Study of Red Algae (Eucheuma denticulatum (Burman) Collins et Harvey) Extract Bioactive Compounds using Liquid Chromatography Mass Spectroscopy - Mass Spectroscopy

 

Syamsuri Syakri^1,5*, Sartini Sartini², Upik A. Miskad³, Aminuddin Aminuddin⁴

¹Doctoral Program, Faculty of Medicine, Universitas Hasanuddin, Makassar, Indonesia.

²Department of Pharmacy, Faculty of Pharmacy, Universitas Hasanuddin, Makassar, Indonesia.

³Department of Anatomical Pathology, Faculty of Medicine, Universitas Hasanuddin, Makassar, Indonesia.

⁴Department of Nutrition, Faculty of Medicine, Universitas Hasanuddin, Makassar, Indonesia.

⁵Department of Pharmacy, Faculty of Medicine and Health Sciences,

Universitas Islam Negeri Alauddin, Makassar, Indonesia.

 

ABSTRACT:

Research has been carried out on the Study of Red Algae (Eucheuma denticulatum (Burman) Collins et Harvey) Extract Bioactive Compounds Using Liquid Chromatography Mass-Mass Spectroscopy (LCMS-MS). This study aims to determine the bioactive compounds produced in this red algae extract by means kinetic maceration of extraction. Reading of bioactive compounds using LCMS-MS to obtain compound results, compound structures, and molecular weights. The results show that there are 22 bioactive compounds contained in this plant. Consist of  : 4-Dodecylbenzenesulfonic acid, NP-009092, 6-Hydroxycaproic acid, Xanthine, 3-Phenyilactic acid, Azelaic acid, Apro barbital, Decosahexaenoic acid, Methil hydrogen Phenylphoshonate, (1S)-1-Carboxy-N,N,N-trimethyl-2-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)ethanaminium, D-(+)-Tryptophan, uracil, ?17-6-keto Prostaglandin F1, Hypoxanthine, 20-hydroxy Prostaglandin F2?, 2-Hydroxy-4-methylthiobutanoic acid, N-(2-Furylmethyl)-3-[(2S,5aS,8aR)-1-methyl-5-oxodecahydropyrrolo[3,2-E][1,4]diazepin-2-yl]propanamide, 6-Hydroxycaproic acid, Methaphenilene, 15(S)-HpEPE, [(4Z)-5-(Methylsulfanyl)-4-penten-2-yn-1-yl]benzene, Suberic acid.

 

 

 

INTRODUCTION: 

Seaweeds are an essential and significant component of the marine ecology. 90% of it is thought to be algae, and they account for more than 50% of all photosynthesis worldwide¹. Secondary metabolites found in seaweed, including flavonoids, alkaloids, polyphenols, and tannins, can lower blood cholesterol levels. Additionally, seaweed has 59.393 IU/kg of vitamin A and 219.96 IU/KG of vitamin E².

 

Because isoflavones and tocopherol operate synergistically to boost each other's antioxidant roles and stop the chain reaction of lipid peroxidation in epithelial cell membranes, they can reduce LDL oxidation and help avoid the buildup of free radicals in tissues. The two primary parts of red seaweed that are extracted industrially and commercially are carrageenan and agar hydrocolloids³. However, this particular macroalga also includes a number of nutritional, water, and mineral components⁴.

 

Around 452 different species of red algae (Rhodophyceae) are found in Indonesian marine waters, followed by 196 species of green algae (Chlorophyceae) and 134 species of brown algae (Phaeophyceae)⁵.  This macroalgae group offers economic potential, specifically as a raw resource for industry and health, in addition to its ecological and biological function in preserving the stability of marine ecosystems, providing a place to live and protection for other biota^6,7.

 

Objectives overview of the pharmacological uses for bioactive components found in red algae. Content Sources of seaweed are becoming more and more popular for medical and therapeutic purposes^8,9. The main group of these red algae contains bioactive substances used in the food industry, manure, cosmetic, pharmaceutical, and other industries. Red algae have been found to contain a wide variety of important bioactive substances, including polysaccharides (aginate, agar, and carrageenan), lipids, polyphenols, steroids, glycosides, flavanoids, tannins, saponins, alkaloids, triterpenoids, antheraquinones, and cardiac glycosides¹⁰.

 

The red algae contain a variety of healthy nutrients. Red algae polysaccharides such agarans, carrageenan, alginate, fucan, laminaran, and naviculan all have the potential to be antiviral¹¹. Red algae's sulfated polysaccharides and carraginans are abundant sources of soluble fibers that, depending on the chemistry of various secondary metabolites and the metabolism of cell line, may have anticancer effects¹². Many red algae's flavon-3-ols, which contain catechins, inhibit the action of telomerase in colon cancer cells^13,14.

 

The primary polysaccharides in the cell walls of red seaweeds that have broad-spectrum medicinal properties are the hydrocolloids galactans, carrageenans, and agars¹⁵. According to the algae species, development stage, environment, and external factors, such as water temperature, light intensity, and nutrient concentrations in the ecosystem, seaweed generally has diverse chemical compositions^16,17.

 

Red algae called Eucheuma denticulatum, also referred to as "Spinosum," thrives in the wild on coral reefs in tropical and subtropical regions with moderately strong currents¹⁸. This species has been extensively farmed because it is the main source of iota-carrageenan, which is highly demanded in the culinary, pharmaceutical, and manufacturing industries^18,19.

 

In bioanalytical laboratories, the hyphenated technology known as LC-MS/MS is utilized for the identification and quantification of organic molecules²⁰. A liquid chromatography unit and a mass spectrometer are basically combined to create the LC-MS device. LCMS-MS, on the other hand, combines two mass spectrometers with liquid chromatography to provide better resolution²¹.

 

MATERIALS AND METHODS:

Materials:

Selection of Plant:

Drug discovery from medicinal plants has evolved to include numerous fields of inquiry and various methods of analysis. The process typically begins with a botanist, ethanopharmacologist who identifies the plant of interest.Collection may involve species with known biological activity. Based on intensive literature survey, Eucheuma denticulatum (Burman) Collins et Harvey) was selected for present study^22,23.

 

Extraction using Kinetica Maceration:

4305g of dry Red Algae samples dissolved in water with a ratio of 1: 2 for 2 repetitions with a temperature of 500 C while stirring for about 1 hour. Then the results of the extraction were filtered using a filter and the results of the extraction were stored in the freezer with a temperature of -300C for 50hours, then put into Freeze Dry for 24hours. The yield of the extract was then calculated by comparing the initial weight of the simplicia with the final weight method of the resulting extract²⁴.

 

Prepration and method used^25-27

Preparation Sample :

500mg of sample was dissolved in 5ml of MeOH, sonicated for 30 minutes and filtered through a 0.2µm PTFE membrane.

Method used:

LCMS: UHPLC Vanquish Tandem Q Exactive Plus Orbitrap HRMS ThermoScientific

Colom: Accucore C18, 100 x 2,1mm, 1,5µm (ThermoScientific)

Flow rate: 0.2ml/min

Eluen: H₂O + 0.1% format acid (A) and asetonitril + 0.1% format acid (B)

Gradien:  0-1 minute (5% B), 1-25 minute (5-95%), 25-28 minute (95%B), 28-30 minute (5%B)

Column temperature: 30⁰C

Injection of volume: 2µl

Range of mass: 100-1500m/z

Ionisation of mode: negative

 

Plant material:

Red algae plants are obtained in the Bantaeng area, South Sulawesi, Indonesia. Then the plant determination was carried out to find out the species of the red algae.

 

RESULT:

Table 1: Red algae of extraction result

Sample	Wet Weight (g)	Dry weight (g)	Extraction of result (g)	Rendamen of result (%)
Red Algae	36000	4305	606.1	14.07

 

Table 2: Red Algae (Eucheuma denticulatum (Burman) Collins et Harvey) Extract Bioactive of Compounds Using Liquid Chromatography Mass Spectroscopy – Mass Spectroscopy

S. No	Compounds	Formula	Annot. Delta Mass [ppm]	Calc. MW	RT [min]	# Chem Spider Results	mz Cloud Best Match	Area Sampel
1	4-Dodecylbenzenesulfonic acid	C18 H30 O3S	-5.87	326.18965	19.934	6	99.1	1051798177
2	NP-009092	C19H22 O3	5.76	298.15861	17.397	0	86.1	389103474
3	6-Hydroxycaproic acid	C6 H12 O3	-12.61	132.07698	6.952	0	98	307238508
4	Xanthine	C5H4N4 O2	-11.21	152.03172	1.748	0	89.8	251069406.2
5	3-Phenyllactic acid	C9H10 O3	-9.79	166.06137	8.119	0	87.1	137239960.1
6	Azelaic acid	C9H16 O4	-8,85	188.10319	9.745	0	97.4	135370990.3
7	Aprobarbital	C10H14 N2O3	0,89	210.10063	9.477	2		120459282.5
8	Docosahexaenoic Acid	C22H32 O2	-5.52	328.23842	24.985	0	84.5	118757281
9	Methyl hydrogen phenylphosphonate	C7H9 O3P	0.88	172.02908	2.075	1		100333814.1
10	(1S)-1-Carboxy-N,N,N-trimethyl-2-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)ethanaminium	C9H16 N3O2S	-0.51	230.0962	11.782	1		93708384.08
11	D-(+)-Tryptophan	C11H12 N2O2	-7.65	204.08832	4,92	0	85.4	85909474.27
12	Uracil	C4H4 N2 O2	-14.2	112.02569	1.632	0	97.6	82408969.47
13	?17-6-keto Prostaglandin F1?	C20 H32 O6	-5.29	368.21794	11.337	0	63.8	73493191.76
14	Hypoxanthine	C5H4 N O	-12.22	136.03685	1.625	4	96	73114106.19
15	20-hydroxy Prostaglandin F2?	C20 H34 O6	-5.25	370.23359	12.219	0	68	70439817.69
16	2-Hydroxy-4-methylthiobutanoic acid	C5H10 O3S	-10.28	150.03352	4.213	0	83.2	69030630.22
17	N-(2-Furylmethyl)-3-[(2S,5aS,8aR)-1-methyl-5-oxodecahydropyrrolo[3,2-E][1,4]diazepin-2-yl]propenamide	C16H24 N4O3	112368.58	356.16356	27.145	31	61.6	66423445.84
18	6-Hydroxycaproic acid	C6H12 O3	-12.61	132.07698	6.82	0	98.2	59730039.38
19	Methaphenilene	C15H20 N2S	4.1	260.13579	2.006	1		55051371.12
20	15(S)-HpEPE	C20H30 O4	-5.14	334.21269	15.525	0	60.9	33654378
21	[(4Z)-5-(Methylsulfanyl)-4-penten-2-yn-1-yl]benzene	C12H12 S	4.58	188.06683	1.718	1		33349973.66
22	Suberic acid	C8H14 O4	-9.34	174.08758	8.169	0	92.7	32099038.25

 

Figure 1: Chromatogram of Red Algae (Eucheuma denticulatum (Burman) Collins et Harvey) Extract

 

DISCUSSION:

Compounds 1. showed a molecular formula of C₁₈H₃₀O₃S, m/z cloud 99.1, with the area sample 1051798177.4-dodecylbenzenesulfonic acid is a member of the class dodecylbenzenesulfonic acids that is benzenesulfonic acid in which the hydrogen at position 4 of the phenyl ring is substituted by a dodecyl group. Compounds 2. showed a molecular formula of C₁₉H₂₂O₃S, m/z cloud 86.1, with the area sampel 389103474 Ostruthin is a terpene lactone. It has a role as a metabolite . Compounds 3. showed a molecular formula of C₆H₁₂O₃,  m/z cloud 98.1, with the area sample 307238508.  Compounds 4. showed a molecular formula of C₅H₄N₄0₂, m/z cloud 89.8, with the area sample 251069406.2 Xanthine belongs to the class of antioxidants. Compounds 5. showed a molecular formula of C₉H₁₀O₃, m/z cloud 87.1, with the area sampel 137239960.1. Compounds 6. showed a molecular formula of C₉H₁₆O₄,  m/z cloud 97.4, with the area sample 135370990.3. Compounds 7. showed a molecular formula of C₁₀H₁₄N₂0₃, with the area sample 120459282.5. Compounds 8. showed a molecular formula of C₂₂H₃₂O2,  m/z cloud 84.5, with the area sample 118757281. Compounds 9. showed a molecular formula of C₇H₉O₃P, with the area sample 10333814.1. Compounds 10. showed a molecular formula of C₉H₁₆N₃O₂S,  with the area sample 93708384.08. Compounds 11. showed a molecular formula of C₁₁H₁₂N₂O₂,  m/z cloud 85.4, with the area sample 85909474.2. Compound 12. showed a molecular formula of C₄H₄N₂O₂,  m/z cloud 97.6, with the area sample 82408969.47. Compounds 13. showed a molecular formula of C₂₀H₃₂O₆,  m/z cloud 63.8, with the area sample 73493191.76.Compounds 14. showed a molecular formula of C₅H₄NO,  m/z cloud 96, with the area sample 73114106.19.Compounds 15. showed a molecular formula of C₂₀H₃₄O₆,  m/z cloud 68, with the area sample 70439817.69.  Compounds 16. showed a molecular formula of C₅H₁₀ O₃S,  m/z cloud 83.2, with the area sample 69030630.22.  Compounds 17. showed a molecular formula of C₁₆H₂₄N₄O₃,  m/z cloud 61.6, with the area sample 66423445.84.  Compounds 18. showed a molecular formula of C₆H₁₂O₃,  m/z cloud 98.2, with the area sample 59730039.38.

 

Compounds 19. showed a molecular formula of C₁₅H₂₀N₂S, with the area sample 55051371.12. Compounds 20. showed a molecular formula of C₂₀H₃₀O₄, m/z cloud 60.9, with the area sample 33654378. Compounds 21. showed a molecular formula of C₁₂H₁₂S, with the area sample 33349973.66. Compounds 22. showed a molecular formula of C₈H₁₄O₄, m/z cloud 92.7, with the area sample 32099038.25.Suberic acid is an alpha,omega-dicarboxylic acid that is the 1,6-dicarboxy derivative of hexane. It has a role as a human metabolite. It is an alpha,omega-dicarboxylicacid and a dicarboxylic fatty acid. It is a conjugate acid of a suberate(2-) and a suberate.

 

CONCLUSION:

The results show that there are 22 bioactive compounds contained in this plant. Consist of : 4-Dodecylbenzenesulfonic acid, NP-009092, 6-Hydroxycaproic acid, Xanthine, 3-Phenyilactic acid, Azelaic acid, Apro barbital, Decosahexaenoic acid, Methil hydrogen Phenylphoshonate, (1S)-1-Carboxy-N,N,N-trimethyl-2-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)ethanaminium, D-(+)-Tryptophan, uracil, ?17-6-keto Prostaglandin F1, Hypoxanthine, 20-hydroxy Prostaglandin F2?, 2-Hydroxy-4-methylthiobutanoic acid, N-(2-Furylmethyl)-3-[(2S,5aS,8aR)-1-methyl-5-oxodecahydropyrrolo[3,2-E][1,4]diazepin-2-yl]propanamide, 6-Hydroxycaproic acid, Methaphenilene, 15(S)-HpEPE, [(4Z)-5-(Methylsulfanyl)-4-penten-2-yn-1-yl]benzene, Suberic acid.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The researcher expresses her highest gratitude to the Indonesia Endowment Fund For Education (LPDP) under the ministry of finance of The Republic Of Indonesia as a sponsor in conducting research and publishing the results of this research.

 

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Accepted on 30.04.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):363-367.