Replication of an experiment on extremely diluted thyroxine and highland amphibians.

INTRODUCTION: The purpose of this study was to reproduce an experiment with diluted thyroxine and amphibians. A detailed account of the difficulties of this line of research has been published by the initial team (Endler and Scherer-Pongratz). One experiment which has been reported reproducible by the initial team and independent researchers is the effect of extremely diluted agitated thyroxine (T30x) versus analogously prepared water (W30x) in amphibians from biotopes above the tree line (highland amphibians). METHODS: (A) The author replicated the experiment. Rana temporaria were taken from an alpine biotope and the methods given in the original protocols were followed. Animals were treated from the 2-legged stage on. (B), the author reanalyzed the results reported by the initial team and by independent researchers (van Wijk, Lassnig, Zausner-Lukitsch, Bach, Harrer). RESULTS: (A) In the author's own experiment, there was a clear trend of T30x animals developing more slowly (i.e. up to 6 h within 3 days) than W30x animals. This is in line with the previous experiments. Due to small numbers of animals, the differences in the frequency of larvae reaching the 4-legged stage and the stage with reduced tail were not statistically significant (p > 0.05). The effect size was large (d > 0.08). (B) In the analysis of all available data with regard to the 4-legged stage, pooled T30x values from the initial team were 10.1% smaller than W30x values (100%) and pooled T30x values from the 5 independent researchers were 12.4% smaller (p < 0.01 and d > 0.08). Analogously, the number of animals entering the juvenile stage with reduced tail was smaller for T30x than for W30x.

Treatment of highland frogs from the two-legged stage with homeopathically prepared thyroxin (10-11 - 10-21).

The influence of moderately diluted, agitated, i.e., homeopathically prepared, thyroxin solutions (10-11 - 10-21, final concentration in the basin water 0.6 10-15 - 0.6 10-25 parts by weight after the first application) on metamorphosis in highland Rana temporaria from the two-legged stage was studied. In accordance with the homeopathic idea of effects of specially prepared dilutions being inverse to those of their mother substances, animals were treated either with thyroxin 10-11 - 10-21 or analogously prepared blank solution (water). Development was monitored by documenting the number of animals that had entered the four-legged stage. It has been found that animals treated with the thyroxin solutions metamorphosed more slowly than the control animals, i.e., the effect of the homeopathically prepared thyroxin was opposed to the usual effect of molecular thyroxin. The number of test animals that reached the four-legged stage at defined points in time was smaller (2-13.5%) in the group treated with homeopathically prepared thyroxin at the points in time, compared to control. The results in this study sustain the previous multiresearcher findings that show that diluted homeopathically prepared thyroxin is able to slow down metamorphosis of R. temporaria.

The effect of homeopathically prepared thyroxine on highland frogs: influence of electromagnetic fields.

BACKGROUND: Previous experiments show that amphibian larvae are responsive to homeopathically prepared thyroxine. METHODS: We studied the effect of a highly diluted and agitated thyroxine solution exposed to various electromagnetic fields on metamorphosis in highland Rana temporaria. The devices tested were: microwave oven, mobile phone, airport X-ray, and a red light barcode scanner. Animals were treated either with homeopathically prepared thyroxine (10(-30) parts by weight, 10(-35) in the water in which the animals were kept), or analogously prepared blank solution, or analogously prepared thyroxine exposed to the electromagnetic field of one of the devices tested. Solutions were administered at 48h intervals according to a standardized protocol. RESULTS: Animals treated with the standard test solution thyroxine 10(-30) metamorphosed more slowly than the control animals, ie the effect of the homeopathically prepared thyroxine was opposed to the usual physiological effect of molecular thyroxine. The cumulative number of test animals that had reached the four-legged stage at defined points in time was smaller in the group treated with homeopathically prepared thyroxine at most of the points in time. This was found independently by all three research teams involved. In contrast, this effect did not occur when the thyroxine solution had been exposed to the field of the early model microwave oven, or mobile phone. There was no difference between aqueous or alcoholic solutions were used, and there was, if any, only a small protective effect from aluminum foil. Airport X-ray and red light barcode scanning did not diminish the effect of the homeopathic solution.

[Determination of cholesteryl palmitate in Oviductus Ranae by HPLC].

OBJECTIVE: To develop a new method for the determination of cholesteryl palmitate in Oviductus Ranae. METHOD: A HPLC method was set up, using Zorbax Silica column and cyclohexane-diethyl ether (40:1) as mobile phase with a flow rate of 1.0 mL x min(-1), and the UV detection wavelength was 203 nm. RESULT: The calibration curve was linear over the range of 0.60-8.92 microg (r = 0.9997), the average recovery of the method was 98.4%. RSD 1.8% (n = 6). CONCLUSION: The results showed that method was reliable and accurate.

[Laboratory study of effects of yifuning soft capsules on postmenopausal osteoporosis].

OBJECTIVE: To study the effects of Yifuning soft capsules on postmenopausal osteoporosis in ovariectomized female rats. METHOD: 60 female sprague-dawley rats in 3-month were used, 50 of them were ovariectomized and randomly divided into 5 groups: ovariectomy (OVX), OVX with diethylstilbestrol tables (DT), OVX with YFN (high dose, middle dose and low dose), the others were sham-operated group. Began to give the rats drugs in the fifth week after the operation. After 12 weeks killed the rats. The blood,the uterus and bones were collected to inspect. The content of estrogen (E2) in serum was detected by radioimmunoassay method. The contents of Ca, P, ALP in serum were detected using the automic biochemistry-analyse device. The uterus were weight and the length, width, dry weight and net weight of the shoulder bone were measured. The contents of Ca, Mg, Zn, Mn in the bone were measured using the automic absorption spectrophotometer. The right femer and the third lumbar vertebra were detected for BMD using the dual-energy X-ray absorptiometry scanner and compression test using electronic testing device. RESULT: After using the drugs,the content of E2, Ca in serum, the weight of uterus, the contents of Ca, Mg, Zn, Mn in the bone and the BMD of right femer increased significantly, the content of P, ALP in serum decreased significantly. CONCLUSION: Yifuning soft capsules has a good effect on postmenopausal osteoporosis, which provides evidence for clinical use.

[Effect of the Yifuning soft gelatin capsules on estrogen receptor and nitric oxide levels in ovariectomized rats].

OBJECTIVE: To investigate the effects of the Yifuning soft gelatin capsules(YFN)on estrogen receptor (ER) and nitric oxide (NO) levels in ovariectomized rats. METHOD: Fifty female mature sprague-dawley rats were randomly divided into 5 groups: normal control; model control; diethylstilbestrol tablets (DT); YFN (high dose and low dose). After 4 weeks' treatment, the serum E2 levels were detected by radioimmunoassay. The estrogen receptor (ER) levels of uterus and artery were detected with method of radioligand binding assay of receptor (RBAR). The uterus pathologic changes were investigated with light microscope. NO and NOS levels of the artery and the uterus index were detected too. RESULT: YFN can obviously improve serum E2, increase index and ER of uterus (P < 0.01 or P < 0.05), and ameliorate the uterus' pathologic changes in OVX rats. It also can increase the artery' ER, NO and NOS levels. CONCLUSION: YFN can cure the climacteric syndrome and prevent the cardiovascular disease after post-menopause.

Pretreatment with thyroxine (10-(8) parts by weight) enhances a 'curative' effect of homeopathically prepared thyroxine (10-(13)) on lowland frogs.

We studied the influence of a moderate homeopathically prepared thyroxine dilution (final concentration in the basin water 10-(13) parts by weight) on the metamorphosis of lowland Rana temporaria which had been hyperstimulated with thyroxine. Two groups of animals were pretreated by immersing them in a molecular thyroxine dilution (10-(8) parts by weight). This pretreatment speeds up development, as is well known. In accordance with the homeopathic/isopathic idea of detoxication or cure, the same hormone was then diluted and agitated in successive steps for further treatment. This homeopathically prepared dilution was administered at 24-hour intervals to one of the groups. An analogously prepared blank solution was used for the control group. Our hypothesis, which was derived from earlier studies, was that animals treated with the test solution would metamorphose more slowly than the control animals, i.e. that the homeopathically prepared thyroxine would have a 'curative' effect. In this new series of experiments this hypothesis was examined by 3 independent researchers. In the experiments carried out by 2 of the 3 researchers the number of animals that reached the four-legged stage at defined points in time was smaller in the group treated with homeopathically prepared thyroxine. In the third laboratory no difference was found between the groups. However, the overall inhibiting effect was statistically significant and more pronounced than in earlier, less promising studies and in parallel experiments in which nonprestimulated animals had been used. Other studies carried out by the 3 researchers involved animals from highland biotopes, where the natural environment probably induces a greater sensitivity towards thyroxine or higher thyroxine levels. These animals reacted to the homeopathically prepared thyroxine with a slowing down of metamorphosis, even when they had not been prestimulated with a molecular dose of the hormone. This effect was observed in all 3 laboratories and is consistent with the results of previous studies.

[Study on the origin of Rana temporaria for quality Oviduetus Ranae].

OBJECTIVE: To determine the origin of Rana temporaria for quality Oviduetus Ranae in the light of historical documents and modern researches on the classification of Rana temporaria chensinensis. METHOD: Works of Chinese meteria medica of all ages, related historical documents and reports from home and abroad on researches of R. temporaria chensinensis were consulted, sorted out, analyzed and summarized. RESULT: The original Shange recorded in the works of Chinese meteria medica is R. temporaria chensinensis, which is the independent species, not one of species of European forest frogs. R. temporaria chensinensis is divided into 4 subspecies: R. temporaria chensinensis, Lanzhou, Kangding, and Changbaishan. The origin of R. temporaria is Changbaishan subspecies of R. temporaria chensinensis. CONCLUSION: Changbaishan subspecies of R. temporaria chensinensis is determined as the origin for quality Oviduetus Ranae.

Isolation and identification of two steroid compounds from Oviductus Ranae.

In this study, cholest-5-en-3ß,7ß-diol and 3ß-hydroxy-cholest-5-en-7-one were isolated from Oviductus Ranae by column chromatographies on Sephadex LH-20, octadecylsilyl (ODS) and pre high-performance liquid chromatography (HPLC). Their structures were elucidated on the basis of chemical and spectral analyses, including electrospray ionisation-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The above two compounds were obtained from Oviductus Ranae for the first time.

Pretreatment with thyroxin 10(-8) and the effect of homeopathically prepared thyroxin 10(-30) on highland frogs--a multi-researcher study.

BACKGROUND: Studies performed in 5 laboratories have shown that homeopathically prepared highly diluted thyroxin (10(-30)=30x) slowed down metamorphosis of highland amphibians. Metamorphosis of lowland amphibians, however, could be slowed down by a low dilution of thyroxin (10(-8)=8x) if animals had been artificially pretreated with thyroxin. OBJECTIVE: To combine the advantages of using animals from highland biotopes and hyperstimulation prior to treatment. ANIMALS AND METHODS: Rana temporaria from an alpine biotope were pretreated in an aqueous molecular thyroxin dilution (10(-8) parts by weight, hyperstimulation). This is supposed to accelerate metamorphosis. In accordance with the homeopathic idea of detoxification or cure, one group of these hyperstimulated animals was then treated with thyroxin 30x, and another group with water 30x. Experiments were performed by 4 independent researchers. RESULTS: As a trend, the thyroxin-30x animals metamorphosed more slowly than the water-30x animals. The number of thyroxin-30x animals that reached the 4-legged stage at defined points in time was slightly smaller at some but not all points in time, compared to control. This is in line with previous findings and can be discussed as an interesting result. Contrary to our working hypothesis, however, differences were not bigger than in the previous experiments in which animals had not been pretreated with thyroxin 10(-8). CONCLUSION: This study supports previous findings but does not prove the assumption that pretreatment of highland animals with molecular thyroxine improves the original protocol.

Re-análise dos dados de 1990-2010 sobre os efeitos da tiroxina altamente diluída na metamorfose de anfíbios de terras altas / Recalculation of data from 1990 to 2010 on the effects of highly diluted thyroxine on the metamorphosis of highland amphibians

Experiments on amphibian metamorphosis can vary considerably in duration. The authors had set themselves the task of defining a generally applicable pooling method for metamorphosis experiments [1]. Normalization with respect to time was done on the assumption that differences in speed of metamorphosis attributable to treatment would override differences in duration between experiments. The problem of artificial differences in variability when comparing and pooling data from several experiments was approached by normalization with respect to time based on the development of both the test and the control animals. The range from 0% to 100% over which the fraction of four-legged animals progresses in the course of an experiment is divided into 10%-intervals and mapped onto a corresponding relative scale. Each measurement is then assigned to the point on the 10%-scale to which it is closest. In this way each reference point is assigned a value giving the number or percentage of four-legged animals at that point. These values are aggregated over all experiments within the test- and control-group. The results of experiments performed over the course of two decades (1990 - 2010) on highland Rana temporaria treated with a homeopathically prepared high dilution of thyroxine (?30x?) are presented in full detail based on this normalization method[1]. It was found that differences between treatment groups thus calculated were in line with those obtained with other pooling methods [2]. Thyroxine 30x does slow down metamorphosis in inert highland amphibians. This was observed by 5 researchers in 20 sub-experiments, and it seems to be the most reliable bio-assay found in amphibian research on homeopathy so far2. When experiments were performed with highland animals pretreated by hyperstimulation with molecular thyroxine, slowing down of metamorphosis was again observed (by 3 of 4 researchers) in most of 10 sub-experiments.(AU)

Experimentos sobre a metamorphose de anfíbios podem variar consideravelmente em termos de duração. Os autores desse trabalho se propuseram a definir um método geral aplicável ao estudo de experimentos commetamorfose [1]. A normalização em relação ao tempo foi realizada considerando que as diferenças na velocidade da metamorfose, atribuídas ao tratamento, minimizariam as diferenças na duração entre experimentos. O problema das diferenças artificiais na variabilidade, quando comparamos e agrupamos dados a partir de vários experimentos foi abordada com a normalização em relação a base temporal no desenvolvimento de ambos os grupos, os tratados e os controles. O intervalo de 0% a 100% para a fração de animais que evoluem para 4 patas durante o experiment é dividido em sub-intervalos de 10% e mapeados em uma escala relativa. Cada ponto é então associado ao ponto mais próximo da escala de 10%. Dessa maneira, a cada ponto da referência é associado um valor correspondente ao número ou porcentagem de animais com 4 patas naquele ponto. Esses valores são agregados sobre todos os experimentos considerando os grupos teste e controle. Os resultados dos experimentos realizados no decorrer de 2 décadas (1990 - 2010) com Rana temporaria tratadas com altas diluições de tiroxina preparadas homeopaticamente (?30x?) são apresentados em detalhes com base nesse método de normalização [1]. Foi encontrada que as diferenças entre os grupos tradados, calculados com o método desenvolvido, estão em concordância com outros métodos de agrupamento de dados [2]. Tiroxina 30x reduz a metamorphose nos anfíbios. Isso foi observado por 5 pesquisadores em 20 sub-experimentos, e parece ser o ensaio biológico mais confiável encontrado em pesquisas com anfíbios pretratados. Quando os experimentos foram realizados com animais pré-tratados por hiperestimulação com tiroxima molecular, novamente foi observada a redução da metamorfose (por 3 dos 4 pesquisadores) na maioria dos 10 sub-experimentos.(AU)

Changes in activity of the organon vasculosum laminae terminalis in the annual cycle in Rana temporaria.

In 70 sexually mature male and femal Rana temporaria frogs captured in natural habitat, mean nuclear volumes for the cells of the pars ependymalis and pars parenchymalis of the organon vasculosum laminae terminalis (OVLT) were determined in seven characteristic stages in life. The mean nuclear volume for the cells of the pars ependymalis and pars parenchymalis of the OVLT showed distinct annual fluctuation. Maximum nuclear volume of the cells in both investigated parts of the OVLT were observed during the breeding period (Ist decade of April), and minimum volume of the nuclei of the pars ependymalis at the beginning of hibernation (IIIrd decade of October), and in the pars parenchymalis near the end of active life (Ist decade of September).

The effect of highly diluted agitated thyroxine on the climbing activity of frogs.

We studied the influence of specially prepared highly diluted thyroxine on the spontaneous tendency of juvenile frogs, which were at the end of thyroxine-controlled metamorphosis, to leave the water and climb onto land. The test dilution with a thyroxine concentration beyond Avogadro's value (dilution thyroxine D30) and the reference (dilution water D30) were prepared according to directions from the literature on homeopathy. A few drops of these solutions were added to tap water of basins containing the frogs. The frogs' climbing activities were monitored immediately after adding the solutions. The hypothesis derived from a preliminary study was that there is less climbing activity in frogs treated with dilution thyroxine D30 than in a reference group. This hypothesis was proven. Climbing activity diminished under the influence of dilution thyroxine D30, with statistical significance both in comparison to the effect of the analogously prepared solvent (dilution water D30) as well as in comparison to control observations before the start of treatment. When in a later step of observation the dilution water D30-control group was treated with dilution thyroxine D30, the diminishing effect on activity also occurred.

Climbing activity in frogs and the effect of highly diluted sucussed thyroxine

The experiments investigate the influence of extremely dilute thyroxine (T4) in special "homoeopathic" preparation (dilution T4.30x) on the spontaneous tendency of juvenile frogs to leave the water and climb on land. Climbing activity was suppressed by dilution T4.30x, with statistical significance both in comparison to the effect of the "potentized" preparation of the solvent (dilution H2O.30x) as well as in comparison to the control observations before the start of the treatment. Finally, in the search for optimal treatment duration, it was shown that exposure to the dilutions for even a few minutes sufficed to cause significant effects