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.

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.

Cytotoxic Activity of Betulinic Acid Derivatives Synthesized Using Enzymes

Betulinic acid, a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. In order to obtain derivatives potentially useful for detailed pharmacological studies, betulinic acid derivatives were synthesized by reaction of betulinic acid with benzoyl chloride and with acetic anhydride using lipase as catalyst. Enzyme-catalyzed of betulinic acid with benzoyl chloride converted betulinic acid into 3β-benzoil-lup-20（29）-ene-28-oic acid ester （BCL） whereas with acetic anhydride converted betulinic acid into 3β-acetoxy-lup-20（29）-ene-28-oic acid ester （BAA）. The BAA then underwent further reaction with l-decanol to produce 3β-acetoxy-lup-20（29）-ene-28 decanoate （BAAD）. Betulinic acid derivatives prepared were tested for cytotoxic activity on three cancer cell lines in vitro： all tested compounds showed stronger cytotoxic activity than 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.

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.

Inhibition of betulinic acid to growth and angiogenesis of human colorectal cancer cell in nude mice

Objective： Angiogenesis plays a major role in the pathogenesis of many disorders. Vascular endothelial growth factor （VEGF） has been shown to be the key regulator of normal and pathological angiogenesis. Many studies showed that decreased expression of VEGF has been inhibited growth and migration of cancer cells. The aim of this study was to explore the effects of Betulinic acid on the VEGF expression and the growth of colorectal cell SW480 xenografts in nude mice. Methods： The xenografts derived from colorectal cell SW480 were established in BALB/C nude mice. Inoculated mice were randomly divided into negative control （corn oil）, low dose betulinic acid group （20 mg/kg/d） and high dose group （40 mg/kg/d）. After 22 days, the animals were sacrificed; tumor volume and weights were measured. The mRNA level of VEGF was analyzed by quantitative real-time polymerase chain reaction. The expression of VEGF protein was detected by immunohistochemistry. Results： The tumor weight was significantly lower in low and high dose groups than in corn oil group （1.12 ＋ 0.04, 0.43 ＋ 0.02 vs 2.08 ＋ 0.07; P 〈 0.05）. The mRNA levels of VEGF was also significantly lower in betulinic acid treated groups （0.72 ＋ 0.02, 0.38 ＋ 0.01; P 〈 0.05） than in control group （1.08 ＋ 0.04）. H＆E staining showed tumor tissue necrosis was observed in treatment groups. The positive expression of VEGF was lower in low and high dose groups than in corn eil group. Gray scale increased in low dose group and high dose group （121.1 ＋ 2.8, 156.2 ＋ 3.3, P 〈 0.05）. Conclusion： Betulinic acid had significant inhibitory effect on VEGF expression and tumors growth of human colorectal cancer xenografts in vivo, and down-regulation of VEGF expression may account for one of the molecular mechanisms of the anticancer effects of betulinic acid.

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.

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.

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.

A clickable photoaffinity probe of betulinic acid identifies tropomyosin as a target

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge,unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid.

Recent progress on betulinic acid and its derivatives as antitumor agents:a mini review

Natural products are one of the important sources for the discovery of new drugs.Betulinic acid(BA),a pentacyclic triterpenoid widely distributed in the plant kingdom,exhibits powerful biological effects,including antitumor activity against various types of cancer cells.A considerable number of BA derivatives have been designed and prepared to remove their disadvantages,such as poor water solubility and low bioavailability.This review summarizes the current studies of the structural diversity of antitumor BA derivatives within the last five years,which provides prospects for further research on the structural modification of betulinic acid.

Betulinic acid accelerates diabetic wound healing by modulating hyperglycemia-induced oxidative stress,inflammation and glucose intolerance

Background:Diabetes significantly delays wound healing through oxidative stress,inflammation and impaired re-epithelialization that lead to defective regulation of the healing process,although the related mechanism remains unclear.Here,we aim to investigate the potential role and mechanism for the beneficial effect of betulinic acid(BA)on diabetic wound healing.Methods:The molecular effect of BA on hyperglycemia-mediated gene expression,oxidative stress,inflammation and glucose uptake was evaluated in endothelial,fibroblast and muscle cells.Burn injury was introduced to streptozotocin-induced diabetic rats and BA administration through either an intraperitoneal(IP)or topical(TOP)technique was used for wound treatment.Glucose tolerance was evaluated in both muscle tissue and fibroblasts,while oxidative stress and inflammation were determined in both the circulatory system and in wound tissues.The effect of BA on the wound healing process was also evaluated.Results:BA treatment reversed hyperglycemia-induced glucose transporter type 4(GLUT4)sup-pression in both muscle and fibroblast cells.This treatment also partly reversed hyperglycemia-mediated suppression of endothelial nitric oxide synthase(eNOS),nuclear factor erythroid 2-related factor 2(Nrf2)signaling and nuclear factor NFκB p65 subunit(NFκB p65)activation in endothelial cells.An in vivo rat study showed that BA administration ameliorated diabetes-mediated glucose intolerance and partly attenuated diabetes-mediated oxidative stress and inflam-mation in both the circulatory system and wound tissues.BA administration by both IP and TOP techniques significantly accelerated diabetic wound healing,while BA administration by either IP or TOP methods alone had a significantly lower effect.Conclusions:BA treatment ameliorates hyperglycemia-mediated glucose intolerance,endothelial dysfunction,oxidative stress and inflammation.Administration of BA by both IP and TOP tech-niques was found to significantly accelerate diabetic wound healing,indicating that BA could be a potential therapeutic candidate for diabetic wound healing.

Betulin protects against isoproterenol-induced myocardial injury by inhibiting NF-κB signaling and attenuating cardiac inflammation and oxidative stress in rats

Objective:To investigate the cardioprotective potential of betulin in isoproterenol(ISO)-induced myocardial injury in rats.Methods:Wistar rats were divided into five groups(n=10):normal,ISO,nebivolol 5 mg/kg,and betulin(20&40 mg/kg).Nebivolol and betulin were administered orally for 29 days.ISO(85 mg/kg)was administered subcutaneously on day 27 and day 28 to induce myocardial injury.On day 29,blood was collected for determination of cardiac markers,and hemodynamic parameters were investigated.The levels of oxidative stress markers and the gene expressions of apoptotic markers and inflammatory mediators were evaluated.Moreover,2,3,5-triphenyltetrazolium chloride staining and histopathological analysis were also performed.Results:Betulin reduced the size of myocardial infarction,decreased elevated levels of cardiac enzymes,and maintained hemodynamic functions.It also inhibited ISO-induced upregulation of Bax,caspase-3,NF-κB,and IL-6,enhanced endogenous antioxidant enzymes,and reduced lipid peroxidation.Additionally,pretreatment with betulin alleviated myocardial ischemic damage,as reflected by reduced myonecrosis,edema,and inflammatory changes.Conclusions:Betulin exhibits strong cardioprotective activity against ISO-induced myocardial injury by anti-inflammatory,anti-apoptotic,and antioxidant activities.

Novel NO-releasing derivatives of betulinic acid with antitumor activity

Thirteen novel NO-releasing derivatives of betulinic acid （BA） bearing two types of NO-donors （nitrates and furoxans） were synthesized and evaluated for their antitumor activity. The results showed that furoxan-based derivatives exhibited higher antitumor activity than nitrate-based derivatives, with compounds lla and llb displaying promising potency against B16 cell lines and HepG2 cell lines （IC50 〈 1 μmol/L）. We supposed that NO-releasing amount of these derivatives which can be detected by Griess method may contribute more to their antitumor activity. As a result, furoxan-based derivatives released larger amount of NO than that of nitrate-based derivatives, which partially explained the higher anti-tumor activity of the former.

Synthetic Analogues of Betulinic Acid as Potent Inhibitors of PS1/BACE1 Interaction to Reduce Aβ Generation

The lupane-type triterpenoids are endowed with a wide range of biological activities such as antiviral, anti-inflammatory and anticancer activity. We describe here its potential application in Alzheimer＇s disease （AD） treatment as an inhibitor of PS1/BACE1 interaction. 3-a-Akebonoic acid, which emanated from a high throughput screening （HTS）, was discovered to interfere with P S 1/BACE 1 interaction and reduce amyloid β-protein （Aβ） production. In view of the limited source, we instead used naturally rich betulinic acid （compound 2） as starting material for lead optimization and a focused library of its derivatives was constructed to gain a better understanding of the structure activity relationship （SAR） of triterpenoid-type inhibitor of PS1/BACE1 interaction. Compound 22 was finally chosen as the most potent PS 1/BACE 1 interaction inhibitor, which reduced Aβgeneration effectively.

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.

三棱的化学成分研究

目的:研究三棱Sparganium stoloniferumBuch.-Ham.的化学成分。方法:采用硅胶柱层析,Sephadex LH-20,ODS C-18,重结晶等方法,根据理化性质和光谱鉴定化合物的结构。结果:从三棱中得到8个化合物,分别为棕榈酸(1),5-羟甲基糠醛(2),6,7,10-三羟基-8-十八烯酸(3),棕榈酸单甘油酯(4),5-Hydroxy-2-(hydroxymethyl)pyridine(5),β-谷甾醇(β-sitosterol)(6),β-胡萝卜苷(daucosterol)(7),Betulinic acid(8)。结论:化合物5,8为首次从该植物中分离得到。

Extraction of betulin from bark of Betula platyphylla by supercritical carbon dioxide extraction

Betulin, which is a medicinal pentacyclic triterpene, is abundant in the bark of white birch (Betula platyphlly). The bark of birch was collected at Tayuan Forest Farm of Jiagedaqi, Heilongjiang Province in September 2000. Supercritical fluid extraction (SFE) that is a new separation technology has been used for the processing pharmaceutical and natural products. In this paper, the extraction of betulin from the bark of birch by supercritical CO2 extraction was studied. The authors investigated and analyzed a few parameters such as modifier dosage, extraction pressure and extraction temperature. The optimal extrac-tion conditions showed that the modifier dosage used for per gram bark powder was 1.5 mL, the extraction pressure was at 20 Mpa, and the extraction temperature was at 55 C. The velocity of flow of liquid CO2 was at 10 kg/h. The pressure and tem-perature in separation vessel were at 5.5 Mpa and 50 C, respectively.

Studies on the Glycosides in the Leaves ofOplopanax elatus NAKAI(Ⅰ)

our new saponins were isolated from the leaves of Oplopanax elatus Nakai.The structures of the four saponins , tentatively named compounds A(1) , B(2) , C(3) and D (4) were elucidated to be 28-O-a-L-rhamnopyranosyl (1-4)-β-D-glu-copyranosyl ( 1-6 )-β-D-glucopyranosyl esters of betulinic acid 3-O-β-D-glucopyra-noside (1) . oleanolic acid 3-O-β-D-glucopyranoside (2) , 3a, 23-dihydroxyolean-12-ene-28-Oic acid ( = 3-epi-hederagenin ) (3) , 3β-hydroxyolean-12-ene-23-al-28-oic acid (=gypsogenin) (4) , respetively.