Effect of Microencapsulation on the Antidiabetic and Antioxidant activities of Elaeocarpus tectorius (Lour.) Poir. Leaf extracts- An In vitro study
1Department of Biochemistry, Sri Ramakrishna College of Arts and Science for Women,
Affiliated to Bharathiar University, Coimbatore, Tamil Nadu, India.
2Centre for Bioscience and Nanoscience Research, Coimbatore, Tamil Nadu, India.
*Corresponding Author E-mail: keerthanamanoharan96@gmail.com
ABSTRACT:
Microencapsulation is a novel, cost-effective technique that helps in protecting the bioactivity of plant extracts. The current study is aimed to prepare chitosan-coated sodium alginate microcapsules containing Elaeocarpus tectorius leaf extracts and to study its antioxidant and antidiabetic properties using in vitro assays. The ethanolic extract of E. tectorius was subjected to UV-VIS and FTIR analysis. The extracts were then subjected to microencapsulation using chitosan and sodium alginate. The synthesized chitosan-sodium alginate microcapsules were characterized using FESEM analysis. Further, the antioxidant and antidiabetic activities of the prepared microcapsules and crude extracts were assessed using in vitro assays. The UV-VIS and FTIR analysis of the leaf extracts revealed the presence of functional groups of phenolics, flavonoids and proteins. The microcapsules exhibited good antidiabetic potential through the inhibition of α- amylase and α- glucosidase enzymes. The antioxidant assays revealed that the microcapsules exhibited significantly higher antioxidant potential compared with the crude extracts. The chitosan-sodium alginate microcapsules containing Elaeocarpus tectorius leaf extracts could be used as an alternative to synthetic antidiabetic drugs and as a rich source of antioxidants.
KEYWORDS: Microencapsulation, Sodium alginate, Chitosan, Medicinal plants, Diabetes, Antioxidants.
INTRODUCTION:
Diabetes mellitus is characterized by prolonged high blood glucose levels resulting from defects in either insulin secretion or action or both. It is also linked to abnormalities in carbohydrates, lipids, and protein metabolism and requires timely diagnosis, treatment and lifestyle changes1. The latest data shows that about 2.8% of the world’s population is affected by diabetes and the count is expected to increase to more than 5.4% by the year 2025. Diabetes is a major health issue in both developed and undeveloped countries. Even though there are many therapeutical options available for the treatment of diabetes, they do not cure the disorder completely and can cause numerous side effects.
This aspect makes the search for more effective and safer antihyperglycemic agents one of the most important areas of investigation. Variable pathogenesis, high prevalence of the disorder, progressive nature of the process, and related complications of diabetes emphasize the urgent need for effective treatment methods2,3.
The enzyme, alpha-amylase found in pancreatic secretion and saliva are in charge of the conversion of starch to simple sugar molecules. Alpha-glucosidase found in the small intestine is responsible for hydrolyzing complex polysaccharide molecules into glucose molecules. The inhibitors of these enzymes can lower carbohydrate absorption and delay carbohydrate digestion which leads to an evident drop in the rate of glucose absorption thereby moderating the postprandial plasma glucose rise4. Medicinal plants have now become an active area of research because they are known to be effective and usually have fewer or no side effects5. Since the pathogenesis of diabetes involves oxidative stress, antioxidant therapies also have great prospects in their treatment. Plants containing antioxidant compounds can protect pancreatic β-cells from oxidative stress and therefore can inhibit oxidative stress6.
Elaeocarpus tectorius (Lour.) Poir belonging to the Elaeocarpaceae family is found in the higher altitude regions of The Nilgiris Mountains, India. The tree bears small green edible fruits that are mainly consumed by the tribal people of the Western Ghats. Pharmacological investigations on various Elaeocarpus species showed that they possess anti-inflammatory, antimicrobial, analgesic, anti-diabetic and anti-hypertensive properties7. The fruits are known to be used in the treatment of rheumatism, leprosy, pneumonia, ulcer, piles and skin allergies8.
The bioactive substances present in the plant extracts are biologically unstable and volatile in nature and are easily prone to oxidation. In recent years, numerous novel strategies are established to overcome the limitations and improve their stability, delivery and bioavailability9. Microencapsulation of plant extracts is one such method in which the plant extracts are entrapped into biodegradable matrix-forming nano or microsystems. Recent research is focused on the use of biopolymers as entrapping materials to encapsulate a variety of therapeutic compounds owing to their biocompatibility, non-toxicity, biodegradability and bio-adhesiveness10,11. The compounds entrapped in biopolymers can be delivered in a sustained manner protected from the gastrointestinal tract and provide numerous advantages including improved bioavailability, increased stability, dose reduction and reduced side effects of the drug12. In the recent past, ionotropic gelation of alginate with calcium chloride to form calcium alginate beads has been widely used in the encapsulation of bioactive ingredients. Sodium alginate derived from brown seaweed and marine algae is a water-soluble polyanionic polymer molecule composed of 1, 4-α-L guluronic acid and β-D mannuronic acid residues. Chitosan is extensively employed as a coating material for calcium alginate beads in order to enhance stability and permeability. Chitosan forms a strong complex with calcium alginate beads through electrostatic interaction between its amino residues and the carboxyl residues of alginate13,14.
In this context, the present study is aimed to formulate chitosan-sodium alginate microcapsules containing Elaeocarpus tectorius leaf extracts and their characterization using FESEM analysis. Further, the effect of microencapsulation on the antidiabetic and antioxidant activities of the leaf extract was evaluated. The present study is the first report on the investigation of the antidiabetic potential of this plant species.
The plant sample was collected from the Nilgiris district, Tamil Nadu, India. The plant was identified and authenticated by a botanist from the Botanical Survey of India, Southern Regional Centre, TNAU Campus, Coimbatore, Tamil Nadu (Reference no. BSI/SRC/5/23/2021/Tech./319). The leaves of Elaeocarpus tectorius were washed with tap water to remove dust particles and shade dried. The dried material was finely powdered using a mortar and pestle and a sample of about 10grams was macerated with 100 mL of ethanol. It was then incubated for 48 hours in a shaker incubator at 40şC after which extracts were filtered and the solvents were evaporated to get the dry extract. The dried extract was stored at -4˚C for further use.
The ethanolic extract of E. tectorius leaves was centrifuged at 3000rpm for 10min and filtered through Whatmann No.1 filter paper. The extract was scanned at wavelengths ranging from 200 to 900nm using PerkinElmer Spectrophotometer and the characteristic peaks were detected. The peak values of the UV-VIS spectrum were recorded.
The dried extract of E. tectorius leaves was used for FTIR analysis. 10mg of the dried extract powder was encapsulated in 100mg of KBr pellet, to prepare translucent sample discs. The powdered sample of each extract was loaded in an FT-IR spectroscope (Shimadzu, Japan), with a scan range from 400 to 4000 cm-1 with a resolution of 4cm-1.
The internal gelation technique was used to form chitosan–alginate microspheres. Briefly, sodium alginate was dissolved in distilled water at a concentration of 4% (w/v), and a pre-calculated quantity of plant extract was added. The solution was stirred thoroughly to ensure the complete mixing of the plant extract. The gelation medium was prepared by dissolving chitosan (1% w/v) in one percent acetic acid, followed by the addition of CaCl2 at the concentration of 4% (w/v). The sodium alginate solution was added dropwise (about 60 drops/min) into the gelation medium under stirring with the speed of 1000r/min. After suspending for half an hour, the microspheres were rinsed with distilled water, filtered, and dried in the oven at 60°C15.
The shape, morphology, and elemental mapping of microcapsules were studied using field emission scanning electron microscopy (FESEM) (MIRA3 TESCAN). For this purpose, the lyophilized sample was sonicated for a sufficient amount of time, the smear was made on a platinum grid, and allowed to dry overnight under vacuum. The grid was then coated with a thin film of palladium and finally subjected to FESEM.
The encapsulation efficiency of the microcapsules was determined by total phenolic content analysis using Folin- Ciocalteu assay16. 10mg of microcapsules were suspended in 5ml of 95ml/ 100ml methanol in water, mixed well, and left in the dark for 1hour at room temperature. The sample then was filtered and 0.25ml of the sample was mixed with 0.25ml Folin-Ciocalteau reagent, 4 ml of water, and 0.5ml of 20% sodium carbonate solution. The samples were then allowed to stand for 2 hours at room temperature protected from light, and the absorbance was measured at 765nm. A standard curve was prepared using gallic acid as the standard to quantify the total phenolic expressed as gallic acid equivalents/100 g. The encapsulation efficiency was calculated according to the formula:
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑎𝑐𝑡𝑖𝑣𝑒 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒𝑑
Entrapment efficiency = ------------------------------- ×100
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑒 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑
The α-amylase inhibition assay was performed by briefly dissolving 200μl of E. tectorius leaf extracts and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts at different concentrations (200- 1000µg) and it was allowed to react with 200μl of porcine pancreatic α- amylase enzyme and 100μl of 200 mM phosphate buffer (pH-6.9). After 20 minutes of incubation, 500μl of 0.5% starch was added. The same was performed for the control where 200μl of the enzyme was replaced by the buffer. After incubation for 5 minutes, 500μl of dinitro salicylic acid was added to both the control and test. The tubes were kept in a boiling water bath for 10 minutes. After cooling to room temperature, the absorbance was recorded at 540nm using a spectrophotometer and the percentage of α- amylase inhibition was calculated using the formula17:
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)−𝐴𝑏𝑠 (𝑡𝑒𝑠𝑡)
Inhibition (%) = -------------------------------× 100
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
Suitable reagent blank and inhibitor controls were also carried out and subtracted. Acarbose was used as a positive control. Further, the IC50 value representing the concentration of the extract that caused 50% inhibition of 𝛼- amylase was calculated by interpolation of linear regression analysis.
The alpha-glucosidase inhibitory activity was performed as follows18. The assay mixtures for this assay contained 300µl of 10mM p-nitrophenyl alpha-D-glucopyranoside, 1ml of 0.1 M potassium phosphate buffer, pH - 6.8, 200µl of α- glucosidase enzyme solution and 200µl of E. tectorius leaf extracts and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts at different concentrations (200-1000µg) all in a final volume of 1.7ml. Following an incubation period of 30 minutes at 37°C, the reaction was terminated by the addition of 2ml of 100mM sodium carbonate. The liberated p-nitrophenol was determined at 400 nm using a spectrophotometer. The percentage inhibition rates were calculated using the formula:
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)−𝐴𝑏𝑠 (𝑡𝑒𝑠𝑡)
Inhibition (%) = -------------------------------------× 100
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
Suitable reagent blank and inhibitor controls were also carried out and subtracted. Acarbose was used as a positive control. Further, the IC50 value representing the concentration of the extract that caused 50% inhibition of 𝛼- glucosidase was calculated by interpolation of linear regression analysis.
The phosphomolybdenum assay was used to estimate the total antioxidant capacity of the extracts19. Briefly, 0.3ml of E. tectorius leaf extracts and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts (50- 250μg/ml) was combined with 3 ml of reagent solution (0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance was measured at 695 nm against a blank. Ethanol (0.3ml) was used as the blank and ascorbic acid was used as a standard. Total antioxidant activity was expressed in relation to ascorbic acid and calculated by the following formula:
𝐴𝑠−𝐴𝑐
% TAC = ----------- ×100
𝐴𝑎𝑎−𝐴𝑐
Where Ac was the absorbance of the control (blank, without extract), As was the absorbance in the presence of the extract and Aaa was the absorbance of ascorbic acid.
The scavenging effects of samples on DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radicals were monitored20. The E. tectorius leaf extracts and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts at various concentrations (200-1000µg) were taken and the volume was adjusted to 100μl with methanol. 5 ml of 0.1 mM methanolic solution of DPPH was added and allowed to stand for 20 min at 27°C. The absorbance of the sample was measured at 517 nm. Ascorbic acid was used as a standard. The percentage radical scavenging activity of the sample was calculated as follows:
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)−𝐴𝑏𝑠 (𝑡𝑒𝑠𝑡)
% DPPH radical scavenging activity = ---------× 100
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
The ABTS free radical scavenging activity of the plant samples was analyzed21. ABTS was produced by reacting 7 mM ABTS aqueous solution with 2.4 mM potassium persulfate in the dark for 12–16 h at room temperature. Before the assay, this solution was diluted in ethanol (1:89 v/v) and equilibrated at 300 C to give an absorbance at 734 nm of 0.700 ± 0.02. The stock solution of the sample extracts was diluted such that after the introduction of 10 μl aliquots into the assay, they produced between 20% and 80% inhibition of the blank absorbance. After the addition of 1 ml of diluted ABTS solution to 10 μl of leaf extracts and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts at different concentrations (200-1000 μg/ml), absorbance was measured at 734 nm at exactly 30 min after the initial mixing. Rutin was used as the standard. The percentage radical scavenging activity of the sample was calculated as follows:
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)−𝐴𝑏𝑠 (𝑡𝑒𝑠𝑡)
% ABTS radical scavenging activity = --------- × 100
𝐴𝑏𝑠 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
The UV-VIS spectrum of the ethanolic leaf extract of Elaeocarpus tectorius is presented in Figure 1. Firm absorption peaks were observed between 200-900 nm. The profile showed peaks at 310nm, 325nm, 365nm and 655nm respectively corresponding to phenolics and flavonoids.
Figure 1- UV-VIS spectra of Elaeocarpus tectorius leaves
The FTIR spectrum of the ethanolic leaf extracts of Elaeocarpus tectorius is presented in Table 1 and Figure 2. The FTIR spectrum was used to identify the functional groups of the active components based on the peak values in the region of IR radiation. The presence of strong bands at 3726, 3595, 3564 and 3533 cm-1 refers O-H group indicating the presence of alcohols and phenols and the peak at 3294.42 cm-1 shows N-H stretching, aliphatic primary amine indicating the presence of protein and peptide groups. The peak at 1921 cm-1 indicates C-H bending aromatic compounds. The peak at 1681 represents the presence of amide groups, which arises due to the carbonyl stretch and NeH vibrations in the amide linkage.
Table 1: Functional groups corresponding to the wavelengths recorded in FTIR spectra
|
Group frequency wavenumber (cm-1) |
Bond and Functional groups |
|
3726.47 |
O-H stretching, alcohol |
|
3595.31 3564.45 |
O-H stretching, alcohol |
|
3533.59 |
O-H stretching, alcohol |
|
3294.42 |
N-H stretching, aliphatic primary amine |
|
2970.38 2885.51 |
C–H stretching, alkanes |
|
2360.87 2337.72 |
O=C=O stretching |
|
1921.10 |
C-H bending, aromatic compound |
|
1681.93 |
C=O stretching, amide |
|
1450.47 |
C-H bending, alkane |
|
1381.03 |
C=H bending alkane, OH bending phenol |
|
1087.85 |
C-O stretching, aliphatic ether |
|
1041.56 |
C-O stretching, aliphatic ether, CO-O-CO stretching anhydride |
|
879.54 |
C-H bending, 1,2,4- trisubstituted |
|
802.39 |
C=C bending, alkene |
Figure 2- FT-IR spectra of Elaeocarpus tectorius leaves
The encapsulation efficiency of the microcapsules was found to be 83.2±0.89%. The microcapsules showed maximum encapsulation efficiency. Field-emission scanning electron micrograph (FESEM) analysis of chitosan- sodium alginate microcapsules containing E. tectorius leaf extracts clearly showed that they were uniform with an average diameter of 5-10mm. The microcapsules were oval and spherical in shape. Most of the microcapsules were individual, and a few aggregated particles were also observed (Figure 3).
Figure 3: Field-emission scanning electron microscope (FESEM) images of the synthesized chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts
The crude and microencapsulated extracts of E. tectorius leaves were evaluated for alpha-amylase inhibition assay using porcine pancreatic alpha-amylase (Figure 4). It was found that the chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts exhibited stronger alpha-amylase inhibition compared to the crude unencapsulated extracts. The microcapsules and plant extracts exhibited enzyme inhibition in a dose-dependent manner as the concentration clearly affects the amount of enzyme inhibited. The IC50 values of microencapsulated and crude extracts for the alpha-amylase inhibition were found to be 471.5µg/ml and 558.2µg/ml respectively whereas, standard acarbose had an IC50 value of 295.8µg/ml.
The alpha-glucosidase inhibitory potential of the crude and chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts was evaluated and the results are presented in Figure 5. Based on the results it was found that the microencapsulated extracts exhibited better inhibitory potential compared with the crude extracts. The extracts exhibited inhibition in a concentration dependant manner. The IC50 values of microencapsulated and crude extracts for the alpha-amylase inhibition were found to be 567.2µg/ml and 608 µg/ml respectively whereas, standard acarbose had an IC50 value of 292.4µg/ml.
Figure 4- Alpha-amylase inhibitory activity (ETL- Eleaocarpus tectorius leaf extracts, M-ETL- Microencapsulated Eleaocarpus tectorius leaf extracts)
Figure 5- Alpha-glucosidase inhibitory activity (ETL- Eleaocarpus tectorius leaf extracts, M-ETL- Microencapsulated Eleaocarpus tectorius leaf extracts)
In vitro antioxidant activity:
The total antioxidant capacity of microencapsulated and crude unencapsulated leaf extracts was analyzed using the phosphomolybdenum method. The microencapsulated leaf extracts of Elaeocarpus tectorius exhibited the highest antioxidant capacity of about 98.22 ±2.68mg/g AAE (Ascorbic acid equivalents) compared to the unencapsulated extracts which exhibited 90.76± 1.34mg/g AAE.
The DPPH (2, 2, diphenyl-1-hydrazyl) radical scavenging activity of the plant extracts was found to be dose- dependent. The dose-dependent increase in DPPH radical scavenging activity of plant extracts is directly proportional to the antioxidant capacity. The results of DPPH radical scavenging activity are shown in Figure 6. The microencapsulated extract exhibited the highest DPPH radical scavenging activity with an IC50 value of 434.18µg/ml followed by crude extracts with 502.03 µg/ml.
Figure 6: DPPH radical scavenging activity (ETL- Eleaocarpus tectorius leaf extracts, M-ETL- Microencapsulated Eleaocarpus tectorius leaf extracts)
ABTS radical scavenging activity was significantly higher in microencapsulated extracts followed by unencapsulated crude extracts and the activity was found to be dose-dependent, as the activity increased with increasing concentration (Figure 7). The IC50 values of microencapsulated and unencapsulated ETL extracts and standard rutin were found to be 227.51 μg, 514.97 μg and 250.35 μg respectively.
Figure 7: ABTS radical scavenging activity (ETL- Elaeocarpus tectorius leaf extracts, M-ETL- Microencapsulated Elaeocarpus tectorius leaf extracts)
Herbal medicine has received enormous interest in recent times due to its reported safety and multiple pharmacological benefits. Traditional folk medicine from wild plants has always guided researchers toward novel medications. The preliminary screening of medicinal plants by spectrometric and chromatographic methods provides vital data on their chemical and pharmacological activities and also helps to select the biologically active plants22. Analytical techniques like Fourier-transform infrared spectroscopy (FTIR) have been commonly employed for the detection of functional groups and the discovery of various bioactive therapeutic compounds that are present in medicinal plants in recent times23. The UV-VIS and FTIR analysis of the E. tectorius leaf extracts revealed the presence of functional groups of phenolics, flavonoids and alkaloid compounds.
The ethanolic extracts of Elaeocarpus tectorius leaves were then microencapsulated using sodium alginate and chitosan. The encapsulation efficiency and the morphology of the chitosan-sodium alginate microcapsules containing E. tectorius leaf extracts were then analyzed. Microencapsulation of plant extracts could help in reducing the dose, facilitate controlled release and also the bioactive constituents are shielded from the external environment24. Biodegradable and biocompatible polymers have been used as nanocarriers in recent times due to their low cost and less toxic nature25.Several studies have shown that nanoencapsulation improves the antioxidant activity of various plant extracts and edible oils26.
The control of postprandial blood glucose levels is the principal component in the management of diabetes. The current treatment involves the use of antihyperglycemic agents like sulfonylureas, meglitinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, and insulin. This drug intervention has been related to unavoidable side effects, mainly hypoglycemia, weight gain and gastrointestinal discomfort. Many medicinal plants exhibit hypoglycemic activity by restoring the function of pancreatic cells leading to an increase in insulin secretion or inhibiting the intestinal absorption of glucose or the facilitation of metabolites in insulin-dependent metabolic processes27. A key approach involved in the treatment of diabetes is the inhibition or lowering of glucose absorption to moderate postprandial hyperglycemia. This can be achieved through the inhibition of carbohydrate metabolizing enzymes like alpha-amylase and alpha-glucosidase which are responsible for the hydrolysis of complex carbohydrates into small absorbable units. The inhibitors of these enzymes thus can delay the rate of carbohydrate digestion and absorption and finally can lower the postprandial glucose levels. Natural plant-based inhibitors of these enzymes can be developed as therapeutic agents for treating diabetes to help control postprandial hyperglycemia28,29.
Hyperglycemic conditions can elicit oxidative stress in diabetic patients through different pathways. Oxidative stress is a condition where there is an imbalance in the production and elimination of reactive oxygen species. Under diabetic conditions, oxidative stress can activate a chain of pathological events leading to diabetic complications such as retinopathy, nephropathy, cardiomyopathy and hypertension. Plants containing antioxidant compounds can protect pancreatic β-cells from reactive oxygen species (ROS) and therefore, can alleviate the complications of diabetes30,31. The alpha-amylase and alpha-glucosidase inhibition assays showed that both the crude and microencapsulated extracts exhibited significant antidiabetic activity with the microencapsulated extract exhibiting higher inhibition. The in vitro antioxidant assays showed that the microencapsulated extracts exhibited better radical scavenging activity in a concentration-dependent manner compared with the crude unencapsulated extracts. Manoharan et al.7 studied the antioxidant activities of E. tectorius leaves and reported that they contain several essential phytochemicals that act as antioxidants. A study on the antioxidant and antidiabetic potential of Elaeocarpus sphaericus leaves revealed that the leaves exhibited strong antidiabetic and antioxidant activities32. From the above results, it was observed that the leaf extracts of E. tectorius exhibit significant antioxidant and antidiabetic activities and the technique of microencapsulation has enhanced the biological effects of the extracts.
CONCLUSION:
The microencapsulation of Elaeocarpus tectorius leaf extracts is a simple, cost-effective technique that improved its biological properties. The antidiabetic and antioxidant effects of the prepared microcapsules were dose- dependent where the activity increases with an increase in concentration. The antidiabetic and antioxidant activities of the chitosan-sodium alginate microcapsules were also higher than the crude extracts. Further in vivo studies on its antidiabetic and antioxidant effects are needed before they can be used clinically. This study, therefore, paves a safer way for employing compounds of botanical origin with nanotechnology for application in healthcare.
The authors declare no conflict of interest.
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Received on 29.12.2021 Modified on 22.12.2022
Accepted on 07.07.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(1):149-155.
DOI: 10.52711/0974-360X.2024.00024