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Selective Pancreatic Beta Cell Activities by Ultra Dynamised Dilutions of Alloxan at Micro-doses: An Experimental Approach

Dr. Sunil Kumar

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The acclimatised animals were subjected for quantitative analysis of blood sugar estimations adopting the Folin-Wu method, by taking 0.5 ml blood sample from the tail vein or through cardio-puncture and measuring absorbance at 620 nm wavelength in a Beckmann model 35 spectrophotometer.

Each animal was weighed and blood was collected from the tail vein for blood sugar and Serum GH-estimations before commencement of the experiments. All the animals were injected intravenous injection 150mg/Kg.b.w. of alloxan monohydrate (Sigma Chemical Co., USA) in citrate buffer of pH 4.5. 10 days after alloxan injection, blood from each of them was again tested and when hyperglycaemic condition (blood glucose more than 250 mg/dil.) was established, the treatments of test drug, alcohol, normal saline were mitigated. The potentised form of Alloxan, 6x, 30X, 200x and 1000x as well as equivalent concentration of vehicle i.e. 90% v/v alcohol, 0.9% physiological saline and dynamised and undynamised preparations were made as per formulations of Homoeopathic Pharmacopoeia Laboratory, Ghaziabad. The parallel studies were conducted with tolbutamide (Rastinon), glibenclamide (Daonil) and insulin, The diabetised rats were divided into groups of 10 each as follows.
The long term experiment was conducted over 45 days but the drug was administered for the first 30 days once in a day to each animal. After that the drug, vehicle, saline administration was stopped and the animals were assessed for blood sugar Stabilization.

Blood samples were collected from the tail vein on 15th & 30th day of treatment for determination of blood glucose and serum GH level. All the blood sugar estimations were done of 14 hours fasted animals using Spectrophotomenter (Systronic) Serum GH estimations were determined by Chemiluminescence immuno assay (Luminometer LB9501/16 using the kits supplied by Nichols Institute Diagnostic, USA. Serum GH 9501/16 using the kits supplied by Nichols Institute Diagnostic, USA. Serum GH assay were performed in duplicate and in a single run.
Body weight lost & gained over initial body weight was recorded. For beta cells study, 2 animals of all the groups were sacrificed on 30th day of treatment and pancreatic tissue, brain and other organs/tissues were dissected out, quickly fixed in Bouin’s fixative. The paraffin section 4um thickness were stained in Haemotoxylin-eosin and Gomoris Aldehyde Fuchsin stain (Cullings, 1963).

The beta cells per islet area in cross section were examined under light microscopy (Olympus). All the data were statistically analysed and level of significance was calculated by student’s ‘t’ test.

Results
The experimental data obtained was statistically analysed using Student’s ‘t’ test. It is evident from the observations that regular administration of dynamised form of Alloxan in its 6x, 30x, 200x, and 1000x potencies at a dose level of 50ul/100g.g.w. once daily for 30 days exhibited a slow and steady fall in the blood sugar accompanied with an increase in growth hormone levels P<0.01 to P<0.001 respectively as compared to normal control and undynamised dilutions of alloxan, dynamised and undynamised alcohol fed control groups as evident from the Table-1 & II. Furthermore, it was also observed that hypoglycaemic potentiality and growth hormone profile of dynamised dilutions of alloxan are more pronounced and perceptible in 200x and 1000x, P<0.001 as compared to 6x, and 30x P<0.01 potencies.

The acute and sub-acute toxicity studies indicate that dynamised control groups of alcohol show more toxic effects and finally lethal to the animal when compared to dynamised and undynamised dilutions of alloxan, vehicle and saline. The revival of degenerated and damaged B-cells were not achieved perceptibly in 30x, 200x and 1000x potencies of dynamised alloxan.

Histomorphometrical studies of brain also discern the involvement of Hypothalamo-hypophysial pancreatic axis. The blood sugar stabilization studies of dynamised dilution of 30x, 200x and 1000x potencies exhibited stabilization of blood sugar after withdrawal of these drug for 10-25 days. The beta cells counts, blood sugar and growth hormone levels in various groups of alloxanised rats are given in Table I & II.

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