http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.960943
ORIGINAL ARTICLE
Effects of a triplex mixture of Peganum harmala, Rhus coriaria, and Urtica dioica aqueous extracts on metabolic and histological parameters in diabetic rats
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Fereydoon Abedi Gaballu1, Yousef Abedi Gaballu2, Omid Moazenzade Khyavy2, Alireza Mardomi3, Kazem Ghahremanzadeh4, Behrooz Shokouhi4, and Himan Mamandy2 1
Young Researchers and Elite Club, Islamic Azad University, Tabriz, Iran, 2Department of Cellular and Molecular Biology, Higher Education Institute of Rab-Rashid, Tabriz, Iran, 3Department of Medical Biotechnology School of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, and 4Department of Pathology, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Keywords
Context: Several therapeutic effects such as antioxidant and blood glucose-lowering activities have been reported for Peganum harmala L (Zygophyllaceae) (PH) seeds, Rhus coriaria L (Anacardiaceae) (RC) fruits, and Urtica dioica L (Urticaceae) (UD) leaves. Objective: This study investigates the effects of a triplex mixture (1:1:1) of these medicinal plants on metabolic and histological parameters in diabetic rats. Materials and methods: Aqueous extracts of PH, RC and UD were administered as either monotherapy or in combination at a final dose of 200 mg/kg to alloxan-induced diabetic rats by daily gavage. Biochemical parameters including blood glucose, liver function-related enzymes, lipid profile, and creatinine were estimated by spectrophotometric methods. Tissues from the liver and kidney stained with hematoxylin/eosin were histologically examined. The results obtained from the exposure groups were compared to either healthy or diabetic control groups. Results: Compared with the diabetic control rats, all aqueous extracts (ED50 ¼ 11.5 ± 2.57 mg/ml) led to significant decreases in the levels of ALP (1.39–2.23-fold, p50.05), low-density lipoprotein cholesterol (LDL-C) (1.79–3.26-fold, p50.05), and blood glucose (1.27–4.16-fold, p50.05). The serum concentrations of TG was decreased only by treatment with UD and triplex mixture (1.25- and 1.20-fold, respectively, p50.05). Among the studied parameters, alanine aminotransferase (ALT), LDL-C, TG, and creatinine recovered to healthy control levels after 4 weeks of treatment with the extract mixture. Conclusion: This study showed that PH, RC, and UD extracts, especially their combination, had significant antidiabetic, hypolipidemic, and liver and renal damage recovering effects.
Antidiabetic drugs, blood glucose, creatinine, herbal remedies, lipid profile, liver function
Introduction Diabetes mellitus is one of the most frequent metabolic disorders, characterized by hyperglycemia due to poor insulin secretion, insulin malfunction, or both of them (Smith, 2003). Diabetes morbidity has been related to increased oxidative stress and defective intracellular antioxidative defense (Khaki et al., 2009). High levels of oxidative free radicals and low levels of endogenous antioxidants are involved in the pathogenesis of diabetes (Jakus, 2000; Rahimi et al., 2005; Vincet et al., 2003). Hyperglycemia is a defining characteristic of diabetes. However, dyslipidemia is regarded as the main cardiovascular risk factor of diabetes. Diabetic dyslipidemia is typically characterized by elevated serum triglycerides (TG), total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol Correspondence: Himan Mamandy, Department of Cellular and Molecular Biology, Higher Education Institute of Rab-Rashid, Tabriz, Iran. Tel: +98 9395930663. Fax: +98 442 2222411. E-mail: [email protected]
History Received 4 May 2014 Revised 10 August 2014 Accepted 28 August 2014 Published online 23 January 2015
(LDL-C), and the low level of high-density lipoprotein (HDL-C) (Brunzell et al., 2008). Although there is a wide range of antidiabetic drugs in use, they often generate undesirable side effects (Suji & Sivakami, 2003). The discovery of more effective and safer remedies is, therefore, critical to the treatment of diabetes (Fakir et al., 2009). In particular, herbal remedies attract considerable research interest because of their less adverse effects comparing with synthetic drug treatments. The medicinal herb Peganum harmala L (Zygophyllaceae) (PH), also known as Syrian rue, grows in Asia, North Africa, and Mediterranean regions (Asghari & Lockwood, 2002). The PH extract has several therapeutic effects such as antitumor, anti-inflammation, antioxidant (Asghari & Lockwood, 2002; Giancarlo et al., 2006; Goel et al., 2009) and blood glucose (BG)-lowering activities. These effects have mainly been attributed to the alkaloid components of the PH, including harmaline, harmine, and harman (Singh et al., 2008). Rhus coriaria L (Anacardiaceae) (RC), also known as Sumac, grows in Asia, North America, and Europe
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(Pashazadeh et al., 2013). It has been reported that this plant shows significant antitumor, anti-inflammation, antioxidant, antifungal, hypoglycemic, and digestive properties (Khaki et al., 2009; Rehman et al, 2000; Smith, 2003). The fruit body of RC contains a complex of bioactive compounds including tannins, quercetin, isoquercetin, myricetin, ellagic acid, gallic acid, and tannic acid (Astulla et al., 2008). The flavonoid quercetin has been shown to have BG-lowering effects similar to a-glucosidase inhibitors through blocking the glucose absorption process in intestine via inhibition of a-amylase enzyme (Giancarlo et al., 2006). Urtica dioica L (Urticaceae) (UD) has long been known in folk medicine (Kavalali et al., 2003; Ur-Rahman & Zaman, 1989). Numerous studies indicate hypoglycemic effect of this plant (Bnouham., 2003; Farzami et al., 2003; Ziyyat et al., 1997). Hypoglycemic effect of UD has been attributed to the presence of lectins (Kavalali et al., 2003). The lectins increase either pancreatic insulin secretion or insulin release from its bond form (Stanely et al., 2000). As mentioned above, several antidiabetic effects of PH, RC, and UD extracts have been reported, but there are no reports yet regarding the combined use of these extracts. The aim of this study was to investigate the effect of a triplex mixture of these medicinal plants on metabolic and histological parameters in normal and experimental diabetic rats.
Materials and methods Experimental animals Male Wistar rats with an average weight of 230 ± 7 g were purchased from the Laboratory Animal Center of Tabriz University of Medical Sciences. Animals were kept under standard laboratory conditions in the Higher Educational Center of Jahad-e-Keshavarzi (Tabriz, Iran). The experiments were initiated after obtaining approval from the Animal Rights Office, Higher Educational Center of Jahad-eKeshavarzi. All protocols were designed and performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
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Table 1. List of animal groups with different treatments. Groups Status and treatment 1 2 3 4 5 6 7
Healthy, treated with saline in equivalent volume to the test treatments Diabetic, treated with saline in equivalent volume to the test treatments Diabetic, treated with aqueous extract of PH (200 mg/kg) Diabetic, treated with aqueous extract of RC (200 mg/kg) Diabetic, treated with aqueous extract of UD (200 mg/kg) Diabetic, treated with triplex mixture of PH, RC and UD (200 mg/kg) Diabetic, treated with glibenclamide (0.16 mg/kg)
in Table 1. Each plant extract or triple mixture (1:1:1) was administered at a dose of 200 mg/kg/d. The treatment was given for 4 weeks by daily gavage. At the end of the experiment, blood samples were obtained from the heart under general anesthesia for biochemical analysis. Then animals were sacrificed and the whole liver and kidney tissues were excised for histopathological examinations. Biochemical analysis The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, TC, VLDL-C, LDL-C, HDL-C, BG, and creatinine were measured at the Danesh Pathobiology Laboratory (Tabriz, Iran) using standard colorimetric enzymatic kits from Bionik (Bionik, Tehran, Iran). Histological evaluation Rat tissue samples were histologically examined by hematoxylin and eosin (H&E) staining. The liver specimens were evaluated for piecemeal necrosis, the degree of portal inflammation and focal lytic necrosis, and the kidney tissue samples for glomerular distension. All histopatjological analyses were performed in the Danesh Pathobiology Laboratory. Statistical analysis
Plant materials and extraction preparation PH seeds, UD leaves, and RC fruits were obtained on April 2013 from the Research Center of Agriculture and Natural Resources (Tabriz, Iran). All plant materials were verified by the Tabriz Reference Herbarium at the Faculty of Agriculture, University of Tabriz, Iran. The extraction procedure was performed as previously described by Clevenger (1928).
All data were expressed as mean ± SD. The one-way analysis of variance (ANOVA) was used to evaluate significance difference. Comparison between the experimental groups of rats was done by the LSD test. All analyses were performed with the SPSS software (SPSS version 9, Chicago, IL). A value of p50.05 was considered statistically significant.
Results Diabetes induction
Metabolic parameters
Diabetes was induced by means of a single dose of alloxan monohydrate (Sigma-Aldrich, St. Louis, MO; 110 mg/kg). Diabetes was confirmed on the third day by determining glucose levels in tail vein blood samples. Glucose concentration of 250 mg/dl was considered as the diabetic threshold.
The ANOVA tests revealed no significant variation among replications, but statistically significant differences among the tested groups at p values between 0.01 and 0.05 (Table 2). Compared with the control group, alloxan administration caused significant increases in ALP, ALT, LDL-C, TG, BG, and creatinine (Table 3). Compared with the saline-treated diabetic rats, all three aqueous extracts led to significant decreases in the levels of ALP, LDL-C, and BG. Both PH and RC treatment led to significant increase in HDL levels. The serum concentration
Experimental groups The rats were randomly divided into seven groups with four rats in each (control, control diabetic, extract-treated groups, and diabetic rats who received glibenclamide) as shown
Effect of triplex mixture on diabetes
DOI: 10.3109/13880209.2014.960943
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Table 2. Summary of the variables and results of ANOVA. Means of square SOV Replication Treatment Error % CV
df 3 6 18 –
ALP
ALT ns
22 802.76 50 658.81** 31 616 22.40
AST ns
712.6 14 235.65** 2533.16 30.06
ns
1566 41 445.65** 5857.86 27.44
VLDL-C ns
6.59 21.18** 2.68 12.78
LDL-C
HDL-C
ns
ns
3.89 66.53** 3.84 18.09
4.72 127.72** 12.90 14
TG ns
88 415.08** 63.95 12.41
TC
BG ns
105.76 252.11* 86.26 11.94
Creatinine ns
1597.76 97 294.5** 2423.92 21.73
0.0002ns 0.033** 0.002 5.2
ns
Non-significant. **Significant at 0.01 p value; *significant at 0.05 p value.
Table 3. Multiple comparison of variables between studied groups of animals using least significance test (LSD). Means ± SD
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U/l Group Group 1 (healthy) Group 2 (diabetic) Group 3 (PH) Group 4 (RC) Group 5 (UD) Group 6 (triple mixture) Group 7 (glibenclamide) 5% LSD
mg/dL
ALP
ALT
AST
VLDL-C
LDL-C
HDL-C
TG
TC
BG
Creatinine
311.8 ± 8.8 1413.3 ± 232.3 631.3 ± 114.3 912.5 ± 232.2 1012.5 ± 165.2 692.5 ± 147.9 581.3 ± 205.5 264.1
83.5 ± 3.1 251.2 ± 45.7 141.7 ± 30.5 144.2 ± 17.6 243.0 ± 93.7 151.2 ± 28.0 156.7 ± 54.6 74.7
178.2 ± 2.0 278.7 ± 66.0 277.0 ± 51.3 284.0 ± 43.3 485.0 ± 145.7 267.0 ± 63.5 182.0 ± 50.6 113.7
22.2 ± 0.8 20.0 ± 0.7 25.6 ± 1.3 33.2 ± 2.5 23.3 ± 1.5 25.0 ± 2.1 30.2 ± 2.5 2.43
67.9 ± 0.4 124.2 ± 0.5 69.6 ± 0.8 61.2 ± 2.2 38.5 ± 0.5 66.5 ± 4.4 103.2 ± 0.9 2.91
41.7 ± 0.8 33.0 ± 2.8 51.8 ± 0.8 57.5 ± 5.5 67.0 ± 2.0 58.5 ± 5.0 49.5 ± 3.4 5.33
54.1 ± 1.5 69.2 ± 8.3 63.2 ± 4.7 82.7 ± 12.6 55.2 ± 8.7 57.2 ± 2.0 69.0 ± 11.6 11.88
70.2 ± 0.9 81.7 ± 4.3 71.5 ± 15.6 82.0 ± 1.4 78.7 ± 34.4 69.2 ± 120.2 91.0 ± 10.6 13.79
112.2 ± 2.2 500.0 ± 8.1 105.0 ± 12.9 392.5 ± 9.5 156.2 ± 34.4 190.7 ± 120.2 128.7 ± 13.1 73.14
0.82±0.0 1.04 ± 0.0 0.90 ± 0.0 1.04 ± 0.0 0.83 ± 0.0 0.89 ± 0.0 0.94 ± 0.0 0.071
of TG was decreased only by treatment with RC and PH. Parameters like ALT, LDL-C, TG, and creatinine recovered to control levels after the treatment with extract mixture (Table 3). Histopathologic findings Histopathology of liver The results obtained from the histopathological study showed that the diabetic group had marked portal inflammation, mild piecemeal necrosis, and 1–4 focal lytic necrosis per 10 microscopic field (Figure 1b). All the applied treatments led to significant improvement in the histological features of the liver (Figures 1c–f and 2). However, the triplex mixture of PH, RC, and UD aqueous extracts caused a more obvious improvement in the liver condition (Figure 1f). Histopathology of kidney The kidney histopathology results showed that diabetic rats had severe glomerular distension (Figure 3b). Treatments with all plant extracts and their mixture recovered glomerular distension (Figures 3c–f and 4). The RC aqueous extract and the triplex mixture were more beneficial in this case as led to absolute remission of glomerular distension (Figure 3d and f).
Discussion In this study, significant changes in the serum lipids, lipoproteins, except VLDL and TC, and markers of liver and kidney function were observed in alloxan-induced diabetic rats, and these changes were modulated with the combination of PH, RC, and UD extracts in the experimental treatments.
The hypoglycemic effect of all three plants has been demonstrated through different mechanisms, including inhibition of glucose absorption (Kavalali et al., 2003; Nuraliev & Avezov, 1992; Ur-Rahman & Zaman, 1989) and stimulation of pancreatic insulin secretion (Stanely et al., 2000). According to our findings, the PH aqueous extract had the most BG-lowering effect in the diabetic rats. The serum level of glucose in animals that received PH treatment alone was even lower than the control. The antidiabetic effect of PH has mainly been attributed to the water-soluble 4-hydroxypipecolic acid (4-HPA) (Singh et al., 2012; Tetko et al., 2005). A synergic effect of 4-HPA and insulin has been reported on stimulation of glucose uptake by skeletal muscle cells (Singh et al., 2008, 2012). Consistent with the current study, several studies have reported a significant improvement in the overall lipid profile in animal models receiving PH, RC, or UD extracts (Anwer et al., 2012; Ozkol et al., 2013; Singh et al., 2012; Soliman et al., 2012). The combined extract treatment was also beneficial with comparable results with those generated separately. The beneficial impacts of the triple mixture were particularly pronounced in the reducing ALT, LDL-C, and creatinine, and in the increasing HDL-C. In the current study, the alloxan-induced increase in TG was reverted by treatment with UD as well as with the triple mixture extract. These results are consistent with a previous report showing TGlowering effect of 4-HPA in diabetic mice (Singh et al., 2012). However, Ahangarpour et al. (2012) found no significant effect of UD extract on the serum TG in fructose-induced insulin-resistant rats. Moreover, reducing effects of PH and RC on serum TC have been previously reported (Anwer et al., 2012; Singh et al., 2012; Tahery et al., 2013). These inconsistencies might be due to differences in the diabetic animal model and the duration of treatment.
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Figure 1. Microscopic structure of liver of a (a) control rat (group 1) and (b) diabetic rat (group 2) showing marked portal areas, mild piecemeal necrosis, and 1–4 foci per field; (c) PH-treated diabetic rat (group 3) showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field; (d) RC-treated diabetic rat (group 4) showing mild portal areas and 1–4 foci per field; (e) UD-treated diabetic rat (group 5) showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field; (f) triplex mixture-treated diabetic rat (group 6) showing mild portal areas. All slides stained with hematoxylin and eosin (original magnification 10).
Figure 2. Microscopic structure of liver of a glibencalmide-treated diabetic rat showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field. The slide stained with hematoxylin and eosin (original magnification 10).
ALP, ALT, and AST are important hepatic enzymes since their levels in serum have been established as specific and reliable biomarkers for liver damage (Ahangarpour et al., 2012). Both beneficial and adverse effects of PH, RC, and UD extracts on liver function tests have been reported earlier (Ahangarpour et al., 2012; Pourahmad et al., 2009; Rayne & Mazza, 2007; Soliman et al., 2012). According to the results of the current study, alloxan-induced diabetic rats had marked increases in the serum levels of ALP and ALT that were consistent with observed changes in histopathology. Although treatments of all the single and mixture significantly improved hepatic necroinflammation, only the triple mixture supplementation significantly reduced two hepatic biomarkers ALP and ALT compared with the diabetic control group. When single treatments were compared with the mixture treatment, the mixture treatment was collectively more potent. High levels of serum creatinine are associated with abnormal renal function, especially decreased glomerular
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DOI: 10.3109/13880209.2014.960943
Effect of triplex mixture on diabetes
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Figure 3. Microscopic structure of kidney of a (a) control rat (group 1) and (b) diabetic rat (group 2) showing sever glomerular distension; (c) PHtreated diabetic rat (group 3) showing only a mild glomerular distension; (d) RC-treated diabetic rat (group 4) showing no pathological condition; (e) UD-treated diabetic rat (group 5) showing only a mild glomerular distension; (f) triplex mixture-treated diabetic rat (group 6) showing no pathological condition. All slides stained with hematoxylin and eosin (original magnification 10).
Figure 4. Microscopic structure of kidney of a glibencalmide-treated diabetic rat showing only a mild glomerular distension. The slide stained with hematoxylin and eosin (original magnification 10).
filtration rate (Gu¨ne¸s et al., 1999). Protective effect of UD on renal injury has been shown previously (Sayhan et al., 2012). However, Kianbakht et al. (2013) have shown that even though UD leaf extract improves glycemic control in type 2 diabetic patients, there was no significant effect on the serum creatinine level. It has also been reported that administration of PH extract induced no changes in the creatinine level in rats (Lamchouri et al., 2002). In the present study, there was a significant reduction in creatinine level with remarkable improvement in the glomerular distension after feeding diabetic rats with the extract combination of PH, RC and UD. There is a rare attention to the effects of combined traditional medicinal plants in the literature. The current study was focused on the triplex mixture of three medicinal plants. To our knowledge, this is the first report showing the curative effects of combined PH, RC, and UD against diabetic complications in an experimental rat model. This study
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included a broad panel of biomarkers and histopathologic characteristics in liver and kidney. Future studies will be required to examine the extracts sub-fractions and to identify the active components.
Conclusion This study showed that PH, RC, and UD extracts had significant antidiabetic, hypolipidemic, and liver and renal damage recovering effects. Moreover, these beneficial effects were more potent with the combination of them compared with that of single treatments.
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Acknowledgements The authors would like to thank Dr. Masoud Darabi for critical revising of the manuscript and Dr. Ahmad Razban Haghighi at the Tabriz Reference Herbarium, University of Tabriz, for providing herbal materials.
Declaration of interest The authors report that they have no declarations of interest. The authors alone are responsible for the content and writing of this article. This study was financially supported by a research grant from the Higher Educational Center of Jahade-Keshavarzi, Tabriz, Iran (Project no.: 7114/261/2)
References Ahangarpour A, Mohammadian M, Dianat M. (2012). Antidiabetic effect of hydroalcholic Urtica dioica leaf extract in male rats with fructoseinduced insulin resistance. Iran J Med Sci 37:181–6. Anwer T, Sharma M, Pillai KK, et al. (2012). Antihyperglycemic, antidyslipidemic and antioxidant activity of Rhus coriaria in STZinduced type 2 diabetic rats. Afr J Pharm Pharmacol 6:2851–5. Asghari G, Lockwood GB. (2002). Stereospecific biotransformation of (±) phenylethyl propionate by cell cultures of Peganum harmala L. Iran Biomed J 6:43–6. Astulla A, Zaima K, Matsuno Y, et al. (2008). Alkaloids from the seeds of Peganum harmala showing antiplasmodial and vasorelaxant activities. J Nat Med 62:470–2. Bnouham M, Merhfour FZ, Ziyyat A, et al. (2003). Antihyperglycemic activity of the aqueous extract of Urtica dioica. Fitoterapia 74: 677–81. Brunzell JD, Davidson M, Furberg CD, et al. (2008). Lipoprotein management in patients with cardiometabolic risk. J Am Coll Cardiol 51:1512–24. Clevenger JF. (1928). Apparatus for the determination of volatile oil. J Am Pharm Assoc 17:345–9. Fakir H, Korkmaz M, Gu¨ller B. (2009). Medicinal plant diversity of western Mediterranean region in Turkey. J Appl Biol Sci 3:30–40. Farzami B, Ahmadvand D, Vardasbi S, et al. (2003). Induction of insulin secretion by a component of Urtica dioica leave extract in perifused islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats. J Ethnopharmacol 89:47–53. Giancarlo S, Rosa LM, Nadjafi F, Francesco M. (2006). Hypoglycaemic activity of two spices extract: Rhus coriaria L, Bunium persicum Boiss. Nat Prod Res 20:882–6. Goel N, Singh N, Saini R. (2009). Efficient in vitro multiplication of Syrian Rue (Peganum harmala L.) using 6-benzylaminopurine preconditioned seedling explants. Nat Sci 7:129–34. _ Aydin M, et al. (1999). The effects of Rumex Gu¨ne¸s Hv, Deg˘irmenci I, patientia L. and Urtica dioica L. on some blood and urine parameters, and liver and kidney histology in diabetic rats. Turk J Med Sci 29: 227–32.
Pharm Biol, Early Online: 1–6
Jakus V. (2000). The role of free radicals, oxidative stress and antioxidant systems in diabetic vascular disease. Bratisl Med J 101: 541–51. Kavalali G, Tuncel H, Goksel S, Hatemi HH. (2003). Hypoglycemic activity of Urtica pilulifera in streptozotocin-diabetic rats. J Ethnopharmacol 84:241–5. Khaki AA, Khaki A, Nouri M, et al. (2009). Evaluation effects of quercetin on liver apoptosis in streptozotocin-induced diabetic rat. J Med Plants 8:70–8. Kianbakht S, Khalighi-Sigaroodi F, Dabaghian FH. (2013). Improved glycemic control in patients with advanced type 2 diabetes mellitus taking Urtica dioica leaf extract: A randomized double-blind placebocontrolled clinical trial. Clin Lab 59:1071–6. Lamchouri F, Settaf A, Cherrah Y, et al. (2002). Experimental toxicity of Peganum harmala seeds. Ann Pharm Fr 60:123–9. Nuraliev IUN, Avezov G.A. (1992). The efficacy of quercetin in alloxan diabetes. Eksp Klin Farmakol 55:42–4. _ (2013). Therapeutic potential of Ozkol H, Tuluce Y, Dilsiz N, Koyuncu I. some plant extracts used in Turkish traditional medicine on streptozocin-induced type1 diabetes mellitus in rats. J Membr Biol 246:47–55. Pashazadeh M, Rezaei A, Pashazadeh M, et al. (2013). Effect of Rhus coriaria consumption on levels of blood glucose and lipids in alloxaninduced diabetic rats. Int J Emerg Trends Pharm Sci 1:34–7. Pourahmad J, Eskandari MR, Shakibaei R, Kamalinejad M. (2009). A search for hepatoprotective activity of aqueous extract of Rhus coriaria L. against oxidative stress cytotoxicity. Food Chem Toxicol 48:854–8. Rahimi R, Nikfar S, Larijani B, Abollahi M. (2005). A review on the role of antioxidants in the management of diabetes and its complications. Biomed Pharmacother 59:365–73. Rayne S, Mazza G. (2007). Biological activities of extracts from sumac (Rhus spp.): A review. Plant Foods Hum Nutr 62:165–75. Rehman A, Jenner A, Halliwel B. (2000). Gas chromatography–mass spectrometry analysis of DNA: Optimization of protocols for isolation and analysis of DNA from human blood. Method Enzymol 319: 401–17. Sayhan MB, Kanter M, Oguz S, Erboga M. (2012). Protective effect of Urtica dioica L. on renal ischemia/reperfusion injury in rat. J Mol Histol 43:691–8. Singh AB, Chaturvedi JP, Narender T, Srivastava AK. (2008). Preliminary studies on the hypoglycemic effect of Peganum harmala L. seeds ethanol extract on normal and streptozotocin induced Diabetic Rats. Ind J Clin Biochem 23:391–3. Singh AB, Khaliq T, Chaturvedi JP, et al. (2012). Anti-diabetic and antioxidative effects of 4-hydroxypipecolic acid in C57BL/KsJ-db/db mice. Hum Exp Toxicol 31:57–65. Smith JF. (2003). Antidiabetic drugs. Med Lib 5:5–6. Soliman AM, Abu-El-Zahab HS, Alswiai GA. (2012). Efficacy evaluation of the protein isolated from Peganum harmala seeds as an antioxidant in liver of rats. Asia Pac J Trop Med 6:285–95. Stanely P, Prince M, Menon VP. (2000). Hypoglycaemic and other related actions of Tinospora cordifolia roots in alloxan-induced diabetic rats. J Ethnopharmacol 70:9–15. Suji G, Sivakami S. (2003). Approaches to the treatment of diabetes mellitus: An overview. Cell Mol Biol 49:635–9. Tahery H, Mehdi M, Fard MH, et al. (2013). Effects of aqueous and ethanolic extracts of Peganum harmala L seeds on lipids profile in rats. J Birjand Univ Med Sci 20:108–14. Tetko IV, Gasteiger J, Todeschini R, et al. (2005). Virtual computational chemistry laboratory – Design and description. J Comput Aided Mol Des 19:453–63. Ur-Rahman A, Zaman K. (1989). Medicinal plants with hypoglycemic activity. J Ethnopharmacol 26:1–55. Vincet AM, Russell JW, Low P, Feldman EL. (2003). Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 25:612–28. Ziyyat A, Legssyer A, Mekhfi H, et al. (1997). Phythotherapy of hypertension and diabetes in oriental Morocco. J Ethnopharmacol 58: 45–54.
ORIGINAL ARTICLE
Effects of a triplex mixture of Peganum harmala, Rhus coriaria, and Urtica dioica aqueous extracts on metabolic and histological parameters in diabetic rats
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Fereydoon Abedi Gaballu1, Yousef Abedi Gaballu2, Omid Moazenzade Khyavy2, Alireza Mardomi3, Kazem Ghahremanzadeh4, Behrooz Shokouhi4, and Himan Mamandy2 1
Young Researchers and Elite Club, Islamic Azad University, Tabriz, Iran, 2Department of Cellular and Molecular Biology, Higher Education Institute of Rab-Rashid, Tabriz, Iran, 3Department of Medical Biotechnology School of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, and 4Department of Pathology, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Keywords
Context: Several therapeutic effects such as antioxidant and blood glucose-lowering activities have been reported for Peganum harmala L (Zygophyllaceae) (PH) seeds, Rhus coriaria L (Anacardiaceae) (RC) fruits, and Urtica dioica L (Urticaceae) (UD) leaves. Objective: This study investigates the effects of a triplex mixture (1:1:1) of these medicinal plants on metabolic and histological parameters in diabetic rats. Materials and methods: Aqueous extracts of PH, RC and UD were administered as either monotherapy or in combination at a final dose of 200 mg/kg to alloxan-induced diabetic rats by daily gavage. Biochemical parameters including blood glucose, liver function-related enzymes, lipid profile, and creatinine were estimated by spectrophotometric methods. Tissues from the liver and kidney stained with hematoxylin/eosin were histologically examined. The results obtained from the exposure groups were compared to either healthy or diabetic control groups. Results: Compared with the diabetic control rats, all aqueous extracts (ED50 ¼ 11.5 ± 2.57 mg/ml) led to significant decreases in the levels of ALP (1.39–2.23-fold, p50.05), low-density lipoprotein cholesterol (LDL-C) (1.79–3.26-fold, p50.05), and blood glucose (1.27–4.16-fold, p50.05). The serum concentrations of TG was decreased only by treatment with UD and triplex mixture (1.25- and 1.20-fold, respectively, p50.05). Among the studied parameters, alanine aminotransferase (ALT), LDL-C, TG, and creatinine recovered to healthy control levels after 4 weeks of treatment with the extract mixture. Conclusion: This study showed that PH, RC, and UD extracts, especially their combination, had significant antidiabetic, hypolipidemic, and liver and renal damage recovering effects.
Antidiabetic drugs, blood glucose, creatinine, herbal remedies, lipid profile, liver function
Introduction Diabetes mellitus is one of the most frequent metabolic disorders, characterized by hyperglycemia due to poor insulin secretion, insulin malfunction, or both of them (Smith, 2003). Diabetes morbidity has been related to increased oxidative stress and defective intracellular antioxidative defense (Khaki et al., 2009). High levels of oxidative free radicals and low levels of endogenous antioxidants are involved in the pathogenesis of diabetes (Jakus, 2000; Rahimi et al., 2005; Vincet et al., 2003). Hyperglycemia is a defining characteristic of diabetes. However, dyslipidemia is regarded as the main cardiovascular risk factor of diabetes. Diabetic dyslipidemia is typically characterized by elevated serum triglycerides (TG), total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol Correspondence: Himan Mamandy, Department of Cellular and Molecular Biology, Higher Education Institute of Rab-Rashid, Tabriz, Iran. Tel: +98 9395930663. Fax: +98 442 2222411. E-mail: [email protected]
History Received 4 May 2014 Revised 10 August 2014 Accepted 28 August 2014 Published online 23 January 2015
(LDL-C), and the low level of high-density lipoprotein (HDL-C) (Brunzell et al., 2008). Although there is a wide range of antidiabetic drugs in use, they often generate undesirable side effects (Suji & Sivakami, 2003). The discovery of more effective and safer remedies is, therefore, critical to the treatment of diabetes (Fakir et al., 2009). In particular, herbal remedies attract considerable research interest because of their less adverse effects comparing with synthetic drug treatments. The medicinal herb Peganum harmala L (Zygophyllaceae) (PH), also known as Syrian rue, grows in Asia, North Africa, and Mediterranean regions (Asghari & Lockwood, 2002). The PH extract has several therapeutic effects such as antitumor, anti-inflammation, antioxidant (Asghari & Lockwood, 2002; Giancarlo et al., 2006; Goel et al., 2009) and blood glucose (BG)-lowering activities. These effects have mainly been attributed to the alkaloid components of the PH, including harmaline, harmine, and harman (Singh et al., 2008). Rhus coriaria L (Anacardiaceae) (RC), also known as Sumac, grows in Asia, North America, and Europe
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(Pashazadeh et al., 2013). It has been reported that this plant shows significant antitumor, anti-inflammation, antioxidant, antifungal, hypoglycemic, and digestive properties (Khaki et al., 2009; Rehman et al, 2000; Smith, 2003). The fruit body of RC contains a complex of bioactive compounds including tannins, quercetin, isoquercetin, myricetin, ellagic acid, gallic acid, and tannic acid (Astulla et al., 2008). The flavonoid quercetin has been shown to have BG-lowering effects similar to a-glucosidase inhibitors through blocking the glucose absorption process in intestine via inhibition of a-amylase enzyme (Giancarlo et al., 2006). Urtica dioica L (Urticaceae) (UD) has long been known in folk medicine (Kavalali et al., 2003; Ur-Rahman & Zaman, 1989). Numerous studies indicate hypoglycemic effect of this plant (Bnouham., 2003; Farzami et al., 2003; Ziyyat et al., 1997). Hypoglycemic effect of UD has been attributed to the presence of lectins (Kavalali et al., 2003). The lectins increase either pancreatic insulin secretion or insulin release from its bond form (Stanely et al., 2000). As mentioned above, several antidiabetic effects of PH, RC, and UD extracts have been reported, but there are no reports yet regarding the combined use of these extracts. The aim of this study was to investigate the effect of a triplex mixture of these medicinal plants on metabolic and histological parameters in normal and experimental diabetic rats.
Materials and methods Experimental animals Male Wistar rats with an average weight of 230 ± 7 g were purchased from the Laboratory Animal Center of Tabriz University of Medical Sciences. Animals were kept under standard laboratory conditions in the Higher Educational Center of Jahad-e-Keshavarzi (Tabriz, Iran). The experiments were initiated after obtaining approval from the Animal Rights Office, Higher Educational Center of Jahad-eKeshavarzi. All protocols were designed and performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
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Table 1. List of animal groups with different treatments. Groups Status and treatment 1 2 3 4 5 6 7
Healthy, treated with saline in equivalent volume to the test treatments Diabetic, treated with saline in equivalent volume to the test treatments Diabetic, treated with aqueous extract of PH (200 mg/kg) Diabetic, treated with aqueous extract of RC (200 mg/kg) Diabetic, treated with aqueous extract of UD (200 mg/kg) Diabetic, treated with triplex mixture of PH, RC and UD (200 mg/kg) Diabetic, treated with glibenclamide (0.16 mg/kg)
in Table 1. Each plant extract or triple mixture (1:1:1) was administered at a dose of 200 mg/kg/d. The treatment was given for 4 weeks by daily gavage. At the end of the experiment, blood samples were obtained from the heart under general anesthesia for biochemical analysis. Then animals were sacrificed and the whole liver and kidney tissues were excised for histopathological examinations. Biochemical analysis The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, TC, VLDL-C, LDL-C, HDL-C, BG, and creatinine were measured at the Danesh Pathobiology Laboratory (Tabriz, Iran) using standard colorimetric enzymatic kits from Bionik (Bionik, Tehran, Iran). Histological evaluation Rat tissue samples were histologically examined by hematoxylin and eosin (H&E) staining. The liver specimens were evaluated for piecemeal necrosis, the degree of portal inflammation and focal lytic necrosis, and the kidney tissue samples for glomerular distension. All histopatjological analyses were performed in the Danesh Pathobiology Laboratory. Statistical analysis
Plant materials and extraction preparation PH seeds, UD leaves, and RC fruits were obtained on April 2013 from the Research Center of Agriculture and Natural Resources (Tabriz, Iran). All plant materials were verified by the Tabriz Reference Herbarium at the Faculty of Agriculture, University of Tabriz, Iran. The extraction procedure was performed as previously described by Clevenger (1928).
All data were expressed as mean ± SD. The one-way analysis of variance (ANOVA) was used to evaluate significance difference. Comparison between the experimental groups of rats was done by the LSD test. All analyses were performed with the SPSS software (SPSS version 9, Chicago, IL). A value of p50.05 was considered statistically significant.
Results Diabetes induction
Metabolic parameters
Diabetes was induced by means of a single dose of alloxan monohydrate (Sigma-Aldrich, St. Louis, MO; 110 mg/kg). Diabetes was confirmed on the third day by determining glucose levels in tail vein blood samples. Glucose concentration of 250 mg/dl was considered as the diabetic threshold.
The ANOVA tests revealed no significant variation among replications, but statistically significant differences among the tested groups at p values between 0.01 and 0.05 (Table 2). Compared with the control group, alloxan administration caused significant increases in ALP, ALT, LDL-C, TG, BG, and creatinine (Table 3). Compared with the saline-treated diabetic rats, all three aqueous extracts led to significant decreases in the levels of ALP, LDL-C, and BG. Both PH and RC treatment led to significant increase in HDL levels. The serum concentration
Experimental groups The rats were randomly divided into seven groups with four rats in each (control, control diabetic, extract-treated groups, and diabetic rats who received glibenclamide) as shown
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Table 2. Summary of the variables and results of ANOVA. Means of square SOV Replication Treatment Error % CV
df 3 6 18 –
ALP
ALT ns
22 802.76 50 658.81** 31 616 22.40
AST ns
712.6 14 235.65** 2533.16 30.06
ns
1566 41 445.65** 5857.86 27.44
VLDL-C ns
6.59 21.18** 2.68 12.78
LDL-C
HDL-C
ns
ns
3.89 66.53** 3.84 18.09
4.72 127.72** 12.90 14
TG ns
88 415.08** 63.95 12.41
TC
BG ns
105.76 252.11* 86.26 11.94
Creatinine ns
1597.76 97 294.5** 2423.92 21.73
0.0002ns 0.033** 0.002 5.2
ns
Non-significant. **Significant at 0.01 p value; *significant at 0.05 p value.
Table 3. Multiple comparison of variables between studied groups of animals using least significance test (LSD). Means ± SD
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U/l Group Group 1 (healthy) Group 2 (diabetic) Group 3 (PH) Group 4 (RC) Group 5 (UD) Group 6 (triple mixture) Group 7 (glibenclamide) 5% LSD
mg/dL
ALP
ALT
AST
VLDL-C
LDL-C
HDL-C
TG
TC
BG
Creatinine
311.8 ± 8.8 1413.3 ± 232.3 631.3 ± 114.3 912.5 ± 232.2 1012.5 ± 165.2 692.5 ± 147.9 581.3 ± 205.5 264.1
83.5 ± 3.1 251.2 ± 45.7 141.7 ± 30.5 144.2 ± 17.6 243.0 ± 93.7 151.2 ± 28.0 156.7 ± 54.6 74.7
178.2 ± 2.0 278.7 ± 66.0 277.0 ± 51.3 284.0 ± 43.3 485.0 ± 145.7 267.0 ± 63.5 182.0 ± 50.6 113.7
22.2 ± 0.8 20.0 ± 0.7 25.6 ± 1.3 33.2 ± 2.5 23.3 ± 1.5 25.0 ± 2.1 30.2 ± 2.5 2.43
67.9 ± 0.4 124.2 ± 0.5 69.6 ± 0.8 61.2 ± 2.2 38.5 ± 0.5 66.5 ± 4.4 103.2 ± 0.9 2.91
41.7 ± 0.8 33.0 ± 2.8 51.8 ± 0.8 57.5 ± 5.5 67.0 ± 2.0 58.5 ± 5.0 49.5 ± 3.4 5.33
54.1 ± 1.5 69.2 ± 8.3 63.2 ± 4.7 82.7 ± 12.6 55.2 ± 8.7 57.2 ± 2.0 69.0 ± 11.6 11.88
70.2 ± 0.9 81.7 ± 4.3 71.5 ± 15.6 82.0 ± 1.4 78.7 ± 34.4 69.2 ± 120.2 91.0 ± 10.6 13.79
112.2 ± 2.2 500.0 ± 8.1 105.0 ± 12.9 392.5 ± 9.5 156.2 ± 34.4 190.7 ± 120.2 128.7 ± 13.1 73.14
0.82±0.0 1.04 ± 0.0 0.90 ± 0.0 1.04 ± 0.0 0.83 ± 0.0 0.89 ± 0.0 0.94 ± 0.0 0.071
of TG was decreased only by treatment with RC and PH. Parameters like ALT, LDL-C, TG, and creatinine recovered to control levels after the treatment with extract mixture (Table 3). Histopathologic findings Histopathology of liver The results obtained from the histopathological study showed that the diabetic group had marked portal inflammation, mild piecemeal necrosis, and 1–4 focal lytic necrosis per 10 microscopic field (Figure 1b). All the applied treatments led to significant improvement in the histological features of the liver (Figures 1c–f and 2). However, the triplex mixture of PH, RC, and UD aqueous extracts caused a more obvious improvement in the liver condition (Figure 1f). Histopathology of kidney The kidney histopathology results showed that diabetic rats had severe glomerular distension (Figure 3b). Treatments with all plant extracts and their mixture recovered glomerular distension (Figures 3c–f and 4). The RC aqueous extract and the triplex mixture were more beneficial in this case as led to absolute remission of glomerular distension (Figure 3d and f).
Discussion In this study, significant changes in the serum lipids, lipoproteins, except VLDL and TC, and markers of liver and kidney function were observed in alloxan-induced diabetic rats, and these changes were modulated with the combination of PH, RC, and UD extracts in the experimental treatments.
The hypoglycemic effect of all three plants has been demonstrated through different mechanisms, including inhibition of glucose absorption (Kavalali et al., 2003; Nuraliev & Avezov, 1992; Ur-Rahman & Zaman, 1989) and stimulation of pancreatic insulin secretion (Stanely et al., 2000). According to our findings, the PH aqueous extract had the most BG-lowering effect in the diabetic rats. The serum level of glucose in animals that received PH treatment alone was even lower than the control. The antidiabetic effect of PH has mainly been attributed to the water-soluble 4-hydroxypipecolic acid (4-HPA) (Singh et al., 2012; Tetko et al., 2005). A synergic effect of 4-HPA and insulin has been reported on stimulation of glucose uptake by skeletal muscle cells (Singh et al., 2008, 2012). Consistent with the current study, several studies have reported a significant improvement in the overall lipid profile in animal models receiving PH, RC, or UD extracts (Anwer et al., 2012; Ozkol et al., 2013; Singh et al., 2012; Soliman et al., 2012). The combined extract treatment was also beneficial with comparable results with those generated separately. The beneficial impacts of the triple mixture were particularly pronounced in the reducing ALT, LDL-C, and creatinine, and in the increasing HDL-C. In the current study, the alloxan-induced increase in TG was reverted by treatment with UD as well as with the triple mixture extract. These results are consistent with a previous report showing TGlowering effect of 4-HPA in diabetic mice (Singh et al., 2012). However, Ahangarpour et al. (2012) found no significant effect of UD extract on the serum TG in fructose-induced insulin-resistant rats. Moreover, reducing effects of PH and RC on serum TC have been previously reported (Anwer et al., 2012; Singh et al., 2012; Tahery et al., 2013). These inconsistencies might be due to differences in the diabetic animal model and the duration of treatment.
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Figure 1. Microscopic structure of liver of a (a) control rat (group 1) and (b) diabetic rat (group 2) showing marked portal areas, mild piecemeal necrosis, and 1–4 foci per field; (c) PH-treated diabetic rat (group 3) showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field; (d) RC-treated diabetic rat (group 4) showing mild portal areas and 1–4 foci per field; (e) UD-treated diabetic rat (group 5) showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field; (f) triplex mixture-treated diabetic rat (group 6) showing mild portal areas. All slides stained with hematoxylin and eosin (original magnification 10).
Figure 2. Microscopic structure of liver of a glibencalmide-treated diabetic rat showing moderate portal areas, mild piecemeal necrosis, and 1–4 foci per field. The slide stained with hematoxylin and eosin (original magnification 10).
ALP, ALT, and AST are important hepatic enzymes since their levels in serum have been established as specific and reliable biomarkers for liver damage (Ahangarpour et al., 2012). Both beneficial and adverse effects of PH, RC, and UD extracts on liver function tests have been reported earlier (Ahangarpour et al., 2012; Pourahmad et al., 2009; Rayne & Mazza, 2007; Soliman et al., 2012). According to the results of the current study, alloxan-induced diabetic rats had marked increases in the serum levels of ALP and ALT that were consistent with observed changes in histopathology. Although treatments of all the single and mixture significantly improved hepatic necroinflammation, only the triple mixture supplementation significantly reduced two hepatic biomarkers ALP and ALT compared with the diabetic control group. When single treatments were compared with the mixture treatment, the mixture treatment was collectively more potent. High levels of serum creatinine are associated with abnormal renal function, especially decreased glomerular
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DOI: 10.3109/13880209.2014.960943
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Figure 3. Microscopic structure of kidney of a (a) control rat (group 1) and (b) diabetic rat (group 2) showing sever glomerular distension; (c) PHtreated diabetic rat (group 3) showing only a mild glomerular distension; (d) RC-treated diabetic rat (group 4) showing no pathological condition; (e) UD-treated diabetic rat (group 5) showing only a mild glomerular distension; (f) triplex mixture-treated diabetic rat (group 6) showing no pathological condition. All slides stained with hematoxylin and eosin (original magnification 10).
Figure 4. Microscopic structure of kidney of a glibencalmide-treated diabetic rat showing only a mild glomerular distension. The slide stained with hematoxylin and eosin (original magnification 10).
filtration rate (Gu¨ne¸s et al., 1999). Protective effect of UD on renal injury has been shown previously (Sayhan et al., 2012). However, Kianbakht et al. (2013) have shown that even though UD leaf extract improves glycemic control in type 2 diabetic patients, there was no significant effect on the serum creatinine level. It has also been reported that administration of PH extract induced no changes in the creatinine level in rats (Lamchouri et al., 2002). In the present study, there was a significant reduction in creatinine level with remarkable improvement in the glomerular distension after feeding diabetic rats with the extract combination of PH, RC and UD. There is a rare attention to the effects of combined traditional medicinal plants in the literature. The current study was focused on the triplex mixture of three medicinal plants. To our knowledge, this is the first report showing the curative effects of combined PH, RC, and UD against diabetic complications in an experimental rat model. This study
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included a broad panel of biomarkers and histopathologic characteristics in liver and kidney. Future studies will be required to examine the extracts sub-fractions and to identify the active components.
Conclusion This study showed that PH, RC, and UD extracts had significant antidiabetic, hypolipidemic, and liver and renal damage recovering effects. Moreover, these beneficial effects were more potent with the combination of them compared with that of single treatments.
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Acknowledgements The authors would like to thank Dr. Masoud Darabi for critical revising of the manuscript and Dr. Ahmad Razban Haghighi at the Tabriz Reference Herbarium, University of Tabriz, for providing herbal materials.
Declaration of interest The authors report that they have no declarations of interest. The authors alone are responsible for the content and writing of this article. This study was financially supported by a research grant from the Higher Educational Center of Jahade-Keshavarzi, Tabriz, Iran (Project no.: 7114/261/2)
References Ahangarpour A, Mohammadian M, Dianat M. (2012). Antidiabetic effect of hydroalcholic Urtica dioica leaf extract in male rats with fructoseinduced insulin resistance. Iran J Med Sci 37:181–6. Anwer T, Sharma M, Pillai KK, et al. (2012). Antihyperglycemic, antidyslipidemic and antioxidant activity of Rhus coriaria in STZinduced type 2 diabetic rats. Afr J Pharm Pharmacol 6:2851–5. Asghari G, Lockwood GB. (2002). Stereospecific biotransformation of (±) phenylethyl propionate by cell cultures of Peganum harmala L. Iran Biomed J 6:43–6. Astulla A, Zaima K, Matsuno Y, et al. (2008). Alkaloids from the seeds of Peganum harmala showing antiplasmodial and vasorelaxant activities. J Nat Med 62:470–2. Bnouham M, Merhfour FZ, Ziyyat A, et al. (2003). Antihyperglycemic activity of the aqueous extract of Urtica dioica. Fitoterapia 74: 677–81. Brunzell JD, Davidson M, Furberg CD, et al. (2008). Lipoprotein management in patients with cardiometabolic risk. J Am Coll Cardiol 51:1512–24. Clevenger JF. (1928). Apparatus for the determination of volatile oil. J Am Pharm Assoc 17:345–9. Fakir H, Korkmaz M, Gu¨ller B. (2009). Medicinal plant diversity of western Mediterranean region in Turkey. J Appl Biol Sci 3:30–40. Farzami B, Ahmadvand D, Vardasbi S, et al. (2003). Induction of insulin secretion by a component of Urtica dioica leave extract in perifused islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats. J Ethnopharmacol 89:47–53. Giancarlo S, Rosa LM, Nadjafi F, Francesco M. (2006). Hypoglycaemic activity of two spices extract: Rhus coriaria L, Bunium persicum Boiss. Nat Prod Res 20:882–6. Goel N, Singh N, Saini R. (2009). Efficient in vitro multiplication of Syrian Rue (Peganum harmala L.) using 6-benzylaminopurine preconditioned seedling explants. Nat Sci 7:129–34. _ Aydin M, et al. (1999). The effects of Rumex Gu¨ne¸s Hv, Deg˘irmenci I, patientia L. and Urtica dioica L. on some blood and urine parameters, and liver and kidney histology in diabetic rats. Turk J Med Sci 29: 227–32.
Pharm Biol, Early Online: 1–6
Jakus V. (2000). The role of free radicals, oxidative stress and antioxidant systems in diabetic vascular disease. Bratisl Med J 101: 541–51. Kavalali G, Tuncel H, Goksel S, Hatemi HH. (2003). Hypoglycemic activity of Urtica pilulifera in streptozotocin-diabetic rats. J Ethnopharmacol 84:241–5. Khaki AA, Khaki A, Nouri M, et al. (2009). Evaluation effects of quercetin on liver apoptosis in streptozotocin-induced diabetic rat. J Med Plants 8:70–8. Kianbakht S, Khalighi-Sigaroodi F, Dabaghian FH. (2013). Improved glycemic control in patients with advanced type 2 diabetes mellitus taking Urtica dioica leaf extract: A randomized double-blind placebocontrolled clinical trial. Clin Lab 59:1071–6. Lamchouri F, Settaf A, Cherrah Y, et al. (2002). Experimental toxicity of Peganum harmala seeds. Ann Pharm Fr 60:123–9. Nuraliev IUN, Avezov G.A. (1992). The efficacy of quercetin in alloxan diabetes. Eksp Klin Farmakol 55:42–4. _ (2013). Therapeutic potential of Ozkol H, Tuluce Y, Dilsiz N, Koyuncu I. some plant extracts used in Turkish traditional medicine on streptozocin-induced type1 diabetes mellitus in rats. J Membr Biol 246:47–55. Pashazadeh M, Rezaei A, Pashazadeh M, et al. (2013). Effect of Rhus coriaria consumption on levels of blood glucose and lipids in alloxaninduced diabetic rats. Int J Emerg Trends Pharm Sci 1:34–7. Pourahmad J, Eskandari MR, Shakibaei R, Kamalinejad M. (2009). A search for hepatoprotective activity of aqueous extract of Rhus coriaria L. against oxidative stress cytotoxicity. Food Chem Toxicol 48:854–8. Rahimi R, Nikfar S, Larijani B, Abollahi M. (2005). A review on the role of antioxidants in the management of diabetes and its complications. Biomed Pharmacother 59:365–73. Rayne S, Mazza G. (2007). Biological activities of extracts from sumac (Rhus spp.): A review. Plant Foods Hum Nutr 62:165–75. Rehman A, Jenner A, Halliwel B. (2000). Gas chromatography–mass spectrometry analysis of DNA: Optimization of protocols for isolation and analysis of DNA from human blood. Method Enzymol 319: 401–17. Sayhan MB, Kanter M, Oguz S, Erboga M. (2012). Protective effect of Urtica dioica L. on renal ischemia/reperfusion injury in rat. J Mol Histol 43:691–8. Singh AB, Chaturvedi JP, Narender T, Srivastava AK. (2008). Preliminary studies on the hypoglycemic effect of Peganum harmala L. seeds ethanol extract on normal and streptozotocin induced Diabetic Rats. Ind J Clin Biochem 23:391–3. Singh AB, Khaliq T, Chaturvedi JP, et al. (2012). Anti-diabetic and antioxidative effects of 4-hydroxypipecolic acid in C57BL/KsJ-db/db mice. Hum Exp Toxicol 31:57–65. Smith JF. (2003). Antidiabetic drugs. Med Lib 5:5–6. Soliman AM, Abu-El-Zahab HS, Alswiai GA. (2012). Efficacy evaluation of the protein isolated from Peganum harmala seeds as an antioxidant in liver of rats. Asia Pac J Trop Med 6:285–95. Stanely P, Prince M, Menon VP. (2000). Hypoglycaemic and other related actions of Tinospora cordifolia roots in alloxan-induced diabetic rats. J Ethnopharmacol 70:9–15. Suji G, Sivakami S. (2003). Approaches to the treatment of diabetes mellitus: An overview. Cell Mol Biol 49:635–9. Tahery H, Mehdi M, Fard MH, et al. (2013). Effects of aqueous and ethanolic extracts of Peganum harmala L seeds on lipids profile in rats. J Birjand Univ Med Sci 20:108–14. Tetko IV, Gasteiger J, Todeschini R, et al. (2005). Virtual computational chemistry laboratory – Design and description. J Comput Aided Mol Des 19:453–63. Ur-Rahman A, Zaman K. (1989). Medicinal plants with hypoglycemic activity. J Ethnopharmacol 26:1–55. Vincet AM, Russell JW, Low P, Feldman EL. (2003). Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 25:612–28. Ziyyat A, Legssyer A, Mekhfi H, et al. (1997). Phythotherapy of hypertension and diabetes in oriental Morocco. J Ethnopharmacol 58: 45–54.
