Volume 5 Issue 2 Apr-Jun 2015 Renal and Hepatic Antioxidant Status of Hyperglycemic
Rats Treated with Single and Combinatorial Herbal
Okey Alphosus Ojiako1, Paul Chidoka Chikezie2, Agomuo Chizaramoku Ogbuji3
1Department of Biochemistry, Federal University of Technology, Owerri, Nigeria
2Department of Biochemistry, Imo State University, Owerri, Nigeria
3Department of Food Science and Technology, Abia State Polytechnic, Aba, Nigeria

Background/Aim: The present study ascertained the comparative capacities of single and combinatorial herbal
formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis and Asystasia gangetica to exert
glycemic control and ameliorate oxidative stress in renal and hepatic tissues of alloxan-induced hyperglycemic rats
(HyGR) following 30 days treatment. Materials and Methods: Phytochemical composition of the herbal extracts
and fasting plasma glucose concentration (FPGC) of the rats were measured by standard methods. The post
mitochondrial supernatant fractions (PMSF) of renal and hepatic tissues homogenates were measured for activity
levels of glutathione peroxidase (GPOx), superoxide dismutase (SOD) and catalase (CAT) as well as glutathione
(GSH) concentration. Results: Comparatively, flavonoids were the most abundant phytochemical present in the
four herbal extracts. FPGC of treated HyGR was with in the range of 66.30±0.71-133.00±0.63 mg/dL compared
to the untreated hyperglycemic rat group=368.20±0.72 mg/dL (p<0.05). At the end of the experimental time,
treated HyGR liver SOD activities were lower than that of normal group (p<0.05). Liver and kidney GPOx activities
of HyGR were 2.36 folds and 3.93 folds lower than the normal rats. Furthermore, liver and kidney CAT activities
of the treated HyGR were significantly (p<0.05) lower than the normal rats, whereas liver and kidney GSH/GSSG
ratios were significantly (p<0.05) increased when compared with treated HyGR. Conclusion: Combinatorial herbal
formulations exerted greater glycemic control than the single herbal formulations and caused a sparing effect on
renal and hepatic antioxidants against reactive oxygen species.
Key words: Antioxidant, Hyperglycemia, Hepatic, Phytochemicals, Renal.
Acanthus montanus (Nees) T. Anderson belongs to the fam- known as mountain thistle or Devil's fig or Bear's Breech. ily of Acanthaceae and is widespread in Africa, the Balkans, The medicinal usefulness of the plant extracts have Romania, Greece and Eastern Mediterranean. The plant been previously reported elsewhere.1,2 Asystasia gangetica is a thinly branched perennial shrub with basal clusters L. T. Anderson is also known as creeping foxglove and of oblong and lance shaped glossy, dark green leaves belongs to the family of Acantheceae. It is a fast-growing that measure about 30 cm long. A. montanus is commonly herbaceous weed of height between 0.3 – 0.4 m. Decoc- tions of A. gangetica are used in African and Asian ethno medicinal practices for the alleviation of stomach ache, *Correspondence author: urethral discharge, inflammation, rheumatism and dry Dr. Paul Chidoka Chikezie
Department of Biochemistry, cough. Also, A. gangetica is used for the management of Imo State University, Owerri, Nigeria diabetes mellitus (DM) and epilepsy as well as antidote to snake bite. Emilia coccinea (SIMS) G. Donis (family: Aster- DOI : 10.5530/pc.2015.2.7 acca) is commonly found in African countries like Congo, Copyright 2015 EManuscript Services, India Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Sudan, Kenya, Tanzanian, Malawi, Uganda and Nigeria.3 level of orthodox anti-diabetic therapeutics, the use sev- In parts of Africa, this multipurpose plant serves as deli- eral plants as herbal remedies by traditional healers for the cacies and herbal concoctions for remedies of numerous treatment of diabetes mellitus is still very common espe- diseases including DM.3,4 Hibiscus rosasinensis L. is an ever- cially in Africa and Asia continents.28-33 Traditional herbs green shrub that grows up to 1.8 – 2.4 m high. The family, are commonly used in the form multi-herb recipe, which malvaceae, is consist of 82 genera and over 1,500 species is believed to possess better pharmacological efficacy than of which over 200 species are found in tropical and sub- the individual composite herb extracts. In that regard, tropical regions of the world. The flowers are hermaph- the present study ascertained the comparative capaci- rodites and vary in color depending on the species and ties of single and combinatorial herbal formulations of location. The medicinal properties of H. rosasinensis have four medicinal plant extracts of A. montanus, E. coccinea, been reported by previous researchers.5-7 H. rosasinensis and A. gangetica to exert glycemic control in alloxan-induced hyperglycemic rats (HyGR). In addition, Hyperglycemia, which is one of the prominent defining hyperglycemia causes glucose auto-oxidation mediated features of DM, has been implicated in promoting over- oxidative organ damage. Previous reports have revealed whelming levels of reactive oxygen and nitrogen species that cellular levels of the antioxidant system could serve as (RONS)8,9 and protein glycation.10,11 Hyperglycemia medi- a reliable biomarker to establishing the pathogenesis and ated generation and accumulation of RONS may occur progression of DM especially with respect to tissue dam- through one or combinations of the following molecular age.34,35 Therefore, studies were carried out to ascertain lev- events: distortions of glycolytic pathways,12,13 intercellular els of renal and hepatic tissues derived primary oxidative activation of sorbitol (polyol) pathways,14 auto-oxidation stress indicators (GPOx, SOD, CAT activities and GSH/ of glucose15 and non-enzymatic proteins and amino acids GSSG ratio) and their correlations, following the admin- glycation.16,17 Antioxidant systems prevent cellular damage istration of the herbal formulations. The outcome of the from the adverse effects of xenobiotics, carcinogens and present investigations will serve to give insights into the toxic RONS. Several reports showed that there are several capacities of the various herbal formulations to ameliorate biologically derived compounds with antioxidant func- hyperglycemia and tissue derived oxidative stress in DM.
tions.18-20 Glutathione peroxidases (GPOx; EC are ubiquitous selenium-containing peroxidases that cata- lyze the reduction of a variety of hydroperoxides (ROOH MATERIALS AND METHODS
and H O ) using GSH as co-factor. GPOx activity, among other functions, protects mammalian cells against oxida- Collection and preparation of herbal samples
tive damage. There are at least five GPOx isoenzymes Fresh leaves of Acanthus montanus (Nees) T. Anderson found in mammals with the levels of each isoform vary (ACMO), Emilia coccinea (SIMS) G. Don (EMCO) and depending on the tissue type.18,21 Superoxide dismutase Hibiscus rosasinensis L. (HIRO) were collected from uncul- (SOD; EC is metallo-enzyme that defines the tivated lands in Umuamacha Ayaba Umaeze, Osisioma basis for their classification; Mn-SOD22 and Cu, Zn- Ngwa LGA, Abia State, Nigeria. Fresh leaves of Asysta- SOD20,23 Ni–SOD.24 SOD activity neutralizes superoxide sia gangetica L. T. Anderson (ASGA) were collected from by transforming oxygen derived reactive species to per- Ubowuala, Emekuku, Owerri North LGA, Imo State, oxide, which can in turn be eliminated by the actions of Nigeria. The four herbs were identified and authenticated catalase or GPOx activities. Catalase (CAT; EC by Dr. M. Ibe, School of Agriculture and Agricultural is a tetrameric haemin-enzyme consisting of four identical Technology (SAAT), Federal University of Technology, tetrahedrally arranged subunits of ≈ 60 kDa.18 CAT activi- Owerri. All the leaves were collected between the months ties prevents H O -induced cellular damage by converting of July and August, 2009.
H O to H O and O . To a large extent, the thiol-redox status of the cell is defined by cellular levels of GSH.25,26 The leaves of individual plants were washed with a con- In addition, the reduced glutathione (GSH)/oxidized glu- tinuous flow of distilled water for 15 min and allowed to tathione (GSSG) ratio (GSH:GSSG) is an important bio- dry at laboratory ambient temperature (24 ± 5°C). A 500 marker of redox environment in biologic systems.25,27 g part of each herbal samples were weighted using a triple beam balance (OHAU 750-50: Burlington, NC, USA) In modern medicine and traditional systems, medicinal and dried in an oven (WTC BINDER, 7200 Tuttlingen, plants have continued to provide valuable therapeutic Germany) at 60°C until a constant weight was achieved. agents for the treatment and management of diseases and The dried leaves were packaged in dark polyethylene disorders. Moreover, in spite of the higher acceptability bags and kept in cold room (7 ± 3°C) for 24 h before Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats pulverization. Next, the separate dried leaves were doni and Ochuka.38 Five grams (5.0 g) of the sample was pulverized using Thomas-Willey milling machine (ASTM mixed with 200 mL of 20% C H OH. The suspension D-3182, INDIA), after which the ground samples were was heated in a water bath at 55°C while stirring for 4 stored in air-tight plastic bottles with screw caps pending h and allowed to cool to 24 ± 5°C. The precipitate was harvested by simple filtration methods using Whatman No 42 filter paper. The filtrate was concentrated by reducing Extraction of herbal samples
the total filtrate volume to 40 mL by evaporation in a Portions of 40 g of each pulverized dried samples of A. steam bath. In a 250 mL capacity separating funnel, the montanus, E. coccinea, H. rosasinensis and A. gangetica were 40 mL concentrated filtrate was mixed properly with 20 subjected to repeated soxhlet extraction cycles for 2 h mL of C H OC H (BDH, U.K). The aqueous layer was using 96% C H OH (BDH, U.K) as solvent to obtain a recovered, whereas the organic layer was decanted. The final volume of 500 mL of each herbal extract. These vol- purification and separation procedure was repeated twice. umes of the extracts were concentrated and recovered in a Finally, 60 mL of n-C H OH (BDH, U.K) was added to rotary evaporator for 12 h at 60°C under reduced pressure. the extract followed by 10 mL of 5% NaCl (BDH, U.K) The extracts were dried in a desiccators for 24 h, wrapped to precipitate saponins. The solution was evaporated to in aluminum foil and stored in air-tight plastic bottles with dryness in a water bath, dried in an oven and cooled in a screw caps at ≤4°C. The yields were calculated to be as follows: A. montanu=16.35% (w/w), E. coccinea=17.99% (w/w), H. rosasinensis=17.23% (w/w) and A. gangetica = Calculations
16.69% (w/w). The separate extracts were reconstituted in The concentrations of flavonoids, alkaloids and saponin phosphate buffered saline (PBS) solution (extract vehicle), (mg/100 g sample weight) in the various herbal extracts osmotically equivalent to 100 g/L PBS (90.0 g NaCI, 17.0 were calculated thus: g Na HPO .2H O and 2.43 g NaH PO .2H O), before appropriated doses were administered to the experimen- tal animals. Portions of the individual extracts were also measured for phytochemical contents.
Phytochemical composition of herbal extracts
Flavonoids content was measured.36 Five grams (5.0 g) of the herbal extract was weighted into 250 mL bea- C : concentrations of flavonoids, alkaloids and sapo- ker, mixed with 100 mL of 96% C H OH for 30 min at 24±5°C and filtered using the Whatman No 1 filter paper. W2: weights of filter paper + precipitate.
The filtrate was dried in an oven to constant weight and cooled in a dessicator.
W1: weight of filter paper.
The concentration of alkaloids of the herbal extracts was Wt: weights of samples.
measured by the methods of Harborne.37 A 5.0 g extract was weighted into 250 mL beaker containing 100 mL of The Van-Burden and Robinson,39 method as reported40 10% CH COOH (Sigma, St. Louis, USA) in C H OH. was used to measure the concentration of tannins of the The content was mixed thoroughly by vortex and allowed herbal extracts. A 5 g of the sample was mixed with 50 to stand with continuous agitation (Shaking Bath; Preci- mL of distilled water for 1 h in a 50 mL capacity plastic sion Scientific Inc., Chicago, IL) at 24±5°C for 4 h. The bottle on a mechanical shaker. The mixture was filtered suspension was separated by simple filtration method with Whatman No 42 filter paper. A 5 mL aliquot of the using Whatman No 1 filter paper and concentrated by filtrate was mixed with 2 mL of 0.1 M FeCl in 0.1 N evaporation to ¼ of its original volume in a steam bath. HCl and 0.008 M K (Fe)CN (Sigma, St. Louis, USA) in a Concentrated NH OH (BDH, U.K) was subsequently test tube. The absorbance of the analyte was measured at added in droplets to the filtrate to form precipitates, maximum absorptivity (λ )=605 nm within 10 min and which were subsequently separated by filtration. The pre- compared with the standards. cipitates were washed with 1% NH OH, dried in an oven to constant weight and cooled in a dessicator.
Experimental animals
Male albino (Wistar) rats weighing between 150-160
Measurement of saponin content of the herbal extracts g were maintained at room temperatures of 24±5°C, was by the methods of Harborne,37 as reported by Oba- 30–55% of relative humidity on a 12-h light/12-h dark Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats cycle, with access to water and standard commercial feed • HrAMEC: HyGR received SCF + water ad libitum + (SCF) (Ewu Feed Mill, Edo State, Nigeria) ad libitum for combined dose (ratio: 1:1 w/w) of A. montanus + E. 2 weeks acclimatization period. The handling of the ani- coccinea (20 mg/kg in PBS; i.p.).
mals was in accordance with the standard principles of laboratory animal care of the United States National • HrAMHR: HyGR received SCF + water ad libitum + Institutes of Health (NIH, 1978).
combined dose (ratio: 1:1 w/w) of A. montanus + H. rosasinensis (20 mg/kg in PBS; i.p.).
Induction of diabetes/experimental design
• HrECHR: HyGR received SCF + water ad libitum + Hypoglycemia was induced in the rats by single intra-peri- combined dose (ratio: 1:1 w/w) of E. coccinea + H. toneal (i.p) injection of 90 mg/kg bw of alloxan monohy- rosasinensis (20 mg/kg in PBS; i.p.).
drate (Sigma, St. Louis, USA) in PBS solution (pH=7.4). The animals with fasting plasma glucose concentration • HrAGEH: HyGR received SCF + water ad libitum + (FPGC)>110 mg/dL for 5 consecutive days were consid- combined dose (ratio: 1:1:1 w/w) of A. gangetica + E. ered diabetic and selected for the study. A total of 102 male coccinea + H. rosasinensis (20 mg/kg in PBS; i.p.).
Wistar rats were allotted into seventeen (17) groups of six • HrAMAE: HyGR received SCF + water ad libitum + (6) rats each. The animals were deprived of food and water combined dose (ratio: 1:1:1 w/w) of A. montanus + A. for additional 16 h before commencement of treatment as gangetica + E. coccinea (20 mg/kg in PBS; i.p.).
described elsewhere.41 The animal groups were designated on the basis of treatments received at regular intervals of • HrAMAH: HyGR received SCF + water ad libitum + 2 days for 30 days. Herbal treatments of the HyGR were combined dose (ratio: 1:1:1 w/w) of A. montanus + A. defined as single herbal formulations (SHF): (HrACMO, gangetica + H. rosasinensis (20 mg/kg in PBS; i.p.).
HrASGA, HrEMCO and HrHIRO), double herbal for- • HrAMEH: HyGR received SCF + water ad libitum + mulations (DHF): (HrAGAM, HrAGEC, HrAGHR, HrA- combined dose (ratio: 1:1:1 w/w) of A. montanus + E. MEC, HrAMHR and HrECHR), triple herbal formulations coccinea + H. rosasinensis (20 mg/kg in PBS; i.p.).
(THF): (HrAGEH, HrAMAE, HrAMAH and HrAMEH) and quadruple herbal formulation (QHF): (HrAAEH).
• HrAAEH: HyGR received SCF + water ad libitum + combined dose (ratio: 1:1:1:1 w/w) of A. montanus + A. gangetica + E. coccinea + H. rosasinensis (20 mg/kg in • NORM: Normal rats received SCF + water ad libitum + 1.0 mL/kg of PBS.
At the end of treatment, the animals were fasted for 12 • DIAB: HyGR received SCF + water ad libitum + 1.0 h28 before their blood samples and organ homogenates were measured for the various biochemical parameters.
• HrACMO: HyGR received SCF + water ad libitum + Fasting plasma glucose concentration
A. montanus (20 mg/kg in PBS; i.p.).
Blood samples (2.0 mL) were drawn from the orbital • HrASGA: HyGR received SCF + water ad libitum + A. sinus42 on the 30th day and measured for FPGC. Deter- gangetica (20 mg/kg in PBS; i.p.).
mination of FPGC was by the glucose oxidase method according to Randox® kit manufacturer's procedure • HrEMCO: HyGR received SCF + water ad libitum + E. (Randox® Laboratories Ltd. Ardmore, United Kingdom).
coccinea (20 mg/kg in PBS; i.p.).
Preparation of renal and hepatic organs
• HrHIRO: HyGR received SCF + water ad libitum + H. rosasinensis (20 mg/kg in PBS; i.p.).
The rats were anaesthetized in CCl chamber and dis- • HrAGAM: HyGR received SCF + water ad libitum + sected. The kidneys and liver were quickly excised and combined dose (ratio: 1:1 w/w) of A. gangetica + A. placed on a blotting paper to remove blood and rinsed in montanus (20 mg/kg in PBS; i.p.).
1.15% of potassium chloride solution to remove residual hemoglobin.43 The two organs were stored in 10% form- • HrAGEC: HyGR received SCF + water ad libitum + aldehyde and preserved at temperature below-8°C before combined dose (ratio: 1:1 w/w) of A. gangetica + E. coc- analyses.43 with minor modifications by Chikezie and cinea (20 mg/kg in PBS; i.p.).
Uwakwe.44 Each organ was homogenized using a Teflon • HrAGHR: HyGR received SCF + water ad libitum + homogenizer in aqueous K PO /KHPO buffer (0.1 M; combined dose (ratio: 1:1 w/w) of A. gangetica + H. pH=7.4); in 4:1 volume of buffer to organ weight. Next, rosasinensis (20 mg/kg in PBS; i.p.).
the crude homogenate was centrifuged at 10,000 ×g for 20 Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats min at 4°C to obtain the post mitochondrial supernatant calculated using Σ=43.6 M-1cm-1 and expressed in terms of fraction (PMSF). The PMSF was finally stored at -8 °C mole H O consumed/min/mg protein (U/mg protein).
before used for analyses. The PMSF was measured for GPOx, SOD and CAT activities. Protein concentra- Reduced glutathione (GSH)/oxidized glutathione
tions of PMSF of hepatic and renal homogenates were (GSSG) ratio
Level of GSH in organ homogenate was determined49 45 using bovine serum albumin (Sigma-Aldrich, St. Louis, USA) as a standard. Also, the PMSF was measured with minor modification by Chikezie and Uwakwe.44 A 100 µL aliquot of the PMSF was mixed with 25% of for glutathione (GSH) concentration.
CHCl and centrifuged at 2000 × g for 15 min to precipi- Glutathione peroxidase activity
tate proteins. The supernatant was aspirated and diluted GPOx activity was measured by the method of Paglia and to 1.0 mL with 0.2 M Na PO /NaHPO buffer (pH=8.0). Valentine46 as reported by Chikezie and Uwakwe.44 Briefly, Next, 2.0 mL of 0.6 mM 5, 5'-dithiobis-(2-nitrobenzoic the reaction mixture contained aliquot of PMSF in 50 acid) (DTNB) was added. The absorbance of the devel- mM K PO /KHPO buffer (pH=7.0), 1.0 mM EDTA oped yellow-colour complex maintained at 24±5°C was (Mayer and Baker, England), 1.0 mM NaN , 0.2 mM measured at λ =405 nm after 10 min. A standard curve NADPH, 1.0 U glutathione reductase, and 1.0 mM GSH. was obtained with GSH standards. The level of GSH was The reaction mixture was allowed to equilibrate at 25°C expressed as μg GSH/mg protein. The GSSG concentra- for 5 min. The reaction was started by introducing 0.1 mL ) of organ homogenates were measured using of 2.5 mM H O . Increase in absorbance at λ =340 nm Bioxytech-412 kits according to manufacturer's procedure was recorded for 5 min. The change in absorbance was (Oxis International Inc., Foster City, CA, USA).
defined as nanomoles of NADPH oxidized to NADP; Σ=6.2 × 103 M-1 cm-1 at λ =340 nm. The levels of GPx were expressed in terms of nmole NADPH consumed/ min/mg protein (U/mg protein).
Accordingly, the ratio of GSH/GSSG of renal and Superoxide dismutase activity
hepatic tissues was evaluated.
SOD activity was measured.47 Briefly, the reaction mixture contained solution A (50 mM Na CO , 0.1 mM EDTA, pH=10.0), solution B (96 µM nitroblue tetrazolium The data collected were analyzed by the analysis of vari- [NBT] in solution A), and solution C (0.6% Triton X-100 ance procedure while treatment means were separated by in solution A) were incubated at 37°C for 10 min. Reac- the least significance difference (LSD) incorporated in the tion was started by introducing 100 µL of solution D (20 statistical analysis system (SAS) package of 9.1 version, mM hydroxylamine hydrochloride, pH=6.0). The rate of (2006).Correlation coefficients and trend lines were deter- NBT dye reduction by O mined using Excel Software (Microsoft, 2010 version).
.- anion generated due to photo-
activation of hydroxylamine hydrochloride was measured at λ =560 nm in the absence of PMSF. Next, 10 µL aliquot of PMSF were added to the reaction mixture and 50% inhibition in the rate of NBT reduction by SOD At the end of the experimental time of 30 days, FPGC present in the enzyme source was measured. A unit (U) of of DIAB group was 4.3 folds > NORM group (Table SOD activity was defined by the 50% inhibition of NBT. 1). Specifically, HrACMO, HrHIRO, HrAGHR and SOD activity was expressed in U/mg protein.
HrAMEH groups exhibited elevated FBGC compared with the NORM group (p<0.05) and were considered to be hyperglycemic after the 30-day treatment. Conversely, Measurement of PMSF CAT activity was according to the FPGC of HrASGA, HrAMEC, HrAMHR, HrECHR, method of Luck,48 as reported by Chikezie and Uwakwe.44 HrAGEH and HrAAEH groups were significantly (p< The final reaction volume of 3.0 mL contained 0.05 M 0.05) higher than the NORM group but not considered to Tris-buffer, 5 mM EDTA (pH=7.0), and 10 mM H O (in be hyperglycemic after the 30-day treatment.
0.1 M K PO /KHPO buffer; pH=7.0). A hundred micro milliliter (100 µL) aliquot of the PMSF was added to the It is worthwhile to note that FPGC of HrAGAM and above mixture. The rate of change of absorbance per min HrAGEC groups were significantly (p<0.05) lower than at λ =240 nm was recorded for 5 min. CAT activity was the NORM group. Specifically, HrAGAM group=66.30 Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats LSOD activity was higher than KSOD activity; p>0.05. Table 1: Fasting plasma glucose concentrations of
normal, diabetic and treated rats
Also, Figure 2 shows that the levels of LSOD and KSOD FPGC (mg/dL)
activities of the DIAB group were in 3.82 and 12.01 folds lower than the NORM group, respectively. At the end of the experimental time, each of the treated diabetic rats LSOD activities were lower than that of NORM group; activity=198.24±1.02 U/mg protein, 105.70 ± 0.71e,f,g,h activity=190.61±1.22 U/mg protein; 118.00 ± 0.92c,d p>0.05. By the same comparison, HrAGAM 112.50 ± 0.70d,e activity were significantly (p<0.05) activity. Peak values of SOD activ- 81.00 ± 0.39n,o,p ities of the treated HyGR were of the following magni- 112.50 ± 0.42d,e,f activity=185.25±1.01 U/mg proteins activity=171.45±0.99 U/mg protein.
105.00 ± 0.62e,f,g,h,i 103.50 ± 0.79f,g,h,i,j However, KSOD activities of other treated HyGR 103.00 ± 0.81f,g,h,i,j,k (HrAGEC, HrAMAH, HrECHR and HrAGEH) were higher than the NORM group, but with no significant dif- ference (p>0.05). Generally, the treated HyGR exhibited 112.00 ± 0.71d,e,f,g increased levels of LSOD and KSOD activities compared with the DIAB group. For instance, HrAMHR group The mean (X) ± S.D of six (n = 6) determinations. Means in the column with the showed 3.97 folds improvement in LSOD activity com- same letter are not significantly different at p > 0.05 according to LSD. FPGC > 110 pared with DIAB group. LSOD and KSOD activities of mg/dL = hyperglycemia.
the treated HyGR showed a very weak positive correla- ± 0.71 mg/dL, HrAGEC group=81.00±0.39 mg/dL and NORM group=86.30±0.15 mg/dL (Table 1). Generally, FPGC of the various treated HyGR was within the range Liver GPOx (LGPOx) activity of the NORM group of 66.30±0.71 mg/dL and 133.00±0.63 mg/dL as against was not significantly (p>0.05) different from the cor- DIAB group=368.20±0.72 mg/dL. At the end of the responding kidney GPOx (KGPOx) activity; LGPOx experimental time, the following animal groups: HrASGA, activity=1.70±0.03 U/mg protein, whereas KGPOx HrAGAM, HrAGEC, HrAMEC, HrAMHR, HrECHR, activity=1.10 ± 0.04 U/mg protein (Figure 3). LGPOx HrAGEH, HrAMAE and HrAAEH registered FPGC < and KGPOx activities of DIAB group were 2.36 folds and 3.93 folds lower than the NORM group. However, LGPOx and KGPOx activities increased following the Figure 1 shows that flavonoids were comparatively the administration of the various herbal formulations to cor- most abundant phytochemical present in the four leaves responding animal groups as against the decreased lev- extracts, in the order: AMCO > ASGA > HIRO > EMCO. els of LGPOx and KGPOx activities of DIAB group. The concentration of alkaloids in HIRO was relatively low LGPOx activity showed a very weak negative correlation compared with other three leaf extracts. Saponin contents (r=-0.088487792) with KGPOx activity in the treated in the four leaf extracts were in the range of 43.50±1.01 animal groups. Figure 3 shows that amongst the various mg/g and 71.01±0.62 mg/g. In addition, saponin contents treated hyperglycemic rat groups, peak LGPOx activ- amongst ASGA, EMCO and HIRO leaf extracts were ity=1.71 ± 0.05 U/mg proteins, occurred following the not significantly (p>0.05); specifically, ASGA=44.50±0.51 administration of AAEH herbal formulation. Similarly, mg/g, EMCO=43.50±0.54 mg/g and HIRO=45.25±0.35 peak value of KGPOx activity=1.99±0.04 U/mg protein, mg/g (Figure 1). Likewise, tannin contents of the four leaf ensued in HyGR consequent upon the administration of extracts was within a narrow range of 25.50±0.18 mg/g AMHR herbal formulation.
and 33.75 ± 0.32 mg/g; p>0.05. In addition, tannin was the lowest phytochemical present in the four leaf extracts.
Liver CAT (LCAT) activity was 2.19 folds > kidney CAT (KCAT) activity in NORM group (Figure 4). DIAB Liver SOD (LSOD) activity of NORM group was sig- activity=23.05±0.82 U/mg protein and DIAB nificantly (p<0.05) higher the corresponding kidney SOD ity=20.78±0.77 U/mg protein, representing 73.93% and (KSOD) activity (Figure 2). Likewise, in the DIAB group, 48.60% reduction in CAT activity compared with NORM Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015

Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Figure 1: Some phytochemical contents of A. montanus, A.
Figure 2: Liver and kidney superoxide dismutase activities of
gangetica, E. coccinea and H. rosasinensis leaf extracts.
experimental rats.
Figure 3: Liver and kidney glutathione peroxidase activities of
Figure 4: Liver and kidney catalase activities of experimental
experimental rats.
group; p<0.05. Comparative analyses showed that DIA- The ratio of liver GSH/GSSG (LGSH: GSSG) levels and activity was not significantly (p>0.05) different from kidney GSH/GSSG (KGSH: GSSG) levels were such activity. Furthermore, at the end of the experi- that in NORM group, KGSH: GSSG > LGSH:GSSG; mental time, LCAT activity and KCAT activity of all the p<0.05 (Figure 5). Also, Figure 4 shows that most values treated hyperglycemic rat groups were significantly (p<0.05) of liver and kidney GSH/GSSG ratios were significantly lower than the NORM group; except HrASGA and (p<0.05) increased when compared with NORM group, activities; p>0.05. LCAT activity of the following the administration of the herbal formulations treated hyperglycemic animal groups showed a very weak to the various hyperglycemic rat groups; except HrA- positive correlation (r=0.000829808) with that of KCAT MAE group which gave LGSH:GSSG=9.74±1.04 and activity. HrAAEH group exhibited peak LCAT activ- KGSH:GSSG=19.00±1.06. LGSH: GSSG and KGSH: ity=83.77±0.82 U/mg protein, whereas HrEMCO group GSSG) of treated hyperglycemic rat groups showed a gave peak KCAT activity = 41.22±0.72 U/mg protein.
very weak negative correlation (r=-0.192889137). Fig- Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015

Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats Figure 5: Liver and kidney ratio of GSH to GSSG concentrations of experimental rats.
ure 5 shows that LGSH: GSSG peak value=15.74±1.05, Previous studies showed that alkaloids and flavonoids occurred in treated hyperglycemic rat groups administered are effective anti-diabetic agents.57,61 In another study, the with AMHR herbal formulation. Correspondingly, peak potency of core structures of flavonoid precursors to act value of KGSH: GSSG = 26.11±1.04, was presented in as anti-hyperglycemic agents was demonstrated in vivo in HyGR administered with AMEC herbal formulation.
HyGR.62 A comprehensive review on natural approach to the treatment of diabetes,7 further confirmed that fla- An overview of Figures 2-5 showed that liver and kid- vonoids are among the listed anti-diabetic compounds, ney levels of antioxidant enzyme activities (SOD, GPOx which exert their hypoglycemic properties via extra pan- and CAT) and GSH/GSSG ratios increased following the creatic mechanism of α-glucosidase modulation.56 Expect- administration of the various herbal formulations, which edly, the relatively high flavonoids contents of the four was order of SHF > DHF > THF > QHF as indicated by medicinal plants (ACMO, ASGA, EMCO and HIRO) their corresponding trend lines.
contributed to the capabilities of the herbal formulations to exert glycemic control in the experimental rats. Like- wise, the relatively high abundance of alkaloids, especially in ACMO, ASGA and EMCO extracts, may have acted as a hypoglycemic agent as previously reported.63 Therefore, From the results of the present study, HyGR treated with combinatorial formulations of the four medicinal plant the various herbal formulations for experimental period extracts promoted synergy amongst the various bioactive of 30 days showed evidence of varied levels of efficacy principles, and thereby potentiated the anti-hyperglycemic of herbal formulations to exert glycemic control (Table capacities of the individual plant extracts in conformity 1). Specifically, HyGR administered with the DHFs (HrA- with previous reports.33,64 Similarly, Brahmbhatt et al.,65 GAM, HrAGEC and HrAMEC), THF (HrAMAE) and demonstrated that combining ginger extracts' with other QHF (HrAAEH) were normoglycemic (FBGC = 66.30 constituents, particularly 6-gingerol, caused significant ± 0.71 - 97.30 ± 0.85 mg/dL) following the 30-day treat- augmentation of anti-proliferative activity of the extracts. ments. In conformity with the present findings, several However, studies have also shown that antagonistic inter- authors had previously reported the use of herbal rem- actions among phytochemicals in medicinal plant extracts edies for the control and treatment of hyperglycemia could, paradoxically, attenuate the therapeutic potentials of the individual bioactive principles.66 28,32,50,51 and individuals suffering from diabetes mellitus.52-56 Furthermore, the modes of actions Saponins and tannins are bioactive principles of medici- of some selected anti-hyperglycemic plants have been nal and toxicity importance and their capabilities to exert extensively discussed elsewhere.32,57,58 In all those reports, glycemic control have been experimentally established.67-69 the quantity and types of phytochemicals present in the Kunyanga et al.,67 reported that condensed tannin extracts extracts were considered to be, to a large extent, respon- of raw and processed indigenous food ingredients from sible for the observed anti-hyperglycemic properties of Kenya exhibited promising anti-diabetic effects; possess- the herbal extracts.32,50,51,53,56,59,60 ing potential α-amylase and α-glucosidase inhibition activ- Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats ities within the range of 23% to 44% and 58% to 88%, Sphaeranthus indicus exhibited protective effect against respectively. In another study Zheng et al.,68, implicated lipid peroxidation and normalized repressed SOD, CAT, the saponins as the anti-diabetic principles of seed extract glutathione S-transferase activities in gentamicin induced of Entada phaseoloides L. They further posited that the nephrotoxic rats.70 Similarly, previous reports showed that therapeutic effect of saponins was facilitated by repres- S-IDR treated with exogenous SOD and GSH exhibited sion of chronic inflammation response pathways of pan- improved antioxidant enzymes activities,11,74,76 which creatic islets that was mediated by its inhibitory actions on implied amelioration of tissue oxidative stress.
interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF- α) and C-reactive protein (CRP) by the seed extracts in Depressed renal and hepatic GPOx and CAT activities are a dose dependent manner. Histopathological examina- associated with the diabetic state as reported here (Figures tion of the pancreatic tissues of streptozotocin-induced 3 and 4). Specifically, previous studies78 had noted that diabetic rats (S-IDR) showed evidence of considerable CAT deficiency accelerated diabetic nephropathy, which quantitative increase in β-cells by 75% when treated with was mediated by peroxisomal dysfunction. However, the saponins.69 The present study showed that saponins cod liver oil intake engenders significant increase in both and tannins are present in approximately equal propor- GPOx and CAT activities in aorta, heart, and liver of tions in extracts of ACMO, ASGA, EMCO and HIRO S-IDR,79 thereby alleviating oxidative stress induced tissue that may have contributed, in part, to the anti-hyperglyce- injuries. Similarly, studies by Shanmugam et al.,75 revealed mic potentials of the experimental plant extracts. that dietary ginger improved renal and hepatic GPOx and CAT activities, alongside other antioxidant enzymes and Generally, the level of enzyme activity in biologic systems oxidative stress indicators of S-IDR. They further noted could serve as a reliable tool in ascertaining health and that treatment of diabetic rats with ginger for 30 days pathologic conditions.20 Also, in the event of exposure of caused a therapeutic protective effect by lowering oxidative biological systems to noxious chemical agents and other stress induced hepatic and renal damage. Also, using environmental insults, previous researchers have applied experimental female Wistar rats, several natural products levels of activity of diverse antioxidant enzymes and obtained from wide varieties of medicinal plants reversed low molecular weight antioxidants (LMWA) as basis for reduced tissue SOD, CAT, GPOx and GST activities ascertaining tissue oxidative stress status.18,20,26,70-73 Find- following exposure to cadmium.72 They further suggested ings from the present study showed that HyGR exhibited that their therapeutic actions were probably connected alterations in tissue antioxidant enzymes (SOD, GPOx with the synergistic effects of the bioactive principles of and CAT) activities and GSH/GSSG ratio, of which the the various herbal extracts. The present study has equally cellular levels these oxidative stress indicators were diag- shown substantial improvements in depressed renal and nostic of oxidative tissue injury.
hepatic GPOx and CAT activities of HyGR following the administration of combinatorial herbal formulations of Decreased SOD activity of untreated HyGR as reported the four medicinal plant extracts.
here conformed with previous studies.11,74,75 In particular, Di Naso et al.,11 noted that decreased SOD activity in DM The physiochemical roles of GSH is indispensible in a is connected with non-enzymatic glycation of intracellular multitude of cellular processes. Therefore, disturbances SOD at the lysine residues located in the heparin-binding in GSH homeostasis are associated with the etiology domain, which indirectly altered extracellular SOD activity and/or progression of a number of human diseases.26 and functionality. In another study Loven et al.,76 posited Furthermore, the redox state of the GSH/glutathione that decreased Cu-Zn SOD activity observed in diabetes disulfide couple (GSH/GSSG) is an important biologic was sequel to the inactivation of the enzyme as a result indicator and regulator of signals of and metabolic of absence or failure to metabolize hydrogen peroxide pathways in normal cell systems27,80 as well as oxidative via GPOx pathway. The decreased levels of renal and stress and glucose metabolism in type 2 DM.81 Accordingly, hepatic SOD activity of HyGR showed evidence of the results of the present investigations have shown restoration of the enzyme activity that were comparable that GSH/GSSG ratio served as a reliable biomarker in to NORM rats following the administration of the various ascertaining the efficacy of herbal remedies in alleviation experimental herbal formulations (Figure 2). The present of hyperglycemia and amelioration of tissue oxidative findings are comparable with that reported by Elmalí stress The present findings corroborated the reports of et al.,77 in which they noted that glibenclamide directly El-Missiry and El Gindy,82 in which it was noted that daily increased renal and hepatic CAT and SOD activities of oral administration of Eruca sativa seeds oil for 2 weeks S-IDR. Reports have shown that ethanolic extract of stimulated and increased hepatic GSH production of Phcog Commn, Vol 5, Issue 2, Apr-Jun, 2015 Ojiako .: Renal and Hepatic Antioxidant Status of Hyperglycemic Rats alloxan-treated rats. Additionally, Zitka et al.,83 used the HyGR suggest that the experimental herbal formulations redox status expressed as GSH:GSSG as a biomarker caused a sparing effect on renal and hepatic antioxidant for ascertaining the level of oxidative stressin paediatric system against reactive oxygen species. Additionally, the tumor patients, which was similar to earlier reports84 on impairment of glutathione metabolism in erythrocytes combinatorial herbal formulations exerted greater glyce- of patients suffering from diabetes mellitus. From the mic control than the single herbal formulations.
present investigations, the antioxidant enzymes activities and GSH:GSSG between renal and hepatic tissues of HyGR treated with the various herbal formulations gave CONFLICT OF INTEREST
poor correlations. This was an obvious indication that the levels of oxidative stress and subsequent capacities The authors declare no conflict of interest.
of the herbal formulations to ameliorate oxidative stress in renal and hepatic tissues of HyGR were not of equal pathophysiological proportions and dimensions.
The authors are thankful to Dr. M. Ibe, School of Agri- Generally, the improvement in renal and hepatic antioxi- culture and Agricultural Technology (SAAT), Federal dant enzymes activities and GSH:GSSG of the treated University of Technology, Owerri.
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