23-hydroxybetulinic Acid Research Articles

Transcription combined metabolic analysis reveals the mechanism of potassium phosphate enhances resistance to walnut anthracnose (Colletotrichum gloeosporioides)

Given the increasingly serious diseases [anthracnose mainly caused by Colletotrichum gloeosporioides (C. gloeosporioides)] of walnut plants in recent years and the environmental pollution caused by chemical pesticides, phosphites had been shown to be new environmental friendly chemical agents for plants. In this study, seven-year-old ‘Xiangling’ and ‘Xifu 4’ walnut trees were treated with different concentrations of potassium phosphite foliar fertilizer. The treatments were T1 (1500-fold dilution fluid of potassium phosphite), T2 (1000-fold dilution fluid of potassium phosphite) and CK (water). The results showed that the disease incidence rates of fruit treated with potassium phosphite were lower than CK. Moreover, the diseased fruit rates of ‘Xiangling’ (4) and ‘Xifu 4’ (14) in T2, lower than they (28 and 38) in CK. Metabolome and transcriptome further analysis revealed that potassium phosphite significantly increased the expression level of genes in the photosynthesis pathway and accumulated terpeniods, phenolic, salicylic acid and other secondary metabolic pathways. In addition, as salicylic acid content significantly increased, the expression level of the gene encoding the disease resistance-related protein (PR1) showed a corresponding trend. In vitro antibacterial tests of six related metabolites on C. gloeosporioides showed that hederagenin, 23-hydroxybetulinic acid, ursolic acid, eriodictyol, and salicylic acid had effect on C. gloeosporioides spores, among which salicylic acid and ursolic acid significantly inhibited the growth of C. gloeosporioides. Further experiments revealed that the minimum inhibitory concentrations of salicylic acid and ursolic acid were 2 and 5 g L−1, respectively. In addition, potassium phosphite could directly inhibit the growth of C. gloeosporioides spores in vitro tests. In conclusion, potassium phosphite enhanced walnut fruit disease resistance through up-regulated the genes related to photosynthetic performance and abundant in secondary metabolites, such as salicylic acid, terpenoids and phenolics, which were antibacterial effects on C. gloeosporioides.

23-Hydroxybetulinic acid attenuates 5-fluorouracil resistance of colorectal cancer by modulating M2 macrophage polarization via STAT6 signaling

Macrophage polarization is closely associated with the inflammatory processes involved in the development and chemoresistance of colorectal cancer (CRC). M2 macrophages, the predominant subtype of tumor-associated macrophages (TAMs) in a wide variety of malignancies, have been demonstrated to promote the resistance of CRC to multiple chemotherapeutic drugs, such as 5-fluorouracil (5-FU). In our study, we investigated the potential of 23-Hydroxybetulinic Acid (23-HBA), a significant active component of Pulsatilla chinensis (P. chinensis), to inhibit the polarization of M2 macrophages induced by IL-4. Our results showed that 23-HBA reduced the expression of M2 specific marker CD206, while downregulating the mRNA levels of M2 related genes (CD206, Arg1, IL-10, and CCL2). Additionally, 23-HBA effectively attenuated the inhibitory effects of the conditioned medium from M2 macrophages on apoptosis in colorectal cancer SW480 cells. Mechanistically, 23-HBA prevented the phosphorylation and nuclear translocation of the STAT6 protein, resulting in the inhibition of IL-10 release in M2 macrophages. Moreover, it interfered with the activation of the IL-10/STAT3/Bcl-2 signaling pathway in SW480 cells, ultimately reducing M2 macrophage-induced resistance to 5-FU. Importantly, depleting STAT6 expression in macrophages abolished the suppressive effect of 23-HBA on M2 macrophage polarization, while also eliminating its ability to decrease M2 macrophage-induced 5-FU resistance in cancer cells. Furthermore, 23-HBA significantly diminished the proportion of M2 macrophages in the tumor tissues of colorectal cancer mice, simultaneously enhancing the anti-cancer efficacy of 5-FU. The findings presented in this study highlight the capacity of 23-HBA to inhibit M2 macrophage polarization, a process that contributes to reduced 5-FU resistance in colorectal cancer.

Pharmacokinetics and metabolite identification of 23-hydroxybetulinic acid in rats by using liquid chromatography-mass spectrometry method

23-hydroxybetulinic acid (23-HA), a main bioactive component isolated from Pulsatilla chinensis (Bunge) Regel, exhibits various pharmacological activities, such as antimelanoma, antileukemia, anti-colon cancer, and antihepatotoxicity. Although the main active ingredient anemoside B4 (AB4) from this plant has been well studied, research on its active metabolite 23-HA is limited. In the present study, a validated HPLC-QQQ-MS/MS method was established for the quantification of 23-HA in rat plasma. Pharmacokinetics analysis showed that the absorption and elimination of 23-HA in rats were rapid, with an oral bioavailability as 12.9 %. After oral administration with 50 mg/kg 23-HA for SD rats, the plasma, urine, feces, and bile samples were collected and analyzed by UPLC-Q Exactive Plus MS and HPLC-QQQ-MS/MS. Seventeen metabolites of 23-HA were identified, and its major metabolic pathways included oxidation, hydration, sulfation, and glucuronidation. This study highlights the first detailed investigation of 23-HA’s pharmacokinetics in rats along with its metabolism in vivo, and will provide robust evidence for further research and clinical application of 23-HA.

LC-MS guided isolation of cytotoxic saponins from the flower buds of Lonicera macranthoides

Two undescribed triterpenoid saponins (1 and 3) along with six known saponins (2, 4-8) were isolated from the flower buds of Lonicera macranthoides by an LC-MS guided fractionation method. Natural products 1, 3, 5 and 7 are the monodesmosidic saponins of ambradiolic acid, 23-hydroxybetulinic acid, hederagenin and oleanolic acid, respectively, sharing the same single sugar chain at C-3 of each aglycone. Furthermore, compounds 2, 4, 6 and 8 are their bisdesmosidic saponins with a further identical sugar chain at C-28 of the aglycone. All structures were identified by interpretation of their spectroscopic data. Compounds 1-8 were evaluated for in vitro cytotoxicity against the HeLa, HTC-116, B16 and MDA-MB-468 cell lines. Results showed that all monodesmosidic saponins exhibited moderate activity with compound 7 showing the most pronounced cytotoxicity against the four human cancer cell lines, with IC50 values of 1.9–13.5 μM. The preliminary structure-activity relationships of the natural products are also discussed.

Pharmacokinetic and metabolite profile of orally administered anemoside B4 in rats with an improved exposure in formulations of rectal suppository

Ethnopharmacological relevancePulsatilla chinensis (Bunge) Regel is a traditional Chinese herbal medicine used to treat intestinal amebiasis, malaria, vaginal trichomoniasis, and bacterial infections. Anemoside B4 (AB4), a pentacyclic triterpenoid saponin, is one of the primary bioactive substances in Pulsatilla chinensis (Bunge) Regel, and gavage administration of AB4 to animals has been demonstrated to exhibit anticancer, anti-inflammatory, and antiviral actions. However, AB4 exposure in plasma is very low after oral administration, and the biotransformation of AB4 in vivo after oral administration remains unknown. Aim of the studyThe reason for conducting this research was to explore at the metabolite profile of AB4 in rats following oral administration. Additionally, we aimed to develop an appropriate extravascular formulation to increase the exposure and duration of AB4 in vivo. Materials and methodsA well-validated HPLC-QQQ-MS/MS method was used for the quantification of AB4 in plasma and was further applied to evaluate and compare the pharmacokinetic properties of AB4 dissolved in a saline solution and AB4 formulations in a rectal suppository or enteric capsule. Reliable UHPLC coupled to Q-Exactive Plus high-resolution MS was used to identify the metabolites in rat plasma, bile, urine, and faeces. ResultsAB4 was extensively metabolized, and a total of 29 metabolites were identified. The primary metabolic routes included deglycosylation, oxidation, dehydrogenation, reduction, sulfation, hydration, acetylation, and glucuronidation. The pharmacokinetic comparison showed that both the rectal suppository and enteric capsule increased the exposures of AB4 and one of its active metabolites, 23-hydroxybetulinic acid (23-HA). Notably, rectal suppositories increased systemic AB4 exposure (AUC0-∞) by approximately 49 and 28 times higher than that of the AB4 saline solution and enteric capsules, respectively. The t1/2 of AB4 was extended to approximately 7 h after rectal administration compared to 2 h after oral administration. ConclusionOverall, our study demonstrated that the mismatched exposure-response relationship of AB4 could result from extensive metabolism in the gastrointestinal and circulatory systems. Thus, a rectal suppository could be an alternative formulation of AB4 to obtain both higher and longer exposure.

23-Hydroxybetulinic Acid, A Natural Compound, Alleviates DSS-induced Colitis by Regulating NF-κB Signaling

Article 23-Hydroxybetulinic Acid, A Natural Compound, Alleviates DSS-induced Colitis by Regulating NF-κB Signaling Shuangli Xiang 1, # , Miaojuan Wang 2, # , and Xiuping Chen 2, * 1 Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou Province, China. 2 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao, China. * Correspondence: xpchen@um.edu.mo, Tel.: +853-88224679, Fax: +853-28841358 # Co-First author. Received: 8 November 2022 Accepted: 2 December 2022 Published: 11 January 2023 Abstract: Ulcerative colitis (UC), an inflammatory intestinal disease, is a growing epidemic affecting people worldwide and requires the development of effective therapeutic drugs. In this study, the effect of 23-hydroxybetulinic acid (23-HBA), a compound isolated from the traditional herb Pulsatilla chinensis (Bunge) Regel, on experimental UC was studied. C57BL/6J male mice were administrated with 3% dextran sodium sulfate (DSS) in drinking water to establish the UC model. 23-HBA was orally administrated at either 3.75, 7.5, or 15 mg/kg for 6 days. Mesalazine was used as a positive control. Examination of the body weight, colon length, disease activity index (DAI), histopathology examination, inflammatory cytokines, oxidative stress, and protein expression was performed. The pathological changes were examined with hematoxylin and eosin (H&E) and Aixian blue-glycogen (AB-PAS) staining. In cultured RAW 264.7 cells, the effects of 23-HBA on lipopolysaccharide (LPS)-stimulated cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and oxidative stress were analyzed. Compared with the colitis model, 23-HBA treatment significantly increased the body weight and colon length and decreased the DAI score. Pathological staining showed that 23-HBA mitigated the damage in intestinal structures, the increase in inflammatory cell infiltration, the increase in submucosa edema, and the decrease in goblet cell number. Furthermore, 23-HBA decreased IL-1β, IL-6, and MDA levels in the colon tissues. In addition, 23-HBA inhibited the protein expressions of COX-2, iNOS, and NF-κB p65 both in the colon tissues and in LPS-stimulated RAW 264.7 cells. In conclusion, these results showed that 23-HBA alleviated DSS-induced acute UC in mice and inhibited LPS-stimulated inflammation in RAW 264.7 cells possibly mediated by regulating the NF-κB pathway.

23-hydroxybetulinic acid reduces tumorigenesis, metastasis and immunosuppression in a mouse model of hepatocellular carcinoma via disruption of the MAPK signaling pathway.

Hepatocellular carcinoma (HCC) shows recurrence and lung metastasis even after treatment. 23-hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis, exhibits potent antitumor activities. We herein investigate the biological effect of 23-HBA on metastasis and immunosuppression in a mouse model of HCC. Microarray-based gene expression profiling was employed to identify the target genes of 23-HBA in the treatment of HCC. The effect of 23-HBA on the progression of HCC was evaluated by in-vitro cell function measurements along with in-vivo xenograft implantation, lung metastasis and CD11b+Gr1+ staining experiments. The potential mechanism involving target signaling pathway was investigated by western blot analysis. Bioinformatics analysis revealed that matrix metalloproteinase 2 (MMP2) was a key target gene mediated by 23-HBA in HCC, whereas Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that MMP2 mainly affects the development and metastasis of HCC. 23-HBA significantly reduced cell malignant functions in vitro while delaying the HCC growth and metastasis in vivo. In addition, the number of myeloid-derived suppressor cells was shown to be reduced following administration of 23-HBA in mice. Mechanistic analysis indicated that these effects of 23-HBA during HCC were involved with the mitogen-activated protein kinase (MAPK) signaling pathway inactivation and resulted in decreased phosphorylation of both mitogen-activated protein kinases 1/2 and extracellular signal-regulated kinase 1/2. Our study reveals that 23-HBA acts as a tumor suppressor agent and suppresses HCC tumorigenesis, metastasis and immunosuppression via blockade of the MAPK signaling pathway, suggesting that 23-HBA may serve as a promising drug target to treat HCC.

Design and synthesis of NAD(P)H: Quinone oxidoreductase (NQO1)-activated prodrugs of 23-hydroxybetulinic acid with enhanced antitumor properties

A series of NQO1 selectively activated prodrugs were designed and synthesized by introducing indolequinone moiety to the C-3, C-23 or C-28 position of 23-hydroxybetulinic acid (23-HBA) and its analogues. Among them, the representative compound 32j exhibited significant antiproliferative activities against NQO1-overexpressing HT-29 cells and A549 cells, with IC50 values of 1.87 and 2.36 μM, respectively, which were 20–30-fold more potent than those of parent compound 23-HBA. More importantly, it was demonstrated in the in vivo antitumor experiment that 32j effectively suppressed the tumor volume and largely reduced tumor weight by 72.69% with no apparent toxicity, which was more potent than the positive control 5-fluorouracil. This is the first breakthrough in the improvement of in vivo antitumor activities of 23-HBA derivatives. The further molecular mechanism study revealed that 32j blocked cell cycle arrest at G2/M phase, induced cell apoptosis, depolarized mitochondria and elevated the intracellular ROS levels in a dose-dependent manner. Western blot analysis indicated that 32j induced cell apoptosis by interfering with the expression of apoptosis-related proteins. These findings suggest that compound 32j could be considered as a potent antitumor prodrug candidate which deserves to be further investigated for personalized cancer therapy.

Synthesis, biological evaluation and mechanism studies of C-3 substituted nitrogenous heterocyclic 23-Hydroxybetulinic acid derivatives as anticancer agents

A series of novel nitrogenous heterocycle substituted 23-Hydroxybetulinic acid (23-HBA) derivatives with amide linkages at the C-3 position were designed, synthesized and evaluated for their antitumor activities. The biological screening results showed that most of the derivatives exhibited more potent antiproliferative activities than 23-HBA. In particular compound II-9 exhibited the most potent activities with IC50 values ranging from 1.96 μM to 6.20 μM against five cancer cell lines (B16, HepG2, A2780, MCF-7 and A549). The preliminary mechanism study showed that compound II-9 caused cell cycle arrest at G1 phase, induced cell apoptosis and depolarized mitochondria of B16 cells in a dose dependent manner. Moreover, western blot analysis indicated that compound II-9 down-regulated the expression of anti-apoptotic protein Bcl-2, up-regulated the expression of pro-apoptotic protein Bad, and activated cytochrome C and caspase 3 to cause cell apoptosis. In summary, II-9 may serve as a promising lead for the development of new natural product-based antitumor agents and deserve further investigation.

Synthesis, Biological Evaluation and Mechanism Studies of C-3 Substituted Nitrogenous Heterocyclic 23-Hydroxybetulinic Acid Derivatives as Anticancer Agents

Synthesis, Biological Evaluation and Mechanism Studies of C-3 Substituted Nitrogenous Heterocyclic 23-Hydroxybetulinic Acid Derivatives as Anticancer Agents

Involvement of P-gp on Reversing Multidrug Resistance Effects of 23-Hydroxybetulinic Acid on Chemotherapeutic Agents.

Betulinic acid (BA) and 23-Hydroxybetulinic acid (23-HBA) are natural products with similar structures, which show a range of biological effects including cytotoxicity activity. The aim of current research was to investigate and evaluate the combinational cytotoxicity of BA and 23-HBA with chemotherapeutic agents in vitro, and to clarify the potential interaction and related mechanism with P-gp. Instead of BA, 23-HBA could increase cytotoxicity of MCF-7/ADR cells to adriamaycin (ADR) and vincristine (VCR). The intracellular accumulation of ADR/VCR in MCF-7/ADR cells was obviously increased in the presence of 23-HBA. Furthermore, 23-HBA could show dose-dependent increase on the transport of VCR and digoxin, which are typical P-gp substrates, in both MDCK-MDR1 and Caco-2 cells. However, the transport of BA and 23-HBA was not influenced by P-gp inhibition in MDCK-MDR1 cells. MDR1 shift assay and molecular docking model suggested that both compounds showed interaction with P-gp, yet the binding affinity and sites are different. In conclusion, 23-HBA could strongly improve the efficacy of anti-tumor agents in multidrug resistance (MDR) cells, which was related to P-gp inhibition. The MDR1 shift assay and molecular docking study further revealed that 23-HBA and BA showed different interaction modes with P-gp.

Acankoreagenin and acankoreosides, a family of lupane triterpenoids with anti-inflammatory properties: an overview.

Acankoreagenin (ACK, also known as acankoreanogenin and HLEDA) and impressic acid are two lupane-type triterpenes that can be isolated from various Acanthopanax and Schefflera species. They efficiently block activation of the NF-κB signaling pathway and the release of proinflammatory cytokines and/or the action of inflammation mediators (HMGB1, iNOS, and NO). These effects are the basis for the antiviral and anticancer activities reported with these pentacyclic compounds or their various glycoside derivatives. More than 15 acankoreosides (Ack-A to -O, and -R) and a few other mono- and bidesmosidic saponins (acantrifoside A and acangraciliside S) derive from the ACK aglycone. Compounds like Ack-A and -B are remarkable anti-inflammatory agents, inhibiting cytokine release from activated macrophages. Despite their effectiveness, ACK and impressic acid are far much less known and studied than the structurally related compounds betulinic acid and 23-hydroxybetulinic acid (anemosapogenin). The structural differences (notably the R/S stereoisomerism of the 3-hydroxyl group) and functional similarities of these compounds are discussed. The complete series of acankoreosides is presented for the first time. These natural products deserve further attention as anti-inflammatory agents, and ACK is recommended as a template for the design of new anticancer and antiviral drugs.

Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth

Epidermal growth factor receptor (EGFR) activation plays a pivotal role in EGFR-driven non-small cell lung cancer (NSCLC) and is considered as a key target of molecular targeted therapy. EGFR tyrosine kinase inhibitors (TKIs) have been canonically used in NSCLC treatment. However, prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs. Therefore, the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance. Here, we identified a 23-hydroxybetulinic acid derivative, namely DPBA, as a novel EGFR small-molecule ligand. It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR. Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.

Identification of cytochrome P450 isoenzymes involved in the metabolism of 23-hydroxybetulinic acid in human liver microsomes

Context 23-Hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), exhibits potential antitumor activity. Its metabolism, however, has not yet been studied. Objective This study focuses on the metabolism of 23-HBA in vitro by human liver microsomes. Materials and methods The metabolic kinetics of 23-HBA (0.5–100 µM) and the effects of selective CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) inhibitors on metabolism of 23-HBA were evaluated in human liver microsomes incubation system and then determined by LC-MS method. The Michaelis–Menten parameters Km and Vmax were initially estimated by analysing Lineweaver–Burk plot. The clearance (CLint ) was also calculated. Results The Vmax, Km, and CLint of 23-HBA were 256.41 ± 11.20 pmol/min/mg, 11.10 ± 1.07 μM, and 23.10 ± 1.32 μL/min/mg, respectively. The metabolism of 23-HBA was significantly inhibited by furafylline (0.05 μM, p < 0.01) and ketoconazole (0.02 μM, p < 0.05). Ticlopidine (1.3 μM, p < 0.05) could inhibit the metabolism of 23-HBA, while the other inhibitors (sulfaphenazole and quinidine) showed nonsignificant inhibition on the metabolism of 23-HBA. Discussion and conclusions This is the first investigation of the metabolism of 23-HBA in human liver microsomes. The in vitro study indicates that CYP1A2 and CYP3A4 are mainly involved in the metabolism of 23-HBA. Special attention should be given to the pharmacokinetic and clinical outcomes when 23-HBA was co-administrated with other compounds mainly undergoing CYP1A2/CYP3A4-mediated metabolism. Further studies are needed to evaluate the significance of this interaction and strengthen the understanding of traditional Chinese medicine.

Synthesis, biological evaluation and mechanism studies of C-23 modified 23-hydroxybetulinic acid derivatives as anticancer agents

A series of C-23 modified 23-hydroxybetulinic acid (HBA) derivatives were synthesized and evaluated for their antiproliferative activity against a panel of cancer cell lines (A2780, A375, B16, MCF-7 and HepG2). The biological screening results showed that most of the derivatives exhibited more potent antiproliferative activity than HBA, and compound 6e exhibited the most potent activity with IC50 values of 2.14 μM, 2.89 μM, and 3.97 μM against A2780, B16, and MCF-7 cells, respectively. Further anticancer mechanism studies revealed that compound 6e induced the generation of intracellular reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) of B16 cells in a dose-dependent manner. Moreover, western blot analysis indicated that compound 6e downregulated the expression of anti-apoptotic protein Bcl-2 and upregulated the expression of proapoptotic protein Bax, activation of caspase 3 to induce cell apoptosis. Meanwhile, compound 6e significantly inhibited the phosphorylation of MEK, ERK, and Akt without affecting the expression of MEK, ERK, and Akt. Furthermore, the in vivo anti-tumor activity of 6e was validated (tumor inhibitory ratio of 68.4% at the dose of 30 mg/kg) in mice with B16 melanoma.

Synthesis, Biological Evaluation of Fluorescent 23-Hydroxybetulinic Acid Probes, and Their Cellular Localization Studies.

23-Hydroxybetulinic acid (23-HBA) is a complex lupane triterpenoid, which has attracted increasing attention as an anticancer agent. However, its detailed mechanism of anticancer action remains elusive so far. To reveal its anticancer mode of action, a series of fluorescent 23-HBA derivatives conjugated with coumarin dyes were designed, synthesized, and evaluated for their antiproliferative activities. Subcellular localization and uptake profile studies of representative fluorescent 23-HBA probe 26c were performed in B16F10 cells, and the results suggested that probe 26c was rapidly taken up into B10F10 cells in a dose-dependent manner and mitochondrion was the main site of its accumulation. Further mode of action studies implied that the mitochondrial pathway was involved in 23-HBA-mediated apoptosis. Together, our results provided new clues for revealing the molecular mechanism of natural product 23-HBA for its further development into an antitumor agent.

Simultaneous determination of eight bioactive compounds by LC-MS/MS and its application to the pharmacokinetics, liver first-pass effect, liver and brain distribution of orally administrated Gouteng-Baitouweng (GB) in rats

Only focusing on the circulating levels is insufficient for the comprehensive understanding of the physiological disposition of herbal medicine in vivo. Therefore, we conducted the comprehensive investigation on the in vivo dynamic process of orally administrated Gouteng-Baitouweng (GB), a classical herb pair with anti-Parkinson potentials. Serving as the technical base, a sensitive and selective liquid chromatography–tandem mass spectrometry method was established and validated in the plasma, liver and brain, for simultaneous determination of five alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine and geissoschizine methyl ether) and three saponins (anemoside B4, anemoside A3 and 23-hydroxybetulinic acid). Following liquid-liquid extraction, favorable chromatographic behaviors of eight analytes were obtained on Waters Xbrigde C18 column within 13 min. This method elicited good linearity for the analytes at the concentration range of 0.3–1000 or 1.8–6000 ng/mL with favorable precision, accuracy and stability. Following oral administration of GB (25 g/kg) in rats, this method was applied to the quantitative analysis in the portal vein plasma, liver, systemic plasma, and brain. Consequently, anemoside B4 was of the highest exposure, followed by 23-hydroxybetulinic acid, anemoside A3, rhynchophylline and isocorynoxeine in vivo. Notably, three saponins were all observed with certain exposure in the brain, along with rhynchophylline at low levels. Besides, five alkaloids and 23-hydroxybetulinic acid underwent serious liver first-pass effect. Hence, the pharmacokinetics, liver first-pass effect, liver and brain distribution of ingredients in GB were clarified, which laid a solid foundation for interpreting its efficacy and safety.

Triterpenoid saponins from the buds of Lonicera similis

Four new lupane triterpenoid saponins, along with one known lupane and eight hederagenin saponins, were isolated from the EtOH extract of the buds of Lonicera similis Hemsl. The structures of the new compounds were established as 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl 23-hydroxybetulinic acid 28-O-β-D-glucopyranosyl ester (lonisimilioside A, 1), 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl 23-hydroxybetulinic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (lonisimilioside B, 2), 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl betulinic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (lonisimilioside C, 3) and 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl betulinic acid 28-O-β-D-glucopyranosyl ester (lonisimilioside D, 4), respectively. The cytotoxic activities of the isolates against human cancer cell lines HepG2, MCF-7 and A-549 were evaluated. Only the monodesmosidic saponin with a free carboxyl group at C-28 (12) exhibited significant cytotoxicities against HepG2, MCF-7 and A-549 cell lines with the IC50 values of 8.98 ± 0.19, 12.48 ± 0.45 and 11.62 ± 0.54 μM, respectively. Furthermore, Hoechst fluorescence 33342 staining was used to demonstrate that 12 could induce HepG2 and A-549 cells apoptosis significantly.

A sensitive HPLC-MS/MS method for the simultaneous determination of anemoside B4, anemoside A3 and 23-hydroxybetulinic acid: Application to the pharmacokinetics and liver distribution of Pulsatilla chinensis saponins.

Pulsatilla chinensis saponins, the major active components in the herb, have drawn great attention as potential hepatitis B virus infection and hepatoma treatments. Here, a sensitive and accurate HPLC-MS/MS method was established for simultaneous determination of three saponins - anemoside B4, anemoside A3 and 23-hydroxybetulinic acid - in rat plasma and liver, and fully validated. The method was successfully applied to a pharmacokinetics and liver distribution study of P. chinensis saponins. Consequently, 23-hydroxybetulinic acid, with an extremely low content in the P. chinensis saponins, exhibited the highest exposure in the liver and in sites before and after hepatic disposition, namely, in the portal vein plasma and systemic plasma, followed by anemoside B4, which showed the highest content in the herb, whereas anemoside A3 displayed quite limited exposure. The hepatic first-pass effects were 71% for 23-hydroxybetulinic acid, 27% for anemoside B4 and 37% for anemoside A3, corresponding to their different extents of liver distribution. To our knowledge, this is the first investigation on the liver first-pass effect and distribution of P. chinensis saponins to date. These results also provide valuable information for the understanding of the pharmacological effect of P. chinensis saponins on liver diseases.

A piperazidine derivative of 23-hydroxy betulinic acid induces a mitochondria-derived ROS burst to trigger apoptotic cell death in hepatocellular carcinoma cells.

BackgroundElevated production of reactive oxygen species (ROS) and an altered redox state have frequently been observed in hepatocellular carcinoma (HCC); therefore, selective killing of HCC cells by chemotherapeutic agents that stimulate ROS generation or impair antioxidant systems may be a feasible approach in HCC chemotherapy. Recently, betulinic acid and its derivatives have attracted attention because they showed anti-cancer effects via a ROS- and mitochondria-related mechanism. However, the source of ROS overproduction and the role of mitochondria were poorly identified, and the weak in vivo antitumour activity of these compounds limits their development as drugs.MethodsCytotoxicity was detected using MTT assays. In vivo anti-HCC effects were assessed using nude mice bearing HepG2 tumour xenografts. Cell cycle analysis, apoptosis rate and mitochondrial membrane potential were measured by flow cytometry. ROS production was detected using a microplate reader or a fluorescence microscope. Changes in gene and protein levels were measured by RT-PCR and western blotting, respectively. Other assays were performed using related detection kits.ResultsB5G9, a piperazidine derivative of 23-hydroxy betulinic acid (23-HBA), showed excellent in vivo anti-HCC effects, with a tumour growth inhibitory rate of greater than 80%, and no significant side effects. B5G9 stimulated the production of ROS, which were derived from the mitochondria, but it had no effect on various other antioxidant systems. Moreover, B5G9 induced mitochondrial dysfunction, which was characterized by morphological changes, membrane potential collapse, membrane permeabilization, and decreases in the O2 consumption rate and ATP production. Furthermore, mtDNA-depleted ρ0 HepG2 cells were less sensitive to B5G9 treatment than wt HepG2 cells, indicating the importance of mitochondria in B5G9-induced cell death.ConclusionWe discovered a piperazidine derivative of 23-HBA, B5G9, with excellent anti-HCC effects both in vivo and in vitro and no obvious toxic effects. The underlying mechanism was associated with mitochondria-derived ROS overproduction, and mitochondria played essential roles in B5G9-induced cell death. This study identified a potential agent for anti-HCC therapy and elucidated the mitochondria-related mechanism of BA and its derivatives.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0457-1) contains supplementary material, which is available to authorized users.