UPLC-MS/MS Profiling, Antioxidant, α-Glucosidase Inhibitory, Cholinesterase Inhibitory, and Cardiovascular Protection Potentials of Jialing 20 (Morus multicaulis Perr.) Mulberry Branch Extract

As a by-product in the sericulture industry, mulberry branches are not currently utilized effectively. Jialing 20 is an artificial triploids mulberry that widely cultivated in southwest China. In this study, the chemical composition of the Jialing 20 mulberry branch extract (MBE) was first analyzed by UPLC-MS/MS, and 42 components, including alkaloids, flavonoids, and coumarins, were obtained. Then, the antioxidant activities, hypoglycemic effect, Alzheimer’s disease inhibition, and cardiovascular protection of MBE were also evaluated in vitro. The IC50 values for the scavenging DPPH and ABTS radicals were, respectively, 31.23 ± 0.57 μg/mL and 8.88 ± 0.36 μg/mL (IC50 values of positive Vc were, respectively, 4.41 ± 0.19 μg/mL and 8.79 ± 0.41 μg/mL). The IC50 value for inhibiting α-glucosidase was 1.90 ± 0.05 μg/mL (IC50 value of positive acarbose was 0.03 μg/mL). The IC50 values for inhibiting acetylcholinesterase and butyrylcholinesterase were, respectively, 179.47 ± 0.38 μg/mL and 101.82 ± 3.37 μg/mL (IC50 values of positive berberine were, respectively, 1.27 ± 0.03 μg/mL and 57.41 ± 0.21 μg/mL). MBE (10 μg/mL and 40 μg/mL) significantly increased the survival rate of oxidized low-density lipoprotein- (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) and significantly decreased the intracellular reactive oxygen species. These results suggest that the extracts of Jialing 20 mulberry branches could be used as a functional food additive.


Introduction
Mulberry is a common cash crop in Asia [1]. Mulberry fruits are usually consumed as fresh fruit and the raw material for the production of delicious juices and jams. Mulberry leaves are used to feed domestic silkworms or are made into mulberry tea. However, mulberry branches, which need to be cut regularly eVery year, are underutilized [2]. Mulberry branches, also known as Ramulus mori (RM), are included in the Chinese Pharmacopoeia as a herbal beverage, and mulberry branch extract (MBE) has been approved as a food source by the Chinese and Korean governments [3,4], indicating that MBE is safe for consumption. Mulberry branches are rich in active components such as flavonoids [5], alkaloids [1], and polysaccharides [6], which have various activities such as antioxidation [7] and hypoglycemic effects [8]. However, the chemical composition and biological activities of MBE of different varieties of mulberry trees vary greatly [7,9,10]. Jialing 20 is an artificially bred triploid mulberry variety and is widely cultivated in southwest China because its leaf and branch yields are significantly better than those of other varieties [11]. In our previous study, 10 components, including mulberroside A, were isolated and identified from MBE of Jialing 20, and the content of mulberroside A in Jialing 20 was found to be significantly

Mass Spectrometry Conditions
A TripleTOFTM 5600+ MS system (AB Sciex, Framingham, MA, USA) with an electrospray ion source and an Analyst TF 1.7.1 workstation (AB Sciex, Framingham, MA, USA) was used. The primary and secondary mass spectrometry data acquisitions were performed by using TOF-MS-Product Ion-IDA with a scan range of 50 to 1500 m/z. The mass spectrometry parameters were set as follows: declustering voltage of 100 eV or −100 eV and focusing voltage of 10 eV or −10 eV; secondary bombardment energy of 40 eV or −40 eV; collision energy spread (CES) of 20 V; and spray voltage of 5500 V (positive ion mode) or −4000 V (negative ion mode).

Mass Spectrometry Data Processing
The data collected by mass spectrometry were first imported into Progenesis QI software (Waters, USA, Milford, MA, USA). Then, corresponding charge forms were selected for peak matching, alignment, and extraction. Then, the obtained results were matched with the secondary mass spectrometry database for screening and identification based on the literature.

Antioxidant Activity 2.4.1. Scavenging of DPPH Radicals
The scavenging capacity of DPPH radicals was determined according to the previous method [30], with minor modifications. Then, 100 µL of 200 µM DPPH solution was dissolved in ethanol, which was then mixed with the same volume of different concentrations of MBE in a 96-well microtiter plate. After the reaction proceeded at room temperature in the dark for 30 min, the absorbance of the mixture was measured at 520 nm three times using an imark microplate reader (Bio-Rad, Hercules, CA, USA), and water-soluble vitamin C was used as a positive control. The DPPH radical scavenging rate was calculated as: where D is DPPH radical scavenging rate (%); A0 is the absorbance of the negative control; and A1 is the absorbance of the sample. The semi-inhibitory concentration (IC 50 ) of DPPH radicals is calculated according to the derivation of the logarithm of the substrate concentration with respect to inhibition rate.

Scavenging of ABTS Radicals
The scavenging capacity of ABTS radicals was determined according to the previous method [31], with minor modifications. First, 7 mM ABTS was mixed with 2.45 mM potassium persulfate and was incubated in the dark for 12 h. Then, the mixture was diluted to a concentration with an absorbance of 0.700 ± 0.05 at 734 nm. Then, 20 µL of the sample was mixed with 180 µL of the radical solution for a 6 min reaction in the dark. Finally, the absorbances were measured at 734 nm three times using an imark microplate reader (Bio-Rad, Hercules, CA, USA), while vitamin C was used as a positive control. The ABTS radical scavenging rate was calculated as: where A is the ABTS radical scavenging rate (%); A0 is the absorbance of the negative control; and A1 is the absorbance of the sample.

Inhibition of α-Glucosidase Activity
The inhibition of α-glucosidase activity was determined according to the previous method [32]. An amount of 50 µL of each sample was to be tested, and 50 µL of αglucosidase (0.5 U/mL) and 50 µL of phosphate buffer (67 mM, pH 6.8) were mixed thoroughly and were kept at 37 • C for 15 min. Then, 50 µL of 6 mM pNPG was added, and the reaction proceeded at 37 • C for 15 min. Then, the reaction was terminated by adding 60 µL of 1 M Na2CO3. Three replicates were set for each group. Then, 200 µL of the solution was added in a 96-well plate, and the absorbance value was measured at 405 nm with an imark microplate reader (Bio-Rad, Hercules, CA, USA) in triplicate (n = 3). The inhibition rate was calculated as: where A1 is the absorbance value of each sample; A2 is the absorbance value of each sample without the enzyme; and A0 is the absorbance value of the group without sample.

Inhibition of Cholinesterase Activity
The inhibition of cholinesterase activity was determined according to the previous method [33], with minor modifications. First, 40 µL of PBS (pH 8.0, 100 mM), 20 µL of DTNB (2 mM), 10 µL of AChE (0.75 U/mL), and 10 µL of samples of different concentrations were mixed followed by the rapid addition of 20 µL of ATC (2 mM) to initiate the reaction. After 2 min, the reaction solution was analyzed with an imark microplate reader (Bio-Rad, Hercules, CA, USA) at 405 nm. The inhibition reaction was determined three times, and the inhibition rate was calculated as: where A1 is the absorbance value of each sample; A2 is the absorbance value of each sample without enzyme; and A0 is the absorbance value of the group without sample.

Cytotoxicity
HUVECs in logarithmic growth phase were inoculated overnight in 96-well plates at a concentration of 5 × 10 3 cells/well and were then treated with different concentrations of samples for 24 h. Six replicates were arranged for each group. Then, 10 µL of CCK-8 solution (CellorLab, Hangzhou, China) was added to each well and was incubated for 1 h in an incubator. The absorbances were measured at 450 nm with an enzyme-labeled instrument to calculate cell viability (n = 6).

Cell Vitality
Cell vitality was determined according to the previous method [21], with minor modifications. First, the HUVECs were inoculated overnight in 96-well plates at a concentration of 5 × 10 3 cells/well and were then treated with different concentrations of samples under the exposure condition of 80 µg/mL ox-LDL (Yiyuanbiotech, Guangzhou, China) for 24 h. Then, 10 µL of CCK-8 solution was added to each well and was incubated for 1 h in an incubator. Six replicates were arranged for each group. The absorbances were measured at 450 nm with an enzyme-labeled instrument to calculate cell viability (n = 6).

Determination of Intracellular Reactive Oxygen Species (ROS)
Intracellular ROS was determined with Dihydroethidium (DHE). HUVECs in logarithmic growth phase were first inoculated for 12 h in 6-well plates at a concentration of 1 × 10 5 cells/well and were then treated for 24 h according to the method in Section 2.7.3. After 10 µM DHE (Beyotime Biotechnology, Shanghai, China) was added, the reaction proceeded for 60 min at 37 • C. Then, the treated HUVECs were rinsed with a blank medium 3 times and were observed with an inverted fluorescence microscope IX73 (Olympus Corporation, Tokyo, Japan) under an excitation wavelength of 518 nm and an emission wavelength of 610 nm.

UPLC-MS/MS Fingerprints
The UPLC-MS fingerprint of MBE is shown in Figure 1. In addition to some interference signals from the fatty acids, more signals of weakly polar components in positive ion mode were acquired. More signals of moderately polar components were obtained in negative ion mode. Through signal processing, the mass spectra of 14,284 positive ions and 14,130 negative ions were obtained. The signal extraction and analysis results indicated that the main components of MBE were flavonoids and polyphenols. These components could be subdivided into stilbene, coumarin, phenylpropanoid compounds, alkaloids, and organic acids. Due to the large interference in the signals of the positive ions, the data obtained in negative ion mode are generally used in the LC-MS analysis of natural products. Therefore, in this study, besides the signals of positive ions, the signals of negative ions were mainly taken into account for data analysis. The resolved compounds are shown in Table 1 In total, 42 obvious compound signals were obtained.    Among the compounds identified by LC-MS/MS, four components, including scopolin, reported in our previous study were also found in this study. Oxyresveratrol (Oxr) is commonly found in mulberry branches, including those from Jialing 20 [15,37]. In summary, 37 new components in MBE of Jialing 20 were found, thus enriching knowledge about the chemical composition of the Jialing 20 mulberry branch.
A preliminary comparison of the mass spectral response intensity indicated that the components with higher contents in MBE might be oxyresveratrol and isoprenoid flavonoids (Kuwanon G). These compounds have also been reported to possess antibacterial [38,39] and tyrosinase inhibitory activities [40,41], suggesting that MBE might be used as antibacterial agents and whitening products.
The application of LC-MS/MS technology when studying mulberry mainly focuses on the composition analysis of mulberry leaves [42,43] and mulberry fruits [44,45], but it is seldom applied in the analysis of the main components in mulberry branches. The compounds identified by UPLC-MS/MS of MBE were quite different from the results of mulberry leaves and mulberry in references [42][43][44][45]. The rapid identification of the main components of MBE by LC-MS/MS can improve our understanding of its chemical composition and can provide theoretical guidance for the development and utilization of its chemical components.

Antioxidant Activity
Free radicals are closely related to the progression of diabetes, neurodegenerative diseases, and atherosclerosis. The scavenging assay of DPPH and ABTS free radicals is a common method to eValuate the free radical scavenging activity in vitro. Figure 2 shows the ability of MBE to scavenge the free radicals DPPH and ABTS. MBE exhibited concentration-dependent DPPH free radical scavenging activity in the concentration range of 0 to 200 µg/mL. The IC 50 value of MBE was 31.23 ± 0.57 µg/mL, and the IC 50 value of positive Vc was 4.41 ± 0.19 µg/mL. In the concentration range of 0 to 50 µg/mL, the ability of MBE to scavenge ABTS free radicals was positively correlated with MBE concentration. The IC 50 value of MBE was 8.88 ± 0.36 µg/mL, and the IC 50 value of positive Vc was 8.79 ± 0.41 µg/mL. The ABTS scavenging ability of the sample was close to that of positive Vc, but the DPPH scavenging ability of the sample was different from that of positive Vc. The DPPH radical scavenging assay displayed the ability of the sample to transfer electrons or hydrogen atoms, whereas the ABTS radical scavenging assay demonstrated the hydrogen supply ability and chain-breaking ability of the sample [46]. The above results indicated that compared to Vc, MBE was more dominated by the hydrogen supply ability and chain-breaking ability rather than the ability to transfer electrons or hydrogen atoms. A preliminary comparison of the mass spectral response intensity indicated that the components with higher contents in MBE might be oxyresveratrol and isoprenoid flavonoids (Kuwanon G). These compounds have also been reported to possess antibacterial [38,39] and tyrosinase inhibitory activities [40,41], suggesting that MBE might be used as antibacterial agents and whitening products.
The application of LC-MS/MS technology when studying mulberry mainly focuses on the composition analysis of mulberry leaves [42,43] and mulberry fruits [44,45], but it is seldom applied in the analysis of the main components in mulberry branches. The compounds identified by UPLC-MS/MS of MBE were quite different from the results of mulberry leaves and mulberry in references 42-45. The rapid identification of the main components of MBE by LC-MS/MS can improve our understanding of its chemical composition and can provide theoretical guidance for the development and utilization of its chemical components.

Antioxidant Activity
Free radicals are closely related to the progression of diabetes, neurodegenerative diseases, and atherosclerosis. The scavenging assay of DPPH and ABTS free radicals is a common method to evaluate the free radical scavenging activity in vitro. Figure 2 shows the ability of MBE to scavenge the free radicals DPPH and ABTS. MBE exhibited concentration-dependent DPPH free radical scavenging activity in the concentration range of 0 to 200 μg/mL. The IC50 value of MBE was 31.23 ± 0.57 μg/mL, and the IC50 value of positive Vc was 4.41 ± 0.19 μg/mL. In the concentration range of 0 to 50 μg/mL, the ability of MBE to scavenge ABTS free radicals was positively correlated with MBE concentration. The IC50 value of MBE was 8.88 ± 0.36 μg/mL, and the IC50 value of positive Vc was 8.79 ± 0.41 μg/mL. The ABTS scavenging ability of the sample was close to that of positive Vc, but the DPPH scavenging ability of the sample was different from that of positive Vc. The DPPH radical scavenging assay displayed the ability of the sample to transfer electrons or hydrogen atoms, whereas the ABTS radical scavenging assay demonstrated the hydrogen supply ability and chain-breaking ability of the sample [46]. The above results indicated that compared to Vc, MBE was more dominated by the hydrogen supply ability and chainbreaking ability rather than the ability to transfer electrons or hydrogen atoms. The DPPH radical scavenging activity of the extracts from the same parts of different species of mulberry (Morus alba L. and Morus nigra L.) differed significantly and was positively correlated with the total content of polyphenols and flavonoids in the extracts, indicating that phenolics might be the main antioxidant components in different Morus extracts [9]. The ethanolic extracts of mulberry branches showed significant antioxidant ac- The DPPH radical scavenging activity of the extracts from the same parts of different species of mulberry (Morus alba L. and Morus nigra L.) differed significantly and was positively correlated with the total content of polyphenols and flavonoids in the extracts, indicating that phenolics might be the main antioxidant components in different Morus extracts [9]. The ethanolic extracts of mulberry branches showed significant antioxidant activities, indicating that oxyresveratrol (Oxr) might be the most effective antioxidant component [47]. Oxr showed a good ability to scavenge DPPH and ABTS radicals. The possible high content of Oxr in MBE of Jialing 20 might be responsible for its better antioxidant activity.

Inhibition of α-Glucosidase Activity
As drugs for the effective control of T2DM, α-Glucosidase inhibitors have relatively few side effects because they do not need to be absorbed into the blood and can function directly in the intestine. These α-glucosidase inhibitor drugs are currently commercially available, such as acarbose and voglibose. We measured the activity of MBE in inhibiting αglucosidase with acarbose as a positive control ( Figure 3A). When the sample concentration increased from 0 to 8 µg/mL, the inhibition activity of MBE on α-glucosidase increased significantly. When the sample concentration further increased from 8 to 16 µg/mL, its inhibition ability increased slowly. When the sample concentration further increased above 16 µg/mL, its inhibition ability mostly remained stable. Its half-inhibitory concentration was 1.90 ± 0.05 µg/mL (the half-inhibitory concentration of acarbose was 0.03 µg/mL).
Foods 2021, 10, x FOR PEER REVIEW 9 of 14 tivities, indicating that oxyresveratrol (Oxr) might be the most effective antioxidant component [47]. Oxr showed a good ability to scavenge DPPH and ABTS radicals. The possible high content of Oxr in MBE of Jialing 20 might be responsible for its better antioxidant activity.

Inhibition of α-Glucosidase Activity
As drugs for the effective control of T2DM, α-Glucosidase inhibitors have relatively few side effects because they do not need to be absorbed into the blood and can function directly in the intestine. These α-glucosidase inhibitor drugs are currently commercially available, such as acarbose and voglibose. We measured the activity of MBE in inhibiting α-glucosidase with acarbose as a positive control ( Figure 3A). When the sample concentration increased from 0 to 8 μg/mL, the inhibition activity of MBE on α-glucosidase increased significantly. When the sample concentration further increased from 8 to 16 μg/mL, its inhibition ability increased slowly. When the sample concentration further increased above 16 μg/mL, its inhibition ability mostly remained stable. Its half-inhibitory concentration was 1.90 ± 0.05 μg/mL (the half-inhibitory concentration of acarbose was 0.03 μg/mL). In the previous study on α-glucosidase inhibitory components in mulberry (Morus alba L.) with molecular docking, three types of potential small-molecule α-glucosidase inhibitory components were identified: alkaloids (1-deoxynojirimycin, DNJ), flavonols (kaempferol, quercetin and rutin), and isoprenoid flavonoids (morin, sanggenon C and kuwanon G) [48]. The LC-MS/MS results showed that DNJ and Kuwanon G in the MBE of Jialing 20 might be the material basis for its inhibition effect on α-glucosidase activity.
Since DNJ is highly polar and is difficult to retain in the C18 column, the LC-MS/MS analysis failed to separate it from other compounds. In addition, it has no UV absorption. This present study determined the DNJ content to be 0.65% in MBE with the reported FMOC-CL method [32]. The IC50 value of DNJ was 1.15 ± 0.13 μg/mL, which was similar to that of MBE. However, the content of DNJ was low, indicating that other α-glucosidase inhibitory components with stronger activity than DNJ existed in MBE. The result was consistent with previous results [32].

Inhibition of Cholinesterase Activities
Acetylcholinesterase and butyrylcholinesterase inhibitors can slow down the hydrolysis of acetylcholine and can increase the acetylcholine (Ach) level in the synaptic cleft, thus delaying the development of symptoms in Alzheimer's patients [49]. Cholinesterase inhibitors such as donepezil and galantamine have been successfully marketed as clinical agents to alleviate the symptoms of Alzheimer's disease. Berberine, a natural product with cholinesterase inhibitory activity, is often used as a positive control in the determination of cholinesterase inhibitory activity of natural products [50][51][52]. In the cholinesterase inhibition assay of MBE ( Figure 3B,C), both MBE and berberine showed the concentrationdependent inhibition activity of AChE and BChE, but the inhibition activity of berberine against AChE was significantly better than that against BChE, as reported in previous In the previous study on α-glucosidase inhibitory components in mulberry (Morus alba L.) with molecular docking, three types of potential small-molecule α-glucosidase inhibitory components were identified: alkaloids (1-deoxynojirimycin, DNJ), flavonols (kaempferol, quercetin and rutin), and isoprenoid flavonoids (morin, sanggenon C and kuwanon G) [48]. The LC-MS/MS results showed that DNJ and Kuwanon G in the MBE of Jialing 20 might be the material basis for its inhibition effect on α-glucosidase activity.
Since DNJ is highly polar and is difficult to retain in the C18 column, the LC-MS/MS analysis failed to separate it from other compounds. In addition, it has no UV absorption. This present study determined the DNJ content to be 0.65% in MBE with the reported FMOC-CL method [32]. The IC 50 value of DNJ was 1.15 ± 0.13 µg/mL, which was similar to that of MBE. However, the content of DNJ was low, indicating that other α-glucosidase inhibitory components with stronger activity than DNJ existed in MBE. The result was consistent with previous results [32].

Inhibition of Cholinesterase Activities
Acetylcholinesterase and butyrylcholinesterase inhibitors can slow down the hydrolysis of acetylcholine and can increase the acetylcholine (Ach) level in the synaptic cleft, thus delaying the development of symptoms in Alzheimer's patients [49]. Cholinesterase inhibitors such as donepezil and galantamine have been successfully marketed as clinical agents to alleviate the symptoms of Alzheimer's disease. Berberine, a natural product with cholinesterase inhibitory activity, is often used as a positive control in the determination of cholinesterase inhibitory activity of natural products [50][51][52]. In the cholinesterase inhibition assay of MBE ( Figure 3B,C), both MBE and berberine showed the concentrationdependent inhibition activity of AChE and BChE, but the inhibition activity of berberine against AChE was significantly better than that against BChE, as reported in previous studies [51,53]. The inhibitory activity of MBE against BChE was significantly stronger than that against AchE. The IC 50 of MBE was 101.82 ± 3.37 µg/mL (IC 50 of positive berberine was 57.41 ± 0.21 µg/mL), whereas the IC 50 of MBE for AChE was 179.47 ± 0.38 µg/mL (IC 50 of positive berberine was 1.27 ± 0.03 µg/mL).
In the only study on the inhibition of the cholinesterase activity of MBE from Morus alba L., the IC 50 values of MBE for AChE and BChE were, respectively, 46.78 ± 6.45 and 50.24 ± 3.01 µg/mL. The IC 50 values of positive berberine for AChE and BChE were, respectively, 0.12 ± 0.00 and 9.45 ± 0.12 µg/mL [53]. The large difference in the IC 50 values between the study and the previous report could possibly be ascribed to the differences in the reaction systems (e.g., final enzyme or substrate concentrations). However, the inhibition activities of berberine against AChE and BchE showed the similar trends. When the inhibition activities were calculated in terms of berberine equivalents, the MBE from Jialing 20 showed the significantly stronger BChE inhibitory activity than the MBE from Morus alba as well as better selectivity for BChE than AChE. Therefore, MBE might contain components with better BChE inhibitory activity.
The cholinesterase inhibitory components in mulberry branches have not been reported upon so far. Kuwanon G from mulberry roots was found to have better cholinesterase inhibitory activity, and its inhibitory activity against BChE was stronger than that against AchE [53]. UPLC-MS/MS analysis in this study showed that Kuwanon G was also contained in the MBE from Jialing 20 and might contribute to the inhibitory activity of MBE against BChE. In addition, the oxyresveratrol in MBE showed high inhibitory activity against BChE, with an IC 50 value of 2.95 µM, but its inhibitory activity against AchE was low (IC 50 > 400 µM) [54]. It was speculated that the inhibition activity of MBE against BchE might be ascribed to multiple components. Corresponding active ingredients and mechanism need to be further investigated.

Protection of MBE against ox-LDL-Induced HUVECs Damage
Atherosclerosis is a common cardiovascular disease, and its development process is closely related to the endothelial cell damage mechanism. ox-LDL can cause vascular endothelial cell damage and dysfunction through various pathways and can promote the development of atherosclerosis. The cytotoxicity results ( Figure 4A) showed that MBE in the range of 10 to 40 µg/mL was not significantly toxic to HUVECs. Therefore, the highest concentration of MBE was subsequently selected to be 40 µg/mL in subsequent assays.
In the MBE protection assay against ox-LDL-induced HUVECs damage ( Figure 4B), the activity of HUVECs exposed to ox-LDL was inhibited compared to those in the blank group. The survival rate of HUVECs exposed to 40 µg/mL MBE and ox-LDL was significantly higher than that of HUVECs exposed to ox-LDL (p < 0.01). eVen 10 µg/mL MBE significantly increased the survival rate of the HUVECs (p < 0.05), indicating that MBE had a certain protective effect on ox-LDL-induced HUVECs damage. In other words, MBE has potential cardiovascular protective activity.
Reactive oxygen species can cause vascular endothelial cell dysfunction and vascular wall cell damage, which, in turn, develops into atherosclerosis. Removing excess ROS from the body may be one of the key points for the prevention and treatment of atherosclerosis [55]. DHE is a commonly used fluorescent detection probe for ROS since it can freely enter cells and can be dehydrogenated under the action of intracellular superoxide anions to form ethidium bromide, which can bind to DNA to produce red fluorescence under the excitation of ultraviolet rays. Within a certain range, the intracellular red fluorescence intensity indicated the concentration of superoxide anions. The red fluorescence intensity of HUVECs exposed to ox-LDL was significantly higher than that in the normal group ( Figure 4D), suggesting the significantly elevated ROS level in HUVECs. In contrast, the intracellular ROS levels were significantly reduced after the HUVECs were treated with 10 µg/mL or 40 µg/mL MBE. In the processed results from Image J ( Figure 4C), MBE effectively reduced the level of ROS in HUVECs under ox-LDL treatment and displayed potential preventive or therapeutic activity against atherosclerosis.
The activity of mulberry branch and its main components to protect ox-LDL-induced HUVECs damage has not been reported upon, but Kuwanon G was reported to reduce atherosclerosis by inhibiting the formation of foam cells and inflammatory response [56]. Kuwanon G might be the active component with protection against ox-LDL-induced HUVECs damage in MBE, but the active components in MBE and the corresponding mechanism remain to be further explored.  In the MBE protection assay against ox-LDL-induced HUVECs damage ( Figure 4B), the activity of HUVECs exposed to ox-LDL was inhibited compared to those in the blank group. The survival rate of HUVECs exposed to 40 μg/mL MBE and ox-LDL was significantly higher than that of HUVECs exposed to ox-LDL (p < 0.01). Even 10 μg/mL MBE significantly increased the survival rate of the HUVECs (p < 0.05), indicating that MBE had a certain protective effect on ox-LDL-induced HUVECs damage. In other words, MBE has potential cardiovascular protective activity.
Reactive oxygen species can cause vascular endothelial cell dysfunction and vascular wall cell damage, which, in turn, develops into atherosclerosis. Removing excess ROS from the body may be one of the key points for the prevention and treatment of atherosclerosis [55]. DHE is a commonly used fluorescent detection probe for ROS since it can freely enter cells and can be dehydrogenated under the action of intracellular superoxide anions to form ethidium bromide, which can bind to DNA to produce red fluorescence under the excitation of ultraviolet rays. Within a certain range, the intracellular red fluorescence intensity indicated the concentration of superoxide anions. The red fluorescence intensity of HUVECs exposed to ox-LDL was significantly higher than that in the normal group ( Figure 4D), suggesting the significantly elevated ROS level in HUVECs. In contrast, the intracellular ROS levels were significantly reduced after the HUVECs were treated with 10 μg/mL or 40 μg/mL MBE. In the processed results from Image J ( Figure  4C), MBE effectively reduced the level of ROS in HUVECs under ox-LDL treatment and displayed potential preventive or therapeutic activity against atherosclerosis.
The activity of mulberry branch and its main components to protect ox-LDL-induced HUVECs damage has not been reported upon, but Kuwanon G was reported to reduce atherosclerosis by inhibiting the formation of foam cells and inflammatory response [56]. Kuwanon G might be the active component with protection against ox-LDL-induced

Conclusions
In this study, the chemical components of the MBE from Jialing 20 were analyzed and identified by UPLC-MS/MS for the first time. Then, the in vitro activities of antioxidation, α-glucosidase inhibition, cholinesterase inhibition, and protection against ox-LDL-induced HUVECs damage were determined. The MBE from Jialing 20 contained various active components such as flavonoids, polyphenols, and alkaloids, and displayed good antioxidant activity and promising applications in terms of hypoglycemic effect, Alzheimer's disease inhibition, and cardiovascular protection. The study indicated that the MBE from Jialing 20 has potential as a food additive for the prevention of age-related diseases.
Author Contributions: W.X.: conceptualization, data curation, formal analysis, investigation, methodology, resources, software, visualization, writing-original draft, writing-review and editing; Z.X.: project administration, software, supervision, validation, visualization; L.X.: conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization. All authors have read and agreed to the published version of the manuscript.