INTRODUCTION
Development of impaired glucose tolerance is an indication of pre-diabetes (1), which later cause a gradual increase in resistance to insulin (2) thus causing the risk of type 2 diabetes mellitus (T2DM) (3) if left unattended, making it the initial avoidable indications of T2DM. The incidence of pre-diabetes is increasing at an alarming rate in the developing countries (4) and as 70% of these will turn into T2DM (5), identification and abolition of pre-diabetes preclude the occurrence of this otherwise impeccable disorder. The fact that obesity and physical inactivity are the most potential causes leading to insulin resistance (6), the precursor to pre-diabetes, can be explained by the role of adipose tissue in the synthesis of various mediators such as Non Esterified Fatty Acids (NEFA’s) and adipocytokines such as leptin, TNF-A, resistin andadiponectin (7), which are involved in the pathogenesis of insulin resistance and β-cell dysfunction (8), the early steps involved in the course of development of whole body insulin resistance in T2DM (9).
Drugs of herbal origin served a major role in the treatment of many ailments since centuries and still continue to be the mainstay in their treatment. Unfortunately, despite extensive research, no alternative drug therapy or herbal medicine has accrued adequate importance in the prevention of progression or treatment of T2DM (10), the most common disorder that affects majority of the population all over the world and considered to be one of the 10 leading causes of morbidity and mortality (11). Though number of plants and phytoconstituents with insulin secretagogue activity were identified (12), they are of minimal use in conditions of hyperinsulinemia, the widely experienced characteristic of T2DM and primary cause of insulin resistance. Provided, very few plants such as Agrimony eupatoria, Helicteres isora and Ipomea batata, etc (13-15), have been mentioned to have an additional insulinomimetic activity along with their insulin secretory effects. Hence search for a potent insulinomimetic with considerable effect on insulin resistance is on, which could be a therapeutic breakthrough in the prevention and treatment of T2DM.
Costus igneus or insulin plant is being used in the traditional folk medicine of various tribes in south East Asia, especially in southern parts of India for the treatment of symptoms such as unexplained weight loss or gain, excessive thirst, hunger, insomnia, weakness or fatigue (16) together which can be characterized as the indicators of pre-diabetes and diabetes. Studies of various researches have reported the therapeutic efficacy of Costus igneus against T2DM (17). Hence the goal of our present study was to report the effect of isolated fractions of various extracts of Costus igneus in neonatal streptozotocin (n5-STZ) model of T2DM in rats (18). The effect on pre-diabetes was studied using oral glucose tolerance test in rats during and after induction of T2DM in the presence and absence of drug treatment. The effect on diabetes was studied after administration of drugs to rats which receive treatment before and after induction of diabetes.
METHODS
Animals and induction of T2DM
Adult wistar albino rats of either sex (350 – 400 g) were procured from the animal house of Bapatla College of pharmacy (1032/ac/07/CPCSEA), Bapatla, India and were housed for breeding, at a constant temperature (22 ± 1°C), relative humidity (40-50%) and 12 – 12 h light/dark cycles were maintained. STZ 90 mg/kg (freshly prepared acetate buffer 0.1 M, pH 4.5) was administered intraperitoneally to the neonatal rats (weighing 10-12 g) on day 5, postnatally (n5-STZ) (19) Buffer was administered accordingly to the neonatal rats which serve as control. These rats were separated from their mothers after 4 weeks and provided with standard pellet feed (Rayan’s Biotech, Hyderabad) and water ad libitum. Animals were acclimatized to laboratory conditions one week prior to initiation of experiments. The experimental protocol was approved by Institutional animal ethics committee (IAEC/IV-22/BCOP/2012). The handling and care of animals was performed in accordance with CPCSEA guidelines for the use and care of experimental animals.
Plant material
Fresh leaves of the plant Costus igneus were collected from ABS botanical gardens, karripatti, (Salem District of Tamilnadu) India. The leaves were identified and authenticated by the botanist Dr. A. Balasubramanian, Executive Director, ABS Botanical Conservation, Research and Training Centre, Salem, Tamilnadu. The voucher specimen AUT/BCP/025 was deposited in the department. The leaves were shade dried and powdered finely for extraction and isolation of various compounds.
Preparation of extract and isolation of the compounds
Costus igneus leaf powder (50 gm) was subjected to soxhlet extraction with petroleum ether for 12 hrs for defatting. The solvent was then evaporated and extract was dried using rotavac at a controlled temperature of 40-50°C. The resulting marc was subjected to soxhlet extraction with 300 ml of methanol for 24 hrs. The solvent was removed using rotavac apparatus and the resulting extract was subjected to extraction with various solvents as previously described by Lui (20). The chloroform extract, which was found to be effective when studied for its effect on fasting blood glucose, was subjected to fractionation by preparative TLC, using 20 × 20 cm glass plates (0.5 mm) coated with silica gel G. The fractions were eluted with the chloroform and were screened for blood glucose lowering effect in normal rats and the biologically active Costus igneus fraction (CIF) was selected for the study.
Acute toxicity studies
Acute toxicity studies of the isolated fraction CIF was administered to male albino mice at doses of 5, 50, 500, 2000 mg/kg body weight orally, to 4 groups of (n=3) overnight fasted mice. They were observed for 24 hours for signs of toxicity and behavioral changes followed by observation for 7 days for morphological changes and mortality.
Grouping of animals
Rats were divided into two sets, one for pre-treatment and other for post-treatment (i.e. non treated for 12 weeks after STZ administration) each containing five groups (n=10), of which treatment starts from 4th week of STZ administration in pre-treated groups and treatment starts after 12th week of STZ administration in post treated groups. Pre-treated set consists of five groups of which Group I served as control, group II served as negative control, group III and group IV were treated with CIF 20 and 40 mg/kg and group V rats were treated with pioglitaozone (PIO) 2.7 mg/kg. CIF and pioglitazone were administered as suspension in 10% tween 20 (vehicle). Dilutions were made accordingly to administer 0.2 ml/100 g intragastric. Negative control group received vehicle alone. Post treated rats were grouped into 5 groups (n=10), but do not receive any drug treatment till 12th week after STZ administration. All treatments were given intragastric.
Oral glucose tolerance test (OGTT)
OGTT was performed in pre-treated and post treated groups on 7th and 12th week after STZ treatment. An additional 4 groups of normal rats of equal age were used to study the effects of these treatments on OGTT. The effect of CIF on glucose overloaded hyperglycemia was studied in all the animals. Overnight fasted (12 h) normal rats were divided into 4 groups (n=6) of which Group I served as control, Group II and Group III were treated with CIF 20 and 40 mg/kg orally administered, Group IV rats were treated with 2.7 mg/kg of pioglitazone intragastric. Group I to V of pre-treatedn 5-STZ rats were treated accordingly as discussed above, whereas post-treated n5-STZ rats did not receive any treatment. Zero hour blood glucose levels were determined by tail vein method. After 30 min of the drug treatment, the animals were fed with glucose (4 g/kg BW) orally and the blood glucose levels were determined after 0.5, 1, 2 and 3 hours after glucose administration (21). Blood glucose levels were estimated using a glucometer (Accu-chekActiveTM Test meter).
Hypoglycemic effect in n5-STZ rats after chronic administration
After performing OGTT on 12 week after STZ administration, rats of both pre and post treated groups were used to study the effect on blood glucose levels. Rats with blood glucose levels >150 mg/dL were considered to be diabetic and are included in the study (n=6) (22). All the rats were treated with CIF and pioglitazone for 21 days as described previously. They were used to study the effect of these treatments on hypoglycemia after treatment for 21 days. Fasting blood glucose levels were determined using glucometer (Accu-chekActive™ Test meter) by tail vein method on day 1, 7, 14 and 21, 30 min after drug administration (23).
Statistical analysis
All data were expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. p<0.05 was considered to be significant.
RESULTS
Preparative TLC fraction
The percentage yield of chloroform extract was 6.52 and its TLC profile had shown the presence of two components with Rf value 0.48 and 0.61 respectively, which were separated using preparative TLC. From 325 mg of chloroform extract, two fractions were obtained namely, CIF-1 (135 mg) and CIF-2 (154 mg). Both the fractions were preliminarily screened for their effect on blood glucose levels in normal rats and CIF-1 was found to have blood glucose lowering effect, hence was selected for the study.
Acute oral toxicity studies
Acute oral toxicity studies of CIF fraction of chloroform extract did not show any abnormal or toxic symptoms in mice treated with 5, 50, 500, 2000 mg/kg of the same and the LD50 value is above 2 g/kg.
Effect on oral glucose tolerance test
In normal rats
Treatment with CIF 20 mg/kg had shown a significant decrease in the elevated blood glucose levels (p<0.01) and (p<0.001) at 2nd and 3rd h respectively and treatment with CIF 40 mg/kg had shown a significant decrease (p<0.001) at 2nd and 3rd h and the observed effects are similar to that of pioglitazone treatment (Figure 1).
FIGURE 1. Effect of CIF on glucose overloaded hyperglycemia in normal rats (n=6). Blood glucose levels of CIF 20, 40 and pioglitazone treatments were compared with that of control group at respective time intervals **p<0.01. #p<0.001.
In STZ treated rats
No significant difference in the basal blood glucose levels were observed in the 7th week of pre-treated and non-treated rats. In OGTT at 7th week, the elevation in blood glucose levels after glucose over loading was more in non-treated rats than that observed in pretreated rats, though not significant. The decrease in the blood glucose levels was significant (p<0.01) in the 2nd and 3rd h CIF 20 and 40 mg/kg treated rats (Figure 2). No significant decrease in the blood glucose levels was observed in non-treated rats. OGTT on 12th week after STZ treatment had shown a significant decrease in the blood glucose levels at 2nd and 3rd h of treatment with CIF 20 (p<0.05) and 40 mg/kg (p<0.01) in pre-treated rats. The increase in blood glucose levels after glucose administration was significant (p<0.01)(Figure 3) and no significant decrease in the blood glucose levels was observed in non-treated rats.
FIGURE 2. Effect on oral glucose tolerance test after 7th week of STZ administration in rats pretreated with CIF (n=10). Blood glucose levels of CIF 20, 40 and pioglitazone treatments were compared with that of negative control group at respective time intervals. *p<0.05, **p<0.01.
FIGURE 3. Effect on oral glucose tolerance test after 12th week of STZ administration in rats pretreated with CIF (n=10). Blood glucose levels of CIF 20, 40 and pioglitazone treatments were compared with that of negative control group at respective time intervals. *p<0.05, **p<0.01.
Effect on hyperglycemia
Pretreatment with CIF 20 mg/kg and 40 mg/kg had shown a significant reduction in blood glucose levels on 1st (p<0.05), 7th (p<0.05), 14th (p<0.01) and 21st day (p<0.001) when compared with that of blood glucose levels of negative control group whereas the reduction in blood glucose levels was significant only on 14th (p<0.05) and 21st (p<0.01) day, when compared with the blood glucose levels of day 1 (Table 1). Basal blood glucose levels were higher in post treated groups when compared with that of pre-treated rats in day 1. Post treatment with CIF 20 had shown a significant decrease in the blood glucose levels on 14th and 21st day (p<0.05) when compared with that of negative control group and 14th (p<0.05) and 21st (p<0.01) when compared with the basal blood glucose levels on day 1, and treatment with CIF 40 mg/kg had shown a significant decrease on 7th (p<0.05), 14th (p<0.01) and 21st day (p<0.001), when compared with that of negative control group and 7th (p<0.05), 14th (p<0.05) and 21st day (p<0.001), when compared with the basal blood glucose levels on day 1 (Table 2).
TABLE 1. Effect of CIF on blood glucose levels in rats pretreated from 4th week after STZ administration
TABLE 2. Effect of CIF on blood glucose levels in post treated rats during 21 days of treatment from 12th week after STZ administration
DISCUSSION
Insulin resistance is the primary cause of development of T2DM, causing significant morbidity and mortality due to various micro and macro vascular changes affecting the quality of life of millions of people all over the world (24). Identification of individuals susceptible to T2DM in their pre-diabetic stage and providing adequate treatment could cut the extent of damage caused. Costus igneus was studied less in terms of its usefulness in the treatment of T2DM despite of its wide usage in many parts of south India. The present study has been designed to explore the possible role of isolated fraction of chloroform extract of Costus igneus on pre-diabetes and diabetes in pre and post treated n5-STZ rats. As n5-STZ rat model produces gradually developing basal hyperglycemia, abnormal glucose tolerance and mild hypoinsulinemia in the initial states of STZ induction up to 7th or 8th week, this could be due to the development of loss of sensitivity of β-cell towards glucose (25), though a compensatory increase in insulin levels to maintain the blood glucose levels occurs initially, there occurs a failure in such response, due to loss of beta cell sensitivity to glucose, which further inhibit insulin action, thus causing elevated blood glucose levels throughout the day leading to development of insulin resistance.
The effect of CIF on normal glucose overloaded rats in oral glucose tolerance test (OGTT) exhibits its potential in reducing the elevated blood glucose levels. OGTT itself has an ability to detect the impaired glucose tolerance (26) an indicator of pre-diabetes. As every 1-5 % of individuals with impaired glucose tolerance, develop into T2DM every year and are at a risk of developing heart diseases (27). Neonatal STZ model of diabetes resemble both pre-diabetic and diabetic states of the human (28). In later stages after 7th or 8th week of age there occurs drastic destruction of β-cells accompanied with peripheral and hepatic resistance of cells against insulin due to prolonged hyperglycemic states (29), which resembles the condition of T2DM in humans. The reduction of blood glucose levels in pre-treated groups after glucose overloading at 7th week indicates the efficacy of CIF on peripheral utilization of glucose levels and could be due to increasing the insulin sensitivity as no such significant decrease in the blood glucose levels were observed in non-treated STZ rats. The effect of OGTT in pre-treated rats is similar to that of observed in normal rats. Whereas, in OGTT after 12 wk of induction pre-treated rats had shown a significant reduction in the blood glucose levels when compared with that of non-treated rats, but was less when compared with that of normal rats. This could be due to the extent of destruction of β-cellsleading to hypoinsulinemia, that might have occurred in the later stages of STZ induction in both pre-treated and non-treated groups (30). Protection against developing resistance to insulin at peripheral and hepatic tissues and increase in the sensitivity of β-cells towards glucose in pre-treated groups (31) could be responsible for the observed beneficial effect.
Whereas, in the effect on hypoglycemia, the reduction in blood glucose levels in the pre-treated groups was less than that observed in normal rats when compared to the observed change in the OGTT performed at 7th week, which once again could be due to the destruction of β-cells in the adult rats. The elevation in the blood glucose levels on 12th week in both pre-treated and non-treated rats could be explained by the destructive phase of pancreatic β-cells after 8th week of STZ administration.
In post treated rats, the basal blood glucose levels are higher than that observed in rats that receive CIF and pioglitazone from 4th week, this protective effect on pre-treatment could be explained by the fact that though there occurs a disruption of pancreatic β-cells, there exists insulin sensitivity, which is evident with the low basal blood glucose levels in them. In conclusion, use of CIF as a prophylactic agent in individuals with basal hyperglycemic states could reduce the risk of developing into T2DM and is also of therapeutic value in the treatment of T2DM.