Isolation of Bioactive compounds from the essential oil of Jambolan (Syzygium cumini) and Invitro evaluation of biological potential

Received 08 Feb, 2023

Accepted 20 Mar, 2023

Published 12 Jul, 2023

The essential oil from leaves of the Syzygium cumini is the subject of this essay, which concentrates on its chemical makeup, antioxidant properties, cytotoxicity (as measured by the mortality and hemolysis of brine shrimp) and antibacterial properties. According to the results of the GC-MS study, 39 substances were discovered. Among these, α-pinene (22.29%), (E)-ocimene (11.68%), D-limonene (7.96%) and α-terpineol (5.26%) are the most noticeable. The free radical scavenging assay (33.17 g/mL), β-carotene in linoleic acid, and percent inhibition in linoleic acid (54.9%) systems were used to test the Syzygium cumini essential oil's antioxidant activity. Oil's antibacterial activity was tested using two-gramme-negative bacteria, four-gramme-positive bacteria, and four pathogenic fungi. The most susceptible fungi and bacteria were Aspergillus niger and Streptococcus Aureus, with MIC values of 2.03 and 3.83 mg/mL, respectively. The LC50 value of the S.cumini leaf essential oil was 29.45 µg/mL for cytotoxicity against Artemia salina nauplii, and the minimum hemolytic activity was 4.26% at a dosage of 0.5 mg/mL

INTRODUCTION

Free radicals bring on the processes taking place in the food systems. Free radicals are typically an essential physiology component and can be found as reactive, transient oxygen and nitrogen species (Di Meo & Venditti, 2020; Kruk et al.,2019). Due to oxidative stress brought on by an unbalanced antioxidant defence system in people, these reactive species are overproduced (Bhatti et al. 2022; Alemany-Cosme et al., 2021).

Despite many food preservation technologies, industrialists and customers are still concerned about how quickly food and other eatables deteriorate. Despite these preservation techniques, food sickness and shorter shelf life are caused by microbial contamination and lipid peroxidation. Additionally, it causes various food-borne illnesses and financial losses (Zubair et al., 2022; Gallo, 2020). Because of this, people are looking for natural ingredients like extracts and essential oils that can be utilized as food preservatives (Rahardiyan, et al., 2020). (Di Meo & Venditti, 2020).

Antioxidants have been overused in the food sector to extend the shelf life of foods containing polyunsaturated fatty acids. Antioxidants can be a very efficient substitute for storing food and reducing the oxidation rate (Gulcin, 2020; Pateiro et al., 2021). Due to their harmful impact on health, the frequent use of synthetic antioxidants is currently prohibited. Therefore, identifying efficient non-carcinogenic antioxidants that can safeguard the human body against free radicals and their deadly consequences through which they become accountable for chronic diseases has been of significant interest to scientists (Chen et al., 2021).

Numerous sources of plant-based antioxidants have been investigated during the investigation that has already been conducted. Additionally, these natural antioxidants can be synthesized into functional meals and nutraceuticals to reduce oxidative damage in our bodies. Scientists are paying attention to essential oils because they can preserve food, pharmaceuticals, complementary treatments, and natural cures (Anjos da Silva et al., 2023). They have also demonstrated several pharmacological properties, including hepatoprotective, antidiabetic, spasmolytic, antibacterial, anti-carcinogenic and antiviral activities. The behaviour of these against bacteria and oxidants, which demonstrated its effect on food security, is another factor drawing attention to them (Drinić et al.2020)

The Jambolan (S.cumini) tree is a sizable, leafy, evergreen plant indigenous to Pakistan, India, China, Indonesia and the West Indies and grown in tropical climates. Jambolan leaf essential oils have a variety of uses along with medicinal uses, including flavour in meat items, alcoholic beverages, and perfumery goods. Numerous studies have been conducted on the biological effects of Jambolan leaf essential oil (Sharmeen et al., 2021; Sarma et al., 2020; Elyemni et al.,2019).

The objective of the current study was to determine the composition and other characteristics of the hydro-distilled essential oil of S. cumini, including its antioxidant, cytotoxic, and antibacterial capabilities.

MATERIALS AND METHODS

Plant collection
The S. cumini leaves sample was collected from the Botanical Garden, University of Agriculture Faisalabad, Pakistan. Fresh material was treated right away to obtain its essential oil.

Extraction of Essential Oil
Bright green colour leaves of Jambolin were collected, washed and weighed. By boiling the plant material in water, the hydrodistillation process was employed to extract essential oils (Drinić et al., 2020; Sarma et al., 2020; Elyemni et al., 2019). 1000 g of fresh leaves were immersed in water and boiled for 3 hours. A separatory funnel was used to separate the oil because of its hydrophobic nature, which caused it to form a separate layer. Additionally, anhydrous sodium sulphate was used to eliminate the water content of the oil, followed by filtration and storage of the filtrate at 4 °C in a sealed glass bottle.

Analysis of Essential Oil
Different physical parameters like colour, refractive index, solubility and oil density were determined using standard methods [3-5]. A Perkin Elmer Clarus 600 GC with an ULTRA 1 column filled with 100% dimethyl polysiloxane (sheet thickness 0.25 m, 30 m 0.25 id) and a Perkin Elmer Clarus 600C MS with an FID detector were used to perform the GC-MS analysis of the S.cumini essential oil.

Antioxidant assay
The antioxidant activity of S.cumini essential oil was discovered with a few modest adjustments. The level of oxidation inhibition was calculated (Mahulette et al., 2020; Bogdan et al., 2021). The IC50 value (g/ml) was used to measure antioxidant activity; the lower the IC50 value, the greater the antioxidant activity (Missouri et al., 2020; Ikeda et al.,2021). Oxidation was measured using the thiocyanate technique (Elazzouzi et al., 2020). Inhibition of the linoleic acid emulsion system with a small adjustment allowed researchers to measure antioxidant activity (Ikeda et al., 2021).

Antimicrobial assays
Bacillus subtilis, Staphylococcus aureus, 2 gram-negative bacteria, Escherichia coli, Pasteurella multocida and 4 pathogenic fungi, Alternaria alternata, Aspergillus flavus, Aspergillus niger and Ganoderma lucidum, were tested for antimicrobial activity against the sample. While fungal strains were cultured for 48 hours at 28°C using potato dextrose agar, bacterial strains were grown overnight at 37°C in nutritional agar. The disc diffusion test was used to assess the antibacterial properties of S.cumini essential oil (Aimad et al., 2021; Krishnan et al., 2019; Abdollahzadeh et al., 2021). After mixing and autoclaving Potato Dextrose Agar and Nutrient Agar, 100 μL/100 mL of inocula were added to a medium, transferred to Petri plates, and hardened. The filter paper disc containing 10 μL sample was deposited and incubated for 24 and 48 hours at 37 and 28 degrees Celsius. Using a zone reader, inhibitory zone diameters were measured in millimetres. Results were contrasted with antibacterial drugs such as Rifampicin and Flucoanazole (Hekmati et al., 2020; Choi et al., 2021; Sun et al., 2021).

The MIC, or the concentration that prevents microorganism growth, was calculated using a microdilution broth susceptibility experiment (Sun et al., 2019; Tibbits et al., 2022; Schön et al., 2020). A 96-well microliter plate generated a series of essential oil dilutions, 160μ L of nutrient broth, potato dextrose broth for bacteria and fungi, and 20μ L of test solution. The next stage was performing an inoculation onto the microplates at a concentration of 105 CFU/mL (as verified by the viable count of a standard microbe suspension). For bacteria, the plates were incubated for 24 hours at 37°C, and for fungus, for 48 hours at 28°C. The antibacterial reference utilized was rifampicin, and the antifungal reference used was fluconazole. The well bottom had a white 'pellet' that represented growth, and the computations were done in mg/mL(Tibbits et al., 2022; Schön et al., 2020; Schug et al., 2020; Kavanagh et al., 2019; Silaban et al., 2022).

Haemolytic and cytotoxic effects
The brine prawn lethality test was conducted according to the protocol (Sharma &. Kharel 2019; Greco et al., 2020). The hemolytic activity was evaluated using human and bovine erythrocytes (Bastos et al., 2022; Santos-Sánchez et al., 2019). Following are the calculations for the hemolysis:

Data from the brine shrimp lethality assay were organized by dose, subject count, and a number of lethal events for statistical analysis. Results contain 95% fiducial limits for all statistically significant data.

RESULTS AND DISCUSSION

Yield of essential oil
The oil was light brown and had a yield of 1.57%. Similar calculations were made for additional physical variables like density (0.83 g/cm³) and refractive index (1.29300).

GC-MS Analysis of Essential Oil
Results of GC-MS analysis showed (Table 1) 39 components, accounting for roughly 98% of the total weight of the S.cumini essential oil sample. The primary constituents of the oil were discovered to include α-pinene (22.29%), (E)-ocimene (11.68%), D-limonene (7.96%), α-terpineol (5.26%), β-Caryophyllene (4.39%) and α-Humulene (2.59%). About 54.32% of the oil's composition was made up of hydrocarbon monoterpenes, followed by oxygenated monoterpenes (10.17%), hydrocarbon sesquiterpenes (5.16%), and oxygenated sesquiterpenes (4.30%).

Table 1:

GC /MS analysis of essential oil from Syzigium cumini leaves

No	Compound	RT	% age area
1.	α-Pinene	6.45	20.97
2.	Camphene	6.755	1.74
3.	Myrcene	8.065	3.78
4.	o-Cymene	9.07	1.12
5.	D-Limonene	9.24	7.96
6.	β-(E)-Ocimene	9.585	11.68
7.	α-(E)-Ocimene	9.86	4.7
8.	γ-Terpinene	10.145	0.36
9.	Isoterpinolene	11.045	0.84
10.	Fenchol	11.835	0.35
11.	Neo-allo-ocimene	12.305	0.46
12.	4(10)-Thujen-3-ol	12.625	0.2
13.	Thujol	12.91	0.19
14.	(E)-Borneol	13.465	0.31
15.	Terpinen-4-ol	13.815	0.41
16.	Crypton	14.11	0.48
17.	α-Terpineol	14.295	5.26
18.	(E )-Myrtenol	14.43	0.18
19.	Carveol	14.605	0.03
20.	(+)-Verbenone	14.815	0.22
21.	Fenchyl acetate	15.085	0.62
22.	Phellandral	16.755	1.34
23.	Bornyl acetate	17.045	2.23
24.	(+)-E-Pinocarvyl acetate	17.335	0.06
25.	(+)-Z-Verbenol, acetate	17.445	0.17
26.	Carvacrol	17.595	0.09
27.	α-Copaene	19.57	0.04
28.	(E)-β-Caryophyllene	20.825	4.39
29.	α-Humulene	21.72	2.59
30.	Alloaromadendrene	21.895	0.07
31.	Eudesma-4(14),11-diene	22.585	0.18
32.	α-Selinene	22.785	0.03
33.	Palustrol	24.72	0.34
34.	(+)-Spathulenol	24.93	1.1
35.	Caryophyllene oxide	25.065	2.2
36.	Epiglobulol	25.48	0.64
37.	Humulenol-II	26.235	0.27
38.	Longipinocarveol, trans-	26.34	0.3
39.	(E)-Guai-11-en-10-ol	26.685	0.33

Antioxidant potential of S.cumini essential oil
DPPH has been utilized as a valuable instrument for assessing the antioxidant capability of S.Cumini oil due to its stable free radical, the. The underlying mechanism is either the transfer of a hydrogen atom to DPPH or the addition of an electron, which neutralizes it in a reaction between DPPH and an antioxidant (Alemany-Cosme et al., 2021; Gulcin et al., 2020; Pateiro et al., 2021). The shift in colour of DPPH from purple to yellow, which was evaluated in terms of absorption taken by spectrophotometer at 515 nm, made it apparent that the concentration had decreased. The stable, purple-coloured radical DPPH was converted into its reduced form, which was yellow, by the S.cumini essential oil under investigation.

Compared to BHT, which had an IC₅₀ of 2.53 g/mL, the S.cumini sample in the current investigation had a good antioxidant potential with an IC50 of 19.83 μg/mL. The essential leaf oil from S.cumini reportedly has stronger antioxidant activity, according to Rabeque (2022). It was thought that the presence of α-pinene, the major component of S.cumini essential oil, which increases the activity of the entire oil, is supposed to be responsible for this potential.

The antioxidant capacity of S.cumini was also examined by S. Temesgen (Xiao et al., 2020) using DPPH and linoleic acid Inhibition tests. The thiocyanate method was employed to examine the antioxidant activity of the samples from S.cumini essential oil. This method inhibits the linoleic acid oxidation pathway. Unsaturated fatty acid linoleic acid oxidizes ferrous and ferric ions using various peroxides, forming a combination with thiocyanate ions derived from ammonium thiocyanate as a result (Ali et al.,2021; Lowry et al., 2020).

Absorbance was measured by spectrophotometer at 500 nm, and the concentration of the produced complex was ascertained. High absorbance values indicate that more peroxides are formed throughout the process, which lowers the oil's antioxidant activity. Compared to BHT, whose inhibition was 79%, all essential oil doses demonstrated considerable inhibition, ranging from 17% to 58%. The highest inhibition for the sample at concentrations of 50μ L/mL and 10μ L/mL is 62% and 12 %, respectively. The percent inhibition increased from 81.8% to 93.3% with changing concentrations from 20-150 μg/mL (Loucif et al., 2020).

The bleaching of β-carotene by S.cumini essential oil as an antioxidant in the linoleic acid system is depicted in Figure 1. Antioxidants prevent the system's β-carotene from being destroyed by linoleate or other free radicals by neutralizing them (Uddin et al.2021; Farahmandfar et al., 2019). The absence of antioxidants rapidly causes β-carotene to be colourless (Szwajgier et al., 2021). The rate of linoleic acid oxidation and antioxidant activity is directly correlated with the decline in β-carotene absorption. The control displayed the maximum colour depletion in the absence of a sample (Nonato et al., 2022; Theofanous et al., 2022; Temesgen et al., 2022)

Fig. 1: Inhibition of linoleic acid per oxidation by Beta carotene bleaching method of S. cumini essential oil

Antimicrobial potential of S.cumini essential oil
The antimicrobial activity of the S.cumini essential oil was tested using eight pathogenic microorganisms. Figures 2 to 5 display the findings of the minimum antibacterial and antifungal potential of S.cumini essential oil made using the disc diffusion method and MBC and MFC measurement by microdilution broth assays. According to the findings, S.cumini essential oil exhibits exceptional activity compared to antibiotics. S. aureus and E. coli were the most susceptible bacteria, with the biggest inhibition zones (11.7 mm and 11.3 mm) and the lowest MIC values (3.83 mg/mL and 2.9 mg/mL), respectively. The most sensitive and dynamic outcome has been shown by A. niger had the greatest inhibition zone (17.5 mm) and the lowest MIC value (2.03 mg/mL). Against A.alternata, A. flavus and G.lucidum, moderate inhibition zones of S.cumini essential oil were found. Setty (2022) have reported similar findings. They discussed the testing of Myristica fragrans' mace, meat extracts, and seeds against various pathogen strains. Regarding action against the pathogens, the flesh extract was superior to seed and mace.

Fig. 2: Antibacterial activity of S.cumini essential oil.

Fig. 3: Antifungal activity of S.cumini essential oil.

Fig. 4: Minimum bactericidal concentration of S.cumini essential oil.

Fig. 5: Minimum fungicidal concentration of S.cumini essential oil.

Haemolytic and cytotoxic potential of S.cumini essential oil
With LC50 values of 29.45 μg/mL and moderate activity in contrast to other essential oils, S. cumini essential oil has emerged as a superior choice for the brine shrimp lethality test. The presence of D limonene and α-β pienene in higher amounts may explain this distinct behaviour. Significant outcomes were discovered to exist. Another investigation was published for the seed and rind fragrant oils of A. subulatum (Nepali cardamom). Both showed moderate activity in the test for cytotoxic behaviour, with LC50 values of 25.2 and 13.1 g/mL, respectively (He et al., 2019)

Results of hemolytic activity showed that 15mg/mL of essential oil of S.cumini had the most hemolytic activity, while 5 mg/mL had the lowest. Bovine and human erythrocytes had maximum values of 8.24% and 9.63%, respectively, whereas these two species' lowest values were 0.29% and 0.43%, respectively. Analyzing a drug's hemolytic potential is crucial. This could serve as a valuable cytotoxicity indicator for assessing the viability of pharmacological applications (Ni,et al., 2019; Mehmood, et al., 2019). When compared to other plant oils and extracts, the S.cumini essential oil displayed less cytotoxicity. This unusual characteristic suggests its usefulness for pharmacological purposes, especially at lower dosages.

CONCLUSIONS

The essential oil was extracted from S.cumini from the Faisalabad district of Pakistan (1.57 g/100 g). It was shown to be enriched with α-pienene (22.29%) by GC-MS analysis. Syzygium cumini oil's antioxidant and antibacterial properties were assessed using various tests. Compared to the control, the results showed it had good potential as an antioxidant and antibacterial agent. Studies on cytotoxicity revealed that it has no cytotoxic impact at lower levels, making it appropriate for use as a medication.

CONFLICT OF INTEREST

The authors say they have no competing interests

REFERENCES

1. Alemany-Cosme, E., Sáez-González, E., Moret, I., Mateos, B., Iborra, M., Nos, P., ... & Beltrán, B. (2021). Oxidative stress in the pathogenesis of Crohn’s disease and the interconnection with immunological response, microbiota, external environmental factors, and epigenetics.Antioxidants,10(1), 64.
2. Anjos da Silva, L., Santos da Silva, R., Rodrigues de Oliveira, M., Guimarães, A. C., & Takeara, R. (2023). Chemical composition and biological activities of essential oils from Myrtaceae species growing in Amazon: an updated review.Journal of Essential Oil Research,35(2), 103-116.
3. Aimad, A., Sanae, R., Anas, F., Abdelfattah, E. M., Bourhia, M., Mohammad Salamatullah, A., ... & Noureddine, E. (2021). Chemical Characterization and Antioxidant, Antimicrobial, and Insecticidal Properties of Essential Oil from Mentha pulegium L.Evidence-based Complementary and Alternative Medicine,2021.
4. Abdollahzadeh, E., Nematollahi, A., & Hosseini, H. (2021). Composition of antimicrobial edible films and methods for assessing their antimicrobial activity: A review.Trends in Food Science & Technology,110, 291-303.
5. Ali, A., Yasir, M., Jilani, M. I., Shoaib, S., Latif, M., Ahmad, A., ... & Nazir, A. (2021). Chemical Composition and in vitro Evaluation of Cytotoxicity, Antioxidant and Antimicrobial Activities of Essential Oil Extracted from Myristica Fragrans Houtt.Polish Journal of Environmental Studies,30(2).
6. Bastos, J. C., Vieira, N. S., Gaspar, M. M., Pereiro, A. B., & Araújo, J. M. (2022). Human Cytotoxicity, Hemolytic Activity, Anti-Inflammatory Activity and Aqueous Solubility of Ibuprofen-Based Ionic Liquids.Sustainable Chemistry,3(3), 358-375.
7. Bhatti, J. S., Sehrawat, A., Mishra, J., Sidhu, I. S., Navik, U., Khullar, N., ... & Reddy, P. H. (2022). Oxidative stress in the pathophysiology of type 2 diabetes and related complications: Current therapeutics strategies and future perspectives.Free Radical Biology and Medicine.
8. Bogdan, M. A., Bungau, S., Tit, D. M., Zaha, D. C., Nechifor, A. C., Behl, T., ... & Copolovici, D. M. (2021). Chemical profile, antioxidant capacity, and antimicrobial activity of essential oils extracted from three different varieties (Moldoveanca 4, Vis Magic 10, and Alba 7) of Lavandula angustifolia.Molecules,26(14), 4381.
9. Chen, G., Sun, F., Wang, S., Wang, W., Dong, J., & Gao, F. (2021). Enhanced extraction of essential oil from Cinnamomum cassia bark by ultrasound assisted hydrodistillation.Chinese Journal of Chemical Engineering,36, 38-46.
10. Choi, J. S., Jung, H. C., Baek, Y. J., Kim, B. Y., Lee, M. W., Kim, H. D., & Kim, S. W. (2021). Antibacterial activity of green-synthesized silver nanoparticles using Areca catechu extract against antibiotic-resistant bacteria.Nanomaterials,11(1), 205.
11. Di Meo, S., & Venditti, P. (2020). Evolution of the knowledge of free radicals and other oxidants.Oxidative Medicine and Cellular Longevity,2020.
12. Drinić, Z., Pljevljakušić, D., Živković, J., Bigović, D., & Šavikin, K. (2020). Microwave-assisted extraction of O. vulgare L. spp. hirtum essential oil: Comparison with conventional hydro-distillation.Food and Bioproducts Processing,120, 158-165.
13. Elyemni, M., Louaste, B., Nechad, I., Elkamli, T., Bouia, A., Taleb, M., ... & Eloutassi, N. (2019). Extraction of essential oils of Rosmarinus officinalis L. by two different methods: Hydrodistillation and microwave assisted hydrodistillation.The Scientific World Journal,2019.
14. Elazzouzi, H., Zekri, N., Zair, T., & El Belghiti, M. A. (2020). Volatiles profiling and antioxidant activity of Moroccan Artemisia ifranensis J. Didier and Anacyclus pyrethrum Link essential oils.Egyptian Journal of Chemistry,63(10), 3937-3947.
15. Farahmandfar, R., Esmaeilzadeh Kenari, R., Asnaashari, M., Shahrampour, D., & Bakhshandeh, T. (2019). Bioactive compounds, antioxidant and antimicrobial activities of Arum maculatum leaves extracts as affected by various solvents and extraction methods.Food science & nutrition,7(2), 465-475.
16. Gallo, M., Ferrara, L., Calogero, A., Montesano, D., & Naviglio, D. (2020). Relationships between food and diseases: What to know to ensure food safety.Food Research International,137, 109414.
17. Greco, I., Molchanova, N., Holmedal, E., Jenssen, H., Hummel, B. D., Watts, J. L., ... & Svenson, J. (2020). Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides.Scientific reports,10(1), 13206.
18. Gulcin, İ. (2020). Antioxidants and antioxidant methods: An updated overview.Archives of toxicology,94(3), 651-715.
19. He, S., Wang, D., Zhang, Y., Yang, S., Li, X., Wei, D., ... & Qin, J. (2019). Chemical components and biological activities of the essential oil from traditional medicinal food, euryale ferox salisb., seeds.Journal of Essential Oil Bearing Plants,22(1), 73-81.
20. Hekmati, M., Hasanirad, S., Khaledi, A., & Esmaeili, D. (2020). Green synthesis of silver nanoparticles using extracts of Allium rotundum l, Falcaria vulgaris Bernh, and Ferulago angulate Boiss, and their antimicrobial effects in vitro.Gene Reports,19, 100589.
21. Ikeda, N. Y., Ambrosio, C. M., Miano, A. C., Rosalen, P. L., Gloria, E. M., & Alencar, S. M. (2021). Essential oils extracted from organic propolis residues: An exploratory analysis of their antibacterial and antioxidant properties and volatile profile.Molecules,26(15), 4694.
22. Kavanagh, A., Ramu, S., Gong, Y., Cooper, M. A., & Blaskovich, M. A. (2019). Effects of microplate type and broth additives on microdilution MIC susceptibility assays.Antimicrobial agents and chemotherapy,63(1), e01760-18.
23. Krishnan, M., Dey, D. K., Sharma, C., & Kang, S. C. (2019). Antibacterial activity of Weissella confusa by disc diffusion method.Bangladesh Journal of Pharmacology,14(3), 117-122.
24. Kruk, J., Aboul-Enein, H. Y., Kładna, A., & Bowser, J. E. (2019). Oxidative stress in biological systems and its relation with pathophysiological functions: the effect of physical activity on cellular redox homeostasis.Free radical research,53(5), 497-521.
25. Loucif, K., Benabdallah, H., Benchikh, F., Mehlous, S., Souici, C. B., & Amira, S. (2020). Total phenolic contents, DPPH radical scavenging and β-carotene bleaching activities of aqueous extract from Ammoides atlantica.Journal of Drug Delivery and Therapeutics,10(3-s), 196-198.
26. Lowry, J. R., Marshall, N., Wenzel, T. J., Murray, T. E., & Klegeris, A. (2020). The dietary fatty acids α-linolenic acid (ALA) and linoleic acid (LA) selectively inhibit microglial nitric oxide production.Molecular and Cellular Neuroscience,109, 103569.
27. Mahulette, A. S., Yahya, S., & Wachjar, A. (2020). Physico-chemical properties of clove oil from three forest clove accession groups in Maluku. InIOP Conference Series: Earth and Environmental Science(Vol. 418, No. 1, p. 012028). IOP Publishing.
28. Manssouri, M., Znini, M., & Majidi, L. (2020). Studies on the antioxidant activity of essential oil and various extracts of Ammodaucus leucotrichus Coss. & Dur. Fruits from Morocco.Journal of Taibah University for Science,14(1), 124-130.
29. Mehmood, T., Afzal, A., Anwar, F., Iqbal, M., Afzal, M., & Qadir, R. (2019). Variations in the composition, antibacterial and haemolytic activities of peel essential oils from unripe and ripened Citrus limon (L.) Osbeck fruit.Journal of Essential Oil Bearing Plants,22(1), 159-168.
30. Nonato, C. D. F. A., Camilo, C. J., Leite, D. O. D., da Nobrega, M. G. L. A., Ribeiro-Filho, J., de Menezes, I. R. A., ... & da Costa, J. G. M. (2022). Comparative analysis of chemical profiles and antioxidant activities of essential oils obtained from species of Lippia L. by chemometrics.Food Chemistry,384, 132614.
31. Ni, J., Mahdavi, B., & Ghezi, S. (2019). Chemical composition, antimicrobial, hemolytic, and antiproliferative activity of essential oils from Ephedra intermedia Schrenk & Mey.Journal of Essential Oil Bearing Plants,22(6), 1562-1570.
32. Pateiro, M., Gómez-Salazar, J. A., Jaime-Patlán, M., Sosa-Morales, M. E., & Lorenzo, J. M. (2021). Plant extracts obtained with green solvents as natural antioxidants in fresh meat products.Antioxidants,10(2), 181.
33. Pisoschi, A. M., Pop, A., Iordache, F., Stanca, L., Predoi, G., & Serban, A. I. (2021). Oxidative stress mitigation by antioxidants-an overview on their chemistry and influences on health status.European Journal of Medicinal Chemistry,209, 112891.
34. Rabeque, S. (2022). A review on phytochemical, antioxidant, and Hypoglycemic activity of traditionally important Syzygium spp.
35. Rahardiyan, D., Poluakan, M., & Moko, E. M. (2020). Physico-chemical properties of nutmeg (Myristica fragrans houtt) of North Sulawesi nutmeg.Fullerene Journal of Chemistry,5(1), 23-31.
36. Santos-Sánchez, N. F., Salas-Coronado, R., Villanueva-Cañongo, C., & Hernández-Carlos, B. (2019). Antioxidant compounds and their antioxidant mechanism.Antioxidants,10, 1-29.
37. Sarma, N., Begum, T., Pandey, S. K., Gogoi, R., Munda, S., & Lal, M. (2020). Chemical composition of Syzygium cumini (L.) Skeels leaf essential oil with respect to its uses from North East region of India.Journal of Essential Oil Bearing Plants,23(3), 601-607.
38. Setty, J. V., Srinivasan, I., Sathiesh, R. T., Kale, M., Shetty, V. V., & Venkatesh, S. (2020). In vitro evaluation of antimicrobial effect of Myristica fragrans on common endodontic pathogens.Journal of Indian Society of Pedodontics and Preventive Dentistry,38(2), 145-151.
39. Schön, T., Werngren, J., Machado, D., Borroni, E., Wijkander, M., Lina, G., ... & Cambau, E. (2020). Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates–the EUCAST broth microdilution reference method for MIC determination.Clinical Microbiology and Infection,26(11), 1488-1492.
40. Schug, A. R., Bartel, A., Scholtzek, A. D., Meurer, M., Brombach, J., Hensel, V., ... & Feßler, A. T. (2020). Biocide susceptibility testing of bacteria: Development of a broth microdilution method.Veterinary microbiology,248, 108791.
41. Sharmeen, J. B., Mahomoodally, F. M., Zengin, G., & Maggi, F. (2021). Essential oils as natural sources of fragrance compounds for cosmetics and cosmeceuticals.Molecules,26(3), 666.
42. Sharma, K. R., & Kharel, R. (2019). Antibacterial, Antidiabetic and Brine Shrimp Lethality Activities of Some Selected Medicinal Plants from Kavrepalanchok District of Nepal.Journal of Institute of Science and Technology,24(1), 57-62.
43. Silaban, S., Nainggolan, B., Simorangkir, M., Zega, T. S., Pakpahan, P. M., & Gurning, K. (2022). Antibacterial activities test and brine shrimp lethality test of Simargaolgaol (Aglaonema modestum Schott ex Engl.) leaves from North Sumatera, Indonesia.Rasayan Journal of Chemistry,15(02), 745-750.
44. Sun, B., Wu, F., Zhang, Q., Chu, X., Wang, Z., Huang, X., ... & Shen, J. (2021). Insight into the effect of particle size distribution differences on the antibacterial activity of carbon dots.Journal of Colloid and Interface Science,584, 505-519.
45. Sun, H., Chan, C. W., Wang, Y., Yao, X., Mu, X., Lu, X., ... & Ren, K. (2019). Reliable and reusable whole polypropylene plastic microfluidic devices for a rapid, low-cost antimicrobial susceptibility test.Lab on a Chip,19(17), 2915-2924.
46. Szwajgier, D., Baranowska-Wójcik, E., Kukula-Koch, W., Kowalik, K., Polak-Berecka, M., & Waśko, A. (2021). Evolution of the anticholinesterase, antioxidant, and anti-inflammatory activity of Epilobium angustifolium L. infusion during in vitro digestion.Journal of Functional Foods,85, 104645.
47. Temesgen, S., Sasikumar, J. M., & Egigu, M. C. (2022). Effect of Extraction Solvents on Total Polyphenolic Content and Antioxidant Capacity of Syzygium Aromaticum L. Flower Bud from Ethiopia.BioMed Research International,2022.
48. Theofanous A, Sarli I, Fragou F, Bletsa E, Deligiannakis Y, Louloudi M. Antioxidant Hydrogen-Atom-Transfer to DPPH Radicals by Hybrids of {Hyaluronic-Acid Components}@SiO₂. Langmuir. 2022 Oct 11;38(40):12333-12345.
49. Tibbits, G., Mohamed, A., Call, D. R., & Beyenal, H.(2022).Rapid differentiation of antibiotic-susceptible and -resistant bacteria through mediated extracellular electron transfer.Biosensors and Bioelectronics,197,113754.
50. Xiao, F., Xu, T., Lu, B., & Liu, R. (2020). Guidelines for antioxidant assays for food components.Food Frontiers,1(1), 60-69.
51. Yau, D., Ahmad, T. Y., Baba, K. I., Lado, A. U., Musbahu, L., & Umar, A. U. (2020). Phytochemical composition of desert date kernel (Balanites aegyptiaca) and the physical and chemical characteristics of its oil.GSC Biological and Pharmaceutical Sciences,11(3), 197-207.
52. Zubair, R., Amin, J., Tuseef, M., Anam, M., Khalid, M. A., Gull, T., & Latif, M. F. (2022). A review on bio-Preservative effect of essential oils as an antioxidant and antimicrobial agents on ready-to-eat chicken sausages.Life Science Journal,19(11).