An epoxide ring at C-14 and C-15 further increases the bioconversion rate; while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low Km value. A molecular docking simulation showed that filling of hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD glycosyltransferases. Furthermore, the glycosylation products showed a stronger inhibitory activity for Na+, K+-ATPase than the corresponding aglycones. This study provides the first stereoselective properties for OleD (ASP) catalyzed glycosylation.The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) are assessed. The results of this study demonstrated that the OleD-catalyze glycosylation of CTS is significantly influenced by the configuration at C-3 and the A/B fusion mode. 3β-OH and A/B ring  The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) was assessed. Streptomyces antibioticus catalyzes the glycosylation of oleandomycin using UDP-D-glucose (UDPG) as the glycosyl donor.1–4 Recent studies revealed an enhanced triple mutant ) that displayed marked improvement in proficiency and substrate promiscuity. The OleD (ASP) was found to show highly permissive properties and is capable of glycosylation on 100 diverse acceptors, e.g. erythromycin,5 flavones,6 indole alkaloids7 and steroids.8 Nowadays, 56% of the drugs in clinics are chiral compounds; however 88% of them are marketed as racemates consisting of an equimolar mixture of two enantiomers.8 Although OleD (ASP) was found to catalyze the glycosylation of cardenolide and bufadienolide aglycone with a bias toward the desired C-3 regiospecificity,7 the stereospecificity of this enzyme remains unreported.The glycosyltransferase OleD from 9,10 such as digitoxin, digoxin and proscillaridin are clinically important drugs for treatment of congestive heart failure for more than two centuries due to their potent inhibition of Na+, K+-ATPase (NKA), which is an integral membrane protein maintaining ionic gradients in all superior eukaryotic cells.11–15 Natural CTS includes cardenolides and bufadienolides. Cardenolides, such as ouabain and digoxin, possess a five-membered lactone ring at position C-17β of the steroidal skeleton. The natural cardenolide can be aglycone or glycosides with one or more sugar groups at C-3.16,17 Bufadienolides, isolated from many plants and animals such as Bufo bufo gargarizans, bears a six-membered lactone ring at C-17β. The bufadienolides can also be aglycone or glycosides in plants but only aglycone in animals.18–21 For both bufadienolides and cardenolides, there is a hydroxy group at C-3, either β- or α-configuration. Normally the CTS with a 3β-OH shows more remarkable activities than the 3α-diastereomer.22 Especially, the sugar groups of C-3 of CTS play a key role for the inhibitory activity of NKA.23Cardiotonic steroids (CTS)Km values of two pairs of epimers representing the bufadienolides and cardenolides were determined. Molecular docking was used to simulate the interactions between the substrate and enzyme. Finally, the inhibitory activities against NKA of CTS aglycone and the corresponding glycosides were compared.In this paper, we use OleD (ASP) to catalyze the glycosylation of four pairs of epimers of cardiotonic steroids. δ) were expressed in ppm with reference to the solvent signals. HR-ESI-MS were determined on a Micromass Q-TOF mass spectrometer . Analytical HPLC is run on Agilent 1200 system  with a Phenomenex Luna C-18 column . Preparative HPLC was performed on a Wu Feng HPLC system  equipped with a preparative reverse phase column .All chemicals and reagents were purchased from Sigma unless otherwise stated. The NMR spectra were recorded on a Bruker AV-400 spectrophotometer  with TMS as internal standard. Chemical shifts (Escherichia coli BL21 (DE3) pLysS  transformed with pET28a/OleD vector was inoculated in Luria Bertani (LB) medium (3 mL) supplemented with 50 μg mL−1 kanamycin. The medium was cultured overnight at 37 °C with shaking (200 rpm). The entire starter culture was then transferred to 1 L LB medium supplemented with 50 μg mL−1 kanamycin and grown at 37 °C with shaking (200 rpm) until the OD600 reached 0.6. Isopropyl β-d-thiogalactoside (IPTG) was subsequently added  and the culture was incubated at 18 °C for 18 h. Then the cell pellets were collected by centrifugation at 10 000g at 4 °C for 20 min and the supernatant was discarded.24 Pellets were resuspended in 10 mL PBS buffer  and then lysed by sonication. Cell debris was removed by centrifugation at 10 000g at 4 °C for 20 min and the clear supernatant was immediately submitted to His60 Ni super flow resin . The resin was balanced by the equilibration buffer . The enzyme was allowed to bind for 1 h at 4 °C with gentle agitation, and the resin was washed with 10 mL mild buffer . Finally, the protein was eluted by with 20 mL strong buffer . The enzyme aliquots were immediately frozen in liquid nitrogen and stored at −80 °C. Protein purity was confirmed by SDS-PAGE to be >95% and protein concentration for all studies was determined using the Bradford Protein Assay Kit from Bio-Rad .The OleD (ASP) glycosyltransferase gene was generated by Genscript Biotechnology  and was cloned into DH5α  and pET28a  expression vector. Single colony of 2Cl2 (35 mL) in a round bottom flask. Pyridinium chloride hydrochloride (PCC) (21.4 mM) was added and stirred constantly to dissolve. The solution was stirred for 2 h at room temperature. Then the solvent was removed under reduced pressure and the residue was dissolved in anhydrous tetrahydrofuran (5 mL) in a round bottom flask. Sodium borohydride (NaBH4) was added and stirred constantly to dissolve. The solution was stirred for 1 h at room temperature. Then 5 mL of water was added slowly to the reaction solution. Ethyl acetate (10 mL) was used to extract the mixture and the solvent was removed under reduced pressure. The final residue was purified by preparative HPLC eluting with acetonitrile to yield C-3α CTS .CTS  was dissolved in CH1H NMR  δ: 8.01 , 7.45 , 6.30 , 3.58 , 2.58 , 2.26–2.11 , 1.92–1.62 , 1.55–1.36 , 1.29–1.19 , 1.09–1.01 , 0.95 , 0.73 ; 13C NMR  δ: 164.8 (C-24), 150.5 (C-21), 149.37 (C-22), 125.0 (C-20), 115.4 (C-23), 86.1 (C-14), 72.3 (C-3), 52.3 (C-17), 49.8 (C-13), 43.2 (C-9), 43.1 (C-5), 41.9 (C-12), 37.7 (C-8), 37.0 (C-4), 36.3 (C-1), 35.9 (C-10), 33.2 (C-15), 31.3 (C-2), 29.9 (C-16), 28.4 (C-6), 23.8 (C-19), 22.8 (C-7), 22.5 (C-11), 17.3 (C-18); HR-ESI-MS: m/z 387.2576 [M + H]+ .1α: 1H NMR  δ: 7.91 , 7.47 , 6.28 , 3.62 , 3.57 , 2.61 , 2.44 , 2.04–1.63 , 1.63–1.28 , 0.99 , 0.79 ; 13C NMR  δ: 164.5 (C-24), 151.8 (C-21), 149.6 (C-22), 124.5 (C-20), 115.3 (C-23), 75.8 (C-14), 72.2 (C-3), 61.1 (C-15), 48.6 (C-17), 46.3 (C-13), 43.1 (C-5), 41.3 (C-9), 40.1 (C-12), 36.9 (C-4), 36.1 (C-1), 36.0 (C-10), 35.2 (C-8), 33.2 (C-16), 31.3 (C-2), 27.5 (C-6), 23.8 (C-19), 22.1 (C-11), 22.0 (C-7), 17.1 (C-18); HR-ESI-MS: m/z 385.2363 [M + H]+ .2α: 1H NMR  δ: 8.04 , 7.39 , 6.26 , 5.51 , 3.76 , 3.58 , 2.95 , 2.20–2.01 , 1.81 , 1.79–1.04 , 0.99 , 0.83 ; 13C NMR  δ: 171.6 (C̲OCH3), 164.0 (C-24), 153.5 (C-22), 150.9 (C-21), 118.4 (C-20), 114.1 (C-23), 76.6 (C-16), 73.4 (C-14), 72.2 (C-3), 60.8 (C-15), 51.4 (C-17), 46.3 (C-13), 43.0 (C-5), 41.1 (C-9), 40.7 (C-12), 36.9 (C-4), 36.1 (C-1), 36.0 (C-10), 34.7 (C-8), 31.2 (C-2), 27.4 (C-6), 23.7 (C-19), 21.9 (C-7), 21.9 (C-11), 20.4 (COC̲H3), 17.5 (C-18); HR-ESI-MS: m/z 443.2445 [M + H]+ .3α: In vitro glycosylation reactions were carried out in 500 μL reaction buffer  containing 250 μg of enzyme, 2.5 mM of UDPG, 1 mM aglycon and 5 mM MgCl2. The mixture was incubated at 37 °C for 16 h. The reaction mixture was subsequently frozen and lyophilized, and the residue was resuspended in 500 μL ice cold methanol and filtered. One portion of each clarified reaction mixture was analyzed by analytical reverse-phase HPLC equipped with a Phenomenex Luna-C18 column . The flow rate was 1 mL min−1. The gradient was consisted of solvent A (0.1% trifluoroacetic acid/H2O) and B (100% acetonitrile): (a) 0–20 min, 10–75% B; (b) 20–21 min, 75–100% B; (c) 21–26 min, 100% B; (d) 26–29 min, 100–10% B; and (e) 29–35 min, 10% B. Detections were set at 220 and 296 nm. Conversion rate was calculated by the corresponding HPLC peak area percentage using the Agilent Chromatography Workstation Software. The LC-ESI-MS analysis was accomplished using standard C-18 reversed-phase chromatography with diode array detection wherein 5% of the flow was diverted to time-of-flight (TOF) mass spectrometer.2). UDPG was added followed by OleD (ASP) catalyst. After 16 h incubation at 37 °C, the reaction was stopped with equal volume of ice cold methanol. Then the reaction mixture was centrifuged at 10 000g for 30 min and the supernatant was concentrated under reduced pressure, and the debris was resuspended in 5 mL of ice-cold methanol and filtered with 0.22 μM membrane. The filtrate was subjected to preparative HPLC  using water/acetonitrile as the eluent to afford the corresponding of glucosides. The compound was then characterized using high resolution MS, 1D and 2D NMR, including 1H, 13C and HSQC.Aglycons (10 mg) were dissolved in 5% of DMSO and transferred to pH 8 buffer solution  δ: 8.03 , 7.45 , 6.30 , 4.37 , 4.12 , 3.88 , 3.71 , 3.40–3.20 , 2.56–2.52 , 2.26–2.11 , 1.92–1.22 , 1.09–1.01 , 0.95 , 0.73 ; 13C NMR  δ: 164.8 (C-24), 150.5 (C-21), 149.37 (C-22), 125.0 (C-20), 115.4 (C-23), 102.7 (sugar C-1), 86.1 (C-14), 78.2 (sugar C-5), 77.84 (sugar C-2), 75.6 (sugar C-4), 75.2 (sugar C-3), 71.8 (C-3), 61.7 (sugar C-6), 52.3 (C-17), 49.8 (C-13), 43.2 (C-9), 43.1 (C-5), 41.9 (C-12), 37.7 (C-8), 37.0 (C-4), 36.3 (C-1), 35.9 (C-10), 33.2 (C-15), 31.3 (C-2), 29.9 (C-16), 28.4 (C-6), 23.8 (C-19), 22.8 (C-7), 22.5 (C-11), 17.3 (C-18); HR-ESI-MS: m/z 549.3035 [M + H]+ .1H NMR  δ: 7.91 , 7.46 , 6.27 , 4.32 , 4.08 , 3.86 , 3.67 , 3.62 , 3.39–3.17 , 2.61 , 2.44 , 2.04–1.43 , 1.40–1.05 , 0.99 , 0.79 ; 13C NMR  δ: 164.5 (C-24), 151.8 (C-21), 149.6 (C-22), 124.5 (C-20), 115.3 (C-23), 102.7 (sugar C-1), 78.2 (sugar C-5), 77.84 (sugar C-2), 75.8 (C-14), 75.4 (sugar C-4), 75.2 (sugar C-3), 71.7 (C-3), 62.8 (sugar C-6), 61.2 (C-15), 48.6 (C-17), 46.3 (C-13), 40.7 (C-12), 40.1 (C-9), 37.4 (C-10), 36.3 (C-5), 35.0 (C-4), 33.2 (C-8), 31.1 (C-1), 30.8 (C-16), 27.5 (C-2), 27.0 (C-6), 24.1 (C-19), 22.2 (C-11), 21.7 (C-7), 17.2 (C-18); HR-ESI-MS: m/z 547.2902 [M + H]+ .1H NMR  δ: 7.91 , 7.47 , 6.28 , 4.34 , 4.10 , 3.87 , 3.76 , 3.69 , 3.58 , 3.45–3.17 , 2.95 , 2.15–2.01 , 1.85 , 1.81–1.04 , 0.99 , 0.83 ; 13C NMR  δ: 171.6 (C̲OCH3), 164.0 (C-24), 153.5 (C-22), 150.9 (C-21), 118.4 (C-20), 114.1 (C-23), 102.7 (sugar C-1), 78.2 (sugar C-5), 77.84 (sugar C-2), 76.6 (C-16), 75.4 (sugar C-4), 75.2 (sugar C-3), 73.4 (C-14), 71.7 (C-3), 62.8 (sugar C-6), 60.8 (C-15), 51.4 (C-17), 46.3 (C-13), 40.5 (C-5), 40.7 (C-9), 37.3 (C-12), 36.3 (C-4), 36.1 (C-1), 36.0 (C-10), 31.7 (C-8), 31.2 (C-2), 27.4 (C-6), 23.7 (C-19), 21.9 (C-7), 21.9 (C-11), 20.4 (COC̲H3), 17.5 (C-18). HR-ESI-MS: m/z 605.2996 [M + H]+ .1H NMR  δ: 5.93 , 5.07 , 4.95 , 4.35 , 4.11 , 3.89 , 3.69 , 3.40–3.21 , 2.95–2.79 , 2.35–2.12 , 2.00–1.43 , 1.40–1.22 , 1.00 , 0.92 ; 13C NMR  δ: 178.5 (C-23), 177.3 (C-20), 117.8 (C-22), 102.7 (sugar C-1), 86.5 (C-14), 78.2 (sugar C-5), 77.8 (sugar C-2), 75.4 (sugar C-4), 75.3 (sugar C-3), 75.2 (C-31), 71.7 (C-3), 62.8 (sugar C-6), 52.2 (C-13), 51.1 (C-17), 42.7 (C-9), 41.0 (C-8), 37.5 (C-5), 36.9 (C-12), 36.3 (C-10), 33.4 (C-4), 31.2 (C-15), 30.9 (C-1), 28.1 (C-2), 27.8 (C-6), 27.5 (C-16), 24.1 (C-7), 22.6 (C-7), 22.4 (C-18), 16.4 (C-19); HR-ESI-MS: m/z 537.8049 [M + H]+ .g for 10 min. Supernatants were analyzed by analytical reverse-phase HPLC. Conversion rate is calculated by the corresponding HPLC peak area percentage using the Agilent Chromatography Workstation Software.25 All experiments were performed in triplicate. Initial velocities were fitted to the Michaelis–Menten equation using Origin Pro 7.0 software.Assays were performed in a final volume of 200 μL 50 mM Tris–HCl (pH 8.0), and contained constant concentrations of OleD (ASP) (40 μg) and UDPG (2.5 mM) while varying the concentration (0.01–1.2 mM) of 2β, 2α, 4β and 4α. Aliquots (100 μL) were removed every 15 min, mixed with an equal volume of ice cold methanol, and centrifuged at 10 0004M60) was retrieved from Protein Data Bank. To create receptor and ligand structures for docking, the following procedure was conducted. Firstly, the 3D structures of the ligands were prepared using the Gaussian 09 program at the B3LYP/6-31G(d) level. Harmonic vibration frequencies were calculated to confirm the stability of these conformers. Then the receptor and optimized structure of the ligands were converted to required pdbqt format using Autodock Tools 1.5.4. The Autodock Vina parameters were set as follow, box size: 15 × 15 × 15 Å, the center of box: x = 38.96, y = 61.05, z = 13.83, the exhaustiveness: 100, and number of output conformations was set to 20. The calculated geometries were ranked in terms of free energy of binding and the best poses were selected for further analysis. All molecular visualizations were carried out in PyMOL software.The program Autodock Vina was used for docking simulations. For docking purpose, the crystal structure of OleD , α-resibufogenin (2α) and α-cinobufagin (3α) were synthesized from β-bufalin (1β), β-resibufogenin (3β) and β-cinobufagin (3β), respectively, by an oxidation with pyridinium chloride hydrochloride followed by a reduction with sodium borohydride. Uzarigenin (4α) and digitoxigenin (4β), two natural cardenolides with a trans and cis A/B fusion mode  could catalyze the generation of monoglycoside of 1β with a conversion rate 30%; while glycosylation product of 1α was not detected (The pilot biotransformation of 1β and 1α were carried out using UDPG as the sugar donor and OleD (ASP) as the catalyst under the standard conditions .detected . Thus thO-β-d-glucoside (1β-glu), a 20 h reaction was carried out at 37 °C. Compound 1β  was dissolved in DMSO (0.625 mL) and diluted with buffer solution . UDPG  was added along with OleD (ASP) (13 mg). The reaction was stopped with 25 mL of ice cold methanol. Then the reaction mixture was centrifuged at 10 000g for 30 min and supernatant was concentrated under reduced pressure. The residue was dissolved in 3 mL methanol, and filtered with 0.22 μM membrane. The filtrate was subjected to preparative HPLC using water/acetonitrile as the eluent. Finally, 3.1 mg of 1β-glu was obtained with a conversion rate of 21%. The structure of 1β-glu was determined by 1D NMR and HR-ESI-MS analysis. The pseudo molecular ion m/z 549.3035 [M + H]+ in the HR-ESI-MS was corresponding to a formula C30H44O9 , which was 162 unit larger than the parent compound bufalin, suggesting the formation of monoglucoside. Compared with 1β, the chemical shift δC-3 of 1β-glu was shifted to downfield by 3.9 ppm and the δH-3 was shifted to downfield by 0.02 ppm; while C-14 was unchanged, confirming the glycosylation site was at 3-OH. The β-configuration was determined by the large coupling constant of the anomeric proton . Based on the above analysis, it was concluded that OleD (ASP) could catalyze the transformation of 1β to its monoglucoside 1β-glu at 3-OH.To maximize the production of bufalin-3-Bufo bufo gargarizans with an 14,15-epoxide ring in contrast to the 14-OH in bufalin. Compound 2β has been reported to exhibit a wide range of activities such as cardiotonic, renal sodium excretion, blood pressure stimulating, antitumor activity and immunoregulatory activity.32 However, the application of resibufogenin is restricted because of its strong toxicity.33 In the past several years, several structure modifications on 2β was performed which generate more than 30 derivatives.23 In order to obtain the glycosylation derivative of 2β and compare the NKA inhibitory activities of these derivatives with different configurations, OleD (ASP) catalysed biotransformation on 2β and 2α were carried out.Resibufogenin (2β) is another type of bufadienolides from the venom of The pilot reaction conditions are the same as those described for 1β and 1α. According to the result of LC-MS analysis, OleD (ASP) catalyzed the formation of monoglucoside (major) and diglucoside (minor) of 2β with a total conversion rate 80% which was much higher than that of 1β; while the conversion rate of monoglucoside 2α-glu is about 1% in contrast to the absence of 1α glucoside . Thus siO-β-d-glucoside (2β-glu), a preparative scale experiment was undertaken. Using the same condition as 1β, while 2β  was served as substrate, we obtain 8.4 mg 2β-glu with a conversion rate 65%. The identification of compounds 2β-glu was determined by 1D NMR and HR-ESI-MS analysis. The pseudo-molecular ion m/z 547.2902 [M + H]+ in the HR-ESI-MS corresponded to a formula C30H42O9  which was 162 unit larger than the parent compound 2β. Compared with 2β, the chemical shift C-3 of 2β-glu was shifted to downfield by 3.9 ppm and the chemical shift H-3 was shifted to downfield by 0.02 ppm, confirming the glycosylation site was also at 3-OH. The β-configuration was determined by the large coupling constant of the anomeric proton . Accordingly, it was concluded that OleD (ASP) could catalyze the transformation of 2β to its glucoside 2β-glu with a much higher rate than 1β. It is noteworthy that bioconversion of 2β also lead to the generation of a diglucoside (2β-diglu) which was confirm by the HR-MS m/z 709.3421 [M + H]+. Due to the small amount, it was not determined by NMR.To maximize the production of resibufogenin-3-Bufo bufo gargarizans with an 14,15-epoxide ring and an acetyl group at C-16.34 Cinobufagin was found to show potent cardiotonic, blood pressure-stimulating, antiviral, local anesthetic and antineoplastic activities.34,35 However, the poor water solubility restricted its clinical use. Though a series of analogues of cinobufagin has been generated by chemical synthesis and cell suspension cultures,33,36 the glycosylation method is rarely reported. Furthermore, difference between the glycosidation profile on the 3β and 3α isomers of cinobufagin is unclear.Cinobufagin is also a major bufadienolides (4–6% dry weight) from the venom of m/z 767.3479 [M + H]+. Due to the small amount, it was not determined by NMR.Using the same reaction conditions as described for 2β and 2α, OleD (ASP) was found to catalyze the glycosylation of 3β leading to both monoglucoside (3β-glu) and diglucoside (3β-diglu) of 3β with a total conversion rate of 75%; while at the same condition as 3β, only the monoglucoside of (3α-glu) was detected with a low conversion rate 2% . Similar1D and 2D NMR and HR-ESI-MS analysis. HR-ESI-MS showed a pseudo molecular ion m/z 605.2996 [M + H]+ corresponding to a formula C32H44O11  which was 162 unit larger than the parent compound 3β. Similar to 2β-glu, the chemical shifts of C-3 and H-3 of 3β-glu were shifted to downfield by 3.9 ppm and 0.02 ppm, respectively, confirming the glycosylation site at 3-OH. The β-configuration was also confirmed by the large coupling constant of the anomeric proton . Accordingly, it was concluded that OleD (ASP) could catalyze the transformation of 3β into 3β-glu.Using the same conditions as 2β, we prepared 6.9 mg 3β-glu with a conversion rate 50% (10 mg 3β was served as substrate). The identification of the product 3β-glu was also confirmed by cis and trans, respectively. Compound 4β was the aglycone of digoxin, a commonly used cardiotonic drug.37 Recently, it is reported that introduction of a sugar unit at C-3 of cardenolides could improves NKA isoform selectivity (α2/α3 over α1).38,39 In order to compare the glycosylation profiles of 4β and 4α with different A/B ring fusion mode, the OleD (ASP) catalyzed bioconversion was carried out.As compared to the three pairs of bufadienolides 1β and 1α, 2β and 2α, 3β and 3α, digitoxigenin (4β) and uzarigenin (4α) were two cardenolides with the same substitution pattern. The only difference between 4β and 4α is the A/B ring fusion modes, which are Using the same reaction conditions as described for the bufadienolides, OleD (ASP) catalyzed the generation of monoglucoside of 4β-glu with a conversion rate of 26%; while the conversion rate for 4α is only 2% . Thus th1D and 2D NMR and HR-ESI-MS analysis . The glycosylation site was again determined at 3-OH by comparison of chemical shifts of C-3 (downfield 3.9 ppm) and H-3 (downfield 0.02 ppm) of 4β-glu with those of the parent compound 4β and the β-configuration of the glycosidic bond was confirmed by the large coupling constant of the anomeric proton . Accordingly, it was concluded that OleD (ASP) could catalyze the transformation of 4β into 4β-glu.We prepared the monoglucoside of 4β (4β-glu) for structural analysis. Compound 4β-glu (3.6 mg) was obtained by preparative HPLC with conversion rate 25% (10 mg 4β of was used as the substrate). Similar to the bufadienolide glycosides 1β-glu, 2β-glu and 3β-glu, the monoglucoside structure of 4β-glu was confirmed by Km value was measured.As we can see that the substrate structure and stereochemistry of cardiotonic steroid epimers significantly affect the final glycosylation rate. In order to compare the dynamic process, Km value was determined based on the Michaelis–Menten equation.25 The result indicated that a high conversion rate corresponded to a low Km value. Particularly, when the conversion rate is less than 2%, Km value is toward a large value more than 100 mM (Epimers 2β and 2α representing the high conversion bufadienolides and 4β and 4α representing the cardenolides were chosen in this study. A series of concentrations of the substrates (0.01–1.2 mM) were incubated with the OleD (ASP) enzyme and UDPG, and the conversion rate was calculated by the corresponding HPLC peak area using the Agilent Chromatography Workstation Software. n 100 mM .Km study, we selected the high conversion enantiomers 2α and 2β to explore the binding mode in the enzyme substrate complex. As can be seen from H-pyran-2-one ring of compound 2α penetrated deeply into the hydrophobic region I defined by His20, Phe85 and Trp74 residues. The epoxide contacted via van der Waals interactions with residues lle112 and Val82. In comparison to 2α binding to OleD (ASP), compound 2β showed an ‘inverse’ binding pose that C-3 aliphatic hydroxyl was placed in the hydrophobic pocket I and the 2H-pyran-2-one moiety bound in a hydrophobic cavity II  glycosyltransferase, since the conversion efficiency is 1% for 2α and 80% for 2β, respectively.Molecular docking studies shed new light on the mechanism of stereoselective glycosylation of cardiotonic steroids derivatives. Similar for the 11,27 As seen in To explore the inhibitory activity of glycosylated products on NKA, the inhibition activity of CTS  and their glycosylation products  were determined using previous reported method.cis fusion are favoured by OleD (ASP), while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low Km value. Molecular docking simulation showed that filling the hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD (ASP) glycosyltransferase. Furthermore, the glycosylation products showed a stronger inhibitory activity for NKA than the corresponding aglycones. This study provided the first stereoselective properties for OleD (ASP) catalyzed glycosylation. It is noteworthy that glycosyltransferase has been used for the glycosylation of a wide variety of natural products;40,41 however, glycosyltransferase for the cardiotonic steroids is rare. Results of this study firstly revealed the stereo-selectivity of OleD-catalyzed glycosylation of cardiotonic steroids.In summary, this study demonstrated that the OleD-catalyze glycosylation of cardiotonic steroids are significantly influenced by the configuration at C-3 and the A/B fusion mode. 3β-OH and A/B ring There are no conflicts to declare.RA-008-C7RA11979H-s001


Penicillium chrysogenum (renamed by some as P. rubens). Through a collaboration with Aotearoa New Zealand Crown Research Institute Manaaki Whenua–Landcare Research we have access to a collection of thousands of fungal cultures known as the International Collection of Microorganisms from Plants (ICMP). The ICMP contains both known and novel species which have not been extensively tested for their antimicrobial activity. Initial screening of ICMP isolates for activity against Escherichia coli and Staphylococcus aureus directed our interest towards ICMP 477, an isolate of the soil-inhabiting fungus, Aspergillus terreus. In our investigation of the secondary metabolites of A. terreus, through extraction, fractionation, and purification, we isolated nine known natural products. We evaluated the biological activity of selected compounds against various bacteria and fungi and discovered that terrein (1) has potent activity against the important human pathogen Cryptococcus neoformans.Antimicrobial compounds, including antibiotics, have been a cornerstone of modern medicine being able to both treat infections and prevent infections in at-risk people, including those who are immune-compromised and those undergoing routine surgical procedures. Their intense use, including in people, animals, and plants, has led to an increase in the incidence of resistant bacteria and fungi, resulting in a desperate need for novel antimicrobial compounds with new mechanisms of action. Many antimicrobial compounds in current use originate from microbial sources, such as penicillin from the fungus  Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Together these bacteria were responsible for 3.57 million of the deaths associated with resistance, and 929,000 of the deaths directly attributable to resistance . Despite previous assumptions that fungi have largely been a tapped resource, the search for antimicrobial compounds from fungi has not been thorough. This is evidenced by the wealth of novel compounds discovered in recent years , a collection of cultures of fungi and bacteria primarily sourced from Aotearoa New Zealand and the South Pacific . The colaetaceae . More reAspergillus terreus, has activity against Mycobacterium abscessus and M. marinum which is retained after extraction (1) has potent activity against the opportunistic human pathogen Cryptococcus neoformans.We have previously reported that ICMP 477, an isolate of the soil-inhabiting fungus traction . Here we1H and 13C NMR spectra were recorded at 298 K on a Bruker AVANCE 400 spectrometer at 400 and 100 MHz, respectively using standard pulse sequences. Proto–deutero solvent signals were used as internal references , δC 77.16). For 1H NMR, the data are quoted as position (δ), relative integral, multiplicity , coupling constant , and assignment to the atom. The 13C NMR data are quoted as position (δ), and assignment to the atom. Flash column chromatography was carried out using Kieselgel 60  silica gel or Diol  bonded silica x (40–63 μm), C8  reversed–phase (40–63 μm) solid support. Gel filtration flash chromatography was carried out on Sephadex LH–20 . Thin layer chromatography was conducted on DC–plastikfolien Kieselgel 60 F254 plates . All solvents used were of analytical grade or better and/or purified according to standard procedures.Mass spectra were acquired on a Bruker micrOTOF Q II spectrometer. Melting points were recorded on an electrothermal melting point apparatus and are uncorrected. A. terreus is supported by GenBank sequence MW862777. A. terreus is a common cosmopolitan saprotrophic soil-inhabiting fungus (A. terreus were grown at room temperature on Potato Dextrose Agar (PDA)  plates.As previously described, culture ICMP 477 was isolated in September 1961 in Auckland, Aotearoa New Zealand, from sheep’s wool incubated at 30 °C . The ideg fungus , althougg fungus . CultureE. coli (ATCC 25922)  or resistant clinical isolates ) or antibiotic-resistant K. pneumoniae  . Similarly, Mueller-Hinton agar plates were inoculated with a lawn of either antibiotic-sensitive S. aureus (ATCC 29213) or antibiotic-resistant S. aureus (ATCC 33593) . ICMP 477 was grown on PDA plates for 36 days and fungal plugs removed using a 6 mm punch biopsy tool . Fungal plugs were placed onto the bacterial lawns, alongside PDA plugs containing no fungus as a control. Plates were incubated inverted at 37 °C for 24 h before measuring any zones of inhibition (in mm) produced. Experiments were performed using three biological replicates with three technical replicates each.Zone of inhibition testing was carried out as previously described . Specifi2Cl2 (2 × 500 mL) overnight. Combined organic extracts were concentrated under reduced pressure to afford an orange oil (2.45 g). The crude product was subjected to C8 reversed-phase column chromatography eluting with a gradient of H2O/MeOH to afford five fractions (F1–F5). Fraction F3 was subjected to purification by Sephadex LH-20, eluting with MeOH/5% CH2Cl2, to afford terrein (1) (3.05 mg), aspulvinone E (2) (4.22 mg) and aspulvinone G (3) (4.43 mg). Further separation of F4 by Sephadex LH-20, eluting with MeOH/5% CH2Cl2, afforded six fractions (A1–A6). Purification of fraction A2 using Diol-bonded silica gel column chromatography, eluting with gradient n-hexane/EtOAc, afforded terretonin (4) (19.60 mg) and terretonin A (5) (4.91 mg) while purification of A4 using the same method afforded asperteretal B (6) (2.39 mg), flavipesolide C (7) (1.44 mg) and butyrolactone II (8) (1.98 mg). Fraction F5 was subjected to purification by Sephadex LH-20, eluting with MeOH/5% CH2Cl2, to afford butyrolactone I (9) (36.12 mg).Compounds were isolated as previously described in 1H NMR  δ 6.86 , 6.46 , 6.03 , 4.71 , 4.11 , 1.96 ; 13C NMR  δ 205.7 (C-1), 170.9 (C-3), 141.9 (C-7), 126.4 (C-6), 124.9 (C-2), 82.4 (C-5), 78.1 (C-4), 19.5 (C-8); (+)-HRESIMS m/z 155.0705 [M+H]+ .1H NMR  δ 7.95 , 7.66 , 6.80 , 6.77 , 6.22 ; 13C NMR  δ 179.3 (C-4), 171.0 (C-2), 158.3 (C-10), 155.0 (C-4′), 147.8 (C-5), 132.6 (C-8), 128.2 (C-2′), 127.5 (C-7), 127.1 (C-1′), 116.4 (C-9), 115.5 (C-3′), 103.8 (C-6), 94.2 (C-3); (+)-HRESIMS m/z 319.0578 [M+Na]+ .m.p. 281–283 °C; 1H NMR  δ 7.76 , 7.65 , 6.81 , 6.33 , 6.29 , 6.18 ; 13C NMR  δ 176.5 (C-4), 170.8 (C-2), 158.7 , 157.7 (C-4′), 146.0 (C-5), 132.7 (C-8), 129.2 (C-6′), 127.1 (C-7), 116.5 (C-9), 113.9 (C-1′), 107.7 (C-5′), 105.4 (C-3′), 104.7 (C-6), 96.3 (C-3); (+)-HRESIMS m/z 335.0536 [M+Na]+ .m.p. 264–265 °C; D21 = −76.2  [lit [α]D25 = −73.7   δ 6.15 , 5.48 , 5.09 , 3.80 , 3.54 , 2.98 , 2.75–2.68 , 2.56–2.48 , 2.41–2.33 , 2.26 , 1.93 , 1.82–1.74 , 1.72 , 1.48 , 1.45 , 1.22 ; 13C NMR  δ 214.1 (C-8), 201.6 (C-1), 197.1 (C-5), 168.7 (C-20), 167.9 (C-4), 140.0 (C-12), 138.8 (C-6), 131.7 (C-6a), 117.3 (C-17), 85.7 (C-2), 77.7 (C-10b), 53.9 (OMe), 52.5 (C-4b), 49.6 (C-12a), 48.0 (C-7), 44.7 (C-4a), 43.3 (C-10a), 35.0 (C-11), 32.8 (C-9), 28.3 (C-10), 23.7 (C-18), 23.6 (C-14), 21.4 , 20.0 (C-13), 18.7 (C-16); (+)-HRESIMS m/z 511.1949 [M+Na]+ .m.p. 288–290 °C [lit m.p. 289–291 °C ]; [α]D219, MeOH) ]; 1H NMRD21 = −60  [lit [α]D20 = −115.7   δ 6.18 , 5.18 , 4.98 , 3.83 , 2.84 , 2.69 , 2.58–2.48 , 2.33 , 2.19 , 1.83 , 1.74 , 1.70 , 1.56–1.52 , 1.47 , 1.46 , 1.44 , 1.11 ; 13C NMR  δ 214.0 (C-8), 201.4 (C-1), 198.1 (C-5), 168.6 (C-20), 167.1 (C-4), 143.2 (C-12), 139.2 (C-6), 137.4 (C-6a), 112.3 (C-17), 86.0 (C-2), 53.9 (OMe), 53.1 (C-10b), 50.7 (C-12a), 49.3 (C-4a), 48.1 (C-7), 46.0 (C-4b), 38.4 (C-10a), 34.5 (C-10), 32.8 (C-9), 29.3 (C-11), 23.9 (C-18), 23.7 (C-14), 22.2 (C-19), 21.1 (C-15), 17.0 (C-16), 16.7 (C-13); (+)-HRESIMS m/z 495.1993 [M+Na]+ .m.p. 231–233 °C [lit m.p. 232–233 °C ]; [α]D21, CHCl3) ]; 1H NMRD21 = −10.9  [lit [α]D20 = −18.6   δ 7.52 , 6.90 , 6.80 , 6.69 , 6.68 , 5.31–5.27 , 3.25 , 1.72 , 1.68 ; 13C NMR  δ 174.0 (C-2), 166.0 (COOH), 160.2 (C-4′), 156.8 (C-4), 154.0 (C-10), 132.4 (C-15), 131.4 (C-2′), 129.8 (C-12), 128.8 (C-11), 128.0 (C-3), 126.8 (C-8), 126.3 (C-7), 123.6 (C-14), 121.9 (C-1′), 115.9 (C-3′), 115.5 (C-9), 102.8 (C-5), 29.8 (C-6), 28.9 (C-13), 25.7 (C-16), 17.5 (C-17); (–)-HRESIMS m/z 409.1287 [M-H]– .[α]2, MeOH) ]; 1H NMR1H NMR  δ 7.53 , 6.97–6.92 , 6.92–6.89 , 6.81 , 6.64 , 3.82–3.77 , 2.74 , 1.80 , 1.26 ; 13C NMR  δ 174.1 (C-2), 166.0 (COOH), 160.7 (C-4′), 156.9 (C-4), 153.6 (C-10), 131.5 (C-2′), 129.2 (C-12), 128.2 (C-3), 127.8 (C-8), 126.3 (C-7), 122.0 (C-11), 121.9 (C-1′), 118.1 (C-9), 116.4 (C-3′), 103.0 (C-5), 75.4 (C-15), 33.8 (C-14), 30.1 (C-6), 27.0 , 23.5 (C-13); (–)-HRESIMS m/z 409.1284 [M-H]– .D23 = +6.2  [lit [α]D29 = +4.8   δ 7.61 , 6.89 , 6.67 , 6.54 , 3.81 , 3.49 ; 13C NMR  δ 170.4 (COOMe), 169.8 (C-2), 157.7 (C-4′), 156.3 (C-10), 137.7 (C-3), 132.4 (C-8), 130.5 (C-2′), 129.2 (C-4), 123.7 (C-7), 120.9 (C-1′), 116.5 (C-3′), 115.5 (C-9), 85.2 (C-5), 53.4 (OMe), 39.3 (C-6); (–)-HRESIMS m/z 355.0819 [M-H]– .[α]acetone) ]; 1H NMRD21 = +46.7  [lit [α]D23 = +68.3   δ 7.61 , 6.89 , 6.56 , 6.52 , 6.44 , 5.12–5.07 , 3.81 , 3.46 , 3.10 , 1.69 , 1.60 ; 13C NMR  δ 171.7 (COOMe), 170.4 (C-2), 159.4 (C-4′), 155.1 (C-10), 139.8 (C-3), 132.4 (C-12), 130.4 (C-2′), 129.8 (C-8), 129.2 (C-11), 128.5 (C-4), 125.1 (C-7), 123.6 (C-14), 123.2 (C-1′), 116.6 (C-3′), 115.1 (C-9), 86.8 (C-5), 53.9 (OMe), 39.7 (C-6), 28.7 (C-13), 26.0 (C-16), 17.8 (C-17); (+)-HRESIMS m/z 447.1406 [M+Na]+ .[α]3, MeOH) ]; 1H NMRS. aureus ) and E. coli (25922 lux (6 colony forming units (CFU)/mL) were then added to all the wells. This gave a maximum concentration of 1 mg/mL and a minimum concentration of 16 µg/mL. The maximum volume/volume concentration of DMSO in all extracts was 4%; therefore, the negative control was tested at an identical concentration.Data were collected as described previously . Specifi5922 lux ), at an Luminescence was measured using a Victor X-3 luminescence plate reader  with an integration time of 1s at 0, 2, 4 and 24 h to determine the minimum inhibitory concentration (MIC), between which times the plates were incubated at 37 °C with shaking at 100 rpm. MIC was defined as causing a 1 log reduction in bacterial light production. After 24 h, 10 µL of liquid from all wells showing inhibition of bacterial growth was pipetted onto a plate of MH agar. Once all liquid had evaporated, the plates were then incubated inverted at 37 °C for 16–20 h, and the minimum bactericidal concentration (MBC) was measured .Acinetobacter baumannii ATCC 19606, Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 208821, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, and S. aureus ATCC 43300 (MRSA) was undertaken at the Community for Open Antimicrobial Drug Discovery at The University of Queensland  according to their standard protocols , nutrient broth (NB)  or MHB at 37 °C overnight   broth to a final concentration of 2.5 × 103 CFU/mL. The compounds were serially diluted 2-fold across the wells of 96-well plates , with compound concentrations ranging from 0.015 to 64 μg/mL and final volumes of 50 μL, plated in duplicate. Then, 50 μL of a previously prepared fungi suspension, in YNB broth to the final concentration of 2.5 × 103 CFU/mL, was added to each well of the compound-containing plates, giving a final compound concentration range of 0.008–32 μg/mL. Plates were covered and incubated at 35 °C for 36 h without shaking. Candida albicans MICs were determined by measuring the absorbance at an optical density (OD) of 530 nm (OD530). For Cryptococcus neoformans, resazurin was added at 0.006% final concentration to each well and incubated for a further 3 h before MICs were determined by measuring the absorbance at OD570–600. Experiments were carried out in duplicate.Fungal strains were cultured for 3 days on yeast potato dextrose (YPD) agar at 30 °C. A yeast suspension of 1 × 10Candida albicans and Cryptococcus neoformans. The antibiotics were provided in four concentrations, with two above and two below their MIC value, and plated into the first eight wells of column 23 of the 384-well NBS plates. The quality control (QC) of the assays was determined by the antimicrobial controls and the Z’-factor (using positive and negative controls). Each plate was deemed to fulfil the quality criteria (pass QC), if the Z’-factor was above 0.4, and the antimicrobial standards showed full range of activity, with full growth inhibition at their highest concentration, and no growth inhibition at their lowest concentration  (M. abscessus was grown at 37 °C and M. marinum at 28 °C. Cultures were grown until they reached the stationary phase (approximately 3–5 days for M. abscessus BSG301 and 7–10 days for M. marinum BSG101) and then diluted in MHB supplemented with 10% Middlebrook ADC enrichment media and 0.05% tyloxapol to give an OD600 of 0.001, which is the equivalent of ~106 bacteria per mL. Pure compounds were dissolved in DMSO and added to the wells of a black 96-well plate  at doubling dilutions with a maximum concentration of 128 μg/mL. Then, 50 μL of diluted bacterial culture was added to each well of the compound containing plates giving final compound concentrations of 0–64 μg/mL and a cell density of ~5 × 105 CFU/mL. Rifampicin  was used as positive control at 1,000 μg/mL for M. abscessus and 10 μg/mL for M. marinum. Between measurements, plates were covered, placed in a plastic box lined with damp paper towels, and incubated with shaking at 100 rpm at 37 °C for M. abscessus and 28 °C for M. marinum. Bacterial luminescence was measured at regular intervals over 72 h using a Victor X-3 luminescence plate reader with an integration time of 1 s. We defined the MIC as causing a 1-log reduction in light production, as previously described  ,2021b anescribed . ExperimE. coli zone of inhibition data was performed using GraphPad Prism version 9.4.1. Data was analysed using a one-way ANOVA using the Kruskal-Wallis test for multiple comparisons. Statistical significance was set at p ≤ 0.05 (adjusted for multiple comparisons).Statistical analysis of A. terreus ICMP 477 was tested for production of zones of inhibition against a panel of antibiotic-sensitive and resistant isolates of E. coli, K. pneumoniae, and S. aureus  and antibiotic-resistant isolate . Interestingly, only two of the three biological replicates of ICMP 477 produced zones of inhibition against E. coli. Of these active cultures, the zones of inhibition produced for the antibiotic-sensitive isolate  were significantly smaller than for the resistant clinical isolates CTX-M-15  and NDM-1 .. aureus . None ofA. terreus ICMP 477 were extracted with a combination of methanol and dichloromethane to afford a crude extract. The crude extract was subjected to C8 reversed-phase column chromatography (H2O/MeOH) which afforded five fractions. The crude extract and five fractions were tested for antimicrobial activity against E. coli and S. aureus. Neither the crude extract or the five fractions showed any significant activity against E. coli  inoculated with  E. coli  while frectively .2Cl2) and Diol-bonded silica gel (hexane/EtOAc) chromatography to afford compounds 1–9  (2) (3) (4) (5) (6) (7) (8) (9)  , aspulvi (1) (2) , aspulvi (2) (3) , terreto (3) (4) , terreto (4) (5) , asperte (5) (6) , flavipe (6) (7) , butyrol (7) (8)  and buty (8) (9) .A. baumannii, Candida albicans, Cryptococcus neoformans, E. coli, K. pneumoniae, P. aeruginosa and methicillin-resistant S. aureus (MRSA) , exhibited potent antifungal activity against C. neoformans. None of the tested compounds exhibited significant activity against any of the bacterial strains.We prioritized testing samples for antimicrobial activity against a panel of important human pathogens: s (MRSA) . These e2–8 were evaluated for their anti-mycobacterial activity against bioluminescent derivatives of M. abscessus and M. marinum at a maximum concentration of 64 µg/mL (1) was not tested as we did not have enough sample. None of the compounds reached the threshold of a 1 log reduction in light output and MBCs were above the tested maximum of 1 mg/mL.Compounds 64 µg/mL . TerreinA. terreus including terrein (1), aspulvinone E (2), flavipesolide C (7), butyrolactone II (8), and butyrolactone I (9) have been reported to exhibit a variety of bioactivities including anti-proliferative, anti-inflammatory, and antioxidant activities (1), terretonin (4), terretonin A (5), butyrolactone II (8) and butyrolactone I (9) have also been previously investigated; 4  against Enterococcus faecalis, K. pneumoniae, S. aureus, MRSA, and methicillin-resistant S. epidermidis along with 9 in broth dilution assays (2) and aspulvinone G (3) display no activity against a range of bacterial and fungal species.Secondary metabolites from tivities . The antgated; 4  and 5  against B. subtillis (MIC 50 µg/mL), E. coli (MIC 50 µg/mL), and S. aureus (MIC 50 µg/mL) (Aeromonas hydrophila (IC50 > 20 µg/mL) and Enterococcus faecalis (IC50 20 µg/mL) (Alternaria brassicae (MIC 8 µg/mL), Edwardsiella tarda (MIC 16 µg/mL), E. coli (MIC 4 µg/mL), Physalospora piricola (MIC 16 µg/mL), and S. aureus (MIC 32 µg/mL)  and P. aeruginosa (MIC >100 µM)  , as well0 µg/mL) , 2020. A2 µg/mL)  for 1. T>100 µM)  and was l growth .8 and 9 has also been investigated. While 9 was found to exhibit potent activity against the protein tyrosine phosphatase B of Mycobacterium tuberculosis (IC50 5.11 ± 0.53 µM) (M. tuberculosis (MIC > 50 µg/mL)  , both na0 µg/mL) . Our datC. neoformans activity for terrein (1), this compound has previously been shown to exhibit activity against a range of fungal plant pathogens, including Alternaria solani (IC50 < 0.1 µg/mL) the causative agent of early blight in tomato and potato plants, Fusarium graminearum (IC50 5.7 µg/mL) the causative agent of head blight in wheat and barley, F. oxysporum f. sp. momordicae (IC50 1.7 µg/mL) a causative agent of fusarium wilt in bitter gourd, and F. oxysporum f. sp. cucumerinum (IC50 0.8 µg/mL) a causative agent of vascular wilt in cucumber plants (Of most relevance to our finding of anti-r plants .1) against the opportunistic human pathogen Cryptococcus neoformans. C. neoformans and the related C. gattii are the major species responsible for life-threatening cryptococcal meningitis, of which there are an estimated 223,100 cases globally each year, leading to 181,100 deaths , aspulvinone E (2), aspulvinone G (3), terretonin (4), terretonin A (5), asperteretal B (6), flavipesolide C (7), butyrolactone II (8) and butyrolactone I (9) upon structure elucidation. One of the limitations of our study is that we had limited amounts of the pure compounds and so were not able to test all compounds against all our target bacterial and fungal species. However, evaluation of the biological activities of a subset of these natural products against a wide range of bacterial and fungal human pathogens showed no activity for any of the tested compound except for terrein (1) which exhibited potent antifungal activity against C. neoformans. While no new natural products were discovered during this project, several known antimicrobial compounds were identified. Transposition of our current methods into a higher throughput screening method will enable the rapid study of far more promising fungal isolates, increasing our chances of identifying novel antimicrobial compounds.Investigation of


Their formation constants, determined by spectrophotometry, are very similar, lgK = 11.6–12.6, suggesting that Fe3+ binds to the common hydroxy-pyranone moiety and is hardly affected by the other aromatic part of the ligand. Accordingly, the characteristic bands in their individual absorption spectra are uniformly red-shifted relative to those of the free ligands. The crystal structures of two new resveratrol–maltol hybrids were recorded, giving additional information on the molecules’ intermolecular hydrogen bonds and packing. In this way, several functionalities of these new resveratrol hybrids were examined as a necessary approach to finding more effective drugs for complicated neurodegenerative diseases.New resveratrol–thiophene and resveratrol–maltol hybrids were synthesized as cholinesterase inhibitors and antioxidants. As with photostability experiments, biological tests also found remarkable differences in the properties and behavior of thiophene and maltol hybrids. While resveratrol–thiophene hybrids have excellent inhibitory and antioxidant properties , maltols have been proven to be weaker inhibitors and antioxidants. The molecular docking of selected active ligands gave insight into the structures of docked enzymes. It enabled the identification of interactions between the ligand and the active site of both cholinesterases. The maltols that proved to be active cholinesterase inhibitors were able to coordinate Fe I), trans-stilbene derivatives , which produces a 3D structure  and C–H⋯π interactions (C5–H5⋯O3→C13), which generate zig-zag chains running in the direction [–1–10] , dichloromethane (DCM), diethylether (E), ethanol (EtOH), ethyl acetate (EtOAc), methanol (MeOH), and toluene were used. All enumerated solvents were purified by distillation and were commercially available. Iron(III) sulfate hexahydrate (Fe2(SO4)3·6H2O), sodium tetraborate decahydrate (Na2B4O7·10H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O), and sodium acetate trihydrate (CH3COONa·3H2O) were purchased from Reanal , and sodium hydroxide > 98% (NaOH) and 98% sulphuric acid from Acros Organics . The water applied was cleaned by Millipure Elix equipment  and completed with a Milli-Q 50 purification system . The column chromatography was performed using silica gel . Thin-layer chromatography was performed using plates coated with silica gel . Nuclear magnetic resonance (NMR) spectroscopic data for 1H and 13C nuclei were recorded at room temperature on a spectrometer at Bruker Avance 300 MHz and 600 MHz. Deuterated chloroform, CDCl3, deuterated methanol, CD3OD, and deuterated dimethyl sulfoxide, DMSO with tetramethylsilane as standard were used for recording NMR spectra. Chemical shifts were reported in parts per million. The 1H NMR spectra for four compounds were also recorded at 5, 15, 25, 35, 45, and 55 °C on a Bruker Advance 600 MHz spectrometer. UV spectra were recorded by UV/Vis spectrophotometer  in ACN. Photochemical irradiations were carried out with 3.0 mL solutions in 1 mL cells at room temperature in photochemical reactor Luzchem equipped with 8 UV lamps at 313 nm. Spectral changes during the irradiation of pyranones were recorded in HPLC grade ACN. Before irradiation, the reaction mixtures were purged with nitrogen, N2, for 15 min. IR spectra were acquired using the Bruker Vertex 70 Fourier transform infrared spectrometer set on attenuated total reflectance (ATR) mode. The samples were pressed on a diamond, and the absorbance data were collected between 400 and 5000 cm−1 with a spectral resolution of 1 cm−1 and an average of 32 scans. HRMS analyses were carried out on a mass spectrometer , equipped with an Nd:YAG laser operating at 355 nm with a fitting rate of 200 Hz in the positive (H+) or negative (-H) ion reflector mode. All solvents were removed from the solutions by rotary evaporator under reduced pressure.Solvents such as acetonitrile (ACN), chloroform (CHCl2 (0.09 mol) was added dropwise to a cooled (0 °C) reaction flask solution of kojic acid (0.05 mol) and DCM (82 mL). The reaction mixture was stirred for 5 h, and the rest of the solvent was evaporated. Crude yellow powder was dissolved in toluene, and triphenylphosphine (0.05 mol) was added and stirred for 3 days and 4 nights at reflux temperature. The reaction mixture was filtrated under reduced pressure, and the beige salt was dried under pressure in an exicator for 12 h. Dried phosphonium salt was used in all further experiments.The 4-pyranone phosphonium salt was synthesized in a three-necked flask (0.25 L) by preparing corresponding pyranone chloride. Thionyl chloride, SOCl1–8 were obtained as mixtures of cis- and trans-isomers (except 1 and 5). The reaction apparatus was purged with N2 for 15 min before adding the reactants. The reaction is carried out in a three-necked flask (100 mL) equipped with a chlorine-calcium tube and an N2 balloon connected. Phosphonium salt (5 mmol) was added to the 40 mL of EtOH, and the mixture was stirred with a magnetic stirrer. The solutions of sodium ethoxides  were added in strictly anhydrous conditions under nitrogen dropwise. The aldehyde (5 mmol) was then added to the reaction mixture, and the reaction mixture was allowed to stir for 24 h at room temperature. The reaction mixture was evaporated on a vacuum evaporator and dissolved in toluene. The mixture was then extracted with toluene (3 × 15 mL). The organic layers were dried under anhydrous magnesium sulfate, MgSO4. Products 1–8 were isolated by repeated column and thin layer chromatography on silica gel using PE/E, PE/DCM, and DCM/E solvent systems. The first isomer to eluate is cis- and then trans-isomer in the last fractions. Spectroscopic characterization of new heterostilbene derivatives of 4-pyranone is given below. The solubility of pyranone derivatives was, in some cases low, especially for corresponding trans-isomers.Compounds H-pyran-4-one (1) (63%). Column chromatography on silica gel using EtOAc/MeOH (50%) afforded 500 mg of a mixture of trans-isomer and triphenylphosphine oxide. Repeated column chromatography on silica gel using EtOAc/MeOH (50%) afforded pure trans-isomer.5-hydroxy-2-(2-hydroxystyryl)-4E)-5-hydroxy-2-(2-hydroxystyryl)-4H-pyran-4-one (trans-1): 88 mg (isolated), orange powder, fR(E) = 0.12; m.p. 185–186 °C; UV  maxλ/nm (ε/dm3mol−1cm−1) 353 (29371); IR vmax/cm−1 (NaCl): 3411, 3248, 1630, 1552, 1421, 1321, 1248, 1040, 963, 939; 1H NMR  δ/ppm: 7.83 , 7.70 , 7.51 , 7.17 , 7.00 , 6.84 ; 13C NMR  δ/ppm: 144.2 (s), 136.4 (s), 131.9 (s), 130.9 (s), 128.5 (d), 124.5 (d), 124.2 (d), 119.6 (d), 116.3 (d), 116.2 (d), 105.1 (s), 107.6 (d), 99.4 (d); MS (ESI) m/z : 231 (100); HRMS (m/z) for C13H10O4: [M+H]+calcd = 230.0579, [M+H]+measured = 230.0583.(H-pyran-4-one (2) (59%). Column chromatography on silica gel using PE/E (0–100%) afforded a mixture of isomers of 2 (cis-2: trans-2 = 1:1.5). Repeated thin-layer chromatography on silica gel using PE/E (30%) as eluent afforded pure cis-2 in the first fractions and trans-2 in the last fractions.5-hydroxy-2-(2-(trifluoromethyl)styryl)-4Z)-5-hydroxy-2-(2-(trifluoromethyl)styryl)-4H-pyran-4-one (cis-2): 14.2 mg (isolated), white powder, fR(E) = 0.45; m.p. 113–115 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 280 (11679); IR vmax/cm−1 (NaCl): 3230, 2920, 2850, 1650, 1580, 1450, 1380, 1250, 930, 860, 770, 690, 580; 1H NMR  δ/ppm: 7.72 , 7.50 , 7.50 , 7.45 , 7.14 , 6.35 , 6.21 ; 13C NMR  δ/ppm: 173.7 (s), 161.8 (s), 145.7 (d), 137.0 (d), 135.1 (d), 134.7 (s), 131.6 (d), 130.3 (d), 128.6 (d), 126.0 (s), 125.9 (s), 123.5 (d), 112.5 (d); MS (ESI) m/z : 283 (100);(E)-5-hydroxy-2-(2-(trifluoromethyl)styryl)-4H-pyran-4-one (trans-2): 21.6 mg (isolated), white powder, fR(E) = 0.45; m.p. 117–120 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 308 (8706); IR vmax/cm−1 (NaCl): 3230, 2920, 2850, 1650, 1580, 1450, 1380, 1250, 930, 860, 770, 690, 580; 1H NMR  δ/ppm: 7.90 , 7.78 , 7.72 , 7.61 , 7.51 , 6.71 , 6.49 ; 13C NMR  δ/ppm: 174.2 (s), 161.8 (s), 145.6 (s), 136.9 (d), 132.3 (d), 131.9 (d), 129.1 (d), 127.3 (d), 126.3 (s), 126.2 (s), 123.4 (d), 111.3 (d); MS (ESI) m/z : 283 (100);(m/z) for C14H9F3O3 : [M+H]+calcd = 282.0504, [M+H]+measured = 282.0505.HRMS (H-pyran-2-yl)vinyl)benzonitrile (3) (44%). Column chromatography on silica gel using PE/E (80%) afforded a pure mixture of isomers of 3  without triphenylphosphine-oxide. Repeated column chromatography on silica gel using PE/E (0–70%) as eluent did not afford pure cis- and trans-isomers due to the very similar Rf values.4-(2-(5-hydroxy-4-oxo-4Z)-4-(2-(5-hydroxy-4-oxo-4H-pyran-2-yl)vinyl)benzonitrile (cis-3): 1H NMR  δ/ppm: 8.93 , 7.74 , 7.4 , 7.10 , 6.84 , 6.79 ; MS (ESI) m/z : 240 (100).(E)-4-(2-(5-hydroxy-4-oxo-4H-pyran-2-yl)vinyl)benzonitrile (trans-3): 1H NMR  δ/ppm: 9.27 , 7.86 , 7.76 , 7.68 , 7.15 , 6.67 ; MS (ESI) m/z : 240 (100);(vmax/cm−1 (NaCl) : 3050, 2910, 2840, 2250, 1715, 1570, 1480, 950, 840, 550; HRMS (m/z) for C14H9NO3 : [M+H]+calcd = 239.0582, [M+H]+measured = 239.0588.IR H-pyran-4-one (4) (74%). Column chromatography was performed on silica gel using PE/E (0–70%) as eluent. Repeated column chromatography on silica gel (PE/E (0–70%)) afforded pure cis-4 in the first fractions and trans-4 in the last fractions.2--5-hydroxy-4Z)-2--5-hydroxy-4H-pyran-4-one (cis-4): 250 mg (isolated), white powder, fR(E) = 0.51; m.p. 149–151 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 291 (14966); IR vmax/cm−1 (NaCl): 3200, 3050, 1600, 1550, 1490, 1380, 1250, 1150, 950, 750, 700; 1H NMR  δ/ppm: 7.58 , 7.45 , 7.21 , 6.90 , 6.36–6.31 ; 13C NMR  δ/ppm: 173.7 (s), 161.8 (s), 145.7 (s), 136.7 (d), 135.1 (s), 134.1 (s), 133.9 (d), 132.8 (s), 130.8 (d), 129.4 (d), 126.9 (d), 123.1 (d), 112.5 (d); MS (ESI) m/z : 283 (100).(E)-2--5-hydroxy-4H-pyran-4-one (trans-4): 40 mg (isolated), white powder, fR(E) = 0.51; m.p. 103–105 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 325 (23654), 288 (20467); 1H NMR  δ/ppm: 8.06 , 7.93 , 7.74 , 7.58 , 7.57 , 6.68 , 6.56 ; 13C NMR  δ/ppm: 174.1 (s), 161.8 (s), 145.7 (s), 137.5 (s), 135.2 (s), 131.1 (d), 130.2 (d), 130.0 (d), 128.4 (d), 127.7 (d), 127.6 (d), 122.1 (s), 111.3 (d); MS (ESI) m/z : 283 (100);(m/z) for C13H8Cl2O3 : [M+H]+calcd = 281.9850, [M+H]+measured = 281.9852.HRMS (N-(4-(2-(5-hydroxy-4-oxo-4H-pyran-2-yl)vinyl)phenyl)acetamide (5) (53%). Repeated column chromatography was performed on silica gel with solvent system EtOAc/MeOH (20%). This derivative gave only cis-isomer as a product of the Wittig reaction, but repeated chromatography was necessary to divide the triphenylphosphine-oxide from cis-5.Z)-N-(4-(2-(5-hydroxy-4-oxo-4H-pyran-2-yl)vinyl)phenyl)acetamide (cis-5): 100 mg (isolated), orange powder, fR(DCM:MeOH = 7:3) = 0.35; m.p. 154–156 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 338 (10321); IR vmax/cm−1 (NaCl): 3372, 1639, 1582, 1534, 1321, 1210, 1178, 1040, 970, 930, 825; 1H NMR  δ/ppm: 6.28 , 6.04 , 5.76 , 5.40 , 4.73 , 3.34 ; 13C NMR  δ/ppm: 174.4 (s), 166.9 (s), 141.7 (s), 139.8 (s), 134.9 (s), 133.2 (d), 132.0 (d), 123.6 (d), 123.2 (d), 121.7 (d), 116.6 (d), 114.6 (s), 26.5 (q); MS (ESI) m/z : 272 (100);(m/z) for C15H13NO4: [M+H]+calcd = 271.0845, [M+H]+measured = 271.0850.HRMS (H-pyran-4-one (6) (38%). Repeated column chromatography was performed on silica gel using PE/DCM (0–90%), DCM/E (30%), and DCM/MeOH (10%) as solvent systems in combination with preparative thin-layer chromatography in a solvent system PE/E (30%). Pure cis-6 was isolated in the first fractions and trans-6 in the last fractions.5-hydroxy-2-(2-(thiophen-3-yl)vinyl)-4Z)-5-hydroxy-2-(2-(thiophen-3-yl)vinyl)-4H-pyran-4-one (cis-6): 7 mg (isolated), white powder, fR(E) = 0.55; m.p. 143–146 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 311 (11679); IR vmax/cm−1 (NaCl): 3220, 2925, 2840, 1740, 1650, 1580, 1470, 1435, 1380, 950, 825, 770, 725; 1H NMR  δ/ppm: 7.77 , 7.39 , 7.29 , 7.09 , 6.81 , 6.47 , 6.10 ; 13C NMR  δ/ppm: 173.9 (s), 162.9 (s), 145.9 (s), 136.7 (d), 136.3 (s), 132.8 (d), 127.8 (d), 127.4 (d), 125.9 (d), 119.2 (d), 111.9 (d); MS (ESI) m/z : 221 (100).(E)-5-hydroxy-2-(2-(thiophen-3-yl)vinyl)-4H-pyran-4-one (trans-6): 9.1 mg (isolated), yellow powder, fR(E) = 0.55; m.p. 111–115 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 314 (20340); IR vmax/cm−1 (NaCl): 3220, 2925, 2840, 1740, 1650, 1580, 1470, 1435, 1380, 950, 825, 770, 725; 1H NMR  δ/ppm: 7.83 , 7.48 , 7.37 , 7.32 , 7.29 , 6.53 , 6.48 ; 13C NMR  δ/ppm: 174.2 (s), 162.8 (s), 145.5 (s), 138.0 (s), 136.8 (d), 130.3 (d), 127.2 (d), 126.9 (d), 124.8 (d), 119.1 (d), 110.1 (d); MS (ESI) m/z : 221 (100);(m/z) for C11H8O3S : [M+H]+calcd = 220.0194, [M+H]+measured = 220.0199.HRMS (H-pyran-4-one (7) (42%). Repeated column chromatography was performed on silica gel with solvent system PE/E (0–50%). After that, repeated thin-layer chromatography of some enriched fractions was performed with a solvent system E/CHCl3 (80%). Only the isolation of the pure trans-7 in the last fractions was successful, while the obtained pure mixture of isomers (13 mg) without triphenylphosphine-oxide confirmed the formation of cis-7 (cis-7:trans-7 = 1:1).5-hydroxy-2-(2-(thiophen-2-yl) vinyl)-4Z)-5-hydroxy-2-(2-(thiophen-2-yl)vinyl)-4H-pyran-4-one (cis-7): 1H NMR  δ/ppm: 7.91 , 7.44 , 7.25 , 7.06 , 6.90 , 6.47 , 6.0 ; MS (ESI) m/z : 221 (100).(E)-5-hydroxy-2-(2-(thiophen-2-yl)vinyl)-4H-pyran-4-one (trans-7): 8 mg (isolated), white powder, fR(E) = 0.35; m.p. 113–116 °C; UV (ACN) maxλ/nm (ε/dm3mol−1cm−1) 340 (30518); IR vmax/cm−1 (NaCl): 3210, 2920, 2840, 1730, 1660, 1580, 1470, 1435, 1390, 1210, 950, 850, 760, 680; 1H NMR  δ/ppm: 7.83 , 7.52 , 7.36 , 7.22 , 7.05 , 6.50 , 6.39 ; 13C NMR  δ/ppm: 174.2 (s), 162.8 (s), 145.5 (s), 140.1 (s), 136.8 (d), 129.9 (d), 129.3 (d), 128.2 (d), 127.2 (d), 118.3 (d), 110.1 (d); MS (ESI) m/z : 221 (100);(m/z) for C11H8O3S : [M+H]+calcd = 220.0194, [M+H]+measured = 220.0200.HRMS (H-pyran-4-one (8) (35%). Repeated column and thin-layer chromatographies (E/EtOAc (0–70%) and EtOAc/MeOH (0–90%)) were performed on silica gel. The pure mixture of isomers was obtained (12 mg) without the presence of triphenylphosphine-oxide (cis-8:trans-8 = 1.3:1).5-hydroxy-2-vinyl)-4Z)-5-hydroxy-2-vinyl)-4H-pyran-4-one (cis-8): 1H NMR  δ/ppm: 8.93 , 8.13–8.08 , 7.92–7.86 , 6.84 , 6.79 , 6.79 ; MS (ESI) m/z : 267 (100).(E)-5-hydroxy-2-vinyl)-4H-pyran-4-one (trans-8): 1H NMR  δ/ppm: 9.27 , 8.13–8.08 , 8.02 , 7.92–7.86 , 7.75 , 7.68 , 6.67 ; MS (ESI) m/z : 267 (100);(vmax/cm−1 (NaCl) : 3320, 2959, 2917, 2847, 1584, 1568, 1377, 1261, 1088, 1014, 794, 749; HRMS (m/z) for C15H10N2O3 : [M+H]+calcd = 266.0691, [M+H]+measured = 266.0697.IR S-butyrylthiocholine iodide (BTChI) and galantamine were purchased from Sigma-Aldrich , 5,50-dithiobis-(2-nitrobenzoic acid)  was purchased from Zwijndrecht (Belgium). The AChE/BChE inhibitory potentials were determined using modified Ellman’s method . The CrysAlisPRO package  was used for data reduction and numerical absorption correction. The structure was solved with SHELXS97 [www.ccdc.cam.ac.uk/conts/retrieving.html accessed on 2 September 2022 . CCDC-2204641 and CCDC-2204642 contain supplementary crystallographic data for this paper.Single crystal measurements were performed on an XtaLAB Synergy diffractometer, using micro-focus sealed X-ray tube CuSHELXS97  and refiSHELXS97 . The modSHELXS97 , and theSHELXS97  and CCDCSHELXS97 . Crystal3+ ion as biometals. In the crystal packing, they are held together by weak intermolecular interactions: hydrogen bonds, C–H⋯π interactions, and dispersion interactions.New resveratrol–thiophene and resveratrol–maltol hybrids were synthesized as cholinesterase inhibitors, metal-chelating agents, and antioxidants. As photostability is the favorable property of resveratrol derivatives, the aim of this study was also to investigate changes in the UV spectra of compounds when exposed to UV light, as well as the capability to complex with biometals. As with photostability experiments, biological tests also found remarkable differences in the properties and behavior of thiophene and maltol hybrids. While resveratrol–thiophene hybrids have excellent inhibitory and antioxidant properties , maltols have been proven to be weaker inhibitors and antioxidants. The results of the docking study indicate that all docked ligands are accommodated by both enzymes, AChE and BChE, mainly through π–π stacking interactions. The compounds proved to be active cholinesterase inhibitors, also prone to coordination with the Fe


Clerodendrum, of the Lamiaceae. Due to their diverse chemical nature, and the type of plant material, a range of extraction techniques are needed with various temperatures, solvent types and extraction times, as well as the use of an ultrasound bath. The diterpenoids isolated from Clerodendrum demonstrate a range of cytotoxic, anti-proliferative, antibacterial, anti-parasitic and anti-inflammatory activities. This review describes the various biological activities of the diterpenoids isolated so far from species of Clerodendrum with the indication of the most active ones, as well as those from other plant sources, taking into account their structure in terms of their activity, and summarises the methods for their extraction.One of the key areas of interest in pharmacognosy is that of the diterpenoids; many studies have been performed to identify new sources, their optimal isolation and biological properties. An important source of abietane-, pimarane-, clerodane-type diterpenoids and their derivatives are the members of the genus  Clerodendrum is derived from two Greek words: kleros (destiny or chance) and dendron (tree) pyran (7H)-7, 12(8H)-dione (bungone A (1)) and 9,10-dihydro-8-hydroxymethyl-3,4,9-trimethylphenanthro pyran(2H)-7,12-dione (bungone B (2)). While these abietane diterpenoids are structurally similar to the royleanones, with both possessing an 11,14-para benzoquinone group, the C-12 has an oxygen enclosed by an additional aliphatic ring instead of a hydroxyl group, which is typical for roylanones. Due to their cytotoxic activities, these compounds are very interesting for further research; like other diterpenes, including horminone or acetyl-horminone, royleanone is able to damage DNA and inhibit topoisomerase I and II [This plant species was first investigated for diterpenoid content by Fan et al. in 1999 . The autI and II ,40,41.Clerodendrum bungei. Five were new structures: 12-O-β-D-glucopyranosyl-3,11,16-trihydroxyabieta-8,11,13-triene (3), 3,12-O-β-D-diglucopyranosyl-11,16-dihydroxyabieta-8,11,13-triene (4), 19-O-β-D-carboxyglucopyranosyl-12-O-β-D-glucopyranosyl-11,16-dihydroxyabieta-8,11,13-triene (5), 11,16-dihydroxy-12-O-β-D-glucopyranosyl-17(15→16),18(4→3)-abeo-4-carboxy-3,8,11,13-abietatetraen-7-one (6) and 19-hydroxyteuvincenone F (7). All are glycosides, apart from compound (7), and all contain aglycone, either as an abietatriene or abietatetraene.Liu et al. (2008)  isolated(8), uncinatone (9) and teuvincenone F (10), first isolated from other plant materials, were also isolated, purified and identified from the aqueous acetone crude extract of C. bungei roots. The identified compounds were tested for their potential cytotoxic activity against three cell lines: B16 murine melanoma, HGC-27 human gastric, and HEK-293 human epithelial kidney. Of the tested compounds, only uncinatone (9), a rearranged abietane derivative containing a 17(15→16), 18(4→3)-diabeo-abietane framework, was found to demonstrate moderate cytotoxicity against tested cell lines: the IC50 value ranged from 1.2 to 6.4 µM depending on the treated cell line, as indicated by MTT, i.e., 3--2,5-diphenyl-2H-tetrazolium bromide. This diterpenoid also inhibited cell proliferation and induced cell-cycle G2/M phase arrest [In addition, the diterpenoids ajugaside A e arrest .O-β-D-glucopyranosyl-3,11,16-trihydroxyabieta-8,11,13-triene (3), 3,12-O-β-D-diglucopyranosyl-11,16-dihydroxyabieta- 8,11,13-triene (4), ajugaside A (8), uncinatone (9) and 19-hydroxyteuvincenone F (7) demonstrated significant anti-complement activity on the classical pathway complement system, as expressed by total hemolytic activity [50 range from 24 µM to 232 µM. The most active compound was found to be 12-O-β-D-glucopyranosyl-3,11,16-trihydroxyabieta-8,11,13-triene (3) [C. bungei: 3β-(β-D-glucopyranosyl)isopimara-7,15-diene-11α,12α-diol (11) and 16-O-β-D- D-glucopyranosyl-3 β-20-epoxy-3-hydroxyabieta-8,11,13-triene (12) together with other known compounds, such as 12-O-β-D-glucopyranosyl-3,11,16-trihydroxyabieta-8,11,13-triene (3) and 3,12-O-β-D-diglucopyranosyl-11,16-dihydroxy-abieta-8,11,13-triene (4) [O-β-D-glucopyranosyl-3β-20-epoxy-3-hydroxyabieta-8,11,13-triene (12) appeared to be active among all tested compounds; it demonstrated moderate cytotoxicity against B16, HGC-27, and BEL-7402 cells, manifested with IC50 values of 8.8, 9.8, and 7.1 µM, respectively [C. bungei roots [In addition, 12-activity . The inhiene (3) . Kim et iene (3) . In addiiene (4) . All isoectively . The autectively ,35,37. IC. bundei resulted in the isolation and identification of the following diterpenoids: bungnate A (13) -abeo-5,8,11,13,15-abietapentaen-7-one-17-carboxylate), bungnate B (14) , 15-dehydrocyrtophyllone A (15) -abeo-5,8,11,13,15-abietapentaen-7-one) 15-dehydro-17-hydroxycyrtophyllone A (16) -abeo-5,8,11,13,15-abietapentaen-7-one), and cyrtophyllone A (17) [(15) demonstrated ACE (Angiotensin Converting Enzyme) inhibition activity, with an IC50 value of 42.7 µM. Among the tested diterpenoids, none inhibited α-glucosidase [Further studies on e A (17) . Of thescosidase .(10), uncinatone (9) and sugiol (18), the triterpenoids friedelin (19) and clerodolone (20) and the phytosteroids stigmasta-5,22,25-trien-3β-ol and clerosterol. In addition, two new abietane derivatives, cyrtophyllone A (17) (16(S)-12,16-epoxy-11,13-dihydroxy-6-methoxy-17(15-16)-abeo-abieta-5,8,11,13-tetraen-7-one) and cyrtophyllone B (21) ((+)-11,12,16-trihydroxy-abieta-8,11,13-trien-7-one) have been isolated from ethanolic extract of the entire C. cyrtophyllum plant following cleaning by water and chloroform mix [abeo-abietane framework.This genus, known in Chinese medicine as “Da quing”, is recommended for treating infectious diseases, common cold and malaria . Many reform mix . The for(18), uncinatone (9) and cyrtophyllone B (21), also isolated from C. cyrtophyllum, have also been identified in Aegiphila lhotzkyan roots. These phytocompounds were tested for antiproliferative activity against leukaemia (CEM and HL-60), breast (MCF-7), colon (HCT-8) and skin (B-16) cancer cell lines in three independent experiments [(21) is able to inhibit the proliferation of all tested tumour cell lines; however, it did not demonstrate strong inhibition  [Caryopteris mongolica roots were found to inhibit acethyl- and butyrylcholineesterase (AChE and BChE) [The diterpenes sugiol eriments . Of thesµg mL−1) . In addind BChE) . The extC. eriophyllum root bark extract demonstrated significant chemosuppressive properties against Plasmodium berghei in infected experimental mice [Clerodendrum eriophyllum was recorded by Machumi et. al. in 2010 [(22). The remaining nine diterpenes had previously been isolated from other plant materials: royleanone (23), taxodione (24), 6-deoxy-taxodione (25) ,13-abietatrien-12-one), sugiol (18), ferruginol (26), 6-hydroxysalvinolone (27), 6,11,12,16-tetrahydroxy-5,8,11,13-abietatetra-en-7-one (28), uncinatone (9) and 11-hydroxy-8,11,13-abietatriene-12-O-β-xylopyranoside (29) [This unusual plant was previously used in malaria treatment in Kenya . An alcotal mice . The fir in 2010 . The extide (29) .(23), was first isolated from Inula royleana roots [Salvia austriaca roots [Salvia officinalis roots [(23), the diterpenoid characterised by the presence of a p-quinone grouping in the C ring, is also well known for its various biological activities. It has been found to demonstrate cytotoxicity against the cancer cell lines HeLa and Hep-2, particularly against Hep-2, with an IC50 value of 34 µg mL−1 [S. aureus strains (MRSA and VRE) compared to other diterpenoids from outside the Clerodendrum genus  [One of the abietane diterpenoids, royleanone na roots . Howeverca roots  and non-is roots . Royleanctively) .(24) is a very well-known abietane-type diterpenoid with a metide-quinone moiety, which was first isolated from entire Taxodium distichum plant [−1) [Taxodione um plant . This coum plant . Its higum plant  and A549um plant . This coum plant . It has ant [−1) .(25), isolated from C. eriophyllum roots, is also detected in various parts of other plant species, e.g., in winter cones of Taxodium distichum and fruits of Cupressus sempervirens [(24), both isolated from Cupressus sempervirens cones, this compound demonstrates potent anti-leishmanial activity, with IC50 values of 0.077 µg mL−1 for 6-deoxy-taxodione (25) and 0.025 µg mL−1 for taxodione (24). The two diterpenoids demonstrated much stronger activity against Leishmania donovani and its promastigotes than the anti-leishmanial drugs used as controls: pentamidine (IC50 1.62 µg mL−1) and amphotericin B (IC50 0.11 µg mL−1) [(25) was found to demonstrate potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), with IC50 values being 0.80 μg mL−1 for (25) and 0.85 μg mL−1 for (26) [6-deoxy-taxodione ervirens ,53,54. L(18), isolated from Clerodendrum eriophyllum roots. This compound has an oxygen atom connected to the B ring and an aromatic C ring. This unusual aromatic diterpene demonstrates various antioxidant, antibacterial, antiviral, anticancer, anti-tumour and anti-inflammatory activities [(18) also demonstrates a concentration-dependent inhibitory effect (72.4%) against NO (nitric oxide), at a concentration of 100 μg mL−1; it also demonstrated similar superoxide radical scavenging activity at a concentration of 250 μg mL−1, to ascorbic acid and α-tocopherol activities  [(18) is also active against various foodborne pathogenic bacteria but neutralises Gram-positive bacteria more effectively than Gram-negative bacteria. When isolated from Metasequoia glyptostroboides cones, the compound was also found to demonstrate stronger antibacterial action against Gram-positive bacteria than the streptomycin used as a control [(18) has also been found to exhibit antiviral activity against the H1N1 virus in infected Madin-Darby canine kidney (MDCK) cells: no cytopathic changes were observed following 72 h of exposure following treatment with 500 μg mL−1 sugiol (18). Hence, sugiol (18) could be a potential antiviral compound that can prevent H1N1-mediated cytopathy in MDCK cells [Another abietane-type diterpenoid is sugiol tivities . Its anttivities . Sugiol ctively) . Sugiol  control . Sugiol CK cells .(18) also demonstrated cytotoxic activity against tumour cell lines, inhibiting the growth of three prostate tumour cell lines  and a non-tumorigenic cell line (MCF10A) [(18) treatment was found to reduce tumour weight and volume by as much as 75% in mice subcutaneously injected with DU145 cells in comparison with the control group. However, sugiol (18) did not affect the body weight of the mouse [The diterpenoid sugiol (MCF10A) . Similarhe mouse .(26) was first isolated in 1939 from the Podocarpus ferruginea tree. Structurally, ferruginol is similar to sugiol (18), although it lacks an oxygen in the B ring. The biologically active ferruginol has been recorded in many plants including those of the Podocarpaceae, Cupressaceae, Lamiaceae and Verbenaceae [Bacillus brevis, B. subtilis and Staphylococcus aureus, with inhibition zone diameters of 18, 10 and 9 mm, respectively. Ferruginol (26) demonstrated fungicidal activity against the pathogenic Paecilomyces variotii, with an inhibition zone of 10 mm [(26) isolated from Chamaecyparis lawsoniana cones also demonstrated antibacterial activity against S. aureus, with MIC values ranging from 4 to 16 μg mL−1 depending on the strain [50 values against Plasmodium falciparum ranging from 2.47 to 19.57 μM, depending on the strain [(26) has displayed moderate cytotoxic activity against NALM-6 human leukaemia lymphoblastic cells (IC50 27.2 μg mL−1) and promyelocytic HL-60 cells (IC50 33.6 μg mL−1) [The abietane diterpenoid ferruginol benaceae . This dibenaceae . It has of 10 mm , and Fere strain . It has e strain , with ECe strain . In addiμg mL−1) .(27), containing oxygen and hydroxyl groups in the B ring, demonstrates strong cytotoxicity against carcinoma cell lines. Following isolation from Salvia chorassanica roots, the compound exhibited strong cytotoxic activity against HL-60 and K562 cell lines with IC50 values of 36.3 and 33.3 μM, respectively. It appeared to demonstrate a substantially less cytotoxic effect on non-cancerous human cell lines. When administered at concentrations of 2.5 and 5.0 μM for 48 h, it also enhanced the expression of the proapoptotic protein Bax, and cleaved caspase-3 and PARP [50 level of 4.5 μg mL−1 [(24), 6-hydroxysalvinolone (27) also demonstrated antifungal activity, especially against Candida neoformans with an IC50 value of 0.96 μg mL−1. In the same assay, the IC50 of taxodione (24) was found to be 0.58 μg mL−1, which is comparable with that of standard amphotericin B (IC50 = 0.44 μg mL−1) [The abietane diterpenoid 6-hydroxysalvinolone and PARP . It also μg mL−1 . Similarμg mL−1) .(28), isolated from Avicennia marina twigs; it differs from 6-hydroxysalvinolone (27) by the presence of a hydroxyl group in the isopropyl moiety. It demonstrated moderate antiproliferative properties against L-929 (mouse fibroblasts) and K562 (human chronic myeloid leukaemia), and cytotoxic activities against the HeLa (human cervix carcinoma) cell line [(28) demonstrated GI50 (concentration causing 50% cell growth inhibition) values of 9.6 and 8.9 μg mL−1, against L-929  and K562 cell lines , and a CC50 (concentration that reduced the cell viability by 50%) of 18 μg mL−1 against the HeLa cell line [Bacillus subtilis ATTC 6 633 (IMET) NA, Bacillus subtilis ATTC 6 633 (IMET) AS, Escherichia coli SG 458, Pseudomonas aeruginosa K 799/61, Mycobacterium vaccae IMET 10 670, Sporobolomyces salmonicolor SBUG 549, Candida albicans BMSY 212 and Penicillium notatum JP [C. albicans) to 25 mm (for B. subtilis ATTC 6 633 (IMET) AS) [Another abietane-type diterpenoid is 6,11,12,16-tetrahydroxy-5,8,11,13-abietatetra-en-7-one ell line . In biolell line . The comtatum JP  found zoMET) AS) .(9), a diterpenoid known for its biological activity, also exhibits potent antileishmanial activity. The IC50 value for L. donovani is 0.2 μg mL−1 [Uncinatone  μg mL−1 .abeo-abietane diterpenoid formidiol (30) was first obtained by methanolic extraction of Clerodendrum formicarum leaves and chromatographic separation of its triterpenoid constituents [(31), which had been previously isolated from a hexane extract of Aegiphila lhotzkiana roots. It was found to demonstrate antiproliferative activity against the leukaemia cell lines HL-60 (IC50 4.4 μM) and CEM (IC50 8.4 μM) [(30), compound (31) should be included in future studies of anti-proliferative activity. The extraction method used for the phytochemical analyses of this plant species is shown in The tituents . It was  8.4 μM) . Due to Clerodendrum inerme resulted in the isolation of cleroinermin (32) a neo-clerodane diterpenoid [Heteroplexis micocephala, showed neuroprotective activity against MPP+ induced PC12-syn cell damage, with a relative cell proliferation rate of 104.32% [neo-clerodane diterpenoids clerodendrin B (33), 3-epicaryoptin (34), clerodendrin C (35), 2-acetoxyclerodendrin B (36) and 15-hydroxyepicaryoptin (37) have since been isolated [Earias vitella at 10 µg cm−3 of diet (30 µg g−1) and against Spodoptera litura at 10 µg cm−2 of leaf mass [Studies on the aerial parts of erpenoid  consisti 104.32% . Elsewheisolated . The exteaf mass .C. inerme has become an interesting subject of research for diterpenoid isolation. The aerial parts are a source of the neo-clerodane-type diterpenoids: clerodermic acid (38), inermes A (39) and B (40), as well as 14,15-dihydro-15β-methoxy-3-epicaryoptin (41) [(38) deserves special attention due to its strong biological activity. The compound, isolated from the dichloromethane extract of the aerial part of Salvia nemorosa, was found to reduce the viability of A549 cells in a concentration-dependent manner, with an IC50 of 35 µg mL−1 at 48 h, based on the MTT assay [(38). DNA ladder, DAPI staining, cell cycle analysis, and annexin V/PI testing indicated that clerodermic acid demonstrates strong geno- and cytotoxicity and is able to induce apoptosis in A549 cells, as evidenced also by DNA fragmentation and chromatin condensation [tin (41) ,71. AmonTT assay . Furtherensation .C. inerme aerial parts have also been found to include a newly rearranged abietane diterpenoid, crolerodendrum B (42), as well as other known diterpenoids, such as crolerodendrum A (43), uncinatone (9) and harwickiic acid (44) [(44) was first isolated from Sindora sumatrana MIQ fruits [Croton sylvaticus, was found to demonstrate significant antileishmanial activity against L. donovani promastigotes with an IC50 of 31.57 µM, as well as cytotoxic activity against RAW 264.7 (CC50 = 247.83 μM) [(44), isolated from C. inerme aerial parts, together with crolerodendrum B (42) and uncinatone (9) also demonstrates strong antioxidant activity measured as DPPH radical-scavenging activity; these compounds have been found to have respective ED50 values of 11.3 µM (44), 17.6 µM (42) and 10.1 µM (9) [cid (44) . HarwickQ fruits . This cl7.83 μM) . Harwick1 µM (9) .(45), 15-methoxy-14,15-dihydroclerodin (46) and 15-hydroxy-14,15-dihyroclerodin (47) [Helicoverpa armigera. Studies on the growth inhibition potential of these diterpenoids found topical application of clerodin (45), 15-methoxy-14,15-dihydroclerodin (46) and 15-hydroxy-14,15-dihyroclerodin (47) to yield GI50 values of 13, 21 and 11 ppm, respectively; in contrast, azadirachtin was found to have a GI50 value of 15 ppm [Crystallization and chromatographic separation of the leaf extract resulted in the isolation and identification of the clerodane diterpenoids clerodin din (47) . The extf 15 ppm .Helicoverpa armigera) [(45) and 15-methoxy-14,15-dihydroclerodin (46) demonstrated significantly higher antifeedant activity compared to high concentration azadirachtin, the key ingredient in many commercial pesticides [(45) has also been found to demonstrate antifeedant activity against Earias vitella and Spodoptera litura [50) values for clerodin (45), 15-methoxy-14,15-dihydroclerodin (46) and 15-hydroxy-14,15-dihyroclerodin (47) were found to be 6, 6, and 8 ppm in the choice tests, and 8, 9, and 11 ppm in the no-choice tests, respectively.The purified diterpenoids, together with their extracts and fractions, also demonstrated insecticidal activity against the highly polyphagous cotton bollworm (rmigera) . The antsticides , with tha litura . The antClerodendrum infortunatum leaf extract offer promise as biopesticides and require further studies [The antifeedant activity of clerodanes has been attributed to the presence of a perhydrofuranofuran moiety and the degree of its unsaturation; a significant role may also be played by the presence of a trans-decalin ring system bearing an epoxide, together with acetate groups ,79. Thes studies .Clerodendrum kaichianum P. S. Hsu is known to be the source of two new abietane-type compounds, viz. 17-hydroxyteuvincenone G (51) and 17-hydroxyteuvincen-5(6)-enone G (52), as well as four known diterpenoids: teuvincenone A (48), 11,14-dihydroxyabieta-8,11,13-trien-7-one (49), dehydroabietan-7-one (50) and sugiol (18) [cis-platin, which was used as a control compound. In addition, 17-hydroxyteuvincenone G (51) yielded IC50 scores of 5.95 and 9.37 µM for HL-60 and A-549 cells, respectively; this activity was slightly higher than that of 17-hydroxyteuvincen-5(6)-enone G (52) (IC50 of 15.91 and 10.35 µM against the same cell lines) [iol (18) . These nl lines) .C. kaichianum stem extract resulted in the isolation of a newly rearranged abietane diterpenoid with five known compounds: villosin A (53), salvinolone (54), 14-deoxyloleon U (55), 5,6-dehydrosugiol (56), and coleon U (57). This new diterpenoid was identified as (16R)-12,16-epoxy-11,14,17-trihydroxy-17(15→16)-abeo-8,11,13-abietatrien-7-one (58) [(53), salvinolone (54) and 5,6-dehydrosugiol (56) were noted in the Clerodendrum genus for the first time. All extraction methods used for the phytochemical analyses of this plant species are shown in (58) with an IC50 value of 18.5 µM, with villosin A (53) and coleon U (57) demonstrating IC50 values of 20.1 and 24.1 µM, respectively. Salvinolone (54), 14-deoxyloleon U (55) and 5,6-dehydrosugiol (56) demonstrated more than two-fold weaker cytotoxic activity, with IC50 values over 40 µM [Further chromatographic separation from one (58) . Villosier 40 µM .C. kiangsiense resulted in the isolation of eight diterpenoids, one of which was a novel abeo-abietane diterpenoid. Spectroscopic analyses resulted in its identification as 12-methoxy-6,11,14,16-tetrahydroxy-17(15→16)-abeo-5,8,11,13-abietatetraen-3,7-dione (59) [(60) -12-hydroxy-8,11,13-abietatriene-37-dione), taxusabietane A (61), 12-O-demethylcryptojaponol (62), cryptojaponol (63), 11,14-dihydroxy-8,11,13-abietatrien-7-one (64), fortunin E (65) and fortunin F (66) [(60) had previously been isolated from Clerodendrum mandarinorum stem [Euonymus lutchuensis roots [A phytochemical study on the aerial parts of one (59) . The remn F (66) . Mandarorum stem  and Euonis roots .C. mandarinorum stem, including mandarone B (67) ((16 S)-12,16-epoxy-11,14-dihydroxy-17(15→16)-abeo-abieta-5,8,11,13-tetraene-7-one), mandarone C (68) -abeo-abieta-2,5,8,11,13,15-hexaene-7-one) [(69) (16S)-12,16-epoxy-l1-hydroxy-17(15→16),18(4→3)-diabeo-abieta-3,5.8,11,13-pentaene-7-one, mandarone E (70) ,18(4→3)-diabeo-abieta-3,5,8,11,13,15-hexaene-7-one), mandarone F (71) ,18(4→3)-diabeo-abieta-3,5,8,11,13,15-hexaene-7-one), mandarone G (72) ,18(4→3)-diabeo-abieta-3,5,8,11,13,15-hexaene-2,7-dione) and mandarone H (73) ,18(4→3)-diabeo-abieta-3,5,8,11,13,15-hexaene-1,7-dione) [Various other mandarones have also been isolated from e-7-one) , mandaro7-dione) . The ext(61), isolated from bark extract of Taxus wallichiana Zucc. (in addition to taxusabietane C and taxamairin F), was found to demonstrate considerable lipoxygenase (LOX) inhibitory activity at an IC50 of 57 μM compared to controls  based on in vitro lipooxygenase inhibition assay and in vivo carrageenan-induced paw oedema model [(63), isolated from extracted Taxodium distichum bark, demonstrated moderate cytotoxic activity against human pancreatic carcinoma (PANC-1) [50 of about 38 μM and selective index (SI) of 7.9 [Taxusabietane A ma model . Cryptoj(PANC-1)  with an ) of 7.9 .(64), an abietane diterpenoid found in Clerodendrum kiangsiense aerial parts, exhibits some interesting biological activities. Costa-Lotufo et al. (2004) found it to demonstrate moderate cytotoxic activity against tumour cell lines, together with as well as carnasol, isolated from Hyptis martiusii roots [Zhumeria majdae and to show promising antiprotozoal activity; the IC50 value was found to be 8.65 μM, with a selectivity index (SI) of 4.6 [In addition, 11,14-dihydroxy-8,11,13-abietatrien-7-one ii roots . Zadali ) of 4.6 . Additio) of 4.6 .(74), 3β,15-dihydroxy-14-hydro-clerodin (75), 2α,15-dihydroxy-3β-(2′-hydroxy-2′-methyl-3′-acetoxy)-butanoyloxy-6α,18-diacetoxy-4α,17-epoxy-clerodan-11,16-lactone (76) and 3β,14S,15-trihydroxy-6α,18-diacetoxy-4α,17-epoxy-clerodan-11,16-lactone (77) [(74) and 2α,15-dihydroxy-3β-(2′-hydroxy-2′-methyl-3′-acetoxy)-butanoyloxy-6α,18-diacetoxy-4α,17-epoxy-clerodan-11,16-lactone (76) exhibit cell growth inhibition activity. In addition, the IC50 values for the two compounds, viz., (76) and (74), were found to be 101 μM and 98 μM, respectively, after 72 h incubation [Scientific research on this species allowed to isolate and identify four new clerodane diterpenoids, namely 2α-acetoxy-3β--butanoyloxy-14-hydro-15-hydroxyclerodin one (77) . The extcubation .(78) was first isolated from Clerodendrum trichotomum roots by careful semi-preparative chromatographical analysis. This diterpenoid is a rare phenolic ketal of a regular abietane derivative, cyrtophyllone B (21), and a rearranged abietane derivative related to uncinatone (9) [(78) demonstrates moderate cytotoxic activity against some tumour cell lines  with IC50 values ranging between 7.51 and 19.38 µM [Trichotomone tone (9) . Trichot19.38 µM .abeo-abietane (6-methoxyvillosin C (79) -12,16-epoxy-11,14,17-trihydroxy-6-methoxy-17(15→16)-abeoabieta-5,8,11,13-tetraene-7-one), 18-hydroxy-6-methoxyvillosin C (80) -12,16-epoxy-6-methoxy-11,14,17,18-tetrahydroxy-17(15→16)-abeo-abieta-5,8,11,13-tetraene-7-one) and -12,16-epoxy-11,14-dihydroxy-6-methoxy-17(15→16)-abeo-abieta-5,8,11,13-tetraene-3,7-dione (81) and 17(15→16),18(4→3)-diabeo-abietane diterpenoids (trichotomone D (82) -12,16-epoxy-11,14-dihydroxy-18-oxo-17(15→16),18(4→3)-diabeo-abieta-3,5,8,11,13-pentaene-7-one, -12,16-epoxy-11,14,17-trihydroxy-17(15→16),18(4→3)-diabeo-abieta-3,5,8,11,13-pentaene-2,7-dione (83) and trichotomone F (84) =-3,4:12,16-diepoxy-11,14-dihydroxy-17(15→16),18(4→3)-diabeo-abieta-5,8,11,13-tetraene-7-one) [(85), 12,16-epoxy-11,14-dihydroxy-6-methoxy-17(15→16)-abeo-abieta-5,8,11,13,15-pentanene-3,7-dione (31), uncinatone (9), mandarone E (70), formidiol (30), teuvincenone E (86), teuvincenone F (10) and Trichotomone H (87) ,18(4→3)-diabeo-abieta-3,5,8,12,15-pentaene-7,11,14-trione) [Wang et al. (2013) report the isolation of various other diterpenoid compounds from the species, including 17(15→16)-e-7-one) . In addi-trione) .R,16S)-12,16-epoxy-11,14-dihydroxy-6-methoxy-17(15→16)-abeo-abieta-5,8,11,13-tetraene-3,7-dione (81) is a newly discovered naturally occurring compound. All the extraction methods used for the phytochemical analyses of this plant species are shown in (82), F (84) and H (87), teuvincenone E and H (88), uncinatone (9) and mandarone E (70) showed cytotoxic activity. IC50 values ranged from 0.83 to 50.99 µM. The most active diterpenoid was found to be Teuvincenone E (86), with IC50 values of 3.95, 5.37, 1.18, 1.27, and 0.83 µM against the BGC-823, Huh-7, KB, KE-97, and Jurkat lines, respectively. The authors attribute the high cytotoxic activity of this compound to its rearranged A ring and intact 2-methyl-3-dihydro-furan fragment [Of the 14 isolated compounds, , teuvincenone A (48), teuvincenone B (89), teuvincenone F (10), teuvincenone H (88), uncinatone (9) and cyrtophyllone B (21) [C. trichotomum stems, the same authors also identified the diterpenoids villosin B (90) and villosin C (85); these demonstrate remarkable cytotoxic activities against tumour cell lines A549, HepG-2, MCF-7 and 4T1 with IC50 values ranging from 14.93 to 29.74 µM [In further phytochemical studies, air-dried stems of e B (21) . In furt29.74 µM .C. trichotomum roots: 15,16-dehydroteuvincenone G (91), 3-dihydroteuvincenone G (92), 17-hydroxymandarone B (93), trichotomin A (94), 15,16-dihydroformidiol (95), 18-hydroxyteuvincenone E (96), 2α-hydrocaryopincaolide F (97), 15α-hydroxyuncinatone (98), 15α-hydroxyteuvincenone E (99), trichotomin B (100), trichotomside A (101) and B (102) [C. trichotomum roots possess anti-inflammatory properties [(85), 15-dehydro-17-hydroxycyrtophyllone A (16), demethylcryptojaponol, 6β-hydroxydemethylcryptojaponol and trichotomone (78), exhibited IC50 values ranging from 6.0 to 16.1 µM, with 15,16-dehydroteuvincenone G being the most active diterpenoid . It is worth adding that all these active compounds acted at non-cytotoxic concentrations and demonstrated stronger activity than aminoguanidine hydrochloride (IC50 26.2 µM) [Hu et al. (2018) isolated twelve new abietane diterpenoids from cenone E , 2α-hydrcenone E , 2α-hydrcenone E , 2α-hydrClerodendrum bracteatum were the plant materials used for the extraction, isolation and purification to identify two new abietane diterpenes compounds, which are defined as: -12,16-epoxy-17(15→16)-abeo-3,5,8,12-abietatetraen-7,11,14-trione (103) and 11,14,16-trihydroxy-6,12-dimethoxy-17(15→16)-abeo-5,8,11,13- abietatetraen-3,7-dione (104) [abeo-abietane structure, the first of which has p-quinone and p-benzoquinone moieties. In compound 104, four methyls, two ketones at C-3 and C-7, as well as the presence of a methine group at C-16 were detected. According to Li et al. (2021), two newly isolated structures (103 and 104) have the strongest antioxidant and cytotoxic activities against HL-60 and A-549 tumour cell lines among seven isolated diterpenes [Woody branches and healthy stems of ne (104) . The extterpenes .Clerodendrum chinense roots, which resulted in the isolation and identification of 6 new diterpenes: Clerodenoids A–F (105–110) (106–108 are the rearranged abietane diterpenoids sharing a 17(15→16)-abeo-abietane skeleton, while compounds 109–110 are 17(15→16),18(4→3)-diabeo-abietane moieties. Furthermore, compound 110 has a persubstituted Δ3 double bond and methylhydroxyl function in the A ring. All six newly isolated diterpenes were examined towards antiproliferative activities against HL-60 and A-549 human tumour cell lines [110 demonstrating IC50 values at 1.36 and 1.00 µM against HL-60 and A-549 cell lines, respectively [Interesting new data on diterpenes were published in the work of Qi et al. in 2021 . The aut105–110) . All of ll lines . The mosectively .Clerodendrum infortunatum aerial parts were used for extraction and isolation of terpenoid compounds [R,10S,16R)-11,16,19-trihydroxy-ompounds . Among vdiabeo-3,8,11,13-abietatetraene-7-one (111) and -11,16-dihydroxy-12-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-17(15→16),18(4→3)-diabeo-4-carboxy-3,8,11,13-abietatetraene-7-one (112) [111 -11,16,19-trihydroxy-12-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-17(15→16),18(4→3)-diabeo-3,8,11,13-abietatetraene-7-one) inhibited these enzymes activity with an IC50 value of 18.5 and 24.6 µM, respectively. -11,16-dihydroxy-12-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-17(15→16),18(4→3)-diabeo-4-carboxy-3,8,11,13-abietatetraene-7-one demonstrated weaker activity (IC50 = 64.6 and 78.3 µM) [50 of 191 and 139 µM, respectively [12-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-17(15→16),18(4→3)-ne (112) . The ext78.3 µM) . Additioectively .C. inerme [Clerodendrum bungei crolerodendrum A (43) and B (42) for the first time in this species [The diterpenes present in . inerme  were iso species ,22.42 is known for its antioxidant properties [50 value of 17 µM [Compound operties  and exhiof 17 µM .Clerodendrum genus are summarised in The biological activities of the diterpenoids isolated from the Clerodendrum, of the family Lamiaceae, are rich in diterpenoid secondary metabolites, both in the aerial parts and the roots. Due to their moderate, and in some cases strong, biological activities, these diterpenoids are interesting experimental objects. This is particularly true for in vivo pharmacological evaluation. Some of the diterpenes isolated from Clerodendrum spp. are structurally similar to the more highly active phytocompounds; however, they have not been tested for their potential biological activities. This is an important area for further study, as both infectious and civilization diseases, such as cancer, require the search for new therapeutically active structures. The new metabolites obtained from Clerodendrum spp. demonstrate high pharmacological potential, and could be an interesting object of further studies, particularly plant in vitro culture aimed at optimizing the cultivation conditions to increase biomass and secondary metabolite production, especially diterpenes. These biotechnological investigations should determine the effect of culture type  and growth conditions such as basal medium and light wavelength, regardless of climatic conditions, season and environmental pollution.The members of the genus Clerodendrum could be the chemical modification of the isolated phytocompounds. These would include the production of semisynthetic analogues with enhanced biological activities, and improved bioavailability or safety [Another interesting area of research concerning the diterpenes from r safety ,100.Another equally interesting area of research into these diterpenes is biotransformation . Biotran