Phytochemical analysis, antioxidant, anti-inflammatory and enzyme inhibitory activities of bean pear (Pyrus calleryana fruit)

Pyrus calleryana fruit (bean pear) is processed into fruit wine and used in traditional Chinese medicine. The present study reported phytochemical constituents, antioxidant, anti-inflammatory, and enzyme inhibitory activities of P. calleryana fruit water extract (WE) and ethanol extract (EE). In the P. calleryana fruit WE and EE, 63 compounds were identified using UHPLC-Q-Orbitrap-MS analysis, including 23 phenols, 13 flavonoids, 14 terpenoids, and 13 other types of compounds. In the antioxidant activity, WE and EE showed marked free radical scavenging effects on both ABTS (2.33 ± 0.15 μg/mL and 2.23 ± 0.15 μg/mL, respectively) and DPPH (5.93 ± 0.55 μg/mL and 7.07 ± 0.23 μg/mL, respectively), especially, their scavenging effects on DPPH free radicals were superior or equivalent to that of BHT (7.47 ± 0.47 μg/mL). In LPS-induced RAW264.7 cells, P. calleryana fruit WE and EE remarkably inhibited the secretion of inflammatory factors, and the inhibitory effect of WE on the release of IL-6, NO, and PGE₂ was superior or equivalent to that of EE. Interestingly, P. calleryana fruit WE and EE exhibited potent inhibition on α-glucosidase (0.60 ± 0.09 μg/mL and 0.48 ± 0.09 μg/mL, respectively) and tyrosinase (210.11 ± 2.59 μg/mL and 45.35 ± 0.96 μg/mL, respectively), which were superior to their respective positive controls acarbose (302.57 ± 22.09 μg/mL) and arbutin (243.07 ± 15.91 μg/mL). Our findings suggested that P. calleryana fruit WE and EE possess significant antioxidant, anti-inflammatory, α-glucosidase, and tyrosinase inhibitory properties. Thus, P. calleryana fruit has great potential for application in functional food products.

1 Introduction

Pyrus calleryana Decne., a deciduous tree of the genus Pyrus in the Rosaceae family, is a native tree species of China (Sapkota et al., 2022) and primarily distributed in Zhejiang, Jiangsu, Jiangxi, Hubei, Guangdong, Guangxi, Yunnan, and Guizhou provinces in China (Jiang et al., 2016). P. calleryana fruit, commonly known as bean pear, is rich in polysaccharides (15-20%) (Liu et al., 2011). It is often eaten after cooking, primarily due to its sour and astringent when raw, and cooking not only eliminates its sour and astringent taste but also makes it very sweet. Besides, it is also a common raw material for brewing fruit wine (Wu and Wu, 2012).

In addition, P. calleryana is often used as a rootstock for pear (Pyrus pyrifolia), and its wood is employed to make furniture (Li et al., 2015). It is also cultivated as an ornamental tree due to its bright white flowers in early spring (Culley and Hardiman, 2007; Santamour and Demuth, 1980; Sapkota et al., 2022). As a folk medicine, various parts of P. calleryana, including its fruit, branches, leaf, and root, are used medicinally to treat stomach pain, cough, indigestion, acute conjunctivitis, and dysentery (China Medical Information Platform, 2024). Past studies of phytochemical constituents and pharmacological activity of P. calleryana have shown that its leaf ethanol extract exhibited strong antioxidant effects (Nassar et al., 2011), and the volatile oil of its fruit possessed antibacterial and anti-inflammatory effects (Tian et al., 2023).

P. calleryana fruit possesses both edible and medicinal values and has great development potential in functional foods. Nevertheless, there are fewer studies on its chemical composition and bioactivities. Hence, in this study, we aimed to analyze the phytochemical composition of P. calleryana fruit WE and EE, explore their antioxidant, anti-inflammatory effects, and enzyme inhibitory activities, and offer a theoretical basis for exploiting P. calleryana fruit in the field of functional foods.

2 Materials and methods

2.1 Chemical and reagents

RAW264.7 cells were purchased at the Kunming Cell Bank (Kunming, China). Folin-Ciocalteu reagent, rutin, arbutin, lipopolysaccharide (LPS), 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), dexamethasone (DXM), galanthamine, dimethyl sulfoxide (DMSO), acarbose and gallic acid were supplied from Solarbio Sciences & Technology (Beijing, China). Mouse TNF-α and IL-6 ELISA kits were obtained from Multi Sciences (Lianke) Biotech, Co., Ltd. (Hangzhou, China). Abnova Corporation (Taiwan, China) provided a prostaglandin E₂ (PGE₂) ELISA kit. Ascorbic acid, 5,5’dithiobis-(2-nitrobenzoic acid), tyrosinase, p-nitrophenyl-α-D-glucopyranoside (p-NPG), L-tyrosine, acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE), butyrylthiocholine chloride (BTCl), butyrylcholinesterase (BChE), butylated hydroxytoluene (BHT), 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), α-glucosidase, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were supplied by Sigma-Aldrich (Germany). Beyotime Biotechnology offered the NO detection kit (Shanghai, China).

2.2 Plant material

P. calleryana fruit was bought from Shansen Reservoir, Yudu County, Ganzhou City, Jiangxi Province, and was identified by Prof. Guoxiong Hu, and preserved in the College of Life Sciences, Guizhou University (herbarium number: PC20210907).

2.3 Extraction of P. calleryana fruit WE and EE

Fresh P. calleryana fruit was chopped, placed in a round-bottomed flask, and extracted with ultrapure water or 70% ethanol solution at a material-liquid ratio of 1:3 (w/v) for 2 h. Afterward, the filtrate was gathered, and the residue was re-extracted according to the above conditions. Subsequently, the two filtrates were merged and condensed by rotary evaporation to obtain the concentrated solution. Finally, the concentrated solution was dried by a freeze-dryer to obtain the WE and EE. The samples were packed into brown bottles and sealed storage at 4°C.

2.4 Chemical composition analysis

Ultra-high-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS) was used to identify phytochemical components of P. calleryana fruit WE and EE. Dionex Ultimate 3000 RSLC liquid phase conditions were as follows: injection volume (5 μL), column (Thermo Fisher Hypersil GOLD aQ, 2.1 mm × 100 mm, 1.9 μm), column temperature (40°C), mobile phase (A: 0.1% formic acid aqueous solution and B: 0.1% formic acid acetonitrile), and flow rate (0.3 mL/min). The gradient elution conditions were as follows: 0~2 min (5% B), 2~42 min (5~95% B), 42~47 min (95% B), 47~47.1 min (5% B), 47.1~50 min (5% B).

Thermo Fisher Scientific’s Q Exactive Focus hybrid quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap-MS) with heated electrospray ionization (HESI-II) was used to acquire MS data. The HESI-II settings were as follows: sheath gas (35 arb), auxiliary gas (10 arb), S-Lens (60), probe heater temperature (350°C), spray voltages (-2.5/+3.0 kV), capillary temperature (320°C), sweep gas (0 arb). The mass spectrometry scanning parameters were as follows: full MS scan range (100 to 1500 m/z), loop count (3), intensity threshold (1.6 e⁵), stepped NCE (20, 40, 60 eV), maximum injection time (full MS: 100 ms, MS/MS: 50 ms), resolution (full MS: 70,000, MS/MS: 17,500), AGC target (full MS: 1 e⁶, MS/MS: 2 e⁵). Thermo Fisher Scientific Xcalibur 4.1 (Waltham, MA, USA) was utilized for the processing of mass spectrometry data on chemical compositions. Phytochemicals were identified by comparing the mzVault and mzCloud databases and references within a 10 ppm threshold.

2.5 Determination of total phenolic acid content and total flavonoid content

2.5.1 Determination of TPC

The Folin-Ciocalteu method was utilized to determine the TPC in the P. calleryana fruit WE and EE, with gallic acid as the standard. WE or EE were dissolved in ultrapure water to prepare sample solution. Folin-Ciocalteu solution (2.5 mL) and sample solution (0.5 mL) were accurately pipetted into a 5 mL tube and reacted for 4 min. Afterward, 7.5% Na₂CO₃ solution (2 mL) was added, mixed, and reacted for 60 min at room temperature. After recording the absorbance at 760 nm, we used linear fitting to create a standard curve with the absorbance values as the vertical coordinate and the concentration of gallic acid as the horizontal coordinate. The TPC of P. calleryana fruit WE and EE was calculated according to the standard curve and expressed as milligrams of gallic acid equivalents per gram of sample (mg GAEs/g sample).

2.5.2 Determination of TFC

The TFC in the WE and EE of P. calleryana fruit was estimated by the NaNO₂-Al(NO₃)₃-NaOH colorimetric method and rutin was used as a standard. The WE or EE dissolved in 70% ethanol (5 mL) and NaNO₂ solution (5%, 0.4 mL) were mixed and reacted for 6 min. Next, we added Al(NO₃)₃ solution (10%, 0.4 mL) and let it react for 6 min. Subsequently, NaOH solution (4%, 4 mL) and ultrapure water (0.2 mL) were added and reacted for 15 min. Then, the absorbance at 510 nm was measured. The standard curve was established using rutin concentration as the horizontal coordinate and absorbance as the vertical coordinate. The standard curve was used to calculate the TFC, which was then expressed as milligrams of rutin equivalents per gram of sample (mg REs/g sample).

2.6 Measurement of antioxidant capacity of P. calleryana fruit WE and EE

The ability of P. calleryana fruit WE and EE to scavenge free radicals was determined by ABTS and DPPH assay. Ascorbic acid and BHT were used as positive controls.

2.6.1 ABTS assay

In the ABTS free radical scavenging capacity assay, ABTS solution (0.7 mM) was prepared by dissolving ABTS (19.2 mg) with dehydrated ethanol (50 mL), and K₂S₂O₄ solution (2.4 mM) was obtained by dissolving K₂S₂O₄ (33 mg) with ultrapure water (50 mL). Subsequently, equal volumes of ABTS and K₂S₂O₄ solution were mixed, protected from light, and incubated at 37°C for 12 h to prepare ABTS•⁺ solution. The ABTS assay included a sample group, a control group, and a blank group. Sample solution (18 μL) was combined with ABTS•⁺ solution (180 μL) in the sample group; in the control group, sample solution (18 μL) was combined with solvent of sample solution (180 μL); in the blank group, 18 μL of the solvent of sample solution and 180 μL of ABTS•⁺ solution were added. Then, all groups were incubated for 10 min and sheltered from light. The absorbance (A) was detected at 734 nm. The findings were displayed as milligrams of ascorbic acid equivalents per gram of sample (mg AEs/g sample) and IC₅₀ values. The following formula was used to determine the scavenging rate:

$$\text{Scavenging rate}(\%)=[1-\frac{{\text{A}}_{\text{sample}}-{\text{A}}_{\text{control}} }{ {\text{A}}_{\text{blank}}}]\times 100\%$$

2.6.2 DPPH assay

In the free radical clearance test for DPPH, DPPH (0.08 mM) solution was prepared by dissolving DPPH (3.15 mg) in dehydrated ethanol (100 mL). The sample group, control group, and blank group were set up. In the sample group, the sample solution (100 μL) was mixed with an equal amount of DPPH solution. Similarly, equal volumes of sample solution and solvent of sample solution were mixed as the control group. In the blank group, the DPPH solution and solvent of the sample solution were mixed in a 1:1 ratio (v/v). Next, these groups were placed under room temperature and light-avoidance conditions for 0.5 h, and their absorbance values were detected at 517 nm. The free radical scavenging capacity was expressed as milligrams of ascorbic acid equivalents per gram of sample (mg AEs/g sample) and IC₅₀ values. The aforementioned formula was used to determine the scavenging rate.

2.7 Anti-inflammatory capacity analysis in LPS-induced RAW264.7 cells

2.7.1 Analysis of cytotoxicity

MTT test was utilized to test the toxicity of P. calleryana fruit WE and EE on RAW264.7 cells (Zhao et al., 2020a). The samples were dissolved in DMSO and diluted half-fold with culture medium (DMSO content was less than 0.05%). Cells (2×10⁵ cells/well) were inoculated and cultured in 96-well plates. Then, the old culture medium was aspirated out and discarded after 24 h incubation. Afterward, the sample solution was pipetted into 96 well plates and incubated for 24 h at 37°C. Next, MTT solution (10 μL, 5 mg/mL) was pipetted into and reacted for 4 h. Subsequently, the old supernatant was discarded, and 150 μL of DMSO was added to each well. Finally, the 96-well plate was shaken at room temperature for 10 min, and the absorbance value was determined at 490 nm.

2.7.2 Measurement of NO, IL-6, TNF-α, and PGE₂ release

In the assay of inflammatory factors release, 100 μL of cell suspensions (2×10⁶ cells/mL) were inoculated in 96-well plates and cultured for 24 h. The sample group, blank group, positive group (DXM), and model group (LPS) were set up. Then, the old supernatants were aspirated out and discarded. In the sample group, 100 μL of sample solution (62.5, 125, and 250 μg/mL) was added; 100 μL of DXM (20 μg/mL) was added in the positive group; 100 μL of culture medium was added in blank and model groups. After 2 h of incubation, the supernatants were discarded; in the sample group, equal volumes of sample solution (100 μL) and LPS (100 μL) were mixed, and the sample solution’s ultimate concentrations were 62.5, 125, and 250 μg/mL; DXM (100 μL) and LPS (100 μL) were added to the positive group, and the ultimate concentration of DXM was 20 μg/mL. In the model group, culture medium (100 μL) and LPS (100 μL) were mixed, and the three groups mentioned above had a final concentration of 1 μg/mL of LPS. In addition, culture medium (200 μL) was added to the blank group. Next, all the groups were placed at a constant temperature of 37°C for 24 h. Then, all the groups were photographed under an inverted fluorescence microscope to observe cell morphological changes. Subsequently, the cell supernatants were collected. Determination of NO release was performed referring to the instructions of the NO detection kit, and the levels of IL-6, TNF-α, and PGE₂ were measured by ELISA kits.

2.8 Enzyme inhibitory activity assay

The inhibitory activities of P. calleryana fruit WE and EE on tyrosinase, α-glucosidase, and cholinesterase were determined by using arbutin, acarbose, and galanthamine as positive controls, respectively.

2.8.1 Tyrosinase inhibitory activity

In the assay of tyrosinase inhibitory activity, we set up four groups: sample group, sample blank group, negative group, and blank group. First, sample solution (70 μL) was added to the sample and sample blank groups, and PBS buffer (70 μL, pH=6.8) was added in the negative and blank groups. Subsequently, tyrosinase (100 μL, 100 U/mL) was added in the sample and negative groups, while PBS buffer (100 μL, pH=6.8) was spiked into the sample blank and blank groups. The above four groups were incubated at a constant temperature of 37°C for 5 min. Then, L-tyrosine (80 μL, 5.5 mM) was added in all groups. After 37°C incubation for 30 min, their absorbance (A) was determined at 492 nm. The following formula was used to get the tyrosinase inhibition rate, and IC₅₀ values were utilized to express the inhibitory activity of tyrosinase.

$$\text{Inhibition rate}(\%)=[1-\frac{{\text{A}}_{\text{sample}}-{\text{A}}_{\text{sample blank}} }{{\text{A}}_{\text{negative}} -{\text{A}}_{\text{blank}}}]\times 100\%$$

2.8.2 α-Glucosidase inhibitory activity assay

In the α-glucosidase inhibitory activity assay, the experimental procedure was divided into four groups: sample group, sample blank group, negative group, and blank group. First, 90 μL of sample solution was added into the sample and sample blank group, whereas 90 μL of PBS buffer (pH=6.8) was utilized in the negative and blank groups. Next, 10 μL of α-glucosidase (0.8 U/mL) was added to the sample and negative groups, while 10 μL of PBS buffer (pH=6.8) was added to the sample blank and blank groups. The four groups were placed in a constant temperature incubator of 37°C for 15 min. Then, 10 μL of the p-NPG (1 mM) was added to all groups and reacted at 37°C for 15 min. Finally, the reaction was terminated by adding 80 μL of sodium carbonate solution (0.2 mM). The absorbance values of each group were measured at 405 nm. The α-glucosidase inhibition rate was calculated as above, and IC₅₀ values represented the inhibitory activity of α-glucosidase.

2.8.3 Cholinesterase inhibitory activity assay

In the determination of cholinesterase inhibitory activity, we also set up sample, sample blank, negative, and blank groups. First, the sample and sample blank groups were given a 50 μL sample solution, while 50 μL of PBS buffer (pH=8) was added to the negative and blank groups. Subsequently, 10 μL of AChE or BChE (0.5 U/mL) was pipetted into the sample and negative groups, and 10 μL of PBS buffer (pH=8) was added to the sample blank and blank groups, which were kept at 4°C for 5 min. Then, 20 μL of ATCI (2 mM) or BTCI (2 mM) and 5,5’dithiobis-(2-nitrobenzoic acid) solution (2 mM) was added to all groups and maintained at 37°C for 30 min. Absorbance values were determined at 405 nm, and the inhibition rate was calculated as above. Finally, the inhibitory activity of the WE and EE on cholinesterase were expressed as IC₅₀ values.

2.9 Statistical analysis

In this study, three independent replications of each experiment were carried out, and their results were represented using mean ± standard deviation (SD). The significant difference was analyzed by one-way analysis of variance (ANOVA) with Fischer’s LSD post-hoc test or two-tailed unpaired t-test using IBM SPSS Statistics 25 software (p < 0.05 indicates a significant difference).

3 Results and discussion

3.1 Phytochemical components of P. calleryana fruit WE and EE

Based on fresh weight, the extraction rates of P. calleryana fruit WE and EE were 8.3% (w/w) and 11% (w/w), respectively. The Folin-Ciocalteu method and the NaNO₂-Al(NO₃)₃-NaOH colorimetric method were used to determine the TPC and TFC of WE and EE, and the results are shown in Figure 1. The TPC of WE and EE were 120.74 ± 0.32 mg GAEs/g sample and 95.37 ± 0.28 mg GAEs/g sample, respectively, and WE exhibited more TPC compared to EE (p < 0.001). The TFC of WE and EE separately were 347.05 ± 6.27 mg REs/g sample and 562.51 ± 10.71 mg REs/g sample, and the TFC in EE was significantly higher than WE (p < 0.001).

Figure 1

Figure 1. Total phenolic and flavonoid contents of P. calleryana fruit WE and EE, *** p < 0.001.

Based on UHPLC-Q-Orbitrap-MS analysis, 63 compounds were characterized in the P. calleryana fruit WE and EE (detailed analysis of fragmentation patterns in Supplementary Material), of which 40 compounds were identified in WE, and 41 compounds were identified in EE (Table 1, Supplementary Figure S1 in Supplementary Materials). A total of 23 phenolic compounds were identified as neochlorogenic acid (6), 3,5-dimethoxy-4-hydroxybenzaldehyde (8), gentisic acid (11), cryptochlorogenic acid (12), aloenin (14), chlorogenic acid (15), vanillic acid (16), 1-caffeoylquinic acid (17), caffeic acid (18), 7-hydroxycoumarin (21), vanillin (23), 2-hydroxy-4-methoxybenzaldehyde (24), protocatechualdehyde (28), ellagic acid (34), sinapyl aldehyde (36), dehydrodiisoeugenol (38), 3,5-dicaffeoylquinic acid (39), isochlorogenic acid B (40), isochlorogenic acid C (44, ethyl caffeate (49), astringin (51), 6-gingerol (53), and (+)-usniacin (60) (Supplementary Figure S2, Supplementary Material). Thirteen flavonoid compounds were identified, including (+)-catechin hydrate (22), procyanidin B1 (26), taxifolin 7-rhamnoside (29), medicarpin (31), hesperetin (32), morin (33), cynaroside (35), isorhamnetin (41), diosmetin-7-O-β-D-glucopyranoside (43), hyperoside (45), isoquercitrin (46), kaempferol-7-O-β-D-glucopyranoside (47), and luteolin (50) (Supplementary Figure S3, Supplementary Material). Fourteen terpenoid compounds were identified as ailanthone (20), sarracenin (25), perillene (37), germacrone (48), glabrolide (52), medicagenic acid (54), quillaic acid (55), 18β-glycyrrhetintic acid (56), ursonic acid (57), echinocystic acid (58), ursolic acid (59), acetyl-11-keto-β-boswellic acid (61), lupenone (62), and roburic acid (63) (Supplementary Figure S4, Supplementary Material). Besides, 13 other types of compounds were identified, including sucrose (1), quinic acid (2), γ-aminobutyric acid (3), L-tryptophan (4), scopolin (5), citric acid (7), o-veratraldehyde (9), mannitol (10), 2-isopropylmalic acid (13), homoveratrumic acid (19), ethyl 4-methoxycinnamate (27), androsin (30), and benzoic acid (42) (Supplementary Figure S5, Supplementary Material). Except for chlorogenic acid (15), vanillic acid (16), and caffeic acid (18), which were previously identified from P. calleryana leaf (Nassar et al., 2011), the remaining 60 compounds were identified for the first time in P. calleryana. The results showed that there were plenty of phenolic, flavonoid, and terpenoid compounds in P. calleryana fruit WE and EE.

Table 1

Table 1. Chemical composition of P. calleryana fruit WE and EE were authenticated using UHPLC-Q-Orbitrap-MS in positive and negative ion modes.

3.2 Antioxidant effects of P. calleryana fruit WE and EE

ABTS and DPPH methods were utilized to determine the antioxidant capacity of P. calleryana fruit WE and EE. As shown in Table 2, both WE (2.33 ± 0.15 μg/mL) and EE (2.23 ± 0.15 μg/mL) exhibited significant ABTS radical scavenging effects. Moreover, WE (5.93 ± 0.55 μg/mL) and EE (7.07 ± 0.23 μg/mL) displayed strong DPPH radical scavenging abilities, which was superior or equivalent to that of BHT (7.47 ± 0.47 μg/mL). The DPPH and ABTS free radical scavenging abilities of WE (601.75 ± 55.63 mg AEs/g and 630.35 ± 40.50 mg AEs/g, respectively) were superior or equivalent to those of EE (503.07 ± 17.34 mg AEs/g and 658.75 ± 44.19 mg AEs/g, respectively).

Table 2

Table 2. Antioxidant properties of P. calleryana fruit WE and EE.

BHT is widely utilized as a common antioxidant and preservative in the cosmetic and food industries (Yehye et al., 2015). However, toxic effects on the liver, kidneys, and lungs have been reported from prolonged exposure to BHT, raising concerns among consumers (Ghosh et al., 2020). In this study, WE and EE exhibited DPPH radical scavenging abilities that were superior to or comparable to BHT. Therefore, P. calleryana fruit WE and EE can be used as antioxidant substitutes for BHT. The determination of TPC and TFC indicated that phenolic and flavonoid compounds were abundant in P. calleryana fruit WE and EE. Plant-derived phenolic and flavonoid compounds have been shown to have potent antioxidant activity (Diaz et al., 2012; Mustafa et al., 2010). In our study, a total of 23 phenols and 13 flavonoids were identified through UHPLC-Q-Orbitrap-MS analysis, among which luteolin (IC₅₀: 0.59 ± 0.02 μg/mL and 2.10 ± 0.06 μg/mL, respectively) has been shown to be superior to that of BHT (IC₅₀: 1.45 ± 0.02 μg/mL and 10.5 ± 0.08 μg/mL, respectively) and ascorbic acid (IC₅₀: 2.16 ± 0.03 μg/mL and 3.03 ± 0.12 μg/mL, respectively) in ABTS and DPPH scavenging effects (Tian et al., 2021a). In the ABTS radical clearance test, vanillin exhibited greater activity than ascorbic acid (Tai et al., 2011). Moreover, ursolic acid (Do Nascimento et al., 2014), vanillic acid (Szwajgier et al., 2005; Tai et al., 2012), isorhamnetin (Gong et al., 2020), morin (Hsu et al., 2022), dehydrodiisoeugenol (Li et al., 2020), 6-gingerol (Mi et al., 2016), caffeic acid (Gülçin, 2006), 3,5-dicaffeoylquinic acid (Hong et al., 2015), ellagic acid (Han et al., 2006), and cynaroside (Zou et al., 2018) have been found to have high ABTS and/or DPPH free radical scavenging capacity. Thus, the strong antioxidant activity of P. calleryana fruit WE and EE may be explained by their high content of flavonoids and phenolic compounds. Our results showed that P. calleryana fruit could be used as a potential antioxidant in functional foods.

3.3 Anti-inflammatory effect in LPS-stimulated RAW264.7 cells of P. calleryana fruit WE and EE

3.3.1 Cytotoxicity of P. calleryana fruit WE and EE

The MTT assay was used to assess the cytotoxicity of WE and EE against RAW264.7 cells. As shown in Figure 2, both WE and EE were non-cytotoxic to RAW264.7 cells in the dose range of 15.625 to 250 mg/mL compared with untreated cells (p > 0.05). Therefore, the concentrations of 62.5, 125, and 250 μg/mL were selected for subsequent experiments.

Figure 2

Figure 2. Cytotoxic activity of P. calleryana fruit WE and EE on RAW264.7 macrophages. ***p < 0.001 versus the untreated cells.

3.3.2 Effects of WE and EE on LPS-induced morphological changes and inflammatory factors release in RAW264.7 cells

The pathogenesis of inflammation is closely related to the excessive secretion of pro-inflammatory factors (IL-6 and TNF-α) and mediators (NO and PGE₂). Inhibiting their production is one of the most important means for treating inflammatory diseases (Wang et al., 2020; Zhao et al., 2021). As shown in Figure 3A, the morphology of RAW264.7 cells changed from round to stretched after LPS stimulation compared with control, while the stretched cells gradually decreased with the gradual increase of WE and EE concentrations, especially at high concentrations (250 μg/mL) there were no stretched cells. As shown in Figures 3B–E, LPS induction significantly increased the release of NO, PGE₂, TNF-α, and IL-6. Compared to the LPS group, different concentrations of P. calleryana fruit WE and EE (62.5, 125, and 250 μg/mL) markedly suppressed the release of NO, PGE₂, and IL-6 in RAW264.7 cells induced by LPS, and the high concentration (250 μg/mL) of WE and EE effectively suppressed the level of TNF-α. Moreover, WE (62.5, 125, and 250 μg/mL) and EE (125 and 250 μg/mL) showed better inhibition on PGE₂ and IL-6 release compared with the positive control DXM (20 μg/mL), and the inhibitory effects of WE on NO, PGE₂, and IL-6 was superior to or equivalent to that of EE at the same concentrations. These results showed that the P. calleryana fruit WE and EE effectively inhibited the level of NO, PGE₂, TNF-α, and IL-6 in RAW264.7 cells induced by LPS.

Figure 3

Figure 3. The influence of P. calleryana fruit WE and EE on LPS-induced RAW264.7 macrophage morphological alterations and pro-inflammatory mediator and cytokine level. (A) P. calleryana fruit WE and EE’s effect on alterations in cellular morphology. (B–E) The NO release was determined by the NO detection kit, while the levels of PGE₂, TNF-α, and IL-6 were measured by ELISA kits. The mean ± SD is used to express data. Significant differences are represented by different alphabets (p < 0.05).

Plant-derived flavonoids, terpenoids, and phenolic compounds have been widely demonstrated to be associated with anti-inflammatory effects (Diaz et al., 2012; Faisal et al., 2022; Jaroš et al., 2022; Maleki et al., 2019; Rathee et al., 2009). Phenols, flavonoids, and terpenoids identified in the P. calleryana fruit, like ellagic acid, caffeic acid, ursolic acid, and luteolin, reduced the levels of IL-6 and TNF-α in mouse models (Aziz et al., 2018; Chao et al., 2010; Zhao et al., 2023). Besides, 18β-glycyrrhetintic acid significantly reduced TNF-α level in serum and NO level in the kidney of MTX-induced rats (Abd El-Twab et al., 2016). Additionally, in the past in vitro studies, vanillin (Zhao et al., 2019), chlorogenic acid (Huang et al., 2023; Hwang et al., 2014), cryptochlorogenic acid (Zhao et al., 2020b), medicarpin (Chern et al., 2021; Mansoori et al., 2020), ethyl caffeate (Chiang et al., 2005), cynaroside (Zou et al., 2018), roburic acid (Islam, 2017), ursonic acid (Son and Lee, 2020), echinocystic acid (Hyam et al., 2013), ursolic acid (Zhao et al., 2023), and 3,5-dicaffeoylquinic acid (Hong et al., 2015) significantly inhibited the release of inflammatory factors, such as TNF-α, IL-6, NO, and PGE₂. Therefore, the strong anti-inflammatory effects of P. calleryana fruit WE and EE may be associated with these flavonoids, terpenoids, and phenolic compounds. Our results suggested that P. calleryana fruit could be exploited greatly as an anti-inflammatory agent in functional foods.

3.4 Enzyme inhibitory activities of P. calleryana fruit WE and EE

Inhibiting the activity of α-glucosidase can suppress oligosaccharide hydrolysis and reduce the absorption of carbohydrates after meals, which can effectively help lower blood glucose (Gao et al., 2022). As shown in Table 3, WE (0.60 ± 0.09 μg/mL) and EE (0.48 ± 0.09 μg/mL) exhibited extremely strong α-glucosidase inhibition, which was stronger than that of acarbose (302.57 ± 22.09 μg/mL) (p < 0.05). Plant-derived flavonoids and phenolic compounds can serve as natural α-glucosidase inhibitors (Goncçalves and Romano, 2017; Şöhretoğlu and Sari, 2020). Phenolic and flavonoid compounds identified in WE and EE of P. calleryana fruit, such as vanillic acid (Singh et al., 2022), vanillin (Zabad et al., 2019), morin (Vanitha et al., 2014), ellagic acid (Chao et al., 2010), isorhamnetin (Alqudah et al., 2023), and luteolin (Zang et al., 2016) have been shown to markedly improved blood glucose levels in a diabetic mouse model. Furthermore, the inhibitory activities of protocatechualdehyde and 6-gingerol on α-glucosidase were superior to those of the positive control acarbose in vitro (Mohammed et al., 2017; Tian et al., 2021b). The above findings indicated that P. calleryana fruit can be utilized in functional foods as an α-glucosidase inhibitor.

Table 3

Table 3. Enzyme inhibition properties of P. calleryan fruit WE and EE.

Tyrosinase is the key enzyme that produces melanin and causes skin pigmentation, and excessive melanin leads to many skin diseases, such as age spots, malignant melanoma, and freckles (Abdul Karim et al., 2014; Brenner and Hearing, 2008). Besides, tyrosinase causes enzymatic browning of beverages, fruits, and vegetables (Loizzo et al., 2012). Therefore, inhibition of tyrosinase activity is useful for achieving skin whitening effects and preventing enzymatic browning of food products. As shown in Table 3, the inhibitory effects of WE (210.11 ± 2.59 μg/mL) and EE (45.35 ± 0.96 μg/mL) on tyrosinase were significantly superior to the positive control arbutin (243.07 ± 15.91 μg/mL) (p < 0.05). Past studies have shown that phenolic and flavonoid compounds can serve as important sources of tyrosinase inhibitors (Goncçalves and Romano, 2017). Luteolin, a compound identified from the fruits of P. calleryana, dose-dependently inhibited tyrosinase activity and melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells (An et al., 2008; Choi et al., 2008). In addition, benzoic acid (Sima et al., 2011), vanillic acid (Smeriglio et al., 2019), ellagic acid (Pillaiyar et al., 2017), and 2-hydroxy-4-methoxybenzaldehyde (Rafiee and Javaheri, 2015) have been well demonstrated to have good tyrosinase inhibitory effects. Several chemical components identified in P. calleryana fruit EE but not detected in WE were proven to have tyrosinase inhibitory effects. For example, ursolic acid exhibited significant anti-tyrosinase effort and inhibited α-MSH-stimulated melanin synthesis in B16F1 cells (Neimkhum et al., 2021; Park et al., 2020). Hesperetin observably inhibited tyrosinase in a competitive manner with IC₅₀ = 11.25 ± 1.73 mM (Si et al., 2012). Besides, isorhamnetin (Yu et al., 2019) and morin (Wang et al., 2014) have been demonstrated to serve as tyrosinase inhibitors. These compounds identified only from P. calleryana fruit EE may be the reason why EE was significantly superior to WE on tyrosinase inhibition. Hence, the strong tyrosinase inhibition activity of P. calleryana fruit WE and EE may be closely related to the presence of these phenolic and flavonoid compounds. The results demonstrated that P. calleryana fruit can be developed as a potential tyrosinase inhibitor for skin whitening and prevention of food browning.

Low levels of acetylcholine in the human brain play a crucial role in the pathogenesis of Alzheimer’s disease, and reducing the catabolism of acetylcholine by inhibiting the activities of acetylcholinesterase and butyrylcholinesterase is an effective treatment for Alzheimer’s disease (Adewusi et al., 2010). As shown in Table 3, EE (40.47 ± 5.22 μg/mL and 26.77 ± 0.89 μg/mL, respectively) and WE (60.75 ± 5.05 μg/mL and 34.31 ± 1.97 μg/mL, respectively) showed moderate inhibitory effects on acetylcholinesterase and butyrylcholinesterase. Past studies have shown that vanillin and vanillic acid inhibited acetylcholinesterase and butyrylcholinesterase activities (Salau et al., 2020). Besides, 2-hydroxy-4-methoxybenzaldehyde and protocatechualdehyde had potential acetylcholinesterase inhibitory activity (Aderogba et al., 2013; Kundu and Mitra, 2013). These compounds may contribute to the moderate cholinesterase inhibitory activity observed in P. calleryana fruit.

4 Conclusions

The present study reports the chemical composition, antioxidant, anti-inflammatory, and enzyme inhibitory activities of P. calleryana fruit WE and EE for the first time. The chemical composition of P. calleryana fruit WE and EE were analyzed by UHPLC-Q-Orbitrap-MS, and the results showed that P. calleryana fruit was rich in phenolic, flavonoid, and terpenoid compounds. Both WE and EE exhibited significant free radical scavenging effects on ABTS and DPPH, and their DPPH radical scavenging effects were superior or equivalent to that of BHT. In addition, WE and EE exhibited remarkable anti-inflammatory activity with significant inhibition on the release of pro-inflammatory cytokines (IL-6 and TNF-α) and mediators (NO and PGE₂) in LPS-stimulated RAW264.7 cells. In enzyme inhibitory activity, P. calleryana fruit WE and EE had significant inhibitory effects on tyrosinase and α-glucosidase, whose inhibitory effect was considerably superior to that of the positive control arbutin and acarbose. Our findings showed that P. calleryana fruit WE and EE possessed significant antioxidant, anti-inflammatory, α-glucosidase and tyrosinase inhibitory properties. Thus, P. calleryana fruit can act as a natural source of antioxidants, anti-inflammatory agents, and tyrosinase and α-glucosidase inhibitors, which has a great potential for application in the field of functional foods. Interestingly, P. calleryana fruit exhibited potent inhibition of tyrosinase and α-glucosidase, indicating that it possesses whitening and hypoglycemic efficacy. Further studies should be conducted on its whitening and hypoglycemic effects in vitro and in vivo to promote its development and utilization in the whitening cosmetics and health food industries.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author/s.

Author contributions

HZ: Investigation, Methodology, Writing – original draft. QW: Investigation, Validation, Writing – original draft. LY: Investigation, Validation, Writing – original draft. YR: Investigation, Validation, Writing – original draft. QH: Formal analysis, Writing – original draft. YH: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. MT: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Natural Science Foundation of China (82360834 and 32260747) and the Science and Technology Program of Guizhou province, China (Qian Ke He Zhi Cheng (2020) 1Y133, Qian Ke He Ji Chu-ZK (2021)).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1521990/full#supplementary-material

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