HR-LCMS and FTIR characterization of bioactive components of Turbinaria ornata

Mohini Salunke¹*, Preeti Mane², Smita Kumbhar³, Balaji Wakure¹

¹Vilasrao Deshmukh Foundation, Group of Institutions, VDF School of Pharmacy, Latur - 413 531,

Maharashtra, India.

²Department of Pharmacy, Terna Public Charitable Trust’s, College of Engineering, Osmanabad, India.

³Sanjivani College of Pharmaceutical Education and Research, Kopargaon, Maharashtra, India.

*Corresponding Author E-mail: mohinisaluke82@gmail.com

ABSTRACT:

Non-specific phytochemical profiling of plant extracts will help in the study of different groups of compounds and discovery of new bioactives, thereby minimising compound identification errors. The pharmaceutical industry has the potential to develop innovative drugs by using the large range of bioactive metabolites that marine seaweed is known to produce. Analysis of complex bioactive ingredients using analytical methods such as HR-LCMS has been demonstrated to be significant. In this research, we used HR-LCMS and FTIR analysis to explore at the metabolic profiles of Turbinaria ornata. The current investigation verifies the existence of significant therapeutic bioactive substances, such as Terpene Glycosides, Beta-Keto Acid, Steroid, Lipids, Glycosides, Triterpenoid, Phenols, Flavonoid, Quinolizidine Alkaloid, Vitamin, and Oligopeptides and shown to have antimicrobial, antioxidant, anti-inflammatory, antitumorproperties. The results of this investigation demonstrate that Turbinaria ornata contains significant phytocompounds and useful for more in-depth research to create marine algae-derived medicines to treat a variety of ailments.

Graphical abstract

KEYWORDS: HR-LCMS, FTIR, Seaweeds, Bioactives.

INTRODUCTION:

Despite synthetic drugs are the crown jewel of the pharmaceutical sector, they have a few negative side effects. However, the bioactive chemicals from both terrestrial and marine plants have been shown to be functionally active, which overcomes the drawback of medications produced chemically¹.

Seaweeds hold more promise than terrestrial plants for isolating unique natural substances of interest for food and health purposes because they are an untapped resource².

Nearly 80% of all plant and animal species in the world are found in the marine environment. Along the intertidal zone, there are over 150,000 different types of seaweed³. The structurally diversified bioactive compounds found in seaweeds have significant potential for use in medicine and pharmaceuticals⁴.

Over the years, people have used seaweed as food and the current global market is over USD 6 billion annually with over 12 million tons in 2018. Based on their colour, seaweeds (macroalgae) are classified in three main groups: Rhodophyta (red algae), Ochrophyta (brown algae), and Chlorophyta (green algae)^5,6. According to estimates, there are 6200 red macroalgae, 1800 brown macroalgae, and 1800 distinct green macroalgae in the marine environment⁷.

In the marine environment, brown seaweed has the potential to be a renewable resource. Along with the Asian coast, it has a long history of usage in traditional medicine and is one of the most cultivated alga species. Brown seaweed has potential for being utilised as functional components for both human and animal health applications due to its abundance of bioactive compounds, such asprotein, carotenoids, vital fatty acids, minerals, dietary fibre, and vitamins^8,9.

The marine alga species Turbinaria ornata (Turner) J. Agardh, 1848 belongs to the Phaeophyceae family. It is abundantly spread in Tamil Nadu's (India's) southeast coast and is reportedly utilised as fertiliser, food, and animal feed. In the central and western Pacific, as well as the Indian Ocean, this alga is extensively dispersed in tropical and subtropical regions. It has been demonstrated that this seaweed possesses a variety of biological properties, including anti-inflammatory, anti-coagulant, antibacterial, and antioxidant properties¹⁰.

The extraction and characterisation of substances with biological activity for the creation of wholesome or functional meals have grown to be significant areas of food science study during the past two decades. According to reports, brown seaweeds are a significant source of bioactive compounds linked to a variety of biological activities in both in vitro and in vivo studies¹¹.

Extraction, assessment, chromatographic separation, and spectroscopic characterization are processes in the traditional techniques of characterisation of bioactive compounds. Unfortunately, despite considerable time and effort, most studies only manage to characterise a small number of known phytochemicals since acceptable phytochemical standards are not readily available. In order to identify the pharmaceutically powerful bio-actives and make the process of understanding their impact on the target easier, it is essential to unravel the intricate chemistry of bioactive crude extracts utilising high throughput and high-resolution approaches¹².

When compared to other Turbinaria species, Turbinaria ornata bioactive components and therapeutic qualities have received less research.As a result, this research sheds light on the many bioactive substances discovered using Turbinaria ornata HR-LCMS and FTIR analyses.

MATERIALS AND METHODS:

Collection of seaweed:

In Rameshwaram, a town close to Mandapam in Tamil Nadu, India, Turbinaria ornata was grown. To get rid of all the undesirable contaminants, clinging sand particles, and epiphytes, the seaweed was extensively washed with sea water and then distilled water. The material was dried in the shade, crushed into powder, and kept for future use in an airtight plastic container in the freezer (-20^o)^13,14.

Authentication of algae:

Dr. B.B. Chaugule, a former Professor of Savitribai Phule University in Pune, verified the authenticity of the algae gathered.

Preparation of extracts:

Using an electric blender, smaller particles of the dried seaweed were crushed. The extract was made by combining 20g of the powdered substance with 200 ml of ethanol. After that, the mixture was incubated in an orbital shaker at 32°C overnight^15,16. The extract was filtered using a funnel and No. 1 Whatmann filter paper. The extract was collected and placed in a 250mL conical flask. A rotating vacuum evaporator was used to dry the collected extract.Following the extraction, the material was subjected to FTIR and HR-LCMS analysis^4,17.

High-Resolution Liquid Chromatography-Mass Spectroscopy (HR-LCMS):

Agilent's (6550 iFunnel Q-TOFs) equipment, which includes a column component, Hip sampler, Q-TOF with dual ion sources, electrospray ion production (ESI with Agilent Jet Stream) (AJS), and binary pumpwas used for HR-LCMS analysis of the ethanolic extract of all samples. An Agilent UHPLC (Ultra High-Performance Liquid Chromatography) and A Hypersil Gold column (C18 100 2.1mm-3 MICRON) equipment were used to perform chromatographic separations utilising 5μl of ethanolic sample¹⁸.Elution was carried out using solvents A (0.1 percent formic acid in water) and B (90 percent acetonitrile + 10 percent H2O + 0.1 percent formic acid) at a flow rate of 300 l/min for up to 30 min. Ionization was accomplished for the MS experiment using a Dual AJS ESI system, with the gas temperature set to 250°C¹⁹, the capillary voltage set to 3500 V, the drying gas flow rate set to 13 l/min, andthe nebulizer pressure set to 35 psi.Agilent Mass Hunter software was used for the acquisition of Q-TOF data and the analysis of the mass spectrometric results^20,21.

FT-IR analysis:

Utilizing a Perkin Elmer Spectrophotometer device with a transmittance range of 400–4000 cm-1, it was used to identify the various peaks and their functional groups²². The FTIR's peak values were captured. Each analysis's findings were checked twice^23–25.

RESULT AND DISCUSSION:

HR-LCMS analysis:

In the ethanolic extract of Turbinaria ornata, 100 compounds were identified using HR-LCMS, in that, 24 main compounds were verified based on their mass, retention time, and molecular formula, as given in Table 1. Figure 1 shows the Turbinaria ornata HR-LCMS chromatogram. Peak resolution was enhanced by the addition of formic acid to the mobile phase. Here, it was noticed that each resolved peak may belong to more than one phytocompound due to having the same retention period, as a result of similarities in their polarity and chemical characteristics. Following compound separation, Agilent iFunnel technology employed electrospray to create various ions fragments, which were then focused on Agilent Jet Stream technology for improved ion sampling and transmission²⁶.

According to the HR-LCMS investigation and extensive literature search, the primary compounds predicted belonged to several categories of secondary metabolites, including Terpene Glycosides, Beta-Keto Acid, Steroid, Lipids, Glycosides, Triterpenoid, Phenols, Flavonoid, Quinolizidine Alkaloid, Vitamin, and Oligopeptides. Figure 2 displays the spectrum of several bioactive compounds isolated from Turbinaria ornata.

Fig 1. HR-LCMS chromatogram of Turbinaria ornata

Table 1 Different bioactive compounds isolated by HR-LCMS analysis of Turbinaria ornata

Sr. No	Name of the Compound	Formula	Mass	RT (Min)	Chemical Nature
1	Neryl Rhamnosyl-Glucoside	C22 H38 O10	462.2561	9.105	Terpene Glycosides
2	Dimethyl 3-Methoxy-4-Oxo-5-(8,11,14-Pentadecatrienyl)-2-Hexenedioate	C24 H36 O6	420.2479	11.263	Beta-Keto Acids
3	Sn-3-O-(Geranylgeranyl) Glycerol 1-Phosphate	C23 H41 O6 P	444.2645	11.423	Lipids
4	11beta,17,21-Trihydroxy-2alpha-Methylpregn-4-Ene-3,20-Dione 21-Acetate	C24 H34 O6	418.2327	11.739	Steroid Ester
5	Schidigeragenin B	C27 H40 O4	424.2734	12.037	Glycosides
6	Neoporrigenin B	C27 H42 O5	446.3	12.426	Triterpenoid
7	Prostaglandin D2-1-Glyceryl Ester	C23 H38 O7	426.254	13.354	Ester
8	1-Octen-3-Ol-3-O-Beta-D-Xylopyranosyl (1->6)-Beta-D-Glucopyranoside	C19 H34 O10	422.2198	13.378	Glucoside
9	Methyl2-(10-Heptadecenyl)-6-Hydroxybenzoate	C25 H40 O3	388.2961	14.396	Phenols
10	(6b,7b,13R)-6,7-Diacetoxy-8,14-Labdadiene-13-Ol	C24 H38 O5	406.2677	15.012	Diterpenoids
11	3'',4''-Diacetylcosmosiin	C25 H24 O12	516.1311	5.764	Flavonoid (Glycosides)
12	Cinegalline	C23 H30 N2 O6	430.2156	7.68	Quinolizidine Alkaloid
13	(4R,5S,7R,11S)-11,12-Dihydroxy-1(10)-Spirovetiven-2-One 11-Glucoside	C21 H34 O8	414.2207	9.365	Terpene Glycosides
14	(9Z,11R,12S,13S,15Z)-12,13-Epoxy-11-Hydroxy-9,15-Octadecadienoic Acid	C18 H30 O4	310.2186	12.187	Fatty Acid
15	7alpha,12alpha-Dihydroxy-3-Oxochol-4-En-24-Oic Acid	C24 H36 O5	404.2619	13.344	Steroid
16	9Z-Octadecenedioic Acid	C18 H32 O4	312.2348	13.681	Fatty Acid
17	Phylloquinone	C31 H46 O2	450.3401	13.736	Vitamin
18	9,10-Dihydroxy Stearic Acid	C18 H36 O4	316.2614	14.444	Fatty Acid
19	17-Hydroxy-Linolenic Acid	C18 H30 O3	294.2195	14.701	Fatty Acid
20	8-Epideoxyloganin Tetraacetate	C25 H34 O13	542.1999	15.123	Terpene Glycoside
21	Hovenine A	C27 H42 N4 O4	486.3206	15.227	Oligopeptides
22	Irigenin 7-O-Glucoside	C24 H26 O13	522.1373	15.83	Polyphenols
23	1-Phenyl-1,3-Eicosanedione	C26 H42 O2	386.3185	15.999	Alkyl-Phenylketones
24	3α,7α-Dihydroxy-5β-Cholestanate	C27 H46 O4	434.3396	17.735	Steroid

CONCLUSION:

For the first time, Turbinaria ornata was taken and subjected to sophisticated methods of phytochemical analysis, including HR-LCMS and FTIR spectroscopy, which shows presence of Terpene Glycosides, Beta-Keto Acid, Steroid, Lipids, Glycosides, Triterpenoid, Phenols, Flavonoid, Quinolizidine Alkaloid, Vitamin, and Oligopeptides. Secondary metabolites derived from natural sources are an effective tool for drug discovery and development. The research also emphasises the possibility of current analytical techniques for documenting chemical components in a genus of plants with great therapeutic value. In order to verify and identify the plant species, this may unquestionably assist in creating a chemo-taxonomical database. This study concluded that seaweeds are a rich source of biogenic compounds that are both structurally and biologically active. These bioactive substances have significant pharmacological properties and may be effective in treating a range of human illnesses. To find potential medication candidates to address different conditions, in vitro and in vivo studies on these bioactive compounds should be performed.

ACKNOWLEDGEMENT:

For the HR-LCMS analysis, the author wants to say thank you to the SAIF at IIT Bombay.

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Received on 18.01.2024 Revised on 14.05.2024

Accepted on 04.07.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):579-584.

DOI: 10.52711/0974-360X.2025.00086

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