SCREENING OF NATURAL PHYTOCONSTITUENTS TO COMBAT DIABETIC CATARACT

Abhinav Temani Rupesh Vishwakarma

Dr. Sadhana Sathaye

Diabetic Cataract

Diabetes mellitus type 1 or juvenile diabetes and Diabetes mellitus type 2 or adult-onset diabetes lead to chronic diabetic complications like neuropathy, nephropathy, angiopathy and retinopathy.

Hyperglycemia is known to instigate these diabetic complications.

With the increased formation of advanced glycation end products (AGE’S).

Enhanced activity of aldose reductase (AR).

Formation of reactive oxygen species (ROS).

Rationale behind DIABETIC CATARACT as therapeutic area

Cataract is the most common cause of blindness, accounting for 51 % of world blindness, as of 2010.

Although the major cause remains to be aging, it is closely related to diabetes as one of its major late complications. Diabetes mellitus is associated with a 5-fold higher prevalence of cataracts.

Some 366 million people worldwide are estimated to have diabetes in 2011, and by 2030, some 552 million people, or every one adult in 10, will have diabetes. So will increase the incidence of diabetic complications like cataract.

Hence, substances are required to prevent or treat cataractogenesis.

RATIONALE BEHIND THE PROJECT

Rationale behind choosing POLYOL PATHWAY as the targetAlthough diabetic cataract is a consequence of cumulative effects of various metabolic processes linked to hyperglycaemia, increased activity of Aldose Reductase in the polyol pathway has been regarded as the initiator of the disease processFor diabetics, maintaining normoglycaemia at all times is not possible.With drugs that decrease blood glucose levels, there is risk of hypoglycaemia. Hence, aldose reductase inhibitors(ARI’s), which keep these complications in check, and work in complete independence to body sugar levels, seem to be an attractive proposition.

Focus on herbal substances

Although many chemical substances showing potent AR inhibition have been reported, only one, epalrestat, is currently marketed .

Toxicity, non-specific activity, limited efficacy, and poor pharmacokinetic properties are some of the severe limitations to therapeutic success of chemical substances.

Shifting focus to herbal substances thus seems to be a good idea under such circumstances.

Choosing Psoralen and Sesamin

Coumarin derivatives like umbelliferone have already been reported for their AR inhibitory activity and as potential therapeutic agents against long term diabetic complications.

The very fact that Psoralen is a coumarin derivative and its structural similarities with umbelliferone made it our subject of interest.

Sesamin is already catered to be a nutritional supplement that confers antioxidant and anti-inflammatory effects or possibly being an estrogen receptor modulator and fat burner.

The very fact that it is already used as a supplement coupled with its antioxidant activity encouraged us to test it for aldose reductase inhibition and its inhibition of diabetic cataract.

Rat Eye LensPellet Resuspend in buffer

Supernatant fraction

Ammonium Sulphate

centrifuge Homogenized buffer

pH 6.6 with beta mercaptanol

Preparation of crude Aldose reductase enzyme from Rat lens:

AR enzyme inhibitory activity of different concentration of Psoralen & Sesamin was investigated by use of in vitro spectrophotometric analysis.

Their anti-cataract activity was investigated by in-vitro cataract induction on goat lens culture.

Protein estimation of goat lens culture was subsequently undertaken.

Biochemical evaluation of goat lens culture was subsequently undertaken.

Antioxidant activity of Psoralen & Sesamin was investigated.

METHODS

IN-VITRO SPECTROPHOTOMETRIC ANALYSIS OF AR INHIBITORY ACTIVITY AR activity and thereby AR inhibition on addition of different concentrations of Psoralen & Sesamin was determined using Continuous Spectrophotometric Rate Determination using a UV spectrophotometer. AR activity was indicated by loss of NADPH absorbancy due to consumption of NADPH in the reaction whereas AR inhibition was indicated by fall in AR activity on addition of drugs.

The reaction mixture contained freshly prepared enzyme, varying concentration of Psoralen or Sesamin, 0.067 M buffer (pH 6.2), 0.125 mM NADPH, 400 mM LiSO4 and 45 mM xylose (substrate) in a 1 mL cuvette. Absorbance data was recorded every second for 3 minutes. Quercetin was used as standard inhibitor.The reference blank contained all the substances except the substrate. DMSO was used as control.Different concentrations of Psoralen & Sesamin were prepared with DMSO as solvent and its % inhibition values were recorded ; % inhibition being given by:

 

In vitro ANTI CATARACT ACTIVITY OF PSORALEN

A total of 36 lenses were divided in the following categories (n = 4 in each category)-  

Lens opacity was then judged by observing no of squares clearly visible through the lens when placed on a mesh. Each lens was graded for its integrity and degree of opacity and results were recorded.

 

72 h

Cataract

Goat Lens

Incubation; 37°c

High Glucose

Biochemical evaluation : REDUCED GLUTATHIONE ESTIMATIONSpectrophotometric method was employed for determination of reduced GSH (Sedlak and Lindsay et al 1979)

PROTEIN ESTIMATIONThe protein content of the samples was determined by the method of Lowry et al using bovine serum albumin as the standard.

ANTIOXIDANT ACTIVITYAntioxidant activity of Psoralen and Sesamin was determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay.

Different concentrations of Psoralen & Sesamin (25-1000 µg/ml) were prepared in methanol and incubated with 200µM DPPH at 37◦C for 20 min. Ascorbic acid was used as the standard antioxidant. 95% ethanol was used as control.

IC50 value for Psoralen & Sesamin was calculated and compared with that of ascorbic acid .

 

RESULTS

IN-VITRO SPECTROPHOTOMETRIC ANALYSIS OF AR INHIBITORY ACTIVITY Both Psoralen & Sesamin exhibited significant concentration dependent aldose reductase inhibition.

IC50 values establishes them as potential AR inhibitors, with Psoralen exhibiting inhibitory activity superior to that of current standard quercetin.

In vitro ANTI CATARACT ACTIVITY OF PSORALEN & SESAMINLenses incubated in Psoralen & Sesamin exhibited concentration dependent resistance to opacity.Those in 1mg Psoralen exhibited slight opacity of ‘+’, while the ones incubated in 3mg exhibited almost complete transparency, i.e ‘0’ opacity. At higher concentrations, Psoralen showed precipitation, hence its results were ignored.Those in 1mg Sesamin exhibited slight opacity of ‘+’, while the ones incubated in 3mg and 10 mg exhibited almost complete transparency, i.e ‘0’ opacity.

NORMAL GLUCOSE 5mM

HIGH GLUCOSE 55mM

QUERCETIN 1mg/ml

PSORALEN 1mg/ml

PSORALEN 3mg/mlSESAMIN 1 mg/mlSESAMIN 3mg/mlSESAMIN 10mg/ml

Integrity: +++opacity: 0

Integrity: +++opacity: +++

Integrity: +++opacity: +

Integrity: +++opacity: +

Integrity: +++opacity: +

Integrity: +++opacity: ++

Integrity: +++opacity: 0

Integrity: +++opacity: +

Reduced Glutathione Estimation Results:

ANTIOXIDANT ACTIVITY

The free radical DPPH was effectively scavenged by Psoralen. The radical scavenging capacity of Psoralen and standard ascorbic acid is dose dependent.

The IC50 of psoralen was found to be 312µg/ml, whereas for sesamin 140 µg/ml. Ascorbic acid showed IC50 of 47 µg/ml.

Thus, Psoralen has inherent antioxidant properties which enhances its anti-cataract potency.

INTERPRETATION OF RESULTS:

Cataract is a consequence of cumulative effects of various metabolic processes linked to hyperglycaemia, namely, osmotic stress due to accumulation of sorbitol, oxidative stress due to ROS , glycative stress due to formation of AGE’s , protein kinase stress due to activation of protein kinase C etc.

Sorbitol generated via polyol pathway is neither efficiently metabolized nor readily diffused through cell membranes. It accumulates, creating hydropic lens fibers that degenerate and form sugar cataract . Psoralen and Sesamin by inhibiting AR, deters this phenomon

The free radical DPPH was effectively scavenged by Psoralen. Thus, Psoralen has inherent antioxidant properties which enhances its anti-cataract potency.

Psoralen and Sesamin, both are thus potential anti-cataract agents acting via multiple mechanisms. Goat lens model established their anti-cataract activity. Lenses incubated with Psoralen or Sesamin along with high glucose clearly resisted onset of cataract as compared to lens in high glucose only, with clearly visible greater transparency in the former.

FUTURE:

Psoralen is found in a variety of plant sources and hence, is abundantly available. It has already established therapeutic importance. It is used immensely in PUVA therapy and is knocking the door of medical field with several activities of therapeutic value- it has shown anti-diabetic, cholinergic and anti-neoplastic properties.

Sesamin is another natural product already employed as a nutritional supplement that confers antioxidant and anti-inflammatory effects or possibly being an estrogen receptor modulator and fat burner.

Hence in-vitro success of Psoralen and Sesamin as AR inhibitor and anti-cataract agent calls for further research on the same and its in-vivo analysis. This compound may find future application in the treatment and prevention of cataract and other diabetic complications.

REFERENCES:

[1] Obrosova, I. G., Chung, S. S. M. and Kador, P. F. (2010), Diabetic cataracts: mechanisms and management. Diabetes Metab. Res. Rev., 26: 172–180. doi: 10.1002/dmrr.1075

 [2]Andreas Pollreisz and Ursula Schmidt-Erfurth, “Diabetic Cataract—Pathogenesis, Epidemiology and Treatment,” Journal of Ophthalmology, vol. 2010, Article ID 608751, 8 pages, 2010. doi:10.1155/2010/608751

[3] Beyer-Mears, A., and E. Cruz. "Reversal of diabetic cataract by sorbinil, an aldose reductase inhibitor." Diabetes 34.1 (1985): 15-21.

[4] Vilasrao J. Kadam, Yadunath M. Joshi, Harshad P. Sawant, Tej A. Jadhav. Free Radical Scavenging Activity of Aqueous Solution of Black Salt. International Journal of Pharmacy and Pharmaceutical Sciences. 2010; 2(suppl 2), 95-96.

[5] J. H. Kinoshita, “Mechanisms initiating cataract formation. Proctor lecture,” Investigative Ophthalmology, vol. 13, no. 10, pp. 713–724, 1974.

 

[6] J. H. Kinoshita, S. Fukushi, P. Kador, and L. O. Merola, “Aldose reductase in diabetic complications of the eye,” Metabolism, vol. 28, no. 4, pp. 462–469, 1979.

[7] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J. Biol Chem 1951:193-265.

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