In-vitro Antioxidant and Antihyperglycemic effect of Muntingia calabura L. fruit extracts


S. Sowmya1, Dr. A. Jayaprakash2*

1Research Scholar, PG and Research Department of Biochemistry, Sacred Heart College (Autonomous),  Tirupattur-635601, Tirupattur District, Tamil Nadu, India.

2Assistant Professor, PG and Research Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur-635601, Tirupattur District, Tamil Nadu, India.

*Corresponding Author E-mail:



This study investigated the in-vitro antioxidant activity and antidiabetic effect of Muntingia calabura fruit extract by in-vitro α-amylase and α-glucosidase inhibitory activity. Muntingia calabura fruit was extracted with aqueous methanol by soxhlet extraction. The total phenols and total flavonoids contents were estimated and evaluated for antioxidant activities (DPPH, ABTS) and in-vitro antidiabetic activity by measuring their inhibitory activity on α-amylase and α-glucosidase levels. The findings showed that the fruit extract had high content of total phenol and exhibited moderate free radical scavenging activity. The fruit extract showed inhibitory effect on α-amylase [IC50Value =61.43 μg/mL] and α-glucosidase [IC50 Value=140.33 μg/mL] compared to standard acarbose. The fruit extract can be used as a potential source for the development of new hypoglycemic agents may be due to the presence of high phenol content.


KEYWORDS: Muntingia calabura, Phytochemicals, Antioxidants, α-amylase and α-glucosidase.




Diabetes mellitus is a chronic metabolic disorder caused by inherited or acquired deficiency of insulin secretion and by decreased sensitivity of the organ to secrete insulin and its deficiency results in increased blood glucose level than normal blood glucose level, which in turn can damage several functions, including blood vessels and nerves1. One of the remedial approaches is to reduce the postprandial hyperglycemia by slowing down the absorption of glucose by inhibition of carbohydrate hydrolyzing enzymes, such as alpha-amylase and alpha-glucosidase2. About 90% of diabetic population is affected by type 2 diabetes, shows mild symptoms like fatigue, frequent urination, hunger, and increased thirst, blurred vision, weight loss and slow healing of wounds and can be controlled with a healthy diet and exercise3.


From this many efforts have been made to look for more effective and safe inhibitors of α-amylase and α-glucosidase from natural materials to develop a physiological system to treat diabetic mellitus4. Many traditional plants have reported in India for diabetes, but only a small number of these have received scientific and medical evaluation to assess their efficacy. The medicinal plants are remedial as well as for the curing of human diseases due to the presence of phytochemical constituents. Phytochemicals have defense mechanisms and protect from various human diseases. Primary and secondary metabolites are biologically active compounds. Proteins, common sugars and Chlorophyll, are included in primary constituents and secondary compounds are terpenes, phenolic compounds and nitrogen-containing secondary metabolites such as alkaloids and non-protein amino acids5. Alkaloids are second-hand as anesthetic agents and are found in medicinal plants6. Phenolic compounds make different vital pharmacological activities i.e., anti-inflammatory, inhibition of cholesterol synthesis, anti-viral, anti-malarial, anti-bacterial activities anti-cancer, anti-diabetes7 and antioxidant substances to inhibiting the formation of free radicals by scavenging radicals. Antioxidants are found in fruits, vegetables and a variety of other foods naturally8.


Based on tribal information Muntingia calabura fruit has been used to treat and prevent diabetes. Muntingia calabura (M. calabura) belonging to the family Muntingiacea is native to Southern Mexico and tropical South America9. It is commonly cultivated and used by many traditional healers in most of the countries like India and Southeast Asia such as Malaysia, Indonesia, and Philippines10. In Tamil Nadu, M. calabura is commonly seen along the road side trees. The plant is further used traditionally for the treatment as an antiseptic, reduce swelling in lower extremities, reduce gastric ulcer and swelling of the prostate gland, alleviate headache, cold and to treat measles, mouth pimples and stomachache 11-12. The leaf decoction is drunk as a tea like beverages13. M. calabura fruit by itself is sweet in taste. Mahmood et al. reported that the different parts of M. calabura possess bioactive compounds and the pharmacological activities on acute toxicity, cytotoxic activity, antiproliferative, insecticidal activity, hypotensive, antinociceptive, cardioprotective, antipyretic, antiplatelet aggregation, antioxidant, anti-inflammation, anti-diabetic, antiulcer, antimicrobial, anthelmintic activity and hepatoprotective14.


There has been no report on the in-vitro antidiabetic effect on M. calabura fruit. So, the present study was aimed to identify the phytochemical constituents and evaluate the in-vitro free radical scavenging ability and antidiabetic activity of M. calabura extract.



Chemicals and reagents:

All the chemicals and solvent were analytical grade and obtained from Hi-media, India.


Collection of plant material:

Muntingia calabura fruits were collected in and around Tirupattur District, Tamil Nadu, India. The fruits were shade dried and properly ground into powder and used for further studies.


Preparation of fruit extract:

Soxhlet extraction method was used to extract 25g of a dried powder sample in 200 mL of solvent (aqueous (20 mL) and (180 mL) methanol). The sample was extracted at ambient temperature for 48 hours and the crude sample was collected and used for further studies.


Quantitative analysis of phytochemical Screening:

Determination of total phenolic content:

Total phenolic content in the extracts were estimated by Folin-Ciocalteu method according to the modified procedure of Tamilventhan et al. using gallic acid as a standard 15. About 500 µg of M. calabura fruit extract was mixed with 0.5 mL of water and 0.2 mL of Folin-Ciocalteu’s phenol reagent (1:1). About 1 mL of saturated Na2CO3 (8 % w/v in water) solution was added after 5 minutes and the volume made up to 5 mL by adding distilled water and kept in dark for 30 min. The absorbance of blue color was measured at 765 nm using UV-vis spectrophotometer. Total phenolic content was expressed in terms of mg/g equivalent to gallic acid (10-320 µg/mL) on the basis of a standard curve. Estimation was repeated thrice and the results were averaged.


Determination of total flavonoid content:

Total flavonoid content in the extracts was determined according to modified method of Ali et al. using quercetin as a standard 16. To 500 µg of M. calabura fruit extract, a volume of 0.5 ml of 2% AlCl3 ethanol solution was added and incubated at room temperature for 1 hour. Absorbance was measured at 420 nm using UV-vis spectrophotometer. Total flavonoids were expressed in terms of mg/g equivalent to quercetin (10-320 µg/mL) on the basis of a standard curve. Estimation was repeated thrice and the results were averaged.


In-vitro antioxidant activity:

DPPH radical scavenging activity:

This test was measured as described by modified method of Perumal et al17. In brief, 0.135 mM DPPH was prepared in methanol. Different Concentration (5, 10, 20, 40, 80, 160 and 320 μg/mL) of M. calabura fruit extract was mixed with 2.5 mL of DPPH solution. The reaction mixture was vortexed thoroughly and kept at room temperature for 30 min. The absorbance was measured at 517 nm. The ability of extract to scavenge DPPH radical and control was calculated from the following formula:

% DPPH inhibition = [(OD of control – OD of test sample) / ( OD of control)] ×100

The concentration of extract that is required for resulting in 50% of inhibition of enzyme activity (IC50) was determined. The standard inhibitor ascorbic acid was used as a positive control at a same concentration range of 5-320 μg/mL.


ABTS radical scavenging activity:

Antioxidant potential of plant extract was determined against ABTS (2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) free radical method. ABTS (7 mM, 25 mL in deionized water) stock solution and potassium persulfate (140 mM, 440 μL) solutions were mixed thoroughly and incubated in dark for the production of ABTS free radical. About different concentrations (5, 10, 20, 40, 80, 160 and 320 μg/mL) of the MCF extract and standard ascorbic acid was mixed with 2.0 mL ABTS working solution and the reaction mixture was incubated for 20 min at room temperature. Finally the absorbance was measured using an UV-vis spectrophotometer at 734 nm. Percentage of ABTS scavenging potential was calculated using the following formula:

ABTS radical scavenging effect (%) = [(A0 - A1)/A0] ×100

Whereas, A0 is the control; A1 is the test.


Evaluation of in-vitro antidiabetic activity:

α-Amylase inhibitory assay:

The inhibition assay of α-amylase starch iodine method was carried out according to the standard method with minor modification according to Unuofin et al18. About 100 μL of α- Amylase (0.1 mg/mL) and the M. calabura fruit extract were mixed at different concentrations (10-320 μg/mL) and incubated at 37°C for 15 min. About 100 μL of soluble starch (1%) was added to each tubes and incubated at 37°C for 60 min and 10 μL of 1M HCl was added to stop the enzymatic reaction, followed by the addition of 100 μL of iodine reagent. The colour change was noted and the absorbance was read at 580 nm in a UV-vis spectrophotometer. A control was prepared using the same method except that the extract was replaced with distilled water. The α-amylase inhibitory activity was calculated as follows:

Percentage of Inhibition (%) = [(Abs Control - Abs Sample) / Abs Control] x 100


The standard inhibitor acarbose was used a positive control at a concentration range of 10-320 μg/mL. The presence of starch indicates formation of dark-blue colour; while no colour complex is developed in the absence of the inhibitor, indicating that starch is totally hydrolysed by a-amylase.


α-Glucosidase inhibition assay:

The inhibitory action of α-glucosidase activity was determined using modified procedure of Apostolidis et al 19. A volume of 200 μL of α-glucosidase and the M. calabura fruit extract at different concentrations (10-640 μg/mL) was incubated in 100 mM phosphate buffer pH 6.8 at 37°C for 15 min. There after reaction mixture, contained 400 μL of 5mM p-nitrophenyl-α-D-glucopyranoside (pNPG) in 100 mM phosphate buffer pH 6.8 at 37°C for 20 min and the reaction was stopped by adding 1 mL of 0.1 M of Na2Co3. The absorbance of the resulting p-nitrophenol was determined at 405 nm using UV–vis spectrophotometer. Acarbose was used as a positive control and the inhibitory activity of α-glucosidase was calculated using the following formula,

Percentage of Inhibition (%) = [(Abs Control - Abs Sample) / Abs Control] x 100


Statistical analysis:

The results were represented as mean ± SD in triplicate experiments. The data were statistically analyzed using GraphPad Prism version 5.



Total phenol content (TPC) and Total flavonoid content (TFC):

Total phenol content in the M. calabura fruit extract using the calibration curve, was found to be 51.84 mg of Gallic acid equivalents/g dry weight and quercetin equivalents/g dry weight of the fruit extract exhibited 23.37 mg of total flavonoid content. The most important phenol content of Gallic acid was found to be higher in aqueous methanolic fruit extract.


In-vitro antioxidant activity:

In the antioxidant activity IC50 values are negatively related, as it expresses the total amount of antioxidant needed to reduce its radical concentration by 50%. The lower IC50 value represents higher antioxidant potential of the tested sample. As shown in Figures 1 and 2, the dose-response curve confirms the free radical scavenging ability of M. calabura fruit extract. In the DPPH method, the scavenging capability of free radicals by the fruit extract was found to be IC50 =169.22 μg/mL and higher than ascorbic acid. The percentage of inhibition increased in concentration dependent manner from 5-320 μg/mL compared to standard ascorbic acid which exhibited 10.53% to 99.20% and fruit extract showed 8.37% to 63.72% respectively. Muntingia calabura fruit extract was found to be more effective in scavenging the ABTS radical. There was a significant increase in percentage of inhibition on concentration dependent manner (5-320 μg/mL). At the concentration of 20 μg/mL, the inhibition of ascorbic acid was 45.21% and fruit extract was 45.28% respectively. The IC50 of ascorbic acid was found to be 24.31 μg/mL while that of fruit extract was 29.58 μg/mL respectively. The findings showed that M. calabura fruit extract exhibited good antioxidant activity in the ABTS assay similar to the standard ascorbic acid may be due to its high phenol levels (Table 1).


Table 1: IC50 Values (μg/mL) for M.calabura and ascorbic acid in Antioxidants assay.

Antioxidants assay

IC50 values (µg/ml)

Ascorbic acid

M. calabura fruit extract







*Values are represented as mean±SD (n = 3); statistical significant level at (P<0.05).


Fig 1: DPPH free radical Scavenging ability of aqueous methanolic extract of M. calabura fruit.



Fig 2: ABTS free radical Scavenging ability of aqueous methanolic extract of M. calabura fruit


In-vitro antidiabetic activity:

Figures 3 and 4 showed a significant increase in the percentage of inhibitory activity was noticed in a concentration-dependent manner. In α-amylase inhibition assay at a minimum concentration 10 μg/mL, the fruit extract exhibited inhibitory percentage of 11.244±2.904% and maximum concentration of M. calabura fruit extract (320 μg/mL) exhibited 94.401±0.011% inhibitory activity. The positive control acarbose exhibited 25.293 ±0.667% to 92.562± 0.023% respectively. Fruit extract IC50 value (61.43 μg/mL) showed higher concentration than standard acarbose. Whereas strong inhibition observed when compared with α-glucosidase inhibition (Table 2). The α-glucosidase inhibitory concentration (IC50) of fruit extract was 140.33 μg/mL. The percentage of inhibition at minimum concentration (10 μg/mL) of the extract showed 18.644±0.363% and maximum concentration of 640 μg/mL exhibited 68.886±1.275% inhibitory activity. The positive control acarbose exhibited 25.036±4.374% to 98.789±0.419% respectively.


Table 2: IC50 Values (μg/mL) for α-amylase and α-glucosidase inhibitory assay

Name of enzyme assay

IC50 values (μg/mL)


M. calabura fruit extract




Alpha- glucosidase



*Values are represented as mean±SD (n = 3); statistical significant level at (P<0.05).


Fig 3: Inhibitory effect of aqueous methanolic extract of M. calabura fruit against α - amylase.


Fig 4: Inhibitory effect of aqueous methanolic extract of M. calabura fruit against α - glucosidase.



Many people with type 2 diabetes mellitus need antidiabetic drugs to manage their condition, but medications may cause fewer side effects. So the currently available drugs are not much use in prevention or reduction of diseases. Herbal drugs have potential bioactive compounds with great medicinal properties to treat diabetes mellitus such as metabolic disorder to inhibit digestive enzymes involved in the digestion of carbohydrates. So, natural products are considered to be safer than synthetic drugs20. The present study comprehensively evaluated the major phytochemical constituents such as total phenols (51.84 mg of gallic acid) and flavonoids (23.37 mg of quercetin) of aqueous methanolic fruit extract. In previous study Preethi et al. investigated the phenol content of M. calabura fruit extract and recorded highest content of phenol (1.486±0.028 mg/100g) followed by the different solvent extractions21. Anusuya et al., carried out with fruit extract with solvent mixture of (methanol/acetone/water) exhibits higher amount of total phenols 22. Phenolic compounds of flavonoids, tannins, anthraquinones, lignins and other compounds of saponins and terpenoids are important bioactive chemical constituents commonly correlated with their antioxidant activities.


The phenolic compounds have redox properties which perform as hydrogen donors, reducing agent and singlet oxygen quenchers. Muntingia calabura fruit was evaluated by the free radical scavenging ability against DPPH and ABTS. This study exhibited the standard antioxidant ascorbic acid showed increased reducing power in a concentration dependent manner found a direct relation between antioxidants as an average proton donating ability. In the DPPH free radical scavenging activity, acceptance of electrons from antioxidant compounds which in turn changes the colour of the solution from violet to yellow23. However, the fruit extract scavenging of DPPH radical was found to be lower than standard ascorbic acid and the ABTS radical scavenging study confirms that fruit extract had antioxidant capability, reacts with potassium persulphate to produce a blue-green chromogen of ABTS radical cation24. In the occurrence of antioxidant reductant, the intensity of the coloured radical is decreased as similar to the ascorbic acid at the concentration of 20 μg/mL. This shows that M. calabura aqueous methanolic extract represents a good antioxidant ability to scavenge ABTS radical. In a previous study Gustavo et al., reported the scavenging possibilities due to the higher contents of total phenolics, flavonoids and anthocyanins25. Although there have been recent studies on M. calabura fruit had appreciable amounts of antioxidants like vitamin C and E26 similar to Begonia versicolor leaves contain vitamin C reported by Ermi et al 27.


Effective antioxidant compound of gallic acid is also present in the fruit of M. calabura28. This beneficial effect of antioxidants in the plant can be attributed to the presence of secondary metabolites especially due to the hydroxyl group containing phenolic compounds such as flavonoid contents29. Thus, it could be concluded that M. calabura fruit can serve as a natural source of antioxidant and play an important role in adsorbing and neutralizing free radicals to prevent cell damages and oxidative stress related diseases like diabetes mellitus and cancer. Diabetes mellitus is a metabolic disorder and one of the major growing health problems. Most of the people have been taking Insulin and oral hypoglycemic agents as the only antidiabetic therapy. Thus, dietary herbal medicine has antioxidant potential to recover the cell damages of pancreatic beta cells and have hypoglycemia potential properties which stimulate the pancreatic insulin secretion and inhibit the carbohydrate hydrolytic enzymes. Alpha-amylase is the key enzyme that is responsible to catalyze the hydrolysis of α-1,4-glycosidic linkages of starch, glycogen and various oligosaccharides and disaccharides. The α-glucosidase enzyme hydrolyzes the disaccharides into simple sugars which are subsequently immersed through the small intestine thus causing postprandial hyperglycemia and may have useful effects on insulin resistance and glycemic index in diabetic people delay the absorption of carbohydrates from dietary sources and decrease postprandial glucose level30-34. By inhibiting the activity of α-glucosidase in the small intestine, this reduces glucose absorption by delaying carbohydrate digestion time may be useful in the management of diabetes35. In the literature there are no in-vitro studies dealing with the antidiabetic activity on M. calabura fruit. So the present in-vitro study proved that fruit aqueous methanolic extract had moderate inhibitory effects on α-amylase and α-glucosidase enzyme when compared to the standard acarbose. Due to the presence of inhibitors in the M. calabura fruit extract, the starch added to the α-amylase enzyme assay mixture is not degraded and gives a dark-blue colour complex in the starch iodine method which is responsible for containing phenol and flavonoid compounds and free radical scavenging ability on inhibitory potential of α-amylase and α-glucosidase. According to Thouri et al., flavonoids and their derivatives had ability to inhibit glucose transporter-1, blocking of glucose absorption and reduce the potency of digestive enzymes36 and fruit extract also have hypoglycaemic action through stimulation of surviving ß-cells of islets of langerhans to release more insulin37 and several flavonoids, glycosides present within the extract reduces the blood glucose reported by Sravan et al38. Also recorded fruit extract decreased the blood glucose level as compared to standard drug, Glibenclamide39. Terpenoids have been found to stimulate insulin secretion or functioning like insulin effect40. Saponin was found to be bioactive against diabetes and reduced fasted blood glucose and regulates the hyperglycemia associated with oxidative stress in type 2 Diabetes mellitus41-43. Muntingia calabura fruit had health promoting properties due to its antioxidant properties reported by Preethi et al44. So natural polyphenols can be used as a drug and as nutritional dietary supplements to cure or prevent various diseases and in extracting some of these compounds appear to be capable of improving the performance of pancreatic tissues by increasing the insulin secretion and inhibiting the carbohydrate hydrolyzing enzymes45.


This study indicated that aqueous methanolic extract of M. calabura fruit contains phenols, possesses free radical scavenging ability and antidiabetic potential. Therefore, the fruit extract might be more effective for type 2 diabetes mellitus and in-vivo studies need to be carried out to confirm these observations of Muntingia calabura fruit for pharmacological and therapeutic properties.



Authors acknowledge the Management of Sacred Heart College (Autonomous), Tirupattur-635601, Tirupattur District, Tamil Nadu for providing laboratory facilities.



The authors declare no conflict of interest.



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Received on 09.09.2020            Modified on 18.02.2021

Accepted on 25.04.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6496-6502.

DOI: 10.52711/0974-360X.2021.01123