Syzygium jambos (SJ), Syzygium malaccense (SM) and Syzygium samarangense (SS) are dicotyledonous species with a high distribution density in the wet zone of Sri Lanka. The leaves, barks and seeds are utilized in traditional medicine to treat different diseases. Despite their prevalence, few studies have been conducted in Sri Lanka to screen the bioactive ingredients. Therefore a comparative study was performed for the first time to investigate the antioxidant, anti-diabetic and antibacterial activities of leaves, fruits and seeds of the three species. Extraction was carried out with hexane, ethyl acetate, methanol and water at room temperature. DPPH free radical scavenging assay, alpha amylase inhibition assay and agar well diffusion method were used for analysis. Highest antioxidant activity was observed in SS methanol seed extract with a maximum percentage inhibition value of 92.07±0.77% at 34.0 (µg/mL) concentration. A noticeable alpha amylase inhibitory activity was seen in SS methanol leaf extract with a maximum percentage inhibition value of 83.10±1.22% at 2.30 (mg/mL) concentration. Hexane extract of SJ leaves depicted the highest inhibition zone, against Escherichia coli which was 21.5±2.5 mm whereas hexane seed extract of SS showed the highest inhibition zones against the bacterial strains Bacillus subtilis and Staphylococcus aureus which was 20±1 mm in diameter. Significant antibacterial activity of SS and SJ seeds were noted against the selected strains for the first time through this study. Due to the appreciable antioxidant, antibacterial and alpha amylase inhibitory activity, these species could be a principal source in nutraceutical and pharmaceutical products.
Keywords: Antioxidant; Antibacterial; Anti-diabetic; Solvents; Alpha amylase; Syzygium
Human beings have been utilizing plants for preventive and curative health care since ancient times [1]. The secondary metabolites in plants have been reported to exert biological effects. As a result, many tropical plants are screened as sources of bioactive molecules that could be incorporated into novel drug discovery targets [2].
According to world health organization up to 80% of world’s population rely on traditional medicinal systems for some aspect of primary health care [3]. Therefore, Sciences, Institute of Chemistry Ceylon, Sri Lanka.
there is a rising demand for plant based novel, inexpensive and effective biomolecules [4].
Sri Lanka being a country with a high degree of biodiversity provides a habitat for a vast number of species [5]. It is recorded that 1414 plant species belonging to different families are being used in traditional medicine within the country [6]. Myrtaceae is one such family of evergreen trees and shrubs, which comprises of Syzygium species [7]. Three Syzygium species selected for this study; SJ, SM and SS have been intimately related with humans due to their seasonal edible fruits and also due to utilization in healthcare practices. Due to characteristic medicinal properties, these plants have been used for therapeutic purposes not only in Sri Lanka but also in other Asian countries such as India, Bangladesh and Nepal [7-10].
In consideration of previous research based on SJ, SM and SS in different parts of the world; antihyperglycemic activity of the leaf and bark extracts of SM and leaf extracts of SS has been established [8-9]. Moreover the antioxidant activity of the fruit extracts of the three species [11-13] and the leaf extract of SJ and SM has been reported [10, 14]. When focusing on Sri Lanka, very limited research has been conducted based on these three species. In spite of the higher consumer acceptability as fruits and known medicinal value, the pharmacological properties of Sri Lankan species remain underestimated. Although there is a global appeal for herbal medicine extensive research is needed to validate the safest and effective plant based biomolecules from the repository of medicinal plants. Experimental evidence reflects significant variations in pharmacological activities based on solvent and on used parts of the plant. Therefore, the bioactivities of leaves, seeds and fruits were evaluated comparatively during this study utilizing four different solvents hexane, ethyl acetate, methanol and water for extraction at room temperature. The most appropriate solvents to establish the respective pharmacological properties and the corresponding plant parts were identified.
Mature leaves of SJ, SM and SS and ripen fruits of SM and SS were collected from Colombo Western Province of Sri Lanka. The leaves of the three species were authenticated by National Herbarium at Royal Botanical Garden, Peradeniya.
Hexane, ethyl acetate, methanol, Folin-Ciocalteu reagent, sodium carbonate, Gallic acid, 1,1-diphenyl-2- picrylhydrazyl(DPPH), Butylated hydroxytoluene (BHT), sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide, alpha amylase, corn starch, 3,5-dinitro salicylic acid (DNSA), Dimethyl sulfoxide (DMSO) and sodium potassium tartrate were used.
Freshly harvested, mature leaves were washed with running water and finally with deionized water. Petiole was removed from the leaves. Selected leaves were air dried at room temperature for three days [9]. Dried leaves were ground using a laboratory grinder to obtain a powder. The leaf powder was soaked in hexane for 24 hours at room temperature on a shaker. Solution was removed from the filtrate by rotary evaporation. The residue was again soaked in ethyl acetate for 24 hours at room temperature while shaking. The solution was filtered, and ethyl acetate was removed from the filtrate by rotary evaporation. The resulting leaf powder was again soaked in methanol for 24 hours at room temperature while shaking. Solution was filtered and methanol was removed from the filtrate by rotary evaporation. This procedure was repeated to prepare the leaf and seed extracts for all the three plants [7].
Aqueous extraction was carried out for fruits as follows. Ripen fruits were collected and washed with running water to remove any dirt and finally with deionized water. The seed from each fruit was separated. The remaining fleshy part of the fruits were cut into small cubes. Next it was homogenized with deionized water using a laboratory homogenizer for 10 minutes [15]. The solution was filtered using a cheesecloth and the filtrate was lyophilized [16]. This procedure was used to prepare the aqueous fruit extracts for SS and SM fruits.
Folin-Ciocalteu assay was used to determine the total phenolic content of all samples, which was expressed as milligrams of gallic acid equivalent per gram of sample (mg GAE/g). Gallic acid /extract (0.5 mL) was mixed with Folin-Ciocalteu reagent (0.5 mL) and was kept in dark at room temperature. After 05 minutes 6% w/v sodium carbonate (0.5 mL) and distilled water (2 mL) were added to the solution. The resulting mixture was again kept in dark at room temperature. After 01 hour, the absorbance was measured at 765 nm using a UV visible spectrophotometer (Hitachi U-2910 UV/Vis) [17].
Antioxidant capacity of all extracts were studied through the evaluation of free radical scavenging effect on the DPPH radical [18]. BHT was used as the standard. DPPH solution was made up to 0.5mg/mL concentration with methanol and the solution (2.0 mL) was mixed with sample series /BHT solution series (2.0 mL) in methanol. The final mixture was incubated at room temperature for 10 minutes in dark.
Absorbance of samples were measured at 517 nm using UV visible spectrophotometer (Hitachi U-2910 UV/Vis). The control was prepared as above without the extract and methanol was used as the blank [19, 20]. Radical scavenging activity which was expressed as the inhibition percentage was calculated using the following formula (1); 𝑨𝒄 − 𝑨𝒔 (1) Percentage inhibition = × 100% 𝑨𝒄 Where, Ac is the absorbance of the control and As is the absorbance of the sample.
Alpha amylase inhibition assay was performed on all the methanol extracts of leaves and seeds and on lyophilized fruit extracts. Commercially available Acarbose tablets were powdered and used as the standard [14]. Series of Acarbose solutions, series of sample solutions, DNSA solution, Sodium phosphate buffer (pH =6.9), 1% w/v starch solution was prepared.
Plant extract/Acarbose solution (0.5 mL) was incubated 0 with alpha amylase enzyme solution (0.5 mL) at 37 C for 10 minutes. After incubation, starch solution (0.5 mL) was added and was incubated for another 10 minutes 0 maintaining the temperature at 37 C. DNSA reagent was added to the above solution and was immediately 0 incubated in a water bath at 100 C. After 5 minutes, the reaction mixture was removed from the water bath and was diluted up to 5mL with deionized water after the assay medium reached to room temperature. For each test sample individual blank was performed by replacing enzyme with buffer whereas the control was performed replacing the extract with buffer. Both the plant extract and alpha amylase solution were replaced by buffer for the preparation of control blank.
Absorbance was measured at 540 nm using UV visible spectrophotometer (Hitachi U-2910 UV/Vis). Percentage inhibition was calculated using the following formula (2)[21,22].
Percentage inhibition Ac − Acb − (At − Atb) = (2) (Ac − Acb) × 100% Where Ac is the absorbance of the control, Acb is the absorbance of the control blank, At is the absorbance of the test sample and Atb is the absorbance of the test blank.
All sample extracts were dissolved in DMSO to obtain a solution with a concentration of 6.8 mg/mL. DMSO was used as the negative control. Clinically used antibacterial drug Azythromycin was used as the positive control [20]. The tablet was powdered and dissolved in DMSO to prepare 6.8 mg/mL concentration. Bacteria from the bacterial subcultures were gently scraped and were mixed with sterilized deionized water inside a test tube to adjust to 0.5 MacFarlane standard [23].
Then the bacterial solution (100.00 μL) was withdrawn from the test tube and was evenly spread in the petri dishes containing solidified agar. After 02 minutes, the agar bed was punched to produce 04 wells of 7 mm diameter. Two wells were filled with sample extract (50.00 μL) another well was filled with DMSO solution (50.00 μL) to be used as the negative standard and the final well was filled with Azythromycin solution. All the samples were maintained at room temperature for about 18 hours [24, 25]. All the plates were examined for growth inhibition and the diameter of the inhibition zone was measured in millimeters.
The highest phenolic content among all leaf, seed and fruit extracts was recorded in SS methanol leaf extract, which was 106±1 (mg GAE/g) as shown in figure 1. Except in SM leaf extract the total phenolic content of all hexane, ethyl acetate and methanol leaf and seed extracts were in a progressively increasing order. Whereas in the SM leaf the highest phenolic content was recorded in the hexane extract. The aqueous fruit extracts exhibited a significant low total phenolic content
All hexane, ethyl acetate, methanol and aqueous extracts were tested for DPPH radical scavenging activity.
Maximum percentage inhibition value was obtained for the methanol seed extract of SS, which was 92.07±0.77% at 34.00 (μg/mL) concentration. All the extracts which depicted significant free radical scavenging activity with respect to BHT are shown in table 1.
When considering the solvents used for extraction; hexane extracts showed least percentage inhibition values, ethyl acetate extracts showed moderate percentage inhibition values and methanol extracts showed the highest percentage inhibition values with respect to the standard BHT. The lyophilized fruit extracts exhibited percentage inhibition values only in higher concentration with respect to the standard BHT. Hence, the radical scavenging activity was progressively higher in water, hexane, ethyl acetate and methanol extracts respectively.
As indicated in table 2, methanol leaf extract of the three plant species showed good alpha amylase inhibition activity in comparison to the standard drug Acarbose, but there was no any significant inhibitory activity in seed extracts and lyophilized fruit extracts.
All the leaf, seed and fruit extracts were tested against Escherichia coli (gram-negative) Staphylococcus aureus (gram positive) and Bacillus subtilis (gram-positive) bacteria. Several leaf and seed extracts showed inhibition zones at least against one type of bacterial species as indicated in table 3. No inhibition zones were observed for the lyophilized fruit extracts against any type of bacteria.
Highest inhibition zone against Escherichia coli was noticed in the hexane leaf extract of SJ whereas the hexane extract of SS seed showed the highest inhibition zone against Staphylococcus aureus and Bacillus subtilis with respect to standard drug Azithromycin.
Presence of anti-diabetic, antioxidant and antibacterial activities in SJ, SS and SM scientifically validates the use of these plants in traditional medicine to treat diseases including diabetes mellitus. But it was observed that the pharmacological properties vary with different parts of the plant. This may be due to the qualitative and quantitative fluctuation of biomolecule distribution throughout the plant.
Out of the three selected plants, SS depicted the highest biological activity with respect to total phenolic content, free radical scavenging activity and antibacterial activity against Staphylococcus aureus and Bacillus subtilis species. Antibacterial activity and free radical scavenging activity were highest in SS seed extracts whereas SS leaves contained the highest phenolic content. Highest alpha amylase inhibition activity was observed in SM leaf.
Escherichia coli SS Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SM Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SJ Leaves Hexane Ethyl acetate Methanol Staphylococcus aureus SS Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SM Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SJ Leaves Hexane Ethyl acetate Methanol Bacillus subtilis SS Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SM Leaves Hexane Ethyl acetate Methanol Seeds Hexane Ethyl acetate Methanol SJ Leaves Hexane Ethyl acetate Methanol When considering about extraction of plant materials; methanol was the most successful solvent for antioxidant and anti-diabetic assays due to the high antioxidant and antidiabetic activity in methanol extracts comparative to the relevant standards. With respect to antibacterial activity, the best solvent was hexane, as the highest
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Apart from consumption as fruits more attention has to be focused on preparing value added products from seeds and leaves of SJ, SS and SM as it can act as a potent source of anti-diabetic, antioxidant and antibacterial developed as plant based nutraceutical and pharmaceutical products.
All the authors have contributed equally in designing, drafting the manuscript as per the journal submission format. All authors read and approved the final manuscript.
The authors declare that there are no competing conflicts of interest.
The authors are thankful to the National Herbarium at Royal Botanical Garden, Peradeniya for conducting the authentication of the plants.