Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima

Chemical investigation of the aerial parts of Phyllanthus acutissima resulted in the isolation of five new dichapetalin-type triterpenoids, acutissimatriterpenes A-E ( 1- 5), and two new lignans, acutissimalignans A ( 6) and B ( 7), along with two known lignans and three known ellagic acid derivatives. The structures of 1- 7 were determined mainly on the basis of spectroscopic methods. The compounds obtained were evaluated for cytotoxic and anti-HIV-1 activities.     

Three novel mutations causing complete T(4)-binding globulin deficiency

Inherited T(4)-binding globulin deficiency is caused by mutations in the T(4)-binding globulin gene located on the X chromosome. We describe herein three novel mutations in three different families producing complete T(4)-binding globulin deficiency. The proposita of a family from Harwichport is a female with XO Turner's syndrome who expressed only the mutant T(4)-binding globulin allele. Her T(4)-binding globulin sequence has a 19-nucleotide deletion in the distal portion of exon 4. This causes a frameshift and a premature stop at codon 384 of the mature protein. Structure analysis with the Swiss PDB-Viewer revealed that this mutation removes beta-strand s5B from the core of the T(4)-binding globulin molecule, leading to a severe folding defect that is likely to prevent synthesis and secretion. The propositi of complete T(4)-binding globulin deficiency 7 and 8 were 7-month-old and 3-wk-old male infants who were identified because of low serum T(4) levels detected during neonatal screening. Sequencing of complete T(4)-binding globulin deficiency 7 revealed a single nucleotide deletion, a G at position 2690 in exon 3. This leads to an alteration of the amino acid sequence starting at codon 283 and a premature stop at codon 301. Complete T(4)-binding globulin deficiency 8 also has a deletion of the first nucleotide of exon 4, a G at position 3358. This leads to a frameshift and a premature stop at codon 374. As in the case of complete T(4)-binding globulin deficiency J, which has also a nucleotide deletion but downstream (position 3421) and a stop at codon 374, these two T(4)-binding globulin mutants undoubtedly have a defect in intracellular transport and therefore fail to be secreted. This explains the lack of T(4)-binding globulin in the hemizygous affected subjects.     

Anti-inflammatory and antipyretic properties of Clerodendrum petasites S. Moore

The methanol extract from Clerodendrum petasites S. Moore (CP extract) was assessed for anti-inflammatory and antipyretic activities on the experimental animal models. It was found that CP extract possessed moderate inhibitory activity on acute phase of inflammation in a dose-related manner as seen in ethyl phenylpropiolate-induced ear edema (ED(50)=2.34 mg/ear) as well as carrageenin-induced hind paw edema (ED(30)=420.41 mg/kg) in rats. However, CP extract did not elicit any inhibitory effect on arachidonic acid-induced hind paw edema in rats. In subchronic inflammatory model, CP extract provoked a significant reduction of transudation but had no effect on proliferative phase when tested in cotton pellet-induced granuloma model. CP extract also reduced the alkaline phosphatase activity in serum of rats in this animal model. Moreover, CP extract possessed an excellent antipyretic effect when tested in yeast-induced hyperthermic rats. It is postulated that the anti-inflammatory and antipyretic effects of CP extract are caused by the inhibition of the prostaglandin synthesis. Anyhow, CP extract did not possess any analgesic activity in acetic acid-induced writhing response in mice. The results obtained show that C. petasites has beneficial properties since it possesses potent antipyretic and moderate anti-inflammatory activities without ulcerogenic effect.     

Antitumor activity of triptolide against cholangiocarcinoma growth in vitro and in hamsters

One of the diverse biological activities of triptolide, a diterpene from Tripterygium wilfordii, is its antitumor effect. We recently reported its in vitro cytotoxicity against several cultured tumor cell lines. Limited availability of purified fraction has prevented detailed investigation on its antitumor activity. In the present study, we showed by in vitro cytotoxicity assay and in vivo inhibition of tumor growth in hamsters that the triptolide was also highly effective against cholangiocarcinoma, a highly fatal tumor predominantly occurring in developing countries. Its ED50 for these hamster cholangiocarcinoma cell lines was found to be as low as 0.05 microg/ml. The compound was highly potent in the induction of apoptotic death in these tumor cells. DNA fragmentation and disintegrating apoptotic cells could be observed within 24 h of exposure to 0.5 microg/ml triptolide. The compound was tested against the growth of cholangiocarcinoma in a hamster model. A significant growth inhibition (P < 0.05) was noted in triptolide-treated hamsters (each of the 10 animals received 10 injections for a total of 1.2 mg/animal). At the time of sacrifice 1 month after the initial injection, the mean tumor mass of the treated group was only 20-25% of that of the control group.     

Long-term effects of triptolide on spermatogenesis, epididymal sperm function, and fertility in male rats

Prior studies had suggested that triptolide, a diterpene triepoxide isolated from a Chinese medicinal plant, might be an attractive candidate as a post-testicular male contraceptive agent. Despite the promise that triptolide would not affect testis function, nagging concerns remained that a delayed onset of testicular effect might exist. The objectives of this study were to assess the effects of relatively longer treatment duration of triptolide on fertility, spermatogenesis, and epididymal sperm pathophysiology; and to evaluate the reversibility of these effects after the cessation of treatment. Adult male Sprague-Dawley rats were fed daily with either 30% gum acacia as a vehicle control (n = 12) or 100 microg/kg body weight (BW) of triptolide for 82 days (n = 12) followed by a recovery period of up to 14 weeks (n = 6). At the end of the treatment period, all rats treated with triptolide were sterile. Cauda epididymal sperm content decreased by 84.8% and sperm motility was reduced to zero. In addition, virtually all cauda epididymal sperm in the triptolide-treated group exhibited severe structural abnormalities. The most striking changes observed were head-tail separation, premature chromatin decondensation of sperm nuclei, a complete absence of the plasma membrane of the entire middle and principle pieces, disorganization of the mitochondrial sheath, and aggregation of many sperm tails. Longer treatment duration of triptolide also affected spermatogenesis, with marked variability in the response of individual animals. The degree of damage ranged from apparently normal-looking seminiferous tubules to flattened seminiferous epithelium lined by a single layer of cells consisting of Sertoli cells and a few spermatogonia. Affected tubules exhibited intraepithelial vacuoles of varying sizes, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. Recovery occurred as early as 6 weeks after cessation of treatment. By 14 weeks, 4 out of 6 triptolide-treated males were fertile and the females that were impregnated by 3 out of 4 triptolide-treated male rats produced apparently normal litters. These results suggest that triptolide has 2 phenotypic effects on mature and maturing germ cells. The first action appears earlier and manifests mainly in epididymal sperm. The second action presumably is directly on germ cells in testis and causes a variable impairment of spermatogenesis that may not be completely reversible. It is unclear if the earlier effect is a delayed manifestation of subtle testicular injury or post-testicular action.     

Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure

A variety of active diterpene epoxides, including the triptolide (isolated from Tripterygium wilfordii) have been reported to cause infertility in male rats. Previously, we showed that oral administration of triptolide at a dosage of 100 microg/kg per body weight for 70 days completely inhibited fertility in male rats, with little or no demonstrable detrimental effect on spermatogenesis and Leydig cell function as determined by testicular light microscopic appearance and serum and intratesticular testosterone levels. Despite the apparent absence of effects on the testes, cauda epididymal sperm were abnormal, with complete cessation of sperm motility and some reduction in sperm numbers. This study was undertaken to provide additional insight into the subcellular sites and possible mechanisms of action of this compound using ultrastructural analysis of the testes and epididymidis. The most striking effect of triptolide treatment was observed in sperm in the epididymis. In rats rendered infertile with 100 microg/kg per body weight of triptolide daily for 70 days, virtually all cauda epididymal sperm exhibited complete absence of plasma membrane over the entire middle and principal piece, premature decondensation of the nuclei, and disorganization of the mitochondrial sheath with many vacuolated mitochondria. No ultrastructural differences in the epididymal epithelium were observed between control and triptolide-treated rats. The testes appeared to be mildly affected after triptolide treatment but exhibited only subtle ultrastructural defects in the germ cells. The findings of severe impairment of cauda epididymal sperm ultrastructure, along with minimal discernible abnormalities in the fine structural cytology of the testes, further suggest that the site of action of this compound is posttesticular and may be confined to the cauda epididymal sperm. However, we cannot rule out an effect of triptolide that occurs during germ cell maturation but is delayed in its manifestation or triggered at the rete testis and epididymal level.     

Topical Antiinflammatory Activity of Two Pimarane Diterpenes from Kaempferia pulchra

Two diterpenes, 2α-acetoxysandraracopimaradien-1α-ol (1) and sandaracopimaradien-1α,2α-diol (2) isolated from Kaempferia pulchra Ridl. (Zingiberaceae), have been assayed for topical antiinflammatory activity in the model of 12-O-tetradecanoylphorbol-13-acetate-induced ear oedema in rats. Both substances were found active and the ID₅₀-values were estimated to 330 and 50 μg/ear, respectively. The ID₅₀-value of the reference drug diclofenac was calculated to 90 μg/ear