In-vitro Inhibitory Activity of Medicinal Plant (Syzygium cumini Seed) against Carbohydrate Metabolizing Enzymes.

 

Madhavi Sahu*, S. Prakash Rao

Department of Pharmacology Columbia Institute of Pharmacy, Tekari, Raipur (CG) pin 493111.

*Corresponding Author E-mail: madhavi3835@gmail.com

 

ABSTRACT:

Purpose- To investigate the carbohydrates metabolizing enzymes of medicinal plant Syzygium cumini seed extract in-vitro. Background- Syzygium cumini and its seed a member of Myrtaceae family acquire potential role in regulating diabetes mellitus and its seeds are moderately rich in protein and contains so many other phytochemicals. Methods- The current study evaluated that the carbohydrates metabolizing enzymes of Syzygium cumini seed via in vitro inhibition of α-amylase and α-glucosidase using the hexane, petroleum ether, ethanol and water extracts. Preliminary phytochemical analysis was performed on the acetone, aqueous and ethanol extracts of Syzygium cumini seed. Results- The extract of Syzygium cumini seed displayed the most effective inhibition of both α-amylase and α-glucosidase activities with inhibitory concentration respectively and the results of phytochemical analysis of the extract revealed the presence of saponins, flavonoids, tannins, terpenoids and reducing sugars. Conclusion- The present study involve in investigation of Syzygium cumini seed possesses in-vitro enzymatic inhibition activities and further scientific validation is essential to understand the therapeutic potential of the Syzygium cumini plant for improving glycaemic control in diabetic subjects. To confirm their antidiabetic mode of action and their inhibition could be useful in the development of new drug strategies.

 

KEYWORDS: α-amylase, α-glucosidase Syzygium cumini, Hyperglycemic, Hexaneand IC50.

 

 


INTRODUCTION:

Diabetes mellitus is a complex metabolic syndrome or carbohydrate metabolism of endocrine system with an absolute or relative deficiency of insulin resulting in disturbed intermediary metabolism and manifestations. Diabetes mellitus affects all body systems and the main brunt is borne by eyes, kidneys, skin and nerves.[1-5] Therefore a therapeutic approach to treat diabetes is to decrease postprandial hyperglycemia.[8]

 

 

This can be achieved by the inhibition of carbohydrate hydrolyzing enzymes like alpha amylase and alpha glucosidase.[9] Alpha glucosidase and alpha amylase are the important enzymes involved in the digestion of carbohydrates. Alpha Amylase is involved in the breakdown of long chain carbohydrates and alpha glucosidase breaks down starch and disaccharides to glucose. They serve as the major digestive enzymes and help in intestinal absorption. Alpha amylase and glucosidase inhibitors are the potential targets in the development of lead compounds for the treatment of diabetes.[10]

 

For a long time natural products from plants have been used for the treatment of diabetes, mainly in developing countries where the resources are limited and affordability and access to modern treatment is a problem.[6] Extensive research has been carried out to screen the bioactivity of these inhibitors because of their significant importance in health care and medicine.[7]

 

Alpha glucosidase inhibitors are used as oral anti diabetic drugs for treating type 2 diabetes mellitus. They act by preventing the digestion of carbohydrates such as starch. Carbohydrates are normally converted into simple sugars which can be absorbed through the intestine.[12] Alpha glucosidase inhibitors act as competitive inhibitors of alpha glucosidase enzyme needed to digest carbohydrates.The intestinal alpha glucosidases hydrolyze complex carbohydrates to glucose and other monosaccharides in the small intestine. Inhibition of these enzyme systems helps to reduce the rate of digestion of carbohydrates.[13]

 

Syzygium cumini is a seasonal berry grows mainly in tropical and sub-tropical parts of the world. Jamun is used for controlling diabetes mellitus. Its seeds are popular amongsalternaive medicine system to control different aliments such as diabetes, cardiovascular and gastrointestinal disorder.[14] Jamun encompasses its use in various traditional medicinal system like ayurveda, unani, siddha and homeopathic and in srilankan and tibetan medicinal system of alternative and complementary medicines.

  Kingdom: Plantae

  Division: Magnoliophyta

  Class: Magnoliosida

  Family: Myrtaceae

  Genus: Eugenia

  Species: Jambolana Lam.

 

Although number of studies have been established the antidiabetic potential of Syzygium cumini seeds in experimental diabetes.

Carbohydrate metabolizing enzymes includes

(1) Alpha amylase

(2) Alpha glucosidase

(3) Sucrase

(4) Dipeptidyl peptidase-IV.

 

 

 

 

Fig : Syzygiumcuminiseed (Jamun)

MATERIALS AND METHODS:

1. Source of plant material:

The fresh Jamun fruits were collected from their natural habitat in Raipur, Chhattisgarh, India. Wash with the normal water to remove the dust particle. The pulp of seeds separate manually. Seed were dried and powdered.

 

2. Preparation of plant extracts:

The powdered extracted with hexane and petroleum ether ratio of 50:50 for 100 ml and filtered. The residue was extracted with water and ethanol ratio of 60:40 for 100 ml using cold maceration. Extract were taken and used for further phytochemical analysis.

 

3.Evaluation of preliminary phytochemical Screenings:

The hydro-alcoholic seed extracts of Syzygium cumini were analyzed for the presence of alkaloids, glycosides, steroids, carbohydrates, proteins, mucilage, tannins and phenolic test, flavonoids according to standard methods.

 

I. Test for alkaloids:

Dragendroff s test:

2 ml of dilute hydrochloric acid was added to the 5 ml of extract then treated with Dragendroffs reagent, appearance of an orange brown precipitate showed the presence of alkaloids.

 

II. Test for glycosides:

Legals test:

To aqueous and alcoholic extract, 1 ml of pyridine and 1 ml of sodium nitroprusside solution were added. Pink colour produced which turn into red indicated presence of glycosides.

 

III. Test for steroids:

Salkowski reaction:

To 2 ml of extract was added 2 ml of chloroform and 2 ml of conc. H2SO4. Shake well. Chloroform layer appears red and acid layer shows greenish yellow fluorescence was not formed which indicates absence of steroids.

 

IV. Test for carbohydrates:

Molichs test:

2-3 ml aqueous extract was added to the few drops of alpha-naphthol solution in alcohol, shake and added conc. H2SO4 from sides of the tube. Violet ring is formed at the junction of two liquids showed the presence of carbohydrates.

 

V. Test for proteins:

Xanthoprotein test:

3 ml of extract was mixed with 1 ml conc. H2SO4. White ppt. is formed. Boiled it. Precipitate turns yellow. Added NH4OH, ppt. turns orange indicated presence of proteins.

 

VI. Test for tannins and phenolic compounds:

5% ferric chloride solution:

In a test tube containing little quantity of extract few drops of 5 % ferric chloride solution were added. Deep blue-black colour obtained it showed the presence of tannins and phenolic compounds.

 

VII. Test for flavonoids:

Few drops of dilute NaOH was mixed with 2 ml of extract. The residue shows colouration, which decolourises after addition of acid showed the presence of flavonoids.

 

ALPHA AMYLASE INHIBITION ASSAY:

1.1.    Extraction of Wheat alpha amylase[61]:

500 g of malted whole wheat flour was added slowly with stirring to 1 litre of 0.2% calcium acetate solution at room temperature and continuously stirred for 2 hours on a stirrer. The suspension was then centrifuged at 40C at 12000g for 10minutes. The resultant clear brown supernatant was stored at 2C to 3C prior to heat treatment.

 

1.2.    Determination of Wheat alpha amylase inhibitor activity:

The assay mixture containing 200 l of 0.02M sodium phosphate buffer, 20 l of enzyme and the plant extracts in concentration range 20-100 g/ml were incubated for 10 minutes at room temperature followed by addition of 200 l of starch in all test tubes. The reaction was terminated with the addition of 400 l DNS reagent and placed in boiling water bath for 5 minutes, cooled and diluted with 15 ml of distilled water and absorbance was measured at 540 nm. The control samples were prepared without addition of any plant extracts and the blank solution used phosphate buffer.

The % inhibition was calculated according to the formula

 

Abs(control)-Abs(extract)

Inhibition (%) = ---------------------------------------------- X 100

(Abs (control)

 

The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and were calculated by non-linear regression values. Acarbose was used as the reference alpha amylase inhibitor. All tests were performed in triplicate.

 

Fungal alpha amylase[62]:

1.1.    Isolation of fungal alpha amylase:

Diastase powder was weighted and kept for 24 hours in temperature 4˚C. then dissolved in 100mM phosphate buffer, pH 6.8 and stirred for 2 hours and centrifuged at 12000 rpm in 4˚C for 10 min. The resultant solution was used as enzyme.

 

1.2.    Inhibition activity of fungal alpha amylase:

The assay mixture containing 200 μl of 0.02M sodium phosphate buffer, 20 μl of enzyme and the plant extracts in concentration range 20-100 μg/ml were incubated for 10 minutes at room temperature followed by addition of 200 μl of starch in all test tubes. The reaction was terminated with the addition of 400 μl DNS reagent and placed in boiling water bath for 5 minutes, cooled and diluted with 15 ml of distilled water and absorbance was measured at 540 nm. The control samples were prepared without any drug sample.

 

The % inhibition was calculated according to to the formula

 

Abs(control)-Abs(extract)

Inhibition (%) =-------------------------------------------X 100

Abs (control)

 

The IC50 values were determine from plots of percent inhibition vs log inhibitor concentration. Acarbose was used as the reference alpha amylase inhibitor. All tests were performed in triplicate.

 

Salivary alpha amylase:

1.1.    Collection and preparation of salivary alpha amylase sample:

Rabbit saliva samples were collected from the rat mouth with the help of cotton bird and dissolve in 0.1 M tris-buffer (pH 7.0) and centrifuged at 13,000 rpm for 20 min. An aliquot of the supernatant was diluted with water. These extract were then used in the assay.

 

1.2.    Salivary alpha amylase inhibitory activity:

Salivary alpha amylase was assayed by the method reported previously with a single modification. the assay mixture containing 200l of 0.02 M sodium phosphate buffer, 20l of enzyme and the plant extract in concentration range 20-100l were incubated for 10min. at room temperature followed by addition of 200l of starch in all test tubes. The reaction was terminated with the addition of 400l DNS reagent and placed in boiling water bath for 5 min. cooled and diluted with 15ml of distilled water and absorbance was measured at 540nm. The control samples were prepared without any plant extract. The inhibition was calculated according to the formula.

 

Abs(control)-Abs(extract))

Inhibition (%) =------------------------------------------ X 100

Abs (control)

The IC50 values were determine from plots of percent inhibition vs log inhibitor concentration and calculated by non-linearregression analysis from the mean inhibitory values.

 

ALPHA GLUCOSIDASE INHIBITION ASSAY:

1.1.    Alpha glucosidase (B. Stearothermophilus) inhibitory activity [63,64]:

 

Preparation of enzyme solution:

α-glucosidase activity was performed according to the method of Lam et al. α-Glucosidase type IV enzyme (Sigma Co., St. Louis, USA) from B. stearothermophilus was dissolved in 0.5 M phosphate buffer (pH 6.5) (3 U/ml).

α-glucosidase inhibitory activity:

The α-glucosidase inhibitory activity was determined by measuring the release of 4-nitrophenol from p-nitrophenyl α-D glucopyranoside. The assay mixtures for these experiments contained 0.3 ml of 10mM p-nitrophenyl α-D-glucopyranoside, 1.0 ml of potassium phosphate (0.1M, pH: 6.8), 0.2 ml of enzyme solution and 0.2 ml of inhibitor test sample , all in a final volume of 1.7 ml. Following an incubation time of 30 min at 37 C, the reaction was terminated by the addition of 2.0 ml of 100 mM sodium carbonate. The liberated p-nitrophenol was determined at 400 nm using spectrophotometer.

 

The % inhibition rates were calculated using the formula,

 

Abs(control)-Abs(extract))

Inhibition (%) =--------------------------------------------- X 100

Abs (control)

 

Suitable reagent blank and inhibitor controls were also carried out and subtracted. Dose dependent variation in the α-glucosidase inhibition was measured using 25 μl to 200 μl of the aqueous extracts.

 

The IC50 values were determine from plots of percent inhibition vs log inhibitor concentration and calculated by non-linearregression analysis from the mean inhibitory values.

 

1.2.    Baker's yeast alpha glucosidase inhibitory activity[65]:

Preparation of enzyme:

The bakers yeast was weighted and kept for 24 hours in temperature 4˚C. then yeast was dissolved in 100 mM phosphate buffer, pH 6.8 and stirred for 2 hours and centrifuged at 12000 rpm in 4˚C for 10 min.

 

Inhibition activity:

The yeast α-glucosidase was dissolved in 100mM phosphate buffer, pH 6.8 was used as enzyme source; 10 mMparanitrophenyl-α-D-glucopyranoside was used as substrate. Syzygium cumini seed extract powder was weighed and mixed with dimethylsulfoxide to get a concentration of 20-100l/ml. The different concentration of plant extract was mixed with 320l of 100mM phosphate buffer (pH 6.8) and 50l of 10mM PNPG in the buffer and then it was incubated at 30C for 5 minutes. After the incubation, 20l of the buffer containing 0.5 mg/ml of the enzyme was added and further incubated at 30C for five minutes. Finally,3.0 ml of 50mM sodium hydroxide was added to the mixture and the absorbance (A) was measured at 410 nm on a spectrophotometer. The enzyme without plant extract was used as a control.

 

Abs(control)-Abs(extract))

Inhibition (%) =------------------------------------ X 100

Abs (control)

The IC50 values were determined from plots of percent inhibition vs log inhibitor concentration and calculated by non-linear regression analysis from the mean inhibitory values.

 

1.3.    Rat small intestinal alpha Glucosidase inhibitory activity[48]:

Preparation of crude alpha Glucosidase from rat small intestine:

Rat small intestine homogenate was prepared according to the method described by Alagesan Kathirvel et. al 2012 with slight modifications. Rats were anaesthesized by using halothane (usually 8-10ml) in a jar. Small intestine was removed and washed with 30ml of 0.9% of NaCl and placed in ice cold 0.9% NaCl. The small intestine was minced with a surgical knife and homogenized using Potter-Elvehjem type of homonizer in 50ml of 0.1 M potassium phosphate buffer of pH 6.8. After 30 min, the homogenates were centrifuged for 30min at 10,000 rpm at 4˚C. The supernatant was used as crude enzyme source.

 

Alpha glucosidase inhibitory activity:

The alpha-glucposidase inhibitory activity was determined by measuring the release of 4-nitrophenol from p-nitrophenyl alpha-D glucopyranoside. The assay mixtures for these experiments contained 0.3 ml of 10 mM p-nitrophenyl alpha glucopyranoside, 1.0 ml of potassium phosphate solution (0.1M, pH 6.8), 0.2 ml of crude enzyme solution and 0.2 ml of inhibitor extract, all in a final volume of 1.7 ml. Following an incubation time of 30 min at 37˚C, the reaction was terminated by the addition of 2.0 ml of 100 mM sodium carbonate solution. The librated p-nitrophenol was determined at 400 nM using spectrophotometer.

 

The % inhibition rates were calculated using formula,

 

Abs(control)-Abs(extract))

Inhibition (%) =---------------------------------------- X 100

Abs (control)

 

The control samples were prepared without addition of any extracts and the blank solution used phosphate buffer and the IC50 values were determine from plots of percent inhibition vs log inhibitor concentration and calculated by non-linearregression analysis from the mean inhibitory values.

 

SUCRASE INHIBITION ASSAY[65,66]:

Isolation of Sucrase enzyme

Rats were sacrificed, intestine removed and chilled with ice cold 80 mM phosphate buffer. The intestine was then cut open, the mucosa scraped off with a piece of glass rod and homogenized with four parts (v/v) of cold buffer. Nuclei and large cell debris were removed by centrifugation at 2000 to 4000 rpm for 10 mins and supernatant aliquoted into 1.5 ml vials and stored at -20C.

 

Determination of sucrase inhibitory assay:

The inhibition assay is carried out as illustrated by Honda and Hara.10 L of the enzyme along with varying concentrations of the sample is incubated for 10 minutes at 37C. Malate buffer (pH 6.0) is used to make up the volume up to 200 L. The enzyme reaction is initiated by adding 100 L sucrose solution (60 mM) and incubated for 30 minutes. The reaction is terminated by the addition of 200 L of 3, 5- dinitrosalysilic acid reagent and treating the mixture in a boiling water bath for 5 minutes. The absorbance of the solution is read at 540 nm. Sucrase inhibition rate is calculated as follows

 

Abs(control)-Abs(extract))

Inhibition (%) =------------------------------------- X 100

Abs (control)

 

Where, AC control is the absorbance of the control reaction (all reagents to be added except for the test sample), and the AS sample is the absorbance of the test sample. Models to target Specific/Particular enzyme.

 

DIPETIDYL PEPTIDASE-IV (GOUT SMALL INTESTINE) ENZYME INHIBITORY ACTIVITY[66]:

Isolation of Dipeptidyl peptidase IV enzyme:

The tissues were weighed (80120 mg) and homogenized in cold PBS (300 μl) containing 1% Triton X-100 and 100 KIU/ml aprotinin (Sigma). The tissue mixtures were centrifuged at 100 g for 10 min. The supernatants were transferred into Eppendorf tubes and centrifuged twice at 20,000 g for 10 min. at 4 C. The final supernatants were incubated with 2 mM glycylprolyl-p-nitroanilidetosylate in PBS for 30 min at 37 C. The reaction was terminated by adding 1 M acetate buffer (pH 4.2). DPP-IV activity was determined by measuring the release of p-nitroanilide absorbed at 380 nm.

 

DPP-IV inhibitory activity:

DPP-IV-inhibitory activity (IC50) was determined using a modified method of Lacroix and Li-Chan (2013). DPP-IV from goat intestine was resuspended in 0.1 M/L TrisHCl buffer, pH 8.0. The test sample (25μL) was pre-incubated with the equal volume of the substrate Gly- Pro-p-nitroanilide (1.6 mM) at 37 C for 10 min. Afterwards, 50μL of DPP-IV (0.01 U/mL, in 0.1 M/L TrisHCl buffer, pH 8.0) was added and the mixture was incubated at 37 C for 60 min. The reaction was stopped by the addition of 100μL of 1 M/L sodium acetate buffer, pH 4.0. The released p-nitroanilide as a hydrolysis product was measured at 405 nm.

 

 

Abs(control)-Abs(extract))

Inhibition (%) =-------------------------------------- X 100

Abs (control)

 

 

The IC50 was defined as the concentration of inhibitor required to inhibit 50 % of the DPP-IV activity under the assay conditions.

 

RESULT AND DISCUSSION:

A. Phytochemical analysis conducted on the plant extracts revealed the presence of constituents which are known to exhibit medicinal as well as physiological activities.

 

Analysis of the plant extracts revealed the presence of phytochemicals such as alkaloids, phenols, tannins, flavonoids, cardiac glycosides, steroids, terpenoids and carbohydrates.

 

B. The inhibitory activity of ethanolic extracts of syzygiumcuminion alpha amylase, alpha glucosidase, sucrose inhibition and Di-peptidyl peptidase inhibition assay was investigated in this study and theresults are shown in Table.

 

1 In the alpha amylase syzygiumcumini showed three different inhibition assay-

i.Wheat alpha amylase was investigated syzygiumcumini seed (73.86 μg/ml) and Acarbose (64.63μg/ml) showed 50% alpha amylase inhibition activity at the varying concentration.

 

ii.Fungal alpha amylase was investigated Syzygium cumini seed (101.26 μg/ml) and Acarbose (73.66 μg/ml) showed 50% alpha amylase inhibition activity at the varying concentration.

 

iii.Salivary alpha amylase was investigated Syzygium cumini seed (126.20 μg/ml) and Acarbose (108.55 μg/ml) showed 50% alpha amylase inhibition activity at the varying concentration.

 

Table:1 The percent inhibition of Wheat alpha amylase by Syzygium cumini (Seed), Acarboseat varying concentrations

Concentration (g/ml)

Syzygium cumini seed extract

Acarbose

% Inhibition

IC50 (g/ml)

% Inhibition

IC50 (g/ml)

20

33.020.125

 

 

73.86

36.090.047

 

 

64.63

40

39.270.047

42.20.046

60

46.860.160

49.860.028

80

53.360.112

55.50.097

100

56.540.135

59.440.082

 

Table:2 The percent inhibition of Fungal Alpha Amylase by Syzygium cumini (Seed), Acarboseat varying concentrations.

Concentration (g/ml)

Syzygiumcumini seed extract

Acarbose

% Inhibition

IC50 (g/ml)

% Inhibition

IC50 (g/ml)

20

32.460.159

 

 

101.26

35.510.092

 

 

73.66

40

37.220.162

40.410.163

60

41.170.183

46.590.148

80

45.30.122

52.960.163

100

49.880.181

56.30.141

 

 

Figure: (A) Percentage inhibition of Wheat alpha-amylase by Syzygium cumini seed

 

 

(B) Percentage inhibition of Wheat alpha-amylase by Acarbose

 

 

Figure 2: (A) Percentage inhibition of Fungal alpha-amylase by Syzygium cumini seed

 

Table:3 The percent inhibition of Salivary alpha amylase by Syzygium cumini (Seed), Acarboseat varying concentrations

Concentration (g/ml)

Syzygiumcumini seed extract

Acarbose

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

28.520.023

 

 

126.20

30.710.051

 

 

108.55

40

31.450.072

35.20.061

60

36.160.064

39.950.060

80

40.120.051

44.50.038

100

45.020.076

47.60.050

 

(B) Percentage inhibition of Fungal alpha-amylase by Acarbose

 

 

 

Figure 3: (A) Percentage inhibition of Salivary alpha-amylase by Syzygium cumini seed

 

 

(B) Percentage inhibition of Salivary alpha-amylase by Acarbose

 

2. In the alpha glucosidase enzymes syzygiumcumini showed three different inhibition assay:

i. TheSyzygium cumini seed extract were found to inhibit Bacillus stearothermophilus alpha-glucosidase inhibition activity at different concentrations and IC50 values of extract activity (117.44 g/ml) and Acarbose (98.31 g/ml ).

 

 

ii. The Syzygium cumini seed extract were found to inhibit baker's yeast alpha-glucosidase inhibition activity at different concentrations and IC50 values of extract activity (75.94 g/ml) and Acarbose (62.08 g/ml).

 

iii.The Syzygium cumini seed extract in rat small intestine alpha glucosidase enzyme inhibition and 50% inhibition showed IC50 values of extract (99.54 g/ml) and acarbose (78.60 g/ml) at varying concentration.

 

Table:1 The percent inhibition of Alpha glucosidase (from Bacillus stearothermophilus) by Syzygium cumini (Seed), Acarboseat varying concentrations

Concentration (g/ml)

Syzygium cumini seed extract

Acarbose

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

28.730.081

 

 

117.44

32.80.081

 

 

98.31

40

33.910.026

36.810.058

60

37.880.026

40.30.082

80

41.280.043

46.860.039

100

46.590.046

50.340.017

 

 

Figure 1:(A) Percentage inhibition of alpha glucosidase (from Bacillus stearothermophilus) by Syzygium cumini seed

 

 

Figure 1:(B) Percentage inhibition of alpha glucosidase (from Bacillus stearothermophilus) by Acarbose.

 

 

 

 

Table:2 The percent inhibition of alpha glucosidase (from Baker's yeast) by Syzygium cumini (Seed), Acarboseat varying concentrations

Concentration (g/ml)

Syzygium cumini seed extract

Acarbose

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

27.730.590

 

 

75.94

34.670.296

 

 

62.08

40

33.660.677

41.490.680

60

44.850.873

51.230.781

80

51.450.873

58.160.733

100

59.730.193

60.960.294

 

 

Figure 2: (A) Percentage inhibition of alpha glucosidase (from bakers yeast) by Syzygium cumini seed

 

 

(B) Percentage inhibition of alpha glucosidase (from bakers yeast) by Acarbose

 

Table:3 The percent inhibition of Rat small intestinal alpha glucosidase by Syzygium cumini (Seed), Acarboseat varying concentrations

Concentration (g/ml)

Syzygium cumini seed extract

Acarbose

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

30.870.903

 

 

99.54

36.691.149

 

 

78.60

40

36.690.931

41.60.682

60

39.660.904

45.730.894

80

44.440.682

51.161.185

100

51.030.560

54.260.447

 

 

Figure 3: (A) Percentage inhibition of Alpha glucosidase enzyme (from rat small intestine) by Syzygium cumini seed

 

 

(B) Percentage inhibition of Alpha glucosidase enzyme (from rat small intestine) by Acarbose

 

3.In the sucrase enzyme and Dipeptidyl peptidase-IV enzyme of syzygiumcumini shows different inhibition assay-

i.Sucrase inhibition assay:

The effectiveness of extract, Acarbose in sucrase enzyme inhibition are show in table and 50% inhibition showed IC50 values of extract (83.79 g/ml) and acarbose (70.18g/ml) at varying concentration.

 

 

Figure 3(i): (A) Percentage inhibition of Sucrase enzyme by Syzygium cumini seed

 

(B) Percentage inhibition of Sucrase enzyme by Acarbose

 

Table:3(i) The percent inhibition ofsucrase by Syzygium cumini (Seed), Acarboseat varying concentrations.

Concentration (g/ml)

Syzygium cumini seed extract

Acarbose

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

27.611.214

 

 

83.79

36.021.214

 

 

70.18

40

36.020.890

42.080.890

60

44.10.890

46.961.05

80

49.321.659

52.520.583

100

53.872.339

58.410.732

 

ii.Dipeptidyl peptidase-IV enzyme inhibition:

The Syzygium cumini seed extract were found to inhibit DPP-4 at different concentrations and IC50 values of summarized in (table-3-ii) extract activity (100.26 g/ml) and Acarbose (95.21 g/ml ).

 

Table:3(ii) The percent inhibition ofDipeptidyl peptidase-IV by Syzygium cumini (Seed), Sitagliptinat varying concentrations

Concentration (g/ml)

Syzygium cumini seed extract

Sitagliptin

% inhibition

IC50 (g/ml)

% inhibition

IC50 (g/ml)

20

29.810.079

 

 

100.26

33.090.079

 

 

95.21

40

33.810.108

36.640.051

60

38.410.167

40.70.103

80

46.110.103

46.170.108

100

49.720.140

51.950.079

 

 

Figure 3(ii): (A) Percentage inhibition of Dipeptidyl peptidase-IV enzyme by Syzygium cumini seed

 

(B) Percentage inhibition of Dipeptidyl peptidase-IV enzyme by Sitagliptin

 

DISCUSSION:

Diabetes mellitus is a common metabolic disorder which may eventually lead to multiple organ damage and syndromes. Synthetic hypoglycemic drugs like acarbose, miglitol and voglibose are used in conjunction with other antidiabetic drugs, but these inhibitors have been found to possess gastrointestinal side effects like abdominal discomfort, flatulence and diarrhoea. The extract was showed their potency in inhibition of carbohydrate metabolizing enzymes and phytochemical screening suggested that Syzygium cumini seed hydro-alcoholic extract contains alkaloids, glycosides, carbohydrates, proteins, tannins and phenolics, flavonoids were present. However steroids and mucilage were not present. The correlation observed between alpha-amylase, alpha-glucosidase, sucrase and dpp-4 inhibitory activities of extract, represents a parallel and effective inhibition of these carbohydrate metabolizing enzymes in the digestive tract.

 

CONCLUSION:

The results of the present study indicate that hydro-alcoholic seed extract of syzygium cumini showed the maximum alpha amylase and alpha glucosidase inhibitory activity. The plants may essentially contain herbal bioactive compounds inhibiting enzyme activity and further structural elucidation and characterization methodologies have to be carried out in order to identify the bioactive constituents. In conclusion, more research is required for developing a potential and valuable anti diabetic therapy using alpha amylase and alpha glucosidase inhibitors of plant origin.

 

 

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Received on 04.07.2018 Modified on 12.07.2018

Accepted on 11.08.2018 A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 311-319.

DOI: 10.5958/0975-4385.2018.00050.X