Sulforaphane is a phytochemical which contains typical isothiocyanates group (NCS group) in cruciferous vegetables1 such as broccoli sprouts. It exists in a bounded form as glucoraphanin a non- glycoside in which Sulforaphane glucosinolate2 is a bounded sugar molecule. It is mainly available in combination with sinigrin3 (metabolized into allyl isothiocyanate), glucotropaeolin (metabolized into benzyl isothiocyanate), gluconasturtiin (metabolized into phenethylisothiocyanate) and glucobrassicin4 (metabolized into diindolylmethane). It has anti oxidant5,6 and anti cancer property and also works as natural detoxifying7 enzyme stimulator. It may reduce the risk of breast, bladder and prostate cancer.8 The present work aims the screening of in vitro anti diabetic activity of Sulforaphane from broccoli extract.
MATERIALS AND METHODS
The study was carried out by using DNS solution which is prepared by dissolving 30g of potassium sodium tartarate in 2N NaOH and made up to 100 ml.
The inhibition assay was performed using the chromogenic DNSA method.9,10 The total assay mixture composed of 1400 μl of 0.05 M sodium phosphate buffer of pH 6.9, 50 μl of amylase and analyte at different concentrations as 100, 250 and 500 μg were incubated at 370C for 10 min. After pre- incubation, 500 μl of 1% (w/v) starch solution in the above buffer was added to each tube and incubated at 370C for 15 min. The reaction was terminated with 1.0ml DNSA reagent,11,12 placed in boiling water bath for 5min, cooled to room temperature and the absorbance measured at 540 nm. The control amylase represented 100% enzyme activity and did not contain any sample of analysis. To eliminate the absorbance produced by analyte, appropriate analyte extract controls with the extract in the reaction mixture in which the enzyme was added after adding DNSA. The maltose liberated was determined by the help of standard maltose curve and activities were calculated according to the following formula.
The inhibitory property shown by the analyte was compared with that of control and expressed as percentage of inhibition.
Analysis of Acarbose as Standard Inhibitor
Acarbose was used as a standard inhibitor and it was assayed at above mentioned test sample concentrations. The assay method was similar to the above mentioned procedure, instead of analyte, acarbose was added. The results were compared to that of analyte.
RESULTS AND DISCUSSION
The results of percentage inhibition of amylase in the study were represented in Table 1 & Figure 1. In this method the percentage inhibition of amylase by the analyte were observed in dose dependent manner and decrease in the absorbance as 1.21 at 540 nm shows increased inhibition of amylase which is noted at 500 μg of analyte. The outcomes of present study suggest that the Sulforaphane exhibited significant inhibition of amylase enzyme which reflects the hypoglycemic activity of Sulforaphane in dose dependent manner. Here the analyte Sulforaphane has clearly displayed significant inhibition of 46% of enzyme at 500 μg concentration.
Hence a proper remedy for diabetes mellitus has to be found before the need reaches to its culmination. Through, many herbal products have been described for the treatment of diabetes mellitus, very few of them have been explored scientifically so far. The existing hypoglycemic drugs encounter many adverse effects and need on prolonged treatment including questionable efficacy in the treatment. This forces the area of research to find improved treatments which will counteract the adverse effects of the existing treatment. Finally here the study on Sulforaphane shows positive stance of having clear hypoglycemic activity. The study of such medicines might offer a natural key to unlock a diabetologist’s pharmacy for the future.
Percentage inhibition of analyte and amylase (control)
|Sample||Absorbance @540 nm||% inhibition||Activity|
|100 µg Sample||1.96||11.65||0.0446|
|250 µg Sample||1.42||36.38||0.0321|
|500 µg Sample||1.21||46.00||0.0273|
Amylase inhibition assay.
Despite of many adverse effects and short shelf life of present drugs here is a proved possibility of antidiabetic effect was found in sulforaphane from broccoli extract.
The authors are gratefully acknowledged to the management of Annamacharya College of Pharmacy, Rajampet for providing the infrastructural facility for carrying out this research work.
, authors. et al. Metabolic activity of radish sprouts derived isothiocyanates in drosophila melanogaster. International Journal of Molecular Sciences. 2016;17(2):251
, authors. Amplification of Sulforaphane Content in Red Cabbage by Pressure and Temperature Treatments. Journal of Applied biological Chemistry. 2011;54(2):183–7
William H. Watson
, authors. Crystal Structure of Sinigrin. Nature- International weekly Journal of Science. 1963;198(4887):1297–8
, authors. Biogenesis of Glucobrassicin, the in vitro Precursor of Ascorbigen. Nature- International weekly Journal of Science. 1962;194(4826):393–4
, authors. Optimization of enzymatic production of Sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days. Journal of Food Science and Technology. 2017;54(1):209–18
, authors. Sulforaphane, polyphenols and related anti-inflammatory and antioxidant activities changes of Egyptian broccoli during growth. Journal of Food Measurement and Characterization. 2017;1–8
, authors. et al. Sulforaphane prevention of diabetes induced aortic damage was associated with the up regulation of Nrf2 and its down-stream antioxidants. Nutrition & Metabolism. 2012;9(1):84
, authors. et al. Prostate, lung, colorectal and ovarian cancer screening trial, prospective study of fruit and vegetable intake and risk of prostate cancer. J Natl Cancer Inst. 2007;99(15):1200–9
Barry V McCleary,
, authors. A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1, 4-β-Xylanase. Journal of Applied Biochemistry and Biotechnology. 2015;177(5):1152–63
, author. et al. Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes. Sci. Trans Med. 2017;9(394):44–77
, authors. Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants. Bio Med Central complementary and alternative medicine. 2011;11(1):5
, authors. A simple micro plate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method). Journal of Food Chemistry. 2016;211:853–9
Mr. Shaik Chand Basha is presently working as Assistant Professor in Dept. of Pharmaceutical Chemistry, Annamacharya College of Pharmacy, Rajampet. He has published 19 research papers in National and International journals. He had received Post Graduate Fellowship from AICTE during M. Pharmacy (PG) course for two years. His area of interest is Natural Product Chemistry.
Mr. Komaragiri Rajesh Babu had completed B.Pharmacy in Annamacharya College of Pharmacy, Rajampet. Presently he is pursuing M Pharmacy in Pharmaceutical Analysis. He has published 03 research papers. His area of interest is Analytical Chemistry.
Mr. Medabalimi Madhu is presently working as Assistant Professor in Dept. of Pharmaceutical Analysis, Annamacharya College of Pharmacy, Rajampet. He has published 22 research papers in National and International journals. He had received Post Graduate Fellowship from UGC during M. Pharmacy (PG) course for two years. His area of interest is Analytical Chemistry.
Prof. Chakka Gopinath is presently working as Professor & Principal in Annamacharya College of Pharmacy, Rajampet. He has published more than 58 research papers in reputed National and International journals. He has received seminar grant from AICTE, New Delhi in year 2017.