Here, to obtain compounds with higher affinity than PS-1 for PM H+-ATPase, we synthesized 34 PS-1 analogs and examined their ability to inhibit PM H+-ATPase activity. The 34 analogs showed varying inhibitory effects on the activity of this enzyme. The strongest effect was observed for the small molecule PS-2, which was approximately five times stronger than PS-1. Compared to PS-1, PS-2 was also a stronger inhibitor of auxin uptake as well as acropetal and basipetal polar auxin transport in Arabidopsis thaliana seedlings. Because PS-2 is a more potent inhibitor of PM H+-ATPase than PS-1, we believe that this compound could be used as a tool to study the functions of this key plant enzyme.We previously identified protonstatin-1 (PS-1) as a selective inhibitor of plasma membrane H The tra+-ATPase . PM H+-An plants .+-ATPase. Therefore, understanding the mechanisms that regulate this pump is important for elucidating the physiological activities of plants. PM H+-ATPases in plants are encoded by a multigene family. For example, there are 11 homologous PM H+-ATPase genes (AHAs) in Arabidopsis thaliana, 10 in rice, 9 in tobacco, and 7 in tomato -5-(furan-2-ylmethylene)-2-thioxothiazolidin-4-one), a selective inhibitor of PM H+-ATPase activity, from a small molecule library. PS-1 binds to the central loop of PM H+-ATPase and inhibits its activity , which is required for the diffusion and active transport of IAA. The transmembrane protein kinase TMK phosphorylates the PM H+-ATPase which is required for H+-ATPase activation and auxin-induced cell wall acidification (The plant hormone auxin (indole-3-acetic acid [IAA]) exhibits polar transport from cell to cell . In the ransport , which iransport . For exaransport . IAA is ransport . Centralfication . Treatmefication . Therefo+-ATPase proton pump is not very high, and the half-maximal inhibitory concentration is 3.9 μM. Therefore, in the current study, we sought out compounds with increased affinity for PM H+-ATPase by synthesizing 34 PS-1 analogs with modified functional groups and testing their effects on PM H+-ATPase activity. The PS-1 analogs showed varying inhibitory effects. PS-2 had the strongest inhibitory effect on PM H+-ATPase activity and was approximately 5-fold stronger than PS-1. PS-2 also inhibited polar auxin transport in Arabidopsis seedlings more strongly than PS-1. These findings suggest that PS-2 represents a more effective tool than PS-1 for studying the functions of PM H+-ATPases in plants.The affinity of PS-1 for the PM H+-ATPase inhibitors.In order to explore the structure-activity relationships of the PS1 analogs, we divided PS-1 into two parts, the furan ring and the rhodanine ring, which are connected by an exocyclic double bond. Modification of PS-1 began with the replacement of the rhodanine ring with various five-membered heterocycle rings (1-5) , PS-1  or 307-2 (10 μM) with increasing concentrations of the substrate ATP (0.25-4 mM). Total PM H+-ATPase hydrolysis activity is shown in +-ATPase. The results showed that the maximum activity of PM H+-ATPase was close to pH 6.5 in the presence or absence of PS-2  promoter. Although yeast contains two PM H+-ATPases, PMA1 and PMA2, unlike PMA1, PMA2 is expressed at low levels and is not required for yeast growth  relative to the AHA2 central loop-PS-1 (Kd=4.88 ± 0.66 μM), while no measurable binding was detected between the 307-2 and AHA2 central loop  had no apparent effect on auxin uptake  at the 5- position improved the inhibitory effect (compounds 12 and 16), while replacement of the furan ring with nitrogen-containing five membrane , especially those with a hydrogen bond donor (24 and 27), markedly increased the potency. The analog 27, named PS-2, had the strongest inhibitory effects on PM H+-ATPase activity and polar auxin transport, and was clearly a stronger inhibitor than PS-1. Because it has a better inhibitory effect than PS-1, PS-2 should be a useful tool to study the biological functions of PM H+-ATPase.We synthesized 34 analogs of the PM H+-ATPase activity on polar auxin transport by treating the roots of Arabidopsis seedlings with selective inhibitor PS-1 of PM H+-ATPase  medium plus 0.6% (w/v) Phytagel  and 2.5% (w/v) sucrose, incubated in the dark at 4°C for 48 h, and vertically grown in a controlled growth chamber under a 16-h-light/8-h-dark cycle (22°C) for 5 d. The seedlings were transferred to control plates (containing 0.1% [v/v] DMSO) or plates containing 2.5 μM PS-1 or PS-1 analogs (in 0.1% [v/v] DMSO) and incubated for 7 d under a 16-h-light/8-h-dark cycle (22°C).600 of 0.5 and diluted at a ratio of 1:5 or 1:25 in fresh medium. A 4 μL aliquot of yeast cells was spotted onto solid medium containing DMSO as a control (0.1% [v/v]) or 10 μM PS-1 or PS-1 analog and photographed after 3 days of incubation at 28°C.We screened PS-I analogs in yeast strain RS72 expressing Arabidopsis AHA2, which was constructed as previously reported . Yeast c600 nm reached approximately 0.4. Yeast cells were then aliquoted into 96-well plates, receiving 200 μL of culture per well in the presence of the DMSO control (0.1% [v/v]) or various compounds . Yeast growth (OD600 nm) was monitored using a microplate reader (Bio-Tek) after shaking at 28°C at 220 rpm/min for 3 h.In the PS-2 dose-dependent inhibition assay against yeast strain RS72, yeast cells were pre-cultured in flasks until OD+-ATPase hydrolytic activity was measured as described previously (Plasma membrane-enriched vesicles were isolated from 3-week-old Arabidopsis seedlings using two-phase partitioning . PM H+-Aeviously  in the peviously .http://www.nanotemper-technologies.com/). The concentration of various compounds  ranged from 0.015 to 1000 μM. The labeled AHA2 central loop peptide was used in PBS buffer  containing 0.05% (v/v) Tween 20 (PBST). The labeled peptide and compound mixture was incubated in PBST buffer for 5 min in the dark and then transferred to standard treated capillaries for measurement.The interaction between the AHA2 central cyclic peptide and PS-2 was detected by MST analysis using the NanoTemper Monolith NT.115 instrument as previously described . RecombiAuxin uptake in Arabidopsis roots was measured as previously described . Assays 1H NMR spectra were performed on a Varian or Bruker 400 MHz spectrometer at ambient temperature with CDCl3 or DMSO-d6 as the solvent unless otherwise addressed. 13C NMR spectra were recorded on a Varian or Bruker 100 MHz spectrometer at ambient temperature (with complete proton decoupling). Chemical shifts are reported in parts per million relative to DMSO-d6  or chloroform . Data for 1H NMR are reported as follows: chemical shift, multiplicity , coupling constants and integration. High-resolution mass spectra were obtained at Peking University Mass Spectrometry Laboratory using Bruker APEX Flash chromatography. All chemical reagents were used as supplied by Alfa Aesar Chemicals, Sigma-Aldrich, J&K, and Acros. EtOH was distilled from calcium hydride; tetrahydrofuran (THF) was distilled from sodium/benzophenone prior to use. Compound 1-2, 4, 6, 8-9, 11, 15-18, 25, 28-31, 33-34 are the same as literature reported.General procedure: To a solution of the aldehyde (1 equiv) and the heterocycle compound (1 equiv) in acetic acid was added ammonium acetate (2 equiv). The mixture was stirred at reflux under an argon atmosphere. After 4 h, the reaction mixture was cooled to room temperature and the precipitates were collected by filtration and washed with distilled water. The title compound was recrystallized in ethanol.(Z)-5-(furan-2-ylmethylene)-2-iminothiazolidin-4-one(3): prepared according to general procedure with furfural and 2-iminothiazolidin-4-one to yield compound 3 . 1H NMR (400 MHz DMSO-d6): δ 6.69 , 6.93 , 7.39 , 7.95 , 9.05 , 9.32 ; 13C NMR (100 MHz DMSO-d6): δ 113.2, 115.8, 116.1, 126.9, 146.1, 149.7, 176.1, 180.1. HRMS (ESI): [M + H]+ calculated for C8H7N2O2S: 195.0223, found: 195.0224.(Z)-4-(furan-2-ylmethylene)-2-thioxothiazolidin-5-one(5): prepared according to general procedure with furfural and 2-thioxothiazolidin-5-one to yield compound 5 . 1H NMR (400 MHz DMSO-d6): δ 6.65, 6.75 , 7.29 , 8.01 ;13C NMR (100 MHz DMSO-d6): δ102.3, 113.8, 119.4, 131.0, 147.5, 148.4, 187.7, 188.2; HRMS (ESI): [M + H]+ calculated for C8H6NO2S2: 211.9835, found: 211.9836.To a solution of PS-1 and (2-iodoethyl)(vinyl)sulfane  in 2 mL of DMF was added K2CO3 . The mixture was stirred under the atmosphere of argon at room temperature overnight. The mixture was then concentrated in vacuo and the residue was purified by thin-layer chromatography to afford compounds 7  and 10 .(Z)-5-(furan-2-ylmethylene)-2-((2-(vinylthio)ethyl)thio)thiazol-4(5H)-one (7): 1H NMR (400 MHz CDCl3): δ 3.12-3.17 , 3.58-3.63, 5.29, 5.31, 6.35 , 6.58 , 6.82 , 7.61 , 7.66 ; HRMS (ESI): [M + H]+ calculated for C12H12NO2S3: 298.0025, found: 298.0029.(Z)-5-(furan-2-ylmethylene)-2-thioxo-3-(2-(vinylthio)ethyl)thiazolidin-4-one (10): 1H NMR (400 MHz CDCl3): δ 3.01 , 4.34, 5.29, 5.35 , 6.36 , 6.60 , 6.85 , 7.48 , 7.71 ; 13C NMR (100 MHz CDCl3): δ 27.6, 43.2, 112.5, 113.6, 118.6, 118.9, 120.5, 130.6, 147.2, 150.0, 167.3, 194.3; HRMS (ESI): [M + H]+ calculated for C12H12NO2S3: 298.0025, found: 298.0027.(Z)-5-((5-bromofuran-2-yl)methylene)-2-thioxothiazolidin-4-one(12): prepared according to general procedure with 5-bromofuran-2-carbaldehyde and rhodanine to yield compound 12 . 1H NMR (400 MHz DMSO-d6): δ 6.91 , 7.20 , 7.42 , 13.75 ; 13C NMR (100 MHz DMSO-d6): δ 116.0, 116.3, 121.7, 123.2, 128.0, 151.5, 168.9,196.1; HRMS (ESI): [M + H]+ calculated for C8H5BrNO2S2: 289.8940, found: 289.8942.(Z)-5-((4-bromofuran-2-yl)methylene)-2-thioxothiazolidin-4-one(13): prepared according to general procedure with 4-bromofuran-2-carbaldehyde and rhodanine to yield compound 12 . 1H NMR (400 MHz DMSO-d6): δ 7.31 , 7.42 , 8.34 , 13.78 ; 13C NMR (100 MHz DMSO-d6): δ 102.5, 116.3, 120.8, 124.5, 146.0, 150.3, 168.9, 196.2, HRMS (ESI): [M + H]+ calculated for C8H5BrNO2S2: 289.8940, found: 289.8942.(Z)-5-((3-bromofuran-2-yl)methylene)-2-thioxothiazolidin-4-one(14): prepared according to general procedure with 3-bromofuran-2-carbaldehyde and rhodanine to yield compound 12 . 1H NMR (400 MHz DMSO-d6): δ 7.06 , 7.17 , 8.20 , 12.92 ; 13C NMR (100 MHz DMSO-d6): δ 108.9, 112.9, 116.8, 124.9, 146.6, 148.9, 169.0,+ calculated for C8H4BrNO2S2: 289.8940, found: 289.8943.196.1;HRMS (ESI): [M + H](Z)-5-(furan-3-ylmethylene)-2-thioxothiazolidin-4-one(19): prepared according to general procedure with furan-3-carbaldehyde and rhodanine to yield compound 19 . 1H NMR (400 MHz DMSO-d6): δ 6.78 , 7.58 , 7.91 , 8.32 , 13.75 ; 13C NMR (100 MHz DMSO-d6): δ 109.4, 121.4, 123.4, 124.9, 146.5, 148.6, 169.5, 195.6; HRMS (ESI): [M + H]+ calculated for C8H6NO2S2: 211.9835, found: 211.9835.(Z)-5-(thiazol-2-ylmethylene)-2-thioxothiazolidin-4-one (20): prepared according to general procedure with thiazole-2-carbaldehyde and rhodanine to yield compound 20 . 1H NMR (400 MHz DMSO-d6): δ 7.91 , 8.09 , 8.21 , 13.79 ; 13C NMR (100 MHz DMSO-d6): δ 119.9, 125.4, 129.3, 145.4, 160.7, 168.9, 199.7; HRMS (ESI): [M + H]+ calculated for C7H5N2OS3: 228.9559, found: 228.9561.(Z)-5-(thiazol-5-ylmethylene)-2-thioxothiazolidin-4-one(21): prepared according to general procedure with thiazole-5-carbaldehyde and rhodanine to yield compound 21 . 1H NMR (400 MHz DMSO-d6): δ 8.03 , 8.05 , 9.46 , 13.95 ; 13C NMR (100 MHz DMSO-d6): δ 121.6, 126.9, 123.1, 149.4, 159.7, 168.6, 194.2; HRMS (ESI): [M + H]+ calculated for C7H5N2OS3: 228.9559, found: 228.9561.(Z)-5-(thiazol-4-ylmethylene)-2-thioxothiazolidin-4-one(22): prepared according to general procedure with thiazole-4-carbaldehyde and rhodanine to yield compound 22 . 1H NMR (400 MHz DMSO-d6): δ 7.70 , 8.40 , 9.34 , 13.63 ; 13C NMR (100 MHz DMSO-d6): δ 122.7, 127.0, 127.4, 150.1, 156.3, 169.4, 199.4; HRMS (ESI): [M + H]+ calculated for C7H5N2OS3: 228.9559, found: 228.9561.(Z)-5-((1H-imidazol-5-yl)methylene)-2-thioxothiazolidin-4-one(23): prepared according to general procedure with 1H-imidazole-5-carbaldehyde and rhodanine to yield compound 23 . 1H NMR (400 MHz DMSO-d6): δ 7.53 , 7.84 , 7.96 , 12.75 , 13.40 ; 13C NMR (100 MHz DMSO-d6): δ 121.3, 124.1, 124.5, 135.7, 138.5, 169.4, 199.7. HRMS (ESI): [M + H]+ calculated for C7H6N3OS2: 211.9947, found: 211.9948.(Z)-5-((1H-imidazol-2-yl)methylene)-2-thioxothiazolidin-4-one(24): prepared according to general procedure with 1H-imidazole-2-carbaldehyde and rhodanine to yield compound 24 . 1H NMR (400 MHz DMSO-d6): δ 7.32 , 7.35 , 7.50 , 12.93 , 13.50 ; 13C NMR (100 MHz DMSO-d6): δ 116.7, 121.0, 125.6, 132.5, 142.0, 169.1, 199.7; HRMS (ESI): [M + H]+ calculated for C7H6N3OS2: 211.9947, found: 211.9948.(Z)-5-(oxazol-5-ylmethylene)-2-thioxothiazolidin-4-one (26): prepared according to general procedure with oxazole-5-carbaldehyde and rhodanine to yield compound 26 . 1H NMR (400 MHz DMSO-d6): δ 7.59 , 7.79 , 8.74 , 13.80 ; 13C NMR (100 MHz DMSO-d6): δ 114.6, 126.0, 133.2, 146.8, 155.0, 168.8, 196.0; HRMS: [M + H]+ calculated for C7H5N2O2S2: 212.9787, found: 212.9789.(Z)-5-((1H-pyrrol-2-yl)methylene)-2-thioxothiazolidin-4-one(27): prepared according to general procedure with 1H-pyrrole-2-carbaldehyde and rhodanine to yield compound 27 . 1H NMR (400 MHz DMSO-d6): δ 6.37 , 6.50 , 7.30 , 11.77 , 13.50 ; 13C NMR (100 MHz DMSO-d6): δ 112.7, 115.0, 116.9, 121.9, 125.8, 127.2, 169.2, 194.8; HRMS: [M + H]+ calculated for C8H7N2OS2: 210.9994, found: 210.9997.5-(furan-2-ylmethyl)-2-thioxothiazolidin-4-one(32): To a solution of PS-1  in THF/pyridine was added 260 uL of LiAlH4 solution in THF(2 M). The mixture was stirred at reflux under the atmosphere of argon overnight. Then, the mixture was quenched by slowly adding 2M HCl solution. The mixture was extracted by ethyl acetate for 3 times. The combined organic layer was washed with brine, dried over anhyd. Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography to give 32 as a brown solid . 1H NMR (400 MHz CDCl3): δ 3.28 , 3.55 , 4.66, 6.18 , 6.32 , 7.36 , 9.39 ; 13C NMR (100 MHz CDCl3): δ 31.0, 53.7, 108.2, 110.6, 142.6, 149.4, 176.1, 200.0; HRMS: [M + H]+ calculated for C8H8NO2S2: 213.9991, found: 213.9993.The original contributions presented in the study are included in the article/YY, LiX, WX, LW, ML, LX, FH, SS, and LeX designed the research. YY, LiX, WX, LW, ML, LX, FH, and SS conducted the research. YY, LiX, and LeX analyzed the data. YY, LiX, and LeX wrote and edited the manuscriptThis work was supported by the National Natural Science Foundation of China , the National Genetically Modified Organisms Breeding Major Projects (2016ZX08009002) and Exploration project of Center for Practical Courses in the Life Sciences of China Agricultural University .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The structures of the undescribed compounds were elucidated by 1D and 2D NMR and high resolution mass spectral analyses. In the case of 2, the absolute configurations of the stereogenic carbons were determined by comparison of their calculated and experimental electronic circular dichroism (ECD) spectra. The absolute configurations of the stereogenic carbons in 6a and 6b were also determined, for the first time, by X-ray crystallographic analysis. Compounds 2, 3a, 3b, 4, 5a, 5b, 5c, 6a, and 6b were assayed for antibacterial activity against four reference strains, viz. two Gram-positive  and two Gram-negative , as well as three multidrug-resistant strains. However, only 5a exhibited significant antibacterial activity against both reference and multidrug-resistant strains. Compound 5a also showed antibiofilm activity against both reference strains of Gram-positive bacteria.An unreported isocoumarin, ( Aspergillus (family Aspergillaceae) is one of the most extensively studied genera of filamentous fungi, mainly due to the medical relevance, food spoilage, and industrial application of some of its species. Aspergilli can grow in a wide range of niches, mainly in soils and on dead matter, but some are also capable of colonizing living animal or plant hosts. However, the increasing numbers of Aspergillus species have been found in different niches in the marine environment, from tropical waters . Inus Curzi . For thiA. stellatus KUFA 2017 by column chromatography of silica gel, followed by purification by preparative TLC, Sephadex LH-20 column and crystallization, led to the isolation of undescribed -4-hydroxy-6-methoxymellein (2), stellatanthraquinone (4), and acetyl carnemycin E (5c), as well as the previously reported p-hydroxybenzaldehyde (1) benzene (5a) benzene-1,3-diol (5a) +  (13C NMR spectrum (δC 169.1), two oxygen-bearing non-protonated sp2 (δC 166.2 and 164.5), two non-protonated sp2 (δC 142.1 and 100.0), two protonated sp2 (δC 106.8 and 101.3), two oxymethine sp3 (δC 77.8 and 67.5), one methoxy (δC 55.8) and one methyl (δC 15.9) carbon, respectively. The 1H NMR spectrum (δH 11.20 (OH-8), a multiplet of a hydroxyl proton at δH 2.28 (OH-4), two doublets of meta-coupled aromatic protons at δH 6.48  and δH 6.45 , a double quartet at δH 4.63 , which was coupled with a double doublet at δH 4.50 , a methoxy singlet at δH 3.85 (δC 55.8) and a methyl doublet at δH 1.55 . The HMBC spectrum (δC 164.5 (C-8), 101.3 (C-7) and 100.0 (C-8a), H-5 to the carbons at δC 166.2 (C-6), C-7, C-8a, C-4, H-7 to C-5, C-6, C-8a, H-3 to Me-9, H-4 to C-4a (δC 142.1), C-5, C-8a, OMe-6 to C-6, Me-9 to C-3, C-4, and a weak correlation from OH-4 to C-3 and C-4. Compound 25.0763) , requirispectrum , exhibitspectrum , can be spectrum , in combspectrum , displayspectrum  exhibite1H and 13C NMR chemical shift values, together with COSY and HMBC correlations, revealed that the planar structure of 2 is the same as that of the enantiomeric mixture of - and -4-hydroxy-6-methoxymellein, previously isolated from the culture extract of the mycobionts of Graphis sp. Since the mixture showed a negative sign of Cotton effect at 267 nm, the authors proposed that the -enantiomer was predominant +  (13C NMR spectrum (δC 184.5 and 183.3), three oxygen-bearing non-protonated sp2 , seven non-protonated sp2 , seven protonated sp2 , two methylene sp3 (δC 33.8 and 23.7), and two methyl (δC 21.9 and 13.7) carbons. The 1H NMR spectrum , 8.85 (J = 6.1 Hz), 7.46 (J = 1.0 Hz), one double doublet at δH 8.16 , one methylene triplet at δH 2.83 (J = 7.4 Hz), one methylene sextet at δH 1.70 (J = 7.4 Hz), one methyl singlet at δH 2.40, and one methyl triplet at δH 0.94 (J = 7.3 Hz). The presence of the 1,8-dihydroxy-6-methyl-1,2,3,6,8-pentasubstitututed anthraquinone scaffold was supported by COSY correlations  to H-5 /δC 120.4) and Me-11 , as well as by HMBC correlations  to C-1 (δC 159.5), C-2 (δC 123.2), C-9a (δC 100.0), OH-8  to C-7 (δC 124.4), C-8 (δC 161.2), C-8a (δC 114.6), H-5  to C-10 (δC 183.3), C-7, C-8a, Me-11 (δC 21.9), H-7  to C-8, C-5 (δC 120.4), C-8a, Me-11, H-4  to C-10, C-2 (δC 123.2), and C-9a. That another portion of the molecule is 3-propylpyridinium was corroborated by COSY correlations  to H-5’  and H-2’ , H-5’ to H-4’ and H-6’ , which was confirmed by HMBC correlations (δC 142.8), C-4’ (δC 146.2) and C-1” (δC 33.8), H-4’ to C-2’ (δC 147.2), C-6’ (δC 145.6) and C-1”, H-5’ to C-3’, and C-6’ and H-6’ to C-4’ and C-5’ + -nona-3,5-dien-1-yl]benzene-1,3-diol moiety, identical to 5a, with a substitution on C-2. The presence of five oxymethine sp3 , one oxymethylene sp3 (δC 61.2) carbons, two hydroxyl groups at δH 4.90 dd  and 4.59 d (J = 5.5 Hz), and the molecular formula C6H11O5 of the substituent on C-2 revealed the presence of a pyranosyl moiety. However, since four oxymethine protons of the sugar moiety appeared as complex multiplets at δH 3.20–3.22 and 3.74, it was not possible to identify the sugar moiety of 5b. Although Wen et al. + , and m/z 459.1989 [M + Na]+   than those of 5b /δC 61.2) as well as the appearance of an acetyl group , suggesting that 5c is a C-21 acetate of 5b.Compound 59.1995) .The 1H ara of 5c  resemblef 5c  and 5.15, d (J = 4.4 Hz), respectively. Furthermore, in the 300 MHz spectra, OH-3 displayed HMBC correlations to C-2 (δC 109.9) and C-3 (δC 157.2) (δH 3.20) and H-4’ (δH 3.18) (J = 9.8 Hz) confirmed the β-anomer of the glucosyl moiety. Since 5c has never been previously reported, it was named acetyl carnemycin EContrary to other proton signals, the signals of OH-3, OH-3’, and OH-4’ appeared as broad signals in the  500 MHz . Moreove 500 MHz , which m 300 MHz , the sigC 157.2) , while OδH 3.18) , respect1H and 13C NMR spectra of 6a and 6b  while the relative configuration of C-20 was suggested by the preferred axial conformation of the isopropyl substituent in the hydrogenated derivatives + .White crystal. Mp 116–118 °C. + .Red solid. Mp. 228–229 °C. For 20D +60 . 1H and 13C spectroscopic data , see m/z 395.2076 [M + H]+ .Pale-yellow viscous mass. [α]20D +260 . 1H and 13C spectroscopic data , see m/z 437.2175 [M + H]+, ; m/z 459.1989 [M + Na]+ .Pale-yellow viscous mass. [α]α radiation (λ = 1.54184 Å). The structures were solved by direct methods using SHELXS-97 and refined with SHELXL-97 benzene (5a), carnemycin E (5b), tajixanthone hydrate (6a), and 15-acetyl tajixanthone hydrate (6b). While the absolute configurations of the stereogenic carbons in 2 were established unambiguously by a combination of NOESY correlations and a comparison of experimental and calculated ECD spectra, the stereostructures of 6a and 6b were established by X-ray analysis for the first time. The EtOAc extract of the culture of a marine-derived fungus, 1 and 3c, were evaluated for their antibacterial activity against four reference strains: two Gram-positive  and two Gram-negative , as well as three multidrug-resistant strains including an extended-spectrum β-lactamase (ESBL)-producing E. coli , a methicillin-resistant isolate (MRSA) S. aureus 74/24 and a vancomycin-resistant (VRE) isolate E. faecalis B3/101. However, only 5c exhibited antibacterial activity against all Gram-positive strains with a MIC value of 16 µg/mL toward E. faecalis ATCC 29212, vancomycin-resistant E. faecalis B3/101, and methicillin-resistant S. aureus 74/24, but with a higher MIC value (32 µg/mL) toward S. aureus ATCC 29213. Since 5a displayed the minimal bactericidal concentration (MBC) equal to or one-fold higher than the MIC, it was suggested that 5a exerted a bactericidal effect towards E. faecalis ATCC 29212, S. aureus 74/24, and S. aureus ATCC 29213. On the contrary, the MBC of 5a was more than two-fold higher than the MIC toward E. faecalis B3/101; therefore, this compound was suggested to have a bacteriostatic effect against this multidrug-resistant species. Interestingly, 5b and 5c, which are C-glucosylated 5a, were void of antibacterial activity against all the tested organisms. These results lead to a conclusion that the polar and bulky glucosyl substituent on the benzene ring can negatively affect the antibacterial activity of this series of compounds. Finally, 5a was also able to completely inhibit biofilm formation in S. aureus ATCC 29213 and E. faecalis ATCC 29212 at both MIC and 2× MIC concentrations. Since 5a possesses interesting antibacterial and potent antibiofilm activities, this compound can be considered as an interesting model for the development of a new type of antibiotics.All the compounds, except


Correction: BMC Ecology and Evolution (2022) 22:90 10.1186/s12862-022-02043-4.LSID urn:lsid:zoobank.org:pub:F29E460E-D072-4C3B-912C-928D8677CC2C.Following the publication of the original article , we wereThe original article  has been


The reaction between dithiomalondianilide  and alkyl 3-aryl-2-cyanoacrylates in the presence of morpholine in the air atmosphere leads to the formation of alkyl 6-amino-4-aryl-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-dithiolo- pyridine-5-carboxylates in 37–72% yields. The same compounds were prepared in 23–65% yields by ternary condensation of aromatic aldehydes, ethyl(methyl) cyanoacetate and dithiomalondianilide. The reaction mechanism is discussed. The structure of ethyl 6-amino-4-(4-methoxyphenyl)-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-dithiolopyridine-5-carboxylate was confirmed by X-ray crystallography. Two of the prepared compounds showed a moderate growth-stimulating effect on sunflower seedlings. Three of the new compounds were recognized as strong herbicide safeners with respect to herbicide 2,4-D in the laboratory and field experiments on sunflower. There are only a few papers that reported the preparation of such compounds. Historically, the first example of the synthesis is probably the preparation of dithiolopyridine ,2Dithiol,4-bpyridth P4S10 . Dithiollycinate  or from lycinate . When niisolated . The for (MfSCl)  -ones B or related products -ethyl 2-cyano-3-(4-methoxyphenyl)acrylate 14a in EtOH were treated with excessive morpholine, a yellow crystal of ethyl 6-amino-4-(4-methoxyphenyl)-7-phenyl- 3-(phenylimino)-4,7-dihydro-3H-dithiolopyridine-5-carboxylate 15a was isolated in 72% yield  gave rather unsatisfactory results. The nature of amine  does not significantly affect the yields of products. Along with Michael addition of 12 to other 2-cyanoacrylates 14a–g  was also accompanied by side processes and led to decreased yields; in this case, compound 15a was obtained in 30% yield.Extended refluxing of the reaction mixture does not favor the formation of dithiolopyridines 12 with 14a was conducted in inert atmosphere under nitrogen stream, dithiolopyridine 15a was not isolated. This fact proves the crucial role of air oxygen for the final step of dithiolopyridine system formation. Nevertheless, the addition of oxidizing agents (hydrogen peroxide or iodine) leads to resinification of the reaction mixture. Aldehydes with both electron-donor and electron-withdrawing substituents are reacted well. However, we failed to obtain any products in the case of furfural or 2-cyano-3-(furan-2-yl)acrylates even if less nucleophilic and milder base triethylamine was taken instead of morpholine. This is probably due to the strong tendency of furan-ring-bearing electron-withdrawing substituents to undergo a nucleophilic attack at the C-5 position to form a complex mixture of furan-ring-cleavage/recyclization products.When the reaction of 15, the absorption bands corresponding to NH2 valence vibrations (ν 3371–3474 cm−1 and ν 3263–3290 cm−1), RO(C=O) group bands (ν 1651–1661 cm−1) and imino group C=N–Ph bands at ν 1622–1638 cm−1 were observed while the bands corresponding to C≡N groups were absent. 1H NMR spectra of 15 revealed characteristic singlets attributed to H-4 protons at δ 5.02–5.94 ppm and a very broadened peak of NH2 protons at δ 7.00–7.19 ppm. It is interesting that in some cases the signal of OCH2 protons is detected not as a typical quartet but as a complex multiplet, probably due to the hindered rotation caused by the intramolecular hydrogen bond C=O...H-NH. Alternatively, the observed splitting of OCH2 signal can be caused by the shielding effect of the aromatic ring at C-4, which affects one of OCH2 protons. 13C NMR spectra lacked signals attributed to C=S carbons and revealed the peaks of C=O (δ 168.7–169.1 ppm), C=N (δ 163.0–163.4 ppm), C-3a (δ 111.1–113.7 ppm), C-5 (δ 76.5–78.4 ppm) and C-4 carbons (δ 33.5–38.6 ppm). The NMR, FTIR and HRMS spectra are shown in In the IR spectra of compounds The new compounds were tested as herbicide safeners with respect to 2,4-dichlorophenoxyacetic acid  and as plant growth regulators. 2,4-D is an herbicide that is widely used for plant protection and was reported to show no significant toxicity to humans . However15, are reported to be effective herbicide safeners pyridines, which can be considered as structural analogs of the prepared 3-imino-3H-dithiolopyridines safeners ,43 and psafeners . We studsafeners  . Sunflower plants of cv. Master in the phase of 10–16 leaves were treated with an aqueous solution of 2,4-dichlorophenoxyacetic acid at a dose of 18 g/ha and 3 days later a safener solution was applied at a dose of 100 g/ha with the working fluid rate of 300 L/ha.The antidote activity of 3H-dithiolopyridines -Control group—untreated plants;-“Herbicide” (reference) group—plants treated with herbicide 2,4-D only;-“Herbicide + antidote” group—plants treated with herbicide 2,4-D and an antidote.The field tests included the following variants:2 with five-fold repetition. Sunflower harvesting was performed at the time of full seed maturity. The field antidote effect FA was determined by the absolute value of the crop yield increase to the herbicide reference by the Equation (2): FA is antidote effect, %; Y1 is crop yield in “herbicide + antidote” group; and Y2 is crop yield in “herbicide” (reference) group.Experiments were conducted in plots of 2.8 mt-test. The field test results are presented in 15a,c,f as herbicide safeners under field conditions provides an antidote effect in the range of 41.4–51.4%.The obtained data were statistically processed using Student’s 15a,c,f was evaluated in laboratory experiments using the known procedure di- thiolopyridine-5-carboxylate (15a). This compound was prepared via Method A in a yield of 388 mg (72%). Alternatively, this compound was prepared via Method B employing ethyl cyanoacetate (0.12 mL), anisaldehyde (0.13 mL) and thioamide 12 (300 mg) in a yield of 351 mg (65%), bright yellow crystals. FTIR, νmax, cm−1: 3400, 3273 (N-H); 1651 (CO2Et); 1628 (C=N). 1H NMR : 1.09 , 3.71 , 3.89–4.03 , 5.02 , 6.84–6.86 , 7.00 , 7.05–7.09 , 7.31–7.34 , 7.60–7.66 . 13C DEPTQ NMR : 14.4* (CH3), 38.0* (C-4), 54.9* (OCH3), 58.6 (OCH2), 78.4 (C-5), 113.2* , 113.5 (C-3a), 120.1* , 124.3* (C-4 Ph), 128.5* , 129.7* , 130.0* , 130.5* , 131.1* (C-4 Ph), 135.6 (C-1 Ph), 138.9 (C-1 Ar), 150.9 (C-1 Ph), 152.7 (C-6), 153.7 (C-8a), 157.6 (C–OMe), 163.3 (C=N), 168.7 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 65.22; H, 4.89; N, 8.15; found (%): C, 65.15; H, 5.00; N, 8.12. HRMS (ESI) m/z: calculated for C28H26N3O3S2 [M + H]+: 516.14156, found 516.1411 (Δ 0.89 ppm).15a  were grown from EtOH–acetone solution. The crystal was kept at 423.15(10) K during data collection. Using Olex2 dithiolopyridine-5-carboxylate ng Olex2 , the strng Olex2  package Ethyl 6-amino-4-(4-nitrophenyl)-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-dithiolo- pyridine-5-carboxylate (15b). This compound was prepared via Method A in a yield of 344 mg (62%), bright-yellow powder. Alternatively, this compound was prepared via Method B employing ethyl cyanoacetate (0.11 mL), 4-nitrobenzaldehyde (160 mg) and thioamide 12 (300 mg) in a yield of 240 mg (43%). FTIR, νmax, cm−1: 3445, 3263 (N-H); 1661 (CO2Et); 1630 (C=N); 1524 (NO2 asym); 1346 (NO2 sym). 1H NMR : 0.92 , 3.81–3.95 , 5.94 , 6.72 , 7.02–7.06 , 7.19 , 7.26–7.30 , 7.38–7.42 , 7.65–7.80 . 13C DEPTQ NMR : 14.1* (CH3), 34.6* (C-4), 58.5 (OCH2), 76.9 (C-5), 111.7 (C-3a), 120.0* , 123.6* (CH Ar), 124.2* (C-4 Ar), 127.2* (CH Ar), 129.4* , 130.3* , 130.5* , 131.3* (C-4 Ph), 133.0* (CH Ar), 135.2 (C-1 Ph), 141.1 (C-1 Ar), 148.8 (C–NO2), 150.6 (C-1 Ph), 153.1 (C-6), 154.6 (C-8a), 163.0 (C=N), 168.7 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 61.12; H, 4.18; N, 10.56; found (%): C, 61.03; H, 4.31; N, 10.41. HRMS (ESI) m/z: calculated for C27H23N4O4S2 [M + H]+: 531.11607, found 531.1169 (Δ −1.56 ppm).Ethyl 6-amino-7-phenyl-3-(phenylimino)-4-(2-thienyl)-4,7-dihydro-3H-dithiolopyridine-5-carboxylate (15c). This compound was prepared via Method A in a yield of 201 mg (39%). Alternatively, this compound was prepared via Method B employing ethyl cyanoacetate (0.11 mL), thiophene-2-carbaldehyde (0.10 mL) and thioamide 12 (300 mg) in a yield of 120 mg (23%), light brown powder. FTIR, νmax, cm−1: 3380, 3269 (N-H); 1653 (CO2Et); 1630 (C=N). 1H NMR : 1.12 , 4.05 , 5.39 , 6.92–6.95 , 7.09–7.12 , 7.28 , 7.34–7.38 , 7.51–7.54 , 7.63–7.65 . 13C DEPTQ NMR : 14.5* (CH3), 33.5* (C-4), 58.7 (OCH2), 78.0 (C-5), 112.7 (C-3a), 120.1* , 122.8* (C-2 thienyl), 123.6* (C-5 thienyl), 124.4* (C-4 Ar), 126.7* (C-4 thienyl), 129.7* , 129.9* , 130.6* , 131.1* (C-4 Ph), 135.5 (C-1 Ph), 150.9 (C-1 Ph), 151.1 (C-1 thienyl), 153.1 (C-6), 154.2 (C-8a), 163.4 (C=N), 168.5 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 61.07; H, 4.31; N, 8.55; found (%): C, 60.98; H, 4.45; N, 8.45. HRMS (ESI) m/z: calculated for C25H22N3O2S3 [M + H]+: 492.0874, found 492.0868 (Δ 1.22 ppm).Ethyl 6-amino-4-(4-chlorophenyl)-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-dithiolo- pyridine-5-carboxylate (15d). This compound was prepared via Method A in a yield of 300 mg (55%). Alternatively, this compound was prepared via Method B employing ethyl cyanoacetate (0.11 mL), 4-chlorobenzaldehyde (150 mg) and thioamide 12 (300 mg) in a yield of 240 mg (46%), yellow powder. FTIR, νmax, cm−1: 3371, 3271 (N-H); 1655 (CO2Et); 1638 (C=N). 1H NMR : 1.07 , 3.96 , 5.04 , 6.83 , 7.05–7.09 , 7.30–7.35 , 7.41 , 7.50–7.55 , 7.64–7.67 . 13C DEPTQ NMR : 14.3* (CH3), 38.6* (C-4), 58.7 (OCH2), 77.7 (C-5), 112.6 (C-3a), 120.1* , 124.3* (C-4 Ph), 127.8* , 129.5* , 129.7* , 130.1* , 130.5* , 131.2* (C-4 Ph), 135.4 (C-1 Ph and C-Cl overlapped), 145.6 (C-1 Ar), 150.8 (C-1 Ph), 152.8 (C-6), 154.2 (C-8a), 163.2 (C=N), 168.6 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 62.36; H, 4.26; N, 8.08; found (%): C, 62.30; H, 4.41; N, 8.05. HRMS (ESI) m/z: calculated for C27H23ClN3O2S2 [M + H]+: 520.09202, found 520.0913 (Δ 1.38 ppm).Ethyl 6-amino-4--7-phenyl-3-(phenylimino)-4,7-dihydro-3H-- dithiolopyridine-5-carboxylate (15e). This compound was prepared via Method A in a yield of 215 mg (37%). Alternatively, this compound was prepared via Method B employing ethyl cyanoacetate (0.11 mL), 2,4-dichlorobenzaldehyde (180 mg) and thioamide 12 (300 mg) in a yield of 175 mg (30%), brownish-yellow powder. FTIR, νmax, cm−1: 3474, 3290 (N-H); 1657 (CO2Et); 1634 (C=N). 1H NMR : 1.05 , 3.87–3.99 , 5.36 , 6.79 , 7.02–7.06 , 7.14 , 7.27–7.31 , 7.35 , 7.42 , 7.52 , 7.55–7.67 . 13C DEPTQ NMR : 14.3* (CH3), 37.9* (C-4), 58.6 (OCH2), 76.5 (C-5), 111.1 (C-3a), 120.1* , 124.2* (C-4 Ph), 126.7* (CH Ar), 128.3* (CH Ar), 129.5* , 130.4* , 130.5* , 130.9 (C–Cl), 131.3* (C-4 Ph), 133.7* (C-3 Ar), 133.8 (C–Cl), 135.3 (C-1 Ph), 142.7 (C-1 Ar), 150.3 (C-1 Ph), 152.9 (C-6), 154.7 (C-8a), 162.2 (C=N), 168.7 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%):C, 58.48; H, 3.82; N, 7.58; found (%): C, 58.60; H, 3.95; N, 7.41. HRMS (ESI) m/z: calculated for C27H22Cl2N3O2S2 [M + H]+: 554.05305, found 554.0532 (Δ −0.27 ppm).Methyl 6-amino-4-(4-methoxyphenyl)-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-di- thiolopyridine-5-carboxylate (15f). This compound was prepared via Method A in a yield of 330 mg (63%). Alternatively, this compound was prepared via Method B employing methyl cyanoacetate (0.09 mL), 4-methoxybenzaldehyde (0.13 mL) and thioamide 12 (300 mg) in MeOH in a yield of 230 mg (44%), bright-yellow powder. FTIR, νmax, cm−1: 3452, 3281 (N-H); 1661 (CO2Me); 1622 (C=N). 1H NMR : 3.51 , 3.71 , 5.04 , 6.84–6.88 , 7.00 , 7.06–7.10 , 7.30–7.35 , 7.55–7.66 . 13C DEPTQ NMR : 37.7* (C-4), 50.5* (CO2CH3), 54.9* (ArOCH3), 78.1 (C-5), 113.4* , 113.7 (C-3a), 120.1* , 124.3* (C-4 Ph), 128.2* , 129.7* , 130.0* , 130.5* , 131.1* (C-4 Ph), 135.6 (C-1 Ph), 138.8 (C-1 Ar), 150.9 (C-1 Ph), 153.0 (C-6), 153.6 (C-8a), 157.6 (C–OMe), 163.2 (C=N), 169.1 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 64.65; H, 4.62; N, 8.38; found (%): C, 64.47; H, 4.70; N, 8.25. HRMS (ESI) m/z: calculated for C27H24N3O3S2 [M + H]+: 502.12591, found 502.1248 (Δ 2.21 ppm).Methyl 6-amino-7-phenyl-3-(phenylimino)-4-(2-thienyl)-4,7-dihydro-3H-dithiolopyridine-5-carboxylate (15g). This compound was prepared via Method A in a yield of 360 mg (72%). Alternatively, this compound was prepared via Method B employing methyl cyanoacetate (0.09 mL), thiophene-2-carbaldehyde (0.09 mL) and thioamide 12 (300 mg) in MeOH in a yield of 326 mg (65%), yellow powder. FTIR, νmax, cm−1: 3440, 3273 (N-H); 1660 (CO2Me); 1627 (C=N). 1H NMR : 3.55 , 5.40 , 6.91–6.95 , 7.10–7.14 , 7.26 , 7.32–7.36 , 7.50–7.54 , 7.60–7.66 . 13C DEPTQ NMR : 34.0* (C-4), 51.1* (CO2CH3), 78.1 (C-5), 112.8 (C-3a), 120.1* , 122.7* (C-2 thienyl), 123.7* (C-5 thienyl), 124.3* (C-4 Ar), 126.6* (C-4 thienyl), 129.7* , 130.0* , 130.5* , 131.0* (C-4 Ph), 135.7 (C-1 Ph), 150.8 (C-1 Ph), 151.0 (C-1 thienyl), 153.3 (C-6), 154.1 (C-8a), 163.4 (C=N), 169.0 (C=O). *Signals with a negative phase. Elemental Analysis : calculated (%): C, 60.35; H, 4.01; N, 8.80; found (%): C, 60.23; H, 4.15; N, 8.75. HRMS (ESI) m/z: calculated for C24H20N3O2S3 [M + H]+: 478.0718, found 478.0722 (Δ −0.84 ppm).–3% by weight) for 1 h to achieve 40–60% inhibition of hypocotyl growth. After treatment, the seedlings were washed with pure water and placed into a solution of the corresponding compound 15a,c,f . After 1 h the seedlings were washed with pure water and placed on paper strips . The strips were rolled and placed into beakers with water (50 cm3). The reference group of seedlings (“herbicide” experiments) was kept in 2,4-D solution (10–3%) for 1 h and then in water for 1 h. The “control” seedlings were kept in water for 2 h. The temperature of all solutions was maintained at 28 °C. The seedlings were then thermostated for 3 days at 28 °C. Each experiment was performed in triplicate; 20 seeds were used in each experiment. The results are given in Germinated sunflower seeds (cv. Master) with 2–4 mm long embryo roots were placed in a solution of 2,4-D (10–2 to 10–5 by weight) for 1 h. After 1 h, the seeds were spread evenly on strips of filter paper, rolled up, placed in beakers with water and thermostated at 28 °C for 3 days. Then stem and root length were measured, and the data were statistically processed using Student’s t-test, p = 0.95. Each experiment was carried out in triplicate with 100 seeds. The results are given in Sunflower seeds were treated with a solution of a test compound at different concentrations  cyanoacetate and dithiomalondianilide. Related reactions of dithiomalondianilide 12 with other Michael acceptors are currently underway in our laboratory. Some compounds showed a strong antidote effect with respect to herbicide 2,4-D accompanied by moderate growth-regulating activity in the experiments on sunflower seedlings. Thus, compounds 15a,c,f reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 34–60% and by 40–55% on sunflower seedling roots in the laboratory experiments and showed an antidote effect of 41.4–51.4% in the field experiments.Generally, a new preparative method for synthesis of alkyl 6-amino-4-aryl-7-phenyl- 3-(phenylimino)-4,7-dihydro-3H-dithiolopyridine-5-carboxylates


O-[β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-β-d-xylopyranosyl]-2β,3β,16α-trihydroxyolean-12-en-23,28-dioic acid-28-O-α-l-rhamnopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-α-l-arabinopyranoside 1 together with a new oleanane triterpene identified as 2β,3β,13α,22α-tetrahydroxy olean-23,28-dioic acid 2 and 6 known compounds (3–8) have been isolated from Gladiolus segetum Ker-Gawl corms. The structural elucidation of the isolated compounds was confirmed using different chemical and spectroscopic methods, including 1D and 2D NMR experiments as well as HR-ESI-MS. Moreover, the in vitro cytotoxic activity of the fractions and that of the isolated compounds 1–8 were investigated against five human cancer cell lines  using doxorubicin as a reference drug. The results showed that the saponin fraction exhibited potent in vitro cytotoxic activity against the five human cancer cell lines, whereas the maximum activity was exhibited against the PC-3 and A-549 cell lines with the IC50 values of 1.13 and 1.98 μg mL−1, respectively. In addition, compound 1 exhibited potent activity against A-549 and PC-3 with the IC50 values of 2.41 μg mL−1 and 3.45 μg mL−1, respectively. Interestingly, compound 2 showed the maximum activity against PC-3 with an IC50 of 2.01 μg mL−1. These biological results were in harmony with that of the molecular modeling study, which showed that the cytotoxic activity of compound 2 might occur through the inhibition of the HER-2 enzyme.A new triterpenoidal saponin identified as 3- Gladiolus segetum Ker-Gawl corms.A new triterpenoidal saponin 1, a new oleanane triterpene 2, and 6 known compounds (3–8) have been isolated from  These herbs have direct medicinal applications as drug entities and there are also many others that can serve as chemical models or templates for synthesis, semi-synthesis and the design of novel drug candidates for the treatment of human diseases.2 The Gladiolus genus is one of the attractive medicinal plants belonging to the horticultural family Iridaceae and comprises 260 species, which are mainly native to South Africa with several species distributed in Western and Central Europe, the Mediterranean region, and Southwest and Central Asia.3 Many Gladiolus species corms have been traditionally used as a remedy for dysentery and rheumatic pain,4 such as G. gandavensis corms, which are used to treat pharyngitis, parotitis and lymphadenitis in Chinese folk medicine.5 In addition, G. dalenii was used in the Kenyan Lake Victoria Basin as a traditional treatment for meningitis, malaria, diarrhoea, ulcers and HIV-related fungal infections. Likewise, the hot water extracts of dried corms are used for the treatment of ulcers.6 Moreover, the phytochemical survey of the genus Gladiolus showed an interesting wide diversity of chemical constituents, such as anthraquinones,4,5,7 flavonoids,8 triterpenes,4 saponins,9 and lignans.10 There are several approaches to drug discovery, such as combinatorial chemistry and computer-based molecular modeling design based on natural products,2 in addition to drug discovery using natural products, which represents a challenging task for designing new leads. Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity,11 which can regulate cell growth, survival, and differentiation via multiple signal transduction pathways and participate in cellular proliferation and differentiation.12 Most studies on HER2 have been carried out in breast cancer and its overexpression occurs in approximately 15–30% of breast cancers.13–15 It has now been documented that HER2 overexpression also occurs in many forms of cancers, such as ovary, stomach, uterine serous endometrial carcinoma, bladder, lung, colon, uterine cervix, head and neck, and esophagus.16,17 Its major role in these tissues is to facilitate excessive/uncontrolled cell growth and tumorigenesis.18–20 Therefore, HER2 has been recognized as a promising anticancer target with many reported inhibitors such as Trastuzumab, Lapatinib, Pertuzumab, Neratinib and Afatinib.11 Therefore, the present study deals with the isolation and structural elucidation of eight compounds from Gladiolus segetum Ker-Gawl corms, which is a herbaceous plant cultivated in Egypt as an ornamental plant and known locally as Seif El-Ghorab.10 Moreover, we evaluated the antiproliferative activity of the pure compounds on five different cell lines. Finally, the docking study of the isolated compounds was carried out against human epidermal receptor 2 (HER-2) at the catalytic ligand binding site (PDBID: 5jeb) to help better understand the binding mode and their possible mechanism of action as potential new anticancer agents.Natural products and their derivatives are of great importance and have been recognized for many years as an important source of therapeutic agents for the treatment of several diseases.2.Gladiolus segetum Ker-Gawl corms was chromatographed using various chromatographic techniques, such as silica gel, Sephadex LH-20, RP18 column chromatography, and the final purification on HPLC led to the identification of two new compounds, in addition to six known compounds, as shown in The crude extract of 2.1.70H112O38 based on the positive ion mode HR-ESI-MS. The 1H-NMR spectrum of compound 1 revealed the presence of six characteristic singlet signals for six methyl groups at δH 0.66, 0.83, 0.90, 1.14, 1.18, and 1.28 , in addition to an olefinic proton at δH 5.21, suggesting the oleanane triterpene skeleton of the aglycon. This was further confirmed from the 13C-NMR spectra that revealed the presence of six tertiary carbon signals, in addition to one olefinic carbon at δC 121.83 and one quaternary carbon signal at δC 143.26, and two carbonyl carbon signals at δC 180.73 (C-23) and 175.34 (C-28). Therefore, the structure of the triterpenoid moiety was obtained principally from the correlations observed in the HSQC and HMBC spectra, and confirmed to be that of 2β,3β,16α-trihydroxyolean-12-en-23,28-dioic acid (zanhic acid) by comparison of the NMR data , glucose II at δH 4.17 , glucose III at δH 4.21 , xylose at δH 4.25 , arabinose at δH 5.19 , rhamnose I at δH 5.40  and rhamnose II at δH 5.26 . The β anomeric configurations of the d-glucose and d-xylose units were determined from their coupling constants (7.0–7.5 Hz). The α-anomeric configurations of l-arabinose and l-rhamnose were determined based on their broad singlet signals of anomeric protons at δH 5.19, 5.26, and 5.40 in the 1H-NMR spectra.Additionally, the δH 3.81 and C-1 of xylose at δC 105.76, and from H-1 of Xyl at δH 4.25 to C-3 at δC 80.40. In addition, the downfield shifted resonances of C-3 at δC 84.20 and H-3 at δH 4.05 of xylose indicated further substitution at this position, which was confirmed by the HMBC correlations of H-1 at δH 4.19 of glucose I to C-3 of xylose at δC 84.20, and H-3 of xylose at δH 4.05 to C-1 of glucose I at δC 103.20. Furthermore, the downfield shifted resonances of C-2 (δC 81.60) and H-2 (δH 3.78) of glucose I indicated further substitution at this position, as confirmed by the HMBC correlations -β-d-glucopyranosyl-(1 → 4)-β-d-xylopyranosyl moiety was O-linked at C-3. The remaining four sugar moieties were present as a tetrasaccharide O-linked at C-28. The primary sugar was arabinose, whereas the HMBC spectrum showed a correlation between its anomeric proton at δH 5.19 and C-28 at δC 175.34. The other sugar sequence was also confirmed from HMBC correlations, whereas a significant correlation was observed between the anomeric proton at δH 4.21 of glucose III and C-2 of arabinose at δC 75.70. Another HMBC correlation was exhibited between H-1 at δH 5.40 of rhamnose I with C-2 of glucose III at δC 81.95, in addition to the correlation of H-1 of rhamnose II at δH 5.26 with C-4 at δC 80.57 of the rhamnose I moiety. From the previously mentioned data and comparison with the literature,9,21,23–29 compound 1 was determined to be 3-O-[β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-β-d-xylopyranosyl]-2β,3β,16α-trihydroxyolean-12-en-23,28-dioic acid-28-O-α-l-rhamnopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-α-l-arabinopyranoside, which is a new compound.The HMBC correlations between the aglycone and the sugar resonances indicated that compound 1 was a bisdesmoside. The long range correlations were observed at C-3 between H-3 of aglycone at elations  from H-230H48O8 from HR-ESI-MS. The IR spectrum of 2 showed absorption bands at 3452 cm−1 for the hydroxyl groups, 1733 cm−1 for the carbonyl groups and 1389 cm−1 for the gem-dimethyl. The 1H-NMR spectrum of compound 2 showed six singlet signals for the six methyl groups at δH 0.69, 0.86, 1.20, 0.83, 1.11, 1.14, which were attributed to H3-30, H3-29, H3-27, H3-26, H3-25 and H3-24, respectively. The 1H-NMR also showed three broad singlet signals at δH 3.91, 3.73 and 3.95 for the three oxygenated methine protons, which were confirmed from the 13C-NMR signals at δC 70.11, 74.65 and 69.81. These signals were assigned to the oxymethines C-2, C-3 and C-22, respectively.Compound 2 was isolated as a white amorphous powder. Its molecular formula was established as C13C-NMR spectrum displayed a signal at δC 69.12, which was attributed to an oxyquaternary carbon (C-13), in addition to two carbonyl carbon signals at δC 179.16 and 177.46 that were assigned to C-23 and C-28, respectively. The 13C-NMR spectrum of compound 2 did not display any olefinic carbons, indicating the absence of unsaturation at C-12. The aforementioned spectral data, together with physicochemical properties, revealed the oleanane triterpene nature of compound 2. The localization of the hydroxyl groups was confirmed from the HMBC spectrum that showed significant correlations between H-1 at δH 1.17, 2.05 with C-2 at δC 70.11 and C-3 at δC 74.65. In addition, H-3 at δH 3.73 was correlated to C-2 at δC 70.11, C-4 at δC 52.30, and C-23 at δC 179.16 and C-24 at δC 12.46. H-24 at δH 1.14 showed a long range correlation with C-3 at δC 74.65, C-4 at δC 52.30, C-5 at δC 51.22 and C-23 at δC 179.16. This established the attachment of the two secondary hydroxyl groups at C-2 and C-3 of ring A, and confirmed the presence of a carboxylic group at C-23. Likewise, H-22 at δH 3.95 showed a correlation with C-21 at δC 44.35, while H-21 at δH 1.18, 2.32 showed correlations with C-17 at δC 48.63, C-20 at δC 33.12 and C-22 at δC 69.81, revealing the presence of another hydroxyl group attached at C-22. The fourth hydroxyl group was deduced to directly attach to C-13 from the HMBC correlations between H-12 at δH  and C-13 at δC 69.12. Other correlations included H-27 at δH 1.20 and C-13 at δC 69.12, and H-18 at δH 1.72 and C-13 at δC 69.12, as shown in δH 3.91 with methine H-3 at δH 3.73 and methylene H-1 protons at δC (1.17 and 2.05). Additionally, significant correlations exhibited between H-18 (δH 1.72)/H-19 , and H-11 /H-12  were also observed. The direct single bond 1H/13C connectivity of each protonated carbon was deduced with the help of the HMQC data , as 2β,3β,16α-trihydroxy olean-23,28-dioic (zanhic acid) (3),22p-hydoxybenzaldehyde (6)33 and spinasterol-3-O-β-d-glucopyranoside (5),34,35 which are reported for the first time in the plant. In addition, 2β,3β-dihydroxyolean-12-ene-23,28-dioic (medicagenic acid) (4),4p-hydroxymethylcinnamate (7),36 and desoxyerytholaccacin (8)10 were identified.The other isolated compounds were identified based on various spectroscopic methods, including 1D and 2D NMR experiments  and breast adenocarcinoma (MCF-7) cell lines with IC50 values of 3.01 and 4.23 μg mL−1, respectively. The biological results indicated that compound 1 was the most active among the isolated compounds. It showed potent activity against A-549 and PC-3 with IC50 values of 2.41 μg mL−1 and 3.45 μg mL−1, respectively. Compound 1 also showed moderate activity against the HePG2 and MCF-7 cell lines with IC50 values of 5.98 and 6.43 μg mL−1, respectively.The saponin fraction and the isolated compounds 1–8 were tested for their tumor cell growth inhibitory activity against five human cancer cell lines, PC-3, A-549, HePG-2, MCF-7 and HCT-116. The saponin fraction exhibited potent cytotoxic activity against all tested cell lines, whereas the maximum activity was observed against prostate cancer cell line (PC-3) and lung carcinoma cell line (A-549) with IC50 value of 2.01 μg mL−1, followed by A-549 (IC50 5.01 μg mL−1), in comparison to the cytotoxic control drug adriamycine (doxorubicin), which exhibited IC50 1.37, 1.63, 2.26, and 2.65 against A-549, PC-3, MCF-7, and HePG2, respectively, as shown in Compound 2 showed the maximum activity against PC-3 with an IC37–39 This clarified the important structural feature of the presence of an oligosaccharide ester at C-3 and a free hydroxyl group at C-16, which were considered to be essential for the cytotoxic activity. Many published studies from literature revealed the correlation of the cytotoxic activity of saponins with the presence of the C-2 glycoside linkage of the first sugar. This could induce the electron deficiency near C-3 of the aglycone more than at the other sites of the first sugar, together with the several types of functional groups. Actually, there are a number of saponins that have occurred in natural sources with a (1 → 2) glycoside linkage between the first and second sugars, rationalizing the wide abundance of this type of saponin.40,41 Concerning triterpene aglycone, compound 2 showed cytotoxic activity higher than 5, which may attributed to the presence of a free carboxyl group at C-28.39,41From these aforementioned results, it is obvious that the cytotoxic potency of the triterpenoid saponin 1 is higher than that of the triterpene aglycone 2, which was in harmony with earlier studies.2.4.via multiple signal transduction pathways and participate in cellular proliferation and differentiation. Therefore, HER-2 receptors overexpressed in tumor cells represent an active target for various cancer therapeutics.18,20,42Molecular modeling studies play an important role in order to construct molecular models that help in better understanding the binding mode of target compounds at the molecular level. Therefore, compounds 2, 3, 4, 5, and 8 were selected to be docked in the active site of human epidermal receptor 2 (HER-2) at the catalytic ligand binding site (PDBID: 5jeb), which is overexpressed in several epithelial-derived tumors including breast, ovarian, lung, renal, colorectal and brain. HER-2 is a family of receptors that plays a vital role in the pathogenesis of several human cancers. They regulate cell growth, survival, and differentiation 43Herein, we report the binding pose of DOX and compounds 2, 3, 4, 5, and 8 with HER-2 protein, as shown in the form of ligplots depicted in i.e., Val 702, Ala 719, Cys 773 and Leu 820). The docking results of compound 2 indicated that it bound nicely to the catalytic binding pocket of the HER-2 protein receptor (PDBID: 5jeb)    via incoLeu 820) .Moreover, the docking results of compounds 3 and 4  and 6 shThe least active compounds 5 and 8  and 8 shIn conclusion, these results indicate that the anticancer activities of these compounds are in agreement with their biological results. Our results strongly suggest that the possible mechanism of action of these compounds, in particular that of compound 2, may be through the inhibition of the HER-2 enzyme.3.3.1.1H-NMR, COSY, NOESY, 13C-NMR, DEPT-13C-NMR HSQC, HMBC) were measured on a Bruker Avance III HD 600 MHz spectrometer (Japan) and a Varian mercury 400 MHz Spectrometer (Japan) using TMS as the internal standard. Column chromatography was carried on Silica Gel 60 N, 40–50 μm (pH: 7.0 ± 0.5), Sephadex LH-20 (20–100 μm) and Wakogel 50C18 (38–63 μm) . HPLC was performed using a Mightysil RP-18 column  on a LC-8A preparative liquid chromatography with a SPD-10A UV detector  and SCL-10A Analytical HPLC using ODS-120T column and Waters 2487 dual UV detector . TLC analyses were performed on precoated Kieselgel 60F254 plates  and TLC precoated Kieselgel 60 RP-18 F254 s plates . The plates were examined under UV light (at 365 and 254 nm) and visualized by spraying with an anisaldehyde reagent, followed by heating at 110–140 °C for 1–2 min, and allowed to dry at room temperature. The following solvent systems were used for TLC:The ESI-MS of compound 1 were measured on ultrahigh resolution mass spectra acquired with a solariX 7.0 T equipped with an ESI/MALDI dual ion source  in the positive ion mode. Compound 2 was measured on a Shimadzu LCMS-IT-TOF (Japan). The FTIR spectra were measured on a Shimadzu Infra-red-470 spectrophotometer (Japan). NMR analyses (3.2.Gladiolus segetum Ker-Gawl were collected from the fields of Giza governorate (2012), and kindly identified and authenticated by Prof. Dr Naeam Keltawy, Professor of Horticultures, Department of Horticulture, Faculty of Agriculture, Assiut University, Assiut, Egypt. A voucher specimen (Gs-002) was kept in the Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt.The corms of 3.3.2CO3, and sugars in the aqueous solution were identified by co-paper chromatography (PC) with authentic materials using n-BuOH–AcOH–H2O  using an aniline phthalate spray as the detection reagent, and their data (Rf) revealed the presence of Glc (Rf 0.13), Arb (Rf 0.16), Xylose (Rf 0.22) and Rha (Rf 0.28) units.Compound 1 (4 mg) was refluxed in 10 mL of 1 N HCl for 4 h. Aglycone was extracted with methylene chloride. The water soluble fraction was neutralized with Nad-Glucose, d-xylose, l-rhamnose and l-arabinose were obtained from Wako Chemical Co., Ltd., Japan.3.4.Human prostate cancer (PC-3), human lung carcinoma (A-549), human hepatocellular carcinoma (HePG-2), human breast adenocarcinoma (MCF-7) and human colon cell line (HC-T116) were purchased from the American Tissue Culture Collection. Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute medium (RPMI 1640), and other chemicals were obtained from Sigma-Aldrich Co. .3.5.Gladiolus segetum Ker-Gawl were extracted by maceration and percolation with ethanol until complete exhaustion. The ethanolic extract (78.0 g) was subjected to successive solvent fractionation over VLC with chloroform (15.0 g), n-butanol saturated with water (40.0 g), and finally washed with methanol (20.0 g) (4 L in each solvent).The air-dried powdered corms (800 g) of The butanolic extract was dissolved in MeOH (400.0 mL), and added portionwise into cold diethyl ether (2 L). The precipitate was removed by filtration on a Buchner funnel, washed with cold diethyl ether, and dried under reduced pressure to give the saponin fraction (32 g).n-butanol (saponin) fraction (500.0 mg) was chromatographed over RP-18, eluted by gradient with MeOH–H2O. Fractions 15–20 were subjected to further purification by HPLC and eluted with CH3CN–H2O (34%), and UV detection at 205 nm resulted in the separation of compound 1 (8.0 mg) with a running time of 33 min.Part of the 2Cl2 and followed by a CH2Cl2–MeOH gradient elution, affording the GC1-GC4 sub-fractions. Sub-fraction GC3 (150.0 mg) was chromatographed over RP-18, and eluted with MeOH–H2O (70 : 30). Fractions 33–42 were subjected to further purification by HPLC and eluted with CH3CN–H2O (59%), and UV detection at 205 nm resulted in the separation of compounds 2 (12.0 mg) with a running time of 16 min. and 3 . Fractions 22–27 afforded compound 4 (15.0 mg). Likewise, sub-fraction GC-4 (100.0 mg) was chromatographed on a silica gel column eluted with CH2Cl2–MeOH, and afforded compound 5 (50.0 mg).A part of the chloroform-soluble fraction (10 g) was chromatographed over a silica gel column, eluted with CH2Cl2–MeOH (1 : 1). Fractions 8–12 were concentrated under reduced pressure afforded compound 6 (10.0 mg), while fractions 16–20 were collected and concentrated under reduced pressure, affording compound 7 (30.0 mg). Fractions 25–32 were rechromatographed over silica gel column chromatography, and eluted isocratically with CH2Cl2–MeOH (9.5 : 0.5), resulting in the separation of 8 (40.0 mg).Sub-fraction GC2 (200.0 mg) was chromatographed over sephadex LH-20 using CH3.5.1.Rf = 0.30  and IR (KBr) cm−1: 3410, 2929, 1737, 1637, 1039. HRESI-MS showed a molecular ion peak at m/z: 1583.6663 [M + Na]+ . MS/MS (MS2) of [M + Na]+ gave fragments at m/z, 997.45753 = [(M + Na)-132-146-146-162]+ attributed to the loss of a tetraglycosidic chain comprising one pentose, two deoxyhexoses and one hexose. The 1H-NMR  and 13C-NMR  spectral data are compiled in Compound 1 was obtained as a white amorphous powder with 3.5.2.Rf = 0.34 . The positive ion mode HR-ESI-MS spectrum showed fragments [M + Na–H2O]+ at m/z = 541.3121 and [M + 1-2COOH–2OH]+ at m/z = 413.2653, while the negative ion mode showed the fragment [M − Na–H2O]− at m/z = 517.3124. The 1H-NMR spectral data  and 13C-NMR spectral data  are listed in Compound 2 was obtained as a white amorphous powder with 3.5.3.Rf = 0.36 . The GC-MS spectrum showed the molecular ion peak at m/z [M]+ = 518, coincident with the molecular formula C30H46O7. 1H-NMR spectral data  δH: 0.67, 0.82, 0.89, 1.16, 1.17 and 1.29 , 3.73 , 3.88 , 4.30 , 5.20 . 13C-NMR spectral data  δC: 44.04 (C-1), 69.99 (C-2), 74.73 (C-3), 52.10 (C-4), 51.10 (C-5), 20.45 (C-6), 34.58 (C-7), 39.92 (C-8), 47.33 (C-9), 35.87 (C-10), 22.97 (C-11), 121.18 (C-12), 144.03 (C-13), 40.04 (C-14), 35.86 (C-15), 72.96 (C-16), 46.67 (C-17), 41.17 (C-18), 46.44 (C-19), 30.26 (C-20), 35.18 (C-21), 32.34 (C-22), 179.50 (C-23), 12.67 (C-24), 16.28 (C-25), 16.77 (C-26), 26.52 (C-27), 178.49 (C-28), 32.93 (C-29) and 24.28 (C-30).22Compound 3 was obtained as a white amorphous powder with 3.5.4.Rf = 0.39 . The GC-MS spectrum showed the molecular ion peak [M]+ at m/z = 502 coincident with the molecular formula C30H46O6. Selected 1H-NMR spectral data  δH: 0.69, 0.86, 1.01, 1.16, 1.86 and 1.91 , 3.73 , 3.88 , 5.16 . 13C-NMR spectral data  δC: 43.95 (C-1), 69.95 (C-2), 74.47 (C-3), 52.15 (C-4), 50.92 (C-5), 20.41 (C-6), 33.81 (C-7), 40.79 (C-8), 47.55 (C-9), 35.87 (C-10), 22.59 (C-11), 121.51 (C-12), 143.74 (C-13), 40.13 (C-14), 27.09 (C-15), 23.35 (C-16), 45.42 (C-17), 41.41(C-18), 45.69 (C-19), 30.37 (C-20), 35.82 (C-21), 32.07(C-22), 179.04 (C-23), 12.52 (C-24), 16.19 (C-25), 16.69 (C-26), 25.61 (C-27), 178.57 (C-28), 32.81 (C-29) and 22.95 (C-30).4Compound 4 was obtained as a white amorphous powder with 3.5.5.Rf = 0.36 . The GC-MS spectrum showed the molecular ion peak at m/z 574 [M]+ corresponding to the molecular formula C35H58O6, 411 [M-glucose] and other peaks at 361, 301, 278, 258, 238, 218, 185, 167, 149, 147, 125, 106 and 77. The selected 1H-NMR spectra  showed the following signals: δH 0.50 ; 0.73 ; 0.76 ; 0.81 ; 0.99 ; 1.04 ; 4.21 ; 4.89 ; 5.11 ; 5.32 ; 5.44 . The 13C-NMR  spectral data showed signals at δC: 36.49 (C-1), 30.86 (C-2), 89.85 (C-3), 34.91 (C-4), 40.11 (C-5), 29.07 (C-6), 117.16 (C-7), 139.05 (C-8), 48,62 (C-9), 33.91 (C-10), 21.12 (C-11), 39.70 (C-12), 42.63 (C-13), 54.23 (C-14), 22.44 (C-15), 28.3 (C-16), 55.18 (C-17), 12.14 (C-18), 13.95 (C-19), 39.89 (C-20), 18.85 (C-21), 137.96 (C-22), 128.88 (C-23), 50.64 (C-24), 31.32 (C-25), 20.99 (C-26), 17.90 (C-27), 24.82 (C-28) and 12.63 (C-29).34,35Compound 5 was obtained as a white amorphous powder, which exhibited 3.5.6.Rf = 0.43 . The GC-MS spectrum showed the molecular ion peak [M]+ at m/z = 138, which was consistent with the molecular formula C6H7O2. 1H-NMR spectral data : δH 6.92 , 7.77  and 9.51 . 13C-NMR spectral data : δC 193.06 (C-1), 130.26 (C-2), 133.49 (C-3), 116.92 (C-4), 165.2 (C-5) 116.92 (C-6) and 133.49 (C-7).33Compound 6 was obtained as a white amorphous powder, which showed 3.5.7.Rf = 0.38 . The GC-MS spectrum showed the molecular ion peak [M]+ at m/z = 178 calculated to the molecular formula C10H10O3. 1H-NMR spectral data : δH 3.3 , 6.33 , 6.81 , 7.45  and 7.60 . 13C-NMR spectral data : δC 169.9 (C-1), 114.9 (C-2), 146.6 (C-3), 127.15 (C-4), 131.15 (C-5) 116.86 (C-6), 161.2 (C-7), 116.86 (C-8), 131.15 (C-9) and 52.08 (OCH3).36Compound 7 was obtained as a white amorphous powder with 3.5.8.Rf = 0.34 . The GC-MS spectrum showed the molecular ion peak [M]+ at m/z = 272 coincident with the molecular formula C15H12O5. 1H-NMR spectral data : δH 2.74 , 6.52 , 6.94 , 7.08  and 7.46 . 13C-NMR spectral data : δC 165.44 (C-1), 109.34 (C-2), 166.34 (C-3), 108.21 (C-4), 113.32 (C-5) 163.20 (C-6), 125.65 (C-7), 138.54 (C-8), 124.50 (C-9), 146.72 (C-10) 136.11 (C-11), 111.79 (C-12) 189.80 (C-13) and 184.38 (C-14).10Compound 8 was obtained as yellow crystals and showed 3.6.3.6.1.−1 potassium penicillin, 10 000 mg mL−1 streptomycin sulphate and 25 mg mL−1 amphotericin B), 1% l-glutamine and 10% fetal bovine serum, according to the cells.Cells were suspended in RPMI 1640 medium (for HePG-2- MCF-7 and HCT-116) and DMEM medium (for A-549 and PC-3). Media were supplemented with a 1% antibiotic-antimycotic mixture . The media was aspirated and fresh medium (without serum) was added, and the cells were incubated either alone (negative control) or with different concentrations of the tested samples to give a final concentration of . After 48 h of incubation, the medium was aspirated, 40 μL MTT salt (2.5 μg mL−1) were added to each well, and the cultures were incubated for four additional hours at 37 °C under 5% CO2. To stop the reaction and dissolve the formed crystals, 200 μL of 10% sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37 °C. A positive control composed of 100 μg mL−1 was used as a known cytotoxic natural agent, which gives 100% lethality under the same conditions.45,46 The absorbance was then measured using a microplate multi-well reader  at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and the negative control (cells with vehicle) using the independent t-test by the SPSS 11 program. DMSO was the vehicle used for the dissolution of the plant extracts, and its final concentration on the cells was less than 0.2%. The percentage of change in viability was calculated according to the formula:Cell viability was assessed by the mitochondrial dependent reduction of yellow 3--2,5-diphenyl tetrazolium bromide (MTT) to purple formazan, which reflects the normal function of mitochondria and cell viability.50 and IC90 determination using the SPSS 11 program.A probit analysis was carried for the IC3.7.5jeb). It was prepared by removing the solvent, adding hydrogens and further minimized in the presence of the bound ligand (6JS) using the protein preparation wizard. Grids for molecular docking were generated with the bound co-crystallized ligand.43,47,48 Force fields were applied on the docked compounds to get the minimum lowest energy structure. The obtained poses were studied and the poses showing the best ligand – HER-2 interactions were selected and used for the CDOCKER energy (protein–ligand interaction energies) calculations. Receptor–ligand interactions of the complexes were examined in 2D and 3D styles.The docking study of DOX and compounds 2, 3, 4, 5, and 8 against the HER-2 receptors was performed using Discovery Studio 2.5 software . The docked compounds were sketched in 3D format using the build panel, and were prepared for docking using the ligprep application. The protein for the docking study was taken from the protein data bank (PDB ID: 4.Gladiolus segetum Ker-Gawl corms, which led to the isolation and characterization of two new compounds and six known compounds. This study revealed the various classes of secondary metabolites in the plant. The structural elucidation was performed using different spectroscopic techniques. The in vitro cytotoxic activity of all isolated compounds were examined against five human cancer cell lines. The results showed that the saponin fraction had potent in vitro cytotoxic activity against the five human cancer cell lines and was particularly potent against the PC-3 and A-549 cell lines. In addition, compound 1 exhibited potent activity against A-549 and PC-3. Moreover, compound 2 showed the maximum activity against PC-3. These biological results together with the molecular modeling study suggested that the cytotoxic activity of these compounds and in particular, compound 2, could occur via the inhibition of the HER-2 enzyme.Our study presented the phytochemical investigation of The authors declare no conflict of interest.RA-010-D0RA02775H-s001


Panax notoginseng. Six transformation products (1–6), named 20(S)-ginsenoside Rg3 (1), 20(R)-ginsenoside Rg3 (2), notoginsenoside SFt3 (3), ginsenoside Rk1 (4), ginsenoside Rg5 (5), and 20(S)-ginsenoside Rh2 (6) were isolated and identified from a microwave processed extract of the stems and leaves of P. notoginseng (MEL). This transformation method was also applied for producing the minor ginsenosides in flowers, seeds and pedicels of P. notoginseng. The extract and compounds 1–6 in MEL were evaluated in vitro for anticancer and anticoagulant activities. The results showed that the MEL extract and transformation products had outstanding inhibitory activities against human cervical cancer Hela and lung cancer A549 cells. The strongest inhibitory effect was observed for 20(S)-Rh2 (6) with an IC50 value of 8.23 μM in Hela cells. Moreover, the results showed that the MEL significantly prolonged prothrombin time in a concentration-dependent manner. The anticoagulant effect of the MEL improved with the increased contents of Rk1, Rg5, and SFt3.A microwave processing technology was applied to degrade saponins from the stems and leaves of  Panax notoginseng.A microwave processing technology was used to produce anticoagulant and anticancer compounds from the stems and leaves of  H. Chen has been widely used as a herbal medicine in China. It was recorded in the Compendium of Materia Medica that the stems and leaves of P. notoginseng (PNL) were traditionally used to treat bone fractures, eliminate swelling, and stop bleeding. The PNL is also used as a functional food in folk medicine, as PNL is rich in nutrients and characterized by high protein, high dietary fiber, and low fat.1 Fresh leaves are suitable for eating as a vegetable and dried as a tea.21, Rc, Rb2, and Rb3 -ginsenosides Rg3 and Rh2 because of more effective anticancer activities.6,7 The Shenyi capsule containing ginsenoside Rg3 was clinically applied for treating non-small cell lung cancer in China.8 The common methods used for producing less-polar saponins are acid–base degradation,9 enzymatic degradation,10 microbial transformation,11 and steam processing.12 At present, microwave processing method as a novel “green” solvent extraction technology is developed to increase the content of minor ginsenosides from white ginseng.13,14 This green extraction process is often used for extracting natural products owing to a faster extraction rate, less consumption of organic solvent, and lower costs of sample preparation,15,16 but more rarely in the case of transformation. The heat processing by microwave method for producing the minor ginsenosides gained high yields within about 60 minutes.13 The microwave time was much shorter than used in the traditional heating method (over 20 hours).12More than 40 chemical constituents have been isolated from the PNL. The main components are protopanaxadiol type saponins, such as notoginsenosides Fa, Fc, and Fe, and the ginsenosides Rb and Rb3 .3 The saP. notoginseng and determined the optimal conversion conditions using response surface methodology.17 The roots of P. notoginseng are traditionally used as medicine, but a huge amount of PNL is often considered waste. In this study, the microwave method was applied to effectively obtain a microwave processing extract from stems and leaves of P. notoginseng (MEL). Six known compounds (1–6) were isolated from the MEL and identified by nuclear magnetic resonance spectroscopic data  were purchased from the wenshan market in Yunnan province of China in October 2016. The samples were identified by Dr Xiuming Cui of Kunming University of Science and Technology. The nuclear magnetic resonance spectra (NMR) was recorded by a Bruker Avance III 500 spectrometer . Formic acid (HPLC grade), acetonitrile (HPLC grade), dimethyl sulfoxide (DMSO), 3--2,5-diphenyl-tetrazolium bromide (MTT), penicillin, streptomycin, l-glutamine and methanol were purchased from Sigma-Aldrich . Fetal bovine serum (FBS) and trypsin were purchased from Gibco BRL . All the other reagents and solvents were of analytical grade.The different parts of 2.2P. notoginseng  were powdered and ultrasonically extracted with 70% aqueous MeOH, three times for 45 min. After filtration and concentration, the extract of stems and leaves of P. notoginseng  was obtained with a yield of 22.5%. In the same way, the yields of the extracts of flowers (EF), seeds (ES), and pedicels (EP) were 36.36%, 6.48% and 25.48%, respectively.The dried stems and leaves of P. notoginseng  was treated using microwave processing method as reported previously.17 160 mg of the extract was added to the water (10 mL) in a microwave extraction system (model no: MDS-6G) manufactured by Sineo microwave chemical technology Co. Ltd. . Considering the influence of microwave temperature, microwave power and time, the temperature ranged from 60 °C to 180 °C, microwave power ranged from 300 W to 1000 W, and time ranged from 5 min to 50 min. After microwave treatment, the sample solution was centrifuged and the supernatant was added to the water for 10 mL. The obtained samples were filtered through a 0.45 μm filter membrane for HPLC analysis. The yields of transformation products were obtained from HPLC data.Mg is the content of transformation products and Ms is total amount of the sample.The extract samples from different parts of 2.3P. notoginseng  was obtained with a yield of 90.4%. The composition of MEL was performed on an Agilent 1290 Infinity II LC system (UHPLC) coupled to an Agilent 6530 quadrupole time-of-flight mass spectrometry system (Q-TOF/MS) . Samples were separated on an Agilent Poroshell 120 EC-C18 column  at a rate of 0.6 mL min−1 using an Agilent 1290 Infinity II LC system. The mobile phases consisted of 0.1% (v/v) formic acid water (A) and 0.1% (v/v) formic acid acetonitrile (B) in the gradient program: 0–10 min, 10–25% (B); 10–50 min, 25–40% (B); 50–65 min, 40–60% (B); 65–70 min, 60–100% (B). For MS detector performed on a Q-TOF/MS, mass spectrometry conditions of electrospray ionization source (ESI) in positive ion detection mode were as follows: capillary voltage 4000 V; gas temperature, 350 °C; during gas N2 flow rate, 10 L min−1; pressure of nebulizer, 35 psi. The mass analyzer scanned over m/z 100–3000 Da.The EL extract (10 g) was processed at a temperature of 150 °C and microwave power of 500 W for 20 min. After removal of the solvent, microwave processing extract of stems and leaves of 2.43  to afford 5 fractions (Fr. 1–5). Fr. 1 and Fr. 2 were purified by recrystallization to yield compound 1 (126.2 mg) and compound 2 (60 mg), respectively. Fr. 3 was subjected to pre-HPLC  to give compound 3 . Fr. 4 was successively purified by pre-HPLC  to yield compounds 4  and 5 . Compounds 6  was obtained from Fr. 5 separated by pre-HPLC .5 g of the MEL extract was subjected to silica gel column chromatography eluted stepwise with MeOH/CHCl2.4.1m/z 807.4663 [M + Na]+. 1H-NMR  δ: 0.94 , 0.82 , 1.40 , 1.60 , 1.60 , 1.30 , 1.11 , 0.99 , 4.96 , 5.38 . 13C-NMR  δ: 39.5(C-1), 27.1(C-2), 89.3(C-3), 40.1(C-4), 56.7(C-5), 18.8(C-6), 35.5(C-7), 40.4(C-8), 50.8(C-9), 37.3(C-10), 32.4(C-11), 71.4(C-12), 48.9(C-13), 52.1(C-14), 31.7(C-15), 27.2(C-16), 55.2(C-17), 16.2(C-18), 16.7(C-19), 73.3(C-20), 27.5(C-21), 36.3(C-22), 23.4(C-23), 126.7(C-24), 131.1(C-25), 26.2(C-26), 17.4(C-27), 28.5(C-28), 17.0(C-29), 18.1(C-30), 105.5 (Glc-I-C-1′), 83.9 (C-2′), 78.4 (C-3′), 72.1 (C-4′), 78.7 (C-5′), 63.2 (C-6′), 106.5 (Glc-II-C-1′′), 77.5 (C-2′′), 78.7 (C-3′′), 72.0 (C-4′′), 78.5 (C-5′′), 63.1 (C-6′′). The structure of compound 1 was identified as 20(S)-ginsenoside Rg3 by comparing the NMR data and MS data with reported data.18White amorphous powder; HRESIMS 2.4.2m/z 807.4652 [M + Na]+. 1H-NMR  δ: 1.01 , 0.82 , 1.39 , 1.69 , 1.65 , 1.30 , 1.11 , 0.99 , 4.94 , 5.38 . 13C-NMR  δ: 39.1(C-1), 26.6(C-2), 88.9(C-3), 39.7(C-4), 56.4(C-5), 18.4(C-6), 35.1(C-7), 39.9(C-8), 50.3(C-9), 36.9(C-10), 31.3(C-11), 70.9(C-12), 49.2(C-13), 51.8(C-14), 32.2(C-15), 26.7(C-16), 50.6(C-17), 15.8(C-18), 16.4(C-19), 73.0(C-20), 22.8(C-21), 43.2(C-22), 22.6(C-23), 126.0(C-24), 130.8(C-25), 25.9(C-26), 17.3(C-27), 28.2(C-28), 16.6(C-29), 17.7(C-30), 105.2 (Glc-I-C-1′), 83.5 (C-2′), 78.0 (C-3′), 71.6 (C-4′), 78.3 (C-5′), 62.9 (C-6′), 106.1 (Glc-II-C-1′′), 77.2 (C-2′′), 78.4 (C-3′′), 71.6 (C-4′′), 78.2 (C-5′′), 62.7 (C-6′′). The structure of compound 2 was identified as 20(R)-ginsenoside Rg3 by comparing the NMR data and MS data with reported data.18White amorphous powder; HRESIMS 2.4.3m/z 921.4989 [M + Na]+. 1H-NMR  δ: 0.95 , 0.79 , 5.14 , 4.90 , 1.65 , 1.59 , 1.28 , 1.10 , 1.00 , 4.94 , 5.52 , 5.41 . 13C-NMR  δ: 39.8(C-1), 26.8(C-2), 88.8(C-3), 39.3(C-4), 56.4(C-5), 18.5(C-6), 35.4(C-7), 40.3(C-8), 48.3(C-9), 37.1(C-10), 32.7(C-11), 72.4(C-12), 52.5(C-13), 51.2(C-14), 32.8(C-15), 30.8(C-16), 50.8(C-17), 16.6(C-18), 15.9(C-19), 155.6(C-20), 108.2(C-21), 33.9(C-22), 27.1(C-23), 125.3(C-24), 131.2(C-25), 25.8(C-26), 17.8(C-27), 28.1(C-28), 16.7(C-29), 17.0(C-30), 104.8 (Glc-I-C-1′), 83.0 (C-2′), 78.7 (C-3′), 71.1 (C-4′), 78.3 (C-5′), 63.0 (C-6′), 103.2 (Glc-II-C-1′′), 84.5 (C-2′′), 78.0 (C-3′′), 71.8 (C-4′′), 77.9 (C-5′′), 62.8 (C-6′′), 106.5 (Xyl-C-1′′′), 76.0 (C-2′′′), 77.8 (C-3′′′), 70.8 (C-4′′′), 67.5 (C-5′′′). The structure of compound 3 was identified as notoginsenoside SFt3 by comparing the NMR data and MS data with reported data.19White amorphous powder; HRESIMS 2.4.4m/z 789.4595 [M + Na]+. 1H-NMR  δ: 1.00 , 0.80 , 5.14 , 4.90 , 1.65 , 1.59 , 1.29 , 1.10 , 0.95 , 4.93 , 5.37 . 13C-NMR  δ: 39.6(C-1), 27.2(C-2), 89.2(C-3), 40.1(C-4), 56.7(C-5), 18.8(C-6), 35.7(C-7), 40.5(C-8), 48.6(C-9), 37.3(C-10), 33.1(C-11), 72.8(C-12), 52.9(C-13), 51.5(C-14), 32.9(C-15), 31.2(C-16), 51.2(C-17), 16.8(C-18), 16.1(C-19), 155.9(C-20), 108.5(C-21), 34.2(C-22), 27.4(C-23), 125.8(C-24), 131.5(C-25), 26.1(C-26), 18.1(C-27), 28.4(C-28), 17.0(C-29), 17.3(C-30), 105.5 (Glc-I-C-1′), 83.8 (C-2′), 78.7 (C-3′), 72.0 (C-4′), 78.3 (C-5′), 63.0 (C-6′), 106.4 (Glc-II-C-1′′), 77.6 (C-2′′), 78.7 (C-3′′), 72.0 (C-4′′), 78.5 (C-5′′), 63.2 (C-6′′). The structure of compound 4 was identified as ginsenoside Rk1 by comparing the NMR data and MS data with reported data.20White amorphous powder; HRESIMS 2.4.5m/z 789.4569 [M + Na]+. 1H-NMR  δ: 1.00 , 0.80 , 1.83 , 5.52 , 5.25 , 1.65 , 1.59 , 1.29 , 1.10 , 0.95 , 4.93 , 5.37 . 13C-NMR  δ: 39.6(C-1), 28.5(C-2), 89.3(C-3), 40.6(C-4), 56.7(C-5), 18.8(C-6), 35.7(C-7), 40.3(C-8), 51.1(C-9), 37.3(C-10), 33.1(C-11), 72.8(C-12), 50.9(C-13), 51.5(C-14), 32.9(C-15), 31.1(C-16), 51.2(C-17), 16.8(C-18), 17.0(C-19), 140.5(C-20), 13.5(C-21), 123.5(C-22), 27.7(C-23), 123.8(C-24), 131.5(C-25), 26.1(C-26), 18.1(C-27), 28.9(C-28), 16.1(C-29), 17.3(C-30), 105.5 (Glc-I-C-1′), 83.8 (C-2′), 78.7 (C-3′), 72.0 (C-4′), 78.3 (C-5′), 63.0 (C-6′), 106.4 (Glc-II-C-1′′), 77.6 (C-2′′), 78.7 (C-3′′), 72.0 (C-4′′), 78.5 (C-5′′), 63.2 (C-6′′). The structure of compound 5 was identified as ginsenoside Rg5 by comparing the NMR data and MS data with reported data.21White amorphous powder; HRESIMS 2.4.6m/z 1245.8682 [2 M + H]+. 1H-NMR  δ: 0.95 , 0.78 , 1.42 , 1.61 , 1.64 , 1.31 , 0.99 , 0.95 , 4.96 . 13C-NMR  δ: 39.1(C-1), 26.9(C-2), 88.8(C-3), 39.7(C-4), 56.4(C-5), 18.5(C-6), 35.2(C-7), 40.0(C-8), 50.3(C-9), 37.0(C-10), 32.1(C-11), 71.0(C-12), 48.5(C-13), 51.8(C-14), 31.3(C-15), 26.7(C-16), 54.8(C-17), 16.4(C-18), 15.8(C-19), 73.0(C-20), 27.1(C-21), 35.9(C-22), 23.0(C-23), 126.3(C-24), 130.8(C-25), 25.8(C-26), 17.7(C-27), 28.2(C-28), 16.8(C-29), 17.1(C-30), 107.0 (Glc-C-1′), 75.8 (C-2′), 78.8 (C-3′), 71.9 (C-4′), 78.4 (C-5′), 63.1 (C-6′). The structure of compound 6 was identified as 20(S)-ginsenoside Rh2 by comparing the NMR data and MS data with reported data.21White amorphous powder; HRESIMS 2.5−1. The mobile phase consisted of distilled water (solvent A) and acetonitrile (solvent B) using the following gradient program: 0–20 min, 20–20% B; 20–45 min, 20–46% B; 45–50 min, 46–55% B; 50–66 min, 55–55% B. The detection wavelength and the column temperature were at 203 nm and 35 °C, respectively. The calibration curves for 20(S)-Rg3 (1), 20(R)-Rg3 (2), SFt3 (3), Rk1 (4), Rg5 (5) and 20(S)-Rh2 (6) showed good linearity (R2 > 0.9990) in the concentration ranges  processed by microwave processing method was determined by high-performance liquid chromatography (HPLC) . The HPLn ranges . The sam2.62.6.1−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mmol L−1l-glutamine. All cultures were maintained with the aeration of 5% CO2 at 37 °C.Human cervical cancer (HeLa) and human lung cancer (A549) cell lines were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. HeLa cells and A549 cells were cultivated in RPMI Media 1640 (RPMI1640) containing 10% fetal bovine serum, 100 U mL2.6.222 The cells were seeded in five-well plates (1 × 104 cells per well) and incubated at 37 °C for 24 h. The cells were then treated with EL, MEL, cisplatin and compounds 1–6 at different concentrations (0.1–100 μmol L−1). Cisplatin was used as the positive control. After 48 h, 20 μL MTT (5 mg mL−1) was added to each well and the cells were incubated for 4 h. The supernatant was removed and then 150 μL of DMSO was added to each well. After the plates were shaken at room temperature for 10 min, the absorbance was measured by PHERA star FSX Microplate Reader  at a wavelength of 570 nm. The half maximal inhibitory concentration (IC50) against cancer cells was obtained by curve fitting of a sigmoidal dosage–response curve using nonlinear regression model.The cytotoxicity of EL, MEL and compounds 1–6 on two cancer cell lines was determined by MTT assay.2.72.7.1Kunming mice (18–22 g) were purchased by Changsha Tianqin Biotechnology Co., China . The mice were maintained at a temperature of 25 ± 2 °C, a humidity of 62 ± 2%, and a 12 h dark/light cycle, with unrestricted access to food and water. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Kunming University of Science and Technology, and were approved by the Animal Ethics Committee of Kunming University of Science and Technology.2.7.2−1 sodium citrate  by centrifugation at 3000 × g for 15 min.The plasma was obtained from mice whole blood mixed with 0.109 mol L2.7.3−1. The concentration of compounds 1–6 is 1 mg mL−1. Purified water was used as negative control. The PT assay reagent (100 μL) was added to the mixed samples, and the clotting time was recorded by the analyzer. All clotting tests were performed with three individual replicates.Prothrombin time (PT) was determined with a coagulation analyzer . 100 μL of a solution containing plasma (50 μL) and EL, MEL and compounds 1–6 (50 μL) were incubated at 37 °C for 3 min. The samples of EL and MEL were diluted with purified water to give the following concentrations: 25, 12.5, 5 mg mL2.8p < 0.05 were considered to be statistically significant, and p < 0.01 and p < 0.001 being very significant.Statistical analyses were performed with SPSS 19.0 software. All data were expressed as mean ± standard deviation (SD). The values of 33.1+, [M + Na]+, or [2 M + H]+. The chemical composition of the MEL was mainly 20(S)-protopanaxadiol (PPD)-type ginsenosides; the PPD skeleton of which displayed characteristic ions at m/z 425.23,24 Eight transformation products in the MEL were identified as ginsenosides, such as F2, 20(S)-Rg3, 20(R)-Rg3, SFt3, Rk1, Rg5, 20(S)-Rh2, and 20(R)-Rh2. The others had already existed in the PNL before microwave transformation, which were identified by matching molecular formulas with reported data.3,4 Six transformation products were purified from the MEL, and their structures were determined by comparing their spectroscopic data with those reported in the literature (S)-ginsenoside Rg3 (1),18 20(R)-ginsenoside Rg3 (2),18 notoginsenoside SFt3 (3),19 ginsenoside Rk1 (4),20 ginsenoside Rg5 (5),20 and 20(S)-ginsenoside Rh2 (6).21The MEL was identified by ultra-high performance liquid chromatography-quantitative time of flight mass spectroscopy (QTOF-MS) in positive mode. The total ion chromatogram of the MEL is shown in terature . The tra3.225 transformation,13,14 and extraction26 in research and industrial fields. The microwave processing technique has advantages of less time and energy as well as fewer solvents.16 In our study, this microwave processing method was applied to degrade the major saponins in the PNL and to produce the rare saponins with fewer sugar moieties. Microwave heating occurs within the molecule, and the glycosidic bonds in the polar molecules break easily.15 The major saponins in PNL were hydrolyzed at the glucosyl residue of C-20 to produce ginsenosides 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh2, Rk1, Rg5, and notoginsenoside SFt3 in the MEL with an increase in microwave power and temperature. The three factors of microwave power, temperature, and time were assessed for yields of the five transformation products by HPLC analysis after microwave processing -Rg3 > 20(S)-Rg3 > 20(S)-Rh2. When the microwave temperature range was from 60–180 °C for 20 min, the yields of the transformation products increased gradually (5 > SFt3 > 20(R)-Rg3 > 20(S)-Rg3 > 20(S)-Rh2. According to the yields of the transformation products, a suitable time for this microwave processing method was 15–30 min (5 > SFt3 > 20(R)-Rg3 > 20(S)-Rg3 > 20(S)-Rh2. It was reported that the contents of the primary degradation products 20(S)-Rh1, 20(R)-Rh1, Rk3, Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 for dried notoginseng steamed at 120 °C increase rapidly after 12 h.12 A similar yield was achieved in 20 min when the microwave processing method was used. Transformation products are obtained by microwave processing with less energy and less waste compared with the steaming method. According to the yields of the transformation products after microwave processing, the optimum conditions of microwave power, temperature, and time were 500 W, 150 °C, and 20 min, respectively.Microwave power was an important factor in the transformation process. When microwave temperature and time were set to 135 °C and 15 min, microwave power was varied from 300 to 1000 W to evaluate its impact on the yields of the transformation products . The higradually . When th5–30 min . The yie3.3P. notoginseng  using microwave processing are shown in P. notoginseng revealed similar typical ginsenosides as the PNL, consisting of notoginsenosides Fa and Fc, and ginsenosides Rc, Rb1, Rb2, and Rb3 (S)-Rg3, 20(R)-Rg3, SFt3, Rk1, Rg5, and 20(S)-Rh2 were generated from the MEL, flowers (MEF), seeds (MES), and pedicels (MEP) of P. notoginseng  the yields of Rg5 in the microwave transformed extracts from different parts of P. notoginseng were in the order of: MEL > MEP > MES > MEF. The 20(S)-Rh2 contents in the MEL and MES were 2.58% and 0.36% respectively, while 20(S)-Rh2 was not detected in the MEP or MEF. Three special saponins of notoginsenoside Fe, ginsenoside Rd2, and gypenoside IX were detected in the PNL (S)-Rh2. This result indicates that 20(S)-Rh2 content in the MEL was much higher than that in the MEP, MES, or MEF.The results of saponin transformation in different parts of  and Rb3 . After moginseng . As show the PNL , which e3.415 If the microwave energy matches with the molecular bond energy, the reactive activity of glycoside bond in the molecule increased and easy to break.15,27 In our paper, major saponins lost the glycosyl residues and produced the minor ginsenosides with increased microwave power and temperature. The proposed transformation pathway of saponins in PNL by the microwave processing were shown in e.g., ginsenosides Rb1, Rd, Rc, Rb2, and Rb3 easily lost the glycosyl residue at C-20 to produce 20(S)/(R)-Rg3, and then formed Rk1 and Rg5 through dehydration at C-20. Notoginsenosides Fa and Fc were hydrolyzed and dehydrated at C-20 to obtain notoginsenoside SFt3. When the microwave temperature was increased, a small amount of 20(S)/(R)-Rg3 was converted to 20(S)/(R)-Rh2 through loss of a glucosyl group at C-3. In addition, the three compounds of notoginsenoside Fe, ginsenoside Rd2 and gypenoside IX firstly removed the arabinose or xylose residues at C-3 to produce ginsenoside F2, and continued to lose one glucose residue at C-20 to obtain 20(S)-Rh2. This indicated that the content of 20(S)-Rh2 was much higher than that of 20(R)-Rh2 in the microwave processing. When the glycosyl residues at C-20 were lost, 20(S/R) epimers such as 20(S/R)-Rg3 and 20(S/R)-Rh2 were obtained by the selective attack of the OH group.28Microwave heating occurs within molecules, which is described as “internal heating”.S/R)-Rg3 and 20(S)-Rh2 had been characterized with NMR. The 13C NMR analysis suggested the obvious changes of the chemical shifts in 20(S/R) epimers at C-17, C-21 and C-22. The chemical shifts of 20(S)-Rg3 were δC 55.2(C-17), 27.5(C-21) and 36.3(C-22) ppm, while those of 20(R)-Rg3 were δC 50.6(C-17), 22.8(C-21) and 43.2(C-22) ppm. The values in 20(S)-Rh2 were also observed in the peaks of δC 54.8(C-17), 27.1(C-21) and 35.9(C-22) ppm. The results were in agreement with the reported data.18 After dehydration at Δ20(21) or Δ20(22), the values were shifted to δC 155.75 ± 0.15(C-20) and 108.35 ± 0.15(C-21) ppm in SFt3 and Rk1, and δC 140.5(C-20) and 123.5(C-22) ppm in Rg5, respectively.The structures of both 20 had no inhibitory effect on human cervical cancer (Hela) or human lung cancer (A549), and no IC50 values were obtained. After the EL microwave treatment, microwave processing of the MEL resulted in better inhibitory activity against Hela and A549 cells, with IC50 values of 42.97 and 50.47 μg mL−1, respectively. Microwave processing of ginseng enhances its anticancer activity due to the increased content of the ginsenosides Rg3, Rg5, and Rk1.13,14 Further research showed that the transformation products of ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5, and 20(S)-Rh2 and notoginsenoside SFt3 in the MEL actively inhibited the Hela and A549 cells. Among compounds 1–6, ginsenoside 20(S)-Rh2 (6) displayed lower IC50 values (8.23 and 12.45 μM) than cisplatin (8.43 and 16.85 μM) in Hela and A549 cells, respectively. The ginsenoside 20(S)-Rh2 effectively inhibits tumor cell growth and survival in both animal models and cell lines.29–31 20(S)-Rh2 directly binds to Annexin A2, which promotes apoptosis by inhibiting NF-kB activity and down-regulating anti-apoptosis gene expression.31 To confirm the antiproliferative effect of the transformation products, the morphologies in Hela and A549 cells treated with ginsenoside 20(S)-Rh2 was observed under a microscope (S)-Rh2 at the concentration of 1 μM had no significant effect on cell morphology. While increasing concentration to 10 or 25 μM, 20(S)-Rh2 induced cell body shrinkage and death in Hela and A549 cells.The antiproliferative activities of the PNL extract before and after microwave processing, and transformation products 1–6 were evaluated in two human cancer cell lines (HeLa and A549) by the MTT assay. The results are expressed as the ICcroscope . In the 3.6P. notoginseng have anticoagulant activity, and the leaves possess stronger anticoagulant activity among them.32 The components of the PNL changed dramatically after the microwave processing treatment (in vitro.33 As shown in p < 0.001). The anticoagulant activity of the MEL was better than that of the EL after comparing the extracts of the PNL before and after the microwave treatment. Further studies showed that the degradation products of ginsenosides Rk1 and Rg5, as well as notoginsenoside SFt3 prolonged coagulation time, while ginsenosides 20 (S/R)-Rg3 shortened coagulation time (S/R)-Rg3) had the hemostatic activities, while Rk1, SFt3 and Rg5 with double bond at Δ20(21) or Δ20(22) played the anticoagulant activities. The anticoagulant activity of the MEL increased due to increases in the concentrations of Rk1, Rg5, and SFt3. This result was consistent with previous findings that ginsenosides Rk1 and Rg5 inhibit arachidonic acid-induced platelet aggregation in a dose dependent manner.34According to previous reports, different parts of reatment , which mreatment . The PT ion time . The str4S)-Rg3, 20(R)-Rg3, Rk1, Rg5, 20(S)-Rh2, and notoginsenoside SFt3 were isolated from the MEL. Microwave power, temperature, and time affected the yields of the six transformation products. This transformation method was widely applied for producing the minor ginsenosides in stems and leaves, flowers, seeds and pedicels of P. notoginseng. The MEL had an obvious inhibitory effect on human cervical cancer and human lung cancer cell lines. The MEL had a stronger anticoagulant effect due to increased contents of ginsenosides Rk1, Rg5, and notoginsenoside SFt3. Therefore, the PNL may be a good source of anticancer and anticoagulant therapeutics after microwave processing.In summary, microwave processing was applied to produce rare saponins in the PNL. Six transformation products of ginsenosides 20(All authors declare no conflicts of interest.