INTRODUCTION:

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia (fasting blood glucose level > 126 mg/100 ml (6.1mmol/l) with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. In accordance with the recommendations of the WHO expert committee on diabetes mellitus, an investigation of anti –diabetic agents of plant origin used in traditional medicine seems important. Many herbs and plant products have been shown to have antidiabetic action¹.

Inflammation is a major component of the damage caused by autoimmune diseases and is also a fundamental contributor to diseases such as cancer, diabetes and cardiovascular disease². Chronic inflammation is the reaction arising when the acute responses is insufficient to eliminate the pro-inflammatory agents. Chronic inflammation includes a proliferation of fibroblast and infiltration of neutrophils with exudation of fluids. It occurs by means of development of proliferation cells which can either spread or form granuloma. Efficacy of anti inflammatory agents in chronic inflammatory states is indicated by their ability to inhibit the increase in the number of fibroblast during granular tissue formation³.

Many herbs and plant products have been shown to have antidiabetic action but very few plants possess anti-diabetic and anti-inflammatory properties together. Inula racemosa⁴ is one such herb. Inula racemosa was traditionally used for different ailments in Chinese and in Ayurveda. The entire plant is used and is considered acrid, sweet, and warm. It is used for wind-dampness pain, broken bones, external injury with bleeding, and clove sores with toxic swelling. Seeds used for optic disorder and pitha and kapha treatment in Ayurveda, it is denoted in the book of Bhavaprakesh nighantu. It possess anti-tumor, anti-diabetic, anti-inflammatory, anti- ulcer, anti-oxidant properties⁵

But till now there is no reported study which witnessed the effect of Inula racemosa in the management of inflammatory conditions and diabetes mellitus. So the present study was designed to evaluate the anti-inflammatory and anti-diabetic activity of ethanolic extract of Inula racemosa.

MATERIALS AND METHOD:

Inula racemosa (F: Composite) (Syn.: Pushkarmoola) is an ayurvedic herb. It grows in the hilly regions in the north –western Himalayas in Kashmir and collected in the spring and autumn. Its roots have been mentioned as bitter, acrid, alterative, aromatic, stimulant and thermo genic. It grows about2 meters long. it is a annual plants stem is leafy smooth and greenish and middle leaf densely, 2.5-4.5*2-3cm, with ovate base chordate and amplexicaul. Colored leaves arranged into alternate phyllotaxy and reticulate venation. Margin coarsely serrate and apex rounded. It has been reported to contain inulin, essential oil, sesquiterpene lactones - Alantolactone, isoalantolactone, isoalloalantolactone, dihydroalantolactone, dihydroisoalantolactone, alantodiene, isoalantodiene, and beta sitosterol, daucosterol, inunal, inunolide.

Animal Selection

Swiss albino rat of Wistar strain weighing 18-30 gm., were used for the present study. The mice were inbred in the central animal house of the Department of Pharmacology, Karavali College of Pharmacy, Mangalore, under suitable conditions of housing, temperature, ventilation and nutrition were used for antidepressant activity. They were kept in clean dry cages week before the beginning of the experiment to acclimatize with the experimental conditions. The animals were fed with standard pelleted diet (Lipton India Ltd., Mumbai) and distilled water ad libitum was maintained at 21⁰C-23⁰C under a constant 12hrs light and dark cycle. The animal care and experimental protocols were in accordance with CPCSEA /IAEC.

Preparation of test solution

Ethanolic extract was prepared from a powder of the whole plant extract of Inula racemosa prepared in an electric grinder. The 200 gm powder was extracted with ethanol. The extract was evaporated to dryness under vacuum and dried in vacuum desiccators (5%w/w).

A. ANTIDIABETIC ACTIVITY

1. Alloxan Induced Diabetes Method

Different groups of rats were used to study the effect of ethanolic extract of Inula racemosa. The rats were divided into five groups each consisting of six rats:

Group I: The rats received 2 ml 1% CMC (Carboxy methyl cellulose). These animals serve as normal controls (NC).

Group II: Received a single dose of (150mg/kg body weight), Alloxan monohydrate in CMC through intraperitoneal route and served as Diabetic control (DC).

Group III: Received the (IRE) Inula racemosa extract 200mg/kg for 21days and served as test 1 (Extract 1).

Group IV: Received the (IRE) Inula racemosa extract 400mg/kg for 21 days and served as test 2 (Extract2).

Group V: Received Glibenclamide 5mg/kg for 21days and served as Standard (STD).

Experimental Procedure

Animals were allowed to fast 24 hr and were injected with freshly prepared aqueous solution of alloxan monohydrate (150 mg/kg i.p.) as reported previously (Kameswara Rao et al., 1999). After a week rats with marked hyperglycemia (fasting blood glucose>200mg/dl) were used for the study.Blood glucose was collected for the measurement of blood glucose from the tail vein at 0, 1, 2 and 3 hr after feeding the plant extracts. The blood glucose levels were determined by using one touch glucometer.

Statistical Analysis:

The collected data were subjected to appropriate statistical test including one way ANOVA, followed by an appropriate Dunnett’s t-test, P-value of less than 0.05, 0.01 and 0.001 were considered as less significant, significant and more significant respectively. The analysis was carried out using graph pad prism software.

2. Streptozocin Induced Diabetes Mellitus⁶

Different groups of rats were used to study the effect of ethanolic extract of Inula racemosa. The rats were divided into seven groups comprising of six animals in each group as follows:

Group I: Normal control and were given only distilled water daily (NC).

Group II: STZ- induced diabetic rats served as diabetic control and were given distilled water only (DC).

Group III: Received the (IRE) Inula racemosa extract 200mg/kg for 21days and served as test 1 (Extract 1).

Group IV: Received the (IRE) Inula racemosa extract 400mg/kg for 21 days and served as test 2 (Extract2).

Group V: Received Glibenclamide 5mg/kg for 21days and served as Standard (STD).

Experimental procedure

Diabetes was induced by STZ⁶ (Sigma-Aldrich, St. Louis, USA). The animals were fasted overnight and diabetes was induced by a single intra peritoneal injection of a freshly prepared solution of STZ -60 mg/kg b.wt in 0.1 M citrate buffer (pH 4.5).Control rats were injected with citrate buffer alone. On the third day of STZ-injection, the rats were fasted for 6 h and blood was taken by sinocular puncture. Rats with moderate diabetes having hyperglycemia (blood glucose of 250–400 mg/dl) were taken for the experiment. The blood glucose levels outside the specified range were excluded from the study. The rats were kept for 15 days to stabilize the diabetic condition. In the experiment, a total of 42 rats (6 normal; 36 STZ-diabetic rats) were used. Blood will be collected for the measurement of blood glucose from the tail vein at 0, 1, 2 and 3 hr after feeding the plant extracts. The blood glucose levels will be determined by using one touch glucometer.

Statistical Analysis:

Statistical analysis was performed using SPSS software package Version 16.0. The values were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). All the results were expressed as mean ± S.D. for six rats in each group. P < 0.05 was considered significant.

ANOVA (Analysis of variance).

In statistics, analysis of variance is a collection of statistical models and their associated procedures, in which the observed variance is partitioned into components due to different explanatory variables. In its simplest form ANOVA gives a statistical test of whether the means of several groups are all equal and therefore generalize Dunnett’s multiple comparison tests to more than two groups.

B. ANTI-INFLAMMATORY ACTIVITY

1 Formalin Induced Paw Edema Model^{7, 8}

Animal Used: Albino Rats of Wistar strain

Chemical agent used to induce inflammation: Formalin (0.1ml injected intra peritoneal to subplanatar region of left hind paw)

Route of Administration: Oral

The method⁷was used for this study. Animals were divided into five groups denoted as Control group, Positive control group (Standard-Ibuprofen 100mg/kg) group), Test group I (IRE-100), Test group II (IRE-200) and Test group III (IRE-400). Each group consisting of 6 albino Wister rats. Control group received orally 0.1ml of 1% suspension in sodium CMC at the dose of 10 ml/kg body weight and Positive control group received orally at the dose of 100mg/kg body weight. Test group I and Test group II, Test group III were treated with test Sample orally at the dose of 100,200 and 500 mg/kg body weight. 0.2 ml of 3 % formalin was injected into the dorsal surface of the left hind paw of rats 1 h after oral administration of the extracts. The time spent by each animal in licking the injected paw was observed for 5 min. (from 0- 5min post formalin injection) and 10 min (from 20-30 min post formalin injection). The mean of the licking time was determined and compared with the mean for the control group.

2. Croton Oil Ear Edema

Albino rats were divided into five groups of six animals each. Animals were treated orally with the extract (TCEE 100, TCEE200 and TCEE 400 mg/kg), Ibuprofen (100 mg/kg) and distilled water (3 ml/kg).Thirty minutes later, edema was induced in each rats group by applying a drop of croton oil to the inner surface of the right ear. After 15 min, the animals were sacrificed under ether anesthesia and both ears cut off, sized and weighed. The anti-inflammatory activity was expressed as the difference in (weight of untreated ear – weight treated ear) of edema in the treated mice in comparison with the control rats.

RESULT:

A. Antidiabetic Activity

1. Alloxan induced Diabetes

Table No 1: Effect of ethanolic whole plant extract of Inula racemosa on fasting blood glucose level in Alloxan induced diabetic rats.

Groups	1^stDay	1^stweek	2^nd week	3^rd Week
NC (Normal control	87.5±3.81	97.33±6.41	104.33±5.28	100.33±2.82
DC (Diabetic control)	131±7.41	253±6.85	275.16±6.11	288±6.53
Extract-1 (IRE-200mg/kg)	106.83±6.93**	164.83±8.75**	157±8.87**	134.5±3.21**
Extract-2 (IRE-400mg/kg)	101.83±6.93**	153.66±5.06**	142.16±4.36**	121.66±5.73**
Standard (Glibenclamide) (5mg/kg)	103.17±4.57**	142.16±7.09**	134.33±5.92**	130±3.48**

Data represents mean ± S.D.[n(number of animals) =6].*p<0.05 significant as compared to normal control.**p<0.01significant as compared to Alloxan control.

***p<0.001 significant as compared to Alloxan control. ns is non-significant as compared to normal control.

Figure 1: Effect of ethanolic whole plant extract of Inula racemosa on fasting blood glucose level in Alloxan induced diabetic rats

2. Streptozocin induced Diabetes

Table No 2: Effect of ethanolic whole plant extract of Inula racemosa on fasting blood glucose level in Streptozocin induced diabetic rats.

Groups	1^stDay	1^stweek	2^nd week	3^rd Week	4^th week
NC	90.5±3.9	97.33±6.41	104.33±5.28	100.33±2.82	98.21±1.85
DC	130±5.21	251±4.74	270.11±4.16	284±3.13	280±1.13
Ext1	106.83±6.93^**	164.83±8.75^**	157±8.87**	134.5±3.21^**	132.1±2.11^**
Ext2	100.83±5.44^**	151.66±5.06^**	140.16±4.36^**	120.66±5.73^**	119±2.51^**
Std	102.17±4.57^**	140.16±7.09^**	120.31±5.92^**	112±3.48^**	103±3.48^**

Data represents mean ± S.D. [n (number of animals) =6].

*p<0.05 significant as compared to normal control.)

**p<0.01significant as compared to diabetic control (DC).

***p<0.001 significant as compared to diabetic control (DC)..

ns is non-significant as compared to normal control.

Figure 2: Effect of ethanolic whole plant extract of Inula racemosa on fasting blood glucose level in Steptozocin induced diabetic rats.

B. Anti-inflammatory Activity

1. Formalin induced Paw Edema Model

Table No 3: Acute anti-inflammatory activity of the extract of whole plant of Inula racemosa and Ibuprofen (reference drug) on formalin induced paw edema in Wistar rats.

Group	N	30 min	60 min	120 min	180 min	240 min	300 min
Control	6	1.26 ±0.014	1.31 ±0.007	1.36 ±0.035	1.32 ±0.056	1.26 ±0.084	1.24 ±0.070
IRE 100	6	1.09 ±0.014	1.15 ±0.049	1.20 ±0.021	1.14 ±0.049	1.12* ±0.070	1.06* ±0.021
IRE 200	6	1.08 ±0.056	1.17 ±0.014	1.25 ±0.042	1.23±0.035	1.10* ±0.014	1.00* ±0.028
IRE 400	6	1.10±0.056	1.20±0.014	1.27±0.042	1.24±0.035	1.11* ±0.014	1.00* ±0.028
Ibuprofen (100mg/kg)	6	1.25 ±0.007	1.31 ±0.028	1.33 ±0.028	1.20 ±0.007	1.02 ±0.014	0.93* ±0.035

Data are the mean ± SEM values for six rats in each group.*p < 0.05, **p < 0.01 as compared to the control. At 400mg/kg dose (1.00±0.028), the activity of the extract showed almost similar activity compare to standard drugs

2. Croton oil Ear edema in Rats

Table No 4 : Acute anti-inflammatory activity of the extract of whole plant of Inula racemosa and Ibuprofen (reference drug) on croton oil ear edema in Wistar rats.

Animal used: Albino Rats of Wistar strain

Route of Administration: External application

Group	Dose (mg/kg)	N	Weight of Untreated ear (Right ear) (mg)	Weight of treated ear (Left ear) (mg)	Difference
Control	1ml/kg	6	37.53 ±1.08	25.02 ±1.17	13.17 ±1.24
IRE 100	100mg/kg	6	37.49 ±0.37	28.14 ±0.28	9.35±0.09
IRE 200	200mg/kg	6	37.02 ±0.51	29.04 ±1.20	7.98* ±0.85
IRE 400	400mg/kg	6	36.82 ±0.44	30.66 ±0.63	6.16**±0.69
Ibuprofen	100mg/kg	6	37.43 ±0.64	32.47 ±0.57	4.95* ±0.11

Data are the mean ± SEM values for six rats in each group.

*p < 0.05, **p < 0.01 as compared to the control.

DISCUSSION:

The present study was aimed to investigate anti-diabetic activity of Inula racemosa Extract (IRE) using Alloxan induced diabetes, Streptozocin induced diabetes models in albino rats of wistar strain and anti-inflammatory activity of Inula racemosa using Formalin induced paw edema model and Croton oil ear edema model.

Antidiabetic Activity

The oxygenated pyrimidine derivative, alloxan monohydrate is the most potent diabetogenic chemical agent in experimental diabetes research. Alloxan induces a multiphasic blood glucose response when injected in an experimental animal, which is accompanied by corresponding inverse changes in the plasma insulin concentration followed by sequential ultra-structural beta cell changes ultimately leading to necrotic cell death. It has been found to be selectively toxic to pancreatic beta cells as it preferentially accumulates in the beta cells as glucose analogues. In addition, the cytotoxic action of alloxan is mediated mainly by the generation of reactive oxygen species (ROS).Alloxan is a hydrophilic and unstable chemical compound which has similar shape as that of glucose, which is responsible for its selective uptake and accumulation by the pancreatic beta cell. Similarity in the shape allows it to transport into the cytosol by the glucose transporter (GLUT2) in the plasma membrane of beta cell. Another biological effect of alloxan has been attributed to the thiol group reactivity that allows selective inhibition of glucose-induced insulin secretion through inhibition of glucokinase^9,10.

Hyperglycemia in diabetic patients is the main factor of diabetic neuropathy which induces oxidative stress through various cellular pathways such as increasing aldose reductase activity, increasing glycation end-products and altering protein kinase C activity. The diabetic neuropathies are heterogeneous, affecting different parts of the nervous system. Chronic hyper glycaemia plays a major role in the initiation of diabetic vascular complications through many metabolic and structural derangements, including the production of advanced glycation end products (AGE), abnormal activation of signaling cascades [such as protein kinase C (PKC)], elevated Long standing hyperglycemia producing a large amount of Reactive Oxygen Species (ROS) which can damage mitochondrial DNA in dorsal root ganglia leading to peripheral nerves dysfunction.²

In the case of Alloxan induced diabetes model, experimental results proved that Inula racemosa extract at 400mg/kg dose (IRE 400) exhibited almost similar antidiabetic activity compared to Standard glibenclamide (5mg/kg) followed by IRE 200 (Inula racemosa extract 200mg/kg). Inula racemosa exhibited dose dependent antidiabetic activity when compared to control.

In the case of Streptozocin induced diabetes model, experimental results proved that Inula racemosa extract at 400mg/kg dose (IRE 400) exhibited almost similar antidiabetic activity compared to Standard glibenclamide (5mg/kg) followed by IRE 200 (Inula racemosa extract 200mg/kg). Inula racemosa exhibited dose dependent antidiabetic activity when compared to control.

The probable mechanism of these actions may be due the cytochrome P450 (CYP) 2C9 inhibitory property (or bio enhancing property) of Inula racemosa on glibenclamide (as glibenclamide is metabolized by CYP2C9 enzyme) which may be responsible for potentiation of glibenclamide. Apart from that earlier studies also reported Inula racemosa is having antidiabetic, antioxidant and protective action on pancreatic β-cells. These beneficial properties of Inula racemosa against diabetic conditions may be contributed to the observed results.

However, further studies are required to understand the exact mechanism behind the antidiabetic activity of Inula racemosa.

Anti-Inflammatory Activity

In the case of Formalin induced paw edema model Inula racemosa extract (IRE 400) (1.27±0.042) exhibited almost similar anti-inflammatory activity compared to standard ibuprofen at (1.33 ±0.028). Anti-inflammatory activity exhibited by Inula racemosa extract was found to be in a dose dependent manner i.e. at lower dose less activity and higher dose more activity. So the order of exhibition of activity was observed as: IRE 400>IRE 200 >IRE 100. Decline in anti-inflammatory activity was observed after 120 minutes.

In the case of Croton oil ear edema model Inula racemosa extract (IRE 400) (6.16±0.69 ) exhibited almost similar anti-inflammatory activity compared to standard ibuprofen at (4.95±0.11). Anti-inflammatory activity exhibited by Inula racemosa extract was found to be in a dose dependent manner i.e. at lower dose less activity and higher dose more activity. So the order of exhibition of activity was observed as: IRE 400>IRE 200 >IRE 100. Decline in anti-inflammatory activity was observed after 120 minutes.

CONCLUSION:

The present study was aimed to investigate the antidiabetic activity of whole plant of Inula racemosa in swiss albino rat using two animal models of diabetes namely, Alloxan induced diabetes and Streptozocin induced diabetes models and to evaluate the anti-inflammatory potential of Inula racemosa using Formalin induced paw edema model and Croton oil ear edema models

The data obtained was satisfactory and conclusive and so as to accomplish our objectives. In conclusion the present data indicate that the administration of Inula racemosa extract to albino rat has shown significant dose dependant antidiabetic activity supporting literature information regarding antidiabetic activity of the plant, relatively sub-chronic study may be necessary to arrive at a better picture.

The exact mechanism underlying antidiabetic effect is not clear but it may be apparently related to active compounds present in Inula racemosa. Hence further studies would be necessary to evaluate the contribution of active chemical constituents for the observed antidiabetic activity as it still remains to be determined which components were responsible for these effects.

The ethanolic extract of Inula racemosa was used to evaluate the anti-inflammatory and it was found that Inula racemosa exhibited dose dependent anti-inflammatory activity. The exact mechanism by which Inula racemosa exhibit anti-inflammatory activity is still to be studied it is presumed that it might be due to the presence of flavonoids in Inula racemosa.

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Received on 16.07.2015 Modified on 25.07.2015

Res. J. Pharmacology & P’dynamics. 7(3): July-Sept., 2015; Page 129-134

DOI: 10.5958/2321-5836.2015.00024.5