Original Article. Free Radical Scavenging Activities and Antidiabetic Properties of Various Extracts of Salacia Reticulata

Transcription

1 Thai Journal of Physiologial Sienes Vol. 21 No. 2 Ot Mar Original Artile Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN 1, Madhyastha S 2, Benjamin S 3, Gopala Krishna K 4 and Srinivasan KK 5 ABSTRACT In the experiment, the root extrat of Selaia retiulata has een found to have hypoglyemi ativity oth in normal and in streptozotoin-indued diaeti rats. The deotion of Selaia retiulata root is used in the treatment of rheumatism, gonorrhea, ithing and swelling, asthma, thirst, amenorrhea and dysmenorrhea. Various onstituents of Salaia retiulata Wight (Hipporataeae) extrats was tested on alino rats (Wistar strain) in whih diaetes were indued y administration of alloxan. The rats were grouped into diaeti ontrol, insulin treated and various extrats of Salaia retiulata wight treated. The effet of extrat on lood gluose level after 15 & 30 days was assessed. Savenging ativity against superoxide anion radial and H2O2 savenging ativity were also tested. Hypoglyemi effet is not onfined to the extrats of either the ark or ore root alone, as it has een oserved that there was signifiant hypoglyemia in many of the extrats of different parts. In vitro studies show savenging ativity of free radials, superoxide and hydrogen peroxide. Our study onfirms the perentage of hypoglyemi effet exerted y all the different frations almost orrespond with the level of ation exerted y insulin. Hene we strongly reommend the use of Salaia retiulata Wight extrat in diaeti ondition. Key words: Salaia retiulata, diaetes mellitus, holesterol, triglyeride, free radial savenging ativities INTRODUCTION Diaetes mellitus is an inreasingly ommon, potentially devastating, expensive, treatale ut inurale life long disease. A estimated 5% of the population in US is affeted y diaetes mellitus 1. The inidene is 1 Department of Physiology Kastura Medial College, International Centre, Manipal, India Department of Anatomy, Kastura Medial College Mangalore, India Department of Physiology, Kastura Medial College Manipal, India Department of Biohemistry, Kastura Medial College Manipal, India College of Pharmaeutial Sienes, Manipal, India Address for Correspondene Department of Physiology, Kastura Medial College, International Centre, Manipal India sheakrdon@yahoo.om almost similar in Orientals as well 2. Insulin and other antidiaeti drugs have een used in allopathi pratie of mediine for the last 75 years. Of late there is an inreased interest to understand the utility of plant and plant produts as therapeuti agents. The utility of the plant and plant produts whih an e taken orally for diaetes is in vogue in different parts of the world. In the experiment, the root extrat of Salaia retiulata (Hipporataeae) has een found to have potent hypoglyemi ativity oth in normal 3 and in streptozotoin-indued diaeti rats 4. Furthermore, the deotion of S.retiulata roots is used in the treatment of rheumatism, gonorrhea, ithing and swelling, asthma, thirst, amenorrhea and dysmenorrhea 3. Root extrats of Salaia olonga Wall showed positive results against diaetes 5. However the hypoglye- 48

2 Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN, et al. mi effet of Salaia retiulata Wight was not studied in an animal model with supporting ontrol groups and also mehanism involved in its ation. Hene the effiay of the plant extrat was tested on alino rats (Wistar strain) in whih diaetes were indued y administration of alloxan. The effet of the extrat after 15 and 30 days of treatment was assessed. We further measures levels of serum holesterol and triglyeride after 30 days of treatment. Savenging ativity against superoxide anion radial and H 2 savenging ativity were also tested. The results of this study have an impat in delivery of etter health are whih is less umersome, more funtional and would e within the reah of ommon man from the eonomy aspet as well. MATERIALS AND METHODS Animals: Wistar male rats aged two to three months old ( g) were used in the study. All the rats were maintained under 12 hr day light environment. For housing the animals, polypropylene ages ( m 3 ) were used (3 animals/age) with paddy husk edding at 26 C ±1 C. All rats were maintained on the standard rat food and water ad liitum. All the experimental proedures were approved y the Institutional Ethis Committee. Maintenane of the animals was done as the guidelines of Government of India for using of Laoratory animals. Government of India notifies the rules for reeding and onduting animal experiments, proposed in the gazette of previous study. 5 Indution of Diaetes: Alloxan solution was prepared at low temperature y plaing ie ues around the eaker, eause alloxan is known to get oxidized at room temperature. Alloxan was dissolved in 0.9% saline. Injetion of either saline or alloxan was arried out on rats that had een deprived of food for aout 24 hours. After aout 30 minutes of injetion, food was provided to animals ad liitum. Confirmation of Diaetes: After seven days of stailization period, lood samples were otained from rats fast overnight. Blood was drawn from intra orital plexus y inserting a muap apillary. Aout 1 ml of lood was drawn into a test tue having sodium fluoride. The sample of lood was mixed with the sodium fluoride and allowed to lot. After an hour, the sample of lood was entrifuged (R&C entrifuge) at 2000 rpm for 15 minutes. After entrifugation, the supernatant serum was olleted and the gluose level was estimated. The alloxan-injeted group, whih had more than 200 mg% of lood gluose, was inluded in the study. The diaetesindued animals were randomly assigned into different groups with eight animals in eah group. The onfirmation of diaetes was taken as day 1 for further ourse of treatment as per the group. The lood sugar estimation was done again on 15 th and 30 th day from rats fast overnight. Preparation of extrat Salaia retiulata roots were otained loally and were authentiated y Dr.Gopalakrishna Bhat, Professor of Botany, Poorna Prajna College, Udupi. A vouher speimen has een deposited in the College of Pharmaeutial Sienes, Manipal. Preparation of extrat using root and ark: Shade dried finely powdered root and ark (2 kg) was soaked in 95% ethanol for four days. The old extrat was deanted off, and the soaked material was refluxed in 5 athes of aout 400 g with 95% ethanol (3 litre x 3 hr). Both the old and warm extrats were oiled and onentrated under redued pressure. It was then spontaneously evaporated to dryness (yield 50.8 g). This was frationated using petroleum ether (PE), diethyl ether (DE) and ethyl aetate (EA) in suession (yield was 4.58 g, 7.01 g and 4.9 g respetively for different frations). Preparation of extrat using ark (2 kg): Ethanol extrat yield (98 g), ethyl aetate extrat yield (2.5 g), diethyl ether extrat yield (5 g) and petroleum ether extrat yield (3.4 g). Preparation of extrat using the ore root alone (2 kg): Ethanol extrat yield (60 g), ethyl aetate extrat yield (2.8 g), diethyl ether extrat yield (2.6 g) and petroleum ether extrat yield (4.9 g). Treatment Shedule The treatment shedule of the various groups is as detailed. On the 15 th and 30 th day of treatment, lood from animals fast overnight was drawn and the iohemial parameters were assessed. There were 8 groups as following: NC - Normal Control - Vehile (propylene glyol) DC - Diaeti Control - Vehile (propylene glyol) 49

3 TJPS Vol. 21 No. 2 Ot Mar IN - Insulin treated - 6 units/ kg ody weight EE - Ethanol extrat treated - 50 mg/ kg ody weight (root+ark/ ark/ ore root) PE - Petroleum ether extrat - 50 mg/ kg ody weight (root+ark/ ark/ ore root) DE - Diethyl ether extrat - 50 mg/ kg ody weight (root+ark/ ark/ ore root) EA - Ethyl aetate extrat - 50 mg/ kg ody weight (root+ark/ ark/ ore root) WE - Water extrat - 50 mg/ kg ody weight (root+ark/ ark/ ore root). The various extrats were dissolved in propylene glyol, and the drug was administered intragastrially using Ryle s tue. While preparing the drug, the onentration of drug was 10 mg/ml of propylene glyol. The weight of was reorded everyday, and drug was administered as per dosage required in relation to ody weight. Normal ontrol and diaeti ontrol groups were also given the vehile as per ody weight range of animals. The insulin group was injeted y insulin suutaneously with a dose of 6 unit/kg ody weight 6. Dilution of insulin to otain the required onentration was done y 0.9% sodium hloride solution. All the solutions (various extrats/insulin) usually lasted for aout 8-10 days one prepared. The various drugs/vehile/insulin were stored in refrigerator from 8 to 10 C. They were taken out of refrigerator and allowed to ome to room temperature efore administration. While preparing the solution of various extrat in propylene glyol, (where the drug did not dissolve at room temperature) the solution was warmed in waterath at aout C for aout minutes to ensure total dissolution. The treatment used to e done etween 8 and 10 AM one daily. The groups would reeive the treatment for 30 days from the day of onfirmation of diaetes. Estimation of lood gluose Done y gluose oxidase - peroxidase method (Kit marketed y Pointe Sientifi In. USA) Test priniple: Gluose oxidase Gluose + + H 2 O Gluoni aid +H 2 Peroxide H amino antipyrine Coloured omplex + H 2 O The red oloured omplex formed is measured at 505 nm. The intensity of the olour formed is diretly proportional to the onentration of gluose in the sample 7. Free radial savenging ativity: Savenging ativity against superoxide anion radial Priniple: Superoxide savenging ativity of the ompound was determined y monitoring the omposition of these with NBT for superoxide anion generated y PMS-NADH system 8. Proedure: Superoxide radials were generated with 1 ml 20 mm Tris HCl uffer ph 8.0 ontaining 0.05 mm Nitrolue tetrazolium (NBT), 0.01mM Phenazine methosulphate (PMS). Test ompounds were preinuated at room temperature for two minutes. The reation was slowed y the addition of mm NADH. Blue hromogen formed due to NBT redution was read at 560 nm. The results are alulated in perentage of inhiition for 10, 25 and 50 µg of the samples. Superoxide savenging ations of Salaia retiulata Wight ethanoli extrat frations of root and ark, ark, and ore root were ompared with known antioxidant asori aid. savenging ativity Reagents: Tris HCl uffer 20 mm ph 8.0, NBT 4 mg/10 ml uffer, NADH 6 mg/10 ml uffer and PMS 0.3 mg/10 ml uffer. NBT, NADH AND PMS were prepared efore the experiment. Blue hromogen read at 560 nm. (C - B) - (T - TB) % inhiition = (T - B) 100 C - Control, B - Blank, T - Test, TB - Test lank Tris Buffer - dissolved 1.21 g Tris in 50 ml DDW. Then add 26.8 ml 0.2 mm HCl and, adjust to ph 8.0. Make up the volume to 200 ml as uffer A. For 10 ml uffer A add 90 ml doule distilled water. H 2 savenging ativity H 2 savenging ativity was measured y peroxidase ased assay system. Horse radish peroxidase (HRP) used H 2 to oxidize guaiaol whih produed rown olour measured at 436 nm. If an antioxidant is inu- 50

4 Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN, et al. Tale 1 Test Test lank Control Blank Buffer 0.7 ml 0.8 ml 0.7 ml 0.8 ml Sample 50 µl 50 µl - - NBT 0.1 ml 0.1 ml 0.1 ml 0.1 ml PMS 0.1 ml 0.1 ml 0.1 ml 0.1 ml Mix inuate at room temperature for 2 min. NADH 0.1 ml ml - Tale 2 Test Test lank Control Blank Buffer 1.8 ml 1.85 ml 1.8 ml 1.85 ml Sample 50 µl 50 µl - - H 2 40 µl 40 µl Mix and inuate at 25 o for 30 minutes Guaiaol 50 µl 50 µl 50 µl 50 µl HRP 100 µl 100 µl 100 µl 100 µl Mix and inuate at room temperature for 15 minutes ated with H 2, the rate of H 2 disappearane an e alulated 9. Proedure: Different onentrations of test ompounds were inuated with 2 mm H 2 for 30 minutes at 25 o C. Remaining H 2 was measured y peroxidase system. Reation mixture 2.0 ml ontains 150 nm KH 2 PO 4 - NaOH uffer ph 7.4, 0.1 µl guaiaol and units of HRP. Perentage inhiition of H 2 y test ompounds was alulated. Ethanoli extrat frations of root and ark, ark, and ore root were tested with known peroxide savenging ativities of toopherol. Perentage inhiition was alulated for 50, 75 and 100 µg of the samples respetively. Reagents: KH 2 PO 4 - NaOH uffer ph 7.4, 150 mm, KH 2 PO 4 / 100ml DDW, 0.6 g NaOH / 100ml DDW, Guaiaol - 10 µl / 5ml DDW, HRP mg / 100 ml uffer, H 2-3 µl /10 ml uffer (Fresh prepared) Blue hromogen read at 436 nm. (C - B) - (T - TB) % inhiition = (T - B) 100 Statistial analysis The data were expressed as mean ± SE. The signifiane of differenes among the groups were assessed using one way analysis of variane (ANOVA) test followed y Bonferroni multiple omparison test using graph pad in stat (GPIS) software, version The p-value < 0.05 were onsidered as signifiant. Comparison of data within the group at different intervals was assessed y paired t test. The differene of freedom has een mentioned at appropriate plaes. RESULTS 1. Blood gluose level in the different groups 1.1 Treatment: Extrat of root and ark In normal ontrol group, whih was not administered alloxan, the lood gluose level was around 80 mg/dl throughout the 30-day study period. The diaeti group that was administered alloxan ut not the extrats or insulin treatment, had sustained lood gluose etween 322 and 332 mg/dl during the same period. In the other groups in whih either insulin or any of the extrats was given, the lood gluose level was as 51

5 Blood gluose level (mg/dl) TJPS Vol. 21 No. 2 Ot Mar *$$$ *$$$ * *$$ * *$ *$$$ * * a $ 50 0 NC DC IN EE PE DE EA WE Day of onfirmation After 15 days After 30 days Figure 1. Comparison of lood gluose level of rats at different intervals treated with Salaia retiulata (Root + ark). ANOVA signifiane for day of onfirmation F=6.81, for 15 days, F=12.19, for 30 days, F= NC vs. others, *p<0.05, p<0.01, *p< DC vs. others, a=p<0.05, =p<0.01, =p< IN vs. others, $=p<0.05, $$=p<0.01, $$$=p< Error ar indiates ± SE. follows (Figure 1). Insulin treatment dereased lood gluose from 275 to 85 mg/dl. Ethanol extrat treatment dereased lood gluose from 345 to 141 mg/dl. Diethyl ether extrat treatment dereased lood gluose from 265 to 116 mg/dl. Ethyl aetate extrat treatment dereased lood gluose from 274 to 57 mg/dl. Water extrat treatment dereased lood gluose from 323 to 140 mg/dl. However, in petroleum ether extrat treated group, the hyperglyemi state inreased further from around 276 to 300 mg/dl (8% inreased). The diaeti group was ompared with the group treated with insulin or any of the extrats. The fall in lood gluose level was highly signifiant (p<0.001) in diethyl ether, ethyl aetate treated groups. It was moderately signifiant (p<0.01) in water and ethanol extrat treated groups (F=19.97) Comparison of perentage in lood gluose level at the end of 30 days When ompared to aout 3% inrease in lood gluose level in diaeti group etween day 1 and day 30, the fall in the various groups were as follows; 69% with insulin, 59% with ethanol extrat, 56% with diethyl ether and water extrat and 79% with ethyl aetate treatment. In petroleum ether extrat treated group, there was aout 8% inrease of lood gluose level during the same period. The fall in lood gluose level was highly signifiant (p<0.001) with insulin, diethyl ether, ethyl aetate and water extrat treated groups. It was moderately signifiant (p<0.01) in ethanol extrat treated group. 1.2 Treatment: Extrats of ark: The normal ontrol group whih was not administered alloxan, the lood gluose level was around 80 mg/dl throughout the 30 day study period. The diaeti group that was administered alloxan ut not the extrats or insulin treatment, had sustained lood gluose etween 322 and 332 mg/dl during the same period. In the other groups in whih either insulin or any of the extrats given the level was as follows (Figure 2). Insulin treatment dereased lood gluose from 275 to 85 mg/dl. Ethanol extrat treatment dereased lood gluose from 344 to 235 mg/dl. Diethyl ether extrat treatment dereased lood gluose from 303 to 200 mg/dl. Ethyl aetate extrat treatment dereased lood gluose from 282 to 77 mg/dl. Petroleum ether extrat treatment dereased from 390 to 78 mg/dl. The diaeti group was ompared with the groups treated with insulin or any of the extrats. The 52

6 Blood gluose level (mg/dl) Blood gluose level (mg/dl) Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN, et al *$$$ $$ * *$$$ * *$$ $$ 50 0 NC DC IN EE PE DE EA Day of onfirmation After 15 days After 30 days Figure 2. Comparison of lood gluose level of rats at different intervals treated with Salaia retiulata (Bark). ANOVA signifiane for day of onfirmation F=10.39, for 15 days F=15.66, for 30 days F=7.08. NC vs. others, *p<0.05, p<0.01, *p< DC vs. others, a=p<0.05, =p<0.01, =p< IN vs. others, $=p<0.05, $$=p<0.01, $$$=p< Error ar indiates ± SE. 450 * * *$$$ *$$$ * * * 300 *$ a a $$ 50 0 NC DC IN EE PE DE EA Day of onfirmation After 15 days After 30 days Figure 3. Comparison of lood gluose level of rats at different intervals treated with Salaia retiulata (Core root). ANOVA signifiane for day of onfirmation F=13.12, for 15 days F=13.47, for 30 days F= NC vs. others, *p<0.05, p<0.01, *p< DC vs. others, a=p<0.05, =p<0.01, =p< IN vs. others, $=p<0.05, $$=p<0.01, $$$=p< Error ar indiates ± SE. 53

7 TJPS Vol. 21 No. 2 Ot Mar fall in lood gluose level was moderately signifiant (p<0.01) with insulin, petroleum ether and ethyl aetate extrat treated groups. There was no signifiant variation in any of the other groups (F7.08). Comparison of perentage hange in lood When ompared to aout 3% inrease in lood gluose level in diaeti group etween day 1 and day 30. The falls in the various groups were as follows; 69% with insulin, 31% with ethanol extrat, 80% with petroleum ether extrat, 34% with diethyl ether extrat and 72% with ethyl aetate extrat treatment. The fall in the lood gluose level was highly signifiant (p<0.001) with insulin, petroleum ether and ethyl aetate extrat treated groups. 1.3 Treatment: Extrats of ore root: In normal ontrol group whih was not administered y alloxan, the lood gluose level was around 80 mg/dl throughout the 30 day study period. The diaeti group that was administered y alloxan ut not the extrat or insulin treatment, had sustained lood gluose etween 322 and 332 mg/dl during the same period. In the other groups in whih either insulin or any of the extrats given the level was as follows (Figure 3). Insulin treatment dereased lood gluose from 275 to 85 mg/dl. Ethanol extrat treatment dereased lood gluose from 346 to 180 mg/dl. Diethyl ether extrat treatment is dereased from 314 to 118 mg/dl, ethyl aetate extrat treatment dereased lood gluose from 320 to 217 mg/dl. Petroleum ether extrat treatment dereased lood gluose from 372 to 79 mg/dl (Figure 3). The diaeti group was ompared with the groups treated with insulin or any of the extrats. The fall in lood gluose level was highly signifiant (p<0.001) with insulin, petroleum ether and diethyl ether extrat treated group. It was moderately signifiant (p<0.01) with ethanol extrat treated group. There was no signifiant fall in lood gluose level in the ethyl aetate treated group (F=16.32). Comparison of perentage hange in lood gluose level at the end of 30 days: When ompared to aout 3% inrease in lood gluose level in diaeti group etween day 1 and day 30, the fall in the various groups were as follows; 69% with insulin, 47% with ethanol extrat, 78% with petroleum ether extrat, 62% with diethyl ether extrat and 32% with ethyl aetate extrat treatment. The fall in lood gluose level was highly signifiant (p<0.001) with insu- Tale 3 Perentage of superoxide savenging ativity Root + Bark Bark Core root Amount of Ethyl Diethyl Petroleum Ethyl Diethyl Petroleum Ethyl Diethyl Petroleum extrat aetate ether ether aetate ether ether aetate ether ether 10 µg µg µg Tale 4 Perentage of H 2 savenging ativity Root + Bark Bark Core root Amount of Ethyl Diethyl Petroleum Ethyl Diethyl Petroleum Ethyl Diethyl Petroleum extrat aetate ether ether aetate ether ether aetate ether ether 50 µg µg µg

8 Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN, et al. Tale 5 Conentration of the extrat needed in (µg) for 50% inhiition (IC 50 values) Extrat Superoxide Hydrogen savenging peroxide savenging Root + Bark (RB) Ethyl aetate (EA) Diethyl ether (DE) Petroleum ether (PE) Bark alone (B) Ethyl aetate (EA) Diethyl ether (DE) Petroleum ether (PE) Core root alone (R) Ethyl aetate (EA) Diethyl ether (DE) Petroleum ether (PE) lin, ethanol, petroleum ether and diethyl ether extrats treated groups. It was least signifiant (p<0.05) with ethyl aetate extrat treated group. 2. Free radial savenging ativity: The hydrogen peroxide and superoxide savenging ativities of various extrats of root and ark of Salaia retiulata Wight is given in tale 1 and 2. Diethyl ether fration of ore root and ethyl aetate fration of ark showed maximum savenging ativity of superoxide anions when ompared with other frations. All other frations exhiited omparale inhiition of generation of superoxide anion. As far as hydrogen peroxide savenging is onerned, maximum ativity was oserved with ethyl aetate extrats of ore root. The ativity of ark extrat was found to e dereased when it was mixed with ore root extrat. In all ases, the ativities were found to e dependent to the onentration through the perfet linearity was not oserved in any ase. These tends may e due to non-homogenous nature of the extrats. The amount of various extrats whih ause 50% inhiition (IC 50 value) of superoxide and hydrogen peroxide formation have een given in tale 3. In other words, the most potent superoxide savenging ativity has een oserved in ethyl aetate extrat of ark and diethyl ether extrat of ore root. The most potent hydrogen peroxide savenging ativity has een oserved in ethyl aetate extrat of ore root. DISCUSSION The urrent treatments for diaetes are far from ideal. Diet ontrol and exerise are diffiult for many people. Oral hypoglyemi agents often eome ineffetive and sometime have side effets. Furthermore, all insulin replaement regimens are grossly imperfet 1. In the present study, it has een oserved that hypoglyemi effet exerted y diethyl ether, ethyl aetate and water extrats and petroleum ether are quite effetive. The perentage hypoglyemi effet exerted y all the different frations almost orresponds with the level of ation exerted y insulin. Petroleum ether extrat from the root ark of Salaia olonga Wall 5, water extrat from the root of Tinospora ordifolia 10, effet of Opuntia megaantha 11, io tea 12, leaf of Hiisus rosa sinesis 13, root of Withania somnifera 14, heral formulation 15, aqueous extrat of Eihostemma littorale Blume 16 are a few studies, however hypoglyemi effets have een demonstrated in the reent past. In general, diaetes is assoiated with aelerated atheroslerosis and predisposes to ertain mirovasular anormalities. It doules the risk for stroke, inreases the risk of heart attak two to three fold and peripheral vasular prolems partiularly in feet y aout fifty fold. There is reasonale to elieve that elevated total holesterol is having equal atherogeni potential in diaeti as in non diaeti sujets. These fators led the Amerian Diaetes Assoiation to onvene a speial onsensus onferene to disuss the role of modifiation of ardiovasular risk fators in prevention and treatment of marovasular hanges in patients with diaetes mellitus. The group ame to the onlusion that it was reasonale to assume that lowering of holesterol in patients with diaetes would have enefiial effet on ardiovasular hanges 17. In our study a highly signifiant (p<0.001) effet has een oserved with petroleum ether extrat followed y ethyl aetate and ethanol extrats. Reently, ethanol extrat of the leaf of Gynura proumens 18, ethanol extrat of the leaves of Averrhoea ilimi 19, seeds of Erua sativa 20, root of Withania somnifera 14 have demonstrated the hypoholesteremi effets of the plant extrats in diaetes. In our study, it has een found that treatment with petroleum ether extrat was ale to register hypotriglyeridemia, Even though there ould not e 55

9 TJPS Vol. 21 No. 2 Ot Mar signifiant inrease in triglyeride level in diaeti group or in any other group. The hypotriglyeridemia oserved in petroleum ether extrat treated group was least signifiant when ompared with normal ontrol. It has een reported that oxidative stress have impliations oth on diaetes and other ompliations assoiated with it. It has een reported that there are evidenes indiating inreased oxidative damage in IDDM oupled with a derease in antioxidant defense enzymes and vitamins 21. Several reports desried elevations in speifi oxidant stress marker in IDDM together with determination of redued total antioxidant defense and depletion in individual antioxidants 22, 23. Free radials and lipid peroxides easily formed in diaeti state play an important role in development of diaeti ompliations There are also reports that any treatments whih try not only to normalize glyemi ondition, ut also enhane the antioxidant defense system, are more enefiial in the treatment. Apart from restoring glyemi state, reduing of oxidative stress would exerise protetion against many of the ompliations of diaetes From our study, the maximum of superoxide savenging ativity was oserved in ethyl aetate frations of ark and diethyl ether fration of ore root. The maximum of hydrogen peroxide savenging ativity was oserved in ethyl aetate fration of ore root. In the onlusion, our study onfirmed that hypoglyemi Salaia retiulata extrats of ark or ore root alone are more effetive in dereasing lood gluose omination of root and ark. In vitro studies onfirmed savenging ativity of free radials, superoxide and hydrogen peroxide. Hene, we strongly support the usage of Salaia retiulata extrats in diaeti onditions in the form of omplimentary and alternative mediine. REFERENCES 1. Creyr PE. We are what we repeatedly do. Diaetes 1998; 47: Lee WW, Ovi BC, Thai AC, et al. The inidene of IDDM in Singapore hildren. Singapore Med 1998; 39(8): Tissera MHA and Tharew MI. Mediinal Plants and Ayurvedi Preparations Used in Sri Lanka for the Control of Diaetes Mellitus. A puliation of the Department of Ayurveda, Ministry of Health and Indigenous Mediine, Sri Lanka Serasinghe S, Serasinghe P, Yamazaki H, et al. Oral hypoglyemi effet of Salaia retiulata in the streptozotoin indued diaeti rat. Phytotherapy Researh 1990; 4: Krishnakumar K, Augusti KT and Vijayammal PL. Hypoglyemi and antioxidant ativity of Salaia olonga Wall extrat in streptozotoin-indued diaeti rats. Indian journal of physiol Pharmaol 1999; 43(4): Ani Das V, Pius Padayatti S and Paulose CS. Effet of leaf extrat of Aegle marmelos (L) Correa Ex Rox on histologial and ultra strutural hanges of Streptozotoin indued diaeti rats. Indian J Exptl Biol 1996; 34: Trinder. Ann Clin Biohem 1969; 6: Liu F, Ooi VEC and Chang T. Life Sienes 1997; 60: Paya M, Halliwell B and Hoult JRS. Biohem Pharmaol 1992;44: Stanley P, Prine M and Menon VP. Hypoglyemi and other related ations of Tinospora Cordifolia roots in alloxan indued diaeti rats. J Ethnopharmaol 2000; 70(1): Bwiti P, Musaayane CT, Nhahi CF. Effets of Opuntia megaantha on lood gluose and kidney funtion in streptozotoin diaeti rats. J Ethnopharmaol 2000; 69(3): Chandrakala Shenoy K. Hypoglyemi ativity of io-tea in mie. Indian J Exptl.Biol 2000; 38: Arhana Sahdeva, Rashmi Nigam and Khemani LD. Hypoglyemi effet of Hiisus rosa sinensis L leaf extrat in gluose and streptozotoin indued hyperglyemi rats. Indian J Exptl Biol 2001; 39: Andallu B and Radhika B. Hypoglyemi, diureti and hypoholeterolmi effet of winter heery (Withania somnifera, Dunal) root. Indian J Exptl Biol 2000; 38: Bhavapriya V, Kalpana S, Govindasamy S et al. Biohemial studies on hypoglyemi effet of Aavirai Kudineer: An heral formulation in alloxan diaeti rats. Indian J Exptl Biol 2001; 39: Ravi Vijayavargia, Monia Kumar and Sarita Gupta. Hypoglyemi effet of aqueous extrat of Eniostemma littorale Blume (Chhota hirayata) on alloxan indued diaetes mellitus in rats. Indian J Exptl Biol 2000; 38: Barara V Howard and Wm James Howard in the Chapter 21 in ook çjoslin s diaetes mellitus. Edited y Alexander M. 1985; Zhang XF and Tan BK. Effets of ethanoli extrat of Gynura proumens on serum gluose, holesterol and triglyeride levels in normal and streptozotoin indued diaeti rats. Singapore Med J 2000; 41(1): Phushparaj P, Tan CH, Tan BK. Effets of Averrhoa ilimi leaf extrat on lood gluose and lipids in streptozotoin-diaeti rats. J of Ethno Pharmaol 2000; 72(1-2), El-Missiry MA and El Gindy AM. Amelioration of alloxan indued diaetes mellitus and oxidative stress in rats y oil of Erua sativa seeds. Ann Nutr Meta 2000; 44(3): West JC. Radials and antioxidative stress in diaetes. Diaet Med 2000; 17(3):

10 Free Radial Savenging Ativities and Antidiaeti Properties of Various Extrats of Salaia Retiulata Chandrashekar CN, et al. 22. Sttinova V and GrossmannV. Effets of known and potential antioxidants on animal models of pathologial proesses (diaetes, gastri lesions, allergi ronhospasm). Exp Toxiol Pathol 2000; 52(5): Manuel Keenoy Y, Vertommen J and De Leeaw I. Divergent effets of different oxidants on glutathione homeostasis and protein damage in erythroytes from diaeti patients: effets of high gluose. Mol Cell Biohem 2001; 225(1): Chen HC, Guh JY, Shin SJ, et al. Insulin and heparin suppress superoxide prodution in diaeti rat glomeruli stimulated with low density lipoprotein. Kidney Int suppl 2001;78: S Mano T, Shinohara R, Nagasak A, et al. Savenging effet of niorandil on free radials and lipid peroxide in streptozotoin indued diaeti rats. Metaolism 2000; 49(4): Laight DW, Carrier MJ and Anggard EE. Antioxidants, diaetes and endothelial dysfuntion. Cardiovas Res 2000; 47(3): Parshad K. Oxidative stress as a mehanism of diaetes in diaeti BB prone rats: effet of seosolariiresinol digluoside (SDG). Mol Cell Biohem (1-2):