Protective effect of lupeol and lupeol linoleate in hypercholesterolemia associated renal damage

The association between hypercholesterolemia and kidney damage has been well known for last few decades. The oxidative stress and inflammatory responses are involved in renal injury, which is upregulated in hypercholesterolemic condition. The present study is aimed to evaluate the possible effect of lupeol and its ester derivative, lupeol linoleate in renal damage associated with hypercholesterolemic rats. Hypercholesterolemia was induced in male Wistar rats by feeding them with a high cholesterol diet (HCD) comprising normal rat chow supplemented with 4% cholesterol and 1% cholic acid for 30 days. Lupeol and lupeol linoleate were supplemented (50 mg/kg body wt/day) to HCD fed rats during the last 15 days. Increased levels of renal total cholesterol, triglycerides and phospholipids, along with altered serum biochemical parameters of tissue injury indices and elevated activities of renal marker enzymes (lactate dehydrogenase and alkaline phosphatase) were noted in HCD fed rats. Elevated lipid peroxidation levels coupled with decreased antioxidant status (enzymatic and non enzymatic antioxidants) were observed in hypercholesterolemic rats, which indicate the onset of oxidative changes in the renal tissue. Renal lysosomal acid hydrolase activities (ACP, beta-Glu, beta-Gal, NAG and Cat-D) and acute phase proteins like C-Reactive protein and fibrinogen were significantly increased in HCD fed rats, which further indicates the heightening of inflammation. In addition, histopathological findings also confirmed the renal damage in hypercholesterolemic condition. Lupeol and lupeol linoleate effectively reverted the above abnormalities and was comparable with that of the control. These observations highlight the protective effect of lupeol and its ester derivative in ameliorating the renal injury associated with hypercholesterolemia.     

Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.     

Mitigating role of lupeol and lupeol linoleate on hepatic lipemic-oxidative injury and lipoprotein peroxidation in experimental hypercholesterolemia

In the present study, the role of pentacyclic triterpenes, lupeol and its ester lupeol linoleate, was studied in relation to hepatic oxidative abnormalities and lipoprotein peroxidation in hypercholesterolemic rats. Hypercholesterolemia was induced in male Wistar rats by feeding them with high cholesterol diet (4% cholesterol + 1% cholic acid; HCD) for 30 days. Pentacyclic triterpenes, lupeol and lupeol linoleate were supplemented (50 mg/kg body wt/day) during the last 15 days. After the experimental period, there was a significant depression in hepatic activities of antioxidant enzymes, SOD (38.39%), CAT (25.03%) and GPx (30.26%) along with a marked fall in the levels of non-enzymic antioxidant molecules GSH (31.39%), vitamin C (46.07%) and vitamin E (42.28%), with a concomitant increase (p<0.001) in lipid peroxidation and in the activities of serum alkaline phosphatase, lactate dehydrogenase and aminotransferases when compared to controls. Treatment with triterpenes decreased lipid peroxidation and reverted the activities of antioxidants (p<0.001 and p<0.01) and marker enzymes to near control. Histopathological findings further confirmed the hepatoprotective nature of triterpenes by showing the normal architecture in treated rats, as against the fatty cellular changes in HCD fed rats. Further, the susceptibility of apo-B containing lipoprotein to oxidation by copper and Fenton’s reagent was increased in in vitro condition in HCD fed rats, whereas the lipoproteins were less susceptible to oxidation in triterpenes treated animals. Therefore, it may be concluded that lupeol and its ester afford protection against the hepatic abnormalities and lipoprotein peroxidation in hypercholesterolemic rats.     

Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis

Pentacyclic triterpenoids are a large group of secondary metabolites found in many different plant species, either as glycoside conjugates or as aglycones. The latter in many cases accumulate to high amounts in the cuticular wax and hence at the surface of plant organs. In the present work, the cuticle-specific formation of triterpenoids was investigated in Ricinus communis stems, combining analytical and molecular genetic methods. Two phenotypes of castor bean could be distinguished based on the glaucous or glossy appearance of the surfaces of all stem portions including the hypocotyls, and were due to the presence or absence of thread-shaped epicuticular wax crystals, respectively. Comparative studies showed that these crystals are formed by the triperpenoid lupeol, present in high amounts on all stem surfaces. On the hypocotyl portion of stems, lupeol was found to accumulate rapidly during early development of the surface (10-15 days after emergence). Mature hypocotyls of glossy individuals were covered with 12.5 microg/cm2 of wax containing approximately 1% of lupeol, whereas the glaucous phenotype had a wax load of 51.9 microg/cm2 with 56% of lupeol. Two oxidosqualene cyclases from castor bean were cloned, functionally expressed in yeast, and characterized as a cycloartenol synthase (RcCAS) and a lupeol synthase (RcLUS). Phylogenetic analyses revealed that RcLUS is similar to two clades of known lupeol synthases, but also exhibits some similarities with beta-amyrin synthases. Both the organ-specific expression of RcLUS and the expression pattern during hypocotyl development exactly matched the accumulation of cuticular lupeol in castor bean. In contrast, RcCAS was constitutively expressed in all organs at various times. We conclude that the RcLUS enzyme is responsible for formation of the cuticular lupeol, and thus for the characteristic surface properties of R. communis stems.     

Lupeol long-chain fatty acid esters and other triterpenoid constituents from Plumeria obtusifolia

From the bark of Plumeria obtusifolia was isolated a series of lupeol fatty esters with the carbon numbers 16, 18,20 and 21–28 in the fatty acid part. Furthermore, lupeol, lupeol acetate, sitosterol, stigmasterol and campesterol were also identified.     

Lupeol esters from the twig bark of Japanese pear (Pyrus serotina Rehd.) cv. Shinko.

Five new lupeol esters, lup-20(29)-ene-3beta-yl eicosanoate, docosanoate, tetracosanoate, hexacosanoate and octacosanoate, were isolated as a mixture from the twig bark of Japanese pear (Pyrus serotina Rehd.) cv. Shinko, together with lupeol and epifriedelinol. Their structures were determined by spectral analyses including 2D-NMR experiments.     

构树皮的化学成分研究

通过反复硅胶柱层析从构树根皮乙醇提取物石油醚部位中分离得到10个化合物,利用波谱方法分别鉴定为β-谷甾醇(1),lupeol acetate(2),6,7-二甲氧基香豆素(3),Broussonin A(4),Broussonin B(5),Broussonin F (6),(+) marmesin(7),(+)-(2...     

Lupeol and its ester ameliorate the cyclophosphamide provoked cardiac lysosomal damage studied in rat

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes fatal cardiotoxicity. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP-induced myocardial toxicity in rats. Male albino rats of Wistar strain were categorized into six groups. Group I served as control. Rats in groups II, V and VI animals were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP-treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight), respectively, dissolved in olive oil for 10 days by oral gavage. CP-administered rats showed a significant increase (p < 0.001) in the activities of lysosomal hydrolases in serum and heart, a decrease (p < 0.001) in the levels of cellular thiols and myofibres were swollen with loss of myofilaments in electron microscopical analysis in heart. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve lysosomal integrity, improve thiol levels, highlighting their protective effect against CP-induced cardiotoxicity.     

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.     

Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.     

Lupeol reduces triglyceride and cholesterol synthesis in human hepatoma cells

Lupeol suppressed triglyceride and cholesterol secretion from HepG2-Lipo human hepatoma cells in a dose-dependent manner. Quantitative real-time RT-PCR analysis demonstrated that lupeol inhibited the expressions of sterol regulatory element-binding protein-1c and -2, fatty acid synthase, 3-hydroxy-3-methylglutaryl-Coenzyme A synthetase-1, and farnesyl-diphosphate farnesyl transferase-1, which are required for lipid synthesis in HepG2-Lipo cells. Furthermore, lupeol markedly inhibited apolipoproteinB-100 and microsomal triglyceride transfer protein in cells at the mRNA level. These results suggest that lupeol lowers lipid secretion from HepG2-Lipo cells by attenuating synthesis within the cells.     

解脂耶氏酵母中羽扇豆醇合成途径的构建与调控

羽扇豆醇因其具有抗癌抗炎等生理活性而广泛应用于医药领域.本研究分别利用源自木榄和蓖麻的羽扇豆醇合酶(LUS)在解脂耶氏酵母(Yarrowia lipolytica)中构建生物合成羽扇豆醇途径(GLU-1、GLU-2),并由对该途径中关键限速酶3-羟基-3-甲基戊二酰辅酶a还原酶(tHMGR)和异戊烯基二磷酸异构酶(ID...     

Lupeol prevents acetaminophen-induced in vivo hepatotoxicity by altering the Bax/Bcl-2 and oxidative stress-mediated mitochondrial signaling cascade

AimsLupeol, a triterpene, possesses numerous pharmacological activities, including anti-malarial, anti-arthritic and anti-carcinogenic properties. The present study was conducted to explore the hepatoprotective potential of lupeol against acetaminophen (AAP)-induced hepatotoxicity in Wistar rats.Main methodsRats were given a prophylactic treatment of lupeol (150 mg/kg body weight, p.o., for 30 consecutive days) with a co-administration of AAP (1 g/kg body weight). The modulatory effects of lupeol on AAP-induced hepatotoxicity were investigated by assaying oxidative stress biomarkers, serum liver toxicity markers, pro/anti apoptotic proteins, DNA fragmentation and by the histopathological examination of the liver.Key findingsLupeol significantly prevented hepatic damage as evident from the histopathological studies and significant decline in serum trans-aminases. The alterations in cellular redox status (p < 0.01) and antioxidant enzyme activities together with the enhanced lipid peroxidation and protein carbonyl levels were also observed in the AAP-treated rats. In addition, significant ROS generation and mitochondrial depolarization were observed in this group. Co-administration of lupeol significantly decreased the level of serum transaminases, MDA and protein carbonyl content. It also prevented ROS generation and mitochondrial depolarization. Furthermore, lupeol enhanced the mitochondrial antioxidant and redox status and inhibited DNA damage and cell death by preventing the downregulation of Bcl-2, upregulation of Bax, release of cytochrome c and the activation of caspase 9/3.SignificanceThe conclusion of this study is that lupeol when co-administered with AAP effectively reduces oxidative stress and prevents AAP-induced hepatotoxicity by inhibiting critical control points of apoptosis.     

Triterpenoids of Amsonia grandiflora

Stems and leaves of Amsonia grandiflora yielded lupeol β-hydroxyoctadecanoate, betulinic acid, oleanolic acid, lupeol, lupeol acetate and 1-O-methyl-myo-inositol.     

Lupeol and its ester inhibit alteration of myocardial permeability in cyclophosphamide administered rats

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes cardiac membrane damage. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP induced alterations in cardiac electrolytes in rats. Male albino rats of Wistar strain were categorized into 6 groups. Group I served as control. Rats in groups II, V and VI were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight) respectively, dissolved in olive oil for 10 days by oral gavage. At the end of the experimental period, urinary risk factors, activities of ATPases and electrolytes were measured using standard procedures. CP administered rats showed a significant decrease (P < 0.001) in the activities of ATPases. It was associated with significant alterations (P < 0.001) of electrolytes both in serum and cardiac tissue. The levels of urea, uric acid and creatinine were also significantly (P < 0.001) altered in the serum and urine. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve membrane permeability, highlighting their protective effect against CP induced cardiotoxicity.     

Lupeol ameliorates aflatoxin B1-induced peroxidative hepatic damage in rats

Aflatoxins are potent hepatotoxic and hepatocarcinogenic agents. Reactive oxygen species and consequent peroxidative damage caused by aflatoxin are considered to be the main mechanisms leading to hepatotoxicity. The present investigation aims at assessing the hepatoprotective effect of lupeol, a pentacyclic triterpene isolated from the stem bark of Crataeva nurvala, on aflatoxin B(1) (AFB(1))-induced hepatotoxicity in a rat model. The hepatoprotection of lupeol is compared with silymarin, a well known standard hepatoprotectant. Lactate dehydrogenase, alkaline phosphatase, alanine and aspartate aminotransferases were found to be significantly increased in the serum and decreased in the liver of AFB(1) administered (1 mg/kg body mass, orally) rats, suggesting hepatic damage. Marked increase in the lipid peroxide levels and a concomitant decrease in the enzymic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) and nonenzymic (reduced glutathione, vitamin C and vitamin E) antioxidants in the hepatic tissue were observed in AFB(1) administered rats. Pretreatment with lupeol (100 mg/kg body mass, orally) and silymarin (100 mg/kg body mass, orally) for 7 days reverted the condition to near normalcy. Hepatoprotection by lupeol is further substantiated by the normal histologic findings as against degenerative changes in the AFB(1) administered rats. The results of this study indicate that lupeol is a potent hepatoprotectant as silymarin.     

Remodeling of actin cytoskeleton in lupeol-induced B16 2F2 cell differentiation

Lupeol induces the formation of dendrites in B16 2F2 melanoma cells. The remodeling of cytoskeletal components contributes to the dendricity of melanoma cells. We studied the effects of lupeol on the remodeling of cytoplasmic filaments in B16 2F2 cells. Western blotting revealed no change in the levels of actin and tubulin. Lupeol attenuated stress fiber assembly, but did not promote the remodeling of microtubular networks. We examined the activation of cofilin, an actin-depolymerizing factor, in lupeol-treated B16 2F2 cells by western blotting. The level of phospho-cofilin was found to decrease in a time-dependent manner. Inhibition of p38 MAPK by SB203580 blocked tyrosinase induction by lupeol, but did not influence the disruption of stress fiber assembly or the dephosphorylation of cofilin. Furthermore, we studied the effects of lupeol on cell migration. At 10 microM, lupeol markedly inhibited the haptotaxis of B16 2F2 cells to fibronectin. Additionally, lupeol strongly inhibited the migration of human melanoma and neuroblastoma cells, and weakly suppressed the migration of lung adenocarcinoma cells. However, lupeol did not affect the motility of other cancer cells. The results suggest that lupeol suppresses the migration of malignant melanoma cells by disassembling the actin cytoskeleton.     

构树皮中三萜类化学成分

对构树皮的化学成分进行进一步研究,通过反复硅胶柱层析从构树皮乙醇提取物的石油醚萃取部位中分离得到六个三萜成分,利用波谱技术及理化性质鉴定其化学结构分别为3β-乙酰氧基-甘遂-7-烯-24S,25二醇(1),3β-乙酰氧基-7,23-甘遂二烯-25-醇(2),羽扇豆醇(3),α-香树脂醇乙酸酯(4),3β,25-二羟基-7,23-甘遂二烯(5)和β-香树脂醇(6)。其中化合物1为新的甘遂烷型三萜,化合物2～5为首次从该植物中分离得到。     

Lupeol supplementation improves blood pressure and lipid metabolism parameters in stroke-prone spontaneously hypertensive rats

Supplementation with lupeol (0.67 g·kg(-1)) of the AIN-93M-based diet fed for 7 weeks to stroke-prone spontaneously hypertensive rats caused significantly decreased blood pressure as compared with a control group. Urinary 8-hydroxy-2'-deoxyguanosine was significantly lower in the lupeol group. Finally, lupeol suppressed the hepatic mRNA expression levels of the genes involved in triglyceride and cholesterol synthesis.