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Antihyperglycemic studies with methanol extract of Annona reticulata L. (Annonaceae) and Carissa carandas L. (Apocynaceae) leaves in Swiss albino mice.

Annona reticulata L. (Annonaceae; local name: ata) is a small deciduous tree, which is cultivated in many parts of the world including Southeast Asia, Taiwan, India, Bangladesh, Australia and West Africa. Carissa carandas L. (Apocynaceae; local name: koromcha) is a species of flowering shrub in the dogbane family producing berry-sized fruits used commonly in the Indian subcontinent (including Bangladesh) as a condiment or additive to spices. Both plants are cultivated in Bangladesh for their edible fruits. Additionally, plant parts of Annona reticulata are used in the folk medicinal system of Bangladesh for treatment of epilepsy, toothache, tumor, fever and dysentery, while plant parts of Carissa carandas are used for treatment of epilepsy, malaria, fever, dysentery and diabetes.

Leaves of Annona species have been reported to possess antioxidant properties (Baskar et al., 2007). Various volatile compounds like a-pinene, b-pinene, myrcene, limonene, terpinen-4-ol, and germacrene D has been reported to be present in fruits of Annona reticulata (Pino et al., 2003). Annonacin, a monotetrahydrofuran acetogenin, isolated from Annona species reportedly arrested cancer cells at the G1 phase and caused cytotoxicity (Yuan et al., 2003). Iso-acetogenins and acetogenins have been reported from seeds of the plant (Yu et al., 1997; Chang et al., 1998). Seeds of the plant also reportedly contain squamocin and rolliniastatin I (Tam et al., 1993). Cytotoxic acetogenins isolated from leaves of the plant include squamone, solamin, annomonicin, and rolliniastatin 2 (Chang et al., 1993).

Histamine releasing activity has been reported for Carissa carandas roots (Joglekar and Gaitonde, 1970). Triterpenoidal constituents have been isolated from leaves (Siddiqui et al., 2003). Extract of unripe fruits of the plant has been shown to possess antidiabetic activity in alloxan-induced diabetic rats (Itankar et al., 2011).

Bangladesh has several types of traditional medicinal practices. Among them folk medicinal practitioners, otherwise known as Kavirajes, are possibly the most numerous and provide primary health-care to substantial segments of the rural and urban population of the country. Kavirajes rely on medicinal plants as the major ingredient in their formulations. We had been conducting extensive ethnomedicinal surveys among the Kavirajes for the last few years. A number of the plants obtained from the Kavirajes have been screened in our laboratory, particularly for their antihyperglycemic and antinociceptive effects, because diabetes is a debilitating disease affecting millions of people worldwide and for which allopathic medicine has no known cure, and pain due to various causes affect millions of people every day and for which the various painkilling drugs used in allopathic medicine has moderate to severe side-effects. Some of the results from these pharmacological activity studies have been reported (Anwar et al., 2010; Jahan et al., 2010; Khan et al., 2010; Mannan et al., 2010; Rahman et al., 2010; Rahman et al., 2011; Shoha et al., 2011; Sutradhar et al., 2011). The objective of the present study was to evaluate the antihyperglycemic potential of methanol extracts of leaves of Annona reticulata and Carissa carandas in oral glucose tolerance tests using glucose-loaded Swiss albino mice.

Materials and Methods

Plant Material and Extraction:

The leaves of Annona reticulata and Carissa carandas were collected from Uttara in Dhaka city in January 2011. The plants were taxonomically identified by the Bangladesh National Herbarium at Dhaka (Accession Numbers: 35,403 and 35,404, respectively). The leaves were separately cut into small pieces and air-dried in the shade for 120 hours, grounded into a fine powder, and were extracted with methanol at a ratio of 1:5 (w/v). The initial weight of dried leaf powder used for extraction was 100g; the final weight of the extract was 8.36g for Annona reticulata and 9.18g for Carissa carandas.

Chemicals and Drugs:

Glibenclamide and glucose were obtained from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals were of analytical grade.

Animals:

In the present study, Swiss albino mice (male), which weighed between 18-22 g were used. The animals were obtained from International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B). All animals were kept under ambient temperature with 12h light followed by a 12h dark cycle. The animals were acclimatized for three days prior to actual experiments. The study was conducted following approval by the Institutional Animal Ethical Committee of University of Development Alternative, Dhaka, Bangladesh.

Anti-hyperglycemic Activity:

Glucose tolerance property of methanol extract of Annona reticulata and Carissa carandas leaves was determined as per the procedure previously described by Joy and Kuttan (1999) with minor modifications. In brief, fasted mice were grouped into ten groups of six mice each. The various groups received different treatments like Group 1 received vehicle (1% Tween 80 in water, 10 ml/kg body weight) and served as control, group 2 received standard drug (glibenclamide, 10 mg/kg body weight). Groups 3-6 received methanol extract of Carissa carandas leaves at doses of 50, 100, 200 and 400 mg per kg body weight. Groups 7-10 received methanol extract of Annona reticulata leaves at doses of 50, 100, 200 and 400 mg per kg body weight. Each mouse was weighed and doses adjusted accordingly prior to administration of vehicle, standard drug, and test samples. All substances were orally administered. Following a period of one hour, all mice were orally administered 2 g glucose/kg of body weight. Blood samples were collected 120 minutes after the glucose administration through puncturing heart. Serum glucose levels were measured by glucose oxidase method (Venkatesh et al., 2004).

Statistical Analysis:

Experimental values are expressed as mean [+ or -] SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.

Results and Discussion

The crude methanol extract of Carissa carandas leaves showed dose-dependent reductions in serum glucose levels when administered to glucose-loaded mice at doses of 50, 100, 200 and 400 mg per kg body weight. At these doses, the extract caused 15.6, 17.8, 20.0 and 47.8% reductions in serum glucose levels. Apart from the 50 mg extract per kg body weight, the reductions at all other doses were statistically significant. However, up till a dose of 200 mg per kg body weight, the antihyperglycemic activity of the extract was not strong. At a dose of 400 mg extract per kg body weight, the percent lowering of serum glucose concentration was high and was comparable to that of the standard antihyperglycemic drug, glibenclamide at the dose administered. At adose of 10 mg per kg body weight, glibenclamide lowered serum glucose level by 43.5%. The results are shown in Table 1.

As also shown in Table 1, the crude methanol extract of Annona reticulata leaves exhibited strong antihyperglycemic activity when administered to glucose-loaded mice. The reductions in glucose levels were both dose-dependent and statistically significant at the administered four different doses of 50, 100, 200 and 400 mg extract per kg body weight. At these four doses, the extract caused, respectively, 34.8, 37.0, 49.6, and 56.1% lowering of serum glucose levels. Thus dose for dose, the leaves of Annona reticulata demonstrated a more potent antihyperglycemic activity than leaves of Carissa carandas. The antidiabetic potential of Carissa carandas fruits has been previously reported (Itankar et al., 2011). Our study demonstrates the antidiabetic potential also of leaves of this plant.

The observed glucose lowering effect by the crude extract of leaves of both plants may occur through several possible mechanisms. The extracts may potentiate the pancreatic secretion of insulin or increase the glucose uptake (Farjou et al., 1987; Nyunai et al., 2009). Alternately, the extracts may inhibit glucose absorption in gut (Bhowmik et al., 2009). Any of the above three mechanisms or a combination of mechanisms can contribute to the observed lowering of blood sugar. However, the exact mechanism of reduction in the concentration of serum glucose remains to be elucidated. The antihyperglycemic activity of extracts of leaves from both plants suggests that further scientific studies need to be conducted towards discovery of possible new antidiabetic compounds. Diabetes is a disease that is increasing with every passing year in the human population throughout the world, possibly caused by a change in food habits and a change in lifestyle. Since the disease is debilitating with advanced stages leading to diabetic retinopathy, diabetic nephropathy and increased risks of cardiovascular disorders, and since the disease cannot be cured with modern allopathic medicine, the plant kingdom may form a useful source for discovery of novel antidiabetic agents.

References

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Corresponding Author: Professor Dr. Mohammed Rahmatullah, Pro-Vice Chancellor and Dean, Faculty of Life Sciences University of Development Alternative House No. 78, Road No. 11A (new) Dhanmondi, Dhaka-1205 Bangladesh Telephone: +88-01715032621 Fax: +88-02-815739 E-mail: rahamatm@hotmail.com

Sk. Mizanur Rahman, Md. Rashedul Islam, Shahnaz Rahman, Tamim Mosaiab, Rasheda Ahmed, Fatema Khatun, Dilruba Nasrin, Nusratun Nahar, Shamima Ahsan, Mohammed Rahmatullah

Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh.
Table 1:   Effect of methanol extract of Carissa carandas and Annona
reticulata leaves on serum glucose level in hyperglycemic mice
following 120 minutes of glucose loading.

                                                          % lowering of
                    Dose (mg/kg      Serum glucose            serum
Treatment           body weight)     level (mg/dl)        glucose level

Control (Group 1)       10 ml      98.29 [+ or -] 5.36        --
Glibenclamide           10 mg      55.56 [+ or -] 4.85        43.5 *
(Group 2)
Carissa carandas        50 mg      82.91 [+ or -] 10.35       15.6
(Group 3)
Carissa carandas       100 mg      80.77 [+ or -] 7.39        17.8 *
(Group 4)
Carissa carandas       200 mg      78.63 [+ or -] 3.72        20.0 *
(Group 5)
Carissa carandas       400 mg      51.28 [+ or -] 3.50        47.8 *
(Group 6)
Annona reticulata       50 mg      64.10 [+ or -] 2.48        34.8 *
(Group 7)
Annona reticulata      100 mg      61.97 [+ or -] 3.47        37.0 *
(Group 8)
Annona reticulata      200 mg      49.57 [+ or -] 5.03        49.6 *
(Group 9)
Annona reticulata      400 mg      43.16 [+ or -] 4.75        56.1 *
(Group 10)

All administrations were made orally. Values represented as
mean [+ or -] SEM, (n = 6); * P < 0.05; significant compared to
hyperglycemic control animals.
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Title Annotation:Original Article
Author:Rahman, Mizanur; Islam, Rashedul; Rahman, Shahnaz; Mosaiab, Tamim; Ahmed, Rasheda; Khatun, Fatema; N
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9BANG
Date:Apr 1, 2011
Words:2497
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