New Lignanamides with Antioxidant and Anti-Inflammatory Activities Screened Out and Identified from Warburgia ugandensis Combining Affinity Ultrafiltration LC-MS with SOD and XOD Enzymes

Warburgia ugandensis, also known as “green heart,” is widely used for the treatment of various diseases as a traditional ethnomedicinal plant in local communities in Africa. In this work, 9 and 12 potential superoxide dismutase (SOD) and xanthine oxidase (XOD) ligands from W. ugandensis were quickly screened out by combining SOD and XOD affinity ultrafiltration with LC-MS, respectively. In this way, four new lignanamides (compounds 11–14) and one new macrocyclic glycoside (compound 5), along with three known compounds (compounds 1, 3, and 7), were isolated and identified firstly in this species. The structures of the new compounds were elucidated by spectroscopic analysis, including NMR and UPLC-QTOF-MS/MS. Among these compounds, compound 14 showed the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) radical scavenging activities, and total ferric-reducing antioxidant power (FRAP) with IC50 values of 6.405 ± 0.362 µM, 5.381 ± 0.092 µM, and 17.488 ± 1.625 mmol TE/g, respectively. Moreover, compound 14 displayed the highest inhibitory activity on cyclooxygenase-2 (COX-2) with IC50 value of 0.123 ± 0.004 µM, and the ranking order of other compounds’ IC50 values was 13 > 11 > 7 > 1 > 12. The present study suggested that lignanamides might represent interesting new characteristic functional components of W. ugandensis to exert remarkable antioxidant and anti-inflammatory activities. Moreover, compound 14, a new arylnaphthalene lignanamide, would be a highly potential natural antioxidant and anti-inflammatory agent from W. ugandensis.


Plant Materials
The stem barks of W. ugandensis were collected in June 2018 from Narok County (latitude: −1.2408 • , longitude: 35.7356 • , altitude: 1851 m), Kenya, and authenticated by Prof. Guangwan Hu, a taxonomist of Wuhan Botanical Garden, Chinese Academy of Sciences. A voucher specimen (No. WBG-ZWHX 201808001) was deposited in the herbarium of the Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture.

Preparation of Samples 2.4.1. Preparation of Extractions and Fractions
Air-dried powders of stem barks from W. ugandensis (4.8 kg) were extracted three times with 95% ethanol (30 L × 3) at room temperature for 6 days, and then concentrated to obtain crude extracts (WUZ, 391.4 g). The crude extracts were dispersed in ultrapure water (1 L) to gain a homogeneous mixture, which was successively partitioned three times with equal volumes of petroleum ether, ethyl acetate, and n-butanol to get four fractions: petroleum ether fraction (WUP), ethyl acetate fraction (WUE), n-butanol fraction (WUN), and H 2 O fraction (WUH). The ethyl acetate fraction (91.1 g) was adsorbed by a macroporous adsorbent resin (AB-8) column, and then washed using gradient elution with EtOH-H 2 O. The resulting fractions were combined based on TLC analysis to give eight fractions for further experiments: Fr. WUE-A-Fr. WUE-H. Fr. WUE-A (22.6 g) was separated by reverse phase silica gel (ODS-A-HG) CC using gradient elution with a solvent system of MeOH-H 2 O (10-55%) to produce seven subfractions: WUE-A1-WUE-A7. The DPPH free radical scavenging activities of extracts and pure compounds from stem barks of W. ugandensis were tested according to previous studies with slight modifications [24,25]. Firstly, 10 µL solutions of the samples, appropriately diluted with MeOH, were homogeneously mixed with 190 µL solution of DPPH (0.1 mM in MeOH) in a 96-well plate. Secondly, the mixtures were gently shaken and put at room temperature for 30 min in the darkness. Finally, the absorbance was recorded at 517 nm with multifunctional microplate reader. Trolox was used as positive control, and MeOH was used as blank control. Each of the samples and controls were tested in triplicate (n = 3). The antioxidant activities of DPPH were expressed as IC 50 (concentration of samples caused 50% inhibition) and TEAC values. The TEAC values were calculated from the standard curve of Trolox and expressed as millimoles of Trolox equivalents per gram of sample (mmol TE/g). The inhibition of the DPPH radical was calculated according to the following formula: DPPH scavenging activity (%) = (1 − absorbance of sample/absorbance of blank control) × 100, where DPPH scavenging activities (%) were plotted against the concentration of samples to obtain the IC 50 .

ABTS Assays
The ABTS free radical scavenging activities of extracts and pure compounds from stem barks of W. ugandensis were tested according to previous studies with slight modifications [24,25]. ABTS + solution was prepared by mixing equal volumes of potassium persulfate (4.9 mM in H 2 O) and ABTS (7.0 mM in H 2 O) and then incubated for 12 h in the darkness. The prepared ABTS + solution was diluted with MeOH to ensure an absorbance value about 0.700 ± 0.005 at 734 nm. Firstly, 10 µL of appropriately diluted solution of samples was added to the 190 µL ABTS + solution in 96-well plate. Secondly, the mixtures were gently shaken and put at room temperature for 30 min in the darkness. Finally, the absorbance was recorded at 734 nm with multifunctional microplate reader. Trolox was used as positive control, and MeOH was used as blank control. Samples and controls were tested in triplicate (n = 3). The antioxidant activities of ABTS were expressed as IC 50 and TEAC values. The inhibition of the ABTS radicals was calculated according to the following formula: ABTS scavenging activity (%) = (1 − absorbance of sample/absorbance of blank control) × 100, where ABTS scavenging activities (%) were plotted against the concentration of samples to acquire the IC 50 .

FRAP Assays
The ferric reducing antioxidant power assays on different extracts and pure compounds of stem barks from W. ugandensis were tested according to previous studies with slight modifications [24,25]. Firstly, freshly prepared FRAP reagent (Fe 3+ -TPTZ solution) was composed of FeCl 3 ·6H 2 O (20 mM in H 2 O), TPTZ (10 mM in 40 mM HCl), and ac-etate buffer (300 mM, pH = 3.6) at a ratio of 1:1:10 (v/v/v), then incubated at 37 • C after preparation, and used within 1-2 h. Secondly, 10 µL of appropriately diluted solution of samples was added to the 190 µL freshly prepared FRAP reagent in 96-well plate. Thirdly, the mixtures were gently shaken and put at 37 • C for 10 min. Finally, the absorbance was recorded at 593 nm with multifunctional microplate reader. Trolox was used as a positive control, and MeOH was used as blank control. Each of the samples and controls were tested in triplicate (n = 3). The antioxidant activities of FRAP were expressed as FRAP values and TEAC values. The FRAP values were calculated and expressed as millimoles of Fe 2+ equivalents per gram of sample (mmol Fe 2+ /g) based on a calibration curve plotted using FeSO 4 ·7H 2 O as standard at a concentration ranging from 0.0185 to 1.5 mM.

In Vitro COX-2 Inhibitory Assays
COX-2 inhibition assay in vitro was performed using COX-2 (human) inhibitor screening assay kits according to the manufacturer's instructions to evaluate the COX-2 inhibition activity of compounds and verify the results of UF-LC-MS/MS [26,27]. Firstly, samples were dissolved in DMSO, and prepared into a series of solutions with different concentrations. COX-2 cofactor working solution, COX-2 working solution, COX-2 probe, and COX-2 substrate were prepared according to manufacturer's instructions, and then diluted 10 times with COX-2 assay buffer, respectively. Secondly, 150 µL Tris-HCl (pH = 7.8), 10 µL COX-2 cofactor working solution, 10 µL COX-2 working solution, and 10 µL sample solution were sequentially added in the 96-well black plates, mixed and incubated at 37 • C for 10 min. The COX-2 working solution of the blank control group was replaced with an equal volume of COX-2 assay buffer, and the sample was replaced with an equal volume of DMSO; the sample of the 100% enzyme activity control group was replaced with an equal volume of DMSO. Thirdly, 10 µL COX-2 probe was added into each well. Finally, 10 µL of COX-2 substrate was quickly added into each well, and incubated at 37 • C in the darkness for 5 min, and followed by the fluorescence measurement. The excitation and emission wavelengths were 560 nm and 590 nm, respectively. Indomethacin was set as a positive control. The experiments were performed in triplicate. The COX-2 inhibitory activity was expressed as IC 50 . The inhibition of COX-2 was calculated according to the following formula: where COX-2 inhibitory activity (%) was plotted against the concentration of samples to acquire the IC 50 . RFU 100%Enzyme , relative fluorescence unit of 100% enzyme control group; RFU Sample , relative fluorescence unit of sample; RFU Blank , relative fluorescence unit of blank group.

2.7.
Screening and Identification of the Potential Ligands of SOD and XOD with UF-LC-MS/MS 2.7.1. Affinity Ultrafiltration with SOD and XOD Potential bioactive components which had a high binding affinity to SOD and XOD were screened by affinity ultrafiltration. The experimental conditions were proposed according to previous relevant studies [27,28]. Briefly, the optimized experiment was composed of three steps. Incubation was the first step, where 100 µL WUE-A4 (10.0 mg/mL), 10 µL SOD (2 U) or XOD (2 U), and 90 µL Tris-HCl (pH = 7.8) were mixed well in 1.5 mL EP tubes, and then incubated at 37 • C in the dark for 1 h. Meanwhile, the incubation operations of inactivated enzymes (inactivated enzymes were obtained by heating enzymes in a 99 • C water bath for 10 min) were the same as the active enzymes. Adsorption was the second step, where the incubated solutions were transferred to ultrafiltration tubes with 30 KDa (for SOD) or 10 KDa (for XOD) ultrafiltration membranes and then centrifuged at 10,000 rpm for 10 min at 25 • C. Simultaneously, components which had no binding affinity to enzymes were washed out. Immediately, 300 µL of Tris-HCl solution (pH = 7.8) was added to the ultrafiltration tubes and centrifuged at 10,000 rpm for 5 min at 25 • C to remove the potential unbound components. Desorption was the third step, where 300 µL of 90% (v/v) MeOH-H 2 O was added and incubated for 10 min at room temperature, and the mixed solutions were centrifuged at 10,000 rpm for another 10 min at 25 • C. After that, the desorption process was repeated two times to release those components with specific bindings to SOD or XOD from the enzyme-ligand complexes. Finally, those ultrafiltrates were dried and reconstituted in 50 µL MeOH for the HPLC-UV/ESI-MS/MS analysis.

HPLC-UV/ESI-MS/MS Analysis
HPLC-UV/ESI-MS/MS was carried out to characterize the components in WUE-A4 before and after ultrafiltration by using a Thermo Accela 600 series HPLC connected with a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). A Waters Symmetry RP-C18 column (4.6 × 250 mm, 5 µm) was used to perform chromatographic analysis at 30 • C, and the mobile phase consisted of H 2 O with 0.1% formic acid (A) and ACN (B). The optimized HPLC elution procedures were as follows: 0-15 min, 17% B; 15-40 min, 17-30% B, 40-42 min: 30-56% B. The flow rate was 0.8 mL/min, the injection volume was 10 µL, and the HPLC-UV chromatograms were detected at a wavelength of 254 nm. The negative ion modes were applied to obtained ESI-MS/MS data. Moreover, the parameters of instrument were set as follows: the vaporizer temperature was 350 • C, the capillary temperature was 250 • C, the spray voltage was 3000 V, the cone voltage energy was 40 V, the collision energy was 10 V, the sheath gas pressure was 40 psi, the aux gas pressure was 10 psi, the drying gas flow rate was 6.0 L/min, and the mass range was set from 50 to 1100 (m/z) in the full-scan mode. Finally, the Thermo Xcalibur ChemStation (Thermo Fisher Scientific) was used for data acquisition and analysis.

Statistical Analysis
All data in this work were expressed as mean ± standard deviation (SD) of triplicate measurements. The percentages of scavenging activities or the inhibition rates were plotted against the sample concentrations (six different concentration gradients in triplicate) to obtain the IC 50 values, defined as the concentrations of samples necessary to cause 50% scavenging or inhibition. Software used for statistical analysis mainly included SPSS 16

Antioxidant Activities of Different Extracts of Stem Barks from W. ugandensis
It is inappropriate to assess the antioxidant activity only by a single method for the reason of the complexity of chemical constituents and their diverse mechanisms of action. Thus, the DPPH, ABTS, and FRAP assays were employed in the present study to assess the antioxidant activities of five extracts of stem barks from W. ugandensis, respectively, according to other relevant reports in our laboratory [29,30]. As shown in Table 1, WUE displayed the highest antioxidant activities among these five extracts, with the IC 50 values of the DPPH radical scavenging activity at 17.800 ± 0.300 µg/mL, ABTS radical scavenging activity at 9.400 ± 0.529 µg/mL, and FRAP at 5.579 ± 0.296 mmol Fe 2+ /g, respectively. Moreover, WUZ also exhibited relatively higher antioxidant activities in comparison with other extracts based on the TEAC assay ( Figure 1). Similarly, the 80% ethanol crude extracts of stem barks of W. salutaris also exerted potential antioxidative activities [31].

Antioxidant Activities of Different Fractions Eluted from WUE
The antioxidant activities of eight fractions obtained from WUE were also tested by three assays to single out the most effective fraction(s). As shown in Table 2 and Figure 2, the results suggested that fraction WUE-B possessed the highest antioxidant activities among these eight fractions, with the IC 50 values of DPPH, ABTS, and FRAP antioxidant activities at 16.400 ± 0.300 µg/mL, 9.633 ± 0.513 µg/mL, and 7.603 ± 0.446 mmol Fe 2+ /g, respectively. Moreover, WUE-A also displayed relatively higher antioxidant activities in comparison with other fractions according to Table 2. To this end, fractions WUE-B and WUE-A were, hence, systematically explored for the affinity ultrafiltration screening, isolation, and purification of the main bioactive constituents with high antioxidant activities in the subsequent operations. antioxidant activities of five extracts of stem barks from W. ugandensis, respectively, according to other relevant reports in our laboratory [29,30]. As shown in Table 1, WUE displayed the highest antioxidant activities among these five extracts, with the IC50 values of the DPPH radical scavenging activity at 17.800 ± 0.300 µg/mL, ABTS radical scavenging activity at 9.400 ± 0.529 µg/mL, and FRAP at 5.579 ± 0.296 mmol Fe 2+ /g, respectively. Moreover, WUZ also exhibited relatively higher antioxidant activities in comparison with other extracts based on the TEAC assay ( Figure 1). Similarly, the 80% ethanol crude extracts of stem barks of W. salutaris also exerted potential antioxidative activities [31].   In previous study, colorotane sesquiternepes extracted from W. ugandensis were speculated as possible antioxidant and antiradical components by using computational tools [11]. However, no substantial evidences for both its active components and their corresponding targets were explored so far. Since UF-LC-MS/MS could be used to rapidly screen out bioactive chemical components from complex plant extracts depending on the binding properties between the enzymes and their ligands [26][27][28]32], we further employed UF-LC-MS/MS to quickly screen out and identify the potential ligands of SOD and XOD in WUE-A4 from Kenya in this work. As a result, it is firstly revealed that W. ugandensis contains components with strong binding affinity to both SOD and XOD.  among these eight fractions, with the IC50 values of DPPH, ABTS, and FRAP antioxidant activities at 16.400 ± 0.300 µg/mL, 9.633 ± 0.513 µg/mL, and 7.603 ± 0.446 mmol Fe 2+ /g, respectively. Moreover, WUE-A also displayed relatively higher antioxidant activities in comparison with other fractions according to Table 2. To this end, fractions WUE-B and WUE-A were, hence, systematically explored for the affinity ultrafiltration screening, isolation, and purification of the main bioactive constituents with high antioxidant activities in the subsequent operations.  As shown in Figures 3 and 4, components in WUE-A4 displayed various binding affinities to SOD and XOD, respectively. Based on the diversities of affinity capabilities between ligand-enzyme complexes after incubation, those potential ligands in groups with the active enzymes displayed bigger peak areas than those of with inactivated enzymes. For further evaluation of the affinity binding strength between the potential ligands and the target enzymes, the relative binding affinity (RBA) was employed to compare the variation of the correlated peak areas in the UF-HPLC-UV chromatograms before and after activation [33]. The RBA formula is expressed as RBA = A active /A inactivated , where the A active and A inactivated represent the peak areas obtained from the WUE-A4 samples with activated and inactivated SOD or XOD, respectively. Table 3 lists the RBAs of the potential ligands in WUE-A4 targeting SOD and XOD. For XOD, peak 14 displayed the highest RBA, with a value of 3.76, followed by peak 8 with a value of 2.35, peak 10 with a value of 2.33, peak 6 with a value of 2.09, peak 2 with a value of 1.76, peak 12 with a value of 1.70, and so on. For SOD, peak 3 exhibited the highest RBA value, with a value of 1.99, followed by peak 1 with a value of 1.93, peak 8 with a value of 1.87, peak 4 with a value of 1.55, peak 14 with a value of 1.51, etc.

WUE-A WUE-B WUE-C WUE-D WUE-E WUE-F WUE-G WUE
between ligand-enzyme complexes after incubation, those potential ligands in groups with the active enzymes displayed bigger peak areas than those of with inactivated enzymes. For further evaluation of the affinity binding strength between the potential ligands and the target enzymes, the relative binding affinity (RBA) was employed to compare the variation of the correlated peak areas in the UF-HPLC-UV chromatograms before and after activation [33]. The RBA formula is expressed as RBA = Aactive/Ainactivated, where the Aactive and Ainactivated represent the peak areas obtained from the WUE-A4 samples with activated and inactivated SOD or XOD, respectively. Table 3 lists the RBAs of the potential ligands in WUE-A4 targeting SOD and XOD. For XOD, peak 14 displayed the highest RBA, with a value of 3.76, followed by peak 8 with a value of 2.35, peak 10 with a value of 2.33, peak 6 with a value of 2.09, peak 2 with a value of 1.76, peak 12 with a value of 1.70, and so on. For SOD, peak 3 exhibited the highest RBA value, with a value of 1.99, followed by peak 1 with a value of 1.93, peak 8 with a value of 1.87, peak 4 with a value of 1.55, peak 14 with a value of 1.51, etc.   As shown in Figure 3, Figure 4, and Table 3, the binding affinities of some bioactive components among the potential bioactive components in WUE-A4 with SOD were consistent with XOD, for example, the RBA values of peaks 8 and 14 were higher than others. In other words, considering the strong affinities of peaks 8 and 14 with both SOD and XOD, it could be assumed that the two components were the main bioactive components in WUE-A4, which were closely related to its noteworthy antioxidant capacity. In addi-   3.8 ± 0.5 a 1.5 ± 0.1 c,d,e As shown in Figure 3, Figure 4, and Table 3, the binding affinities of some bioactive components among the potential bioactive components in WUE-A4 with SOD were consistent with XOD, for example, the RBA values of peaks 8 and 14 were higher than others. In other words, considering the strong affinities of peaks 8 and 14 with both SOD and XOD, it could be assumed that the two components were the main bioactive components in WUE-A4, which were closely related to its noteworthy antioxidant capacity. In addition, for the specific components, the binding affinities of peaks 2, 6, 10, and 12 with XOD were significantly stronger than that of SOD with higher RBA values, while peaks 1, 3, and 4 exhibited stronger binding affinities with SOD than that of XOD with higher RBA values. In this regard, each potential peak (component) in WUE-A4 exerted diverse binding affinities to these two enzymes. However, peaks 9 and 11 exhibited extremely low or no binding affinities to both SOD and XOD, due to no visible chromatographic peaks in Figures 3 and 4 after ultrafiltration screening.

Antioxidant Activities of Compounds Isolated from WUE-A4
Antioxidant activities of WUE-A4 and compounds isolated in WUE-A4 were evaluated with DPPH, ABTS, and FRAP assays to further explore their potential total antioxidant capacities. As shown in Table 4 and Figure 6, compound 14 exhibited the highest DPPH and ABTS radical scavenging activities with the IC 50 values of 6.405 ± 0.362 µM and 5.381 ± 0.092 µM, which were lower than the IC 50 values of Trolox (35.973 ± 1.102 µM and 22.353 ± 0.568 µM). Moreover, compound 14 also showed the highest ferric reducing antioxidant capacities with a FRAP value of 17.488 ± 1.625 mmol Fe 2+ /g, which was higher than the FRAP value of Trolox (16.212 ± 1.271 mmol Fe 2+ /g). Moreover, compound 1 also displayed excellent antioxidant activities. It is a C-glycosyl flavonoid that has been

Anti-inflammatory Activities of Compounds Isolated from WUE-A4
Previous studies have shown that free oxygen radicals participate in the pathogenes of many diseases by damaging important biological macromolecules such as proteins an DNA, and then cause pathological reactions such as cancer and inflammation [42]. A cordingly, inflammation is one of the most important symptoms imposed by oxidativ stress. COX-2 is a key enzyme which catalyzes the conversion of arachidonic acid (AA) t

Anti-inflammatory Activities of Compounds Isolated from WUE-A4
Previous studies have shown that free oxygen radicals participate in the pathogenesis of many diseases by damaging important biological macromolecules such as proteins and DNA, and then cause pathological reactions such as cancer and inflammation [42]. Accordingly, inflammation is one of the most important symptoms imposed by oxidative stress. COX-2 is a key enzyme which catalyzes the conversion of arachidonic acid (AA) to prostaglandin G 2 (PGG 2 ), and then catalyzes a sequential enzyme reaction to transform PGG 2 into prostaglandin H 2 (PGH 2 ). Meanwhile, COX-2 can also be induced to overexpress when macrophages, fibroblasts, endothelial cells, and monocytes undergo inflammation, which are closely associated with inflammation and cancer [43]. As shown in Figure 7, compound 14 also exhibited the highest inhibitory activities against COX-2 with an IC 50 value of 0.123 ± 0.004 µM, which was lower than that of indomethacin (1.248 ± 0.158 µM). Meanwhile, compound 13 also displayed relatively higher inhibitory capacities on COX-2 with an IC 50 value of 0.713 ± 0.322 µM, which was lower than the positive control of indomethacin. In addition, compounds 1, 7, and 11 exerted almost the same inhibitory activities on COX-2 compared with the positive control of indomethacin. In this regard, many studies reported the anti-inflammatory activities of compound 7; for example, it showed good inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages [44][45][46]. overexpress when macrophages, fibroblasts, endothelial cells, and monocytes undergo inflammation, which are closely associated with inflammation and cancer [43]. As shown in Figure 7, compound 14 also exhibited the highest inhibitory activities against COX-2 with an IC50 value of 0.123 ± 0.004 µM, which was lower than that of indomethacin (1.248 ± 0.158 µM). Meanwhile, compound 13 also displayed relatively higher inhibitory capacities on COX-2 with an IC50 value of 0.713 ± 0.322 µM, which was lower than the positive control of indomethacin. In addition, compounds 1, 7, and 11 exerted almost the same inhibitory activities on COX-2 compared with the positive control of indomethacin. In this regard, many studies reported the anti-inflammatory activities of compound 7; for example, it showed good inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages [44][45][46].

Conclusions
To date, as a traditional medicinal plant in local communities in Africa, the potential bioactive components with noteworthy antioxidant and anti-inflammatory activity in W. ugandensis and its correlated mechanisms have not been explored. To meet and solve this challenge, three different antioxidant assays, including DPPH, ABTS, and FRAP, were firstly used to trace the antioxidant activities of crude extracts and fractions from stem barks of W. ugandensis. Then, the bio-affinity ultrafiltration combining SOD and XOD with LC-MS/MS was used to rapidly screen out 9 and 12 bioactive components against SOD and XOD from the antioxidant effective fraction WUE-A4, respectively. As a result, eight compounds, including four new lignanamides, one new macrocyclic glycoside, and three known compounds, were successfully isolated and identified from WUE-A4, which

Conclusions
To date, as a traditional medicinal plant in local communities in Africa, the potential bioactive components with noteworthy antioxidant and anti-inflammatory activity in W. ugandensis and its correlated mechanisms have not been explored. To meet and solve this challenge, three different antioxidant assays, including DPPH, ABTS, and FRAP, were firstly used to trace the antioxidant activities of crude extracts and fractions from stem barks of W. ugandensis. Then, the bio-affinity ultrafiltration combining SOD and XOD with LC-MS/MS was used to rapidly screen out 9 and 12 bioactive components against SOD and XOD from the antioxidant effective fraction WUE-A4, respectively. As a result, eight compounds, including four new lignanamides, one new macrocyclic glycoside, and three known compounds, were successfully isolated and identified from WUE-A4, which greatly improved the phytochemical knowledge on the bioactive constituents from W. ugandensis. Then, the antioxidant activities revealed that compounds 14 and 1 showed higher antioxidant activities than the positive control of Trolox, suggesting they would be the potential natural antioxidants from W. ugandensis. In addition, the potential antioxidant activities of compounds 14 and 1 might be highly related to their strong binding affinities with SOD and XOD. More strikingly, compounds 14, 13, 1, 7, and 11 expressed noteworthy inhibitory activities on COX-2, comparable to or even better than that of indomethacin, a positive drug control, which is in common clinical use. Especially, the new compound 14 also displayed the best anti-inflammatory activity, much better than that of clinical indomethacin. In summary, this study showcased an alternative and integrative strategy to quickly screen for and to subsequently identify the most potential natural antioxidants combing multiple target enzymes with affinity ultra-filtration LC-MS from the crude extracts of a medicinal plant of interest, and would provide a good guidance to search for other natural antioxidants from other natural products. Moreover, it was also revealed for the first time that new lignanamides could be the bioactive components of W. ugandensis with the potentials to exert prominent antioxidant and anti-inflammatory activities, which is of great interest for further exploration in the near future. On the other hand, besides the individual potentials of the singular chemical moieties that were isolated and discussed, the bouquet of the compounds identified together in their entirety may work synergistically to unfold the discussed biological effects of W. ugandensis.