doi:10.5455/vetworld.2013.502-507 Haematological and hypoglycemic potential Anethum graveolens seeds extract in normal and diabetic Swiss albino mice

Aim: The present study investigates the hypoglycemic and hematopoetic potential of the seed extracts of Anethum graveolens. Context: A. graveolens (Apiaceae) is an Indian traditional herb. Leaves and seeds of this plant are known for their medicinal properties. Leaves are best used in fresh form while seeds are used in dried form. Materials and Methods: Control and alloxan-induced diabetic mice were orally treated once in a day with aqueous and ethanol extracts for the period of 15 days and the effect of both extracts on body weight, organ weight and blood glucose level were determined. For the hematological study, aqueous (3.04 g/kg), ethanol extracts (6.98 g/kg) and carvone (100 mg/kg body weight) were orally administered once in a day to male Swiss albino mice. While control mice were supplemented with normal saline. Results: A significant decrease (P<0.05) in blood glucose level after administration of aqueous extract was observed along with a significant increase in body and organ weight in alloxan induced diabetic mice after the treatment with plant extracts. Moreover carvone and the aqueous extract significantly increased (P<0.05) red blood cell count (11.94 and 10.42%), hemoglobin (15.55 and 15.06%), Mean Corpuscular Haemogolobin (MCH) (2.80 and 4.80%), Mean Corpuscular Haemoglobin Content (MCHC) (8.16 and 9.57%) when compared with control. While in ethanol extract no such changes were observed except in WBC, which was increased by 9.43%. Conclusion: The result suggests an anti-diabetic property of the aqueous and ethanol extracts. Both extract and carvone also have beneficial effect on hematological parameters.


Introduction
such as anti-microbial [9][10][11], anti-spasmodic, antisecretary, and mucosal protective effects [3], A. graveolens Anethum graveolens L. (Apiaceae), commonly has significant lipid lowering effects and is a promising known as "dill seed", is an annual herb found in cardioprotective agent [12] and antioxidant properties Mediterranean region, Europe, and Central and South have also been reported [13].Asia. A. graveolens is used both as a medicine and an Ingestion of some plant material (either in the raw aromatic herb and is shown to have therapeutic form or their extracts) has been reported to cause properties [1].It is used in Iranian folk medicine as an anaemia, which may result from confiscation of RBC anti-hypercholesterolaemic plant [2].It has traditionally in the spleen, impaired red blood cell production or been used for gastrointestinal ailments, such as primary bone marrow dysfunction [14,15].Diabetes flatulence, indigestion, stomachache colic, and to tract mellitus is a chronic disorder of carbohydrate, fat and intestinal gas [3].Various different compounds have protein metabolism.It represents a heterogeneous group been isolated from the seeds, leaves, and the of disorders that have hyperglycemia as a common inflorescence of this traditional herb; For example; feature [16].It affects over 100 million people worldwide seventeen different volatile compounds have been [17].Its conventional treatment is oral hypoglycemic isolated from this herb [4].Carvone and limonene are agent/insulin therapy [18].However, several herbs are monoterpenes, which are present as the main now being used in the management of diabetes constituent of dill oil from fruits [5].á-Phellandrene, mellitus, although the active principles have been dill ether and myristicin are the compounds, which isolated [19][20][21].In this study, we sought to verify the form the important odor of dill herb [6,7].The presence effect of the administration of aqueous and ethanol of flavonoids, phenolic compounds and essential oil extracts of Anethum graveolens seeds on blood glucose constituents in seeds of A. graveolens make it an level and few haemoglobin parameters, which could important component for the preparation of gripe water serve as indices of anaemia and bone marrow function.[8] A. graveolens has some pharmacological effects, testing was performed for inter-group comparisons Experimental design and treatment schedule: Twenty using the Tukey multiple comparison test at P<0.05.mice selected for study were divided into 5 groups of 4 Whenever sphericity was significant, degree of animals each.Groups were as Group 1: Untreated freedom and F-value are corrected by Huynh Feldt animals (control), Group 2: Alloxan Treated (70 mg/kg epsilon.

Results
Whereas ethanol extract showed no significant change in RBCs and its relating indices.Only HGB, WBC was Effect of extract on body and organ weight: Adminisslightly increased although number of platelets decreased.tration of vehicle (distilled water) in alloxan induced diabetic mice resulted in gradual decrease in body Phytochemical analysis: HPLC chromatogram showed weight during the period of 15 days (Figures 1A and prominent peaks at retention time 17.188 and 46.71% 1B).Aq.Ex and Et.Ex of Anethum did not cause any area (Figure -5).NMR results shows 6.9 ppm due to decrease in body weight and doses were helpful for aromatic protons formed as multiplet, 4.8 ppm doublet alloxan induced diabetic mice for regaining their body due to olefinic proton, 2.7 ppm due to the saturated ring weight.Organ weight was also significantly recovered protons A-broad spectrum, 1.76 ppm two singlet due to (P<0.05) due to treatment (Figure -2).two methyl protons.An IR spectrum exhibits a peak at

Discussion
Experimental studies revealed that the aqueous extract Herbal medicine basic tenets are the interactions of Anethum seeds shows a significant decrease in blood th between different constituents occur in such a fashion glucose level in alloxan induced diabetic mice from 5 that their activity is enhanced and the likelihood of day onwards.
adverse effects is reduced.Such interaction is generally Effect of extract on hematological parameters: The additive or truly synergistic because the compounds effect of aqueous, ethanol extract of A. graveolens and present in the plant interact and produce an effect of carvone on hematological parameters of control and greater than the contribution to each of them singly.For experimental mice is shown in Table-1.Results inducing diabetes alloxan was used which is âindicates, that the administration of plant aqueous cytotoxic to pancreatic beta cells.Induction of diabetes extract and carvone produced significant increase decreases the weight of mice.(P<0.05) in the RBC and factors relating to it (HGB, Dose of alloxan varies with the species for HCT, MCH and MCHC).Though it significantly induction of diabetes.From the results summarized in decreased (P<0.05)platelets and MCV number.
Figure 1 and 2, it is clear that the extracts of the plant    taken for the study have no affect on the body weight of RBC and HGB play a major role in transport of the control mice but it has significant effect on the respiratory gases [36].Aqueous extract and carvone alloxan induced diabetic mice, however the weight of also significantly increased WBC count as compared to control, which indicates that plant aqueous extract and testis and epididymis inclined significantly in treated carvone, is working as a booster dose for immune animals.Moreover, after the treatment, the weight of system.It was found that MCV was not showing any kidney, liver and spleen, increased in the case of A.
significant change while MCH and MCHC were graveolens.
increased from 13.53 to 14.18 pg and 28.41 to 31.13According to Adebayo et al [25], an increase in g/dL, respectively.But there was a decrease in platelets the body weight and organs is an indication of count, which might be results from the inhibitory effect inflammation and decrease is an indication of cell on thrombopoietin [37,38].MCV, MCH and MCHC constriction.According to this statement, the increase values have meticulous significance in anaemia in the weight of testis, epididymis and other organ diagnosis in most animals [39].Increased count of observed in present study might be due to the RBC and its associated parameters suggest that stage as phenomena of inflammation.
polycythemia [40].Therefore it can be concluded that Alloxan causes diabetes through its ability to aqueous extract of Anethum may not have any destroy the insulin producing â cells of the pancreas unfavorable effect on the bone marrow, kidney and [26,27].It leads to cell necrosis and also cause time and haemoglobin metabolism, since the value for RBC are concentration dependent degenerative lesions of the not greatly affected [41].While in the case of ethanol pancreatic â cells which results in the increased blood extract no change was observed on RBCs and its glucose level [28,29].related indices except HGB, which 12.14 g/dL was for Administration of aqueous extract of A. graveolens control and 13.51 g/dL was seen in case of ethanol orally for the period of 15 days produced significant extract treated mice.Ethanol extract showed slight (P<0.05)decrease in blood glucose level of alloxan increase in WBC count whereas no significant effect induced diabetic male mice, as it was earlier reported.
was found on RBC and its indices.Similarly platelets While the effect of ethanolic extract exhibits slight count was also significantly decreased.This implies lesser antidiabetic activity [30][31][32].
that the ethanol extract does not posses any potential of For Haematological parameters, it was also found inducing anaemia throughout the 15 days period of that aqueous extract has positive effect on the administration.HPLC, NMR and IR peaks depict the haemopoeitic system of mus musculus.It was showing presence of carvone in aqueous extract of seeds.The significant increase in RBC, HCT.The rise in production phytochemical screening of the seed showed the of RBCs (erythropoiesis) shows that aqueous extract of presence of, tannins terpinoids saponins flavonoids, anethum and carvone have the probablility of anthraquinones and alkaloids [42,43].provoking erythropoietic release from kidney, which Conclusion act as a humoral regulator for RBC production [33,34], as earlier reported in case of Mangifera indica L. [35].
From the above results, it may be concluded that The percentage increase in HCT was from 42.73% for Anethum graveolens aqueous extract is constitute of th control to 46.57% for group 3 and 46.67% in 5 group carvone which is responsible for its potency for curing mice.The raised haematocrit is an indication of increased diabetes and can be used for the treatment of anaemia.haemoconcentration.Administration of aqueous While ethanol extract is having lesser anti-diabetic extract and carvone also significantly increased the activity and no significant affect on Haematological haemoglobin level which shows that it will help in parameters.Thus, aqueous extract is more potent than increasing the oxygen carrying capacity of the blood.ethanol extract.

Figure- 1 .
Figure-1.Effect of Aq.Ex and Et.Ex of A graveolens L. on body weight expressed as mean ±SD.

Figure- 2 .
Figure-2.Effect of Aq.Ex and Et.Ex of A graveolens L. on organ weight expressed as mean ±SD.

Figure- 3 .
Figure-3.Effect of Aq.Ex and Et.Ex of A graveolens L. on blood glucose level (fasting) expressed as mean ±SD.

Figure- 4 .
Figure-4.Effect of Aq.Ex and Et.Ex of A graveolens L. on Blood glucose level (PP) expressed as mean ±SD.

a
-mean value of six readings (Mean ± SD) b -Data significant at P<0.05 as compared to control c -Data siginificant at P<0.05 as compared to aqueous extract treated groups d -Data significant at P<0.05 as compared to ethanolic extract treated groups doi:10.5455/vetworld.2013.502-507

MCH (Mean Corpuscular Haemogolobin) fed
Kendra Jabalpur, M.P., India.Rajasthan University of B.W), Group 3: Diabetic Mice given with Aq.Ex at Herbarium incharge Roop Singh properly identified 3.04 g/kg for 15 days, Group 4: Diabetic Mice given the plant with voucher specimen in the herbarium of with Et.Ex at 6.98 g/kg for 15 days, and Group 5: Department of Botany, Rajasthan University, Jaipur.Diabetic mice given with Carvone 100 mg/kg for 15 days.All the samples were air dried in shade and then Fasting and PP blood glucose level, body weight powdered.The seed powder was extracted using were recorded periodically.The blood samples were maceration with ethanol (80 v/v) and water for 3 days collected from tail vein for measuring blood glucose and, subsequently, the mixture was filtered (Wattman level and for hematological parameters.Blood sample filter paper 1) and concentrated under reduced pressure was collected from retro-orbital plexus using micro- doi:10.5455/vetworld.2013.502-507Experimental animals : A total 20 adult male Swiss hematocrit tubes, Haemoglobin by Sahli's method [24] albino mice (age 12-14 weeks) weighing 30 g were and certain hematological indices (MCV, MCH and used in this study.The mice were housed in university MCHC) were calculated with formula; animal house facility with controlled temperature (22 ± o MCV (Mean Corpuscular Volume) 2 C) under a 12/12 h light/dark schedule.Mice were MCV in cubic microns=PCV X 10/RBC (in maintained under hygienic conditions in wellmillion per cubic mm) ventilated room of animal house.All the animals were twice a day with animal pellet feed (Hindustan MCH in Picograms=Haemoglobin in g/100 ml x Lever Limited, Mumbai).Tap water was provided ad 10/RBC (in million per cubic mm) libitum.Animals in each group were housed in

MCHC (Mean Corpuscular Haemoglobin Content) polypropylene
cages.General body weight of the MCHC in (g/dl)=Haemoglobin in g/100 x 100/ PCV animals was monitored regularly during entire tenure of the experiment.Characterization of phytoconstituents: For characterization of compounds HPLC, IR and NMR were performed.Variance (ANOVA) using the SPSS (Ver.16).Post hoc glucometer (Dr.Morepen Gluco Monitor).

Table - 1
. Effect of Aq.Ex and Et.Ex of Anethum graveolens L. on hematological parameter expressed as mean ±SD.