Studies on the Constituents of Chinese Traditional Drug Suan Zau Ren

Suan Zau Ren (the seeds of Zizyphus spinosus Hu) is a widely used chinese traditional drag for treatment of insomnia, neurasthenia and sometimes for sleepiness caused by physical emaciation in chinese traditional medicine. Its dichloromethane extract afforded four pentacyclic triterpenes of the lup-20(29)-en group, namely betulin (Ⅱ), betulinic acid(Ⅰ), ceanothic acid(Ⅲ), and alphitolic acid (Ⅳ), and one sterol, daucosterol (Ⅴ). Their structures were identified by the spectroscopic analysis. The later three compounds were isolated from Suan Zau Ren for the first time.     

Zizimauritic acids A–C,three novel nortriterpenes from Ziziphus mauritiana

Zizimauritic acids A–C (1–3), three novel nortriterpenes with a unique A-nor-E-seco spiro-lactone ceanothane-type triterpene skeleton, together with 3 known triterpenes ceanothenic acid (4), betulinic acid (5), and ceanothic acid (6), were isolated from the roots of Ziziphus mauritiana. Compounds 1–4 showed cytotoxicities with the IC₅₀ values ranging from 5.05 to 11.94 μg/ml, and compounds 1 and 3 showed an inhibitory effect on the growth of Staphylococcus aureus with the IC₅₀ values 2.17 and 12.79 μg/ml. A plausible biosynthetic pathway of compounds 1-3 was proposed.     

Lipid, fatty acid and protein utilization during lecithotrophic larval development of Lithodes santolla (Molina) and Paralomis granulosa (Jacquinot)

During the larval development of the subantarctic king crab, Lithodes santolla, and stone crab, Paralomis granulosa, we compared changes in the carbon, fatty acid and protein contents of larvae reared under constant conditions from hatching to metamorphosis, either in presence or absence of food (Artemia spp. nauplii). In both species the feeding condition had no influence on any of the chemical parameters studied, indicating a fully lecithotrophic (i.e. non-feeding) mode of development from hatching of the first zoea to metamorphosis of the late megalopa. Dry mass and carbon contents at hatching were similar in the larvae of both species, but L. santolla contained initially higher total amounts of fatty acids and protein than P. granulosa. Both species utilized considerable portions of their total fatty acid pool which decreased logarithmically throughout the time of development. At metamorphosis, it was almost exhausted in P. granulosa, while L. santolla had consumed only about 60%. Protein utilization, in contrast, was higher in L. santolla (40%) than in P. granulosa (20%). Triacylglycerol was the principal storage lipid in both species, accounting initially for about 75% of the lipid fraction; it was strongly utilized during larval development. Phospholipid constituted the second largest lipid class; it also decreased in P. granulosa, but to a lesser extent in L. santolla. The major fatty acids of both species were 18:1(n−9), 20:5(n−3) and 16:0 as well as, in lower proportions, 18:1(n−7), 22:6(n−3), 16:1(n−7) and 18:0. Monounsaturated fatty acids represented the dominant group in L. santolla, whereas P. granulosa contained similar amounts of mono- and polyunsaturated fatty acids. In L. santolla, monounsaturated fatty acids, especially 16:1(n−7), were preferentially utilized as compared to polyunsaturates. Due to a particularly strong lipid utilization in P. granulosa, all individual fatty acids were largely depleted at metamorphosis, showing similar extents of consumption. L. santolla had higher initial lipid and protein stores that seem to be used more economically as compared to P. granulosa.     

Synthesis of new betulinic acid/betulin-derived dimers and hybrids with potent antimalarial and antiviral activities

Severe malaria and viral infections cause life-threatening diseases in millions of people worldwide every year. In search for effective bioactive hybrid molecules, which may possess improved properties compared to their parent compounds, a series of betulinic acid/betulin based dimer and hybrid compounds carrying ferrocene and/or artesunic acid moieties, was designed and, synthesized de novo. Furthermore, they were analyzed in vitro against malaria parasites (growth inhibition of 3D7-strain P. falciparum-infected erythrocytes) and human cytomegalovirus (HCMV). From this series of hybrids/dimers, the betulinic acid/betulin and artesunic acid hybrids 11 and 12 showed the most potent activities against P. falciparum and HCMV. On the strength of results, additive and/or synergistic effects between the natural or semisynthetic products, such as betulinic acid-/betulin- and artesunic acid-derived compounds, are suggested on the basis of putatively complex modes of antimicrobial action. This advantage may be taken into account in future drug development.     

Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents

C-3 esterifications of betulinic acid (BA, 1) and its A-ring homolog, ceanothic acid (CA, 2), were carried out to provide sixteen terpenoids, 4–19, including nine new compounds (4–12). All synthesized compounds were evaluated in an in vitro antitumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among them, compounds 4–6, 11–14, 16, and 17 displayed remarkable inhibitory effects of EBV-EA activation. BA analog 6, which contains a prenyl-like group, showed the most potent inhibitory effect (100%, 76%, 37%, and 11% inhibition of EBA activation at 1000, 500, 100, and 10 mol ratio/TPA, respectively, with IC₅₀ value of 285 mol ratio/32 pmol TPA). Compound 6 merits further development as a cancer preventive agent.     

Effect of nitrogen-containing derivatives of the plant triterpenes betulin and glycyrrhetic acid on the growth of MT-4, MOLT-4, CEM,and Hep G2 tumor cells

Derivatives of the available plant triterpenes glycyrrhetic acid and betulin (betulin succinates and amides of betulonic and 18β-glycyrrhetic acids containing fragments of long-chain amino acids and a peptide) were synthesized. The inhibitory action of these compounds on the growth of MT-4, MOLT-4, CEM, and Hep G2 tumor cells and their effect on the apoptosis of these cells were studied. It was shown that betulonic acid amides are more effective inhibitors of the tumor cell growth than the corresponding amides of glycyrrhetic acid. It was also found that betulonic acid amides containing fragments of caprylic, pelargonic, and undecanoic acids are more effective inhibitors of tumor cell growth than betulinic acid. The 17-dipeptide derivative of betulonic acid N-{N-[3-oxo-20(29)-lupen-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid exhibited the maximum inhibitory activity toward the tumor cells studied. Data on the induction of apoptosis in tumor cells by betulin derivatives at a concentration of 10 μg/ml were obtained by flow cytometry. The amides of betulonic acid proved to be the most effective inducers of apoptosis.     

Distribution of some triterpenes and phenolic compounds in the extractives of endemic dipterocarpaceae species of Sri Lanka

Extractives of bark and/or timber of 11 species belonging to the genera Cotylelobium, Hopea, Shorea, Vateria and Vatica yielded a fatty-acid ester, a sitosteryl ester, β-amyrin acetate, β-amyrin, dipterocarpol, ursolic acetate, lupeol, sitosterol, ursolic acid, betulinic acid, hexamethyl-coruleoellagic acid, tetramethylellagic acid, chrysophanol and scopoletin. The distribution of these compounds in 18 other species was examined by TLC screening.     

Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus

Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome (http://www.ncbi.nlm.nih.gov/dbEST), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.     

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5?mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000?μg/mL respectively. The compound (50 and 100?mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90?min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100?mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4?h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.     

Synthesis and Antiviral Activity of Hydrazides and Substituted Benzalhydrazides of Betulinic Acid and Its Derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N"-benzalhydrazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

NMR-based metabolomic analysis of the seasonal and regional variability of phytochemical compounds in Curtisia dentata stem bark

Traditional medicine markets are provided with medicinal plant material throughout the year, however, internal (e.g. plant age, genetic variability and differential expression of genes) and external factors (e.g. water and nutrient availability, rainfall, photoperiod and herbivory), affect secondary metabolite production in plants. In this study, seasonal variability in metabolite production in Curtisia dentata trees from two geographically separated regions in South Africa are compared. NMR analysis of C. dentata stem bark samples yielded spectral data which were processed in MestReNova to perform multivariate data analysis using Soft Independent Modeling of Class Analogy (SIMCA) software. This study shows that there are not only seasonal, regional and yearly differences in secondary metabolite production in C. dentata trees, but that production patterns of hydrophilic and lipophilic chemical compounds in individual trees also vary. Sucrose, isoeugenol and betulinic acid have been used in a targeted analysis to show the variation in individual compounds in the individual trees as a response to seasonal and geographical differences. Therefore, the season and year, as well as the region, harvesting site and specific trees from which plant material is collected affect the concentrations of chemical compounds extracted from C. dentata stem bark for the preparation of remedies.     

Topical anti-inflammatory activity of 2alpha-hydroxy pentacyclic triterpene acids from the leaves of Ugni molinae

Leaf extracts of Ugni molinae Turcz. are used in the Chilean cosmetic industry on the assumption that they have decongestant, regenerative, and anti-aging properties. A bioassay-guided fractionation of this plant material showed that some extracts have potent anti-inflammatory activities. Further fractionation led to the isolation and identification of betulinic acid, a mixture of ursolic and oleanolic acids, and the 2alpha-hydroxy derivatives alphitolic, asiatic, and corosolic acids. The latter three were evaluated in vivo in the mouse ear assay for their topical anti-inflammatory activity, inducing inflammation with either arachidonic acid (AA) or 12-O-tetradecanoylphorbol-13 acetate (TPA). Only corosolic acid was active in the AA assay, with similar potency to nimesulide, but all three triterpene acids inhibited TPA-induced inflammation with potencies comparable to that of indomethacin.     

Semisynthesis,cytotoxicity, antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives

In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was the most active compound (IC₅₀?=?3.4?µM) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC₅₀?>?400?µM), showing selectivity towards parasites (selectivity index?>?117.47). In combination with artemisinin, compound 7b showed an additive effect (CI?=?1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of ?7.02?kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC₅₀?=?93?µM) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3β-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.     

Synthesis and activity of new triphenylphosphonium derivatives of betulin and betulinic acid against Schistosoma mansoni in vitro and in vivo

We studied the antischistosomal activity of betulin, betulinic acid and its 9 triphenylphosphonium derivatives characterized by a covalently linkage of the hydrophobic fragment of triterpenoid at C(2)- or C(30)-position with the triphenylphosphonium moiety via a hydrocarbon bridge. The triphenylphosphonium salts showed in vitro antischistosomal activity against newly transformed schistosomula (NTS) and adult worms of Schistosoma mansoni at low micromolar concentrations. In contrast betulin and betulinic acid were inactive against NTS and adult S. mansoni. Of the 9 triphenylphosphonium derivatives tested, the allyl salts 10 (IC₅₀ of 0.76 μg/mL) and 11 (IC₅₀ of 0.64 μg/mL) demonstrated the highest antischistosomal activity against adult S. mansoni. Low worm burden reductions of 22% were observed in vivo for these two compounds. In conclusion, triphenylphosphonium derivatives were obtained from available natural betulin by simple transformations, rendering it practical and useful for large scale application. However, further structural modifications are necessary to translate the promising antischistosomal in vitro activities into in vivo.     

Two new triterpenoids from Rhodomyrtus tomentosa

Repetition of an investigation of the petrol extracts of Rhodomyrtus tomentosa has led to the isolation of two new triterpenoids, R₄ from the leaves and R₅ from the stems besides R₁, R₂, R₃ and the other known compounds already reported. R₁ and R₄ were proved to be 21α$\text{H}$-hop-22(29)en-3β,30-diol and 3β-hydroxy-21α$\text{H}$-hop-22(29)-en-30-al respectively, and R₂, R₃ and R₅ are 3β-acetoxy-11α,12α-epoxyoleanan-28,13β-olide, 3β-acetoxy-12α-hydroxyoleanan-28, 13β-olide and 3β-acetoxy-12-oxo-oleanan-28,13β- olide respectively. The ethanol extract of the leaves contained betulinic, ursolic and aliphitolic acids and that of the stems betulonic, betulinic and oleanolic acids.     

A new Alokistocaridae Resser, 1939 (Trilobita) from the middle Cambrian of Spain

A new trilobite species, Schopfaspis? graciai, from the middle Cambrian of Spain is the first member of Alokistocaridae reported from west Gondwana. A cladistic analysis of this trilobite and other Gondwanan trilobites of possible alokistocarid affinities (Schopfaspis granulosa, Chelidonocephalus anatolicus, Derikaspis toluni, Kounamkites multiformis) suggests that this family can be divided into two subfamilies: Alokistocarinae and Altiocculinae. Schopfaspis? graciai nov. sp. and Schopfaspis granulosa are assigned to the subfamily Altiocculinae laying in a more basal position than Altiocculus species. The cladistic analysis also demonstrates a possible relationship of Chelidonocephalus anatolicus and Derikaspis toluni with Alokistocaridae. Enrolment is analysed in this new trilobite describing novelties in the ventral surface of the cephalon which allowed interlocking of the trunk and cephalon in a discoidal enrolment-type.     

Conjugation of antifungal benzoic acid derivatives as a path for detoxification in Penicillium brasilianum,an endophyte from Melia azedarach

In this study, the consumption of 4-bromobenzoic acid and 4-chlorobenzoic acid by the fungus Penicillium brasilianum, an endophyte from Melia azedarach is evaluated. This fungus metabolizes these halobenzoic acids to produce three new brominated compounds, which have been isolated and characterized, and three new chlorinated derivatives identified by HRMS. Among these products, (4-bromobenzoyl)proline has been also chemically synthesized and employed in biological assays, thus providing insights for the elucidation of the defense mechanism of P. brasilianum towards these halobenzoic acids.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

New unsaturated fatty acid and other chemical constituents from the roots of Cola rostrata K. Schum. (Malvaceae)

Phytochemical investigation of the roots of Cola rostrata K. Schum. led to the isolation of a new unsaturated fatty acid, named rostratanic acid (1), together with fourteen known compounds, lignoceric acid, friedelan (7), friedelanone (8), bauerenol (3), lupeol (4), herranone (9), acotatarone A (11), betulinic acid (6), betulin (5), nonanedioc acid (2), arjunolic acid (10) stigmasterol, β−sitosterol, and β−sitosterol-3-O-β-D-glucopyranoside. The structure of the new compound as well as those of the known compounds were established by means of spectroscopic methods: NMR analysis (¹H and ¹³C NMR, ¹H–¹H–COSY, HSQC and HMBC) and high-resolution mass spectrometry (HR-ESI-MS), and by comparison with previously reported data. Two of those known compounds were modified chemically to afford three new derivatives. All those compounds were tested for their cytotoxic activity against the human cervix carcinoma KB-3-1 cells and their antibacterial activity against Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Staphylococcus aureus. Although the crude extract gave weak antibacterial activity, none of the isolated compounds showed antibacterial activity, and, only the prenylated derivative showed weak cytotoxicity. In addition, the chemotaxonomic significance of the species Cola rostrata is discussed.     

Synthesis and Antiviral Activity of Ureides and Carbamates of Betulinic Acid and Its Derivatives

Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by interaction of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols. Ureides of betulonic acid containingL-Val and L-Met residues were found to be effective against herpes simplex type 1 virus.