Comparison of Alpha Amylase Inhibitory Potentials of Different Fractions of Cassia auriculata Leaf Extract

Shambhulingaiah H M, Asma Jaben, Sahana S C, Vinuta P C, Traveni T, Karthik P,

doi:10.5281/zenodo.17502374

Research Paper | Open Access
Volume 03 | Issue 11 | Article Id IJPS/250310489

Comparison of Alpha Amylase Inhibitory Potentials of Different Fractions of Cassia auriculata Leaf Extract
Shambhulingaiah H M* Asma Jaben Sahana S C Vinuta P C Traveni T Karthik P
Department of Pharmacology, SCS College of pharmacy, Harapanahalli -583131, Karnataka

Abstract

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia caused due to defects in insulin production, insulin sensitivity or both, Type 2 diabetes mellitus (T2 DM) represents the most frequent type of DM, accounting for more than 90?ll DM cases globally, One therapeutic approach which may prove to be beneficial for treatment of diabetes is to decrease the post-prandial hyperglycemia. This can be achieved by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes in the digestive tract. Many plants and their products have been widely prescribed and used for diabetic treatment all around the world, Tanner's Cassia or Cassia auriculata is one such traditionally used plant belongs to Fabaceae Family has been used in the present work with aim of comparison of alpha amylase inhibitory properties of different fractions of alcoholic extract of Tanner's Cassia leaves and findings from the studies revels that all the fractions shows the inhibitory properties on carbohydrate metabolism with increasing order of concentration but alcoholic fractions shows significant increase in the alpha amylase inhibitory properties may be due the presence of varying concentration of phytoconstituents including polyphenols.

Keywords

Cassia auriculata; Type 2 diabetes mellitus; Hyperglycemia; alpha amylase; Polyphenols

Introduction

Diabetes mellitus is a metabolic disorder characterized by hyperglycaemia caused due to defects in insulin production, insulin sensitivity or both. The major complications of diabetes include abnormally high blood sugar levels and blood vessel diseases, which may further cause long-term damage to vital organs such as the eye, kidney, nerves, and heart.¹ In recent years, the prevalence of diabetes has increased worldwide. The global prevalence of diabetes is estimated to be 9.3% in 2019, rising to 10.2% by 2030 and 10.9% by 2045.² The International Diabetes Federation (IDF) has reported that approximately 463 million people (20–79 years) are living with diabetes in 2019, and the number is projected to rise to 578 million by 2030 and 700 million by 2045. Diabetes has caused around 4.2 million deaths worldwide.

Diabetes mellitus popularly known as diabetes, is a global health problem and one of the leading causes of death worldwide. The latest data shows that around 463 million adults are surviving with diabetes. Controlling blood glucose levels is an essential intervention for treating and managing diabetes and related complications.³

India currently has the second-highest number of diabetics globall, with 66.84 million, behind China, which had 96.28 million in 2016, However recent data suggested that India will become the first country with a high number of diabetics due to fast food consumption and an uncontrolled lifestyle.⁴

Type 2 diabetes mellitus (T2 DM) represents the most frequent type of DM, accounting for more than 90% fall DM cases globally. Even though DM is not infectious, this disease is one of the significant reasons for mortality, as well as creating additional medical issues such as high blood pressure, heart disease, stroke, and chronic renal disease.⁵

Over the years, the diabetes prevalence has been increasing in India. The age-adjusted prevalence of diabetes is expected to increase from 9% in 2011 to 10.8% by 2045.

Moreover, more than half of the diabetic individuals in India are undiagnosed. A recent study reported that there are 101 million individuals with diabetes and 136 million individuals with pre diabetes in India. In 2019, India had an age-standardized incidence of diabetes at 317.02 per 100,000 populations and a mortality of 27.35 deaths per 100,000 population.⁶

The risk of diabetes can be either due to modifiable risk factors, non-modifiable ones, or both. Studies on the epidemiology of type-2 diabetes in India identified that genetics, family history, age, ethnicity, unhealthy diet, physical inactivity, use of tobacco and alcohol, high body mass index, raised blood sugar, and blood lipid levels are major risk factors for diabetes. Further, it has been found that high blood pressure, heart disease, and stroke are associated with diabetes.

The number of people in the world with diabetes has increased dramatically over recent years. It is also predicted that by 2030, India, China and the United States will have the largest number of people with diabetes.⁷ Currently treatments of diabetes, in addition to insulin supplement includes many oral hypoglycemic agents along with appropriate diet and exercise. One therapeutic approach which may prove to be beneficial for treatment of diabetes is to decrease the post-prandial hyperglycemia. This can be achieved by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes in the digestive tract, like The alpha glucosidase and α-amylase, these are responsible for the breakdown of oligo and/or disaccharide to monosaccharides. Inhibitors of these enzymes delay carbohydrate digestion and prolong overall carbohydrate digestion time causing a marked decrease in the rate of glucose absorption thereby blunting the post prandial plasma glucose rise.⁸ Examples of such inhibitors which find application in the clinical practice for management of diabetes are acarbose, miglitol and voglibose.⁹However, these drugs are known to be associated with various gastrointestinal side effects such as abdominal pain, flatulence and diarrhoea in the patients.^10,11Therefore, it is the need of time to identify and explore the amylase inhibitors from natural sources having fewer side effects. The Indian traditional system of medicine practiced for over thousands of years have reports of numerous anti- diabetic plants with no known side effects. Many plants and their products have been widely prescribed and used for diabetic treatment all around the world with less known mechanistic basis of their functioning. Thus, these natural products need to

be evaluated scientifically in order to verify for their anti-diabetic properties.

Cassia auriculata or Tanner's Cassia is one such plants belongs to Fabaceae Family, various parts of plants consists of different phytocnstituents like polyphenols, alkaloids tannins etc. Exhibit different pharmacological actions and are witnessed in traditional as well as modern health care professionals so present project work has taken to evaluate the in-vitro antidiabetic potentials of different fractions of the plant extract on alpha amylase enzyme inhibition and to establish the potential in- vitro mechanism to justify the traditional and modern claim.

MATERIALS AND METHODS

Collection and authentication

The leaves of Cassia auriculata Linn were collected around the fields of Harapanahalli, Vijayanagara(D) Karnataka, Authentication of Raw Plant Material is the basic Starting Priority in Developing of Botanical/Herbal Product. Cassia auriculata Linn Plant Material was authenticated by Prof. K Prabhu Sir, Dept. Of Pharmacognacy, S.C.S College of Pharmacy. Harapanahalli Herbarium ( 10 UG 2025) is deposited in the Department of Pharmacognocy.

Preparation of Cassia auriculata Linn leaves extract.

In this study shade dried leaves material of Cassia auriculata Linn extract were obtained by hot extraction method in SOXHLET APPARATUS..

Extraction procedure ¹²

Collected Leaves of Cassia auriculata Linn were dried under shade, mixed together & then made in to coarse powder with mechanical grinder.
The powder was pass through Sieve No.60 and stored in an air-tight container for future use
The dried powder material (185 gms) was extacted with 95% ethanol in a Soxhlet extractor or 72hrs
The solvent was then distilled off and the resulting semisolid mass was dried in a vacuum evaporator& yield was calculated.

Fig.01: Extraction Process

The percentage yield was calculated for the extracts with reference to the crude material taken using the formula given below. The percentage yield of the each extract is tabulated in table no 01.

% of yield = Weight in grams of extract obtained X100

Weight in grams of plant material taken

Fractionation ¹³

Fractionation of crude extract was carried out by suspending 73.54gm of extract in 200ml of water and then partitioning with hexane, chloroform , acetate and alcohol in order of increasing polarity by using separating funnel. All the five fractions including aqueous fraction were condensed using Rotary evaporator or water bath. Fractions so obtained were hexane (13.61gm), choloroform (18.3 gm), ethyl acetate (4.23gm), ethanol (18.07gm) and aqueous (3.54gm). Scheme used in fraction of crude ethanol extract is summarized in Fig.8.

Fig.02 : Summary of scheme used for fractionation process

Fig.03 : Separation of different solvent layers used for fractionation

Preliminary phytochemical screening

The solution of various fractions were prepared using same solvents and subjected to the following investigations. The obtained extract will be subjected to preliminary phytochemical screening following the standard procedures described in the practical Pharmacognosy by C.K.Kokate¹⁴and R.K.Khandelwal ¹⁵results are summarized in table no 03.

1. Detection of Carbohydrates:

Molish test : Few drops of alcoholic a – naphthol solution were added to 2ml of extract. Later, few drops of concentrated H2SO4 were added along the walls of test tube. At the junction of two liquids, a violet colour ring appeard, indicating that carbohydrates were present.
Benedict’s test : To 5ml of Benedict’s reagent, 8 – 10 drops extract were added, then heated for five minutes; the resulting dark red precipitate indicated the presence of carbohydrates.
Fehling’s test: to 2ml of extract, an equal volume of Fehling’s (A & B) solution was added and heated for five minutes, the resulting red / dark red precipitate indicating the presence of Carbohydrates.

2. Detection of Glycosides :

Take small quantity of ethanol extract make it into two portion solution A and solution B. To the solution A add Fehling’s solution A and B, boli. To the solution B add sulphuric acid and Fehling’s solution A and B and boil. Formation of red colour in test tube containg solution A and B. If test tube contain solution B shows dark red colour than the solution A. It indicates presence of Glycosides.

Test for Saponin and steroid glycosides: Preparation of test solution: take small quantity of ethanol extract and add dil . Hcl, heat for few minutes. Use the smaple for following test:

Liberman Burchard Test: Mix 2ml test solution (extract) with 1ml of choloroform, 1ml of acetic anhydride and add one drop of concentrated H2SO4. Blue green to red colour indicates presence of saponins.
Libermann Test: Mix 2ml of extract with 2ml of acetic anhydride, boil and add 0.5ml of H2SO4. Blue green to red colour indicates presence of steroid saponins.

3. Test For Anthroquinones:

Preparation of test solution:

Take small quantity of ethanol extract with Hcl heat on water bath foe few minutes. Use this solution for following tests:

Born Trager’s Test: Take little quantity of test sample add H2SO4 and add CCl4 or Benzene separate the organic layer and shake with dil.ammonia. Rose pink colour formed in organic layer indicates the presence of Anthroquinone o glycosides.
Modified Borntrager’s test: Take the little quantity of test solution of above (aqueous) and treat with ferric chloride solution, and Hcl .Heat in water bath for five minutes, cool shake the cooled mixture add benzene or CCL4, separate the organic layer and shake it with half of its volume of ammonia. Rose pink to cherry red colour is formed anthroquinone c glycosides are present.

4. Test For cardiac Glycosides:

Keller- killiany test: Take a sample and add 5ml of water and 0.5ml of strong solution of lead acetate shake well and filter. The clear filtrate is treated with equal volume of chloroform and choloroform layer is evaporated. The residue is dissolved in 3ml of glacial acetic acid and to this add two drops of ferric chloride solution. The contents are transferred to a test tube containing 2ml of conc. Sulphuric acid. Formation of reddish brown layer acquiring bluish green colour after standing due to digitaxose sugar.
Kedde’s test: Mix 1 ml of test solution(extract)with 2 ml test reagent i.e. kedde’s reagent (dissolve 3,5,dinitrobenzoic acid of 2 grams in 90% ethanol 100 ml). blue to purple colour indicates the presence of cardiac glycosides.
Legal test : Mix 1 ml of alcoholic test solution with 2 ml of pyridine and sodium nitroprusside. Formation of pink or red colour indicated present of cardiac glycosides.

5. Test For Flavonide Glycosides :

Preparation of test solution: solution of ethanol extract with dil.Hcl extract filter for 20 minutes, take filtrate as test solution for following test:

Shinoda test : To the test solution, add 5ml of 95% alcohol, few drops of conc. Hcl and 0.5 gram of magnisum turings. Formation of pink colour indicate the presence of flavanoids.
Lead acetate test : Mix test solution with lead acetate. Formation of yellow ppt indicates presence of flavanoids.

6. Detection of Phenolic compounds and Tannins:

Ferric chloride test : Two ml of 5% neutral ferric chloride solution were added to 1ml of extract, the dark blue colouring indicating the presence of phenolic compounds and tannins.
Lead tetra acetic acid test : 1ml of lead tetra acetate solution was treated with 0.5 ml of extract, precipitate formation indicating the presence of phenolic compounds and tannins.

7. Detection of Alkaloids:

Dragendorff’s test : By adding 1ml of Dragendorff’s reagent to 2ml of extract, an ornge red precipitate was formed, indicating the presence of alkaloids.
Mayer’s test : Few drops of Mayer’s reagent were added to 1ml of extract. A yeelowish or white precipitate was formed, indicating the presence of alkaloids.
Hager’s test : 2ml of extract were treated with few drops of Hager’s reagent. A yellow precipitate was formed, indicating the presence of alkaloids.

8. Detection of Proteins:

Biuret test : Two drops of 3% copper sulphate and few drops of 10% sodium hydroxide were added to 1ml of extract, violet or red colour formation indicating that proteins are present.
Ninhydrin test : two drops of 0.2% freshly prepared ninhydrin solution added to 1ml of extract. Production of purple colour shows the presence of proteins.

Αlpha- Amylase inhibition

Alpha amylase is an enzyme that hydrolyses alpha-bonds of large alpha linked polysaccharide such as glycogen and starch to yield glucose and maltose.Alpha amylase inhibitory activity was based on the starch iodine method that was originally developed by Fuwa (1954)¹⁶and later employed by others for determination of amylase activity in plant extracts with some modifications.¹⁷

In alpha amylase inhibition method 1ml substrate- potato starch (1%w/v), 1 ml of drug solution (Acarbose std drug/Different fractions of ethanol extract) of four different concentration such as 250, 500, 750 and 1000 μg/ml, 1ml of alpha amylase enzyme (1% w/v) and 2ml of acetate buffer (0.1 M, 7.2 pH) was added.

(NOTE- Potato starch solution, alpha amylase solution and drug solution was prepared in acetate buffer (820.3 mg Sodium acetate and 18.7mg sodium chloride in 100ml distilled water).

The above mixture was incubated for 1 hr. Then 0.1 ml Iodine-iodide indicator (635mg Iodine and 1gm potassium iodide in 250ml distilled water) was added in the mixture. Absorbance was taken at 565 nm in UV-Visible spectroscopy.

Inhibition of alpha- Amylase (%) =

Abs control – Abs sample X 100

Abs control

Where, Abs control is the absorbance of the control reaction (containing all reagents except the test sample) and Abs sample is the absorbance of the test sample. All the experiments were carried out in triplicates.

All values were expressed mean ± SD. Statistical difference and linear regression analysis were performed using Graph pad prism 5 statistical software.

5. RESULTS

I. Preparation of extract and percentage of yield

The percentage yield of 95 % etanol extract was given in the following table no.01

Percentage and Color of the Extract:

Table No 01 The percentage yield of 95 % etanol extract

Sr. No	Solvent type	Wt. of the crude sample before extraction (gm)	Wt. of the extract (gm)	Percentage of yield (%)	Colour of the extract	Consistency
01	95% Ethanolic extract	550 gm	180.25 gm	32.77 %	Bluish Green	Sticky and Viscous

II. Fractionation

Fractionation of 95 % Ethanol extract was carried out with different solvents with increasing order of polarity and fractions so obtained were given in the table no.02.

Table no.02. Weight of Ethanol fractions.

Sr.no	Solvent fraction	Qut in gms
01	n- Hexane fractions	13.61
02	Chloroform fractions	18.34
03	Ethyl acetate fractions	04.6
04	Ethanol fractions	18.07
04	Aqueous fractions	03.54

III. Qualitative analysis of phytochemicals

The obtained extracts were tested for various chemical constituents according to standard procedure and results are shown in the following table no 03.

Table no.03. Phytochemical analysis of different fractions of Cassia auriculata leaves extract

Sr. no	Name of phytochemicals	n- Hexane fractions	Chloroform fractions	Ethyl acetate fractions	Ethanol fractions	Aqueous fractions
01.	Carbohydrates	+ +	+ +	+ +	+ +	+
02.	Glycosides	+ +	+ +	+ +	+ +	+ +
04.	Alkaloids’	+ +	+ +	+ +	-	-
05.	Phenols and Tannins	+	+	+ +	+ + +	+ +
06.	Saponins	+	+ +	+ +	+ +	-
07.	Flavonoids	+ +			+ + + +	+ + + +
08.	Proteins and Amino acids	_			++ ++

Present + Absent - Intensified results +

IV. In-vitro alpha amylase inhibition activity

In-vitro Alpha amylase inhibition activity of different fractions of plant ethanol extract carried out according to the procedure and % inhibition were calculated and results are given in the no.04 and fig no 04.

Sr. No	Concn. (μg/m)	Percentage of inhibition %
Sr. No	Concn. (μg/m)	STD	n-Hex fractions	Chlo fractions	Ethyl fractions	Ethan fractions	Aqu fractions
1	250	63.40	32.11	28.43.	32.12	48.01	41.02
2	500	69.84	36.23	33.45	38.43	53.34	43.23
3	750	75.26	40.23	37.35	46.24	55.65	47.34
4	1000	82.12	47.12	40.08	51.45	59.23	53.13

Fig no 04 . Graphical representation of % inhibition alpha amylase inhibition activity

DISCUSSION & CONCLUSION

Diabetes mellitus, popularly known as diabetes, is a global health problem and one of the leading causes of death worldwide. In diabetes high postprandial blood glucose leads to micro vascular complications like retinopathy, nephropathy, neuropathy, and macrovascular complications include the increased atherosclerosis-related events such as myocardial infarction and stroke are the leading cause of death and economical burden among the populations.¹⁸

Management of Diabetes Mellitus is a global problem. Successful treatment is very important for preventing or at least delaying the onset of long-term complications. Regulation of glucose level in the blood of the diabetic patient can prevent the various complications associated with the disease. The maintenance of plasma glucose concentration for a long term under a variety of dietary conditions is one of the most important and closely regulated processes observed in the mammalian species.¹⁹

One of the therapeutic approaches for controlling postprandial hyperglycemia in diabetic patient is to prevent or decreasing absorption of carbohydrate after food intake. Complex starches, oligosaccharides, and disaccharides must be broken down into monosaccharides by carbohydrate metabolising enzymes like α-amylase and α-glucosidases before they are absorbed in the duodenum and upper jejunum.²⁰

The diet usually contains a mixture of mono-, di- and polysaccharides such as glucose, sucrose and starch. Within the digestive tract, complex carbohydrates are initially converted into monosaccharides before they are absorbed and lead to a postprandial elevation of blood glucose level. The first step of dietary starch breakdown is accomplished by the enzyme α-amylase, which is predominantly secreted by salivary glands and the pancreas . As a member of endoenzymes, α-amylase degrades complex starch molecules into oligosaccharides of 6 - 8 glucose units by catalysing the hydrolysis of internal α-1,4 glycosidic bonds.

Recent advances in understanding the activity of intestinal enzymes helped in the development of newer pharmacological agents.²¹ these inhibitors reduce intestinal absorption of starch, dextrin, and disaccharides by inhibiting the action of carbohydrate metabolizing enzymes in the intestinal brush border. Inhibition of this enzyme slows the absorption of carbohydrates from the GI tract and decreases the rate of rise of postprandial glucose (PP hyperglycemia). This delay in digestion and breakdown of starch may have beneficial effects on insulin resistance and glycemic index control in people with diabetes.²²

Natural substances and plant extracts in particular, already play an important role in the control of glucose homeostasis. It is therefore promising to expand our portfolio of natural substances in this field and to find even more effective drugs with fewer side effects.

In our present studies we selected traditionally well-known plant Cassia auriculata or Tannirs cassia of Fabaceae Family to compare the inhibitory properties of carbohydrate metabolising enzymes like alpha amylase, Extraction is the crucial step for the analysis of medicinal plants and choice of solvents is crucial in solvent extraction, solvent should have low toxicity and preservation capabalities, promote extract absorption and low temperature evaporation and also consider the quality of phytochemicals and metabolites recovered, to extract polar and non polar components polar and non polar solvents are typically utilized based on polarity solvents from least polar to polar like n-Hexane to water are used for extraction and usually aqueous mixture of ethanol and methanol are used in extraction as they have better efficiency of salvation

In the current investigation, the extraction process was carried out using ethanol as the solvent because it was readily available and less hazardous than methanol. Because of its high dielectric constant, ethanol can extract the maximum amount of Bioactive components from the plant, including polyphenols, tannins, flavonoids, terpenoids, and alkaloids ²³

For this reason, ethanol was chosen as the solvent for preparing the extract in the current investigation. So in our studies we selected 95 % alcohol as extraction solvent and we got percentage of yield of 25.20 gm % revels the presence of phytoconstituents in the extract.

In fractionation selected solvent is added according to the increasing order of polarity starting from n-Hexane to water, in all combination water is present because of its high polarity and miscibility with organic solvents and we found that highest concentration in ethanol (Quantity) fractions and in Qualitative phytochemcal analysis of various solvent fractions shows the presence of various secondary metabolites like alkaloids, saponins proteins except in aqueous fractions and other metabolites like phenolic compounds and Flavanoids are present in all fractions but in ethanolic fractions more intinsfied results indicate the presence of more concentration of these compounds our research finding revels that all the fractions of plant extract shows the increasing order of alpha amylase inhibition with increasing order of concentrations and in that ethanol fractions shows higher percentage of inhibition from 48.01 to 59.23% when compared to other fractions but less when compared to standard drug Acarbose which shows inhibition percentage from 63.40 to 82.12% from 250 microgram to 1000 micro gram per ml concentration respectively, Indicating that ethanol fractions are having significnt inhibitory properties may be due to the presence of higher concentration phytoconstituents including polyphenols. But further more detailed quantification studies and various in-vitro and In-vivo studies are required to justify the claim.

ACKNOWLWDGEMENT

We are thankful to management, principal and other staff members for their support and guidance to carry out this research work.

CONFLICTS OF INTEREST

Authors declare that no conflict of interest

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