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Mice belonging to the benzodiazepine standard treatment group were also injected with the control solution for 9 days, but on the testing days the control solution was supplemented with diazepam see below. Apart from the drug injections and testing, the animals were not subjected to pain or other forms of emotional or physical stress. The animals were used only once in any given test to avoid the confounding effects of learning and habituation.

After completing the tests, the mice were put back into their original home cages and then sacrificed using CO2-saturated atmosphere. The mother tincture was assayed to determine its gelsemine content, which proved to be 0. On the morning of the first day of drug delivery, before starting the treatment, 0. Blinding of all procedures of injection and testing was performed by randomizing and coding the tubes containing the drug and control solutions.

The person who coded the solutions did not belong to the research group and signed a confidentiality agreement form; the codes were recorded on a sheet that was kept sealed inside an envelope until all the tests and calculations were completed. The coded solutions were distributed in 15 ml sterile Falcon plastic tubes 7. Before administering the contents, each tube was manually shaken with 20 strokes.

On the days of the behavioral tests, the mice were injected 60 min before the start of the tests, which were performed between 11 a. Each day, 0. The camera viewed four test arenas, each one of which, in turn, was divided by the software into two different zones according to the test to be performed LD or OF. All the sessions were recorded and stored as DVD. The video outcome signals were converted through the image processor into binary images in such a manner that the animal was tracked as a black spot with a white background.

The movement of the spot was recorded to track the position of the animals, the time they stayed in different zones and the distance traveled. The number of transitions between light and dark compartments in the LD test was evaluated on the video by an operator who was unaware of the group assignment of the mice.

The experiments were performed on each group of animals in the following order: LD choice test on day one 8th day of drug administration and OF exploration test on the following day 9th day of drug administration. Mice were tested individually in each test arena and the operators stayed outside the testing room during the recording of the experimental session.

Just before testing, the animals were allowed to acclimate to the room inside their cages for 3 min after being moved from their usual housing area. Both compartments had walls that were 25 cm high. The white field was brightly lit at lux and the mice were left to explore the space for a 5 min testing period. White light lux was present in the room. The 10 min test began with the subject being placed inside the OF arena, in a corner near the walls.

The tendency to enter the central zone and to travel inside it, instead of running along the walls or staying in the corners, is considered to be a sign of a decrease in the anxiety induced by a novel environment neophobia and of an increase in exploratory attitude, typical of mice that consider the experimental setting to be familiar. Thigmotaxis is particularly apparent upon the first exposure to a novel space, and helps the animal to define the boundaries of an unfamiliar environment.

In unconditioned behavior tests in which an animal is placed for the first time in an arena, it also reflects novelty induced anxiety, general activity, exploratory behavior and decision-making [ 12 ].

Extreme behavioral responses were considered as outlier data and excluded when their value exceeded 2 standard deviations SDs. This occurred in very few cases see Results section. The latter factor was included in the ANOVA analysis because the control values of activity varied considerably between experiments.

Post-hoc t-tests were performed assuming equal variances with least significant difference LSD corrections to adjust for multiple comparisons.

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When the distribution was not normal data from LD tests , the groups were compared by the non-parametric Kruskal-Wallis analysis of variance test, followed by the Mann—Whitney test for two independent samples to determine whether the activities of the control and drug-treated groups differed from each other. This allowed the effects of the various dilutions tested in the two series of experiments, standardized as a percentage of their internal control values for untreated animals, to be compared and statistically evaluated.

Results 3. LD Choice Table 1 reports the mouse behavior data in the LD test, in terms of the two main parameters: percentage of time spent in the illuminated area and the number of transitions between the two compartments. The mean time spent by control placebo-treated animals in the illuminated area was The number of transitions between the two compartments increased little in the G. Table 1: Effects of G.

The placebo-treated control animals spent 6. In the G. The distance traveled by mice in the central zone of the arena was also positively influenced by treatment with G. During the OF test the total distance traveled by the mice in the entire arena was also analyzed.

No significant effect was found in drug-treated versus placebo-treated animals, indicating that the observed differences in time and distance spent in the central zone were not due to changes in the general, unspecific, locomotor activity of the mice. Table 2: Effects of G. The weight of the animals before and after the treatment was similar in the three treatment groups, suggesting no differences in feeding, metabolism or growth attributable to drug effects data not shown.

There was found to be a significant difference between the mean values for the five experiments see notes to Tables 1 and 2 , which was taken into account to control for its possible confounding effects. Finally, no significant interactions between groups and experiments were noted. This yielded results qualitatively similar to those for the CD1 mice in both test models, confirming an increase in the time spent and distance traveled in the center of the OF induced by G. However, due to the variability of responses between animals, the differences between control- and G.

Diazepam was not included here to avoid needlessly sacrificing animals, since the results for this control drug were sufficiently clear from the first series of experiments. In the OF test Table 3 , the percentage time spent in the center of the arena and the distance traveled in the center were increased by all the G. In this series of experiments, too, the tested dilutions were found to have no significant effect on the unspecific locomotor activity, as indicated by the total distance traveled in the arena.

Table 3: Effects of G. Summary of All the Experimental Data A summary of the main results of this investigation is given in Figure 1.

The mean percentage values for all the mice of the treated groups confirms the increase due to diazepam but not due to G. Figure 1: Summary of the effects of G. In each experiment the functional activity of mice was calculated as a percentage with reference to the mean value of the placebo group control vehicle. Discussion In the field of psychopathology, animal models have become an invaluable tool for analyzing the mechanisms of various disorders, and have aided in developing and predicting therapeutic responses to pharmacological agents such as benzodiazepines.

In this work we investigated the effects of G s.

Traditional difficulties in accepting these models stem from the argument that there is no conclusive evidence that what occurs in animals is equivalent to what occurs in humans. On the other hand, most conventional drugs and, recently, several homeopathic medicines have been tested in animal models, whose main advantages are that they allow multiple testing under controlled conditions and easier access to studying the mechanism of drug s activity.

Different Symptoms Modulation According to the Model Most behavioral procedures for studying the pharmacology of anxiety use models involving non-conditioned behavior, that are usually based on novelty induced variations in exploratory activity.

Considering the LD model, diazepam was active, as expected, significantly increasing the time spent in the light arena and the number of transitions. In the same assays, G. Our data, showing small, but not significant, effects of G. Differences between the type and severity of external stressors or the animal strain or in the experimental setup might account for the high variability of results reported in different experimental conditions and by different laboratories [ 21 , 22 ].

It has been noted that the extent to which an anxiolytic compound can facilitate exploratory activity depends on its baseline level in the control group [ 23 ].

Since our experimental setting did not involve prior exposure to stress, it is conceivable that in those conditions the response of the mice to G. The effects of G.

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Thigmotaxis is a very conspicuous behavior in anxious animals, and plays a role in the formation of avoidance behavior and cognition. It is therefore a primordial behavior, with a genetic basis, that is ecologically important and used by both animals and humans for exploring the environment.

The finding of a drug-induced increase in the distance traveled in the center of the arena, but not in the total distance traveled, suggests that the effects of G. Since we used a control vehicle as a placebo in all experiments, the effects cannot be attributed to the very low dose of ethanol present in the final operating solutions, unlike in certain previous studies where the use of alcohol as vehicle for homeopathic preparations might have been able to produce some background effects in behavioral tests [ 24 ].

A further methodological issue that reinforces our findings is the systematic use of blinding, a procedure rarely employed in animal research, but which is worthwhile especially when testing hypotheses that are apparently in contrast with conventional scientific wisdom, such as the very existence of biological activity of solutions diluted beyond the Avogadro limit.

Since the reference benzodiazepine drug was inactive in the OF test, the results indicate that, at least in our experimental conditions, OF and LD explore different emotional responses, with different sensitivity to drugs and neurological mechanisms.

This suggestion is in agreement with reports showing that anxiolytic treatments do not by themselves increase exploration in the central zone of the OF, but that they do decrease the stress-induced inhibition of exploration behavior [ 14 ].

To the best of our knowledge open field behaviors of mice were reduced by diazepam at doses which may be within the sedative—hypnotic range [ 16 , 28 ]. These conflicting drives could be influenced in various ways by drugs and small differences in the experimental conditions may have marked effects on the outcomes.

Ethological models present individual differences and variable behavioral baseline levels and this requires strong care for variable parameters linked to environment, handling and testing [ 14 ]. Doses and Dilutions Due to the complexity of the experimental setting, and in particular the need to use many animals in each group, in this study we started with a single dose potency of G.

If confirmed, these effects on mouse behavior would be much more coherent with the homeopathic paradigm and with traditional homeopathic medicine than initially expected. A number of observations—coming from several research fields [ 11 , 24 , 31 — 34 ]—suggest that biologically active compounds may indeed have high-dilution effects which mimics those of lower dilutions higher doses : in homeopathy there does not exist linearity or proportionality between molecular concentration of active principles and therapeutic effect [ 33 — 41 ].

So far there is no satisfactory or uniting theoretical explanation for these observations, but recent evidence seems to point to organization of the solvent water on a mesoscopic scale: the nano-heterogenous structure of water can be determined by interactive phenomena such as coherence [ 42 — 44 ], epitaxy [ 45 , 46 ], temperature-pressure processes during strong agitation and formation of colloidal nanobubbles containing gaseous inclusions of oxygen, nitrogen, carbon dioxide, silica and possibly the remedy source material [ 46 — 51 ].

These unusual properties of high dilutions, which merit further investigation, are potentially relevant not just to homeopathic pharmaceutical practice, but also to basic research into cell sensitivity to regulation. Hypothetical Mechanisms A hypothetical diagram showing the possible action mechanism s of G. A possible target of G.

This effect could be due to a specific interaction at the level of the glycinergic receptors since this was antagonized by strychnine. However, since we noted differences in the action of conventional and homeopathic anxiolytic-like effects, it is highly conceivable that the multicomponent nature of the active principles of G.

Figure 2: Hypothetical mechanism of action of G. Benzodiazepines act by enhancing the inhibitory effects of the neurotransmitter GABA. The neurosteroid allopregnanolone is produced from progesterone and acts both on the GABA receptor—at a binding site different from benzodiazepines—and on other receptors including the serotonine 5HT and nicotinic-acetylcholine receptor, thus reducing the impulse generation in postsynaptic neurons.

By fine tuning inhibitory transmission through glycinergic system and allopregnanolone synthesis [ 19 , 20 ], G.

Due to the multicomponent nature of G. Beneficial, therapeutical effects of extremely low doses of agents which are toxic at high doses are defined hormesis. The development of interventions that activate hormetic signaling pathways in neurons is a promising new approach for the prevention and treatment of a range of neurological disorders [ 57 ].

Conclusions Taken together, our data suggest that the effects of G. Mouse behavior in the OF test is affected by various factors such as individual testing since mice are social animals , neophobia emotional response to a novel environment and agoraphobia being in an exposed setting from which there is no easy means of escape.

In our experimental conditions G. Materia Medica, according to which individuals who respond to this medicine are characterized by experiencing strong discomfort when confronted with novelty or unfamiliar situations. The study provides a basic knowledge of the pharmacological effects of G. Funding Grants from Laboratoires Boiron s.

Notation Recently the same group showed that following small methodological changes a significant anxiolytic-like effect of Gelsemium s. Dose-effect study of Gelsemium sempervirens in high dilutions on anxiety-related responses in mice. Psychopharmacology 4 , Acknowledgments The authors wish to thank Prof.

Cristiano Chiamulera, Prof. Andrea Sbarbati, Prof. The authors have no conflicts of interest. References W. Pilkington, G. Kirkwood, H. Rampes, P. Fisher, and J.


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Desislava August 15, Amir January 20, Nic Dhonnchadha, and M. Therapeutic pocket book. Read Books Online http: We performed the study on the unconditioned responses, using ethologically based paradigms which involve the spontaneous reactions to non-painful stimuli.

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