PHARMACOLOGICAL SCREENING OF UDESALEEB ( PAEONIA, EMODILINN.) (AN ANTIEPILEPTIC UNANI DRUG)
A THESIS SUBMITTED TO THE THE FACULTY OF MEDICINE ALIGARH MUSLIM UNIVERSITY
ALIGARH FOR M.D. (UNANI) IN ILMUL ADVIA (PHARMACOLOGY)

  Musthaq Ahmad 1979

SUMMARY

     The present study was under taken to investigate phytochemical and pharmacological properties of tubers of Paeonia emodi Linn. was obtained from Dawakhana Tibbiya College, Aligarh Muslim University, Aligarh.

      The phytochemical studies include macroscopy qualitative tests for various organic compounds, fluorescence analysis of the powdered drug, thin layer chromatography of aqueous and alcoholic extracts of the drug, ash values, extractive values and the quantitative estimation of reducing sugar, nitrogen and moisture content.

     Biological studies includes the effect of Paeonia emodi Linn. on chemically and electrically induced seizures, behavioural studies in rats, body  temperature of rats, frog's heart ( in vovo and vitro), blood pressure and respiration of dog, smooth muscle of guinea pig ileum, skeletal muscle of frog rectus abdominis and experimentally carrageenin induced acute inflammation in rats.

    Macroscopy: The tubers of Paeonia emodi Linn. are greyish brown in colour, size varies from 2.0 - 15.5 cm in length and 3.0- 9.5 cm. in diameter. They vary in shape may be eliptical, round or elongated. The surface is rough and multiple elongated grooves are present in elngated tubers. Round tuberrs possess plane surface. Tubers show longitudinal scars and are bitter in taste.

    Qualitative tests for the organic constituents of this drug show, the presence of reducing and non - reducing sugars, proteins, tannins, glycosides and carbohydrates. Tests of alkaloids, sterol and resin show their absence in both the extracts.

    The fluorescence characteristics of the dry powder (tubers of Paeonia emodi Linn.) was studies, and these findings from the physical standard of the powdered drug. The powdered drug observed under ultraviolet light appears light green in colur. Power treated with N-sodium hydroxide in methanol and observed under ultraviolet light appears dark green in colour. Powder mounted in nitrocellulose and observed under ultraviolet light appears bright green. Power treated with sodium hydroxide in methanol and mounted in nitrocellulose appears bright green when observed under ultraviolet light.

    The thin layer chromatography of the ethyl alcohol extract using n-butanol, acetic acid and water(4:1:5) solvant system showed four spots having Rf values 0.19, 0.48, 0.81 and 0.97. The aqueous extract also shows four spots having Rf values of 0.18, 0.44, 0.75 and 0.88.

    Total percentage of ash, acid insoluble and water soluble ash was determined as 3.87% ± 0.11, 0.67% ± 0.02, and 0.98% ± 0.05 respectively.
    The present extractive value was 0.688%  ± 0.002 in pet-ether 0.232% ± 0.002 in ether, 0.188 ± 0.002 in chloroform, 0.012% ± 0.002 in benzene, 14.76 ± 0.002 in ethyl alcohol, 27.804% ± 0.0024 in water.

   The percentage of nitrogen and reducing sugar of tubers were estimated. They are 3.88% ± 0.14 and 3.03 ± 0.04 respectively. The percentage of moisture in dry tubers of Paeonia emodi Linn. was estimated as 14% ± 0.000.

    The anti -convulsant activity of the tubers of Paeonia emodi Linn. was studied against electrically and chemically induced convulsions in rats.

    The aqueous extract of Paeonia emodi Limn. in the dose of 1 gm/100 gm of body weight one, two and three hours before electrically induced convulsions ( supramaximal electroshock) showed average flexion phase 4.66 ± 2.30 sec. ( P < .05), 8.50 ± 1.40 sec. ( P < .05) and 8.67 ± 1.99 sec. ( P < .05) average extension phase 5.00 ± 1.31 sec. ( P < .05) 2.17 ± 1.28 sec. ( P < .05) and 2.50 ± 1.59 sec. ( P < .01) respectively as compare to control group ( flexion phase 3.50 ± 0.84 sec. and extension phase 11.00 ± 1.41 sec.). The values in the standard group treated with phenobarbitone sodium ( 6 mg/100 gm of body weight) two hours before the electroshock were, flexion phase 11.25 ± 4.57 sec. ( P > .05) and extension phase 1.33 ± 1.33 sec. ( P < .001). The alcoholic extract in the dose of 1 gm/ 100 gm of body weight and shock given one, two and three hours after drug administration showed flexion phase 7.0  ± 0.26 sec.  ( p< .001), 7.0 ± 0.26 sec. ( p < .001 and 6.66 ± 0.91 sec. ( p < .05) and extension phase 0.0 ± 0.0 sec. ( p < .001), 0.0 ± 0.00 sec. ( < .001) and 1.50 ± 1.02 sec. ( p < .001) respectively as compared to untreated group. These findings suggest that aqueous extract of Paeonia emodi Linn, reduce the the extension phase and increases the flexion phase in supra maximal electroshock seiqures of albino rats. Further that the alcholic extract of the test drug completely abolishes the extension phase and increases flexion phase appreciably in the supra maximal electroshock seizures of albino rats when shock is given one and two hours after drug ingestion. The peak effect in both extracts is observed after two hours of drug ingestion. These findings also suggest that the alcholic extract of the test drug is more potent than its aqueous extract in this study.

     Protection against chemically induced seizures was studied. Alcholic extract of Paeonia emodi Linn. in the dose of 1 gm / 100 gm of body weight before the injection of leptazol ( 10 mg/ 100 gm of body weight of rat subcutaneously) showed significant protection 83% ( P < .05) as   compared to control group ( leptazol only) and the aqueous extract of Paeonia emodi Linn, in the same dose showed 17% protection as compared to control group while standard drug ( phenobarbitone sodium 6 mg / 100 gm of body weight) showed 83%   protection ( p < .05) against leptazol induced convulsions.

     Protection from the death within two hours after leptazol injection was also observed. Alcholic extract of Paeonia emodi Linn, in the dose of 1 gm/ 100 mg of body weight showed significant protection form death 83% ( p < .05) as compared to control group ( leptazol only). Aqueous extract in the same dose showed 50% ( p > .05) protection from death as compared to control group while standard drug ( phenoharbitone sodium 6 mg / 100 mg of body weight) showed 100% protection ( p < .05) against death caused by leptazol induced convulsions. These findings suggested that alcholic extract of Paeonia emodi Linn. is more potent than aqueous extract against leptazol induced convulsion of albino rats.

     Behaviourl studies on aqueous extract of PAEONIA EMODDI Linn.  ( 500  mg/ 100 gm of the body weight) were made on spontaneous activity of rats. The mean number of counts in control ( untreated) group was 266.38 ± 26.83, in the standard group ( treated with chlorodiazipoxide, 2.5 mg/100 of the body weight) the counts were 138.25 ± 22.10 ( p < .01), while in the test group ( Paeonia emodi Linn.) the counts were significantly less 118.75 ± 20.11 ( p << .01). The mean distance travelled ( motor activity ) by rats in 30 minutes in control group was 68.63 ± 6.02, the counts were significantly less in test group treated with aqueous extract of Paeonia emodi Linn. 25.75 ± 4.33, ( P < .001). The values in the standard group ( treated with chlorodiazipoxide 2.5 mg/100 gm of body weight) was also less as compared to control group ( 43.25 ± 7.07), ( P < .05). These finding  suggest that the drug posses significant effect on spontaneous and motor activity of albino rats.

    The effect of aqueous and alcholic extract of Paeonia emodi Linn.. was studied on body temperature of albino rats in the dose of 1 gm / 100 gm of body weight orally. Before drug ingestion the mean rectal temperature was 35.25 oC ± 0.67. The temperature after giving aqueous extract of drug decreased to 33.5 oC ± 0.37 ( p < .05) , 33.5 oC ± 0.84 ( p < .5),  32.62 oC ± 0.78 ( p < .05) and 32.62 oC ± 0.82 ( p < .05) after 30,60,90 and 120 minutes respectively. In the second group of rats mean rectal temperature before drug ingestion was 37.38 oC ± 0.22. After treating the animals with alcholic extract of Paeonia emodi Linn. The temperature decreased to 36.06 oC ± 0.44 ( P < .001), 35.38 oC ± 0.35 ( p < .001), 34.88 oC ± 0.28 ( P < .001) and 34.38 oC ± 0.26 ( p < .001), at 30,60,90 and 120 minutes respectively.

     The mean rectal temperature of the control group at 0, 30,60,90 and 120 minute levels was recorded as 35.31 oC ± 0.33, 35.31 oC ± 0.26 , 35.81 oC ± 0.38, 36.31oC ± 0.34 and 36.31 oC ± 0.26 respectively. The P values of group treated with aqueous extract of Paeonia emodi at levels of 30, 60, 90 and 120 minutes as compared to contro9l group are < .01, <.05, <.001 and <.001 respectively and that of the group treated with alcholic extract of Paeonia emodi are >.05, > .05, <.01 and < .001 respectively.

    The effect of aqueous and alcholic extract of Paeonia emodi Linn, was studied on frog's heart. On isolated frog heart, the aqueous extract in the doses of 1 mg to 10 mg did not produce and effect on heart. However, in one higher doses it decreased heart rate and force of contraction. In the doses of 50 mg and 100 mg the heart rate decreased from 26/ min to 19/ min and 26/ min to 15/ min respectively. The alcholic extract of the drug in 1 mg dose significantly increases the heart rate and force of contraction of heart, the effect being more marked on sysstolic phase. In the higher doses this effect gradually diminishes. In the dose of 50 mg the heart rate was observed as 19/ min. in comparison to normal rate of 45/ min. while in the dose of 100 mg. it produced heart block for 30 seconds.

    On continuous heart perfusion ( invivo) of frog aqueous extract in the lower doses from ( 5-25 mg) did not produce any significant effect on the heart. However, in the higher dose ( 50-100 mg) drug has a depressant effect on heart. The heart rate and cardiac out put was recorded as 18/min.   and 2.2 ml/min. respectively in the dose of 50 mg when the normal out put was 3.5 ml/ min. and normal heart rate as 30/min. In the dose of 100 mg. heart was blocked for 30 seconds. The alcholic extract of the drug in the dose of 1 mg produced significant increase in ionotropic and chronotropic activity of the heart and in cardiac out put. In the higher doses this effect was gradually decreased causing partial block for 30 seconds in the dose of 50 mg.

   The aqueous extract of Paeonia emodi Linn. in doses of 5 to 100 mg/kg of body weight produced no effect on arterial blood pressure of anaesthetised dog. No effect on dog's respiration was observed by aqueous extract of the drug also.

   The studies on isolated guinea pig ileum revealed that the aqueous and alcholic extract of the drug has no effect on smooth muscle in the dose of 5 to 100 mg. Further the drug did not protentiate or antagonise the effect of acetylcholine and histamine in this muscle. The rectus abdominis muscle of frog also revealted that the drug has no effect on skeletal muscle in the dose of 5 to 100 mg and did not potentiate or antagonise the effect of acetylcholine.

   The anti-inflammatory activity ( in acute inflammation induced by carrageenin in the paw of albino rats) of Paeonia emodi Linn. was studied. The volume of the paw was measured before and after carrageenin at zero and three hours with the help of a plethyamometer. The treatment with the drug was given one hour before the induction of carrageenin. Aqueous extract of "Paeonia emodi" Linn. in the dose of 20 mg/ 100 gm inhibited the oedema ( 59%) after three hours following carrageenin injection as compared to the control. The alcholic extract of Paeonia emodi Linn. in the same dose inhibited odema by 66% as compared to control group. The percentage of inhibition of inflamation in the group treated with oxyphenyl butazone ( standard drug) in the dose of 10 mg / 100 gm was 68.96%.

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