|| Alkaline Phosphatase
in Actinomyces sp by Potencies of Lycopodium
|- A. S. Paranjpe & S. P.
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.
|| 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.
||Culture: Stationery phase culture
of Actinomyces sp.
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.
|| 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.
|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.
|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.
|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.