Preventive effect of Sphaeranthus indicus during progression of glucocorticoid-induced insulin resistance in mice

Abstract

Context: The whole plant of Sphaeranthus indicus Linn. (Asteraceae) is traditionally used in the treatment of diabetes mellitus.

Objective: The present study investigated the effect of the methanol extract of Sphaeranthus indicus whole plant in dexamethasone-induced insulin resistance in mice.

Materials and methods: The mice were treated with dexamethasone for 22 days and effects on plasma glucose level, serum triglyceride level, glucose uptake, levels of hepatic enzymes like glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), lipid peroxidase (LPO), and body weight was observed.

Results: The Sphaeranthus indicus extract (SI) showed significant decrease in plasma glucose and serum triglyceride levels (p <0.01) at doses, of 400 and 800 mg/kg, p.o., and stimulated insulin assisted and non-insulin assisted glucose uptake in skeletal muscle. The levels of antioxidant enzymes GSH, SOD, and CAT were significantly increased (p <0.01) and there was a significant decrease in level of LPO (p <0.01). SI significantly restored (p <0.01) dexamethasone induced body weight loss.

Discussion and conclusion: Sphaeranthus indicus may prove to be effective in the treatment of type II diabetes mellitus owing to its ability to decrease insulin resistance.

Keywords::

• Antioxidant
• diabetes
• dexamethasone
• glucose uptake
• hemidiaphragms
• insulin resistance
• mice
• Sphaeranthus indicus

Introduction

Insulin resistance is a condition where normal or elevated insulin level produces an attenuated biological response (Wilcox, 2005). In subjects with type 2 diabetes, insulin resistance is the central pathophysiological event (Schinner et al., 2005). Currently, there is an epidemic expansion of type 2 diabetes, i.e., non-insulin dependent diabetes mellitus (NIDDM), and clinical implication of insulin resistance provides the rationale and means for developing an anti-insulin resistance approach to the treatment of type 2 diabetes mellitus.

Sphaeranthus indicus Linn. (Asteriaceae) common name: Sphaeranthus is distributed throughout India and commonly known as gorakhmundi. In folk medicine, all parts of the plant have medicinal uses. Gorakhmundi is traditionally used in the treatment of diabetes, jaundice, chronic bronchitis, asthma, leukoderma, tuberculosis, spleen diseases, and piles (Gogate, 2000; Warrier et al., 2004; Kirtikar & Basu, 1998). Recently, many medicinal properties have been reported for this plant including antidiabetic (Prabhu et al., 2008), antioxidant (Shirwaikar et al., 2006), hepatoprotective (Tiwari & Khosa, 2008), wound healing (Sadaf et al., 2006), immunomodulatory (Bafna & Mishra, 2004), and anxiolytic (Ambavade et al., 2006). A large number of constituents have been isolated from extracts of the whole herb, flowers, and leaves of this plant. The essential oil, obtained by steam distillation of the whole herb, contains ocimene, α-terpinene, methyl-chavicol, α-citral, geraniol, α-ionone, δ-cadinene, p-methoxycinnamaldehyde (Baslas, 1959), and an alkaloid, sphaeranthine (Basu & Lamsal, 1946). Alcoholic extract of powdered capitulum contains β-sitosterol, β-d-glucoside of β-sitosterol, stigmasterol (Gupta et al., 1967), sesquiterpenoids (Gogte et al., 1986), sesquiterpene lactone (Rahman et al., 1989), and sesquiterpene glycoside (Shekhani et al., 1990). The alcohol extract of the leaves contains isoflavone glycoside (Yadav & Kumar, 1998); the alcohol extract of aerial parts contains flavonoid C-glycoside (Mishra et al., 2007).

The present study is an effort to validate scientifically the preventive effect of Sphaeranthus indicus during progression of glucocorticoid-induced insulin resistance in mice.

Materials and methods

Plant material and preparation of extract

Whole plant of Sphaeranthus indicus was collected in the month of June 2007 from Pune city, Maharashtra, India. A. M. Mujumdar, Head of the Plant Sciences Division, Agharkar Research Institute, Pune, authenticated and catalogued the specimen (voucher specimen no. 08-52). The herb was dried in shade, powdered and passed through 40-mesh sieve. Powdered material (1 kg) was macerated with AR grade 95% methanol (Merck, Mumbai, India) for 72 h with occasional shaking. The extract was filtered and the solvent was evaporated under vacuum. The yield of methanol extract of Sphaeranthus indicus whole plant (SI) was 5.7% w/w.

Animals

Swiss Albino mice (25–30 g) of either sex were procured from the Serum Institute of India Ltd., Pune, India. The animals were housed for 2 weeks prior to the experiment for acclimatization in the animal house of Padm. Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune, India. The animals were kept under controlled conditions of temperature 26° ± 2°C, relative humidity 44–56%, and photo-schedule (12 h light/dark). The animals were provided with standard diet (Amrut Feeds, Mumbai, India) and water ad libitum. The food was withdrawn 18 h before the start of the experiment. The Institutional Animal Ethics Committee approved the experimental protocol (198/99/CPCSEA). All the pharmacological work was carried out as per guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).

Experimental design

Acute toxicity study

Acute toxicity of methanol extract of Sphaeranthus indicus was determined using female Swiss albino mice (25–30 g) according to the procedure of the Organization for Economic Co-operation and Development (OECD) guideline no. 425 (OECD, 2001). Initially, the limit test at 2000 mg/kg was performed, in which all the five animals survived, hence the limit test was terminated and the main test was performed. In the main test, animals were dosed in sequence. The animals were observed for mortality for 48 h, and SI was found to be safe up to a dose of 4000 mg/kg bw.

Dexamethasone-induced insulin resistance in mice

Seventy-two mice were weighed before treatment. Group I (Normal) received 1% gum acacia (1 mL/kg p.o.), and 36 mice were rendered hyperglycemic by daily administration of dexamethasone [1 mg/kg, intra muscular (i.m.)] for 7 consecutive days and then divided into six groups of six each. Group II (D-control) continued to receive only dexamethasone and 1% gum acacia (1 mL/kg, p.o.). Group III (D + keto) received ketoconazole (24 mg/ kg, p.o.) as an antiglucocorticoid standard along with dexamethasone treatment. Group IV (D + pio) received pioglitazone (2 mg/kg, p.o.) as an antidiabetic standard along with dexamethasone treatment. Groups V, VI, VII were treated with dexamethasone along with three different doses of SI: 200, 400, 800 mg/kg, p.o., respectively. Simultaneously five other groups (Groups VIII, IX, X, XI, and XII), each with six normoglycemic animals, were administered equivalent amounts of ketoconazole and pioglitazone and three different doses of SI: 200, 400, and 800 mg/kg, p.o., respectively (Gholap & Kar, 2005).

Biochemical estimations

On day 23, after overnight fasting, all the animals were weighed and later sacrificed by cervical dislocation. Blood samples were collected and used for the estimation of glucose and triglyceride. Biochemical estimation of plasma glucose and serum triglyceride was done by GOD/POD and GPO/PAD methods, respectively, using standard diagnostic kits (Shalam et al., 2006).

Hepatic GSH, SOD, CAT, and LPO estimations

Liver samples were dissected out and washed immediately with ice-cold saline to remove as much blood as possible. Liver homogenates (5% w/v) were prepared in cold 50 mM Tris buffer (pH 7.4) using a Remi homogenizer. The unbroken cells and cell debris was removed by centrifugation at 5000 rpm for 10 min using a Remi refrigerated centrifuge. The supernatant was used for the estimation of GSH (Ellaman, 1959), LPO (Slater & Sawyer, 1971), SOD (Mishra & Fridovich, 1972) and CAT (Aebi, 1974; Colowick et al., 1984) levels.

Glucose uptake estimations

Glucose uptake in mice hemidiaphragms was estimated by the method described by Ghosh et al. (2004) with some modification. Fourteen sets containing graduated test tubes (n = 6) each, were taken, seven sets were used for non-insulin assisted glucose uptake and remaining seven sets were used for insulin assisted glucose uptake study. The diaphragms were taken out quickly avoiding trauma, and divided into two halves. The hemidiaphragms were rinsed in cold Tyrode’s solution (without glucose) to remove any blood clots. In non-insulin assisted glucose uptake study, one hemidiaphragm of each animal from group I to VII was exposed to 2 mL Tyrode’s solution with glucose (2000 mg/dL) in respective graduated test tubes. In insulin assisted glucose uptake study, the remaining hemidiaphragms of each animal from group I to VII was exposed to 2 mL Tyrode solution with glucose (2000 mg/dL) + insulin (0.25 IU/ mL) in respective graduated test tubes. All the graduated test tubes were incubated for 30 min at 37°C in an atmosphere of 95% O₂–5% CO₂ with shaking at 140 cycles per minute. Following incubation, the hemidiaphragms were taken out and weighed. The glucose content of the incubated medium was measured by GOD/POD, enzymatic method. Glucose uptake was calculated as the difference between initial and final glucose content in the incubation medium.

Statistical analysis

The results were expressed as mean ± SEM and statistically analyzed by one way ANOVA followed by Dunnett’s test, with level of significance set at p <0.05.

Results

Effects of SI on plasma glucose, serum triglyceride levels and body weight change

In D-control group, there was significant increase in plasma glucose level (p <0.01) and serum triglyceride level (p <0.01) when compared with normal. All mice treated with dexamethasone along with SI showed significant decrease (p <0.01) in the levels of plasma glucose and serum triglyceride when compared with D-control. The mice treated with dexamethasone along with ketoconazole or pioglitazone showed a significant decrease in plasma glucose level (p <0.01) and serum triglyceride level (p <0.01) when compared with D-control. In normoglycemic groups, ketoconazole showed significant decrease in plasma glucose level (p <0.01), while SI at the dose of 800 mg/kg, p.o., showed marginal hypoglycemia (p <0.05) when compared with normal. Significant reduction in body weight (p <0.01) was observed in D-control group when compared with normal. SI, ketoconazole and pioglitazone treatment significantly restored the dexamethasone induced decrease in body weight (p <0.01) when compared to D-control (Table 1).

Table 1.  Effect of SI on plasma glucose, serum triglyceride levels and body weight change.

Groups	Plasma glucose (mg/dL)	Serum triglyceride (mg/dL)	Body weight change (g)
Normal	54.42 ± 1.15	83.68 ± 1.17	+0.65 ± 0.06
D-control	76.4 ± 0.84^a**	144.38 ± 2.42^a**	-2.09 ± 0.19^a**
D + keto	66.87 ± 1.3^b**	92.65 ± 1.96^b**	+0.3 ± 0.02^b**
D + pio	55.29 ± 0.94^b**	85.48 ± 1.91^b**	+0.54 ± 0.08^b**
D + SI-200	71.46 ± 1.9^b*	140.87 ± 1.78	-1.96 ± 0.09
D + SI-400	65.08 ± 0.79^b**	136.76 ± 1.9^b*	-0.16 ± 0.03^b**
D + SI-800	55.88 ± 0.96^b**	96.98 ± 1.85^b**	+0.55 ± 0.07^b**
Keto	48.2 ± 0.91^a**	85.75 ± 1.92	+0.52 ± 0.05
Pio	53.51 ± 0.51	81.87 ± 1.83	+0.44 ± 0.04
SI-200	54.55 ± 1.03	84.38 ± 0.82	+0.62 ± 0.07
SI-400	52.63 ± 1.67	85.08 ± 0.79	+0.69 ± 0.09
SI-800	48.93 ± 0.77^a*	82.73 ± 1.3	+0.76 ± 0.09

Values are expressed as mean ± SEM, n = 6, analyzed by one way ANOVA followed by Dunnett’s test.

^a*p<0.05, ^a**p<0.01 when compared with normal group; ^b*p<0.05, ^b**p<0.01 when compared D-control group; (+) and (–) indicates increase and decrease in body weight.

D, dexamethasone (1 mg/kg i.m.); keto, ketoconazole (24 mg/kg, p.o.); pio, pioglitazone (2 mg/kg, p.o.); SI, methanol extract of Sphaeranthus indicus (200, 400, 800 mg/kg, p.o.).

Effects of SI on hepatic GSH, SOD, CAT, and LPO levels

In D-control group, there was increase in the level of LPO (p <0.01) when compared with normal; treatment with SI significantly prevented this rise (p <0.01). D-Control group showed significant decrease (p <0.01) in GSH, SOD and CAT levels when compared with normal, whereas there was significant restoration (p <0.01) in the levels of these enzymes in the SI treated groups when compared with D-control group (Table 2).

Table 2.  Effect of SI on hepatic GSH, SOD, CAT, and LPO levels.

Groups	GSH (µg GSH/g of tissue)	SOD (units/g of tissue)	CAT (µM H2O2/g of tissue)	LPO (nM MDA/g of tissue)
Normal	24.42 ± 2.43	73.65 ± 2.95	6.83 ± 0.03	10.58 ± 0.94
D-control	14.77 ± 1.67^a**	22.98 ± 1.02^a**	3.07 ± 0.02^a**	20.94 ± 1.27^a**
D+keto	16.81 ± 1.87^b*	34.85 ± 1.91^b**	4.21 ± 0.04	18.75 ± 0.99^b**
D+pio	18.58 ± 1.76^b**	72.55 ± 3.62^b**	5.83 ± 0.03^b**	10.29 ± 0.87^b**
D+SI-200	16.27 ± 0.32	47.14 ± 0.96^b**	3.85 ± 0.35	16.66 ± 0.49^b**
D+SI-400	17.84 ± 0.66^b*	54.72 ± 1.21^b**	4.6 ± 0.25^b*	14.64 ± 0.34^b**
D+SI-800	20.47 ± 0.57^b**	75.72 ± 1.62^b**	6.14 ± 0.09^b**	11.38 ± 0.64^b**
Keto	23.64 ± 2.11	73.6 ± 4.32	6.82 ± 0.04	12.02 ± 1.09^a*
Pio	25.73 ± 2.31	72.37 ± 3.21	6.81 ± 0.04	10.95 ± 1.1
SI-200	23.77 ± 0.39	71.62 ± 0.6	6.51 ± 0.3	12.06 ± 0.27 ^a*
SI-400	26.33 ± 0.64	73.19 ± 0.37	6.98 ± 0.09	11.69 ± 0.61
SI-800	26.84 ± 1.39	76.2 ± 0.89	7.51 ± 0.35	10.19 ± 0.24

Values are expressed as mean ± SEM, n = 6, analyzed by one way ANOVA followed by Dunnett’s test.

^a*p<0.05, ^a**p<0.01 when compared with normal group; ^b*p<0.05, ^b**p<0.01 when compared with D-control group.

D, dexamethasone (1 mg/kg, i.m.); keto, ketoconazole (24 mg/kg, p.o.); pio, pioglitazone (2 mg/kg, p.o.); SI, methanol extract of Sphaeranthus indicus (200, 400, 800 mg/kg, p.o.); GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; LPO, lipid peroxidase.

Effect of SI on glucose uptake in insulin resistant isolated hemidiaphragms of mice

In insulin assisted and non-insulin assisted glucose uptake, hemidiaphragms of the mice treated with dexamethasone showed a significant decrease (p <0.01), (p <0.01) in glucose uptake when compared with normal. In non-insulin assisted glucose uptake, SI treatment showed significant increase in glucose uptake (p <0.01) while ketoconazole and pioglitazone did not show significant increase in glucose utilization by mice isolated hemidiaphragms when compared with D-control. In insulin assisted glucose uptake the pioglitazone showed significant increase in glucose uptake (p <0.01) while, SI showed significant increase in glucose uptake (p <0.01) when compared with D + insulin group (Table 3).

Table 3.  Effect of SI on glucose uptake in isolated insulin resistant hemidiaphragms of mice.

Non-insulin assisted glucose uptake		Insulin assisted glucose uptake
Groups	mg/g/30 min	Groups	mg/g/30 min
Normal	8.89 ± 0.31	Normal + I	14.47 ± 0.77
D-control	5.12 ± 0.33^x**	D-control + I	08.74 ± 0.344^y**
D + keto	7.99 ± 0.37^a*	D + keto + I	10.77 ± 0.37
D + pio	7.44 ± 0.4	D + pio + I	23.46 ± 1.23^b**
D + SI-200	5.82 ± 0.72	D +SI-200 + I	13.18 ± 0.89^b*
D +SI-400	7.84 ± 0.49^a*	D +SI-400 + I	18.39 ± 0.79 ^b**
D +SI-800	8.59 ± 1.24^a**	D +SI-800 + I	28.43 ± 1.51^b**

Values are expressed as mean ± SEM, n = 6, analyzed by one way ANOVA followed by Dunnett’s test.

^xp <0.01 when compared with normal group. ^y*p <0.01 when compared with normal + insulin group. ^a*p <0.05, ^a**p <0.01 when compared with D-control group. ^b*p <0.05, ^b**p <0.01 when compared with D-control + insulin group.

D, dexamethasone (1 mg/kg, i.m.); keto, ketoconazole (24 mg/kg, p.o.); pio, pioglitazone (2 mg/kg, p.o.); I, insulin (0.62 mL of 0.4 IU per mL solution); SI, Methanol extract of Sphaeranthus indicus (200, 400, 800 mg/kg, p.o.).

Discussion

Glucocorticoids are widely used therapeutic tools, particularly for anti-inflammatory and immunomodulatory purposes. Side effects of glucocorticoid treatment include steroid diabetes (Hoogwerf & Denese, 1999; Schacke et al., 2002). Different mechanisms for corticosteroid induced diabetes mellitus have been postulated from time to time. One of those is insulin resistance, caused by the alteration in binding of insulin to its receptor (receptor defect) or by the impairment of the intracellular response to insulin (post-receptor defect) (Gholap & Kar, 2005).

Excess of either endogenous or exogenous glucocorticoids has been shown to cause insulin resistance, increase gluconeogenesis and decrease tissue glucose uptake, thus resulting in hyperglycemia (Gholap & Kar, 2005). In the present study, Sphaeranthus indicus significantly prevented dexamethasone induced hyperglycemia as well as hypertriglyceridemia. The methanol extract of Sphaeranthus indicus is rich in stigmasterol, β-sitosterol, β-d-glucoside of β-sitosterol, sesquiterpenes and flavonoids (Ambavade et al., 2006). The effect of Sphaeranthus indicus on the plasma glucose level and triglyceride level may be due to presence of chemical constituents such as β-sitosterol and the β-d-glucoside of β-sitosterol, reported to possess antihyperglycemic and insulin releasing effects (Ivorra et al., 1988; Prabhu et al., 2008).

Hyperglycemia lowers the activity of antioxidant enzymes such as glutathione reductase, superoxide dismutase and catalase, and increases glycation and oxidation rate whose end points are diabetic complications (Piconi & Ceriello, 2007). Lipid peroxide measurements are used to quantify hydroxyl radical damage in cells, which produces malondialdehyde (MDA) by lipid peroxidation (West, 2000). In the present study, Sphaeranthus indicus significantly prevented oxidative damage caused by hyperglycemia as well as also significantly raised the endogenous antioxidant levels. Phytochemical study of the methanol extract of Sphaeranthus indicus revealed the presence of sterols, phenols and flavonoids. Flavonoids play a major role in reducing oxidative stress associated with diabetes (Shirwaikar et al., 2006), probably scavenge the free radicals and prevent the depletion of endogenous antioxidants.

Glucocorticoids affect the PI3-kinase/PKB pathway involved in the regulation of insulin sensitive glucose uptake by recruiting GLUT-4 (glucose transporter 4) from intracellular pool to plasma membrane (Schinner et al., 2005). In the present study, two sets were used for glucose uptake estimation, to determine whether SI depends on insulin or is independent of insulin assistance for glucose uptake. SI exhibited significant increase in glucose uptake in presence as well as in absence of insulin. The results of the glucose uptake study indicate that SI probably causes increase in the insulin sensitivity. However, the probable mechanism by which SI increased the glucose uptake in the absence of insulin needs to be further explored.

The results of the present investigation indicate that Sphaeranthus indicus has the ability to decrease blood glucose and triglyceride level, increase antioxidant enzymes and increase peripheral glucose uptake, indicating the preventive effect of Sphaeranthus indicus during progression of glucocorticoid induced insulin resistance in mice and its utility in NIDDM.

Declaration of interest

There is no conflict of interest.