Indian J Physiol Pharmacol 1998; 42 (3) : 401-406

HYPOGLYCEMIC EFFECT OF BIOPHYTUM SENSITIVUMIN THE ALLOXAN DIABETIC RABBITS

DINESH PURI* AND NIRMAL BARAL

Department of Biochemistry,B. P. Koirala Institute of Health Sciences,Dharan, Nepal

(Received on September 29, 1997)

Abstract : Hypoglycaemic effect of a N eapalese plant Biophytumsensitivum was investigated in the alloxan diabetic male rabbits of differentseverities: subdiabetic (Alloxan recovered; AR), mild diabetic (MD) andseverely diabetic (SD). Assessment of activity of the extract, preparedfrom the plant leaves, was done by fall in fasting plasma glucose (FPG)and improvement in the oral glucose tolerance test (OGTT), followingsingledose and prolonged administrations. Following single dose administrationthere was fall in 1 hour and 2.5 hour glucose values by 25.9% and 27.4%respectively in the subdiabetic rabbits, and by 36.9% and 37.7% in themild diabetic rabbits. Improved GTT response is shown by fall in areaunder curve (AUC) from 16138 mg/dl to 12355 mg/dl (23.4%) in thesubdiabetic rabbits, and from 19258 to 12238 mg/dl in the MD rabbits.More significant improvements occurred following one week of abovetreatment. The results prove that the plant material has significanthypoglycaemic effect, which is possibly due to pancreatic beta-cellstimulating action. To investigate its possible role in correction of othermetabolic abnormalities of diabetes further long term studies are required.

Key words: hypoglycaemia biophytum sensitivumalloxan-diabetic glucose tolerance test

INTRODUCTION diversity which were used by the traditionalhealers for the treatment of many

Despite considerable progress in the diseases, including diabetes (6). Biophytumtreatment of diabetes by oral hypoglycaemic sensitivitum D. C. syn Oxalis sensitiva Lagents, search for newer drugs continues (family Oxalidaceae; local name Nagbeli) isbecause the existing synthetic drugs have one such plant that is traditionally used forseveral limitations. In recent times there the treatment of "Madhumeha". It is anhas been a renewed interest in the plant annual herb that grows in the inner Tarairemedies (1, 2, 3). In an attempt to discover region, east of Koshi river. In spite of itsnatural hypoglycaemic compounds, various widespread use by the traditional healers,plants having "folk-medicine reputation" there are no systematic scientific studies tohave been screened by biochemical tests for validate effects of this potentially usefultheir hypoglycaem'ic effects (4, 5). Nepal has plant. The present study was undertakena rich heritage of medicinal plants of wide to scientifically investigate its possible*Corresponding Author and address : B-163, Yojna Vihar, Delhi - 110 092

402 Purl and Baral

usefulness in diabetes mellitus.

METHODS

Plant material: Biophytum sensitivumwas collected during August-September,1996 on the bank of Koshi river, in the innerTarai region (Sunsari, Nepal). For reliableidentification a sample was sent toMahender Morang Campus (BotanyDepartment, P. G. Institute, Bir atnagar).Leaves were separated and dried, and weresoaked in water for about 16 hours.Thereafter they were homogenized andcentrifuged at 3000 g for 15 minutes. Theextract was force fed to the rabbits usingfeeding tubes, number seven.

Animals: Inbred male Angora rabbits ofbody weight 700 to 1000 gm, and age 2-2.5months were purchased from the PakhribasAgricultural Centre, Dhankuta district.They were given pallet diet and water ad-libitum and were acclimatized to thelaboratory conditions and handling for atleast two weeks before carrying out anyexperimental work. The oral glucosetolerance test (OGTT) was performed inthem to rule out diabetes and other formsof carbohydrate intolerance.

Induction of alloxan diabetes: Followingan overnight fast of 14-16 hrs, alloxan wasinjected intravenously at a dose of 80 mg/kg body weight for the induction of diabetes.

During next few days the fasting plasmaglucose (FPG) levels were estimated atintervals of five days for one month, in orderto follow the course of hyperglycaemic state.The animals were arbitrarily divided intothe following three types, depending on theresponse:

1. Alloxan Recovered (AR; Subdiabetic)In such rabbits initial rise in the FPG

Indian J Physiol Pharmacal 1998; 42(3)

level was observed for about 15 daysfollowed by a period of recovery when itgradually came down. By one month theFPG returned to normal or slightly elevated(upto 120 mg%) but still on performance oforal glucose tolerance test (OGTT), theglucose tolerance was found abnormal andthe OGTT curve was substantially elevated.In the earlier studies, conducted in our lab.with the male albino rabbits, the AR animalswere found to be better animal modelsfor the experimental evaluation of thehypoglycaemic effect of compounds (7).

2. Mild diabetic (MD) : The hypergl-ycaemic state persisted in this group ofanimals and FPG levels stabilized between120-250 mg/dl.

3. Severely diabetic (SD): The hyper-glycaemic rabbits with FPG levels above250 mg/dl were included in this group.

Subsequently, before carrying out anyexperiments, a period of 45 days wasallowed after the day of alloxan injectionfor the stabilization of rabbits into one ofthe three diabetic states (7, 8).

Assessment of the hypoglycaemic activity

The hypoglycaemic effect of the plantmaterial was assessed by evaluating thefasting plasma glucose and/or glucosetolerance test.

1. Glucose tolerance test: Basal GTT wasdone in all the animals with 3 gm/kg bodyweight glucose load, following an over-nightfast of 14-16 hours. Next morning thefasting, 1 hour and 2.5 hour samples werecollected, as recommended by some earlierworkers (9). Five days later, fasting bloodsample was drawn and immediately theplant material was given orally at a dosefound optimum in preliminary trials

Indian J Physiol Pharmacal 1998; 42(3)

(200 mg/kg body weight). Ninety minuteslater, GTT was performed (on the samerabbit) with same oral glucose load. Themean GTT curve obtained after thetreatment was compared with thepretreatment basal curve. The activity wasassessed quantatively by the extent of fallin area under curve (AUC). This method hadearlier been reported to be more sensitiveand meaningful because the animals servedas their own control, thus minimizing thebiological variations (7, 8).

2. Fasting plasma glucose (FPG) : Afteran overnight fast, the fasting blood samplewas collected and immediately thereafterthe plant material was administered, asdiscussed above. Ninety minutes later nextblood sample was withdrawn. The FPGvalues before and after the administrationof the plant material reflected itshypoglycemic effect. As above, the animalsserved as their own controls.

In the sub-diabetic (AR) animals theactivity was assessed by improvement in theOGTT response but not by FPG since it wasnearly normal or only slightly elevated even

Hypoglycemic Effect of Biophytum Sensitivum 403

before treatment; in the severely diabeticanimals by a fall in FPG and in the milddiabetics by both FPG and GTT. OGTTwas rarely performed in the severelydiabetic rabbits as they show high mortalitywith it.

RESULTS AND DISCUSSION

Effect of single dose administration ofthe plant material on fasting plasma glucoselevels and GTT pattern in diabetic rabbits:

The effect of a single administration ofthe plant material in a dose of 200 mg/kgbody weight was assessed in the alloxanatedsubdiabetic, mild diabetic and severelydiabetic rabbits. Five animals in each groupwere taken.

In the subdiabetic rabbits (Table I), theone hour peak value in the treated animalsshowed a significant fall of 25.9% from268.1 ± 11 mg/dl to 198.6 ± 6 mg/dl. The twoand half hour value in the treated animals(160.4 ± 4 mg/dl) was 27.4% lower than thecorresponding pre-treated value of 220.8 ± 8mg/dl. These were significant (P<O.Ol). The

TABLE I : Effect of single dose administration of the plant material onGTT in the subdiabetic and mild diabetic rabbits (n=5).

Fasting AUC (mg l dl)

Plasma glucose (mg / dl)

I-hour 2.5-hours

Normal-PretreatedTreated

808 ± 684.6 ± 11(NS)

90.8 ± 4

92.1 ± 4-1.43

NS140.4 ± 10

140.8 ± 6-0.3

NS

Subdiabetic-Pretreated

Subdiabetic-Treated% Change

SignificanceMild diabetic-pretreatedMild diabetic-Treated

%ChangeSignificance

152.2 ± 5143.7 ± 10(NS)

268.1 ± 11

198.6 ± 6-25.9P<O.OI

298.8 ± 12188.4 ± 12

-36.9

P<O.OI

130.4 ± 6121.2 ± 7(NS)

220.8 ± 8160.4 ± 4-27.4

P<O.OI

272.8 ± 9169.8 ± 6-37.7

P<O.OI

1925812238-36.4

16138

12355-23.4

NS - Not significant

404 Purl and Baral

mean area under curve (AUC) in the treatedanimals (12355 mg/dl) was lower than thatbefore treatment (16138 mg/dl) by 23%.However, no significant alteration in theFPG occurred either in this group of theAR or the MD rabbits, in course ofperforming the GTT. In normal controls nochange in FPG and a slight attenuation ofthe glucose induced hypoglycemic responsewas observed. In the mild diabetic treatedanimals significant reductions wereobserved in the one hour peak value from298.8 ± 12 mg/dl to 188.4 ± 12 mg/dl (36.9%fall), two and half hour value from 272,.8 ± 9mg/dl to 169'.8 ± 6 mg/dl (37.7% fall)and AUC from 19258 mg/dl to 12238mg/dl (36.4% fall). These were significant(P<O.OI).

Fasting plasma glucose levels in thesubdiabetic; mild diabetic and severelydiabetic rabbits were separately estimatedbefore and after single dose administrationof the plant material. No significant fallwas, however, observed in any of the threegroups (Table II).

Effect of one week administration of theplant material, on fasting plasma glucoselevels and GTT pattern: The plant materialwas daily administered to the subdiabetic,mild diabetic and severely diabetic rabbitsin a daily oral dose of 200 mg/kg/day for aperiod of one week. The experiment wasaimed at finding out whether suchtreatment would improve the GTT curve any

Indian J Physiol Pharmacol 1998; 42(3)

further, and whether it would produce anyfall in the FPG also, where single dose wasfound inadequate. At the end of one weektreatment, the GTT curves obtained in thesubdiabetic and the MD rabbits wereevaluated in relation to the correspondingpre-treatment curves. GTT was not repeatedin the severely diabetic rabbits as manyanimals had died with it initially.

In the subdiabetic rabbits there was nochange in FPG values which were normaleven in untreated rabbits. GTT returned tonormal after 7 days (Table III), as judgedby the post glucose load plasma glucosevalues and the AUCs, which werecomparable with those of the normalanimals (Table I). In the mild diabeticrabbits fasting plasma glucose returned to98.6 ± 6 mg/dl from the pretreatment valueof 153.1 ± 14 mg/dl after the abovetreatment, whereas the post treatment GTTresponse, though significantly attenuated,was still impaired compared to a normalresponse (Table Ill). When the abovetreatment was given to the severely diabeticrabbits with FPG level of 438.9 ± 41 mg/dI,the reduction after the third and theseventh days were 0.09% and 7.2%respectively. However, the post treatmentvalues were still markedly elevated(Table IV).

Effect of the plant product on bodyweight of the diabetic rabbits: Body weightsof the subdiabetic, mildly diabetic and

TABLE II : Effect of single dose administration of the plant material onFPG in the subdiabetic, mild diabetic and SD rabbits (n=fi),

Plasma glucose (mg / dl)

Fasting % Change90 minutes' afteradministration

Significance

SubdiabeticMild DiabeticSeverely Diabetic

88.4±5154.6±9488.1±46

89.4±9146.6±16

, 456.4±44

+1.1-5.2-6.4

NSNSNS

NS = Not significant

Indian J Physiol Pharmacol 1998; 42(3) Hypoglycemic Effect of Biophytum Sensitivum 405

TABLE III: Studies with one week administration of the plant materialin the subdiabetic and mild diabetic rabbits. (n=5).

Plasma glucose (mg I dl)

Fasting I-hour 2.5-hours AUC (mg I dl)

SubdiabeticDay 0 90.6 ± 6 266.6±36 218.4 ± 22 13446Day 7 91.6 ± 5 150.4± 13 132.4 ± 17 10352

% Change +1.1 - 43.6 - 39.4 - 23.01Significance NS P<0.01 <0.01

Mild DiabeticDay 0 153.1 ± 14 298.4± 41 217.5±15 17838Day 7 98.6 ± 6 184.7±12 156.6 ± 14 11844

% Change -35.6 -38.1 -28.0 -33.6Significance P < 0.01 P < 0.01 P < 0.01

NS - Not significant; (% Change compared with day 0 value)

TABLE IV : Studies with one week administration in the SD rabbits (n=5).

Plasma glucose (mg I dl)

FPG significance% change

Day 0Day 3Day 7

438.9 ± 41436.6 ± 42405.4 ± 28

NS - Not significant

-0.09-7.2

NSNS

severely diabetic animals were taken beforeand after the treatment. Weight gains inthe AR, mild diabetic and severely diabeticrabbits were from 1.06 ± 0.10 kg to1.12 ± 0.17 kg (5.6%), from 0.84 ± 0.08 kgto 0.90 ± 0.11 kg (7.1%) and from 0.76 ± 0.11kg to 0.80 ± 0.12 kg (5.2%) respectively with7 days of the treatment; whereas weightloss in the three groups without thetreatment were 1.8%, 4.5% and 10.5%respectively in 7 days (Table V), possiblydue to metabolic alterations associated withthe diabetic state. In the AR and MD rabbitsthe weights reached the normal value (i.e.,value before induction of diabetes). In theSD rabbits also there was considerable gainin the body weight, which reached almostnormal value of 0.8 kg which is only slightlylower than the average normal (0.85 kg).The treatment, therefore, not only arrests

loss of weight due to diabetic state but alsoresults in weight gain, probably due to itsrole in initiating an improvement in theoverall metabolic status.

The above results clearly show that theplant extract has a significant and sustainedhypoglycaemic action. It is evident from thehypoglycaemic effect elicited in thesubdiabetic and the MD rabbits.Furthermore, the above treatment possiblyinitiates improvement in the metabolicalterations associated with diabetes mellitus,as suggested by gain in weight in the treatedgroup of the animals. However, nosignificant biochemical alteration wasobserved, such as in lipid profile with oneweek of treatment. This period is too shortto bring about changes in the lipid profilewhic.h, once elevated, takes a much longer

406 Puri and Baral Indian J Physiol Pharmacol 1998; 42(3)

TABLE V: Effect of the diabetic state and 15 days of the treatment on bodyweights (kg) of the AR, mild diabetic and severely diabetic rabbits.

AR Mild diabetic Severely diabetic

Untreated 1.08 ±0.10 0.88 ± 0.07 0.85 ±0.08(0 Week)Untreated 1.06 ±0.10 0.84 ± 0.08 0.76 ±0.11(1 Week)%change -1.8% -4.5 -10.5Treated 1.12 ±0.17 0.90 ± 0.11 0.80 ±0.12(2 Week)%Change +5.6 +7.1 +5.2Significance NS P<.Ol NS

time than one week to fall. Studies of longerduration are, therefore, necessary todelineate the metabolic alterations, if any.Weight gain in the treated rabbits is incontrast with the untreated diabetic animalswhich lost considerable weight during thisperiod. However, in the SD rabbits, whichare reasonable analogues of human IDDM,having near complete beta cell destruction,single dose as well as prolongedadministrations were found inadequate todecrease the FPG. The results suggest apredominant insulinotropic (beta cellstimulating) effect, because improvement inthe glycaemic state was observed in thesub diabetic and MD rabbits only, whichhave functional beta cell reserve. In the SDrabbits, where beta cell damage is extensive,this effect can not be elicited. However,extra-pancreatic mode of action can not be

ruled out on the basis of study of thisduration.

In view of the fact that the crude plantextract was administered in this preliminarystudy, the purified and homogenousmaterial, if isolated from it, is expected tobe effective at a much lower dose. Attemptstowards purification of the extracts arebeing planned at present, and the aboveresults are encouraging for carrying outmore detailed biochemical studies in orderto observe other beneficial effects and toelucidate the mode of action.

ACKNOWLEDGEMENTS

The research project was supported bythe BPKIHS research grant scheme. Theauthors acknowledge the contribution byShri Sunil Dhungel, the Botonist, for theinvaluable help.

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