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Journal of Ethnopharmacology 123 (2009) 392–396

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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ffect of Moringa oleifera Lam. leaves aqueous extract therapyn hyperglycemic rats

olly Jaiswal, Prashant Kumar Rai, Amit Kumar, Shikha Mehta, Geeta Watal ∗

lternative Therapeutics Unit, Drug Development Division, Medicinal Research Lab, Department of Chemistry, University of Allahabad, Allahabad, U.P. 211 002, India

r t i c l e i n f o

rticle history:eceived 10 October 2007eceived in revised form 2 February 2009ccepted 19 March 2009vailable online 5 April 2009

eywords:ntidiabeticrumstick treeoringaceaeoringa oleifera

a b s t r a c t

Ethnopharmacological relevance: In Indian traditional system of medicine, Moringa oleifera Lam. Syn.Moringa pterygosperma Gaerth (Moringaceae) is commonly used as healing herb to treat diabetes.Aim of the study: The purpose of the present study was to assess the effect of M. oleifera leaves aqueousextract therapy on glycemic control, haemoglobin, total protein, urine sugar, urine protein and bodyweight.Materials and methods: Variable doses of 100, 200 and 300 mg kg−1 of aqueous extract were administeredorally by gavage for evaluating their hypoglycemic and antidiabetic effects on fasting blood glucose (FBG),oral glucose tolerance test (OGTT) and post prandial glucose (PPG) of normal and streptozotocin (STZ)induced sub, mild and severely diabetic rats.Results: The dose of 200 mg kg−1 decreases blood glucose level (BGL) of normal animals by 26.7 and 29.9%

during FBG and OGTT studies respectively. In sub and mild diabetic animals the same dose produced amaximum fall of 31.1 and 32.8% respectively, during OGTT. In case of severely diabetic animals FBG andPPG levels were reduced by 69.2 and 51.2% whereas, total protein, body weight and haemoglobin wereincreased by 11.3, 10.5 and 10.9% respectively after 21 days of treatment. Significant reduction was foundin urine sugar and urine protein levels from +4 and +2 to nil and trace, respectively.Conclusion: The study validates scientifically the widely claimed use of M. oleifera as an ethnomedicine

.

to treat diabetes mellitus

. Introduction

Moringa oleifera Lam. Syn. Moringa pterygosperma Gaerth (fam-ly Moringaceae) is commonly known as Drumstick tree, indigenouso Northwest India. Most of the parts of the plant possess antimi-robial activity (Bhavasar et al., 1965; Caceres et al., 1991). Theyre well known for their pharmacological actions too and are usedor the traditional treatment of diabetes mellitus (Bhishagratna,991; Sharma, 1981; Babu and Chaudhuri, 2005) hepatotoxicityRuckmani et al., 1998), rheumatism, venomous bites and also forardiac stimulation (Chaudhary and Chopra, 1996). In India M.leifera is incorporated in various marketed herbal formulations,

uch as Rumalya and Septilin (The Himalya Drug Company, Banga-ore, India), Orthoherb (Water Bush-nell Ltd., Mumbai, India), whichre available for various disorders.

Abbreviations: bw, body weight; BGL, blood glucose level; FBG, fasting bloodlucose; LD50, lethal dose50; M. oleifera, Moringa oleifera; OGTT, oral glucose toleranceest; STZ, streptozotocin; WHO, World Health Organization.∗ Corresponding author. Tel.: +91 532 2462125; mobile: +09450587750.

E-mail address: geetawatal@gmail.com (G. Watal).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.03.036

© 2009 Elsevier Ireland Ltd. All rights reserved.

Leaves of M. oleifera are lopped for fodder (Sastri, 1962) and havebeen used as antiulcer, diuretic, anti-inflammatory and for woundhealing (Kirtikar and Basu, 1935; Caceres et al., 1992; Udupa etal., 1994; Pal et al., 1995). Ethanolic extract of leaves have shownantifungal activity against a number of dermatophytes (Chuanget al., 2007), whereas methanol extract has a potent CNS depres-sant action (Pal et al., 1996). The aqueous extract of the leaveshas been found to possess antifertility activity (Shukla et al., 1981;Prakash, 1998) and is very useful in regulating the thyroid hor-mone status in adult Swiss rats (Tahiliani and Kar, 2000). Its leavesare also used as nutritional supplement and growth promotersdue to the significant presence of protein, Se, P, Ca, ˇ-caroteneand ˛-tocopherol (Makkar and Becker, 1996; Freiberger et al.,1998; Nambiar and Seshadri, 2001; Lakshminarayana et al., 2005;Sanchez et al., 2006).

N-Benzyl thiocarbamates, N-benzyl carbamates, benzyl nitrilesand a benzyl ester isolated from methanol extract of its driedfruit powder has been shown to stimulate significantly insulin

release from the rodent pancreatic beta cells and have cycloxyge-nase enzyme and lipid peroxidation inhibitory activities (Francis etal., 2004).

Hyperglycemia resulting either due to defective production oraction of insulin leads to a number of complications; cardiovascular,

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enal, neurological, ocular etc (Park, 2007). WHO has emphasizedtrongly on the rational use of traditional and natural indigenousedicines, for treating diabetes mellitus (WHO, 1994). In Indian

thnotherapeutic system of medicine M. oleifera is reported to pos-ess hypoglycemic activity (Sharma, 1981; Bhishagratna, 1991; Kart al., 2003). Its leaves, fruits and stem bark have been scientifi-ally examined for their use in hypercholesterolaemia (Ghasi et al.,000; Mehta et al., 2003), but there are no reports on hypoglycemicnd antidiabetic actions of its leaves. However, fruits and stem barkave been reported to have antidiabetic action (Kar et al., 2003).

Thus, present study deals with the scientific validation oflycemic potential of aqueous extract of M. oleifera leaves in nor-al, sub, mild and severely diabetic rats since M. oleifera have been

laimed to possess hypoglycemic effect in Indian traditional systemf medicine (Sharma, 1981; Bhishagratna, 1991; Kar et al., 2003).he leaves of other species of Moringa stenopetala are used tradi-ionally in Ethiopia for treating diabetes mellitus and had alreadyeen explored for their hypoglycemic action (Makonnen et al.,997). It was considered therefore worthwhile to investigate thequeous extract of M. oleifera leaves for its glycemic potential byvaluating its effect on blood glucose level of normal, sub, mildnd severely diabetic rats. Its effect on haemoglobin, total protein,rine sugar, urine protein and body weight were also examined

n the severely diabetic models. Thus, this is the first reporting ofypoglycemic and antidiabetic effect of aqueous extract of Moringaleifera leaves.

. Materials and methods

.1. Plant material

Fresh leaves of M. oleifera (5 kg) were collected from the Botan-cal garden of University of Allahabad, Allahabad, India. It wasdentified and authenticated by Dr Satya Narain, Taxonomist,otany Department, University of Allahabad, Allahabad, India, inhe month of March 2007. A voucher specimen no. AD/428/07 haseen submitted. The leaves were washed thoroughly with distilledater, crushed and extracted twice with distilled water at tempera-

ure 60–70 ◦C repeatedly, for 48 h. The resulting extract was filteredsing Whatman no. 1 filter paper and concentrated in rotavapournder reduced pressure to give a semisolid residue, which was then

yophilized to get powder (yield: 11.7%, w/w) for further explo-ation.

.2. Experimental animals

More than hundred twenty five, male albino Wistar rats of samege group and body weight 150–200 g were selected for all thexperiments. Animals obtained from National Institute of Commu-icable Disease, New Delhi, India, were housed in polypropyleneages at an ambient temperature of 25–30 ◦C and 45–55% relativeumidity with a 12 h each of dark and light cycle. Animals were

ed pellet diet (Pashu Aahar Kendra, Varanasi, India) and waterd libitum. The Institutional Ethical Committee has approved thetudy.

.3. Induction of diabetes

Diabetes was induced by a single intraperitonial injection of

reshly prepared streptozotocin (STZ) at the dose of 55 mg kg−1 in.1 M citrate buffer (pH 4.5) to a group of overnight fasted ratsShanmugasundaram et al., 1990). After 3 days of streptozotocindministration, depending on their blood glucose levels (BGLs) thenimals were divided into three groups sub, mild and severely dia-etic. (Singh et al., 2007)

macology 123 (2009) 392–396 393

2.4. Estimation

BGL was estimated by glucose oxidase method (Barham andTrinder, 1972) using standard kit of Bayer Diagnostics India Lim-ited. Total protein (Stricklad et al., 1961) in serum and Totalhaemoglobin (Nonfon et al., 1990) in blood were also estimatedweekly in severely diabetic rats. Urine sugar and urine protein weredetected by reagent based Uristix from Bayer Diagnostics and bodyweight of severely diabetic models was measured every week up to21 days.

2.5. Experimental design

Initial screening of the aqueous extract of leaves for evaluatingits glycemic potential was done with a range of variable doses of100, 200 and 300 mg kg−1 given orally by gavage in normal anddiabetic rats by conducting fasting blood glucose (FBG) and oralglucose tolerance test (OGTT) studies. The antidiabetic effect of theextract was also assessed in severely diabetic models, with a doseof 200 mg kg−1which was identified as the most effective dose byinitial screening.

2.5.1. Assessment of hypoglycemic activity in normal healthy ratsTwenty four normal rats fasted over night were divided in to

four groups of six rats each, and used in the experiment. Group Iserved as control received vehicle (distilled water only) and ani-mals of group II, III and IV received variable doses of 100, 200and 300 mg kg−1 respectively, of extract powder suspended in dis-tilled water. FBG was taken initially and then blood samples werecollected from tail vein at 2, 4, 6 and 8 h after administering theextract.

2.5.2. Assessment of hypoglycemic activity by OGTT in normalhealthy rats

A different group of twenty four normal rats was divided andtreated on the same pattern as mentioned above. FBG was checkedinitially, and then BGL was taken after 90 min of treatment con-sidered as ‘0’ h value. The rats were then orally administered with2 g kg−1 of glucose and their glucose tolerance was studied up to3 h at regular interval of 1 h each.

2.5.3. Assessment of hypoglycemic activity by OGTT in subdiabetic and mild diabetic rats

Thirty overnight fasted rats of each diabetic model (sub andmild) were divided into five groups of six rats each. Group I servedas control received vehicle (distilled water only), whereas variabledoses of 100, 200 and 300 mg kg−1 of extract were given to group II,III and IV respectively. Group V serving as a positive control receiveda dose of 2.5 mg kg−1 of a known antidiabetic drug Glipizide, asreference drug (El-Hilaly et al., 2006). FBG was checked initiallyand then BGL was taken after 90 min of treatment considered as‘0’ h value. A dose of 2 gkg−1of glucose was then given orally to allthe groups. BGLs were further checked, upto three hours at regularintervals of 1 h each, considered as 1, 2 and 3 h values.

2.5.4. Assessment of antidiabetic activity in severely diabetic ratsLong term study of 21 days was conducted in severely diabetic

rats. Eighteen rats were divided in to three groups of six rats each.Group I served as diabetic control received vehicle (distilled water

only), whereas group II and III were treated with a single dose of200 mg kg−1 of extract and 2.5 mg kg−1 of Glipizide respectively,once a day upto 21 days. Blood and urine samples were collectedinitially and then weekly upto 21 days. The levels of blood glucose,hemoglobin, total protein were estimated and body weight, urineprotein, urine sugar were checked regularly.

394 D. Jaiswal et al. / Journal of Ethnopharmacology 123 (2009) 392–396

Table 1Effect of graded doses of aqueous extract of Moringa oleifera leaves on BGL of normal rats (mean ± S.D.).

Groups/Treatment Doses Blood glucose levels (mgdl−1)

FBG 2 h 4 h 6 h 8 h

Gp I (control) D W 78.4 ± 2.1 78.1 ± 2.2 77.5 ± 1.9 76.9 ± 3.9 76.3 ± 2.8Gp II (extract) 100 mg kg−1 74.4 ± 4.1 73.6 ± 2.7a 71.2 ± 4.1c 63.9 ± 4.3b 64.7 ± 1.3Gp III (extract) 200 mg kg−1 78.1 ± 3.6 72.6 ± 3.9a 67.4 ± 3.8a 56.4 ± 3.6c 58.5 ± 2.4Gp IV (extract) 300 mg kg−1 76.1 ± 3.2 73.8 ± 4.3 69.1 ± 2.8a 57.7 ± 2.4 a 59.1 ± 3.4Gp V (glipizide) 2.5 mg kg−1 77.5 ± 3.6 70.1 ± 4.1 63.4 ± 3.9b 51.5 ± 3.7 b 50.3 ± 2.9

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3.5. LD50

No toxic effect was observed on treatment with upto 10 and15 times the effective dose of the extract as the behavior of the

a P < 0.05 as compared with control.b P < 0.01 as compared with control.c P < 0.001 as compared with control.

.5.5. LD50 experimentToxicity of the extract was also studied by LD50 experiment. Two

roups of six rats each of both the sex (3 females and 3 males)eighing about 150–200 g were orally administered a dose of ten

nd fifteen times the most effective dose of aqueous extract of. oleifera. Rats were then observed continuously for their gross

ehavioural, neurologic, autonomic and toxic effects up to 24 h.ood consumption, faeces and urine were also examined at 2 h andhen at 6 h intervals for 24 h.

.6. Statistical analysis

The results are expressed as mean ± S.D. and all statistical com-arisons were made by means of one-way analysis of varianceANOVA) followed by Newman–Keuls Multiple Comparison Test.he data were analyzed with Graph Pad Prism 4.0 v for WindowsGraph Pad Software, San Diego, CA, USA). The difference showing alevel of 0.05 or lower was considered to be statistically significant.

. Results

.1. Effect on fasting blood glucose level of normal healthy rats

Table 1 describes the hypoglycemic effect of graded doses ofqueous extract of M. oleifera leaves on FBG of normal rats. Ratsreated with 200 mg kg−1 showed a maximum fall of 26.7% in FBGfter 6 h of oral administration, whereas the fall of 16.9 and 25.1%as observed with the doses of 100 and 300 mg kg−1 respectively.

.2. Effect on oral glucose tolerance of normal healthy rats

Fig. 1 depicts the hypoglycemic effect of a single oral adminis-ration with variable doses of 100, 200 and 300 mg kg−1 on OGTT oformal rats. The dose of 200 mg kg−1 produced a maximum the fallf 29.9% at 3 h after glucose administration, whereas fall of 21.1nd 27.9% was observed with the dose of 100 and 200 mg kg−1,espectively.

.3. Effect on oral glucose tolerance of sub diabetic and mildiabetic rats

In order to choose the optimum dose for the severely dia-etic animals, different doses of aqueous extract 100, 200 and00 mg kg−1 were evaluated on glucose tolerance of sub diabetics well as mild diabetic rats. Table 2 demonstrates the effect ofraded doses of aqueous extract of M. oleifera leaves on BGL of subiabetic and mild diabetic rats during OGTT studies. After 3 h of

lucose administration the fall observed with the dose of 100, 200nd 300 mg kg−1 was 15.9, 31.1 and 29.7% in case of sub and 15.5,2.8 and 30.9% in case of mild diabetic rats respectively. Moreover,ven Glipizide, the reference drug produced a fall of only 26.7 and0.5% in sub diabetic and mild diabetic cases respectively, which

was lesser than the fall produced by the most effective dose of theextract.

3.4. Effect on blood glucose level and other parameters of severelydiabetic rats

Figs. 2 and 3 reveals the effect of 21 days long treatment withextract of M. oleifera leaves on FBG, PPG, haemoglobin, and totalprotein of severely diabetic rats. The fall observed after 7, 14 and 21days treatment with the dose of 200 mg kg−1of the extract was 25.9,53.5, 69.2% in FBG and 21.4, 37.8, 51.2% in PPG levels respectively.An increase of 10.9% in haemoglobin and 11.3% in total protein wasobserved after 21 days treatment.

As far as urine protein and urine sugar levels are concernedamazing results were noticed as both were nil only after 14 days oftreatment. Though, similar results were found with Glipizide treat-ment yet the aqueous extract of M. oleifera was found to be moreeffective in all the parameters. The body weights of all the groups(control and treated) were estimated before the treatment and thenweekly during the treatment. A significant increase of about 21 gafter 21 days treatment was observed in body weight of extracttreated animals.

Fig. 1. Hypoglycemic effect of graded doses of aqueous extract of M. oleifera leaveson BGL of normal rats during OGTT, each value shown in mean ± S.D. (n = 6). Group I:control (distilled water); Group II: extract treated (100 mg kg−1); Group III: extracttreated (200 mg kg−1); Group IV: extract treated (300 mg kg−1); Group V: Glipizidetreated (2.5 mg kg−1), * P < 0.05, ** P < 0.01 compared to the control at the corre-sponding time.

D. Jaiswal et al. / Journal of Ethnopharmacology 123 (2009) 392–396 395

Table 2Effect of variable doses of Moringa oleifera on OGTT of sub diabetic and mild diabetic rats (mean ± S.D.).

Groups (treatment and doses) FBG 0 h 1 h 2 h 3 h

BGL of sub diabetic animals (mg dl−1)I (control, D W) 89.2 ± 4.6 88.7 ± 4.4 217.5 ± 3.8 139.2 ± 4.4 124.1 ± 3.9II (extract, 100 mg kg−1) 85.6 ± 5.2 84.3 ± 4.8a 200.7 ± 4.5a 121.7 ± 4.1a 104.3 ± 4.6a

III (extract, 200 mg kg−1) 87.2 ± 3.9 79.6 ± 3.7 182.2 ± 3.3a 103.8 ± 4.3b 85.7 ± 4.1c

IV (extract, 300 mg kg−1) 89.4 ± 4.5 81.9 ± 5.1 186.7 ± 3.8a 105.4 ± 4.7b 87.2 ± 4.6b

V (glipizide, 2.5 mg kg−1) 88.1 ± 4.5 86.3 ± 4.5 190.2 ± 3.9a 104.1 ± 4.8b 88.5 ± 4.3b

BGL of mild diabetic animals (mg dl−1)I (control, D W) 189.7 ± 3.8 189.9 ± 5.1 375.5 ± 4.9 318.9 ± 5.5 264.7 ± 5.2II (extract, 100 mg kg−1) 190.4 ± 4.8 180.5 ± 5.1 338.1 ± 4.5a 277.3 ± 4.4a 223.8 ± 4.6a

III (extract, 200 mg kg−1) 188.2 ± 4.5 168.2 ± 4.6 300.9 ± 5.3a 238.5 ± 5.5b 177.9 ± 5.4c

IV (extract, 300 mg kg−1) 190.6 ± 5.3 170.6 ± 5.5 307.1 ± 4.9b 242.4 ± 4.8b 182.8 ± 5.3a

V (glipizide, 2.5 mg kg−1) 191.3 ± 5.3 167.7 ± 5.0 229.6 ± 5.2 240.1 ± 4.6a 183.9 ± 4.8b

a P < 0.05 as compared with control.b P < 0.01 as compared with control.c P < 0.001 as compared with control.

Fig. 2. Effect of aqueous extract of Moringa oleifera leaves on FBG and PPG inseverely diabetic rats Group I: control (distilled water); Group II: extract treated(300 mg kg−1); Group III: Glipizide treated (2.5 mg kg−1), * P < 0.05, ** P < 0.01 com-pared to the control at the corresponding time.

Fig. 3. Effect of aqueous extract of Moringa oleifera leaves on Haemoglobin and totalprotein in severely diabetic rats Group I: control (distilled water); Group II: extracttreated (300 mg kg−1); Group III: Glipizide treated (2.5 mg kg−1), * P < 0.05, ** P < 0.01compared to the control at the corresponding time.

treated rats appeared normal and no death occurred in any of thesegroups.

4. Discussion

STZ-induced hyperglycemia has been described as a usefulexperimental model to study the activity of hypoglycemic agents(Junod et al., 1969). The purpose of the present study was to assessthe effect of M. oleifera leaves aqueous extract therapy on glycemiccontrol, haemoglobin, total protein, urine sugar, urine protein andbody weight.

Aqueous extract of M. oleifera leaves reduces the blood glucoselevel in normal rats and normalizes the high blood glucose levels insub, mild and severely diabetic rats. It also improves glucose toler-ance in normal, sub and mild diabetic animals. Glipizide was used asreference drug in diabetic models for positive control. It is interest-ing to note that the extract was more effective than reference drug.However, the higher concentration of the extract used, against thereference drug Glipizide could be because of only a small amountof active substance present in the extract. Since, good activity hasbeen seen in severely diabetic rats with damaged islets therefore, itis likely to be expected that the aqueous extract of leaves has somedirect effect by increasing the tissue utilization of glucose (Ali etal., 1993; Gray et al., 2000), by inhibiting hepatic gluconeogene-sis or absorption of glucose into the muscles and adipose tissues(Kamanyi et al., 1994).

The FBG decreases by 26.7 and 25.1% after 6 h, in normalrats treated with a single dose of 200 mg kg−1 and 300 mg kg−1

respectively. Such a phenomenon of less hypoglycemic response athigher doses is common with indigenous plants and has alreadybeen observed in Psidium guajava (Rai et al., 2007), Trichosan-thes dioica (Rai et al., 2008), Cynodon dactylon (Singh et al.,2007, 2008) and Cinnamomum tamala (Sharma et al., 1996). Thedaily treatment with 200 mg kg−1 of extract for 21 days broughtFBG and PPG levels to nearly normal range in severely diabeticanimals.

Significant improvement in haemoglobin and total protein lev-els on long term treatment with extract for 21 days indicated that ithas favourable effect in bringing down the severity of diabetes. Usu-ally elevated levels of urine sugar and urine protein are associatedwith diabetes mellitus and complete elimination of these within 14days from urine of severely diabetic animals through extract ther-

apy, is an additional advantage, indirectly confirming thereby theantidiabetic activity of the extract.

STZ- induced diabetes is characterized by severe loss in bodyweight (Ravi et al., 2004). Hence, the weight gain after adminis-tration of the extract in severely diabetic rats is simply due to the

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96 D. Jaiswal et al. / Journal of Ethn

bility of the extract to reduce hyperglycemia. High LD50 of thextract indicates its high margin of safety.

In conclusion, the present study using biochemical assays per-aining to BGL of different animal models reveals that aqueousxtract of leaves has significant hypoglycemic and antidiabeticotential. This is the first report of its type. Further pharmacolog-

cal and biochemical investigations are underway to elucidate theechanism of the hypoglycemic and antidiabetic actions of Moringa

leifera leaves.

cknowledgement

The authors are thankful to National Medicinal Plants BoardNMPB) for financial assistance.

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