An efficient method for synthesizing hydroxy benzoin compounds (1-7) was prepared from four different benzaldehydes using an ultrasonic bath. Then, antioxidant , antimicrobial (3 Gram (-), 4/6 Gram (+), one tuberculosis and one fungus), and enzyme inhibition  for the all synthesized compounds (1-25) were evaluated. And also, four most active compounds  from each group were evaluated to the human cervical cancer cell line (HeLa) and anticancer screening tests against the human retinal normal cell line (RPE). Compound4showed HeLa and RPE cancer cell activities as much as cisplatin. The synthesized compounds were characterized by spectroscopic methods  and the ACD NMR program’s help. In this study, hydroxy benzoin ( It is known that drugs containing phenolic compounds are frequently used to treat diseases such as diabetes, Alzheimer’s, and cardiovascular diseases. One of the most abundant secondary metabolites in plants is phenolic compounds, such as simple phenols, phenolic acids, flavones, flavanones, and stilbenes. The main sources of phenolic compounds are fruits and vegetables, making up an important part of the human diet. According to the studies of the national health organization, due to the antioxidant effects of phenolic compounds found in herbal products, it has been revealed that rich fruit and vegetable consumption reduces the risks of diseases such as cancer, diabetes, Alzheimer’s, and cardiovascular diseases [1]. Some benzoin compounds with the phenolic structure are found in fruits and vegetables in nature.The carbon-carbon bond formation is an important reaction in organic chemistry and studied extensively in the literature [2–5]. The benzoin condensation reaction is an important type of C-C bond formation reaction and is widely used to synthesize natural compounds and analogs. Symmetric and asymmetric benzoin derivative synthesis using different catalysts in benzoin condensation have been studied under milder reaction conditions [2–15]. However, the condensation of two different benzaldehydes may have a widely different character; only the more stable form of the isomeric mixed benzoins could be isolable in excess. When the carbonyl group is adjacent to the phenyl ring with the more electron-donating substituent, it is consistent with the reversibility of the reaction and the relative stability of the carbonyl groups in the possible products [16]. In the literature, the synthesis of mixed benzoin had been made by a variety of methods involving the generation of a “masked” acyl carbanion, which reacts with aromatic aldehydes [17], the addition of an excess of the Grignard reagent to a cyanohydrin or a protected cyanohydrin of an aromatic aldehyde [18], and reduction of unsymmetrical benzils [19]. Thus, all of the mixed benzoin synthesis involve masking or unmasking steps.A literature search showed that various synthetic methods were reported for the benzil syntheses [20-22]. Diphenyl alkynes were oxidized efficiently to yield the corresponding benzil [23]. In another work, the selective addition of organomagnesium reagents to 2,4,6-trichlorophenyl isocyanide then following reactions leading to an efficient synthesis of benzil compounds [24]. Facile oxidation of benzylic alcohols and benzoin to give benzil compounds with various oxidation reagents had been reported [24–32].Carbohydrates play important functional roles in numerous physiological processes, including various disease states [33–34]. Synthetic carbohydrates-based small molecule selective inhibitors are thereof being pursued as potential medicinal agents [35–38]. β-D-glucosides, and we decided to study their pharmacological activities. Due to the biological activities’ evaluation, we wish to report the synthesis of hydroxy benzoins (1-7) from hydroxy benzaldehydes, hydroxy benzils (8-14) from the oxidation of benzoins (1-7), and benzoin/benzil-O-β-D-glucosides (15-25) from the glycosylation of hydroxy benzoins/benzils (1-14). Then, their antioxidant, antimicrobial, enzyme inhibitions, and cytotoxic activity investigations were reported.The significance of benzoin/benzil and carbohydrate-based agents caught our attention for the synthesis of benzoin/benzil-O-n-hexane, chloroform, ethyl acetate, acetone, methanol, and dimethyl sulfoxide), aldehyde compounds , and any used reagent were purchased from by Sigma-Aldrich , Fluka, or Merck  unless otherwise stated. 1H and 13C NMR spectra were obtained on a Bruker 400 MHz NMR spectrometer , using tetramethylsilane (TMS) as an internal standard. CDCl3, CD3OD, and acetone-d6 were used as NMR solvents. 13C and APT spectra were adjusted according to deutero solvent peaks. Chemical shifts were expressed in δ (ppm), and coupling constants (J) were reported in hertz (Hz). ACD NMR program was used for the interpretation of spectra. Ultrasonic bath  was used for the benzoin synthesis. FT-IR spectra were taken using the Perkin-Elmer 1600 (ATR) (4000–400 cm–1) spectrophotometer . Melting points were determined using the Thermo-var apparatus fitted with a microscope. Normal phase silica gel (230–400 mesh) was used in vacuum column chromatography (VLC). TLC was carried out on silica gel 60 F254, and the spots were visualized by ultraviolet (UV) lamp (254 nm and 366 nm) or spraying with 20% H2SO4 and heating. Solvents (Synthesis of hydroxy benzoins (1-7): Hydroxy benzaldehydes (0.001 mol) in dry DMSO (10 mL) were reacted with KCN (0.001 mol) in an N2 environment using an ultrasonic bath  at 70–85 °C. The reactions were terminated after the TLC control. Water (30 mL) was added to the flask, extracted with ethyl acetate (3×30 mL) to give a crude mixture then compounds1-7were purified as a racemic mixture with repeated vacuum liquid chromatography  using the increasing polarity ofn-hexane, chloroform, ethyl acetate, and methanol solvent mixtures, and the fractions were checked by TLC . The synthesis of compounds1[39],2, and4[42–43] had been mentioned in the literature.Compound 1 (2-Hydroxy-1-(3-hydroxyphenyl)-2-phenylethanone):Yield: 45%; Rf = 0.5 (chloroform-ethyl acetate-acetic acid: 25:10:1); UV (MeOH) λ max nm (logɛ): 203; FT-IR (cm–1): 3198, 2924, 1682, 1597, 1584, 1485, 1450, 1285, 1240, 1068, 1014, 950, 787, 762, 700; 1H-NMR : 5.82 , 7.37–7.02 , 4.80 ; 13C-NMR : 198.86 (C-1), 76.16 (C-2), 130.34 (C-1’), 115.71 (C-2’), 156.74 (C-3’), 121.07 (C-4’), 134.64 (C-5’), 121.55 (C-6’), 138.60 (C-1’’), 127.79 (C-2’’), 129.13 (C-3’’), 119.28 (C-4’’), 129.13 (C-5’’), 128.67 (C-6’’). Compound 2 (2-Hydroxy-1-(4-hydroxyphenyl)-2-phenylethanone): Yield: 48%; Rf = 0.48 (chloroform-ethyl acetate-acetic acid: 25:10:1); FT-IR (cm–1): 3371, 3040, 2920, 1661, 1584, 1514, 1455, 1388, 1260, 1065, 971, 836, 763, 701; 1H-NMR : 6.05 , 7.90 , 6.78 , 7.43 , 7.34 , 7.26 , 8.52 ; 5.02 ; 13C-NMR : 197.34 (C-1), 75.45 (C-2), 125.90 (C-1’), 131.35 (C-2’), 114.83 (C-3’), 162.65 (C-4’), 114.83 (C-5’), 131.35 (C-6’), 139.74 (C-1’’), 127.42 (C-2’’), 128.45 (C-3’’), 127.88 (C-4’’), 128.45 (C-5’’), 127.42 (C-6’’). Compound 3 -2-phenylethanone): Yield: 40%; Rf = 0.48 (chloroform-ethyl acetate-acetic acid: 25:10:1); brown oil; UV (MeOH) λ max nm (logɛ): 213 ; FT-IR (cm–1): 3367, 3028, 2960, 1681, 1598, 1452, 1341, 1304, 1164, 1082, 1036, 1004, 699; 1H-NMR : 5.80 , 6.80 , 6.40 , 7.22-7.15 , 8.79 , 5.80 ; 13C-NMR : 199.26 (C-1), 75.92 (C-2), 135.31 (C-1’), 107.91 (C-2’), 157.95 (C-3’), 108.54 (C-4’), 157.95 (C-5’), 107.91 (C-6’), 138.53 (C-1’’), 127.66 (C-2’’), 129.05 (C-3’’), 128.59 (C-4’’), 129.05 (C-5’’), 127.66 (C-6’’); Positive LC-QTOF-MSm/z(%): [M+K-H]+ 282.2693(67), calc. 282.2670.Compound 4 -2-hydroxyethanone): Yield: 68%; Rf = 0.45 (chloroform-ethyl acetate-acetic acid: 25:10:1); FT-IR (cm–1): 3320, 2946, 1678, 1586, 1486, 1452, 1277, 1234, 1069, 1016, 996, 876, 779; 1H-NMR : 5.84 , 6.79 , 6.70-6.67 , 7.36-7.31 , 7.14 , 6.77 , 6.96-6.94 , 7.05 , 5.07 ; 13C-NMR : 199.15 (C-1), 75.89 (C-2), 134.66 (C-1’), 115.87 (C-2’), 156.92 (C-3’), 115.40 (C-4’), 130.31 (C-5’), 121.47 (C-6’), 139.83 (C-1’’), 114.61 (C-2’’), 156.83 (C-3’’), 119.28 (C-4’’), 129.83 (C-5’’), 120.77 (C-6’’). Compound 5 (2-Hydroxy-2-(3-hydroxyphenyl)-1-(4-hydroxyphenyl)ethanone): Yield: 39%; Rf = 0.46 (chloroform-ethyl acetate-acetic acid: 25:10:1); light yellow oil; FT-IR (cm–1): 3292, 3045, 2211, 1740, 1668, 1590, 1514, 1453, 1239, 1171, 982; 1H-NMR : 5.98 , 7.89 , 6.80 , 6.89 , 6.73 , 7.14 , 6.90 , 5.21 ; 13C-NMR : 197.43 (C-1), 75.42 (C-2), 125.89 (C-1’), 131.46 (C-2’), 115.00 (C-3’), 162.60 (C-4’), 115.00 (C-5’), 131.46 (C-6’), 140.96 (C-1’’), 114.20 (C-2’’), 157.41 (C-3’’), 115.18 (C-4’’), 129.75 (C-5’’), 118.90 (C-6’’); Positive LC-QTOF-MSm/z(%): [M+K+Na+CH3OH-H]+ 337.2229(85), calc.337.2214.Compounds 6a and 6b -2-(3-hydroxyphenyl)ethanone)and (2-Hydroxy-1-(3-hydroxyphenyl)-2-ethanone): Yield: 55%; Rf = 0.45 (chloroform-ethyl acetate-acetic acid: 25:10:1). Mix. m.p. (oC): 110-112; UV (MeOH) λ max nm (logɛ):210 ; FT-IR (cm–1): 3363, 2915, 1682, 1600, 1457, 1339, 1283, 1165, 999, 722; 1H-NMR 2CO, d, ppm): 7.89-6.70 , 6.10, 5.98 ; 9.60 ; 13C-NMR 2CO, d, ppm): 198.73, 193.62 (C=O), 77.78, 75.76 (C-2), 165.77, 158.51, 158.25, 158.19, 157.40, 156,94 136.26, 135.72, 135.40, 134.88, 130.80, 130.41 (Ar-C), 130.01, 129.67, 129.44, 121.10, 120.97, 120.74, 120.61, 120.14, 119.78, 116.39, 108.33, 107.56, 107.28, 103.79 (Ar-CH); Positive LC-QTOF-MSm/z(%): [M-H2O+CH3OH]+ 274.2644(100), calc. 274.2647.Compound 7 -2-hydroxyethanone): Yield: 65%; Rf = 0.35 (chloroform-ethyl acetate-acetic acid: 25:10:1); light brown oil; UV (MeOH) λ max nm (logɛ):220; FT-IR (cm-1): 3360, 3160, 3037, 2917, 1687, 1594, 1453, 1343, 1306, 1166, 1006, 951, 707.; 1H-NMR : 5.75 , 6.80 , 6.40 , 6.31 , 6.22 ; 13C-NMR : 199.02 (C-1), 75.87 (C-2), 136.08 (C-1’), 107.49 (C-2’), 158.62 (C-3’), 108.29 (C-4’), 158.62 (C-5’), 107.49 (C-6’), 141.33 (C-1’’), 106.44 (C-2’’), 158.50 (C-3’’), 102.90 (C-4’’), 158.50 (C-5’’), 106.44 (C-6’’); Positive LC-QTOF-MSm/z(%): [M+CO2-H2O+2H]+ 304.2571(85), calc. 304.2500, [M+CO]+ 304.2526(80), calc. 304.2500.Synthesis of hydroxy benzils (8-14): Hydroxy benzoins (100–400 mg) were dissolved in acetone (5 mL) and conc. HNO3 (2-3 mL) was added, and the reactions were stirred at 50–70 °C for 30–120 min [31]. The reactions were terminated after the TLC control. Acetone was evaporated, then water (30 mL) was added to the flask, extracted with ethyl acetate (3×30 mL) to give crude mixture then compounds8-15were purified with repeated VLC (Silica gel 230–400 mesh) using the increasing polarity ofn-hexane, chloroform, ethyl acetate, and methanol solvent mixtures, and the fractions were checked by TLC . The synthesis of compounds8[23],9[9],11[44], and15[commercial product] had been mentioned in the literature.Compound 8 (1-(3-Hydroxyphenyl)-2-phenylethane-1,2-dione): Yield: 25%;Rf = 0.55 (chloroform-ethyl acetate-acetic acid: 25:10:1); light yellow oil; FT-IR (cm–1): 3396, 2933, 1671, 1597, 1450, 1303, 1263, 1176, 942, 840, 780, 749, 635; 1H-NMR : 7.55 , 7.35 , 7.28 , 7.13 ; 7.91 , 7.54 , 7.69 ; 13C-NMR : 196.58 (C-1), 202.50 (C-2), 134.40 (C-1’), 116.36 (C-2’), 159.60 (C-3’), 122.42 (C-4’), 131.53 (C-5’), 123.62 (C-6’), 135.62 (C-1’’), 130.81 (C-2’’), 130.40 (C-3’’), 136.30 (C-4’’), 130.40 (C-5’’), 130.81 (C-6’’).Compound 9 (1-(4-Hydroxyphenyl)-2-phenylethane-1,2-dione): Yield: 16%; Rf = 0.53 (chloroform-ethyl acetate-acetic acid: 25:10:1); light yellow oil; FT-IR (cm-1): 3368, 3027, 2927, 2856, 1740, 1678, 1599, 1582, 1448, 1369, 1267, 1213, 1164, 1043, 879, 719, 611; 1H-NMR 2CO, d, ppm): 7.83 , 6.90 , 7.94 , 7.47 , 7.62 ; 13C-NMR 2CO, d, ppm): 193.49 (C-1), 195.35 (C-2), 125.15 (C-1’), 132.72 (C-2’), 116.15 (C-3’), 163.40 (C-4’), 116.15 (C-5’), 132.72 (C-6’), 133.12 (C-1’’), 129.88 (C-2’’), 128.98 (C-3’’), 134.83 (C-4’’), 128.98 (C-5’’), 129.88 (C-6’’).Compound 10 -2-phenylethane-1,2-dione): Yield: 35%; Rf = 0.48 (chloroform-ethyl acetate-acetic acid: 25:10:1); light brown oil; FT-IR (cm-1): 3434, 2964, 1747, 1598, 1450, 1368, 1227, 1166, 1035; UV (MeOH) λ max nm (logɛ): 220; 1H-NMR 2CO, d, ppm): 6.94 , 7.35 ; 7.92 , 7.50 , 7.65 , 9.08 ; 13C-NMR 2CO, d, ppm): 195.04 (C-1), 195.15 (C-2), 132.96 (C-1’), 108.30 (C-2’), 158.88 (C-3’), 110.04 (C-4’), 158.88 (C-5’), 108.30 (C-6’), 134.42 (C-1’’), 129.75 (C-2’’), 128.95 (C-3’’), 134.77 (C-4’’), 128.95 (C-5’’), 129.75 (C-6’’); Positive LC-QTOF-MSm/z(%): [M+Na+K]+ 304.2539(100), calc. 304.2580; [M+K+H]+ 282.2722(100), calc. 282.2753.Compound 11 ethane-1,2-dione): Yield : 45%; Rf = 0.45 (chloroform-ethyl acetate-acetic acid: 25:10:1); FT-IR (cm–1): 3380, 2960, 2931, 2874, 1736, 1646, 1618, 1582, 1452, 1350, 1225, 1194, 1108, 983, 865, 785, 684; 1H-NMR 2CO, d, ppm): 7.72 , 7.25 , 9.00 ; 13C-NMR 2CO, d ppm): 194.95 (C-1/2) 134.32 (C-1’/1’’), 115.03 (C-2’/2’’), 158.10 (3’/3’’), 121.25 (4’/4’’), 130.55 (5’/5’’), 122.41 (6’/6’’).Compound 12 (1-(4-Hydroxyphenyl)-2-(3-hydroxyphenyl)ethane-1,2-dione:Yield: 23%;Rf = 0.40 (chloroform-ethyl acetate-acetic acid: 25:10:1); m.p. (oC): 60–62; UV (MeOH) λ max nm (logɛ): 203; FT-IR (cm-1): 3436, 2947, 1751, 1598, 1450, 1369, 1232, 1166, 1034; 1H-NMR 2CO, d, ppm): 7.05 , 7.38–7.24 , 7.22 , 7.55/7.51 , 6.70/6.66 , 5.14 ; 13C-NMR 2CO, d, ppm): 200.15 (C-1/2), 163.74, 157.64, 135.73, 129.73 (Ar-C), 149.93, 144.61, 132.15, 129.73, 126.36, 119.89, 117.64, 115.54, 114.19 (Ar-CH), 191.58 (-CHO); Positive LC-QTOF-MSm/z(%): [M+CH3OH] + 274.2679(90), calc. 274.2695.Compound 13 -2-(3-hydroxyphenyl)ethane-1,2-dione): Yield: 18%; Rf = 0.42 (chloroform-ethyl acetate-acetic acid: 25:10:1); light brown oil; UV (MeOH) λ max nm (logɛ): 211; FT-IR (cm-1): 3372, 2957, 1675, 1603, 1453, 1279, 1245, 1171; 1H-NMR 2CO), d, ppm): 7.15 , 6.93 , 7.68-7.25 , 8.86/8.75 ; 13C-NMR 2CO), d, ppm): 191.67 (C-1), 191.95 (C-2), 133.90 (C-1’), 107.97 (C-2’), 158.32 (C-3’), 109.31 (C-4’), 158.32 (C-5’), 107.97 (C-6’), 134.35 (C-1’’), 115.26 (C-2’’), 157.22 (C-3’’), 121.19 (C-4’’), 129.75 (C-5’’), 122.00 (C-6’’); Positive LC-QTOF-MSm/z(%): [M-H2O+CH3OH+2H]+ 274.2711(100), calc. 274.2720.Compound 14 ethane-1,2-dione):Yield: 28%;Rf = 0.38 (chloroform-ethyl acetate-acetic acid: 25:10:1); FT-IR (cm-1): 3369, 2938, 1726, 1602, 1366, 1267, 1221, 1165, 1034; 1H-NMR 2CO, d, ppm): 6.85 , 6.69 ; 13C-NMR 2CO, d, ppm): 195.48 (C-1/2), 134.79 (C-1’/1’’), 107.45 (C-2’/6’/2’’/6’’), 159.58 (C-3’/5’/3’’/5’’), 109.39 (C-4’/4’’). Synthesis of benzoin/benzil-D-glucosides (15-25): Hydroxy benzoins  or benzils  were dissolved in anhydrous methanol (10 mL) under the inert nitrogen atmosphere. KOH (2-4 equiv.) dissolved in methanol (5 mL) and added to the reaction mixtures, which were stirred in an ice bath for half an hour. Then, tetra-O-acetyl-α-D-bromoglucose  in acetone was added to the reaction medium and stirred at room temperature for 12 h [36–38]. As a result of the TLC control of the reactions, NaOMe (5 equiv.) was added to the medium, and the reactions were terminated after 12–24 h with the control of TLC. Excess of NaOMe was killed by the addition of MeOH. The solvent was evaporated, then water (15 mL) was added to the flask, extracted with ethyl acetate (3×20 mL) to give crude mixture then compounds15-25were purified with repeated VLC (Silica gel 230–400 mesh) using the increasing polarity ofn-hexane, chloroform, ethyl acetate, and methanol solvent mixtures, and the fractions were checked by TLC .Compound 15 (2-Hydroxy-1-(3-O-β-D-glucopyranosylphenyl)-2-phenylethanone): Yield: 15%; diastereomer; Rf = 0.5 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 210; FT-IR (cm–1): 3342, 3020, 2924, 1676, 1641, 1596, 1448, 1400, 1256, 1072, 1040, 892; 1H-NMR 2CO, d, ppm): 7.67–7.21 , 6.13, 6.12 , 5.09 , 5.01, 4.75-3.22 ; 13C-NMR 2CO, d, ppm): 191.83 (C = O), 163.53, 144.58, 133,81, 133,49 (Ar-C), 131.43, 130.21, 129,64, 128.65, 123.29, 122.87, 122.53, 121.02, 117.55, 116.58 (Ar-CH), 101.23, 101.03 (anomeric CH), 76.95, 76.92 (C-2), 77.0, 73.80, 73.76, 70.73 (glucose CH), 61.67 (glucose CH2). Positive LC-QTOF-MSm/z(%): [M+Na]+ 413.2563(20), calc. 413.2549.Compound 16 (2-Hydroxy-1-(4-O-β-D-glucopyranosylphenyl)-2-phenylethanone): Yield: 17%; diastereomer; Rf = 0.68 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 210; FT-IR (cm–1): 3374 3018, 2927, 1582, 1410, 1348, 1313,1160, 1078, 1048, 610; 1H-NMR : 7.64 , 6.40 , .7.42–7.10 , 4.80 (anomeric CH remained within the water peak), 4.62-3.12 ; 13C-NMR : 196.29 (C=O), 165.07, 140.89, 119.28 (Ar-C), 131.98, 128.35, 127.63, 127.39, 118.76 (Ar-CH), 103.99 (anomeric CH), 76.53 (benzoin CH), 76.41, 74.41, 73.60, 69.99 (glucose CH), 60.69 (glucose CH2). Positive LC-QTOF-MSm/z(%): [M+Na]+ 413.1121(9), calc. 413.1141; [M+Na-H]+ 412.1015(23), calc. 412.1063.Compound 17 -2-phenylethanone): Yield: 14%; diastereomer (2:1); Rf = 0.74 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 211; FT-IR (cm–1): 3367, 2972, 2270, 1720, 1269, 1057; 1H-NMR 2CO, d, ppm): 8.05-6.48 , 6.06/6.00 , 5.03 , 4.98 , 4.46–3.32 ; 13C-NMR 2CO, d, ppm): 199.00 (C=O), 159.02, 158.57, 141.90, 135.09 (Ar-C), 129.46, 129.02, 128.72, 128.65, 127.64, 127.52, 110.16, 109.95, 108.21, 108.02 (Ar-CH), 100.89/100.83 (anomeric CH), 76.90, 76.84, 76.06, 74.12, 73.84, 73.65, 70.38, 70.17 (benzoin CH and glucose CH), 63.56/63.38 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M+K-CH3OH-2H]+ 573.1286(100), calc. 573.1249.Compounds 18a+b (2-Hydroxy-1-(3-O-β-D-glucopyranosylphenyl)-2-(3-hydroxyphenyl)-ethanone; 2-Hydroxy-1-(3-hydroxyphenyl)-2-(3-O-β-D-glucopyranosylphenyl)ethanone): Yield: 11%; diastereomer (2:1); Rf = 0.80 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 220; FT-IR (cm–1): 3343, 3030, 2923, 1636, 1586, 1447, 1397, 1251, 1067, 1033, 1014, 892, 786; 1H-NMR : 7.48–6.55 , 6.00, 5.95 , 4.84–4.74 (anomeric CH remained within the water peak), 4.43-3.17 ; 13C-NMR : 198.97, 198.94, 198.73, 198.50 (C=O), 157.80, 157.59, 157.54, 157.44, 140.64, 140.59, 140.37, 140.32, 135.97, 135.86, 135.84, 135.74 (Ar-C), 129.75-114.29 (Ar-CH), 103.93, 103.78, 103.1, 100.64 (anomeric -CH), 76.45, 76.37, 76.25, 76.13, 73.98, 73.63, 73.65, 73.56, 70.65, 70.13 (benzoin CH and Glucose CH (C-2-5)), 63.45, 63.38 (Glucose -CH2OH); Positive LC-QTOF-MS m/z (%): [M+C6H12O6-CH3OH-H]+ 563.5404(100), calc. 563.5404.Compound 19 (2-Hydroxy-1-(4-O-β-D-glucopyranosylphenyl)-2-(3-O-β-D-glucopyranosyl-phenyl)ethanone): Yield: 18%; diastereomer (1:2); Rf = 0.45 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 213; FT-IR (cm-1): 3380, 3032, 2924, 1734, 1596, 1450, 1376, 1250, 1053; 1H-NMR : 8.02 , 7.34 , 7.80-7.45 , 6.11, 6.08 , 5.13–5.08 (anomeric CH beside the water peak), 4.52–3.38 ; 13C-NMR : 193.74, 192.80 (C=O), 76.50, 76.03 (C-2), 164.01, 159.30, 139.28, 114.59 (Ar-C), 130.28, 129.8, 122.25, 122.15, 122.06, 119.88, 116.14 (Ar-CH), 102.05, 101.36 (anomeric -CH), 77.90, 74.63, 74,55, 73.45, 73.33, 73.21, 69.89, 69.67, 69.54, 68.52 (Glucose C2-C5), 62.92, 60.26 (Glucose -CH2); Positive LC-QTOF-MSm/z(%): [M+Na]+ 575.2733(75), calc. 575.2740.Compound 20 -2-(3-O-β-D-glucopyranosylphenyl)ethanone): Yield: 12%; diastereomer; Rf = 0.5 (chloroform-methanol: 8:2); light yellow oily; UV (MeOH) λ max nm (logɛ): 213; FT-IR (cm–1): 3385, 3028, 2923, 2568, 1688, 1597, 1456, 1287, 1075, 1034; 1H-NMR : 7.74-6.87 , 5.84, 5.71 , 5.04–4.8 (anomeric CH beside the water peak), 4.65-3.30 ; 13C-NMR : 198.73, 194.24 (C=O), 76.74 (C-2), 166.90, 160.50, 158.48, 158.27, 158.20, 157.89, 157.84 139.25, 138.64, 134.41, 134.28, 131.85, 131.22 (Ar-C), 131.22, 129.88, 129.63, 124.10, 123.65, 120.32, 120.11, 120.08, 119.87, 116.59, 116.20, 116.06, 109.33, 109.16, 108.78 (Ar-CH), 103.97, 100.72 (anomeric CH), 76.48, 76.16, 74.98, 72.56, 69.81 (Glucose CH (C-2-5)), 61.31, 61.01 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M+Na-H2O-H]+ 750.5818(74), calc. 750.5836; [M+Na-CH3OH-H]+ 736.5696(100), calc. 736.5600.Compound 21 (1-(3-O-β-D-Glucopyranosylphenyl)-2-phenylethane-1,2-dione): Yield: 18%; Rf = 0.77 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 205;FT-IR (cm–1): 3364, 2938, 1739, 1665, 1445, 1227, 1074; 1H-NMR : 7.96-7.31 , 4.99 , 4.49-3.33 ; 13C-NMR : 194.51, 192.42 (C=O), 158.10, 134.94, 132.82 (Ar-C), 134.16, 130.07, 129.39, 128.98, 124.20, 123.60, 115.90 (Ar-CH), 100.68 (anomeric CH), 76.26, 74.12, 73.32, 70.21 (glucose CH), 63.42 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M-2CH3OH+H]+ 325.2283(100), calc. 325.2280.Compound 22 (1-(4-O-β-D-Glucopyranosylphenyl)-2-phenylethane-1,2-dione): Yield: 12%;Rf = 0.77 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 210; FT-IR (cm-1): 3385, 2972, 1710, 1603, 1445, 1270, 1058; 1H-NMR 2CO, d, ppm): 7.94 , 7.63 , 7.74 , 7.22 , 5.20 , 4.43-3.43 ; 13C-NMR 2CO, d ppm): 198.00, 194.58 (C=O), 161.59, 133.36, 126.93 (Ar-C), 134.98, 131,88, 129.57, 129.24, 116.72 (Ar-CH), 100.11 (anomeric CH), 76.80, 74.26, 73.58, 70.15 (glucose CH), 63.24 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M+K+Na+3H]+ 453.1011(100), calc. 453.1016.Compound 23 -2-phenylethane-1,2-dione): Yield: 9%; Rf = 0.75 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 215; FT-IR (cm–1): 3627, 2975, 2256, 1713, 1524, 1386, 1058; 1H-NMR 2CO, d, ppm): 7.80 , 7.80-7.40 , 7.08 , 6.95 , 4.98 , 4.30-3.44 ; 13C-NMR 2CO, d, ppm): 194.98, 193.68 (C=O), 159.27, 134.73, 133.96 (Ar-C), 135.21, 129.55, 129.45, 129.33, 124.71, 110.25, 109.18 (Ar-CH), 101.05 (anomeric CH), 77.08, 73.67, 70.21 (glucose CH), 61.57 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M-CH3OH-CO2-3H]+ 325.2162(100), calc. 325.2162.Compound 24 (1-(3-O-β-D-Glucopyranosylphenyl)-2-(3-hydroxyphenyl)ethane-1,2-dione): Yield: 42%; Rf = 0.75 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 210; FT-IR (cm–1): 3364, 2938, 1739, 1665, 1445, 1227, 1075; 1H-NMR : 7.78-6.77 , 4.97 , 3.96-3.17 ; 13C-NMR : 193.42, 193.35, (C=O), 156.56, 135.01, 134.50, 132.58 (Ar-C), 128.54, 128.60, 126.72, 123.45, 125.82, 121.65, 120.91, 114.28 (Ar-CH), 102.43 (anomeric -CH), 75.20, 74.96, 72.10, 68.68 (glucose CH), 59.77 (glucose CH2); Positive LC-QTOF-MSm/z(%) : [M+Na-H]+ 588.4345(100), calc. 588.4387.Compound 25 (1-(3-O-β-D-Glucopyranosylphenyl)-2-(4-hydroxyphenyl)ethane-1,2-dione): f = 0.60 (chloroform-methanol: 8:2); light yellow oil; UV (MeOH) λ max nm (logɛ): 206; FT-IR (cm-1): 3748, 3620, 2973, 2302, 1732, 1386, 1228, 1057; 1H-NMR : 7.42 , 7.38–7.24 , 7.18 , 7.66/7.64 , 6.83/6.79 , 4.96 , 4.31–3.37 ; 13C-NMR : 192.34, 191.98 (C=O), 158.20, 143.92, 135.94, 126.86 (Ar-C), 129.67, 129.18, 126.33, 122.95, 119.40, 117.31, 115.17, 114.28 (Ar-CH), 100.95 (anomeric CH), 76.90, 76.57, 73.51, 70.04 (glucose CH), 61.14 (glucose CH2); Positive LC-QTOF-MSm/z(%): [M+H+C6H12O6]+ 585.5184(15), calc. 585.5156.Yield: 12%; R1-25were tested against iron (III) / ferric reducing antioxidant power (FRAP), Cu (II) reducing antioxidant capacity (CUPRAC), and 2,2-Diphenyl-1-picrylhydrazyl radical quenching capacity (DPPH) methods according to the literature [45–50] (Table 2). Butylated hydroxytoluene for DPPH and Trolox for CUPRAC and FRAP was used as standard.Antioxidant activities of the synthetic compoundsFerric reducing antioxidant power (FRAP) assay: The method was carried out based on the determination of the iron ions reducing the samples’ power. First, 2,4,6-tripyridyl-s-triazine  was dissolved in a mixture of hydrochloric acid (50 μL) and distilled water (10 mL). Then, FeCl3 (32 mg) was dissolved in distilled water (10 mL). Finally, distilled water (250 mL) was added to acetic acid , and sodium acetate (0.66 g) was completely dissolved in this solution. Buffer, TPTz, and FeCl3 were mixed at 10/1/1 ratios, and 2 mL of this mixture was mixed with 0.1 mL of compounds1-25(2 mg/mL) and incubated at 30oC for 30 min. As a standard, different concentrations of Trolox solution  were used instead of the sample. At the end of the incubation, the samples’ absorbance was read at 595 nm, and the results are given as Trolox equivalents. Results were expressed as μmol Trolox/g dry weight of compounds1-25(μg/mL Trolox/g DW)  (Table 2). Copper ions reducing activity (CUPRAC): In a test tube, ammonium acetate , CuCl2 , and neocuproin  solutions were taken, and 0.5 mL of compounds1-25and standards (Trolox) at different concentrations  were mixed, and 1 mL of distilled water was added to each tube. After 30 min in a dark environment at room temperature, it was read against blank at 450 nm using Shimadzu UV-1600 spectrophotometer [49], and results are given in Table 2.DPPH radical scavenging activity: In vitro antioxidant properties of compounds,1-25 were tested using 2,2-diphenyl-1-picrylhydrazyl scavenging (DPPH). 0.75 mL of compounds1-25and standard (BHT) at varying concentrations (mg/mL) and 0.75 mL of 0.1 mM DPPH solution were mixed. All tubes were left in the dark for 50 min at room temperature. The absorbance was read 517 nm using Shimadzu UV-1600 spectrophotometer, and results are given as SC50 value (mg/mL) in Table 2 [45–47].Escherichia coliATCC 25922 (Ec),Yersinia pseudotuberculosisATCC911 (Yp),Pseudomonas aeruginosaATCC27853 (Pa),Staphylococcus aureusATCC25923 (Sa), Streptococcus mutansRSKK07038 (Sm), Enterococcus faecalis ATCC29212 (Ef),Paenibacillus larvae DSM7030 (PSP),Bacillus cereus Roma709 (Bc),Bacillus subtilisATCC1266 (Bs),Mycobacterium smegmatisATCC607 (Ms),Candida albicansATCC60193 (Ca). Inhibition diameters were measured by the agar well diffusion method [51–53], and the MIC value was determined as microgram-milliliter (µg / mL) to the microdilution technics (Table 3). The test microorganisms used in the study were obtained from Refik Saydam Hıfzısıhha Institute  and are as follows.Antimicrobial activity assessment (agar-well diffusion method): The antimicrobial screening test using the agar-well diffusion method as adapted was used earlier [53–54]. Each microorganism was suspended in Mueller-Hinton broth  and diluted approximately 106 colony-forming units (CFU) per mL. They were “flood-inoculated” onto the surface of Mueller–Hinton agar, brain heart infusion agar, and potato dextrose agar (PDA)  and then dried. Brain heart infusion agar was used for M. smegmatisandS. mutans. ForC. albicans,PDA was used. Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 μL of the compound substances were delivered into the wells. The plates were incubated for 24–48 h at 36 °C. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. Compound stock solutions were prepared at different concentrations (1.100–80.200 μg/mL) according to the amount of material obtained. The 1/10 dilution of each solvent was used as a control.Minimal inhibition concentration (MIC) assay: The antimicrobial properties of compounds1-25were investigated quantitatively in respective broth media by using the microdilution method, and the minimal inhibition concentration (MIC) values (μg/mL) were examined [53]. The antibacterial activity assays were carried out in Mueller–Hinton broth (MHB) at pH = 7.0±0.2 and 18–24 h at 36 °C incubated. For the antifungal activity test were used yeast extract peptone dextrose (YEPD) broth (pH = 6.5 ± 0.2) and 48 h at 36 °C incubated. Brain heart infusion broth (BHI)  was used forM. smegmatis andS. mutansand incubated for 72 h at 36 °C. The minimal inhibition concentration value was defined as the lowest concentration that showed no growth. Ampicillin (10 mg/mL), streptomycin (10 mg/mL) and fluconazole (5 mg/mL) were used as standard antibacterial and antifungal drugs, respectively (Table 3). The 1/10 dilution of each solvent was used as a control.Acetylcholinesterase (AChE) inhibition: The acetylcholinesterase method is based on the principle that thiocholine released by a chromogenic reagent 5,5-dithio-bis-(2-nitrobenzoic acid) gives a colored product. The sample solution (10 μL) and acetylcholinesterase solution (20 μL) were mixed in Tris-HCl buffer . It was then incubated at 25 °C for 10 min in a 96-well microplate. Then, DTNB (20 μL) and acetylthiocholine iodide (20 μL) were mixed. Similarly, an enzyme-free tube was prepared blindly. Sample and blank absorbances were read after 10 min of incubation at 25 °C at 405 nm. Acetylcholinesterase inhibitory activity was given equivalent to galantamine [55], and the results were given in Table 4.Butyryl cholinesterase (BChE) inhibition: Butyrylcholinesterase inhibition is based on acetylcholine’s hydrolysis by cholinesterase to 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) into yellow colored 5-thio-2-nitrobenzoic acid. The sample solution (10 μL) and butyrylcholinesterase solution (20 μL) were mixed in Tris-HCl buffer . It was then incubated at 25 °C for 10 min in a 96-well. Similarly, an enzyme-free tube was prepared blindly. Sample and blank absorbances were read after 10 min of incubation at 25 °C at 405 nm. Butyrylcholinesterase inhibitory activity was given equivalent to galantamine [55], and results were given in Table 4.Tyrosinase inhibition: Tyrosinase inhibitor activity was performed by the dopachrome method using L-DOPA as a substrate. The sample solution (25 μL) was mixed with tyrosinase solution (40 μL) and phosphate buffer  in a 96-well microplate and incubated at 25 °C for 15 min. The reaction was initiated by the addition of L-DOPA (40 μL). Similarly, the enzyme-free blank solution was prepared, and the sample and blank absorbance were read at 492 nm after incubating at 25 °C for 10 min. Tyrosinase inhibitory activity results were given as equivalent to kojic acid [56], and results were given in Table 4.α-Amylase inhibition: α-Amylase inhibitor activity was applied using the Caraway-Somogyi iodine/potassium iodide (I2/KI) method. Sample solutions (25 μL) were mixed with the α-amylase solution (50 μL) in phosphate buffer  in a 96-well microplate. The mixture was incubated at 37 °C for 10 min. After pre-incubation, the reaction was initiated when the starch solution  was added. Similarly, the enzyme-free blank solution was prepared. The reaction mixture was incubated for 10 min at 37 °C, and the reaction was stopped by adding HCl . Following this, iodine -potassium iodide solution (100 μL) was added. Sample and blank absorbance were read at 630 nm, and α-amylase inhibitor activity results were given as acarbose equivalent [57–58], and results were given in Table 4.α-Glucosidase inhibition: α-Glucosidase inhibitor activity was applied according to the method of Palanisamy et al. Sample solution (50 μL), glutathione (50 μL), α-glucosidase solution (50 μL) phosphate buffer (pH = 6.8) and PNPG (4-Nitrophenylβ-D-glucuronide) (50 μL) solution mixed in a 96-well microplate. It was incubated at 37 °C for 15 min. Similarly, an enzyme-free blank was prepared. The reaction was stopped when sodium carbonate  was added. Sample and blank absorbance were read at 400 nm. α-Glucosidase inhibitor activity was given as acarbose equivalent [57–58], and results were given in Table 4.Anticancer cell lines and cell culture: The human cervical cancer cell line (HeLa) and human retinal normal cell line (RPE) were used to determine the anticancer activities of molecules synthesized within the scope of the project [59–61]. All cell preparation processes were carried out in a sterile environment in a laminar cabinet. HeLa cell line was used in DMEM medium supplemented with 10% FBS  and 2% PenStrep (Penicillin-Streptomycin) solution at 37 °C, 5% CO2 conditions, after achieving sufficient concentration (confluent). DMEM/F12 medium was used for RPE cell lines. Inoculation was carried out on the measurement plates with 10,000 cells per well. After approximately 16 h of pre-incubation, test molecules were added, and measurements were made after 24 h of incubation, and results were given in Table 5.Cell proliferation measurement, determination of GI50 and IC50 values: MTT  test was used to measure the effects of synthesized test compounds4,12,18a+b, and25on cell proliferation . This test protocol was applied after the cancer cell lines were incubated for 24 h with test substances. The results were reported as % cell inhibition, with the solvent’s optical density (DMSO) treated cells considered to be 100%. Accordingly, the % inhibition was calculated according to the formula [1-(test substance A/solvent control A)×100. MTT on cells of increasing concentrations  of each test substance over a certain range to determine the IC50 concentrations of test substances (concentration that inhibits the proliferation of 50% of cells in the medium) were prepared. Results were analyzed using a logarithmic function with the help of the Excel program over the logarithmic curve prepared from the absorbance values obtained after the test (Table 5). The following formula is used for the GI50 value calculation as cell proliferation: [(Ti-Tz)/(C-Tz)]x100 if Ti>/=Tz (cytostatic effect) or [(Ti-Tz)/Tz]x100 if Ti<Tz (cytotoxic or cytotoxic effect) . GI50: Concentration value that reduces growth by 50% ([(Ti-Tz)/(C-Tz)]×100 = 50). The following formula is used for the IC50 value calculation. Accordingly, the % inhibition was calculated according to the formula [1-(A test substance/A solvent control)×100 [59-61].1-7  were obtained as a racemic mixture  .In this study, self or cross-benzoin reaction of benzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, and 3,5-dihydroxy benzaldehyde was carried out by using different methods. Substituted groups are very effective for the synthesis of benzoin when the electron donating groups are present on benzaldehyde; it is difficult to synthesize the benzoin as known in the literature . For this purpose, MW, US, and reflux methods were investigated to find out the method to synthesize electron donating substituted group contains asymmetric or symmetric benzoin according to known methods. MW and reflux methods gave low yield or no reactions, and HCN gas evolution was observed. Dozens of trials have been made. However, for the most part, hydroxy substituted benzoin compounds could not be difficult to be synthesized. The US method was the best method to synthesize some of the hydroxy substituted benzoin even in low yield. In the case of using different aldehyde compounds, it is possible to form four alternative benzoins (Table 1). After chromatographic purification, compounds1H NMR spectra of benzoin compounds gave benzylic -CH(OH) at δ 5.8-6.4  ppm and 13C NMR spectra revealed peaks at δ 195-198 (C = O) for C-1 and δ 75-78 ppm for C-2, which indicates benzoin structures .8-14) were synthesized from hydroxy benzoins’ oxidation (1-7) [72–74] with conc. nitric acid, PCC, and Fehling reagent. It was observed that during the oxidation of benzoin compounds, they decomposed to aldehydes or benzil compounds rearranged to benzylic acids. It has been known that substituted benzoin/benzil compounds decompose during the oxidation . Therefore, the yields of benzil reactions were found in the range of 18%–45%. The disappearance of the benzylic proton peak at δ 5.8–6.4 ppm and benzylic carbon peak at δ 75–78 ppm for the hydroxy benzoin compounds, and the appearance of 1,2-dione carbon peaks at δ 192-195 ppm (C = O) for C-1 and C-2 indicated the hydroxy benzil structures  .Hydroxy benzil compounds (1-7) and benzils (8-14) with TABG then hydrolysis of acetyl group yielded benzoin-D-glucosides (15-20) and benzil-D-glucosides (22-25), respectively [21-22]. Observation of the anomeric proton coupling constant value aroundJ= 7-8 Hz in the 1H NMR spectrum of the synthesized benzoin/benzil-O-β-D-glucosides shows that the D-glucose unit was in theβform. 13C NMR spectra of benzoin/benzil-O-β-D-glucoside compounds indicate anomeric carbon peaks at δ 100–105 ppm, which showed that one or more D-glucose units were attached to benzoin/benzil compounds . Benzoin-O-β-D-glucoside compounds were observed as diastereomeric mixtures, as seen in NMR spectra. Compounds6and18were observed as an isomeric mixture. In total, 7 benzoin, 7 benzil, and 6/5 benzoin/benzil-D-glucoside compounds were synthesized, respectively. In the synthesis of the D-glucoside derivative of compounds7,13,and14, highly mixed products were obtained and could not be purified. All the synthetic compounds were characterized by NMR (1D and 2D) and ACD NMR program. According to our literature survey, compounds3,5-7,10,12-13, and15-25have not been found in the literature.The reaction of hydroxy benzoins  were 2237±58.3 and 1113.33±64 (μg/mL Trolox/gram DW) in compounds6 and5, and the lowest DPPH values for compounds5and4were found to be 8.12 ± 1.2 mg/mL and 8.16 ± 0.3 mg/mL, respectively. Among the benzil compounds (8-14), compound14(1974 ± 76.9 μg/mL Trolox/gram DW) to FRAP, compounds8and13(1095.00 ± 18.1 μg/mL Trolox/gram DW) to CUPRAC, and compound14(7.38 ± 1.0 mg/mL) to DPPH methods, were found to be the most active compounds. In the benzoin-D-glucoside compounds(15-20), the highest FRAP, CUPRAC, and lowest DPPH values for benzoin-D-glucoside were 2918.75±36.14, 775.58±12.34 (μg/mL Trolox/gram DW), and 7.78±0.12 mg/mL for compound20, respectively. When the activities of all compounds according to FRAP, CUPRAC and DPPH were examined, it was seen that compound20for FRAP, compound5for CUPRAC, and compound14for DPPH were the most effective antioxidant compounds. The antioxidant activities for the benzil-O-β-D-glucoside (21-25) showed that compound25was the most effective for FRAP (2570.42 ± 25.01 μg/mL Trolox/gram DW) and the compound23was the most active to CUPRAC  and DPPH  methods. When looking at the substitution positions of these compounds, it has been found that they are generally more effective when they are substituted at the 3-position.Antioxidant properties of synthesized compounds1-14against eight bacteria and one yeast, and compounds16-25against ten bacteria and one yeast were evaluated. After the inhibition diameters were observed in mm (data are not shown), the MIC values (µg/mL) were calculated [53–54] (Table 3). The best MIC values for hydroxy benzoin/benzil compounds6a+b,8,10,and13were found within the range of 9–52 µg/mL against bacteriaY. pseudotuberculosis, P. aeruginosa, S. aureus, S. mutans, E. faecalis, and B. cereus. Compounds6a+b,8,10,11,12,and13showed the best antituberculosis activity with the MIC value in the range of 9–18 µg/mL againstM. smegmatis. Considering the antifungal activity results, compounds8and10-15showed closer activity againstC. albicanswith 17-51 µg/mL MIC values. The antibacterial activity results of compounds15-25 showed that compounds (15-25) were generally the most effective against Gram (+) nonpathogenic spore forming bacteriaP. larvaeandB. suptilis. None of the tested compounds15-25 gave any activity againstY. pseudotuberculosis,P. aeruginosa, andS. mutans. Compound25showed MIC values in the range of 8–15 µg/mL against bacteria . Among the glycoside compounds, compounds21and22were only active against nonpathogenicB. subtilisandP. larvaewith 12–27 µg/mL MIC value. Only compound 15 was found to be active againstC. albicanswith 25 µg/mL MIC value among the glycoside compounds. It has been observed that compounds carrying –OH/-H as the -R group in the meta position compared to the carbonyl in their structure and the meta position in the other phenyl ring are more active. The antimicrobial activities of the synthesized compounds50 values were calculated and given in Table 4. The result of enzyme inhibition  for hydroxy benzoin/benzil (1-14) gave that compound4/10,2,6a+b /10,7/9, and1/9were the most active. Their IC50 values were within the range of 25.45–110.96 µg/mL, and 38.90–219.82 µg/mL, respectively. The five different enzyme inhibition  of benzoin/benzil-O-β-D-glucosides (15-25) resulted that compounds19/23,16/25,19/21,19/23, and20/24were the most active. Their IC50 values were within the range of 10.52 –188.93 µg/mL, and 58.90–133.86 µg/mL, respectively. In the enzyme inhibition study, compounds19 and2against ACh and BCh, compounds7and12against α-amylase and α-glucosidase, and compound19against tyrosinase showed activity as much as galantamine, acarbose, and kojic acid standards used, respectively. When these results are compared with the used standards, compounds19,2,7,12, and19could be used as ACh, BCh, tyrosinase, α-amylase, and α-glucosidase inhibitors, respectively. The highest activities were seen by the compounds bearing the 4-OH, 3-OH substituent on the phenyl rings considering the structure-activity relationship. It is known in the literature that many different hydroxy phenolic compounds show biological activity in a wide spectrum range .Acetylcholinesterase [55], butyrylcholine esterase [55], tyrosinase [56], α-amylase [58], α-glucosidase inhibition [58] activity results were made according to spectrophotometric methods. IC4,12,18a+b, and25showed that only compound4(IC50 42.67 µg/mL) had a strong antiproliferative effect on cancer cells (Table 5). However, the toxicity caused by compound4on the normal cell line was examined (IC50 30.27 µg/mL), it was found to be toxic. The inhibitor concentrations (IC50) of the compounds4,12,18a+b, and25were compared on the HeLa cancer cell, compound4(IC50 42.67 µg/mL) had a strong antiproliferative effect as cisplatin (IC50 42.76 µg/mL). The growth inhibition (GI50) of compound4(GI50 1.12 µg/mL) on the HeLa cancer cell was as the control compound cisplatin (GI50 1.13 µg/mL) [59–61]. The inhibitor concentrations (IC50) of the compounds4,12,18a+b, and25on the RPE normal cell line gave that compound4(IC50 30.27 µg/mL) had better antiproliferative activity than control compound cisplatin (IC50 58.77 µg/mL). Comparison of growth inhibition (GI50) on the RPE cancer cells, compound4(GI50 1.15 µg/mL) showed growth inhibition as much as cisplatin (GI50 1.56 µg/mL).HeLa test results of compounds21(2918.75±36.14 μg/mL Trolox/gram DW) for FRAP, compound5(1113.33 ± 64.9 μg/mL Trolox/gram DW) for CUPRAC, and compound15 (7.38±1.0 mg/mL) for DPPH were the most effective antioxidant compounds. Hydroxy benzoin/benzil compounds1-14were more effective against test microorganisms in the antimicrobial activity among the synthesized compounds1-25. Compound24showed the only antitubercular activity with the MIC value of 15 µg/mL againstM. smegmatis,and compound15was found to be active against onlyC. albicanswith 25 µg/mL MIC value. Thus, compounds24and15could be used as the standard forM. smegmatisandC. albicans, respectively. Enzyme inhibition study showed that compounds19 and2against ACh and BCh, compounds7and12against α-amylase and α-glucosidase, and compound19against tyrosinase gave the activity as much as galantamine, acarbose, and kojic acid standards used, respectively. However, it has been observed that all compounds synthesized show different levels of activity against tested enzymes. HeLa and RPE cancer cell activities of compound4were observed as much as cisplatin. Biological activity studies of compounds1-25showed benzoin and benzil analogs were more active when the substituents in themetapositions. Compounds3,5-7,10,12-13, and15-25are new, and their biological evaluation was made first time in this work.In this study, benzoin reaction was carried out in a modified ultrasonic bath. Antioxidant activities of all compounds were compared according to FRAP, CUPRAC, and DPPH methods. It was found that compound


The structures of these new compounds were identified by spectroscopic and MS analyses. Compounds 3, 5, 19, and 20 were evaluated for inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells with IC50 values of 17.03, 18.21, 16.30, and 12.64 μM, respectively. Compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 cells with IC50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 μM, respectively.Three new triterpenoids—spergulagenin B ( Glinus oppositifolius (L.) Aug. DC. , spergulagenin C (2), and spergulagenin D (3)—together with 17 known compounds. This article describes the structural elucidation of 1–3 and the anti-inflammatory activity of the isolated compounds.Molluginaceae has about 13 genera and more than 120 kinds of plants in the world, mainly distributed in tropical and subtropical regions. Aug. DC.  is an andiseases , triterpdiseases , naphthadiseases , and thediseases , antifundiseases . Macrophdiseases . Tumor ndiseases . Inhibit3. Electronic circular dichroism (ECD) spectra were recorded on a Chirascan CD spectrometer . High resolution electron ionization mass spectrometry (HR-EI-MS) was measured at Chung Hsing University . Ultraviolet (UV) spectra were measured using a Shimadzu Pharmaspec-1700 UV-Visible spectrophotometer . Nuclear magnetic resonance (NMR) spectra—including heteronuclear single-quantum coherence (HSQC), correlation spectroscopy (COSY), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectrometry (NOESY) experiments—were measured using a Bruker DRX-500 FT-NMR  operating at 125 MHz (13C) and 500 MHz (1H), respectively. Chemical shifts are given in ppm (δ) using tetramethylsilane (TMS) as internal standard. HPLC separations were carried out utilizing a P230 HPLC system  equipped with P230 HPLC Pump and an IOTA 2 detector, utilizing ChromNav software . TLC analysis was performed utilizing aluminum pre-coated Si plates . Column chromatography was carried out utilizing LiChroCART Si gel (5 μM) .Infrared (IR) spectra (KBr or neat) were measured using a Shimadzu IR prestige-21 Fourier transform infrared spectrophotometer . Optical rotations were recorded on a Jasco P-1020 polarimeter  in MeOH and CHCln-hexane, acetone, and chloroform), HPLC grade solvents  and deuterated solvents  for NMR measurements were procured from Merck, Taipei, Taiwan. LPS , Carr (type IV), and quercetin were purchased from MedChemExpress . MTT  was acquired from Sigma Chemical Co. .ACS grade solvents  was deposited in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan.Glinus oppositifolius was extracted 3 times with methanol (80 L each) for 7 days. The extract was concentrated under reduced pressure at 38 °C, and the residue (1.48 kg) was partitioned between H2O and EtOAc (1:1) to provide the EtOAc-soluble fraction . Fraction A (285 g) was separated by column chromatography (CC)  to afford 20 fractions: A1–A20.The dried whole plant (10.58 kg) of n-hexane:acetone = 8:3), and then HPLC (chloroform:acetone = 6:1) to obtain 4 (12.8 mg), 5 (27.4 mg), 6 (12.2 mg), 7 (8.4 mg), and 5 (7.3 mg). Fraction A14 (16.7 g) was purified by silica gel CC (ethyl acetate: n-hexane = 1:6) and HPLC (acetone:n-hexane = 1:8) to obtain 6 (12.6 mg), 7 (6.4 mg), and 8 (13.4 mg). Fraction A16 (15.5 g) was purified by silica gel CC (n-hexane:ethyl acetate = 4:1) and HPLC (n-hexane:acetone = 3:1) to obtain 9 (8.2 mg), 10 (27.5 mg), 11 (25.0 mg), 12 (8.4 mg), 13 (13.4 mg), 14 (24.5 mg), 15 (7.8 mg), 16 (6.2 mg), 17 (4.3 mg), 18 (32.4 mg), 19 (4.5 mg), and 20 (32.4 mg). Fraction A18 (13.3 g) was purified by Sephadex LH 20 CC (chloroform:methanol = 3:7), silica gel CC (n-hexane:acetone = 5:1), and then semi-preparative HPLC (chloroform: ethyl acetate = 3:2) to obtain 1 (6.6 mg), 2 (4.2 mg), and 3 (3.6 mg).Fraction A13 (7.69 g) was purified by Sephadex LH 20 CC (chloroform:methanol = 3:7), silica gel CC (1): colorless needle; mp 306.2–307.6 °C; IR (KBr) υmax: 3423 (OH), 2943, 1694 (C=O), 1458, 1385, 1155, 1113, 1061 cm−1  λma (Δε) 284 (+0.88), 250 (–0.12), 217 (+0.98), 198 (–1.34) nm; HI-EI-MS: 472.3549 [M]+ .Spergulagenin B : colorless needle; mp 305.4–306.8 °C; UV (MeOH) λmax nm (log λ): 229 (3.73); IR (KBr) υmax: 3493 (OH), 3416 (OH), 2972, 1690 (C=O), 1458, 1385, 1355, 1254, 1076 cm−1  λmax (Δε) 283 (+0.96), 249 (–0.14), 219 (+1.05), 198 (–1.09) nm; HI-EI-MS: 470.3409 [M]+ .Spergulagenin C : colorless needle; mp 286.4–287.0 °C; IR (KBr) υmax: 3447 (OH), 2938, 1697 (C=O), 1558, 1420, 1387, 1354, 1327 cm−1  λmax (Δε) 284 (+1.02), 249 (–0.20), 218 (+1.02), 197 (–0.91) nm; HI-EI-MS: 470.3407 [M]+ .Spergulagenin D : yellow powder; mp 274~276 °C; IR (KBr) υmax: 3348, 3278~2509, 1661, 1616, 1570, 1089, 1010 cm−1; 1H -NMR  δ (ppm): 6.26 , 6.52 , 7.01 , 8.14 , 12.15 .Kaempferol (5): yellow powder; mp 220~225 °C; IR (KBr) υmax: 3427, 3704~2509, 1654, 1611, 1576, 1555 cm−1; 1H-NMR  δ (ppm): 2.13 , 2.36 , 6.64 , 7.05 , 7.98 , 13.24 .6,8-Dimethyl-5,7,4′-trihydroxyflavone (6): yellow powder; mp 289~290 °C; IR (KBr) υmax: 3400, 3587~2403, 1650, 1602, 1486 cm−1; 1H-NMR  δ (ppm): 2.19 , 2.37 , 6.68 , 7.54 , 7.91 , 12.95 .5,7-Dihydroxy-6,8-dimethylflavone (7): yellow powder; mp 286~287 °C; IR (KBr) υmax: 3502~2423, 3430, 3072, 2920, 1650, 1612, 1585, 1466 cm−1; 1H-NMR  δ (ppm): 2.18 , 2.36 , 3.90 , 5.40 , 6.89 , 7.03 , 7.87 , 13.03 .5,4′-Dihydroxy-7-methoxy-6,8-dimethylflavone (8): white solid; mp 210~212 °C; IR (KBr) υmax: 3300~2500, 1696 cm−1; 1H-NMR  δ (ppm): 6.33 , 6.81 , 7.87 .4-Hydroxybenzoic acid (9): white solid; mp 110~112 °C; IR (KBr) υmax: 3170, 1676, 1600, 1519, 1454 cm−1; 1H-NMR  δ (ppm): 5.81 , 6.95 , 7.81 , 9.87 .4-Hydroxybenzaldehyde (10): white solid; mp 106~107 °C; IR (KBr) υmax: 3312, 1664, 1602, 1578 cm−1; 1H-NMR  δ (ppm): 2.56 , 6.09 , 6.89 , 7.91 .4-Hydroxyacetophenone (11): white solid; mp 124~125 °C; IR (KBr) υmax: 3358, 1689, 1608, 1585, 1514 cm−1; 1H-NMR  δ (ppm): 3.89 , 5.37 , 6.95 , 7.95 .Methyl 4-Hydroxybenzoate (p-Anisic acid (12): white solid; mp 182~184°C; IR (KBr) υmax: 3307~2503, 2926, 1686, 1605, 1578, 1516 cm−1; 1H-NMR  δ (ppm): 3.85 , 6.97 , 7.96 .13): white solid; mp 210~212 °C; IR (KBr) υmax: 3213, 2724, 2858, 1667, 1589, 1510 cm−1; 1H-NMR  δ (ppm): 3.97 , 6.21 , 7.04 , 7.42 , 7.43 , 9.83 .Vanillin (14): white solid; mp 182~184 °C; IR (KBr) υmax: 3323, 2912, 1658, 1575, 1518 cm−1; 1H-NMR  δ (ppm): 2.56 , 3.96 , 6.05 , 6.95 , 7.54 .4-Hydroxy-3-methoxyacetophenone (15): white solid; mp 105~107 °C; IR (KBr) υmax: 3307, 1672, 1608 cm−1; 1H-NMR  δ (ppm): 2.57 , 3.96 , 6.05 , 7.25 .Acetosyringone (16): white solid; mp 112~114 °C; IR (KBr) υmax: 3410, 2727, 1685, 1605, 1514 cm−1; 1H -NMR  δ (ppm): 3.98 , 5.91 , 7.15 , 9.82 .4-Hydroxy-3, 5-dimethoxybenzaldehyde (17): white solid; mp 116–117 °C; IR (KBr) υmax: 3370, 1585, 1512 cm−1; 1H-NMR  δ (ppm): 4.62 , 4.79 , 6.82 , 7.25 .4-Hydroxybenzyl alcohol (18): white solid; mp 92~93 °C; IR (KBr) υmax: 3392, 1599, 1514 cm−1; 1H-NMR  δ (ppm): 2.80 , 3.83 , 4.75 , 6.79 , 7.10 .2-(4-Hydroxyphenyl)ethanol (19): white solid; mp 133~135 °C; IR (KBr) υmax: 3267~2582, 2962, 1684, 1629 cm−1; 1H-NMR  δ (ppm): 6.49 , 7.41 , 7.60 , 7.68 .Cinnamic acid (trans-Ferulic acid (20): white solid, mp 168~169 °C; IR (KBr) υmax: 3435, 3481~2750, 1690, 1662, 1515 cm−1; 1H-NMR  δ (ppm): 3.89 , 6.30 , 6.80 , 7.05 , 7.17 , 7.59 , 8.17 .2 + ion at m/z 472.3549  and supported by the 1H- and 13C-NMR data. The IR absorption bands implied the presence of OH (3423 cm−1) and acetyl group (1694 cm−1). The 1H- and 13C-NMR data of 1 showed the acetyl group  and seven methyl signals . Comparison of the 1H- and 13C-NMR data of 1 with those of spergulagenin A (1a) [C 217.3 (C-3)] at C-3 of 1 replaced the 3β-hydoxyl group of spergulagenin A (1a) chrysen-9-one, named spergulagenin B.Spergulagenin B  was obtained as colorless needle crystal. Its molecular formula, C30H46O4, was confirmed by the positive HR-ESI-MS at m/z 470.3409 [M]+  and supported by the 13C, 1H, and DEPT NMR data. IR absorptions for OH (3493 and 3416 cm−1) functions were observed. The presence of the acetyl group was supported by a band at 1690 cm−1 in the IR spectrum and was affirmed by signal at δ 25.9, and δ 217.0 in the 13C-NMR spectrum. The 13C- and 1H-NMR data of 2 revealed the acetyl group  and seven methyl signals . The 1H- and 13C-NMR data of 2 were similar to those of 1, except that the double bond at C-1,2  of 2 replaced C-1,2 single bond  of 1. This was supported by the HMBC correlations between H-1 (δH 7.10) and C-3 (δC 205.3), C-4 (δC 39.2), C-5 (δC 53.3), and C-9 (δC 42.7); and between H-2 (δH 5.83) and C-4 (δC 39.2) and C-10 (δC 44.6). The NOESY cross-peaks between H-5/H-9, H-9/H-12, H-12/H-27, H-13/H-17, H-13/H-26, H-16/H-29, H-23/H-25, H-25/H-26, H-27/H-28, and, H-28/H-29 suggested that H-13, H-17, Me (23), Me (25) and Me (26) are on the β-side and H-5, H-9, H-12, H-16, Me (27), Me (28) and Me (29) are on the α-side of 1. The full assignment of 13C- and 1H-NMR resonances was confirmed by 13C-DEPT (1H–1H COSY (2 was evidenced by the ECD Cotton effects at 283 (Δε +0.96), 249 (Δε −0.14), 219 (Δε +1.05), and 198 (Δε −1.09) nm, in analogy with those of 1 and glinusopposide D  chrysen-9-one, named spergulagenin C.Spergulagenin C  was obtained as colorless needle. Its molecular formula, C30H46O4, was determined on the basis of the positive HR-EI-MS at m/z 470.3407 [M]+  and supported by the 1H, 13C, and DEPT NMR data. IR absorptions for OH (3447 cm−1) and carbonyl (1697 cm−1) functions were observed. The 1H- and 13C-NMR data of 3 showed the acetyl group  and seven methyl signals . The 1H- and 13C-NMR data of 3 were similar to those of 1, except that the carbonyl group at C-12 [δC 210.9 (C-12)] of 3 replaced the 12β-OH group  of 1. This was supported by the HMBC correlations between H-11  and C-9 (δC 49.6), C-12 (δC 210.9); and between H-9 (δH 1.70) and C-10 (δC 37.0) and C-12 (δC 210.9). The relative stereochemistry of 3 was elucidated on the basis of NOESY experiments (3. The full assignment of 13C- and 1H-NMR resonances was determined by 13C-DEPT (1H–1H COSY (3 was evidenced by the ECD Cotton effects at 284 (Δε +0.76), 249 (Δε −0.09), 218 (Δε +1.00), and 197 (Δε −0.91) nm, in analogy with those of 1 and glinusopposide D chrysene-9,13(8H)-dione, named spergulagenin D.Spergulagenin D  with the literature values or corresponding authentic samples, and this included four flavonoids, kaempferol (4) [5) [6) [7) [8) [9) [10) [11) [p-anisic acid (12) [13) [14) [15) [16) [17) [18) [19) [20) [The known isolated compounds were readily determined by a comparison of physical and spectroscopic data (erol (4) , 6, 8-di [5) [6) , and 5,4 [6) [7) , and thi [7) [8) , 4-hydro [8) [9) , 4-hydro[9) [10) , methyl 10) [11) , p-anisicid (12) , vanilli12) [13) , 4-hydro13) [14) , acetosy14) [15) , 4-hydro15) [16) , 4-hydro16) [17) , 2-(4-hy17) [18) , cinnami18) [19) , and tra19) [20) .G. oppositifolius were also evaluated by suppressing lipopolysaccharide (LPS)-induced NO generation in macrophage cell line RAW264.7. The inhibitory activity data of the isolates 1–20 on NO generation by macrophages are shown in 3, 5, 19, and 20 exhibited inhibitory effects on lipopolysaccharides (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values of 17.03 ± 1.28, 18.21 ± 1.15, 16.30 ± 1.41, and 12.64 ± 1.14 μM, respectively (3)  exhibited more effective inhibition than its analogues, spergulagenin B (1) (with 3-oxo-12β-hydroxy groups) and spergulagenin C (2)  against LPS-induced NO generation. (c) Among the flavonoids, 6,8-dimethyl-5,7,4′-trihydroxyflavone (5)  exhibited more effective inhibition than its analogues, kaempferol (4) , 5,7-dihydroxy-6,8-dimethylflavone (6) , and 5,4′-dihydroxy-7-methoxy-6,8-dimethylflavone (7)  against LPS-induced NO generation. (d) trans-ferulic acid (20) is the most effective among the isolated compounds against LPS-induced NO generation. In addition, compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced TNF-α production in RAW 264.7 cells with IC50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 μM, respectively (Nitric oxide (NO) is derived from the oxidation of L-arginine by NO synthase (NOS) and is a mediator in the inflammatory response involved in host defense . In inflectively ; (b) Amoectively .G. oppositifolius and its isolates could stimulate future development of new anti-inflammatory agents.The above findings indicated that the promising inhibitory activity against LPS-induced NO and TNF-α generation of 1), spergulagenin C (2), and spergulagenin D (3)—were isolated from aerial part of G. oppositifolius. The structures of these new compounds were elucidated on the basis of spectral data. The effects on macrophage pro-inflammatory responses of isolated compounds were evaluated by suppressing LPS-induced NO generation by macrophage RAW264.7 cells. The results of anti-inflammatory assays show that compounds 3, 5, 19, and 20 can obviously inhibit LPS-induced NO generation. Trans-ferulic acid (20) is the most effective among the isolated compounds, with IC50 value of 12.64 ± 1.14 μM, against LPS-induced NO generation. Furthermore, compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced TNF-α production in RAW 264.7 cells with IC50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 μM, respectively. Our research indicates G. oppositifolius and its isolates  are worth further research and may be expectantly developed as candidates for the treatment or prevention of various inflammatory diseases (such as dermatitis and arthritis). This study also provides anti-inflammatory scientific evidence for the use of traditional herbal medicine (G. oppositifolius) in the treatment of dermatitis and chronic inflammatory diseases [Twenty compounds, including three new triterpenoids—spergulagenin B (agenin C , and spe


Euphorbia genus are known for their ability to regulate the protein kinase C (PKC) family, which mediates their ability to promote the proliferation of neural precursor cells (NPCs) or neuroblast differentiation into neurons. In this work, we describe the isolation from E. resinifera Berg latex of fifteen 12-deoxyphorbol esters (1–15). A triester of 12-deoxy-16-hydroxyphorbol (4) and a 12-deoxyphorbol 13,20-diester (13) are described here for the first time. Additionally, detailed structural elucidation is provided for compounds 3, 5, 6, 14 and 15. The absolute configuration for compounds 3, 4, 6, 13, 14 and 15 was established by the comparison of their theoretical and experimental electronic circular dichroism (ECD) spectra. Access to the above-described collection of 12-deoxyphorbol derivatives, with several substitution patterns and attached acyl moieties, allowed for the study of their fragmentation patterns in the collision-induced dissociation of multiple ions, without precursor ion isolation mass spectra experiments (HRMSE), which, in turn, revealed a correlation between specific substitution patterns and the fragmentation pathways in their HRMSE spectra. In turn, this allowed for a targeted UHPLC-HRMSE analysis and a biased non-targeted UHPLC-HRMSE analysis of 12-deoxyphorbols in E. resinifera latex which yielded the detection and identification of four additional 12-deoxyphorbols not previously isolated in the initial column fractionation work. One of them, identified as 12-deoxy-16-hydroxyphorbol 20-acetate 13-phenylacetate 16-propionate (20), has not been described before.Diterpenes from the  Euphorbia is the largest genus in the Euphorbiaceae family, and it is well known for the extraordinary chemical diversity and intriguing biological activities of its constituents + molecular ion in its HRMSE spectrum at m/z 609.2463   and DPPT (2) , in turn, originated from the loss of ester groups at C-13 and C-16 from the parent molecular ion. The proposed fragmentation pathways leading to the above-mentioned ions can be found in 1), DPPT (2) and compound 3.On the other hand, compound 09.2464) c, which 47.1486, c, which DPPT (2) a,b, and 3 showed similar 1H and 13C NMR data to those of DPPI (1) and DPPT (2) but displayed the presence of signals characteristic of a benzoate ester group , consistent with a loss of phenylacetic acid from the parent molecular ion  was identical to the one previously observed for 1 (DPPI) and 2 (DPPT).Furthermore, compound er group , whose c signals . These dular ion c, pointend H3-17  confirme3 (DPPBz) was previously identified by Hergenhahn et al. as a component of E. resinifera latex (RL22) + molecular ions in their HRMSE spectra at m/z 617.2755 , m/z 629.2745  and at m/z 651.2601   (1), DPPT (2) and DPPBz (3) ) and m/z 411.1784, 393.1678 and 333.1467  , which, in turn, originates from the loss of an ester group at C-16 from the parent molecular ions of compounds 4, 5 and 6 (4), AcDPPT (5) and Ac DPPBz (6).Compounds 51.2570) , which pculated) a–c, whicculated) a were al 5 and 6 . The pro1H and 13C NMR data of compounds 4, 5 and 6 were very similar with those of DPPI (1), DPPT (2) and DPPBz (3), respectively, except for the presence of signals corresponding to an extra acetate group in each compound  could be understood to originate from a loss of acetic acid from the precursor at m/z 393.1678  in compounds 4, 5 and 6  for compound 4, m/z 493.2221  for compound 5 and m/z 515.2083  for compound 6 were consistent with a loss of phenyl acetic acid from a molecular ion in each compound  in the HRMSE spectra of compounds 4, 5 and 6, which would be consistent with losses of isobutyric, tiglic and benzoic acids from the molecular ions of previously mentioned compounds, respectively , DPPT (2) + ion) not only in the HRMSE spectra of compounds 13 and 14, (E spectra of AcDPB (10), AcDPA (11) and AcDPP (12) (+ ions for the compounds AcDPB (10), AcDPA (11) and AcDPP (12) and by the loss of carboxilic acids of the formulas C5H10O2 and C9H10O3, respectively, from [M + Na]+ ions for compounds 13 and 14. The proposed fragmentation pathways leading to previously described ions can be found in Compounds ectively , which pculated) . Alterna and 14,  but as wDPP (12) . The for13 and 14 exhibited 1H and 13C NMR spectra (10), AcDPA (11) and AcDPP (12), with differences in the nature of the acyloxy fragment at C-13. The 1H NMR spectra of compound 13 showed a doublet at δH 0.96 ppm , correlated in the 1H-1H COSY spectrum with a multiplet at δH 2.08 ppm (1H), which, in turn, correlated with a signal at δH 2.21 ppm , which is consistent with a 3-methylbutanoate moiety (13C NMR spectrum at δc 22.8 and 22.7 ppm (q), 27.0 ppm (d) and 44.4 ppm (t), which were assigned to C-4′/C-5′, C-3′ and C-2′, respectively  and δC 172.6 ppm (RCOO), which, in turn, was further correlated with a singlet at δH 2.02 ppm (3H); this, conversely, supported a C-13 location for 3-methylbutanoate substituent. Therefore, a structure of 12-deoxyphorbol 20-acetate 13-(3-methyl)butanoate (AcDPiPn) was assigned to compound 13.Furthermore, compounds  spectra  very sime moiety . On the ectively . Correla to C-1′ , confirm1H and 13C NMR spectra for compound 14 showed close similarities with those of compound 13; the main differences being the presence of a group of 1H and 13C resonances correlated in the HMBC spectrum  phenylacetate (AcDPMeOP). NOESY correlations of H3-19/H-1/H3-18α and H-11β/H-8β/H3-17β, on the one hand, and of H2-20/H-7/H-14α/H3-16α, on the other (13)), were consistent with a relative configuration 4R(S),8S(R),9S(R),10S(R),11R(S),13S(R),14R(S) for both AcDPiPn (13) and AcDPMeOP (14).On the other hand, the analysis of the  (OCH3)]  and assi13 and 14 was determined by the comparison of their experimental electronic circular dichroism (ECD), with the computed ECD spectrum for their 4R,8S,9R,10S,11R,13S,14R steroisomers, calculated from quantum mechanical time-dependent density functional theory (TDDFT) calculations with a 6–31 + G level of theory, using the Gaussian 16 program + molecular ion in its HRMSE spectrum at m/z 425.2322   which could be assigned to losses of a neutral fragment of formula C4H8O2 and further losses of water, 2 molecules of water and a loss of water and CO, respectively   , which aectively . A propo, 7) see .1H and 13C NMR spectra (7)); the main differences were the absence of a signal corresponding to a hydroxymethylene group at C-20 and the presence of a new methyl group . This 1H-NMR signal correlates, on the one hand, with the 13C-NMR resonances for C-5, C-6 and C-7 in the HMBC experiment . NOESY correlations of H3-19/H-1/H3-18α, H-11β/H-8β/H3-17β and H3-20/H-7/H-14α/H3-16α (R(S),8S(R),9R(S),10S(R),11R(S),13S(R),14R(S) for compound 15, which is in accordance with the one previously described for other tigliane derivatives 3); UV (MeOH) λmax (log ε) 291 (−0.55); ECD  λ max (Δε) 217 (0.35), 225 (0.04), 246 (1.55), 267 (−1.04), 299 (−0.01), 337 (−0.48); IR (film) νmax 3416.78, 2925.08, 1712.19, 1633.64, 1454.77, 1386.12, 1335.45, 1244.85, 1100.33, 1015.65, 947.54, 927.99, 754.5, 699.95, 665.06, 626.32, 534.64, 469.85, 456.65 cm−1; 1H and 13C NMR data, see m/z 609.2463 [M + Na]+ ; for further details, see -12-Deoxy-16-hydroxyphorbol 20-acetate 16-isobutyrate 13-phenylacetate (AcDPPI (4)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 291 (0.05), 319 (−0.17); ECD  λ max (Δε) 233 (−0.19), 245 (1.37), 248 (1.31), 251 (1.19), 272 (−0.85), 297 (−0.06), 336 (−0.71); IR (film) νmax 3411.04, 3124.62, 2924.66, 2578.29, 1711.84, 1535.18, 1378.03, 1364.35, 1332.08, 1296.70, 1242.28, 1200.80, 1135.05, 1107.46, 1020.51, 820.26, 755.8, 498.17, 474.99 cm−1; 1H and 13C NMR data, see m/z 617.2755 [M + Na]+ ; for further details, see -12-Deoxy-16-hydroxyphorbol 20-acetate 13-phenylacetate 16-tigliate (AcDPPT (5)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 289 (−0.25), 318 (−0.55); ECD  λ max (Δε) 205 (0.15), 220 (−0.14), 227 (0.05), 232 (−0.07), 244 (1.58), 267 (−1.06), 299 (−0.18); IR (film) νmax 3408.38, 3099.26, 2925.57, 2582.32, 1705.14, 1526.07, 1380.41, 1362.73, 1270.85, 1200.37, 1141.67, 1107.62, 1077.14, 1056.42, 1017.71, 821.66, 755.56, 498.35, 493.63, 470.18, 457.25 cm−1; 1H and 13C NMR data, see m/z 629.2745 [M + Na]+ ; for further details, see -12-Deoxy-16-hydroxyphorbol 20-acetate 16-benzoate 13-phenylacetate (AcDPPBz (6)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 290 (−0.49); ECD  λ max (Δε) 205 (0.15), 220 (−0.14), 227 (0.06), 232 (−0.07), 244 (1.58), 267 (−1.06), 299 (−0.18); IR (film) νmax 3479.26, 3187.11, 2983.15, 2629.86, 1746.08, 1565.88, 1405.59, 1391.64, 1358.72, 1322.63, 1267.13, 1224.82, 1157.75, 1129.61, 1040.92, 836.67, 770.92, 508.13, 484.49, 450.85 cm−1; 1H and 13C NMR data, see m/z 651.2601 [M + Na]+ ; for further details, see -12-Deoxyphorbol 20-acetate 13-(3-methyl)butanoate (AcDPiPn (13)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 289 (−0.56); ECD  λ max (Δε) 213 (−0.05). 232 (2.52), 272 (−0.26), 299 (−0.03). 337 (−0.23), 391 (0.03); IR (film) νmax 3395.34, 3078.68, 2930.26, 2318.33, 1712.41, 1530.44, 1514.07, 1492.18, 1246.98, 845.9, 756.19, 483.77, 468.75, 454.88 cm−1; 1H and 13C NMR data, see m/z 497.2527 [M + Na]+ ; for further details, see -12-Deoxyphorbol 20-acetate 13-(p-methoxy)phenylacetate (AcDPMeOP (14)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 290 (−0.34); ECD  λ max (Δε) 213 (−0.05), 232 (2.52), 272 (−0.26), 299 (−0.03), 337 (−0.23), 391 (0.03); IR (film) νmax 3364.12, 3085.33, 2926.02, 2344.07, 1698.80, 1544.00, 1380.39, 1347.06, 1331.46, 1301.59, 1243.45, 1203.83, 1165.4, 1106.33, 1014.30, 821.79, 756.00, 498.08, 486.81, 480.33, 476.94, 465.02, 457.12 cm−1; 1H and 13C NMR data, see m/z 561.2455 [M + Na]+ ; for further details, see -12,20-Dideoxyphorbol 13-isobutyrate (diDPB (15)): amorphous powder; [α]3); UV (MeOH) λmax (log ε) 288 (−0.61); ECD  λ max (Δε) 209 (−0.01), 225 (1.92), 238 (1.06), 245 (1.45), 269 (−0.85), 303 (−0.14), 335 (−0.43); IR (film) νmax 3361.39, 3047.89, 2900.96, 2295.15, 1695.29, 1515.14, 1498.93, 1477.26, 1234.51, 837.44, 748.63, 478.93, 464.06, 450.33 cm−1; 1H and 13C NMR data, see m/z 425.2322 [M + Na]+ ; for further details, see , AcDPPU2 (20), AcDPPU3 (21) and diDPU4 (22)  , which w1 (19): HRESIMS m/z 589.2787 [M + Na]+ ; for further details, see DPPU2 (20): HRESIMS m/z 603.2540 [M + Na]+ ; for further details, see AcDPPU3 (21): HRESIMS m/z 631.2911 [M + Na]+ ; for further details, see AcDPPU4 (22): HRESIMS m/z 437.2304 [M + Na]+ ; for further details, see diDPU1–15) have been isolated from E. resinifera Berg latex, together with resiniferatoxin (16) and ingol derivatives EOF1 (17) and EOF2 (18). AcDPPI (4), a triester of 12-deoxy-16-hydroxyphorbol, and AcDPiPn (13), a 12-deoxyphorbol 13,20-diester, are described here for the first time. Additionally, detailed structural elucidation is provided for DPPBz (3), AcDPPT (5), AcDPPBz (6), AcDPMeOP (14) and diDPB (15), including analysis of their HRMSE. The absolute configuration for compounds 4, 6, 13, 14 and 15 was established by a comparison of their theoretical and experimental electronic circular dichroism (ECD) spectra. Access to the above-described collection of 12-deoxyphorbol derivatives, with several substitution patterns and attached acyl moieties, allowed for the study of their fragmentation patterns in the collision-induced dissociation of multiple ions, without precursor ion isolation mass spectra experiments (HRMSE) [E spectra. In turn, this allowed for a targeted UHPLC-HRMSE analysis and a biased non-targeted UHPLC-HRMSE analysis [E. resinifera latex which yielded the detection and identification (level 2 or 3 [1 (19), AcDPPU2 (20), AcDPPU3 (21) and diDPU4 (22), not previously isolated in the initial column fractionation work. To our knowledge, AcDPPU2 (20), identified as 12-deoxy-16-hydroxyphorbol 20-acetate-13-phenylacetate-16-propionate  of four level 2 ), has no


V. tetrasperma are consumed daily as cooked vegetables and can provide various health benefits. The antioxidant and anti-inflammatory capacities of its total extract and fractions were accessed for the first time in this study. The bioactivities guided the separation of the active fraction (EtOAc), leading to the identification of nine flavonoid glycoside compositions from this plant for the first time. In addition, the fractions and all isolates were evaluated for their inhibition against NO and IL-8 production in LPS-stimulated RAW264.7 and HT-29 cell lines, respectively. The most active ingredient was further assayed for its inhibitory abilities to iNOS and COX-2 proteins. Indeed, its mechanisms of action modes were confirmed by Western blotting assays through the reduction in their expression levels. An in silico approach revealed the substantial binding energies of docked compounds into established complexes to verify their anti-inflammatory properties. In addition, the presence of active components in the plant was validated by an established method on the UPLC-DAD system. Our research has boosted the value of this vegetable’s daily use and provided a therapeutic approach for the development of functional food products for health improvement regarding the treatment of oxidation and inflammation.The young leaves and shoots of  Antioxidants and inflammatory inhibitors are served as therapeutic interventions for the treatment of disease development. For example, antioxidant therapy could delay the progression of atherosclerotic lesions . AntioxiV. faba, V. sativa, V. sativum, V. parviflora, and V. tenuifolia have shown antioxidant capacity -), was used to ensure accuracy throughout the mass spectrometry analysis. All data acquisition and processes were performed for qualitative analysis by using MassLynx V4.1 and UNIFI V1.9 . The gradient elution of the mobile phase was conducted following the above established gradient elution.The mass fragmentation of isolated compounds : δH 7.45 , 7.41 , 6.90 , 6.76 , 6.75 , 6.42 , 5.35 , 5.18 , 3.91 , 3.65 , 3.71 , 3.54 , 3.46–3.64 , 3.30 , 3.15 . 13C NMR : δC 182.0 (C-4), 164.6 (C-2), 162.7 (C-7), 161.2 (C-5), 157.0 (C-9), 150.0 (C-4′), 145.9 (C-3′), 121.4 (C-1′), 119.3 (C-6′), 116.1 (C-5′), 113.6 (C-2′), 108.8 (C-1‴), 105.4 (C-10), 103.2 (C-3), 99.4 (C-6), 98.1 (C-1″), 94.7 (C-8), 79.4 (C-3‴), 77.1 (C-5″), 76.8 (C-2‴), 76.1 (C-2″), 75.7 (C-3″), 74.1 (C-4‴), 69.8 (C-4″), 64.3 (C-5‴), 60.6 (C-6″). Mass m/z 579.1371 [M − H]− .Compound 2: yellowish powder; 1H NMR : δH 7.45 , 7.41 , 6.90 , 6.79 , 6.75 , 6.44 , 5.07 , 3.71 , 3.51 , 3.27–3.29 , 3.10 . 13C NMR : δC 182.0 (C-4), 164.6 (C-2), 163.0 (C-7), 161.2 (C-5), 157.0 (C-9), 150.1 (C-4′), 145.9 (C-3′), 121.4 (C-1′), 119.2 (C-6′), 116.0 (C-5′), 113.6 (C-2′), 105.4 (C-10), 103.2 (C-3), 99.9 (C-6), 99.6 (C-1″), 94.8 (C-8), 77.2 (C-3″), 76.4 (C-5″), 73.2 (C-2″), 69.6 (C-4″), 60.7 (C-6″). HR-ESI-MS m/z 447.0921 [M − H]− .Compound 3: yellowish powder. 1H NMR : δH 7.96 , 6.94 , 6.87 , 6.81 , 6.43 , 5.35 , 5.15 , 3.91 , 3.73 , 3.71 , 3.66 , 3.45-3.52 , 3.30 . 13C NMR : δC 182.1 (C-4), 164.3 (C-2), 162.7 (C-5), 161.4 (C-7), 161.2 (C-4′), 157.0 (C-9), 128.7 , 121.05 (C-1′), 116.1 , 108.8 (C-1‴), 105.4 (C-10), 103.0 (C-3), 99.4 (C-6), 98.1 (C-1″), 94.8 (C-8), 79.3 (C-3‴), 77.1 (C-3″), 76.8 (C-2″), 76.1 (C-2‴), 75.7 (C-5″), 74.0 (C-4‴), 69.8 (C-4″), 64.2 (C-5‴), 60.5 (C-6″). HR-ESI-MS m/z 563.1428 [M − H]− .Compound 4: yellowish powder; 1H NMR : δH 7.46 , 7.44 , 6.89 , 6.74 , 6.73 , 6.41 , 5.35 , 5.21 , 4.40 , 4.10 , 3.93 , 3.73–3.80 , 3.66 , 3.50–3.52 , 3.30 , 3.20 . 13C NMR : δC: 181.9 (C-4), 167.9 (C-3⁗), 167.1 (C-1⁗), 164.6 (C-2), 162.4 (C-7), 161.2 (C-5), 157.0 (C-9), 150.0 (C-4′), 146.0 (C-3′), 121.4 (C-1′), 119.1 (C-6′), 116.0 (C-5′), 113.6 (C-2′), 108.8 (C-1‴), 105.5 (C-10), 103.2 (C-3), 99.4 (C-6), 97.9 (C-1″), 94.6 (C-8), 79.3 (C-3‴), 76.4 (C-3″), 76.1 (C-2″), 75.5 (C-2‴), 74.0 (C-4‴), 73.7 (C-5″), 69.8 (C-4″), 64.2 (C-5‴), 64.2 (C-6″), 41.2 (C-2⁗). HR-ESI-MS m/z 665.1373 [M − H]− .Compound 5: yellowish powder; 1H NMR : δH 7.57 , 7.46 , 7.10 , 6.84 , 6.79 , 6.43 , 5.35 , 5.18 , 3.91 , 3.95 , 3.74 , 3.71 1H, m, H2-6″), 3.66 , 3.45–3.50 , 3.30 , 3.17 . 13C NMR : δC 182.0 (C-4), 164.2 (C-2), 162.8 (C-7), 161.2 (C-5), 157.0 (C-9), 151.4 (C-4′), 146.9 (C-3′), 122.9 (C-1′), 118.9 (C-6′), 113.1 (C-2′), 112.2 (C-5′), 108.9 (C-1‴), 105.5 (C-10), 103.9 (C-3), 99.6 (C-6), 98.1 (C-1″), 94.8 (C-8), 79.3 (C-3‴), 76.1 (C-5″), 77.0 (C-3″), 76.1 (C-2‴), 75.7 (C-2″), 74.0 (C-4‴), 69.8 (C-4″), 64.2 (C-5‴), 60.5 (C-6″), 55.8 (C-OCH3). HR-ESI-MS m/z 593.1509 [M − H]− .Compound 6: yellowish powder; 1H NMR : δH 7.96 , 6.94 , 6.87 , 6.78 , 6.42 , 5.35 , 5.21 , 4.39 , 4.12 , 3.92 , 3.78 , 3.75 , 3.67 , 3.48–3.57 , 3.21 , 2.01 . 13C NMR : δC 182.0 (C-4), 167.9 (C-7″), 164.4 (C-2), 162.4 (C-4′), 161.4 (C-7), 161.2 (C-5), 157.0 (C-9), 128.7 , 121.0 (C-1′), 116.0 , 108.8 (C-1‴), 105.5 (C-10), 103.1 (C-3), 99.4 (C-6), 97.8 (C-1″), 94.7 (C-8), 79.3 (C-3‴), 76.5 (C-3″), 76.81 (C-2″), 76.1 (C-2‴), 75.5 (C-5″), 74.0 (C-4‴), 69.8 (C-4″), 64.2 (C-5‴), 64.0 (C-6″), 20.6 (C-OCH3). HR-ESI-MS m/z 605.1528 [M − H]− .Compound 7: yellowish powder; 1H NMR : δH 7.56 , 7.55 , 7.05 , 6.84 , 6.60 , 6.39 , 5.38 , 5.17 , 4.45 , 4.04 , 3.93 , 3.85 , 3.78 , 3.67 , 3.75 , 3.50–3.56 , 3.30 , 3.19 , 2.01. . 13C NMR : δC 182.0 (C-4), 167.9 (C-CH3CO), 164.5 (C-2), 162.6 (C-7), 161.1 (C-5), 157.1 (C-9), 151.4 (C-4′), 147.4 (C-3′), 122.9 (C-1′), 118.7 (C-6′), 113.2 (C-2′), 112.2 (C-5′), 108.8 (C-1‴), 105.6 (C-10), 103.9 (C-3), 99.7 (C-6), 98.1 (C-1″), 94.5 (C-8), 79.3 (C-3‴), 76.4 (C-3″), 76.1 (C-2‴), 75.6 (C-2″), 74.0 (C-5″), 73.9 (C-4‴), 69.9 (C-4″), 64.1 (C-5‴), 63.9 (C-6″), 55.7 (5′-OCH3), 20.3 (CH3). HR-ESI-MS m/z 635.1634 [M − H]− .Compound 8: yellowish powder; 1H NMR : δH 7.70 , 7.27 , 6.88 , 6.84 , 6.73 , 6.61 , 6.60 , 6.38 , 6.31 , 6.01 , 5.50 , 5.18 , 4.29 , 4.22 , 4.18 , 3.92 , 3.86–3.88 , 3.66–3.76 , 3.74 , 3.74 , 3.68 , 3.56 , 3.43 . 13C NMR : δC 183.8 (C-4), 168.7 (CO-5‴), 166.4 (C-2), 164.2 (C-7), 163.0 (C-4′), 162.8 (C-5), 158.8 (C-9), 150.6 (C-4⁗), 149.1 (C-3⁗), 146.9 (C-β), 129.5 , 127.3 (C-6′), 123.0 (C-1⁗), 124.2 (C-6⁗), 116.9 , 116.3 (C-5⁗), 114.3 (C-α), 111.1 (C-2⁗), 109.9 (C-1‴), 107.0 (C-10), 104.1 (C-3), 100.4 (C-1″), 99.6 (C-6), 95.9 (C-8), 79.2 (C-3‴), 78.7 (C-5″), 78.6 (C-2‴), 78.3 (C-3″), 77.3 (C-2″), 75.1 (C-4‴), 71.3 (C-4″), 67.8 (C-5‴), 62.4 (C-6″), 56.2 (3⁗-OCH3). HR-ESI-MS m/z 739.1878 [M − H]− .Compound 9: yellowish powder; 1H NMR : δH 7.29 , 7.28 , 7.25 , 6.86 , 6.84 , 6.75 , 6.64 , 6.60 , 6.40 , 6.29 , 6.03 , 5.50 , 5.18 , 3.75 , 3.47–3.91 , 3.40 . HR-ESI-MS m/z 755.1856 [M − H]− .Compound 2. RAW264.7 cells were seeded into 96-well plates followed by preincubation for 24 h before addition with samples for 1 h, and then stimulation with LPS (1 μg/mL) for 16 h, whereas HT-29 cells were seeded into 96-well plates, preincubated for 24 h, then treated with 100 μM of compounds (1–9) for 2 h, and stimulated with LPS (100 ng/mL) for 12 h. The cultured cells were incubated with MTT (5 mg/mL) at 37 °C for four hours. The supernatants were removed and 100 µL dimethyl sulfoxide (DMSO)  was added. After five minutes, the absorbance of the formazan crystals was measured at 570 nm in a microplate reader .Mouse macrophages (RAW264.7) and human colon epithelial (HT-29) cells were acquired from the Korean Cell Lines Bank . These cell lines were grown at 37 °C and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, streptomycin sulfate (100 µg/mL), and penicillin (100 IU/mL) in a humidified atmosphere of 5% CO4 cells/well for 20 h and then treated with positive control  and samples  at a concentration of 100 μg/mL. Subsequently, 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA) diluted with 1/1000 in a serum-free medium was added after removing all the cell culture medium. After incubation for 20 min at 37 °C in dark, the cells were washed 3 times with PBS to remove DCFH-DA, which had not been entered into cells. The cells were visualized by an inverted fluorescence microscope and further detected by a fluorescent microplate reader at 485 and 530 nm.RAW264.7 were seeded in a 6-well plate at concentration of 1 × 105 cells/well) were pretreated with samples  for 1 h and stimulated with LPS (1 μg/mL) for 20 h. Then, the collected supernatant was incubated with an equal volume of Griess reagent by using equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) N-(1-naphtyl) ethylenediamine at room temperature for 10 min. The absorption was measured at 550 nm by microplate reader . The LPS-induced NO production was measured in the treated samples and controls (with and without LPS in the absence of sample addition).The level of NO production was determined by measuring the amount of secreted nitrite from the cell culture supernatants, as described previously . BrieflyThe scavenging abilities of samples on DPPH and ABTS radicals were assayed following a previous method  in which6 and total extract at concentrations of 100, 200 μM and 100, 200 μg/mL, respectively, for 1 h, then stimulated with LPS (1 μg/mL). After 24 h, (iNOS and COX-2) cells were washed with cold phosphate-buffered saline (PBS) twice. The cell lysates were extracted using a protein extraction solution . The protein concentration was determined by the Bradford assay method. Equal amounts of control, LPS, and compound-treated samples (30 µg) were separated by 10% SDS-PAGE gels and transferred on PVDF membranes. The membranes were blocked with 5% skim milk powder in plain buffer [20 mM Tris-HCl (pH 7.4) and 4 M NaCl] for one hour at room temperature. The membranes were incubated with primary antibodies  at 4 °C overnight, and then washed three times with a wash buffer  for 10 min. They were incubated in specific secondary antibodies (1:2000 dilution) conjugated with horseradish peroxidase at RT for two hours and washed. The protein signals were obtained using chemiluminescence detection reagents  and imaged using a bio-imaging system .The reduction ability of iNOS and COX-2 protein expression on RAW264.7 cells of the strong Active Compound 6 and total extract was examined, following our previous study . Briefly5 cells/well) in 96-well plates using ELISA kit  following the manufacturer’s instructions.The IL-8 production assay was used on HT-29 cells . Proteins and ligand were prepared using MGL tools 1.5.6. The structures of receptors were processed by removing water, adding polar hydrogen atoms, and Kollman charges. Structure  was downloaded from PubChem  in sdf formats, and structures (apigenin-7-O-[2″-O-(5‴-O-feruloyl)-D-apiofuranosyl]-D-glucopyranoside, 8; luteolin-[2″-O-(5‴-O-feruloyl)-D-apiofuranosyl]-D-glucopyranoside, 9) were prepared using by Avogadro package via force field method-MMFF94. Then, the geometries of these structures were transformed into pdbqt format using the Open Babel. The ligand conformations were performed by adding Gasteiger charges. A grid box of coordinates was determined using Pymol. A total of 100 runs were conducted under default parameters using the Lamarckian genetic algorithm. The protein–ligands docking calculations were performed using AutoDock 4.2. Residues–ligand interactions were visualized with Discovery studio 2021.The 3D structures of iNOS (PDB ID: 3E7G), COX-2 (PDB ID: 5IKQ), and IL-8 (PDB ID: 5D14) proteins were obtained from the RCSB protein data bank  equipped with an autosampler, column temperature controller, and DAD detector. A Waters Acquity UPLC HSS T3 column  was used to carry out the experiment at 40 °C. The mobile phases were buffered with 0.1% formic acid for both aqueous  and organic  elements. The flow rate was set to 0.4 mL/min, and the injection volume was 2 μL. The gradient elution of the mobile phase was conducted as follows: 0–1 min (B: 5 %), 1–4 min (B: 5–15%), 4–12 min (B: 15–35%), 12–17 min (B: 35–45%), 17–23 min (B: 45–100%). Then, it was held at 100% B for 3 min and then returned to the initial conditions for re-equilibration. The range of DAD detection wavelength was set to 200–400 nm and chromatograms were recorded at 254 nm.1 and 3–7) were isolated from V. teterasperma, and further purified with purity of 93.3, 95.2, 98.0, 97.2, 95.3, and 97.4%, respectively.The marker compounds  was extracted with 100% ethanol (10 mL) by sonication for 90 min. The extracted solution was filtered through filter paper. Its solvent was removed at room temperature using an evaporator under nitrogen gas. For a quantitative analysis of the V. terterasperma, a total extract (13 mg) was dissolved in 1 mL of methanol and sonicated for 5 min. The sample solutions were then filtered through a 0.22 µm PTFE syringe filter and the filtrates were collected and stored at 4 °C prior to use.Sample preparation: a dried herb of r2) were calculated. The LOD values were calculated using LOD = 3.3 × SD/S. In addition, the LOQ values were calculated using LOQ = 10 × SD/S, while SD was the standard deviation and S was the slope of the calibration curve.Linearity, LODs, LOQs: the identification of standards in the sample was accessed by calculating relative retention time and UV absorption maxima in comparison to those of standard mixture at the same UPLC analytical conditions. The UPLC-DAD method was applied to conduct the experiment with validation parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy, in accordance with the guidelines described at the international conference on Harmonisation, whereas the calibration curves were built using the regression equation based on the peak areas (y) in response to the corresponding concentrations  of six markers in the standard solutions at eight different concentrations, and the correlation coefficients  was obtained using the following equation: RSD (%) = SD × 100/mean measured concentration.1 and 3 , Compounds 4 and 5 , and Compounds 6  and 7 . Then, a recovery test was performed using the above analytical condition. The recovery (%) was calculated using the following equation:To prove the repeatability of the analytical method, the accuracy was experimented with by spiking the known amounts of the standards into a working solution. Their concentrations were prepared at three  levels such as Compounds n = 3) of at least three replicates. The nonparametric one-way ANOVA followed by Turkey’s multiple comparison test using the Graphprism version 8.0.1 software  was used for statistical analyses. * p < 0.05, ** p < 0.01, compared to controls, were considered significant values.Data were presented as mean ± SD , without cytotoxic effects (cell viability > 90%). Thus, the total extract was suspended in water and partitioned into n-hexane, CH2Cl2, EtOAc, n-BuOH, and distilled water (DW) fractions. The cell viability of the total extract and fractions were conducted on RAW264.7 cells. The result revealed that the n-hexane fraction showed some toxic effects on cells. Thus, this fraction was eliminated for the ROS experiment. Subsequently, CH2Cl2, EtOAc, n-BuOH, and DW fractions were examined for their antioxidant effect using an ROS assay. Among the tested samples, EtOAc and MC fractions showed potential for antioxidant effects with inhibition rates at 95.4% and 91.8%, respectively, stronger than those of positive control  at the tested concentration of 100 μg/mL. n-BuOH and DW fractions showed weak antioxidant effects , respectively, at 100 μg/mL. Additionally, the EtOAc showed the highest antioxidant capacities among all the fractions for both radical scavenging activities on DPPH• and ABTS•+  (The most active fraction (EtOAc), which has potent antioxidant and anti-inflammatory capacities, was separated using multiple chromatographic methods to yield nine compounds (1–9) .1 was obtained as a yellowish powder. Its molecular formula was identified as C26H28O15 based on the HR-ESI-MS ion peak [M − H]− at m/z 579.1371 . 1H NMR spectrum of 1 displayed the signals of an ABX spin-coupling system at δH 7.45 , 7.41 , and 6.90 , and an AX coupling system at δH 6.76  and 6.42 , revealing a flavonol bearing an asymmetrical B-ring substitution. The HMBC cross-peak of H-3 (δH 6.75) to C-2 (δC 164.6)/C-4 (δC 182.0)/C-1′ (δC 121.4) together with an MS negative ion peak [M − H]− at m/z 285  and 5.18 , suggesting two glycoside units. Indeed, the MS peaks produced secondary dissociation mass fragment ion peaks [M − H − Api]− at m/z 447 and [M − H − Api − Glc]– at m/z 285 (δH 5.18) to C-7 (δC 162.7), and those of H-1‴ (δH 5.35) to C-2″ (δC 76.1), confirmed the glucopyranosyl linked to luteolin aglycone through C-7 and apiopyranosyl attached to C-2″ of glucose unit. Thus, the structure of luteolin 7-O-− at m/z 447.0917 of 2 indicated that structural formula of 2 is less of an apiopyranosyl unit than that of 1  and 4.10  coupled to a secondary carbon C-6″ (δC 64.2), which were confirmed by the HSQC spectrum. This information suggested an esterification of the Glc-6 position. Indeed, the identification of the ester produced as Glc (C-6)-OCOR was supported by the HMBC cross-peaks of H2-6″ at δH 4.10 to C-1⁗ (δC 167.9), and those of OC-CH2-CO at δH 3.51 to C-1⁗ (δC 167.9)/C-3⁗ (δC 167.1) revealed the presence of malonyl group. Furthermore, the negative HR-ESI-MS of 4 produced a deprotonated ion peak [M − H]− at m/z 665.1373 -glucoside (4) − at m/z 593.1493 and [M − H − Glc − Api]− at m/z 299.0574 proving the above suggestion (5 was identified as chrysoeriol 7-O-(2-apiosyl)-glucoside − at m/z 563.1428 (3 (apiin) − at m/z 605.1528, identified the structure of 6″-acetylapiin − at m/z 563.1420 (8 was determined as apigenin-7-O-[2″-O-(5‴-O-feruloyl)-D-apiofuranosyl]-D-glucopyranoside − at m/z 755.1857. The 1H NMR spectrum of 9 also exhibited an asymmetric flavone structure of luteolin aglycone with two ABX spin system at δH 7.25 , 6.86 , 6.84 , 6.75 , 6.64 , an AX spin coupling system at δH 6.60 , 6.40 , and an olefinic proton at δH 6.29 . Therefore, the structure of 9 was determined as luteolin-[2″-O-(5‴-O-feruloyl)-D-apiofuranosyl]-D-glucopyranoside by comparison to the data reported in reference -D-glucopyranoside (8), and luteolin-[2″-O-(5‴-O-feruloyl)-D-apiofuranosyl]-D-glucopyranoside (9), were isolated for the first time from this plant. These isolated compounds displayed a significant inhibition on NO production in LPS-stimulated RAW264.7 cells with different inhibition rates at the tested concentration of 100 μM. Based on the NO-producing activity of the isolates and compounds  could be distinguished by 4′-OCH3 and 6‴-feruloyl-functional groups. However, Compound 6 exhibited the strongest inhibitory effect on NO production derived from LPS-mediated RAW264.7 cells at the same concentration, and Compound 8 exhibited the weakest inhibition of NO production. The results indicated that the 4′-OCH3 and 6‴-feruloyl-functional groups were not favored by these flavonoid glycoside compounds. Compounds 1, 2, 4, and 9 displayed the same luteolin-glucoside derivatives. However, Compound 4 exhibited stronger NO inhibition than Compound 1, followed by Compounds 2 and 9. Therefore, malonyl and apiosyl moieties were important in promoting the inhibition of NO production under the tested conditions. Our study is the first to report the inhibitory effect of Compounds 1 and 4–7 on LPS-induced NO production in RAW264.7 cells.The antioxidant and anti-inflammatory effects of the total extract and fractions derived from 3 and 4 had been reported to have antioxidant [The strong active compound 6″-acetylapiin exhibited a reduction in the levels of expression of both catalyst enzymes, iNOS and COX-2. The result suggested that these inhibitors may affect NO production by inhibiting catalyst enzymes iNOS and COX-2 in the rate-limiting steps to produce NO and PGE2, respectively. 6″-Acetylapiin first expressed the inhibitory mechanism of suppressing (iNOS and COX-2) isoform expression in this study. Previously, Active Compounds ioxidant ,33 and aioxidant ,35 effecioxidant  studies.1–9) also inhibited IL-8 production in LPS stimulated HT-29 cells with different inhibition rates. A structural activity relationship indicated that the key functional group inhibiting IL-8 production was correlated with the presence of a feruloyl moiety in the structures of the compounds (8 and 9). Thus, this functional group was important in the inhibition of LPS-induced IL-8 production in HT-29 cells. This is the first report of the inhibitory effect of Compounds 1 and 4–7 on LPS-induced IL-8 production in HT-29 cells.In addition, the isolated compounds . In detail, Compound 6 docked to both iNOS and COX-2 receptors with low binding energies  surrounded by hydrogen and hydrophobic interactions (8 and 9 occurred in the same pocket of the IL-8 (PDB ID: 5D14) receptor  of correlation coefficients also displayed the linearity regression. This established method was then employed to quantitate the marker content from the extract. As a result, the active compounds, Compounds 1 and 3, expressed high levels with amounts of 2.18 and 3.02 mg/g, respectively, reasonably considered as important components promoting the anti-inflammatory capacity of the extract of this plant. On the other hand, the content of other active components, Compounds 4 and 6, with amounts of 0.39 and 0.09 mg/g, respectively, may contribute to the enhancement of the anti-inflammatory property of this plant.An analytical method was successfully established with sensitivity , showing the resolution of peak separations from each of the six maker compounds presented in the total extract of this plant in the chromatogram. The appreciate values -glucoside was reported for the first time in NMR data. This is also the first report on the antioxidant and anti-inflammatory properties of this herbal food by inhibiting NO and IL-8 production in RAW264.7 and HT-29 cells, respectively. We firstly reported on the inhibitory effects of Compounds 1 and 4–7 in LPS-induced NO production on RAW264.7 cells and in LPS-induced IL-8 production on HT-29 cells, respectively. Further experiments with the strongly active compound were conducted to assess the inhibitory mechanism of suppressing (iNOS and COX-2) isoform expression for the first time. The active compounds with anti-inflammatory effects and their content in the herbal extract clarified the inhibitory capacity of V. tetrasperma. The molecular docking analysis further proved the anti-inflammatory mode of action by inhibiting the enzymatic activities of iNOS, COX-2, and of the IL-8 protein during the reaction process to produce mediators and cytokine production. The interesting activities this study has found indicate the new value of V. tetrasperma, which is consumed as a vegetable daily, and offered some evidence for the usage and development of this plant in the field of functional food to guide the treatment of inflammatory diseases in the future. This study also provided an effective tool for assessing the quality of this herbal food on the market through a UPLC analytical method using the above six standards. Our findings highlight the significance of this herb as a potential source of previously unrecognized health-promoting substances.In this study, we firstly reported antioxidant and anti-inflammatory activities through DPPH/ABTS radical scavenging activities, as well as inhibitory effects against ROS, and NO production, respectively. The current study also revealed the separation of flavonoid glycosides obtained from