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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Liquid Chromatography-Mass Spectrometry based Phytochemical Profiling of Marine Macroalga Ulva compressa Methanol Extract

Shivam Kumar Jhoomuck *, Prerana Mund *

GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India 

 Graphical Abstract

 

 

 

INTRODUCTION

Phytochemicals, which are non-nutritive molecules originating from plants, have been shown to alter biochemical pathways related to health and illness, making them key targets in natural product research. Although the phytochemical profiles of terrestrial plants have long been investigated, marine algae are now known for their structurally distinct and functionally powerful metabolites, especially phenolics, fatty acids, alkaloids, and terpenoids¹. Seaweeds, commonly known as marine macroalgae, have become a vital source of biologically active compounds with immense ecological, nutritional, and pharmaceutical significance, among them, Ulva compressa, a green macroalga belonging to the genus Ulva and class Chlorophycota, has gathered significant interest for its potential as a source of secondary metabolites, exhibiting numerous bioactivities, such as antimicrobial, antioxidant, and anti-inflammatory². Given the chemical complexity of marine extracts, Liquid Chromatography-Mass Spectrometry (LC-MS) has emerged as the most effective method for phytochemical profiling, allowing high-resolution separation and sensitive detection of a wide range of molecules, even at low concentrations³. Comprehensive phytochemical investigation of the marine macroalga, Ulva compressa, via LC-MS is relatively unexplored, despite its potential. The precise chemical components of Ulva compressa’s methanolic extract are unknown, as previous research has concentrated more on species like Ulva lactuca and Ulva rigida^4,5. Thus, a thorough phytochemical characterisation of UCME using an advanced technique, LC-MS, is the aim of this investigation. Establishing a biochemical foundation for its possible therapeutic uses and clarifying the existence of bioactive compounds will support the growing field of natural products produced from marine sources.

MATERIALS AND METHODS

Collection and authentication of Ulva compressa

Fresh green alga, identified as Ulva compressa, was hand-harvested from the Visakhapatnam coast (Andhra Pradesh), particularly around Tenneti Park during low tide to access healthy and mature alga. It was washed immediately with seawater to eliminate epiphytes and kept in a clean, moisture-free bag for further processing. The collected alga was submitted to the Department of Marine Living Resources at Andhra University, where it was authenticated based on taxonomical characters as Ulva compressa (formerly known as Enteromorpha compressa).

Drying of Ulva compressa

In the laboratory, Ulva compressa was rinsed several times with tap water, followed by distilled water to remove any residual salts, adhering sand particles, and possible contaminants. After cleaning, the alga was spread out on a filter paper in a well-ventilated area and shade dried at room temperature for 72 hours and kept in an air-tight container for further processing⁶.

Extraction using methanol

Dried Ulva compressa was macerated in pure methanol at a ratio of 1:20 (w/v) in a long-necked round-bottom flask. The extraction was conducted at ambient temperature for 72 hours, with periodic stirring. The mixture was gently decanted to isolate the solvent extract from the residual alga. The resulting filtrate was then concentrated under reduced pressure at 400°C and 120 rpm, using a rotary evaporator (Hei-VAP Core, motor lift model, equipped with G3 vertical glassware)⁷. 

The dried residue was weighed, and the extraction yield was evaluated using:

 

Preliminary Qualitative phytochemical analysis

A small amount of the dried Ulva compressa’s methanolic extract (UCME) was reconstituted with methanol, and standard protocols were used to identify the presence of alkaloids, flavonoids, tannins, phenolics, fatty acids, saponins, glycosides, and lipids^8,9.

Liquid chromatography-Mass spectrometry analysis

About 1 ml of the reconstituted UCME was transferred into a sterile Eppendorf tube and sent for analysis at the MURTI Lab, GITAM School of Pharmacy. The analysis was conducted using the AB Sciex Qtrap 5500+ LC-MS system. A volume of 5µl was drawn by an autosampler syringe and subjected to ionisation via Electrospray Ionisation (ESI). The resulting ions were then detected via Channel Electron Multiplier detector. The phytochemical profile was examined in both positive (ESI⁺) and negative ionisation modes (ESI^-). 

RESULTS

Extraction percentage yield

The weight of the dried extract was calculated by subtracting the initial crucible weight from the final weight of the crucible plus dried extract. The percentage yield of Ulva compressa using methanol was found to be 3.38%. 

Preliminary phytochemical screening

Preliminary tests revealed the presence of several major classes of bioactive compounds such as alkaloids, flavonoids, amino acids, phenolic compounds, and carbohydrates. The result of the qualitative analysis of UCME is presented in Table 1.

Phytochemicals identified by LC-MS

The LC-MS analysis identified many phytochemicals based on their retention times. The mass spectrum for ESI+ and ESI- is depicted in Figs. 1 and 2, respectively. The peaks obtained in the spectra were identified using the NIST database. A total of 66 phytochemical compounds were identified via ESI^- and 29 via ESI⁺, depicted in Tables 2 and 3, respectively. The peaks are described as tentative due to the presence of natural products in isomeric forms, or as isobaric compounds sharing the same molecular weight but differing in elemental composition.

Table 1: Phytochemical analysis results in UCME

 

Figure 1: Mass spectrum of UCME acquired in ESI^‑

Figure 2: Mass spectrum of UCME acquired in ESI⁺

Table 2: List of Phytoconstituents identified using ESI^‑ by the NIST database

Serial No.	RT (min)	MW	Adduct   formed	Theoretical (m/z)	Observed (m/z)	Proposed Phytocompounds
1	1.7	254.02387	[M-H]-	253.017	253.018	Palmitelaidic acid
2	1.7	155.10785	[M-H]-	154.101	154.0932	3-Hydroxyanthranilic acid
3	1.97	230.92673	[M-H]-	229.92	229.9246	1H-Indole-3-carboxylic acid, 1-pentyl
4	2	186.88673	[M-H]-	185.88	185.9239	3-Indoleacrylic acid
5	2.26	144.15573	[M-H]-	143.149	143.1538	γ-Octalactone
6	4.3	210.95026	[M-H]-	209.944	209.9432	Glycine-Tryptophan-Arginine
7	6.43	127.96397	[M-H]-	126.957	126.962	Maltol
8	6.52	186.0415	[M-H]-	185.035	184.9956	Chamazulene
9	7.28	123.99151	[M-H]-	122.985	122.9896	4-Methylcatechol
10	8.05	117.88673	[M-H]-	116.88	116.8757	Succinamide
11	8.12	192.04673	[M-H]-	191.04	191.0362	Citric acid
12	8.16	129.04673	[M-H]-	128.04	128.0436	DL-Pyroglutamic acid
13	8.85	188.11565	[M-H]-	187.109	187.1001	Azelaic acid
14	9.18	172.12673	[M-H]-	171.12	171.1263	1-Naphthoic acid
15	9.18	253.95339	[M-H]-	252.947	253.0083	6,4'-Dihydroxyflavone
16	9.31	163.96673	[M-H]-	162.96	162.9551	p-Coumaric acid
17	9.57	152.08673	[M-H]-	151.08	151.0856	3-Hydroxyphenylacetic acid
18	9.7	114.04673	[M-H]-	113.04	113.0381	(2E,4E)-Hexa-2,4-dienoic acid
19	9.82	138.04673	[M-H]-	137.04	137.0427	Salicylic acid
20	9.82	376.36673	[M-H]-	375.36	375.33	Digitoxigenin
21	9.85	216.16673	[M-H]-	215.16	215.1623	Undecanedioic acid
22	10.24	266.08673	[M-H]-	265.08	265.0817	4-Isopropyl-4'-methylchalcone
23 24	10.35 10.35	290.08673 220.12673	[M-H]- [M-H]-	289.08 219.12	289.0797 219.1218	Shikonin  Ethyl coumarin-3-carboxylate
25	10.42	178.36673	[M-H]-	177.36	177.3397	Glycine-Cysteine
26	10.42	208.12673	[M-H]-	207.12	207.1526	p-Methoxycinnamic acid ethyl ester
27	10.42	180.16673	[M-H]-	179.16	179.1368	Olivetol
28	10.42	182.20673	[M-H]-	181.2	181.2026	2,5-Dimethoxybenzoic acid
29	10.52	334.12673	[M-H]-	333.12	333.1204	Estrone sulfate
30	10.59	254.08673	[M-H]-	253.08	253.078	Chrysin
31	10.59	276.04673	[M-H]-	275.04	275.0383	Methysticin
32	10.73	428.44673	[M-H]-	427.44	427.4419	Oleoside 11-methyl ester
33	10.73	412.36673	[M-H]-	411.36	411.3552	L-Arginine, N2-2-(2,2-diphenylethoxy)acetyl
34	10.82	256.12673	[M-H]-	255.12	255.1192	Purpurin
35	11.16	194.20673	[M-H]-	193.2	193.1988	Alanine-Cysteine
36	11.32	264.04673	[M-H]-	263.04	263.0761	Aspartic Acid-Methionine
37	11.39	295.97785	[M-H]-	294.971	294.9881	9R-Hydroxy-10E,12Z-octadecadienoic acid
38	11.39	294.16673	[M-H]-	293.16	293.1678	13S-Hydroxy-6Z,9Z,11E-octadecatrienoic acid
39	11.53	286.01747	[M-H]-	285.011	285.0242	Hexadecanedioic acid
40	11.88	222.16673	[M-H]-	221.16	221.1707	Benzyl cinnamate
41	11.99	234.04673	[M-H]-	233.04	233.095	7-Hydroxy-4-methylcoumarin-3-acetic acid
42	12.1	248.20673	[M-H]-	247.2	247.1954	Aspartic Acid-Aspartic Acid
43	12.39	224.20673	[M-H]-	223.2	223.2089	4'-Hydroxychalcone
44	12.73	276.16673	[M-H]-	275.16	275.1662	Stearidonic acid
45	13.25	278.20673	[M-H]-	277.2	277.2139	6-Gingerol
46	13.25	474.52673	[M-H]-	473.52	473.5319	Lauroyl coenzyme A
47	13.25	466.60673	[M-H]-	465.6	465.5975	2'-Deoxycytidine 5'-triphosphate
48	13.69	304.12673	[M-H]-	303.12	303.1421	cis-5,8,11,14-Eicosatetraenoic acid
49	13.74	220.12673	[M-H]-	219.12	219.125	5-Sulfosalicylic acid
50	14.23	305.99028	[M-H]-	304.984	304.9832	cis-8,11,14-Eicosatrienoic acid
51	14.81	326.08673	[M-H]-	325.08	325.0019	Chamissonolide
52 53	14.85 15.41	256.12673 310.24673	[M-H]- [M-H]-	255.12 309.24	255.1771 309.3018	Apigeninidin cation  Gastrodin
54	15.41	310.96673	[M-H]-	309.96	309.9646	9-Nitrooleic acid
55	15.64	414.52673	[M-H]-	413.52	413.4969	Loganin
56	15.97	298.00673	[M-H]-	297	297.0028	Ricinoleic acid
57	16.05	398.32673	[M-H]-	397.32	397.2973	Geniposidic acid
58	16.05	1332.86018	[M-3H]3-	443.28	443.3051	D-myo-Inositol-1,3,4-triphosphate
59	16.5	284.08673	[M-H]-	283.08	283.059	D-Mannose 6-phosphate
60	16.75	496.36673	[M-H]-	495.36	495.3753	1,2-Dimyristin
61	17.39	124.96673	[M-H]-	123.96	123.9923	Taurine
62	18.36	327.88673	[M-H]-	326.88	326.9007	17(R)-Hydroxydocosahexaenoic acid
63	18.43	310.48673	[M-H]-	309.48	309.4603	Neohesperidose
64	18.7	121.00673	[M-H]-	120	119.9994	O-Methyl-DL-serine
65	18.9	198.88673	[M-H]-	197.88	197.8749	N5-(1-Imino-3-butenyl)-L-ornithine
66	19.1	205.00673	[M-H]-	204	203.9743	Indole-3-butyric acid

RT: Retention time, min: minute, MW: Molecular weight, m/z: mass to charge ratio
 [Peaks are described as tentative due to the presence of natural products in isomeric forms, or as isobaric compounds sharing the same molecular weight but differing in elemental composition]

 

Table 3: List of Phytoconstituents identified using ESI⁺ by the NIST database

  RT: Retention time, min: minute, MW: Molecular weight, m/z: mass to charge ratio
   [Peaks are described as tentative due to the presence of natural products in isomeric forms, or as isobaric compounds sharing the same molecular weight but differing in elemental composition]

 

 

DISCUSSION

The diverse phytochemical profile observed in the study supports the growing evidence of the marine macroalga, Ulva compressa, as a rich source of bioactive compounds with pharmaceutical and nutraceutical potential. 

The identification of a broad range of flavonoids, like 4’-Hydroxychalcone, 6, 4’-Dihydroxyflavone, and Nobiletin, from UCME highlights the significant phytochemical complexity and possible bioactivity of Ulva compressa. Previous research on Ulva species confirms a strong flavonoid content, with Ulva compressa having the highest values in flavonoid content among other green macroalgae, validating its antioxidant activity¹⁰. This aligns with earlier findings where Ulva clathrata and other species had high levels of flavonoids and phenolics, corresponding to significant radical scavenging activity¹¹. The presence of polymethoxylated flavones, Nobiletin, has been reported to have cytoprotective and anti-inflammatory actions¹².

The detection of a broad range of phenolic and aromatic compounds, such as salicylic acid, shikonin, p-coumaric acid, and 6-gingerol, highlights the chemical intricacy of Ulva compressa. These compounds have been well-documented to possess antioxidant, anti-inflammatory, and antimicrobial activities, which further validates Ulva compressa’s therapeutic potential. Ulva compressa has been found to have one of the highest levels of phenolic content among studies of seaweeds^10,11. Phytochemicals like p-coumaric acid and methylcatechol exhibit a protective role against oxidative stress as well as microbial invasion¹³. In addition, gingerol and coumarin derivatives are likely to be involved in interspecies communication and UV protection within the marine ecosystem¹².

The presence of various dipeptides and amino acid derivatives like Ala-Cys, Trp-Glu-Arg, and DL-pyroglutamic acid in UCME makes it a rich source of bioactive peptides. Marine macroalgae, especially Ulva species, are gaining prominence for their ability to produce protein-derived peptides having multifunctional activities, such as antioxidant, antihypertensive, and antimicrobial¹⁴. The presence of peptides like Gly-Cys and His-Cys-Arg contains thiol and arginine residues responsible for radical scavenging and nitric oxide-mediated vasodilatory activity. In silico and biochemical studies on Ulva lactuca and Ulva rigida have also indicated the occurrence of angiotensin-converting enzyme inhibitory peptides, indicating potential cardiovascular use^15,16. 

The identified fatty acids and lipid derivatives in UCME, including 5-oxo-eicosatetraenoic acid, 17(R)-hydroxydocosahexaenoic acid, and stearidonic acid, highlight its nutritional and biochemical significance. These phytochemicals include saturated, monounsaturated, and polyunsaturated fatty acids, many of which exhibit anti-inflammatory, antimicrobial, and cardioprotective properties. Many polyunsaturated fatty acids, such as stearidonic acid and arachidonic acid, produced by marine Ulva species, play an essential role in modulating immune responses and have potential nutraceutical value^13,17. Moreover, hydroxylated fatty acids like 13s-hydroxy-octadecatrienoic acid and ricinoleic acid may contribute to the alga's defence system against oxidative stress and pathogen invasion¹⁸. The presence of medium and long-chain dicarboxylic acids like azelaic and hexadecanedioic acids also highlights Ulva compressa’s multifaceted lipid profile and its potential for green bioactive lipid production¹⁹.

CONCLUSION

Ulva compressa is a potential marine biological resource with pharmaceutical and nutraceutical applications. The LC-MS analysis identified the presence of many phytochemical compounds, such as flavonoids, phenolics, peptides, fatty acids, and lipids, which contribute to the therapeutic potential of the marine macroalga. The presence of oligopeptides in UCME distinguishes it from other Ulva species and suggests that it may be a source of novel bioactive compounds. Its potential can be further developed as an economically sustainable and viable marine macroalga through further research. Further studies could include purification techniques, evaluation, and structural characterisation of the aforementioned Phytoconstituents. 

Acknowledgement: All authors are grateful to the MURTI Laboratory at GITAM Deemed to be University for providing the necessary analytical facilities and technical support for conducting the research analysis.

Conflict of interest: None

Funding: Nil

Author Contributions: All authors have equal contribution in the preparation of manuscript and compilation.

Source of Support: Nil

Informed Consent Statement: Not applicable. 

Data Availability Statement: The data presented in this study are available on request from the corresponding author. 

Ethical approval: This study does not involve experiments on animals or human subjects.

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