Study on extraction process of total flavonoids from polyscias fruticosa root and leaf

Vu Thi Hanh Supervisor's Signature: This study aimed to investigate the effects of some factors on the extraction process of total flavonoid content from Polycias fruticosa root and leaf

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURAL AND FORESTRY

TRAN DIEU LINH

STUDY ON EXTRACTION PROCESS OF TOTAL FLAVONOIDS FROM

POLYSCIAS FRUTICOSA ROOT AND LEAF

BACHELOR THESIS

Study Mode : Full time

Major : Food Technology

Faculty : Advanced Education Program Office

Batch : 2018 – 2022

Supervisor : Dr Vu Thi Hanh

Thai Nguyen, 2022

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program: Bachelor of Food Science and Technology

Student name: Tran Dieu Linh

Thesis Title: Study on extraction process of total flavonoids from

Polyscias fruticosa root and leaf

Supervisor(s): Dr Vu Thi Hanh

Supervisor's Signature:

This study aimed to investigate the effects of some factors on the extraction

process of total flavonoid content from Polycias fruticosa root and leaf, including:

solvent type, concentration of extraction solvent, extraction time, ratio between solvent and material Moreover, the purpose of this study is also to determine the effects of magnetic stirring on total flavonoid extraction process Finally, after

investigating the factors influencing the extraction of flavonoids from the Polycias fruticosa root and leaf, the suitable solvent, concentration of solvent, extraction

time, solvent/material ratio, material size, and magnetic stirring capability are detemined Before process of total flavonoid content, root and leaf were washed carefully and dried at 60oC for 8 hours until the moisture of sample was under 10% After drying, the samples were chopped into small pieces and ground in a mill The samples were crushed to increase the permeability of the solvent into the raw components and therefore the extraction process’s effectiveness In these experiments, the samples were soaked at different solvent/material ratio, solution concentration, size, time, and tested to aid in stirring to determine the factors affecting extraction efficiency The results show that, the suitable solvent is ethanol (C2H5OH) at concentration of 90%, with a ratio of 10:1(ml/g) for root; 30:1(ml/g) for leaf, size1mm and needs the support of magnetic stirrer for 20 hours for high efficiency extraction process

Keywords: Polyscias fruticosa, flavonoid content, extraction, solvent,

ratio, concentration, size, room temperature, root and leaf

Number of pages: 48 pages

Date of submission November 2nd, 2022

Secondly, I would want to express my sincere gratitude to my family and friends for always being there for me, supporting me whenever I've encountered

a challenge and assisting me in getting through the tough period of finishing this bachelor thesis

Finally, due to my lack of specific expertise, I continue to have several weaknesses while studying, assessing, and presenting on the topic I am looking forward to obtaining feedback and recommendations from professors and friends

to help me enhance my thesis

TABLE OF CONTENT

CHAPTER 1 INTRODUCTION 1

1.1 Research rationale 1

1.2 Research’s objective 2

1.3 Research question 3

1.4 Limitation 3

CHAPTER 2 LITERATURE REVIEW 4

2.1. Overview about Polycias Fruticosa (L.) Harms 4

2.1.1 Characteristics of Polycias Fruticosa (L.) Harms 4

2.1.2 Overview of the chemical composition of Polycias fruticosa (L) Harms 4

2.1.3 Overview of the pharmacological composition of Polycias fruticosa (L) Harms 5

2.2 Overview of flavonoids 6

2.2.1 Definition 6

2.2.2 The origin of flavonoids 6

2.2.3 Structure and classification of flavonoids 6

2.2.4 Overview of properties of flavonoids 8

2.2.5 The biological values of flavonoid 10

2.3 Overview of methods for the total flavonoid extraction 11

2.3.1 The traditional extraction methods 12

2.3.2 The mordern extraction methods 13

2.4 Factors influencing flavonoid extraction process 15

2.5 Research situation in the world and Vietnam 17

2.5.1 Research situation in the world 17

2.5.2 Research in Vietnam 17

CHAPTER 3 MATERIALS, RESEARCH CONTENTS AND METHODOLOGY 18 3.1 Material and research scope 18

3.1.1 Material 18

3.1.2 Research scope 18

3.2 Workplace and time to proceed 18

3.3 Chemicals, equipment 18

3.4 Research content 19

3.5 Research methods 20

3.5.1 Experimental design method 20

3.5.2 Analytical methods 24

3.5.3 Data processing methods 26

CHAPTER 4 RESULTS AND DISCUSSION 27 4.1. The result of qualitative analysis of the presence of total flavonoids in the Polyscias fruticosa

4.2. Effect of some factors on the extraction process of Polycias fruticosa root and leaf 27

4.2.1 Effect of solvents for extracting total flavonoids from the Polyscias fruticosa root and leaf 27 4.2.2 Effect of solvent concentration on extraction capability of total flavonoid content 30

4.2.3 Influence of solvent time on extraction capability of total flavonoid content 31

4.2.4 Effect of solvent/material ratio on extraction capability of total flavonoid content 33

4.2.5 Effect of material size on extraction capability of total flavonoid content 34

4.2.6 Effect of magnetic stirring on extraction capability of total flavonoid content 36

CHAPTER 5 CONCLUSION AND RECOMMENDATION 38

5.1 Conclusion 38

5.2 Recommendation 38

REFERENCE 39

CHAPTER 6 APPENDIX 41

6.1 Appendix ANOVA data analysis 41

6.2 Appendix picture 58

LIST OF FIGURES

Figure 2.1 Structure of flavonoids 7 Figure 2.2 Structure of flavonoid aromatic ring 7 Figure 2.3 Basic skeleton structure of flavonoids and their classes 8 Figure 4.1 The results of qualitative analysis of flavonoid presence in the

Polycias fruticosa root and leaf 27

Figure 4.2 The effect of extraction solvent on total flavonoid extraction 29 Figure 4.3 The effect of solvent concentration on total flavonoid extraction 31 Figure 4.4 The effect of extraction time on total flavonoid extraction

capability 32 Figure 4.5 The effect of the solvent/material ratio on the total flavonoid content extraction capability 33 Figure 4.6 The effect of the material size on the total flavonoid content extraction capability 35 Figure 4.7 The effect of the magnetic stirrer on the total flavonoid content extraction capability 36

LIST OF TABLE

Table 3.1 Experiment chemicals 18 

Table 3.2 Laboratory instruments 19 

Table 3.3 The investigation of the appropriate solvents for total flavonoid extraction 20 

Table 3.4 The investigation of the influence of solvent concentration on total flavonoid extraction 21 

Table 3.5 The investigation of the influence of solvent time on total flavonoid extraction 22 

Table 3.6 The investigation of the influence of solvent/material ratio on total flavonoid extraction 22 

Table 3.7 The investigation of the influence of sample size on total flavonoid extraction 23 

Table 3.8 The investigation of the influence of magnetic stirring on total flavonoid extraction 24 

LIST OF ABBREVIATIONS

QE Quercetine Ultrasonication Assisted Extraction UAE

Enzyme – assistant extraction EAE

Microwave – assisted extraction MAE

Pressurized liquid extraction PLE

Supercritical fluid extraction SFE

CHAPTER 1 INTRODUCTION

1.1 Research rationale

Life is more and more contemporary, people are becoming increasingly concerned about their personal health, thus they are keenly interested in the helpful substances found in nature As a result, scientists have concentrated their efforts on discovering such molecules and determining how to extract them so that they might be utilized in food and medication to benefit human health Scientists have recently focused on foods that contain antioxidants, a healthful component that can help prevent cardiovascular disease and combat aging Flavonoids are one type of chemical that is useful as an antioxidant Flavonoids also have antibacterial, anti-inflammatory, analgesic, sedative, and other biological effects Flavonoids are found in many plants, fruits and vegetables Vietnam is located in the tropical monsoon hot and humid belt of Asia with ¾ of the continent's area of hills and mountains stretching from North to South These natural conditions have really favored our country with a rich and diverse forest ecological system Our country has up to 12,000 species of higher plants that not only regulate the climate but also have great potential for medicinal resources

From distant past to present day, medicinal plants have always played an important role in maintaining human health, so the research on medicinal plants has been conducted very early Herbal medicine is known to be suitable for the body's physiology, effectively treating many complex diseases with little or no side effects Therefore, the search for natural active ingredients with high biological activity is a trend that is of great interest to scientists

Polyscias fruticosa (L.) Harms (Araliaceae) is an herbal plant having a

myriad of medicinal purposes In Vietnam, this plant has been used for centuries

in traditional medicine, which is seen as being analgesic, febrifuge, and diuretic Polyscias fruticosa is widely cultivated in Vietnam, China, and other tropical countries Polycias fruticosa has been utilized as folk medicine in Vietnam to

cure ischemia and inflammation and to accelerate blood flow in the brain (Tran

Phuong Thao, 2019) Moreover, Polyscias fruticosa has long been widely known

for promoting health, increasing blood circulation, reducing rheumatic pain The

roots of Polycias fruticosa are used as a tonic to strengthen the body, treat

weakness, weak digestion, postpartum women have little milk In some places, it

is also used to treat cough, hemoptysis, uterine pain, dysentery, and as a diuretic and anti-toxic

The most active components in Polyscias fruticosa are total phenolics, flavonoids, saponin, triterpenoides Flavonoid compounds in Polycias fruticosa

have been used in medicine to treat a number of diseases such as allergic inflammation, gastric ulcer, and duodenal ulcer, helping the body to regulate metabolic processes, anti-aging, strengthen blood vessels and reduce blood cholesterol

Currently, research on the active ingredients in Polyscias fruticosa is still

limited, usually only used in traditional folk remedies It is necessary to study the method of extracting the important active ingredient total flavonoids from

Polyscias fruticosa root and leaf as a basic premise in the preparation of active

ingredients for application in medicine, pharmaceuticals, and health foods

Considering the abovementioned problems, the topic of bachelor’s thesis

is chosen as: "Study on extraction process of total flavonoids from Polyscias

fruticosa root and leaf"

1.2 Research’s objective

1.2.1 Overall objectives

The overall objective of this research is to find out the factors affecting the

extraction process of Polycias Fruticosa root and leaf with the aim towards improving the pure flavonoid extraction capacity of Polycias Fruticosa root and leaf

1.2.2 Detail objectives

The main objectives of this thesis are briefly summarized in the followings:

- To determine total flavonoid content in Polycias Fruticosa

- Qualitative analysis of total flavonoid content found in Polyscias fruticosa

root and leaf

- To Investigate potential factors that could influence the extraction process

of total flavonoids in Polyscias fruticosa root and leaf including extraction

temperature, type of solvent, concentration and extraction time, size of material

1.3 Research question

- Do the roots and leaves contain total flavonoids?

- If having, what factors can influence the extraction process from Polyscias fruticosa root and leaf?

CHAPTER 2 LITERATURE REVIEW

2.1 Overview about Polycias Fruticosa (L.) Harms

2.1.1 Characteristics of Polycias Fruticosa (L.) Harms

Polyscias fruticosa is also known as Ming aralia, which is a member of the

Subfamilia Aralioideae; the Genus Polyscias; the Familia Araliaceae and the

Regnum Plantae Polyscias fruticosa is a perennial plant, a small dicot evergreen

shrub with smooth spines that grows to be 0.8 to 1.5 meters tall The leaves are dark green in color, have a glossy texture, and are tripinnately split Flat fruit is 3-4mm length, 1mm thick and have a spout

Polyscias fruticosa is a popular plant grown as an ornamental throughout in

Vietnam, growing both in Laos and southern China The roots, stems, leaves and branches are all harvested from over 3-years-old trees in autumn, then sliced and dried for later use As a perennial tree, it can live up to few decades and often

planted in temples, family garden or hospital Polyscias fruticosa is a hygrophyte

and phototropic tree which thrives when the temperature is below 28oC

Polyscias fruticosa is also used as an ornamental plant, which is a type of plant

that many people are looking for today, not only to treat diseases, but also to grow as decorative ornamental plants in home gardens, office bonsai, and interiors (Do Tat Loi, 2004) In addition, it also has many benefits for medicine such as: improving health, increasing blood circulation, and reducing rheumatic pain (Pham Hoang Ho, 2003) It also has diuretic, anti-depressant, anti-inflammatory, antipyretic, enzyme inhibitory effects (Do Tat Loi, 2004) In

Polyscias fruticosa, there are alkaloids, glucosides, saponins, flavonoids, tannins,

vitamin B1, amino acids including lysine, xystei, and methionine which are irreplaceable amino acids

2.1.2 Overview of the chemical composition of Polycias fruticosa (L) Harms

Polycias fruticosa contains alkaloids, glucosides, saponins, flavonoids,

tannins, vitamin B1, and essential amino acids such as lysine, cysteine, and

methionine The Polycias fruticosa root has the greatest chemical components,

whereas the leaves, branches, and stems have smaller quantities (Do Tat Loi, 2004)

Root bark and leaves contain saponins, alcoloids, vitamins B1, B2, B6, vitamin C, 20 amino acids, glycocids, alkaloids, phytosterols, tannins, organic acids, essential oils, several trace elements, and 21% sugar The leaves also contain triterpene saponins (1.65%), a genin known as oleanolic acid (Do Huy Bich et al, 2006)

The National Institute of Medicinal Materials' Research Center of Ginseng and Medicinal Materials identified 5 polyacetylene chemicals from the leaves, including panaxynol, panoxydol, heptadeca - 1,8 (E) - diene - 4.6 diyn - 3,10 diol, heptateca - 1,8 (E) - diene - 4.6 diyn - 3 ol - 10 on The following two chemicals are found solely in the leaves of Panax ginseng and not in other Panax and Araliaceae plants Although 5 polyacetylene chemicals were discovered in the roots, only panoxydol, panaxynol, and heptadeca - 1,8 (E) - diene - 4,6 diyn - 3,10 diol were determined to be congruent with the substances identified in the leaves These three compounds have potent antibacterial and anti-cancer properties (Do Huy Bich et al, 2006)

2.1.3 Overview of the pharmacological composition of Polycias fruticosa (L) Harms

According to contemporary medicine, the plant has several key benefits, including: general tonic impact, improved appetite, ease of sleep and weight gain, increased vitality, enhanced capacity to work hard and recover good health, nerve cell activation, and memory enhancement (Tran Thi Kim Tuyen, 2017)

Polycias fruticosa has the following effects in Oriental Medicine:

detoxification, diagnostic, typhoid, urinary tract, lung cooling, hemoptysis, dysentery, rheumatism, limb aches and pains (Tran Thi Kim Tuyen, 2017)

The Polycias fruticosa root is used as a tonic to strengthen the body and

cure weakness, thinness, poor digestion, and women who have little milk after giving birth It is also used to treat cough, uterine discomfort, diuretic, anti-toxic, and uterine contractions in some locations (Vietnamese Ministry of Health, 2009) Colds, fevers, swelling boils, enlarged breasts, allergic rashes, and wounds

are all treated using Polycias fruticosa leaves (by peeling and rubbing)

Rheumatism and back pain are relieved by stems and branches It is used as an astringent and antimalarial in India The roots and leaves are used to treat kidney

stones, bladder stones, and dysuria as a diuretic The wound is treated using leaf powder pounded with salt (Vietnamese Ministry of Health, 2009; Do Tat Loi, 2004)

2.2 Overview of flavonoids

2.2.1 Definition

Flavonoids is a group of natural substances with variable phenolic structures, are found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine (A N Panche, 2016) Flavonoids are phenolic chemicals having a C6-

C3-C6 structure In other terms, it is a fundamental framework composed of two benzene rings A and B linked by a three-carbon chain It is a class of natural chemicals that are typically found in medicinal plants (Bui Hong Hanh, 2013)

2.2.2 The origin of flavonoids

Flavonoids were discovered in 1938 by a Hungarian scientist named Dr Albert Szent-Gyorgyi, who referred to them as “vitamin P”

In the 1930s, Albert Szent-Gyorgyi and colleagues determined that only vitamin C was not as efficient in preventing scurvy as fresh yellow extract from oranges, lemons, or paprika They ascribed the enhanced action of this extract to other chemicals in the combination, which they referred to as "citrin" (relating to citrus) or "Vitamin P." (referring to its effect in decreased capillary permeability) The compounds including hesperidin, eriodictyol, hesperidin methyl chalcone, and neohesperidin were later demonstrated to not fit the requirements for a vitamin, hence the word is now defunct (Bui Hong Hanh, 2013)

2.2.3 Structure and classification of flavonoids

2.2.3.1 Structure of flavonoids

Flavonoids are polyphenolic chains made up of 15 Carbon atoms and two benzene rings joined by a three-carbon line

The above structure may be represented as a C6-C3-C6 system

Flavonoids have a chemical structure that is based on a 15 Carbon framework with a 2nd, 3rd, or 4th B aromatic chromane

Figure 2.2 Structure of flavonoid aromatic ring Flavonoids are made up of two aromatic rings and one pyran ring:

 Aromatic ring A is the aromatic ring on the left

 Aromatic ring B is the aromatic ring on the right

 A pyran ring is an intermediate ring that contains an oxygen atom (Bui Hong Hanh, 2013)

2.2.3.2 Classification of flavonoids

Flavonoids can be subdivided into different subgroups depending on the carbon of the C ring on which the B ring is attached and the degree of unsaturation and oxidation of the C ring Isoflavones are flavonoids in which the

B ring is connected in position 3 of the C ring Those with the B ring linked in position 4 are referred to as neoflavonoids, while those with the B ring linked in position 2 can be further classified into many subgroups based on the structural characteristics of the C ring Flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones are the subgroups (A N Panche, 2016)

Flavonoids are classified into three major groups based on the structure of the Carbon chain in the C6C3C6 skeleton:

Figure 2.1 Structure of flavonoids

 Flavan, flavan 3-ol (catechin), flavan 4-ol, flavan 3,4-diol, flavone, flavanol, flavonol, chalcon, anthocyanin, anthocyanidin, aurone are examples of eucaflavonoid (2-phenylbenzopyrans)

 Isoflavan, isoflavan-4-ol, isoflavones, isoflavanone, rotenoid are all isoflavonoid (3-benzopyrans)

 Calophylloid, neoflavan, and other neoflavonoid (4-benzopyrans) (Bui Hong Hanh, 2013)

Figure 2.3 Basic skeleton structure of flavonoids and their classes

2.2.4 Overview of properties of flavonoids

2.2.4.1 The physical properties of flavonoids

Physical qualities are used to determine procedures for isolating, analyzing, and identifying flavonoid molecules Flavon derivatives are light yellow to yellow, flavols are light yellow to yellow, chalcones and auros are dark yellow to orange red, and isoflavones, flavanones, isoflavanols, flavanonols, leucoantoxyanidins, and catechins are colorless Anthocyanidins come in a

variety of colors, including yellow-orange, red and purple, depending on the pH

of environment (Nguyen Minh Thang, 2009)

Solvent solubility: Flavonoid' solubility varies based on the OH group and other substituents Flavonoids are found in plants mostly in two forms:

 Flavonoid aglycol is a flavonoid free form The property of flavonoid aglycol is dissolved in organic solvents such as ether and ethanol Water does not dissolve flavonoid aglycol

 Flavonoid glycosides are flavonoids that have been connected to sugar - glucid Flavonoid glycosides are dissolved in water Flavonoid glycosides are insoluble in non-polar organic solvents

The Ultraviolet (UV) radiation absorption capacity of flavonoids is an essential characteristic The conjugated double bond system formed by two benzene rings A, B, and pyran C rings is responsible for this absorption Flavonoids have two maximal absorption bands: band 1 at wavelengths more than 290 nm and band 2 at wavelengths between 220 and 280 nm (Nguyen Minh Thang, 2009)

In the flavonoid study areas that we researched, ethanol is the solvent chosen for extraction because it has a quick denaturing impact, breaks cell membranes, and produces ideal circumstances for invasion and antioxidant exposure Furthermore, ethanol has one polar and one non-polar end, which is comparable to the flavonoid structure

2.2.4.2 The chemical propeties of flavonoids

Flavonoids have a complex chemical structure, therefore their chemical reactivity varies greatly based on several aspects, including OH group location, conjugated double bond system, and substituents (Dias et al, 2021) The following are the fundamental responses of flavonoids:

 The OH- group reacts in three ways: oxidation, hydrogen bond creation, and esterification

 Diazotization is an aromatic ring reaction

 Complexation of the carbonyl group with metals This is a reduction reaction that related to metals like Fe, Zn, and Mg The product

oxidation-after reaction have orange, pink, or red color This reaction is only applicable to flavonoids with a C=O group at C4 and a double bond between C2 and C3 (Nguyen Minh Thang, 2009)

2.2.5 The biological values of flavonoid

Antioxidant activity

Many studies show that flavonoids have antioxidant effects on human health, and many ways by which flavonoids can directly or indirectly exhibit antioxidant activity are recognized This effect is connected to the flavonoids’ structure, depending on the amount of hydroxyl substituents it contains, with a direct relationship between the maximum number of these substituents and the higher activity of the flavonoid molecule (Mercia Marques Juca, 2018) Flavonoids in plant have been used in Eastern medicine for centuries because of their antioxidant and protecting characteristics Flavonoids in foods that are commonly utilized in Eastern medicine include scultellaria root, fennel berries, licorice, and green tea(Falcones Ferreyra et al, 2012)

Anti – inflammatory activity

While inflammation is a natural reaction to danger or harm, it must be carefully managed to avoid immune system overactivity and undesirable immunological responses Chemical flavonoids appear to have a major function

in reducing excessive inflammation with a range a variety of substances, such as polysaccharides, lectins, peptides, saponins, oils and others, from plants are able

to stimulate the immune system, exhibiting immuno- modulatory activity The effect of flavonoids on B and T lymphocytes, macrophages, natural killer (NK) cells, basophils, neutrophils, eosinophils and monocytes has been investigated (Mercia Marques Juca, 2018)

Antimicrobian activity

Among the different physiological activities of flavonoids, antimicrobial defense is still provided by polyphenols present in plants In vitro experiments have shown that flavonoids have antibacterial action against a wide range of pathogens It also has antioxidant properties and has shown significant anticancer activity (Mercia Marques Juca, 2018)

Antiviral activity

Flavonoids antiviral activity is based on their apparent effect as antioxidants, blocking enzymes, damaging cell membranes, limiting virus penetration and attaching to cells, and activating host self-defense systems Flavonoids from the flavone class, such as catechins, quercetin, epicatechins, and theaflavins, are the most sought after for their antiviral properties (Mercia Marques Juca, 2018)

Antibacterial activity

Flavonoids have antibacterial properties due to the presence of hydroxyl phenolic groups, which have affinity for proteins and function as inhibitors of bacterial enzymes as well as interfering with their manufacturing routes

Biofilms have a significant role in antibacterial action These are generated

by pathogenic bacteria and cause a variety of health issues They are vital in bacterial pathogenesis and antibiotic resistance; biofilm inhibitors will assist in the control of infectious illnesses Flavonoids have been demonstrated to inhibit the production of biofilms by Streptococcus mutans, Aeromonas shydrophila, and Escherichia coli Some flavonoids, including naringenin, kampferol, and quercetin, have been shown to suppress biofilm development in Escherichia coli Lee et al (2011) discovered that afloretin, a natural flavonoid, was a non-toxic inhibitor of the Escherichia coli biofilm

2.3 Overview of methods for the total flavonoid extraction

The flavonoid extraction process is designed using the same principles of polyphenol extraction This process is commonly carried out with methanol, ethanol, acetonitrile, acetone, or a combination of these chemical and water The needed solvent polarity depends on the kind of flavonoids (Milena Tzanova et al, 2020) For less polar flavonoid structures such as isoflavones, flavonones, and flavones, appropriate solvents include acetone, chloroform, methylene chloride, and diethyl ether; for more polar flavonoid fractions, the solvent is commonly alcohol or an alcohol-water combination

The extraction process is carried out by different methods including:

 The traditional extraction methods: Soxhlet extraction apparatus, thorough extraction, and progressive infiltration

 The mordern extraction methods: Extraction with supports from

- Ultrasonication Assisted Extraction (UAE)

- Pulsed Electric Field (PEF)

- Enzyme – assistant extraction (EAE)

- Microwave – assisted extraction (MAE)

- Pressurized liquid extraction (PLE)

- Supercritical fluid extraction (SFE)

2.3.1 The traditional extraction methods

2.3.1.1 The Soxhlet extraction apparatus

One of the most popular used techiques for extracting analytes from solid materials is Soxhlet extraction apparatus The conventional Soxhlet procedure has been used in practically every analytical laboratory since its development in

1879 To this day, the Soxhlet extraction technique is still used to compare the effectiveness of current extraction techniques

It is a high-temperature continuous extraction method A Soxhlet apparatus

is used for Soxhlet extraction: The soil sample is inserted in a chamber-housed porous "measuring tube." In the bottom flask, the extraction solvent is heated, evaporated into the sample tube, condensed in the condenser, and dripped back This approach needs less solvent and a shorter processing duration Soxhlet extract has the common drawbacks: The flavonoid extraction must be heat stable, plant samples must be dried, and poisonous and flammable liquid organic solvents must be utilized

2.3.1.2 The thorough extraction

Medicinal herbs are soaked the in the solvent and then after a certain amount of time (depending on the kind of medicinal herb), drip the extract at the bottom while simultaneously adding more solvent at the top by letting the solvent

to flow very slowly and constantly through the layer of medicinal herbs (do not stir) The solvent layer in the extraction vessel is normally buried approximately 3-4 cm above the medicinal material's surface

 Simple thorough method: A thorough method in which the active components in medicinal plants are extracted using a fresh solvent until they are exhausted

 Fractional thorough method (re-thorough): This is a method approach that uses dilute fresh batches (new medicinal herbs) or extract batches with varying degrees of extraction

Advantage of thorough method:

 Medicinal herbs are exhausted

 Conserve solvents (re-exhausted)

Disadvantage of thorough method:

 There are general drawbacks of fractional thorough method, including low productivity and physical labor

 The process is more difficult than the immersion method

 Consumption of solvents (simple thorough method)

2.3.1.3 The maceration extraction method

The ground plant material is immersed in a sealed container with a suitable solvent during the process The samples are kept at room temperature for

at least three days and are shaken frequently Solvents soften and break down the plant's cell walls during this process, releasing soluble phytochemicals

Advantage of the maceration extraction method: The simplest technique, requiring no specific laboratory equipment

Disadvantage of the maceration extraction method: Massive solvent volume, lengthy processing time, and future purification required When it comes

to purity, superior extraction technologies should be considered

2.3.2 The mordern extraction methods

There are four common types of modern extraction methods (Celeste De

Monte et al, 2014)

2.3.2.1 Ultrasonication Assisted Extraction (UAE)

Ultrasonic waves have frequencies ranging from 20 kHz to 10 MHz and can pass through solids, liquids, and gases without being sensed by humans High-energy gas bubbles are created in this extraction procedure, which disrupts

the material's cell wall structure, allowing for greater release of intracellular materials To extract phenolic chemicals from plants, ultrasonic waves are applied and utilised in two types of devices: transducers and ultrasonic baths Aside from the inherent parameters of ultrasonic devices (such as amplitude, frequency, and wavelength), their power and intensity have a significant impact

on the extraction process and must be tuned The extraction process can be influenced by the design and form of the ultrasonic bath, as well as the shape of the transducer

Advantage of Ultrasonication Assisted Extraction (UAE)

 Ease of use, low cost, high efficiency, low organic solvent consumption and shorter extraction time

 On a large scale and industrial level, it may be employed as a simple and dependable process in a wide range of organic solvents for diverse phenolic compounds

Disadvantage of Ultrasonication Assisted Extraction (UAE): It must be done on a huge scale

2.3.2.2 Microwave – assisted extraction (MAE)

Microwaves are electromagnetic waves with frequencies ranging from 30 to

300 MHz The heating effect is created by the continual movement of polar molecules in matter induced by electromagnetic induction, which causes the cell wall to break down and the active chemicals in the cell to be released into the environment

Advantage of Microwave – assisted extraction (MAE)

 Reduced time and expense, great efficiency, and little usage of organic solvents

 Extract many compounds concurrently in a short amount of time

Disadvantage of Microwave – assisted extraction (MAE):

 Solvents must absorb

 Microwave radiation can be reactive and are difficult to implement on a wide scale

2.3.2.3 Supercritical fluid extraction (SFE)

The solvent is at a temperature and pressure above its critical point in this process, and there is no surface tension Therefore, it possesses both liquid and gas qualities, which make it more efficient for the extraction of phenolic chemicals from plants Supercritical fluids' low viscosity and high diffusivity allow them to extract diverse phenolic chemicals fast and effectively

Advantage of Supercritical fluid extraction (SFE)

 It is speedy, simple to operate, and selective

 Heat-labile chemicals are permanent in this process, that needs little or no solvent

Disadvantage of supercritical fluid extraction (SFE): Expensive and only appropriate for high-value materials

2.3.2.4 Pressurized liquid extraction (PLE)

PLE uses high pressure to keep solvents liquid above their boiling point

As a result, hydrophobic molecules in the solvent have a high solubility and diffusion rate, and the solvent penetrates deeply into the substrate

Advantages:

- Reduced extraction time and solvent consumption, as well as improved repeatability

- Automation, which allows for faster extraction with less solvent

Disadvantages: Must have appropriate equipment, which is more expensive

2.4 Factors influencing flavonoid extraction process

Type of solvent

To extract the desired compounds from various source materials, different solvents are required Depending on the type of the substance to be extracted, organic or inorganic, polar or non-polar solvents ought to be used As a result, the kind of solvent must be chosen carefully in order to produce the greatest concentration of extract

Solvent concentration

Each material is extracted using one or more specific solvents throughout the extraction process The amount of substances extracted from the material can

be affected by the concentration of the solvent High or low concentrations may result in extraction of less of the desired amount or further separation of unwanted chemicals As a result, the solvent chosen will typically be of relevance

to the solvent's solubility, degree of polarity, and concentration

Extraction time

Extraction time has a significant impact on the extraction process, causing the amount of material to be removed to increase or decrease The extraction time should be long enough to allow the compounds to separate from the substance

As a result, the extraction time is chosen based on the nature of the extraction and the kind of solvent

Solvent/material ratio

The solvent/material ratio influences extraction capacity by influencing the thorough diffusion of the extracted component into the solvent When the ratio of solvent to raw materials is increased, the concentration difference increases, making it easier for soluble components to diffuse into the solvent However, because of the huge volume of solvent utilized, raising the solvent/material ratio to a certain limit would make it difficult to process after extraction As a result, the appropriate solvent ratio is utilized to optimize extraction efficiency based on the type of the substance to be collected in the raw materials

Material size

Crushing the material breaks down the tissue structure, making it easier to extract the chemical The fineness of the raw material influences the contact area between the material and the solvent, lowering extraction efficiency The better the extraction efficiency, the smaller the size of the substance However, the size

of the material is a limiting issue because if the material is too tiny, the chemicals

in the cells will be deposited in the material layer, blocking the capillary tubes and contaminating the extract, producing problems and problematic in the following processes

2.5 Research situation in the world and Vietnam

2.5.1 Research situation in the world

In 1998, Bensita Mary Bernard et al discovered that n-Butanol extract from

Polycias fruticosa leaves had anti-inflammatory properties and can decrease

edema in mice

M.B Bensita et al discovered the antibacterial activity of polyacetylene

chemicals in Polycias fruticosa leaves in a paper published in the journal Ancient

Science of Life in 1999 This antibacterial ability outperforms saponins

George Asumeng Koffuor et al wrote in 2016 on the potential of ethanol

extracts from Polycias fruticosa leaves to cure asthma

2.5.2 Research in Vietnam

Vo Xuan Minh investigated and suggested the technology of extracting

saponins from Polycias fruticosa and generating a number of dosage-form

products from this substance in 1992

Nguyen Thi Ngoc Thuy et al (2020) investigated the extraction of total

triterpenoid saponins from Polycias fruticosa leaves using cellulose enzymes

The results demonstrated that, under the identical settings, the samples with enzyme treatment performed better than the samples without treatment The material: solvent/material ratio is an appropriate parameter for the extraction of

triterpenoid saponins and the capacity to inhibit-amylase from Polycias fruticosa

The water-to-enzyme ratio is 1:30, the enzyme-to-substance ratio is 1%, and the processing duration is 40 minutes Clearly, the ultrasonic approach has aided in

the extraction of components from Polycias fruticosa

CHAPTER 3 MATERIALS, RESEARCH CONTENTS

AND METHODOLOGY

3.1 Material and research scope

3.1.1 Material

Polycias fruticosa (L) Harms root and leaf were collected in Thai Nguyen

province after 3 years of cultivation The fresh root and leaf samples are cleaned

and removed from broken slides After that these materials have been drying for

8 hours at 60oC until the moisture content reaches under 10% The root and leaf

were preserved in PE bags in the refrigerator and used during the period of this

research

3.1.2 Research scope

Research was carried out in the laboratory scale

3.2 Workplace and time to proceed

 Location: Laboratory under the Institute of Life Sciences, Thai Nguyen

University

 Implementation time: February 2022 to October 2022

3.3 Chemicals, equipment

Table 3.1 Experiment chemicals

Table 3.2 Laboratory instruments

3.4 Research content

Research content 1: Qualitative analysis of total flavonoid content found

in the Polyscias fruticosa root and leaf

Research content 2: Determination on factors that affect to extraction

process of total flavonoid content in the Polyscias fruticosa root and leaf

 To choose the most appropriate solvents for extraction process of total

flavonoid content in the Polyscias fruticosa root and leaf

 To determine of concentration of solvent on total flavonoid content extraction process

 To investigate on the effect of solvent/material ratio on the total flavonoid content extraction process

 To investigate on the effect of extraction time on the total flavonoid content extraction process

 To investigate on the influence of raw material size on the total flavonoid

content extraction process

 To investigate on the effect of magnetic stirrer on the total flavonoid

content extraction process

3.5 Research methods

3.5.1 Experimental design method

Experiment 1: Qualitative analysis of total flavonoids found in the Polyscias fruticosa root and leaf

The ground powder from the Polycias fruticosa root and leaf are extracted

with 70% ethanol for 20 hours at room temperature Filter and collect 1ml of extract at the conclusion of the extraction, then gently add 1ml of 10% Pb(CH3COO)2 solution to the collected extract and let stand for 1-2 minutes for

the reaction to occur Completely record the presence or absence of flavonoids in

the extract by observing the color phenomena before and after the reaction If the extract forms a yellow precipitate after being instilled with Pb(CH3COO)2reagent, it includes flavonoid components

Experiment 2: To investigate the most appropriate solvent for total flavonoid extraction process

To investigate the influence of extraction solvent type, two different solvents are used: water (H2O) and ethanol (C2H5OH) The experiments are set up according to the following table:

Table 3.3 The investigation of the appropriate solvents for total flavonoid

extraction

Formula Experimental criteria Extraction conditions

1 Extraction of 0.1g of leaf ground

powder and 0.5g of root ground

powder with distilled water

(H2O) and ethanol (C2H5OH)

Ratio: 100:1 (ml/g) for leaf and 50:1 (ml/g) for root Time: 20 hours

Temperature: room temperature

At the end of the process, determine the total flavonoid content, and select the optimal solvent The results of experiment 2 are used for the next experiment

Experiment 3: To investigate the influence of solvent concentration on total flavonoid extraction process

To investigate the influence of extraction solvent concentration on the flavonoid content at 70%, 80%, and 90% concentrations The experiments are set

up according to the following table:

Table 3.4 The investigation of the influence of solvent concentration on total flavonoid extraction

Formula Experimental criteria Extraction conditions

1 0.1g of leaf ground powder

and 0.5g of root ground

powder were soaked with

solvent determined at different

concentration of 70%, 80%

and 90%

Ratio: 100:1 (ml/g) for leaf and 50:1 (ml/g) for root Time: 20 hours

The solvent is determined in experiment 2

Temperature: room temperature

At the end of the process, determine the total flavonoid content, select the optimal solvent concentration The results of experiment 3 are used for the next experiment

Experiment 4: To investigate the effect of solvent time on total flavonoid extraction process

To investigate the influence of extraction solvent time on the flavonoid content at for 16; 18 20; 22 and 24 hours The experiments are set up according

to the following table:

Table 3.5 The investigation of the influence of solvent time on total flavonoid

extraction

1 0.1g of leaf ground powder or

0.5g of root ground powder

samples were soaked with

solvent for 16, 18, 20, 22, and

24 hours

Ratio: 100:1 (ml/g) for leaf and 50:1 (ml/g) for root Time: 20 hours

The solvent is determined in experiment 2

The solvent concentration is determined in experiment 3 Temperature: room

temperature

At the end of the process, determine the total flavonoid content, select the optimal extraction time The results of experiment 4 are used for the next experiment

Experiment 5: To investigate the influence of solvent/material ratio on total flavonoid extraction process

To investigate the influence of solvent/material ratio on the flavonoid content at 5:1, 10:1, 30:1, 50:1, 70:1 and 100:1 The experiments are set up according to the following table

Table 3.6 The investigation of the influence of solvent/material ratio on total

flavonoid extraction process

Formula Experimental criteria Extraction conditions

1 Extraction of 0.1g of leaf and

0.5g of root ground powder

with solvent/material ratio

At the end of the process, determine the total flavonoid content, select the optimal solvent/material ratio The results of experiment 5 are used for the next experiment

Experiment 6: To investigate the influence of raw material size on total flavonoid extraction process

To investigate the influence of raw material size with size  1mm, 1 < size  3mm, 3 <size 5mm The experiments are set up according to the following table:

Table 3.7 The investigation of the influence of sample size on total

flavonoid extraction

Formula Experimental criteria Extraction conditions

1 Extraction of 0.1g of leaf and

0.5g of root ground powder with size  1mm, 1mm < size

 3mm and 3mm < size  5mm

The solvent is determined in experiment 2

The solvent concentration is determined in experiment 3 The extraction time is determined in experiment 4 The solvent/material ratio is determined in experiment 5 Temperature: room temperature

At the end of the process, determine the total flavonoid content, select the optimal sample size The results of experiment 6 are used for the next experiment

Experiment 7: To investigate on the effect of magnetic stirring on the flavonoid content extraction process

To investigate the influence of magnetic stirring on the extraction capacity of total flavonoid content The experiments are set up according to the following table:

Table 3.8 The investigation of the influence of magnetic stirring on total

flavonoid extraction

Formula Experimental criteria Extraction conditions

1 Extraction of 0.1g of leaf

and 0.5g of root ground

powder by using magnetic

stirring and when not

using magnetic stirring

The solvent is determined in experiment 2

The solvent concentration is determined in experiment 3 The extraction time is determined in experiment 4

The solvent/material ratio is determined in experiment 5 The sample size is determined in experiment 6

Temperature: room temperature

At the end of the process, determine the total flavonoid content

3.5.2 Analytical methods

3.5.2.1 Moisture analysis method

The essence: The method is drying the Polycias fruticosa sample to

constant mass under identified conditions

Process: Weighing the raw material and sample basket before drying Increasing the temperature of the oven to 60oC After that, bake the sample basket for 2 hours Repeat the drying procedure at the above temperature after the first time of weighing until the moisture content is less than 10%

Moisture (W) is computed as a mass percentage using a formula

where:

m0: Weight of sample basket (g)

m: Mass of sample before drying (g)

m1: Weight of basket and sample after drying (g)

3.5.2.2 Total flavonoid quantification

The total flavonoid content was calculated using the AlCl3 chromogenic technique and a calibration curve using quercetine (QE) The total flavonoid content is given in milligrams of quercetin equivalent (mg QE/g extract) (Chang

C et al, 2002)

Construct the quercetine standard curve as follows:

Prepare a 1mg/ml quercetin solution in 80% alcohol solution (Preparation method: Weighing 0.100g QE precisely, then place it in a 100 ml volumetric flask and top it with 80% alcohol, shake vigorously, and cover with paper Avoid direct sunlight by silver paper)

Continue to prepare solutions with concentrations of 0 g/ml, 20 g/ml, 40 g/ml, 60 g/ml, 80 g/ml, and 100 g/ml from standard quercetin solution 1 mg/ml Draw 0.5 ml into a test tube for each dilution, then add 1.5 ml of 95% alcohol, 0.1 ml of 10% AlCl3, 0.1 ml of 1M CH3COOK, and 2.8 ml of water Shake thoroughly after adding all of the aforementioned solutions and set aside at room temperature for 30 minutes

At the specified wavelength of 415 nm, calculate the absorbance A of the reference series

Excel graphs depicting conventional curves and equations:

Total flavonoid content determination Take 0.5 ml of Polycias fruticosa extract and follow the same steps to

construct a standard curve Determine the TFC in the sample:

TFC = . 10 (mg/g) where:

a: Quercetin content calculated using the calibration curve (ppm)

V: Extraction volume (ml)

n: Dilution factor

m: Weight of sample (g)

3.5.3 Data processing methods

Each experiment in this study was conducted three times The results were computed in Microsoft Office Excel and provided as mean standard deviation ANOVA analysis results with 95% confidence in one-way Tukey

y = 0.0063 x - 0.0036 R² = 0.9958

‐0,1

0 0,1

CHAPTER 4 RESULTS AND DISCUSSION

4.1 The result of qualitative analysis of the presence of total flavonoids in

the Polyscias fruticosa root and leaf

Polycias fruticosa root and leaf crushed powder (raw material after drying) is

extracted for 20 hours at room temperature with an 80% ethanol solvent Completing the extraction procedure by filtering and collecting the extract We aspirate 1ml of extract, then slowly add 1ml of 10% Pb(CH3COO)2 solution, leave for 1-2 minutes to allow the reaction to completely, and note the color phenomena before and after the reaction

The results are shown in Figure 4.1

Figure 4.1 The results of qualitative analysis of flavonoid present in the Polycias

fruticosa root and leaf

The experimental results in Figure 4.1 reveal that the extraction solution before Pb(CH3COO)2 instillation has a clear hue (A), but the extraction solution after Pb(CH3COO)2 instillation appears as a yellow hazy precipitate (B) may be visible with the naked eye, suggesting the presence of flavonoids in the root and leaf

4.2 Effect of some factors on the extraction process of Polycias fruticosa

root and leaf

4.2.1 Effect of solvents for extracting total flavonoids from the Polyscias

fruticosa root and leaf

Solvent affects the solubility and stability of molecules having antioxidant action, such as flavonoids and polyphenols Some undesirable components may

Precipitate Precipitate

dissolve in the extract during the extraction process, influencing the experimental results As a consequence, selecting the appropriate solvent is essential for the greatest and most effective extraction of flavonoids

To investigate the capacity to extract total flavonoids from Polycias fruticosa root and leaf, distilled water and ethanol were used in this experiment These are two types of solvents that are often used to dissolve the components to

be separated due to their benefits such as low cost, ease of availability, and ease

of recovery for reuse Experiments were carried out using two solvent formulations, respectively distilled water (H2O), ethanol (C2H5OH) 70% for 20 hours at room temperature and a solvent/material ratio of 100/1 (ml/g) for leaf and 50/1 (ml/g) for root, to assess the flavonoid concentration The results are shown in Figure 4.2: