ANTI-OXIDANT AND ANTI-DIABETIC ACTIVITIES OF ETHANOLIC EXTRACT OF Primula denticulata FLOWERS

Email: bhawana.sati@gmail.com ABSTRACT The present investigation was carried out to focus on the anti-oxidant and anti-diabetic effect of ethanolic extract of flowers of Primula denticulata by DPPH, ferrous chelating, reducing power assay and streptozotocin induced diabetes respectively. Dose selection was made on the basis of acute oral toxicity study (100mg/kg, 200mg/kg, 400mg/kg bodyweight) as per OECD guidelines 423. The blood glucose levels were measured by using blood glucose measuring strips based on glucose-oxidase method. The extract showed significant anti-oxidant and anti-diabetic activity when compared with standard drug.


INTRODUCTION
Diabetes mellitus is a chronic disease caused by deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced.Due to diabetes numerous complications occurs such as retinopathy, neuropathy and peripheral vascular insufficiencies (Chehade et al., 2000).It is considered as one of the five leading causes of death in the world.(Ahmed et al., 2010)Traditional plants are used throughout the world for the treatment of diabetes.The World Health Organization has also recommended the evaluation of traditional plant treatments for diabetes (Day, 1998).Oxidative stress which is defined as an imbalance between the generation of oxidants and antioxidant defense capacity of the body is suggested as a mechanism underlying diabetes and diabetic complications like many other diseases (Atalay et al .,2002 ).There is also evidence that higher glucose concentration causes depression on natural antioxidant defense agents such as glutathione or vitamin C (Gumieniczek et al., 2002).The increase in the levels of reactive oxygen species and free radicals cause damage in the biological structures such as cell wall, genetic material and enzymes.These oxidants also cause microvascular and macrovascular complications, cardiovascular diseases, kidney and nerve damage (Aydın et al., 2001).
P. denticulata belongs to the family Primulaceae is an easy-growing, rumbustious species originating from meadows and the light woodland of the Himalayas.It is also called as drumstick primula.Primula denticulata grows best in a partly shaded spot with soil that does not dry out in summer.Primula species have medicinal properties and used as antiinflammatory, bactericidal, diuretic, antitumour and hepatoprotective.(Tokalov et al., 2004) Primula denticulata contains triterpene glycosides denticin and denticulatin and flavanoid.(Ahmad et al., 1990).As there is no scientific data available regarding anti-diabetic activity of plant, the aim of the study was to investigate anti-diabetic effect of ethanolic extract by streptozotocin induced diabeties in rats, as well as to evaluate the anti-oxidant effect of ethanolic extract by three different models DPPH, ferrous chelating and reducing power assay.

Collection and authentification of plant material
P. denticulata was authenticated at Department of Botany H.N.B Garhwal University, Srinagar Garhwal, Uttarakhand by taxonomist Dr. J. K. Tewari.The flowers of Primula denticulata were collected from Rudraprayag, Garhwal, Uttarakhand, India during July-August 2010.

Extraction of plant material
The flowers of P. denticulata was collected and dried by means of a natural sundry.The dried plant material was powdered and subjected to hot extraction by means of soxhlet apparatus using 95% ethanol.The solvent from total extract was distilled off and concentrate was evaporated on water bath to syrupy consistency and then evaporated to dryness.

Preliminary phytochemical screening
The qualitative chemical tests of ethanolic extract of P. denticulata were carried out by using standard procedure to determine the presence of various phytochemicals (Kokate, 2005),

In-vitro antioxidant studies DPPH method
The stable 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radical-scavenging activity of the extracts.On accepting hydrogen from a corresponding donor (antioxidant), its solutions lose the characteristic deep purple (λmax 515-517nm) colour.DPPH is very popular for the study of natural antioxidants.Antioxidants reacts with DPPH, which is reduced to the DPPHH and as consequence the absorbance's decreased from the DPPH radical to the DPPH-H form.The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.75µM DPPH solutions in methanol were freshly prepared.Different concentrations of extract and standard ascorbic acid were added to the equal volume of methanolic solution of DPPH.After 30min at room temperature, the absorbance was recorded at 517nm.A control reaction was carried out without the test sample.Radical scavenging activity was calculated by the following formula.% Inhibition=(A0-A1)/ A0 x100 where A0 is the absorbance of control and A1 is the absorbance of extract or standard.IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals.IC50 value was determined by linear regression analysis from the graph plotted between % inhibition and the different concentrations of P. denticulata extracts as well as standarad.(Curcic et al., 2012).

Ferrous chelating activity
On 1mL of each ethanolic extract (200-1000µg/mL) and standard BHT dilutions (100-400µg/mL) was added to a solution of 2mM ferrous chloride (0.2mL).The reaction was initiated by addition of 5mM ferrozine (0.4mL).The total volume was adjusted to 4mL with ethanol.Then, the reaction mixtures were shaken vigorously and kept at room temperature for 10min.Absorbance of all the solutions were measured spectrophoto-metrically at 562nm (Robu et al., 2012).Ferrous chelating activity was calculated by the following formula.% ferrous chelation=(A0-A1)/A0x100 where A0 is the absorbance of control and A1 is the absorbance of extract or standard.

Reducing power Method
Substances, which have reduction potential, react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700nm.1mL of each ethanolic extract (100-400µg/mL), standard ascorbic acid dilutions (20-100 µg/mL) and control sample (1mL distilled water) was mixed with 2.5mL phosphate buffer solution (pH 6.6) and 2.5mL potassium ferricyanide (1%).The final reaction mixtures were properly mixed and incubated at 50ºC for 20min.After incubation, the reaction mixtures were rapidly cooled and mixed with 2.5mL of 10% tri chloroacetic acid.All solutions were centrifuged at 3000rpm for 10min.About 2.5mL of the supernatant was taken from each reaction mixture and 2.5mL distilled water and 0.5mL of ferric chloride (0.1%) was added, mixed well and allowed to stand for 10min.The absorbance was measured at 700nm (Maisarah et al., 2013).

Acute toxicity testing
Acute toxicity was performed for all extract according to the acute toxic classic method (as per OECD guidelines 423).
Female albino rats were used and the animals were kept fasting for overnight providing only water, after which the extract was administered orally at the dose of 300mg/kg and observed for 24h.If the mortality was observed in two out of three animals, then the dose administered was assigned as toxic dose.If the mortality was observed in one animal, then the same dose was repeated to confirm the toxic dose.If mortality was not observed, the procedure was repeated for further higher dose i.e. 2000mg/kg.One tenth of maximum dose of the extract tested for acute toxicity was selected as middle dose i.e. 200mg/kg and double and half of middle dose was selected for higher and lower dose i.e. 400, 100mg/kg.

Experimental induction of diabetes
The rats were injected intraperitonially with streptozotocin 55 mg/kg b.w.dissolved in sterile citrate buffer (1M, pH 4.5).(Aslan et al., 2000, Aslan et al., 2003, and Nilufer et al.,2006)After 72h treatment blood glucose levels of all the rats were determined and rats with blood glucose level above 250mg/dL were selected for the acute study.The animals were randomly divided into six groups after the induction of diabetes.Group I contained normal animals and served as normal control.Group II served as diabetic control.Groups I and II received vehicle1% CMC during the experiment, while the Group III received the reference standard drug glimeperide (0.1mg/kg) and groups from IV to VI received the extract of (100mg/kg, 200mg/kg and 400mg/kg ) respectively.Blood samples were collected just prior to 1, 3, 5 and 7h after drug administration.

RESULTS AND DISCUSSION
Phytochemical analysis of crude extract revealed the presence of carbohydrates, flavanoids, triterpenoids, proteins and steroids (Table I).The ethanolic extract of flowers of P. denticulata shows significant anti-oxidant activity by DPPH method, Reducing power assay and Ferrous chelating method.The IC50 values of extract and ascorbic acid are found to be 127.68 μg/ mL and 6.727 μg/ mL respectively by DPPH method (Table II).The IC50 values of extract and ascorbic acid are found to be 221.5μg/mLand 43.7μg/mL respectively by reducing power assay method (Table III).The IC50 values of extract and BHT are found to be 527.5μg/mLand 165.63μg/mL respectively by ferrous chelating method (Table IV).
This study showed no mortality up to the dose of 2000mg/kg body weight.So, the extract is found to be safe for the experiment.In this study, the plant extract, up to the highest dose (2000mg/kg, p.o.) used in the acute toxicity test, did not cause any death or acute toxicity symptoms in the rats.The LD50, therefore, may be greater than 4000mg/kg (p.o.).This relatively high LD50 shows that the plant extract is non-toxic and/or safe in rats for the study.However, this study did not ascertain the doses used by the practitioners for such treatments.Streptozotocin shows diabetogenic properties by pancreatic beta cell destruction, beta cells normally regulate blood glucose levels  (Junod et al., 1969).STZ also induces oxidative stress or relative overload of oxidants i.e. reactive oxygen species (Wright et al., 1999).P.denticulata flower extract shows very significant invitro anti-oxidant activity.Various studies have shown that diabetes is associated with increased formation of free radicals and a decrease in antioxidant potential.The antioxidant and antidiabetic activity may be due to the presence of flavanoids and triterpenoid saponins.( Tokalov et al., 2004 andAhmad et al.,1990)

CONCLUSION
The results revealed that P.denticulata possess significant antidiabetic and anti-oxidant activity by streptozotocin induced diabetic rats and DPPH method, reducing power assay and ferrous chelating method respectively.Further studies are necessary to elucidate the mechanism of action at cellular levels and components present it.

Table I .
Preliminary phytochemical investigation of P. denticulata

Table II .
Antioxidant activity of ethanolic extract of flower of Primula denticulata by DPPH method.

Table III .
Antioxidant activity of ethanolic extract of flower of Primula denticulata by reducing power assay.

Table IV .
Antioxidant activity of ethanolic extract of flower of Primula denticulata by ferrous

Table V .
Effect of ethanolic extract of the flowers of Primula denticulata on blood glucose level in streptozotocin induced diabetic rats (mg/dl) (Acute Study) *p<0.05, **p<0.01significant compared to diabetic control