Evaluation of the effect of background electrolyte composition and independence of parameters in determining binding constants of betulin derivatives to ß- and dimethyl-ß-cyclodextrins by affinity capillary electrophoresis.

The values of the apparent binding constants for ß-cyclodextrin complexes of betulin derivatives determined by mobility shift affinity capillary electrophoresis were found to be independent of the composition of the two background electrolytes used (tetraborate buffer, pH 9.18, and phosphate buffer, pH 8.00, both of them with 20 mM ionic strength). It has been found that if there is not a constant plateau on the binding curve then four independent parameters can be determined: binding constants (also referred to as stability, association, or formation constants) and ionic mobilities of 1:1 and 1:2 complexes. However, at least 10-12 data points in the binding curve should be used to reliably estimate the parameters. For the first time, the apparent binding constants for complexes of ester betulin derivatives with dimethyl-ß-cyclodextrin have been determined by mobility shift affinity capillary electrophoresis. The logarithms of the constants for 1:1 and 1:2 complexes at 25°C for betulin 3,28-diphthalate with a 95% confidence interval are 4.98 (4.95-5.01) and 7.52 (7.26-7.68); for betulin 3,28-disulfate, the values are 4.97 (4.89-5.03) and 8.24 (6.82-8.52). It has been found that betulin 3,28-disuccinate forms only a 1:1 complex and the binding constant logarithm is 5.25 ± 0.02.

Cytotoxic and anti-tumor effects of 3,4-seco-lupane triterpenoids from the leaves of Eleutherococcus sessiliflorus against hepatocellular carcinoma.

A rich of 3,4-seco-lupane triterpenoids (3,4-SLT), including chiisanoside (CSS), divaroside (DVS), sessiloside-A1 (SSA), chiisanogenin (CSG), sessiligenin (SSG), were isolated from the ethanol extract of the leaves of Eleutherococcus sessiliflorus (LES). The present study was performed to explore the cytotoxic and anti-tumor effects of the isolated five ones, as well as potential molecular mechanisms. The results of a CCK-8 assay demonstrated that these 3,4-SLT can inhibit the growth of HepG2 cells, and SSG showed the most significant cytotoxicity. Hoechst 33258 fluorescence staining and Annexin V-FITC/PI staining indicated that 3,4-SLT in LES can induce HepG2 cell apoptosis effectively. The AutoDock Vina program was used to simulate molecular docking of drugs and targets to discuss possible pharmacological mechanisms. Besides, western blot and qRT-PCR results indicated that SSG can inhibit PI3K/AKT signaling pathway through controlling multi-targets. This study suggested that 3,4-SLT might become a new research hotspot for antineoplastic drugs.

Electrophoretic mobility of ester betulin derivatives and their complexation with γ-cyclodextrin studied by capillary electrophoresis in aqueous solutions at different pH values.

In this article, capillary electrophoresis was used to measure the effective electrophoretic mobility of ester betulin derivatives as a pH function and to study their complexation with γ-cyclodextrin (γ-CD). The electrophoretic mobility of betulin 3,28-diphthalate (DPhB) and 3,28-disuccinate (DScB) changed unusually with decreasing pH: instead of decreasing, it first increased and then decreased. This fact as well as the turbidity of sample solutions at pH from 2.5 to 6, broadening of electrophoretic peaks and a decrease in the surface tension of the solutions indicates that these betulin derivatives, being amphiphilic compounds and weak acids, exist as micelles in aqueous solutions at pH 6 and below. The inclusion complexation of betulin derivatives with γ-CD at pH 9.18 and 4.5 was studied by mobility shift affinity capillary electrophoresis. At pH 9.18, the apparent binding (stability) constant logarithms for 1:1 γ-CD complexes of DPhB, betulin 3,28-disulfate (DSB) and DScB with 95% confidence interval limits were equal to 7.44 ± 0.02, 7.09 (7.01-7.19), and 6.97 (6.87-7.08) at 25°C, respectively. At pH 4.5, the binding constant for the DSB complex was slightly lower, while the micelle formation did not allow determining the exact values of the constants for the DPhB and DScB complexes.

Antibacterial and synergistic activity of 6ß-hydroxy-3-oxolup-20(29)-en-28-oic acid (6ß-hydroxy betunolic acid) isolated from Schumacheria castaneifolia vahl.

Multi- drug resistant microbial pathogens are a serious global health problem and thus new antibacterial agents, which are effective both alone and in combination with traditional antibiotics, are urgently needed. Hence, the objective of the present study was to investigate the antibacterial activity of 6ß-hydroxy-3-oxolup-20(29)-en-28-oic acid (6ß-hydroxy betunolic acid) isolated from the bark of Schumacheria castaneifolia and its effect when combined with oxacillin. Antibacterial potential of 6ß-hydroxy betunolic acid alone was performed using broth micro dilution assay against sixteen bacterial strains which included eight standard strains [Staphylococcus aureus (ATCC 29213 and ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 35218 and ATCC 25922), carbapenemase producing Kebsiella pneumonia (ATCC BAA 1705), carbapenemase non-producing K. pneumonia (ATCC BAA 1706) and Pseudomonas aeruginosa (ATCC 27853)] and four strains each of clinically isolated meropenem resistant Acinetobactor sp. and methicillin resistant S. aureus (MRSA) which were included in the urgent threat list and serious threat list, respectively in 2019 by the Centers for Disease Control and Prevention in the United States. Its effect when combined with oxacillin was tested against S. aureus (ATCC 29213) and MRSAs using a checkerboard dilution method. The results indicated that 6ß-hydroxy betunolic acid had antibacterial activity against the tested Gram positive organisms with MICs ranging from 16 to 32 mg L-1 (MIC of oxacillin and meropenem ranged from 0.25-16 and 0.03-128 mg L-1 respectively). The high MIC values (>1024 mg L-1) of 6ß-hydroxy betunolic acid against Gram negative strains indicated a likely lack of activity. Further, 6ß-hydroxy betunolic acid exhibited synergistic effect with oxacillin against Staphylococcus aureus (0.49) and showed an additive effect against all the tested MRSAs. The present study suggested that the antibacterial activity of the 6ß-hydroxy betunolic acid is restricted to Staphylococcus isolates and possibly Enterococcus faecalis. Further testing on different types of Gram positives and identification of the exact mechanism of action would be of importance.

Determining binding constants for 1:1 and 1:2 inclusion complexes of ester betulin derivatives with (2-hydroxypropyl)-ß-cyclodextrin by affinity capillary electrophoresis.

The complexation of ester betulin derivatives with (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD) was studied by mobility shift affinity CE. Electrophoretic mobility for triangular peaks was calculated using the parameter a1 of the Haarhoff-Van der Linde function instead of the peak top time. Dependences of the viscosity corrected electrophoretic mobility on HP-ß-CD concentration were not described on the basis of only complexes with 1:1 stoichiometry due to the fact that these binding curves did not reach a plateau. However, the dependences were well described taking into account both 1:1 and 1:2 complexes. The presence of higher order equilibria was also revealed by x-reciprocal plots. The values of apparent binding constant logarithm, obtained for the first time, for 1:1 and 1:2 HP-ß-CD complexes of betulin 3,28-diphthalate and betulin 3,28-disuccinate with 95% confidence interval limits in brackets are the same within error and are equal to 4.85 (4.73-4.95), 8.56 (7.75-8.82), 4.92 (4.86-4.97), and 8.54 (8.23-8.72) at 25°C, respectively. These values for 1:1 and 1:2 HP-ß-CD complexes of betulin 3,28-disulfate at 25°C are 4.61 (4.57-4.64) and 7.11 (6.57-7.34), respectively. The binding constants for betulin 3,28-disulfate agree with the previously obtained results from the separation in the thermostatted capillary segment.

Potential Myocardial Protection of 3,4-seco-Lupane Triterpenoids from Acanthopanax sessiliflorus Leaves.

A rich of 3,4-seco-lupane triterpenoids including chiisanoside (CSS), divaroside (DVS), sessiloside-A1 (SSA) and chiisanogenin (CSG) were isolated from the ethanol extract of the leaves of Acanthopanax sessiliflorus. On the basis of previous studies, this article focused on four important components of 3,4-seco-lupane triterpenoids in Acanthopanax sessiliflorus leaves and explored their protective effects against aconitine-induced cardiomyocyte injury and their molecular mechanisms. The results showed that pretreatment with 3,4-seco-lupane triterpenoids could effectively increase cell viability, reduce CK-MB and LDH activities, reduce ROS production, maintain calcium concentration balance, and inhibit apoptosis, with divaroside having the best effect. In addition, Western blot results showed that divaroside down-regulated Cleaved caspase-3 and Bax and up-regulated Bcl-2 expression through activating the PI3 K/AKT pathway. However, the LY294002 inhibitor reversed this situation. This suggests that 3,4-seco-lupane triterpenoids may be a new hotspot for potential myocardial protective drugs research.

Evaluation of Cytotoxicity and α-Glucosidase Inhibitory Activity of Amide and Polyamino-Derivatives of Lupane Triterpenoids.

A series of two new and twenty earlier synthesized branched extra-amino-triterpenoids obtained by the direct coupling of betulinic/betulonic acids with polymethylenpolyamines, or by the cyanoethylation of lupane type alcohols, oximes, amines, and amides with the following reduction were evaluated for cytotoxicity toward the NCI-60 cancer cell line panel, α-glucosidase inhibitory, and antimicrobial activities. Lupane carboxamides, conjugates with diaminopropane, triethylenetetramine, and branched C3-cyanoethylated polyamine methyl betulonate showed high cytotoxic activity against most of the tested cancer cell lines with GI50 that ranged from 1.09 to 54.40 µM. Betulonic acid C28-conjugate with triethylenetetramine and C3,C28-bis-aminopropoxy-betulin were found to be potent micromolar inhibitors of yeast α-glucosidase and to simultaneously inhibit the endosomal reticulum α-glucosidase, rendering them as potentially capable to suppress tumor invasiveness and neovascularization, in addition to the direct cytotoxicity. Plausible mechanisms of cytotoxic action and underlying disrupted molecular pathways were elucidated with CellMinner pattern analysis and Gene Ontology enrichment analysis, according to which the lead compounds exert multi-target antiproliferative activity associated with oxidative stress induction and chromatin structure alteration. The betulonic acid diethylentriamine conjugate showed partial activity against methicillin-resistant S. aureus and the fungi C. neoformans. These results show that triterpenic polyamines, being analogs of steroidal squalamine and trodusquemine, are important substances for the search of new drugs with anticancer, antidiabetic, and antimicrobial activities.

The New Pharmaceutical Compositions of Zinc Oxide Nanoparticles and Triterpenoids for the Burn Treatment.

We studied oleogels containing zinc oxide nanoparticles (ZnO NPs) and lupane triterpenoids in sunflower oil for the treatment of burns. The modification of ZnO was carried out by treatment with alcohol solutions of betulin, betulonic acid, betulin diacetate and betulin diphosphate. The properties of modified ZnO NPs were studied by powder XRD (average sizes of 10-20 nm), FTIR (νZnO 450 cm-1), UV-vis (345-360 nm), and blue-violet emission (380-420 nm). The identification and assay of modified ZnO NPs and triterpenoids were estimated. The treatment by oleogels of deep II-degree burns was studied on rats using histological studies, Doppler flowmetry and evaluation of enzymes activity and malonic dialdehyde (MDA) level. After the action of oleogels, burn wound area, and the necrosis decreased twice on the 10th day in comparison with the 1st day after burn. The microcirculation index in the near-wound zone by 20-30% improved compared with the group without treatment. Evaluation of the enzyme activity and the MDA level after treatment by oleogels during the course of 10 days showed them returning to normal. The improvement of antioxidant biochemical indexes, as well as wounds' healing, was mainly determined by the influence of zinc oxide nanoparticles.

Anti-inflammatory Activities of Lupane-triterpenoids In Vitro and Their Phytochemical Fingerprinting from Leaves of Acanthopanax gracilistylus

The activities on the inhibition of NO on LPS-induced RAW 264.7 macrophages were investigated in this work. A simple and sensitive method has been developed and validated for fingerprinting analysis of leaves of Acanthopanax gracilistylus W.W. Smith (AGS). The cytotoxicity and inhibition of NO on LPS-induced RAW 264.7 cells of the extract and triterpenoids were determined. Optimal conditions of HPLC analysis were established as follows. The separation was performed with an ODS-C18 column at 30 degrees C, the detected wavelength was 210 nm, the flow rate was 1 mL/min, and the mobile phase consisted of acetonitrile (0.05% phosphoric acid) -0.05% phosphoric acid solution with gradient elution. Our results showed that impressic acid and acankoreaogenin was more effective on the inhibition of NO than the methanol extract and other compounds. There were seventeen peaks coexisted with similarities above 0.95 and nine lupane-triterpenoids including acankoreaogenin and impressic acid detected and identified. The result of anti-inflammatory activities provides a potential explanation for the use of AGS leaves as a herbal medicine in the treatment of inflammatory diseases. Our results also show that acankoreanogenin and impressic acid may be potentially useful in developing new anti-inflammatory agents. In addition, the fingerprint chromatography clearly illustrated and confirmed the material basis for the anti-inflammatory activities of this plant.

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death.

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.

Chemical constituents in leaves of Acanthopanax gracilistylus / 中草药

Objective To study chemical constituents in the leaves of Acanthopanax gracilistylus.Methods The chemical components were isolated and purified by silica gel,ODS C-18,and Sephadex LH-20 column chromatogram.The chemical structures were elucidated on the basis of physicochemical properties and spectral data.Results Seventeen compounds were isolated and identified as(-)-kaur-16-en-19-oic acid(Ⅰ),quercetin(Ⅱ),kaempferol(Ⅲ),protocatechuic acid(Ⅳ),acankoreoside A(Ⅴ),acantrifoside A(Ⅵ),3?,11?-dihydroxy-20(29)-lupene-23,28-dioic acid(Ⅶ),?-sitosterol(Ⅷ),daucosterol(Ⅸ),palmitic acid(Ⅹ),rutin(Ⅺ),stigmast-5,22-dien-3-O-?-D-glucopyranoside(ⅩⅡ),acankoreagenin(ⅩⅢ),3,11-dihydroxy-23-oxo-20(29)-lupen-28-oic acid(ⅩⅣ),3-hydroxy-23-oxo-20(29)-lupen-28-oic acid(ⅩⅤ),myristin(ⅩⅥ),and acanthopanaxgric acid(ⅩⅦ).Conclusion Compounds Ⅱ-Ⅳ,Ⅶ,Ⅺ,ⅩⅡ,ⅩⅣ,and ⅩⅤ are obtained from the leaves of the plant for the first time and compounds ⅩⅦ is a new proved compound named acanthopanaxgric acid.