Actions Underlying Antidiabetic Effects of Ocimum sanctum Leaf … · 2016-07-07 · 1986 and EC...

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*Corresponding author: Email: [email protected], [email protected], [email protected];

European Journal of Medicinal Plants5(1): 1-12, 2015, Article no.EJMP.2015.001

ISSN: 2231-0894

SCIENCEDOMAIN internationalwww.sciencedomain.org

Actions Underlying Antidiabetic Effects ofOcimum sanctum Leaf Extracts in Animal Models of

Type 1 and Type 2 Diabetes

J. M. A. Hannan1, O. O. Ojo2,3*, L. Ali4, B. Rokeya4, J. Khaleque1, M. Akhter4,P. R. Flatt2 and Y. H. A. Abdel-Wahab2

1Department of Pharmacy, North South University, Dhaka, Bangladesh.2School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, BT52 1SA,

United Kingdom.3Chevron Biotechnology Centre, Modibbo Adama University of Technology, Yola, Nigeria.

4Research Division, BIRDEM, Dhaka, Bangladesh.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors JMAH, LA, BR, JK, MA, PRFand YHAAW designed the study. Authors JMAH, LA, BR, JK and MA conducted the experiment.

Authors PRF and YHAAW supervised the study. Authors JMAH and OOO performed data analysisand wrote the first draft of the manuscript. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/EJMP/2015/11840Editor(s):

(1) Thomas Efferth, Chair, Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, JohannesGutenberg University, Germany.

(2) Marcello Iriti, Plant Biology and Pathology, Department of Agricultural and Environmental Sciences, Milan State University,Italy.

Reviewers:(1) Anonymous, Chang Jung Christian University, Tainan.

(2) Nahla S. EL-Shenawy, Zoology, Faculty of Science, Suez Canal University, Ismailia, Egypt.(3) Ng Zhi Xiang, Department of Biomedical Science, Faculty of Medicine, MAHSA University, Malaysia.

Peer review History: http://www.sciencedomain.org/review-history.php?iid=648&id=13&aid=5979

Received 5th June 2014Accepted 3rd July 2014

Published 6th September 2014

ABSTRACT

Aim: This study investigated mechanisms by which O. sanctum leaf extracts amelioratehyperglycaemia using animal and cellular models of diabetes.Place and Duration of Study: Diabetes Research Laboratory, University of Ulster, Coleraine,United Kingdom and Research Division, BIRDEM, Dhaka, Bangladesh; 8 months.Methodology: Acute anti-diabetic effects of ethanolic extracts of O. sanctum were examined innormal and chemically-induced type 1 and 2 diabetic rats. Effects of extracts on glucoseabsorption, intestinal disaccharidase activity and gastrointestinal motility in type 2 diabetic rats and

Original Research Article

Hannan et al.; EJMP, 5(1): 1-12, 2015; Article no. EJMP.2015.001

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on glucose uptake and insulin action in 3T3-L1 cells were assessed.Results: Treatment with the extract (1.25 g/kg bw) significantly improved oral glucose tolerance innormal and type 2 diabetic rats and suppressed blood glucose elevation after oral sucrose (2.5g/kg bw) administration. The extract significantly reduced glucose absorption, gastrointestinalmotility and disaccharidase activity. A 28-day treatment with O. sanctum decreased serum glucose,increased liver glycogen and enhanced circulating insulin and total oxidant status in type 2 diabeticrats. Glucose transport and insulin action in 3T3-L1 were increased by extract.Conclusion: O. sanctum represents a useful as a source for discovery of novel antidiabeticcompounds and as a dietary adjunct for the management of type 2 diabetes and its complications.

Keywords: Ocimum sanctum; 3T3 adipocytes; intestinal disaccharidase activity; hepatic glycogen;glucose uptake.

1. INTRODUCTION

Although, classical oral antihyperglycaemicagents are the mainstay treatment of type 2diabetes, they fail to prevent diabeticcomplications [1]. This, therefore, justifies thesearch for more efficacious drugs. Althoughmodern medicine has provided drugs belongingto classes including thiazolidinediones, GLP-1mimetics and DPP-IV inhibitors [2], there is still aneed for new agents with better potential andphysiological antidiabetic actions for thetreatment of diabetes. Therapeutic potential ofphytochemicals in the management of manyhuman diseases, including diabetes, have beenwidely acknowledged. However, scientific studiesassessing the potentially important benefits ofplants used traditionally for the treatment ofdiabetes are still limited [3,4].

Ocimum sanctum Linn (Labiatae), commonlyknown as ‘Holy basil’ is a herbaceous plant foundthroughout the Southern Asian region. It growswild in India but is also widely cultivated for foodin many homes and temple gardens due to itsreligious significance. O. sanctum has a longhistory of medicinal use and was mentioned inCharak Samhita, the ancient textbook ofAyurveda. The leaf of O. sanctum has beenreported to contain bioactive phytochemicalssuch as saponin, flavonoids, triterpenoids, andtannins [5]. Specific bioactive compoundspreviously isolated from O. sanctum leaf arepresented in (Table 1) [6-8]. The use ofO. sanctum leaves in conditions, includingcatarrhal bronchitis, bronchial asthma, dysentery,dyspepsia, skin diseases, chronic fever,haemorrhage, helminthiasis and ring worms havebeen reported [9,10]. Fresh leaves of basil takentogether with black pepper are used as aprophylactic treatment for malaria [11].

Extracts of O. sanctum leaves have been shownto exert hypoglycaemic effects in variousexperimental animals [12,13]. Ethanol extracts

reduced blood glucose in normal, glucose-fedhyperglycaemic and streptozotocin-diabetic rats[12]. A diet containing O. sanctum leaf powderfed to diabetic rats for 1 month also significantlydecreased fasting blood glucose [13]. Ethanolextracts of O. sanctum have also been reportedto exhibit acute and chronic beneficial effects inalloxan-induced diabetes in rats [14]. Finally, in arandomized, placebo-controlled, clinical trial, leafextract of O. sanctum caused a significantdecrease in fasting and post-prandial glucose[15]. These observations plus the evidence thatcompounds isolated from ethanol extracts of O.sanctum exert antioxidant effects [6], illustratethe value of further studies to elucidate theantidiabetic actions of this plant.

We have reported that extracts of leaves of O.sanctum enhance insulin secretion in isolatedperfused pancreas, isolated islets and clonalinsulin secreting cells [16]. In the present study,we have investigated other possible mechanismsby which O. sanctum leaf extracts amelioratehyperglycaemia using animal and cellular modelsof diabetes.

2. MATERIALS AND METHODS

2.1 Chemicals and Instrumentation

Reagents of analytical grades and deionizedwater (Purite, Oxon, UK) were used for the study.Steptozotocin and sodium pentobarbital werepurchased from Sigma-Aldrich (St Louis, MO,USA). Sodium chloride, D-glucose, sucrose,ethanol, barium sulphate, carboxy methylcellulose, calcium chloride, potassium chlorideand sodium hydrogen carbonate were obtainedfrom BDH Chemical Ltd (Poole, Dorset, UK). Ratinsulin standard was obtained from NovoIndustrial (Copenhagen, Denmark) and tritiated2-deoxyglucose was supplied by AmershamInternational Plc (Burkinghamshire, UK). Hanksbalanced salt solution; trypsin, RPMI-1640 tissueculture medium, foetal bovine serum and


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penicillin/streptomycin were ontained from GibcoLife Technologies Ltd (Paisley, UK). The DCprotein kit was procured from Bio-Rad, USAwhile all other kits were purchased fromBoehringer Mannheim GmbH, Germany. Wallac1409 scintillation counter was supplied byWallac, Turke, Finland while the microwell plateELISA reader was obtained from Bio-Tek, USA.

2.2 Plant Material Preparation

O. sanctum leaves were obtained fromRamkrishna Mission, India and voucher

specimens were deposited in the NationalHerbarium, Bangladesh after botanicalauthentication. Leaves were dried at roomtemperature and pulverized prior to extraction ofpowdered samples (2 kg) with ethanol (80%, 10l) for approximately 4 days at room temperature.The extraction solvent was changed daily andthe combined extract was filtered, evaporated todryness using a rotary evaporator and freezedried (yield = 275 g, 13.8%). The sample waskept at 4ºC until used for assays.

Table 1. Active constituents isolated from Ocimum sanctum leaf

S/no Common name Chemical structure IUPAC name1 Cirsilineol 4',5-Dihydroxy-3',6,7-trimethoxyflavone

2 Cirsimaritin 5,4’dihydroxyl-6,7-dimethoxyflavone

3 Isothymusin 6,7-dimethoxy-5,8,4’-trihydroxyflavone

4 Isothymonin 4',5,8-Trihydroxy-3',6,7-trimethoxyflavone

5 Apigenin 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one

6 Rosmarinic acid (2R)-2-[[(2E)-3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]]oxy]-3-(3,4-dihydroxyphenyl)propanoic acid

7 Eugenol 4-Allyl-2-methoxyphenol

8 Urosolic acid 3-beta-3-hydroxy-urs-12-ene-28-oic-acid

9 Carvacrol 2-Methyl-5-(1-methylethyl)-phenol

10 Linalool 3,7-dimethylocta-1,6-dien-3-ol

11 Caryopylline 4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene

12 Estragol 1-methoxy-4-(2-propenyl)-benzene

Kelm et al. [6]; Hakkim et al. [7]; Rahman et al. [8]


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2.3 Laboratory Animals and Induction ofDiabetes

Male Long-Evans rats (14 weeks old, weighing180 - 220 g) maintained on a standard rodentdiet (containing 36.2% carbohydrate, 20.9%protein, 4.4% fat and 38.5% fibre with energycontent of 282 Kcal/100 g) with 12 h light-darkcycle at 21±2ºC and water ad libitum were usedin the present study. All animal experiments werecarried out in accordance with local regulationsand the UK Animals (Scientific Procedures) Act1986 and EC Directive 86/609/EEC for animalexperiments. Type 1 diabetes was induced inadult rats by a single i.p. injection ofstreptozotocin (STZ, 65 mg/kg bw) as previouslydescribed by Hannan et al. [22] while type 2diabetes was induced by a single i.p. injection ofSTZ (90 mg/kg bw) 48h-old rats as previouslydescribed [17-19]. For type 1 diabetes, animalswith blood glucose level >20 mM were includedin the experiment. For type 2 diabetes, animalswith clearly manifested features of obesity (bodyweight = 175 – 180 g), hyperglycaemia (glucoselevel >8mmol/l) and insulin resistance (adjudgedby results of a prior insulin sensitivity test) wereused for the experiments.

2.4 Oral Glucose Tolerance Tests

Acute glucose tolerance (oral, 32% w/v) testswere performed in overnight fasted non-diabeticand diabetic (type 1 and type 2, n=8) in thepresence or absence of ethanol extract of O.sanctum (1.25 g/kg bw) as previously described[20]. For chronic studies, type 2 diabetic rats (n= 8) were treated with ethanol extracts of O.sanctum (1.25 g/kg body weight, twice-daily) orsaline vehicle (0.9%, w/v, NaCl) over a 28-dayperiod. Blood samples were collected at thetimes indicated in the (Fig. 1) for biochemicalanalysis as described previously [21,22].Pancreas and liver were excised fordetermination of insulin content by ELISA [23]using commercial rat insulin kit from CrystalChemical Inc., USA according to themanufacturer’s protocol. Rat insulin standardswere prepared over a concentration range of 0 –2500 ρg/ml.

2.5 Sucrose Absorption in the Gut

Normal and type 2 diabetic rats wereadministered sucrose solution (2.5 g/kg bodyweight) alone or in combination with O. sanctumextract (2.5 g/kg body weight) after an overnightfast. Blood samples were collected by tail vein

puncture at prior to sucrose administration and attimes indicated in (Figs. 1G and 1H) for bloodglucose measurement. Another set of rats werekilled at intervals indicated in (Fig. 2) aftersucrose administration and their gastrointestinaltract excised were divided into 6 segments asshown in the figure. Glucose liberated from eachsegment was measured as previously described[22,24]. Gastrointestinal sucrose content wascalculated from the amount of glucose liberatedas described by Goto et al. [24].

2.6 Intestinal Glucose Absorption

Effects of O. sanctum extracts (25 mg/ml) onintestinal glucose absorption were assessed byIn situ intestinal perfusion as previouslydescribed [22] in non-diabetic rats. The perfusatewas collected from a catheter set at the end ofileum at room temperature and a perfusion rate0.5 ml/min for 30 min. Absorbed glucose wasestimated from the concentration of glucose inthe solution before and after the perfusion.

2.7 Intestinal Disaccharidase Activityand Gastrointestinal Motility

Intestinal disaccharidase activity in normal rats inthe presence of ethanol extract ofO. sanctum (1.25 g/kg) or vehicle was assessedas previously described [22]. Glucose andprotein concentrations in the homogenate ofsmall intestines excised from treated animals 1hafter administration was measured as previouslyreported [22]. Gastrointestinal motility wasevaluated using barium sulfate milk containing10% w/v BaSO4 in 0.5% carboxy methylcellulose (CMC) [25]. The distance traversed byBaSO4 milk was measured and reported as apercentage of the total length of small intestine[22].

2.8 Measurement of Total AntioxidantStatus (TAS)

Total antioxidant status was measured usingserum of treated and control animals with 6-hydroxy-2, 5, 7, 8-tetramethylchriman-2-carboxylic acid as standard as describedpreviously [22]. Following 3 min incubation ofstandard or serum samples (5µl) with thechromogen reagent (200µl, containingmetmyoglobin and 2, 2’-Azino-di-[3-ethylbenzthiazoline sulphonate]) plus H2O2,absorbance was measured at 630nm. Unknownconcentration of TAS was estimated from thestandard curve.


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2.9 Glucose Uptake and Insulin Action

Effects of O. sanctum extracts on glucose uptakeand insulin action were examined using 3T3-L1cells as reported earlier [20,22]. Glucose uptakewas initiated by the addition of tritiated 2-deoxyglucose (0.5 Ci/well, 50 l) at glucoseconcentration of 50mM. Radioactivity wasmeasured on a Wallac 1409 scintillation counter(Wallac, Turke, Finland) and result expressed asdisintegration per min (DPM) [26].

2.10 Statistical Analysis

Results are expressed as the mean ± SD. Valueswere compared by Student’s unpaired t-test andMann-Whitney U test where appropriate. Data atdifferent time points were analysed by one wayANOVA with Bonferroni adjustment. Pair-wisecomparisons to the control group were performedusing Dunnett’s test. Groups of data wereconsidered to be significantly different if P<0.05.Analyses were carried out using GraphPad Prism(GraphPad Software, Inc., USA).

3. RESULTS

3.1 Acute and Long-term Effects ofEthanol Extract of O. sanctum onGlucose Tolerance

Fasting serum glucose concentrations in normaland type 2 diabetic rats were reducedsignificantly following oral administration ofethanol extracts of O. sanctum (Fig. 1). Co-administration of glucose and the extracts alsoproduced a significant improvement in glucosetolerance in normal and type 2 diabetic rats.Significantly lower serum glucose levels wereobserved at 30min (p<0.01 normal, p<0.001 type2 diabetic) and 75 min (p<0.05) in both groups ofrats (Figs. 1A, C). The extracts produced no anti-hyperglycaemic effect in rats with type 1 diabetes(Fig. 1B).

Twice-daily administration of O. sanctum extractfor 28 days resulted in reduced serum glucose by29% (p<0.01) and elevated serum insulin by 41%(p<0.001) in rats with type 2 diabetes comparedwith their saline controls. While pancreatic insulincontent remain unchanged, hepatic glycogenconcentration and total antioxidant status (TAS)were increased by 1.7-fold and 1.4-foldrespectively after the administration of O.sanctum extract to type 2 diabetic rats (Table 2).

3.2 Effects of O. sanctum on SucroseTolerance and Absorption in the Gut

A sharp rise followed by a gradual release inblood glucose concentration was observed at 30min post sucrose administration in normal andtype 2 diabetic control rats (Figs. 1G and 1H).This rapid increase was significantly inhibited inboth normal and type 2 diabetic rats treated withethanol extracts of O. sanctum with markeddifferences observed at 30 min (p<0.05) and 60min (p<0.01) post sucrose administration.Following a 20 h fast and a sucrose load of 425mg/rat to non-diabetic control rats, significantlyhigh concentration of sucrose was detected in thestomach and the upper and middle small intestinewithin 30 min of sucrose administration (Figs. 2A–C). Sucrose concentration in all these regionwere rapidly reduced within 2h post sucroseadministration except in lower small intestinewhere an increase was observed at 1h postsucrose administration (Fig. 2D). Co-administration of sucrose and O. sanctum extractdid not affect sucrose concentration at 30min inthe stomach and lower part of small intestine butproduced a slower rate of absorption of sucroseacross the small intestine (Fig. 2A-D). Comparedwith non-diabetic controls, significantly (p<0.05)higher concentration of sucrose was observed allthe different regions of the GI tract in non-diabeticmice administered O. sanctum extracts withsucrose. Compared to other parts of the GI tract,significantly lower concentration of sucrose wasobserved in the caecum and larger intestine.Higher inhibition of sucrose absorption wasobserved in the caecum of non-diabetic ratsadministered O. sanctum while a relatively similarpattern of absorption was observed in the largeintestine of these rats and control rats (Figs. 2E-F). In type 2 diabetic control rats, a more rapidrate of sucrose clearance was observedcompared with their non-diabetic counterparts.Lower concentration of sucrose was observed,particularly in the caecum of type 2 diabeticcontrol rats compared with non-diabetic rats (Fig.2). Administration of O. sanctum extractssignificantly inhibited sucrose absorption in type 2diabetic rats with significant accumulationobserved in all the regions of small intestine andcaecum (p<0.05, Figs. 2H – K) for up to 2h postsucrose administration. Sucrose absorption in thelarge intestine of both groups of type 2 diabeticrats were not significant different except at timepoint 2h post injection where sucroseaccumulation was observed in rats treated with O.sanctum extracts (p<0.05, Fig. 2L).


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Table 2. Effects of ethanol extract of Ocimum sanctum leaves on serum levels of glucose andother parameters in type 2 diabetic rats after 28 days of feeding

Parameters Day 0 Day 28Control O sanctum Control O sanctum

Glucose (mmol/l) 8.84±1.3 8.76±1.2 8.90±1.02 6.18±0.9**

Insulin (ng/ml) 0.434±0.1 0.446±0.2 0.442±0.1 0.628±0.1*

Pancreatic insulin (ng/g pancreas) 0.88±0.34 0.96±0.61Liver glycogen (g/100g tissue) 0.79±0.2 1.34±0.4*

Total antioxidant status (mmol/l) 0.87±0.1 0.92±0.2 0.89±0.1 1.23±0.2**

Diabetes was induced by injection of neonatal rats with 90 mg/kg STZ approximately three months previously. Dose ofO sanctum was administered orally by gavage (1.25g/kg body weight) twice daily for 28 days. Data are presented asmean ± SD (n=12). Significance differences were compared by unpaired t test; *P<0.05, **P<0.01 compared to type 2

diabetic control rats

Fig. 1. Effects of ethanol extract of O. sanctum on fasting glucose (A – C) and glucosetolerance (D – F) in non-diabetic, type 1 and type 2 diabetic rats as well as serum glucose after

sucrose load in non-diabetic (G) and type 2 diabetic rats (H)Results are mean ± SD depicted by vertical bars (n = 8). Fasted rats were given ethanol extract by gavage (1.25 g/kg

body weight) with or without glucose (2.5g/kg body weight). For (G) and (H), rats were fasted for 20 h and administeredsucrose solution (2.5 g/kg body weight) by gavage with or without ethanol extract of O. sanctum (1.25 g/kg body

weight). Significances are derived from repeated measures ANOVA and adjusted using Bonferroni correction; *P<0.05,**P<0.01, ***P<0.001 compared to respective control rats


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Glucose (mmol/l) 8.84±1.3 8.76±1.2 8.90±1.02 6.18±0.9**

Insulin (ng/ml) 0.434±0.1 0.446±0.2 0.442±0.1 0.628±0.1*










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Glucose (mmol/l) 8.84±1.3 8.76±1.2 8.90±1.02 6.18±0.9**

Insulin (ng/ml) 0.434±0.1 0.446±0.2 0.442±0.1 0.628±0.1*










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3.3 Effects of O. sanctum Extracts onIntestinal Glucose Absorption

Intestinal glucose absorption was almostconstant during 30 min of in situ perfusion with

glucose. However, 13 - 19% reduction in thepercentage glucose absorption was observed inthe presence of O. sanctum extract during wholeperfusion period (p<0.05, Fig. 3A).

Fig. 2. Effects of ethanol extract of O. sanctum on gastrointestinal sucrose content after oralsucrose loading in non-diabetic (A – F) and type 2 diabetic rats (G – L)

Rats were fasted for 20 h prior to administration of a sucrose solution (2.5 g/kg body weight) by gavage with orwithout ethanol extract (1.25 g/kg body weight). Results are mean ± SD (n = 8); *P<0.05 compared to control


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Fig. 3. Effects of ethanol extract of O. sanctum on intestinal glucose absorption (3A), intestinaldisaccharidase activity (3B) and gastrointestinal motility (3C) in non-diabetic rats as well as

glucose uptake by 3T3-L1 adipocytes (3D)For (A), rats were fasted for 36 h prior to glucose (54 g/l) perfusion with or without extract of O. sanctum (25 mg/ml)

while rats were fasted for 20 h prior to administration of plant extract by gavage (1.25g/kg body weight) for 3B and 3C.Enzyme activity determination and BaSO4 administration was at 60 min (3B). Motility was measured over the following15 min (3C). Values are mean ± SD (n = 8). Significances are derived from repeated measures ANOVA and adjusted

using a Bonferroni correction. *P<0.05, **P<0.01 compared to non-diabetic control (3A-C). For 3D, *P<0.05, ***P<0.001compared to no insulin incubation, P<0.001 compared to plant ethanol extract incubation without insulin and

+++P<0.0001 compared to 10-9M insulin alone

3.4 Effects of O. sanctum on IntestinalDisaccharidase Activity andGastrointestinal Motility

Intestinal disaccharidase activity in non-diabeticrats was significantly inhibited (p<0.05) in thepresence of the plant extract (Fig. 3B) andgastric mobility, measured by the percentage ofintestinal length traversed by barium milk,decreased by 23% in rats treated with O.sanctum extracts compared to control rats(p<0.05, Fig. 3C).

3.5 Effects of O. sanctum on GlucoseUptake in 3T3 Adipocytes

The ethanol extract of O. sanctum significantlyenhanced glucose transport compared with

control incubations in the absence of insulin(p<0.001, Fig. 3D). The positive effects of O.sanctum extract were further increased by thepresence of insulin (10-9 M, p<0.001, Fig. 3D).

4. DISCUSSION

The use of O. sanctum leaves in traditionalmanagement of diabetes has been widelyreported and the traditional claims of theantihyperglycaemic effects of O. sanctum leafextracts have been verified by a number ofscientific studies [12,13,15]. To furtherunderstand the mechanisms of actions andtherapeutic efficacy of O. sanctum extracts in themanagement of diabetes, our study examinedacute and chronic antidiabetic actions ofO. sanctum using non-diabetic and diabetic


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4. DISCUSSION



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4. DISCUSSION



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animal models. We also uniquely investigatedthe extrapancreatic actions of O. sanctum andstudied its effects on gastrointestinal tractmotility, intestinal carbohydrate digestion andabsorption, liver glycogen content and cellularglucose uptake.

Oral administration of ethanol extracts of O.sanctum leaves prevented elevated levels offasting blood glucose and resulted in significantimprovement of glucose tolerance in non-diabeticand type 2 diabetic rats with no beneficial effectsobserved in type 1 diabetic rats. Moreover,enhanced insulin secretion was observed in type2 diabetic rats treated with O. sanctum extractsfor 28 days. These observations and our earlierreport of the insulinotropic effects of O. sanctumextracts on perfused pancreas, isolated isletsand clonal pancreatic cells [16,27] suggest thatthe glucose lowering action is mediated partlythrough the stimulation of insulin release.Moreover, the lack of antihyperglycaemic effectsin type 1 diabetic rats characterized by beta celldestruction further corroborates the assertionthat O. sanctum extracts act, at least in part, byimproving insulin secretion. Insulinotropic actionsobserved for O. sanctum extracts is consistentwith observations reported for extracts of otherplants, such as Agrimony eupatoria [28],Terminalia bellirica [29], Asparagus adscendens[4], Sambucus nigra [30], Coriadrum sativum[31], Viscum album [32] and many others [33]with insulin-releasing actions.

Consistent with the growing body of evidenceshowing that leaves of O. sanctum containnatural agents with reproducible antidiabeticeffects, acute In vivo experiments conducted inthis study revealed that oral administration ofethanol extracts of O. sanctum inhibitedincreased serum glucose level following an oralglucose load. This observation raises thepossibility that O. sanctum may also act byinterfering with intestinal glucose absorption inthe postprandial state. Our previous studies withseed extracts of Trigonella foenum-graecum [34],root extracts of Asparagus racemosus [22] andhusk extracts of Plantago ovata [20]produced a similar inhibition of intestinal glucoseabsorption as observed in this study.

It is known that improved glucose tolerance couldbe achieved in people suffering from diabetes byeither slowing down gastric emptying, increasingintestinal nutrient transit or modification of therelease or actions of key digestive enzymes [35].It has also been shown that high-fibre diets and

some plant products could help achieveimproved tolerance through one or a combinationof these approaches. We examined the effects ofO. sanctum extract on sucrose digestion andabsorption in the gut of normal and type 2diabetic rats. Significant inhibition of postprandialhyperglycaemia and increased concentration ofresidual sucrose was observed, suggesting thatthe carbohydrate digestion or absorption wasinhibited by the extract. While this result isconsistent with previous findings [16], low levelsof sucrose detected in the large intestinessuggests that O. sanctum extracts may act byslowing down but not totally preventing sucroseabsorption.

The hypothesis that antihyperglycaemic effectsof the extract may be, at least partly, mediatedthrough the retardation of carbohydrate digestionand absorption was further investigated by gutperfusion experiments, which showed that O.sanctum extracts significantly reduced intestinalglucose absorption. Consistent with otherstudies, intestinal disaccharidase activity wasalso significantly, suggesting that the reduction ofabsorption may also partly related to inhibition ofintestinal disaccharidase activity [16,35]. Agentsthat inhibit carbohydrate digestion, such as α-glucosidase inhibitors, are currently in use fordiabetes management [36]. However, it is not yetclear if the active principle in the ethanol extractof O. sanctum can be considered as a memberof this group of agents. As indicated byexperiments in non-diabetic rats, reduced gastricmotility in the presence of O. sanctum extractmay also contribute to the reduced rate ofcarbohydrate digestion and absorption observedin this study. While further investigations may berequired to evaluate such effects in diabeticanimal models, agents such as exanatide [37]and pramlinitide [38] that act partly by reducinggastric emptying are currently in use for diabetesmanagement.

Impaired glucose uptake and utilization byperipheral tissues is one of the majorderangements of type 2 diabetes [39]. Weevaluated the effects of O. sanctum leaf extracton glucose uptake in the absence and presenceof insulin using 3T3 adipocytes; a cell line thathas been successfully used in previous studiesto investigate effects of novel agents on glucoseuptake [16,22]. Results revealed that O. sanctumextracts significantly enhanced glucose transportand that its effects were augmented in thepresence of insulin. While further investigationsare necessary to understand In vivo molecular


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mechanisms through which O. sanctum extractsfacilitate glucose uptake, its lack of effect inanimal models of type 1 diabetes makes thiseffect of O. sanctum extracts less exciting.

Longer term In vivo studies were performed inrats with type 2 diabetes induced by a singleintraperitoneal injection of streptozotocin (STZ) at48 hours of age [40]. Increased total antioxidantstatus observed in rats treated with O. sanctumextracts for 28 days (Table 2) suggests that theextract may afford protection to pancreatic islets[41] against free radical mediated pancreatic cells damage [42]. In addition, this action of theextract may be beneficial in preventing oxidativestress mediated diabetic complications [43].Though the pancreatic insulin contents of controland O. sanctum treated rats were similar,increased hepatic glycogen content wasobserved in the group treated with the plantextract. Further investigations are necessary toexamine if there is link between the actions of O.sanctum extracts on insulin-release, glucoseuptake and hepatic glycogen deposition indiabetic rats [44].

5. CONCLUSION

Ethanol extracts of O. sanctum leaves improvedglucose homeostasis in type 2 diabetes rats byenhancing circulating insulin and delayingcarbohydrate digestion. O. sanctum thereforerepresents a useful as a source for discovery ofnovel antidiabetic compounds and as a dietaryadjunct for the management of type 2 diabetesand its complications.

CONSENT

Not applicable.

ETHICAL APPROVAL

All authors hereby declare that "Principles oflaboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as theUK Animals (Scientific Procedures) Act 1986 andEU Directive 2010/63EU for animal experiments.All experiments were examined and approved bythe appropriate ethics committee.

ACKNOWLEDGEMENTS

These studies were supported by the SAADTrading and Contracting Company andUniversity of Ulster Strategic Research Funding.

COMPETING INTERESTS

Authors have declared that no competinginterests exist.

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_________________________________________________________________________________© 2015 Hannan et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.

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