Asian Journal of Plant Sciences

Year: 2023 | Volume: 22 | Issue: 1 | Page No.: 104-112
DOI: 10.3923/ajps.2023.104.112

Antioxidant, Antimicrobial Activity and Phytochemical Screening of Syzygium cumini L. Leaves in Tropical Region from Surabaya, East Java, Indonesia

Junairiah , Fatimah , Tri Nurhariyati and Nabilah Istighfari Zuraidassanaaz

Abstract: Background and Objective: Syzygium cumini is a plant of the Myrtaceae family. This plant has multifunctionality, due to its utilization as medicine, food, dye and building. This study aimed to determine the activities of antioxidant, antimicrobial, bioactive compounds and secondary metabolites contained in S. cumini leaves. Materials and Methods: The powder of S. cumini leaves was extracted with n-hexane, ethyl acetate and methanol as solvents. Three extracts were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and phytochemical screening to determine the type of bioactive compound and secondary metabolite. Antioxidant activity was tested with the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method and the antimicrobial test by diffusion method. Results: The results showed that n-hexane, ethyl acetate and methanol extracts of S. cumini contained 69, 57 and 64 bioactive compounds. Phytochemical screening displayed flavonoids, terpenoids and steroids from n-hexane extracts. Ethyl acetate extracts showed only flavonoid and methanol extracts contained flavonoid and saponin. N-hexane extracts of S. cumini had very strong antioxidant activity content with 23.79% of IC₅₀ when compared with the other two extracts. Conclusion: The results of the diffusion test exhibited three types of extracts that could inhibit the microbial pathogen such as Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10231.

Keywords: Antioxidant, antimicrobial, Indonesia, phytochemical screening and Syzygium cumini

INTRODUCTION

Plants have secondary metabolites and bioactive compounds which provide benefits for human health such as antioxidants, antimicrobials, anticancer, anti-inflammatory and antidiuretic^1-4. Medicinal materials of the plant have the advantage for easy to obtain, cheap, safe, efficient and rarely causing side effects⁵.

One type of plant that has potential as a medicinal ingredient is Syzygium cumini. The local name of this plant is Juwet which is also a member of the Myrtaceae family. The presence of this plant in the community is increasingly difficult to find, due to not many cultivating it. The shape of S. cumini ’s stem is round, branched and rooted. Moreover, its leaves are opposite which are generally oval, the base of the leaves is peg-shaped or round with the tips of the leaves that are blunt, pointed and rounded, then the texture of the leaves is smooth and the colour is green, light green, dark green. The fruit of this plant is buni type, oval in shape, purple in colour, black to dark purple in the skin fruit and purple to whitish purple in flesh. The texture of fruit provides smoothness, sweet taste and sour taste⁶.

People use this plant as a shade plant, fruit producer, building material and medicinal ingredient for antidiabetic, anti-inflammatory, antidiarrheal and wound. However, little information has been revealed about the content of secondary metabolites and bioactive compounds from leaf extract of S. cumini. Information about its potential as an antioxidant and antimicrobial has also not been widely reported. Therefore, the objective of this study was to identify the bioactive compounds and secondary metabolites contained in n-hexane, ethyl acetate and methanol extracts of S. cumini and evaluate the antioxidant and antimicrobial activities of three extracts.

MATERIALS AND METHODS

Plant collection and extraction preparation: The leaves of S. cumini were collected from Surabaya, Indonesia. This plant was identified at the Plant Physiology Laboratory, Department of Biology, Faculty of Science and Technology, Universitas Airlanggga from April to November, 2021. Leaves with healthy ones, free of pests and disease were used as research material. The leaves are washed with tap water, then dried and made into powder. Each leaf powder of S. cumini weighing 70 g was extracted with 900 mL of n-hexane (Fulltime, China), ethyl acetate (Fulltime, China) and methanol (Fulltime, China). Extraction was done by the maceration method. The extraction time for each solvent was 3 days and repeated 3 times.

Phytochemical screening and GC-MS analysis: The extract was treated by a phytochemical screening method to identify the type of secondary metabolites. Moreover, the alkaloid test used Mayer, Wagner and Dragendorff reagents. The terpenoid test utilized acetic anhydride and sulfuric acid. Flavonoid test with hydrochloric acid and magnesium (Mg) band. The saponin test used hot water and hydrochloric acid. Identification of the number and types of bioactive compounds were analyzed by GC-MS (Agilent Technologies 7890A, United States) and software GC-MS D5975C for data analysis.

Antioxidant activity: Antioxidant activity assay using DPPH (1,1-diphenyl-2-picrylhydrazyl) (Himedia, India) method with variant concentration at 6.25, 10, 12.5, 15, 25, 35, 50, 75 and 100 ppm. The percentage of antioxidant activity was calculated using formula⁷:

Calculation of inhibition concentration 50% (IC₅₀) was the concentration of the extract that inhibited 50% of DPPH free radicals. The IC₅₀ value was calculated using the linear regression equation:

where, the x-axis was the concentration value (ppm) and the y-axis was the value of antioxidant activity (%).

Antimicrobial activity: This study also used three types of microbes such as Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10231 each grown on Eosin Methylene Blue (EMB) (Oxoid, United Kingdom), Mannitol Salt Agar (MSA) (Oxoid, United Kingdom) and Potatoes Dextrose Agar (PDA) (Oxoid, United Kingdom) mediums. The paper blank disc (Oxoid, United Kingdom) was used to determine antimicrobial activity with Mueller Hinton Agar (MHA) medium (Oxoid, United Kingdom). The microbial suspension was prepared by determining the turbidity of microbial suspension at optical density (OD) 0.1 with the wavelength of 625 nm for bacteria and OD 0.1 with the wavelength of 600 nm for fungi.

A total of test microbial suspensions was put into the sterile petri dish and then 15 mL of MHA medium was added to the dish to be homogenized. A total of three sterile paper blank discs with a diameter of 6 mm and the same thickness were placed on the agar surface at an equal distance apart. In the paper disc, 25 μL of each extract was injected with the concentration of 0, 250, 500, 750 and 1000 ppm. Growth inhibition was indicated by the formation of a clear inhibition area (Halo) around the paper disc. The diameter of the inhibition zone was measured with a calliper (LC = 0.05 mm).

RESULTS

Identification of secondary metabolites and bioactive compounds on n-hexane, ethyl acetate and methanol of S. cumini leaves extracts: Table 1 showed that the extracts of n-hexane, ethyl acetate and methanol contain flavonoids. Terpenoids were only present in n-hexane extract. Saponins are only present in methanol extract. Meanwhile, Table 2 showed that the n-hexane extract of S. cumini leaves identified 69 compounds with two dominating compounds, namely 3-Buten-1-ol, 3-methyl and Furan, tetrahydro-3-methyl. While, Table 3 showed that 57 compounds were identified in the ethyl acetate extract of S. cumini leaves with the two compounds having the highest (%) area being 2E, 4E, 14E-N-Isobutylicosa and squalene. Table 4 shows that the methanol extract of S. cumini leaves has 64 compounds with 3 dominating compounds, namely acetic acid, prolol 92,1,5-cdoindolizine-1-crab and delta tocopherol.

Antioxidant activity observation: Table 5 shows that n-hexane extract of S. cumini leaves has the highest OD value at concentration 6.25 μg mL^–1 with 0.333 and the highest IC (%) at 100 μg mL^–1 with 80.65611%. While in the ethyl acetate extract of S. cumini leaves, a concentration of 15 μg mL^–1 has the highest OD value (0.439) and in concentration of 100 μg mL^–1 has the highest IC (%) with 29.63801% and the lowest OD value with 0.311. The concentration of 100 μg mL^–1 in the methanol extract of S. cumini leaves has the highest IC (%) with 59.27602%, while a concentration of 50 μg mL^–1 in the methanol extract of S. cumini leaves has the highest OD value (0.283). Table 5 also shows that n-hexane extracts of S. cumini leaves have the highest antioxidant activity with IC₅₀ 23.79 (very strong) compared between ethyl acetate and methanol extracts of S. cumini with IC₅₀ 171.53 (weak) and 64.44 (strong), respectively.

Antimicrobial activity evaluation: Table 6 shows that all various extracts of S. cumini leaves can inhibit S. aureus ATCC 25923 and C. albicans ATCC 10231 in all concentrations with an inhibition diameter was 6 mm, except for a concentration 750 ppm in n-hexane extract with an inhibition diameter were 6.7 mm. While the growth of E. coli ATCC 25922 can be the best inhibited by n-hexane extracts of S. cumini with the highest inhibition diameter being 8.8 mm at a concentration of 250 ppm. Meanwhile, in ethyl acetate extract of S. cumini the highest inhibition diameter was 8.1 mm at a concentration of 1000 ppm. The highest inhibition diameter at the methanol extract of S. cumini was 7.6 mm at a concentration of 250 ppm.

DISCUSSION

According to the results of phytochemical screening, flavonoids were present in three types of extracts (Table 1). Flavonoids are part of polyphenol compounds that have a benzo-γ-pyran structure and generally were found in plants. These metabolites are synthesized by the phenylpropanoid pathway and have various pharmacological activities^8,9. Flavonoids are a group of phenolic compounds found in all parts of plants, especially in photosynthetic plant cells. These flavonoids have various biological activities such as antioxidants, hepatoprotective, antimicrobial, anti-inflammatory, anticancer and antiviral^10-16.

In addition to a phytochemical screening test and bioactive compounds were identified using GC-MS. The identification of n-hexane, ethyl acetate and methanol extracts from S. cumini were shown in Table 2-4. The n-hexane extracts of S. cumini leave contained 69 compounds. This extract contained 37.39% furan compounds which these compounds are known to have antimicrobial activity¹⁷. The extract also contained 14.27% boronic acid compounds, which have several biological activities such as anticancer, antibacterial and antiviral¹⁸ (Table 2). The ethyl acetate extracts of S. cumini contained 57 compounds. One of the dominant compounds was 2E, 4E,14E-N-Isobutylicosa (3.51%). This compound has the potential of anti-inflammatory and anticancer¹⁹. Another compound found quite a lot was piperine. This compound has antibacterial activity against Salmonella typhi ²⁰ (Table 3). The methanolic extracts of S. cumini contained 64 compounds. One of the dominant compounds is tocopherol, which is a group of vitamin E. This compound has the potential as an antioxidant²¹. In addition, there are also acetic acid compounds that have the ability as antioxidants and antibacterial agents against Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231²² (Table 4).

Antioxidant activity test of n-hexane, ethyl acetate and methanol extracts using the DPPH method. Value of inhibition concentration 50% (IC₅₀) using DPPH method (1,1-diphenyl-2-picrylhydrazyl). The IC₅₀ is the concentration of substrate or sample solution that can reduce the activity of

DPPH (1,1-diphenyl-2-picrylhydrazyl) by 50% which indicates the concentration of extract (ppm) capable of inhibiting the oxidation process²³. In the DPPH test, antioxidants will react with 1,1-diphenyl-2-picrylhydrazyl (DPPH) which stabilizes free radicals and reduces DPPH. Furthermore, DPPH will react with hydrogen atoms from free radical scavenging compounds to form 1,1-diphenyl-2-picrylhydrazine (DPPH-H) which is more stable. The colour change from purple to yellow will occur due to the reaction of DPPH with antioxidants. The ability of antioxidants will determine the intensity of colour change²³. The results of the antioxidant activity from n-hexane, ethyl acetate and methanol extracts were shown in Table 5. Based on the results, the antioxidant activity of n-hexane extracts of S. cumini was stronger than that of the ethyl acetate and methanol extracts. The IC₅₀ of n-hexane extracts was 23.79 ppm, while IC₅₀ of silymarin solution was 29.63 ppm. The IC value of n-hexane, ethyl acetate and methanol extracts of Syzygium cumini collected from the Bogor Botanical Gardens, Bogor, West Java, Indonesia was 12.58 g mL^–1, 48.06 g mL^–1 and 16.91 μg mL^{–1 24}. This is also due to the flavonoid content contained in extracts. Flavonoids compound as an antioxidant, the mechanism is to capture reactive oxygen species (ROS) directly, prevent ROS regeneration and indirectly increase the antioxidant activity of cellular antioxidant enzymes²⁵.

Based on results of antimicrobial activity of n-hexane, ethyl acetate and methanol extracts of S. cumini using diffusion test were performed in Table 6. The three types of extracts inhibited S. aureus, E. coli and C. albicans. In general, the inhibition of E. coli was better when compared to other microbes. The antibacterial mechanism of flavonoids is to inhibit nucleic acid synthesis, cytoplasmic membrane function, energy metabolism, attachment and biofilm formation, inhibition of porins in cell membranes, change in membrane permeability and attenuation of pathogenicity²⁶. Factors affecting the diameter of the inhibition zone were extracts concentration, type of pathogenic microbe, the volume of extracts inserted into the paper disc, the thickness of the agar medium and the diffusion rate of antimicrobial substance through agar. The conclusion of this study is n-hexane, ethyl acetate and methanol extracts from S. cumini leaves contain bioactive compounds and secondary metabolites that have antioxidant and antimicrobial activity.

CONCLUSION

It can be concluded that the n-hexane extract of S. cumini leaves contains flavonoids and terpenoids with 69 bioactive compounds. The ethyl acetate extract of S. cumini leaves only contains flavonoids with 57 bioactive compounds. The methanol extract of S. cumini contains flavonoids and saponins with 64 identified bioactive compounds. The n-hexane extract of S. cumini leaves has the strongest antioxidant activity with IC₅₀ 23.79, it is also the highest of inhibiting the growth of E. coli ATCC 25922 with an inhibitory diameter was 8.8 mm at a concentration of 250 ppm.

SIGNIFICANCE STATEMENT

This study discovered the bioactive compounds, antioxidant and antimicrobial activity of S. cumini leaves that can be beneficial for providing information in the form of bioactive compounds that have the potential to be used as an alternative to developing drug-based sources in the pharmaceutical field. This study will help the researchers to uncover the critical areas of biological activities of S. cumini from Surabaya, Indonesia that many researchers were not able to explore.

ACKNOWLEDGMENT

The author thanks the Chancellor of Universitas Airlangga who has provided research funding (Faculty RKAT) through the 2021 Faculty Leading Research Grant (212/UN3/2021).

REFERENCES