LITERATURE REVIEW
Gac (Momordica cochichinensis Spreng)
Gac (Momordica cochinchinensis Spreng) is botanically classified as: Family
Cucurbitaceae, Genus Momordica, Species Cochichinensis Gac has an Asian origin
Schaefer et al (2009) highlight that this plant is found in the wild across India, the Philippines, Vietnam, and Thailand, while it is cultivated in Vietnam, Japan, and other Asian nations for its fruit, which is believed to possess numerous medicinal properties (Behera et al., 2011).
Gac, a fruit native to Vietnam, is commonly referred to in English as Chinese Cucumber, Cochinchin Gourd, Giant Spine Gourd, Spiny Bitter Cucumber, Spiny Bitter Gourd, and Sweet Gourd (Lim, 2012).
The plant is a vine, which can be cultivated either from seeds or root tubers (Parks et al.,
The flowering of the plant begins approximately two months after planting root tubers, typically occurring in Vietnam from April to August, and occasionally extending into September The average maturation period for the fruit, following the emergence of the female flower bud, is about 18-20 days.
As Gac fruit matures, it initially grows in size and eventually turns a dark red/orange color when ripe Typically round or oblong, it reaches approximately 13 cm in length and 10 cm in diameter In certain countries, the immature green fruit can be utilized as a vegetable for cooking, while in Vietnam, fully ripe fruits weighing between 350-600 g or more are commonly harvested.
The bright red fruit, which contains hardened seeds, is utilized for both food and medicinal purposes (Behera et al., 2011; Lim, 2012) On average, a single plant can produce between 30 to 60 fruits in a season (Wimalasiri et al., 2016).
The Gac fruit features a spiny exterior and a fleshy yellow pulp that makes up 49% of its weight, while the dark red aril, known for its high carotenoid content, averages 18% of the fruit's weight, with variations ranging from 10% to 24.6% The skin and seeds contribute 17% and 16% to the total weight, respectively These proportions can fluctuate based on the fruit's variety, growth stage, and storage duration.
Figure 1: Components of Gac fruit
(A) Green Gac fruit; (B) Ripe Gac fruit; (C) Yellow pulp and red aril; (D) Gac seeds
Figure 2: Components of Gac fruit
(Data from Kha et al (2013a))
The Gac aril, a component of the ripe Gac fruit, is highly valued for its rich content of carotenoids, particularly β-carotene and lycopene, as well as polyunsaturated fatty acids Traditionally, the vibrant red aril is used as a natural food colorant, especially in Vietnam where it enhances the popular dish 'xôi gấc,' made by cooking glutinous rice with the aril In Thailand, immature Gac fruit is utilized as a vegetable, often cooked with chili paste or in curries Recently, Gac aril has gained commercial popularity, being available as juice, dietary supplements, frozen aril, and dried powder.
Recent studies have focused on improving the application of Gac aril in the food industry by processing it into more dispersible forms (Kha et al., 2010; Kha et al., 2014b) Additionally, significant attention has been given to pre-treatment and extraction techniques to optimize its usage.
Recent studies have shown a 30% increase in the yield of oil and bioactive compounds extracted from Gac aril, alongside advancements in encapsulation techniques that enhance the storage longevity of these extracts (Kha et al., 2013b, 2014a; Kha et al., 2014c).
The pulp and skin of the Gac fruit are rich in carotenoids, particularly lutein, making them promising sources for carotenoid extraction, as highlighted in various studies (Ishida et al., 2004; Kubola & Siriamornpun, 2011; Chuyen et al., 2017).
Gac seeds
Gac seeds, derived from the dried ripe Gac fruit, are large, compressed, and ovoid with a distinctive wavy outline Encased in a protective aril, these seeds are blackish-brown and feature an irregularly sculptured seed coat Traditionally, fully ripened Gac seeds are utilized for their medicinal properties (Lim, 2012).
Each fruit has on average between 15 to 20 round, compressed and sculptured seeds The seeds, which represent about 16% of the total weight (Figure 2) of the fruit (Ishida et al.,
In the process of recovering the red pulp (aril) from Gac seeds, it is important to note that the kernel constitutes approximately 80% of the seed's total weight.
In Vietnam, approximately 760 tons of Gac seeds are produced annually, representing 16% of the total Gac fruit yield of around 4,750 tons Despite their potential benefits, these seeds are often discarded, although some factories do extract oil from them through pressing This Gac seed oil is utilized for massage therapy and the treatment of certain skin conditions.
31 to traditional uses However, the resulting residue, including the hydrophilic components of the seeds, is discarded
Gac seeds are rich in oil, comparable to sesame seeds and peanuts, with fresh seeds containing up to 60% oil The primary components include stearic acid, a long-chain saturated fatty acid, making up 60%, and linoleic acid, an essential ω-6 polyunsaturated fatty acid, constituting 20%.
Gac seed oil's unique fatty acid composition makes it a promising alternative for the confectionery and frying industries due to its high melting point, which helps maintain the integrity of confectionery products and enhances oil stability at elevated frying temperatures While hydrogenated oils are commonly used in these applications, they are a significant source of trans fatty acids that can raise blood cholesterol levels and increase the risk of coronary heart disease In contrast, Gac oil contains beneficial fatty acids such as stearic and linoleic acid, offering a healthier option for consumers.
1991) have no effect or lower blood cholesterol, respectively Therefore, Gac seed oil has the potential of being a good replacement for hydrogenated oils in these areas of the food industry
Gac seeds contain hydrophilic components, particularly a group of low molecular weight trypsin inhibitors, many of which are cyclotides These compactly structured compounds are effective in penetrating tumor cells, contributing to their anticancer properties Numerous studies have validated the anticancer activity of trypsin inhibitors derived from Gac seeds (Zhao et al., 2010a; Zheng et al., 2013; Zhizhong, 2007).
Cyclotides are bioactive mini-proteins derived from plants, characterized by a unique cyclic backbone and a cystine knot, which contribute to their remarkable stability This stability has sparked interest in their potential as peptide-based templates for drug design Cyclotides exhibit a variety of pharmaceutical activities, such as anti-HIV, antimicrobial, and uterotonic effects, making them valuable candidates for developing new therapeutics Their structural resilience allows for the incorporation of bioactive peptide sequences, enhancing their applicability in pharmaceutical research (Smith et al., 2011).
Gac seeds are rich in bioactive compounds, including trypsin inhibitors, cyclotides, saponins, phenolics, flavonoids, and tocopherols, making them a valuable source of nutrients (Lin et al., 2012; Matthaus et al., 2003) Numerous studies have demonstrated the biological activities associated with these components (Jung et al., 2013a; Jung et al., 2013b) Traditionally, Gac seeds have been utilized in medicine across various Asian countries, especially in China, due to their diverse health benefits.
The discarded Gac seed residue contains valuable hydrophilic components such as trypsin inhibitors, phenolics, flavonoids, and certain saponins, leading to the wastage of potential bioactive compounds The high concentration of these bioactives poses risks for animal feed, as they can be toxic in large amounts Additionally, if the waste is improperly disposed of, it can cause environmental harm to land and waterways, making it unsuitable for use as fertilizer.
Utilizing Gac seeds for the extraction of bioactive compounds and oil can significantly enhance the value of the Gac fruit industry by creating new marketable products.
The project focuses on extracting 33 bioactive compounds and oil products from Gac fruit, aiming to reduce environmental issues linked to industry waste By removing these bioactive compounds, the remaining waste can potentially be repurposed as a resource for the fertilizer and animal feed sectors Consequently, the findings from this initiative could provide advantages not only for Vietnam but also for other nations, such as Australia, that are developing their Gac fruit industries.
1.2.1 Oil and fatty acids in Gac seeds
Numerous studies indicate that Gac seeds have a high oil content, comprising various fatty acids The oil concentration in Gac seeds varies across research due to factors such as seed variety, growing conditions, sample preparation, extraction methods, and measurement techniques Generally, the oil content of Gac seeds is comparable to that of other oil-rich seeds, including peanuts, sunflower, and sesame Table 1 summarizes the oil content and fatty acid composition of Gac seeds as reported in different studies.
Gac seeds are unique among tropical plants as they contain a higher level of stearic acid compared to the more common palmitic acid found in other seeds This higher stearic acid content increases the oil's melting point, making it suitable for creating solid and oxidatively stable fats for use in shortenings and margarines Such properties can help minimize the need for partial hydrogenation of liquid oils, a process that often leads to the formation of unhealthy trans fatty acids.
34 seeds has also been reported to have a reasonable amount of Vitamin E (274 mg/kg oil) and the omega-3 linolenic acid (Matthaus et al., 2003)
Some unsaponifiable compounds have also been reported to present in Gac seed oil, including karounidiol, β-sitosterol, pentacyclic triterpene and their derivatives (Kan et al.,
2006) Another study by Akihisa et al (1986) showed that there was 218 mg of sterol in
100 g of dried Gac seeds, and most of these were ∆7-sterols
Table 1 Oil content and composition of main fatty acids (as % of total fatty acid) in Gac seeds
Gac seeds are abundant in oil and phytochemicals, including trypsin inhibitors, saponins, phenolics, and flavonoids While these compounds were once viewed as antinutritional or toxic (Anderson & Wolf, 1995; George, 1965), recent studies suggest they may play a significant role in medicinal and antioxidant activities, contributing to the health benefits of Gac seeds However, there is a lack of research on how growing, storage, and processing conditions affect the quantity and quality of these phytochemicals, and further development of techniques and varieties is necessary to enhance their bioactive content and quality.
1.2.2 Bioactive compounds in Gac Seeds
Trypsin inhibitors (TIs) are low molecular weight peptides that inhibit the hydrolase activity of various serine proteases, primarily found in the storage organs of plants like seeds, roots, and tubers There are three major sub-types of TIs: Bowman-Birk-type inhibitors, Kunitz-type inhibitors, and squash family inhibitors, with molecular weights of approximately 7500, 20000, and 3500 kDa, respectively The first two types are derived from leguminous plants, while the squash family inhibitors are obtained from Cucurbitaceous species Recent studies have identified these protease inhibitors, highlighting the squash inhibitor family as the latest addition to this classification.
Table 2 Families of protease inhibitors in plant tissues
(Source: De Leo et al (2002))
1 Bowman-Birk serine proteinase inhibitors
7 Potato type II proteinase inhibitors
The anticancer activity of protease inhibitors was first reported by Troll and Kennedy
(1993) in relation to trypsin inhibitors suppressing two-stage carcinogenesis and breast cancer Since then, extensive investigations on trypsin inhibitors as cancer chemo-
Extraction of oil and bioactives from Gac seeds
Various methods have been employed for oil extraction from plant seeds, but traditional techniques that utilize potentially harmful organic solvents are being phased out due to health and environmental concerns, as well as quality degradation Therefore, it is crucial to explore alternative extraction methods that utilize non-organic food-grade solvents.
Traditional methods for producing seed oil typically involve expeller pressing and conventional solvent extraction with hexane While expeller pressing produces high-quality extracts, it often results in low yields and can lead to thermal degradation of active components On the other hand, solvent extraction maximizes oil recovery but poses risks of solvent contamination, which can be detrimental to human health and the environment, limiting its application in food, cosmetic, and pharmaceutical sectors.
Extraction of phenolics Extraction of trypsin inhibitors
Figure 8: A potential processing scheme for Gac seeds
Various environmentally-friendly methods for extracting plant oils have been identified, including supercritical carbon dioxide (SC-CO2), aqueous enzymatic extraction, microwave-assisted extraction, and ultrasound-assisted extraction These techniques are notable for being free of organic solvents The ultrasound-assisted and microwave-assisted pressing extraction methods have been studied extensively, highlighting both their advantages and limitations in the extraction process.
2011) in food extraction have been reviewed
The Soxhlet leaching technique has been a standard method for over a century, serving as the primary reference point for evaluating the performance of other leaching methods.
In the traditional Soxhlet extraction method, a sample is contained in a thimble-holder, which is gradually filled with fresh solvent from a distillation flask Once the liquid reaches the overflow level, a siphon extracts the solute from the thimble-holder and returns it to the distillation flask, effectively transferring the extracted analytes into the bulk liquid This process continues until complete extraction is accomplished.
Applications of Soxhlet extraction for vegetable oil
For decades, this traditional method of Soxhlet extraction have been used everywhere for many different purposes In terms of efficiency, the Soxhlet extraction is described as an
59 universal chemical extraction process However, this method requires large extraction time and quantity of solvent
1.3.1.2 Supercritical carbon dioxide (SC-CO 2 ) extraction
Supercritical fluid extraction (SFE) utilizes fluids in a supercritical state, where pressure and temperature exceed critical values, resulting in unique properties that blend characteristics of both gases and liquids This state is marked by lower viscosity and higher diffusivity compared to liquids, while the density resembles that of a liquid These properties are influenced by the fluid's pressure, temperature, and composition, allowing for adjustments in density and dissolving power through temperature and pressure changes SFE presents several advantages over traditional extraction methods, such as reduced extraction time, minimized solvent usage, and enhanced selectivity in extractions.
1996) In addition, it is also considered to offer benefits such as non-solvent residues, higher extraction yields and better retention of nutritional and valuable bioactive compounds (Herrero et al., 2006)
It is important to select the most suitable supercritical fluid in this extraction technique
Supercritical fluids, including solvents like ethylene, methane, nitrogen, xenon, and fluorocarbons, play a crucial role in extraction processes Among these, carbon dioxide (CO2) stands out as the preferred solvent for supercritical fluid extraction (SFE), specifically known as supercritical carbon dioxide (SC-CO2) extraction This preference is largely due to CO2's low critical temperature of 31°C and critical pressure of 74 bar, allowing for extraction at low temperatures and moderate pressures Additionally, CO2's low polarity makes it particularly effective for extracting low or non-polar compounds.
To enhance the solubility of polar compounds in supercritical carbon dioxide (SC-CO2), the addition of a small quantity of chemical modifiers or co-solvents is effective Figure 9 illustrates the phase transitions of carbon dioxide, highlighting its behavior in various conditions.
In the SC-CO2 extraction process, carbon dioxide is vented post-extraction, ensuring that the final extract is free from CO2, which contributes to its status as Generally Recognized As Safe (GRAS) for food production (Gerard & May, 2002) Additionally, the quality of the extracted products is typically high, often eliminating the need for extensive refining processes.
Figure 9: Phase diagram of carbon dioxide
Applications of SC-CO 2 extraction for vegetable oil
Supercritical carbon dioxide (SC-CO2) technology has gained traction in the extraction of oils from various materials in the food and pharmaceutical industries, showcasing significant potential for future applications Recent studies have demonstrated its effectiveness in extracting essential oils, fatty acids, carotenoids, and vitamin E from fruits and vegetables Comparisons between SC-CO2 and traditional solvent extraction methods highlight the advantages of SC-CO2 in oil extraction processes.
61 extracted Generally, oils extracted by SC-CO2 is reported to have a lighter colour, a milder odour, more tocopherols and less impurities such as unsaponifiables, gossypol and phosphorous (Friedrich & Pryde, 1984)
Traditional methods for extracting vegetable oils, such as hydraulic pressing and solvent extraction, often face challenges While solvent extraction offers high efficiency, it involves hazardous chemicals and complicated solvent removal processes Additionally, this method can lead to thermal degradation of valuable bioactive compounds and may not fully eliminate solvents, posing potential quality concerns.
Supercritical carbon dioxide (SC-CO2) extraction is an effective method for isolating nonpolar compounds, allowing for easy separation of the solute by depressurizing the CO2 This technique serves as a safer alternative to traditional extraction methods that utilize hazardous solvents Recent studies have demonstrated the successful extraction of vegetable oils from plant materials using SC-CO2, highlighting that factors such as particle size, moisture content, and extraction conditions (pressure, temperature, time, and flow rate) significantly affect the yield Smaller particle sizes enhance CO2 diffusion rates due to a greater surface area to volume ratio and the disruption of cell membranes, making it advisable to grind samples to an optimal size to prevent channelling issues within the extraction bed.
To ensure effective SC-CO2 extraction, it is crucial to maintain a homogeneous bed of material within the vessel to prevent channelling Additionally, monitoring the moisture content of the material is vital, as high moisture levels can negatively impact the extraction process.
To prevent mechanical issues like restrictor clogging due to ice formation, it is essential to manage the moisture content in plant samples Adding anhydrous Na2SO4 and silica gel can help capture moisture, but the ideal pre-treatment involves drying the plant materials to a moisture content below 12% High moisture levels can lead to complications, including ice formation in pipelines (Lang & Wai, 2001; Fornari et al., 2012).
Extraction efficiency is influenced by various conditions, including pressure, temperature, time, and flow rate Higher pressure and temperature generally enhance mass transfer and the release of bioactive compounds from plant matrices, but they can also lead to the formation of undesirable compounds in the extract Optimal oil yield is typically achieved at extraction pressures between 30 and 40 MPa across different plant matrices Studies have shown that a lower extraction temperature of 60°C yields the highest efficiencies for saponins, while a higher temperature of 80°C also supports saponin extraction However, temperatures exceeding 80°C may cause degradation, suggesting that lower temperatures are preferable Therefore, it is essential to investigate and optimize the key factors affecting extraction efficiency.
Bioactive compounds phytochemicals found in plant materials that are capable of modulating metabolic processes and resulting in the promotion of better health
Spectrophotometry method for quantification of bioactive compounds
Spectrophotometry is a technique used to quantify how much light a chemical substance absorbs by analyzing the intensity of light passing through a sample solution Each compound uniquely absorbs or transmits light within specific wavelength ranges, allowing for precise measurements This method is essential for determining the concentration of known chemical substances and is widely applied in various fields, including chemistry, physics, biochemistry, materials science, chemical engineering, and clinical diagnostics Additionally, colorimetric reactions play a significant role in enhancing the effectiveness of spectrophotometric analysis.
UV/VIS spectrophotometric method, which is easy to perform, rapid and applicable in routine laboratory use, and low-cost (Pelozo et al., 2008)
Every chemical compound interacts with light by absorbing, transmitting, or reflecting it across specific wavelengths Spectrophotometry measures the extent to which a chemical substance absorbs or transmits light, making it an essential technique for estimating the concentration of an analyte in a solution.
Beer’s Law explains that the absorbance of light at a specific wavelength by a substance is directly proportional to its concentration over a consistent distance This relationship can be expressed by the formula An = L × C, where An represents the absorbance at n nm, L denotes the light path in centimeters, and C indicates the concentration of the analyte in the solution.
Measuring the absorbance of a solution with a known analyte concentration allows for the estimation of the analyte concentration in an unknown solution by comparing absorbance values The proportional range of absorbance to concentration depends on the analyte and the light wavelength used To establish a direct relationship between absorbance and concentration, a standard curve must be created using a reference substance, with the linear portion of the curve indicating the proportional relationship.
To create a standard curve, a sequence of dilutions is made from a standard solution with a known concentration of the reference substance The initial tube serves as a blank, containing no standard (0 g/L), and is essential for calibrating the spectrophotometer to account for the natural absorbance.
77 the diluents Each of the following tubes contain increasing concentrations of the analyte The absorbance of each of the standards are read using the spectrophotometer
To establish the standard curve, a line graph is created plotting Absorbance on the Y-axis against Concentration on the X-axis for each standard Subsequently, a line of best fit is drawn to connect the data points.
To determine the concentration of an unknown analyte solution, the absorbance of the sample is measured, and the standard curve's linear equation is applied to estimate the analyte's concentration in the extract.
1.4.2 Devices and mechanism for spectrophotometry measurement
A spectrophotometer is a device which measures the absorbance of a solution as light of a specified wavelength is passed through it (Figure 10)
Figure 10: The mechanism for spectrophotometry measurement
The difference between the incident and transmitted light indicates the absorbance
(Source: https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-services/spectrophotometry)
A spectrophotometer is an analytical instrument that combines two key components: a spectrometer and a photometer The spectrometer generates and disperses light, while the photometer measures the intensity of that light using a photoelectric detector.
The spectrometer produces a desired range of wavelength of light First a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism)
78 to split it into several component wavelengths (spectrum) Then a wavelength selector (slit) transmits only the desired wavelengths, as shown in Figure 10
When light of a specific wavelength passes through a sample solution in a cuvette, the photometer measures the absorbed photons and transmits the data to a galvanometer or digital display, as shown in Figure 10.
Oxidation and antioxidant activity of medicinal plants
The oxidation process in biological systems heavily relies on oxygen, the primary electron acceptor, resulting in the generation of active oxygen and free radical species Free radicals are defined as molecular entities that possess one or more unpaired electrons, allowing them to exist independently (Naumovski, 2014).
Oxygen is derived from two main types of reactive species: Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) ROS includes superoxide anion, hydroxyl radicals, peroxyl and alkoxyl radicals, hydrogen peroxide, organic hydroperoxide, and singlet oxygen RNS comprises nitric oxide, peroxynitrite, peroxynitrous acid, and nitrogen dioxide These reactive species can damage essential biological macromolecules, leading to protein, lipid, nucleic acid, and DNA harm This damage contributes to cellular aging and the development of oxidative stress-related diseases, including cardiovascular issues, diabetes, neurodegenerative disorders, and cancer.
To effectively delay the oxidation process and prevent radical chain reactions, it is essential to inhibit the initiation and propagation steps of oxidation (Doughari, 2012) A widely recognized method for achieving this is through the use of natural antioxidant compounds or bioactive substances, which can be incorporated into the body through daily dietary intake.
79 consumption of vegetables, fruits, drinks, or may be extracted and purified from various plant materials in either an individual form or in a combined form
Recent studies have highlighted the significant antioxidant activity of various bioactive compounds Notably, Chen et al (2014) reported on the antioxidant properties of Radix Trichosanthis saponins, while Dai and Mumper investigated the antioxidant effects of plant phenolics.
(2010), or a wide variety of free radical scavenging molecules such as phenols, flavonoids, flavanols, proanthocyanidins, vitamins, terpenoids, and carotenoids with potential antioxidant activity have been identified in the plants (Doughari, 2012)
Researchers are increasingly interested in evaluating the total antioxidant capacity of bioactive compounds Recent advancements have led to the development of various chemical assay techniques for assessing antioxidant capacity However, since each assay reveals only a limited aspect of total antioxidant capacity, it is essential to employ multiple antioxidant assays for a comprehensive evaluation.
The total antioxidant capacity is commonly evaluated using two main reaction types: single electron transfer (SET) and hydrogen atom transfer (HAT) SET reactions can be identified by the color change in the reagent solution when the oxidant is reduced, as seen in assays like ABTS and DPPH radical scavenging, where antioxidants transfer an electron, resulting in a lighter color proportional to their concentration In contrast, assays such as cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) measure the ability of antioxidants to reduce copper or ferric ions, leading to a darker color in the reagent solution, also dependent on antioxidant concentration.
Antioxidants play a crucial role in neutralizing free radicals, particularly peroxyl radicals, through hydrogen atom donation Key methods to assess antioxidant capacity include crocin bleaching, β-carotene bleaching, oxygen radical absorbance capacity (ORAC), and total peroxyl radical-trapping antioxidant parameter (TRAP) assays Additionally, other innovative techniques such as total oxidant scavenging capacity (TOSC), chemiluminescence, and electrochemiluminescence assays have been developed to further evaluate antioxidant effectiveness (Apak et al., 2013; Apak et al., 2007).
Cancer and anticancer activity of medicinal plants
Cancer is a disease characterised by uncontrolled cell growth and proliferation initiated by inappropriate cell division It is categorised as the second leading cause of death, with
In 2018, there were an estimated 18 million new cancer cases globally, posing significant health challenges in both developed and developing nations (Bray et al., 2018) Melanoma ranks as the fourth most commonly diagnosed cancer in Australia, which, alongside New Zealand, has the highest incidence rates worldwide, largely due to high UV radiation exposure (Cancer Council Australia, 2017) The rising incidence and mortality rates of melanoma have intensified the focus on drug development, public health policies, and awareness initiatives aimed at cancer prevention (Stracci et al., 2005).
Over 80% of the global population relies on traditional plant-derived medicine as their primary healthcare source, highlighting the ongoing need for innovative anticancer drugs and combinations through systematic exploration of plant-based products Current cancer treatment methods, including chemotherapy, surgery, and radiotherapy, often involve non-surgical therapies that can lead to toxicity due to their non-selective targeting.
Plant-derived medicines have a long history of use in the treatment of cancer and over 60% of currently used anti-cancer agents are from natural sources (Bhanot et al., 2011)
Medicinal plants are rich sources of phytochemicals with antioxidant, immune- modulatory and anti-cancerous properties (Greenwell & Rahman, 2015)
Gac seeds are abundant in phytochemicals, including saponins, trypsin inhibitors, and tocopherols, which may offer significant health benefits such as vitamin E, antimicrobial, and anticancer properties These bioactive compounds are known for their antioxidant, anti-inflammatory, and anticancer effects (Lim, 2012).
Ester extracts from Gac seeds have demonstrated anticancer properties against melanoma cells, while ethanolic extracts have been shown to inhibit the proliferation of various carcinoma cells, including those of the lung, breast, and esophagus (Zhao et al., 2010a; Zhao et al., 2010b) However, the effects of water extracts from defatted Gac seeds on cancer cells, particularly melanoma, remain unexplored Identifying the compounds responsible for the anticancer activity in Gac seeds is crucial for optimizing extraction, purification, and isolation processes to develop medicinal products with enhanced anti-cancer efficacy.
Experimental rationale
Plants and their extracts have long been used in traditional medicine globally, leading to a resurgence of interest in secondary plant metabolites for their potential to prevent and treat chronic diseases like cardiovascular issues and cancer Recent efforts have focused on isolating, identifying, and quantifying phytochemicals in various plant resources while evaluating their health benefits However, in vitro and animal studies indicate that the effects of certain isolated phytochemicals may require doses significantly higher than what can be obtained through a typical plant-based diet.
(Rowland, 1999) Therefore, the extraction of active ingredients is essential if these kinds of preparations are to be of prophylactic or therapeutic value in human subjects (Rowland,
Gac is a tropical Asian fruit increasingly cultivated worldwide, primarily valued for its nutrient-rich aril, which is high in carotenoids, vitamin E (α-tocopherol), and polyunsaturated fatty acids However, the seeds, constituting 16-18% of the fruit's weight, are often discarded, leading to significant environmental waste—approximately 750 tons annually in Vietnam alone Utilizing these seeds for oil extraction and bioactive compounds could enhance the Gac fruit industry's value while addressing environmental concerns.
Gac seeds are rich in oil, comprising 15.7% to 36.6% of their total weight, highlighting their potential as a source of both edible and medicinal oils Further research is essential to explore their applications in these areas.
The fatty acid composition of Gac seed oil includes a high percentage of stearic acid (54.5–71.7% by weight), alongside linoleic acid (11.2–25.0%) and trace amounts of α-linolenic acid (0.5–0.6%), as well as other fatty acids in smaller quantities (Ishida et al., 2004) Optimizing the extraction process from Gac seeds is essential for maximizing the yield of this oil.
Gac seeds have a rich history in traditional Chinese medicine, where they have been utilized to treat various ailments, including skin disorders Recent research has highlighted their potential antitumor and anticancer properties, suggesting that Gac seeds are rich in medicinal compounds Notably, bioactive compounds such as trypsin inhibitors have been identified in Gac seeds, further supporting their therapeutic potential.
83 saponins as described in detail in Section 1.2.2 These bioactive compounds are enriched in Gac seeds and therefore, it is worth optimising their extraction
The efficacy of extracting oil and bioactives from plant materials, particularly seeds, is significantly influenced by several factors, including the extraction method, solvent composition, extraction time, temperature, solid-to-solvent ratio, and extraction pressure.
Supercritical carbon dioxide (SC-CO2) extraction has emerged as a highly regarded alternative to traditional solvent extraction and mechanical pressing for vegetable oil extraction This method is favored due to its superior extraction rates compared to mechanical processes Additionally, SC-CO2 utilizes carbon dioxide, which is non-flammable, non-explosive, cost-effective, and readily available Its gaseous state also simplifies the removal process from the extracted oil, enhancing its appeal in the industry.
Conventional solvent extraction of bioactive compounds from plant materials often involves prolonged heating times, which can degrade these valuable compounds To enhance extraction efficiency, advanced techniques like microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) are being utilized MAE utilizes microwave heating to disrupt plant cell structures by increasing internal pressure, effectively releasing bioactive compounds Similarly, UAE employs ultrasonic cavitation to achieve improved extraction results, reducing both extraction time and solvent consumption.
Advanced extraction methods, such as shockwave techniques, effectively disrupt plant cell structures and release bioactive compounds, making them prominent in green chemistry practices (Pingret et al., 2013; Wang & Weller, 2006) These methods have demonstrated superior efficiency in recovering carotenoids from Gac peel compared to traditional extraction techniques (Chuyen et al., 2018).
To maximize the yield of Gac seed oil and its bioactive compounds, optimizing extraction processes is essential Traditional empirical methods, particularly the one-factor-at-a-time approach, are limited as they are time-consuming and fail to account for interactions among multiple factors, making it unlikely to achieve true optimization This method assumes that parameters do not interact, leading to results that only reflect the influence of a single variable In contrast, a statistical optimization procedure, such as Response Surface Methodology (RSM), is preferable as it considers the interactive effects of various variables, ultimately enhancing the optimization process (Haaland, 1989).
Response Surface Methodology (RSM), first introduced by Box and Wilson in 1951, is a set of statistical and mathematical techniques designed to evaluate the effects of multiple process variables and their interactions on response variables This methodology has proven effective for developing, improving, and optimizing various processes, as highlighted by Myers and Montgomery in 2002 RSM has been particularly successful in modeling and optimizing biochemical and biotechnological processes within food systems, including the extraction of phenolic compounds from berries, as demonstrated by studies from Cacace and Mazza (2003a, 2003b) and Parajó et al (1995).
& Mazza, 2003a) and evening primrose meal (Wettasinghe & Shahidi, 1999b), anthocyanins from black currants (Cacace & Mazza, 2003b) and sunflower hull (Gao & Mazza, 1996) and vitamin E from wheat germ (Ge et al., 2002), among others
This thesis explores the optimization of supercritical carbon dioxide (SC-CO2) extraction conditions to enhance the yield and quality of Gac seed oil, utilizing Response Surface Methodology (RSM) It compares the efficiency of the SC-CO2 extraction method with traditional Soxhlet extraction regarding oil yield and characteristics Additionally, the study examines the impact of various solvents and assisted-extraction methods, such as Microwave-Assisted Extraction (MAE) and Ultrasound-Assisted Extraction (UAE), on the yield of trypsin inhibitors, saponins, and phenolic compounds The extraction processes for trypsin inhibitors and saponins were optimized through one-factor-at-a-time and RSM methodologies Furthermore, the antioxidant activity and anticancer potential of extracts from different solvents were assessed Ultimately, a freeze-dried powder enriched with trypsin inhibitors was developed using the optimal extraction conditions for Gac seed trypsin inhibitors.
Hypotheses, Aims and Objectives
The working hypotheses for this study were that:
1) Gac seeds contain high levels of extractable oil, trypsin inhibitors and saponins and their yield can be optimised using different extraction methods and solvents
2) The Gac seed extracts are of high quality and possess biological activity, including antioxidant and anticancer properties
The aim of the thesis was to extract oil, trypsin inhibitors and saponins from Gac seeds with high yields
In order to test the hypotheses and achieve the aim, the objectives of this thesis were as follows:
1 To optimise the extraction of oil from the dried Gac seeds using SC-CO2 The conditions evaluated were the critical temperature, pressure and flow rate of the fluid
2 To characterise the Gac seed oil extracted by SC-CO2 and compare it with the oil extracted by the conventional Soxhlet method
3 To determine suitable extraction methods for Gac seed trypsin inhibitors and saponins
4 To optimise the extraction of Gac seed trypsin inhibitors and produce a freeze dried trypsin inhibitor-enriched powder The conditions evaluated were 1) type of solvent, 2) time of extraction and 3) the ratio of Gac seed material to solvent
5 To determine the optimal method and conditions for the ethanol extraction of Gac seed saponins The conditions evaluated were 1) microwave conditions (power, irradiation time) and 2) the ratio of ethanol to Gac seed material
6 To evaluate the antioxidant activity of the Gac seed oils and bioactive extracts from different extraction methods and solvents
7 To evaluate the anticancer potential of Gac seed bioactives extracted using various solvents
A summary of the research design used to achieve the aims and objectives of this thesis is shown in Figure 11
Figure 11: Diagram summarizing the research design compare
Vacuum drying De-coating Kernel
Oil extraction by SC-CO 2
Extraction of MCoTIs and saponins: