EXCLI Journal 2012;11:291-308 – ISSN 1611-2156 Received: February 14, 2012, accepted: June 11, 2012, published: June 18, 2012

Original article:

ALTERATION OF PLASMA BIOCHEMICAL, HAEMATOLOGICAL AND OCULAR OXIDATIVE INDICES OF ALLOXAN INDUCED DIABETIC RATS BY AQUEOUS EXTRACT OF LINN ()

Jude Chigozie Ikewuchi

Department of Biochemistry, Faculty of Science, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria E-mail: [email protected]

ABSTRACT In this study, the effects of an aqueous extract of the leaves of Tridax procumbens on the haematology, plasma biochemistry and ocular indices of oxidative stress was investigated in alloxan induced diabetic rats. Diabetes mellitus was induced by injection of alloxan (80 mg/kg body weight), via the tail vein. The extract was administered orally at 100, 200 and 300 mg/kg (both to normal and diabetic rats), and metformin at 50 mg/kg. On gas chromato- graphic analysis of the alkaloid fraction of the aqueous extract, thirty nine known alkaloids were detected, consisting mainly of 73.91 % akuamidine, 22.33 % voacangine, 1.27 % echi- tamine, 0.55 % echitamidine, 0.36 % lupanine, 0.27 % crinamidine, 0.23 % augustamine and 0.10 % 6-hydroxypowelline. Tannic acid and β-sitosterol were detected in high quantities. Compared to Test control, the treatment dose-dependently, significantly lowered (P<0.05) plasma glucose, triglyceride, very low density lipoprotein cholesterol, total bilirubin, urea, blood urea nitrogen; plasma alkaline phosphatase, alanine and aspartate transaminases, and ocular superoxide dismutase activities, and lymphocyte count. It also significantly increased (P<0.05) plasma calcium and ocular ascorbic acid contents, haemoglobin concentration and neutrophil count. This study showed that the extract was hypoglycemic, positively affected the haemopoietic system and integrity and function (dose dependently) of the liver and kidney of the diabetic rats; improved the lipid profile and had no deleterious effect on red cell mor- phology and protected against oxidative stress in ocular tissues. This study also revealed the presence of pharmacologically active compounds in the leaf extract. All of these, highlight the cardioprotective potential of the leaves of Tridax procumbens, and support its use in tradition- al health care practices for the management of diabetes mellitus.

Keywords: β-sitosterol, hypoglycemia, lipid profile, ocular oxidative stress, tannic acid, Tridax procumbens Linn

INTRODUCTION African populations, the past two decades has witnessed the emergence of type 2 dia- Diabetes mellitus is a group of metabol- betes mellitus as a major health problem, ic diseases characterized by elevated glu- affecting about 2-7 % of these populations cose in the plasma resulting from defects in (Rolfe et al., 1992; Amos et al., 1997). The insulin secretion, insulin action, or both WHO 2004 report estimated that 1.7 mil- (American Diabetes Association, 2004; lion people in Nigeria had diabetes, with Centers for Disease Control and Prevention, the projection that the number will triple by 2008; Wardlaw, 1999). In Nigeria and other 2030 (World Health Organization, 2004). 291 EXCLI Journal 2012;11:291-308 – ISSN 1611-2156 Received: February 14, 2012, accepted: June 11, 2012, published: June 18, 2012

Presently, there is renewed interest in well as its weight reducing (Ikewuchi et al., the use of herbal products. This may be at- 2011a) and hypotensive (Ikewuchi et al., tributable to the down turn in the economy, 2011b) activities have also been reported. as herbal medicine is perceived to be a Prabhu et al. (2011) reported the analgesic cheaper means of treatment (Kamboj, 2000; activity of the leaves. In this study, the ef- Acuff et al., 2007). Herbal products can im- fects of an aqueous extract of the leaves of prove glucose metabolism and the overall Tridax procumbens Linn on haematology, condition of individuals with diabetes, not plasma biochemistry and ocular indices of only by hypoglycemic effects but also by oxidative stress in normal and alloxan- improving lipid metabolism, antioxidant induced diabetic Wistar rats were investi- status, and capillary function (Bailey and gated. Day, 1989). Tridax procumbens is one of a number of medicinal herbs that is used in MATERIALS AND METHODS traditional health care practices for the Preparation of extract management of diabetes mellitus. Samples of the fresh Tridax procum- Tridax procumbens Linn (Family Aster- bens (Figure 1) were collected from aceae) is native to Central America and within the Choba and Abuja Campuses of tropical , but has spread University of Port Harcourt, Nigeria. After throughout the tropical and subtropical due identification at the University of Port parts of the world. Its common names are Harcourt Herbarium, Port Harcourt, Nige- coat buttons, tridax daisy, tridax (United ria, the identity was confirmed/authent- States Department of Agriculture, Agricul- icated by Dr. Michael C. Dike of the Tax- tural Research Service, 2011). The Ibo peo- onomy Unit, Department of Forestry and ple of South Eastern Nigeria call it “mbuli”. Environmental Management, Michael Ok- It has daisy-like yellow centered white or para University of Agriculture, Umudike, yellow flowers with three toothed ray floret. Abia State, Nigeria; and Mr. John Ibe, the The leaves are short, hairy and arrow Herbarium Manager of the Forestry De- shaped (Jahangir, 2001). It produces a hard partment, National Root Crops Research achene fruit that is covered with stiff hairs Institute (NRCRI), Umuahia, Nigeria. The (Fosberg and Sachet, 1980). It is used as an samples were rid of dirt and the leaves re- ornamental or fodder plant, and its leaves moved, oven dried at 55°C and ground into are cooked as vegetables (Acharya and Sri- powder. The resultant powder was soaked vastava, 2010; Prajapati et al., 2008). Tradi- in hot, boiled distilled water for 12 h, after tionally, it is used for the treatment of bron- which the resultant mixture was filtered and chial catarrh, malaria, stomach ache, diar- the filtrate, hereinafter referred to as the rhoea, epilepsy, diabetes, high blood pres- aqueous extract was stored in a refrigerator sure, haemorrhage, liver problems, and as a for subsequent use. A known volume of this hair tonic (Agrawal et al., 2010; Ahirwar et extract was evaporated to dryness, and the al., 2010; Hemalatha, 2008; Jahangir, 2001; weight of the residue used to determine the Ravikumar et al., 2005). It possesses anti- concentration of the filtrate, which was in septic, insecticidal, parasiticidal properties turn used to determine the dose of admin- and has marked depressant action on respi- istration of the extract. The resultant residue ration (Edeoga et al., 2005; Salahdeen et al., was used for the phytochemical study. 2004; Saxena and Albert, 2005).

Ikewuchi et al. (2009) and Ikewuchi and

Ikewuchi (2009a, b) reported the nutri- ent/nutraceutical potential of the leaves. The protective effects of aqueous extract of the leaves against cholesterol and salt load- ing in Wistar albino rats (Ikewuchi and Ikewuchi, 2009c: Ikewuchi et al., 2010), as

292 EXCLI Journal 2012;11:291-308 – ISSN 1611-2156 Received: February 14, 2012, accepted: June 11, 2012, published: June 18, 2012

Determination of the phytochemical con- tion), extraction of the non-saponifiables, tent of the crude aqueous leaf extract clean-up of the extract, derivatisation of the sterols, and separation and quantification of Calibration, identification and quantifica- the sterol derivatives by gas chromato- tion graphy (GC) using a capillary column. Standard solutions were prepared in Chromatographic analyses were carried out methanol for alkaloids and tannins, and on an HP 6890 (Hewlett Packard, Wilming- methylene chloride for phytosterols. The ton, DE, USA), GC apparatus, fitted with a linearity of the dependence of response on flame ionization detector (FID), and pow- concentration was verified by regression ered with HP Chemstation Rev. A 09.01 analysis. Identification was based on com- (1206) software, to quantify and identify parison of retention times and spectral data compounds. The column was HP with standards. Quantification was per- INNOWax Column (30 m × 0.25 mm × formed by establishing calibration curves 0.25 μm film thickness). The inlet and de- for each compound determined, using the tection temperatures were 250 and 320 °C. standards. Split injection was adopted with a split ratio of 20:1. Nitrogen was used as the carrier Determination of alkaloid composition gas. The hydrogen and compressed air pres- The extraction was carried out accord- sures were 22 psi and 35 psi. The oven was ing to the method of Tram et al. (2002). The programmed as follows: initial temperature alkaloid fraction of the crude aqueous ex- at 60 °C, first ramping at 10 °C/min for tract was extracted with methanol and sub- 20 min, maintained for 4 min, followed by jected to gas chromatographic analysis. a second ramping at 15 °C/min for 4 min, Chromatographic analyses were carried out maintained for 10 min. on an HP 6890 (Hewlett Packard, Wilming- ton, DE, USA), GC apparatus, fitted with a Determination of tannin composition flame ionization detector (FID), and pow- Extraction was carried out according to ered with HP Chemstation Rev. A 09.01 the method of Luthar (1992). The tannin (1206) software, to quantify and identify fraction of the crude aqueous extract above compounds. The column was a capillary was extracted with methanol and subjected DB-5MS (30 m × 0.25 mm × 0.25 μm film to gas chromatographic analysis. Chroma- thickness). The inlet and detection tempera- tographic analyses were carried out on an tures were 250 and 320 °C. Split injection HP 6890 (Hewlett Packard, Wilmington, was adopted with a split ratio of 20:1. Ni- DE, USA), GC apparatus, fitted with a trogen was used as the carrier gas. The hy- flame ionization detector (FID), and pow- drogen and compressed air pressures were ered with HP Chemstation Rev. A 09.01 28 psi and 38 psi. The oven was pro- (1206) software, to quantify and identify grammed as follows: initial temperature at compounds. The column was HP 5 Column 60 °C for 5 min. First ramping at 10 °C/min (30 m × 0.25 mm × 0.25 μm film thick- for 20 min was followed by a second ramp- ness). The inlet and detection temperatures ing at 15 °C/min for 4 min. were 250 and 320 °C. Split injection was adopted with a split ratio of 20:1. Nitrogen Determination of phytosterol composition was used as the carrier gas. The hydrogen Extraction of oil was carried out accord- and compressed air pressures were 28 psi ing to AOAC method 999.02 (AOAC Inter- and 40 psi. The oven was programmed as national, 2002), while the analysis of sterols follows: initial temperature at 120 °C, fol- was carried out according to AOAC method lowed by ramping at 10 °C/min for 20 min. 994.10 (AOAC International, 2000). This involved extraction of the lipid fraction from homogenized sample material, fol- lowed by alkaline hydrolysis (saponifica-

293 EXCLI Journal 2012;11:291-308 – ISSN 1611-2156 Received: February 14, 2012, accepted: June 11, 2012, published: June 18, 2012

Experimental design for the anti-diabetic lowed normal feed and water ad libitum. At study the end of the treatment period, they were Male Wistar albino rats (weighing 180- weighed, fasted overnight and anaesthetized 200 g at the start of the study) were collect- by exposure to chloroform. While under ed from the animal house of the Department anesthesia, they were sacrificed and blood of Physiology, University of Nigeria, Enu- was collected from each rat into heparin gu Campus. All the experiments were con- and EDTA sample bottles. Whole blood ducted in accordance with the international- was immediately used to determine the tri- ly accepted principles for laboratory animal glycer