Inhibitory activities of some Folklore remedies on Aldose reductase of rat lens and generation of advanced glycation end products

 

Gaviraj EN^1*, Ramarao A², Veeresham C², Shivakumar B¹, Kalyane NV¹, Biradar SM¹

¹BLDEAs SSM College of Pharmacy and Research Centre, BLDE (Deemed to be University) Campus, Vijayapura-586103, Karnataka, India

²University College of Pharmaceutical Sciences, Kakatiya University, Warangal-506009, Telangana, India

 

ABSTRACT:

Diabetes mellitus is a group of metabolic disorders defined by elevated blood glucose levels because of insufficient insulin, inefficient insulin or both and serious complications can potentially occur in both type 1 and type 2 diabetics. Plants are known to inhibit polyol pathway and they can be a potential source of unique chemicals for drug development of inhibitors of aldose reductase (AR) and formation of advanced glycation end products (AGE).   In this context, some folklore remedies for diabetes viz. roots of Hibiscus arnottianus L. (HA), seeds of Ziziphus jujuba Mill. (ZJ), seeds of Strychnos potatorum L. (SP) and roots of Erythrina variegata L were selected.  The aqueous alcoholic extracts at 100 µg/ml exhibited 75.6 (HA), 83.63 (ZJ), 75.97 (SP) and 63.8 (EV) percentage of inhibitory activity on AR and 26.5 (HA), 79.3 (ZJ), 20.8 (SP) and 22.2 (EV) percentage of inhibitory activity on AGE.  ZJ showed best inhibitory activity with an IC₅₀ of 49.27 µg/ml and 60.63 µg/ml for AR and AGE, respectively. The fractions of ZJ at 50 µg/ml exhibited 49.85 (ZJ1), 54.95 (ZJ2), 56.1 (ZJ3) and 50.1 (ZJ4) percentage of inhibitory activity on AR and 30.4 (ZJ1), 76.8 (ZJ2), 54.6 (ZJ3) and 43.7 (ZJ4) percentage of inhibitory activity on AGE. Among all fractions, ZJ3 showed best inhibitory activity with an IC₅₀ of 42.66 µg/ml and 31.72 µg/ml for AR and AGE, respectively. In the in vivo studies, galactitol levels in the lens were significantly reduced by ZJ3 (p<0.05) when compared to control. The efficacy of the fraction could be due to the presence of phenolic compounds and triterpenoids. The present work indicates the potential of folklore remedies for treating diabetic complications.

 

 

 

INTRODUCTION:

Diabetes mellitus is a group of metabolic disorders 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 hyperglycemia¹.

 

The microvascular complications include neuropathy, nephropathy, retinopathy, and cataracts whereas macrovascular complications comprise the defects of cardiovascular and cerebrovascular systems which can ultimately cause myocardial infarction and strokes, respectively².

 

Several mechanisms have been proposed but the involvement of hyperglycemia-activated polyol pathway is particularly important in the pathogenesis of diabetic complications³. Aldose reductase (AR) is the main oxidoreductase responsible for the conversion of glucose into sorbitol. Increased accumulation of sorbitol inside the cells causes osmotic damage and is associated with microvascular complications. Sorbitol undergoes further metabolism to form fructose. Accumulation of fructose ultimately leads to the increased synthesis of advanced glycation end products (AGE). Such modified proteins when bound to AGE receptors produce cytokines and growth factors which cause vascular complications². 

 

Diabetes is considered as one of the major causes of death across the world as every six seconds one person is killed⁴.The treatment costs for diabetes and its complications can be huge and this can create an economic burden to a nation. As a result, there is a genuine interest on the research on inhibition of AR and AGE formation. Plants have been shown to inhibit polyol pathway and they can be a potential source of unique chemicals for the drug development of inhibitors of AR and AGEs formation^1,3,5,6. In this context, some folklore remedies for diabetes were selected for studying the inhibitory activity on AR and AGE formation. Two teaspoonful of root paste of Hibiscus arnottianus L. (Fam: Malvaceae) is given twice a day and half a teaspoon of seed powder of Ziziphus jujuba Mill. (Fam: Rhamnaceae) is taken with little honey for managing elevated blood glucose levels^7-10. Some of the common forest plants of Karnataka viz. seed of Strychnos potatorum L. (Fam: Strychnaceae) and root of Erythrina variegata L. (Fam: Fabaceae) have been reported to be useful in diabetes^11-13. Because of easy availability and low cost, most of economically weaker populations are dependent on folklore remedies for their health care. The above plants were selected with an intention of scientific validation of their folklore use.

 

MATERIALS AND METHODS:

Plant Material:

The selected plant materials namely root of H. arnottianus L. (HA), seed of Z. jujuba Mill. (ZJ) and root of E. variegata L (EV) were collected from the fields in and around Vijayapura in from November to February 2016. Seed of S. potatorum L. (SP) was purchased from Yucca Enterprises, Mumbai. The collected plant materials were identified by Dr. M. B. Moolimani, Professor and Head, Department of Botany, BLDEAs S.B. Arts K.C.P Science College, Vijaypura, Karnataka.   

 

Extraction and Fractionation:

The extraction and fractionation were done according to Telagari and Hullatti with minor modifications¹⁴. Dried powdered plant material (500 g) was first subjected to cold maceration with 70% v/v ethanol for 24 h to extract heat sensitive constituents if any. The extract was collected and the marc was further subjected to soxhlet extraction with ethanol (95% v/v). Extracts from both procedures were combined and concentrated using a rotary evaporator (IKA RV 10) at 40°C under reduced pressure.

 

 

The active Z. jujaba extract was subjected to fractionation. The alcoholic extract 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 adjusted to 9.0 with 10% ammonium hydroxide. This was washed with dichloromethane to get dichloromethane fraction (ZJ1, 2.5 g) and aqueous fraction (ZJ2, 20.4 g). Dichloromethane layer was similarly concentrated to one-third of the original volume. This was further partitioned with 90% v/v methanol and petroleum ether (1:1) to get methanolic fraction (ZJ3, 17.8 g) and petroleum ether fraction (ZJ4, 5.1 g).

 

Animals:

Wistar albino rats were housed under standard laboratory conditions and fed commercial rat feed and tap water ad libitum. All the animal work was approved by Institutional Animal Ethics Committee (IAEC No: BLDE/BPC/157A/2014-15).  

 

In vitro inhibitory studies on aldose reductase (AR):

Preparation of rat lens homogenate:

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 homogenized with 0.1 M sodium phosphate buffer, pH 6.2 (Mikro 220R, Hettich, Germany). The supernatant which was collected after centrifugation 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^15,16.

 

In vitro AR inhibitory activity of extracts/fractions:

Spectrophotometric method was used to assay AR Inhibitory activity¹⁶. 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 consisted 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 absorbance 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)¹⁷. 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 37^oC for 7 days. The intensity of fluorescence was determined at the excitation wavelength of 350 nm and the emission wavelength of 450 nm (CL-53, Elico fluorimeter). The standard for these studies was aminoguanidine and AGE inhibitory activity was assessed using the formula,    

 

 

 

In vivo inhibitory studies on aldose reductase (AR):

In vivo galactosemic 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 15^th 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 ethanol¹⁸. Centrifugation was done for 30 min at 4^oC at 16,000 rpm and lyophilized at -40^oC (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 Tri-sil 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 295ºC¹³. The internal standard was Methyl-α-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, aqueous alcoholic extracts of roots of H. arnottianus L. (HA), seeds of Z. jujuba Mill. (ZJ), seeds of S. potatorum L. (SP) and roots of E. variegata L.  (EV) were subjected to evaluation of inhibitory activity on rat lens aldose reductase (AR) and advanced glycation end product (AGE) formation. The extracts at 100 µg/ml exhibited 75.6 (HA), 83.63 (ZJ), 75.97 (SP) and 63.8 (EV) 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.

 

 

Fig. 1: Aldose reductase (AR) inhibitory activity of plant extracts and Quercetin (standard).

HA: H. arnottianus L; ZJ: Z. jujuba  Mill; SP: S. potatorum L; EV: E. variegata L;

 

The extracts at 100 µg/ml exhibited 26.5 (HA), 79.3 (ZJ), 20.8 (SP) and 22.2 (EV) AGE inhibitory activity (Fig. 2). 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.

 

 

Fig. 2: Advanced glycation end product (AGE) inhibitory activity of plant extracts and Aminoguanidine (AG; standard).

HA: H. arnottianus L; ZJ: Z. jujuba  Mill; SP: S. potatorum L; EV: E. variegata L.

Table 1 shows IC₅₀ of extracts for inhibitory activities on AR and advanced glycation end product formation. Among all, ZJ showed best inhibitory activity with an IC₅₀ of 49.27 µg/ml and 60.63 µg/ml for AR and AGE, respectively. Therefore, ZJ was fractionated and evaluated for inhibitory activities.

 

Table 1: IC₅₀ (µg/ml) of extracts and standards on inhibitory activities on aldose reductase (AR) and advanced glycation end product (AGE). HA: H. arnottianus L; ZJ: Z. jujuba  Mill; SP: S. potatorum L; EV: E. variegata L.

Extract	IC50 (µg/ml)
	AR inhibitory activity	AGE inhibitory activity
HA ZJ SP EV Quercetin Aminoguanidine	58.26±0.42 49.27±0.45 53.35±0.38 71.73±0.53 4.5±0.05 -	193.68±7.42 60.63±4.52 253.29±9.38 242.72±4.64 - 4.91±1.26

 

The fractions at 50 µg/ml exhibited 49.85 (ZJ1), 54.95 (ZJ2), 56.1 (ZJ3) and 50.1 (ZJ4) AR inhibitory activity (Fig. 3). The fractions at 50 µg/ml exhibited 30.4 (ZJ1), 76.8 (ZJ2), 54.6 (ZJ3) and 43.7 (ZJ4) AGE inhibitory activity (Fig. 4).

 

 

Fig. 3: Aldose reductase (AR) inhibitory activity of Z. jujuba (ZJ) fractions (ZJ1 to ZJ4) and Quercetin (standard).

 

 

Fig. 4: Advanced glycation end product (AGE) inhibitory activity of Z. jujuba (ZJ) fractions (ZJ1 to ZJ4) and AG, Aminoguanidine (standard).

Among all, ZJ3 showed best inhibitory activity with an IC₅₀ of 42.66 µg/ml and 31.72 µg/ml for AR and AGE, respectively (Table 2). Therefore, ZJ3 was evaluated for in vivo AR inhibitory activity.

 

Table 2: IC₅₀ (µg/ml) of Z. jujuba (ZJ) fractions and standards on inhibitory activities on aldose reductase (AR) and advanced glycation end product (AGE).

 

After derivatization, the 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-D-mannopyranoside, respectively. Fig. 5 shows the comparison of galactitol concentration of test group and the control.  When compared to control, galactitol levels in the lens were significantly reduced by the standard, quercetin and ZJ3 (p<0.05).

 

 

Fig. 5: Rat lens galactitol levels of treated and control groups as measured by GLC in the in vivo studies.

 

GL Science GC 353; Supelco DB-1 capillary column: 30 x 0.25 mm x 0.25 μM coated with cross linked methyl silicone;

Temperature of the column was increased at 5ºC/min from 120 to 265 ºC and then to 295ºC.

 

DISCUSSION:

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 affected with serious complications including heart attacks, blindness, kidney failure and neuropathy¹.  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 2030^1,19. Diabetes requires early diagnosis, treatment, and lifestyle changes. The role of four molecular mechanisms in causing diabetic complications has been extensively studied¹. They are  i) enhanced glucose flux through polyol pathway, ii) enhanced accumulation of advanced glycation end-products (AGE), iii) Stimulation of protein kinase C (PKC) and iv) enhanced flux through hexosamine pathway.

 

Polyol pathway seems to be particularly prominent in the pathogenesis diabetic complications³. 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. In recent years, herbal drugs are increasingly used to in combination or even alone for the management of diabetes and its complications^1,19. No information was available in the literature about the use of selected folklore remedies for treating diabetic complications. Hence, H. arnottianus L., Z. jujuba Mill., S. potatorum L. and E. variegata L. were screened for AR and AGE inhibition by in vitro and in vivo methods. 

 

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 1, ZJ exhibited the best inhibitory activity with an IC₅₀ of 49.27 µg/ml and 60.63 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 increases the susceptibility of cells by highly damaging reactive oxygen species^18,20.  Accumulation of fructose is highly conducive for initiation for glycation reactions. Such glycated proteins activate AGE receptors and lead to the development of vascular complications². Literature reveals the potential beneficial effects of several plant extracts and their constituents in alleviating diabetic complications through AR inhibitory activity^1,6,19. The phytochemical investigation of the plant extracts is given in the Table 3. Alkaloids, steroids, triterpenoids, tannins/phenolic compounds and flavonoids were found to be the secondary metabolites in the ZJ extract.

 

After fractionation of the aqueous alcoholic extract, the fractions were studied for AR and AGE inhibitory activities. ZJ3 showed the highest inhibitory potential with an IC₅₀ of 42.66 µg/ml and 31.72 µg/ml for AR and AGE, respectively (Table 2). Alkaloids and flavonoids were identified in ZJ1 and ZJ2 fractions, respectively. ZJ3 was found to contain phenolic compounds and triterpenoids whereas ZJ4 gave positive results for steroids and lipids.

Table 3: Phytochemical investigation of plant extracts.

Phytoconstituents*	HA	ZJ	SP	EV
Alkaloids Cardiac glycosides Anthraquinone glycosides Saponins Cyanogenetic glycosides Steroids Triterpenoids Tannins/Phenolics Flavonoids Carbohydrates Fats and oils Amino acids/ Proteins	- - + - - - + + + + - -	+ - - - - + + + + + - +	+ - - - - + - + - + - +	+ - - - - - - + - + - -

(HA: H. arnottianus L; ZJ: Z. jujuba  Mill; SP: S. potatorum L; EV: E. variegata L.)

*+: present; -: absent;

 

Phenolic compounds and triterpenoids do possess AR inhibitory activity^1,19. These phytoconstituents also possess antioxidant and antiglycation properties^20-26. Severe cataract is more easily produced in a short duration of time by galactosemia than by hyperglycemia²⁷. 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, ZJ3 significantly decreased the galactitol levels in the lens in glactose-fed rat model (Fig. 5). The effectiveness of the active fraction in the present study could be due to the presence of secondary metabolites like phenolic compounds and and triterpenoids. There is a renewed interest among scientific community to prevent and treat diabetic complications. The present work correlates well with earlier reports and indicates the potential of folklore remedies for treating diabetic complications.

 

Acknowledgements:

Authors are thankful to Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru for the award of research grant and the Principal and Management of BLDEA's SSM College of Pharmacy and Research Centre, Vijayapur, Karnataka for the facilities and support.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 27.12.2018           Modified on 30.01.2019

Accepted on 27.02.2019         © RJPT All right reserved