DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2017.28

Research Article

Aldose reductase and advanced glycation end products formation inhibitory activity of standardized extracts of Picrorhiza kurroa (Royle ex benth) and Hibiscus rosa-sinensis (Linn.)

Anasuri Santhosh*, C. Veeresham, A. Rama Rao

Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical Sciences, Kaktiya University, Warangal, Telangana, India

*For correspondence

Anasuri Santhosh,

Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical Sciences, Kaktiya University, Warangal, Telangana, India.

Email: santupharmacy @gmail.com

 

 

 

 

 

 

Received: 11 June 2017

Accepted: 04 July 2017

ABSTRACT

Objective: The objectives were to study the in-vitro and in-vivo aldose reductase and in-vitro advanced glycation end products formation inhibitory activities of the standardized extracts of Picrorhiza kurroa roots and Hibiscus rosa-sinensis flowers.

Methods: In-vitro Aldose reductase inhibitory activity was studied by using isolated rat lens and kidney Aldose reductase by UV-Visible spectro photo metric method by using Quercetin as reference compound. In-vivo Aldose reductase inhibitory activity was evaluated by using experimental rat models of galactosemia and the final lens galactitol was evaluated by High performance liquid chromatography and Gas chromatography methods by using Quercetin as reference compound. In-vitro advanced glycation end products formation inhibitory activity was estimated by using laboratory test reaction with protein and sugars by spectro fluorimetric method by sung aminoguanidine as reference compound. Statistical analysis of the results was done by using Analysis of the variance method.

Results: The plant extracts were found to possess significant aldose reductase and advanced glycation end products formation inhibitory activity.

Conclusions: More study is required for isolation and characterization of the chief chemical constituents responsible for the biological activity of the plant extracts.

Keywords: Aldose reductase, Advanced glycation end products, Complications of diabetes mellitus, Picrorhiza kurroa, Hibiscus rosa-sinensis, Standardized extracts, Quercetin, Aminoguanidine

Introduction

Diabetes mellitus (DM) is an important metabolic disorder leading to several complications such as Diabetic-neuropathy, nephropathy, retinopathy and cataracts in severe and untreated conditions which require a special medical attention. Most of these occur in the tissues of insulin independent uptake of glucose such as nerves, kidneys, lens etc. Via polyol pathway (POP) of glucose metabolism. Aldose reductase (AR, ALR2, EC 1.1.1.21) is a NADP+, alditol, oxidoreductase, a member of aldoketoreductase superfamily that plays a key role in POP. ALR2 converts the glucose in the first step of POP into sorbitol which gets accumulated in such tissues due to the poor permeability of the tissues and the slow rate of the second step of POP where, the sorbitol is converted to fructose by another enzyme sorbitol dehydrogenase (SD, EC 1.1.1.2). As a result of accumulation of sorbitol, the normal physiology of the tissues was altered and leading to several complications.1 Advanced glycation end products (AGE) are the reactive, fluorescent compounds produced in severe and untreated conditions of DM. These are mainly derived from the products of POP such as fructose and even from the sugars like glucose via non enzymatic glycation reaction with tissue proteins and aminoacids- Millard's reaction and Amodari rearrangement and Schiff's base formation. The AGE's are further more reactive with tissue proteins and cause irreversible damage to such tissues and increase the severity of the complications of DM.2

Inhibition of ALR2 and the formation of AGE's is a promising and novel approach for the treatment of such complications of DM. Certain synthetic compounds such as spirohydantoin derivatives like sorbinil and carboxylic acid derivatives like epalrestat are well reported for AR inhibitory activity. But most of the synthetic compounds are also found to possess unwanted effects and poor pharmacokinetic parameters and less effective in-vivo.1

Several natural products are reported to be the safe and effective as AR, AGE formation inhibitors such as, Nelumbo nucifera-Leaves, Extracts from Hops-Humulus lupulus etc.2,3 Most of these are also reported for antidiabetic activity and contains flavanoids or other antioxidant chemicals.

The primary objective of the present work is to study the AR and AGE formation inhibitory potential of the standardized extracts of two Ayurvedic medicinal plants including Picrorhiza kurroa and Hibiscus rosa-sinensis in an attempt to discover safe and effective natural products as AR and AGE formation inhibitors. Picrorhiza kurroa was known to possess several constituents like Picrosides, Kutkosides and antioxidant substances and reported for antidiabetic, other pharmacological activities like antihepatotoxic, nephroprotective, and antioxidant.4-9 Hibiscus rosa-sinensis is reported to contain flavanoids like Hibiscitin and mentioned to possess antidiabetic, several other Pharmacological activities such as antihepatotoxic, antioxidant and hypoli-pidemic.10-15

Quercetin possesses significant AR inhibitory and AD activities.16,17 Aminoguanidine well known to be an AGE inhibitor and AD agent were taken as the reference compounds for the study.18,19

Materials and Methods

Plant materials

The standardized extracts of the plants were procured from authentic source- Amsar Extracts Pvt.Ltd. Indore, India. All the extracts were stored under cool and dry conditions until use. These are reconstituted in the appropriate medium when used.

Animals

Male wistar rats of weight 200-250 g are procured from authentic source and maintained under standard conditions of diet and water ad libitum at standard temperatures at the animal house of the college. All the animal work was approved by Institutional Animal Ethics Committee (IAEC).

Aldose reductase inhibitory activity

In-vitro assay

The AR inhibitory activity of the plant extracts was determined in-vitro by spectrophotometric method using isolated rat lens and kidney AR enzyme by the method mentioned by Jung et al., 2008 with minor modifications.2

Isolation of rat lens and kidney AR

Rats were sacrificed by spinal nerve dislocation after ether anesthesia. The lenses were separated from the eyes by posterior approach and kidneys were separated and made into small pieces by surgical procedure. Lenses and Kidneys were immediately placed in an appropriate tubes containing phosphate buffer of PH 6.2 (100 mM). Lenses and kidneys were immediately stored in an ultra deep freezer at -4°C. Each lens was homogenized in 1 ml of same buffer using tissue homogenizer and immediately centrifuged using cooling centrifuge at 4°C at 16000 rpm for 30 min. The supernant thus obtained from each lens preparation was pooled and taken as the crude rat lens AR. Each kidney preparation was homogenized in the same way as lens in 6 ml buffer and immediately centrifuged by cooling centrifuge at 8°C at 4000 rpm for 30 min. The supernant thus obtained from each kidney preparation was pooled and taken as crude kidney AR. The enzyme preparations were stored at -4°C until use.

Total protein content

The total protein content of the isolated rat lens and kidney AR was determined by the method of Lowry et al, with the standard graph of Albumin using UV-Visible Spectrophotometer and expressed in mg/ml.20

Enzyme and specific activity

These were assessed for both rat lens and kidney AR by spectrophotometric method based on the change in the absorbance of the enzyme co-factor-NADPH (0.15 mM) against blank in the presence of enzyme, substrate-DL-Glyceraldehyde (10 mM) at 340 nm within 1 min. The reaction mixture consists of, 0.3 ml of 10% DMSO, 0.3 ml of 10 mM DL-Glyceraldehyde, 0.3 ml of rat lens or kidney AR, 0.3 ml of 15 mM NADPH.

Make up the volume to 3 ml with phosphate buffer of pH 6.2. The change in the absorbance of the co-factor in presence of substrate is dependent on the enzyme activity. The enzyme activity is determined by following formula-

Enzyme activity (U/ml) = ((∆A test / min-∆A control / min) / (6.2 × volume of the enzyme taken (ml) × total volume of the solution (ml))

∆A test- Change in the absorbance of control solution

∆A control- Change in the absorbance of blank solution

The specific activity of the enzyme preparations was determined by following formula:

Specific activity (U/ml) = ((enzyme activity (U/mg)/protein content of the enzyme preparation (mg/ml))

AR inhibitory potential of the extracts

The AR inhibitory activity of the samples was determined against both rat lens and kidney AR separately. It was based on the spectrophotometric method in which, the inhibitory potential of the extracts and standard were assessed based on the change in the absorbance of NADPH at 340 nm within 1 min in the presence of substrate and enzyme, samples, that is dependent on their potential to inhibit enzyme. Quercetin was used as a reference compound for the study. The reaction mixture consists of, 0.3 ml of Quercetin ( 0.1, 1, 5, 10 µg/ ml ) or extracts (1, 10, 50, 100 µg/ ml), 0.3 ml of 15 mM NADPH, 0.3 ml of rat lens or kidney AR and 0.3 ml of 10 mM DL-glyceraldehyde. The total volume was made upto 3ml with phosphate buffer of pH 6.2. Quercetin and extracts were prepared in 10% DMSO as a vehicle. The percentage inhibition of standard or samples was determined by using the following formula:

Percentage Inhibition= ((1- (∆A sample - ∆A blank))/ (∆A control - ∆A blank) ×100])

∆A sample- Change in the absorbance of test solution

∆A blank- Change in the absorbance of the blank solution

∆A control- Change in the absorbance of the control solution.

In-vivo study

Rat experimental models of galactosemia are used for in-vivo estimation of AR inhibitory activity of the plant extracts by both HPLC and GC methods with minor modifications.21-23 The animal models possess advantage over Diabetic models as galactose have equal or more affinity to AR in POP over glucose in the condition of galactosemia, it is converted to galactitol by AR in the tissues of POP like lens and not further metabolized and accumulated in such tissues and can be easily determined by HPLC or GC methods. The content of galactitol depends on the AR activity. So, based on the lens galactitol content, one can assess the AR activity.

Body weights and blood glucose levels

The body weights of the animals were determined by animal balance at 1st and last day of the experiment. The blood glucose levels of the animals was determined by Trinder's (GOD-PAP) method with minor modifications using commercial glucose kit by spectrophotometric method at 505 nm at 1st and last day of the experiment and expressed in mg/dl by following formula- Concentration of glucose (mg/dl) = ((Absorbance of the test against blank)/(Absorbance of the standard against blank) × 100).

Administration of samples

The animals were grouped into 4 groups 6 animals each. Galactosemia was induced to all the groups of animals by giving 1 ml of galactose suspended in 1% gum acacia in normal saline as a vehicle with a dose of 10 mg/kg of the body weight once a day for 14 days by oral route. Quercetin and the extracts were suspended in 1% gum acacia in normal saline as vehicle. Control group taken only 1 ml vehicle. Reference group given 1 ml Quercetin 10 mg/kg body weight, P. kurroa std extract 200 mg/kg body weight and H. rosa-sinensis std extract 500 mg/ kg body weight were taken as test samples and administered to the respective groups of animals at 1 ml dose by oral route simultaneously 12 hours after administration of galactose for 14 days. Doses of the samples were selected based on the literature for their AR inhibitory activity for Quercetin and anti diabetic activity for extracts.

Final lens galactitol levels

The final lens galactitol levels of all groups of animals were determined by HPLC and GC methods as follows

HPLC method

Derivatization of galactitol by Phenyl isocyanate (PHI) in the presence of pyridine. Galactitol reacts with PHI in presence of Pyridine forms strongly UV absorbing and stable derivatives of galactitol can be assessed by HPLC - UV detectors at 240 nm. The final lens galactitol levels were determined from the standard graph of galactitol prepared by HPLC method. HPLC system used consists of LC-10 AT, LC pump, SPD-M 10 A VP, DAD detector, Hibar-250-4, 6, Lichrosphere-100 column, LC-Solution, Shimadzu software. Acetonitrile and water (60:40) was used as mobile phase at 2ml/min flow rate, Photo diode array detector setted at 240 nm, C18 reverse phase, nucleosil column, and Loop injector of 20 µl capacity was used.

Derivatization

Add 250 µl of pyridine, 500 µl of phenyl isocyanate (PHI) to each bottle of lyophilized powders of the samples. Then the samples were incubated at 55°c in water bath with mechanical shaking for 30 min. After, the bottles were cooled to room temperature and add 250 µl of methanol for eliminating the excess phenyl isocyanate. Otherwise, the PHI reacts with water and eluant. The samples were finally, diluted twice with pyridine to minimize the interference of the reagents.

Preparation of biological samples

After 14 days of study, all the rats were sacrificed by spinal nerve dislocation after ether anesthesia. The lenses from each rat in each group were removed by posterior approach. The lenses were taken into separately labeled centrifuge tubes each containing 1 ml of 20% ice cold ethanol. The lenses were immediately stored in freezing conditions until use. All the lenses were homogenized using tissue homogenizer. 70% Ethanol was added to each lens homogenate for precipitation of the proteins. 0.1 ml of glucose (10 µg/ml) was added to each lens homogenate as an internal standard. Each tube was centrifuged at 4°C using cooling centrifuge. The supernant thus obtained after centrifugation from each tube was separated and stored at freezing temperatures. The supernant from each tube was taken into separate lyophilyzation bottles. These are lyophilized for 24 hrs at -40°C using freeze drier. The freeze dried powders of the respective samples were used for derivatization by PHI. All the derivatized samples were analyzed by HPLC.

Estimation of lens galactitol levels

The galactitol content of each sample was determined by interpreting the value of peak height of galactitol in each sample with standard graph of galactitol prepared by HPLC method.

Analysis of results

The AR inhibitory activity of Quercetin and extracts was determined with respect to their effect on the lens galactitol levels in comparison with control group.

GC method

Derivatization of galactitol by Trisil-Z (Mixture of 1(Trimethyl sily)-Imidazole and Pyrimidine) reagent to form trimethylsilylated derivatives of galactitol that can be easily analyzed by GC. The system used consists of GL-Science 353 – GC with DB-1, 30×0.250 mm, 0.25 µm -column, Nitrogen as a mobile phase, temperature programming-120-0-5-265-0-10-295-0 and injection temperature 280°C, detection temperature 300°C, flow rate 1 ml/min, split ratio 1:100. The final lens galactitol levels were determined by using the standard graph of galactitol prepared by GC method.

Derivatization

The lyophilized samples were treated with 1 ml Trisil-Z reagent at 60°C for 30 min.

Biological sample preparation

After 14 days of the study, the rats from different groups were sacrificed and the lenses of such were removed by posterior approach into tubes containing 1 ml 20% ice cold Acetonitrile. The lenses were homogenized using tissue homogenizer and added 0.1 µM α-D Mannopyranoside as an internal standard to each lens and centrifuged by using cooling centrifuge at 4°C for 30 min. The supernant from each tube was taken into separate lyophilyzation bottles. These are lyophilized for 24 hrs at -40°C using freeze drier. The freeze dried powders of the respective samples were used for derivatization. These are used for GC analysis for galactitol content.

Estimation of lens galactitol levels

The galactitol content of each sample was determined by interpreting the value of peak area of galactitol in each sample with standard graph of galactitol.

Analysis of results

The AR inhibitory activity of Quercetin and extracts was determined with respect to their effect on the lens galactitol levels in comparison with control group.

Assay for inhibition of AGE formation

It is based on the method of Jung et al, with minor modifications.2 The sugars react with the proteins in the presence of test samples undergoes to a series of non-enzymatic glycation reactions like Millard's reaction to form fluorescent AGE's that can be determined based on the fluorescence of the reaction mixture by spectrofluorimeter. So, based on the fluorescence, one can assess the inhibitory potential of the samples on AGE inhibition. The conditions of elevated temperatures, darkness and alkalinity are known to promote the formation of AGE's.

Preparation of test

The reaction mixture consists of, 2.85 ml of 10 mg/ml bovine serum albumin (BSA) solution-Protein in 50 mM phosphate buffer of pH 7.4, 0.2 M glucose (0.108 g), 0.2 M fructose (0.108 g), 0.3% of sodium benzoate (0.009 g)-Preservative and 0.15 ml of sample- 10% DMSO as control, 1, 5, 10 µg/ml of aminoguanidine-as standard, 10, 50, 100 µg/ml extracts of H. rosa-sinensis and P. kurroa as test sample into separate flasks. Total volume of the reaction mixture in each flask is 3 ml. All the flasks were closed and incubated in an incubator at 37°C for 7 days in the dark conditions. Standard and extracts were prepared in 10% DMSO as vehicle. After 7 days of incubation, the fluorescence intensity of the control and the samples was determined against blank using spectrofluotimeter. The percentage AGE inhibition is determined by the following formula.

((Fluorescence of control-Fluorescence of the test) / (Fluorescence of the control) × 100).

Results and Discussion

Aldose reductase inhibitory activity

In-vitro assay

The total protein content of the rat lens and kidney AR preparation was found to be, 1.23 mg/ml and 1.76 mg/ml respectively. The enzyme activity of the rat lens and kidney AR was calculated as, 3.58 U/ml and 22.04 U/ml respectively. The specific activity of the rat lens and kidney AR was determined as, 2.91 U/mg and 12.52 U/mg respectively.

The AR inhibitory potential of Quercetin and the plant extracts against rat lens and kidney AR were presented in Table 1.

Table 1: In-vitro AR inhibitory potential of quercetin and extracts.

S. No. Sample and Conc.(µg/ml) Rat lens AR Rat kidney AR
Percentage inhibition IC50 value (µg/ml) Percentage inhibition IC50 value (µg/ml)
1. Quercetin        
0.1 8.73±0.25 4.37±0.56 8.40±0.10 4.61±0.78
1 39.17±0.76 38±0.4
5 54.12±0.82 52.3±0.5
10 88.34±0.91 85.5±1.5
2. Picrorhiza- Extract        
1 7.42±0.10 52.84±0.01 5.67±0.06 46.11±0.81
10 37.47±0.55 36.3±0.61
50 55.13±1.01 66.42±0.45
100 71.93±0.40 76.64±0.31
3. Hibiscus- Extract        
1 4.8±0.26 51.86±0.71 4.84±0.04 47.13±0.80
10 35.37±0.72 36.1±0.9
50 60.03±0.95 64.1±0.26
100 72.17±0.76 77.18±0.25

All the values of percentage inhibition and IC50 are expressed as mean±SD.

Table 2: Effect of quercetin and extracts on body weights and blood glucose levels.

S. No. Group Body weight (grams) Serum glucose levels (mg/dl)
Initial Final Initial Final
1. Control 175±0 162.33±1.50 a 79.29±0.92 144.18±1.73 a
2. Standard 175±0 187.33±1.50 a 103.01±1.57 90.3±0.50 a
3. Picrorhiza extract 200±0 212.33±1.50 a 119.35±1.27 110.56±1.21 a
4. Hibiscus extract 225±0 232.33±1.50 a 99.32±0.95 98.55±0.77 a

All the values are expressed as mean±SD, n=4, ap<0.0001, when compared with control.

Table 3: Final lens galactitol levels of the experimental animals.

S. No.

Group

Galactitol level (µg/ml)

HPLC Method GC Method

1.

Control

77.7±0.82 a

72.3±0.8 a

2.

Standard

33.5±1.28 a

30.7±1.47 a

3.

Picrorhiza extract

55.57±0.71 a

51.33±0.86 a

4.

Hibiscus exract

58.47±0.94 a

54.47±1.40 a

All the values are expressed as mean±SD, n=4, ap<0.0001 when compared to control.

The plant extracts were found to possess significant AR inhibitory activity in-vitro against both rat lens and kidney AR. Querecetin was proved to be a significant inhibitor of AR in-vitro.16,17

In-vivo study

Body weight and serum glucose levels

The effect of Quercetin and the extracts on the body weight and blood glucose levels of the Experimental rat models of galactosemia were presented in Table 2. The initial and final body weights and serum glucose levels were expressed in grams as mean±SD. These are compared by one-way ANOVA for Dunnet's test for multiple comparisons. All the values are found to be significant with p<0.0001 when compared to control. These are calculated by using Graph Pad prism software, version 5.0.

From the results, it was found that both the plant extracts and Quercetin no major effect on the body weights and the serum glucose levels of the experimental rat models of galactosemia during the study.

Lens galactitol levels

The final lens galactitol levels of the experimental animals determined by both HPLC and GC methods were presented in the Table 3. All the values are expressed in µg/ml as mean±SD. These were calculated by using graph pad prism software, version-5.0.

From the results, it was found that, the plant extracts possess significant AR inhibitory activity in-vivo. It was proven that, Quercetin possess significant AR inhibitory activity in-vivo.16,17

AGE assay in-vitro

The AGE formation inhibitory potential of reference aminoguanidine and the plant extracts was presented in Table 4. All the values were expressed as mean±SD and calculated by using graph pad prism software, version 5.0.

Table 4: AGE formation inhibitory potential of aminoguanidine and extracts.

S. No. Sample and Conc.(µg/ml) Percentage of inhibition IC50 value (µg/ml)
1. Aminoguanidine    
1 7.5±1 6.01±0.00
5 55.2±0.36
10 72.87±0.71
2. Picrorhiza extract    
10 5.3±0.46 67.97±1.52
50 49.7±0.57
100 66.0±0.61
3. Hibiscus extract    
10 5.4±1.34 66.93±1.33
50 48.8±0.7
100 67.97±0.85

All the values are expressed as mean±SD.

From the results it was found that, both the plant extracts possess significant activity in-vitro. It was proven that, aminoguanidine as a potent AGE formation inhibitor in-vitro.18

Several natural products, extracts and their pure phytochemicals mainly flavonoids and antioxidant substances are known as safe and effective as AR and AGE formation inhibitors. Most of them are also reported for antidiabetic (AD) activity.2,3 It was taken as a criteria for the selection of plants for the present study. The extracts of P. kurroa contains Picrosides and reported for AD activity.4,5 The extracts of H. rosa-sinensis was known to contain flavonoids and reported for AD activity.9,10 The present study included the assessment of the AR and AGE formation inhibitory potential of the standardized plant extracts not reported earlier. From the study, it was found that the plant extracts possess significant activity and proved that Quercetin as an effective AR inhibitory flavonoid in-vitro and in-vivo.16,17 The plant extracts were also found to possess significant AGE formation inhibitory activity in-vitro and aminoguanidine was proved to be a potent AGE inhibitor.18 However, it is a preliminary report of the extracts for AR and AGE studies.

Conclusion

From the study, it was found that the plant extracts possess significant AR and AGE formation inhibitory potential. However, more study is essential to characterize the specific fractions and chemical constituents from these extracts as AR and AGE formation inhibitors.

Acknowledgements

We are especially thankful to Amsar extracts Private Limited for providing the samples of the plant extracts and AICTE for financial assistance.

Funding: Financial assistance by AICTE Conflict of interest: None declared

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