PHYTOCHEMICAL SCREENING OF AQUEOUS AND ETHANOL EXTRACTS OF SOME MEDICINAL PLANTS AND IN -VITRO STUDY OF INHIBITION OF α- AMYLASEHTML Full Text
Received on 23 March, 2014; received in revised form, 14 June, 2014; accepted, 28 July, 2014; published 01 August, 2014
PHYTOCHEMICAL SCREENING OF AQUEOUS AND ETHANOL EXTRACTS OF SOME MEDICINAL PLANTS AND IN -VITRO STUDY OF INHIBITION OF α- AMYLASE
Rita P. Mahajan, Rahul R. Mahire and Dhananjay H. More*
School of Chemical Sciences, North Maharashtra University, Jalgaon-425 001. (M.S.), India
ABSTRACT: In present study we screened four medicinal plant for the α-amylase inhibitory activity of each extracts phytochemicals and it was determined based on the colorimetric assay using acarbose as a reference compound. The aqueous and ethanol extracts of the medicinal plants viz. Enicostemma littorale, Acheranthus aspera, Abutilon indicum and Tridax procumbens were shows moderate α-amylase inhibitory activity against reference, account of that Acheranthus aspera shows potent activity towards remaining. Not only leaves but also whole plant is used for comparative in -vitro study of inhibition of α- amylase activity.
Medicinal plants; Aqueous extract; ethanol extract; Phytochemicals; α-amylase inhibitoryactivity
INTRODUCTION: The metabolic disorder is one of the common ailment in living organism. In human being numbers of diseases are well known which associated with metabolism. Diabetes mellitus is most common disease of metabolic malfunction depicted by chronic hyperglycemia or increased blood glucose levels. The malfunctioning associated with disturbances in carbohydrate, fat and protein metabolism resulting lack of insulin secretion 1. It has been observed that both genetic and environmental factors are responsible for the disease 2.Much subclinical complication may occur due long tern persistence of metabolic disorder including arteriosclerosis. In some cases diabetes mellitus may remain asymptomatic till ketoacidosis or coma, depending up on the severity of metabolic malfunction. 3
The diabetes mellitus may be classified into major as Type 1, Diabetes Mellitus and Type 2, Diabetes Mellitus. The classification is based on clinical observations. Type 1 is characterized by destruction of pancreatic beta cell whereas type 2 is due to decreased insulin sensitivity (insulin resistance) 4.
Alpha-amylase are distributed all over various organisms and show diverse substrate specificities, while possessing a common topology formed from three domain, one of which being a typical alpha-beta barrel. Inhibition of insect’s alpha-amylase is a proposed method of crop protection. On the other hand, inhibition of mammalian alpha-amylase is a proven therapeutic approach in diabetes and related disorders 5.
Widespread species of plants have been described in the scientific and popular literature as having hypoglycemic activity. Because of their perceived effectiveness, minimal side effects in clinical experience and relatively low costs, herbal drugs are prescribed widely even when their biologically active compounds are unknown 6-8.
Acarbose currently marketed as a medicine in the treatment of diabetes, lowering post prandial peaks of glucose. However, acarbose is principally known as an alpha-glucosidase inhibitor, and cause side effects such as, abdominal distension, flatulence, meteorism and possibly diarrhea. Therefore it is attractive to find a substance that has strong inhibitory activity against α-glucosidase. But minor effect on α-amylase activity. A problem that seem not to occur in the case of alpha-amylase inhibitors 5, 9.
MATERIAL AND METHOD:
Collection of plants:
Enicostemma littorale, Acheranthus aspera, Abutilon indicum and Tridax procumbens was collected from Satpuda Mountain in Maharashtra state of India and used freshly for extraction and isolation of their phytochemicals after grinding and the both extracts stored in the refrigerator and used when needed 10, 11.
Extraction and isolation:
The crushed plant material was placed in thimble of soxhlet apparatus 7, 12, 13 100g and 50g for whole plant and leaves respectively and extraction carried out by using ethanol as solvent (for 12-14 hrs). The extracts were filtered; ethanol was distilled off using rotary evaporator to furnish the desired brownish-green residue, the yield of which was 3.9% and 2.63% for whole plant and leaves respectively. The residues were dissolved in ethanol at 100, 200, and 500 µg/mL concentration14.
The 25g of crushed whole plant material was soaked in 25ml, 50ml, and 100ml each in distilled water for 24 hrs. The extract was filtered by using muslin cloth. The final volume were corrected to viz. 25mL, 50mL, and 100mL by washing residue with distilled water (13-14) and used for α-amylase inhibitory activity. Same procedure adopted for leaves.
Isolation of Phytochemicals:
Whole fresh plant of 35g E. littorale was ground in a mixure and homogenized by methanol: water mixture 4:1 (10 X Wt.) for 5 minute. Then it was filtered and the filtrate was evaporated. 2M H2SO4 was added and extracted with chloroform yielded terpenoids. Aqueous acid layer was made alkaline with NH4OH and then extracted with chloroform-methanol (3:1, 60 ml) twice, this extract afforded most of the alkaloids whereas remaining aqueous basic layer was evaporated and extracted with methanol yielded quaternary alkaloids. During purification of quaternary alkaloids, tannins was separated and analyzed 16. Similarly phytochemicals of remaining allthree plants isolated.
Tannins (A): (200 mg Compound in 10mL distilled water, filtered); 2ml filtrate + 2ml FeCl3 blue-black precipitate indicate the presence of Tannins. Terpenoids (B): 200mg Compound + 2ml acetic anhydride + Conc. H2SO4 red coloration of solution indicate the presence of Terpenoids. Alkaloids(C) and Quaternary alkaloids (D): (200mg Compound in 10ml methanol, filtered); a 2ml filtrate + 1% HCl + Steam, 1ml filtrate + 6 drops of Wagner reagent/ Dragendroff reagent, brownish precipitate/ orange precipitate indicate the presence of respective alkaloids 17, 18.
Dilution of Phytochemicals:
The 10, 50 and 100 µg/mL concentrations of isolated phytochemicals were prepared by dilution with appropriate solvents of each plant
α-amylase inhibitory activity:
The α-amylase inhibitiory activity of each extracts and phytochemicals was determined based on the colorimetric assay using acarbose as a reference compound (1-2). The Starch solution (0.5% w/v) was obtained by boiling and stirring 0.25g of potato starch in 50 ml of deionized water for 15 min. The enzyme solution (0.5 unit/mL) was prepared by mixing 0.001g of α-amylase in 100 ml of 20 M sodium phosphate buffer (pH 6.9) containing 6.7mM sodium chloride. The both extracts and isolated phytochemicals were dissolved in respective solvent to give concentrations for aqueous extracts are 25 mL, 50 mL and 100 mL, for ethanol extracts concentrations are 100 µg/mL, 200µg/mL and 500µg/mL. 10µg/mL, 50µg/mL and 100µg/mL for isolated phytochemicals. The color reagent was a solution containing 96 mM 3, 5-dinitrosalicylic acid (20 mL), and 5.31 M sodium potassium tartrate in 2 M sodium hydroxide (8mL) and deionized water (12 mL). 1 ml of each plant extract and 1 ml enzyme solution were mixed in a tube and incubated at 25°C for 30 min. To 1 ml of this mixture was added 1 ml of starch solution and the tube incubated at 25°C for 3 min. Then, 1 mL of the color reagent was added and the closed tube placed into an 85°C water bath.
After 15 min, the reaction mixture was removed from the water bath and cooled thereafter, diluted with 9 mL distilled water and the absorbance value determined at 540 nm. Individual blanks were prepared for correcting the background absorbance. In this case, the color reagent solution was added prior to the addition of starch solution and then the tube placed into the water bath. The other procedures were carried out as above. Controls were conducted in an identical fashion replacing plant extracts and isolated phytochemicals with 1 ml respective solvents. Acarbose solution (at the concentrations of 10µg/mL, 50µg/mL and 100µg/mL) was used as positive control. The inhibition percentage of α-amylase was assessed by the following formula:
Inhibition (%) = X 100
RESULTS AND DISCUSSION: All four plants showed satisfactory α-amylase inhibitory activity. This study deals with α-amylase inhibition activity of aqueous, ethanol extracts of whole plant and leaves as well as isolated phytochemicals of four plants. Both extracts and isolated phytochemicals found to have capacity better inhibition of α-amylase.In vitro α-amylase inhibition activity of aqueous extracts of whole plant and leaves.
FIG. 1 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF AQUEOUS EXTRACTS OF LEAVES
FIG. 2 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF AQUEOUS EXTRACTS OF WHOLE PLANT
Aqueous extracts of leaves and whole plant of four plants as shown in Table 1. Both extracts showed activity at higher concentration tested. Leaves of Enicostemma littorale exhibited grater activity i.e. it inhibits α-amylase activity by about 88.48 %, 84.96 % and 81.67 % at 25 mL, 50 mL and 100 mL concentrations respectively, followed by plant Acheranthus aspera and Abutilon indicum. Natural health products were clearly indicates as a promising avenue for the prevention of chronic diseases.
TABLE 1: IN - VITRO Α-AMYLASE INHIBITION ACTIVITY OF AQUEOUS EXTRACTS OF WHOLE PLANT AND LEAVES.
Dilutions in (mL)
|In -vitro α-amylase inhibition activity (% inhibition)|
E-1, E-2 and E-3 represent that 25 g crushed plant material in 25 mL, 50 mL and 100 mL distilled water respectively.
Similarly in aqueous extracts of whole plant, four plant extracts showed activity at higher concentration tested. In whole plant extracts the maximum inhibition (%) of Enicostemma littorale was 86.83 %, 83.74 % and 80.25 % at 25 mL, 50 mL and 100 mL concentrations respectively, followed by plant Abutilon indicum and Acheranthus aspera so, in conclusion both leaves and whole plant (aqueous) extracts Enicostemma littorale demonstrated grater activity but plant Tridax procumbens disappointing activity.
TABLE 2: IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF ETHANOL EXTRACTS OF WHOLE PLANT AND LEAVES
|In-Vitro α-amylase inhibition activity (% inhibition)|
FIG. 3 IN -VITRO α-AMYLASE INHIBITION ACTIVITY OF ETHANOL EXTRACTS OF LEAVES
FIG. 4 IN -VITRO α-AMYLASE INHIBITION ACTIVITY OF ETHANOL EXTRACTS OF WHOLE PLANT
Ethanol extract of leaves and whole plant of four plants are shown in Table 2. All four plants exhibited activity at higher concentration. In ethanol leaves extracts of Enicostemma littorale showed grater activity i.e. it inhibits α-amylase activity by about 77.55 %, 81.93 % and 85.59 % at 100, 200 and 500 µg/mL concentrations respectively, followed by plant Acheranthus aspera and Abutilon indicum. Similarly in ethanol extracts of whole plant, also plant Enicostemma littorale showed grater activity i.e. 66.39 %, 72.16 % and 85.95 % at 100, 200 and 500 µg/mL concentrations respectively, followed by plant Tridax procumbens and Acheranthus aspera. In overall conclusion of both extracts aqueous and ethanol of leaves and whole plant, Enicostemma littorale showed grater inhibition of α-amylase activity of all remaining three plants
TABLE 3: IN -VITRO α-AMYLASE INHIBITION ACTIVITY OF ISOLATED PHYTOCHEMICALS
|In- vitro α-amylase inhibition activity (% inhibition)|
(A – Tannins, B- Terpenoids, C- Alkaloids, D- Quaternary alkaloids)
FIG. 5 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF PHYTOCHEMICAL (A)
FIG. 6 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF PHYTOCHEMICAL (B)
FIG. 7 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF PHYTOCHEMICAL (C)
FIG. 8 IN - VITRO α-AMYLASE INHIBITION ACTIVITY OF PHYTOCHEMICAL (D)
Compound A of Acheranthus aspera inhibits the α-amylase having (%) 25.59, 48.55 and 57.62 % at 10, 50 and 100 µg/mL concentrations respectively, followed by plant Enicostemma littorale and Tridax procumbens. In Compound B the maximum inhibition (%) of Acheranthus aspera was 14.37, 28.41 and 43.04 % at 10, 50 and 100 µg/mL concentrations respectively, followed by plant Abutilon indicum and Enicostemma littorale. Compound C the maximum inhibition (%) of Tridax procumbens was 39.63, 47.80 and 53.04 % at 10, 50 and 100 µg/mL concentrations respectively, followed by plant Abutilon indicum and Enicostemma littorale. Compound D of Tridax procumbens inhibits α- amylase activity having 12.94, 19.12 and 31.16 % at 10, 50 and 100 µg/mL concentrations respectively, followed by plant Abutilon indicum and Enicostemma littorale
In overall conclusion, plant Acheranthus aspera showed maximum inhibition in compound A and B. similarly plant Tridax procumbens demonstrated maximum inhibition in compound C and D.
- World Health Organisation Consultation: Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. In Report of a WHO Consultation. Geneva; 1999.
- Nickavar B and Yousefiana N; Inhibitory Effects of Six Allium Species onα-Amylase Enzyme Activity. Iranian Journal of Pharmaceutical Research. 2009; 8 (1):53-57
- Nickavar B, Abolhasani L and Izadpanah H; α-Amylase Inhibitory Activities of Six Salvia Species. Iranian Journal of Pharmaceutical Research, 2008;7 (4):297-303.
- Christidus S, Savarimuthu I and Paul A. Antidiabetic effect of Symplocos cochinchinensis (Lour) S. Moore in type 2 diabetic rats. Journal of Ethnopharmacology. 2011; 134:298-304.
- Mahmoud N, Habibi AE, Hazareh N, Parichehreh Y, Parivar K and Larijani B. Trans Chalcone: a novel small molecule inhibitor of mammalian alpha-amylase; Journal of molecular Boilogy Rep. 2010:1-3.
- Daisy P, Jasmine R, Ignacimuthu S and Murugan E. A novel steroid from Elephantopus Scaber L; an Ethnomedicinal plant with antidiabetic activity. Journal of Phytomedicine. 2009:16; 252-257.
- Shirwaikar A, Rajendran K and Punitha I S R. Antidiabetic activity of alcoholic stem extract of Coscinium fenestratum in streptozotocin-nicotinamide induced type 2 diabetic rats. Journal of Ethnophaacology. 2005; 97:369-374.
- Akerele O. Traditional Medicine: Nature's medicinal bounty; don't throw it away; world health forum. 1993; 14:390-395.
- Al-Zuhair S, Dowaidar A and Kamal H. Inhibitory effect of dates extracts on α-amylase and α-glucosidase enzymes relevant to non-insulin dependent diabetes mellitus; Journal of Biochemistry Technology. 2010,2(2):158-160.
- Afonne OJ, Orisakwe OE, Obi E, Orish C and Akumka DD. Some pharmacological properties of Synclisia scabrida III. Indian journal of pharmacology, 2000; 32:239-241.
- Anyasor GN, Aina DA, Olushola M and Aniyikaye AF. Phytochemical constituent, proximate analysis, antioxidant, antibacterial and wound healing properties of leaf extracts of Chromolaena Odoratk. Annals of Biological Research. 2011; 2 (2):441-451.
- Vogel AI. Text book of Practical Organic Chemistry, 5th ed, 1994; 164.
- Karodi R, Jadhav M, Rub R and Bafna A. Evaluation of the wound healing activity of a Crude extract of Rubia cordifolia L. (Indian madder) in mice. International Journal of Applied Research in Natural Products. 2009; 2(2):12-18.
- Shimpi SR, Chaudhari LS, Bharambe SM, Kharche AT, Patil KP, Bendre RS and Mahulikar PP. Evaluation of Antimicrobial Activity of Organic Extract of Leaves of Aristolochia bracteata; Pesticide Research Journal, 2005;17(1):16-18.
- Chavan KM, Tare VS and Mahulikar PP. Studies on Stability and Antibacterial Activity of Aqueous Extracts of Some Indian Medicinal Plants; Orient Journal of Chemistry, 2003; 19 (2):387-392.
- Harborne J. B. A Guide to Modern Techniques of Plant Analysis, 3rd ed, London,UK. Published by Chapman and Hall. 1998; 6-7.
- Kumar GS, Jayaveera KN, Ashok Kumar CK, Umachigi PS, Vrushabendra Swamy BM and Kishore Kumar DV. Antimicrobial effects of Indian Medicinal Plants against acne including bacteria; Tropical Journal of pharmaceutical research, 2007; 6(2):717-723.
- Faraz M, Mohammad K, Naysaneh G and Hamid RV. Phytochemical Screening of Some Species of Iranian Plants; Iranian Journal of Pharmaceutical Research, 2003; 77-82.
- Gupta OP, Phatak S: Pandemic Trends in Prevalence of Diabetes Mellitus and Associated Coronary Heart Disease in India - Their Causes and Prevention. Int J Diabetes Dev Countries 2003; 23:37-50.
- Mahajan R. and More D. Evaluation of anticoagulant activity aqueous and ethenolic extracts and their isolated phytochemicals of some medicinal plants.International Journal of Pharmacy and Pharmaceutical Sciences, 2012; 4 (4), 498-500.
- Sudha P, Smita S Zinjarde, Shobha Y Bhargava and Ameeta R Kumar. Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants. BMC Complementary and Alternative Medicine 2011; 11-5.
- Mohan V, Spiegelman D, Sudha V, Gayathri R, Hong B, Praseena K, Anjana RM, Wedick NM, Arumugam K, Malik V, Ramachandran S, Bai MR, Henry JK, Hu FB, Willett W, Krishnaswamy K. Effect of Brown Rice, White Rice, and Brown Rice with Legumes on Blood Glucose and Insulin Responses in Overweight Asian Indians: A Randomized Controlled Trial. Diabetes Technology and Therapeutics. 2014 Jan 21.
- Anjana RM, Pradeepa R, Deepa M, Datta M, Sudha V, Unnikrishnan R, Nath LM, Das AK, Madhu SV, Rao PV, Shukla DK, Kaur T, Ali MK, Mohan V. The Indian Council of Medical Research – India Diabetes (ICMR – India B) Study: Methodological details. Journal of Diabetes Science and Technology. 2011; 5:906-914.
How to cite this article:
Mahajan RP, Mahire RR and More DH:Phytochemical Screening of Aqueous and Ethanol Extracts of Some Medicinal Plants and In -Vitro Study of Inhibition of α- Amylase. Int J Pharmacognosy 2014; 1(8): 501-506: .doi: 10.13040/IJPSR. 0975-8232.IJP.1(8).501-506.
All © 2014 are reserved by International Journal of Pharmacognosy. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Rita P. Mahajan, Rahul R. Mahire and Dhananjay H. More*
School of Chemical Sciences, North Maharashtra University, Jalgaon-425 001. (M.S.), India.
23 March, 2014
14 June, 2014
28 July, 2014
01 August, 2014