INTRODUCTION
Introduction
As land resources diminish, the ocean, covering over 70% of the Earth, emerges as a vital alternative for food, medicine, and essential human needs With the global population rising and facing food shortages, new disease outbreaks, and reduced land space due to seawater encroachment from global warming and urban development, the ocean has consistently proven to be a reliable resource for humanity.
Seaweeds (macroalgae) abundant on the seashores, ocean beds and solid structures, have been essential to humans (coastal dwellers) for as far as prehistoric time
The modern applications of the genus Laurencia in food, medicine, engineering, and cosmetics are significant, as highlighted in this study Each year, approximately 1.3 billion tons of food are lost to waste due to microbial, chemical, and physical deterioration, with microbes and enzymatic browning driven by oxygen being major contributors Additionally, oxidation is associated with many chronic diseases Laurencia is recognized as a valuable source of antioxidants and tyrosinase inhibitors, which are crucial in addressing these challenges.
The genus Laurencia is known for its rich bioactive compounds, yet optimizing extraction conditions to achieve high yields, quality, low cost, and environmentally friendly techniques remains a significant challenge This study aims to enhance existing research on seaweeds by establishing optimized extraction conditions using a novel technique, Ultrasound-Assisted Extraction (UAE), to evaluate brominated compounds The research successfully separates and characterizes two compounds from L intermedia Yamada extract, while also assessing their total phenolic content, antioxidant activity, and tyrosinase inhibitory activity This innovative approach is crucial for paving the way for future studies on this species.
Background
The ocean floor is home to over 11,017 species of macroalgae, including 1,901 green algae (Ulvophyceae), 7,083 red algae (Rhodophyceae), and 2,033 brown algae (Phaeophyceae) These remarkable algae have been a food source for prehistoric populations in southeastern Asia, particularly in Japan, China, Korea, and Polynesia In Vietnam, macroalgae are plentiful along the coast and are utilized for culinary, medicinal, and cosmeceutical applications.
Macroalgae are classified into three divisions based on color: red, green, and brown, which are influenced by pigments such as phycobilins, chlorophyll, and fucoxanthin Vietnam's extensive coastline, spanning 3,260 kilometers in an 'S' shape, is home to a diverse range of seaweed species A study by Nguyen et al in 2013 revealed that there were 827 publications focused on Vietnamese seaweeds, with 412 (49.8%) specifically addressing red seaweeds, as illustrated in the distribution map of seaweeds by province along the coast of Vietnam.
Seaweeds have emerged as vital contributors to global food security due to their rich content of primary metabolites, including carbohydrates, proteins, amino acids, and vitamins, which are essential for plant growth and cellular function Additionally, seaweeds contain secondary metabolites, known as phytochemicals, categorized into terpenoids, phenolic compounds, alkaloids, and sulfur-containing compounds, which protect them from herbivores and environmental stressors These phytochemicals are not only crucial for food preservation and modification but also serve as active ingredients in the development of new drugs and treatments for skin diseases.
[19] Seaweeds have gained their importance among several sea species because of the diversity of their application in the food, cosmetic and pharmaceutical industries
The genus Laurencia, part of the Rhodophyta family of red algae, is a significant source of diverse halogenated secondary metabolites, exhibiting notable biological activities such as antibacterial, antifungal, anti-predatory, and anticancer properties These algae are also rich in natural antioxidant compounds, enabling them to withstand various stress-inducing factors Predominantly found in tropical oceans, Laurencia species are recognized for their complex chemical composition, including brominated compounds, diterpenes, halogenated sesquiterpenes, and acetogenins Since its classification by Lamouroux in 1813, numerous red macroalgal species within this genus have been identified.
In our study evaluating the antioxidant and tyrosinase inhibitory activities of brominated compounds from Vietnamese red seaweeds, we screened three species: Galaxaura arborea, Mastophora rocea, and L intermedia Yamada Among these, L intermedia Yamada exhibited the highest yield and activity for total phenolic content (TPC), DPPH radical scavenging capacity (DRSC), and Ferric Reducing Antioxidant Power (FRAP), leading to its selection for further research.
Figure 1.1 Pictorial of L intermedia Yamada blade stipe holdfast frond
The selected species (L intermedia Yamada) belongs to the genus Laurencia
Classification of L intermedia Yamada, according to World Register of Marine Species [24]:
Bromo-containing compounds from the genus Laurencia exhibit strong properties that inhibit tyrosinase, combat cancer, and slow down oxidation, which contributes to aging in humans as well as enzymatic browning that leads to discoloration in fruits, vegetables, and seafood Melanin, the pigment responsible for color in skin, eyes, and hair, plays a crucial protective role against ultraviolet (UV) radiation and is produced through melanogenesis in melanosomes by melanocytes located in the basal layer of the epidermis However, excessive melanin production can result in various skin disorders, including melasma, freckles, and age-related spots.
Tyrosinase is a crucial enzyme in melanin synthesis, facilitating the conversion of L-tyrosine to L-Dopa and subsequently to dopaquinone This enzyme is also significant in the browning process of fruits, vegetables, and seafood In cosmetology, tyrosinase inhibitors are commonly used in creams aimed at achieving skin whitening, as well as in food preservation to prevent enzymatic browning The inhibition of tyrosinase is a widely adopted strategy due to its role in regulating melanin biosynthesis.
Laurencia is well able to retard browning in food and human skin
Oxygen plays a crucial role in metabolism, but under certain conditions, it can be detrimental, leading to oxidative rancidity that deteriorates food quality and results in undesirable aromas, off-flavors, and color degradation This process not only affects food but also impacts human health, as free radicals contribute to various diseases, including arteriosclerosis, diabetes, and neurodegenerative disorders, due to oxidative stress Oxidative stress primarily damages membrane lipids, proteins, and nucleic acids when the body's ability to counteract reactive oxygen species is overwhelmed by external factors like radiation, industrial chemicals, and pollution However, the genus Laurencia, particularly L intermedia Yamada, has a rich antioxidant profile that can help mitigate food spoilage and combat oxidation-related health issues.
To meet the growing demand for natural products derived from macroalgae, it is essential to employ advanced extraction methodologies Key factors influencing the extraction process include the chosen methods, solvents, temperature, duration, and solid-to-solvent ratios Although these factors are influenced by the solubility, volatility, and stability of the target compounds, a comprehensive understanding of their interactions remains incomplete Therefore, it is crucial to identify appropriate extraction techniques and parameters specifically for important species like L intermedia Yamada.
Statement of Problem
Seaweeds are abundant in marine environments and are recognized as a rich source of bioactive compounds vital for the food, pharmaceutical, and cosmeceutical industries However, there is a lack of studies focused on effective extraction techniques for recovering bioactive compounds and antioxidants from L intermedia Yamada Most research has concentrated on isolating, purifying, and identifying new compounds, leading to the prediction that this species is under-exploited due to the absence of optimized extraction conditions This lack of established parameters not only results in low extraction yields but also compromises extract quality, creating challenges for future research on extraction methods.
The food, cosmeceutical, and pharmaceutical industries are increasingly relying on compounds for additives, preservatives, and drugs to address post-harvest losses, new diseases, and skin radiation issues However, the use of synthetic products poses significant health risks, including cancer, allergies, and hyperactivity in children Despite their benefits in extending food shelf life and serving as raw materials for new drugs, the adverse health effects associated with synthetic compounds highlight the urgent need for safer alternatives.
Justification of the Study
The combined effects of population growth, climate change, and the Covid-19 pandemic are significantly threatening global food security and exacerbating food shortages Additionally, environmental pollution and global warming are challenging human survival against various incurable diseases It is therefore counterproductive to increase health risks through the consumption of food, drugs, and cosmetics containing synthetic compounds This study aims to build on previous research by sourcing products from natural origins, with seaweeds being identified as a promising alternative that offers minimal environmental impact.
Due to the limited research on extraction techniques for recovering bioactive compounds and antioxidants from L intermedia Yamada, optimizing extraction conditions is crucial for its economic significance This optimization aims to select eco-friendly methods and solvents, minimize extraction time, enhance yield, and produce high-quality extracts Therefore, employing ultrasound-assisted extraction to optimize conditions while analyzing total phenolic content, antioxidant capacity, and tyrosinase inhibitory activities of the extracted compounds is a strategic approach The study utilizes Response Surface Methodology (RSM) with Box-Behnken Design, incorporating two levels, four factors, and three center points.
JMP 15 statistical software was used to design optimization experiments, to generate the model equations, to graph the 3D and 2D contour plots of the responses and to predict the optimum values for the independent variables Minitab 16 and excel statistical tools were used for data analysis This specific study is the first in literature about the species and would serve as a roadmap for subsequent studies.
Research Question
Recent research highlights the significance of seaweeds, particularly the genus Laurencia, which has been utilized in various applications, including food and pharmaceuticals The study explores the roles of oxygen and tyrosinase in food spoilage, disease retardation, and skin aging, addressing these critical issues through innovative approaches Additionally, it introduces a new extraction method that enhances the yield and quality of bioactive compounds, offering a promising alternative to conventional extraction techniques.
Hypothesis
Red seaweeds are rich in brominated compounds that exhibit potent antioxidant properties and inhibit tyrosinase activity These characteristics make them valuable in the cosmeceutical, pharmaceutical, and food processing industries for combating skin aging, reducing oxidative stress in humans, and preventing food deterioration.
Objective
This study aims to utilize response surface methodology to optimize ultrasonic-assisted extraction conditions for maximizing the phenolic content and antioxidant activity of L intermedia Yamada Additionally, it investigates the total phenolic content, antioxidant properties, and tyrosinase inhibitory activities of the extracts and fractions obtained from L intermedia Yamada.
1.7.2 Specific Objective a Screen three seaweeds species b Investigate varying extraction conditions for the selected species (L intermedia Yamada) c Optimize the appropriate ultrasound-assisted extraction conditions d Separate fractions of L intermedia Yamada extracts using n-hexane and ethyl acetate e Isolate, classify and purify compounds from L intermedia Yamada f Investigate the total phenolic content and biological activities (antioxidant and tyrosinase inhibitory activities) of different fractions and isolated compounds
Scope of Study
A study was conducted in Nha Trang, Vietnam, situated in the southern and eastern regions of the Indochinese peninsula in Southeast Asia The research focused on farmed species collected from Nha Trang Bay between March and April.
Figure 1.2 Distribution of seaweeds by province along the coast of Vietnam
Nha Trang Bay, covering an area of 507 square kilometers with a coastline of 103 kilometers, is situated between Cay Cape to the north and Dong Ba Cape to the south As the capital of Khanh Hoa Province, Nha Trang is one of the fifty-eight provinces and coastal cities along Vietnam's south-central coast This region is notable for having the highest concentration of seaweeds along the entire Vietnamese coastline, with experimental analyses of selected species conducted at Nha Trang University and Ho Chi Minh University of Medicine and Pharmacy.
Limitation of the study
The study faced significant challenges, primarily due to the impact of the global Covid-19 pandemic, which hindered the timeline and resulted in only half of the proposed objectives being met Additionally, the scarcity and seasonal variation of the species L intermedia Yamada made it difficult to obtain fresh samples for the research.
The challenges faced negatively affected the study's objectives, leading to limitations that hindered the thorough investigation of key goals, including the isolation, purification, identification, and characterization of the extracted compounds.
LITERATURE REVIEW
Introduction
The growing demand for bioactive compounds from natural sources has sparked increased research interest in exploring a wide range of plant species, both aquatic and terrestrial This study focuses on evaluating the antioxidant and tyrosinase inhibitory activities of brominated compounds derived from Vietnamese red seaweeds, marking a significant contribution to existing research By reviewing relevant literature, this study aims to identify research gaps and clearly articulate how it differs from and relates to previous work in the field.
This review provides an overview of seaweeds, emphasizing the importance of studying them, particularly the phenolic content, tyrosinase inhibitory, and antioxidant activities of extracts and compounds from the genus Laurencia It connects these findings to specific species, while also discussing modern applications of seaweeds The study aims to optimize extraction conditions for L intermedia Yamada, highlighting various extraction methods and experimental designs from related species Additionally, it evaluates the Ultrasound-assisted Extraction method used in this research.
Scope
The literature selection for this study was guided by specific criteria, focusing on keywords such as brominated compounds, seaweeds, Laurencia, fractionation, ultrasound-assisted extraction, antioxidants, and tyrosinase inhibitors The reviewed materials included journals, research papers, review articles, conference proceedings, and books Additionally, relevant resources were sourced from reputable databases like DBLP, Google Scholar, ISI Proceedings, JSTOR, Medline, and Scopus to ensure comprehensive coverage of the topic.
The Web of Science database includes a comprehensive review of sources from the past fifteen years (2005-2020), which is crucial for analyzing trends in reported data and findings during this timeframe However, it is important to note that certain limits were surpassed under specific conditions.
Overview of Seaweeds
Covering about 71 percent of the Earth's surface, oceans are home to invaluable renewable resources and culinary treasures As land resources dwindle, our quest for new foods and pharmaceuticals has turned our attention to the sea, particularly seaweeds, which have been a focus of interest in recent years Historically, seaweed consumption dates back to prehistoric times, with evidence suggesting that coastal communities recognized its nutritional value as early as 14,000 years ago in Chile Archaeological studies indicate that seaweeds were commonly used by ancient populations in southeastern Asia, including Japan, China, Korea, and Polynesia, a tradition that continues today The earliest written records of seaweed consumption in Japan date back approximately 1,500 years to the Asuka and Nara Era.
Mouritsen et al [2] highlight the absence of archaeological evidence supporting the use of seaweeds as food, suggesting that their physical structure is more indicative of other uses, such as in prehistoric human dwellings, firewood, medicine, and shelter Seaweeds are categorized into three divisions based on color: Rhodophyceae (red), Chlorophyceae (green), and Phaeophyceae (brown), with their distinct hues attributed to pigments like phycobilins in red algae, chlorophyll in green, and fucoxanthin in brown algae [11].
Seaweeds, like other plants, contain essential primary metabolites such as carbohydrates, proteins, and vitamins, which are crucial for growth and cellular functions In addition, they produce secondary metabolites, classified into terpenoids, phenolic compounds, alkaloids, and sulfur-containing compounds, that serve as phytochemicals Due to their sessile nature, seaweeds are more susceptible to herbivores and environmental threats These secondary metabolites exhibit antimicrobial and antifungal properties, acting as attractants, repellents, and deterrents against pathogens, insects, viruses, and herbivores Recent studies in ecological biochemistry have shown that plants with high concentrations of secondary metabolites have enhanced resistance to biotic and abiotic stresses, highlighting their ecological and chemical defensive roles.
Significance of Investigating Seaweeds
The selection of sources for bioactive compounds is vast, encompassing various plant parts such as leaves, roots, stems, seeds, and nuts Numerous studies have highlighted terrestrial plants and edible seeds as excellent sources of essential bioactive compounds However, choosing seaweeds over terrestrial plants carries significant environmental advantages Investigating the bioactivity of seaweeds helps source highly sought-after natural compounds and food while reducing the impact on nutrient recycling within the phytoplankton food chain, making it a more environmentally friendly approach.
Even though the investigation of nutrient depletion in the marine ecosystem is still in its early stage, but evidences have supported the link to seaweeds overpopulation
Research in South Korea and Japan indicates a strong relationship between nutrient levels and Undaria production, highlighting that nutrients absorbed by macroalgae may influence nutrient recycling and secondary productivity However, Staff et al warn that excessive nutrient enrichment can lead to detrimental effects, including toxic algal blooms, shellfish poisoning, and coral reef degradation.
Harvesting seaweeds positively influences the survival of seagrasses, which serve as crucial habitats for various marine species and help mitigate ecosystem threats posed by bloom-forming seaweeds Excessive seaweed growth can outcompete seagrasses, negatively impacting physiochemical properties such as turbidity and biological oxygen demand, leading to eutrophication and endangering fish and invertebrate populations Furthermore, seaweeds are increasingly recognized for their role in the destruction of seagrass beds, especially in areas with high nutrient pollution, reduced top-down control from fishing, or the introduction of invasive seaweed species.
Rose et al [57] highlighted that seaweed growth plays a crucial role in reducing excess nitrogen and phosphorus levels, while simultaneously generating and releasing oxygen through photosynthesis This underscores the need for state-regulated farming practices and suggests further research to explore the nutrient absorption rates of seaweeds.
Biological Potential of Seaweeds (Laurencia)
Phenolic compounds are crucial plant constituents known for their antioxidant properties due to their redox capabilities Total phenolic content (TPC) is often utilized to predict the antioxidant potential of various species, with many studies indicating a positive correlation between phenolics and antioxidant activity However, it is important to recognize that the species orientation and extraction parameters can significantly affect this relationship Conversely, several studies have reported no correlation between phenolic compounds and antioxidant properties, highlighting the complexity of this interaction.
In the study done by Topuz et al [63], phenolic compounds in Laurencia obtuse were investigated using ethanol as extraction solvent Against these parameters:
At a temperature of 50°C and a solid-to-solvent ratio of 30:1 mL/g for 45 minutes, the maximum total phenolic content (TPC) recorded was 25.95 mg GAE/g of seaweed, indicating a strong correlation with antioxidant activity Additionally, Al-Amro and colleagues conducted research on the total phenolic content in Laurencia collected from various sources.
A study on the extraction of compounds from the Arabian Gulf using ethanol and aqueous solvents revealed that ethanol yielded 18.99 mg GAE/g, while aqueous extraction resulted in 0.83 mg GAE/g The specific concentration of ethanol used was not disclosed, complicating comparisons with other research Nevertheless, our current study supports the use of water for extraction, as evidenced by increased yield with lower ethanol concentrations.
Enzymatic browning in fruits, vegetables, and seafood has been extensively researched, revealing its association with melanosis, a condition characterized by hyperpigmentation due to melanin, leading to unsightly discoloration or black spots on these products This aesthetic change can significantly diminish consumer appeal, resulting in rejection and reduced market value Notably, over 80% of the global population has lighter skin tones, which contain pheomelanin that is less effective at blocking ultraviolet (UV) radiation In fact, pheomelanin may enhance the effects of UV exposure, contributing to free radical formation and increasing the risk of skin carcinogenesis.
2.5.2.2 Role of Tyrosinase in Melanin Biosynthesis
Melanin is the key pigment that determines the color of human skin, hair, and eyes, and is also responsible for the browning of certain fruits and vegetables It is produced by melanocytes through a process called melanogenesis This process, along with skin pigmentation, serves as a crucial photoprotective mechanism against harmful ultraviolet radiation, playing a significant role in preventing skin damage and photo-carcinogenesis.
A significant lack of melanin and depigmentation can pose serious aesthetic and dermatological challenges for individuals, leading to psychological effects that impact their personality and societal interactions Various skin conditions, such as acanthosis nigricans, cervical poikiloderma, melasma, periorbital hyperpigmentation, lentigines, and neurodegeneration linked to Parkinson's disease, are associated with decreased melanin production, which can also increase the risk of skin cancer.
Melanogenesis is a complex process where enzymes like tyrosinase, along with proteins such as TYRP1 and TYRP2, are essential for melanin synthesis TYRP1, a product of the melanocyte gene, contributes to this process by stabilizing tyrosinase and modulating its activity While the function of mouse Tyrp1 is well understood, its role in human melanocytes remains less clear Additionally, TYRP1 is crucial for maintaining melanocyte formation and influencing their proliferation Tyrosinase, a key metalloenzyme containing dinuclear copper ions, serves as the rate-limiting factor in melanin production.
Tyrosinase plays a crucial role in the unwanted browning of fruits and vegetables, as well as in diseases linked to excessive melanin production The process of melanogenesis involves the inhibition of tyrosinase by L-Tyrosine and L-DOPA, as illustrated in Figure 2.1.
Tyrosinase is essential for melanogenesis and browning, with various studies identifying inhibitors from natural sources like fungi, bacteria, and plants, as well as synthetic origins These inhibitors are tested using monophenolic substrates like tyrosine or diphenolic substrates such as L-dopa, with their activity measured through dopachrome formation.
Quinones, the key agents in enzymatic browning, contribute to food deterioration during storage and processing This biochemical process occurs in fruits, vegetables, and beverages when tyrosinase interacts with polyphenolic substrates, particularly after operations like brushing, peeling, and crushing The disruption of cell structures enhances melanin formation, leading to undesirable changes in food quality.
Pheomelanin enzymatic reaction [26] Quinones cause food deterioration during an enzymatic browning reaction in the presence of oxygen, a form of oxidation [26], while melanin causes pigmentation in humans
The tyrosinase inhibitory potential of the genus Laurencia has been largely overlooked, with only one notable study identified In research by Qin et al., five new highly brominated metabolites were extracted from the red alga Laurencia similis, revealing that two compounds—3',5',6',6-tetrabromo-2,4-dimethyldiphenyl ether and 2',5',6',5,6-pentabromo-3',4',3,4-tetramethoxybenzo-phenone—exhibited strong inhibitory effects against protein tyrosine phosphatase 1B (PTP1B), with IC50 values of 2.97 and 2.66 μM, respectively Our study further investigates the tyrosinase inhibitory activity of fractions and two isolated compounds from L intermedia Yamada, making a valuable contribution to this under-researched area.
Oxygen plays a crucial role in the life processes of fruits and vegetables, particularly in post-harvest respiration However, its high chemical reactivity can lead to the production of toxic metabolic by-products, contributing to food deterioration Humans are exposed to various free radicals from both internal and external sources, with intrinsic sources stemming from normal metabolic processes and extrinsic sources including UV light, ionizing radiation, industrial activities, cigarette smoke, chemicals, and air pollution.
2.5.3.1 Effect of Reactive Oxygen Species
Reactive oxygen species (ROS) are oxygen-containing radicals with unpaired electrons, including compounds like hydroxyl radicals, superoxide anions, hydrogen peroxide, and nitric oxide These ROS play a significant role in various disease states The body's antioxidant defense system helps maintain steady levels of ROS, but under stress conditions, excessive ROS production can lead to oxidative stress, disrupting this balance This oxidative stress damages crucial biomolecules such as proteins, DNA, and lipids, and has been linked to several health issues, including cancer, diabetes, cardiovascular diseases, and aging.
Antioxidants are crucial in preventing oxidation by neutralizing free radicals, binding with free catalytic metals, and donating electrons They are commonly utilized as food additives to protect against the oxidative degradation of foods and oils.
Figure 2.2 Inhibition of polyphenol oxidase activity (natural extract and synthetic additive)
Oxidation ranks as the second leading cause of food deterioration, following microbiological spoilage Post-harvest handling introduces physical and mechanical stresses that promote enzymatic browning in fruits and vegetables The primary enzyme responsible for this reaction is polyphenol oxidase (PPO), an intracellular o-diphenol oxidase commonly found in higher plants and fungi.
Effect of extraction methods and parameters on the biological activity of
Choosing the right extraction methods and parameters—such as temperature, time, solvent-solute ratio, solvent type, and concentration—is crucial for maximizing both the yield and quality of metabolites from phytochemical sources These parameters can significantly affect the extraction efficiency of bioactive compounds, with their impact varying based on the species and the specific analytical assay used It is important to note that variations in extract yield or activity may occur due to changes in a single variable while keeping all other factors constant.
The rising interest in phytochemical studies has transformed the extraction techniques for bioactive compounds, revealing significant variations under identical conditions when employing different methods Environmental factors can greatly affect the composition of seaweed species across various regions Additionally, discrepancies in the estimated antioxidant capacity and total phenolic content reported in different studies stem from the diverse methodologies and conditions utilized.
An investigation by Ge et al revealed a significant variation of 11.5 ± 0.5% in total polyphenol content (TPC) and total flavonoid content (TFC) when comparing conventional extraction methods to ultrasound-assisted methods Similarly, Nguyen TH et al examined four extraction techniques—Conventional Extraction (CE), Microwave Assisted Extraction (MAE), Ultrasound Assisted Extraction (UAE), and Heat Reflux Extraction (HRE)—to evaluate α-glucosidase inhibitory activity and antioxidant properties from L dendroidea The study found notable differences between conventional methods and others, with UAE and HRE showing consistent results across all assays In contrast, the conventional method exhibited varying IC50 values, highlighting the significant impact that different extraction techniques have on the extract's capacity.
The polarity of solvents used for extraction significantly influences the content and activity of the extracts, as demonstrated by numerous studies Water and perfluorohydrocarbons represent the extremes of solvent polarity, with water being the most polar and perfluorohydrocarbons among the least Recently, both have gained attention for various applications; water is favored for its environmental safety and ability to hydrate polar solutes, while perfluorohydrocarbons are valued for their nonpolar, hydrophobic, and chemically inert properties, making them nontoxic solvents with a density greater than that of hydrocarbons.
The Latin phrase "similia similibus solvuntur," meaning "like dissolves like," highlights the principle that polar solvents effectively dissolve polar solutes, while non-polar solvents dissolve non-polar solutes This solubility rule categorizes solvents based on their chemical composition, enabling qualitative predictions about solute behavior Compounds are more likely to dissolve in solvents with similar functional groups, such as polar compounds favoring water for extraction Thus, selecting the appropriate solvent requires understanding its chemical polarity and reactivity with the solute to prevent unwanted reactions This study focuses on optimizing extraction conditions for L intermedia.
Yamada, preliminary investigation of the appropriate solvent and other conditions were studied prior to determine the most influential parameters
In various studies investigating a variety of seaweed species [L dendroidea,
Ascophyllum nodosum (AN), Fucus vesiculosus (FV) and Fucus serratus (FS),
Different extraction solvents significantly affect the extract activities of Laver (Porphyra tenera) [7, 110, 111] Similarly, Michiels et al (104) investigated the phenolic content and antioxidant capacities of orange, apple, leek, and broccoli using various solvents (acetone, water, and acetic acid) and solvent ratios Their findings revealed a notable variation in total phenolic content and antioxidant capacity, with differences of approximately 25% and 30%, respectively, due to changes in solvent and solvent ratio [104].
Time is a crucial factor in the extraction of bioactive compounds, as the stability of these compounds can be compromised at high temperatures Typically, a balance is struck between temperature and extraction duration, with higher temperatures necessitating shorter extraction times and lower temperatures allowing for longer durations Gope et al [112] highlighted the detrimental effects of extraction time and solvents on the phenolic content and antioxidant properties derived from the pulp of Citrus macroptera.
Temperature significantly influences the kinetic energy of molecules, leading to increased collisions and facilitating the extraction of bioactive compounds In the context of seaweeds, their structure comprises polysaccharic chains with hydroxyl groups of phenol linked by hydrogen bonds, which require activation energy (Ea) for chemical reactions to occur This activation energy enables molecular movement and proper collision orientation, allowing the kinetic energy during collisions to surpass the energy barrier that typically hinders reactions Consequently, temperature is crucial for providing the necessary activation energy to dissociate these bonds and release phenolic compounds during the extraction process.
Temperature can significantly influence the biological activities of seaweed extracts, with effects varying based on the species' age, the condition of the raw materials, and the biochemical properties of the active ingredients Research, including a study by Hwang and Nhuan (2014), highlights this variability, demonstrating that the total phenolic (TPC) and total flavonoid contents (TFC) of Laver (Porphyra tenera) were maximized when using water as a solvent, achieving a 100% yield with temperature adjustments from 37°C to 100°C.
2.7.5 Solute-solvent (volume/mass) ratio
The solute-solvent ratio plays a crucial role in determining the reaction rate and extract yield, as a higher solvent volume enhances the concentration gradient, facilitating the transfer of solutes from the sample matrix to the solvent Increased reactant concentration typically results in more effective collisions, thereby accelerating the reaction rate, except in zero-order reactions While it is generally observed that higher product concentrations correlate with reduced reaction rates, this study's findings indicate that the scavenging capacity of L intermedia Yamada actually increases with higher solute concentrations.
Mechanism of Ultrasound-Assisted Extraction
Modern extraction techniques are replacing traditional methods like maceration, Soxhlet extraction, and reflux extraction due to their ability to minimize associated drawbacks Conventional methods often require high temperatures, extended extraction times, and excessive solvents, leading to increased costs, lower yields, and negative environmental effects Prolonged exposure to high temperatures can cause thermal decomposition of sensitive components, ultimately resulting in poor extract quality.
Ultrasound-assisted extraction (UAE) is a modern extraction technique that, along with methods like pulsed electric field (PEF) extraction and supercritical fluid extraction (SFE), is gaining popularity due to the limitations of traditional extraction methods These innovative techniques are utilized to extract nutraceuticals from plants, enhancing the quality of extracts, increasing yield, reducing extraction time, and minimizing solvent usage.
Figure 2.4 Mechanism of ultrasound-assisted extraction process
Ultrasound-assisted Extraction (UAE) utilizes the cavitation phenomenon, where high-frequency ultrasound pulses (20 kHz) generate local hotspots with significant shear stress and temperature by creating cavitational bubbles This process enhances mass transfer of analytes into the extraction solvent due to the pressure and temperature changes caused by the collision of these bubbles UAE is recognized as a novel extraction technique, demonstrating notable efficiency in various applications.
• extremely high temperature and pressure
Ultrasound enhancement of Extraction Process
• promotion of softening and swelling of raw materials
• disruption of raw material surface
The acceleration of mass transfer shows similarities with MAE, which is typically 30 minutes faster than UAE However, the use of ultrasounds is favored due to its gentler approach, making it more suitable for extracting unstable compounds.
Figure 2.5 Pictorial mechanism of ultrasound-assisted extraction
Response Surface Methodology (RSM)
Response surface methodology (RSM) is an optimization technique that employs statistical methods derived from the factorial designs of Box and Behnken, as well as Box and Wilson This approach combines mathematical and statistical tools to effectively fit models and analyze complex problems involving multiple independent parameters that influence a dependent variable.
Modern Application of Seaweeds
In recent years, seaweeds (macroalgae) have become increasingly significant due to their diverse applications in the food, cosmeceutical, and pharmaceutical industries Recent research findings on the uses of seaweeds are summarized in Table 2.1.
Table 2.1 Modern application of seaweeds
Environmental factors like UV radiation, wind, and smoke, along with natural aging and skin barrier deterioration, lead to fine lines, wrinkles, pigmentation, and increased skin coarseness As a response to skin aging issues, the cosmeceutical industry has increasingly relied on synthetic active ingredients, which have been shown to harm the skin This has sparked a growing interest in sourcing compounds from natural sources, such as alguronic acid, a mixture of exo polysaccharides derived from microalgae, known for its anti-aging benefits and bioactive properties.
Seaweeds have long been a staple in Asian cuisine, commonly used in various dishes Popular species include Nori (Porphyra spp.), utilized in sushi, and aonori (Monostroma spp and Enteromorpha spp.), often found in soups and salads Kombu is another significant variety, featured in teas, mustards, pasta, and breads, highlighting the versatility of seaweeds in enhancing flavors and nutrition in everyday meals.
Laminaria japonica, commonly known as wakame, along with Undaria pinnatifida, Hizikia fusiforme (hiziki), Cladosiphon okamuranus (mozuku), Caulerpa lentillifera (sea grapes or green caviar), Palmaria palmata (dulse), Chondrus crispus (Irish moss), Alaria esculenta (winged kelp), Gracilaria spp (ogo), and Callophyllis variegata are all notable types of edible seaweeds These marine plants are rich in nutrients and offer various health benefits, making them popular choices in culinary dishes around the world.
Marine macroalgae are rich in a variety of biologically active metabolites, including acrylic acid, fatty acids, phenolic compounds, steroids, terpenoids, ketones, phlorotannins, and alkanes These bioactive compounds are responsible for the diverse biological activities exhibited by macroalgae.
Rich in antioxidants like alkaloids, flavonoids, phenols, tannins, phlorotannins, terpenoids, pigments, glycosides, and steroids, these substances provide a defense mechanism that protects against reactive oxygen species (ROS) caused by harsh environmental conditions.
MATERIALS AND METHODS
Methods
The entire study followed this trend keeping track of the most important activities performed
Figure 3.1 General order of major activities
3.2.2 Screening test to select the starting sample
A modified conventional extraction method was employed to screen three seaweed species—Galaxaura arborea, Mastophora rocea, and L intermedia Yamada—for optimal total phenolic content (TPC) and antioxidant activity In this process, 1 g of dried seaweed was mixed with 50 mL of 75% methanol, and the samples were subjected to orbital mixing to enhance extraction efficiency.
FRAP and TIA in fractions and isolated compounds
Dried Seaweeds Milling and Storage (-2C)
Species Screening Single Factor Optimization
The samples were incubated in a Shaking Water Bath (VS—1205 SW2) at 60°C for 2 hours Following incubation, they were cooled in a cold-water bath and centrifuged using a MEGA 17R Small High-Speed refrigerated centrifuge at 8500 rpm and 4°C for 20 minutes to obtain the extract The total phenolic content and antioxidant capacity of the extract were analyzed, leading to the selection of the species with the highest activity.
The single factor tests evaluate the effects of various solvents, including ethanol and methanol, in different concentrations (25%, 50%, 75%, and 100%) mixed with water, while keeping other factors constant Following this, the tests examine the impact of temperature at levels of 30°C, 40°C, 50°C, and 60°C, and subsequently assess the influence of time across intervals of 10, 20, 30, 40, and 50 minutes.
60 min) and finally solid to solvent ratio (0.1, 0.2, 0.4 and 0.6 g/ 10 mL)
In this study, four critical variables were identified for Response Surface Methodology (RSM) optimization based on single factor tests: solvent concentration (0-75% ethanol), temperature (40-60°C), extraction time (30-60 min), and the solid-to-liquid ratio (0.1-0.6 g/10 mL) To explore the interactions among these variables and their impact on extraction efficiency, a two-level, three-factor Box-Behnken design was employed, comprising 27 experimental runs Each parameter was assessed at low and high levels, with middle terms and three center points, and the resulting data were analyzed using a second-order polynomial model.
In this regression model, k denotes the number of independent variables (Xi) that influence the response variables Y, which include TPC, DPPH FRAP, and TIA The coefficients βo, βi, βii, and βij represent the intercept, linear, quadratic, and interaction effects, respectively.
Ultrasound-assisted extraction was performed in the ultrasonic bath (Branson
Seaweed samples were combined with ethanol at suitable concentrations and subjected to sonication using a Branson Ultrasonic Corp device (2510, 50/60 Hz, 100 W) for varying durations and temperatures Following the extraction process, the flasks were promptly removed from the ultrasonic bath and allowed to cool to room temperature with the aid of cooling water The resulting seaweed extracts were then centrifuged at 8500 rpm and 4°C for 20 minutes using a MEGA 17R small high-speed refrigerated centrifuge The collected extracts were subsequently analyzed for total phenolic content (TPC) and antioxidant capacity.
3.2.6 Fractionation, Isolation and characterization of compounds from L intermedia Yamada
Nuclear Magnetic Resonance (NMR) experiments, both one-dimensional (1D) and two-dimensional (2D), were conducted using a Bruker Advance III 500 FT-NMR, with tetramethylsilane as the internal standard and chloroform-d (CDCl3) as the solvent High-resolution mass spectrometry (HRMS) was performed on an X500R QToF-MS from Sciex, USA, while APCI-MS/ESI-MS analyses were carried out using a single quadrupole MSQ Plus Mass Spectrometer from Thermo Fisher Scientific, USA Additionally, infrared (IR) spectra were recorded using a Shimadzu IR Affinity 1-S from Japan, and optical rotations were measured with a P8000.
A Krüss polarimeter (Germany) Column chromatography was performed with silica gel (40 – 63 àm, Merck) and Sephadex LH-20 (GE Healthcare Life) Thin layer chromatography (TLC) was carried out on silica gel 60 F 254 plates (Merck) Spots were detected by spraying with the vanillin – sulfuric reagent followed by heating to 105C
L intermedia was extracted with ethanol 30% under optimum UAE conditions determined as above to obtain the extract, which was condensed using a vacuum rotary evaporator (Laborota 4001, Heidolph, Germany) Portion of the crude extract was stored at -20Cfor analysis After partial removal of solvent (water and ethanol) under reduced pressure, concentrated extract (0.5 L) was successively liquid — liquid fractionated with n-hexane (0.5 L × 3 times), ethyl acetate (0.5 L × 3 times) and the remaining aqueous fraction The obtained fractions were evaporated to dryness, affording n-hexane (6.24 g), ethyl acetate (1.43 g) and aqueous (1.41 g) fractions
Portion of the fractions and the crude extract were further dehydrated to dryness and the phenolic content and antioxidant activities were evaluated
The n-hexane extract underwent separation using silica gel column chromatography with a gradient solvent system of n-hexane and ethyl acetate (95:5 to 70:30, v/v), resulting in 31 fractions (A.1 – A.31) Fraction A.3 (0.9 g) was further purified through isocratic elution with n-hexane and dichloromethane (6:4, v/v), yielding 6 sub-fractions (A.3.1 — A.3.6) Compound 1 (1.7 g) was isolated from fraction A.18 (2.3 g) via recrystallization in n-hexane, while Compound 2 (163.7 mg) was obtained from sub-fraction A.3.3 through recrystallization in a chloroform and methanol mixture (5:5, v/v).
Aplysistatin (1): C 15 H 21 BrO 3 , colorless crystals, -30 o (c 0.5, CHCl 3 ) HRESIMS m/z 329.0719/331.0698 [M+H] + ; IR: 1759, 1672, 1386, 1016, 704, 590 cm -
1; 1 H-NMR (CDCl 3 , 500 MHz) δ H : 0.97 (3H, s, H-13), 1.18 (3H, s, H-15), 1.30 (3H, s, H-14), 1.63 (1H, t, J = 3.5 Hz, H-8α), 1.79 (1H, td, J = 4.0, 13.5 Hz, H-8β), 2.05 (1H, dd, J = 2.0, 9.0 Hz, H-6), 2.12 (1H, dd, J = 3.5, 13.0 Hz, H-9), 2.29 (1H, dd, J = 3.5, 14.0 Hz, H-9), 2.55 (2H, m, H-5), 3.87 (1H, dd, J=7.5, 9.0 Hz, H-1α), 3.93 (1H, dd, J
= 4.5, 13.0 Hz, H-10), 4.49 (1H, t, J = 9.0 Hz, H-1β), 5.13 (1H, br s, H-2), 6.95 (1H, dt, J = 2.5, 5.4 Hz, H-4) 13 C-NMR (CDCl 3 , 150 MHz) δ C: 69.9 (t, C-1), 66.8 (d, C-2), 132.0 (s, C-3), 143.1 (d, C-4), 27.2 (t, C-5), 51.2 (d, C-6), 79.0 (s, C-7), 37.7 (t, C-8), 32.4 (t, C-9), 65.1 (d, C-10), 41.0 (s, C-11), 169.1 (s, C-12), 18.0 (q, C-13), 21.7 (q, C-
Palisadin B (2): C 15 H 24 Br 2 O, colorless crystals, +10 o, ESI-MS (negative): 361.29/363.13/365.20 [M-OH]¯; IR: 1383, 1082, 1055, 667, 602 cm -1 ; 1 H-NMR (CDCl 3 , 500MHz) δ H: 0.90 (3H, s, H-13), 1.12 (H, s, H3-15), 1.30 (3H, s, H-14), 1.65 (1H, m, H-8), 1.77 (1H, d, J = 10.0 Hz, H-6), 1.79 (2H, m, H-8), 2.06 (1H, dd, J = 8.0, 17.5 Hz, H-4), 2.13 (1H, dd, J = 3.5; 13.0 Hz, H-9), 2.28 (1H, dd, J = 4.0, 12.5 Hz), 3.37 (1H, dd, J = 8.5, 10.5 Hz, H-1α), 3.69 (1H, dd, J = 3.0, 10.5 Hz, H-1β), 4.49 (1H, m, H-2), 5.61 (1H, m, H-4), 2.13 (2H, m, H-9), 2.28 (2H, m, H-9), 3.91 (1H, dd, J 4.0, 12.5 Hz, H-10); 13 C-NMR(CDCl 3 ,125MHz) δ C : 36.2 (t, C-1), 70.7 (d, C-2), 136.2 (s, C-3), 129.4 (d, C-4), 25.9 (t, C-5), 52.9 (d, C-6), 77.5 (s, C-7), 36.7 (t, C-8), 33.0 (t, C-9), 66.4 (d, C-10), 40.8 (s, C-11), 21.1 (q, C-12), 18.0 (q, C-13), 22.1 (q, C-14), 30.7 (q, C-15)
3.2.7 Determination of total phenolic content, antioxidant and tyrosinase inhibitory activities
The total phenolic content (TPC) of the extracts was assessed using the method outlined by Pham et al [157] A 0.5 mL sample of the extract was combined with 2.5 mL of 10% Folin-Ciocalteu reagent and allowed to sit at room temperature for 8 minutes This mixture was then mixed with 2 mL of 7.5% sodium carbonate and incubated in the dark at room temperature for 1 hour The absorbance was measured at 765 nm with a UV-VIS spectrophotometer (Biochrom Libra S50, Biochemical Ltd., Cambridge, UK), using gallic acid as a standard The results were expressed in milligrams of gallic acid equivalents per 100 grams of dried material (mg GAE/100 g).
3.2.7.2 DPPH radical scavenging assay (DRSC)
The DPPH stock solution, essential for assessing DRSC as outlined by Pham et al., was created by dissolving 24 mg of DPPH in 100 mL of methanol This solution was stored in the dark at −20°C for future applications Subsequently, 10 mL of the stock solution was utilized for further experimentation.
A working solution was prepared by adding 45 mL of methanol to achieve an absorbance of 1.1 ± 0.02 at 515 nm Subsequently, 0.15 mL of the sample was mixed with 2.85 mL of the working solution and incubated in the dark at room temperature for 3 hours The absorbance was then measured at 515 nm using a Biochrom Libra S50 UV–VIS spectrophotometer Results were quantified using trolox as a standard and expressed as mg trolox equivalents per 100 g of dried weight (mg TE/100 g).
3.2.7.3 Ferric reducing antioxidant power (FRAP)
FRAP was assessed following the modified method of Pham et al [158] The working solution was created by combining 300 mM acetate buffer, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3 in a 10:1:1 ratio For the measurement, 0.15 mL of the sample was mixed with 2.85 mL of the FRAP working solution, and the absorbance was measured at 593 nm after a 30-minute incubation in the dark at room temperature Results were reported as mg trolox equivalents per 100 g of dried weight (mg TE/100 g).
The modified method by Luisi et al involved mixing 0.1 mL of the sample solution with 0.3 mL of tyrosinase solution (approximately 300 units/mL) and 2.3 mL of phosphate buffer at pH 6.8 in test tubes, followed by incubation for 5 minutes at 37°C The reaction was initiated by adding 0.3 mL of L-tyrosine at a concentration of 2 mM A blank control was prepared by adding the sample solution to all reagents except the tyrosinase enzyme Absorbance readings for both the sample and blank were subsequently taken.
RESULTS AND DISCUSSION
Screening
The data in Table 4.1 compare the antioxidant capacity and total phenolic content of three screened red seaweed‘s species (Galaxaura arborea, Mastophora rocea and
L intermedia Yamada) According to the results obtained, L intermedia Yamada demonstrated the highest total phenolic content and antioxidant activity It also shows no significant difference between the DRSC and TPC of both G arborea and M rocea L intermedia Yamada was therefore selected for subsequent investigation
Table 4.1 TPC and antioxidant activity of three screened red seaweed’s species
The results are presented as means ± standard deviations (n = 3), with statistically significant differences indicated by different letters (p < 0.05) Key metrics include Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), Ferric Reducing Antioxidant Power (FRAP), and Trolox Equivalents (TE) Additionally, Gallic Acid Equivalents (GAE) are measured using the Fisher method (n=3).
Single factor optimization
Data in Table 4.2 present the total phenolic content and antioxidant activity of
L intermedia Yamada at varying solvents and concentrations Ethanol, methanol, water and their mixture with water at 100, 75, 50 and 25% were studied The table further shows a significant decrease in the total phenolic content at concentration
Ethanol at 50% concentration emerged as the most effective solvent for extracting total phenolic content from L intermedia Yamada, yielding 173.90 ± 8.01 mg GAE/100 g of dried sample Additionally, water and lower ethanol concentrations exhibited significant phenolic activity, indicating that most components in the plant are highly polar However, lower activities were observed for DPPH radical scavenging capacity (DRSC) at concentrations above 75% and below 50% for both ethanol and methanol No specific trends were identified for the ferric reducing antioxidant power (FRAP) assay, with ethanol at 75% and water also providing notable results at 63.30 ± 3.37 mg TE/100 g dry sample and 106.04 ± mg TE/100 g dry sample, respectively.
4.04 mg TE/100 g dry sample) for DRSC and FRAP respectively Ethanol demonstrated the highest activity on average for the three assays and was selected for further range test A further verification test for solvent concentration was done at 80,
In a study examining the effectiveness of ethanol and methanol as solvents for extraction, a 60, 40, and 20% ratio of ethanol and methanol was tested, revealing no significant differences (p-value < 0.005) compared to previous tests This evaluation was crucial, as existing literature often cites methanol as the preferred solvent for extraction Notably, the total phenolic content and FRAP values varied significantly depending on the solvent concentration, leading to the selection of a 0-75% ethanol concentration range for optimization Research on L dendroidea further supports that different extraction solvents can produce significant variations in extract activities.
The findings regarding ethanol concentration align with the prediction profiler derived from the optimization of L intermedia Yamada Additionally, the results from the optimization correlate with the outcomes of the single factor experiment.
Table 4.2 Effect of various solvents on the TPC and antioxidant activities of L intermedia Yamada
The results are presented as means ± standard deviations (n = 3), with significant differences indicated by different letters (p < 0.05) Key metrics include Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), Ferric Reducing Antioxidant Power (FRAP), Trolox Equivalents (TE), and Gallic Acid Equivalents (GAE) using the Fisher method (n=3).
Temperature plays a crucial role in the extraction process, significantly affecting sensitive bioactive compounds Our assessment of the impact of temperature on total phenolic content (TPC) and antioxidant activities revealed an increase in both TPC and DPPH radical scavenging capacity with rising temperatures However, Fisher mean comparison indicated no significant differences in TPC, FRAP, and DRSC between 30 to 40°C and 50 to 60°C Therefore, we deemed it essential to continue the experiment by increasing the temperature beyond 60°C.
The study established a maximum temperature of 60°C for the UAE process, with optimal yields observed at 50°C and 60°C Given that no significant differences were found between 30°C and 40°C, the optimal temperature range was determined to be 40°C to 60°C The prediction profiler indicated that 57°C yields the highest DPPH scavenging activity In contrast, the optimal temperature for DRSC during the optimization phase was notably higher than that identified in the single factor experiment The temperature's effect on total phenolic content aligns with findings from Santos et al [161].
Table 4.3 Effect of temperature on the TPC and antioxidant activities of L intermedia Yamada
The results are presented as means ± standard deviations (n = 3), with significant differences indicated by different letters (p < 0.05) The study evaluates Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), Ferric Reducing Antioxidant Power (FRAP), and Trolox Equivalents (TE), using Gallic Acid Equivalents (GAE) through the Fisher method (n = 3).
The extraction yield of total phenolic compounds (TPC) and antioxidant capacity is significantly influenced by time, as shown in Table 4.4 While longer extraction times can enhance yields, excessive duration combined with high temperature, frequency, and power may compromise the quality of the extracted compounds The data indicate no significant differences in TPC and DPPH radical scavenging capacity (DRSC), except for an unusual trend observed at 30 minutes Although TPC levels fluctuated with time, there was no impact on the Ferric Reducing Antioxidant Power (FRAP) Consequently, time was included as a factor for optimization, with a temperature range of 20-60°C selected based on the findings The prediction profiler (Figure 4.5) illustrates a decrease in DRSC from 20°C to 40°C, followed by an increase from 40°C to 60°C Overall, the effect of time on the yield and activity of TPC and FRAP was not significant, aligning with similar findings reported by Mokrani & Madani [107].
Table 4.4 Effect of time on the TPC and antioxidant activities of L intermedia Yamada Time
The study presents the results as means ± standard deviations (n = 3), indicating significant differences among groups with different letters (p < 0.05) The total phenolic content (TPC) was measured alongside the DPPH radical scavenging capacity (DRSC) and Ferric reducing antioxidant power (FRAP), expressed in Trolox equivalents (TE) and Gallic acid equivalents (GAE) using the Fisher method.
The analysis of total phenolic content reveals a significant decrease as the solid-to-solvent ratio increases, while the FRAP shows a slight decline In contrast, the DPPH Radical Scavenging Capacity (DRSC) exhibits a slight increase, with optimal yields and activities recorded at 0.1 g/10 mL for total phenolic content (TPC), 0.2 g/10 mL for FRAP, and 0.1 g/10 mL for DRSC According to mass transfer principles, a higher solvent volume enhances the concentration gradient, facilitating solute transfer from the sample to the solvent This trend is evident in the decreasing TPC and FRAP values with an increasing solid-to-solvent ratio Interestingly, the peak activity for DRSC was observed at 0.2 g/10 mL, which deviates from the optimization prediction of 0.6 g/10 mL.
Several studies have mentioned the influence of solid-to-solvent ratio similar to findings in this study [162, 163]
Table 4.5 Effect of solid-to-solvent ratio on the TPC and antioxidant activities of L intermedia Yamada
TPC (mg GAE/100 g dry sample)
DRSC (mg TE/100 g dry sample)
FRAP (mg TE/100 g dry sample)
The results are presented as means ± standard deviations (n = 3), with significant differences indicated by different letters (p < 0.05) Key metrics include Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), Ferric Reducing Antioxidant Power (FRAP), and Trolox Equivalents (TE), along with Gallic Acid Equivalents (GAE) calculated using the Fisher method (n=3).
Optimization
Table 4.6 presents the Box-Behnken design (BBD) results, highlighting a strong correlation between actual and estimated values, evidenced by R² values of 0.95, 0.97, and 0.99 for Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), and Ferric Reducing Antioxidant Power (FRAP), respectively The p-values for the lack of fit for all assays (TPC, DRSC, and FRAP) were above 0.05, indicating no significant lack of fit, with values of 0.0909, 0.0768, and 0.1951 Additionally, Table 4.5 details the estimated regression coefficients and analyses of variance for TPC, DRSC, and FRAP, confirming the reliability of the developed models in predicting antioxidant activity responses through second-order polynomial equations.
The model's fit is crucial for establishing the reliability of optimization and the predicted conditions, confirming the correlation between predicted and experimental values of total phenolic content and antioxidant activity in L intermedia Yamada To optimize parameters such as extraction temperature (X1), extraction time (X2), ethanol concentration (X3), and solid-to-solvent ratio (X4), a Response Surface Methodology (RSM) design with 27 experimental runs was utilized The statistical significance of the regression equation was assessed using p-values and R².
The R² (determination coefficient) indicates the ratio of explained variation to total variation, measuring the degree of fit For Total Phenolic Content (TPC), the model demonstrated a high R² of 0.95, with a lack of fit test result of 0.091 and a p-value of less than 0.0001, confirming model adequacy as shown in the Box-Behnken plot (Figure 4.1a) Similarly, the R² for the Dried Residue Soluble Content (DRSC) was 0.97, indicating a strong fit between actual and predicted values, with a lack of fit of 0.077 and a p-value below 0.0001, reinforcing the model's reliability (Figure 4.1b) For the Ferric Reducing Antioxidant Power (FRAP), the insignificant lack of fit (0.1951) and a p-value of less than 0.0001 also indicated a strong model fit, with an impressive R² of 0.99, reflecting a 99% correlation between actual and predicted values (Figure 4.1c).
Figure 4.1 Actual by predicted plot of Box-Behnken Design (BBD) un-coded variables for TPC (a), DRSC (b) and FRAP (c)
Table 4.6 Box-Behnken Design (BBD) with experimental versus estimated data for responses ( n =3)
X 1 X 2 X 3 X 4 Exp Est Exp Est Exp Est
The study investigates the effects of temperature (°C), time (min), ethanol concentration (%), and sample to solvent ratio (g/100 mL) on total phenolic content (TPC), DPPH radical scavenging capacity (DRSC), and ferric reducing antioxidant power (FRAP) The findings are expressed in terms of gallic equivalents (GAE) and trolox equivalents (TE), with results categorized as experimental (Exp.) and estimated (Est.).
4.3.2 Influence of extraction parameters on TPC and antioxidant capacity
Table 4.7 presents the estimated regression coefficients for the quadratic polynomial model and ANOVA analysis of the experimental results for Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), and Ferric Reducing Antioxidant Power (FRAP) The findings indicate a minor interaction effect among the variables, specifically between temperature and solvent concentration for FRAP, and concentration and solid-to-solvent ratio for DRSC Both FRAP and TPC exhibit strong linear and quadratic effects ANOVA results identify temperature, solvent concentration, and solid-to-solvent ratio as significant variables for DRSC, while temperature, time, and solvent concentration are significant for FRAP Notably, the significant variables for the linear effect of FRAP align with those of the quadratic effect, revealing similar significance for DRSC and FRAP at the quadratic level.
The study investigated the impact of four extraction parameters—temperature, time, ethanol concentration, and sample-to-solvent ratio—on the phenolic content of L intermedia Yamada Results indicated that temperature, time, and solvent concentration did not significantly affect total phenolic content (p > 0.05) However, the sample-to-solvent ratio emerged as a statistically significant factor influencing total phenolic content (TPC), with a notable increase in TPC observed when the solid-to-solvent ratio was decreased Other parameters showed no significant effects (p > 0.07) These findings align with previous research, including studies conducted by Ahmed et al.
Bamba et al reported that an increase in total phenolic content correlates with a decrease in the solid-to-solvent ratio This observation aligns with the mass transfer principle, which states that a higher solvent volume enhances the concentration gradient, thereby facilitating the transfer of solutes from the sample matrix to the solvent.
The linear (X 1 , X 3 , X 4 ), quadratic (X 1 2 , X 2 2 , X 3 2 ) and interaction (X 3 X 4 ) effects had significant impact on the DRSC (p 0.05) It was found that the DRS concentration increased as the interaction between concentration and solid-to-solvent ratio decreased, alongside the quadratic effects of temperature and ethanol concentration, which enhanced the scavenging capacity of L intermedia Yamada, as indicated by negative coefficients This finding aligns with the results reported by Ahmed et al.
The findings from the FRAP analysis indicate that the linear effects (X1, X2, X3), quadratic effects (X1², X2², X3²), and interaction effect (X1X3) significantly influence the ferric reducing antioxidant ability This suggests that these variables are the most appropriate for assessing FRAP in L intermedia.
Yamada The rest of the terms were insignificant with p-values higher than 0.05
Negative coefficients for the linear effect of ethanol concentration and its interactions with temperature and time indicate that decreases in these variables lead to an increase in FRAP value This aligns with the findings of Mokrani & Madani, who reported similar responses of FRAP under these conditions.
Table 4.7 Estimated regression coefficients for the quadratic polynomial model and analyzes of variance (ANOVA) for the experimental results for TPC, DRSC and FRAP
TPC (mg GAE/100 g dry sample)
Effects DF Estimate F-Value p-value Estimate f-value p-value Estimate f-value p-value
RMSE 9.0393 2.1092 1.833 a Stands for statistical significance (p < 0.05) TPC, DRSC and FRAP represent total phenolic content, DPPH radical scavenging capacity and ferric reducing antioxidant power, respectively GAE and TE mean gallic equivalents and trolox equivalents, respectively DF and RMSE mean degree of freedom and root mean square, respectively
The prediction profiler for the combined assays (TPC, DRSC, and FRAP) is illustrated in Figure 4.5, showcasing the optimal conditions at 50°C, 40 minutes, 37.5% ethanol, and 0.35 g/10 mL Additionally, Figures 4.2-4.4 present response surface plots that depict interactions among various factors: (a) temperature and time, (b) temperature and concentration, (c) temperature and ratio, (d) time and concentration, (e) time and ratio, and (f) concentration and ratio These graphical representations effectively highlight the relationships between the variables in the optimization process.
The 3D response surface plots for Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), and Ferric Reducing Antioxidant Power (FRAP) were created using Equation (2-4) to illustrate the interactions between various factors These graphs, generated in JMP 15 statistical software, plot responses on the z-axis against two independent variables while keeping others constant Significant interactions were observed between the ratio and solvent concentration for DRSC, and temperature and solvent concentration for FRAP An optimization study was conducted to determine the ideal extraction conditions for TPC, DRSC, and FRAP, focusing on suitable Ultrasound-Assisted Extraction (UAE) conditions for each response and their combinations Notably, some factors that enhanced phenolic content negatively impacted antioxidant activity; for example, while DRSC increased with a higher solid-to-solvent ratio, TPC significantly decreased.
Figure 4.2 Response surface plots for TPC (mg GAE/100 g dry seaweed) of L intermedia Yamada
Figure 4.3 Response surface plots for DRSC (mg TE/100 g dry seaweed) of L intermedia Yamada
Figure 4.4 Response surface plots for FRAP (mg TE/100 g dry seaweed) of L intermedia Yamada
4.3.3 Optimization and validation of the models
The optimal UAE conditions for maximum recovery of TPC from L intermedia
Yamada established optimal extraction conditions for total phenolic content (TPC), with a fixed power of 100 W, frequency of 50/60 kHz, temperature of 60°C, time of 60 minutes, 5% ethanol concentration, and a solid-to-solvent ratio of 0.1 g/10 mL, resulting in a yield of 199.16 ± 28.20 mg GAE/100 g For the DPPH radical scavenging capacity (DRSC), the best conditions were found to be 57°C, 20 minutes, 64% ethanol concentration, and a solid-to-solvent ratio of 0.6 g/10 mL, yielding 47.29 ± 5.22 mg TE/100 g Additionally, the highest values for ferric reducing antioxidant power (FRAP) were achieved at 53°C, 60 minutes, 1.95% ethanol concentration, and a solid-to-solvent ratio of 0.14 g/10 mL, resulting in 94.86 ± 4.07 mg TE/100 g.
The optimal conditions for ultrasonic-assisted extraction (UAE) of phenolic compounds and antioxidant properties from L intermedia Yamada were established with a fixed power of 100 W and frequency of 50/60 kHz, at a temperature of 50°C for 60 minutes Using a solvent concentration of 30% ethanol and a solid-to-solvent ratio of 0.2 g/10 mL, the total phenolic content (TPC) achieved was 161.79 ± 3.52 mg GAE/100 g, with a DPPH radical scavenging capacity (DRSC) of 32.30 ± 1.20 mg TE/100 g and a Ferric Reducing Antioxidant Power (FRAP) of 87.77 ± 3.17 mg TE/100 g.
Correlation between the total phenolic content and antioxidant activities
Phenolic compounds play a crucial role in scavenging free radicals, thereby shielding algae thalli from harmful UV radiation While high phenolic content is generally associated with antioxidant and antibacterial properties, this study revealed no strong correlation between total phenolic content and DPPH Radical Scavenging Capacity, indicated by an R² value of 0.865 The complexity and variety of compounds within single macroalgae species complicate the prediction of this correlation due to their interactions under different extraction conditions Previous research has documented variations in the relationship between phenolic content and antioxidant properties based on extraction parameters Notably, a strong correlation was found between Total Phenolic Content (TPC) and Ferric Reducing Antioxidant Power (FRAP), with an R² value of 0.956, suggesting that low solvent concentration (water) maximizes yield for both assays, although it negatively impacts DPPH Radical Scavenging Capacity yield Similar findings were reported by Terpinc et al in their investigation of oil cake extract's phenolic content and antioxidant capacity.
No correlation was found between phenolic content and antioxidant activity over a storage period Further research is recommended to explore the lack of correlation between total phenolic content (TPC) and DPPH radical scavenging capacity (DRSC) It is noteworthy that TPC and FRAP were measured in an aqueous solution under acidic conditions, while DPPH was assessed in ethanol.
Total phenolic content and biological activities of the fractions and isolated
The biological activities and total phenolic content (TPC) of fractions and isolated compounds, specifically aplysistatin and palisadin B from L intermedia Yamada, were evaluated, revealing a yield order of aqueous fraction (13.40%), n-hexane (2.38%), and ethyl acetate (0.20%) Notably, the ethyl acetate fraction exhibited a phenolic content yield sixteen times greater than the aqueous fraction and ten times higher than n-hexane Given that bromine is a non-polar, volatile halogenated compound, the study focused on n-hexane for isolation, as it is the most suitable non-polar solvent for containing brominated compounds, despite ethyl acetate showing superior biological activity.
Table 4.9 Biological activities of fractions and compounds from L intermedia Yamada
TPC (mg GAE/ g dry fraction)
DRSC (mg TE/ g dry fraction)
FRAP (mg TE/ g dry fraction)
TIA (mg AAE/compd Fraction) n-hexane 4.61 ± 0.13 b 0.17 ± 0.03 b 1.59 ± 0.06 b 3.65 ± 0.13 a EtOAc 46.97 ± 0.71 a 13.48 ± 0.18 a 36.95 ± 0.87 a NA
The results are presented as means ± standard deviations (n = 3), with means denoted by different letters indicating significant differences (p < 0.05) Key metrics include Total Phenolic Content (TPC), DPPH Radical Scavenging Capacity (DRSC), Ferric Reducing Antioxidant Power (FRAP), and Tyrosinase Inhibitory Activity (TIA) Additionally, values are expressed in Trolox equivalents (TE), Gallic acid equivalents (GAE), and Ascorbic acid equivalents (AAE), with "NA" indicating no activity and "ND" for not determined.
The FRAP assessment revealed that the ethyl acetate fraction had a FRAP value twenty-three times greater than n-hexane and thirty-seven times higher than the aqueous fractions Additionally, the DRSC for ethyl acetate was eighty-one times higher than that of n-hexane and fifty-three times higher than the aqueous fraction Among the three fractions, only n-hexane exhibited tyrosinase inhibitory activity (TIA), with compounds showing varying levels of TIA Aplysistatin demonstrated a significantly higher TIA (3.18 ascorbic acid/g dry compound) compared to palisadin B (2.12 ascorbic acid/g dry compound), while results for n-hexane matched those of aplysistatin However, no antioxidant activity was detected in the compounds.
Solvent fractionation plays a crucial role in separating compounds based on polarity, particularly for extracting phenolic compounds and antioxidants In this study, the ethyl acetate fraction yielded the highest levels of phenolic content and antioxidant activity, despite ethyl acetate being a less polar solvent than water This suggests that the majority of compounds in the species studied are polar, supporting the observation that lower solvent concentrations enhance phenolic content and ferric reducing antioxidant power Similar findings were reported by Hacke et al and Nguyen et al., who noted that ethyl acetate contained the highest amounts of phenolic compounds in red seaweed from L dendroidea However, Nguyen et al.'s findings on DRSC were notably lower and differed from those of the current study.
Purification and structural identification of aplysistatin and palisadin b
The n-hexane fraction of L intermedia Yamada underwent treatment with methanol to eliminate impurities Following this, the methanol extract was subjected to various chromatography techniques, resulting in the isolation of two distinct compounds The chemical structures of these isolated compounds were determined through analytical methods.
The compounds aplysistatin (1) and palisadin B (2) were identified through MS and NMR data, alongside comparisons with existing literature Figures 4.12-4.16 and 4.7-4.11 illustrate the NMR, HSQC, and COSY spectra for aplysistatin and palisadin B, respectively, showcasing the structural details of the isolated compounds.
Aplysistatin (1) and palisadin B (2) are brominated snyderane sesquiterpenes found in various Laurencia species, including L snackeyi and L luzonensis Recent research by Trung et al identified these compounds, along with 3,4-epoxypalisadin A and two forms of 2-hydroxyluzofuranone, in L intermedia Yamada from Ly Son island, Quang Ngai province, Vietnam; however, their bioactivities remain unexamined Notably, the concentration of aplysistatin in samples from Khanh Hoa province was significantly higher at 0.17% compared to just 0.015% in those from Quang Ngai, suggesting that environmental factors such as temperature and salinity may influence the levels of these brominated compounds.
Figure 4.6 Structure of aplysistatin (1) and palisadin B (2) isolated from L intermedia Yamada
The NMR spectrum of aplysistatin is shown in Figure 4.7 The resonates generated by these protons are more up field as bulk of the resonates fall between 4.5-
The NMR spectrum analysis reveals that the chemical shifts between 1.5-1 ppm indicate the presence of many chemically equivalent protons, resulting in longer peaks In the spectrum, R-OH groups are identified around 5-1 ppm, while halogens, which are of primary interest, appear between 5-3 ppm Notably, the proton resonances for palisadin, illustrated in Figure 4.12, predominantly fall between 4-1 ppm, suggesting they are located further from electronegative elements, as indicated by their shift to the right on the spectrum.
The graph indicates that R-OH groups are located between 5-1 ppm, while halogens, which are the primary focus of this analysis, are found in the range of 5-3 ppm The peak lengths suggest a concentration between 1.7-1 ppm.
Figure 4.7 1 H NMR spectrum of Aplysistatin (compound 1) in CDCl3 (500 MHz)
Figure 4.8 13 C NMR spectrum of Aplysistatin (compound 1) in CDCl3 (125 MHz)
Figure 4.9 HSQC spectrum of Aplysistatin (compound 1) in CDCl3 (500
Figure 4.10 HMBC spectrum of Aplysistatin (compound 1) in CDCl3 (500
Figure 4.11 COSY spectrum of Aplysistatin (compound 1) in CDCl3 (500 MHz)
Figure 4.12 1 H NMR spectrum of Palisadin B (compound 2) in CDCl3 (500 MHz)
Figure 4.13 13 C NMR spectrum of Palisadin B (compound 2) in CDCl3 (125 MHz)
Figure 4.14 HSQC spectrum of Palisadin B (compound 2) in CDCl3 (500
Figure 4.15 HMBC spectrum of Palisadin B (compound 2) (500 MHz/125 MHz)
Figure 4.16 COSY spectrum of Palisadin B (compound 2) (500 MHz)