A rapid HPLC-ESI-MS/MS for qualitative and quantitative analysis of saponins in "XUESETONG" injection.

'XUESETONG' injection, one of the most widely used proprietary medicines in traditional Chinese medicine, consists of total saponins made from Panax notoginseng, which is a highly valued and important Chinese medicinal herb. It is used to treat cardiovascular diseases. In order to control the quality of XUESETONG injection, a rapid HPLC-ESI-MS/MS method was developed for qualitative and quantitative determination of the saponins. The analyses were performed on SB-C18 column using gradient elution in 25 min. Full scan and time programmed selected reaction monitoring (SRM) were used for qualitative and quantitative analysis of saponins, respectively. Twenty-seven saponins were identified and nine of them including notoginsenoside R1, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf and Rg1 were quantified. Ten XUESETONG injections were analyzed and compared. The results showed that there is a great variation among different samples. In conclusion, the developed method is rapid, accurate and sensitive for qualitative and quantitative analysis of saponins in XUESETONG injection. Moreover, it also can be used for the quality control of P. notoginseng raw material and its preparations.

Novel, potent, semisynthetic antimalarial carba analogues of the first-generation 1,2,4-trioxane artemether.

Ten novel, second-generation, fluorinated ether and ester analogues of the potent first-generation analogues artemether (4a) and arteether (4b) have been designed and synthesized. All of the compounds demonstrate high antimalarial potency in vitro against the chloroquine-sensitive HB3 and -resistant K1 strains of Plasmodium falciparum. The most potent derivative 8 was 15 times more potent than artemisinin (2) against the HB3 strain of P. falciparum. In vivo, versus Plasmodium berghei in the mouse, selected derivatives were generally less potent than dihydroartemisinin with ED(50) values of between 5 and 8 mg/kg. On the basis of the products obtained from the in vitro biomimetic Fe(II)-mediated decomposition of 8, the radical mediator of biological activity of this series may be different from that of the parent drug, artemisinin (2).

Gestational changes of thyroid hormone action in the developing fetal bovine epiphysis.

Thyroid hormones are essential for cartilage growth and maturation. In order to assess their actions during different periods of skeletal development, [125I]-triiodothyronine (T3) binding capacity in epiphyseal cartilage and triiodothyronine concentrations in serum were quantitated in bovine fetuses of the second and third trimesters of gestation (equivalent to fetal sizes of 10-90 cm crown-rump (CR) length), and related to the alkaline phosphatase activities in the same cartilaginous tissues. Nuclear T3 binding levels, which were initially low during 10-30 cm CR, rose to a peak value (1.7 pmol/mg DNA) at the end of the second trimester (40-50 cm CR). Then, following a sharp decline at 50-60 cm CR, T3 binding rose to a moderate level in the later gestational period (60-90 cm CR). Serum total triiodothyronine rose transiently in fetuses of 30-50 cm CR to a peak level (34 ng/100 ml), and subsequently increased continuously in the later period (60-90 cm CR). Alkaline phosphatase activities measured in epiphyseal chondrocytes rose significantly in fetuses of 60-90 cm CR. The coincident rise of cartilage triiodothyronine binding capacity and serum circulating triiodothyronine levels in the late second trimester suggests that this thyroid hormone induces its own binding sites in bovine epiphyseal cartilage; the dramatic increase in the level of serum triiodothyronine during the third trimester of gestation is temporally related to the increase in alkaline phosphatase activity of chondrocytes, as well as other recognized developmental changes in the fetal bovine skeletal tissues.

Temporal relationship between fetal bovine skeletal growth and circulating hormonal levels.

Skeletal growth and serum hormonal levels in bovine fetuses were studied cross-sectionally from late first trimester to late third trimester of gestation (corresponding approximately to 75-275 days of age, and a crown-rump (CR) 5-105 cm/in size). Measurements of tibial and femoral lengths showed that bone growth proceeds at a 30% faster rate in fetuses of 50-105 cm CR, coincident with the appearance and exponential growth (y = 0.000207 e 0.12522 x; y, dried weights of ossification in grams and x, CR in cm) of a secondary center of ossification in the epiphysis. During this period there is an increase in the proportion of [3H]-thymidine-labeled nuclei (measured by autoradiography) in the proliferative zone of the epiphyseal growth plate and a progressive hypertrophy of chondrocytes in the epiphysis; in serum there is a rise in alkaline phosphatase activity, a rise in the calcium, and a decrease in the phosphorus concentrations. Cellularity (nuclei/area) and the proportion of [3H]-thymidine labeled nuclei in epiphyseal cartilage decline during the period of 15-105 cm CR, except at 25-45 cm CR when both parameters of chondrocyte growth have transiently stabilized. [35S]-Sulfate and [3H]-proline incorporation (cpm/100 micrograms DNA) in epiphyseal cartilage also decline initially during 10-25 cm CR, then attain a stable level during 25-50 cm CR; subsequently, [35S]-sulfate incorporation gradually increases and [3H]-proline incorporation remains at a constant level. The proportion of [3H]-thymidine-labeled nuclei in the epiphyseal growth plate also declines in early gestation, then becomes stabilized at 20-50 cm CR. The whole growth plate thickness varies during gestation and is maximal during 20-50 cm CR. The proliferative zone attains maximal thickness at 20-50 cm CR while the hypertrophied and degenerative zone has maximal thickness at 40-80 cm CR. Gestational changes of hormone levels were quantitated in fetal serum. Glucocorticoids and thyroxine were measured by radioimmunoassay; somatomedinlike bioactivity was measured as the capacity of a serum sample to stimulate [3H]-thymidine incorporation in chondrocytes compared to that of a control serum. Temporally related to the changes occurring in the skeletal tissues, there is a high serum level of glucocorticoids at 10-20 cm CR when the cartilaginous activities are declining, a peak level of serum somatomedinlike bioactivity at 20-50 cm CR when cartilage growth and metabolism become stabilized, and a rise of thyroxine level after 45 cm CR during which time there is an increasing rate of bone formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Gestational changes in the level of glucocorticoid-binding sites in fetal bovine cartilaginous tissues.

In order to assess glucocorticoid actions on fetal cartilage development, [3H]dexamethasone binding site levels in fetal bovine cartilaginous tissues from long bones were measured, using a whole cell assay at 37 degrees C. Displaceable [3H]dexamethasone binding in epiphysial growth cartilage was maximal (16.2 fmol/10(6) cells) in fetuses of 10-15 cm crown-rump length (CR), and declined to 22% of the maximum in fetuses of 20-30 cm CR. Subsequently, [3H]dexamethasone binding rose to a plateau (13.0 fmol/10(6) cells) in fetuses of 30-80 cm CR and declined in those of 80-100 cm CR. When measured in growth plate cartilage, [3H]dexamethasone binding was significantly higher in fetuses of 40-80 cm CR (39 fmol/10(6) cells) than in those of 80-100 cm CR. There was no significant change of [3H]dexamethasone binding affinities in epiphysial chondrocytes of 5-100 cm CR fetuses or in growth plate chondrocytes of 40-100 cm CR fetuses. These results demonstrate that fetal cartilaginous tissues during development possess varying cellular levels of glucocorticoid binding and may thus have temporal changes in sensitivity to glucocorticoid hormones.

Hormone receptors in the epiphysial cartilage.

In order to assess which hormones may exert direct effects on skeletal growth at the epiphysial growth plate, the specific binding of hormones to the epiphysial cartilage of growing dogs and rabbits was studied. Membrane fractions obtained by centrifugation of homogenates prepared from dog and rabbit growth plate cartilage at 600, 15 000 and 105 000 g showed significant specific binding of serum insulin-like activity and insulin. Binding of growth hormone and prolactin by the three membrane fractions was negligible. Saturable binding sites for triiodothyronine could be demonstrated in nuclei from the dog growth plate. Nuclear binding showed an apparent Kd of 11 +/- 3.6 nmol/l and a maximum binding capacity of 4.1 +/- 1.6 pmol/mg DNA, a level comparable to dog liver. Using a viable chondrocyte suspension prepared from dog epiphysial cartilage, specific steroid binding in the cells could be demonstrated for [3H]dexamethasone but not 17 alpha-methyltrienolone, oestradiol-17 beta or 1 alpha, 25-dihydroxycholecalciferol. Scatchard analysis of dexamethasone binding showed high affinity binding sites having a Kd of 1.2 +/- 0.35 nmol/l and a capacity of 1700 sites/cell, and a low affinity binding with a Kd of 109 +/- 57 nmol/l and a capacity of 24 000 sites/cell. Steroid competition for the specific binding showed the following sequence of affinity: dexamethasone greater than corticosterone greater than 11-deoxycortisol greater than testosterone greater than oestradiol-17 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Gestational changes in the metabolism of progesterone by the fetal calf adrenal.

The metabolism of [3H]progesterone by the fetal calf adrenal was examined in homogenates and microsomal fractions of adrenals obtained from mid- or late gestational fetal calves. Throughout the course of gestation, there was no detectable difference in the formation of corticosterone from progesterone by adrenal homogenates. In contrast, cortisol production in these same homogenates increased 7-fold from 0.3-2.2% at the end of gestation. Similar results were obtained using microsomal fractions prepared from these homogenates: the 17 alpha-hydroxylation of progesterone (i.e. 17 alpha-hydroxyprogesterone and 11-deoxycortisol) increased from 0.7% to 26% at the end of gestation. The results suggest that the increased cortisol observed in the fetal circulation toward the end of gestation is the result of a rapid increase in the capacity of the fetal calf adrenal to form 17 alpha-hydroxycorticosteroids. The underlying mechanism for this change is discussed.

Zonal analysis of cytoplasmic steroid hormone receptor levels: replication and synthetic activity in embryonic calf growth cartilage.

Using the dextran charcoal method, a specific steroid receptor for dexamethasone, but not 17 beta-estradiol or 17 alpha-methyltrienolone, was demonstrated in cytosol prepared from mid-gestation fetal calf growth cartilage. Zonal analysis of cytoplasmic receptor levels showed that tissue sections from the center, the peripheral surface, and the palisade section bound 229 +/- 111, 173 +/- 64, and 15 +/- 28 femtomole dexamethasone per mg of protein, respectively. Incorporation of 3H-thymidine, 35S-sulfate, and 3H-proline into cartilaginous tissues was used as an index of replication and synthetic activity. The percentage of 3H-thymidine labeled nuclei, as determined by radioautography, showed that the palisade zone has a significantly lower replicating activity (P less than 0.001). Values were 5.6 +/- 1.2, 5.4 +/- 1.2, 3.8 +/- 1.3, and 1.1 +/- 0.2 for the center, peripheral surface, and upper and lower half of the palisade section. The rate of sulfate and proline incorporation into the palisade zone was over seven times higher than the central and peripheral surface (P less than 0.001). The difference in the rate of matrix synthesis and proliferating activity in these regions may be related to the zonal heterogeneity of receptor levels of glucocorticoids in fetal growth cartilage.

Role of pro-opiomelanocortin-derived peptides in the regulation of steroid production by human fetal adrenal cells in culture.

Synthetic (1-39)ACTH, (1-24)ACTH, (18-39)ACTH, alpha-MSH, met-enkephalin and alpha-, beta- and gamma-endorphin were tested for their ability to stimulate steroidogenesis by human fetal adrenal cells in culture. Adrenal cells were incubated with peptide hormones for two periods of 24h. On the third day of the experiment the cells were incubated with progesterone (4 micrograms/2 ml) for 8 h. At the doses tested only (1-39)ACTH, (1-24)ACTH and alpha-MSH stimulated steroidogenesis. None of the other peptides had any corticotrophic effect on the formation of cortisol, corticosterone or dehydroepiandrosterone sulphate (DHAS). At the highest doses tested, alpha-MSH (100 micrograms/2 ml) had a corticotrophic effect that was not different from that obtained with 20 ng (1-39)ACTH or (1-24)ACTH. At the lower doses (0.2-2 micrograms/2 ml), alpha-MSH stimulated the formation of DHAS (P less than 0.01) without stimulating the formation of cortisol.

Androstenedione-mediated inhibition of 11 beta-hydroxylation in monolayer cell cultures of fetal calf adrenals.

The possibility that the formation of androstenedione by fetal calf adrenal cells in culture is linked to their decreased ability to form cortisol and corticosterone was investigated. Fetal calf adrenal cells metabolise radioactive adrostenedione to two major products which coelute on thin layer chromatography with 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone. When the cells are incubated with 11-deoxycortisol or 11-deoxycorticosterone in the presence of androstenedione there is a dose dependant inhibition of cortisol and corticosterone formation. Further studies with progesterone showed an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in cells incubated simultaneously with androstenedione. The results demonstrate that exogenous androstenedione can have dramatic effects on steroidogenesis in the fetal calf adrenal and suggest that the accumulation of androstenedione in the medium of cultured andrenocortical cells is responsible, at least in part, for the decreased formation of cortisol and corticosterone.