i DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Thesis Title: Research on extraction proces
INTRODUCTION
Research rationale
Vietnam's tropical climate fosters a rich and diverse flora, boasting approximately 12,000 species of higher plants, nearly 5,000 of which have medicinal uses (Vo Van Chi, 2003) This biodiversity presents significant opportunities for researching natural compounds for pharmaceutical applications in the country.
Polyscias fruticosa, also known as Nam Duong Lam, is a member of the ginseng family (Araliaceae) and has been utilized in traditional Vietnamese medicine for its health benefits, including promoting health, enhancing blood circulation, and alleviating rheumatic pain This plant contains various bioactive compounds, such as glucosides, alkaloids, tannins, vitamin B1, and approximately 20 amino acids, including arginine, alanine, asparagine, glutamic acid, leucine, lysine, phenylalanine, proline, threonine, tyrosine, cysteine, tryptophan, and methionine, as well as oleanolic acid.
1992), Flavonoids (Nguyen Thi Luyen et al., 2012), essential oils (Brophy JJ et al , 1990), Polyacetylene (Luto mski J, and Tran Cong Luan, 1992; Tran Cong Luan, 1996), Saponin (Chaboud A…, 1996; Vo Duy Huan, 1998)”
Research on natural active ingredients is gaining significant attention for their healing properties Polyscias fruticosa is particularly noted for its pharmacological effects and biological potential, leading to increased interest in its breeding and cultivation In our country, the bark of Polyscias fruticosa is traditionally used to aid in the removal of the placenta post-childbirth and to alleviate various aches and pains, including back pain and rheumatism.
Flavonoids in Polyscias fruticosa are effective in treating various conditions such as allergic inflammation and gastric and duodenal ulcers, while also aiding the body in regulating metabolic processes.
2 anti-aging, and sustaining endurance, blood vessels, and lowering cholesterol in the blood
Research on the active ingredients in Polyscias fruticosa remains limited, primarily due to its traditional use in folk remedies To advance the understanding of its medicinal properties, it is essential to investigate the extraction process of total flavonoids, a significant active component found in the trunk of Polyscias fruticosa, for applications in medicine, pharmaceuticals, and food.
Based on the above concerns, I have chosen the topic: “Study on extraction process of total flavonoid from Polyscias fruticosa trunk”.
Research’s objectives
Qualitative analysis of the presence of total flavonoids in the trunk of
The extraction of total flavonoids from the trunk of Polyscias fruticosa is influenced by several key factors, including extraction temperature, solvent type, concentration, extraction duration, trunk size, and the application of a magnetic stirrer.
Propose the total flavonoid extraction process in Polyscias fruticosa trunk.
Research questions and hypotheses
Is it probable to obtain total flavonoid content from the trunk of Polyscias fruticosa?
What variables can greatly affect the extraction process from the trunk of
Limitation
Polyscias fruticosa is widely grown in Thai Nguyen, favored as a medicine
Transporting and harvesting materials for laboratory experiments in Thai Nguyen city is made convenient However, variations in seasonal conditions and the specific areas where the Polyscias fruticosa trunk is harvested affect the quality of the prepared materials.
The research revealed uneven concentrations of flavonoids, which are compounds known for their numerous pharmacological benefits and are found in the trunk of Polyscias fruticosa Efficient extraction methods for flavonoids were specifically chosen to enhance the study's outcomes.
LITERATURE REVIEW
Overview about Polyscias Fruticosa
Polyscias fruticosa is a species of small tree within the genus Polyscias of the Araliaceae familia
The small shrub, which remains green throughout the year, typically reaches a height of 1.5 to 2 meters Its rough, spineless body is less branched and features numerous large gray scars, while young branches exhibit a convex dermis The compound leaves are staggered, measuring 20 to 40 cm in length with 2 to 3 leaflets that are irregularly serrated and occasionally lobed The oblong root and chop emit a fragrant aroma when crushed, and the long petioles develop into large sheaths at the end, with all segments having stalks The inflorescence is a panicle at the top, adorned with small grayish-white flowers, and the fruit is ovoid, flat, and silvery white (Vo Van Chi, 1997).
Polyscias fruticosa is a moisture-loving, slightly shade-tolerant plant that thrives in various soil types, even in minimal soil conditions, resembling a bonsai When propagated by cuttings, it can bear fruit within 2-3 years This species exhibits strong clonal regeneration, allowing a stem or branch embedded in the ground to develop into a new tree (Do Huy Bich et al., 2006).
Polyscias fruticosa has numerous applications, including improving health, increasing blood circulation, and reducing rheumatic pain (Pham Hoang Loc,
2003) It also has diuretic, anti-depressant, anti-inflammatory, antipyretic, and enzyme inhibitory properties (Do Tat Loi, 2004)
Research on the chemical composition of the genus Polyscias began in the 1980s, leading to the isolation of substances from 12 of its 176 accepted species, including P amplifolia, P australiana, P balfouriana, P dichroostachya, P duplicata, P fulva, and P fruticosa To date, there have been over 200 documented cases related to this genus.
P guilfoylei, P murrayi, P nodosa, P scutellaria, P serrata)”
The isolated compounds were mainly identified as triterpenoids and triterpenoid saponins, along with polyacetylenes, essential oils, flavonoids, phenolic compounds, sterols, ceramides, cerebrosides, propanoic acid derivatives, lignans, and various cyanogen production derivatives.
Alkaloids, glucosides, saponins, flavonoids, tannins, and vitamin B1 amino acids, alike as lysine, cysteine, and methionine, are all-important amino acids in
Polyscias fruticosa (Ngo Ung Long- Military Institute, 1985) Polyscias fruticosa trunk contains the most chemical elements, while the leaves and branches have lower attention
The roots and leaves contain a variety of beneficial compounds, including saponins, alkaloids, vitamins B1, B2, B6, and C, 20 amino acids, glycosides, phytosterols, tannins, organic acids, essential oils, and numerous trace elements Additionally, the leaves are rich in triterpene saponins (1.65) and oleanolic acid, a notable genin (Do Huy Bich et al., 2006; P Corcoran, L McKay, & B Blumberg, 2012).
According to modern medicine, Polyscias fruticosa has the main effects such as:
- Improve physical strength and reduce stress by stimulating brain activity, fighting fatigue and anxiety, and boosting immunity
- Anti-inflammatory and anti-swelling
- Antihistamine and asthma treatment with plant-derived alcohol extract
- Improve memory and prolong life (tested on old mice)
According to traditional medicine, Polyscias fruticosa has the following effects:
- Leaves treat fever, swelling, and boils
- Roots are used as a tonic and diuretic
The Polyscias fruticosa trunk and branches relieve low back pain and numbness (Nguyen Thi Thu Huong, 2007)
Polyscias fruticosa roots serve as a tonic to enhance physical strength and address issues such as weakness, fatigue, and poor digestion They are particularly beneficial for postpartum women experiencing low milk production Additionally, these roots are utilized to alleviate cough, hemoptysis, uterine pain, dysentery, and function as a diuretic and anti-toxic agent.
Leaves treat colds, fevers, swollen boils, swollen breasts, allergic rashes, wounds (peel and rub) Stems and branches cure rheumatism, back pain
Polyscias fruticosa, commonly found in Southeast Asia and tropical Pacific islands, thrives in medium humidity and temperatures between 16 to 29 degrees Celsius (60 to 85 degrees Fahrenheit) This plant is widely cultivated in countries such as Malaysia, Indonesia, Cambodia, and Laos In Vietnam, cloves have a long-standing presence and are popularly grown in family gardens, pagodas, clinics, hospitals, and are used in both medicine and spices.
2.1.6 Polyscias Fruticosa part for research use
Polyscias Fruticosa trunk (Polyscias Fruticosa (L.) Harms)
Flavonoid Overview
Flavonoids are natural compounds with diverse phenolic structures found in various sources such as fruits, vegetables, grains, and beverages like tea and wine Known for their significant health benefits, flavonoids are increasingly isolated for use in nutraceutical, pharmaceutical, medicinal, and cosmetic products Their effectiveness stems from their ability to regulate essential cellular enzyme activity and their potent anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties.
Flavonoids, first identified in 1938 by Hungarian scientist Dr Albert Szent-Gyorgyi, were originally referred to as vitamin P These compounds are abundant in various foods and beverages, particularly in fruits, vegetables, flowers, and seeds, with notable sources including wine, beer, and tea The concentration of flavonoids in these plants is influenced by multiple factors such as the plant cultivar, growing conditions, soil characteristics, harvesting methods, and storage practices.
Flavonoid compounds are plant-derived substances present in various parts of plants, playing a crucial role in growth and protection against plaques These low-molecular-weight phenolic compounds are among the most distinctive classes found in higher plants, with many serving as identifiable flower pigments in angiosperms However, flavonoids are not confined to flowers; they are distributed throughout all parts of the plant.
Flavonoids, known as dietary flavonoids, are plentiful in plant-based foods and beverages, including fruits, vegetables, tea, cocoa, and wine They are categorized into various subgroups, such as chalcones.
8 flavones, flavonols, and isoflavones These subgroups each have their own major sources Onions and tea, for example, are excellent sources of flavonols and flavones
2.2.3 Structure and classification of Flavonoids
The fundamental structure of Flavonoids is C6-C3-C6, which consists of two benzene rings A and B connected by a three-carbon chain The structure can be closed or open
In many cases, the 3-carbon chain creates an oxygenated heterocycle by connecting with the A ring (the C ring) Chalcones, anthocyanidins, and auronons are identified as pigments within a diverse range of flavonoids The numbering system for the flavonoid carbon skeleton is illustrated below, with a distinct numbering for the chalcone bracket.
The oxidation level and substitution pattern of the C ring vary among different flavonoid classes, while individual compounds within each class exhibit distinct substitution patterns in the A and B rings.
Flavonoids are classified according to their biosynthetic origin and are characterized by a basic structure consisting of 15 carbon atoms arranged in a C6-C3-C6 formation, which includes two aromatic rings, A and B.
B linked by a unit of three carbon atoms, which may or may not give rise to a third ring
Classification of flavonoid includes 6 classes, 6 subclasses and 6 main natural sources
Figure 4 Structure of flavonoid subgroups
Flavonoids are classified into three major families based on the structure of the C chain in the C6C3C6 skeleton:
+ Eucoflavonoid (2-phenylbenzopyrans): flavan, flavan 3-o (catechin), flavan 4-ol, flavan 3,4-diol, flavone, flavanol, flavonol, chalcon, anthocyanin, anthocyanidin, aurone
+ Isoflavonoid (3-benzopyrans): isoflavan, isoflavan-4-ol, isoflavones, isoflavanone, rotenoid,
The flavonoid family contains over 6000 low-molecular-weight phenolic compounds that are flavan derivatives Flavones, flavonols, flavonones, flavononols, flavan-3-ols, anthocyanines, isoflavones, and chalcones are the main subgroups
The flavan core can be found in every flavonoid structure It is made up of
The structure of flavonoids typically consists of 15 carbon atoms arranged in two aromatic rings, known as A and B, which are connected by a three-carbon chain This chain is integrated into a heterocyclic central ring, referred to as C, and is characteristic of most flavonoids However, chalcones are an exception, as they feature a linear carbon chain between the A and B rings, classifying them as open-chain flavonoids.
Flavonoids, including flavones, flavonols, flavonones, flavononols, flavon-3-ols, and anthocyanins, are classified as 2-phenylbenzopyrans, distinguished by the position of the B-ring linked to the benzopyrano (or chromano) structure In contrast, isoflavonoids are categorized as 3-phenylbenzopyrans, making them positional isomers Additionally, the oxidation and saturation levels of the heterocyclic C-ring differ among the flavonoids within the 2-phenylbenzopyran subgroup.
Flavonoids are a large group of naturally occurring compounds found in plants that are mostly yellow in color but can also be green, purple, red, or colorless
Methods for isolating, analyzing, and identifying flavonoid compounds rely on their physical properties Flavon derivatives and flavols exhibit light yellow to yellow hues, while chalcones and auros display dark yellow to orange-red colors In contrast, isoflavones, flavanones, isoflavanols, flavanonols, leucoantocyanidins, and catechins are typically colorless Anthocyanidins can appear in yellow, orange, red, or purple shades, influenced by the pH of their environment (Nguyen Minh Thang, 2009).
Flavonoid glycosides and flavonoid sulfates are polar compounds that are generally insoluble in water but dissolve well in alcohol In contrast, flavonoid aglycones are soluble in organic solvents while remaining insoluble in water.
Solvent solubility: the solubility of flavonoid is not the same, which is based on the OH group and their other substituents Flavonoids in plants mainly exist in 2 forms:
Flavonoid aglycol is a free form of flavonoid known for its solubility in organic solvents like ether, acetone, and ethanol, while being insoluble in water.
Flavonoid glycosides are compounds formed by the linkage of flavonoids to sugars, making them water-soluble However, they are insoluble in non-polar organic solvents like acetone, benzene, and chloroform.
Flavonoids are notable for their capacity to absorb ultraviolet light, a property attributed to their conjugated double bond system formed by two benzene rings (A and B) and a pyran ring (C) They exhibit two primary absorption bands: Band 1, which occurs at wavelengths greater than 290 nm, and Band 2, which is observed in the range of 220 to 280 nm (Nguyen Minh Thang, 2009).
Flavonoids exhibit a varied chemical structure, leading to significant differences in their reactivity based on factors such as the position of the OH group, the presence of a conjugated double bond system, and various substituents The primary reactions of flavonoids include oxidation, hydrogen bond formation, and esterification of the OH group.
Methods for extraction of total flavonoids
Extraction is a crucial initial step in studying the biological activities of naturally occurring compounds from plants, significantly influencing the outcomes and overall success of the research.
The extraction process is carried out by different methods including:
Traditional methods : extraction by Soxhlet apparatus, exhaustive extraction and gradual infiltration
Modern methods : Extraction assisted by:
Soxhlet extraction is a continuous high-temperature process where a solid sample is placed in a porous measuring tube within a chamber The extraction solvent is heated in a flask, vaporizes into the sample tube, condenses in a condenser, and then drips back into the sample, facilitating efficient extraction.
This method is efficient as it requires less solvent and reduces processing time However, the Soxhlet extraction method has notable drawbacks, including the necessity of using toxic and flammable organic solvents, as well as the requirement for extracted flavonoids to be heat stable Additionally, several critical factors must be taken into account, such as temperature, solvent-to-sample ratio, and stirring speed.
To prepare herbal remedies, soak the medicinal herbs in a solvent within a drained water jar After a specified duration, depending on the type of herbs, allow the extract to settle at the bottom while slowly adding more solvent on top, ensuring it flows continuously through the layer of herbs without stirring The solvent should typically cover the medicinal substance by about 3-4 cm.
Simple percolation: It is a method of exhaustion that always uses a new solvent to extract the active ingredients in medicinal herbs until they are exhausted
Fractional percolation : is a technique of depletion that employs dilute extracts to extract new batches (new herbs remedies) or to extract batches with different degrees of extraction
- There are common disadvantages of the fractional extraction method: low productivity, manual labor
- The procedure is more complicated than the immersion method
The ground plant material is immersed in a sealed container with a suitable solvent and kept at room temperature for at least three days while being agitated continuously This process allows the solvents to soften and break down the plant's cell walls, releasing soluble phytochemicals Subsequently, the mixture must be filtered to separate the components.
- The uncomplicated method, no special requirement for the laboratory equipment
- Large solvent volume, lengthy processing duration, and the need for further purification When it comes to pureness, advanced extraction technology should be considered
Ultrasonication assisted extraction utilizes sound waves with frequencies ranging from 20 kHz to 10 MHz, capable of penetrating solids, liquids, and gases without being perceptible to humans This method generates high-energy gas bubbles that break down the cell wall structure of the material, enhancing the release of intracellular components.
Ultrasonic waves are utilized in transducers and ultrasonic baths to extract phenolic compounds from plants The extraction process is significantly influenced by the inherent parameters of ultrasonic devices, including amplitude, frequency, and wavelength, as well as their power and intensity, which must be optimized Additionally, the design and shape of both the ultrasonic bath and the transducer play a crucial role in enhancing the extraction efficiency.
- Easy to use, low cost, high efficiency, low organic solvent consumption and reduced extraction time
- It can be used as a simple and reliable process in a wide range of organic solvents for various phenolic compounds at large scale and industrial level
Disadvantages: Must be applied on a large scale
2.3.2.2 Pulsed Electric Microwave-assisted (PEF)
Pulsed Electric Field (PEF) technology is a growing area within High Voltage Engineering, known for its ability to generate transmembrane potential in cell membranes, thereby enhancing their conductivity and permeability This increased permeability can lead to either reversible or permanent cell membrane disintegration, which has significant applications in biomedicine, environmental solutions, and the food industry.
- Does not involve dewatering or drying process and no addition of chemicals, thus reduce the operational price
- No heating (non-thermal equipment) hence, use less energy
- The presence of air bubbles in the treatment chamber can impact dielectric breakdown, leading the PEF treatment to become less uniform
- Depending on the intensity of the applied electric field, cell membranes might be reversible or irreversible during the electroporation mechanism
- The PEF's efficiency is strongly dependent on the quantity of electric field intensity and electrode gap
The enzyme-assisted extraction approach breaks the cell wall of the source material to enhance extraction yield This approach can be used with other
18 procedures to improve total bioactive recovery from source materials
The effectiveness of enzyme-assisted extraction (EAE) depends on several key factors, including the type and dosage of enzymes used, the specific conditions required for their activity, and the optimal time-temperature combination during the process Additionally, the characteristics of the plant material, such as particle size, water content, and chemical composition, play a crucial role, along with the solvent-to-solid ratio (Azmir et al., 2013).
Enzymes have been utilized to improve flavonoid release from plant material while using as few solvents and heat as possible
Microwaves are electromagnetic waves that range in frequency from 30 to
300 MHz The thermal effect is caused by the continuous movement of polar molecules in matter under the influence of electromagnetic induction
- Time and cost savings, high efficiency, and low organic solvent consumption
- Extract multiple substances at the same time in a short period of time
- Microwave energy-absorbing solvents are potentially explosive and difficult to apply on a large scale.
Researches in our country and international researches
In 2015, George Asumeng Koffuor and colleagues conducted a research on Evaluating Muco-suppressant, anti-tussive and safety Profile of Polyscias fruticosa (L.) Harms (Araliaceae) in Asthma Management
In 2020, Rodríguez De Luna, S L., Ramírez-Garza, R E., and Serna Saldívar, S O conducted a study published in The Scientific World journal, focusing on environmentally friendly methods for extracting flavonoids from plant material Their research examined how various operating conditions affect both the yield of flavonoids and their antioxidant properties.
In 2001, Nguyen Thi Thu Huong did the research on the antidepressant and stress effects of Polyscias Fruticosa It posted on Journal of Medicine
In 2019, Nguyen Manh Tuyen at Hanoi University of Pharmacy did a study on the botanical characteristics and chemical composition of the Polyscias Fruticosa trunk
In 1990, Nguyen Thoi Nham revealed that the trunk roots, trunks, and leaves of Polyscias fruticosa are rich in glycosides, alkaloids, tannins, vitamin B1, and approximately 20 amino acids, including arginine, alanine, asparagine, glutamic acid, leucine, lysine, phenylalanine, proline, threonine, tyrosine, cysteine, and tryptophan.
In 1991, Vo Xuan Minh investigated the total saponin content in parts of
Polyscias Fruticosa with the following results: Root (0.49%), root bark (1%), root core (0.11%) and leaves (0.38%)
RESEARCH CONTENT AND METHODS
Research subject
Polyscias fruticosa (L.) Harms trunks are sourced from Thai Nguyen city, thoroughly washed, and then dried at 60°C until the moisture content is below 10% The dried samples are subsequently stored at room temperature.
Research scope
The experiments are conducted in the laboratory at the Institute of Life Sciences, Thai Nguyen University
Conducting the research from December 2021 to October 2022
Table 1 Equipments used in laboratory
3 Chemical laboratory glassware 14 Glass-stirring rods
7 Test tube rack 18 Distilled water wash bottle
10 Digital laboratory scale 21 Measuring cylinder
Table 2 Chemical used in laboratory
No Chemical name Production place
16 Distilled water wash bottle Vietnam
Chemicals must be fully prepared before conducting experiments, stored in bottles and stored properly Chemicals be stored in containers made of materials that will not react.
Research content
Content 1 : Qualitative analysis of the presence of total flavonoid in the polyscias fruticosa trunk
Content 2 : Determination on factors that influence the ability to extract total flavonoid from the Polyscias fruticosa trunk as follows:
To choose the best solvent for extracting total flavonoid from the polyscias fruticosa trunk
To evaluate of solvent concentration on extraction capability of total flavonoid content
To study on the influence of extraction duration on extraction capability of total flavonoid content
To investigate on the influence of solvent/material ratio on total flavonoid extraction content
To determine the effect of raw material size on extraction capability of total flavonoid content
To investigate on the influence of magnetic stirring on the extraction capacity of total flavonoid content.
Research method
Experiment 1: Qualitative analysis of the presence of total flavonoid in Polyscias fruticosa trunk:
The extraction of powder from the trunk of Polyscias fruticosa is performed using 70% ethanol for 20 hours at room temperature After filtering the extract, 1 ml is collected and mixed with 1 ml of a 10% Pb(CH3COO)2 solution The mixture is allowed to react for 1-2 minutes, and the presence of flavonoids in the extract is assessed by observing the color changes before and after the reaction.
23 forms a yellow precipitate after being instilled with Pb(CH3COO)2 reagent, it contains flavonoid components
Experiment 2: The effect of solvent type on extraction capability:
To investigate the influence of solvent type on extraction capability, there are 2 distinct solvents are used: distilled water (H2O), and ethanol (C2H5OH), and the experimental designs are based on 2 formulas:
Formulation 1: Extracting 1g powder of polyscias fruticosa trunk by distilled water H2O at ratio of 10/1 (ml/g)
Formulation 2: Extracting 1g powder of polyscias fruticosa trunk by ethanol
(C2H5OH) at ratio of 10/1 (ml/g)
Table 4 The effects of solvent type on extraction capability of total flavonoid content
CT Experimental Criteria Extraction Conditions
Extracting 1g powder of polyscias fruticosa trunk by distilled water (H2O)
Ratio: 10/1 (ml/g) Extracted time: 20 hours Extracted temperature: room temperature
Extracting 1g powder of polyscias fruticosa trunk by ethanol (C2H5OH)
At the conclusion of the extraction process, it is essential to assess the total flavonoid content and select the most suitable solvent The findings from this experiment will inform the design of subsequent experiments.
Experiment 3: The effect of extraction solvent concentration on the total flavonoid content
The influence of extraction solvent concentration on total flavonoid content of the research at concentration of 70%, 80% and 90% is arranged according to the following fomulas:
Formulation 3: Extracting 1g powder of polyscias fruticosa trunk by determined solvent in experiment 1 at concentration of 70%
Formulation 4: Extracting 1g powder of polyscias fruticosa trunk by determined solvent in experiment 1 at concentration of 80%
Formulation 5: Extracting 1g powder of polyscias fruticosa trunk by determined solvent in experiment 1 at concentration of 90%
Table 5 The effect of concentration of extraction solvent on the total flavonoid content
CT Experimental Criteria Extraction Conditions
Extracting 1g powder of polyscias fruticosa trunk by determined solvent in experiment 2 at different concentration of 70% (CT3);
Ratio: 10/1 (ml/g) Extracted time: 20 hours Solvent determined in experiment 2
At the end of the extraction process, determine the total flavonoid content and choose the best solvent concentration The results of experiment 3 are used to design the next experiment
Experiment 4: The effects of extraction time on the total flavonoid content:
To evaluate the effect of the extraction time on the total flavonoid content, the experiments are set up according to the following formulas:
Table 6 The effects of extraction time on the total flavonoid content
CT Experimental Criteria Extraction Conditions
1 Extracting 1g powder of poluscias fruticosa trunk by determined solvent concentration in experiment 2 and experiment 3 for
16 (CT6); 18 (CT7); 20 (CT8); 22 (CT9); and 24 hours (CT10)
Solvent and solvent concentration determined in experiment 2 and experiment
Ratio: 10/1 (ml/g) Extracted temperature: room temperature
At the end of the extraction process, determine the total flavonoid content and choose the best solvent concentration The results of experiment 4 are used to design the next experiment
Experiment 5: The effects of ratio between solvent and material on the total flavonoid content:
To evaluate the influence of ratio between solvent and material, the experiments are set up according to the following formulas:
Table 7 The effects of ratio between solvent and material on the total flavonoid content
CT Experimental Criteria Extraction Conditions
1 Extracting 1g powder of poluscias fruticosa trunk by determined solvent concentration in experiment 2 and experiment 3 at suitable duration in experiment 4 with solvent:material ratio of 5:1
Solvent and solvent concentration determined in experiment 2 and experiment 3 Suitable duration in experiment 4
At the conclusion of the extraction process, the total flavonoid content is assessed to identify the optimal solvent-to-material ratio, with the findings from experiment 5 guiding the design of subsequent experiments.
Experiment 6: The influence of raw material size on extraction capability of total flavonoid content
To evaluate the influence of raw material size on extraction capability of total flavonoid content, the experiments are set up according to the following formulas:
Table 8 Effects of raw material size on extraction capability on total flavonoid content
CT Experimental Criteria Extraction Conditions
1 Extracting 1g powder of poluscias fruticosa trunk with the raw material size at 1mm or lower (CT17); from
1 to 3 mm (CT18) and ranging from 3 to 5 mm (CT19)
Solvent and solvent concentration determined in experiment 2 and experiment 3 Suitable time in experiment 4 Determined ratio in experiment 5
At the end of extraction process, the total flavonoid content is determined and choose the best material size The results of experiment 6 are used to design the next experiment
Experiment 7: The effect of magnetic stirring on the extraction capacity of total flavonoid content
To evaluate the influence of magnetic stirring on extraction capability, the experiments are set up according to the following formulas:
Table 9 The effect of magnetic stirring on the extraction capacity of total flavonoid content
CT Experimental Criteria Extraction Conditions
1 Extracting 1g powder of poluscias fruticosa trunk using a magnetic stirrer (CT 20) and not using magnetic stirrer (CT 21)
Solvent and solvent concentration determined in experiment 2 and experiment 3 Suitable duration in experiment 4 Determined ratio in experiment 5 Suitable size in experiment 6 Extracted temperature: : room temperature
In fact, the method is based on drying a sample of Polyscias fruticosa trunk to constant weight under defined conditions
To determine the moisture content of Polyscias fruticosa trunk samples, weigh the samples and bottles prior to drying Set the oven temperature to 60 degrees Celsius and place the sample bottles in the Laboratory Dryer for 2 hours After the initial weighing, continue the drying process at the same temperature until the moisture content reaches 10% or less.
Moisture (W) is calculated as a percentage by mass determined by the formula:
X(%) mo: Weight of sample bottle (g) m: Mass of test piece before drying (g) m1: Weight of basket and sample after drying (g)
The total flavonoid content was assessed using the AlCl3 chromogenic method, establishing a calibration curve with quercetin (QE) Results are expressed as milligrams of quercetin equivalent per gram of extract (mg QE/g extract) (Chang C et al., 2002).
To prepare a quercetin solution at a concentration of 1 mg/ml, accurately weigh 0.100 g of quercetin and transfer it to a 100 ml volumetric flask Fill the flask to the mark with an 80% alcohol solution, ensuring thorough mixing Finally, cover the flask with silver paper to protect the solution from exposure to natural daylight.
To prepare a series of quercetin solutions from a standard concentration of 1 mg/ml, create dilutions of 0 μg/ml, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml For each dilution, combine 0.5 ml of the solution with 1.5 ml of 95% alcohol, 0.1 ml of 10% AlCl3, 0.1 ml of 1M CH3COOK, and 2.8 ml of water for extraction After mixing the components thoroughly, allow the mixture to stand at room temperature for 30 minutes.
Measure the absorbance A of the reference series at the selected wavelength of 415 nm Graphs representing standard curves and equations in Excel:
Figure 5 Graph showing the standard curve and the standard curve equation
Determination of total flavonoid content
Take 0.5 ml of Polyscias fruticosa trunk extract and follow the same steps to create a standard curve Define TFC in formualtion:
TFC = 10 -3 (mg/g) a: Quercetin content determined from the calibration curve (ppm)
V: Volume of extract (ml) n: Dilution factor m: Weight of sample (g)
Medology of data processing
All experiments in this research were conducted in triplicate The results were analyzed using Microsoft Excel and Minitab, presenting the data as mean with standard deviation ANOVA analysis was performed, yielding 95% confidence intervals through one-way Tukey comparison.
EXPECTED RESULTS
The result of qualitative analysis of total flavonoid found in the Polyscias
Crushed powder from Polyscias fruticosa trunk (raw material after drying) was extracted with 70% ethanol solvent during 20 hours at room temperature Finish the extraction process, filter and collect the extract
To test for the presence of flavonoids in the extract, aspirate 1ml of the extract and add 1ml of a 10% Pb(CH3COO)2 solution Allow the mixture to stand for 1-2 minutes to ensure the reaction occurs completely, and then observe the color change before and after the reaction This will help determine whether flavonoids are present in the extract.
Qualitative results of the presence of flavonoids in Polyscias fruticosa trunk
Figure 7 Extract solution (A) Figure 6 Extraction solution + Pb(CH3COO)₂
Experimental results depicted in Figure 2 reveal that the extract solution prior to the instillation of Pb(CH3COO)2 is yellow in color (A) Following the instillation, the extract solution (B) exhibits a yellow cloudy precipitate visible to the naked eye, suggesting the presence of flavonoids in the stem.
Determination on factors that influence the ability to extract total
4.2.1 Selecting the best solvents for extracting total flavonoid from the Polyscias fruticosa trunk
The solubility and stability of compounds are significantly affected by solvents with antioxidant properties, such as flavonoids and polyphenols During the extraction process, undesirable components can dissolve in the extract, potentially skewing experimental outcomes Therefore, selecting the right solvent is essential for optimizing the extraction of flavonoids.
This study investigates the extraction efficiency of total flavonoids from the trunk of Polyscias fruticosa using distilled water and 70% ethanol as solvents These solvents were chosen for their cost-effectiveness, availability, and ease of recovery The experiments utilized a solvent-to-material ratio of 10:1 (ml/g) at room temperature, with a duration of 20 hours The flavonoid content was subsequently measured, and the results are illustrated in Figure 8.
Figure 8 Effect of extraction solvent on the extraction of total flavonoids
Note: the letters a, b represent the significant difference at the 5% significance level
The flavonoid content varies significantly across different solvents, with ethanol yielding the highest concentration at 0.293±0.007 mg/g, while distilled water results in the lowest flavonoid content at 0.132±0.003 mg/g.
The study reveals a significant difference at the 5% level between the extraction of total flavonoids using ethanol and distilled water This difference arises because total flavonoids often contain non-polar components that do not dissolve well in polar solvents Ethanol proves to be the superior solvent due to its rapid denaturation effect, which disrupts cell membranes and enhances extraction conditions for antioxidant substances Additionally, ethanol is recognized as a safe and non-toxic solvent, making it the most suitable choice for extracting total flavonoids from the trunk of Polyscias fruticosa.
4.2.2 The effect of concentration of extraction solvent on the total flavonoid content
Diffusion plays a vital role in the extraction process, facilitating the movement of dissolved molecules from the center of materials to the surface and subsequently into the solvent This process also allows solvent molecules to penetrate from the outside into the material, enhancing the extraction of components The efficiency of diffusion is primarily driven by concentration differences Research indicates that ethanol concentrations significantly influence flavonoid solubility and overall flavonoid content (Le Van Viet Man et al., 2011) In this experiment, ethanol solvents at concentrations of 70%, 80%, and 90% were utilized to assess their effectiveness in extracting total flavonoid content from dried trunk material.
20 hours at room temperature in ethanol at 70%, 80% and 90% The results are depicted in Figure 9 below:
Figure 9 The effect of extraction time on total flavonoid extraction
Note: the letters a, b, c represent the significant difference at the 5% significance level
The results indicate that increasing the ethanol concentration in the solvent leads to a corresponding rise in flavonoid content Specifically, the total flavonoid content extracted using ethanol at concentrations of 70%, 80%, and 90% (v/v) were measured at 0.296±0.002, 0.331±0.008, and 0.419±0.021 mg/g, respectively.
The highest flavonoid concentration was obtained at 90% ethanol concentration, much higher than 80% concentration Meanwhile, when the concentration was 70%, the flavonoid content began to show a slight decrease
As a result, ethanol with concentration at 90% is suitable for flavonoid extraction with the highest yield
4.2.3 The effects of extraction time on the total flavonoid content
Extraction time plays a crucial role in flavonoid extraction; insufficient time can lead to incomplete dissolution of the solute in the solvent Once a specific time threshold is surpassed, the yield of the product increases minimally, potentially compromising product quality due to the risk of reverse osmosis of the solute back into the raw materials or the decomposition of compounds.
In this experiment, the stem powder was soaked in ethanol at 90% for different periods of 16, 18, 20, 22 and 24 hours at room temperature The results of the survey are illustrated in Figure 10:
Figure 10 The effect of extraction time on the total flavonoid content Note: the letters a, b, c represent the significant difference at the 5% significance level
The flavonoid content reached its peak at 20 hours of soaking, measuring 0.424±0.005 mg/g, while the lowest content was observed at 24 hours, with a value of 0.318±0.003 mg/g Statistical analysis indicated significant differences at a 5% significance level when comparing the 20-hour extraction to the 16, 18, 22, and 24-hour time points, which had flavonoid contents of 0.383±0.003, 0.388±0.003, and 0.374±0.015 mg/g, respectively These findings suggest that soaking for 20 hours in 90% ethanol enhances the efficiency of total flavonoid extraction.
4.2.4 The effects of ratio between solvent and material on the total flavonoid content
The flavonoid content extracted is significantly influenced by the solvent-to-material ratio A small difference between the solvent amount and material quantity restricts their interaction, resulting in limited diffusion and preventing the extraction of all compounds from the raw materials Once the extraction solution reaches equilibrium, the yield of extracted compounds stabilizes and does not increase further (Herode et al., 2003) To maximize flavonoid content, we conducted experiments using various solvent/material ratios: 5/1, 10/1, 30/1, 50/1, 70/1, and 100/1 (ml/g) The results, shown in Figure 11, illustrate the effects of soaking 90% ethanol for 20 hours at room temperature.
Figure 11 The effect of solvent/material ratio on the total flavonoid content Note: the letters a, b, c, d represent the significant difference at the 5% significance level
Figure 11 illustrates that the optimal flavonoid content was achieved with a solvent/material ratio of 10/1 (ml/g), resulting in a total flavonoid concentration of 0.432 ± 0.004 (mg/g).
The flavonoid content significantly decreased when extracted at various solvent-to-material ratios of 1/5, 1/30, 1/50, 1/70, and 1/100, yielding values of 0.348 ± 0.005, 0.320 ± 0.003, 0.320 ± 0.005, 0.313 ± 0.004, and 0.297 ± 0.005 (mg/g), respectively Notably, at a 5% significance level, the extraction ratio of 10/1 (ml/g) showed a significant difference compared to the other ratios.
As a result, we chose a solvent/material ratio of 10/1 (ml/g) to achieve both extraction efficiency and solvent savings
4.2.5 The influence of raw material size on extraction capability of total flavonoid content
The size and shape of the raw material significantly impact the extraction process, as a smaller material size increases the contact area with the solvent, enhancing extraction efficiency In this study, we examined three material sizes—1mm or less, 1 to 3mm, and 3 to 5mm—by immersing the samples in 90% ethanol for 20 hours at a 10/1 (ml/g) ratio to evaluate the differences in extraction outcomes The results are depicted in Figure 12 below.
Figure 12 The influence of Material size on extraction capability of total flavonoid content Note: the letters a, b represent the significant difference at the 5% significance level
Figure 12 illustrates that flavonoid content decreases with increasing particle size Specifically, the flavonoid content for different sizes was measured at 0.450±0.012, 0.351±0.02, and 0.385±0.012 (mg/g), with the highest concentration of 0.450±0.012 (mg/g) found in fine powder sized at or below 1 mm Statistical analysis indicates a significant difference in extraction efficiency between sizes less than or equal to 1 mm and those ranging from 1 to 3 mm or 3 to 5 mm at a 5% significance level.
As a result, in order to maximize extraction efficiency, size of 1 mm or less is the best choice for the next experiment
4.2.6 The effect of magnetic stirring on the extraction capacity of total flavonoid content
The efficiency of extraction is significantly influenced by the mixing of raw materials, as frequent agitation enhances the diffusion of essential oils into the solvent To explore this effect, we examined the impact of magnetic stirring on flavonoid extraction at a ratio of 10/1 ml/g, with a 20-hour immersion at room temperature and stirring for 5 hours at a time The results, depicted in Image 13, indicate whether the total flavonoid content was increased or decreased.
Figure 13 The influence of magnetic stirrer on the total flavonoid content
Note: the letters a, b represent the significant difference at the 5% significance level
The total flavonoid content with the use of a magnetic stirrer is 0.557 ± 0.005 mg/g, while without it, the content is 0.452 ± 0.018 mg/g Statistical analysis indicates a significant difference between the effects of magnetic stirring and no stirring at the 5% significance level.
As a result, in order to maximize extraction efficiency, we chose to use magnetic stirrer to contribute to the extraction process of total flavonoid
CONCLUSION & RECOMMENDATIONS
Conclusion
According to the result of experiments, we conclude the following conclusions:
- The presence of flavonoids has been determined in the extract from the trunk of Polyscias fruticosa
- Ethanol is the best solvent for getting the greatest flavonoid content during the extraction process
- The best solvent concentration for attaining optimum flavonoid content during the extraction process is 90%
- The most suitable extraction time and to achieve the greatest flavonoid content during extraction is 20 hours
- The most suitable solvent/material ratio and to obtain the highest flavonoid content during extraction is 10/1 (ml/g)
- The most suitable material size to obtain the highest flavonoid content is 1 mm
- Magnetic stirring helps to improve the extraction of flavonoid content.
Recommendations
Due to the time constraint accessible to conduct the research and, the experimental procedures do not permit it Therefore, we propose to investigate several additional issues:
- Research extraction following by various temperature conditions
- Using other methods to extract flavonoids found in Polyscias fruticosa, such as the microwave method
- Investigate and apply flavonoid preparations in food and medicine, etc to make efficient use of waste resources
APPENDIX LIST
Appendix statistical analysis
One-way ANOVA: Content versus Solvent Type
Individual 95% CIs For Mean Based on
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Solvent Type
Solvent Type = Ethanol subtracted from:
One-way ANOVA: Content versus Solvent Concentration
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev + -+ -+ -+ -
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Solvent Concentration
One-way ANOVA: Content versus Extraction Time
Individual 95% CIs For Mean Based on Pooled StDev
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Extraction Time Individual confidence level = 99.18%
One-way ANOVA: Content versus Ratio
Individual 95% CIs For Mean Based on Pooled StDev
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Ratio
One-way ANOVA: Content versus Size
Individual 95% CIs For Mean Based on Pooled StDev
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Size
One-way ANOVA: Content versus Magnetic stirrer
Individual 95% CIs For Mean Based on
Grouping Information Using Tukey Method
Means that do not share a letter are significantly different
All Pairwise Comparisons among Levels of Magnetic stirrer
Magnetic stirrer = magneticstirring subtracted from:
Magnetic stirrer Lower Center Upper