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Trigonella foenum-graecum seeds extract plays a beneficial role on brain antioxidant and oxidative status in alloxan-induced Wistar rats

Trigonella foenum-graecum seeds extract plays a beneficial role on brain antioxidant and... Key words: Trigonella foenum-graecum; hyperglycaemia; brain; antioxidants; rats. The central complications of hyperglycaemia also include the Introduction damage of neuronal circuits and the damage is more augmented due Diabetes mellitus (DM) is a major health problem all over the world to hyperglycaemic-induced oxidative stress. Oxidative stress, leading today. It is a metabolic disorder of carbohydrate, fat, and protein to an increased production of ROS, as well as LPx, is increased in metabolism characterized by the elevation of both fasting and post- diabetes and also by stress in euglycemic animals. Similarly, the oxi- prandial blood glucose levels. DM has been shown to be a state dative damage in rat brain is increased by experimentally induced of increased lipid peroxidation (LPx). LPx of cellular structures, a hyperglycaemia. Under experimental conditions, hyperglycaemia free radical-induced activity, is thought to play an important role in dramatically increases neuronal alterations and glial cell damage ageing, atherosclerosis, and late complications of DM (Klein et  al., caused by temporary ischaemia. Several lines of evidence indicated 2015). An impaired radical scavenger function has been linked to that the modified oxidative state induced by chronic hyperglycaemia the altered activity of enzymes (catalase, superoxide dismutase, and may contribute to nerve tissue damage; free radical species impair glutathione peroxidase) and non-enzymatic (glutathione) free rad- the central nervous system, attacking neurons and Schwann cells and ical scavengers. The increased production of reactive oxygen spe- the peripheral nerves. Because of its high polyunsaturated lipid con- cies (ROS) has been attributed to protein glycation and/or glucose tent, Schwann cells and axons are particularly sensitive to oxygen auto-oxidation due to a hyperglycaemic environment. It has been when exposed free radicals impending the damage. LPx may increase suggested that during DM almost all vital organs, including brain, cell membrane rigidity and impair cell function, which lead to many are adversely affected (Nedzvetsky et  al., 2012). The neurological metabolic disorders. It has been claimed that synthetic oral hypo- consequences of DM in the central nervous system are now receiving glycaemic agents and insulin that are used in the management of greater attention. The cognitive deficits, along with morphological DM often lead to side effects (Yamamoto et al., 2001; Tolman and and neurochemical alterations, illustrate that the neurological com- Chandramouli, 2003; Weijers, 2015). Hence, the demands for safer plications of diabetes are not limited to peripheral neuropathies. and more effective agents were required to treat diabetes. Moreover, DM also contributes to cerebrovascular complications, Traditional medicinal plants have been extensively used for the reductions in cerebral blood flow, disruption of the blood-brain bar - treatment of diabetes and therein exist as a hidden wealth of poten- rier, and cerebral oedema (Prasad et  al., 2014). All of these neuro- tially useful natural products for diabetes control (Gray and Flatt, chemical and neurophysiological changes ultimately contribute to 1997). Trigonella foenum-graecum (TriFG) is commonly known as the long-term complications associated with diabetes, including fenugreek. It is an annual herb that belongs to the family Fabaceae morphological abnormalities, cognitive impairments, and increased and its active ingredient is Trigonelline. Fenugreek seeds are used vulnerability to the pathophysiological event (Sandeep et al., 2004). Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 Effect of TriFG on lipid peroxidation levels and antioxidant status in Wistar rats, 2020, Vol. 4, No. 2 85 as a traditional remedy for the treatment of diabetes (Miraldi administered orally to rats to know whether they have any influence et al., 2001; Basch et al., 2003). Supplementation of fenugreek seed on the antioxidant status in the brain tissue of diabetic rats. powder in the diet leads to a reduction in biomarkers of oxidative damage in alloxan diabetic rats (Ravikumar and Anuradha, 1999). Experimental design These fenugreek  seeds have been shown to lower blood glucose Group 1 - Normal untreated rats—Normal (N) levels and partially restore the activities of key enzymes of carbohy- Group 2 - Diabetic untreated rats—Diabetic(D) drate and lipid metabolism close to normal values in various animal Group 3 - Normal rats treated—N + TriFG with 0.25 g/kg/BW of model systems (Raju et  al., 2001). In addition to its antidiabetic TriFG properties, Trigonella also has antioxidant properties (Genet et  al., Group 4 - Diabetic rats treated—D + TriFG with 0.25 g/kg/BW of 2002). However, its antioxidant potential in the brain tissue of dia- TriFG betic condition is poorly understood. Considering the facts that (a) Group 5 - Diabetic rats treated—D + Glibenclamide with 0.2 g/kg/ DM at least in part targets pro- and /antioxidant status in brain and BW of Glibenclamide (b) TriFG is widely used to treat DM, the present study was aimed to investigate the effect of TriFG on antioxidant enzyme levels and LPx The animals were killed after the last treatment by cervical disloca- in the brain tissue of diabetic rats. tion and the brain tissues were isolated. These tissues were washed with ice-cold saline and immediately stored at −80°C for biochem- ical analysis and enzymatic assays. Materials and Methods Extraction preparation Enzymatic assays The seeds of TriFG were purchased from local market, dried in The antioxidant enzymes that include glutathione-S-transferase shade, and powdered. The powder was used for the extraction of (GST), catalase, superoxide dismutase (SOD), glutathione perox- antidiabetic principle/s using ethanol as solvent. The active prin- idase (GPx) and LPx levels were estimated with respective proced- ciples of the above-mentioned seeds were extracted into ethanol. ures as follows. GST activity in the cytosol fraction of the brain was The powdered material was soaked in water in a glass jar for 2 days assayed by using 1-chloro-2,4-dinitro benzene (at 340  nm) as de- at room temperature and the solvent was filtered. This process was scribed by Habig et al. (1974). Catalase activity was assayed by the repeated three to four times until the extract gave no coloration. The method of Chance and Maehly (1955) and SOD activity in brain extract was distilled and concentrated under reduced pressure in the homogenate was determined according to the method of Minami Buchi, rotavapor R-114 and finally freeze dried. This extract was and Yoshikawa (1979). GPx activity was measured by the method used for the investigation. described by Rotruck et al. (1973); the level of LPx in the brain was measured in terms of malondialdehyde (a product of LPx content Animals and determined by using the thiobarbituric acid reagent (TBAR). Male albino Wistar rats, aged 4  months (body weight: 180  ± The reactivity of TBAR is determined with minor modifications of 10  g) were used for the present study, procured from Indian the method adopted by Hiroshi et al. (1979). Institute of Science, Bangalore, India. All the animals were main- Brain tissue homogenates (10% W/W) were prepared in 0.25 mol/L tained under regulated laboratory conditions (12L:12D; hu- ice-cold sucrose solution. The mitochondrial and cytosol fractions midity: 76% and temperature: 28  ± 20°C) in the Department were separated by centrifugation and used for biochemical analysis. of Zoology, S.V. University, Tirupati. All the animals main- Succinate dehydrogenase (SDH) (E.C. 1.3.99.1) activity was estimated tained under regulated laboratory conditions in polypropylene by the method of Nachlas et al. (1960). Lactate dehydrogenase (LDH) cages (as per the Institutional Animal Ethical Committee, S.  V. (E.C. 1.1.1.27) activity was estimated by the method of Srikanthan University (No.01/2011–2012/(i)/a/CPCSEA/IAEC/SVU/MB SR/ and Krishnamurthy (1955). Glucose-6-phosphate dehydrogenase (G-6- Dt20/06/2011), Tirupati) for 1-week study provided the standard PDH) (E.C. 1.1.1.49) activity was assayed by the method of Bergmeyer rat pellet and water ad libitum. and Bruns (1965). Glutamate dehydrogenase (GDH) (E.C. 1.4.1.3) ac- tivity was determined by the method of Lee and Lardy (1965). The enzymatic values of antioxidative enzymes and oxidative Induction of diabetes enzymes of control and experimental values were calculated to de- Diabetes was induced in male albino Wistar rats aged 4  months termine the significance of the effect of the treatment on the experi- (body weight: 180  ± 10  g) by intraperitoneal administration of mental group of animals. ice cold aqueous alloxan monohydrate (120  mg/kg body weight). Alloxan is associated with hypoglycaemia as a result of massive Statistical analysis pancreatic insulin release, 8 h after alloxan administration the rats The mean, standard deviation (SD) were carried out according to were with 15% glucose provided for the next 24 h to prevent hypo- Steel and Torrie (1960) using basic programming techniques on SPSS glycaemia. To know the induction of diabetes by alloxan, from the for different parameters. The P-value of more than 0.01 was con- second day onwards fasting blood samples were collected by cut- sidered as not significant. ting the tails of rats and the blood glucose levels were measured by using an AccuCheck sensor comfort glucometer (Roche, Germany). After 48 h of injection of alloxan fasting blood glucose levels were Results increased to a level higher than normal. After fourth night rats with General toxicity marked hyperglycaemia (blood glucose: 250  mg/dl) were selected No clinical signs of toxicity were observed in any of the control and and used for the study. Diabetic rats were allowed free access to experimental groups. None of the animals were excluded from the tap water and the pellet diet and after selection of dose the seed present study. extract TriFG extracts (0.25  g/Kg BW) orally gavaged daily  was Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 86 P. Jangampalli Adi et al., 2020, Vol. 4, No. 2 TriFG effect on blood glucose levels decreased as compared to the normal control rats, interestingly in TriFG extract treated diabetic rats GDH activity was significantly The blood glucose levels in the control and experimental groups (P < 0.05) increased when compared to diabetic rats. Brain G-6-PDH were checked at different time intervals. The blood glucose level was activity was significantly (P < 0.05) decreased in diabetic rats when significantly increased in diabetic untreated rats. Oral administration compared to normal control. However, in TriFG extract-treated dia- of plant extract of TriFG and glibenclamide to diabetic rats signifi- betic rats, G-6-PDH activity was significantly (P  <  0.05) increased cantly reversed all these changes to near normal levels. TriFG seeds when compared with diabetic control rats. powder for diabetic animals has been shown to lower blood glucose When TriFG extract alone treated to normal control, no sig- levels and partially restore the activities of key enzymes of carbo- nificant change was observed when compared with normal control hydrate and lipid metabolism close to normal blood glucose values rats. When compared to Aloe vera extract, glibenclamide treatment (Figure 1). showed the similar results regarding restoration of biochemical, anti- oxidant enzyme changes against diabetic-induced depletion. Effect of TriFG on LPx and antioxidant status The changes in LPx and antioxidant status in the brain tissue of the control and experimental groups were given in Table 1. In alloxan- Discussion treated diabetic rats, the levels of TBARS in the brain tissue were The present findings of the present study demonstrate that an imbal- significantly increased (77.27) as compared to control rats. On the ance in the oxidative status of the nervous tissue leads to increased other hand, oral gavage of TriFG (7.272) and glibenclamide treat- generation of ROS as a consequence of perturbations in the brain ment (20.00) compared to diabetic rats significantly decreased. The tissue of alloxan-treated rats; and on the other hand, surprisingly, levels of lipid peroxidative marker in the brain tissue where the ef- oral administration of TriFG reversed the alloxan-induced effects in fect is more in the TriFG-exposed diabetic rats (−14.66). Whereas the brain tissue of rats. It is well known that under normal conditions, no significant change was observed in the LPx levels in the brain the generation of free radicals or of active species in the brain, as in of normal rats exposed to TriFG (Table  1). Alloxan treatment sig- other tissues, is maintained at extremely low levels (Chan, 1996). nificantly reduced the activity levels of antioxidant enzymes such as Earlier studies have demonstrated a defective metabolism of lipid SOD (−37.293) and catalase (−70.19), GST (−82.251) and reduced peroxides in other tissues of diabetic animal (Pari and Latha, 2004). glutathione levels (−59.647) in the brain of rats when compared to Hyperglycaemia has been shown to generate free radicals from auto- controls. Exposure of plant extracts of TriFG significantly increased oxidation of glucose, the formation of advanced glycated end prod- the levels of antioxidant enzymes such as SOD (−4.962), catalase ucts and increased polyol pathway, with a concomitant increase in (−9.294), GST (−75.541) and reduced glutathione levels (−25.433) cellular LPx and damage of membrane in diabetes. One of the conse- in the brain of rats as compared to controls. No significant changes quences of LPx degenerative processes can result in enzyme activity were observed in the brain tissue of normal rats exposed to plant changes. This increased lipid peroxides formation during diabetes extract alone. disturbs the anatomical integrity of the membrane, leading to the The effects of TriFG extract on oxidative enzymes in brain tis- inhibition of several membrane-bound enzymes. Previously, studies sues are given in Table 2. The activity levels of mitochondrial marker + + reported that the mouse cerebral enzymes pump Na /K ATPase ac- enzyme SDH were significantly (P  <  0.05) decreased in renal and tivity inhibition of by ultraviolet C (UV-C) generated OH and a hepatic tissues of diabetic rats when compared to normal rats. The peroxyl (ROO ) radical is mediated via LPx induced disturbances of SDH activity was significantly (P < 0.05) increased in TriFG extract membrane integrity (Jamme et al., 1995). The decreased activity of treated diabetic rats when compared to diabetic rats. Brain tissue + + Na /K ATPase observed in diabetic brain tissue may be due to the homogenate LDH activities were significantly (P  <  0.05) increased membrane fluidity and peroxidative damage induced by increased in diabetic rats when compared to normal control rats, in contrast lipid peroxidative status. The statuses of elevated ROS may also af- TriFG extract to diabetic rats significantly decreased (P < 0.05) was fect the membrane-linked enzyme activity through modifications of observed in LDH activity levels when compared to diabetic rats. In membrane fluidity because the activity of most membrane-bound diabetic rats brain tissue GDH activity was significantly (P < 0.05) Figure 1. Effect of Trigonella on blood glucose levels and changes observed in 4 weeks in all experimental groups: Normal (N), Diabetic (D), N + TriFG, D + TriFG, D+ TriFG groups for complete 30 days period of experiment. Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 Effect of TriFG on lipid peroxidation levels and antioxidant status in Wistar rats, 2020, Vol. 4, No. 2 87 enzymes is regulated by the physiochemical state of their membrane lipid environment. The membrane transporters that include Na / K ATPase are reported to play a sensitive and key role in changes in membrane fluidity (Sutherland et  al., 1988). Another plausible mechanism of increased LPx in the brain tissue might be due to the fact that the brain contains relatively high concentration of easily peroxidizable fatty acids (Saravanan and Pari, 2005). In addition, it is known that certain regions of the brain are highly enriched in iron and in its free state it has the ability to catalytically partici- pate in the production of ROS (Droge, 2002). Increased oxidative stress, which contributes substantially to the pathogenesis of diabetic complications, is the consequence of either enhanced ROS produc- tion or attenuated ROS scavenging capacity. Several reports have shown alterations in the antioxidant enzymes during diabetic condi- tion (Genet et al., 2002; Preet et al., 2005). In the present study, al- tered antioxidant status is a physiological adaptation in the diabetic rats. This is in agreement with the earlier published data (Mayanil et  al., 1982). The antioxidative defense system like SOD and cata- lase showed lower activities in the brain during diabetes and the results agree well with the earlier published data (El-Missiry et  al., 2004). The decreased activities of SOD and catalase may thus indi- cate improper dismutation of superoxides and response to increased production of H O , respectively which is a consequence of auto- 2 2 oxidation of excess glucose and non-enzymatic glycation of proteins (Hodgson and Fridovich, 1975; Aragno et al., 1997). The decreased activity of SOD and catalase could also be due to their decreased protein expression levels in the diabetic condition as reported re- cently in liver (Sindhu et  al., 2004). However, the activity of GPx is enhanced in diabetic rat brains. This result is consistent with the studies of Ulusu et al. (2003): the increased GPx activity represents a counter-attack mechanism to degrade H O , which is produced in 2 2 excess during the metabolism of catecholamines. In the present study, treatment with ethanolic extracts of TriFG showed significant antihyperglycemic activity in diabetic  rats. The anti-hyperglycemic activity of these seed extracts may be participating in the release of insulin from the pancreas as the blood glucose levels were significantly lower than diabetic untreated rats. It has been postulated that TriFG plant extracts release the insulin from the beta cells of the pancreas in view of the measured increase in the plasma insulin concentrations (Rizwan Siddiqui et al., 2005). Trigonella may exhibit its therapeutic effects through modulation of insulin secretion, 4-hydroxyisoleucine, and an amino acid extracted and purified from Trigonella seed displays an insulinotropic prop- erty in vitro and stimulates insulin secretion (Broca et  al., 1999). Furastanol, saponins in Trigonella seeds, increases food consump- tion and induces hypocholesterolemia in streptozotocin (STZ) dia- betic rats (Petit et  al., 1995). It has been suggested that Trigonella and Momordica charantia are known to rejuvenate the beta cells in the islets of Langerhans, thus increasing the capacity of insulin secre- tion in type 1 diabetes (Ahmed et al., 1998). The active component of TriFG seeds has been found to be associated with the defaulted part of the seeds, rich in fibre containing steroidal saponins and proteins comparable to those of soybean (Al-Habori et  al., 2001). Another possible mode of action of seed powder of Trigonella was an effect on intestinal carbohydrate digestion and was found to de- crease digestion of starch and also glucose absorption both in vivo and in vitro, elucidating a direct inhibitory effect on the digestive enzymes (Baquer et al., 2011). One of the important findings of the present study reveals that oral treatment of seed powder of Trigonella reversed alloxan- induced perturbations in the pro- and antioxidant status in the brain Table 1. Effect of plant extracts of TriFG on lipid peroxidation, activities of enzymatic and non-enzymatic antioxidants status in the brain tissue of diabetic rats. Groups TBARS (mM/100 g Catalase (μmoles of H O SOD (units A SOD/mg/ GPx (μg of GSH consumed/ GST (μmoles of CDMB-GSH/conjugate Reduced glutathione 2 2 tissue) consumed/min/mg) protein) min/mg/protein) formed/min/mg protein) (mg/100 g tissue) a a a a a a Normal (N) 1.10 ± 0.08 3.12 ± 0.21 6.65 ± 0.38 3.41 ± 0.2 4.62 ± 0.28 35.19 ± 3.31 b b b b b b Diabetic(D) 1.95 ± 0.05 (77.272) 0.93 ± 0.05 (−70.19) 4.17 ± 0.3 (−37.293) 1.01 ± 0.04 (−70.381) 0.82 ± 0.02 (−82.251) 14.2 ± 2.14 (−59.647) a c a a a a N + TriFG 1.03 ± 0.06 (−6.363) 4.00 ± 0.23 (28.205) 6.05 ± 0.28 (−9.022) 3.8 ± 0.19 (11.436) 4.9 ± 0.28 (6.060) 36.12 ± 3.21 (2.642) c d c c c c D + TriFG 1.18 ± 0.07 (7.272) 2.83 ± 0.29 (−9.294) 6.32 ± 0.2 (−4.962) 2.91 ± 0.1 (−14.662) 1.13 ± 0.12 (−75.541) 26.24 ± 2.12 (−25.433) d e d d c c D + 1.32 ± 0.09 (20.00) 1.98 ± 0.13 (−36.538) 5.32 ± 0.3 (20.00) 1.99 ± 0.12 (−41.642) 1.04 ± 0.13 (−77.489) 25.5 ± 2.10 (−27.536) Glibenclamide Values are mean ± SD of six rats in each group and values not sharing common superscript letter are significantly different at P < 0.05. Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 88 P. Jangampalli Adi et al., 2020, Vol. 4, No. 2 Table 2. Effects of TriFG extract on oxidative enzymes activities in brain tissue of control and experimental group rats. Enzyme SDH LDH GDH G-6-PDH Normal (N) 0.591 ± 0.06 0.412 ± 0.04 0.631 ± 0.06 0.534 ± 0.05 Diabetic(D) 0.362* ± 0.03 (−38.64) 0.542* ± 0.04 (32.19) 0.361* ± 0.03 (−42.85) 0.328* ± 0.03 (−38.11) N + TriFG 0.609 ± 0.06 (3.22) 0.419 ± 0.04 (2.19) 0.649 ± 0.06 (3.04) 0.548 ± 0.05 (3.39) D + TriFG 0.459** ± 0.03 (−22.20) 0.389** ± 0.03 (−5.12) 0.502** ± 0.05 (−20.31) 0.491** ± 0.05 (−7.34) D + Glibenclamide 0.448** ± 0.02 (−24.06) 0.372** ± 0.03 (−9.76) 0.482** ± 0.04 (−23.80) 0.481** ± 0.04 (−9.24) Values are mean ± SD of six individual rats in each group. Values in the parenthesis are % change from that of the control. *P < 0.01 as compared with the control group.  **P <0.01 as compared with the diabetic control group. tissue of rats. Earlier, it has been indicated that Trigonella restored 1992). In our study, administration of Trigonella extract significantly the altered activities of SOD, catalase, and GPx (Kumar et al., 2011). increased the activity of G-6-PDH in diabetic rats when compared In the present study, treatment of diabetic rats with plant extracts with the control group rats of brain oxidative status. of TriFG reduced the TBARS, a marker used to monitor LPx levels in the brain of rats. These results are consistent with the results of Conclusions Kumar et al. (2011). The mechanism, by which Trigonella exerts its effects, is still not clear; however, it is well recognized that Trigonella In diabetic rats brain  the decreased antioxidant and increased also has antioxidant properties (Genet et al., 2002). It has been re- oxidative stress observed. In TriFG diabetic treated rats antioxi- ported that the reducing power of bioactive compounds is associated dants like  SOD, CAT,GPx, GST and GSH was significantly  im- with antioxidant activity. proved.  Oxidative enzymes like SDH, GDH, and G-6-PDH as Thus, the antioxidant properties of Trigonella can be attrib- significant decrease observed in alloxon-induced diabetic rats. The re- uted to its phenolic content. Moreover, the chemical composition versed action of Trigonella by the oral administration of TriFG seeds of Trigonella indicates the presence of phenolic compounds such as extract maintained and near to normal levels  both in antioxidant tannins and flavonoids, which are known to possess antioxidative and oxidative enzymes. The levels of TBARS and hydroperoxides properties (Prieto et al., 1999). Therefore, the high phenolic and fla- were significantly increased in diabetic rats when compared to the vonoid contents in the Trigonella might be responsible for decreased normal and TriFG-treated rats. Oral administration of TriFG to LPx content in the brain tissue homogenate. Piecing these data, it can diabetic rats significantly decreased the levels of lipid peroxidative be concluded that a decrease in the intrinsic defense system induced markers and increased oxidative stress. The effects produced by generation of free radicals as evidenced by high LPx content; and on TriFG were more significant than glibenclamide. The above observa- the other hand, oral treatment of Trigonella significantly enhanced tions show that the aqueous extract of TriFG possesses antioxidant antioxidant system thereby neutralized free radical generation in the and oxidative property, which could exert a beneficial role against brain tissue of alloxan-induced diabetic rats. pathological alterations caused by the presence of free radicals in SDH is a vital enzyme of citric acid cycle that catalyses the revers- diabetes. Moreover, the molecular-level investigation and phyto- ible oxidation of succinate to fumarate. In the present study, SDH ac- chemical investigations may need to find the beneficial activities of tivity is significantly decreased in diabetic rats, and it clearly indicates plant extracts of TriFG is further needed. Our future studies to give that the energy production through aerobic oxidation is decreased in more information to find the bioactive compounds which influences diabetic rats. Similar to our studies, Shanmugam et al. (2011) studies the antioxidant and antidiabetic properties of TriFG. decreased the SDH activity in STZ-induced diabetic rats. Increased Conflict of interest statement. The authors declare that they have no com- peting interests. SDH activity in hepatic and renal tissues of Aloe vera extract-treated diabetic rats indicates that increased mitochondrial oxidative poten- tial, and utilization of carbohydrates and fats, may be due to some References compounds like flavonoids, carotenoids, and tannins present in the Abdel-Rahim, E. A. et al. (1992). The effect of sammo administration on some Aloe vera. In the present study, increased LDH activity in brain of fundamental enzymes of pentose phosphate pathway and energy metabol- diabetic rats indicates that the conversion of pyruvate to lactate in- ites of alloxanised rats. 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Trigonella foenum-graecum seeds extract plays a beneficial role on brain antioxidant and oxidative status in alloxan-induced Wistar rats

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Abstract

Key words: Trigonella foenum-graecum; hyperglycaemia; brain; antioxidants; rats. The central complications of hyperglycaemia also include the Introduction damage of neuronal circuits and the damage is more augmented due Diabetes mellitus (DM) is a major health problem all over the world to hyperglycaemic-induced oxidative stress. Oxidative stress, leading today. It is a metabolic disorder of carbohydrate, fat, and protein to an increased production of ROS, as well as LPx, is increased in metabolism characterized by the elevation of both fasting and post- diabetes and also by stress in euglycemic animals. Similarly, the oxi- prandial blood glucose levels. DM has been shown to be a state dative damage in rat brain is increased by experimentally induced of increased lipid peroxidation (LPx). LPx of cellular structures, a hyperglycaemia. Under experimental conditions, hyperglycaemia free radical-induced activity, is thought to play an important role in dramatically increases neuronal alterations and glial cell damage ageing, atherosclerosis, and late complications of DM (Klein et  al., caused by temporary ischaemia. Several lines of evidence indicated 2015). An impaired radical scavenger function has been linked to that the modified oxidative state induced by chronic hyperglycaemia the altered activity of enzymes (catalase, superoxide dismutase, and may contribute to nerve tissue damage; free radical species impair glutathione peroxidase) and non-enzymatic (glutathione) free rad- the central nervous system, attacking neurons and Schwann cells and ical scavengers. The increased production of reactive oxygen spe- the peripheral nerves. Because of its high polyunsaturated lipid con- cies (ROS) has been attributed to protein glycation and/or glucose tent, Schwann cells and axons are particularly sensitive to oxygen auto-oxidation due to a hyperglycaemic environment. It has been when exposed free radicals impending the damage. LPx may increase suggested that during DM almost all vital organs, including brain, cell membrane rigidity and impair cell function, which lead to many are adversely affected (Nedzvetsky et  al., 2012). The neurological metabolic disorders. It has been claimed that synthetic oral hypo- consequences of DM in the central nervous system are now receiving glycaemic agents and insulin that are used in the management of greater attention. The cognitive deficits, along with morphological DM often lead to side effects (Yamamoto et al., 2001; Tolman and and neurochemical alterations, illustrate that the neurological com- Chandramouli, 2003; Weijers, 2015). Hence, the demands for safer plications of diabetes are not limited to peripheral neuropathies. and more effective agents were required to treat diabetes. Moreover, DM also contributes to cerebrovascular complications, Traditional medicinal plants have been extensively used for the reductions in cerebral blood flow, disruption of the blood-brain bar - treatment of diabetes and therein exist as a hidden wealth of poten- rier, and cerebral oedema (Prasad et  al., 2014). All of these neuro- tially useful natural products for diabetes control (Gray and Flatt, chemical and neurophysiological changes ultimately contribute to 1997). Trigonella foenum-graecum (TriFG) is commonly known as the long-term complications associated with diabetes, including fenugreek. It is an annual herb that belongs to the family Fabaceae morphological abnormalities, cognitive impairments, and increased and its active ingredient is Trigonelline. Fenugreek seeds are used vulnerability to the pathophysiological event (Sandeep et al., 2004). Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 Effect of TriFG on lipid peroxidation levels and antioxidant status in Wistar rats, 2020, Vol. 4, No. 2 85 as a traditional remedy for the treatment of diabetes (Miraldi administered orally to rats to know whether they have any influence et al., 2001; Basch et al., 2003). Supplementation of fenugreek seed on the antioxidant status in the brain tissue of diabetic rats. powder in the diet leads to a reduction in biomarkers of oxidative damage in alloxan diabetic rats (Ravikumar and Anuradha, 1999). Experimental design These fenugreek  seeds have been shown to lower blood glucose Group 1 - Normal untreated rats—Normal (N) levels and partially restore the activities of key enzymes of carbohy- Group 2 - Diabetic untreated rats—Diabetic(D) drate and lipid metabolism close to normal values in various animal Group 3 - Normal rats treated—N + TriFG with 0.25 g/kg/BW of model systems (Raju et  al., 2001). In addition to its antidiabetic TriFG properties, Trigonella also has antioxidant properties (Genet et  al., Group 4 - Diabetic rats treated—D + TriFG with 0.25 g/kg/BW of 2002). However, its antioxidant potential in the brain tissue of dia- TriFG betic condition is poorly understood. Considering the facts that (a) Group 5 - Diabetic rats treated—D + Glibenclamide with 0.2 g/kg/ DM at least in part targets pro- and /antioxidant status in brain and BW of Glibenclamide (b) TriFG is widely used to treat DM, the present study was aimed to investigate the effect of TriFG on antioxidant enzyme levels and LPx The animals were killed after the last treatment by cervical disloca- in the brain tissue of diabetic rats. tion and the brain tissues were isolated. These tissues were washed with ice-cold saline and immediately stored at −80°C for biochem- ical analysis and enzymatic assays. Materials and Methods Extraction preparation Enzymatic assays The seeds of TriFG were purchased from local market, dried in The antioxidant enzymes that include glutathione-S-transferase shade, and powdered. The powder was used for the extraction of (GST), catalase, superoxide dismutase (SOD), glutathione perox- antidiabetic principle/s using ethanol as solvent. The active prin- idase (GPx) and LPx levels were estimated with respective proced- ciples of the above-mentioned seeds were extracted into ethanol. ures as follows. GST activity in the cytosol fraction of the brain was The powdered material was soaked in water in a glass jar for 2 days assayed by using 1-chloro-2,4-dinitro benzene (at 340  nm) as de- at room temperature and the solvent was filtered. This process was scribed by Habig et al. (1974). Catalase activity was assayed by the repeated three to four times until the extract gave no coloration. The method of Chance and Maehly (1955) and SOD activity in brain extract was distilled and concentrated under reduced pressure in the homogenate was determined according to the method of Minami Buchi, rotavapor R-114 and finally freeze dried. This extract was and Yoshikawa (1979). GPx activity was measured by the method used for the investigation. described by Rotruck et al. (1973); the level of LPx in the brain was measured in terms of malondialdehyde (a product of LPx content Animals and determined by using the thiobarbituric acid reagent (TBAR). Male albino Wistar rats, aged 4  months (body weight: 180  ± The reactivity of TBAR is determined with minor modifications of 10  g) were used for the present study, procured from Indian the method adopted by Hiroshi et al. (1979). Institute of Science, Bangalore, India. All the animals were main- Brain tissue homogenates (10% W/W) were prepared in 0.25 mol/L tained under regulated laboratory conditions (12L:12D; hu- ice-cold sucrose solution. The mitochondrial and cytosol fractions midity: 76% and temperature: 28  ± 20°C) in the Department were separated by centrifugation and used for biochemical analysis. of Zoology, S.V. University, Tirupati. All the animals main- Succinate dehydrogenase (SDH) (E.C. 1.3.99.1) activity was estimated tained under regulated laboratory conditions in polypropylene by the method of Nachlas et al. (1960). Lactate dehydrogenase (LDH) cages (as per the Institutional Animal Ethical Committee, S.  V. (E.C. 1.1.1.27) activity was estimated by the method of Srikanthan University (No.01/2011–2012/(i)/a/CPCSEA/IAEC/SVU/MB SR/ and Krishnamurthy (1955). Glucose-6-phosphate dehydrogenase (G-6- Dt20/06/2011), Tirupati) for 1-week study provided the standard PDH) (E.C. 1.1.1.49) activity was assayed by the method of Bergmeyer rat pellet and water ad libitum. and Bruns (1965). Glutamate dehydrogenase (GDH) (E.C. 1.4.1.3) ac- tivity was determined by the method of Lee and Lardy (1965). The enzymatic values of antioxidative enzymes and oxidative Induction of diabetes enzymes of control and experimental values were calculated to de- Diabetes was induced in male albino Wistar rats aged 4  months termine the significance of the effect of the treatment on the experi- (body weight: 180  ± 10  g) by intraperitoneal administration of mental group of animals. ice cold aqueous alloxan monohydrate (120  mg/kg body weight). Alloxan is associated with hypoglycaemia as a result of massive Statistical analysis pancreatic insulin release, 8 h after alloxan administration the rats The mean, standard deviation (SD) were carried out according to were with 15% glucose provided for the next 24 h to prevent hypo- Steel and Torrie (1960) using basic programming techniques on SPSS glycaemia. To know the induction of diabetes by alloxan, from the for different parameters. The P-value of more than 0.01 was con- second day onwards fasting blood samples were collected by cut- sidered as not significant. ting the tails of rats and the blood glucose levels were measured by using an AccuCheck sensor comfort glucometer (Roche, Germany). After 48 h of injection of alloxan fasting blood glucose levels were Results increased to a level higher than normal. After fourth night rats with General toxicity marked hyperglycaemia (blood glucose: 250  mg/dl) were selected No clinical signs of toxicity were observed in any of the control and and used for the study. Diabetic rats were allowed free access to experimental groups. None of the animals were excluded from the tap water and the pellet diet and after selection of dose the seed present study. extract TriFG extracts (0.25  g/Kg BW) orally gavaged daily  was Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 86 P. Jangampalli Adi et al., 2020, Vol. 4, No. 2 TriFG effect on blood glucose levels decreased as compared to the normal control rats, interestingly in TriFG extract treated diabetic rats GDH activity was significantly The blood glucose levels in the control and experimental groups (P < 0.05) increased when compared to diabetic rats. Brain G-6-PDH were checked at different time intervals. The blood glucose level was activity was significantly (P < 0.05) decreased in diabetic rats when significantly increased in diabetic untreated rats. Oral administration compared to normal control. However, in TriFG extract-treated dia- of plant extract of TriFG and glibenclamide to diabetic rats signifi- betic rats, G-6-PDH activity was significantly (P  <  0.05) increased cantly reversed all these changes to near normal levels. TriFG seeds when compared with diabetic control rats. powder for diabetic animals has been shown to lower blood glucose When TriFG extract alone treated to normal control, no sig- levels and partially restore the activities of key enzymes of carbo- nificant change was observed when compared with normal control hydrate and lipid metabolism close to normal blood glucose values rats. When compared to Aloe vera extract, glibenclamide treatment (Figure 1). showed the similar results regarding restoration of biochemical, anti- oxidant enzyme changes against diabetic-induced depletion. Effect of TriFG on LPx and antioxidant status The changes in LPx and antioxidant status in the brain tissue of the control and experimental groups were given in Table 1. In alloxan- Discussion treated diabetic rats, the levels of TBARS in the brain tissue were The present findings of the present study demonstrate that an imbal- significantly increased (77.27) as compared to control rats. On the ance in the oxidative status of the nervous tissue leads to increased other hand, oral gavage of TriFG (7.272) and glibenclamide treat- generation of ROS as a consequence of perturbations in the brain ment (20.00) compared to diabetic rats significantly decreased. The tissue of alloxan-treated rats; and on the other hand, surprisingly, levels of lipid peroxidative marker in the brain tissue where the ef- oral administration of TriFG reversed the alloxan-induced effects in fect is more in the TriFG-exposed diabetic rats (−14.66). Whereas the brain tissue of rats. It is well known that under normal conditions, no significant change was observed in the LPx levels in the brain the generation of free radicals or of active species in the brain, as in of normal rats exposed to TriFG (Table  1). Alloxan treatment sig- other tissues, is maintained at extremely low levels (Chan, 1996). nificantly reduced the activity levels of antioxidant enzymes such as Earlier studies have demonstrated a defective metabolism of lipid SOD (−37.293) and catalase (−70.19), GST (−82.251) and reduced peroxides in other tissues of diabetic animal (Pari and Latha, 2004). glutathione levels (−59.647) in the brain of rats when compared to Hyperglycaemia has been shown to generate free radicals from auto- controls. Exposure of plant extracts of TriFG significantly increased oxidation of glucose, the formation of advanced glycated end prod- the levels of antioxidant enzymes such as SOD (−4.962), catalase ucts and increased polyol pathway, with a concomitant increase in (−9.294), GST (−75.541) and reduced glutathione levels (−25.433) cellular LPx and damage of membrane in diabetes. One of the conse- in the brain of rats as compared to controls. No significant changes quences of LPx degenerative processes can result in enzyme activity were observed in the brain tissue of normal rats exposed to plant changes. This increased lipid peroxides formation during diabetes extract alone. disturbs the anatomical integrity of the membrane, leading to the The effects of TriFG extract on oxidative enzymes in brain tis- inhibition of several membrane-bound enzymes. Previously, studies sues are given in Table 2. The activity levels of mitochondrial marker + + reported that the mouse cerebral enzymes pump Na /K ATPase ac- enzyme SDH were significantly (P  <  0.05) decreased in renal and tivity inhibition of by ultraviolet C (UV-C) generated OH and a hepatic tissues of diabetic rats when compared to normal rats. The peroxyl (ROO ) radical is mediated via LPx induced disturbances of SDH activity was significantly (P < 0.05) increased in TriFG extract membrane integrity (Jamme et al., 1995). The decreased activity of treated diabetic rats when compared to diabetic rats. Brain tissue + + Na /K ATPase observed in diabetic brain tissue may be due to the homogenate LDH activities were significantly (P  <  0.05) increased membrane fluidity and peroxidative damage induced by increased in diabetic rats when compared to normal control rats, in contrast lipid peroxidative status. The statuses of elevated ROS may also af- TriFG extract to diabetic rats significantly decreased (P < 0.05) was fect the membrane-linked enzyme activity through modifications of observed in LDH activity levels when compared to diabetic rats. In membrane fluidity because the activity of most membrane-bound diabetic rats brain tissue GDH activity was significantly (P < 0.05) Figure 1. Effect of Trigonella on blood glucose levels and changes observed in 4 weeks in all experimental groups: Normal (N), Diabetic (D), N + TriFG, D + TriFG, D+ TriFG groups for complete 30 days period of experiment. Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 Effect of TriFG on lipid peroxidation levels and antioxidant status in Wistar rats, 2020, Vol. 4, No. 2 87 enzymes is regulated by the physiochemical state of their membrane lipid environment. The membrane transporters that include Na / K ATPase are reported to play a sensitive and key role in changes in membrane fluidity (Sutherland et  al., 1988). Another plausible mechanism of increased LPx in the brain tissue might be due to the fact that the brain contains relatively high concentration of easily peroxidizable fatty acids (Saravanan and Pari, 2005). In addition, it is known that certain regions of the brain are highly enriched in iron and in its free state it has the ability to catalytically partici- pate in the production of ROS (Droge, 2002). Increased oxidative stress, which contributes substantially to the pathogenesis of diabetic complications, is the consequence of either enhanced ROS produc- tion or attenuated ROS scavenging capacity. Several reports have shown alterations in the antioxidant enzymes during diabetic condi- tion (Genet et al., 2002; Preet et al., 2005). In the present study, al- tered antioxidant status is a physiological adaptation in the diabetic rats. This is in agreement with the earlier published data (Mayanil et  al., 1982). The antioxidative defense system like SOD and cata- lase showed lower activities in the brain during diabetes and the results agree well with the earlier published data (El-Missiry et  al., 2004). The decreased activities of SOD and catalase may thus indi- cate improper dismutation of superoxides and response to increased production of H O , respectively which is a consequence of auto- 2 2 oxidation of excess glucose and non-enzymatic glycation of proteins (Hodgson and Fridovich, 1975; Aragno et al., 1997). The decreased activity of SOD and catalase could also be due to their decreased protein expression levels in the diabetic condition as reported re- cently in liver (Sindhu et  al., 2004). However, the activity of GPx is enhanced in diabetic rat brains. This result is consistent with the studies of Ulusu et al. (2003): the increased GPx activity represents a counter-attack mechanism to degrade H O , which is produced in 2 2 excess during the metabolism of catecholamines. In the present study, treatment with ethanolic extracts of TriFG showed significant antihyperglycemic activity in diabetic  rats. The anti-hyperglycemic activity of these seed extracts may be participating in the release of insulin from the pancreas as the blood glucose levels were significantly lower than diabetic untreated rats. It has been postulated that TriFG plant extracts release the insulin from the beta cells of the pancreas in view of the measured increase in the plasma insulin concentrations (Rizwan Siddiqui et al., 2005). Trigonella may exhibit its therapeutic effects through modulation of insulin secretion, 4-hydroxyisoleucine, and an amino acid extracted and purified from Trigonella seed displays an insulinotropic prop- erty in vitro and stimulates insulin secretion (Broca et  al., 1999). Furastanol, saponins in Trigonella seeds, increases food consump- tion and induces hypocholesterolemia in streptozotocin (STZ) dia- betic rats (Petit et  al., 1995). It has been suggested that Trigonella and Momordica charantia are known to rejuvenate the beta cells in the islets of Langerhans, thus increasing the capacity of insulin secre- tion in type 1 diabetes (Ahmed et al., 1998). The active component of TriFG seeds has been found to be associated with the defaulted part of the seeds, rich in fibre containing steroidal saponins and proteins comparable to those of soybean (Al-Habori et  al., 2001). Another possible mode of action of seed powder of Trigonella was an effect on intestinal carbohydrate digestion and was found to de- crease digestion of starch and also glucose absorption both in vivo and in vitro, elucidating a direct inhibitory effect on the digestive enzymes (Baquer et al., 2011). One of the important findings of the present study reveals that oral treatment of seed powder of Trigonella reversed alloxan- induced perturbations in the pro- and antioxidant status in the brain Table 1. Effect of plant extracts of TriFG on lipid peroxidation, activities of enzymatic and non-enzymatic antioxidants status in the brain tissue of diabetic rats. Groups TBARS (mM/100 g Catalase (μmoles of H O SOD (units A SOD/mg/ GPx (μg of GSH consumed/ GST (μmoles of CDMB-GSH/conjugate Reduced glutathione 2 2 tissue) consumed/min/mg) protein) min/mg/protein) formed/min/mg protein) (mg/100 g tissue) a a a a a a Normal (N) 1.10 ± 0.08 3.12 ± 0.21 6.65 ± 0.38 3.41 ± 0.2 4.62 ± 0.28 35.19 ± 3.31 b b b b b b Diabetic(D) 1.95 ± 0.05 (77.272) 0.93 ± 0.05 (−70.19) 4.17 ± 0.3 (−37.293) 1.01 ± 0.04 (−70.381) 0.82 ± 0.02 (−82.251) 14.2 ± 2.14 (−59.647) a c a a a a N + TriFG 1.03 ± 0.06 (−6.363) 4.00 ± 0.23 (28.205) 6.05 ± 0.28 (−9.022) 3.8 ± 0.19 (11.436) 4.9 ± 0.28 (6.060) 36.12 ± 3.21 (2.642) c d c c c c D + TriFG 1.18 ± 0.07 (7.272) 2.83 ± 0.29 (−9.294) 6.32 ± 0.2 (−4.962) 2.91 ± 0.1 (−14.662) 1.13 ± 0.12 (−75.541) 26.24 ± 2.12 (−25.433) d e d d c c D + 1.32 ± 0.09 (20.00) 1.98 ± 0.13 (−36.538) 5.32 ± 0.3 (20.00) 1.99 ± 0.12 (−41.642) 1.04 ± 0.13 (−77.489) 25.5 ± 2.10 (−27.536) Glibenclamide Values are mean ± SD of six rats in each group and values not sharing common superscript letter are significantly different at P < 0.05. Downloaded from https://academic.oup.com/fqs/article/4/2/83/5834589 by DeepDyve user on 27 August 2020 88 P. Jangampalli Adi et al., 2020, Vol. 4, No. 2 Table 2. Effects of TriFG extract on oxidative enzymes activities in brain tissue of control and experimental group rats. Enzyme SDH LDH GDH G-6-PDH Normal (N) 0.591 ± 0.06 0.412 ± 0.04 0.631 ± 0.06 0.534 ± 0.05 Diabetic(D) 0.362* ± 0.03 (−38.64) 0.542* ± 0.04 (32.19) 0.361* ± 0.03 (−42.85) 0.328* ± 0.03 (−38.11) N + TriFG 0.609 ± 0.06 (3.22) 0.419 ± 0.04 (2.19) 0.649 ± 0.06 (3.04) 0.548 ± 0.05 (3.39) D + TriFG 0.459** ± 0.03 (−22.20) 0.389** ± 0.03 (−5.12) 0.502** ± 0.05 (−20.31) 0.491** ± 0.05 (−7.34) D + Glibenclamide 0.448** ± 0.02 (−24.06) 0.372** ± 0.03 (−9.76) 0.482** ± 0.04 (−23.80) 0.481** ± 0.04 (−9.24) Values are mean ± SD of six individual rats in each group. Values in the parenthesis are % change from that of the control. *P < 0.01 as compared with the control group.  **P <0.01 as compared with the diabetic control group. tissue of rats. Earlier, it has been indicated that Trigonella restored 1992). In our study, administration of Trigonella extract significantly the altered activities of SOD, catalase, and GPx (Kumar et al., 2011). increased the activity of G-6-PDH in diabetic rats when compared In the present study, treatment of diabetic rats with plant extracts with the control group rats of brain oxidative status. of TriFG reduced the TBARS, a marker used to monitor LPx levels in the brain of rats. These results are consistent with the results of Conclusions Kumar et al. (2011). The mechanism, by which Trigonella exerts its effects, is still not clear; however, it is well recognized that Trigonella In diabetic rats brain  the decreased antioxidant and increased also has antioxidant properties (Genet et al., 2002). It has been re- oxidative stress observed. In TriFG diabetic treated rats antioxi- ported that the reducing power of bioactive compounds is associated dants like  SOD, CAT,GPx, GST and GSH was significantly  im- with antioxidant activity. proved.  Oxidative enzymes like SDH, GDH, and G-6-PDH as Thus, the antioxidant properties of Trigonella can be attrib- significant decrease observed in alloxon-induced diabetic rats. The re- uted to its phenolic content. Moreover, the chemical composition versed action of Trigonella by the oral administration of TriFG seeds of Trigonella indicates the presence of phenolic compounds such as extract maintained and near to normal levels  both in antioxidant tannins and flavonoids, which are known to possess antioxidative and oxidative enzymes. The levels of TBARS and hydroperoxides properties (Prieto et al., 1999). Therefore, the high phenolic and fla- were significantly increased in diabetic rats when compared to the vonoid contents in the Trigonella might be responsible for decreased normal and TriFG-treated rats. Oral administration of TriFG to LPx content in the brain tissue homogenate. Piecing these data, it can diabetic rats significantly decreased the levels of lipid peroxidative be concluded that a decrease in the intrinsic defense system induced markers and increased oxidative stress. The effects produced by generation of free radicals as evidenced by high LPx content; and on TriFG were more significant than glibenclamide. The above observa- the other hand, oral treatment of Trigonella significantly enhanced tions show that the aqueous extract of TriFG possesses antioxidant antioxidant system thereby neutralized free radical generation in the and oxidative property, which could exert a beneficial role against brain tissue of alloxan-induced diabetic rats. pathological alterations caused by the presence of free radicals in SDH is a vital enzyme of citric acid cycle that catalyses the revers- diabetes. Moreover, the molecular-level investigation and phyto- ible oxidation of succinate to fumarate. In the present study, SDH ac- chemical investigations may need to find the beneficial activities of tivity is significantly decreased in diabetic rats, and it clearly indicates plant extracts of TriFG is further needed. Our future studies to give that the energy production through aerobic oxidation is decreased in more information to find the bioactive compounds which influences diabetic rats. 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