Melatonin stimulates proliferation and type I collagen synthesis in human bone cells in vitro.

The pineal secretory product melatonin reportedly regulates release of growth hormone in humans and prevents phototherapy-induced hypocalcemia in newborn rats, suggesting that melatonin affects bone metabolism. Little is known about the effects of melatonin on bone in vitro or in vivo. The present study was undertaken to examine whether melatonin acts directly on normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO cells) to affect osteogenic action in vitro. The effect of melatonin on bone cell proliferation was determined using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xanilide (XTT) assay after a 24 hr incubation with melatonin. Melatonin significantly and dose-dependently increased the proliferation in HOB-M cells and SV-HFO cells by 215 +/- 22.1%, and 193 +/- 6.4%), respectively, with a maximal effect at a concentration of 50 microM. To evaluate the effect of melatonin on bone cell differentiation, alkaline phosphatase (ALP) activity, osteocalcin secretion and procollagen type I c-peptide (PICP) production (a measure of type I collagen synthesis) were measured after a 48 hr treatment. While melatonin at micromolar concentrations did not significantly affect either the ALP activity or the osteocalcin secretion, it significantly and dose-dependently increased the PICP production in HOB-M cells and SV-HFO cells by 983 +/- 42.2%, and 139 +/- 4.2%, respectively, with the maximal stimulatory doses between 50 and 100 microM. These results provide new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells in vitro, suggesting that melatonin may act to stimulate bone formation.

Isolation and characterization of ginsenoside-Rg2, 20R-prosapogenin, 20S-prosapogenin and delta 20-prosapogenin. Chemical studies on saponins of Panax ginseng C. A. Meyer, Third report.

In the course of a chemical study on pure ginsenosides, a ginsenoside-Rg2 was isolated as colorless needles, from the lateral root of Panax ginseng C. A. Meyer. For the characterization of Rg2, a chikusetsusaponin-I (ginsenoide-Rg2) was isolated as colorless needles from rhizome of Panax japonicus C. A. Meyer. The optical rotation value of both saponins were opposite to the published data. On the large scale isolation of ginsenosides, trace amount of ginsenoside-Rg2 was isolated as crystals. Three compounds, A, B and C, were isolated from the reaction mixture on hydrolysis of ginsenoside-Rb1, Rb2 and Rc with 50% aqacetic acid. A was 20R-prosapogenin and it was identical with Rg3. On the other hand, B was found to be 20S-prosapogenin. And C was estimated to be prosagenin dehydrated at C20-OH and named as delta 20-prosapogenin. It was found that an equilibration is present among A, B and C at the earlier stage on acid hydrolysis.

Chemico-pharmacological studies on saponins of Panax ginseng C. A. Meyer. II. Pharmacological part.

The pharmacological properties of seven pure saponins isolated from Panax ginseng C. A. Meyer were studied. 1. The ginseng saponins showed weak toxicities in mice. Especially, Rg1, Rf and Rb 1, which contained glucose as a sugar component, were weaker in their toxicities than the rest, which contained arabinose and/or rhamnose. It was also noted that the saponins containing protopanaxadiol as sapogenin were more toxic than those containing protopanaxatriol. 2. All the saponins diminished ACh-induced contraction of the isolated ileum of the guinea pig. On the other hand high concentrations of Rb 2 caused contraction of the ileum by itself. 3. All of the saponins induced a decrease in heart rate and showed biphasic actions on the blood pressure in rats, while they little affected respiration. They caused blood pressure fall preceded by slight rise. Among them, Rg 1 showed the most prominent action and it produced a blood pressure rise with doses of 30 to 100 mg/kg. The pressor as well as depressor action was not influenced by the pretreatment with any of atropine, diphenhydramine, phentolamine and propranolol. 4. Rg 1 and Re showed vasodilator action in dogs, the potencies of which were 1/20 and 1/50 of that of papaverine, respectively. Rc and Rb 2 showed very weak vasodilator actions but Rb 1 did not. 5. Among the 7 saponins, Rd, Re and Rb 2 showed more potent hemolytic actions than those of the rest and the potencies were proportional to their toxicities. 6. Whereas single administration of Rf, Re and Rd significantly suppressed the conditioned avoidance response, repeated administration of them caused facilitation of the response. On the other hand, Rb 2 always showed very weak suppressant action. 7. Rg 1, Rf, Re and Rd significantly suppressed the fighting of mice induced by foot shock, while Rb 1, Rb 2 and Re little affected the fighting. 8. All the saponins showed antifatigue action. They markedly increased the movement after compulsory gait and the action was consistent and independent of their action on the movement before compulsory gait. 9. The saponins showed moderate depressant actions on the EEG and the behavior in cats. They were qualitatively similar in their actions, although Rg 1, Re and Rb 2 were more potent than the rest. They also suppressed EEG arousal response induced by electrical stimulation of the mid brain in cats.

Chemico-pharmacological studies on saponins of Panax ginseng C. A. Meyer. I. Chemical part.

For the purpose of clarification of pharmacological actions of ginseng saponins we tried the isolation of ginseng saponins on a larger scale and succeeded in the isolation of eight saponins in a pure state and in sufficient amounts for the pharmacological assay. These eight saponins agreed all completely with the authentic samples of Shibata-Shoji and co-workers in molecular formula of saponins and sapogenins, sugars and TLC. The physical properties of saponins were shown. As other components of ginseng, there were isolated small amounts of panaxadiol, panaxatriol, daucosterol from the first running of column chromatography and mannitol, sucrose and glucose from the ethanol insoluble aqueous extract.