African Journal of
Pharmacy and Pharmacology

  • Abbreviation: Afr. J. Pharm. Pharmacol.
  • Language: English
  • ISSN: 1996-0816
  • DOI: 10.5897/AJPP
  • Start Year: 2007
  • Published Articles: 2243

Full Length Research Paper

Effect of Citrus paradisi and Citrus sinensis on glycemic control in rats

Neelam Mallick
  • Neelam Mallick
  • Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi 75270-Pakistan.
  • Google Scholar
and Rafeeq Alam Khan*
  • and Rafeeq Alam Khan*
  • Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi 75270-Pakistan.
  • Google Scholar


  •  Received: 22 September 2014
  •  Accepted: 21 January 2015
  •  Published: 22 January 2015

 ABSTRACT

This study was conducted to explore the effects of Citrus sinensis (orange juice) and Citrus paradisi (grapefruit juice) at three different doses alone and their two combinations on plasma insulin and blood glucose levels in healthy and diabetic rats. Diabetes was induced by alloxan after which rats were treated with C. sinensis and C. paradisi juices for six weeks, blood glucose and plasma insulin concentration was estimated. C. sinensis showed significant reduction in blood glucose and a significant rise in plasma insulin at all three doses. However C. paradisi revealed highly significant fall in blood glucose and highly significant rise in plasma insulin levels only at 0.5 ml/kg. Whereas combination dose group CSP-2 (5 + 0.3 ml/kg) showed highly significant reduction in blood glucose and highly significant rise in plasma insulin levels as compared to diabetic control. These results suggest that flavonoids and other essential nutrients present in citrus fruits juices might be responsible for these effects. Hence, it may be concluded that these juices may be used in combination to produce a synergistic effect in decreasing blood glucose and elevating plasma insulin levels.

Key words: Grape fruit, orange, glucose level, insulin.


 INTRODUCTION

Diabetes mellitus is a metabolic disorder resulting in severe immediate and continuing consequences extending from brain damage to heart disease (American Diabetes Association, 2009). According to the report of International Diabetes Federation, the estimated number of people with diabetes increased universally in recent years and is expected to reach 380 million in 2025 (Samreen, 2009). Large number of agents is available for treatment but is associated with various serious adverse effects (Tahrani et al., 2010; Patel et al., 2012). Plants have been used over a long time for the treatment of different ailments. Fruits and vegetables have gained importance for the management of diabetes mellitus, for example pomegranate, grapes, guava, lemon, tomatoes and grape fruits. They have the capacity to re-establish function of pancreatic tissues by increasing insulin output, inhibiting intestinal absorption of glucose or helping metabolites in insulin dependent processes (Javascript, 2002; Free encyclopedia, 2002; Punitha et al., 2006; Malviya et al., 2010; Riaz et al., 2013).

This study was conducted on fresh juices of two fruits, Citrus sinensis (orange) and Citrus paradisi (grapefruit) belonging to Rutaceae family, since plants and plant derived phytochemical have great potential to treat and control diabetes (Behera and Yadev, 2013). Grapefruit contains phytochemicals including limonoids and lycopene. It is also an exceptional source of vitamin C, dietary fiber, vitamin A, potassium, folate and vitamin B5 (Mateljan, 2006). It also contains high levels of iron, calcium and other minerals. Pink and red varieties of grapefruits are rich in beta carotene, high in fiber and low in calories. They possess protective plant chemicals like phenolic acid, limonoids, terpenes, monoterpenes and bioflavonoids which protect against cancer and heart disease. The major bioflavonoid in grapefruit is naringin that gives grapefruit juice bitter taste (Giovannucci et al., 2002; Armando et al., 1998). Naringin exerts diverse pharmacological effects like antioxidant activity, blood lipid-lowering effect (Gorinstein et al., 2006), anti-carcinogenic activity (Armando et al., 1998) and inhibition of selected cytochrome P450 enzymes including CYP3A4 and CYP1A2 (Gao et al., 2006). Naringin being one of the components of these fruits have been reported for its antidiabetic effects (Pari and Suman, 2010). Grapefruit enhance appetite and is employed for its digestive, stomachic, antiseptic and diuretic properties (Herbal medicine, 2000).

Orange being a rich source of nutrients has also been known for a number of health and nutritional benefits. Orange contains nearly two hundred phytonutrients and flavonoids, which have shown activity against different types of cancers. They also have been reported to have strong anti-inflammatory and anti-oxidant properties and prevent bone loss (Liu et al., 2012; Peluso, 2006; Gao et al., 2006; Chiba et al., 2003). The essential oils in orange juice (C. sinensis) contain many constituents, including monoterpenes and sesquiterpenes with d-limonene as a major constituent (Graciela et al., 2003). Orange juice has been reported for cholesterol lowering effect in animal models (Kurowska et al., 2000) as well as humans (Roza et al., 2007). Its anti-inflammatory role in different disease is also well documented (Buscemi et al., 2012; Ghanim et al., 2010). Narirutin or Naringenin 7-O-rutinoside is another important flavonoid present abundantly in orange juice (Sawalha et al., 2009). It is absorbed well and shows good bioavailability (Manach et al., 2003). It is also shown to possess anti-inflammatory (Ha et al., 2012; Funaguchi et al., 2007), anti-allergic and anti-asthmatic effects (Rogerio et al., 2010; Funaguchi et al., 2007). Orange and grape fruit are both rich in phytochemical, flavonoids and vitamins which revealed to have strong anti-inflammatory and antioxidant properties (Liu et al., 2012; Peluso, 2006; Gao et al., 2006; Chiba et al., 2003). 


 MATERIALS AND METHODS

Animals

Present study was conducted after approval of the proposal by board of advance studies and research, University of Karachi on adult Wister rats with mean body weight of 220 ± 10 g. Animals were kept under controlled condition of temperature 23 ± 2°C and humidity 50 to 60%, with free access to food and water. Five rats were housed in each plastic cage measuring 32" × 18" × 16".

Citrus sinensis

C. sinensis was obtained from local market and recognized by Center of Plant Conservation, University of Karachi. The voucher specimen no.CS-10-10 was placed in the Department of Pharmacognosy, University of Karachi. C. sinensis was peeled and pressed by hand to yield fresh juice which was then used immediately after filtration. C. sinensis juice was administered in three different doses that is, 2, 5 and 8 ml/kg according to body weight through oral route.

Citrus paradisi

C. paradisi was also bought from local market, recognized by Center of Plant Conservation, University of Karachi. The voucher specimen no. CP-09-10 was kept in the Department of Pharmacognosy, University of Karachi. The fruits were peeled, pressed and fresh juice so obtained was filtered and used soon after yielding. C. paradisi was given in three doses that is, 0.1, 0.3 and 0.5 ml/kg according to body weight through oral route.

Combinations of C. sinensis and C. paradisi

C. sinensis and C. paradisi were also given orally in two combined doses that is, 2 + 0.1 and 5 + 0.3 ml/kg, and were abbreviated as CSP-1 and CSP-2.

Induction of diabetes

Diabetes was induced in overnight fasted rats by means of a single dose of alloxan monohydrate (Sigma chemicals, USA). Alloxan was administered in the dose of 180 mg/kg in normal saline by intraperitoneal injection. The glucose level in plasma was determined at 72 h after the administration of alloxan (Rohilla and Ali, 2012). Animals with blood glucose concentration more than 250 mg/kg were considered diabetic and used for the study.

Design of experiment

All rats were distributed in eleven groups each comprising of ten animals. One group received saline and served as normal control, while remaining ten groups induced diabetes received C. sinensis (three groups), C. paradisi (three groups), while two groups received C. sinensis and C. paradisi in combination, one group received standard drug glibenclamide and one group received saline served as diabetic control. All doses were given by gastric intubation on once daily basis for a period of six weeks. Entire study was performed under NACLAR guideline (Bernard, 2004). At the end of the investigational period, animals were deprived of food overnight, sacrificed by amputation and blood samples were collected in gel tubes and 3.2% sodium citrate containing tubes (9:1 v/v).

Estimation of blood glucose

Blood glucose was estimated by two methods, first through Accu-Chek glucometer by taking blood from tail vein of rats, after 72 h of intraperitoneal injection of alloxan to check the induction of diabetes. Second through blood collected in gel tubes.  Serum  was separated by Humax 14K centrifuge at 2000 rpm for 10 min and then commercial kit of glucose (Human Diagnostic, Germany) was used to estimate blood glucose by Humalyzer 3000 Human Germany.

Estimation of plasma insulin

Blood was collected in 3.2% sodium citrate tubes and plasma was parted by Humax 14 K centrifuge at 3000 rpm for 15 min. Insulin was estimated using commercial ELISA insulin kit (Accu-Bind, Elisa Microwells, USA).

Statistical analysis

Entry of data and analysis was performed using 17th version of superior performance statistical software (SPSS). Quantities were presented as mean ± SD with 95% confidence interval. Analysis of variance (ANOVA) followed by post hoc was performed for comparisons of values with control. Values of p < 0.05 were considered statistically significant and p < 0.005 highly significant.

 


 RESULTS

Table 1 reveals the effect of C. sinensis on blood glucose and plasma insulin levels in diabetic and control rats. There was significant decrease in blood glucose at 2, 5 and 8 ml/kg of C. sinensis in a dose dependent manner than diabetic control, while there was significant rise in insulin level at all three dose as compared to diabetic control. Table 2 displays the effect of C. paradisi on blood glucose and plasma insulin levels in diabetic and control animals. There was highly significant decrease in blood glucose at 0.1, 0.3 and 0.5 ml/kg of C. paradisi in a dose dependent manner than diabetic control. While there was highly significant increase in plasma insulin levels at 0.5 ml/kg and significant increase at 0.3 ml/kg. However, there was no change in plasma insulin level at 0.1 ml/kg. Table 3 shows the comparative effect of combination doses of C. sinensis and C. paradisi with standard drug glibenclamide on plasma glucose and insulin levels in diabetic control and normal control animals. There was highly significant decrease in blood glucose in animals treated by CSP-2 combination and glibenclamide. While significant decrease was observed in animals treated with CSP-1 combination as compared to diabetic control. There was also highly significant rise in plasma insulin at CSP-2 and glibenclamide treated  animals  as  compared to diabetic control. However, there was no change in plasma insulin level at CSP -1.

 

 

 


 DISCUSSION

Type-I diabetes is categorized by a loss of insulin-producing beta cells islets of Langerhans in the pancreas leading to decrease in insulin. Diabetes was induced by the administration of alloxan to the experimental animals causing destruction of beta cells, a result consistent with several studies in rats (Mohammed et al., 2010; Prem et al., 2012). Findings of this study clearly indicates that diabetic animals treated with C. sinensis and C. paradisi had a reduced blood glucose concentration when compared to the diabetic control animals, a result almost similar to reference drug glibenclamide (Table 3).

Present study showed significant blood glucose lowering effect by C. sinensis at all three doses, while there was also significant increase in plasma insulin levels. On the basis of these results, it could be postu-lated that C. sinensis induced hypoglycemic effect may be due to the presence of high contents of flavonoids and monoterpenes in C. sinensis. The hypo-glycemic effect of monoterpenes had been previously demonstrated by Tavafi et al. (2011). Monoterpenes may have insulin mimetic properties or may be able to induce insulin production from the surviving beta cells enough to facilitate glucose uptake from the blood.

C. paradisi showed highly significant increase in plasma insulin levels at 0.5 ml/kg. This effect may be due to the presence of vitamin C in C. paradisi which acts as strong antioxidant. This effect might be also due to the presence of naringin. Since naringin is reported to produce hypoglycemic effect due to its strong anti-oxidation property (Pari et al., 2010). Several flavonoids and terpenoids are present in C. paradisi. It is therefore, logical to conclude that the hypoglycemic effect of grapefruit juice (C. paradisi) may be due to flavonoids and/or terpenoids content of C. paradisi (Cerda et al., 1988). While there was highly significantly decrease in glucose level by CSP-2. This might be due to synergic effect of naringin in C. sinensis and polyphenols in C. paradisi. Moreover naringin and vitamin C had been reported to produce hypoglycemic and hypo-cholesterolemic effects due to their strong anti-oxidant properties (Pari and Suman, 2010). While polyphenols and vitamin C in C. paradisi may be responsible for these beneficial effects due to their strong antioxidant property (Violi et al., 2010).

All antioxidants may produce a synergistic effect which may provide strengthening to the B-cells of pancreas to release more insulin. Combined management with naringin and vitamin C had been demonstrated to ameliorate streptozotocin-induced diabetes in rats (Punithavathi et al., 2008). It has also been reported that hesperidin and naringin are useful for improving hyperlipidemia   and   hyperglycemia  in  type - II  diabetic animal models by partially regulating fatty acid and cholesterol metabolism and affecting gene expression of glucose-regulating enzymes (Jung et al., 2004; Jung et al., 2006). Though grapefruit juice has potent hypo-glycemic effects in experimental animals, however its use should be cautioned due to its enzyme inhibitory effects on the metabolism of many drugs (Bailey et al., 2000), which can lead to adverse effects from these medications (Gao et al., 2006).


 CONCLUSION

From the results of the present study it may be concluded that combination of C. sinensis and C. paradisi is most effective in high doses, however further studies on more combination doses of C. sinensis and C. paradisi are required to reveal the role of these substances on reduction of blood glucose levels and combating diabetes mellitus. This study suggests that grapefruit juice and orange juice plays a good role in controlling the glucose level of experimental animals and can be applied clinically on patients with diabetes and lower cholesterol level.


 CONFLICT OF INTEREST

There are no conflicts of interests



 REFERENCES

 

American Diabetes Association. (2009). Standards of medical care in diabetes-2009. Diabetes care 32(1):S13-S61.
 
Armando C, Maythe S, Beatriz NP (1998). Antioxidant activity of grapefruit seed extract on vegetable oils. J Sci. Food Agric. 77:463-467.
crossref
 
Bailey DG, Dresser GK, Kreeft JH, Munoz C, Freeman DJ and Bend JR (2000). Grapefruit-felodipine interaction: effect of unprocessed fruit and probable active ingredients. Clin. Pharmacol. Ther. 68:468-477.
crossref
 
Behera B, Yadav D (2013). Current research on plants having antidiabetic potential: An overview. Research and Reviews. J. bot. Sci. 2(2):4-17.
 
Bernard T (2004). Guidelines on the care and use of animals for scientific purposes. National Advisory Committee for Laboratory Animal Research (NACLAR). Available at: www3.ntu.edu.sg/Research2/.../NACLAR-guide% Z0Lines.pdf.
 
Buscemi S, Rosafio G, Arcoleo G, Mattina A, Canino B, Montana M, Verga S, Rini G (2012). Effects of red orange juice intake on endothelial function and inflammatory markers in adult subjects with increased cardiovascular risk. Am. J. Clin. Nutr. 95(5):1089-1095.
crossref
 
Cerda JJ, Robbins FL, Burgin CW, Baumgatner TG, Rice RW (1988). The effects of grapefruit pectin on patients at risk for coronary heart disease without altering diet or lifestyle. Clin. Cardiol. 11:589-94.
crossref
 
Chiba H, Uehara M, Wu J, Wang X, Masuyama R, Suzuki K, Ishimi Y (2003). Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice. J. Nutr. 133(6):1892-1897.
 
Free encyclopedia (2002). Grapefruit. Available at: http://en.wikipedia.org/wiki/Grapefruit.
 
Funaguchi N, Ohno Y, La BLB, Asai T, Yuhgetsu H, Sawada M, Takemura G, Minatoguchi S, Fujiwara T, Fujiwara H (2007). Narirutin inhibits airway inflammation in an allergic mouse model. Clin Exp. Pharmacil. Physiol. 34(8):766-770.
crossref
 
Gao K, Henning SM, Niu Y, Youssefian AA, Seeram NP, Xu A, Heber D (2006). The citrus flavonoid naringenin stimulates DNA repair in prostate cancer cells. J. Nutr. Biochem. 17(2):89-95.
crossref
 
Ghanim H, Sia CL, Upadhyay M, Korzeniewski K, Viswanathan P, Abuaysheh S, Mohanty P, Dandona P (2010). Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression. Am. J. Clin. Nutr. 91(4):940-949.
crossref
 
Giovannucci E, Rimm EB, Liu Y, Stampfer MJ, Willett WC (2002). A Prospective study of tomato products, Lycopene, and prostate cancer risk. J. Natl. Cancer Inst. 94:391-8.
crossref
 
Gorinstein S, Caspi A, Libman I, Lerner HT, Huang D, Leontowicz H, Leontowicz M, Tashma Z, Katrich E, Feng S, Trakhtenberg S (2006). Red Grapefruit Positively Influences Serum Triglyceride Level in Patients Suffering from Coronary Atherosclerosis: Studies in Vitro and in Humans. J. Agric. Food Chem. 54(5):1887-1892.
crossref
 
Graciela OR, Fredy Y, Betzabe SF, Lilibeth C (2003). Volatile fraction composition of Venezuelan sweet orange essential oil (Citrus sinensis (L.) Osbeck). Ciencia 11(1):55-60.
 
Ha SK, Park HY, Eom H, Kim Y, Choi I (2012). Narirutin fraction from citrus peels attenuates LPS-stimulated inflammatory response through inhibition of NF-κB and MAPKs activation. Food Chem. Toxicol. 50(10):3498-504.
crossref
 
Herbal Medicine (2000). Grapefruit. Available at: http://www.holistic-online.com/HerbalMed/_Herbs/h_grapefruit.htm.
 
Javascript AG (2002). Grapefruit, J. Altern Complement. Med. 8: 333-40.
 
Jung UJ, Lee MK, Jeong KS, Choi MS (2004). Hypoglycemic effects of hesperidin and naringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJ-db/db mice. J. Nutr. 134:2499–2503.
 
Jung UJ, Lee MK, Park YB, Kang MA, Choi MS (2006). Effect of citrus flavonoids on lipid metabolism and glucose-regulating enzyme mRNA levels in type-2 diabetic mice. Int. J. Biochem. Cell Biol. 38(7):1134–1145.
crossref
 
Kurowska EM, Spence JD, Jordan J, Wetmore S, Freeman DJ, Piché LA, Serratore P (2000). HDL-cholesterol-raising effect of orange juice in subjects with hypercholesterolemia. Am. J. Clin. Nutr. 72(5):1095-1100.
 
Liu Y, Heying E, Tanumihardjo SA (2012). History, global distribution, and nutritional importance of citrus fruits. Compr. Rev. Food Sci. Food Saf. 11(6):530-545.
crossref
 
Malviya N, Jain S, Malviya S (2010). Antidiabetic potential of medicinal plants. Acta. Pol. Pharm. 67(2):113-118.
 
Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C (2003). Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 57(2):235-242.
crossref
 
Mateljan G (2006). The world's healthiest foods-Grapefruit. Available at: http://www.whfoods.com/genpage.php?tname=foodspice&dbid25#healthbenefits.
 
Mohammed FA, Mohammed SK, Syed SG, Syeda S, Shaik RA, Shaik MA, Mohammed I (2010). Antidiabetic Activity of Vinca rosea Extracts in Alloxan-Induced Diabetic Rats. Int. J. Endocrinol. 841090:10-16.
 
Pari L, Suman S (2010). Antihyperglycemic and antilipidperoxidative effect of flavonoid naringin in streptozotocin-nictonamide induced diabetic rats. Int. J. Biol. Med. Res. 1(3):206-210.
 
Patel DK, Prasad SK, Kumar R, Hemalatha S (2012). An overview on antidiabetic medicinal plants having insulin mimetic property. Asian Pac. J. Trop. Biomed. 2(4):320-330.
crossref
 
Peluso MR (2006). Flavonoids attenuate cardiovascular disease, inhibit phosphodiesterase, and modulate lipid homeostasis in adipose tissue and liver. Exp. Biol. Med. 231(8):1287-1299.
 
Prem KS, Darshee B, Sudip B, Ramachandran AV (2012). Therapy with methanolic extract of Pterocarpus marsupium Roxb and Ocimum sanctum Linn reverses dyslipidemia and oxidative stress in alloxan induced type I diabetic rat model. Exp.Toxicol. Pathol. 64(5):441-448.
crossref
 
Punitha R, Vasudevan K, Monoharan S (2006). Effect of Pongamia pinnata flowers on blood glucose and oxidative stress in alloxan induced diabetic rats. Indian J. Pharmacol. 38:62-63.
crossref
 
Punithavathi VR, Anuthama R, Prince PS (2008). Combined treatment with naringin and vitamin C ameliorates streptozotocin-induced diabetes in male Wistar rats. J. Appl. Toxicol. 28(6):806-813.
crossref
 
Riaz A, Khan RA, Ahmed M (2013). Glycemic response of Citrus Limon, Pomegranate and their combinations in Alloxan induced diabetic rats. Aust. J. Basic Appl. Sci. 7(10):215-219.
 
Rogerio AP, Sá-Nunes A, Faccioli LH (2010). The activity of medicinal plants and secondary metabolites on eosinophilic inflammation. Pharmacol. Res. 62(4):298-307.
crossref
 
Rohilla A, Ali S (2012). Alloxan induced diabetes: Mechanism and effects. Int. J. Res. Pharmaceut. Biomed. Sci. 3(2):819-823.
 
Roza JM, Xian-Liu Z, Guthrie N (2007). Effect of citrus flavonoids and tocotrienols on serum cholesterol levels in hypercholesterolemic subjects. Altern. Ther. Health Med. 13(6):44.
 
Samreen R (2009). Diabetes mellitus. Sci. Res. Essay 4(5):367-373.
 
Sawalha S, Arráez-Román D, Segura-Carretero A,Fernández-Gutiérrez A (2009). Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS. Food Chem. 116(2):567-574.
crossref
 
Tahrani AA, Piya MK, Kennedy A, Barnett AH (2010). Review Glycemic control in type 2 diabetes: targets and new therapies. Pharmacol. Ther. 125(2):328-61.
crossref
 
Tavafi M, Ahmadvand H, Tamjidipoor A, Delfanc B. Khalatbarid AR Satureja khozestanica (2011). Essential oil ameliorates progression of diabetic nephropathy in uninephrectomized diabetic rats. Tissue Cell J. 43:45-51.
crossref
 
Violi F, Pignatelli P, Basili S (2010). Nutrition, Supplements and Vitamins in Platelet Function and Bleeding. Circulation 121:1033-1044.
crossref

 




          */?>