Betulin and betulinic acid attenuate ethanol-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS), cytokine (TNF-α, TGF-β) production and by influencing intracellular signaling

Background/aims

Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids. However, the mechanisms of the action of those triterpenoids are poorly understood. In this study, we aimed to determine the antifibrotic potential of other triterpenes, betulin and betulinic acid, and to characterize their influence on the signal transduction pathways involved in ethanol-activated hepatic stellate cells (HSCs).

Methods

Investigated was the influence of preincubation of rat HSCs with betulin and betulinic acid, at non-toxic concentrations, on ethanol-induced toxicity, migration, and several markers of HSC activation such as smooth muscle α-actin (α-SMA) and procollagen I expression, release of reactive oxygen species (ROS) and cytokines: tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1), and production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). To assess the mechanism of the action of those triterpenes, intracellular signals such as nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol were examined.

Results

In vitro, betulin, but not betulinic acid, protected HSCs against ethanol toxicity. However, both betulin and betulinic acid inhibited the production of ROS by HSCs treated with ethanol and inhibited their migration as well as ethanol-induced TNF-α, and TGF-β1, production. Betulin and betulinic acid down-regulated ethanol-induced production of TIMP-1 and TIMP-2. Betulin and betulinic acid, also decreased ethanol-induced activity of MMP-2. In ethanol-induced HSCs, betulin inhibited the activation of the p38 MAPK and the JNK transduction pathways, while betulinic acid inhibited the JNK transduction pathway only. They also significantly inhibited phosphorylation of IκB and Smad 3 and attenuated the activation of TGF-β1 and NFκB/IκB transduction signaling.

Conclusion

The results indicated that betulin and betulinic acid inhibited ethanol-induced activation of HSCs on different levels, acting as antioxidants, inhibitors of cytokine production, and inhibitors of TGF-β, and NFκB/IκB transduction signaling. Betulin was also inhibitor of both JNK and p38 MAPK signal transduction, while betulinic acid inhibited only JNK. The remarkable inhibition of several markers of HCS activation makes triterpenes, especially betulin, promising agents for anti-fibrotic combination therapies.     

Betulin, betulinic acid and butein are inhibitors of acetaldehyde-induced activation of liver stellate cells

Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids; however, the activity of other triterpenes like betulin and betulinic acid has not been examined. Butein has also been reported to prevent and partly reverse liver fibrosis in vivo, although its mechanism of action is poorly understood. Therefore, the aim of this study was to determine the antifibrotic potential of butein, betulin, and betulinic acid and examine their mechanisms of action in vitro. This study was conducted in rat stellate cells (HSCs) that were treated with acetaldehyde, which is the most reactive product of ethanol metabolism. Butein, betulin, and betulinic acid were preincubated with rat HSCs at non-toxic concentrations. Treatment effects were measured in regard to acetaldehyde-induced toxicity and cell migration, and several markers of HSC activation were evaluated, including smooth muscle α-actin (α-SMA) and procollagen I expression. In addition, changes in the release of reactive oxygen species (ROS) and cytokines such as tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1) and changes in the production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined. In vitro, HSCs were protected against acetaldehyde-induced toxicity by betulin but not by betulinic acid and butein. However, butein, betulin, and betulinic acid inhibited the production of ROS by HSCs treated with acetaldehyde and inhibited their migration. Butein also inhibited acetaldehyde-induced TGF-β1 production. Butein, betulin, and betulinic acid down-regulated acetaldehyde-induced production of TIMP-1 and TIMP-2. Betulin decreased the acetaldehyde-induced activity of MMP-2, but butein and betulinic acid did not. The results indicated that butein, betulin, and betulinic acid inhibited the acetaldehyde-induced activation of HSCs. Each drug functioned in a different manner, whereby some were acting as either antioxidants or inhibitors of TIMPs expression and butein additionally acted as an inhibitor of TGF-β production.     

Synthesis and cytotoxicity of triterpenoids derived from betulin and betulinic acid via click chemistry

In this study, a series of triazole substituted betulin and betulinic acid derivatives was designed and synthesized via click chemistry at C-30 position. Eighteen target compounds were evaluated in vitro for their antitumor activities against leukemia cell-line HL-60. Seventeen compounds have not reported before. The cytotoxic experiment showed that most of betulinic acid derived triazoles have higher cytotoxic profile than betulinic acid. Among them, compound 30-[4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl] betulinic acid (7b) showed the best IC₅₀ value (1.3 μM) against leukemia cell-line HL-60 (eight- to ninefold higher potency than betulinic acid).     

Betulin binds to γ-aminobutyric acid receptors and exerts anticonvulsant action in mice

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter γ-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA_A-receptor sites in mice brain in vitro and antagonised the GABA_A-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA_A receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA_A-receptor-related properties of lupane type triterpenes.These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.     

Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter γ-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA_A-receptor sites in mice brain in vitro and antagonised the GABA_A-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA_A receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA_A-receptor-related properties of lupane type triterpenes.These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.     

Synthesis and Anticancer Activity of Novel Betulinic acid and Betulin Derivatives

A series of novel betulinic acid derivatives 3–11 and betulin derivatives 12–17 were synthesized. The compounds were characterized by the means of ¹H‐ and ¹³C‐NMR spectroscopy as well as mass spectrometry. The compounds have been tested on ten tumor cell lines of different histogenic origin. The most active derivatives, containing a chloroacetyl group on C‐3 in betulinic acid 9 and C‐28 in betulin 15 , were up to ten times more cytotoxic and many fold more selective towards tumor cells in comparison to normal cells (fibroblasts) than betulinic acid. Furthermore, compound 15 was found to possess cell growth inhibition even when treated for a short time on anaplastic thyroid cancer cells (SW1736).     

Protective effects of betulin and betulinic acid against ethanol-induced cytotoxicity in HepG2 cells

Plant triterpenes, such as oleanolic acid and betulin were described as hepatoprotectants active against cytotoxicity of acetaminophen or cadmium. The aim of this paper is to compare the cytoprotective activity of betulin, betulinic acid and oleanolic acid against ethanol-induced cytotoxicity in HepG2 cells. The influence of three triterpenes on ethanol-induced production of superoxide anion and hydrogen peroxide was also examined. Among the examined triterpenes, betulin was the most active protectant of HepG2 cells against ethanol-induced cytotoxicity. Betulin and betulinic acid significantly decreased ethanol-induced production of superoxide anion. Oleanolic acid inhibited only ethanol- and phorbol ester-induced production of hydrogen peroxide. The results indicate that cytoprotective or antioxidative activity of triterpenes depends on their chemical structure.     

Advances in the study of structural modifications and biological activities of betulinic acids

Betulinic acids are lupine-type pentacyclic triterpenoid saponins commonly found in some plants of Betulaceae family, especially in the bark of betula alba (birch). The potent anti-HIV and anti-tumor activities of betulinic acids have been greatly concerned. The natural betulinic acids include betulinic acid, 23-hydroxy betulinic acid, betulin and so on. Some investigations on the structural modifications of betulinic acids were carried out, and many derivatives with excellent biological activity have been obtained nowadays. In this paper, the research advances of the structural modification of betulinic acids, as well as their anti-HIV and anti-tumor activities are reviewed.     

Simultaneous determination of betulin and betulinic acid in white birch bark using RP-HPLC

A simple procedure is described for the simultaneous extraction and determination of betulin and betulinic acid in white birch bark. The extraction was checked using different solvents: dichloromethane, ethyl acetate, acetone, chloroform, methanol and 95% ethanol (aqueous solution, v/v). It was found 95% ethanol was a good extraction solvent that allowed extraction of triterpenoid with a highest content. Separation was achieved on a reversed phase C(18) column with acetonitrile-water 86:14 (v/v). Detection was accomplished with UV detection at lambda=210 nm. Using this method, the bioactive triterpenoid in white birch bark were simultaneously determined. Significant variations in the content of betulin and betulinic acid in white birch bark growing in different locations of China were also observed.     

Differential effect of betulin and betulinic acid on cytokine production in human whole blood cell cultures

Betulin and betulinic acid, plant-derived triterpenoid compounds, have been described to possess anti-inflammatory activity. In this paper, we examine the ability of both compounds to induce and modulate cytokine production in human whole blood cell cultures. The results indicate that betulin is a modest TNF-alpha inducer and also an enhancer of mitogen-induced TNF-alpha production. In contrast to betulin, betulinic acid is a modulator of cytokine production by Th1/Th2 cell subpopulations which slightly enhances IL-10 formation and inhibits IFN-gamma production, reducing IFN-gamma/IL-10 ratio from 3.6 to 2.6.     

白桦酸类化合物的研究进展

白桦酸类化合物是一类可以从多种植物中分离得到的天然产物，包括白桦酸、23-羟基白桦酸、白桦素及其衍生物等，是一类结构及作用机制相对新颖、具有抗HIV-1和抗肿增活性的天然化合物。对这类化合物的结构修饰和改造，目前已经获得大量具有良好生物活性的衍生物。现对这方面的研究进展进行综述。     

3D-QSAR studies on betulinic acid and betulin derivatives as anti-HIV-1 agents using CoMFA and CoMSIA

3D-QSAR studies were performed on a series of betulinic acid and betulin derivatives with anti-HIV-1 activities. 3D-QSAR models of analysis A and analysis B, which were related to two activity indexes EC₅₀ and TI, respectively, were built by using CoMFA and CoMSIA. The analysis A resulted from 41 molecules gave r _cv² values of 0.664 and 0.718, r ² values of 0.979 and 0.955, respectively. The analysis B also resulted from 41 molecules gave r _cv² values of 0.570 and 0.559, r ² values of 0.938 and 0.933, respectively. The contour maps generated from the two analyses provided the regions in space where interactive fields may influence the activity. The results could serve as a useful guide for the design of potential betulinic acid and betulin derivatives with better anti-HIV-1 activity.     

Aryl Hydrocarbon Receptor Agonists in European Herring Gull (Larus argentatus) Eggs From Norway

Herring gull eggs from two locations in Norway, an island situated in the north (Musvær, 69.88° N, 18.55° E) and an island in the southeast (Reiaren, 59.15° N, 10.46° E) of the country, were analyzed for the presence of aryl hydrocarbon receptor (AhR) agonists. AhR agonist activity was determined using the dioxin-responsive chemically activated luciferase expression (DR-CALUX) assay to calculate the toxic equivalent quotient (TEQ)_CALUX. TEQ_CALUX ranged from 16 to 401 pg TEQ/g lipid in the samples from the north (n = 11) and between 6 and 360 pg TEQ/g lipid (n = 12) in the southeastern samples. The large variance between the individual samples is postulated to be due to different feeding habits of individual birds. The levels of AhR agonists detected might lead to adverse effects for the developing embryo or to a significant increase of contaminant load for human consumers of eggs.     

接骨木中的三萜类化合物及其对类成骨细胞UMR106的作用

目的用大鼠类成骨细胞UMR106的增殖作为活性追踪指标,对接骨木茎枝的体积分数为60%的乙醇提取物进行抗骨质疏松活性成分的追踪分离;初步考察化合物对UMR106细胞增殖、分化的影响。方法运用各种现代分离手段对活性部位进行追踪分离,用光谱学方法对得到的化合物进行结构鉴定;用比色法测定UMR106细胞的增殖和碱性磷酸酶活性。结果分离得到了5个三萜苷元类化合物,包括白桦醇(betulin,1)、白桦酸(betulinic acid,2)、齐墩果酸(oleanolic acid,3)、熊果酸(ursolic acid,4)、α香树脂醇(αamyrin,5),同时还分离到了2个甾醇类化合物,豆甾醇(stigmasterol,6)、胡罗卜苷(sitosterol 3 glucoside,7)。结论化合物1~3均为首次从接骨木中分离得到,其中化合物1、2可以促进大鼠类成骨细胞UMR106的增殖,化合物1对UMR106细胞碱性磷酸酶的活性也有促进作用。     

HPLC法测定西伯利亚接骨木中白桦脂酸含量

目的建立西伯利亚接骨木中白桦脂酸的含量测定方法,为完善西伯利亚接骨木的质量标准提供理论依据。方法采用HPLC法。色谱柱:Agilent?C18(250mm×4.6mm,5μm);流动相:乙腈-1mL·L~(-1)磷酸水溶液(90∶10);柱温:25℃;流速:1.0mL·min~(-1);检测波长:210nm。结果白桦脂酸质量浓度在0.02~0.32mg·mL~(-1)范围内线性关系良好(r=0.999 9),平均回收率为99.6%,RSD值为1.34%(n=9),白桦脂酸平均含量为0.31mg·g-1。结论该方法简便、准确、可靠,可作为西伯利亚接骨木药材白桦脂酸的含量测定方法。     

Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line

The mitochondrion plays a crucial role in the process of apoptosis and has thus become one of the targets for the search for potential chemotherapeutic agents. Betulinic acid [3beta-hydroxy-lup-20(19)lupaen-28-carbonic acid], a lupane-type triterpene which is abundant in many plant species, has been shown to exert a direct effect on the mitochondria and subsequent apoptosis in melanoma cells. Chemical synthesis and modification of betulinic acid are being explored to develop more potent derivatives. We present here the apoptotic activity of several natural derivatives of betulinic acid which were isolated from the roots of a Chinese medicinal herb, Pulsatilla chinensis (Bge) Regel [Ye, W., Ji, N.N., Zhao, S.X., Liu, J.H., Ye, T., McKervey, M.A., Stevenson, P., 1996. Triterpenoids from Pulsatilla chinensis. Phytochemistry 42, 799-802]. Of the five compounds tested, 3-oxo-23-hydroxybetulinic acid was the most cytotoxic on murine melanoma B16 cells (IC50=22.5 microg/ml), followed by 23-hydroxybetulinic acid and betulinic acid (IC50=32 and 76 microg/ml, respectively), with lupeol and betulin exhibiting the weakest cytotoxicity (IC50> or =100 microg/ml). Exposure of B16 cells to betulinic acid, 23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid caused a rapid increase in reactive oxidative species production and a concomitant dissipation of mitochondrial membrane potential in a dose- and time-dependent manner, which resulted in cell apoptosis, as demonstrated by fluorescence microscopy, gel electrophoresis and flow-cytometric analysis. Cell cycle analysis further demonstrated that both 3-oxo-23-hydroxybetulinic acid and 23-hydroxybetulinic acid dramatically increased DNA fragmentation at the expense of G1 cells at doses as low as 12.5 and 25 microg/ml, respectively, thereby showing their potent apoptotic properties. Our results showed that hydroxylation at the C3 position of betulinic acid is likely to enhance the apoptotic activity of betulinic acid derivatives (23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid) on murine melanoma B16 cells.     

Plant Anticancer Agents XXXVII. Constituents of Amanoa oblongifolia1

4'-Demethyldeoxypodophyllotoxin was isolated as the sole cytotoxic constituent of a chloroform-soluble extract of the stem bark of AMANOA OBLONGIFOLIA. Also identified from this extract were a number of noncytotoxic isolates that comprised the lignans, (+)-sesamin and paulownin, the triterpenes, friedelin, canophyllol, betulin, betulinic acid, and ursolic acid, and the sterols, beta-sitosterol and daucosterol.     

Further triterpenes from Melissa officinalis L (author's transl)

Further Triterpenes from Melissa officinalis L. Melissa off. contains as further triterpenes pomolic acid, three 2-hydroxy- and one 29-hydroxytriterpenic acid. The root contains betulinic acid, betulin and three 2-hydroxytriterpenic acids. The configuration of the 2,3-dihydroxytriterpenic acids was proved by synthesis of the four isomeric 2-hydroxy-derivatives of oleanolic acid, ursolic acid and α-amyrin.     

冬凌草的化学成分研究

目的 研究冬凌草的化学成分。方法 利用硅胶柱色谱、中压液相色谱、高效液相色谱等色谱技术对冬凌草甲醇提取物进行分离纯化,通过波谱数据分析,鉴定了化合物的结构,利用噻唑蓝(MTT)法测试化合物抗肿瘤活性。结果 从冬凌草中共分离鉴定6个化合物,分别为：rabdosianin A（1）、parvifoline G（2）、rabdoternin C（3）、betulin（4）、betulinic acid（5）、古柯二醇（6）。结论 化合物6为首次从香茶菜属中分离得到,化合物1、2、4、5为首次从冬凌草中分离得到。化合物5对人白血病细胞K562有较强的抑制作用,IC₅₀值为1.45 µmol·L^-1。     

Toxicological,chemopreventive, and cytotoxic potentialities of rare vegetal species and supporting findings for the Brazilian Unified Health System (SUS)

ABSTRACT

Caatinga flora which are found in a poor Brazilian region contain a substantial number of endemic taxa with biomedical and social importance for regional communities. This study examined the antioxidant and cytotoxic potential of 35 samples (extracts/fractions) from 12 Caatinga species and determined the antiproliferative and genotoxic action of dichloromethane fraction from Mimosa caesalpiniifolia stem bark (DC-Mca) on human and vegetal cells. Samples were assessed for chemopreventive ability, toxic effects on Artemia salina shrimp as well as cytotoxicity on tumor cell lines and erythrocytes. DC-Mca was also tested with respect to antiproliferative and genotoxic effects upon normal leukocytes and meristematic cells from A. cepa roots. Some extracts reduced free radical levels >95% and 7 samples exhibited a lethal concentration (LC) ₅₀ < 100 µg/ml upon Artemia salina larvae. Eight samples displayed in vitro antitumor effects and three produced hemolysis. Data also demonstrated the pharmacological significance of bioactive extracts from Brazilian semi-arid region. There was no significant relationship between antioxidant, toxic, and antiproliferative activities, and that these properties were dependent upon the extractant. DC-Mca contained betulinic acid as main compound (approximately 70%), which showed higher (1) cytotoxic activity on cancer cell lines and dividing leukocytes, (2) reduced mitotic index of Allium cepa roots, and (3) induced cell cycle arrest and chromosomal bridges, thereby providing native promising sources for phytotherapy development.