Inhibitory Activity of Unripe Fruits of Linseed on Aldose Reductase and Production of advanced glycation end Products

INTRODUCTION

Diabetes mellitus is associated with insulin resistance or insulin deficiency and altered metabolism of carbohydrates, lipids and proteins. People with diabetes are more likely to be af-fected with serious complications including heart attacks, blindness, kidney failure and neuropathy.¹ The prevalence of diabetes across the world was approximately 285 million in 2010 and can become a major epidemic as 7.7% of the population is expected to be affected by 2030.²

Several mechanisms have been proposed but the involvement of hyperglycemia-activated polyol pathway is particularly important in the origin of diabetic complications.¹ The transformation of glucose into sorbitol is because of the catalytic activity of aldose reductase (AR). Increased accumulation of sorbitol inside the cells causes osmotic damage and is associated with microvascular complications. Sorbitol undergoes further metabolism to form fruc-tose. Accumulation of fructose ultimately results in the increased synthesis of advanced gly-cation end products (AGE). Such modified proteins when bound to AGE receptors produce cytokines and growth factors which cause vascular complications.³

As synthetic drugs possess multiple adverse effects, herbal drugs are increasingly used to control diabetes and its complications in recent years^1,4. Linum usitatissimumL. which belongs to the family Linaceae, is a widely used plant food. Linseed or flaxseed, the more common names for the selected plant, has been reported to be beneficial in the treatment of diabetes, cardiac disease and oxidative stress.^5-8 Petroleum ether and hydro-alcoholic extract of L. usitatissimum seeds was reported to be effective in the treatment of STZ-nicotinamide in-duced diabetic nephropathy.⁹ As literature indicated no previous reports, unripe fruits of L. usitatissimum were chosen to study the AR inhibitory potential by in vitro and in vivo methods.

MATERIALS AND METHODS

Plant Material

Unripe fruits of Linum usitatissimum L. (LU) (Fam: Linaceae) were collected from the fields in and around Vijayapura. The collected plant material was identified by Dr. M. B. Moolimani, Professor and Head, Department of Botany, BLDEAs S.B. Arts K.C.P Science College, Vijayapura, Karnataka.

Extraction and Fractionation

The extraction and fractionation were done according to Telagari and Hullatti with minor modifications.¹⁰ Dried powdered plant material (500 g) was first treated with 70% v/v ethanol for 24 h to extract heat sensitive constituents if any. The extract was collected and the marc was further extracted with ethanol (95% v/v) using a soxhlet extractor. Extracts from both procedures were mixed and evaporated at 40°C under reduced pressure (Rotary evaporator, IKA RV 10).

The alcoholic extract (50 g) was dispersed in citric acid (5% w/v), treated with dichloromethane to get aqueous alcohol layer and dichloromethane layer. Aqueous alcohol layer was subjected to concentration under reduced pressure to half of the original volume and pH was increased to 9.0 using 10% ammonium hydroxide. This was washed with dichloromethane to get dichloromethane fraction (LU1; 3.2 g) and aqueous fraction (LU2; 18.8 g). Dichloromethane layer was similarly concentrated to one-third of the original volume. This was further treated with 90% v/v methanol and petroleum ether (1:1) to get methanolic fraction (LU3; 16.8 g) and petroleum ether fraction (LU4; 6.2 g).

Animals

Wistar albino rats were maintained according to standard guidelines in the laboratory. Approval from the Institutional Animal Ethics Committee (IAEC No: BLDE/BPC/157A/2014-15) was taken before conducting studies.

In vitroinhibitory studies on aldose reductase (AR)

Homogenization of rat lens

Male Wistar albino rats, around 150 g in weight, were selected and were subjected to physical euthanasia by damaging spinal nerve. The eye balls were collected and enucleation of the lenses was done through the posterior part. Lenses were disintegrated with 0.1 M sodium phosphate buffer, pH 6.2 (Mikro 220R, Hettich, Germany). The supernatant which was col-lected after spinning for 30 min at 16,000 rpm at 4 °C served as crude enzyme. The enzyme was characterized with respect to activity and specific activity.¹¹

In vitro AR inhibitory activity of extracts/fractions

Spectrophotometric method was used to assay AR Inhibitory activity.¹¹ The test extracts, dissolved in 10% DMSO were used at concentrations of 10, 50 and 100 µg/ml and quercetin was the standard. Crude enzyme preparation, 0.15 mM NADPH, double distilled water and DMSO were added 300 µl each to make up the blank reaction mixture. Sodium phosphate buffer (pH 6.2) was added to make the volume to 3 ml and absorbance was recorded at 340 nm (SL 210, Elico, India). 300 µl of 10 mM DL-glyceraldehyde replaced distilled water in the control reaction mixture. The reaction mixture for testing plant extracts and fractions con-sisted of the same ingredients of the control and 300 µl of sample. Initiation of the reactions was done by adding the substrate and absorbance was recorded for 1 min at 5 sec intervals. For all concentrations, triplicate readings were taken and the percentage of

inhibition was calculated using the formula (∆A sample/min is the reduction of the absor-bance for a min; ∆A blank/min is the reduction of the absorbance for a min with blank; ∆A control/min is the reduction of the absorbance for a min with control),

Inhibition of Advanced glycation end products (AGE) formation

AGE Inhibitory potential was assessed by mixing 0.2 M fructose and 0.2 M glucose solutions with bovine serum albumin (10 mg/ml).¹² The protein solution was prepared in 50 mM sodium phosphate buffer of pH 7.4 and contained 0.02% sodium benzoate for preventing microbial growth. Different concentrations of the plant material (1.25 ml; prepared in 10% DMSO) was added to the reaction mixture (2.75 ml). Incubation of the samples was done at 370 C for 7 days. The fluorescence intensity was recorded at 350 nm (excitation wavelength) and 450 nm (emission wavelength) (CL-53, Elico fluorimeter). The standard for these studies was aminoguanidine and AGE inhibitory activity was assessed using the formula,

In vivoinhibitory studies on aldose reductase (AR)

In vivogalactosemic animal model

6-week-old male Wistar albino rats weighing 180–200 g were utilized for in vivo studies. 3 groups were made with each consisting of six animals. Test sample (ZJ3 fraction) and the standard (quercetin) were given to two groups of rats. The remaining two groups served as control. Test solutions were prepared in distilled water and were administered for 14 days. Galactose, ZJ3 and quercetin were given at a dose of 10 mg/kg body weight to all the groups. All the rats were sacrificed on the 15th day and lenses collected as described above. Lenses were homogenized with 1 ml of ice cold water and precipitation of the proteins was done with ethanol.¹³ Centrifugation was done for 30 min at 40 C at 16,000 rpm and lyophilized at -400 C (Lyodel freeze-drier, Delvac Pumps Pvt. Ltd, Chennai, Tamil Nadu, India).

Determination of lens galactitol levels by GLC

The sample was treated with 1 ml of Trisil HTP reagent at 60°C for 10 min. Then, the cooled samples were subjected analysis by gas liquid chromatography. The carrier gas was nitrogen and temperature of the column (GL Science GC 353; Supelco DB-1 capillary column: 30 x 0.25 mm x 0.25 μM coated with cross linked methyl silicone) was increased at 5ºC/min from 120 to 265 ºC and then to 295ºC.¹³ The internal standard was Methylα-D-mannopyranoside.

Statistical analysis

One-way analysis of variance was used find out the significance of difference between the groups. All the results were recorded in triplicate and represented as mean±SD.

RESULTS

In the present study, hydro-alcoholic extract of unripe fruits of L. usitatissimum L. (LU) was subjected to evaluation of inhibitory activity on rat lens aldose reductase (AR) and advanced glycation end product (AGE) formation. The extract exhibited 24.36, 55.5 and 85.4 percentage of AR inhibitory activity (Fig. 1). Quercetin was used as a standard, which showed 25, 57.33 and 85 percentage of inhibition at 1, 5 and 10 µg/ml, respectively. LU showed 28.8, 61.9 and 88.22 percentage of AGE inhibitory activity (Fig. 1). Aminoguanidine was used as a standard, which showed 15.24, 47.8 and 98.9 percentage of inhibition at 1, 5 and 10 µg/m, respectively.

Table No.1 shows IC50 of LU for inhibitory activities on AR and advanced glycation end product formation. LU showed significant inhibitory activity with an IC50 of 45.79 µg/ml and 38.6 µg/ ml for AR and AGE, respectively. Therefore, LU was fractionated and evaluated for inhibitory activities. The fractions at 50 µg/ml exhibited 48.4 (LU1), 52.4 (LU2), 60.25 (LU3) and 55.2 (LU4) AR inhibitory activity (Fig. 2). The fractions at 50 µg/ ml exhibited 34.7 (LU1), 44.35 (LU2), 92.5 (LU3) and 87.9 (LU4) AGE inhibitory activity (Fig. 3). Among all, LU3 showed best inhibitory activity with an IC50 of 42.66 µg/ml and 31.72 µg/ml for AR and AGE, respectively (Table No. 2). Therefore, LU3 was evaluated for in vivo AR inhibitory activity.

After derivatization, the rat lens homogenates of test, standard and control subjected to analysis by GLC. 22.11 min and 6.41 min were the retention times of galactitol and methyl-α-Dmannopyranoside, respectively. Fig. 4 shows the comparison of galactitol concentration of test group and the control. When compared to control, galactitol levels in the lens were sig-nificantly reduced by the standard, quercetin and LU3 (p<0.05).

DISCUSSION

Diabetes mellitus disorders are defined by elevated blood glucose levels because of insufficient insulin, inefficient insulin or both. Serious complications can potentially occur in both type 1 and type 2 diabetics because of the insidious and chronic nature of hyperglycemia. Neuropathy, nephropathy, retinopathy, and cataracts are the microvascular complications of diabetes. The defects of cardiovascular and cerebrovascular systems which can ultimately cause myocardial infarction and strokes respectively are the macrovascular complications of diabetes. Polyol pathway seems to be particularly prominent in the pathogenesis diabetic complications^1,3. Hyperglycemia activates AR resulting in the synthesis of sorbitol from glucose. Reduction of fructose by sorbitol dehydrogenase leads to the synthesis of sorbitol. Hence, inhibition of AR has long been viewed as significant in overcoming diabetic complications. As there were no reports, the present work of investigating the potential of unripe fruits of L. usitatissimum L. (LU) in the inhibition of rat lens aldose reductase by in vitro and in vivo methods was taken up.

Characterization studies of the crude enzyme were done and protein concentration (2 mg/ml), enzyme activity (14.11 U/ml) and specific activity (7.06 U/mg) were calculated. As shown in the Table No. 1, LU exhibited significant inhibitory activity with an IC50 of 45.79 µg/ml and 38.6 µg/ml for AR and AGE, respectively. The increased production of an organic osmolyte, sorbitol through the polyol pathway and the associated exhaustion of NADPH cell stores in-creases the susceptibility of cells by highly damaging reactive oxygen species.¹⁴ Accumulation of fructose is highly conducive for initiation for glycation reactions. Such glycated proteins activate AGE receptors and lead to the development of vascular complications.³ Literature reveals the potential beneficial effects of several plant extracts and their constituents in alleviating diabetic complications through AR inhibitory activity.^1,4 The phytochemical in-vestigation indicated the presence of alkaloids, steroids, triterpenoids, cyanogenic glycosides, tannins/phenolic compounds and flavonoids in the hydro-alcoholic extract of LU. After fur-ther processing of the hydro-alcoholic extract, the fractions were studied for AR and AGE inhibitory activities. LU3 showed the highest inhibitory potential with an IC50 of 39.81 µg/ml and 22.42 µg/ ml for AR and AGE, respectively (Table No. 2) and therefore, it was studied for in vivo AR inhibitory activity.

Severe cataract is more easily produced in a short duration of time by galactosemia than by hyperglycemia.¹³ AR exhibits higher affinity for galactose than for glucose and it is more difficult to further metabolize galactitol. Hence, galactosemic cataract has been commonly used as a model to investigate the mechanism or drugs on diabetic complications. In the present study, LU3 significantly decreased the galactitol levels in the lens in glactose fed rat model (Fig. 4). Phenolic compounds and triterpenoids do possess AR inhibitory, antioxidant and antiglycation properties.^1,4,15-17 Alkaloids and flavonoids were detected LU1 and LU2 fractions, respectively. LU3 was found to contain phenolic compounds and triterpenoids whereas LU4 gave positive results for steroids. Ghule et al. reported the significant influence of linseed in neutralizing reactive oxygen species formation and stimulating the synthesis of antioxidant enzymes of pancreas in alloxan induced diabetic rat¹⁸. Dusane and Joshi evaluated the efficiency of L. usitassimum active fraction in enhancing glucose utilization and synthesis of glycogen in liver and muscle tissues.¹⁹ The active fraction was found to help in the forma-tion of new cells of Islets, thereby stimulating the functional role of pancreas. Tupe et al. studied the antiglycation property of common food plants and flaxseed was found to show strong inhibitory activity towards generation of advanced glycation end products.²⁰ The secondary metabolites like phenolic compounds and triterpenoids, which were detected in the fraction, could be responsible the observed activity.

CONCLUSION

Diabetes mellitus affects millions of people worldwide. There is a renewed interest among scientific community to prevent and treat diabetic complications. The well known plant food, unripe fruits of L. usitatissimumL., was found to be significantly effective in lowering rat lens aldose reductase activity and formation of advanced glycation end products. The present work correlates well with earlier reports and indicates the potential of common plant foods for treating diabetic complications.

CONFLICT OF INTEREST

The authors declare no conflict of interest.