AN EVALUATION OF ANTI-DIABETIC ACTIVITY OF CALOTROPIS PROCERA LEAF EXTRACTION (AAK PALNT)
HTML Full TextAN EVALUATION OF ANTI-DIABETIC ACTIVITY OF CALOTROPIS PROCERA LEAF EXTRACTION (AAK PALNT)
Mohd Aalam *, Gulafshan Parveen and Chavi Mittal
Guru Nanak College of Pharmaceutical Sciences, Jhajra, Dehradun, Uttarakhand, India.
ABSTRACT: Aim: This study reviews the anti-diabetic property of Calotropis procera leaf extract. Calotropis procera used for different pharmacological activities. Calotropis procera has a number of phytoconstituents which are: Cardenolides, Terpenes, Flavonoids, Enzyme and other more components which are used to treat a number of diseases. They have some pharmacological activities like: cytotoxic activity, anti-diabetic, antioxidant, analgesics, anti-inflammatory, anti-arthritic, anthelmintic, antimicrobial, wound healing, hepato-protective, immune response, myocardial infarction and so on. Calotropis procera plant which is used to treat diabetes, it inhibit the α-amylase and α- glycosidase enzyme that are responsible for increasing the blood glucose level. Methanolic Extraction and Aqueous extraction of the Calotropis procera leaf is used as a drug. We also can use these extractions in novel technologies to treat the disease. It was studied by in-vitro testing of the extraction, which inhibits the particular enzyme by the non-competitive enzyme inhibition mechanism. Calotropis procera having a major effect of anti-diabetic inhibits these enzymes. Calotropis procera is the major source of anti-diabetic agents acting on the carbohydrate enzymes that are α- amylase and α- glycosidase. This study is revealed the anti-diabetic effect of Calotropis procera.
Keywords: Calotropis procera, α- amylase, α- glycosidase, Diabetes, Drug non- competitive
INTRODUCTION: Calotropis procera Linn (Apocynacea) have a several number of important medicinal properties that have been mentioned in Ayurveda, Madar (Hindi), Alarka (Sanskrit), and Ark; swallow wart or milkweed are some of the common names for this plant. It is a common wasteland weed in Indonesia, Malaysia, China and India, and it is found in different regions of the world. Plants as a source of new drugs have focus for major pharmaceutical companies, and they are continuously conducting or performing the many researches on plants material to introduce new drugs into medical practices.
As per the literature review, Calotropis procera contains various physiologically active chemical groups such as Tannins, Steroids, Glycosides, Phenols, Flavonoids, Alkaloids, etc. These phytoconstituents are effective in many pharmacological actions as antimicrobial, anti-inflammatory, anthelmintic, analgesic, fever-reducing agents, anti-cancer, anti-angiogenic, immuno-logical, anti-diabetic, cardiovascular, hypolipidemic, gastro-protective, hepatic protective, anti-oxidant, anti-convulsant, and wound healing enhancement, anti-fertility and effect of smooth muscle relaxant. This review is designed to deliver the anti-diabetic activity of Calotropis procera.
Plant Profile: C. procera is an erect, tall, large, highly branched perennial shrub or small tree that grows to 5.4 min-height and has milky latex throughout the plant.
FIG. 1: CALOTROPIS PROCERA
Synonyms: Calotropis gigantean var procera (Aiton), Calotropis inflexachiov, Calotropis heterophyalla wall, ex wight, Calotropis persica gand, Calotropis wallichii wight, madorius procerus (Aiton) Kuntze, Asclepia spetula decne, Apocynum sariacum Garsault, Asclepias procera aiton, etc. There is little information in the literature about the potential of the anti-diabetic activity of the secondary metabolites found in the leaves of this species. Etuk and Mohammed (2009) found that an aqueous extract of C. procera have anti-diabetic activity to the Alloxan-induced rat.
On the other hand, Rehmatullah et al. (2010) tested the anti-diabetic activity of a methanol extract of C. procera leaves on diabetic mice and found no effects. In this way, the current review was designed to look into the effects of long term therapy with a hydro alcoholic extract of C. procera leaves on physic 3 chemical blood parameters, the oral glucose tolerance test and other diabetic disorders in Streptozotocin-induced diabetic rats. The current study concentrated for the Anti-diabetic activity of Calotropis procera. C. procera has been reported to have a number of medicinal uses. The plant was fully used to treat many common diseases such as fever, rheumatism, indigestion, cold, eczema, and Diarrhoea, the treatment of elephantiasis was done by applying the paste of root bark topically and the powder of root bark was used to treat Diarrhoea and Dysentry and it’s a good substitute of ipepac. This plant has many traditional uses such as cholera, extract guinea worms and treat indigestion. To promote healing, just dried powdered leaves on wounds, ulcers, and old sores. Cold, cough, asthma, ear ache, eczema, scars on face, skin eruption, pain of body, syphilis, leprosy, and edema are all treated with Calotropin which is isolated from latex. Milky latex is used locally to treat cutaneous diseases like ringworm, syphilitic sores, and leprosy. Calotropis procera flower extracts were tested for cytotoxicity against human colorectal carcinoma cell lines and demonstrated strong cytotoxic activity. Researchers have made significant efforts to demonstrate its efficacy as a curative agent through.
Phytochemical Constituents: The study of phytochemicals on Calotropis procera exposed that various types of compounds are acquire from various parts of the plant and from plant latex. Table shows the list of chemical compounds which are isolated from C. procera, including Cardenolides, Triterpenoids, Proteolytic enzymes, resins, flavonoids, tannins, sterol, and Terpenes.
Antidiabetic and Antioxidant Properties: The latex of Calotropis procera in a dry form tested for the antioxidant and anti-hyperglycemic effects in Alloxan induced diabetic rats. The dose of dry latex on a daily oral administration is 100 and 400 mg/kg, which resulted in a dose dependent in the decrease in the blood sugar level and enhance in the hepato-glycogen content. Dry latex also prevented many diabetic rats from losing body weight and reduced daily water intake to levels as compared to normal rats. In that rat in which the Alloxan induces, Dry latex increase the level of endogenous antioxidants such as superoxide dismutase, catalase and glutathione, rather, it decreases the level of thiobarbituric acid reactive substances. The anti-diabetic and antioxidant effects of dry latex are compared to that of the standard anti-diabetic glibenclamide. The Calotropis procera antioxidant activity by methanol extract was determined according to the scavenging activity of the stable 1, 1-diphenyl-2-picryl hydrazyl (DPPH) free radical. The IC50 of the methanol extract revealed that the plant has high antioxidant activity. The aqueous extract, on the other hand, demonstrated mild antioxidant activity.
METHODOLOGY:
Plant Extraction: Cut the Fresh Calotropis procera leaves and wash them with water to dismiss all the impurities before being dried at room temperature and powdered.
The powdered leaves were then divided into two parts and dissolved in ethanol and water.
They all kept a side incoveredcontainerfor1 day before the decantation, filtration, and evaporation rotatory evaporator. A freeze dryer was used to dry the extracts then the dry extract was weighed and dissolved into 10% of the dimethyl sulfoxide to create a stock solution for the preparation of lower concentrations.
FLOW CHART OF EXTRACTION PROCESS:
FIG. 2: SOXHLET ASSEMBLY
M.O.A of Alpha Amylase Inhibitors: The mechanism of inhibition of the enzyme through the leaf extraction was investigated by using that extract by the lowest IC50. 250 µl of the 5mg/l extract was preincubated for 10 minutes at 25oC with 250 µl of amylase solution in one set of tubes. Alpha amylase waspre-incubated of 250 µl of phosphate buffer (pH 6.9) in another set of tubes. To begin there action, 250 µl of starch solution with enhancing concentration (0.30-5.0 mg/ml) was added in both reaction mixtures. Then the mixture was incubated for 10 minutes at 25oC before being boiled for 5 minutes and 500 L of DNS added to stop the reaction.
The reducing sugars amount was released and measured spectrophotometrically, and the reaction velocities were extrapolated using a standard maltose curve. A double reciprocal plot (1/v vs 1/s) was created, where did S. represent presents reaction velocity and substrate concentration The type of alpha-amylase inhibition activity the extract was determined by using Michaelis- Menten kinetics by the analytical test of the double reciprocal (Line weaver- Burke Plot).
M.O.A of Alpha Glycosidase Inhibitors: Using the modified method, the mechanism of inhibition of the enzyme by the leaf extraction was investigated used the extraction of Calotropis procera with the lowest IC50. 50 µl of the (5mg/ml) extract was pre-incubated with 100 L of glycosidase solution (1.0 U/ml) for 10 minutes at 25o C in one set of tubes. Alpha-glycosidase was pre-incubated with 50 µl of phosphate buffer (pH 6.9) in another set of tubes. To initiate the reaction, 50 µl of P- nitro-phenyl glucopyranosideat increasing conc. (0.63-2.0mg/ml) were added to both sets of the reaction mixture.
The mixture then incubated for 10 minutes at 25oC before adding 500 µl of Na2CO3 to stop the reaction. Using a p-nitro-phenol standard curve, the amount of reducing sugars released was determined spectrophotometrically and converted to reaction velocities. A double reciprocal curve plot was created (1/v v/s 1/s), where v represents reaction velocity and S represents substrate concentration. The mode of alpha glycosidase inhibition mechanism by the extract was determined by using Michaelis-Menten kinetics and an analytical test of the double reciprocal (Line weaver burke) plot.
Alpha Glycosidase Inhibitors Assay: Assay was carried out using the method described by Kim et al (30), with a conc. of 1.0 µ/ml of glycosidase solution used. The P-nitro-phenyl glycol-pyranoside substrate solution prepared in 20m M phosphate buffer, pH 6.9. It was pre-incubated with 50µl of the different extract concentrations (acetone, ethanol and water) for 10 min. 100 µl of glycosidase. The reaction was started with50 µL of 3mM p-nitro-phenyl glycol-pyranoside dissolved in 20 mm phosphate buffer, pH 6.9. Add 2 ml of 0.1M Na2CO3 after 20 min. at 37oC for reaction stopped. The glycosidase activity was measured at 405 nm yellow-colored p-nitro-phenol released from the reaction. Control was made. Using the same procedure but substituting the extract with 10% DMSO the result were expressed as a percentage of the control group.
Percentage Inhibition Determined in a Percentage Inhibition:
% Inhibition = (Abs control - Abs extract) / Abs control × 100
Enzyme activity (IC50) were determined dgraphically by the conc. of extracts resulting in 50% inhibition.
Alpha Amylase Inhibitor Assay: Assay was performed using a modified McCue and Shetty procedure and the conc. of alpha-amylase solution used was 0.5 mg/ml. 250 µL of 0.02M sodium phosphate buffer (pH 6.9) was added 250 µL extract in a tube containing alpha-amylase solution. This solution was preincubated at 28 oC for 10 minutes before adding 250 µL of 1% starch solution in 0.02 M sodium phosphate buffer (6.9 pH) at time intervals and incubating at 25oC for another 10 minutes.
After incubation, 500Lofdinitro- salicylic acid reagent was added to stop the reaction. Before the cooling process, the tubes were boiled for 5 min. The reaction mixture was diluted with 5 ml of distilled water, and at 540 nm the help of a spectrophotometer measured it. Control was made by repeating the procedure but replacing the extract with the 10% DMSO. The inhibitory activity of alpha-amylase was determined in a percentage of inhibition.
% Inhibition= (Abs control-Abs extract) / Abs control x 100
Statistical Method: Statistical methods are used to analyze the anti-diabetic properties of Calotropis procera as a one-way ANOVA test.
Evaluation Studies:
TABLE 1: PHYTOCHEMICAL PRESENTS IN ETHANOL AND AQUEOUS EXTRACTION
S. no. | Phytochemicals | Extraction interference Ethanol | Extraction interference Water |
1 | Anthraquinones | - | - |
2 | Flavonoids | + | + |
3 | Reducing Sugars | + | + |
4 | Saponins | - | + |
5 | Steroids | + | + |
6 | Tanins | - | + |
7 | Terpenoids | - | - |
After testing this extraction, the α-amylase enzyme activity is inhibited with ethanol extract and aqueous extract on α-glycosidase enzyme by the non-competitive enzyme. The concentration of aqueous extract inhibition on α-glycosidase enzyme with 10mgml concentration. And ethanol α-amylase inhibition with 7.8 mgml. These are shown graphically in Fig. 3 and Fig. 4. Mechanism of inhibition of α-amylase enzyme by the ethanol extract of Calotropis procera leaf extract determined with the help of Lineweaver-Burke plot is shown in Fig. 5 & 6.
FIG. 3: INHIBITION OF ETHANOL EXTRACT
FIG. 4: INHIBITION OF AQUEOUS EXTRACT
TABLE: 2: IC50 INHIBITION OF ETHANOL AND AQUEOUS EXTRACT ON ALPHA-AMYLASE AND ALPHA-GLYCOSIDASE ENZYME
Extract | Alpha-Amylase IC50 | Alpha-Glycosidase IC50 |
Ethanol | 7.80 | 11.20 |
Aqueous | 15.75 | 3.25 |
FIG. 5: MECHANISM OF INHIBITION Α-AMYLASE BY ETHANOL EXTRACTION OF CALOTROPIS PROCERA
FIG. 6: MECHANISM OF INHIBITION Α-GLYCOSIDASE BY AQUEOUS EXTRACT OF CALOTROPIS PROCERA
DISCUSSION: In body blood glucose level increase it is characterized by Hyperglycemia. It is just Because of the continuous breakdown of glucose by the pancreatic α-amylase and by the absorption of glucose with the help of intestine Alpha-glycosidase enzymes. This extraction of the Calotropis procera leaf is an approach to decrease the postprandial hydrolysis of the glucose, and it inhibits these carbohydrate enzymes that, are Alpha-amylase and Alpha-glycosidase. After the inhibition of these carbohydrate enzymes that lowest the postprandial glucose increment with the mixture of carbohydrate diet helps to manage diabetes.
CONCLUSION: This study concluded that both extractions inhibit Alpha-amylase and Alpha-glycosidase enzymes. These enzymes are non-competitive enzymes. In-vitro inhibition concluded that they decreased the glucose level in the body act as an anti-diabetic agent. These extractions can be used in the future for further research on anti-diabetic. Calotropis procera extraction can be used in the replacement of anti-diabetic agents and also that extraction used for synergism.
ACKNOWLEDGEMENTS: Nil
CONFLICTS OF INTEREST: Nil
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How to cite this article:
Aalam M, Parveen G and Mittal C: An evaluation of anti-diabetic activity of Calotropis procera leaf extraction (Aak palnt). Int J Pharmacognosy 2023; 10(6): 330-35. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.10(6).330-35.
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English
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Mohd Aalam *, Gulafshan Parveen and Chavi Mittal
Guru Nanak College of Pharmaceutical Sciences, Jhajra, Dehradun, Uttarakhand, India.
gulafshanmirza12345@gmail.com
05 June 2023
27 June 2023
29 June 2023
10.13040/IJPSR.0975-8232.IJP.10(6).330-35
30 June 2023