Betulinic acid protects against ovarian impairment by decreasing F-2 toxin-induced oxidative stress and inflammation associated with the downregulation of p38 expression in mice

F-2 toxin is an estrogenic mycotoxin that causes reproductive disorders in animals.Betulinic acid(BA)is a natural pentacyclic lupane-structure triterpenoid that has diverse pharmacological activities.In this study,the antioxidative and anti-inflammatory effects of BA and its underlying mechanism are explored in F-2 toxin-triggered mouse ovarian damage.We found that BA alleviated the F-2 toxin-induced ovarian impairment by stimulating follicle growth,reducing inflammatory cell infiltration,repairing damaged mitochondria and endoplasmic reticulum.Simultaneously,BA not only reversed F-2 toxin-induced reduction of follicle stimulating hormone(FSH)and luteinizing hormone(LH)levels in the serum,but also restrained the protein expression of the estrogen receptors a(ERa)and ERβ.Moreover,BA restored the balance of F-2 toxin-induced ovarian redox system disorders.Subsequently,we found that 0.25 mg/kg BA played an anti-inflammatory role in the F-2 toxin-induced ovarian impairment by decreasing interleukin-1β(IL-1β).IL-6,and tumor necrosis factor-α(TNF-α)mRNA expression,as well as inhibiting p38 protein expression.These data demonstrated that BA exerts its protective effect on F-2 toxin-induced ovarian oxidative impairment and inflammation by inhibiting p38 expression,which implies a natural product-based medicine to ameliorate F-2 toxin-caused female reproductive toxicity and provides a detoxifying method for food contaminated by mycotoxin.

Epigenetic modification associated with climate regulates betulin biosynthesis in birch

The Betula genus contains pentacyclic triterpenoid betulin known for its environmental adaptation and medicinal properties.However,the mechanisms underlying betulin biosynthesis responding to climate change remain unclear.In this study,the role of epigenetic modification(DNA methylation) in betulin biosynthesis was examined and how climatic factors influence it.Whole-genome bisulfite sequencing was performed for greenhouse-grown Chinese white birch(Betula platyphylla Sukaczev) treated with DNA methylation inhibitor zebularine(ZEB) and a natural birch population in Northeast China.ZEB treatment significantly affected the CHH methylation level of transposable elements and betulin content in a hormesis dose-dependent manner.The methylation and expression of bHLH9,a key transcriptional factor controlling betulin biosynthesis,were also consistently affected by ZEB treatment as a hormetic dose-response.In the natural population,there was a positive correlation between promoter methylation of bHLH9 and summer precipitation,while winter temperature was negatively correlated.Thus climate-dependent methylation of bHLH9 regulates the expression of downstream genes involved in betulin biosynthesis.This study highlights the role of environmental signals to induce epigenetic changes that result in betulin production,possibly helping to develop resilient plants to combat ongoing climate change and enhance secondary metabolite production.

Betulinic acid-mediating miRNA-365 inhibited the progression of pancreatic cancer

Background:The dilemma of pancreatic cancer treatment has become a global challenge.For this reason,effective,feasible,and new medical methods are currently much-needed.Betulinic acid(BA)has been valued as a potential therapy for pancreatic cancer.However,the mechanism by which BA exerts an inhibitory effect on the development of pancreatic cancer remains elusive.Methods:A rat model and two cell models of pancreatic cancer were established,and the effect of BA on pancreatic cancer was verified in vivo and in vitro by using MTT,Transwell,flow cytometry,RT-PCR,Elisa and immunohistochemistry.At the same time,miR-365 inhibitors were introduced to test whether BA played a role in mediating miR-365.Results:BA can significantly inhibit the proliferation and invasion of pancreatic cancer cells and promote apoptosis.In vivo experiments,BA can significantly lower the number of cancer cells and tumor volume in the rat model of pancreatic cancer.In vitro,it was found that BA inhibited the protein level and phosphorylation level of AKT/STAT3 by mediating the expression of miR365/BTG2/IL-6.Like BA,miR-365 inhibitors also significantly inhibited cell viability and invasion ability,and inhibited the protein level and phosphorylation level of AKT/STAT3 by changing the expression of BTG2/IL-6,and their combination had a synergistic effect.Conclusion:BA inhibits AKT/STAT3 expression and phosphorylation by modulating miR-365/BTG2/IL-6 expression,and BA inhibits the progression of pancreatic cancer through the aforementioned mechanism.

Biological Activity of Betulinic Acid: A Review

Betulinic acid is a known natural product which has gained a lot of attention in the recent years since it exhibits a variety of biological and medicinal properties. This review provides the most important biological properties of betulinic acid.

Effects of Betulinic Acid on Proliferation and Apoptosis in Jurkat Cells and Its In Vitro Mechanism

The anti-cancer effects of betulinic acid （BA） on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-Ⅴ/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time-and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G0/G1 phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from （31.00±1.25）% to （58.84±0.32）% in G0/G1 phase, whereas it was decreased from （61.45±1.04）% to （35.82±1.95）% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G0/G1 phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.

Down-regulation of NOX4 by Betulinic Acid Protects against Cerebral Ischemia-reperfusion in Mice

Ischemic stroke leads to high potentiality of mortality and disability. The current treatment for ischemic stroke is mainly focused on intravenous thrombolytic therapy. However, ischemia/reperfusion induces neuronal damage, which significantly influences the outcome of patients with ischemic stroke, and the exact mechanism implicated in ischemia/reperfusion injury remains unclear, although evidence shows that oxidative stress is likely to be involved. Betulinic acid is mainly known for its anti-tumor and anti-inflammatory activities. Our previous study showed that betulinic acid could decrease the reactive oxygen species（ROS） production by regulating the expression of NADPH oxidase. Thus, we hypothesized that betulinic acid may protect against brain ischemic injury in the animal model of stroke. Focal cerebral ischemia was achieved by using the standard intraluminal occlusion method and reperfusion enabled after 2 h ischemia. Neurological deficits were scored. Infarct size was determined with 2,3,5-triphenyltetrazolium chloride monohydrate（TTC） staining and the mRNA expression of NADPH oxidase 4（NOX4） was determined by RT-PCR in infarct tissue. ROS generation and apoptosis in ischemic tissue were analyzed by measuring the oxidative conversion of cell permeable 2＇,7＇-dichloro-fluorescein diacetate（DCF-DA） to fluorescent dichlorofluorescein（DCF） in fluorescence microplate reader and TUNEL assay, respectively. In Kunming mice, 2 h of middle cerebral artery（MCA） occlusion followed by 24 or 72 h of reperfusion led to an enhanced NOX4 expression in the ischemic hemisphere. This was associated with elevated levels of ROS generation and neuronal apoptosis. Pre-treatment with betulinic acid（50 mg/kg/day for 7 days via gavage） prior to MCA occlusion prevented the ischemia/reperfusion-induced up-regulation of NOX4 and ROS production. In addition, treatment with betulinic acid could markedly blunt the ischemia/reperfusion-induced neuronal apoptosis. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this stroke mouse model. Our results suggest that betulinic acid protects against cerebral ischemia/reperfusion injury in mice and the down-regulation of NOX4 may represent a mechanism contributing to this effect.

Purity and Uncertainty Study of CRM Betulin by DSC

Betulin(BE)can be obtained from many plants,such as those belonging Betulaceae family,and pharmacological investigations showed its notable biological properties and good potential for food and pharmaceutical development.We investigated the homogeneity,stability,purity,and uncertainty of a newly certified reference material(CRM)of BE.The certified purity value for the CRM of BE was 99.56%with an extended uncertainty of 0.07%(k=2,P=0.95),as determined by differential scanning calorimetry(DSC).In this study,DSC was used for the first time for purity determination of BE.Given its high accuracy,precision,and reproducibility,DSC can be used as an alternative technique for purity determination of CRMs in the pharmaceutical and food industry.

Caspase-9 Activation—Critical for Betulin-induced Apoptosis of Human Hepatoma Cells

Betulinic acid and its derivatives have been extensively studied in the past for their anti-tumor effects, but relatively little is known about its precursor betulin. In this study we showed that betulin, an abundant natural product, significantly inhibits the cell growth of human hepatoma HepG2 cells in a dose-dependent manner. In the presence of 10 μg/mL betulin, HepG2 cells undergo an apoptosis, as evidenced by apoptotic morphology such as cell shrinkage, membrane blebbing, nuclear condensation and fragmentation, apoptotic body formation, and caspase activation. Kinetics analysis shows that the depolarization of the mitochondrial membrane potential and the release of the mito- chondrial apoptotic protein cytochrome c occurred as early as 2 h post treatment of HepG2 cells with 10 μg/mL betulin. Proteolytic activation of caspase-9, but not caspase-8, was observed in this apoptosis process. Moreover, the inactivation of caspase-9 by its specific siRNA dramatically reduced betulin-induced caspase-3 activation and apoptosis. Taken together, our observations indicate that the activation of caspase-9 is critical for betulin-induced apoptosis of human hepatoma HepG2 cells.

Structure and Anti-HIV Activity of Betulinic Acid Analogues

Firstly discovered in 1980s, human immunodeficiency virus （HIV） continues to affect more and more people. However, there is no effective drug available for the therapy of HIV infection. Betulinic acid existing in various medicinal herbs and fruits exhibits multiple biological effects, especially its outstanding anti-HIV activity, which has drawn the attentions of many pharmacists. Among the derivatives of betulinic acid, some compounds exhibited inhibitory activities at the nanomolar concentration, and have entered phase II clinical trials. This paper summarizes the current investigations on the anti-HIV activity of betulinic acid analogues, and provides valuable data for subsequent researches.

Antitumor Effect of Betulinic Acid on Human Acute Leukemia K562 Cells in vitro

The effects of betulinic acid (BA), a pentacyclic lupane-type triterpene, on the cell viability, cell cycle and apoptosis in human leukemia K562 cells were investigated. The effects of BA on the growth of K562 cells were studied by MTT assay. Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry. The effects of BA on the cell cycle of K562 cells were studied by a PI method. The expression of Bax and capase-3 was detected by using Western blot. The results showed that BA was cytotoxic to K562 cells with an IC50 of 21.26 μg/mL at 24 h. After treating K562 cells with 10 μg/mL BA for 72 h, the number of cells was reduced by 58%. BA induced apoptosis of K562 cells in a time-and dose-dependent manner. The proportion of cells in G0/G1 and G2/M phases was decreased and that in S phase was increased after K562 cells were treated with BA for 24 h. BA treatment also increased the expression of the pro-apoptotic proteins Bax and caspase-3. It suggested that BA could inhibit the proliferation of K562 cells through the induction of cell cycle arrest and apoptosis. The antitumor effects of BA were related with up-regulation of the expression of Bax and caspase-3 proteins. BA may qualify for the development of new therapies for leukemia.

Effect of MeJA and Light on the Accumulation of Betulin and Oleanolic Acid in the Saplings of White Birch (<i>Betula platyphylla</i>Suk.)

In this study, we investigated the effect of different types of light and MeJA treatment on the accumulation of betulin and oleanolic acid in various organs of white birch. Our results showed that betulin and oleanolic were accumulated mainly in the stalk skin. The content of both substances in the stalk skin was significantly affected by seasons with a peak accumulation in August. The content of oleanolic and betulin was significantly decreased in the stem skin treated with 4 types of light (red, yellow, blue and green) compared with the plant with normal illumination. In contrast, oleanolic acid in leaves was increased by 13.28 folds when the white birch was treated with green light. Betulin was increased by 1.959 folds in leaves of white birch treated with blue light. The highest content of betulin and oleanolic acid in various organs of birch with appropriate shading treatment (light transmittance: 50%) was increased by 45.09% and 30.50%, respectively, in comparison with those with non-shading treatment. Content of oleanolic acid and betulin can be significantly improved in various parts of birch after treatment with different concentration of MeJA. The study lays the foundation to metabolic regulation of oleanolic acid and betulin in birch.

Analysis of Four Solvatomorphs of Betulin by TG-DTA-EI/PI-MS System Equipped with the Skimmer-Type Interface

Betulin(BE)has exceedingly become a potential natural product,providing multiple pharmacological and biological activi-ties,including anti-cancer,anti-viral,and anti-inflammatory benefits.Previous research indicated that the solvatomorphism of BE can easily occur through crystallization with different organic solvents.This property of BE can directly affect its extraction,isolation,and preparation process.In this study,a system of thermogravimetry(TG)-differential thermal analysis(DTA)coupled with mass spectrometry(MS)with electron ionization(EI)and photoionization(PI)capability,equipped with the skimmer-type interface(i.e.,skimmer-type interfaced TG-DTA-EI/PI-MS system),as a real-time and onsite analysis technique,was employed.Then,four solvatomorphs of BE,namely,with pyridine and water(A),sec-butanol(B),n,n-dimethylformamide(DMF)(C),and isopropanol(V),were analyzed for the first time.Finally,five kinds of the main volatile gaseous species,including H2O,pyridine,sec-butanol,DMF,and isopropanol,were identified clearly.Furthermore,the multi-step desolvation processes of the four solvatomorphs of BE were revealed by this system for the first time.This system showed great potential for the rapid and accurate analysis of various solvatomorphs of natural products.

Research Progress on Pharmacological Effects of Betulin

Betulin is main component of triterpenoids in bark extract of Betula platyphylla,and has antibacterial,antiviral,liver protecting,cholagogic,antitumorus and other functions.This paper reviews the pharmacological effects and mechanisms of betulin.

Current advances in the biotechnological synthesis of betulinic acid:new findings and practical applications

Betulinic acid(BA),a penta-cyclic triterpenoid found as a ubiquitous secondary metabolite throughout the plant kingdom,has aroused tremendous interests due to its different pharmacological properties,which lead to large market demand.However,the content of BA in plant is low for phytoextraction.Although chemical semi-synthesis or biotransformation of BA from betulin with high conversion efficiency is achieved,it still relies on phytoextraction from the bark of medicinal trees.To circumvent this issue,the biotechnological synthesis of BA in engineered yeasts has been developed.In this review,the pharmacological properties of BA are first summarized,including antitumor,anti-HIV,antiprotozoal,anti-inflammatory,apoptosis activator and anti-metabolic syndrome.Then,the traditional phytoextraction,semi-synthesis and biotechnological synthesis of BA are discussed.Particularly,current advances in its biotechnological synthesis and strategies to improve BA production are focused.Moreover,potential strategies for further promotion of BA yield,including the introduction of artificial isopentenol utilization pathway,semi-rational mutagenesis of lupeol synthase and cytochrome P450,and subcellular morphology and compartmentalization,are discussed.

白桦脂酸通过RRAGD/mTOR/ULK1促进自噬改善血管平滑肌细胞钙化

目的 探讨白桦脂酸(Betulinic acid, BA)对高磷诱导血管平滑肌细胞钙化的作用机制。方法 采用2.6μmol·g^(-1)高磷诱导小鼠主动脉平滑肌细胞体外钙化模型,CCK8检测白桦脂酸对细胞活力的影响;加入白桦脂酸低、中、高剂量(5μmol·L^(-1)、10μmol·L^(-1)、20μmol·L^(-1))及自噬抑制剂3-甲基腺嘌呤(3-MA,5μmol·g^(-1))。慢病毒过表达Ras相关GTP结合蛋白D(Ras-related GTP-binding Protein D,RRAGD)转染血管平滑肌细胞,通过荧光显微镜、RT-PCR验证转染效率。采用茜素红染色、细胞内钙含量、碱性磷酸酶活性检测细胞钙化程度;Western blot检测血管平滑肌细胞钙化指标RUNT相关转录因子2(Runt-related transcription factor 2,RUNX2),骨形态发生蛋白2(Bone morphogenetic protein, BMP2),平滑肌蛋白22α(Smooth muscle protein 22α,SM22α)和平滑肌肌动蛋白(α-smooth muscle actin, α-SMA);UNC-51样激酶1(UNC-51-like kinase 1,ULK1),自噬蛋白微管相关蛋白1轻链3(LC3Ⅱ/LC3Ⅰ),泛素结合蛋白P62(P62),哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin, mTOR)以及RRAGD的表达。结果 根据CCK8细胞活力结果选择白桦脂酸5、10、20μmol·L^(-1)剂量进行后续实验;钙水平、碱性磷酸酶活性及茜素红染色实验结果显示白桦脂酸改善高磷诱导血管平滑肌细胞钙化;Western blot结果显示白桦脂酸下调RUNX2、BMP2、RRAGD、p-mTOR、P62(P<0.05),上调SM22α、α-SMA、LC3Ⅱ/Ⅰ、p-ULK1的蛋白表达(P<0.05)。白桦脂酸减轻高磷诱导血管平滑肌细胞钙化的作用被自噬抑制剂3-MA和过表达RRAGD阻断。结论 白桦脂酸可能通过RRAGD/mTOR/ULK1信号通路促进自噬抑制血管平滑肌细胞钙化。

白桦脂醇纳米乳的制备及其在S180荷瘤小鼠体内分布研究

目的制备白桦脂醇纳米乳,研究其在S180荷瘤小鼠体内的分布情况。方法通过相转变法制备白桦脂醇纳米乳,腹腔注射白桦脂醇纳米乳和白桦脂醇注射液,采用HPLC方法测定小鼠各组织中的药物浓度并计算药动学参数,分析比较两种制剂在小鼠体内组织分布情况。结果白桦脂醇纳米乳在各组织中Cmax和AUC依次为:肿瘤组织>肝>肾>脾>肺>心脏,且均高于白桦脂醇注射液组(P<0.05);肿瘤组织和肝脏的峰浓度比Ce分别为1.53和1.54;肿瘤的靶向效率Te值最大。结论与白桦脂醇注射液相比,白桦脂醇纳米乳在肿瘤组织和肝脏中的分布显著增加(P<0.01),白桦脂醇纳米乳对肿瘤组织具有明显的靶向性。

桦木酸通过Wnt/β-catenin信号通路调控瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成

目的研究桦木酸(betulinic acid,BA)对瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成的影响并探讨分子作用机制。方法收集临床切除的瘢痕疙瘩组织,采取组织块贴壁法分离培养成纤维细胞。细胞分为3组:空白对照组、二甲基亚砜(dimethyl sulfoxide,DMSO)处理组(溶剂对照组)和BA处理组。细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测细胞生长。5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)法检测细胞增殖。流式细胞术检测细胞凋亡。Transwell法测细胞侵袭。荧光素酶报告基因实验检测Wnt/β-catenin信号通路活性。实时定量PCR(real-time quantitative PCR,RT-qPCR)检测c-myc和cyclin D1 mRNA表达。Western blot检测Ⅰ型胶原(Collagen typeⅠ,ColⅠ)、β-catenin、c-myc和cyclin D1蛋白表达。结果与空白对照组和DMSO处理组相比,BA处理组瘢痕疙瘩成纤维细胞增殖降低,细胞凋亡率上升,细胞侵袭水平下降,胶原合成减少,差异有统计学意义(P<0.05)。与空白对照组和DMSO处理组相比,BA处理组瘢痕疙瘩成纤维细胞中Wnt/β-catenin信号通路活性降低,β-catenin、c-myc和cyclin D1表达水平减少,差异有统计学意义(P<0.05)。结论桦木酸通过下调Wnt/β-catenin信号通路抑制瘢痕疙瘩成纤维细胞增殖、侵袭和胶原合成。