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ORIGINAL ARTICLE
Year : 2012  |  Volume : 2  |  Issue : 2  |  Page : 105-110

Antidiabetic activity of Pandanus fascicularis Lamk - aerial roots in alloxan-induced hyperglycemic rats


1 Institute of Pharmacy, Bundelkhand University, Kanpur Road, Jhansi, Uttar Pradesh, India
2 National Research Institute for Ayurveda Siddha Human Resource Development Aamkho, Gwalior, Madhya Pradesh, India

Date of Submission03-Jun-2011
Date of Acceptance21-Aug-2011
Date of Web Publication9-May-2012

Correspondence Address:
Savita Kumari
Institute of Pharmacy, Bundelkhand University, Kanpur Road, Jhansi, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.95943

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   Abstract 

Background: The aerial roots of Pandanus fascicularis are used traditionally in treatment of diabetes. Objective: This study evaluated the antidiabetic activity of methanolic extract of aerial roots of P. fascicularis. Materials and Methods: Effect of methanolic extract of roots of P. fascicularis on normal blood glucose levels and oral glucose tolerance test were studied in normoglycemic rats while antidiabetic effect was evaluated in alloxan-induced hyperglycemic rats. The extract (250, 500, and 1000 mg/kg) was administered orally for 7 days. Glibenclamide (3 mg/kg, orally for 7 days) was used as reference standard. Results: Administration of the methanolic extract of aerial roots of P. fascicularis caused significant dose-dependent reduction in serum glucose in both normoglycemic and hyperglycemic rats and also improved glucose tolerance test. The treatment also showed the enhanced beta cell function in histological studies. Conclusion: Therefore, this study suggests that methanolic extract of aerial roots of P. fascicularis exhibits antidiabetic activity possibly through increased secretion of insulin and the effect may be due to the presence of flavonoids and phenolic compounds.

Keywords: Alloxan monohydrate, diabetes, glibenclamide, Pandanus fascicularis Lamk


How to cite this article:
Kumari S, Wanjari M, Kumar P, Palani S. Antidiabetic activity of Pandanus fascicularis Lamk - aerial roots in alloxan-induced hyperglycemic rats. Int J Nutr Pharmacol Neurol Dis 2012;2:105-10

How to cite this URL:
Kumari S, Wanjari M, Kumar P, Palani S. Antidiabetic activity of Pandanus fascicularis Lamk - aerial roots in alloxan-induced hyperglycemic rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2019 Sep 21];2:105-10. Available from: http://www.ijnpnd.com/text.asp?2012/2/2/105/95943


   Introduction Top


Diabetes mellitus is a metabolic disorder of carbohydrate metabolism attributed to insulin deficiency or insulin resistance and long-term diabetes mellitus often leads to various serious complications such as retinopathy, neuropathy, nephropathy, and cardiomyopathy. [1],[2],[3] It is considered a major health problem in the world today. Despite appreciable progress made in the management of diabetes mellitus using conventional antidiabetic management strategies, the search for plant-based products continues.

Pandanus fascicularis Lamk. (Pandanaceae) is a densely branched shrub indigenous to seacoast of the Indian Peninsula, Andaman, and other humid parts of India. It is commonly known as Kewda in Hindi and the flowers are widely used for medicinal purpose. The flower oil is used in earache, headache, disorders of blood, and as antispasmodic and stimulant. Roots are widely used in treatment of osteoarthritis and skin diseases like leprosy. [4],[5] In the Ayurvedic system of medicine, the roots of P. fascicularis are indicated in Prameha and employed for their hypoglycemic action. [5],[6] Further, decoction of the roots and rhizomes of other species of Pandanus are traditionally used in treatment of diabetes. Patients and medical practioners believe that the roots and rhizomes of this plant are effective in the treatment of diabetes. [7],[8] The aqueous extract of Pandanus odorus, another species of Pandanus, also showed hypoglycemic activity in normal and streptozotocin diabetic rats. [9]

Despite the traditional use of roots of P. fascicularis as hypoglycemic agent, no attempts have been made to study the antidiabetic activity of aerial roots of P. fascicularis. Therefore, this study evaluated the antidiabetic activity of aerial roots of P. fascicularis in alloxan-induced hyperglycemic rats. Alloxan-induced hyperglycemia simulates to type-I diabetes (insulin-dependent diabetes mellitus). It has two distinct pathological effects: Selective inhibition of glucose-induced insulin secretion through specific inhibition of glucokinase, the glucose sensor of the beta cell, and generation of free radicals through redox cycling between alloxan and its reduction product dialuric acid, resulting in the selective necrosis of beta cells. These two effects can be attributed to selective cellular uptake and accumulation of alloxan by the beta cell. [10],[11]


   Materials and Methods Top


Plant material

The aerial roots of the plant P. fascicularis were collected during September 2006 from Narayan Bagh, Jhansi, Uttar Pradesh, India. The plant was identified by Dr. N. K. Pandey, Research Officer (Botany), Central Research Institute (Ayurveda), Aamkho, Gwalior and a voucher specimen CRI-Gwl/Pandey/147/184 has been deposited in the herbarium of the Institute.

Preparation of methanolic extract of aerial roots of Pandanus fascicularis

The shade-dried powdered roots (300 g) were defatted with petroleum ether (60-80°C) using a Soxhlet apparatus and subsequently extracted with 95% methanol. The total extract was filtered, concentrated, dried in Buchi type rotary vacuum evaporator, and the residue (yield 5%, w/w) was stored in a dessicator for use in subsequent experiments. For administration to the animals, the Preparation of methanolic extract of aerial roots of Pandanus fascicularis (MEPF) was suspended in distilled water with 4% gum acacia.

Preliminary phytochemical screening

MEPF was subjected to preliminary phytochemical screening. [12]

Drugs and chemicals

Alloxan monohydrate was procured from CDH Chemicals, Mumbai, India, while glibenclamide was gift sample from Sun Pharma Advance Research Centre, Vadodara, India. Glucose oxidase peroxides glucose estimation kit was procured from Span Diagnostic Ltd., Surat, India. All other reagents used in the experiments were of analytical grade and procured from Glaxo-Qualigens, Mumbai, India.

Experimental animals

Healthy adult Wistar albino rats (200-250 g) of either sex between 2-3 months of age were used for the investigations. Animals were bred and maintained at Central Animal Facility of the Institute under standard housing conditions of temperature 25 ± 2°C, relative humidity 55-65%, and light and dark cycles of 12 h, respectively. Animals were provided with standard pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. Principles of laboratory animal care guidelines were followed and prior permission was sought from the Institute Animal Ethics Committee for conducting the experiments (Regd no. 716/02/a/CPCSEA).

Acute toxicity study

Healthy Wistar rats of either sex, starved overnight were subjected to acute toxicity studies to determine the safe dose by acute toxic class method of oral toxicity as per OECD 423 guidelines. The rats were observed continuously for 2 h for behavioral, neurological, and autonomic profiles and, after a period of 72 h up to 14 days, for any lethality, moribund state or death. [13]

Induction of hyperglycemia

Hyperglycemia was induced by a single dose of alloxan monohydrate. It was prepared freshly in normal saline and administered to rats in the dose of 70 mg/kg, intravenously through tail vein. [14] Glucose solution (1%, w/v) 10 ml/kg was immediately administered orally to alloxan-treated rats in order to prevent transient hypoglycemia. In overnight fasted rats 48 h after the administration of alloxan, blood was collected from retro-orbital plexus [15] under light ether anesthesia and clear serum was obtained after centrifugation at 3000 rpm. Fasting serum glucose levels were estimated using a glucose oxidase peroxidase glucose estimation kit. [16] The rats exhibiting serum glucose more than 250 mg/dl were considered hyperglycemic and included in the study.

Assessment of serum glucose in normoglycemic rats

Rats were divided in five different groups (five rats/group). Rats were fasted overnight and optimum care was exercised to avoid coprophagia. Vehicle treated group received 4% gum acacia in distilled water while extract treated groups received MEPF (250, 500, and 1000 mg/kg). The reference standard-treated group received glibenclamide (3 mg/kg). [17] All the treatments were given orally for 7 days. Blood was withdrawn from the retro-orbital sinus at 0, 1, 2, and 3 h after administration of drugs on day 1 and later on day 3 and 7. The serum was separated and glucose was estimated as described earlier.

Oral glucose tolerance test

The oral glucose tolerance test was performed on overnight fasted (18 h) normal rats as per the procedures described previously. [18],[19] Rats were divided into five groups (five rats/group) and administered orally vehicle (10 ml/kg), MEPF (250, 500, and 1000 mg/kg), and glibenclamide (3 mg/kg) for 7 days, respectively. On day 7, glucose (4 g/kg) was fed orally, 3 h after the administration of extract/vehicle. Blood was withdrawn from the retro-orbital sinus at 0, 30, 60, and 120 min after the administration of glucose load and the serum was estimated for glucose levels as mentioned earlier.

Assessment of serum glucose in hyperglycemic rats

The rats were divided into different groups (five rats/group). They were fasted overnight and optimum care was exercised to avoid coprophagia. Nondiabetic control group rats received saline. All other alloxan diabetic rats received vehicle (4% gum acacia) of the extract or MEPF (250, 500, and 1000 mg/kg, orally) or glibenclamide (3 mg/kg, orally). The drugs were given every day for 7 days. Blood was withdrawn from the retro-orbital sinus at 0, 1, 2, and 3 h after administration of drugs on day 1 and later on day 3 and 7. The serum was separated and glucose was estimated as described earlier.

Histology of pancreas

Immediately after the withdrawal of blood, rats treated with normal saline (untreated), alloxan+vehicle, and MEPF were sacrificed by cervical dislocation at the end of experiment. Pancreas were removed, washed with cold saline and preserved in 10% formalin in buffered form and subsequently dehydrated in graded alcohol. After embedding in paraffin 4 μM sections were cut and mounted on aminosilane-coated glass slides. After drying overnight, paraffin sections were warmed for 1 h at 15°C. Sections were then deparaffinized in four changes xylene, 10 min each, followed by one change on 100% ethanol for 1 min. The slides were rehydrated by 1 min change, each of 100%, 95%, 75%, and 50% ethanol, and then held in distilled water. After staining with haematoxylin for 4 min, slides were rinsed in distilled water, decolorized with lithium carbonate solution, and rinsed with distilled water again. Following a 1 min wash in 70% ethanol, slides were stained with eosin for 1 min and then dehydrated. Coverslips were mounted with Premount. The tissues were examined by light microscopy using coded slides by investigators who were blind to the treatment group.

Statistical analysis

The data were analyzed with two-way ANOVA followed by Bonferroni multiple comparison post hoc test. A statistical difference of P<0.05 was considered significant in all cases.


   Results Top


Preliminary phytochemical screening

The preliminary phytochemical screening of MEPF revealed the presence of carbohydrates, sterols, flavonoids, phenolic compounds, and the absence of proteins, alkaloids, and glycosides.

Acute toxicity study

Acute toxicity studies revealed that MEPF was safe up to 2000 mg/kg of body weight (limit test) and approximate LD 50 is more than 2000 mg/kg. No lethality or any toxic reactions or moribund state was observed up to the end of the study period.

Effect of MEPF on serum glucose in normoglycemic rats

Two-way ANOVA showed significant effect of MEPF on serum glucose levels in normal rats [MEPF-time interaction F (20, 120) = 5.67, P<0.0001; MEPF effect F (4, 120) = 46.78, P<0.0001; and time effect F (5, 120) = 54.43, P<0.0001] [Table 1]. Post hoc test indicated that MEPF at 250, 500, and 1000 mg/kg exhibited significant reduction in the blood glucose in normal rats. The onset of hypoglycemic effect was observed on day 3 onwards while early onset (after 3 h) of hypoglycemia was observed with higher doses of MEPF. The highest dose of MEPF (1000 mg/kg) caused maximum 47.7% reduction from day 0 to 7. The reduction in the blood glucose was very much comparable with that of standard antidiabetic, glibenclamide that also showed hypoglycemic effect and caused 42.5% reduction on day 7 [Table 1].
Table 1: Effect of MEPF on normoglycemic rats

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Effect of MEPF on oral glucose tolerance test

Two-way ANOVA showed significant effect of MEPF on oral glucose tolerance test (OGTT) [MEPF-time interaction F (12, 80) = 6.14, P<0.0001; MEPF effect F (4, 80) = 34.52, P<0.0001; and time effect F (3, 80) = 145.5, P<0.0001] [Table 2]. Post hoc test indicated that glucose load has caused significant sudden increase in glucose at 30 min to 1 h after administration and MEPF (250, 500, and 1000 mg/kg) for 7 days showed a significant reduction in serum glucose levels up to 120 min on glucose administration in oral glucose tolerance test. The effect was comparable to that of standard antidiabetic (P>0.05), glibenclamide [Table 2]. The difference observed at 120 min in the mean blood glucose levels of MEPF and glibenclamide treated animals with respect to vehicle control indicates that reduction observed was dose dependent. The maximum difference observed was 62.2 mg/dl at MEPF (1000 mg/kg) while glibenclamide-treated animals exhibited the difference of 48.8 mg/dl [Table 2].
Table 2: Effect of MEPF on oral glucose tolerance test

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Effect of MEPF on alloxan-induced hyperglycemia

Two-way ANOVA showed significant effect of MEPF on alloxan-induced hyperglycemia [MEPF-time interaction F (25, 144) = 7.80, P<0.0001; MEPF effect F (5, 144) = 280.5, P<0.0001; and time effect F (5, 144) = 58.69, P<0.0001] [Table 3]. Post hoc test indicated that alloxan has caused significant increase in serum glucose compared to saline-treated animal which indicates dependable hyperglycemia. Treatment of hyperglycemic rats with MEPF (250, 500, and 1000 mg/kg) exhibited significant reduction in the blood glucose. The highest dose of MEPF (1000 mg/kg) caused maximum reduction in blood glucose of 65.3% from day 0 to 7. The glucose lowering effect of MEPF was comparable (P>0.05) to that of standard drug, glibenclamide that showed reduction of 63.6% from day 0 to 7 [Table 3].
Table 3: Effect of MEPF on alloxan-induced hyperglycemia

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Effect of MEPF on histology of pancreas

Photomicrographs of pancreas from untreated rats [Figure 1]a showed normal architecture with normal acini and normal population of the  Islets of Langerhans More Details. Pancreas from alloxan-treated rats exhibited degeneration and necrosis of pancreatic tissue and exhibited damage to islets of Langerhans and reduced dimension of islets [Figure 1]b. Pancreas from rats treated with MEPF (500 and 1000 mg/kg) exhibited restoration of normal cellular population and enlarged size of β-cells with hyperplasia compared to alloxan-treated pancreas [Figure 1]c and d.
Figure 1: Effect of MEPF on histology of pancreas. Rats were treated with alloxan to induce hyperglycemia and hyperglycemic rats were treated with vehicle or MEPF (250, 500, and 1000 mg/kg, orally) or glibenclamide (3 mg/kg, orally) for 7days. On day7, the pancreas were excised from the rats, fixed in formalin and processed for histopathology. Figure illustrates the photomicrographs of pancreas (haematoxylin and eosin staining) of untreated rats (a), alloxan treated rats (b), MEPF (500 mg/kg) treated rats (c), and MEPF (1000 mg/kg) treated rats (d). Microscope magnification (×100)

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   Discussion Top


The results of the investigations revealed that treatment with methanolic extract of aerial roots of P. fascicularis (MEPF) produced significant hypoglycemia in normoglycemic (euglycemic) rats. This suggests that MEPF has per se hypoglycemic effect and it is in comparison with the standard hypoglycemic agent, glibenclamide. In concordance with this, hypoglycemia was observed up to 120 min after the administration of the MEPF in glucose-loaded normal rats. This further indicates the efficacy of the MEPF to control elevated blood sugar levels. From these findings, it can be speculated that MEPF treatment may have secretogouge activity on pancreatic beta cells.

It was observed that single intravenous dose of alloxan exhibited significant hyperglycemia. Excessive hepatic glycogenolysis and gluconeogenesis associated with decreased utilization of glucose by tissues is the fundamental mechanism underlying hyperglycemia in the diabetic state. It is a well-known fact that alloxan destroys the beta cells of the pancreas and causes hyperglycemia in rats. [11] As the hyperglycemia induced by alloxan falls under category of mild diabetes and may reverse after a week, [20] the hypoglycemic effect of the MPEF in hyperglycemic rats was studied during 7 days treatment. The difference observed between the initial and final fasting serum glucose levels of MEPF treated hyperglycemic rats revealed hypoglycemic effect of MEPF throughout the period of study. The marked antidiabetic effect could be observed with prolong treatment for 7 days. The effect of MEPF was comparable to that of reference standard, glibenclamide.

The mechanism of the hypoglycemic effect of MEPF is not elucidated in the study. Some medicinal plants with antidiabetic properties are known to increase circulating insulin levels in euglycemic [21] and hyperglycemic [22],[23] rats. Therefore, it is possible that the extract might have stimulated residual pancreatic mechanism, probably increasing peripheral utilization of glucose. [24]

It is reported that aqueous extract of root of P. odorus, another species of Pandanus, exhibited hypoglycemic activity in normal and streptozotocin diabetic rats. The hypoglycemic activity of P. odorus has been attributed to a phenolic compound, 4-Hydroxybenzoic acid by increasing peripheral glucose consumption. [9] As P. fascicularis is closely related to P. odorus, 4-hydroxybenzoic acid-like compounds may be present in the aerial roots of P. fascicularis and showing the hypoglycemic activity. The phytochemical data on MEPF revealed the presence of phenolic compounds and supports such possibility. The phenolic compounds are known to help in the regulation of plasma glucose concentration, [25] and therefore it is suggested that MEPF exhibited hypoglycemic effect through increased insulin secretion due to the presence of phenolic compounds. A recent study has also reported the antidiabetic activity of P. fascicularis roots and strengthens the present findings on aerial roots of the plant. [26]

Previous phytochemical investigations on root extract of P. fascicularis revealed the presence of strong antioxidant compounds such as pinoresinol and 3,4-bis(4-hydroxy-3-methoxybenzyl) tetrahydrofuran etc. [27] The phytochemical studies carried out on the extract revealed the presence of polyphenols (flavonoids), and the flavonoids are known to regenerate the pancreatic beta cells in alloxan diabetic animals. [28] Thus, the significant antidiabetic activity of MEPF in our study may be attributed to the presence of flavonoids and other phenolic compounds in the extract. The further phytochemical investigations with regard to the presence of 4-hydroxybenzoic acid and flavonoids in the aerial roots of P. fascicularis need to be carried out to comment on the exact mechanism and effect of the drug.

 
   References Top

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22.Syiem D, Syngai G, Khup PZ, Khongwir BS, Kharbuli B, Kayang H. Hypoglycemic effects of Potentilla fulgens L in normal and alloxan-induced diabetic mice. J Ethnopharmacol 2002;83:55-61.  Back to cited text no. 22
    
23.Singab AN, El-Beshbishy HA, Yonekawa M, Nomura T, Fukai T. Hypoglycemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats. J Ethnopharmacol 2005;100:333-8.  Back to cited text no. 23
    
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25.Olmedilla B, Granado F, Gil-Martinez E, Blanco I, Rojas-Hidalgo E. Reference values for retinol, tocopherol and main carotenoids in serum of control and insulin-dependent diabetic Spanish subjects. Clin Chem 1997;43:1066-71.  Back to cited text no. 25
    
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28.Chakkravarthy BK, Gupta S, Gambir SS, Gode KD. Pancreatic beta cell regeneration - a novel antidiabetic mechanism of Pterocarpus marsupium roxb. Indian J Pharmacol 1980;12:123-7.  Back to cited text no. 28
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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