Alkaline Phosphatase in Actinomyces sp by Potencies of Lycopodium
– A. S. Paranjpe & S. P. Kale.
Abstract
The activity of the alkaline phosphatase enzyme has been tested in micro-organism Actinomyces with different potencies of Lycopodium. There is a significant increase in the enzyme activity of all potencies used. This suggests that the possible mode of action of homoeo potencies may be through a modification in gene function.
Introduction
Homoeo potencies offer a virgin field for scientific research. The very first scientific task is to demonstrate that there is a difference between a potentised solvent and an unpotentised one. There is abundant clinical evidence to accept the potentised solvent have medicinal values. However, this does not convince the skeptics. This is because chemically no material difference is expected between them beyond the potency of 12. From the time of conception of homoeopathy, several workers have undertaken the task of experimentally demonstrating the action of homoeo potencies. So far, there is no result, which is reportedly reproducible. In many physical and biological experiments, the action of homoeo potencies with increasing potencies is observed to be cyclic, occasionally varying between inhibitory and stimulating. Such cyclic variations around the normal activity do not establish the action unequivocally. Further, there are no experiments to suggest the path of action of a medicine. In the present paper, using a microbial system, we give a simple laboratory experiment, which demonstrates that the biological activity of a potentised solvent is different from that of the unpotentised one. The experiment also suggests that the path of action of the medicine is by triggering certain genes which otherwise do not express.
Materials
Culture: Stationery phase culture of Actinomyces sp.
Chemicals:
a) P-nitrophenol phosphate (0.25mol).
b) CaCl2 (0.5mol).
c) Tris buffer.
d) P-nitrophenol (sigma) standard.
e) 0.5N NaOH.
f) Blank sugar pills No. 30 (Zorastrian Homoeo Pharmacy).
g) Lycopodium potencies: 15 (prepared in the laboratory), 30, 200 and 1000 (Zorastrian Homoeo Pharmacy).
Materials used for the growth of cultures are standard laboratory materials. Sugar pills of the same size are used as solvent carrier for blanks and different potencies. These are first soaked in alcohol and dried over paper in order to remove excess alcohol. A pill of weight 1.9mg prepared in this manner contains about 1017 alcohol molecules. One pill of blank and of respective potencies is dissolved in about 4-ml distilled water which represents the samples to be used in further experiments.
Method
The culture of Actinomyces sp. was grown in minimal medium with 1% glucose as shake for 48 hours at 30 degrees celcius. One ml of sample or control is added to a flask of 50 ml. culture, shaken and incubated further under similar conditions. Three flasks per sample are used in a typical experiment. 3ml of cultures were taken periodically for assay of enzyme activity.
Enzyme assay
The method of Eivazi and Tabetabai was followed for the assay. To 1 ml substrate and 4ml of tris HCL buffer, 3ml of culture was added. The tubes were mixed in a vortex mixer and incubated at 37°C for 30 minutes. The p-nitrophenol formed was extracted with 1ml of CaCl2, and 4ml of 0.5N NaOH. The yellow colour developed was measured at 410nm using a spectronic – 20 (Bausch & Lomb) spectrometer. The amount of p-nitrophenol formed was determined using a standard curve obtained by use of p-nitrophenol standard.
Results
In an open experiment, there was an increasing stimulation of enzyme activity over that of control with increasing potency of Lycopodium. The stimulation is 85%, 161% and 215% for n = 30, 200 and 1000 respectively. This stimulation was observed over a period of 48 hours. It is interesting to note that the stimulation has occurred in the first three hours only. Thereafter, there is no significant stimulation by the drug and the pattern of stimulation remains the same. The double blind experiment also shows a significant stimulation by the drug, 81 %, 50% and 94% for n = 30, 200 and 1000 respectively. A well-defined trend with respect to change in potency is not observed. Though stationary phase culture was used in the experiments, it is known that Actmomycetes does not form a synchronous culture due to its mycelial character. This may account for the lack of systematic in the results. In all these experiments, the enzyme activity in control experiments ~30 micromol of p-nitrophenol/50ml of culture medium, is comparable with the normal enzyme activity of the microbe.
We have also measured the enzyme activity with 15th potency prepared in the Hahnmannian manner. This was with an aim to compare the action of Hahnemannian potencies with the commercial ones. There is a significant difference between the enzyme activity of the drug and the control, 69% in the open experiment and 134% in the blank experiment.
Discussions
In these experiments, the microbes Actinomices were used as an individual biological system. The aim was to see if the potentised medium has any biological activity. Experiments were not intended to test the effect of a medicine on a microbe as is done in in-vitro pathological experiments. The philosophy behind such a choice is that if an nth potency can be used to dynamise a solvent, that is a chemical, to (n+1)th potency is capable of acting on a chemical system. Hence, it should be capable of acting on any biological system as well. It was decided to monitor Alkaline Phosphatase activity as a parameter. The drug Lycopodium was chosen because it is a deep acting medicine known to have an affinity for liver and enzyme activity and is known to be perverted during liver disorders. Lycopodium potencies show a definite stimulating effect on activity of alkaline phosphatase in Actinomyces sp. The primary action appears to enhance the alkaline phosphatase activity which is in agreement with the expected primary action of the medicine. No systematic relationship seems to evolve between the activities of different potencies. The stimulation is different in different experiments with the same potency. At this stage, it is too early to expect some systematics with potencies since synchronous culture cannot be obtained for the microbe used and nature of potencies and their inter-relationship is unknown. However, the average stimulation by different potencies of Lycopodium-111 % over that of control, is quite significant. The action of the medicine appears to be instantaneous and single shot and medicine does not seem to cause any further effect as a function of time. This is in contrast with the action of the medicine in human system, where the secondary action, which is the reflex action of the system to oppose any external influence, and hence is by nature opposite to the primary action, is curative. Enzyme activity can increase only if the genes, which are responsible for this activity, are triggered. Thus the medicine appears to modify the function of genes.
Conclusions
We have demonstrated that alcohol potentised with Lycopodium can alter biological activity in Actinomyces sp. The mode of action appears to be by modifying the gene response.
In future, we plan to see if the enhanced alkaline pnosphatase activity remains after a large number of generations of the microbe. By using successive potencies systematically prepared in the laboratory in Hahnemannian way, we plan to study the systematic effect of potencies on enzyme activity. We also plan to investigate if in these experiments, the activities of other well-known enzymes are also altered by the potencies of Lycopodium. We do believe that apart from enhancing the image of homoeopathy as a system of medicines, these experiments will trigger activity in biological sciences.