Phytochemical Characterization and Antioxidant and Enzyme Inhibitory Activities of Different Parts of Prinsepia utilis Royle

The objective of this study was to evaluate the phenolic composition and antioxidant and enzyme inhibitory activities of the ﬂowers, leaves, and stems of Prinsepia utilis Royle. In the work, their total phenol content and ﬂavonoid content were determined. In addition, the scavenging eﬀects of DPPH and ABTS free radicals and ferric reducing antioxidant power were measured. The results showed the ﬂowers had the highest total phenol and ﬂavonoid content, followed by the leaves and stems. A total of 11 phenolic substances were identiﬁed and quantiﬁed using UHPLC-ESI-HRMS/MS, of which rutin was the dominant phenolic compound in all samples. All three samples had good antioxidant activity and dose dependently inhibited the activity of α -glucosidase, pancreatic lipase, and tyrosinase. In summary, the ethanol extracts of the ﬂowers have the best antioxidant and enzyme inhibitory ability among three samples. The outcome could provide support for the development and utilization of P. utilis.


Introduction
Polyphenols are naturally occurring compounds that are widely distributed in different parts of plants including flowers, leaves, stems, fruits, and bark. Polyphenols play an important role in plant growth and development and protecting plants from insect damage, ultraviolet light, and wound infections [1]. In addition, they have biological functions and benefits to human health [2]. Humans ingest polyphenols mainly from fruits, vegetables, and beverages, especially tea, coffee, and red wine for good health, whereas the body cannot synthesize them by its own [3]. erefore, it is important to adhere to daily eat a certain amount of polyphenol-rich food.
Nowadays, the incidence of chronic noncommunicable diseases (CNCDs) is increasing and the age of patients has shown a trend of getting younger, resulting from changes of diet habits, living environment, and lifestyle, for example, high-calorie food intake, industrial pollution, smoking, alcoholism, staying up late, and lack of exercise [4,5].
Researchers have increasingly focused on the prevention and alleviation of the diseases through diets with little side effects. More and more research projects indicated that polyphenols have the potential to prevent or treat metabolic syndromes and many noninfectious diseases in vivo [4], so as to enhance human health. According to the previous reports, polyphenols could exhibit various biological activities including antioxidant [6,7], anti-inflammatory [8], and anticancer [9], as well as the prevention of cardiovascular and Parkinson's diseases [10].
Prinsepia utilis Royle is a deciduous shrub of the Rosaceae family. It is perennial and mainly distributed in high-altitude areas, such as some areas in China and India [11]. e P. utilis usually grow among bushes, along depressions and roadsides, and on both sides of mountain slopes or valleys. ey are cultivated as windproof and sandfixing plants that show strong adaptability to the environment. Residents in Yunnan, China, usually consume P. utilis as vegetables. Many parts of P. utilis (e.g., leaves, fruits, and roots) can be used as traditional Chinese medicine to treat various diseases, such as skin-related diseases, fracture, and rheumatism [12]. Previous studies have separated and identified the chemical constituents of P. utilis, which reported it contains hydroxybutyronitrile glucoside, diterpene glucoside, hydrocyanic acid, triterpenoids, etc [13]. However, there are few reports on the phenolic composition and biological activities of P. utilis flowers, leaves, and stems, which hinders the development and utilization of P. utilis. erefore, the objective of this study is to compare the phenolic composition and antioxidant and enzyme (e.g., glycosidase, pancreatic lipase, and tyrosinase) inhibitory properties of different parts of P. utilis, namely flowers, leaves, and stems.

Materials and
Treatment. P. utilis flowers, leaves, and stems were collected from Kunming, Yunnan Province, in April 2019. After picking, they were dried and ground with a grinder (30,000 rpm; Lingdan LD-T300; Shanghai, China). A total of 100 g powders of the flowers, stems, and leaves were defatted with petroleum ether at a material/solvent ratio of 1 : 5 (w/v) for five times and then extracted three times (30 min for each time) with 80% ethanol (v/v) in an ultrasonic bath. After extraction, the mixture was centrifuged at 25°C and 4000g for 15 min. e supernatant was collected and evaporated using a rotary evaporator (Hei-VAP, Heidolph, Germany) under vacuum at −40°C to remove the organic reagent and excess water. en, the concentrated solution was lyophilized to obtain the ethanol extracts of the flowers, stems, and leaves.

Determination of Total Phenol Content (TPC).
e total phenol content of the ethanol extracts of P. utilis flowers, leaves, and stems was determined by using the Folin-Ciocalteu method [12]. e absorbance was measured with a microplate reader (SpectraMax M5; Molecular Device, San Jose, CA, USA) at 765 nm, and gallic acid was used to establish the standard curve. e TPC of each sample was expressed as mg of GAE/g, where GAE was gallic acid equivalents.

Determination of Total Flavonoid Content (TFC).
e total flavonoid content of three ethanol extracts was determined according to the methods reported in the literature [12]. e absorbance value was measured with a SpectraMax M5 microplate reader at 510 nm, and rutin was used to establish the standard curve. e TFC of each sample was expressed as mg RE/g, where RE was rutin equivalents.

Identification and Quantification of Phenolics with UHPLC-ESI-HRMS/MS.
e ermo Fisher Ultimate 3000 UHPLC system ( ermo Fisher Scientific, Bremen, Germany) with an Agilent Zorbax SB-C18 column (2.1 × 100 mm, 1.7 μm) was utilized for the qualitative and quantitative analysis of phenolic substances in the ethanol extracts of flowers, leaves, and stems from P. utilis. In the current study, the identified phenolic compounds were quantified by the calibration curve of the corresponding authentic standard or by the calibration curve of the standard shared a similar aglycone. e injection volume was 3 μL, the flow rate was 0.2 mL/min, and the column temperature was 35°C. Acidified ultrapure water (0.1% formic acid, phase A) and acetonitrile (phase B) were used as the mobile phases. e gradient elution condition was 0-2 min, 5% B; 2-15 min, 50% B; 15-16 min, 70% B. e mass spectrometry conditions were set according to the study of Sun et al. [14].

DPPH Free Radical Scavenging Experiment.
e DPPH free radical scavenging ability of ethanol extracts was evaluated according to the methods of Sun et al. [14]. e absorbance of each sample was measured at 517 nm with a SpectraMax M5 microplate reader. e free radical scavenging capacity of DPPH is calculated by using equation (1).
DPPH free radical scavenge ratio(%) where A 1 represents the absorbance of sample solution and DPPH free radical, A 1 ′ represents the absorbance of sample solution and methanol, A 0 is the absorbance of methanol and DPPH free radical, and A 0 ′ is the absorbance of methanol and distilled water.

ABTS Free Radical Scavenging Experiment.
e ABTS free radical scavenging ability of ethanol extracts was evaluated by the methods of Sun et al. [14]. e absorbance of each group was measured at 734 nm with a SpectraMax M5 microplate reader. e following is the calculation formula of ABTS (equation (2)): ABTS free radical scavenge ratio (%) where A 1 represents the absorbance of sample solution and ABTS free radical, A 1 ′ represents the absorbance of sample solution and methanol, A 0 is the absorbance of methanol and ABTS free radical, and A 0 ′ is the absorbance of methanol and distilled water.

Ferric Reducing Antioxidant Power (FRAP) Method.
e FRAP values of three ethanol extracts were evaluated according to the reported method with some modifications [15]. Trolox FeSO 4 ·7H 2 O was used to prepare the calibration curve, ranging from 0.1 mmol/L to 0.5 mmol/L. is curve was used to calculate the antioxidant values of each sample.

Determination of α-Glucosidase Inhibitory Effect.
e α-glucosidase inhibitory activities of the ethanol extracts of P. utilis spikes, leaves, and stems were measured with the methods reported by Zhang et al. [16]. e absorbance of each reaction solution was measured at 405 nm with a SpectraMax M5 microplate reader. e following is the calculation formula for the α-glucosidase inhibition (%) (equation (3)):

Determination of Pancreatic Lipase Inhibitory
Effect. e pancreatic lipase inhibitory activities of the extracts of P. utilis flowers, leaves, and stems were measured according to the study of Cai et al. [17]. e absorbance of each reaction solution was measured at 405 nm with a SpectraMax M5 microplate reader. e following is the calculation formula for the pancreatic lipase inhibition (%) (equation (4)):

Determination of Tyrosinase Inhibitory
Effect. e tyrosinase inhibitory activity of ethanol extracts of P. utilis flowers, leaves, and stems were measured according to the previously reported method with some modifications [18]. e volume of the reaction system was reduced from 1 mL to 200 μL, and the added volume of each reagent was reduced in proportion.

Statistical Analysis.
All experiments were measured in triplicate, and the experimental data results were expressed as mean ± standard deviation (SD). One-way analysis of variance and Tukey's test were used to determine the significance of difference (p < 0.05). All analyses were performed using Origin 8.5 software (OriginLab, Northampton, MA, USA).

Determination of Total Phenolic and Total Flavonoid
Content. Table 1 shows the total phenolic and total flavonoid contents in the P. utilis flowers, leaves, and stems. Sánchez et al. [19] reported that the positive correlations between total phenol content and antioxidant capacity measured according to ABTS •+ , DPPH • , ORAC, and β-carotene-linoleate model system. It indicates that the total phenol content can reflect the strength of antioxidant activity of extract to a certain extent. In this study, the TPCs in the ethanol extracts of the flowers, leaves, and stems of P. utilis were not significantly different (p > 0.05), while the TPCs of dried flowers and leaves were significantly higher than that of dried stems (p < 0.05). e dried flowers of P. utilis had the highest TPC, which was 1.7 times of that of the stems.
As shown in Table 1, the TFC of P. utilis flower ethanol extract was significantly higher than that of leaves and stems (p < 0.05). e dried flowers, leaves, and stems of P. utilis have significant differences in TFC (p < 0.05), and the dried flowers had the highest TFC, followed by leaves. e TFC of the dried flowers was as about twice as that of the dried stems. According to previous reports [20], the ethyl acetate component extracted from tubers of Stachys affinis had good antioxidant activity, and the concentrations of phenols and flavonoids are positively correlated with the antioxidant activity.

Identification and Quantitation of Phenolic Compounds.
e phenolic components of the ethanol extracts of flowers, leaves, and stems of P. utilis were analyzed using UHPLC-ESI-HRMS/MS in a negative ion mode. e ion flow chromatogram is shown in Figure 1. In addition, the relevant data such as retention time, molecular mass, molecular formula, and secondary ion fragments are shown in Table 2.
e composition and content of polyphenols in the ethanol extracts of three samples were investigated by comparing the information, including secondary MS fragments with a large number of references or public mass spectrometry databases (MassBank, http://www.massbank.jp/QuickSearch.htmL). e extracts were subjected to preliminary qualitative and quantitative analysis. According to the qualitative data, appropriate standards were selected to perform further quantitative analysis on the corresponding substances. As known, the negative ion mode is usually used to detect phenolic acids and flavonoids [22]. In negative ion mode, a total of 11 compounds were analyzed including 1 phenolic acid, 10 flavonoids, and their derivatives ( Table 2 and Figure 1). e retention time of flavonoids was mainly between 7 and 15 min. In the flower extracts, the peak areas of compounds 6 and 10 were relatively high, indicating that the     Table 3. Among them, there were 8 compounds (1, 2, 4, 5, 6, 7, 10, and 12) that can be analyzed by comparison with the corresponding standards. e rest of the phenolic substances (8, 9, and 11) can use similar aglycone standards for their quantitation. As shown in Table 3, rutin was the dominant phenolic compound in all the extracts. However, the rutin content was different among three extracts, that the highest content was detected in flowers, followed by leaves and stems. It indicates that phenol contents and species in the ethanol extracts of P. utilis flowers, leaves, and stems were abundant, which may have good biological activity. Zhang et al. [16] studied the composition and biological activity of the total phenols, total flavonoids, and anthocyanin components in P. utilis fruits. A total of 20 phenolic compounds were identified, of which composition was similar to that of ethanol extracts of P. utilis flowers, leaves, and stems. Zhang et al. found that three main components (rutin, isorhamnetin-3-O-rutinoside, and cyanidin-3-O-glucoside) in P. utilis fruits have a good inhibitory effect on α-glycosidase and lipase. e ethanol extracts of the flowers, leaves, and stems of P. utilis also contain rutin and isorhamnetin-3-O-rutinoside.

In Vitro Antioxidant Activity.
Free radicals are inevitably produced in biological systems and taken into the body from the environment. ey are known to cause a variety of degenerative diseases, such as mutagenesis, carcinogenesis, cardiovascular disorders, and aging [23]. An antioxidant is a compound that inhibits free radicals by interfering with the three main steps of the free radical-mediated oxidation process, namely initiation, proliferation, and termination [24]. e balance between oxidants and antioxidants determined inside the human body is important to protect against the occurrence of many diseases [25].
Generally, plant polyphenols have been reported to show many important biological activities, which is believed to be partially or largely related to their antioxidant activity [15]. At present, some in vitro antioxidant methods (e.g. DPPH, ABTS, and FRAP) are usually used to measure the antioxidant capacity of a substance. e obtained results are used as a reference index for the antioxidant effect in vivo. In the work, DPPH, ABTS, and FRAP methods were used to evaluate the antioxidant activity of the ethanol extracts of P. utilis flowers, leaves, and stems.

DPPH Free Radical Scavenging Activity.
e DPPH free radical scavenging activity of ethanol extracts from different parts of P. utilis is shown in Figure 2(a). In the concentration range of 20-100 μg/mL, the DPPH free radical scavenging activity of all samples was concentration-dependent. e DPPH free radical scavenging activity of the flower extract of P. utilis was significantly stronger than that of the other two samples (p < 0.05). e IC 50 values of the ethanol extracts of P. utilis flowers, leaves, and stems were 46.07 ± 2.42, 82.08 ± 0.97, and 71.46 ± 2.85 μg/mL, respectively. e scavenging rate of DPPH free radicals of three samples is lower than that of Vc (positive control, IC 50 � 3.75 ± 0.02 μg/mL).
e flower extract has the strongest DPPH scavenging ability among the three components (p < 0.05), which may be attributed to its highest polyphenol content. Figure 2(b) shows the ABTS free radical scavenging ability of the ethanol extract of P. utilis flowers, leaves, and stems. e IC 50 values of the ethanol extracts of P. utilis flowers, leaves, and stems for scavenging ABTS free radicals were 22.07 ± 0.94, 27.14 ± 0.28, and 20.29 ± 0.57 μg/mL, respectively. e IC 50 values of the ethanol extracts of flowers, leaves, and stems of P. utilis for ABTS scavenging assay were 22.07 ± 0.94, 27.14 ± 0.28, and 20.29 ± 0.57 μg/mL, respectively. It shows that the ethanol extract of leaves had the best scavenging effect on ABTS free radicals among all ethanol extracts. e IC 50 value of Vc was 3.56 ± 0.07 μg/mL, and its ABTS scavenging effect is better than that of the P. utilis extracts. Overall, the ethanol extracts of flowers, leaves, and stems from P. utilis had strong free radical scavenging activity. ree extracts had different IC 50 values for their DPPH and ABTS free radical scavenging activities, which may be caused by the differences in free radical species, polyphenol compositions, and contents.

FRAP Evaluation.
FRAP is a relatively efficient and sensitive method to determine the antioxidant capacity of samples, which is widely used in food, chemical, and other fields. e antioxidant capacity of a sample evaluated using this method is expressed by the equivalent value of FeSO 4 . e concentration of FeSO 4 was in the range of 0.1∼0.5 mmol/L, and the concentration has a good linear relationship with the absorbance at 593 nm. Figure 2(c) shows the ferrous reduction capacity of the ethanol extracts of P. utilis. e concentration of three group samples filled in the range of 20-60 μg/mL. e flower extract had the highest FRAP value (p < 0.05), followed by the leaf extract and the stem extract. For example, the ferrous reduction activity of flower extract was significantly higher than that of leaf and stem extracts (p < 0.05) at 60 μg/mL.  All the values are expressed as mean ± SD (n � 3). Values with different superscript letters in each column indicate significant differences among samples. Compounds 1, 2, 4, 5, 6, 7, 10, and 12 were quantified by the calibration curve of the corresponding commercial standard; compounds 8 and 11 were quantified by the calibration curve of compound 5; and compound 9 was quantified by the calibration curve of compound 7. e result is expressed as μg/g dry matter. ND � not detected.
0.25 ± 0.003, and 0.19 ± 0.01 mmol/L, respectively. e ferrous reduction activity of flower extract was 3 times higher than that of stem extract and 2.4 times higher than that of leaf extract. In addition, it can be observed that the samples with high total phenol content have high FRAP values. In general, the antioxidant activity of the P. utilis flower ethanol extract was the highest among all three samples. It may be due to the fact that the total phenol and total flavonoid contents of flower extract was higher than other samples. Huang et al. [11], Zhang et al. [26], and Zhang et al. [16] reported that P. utilis fruits showed a higher antioxidant capacity than flowers, leaves, and stems. Although the total phenol and total flavonoid contents in leaves are higher than those in stems, the ethanol stem extract had a stronger free radical scavenging ability than that of leaf extract. It was found that the content of isoschaftoside in the ethanol extract of stem was higher than that of leaf, but the other components of the ethanol extract of stems were lower than that of the leaf extract. Compared with the leaf extract, the better scavenging effect of stem extract against DPPH and ABTS free radicals may attribute to its higher content of isoschaftoside. A previous study reported a similar finding that the DPPH scavenging activity of ethanol extract from Okinawa Taumu (Colocasia esculenta S.) was higher than that of the water extract [27]. In addition to phenolic compounds, some other components also exhibited a good antioxidant activity, such as peptides [28]. erefore, the total antioxidant activity of a sample was contributed from a combination of several components. Figure 3(a) shows the inhibitory effect of ethanol extracts of P. utilis flowers, leaves, and stems on α-glucosidase. e α-glucosidase inhibitory effects of three extracts were concentration-dependent. As shown in Figure 3(a), the IC 50 values for the α-glucosidase inhibitory activity of ethanol extracts from P. utilis spike flowers, leaves, and stems were 35.71 ± 0.58, 231.12 ± 6.59, and 37.29 ± 1.59 μg/mL, respectively. e IC 50 value of the positive control acarbose was 0.16 ± 0.04 μg/mL, which was significantly lower than that of three extracts (p < 0.05). e flower extracts had the highest inhibitory effect on α-glucosidase, followed by stem and leaf extracts, which may be due to the different polyphenol composition. Several studies [29,30] reported that plants rich in polyphenols have good inhibitory activity on α-glucosidase. It suggested that phenolic compounds are considered to be the main compounds that contribute to the α-glucosidase inhibitory activity. Zhang et al. [16] have reported that the P. utilis fruits were rich in rutin and isorhamnetin-3-O-rutinoside, and those phenolic compounds exhibited a good inhibitory activity towards α-glucosidase.

α-Glucosidase Inhibitory Activity.
3.5. Pancreatic Lipase Inhibitory Activity. Figure 3(b) shows the pancreatic lipase inhibitory activity of P. utilis flower, leaf, and stem ethanol extracts. As shown in Figure 3(b), each extract had a good inhibitory effect on pancreatic lipase. As the concentration increased, the pancreatic lipase inhibition rates of three extracts gradually increased. e IC 50 values for pancreatic lipase inhibitory activity of the ethanol extracts from P. utilis flowers, leaves, and stems were 1.72 ± 0.03, 1.82 ± 0.15, and 2.56 ± 0.02 mg/mL, respectively. It can be seen that the flower extract had the highest inhibitory activity on pancreatic lipase, followed by the leaf and stem extracts. However, orlistat, as a positive control, inhibited almost 50% of lipase activity at 40.00 μg/mL in the current work, indicating that orlistat may possess much stronger inhibitory effect against lipase than the three extracts. Considering the total phenol content of three samples, it was found that the sample with higher polyphenol content had better inhibitory activity on pancreatic lipase. Many studies have shown that plant food extracts rich in phenols generally have good inhibitory activity against lipases [31]. Dorota et al. reported the inhibitory effects of the crude extract of black chokeberry fruit and its ethyl acetate fraction and polyphenol-rich fraction on pancreatic lipase activity [31]. e results showed that the polyphenol-rich fraction exhibited higher antilipase activity than other components. In general, the composition and content of phenolics in the extract may play an important role in pancreatic lipase inhibitory activity.
3.6. Tyrosinase Inhibitory Activity. Figure 3(c) shows the inhibition of tyrosinase activity of ethanol extracts of P. utilis flowers, leaves, and stems. As the concentration increased, the inhibitory effect of each extract on tyrosinase gradually increased. e IC 50 values of the P. utilis flower, leaf, and stem extracts for tyrosinase inhibitory activity were 4.11 ± 0.11, 4.33 ± 0.04, and 4.58 ± 0.11 mg/mL, respectively, which were higher than that of the positive control kojic acid (IC 50 � 0.39 ± 0.02 μg/mL) (p < 0.05). It can be seen that the tyrosinase inhibitory activity of the P. utilis flower extract is the highest, followed by the leaf extract, and the stem extract had the lowest tyrosinase inhibitory activity. In addition, the inhibitory activity of three samples on tyrosinase was related to the total phenol and total flavonoid content, by comparing their corresponding total phenol and total flavonoid content. Our results showed that the ethanol extracts of flowers, leaves, and stems contained p-coumaric acid, which has been reported to have a good inhibitory effect on mushroom polyphenol oxidase, human tyrosinase, and cell melanin [32]. erefore, the utilization of flower extracts with high content of coumaric acid may be helpful for fruit browning prevention and human skin whitening.

Conclusions
is study investigated the total phenols, total flavonoids, and polyphenol composition of ethanol extracts from flowers, leaves, and stems of P. utilis. eir antioxidant capacity and inhibitory activities against α-glycosidase, lipase, and tyrosinase were also determined in vitro. e total phenols and total flavonoids were detected with the highest contents in flowers followed by leaves and stems. Among polyphenol compounds, rutin and isorhamnetin-3-O-rutinoside were the dominant compounds in flower and leaf ethanol extracts. As for the stem ethanol extract, rutin and isoschaftoside were the most abundant substances. Moreover, the antioxidant capacity and enzyme inhibitory capacity were detected with the highest value in the flower ethanol extract compared with others. e outcome of the work could provide support to the development and utilization of P. utilis in food and health products.

Data Availability
All data included in this study are available upon request by contacting the corresponding author.

Conflicts of Interest
ere are no conflicts of interest regarding the publication of this article.