Potential antidiabetic effect of ethanolic and aqueous-ethanolic extracts of Ricinus communis leaves on streptozotocin-induced diabetes in rats

Potential antidiabetic effect of ethanolic and aqueous-ethanolic extracts of Ricinus communis leaves on streptozotocin-induced diabetes in rats

 

Mohamed A. M. Gad-Elkareem, Elkhatim H. Abdelgadir, Ossama M. Badawy,

Adel Kadri

ABSTRACT

Recently, herbal drugs and their bioactive compounds have gained popularity in the management of diabetes mellitus (DM) which has become an epidemic disease all over the world, especially prevalent in the Kingdom of Saudi Arabia (KSA). This study aimed to investigate the antidiabetic effect of ethanolic and aqueous-ethanolic extracts of wild Ricinus communis (R. communis) leaves in streptozotocin (STZ) induced diabetic rats. Diabetic rats were administered orally with the mentioned extracts at doses of 300 and 600 mg/kg/BW for 14 days, and the obtained results of different biochemical parameters were compared with normal control, diabetic control and standard drug glibenclamide (5 mg/kg/BW). The obtained results revealed a remarkable and significantly (P<0.05) reverse effect of the body weight loss, observed when diabetic rats were treated with ethanol and aqueous-ethanol extracts at 300 mg/kg/BW. Administration of the ethanol extract at 600 mg/kg/BW significantly (P<0.05) reduced the blood glucose level. A significant increase in the AST, ALT and ALP levels (P<0.05) was observed in the diabetic control and in the experimental groups with glibenclamide which was also significantly (P<0.05) lowered after treatment with extracts at special doses. Total proteins, albumin, total bilirubin, direct bilirubin, creatinine and urea were also investigated and compared to the corresponding controls. We showed that administration of R. communis extract generally significantly (P<0.05) ameliorated the biochemical parameters of diabetic rats. Also, the changes in serum electrolyte profile were assessed and the results demonstrate that administration of  extracts at concentration of 600 mg/kg/BW generally inhibits the alteration maintain their levels. The obtained data imply the hypoglycemic effects of this plant, which may be used as a good alternative for managing DM and therefore validating its traditional usage in KSA.

 

Collection of plant materials

The leaves of R. communis were harvested in November 2017 from a mountain in Buljurashi City, Al Baha, Saudi Arabia, with coordinates 19°51′34″N 41°33′26″E.  Voucher specimens with the corresponding number BRC100 were deposited at the Chemistry Department, College of Science and Arts in Baljurashi, Al Baha University. This work was supported by Deanship of Scientific Research, Project number: 71/1438, Albaha University, Kingdom of Saudi Arabia.

Preparation of plant extract

The leaves of R. communis were dried at room temperature, ground into a fine powder and stored at 5°C until needed. 200 g of R. communis powder were added to 500 mL ethanol (96%) and a mixture of aqueous-ethanol (with ratio 60:40). When obtaining the plant extract, we followed the same method as done by Bakari et al. (2015), and then the plant extract was reconstituted with distilled water for oral administration.

Experimental Animals 

Forty two Male Wistar rats 12-week-old (150-160g) were obtained from the Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Animals were maintained on a 12 h light/dark in cycle polypropylene cages (six rats in each) at the ambient temperature of 2 3 ± 2 °C and relative humidity of 50-60% with food and water provided ad libitum. All experiments were carried out according to the recommendation of Experimental Animals Ethics Committee of The King Saud University in accordance with the international standards for the handling of experimental animals. The rats were acclimatized for 1 week before the start of the experiment.

 

Toxicity profile

In this study, acute toxicity study was carried according to Organization for Economic Cooperation and Development, guideline 423. A limit dose of 2 000 mg/ kg body weight /oral was used. The signs of toxic effects and/or mortality were observed 3 h after administration then for the next 48 h. The body weight was recorded for consecutive 14 days. Since the extracts were found safe up to the dose level of 2000 mg/kg body weight, a dose of 300 and 600 mg/kg body weight of the two extracts was selected for screening of the antidiabetic activity.

Induction of diabetes

Rats were fasted overnight and experimental diabetes was induced by intraperitoneal injection of 55 mg/kg body weight of streptozotocin (STZ, Sigma, St Louis, MO, USA) dissolved in freshly prepared citrate buffer (0.1 mol/L, pH 4.5) (Arora et al., 2010). Fasting blood sugar for the animals was measured after 72 h using Medisafe Mini Blood Glucose Reader (TERUMO Corporation Ltd., Hatagaya, Tokyo, Japan). Rats with fasting blood sugar level more than 200 mg/dL (11.1 mmol/L) were considered as diabetic and used for the study. Rats were then allowed to develop diabetes for 14 days (Arora et al., 2010).

Experimental design

Oral glucose tolerance test with extracts in diabetic rats: Forty two rats (36 diabetic surviving rats and 6 normal rats) were divided into seven groups of six rats each. The animals were treated orally once daily for 14 consecutive days as follows.

- Group 1, normal rats were treated with distilled water and used as the negative control.

- Group 2, diabetic control rats were treated with distilled water.

- Group 3, diabetic rats were given standard drug glibenclamide (5 mg/kg body weight) (Alamin et al., 2015).

- Groups 4 and 5, served as diabetic rats given ethanolic R. communis extracts at doses of 300 mg/ kg and 600 mg/ kg respectively, once daily for 14 days,

- Groups 6 and 7, served as diabetic rats given aqueous-ethanol extract at doses of 300 mg/ kg and 600 mg/ kg respectively, once daily for 14 days.

Normal control rats and untreated diabetic rats received equal volumes of water in place of the extract. The body weight was measured every day and the dose was calculated accordingly.

 

Collection of blood samples and estimation of biochemical parameters

Blood samples were collected from overnight fasted rats (only water allowed) after 2 weeks of treatment under diethyl ether anesthesia by cardiac puncture (good quality and large volume of blood from the experimental animals) into heparinized and non-heparinized tubes for hematological and biochemical analyses.  For serum samples, blood was allowed to coagulate, followed by centrifugation at 3000 r/min for 15 min at 4 °C to separate serum. Sera were divided into aliquots and stored at -80 °C for biochemical assay.

Biochemical analysis

For biochemical analysis we used standard commercial kits according to the manufacturer's instructions. Fasting serum glucose level was determined on day 14 by glucose oxidase-peroxidase method using the kit from RANDOX Laboratories Ltd, UK. Alanine and aspartate aminotransferase (ALT and AST) (Schmidt and Schmidt, 1963) and alkaline phosphatase (ALP) (Wright, 1972) were measured using kits from Randox Laboratory Ltd., UK. Serum creatinine (Owen et al., 1954), serum sodium, potassium, chloride, phosphorus and carbon dioxide (Tietz et al., 1994), bilirubin (Malloy and Evelyn, 1937) total protein, albumin (Spencer and Price, 1977) and urea (Marsh et al., 1965) were determined using a commercial kit from QUIMICA Clinica Aplicada, Amposta, Spain.

Statistical analysis

A one-way analysis of variance (ANOVA) and Tukey's post-hoc test were performed to determine significant differences between the parameters using the SPSS 19 statistical package (SPSS Ltd. Woking, UK). Means and standard errors were calculated. Differences among the mean values of the various parameters were determined by the least significant difference test. A probability level of P<0.05 was used in testing the statistical significance of all experimental data.

 

REFERENCES

 

Abraham Z, Bhakuni SD, Garg HS, Goel AK, Mehrotra BN, Patnaik GK. 1986. Screening of Indian plants for biological activity. Indian Journal of Experimental Biology 12(24): 48-68.

Afrisham R, Aberomand M, Ghaffari MA, Siahpoosh A, Jamalan M. 2015. Inhibitory effect of Heracleum persicum and Ziziphus jujuba on activity of alpha-amylase. Journal of Botany 1-8.

Agoramoorthy G, Chen F, Venkatesalu V, Kudo DH, Shea PC. 2008. Evaluation of antioxidant polyphenols from selected medicinal plants of India. Asian Journal of Chemistry 20: 1311-1322.

Akinyemi O, Iyebor EW, Osadebe CO, Oniroko NS. 2016. Proximate and phytochemical compositions of Ricinus communis in Ibadan, South-Western Nigeria. Nutrition Research and Food Science 3(5): 96-101.

Alamin MA, Yagi AI, Yagi SM. 2015. Evaluation of antidiabetic activity of plants used in Western Sudan. Asian Pacific Journal of Tropical Biomedicine 5(5): 395-402.

Alqahtani N, Khan WA, Alhumaidi MH, Ahmed YA.  2013. Use of glycated hemoglobin in the diagnosis of diabetes mellitus and pre-diabetes and role of fasting plasma glucose, oral glucose tolerance test. International Journal of Preventive Medicine 4: 1025-1029. 

Arora MK, Reddy K, Balakumar P. 2010. The low dose combination of fenofibrate and rosiglitazone halts the progression of diabetes-induced experimental nephropathy. European Journal of Pharmacology 636: 137-144.

Arthur FKN, Woode E, Terlabi EO, Larbie C. 2012. Bilirubin lowering potential of Annona muricata (Linn.) in temporary jaundiced Rats. American Journal of Pharmacology and Toxicology 7 (2): 33-40.

Atangwho IJ, Ebong PE, Egbung GE, Ani IF. 2009. Effects of co-administration of extracts of Vernonia amygdalina and Azadirachta Indica on serume profile of diabetic and non-diabetic rats. Australian Journal of Basic and Applied Sciences 3(3): 2974-2978.

Atangwho IJ, Ebong PE, Eyong EU, Asmawi MZ, Ahmad M. 2012. Synergistic antidiabetic activity of Vernonia amygdalina and Azadirachta indica: biochemical effects and possible mechanism. Journal of Ethnopharmacology 141(3):878-887.

Bakari S, Ncir M, Felhi S, Hajlaoui H, Saoudi M, Gharsallah N, Kadri A. 2015. Chemical composition and in vitro evaluation of total phenolic, flavonoid, and antioxidant properties of essential oil and solvent extract from the aerial parts of Teucrium polium grown in Tunisia. Food Science and Biotechnology 24(6): 1943-1949.

Cai1 Y, Qiu R, Yu Lu, Huang C, Wang J, Ji Y, Wang A. 2016. Hypoglycemic activity of two anthraquinone derivatives from Juncus setchuensis Buchen. International Journal of Clinical and Experimental Medicine 9(10): 19664-19672.

Capasso F, Mascolo N, Izzo AA, Gaginella TS. 1994. Dissociation of castor oil induced diarrhoea and intestinal mucosal injury in rat, effect of NG-nitro-Larginine methyl ester. British Journal of Pharmacology 113: 1127–1130.

Dimo T, Rakotonirina SV, Tan PV, Azay J, Dongo E, Kamtchouing P, Cros G. 2007. Effect of Sclerocarya Birrea (Anacardiaceae) stem bark methylene chloride/methanol extract on streptozotocin-diabetic rats. Journal of Ethnopharmacology 110(3): 434-438.

Helal EGE, NA Aouf, Khattab AM, Zoair MA. 2014. Antidiabetic effect of Artemisia annua (kaysoum) in alloxan-induced diabetic rats. The Egyptian Journal of Hospital Medicine 57: 422-430.

Kadri A, Gharsallah N, Damak M, Gdoura R. 2011. Chemical composition and in vitro antioxidant properties of essential oil of Ricinus communis L. Journal of Medicinal Plants Research 5(8):1466-1470.

Khavandi K, Amer H, Ibrahim B, Brownrigg J. 2013. Strategies for preventing type 2 diabetes: an update for clinicians. Therapeutic Advances in Chronic Disease: 4(5): 242–261.

Kibiti CM, Afolayan AJ. 2015. Herbal therapy: a review of emerging pharmacological tools in the management of diabetes mellitus in Africa. Pharmacognosy Magazine 11(2): 58-74.

Kirtikar KR, Basu BA. 1991. Indian Med. Plants 3: 2274–2277.

Kumar M. 2017. A review on phytochemical Constituents and pharmacological activities of Ricinus communis L. Plant. International Journal of Pharmacognosy and Phytochemical Research 9(4): 466-472.

Lenzen S. 2008. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia  51:216–226.

Liu Y, Cao Y, Fang S, Wang T, Yin Z, Shang X, Yang W, Fu X. 2018. Antidiabetic effect of Cyclocarya paliurus leaves depends on the contents of antihyperglycemic flavonoids and antihyperlipidemic triterpenoids. Molecules 23: 1042-1059.

Machry RV, Pedroso HU, Vasconcellos LS, Nunes RR, Evaldt CA, Yunes Filho EB, Rodrigues TDC. 2018. Multifactorial intervention for diabetes control among older users of insulin. Revista de Saude Publica 52-60

Mahmoodzadah Y, Mazani M, Rezagholizadeh L. 2017. Hepatoprotective effect of mrhanolic tanacetum parthenium extract on CCl4-induced liver damage in rats. Toxicology Reports 4: 455-462.

Malloy HT, Evelyn KA. 1937. The determination of bilirubin with the photoelectric colorimeter. The Journal of Biological Chemistry 119(2): 481–490.

Man S, Singh PK, Gubta AAA. 2013. Antidiabetic effects of Ricinus communis on the blood biochemical parameters in streptozotocin induced rat. International Journal of Pharma and Biosciences 4(2): 382-388.

Marsh WH,  Fingerhut B, Miller H. 1965. Automated and manual direct methods for the determination of blood urea. Clinical chemistry 11:624–627.

Nandkarni KM, 1954. Indian Materia Medica, third ed. The Popular Book Depot,

Bombay. pp. 1065–1070.

Owen JA Iggo B,  Scandrett F J, Stewart CP. 1954. The determination of creatinine in plasma or serum, and in urine; critical examination. The Biochemical Journal 58(3): 426–437.

Pullaiah T, Naidu KC. 2003. Antidiabetic plants in India and herbal based antidiabetic research. Regency Publications, New Delhi.

Qaid MM, Abdelrahman MM.  2016. Role of insulin and other related hormones in energy metabolism-A review. Cogent Food and Agriculture 2: 1-18.

Rajendra A, Narayan V, Granavel I. 2007. Study on the analysis of trace elements in Aloe vera and its biological importance. Journal of Applied Science Research 3:1476-1478.

Robert AA, Al Dawish MA, Braham R, Musallam MA, Al Hayek AA, Al Kahtany NH. 2016. Type 2 diabetes mellitus in Saudi Arabia: Major challenges and possible solutions. Current Diabetes Reviews 12: 1-6.

Schmidt E, Schmidt FW. 1963. Determination of serum GOT and GPT activities. Enzymologia Biologica Et Clinica 3(1): 1–5.

Shalev A. 1999. Hope for insulin mimetic oral antidiabetic drugs.  European Journal Endocrinology 8:561-562.

Shokeen P, Anand P, Murali YK, Tandon V. 2008. Antidiabetic activity of 50% ethanolic extract of Ricinus communis and its purified fractions. Food and Chemical Toxicology 46: 3458-3466.

Spencer K, Price CP. 1977. Influence of reagent quality and reaction conditions on the determination of serum albumin by the bromcresol green dye binding method. Annals of Clinical Biochemistry 14(2): 105–115.

Stryer L, Tymoczko JL, Berg J. 1997. Biochemistry, 5th edition, W.H. Freeman and company, New York, pp. 324- 50.

Swamy SK, Nagalakshmi NC, Santhosh K, Yogesh HS. 2018. Hypoglycemic activity of ethanol extract of Jasminum grandiflorum flowers in vivo and cytotoxicity of its chloroform isolate in vitro. Journal of Diabetes and Metabolic Disorders 3(2):1-9.

Tietz NW, Prude EL, Sirgard-Anderson O. 1994. Tietz Textbook of Clinical Chemistry, WB Saunders Company, London, UK.

Visen P, Shukla B, Patnaik G, Tripathi S, Kulshreshtha D, Srimal R, Dhawan B. 1992. Hepatoprotective activity of Ricinus communis leaves. International Journal of Pharmacology 30:241–250.

Wright PJ, Leathwood PD, Plummer DT. 1972. Enzymes in rat urine: alkaline phosphatase. Enzymologia 42(4): 317–327.

Yuan H, Ma Q, YeL, Piao G. 2016. The traditional medicine and modern medicine from natural products. Molecules 2:E559.  

Zarai Z, Chobba IB, Mansour RB, Békir A, Gharsallah N, Kadri A. 2012. Essential oil of the leaves of Ricinus communis L. In vitro cytotoxicity and antimicrobial properties. Lipids in Health and Disease 11:102

 

 

 

 

 

Figure: The effect of Ricinus communis leaves extracts on the blood glucose in STZ induced diabetic rats after 2 weeks of treatment.

 

STZ: Control diabetic rats; STZ+EE300 mg/kg/BW: STZ + Ethanol extract at 300 mg/kg/BW; STZ+AE-300 mg/kg/BW: STZ + Aqueous-ethanol at 300 mg/kg/BW; STZ+ E600mg/kg/BW: STZ + Ethanol at 600 mg/kg/BW; STZ+AE600 mg/kg/BW: STZ + Aqueous-ethanol at 600 mg/kg/BW

 

*Values are statistically significant at p <0.05

**Values are statistically significant at p <0.01

The data were analysed using the parametric method, ANOVA followed by Tukey's post-hoc test.