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Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model
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Research Article
Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model Uduak Akpan Okon, Idorenyin Udo Umoren Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, University of Uyo, Akwa Ibom State 520001, Nigeria ABSTRACT OBJECTIVE: Diabetes mellitus (DM) is known to be associated with increase of oxidative stress products. The direction of effect of any treatment on these products could therefore be a reliable measure of its efficacy on DM. So the aim of this study was to investigate the activity of insulin, Ocimum gratissimum L. (OG) and Vernonia amygdalina L. (VA) on oxidative stress products. METHODS: Thirty-six female Wistar rats weighing 150–200 g were randomly divided into six groups of six rats each. Thirty rats were induced for type 1 DM (DM1) with a single intraperitoneal administration of 65 mg/ kg body weight of streptozotocin. Group 1 was normal control and was administered distilled water while Group 2 served as DM1 control group; Groups 3, 4, 5 and 6 were diabetic rats treated with 208 mg/kg OG (DM1 + OG), 52 mg/kg VA (DM1 + VA), 208 mg/kg OG + 52 mg/kg VA (DM1+OG +VA) and 0.16 IU insulin (DM1 + insulin) respectively. Determination of methemoglobin and sulfhemoglobin was achieved by the absorption spectrum principle. Red blood cell (RBC) catalase was assayed by continuous spectrophotometric method. RESULTS: The RBC catalase concentration was significantly decreased in the DM1 and DM1+VA groups when compared with the normal control. DM1 + OG significantly increased RBC-catalase when compared to DM1. The methemoglobin concentration was significantly reduced in the DM1, DM1 + VA, DM1 + OG + VA and DM1 + insulin groups when compared to the normal control group. The sulfhemoglobin concentration was significantly increased in the diabetic control and the diabetic treated groups when compared to the normal control. DM1 + OG reduced the sulfhemoglobin concentration when compared to DM1. The blood glucose concentration of all the diabetic groups was significantly raised compared to normal control. OG, VA and insulin significantly reduced the blood glucose concentration with the efficacy of OG and VA higher than insulin. CONCLUSION: Adverse alteration of oxidative indices were observed in type 1 DM model. Treatment with OG and insulin showed potent antioxidant activity, while the hypoglycemic efficacy of OG and VA were higher than insulin. Keywords: oxidative stress; diabetes mellitus, type 1; insulin; Ocimum gratissimum L.; Vernonia amygdalina L. Citation: Okon UA, Umoren IU. Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model. J Integr Med. 2017; 15(4): 302–309.
https://dx.doi.org/10.1016/S2095-4964(17)60332-7 Received November 14, 2016; accepted January 4, 2017. Correspondence: Uduak Akpan Okon, PhD; E-mail: [email protected]
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1 Introduction Ocimum gratissimum L. (OG) and Vernonia amygdalina L. (VA) occupy prominent positions in the diets of several Nigerian ethnic groups and are traditional ingredients in some African cuisine. Leaves of both plants are also reported to possess medicinal values in ethnotraditional medicine and various scientific research studies.[1] OG and VA are used in the treatment of diabetes mellitus (DM) by traditional medical practitioners and natives of South Eastern Nigeria.[2] Their hypoglycemic effect has been widely reported.[1,3] Insulin is principally responsible for the maintenance of normal blood glucose concentration. Almost all acute cases of DM are treated with insulin. Insulin deficiency or defective activity usually results in DM with hyperglycemia as its primary defect. Persistent hyperglycemia is associated with derangement in metabolic signal transduction pathway and generation of reactive oxygen species (ROS). Excess ROS may overwhelm the normal antioxidant enzymes such as super oxide dismutase (SOD), glutathione peroxidase and catalase that normally checkmate oxidative stress mediators.[4] Oxidative stress causes a complex dysregulation of cell metabolism and cell homeostasis. Recent findings show that oxidative stress plays a vital role in the pathogenesis of insulin resistance and β-cell dysfunction. There is growing evidence that oxidative stress (i.e., imbalance between free radical production and antioxidant defense) is involved in the pathogenesis of cardiovascular disease in diabetes.[5,6] Enzymatic sources of increased generation of ROS in DM include nitric oxide synthase (NOS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase.[7] It has been reported that NOS may produce O2- instead of NO and this is referred to as the uncoupled state of NOS.[7] Most findings show that NADPH oxidase, a membrane-associated enzyme that consists of five subunits, is a major source of O 2production. It has also been reported that NADPH oxidase activity is significantly higher in vascular tissues (saphenous vein and internal mammary artery) obtained from diabetic patients. [8] There is plausible evidence that protein kinase C, which is stimulated in diabetes via multiple mechanisms (i.e., polyol pathways and angiotensin II), activates NADPH oxidase.[9] Oxidative stress in type 1 DM (DM1) can be reduced by enhancing antioxidant defense.[10] This can be achieved by eliminating ROS. They can be eliminated by a number of enzymatic and nonenzymatic antioxidant mechanisms. Maritim et al. [10] recently reported that diabetes has multiple effects on the activity of these enzymes, which further augment oxidative stress by causing a suppressed defense response. Journal of Integrative Medicine
Significant to oxidative stress, alteration of normal hemoglobin molecule gives rise to abnormal forms which do not have oxygen-carrying capacity with complications such as anemia. [11] Variant forms of hemoglobin are oxyhemoglobin (OxyHb), methemoglobin, carboxyhemoglobin and sulfhemoglobin (SulfHb). Methemoglobin (MetHb) is a bluish chocolate brown color molecule of which a trace amount (1%–2% of MetHb) is found in the body.[12] Levels of MetHb greater than 2% can be due to genetic factors, caused by exposure to various chemicals, due to deficiency in some enzyme activities or to a disease condition.[13] SulfHb is found in the blood in a minimal level (< 1%). Increases in the level of SulfHb may be caused by taking medications containing sulfonamides, occupational exposure to sulfur compounds and exposure to chemical compounds such as acetanilide, phenacetin, nitrates and sulfur compounds. Such increases of SulfHb can result in a clinical condition known as sulfhemoglobinemia.[14] Since the degree of oxidative stress is strongly linked to the level of altered hemoglobin derivatives and the depletion of red blood cell (RBC) catalase, resulting from hyperglycemia, the comparative estimation of antioxidant activity of insulin, OG and VA could therefore be a reliable measure of their efficacy in the management of DM and its complications. 2 Materials and methods 2.1 Drugs Streptozotocin (STZ) and insulin (Actrapid, Novo Nordisk A/S, Denmark, 100 IU/mL ) were purchased from a reputable pharmaceutical company in Uyo, Akwa Ibom State, Nigeria. 2.2 Plant material 2.2.1 Collection and identification Fresh leaves of OG and VA were collected from the medicinal farm of the Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo. Collection took place in the early rainy season of June, 2014. Uyo is in the sub-tropical region. The soil type used is predominantly loamy and OG is known to grow in well-drained soil. The plants were identified by a taxonomist in the Department of Botany and Ecological Studies, University of Uyo. Samples of VA and OG were stored in the lab respectively. 2.2.2 Extract preparation The fresh leaves of OG and VA were rinsed with water to remove sand and debris and allowed to air dry. The leaves were cut into small pieces and sundried for two days and then transferred into an oven (Astell Hearson, England) and dried at a temperature range of 40–45 ºC. The dried leaves were pulverized into fine powder. Samples
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from each pulverized leaf type (425 g) were macerated in 3 000 mL of distilled water for about 12 h and stirred at regular intervals. These mixtures were filtered and their filtrates were concentrated to dryness in a water bath at 45 ºC to obtain a brown gummy paste. The weight of each dried extract was 57 g; stock solutions of the extracts were prepared by dissolving 15 g of each extract in 10 mL of water to give a concentration of 1 500 mg/mL, and stored at 4 ºC until they were required for use. The median lethal dose (LD50) was determined using the method presented by Lorke.[15] The LD50 for VA and OG was 52 and 208 mg/kg respectively. 2.2.3 Determination of phytoconstituents The phytoconstituents of the extracts were determined and screened to reveal the presence of carbohydrates, tannins, alkaloids, saponnins, phenolics, anthraquinines, cardiac glycosides and other compounds, as described by Sofowora.[16] The analysis was limited to identification of phytoconstituents only. 2.3 Animal preparations, experimental groupings and treatment Thirty-six 7–8 weeks old, female, albino Wistar rats, weighing between 150 and 200 g, were used for the study. The animals were divided into six groups of six rats each. Administration of OG and VA was done daily with a syringe and orogastric tube. The animal groups were as listed in Table 1. All animal groups were allowed access to water ad libitum. The experimental procedures involving the animals and their care met guidelines approved by the University of Uyo Research and Ethical Committee, established in line with the regulations of the Declaration of Helsinki on animal research. 2.4 Induction of diabetes DM1 was induced in 30 female Wistar rats by a single intraperitoneal injection of 65 mg/kg of STZ. Successful induction of diabetes was verified one week after STZ injection by symptoms of polyuria, polyphagia, polydipsia, loss of body weight, emaciation and wetting of their beddings and by testing blood glucose levels using a glucometer (Accu-Check Advantage II, Roche Diagnostic,
Germany). 2.5 Blood glucose levels Blood samples were collected by tail prick at weekly intervals. Blood glucose levels in mmol/L were measured in the animals with the aid of glucometer using compatible glucose test strips according to prescribed instructions by pricking the tail tip and dropping blood gently on the glucometer. 2.6 RBC catalase activity Determination of RBC catalase was done at the University of Uyo Teaching Hospital, Uyo. The activity of catalase in the blood serum was assayed using a continuous spectrophotometric method. The catalase-520 assay is a two-step procedure. The rate of decomposition of hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen is proportional to the concentration of catalase. Therefore, the sample containing catalase is incubated in the presence of a known concentration of H2O2. After incubation for 1 min the reaction is quenched with sodium oxide. The amount of H2O2 remaining in the reaction mixture is then determined by the oxidative coupling reaction of 4-aminophenazone and 3,5-clhoro-2-hydroxybezenesulphuric acid in the presence of H2O2 and catalyzed by horse radish peroxidase (HRP). The resulting quinoneimine dye was measured at 2 520 nm. Reagents involved in the estimation of the activity of catalase are listed in Table 2. To preparae HRP/chromogen, 1 volume of HRP was added to 1 000 volumes of chromogen, e.g., 110 μL in 110 mL. To prepare 10 mm hydrogen peroxide in bugger, 1 volume of 30% is added to 1 000 volumes of substrate diluent (e.g., 30 μL in 30mL). Then, the sample (30 μL) of diluted standards was added to tubes. Then 500 mL of substrate (H2O2) was added into each tube. Each tube was incubated for exactly 1 min at room temperature, before 500 mL stop reagent was added. Tubes were capped and mixed by inversion. A 20 mL aliquot of each reaction mixture was added into cuvettes. Next, 2 mL of HRP/ chromogen reagent was added into each cuvette, and they were mixed by inversion. The mixtures were then incubated for 10 min at room temperature. Absorbance was read at 520 nm.
Table 1 The experimental groupings and treatment of Wistar rats Number
Group
1 2 3 4 5 6
Control DM1 DM1 + OG DM1 + VA DM1 + OG + VA DM1 + insulin
Treatment Normal rats which received distilled water only STZ-induced diabetic rats left untreated STZ-induced diabetic rats treated with 208 mg/kg of OG for 28 d STZ-induced diabetic rats treated with 52 mg/kg of VA for 28 d STZ-induced diabetic rats treated with 208 mg/kg of OG and 52 mg/kg of VA for 28 d STZ-induced diabetic rats treated with 0.16 IU of insulin for 28 d
Six groups of six rats each. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; STZ: streptozotocin.
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www.jcimjournal.com/jim Table 2 Reagents involved in the estimation of the activity of catalase Reagents Chromogen
Quantity 4-Aminoantipyrene, 3,5-clhoro-2-hydroxybezenesulphuric acid in phosphate buffer, 2 × 110 mL
Substrate
30% Hydrogen peroxide, 300 μL
Horse radish peroxidase
Horse radish peroxidase in phosphate buffer, 400 μL
Buffer
Phosphate buffer, 60 mL
Sample diluents
Surfactant in phosphate buffer, 250 mL
Stop reagent
Sodium azide, 2 × 30 mL
Standard
Catalase, approximately 160 μ/vial
3.1 Comparison of RBC catalase concentrations in experimental type 1 diabetic rats Figure 1 shows that the RBC catalase concentration was significantly (P < 0.01) decreased in the DM1 group and the DM1 + VA group when compared with the normal control group. DM1 + OG significantly (P < 0.05) increased RBC catalase when compared with DM1 group. DM1+ insulin and DM1 + OG + VA closely followed the DM1 + OG direction, though their efficacies were not significant (Figure 1). 70 60 50 40
10
SulfHb (g/mL) = F3×A3×8 000－ (F4×(A1－A2)－F5×A4)×100 MetHb (g/mL) Total Hb (g/mL)
×100%
SulfHb (g/mL) Total Hb (g/mL)
×100%
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3 Results
20
MetHb (g/mL) = F2×(A1－A2)×100
SulfHb percent =
2.8 Statistical analysis Data collected during the study were expressed as mean ± standard error of mean. The data were statistically analyzed using analysis of variance with multiple comparisons made against the control group. Computer software SPSS Version 20.0 (IBM Corp, NY, USA) and Excel 2007 analyzer (Microsoft, USA) were used for the analysis.
30
Total Hb (g/mL) = F1×A4×100
MetHb percent =
F1, F2, F3, F4 and F5 are constant taken directly from the work of Drabkin and Austin.[17]
RBC catalase concentration (μmol/(mL·min))
2.7 MetHb and SulfHb assay MetHb and SulfHb quantification was done at the chemical pathology laboratory, University of Uyo Teaching Hospital, Uyo. They were assayed using the absorption spectrum principle. The procedures were as following. Firstly, the fresh blood from the animals was collected into EDTA bottles to prevent coagulation. Then, the sample (0.1 mL) of whole blood was delivered into a 10 mL test tube containing 3.9 mL of distilled water and swirled to mix. A 4.0 mL aliquot of potassium phosphate was added to the same tube and mixed thoroughly. A cuvette containing 1.5 mL phosphate buffer and 1.5 mL water was prepared and designated C1. Three millilitres of hemolysate was transferred to each of the two cuvettes, designated C2 and C3. K3Fe(CN)6 solution (0.1 mL) was added into cuvette C3. Then the cuvette was covered with parafilm and mixed by inversion three times. This cuvette was allowed to stand for 2 min before the absorbance was measured. The absorbance was measured at 630 nm for cuvettes C2 and C3; C1 was used as a blank. The values were recorded as A2a and A3a. Next, 0.1 mL of potassium cyanide was added to all cuvettes, which were then covered with parafilm, mixed by inversion three times and allowed to stand for 5 min. The absorbance was then measured at 630 nm for cuvettes C2 and C3, with C1 serving as a blank. The values were recorded as A2b and A3b. The concentrations of MetHb and SulfHb were thus calculated. (A2a－A2b) MetHb percent = ×100% (A3a－A3b)
0
Control
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 1 Comparison of RBC catalase concentrations in experimental type 1 diabetic rats Values are expressed as mean ± standard error of mean, n = 6. **P < 0.01, vs control; △ P < 0.05, vs DM1. RBC: red blood cell; DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.
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0.35
SulfHb concentration (g/dL)
3.2 Comparison of MetHb concentrations in experimental type 1 diabetic rats The MetHb concentration was significantly (P < 0.001) decreased in the DM1 and DM1 + VA group when compared to normal control. DM1+ OG + VA and DM1 + insulin significantly (P < 0.01) decreased MetHb concentration when compared to normal control. DM1 + OG marginally raised MetHb concentration close to the normal control (Figure 2).
0.25 0.2 0.15 0.1 0.05 0
1
Control
0.9
MetHb concentration (g/dL)
0.3
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 3 Comparison of SulfHb concentrations in experimental type 1 diabetic rats
0.8 0.7
Values are expressed as mean ± standard error of mean, n = 6. * P < 0.05, ** P < 0.01, vs control; △△ P < 0.01, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; SulfHb: sulfhemoglobin.
0.6 0.5 0.4 0.3 0.2 0.1 0 Control
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 2 Comparison of MetHb concentrations in experimental type 1 diabetic rats
Values are expressed as mean ± standard error of mean, n = 6. ** P < 0.01, vs control; △ P < 0.05, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; MetHb: Methemoglobin.
3.3 Comparison of SulfHb concentrations in experimental type 1 diabetic rats Figure 3 shows that the SulfHb concentration was significantly increased in the diabetic control group and diabetic groups treated (DM1, DM1 + OG, DM1 + VA, DM1 + OG + VA, and DM1 + insulin) when compared to the normal control (P < 0.05 or P < 0.01). DM1 + OG significantly (P < 0.01) reduced SulfHb concentration when compared to DM1. However, attempt at reduction of SulfHb concentration by DM1 + OG + VA and DM1 + insulin were not significant.
3.4 Comparison of weekly blood glucose level in experimental type 1 diabetic rats Table 3 shows that from weeks 1 to 4, the blood glucose concentrations of all the diabetic groups (DM1, DM1 + OG, DM1 + VA, DM1 + OG + VA, DM1 + insulin) were significantly increased compared to the normal control (P < 0.05 or P < 0.01). A sequential duration-dependent reduction of the blood glucose concentration was observed for DM1 + OG, DM1 + VA and DM1 + insulin, while DM1 + OG + VA showed no significant activity compared to DM1. DM1 + OG, DM1 + VA and DM1 + insulin significantly reduced the blood glucose concentration compared to DM1 (P < 0.01). This shows a higher hypoglycemic activity of OG and VA over insulin. DM1 + OG + VA had no significant effect. 4 Discussion As a metabolic disorder, DM is known to be associated with oxidative stress. This may either be due to excess generation of ROS or depletion of antioxidant defenses.
Table 3 Comparison of weekly blood glucose level in experimental type 1 diabetic rats Group
Before induction
After induction
Control
5.30 ± 0.30
–
DM1
3.56 ± 0.20** **
Glucose level (mmol/L) Week 1 Week 2
Week 3
Week 4
4.58 ± 0.20
4.72 ± 0.26
4.68 ± 1.15
4.58 ± 0.20
26.48 ± 1.67
24.86 ± 1.82**
24.40 ± 2.59**
31.40 ± 1.11**
25.18 ± 1.03**
DM1 + OG
3.40 ± 0.32
18.34 ± 1.28
16.28 ± 1.23
14.26 ± 1.33
9.84 ± 2.62
DM1 + VA
2.85 ± 0.16**△
23.38 ± 1.00△
14.98 ± 1.84**△△
12.30 ± 2.08**△ 25.70 ± 1.07
9.58 ± 2.96△△
25.10 ± 1.32
23.63 ± 3.99**
21.17 ± 2.97**
18.00 ± 3.24**
14.55 ± 2.80*△△
12.00 ± 2.69*△△
△
DM1 + OG + VA
4.18 ± 0.46
26.23 ± 3.10
DM1 + insulin
3.55 ± 0.33**
27.78 ± 2.81△△
△
**△ **
26.58 ± 2.42
**△△ **
△△ **△△
6.86 ± 1.83△△ 5.18 ± 1.01△△
Values are expressed as mean ± standard error of mean, n = 6. * P < 0.05, ** P < 0.01, vs control; △ P < 0.05, △△ P < 0.01, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.
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This basic etiologic mechanism of oxidative stress in DM was tested in the present study. Both standard and nonstandard hypoglycemic agents, including insulin, OG and VA, were employed to assess and compare their potential in the handling of some oxidative stress indices, including RBC catalase, MetHb and SulfHb. The diabetic state induced in study animals was characterized by reduced expression of RBC catalase; the various treatments administered tended to return it to the normal level. For RBC catalase, OG was the most potent, followed by insulin and VA as the least. The combination treatment of OG and VA increases RBC catalase concentration more than VA alone, but not up to the level of OG single treatment. Interestingly, the MetHb derivative concentration was also reduced in the diabetic state, with insulin and OG being more efficacious in its elevation towards the normal range. VA treatment further reduced the MetHb below the diabetic control level. Here again, the combination treatment of OG and VA improves the concentration above VA single treatment. SulfHb concentration was increased in all diabetic groups, unlike RBC catalase and MetHb. OG significantly decreased SulfHb concentration when compared to diabetic control while OG + VA, insulin and VA in the order of efficacy, attempted to marginally reduce the SulfHb concentration towards normal. These effects were however not significant. Most studies have shown that continuation of high glucose levels after the onset of either type 1 or type 2 diabetes has secondary adverse effects on the β cells themselves, not that glucose toxicity is the initial cause of either disease. In the case of DM1, it has been observed that increase of glucose levels is associated with increase of β cell generation of cytokines, which are prooxidants.[18] In a model of type 2 DM, high glucose concentrations increased intracellular peroxide levels in the islets.[19] This raises question about the state of antioxidant host defenses within the islets and whether augmentation of these defenses might be an appropriate therapeutic strategy to lessen the impact of hyperglycemia on the β cells. In this study, OG and VA exhibited significant antioxidant effects in type 1 diabetes. Oxidative stress is found to be increased in DM patients, and evidence suggests that oxidative cellular injury caused by free radicals can contribute to the development of diabetes.[20] Antioxidant enzymes scavenge the free radicals and protect the organs and membranes from oxidative damage. Moreover, diabetes also induces changes in the tissue content and activity of the antioxidant enzymes. [21] SOD protects tissues against oxygen free radicals by catalyzing the removal of the superoxide radical, converting it into H2O2 and molecular oxygen, which both damage the cell membrane and other biological structures.[22] RBC catalase is a heme-protein, which is responsible for Journal of Integrative Medicine
the detoxification of significant amounts of H2O2.[23] RBC catalase has a major role in controlling the concentration of H2O2[24] and consequently protects pancreatic β cells from damage by H 2O 2. In this study, type 1 diabetic rats showed a significant decrease in RBC catalase as compared to normal control. Our results are in harmony with those obtained in previous studies, which suggest that hyperglycemia induces a depletion of the antioxidant system due to the increased lipid peroxidation and formation of free radicals. [25–27] .This also confirms the suggestions of many studies that have shown that individuals with low RBC catalase lack the ability to protect their β cells and are consequently at risk of developing diabetes.[28] Although in this study, the direct cause of the DM was the destruction of β cells by STZ, the subsequent depletion of RBC catalase could be an aggravating mechanism. Low catalase activities can cause methemoglobinemia and hemolytic anemia, which may be attributed to deficiency of glucose-6-phosphate dehydrogenase or other unknown circumstances and also may damage hemeproteins, cause cell death, and together with redox active metal ions, produce highly toxic hydroxyl radicals.[29,30] Treatment of type 1 diabetic rats with OG and VA also showed an increase in RBC catalase as compared to diabetic control. The increased RBC catalase may be attributed to the presence of some phytochemical compounds, including flavonoids, terpenes, polyphenols, alkaloids, saponin, steroids and cardiac glycosides, which are known to possess antioxidant properties. [31,32] The phytochemical constituents may in part be responsible for the observed significant antioxidant activity of these extracts either singly or in synergy with one another.[33] In a future study, it will be expedient to investigate why VA did not show a similar effect to OG on RBC catalase even though they have similar phytochemical constituents. Hyperglycemia will promote the conversion of OxyHb to MetHb, and consequently the fractions of unstable hemoglobin molecules that undergo abnormal dissociation (auto-oxidation) to MetHb, SulfHb and carboxyhemoglobin with increasing hyperglycemia. [34] It is expected that MetHb concentration should increase in diabetic rats relative to normal control, but in this study, it decreased significantly. However, the SulfHb was increased. The reason for this discrepancy is not fully understood. Nevertheless, the disruption of the protective enzyme systems (e.g., NADH-MetHb reductase, cytochrome-b5 reductase) which normally reduce spontaneously formed MetHb to normal hemoglobin may be related. As expected, insulin treatment of DM1 model group (DM1 + insulin) was found to reduce the blood glucose levels. Remarkably, when compared to OG and
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VA treatment the extracts were found to have greater reduction in blood glucose levels at all times in the course of treatment. We also observed in the study that the combination treatment of OG + VA, did very little to reduce blood glucose levels relative to the DM1 control; it only showed a small, but statistically significant, reduction in blood glucose levels in weeks 3 and 4 of treatment. Some plant extracts are reported to exert hypoglycemic action by potentiating the insulin effect, either by stimulating the pancreatic secretion of insulin from the cells of islets of Langerhans or its release from bound insulin,[35] Others act through related mechanisms like inhibition of hepatic glucose production or correction of insulin resistance.[36] Antidiabetic effects of a majority of plants are attributed to their ability to restore the function of pancreatic tissues by causing an increase in insulin secretion or to inhibit absorption of glucose.[33,38] These leaf extracts may have acted through one of the above mechanisms resulting in anti-oxidative stress activity.
3
4
5 6 7
8
5 Conclusion These findings indicate that decreased production of RBC catalase may be one of the principal mechanism by which the diabetic state produces oxidative stress. The beneficial concomitant reduction in the MetHb concentration was counteracted with the gross elevation of SulfHb concentration. This supports our earlier hypothesis that antioxidant depletion and generation of oxidative stress products may be the basic underlying mechanism in generation of oxidative stress in DM, vis-à-vis RBC catalase, MetHb and SulfHb. Treatment with OG and insulin was very potent in the relief of this derangement, while VA and its combination with OG were less potent. The hypoglycemic efficacy of OG and VA were however, higher than that of insulin. Some aspect of the result obtained from this study appears to be novel and will serve as a reference for further research work.
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Okon U, Owu D, Udokang N, Udobang J, Ekpenyong C. Oral administration of aqueous leaf extract of Ocimum gratissimum ameliorates polyphagia, polydipsia and weight loss in streptozotocin-induced diabetic rats. Am J Med Sci. 2012; 2(3): 45–49. Asuquo O, Edet A, Mesembe O, Atanghwo J. Ethanolic extracts of Vernonia amygdalina and Ocimum gratissimum enhance testicular improvement in diabetic Wistar rats. Int J
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The authors have declared no conflict of interests.
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Alt Med. 2009; 8(2): 432–440. Egesie U, Adelaiye A, Ibu J, Egesie O. Safety and hypoglycaemic properties of aqueous leaf extract of Ocimum gratissimum in streptozotocin induced diabetic rats. Niger J Physiol Sci. 2006; 21 (1–2): 31–35. Fatmah A, Siti B, Zariyantey A, Nasar A, Jamaludin M. The role of oxidative stress and antioxidants in diabetic complications. Sultan Qaboos Univ Med J. 2012; 12(1): 5–18. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care. 1996; 19(3): 257–267. Pandey A, Tripathi P, Pandey R, Srivatava R, Goswami S. Alternative therapies useful in the management of diabetes: a systematic review. J Pharm Bioall Sci. 2011; 3: 504–512. Guzik T, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon K. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662. Ergul A, Schultz J, Stromhang C, Harris A, Tawfik A, Wells B, Caldwell R. Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes: role of endothelium-1. J Pharmacol Res Ther. 2004; 313: 70–77. Amiri F, Shaw S, Wang X, Tang J, Waller J, Eaton D, Marrero M. Angiotensin II activation of JAK/STAT pathway in mesangial cells is altered by high glucose. Kidney Int. 2002; 61: 1605–1616. Maritim A, Sanders R, Watkins J. Diabetes, oxidative stress and antioxidants: a review. J Biochem Mol Toxicol, 2003; 17: 24–38. Shihana F, Dawson AH, Buckley NA. A bedside test for methemoglobinemia, Sri Lanka. Bull World Health Organ. 2016; 94(8): 622–625. Sato K, Tamaki K, Okajima H, Katsumata Y. Longterm storage of blood samples as whole blood at extremely low temperature for methemoglobin determination. Forensic Sci Int. 1988; 37: 99–104. Adams V, Marley J, McCarrol C. Prilocaine induced methemoglobinemia in a medically compromised patients. Was this an inevitable consequence of the dose administered? Br Dent J. 2007; 203(10): 585–587. Flexman AM, Del Vicario G, Schwarz SK. Dark green blood in the operating theatre. Lancet. 2007; 369(9577): 1972. Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol. 1983; 54(4): 275–287. Sofowora A. Medicinal plants and traditional medicine in Africa. 2nd ed. Ibadan: Spectrum Book Ltd. 1993. Drabkin DL, Austin JR. Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhaemoglobin. J Biol Chem 1935;112:5165. Maedler K, Sergeev P, Ris F, Oberholzer J, Joller-Jemelka HI, Spinas GA, Kaiser N, Halban PA, Donath MY. Glucose-induced β cell production of IL-1β contributes to glucotoxicity in human pancreatic islets. J Clin Invest. 2002; 110(6): 851–60. Tanaka Y, Tran PO, Harmon J, Robertson RP. A role of Journal of Integrative Medicine
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glutathione peroxidase in protecting pancreatic β cells against oxidative stress in a model of glucose toxicity. Proc Natl Acad Sci U S A. 2002; 99: 12363–12368. Gomathi D, Ravikumar G, Kalaiselvi M, Devaki K, Uma C. Efficacy of Evolvulus alsinoides (L.) L. on insulin and antioxidants activity in pancreas of streptozotocin induced diabetic rats. J Diabetes Metab Disord. 2013; 12(1): 39. Jung M, Park M, Lee HC, Kang YH, Kang ES, Kim SK. Antidiabetic agents from medicinal plants. Curr Med Chem. 2006; 13(10): 1203–1218. Arivazhagan P, Thilagavathy T, Pannerselvam C. Antioxidant lipoate and tissue antioxidants in aged rats. J Nutr Biochem. 2000; 11(3): 122–127. Cheng L, Kellogg EW 3rd, Packer L. Photo inactivation of catalase. Photochem Photobiol. 1981; 34(1): 125–129. Gaetani GF, Ferraris AM, Rolfo M, Mangerini R, Arena S, Kirkman HN. Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood. 1996; 87(4): 1595–1599. Shabeer J, Srivastava RS, Singh SK. Antidiabetic and antioxidant effect of various fractions of Phyllanthus simplex in alloxan diabetic rats. J Ethnopharmacol. 2009; 124(1): 34–38. Punitha R, Manoharan S. Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats. J Ethnopharmacol. 2006; 105(1–2): 39–46. Vina J, Borras C, Gomez-Cabrera MC, Orr WC. Part of the series: from dietary antioxidants to regulators in cellular signalling and gene expression. Role of reactive oxygen species and (phyto)oestrogens in the modulation of adaptive response to stress. Free Radic Res. 2006; 40(2): 111–119. Góth L, Lenkey A, Bigler WN. Blood catalase deficiency and diabetes in Hungary. Diabetes Care. 2001; 24(10): 1839–1840.
29 Góth L, Bigler NW. Catalase deficiency may complicate urate oxidase (rasburicase) therapy. Free Radic Res. 2007; 41(9): 953–955. 30 Góth L, Tóth Z, Tarnai I, Bérces M, Török P, Bigler WN. Blood catalase activity in gestational diabetes is decreased but not associated with pregnancy complications. Clin Chem. 2005; 51(12): 2401–2404. 31 Afolabi C, Akanimoladun E, Ibukun I, Emmanuel A, Obutor E, Foramb E. Phytochemical constituent and antioxidant activity of extract from the leaves of Ocimum gratissimum. Sci ResEssay. 2008; 2(5): 163–166. 32 Villines TC, Kim AS, Gore RS, Taylor AJ. Niacin: the evidence, clinical use, and future directions. Curr Atheroscler Rep. 2012; 14(1): 49–59. 33 Patil RN, Patil RY, Ahirwar B, Ahirwar D. Evaluation of antidiabetic and related actions of some Indian medicinal plants in diabetic rats. Asian Pac J Trop Med. 2011; 4(1): 20–23. 34 Moussa SA. Oxidative stress in diabetes mellitus. Romanian J Biophys. 2008; 18(3): 225–236. 35 Xu Z, Wang X, Zhou M, Ma L, Deng Y, Zhang H, Zhao A, Zhang Y, Jia W. The antidabetic activity of total lignin from Fructus Arctii against alloxan-induced diabetes in mice and rats. Phytother Res. 2008; 22(1): 97–101. 36 Hu X, Sato J, Oshida Y, Xu M, Bajotto G, Sato Y. Effect of Gosha-jinki-gan (Chinese herbal medicine) on insulin resistance in streptozotocin-induced diabetic rats. Diabetes Res Clin Pract. 2003; 59(2): 103–111. 37 Kujur RS, Singh V, Ram M, Yadava HN, Singh KK, Kumari S, Roy BK. Antidiabetic and phytochemical screening of crude extract of Stevia rebaudiana in alloxan-induced diabetic rats. Pharmacognosy Res. 2010; 2(4): 258–263. 38 Akpan OU, Effiom OE. Ocimum gratissimum impairs gut glucose absorption but enhances water absorption in streptotozocin induced diabetic rats. Gaziantep Med J. 2015; 21(1): 43–50.
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Research Article
Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model Uduak Akpan Okon, Idorenyin Udo Umoren Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, University of Uyo, Akwa Ibom State 520001, Nigeria ABSTRACT OBJECTIVE: Diabetes mellitus (DM) is known to be associated with increase of oxidative stress products. The direction of effect of any treatment on these products could therefore be a reliable measure of its efficacy on DM. So the aim of this study was to investigate the activity of insulin, Ocimum gratissimum L. (OG) and Vernonia amygdalina L. (VA) on oxidative stress products. METHODS: Thirty-six female Wistar rats weighing 150–200 g were randomly divided into six groups of six rats each. Thirty rats were induced for type 1 DM (DM1) with a single intraperitoneal administration of 65 mg/ kg body weight of streptozotocin. Group 1 was normal control and was administered distilled water while Group 2 served as DM1 control group; Groups 3, 4, 5 and 6 were diabetic rats treated with 208 mg/kg OG (DM1 + OG), 52 mg/kg VA (DM1 + VA), 208 mg/kg OG + 52 mg/kg VA (DM1+OG +VA) and 0.16 IU insulin (DM1 + insulin) respectively. Determination of methemoglobin and sulfhemoglobin was achieved by the absorption spectrum principle. Red blood cell (RBC) catalase was assayed by continuous spectrophotometric method. RESULTS: The RBC catalase concentration was significantly decreased in the DM1 and DM1+VA groups when compared with the normal control. DM1 + OG significantly increased RBC-catalase when compared to DM1. The methemoglobin concentration was significantly reduced in the DM1, DM1 + VA, DM1 + OG + VA and DM1 + insulin groups when compared to the normal control group. The sulfhemoglobin concentration was significantly increased in the diabetic control and the diabetic treated groups when compared to the normal control. DM1 + OG reduced the sulfhemoglobin concentration when compared to DM1. The blood glucose concentration of all the diabetic groups was significantly raised compared to normal control. OG, VA and insulin significantly reduced the blood glucose concentration with the efficacy of OG and VA higher than insulin. CONCLUSION: Adverse alteration of oxidative indices were observed in type 1 DM model. Treatment with OG and insulin showed potent antioxidant activity, while the hypoglycemic efficacy of OG and VA were higher than insulin. Keywords: oxidative stress; diabetes mellitus, type 1; insulin; Ocimum gratissimum L.; Vernonia amygdalina L. Citation: Okon UA, Umoren IU. Comparison of antioxidant activity of insulin, Ocimum gratissimum L., and Vernonia amygdalina L. in type 1 diabetic rat model. J Integr Med. 2017; 15(4): 302–309.
https://dx.doi.org/10.1016/S2095-4964(17)60332-7 Received November 14, 2016; accepted January 4, 2017. Correspondence: Uduak Akpan Okon, PhD; E-mail: [email protected]
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1 Introduction Ocimum gratissimum L. (OG) and Vernonia amygdalina L. (VA) occupy prominent positions in the diets of several Nigerian ethnic groups and are traditional ingredients in some African cuisine. Leaves of both plants are also reported to possess medicinal values in ethnotraditional medicine and various scientific research studies.[1] OG and VA are used in the treatment of diabetes mellitus (DM) by traditional medical practitioners and natives of South Eastern Nigeria.[2] Their hypoglycemic effect has been widely reported.[1,3] Insulin is principally responsible for the maintenance of normal blood glucose concentration. Almost all acute cases of DM are treated with insulin. Insulin deficiency or defective activity usually results in DM with hyperglycemia as its primary defect. Persistent hyperglycemia is associated with derangement in metabolic signal transduction pathway and generation of reactive oxygen species (ROS). Excess ROS may overwhelm the normal antioxidant enzymes such as super oxide dismutase (SOD), glutathione peroxidase and catalase that normally checkmate oxidative stress mediators.[4] Oxidative stress causes a complex dysregulation of cell metabolism and cell homeostasis. Recent findings show that oxidative stress plays a vital role in the pathogenesis of insulin resistance and β-cell dysfunction. There is growing evidence that oxidative stress (i.e., imbalance between free radical production and antioxidant defense) is involved in the pathogenesis of cardiovascular disease in diabetes.[5,6] Enzymatic sources of increased generation of ROS in DM include nitric oxide synthase (NOS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase.[7] It has been reported that NOS may produce O2- instead of NO and this is referred to as the uncoupled state of NOS.[7] Most findings show that NADPH oxidase, a membrane-associated enzyme that consists of five subunits, is a major source of O 2production. It has also been reported that NADPH oxidase activity is significantly higher in vascular tissues (saphenous vein and internal mammary artery) obtained from diabetic patients. [8] There is plausible evidence that protein kinase C, which is stimulated in diabetes via multiple mechanisms (i.e., polyol pathways and angiotensin II), activates NADPH oxidase.[9] Oxidative stress in type 1 DM (DM1) can be reduced by enhancing antioxidant defense.[10] This can be achieved by eliminating ROS. They can be eliminated by a number of enzymatic and nonenzymatic antioxidant mechanisms. Maritim et al. [10] recently reported that diabetes has multiple effects on the activity of these enzymes, which further augment oxidative stress by causing a suppressed defense response. Journal of Integrative Medicine
Significant to oxidative stress, alteration of normal hemoglobin molecule gives rise to abnormal forms which do not have oxygen-carrying capacity with complications such as anemia. [11] Variant forms of hemoglobin are oxyhemoglobin (OxyHb), methemoglobin, carboxyhemoglobin and sulfhemoglobin (SulfHb). Methemoglobin (MetHb) is a bluish chocolate brown color molecule of which a trace amount (1%–2% of MetHb) is found in the body.[12] Levels of MetHb greater than 2% can be due to genetic factors, caused by exposure to various chemicals, due to deficiency in some enzyme activities or to a disease condition.[13] SulfHb is found in the blood in a minimal level (< 1%). Increases in the level of SulfHb may be caused by taking medications containing sulfonamides, occupational exposure to sulfur compounds and exposure to chemical compounds such as acetanilide, phenacetin, nitrates and sulfur compounds. Such increases of SulfHb can result in a clinical condition known as sulfhemoglobinemia.[14] Since the degree of oxidative stress is strongly linked to the level of altered hemoglobin derivatives and the depletion of red blood cell (RBC) catalase, resulting from hyperglycemia, the comparative estimation of antioxidant activity of insulin, OG and VA could therefore be a reliable measure of their efficacy in the management of DM and its complications. 2 Materials and methods 2.1 Drugs Streptozotocin (STZ) and insulin (Actrapid, Novo Nordisk A/S, Denmark, 100 IU/mL ) were purchased from a reputable pharmaceutical company in Uyo, Akwa Ibom State, Nigeria. 2.2 Plant material 2.2.1 Collection and identification Fresh leaves of OG and VA were collected from the medicinal farm of the Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo. Collection took place in the early rainy season of June, 2014. Uyo is in the sub-tropical region. The soil type used is predominantly loamy and OG is known to grow in well-drained soil. The plants were identified by a taxonomist in the Department of Botany and Ecological Studies, University of Uyo. Samples of VA and OG were stored in the lab respectively. 2.2.2 Extract preparation The fresh leaves of OG and VA were rinsed with water to remove sand and debris and allowed to air dry. The leaves were cut into small pieces and sundried for two days and then transferred into an oven (Astell Hearson, England) and dried at a temperature range of 40–45 ºC. The dried leaves were pulverized into fine powder. Samples
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from each pulverized leaf type (425 g) were macerated in 3 000 mL of distilled water for about 12 h and stirred at regular intervals. These mixtures were filtered and their filtrates were concentrated to dryness in a water bath at 45 ºC to obtain a brown gummy paste. The weight of each dried extract was 57 g; stock solutions of the extracts were prepared by dissolving 15 g of each extract in 10 mL of water to give a concentration of 1 500 mg/mL, and stored at 4 ºC until they were required for use. The median lethal dose (LD50) was determined using the method presented by Lorke.[15] The LD50 for VA and OG was 52 and 208 mg/kg respectively. 2.2.3 Determination of phytoconstituents The phytoconstituents of the extracts were determined and screened to reveal the presence of carbohydrates, tannins, alkaloids, saponnins, phenolics, anthraquinines, cardiac glycosides and other compounds, as described by Sofowora.[16] The analysis was limited to identification of phytoconstituents only. 2.3 Animal preparations, experimental groupings and treatment Thirty-six 7–8 weeks old, female, albino Wistar rats, weighing between 150 and 200 g, were used for the study. The animals were divided into six groups of six rats each. Administration of OG and VA was done daily with a syringe and orogastric tube. The animal groups were as listed in Table 1. All animal groups were allowed access to water ad libitum. The experimental procedures involving the animals and their care met guidelines approved by the University of Uyo Research and Ethical Committee, established in line with the regulations of the Declaration of Helsinki on animal research. 2.4 Induction of diabetes DM1 was induced in 30 female Wistar rats by a single intraperitoneal injection of 65 mg/kg of STZ. Successful induction of diabetes was verified one week after STZ injection by symptoms of polyuria, polyphagia, polydipsia, loss of body weight, emaciation and wetting of their beddings and by testing blood glucose levels using a glucometer (Accu-Check Advantage II, Roche Diagnostic,
Germany). 2.5 Blood glucose levels Blood samples were collected by tail prick at weekly intervals. Blood glucose levels in mmol/L were measured in the animals with the aid of glucometer using compatible glucose test strips according to prescribed instructions by pricking the tail tip and dropping blood gently on the glucometer. 2.6 RBC catalase activity Determination of RBC catalase was done at the University of Uyo Teaching Hospital, Uyo. The activity of catalase in the blood serum was assayed using a continuous spectrophotometric method. The catalase-520 assay is a two-step procedure. The rate of decomposition of hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen is proportional to the concentration of catalase. Therefore, the sample containing catalase is incubated in the presence of a known concentration of H2O2. After incubation for 1 min the reaction is quenched with sodium oxide. The amount of H2O2 remaining in the reaction mixture is then determined by the oxidative coupling reaction of 4-aminophenazone and 3,5-clhoro-2-hydroxybezenesulphuric acid in the presence of H2O2 and catalyzed by horse radish peroxidase (HRP). The resulting quinoneimine dye was measured at 2 520 nm. Reagents involved in the estimation of the activity of catalase are listed in Table 2. To preparae HRP/chromogen, 1 volume of HRP was added to 1 000 volumes of chromogen, e.g., 110 μL in 110 mL. To prepare 10 mm hydrogen peroxide in bugger, 1 volume of 30% is added to 1 000 volumes of substrate diluent (e.g., 30 μL in 30mL). Then, the sample (30 μL) of diluted standards was added to tubes. Then 500 mL of substrate (H2O2) was added into each tube. Each tube was incubated for exactly 1 min at room temperature, before 500 mL stop reagent was added. Tubes were capped and mixed by inversion. A 20 mL aliquot of each reaction mixture was added into cuvettes. Next, 2 mL of HRP/ chromogen reagent was added into each cuvette, and they were mixed by inversion. The mixtures were then incubated for 10 min at room temperature. Absorbance was read at 520 nm.
Table 1 The experimental groupings and treatment of Wistar rats Number
Group
1 2 3 4 5 6
Control DM1 DM1 + OG DM1 + VA DM1 + OG + VA DM1 + insulin
Treatment Normal rats which received distilled water only STZ-induced diabetic rats left untreated STZ-induced diabetic rats treated with 208 mg/kg of OG for 28 d STZ-induced diabetic rats treated with 52 mg/kg of VA for 28 d STZ-induced diabetic rats treated with 208 mg/kg of OG and 52 mg/kg of VA for 28 d STZ-induced diabetic rats treated with 0.16 IU of insulin for 28 d
Six groups of six rats each. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; STZ: streptozotocin.
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Quantity 4-Aminoantipyrene, 3,5-clhoro-2-hydroxybezenesulphuric acid in phosphate buffer, 2 × 110 mL
Substrate
30% Hydrogen peroxide, 300 μL
Horse radish peroxidase
Horse radish peroxidase in phosphate buffer, 400 μL
Buffer
Phosphate buffer, 60 mL
Sample diluents
Surfactant in phosphate buffer, 250 mL
Stop reagent
Sodium azide, 2 × 30 mL
Standard
Catalase, approximately 160 μ/vial
3.1 Comparison of RBC catalase concentrations in experimental type 1 diabetic rats Figure 1 shows that the RBC catalase concentration was significantly (P < 0.01) decreased in the DM1 group and the DM1 + VA group when compared with the normal control group. DM1 + OG significantly (P < 0.05) increased RBC catalase when compared with DM1 group. DM1+ insulin and DM1 + OG + VA closely followed the DM1 + OG direction, though their efficacies were not significant (Figure 1). 70 60 50 40
10
SulfHb (g/mL) = F3×A3×8 000－ (F4×(A1－A2)－F5×A4)×100 MetHb (g/mL) Total Hb (g/mL)
×100%
SulfHb (g/mL) Total Hb (g/mL)
×100%
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20
MetHb (g/mL) = F2×(A1－A2)×100
SulfHb percent =
2.8 Statistical analysis Data collected during the study were expressed as mean ± standard error of mean. The data were statistically analyzed using analysis of variance with multiple comparisons made against the control group. Computer software SPSS Version 20.0 (IBM Corp, NY, USA) and Excel 2007 analyzer (Microsoft, USA) were used for the analysis.
30
Total Hb (g/mL) = F1×A4×100
MetHb percent =
F1, F2, F3, F4 and F5 are constant taken directly from the work of Drabkin and Austin.[17]
RBC catalase concentration (μmol/(mL·min))
2.7 MetHb and SulfHb assay MetHb and SulfHb quantification was done at the chemical pathology laboratory, University of Uyo Teaching Hospital, Uyo. They were assayed using the absorption spectrum principle. The procedures were as following. Firstly, the fresh blood from the animals was collected into EDTA bottles to prevent coagulation. Then, the sample (0.1 mL) of whole blood was delivered into a 10 mL test tube containing 3.9 mL of distilled water and swirled to mix. A 4.0 mL aliquot of potassium phosphate was added to the same tube and mixed thoroughly. A cuvette containing 1.5 mL phosphate buffer and 1.5 mL water was prepared and designated C1. Three millilitres of hemolysate was transferred to each of the two cuvettes, designated C2 and C3. K3Fe(CN)6 solution (0.1 mL) was added into cuvette C3. Then the cuvette was covered with parafilm and mixed by inversion three times. This cuvette was allowed to stand for 2 min before the absorbance was measured. The absorbance was measured at 630 nm for cuvettes C2 and C3; C1 was used as a blank. The values were recorded as A2a and A3a. Next, 0.1 mL of potassium cyanide was added to all cuvettes, which were then covered with parafilm, mixed by inversion three times and allowed to stand for 5 min. The absorbance was then measured at 630 nm for cuvettes C2 and C3, with C1 serving as a blank. The values were recorded as A2b and A3b. The concentrations of MetHb and SulfHb were thus calculated. (A2a－A2b) MetHb percent = ×100% (A3a－A3b)
0
Control
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 1 Comparison of RBC catalase concentrations in experimental type 1 diabetic rats Values are expressed as mean ± standard error of mean, n = 6. **P < 0.01, vs control; △ P < 0.05, vs DM1. RBC: red blood cell; DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.
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0.35
SulfHb concentration (g/dL)
3.2 Comparison of MetHb concentrations in experimental type 1 diabetic rats The MetHb concentration was significantly (P < 0.001) decreased in the DM1 and DM1 + VA group when compared to normal control. DM1+ OG + VA and DM1 + insulin significantly (P < 0.01) decreased MetHb concentration when compared to normal control. DM1 + OG marginally raised MetHb concentration close to the normal control (Figure 2).
0.25 0.2 0.15 0.1 0.05 0
1
Control
0.9
MetHb concentration (g/dL)
0.3
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 3 Comparison of SulfHb concentrations in experimental type 1 diabetic rats
0.8 0.7
Values are expressed as mean ± standard error of mean, n = 6. * P < 0.05, ** P < 0.01, vs control; △△ P < 0.01, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; SulfHb: sulfhemoglobin.
0.6 0.5 0.4 0.3 0.2 0.1 0 Control
DM1
DM1+OG
DM1+VA DM1+OG+VA DM1+Insulin
Figure 2 Comparison of MetHb concentrations in experimental type 1 diabetic rats
Values are expressed as mean ± standard error of mean, n = 6. ** P < 0.01, vs control; △ P < 0.05, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.; MetHb: Methemoglobin.
3.3 Comparison of SulfHb concentrations in experimental type 1 diabetic rats Figure 3 shows that the SulfHb concentration was significantly increased in the diabetic control group and diabetic groups treated (DM1, DM1 + OG, DM1 + VA, DM1 + OG + VA, and DM1 + insulin) when compared to the normal control (P < 0.05 or P < 0.01). DM1 + OG significantly (P < 0.01) reduced SulfHb concentration when compared to DM1. However, attempt at reduction of SulfHb concentration by DM1 + OG + VA and DM1 + insulin were not significant.
3.4 Comparison of weekly blood glucose level in experimental type 1 diabetic rats Table 3 shows that from weeks 1 to 4, the blood glucose concentrations of all the diabetic groups (DM1, DM1 + OG, DM1 + VA, DM1 + OG + VA, DM1 + insulin) were significantly increased compared to the normal control (P < 0.05 or P < 0.01). A sequential duration-dependent reduction of the blood glucose concentration was observed for DM1 + OG, DM1 + VA and DM1 + insulin, while DM1 + OG + VA showed no significant activity compared to DM1. DM1 + OG, DM1 + VA and DM1 + insulin significantly reduced the blood glucose concentration compared to DM1 (P < 0.01). This shows a higher hypoglycemic activity of OG and VA over insulin. DM1 + OG + VA had no significant effect. 4 Discussion As a metabolic disorder, DM is known to be associated with oxidative stress. This may either be due to excess generation of ROS or depletion of antioxidant defenses.
Table 3 Comparison of weekly blood glucose level in experimental type 1 diabetic rats Group
Before induction
After induction
Control
5.30 ± 0.30
–
DM1
3.56 ± 0.20** **
Glucose level (mmol/L) Week 1 Week 2
Week 3
Week 4
4.58 ± 0.20
4.72 ± 0.26
4.68 ± 1.15
4.58 ± 0.20
26.48 ± 1.67
24.86 ± 1.82**
24.40 ± 2.59**
31.40 ± 1.11**
25.18 ± 1.03**
DM1 + OG
3.40 ± 0.32
18.34 ± 1.28
16.28 ± 1.23
14.26 ± 1.33
9.84 ± 2.62
DM1 + VA
2.85 ± 0.16**△
23.38 ± 1.00△
14.98 ± 1.84**△△
12.30 ± 2.08**△ 25.70 ± 1.07
9.58 ± 2.96△△
25.10 ± 1.32
23.63 ± 3.99**
21.17 ± 2.97**
18.00 ± 3.24**
14.55 ± 2.80*△△
12.00 ± 2.69*△△
△
DM1 + OG + VA
4.18 ± 0.46
26.23 ± 3.10
DM1 + insulin
3.55 ± 0.33**
27.78 ± 2.81△△
△
**△ **
26.58 ± 2.42
**△△ **
△△ **△△
6.86 ± 1.83△△ 5.18 ± 1.01△△
Values are expressed as mean ± standard error of mean, n = 6. * P < 0.05, ** P < 0.01, vs control; △ P < 0.05, △△ P < 0.01, vs DM1. DM1: type 1 diabetes mellitus; OG: Ocimum gratissimum L.; VA: Vernonia amygdalina L.
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This basic etiologic mechanism of oxidative stress in DM was tested in the present study. Both standard and nonstandard hypoglycemic agents, including insulin, OG and VA, were employed to assess and compare their potential in the handling of some oxidative stress indices, including RBC catalase, MetHb and SulfHb. The diabetic state induced in study animals was characterized by reduced expression of RBC catalase; the various treatments administered tended to return it to the normal level. For RBC catalase, OG was the most potent, followed by insulin and VA as the least. The combination treatment of OG and VA increases RBC catalase concentration more than VA alone, but not up to the level of OG single treatment. Interestingly, the MetHb derivative concentration was also reduced in the diabetic state, with insulin and OG being more efficacious in its elevation towards the normal range. VA treatment further reduced the MetHb below the diabetic control level. Here again, the combination treatment of OG and VA improves the concentration above VA single treatment. SulfHb concentration was increased in all diabetic groups, unlike RBC catalase and MetHb. OG significantly decreased SulfHb concentration when compared to diabetic control while OG + VA, insulin and VA in the order of efficacy, attempted to marginally reduce the SulfHb concentration towards normal. These effects were however not significant. Most studies have shown that continuation of high glucose levels after the onset of either type 1 or type 2 diabetes has secondary adverse effects on the β cells themselves, not that glucose toxicity is the initial cause of either disease. In the case of DM1, it has been observed that increase of glucose levels is associated with increase of β cell generation of cytokines, which are prooxidants.[18] In a model of type 2 DM, high glucose concentrations increased intracellular peroxide levels in the islets.[19] This raises question about the state of antioxidant host defenses within the islets and whether augmentation of these defenses might be an appropriate therapeutic strategy to lessen the impact of hyperglycemia on the β cells. In this study, OG and VA exhibited significant antioxidant effects in type 1 diabetes. Oxidative stress is found to be increased in DM patients, and evidence suggests that oxidative cellular injury caused by free radicals can contribute to the development of diabetes.[20] Antioxidant enzymes scavenge the free radicals and protect the organs and membranes from oxidative damage. Moreover, diabetes also induces changes in the tissue content and activity of the antioxidant enzymes. [21] SOD protects tissues against oxygen free radicals by catalyzing the removal of the superoxide radical, converting it into H2O2 and molecular oxygen, which both damage the cell membrane and other biological structures.[22] RBC catalase is a heme-protein, which is responsible for Journal of Integrative Medicine
the detoxification of significant amounts of H2O2.[23] RBC catalase has a major role in controlling the concentration of H2O2[24] and consequently protects pancreatic β cells from damage by H 2O 2. In this study, type 1 diabetic rats showed a significant decrease in RBC catalase as compared to normal control. Our results are in harmony with those obtained in previous studies, which suggest that hyperglycemia induces a depletion of the antioxidant system due to the increased lipid peroxidation and formation of free radicals. [25–27] .This also confirms the suggestions of many studies that have shown that individuals with low RBC catalase lack the ability to protect their β cells and are consequently at risk of developing diabetes.[28] Although in this study, the direct cause of the DM was the destruction of β cells by STZ, the subsequent depletion of RBC catalase could be an aggravating mechanism. Low catalase activities can cause methemoglobinemia and hemolytic anemia, which may be attributed to deficiency of glucose-6-phosphate dehydrogenase or other unknown circumstances and also may damage hemeproteins, cause cell death, and together with redox active metal ions, produce highly toxic hydroxyl radicals.[29,30] Treatment of type 1 diabetic rats with OG and VA also showed an increase in RBC catalase as compared to diabetic control. The increased RBC catalase may be attributed to the presence of some phytochemical compounds, including flavonoids, terpenes, polyphenols, alkaloids, saponin, steroids and cardiac glycosides, which are known to possess antioxidant properties. [31,32] The phytochemical constituents may in part be responsible for the observed significant antioxidant activity of these extracts either singly or in synergy with one another.[33] In a future study, it will be expedient to investigate why VA did not show a similar effect to OG on RBC catalase even though they have similar phytochemical constituents. Hyperglycemia will promote the conversion of OxyHb to MetHb, and consequently the fractions of unstable hemoglobin molecules that undergo abnormal dissociation (auto-oxidation) to MetHb, SulfHb and carboxyhemoglobin with increasing hyperglycemia. [34] It is expected that MetHb concentration should increase in diabetic rats relative to normal control, but in this study, it decreased significantly. However, the SulfHb was increased. The reason for this discrepancy is not fully understood. Nevertheless, the disruption of the protective enzyme systems (e.g., NADH-MetHb reductase, cytochrome-b5 reductase) which normally reduce spontaneously formed MetHb to normal hemoglobin may be related. As expected, insulin treatment of DM1 model group (DM1 + insulin) was found to reduce the blood glucose levels. Remarkably, when compared to OG and
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VA treatment the extracts were found to have greater reduction in blood glucose levels at all times in the course of treatment. We also observed in the study that the combination treatment of OG + VA, did very little to reduce blood glucose levels relative to the DM1 control; it only showed a small, but statistically significant, reduction in blood glucose levels in weeks 3 and 4 of treatment. Some plant extracts are reported to exert hypoglycemic action by potentiating the insulin effect, either by stimulating the pancreatic secretion of insulin from the cells of islets of Langerhans or its release from bound insulin,[35] Others act through related mechanisms like inhibition of hepatic glucose production or correction of insulin resistance.[36] Antidiabetic effects of a majority of plants are attributed to their ability to restore the function of pancreatic tissues by causing an increase in insulin secretion or to inhibit absorption of glucose.[33,38] These leaf extracts may have acted through one of the above mechanisms resulting in anti-oxidative stress activity.
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5 Conclusion These findings indicate that decreased production of RBC catalase may be one of the principal mechanism by which the diabetic state produces oxidative stress. The beneficial concomitant reduction in the MetHb concentration was counteracted with the gross elevation of SulfHb concentration. This supports our earlier hypothesis that antioxidant depletion and generation of oxidative stress products may be the basic underlying mechanism in generation of oxidative stress in DM, vis-à-vis RBC catalase, MetHb and SulfHb. Treatment with OG and insulin was very potent in the relief of this derangement, while VA and its combination with OG were less potent. The hypoglycemic efficacy of OG and VA were however, higher than that of insulin. Some aspect of the result obtained from this study appears to be novel and will serve as a reference for further research work.
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The authors have declared no conflict of interests.
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