INTRODUCTION:

Natural products still contribute to drug development nowadays, where up to 50% of approved drugs in the last 30 years isolated from natural products.¹ Myrtaceae families consist of 3,800-5,800 species in 140 genera. Meanwhile, syzygium is the largest woody genus of flowering plants and the 16^th most diverse genus with 1,100-1,200 species widely spread in the tropical and subtropical regions of the world. Samarangense is a common fruit plant in the home spreads of Kerala with good nutritive and chemical properties called rose apple or chambanga. Myrtaceae family consist of flowering plants native to an area that includes the Greater Sunda Islands, Malay Peninsula, and the Andaman and Nicobar Islands, but now widely cultivated in the tropics.

Ethyl acetate fraction of Syzygium samarangense leaves showed the highest antioxidant activity.² n studied Syzygium samarangense leaves contain flavonoids and saponins that can increase the activation of macrophages which is important to accelerate the process of collagen formation.³

Two flavonol glycosides have been isolated and characterized from the leaves Szygium samarangense.⁴ The ethanolic extract of Syzygium samarangense leaves contains, phenol, flavonoid, alkaloid, tannin, and terpenoid as chemical constitutents.⁵ The leaves of Syzygium samarangense have many compounds, which are flavonoids, phenolic compounds, tannins, terpenoids, and sterols.⁶ Ethanol extract of Syzygium samarngense leaves has been examined for antibacterial activity by using the broth dilution method.⁷ The extract was tested against E.coli, B.cereus, Enterobacter aerogenes, and Salmonella enterica and was more effective against B. cereus and S. enterica than others when compared with chloramphenicol.⁸Acylphloroglucinol, samarons, derivatives bearing a C₁₇alkyl side chain, along with five known analogs (5-9) are isolated from the leaves of Syzygium samarngense.⁹ Syzygium samarangense leave has beneficial effects together with drugs known for a remarkable analgesic, moderate anti-inflammatory as well as antidepressant effects.¹⁰ Secondary metabolites present in the plants are responsible for biological activities such as hypoglycaemic, antidiabetic, antioxidant, antimicrobial, anti-inflammatory anticarcinogenic¹¹ Rose apples are usually bell-shaped in appearance and can be eaten raw or preserved in the form of jellies and jams. They are also added to desserts and can be used to make fruit sauce or syrup. The rich vitamin C, dietary fiber, vitamin A, calcium, thiamin, niacin, iron, sulfur, and potassium are contained in rose apples. In terms of organic compounds, the rose apples also contain jambosine, betulinic and friedelolactone.

Moreover, some of the health benefits of rose apples include their ability to detoxify the liver, improve digestion, protect against diabetes, boost the immune system, lower cholesterol, prevent certain types of cancers. Also, eliminate fungal and bacterial infections and reduce fevers and even halt epileptic seizures. Leaves of Syzygium samarangense have been used by the local residence as medicine for various diseases caused by free radical agents in the human body. Myrtaceae plants are mostly used as medicinal plants for the treatment of bronchitis, asthma, diabetes mellitus, and inflammation syndromes.¹² Leaves and barks of Syzygium samarngense are used for various health issues like cough, cold, and amenorrhoea. Fruits are used as stomatitis aphthous, diuretic, emmengogue, abortifacient, and febrifuge.¹³ Ethanolic leaf extract of wax apple exhibited immunostimulant activity.¹⁴

MATERIALS AND METHODS:

Preparation of extract:

Syzygium Samarangense leaves (Figure1(a)) were collected at Kollemcode, Kanyakumari District, Tamilnadu, India. The leaves were dried in shadow for 10 to 15 days and ground by using the mechanical blender. About 20g of leaf powder (Figure 2(b)) was weighed and soaked in 200mL methanol for 72 h. The supernatant was collected and condensed by rotor evaporator at 50^oC, which yields greenish gummy extract.

Figure 1(a) Syzygium Samarangense leaves

Figure 1(b) Syzygium Samarangense leaves powder

Determination of total phenols:

The total phenol content of methanol leaves extract of Syzygium samarangense was determined by the Folin-Ciocalteau reagent method. One hundred μL of methanol leaves extract (1 mg/mL) was mixed with 900 μL of methanol and 1 ml of Folin-Ciocalteau reagent (1:10 diluted with distilled water). Then, 1 mL of 20% (w/v) of Na₂CO₃ solution was added and mixed well. Then it was allowed to stand for 30 min incubation in dark at room temperature. The absorbance was measured at 765 nm. The total phenolic content was expressed in terms of gallic acid and equivalent (μg/mg of extract), which is a common reference compound.

Determinations of total flavonoids:

The total flavonoid content of methanol leaves extract was determined by the aluminium chloride reagent method.^[15] Five hundred μL of leaf extract (1 mg/mL) was mixed with 0.5 mL of methanol and 1 mL of 5% (w/v) sodium nitrite solution. Then 1 mL of 10% (w/v) aluminium chloride solution was added followed by 100 μL of NaOH solution was added and shaken well. The absorbance was measured at 510 nm and the result was expressed as (μg/mg) of extract) quercetin equivalent, which is a common reference compound.

In vitro antioxidant assays:

DPPH radical scavenging activity:

The antioxidant activity of methanol leaves extract was measured by DPPH (I, I-diphenyl 2-picrylhydrazyl) free radical scavenging assay. One ml of 0.1 mM DPPH solution in methanol was mixed with 1ml of various concentrations (20-120 μg/mL) of aqueous leaves extract. The mixture was then allowed to stand for 30min incubation in dark. One ml of DPPH solution mixed with 1ml of methanol was used as the control. The absorbance was measured at 517 nm. Ascorbic acid was used as the positive control. The percentage of inhibition was calculated as follows:

Superoxide radical scavenging activity

Superoxide radical scavenging activity was carried by the Riboflavin method. The reaction mixture contains different concentrations (20-120 μg/ml) of methanol leaves extract, 1.5 mM of Riboflavin (200 μL), 12 mM of EDTA (100 μL),and 50 mM of NBT (50 μL). All the reagents should be prepared in 50 mM of phosphate buffer (pH 7.8) and the reaction was started by illuminating the reaction mixture for 90 seconds. Immediately, the absorbance was measured at 590 nm and ascorbic acid was used as the positive control. The percentage of inhibition was calculated as follows:

Phosphomolybdenum reduction assay:

The antioxidant capacity of methanol leaves extract of Syzygium samarangense was assessed by the phosphomolybdenum reduction assay method.^[16] Extract with different concentrations (20-120 μg/mL) was combined with 1 mL of reagent solution containing ammonium molybdate (4 mM), sodium phosphate (28 mM), and sulphuric acid (600 mM). The reaction mixture was incubated in a water bath at 95^oC for 90 min. The absorbance with the coloured complex was measured at 695nm. Ascorbic acid was used as a standard reference for the phosphomolybdenum reduction essay. The percentage of reduction was calculated as follows:

Ferric (Fe ³⁺) reducing power assay:

The reducing power of methanol leaves extract of Syzygium samarangense was determined by the slightly modified method.^[17] One mL of extract with different concentrations (20-120 μg/mL) was mixed with 1 mL of 1% (w/v) potassium ferricyanide [K₃Fe (CN)₆] solution add 1 mL of phosphate buffer (0.2 M, pH 6.6). The mixtures were then incubated at 50^oC in a water bath for 20 min. Five hundred μL of 10% (w/v) trichloroacetic acid solution was added to each mixture followed by 1 mL of 0.1% (w/v) freshly prepared FeC1₃ solution was added and shaken well. The absorbance is measured at 700 nm and ascorbic acid is used as the standard reference. The percentage of reduction was calculated as follows:

Antibacterial activity:

Microbial strains:

The microorganisms of Gram-positive bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Gram-negative bacteria such as Escherichia Coli and Enterococcus faecalis were used for the evaluation of the antibacterial activity.

Reference and control:

Tetracycline was chosen as the standard reference. The controls consist of solidifying agar onto which, solvent and the test compounds were soluble in it.

Aseptic conditions:

The aseptic chamber which consists of a wooden box (1.3m x 1.6m x 0.6m) with the door was, cleaned with 70% ethanol and irradiated with short wave UV light.

Nutrient broth agar medium:

Nutrient broth agar medium is prepared according to the standard methods (peptone-5 g, yeast extract-3 g, NaC1-5g distilled water- 1000 mL, agar-20 g). The required amount of ingredients were weighed and suspended in the required volume of 100 mL of distilled water and then autoclaved at 15 lbs and 121^oC for 15 min. The hot medium is poured in sterile petri plates which were kept in the aseptic Laminar chamber. The medium is allowed to solidify for 15 min.

Agar well diffusion method:

Antibacterial activity of methanol leaf extract was carried out using the agar well diffusion method. The solidified nutrient agar in the petri plates was inoculated by dispensing the inoculums using sterilized cotton swabs which are previously immersed in the inoculums containing test tubes and spread evenly onto the solidified agar medium. Five wells are created in each plate with the help of a sterile well-borer of 8 mm diameter. The root tuber extract was then poured into each well containing 250,375 and 500 μg/mL concentrations. All the plates with extract-loaded wells were incubated at 37^oC for 24H and the antibacterial activity was assessed by measuring the diameter of the inhibition zone formed around the well. Tetracycline (25 μg) was used as the positive control.

Gas chromatography-Mass Spectrometry (GC-MS):

The methanol leaf extract was injected into an HP-5 column (30 m X 0.25 mm i.d with 0.25 μm film thickness), Agilent technologies 6890 N JEOL GC Mate II GC-MS model. Following chromatographic conditions were used: Helium as the carrier gas, the flow rate of 1 mL/min; and the injector was operated at 200^oC and column oven temperature was programmed as 50-250^oC at a rate of 10^oC/min injection mode. Following MS conditions were used: ionization voltage of 70 eV; ion source temperature of 250^oC; interface temperature of 250^oC; the mass range of 50-600 mass units.

Identification of components:

The database of the National Institute of Standard and Technology (NIST) having more than 62,000 patterns was used for the interpretation of the mass spectrum of GC-MS. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library.

RESULTS AND DISCUSSION:

Total phenols and flavonoids:

Flavonoids and phenolic acids are making up one of the important pervasive groups in plant phenolics. Due to the tremendous biological of phenolic acids and flavonoids, they are used as therapeutic agents. Humans may contain thousands of different phenolic acid flavonoid components due to the consumption of plants and herbs. The antioxidative and possible anticarcinogenic activities are important in dietary phenolics. Reducing agents, free radical scavengers, and quenchers of singlet oxygen formation are the functions of Phenolic acids and flavonoids.¹⁸ In cancer and other human diseases, flavonoids and phenolic acids components are a very important role to control the diseases.¹⁹ Oxidative stress is considered to be substantial, if not crucial, in the initiation and development of many diseases, including inflammation, autoimmune diseases, cataracts, cancer, Parkinson’s disease, arteriosclerosis, and aging. Oxidative stress plays a role in heart diseases, neurodegenerative diseases, cancer, and the aging process. Phenolic compounds are physiologically active against herbivores or pathogens and degenerative diseases. Also, flavonoids can prevent injury caused by the free radicals and are oxidized by radicals, resulting in a more stable, less-reactive radical. The flavonoids and radicals are made to be inactive due to the high reactivity of the hydroxyl group. Antioxidants are specific compounds that protect human, animal, and plant cells against the damaging effects of free radicals. The phenols and flavonoids are good antioxidants, quantified in the methanol leaves extract of Syzygium samarangense. The Quantitative estimations of methanol leaves extract of Syzygium samarangense are presented in Table1.

Table 1: Quantitative estimations of methanol leaves extract of Syzygium samarangense

S. No	Phytochemicals	Amount (µg/mg)
1.	Phenols	643.58±3.90
2.	Flavonoids	20.30±1.14

DPPH radical scavenging activity:

Scavenging of DPPH free radicals is one of the most popular antioxidant methods. DPPH radical scavenging assay is a decolorization assay. It will measure the capacity of antioxidants to directly scavenge DPPH radicals. The ability of methanol leaves extract of Syzygium samarangense for scavenging free radicals was assessed by DPPH method. The maximum DPPH radical scavenging activity of methanol leaves extract of Syzygium samarngense was 66.19±3.27% at 120 µg/ml concentration (Table 2 Fig 2) and the IC₅₀ was 88.90 µg/ml concentration. It was compared to the standard of ascorbic acid.(IC₅₀ = 11.98 µg/ml concentration). In this method, the purple colour I,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was reduced to yellow colour 1, 1-diphenyl-2-picrylhydrazine , and the reducing capacity increases with increasing concentration of the extract.²⁰

Superoxide radical scavenging activity:

Superoxide anion is risky to cellular components and their effects can be magnified because it produces other kinds of free radicals and oxidizing agents. Flavonoids are effective antioxidants due to the scavenge superoxide anions. Superoxide anion is derived from dissolved oxygen by the riboflavin-light-NBT system will produce in the NBT system. In this system, the superoxide anion is reduced from yellow dye (NBT²⁺) to blue formazan, which is measured at 590 nm. Antioxidants can inhibit the blue NBT formation and the decrease of absorbance with antioxidants indicates the consumption of superoxide anion in the reaction mixture. The maximum superoxide radical scavenging activity of methanol leaves extract of Syzygium samarangense was 84.10±6.50% at 120 µg/ml concentration (Table 2, Figure 2) and the IC₅₀was 62.24 µg/ml concentration. It is compared with the standard of ascorbic acid. (IC₅₀was 9.65 µg/ml concentration).

Table 2: DPPH radical and superoxide radical (O2^•–) scavenging activities of methanol leaves extract Syzygium samarangense.

S. No	Concentration (µg/mL)	Methanol leaves extract of Syzygium samarangense
		% of inhibition
		DPPH. Radical	Superoxide (O2•–) radical
1	20	12.69±2.22	66.99±4.45
2	40	25.39±3.18	69.17±11.26
3	60	30.88±2.48	73.71±12.75
4	80	37.65±2.02	76.88±10.07
5	100	56.24±1.78	80.33±04.63
6	120	66.19±3.27	84.10±06.50

Figure 2: DPPH radical and superoxide (O2’^-) radical scavenging activities of methanol leaves extract of Syzygium samarangense.

Phosphomolybdenum (PM) reduction activity:

The total antioxidant activity of methanol leaves extract of Syzygium samarangense was measured by phosphomolybdenum reduction assay method. In this method, Mo (VI) ion is reduced to Mo(V) by the extract and the formation of green phosphate/Mo(V) complex at acidic ph¹⁹. The maximum Phosphomolybdenum reduction was 95.69±1.17% at 120 µg/ml concentration (Table 3, Figure 3) and the RC₅₀was 52.56µg/ml concentration. It was compared with the standard ascorbic acid (RC₅₀=6.34µg/ml concentration). Phosphomolybdenum assay is a quantitative method to investigate the reduction reaction rate among antioxidant, oxidant, and molybdenum ligand. It involves thermally generating auto-oxidation during the prolonged incubation period at a higher temperature.

Ferric (Fe³⁺) reducing power activity:

The reducing power activity was carried out by the reduction of Fe³⁺to Fe²⁺ by the methanol leaves extract of Syzygium samarangense and the subsequent formation of the Ferro-Ferric complex. The reduction increases with an increase in the concentration of the extract. The maximum Fe³⁺ reduction was 67.49±0.37% at 120µg/ml concentration and the RC₅₀ was 74.12 µg/mL concentration. It is compared with the standard ascorbic acid (RC₅₀ = 7.72µg/mL concentration). Also in this assay, higher absorbance of the reaction mixture indicates higher reduction potential and a significant indicator of its potential antioxidant activity. The educing capacity of the extract was performed using Fe³⁺ to Fe²⁺ reduction assay as the yellow colour changes to green or blue colour depending on the concentration of the antioxidants. The antioxidants such as phenolic acids and flavonoids were present, a considerable amount in the methanol leaves extract of Syzygium samarangense , and showed the reducing capacity in a concentration dependant manner.

Table 3: Phosphomolybdenum reduction and ferric reducing power activities of methanol leaves extract of Syzygium samarangense

S. No	Concentration (µg/mL)	Methanol leaves extract of Syzygium samarangense
		% of reduction
		Mo⁶⁺ reduction	Fe³⁺ reduction
1	20	72.97±6.24	23.32±0.45
2	40	88.96±2.91	44.6±0.61
3	60	92.93±1.85	44.29±0.62
4	80	95.04±1.34	50.58±0.44
5	100	95.12±1.36	67.45±1.45
6	120	95.69±1.17	67.49±0.37

Antibacterial activity by agar well diffusion method:

The methanol leaves extract of Syzygium samarangense was tested for its antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus (ATCC-25923) Pseudomonas aeruginosa, (ATCC-27853) and Gram-negative bacteria such as Escherichia coli, (ATCC-25922) and Enterococcus faecalis, (ATCC-29212). The antibacterial activity showed a maximum zone of inhibition of 20mm against Staphylococcus aureus at 500 µg/mL concentration of extract.²¹ The antibacterial activity may be due to the presence of phenolic compounds, terpenoids, which inhibit bacterial cell wall biosynthesis by binding to a highly conserved motif to lipid II and lipid III, two key precursors of bacterial cell-wall polymers as peptidoglycan and teichoic acid that adversely affect the growth of microbes.²² Antibacterial activity of methanol leaves extract of Syzygium samarangense as shown below (Figure 4).

Table 4: Antibacterial activity of methanol leaves extract of Syzygium samarangense

S. No	Organism	Zone of inhibition Mm			Standard (Tetracycline)
S. No	Organism	250 µg	375 µg	500 µg	Standard (Tetracycline)
1.	Escherichia coi (ATCC-25922)	16	17	18	19
2.	Enterococcus faecalis (ATCC-29212)	16	17	18	17
3.	Pseudomonas aeruginosa (ATCC-27853)	15	16	18	19
4.	Staphylococus aureus (ATCC-25923)	17	18	19	18

Figure 3: Phosphomolybdenum reduction and ferric (Fe³⁺) reducing power activity of methanol leaves extract of Syzygium samarangense.

Figure 4: Antibacterial activity of methanol leaves extract of Syzygium samarangense.

Figure 5: Gas chromatogram of methanol leaves extract of Syzygium samarangense.

Table 5: GCMS analysis of methanol leaves extract of Syzygium samarangense

S. No	Compound name	RT	Compound structure	Mol. weight g/mol	Mol. formula
1.	Phenol, 2-Propyl	11.28		136.19	C₉H₁₂O
2.	Bicyclo [3,3,1] nonan -9-ol, 9- methyl-	12.13		154.25	C₁₀H₁₈O
3.	Eugenol	13.47		164.20	C₁₀H₁₂O₂
4.	Benzeneacetic acid, a, 4- dihydroxy	15.25		168.14	C₈H₈O₄
5.	Thujopsene -(12)	16.18		204.35	C₁₅H₂₄
6.	Flavone	17.22		222.24	C₁₅H₁₀O₂
7.	2,6,10 – Dodecatriene, 3(E), 7(E), 11-trimethyl -1-methoxy	17.93		236.39	C₁₆H₂₈O
8.	n-Hexadecanoic acid	18.92		256.42	C₁₆H₃₂O₂
9.	Pentadecanoic acid,3-oxo-; methyl ester	19.52		270.41	C₁₆H₃₀O₃
10.	Oleic acid	21.68		282.5	C₁₈H₃₄O₂
11.	Pyrimidine,5 ethyl 1-2-[4-4-ethylcyclohexyl) Phenyl]-	23.27		294.43	C₂₀H₂₆N₂
12.	Morin	25.35		302.23	C₁₅H₁₀O₇
13.	Pregn-5-en-20-one,3 hydroxy-	27.73		316.5	C₂₁H₃₂O₂

GC-MS analysis:

GC-MS analysis of methanol leaves extract of Syzygium samarangense was shown in Table 5 and the chromatogram was represented in Figure 5. Antioxidant compounds such as flavone, Eugenol,^23,24 n-Hexadecanoic acid, oleic acid,^25,26 Morin and antibacterial compound Pyrimidine,5 ethyl 1-2-[4-4-ethylcyclohexyl phenyl]^27,28 were eluted and recorded.

CONCLUSION:

Antioxidants are substances that significantly delay or prevent the oxidation of an oxidizable substrate when present in low concentrations. Antioxidants help protect human body cells from the formation of radicals, also known as free are aggressive and toxic and are produced typically during cell metabolism. They are common transient intermediaries in chemical reactions with cell components, causing permanent damage and they are believed to be the source of aging and the cause of several degenerative diseases. Plants are potential sources of natural invaluable food antioxidants. Antioxidants react with free radicals and form a complex that prevents cell destruction and is easily cleaned out. The results of the study indicate that methanol leaves extract of Syzygium samarangense has significant antioxidant activities to reduce the harmful effect of radicals. The result of the present study provides promising hope to use Syzygium samarangense leaf extracts as a natural antioxidant as well as antibacterial agents. As an outcome of its antibacterial activity, Syzygium samarangense leaves are regarded as a very promising antibiotic candidate for the treatment of Gram-positive infections.

ACKNOWLEDGMENT:

The authors are thankful to SAIF, IIT Madras for helping in GCMS analysis.

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Received on 06.01.2022 Modified on 08.06.2022

Research J. Pharm. and Tech 2023; 16(5):2154-2160.

DOI: 10.52711/0974-360X.2023.00354