Journal of Ethnopharmacology 123 (2009) 392–396

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Journal of Ethnopharmacology
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Effect of Moringa oleifera Lam. leaves aqueous extract therapy on hyperglycemic rats
Dolly Jaiswal, Prashant Kumar Rai, Amit Kumar, Shikha Mehta, Geeta Watal ∗
Alternative Therapeutics Unit, Drug Development Division, Medicinal Research Lab, Department of Chemistry, University of Allahabad, Allahabad, U.P. 211 002, India

a r t i c l e

i n f o

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 aqueous extract therapy on glycemic control, haemoglobin, total protein, urine sugar, urine protein and body weight. Materials and methods: Variable doses of 100, 200 and 300 mg kg−1 of aqueous extract were administered orally 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 a maximum fall of 31.1 and 32.8% respectively, during OGTT. In case of severely diabetic animals FBG and PPG levels were reduced by 69.2 and 51.2% whereas, total protein, body weight and haemoglobin were increased by 11.3, 10.5 and 10.9% respectively after 21 days of treatment. Significant reduction was found in 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. © 2009 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 10 October 2007 Received in revised form 2 February 2009 Accepted 19 March 2009 Available online 5 April 2009 Keywords: Antidiabetic Drumstick tree Moringaceae Moringa oleifera

1. Introduction Moringa oleifera Lam. Syn. Moringa pterygosperma Gaerth (family Moringaceae) is commonly known as Drumstick tree, indigenous to Northwest India. Most of the parts of the plant possess antimicrobial activity (Bhavasar et al., 1965; Caceres et al., 1991). They are well known for their pharmacological actions too and are used for the traditional treatment of diabetes mellitus (Bhishagratna, 1991; Sharma, 1981; Babu and Chaudhuri, 2005) hepatotoxicity (Ruckmani et al., 1998), rheumatism, venomous bites and also for cardiac stimulation (Chaudhary and Chopra, 1996). In India M. oleifera is incorporated in various marketed herbal formulations, such as Rumalya and Septilin (The Himalya Drug Company, Bangalore, India), Orthoherb (Water Bush-nell Ltd., Mumbai, India), which are available for various disorders.

Abbreviations: bw, body weight; BGL, blood glucose level; FBG, fasting blood glucose; LD50 , lethal dose50 ; M. oleifera, Moringa oleifera; OGTT, oral glucose tolerance test; STZ, streptozotocin; WHO, World Health Organization. ∗ Corresponding author. Tel.: +91 532 2462125; mobile: +09450587750. E-mail address: geetawatal@gmail.com (G. Watal). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.03.036

Leaves of M. oleifera are lopped for fodder (Sastri, 1962) and have been used as antiulcer, diuretic, anti-inflammatory and for wound healing (Kirtikar and Basu, 1935; Caceres et al., 1992; Udupa et al., 1994; Pal et al., 1995). Ethanolic extract of leaves have shown antifungal activity against a number of dermatophytes (Chuang et al., 2007), whereas methanol extract has a potent CNS depressant action (Pal et al., 1996). The aqueous extract of the leaves has been found to possess antifertility activity (Shukla et al., 1981; Prakash, 1998) and is very useful in regulating the thyroid hormone status in adult Swiss rats (Tahiliani and Kar, 2000). Its leaves are also used as nutritional supplement and growth promoters due to the significant presence of protein, Se, P, Ca, ˇ-carotene and ˛-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 nitriles and a benzyl ester isolated from methanol extract of its dried fruit powder has been shown to stimulate significantly insulin release from the rodent pancreatic beta cells and have cycloxygenase enzyme and lipid peroxidation inhibitory activities (Francis et al., 2004). Hyperglycemia resulting either due to defective production or action of insulin leads to a number of complications; cardiovascular,

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renal, neurological, ocular etc (Park, 2007). WHO has emphasized strongly on the rational use of traditional and natural indigenous medicines, for treating diabetes mellitus (WHO, 1994). In Indian ethnotherapeutic system of medicine M. oleifera is reported to possess hypoglycemic activity (Sharma, 1981; Bhishagratna, 1991; Kar et al., 2003). Its leaves, fruits and stem bark have been scientifically examined for their use in hypercholesterolaemia (Ghasi et al., 2000; Mehta et al., 2003), but there are no reports on hypoglycemic and antidiabetic actions of its leaves. However, fruits and stem bark have been reported to have antidiabetic action (Kar et al., 2003). Thus, present study deals with the scientific validation of glycemic potential of aqueous extract of M. oleifera leaves in normal, sub, mild and severely diabetic rats since M. oleifera have been claimed to possess hypoglycemic effect in Indian traditional system of medicine (Sharma, 1981; Bhishagratna, 1991; Kar et al., 2003). The leaves of other species of Moringa stenopetala are used traditionally in Ethiopia for treating diabetes mellitus and had already been explored for their hypoglycemic action (Makonnen et al., 1997). It was considered therefore worthwhile to investigate the aqueous extract of M. oleifera leaves for its glycemic potential by evaluating its effect on blood glucose level of normal, sub, mild and severely diabetic rats. Its effect on haemoglobin, total protein, urine sugar, urine protein and body weight were also examined in the severely diabetic models. Thus, this is the first reporting of hypoglycemic and antidiabetic effect of aqueous extract of Moringa oleifera leaves. 2. Materials and methods 2.1. Plant material Fresh leaves of M. oleifera (5 kg) were collected from the Botanical garden of University of Allahabad, Allahabad, India. It was identified and authenticated by Dr Satya Narain, Taxonomist, Botany Department, University of Allahabad, Allahabad, India, in the month of March 2007. A voucher specimen no. AD/428/07 has been submitted. The leaves were washed thoroughly with distilled water, crushed and extracted twice with distilled water at temperature 60–70 ◦ C repeatedly, for 48 h. The resulting extract was filtered using Whatman no. 1 filter paper and concentrated in rotavapour under reduced pressure to give a semisolid residue, which was then lyophilized to get powder (yield: 11.7%, w/w) for further exploration. 2.2. Experimental animals More than hundred twenty five, male albino Wistar rats of same age group and body weight 150–200 g were selected for all the experiments. Animals obtained from National Institute of Communicable Disease, New Delhi, India, were housed in polypropylene cages at an ambient temperature of 25–30 ◦ C and 45–55% relative humidity with a 12 h each of dark and light cycle. Animals were fed pellet diet (Pashu Aahar Kendra, Varanasi, India) and water ad libitum. The Institutional Ethical Committee has approved the study. 2.3. Induction of diabetes Diabetes was induced by a single intraperitonial injection of freshly prepared streptozotocin (STZ) at the dose of 55 mg kg−1 in 0.1 M citrate buffer (pH 4.5) to a group of overnight fasted rats (Shanmugasundaram et al., 1990). After 3 days of streptozotocin administration, depending on their blood glucose levels (BGLs) the animals were divided into three groups sub, mild and severely diabetic. (Singh et al., 2007)

2.4. Estimation BGL was estimated by glucose oxidase method (Barham and Trinder, 1972) using standard kit of Bayer Diagnostics India Limited. Total protein (Stricklad et al., 1961) in serum and Total haemoglobin (Nonfon et al., 1990) in blood were also estimated weekly in severely diabetic rats. Urine sugar and urine protein were detected by reagent based Uristix from Bayer Diagnostics and body weight of severely diabetic models was measured every week up to 21 days. 2.5. Experimental design Initial screening of the aqueous extract of leaves for evaluating its glycemic potential was done with a range of variable doses of 100, 200 and 300 mg kg−1 given orally by gavage in normal and diabetic rats by conducting fasting blood glucose (FBG) and oral glucose tolerance test (OGTT) studies. The antidiabetic effect of the extract was also assessed in severely diabetic models, with a dose of 200 mg kg−1 which was identified as the most effective dose by initial screening. 2.5.1. Assessment of hypoglycemic activity in normal healthy rats Twenty four normal rats fasted over night were divided in to four groups of six rats each, and used in the experiment. Group I served as control received vehicle (distilled water only) and animals of group II, III and IV received variable doses of 100, 200 and 300 mg kg−1 respectively, of extract powder suspended in distilled water. FBG was taken initially and then blood samples were collected from tail vein at 2, 4, 6 and 8 h after administering the extract. 2.5.2. Assessment of hypoglycemic activity by OGTT in normal healthy rats A different group of twenty four normal rats was divided and treated on the same pattern as mentioned above. FBG was checked initially, and then BGL was taken after 90 min of treatment considered as ‘0’ h value. The rats were then orally administered with 2 g kg−1 of glucose and their glucose tolerance was studied up to 3 h at regular interval of 1 h each. 2.5.3. Assessment of hypoglycemic activity by OGTT in sub diabetic and mild diabetic rats Thirty overnight fasted rats of each diabetic model (sub and mild) were divided into five groups of six rats each. Group I served as control received vehicle (distilled water only), whereas variable doses 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 received a dose of 2.5 mg kg−1 of a known antidiabetic drug Glipizide, as reference drug (El-Hilaly et al., 2006). FBG was checked initially and then BGL was taken after 90 min of treatment considered as ‘0’ h value. A dose of 2 gkg−1 of glucose was then given orally to all the groups. BGLs were further checked, upto three hours at regular intervals of 1 h each, considered as 1, 2 and 3 h values. 2.5.4. Assessment of antidiabetic activity in severely diabetic rats Long 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 of 200 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 collected initially and then weekly upto 21 days. The levels of blood glucose, hemoglobin, total protein were estimated and body weight, urine protein, urine sugar were checked regularly.

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Table 1 Effect 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 Gp I (control) Gp II (extract) Gp III (extract) Gp IV (extract) Gp V (glipizide)
a b c

2h ± ± ± ± ± 2.1 4.1 3.6 3.2 3.6 78.1 73.6 72.6 73.8 70.1 ± ± ± ± ± 2.2 2.7a 3.9a 4.3 4.1

4h 77.5 71.2 67.4 69.1 63.4 ± ± ± ± ± 1.9 4.1c 3.8a 2.8a 3.9b

6h 76.9 63.9 56.4 57.7 51.5 ± ± ± ± ± 3.9 4.3b 3.6c 2.4 a 3.7 b

8h 76.3 64.7 58.5 59.1 50.3 ± ± ± ± ± 2.8 1.3 2.4 3.4 2.9

DW 100 mg kg−1 200 mg kg−1 300 mg kg−1 2.5 mg kg−1

78.4 74.4 78.1 76.1 77.5

P < 0.05 as compared with control. P < 0.01 as compared with control. P < 0.001 as compared with control.

2.5.5. LD50 experiment Toxicity of the extract was also studied by LD50 experiment. Two groups of six rats each of both the sex (3 females and 3 males) weighing about 150–200 g were orally administered a dose of ten and fifteen times the most effective dose of aqueous extract of M. oleifera. Rats were then observed continuously for their gross behavioural, neurologic, autonomic and toxic effects up to 24 h. Food consumption, faeces and urine were also examined at 2 h and then at 6 h intervals for 24 h. 2.6. Statistical analysis The results are expressed as mean ± S.D. and all statistical comparisons were made by means of one-way analysis of variance (ANOVA) followed by Newman–Keuls Multiple Comparison Test. The data were analyzed with Graph Pad Prism 4.0 v for Windows (Graph Pad Software, San Diego, CA, USA). The difference showing a P level of 0.05 or lower was considered to be statistically significant. 3. Results 3.1. Effect on fasting blood glucose level of normal healthy rats Table 1 describes the hypoglycemic effect of graded doses of aqueous extract of M. oleifera leaves on FBG of normal rats. Rats treated with 200 mg kg−1 showed a maximum fall of 26.7% in FBG after 6 h of oral administration, whereas the fall of 16.9 and 25.1% was observed with the doses of 100 and 300 mg kg−1 respectively. 3.2. Effect on oral glucose tolerance of normal healthy rats Fig. 1 depicts the hypoglycemic effect of a single oral administration with variable doses of 100, 200 and 300 mg kg−1 on OGTT of normal rats. The dose of 200 mg kg−1 produced a maximum the fall of 29.9% at 3 h after glucose administration, whereas fall of 21.1 and 27.9% was observed with the dose of 100 and 200 mg kg−1 , respectively. 3.3. Effect on oral glucose tolerance of sub diabetic and mild diabetic rats In order to choose the optimum dose for the severely diabetic animals, different doses of aqueous extract 100, 200 and 300 mg kg−1 were evaluated on glucose tolerance of sub diabetic as well as mild diabetic rats. Table 2 demonstrates the effect of graded doses of aqueous extract of M. oleifera leaves on BGL of sub diabetic and mild diabetic rats during OGTT studies. After 3 h of glucose administration the fall observed with the dose of 100, 200 and 300 mg kg−1 was 15.9, 31.1 and 29.7% in case of sub and 15.5, 32.8 and 30.9% in case of mild diabetic rats respectively. Moreover, even Glipizide, the reference drug produced a fall of only 26.7 and 30.5% in sub diabetic and mild diabetic cases respectively, which

was lesser than the fall produced by the most effective dose of the extract. 3.4. Effect on blood glucose level and other parameters of severely diabetic rats Figs. 2 and 3 reveals the effect of 21 days long treatment with extract of M. oleifera leaves on FBG, PPG, haemoglobin, and total protein of severely diabetic rats. The fall observed after 7, 14 and 21 days treatment with the dose of 200 mg kg−1 of 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 was observed after 21 days treatment. As far as urine protein and urine sugar levels are concerned amazing results were noticed as both were nil only after 14 days of treatment. Though, similar results were found with Glipizide treatment yet the aqueous extract of M. oleifera was found to be more effective in all the parameters. The body weights of all the groups (control and treated) were estimated before the treatment and then weekly during the treatment. A significant increase of about 21 g after 21 days treatment was observed in body weight of extract treated animals. 3.5. LD50 No toxic effect was observed on treatment with upto 10 and 15 times the effective dose of the extract as the behavior of the

Fig. 1. Hypoglycemic effect of graded doses of aqueous extract of M. oleifera leaves on 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: extract treated (200 mg kg−1 ); Group IV: extract treated (300 mg kg−1 ); Group V: Glipizide treated (2.5 mg kg−1 ), * P < 0.05, ** P < 0.01 compared to the control at the corresponding time.

D. Jaiswal et al. / Journal of Ethnopharmacology 123 (2009) 392–396 Table 2 Effect of variable doses of Moringa oleifera on OGTT of sub diabetic and mild diabetic rats (mean ± S.D.). Groups (treatment and doses) I (control, D W) II (extract, 100 mg kg−1 ) III (extract, 200 mg kg−1 ) IV (extract, 300 mg kg−1 ) V (glipizide, 2.5 mg kg−1 ) I (control, D W) II (extract, 100 mg kg−1 ) III (extract, 200 mg kg−1 ) IV (extract, 300 mg kg−1 ) V (glipizide, 2.5 mg kg−1 )
a b c

395

FBG

0h
−1

1h 217.5 ± 3.8 200.7 ± 4.5a 182.2 ± 3.3a 186.7 ± 3.8a 190.2 ± 3.9a 375.5 ± 4.9 338.1 ± 4.5a 300.9 ± 5.3a 307.1 ± 4.9b 229.6 ± 5.2

2h 139.2 ± 4.4 121.7 ± 4.1a 103.8 ± 4.3b 105.4 ± 4.7b 104.1 ± 4.8b 318.9 ± 5.5 277.3 ± 4.4a 238.5 ± 5.5b 242.4 ± 4.8b 240.1 ± 4.6a

3h 124.1 ± 3.9 104.3 ± 4.6a 85.7 ± 4.1c 87.2 ± 4.6b 88.5 ± 4.3b 264.7 ± 5.2 223.8 ± 4.6a 177.9 ± 5.4c 182.8 ± 5.3a 183.9 ± 4.8b

BGL of sub diabetic animals (mg dl ) 89.2 ± 4.6 88.7 ± 4.4 85.6 ± 5.2 84.3 ± 4.8a 87.2 ± 3.9 79.6 ± 3.7 89.4 ± 4.5 81.9 ± 5.1 88.1 ± 4.5 86.3 ± 4.5 BGL of mild diabetic animals (mg dl−1 ) 189.7 ± 3.8 189.9 ± 5.1 190.4 ± 4.8 180.5 ± 5.1 188.2 ± 4.5 168.2 ± 4.6 190.6 ± 5.3 170.6 ± 5.5 191.3 ± 5.3 167.7 ± 5.0

P < 0.05 as compared with control. P < 0.01 as compared with control. P < 0.001 as compared with control.

treated rats appeared normal and no death occurred in any of these groups. 4. Discussion STZ-induced hyperglycemia has been described as a useful experimental model to study the activity of hypoglycemic agents (Junod et al., 1969). The purpose of the present study was to assess the effect of M. oleifera leaves aqueous extract therapy on glycemic control, haemoglobin, total protein, urine sugar, urine protein and body weight. Aqueous extract of M. oleifera leaves reduces the blood glucose level in normal rats and normalizes the high blood glucose levels in sub, mild and severely diabetic rats. It also improves glucose tolerance in normal, sub and mild diabetic animals. Glipizide was used as reference drug in diabetic models for positive control. It is interesting to note that the extract was more effective than reference drug. However, the higher concentration of the extract used, against the reference drug Glipizide could be because of only a small amount of active substance present in the extract. Since, good activity has been seen in severely diabetic rats with damaged islets therefore, it is likely to be expected that the aqueous extract of leaves has some direct effect by increasing the tissue utilization of glucose (Ali et al., 1993; Gray et al., 2000), by inhibiting hepatic gluconeogenesis 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 normal rats treated with a single dose of 200 mg kg−1 and 300 mg kg−1 respectively. Such a phenomenon of less hypoglycemic response at higher doses is common with indigenous plants and has already been observed in Psidium guajava (Rai et al., 2007), Trichosanthes dioica (Rai et al., 2008), Cynodon dactylon (Singh et al., 2007, 2008) and Cinnamomum tamala (Sharma et al., 1996). The daily treatment with 200 mg kg−1 of extract for 21 days brought FBG and PPG levels to nearly normal range in severely diabetic animals. Significant improvement in haemoglobin and total protein levels on long term treatment with extract for 21 days indicated that it has favourable effect in bringing down the severity of diabetes. Usually elevated levels of urine sugar and urine protein are associated with diabetes mellitus and complete elimination of these within 14 days from urine of severely diabetic animals through extract therapy, is an additional advantage, indirectly confirming thereby the antidiabetic activity of the extract. STZ- induced diabetes is characterized by severe loss in body weight (Ravi et al., 2004). Hence, the weight gain after administration of the extract in severely diabetic rats is simply due to the

Fig. 2. Effect of aqueous extract of Moringa oleifera leaves on FBG and PPG in severely 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 compared to the control at the corresponding time.

Fig. 3. Effect of aqueous extract of Moringa oleifera leaves on Haemoglobin and total protein in severely 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 compared to the control at the corresponding time.

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