MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY HASSAN IYUNADE HASSANAT OPTIMISATION OF ULTRASOUND-ASSISTED EXTRACTION CONDITIONS FOR ANTIOXIDANT AND TYROSINASE INHIBITORY
INTRODUCTION AND LITERATURE REVIEW
Introduction
Melanin, a dark pigment produced by the skin cells in the innermost layer of the epidermis, is the primary skin pigment that plays a very crucial role in shielding the human skin from ultraviolet (UV) radiation (Tu &Tawata, 2015) However, the excessive accumulation of melanin in the human skin results in hyperpigmentation disorders such as lentigo, naevus, freckles, age spots, chloasma and melanoma (Kim et al., 2012; Tu and Tawata, 2015; De Morais et al., 2018) Tyrosinase, a copper enzyme, is a vital regulator and the rate-limiting enzyme for melanogenesis (Jimenez-cervantes et al., 1993) It regulates browning reactions in fruits and vegetables Tyrosinase has also been implicated in Parkinson’s disease, a progressive neurodegenerative disorder (Hasegawa, 2010) Therefore, it is crucial to find new natural substances that can effectively inhibit the enzyme’s activity
Free radicals and other oxidants are by-products of normal physiological processes, such as cellular respiration, cell growth regulation and synthesis of biological substances They play an essential role in different physiological processes and have been found to cause a wide range of illnesses (Phaniendra et al., 2015) The excessive accumulation of free radicals in the body causes oxidative stress This phenomenon can lead to several degenerative diseases, such as diabetes, cardiovascular diseases (CVD), various types of cancer and neurological disorders (Phaniendra et al., 2015) Antioxidants are compounds that are used to control the production of these substances They act by neutralising or capturing oxidative species, thus minimising oxidative tissue damage (Rodrigues et al., 2019) Generally, natural antioxidants have considerably low toxicity in addition to being readily biodegradable, making them an effective substitute for synthetic antioxidants
Seaweeds are divided into three main classes viz; green (Chlorophytes), brown (Phaeophytes) and red (Rhodophytes) (Kumar et al., 2008) They have long been cultivated in Vietnam to be used as food, traditional medicine and more recently, ingredients for bio-industries Most brown seaweeds are so-called because they contain the pigments, fucoxanthin and various pheophycean tannins which mask other pigments, giving them their characteristic greenish-brown colour (Kumar et al., 2008) Some certain brown seaweeds have been found to possess antihypertensive, antiobesity, anticancer, antiviral, anti-coagulating and anti-hemorrhagic activities (Iwai, 2008; Zandi et al., 2010; Soares et al., 2012; Mohamed et al., 2012; Xu et al., 2012)
UAE has proved to be the most economically feasible technology, that is suitable for the extraction of bioactive compounds due to its low equipment cost, low energy requirements, low solvent consumption (Chemat et al., 2011; Dang et al., 2017) Additionally, the use of moderate temperatures makes it suitable for thermolabile compounds (Bamba et al., 2018) Some factors such as extraction time, ultrasound temperature, solvent composition, solid-to-solvent ratio, particle size of the raw material, matrix parameters as well as ultrasonic irradiations (power, frequency) affect the quality, bioactivity, and composition of extracts obtained by UAE, (Esclapez et al., 2011; da Silva et al., 2016; Bamba et al., 2018) Recently, some studies have reported the effect of key ultrasonic factors on antioxidant activity of brown seaweed species Rodrigues et al
(2015) determined the impact of UAE on the biological activities of Osmundea pinnatifid from the central west coast of Portugal Kadam et al., (2015) investigated the effect of optimisation of key parameters on the yield of total phenolics, fucose and uronic acid from Ascophyllum nodosum Garcia-Vaquero et al., (2018) studied the impact of some UAE parameters on the yields of fucose, total glucans and antioxidant activities from Laminaria digitata Dang et al., (2017) optimised UAE for maximum antioxidant activities and total phenolic content (TPC) of the H banksii No study has however been conducted on the effect of key UAE parameters on the total phenol content and antioxidant activity of Vietnamese brown seaweed species, particularly Padina australis Therefore, this study aimed to investigate the effect of UAE variables (ultrasound temperature, extraction time, solvent concentration and solid-to-solvent ratio) on the total phenol content and antioxidant activities and to optimise the UAE variables to obtain high yields of phenolic compounds, antioxidant and tyrosinase inhibitory activities of Padina australis
The overproduction of melanin in different specific parts of the skin can result in hyper-pigmentary disorders, and aesthetic problems in humans (Chang, 2009) Melanosis, one of the most common forms of acquired hyperpigmentation, is characterised by irregular brown patches on sun-exposed skin It often affects the populations with darker skin complexion, with greater severity and higher frequency in
Hispanics and Asians (Stratigos & Katsambas, 2004) In these populations, there is an increasing demand for skin whitening agents and other related cosmetics product
Several tyrosinase inhibitors have been reported from natural sources, but only a few of them are used as skin-whitening agents, primarily due to various safety concerns For example, linoleic acid, hinokitiol, kojic acid, arbutin, naturally occurring hydroquinones, and catechols were reported to inhibit enzyme activity but also exhibited side effects Therefore there is the constant search for cosmetic products that are not only safe but contain biologically active ingredients that possess medical or drug-like benefits in addition to their skin whitening effect
This research seeks to answer the following questions:
Does Vietnamese brown seaweed contain phenolics and possess antioxidant and tyrosinase inhibitory activities?
What UAE optimisation condition is best for maximum yield of phenolics and antioxidant activity?
Brown seaweed species may possess tyrosinase inhibitory activity
Brown seaweed species extracted by UAE may possess high phenolics, antioxidant and tyrosinase inhibitory activities
Extraction of Vietnamese brown seaweed bioactive compounds using UAE will improve the tyrosinase inhibitory activity, thus making it suitable for use in the cosmeceutical and food industry
The main objective of this study is to investigate the phenolic content, antioxidant and tyrosinase inhibitory activities of some brown seaweed species harvested in Vietnam
To screen five Vietnamese brown seaweed species based on their phenolics and antioxidant activity in order to select the starting material
To optimise key UAE parameters to obtain high yields of phenolics and antioxidant activity from selected brown seaweed
To fractionate crude extract of selected brown seaweed obtained from optimised UAE conditions with different solvents
Literature review
The marine environment covers over 70% of the earth’s surface and provides a broad range of highly ecological, chemical, and, biological diversity starting from microorganisms to vertebrates (Karupanan & Sutlana, 2017) One of these organisms is marine algae, one of the largest producers of important marine living, renewable
FRACTIONATION solvents (ethyl acetate, n-hexane, water) Tyrosinase inhibitory activity, TPC, DPPH, FRAP
UAE (Time, temperature, solvent concentration, solid-to-solvent ration) TPC, DPPH, FRAP
UAE (Time, temperature, solvent concentration, solid-to-solvent ration) TPC, DPPH, FRAP
Conventional extraction TPC, DPPH, FRAP resources (Saranraj et al., 2014) Marine algae are a diverse group of oxygen- generating, chlorophyll-containing, photosynthetic organisms which inhabit the marine environment and are classified as either microalgae or macroalgae Microalgae are small (àM) unicellular organisms while macroalgae, on the other hand, are large, multicellular organisms
Seaweeds are macroscopic algae found freely floating or attached to the bottom of rocks, dead corals, pebbles, shells and, other plant materials (Rindi et al., 2012; Pati et al., 2016) They are so-called because of their abundance in seas and oceans They are found in relatively shallow coastal waters, estuaries, intertidal and deep-sea areas up to 180 meters depth (Pati et al., 2016) They are primitive plants with little tissue differentiation, no roots, no true vascular tissues, stems or leaves, and flowers (Chia, 2010; Zandi et al., 2010) They belong to the division of Thallophyta in the plant kingdom They are not only frequently classified based on their photosynthetic pigments but also by differences in many ultra-structural and biochemical features including the type of storage material, cell wall composition, presence/absence of flagella, and the fine structure of the chloroplasts (Rindi et al., 2012; Mekinić et al.,
2019) Based on these features, marine algae are broadly classified into four groups namely Chlorophyceae (green algae), Phaeophyceae (brown algae), Rhodophyceae (red algae), and, Cyanophyceae (blue-green algae) (Lavanya et al., 2017) The group brown, green, and, red algae consist of approximately 1,800 1,200 and 6000 species, respectively
Seaweeds contain a large variety of beneficial compounds, and for this reason, they have been used in many parts of the world as a source of essential nutrients and ingredient in cosmeceutical and industrial applications They are also known to be an extremely rich source of biologically active substances with health-promoting ability (Wijesinghe & Jeon, 2011) Their biological activities are correlated to the presence of secondary metabolites, present as the plants’ defence mechanism against extreme marine environmental conditions These bioactive compounds include soluble polysaccharides, sulfated polysaccharides, carotenoids, omega-3 fatty acids, vitamins, tocopherols, and, phycocyanins and have been extracted using different extraction techniques Figure 1.2 shows the structure of some seaweed
Figure 1.2 Structure of some marine algae
Vietnam’s coastline is estimated to be around 3260 km with her climate varying from subtropical in the northern part which gradually becomes tropical in the southern part of the country (Hong et al., 2007) These physical and climatological characteristics are suitable for the cultivation of seaweed Vietnam has an abundance of algae floral with the total number of species estimated to be nearly 1000 spp with about 827 species already identified (Tu et al., 2013) The traditional Vietnamese coastal people have harvested and utilised seaweeds for over one hundred years However, the use of seaweeds is limited to people living in coastal areas (Hong et al.,
2007) Seaweeds play a vital role as a source of food and ingredients in traditional Vietnamese medicine Vietnamese consume seaweed as fresh vegetables, salad, soups, or snacks Apart from food uses, seaweeds are widely used as ingredients in various industries such as cosmetology, pharmaceutical, animal feed, and fertiliser industries The schematic representation of the common application of seaweed in Vietnam is shown in Figure 1.3
Large scale commercial cultivation for Harvesting in the wild for industrial industrial and traditional applications and traditional applications
Figure 1.3 Common applications of seaweed (Sanjeewa & Jeon, 2018)
Brown algae (Phaeophyceae) have received interest from researchers due to their large variety of bioactive compounds and nutritional value The brown algae are described as the largest and most diverse class of macroalgae They range from small filamentous forms to large, complex seaweed (Rindi et al., 2012) Most brown seaweeds are so-called because they contain the xanthophyll pigment viz., fucoxanthin and various pheophycean tannins which mask other pigments such as chlorophyll a and chlorophyll b, thus giving them their characteristic greenish-brown colour (Kumar et al., 2008)
All members of the phaeophyceae are multicellular, none are unicellular in the vegetative phase, this is unlike other groups in the same phyllum (Wehr, 2015) Most species have an alternation of haploid and diploid generations, which may be either isomorphic or heteromorphic (Wehr, 2015) Many are macroscopic seaweeds with complex tissues and reproductive structures, although many simpler filamentous forms exist There are approximately 1,800 species in this class of macroalgae
Figure 1.4 Macroscopic appearance of freshwater brown seaweed (Wehr, 2015) 1.2.4 Major antioxidant constituents of brown seaweed
Oxidative stress in humans has been found to be the result of an imbalance between the homeostasis of prooxidants and antioxidants thus leading to the generation of free radicals and other toxic reactive oxygen or nitrogen species (ROS and RNS) (Vadlapudi et al., 2012) Researches done over the past two decades have demonstrated that oxidative stress is involved in the pathogenesis and pathophysiology of many chronic diseases and inflammation (Blomhoff et al., 2006) Antioxidants, also known as free radical scavengers, in simplest terms are defined as redox-active compounds that can reduce oxidative stress by reacting non-enzymatically with free radicals and reactive oxygen or nitrogen species thus helping the body maintain normal physiological balance Epidemiological evidence has shown that high antioxidant activity in plant materials are highly correlated with their potential health benefits
Studies of the antioxidant constituents of brown seaweed have been conducted over the years and have been primarily classified as pigments, polysaccharides, polyphenols, vitamins, and enzymes (Holdt & Kraan, 2011) The bioactive compounds all exhibit some degree of antioxidant, prooxidant or synergistic activity The following section will discuss the properties of fucoidans, carotenoids, polyphenolic compounds and enzymes found in brown seaweed and their influence on the antioxidant efficacy of brown seaweed extracts
Brown seaweed contains a diverse number of photosynthetic pigments such as carotenoids and xanthophyll, which are responsible for its characteristic golden-brown colour and serve as an important source of antioxidant Fucoxanthin, a xanthophyll, found in the chloroplast of algal cells is the primary biologically active pigment present in brown seaweed It accounts for more than 10% of the estimated total production of carotenoids in the marine environment (Peng et al., 2011) Fucoxanthin, shown in Figure 1.5, has been found to act as an antioxidant under anoxic conditions, i.e in the presence of meagre amounts of oxygen typical of most animal tissues under normal physiologic conditions, thus making it a potent free radical scavenger (D’Orazio et al., 2012) Industrially
Undaria pinnatifida is the most widely utilised brown seaweed for fucoxanthin extraction of because of the high concentration of the pigment in its lipid extract (Abu-Ghannam & Shannon, 2017) It has also been extracted and isolated from other species like Undaria pinnatifida, Hijikia fusiformis, Laminaria japonica, Undaria pinnatifida, and, Sargassum fulvellum ( Roh et al., 2008; D’Orazio et al., 2012;) Studies have shown that brown algae pigments possess anti-cancer, anti-obesity, and, anti-diabetic properties which have been found to be mainly because of their antioxidant capacity (Gammone & D’Orazio, 2015; Usoltseva et al., 2018)
Figure 1.5 Chemical structure of fucoxanthin (Abu-Ghannam & Shannon, 2017)
Seaweeds contain a significant amount of soluble polysaccharides and have the potential function as dietary fibre They are the main component of seaweeds, constituting about 70% of their dry weight (Holdt & Kraan, 2011) The polysaccharide content of some major brown seaweed is presented on Table 1.1 Biologically, polysaccharides function to provide structure to the plants and physically support their thallus in water (Venugopal, 2019) They also act as antioxidants thus protecting the plant from oxidative stress Polysaccharides consist of several units of monosaccharides linked together by glycosidic bonds and they usually differ from one another by the number of glycosidic bonds, type of functional group, type of branching, molecular weight, and, chain length In a broad sense, algae polysaccharides are classified as sulfated and non-sulfated Sulfated polysaccharides include fucoidans, agar and carrageenans and ulvans, while the most commercially important non-sulfated polysaccharide is alginate Fucoidan, laminarin, and, alginic acid are the significant polysaccharides found in brown seaweed (Okolie et al., 2017)
They are a group of fucose-rich, sulfated polysaccharides, consisting of α- linked l-fucose residues having various substitutions (Ale & Meyer, 2013) They are found in the cell wall of cell walls of brown seaweeds including Sargassum spp and Fucus vesiculosus (Venugopal, 2019) They are water-soluble and are constituted mainly of sulfated α-L-fucopyranose residues and may contain monosaccharides such as glucose, mannose, galactose, xylose and some acetylated groups (Kumar et al.,
2008) The chemical structure of fucoidan shown in Figure 1.7, differ according to the seaweed species, but they are mainly composed of fucose and sulphates
Tyrosinase
Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is a multifunctional copper- containing enzyme prevalent in plants and animal kingdom (Chen et al.,, 2016) It is the key enzyme that regulates the process of melanogenesis within special organelles, in the melanocytes and melanoma cells (Zolghadri et al., 2019) The melanin produced during melanogenesis determines the colour of the skin, in addition to protecting the human skin from harmful UV radiation, however, the overproduction and accumulation of melanin pigments in the skin lead to a number of dermatological conditions such as solar lentigo, melisma and, linea nigra (Chang, 2009) Tyrosinase is also reported to be a cause of Parkinson’s disease, a progressive neurodegenerative disorder characterised by selective degeneration of dopamine (Hasegawa, 2010) Furthermore, tyrosinase is also responsible for enzymatic browning reactions in fruits and vegetables Browning usually damages the colour attribute of plant-derived food products, this may indicate spoilage of its nutritional quality Therefore, research on new agents, particularly natural products that inhibit the tyrosinase activity and that are effective in the treatment of hyperpigmentation and retardation of enzymatic browning, is of utmost relevance
1.3.1 Role of tyrosinase in melanogenesis
Melanogenesis refers to the entire process leading to the formation of dark macromolecular pigments, i.e., melanin (Chang, 2009) Melanin consisting of two types, pheomelanin and eumelanin, is formed by a combination of enzymatically catalysed and chemical reactions The biosynthetic pathway for melanin formation in various life forms is called the Raper-Mason pathway as shown in Figure 1.11 This pathway is initiated by the conversion of monophenol L-tyrosine to L-3,4- dihydroxyphenylalanine (L-dopa) and the subsequent oxidation of L-dopa to the L dopaquinone, the resulting quinone then serves as a substrate for the formation of melanin resulting in the development of brown or black colour in human skin and browning reactions some plants (Zolghadri et al., 2019) This step is a rate-limiting process as other reactions can proceed under normal physiological conditions The dopaquinone produced by tyrosinase from the first stage is converted into dopa and dopachrome by autoxidation The reaction products of dopachrome, dihydroxyindole (DHI) and dihydroxyindole-2-carboxylic acid (DHICA) are finally oxidised and polymerised to form eumelanin (brown-black pigment) while dopa, a substrate for tyrosinase is converted back to dopaquinone (Chang, 2012) The dopaquinone is converted to cysteinyldopa or glutathionyldopa in the presence of cysteine or glutathione Pheonelanins (red-yellow) pigments are formed through the conjugation of cysteinyldopa or glutathionyldopa (Pillaiyar et al., 2017) Mixed-type melanins are formed from the interaction between the eumelanin and pheomelanin
Figure 1.11 The pathway for melanin synthesis in mammals (Islam, 2018)
Fruits, fungi and vegetables: Melanin pigments are widespread in fungi, although melanogenesis is restricted to specific developmental stages on mycelium, sporulation, or defensive reactions to wounding (Solano, 2014) Fungal melanin is quite abundant and appears in the cell wall rather than in specialised subcellular organelles such as the animal melanosomes The enzymatic activity of tyrosinase in fruits and vegetables results in the production of melanin pigments, consequently leading to enzymatic browning During peeling, crushing and milling operations, the cell structure is disrupted, leading to the mixture of tyrosinase and its polyphenolic substrate thus, resulting in enzymatic browning This results in undesirable changes in the nutrient composition, shelf-life and loss of customer acceptance in fruits and vegetables Enzymatic browning is one of the major concerns in the food industry Figure 1.11 shows enzymatic browning in banana
Figure 1.12 Enzymatic browning in banana
Human skin: in humans, melanin is responsible for the colour of the skin, hair and eye Melanocytes, special pigment-producing cells located in the deep layer of the human skin are responsible for the production of melanin These cells originate during embryogenesis and are distributed throughout the organism during development (Dolorosa et al., 2019; Radhakrishnan, 2016) The main action of melanin in human skin appears to be attenuation of UV penetration to blood in dermal vessels Thus high melanin content (racial pigmentation) protects the skin against ultraviolet (UV) induced skin damage through its optical and chemical filtering properties Melanin formation (through browning reactions) in human skin occurs in the presence of a catalyst (a tyrosinase enzyme form) and UV light
The overproduction of melanin in different specific parts of the skin can result in hyperpigmentation an esthetic problem in humans (Chang, 2009) Hyper-pigmentary disorders such as actinic and senile lentigines, melasma and post-inflammatory hyperpigmentation are major cosmetic problems, and sufferers often pursue medical advice Melanosis is one of the most common forms of acquired hyperpigmentation, characterised by irregular brown patches on sun-exposed skin These conditions affect the populations with darker skin complexion, particularly Hispanics and Asians with greater severity and frequency (Stratigos & Katsambas, 2004)
It is one of the most researched tyrosinase inhibitors and is widely used positive control in tyrosinase inhibitory assays Kojic acid is a fungal metabolite, currently functional as a cosmetic skin-whitening agent and as a food additive for preventing enzymatic browning Kojic acid inhibits mushroom tyrosinase through competitive and mixed modes of inhibition on monophenolase activity and diphenolase activity respectively (Chang, 2009) Its uses are however restricted due to its severe side effects (Burnett et al., 2010)
This is able to inhibit both the mono- and di-phenolase activity of the enzyme tyrosinase It is a slow-binding inhibitor that is able to form complexes with the enzyme thus inhibiting it Tropolone binds near the binuclear copper site without directly coordinating the copper ions
Hydroquinone, a ubiquitous molecule, is a hydroxyphenol, which is naturally present in plants and foods, such as coffee, cranberries and blueberries Because of its structural analogy with tyrosine (TYR), HQ can act as an alternative substrate for
TYR This reaction generates ROS, which are thought to be responsible not only for some of the side-effects seen with HQ, but also oxidative damage of TYR, which explains the skin lightening effect It has been used for depigmentation purposes since the 1960s but its use is highly controversial because of its perceived side effects
Ascorbic acid or vitamin C has been known over the years to play a vital role in helping to maintain skin health and reducing melanin production (Wang et al., 2018)
It was extensively utilised as tyrosinase inhibitors because of its anti-browning effect as it has been found to be an effective antioxidant Ascorbic acid causes chemical reduction of dopaquinone, and reduces o-dopaquinone to L-DOPA, thus avoiding the formation of dopachrome and melanin (Chang, 2009)
Figure 1.13 Chemical structures of a) L-ascorbic acid b) Kojic acid c) Tropolone d)
1.3.4 Potential Applications of Tyrosinase Inhibitors from Brown Seaweed
One of the attributes that are considered by consumers when they choose a food product is appearance Amongst the characteristics that define appearance, colour is a critical determinant for the appearance of fruits, vegetables, and crustaceans Browning usually impairs the colour attribute together with sensory properties such as flavour and texture (softening) As discussed earlier, enzymatic browning of fruits, vegetables, and beverages takes place in the presence of oxygen when tyrosinase and its polyphenolic substrates are mixed after brushing, peeling, and, crushing operations, which lead to the rupture of cell structure The current conventional techniques to avoid browning include the use of autoclave and blanching or other heat treatment methods to inactivate tyrosinase, but these processes cause important weight and nutrient losses in the product Various chemicals such as halide salts, aromatic carboxylic acids and other compounds with reducing properties such as sulfite, citric acid, ascorbic acid and its derivatives are known to inhibit tyrosinase However, the effect of ascorbic acid, sodium bisulfate and other reducing agents on tyrosinase has been controversial over the years, moreover, the use of sulfites is becoming more and more restricted due to potential health hazards
Since safety is of prime concern for an inhibitor to be used in the food industry, there is a constant search for better inhibitors from natural sources as they are largely free of any harmful side effects, brown seaweed is a potential source of these inhibitors Brown seaweed extract is rich in many phenolic and other bioactive compounds that are considered to possess the ability to inhibit tyrosinase When used as inhibitors in the food industry, in addition to retarding the enzyme tyrosinase and inhibiting browning, they have the ability to supply consumers with added nutritional benefits
The term cosmeceutical, derived from ―cosmetic and pharmaceutical‖ is used to describe a cosmetic product that contains biologically active ingredients thus exerting therapeutic benefits (Choi & Berson 2006) Cosmetics are products that are used to enhance, alter or maintain the appearance of the human hair and skin (Pereira, 2018)
There is presently a wide range of cosmetics marketed and sold worldwide however, because of the complex relationship between the human skin and cosmetics, the toxicological safety of these products and their ingredients has attracted increasing attention Like food, there is an increasing demand for personal care products that contain natural ingredients and pose no side effects Linoleic acid, hinokitiol, kojic acid, arbutin are some natural tyrosinase inhibitors, however, their use has been associated with a number of side effects therefore, there is a constant search for natural and safe ingredients from natural sources that will exhibit no adverse effect on the skin or health
Cosmeceuticals as stated earlier contain biologically active ingredients that provide health benefits such as anti-aging, anti-wrinkling, in addition to their skin altering or enhancing effect There is an increasing number of novel cosmeceutical products with functional ingredients such as fucoidan, fucoxanthin, and alginate from brown seaweeds Since these compounds are from marine resources, they present an ideal addition as ingredients for cosmeceutical products (Fitton et al., 2007) The use of these brown seaweed extracts is in line with the recent trend for natural product formulations.
Extraction of bioactive compounds from seaweed
The extraction of bioactive compounds from plant or animal materials is a crucial food technology process and several methods have been developed and well exploited over the years for this purpose Simple conventional extraction is the most popular of these methods, however, it has been found to be time consuming, laborious, in addition to consuming very high volumes of high purity solvents and having low extraction efficiency (Kadam et al., 2015) Innovative and novel technologies for the extraction of bioactive compounds from biological materials have been developed to mitigate the limitations of conventional extraction methods These methods include enzyme-assisted extraction (EAE) Supercritical-fluid Extraction (SFE), microwave- assisted extraction (MAE) and, ultrasound-assisted extraction (UAE) (Sosa-Hernández et al., 2018) These novel techniques are regarded as ―green‖ technologies as they comply with standards set by the U.S Environmental Protection Agency (EPA) (García‐Pérez et al., 2017) UAE has proven to be the most economically feasible method amongst these novel techniques due to its low equipment cost, low solvent consumption, low energy requirements and use of moderate temperatures thus making it suitable for use on thermo-labile compounds (Chemat et al., 2011; Dang et al., 2017; Bamba et al., 2018)
The term ultrasound can be described as acoustic waves with frequencies in within a threshold that exceeds those audible to the human ears High-frequency and power (high-intensity) ultrasound are two main approaches to the use of UAE in food processing, these approaches are based on the differences in frequency and sound intensity of the process (Bimakr et al., 2017) UAE, regardless of the approach, takes advantage of acoustic or sonic waves since they are able to travel through the solvent in contact with a material thus producing cavitation bubbles (Al Jitan et al., 2018) The rise and fall of these bubbles causes pressure and thermal changes which in turn reduce particle size, cause cell wall destruction and improve the rate of mass transfer of biological compounds from cellular membranes of the material into the solvent (Wen et al., 2018; Pacheco-Fernández & Pino, 2019) The cavitation bubbles can also induce chemical changes within the system by modifying existing chemical reactions or initiating new ones by the formation of free radicals (Esclapez et al., 2011) The major advantage of UAE over other methods is its inexpensiveness, simplicity and effectiveness The schematic diagram of a typical ultrasound equipment is shown in Figure 1.15
There are several variables that directly or indirectly affect the effectiveness and yield of a UAE process, they include the approach used, the design of the system (ultrasonic probe or ultrasonic bath), ultrasound process parameter (amplitude, frequency, or power), particle size of the material, and extraction process parameters such as time, temperature, solvent concentration etc The particle size of a material greatly influences the diffusion of solvent into its solid matrix due to an increase in the surface area of contact thus affecting the extraction yield and efficiency (Medina-Torres et al., 2017) The choice of the extraction solvent affects the yield of bioactive compounds from a biological material Water is often used as the extraction solvent of choice for UAE because of its high polarity, availability and low cost, however, other volatile solvents, such as ethanol and methanol have been used in combination with water to achieve high yields Temperature is one of the most important factors that affect the effectiveness of a UAE process, an increase in this extraction parameter is directly proportional to an improvement in extraction yield Solid-to-solvent ratio is also a vital parameter as it helps determine the diffusion rate of the solvent, lower solid-to-solvent ratio will imply higher diffusion of solvent in the materials solid matrix and consequently an improvement in the extraction efficiency and yield ( Muủiz-Mỏrquez et al., 2013; Medina-Torres et al., 2017) Extraction time is also a crucial factor, the amount of time the material stays in contact with the extracting solvent will determine the effectiveness of the process
As discussed earlier, a number of important factors affect the efficiency and yield of a UAE process, perhaps, what is more important is the interaction between this factors, hence the importance to optimise a UAE process Optimisation is usually done to determine the best combination of process conditions for an effective food processing method, in this case, UAE process RSM is a widely used method for optimising experimental factors such as temperature, time, frequency, power etc that directly affect a process It uses regression analysis to determine the impact of each parameter (independent variable) on the experimental response (dependent variable), the interaction between these parameters and uses mathematical models in form of second order polynomial equations to predict optimum extraction conditions, the reliability and repeatability of the process Amongst the different methods of RSM, Box-Behnken design is the commonly used form (Medina-Torres et al., 2017), its main advantage is the small number of experimental trials or runs needed to evaluate the effect of several process parameters
Figure 1.14 Schematic representation of an ultrasound equipment (Rojas et al., 2016)
MATERIALS AND METHODS
Materials
Five brown seaweed species (Sargassum mcclurei, Turbinaria ornate, Sargassum swartzi, Padina australis and Sargassum duplicatum) were freshly harvested from the Nha Trang Bay, Nha Trang, Vietnam in May 2020 They were washed lightly to remove salt, sand and epiphytes and then sun-dried for six hours The dried samples were pulverised into fine powder (0.75mm) and stored at -20 C for further investigation
Folin-Ciocalteu reagent, 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), mushroom tyrosinase were purchased from Sigma-Aldrich, (USA) L-tyrosine, 2,2-Diphenyl-1- picrillhydrazyl (DPPH) were purchased from Alfa Aesar (UK) All other chemicals and reagents used were of analytical grade.
Experimental design
The extracts of five brown seaweed species shown in Figure 2.2, were prepared by conventional extraction and then investigated for their TPC and antioxidant power The sample that contained the highest level of phenolics and antioxidant capacity was selected for further experiments Conventional extraction was done according to the method described in a previous study (Tierney et al., 2013) with some alterations 1g of dried seaweed was added to 30 ml of 80% ethanol (1:30 w/v) and placed in a shaking water bath at 60 °C for 2 hours, upon the completion of extraction, the samples were immediately cooled on ice to terminate the extraction process The suspension was filtered through Whatman No 1 filter paper to remove the residue, the supernatant was collected and kept at -20 C for further analysis
This was done as a preliminary test to serve as a guide for the optimisation using RSM In the test, the influence of four factors including temperature (X1), time (X2), solvent concentration (X3), and solvent ratio (X4) on the total phenol content, DPPH radical scavenging ability and ferric reducing antioxidant power was determined To investigate the influence of these factors on the responses, one factor was varied while the others were kept constant
To establish the range for ultrasonic temperature, samples were extracted in 80% ethanol at different extraction temperatures (30, 40, 50 and 60 C) for 30 minutes with a sample-to-solvent ratio of 1:30 g/ml in an ultrasonic bath (Brasonic ultrasonic bath, Model 2510E – DTH, 42kHz, United States) After ultrasonic extraction, the extracts were immediately cooled on ice to room temperature and centrifuged at 8500 rpm for
20 minutes The supernatant was collected after centrifugation and used for further quantitative analysis The range of ultrasound temperature was selected based on the yield of phenolics and antioxidant activities of the extracts and was employed for the optimisation process
To determine the parameter of ultrasonic time, samples were extracted in 80% ethanol for a range of extraction time from 20 to 90 min at the suitable ultrasonic temperature selected from the previous experiment with a sample-to-solvent ratio of 1:30 g/ml The range of extraction time was selected based on the yield of phenolics and antioxidant activities of the extracts and was employed for the optimisation process
The suitable ultrasonic temperature (50 C) and time (60 min) obtained in previous experiments were employed to determine the range of ethanol concentration (0,
20, 40, 60, 80 and 100%) Similarly, the range of ethanol concentration was chosen based on the TPC and antioxidant activities of the extracts, and used for the subsequent optimisation experiments
Regarding the sample-to-solvent ratio range, 40% ethanol was used as the extraction solvent with the ratios of sample-to-water of 1/100, 1/80, 1/60, 1/40, 1/20 and 1/10 (g/ml) at temperature of 50 °C for 60 min The sample-to-solvent range was chosen based on the TPC and antioxidant activities of the extracts and used for optimisation experiments
The ultrasonic extraction conditions were optimised using RSM with Box-Behnken design The ranges of temperature (40 – 60 °C), time (50 – 80 min), ethanol concentration (0 – 60%) were determined based on the single factor experiments A total of 27 experiments were carried out in random order Regression analysis was performed to fit the following quadratic polynomial model:
(1) where β 0 is a constant intercept, and β i , β ii , and β ij are linear, quadratic, and interactive regression coefficients of the model, respectively and k is the number of variables
2.2.4 Preparation of crude extract and fractions
UAE was performed in an ultrasonic apparatus (Brasonic ultrasonic bath, Model 2510E – DTH, United States) with a working frequency and power fixed at 50/60 kHz and 100W, respectively The dried seaweed was extracted using optimal extraction conditions found in previous experiments (ultrasonic temperature of 60 C, ultrasonic time of 60 min, solvent concentration of 60% (v/v) aqueous ethanol) with the solid-to- solvent ratio of 2:100 g/ml After ultrasonic extraction, the extracts were immediately cooled on ice to room temperature and centrifuged at 8500 rpm for 20 minutes (HERMLE Z 323, made in Germany) The supernatant was collected after centrifugation and vacuum evaporated (45C) to remove the solvent The crude extract of the sample was fractionated using n-hexane and ethyl acetate in the order of their polarities After exhaustive removal of each solvent fractions, they were condensed in a rotary evaporator (LABOTA 4001 OB, Heidolph, Germany) and stored at -20 C until further analysis The flow chart of the process is shown in Figure 2.1
Figure 2.1 Flow chart for UAE
Time Temperature Solvent concentration Solid-to-solvent ratio
Solvent fractionation (EA, n-hexane, water)
Figure 2.2 Brown seaweed samples (a) Sargassum mcclurie (b) Turbinaria ornata (c)
Sargassum duplicatum (d) Sargassum nipponicum (e) Padina australis
Preparation of calibration curve of gallic acid
The calibration curve of gallic acid was determined by Folin-ciocalteu reagent Dilute Folin-coicalteu reagent (10% v/v) was first prepared by adding 10 ml of FC solution into 90 ml of distilled water Different concentrations of gallic acid solution
(10, 20, 30, 40, 50, 75, 100 àg/ml) were prepared by serial dilution and 0.5 ml each taken into clean test tubes Reagent blank using distilled water was also prepared 2.5 ml of 10% Folin was added and allowed to stand for 8 minutes before the addition of 2 ml 7.5% sodium bicarbonate (Na2CO3) solution The mixture was mixed thoroughly on a vortex vibrator and allowed to incubate for 1 hour at room temperature The absorbance of the samples against reagent blank was determined at 765 nm using UV- Vis spectrophotometer The linear regression equation was calculated, with sample concentrations as the X coordinate axis and the absorbance of the sample solution at
765 nm as the Y coordinate axis
TPC was determined according to the method of Vuong et al (2013) Briefly, 0.5 ml of the diluted sample, 2.5 ml of 10% (v/v) Folin–Ciocalteu reagent was added into a clean test tube, followed by the addition of 2 ml of Na2CO3 7.5% (w/v), the solution was mixed well on a vortex vibrator for 2 min and incubated at room temperature (RT) for 1 hour The absorbance of the resulting blue-coloured solution was measured at 765 nm using UV-Vis spectrophotometer (LIBRA S50) Gallic acid was used as a standard, and the results expressed as mg of gallic acid equivalents per gram of dried material (mg GAE/gdm)
Preparation of calibration curve of trolox for DPPH
DPPH stock solution was first prepared by dissolving 0.024 g of DPPH in 100 ml of methanol Working DPPH solution was obtained by mixing 10 ml of the stock solution in 45 ml of methanol, the absorbance value of the solution was adjusted to 1.1 at 515 nm, on a UV-Vis spectrophotometer Different concentrations of trolox standard (12.5, 25 50, 100, 200, 400, 500, 800, 100 àM) were prepared by serial dilution 0.15 ml of each concentration was taken into clean test tubes; 2.85 ml of the DPPH working solution was added Reagent control was prepared with methanol The resulting deep violet coloured solution was shaken vigorously on a vortex vibrator and allowed to stand in the dark for 3 hours The absorbance of the solution was taken at
515 nm on a UV-Vis spectrophotometer The linear regression was calculated, with sample concentrations as the X-axis and sample absorbance as the Y-axis
Determination of DPPH radical scavenging ability
The effect of the seaweed crude extracts and their fractions on 1,1-diphenyl-2- picrylhydrazyl (DPPH) was examined as described by Yen & Chen (1995) 2.85 ml of 0.16 mM DPPH solution in methanol was added to the test tube containing 0.15 ml aliquot of the diluted sample The mixture was mixed thoroughly on a vortex vibrator for 1 min and kept in the dark, at room temperature for 3 hours The absorbance of all the sample solutions was then measured at 515 nm on a UV-Vis spectrophotometer (LIBRA S50) Results were compared with the standard curve of Trolox and expressed as mg of trolox equivalents per gram of dried material (mg TE/gdm)
Preparation of calibration curve of trolox for FRAP
Acetate buffer (pH 3.6) was first prepared by dissolving 3.1 g of sodium acetate trihydrate in 16 ml of glacial acetic acid The volume of the resulting solution was made up to 1 L with distilled water 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) reagent was prepared by diluting 0.3123 g of TPTZ in 100 ml of dilute hydrochloric acid (HCl) Ferric chloride reagent was also prepared by dissolving 5.406 g of ferric chloride in 1
L of distilled water Working FRAP reagent was prepared by mixing Acetate buffer, TPTZ and ferric chloride reagents in the ratio of 10:1:1 Different concentrations of trolox standard (12.5, 25 50, 100, 200, 400, 500, 800, 100 àM) were prepared by serial dilution 0.15 ml of each concentration was taken into clean test tubes, 2.85 ml of the FRAP working solution was added Reagent blank was prepared with distilled water The solution was mixed thoroughly on a vortex vibrator and allowed to incubate in the dark for 30 minutes after which the absorbance of the resulting blue coloured solution was taken at 593 nm on a UV-Vis spectrophotometer Linear regression was calculated by plotting a graph of sample concentrations against the sample absorbance
Determination of ferric reducing antioxidant power (FRAP)
Statistical analysis
RSM experimental design and analysis was conducted using JMP software (Version 15) The software was also used to establish the model equation to graph the 3D surface and 2D contour plots of variable response and to predict optimum values for the three response variables All measurements were taken in triplicate, and results expressed as mean values ± standard deviations One-way analysis of variance and Tukey’s post hoc test using SPSS version 16 was used to determine any significant difference (p 0.05) Similar results were obtained by O’Sullivan et al
(2011) in five Ireland brown seaweed species, where the total phenolic content of ranged from 1.5 (Laminaria hyperborea) to 4.5 mg GAE/gdm (Ascophyllum nodosum) Chandini et al., (2008) found much lower phenolic contents of 0.29 and 0.86 mg GAE/gdm in two Indian brown seaweeds, Sargassum marginatum and Turbinaria conoides, respectively However, higher phenolic contents of 24.30 mg GAE/gdm
(Padina antillarum) and 33.8 mg GAE/gdm (Dictyopteris membranacea) were reported by Chew et al., (2008) and Aoun et al., (2010), respectively Mekinić et al., (2019) has previously reported that phenolic content varies considerably depending on the seaweed species
DPPH assay’s principle is based on the scavenging ability of the antioxidant towards DPPH, a stable free radical It is the most common used assay as it provides a rapid and straightforward way to determine the antioxidant capacity of samples by spectrophotometry (Garcia et al., 2012) The DPPH scavenging capacity of the extracts were in the following order Turbinaria ornata > Padina australis >
Sargassum mcclurei > Sagassum nipponicum > Sargassum duplicatum The highest
DPPH scavenging capacity was found in Turbinaria ornata (5.81 mg TE/gdm) followed by Padina boryana at 5.63 mg TE/gdm The DPPH scavenging activity of the ethanolic extracts of all the seaweed species were significantly different (p < 0.05) The high DPPH scavenging capacity observed in Turbinaria ornata may be attributed to its being rich in quercetin and salicylic acid (Chakraborty & Joseph, 2016)
Table 3.1 The TPC and antioxidant capacity of five different brown seaweed species
Antioxidant capacity (mg TE/gdm)
The results are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents
The ferric reducing antioxidant power (FRAP) mechanism is based on the ability of the extract to reduce Fe 3+ to Fe 2+ in a solution of 2,4,6-trypyridyl-s-triazine (TPTZ) (Cerretani and Bendini, 2010) The FRAP varied between 3.06 and 6.93 mg TE/gdm The highest FRAP was found in the crude extract of Padina australis extract with 6.93 ± 0.19 mg TE/gdm, followed by Padina australis with 5.24 ± 0.35 mg TE/gdm while the extract from Sargassum duplicatum had the lowest FRAP In this study, the FRAP of the brown seaweed species were significantly different (p < 0.05) Overall, the result shows that higher total phenolic content resulted in higher antioxidant capacity, indicating that the phenolics of some selected brown seaweed species have close correlation with their antioxidant power Padina australis was selected for further analysis since its extract had the highest amount of TPC and FRAP.
The effect of single factors on the TPC and antioxidant power of Padina
To determine the influence of extraction temperature on TPC and antioxidant capacity of Padina australis extract, ultrasonic temperature was varied between 30 to
60 o C while ultrasonic time, solvent concentration and solid-solvent ratio were kept constant at 30 mins, 80% ethanol and 1/30 (g/ml), respectively The effect of temperature on TPC and antioxidant capacity is shown in Table 3.2 It can be observed that increasing temperature from 30 to 60 C resulted in a considerable increase in the TPC and antioxidant capacity of the extract While there was no significant difference of TPC and antioxidant power of the extract in using 30 or 40 C (p < 0.05), there was a significant difference in using 60 C compared to the other temperatures The result also indicated that there was about 19.80%, 21.18%, and 39.84% improvement in the TPC, DPPH and FRAP, respectively when the temperature was increased from 30 to
60 C This corresponds to the result of Dang et al., (2017) and Fernández-Barbero et al., (2019) where the extraction temperature was found be very crucial factor for obtaining higher total phenolic content and antioxidant capacity Temperature may have a significant effect on bioactivity because an increase in temperature is related to a proportional increase in the number of cavitation bubbles, thus improving the extraction of bioactive compounds (Febriana et al., 2016) Consequently, the range of ultrasonic temperature (40 – 60 C) was selected for the optimisation experiment
The influence of extraction time was determined by varying extraction time from 20 – 90 minutes while the temperature, solvent concentration and solid-solvent ratio were kept constant at 50 C, 80% ethanol and 1/30 (g/ml), respectively The effect of extraction time on the bioactivity of Padina australis extract is shown in
Table 3.3 From the results, it can be seen that the TPC and antioxidant capacity of the extract increased with increasing time until a peak was reached at 70 minutes, after which the values reduced slightly According to Bi et al., (2019), the gradual increase in the bioactivity of the extract with time may be attributed to the fact that polyphenols and other bioactive compounds are still bound within the cell matrices during the early stage of extraction and sufficient time is required to allow for their release The subsequent decrease in bioactivity may be because longer time of exposure to ultrasonic conditions induces the degradation or oxidation of these bioactive compounds (Tiwari et al., 2009) Therefore, the suitable range of extraction time for the subsequent optimisation process was chosen to be from 60 to 80 minutes
Table 3.2 Influence of ultrasound temperature on the TPC and antioxidant capacity of
Antioxidant capacity (mg TE/gdm) Temperature
The results are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents
The extraction of bioactive compounds from natural materials is not only affected by the extraction method, it is also affected by choice of solvent Organic solvents such as water, ethanol, methanol, butanol etc have been used for the extraction of bioactive compounds from brown seaweed Ethanol has been found to be a solvent of choice because of its high polarity and non-toxicity (Franco et al., 2008) Also, it was determined to be the best solvent for the extraction of brown seaweed in previous research (O’Sullivan et al., 2013; Do et al., 2014; Sánchez-Camargo et al.,
2016) The influence of solvent (ethanol) concentration on TPC and antioxidant capacity of Padina australis was determined by varying solvent concentration from
0% (water) to 100% (absolute ethanol), temperature, time and solvent ratio were kept constant at 50 o C, 60 mins and 1/30 (g/ml), respectively From the results shown on Table 3.4, it can be seen that the DPPH and FRAP of the extract increased with increasing concentration until it reached a peak at 40% (13.42 and 8.14 mg TE/gdm respectively), after which it began to reduce The highest TPC value was obtained at the ethanol concentrations of 20% and 40% (8.63 and 8.56 mg GAE/gdm, respectively
Similarly, increasing the solvent concentration to 100% caused a significant decrease in TPC It was assumed that aqueous mixtures of ethanol with less than 40% water may not be efficient for the extraction of bioactive compounds from Padina australis Hence, the suitable range of ethanol concentration was determined to be from 0 – 60%
Table 3.3 Effect of extraction time on the TPC and antioxidant capacity of Padina australis extract
Antioxidant capacity (mgTE/gdm) Time
The results are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents
3.2.4 Influence of solid-to-solvent ratio
The effect of solid-to-solvent ratio on the bioactivity of the ethanolic extract of
Padina australis was determined by varying the ratio from 1/100 – 1/10 (g/ml) while fixing temperature, time and solvent concentration at 50 o C, 60 mins and 40% ethanol, respectively It can be observed that solid-to-solvent ratio had a significant effect (p < 0.05) on the TPC and antioxidant capacity of the extract (Table 3.5) Notably, increasing solid to solvent ratio led to a remarkable fall in TPC and antioxidant capacity of Padina australis extract The solid-to-solvent ratio of 1/100 (g/ml) showed the highest TPC, DPPH and FRAP of 9.71 mg GAE/gdm, 15.32 and 8.25 mgTE/gdm, respectively This may be attributed to the fact that an increase in solvent-to-solid ratio increases the concentration gradient and consequently increase the rate of diffusion of the compounds from solid to the solvent, thus resulting in increased extraction efficiency of bioactive materials Additionally, an increased amount of solvent could improve leaching-out rates of bioactive compounds from the raw materials Therefore, it can be assumed that low solid-to-solvent ratio favours the extraction of bioactive compounds from Padina australis Therefore, the range of solid-to-solvent of 1 – 5 g/100 ml was chosen for subsequent experiment
Table 3.4 Effect of solvent concentration on the TPC and antioxidant capacity of Padina australis extract
Antioxidant capacity (mgTE/gdm) DPPH scavenging ability
The results are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents
Table 3.5 Effect of solid-to-solvent ratio on the TPC and antioxidant capacity of
Antioxidant capacity (mgTE/gdm) Solid-to-solvent ratio
The results are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents.
Modeling of the UAE process
The combined effects of extraction variables including temperature, time, solvent concentration and solid-to-solvent ratio on the total phenol content and antioxidant capacity of Padina australis extract was determined by performing experiments at different combinations of the variables using the Box-Behnken design (Table 3.6) The experimental results of TPC, DPPH and FRAP in the extract varied from 5.85 - 10.93 mg GAE/gdm, 9.01 – 16.80 and 5.36 – 9.31 mg TE/gdm, respectively
In addition to determining the effects of extraction variables on the responses, it is essential to check the adequacy of a model as the exploration of fitted response surface models may produce poor or misleading results, unless the model exhibits a good fit (Prakash et al., 2013) Two different tests, actual versus predicted plot and analysis of variance (ANOVA) were used as diagnostic plots to determine the reliability of the RSM mathematical model From the actual versus predicted plots (Figure 3.1), it can be observed that the points lie closely to the fitted line, indicating that there is a strong correlation between the actual values and the predicted values obtained from the model R 2 values of 0.96, 0.96, and 0.86 (Table 3.7) were obtained for the TPC, DPPH and FRAP respectively, implying that 86% - 96% of the data match (Krishna et al.,
2017) RMSE, the square root of the variance of residuals, also indicates how accurately the model predicts the response variables Lower RMSE values imply a better fit The RMSE values for the TPC, DPPH and FRAP were 0.38, 0.60 and 0.62, respectively, indicating that the model can accurately predict the response Importantly, the lack of fit for TPC, DPPH and FRAP were determined to be 0.32, 0.08 and 0.41, respectively, indicating that the values of lack of fit were not significant (p > 0.05), confirming that the model could adequately fit the experimental data The p-values (Table 3.7) of the responses were determined by ANOVA to further test the adequacy of the model The p-values for TPC, DPPH and FRAP were 0.0001, 0.0001 and 0.0071, respectively, showing a significant difference of the model at a confidence interval of 95% The F-ratio of TPC, DPPH and FRAP were 18.97, 20.55 and 4.67, respectively The high F-ratio of the response variables further revealed the adequacy of the mathematical model and thus, provided good reproducibility and precision The second order polynomial equation (Equation 3.1-3.3) described the relationship between ultrasonic temperature, extraction time, solvent concentration and solid-to- solvent ratio and the response variables, TPC, DPPH and FRAP
Table 3.6 Box-Behnken design and experimental results
27 50 65 30 3 7.48 12.56 6.16 α The units of the response variables are expressed as follows: TPC (mg GAE/g dm ), DPPH and FRAP (mg TE/g dm ) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; DPPH = DPPH scavenging ability; GAE = gallic acid equivalents; TE = trolox equivalents X 1 : ultrasonic temperature (C); X 2 : ultrasonic time (min); X 3 : ethanol concentration (%), and X 4 : solid to solvent ratio (g/100ml)
Figure 3.1 Actual versus predicted plots for TPC (A), DPPH (B) and FRAP (C)
Table 3.7 Analysis of variance for the determination of model adequacy (TPC, DPPH and
F ratio 18.97 20.55 4.67 α The units of the response variables are expressed as follows: TPC (mg GAE/g dm ), DPPH and FRAP (mg TE/g dm ) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; DPPH = DPPH scavenging ability; GAE = gallic acid equivalents; TE = trolox equivalents.
Effect of extraction variables on experimental responses of TPC, DPPH and
The least square method was used to estimate the regression coefficients of the intercept, linear, quadratic and interaction terms (Table 3.8) Regression coefficients are useful for understanding the impact of extraction variables on the responses 3D surface and 2D contour plots (Figure 3.3) were constructed using JMP (version 15) to help visualise the response and variable interaction They are graphical representations that not only provide at a glance the relationship between responses and extraction variables at each experimental level but also the interaction between two different variables (Minjares-Fuentes et al., 2014) The response surface plot shows the relationship between independent variables, whereas the response surface shape is shown by a contour plot (Dang et al., 2017) The graphs were constructed by plotting the response against two independent extraction variables while keeping the other two variables fixed at the maximum Linear parameters, temperature (40 – 60 o C) and solvent concentration (0 – 60%) had significant effects on the total phenol content and DPPH scavenging activity of the extract (p < 0.05) Solvent-to-solid ratio (1 – 5 g/100 ml) on the other hand had the most significant effect on TPC, DPPH scavenging activity and FRAP of the extract (p < 0.0001), further confirming the results of the single factor experiment where the biological activity decreased significantly with increased solid-to-solvent ratio Extraction time (50 – 80 mins) had no impact on any of the response variables (p > 0.05) This was in agreement with the result obtained by
Dang et al., (2017) where extraction time was found to have no significant effect on the DPPH free radical scavenging activity and FRAP of the extract from brown seaweed, Hormosira banksii This implies that solid-to-solvent ratio, temperature and solvent concentration are the main factors affecting the biological activity of Padina australis None of the interaction terms had any significant effect on the response variables (p > 0.05) Regarding the quadratic terms, X2
2 (Time and Time) had a significant effect on the FRAP of the extract (p < 0.01), X4
2 had a significant effect on the TPC and DPPH scavenging activity of the extract (p < 0.05) However, the terms
2 were insignificant (p>0.05) The maximal yield of TPC predicted by the model was 11.04 mg GAE/gdm at a temperature of 60 o C, time of 60 mins, solvent concentration of 0% (water) and solid-to-solvent ratio of 1 g/100 ml For DPPH, the maximal yield was predicted to be 16.51 mg TE/gdm at extraction conditions of 60 o C,
50 minutes, 60% ethanol and 1 g/100 ml while for FRAP, it was predicted that a maximum yield of 9.68 mg TE/gdm could be obtained at temperature of 60 o C, time of
65 mins, ethanol concentration of 60% and solid-to-solvent ratio of 1 g/100 ml
Table 3.8 Results of regression analysis of experimental values for TPC, DPPH and FRAP
Term Coefficient Prob>|t| Coefficient Prob>|t| Coefficient Prob>|t| b0
*Significant at the p < 0.05 TPC = total phenolic content; FRAP = ferric reducing antioxidant power; DPPH = DPPH scavenging ability; GAE = gallic acid equivalents; TE = trolox equivalents
Optimisation and validation
The current study aimed to optimise ultrasound extraction conditions for maximum yield of both total phenol content and antioxidant activity of Padina australis extract The optimal conditions, chosen based on the model and prediction profiler (Figure 3.2) constructed by JMP (version 15), were as follows: Ultrasonic temperature of 60 o C, Extraction time of 60 minutes, ethanol concentration of 60% and solid-to-solvent ratio of 1 g/100 ml Validation experiments were performed in triplicate at the chosen optimal conditions to verify reliability of the model The TPC, DPPH and FRAP of the extract were found to be 9.07 ± 0.49 mg GAE/gdm, 16.11 ± 1.69 mg TE/gdm and, 9.03 ± 0.58 mg TE/gdm, respectively, which were insignificantly different (p > 0.05) with the predicted values of 10.28 mg GAE/gdm, 16.42 mg TE/gdm, and 9.59 mgGAE/gdm, respectively, implying that the values were well correlated Thus, we can deduce that the regression model obtained by RSM could be reliably used to predict the yields of TPC, DPPH and FRAP of extracts from Padina australis at different combinations of the extraction parameters
To further establish the optimal conditions for the UAE process of Padina australis, another comparable condition (temperature and time were kept constant at
60 o C and 60 minutes, respectively, while sample-to solvent ratio was 2 g/100 ml) was tested to determine the significant difference between this condition and the chosen optimal conditions for UAE The results for TPC and antioxidant activity of the extracts obtained at these conditions are shown in Table 3.9 Although, there was an 15.87%, 24.52% and 5.21% of decrease in the yields of TPC, DPPH scavenging activity and FRAP, respectively when the solid-to-solvent ratio was increased from 1 g/100 ml to 2 g/100 ml, solid-to-solvent ratio of 2 g/100 ml was suggested for preparing the crude extract of Padina australis as it will help to reduce 50% volume of solvent thus saving cost as well as time and energy required for the removal of solvent after ultrasound extraction
Figure 3.2 Prediction profiler showing the effect of X1 (Temperature: 40 – 60 o C), X2
(Time: 50 – 80 mins), X3 (Solvent concectration: 0 – 60%), and X4 (Solid-to-Solvent ratio: 1 – 5 g/100 ml) on the TPC, DPPH and FRAP of Padina australis extract
Table 3.9 Comparisons between two various ratios of sample to solvent for the selection of optimum UAE conditions
DPPH scavenging ability (mg TE/g)
The results are displayed as means and standard deviation (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05) TPC = total phenolic content; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; TE = trolox equivalents.
Figure 3.3 3D surface plots and 2D contour plots constructed by JMP software
(version 15) of I –TPC, II – DPPH and III – FRAP of Padina australis extract as a function of (a) Temperature and Time, (b) Temperature and Solvent concentration, (c) Temperature and Solid-to-Solvent ratio, (d) Time and Solvent concentration, (e) Time and Solid-to-Solvent ratio, (f) Solvent concentration and Solid-to-Solvent ratio.
Recovery yield, TPC, antioxidant and tyrosinase inhibitory activities of Padina
Padina australis crude extract and fractions
Crude extracts of Padina australis was fractionated by n-hexane and ethyl acetate chosen on the basis of their polarities Solvent fractionation involves separating compounds based on their solubility in different solvents (Chatterjee & Saito, 2014) Polar compounds like organic acids, will be eluted to the more polar phase while other non-polar compounds have an affinity for non-polar compounds The percentage yield of the fractions are presented in Table 3.10, the yield of the crude extract (17.48%) was the highest amongst the other fractions while that of n-hexane fraction (0.09%) was the lowest The yield of crude Padina australis extract (17.48%) was comparatively higher than that of Catharanthus roseus stem extract (14.48) (Pham et al., 2018), but lower than that of Ascophyllum nodosum (24.4%) obtained by ultrasound-assisted extraction (Ummat et al., 2020) The yields of the ethyl acetate (0.21%) and n-hexane (0.09%) fractions were comparable with results obtained by
Kim et al., (2018), where the yield ethyl acetate and n-hexane fractions were found to be 0.17% and 0.36%, respectively in Sargassum horneri
The fractions and condensed crude extract were analysed for their total phenolic content and antioxidant activities The levels of TPC in the crude extract and fractions varied significantly The ethyl acetate (EA) fraction had a remarkably high level of TPC of 807.20 mg GAE/g, followed by the n-hexane fraction of 71.92 mg GAE/g The results were in agreement with the previous study of Duan et al., (2006)where higher total phenolic content (73.7 mg GAE/g) was found in the EA fraction of red seaweed
Polysiphonia urceolata compared to the other fractions Similarly, the EA fraction had the highest DPPH scavenging activity and FRAP of 1417 mg TE/g and 615.07 mg TE/g, respectively These results were consistent with those obtained by Taheri (2016), where the EA fractions of Ulva faciata and Gracilaria corticata had the most effective antioxidant properties The high antioxidant activity of EA fraction can be explained by the fact that this fraction contained a higher concentration of phenolic compounds than others
The tyrosinase inhibitory activity of the extract and fractions of Padina australis was also investigated using mushroom tyrosinase (Table 3.10) The major objective of this analysis is to determine the inhibitory effect of Padina australis extract and its fractions on tyrosinase, which is responsible for melanogenesis in humans and browning reactions The tyrosinase inhibitory activity of Padina australis extract and its fraction will determine its potential application in the cosmeceutical and food industries The result shows that the EA fraction possessed the highest tyrosinase inhibitory activity at 29.9 mg AAE/g amongst the other fractions and crude extract The crude extract, n-hexane and aqueous fractions exhibited lower inhibitory activities of 0.09 mgAAE/g, 0.20 and 3.64 mgAAE/g, respectively Similar results obtained by Namjooyan et al., 2019 showed that Padina australis possessed significant inhibitory effect against tyrosinase The high amount of TPC found in EA fraction may have a link with its strong tyrosinase inhibitory activity (Yoon et al., 2009; Xue et al., 2011; Wang et al., 2014)
Table 3.10 Recovery yield (% w/w on a dry weight basis), TPC, antioxidant capacity and tyrosinase inhibitory activity of crude extract and fractions of Padina australis
Antioxidant capacity (mg TE/g) Fraction/
The results of TPC, DPPH, FRAP and tyrosinase inhibitory activities are displayed as means and standard deviations (n = 3) Different superscript letters in the same column indicate significant difference (p < 0.05)
HF = n-hexane fraction; EAF = ethyl acetate fraction; AQ = aqueous fraction; CRE = crude extract TPC = total phenolic content; FRAP = ferric reducing antioxidant power; TIA = tyrosinase inhibitory activity; GAE = gallic acid equivalents; TE = trolox equivalents; AAE = ascorbic acid equivalents.