Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix.

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.

Purification of triterpenoid saponins and 25R/25S-inokosterone from Achyranthes bidentata Bl. by high-speed countercurrent chromatography coupled silver nitrate coordination.

An effective method by high-speed countercurrent chromatography coordinated with silver nitrate for the preparative separation of sterones and triterpenoid saponins from Achyranthes bidentata Blume was developed. Methyl tert-butyl ether/n-butanol/acetonitrile/water (4:2:3:8, v/v/v/v) was selected for 20-hydroxyecdysone (compound 1), chikusetsusaponin IVa methyl ester (compound 4), 2'-glycan-11-keto-pigmented saponin V (compound 5), as well as a pair of isomers of 25S-inokosterone (compound 2) and 25R-inokosterone (compound 3), which were further purified by silver nitrate coordinated high-speed countercurrent chromatography. What is more, dichloromethane/methanol/isopropanol/water (6:6:1:4, v/v/v/v) was applied for calenduloside E (compound 6), 3ß-[(O-ß-d-glucuronopyranosyl)-oxy]-oleana-11,13-dien-28-oic acid (compound 7), zingibroside R1 (compound 8) and chikusetsusaponin IVa (compound 9). Adding Ag+ to the solvent system resulted in unique selectivity for 25R/25S isomers of inokosterone, which increased the complexing capability and stability of Ag+ coordinated 25S-inokosterone, as well as the α value between them. These results were further confirmed by the computational calculation of geometry optimization and frontier molecular orbitals assay. Comprehensive mass spectrometry and nuclear magnetic resonance analysis demonstrated the structures of the obtained compounds.

Effects of Saikosaponin-A on Insulin Resistance in Obesity: Computational and Animal Experimental Study.

Obesity is known to be associated with increased inflammation and dysregulated autophagy, both of which contribute to insulin resistance. Saikosaponin-A (SSA) has been reported to exhibit anti-inflammatory and lipid-lowering properties. In this research, we employed a combination of computational modeling and animal experiments to explore the effects of SSA. Male C57BL/6 mice were categorized into four groups: normal diet, high-fat diet (HFD), HFD + atorvastatin 10 mg/kg, and HFD + SSA 10 mg/kg. We conducted oral glucose and fat tolerance tests to assess metabolic parameters and histological changes. Furthermore, we evaluated the population of Kupffer cells (KCs) and examined gene expressions related to inflammation and autophagy. Computational analysis revealed that SSA displayed high binding affinity to tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, fibroblast growth factor 21 (FGF21), and autophagy-related 7 (ATG7). Animal study demonstrated that SSA administration improved fasting and postprandial glucose levels, homeostatic model assessment of insulin resistance (HOMA-IR) index, as well as triglyceride, free fatty acid, total cholesterol, low-density lipoprotein cholesterol (LDL-C)-cholesterol, and high-density lipoprotein cholesterol (HDL-C)-cholesterol levels in HFD-fed mice. Moreover, SSA significantly reduced liver weight and fat accumulation, while inhibiting the infiltration and M1 activation of KCs. At the mRNA level, SSA downregulated TNF-α and NF-κB expression, while upregulating FGF21 and ATG7 expression. In conclusion, our study suggests that SSA may serve as a therapeutic agent for addressing the metabolic complications associated with obesity. This potential therapeutic effect is attributed to the suppression of inflammatory cytokines and the upregulation of FGF21 and ATG7.

[Saikosaponin a alleviates pentylenetetrazol-induced acute epileptic seizures in mouse models of depression by suppressing microglia activation-mediated inflammation].

OBJECTIVE: To explore the inhibitory effect of saikosonin a (SSa) on pentylenetetrazol-induced acute epilepsy seizures in a mouse model of depression and explore the mechanism mediating this effect. METHODS: Male C57BL/6J mouse models of depression was established by oral administration of corticosterone via drinking water for 3 weeks, and acute epileptic seizures were induced by intraperitoneal injection of a single dose of pentylenetetrazole. The effect of intraperitoneal injection of SSa prior to the treatment on depressive symptoms and epileptic seizures were assessed using behavioral tests, epileptic seizure grading and hippocampal morphology observation. ELISA was used to detect blood corticosterone levels of the mice, and RTqPCR was performed to detect the pro- and anti-inflammatory factors. Microglia activation in the mice was observed using immunofluorescence staining. RESULTS: The mouse model of corticosterone-induced depression showed body weight loss and obvious depressive behaviors with significantly increased serum corticosterone level (all P < 0.05). Compared with those with pentylenetetrazole-induced epilepsy alone, the epileptic mice with comorbid depression showed significantly shorter latency of epileptic seizures, increased number, grade and duration of of seizures, reduced Nissl bodies in hippocampal CA1 and CA3 neurons, increased number of Iba1-positive cells, and significantly enhanced hippocampal expressions of IL-1ß, IL-10, TNF-α and IFN-γ. Pretreatment of the epileptic mice with SSa significantly prolonged the latency of epileptic seizures, reduced the number, duration, and severity of seizures, increased the number of Nissl bodies, decreased the number of Iba1-positive cells, and reduced the expression levels of IL-1ß, IL-10, TNF-α, and IFN-γ in the hippocampus (P < 0.05). CONCLUSION: Depressive state aggravates epileptic seizures, increases microglia activation, and elevates inflammation levels. SSA treatment can alleviate acute epileptic seizures in mouse models of depression possibly by suppressing microglia activation-mediated inflammation.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Background: Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. Methods: The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. Results: In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Conclusion: Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Anti-Biofilm Activity of Assamsaponin A, Theasaponin E1, and Theasaponin E2 against Candida albicans.

Biofilm formation plays a crucial role in the pathogenesis of Candida albicans and is significantly associated with resistance to antifungal agents. Tea seed saponins, a class of non-ionic triterpenes, have been proven to have fungicidal effects on planktonic C. albicans. However, their anti-biofilm activity and mechanism of action against C. albicans remain unclear. In this study, the effects of three Camellia sinensis seed saponin monomers, namely, theasaponin E1 (TE1), theasaponin E2 (TE2), and assamsaponin A (ASA), on the metabolism, biofilm development, and expression of the virulence genes of C. albicans were evaluated. The results of the XTT reduction assay and crystal violet (CV) staining assay demonstrated that tea seed saponin monomers concentration-dependently suppressed the adhesion and biofilm formation of C. albicans and were able to eradicate mature biofilms. The compounds were in the following order in terms of their inhibitory effects: ASA > TE1 > TE2. The mechanisms were associated with reductions in multiple crucial virulence factors, including cell surface hydrophobicity (CSH), adhesion ability, hyphal morphology conversion, and phospholipase activity. It was further demonstrated through qRT-PCR analysis that the anti-biofilm activity of ASA and TE1 against C. albicans was attributed to the inhibition of RAS1 activation, which consequently suppressed the cAMP-PKA and MAPK signaling pathways. Conversely, TE2 appeared to regulate the morphological turnover and hyphal growth of C. albicans via a pathway that was independent of RAS1. These findings suggest that tea seed saponin monomers are promising innovative agents against C. albicans.

[Comparison of in vivo pharmacokinetics of twelve constituents in Qihe Fenqing Yin in normal rats and diabetic rats].

This paper aims to study the pharmacokinetic differences of twelve effective constituents(succinic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, protocatechuic aldehyde, caffeic acid, 5-O-ferulogeninic acid, p-coumaric acid, nuciferine, quercetin, oleanolic acid, and ursolic acid) in Qihe Fenqing Yin in normal and diabetic rats. The diabetic rat model was established by a high-fat diet combined with intraperitoneal injection of streptozocin. A UHPLC-QTRAP-MS/MS method was established for the simultaneous determination of 12 constituents in the plasma of normal rats and model rats after a single intragastric administration of Qihe Fenqing Yin. The results show that the established analytical method has a good linear relationship with the 12 components, and the specificity, accuracy, precision, and stability meet the requirements. The computational pharmacokinetic parameters are fitted by DAS 3.2.8 software, and the results show that the half-life time(t_(1/2)) of the other nine components in the model group was longer than that in the normal group except for caffeic acid, 5-O-ferulogeninic acid, and oleanolic acid. The area under curve(AUC_(0-t)) of cryptochlorogenic acid, p-coumaric acid, ursolic acid, and oleanolic acid increases compared with the normal group. Meanwhile, mean residence time(MRT) delays. The "double peaks" of quercetin and nuciferine in the normal group are not observed in the model group, suggesting that the pharmacokinetic parameters of the drugs in the disease state are significantly different.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-ß-D-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s-1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: • The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. • Mechanisms to interpret PlGH3 substrate specificity by molecular docking. • Case of preparation of low-sugar alternative saponins by directed hydrolysis.

Structure-activity relationship and biofilm formation-related gene targets of oleanolic acid-type saponins from Pulsatilla chinensis against Candida albicans.

In the course of our investigations of antifungal natural products, the structure-activity relationship and antifungal activities of oleanolic acid-type saponins (1-28) from Pulsatilla chinensis against human and plant pathogenic fungi were elucidated. The analysis of structure-activity relationship of oleanolic acid-type saponins showed that the free carboxyl at C-28 was essential for their antifungal activities; the free hydroxyl group at the C-23 site of oleanolic acid-type saponins played a crucial role in their antifungal activities; the oligosaccharide chain at C-3 oleanolic acid-type saponins showed significant effects on antifungal efficacy and a disaccharide or trisaccharide moiety at position C-3 displayed optimal antifungal activity. The typical saponin pulchinenoside B3 (16, PB3) displayed satisfactory antifungal activity against human and plant pathogenic fungi, especially, C. albicans with an MIC value of 12.5 µg/mL. Furthermore, PB3 could inhibit the biofilm formation of C. albicans through downregulating the expression of the integrated network of biofilm formation-associated transcription factors (Bcr1 Efg1, Ndt80, Brg1, Rob1 and Tec1) and adhesion-related target genes (HWP1, ALS1, and ALS3). Meanwhile, we found that PB3 could effectively destroy the mature biofilm of C. albicans by the oxidative damage and inducing mitochondria-mediated apoptosis in cells.

In Vitro Anti-Toxoplasma Activity of Extracts Obtained from Tabebuia rosea and Tabebuia chrysantha: The Role of ß-Amyrin.

Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii that is highly prevalent worldwide. Although the infection is asymptomatic in immunocompetent individuals, it severely affects immunocompromised individuals, causing conditions such as encephalitis, myocarditis, or pneumonitis. The limited therapeutic efficacy of drugs currently used to treat toxoplasmosis has prompted the search for new therapeutic alternatives. The aim of this study was to determine the anti-Toxoplasma activity of extracts obtained from two species of the genus Tabebuia. Twenty-six extracts, 12 obtained from Tabebuia chrysantha and 14 from Tabebuia rosea, were evaluated by a colorimetric technique using the RH strain of T. gondii that expresses ß-galactosidase. Additionally, the activity of the promising extracts and their active compounds was evaluated by flow cytometry. ß-amyrin was isolated from the chloroform extract obtained from the leaves of T. rosea and displayed important anti-Toxoplasma activity. The results show that natural products are an important source of new molecules with considerable biological and/or pharmacological activity.

Effective separation of maslinic acid and oleanolic acid from olive pomace using high-speed shear off-line coupled with high-speed countercurrent chromatography and their antibacterial activity test.

In this work, a high-speed shear extraction off-line coupling high-speed countercurrent chromatography method was developed to separate maslinic acid and oleanolic acid from olive pomace. To improve extraction efficiency, the polar disparity between maslinic acid and oleanolic acid necessitated the concurrent utilization of both polar and non-polar solvents during high-speed shear extraction. Then, the high-speed shear extraction was directly feed to high-speed countercurrent chromatography for subsequently separation. A total of 250 min were needed to complete the extraction and separation process. This yielded two molecules from 3.3 g of defatted olive pomace: 7.2 mg of 93.8 % pure maslinic acid and 2.3 mg of 90.1 % pure oleanolic acid, both determined by HPLC at 210 nm. Furthermore, the compounds exhibited inhibitory activity against Escherichia coli and Staphylococcus aureus. At a concentration of 100 µg/mL, its efficacy in inhibiting hyaluronidase was comparable to that of the standard drug indomethacin. Compared with the conventional separation method, this coupled technique reduced the whole time due to the direct injection of sample extraction solution. This technique provides a useful approach for the separation of natural products with significant polarity differences.

Saikosaponin D mitigate pilocarpine-induced astrocyte injury by regulating the NLRP3/caspase-1 signaling pathway.

Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 µM) had no toxicity to astrocytes. Besides, SSD (4 µM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1ß, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.

Diethoxyphosphoryloxy-oleanolic acid is a nanomolar-inhibitor of butyrylcholinesterase.

A series of new betulin, lupeol, erythrodiol, and oleanolic acid phosphoryloxy- and furoyloxy-derivatives has been synthesized and their structure was confirmed by NMR spectroscopy. Synthesized compounds were subjected to Ellman's assays to determine their ability to inhibit the enzymes AChE and BChE. Among them, diethoxyphosphoryloxy-oleanolic acid inhibited BChE with a value of 99%, thereby acting as a mixed-type inhibitor holding very low Ki values of Ki = 6.59 nM and Ki ' = 1.97 nM, respectively.

Late-Stage Derivatization of Oleanolic Acid-Based Anti-HIV-1 Compounds.

A 12-keto-type oleanolic acid derivative (4) has been identified as a potent anti-human immunodeficiency virus type-1 (HIV-1) compound that demonstrates synergistic effects with several types of HIV-1 neutralizing antibodies. In the present study, we used a common key synthetic intermediate to carry out the late-stage derivatization of an anti-HIV compound based on the chemical structure of a 12-keto-type oleanolic acid derivative. To execute this strategy, we designed a diketo-type oleanolic acid derivative (5) for chemoselective transformation, targeting the carboxy group and the hydroxyl group on the statine unit, as well as the 3-carbonyl group on the oleanolic acid unit, as orthogonal synthetic handles. We carried out four types of chemoselective transformations, leading to identification of the indole-type derivative (16) as a novel potent anti-HIV compound. In addition, further optimization of the ß-hydroxyl group on the statine unit provided the R-4-isobutyl γ-amino acid-type derivative (6), which exhibited potent anti-HIV activity comparable to that of 4 but with reduced cytotoxicity.

Saikosaponin F ameliorates depression-associated dry eye disease by inhibiting TRIM8-induced TAK1 ubiquitination.

AIMS: Saikosaponin F (SsF) is one of the major active ingredients of Radix Bupleuri, an herb widely used in the treatment of depression. Studies have shown that dry eye disease often occurs together with depression. The aim of this study is to investigate whether SsF can improve depression-associated dry eye disease and explore the underlying mechanism. METHODS: Behavioral test was used to verify the effect of SsF on CUMS-induced depression-like behaviors in mice. Corneal fluorescein staining, phenol red cotton thread test and periodic acid-Schiff (PAS) staining were used to observe the effect of SsF on depression-associated dry eye disease. Western blot (WB) was performed to observe the expression of TAK1 protein and key proteins of NF-κB and MAPK (P38) inflammatory pathways in the hippocampus and cornea. Immunohistochemical staining was used to observe the expression of microglia, and immunoprecipitation was used to observe K63-linked TAK1 ubiquitination. Subsequently, we constructed a viral vector sh-TAK1 to silence TAK1 protein to verify whether SsF exerted its therapeutic effect based on TAK1. The expression of inflammatory factors such as IL-1ß, TNF-α and IL-18 in hippocampus and cornea were detected by ELISA. Overexpression of TRIM8 (OE-TRIM8) by viral vector was used to verify whether SsF improved depression-associated dry eye disease based on TRIM8. RESULTS: SsF treatment significantly improved the depression-like behavior, increased tear production and restored corneal injury in depression-related dry eye model mice. SsF treatment downregulated TAK1 expression and TRIM8-induced K63-linked TAK1 polyubiquitination, while inhibiting the activation of NF-κB and MAPK (P38) inflammatory pathways and microglial expression. In addition, selective inhibition of TAK1 expression ameliorated depression-associated dry eye disease, while overexpression of TRIM8 attenuated the therapeutic effect of SsF on depression-associated dry eye disease. CONCLUSION: SsF inhibited the polyubiquitination of TAK1 by acting on TRIM8, resulting in the downregulation of TAK1 expression, inhibition of inflammatory response, and improvement of CUMS-induced depression-associated dry eye disease.

Oleanolic acid exerts bone anabolic effects via activation of osteoblastic 25-hydroxyvitamin D 1-alpha hydroxylase.

Oleanolic acid (OA) is previously shown to exert bone protective effects in aged animals. However, its role in regulating osteoblastic vitamin D bioactivation, which is one of major causes of age-related bone loss, remains unclear. Our results revealed that treatment of OA significantly increased skeletal CYP27B1 expression and circulating 1,25(OH)2D3 in ovariectomized mice (p <0.01). Moreover, OA upregulated CYP27B1 protein expression and activity, as well as the vitamin D-responsive bone markers alkaline phosphatase (ALP) activity and osteopontin (OPN) protein expression, in human osteoblast-like MG-63 cells (p<0.05). CYP27B1 expression increased along with the osteoblastic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). CYP27B1 expression and cellular 1,25(OH)2D3 production were further potentiated by OA in cells at mature osteogenic stages. Notably, our study suggested that the osteogenic actions of OA were CYP27B1 dependent. In summary, the bone protective effects of OA were associated with the induction of CYP27B1 activity and expression in bone tissues and osteoblastic lineages. Hence, OA might be a potential approach for management of age-related bone loss.

Hepatotropic activity of a betulonic acid based compound.

Using the model of cyclophosphamide (CP)-induced immunosuppression in C57BL/6 mice, the hepatotropic effects of a conjugate of betulonic acid with 9-(4-methylpiperazin-1-ylmethyl)-2-(1,2,3-triazolyl) oreozelone (BABC) have been studied. In the liver of treated animals the expression of genes for cytochromes (CYP 1A1, CYP 1A2, CYP 3A44, CYP 2B10, CYP 2C29, CYP 17A1), PPARA, and cytokines (TNF-α, IL-1ß, IL-12α, IL-10) and the relative levels of NF-κB p65, GST-π, and NAT-1 proteins were determined. On day six after administration of the compound and CP to animals a significant (3.2-fold) increase in the expression of the CYP 2B10 as compared to the control group was observed. Treatment of mice with the compound and CP also caused a 2.4-fold increase in the mRNA level of the pro-inflammatory TNF-α gene as compared to the group of animals receiving CP. Administration of the studied compound to intact animals was accompanied by a 2.5-fold increase in the IL-1ß expression and a 1.8-fold decrease in the IL-10 expression as compared to the control group. An increase in the expression of pro-inflammatory cytokine genes in the liver of animals treated with the compound was accompanied by an increase in the content of NF-κB p65 (by 1.6 times), as well as an increase in the relative amount of NAT-1 protein (by 2.7 times) as compared to control animals.

CDDO regulates central and peripheral sensitization to attenuate post-herpetic neuralgia by targeting TRPV1/PKC-δ/p-Akt signals.

Postherpetic neuralgia (PHN) is a notorious neuropathic pain featuring persistent profound mechanical hyperalgesia with significant negative impact on patients' life quality. CDDO can regulate inflammatory response and programmed cell death. Its derivative also protects neurons from damages by modulating microglia activities. As a consequence of central and peripheral sensitization, applying neural blocks may benefit to minimize the risk of PHN. This study aimed to explore whether CDDO could generate analgesic action in a PHN-rats' model. The behavioural test was determined by calibrated forceps testing. The number of apoptotic neurons and degree of glial cell reaction were assessed by immunofluorescence assay. Activation of PKC-δ and the phosphorylation of Akt were measured by western blots. CDDO improved PHN by decreasing TRPV1-positive nociceptive neurons, the apoptotic neurons, and reversed glial cell reaction in adult rats. It also suppressed the enhanced PKC-δ and p-Akt signalling in the sciatic nerve, dorsal root ganglia (DRG) and spinal dorsal horn. Our research is the promising report demonstrating the analgesic and neuroprotective action of CDDO in a PHN-rat's model by regulating central and peripheral sensitization targeting TRPV1, PKC-δ and p-Akt. It also is the first study to elucidate the role of oligodendrocyte in PHN.

ß-keto amyrin isolated from Cryptostegia grandiflora R. br. inhibits inflammation caused by Daboia russellii viper venom: Direct binding of ß-keto amyrin to phospholipase A2.

The search for mechanism-based anti-inflammatory therapies is of fundamental importance to avoid undesired off-target effects. Phospholipase A2 (PLA2) activity is a potential molecular target for anti-inflammatory drugs because it fuels arachidonic acid needed to synthesize inflammation mediators, such as prostaglandins. Herein, we aim to investigate the molecular mechanism by which ß-keto amyrin isolated from a methanolic extract of Cryptostegia grandiflora R. Br. Leaves can inhibit inflammation caused by Daboia russellii viper (DR) venom that mainly contains PLA2. We found that ß-keto amyrin neutralizes DR venom-induced paw-edema in a mouse model. Molecular docking of PLA2 with ß-keto amyrin complex resulted in a higher binding energy score of -8.86 kcal/mol and an inhibition constant of 611.7 nM. Diclofenac had a binding energy of -7.04 kcal/mol and an IC50 value of 620 nM, which predicts a poorer binding interaction than ß-keto amyrin. The higher conformational stability of ß-keto amyrin interaction compared to diclofenac is confirmed by molecular dynamics simulation. ß-keto amyrin isolated from C. grandiflora inhibits the PLA2 activity contained in Daboia russellii viper venom. The anti-inflammatory property of ß-keto amyrin is due to its direct binding into the active site of PLA2, thus inhibiting its enzyme activity.

Oleanolic acid promotes liver regeneration after partial hepatectomy via regulating pregnane X receptor signaling pathway in mice.

Liver regeneration after liver tumor resection or liver transplantation is crucial, the remaining liver frequently fails to regenerate in some patients. Oleanolic acid (OA), a pentacyclic triterpenoid compound which has been shown to protect against various liver diseases. However, the effect of OA on liver regeneration after partial hepatectomy (PHx) is still unclear. In this study, the results showed that OA (50 mg/kg, twice daily) treatment induced liver mass restoration and increased the liver-to-body weight ratio of mice following PHx. Meanwhile, OA promoted hepatocyte proliferation and increased the number of BrdU-, Ki67-and PCNA-positive cells. Furthermore, OA increased the nuclear accumulation of PXR and induced the expression of PXR downstream proteins such as CYP3A11, UGT1A1 and GSTM2 in mice, as well as in AML12 and HepRG cells. Luciferase reporter assay and nuclear localization of PXR further demonstrated the effect of OA on PXR activation in vitro. Molecular docking simulation showed that OA could interact with the PXR active sites. Moreover, OA inhibited the expression of FOXO1, RBL2 and CDKN1B, and increased the expression of PCNA, CCND1 and CCNE1 in vivo and in vitro. Silencing of Pxr further confirmed that OA-mediated upregulation of proliferation-related proteins depended on PXR. The current study illustrated that OA exhibited a significant promoting effect on liver regeneration following PHx, potentially through regulation of the PXR signaling pathway to accelerate liver recovery.