Journal of Food Quality
Volume 2021 (2021), Article ID 5513717, 11 pages
https://doi.org/10.1155/2021/5513717

Research Article

Evaluation of the Physicochemical, Antioxidant, and Antibacterial Properties of Tunichrome Released from Phallusia nigra Persian Gulf Marine Tunicate

Zohreh Marhamati,¹ Mohammad Hossein Marhamatizadeh,¹ and Gholamhossein Mohebbi²

¹Department of Food Hygiene, Faculty of Veterinary Medicine, Kazerun Branch, Islamic Azad University, Kazerun, Iran
²The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran

Correspondence should be addressed to Mohammad Hossein Marhamatizadeh; drmarhamati@gmail.com

Received 11 February 2021; Revised 26 March 2021; Accepted 23 April 2021; Published 28 April 2021

Academic Editor: Laura Arru

Copyright © 2021 Zohreh Marhamati et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The aim of this study was to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Persian Gulf tunicate (Phallusia nigra). For this purpose, molecular weight (SDS-PAGE), amino acid profile, chemical composition (GC-MS), mineral composition, functional groups (FTIR), total phenol content (TPC), total flavonoid content (TFC), antioxidant activity, and antimicrobial properties were investigated. The results showed that tunichrome contained a high amount of essential amino acids (i.e., Lys = 32.24 mg/100 g) and essential minerals. According to GC-MS results, tunichrome had different antioxidant and antimicrobial components. The TPC and TFC of tunichrome were 0.55 mg GA/g and 0.21 mg quercetin/100 g, respectively. Tunichrome showed higher antioxidant activity than ascorbic acid, and its radical scavenging activity values were increased from 30.28 to 82.08% by increasing concentration from 50 to 200 ppm. Inhibition zones of Staphylococcus aureus, Bacillus cereus, Salmonella enterica, and Escherichia coli O157:H7 were 14, 18, 17, and 15 mm, respectively. Moreover, the minimum inhibitory concentration values of tunichrome for S. aureus, Bacillus cereus, S. enterica, and E. coli O157:H7 were 1.17, 0.59, 0.59, and 1.17 mg/ml, respectively. The minimum bacterial concentrations were 2.34, 1.17, 1.17, and 2.34 mg/ml for S. aureus, Bacillus cereus, S. enterica, and E. coli O157:H7, respectively. These results showed that tunichrome of Phallusia nigra has excellent biological effects as a bioactive compound for food fortification.

1. Introduction

Since the synthetic antioxidants and antimicrobial components could exert several side effects, the interest of consumers and producers for safe and natural ingredients is growing [1]. Bioactive compounds are the most attractive ingredients in the design and development of functional foods. The tunicates are a wide marine animal group whose bodies are covered by the cellulose-containing tunic. They are marine filter invertebrates that exhibit the properties of the vertebrates. The accumulation of a high level of metals (i.e., either vanadium or iron) in seawater can be possible by intracellular polymer matrices [2, 3]. Thus, in the presence of metal chelators such as catechol and pyrogallol groups, tunichromes are associated with metal reduction. Moreover, tunichrome can form covalent crosslinking interactions or complexes with different types of multivalent ions in seawater. This ability can affect its functional properties such as swelling, solubilization, coagulation, and precipitation behavior.

The ascidians, thaliaceans, and appendicularians are the three main groups of tunicates [4]. These animals are consumed in Asia, Chile, and the Mediterranean in the past. These products come from sea wild and cultured populations when the demand is high, especially for Halocynthia and Styela species. Edible species are usually from solitary stolidobranchs. Halocynthia aurantium, H. roretzi, Microcosmus hartmeyeri, M. sabatieri, M. vulgaris, Polycarpa pomaria, Pyura chilensis, Styela clava, and S. plicata are the important species eaten. These groups are presented in fresh and dried forms in the markets. High-quality processes of H. aurantium, H. roretzi, P. chilensis, S. clava, and S. plicata are the main seafood exports to Europe and America [5].

Generally, tunicates have a high nutritional value due to the presence of a high level of bioactive components and protein and low calories [6–10]. Moreover, some of them contain different vitamins (i.e., vitamin E, vitamin B12, and vitamin C), minerals (i.e., Na, K, Ca, Mg, P, Fe, Zn, and Cu), amino acids, folic acid, fatty acid, and pantothenic acid [11]. Some groups of tunicates have different low molecular weight peptides in their bodies such as styelins, plicatamide, halocyamines, lamellarins, and ferreascidin from Styela clava, Styela plicata, Halocynthia roretzi, Didemnum chartaceum, and Pyura stolonifera, respectively, as well as tunichromes from different tunicates species [5, 12, 13].

Some tunicates species can accumulate low molecular weight oligopeptides in their blood cell which are known as tunichromes. Tunichromes could play an important role in defense mechanism, mainly due to the phenoloxidase which can attack easily tunichrome. It is considered as a key component for both tunic formation and tunic wound healing which use the same primary biochemical mechanisms [13]. Tunichromes have different colors such as pink, red, and blue [14]. These pigments are ingredients in the blood cells of tunicates. Tunichromes were identified in 11 groups of different tunicates [5]. The broad biological activity of tunichromes remains mainly unknown. However, some researches offer unique functionalities for tunichrome in terms of antioxidant [15–17], antimicrobial [5, 18], and anticancer activities [19].

To the best of our knowledge, there is no information on the biological activities of the amazing tunichrome released from the Phallusia nigra Persian Gulf marine tunicate. Therefore, this research is a first attempt to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

2. Materials and Methods

2.1. Materials

Phallusia nigra tunicate was randomly collected from Nayband Bay situated in the north (27° 30′S, 52° 35′E) of Bushehr, Iran, using SCUBA diving at the depth ranging from 0.5 to 1.5 m in spring of 2019. Media cultures and all other chemical materials were prepared in analytical grade from Merck and Sigma-Aldrich.

2.2. Methods

2.2.1. Sampling and Sample Preparation

50 samples of Phallusia nigra tunicates were washed by double distilled water (DDW) to remove contaminants. They were immediately transported to the laboratory in a cool box (in ice-cold condition) and kept at room temperature for 30 min to release a pink color solution (tunichrome). The obtained suspension was centrifuged (4000 × g for 5 min) and then filtrated by Whatman No. 1 filter paper. The final solution was freeze-dried and powdered for further studies.

2.2.2. Total Protein Content

Total protein content was measured based on the AOAC method by the Kjeldahl device (Buchi, Switzerland).

2.2.3. Molecular Weight Measurement (SDS-PAGE)

For the molecular weight determination, the tunichrome (5 mg/mL) was dissolved in 1% SDS solution prepared by phosphate buffer (pH 7.0) and stirred for 24 h followed by centrifugation at 5000 × g for 6 min at 20°C [20]. The supernatant (20 μl) was added to 10 μL Tris-HCl buffer (10 mM Tris-HCl, 10% (w/v) SDS, 0.1% (w/v) bromophenol blue, and 10% (v/v) glycerol) with or without 2% (v/v) β-mercaptoethanol. The suspension was then heated at 95°C for 11 min, and 20 μL of the suspension was developed on the gel slab which was contained resolving gel (12.5%, pH 8.8) and stacking gel (6%, pH 6.8). PowerPac 1000 (Bio-Rad, USA) was used for running the electrophoresis. The running buffer was made by diluting 100 mL 10 X Tris/Glycine/SDS buffer with the DDW at the constant voltage 220 V. The Coomassie Brilliant Blue was used for staining the gel for 60 min, and water was used for destaining for 24 h. The molecular weights were evaluated by comparison to Sinaclon markers (PR901641, CinnaGen Co., Tehran, Iran) at the ranges of 245–11 kDa.

2.2.4. Amino Acids’ Profile

To evaluate amino acid profile in tunichrome, peptide bonds of protein were broken by hydrolysis. The hydrolysis process was conducted by heating the sample in an oxygen-free condition containing 6 M HCl and 0.1% phenolphthalein at 110°C overnight. Amino acids were measured by a Varian chromatographic system, containing a 1525 pump, a 9100 autoinjector, and a UV-vis detector. The hydrolyze suspension was injected into an automatic precolumn reaction by 0.1 mL of derivatizing reagent. This chromatographic process contained a solvent mixture (PBS buffer (10 mM, pH 4): acetonitrile (25 : 75) at a flow rate of 1 mL/min. In this system, a C18 Waters Nova-Pack reverse phase column (particle size 5 μm, 250 × 4.6 mm internal diameter) was applied. All the chromatographic data were processed in a V. 4.5 Star workstation supplied by Varian [21].

2.2.5. Na, Ca, K, Mg, Mn, Cu, Fe, and Zn Measurements

The concentrations of Na, Ca, K, Mg, Mn, Cu, Fe, and Zn of tunichrome were analyzed by a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, Netherlands). To this end, 1 g of lyophilized powder was dissolved in the 5 g of DDW for 24 h and then centrifuged.

2.2.6. Heavy Metal (Cd, As, Pb, and Hg) Measurements

The lyophilized powder of tunichrome (0.2 g) was weighed and mixed with concentrated HNO₃ (7 mL, 65% v/v) and H₂O₂ (1 mL, 30% v/v) in the polytetrafluoroethylene vessel. The vessel temperature was raised to 160°C for 21 min by mixing at 1500 W in the magnetron (ETHOS One, Milestone, Italy). After this digestion process, the sample was cooled for 120 min, and then, the final volume of the sample reached 50 mL with DDW. The determination of heavy metals was conducted by an atomic absorption spectrometry (Atomic Absorption Spectrophotometer, Varian AA240 FS) system which contained an electrode discharge lamp for evaluating volatile and nonvolatile toxic heavy metals by using argon gas. A graphite furnace was applied to determine nonvolatile compounds (Pb and Cd), and a flow injection-mercury hydride system was applied to calculate volatile (As and Hg) compounds.

2.2.7. Fluoride, Chloride, Bromide, Nitrite, Nitrate, Phosphate, and Sulfate Content Measurement

This measurement was done by an ion chromatograph 761 Compact IC (Metrohm, Herisau, Switzerland) with anion self-regenerating suppressor Metrohm Suppressor Module MSM and conductivity detector. Anion separation was performed by a “Star-Ion-A300” column (100 mm × 4.60 mm, Phenomenex, Torrance, USA). A “Metrosep A PCC 1 HC” column (12.5 mm × 4.0 mm, Metrohm, Herisau, Switzerland) was applied for preconcentration. The volume sample loop was 20 μl. Sample injection to the ion chromatography was done by a 5 ml Becton Dickinson syringe (Fraga, Spain) [22].

2.2.8. FTIR Spectroscopy

FTIR spectroscopy (WQF-510 FTIR Rayleigh, Beijing Rayleigh, China) was used for the evaluation of the peak absorbance intensity of the functional groups in the structure of tunichrome. To this end, dried powder of tunichrome was mixed with KBr to form a compact plate. The FTIR spectra were recorded in the wavenumber range of 4000–400 cm⁻¹.

2.2.9. Determination of Chemical Composition by GC-MS

The chemical composition of tunichrome was investigated by a GC (7890B, Agilent Technologies, Santa Clara, CA, United States) system containing MS (5977ANetwork, Agilent Technologies). Briefly, 200 mg of tunichrome was homogenized with 200 ml of methanol: chloroform: hexane (1 : 1 : 1), for 24 h in 200 rpm and then centrifuged for 20 min at 4000 rpm. The final supernatant was used for evaluating the chemical composition. The GC system was equipped with an HP5 MS column (nonpolar column, Agilent Technologies, internal diameter: 30 m × 250 μM, film thickness: 0.25 μm). The flow rate of carrier gas (Helium) was 1 mL/min. The temperature of the injector was set at 120°C. The oven temperature program included three steps: (1) 50°C for 1 min, (2) temperature raising to 300°C at a rate of 15°C/min, and (3) holding at 300°C for 20 min. The total processing time was 37.66 min. The scanning range of mass spectra was 50–550 m/z in the EI mode at 70 eV. Components were identified based on mass spectra in comparison with those deposited in the database of NIST11 (U.S. Department of Commerce, Gaithersburg, MD, United States) and literature data [23].

2.2.10. Antioxidant Properties

Antioxidant Activity. The radical scavenging activity (RSA) (%) was determined by the DPPH° radical scavenging method according to the technique of Ruengdech and Siripatrawan [24]. First, 200 mg of tunichrome with different concentrations within the range of 50, 100, 150, and 200 μg/mL was mixed with 800 μL methanol. Then, 400 μL of each diluted tunichrome was homogenized with 1.6 mL of DPPH° solution (0.1 mM). The suspension was kept at 25°C for 1 h under dark conditions, and finally, the absorbance of the suspension was recorded at 517 nm. A sample without tunichrome was used as a control. The RSA (%) was determined based on the following equation:

where Ac and As express the absorbances of control and test samples, respectively.

Total Phenol Content (TPC). The TPC was determined by Folin-Ciocalteu reagent assay based on the method of Majzoobi et al. [25]. In brief, samples were mixed with 750 μL of 10% w/w Folin-Ciocalteu (diluted in DDW) and then hold at 20°C for 15 min. Afterward, 750 μL of 0.2% sodium carbonate was mixed with the suspension, and the absorbance of the sample was determined at 765 nm after 1 h incubation in a dark place. The TPC was calculated via a calibration curve of different concentrations of a gallic acid solution (0 to 150 μg/mL) and reported as mg gallic acid/g of tunichrome weight.

Total Flavonoid Content (TFC). The method developed by Bagheri et al. [26] was used to measure TFC. For this purpose, 0.5 g of samples and 500 μL of methanol were homogenized with 100 μL of AlCl₃ (10% w/v), 100 μL of CH₃CO₂K (1 M), and 2800 μL of DDW. Absorbance at 415 nm was measured after incubation at 23°C for 35 min. The standard calibration curve was plotted at the same wavelength for various quercetin concentrations (5 to 30 μg/mL methanol). The TFC was reported as mg quercetin/g of the sample weight.

2.2.11. Antimicrobial Properties

Well Diffusion Agar Method. The tunichrome sample antimicrobial activity was measured against selected Gram-positive and Gram-negative bacteria including Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), Salmonella enterica (ATCC14028), and Escherichia coli (ATCC 35218) by well diffusion agar method. Muller Hinton agar plates were cultured using 0.1 mL of bacterial suspension with a cell density of 0.5 Mc-Farland standard (≈1.5 × 10⁸ CFU/ml). Wells with 4 mm diameter were created by a sterile punch and filled with 50 μl of the sample (150 mg/ml). After 24 h incubation at 37°C, the diameter inhibition zone (DIZ) was determined in mm.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Method. The broth microdilution technique was used for the evaluation of MIC. First, broth subcultures were produced by inoculating one colony of each bacteria grown 24 h in the 50 ml flask with 20 ml Mueller Hinton Broth in a shaker incubator (Jal, Tehran, Iran) at 150 rpm. Then, the final concentration of bacterium was set at 1.5 × 10⁶ CFU/ml followed by separating the cells using centrifugation at 6000 × g for 5 min. Then, the pellet was dispersed in sterile saline (0.9% NaCl) and was applied for inoculation by 96-well microplates with tunichrome serial dilutions from 0 to 150 mg/ml. The microplates were held at 37°C overnight. After incubation, bacterial growth was determined by turbidity methods. The concentration around MIC was cultured on the MRS agar for determining the MBC.

2.2.12. Statistical Analysis

All data were determined in triplicate. Analysis of variance (ANOVA), and the Duncan multiple range tests were done to evaluate the significant differences ( < 0.05) among the average values (SAS ver. 9.1, 2002–2003 by SAS Institute Inc., Cary, NC, USA).

3. Results and Discussion

3.1. Molecular Weight Measurement

SDS-PAGE of tunichrome released from Persian Gulf marine tunicate is shown in Figure 1. The bands at 245, 180, 135, 100, and 75, between 75 and 63, between 63 and 48, 48, 35, between 35 and 25, 25, 20, between 20 and 17, 17, between 17 and 11, and 11 kD were identified in the sample. Two main bands were between 63 and 48 and 20 kD. This result showed that there were different proteins and peptides with antioxidant and antimicrobial properties in the tunichrome.

(a)

(b)

Figure 1: SDS-PAGE: (a) sinaclon marker (Tris-Glycine) and (b) tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

3.2. Amino Acids’ Profile

The protein content of tunichrome released from Phallusia nigra Persian Gulf marine tunicate was 0.7 ± 0.02%. Amino acids’ profile of tunichrome by HPLC is reported in Table 1. The main amino acids were lysine, arginine, glycine, aspartic acid, and proline with concentrations of 32.24, 14.34, 12.44, 10.62, and 9.47 mg/100 g, respectively. Therefore, tunichrome can be considered as a suitable source of lysine essential amino acid (EAA). Kumaran and Bragadeeswaran [27] reported that the release of E. viride is containing leucine (582.3 μg/g), arginine (365.4 μg/g), lysine (344.5 μg/g), threonine (295.6 μg/g), and isoleucine (231.2 μg/g) and the release of D. psammathodes contains leucine (540.9 μg/g), arginine (401.2 μg/g), lysine (385.4 μg/g), threonine (312.5 μg/g), and isoleucine (254.1 μg/g). Karthikeyan et al. [28] evaluated the amino acid profile of solitary ascidian Microcosmus exasperatus and reported the presence of a total of seventeen essential and nonessential amino acids (nEAA). Among them, ten essential and seven nonessential amino acids were reported in ascidians mussel. The maximum recorded level of the essential amino acid (567.3 mg) was leucine, and the least level of nonessential amino acids (0.212 mg) was aspartic acid. Tabakaeva and Tabakaev [29] also evaluated the different amino acids of the ascidian Halocynthia aurantium in the Japan Sea. Among eighteen identified amino acids, eight of them were essential and the others were nonessential. Internal organs had the highest essential amino acid (50.61%) content and tunic had the lowest (35.01%) content. The common nonessential amino acid in all parts (5.84–10.16%) was aspartic acid.

Table 1: Amino acids’ profile of tunichrome released from Phallusia nigra Persian Gulf marine tunicate by HPLC.

Amino acid
Retention time (min)
Concentration (mg/100 g)
Content (%)

1
Asp
5.56
10.62 ± 0.004
9.2 ± 0.04

2
Glu
6.63
6.55 ± 0.003
6.7 ± 0.03

3
H-Pro
10.79
0.29 ± 0.002
0.5 ± 0.02

4
Ser
14.40
2.79 ± 0.003
7.7 ± 0.03

5
Gly
15.55
12.44 ± 0.003
9.7 ± 0.02

6
His
17.19
0.87 ± 0.002
3.2 ± 0.02

7
Arg
19.11
14.34 ± 0.002
7.8 ± 0.02

8
Thr
19.62
4.45 ± 0.002
4.6 ± 0.02

9
Ala
19.83
5.83 ± 0.004
5.4 ± 0.04

10
Pro
20.38
9.47 ± 0.002
6.6 ± 0.02

11
Tyr
26.00
0.72 ± 0.003
2.2 ± 0.03

12
Val
27.22
1.97 ± 0.002
3.5 ± 0.02

13
Met
28.43
5.32 ± 0.003
5.6 ± 0.03

14
Ile
31.17
4.02 ± 0.035
2.3 ± 0.04

15
Leu
31.55
7.72 ± 0.002
5.1 ± 0.02

16
Phe
33.92
5.87 ± 0.002
3.1 ± 0.02

17
Lys
34.72
32.24 ± 0.003
17.0 ± 0.03

Data represent mean ± standard deviation of three independent repeats.

The contents of EAA, semiessential amino acids (SEAA), and nEAA of tunichrome released from Persian Gulf marine tunicate are reported in Figure 2. It contained 44.4, 21.3, and 34% of EAA, SEAA, and NEAA, respectively. Cho et al. [7] and Kang et al. [10] reported that the protein content and amino acid type are related to the age and type of tunicate, environmental condition, pH of water, and the salt type and concentration.

Figure 2: Essential amino acids (EAA), semiessential amino acids (SEAA), and nonessential amino acids (nEAA) of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

3.3. Mineral Content

Table 2 shows the Na, K, Mg, Ca, Mn, Zn, Fe, and Cu contents of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. The main cation ions of tunichrome were sodium, potassium, and magnesium with concentrations of 858.4, 778.5, and 94.7 mg/100 g, respectively. Based on these results, this sample is a good source of essential minerals for the human body. Lee et al. [30] also reported that the internal part of tunicate from Korea was containing Na, K, Mg, Ca, Mn, Zn, Fe, and Cu, in the range of 1471.1–1257.9, 39–98.3, 42.8–78, 129.1–273.3, 5.2–0.9, 0.2–0.4, 2.5–0.9, and 0–0.2 mg/100 g, respectively. Papadopoulou and Kanias [31] reported that Zn and Fe of tunic of Ciona intestinalis were 110 and 610 mg/kg. Cho et al. [7] reported that the mineral type is related to the age and type of tunicate, environmental condition, and pollution.

Table 2: Na, K, Mg, Ca, Mn, Zn, Fe, and Cu contents of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Name
Concentration (mg/100 g)

Na
858.4 ± 0.02

K
778.5 ± 0.03

Mg
94.7 ± 0.02

Ca
2.1 ± 0.02

Mn
0.3 ± 0.03

Zn
0.6 ± 0.02

Fe
0.3 ± 0.03

Cu
0.1 ± 0.04

Data represent mean ± standard deviation of three independent repeats.

Table 3 shows the heavy metals of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. This source contained high amounts of heavy metals such as Pb and Hg with concentrations of 36.35 and 8 μg/g, respectively. The main idea is culturing this tunicate in the pure water instead of the sea. Lee et al. [30] evaluated the internal part of tunicate from Korea and reported a trace content of chromium, lead, silver, and arsenic heavy metals. Papadopoulou and Kanias [31] reported 1, 1.9, 0.011, 0.021, 0.041, 3.7, 0.44, and 0.15 ppm of Se, Cr, Ag, Cs, Sc, Rb, Co, and Sb for the tunic of Ciona intestinalis, respectively.

Table 3: Heavy metals of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Name
LOQ
Concentration (μg/g)

Cd
0.3
0.013 ± 0.003

As
0.025
0.013 ± 0.002

Pb
4.2
36.35 ± 0.010

Hg
3
8.00 ± 0.001

Data represent mean ± standard deviation of three independent repeats.

Table 4 shows the concentrations of fluoride, chloride, bromide, nitrite, nitrate, phosphate, and sulfate of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. Chloride (1750 μg/g), nitrate (850 μg/g), and fluoride (17.50 μg/g) were the main anions of tunichrome. Since a high amount of nitrite and nitrate in the processed food can lead to different cancers, the low content of nitrite and nitrate in tunichrome showed its beneficial health effects [32].

Table 4: Fluoride, chloride, bromide, nitrite, nitrate, phosphate, and sulfate contents of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Name
RT (min)
Concentration (μg/g)

Fluoride
3.75
17.50 ± 0.2

Chloride
5.68
1750 ± 0.3

Bromide
8.59
15 ± 0.2

Nitrite
7.33
5 ± 0.1

Nitrate
10.39
850 ± 0.2

Phosphate
11.72
5 ± 0.1

Sulfate
14.41
15 ± 0.1

Data represent mean ± standard deviation of three independent repeats.

3.4. FTIR

Infrared spectroscopy is a valuable analytical technique for obtaining rapid information about the structure and chemical changes of compounds during the different processes. The FTIR spectra of tunichrome released from Phallusia nigra Persian Gulf marine tunicate are presented in Figure 3. The spectrum had two low-intensity peaks at 1624 and 1728 cm⁻¹. The signal around 1624 cm⁻¹ was due to the symmetric deformation vibration of adsorbed water molecules [33]. The peak at 1728 cm⁻¹ was attributed to the C=O stretching vibration of formate ester moieties. The low intensity of this band suggested that only small amounts of this specific band are formed [34]. Tunichrome showed a strong O–H stretching absorption band around 3300 cm⁻¹ and C–H stretching absorption band around 2962 cm⁻¹ (Table 5). Similar results were reported by Núñez-Pons et al. [35] and Song et al. [36]. Moreover, these results were in agreement with those obtained by GC-MS for the detection of carbocyclic, acidic, and steric groups.

Figure 3: FTIR spectrum of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Table 5: FTIR of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Wavenumber (cm⁻¹)

1
2962
C-H stretching

2
2573
O-H stretch vibration

3
1728
C=O stretch vibration

4
1624
C=C symmetric deformation vibration

5
1608
N-O stretch vibration

6
1388

7
1228

8
1076

9
899

10
852

11
652

12
536
C-Br stretch vibration

3.5. Chemical Composition

The chemical composition of tunichrome released from Phallusia nigra Persian Gulf marine tunicate by GC-MS is reported in Table 6. Octadecanoic acid methyl ester (25.74%), nonanal (14.08%), hydrazinecarbothioamide (12.38%), octadecanoic acid (9.67%), and hexadecanoic acid (9.06%) were the main components extracted by methanol: hexane: chloroform (1 : 1 : 1). Similar components in tunichrome of a marine ascidian, Lissoclinum bistratum, were reported by Karthi et al. [37] after analysis by the GC-MS method.

Table 6: Chemical composition of tunichrome released from Phallusia nigra Persian Gulf marine tunicate by GC-MS.

RT (min)
Name
Formula
MW (g/mol)
Content (%)

1
3.22
4-Phenyltetrahydro-1,3-oxazine-2-thione
C₁₀H₁₁NOS
193.27
0.71

2
5.92
Nonanal
C₉H₁₈O
142.24
14.08

3
8.39
3,3-Dimethyl-1-(2-carboxyphenyl)triazene
C₉H₁₁N₃O₂
193.20
0.87

4
8.48
3-Methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid
C₁₇H₂₅NO₄
307.40
0.87

5
8.68
p-Cynophenyl p-(2-propoxyethoxy)benzoat
C₁₉H₁₉NO₄
325.40
0.87

6
8.88
(+)-trans-3,4-Dimethyl-2-phenyltetrahydro-1,4-thiazine
C₁₂H₁₇NS
207.30
0.87

7
17.06
Phenol, 2,4-bis(1,1-dimethylethyl)-
C₁₄H₂₂O
206.32
0.73

8
17.68
Hexadecanoic acid, methyl ester
C₁₇H₃₄O₂
270.50
9.06

9
18.77
n-Hexadecanoic acid
C₁₆H₃₂O₂
256.42
5.84

10
21.96
Hydrazinecarbothioamide, 2-[1-(4-nitrophenyl)ethylidene]-
C₉ H₁₀N₄O₂S
238.27
12.38

11
23.02
Octadecanoic acid, methyl ester
C₁₉H₃₈O₂
298.50
25.74

12
24.02
Octadecanoic acid
C₁₈H₃₆O₂
284.50
9.67

13
27.32
Spirost-8-en-11-one,3-hydroxy-,(3β, 5α, 14β.20β, 22β, 25R)-
C₂₇H₄₀O₄
428.00
0.87

14
29.37
Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
C₁₉H₃₈O₄
330.50
17.43

This important analysis showed that tunichrome released from Phallusia nigra Persian Gulf marine tunicate contained a large number of bioactive compounds which are known as antioxidant and antimicrobial natural products. Several amazing medicinal and pharmacological activities were also reported for tunichrome. According to the previous studies, some identified components such as 3-methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid [38], hexadecanoic acid [39], and phenol, 2,4-bis(1,1-dimethylethyl)- [40] exhibit high antioxidant activity.

The antimicrobial activities of oxazine [41], 1,3-oxazine [42], nonanal [43], hydrazinecarbothioamide [44], triazene [45], butyric acid [46, 47], (+)-trans-3,4-Dimethyl-2-phenyltetrahydro-1,4-thiazine [48–50], hexadecanoic acid [47], octadecanoic acid [51], spirost-8-en-11-one,3-hydroxy-, (3β, 5α, 14β.20β, 22β, 25R)- [52, 53], and phenol, 2,4-bis(1,1-dimethylethyl)- [40] were also well documented.

In addition, other pharmaceutical properties such as sedative, analgesic, antipyretic, anticonvulsant, antitubercular, antitumor, and antimalarial properties of 4-phenyltetrahydro-1,3-oxazine-2-thione [41], antitumor and anti-inflammatory properties of (+)-trans-3,4-dimethyl-2-phenyltetrahydro-1,4-thiazine [54], antitrypanosomal properties of hydrazinecarbothioamide, 2-[1-(4-nitrophenyl)ethylidene]- [55], and anticancer, antiproliferative, and anti-inflammatory properties of spirost-8-en-11-one,3-hydroxy-,(3β, 5α, 14β.20β,22β, 25R)- [56, 57] were reported previously.

3.6. Antioxidant Properties

DPPH is a stable radical with an absorbance at 517 nm which can react with any antioxidant. This interaction can decrease the absorbance due to the color change from purple to white or yellow [58, 59]. TPC and TFC of the sample were 0.55 mg GA/g and 0.21 mg quercetin/100 g, respectively. The RSA (%) values of tunichrome released from Phallusia nigra Persian Gulf marine tunicate and ascorbic acid are reported in Table 7. Tunichrome showed higher antioxidant activity than ascorbic acid, and RSA values were increased from 30.28 to 82.08% by increasing the concentration from 50 to 200 ppm, indicating its dose-dependent activity. These results showed that tunichrome released from Phallusia nigra Persian Gulf marine tunicate can be an appropriate source of natural antioxidants for food and pharmaceutical purposes.

Table 7: RSA (%) of tunichrome released from Phallusia nigra Persian Gulf marine tunicate and ascorbic acid.

Concentration (ppm)
Tunichrome
Ascorbic acid

50
30.28 ± 0.2Ca
17.86 ± 0.1Db

100
47.59 ± 0.1Ca
22.04 ± 0.2Cb

150
66.28 ± 0.2Ba
42.47 ± 0.1Bb

200
82.08 ± 0.1Aa
53.83 ± 0.1Ab

Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences ( < 0.05).

The antioxidant activities of 3-methyl-2-[4-(3-methyl-butoxy)-benzoylamino]-butyric acid [38] and hexadecanoic acid [39] and phenol, 2,4-bis(1,1-dimethylethyl)- [40] were also reported previously. Lee et al. [15] evaluated the antioxidant activity of extracts of Stalked sea squirt Styela clava tunic. They reported that this extract showed high dose-dependent antioxidant activity, and water extract had 0.192 mg/ml. Also, Lee et al. [60] showed that the RSA of the water and ethanol Styela clava tunic extracts was 31 and 48.6% at 10 mg/ml, respectively. Lee et al. [61] also reported high scavenging activities (50%) of starfish Acanthaster planci extracts at concentrations of 1.62 mg/ml, >10 mg/ml, and 4.03 mg/ml for the ethanol, ethyl acetate, and n-butanol extracts, respectively. Kim [14] reported that hydrolysates of solitary tunicate (Styela clava) had high antioxidant activity.

3.7. Antimicrobial Content

3.7.1. Well Diffusion Agar

Antimicrobial activity of tunichrome was reported by Sugumaran and Robinson [62] previously. Inhibition zones (mm) of tunichrome released from Phallusia nigra Persian Gulf marine tunicate (150 μg/ml) and gentamicin (10 μg/ml) are reported in Table 8. Inhibition zones of Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were 14, 18, 17, and 15 mm, respectively. This sample had the highest antimicrobial activity against Bacillus cereus. Independent of bacterial strain, the antimicrobial activity of tunichrome was lower than gentamicin, as shown in Table 8. These results showed the significant antimicrobial activity of tunichrome released from Phallusia nigra Persian Gulf marine tunicate as a natural compound in food application.

Table 8: Inhibition zones (mm) of tunichrome released from Phallusia nigra Persian Gulf marine tunicate (150 μg/ml) and gentamicin (10 μg/ml).

Bacteria
Tunichrome (mm)
Gentamicin (mm)

Staphylococcus aureus
14 ± 0.3Db
16 ± 0.2Da

Bacillus cereus
18 ± 0.3Ab
19 ± 0.2Aa

Salmonella enterica
17 ± 0.2Bb
18 ± 0.3Ba

Escherichia coli O157:H7
15 ± 0.2Cb
17 ± 0.3Ca

Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences ( < 0.05).

The antimicrobial activities of oxazine and its derivatives were reported by Sindhu et al. [41] against S. aureus and E. coli. The antimicrobial activities of 1,3-oxazine against Enterococcus faecalis and Listeria monocytogenes were also reported by Hamza et al. [42]. The antimicrobial activity of nonanal was reported by Zhang et al. [43]. The antimicrobial potential of hydrazinecarbothioamide against Bacillus subtilis, E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Paenibacillus macerans, and Salmonella typhimurium was reported by Shim et al. [44]. Mohammadi [45] studied the antimicrobial activities of triazene against Bacillus subtilis, Pseudomonas aeruginosa, Salmonella enterica, and Micrococcus luteus. The butyric acid activities against Salmonella enterica, E. coli and Campylobacter jejuni [46], Candida albicans, Streptococcus mutans, and Streptococcus sanguinis [47] were also documented. Moreover, the antimicrobial activities of (+)-trans-3,4-dimethyl-2-phenyltetrahydro-1,4-thiazine against Mycobacterium tuberculosis [48], Staphylococcus aureus and E. coli [49], Staphylococcus aureus, E. coli, Bacillus subtilis, and Pseudomonas aeruginosa were reported previously [50].

The antimicrobial activities of hexadecanoic acid against Candida albicans, Streptococcus mutans, and Streptococcus sanguinis were shown by Huang et al. [47]. According to da Silva et al. [51], octadecanoic acid had significant antimicrobial activities against Bacillus subtilis and Bacillus cereus. The antimicrobial activities of spirost-8-en-11-one,3-hydroxy-,(3β, 5α, 14β.20β, 22β, 25R)- against E. coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were reported in [52, 53]. The antimicrobial activities of phenol, 2,4-bis(1,1-dimethylethyl)- against Aspergillus niger, Fusarium oxysporum, and Penicillium chrysogenum were reported by Varsha et al. [40].

Galinier et al. [18] evaluated the two antimicrobial peptides from hemocytes of the Halocynthia papillosa tunicate. They reported that these components had high antimicrobial activities against S. aureus and E. coli. Cai et al. [5] reported that tunichromes showed antimicrobial activity against E. coli and Photobacterium phosphoreum.

3.7.2. MIC and MBC

Table 9 shows the MIC and MBC of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. MIC values of tunichrome for Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were 1.17, 0.59, 0.59, and 1.17 mg/ml, respectively. Moreover, the values of MBC for the Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were 2.34, 1.17, 1.17, and 2.34 mg/ml, respectively. It was reported that styelins, clavanins, halocyamine, and plicatamide of tunichrome separated from ascidian blood cells exhibit antimicrobial properties [62, 63]. Cai et al. [5] also reported that there are different components in the tunichrome with in vitro antibiotic properties.

Table 9: MIC and MBC values of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Bacteria
MIC (mg/ml)
MBC (mg/ml)

Staphylococcus aureus
1.17 ± 0.00Ab
2.34 ± 0.00Aa

Bacillus cereus
0.59 ± 0.00Bb
1.17 ± 0.00Ba

Salmonella enterica
0.59 ± 0.00Bb
1.17 ± 0.00Ba

Escherichia coli O157:H7
1.17 ± 0.00Ab
2.34 ± 0.00Aa

Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences ( < 0.05).

4. Conclusion

The aim of this study was to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. The results showed that this release contained a high amount of essential amino acids, especially lysine. Also, the GC-MS profile showed that this compound included the functional, antioxidant, and antimicrobial components. Tunichrome had high antioxidant activity, TPC and TFC. Also, the strong antimicrobial activities against Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were observed. Therefore, tunichrome can be considered as a good source of natural antioxidants and antimicrobials for future food and pharmaceutical applications.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

References

V. Silva, G. Igrejas, V. Falco et al., “Chemical composition, antioxidant and antimicrobial activity of phenolic compounds extracted from wine industry by-products,” Food Control, vol. 92, pp. 516–52, 2018.

A. D. Dwivedi, R. Permana, J. P. Singh et al., “Tunichrome-inspired gold-enrichment dispersion matrix and its application in water treatment: a proof-of-concept investigation,” ACS Applied Materials & Interfaces, vol. 9, no. 23, pp. 19815–19824, 2017.

Q. F. Kuang, A. Abebe, J. Evans, and M. Sugumaran, “Oxidative transformation of tunichromes-model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine,” Bioorganic Chemistry, vol. 73, pp. 53–62, 2017.

P. Lemaire, “Evolutionary crossroads in developmental biology: the tunicates,” Development, vol. 138, no. 11, pp. 2143–2152, 2011.

M. Cai, M. Sugumaran, and W. E. Robinson, “The crosslinking and antimicrobial properties of tunichrome,” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, vol. 151, no. 1, pp. 110–117, 2008.

G. Ananthan, M. Karthikeyan, P. A. Selva, and C. Raghunathan, “Studies on the seasonal variations in the proximate composition of ascidians from the Palk Bay, southeast coast of India,” Asian Pacific Journal of Tropical Biomedicine, vol. 2, no. 10, pp. 793–797, 2012.

N.-D. Cho, J. Zeng, B.-D. Choi, and H.-S. Ryu, “Shelf life of bottled sea squirt Halocynthia roretzi meat packed in vegetable oil (BSMO),” Fisheries and Aquatic Sciences, vol. 17, pp. 37–46, 2014.

R. Roje-Busatto and I. Ujević, “PSP toxins profile in ascidian Microcosmus vulgaris (Heller, 1877) after human poisoning in Croatia (Adriatic Sea),” Toxicon, vol. 79, pp. 28–36, 2014.

M. Tamilselvi, V. Sivakumar, H. Abdul Jaffar Ali, and R. Thilaga, “Preparation of pickle from Herdmania pallida, simple ascidian,” World Journal of Dairy & Food Sciences, vol. 5, pp. 88–92, 2010.

C.-K. Kang, E. J. Choy, W. C. Lee, N. J. Kim, H.-J. Park, and K.-S. Choi, “Physiological energetics and gross biochemical composition of the ascidian Styela clava cultured in suspension in a temperate bay of Korea,” Aquaculture, vol. 319, no. 1-2, pp. 168–177, 2011.

G. Lambert, R. Karney, W. Rhee, and M. Carman, “Wild and cultured edible tunicates: a review,” Management of Biological Invasions, vol. 7, no. 1, pp. 59–66, 2016.

N. D. Sanandiya, S. Lee, S. Rho, H. Lee, I. S. Kim, and D. S. Hwang, “Tunichrome-inspired pyrogallol functionalized chitosan for tissue adhesion and hemostasis,” Carbohydrate Polymers, vol. 208, pp. 77–85, 2019.

A. Abebe, Q. F. Kuang, J. Evans, W. E. Robinson, and M. Sugumaran, “Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes,” Bioorganic Chemistry, vol. 71, pp. 219–229, 2017.

A. D. Dwivedi, R. Permana, J. P. Singh et al., “Tunichrome mimetic matrix, its perspective in abatement for carcinogenic hexavalent chromium and specific coordination behavior,” Chemical Engineering Journal, vol. 328, pp. 629–638, 2017.

S.-M. Lee, Y.-R. Lee, K.-S. Cho et al., “Stalked sea squirt (Styela clava) tunic waste as a valuable bioresource: cosmetic and antioxidant activities,” Process Biochemistry, vol. 50, no. 11, pp. 1977–1984, 2015.

A. Sato, T. Shindo, N. Kasanuki, and K. Hasegawa, “Antioxidant metabolites from the tunicate Amaroucium multiplicatum,” Journal of Natural Products, vol. 52, no. 5, pp. 975–981, 1989.

H.-J. Lee, S.-J. Chae, P. S. Saravana, and B.-S. Chun, “Physical and functional properties of tunicate (Styela clava) hydrolysate obtained from pressurized hydrothermal process,” Fisheries and Aquatic Sciences, vol. 20, pp. 1–8, 2017.

R. Galinier, E. Roger, P.-E. Sautiere, A. Aumelas, B. Banaigs, and G. Mitta, “Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa,” Journal of Peptide Science, vol. 15, no. 1, pp. 48–55, 2009.

S. M. Kim, “Antioxidant and anticancer activities of enzymatic hydrolysates of solitary tunicate (Styela clava),” Food Science and Biotechnology, vol. 20, p. 1075, 2011.

Y. Shen, X. Tang, and Y. Li, “Drying methods affect physicochemical and functional properties of quinoa protein isolate,” Food Chemistry, vol. 339, p. 127823, 2020.

P. B. Zamudio-Flores, M. H. Gonzaléz, and V. G. G. Cano, “Food supplements from a Grasshopper: a developmental stage-wise evaluation of amino acid profile, protein and vitamins in Brachystola magna (Girard),” Emirates Journal of Food and Agriculture, vol. 31, pp. 561–568, 2019.

E. Kapinus, I. Revelsky, V. Ulogov, and Y. A. Lyalikov, “Amino acids and related compounds of the ascidian Halocynthia aurantium from the sea of Japan,” Journal of Chromatography B, vol. 800, pp. 321–323, 2004.

M. Mollah, M. Imam, M. C. Roy et al., “Variations of indole metabolites and NRPS-PKS loci in two different virulent strains of Xenorhabdus hominickii,” Frontiers in Microbiology, vol. 11, p. 2870, 2020.

A. Ruengdech and U. Siripatrawan, “Application of catechin nanoencapsulation with enhanced antioxidant activity in high pressure processed catechin-fortified coconut milk,” LWT, vol. 140, p. 110594, 2021.

M. Majzoobi, F. Ghiasi, and A. Farahnaky, “Physicochemical assessment of fresh chilled dairy dessert supplemented with wheat germ,” International Journal of Food Science & Technology, vol. 51, no. 1, pp. 78–86, 2016.

N. Bagheri, H. A. Al Lawati, and J. Hassanzadeh, “Simultaneous determination of total phenolic acids and total flavonoids in tea and honey samples using an integrated lab on a chip device,” Food Chemistry, vol. 342, 2021.

N. S. Kumaran and S. Bragadeeswaran, “Nutritional composition of the colonial ascidian eudistoma viride and Didemnum psammathodes,” Biosciences Biotechnology Research Asia, vol. 11, no. SE, pp. 331–338, 2014.

M. Karthikeyan, G. Anathan, and A. J. Ali, “Nutritional values of solitary ascidian Microcosmus exasperatus heller, 1878 (ascidiacea: pyuridae),” Global Veterinaria, vol. 4, pp. 255–259, 2010.

O. V. Tabakaeva and A. V. Tabakaev, “Amino acids and related compounds of the ascidian Halocynthia aurantium from the sea of Japan,” Chemistry of Natural Compounds, vol. 53, no. 4, pp. 722–725, 2017.

J.-S. Lee, S.-J. Kang, and B.-D. Choi, “Seasonal variation in the nutritional content of Mideodeok Styela clava,” Fisheries and Aquatic Sciences, vol. 9, pp. 49–56, 2006.

C. Papadopoulou and G. D. Kanias, “Tunicate species as marine pollution indicators,” Marine Pollution Bulletin, vol. 8, no. 10, pp. 229–231, 1977.

F. Hosseini, M. Majdi, S. Naghshi, F. Sheikhhossein, K. Djafarian, and S. Shab-Bidar, “Nitrate-nitrite exposure through drinking water and diet and risk of colorectal cancer: a systematic review and meta-analysis of observational studies,” Clinical Nutrition, vol. 20, 2020.

Q. G. Fan, D. M. Lewis, and K. N. Tapley, “Characterization of cellulose aldehyde using Fourier transform infrared spectroscopy,” Journal of Applied Polymer Science, vol. 82, no. 5, pp. 1195–1202, 2001.

H. Yuan, Y. Nishiyama, M. Wada, and S. Kuga, “Surface acylation of cellulose whiskers by drying aqueous emulsion,” Biomacromolecules, vol. 7, no. 3, pp. 696–700, 2006.

L. Núñez-Pons, M. Carbone, J. Vázquez et al., “Natural products from Antarctic colonial ascidians of the genera Aplidium and Synoicum: variability and defensive role,” Marine Drugs, vol. 10, no. 12, pp. 1741–1764, 2012.

G. Song, J. Delroisse, D. Schoenaers et al., “Structure and composition of the tunic in the sea pineapple Halocynthia roretzi: a complex cellulosic composite biomaterial,” Acta Biomaterialia, vol. 111, pp. 290–301, 2020.

S. Karthi, B. Somanath, and A. Abdul Jaffar, “Efficacy of methanolic extract of a marine ascidian, Lissoclinum bistratum for antimicrobial activity,” Journal Of Chemical, Biological and Physical Sciences. B, vol. 5, pp. 4119–4125, 2015.

A. P. Kumar, M. Chougala, C. D. Nandini, and P. V. Salimath, “Effect of butyric acid supplementation on serum and renal antioxidant enzyme activities in streptozotocin-induced diabetic rats,” Journal of Food Biochemistry, vol. 34, pp. 15–30, 2010.

G. Belakhdar, A. Benjouad, and E. Abdennebi, “Efficacy of methanolic extract of a marine ascidian, Lissoclinum bistratum for antimicrobial activity,” Journal of Materials and Environmental Science, vol. 6, pp. 2778–2783, 2015.

K. K. Varsha, L. Devendra, G. Shilpa, S. Priya, A. Pandey, and K. M. Nampoothiri, “2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp,” International Journal of Food Microbiology, vol. 211, pp. 44–50, 2015.

T. Sindhu, S. Arikkatt, G. Vincent, M. Chandran, A. Bhat, and K. Krishnakumar, “Biological activities of oxazine and its derivatives: a review,” International Journal of Pharma Sciences and Research, vol. 4, pp. 134–143, 2013.

A. Hamza, H. A. El-Sayed, M. G. Assy, N. H. Ouf, and M. E. Farhan, “Synthesis and antimicrobial activity of some new triazine, 1, 3-oxazine, fused pyridine and pyrimidine derivatives,” World Applied Sciences Journal, vol. 36, pp. 637–645, 2018.

J. Zhang, L. Zeng, H. Sun, S. Chen, T. Wang, and S. Ma, “Use of nonanal-wax as postharvest fungicide of tomato against botrytis cinerea,” Journal of Food and Nutrition Research, vol. 5, no. 7, pp. 458–466, 2017.

J. Shim, N. R. Jyothi, and N. A. M. Farook, “Biological applications of thiosemicarbazones and their metal complexes,” Asian Journal of Chemistry, vol. 25, no. 10, pp. 5838–5840, 2013.

A. Mohammadi, “Novel triazene dyes based on N-phenylpiperazine: synthesis, anti-bacterial activity and solvatochromic properties,” Journal of Molecular Liquids, vol. 193, pp. 69–73, 2014.

L. Kovanda, W. Zhang, X. Wei et al., “In vitro antimicrobial activities of organic acids and their derivatives on several species of gram-negative and gram-positive bacteria,” Molecules, vol. 24, no. 20, p. 3770, 2019.

C. B. Huang, Y. Alimova, T. M. Myers, and J. L. Ebersole, “Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms,” Archives of Oral Biology, vol. 56, no. 7, pp. 650–654, 2011.

S. Indumathi, S. Perumal, D. Banerjee, P. Yogeeswari, and D. Sriram, “l-Proline-catalysed facile green protocol for the synthesis and antimycobacterial evaluation of [1,4]-thiazines,” European Journal of Medicinal Chemistry, vol. 44, no. 12, pp. 4978–4984, 2009.

S. Badshah and A. Naeem, “Bioactive thiazine and benzothiazine derivatives: green synthesis methods and their medicinal importance,” Molecules, vol. 21, no. 8, p. 1054, 2016.

S. S. Didwagh and P. B. Piste, “Synthesis and antimicrobial activity of thiazine derivatives,” Journal of Chemical and Pharmaceutical Research, vol. 55, pp. 171–174, 2013.

L. L. D. da Silva, M. Nascimento, D. H. S. Silva, M. Furlan, and V. da Silva Bolzani, “Antibacterial activity of a stearic acid derivative from Stemodia foliosa,” Planta Medica, vol. 68, pp. 1137–1139, 2002.

A. Doğan, S. Otlu, Ö. Çelebi, and P. A. Kilicle, “An investigation of antibacterial effects of steroids,” Turkish Journal of Veterinary and Animal Sciences, vol. 41, pp. 302–305, 2017.

J. H. Hussein, Y. H. Mohammed, and H. H. Imad, “Study of chemical composition of Foeniculum vulgare using Fourier transform infrared spectrophotometer and gas chromatography-mass spectrometry,” Journal of Pharmacognosy and Phytotherapy, vol. 8, no. 3, pp. 60–89, 2016.

P. K. Sharma and R. Makkar, “A review: thiazines derivatives treated as potential antimicrobial agents,” Asian Journal of Pharmaceutical and Clinical Research, vol. 10, pp. 43–46, 2017.

A. Pérez-Rebolledo, L. R. Teixeira, A. A. Batista et al., “4-Nitroacetophenone-derived thiosemicarbazones and their copper(II) complexes with significant in vitro anti-trypanosomal activity,” European Journal of Medicinal Chemistry, vol. 43, no. 5, pp. 939–948, 2008.

J. Raju and R. Mehta, “Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin,” Nutrition and Cancer, vol. 61, pp. 27–35, 2009.

L. Xiang, X. Yi, Y. Wang, and X. He, “Antiproliferative and anti-inflammatory polyhydroxylated spirostanol saponins from Tupistra chinensis,” Scientific Reports, vol. 6, pp. 1–13, 2016.

Q. Liu, B. Kong, Y. L. Xiong, and X. Xia, “Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis,” Food Chemistry, vol. 118, no. 2, pp. 403–410, 2010.

X. Peng, Y. L. Xiong, and B. Kong, “Antioxidant activity of peptide fractions from whey protein hydrolysates as measured by electron spin resonance,” Food Chemistry, vol. 113, no. 1, pp. 196–201, 2009.

D.-W. Lee, D.-H. You, E.-K. Yang et al., “Antioxidant and ACE inhibitory activities of Styela clava according to harvesting time,” Journal of the Korean Society of Food Science and Nutrition, vol. 39, no. 3, pp. 331–336, 2010.

C.-C. Lee, H. J. Hsieh, C.-H. Hsieh, and D.-F. Hwang, “Antioxidative and anticancer activities of various ethanolic extract fractions from crown-of-thorns starfish (Acanthaster planci),” Environmental Toxicology and Pharmacology, vol. 38, no. 3, pp. 761–773, 2014.

M. Sugumaran and W. E. Robinson, “Structure, biosynthesis and possible function of tunichromes and related compounds,” Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, vol. 163, no. 1, pp. 1–25, 2012.

J. A. Tincu, L. P. Menzel, R. Azimov et al., “Plicatamide, an antimicrobial octapeptide from Styela plicata hemocytes,” Journal of Biological Chemistry, vol. 278, no. 15, pp. 13546–13553, 2003.