, added the ethanol with a final concentration of 80 % to precipitate at 4 °C, allowed to stand for 24 h, followed by centrifugation. Then the precipitation was collected and freeze-dried with a freeze drier (Labconco, FreeZone®, American) to obtain Patriniae polysaccharide (Hui, & Gao, 2022). The polysaccharide extraction yield (%) was calculated as the following formula:

Polysaccharide extraction yield (%) = (W₁/W₀) × C × 100 （1）.

Where W₀ is the quality of weight of pretreated Patriniae powder, g; W₁ is the weight of the extracted polysaccharide after freeze-drying, g; C is the content of total polysaccharide, mg/mL.

2.2.3 Ultrasonic assisted hot water extraction (WU)

According to the literature method (Zhang et al., 2022), the treated Patriniae powder was mixed with 15 times its volume of distilled water, ultrasonically treated at 70 °C, 120 W power for 90 min, followed by filtered. The resulting filtrate was concentrated under reduced pressure to 1/10 of the original volume, and then precipitated by ethanol with a final concentration of 80 % at 4 °C, standing overnight and following centrifugation (5000 rpm, 5 min). The obtained precipitation was collected and further freeze-dried with a freeze drier (Labconco, FreeZone®, American) to obtain Patriniae polysaccharide, whose extraction yield was calculated according to formula 1.

2.2.4 Ultrasonic assisted papain mixed cellulase extraction (PCU)

The treated Patriniae powder was mixed with 20 times its volume of sodium acetate buffer (pH 6.5), then added a 2.0 % composite enzyme solution (papain: cellulase = 1:1/ m:m), shaken vigorously and left to stand for 30 min. Subsequently, the mixture was ultrasonically extracted at 90 W power and 55 °C for 20 min, then quickly transferred the mixture to a 95 °C oven and heated for 10 min, taken out, cooled to room temperature, and filtered, added ethanol with a final concentration of 80 % to the filtrate, placed in a refrigerator overnight at 4 °C and then centrifuged (3000 rpm, 5 min). The resulting precipitate was collected, washed with anhydrous ethanol and acetone in turn, and freeze-dried to obtain Patriniae polysaccharide (Hui, Jin, Zhao, et al., 2022), whose extraction yield was calculated according to Formula 1.

2.2.5 Ultrasonic assisted cellulase extraction (CU)

2.2.6 Ultrasonic assisted papain extraction (PU)

2.2.7 Papain mixed cellulase assisted hot water extraction (WPC)

2.2.8 Cellulase assisted hot water extraction (WC)

2.2.9 Papain assisted hot water extraction (WP)

2.3.1 Determination of chemical composition of HPs

The contents of reducing sugar were determined by the 3,5-dinitrosalicylic acid method (DNS), using glucose as standard (Hui, Wang, Zhang, et al., 2022), and the protein contents were determined by Bradford’s method, using bovine serum albumin as the standard (Hui, & Gao, 2022), while the total polysaccharide contents were determined by phenol–sulfuric acid method with glucose as standard (Hui, Jin, Zhao, et al., 2022).

2.3.2 Determination of molecular weight of HPs

According to the previous method with slight modifications (Hui et al., 2019), the absolute molecular weight (Mw) and polydispersity (Mw/Mn) of Pariniae polysaccharides were measured by high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID). HPSEC-MALLS-RID measurements were carried out on a multi-angle laser light scattering detector with an Agilent 1260 series LC system. TSK-Gel G5000PWXL (300 mm × 7.8 mm, i.d.) and TSK-Gel G3000PWXL (300 mm × 7.8 mm, i.d.) were used in series at 30℃. The MALLS instrument was equipped with a He-Ne laser (λ = 658 nm), and an Optilab rEX refractometer was simultaneously connected. The sample concentration was about 1.0 mg/mL with an injection volume 5 μL, and the mobile phase was distilled water at a flow rate of 0.8 mL/min. The Mw was calculated by the Zimm method of static light scattering based on the basic light scattering equation.

2.3.3 Determination of monosaccharide composition of HPs

Monosaccharides composition of Pariniae polysaccharides were measured by GC (Hui, & Gao, 2022). Briefly, each sample (5.0 mg) was hydrolyzed with 2.0 M trifluoracetic acid (2.0 mL) at 110 °C for 6 h, then mixed with 10 mg of hydroxylamine hydrochloride and 1 mL of pyridine in the reaction bottle, continued to react in an oven at 90 °C for 0.5 h. After that, removed from the oven and cooled, added 1 mL acetic anhydride to the mixture and continued to react at 90 °C for 0.5 h. Subsequently, 1 mL of distilled water and chloroformed were added into the reaction solution, shaken evenly, and stood for layering, and then transferred the chloroform layer to the evaporating dish and evaporated to dryness. The residue was dissolved into 0.5 mL of chloroform for GC analysis. A mixed standard solution, containing rhamnose, mannose, glucose, galactose, xylose, and arabinose, was derivatized by the same method as described above.

All the hydrolysates were modified to acetate derivative and analyzed by Agilent 6890n gas chromatograph (Agilent, Santa Clara, CA, USA) in flame ionization detector (FID) and quartz capillary column DB-1 (30 m × 0.32 mm × 0.3 μm). The temperatures of injection port and detector were 250 °C and 260 °C, respectively. The column temperature was programmed to rise from 100 °C (2 min) to 220 °C (5 min) at a rate of 3 mL/min, while the diffusion ratio was 50:1, and the carrier gas was N₂ at a flow rate of 2 mL/min (Hui, Jin, Zhao, et al., 2022).

2.4.1 The clearance activity of DPPH of HPs

The DPPH clearance activity of Pariniae polysaccharides was measured according to the previous method with minor modification (Xiong et al., 2022; Hui, & Gao, 2022). Briefly, 2 mL of polysaccharide solutions with different concentrations was mixed with 2 mL of DPPH solution (0.1 mM with 50 % ethanol), shaken vigorously, and incubated in the dark at room temperature for 30 min. Then, the absorbance value of the reaction solution was quickly measured at 517 nm. Each concentration gradient was operated in parallel three times, and the DPPH radical clearance capacity of polysaccharide was calculated as the following formula:

Where: A_s is the absorbance of 2 mL of polysaccharide solution mixed with 2 mL of DPPH solution, A_b is the absorbance of 2 mL of water mixed with 2 mL of DPPH solution, A_c is the absorbance of 2 mL of polysaccharide solution mixed with 2 mL of anhydrous 50 % ethanol.

2.4.2 The clearance activity of hydroxyl radical of HPs

Hydroxyl radical clearance capacity was carried out as reference with slight modifications (Sun et al., 2022; Hui, & Gao, 2022). In short, 1 mL of polysaccharide solution with different concentrations was fully mixed with 1 mL of FeSO₄·7H₂O solution (9 mM) and 1 mL of H₂O₂ solution (10 mM), and incubated at 37 °C for 10 min. Then, 1 mL of salicylic acid solution (9 mM) was added, mixed well, and continued to incubate at 37 °C for 30 min. Thereafter, the absorbance value was measured at 510 nm. Each concentration gradient was operated in parallel three times, and the hydroxyl radical clearance capacity of polysaccharide was calculated as the following formula:

Where: A_s is the absorbance of polysaccharide solution with different concentrations mixed with FeSO₄·7H₂O solution and salicylic acid solution; A_c is the absorbance value of polysaccharide solution with different concentrations mixed with FeSO₄·7H₂O solution and distilled water; A_b is the absorbance value of distilled water mixed with FeSO₄·7H₂O solution and salicylic acid solution.

2.5.1 The HPs inhibitory effect of α-amylase

α-amylase inhibitory effect of Pariniae polysaccharides was measured according to a previous report (Hui, & Gao, 2022a). In brief, 0.04 mL of polysaccharide with the concentrations of 0.125, 0.25, 0.5, 1, 2 and 4 mg/mL were transferred into 96 well plates, added 0.04 mL of phosphate buffer (0.1 mol/L, pH 6.8) and 0.02 mL of α-amylase solution, mixed thoroughly. After 15 min of warm bath at 37 °C, 0.04 mL of PNPG substrate (10 mmol/L) was added and mixed well again. After water bath at 37 °C for 20 min, the absorbance value was measured at 405 nm. The experiment was repeated three times, and the calculation formula was as follows:

Where: A₁ is the absorption of sample; A₂ is the absorption of sample mixed with substrate; A₃ is the absorption of enzyme mixed with substrate; A₀ is the absorption value of substrate.

2.5.2 The HPs inhibitory effect of α-glucosidase

α-glucosidase inhibitory effect of Pariniae polysaccharides was measured by the reported method with slight modifications (Sun et al., 2022). In brief, 0.03 mL of polysaccharide with the concentration of 0.125, 0.25, 0.5, 1, 2 and 4 mg/mL were transferred into 96 well plates, added 0.03 mL of α-glucosidase (10 U/mL) and mixed completely. After 15 min of warm bath at 37 °C, 0.03 mL of soluble starch (1 %) was added and mixed well again, then heated in a water bath at 37 °C for 15 min, added 0.05 mL of DNS. After boiling water bath for 15 min, 0.11 mL of distilled water was added, and the absorbance was measured at 660 nm. Repeat the test three times, and the calculation formula was as follows:

Where: A₁ is the absorption of sample; A₂ is the absorption of sample mixed with substrate; A₃ is the absorption of enzyme mixed with substrate; A₀ is the absorption value of substrate.

3.3.1 Chemical composition analysis of HPs

The results were shown in Table S3. It was found that the reducing sugar content of polysaccharides extracted by ultrasonic or enzyme treatment was significantly increased. Among them, the PCU-HPs exhibited the highest reducing sugar content, reaching up to 8.8 %, which was nearly 30 times that of HW-HPs. This might be due to the degradation of some bound proteins and lipids by ultrasound or enzyme, which promoted the depolymerization of polysaccharide molecular structure, so that more hydroxyl groups at the reducing end were freed, resulting in an increase in reducing sugar content (Al-Shawi et al., 2021; Wu et al., 2019; Fu et al., 2019). Whereas, the protein content of polysaccharides extracted by different methods basically kept except for PCU extraction, which was due to the synergistic degradation of cellulase and papain into small molecular proteins (Feng et al., 2022).

3.3.2 Monosaccharide composition analysis of HPs

The results were showed in Fig. 2. and Table 4. All HPs extracted by different methods were mainly composed of arabinose, glucose, mannose, and galactose with varying molar ratios. Whereas, the WP-HPs, CU-HPs and HW-HPs contained a small amount of xylose. This might be related with the degradation or conversion of xylose during processing, suggesting that xylose was mainly located at the end of the branch or main chain of the skeleton of Patriniae polysaccharide. Compared with the HW-HPs, enzyme assisted ultrasound method increased the content of mannose and glucose in HPs, and decreased the content of galactose and xylose. While the ultrasonic method reduced the content of mannose, glucose, and xylose in HPs, but increased the content of galactose. This might be the rearrangement of atoms caused by ultrasound and conversion of some mannoses and glucoses into galactose (Hu et al., 2020). In contrast, the enzymatic method generally increased the content of mannose and glucose in HPs, and reduced the content of galactose except for the cellulase method. In general, mannose was derived from cellulose and hemicellulose in plant cell walls. The cellulase used in current experiment mainly contained endoglucanase, which randomly cleaved the β-1,4 glycosidic bonds within cellulose molecules in the cell wall to generate cellobiose and cellooligosaccharides, which caused them to dissolve in the solvent with the polysaccharides, thereby altering the composition ratio of monosaccharides in the polysaccharides. (Huang et al., 2019; Chen et al., 2018).

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Fig. 2

(a): GC profile of monosaccharide standards (1. Rha, 2. Ara, 3. Xyl, 4. Man, 5. Glc, 6. Gal). (b–i): GC profiles of the Patriniae polysaccharides extracted by WU, PCU, WPC, PU, WP, HW, CU, and WC methods.

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3.3.3 Molecular weight analysis of HPs

Generally, the bioactivities of polysaccharides are closely related to their molecular weight (Hui et al., 2019; Feng et al., 2022; Hui et al., 2023). As shown in Fig. 3a-h. and Table S4, the polysaccharides extracted by different methods contained about three kinds of polysaccharides with different molecular weights, indicating that these polysaccharides were heterogeneous polysaccharides. The proportion of high molecular weight polysaccharides in PU, HW and WU were basically the same as that of low and medium molecular weight polysaccharides. Whereas, the high molecular weight polysaccharides contained in WP, CU, WC, WPC and PCU were dominant, accounting for 88.56 %, 79.93 %, 74.30 %, 73.27 %, 67.61 %, respectively, while low and medium molecular weight polysaccharides were contained secondarily. This might be due to the different depolymerization and degradation ability of enzyme and ultrasound. The depolymerization and degradation ability of papain for HPs was weaker than that of cellulase, which resulted in the higher content of high molecular weight HPs. The order of average molecular weight was PCU-HPs > CU-HPs > WPC-HPs > WC-HPs > HW-HPs > WP-HPs > WU-HPs > PU-HPs.

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Fig. 3

Profile of SEC-MALLS assay of Patriniae polysaccharides from different extraction methods (a. PU; b. WP; c. CU; d. WC; e. WPC; f. PCU; g. HW; h. WU). Light scattering signal (red), Refractive index signal (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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In addition, the ultrasonic and enzymatic extraction increased the molecular weight distribution range (DR) of HPs, further showing that enzymes and ultrasound had different depolymerization and degradation abilities on HPs. The DR value of polysaccharides was close to 1, which belonged to the narrow distribution of width sample, and the DR value was greater than 2, which belonged to the wide distribution of width sample (Tang, He, Liu, et al., 2023; Bai et al., 2023; Sheng et al., 2014). Looking from the Table S4, WP-HPs was a wide distribution of width sample, and the HPs extracted by other methods was a narrow distribution of width sample. Enzymatic method, ultrasonic method or their combination can cause the degradation, grafting and crosslinking reaction of polysaccharides, and then change the physicochemical characteristics of polysaccharides, such as molecular weight and its distribution as well as monosaccharide composition, thus affecting the biological activity of polysaccharides (Ji, et al.,2023; Xue,et al., 2022).

3.4.1 DPPH free radical clearance activity

DPPH as a stable nitrogen-centered free radical and is widely employed to screen various antioxidant products. As shown in Fig. 4a, all polysaccharides extracted by different methods presented a good effect on DPPH free radical, which was much better than that of comfrey root and Ginkgo biloba seed polysaccharides (Chen et al., 2018; Hu et al., 2020). With the increase of concentration, the DPPH free radical clearance ability of CU-HPs and PU-HPs initially demonstrated a decrease as the concentrations < 0.5 mg/mL, followed by an increase trend within the range of 0.5–4 mg/mL. In contrast, the DPPH free radical clearance ability of WPC-HPs exhibited an initial increase with concentrations < 2 mg/mL, but decreased within the 2–4 mg/mL range. However, the WC-HPs showed a positive concentration-dependent manner for DPPH free radical clearance ability, and the WP-HPs showed a negative concentration-dependent pattern for DPPH free radical clearance ability. This positive or negative trend gradually leveled off after the polysaccharide concentration reaching to 2 mg/mL. At a concentration of 0.25 mg/mL, the DPPH free radical clearance rate of all HPs was approximately 60 %. When the concentration climbed 4 mg/mL, the DPPH free radical clearance rate of WP-HPs, WC-HPs and CU-HPs exceeded 80 %. Conversely, the PU-HPs and WPC-HPs yielded clearance rate of 62 % and 78 % at 4 mg/mL, respectively. Compared the findings with prior literature (Hui, & Gao, 2022; Hui, Jin, Zhao, et al., 2022) and current data, cellulase-assisted extraction improved much more clearance ability of HPs on DPPH free radical than that of papain-assisted extraction. Interestingly, ultrasound-assisted extraction without enzyme addition led to a reduction of the clearance ability of HPs on DPPH free radical. In terms of DPPH free radical clearance ability, the orders were as follows: WC-HPs > WP-HPs > CU-HPs > WPC-HPs > PCU-HPs > HW-HPs > WU-HPs > PU-HPs.

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Fig. 4

Clearence ability of Patriniae polysaccharides with different extraction methods on DPPH (a) and hydroxyl radical (b).

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3.4.2 Hydroxyl radical clearance activity

To date, hydroxyl radicals is the most potent oxidant, capable of reacting with biomacromolecules such as proteins, lipoproteins, and nucleic acids within living cells. This reaction triggers tissue and cellular damage, leading to various pathological and physiological phenomena. As depicted in Fig. 4b, the hydroxyl radical clearance ability of HPs extracted by different methods in current study was weaker than that of HW-HPs and WU-HPs (Hui, & Gao, 2022; Zhang et al., 2022). At a concentration 4 mg/mL, the hydroxyl radical clearance rate of HW-HPs and WU-HPs reached 90 % and 82 %, respectively. Whereas, the PU-HPs, WP-HPs, WPC-HPs, WC-HPs, and CU-HPs showed weak clearance effect on hydroxyl radicals, with clearance rate of 40 %, 21 %, 14 %, 20 %, and 6 % at 4 mg/mL, respectively. In view of the above analysis, the ultrasound and enzyme assisted extraction were did not significantly enhance the inhibitory ability of HPs on hydroxyl radicals. However, it was observed that the hydroxyl radical clearance ability of HPs extracted by different methods gradually increased with the increasing concentration, which was in accordance with the earlier reports (Feng et al., 2022; Bai et al., 2023; Hui, & Gao, 2022; Tang, He, Liu, et al., 2023). In terms of hydroxyl radical clearance ability, the orders were: HW-HPs > WU-HPs > PCU-HPs > PU-HPs > WP-HPs > WPC-HPs > WC-HPs > CU-HPs. Combined with DPPH free radical clearance ability, CU-HPs exhibiting a moderate and mild antioxidant activity compared to other extraction methods.

3.5.1 α-amylase inhibition activity

As shown in Fig. 5a, it could be seen that the rate of inhibition on α-amylase by WPC-HPs decreased with the increase of concentration. In contrast, WP-HPs, WC-HPs, and CU-HPs showed an increasing concentration-dependent inhibitory trend, while PU-HPs exhibited the highest inhibition rate at 2 mg/mL, with a value of 68.51 %. Notably, at a concentration of 0.0625 mg/mL, the inhibition rate of WPC-HPs was close to 100 %, then reduced to 48.88 % at 4 mg/mL. However, the WP-HPs, WC-HPs, and CU-HPs at 4 mg/mL presented inhibition rates of 76.36 %, 82.78 %, and 62.21 %, respectively. Preliminary analysis suggested that the mixed enzyme method for inhibiting α-amylase was more effective than the single enzyme method, and the α-amylase inhibitory activity of HPs using papain and cellulase-assisted extraction were superior to that of water extraction alone. These findings were consistent with the results of previous research (Bai et al., 2023; Guo et al., 2020; Feng et al., 2022; Tang, He, Liu, et al., 2023; Hu et al., 2020).

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Fig. 5

Effect of Patriniae polysaccharides with different extraction methods on inhibition of α- amylase (a) and α- glucosidase (b).

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3.5.2 α-glucosidase inhibition activity

The results presented in Fig. 5b demonstrated the inhibition rates of HPs extracted by various methods. Specifically, the inhibition rates of WPC-HPs and HW-HPs for α-glucosidase increased with increasing concentration, while the inhibition rates of WP-HPs, WC-HPs, and CU-HPs decreased. Within the test concentration range, the maximum inhibition rates of WPC-HPs, HW-HPs, WP-HPs, WC-HPs, and CU-HPs on α-glucosidase were 97.69 %, 96.87 %, 78.38 %, 67.23 % and 66.29 %, respectively. Apparently, the α-glucosidase inhibition rate of HPs extracted using the compound enzyme was superior to that of a single enzyme extraction, suggesting that papain and cellulase extraction had a synergistic effect on the inhibition of α-glucosidase by HPs. Intriguingly, the inhibition rates of α-glucosidase by PCU-HPs and PU-HPs reached their maximum at a concentration of 1 mg/mL, with values of 86.33 % and 88.95 %, respectively, indicating that ultrasound-assisted extraction had a promoting inhibitory effect on α-glucosidase by HPs. These results implied that different extraction methods might influence the physicochemical characteristics of HPs, such as total polysaccharide content, protein content, reducing sugar content, molecular weight, and monosaccharide composition, thereby impacting their inhibitory effect on α-glucosidase (Xiong et al., 2022; Sun et al., 2022), and the specific relationship will be discussed later. Combined with the ability of inhibition on α-amylase, CU-HPs exhibited a mild hypoglycemic activity compared to other extraction methods.