Journal of Ethnopharmacology 151 (2014) 618–623
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Evaluation of antidiabetic and antihyperlipidemic activity of Artemisia indica linn (aeriel parts) in Streptozotocin induced diabetic rats Waqar Ahmad a, Imran Khan a, Mir Azam Khan a, Manzoor Ahmad b, Fazal Subhan c, Nasiara Karim a,n a
Department of Pharmacy, University of Malakand, Chakdara, Pakistan Department of Chemistry, University of Malakand, Chakdara, Pakistan c Department of Pharmacy, University of Peshawar, Peshawar, Pakistan b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 July 2013 Received in revised form 28 October 2013 Accepted 9 November 2013 Available online 16 November 2013
Ethnopharmacological relevance: Diabetes mellitus is a major metabolic disorder affecting a huge population all over the world. Artemisia species have been extensively used for the management of diabetes in folkloric medicine. The present study is designed to investigate the antidiabetic and antihyperlipidemic effects of aeriel parts of Artemisia indica. Materials and methods: Hydromethanolic crude extracts, chloroform, ethyl acetate and n-butanol fractions of aerial parts of Artemisia indica were tested for their antidiabetic potential in Streptozotocin (STZ) (50 mg/kg, i.p.) induced diabetic Sprague-Dawley rats. Blood glucose level, body weight, serum lipid profile and activities of liver enzymes were determined. The extracts were further subjected to preliminary phytochemical analysis. Results: A daily oral dose of hydromethanolic crude extracts (200 and 400 mg/kg b.w.) and chloroform fraction (200 mg/kg b.w.) of Artemisia indica for 15 days showed a significant reduction in blood glucose level which was comparable to that of the standard antidiabetic drug, glibenclamide (500 μg/kg, p.o.). Artemisia indica extracts also showed reduction in total cholesterol, triglycerides and low density lipoproteins as well as serum creatinine level, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in diabetic rats. Conclusion: According to the results Artemisia indica possesses hypoglycemic, antihyperlipidemic and valuable effects on liver and renal functions in diabetic rats, which seems to validate its traditional usage. & 2013 Published by Elsevier Ireland Ltd.
Keywords: Hypoglycemic Artemisia indica Asteraceae Glibenclamide Streptozotocin-induced diabetes
1. Introduction Diabetes mellitus is one of the most severe and incurable metabolic disorders characterized by increased blood glucose level as a result of an absolute or relative lack of insulin and failure of insulin to act on its targets tissue (Valiathan, 1998). The acute and chronic manifestations of hyperglycemia cause microvascular (retinopathy, neuropathy and nephropathy) and macrovascular complications (Coronary artery disease leading to myocardial infarction, diabetic ketoacidosis, nonketotic hyperosmolar coma and diabetic coma; Fowler, 2008). It is a leading factor of multiple disorders due to chronic hyperglycemia and causes abnormalities in glucose, lipids and protein metabolism (Kahn et al., 1976). India, China and United States are the largest countries presenting more than 30 million diabetic people and the incidence is increasing day by day (Wild et al., 2004). Several workers described the treatment
n
Corresponding author. Tel.: þ 92 300 957 3590. E-mail address: [email protected] (N. Karim).
0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2013.11.012
of diabetes mellitus based on the interaction of nutritional agents. These agents limit the availability of fatty acids, inhibit gluconeogenesis (Moneva and Dagogo-Jack, 2003) and enhance lipolysis (Bebernitz and Schuster, 2002). Furthermore, they promote fatty acid oxidation in a futile cycle that does not yield metabolic energy and stimulate peripheral glucose utilization (Bebernitz and Schuster, 2002). More than 800 plants have been reported to have antihyperglycemic effects with less adverse effects and low toxicity as compared to synthetic compounds (Kirithikar and Basu, 1995; Nadkarni, 1976). According to the World Health Organization (WHO), almost 70% of the diabetic patients use plants as a primary source of antidiabetic agents in order to satisfy their principal health needs (Bailey and Day, 1989). Artemisia indica also known as “mugwort” belongs to family Astereaceae. The plant is a perennial shrub with a height of 2–8 m and is found in northe rn areas of Pakistan, as well as in cold temperate zones of Asia. Artemisia species have been reported to exhibit antiplasmodial (Tran et al., 2003), antispasmodic and bronchodilator (Khan and Gilani, 2009), antihypertensive (Tigno et al., 2000), antiallergic
W. Ahmad et al. / Journal of Ethnopharmacology 151 (2014) 618–623
(Rodrigues-Alves et al., 2008), hepato-protective (Gilani et al., 2005), antibacterial (Chen et al., 1989) and antinociceptive effects (Pires et al., 2009). Furthermore, various species of Artemisia have been traditionally used for their antihyperglycemic effects in Mexico (Andrade-Cetto and Heinrich, 2005). Therefore, keeping in view the medicinal importance of the plant and extensive folkloric use in rural Mexican communities as an antidiabetic plant, this study was planned to explore the antidiabetic activity of Artemisia indica on scientific grounds.
2. Materials and methods 2.1. Chemicals Streptozotocin (Sigma-Aldrich), glibenclamide (Sanofi Aventis Pharma (Pvt.) Ltd., Pakistan) and glucose estimation kits (S.D Chek Gold Germany) were used in this study. The different organic solvents and chemicals used for extraction were purchased from local suppliers of Merck, Germany. Other reagents used in this study were Tween-80 (Scharlau chem., Spain), normal saline (Utsoka Pharma (Pvt.) Ltd., Pakistan), biochemical reagents for lipid profile, LFTs Kits (Human, Germany) and RFTs kit (Bioneed Germany diagnostic).
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identification of different constituents like alkaloids, flavonoids, terpenoids, glycosides, saponins and tannins by using standard qualitative methods described by Trease and Evans (1983). 2.6. Acute toxicity study of the crude methanolic extract of Artemisia indica The acute toxicity of the crude methanolic extract of Artemisia indica was determined by using Sprague-Dawley rats (150–200 g), according to the method described by Irwin (1968) and described elsewhere (Karim et al., 2012). The animals were divided into six groups (n¼ 6). One group served as a control and received Tween80 suspension orally. The crude methanolic extract was given in a dose of 250, 500, 1000, 1500 and 2000 mg/kg b.w. to each rat orally. All the doses of the extracts were prepared by dissolving the extract in Tween-80 suspension prior to administration. The animals were observed at 0, 30 and 60 min, 24, 48 and 72 h and 1 week after administration for any kind of behavioral, physical and pharmacological toxic effects, respectively. Since the extract was found safe up to the dose level of 2000 mg/kg b.w., a dose of 200 mg/kg b.w. (1/10 of 2000 mg/kg b.w.) of the extracts was selected according to OECD guidelines for screening of the antidiabetic activity (Sharma et al., 2010). 2.7. Induction of hyperglycemia
2.2. Animals Adult Sprague-Dawley rats in the weight range of 150–200 g were purchased from the Department of Pharmacy, University of Peshawar. Animals were housed in the Department's animal house with fresh water and standard food available ad libitum. The animals were maintained at 12 h light and dark cycles and with room temperature maintained at 22–25 1C in the animal house. All animal procedures have been approved by the Departmental Animal Ethical Committee (DAEC/PHARM/2012/10) and were conducted according to the UK Animal Scientific Procedure Act, 1986. 2.3. Plant material The aerial parts of the plant were collected from upper Dir (Sherin Gal) Khyber Pakhtunkhwa, Pakistan, in the month of July 2008 and were authenticated by a taxonomist. The plant specimen was deposited in the Department of Pharmacy, University of Malakand, Pakistan. A voucher specimen number (200300106) was assigned at the same institution. 2.4. Preparation of extracts The shade dried aerial parts of the plant were coarsely ground with the help of a dry grinder followed by extraction with 70% methanol (three times) and filtered through a piece of cloth (plant material to solvent ratio was 1:5 w/v). The extract was concentrated to a semisolid mass using a rotary evaporator under reduced pressure at 45 1C for a final extract yield of 20% (w/w). The semisolid mass was fractionated with various organic solvents: ethyl acetate, chloroform and n-butanol. Each fraction was finally evaporated to semisolid masses by the rotary evaporator. The yields of ethyl acetate, chloroform and n-butanol were 3.8%, 5.5% and 4.3% w/w respectively. For antidiabetic activity, the extract and its fractions were formulated as suspensions in normal saline with 5% Tween-80 as the suspending agent. 2.5. Preliminary phytochemical tests The crude methanolic extract, chloroform and ethyl acetate fractions of Artemisia indica were subjected to qualitative tests for
Hyperglycemia was induced in Sprague-Dawley rats by a single intra peritoneal (i.p.) injection of 50 mg/kg of Streptozotocin (STZ) reconstituted in normal saline (0.9%) after overnight fasting. After 72 h of STZ administration, blood glucose levels were measured in blood samples collected from tail vein puncture with one touch Glucometer strips using an SD glucometer (Germany). Rats with fasting blood glucose levels more than 300 mg/dl were considered diabetic and selected for the study. 2.8. Experimental design Rats that fasted overnight for 12 h were randomly divided into six groups (eight rats in each group). The first group served as normal control (non-diabetic) and received normal saline whereas the second group served as diabetic control and received 5% Tween-80 suspension. The third group received standard drug glibenclamide (500 μg/kg, p.o.). The fourth and fifth groups received hydromethanolic extracts of Artemisia indica at dose levels of 200 and 400 mg/kg (p.o.) respectively, whereas the sixth, seventh and eighth groups received chloroform, ethyl acetate and n-butanol fractions of Artemisia indica respectively, at a dose of 200 mg/kg orally. The treatment of crude extract and fractions was continued once daily at 09:00 a.m. for 15 days. Body weight and blood glucose levels were estimated on the 0th, 4th, 7th, 10th and 15th day of treatment (Gupta et al., 2004). 2.9. Estimation of serum lipid and liver physiological profile After completion of antidiabetic assay on the 15th day, all animals were anesthetized by pentobarbital sodium (35 mg/kg) and euthanized by cervical decapitation using the method described in schedule 1 of the Animal Scientific Procedure Act 1986 and blood samples were collected through cardiac puncture for biochemical parameters studies (Nagappa et al., 2003). Collected blood was centrifuged at 1500g for 10 min for serum separation. The serum sample was then analyzed by a spectrophotometer (Perkin-Elmer, Germany) for determination of serum SGPT, serum SGOT and serum alkaline phosphatase (ALP) using a standard IFCC kinetic Method (Bioneed kit, Germany). Total cholesterol (TC), triglycerides (TGs), low
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density lipoproteins (LDL), high density lipoprotein (HDL) and serum creatinine were determined by CHOD-PAP and GPO-PAP methods on a UV-Spectrophotometer using a Human kit, Germany.
well with no observable changes in behavior or appearance. No deaths were observed up to 1 week of study.
2.10. Statistical analysis
3.3. Antihyperglycemic effect of Artemisia indica
All the values of blood glucose, body weight, and biochemical parameters were represented as mean7S.E.M. When two groups were compared, Student's t-test was used and when more than two groups were compared, one way ANOVA followed by Dunnett's posthoc multiple comparison test was used. Differences between groups were considered significant at po0.05.
Changes in blood glucose level in normal, diabetic and on treatment for diabetes rats with the crude methanolic extract and various fractions of Artemisia indica and glibenclamide are presented in Table 1. Oral administration of the crude methanolic extract (200 and 400 mg/kg) caused a significant (po 0.01, n ¼8; one way ANOVA with Dunnett's posthoc test) reduction in blood glucose level compared to diabetic control. Crude methanolic extract 200 and 400 mg decreased blood glucose level from 477.77 18 to 244.7 712 mg/dl and from 476.7 753 to 240.6 7 10 mg/dl respectively. The effect was evident from the 7th day onwards. The chloroform fraction (200 mg/kg) retained the antihyperglycemic effect of the crude methanolic extract and significantly (p o0.01, n ¼8; one way ANOVA with Dunnett's posthoc test) reduced blood glucose level from 473 752 to 220.8 7 13.1 mg/dl. With the chloroform fraction, the effect was evident from the 4th day onwards. Group treated with standard drug glibenclamide (500 μg/kg, p.o.) showed similar results and caused significant reduction (p o0.01, n¼ 8; one way ANOVA with Dunnett's posthoc test) in blood glucose level, decreasing the mean blood glucose level from 460.8 743 to 206.2 7 40.8 mg/dl (Table 1). The antihyperglycemic effect of the crude methanolic extract and chloroform fraction of Artemisia indica was comparable to that of glibenclamide. The groups treated with the ethyl acetate and n-butanol (200 mg/kg) fractions showed no significant changes in blood glucose level (p 40.05, n ¼8; one way ANOVA with Dunnett's posthoc test) (Table 1).
3. Results 3.1. Phytochemical tests Preliminary phytochemical analysis of the crude methanolic extract and chloroform fraction showed the presence of alkaloids, flavonoids, glycosides, terpenoids, saponins and tannins whereas alkaloids were absent in the ethyl acetate fraction. 3.2. Acute toxicity tests Acute toxicity studies revealed that the administration of crude methanolic extract (250–2000 mg/kg) of Artemisia indica did not produce significant changes in the behavior of the animals as observed by lack of convulsions, respiratory distress, writhing, changes to reflex activity or mortality. A slight increase in irritability and escape behavior was observed at 2000 mg/kg in three out of eight animals. At 24 h–1 week, all animals seemed
Table 1 Effect of daily oral administration of Artemisia indica extracts or glibenclamide on blood glucose level of STZ-induced diabetic rats. Sl. no.
Groups
Dose (mg/kg)
1st day
4th day
7th day
10th day
15th day
1 2 3 4 5 6 7 8
Diabetic control Normal control saline Glibenclamide Crude methanolic extract Crude methanolic extract Chloroform fraction Ethyl acetate fraction n-butanol fraction
0.4 ml 0.4 ml 0.5 200 400 200 200 200
471.3 7 37 1007 11 460.8 7 43 477.7 7 18 476.7 7 53 4737 52 4877 38 4727 32
484.77 30 92.3 7 8 300.7 7 45nn 476.3 7 22 489.27 8 259.8 7 58nn 458 7 23 476.5 7 58
500.8 7 50 92.3 7 6 294.5 7 50 345.7 7 20nn 269.8 7 10nn 242.17 16nn 507.3 7 21 493.87 30
506 7 34 91.3 7 7 248.8 7 53nn 309.5 7 12nn 254.8 7 7nn 226.2 7 14nn 495.37 24 4977 18
516.7 7 50 91.5 7 5 206.27 40nn 244.37 12nn 240.67 10nn 220.8 7 13nn 510.3 7 32 490.27 58
The values are expressed as mean 7 SEM. n¼ 8 in each group. nnpo 0.01 as compared with diabetic control at the same time (one way ANOVA followed by Dunnett's multiple comparison test).
Table 2 Effects of various extracts of Artemisia indica on body weight in STZ -induced diabetic rats. Sl. no.
1 2 3 4 5 6 7 8
Change in body weight (g) at days
% change in body weight
Treatments
Dose (mg/kg)
0
4
7
10
15
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
153 70.5 152 77 153 74 152 73 154 74 151 75 153 77 154 75
155 79 148 75n 157 73n 155 75n 155 76n 154 75n 147 74 149 73
165 70.9 145 75nn 160 76n 157 77n 158 74n 156 73n 142 72 145 74
1707 0.9 1407 3nn 164 7 4nn 1607 5nn 1627 3nn 1627 4nn 1397 4 1407 3
1787 3 1337 4nnn 168 7 5nn 164 7 4nn 1657 6nn 1677 5nn 1357 4 1347 4
Normal (control) Diabetic (control) c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol fraction b
12.5 þ 9.8 þ 7.8 þ 7.1 þ 10.6 11.7 13.0
The values are expressed as mean7 SEM. Each value corresponds to a mean of eight animals. np o0.05, nnp o 0.01, nnnp o 0.001; comparison of a(normal control) vs b(diabetic control) (Student t-test), np o 0.05, nnpo 0.01; comparison of b(diabetic control) vs c(Glibenclamide and extracts treated groups) (one way ANOVA followed by Dunnett's posthoc multiple comparison test). f inalweightðgÞ %changeinb:w: ¼ initialweightðgÞ 100 inititalweightðgÞ
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3.4. Effects of Artemisia indica on body weight in diabetic rats The results of changes in body weight of control and experimental rats treated with extracts and glibenclamide are shown in Table 2. STZ-induced diabetic rats produced significant (po 0.001, n ¼8; Student's t test) loss in body weight as compared to normal rats during the study (Table 2). In diabetic control rats, continued weight loss was observed till the end of the study (15 days treatment). During this period, 12.5% reduction in their body weight (Table 2) was recorded. STZ mediated body weight reduction was reversed by crude methanolic extract (200 and 400 mg/ kg b.w.) and chloroform fraction (200 mg/kg b.w.). At the end of 15 days treatment, the crude methanolic extract 200 and 400 mg/kg caused increases in body weight by 7.1% and 7.8% respectively. Similarly, the chloroform fraction (200 mg/kg) also caused an increase in body weight by 10.6%. The ethyl acetate and nbutanol fractions (200 mg/kg b.w.) did not cause any improvement in body weight compared to diabetic control (Table 2). 3.5. Effects of Artemisia indica on lipid profile in Streptozotocin induced diabetic rats The lipid profiles in control and experimental rats are depicted in Table 3. In STZ-induced diabetic control rats, there was a significant (po 0.01, n ¼8; Student's t test) increase in total cholesterol, TG, LDL cholesterol and serum creatinine compared to normal control. In addition, there was a significant decrease (p o0.05, n¼ 8; Student's t test) in HDL cholesterol in diabetic control rats in comparison to normal control. Administration of the crude methanolic extract (200 and 400 mg/ kg) and chloroform fraction (200 mg/kg) for 15 days showed a significant (po0.01, po0.001, n¼ 8; one way ANOVA with Dunnett's posthoc test) reduction in total cholesterol, TGs and LDL cholesterol
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in comparison to diabetic control rats. The effect was comparable to that of the standard drug glibenclamide (500 μg/kg). The ethyl acetate fraction also significantly (po0.05, n¼8; one way ANOVA with Dunnett's posthoc test) reduced total cholesterol and LDL cholesterol but had no effect on TG. Furthermore, the crude methanolic extract (200 and 400 mg/kg), chloroform, n-butanol, and ethyl acetate fractions (200 mg/kg) and glibenclamide (500 μg/kg) significantly (po0.05, po0.01, n¼8; one way ANOVA with Dunnett's posthoc test) increased HDL cholesterol in diabetic rats.
3.6. Effect of Artemisia indica on liver function in Streptozotocin induced diabetic rats Table 4 summarized the effects of STZ on the activity of hepatic marker enzymes in serum. In the present study the levels of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in STZ-induced diabetic rats were elevated. Administration of the crude methanolic extract (200 and 400 mg/kg) and the chloroform (200 mg/kg) fraction of Artemisia indica significantly reduced (p o0.01, n¼ 8; one way ANOVA with Dunnett's posthoc test) serum SGPT, SGOT and ALP in rats intoxicated with STZ. The restoration of hepatic enzymes observed with the crude methanolic (200 and 400 mg/kg) and chloroform (200 mg/kg) fractions of Artemisia indica was comparable to standard drug glibenclamide (p o0.01, n ¼ 8; one way ANOVA with Dunnett's posthoc test). The crude methanolic extract (200 and 400 mg/kg) and the chloroform fraction (200 mg/kg) also caused a significant reduction (p o0.001, n ¼8; one way ANOVA with Dunnett's posthoc test) in serum creatinine comparable to standard drug glibenclamide. The n-butanol (200 mg/kg) and ethyl acetate (200 mg/kg) fractions slightly decreased serum SGPT, SGOT, ALP and creatinine;
Table 3 Antihyperlipidemic effect Artemisia indica on STZ-induced diabetic rats. Sl. no.
Treatment dose (mg/kg)
Dose (mg/kg)
Total cholesterol (mg/dl)
TG (mg/dl)
HDL (mg/dl)
LDL (mg/dl)
1 2 3 4 5 6 7 8
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
120 7 9.12 185.3 7 4.4nn 137.7 7 5.3nn 144.2 7 4.3nn 140.5 7 7.5nn 134.4 7 2.7nn 168.27 3.6n 167.9 7 9.8n
1257 8.80 169.0 7 7.9nn 128.3 7 6.5nn 138.5 7 4.7nn 136.8 7 4.5nn 136.2 7 8.1nn 165.8 7 9.0 146.8 7 3.2n
347 2.2 30.2 7 2.4n 39.17 2.3nn 37.5 7 3.1nn 35.67 5.5nn 38.2 7 1.5nn 36.17 2.5n 36.2 7 2n
817 2.5 177.4 7 8.9nn 90.3 7 3.5nnn 95.6 7 3.2nnn 93.5 7 3.6nnn 81.3 7 4.0nnn 151.8 7 8.0n 149.87 7.7n
Normal control Diabetic Control c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol b
Each value is mean7 SEM of eight animals. Comparisons were made between anormal control and bdiabetic control using Student t-test (np o 0.05, nnp o 0.01) and between b diabetic control and c(Glibenclamide/extracts) treated groups using one way ANOVA followed by Dunnett's posthoc multiple comparison test (np o 0.05, nnp o 0.01, nnn p o 0.001).
Table 4 Effect of various extracts of Artemisia indica on serum profile in STZ-induced diabetic rats. Sl. no.
Treatment
Dose (mg/kg)
(SGPT) IU
(SGOT) IU
(ALP) IU
Serum creatinine (mg/dl)
1 2 3 4 5 6 7 8
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
157 4.4 52.2 7 11.8 17.6 7 4.5nn 22.4 7 9.0nn 19.4 7 9.0nn 18.8 7 1.5nn 50.5 7 3.5 48.87 4.0
18 73.5 41.4 742.1nn 17.5 72.6nn 21.9 74.9nn 18.9 74.9nn 19.9 74.1nn 39.5 73.8 37.0 73.9
180 720.5 290.8 716.3nn 195.9 75.3nn 203.2 714.52nn 199.2 714.52nn 183.6 712.5nn 281.3 731.2 272.5 710.3
0.52 7 0.2 2.5 7 0.2nn 0.5 7 0.1nnn 0.78 7 0.1nnn 0.6 7 0.2nnn 0.8 7 0.1nnn 2.2 7 0.2 2.3 7 0.3
Normal control Diabetic control c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol fraction b
Each value is mean 7 SEM of eight animals; comparisons were made between anormal control and bdiabetic control using Student t-test (nnp o 0.01) and between bdiabetic control and cGlibenclamide/extract treated groups using one way ANOVA followed by Dunnett's posthoc multiple comparison test (np o 0.05, nnp o 0.01, nnnp o0.001).
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however the effect was not significant (p 40.05, n ¼8; one way ANOVA with Dunnett's posthoc test; Table 4).
bioactive compounds present in the plant extracts and for elucidation of their molecular mechanisms.
Acknowledgments 4. Discussion Despite the presence of known antidiabetic prescription medicines, herbal drugs and preparations are of considerable interest for the ethno-botanical community and are considered to be less toxic and free from adverse effects than synthetic agents (Atmakuri and Dathi, 2010; Pari and Umamaheswari, 2000). STZ is an antibiotic produced by Streptomyces achromogenes. In experimental animals it causes irreversible destruction of pancreatic beta cells, and is mostly used to induce diabetes mellitus in experimental animals through its toxic effects (Kim et al., 2003; Matteucci and Giampietro, 2008). The results of the present study indicate that the extracts of Artemisia indica reduced blood glucose levels in the diabetogenic animal model with STZ (50 mg/kg). The crude methanolic extract and the chloroform fraction of Artemisia indica plant demonstrated a significant lowering of blood glucose level (BGL). The antihyperglycemic effect of Artemisia indica may be due to prevention of free radical formation induced by STZ. Elevated plasma total cholesterol, triglycerides and LDL cholesterol are the major risk factors of cardiovascular diseases. Diabetic rats showed elevated plasma total cholesterol, triglycerides and LDL cholesterol. The crude methanolic extract (200 and 400 mg/kg) and the chloroform fraction (200 mg/kg) reduced the lipid profile and caused a reduction in blood glucose level. STZ-induced diabetes was characterized by severe loss in body weight (Chen and Ianuzzo, 1982). The crude methanolic extract at the dose level of 200 and 400 mg/kg and the chloroform fraction (200 mg/kg) also showed improvement in body weight. Improvement in body weight could be due to the ability of Artemisia indica extracts to reduce hyperglycemia (Pillai et al., 2012). Reports have shown that the liver cells are irreversibly destroyed in STZ-induced diabetic rats, causing the liver microsomal cells to release different enzymes, including SGPT, SGOT and serum ALP in the blood (Daisy et al., 2008). It leads to an increased level of these enzymes in the plasma. The crude extract and the chloroform fraction of Artemisia indica significantly lowered the SGPT, SGOT and ALP levels. Chronic hyperglycemia induces elevation of serum creatinine and serum uric acid leading to renal dysfunction (Almdal and Vilstrup, 1988). The results from the present study show that Artemisia indica can also reduce the level of serum creatinine in STZ-induced diabetic rats that further helps in treating and preventing the renal system from damage. These data confirmed the possibilities that the major function of these extracts is on protective effects of major tissues such as kidney, liver and pancreas, thereby reducing the incidence of diabetes in experimental animals.
5. Conclusion We conclude from the above results and discussion on the plant Artemisia indica that the crude methanolic and chloroform extracts have significant hypoglycemic and antidiabetic potentials. These extracts improve effects on body weight. They can also improve the condition of diabetes mellitus as indicated by parameters such as body weight, lipid profiles and a reduction in serum creatinine, serum SGPT, serum SGOT and ALP in STZ-induced diabetic rats. Furthermore, this study provides scientific evidence for the ethnobotanical use of Artemisia indica in the treatment of diabetes mellitus. However, further studies are warranted for the isolation and purification of
The authors are thankful to the Higher Education Commission of Pakistan for financing this research from beginning till completion. They would also like to thank Professor Mary Collins and Graham AR Johnston, University of Sydney, for helping with the revision of this manuscript. References Andrade-Cetto, A., Heinrich, M., 2005. Mexican plants with hypoglycaemic effect used in the treatment of diabetes. J. Ethnopharmacol. 99, 325–348. Almdal, T.P., Vilstrup, H., 1988. Strict insulin therapy normalises organ nitrogen contents and the capacity of urea nitrogen synthesis in experimental diabetes in rats. Diabetologia 31, 114–118. Atmakuri, L.R., Dathi, S., 2010. Current trends in herbal medicines. J. Pharm. Res. 3, 109–113. Bailey, C.J., Day, C., 1989. Traditional plant medicines as treatments for diabetes. Diabetes Care 12, 553–564. Bebernitz, G.R., Schuster, H.F., 2002. The impact of fatty acid oxidation on energy utilization: targets and therapy. Curr. Pharm. Des. 8, 1199–1227. Chen, C.P., Lin, C.C., Namba, T., 1989. Screening of Taiwanese crude drugs for antibacterial activity against Streptococcus mutans. J. Ethnopharmacol. 27, 285–295. Chen, V., Ianuzzo, C.D., 1982. Dosage effect of Streptozotocin on rat tissue enzyme activities and glycogen concentration. Can. J. Physiol. Pharmacol. 60, 1251–1256. Daisy, P., Eliza, J., Ignacimuthu, S., 2008. Influence of Costus speciosus (Koen.) rhizome extracts on biochemical parameters in Streptozotocin induced diabetic rats. J. Health Sci. 54, 675–681. Fowler, M.J., 2008. Microvascular and macrovascular complications of diabetes. Clin. Diabetes 26, 77–82. Gilani, A.H., Yaeesh, S., Jamal, Q., Ghayur, M.N., 2005. Hepatoprotective activity of aqueous-methanol extract of Artemisia vulgaris. Phytother. Res. 19, 170–172. Gupta, S., Kataria, M., Gupta, P.K., Murganandan, S., Yashroy, R.C., 2004. Protective role of extracts of neem seeds in diabetes caused by Streptozotocin in rats. J. Ethnopharmacol. 90, 185–189. Irwin, S., 1968. Comprehensive observational assessment: a systematic, quantitative procedure for assessing the behavioural and physiological state of the mouse. Psychopharmacologia 13, 222–257. Kahn, C.R., Flier, J.S., Bar, R.S., Archer, J.A., Gorden, P., Martin, M.M., Roth, J., 1976. The syndromes of insulin resistance and acanthosis nigrican-insulin-receptor disorders in man. N. Engl. J. Med. 294, 739–745. Karim, N., Curmi, J., Gavande, N., Johnston, G.A., Hanrahan, J.R., Tierney, M.L., Chebib, M., 2012. 2′-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors. Br. J. Pharmacol. 165, 880–896. Khan, A.U., Gilani, A.H., 2009. Antispasmodic and bronchodilator activities of Artemisia vulgaris are mediated through dual blockade of muscarinic receptors and calcium influx. J. Ethnopharmacol. 126, 480–486. Kim, M.J., Ryu, G.R., Chung, J.S., Sim, S.S., Min, D.S., Rhie, D.J., Yoon, S.H., Hahn, S.J., Kim, M.S., Jo, Y.H., 2003. Protective effects of epicatechin against the toxic effects of Streptozotocin on rat pancreatic islets: in vivo and in vitro. Pancreas 26, 292–299. Kirithikar, K.R., Basu, B.D., 1995. Indian Medicinal Plants. International Book Distributors, Dehradun, India. Matteucci, E., Giampietro, O., 2008. Proposal open for discussion: defining agreed diagnostic procedures in experimental diabetes research. J. Ethnopharmacol. 115, 163–172. Moneva, A.A., Dagogo-Jack, S., 2003. Multiple targets in the management of type 2 diabetes mellitus. Curr. Drug Targets 3, 203–221. Nadkarni, K.M., 1976. Indian Material Medica. Popular Prakashan, Bombay, India. Nagappa, A.N., Thakurdesai, P.A., Venkat Rao, N., Singh, J., 2003. Antidiabetic activity of Terminalia catappa Linn fruits. J. Ethnopharmacol. 88, 45–50. Pari, L., Umamaheswari, J., 2000. Antihyperglycaemic activity of Musa sapientum flowers: effect on lipid peroxidation in alloxan diabetic rats. Phytother. Res. 14, 136–138. Pillai, K.K., Chidambaranathan, N., Halith, M.M., Jayaprakash, S., Narayanan, N., 2012. Antihyperglycemic effect of alcoholic extracts of cnidoscolus chayamansa in experimental diabetes and their effects on key metabolic enzymes involved in carbohydrate metabolism. Int. J. Res. Pharm. Chem. 2, 179–187. Pires, J.M., Mendes, F.R., Negri, G., Duarte-Almeida, J.M., Carlini, E.A., 2009. Antinociceptive peripheral effect of Achillea millefolium L. and Artemisia vulgaris L.: both plants known popularly by brand names of analgesic drugs. Phytother. Res. 23, 212–219. Rodrigues-Alves, R., Pregal, A., Pereira-Santos, M.C., Branco-Ferreira, M., Lundberg, M., Oman, H., Pereira-Barbosa, M., 2008. Anaphylaxis to pine nut: crossreactivity to Artemisia vulgaris. Allergol. Immunopathol. 36, 113–116. Sharma, B., Salunke, R., Balomajumder, C., Daniel, S., Roy, P., 2010. Anti-diabetic potential of alkaloid rich fraction from Capparis decidua on diabetic mice. J. Ethnopharmacol. 127, 457–462.
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Tigno, X.T., Guzman, F., Ma, A., Flora, T.V., 2000. Phytochemical analysis and hemodynamic actions of Artemisia vulgaris L. Clin. Hemorheol. Microcirc. 86, 249–252. Tran, Q.L., Tezuka, Y., Ueda, J.Y., Nguyen, N.T., Maruyama, Y., Begum, K., Kim, H.S., Wataya, Y., Tran, Q.K., Kadota, S., 2003. In vitro antiplasmodial activity of antimalarial medicinal plants used in Vietnamese traditional medicine. J. Ethnopharmacol. 86, 249–252.
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Trease, G.E., Evans, W.C., 1983. Text Book of Pharmacognosy. Tindall and Company Publisher, London. Valiathan, M.S., 1998. Healing plants. Curr. Sci. 75, 1122–1127. Wild, S., Roglic, G., Green, A., Sicree, R., King, H., 2004. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27, 1047–1053.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Evaluation of antidiabetic and antihyperlipidemic activity of Artemisia indica linn (aeriel parts) in Streptozotocin induced diabetic rats Waqar Ahmad a, Imran Khan a, Mir Azam Khan a, Manzoor Ahmad b, Fazal Subhan c, Nasiara Karim a,n a
Department of Pharmacy, University of Malakand, Chakdara, Pakistan Department of Chemistry, University of Malakand, Chakdara, Pakistan c Department of Pharmacy, University of Peshawar, Peshawar, Pakistan b
art ic l e i nf o
a b s t r a c t
Article history: Received 21 July 2013 Received in revised form 28 October 2013 Accepted 9 November 2013 Available online 16 November 2013
Ethnopharmacological relevance: Diabetes mellitus is a major metabolic disorder affecting a huge population all over the world. Artemisia species have been extensively used for the management of diabetes in folkloric medicine. The present study is designed to investigate the antidiabetic and antihyperlipidemic effects of aeriel parts of Artemisia indica. Materials and methods: Hydromethanolic crude extracts, chloroform, ethyl acetate and n-butanol fractions of aerial parts of Artemisia indica were tested for their antidiabetic potential in Streptozotocin (STZ) (50 mg/kg, i.p.) induced diabetic Sprague-Dawley rats. Blood glucose level, body weight, serum lipid profile and activities of liver enzymes were determined. The extracts were further subjected to preliminary phytochemical analysis. Results: A daily oral dose of hydromethanolic crude extracts (200 and 400 mg/kg b.w.) and chloroform fraction (200 mg/kg b.w.) of Artemisia indica for 15 days showed a significant reduction in blood glucose level which was comparable to that of the standard antidiabetic drug, glibenclamide (500 μg/kg, p.o.). Artemisia indica extracts also showed reduction in total cholesterol, triglycerides and low density lipoproteins as well as serum creatinine level, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in diabetic rats. Conclusion: According to the results Artemisia indica possesses hypoglycemic, antihyperlipidemic and valuable effects on liver and renal functions in diabetic rats, which seems to validate its traditional usage. & 2013 Published by Elsevier Ireland Ltd.
Keywords: Hypoglycemic Artemisia indica Asteraceae Glibenclamide Streptozotocin-induced diabetes
1. Introduction Diabetes mellitus is one of the most severe and incurable metabolic disorders characterized by increased blood glucose level as a result of an absolute or relative lack of insulin and failure of insulin to act on its targets tissue (Valiathan, 1998). The acute and chronic manifestations of hyperglycemia cause microvascular (retinopathy, neuropathy and nephropathy) and macrovascular complications (Coronary artery disease leading to myocardial infarction, diabetic ketoacidosis, nonketotic hyperosmolar coma and diabetic coma; Fowler, 2008). It is a leading factor of multiple disorders due to chronic hyperglycemia and causes abnormalities in glucose, lipids and protein metabolism (Kahn et al., 1976). India, China and United States are the largest countries presenting more than 30 million diabetic people and the incidence is increasing day by day (Wild et al., 2004). Several workers described the treatment
n
Corresponding author. Tel.: þ 92 300 957 3590. E-mail address: [email protected] (N. Karim).
0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2013.11.012
of diabetes mellitus based on the interaction of nutritional agents. These agents limit the availability of fatty acids, inhibit gluconeogenesis (Moneva and Dagogo-Jack, 2003) and enhance lipolysis (Bebernitz and Schuster, 2002). Furthermore, they promote fatty acid oxidation in a futile cycle that does not yield metabolic energy and stimulate peripheral glucose utilization (Bebernitz and Schuster, 2002). More than 800 plants have been reported to have antihyperglycemic effects with less adverse effects and low toxicity as compared to synthetic compounds (Kirithikar and Basu, 1995; Nadkarni, 1976). According to the World Health Organization (WHO), almost 70% of the diabetic patients use plants as a primary source of antidiabetic agents in order to satisfy their principal health needs (Bailey and Day, 1989). Artemisia indica also known as “mugwort” belongs to family Astereaceae. The plant is a perennial shrub with a height of 2–8 m and is found in northe rn areas of Pakistan, as well as in cold temperate zones of Asia. Artemisia species have been reported to exhibit antiplasmodial (Tran et al., 2003), antispasmodic and bronchodilator (Khan and Gilani, 2009), antihypertensive (Tigno et al., 2000), antiallergic
W. Ahmad et al. / Journal of Ethnopharmacology 151 (2014) 618–623
(Rodrigues-Alves et al., 2008), hepato-protective (Gilani et al., 2005), antibacterial (Chen et al., 1989) and antinociceptive effects (Pires et al., 2009). Furthermore, various species of Artemisia have been traditionally used for their antihyperglycemic effects in Mexico (Andrade-Cetto and Heinrich, 2005). Therefore, keeping in view the medicinal importance of the plant and extensive folkloric use in rural Mexican communities as an antidiabetic plant, this study was planned to explore the antidiabetic activity of Artemisia indica on scientific grounds.
2. Materials and methods 2.1. Chemicals Streptozotocin (Sigma-Aldrich), glibenclamide (Sanofi Aventis Pharma (Pvt.) Ltd., Pakistan) and glucose estimation kits (S.D Chek Gold Germany) were used in this study. The different organic solvents and chemicals used for extraction were purchased from local suppliers of Merck, Germany. Other reagents used in this study were Tween-80 (Scharlau chem., Spain), normal saline (Utsoka Pharma (Pvt.) Ltd., Pakistan), biochemical reagents for lipid profile, LFTs Kits (Human, Germany) and RFTs kit (Bioneed Germany diagnostic).
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identification of different constituents like alkaloids, flavonoids, terpenoids, glycosides, saponins and tannins by using standard qualitative methods described by Trease and Evans (1983). 2.6. Acute toxicity study of the crude methanolic extract of Artemisia indica The acute toxicity of the crude methanolic extract of Artemisia indica was determined by using Sprague-Dawley rats (150–200 g), according to the method described by Irwin (1968) and described elsewhere (Karim et al., 2012). The animals were divided into six groups (n¼ 6). One group served as a control and received Tween80 suspension orally. The crude methanolic extract was given in a dose of 250, 500, 1000, 1500 and 2000 mg/kg b.w. to each rat orally. All the doses of the extracts were prepared by dissolving the extract in Tween-80 suspension prior to administration. The animals were observed at 0, 30 and 60 min, 24, 48 and 72 h and 1 week after administration for any kind of behavioral, physical and pharmacological toxic effects, respectively. Since the extract was found safe up to the dose level of 2000 mg/kg b.w., a dose of 200 mg/kg b.w. (1/10 of 2000 mg/kg b.w.) of the extracts was selected according to OECD guidelines for screening of the antidiabetic activity (Sharma et al., 2010). 2.7. Induction of hyperglycemia
2.2. Animals Adult Sprague-Dawley rats in the weight range of 150–200 g were purchased from the Department of Pharmacy, University of Peshawar. Animals were housed in the Department's animal house with fresh water and standard food available ad libitum. The animals were maintained at 12 h light and dark cycles and with room temperature maintained at 22–25 1C in the animal house. All animal procedures have been approved by the Departmental Animal Ethical Committee (DAEC/PHARM/2012/10) and were conducted according to the UK Animal Scientific Procedure Act, 1986. 2.3. Plant material The aerial parts of the plant were collected from upper Dir (Sherin Gal) Khyber Pakhtunkhwa, Pakistan, in the month of July 2008 and were authenticated by a taxonomist. The plant specimen was deposited in the Department of Pharmacy, University of Malakand, Pakistan. A voucher specimen number (200300106) was assigned at the same institution. 2.4. Preparation of extracts The shade dried aerial parts of the plant were coarsely ground with the help of a dry grinder followed by extraction with 70% methanol (three times) and filtered through a piece of cloth (plant material to solvent ratio was 1:5 w/v). The extract was concentrated to a semisolid mass using a rotary evaporator under reduced pressure at 45 1C for a final extract yield of 20% (w/w). The semisolid mass was fractionated with various organic solvents: ethyl acetate, chloroform and n-butanol. Each fraction was finally evaporated to semisolid masses by the rotary evaporator. The yields of ethyl acetate, chloroform and n-butanol were 3.8%, 5.5% and 4.3% w/w respectively. For antidiabetic activity, the extract and its fractions were formulated as suspensions in normal saline with 5% Tween-80 as the suspending agent. 2.5. Preliminary phytochemical tests The crude methanolic extract, chloroform and ethyl acetate fractions of Artemisia indica were subjected to qualitative tests for
Hyperglycemia was induced in Sprague-Dawley rats by a single intra peritoneal (i.p.) injection of 50 mg/kg of Streptozotocin (STZ) reconstituted in normal saline (0.9%) after overnight fasting. After 72 h of STZ administration, blood glucose levels were measured in blood samples collected from tail vein puncture with one touch Glucometer strips using an SD glucometer (Germany). Rats with fasting blood glucose levels more than 300 mg/dl were considered diabetic and selected for the study. 2.8. Experimental design Rats that fasted overnight for 12 h were randomly divided into six groups (eight rats in each group). The first group served as normal control (non-diabetic) and received normal saline whereas the second group served as diabetic control and received 5% Tween-80 suspension. The third group received standard drug glibenclamide (500 μg/kg, p.o.). The fourth and fifth groups received hydromethanolic extracts of Artemisia indica at dose levels of 200 and 400 mg/kg (p.o.) respectively, whereas the sixth, seventh and eighth groups received chloroform, ethyl acetate and n-butanol fractions of Artemisia indica respectively, at a dose of 200 mg/kg orally. The treatment of crude extract and fractions was continued once daily at 09:00 a.m. for 15 days. Body weight and blood glucose levels were estimated on the 0th, 4th, 7th, 10th and 15th day of treatment (Gupta et al., 2004). 2.9. Estimation of serum lipid and liver physiological profile After completion of antidiabetic assay on the 15th day, all animals were anesthetized by pentobarbital sodium (35 mg/kg) and euthanized by cervical decapitation using the method described in schedule 1 of the Animal Scientific Procedure Act 1986 and blood samples were collected through cardiac puncture for biochemical parameters studies (Nagappa et al., 2003). Collected blood was centrifuged at 1500g for 10 min for serum separation. The serum sample was then analyzed by a spectrophotometer (Perkin-Elmer, Germany) for determination of serum SGPT, serum SGOT and serum alkaline phosphatase (ALP) using a standard IFCC kinetic Method (Bioneed kit, Germany). Total cholesterol (TC), triglycerides (TGs), low
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density lipoproteins (LDL), high density lipoprotein (HDL) and serum creatinine were determined by CHOD-PAP and GPO-PAP methods on a UV-Spectrophotometer using a Human kit, Germany.
well with no observable changes in behavior or appearance. No deaths were observed up to 1 week of study.
2.10. Statistical analysis
3.3. Antihyperglycemic effect of Artemisia indica
All the values of blood glucose, body weight, and biochemical parameters were represented as mean7S.E.M. When two groups were compared, Student's t-test was used and when more than two groups were compared, one way ANOVA followed by Dunnett's posthoc multiple comparison test was used. Differences between groups were considered significant at po0.05.
Changes in blood glucose level in normal, diabetic and on treatment for diabetes rats with the crude methanolic extract and various fractions of Artemisia indica and glibenclamide are presented in Table 1. Oral administration of the crude methanolic extract (200 and 400 mg/kg) caused a significant (po 0.01, n ¼8; one way ANOVA with Dunnett's posthoc test) reduction in blood glucose level compared to diabetic control. Crude methanolic extract 200 and 400 mg decreased blood glucose level from 477.77 18 to 244.7 712 mg/dl and from 476.7 753 to 240.6 7 10 mg/dl respectively. The effect was evident from the 7th day onwards. The chloroform fraction (200 mg/kg) retained the antihyperglycemic effect of the crude methanolic extract and significantly (p o0.01, n ¼8; one way ANOVA with Dunnett's posthoc test) reduced blood glucose level from 473 752 to 220.8 7 13.1 mg/dl. With the chloroform fraction, the effect was evident from the 4th day onwards. Group treated with standard drug glibenclamide (500 μg/kg, p.o.) showed similar results and caused significant reduction (p o0.01, n¼ 8; one way ANOVA with Dunnett's posthoc test) in blood glucose level, decreasing the mean blood glucose level from 460.8 743 to 206.2 7 40.8 mg/dl (Table 1). The antihyperglycemic effect of the crude methanolic extract and chloroform fraction of Artemisia indica was comparable to that of glibenclamide. The groups treated with the ethyl acetate and n-butanol (200 mg/kg) fractions showed no significant changes in blood glucose level (p 40.05, n ¼8; one way ANOVA with Dunnett's posthoc test) (Table 1).
3. Results 3.1. Phytochemical tests Preliminary phytochemical analysis of the crude methanolic extract and chloroform fraction showed the presence of alkaloids, flavonoids, glycosides, terpenoids, saponins and tannins whereas alkaloids were absent in the ethyl acetate fraction. 3.2. Acute toxicity tests Acute toxicity studies revealed that the administration of crude methanolic extract (250–2000 mg/kg) of Artemisia indica did not produce significant changes in the behavior of the animals as observed by lack of convulsions, respiratory distress, writhing, changes to reflex activity or mortality. A slight increase in irritability and escape behavior was observed at 2000 mg/kg in three out of eight animals. At 24 h–1 week, all animals seemed
Table 1 Effect of daily oral administration of Artemisia indica extracts or glibenclamide on blood glucose level of STZ-induced diabetic rats. Sl. no.
Groups
Dose (mg/kg)
1st day
4th day
7th day
10th day
15th day
1 2 3 4 5 6 7 8
Diabetic control Normal control saline Glibenclamide Crude methanolic extract Crude methanolic extract Chloroform fraction Ethyl acetate fraction n-butanol fraction
0.4 ml 0.4 ml 0.5 200 400 200 200 200
471.3 7 37 1007 11 460.8 7 43 477.7 7 18 476.7 7 53 4737 52 4877 38 4727 32
484.77 30 92.3 7 8 300.7 7 45nn 476.3 7 22 489.27 8 259.8 7 58nn 458 7 23 476.5 7 58
500.8 7 50 92.3 7 6 294.5 7 50 345.7 7 20nn 269.8 7 10nn 242.17 16nn 507.3 7 21 493.87 30
506 7 34 91.3 7 7 248.8 7 53nn 309.5 7 12nn 254.8 7 7nn 226.2 7 14nn 495.37 24 4977 18
516.7 7 50 91.5 7 5 206.27 40nn 244.37 12nn 240.67 10nn 220.8 7 13nn 510.3 7 32 490.27 58
The values are expressed as mean 7 SEM. n¼ 8 in each group. nnpo 0.01 as compared with diabetic control at the same time (one way ANOVA followed by Dunnett's multiple comparison test).
Table 2 Effects of various extracts of Artemisia indica on body weight in STZ -induced diabetic rats. Sl. no.
1 2 3 4 5 6 7 8
Change in body weight (g) at days
% change in body weight
Treatments
Dose (mg/kg)
0
4
7
10
15
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
153 70.5 152 77 153 74 152 73 154 74 151 75 153 77 154 75
155 79 148 75n 157 73n 155 75n 155 76n 154 75n 147 74 149 73
165 70.9 145 75nn 160 76n 157 77n 158 74n 156 73n 142 72 145 74
1707 0.9 1407 3nn 164 7 4nn 1607 5nn 1627 3nn 1627 4nn 1397 4 1407 3
1787 3 1337 4nnn 168 7 5nn 164 7 4nn 1657 6nn 1677 5nn 1357 4 1347 4
Normal (control) Diabetic (control) c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol fraction b
12.5 þ 9.8 þ 7.8 þ 7.1 þ 10.6 11.7 13.0
The values are expressed as mean7 SEM. Each value corresponds to a mean of eight animals. np o0.05, nnp o 0.01, nnnp o 0.001; comparison of a(normal control) vs b(diabetic control) (Student t-test), np o 0.05, nnpo 0.01; comparison of b(diabetic control) vs c(Glibenclamide and extracts treated groups) (one way ANOVA followed by Dunnett's posthoc multiple comparison test). f inalweightðgÞ %changeinb:w: ¼ initialweightðgÞ 100 inititalweightðgÞ
W. Ahmad et al. / Journal of Ethnopharmacology 151 (2014) 618–623
3.4. Effects of Artemisia indica on body weight in diabetic rats The results of changes in body weight of control and experimental rats treated with extracts and glibenclamide are shown in Table 2. STZ-induced diabetic rats produced significant (po 0.001, n ¼8; Student's t test) loss in body weight as compared to normal rats during the study (Table 2). In diabetic control rats, continued weight loss was observed till the end of the study (15 days treatment). During this period, 12.5% reduction in their body weight (Table 2) was recorded. STZ mediated body weight reduction was reversed by crude methanolic extract (200 and 400 mg/ kg b.w.) and chloroform fraction (200 mg/kg b.w.). At the end of 15 days treatment, the crude methanolic extract 200 and 400 mg/kg caused increases in body weight by 7.1% and 7.8% respectively. Similarly, the chloroform fraction (200 mg/kg) also caused an increase in body weight by 10.6%. The ethyl acetate and nbutanol fractions (200 mg/kg b.w.) did not cause any improvement in body weight compared to diabetic control (Table 2). 3.5. Effects of Artemisia indica on lipid profile in Streptozotocin induced diabetic rats The lipid profiles in control and experimental rats are depicted in Table 3. In STZ-induced diabetic control rats, there was a significant (po 0.01, n ¼8; Student's t test) increase in total cholesterol, TG, LDL cholesterol and serum creatinine compared to normal control. In addition, there was a significant decrease (p o0.05, n¼ 8; Student's t test) in HDL cholesterol in diabetic control rats in comparison to normal control. Administration of the crude methanolic extract (200 and 400 mg/ kg) and chloroform fraction (200 mg/kg) for 15 days showed a significant (po0.01, po0.001, n¼ 8; one way ANOVA with Dunnett's posthoc test) reduction in total cholesterol, TGs and LDL cholesterol
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in comparison to diabetic control rats. The effect was comparable to that of the standard drug glibenclamide (500 μg/kg). The ethyl acetate fraction also significantly (po0.05, n¼8; one way ANOVA with Dunnett's posthoc test) reduced total cholesterol and LDL cholesterol but had no effect on TG. Furthermore, the crude methanolic extract (200 and 400 mg/kg), chloroform, n-butanol, and ethyl acetate fractions (200 mg/kg) and glibenclamide (500 μg/kg) significantly (po0.05, po0.01, n¼8; one way ANOVA with Dunnett's posthoc test) increased HDL cholesterol in diabetic rats.
3.6. Effect of Artemisia indica on liver function in Streptozotocin induced diabetic rats Table 4 summarized the effects of STZ on the activity of hepatic marker enzymes in serum. In the present study the levels of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in STZ-induced diabetic rats were elevated. Administration of the crude methanolic extract (200 and 400 mg/kg) and the chloroform (200 mg/kg) fraction of Artemisia indica significantly reduced (p o0.01, n¼ 8; one way ANOVA with Dunnett's posthoc test) serum SGPT, SGOT and ALP in rats intoxicated with STZ. The restoration of hepatic enzymes observed with the crude methanolic (200 and 400 mg/kg) and chloroform (200 mg/kg) fractions of Artemisia indica was comparable to standard drug glibenclamide (p o0.01, n ¼ 8; one way ANOVA with Dunnett's posthoc test). The crude methanolic extract (200 and 400 mg/kg) and the chloroform fraction (200 mg/kg) also caused a significant reduction (p o0.001, n ¼8; one way ANOVA with Dunnett's posthoc test) in serum creatinine comparable to standard drug glibenclamide. The n-butanol (200 mg/kg) and ethyl acetate (200 mg/kg) fractions slightly decreased serum SGPT, SGOT, ALP and creatinine;
Table 3 Antihyperlipidemic effect Artemisia indica on STZ-induced diabetic rats. Sl. no.
Treatment dose (mg/kg)
Dose (mg/kg)
Total cholesterol (mg/dl)
TG (mg/dl)
HDL (mg/dl)
LDL (mg/dl)
1 2 3 4 5 6 7 8
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
120 7 9.12 185.3 7 4.4nn 137.7 7 5.3nn 144.2 7 4.3nn 140.5 7 7.5nn 134.4 7 2.7nn 168.27 3.6n 167.9 7 9.8n
1257 8.80 169.0 7 7.9nn 128.3 7 6.5nn 138.5 7 4.7nn 136.8 7 4.5nn 136.2 7 8.1nn 165.8 7 9.0 146.8 7 3.2n
347 2.2 30.2 7 2.4n 39.17 2.3nn 37.5 7 3.1nn 35.67 5.5nn 38.2 7 1.5nn 36.17 2.5n 36.2 7 2n
817 2.5 177.4 7 8.9nn 90.3 7 3.5nnn 95.6 7 3.2nnn 93.5 7 3.6nnn 81.3 7 4.0nnn 151.8 7 8.0n 149.87 7.7n
Normal control Diabetic Control c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol b
Each value is mean7 SEM of eight animals. Comparisons were made between anormal control and bdiabetic control using Student t-test (np o 0.05, nnp o 0.01) and between b diabetic control and c(Glibenclamide/extracts) treated groups using one way ANOVA followed by Dunnett's posthoc multiple comparison test (np o 0.05, nnp o 0.01, nnn p o 0.001).
Table 4 Effect of various extracts of Artemisia indica on serum profile in STZ-induced diabetic rats. Sl. no.
Treatment
Dose (mg/kg)
(SGPT) IU
(SGOT) IU
(ALP) IU
Serum creatinine (mg/dl)
1 2 3 4 5 6 7 8
a
0.4 ml 0.4 ml 0.5 200 400 200 200 200
157 4.4 52.2 7 11.8 17.6 7 4.5nn 22.4 7 9.0nn 19.4 7 9.0nn 18.8 7 1.5nn 50.5 7 3.5 48.87 4.0
18 73.5 41.4 742.1nn 17.5 72.6nn 21.9 74.9nn 18.9 74.9nn 19.9 74.1nn 39.5 73.8 37.0 73.9
180 720.5 290.8 716.3nn 195.9 75.3nn 203.2 714.52nn 199.2 714.52nn 183.6 712.5nn 281.3 731.2 272.5 710.3
0.52 7 0.2 2.5 7 0.2nn 0.5 7 0.1nnn 0.78 7 0.1nnn 0.6 7 0.2nnn 0.8 7 0.1nnn 2.2 7 0.2 2.3 7 0.3
Normal control Diabetic control c Glibenclamide c Crude methanolic extract c Crude methanolic extract c Chloroform fraction c Ethyl acetate fraction c n-butanol fraction b
Each value is mean 7 SEM of eight animals; comparisons were made between anormal control and bdiabetic control using Student t-test (nnp o 0.01) and between bdiabetic control and cGlibenclamide/extract treated groups using one way ANOVA followed by Dunnett's posthoc multiple comparison test (np o 0.05, nnp o 0.01, nnnp o0.001).
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however the effect was not significant (p 40.05, n ¼8; one way ANOVA with Dunnett's posthoc test; Table 4).
bioactive compounds present in the plant extracts and for elucidation of their molecular mechanisms.
Acknowledgments 4. Discussion Despite the presence of known antidiabetic prescription medicines, herbal drugs and preparations are of considerable interest for the ethno-botanical community and are considered to be less toxic and free from adverse effects than synthetic agents (Atmakuri and Dathi, 2010; Pari and Umamaheswari, 2000). STZ is an antibiotic produced by Streptomyces achromogenes. In experimental animals it causes irreversible destruction of pancreatic beta cells, and is mostly used to induce diabetes mellitus in experimental animals through its toxic effects (Kim et al., 2003; Matteucci and Giampietro, 2008). The results of the present study indicate that the extracts of Artemisia indica reduced blood glucose levels in the diabetogenic animal model with STZ (50 mg/kg). The crude methanolic extract and the chloroform fraction of Artemisia indica plant demonstrated a significant lowering of blood glucose level (BGL). The antihyperglycemic effect of Artemisia indica may be due to prevention of free radical formation induced by STZ. Elevated plasma total cholesterol, triglycerides and LDL cholesterol are the major risk factors of cardiovascular diseases. Diabetic rats showed elevated plasma total cholesterol, triglycerides and LDL cholesterol. The crude methanolic extract (200 and 400 mg/kg) and the chloroform fraction (200 mg/kg) reduced the lipid profile and caused a reduction in blood glucose level. STZ-induced diabetes was characterized by severe loss in body weight (Chen and Ianuzzo, 1982). The crude methanolic extract at the dose level of 200 and 400 mg/kg and the chloroform fraction (200 mg/kg) also showed improvement in body weight. Improvement in body weight could be due to the ability of Artemisia indica extracts to reduce hyperglycemia (Pillai et al., 2012). Reports have shown that the liver cells are irreversibly destroyed in STZ-induced diabetic rats, causing the liver microsomal cells to release different enzymes, including SGPT, SGOT and serum ALP in the blood (Daisy et al., 2008). It leads to an increased level of these enzymes in the plasma. The crude extract and the chloroform fraction of Artemisia indica significantly lowered the SGPT, SGOT and ALP levels. Chronic hyperglycemia induces elevation of serum creatinine and serum uric acid leading to renal dysfunction (Almdal and Vilstrup, 1988). The results from the present study show that Artemisia indica can also reduce the level of serum creatinine in STZ-induced diabetic rats that further helps in treating and preventing the renal system from damage. These data confirmed the possibilities that the major function of these extracts is on protective effects of major tissues such as kidney, liver and pancreas, thereby reducing the incidence of diabetes in experimental animals.
5. Conclusion We conclude from the above results and discussion on the plant Artemisia indica that the crude methanolic and chloroform extracts have significant hypoglycemic and antidiabetic potentials. These extracts improve effects on body weight. They can also improve the condition of diabetes mellitus as indicated by parameters such as body weight, lipid profiles and a reduction in serum creatinine, serum SGPT, serum SGOT and ALP in STZ-induced diabetic rats. Furthermore, this study provides scientific evidence for the ethnobotanical use of Artemisia indica in the treatment of diabetes mellitus. However, further studies are warranted for the isolation and purification of
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