Introduction

Medicinal plants play an important role in allopathic medicine, herbal medicine, homoeopathy and aromatherapy by being the sources of a variety of drugs in the modern world [26]. The use of plant extract as therapeutic agents is cheaper and easily available to most people in the developing countries. Currently, the highest attention is given to the medicinal values including the antimicrobial properties of plants from naturally occurring antioxidants of plant origin. Antioxidants have been reported to prevent oxidative damage by free radicals and reactive oxygen species (ROS) and prevent the occurrence of disease such as cancer and ageing.

Antioxidant molecules interfere with the oxidation process by reacting with free radicals and chelating and catalytic metals and also by acting as oxygen scavengers [8, 31]. Natural antioxidants, particularly in fruits, vegetables and beverages, have gained interest among consumers. Medicinal plants are mostly used as an alternative treatment for some diseases by producing a variety of biologically active compounds of known therapeutic properties [14].

Plant extract which is known to contain many phenolic compounds, including flavonoids, have attracted considerable attention because its antioxidant activity is more powerful than vitamins, C and E [6, 35]. The use of plants and herbs as antioxidants in processed foods is of importance in the food industry as an alternative to synthetic antioxidants [4, 30].

Effective determination of biologically active compounds from plant material is basically dependent on solvents polarity. Traditional therapists use water for extraction but found out that plant extracts extracted using organic solvents contained more constant biological activities compared to water extracts [28]. In this study, solvents of different polarities, viz. hexane, dichloromethane (DCM) and methanol, have been used for the extraction procedure. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarities [7].

Microorganisms are common pathogens causing severe ailments and fatal infections in humans. Natural products of higher plants may provide a new source of antimicrobial agents with potentially novel mechanisms of action [23].

Artocarpus altilis (Family Moraceae) commonly known as breadfruit originated from New Guinea and grows extensively in the southern parts of India. Breadfruit (A. altilis) is a multipurpose agroforestry tree crop which is primarily used for its nutritious, starchy fruit which is a rich source of carbohydrates, calcium and phosphorus [29]. The multifarious uses of breadfruit includes food, medicine, clothing and animal feed.

The main objectives of the present study were to determine the total phenolic and flavonoid contents and to determine antioxidant and antimicrobial activities of the crude extracts, then correlating the antioxidant assays with consideration of the parts of the fruit as well as the solvents (hexane, dichloromethane (DCM) and methanol) of the crude extracts.

Materials and Methods

Collection of the Plant Material

The fresh fruits were collected in Taman Pertanian, Kuantan, Malaysia, in July 2013. After collection, the dried fruits were ground, then stored in cold room at 4 °C until further analyses.

Extraction

The ground plant material was extracted using Soxhlet apparatus according to Green [7]. The pulp, peel and whole fruit of A. altilis were extracted using three solvent systems (hexane, dichloromethane and methanol) in order of increasing polarity.

Determination of Total Phenolic content

Total phenolic content was determined by [10] Folin-Ciocalteu reagent [19]. One millilitre of each sample extract (4 mg/ml) were added to test tubes and made up to the volume of 10 ml by adding 5 ml of diluted Folin-Ciocalteu phenol reagent (1:10 distilled water) and 4 ml of sodium carbonate solution (7 %) after 4 min sequentially. Soon after homogenizing the reaction mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725 nm against a reagent blank. All tests were carried out in triplicate. Total content of phenolic compounds in extracts was expressed as gallic acid equivalent (GAE) milligramme per gramme of dry sample extract. Gallic acid was used as standard for the calibration curve. Gallic acid of different concentrations, viz. 0.02, 0.04, 0.06, 0.08 and 0.10 mg/ml, was prepared in 50 % (v/v) methanol. The optical density was measured using ELISA Reader (Versa max, microplate reader).

Determination of Total Flavonoid Content

Flavonoid content was determined according to the aluminium chloride colorimetric method with some modifications [5]. The sample (4 mg/ml) solution (0.5 ml) was mixed with 0.1 ml of 5 % C4H4O6KNa·4H2O (potassium sodium l-(+)-tartrate tetrahydrate). After 5 min, 0.1 ml of 10 % aluminium chloride hexahydrate was added to the mixture and make up to 3 ml using distilled water. After incubation at room temperature for 40 min, the absorbance of the reaction mixture was measured at 430 nm. A blank was prepared substituting 0.1 ml of distilled water in place of 10 % aluminium chloride in the above reaction mixture. All tests were performed in independent triplicates. The total flavonoid content was expressed in milligramme Quercetin equivalent (QE) per gramme extract. Crude extracts that have been attuned to come under the linearity range and different dilution of standard solution of Quercetin (20–100 μg/ml).

DPPH Radical Scavenging Assay

The antioxidant effect of the extracts on 2,2-diphenyl,1-picryl hydrazyl (DPPH) radical was assayed using method described by Mensor et al. [20] with some modifications. Three millilitres of DPPH at concentration of 0.004 % (w/v) was added to 1 ml of the plant extracts at concentrations which are ranged from 4000 to 31 μg/ml. DPPH solution in methanol was used as control and methanol alone acted as blank. After 30 min, the discoloration from deep violet to yellow colour was measured at 517 nm using spectrophotometer.

The percentage inhibition was calculated by the following formula:

$$ \mathrm{Scavenging}\ \mathrm{activity}\ \left(\%\ \mathrm{of}\ \mathrm{inhibition}\right) = \left(A\mathrm{c}\ \hbox{--}\ A\mathrm{s}/A\mathrm{c}\right) \times 100 $$

where

A c :

Absorbance of DPPH radical as control

A s :

Absorbance of DPPH radical in the presence of the sample of plant extract

β-Carotene Bleaching Inhibition Assay

The antioxidant activity of plant extract was also evaluated using β-carotene linoleic acid model system [12]. β-carotene (0.5 mg) in 1 ml of chloroform was added to 25 μl of linoleic acid and 200 mg of Tween 80 (emulsifier mixture). Chloroform was evaporated at 40 °C using a rotary evaporator. One hundred millilitres of distilled water was slowly added to the residue, and the solution was vigorously agitated to form a stable emulsion. Four millilitres of this mixture was added into the test tubes containing 200 μl of plant extracts (4 mg/ml). As soon as the emulsified solution was added to the tubes, zero time absorbance was recorded at 470 nm. Then, the tubes were incubated for 2 h at 50 °C. Trolox was used as standard. Antioxidant activity was calculated as percentage of inhibition (%) relative to the control using the following equation:

$$ \mathrm{Inhition}\ \% = \left[1-\left({A}_{\mathrm{s}0}-{A}_{\mathrm{s}120}\right)/{A}_{\mathrm{c}}-{A}_{\mathrm{c}120}\right)\left.x100\right] $$

A s0 was initial absorbance, A s120 was the absorbance of the sample after 120 min, A c was initial absorbance of negative control and A c120 was the absorbance of the negative control after 120 min. The test was done in three replicates.

Antimicrobial Activity Tests

Microorganism

A total of seven microorganisms were selected for this study. Clinically isolated Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae and Candida albicans were obtained from medical microbiology laboratories, School of Medicine, Hospital Universiti Sains Malaysia.

Disc Diffusion Assay

The extracts were tested for their antimicrobial potential against the microorganisms using the standardised disc diffusion method ( British Society for Antimicrobial Chemotherapy (BSAC). The experiments were carried out in triplicates. The diameter of the inhibition zone was measured and recorded for each organism. Prior to test, the preparation of standard suspension was carried out according to the BSAC. Briefly, morphologically identical colonies of selected microorganisms were suspended from a plate of 18–24-h growth in sterile Mueller Hinton and Sabouraud dextrose broth (Oxoid) for bacteria and fungus, respectively, then, adjusted to a density of 0.5 McFarland standard units using McFarland densitometer (DEN-1B, BioSan) which corresponds to 108 CFU/ml for bacteria and 106 CFU/ml for fungus [33]. Within 15 min, 100 μl of prepared inoculum was then plated onto agar surface followed by impregnated disc application and incubation at 37 °C for 24 h and 30 °C for 48 h for bacteria and fungus, respectively. The antimicrobial activity of A. altilis fruit extracts was indicated by measuring the diameter of the inhibition surrounding each disc. All tests were done in triplicate.

Determination of MIC and MBC

The antimicrobial activity was carried out in vitro using microplate method (microwell dilution) according to the Wilkinson [36] procedure with some modifications. 2,3,5-Triphenyltetrazolium chloride (TTC) was used as visual indicator of microorganisms’ growth. The extract concentrations ranged from 4000 to 31 μg/ml by twofold dilution. The 96 well plates were set by dispensing the inoculum suspensions which was prepared as previously mentioned for disc diffusion assay. Into each well, 95 μl of Mueller Hinton broth (Oxoid, England) and 5 μl of the suspensions were added and then made up to a final volume of 200 μl for each well by adding 100 μl of extract. The growth control was broth and suspensions. The plates were incubated at 37 °C for 24 h for bacteria and 30 °C for 48 h for fungus. The minimum inhibitory concentration (MIC) value is considered as the lowest concentration of the sample extract which inhibits the growth of a microbe. To indicate the growth of microorganisms in well, a red colouration would be formed after adding TTC (10 μl/well, 20 mg/ml, dissolved in distilled water) and incubated for 30 min in the dark whereas the clear well can be indicated as inhibition of the growth [13].

The minimum bacterial concentration (MBC)/minimal fungicidal concentration (MFC) were determined by subculturing the clear wells from the MIC 96 well plates onto free microorganisms agar plates in order to determine the lowest concentration required to kill the microorganisms by incubating the plates at 37 °C for 24 h and 30 °C for 48 h for bacteria and fungus, respectively.

Statistical Analysis

Data of replicates were analysed using a one way ANOVA analysis and were expressed as means ± standard deviation (SD). Statistical differences between the reference and the sample groups were evaluated by ANOVA (one way) with Duncan’s multiple comparison tests p < 0.05 using SPSS 20.0 software. Correlations among data were calculated using Pearson’s correlation coefficient (R).

Results and Discussions

Determination of Total Phenols and Total Flavonoids Content

All plants produce a remarkable range of secondary metabolites. One of the most important groups of these metabolites are phenolic compounds. Phenolic compounds can be simple, low molecular weight, single aromatic ringed compounds to large and complex polyphenols. The antioxidant action of phenolic compounds is due to their high susceptibility to chelate metals. Phenolics possess hydroxyl and carboxyl groups, able to bind mostly iron and copper [11], and the concept of antioxidant action of phenolic compounds is not new. Total phenol content per 0.004 g of each extract of pulp, peel and whole fruit of A. altilis results are shown in Fig. 1; they were quantified based on the linear equation obtained from gallic acid standard calibration curve. Values of total phenol content of A. altilis fruit were in range from 203.17 ± 7.65 to 781 ± 52.97 mg GAE/g per g dry weight bases of extracts. Out of all extracts, methanol extract of pulp part had the highest content of phenols of 781 ± 52.97 mg GAE/g exhibiting relatively high levels of polyphenols phytochemicals. This is followed by methanol extract of peel part and whole of fruit of A. altilis (755.43 ± 22.02 and 717.82 ± 11.37 mg GAE/g), respectively, whereas the DCM (dichloromethane) extract of whole fruit had a phenol content of 335.65 ± 14.46 mg GAE/g. Statistical analysis showed that these variations are significantly different. From the results, it was also found that methanol extracts of all parts had higher TPC content than the others (hexane and DCM) due to the difference in solvent polarity. Methanol being a more polar solvent during the extraction process dissolved phytochemicals with higher polarity [38]. Phenolic compounds also play an important role which serve in defense mechanisms of plants by stabilizing the reactive oxygen species and prevent molecular damage [32].

Fig. 1
figure 1

Total phenolic contents of the crude extracts (mg GAE/g) are presented as mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 level compared to other varieties determined by Duncan’s multiple range test. GAE gallic acid equivalents (mg GAL/g dry sample)

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The total flavonoids content of different parts of A. altilis are shown in Fig. 2. They were quantified based on the linear equation obtained from Quercetin standard calibration curve. Total flavonoids (TFC) of A. altilis varied from 913.33 ± 24.44 to 6213.33 ± 142.22 in terms of milligramme Quercetin equivalents per gramme of dried sample (mg QE/g). The highest flavonoids found in content methanol extract of pulp are part of A. altilis fruit (6213.33 ± 142.22 mg QE/g per dry weight of extract).

Fig. 2
figure 2

Total flavonoid contents of the crude extracts (mg QE/g) are presented as mean ± SD (n = 3). Different letters indicate significant differences at p < 0.05 level compared to other varieties determined by Duncan’s multiple range test. QE Quercetin equivalents (mg QE/g dry sample)

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Antioxidant Activity

Determination of DPPH and BCB

The crude extracts of A. altilis were analysed for their antioxidant activities using the DPPH radical test, and the values are expressed as IC50 (Table 1).

Table 1 IC50 (μg/ml) values of the crude extracts
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One-way (ANOVA) analysis showed significant differences between the values of methanol extract of the pulp part of the fruit (55 ± 5.89 μg/ml) and the positive control (ascorbic acid, 61 ± 4.51 μg/ml). Methanol extract of pulp part (55 ± 5.89 μg/ml) inhibited the 50 % of DPPH free radicals, which is significantly lower than the standard (ascorbic acid). This indicated that maximum antioxidant activity is present in the pulp than in other parts of the fruit.

Beside the DPPH test, β-carotene bleaching assay was also carried out and the results are shown in Fig. 3. Trolox was used as a positive control which had the highest antioxidant activity (90.02 ± 1.51 %) compared to all extracts. Methanol extract of pulp part demonstrated antioxidant value of 88.34 ± 1.31 % followed by methanol extracts of whole fruit and peel part with 78.83 ± 1.38 and 76.93 ± 1.36 %, respectively. When seen together with the values of total flavonoid content, it shows that non-phenolic flavonoids are contributing to a large extent to the antioxidant activity of methanolic extract of all parts of A. altilis fruit.

Fig. 3
figure 3

The end point total antioxidant activities of the extracts of three parts of A. altilis fruit against β-carotene oxidation by linoleate radical. Data were expressed as antioxidant activities (% AA), mean ± SD (n = 3). Different letters showed significant difference compared to positive control (Trolox)

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Antimicrobial Activity Tests

Disc Diffusion Test

Natural product chemistry is a field that search for phytochemicals which could be synthesized and used as treatment for infectious ailments [34]. Due to the development of drug resistance in microorganisms, the demand of more effective antimicrobial agents is increasing. Mathekaga and Meyer [18] reported that in vitro screening method of plant crude extracts could help to find the alternative of commercial antibiotics by further investigations. This study investigated the antimicrobial activities of Artocarpus altilis fruit extracts against seven human pathogenic microorganisms. Two methods, viz. the disc diffusion method and microtitre plate method, were used to determine the antimicrobial activity of the fruit extracts in terms of MIC and MBC/MFC. The results of antimicrobial activity of the crude extracts of A. altilis were shown in Table 2. In this study, it was found that the methanol extract of pulp part was the most active extract against Gram-positive and Gram-negative bacteria. In this experiment, seven selected microorganisms were used. Methanol extracts of pulp part showed the highest zone of inhibition (14.83 ± 0.28 to 20.50 ± 0.76 mm) against all the species of microorganisms tested. The hexane and DCM extracts of both pulp and peel parts of A. altilis fruit showed antimicrobial activity only against Gram-positive bacteria such as S. aureus. Results in Table 2 indicated that methanol extracts of three parts of fruit exhibited the highest activity against Gram-positive and Gram-negative bacteria among other extracts (hexane and DCM), and this may be due to the correlation between the total phenolic content and the antimicrobial activity.

Table 2 Antimicrobial activity of A. altilis fruit extracts (500 μg g/ml) showing zone of inhibitions
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MIC and MBC/MFC

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of the extracts that inhibit growth of microorganisms. It is an essential test in terms of confirming resistance of microorganism to an antimicrobial agent by observing the activities of the extracts [15]. Minimum bactericidal concentration (MBC) and MFC was determined by subculturing the tested broth dilution (MIC) onto an agar plate and incubated further for 24 and 48 h for bacteria and fungus, respectively. Omar et al. [25] reported that most of the antimicrobial properties in different plant part extractions shows MBC/MFC value that is almost twofold higher than the corresponding MIC.

Results in Table 3 show the MIC concentrations of the various parts of A. altilis fruit with various types of solvents which have been used in this study. Methanol extract of pulp part of fruit revealed the lowest concentration value of 63 μg/ml, whereas methanol extracts of peel and whole fruit showed a value of 125 μg/ml against S. aureus. The highest concentration of methanol extract of pulp part was 1000 μg/ml against E. coli, whereby DCM extract of whole fruit showed comparatively efficient MIC value of 125, 250, 500 and 1000 a μg/ml against S. aureus, S. epidermidis, S. typhimurium and C. albicans, respectively. The lowest concentration against C. albicans among the extracts was 500 μg/ml demonstrated by methanol extract of pulp part. MBC and MFC results are shown in Table 4. The lowest concentration that killed 99.9 % of pathogen was 250 and 500 μg/ml of methanol extract of pulp part of the fruit against S. aureus and S. typhimurium, respectively, while the highest concentration was 4000 μg/ml by DCM extract of peel part against Gram-positive bacteria and fungus (S. aureus, B. cereus, S. epidermidis and C. albicans). The lowest MFC was 2000 μg/ml demonstrated by methanol extracts of pulp, peel and whole fruit and the DCM extract of whole fruit against C. albicans. It was also observed that extracts which have been tested via broth microdilution assay method demonstrated antimicrobial activity at lesser concentrations than those tested via disc diffusion methods, and this refer to many factors such as the slow diffusion of extracts into agar, highly affected by temperature and pH [17]. It enables the use of small quantities of extract which is important if the antimicrobial is limited as is the case for many natural products, ability to distinguish between bacteriostatic and bactericidal effects, and quantitative determination of the MIC [15]. It is reported that disc diffusion and serial dilution methods do not always give parallel results while measuring their antimicrobial activity [22].

Table 3 Minimum inhibitory concentration (MIC) (μg/ml) of A. altilis fruit extracts on different microorganisms
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Table 4 Minimum bactericidal/fungicidal (MBC)/(MFC) concentration (μg/ml) of A. altilis fruit extracts on different microorganisms
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As an overall insight of the study, the methanol extracts of various parts of A. altilis fruit showed the highest antioxidant and antimicrobial activity compared to other extracts. This enhances the correlations between the antioxidants assays and with antimicrobial assays. Ncube et al. [21] reported that the phenolics and polyphenols exhibited antimicrobial activity including essential subclasses of this group such as phenols, phenolic acids, quinones, flavones, flavonoids, flavonols and tannins and generally are soluble in polar solvents such as methanol [9]. There are also reports in the literature that the most commonly used solvents for investigations of antimicrobial activity in plants are methanol, ethanol and water [28]. Significant correlations were found between and within assays. The correlation coefficient of methanol extract of pulp part of the fruit for total phenol content and antioxidant activities using DPPH was 0.999, (p < 0.05), and using BCB was 0.997. Correlation between DPPH and BCB for methanol extract of pulp part was 0.999, (p < 0.05). The total flavonoids of the pulp part extracts also showed strong correlations using DPPH (0.978, p < 0.01) and BCB (0.992, p < 0.01). Methanol extract of whole fruit also showed a strong correlation for total phenols content using DPPH (0.998, p < 0.05) and BCB (0.998, p < 0.05), while for total flavonoids whole fruit extracts using DPPH showed a correlation coefficient of 0.876 and using BCB showed 0.910. These results are in concurrence with that of Yen et al. [37] and Norshazila et al. [24] who suggested strong correlations between the extracts with high amounts of total phenolic content and high antioxidant activity. On the other hand, the correlations between the antioxidant content and the antimicrobial activity of methanol extract of pulp part against Gram-positive bacteria such as S. aureus (0.998) and Gram-negative bacteria such as S. typhimurium (0.999, p < 0.05) showed high correlation of 0.998 and 0.999 (p < 0.05), respectively. There were no significant correlations between the antioxidant assays of peel part of the fruit. Similar findings were reported by Akinmoladun et al. [2] and Aboaba and Efuwape [1]. Levy [16] also stated that flavonoid, phenolic compound, tannins and alkaloid are the most important antimicrobial agents and bioactive constituents in plant. In recent years, studies of A. altilis have increased due to its importance in pharmaceutical applications [27]. It is important to bring underutilized plants to light not only for their potential bioactivities but also for the discovery of new sources of economic resources such as tannins, oil, gums, flavonoids and saponins [3] in line with current study, from the results which showed that A. altilis fruit extracts have high phenol and flavonoid content which make it valuable. Present study shows that A. altilis fruit contains high levels of phenols and flavonoids making it a potential source of antioxidants and antimicrobials.

Conclusion

Antioxidant and antimicrobial properties of various parts of plants extracts have recently been of abundant interest in both research and food industry because of their possible use as natural additives. Based on the results which were obtained in this research, it was concluded that the pulp, peel parts and whole fruit extracts of A. altilis have a high antioxidant and antimicrobial activities. The methanol extracts of pulp, peel and whole fruit showed considerably higher antioxidant and antimicrobial bioactivities, thus making them potential sources of antioxidant and antimicrobial agents.