Hypoglycemic and Antihyperglycemic Activity of Nymphaea stellata Flowers in Normal and Alloxan Diabetic Rats

Abstract

Flowers of Nymphaea stellata Willd. (Nymphaeaceae) are used in the Indian traditional system of medicine to treat diabetes mellitus but have not been scientifically investigated. Hence, the current study was aimed to evaluate the hypoglycemic and antihyperglycemic effect on normal and alloxan-induced diabetic rats. Hydroethanol extract (HEE) of Nymphaea stellata at an oral dose of 200, 300, and 400 mg/kg was given, and blood glucose level (BGL) on normoglycemic and, diabetic rats and oral glucose tolerance test (OGTT) were evaluated. HEE of the flowers did not show significant reduction on BGL in normoglycemic rats but significantly (p < 0.001) reduced the BGL in hyperglycemic animals by improving OGTT. These results clearly show that flowers of N. stellata do not have hypoglycemic activity in normoglycemic rats but have an antihyperglycemic activity in alloxan-induced diabetic rats.

Keywords:

• Alloxan
• BGL
• diabetes mellitus
• hypoglycemia
• Nymphaea stellata
• OGTT

Introduction

Nymphaea stellata Willd. (Nymphaeaceae) is a perennial aquatic herb generally found in tanks and ponds throughout the warmer parts of India and Africa. In Sanskrit it is called “kumuda” and in southern India it is well-known as “alli” or “nilotpalam” (Anon., 2001). Roots, flowers, seeds, rhizomes, stem, and leaves are used in folk medicine. The powder of rootstock is given to treat dyspepsia, diarrhea, and piles. An infusion of the rhizomes and stem is considered to be an emollient, diuretic, and used for treatment of blennorrhagia and diseases of the urinary tract. The flower has an acrid, bitter-sweet taste, removes impurities from blood, cools, and alleviates cough, is used for biliousness, as an aphrodisiac, for vomiting, giddiness, worm infestation, and burning of the skin. The decoction of the flower is used in palpitation of the heart and as a narcotic; syrup of the flower is used in high fever, apoplexy, inflammatory diseases of the brain, and also in dysuria. The filaments of the plants are used as an astringent and a cooling agent in burning sensation of the body, bleeding piles, and menorrhagia. Leaves are applied topically in erysipelas, whereas the macerated leaves are used as a lotion in eruptive fevers. The seeds are said to be stomachic and restorative (Satyavati et al., 1987; Kirtikar & Basu, 2001).

Protein, pentosan, and tannins were reported to present in seeds of N. stellata (Gujral et al., 1955). N. stellata seeds are prescribed as diet in diabetes mellitus in the Aurvedic system of medicine (Achariya et al., 1996). Phenolic constituents were found to be present in flowers of N. stellata (Kizu & Tamimori, 2003). The alcohol extract of defatted fruits of N. stellata produced mild sedation and ataxia, potentiated hexobarbitone-induced hypnosis in mice, and also produced a sharp and transient hypotension blocked by pretreatment with atropine. In large doses, when given after atropinization, it produced a rise in blood pressure and also a stimulant effect on guinea pig ileum indicating the presence of some unstable cholinergic principle (Satyavati et al., 1987). The extract had a significant analgesic activity as revealed by aconitine-induced writhing in mice and antipyretic activity against carrageenin-induced rat paw edema. The anti-inflammatory activity compared well with that of hydrocortisone. The approximate LD₅₀ of the extract was 1250 ± 30 mg/kg i.p. in albino mice (Singh et al., 1977). The petroleum ether extracts of N. stellata seeds tested against carbon tetrachloride (CCl₄) induced hepatotoxicity in rats and mice at a dose of 300 mg/kg i.p. The extract markedly reduced the prolongation of sleeping time and significantly prevented the CCl₄-induced increase in weight and volume of the liver, and mortality. The extract also was found to prevent necrosis of the liver and promoted to some extent liver generation (Singh et al., 1978). The LD₅₀ of the 50% ethanol extract of N. stellata was found to be 681 mg/kg in albino mice. It was found to be inactive as an antibacterial, antifungal, antiprotozoal, antiviral, diuretic, and with no effect on cardio vascular system and central nervous system (Aswal et al., 1984). Recently, N. stellata flowers have also been reported to have hepatoprotective activity against CCl₄-induced hepatic damage (Bhandarkar & Khan, 2004).

The decoction of flowers of N. stellata was used to treat diabetes mellitus in the Siddha system of medicine, which is practiced locally and is popular in peninsular India. One of the reasons might be that the original texts of the Siddha system of medicine are written in the Tamil language, translations of which are not readily available in other parts of the country. Thus far, no systematic study has been reported on the hypoglycemic and antihyperglycemic effect of this plant. Hence, the current study was aimed to determine the hypoglycemic and antihyperglycemic effects of hydroalcohol extract of Nymphaea stellata flowers in normoglycemic and alloxan-induced diabetic rats.

Materials and Methods

Plant material

Fresh, white-colored flowers of Nymphaea stellata, abundant during the rainy season, were collected in the year 2004 from Vadakara district, Kerala, India. They were carefully identified and authenticated by Dr. P. Daniel, Professor of Botany, Botanical Survey of India, Tamil Nadu Agricultural University (Coimbatore, India). A voucher specimen (BSI-4523) of this plant was deposited in the herbarium of the university.

Preparation of the plant extract

About 3 kg of flowers of N. stellata were shade-dried at room temperature and pulverized using a mixer grinder. About 1 kg of coarse powder was chopped in (1:1 v/v) ethanol and cold macerated for 3 days. During the maceration period, occasional stirring was done. After 3 days, the suspension was filtered through a fine muslin cloth. The residue was removed and the extract was concentrated on rotavapor under reduced pressure and then lyophilized. Finally, a dark brown–colored crystal was obtained (yield: 6.8% w/w, dry weight basis).

Animals

Male rats of the Wistar strain, weighing about 150–200 g, were obtained from the small animal breeding center of Kerala Agricultural University (Mannuthy, Trichur, Kerala, India) for study. Animals were kept in the animal house at room temperature of 25–30°C, 45–55% relative humidity, and 12-h light-dark cycle. The animals were fed with rat pellets feed (Hindustan Lever Limited, Bangalore, India) and filtered water ad libitum. Animal studies have been strictly performed as per the Institutional Animal Ethical Committee (IAEC) constituted under the guidelines of Committee for the Purpose of Control and Supervision on Experimental Animal (CPCSEA), Ministry of Environment, Government of India, New Delhi.

Study of N. stellata HEE in normoglycemic rats

Initial screening of the flower extract for the hypoglycemic activity was done in normal healthy rats with different dose levels. Overnight fasted animals were randomly divided in to five groups.

• Group I received distilled water only (control).

• Group II, group III, and group IV received hydroethanol/extract (HEE) dissolved in distilled water orally at a dose of 200, 300, and 400 mg/kg, respectively.

• Group V received glibenclamide (Sun Pharma, Ahmadabad, India) orally at 2 mg/kg.

Doses selected were comparable with what has generally been used for investigating pharmacologic activities of herbal extracts (Grover et al., 2000; Saravanan et al., 2002). For blood glucose determination, the blood was obtained by snipping the tail with a sharp razor. Blood glucose level was determined before and after 1, 2, and 3 h of flower extract administration. Blood glucose level (BGL) was determined by using a one-touch glucometer (Accu-chek sensor; Roche Diagnostics, Germany).

Study of N. stellata HEE on oral glucose tolerance

Oral glucose tolerance test (OGTT) was investigated in normoglycemic fasted rats. The animals were fasted for 16 h and randomly assigned into five groups with six in each group.

• Group I received glucose 3 g/kg only (control).

• Group II, group III, and group IV received glucose 3 g/kg + HEE dissolved in distilled water orally at a dose of 200, 300, and 400 mg/kg, respectively.

• Group V received glucose 3 g/kg + glibenclamide orally at 2 mg/kg.

Blood samples from the tail were collected immediately prior to commencement of treatment and at 30, 60, 90, and 180 min after the glucose loading, and BGL was measured immediately.

Study of N. stellata HEE in diabetic rats

Diabetes was induced by single intraperitoneal injection of freshly prepared 120 mg/kg of alloxan monohydrate (Sigma-Aldrich Co., St. Louis, MO, USA) dissolved in sterile 0.9% saline (El-Demerdash, 2005).

Development of hyperglycemia was confirmed after 72 h of alloxan injection by determining glucose levels. The animals having BGL > 300 mg/dL showing polydipsia and polyuria were selected for the study. These rats were assigned randomly into five groups (n = 6). Different doses such as 200, 300, 400 mg/kg of HEE and reference drug glibenclamide (2 mg/kg) were assessed to find out the effective dose.

• Group I received distilled water only (diabetic control).

• Group II, group III, and group IV: Diabetic animals received HEE dissolved in distilled water orally at a dose of 200, 300, and 400 mg/kg, respectively.

• Group V: Diabetic rats received glibenclamide orally at 2 mg/kg.

Blood samples were collected from the tail vein at 1, 2, and 4 h after treatment with HEE of N. stellata.

Statistical analysis

All values are expressed as the mean of six experiments ± SEM. Statistical significance was estimated by one-way analysis of variance (ANOVA) followed by Bonferroni's post-test. p < 0.05 implies significance.

Results

Effect of N. stellata HEE in normoglycemic rats

Results of the effect of HEE of N. stellata flower and reference drug glibenclamide on BGL of normal healthy rats are presented in Table 1. The flower extract in all three doses of 200, 300, and 400 mg/kg produced no significant hypoglycemic effect in normal rats, whereas glibenclamide, the reference drug, showed a marked reduction on BGL in normoglycemic rats.

Table 1  Effect of N. stellata HEE in normoglycemic rats.

	BGL (mg/dL)
Treatment groups	0 h	1 h	2 h	3 h
I. Control	82.2 ± 3.81	82.9 ± 3.1	83.4 ± 2.9	81.9 ± 3.5
II. Diabetic group treated with HEE 200 mg/kg	79.8 ± 3.9	85.23 ± 6.15	87.28 ± 6.15	82.83 ± 4.71
III. Diabetic group treated with HEE 300 mg/kg	81.43 ± 4.6	78.53 ± 4.8	79.58 ± 4.4	83.66 ± 4.6
IV. Diabetic group treated with HEE 400 mg/kg	85.58 ± 2.81	84.39 ± 3.98	86.81 ± 4.32	84.2 ± 4.8
V. Diabetic group treated with glibenclamide 2 mg/kg	99.46 ± 6.8	95.57 ± 6.2	87.28 ± 4.9***	82.1 ± 5.87***

Values are mean ± S. E. M (n = 6).*** P < 0.001 compared with initial value.

Effect of N. stellata HEE on oral glucose tolerance

The blood glucose concentration of control, and drug-treated animals (different doses of N. stellata flower extract) and the effect of reference drug glibenclamide on glucose tolerance are represented in Table 2. Administration of glucose (3 g/kg body weight) to normal rats showed significant (p < 0.001) increase in fasting BGL after 30 min of glucose challenge. The rats treated with HEE of N. stellata prevented the rise in BGL significantly (p < 0.001) after 60 min of glucose load. Glibenclamide also showed significant reduction in BGL from 60 min of glucose administration until 180 min.

Table 2  Effect of N. stellata HEE on oral glucose tolerance.

	BGL (mg/Dl)
Treatment Groups	0 h	30 min	60 min	90 min	180 min
I. Control	82.1 ± 3.2	138.8 ± 3.31	129 ± 4.9	118 ± 3.12	98.6 ± 3.7
II. Diabetic group treated with	83.2 ± 2.7	129.78 ± .64***	148.62 ± .82***	138.89 ± .97***	112.5 ± 3.28***
HEE 200 mg/kg
III. Diabetic group treated with	91.25 ± 4.36	158.67 ± 4.8***	167.28 ± .75***	112.5 ± 2.7***	91.58 ± 4.1***
HEE 300 mg/kg
IV. Diabetic group treated with	89.53 ± 2.85	139.25 ± 2.8***	143.42 ± .98***	136.7 ± 4.82***	109.87 ± 3.9***
HEE 400 mg/kg
V. Diabetic group treated with	92.42 ± 5.25	153.84 ± 3.2***	128.58 ± .87***	105.8 ± 7.2***	85.45 ± 5.2***
glibenclamide 2 mg/kg

Values are mean ± SEM (n = 6).*** P < 0.001 compared with initial value.

Effect of N. stellata HEE in diabetic rats

Table 3 summarize the antihyperglycemic effect of different doses of HEE on diabetic rats at different time intervals. Administration of alloxan significantly increased (p < 0.001) BGL in rats. Dose-dependent antihyperglycemic activity was assessed by comparing the BGL (with zero time) at all time points. At a dose of 200 mg/kg, HEE of N. stellata produced a significant (p < 0.001) reduction in BGL at 1 h (5.37%), 2 h (14%), and 4 h (33.27%) after administration.

Table 3  Effect of N. stellata HEE in diabetic rats.

	BGL (mg/Dl)
Treatment Groups	0 h	30 min	60 min	120 min	240 min
I. Control	321.68 ± 10.24	343.27 ± 8.27	337.52 ± 6.8	307.69 ± 8.45	312.69 ± 7.20
II. Diabetic group treated with	318 ± 5.93	327.48 ± 10.98***	301.48 ± 6.72***	273.68 ± 7.48***	212.6 ± 10.3***
HEE 200 mg/kg
III. Diabetic group treated with	327.57 ± 5.94	338.94 ± 9.48***	277.84 ± 5.26***	210.82 ± 7.98***	178.68 ± 11.3***
HEE 300 mg/kg
IV. Diabetic group treated with	308.59 ± 3.92	322.57 ± 6.48***	289.43 ± 4.95***	258.78 ± 8.23***	237.73 ± 9.82***
HEE 400 mg/kg
V. Diabetic group treated with	317.82 ± 4.58	328.72 ± 5.47***	294.99 ± 10.82***	278.39 ± 8.9***	259.90 ± 9.52***
glibenclamide 2 mg/kg

Values are mean ± SEM (n = 6).*** P < 0.001 compared with initial value.

However, a more pronounced activity was shown by HEE at a dose of 300 mg/kg. At this dose, a significant (p < 0.001) reduction in BGL at 1 h (15.18%), 2 h (35%), and 4 h (45.45%) was observed. At 400 mg/kg dose, the fall in BGL was 22.97% after 4 h. Glibenclamide showed only a weak reduction (18.22%) at 4 h. Therefore, 300 mg/kg, of HEE of N. stellata flower is the most effective dose as it is evaluated from different doses, which possess a significant antihyperglycemic effect.

Discussion

Diabetes mellitus characterized by hyperglycemia usually produces many complications such as hyperlipidemia, hyperinsulinemia, hypertension, obesity, atherosclerosis, and even cardiovascular disease (Defronzo et al., 1992). Hyperglycemia can lead to the glycation of tissue proteins (Luo et al., 2004). The existence of diabetes was mentioned in Rig Veda, a type of ancient book written in regional languages in India that deals with diseases and their treatment (2500 to 1800 BC). Charaka Samhita and Sushruta Samhita gave an extensive description on the treatment of diabetes with medicinal herbs (Ali Hussain, 2002), which is regarded as a noncurable but controllable disease (Schoenfelder et al., 2006). Several authors reviewed the potential role of medicinal plants as hypoglycemic agents, which is supported by various ethnobotanical surveys (Roy & Agrawal, 2001; Li et al., 2004; Afifi et al., 2005). Such study might provide a natural key to unlock a diabetologist's pharmacy for the future.

The current investigation evaluated the hypoglycemic and antihyperglycemic effect of the hydroethanol extract of Nymphaea stellata flowers in normoglycemic and alloxan-induced diabetic rats. HEE of N. stellata flowers has no hypoglycemic effect in normoglycemic animals, but was found to possess statistically significant antihyperglycemic activity by improvement of OGTT. The flower extract causes significant reduction in BGL of diabetic rats. Alloxan induces chemical diabetes through damage of insulin-secreting cells (Rerup, 1970) and also causes permanent destruction of β -cells (Drews et al., 2000). It is conceivable that the significant antihyperglycemic activity of N. stellata flowers may be due to an extra-pancreatic mechanism, such as enhancement of the peripheral utilization of glucose and/or potentiation of the biological effect of insulin. An emphasis is placed on glucose homeostasis as severe hypoglycemia can result in a life-threatening situation (Murthy et al., 2004). Therefore, no hypoglycemic effect with the flower extract of N. stellata in normoglycemic animals compared with hyperglycemic animals is a desirable feature. Our report is in context with earlier studies of Tremamicrantha (Schoenfelder et al., 2006), Butea monosperma (Somania et al., 2006), and Urtica dioica (Bnouham et al., 2003). Different does of HEE (200, 300, and 400 mg/kg) were assessed to find the effective optimum dose. The dose of 300 mg/kg gave significant BGL reduction (45%) at 4 h after administration of the flower extract. This effect was found to be greater than that of the reference drug, glibenclamide. The effect of HEE of N. stellata flowers was dose-dependent up to 300 mg/kg and showed decreased response at the higher dose of 400 mg/kg. This phenomenon is common with indigenous plants and has been observed with Cinnamomum tamala Nees and Eberm. (Sharma et al., 1996), Aegle marmelose Linn. (Rao et al., 1995), and Murraya koenigii (L.) Spreng (Kesari et al., 2005).

In conclusion, our results have shown that flowers of N. stellata HEE possess promising value of blood glucose lowering effect in alloxan-induced hyperglycemic rats. Studies have to be done with the effective dose of 300 mg/kg to evaluate antihyperlipidemic, carbohydrate metabolism enzymes, and enzymic and nonenzymic antioxidants to confirm its usefulness as an antidiabetic drug. The active component of the flowers should be isolated for further comprehensive pharmacologic investigations to elucidate the mechanism of action of Nymphaea stellata.

Acknowledgments

We are thankful to Mr. P.K.S, K.K.K, Mrs. Meenatchi, and Mrs. Rajeshwari for their great help in ethnobotanical and ethnopharmacologic aspects. We are also thankful to R. Rajkumar and R. Naveenkumar for their help in preparing the manuscript.