a study on the extraction of polysaccharides from ganoderma lucidum

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

Mohammad Mazbah Uddin Bahar

A STUDY ON THE EXTRACTION OF POLYSACCHARIDES FROM

Degree Program Bachelor of Food Technology Student name Mohammad Mazbah Uddin Bahar

Ganoderma lucidum is a pharmaceutical fungus that offers plentiful bioactive

compounds, which take part in enhancing human’s health This study aimed to provide more scientific insight toward the extraction of the fungus, thus creating a new

foundation for further development of specialized products based on G lucidum There

were 3 objectives, corresponding to 3 research contents The first was to determine the moisture and the ash contents of the input material In this study, the moisture content

of G lucidum was 9.51% while the ash content was 2.06% of the dry weight For

extraction, this study employed the ultrasonically-assisted extraction (UAE) and

examined the best operational conditions for extracting G lucidum Optimization was

conducted using Response Surface The results were an ultrasonically-processing time of 15 minutes, a temperature of 68oC, time of 158 minutes, and the solid-liquid ratio of 1:28 g/ml with ethanol 80% as the primary solvent This operation produced 1.55% of polysaccharides extracted from the fungus

Keywords: Ganoderma lucidum, fungal extraction, polysaccharides

Number of pages: 60 Date of Submission:

Acknowledgment

The accomplishment of this graduation thesis is one of the greatest achievements that I have had in years However, it came with much effort and the precious enthusiasm from not only myself but also others

Thank you, Doctor Luong Hung Tien, for being my supervisor and my lecturer You have always been intelligent, visionary, and you express that in really cool styles, which I love and take much inspiration from I wish you strength, success, and all the best in the future, sir

Many thanks to the lecturers who have been teaching me and my friends Without your lessons and your thorough guides, I am certain that I would not have been able to work on not just this, but also my future

The faculty of Advanced Education Program definitely helped me a lot with my progress Thanks to their schedules and information, I was able to arrange the work, which led to not just this thesis but also the completion of other plans I am grateful for that

May we all be blessed!

Muhammad Mazbah Uddin Bahar

II LITERATURE REVIEW 4

2.1 The research subject – Ganoderma lucidum 4

2.1.1 Overview of Ganoderma lucidum 4

2.1.2 Chemical composition and bioactivity 5

2.1.3 Pharmacological effects on human health 6

2.2 Usage on the global scale 7

2.2.1 Oriental herbal therapies 7

2.2.2 Modern medicine and pharmacy 8

2.3 Extraction and compound isolation techniques 9

3.3 Experiment design and methods 13

3.3.1 The quality of input materials 13

3.3.2 The extraction of Ganoderma lucidum 15

3.3.2 Process optimization 20

3.4 Data analysis 22

IV RESULTS AND DISSCUSION 23

4.2 The effects of environmental factors on extracting G lucidum 24

4.2.1 Ultrasonically processing time 24

4.2.2 Type of solvent 26

4.2.3 Solvent concentration 28

4.2.4 Extraction temperature 30

4.2.5 Extraction time 32

4.2.6 Solid – liquid ratio 34

4.3 Optimal extraction conditions for G lucidum 36

LIST OF TABLES

Table 3.1 Laboratory instruments 12

Table 3.2 Chemicals involved in the process 13

Table 3.3.1 Investigated range for ultrasound 16

Table 3.3.2 Investigated types of solvent 16

Table 3.3.3 Investigated concentrations of solvent 17

Table 3.3.4 Investigated temperature levels 17

Table 3.3.5 Investigated range of extraction time 17

Table 3.3.6 Investigated solid-liquid ratios 18

Table 3.3.7 Dilution formulas of the control solution 18

Table 3.3.8 Response surface method’s design and investigated parameters 20

Table 3.3.9 Experiment design for optimization experiments 21

Table 4.1 Moisture content and ash content of G lucidum 23

Table 4.2 Total polysaccharides from different ultrasonically processing time 24

Table 4.3 Total polysaccharides from different types of solvent 26

Table 4.4 Total polysaccharides from different solvent concentrations 28

Table 4.5 Total polysaccharides from different temperatures 30

Table 4.6 Total polysaccharides from different time 32

Table 4.7 Total polysaccharides from different solid-liquid ratio 34

Table 4.8 Input values of optimization matrix 36

LIST OF FIGURES

Figure 3.1 Standard curve of diluted glucose 19

Figure 4.1 Polysaccharides from ultrasonically-processing time 25

Figure 4.2 Polysaccharides from types of solvent 27

Figure 4.3 Polysaccharides from solvent concentration 29

Figure 4.4 Polysaccharides from temperatures 31

Figure 4.5 Polysaccharides from extraction time 33

Figure 4.6 Polysaccharides from solid-liquid ratios 35

Figure 4.7 3D interaction simulation 37

Figure 4.8 Solution 28 – Desirability 1.000 38

I INTRODUCTION 1.1 Rationale

Ganoderma is a common name of various fungi that are relatively popular as a pharmaceutical ingredient in Asian countries, especially Eastern – Southeast Asia Particularly, they take part in herbal therapies (Hijikata et al., 2005; Shanshoury et al.,

2021) The number of species in genus Ganoderma is also impressive with notable names such as G lingzhi, G subresinosum, G sinense, and G lucidum Depending

on the specific species, there are distinct profiles of chemical composition and bioactivity, thus the pharmacological effects vary accordingly (Du et al., 2019) That is why comprehension over these subjects or one of them in particular is much needed for using them in the most proper way

Despite much appreciation and acknowledgement throughout the communities

of medicine and pharmacy, Ganoderma species, particularly Ganoderma lucidum,

have not been utilized up to their potential In modern countries, there are several specialized products taking form as dietary supplements such as capsules (Wicks et al., 2007; Rossi et al., 2014) Many respected pharmaceutical companies in China have developed products with health effects of blood and heart regulation, anti-fatigue and depression, as well as anti-cancer and tumor Nevertheless, there are still many

markets remaining for the development of a specialized product from Ganoderma lucidum like Russia and Bangladesh, where this material is relatively uncommon Even in Southeast Asian countries like Vietnam, the use of Ganoderma lucidum has

only been limited with the raw form infused in hot water This technique ultimately leads to inefficient extraction, thus resulting in waste in terms of material and cost

From these facts, it is comprehensible that such a miraculous material as Ganoderma lucidum needs to be further promoted through studying and developing

That is why the study on “The extraction of Polysaccharides from Ganoderma

lucidum” was implemented This study promised to examine the Ganoderma lucidum

in terms of the total polysaccharides as well as the proper extraction method in order to optimize both the operation and the cost

1.2 Research hypotheses

Based on the rationale and the literature review, there were the following

hypotheses set for the subject of Ganoderma lucidum:

1 Ganoderma lucidum can be further promoted and disseminated in other

regions like Russia, Bangladesh, and Vietnam

2 There is a way to specialize G lucidum instead of consuming it daily as water

1.3 Research objectives

This study had 3 primary objectives as follows:

• Identify the quality of input Garnoderma lucidum

• Identify the technique for extraction as well as conditions in the technique • Optimize the extraction process, particularly the operational conditions These objectives led to the research contents of the study

1.4 Limitations

Research capability and practice: Capability exists in not only instruments,

techniques, and protocols but also in humans, who are responsible for executing the research Such a limit, although it could be minimized in many ways, still remains to

Literature inheritance: It is expected from any scientific project to employ the

results of others to discuss and further fortify that project However, there are many reasons that make such an act a limitation Prominently, the variability in terms of practice, equipment, and materials often results in values that possibly contradict the findings of others Another reason is that the accessibility to scientific resources is limited, which leads to less information and discussion

II LITERATURE REVIEW 2.1 The research subject – Ganoderma lucidum

2.1.1 Overview of Ganoderma lucidum

The classification of Ganoderma fungi depends on the color of the species and lucidum is the species with the red color It is possibly the most commonly found

variation and is considered as the best type in terms of bioactivity According to the

classification, biological characteristics, and cultivations of Ganoderma documented by Du et al (2019), Ganoderma lucidum is a fungus with a complete compositional

structure like a regular one, which means there are the mycelium, the stem, the ring,

the gill, the cap, and the spore Although the mycelium and the spore of G lucidum

are deemed usable with various beneficial bioactive compounds, the fruiting body consisting primarily of the cap and the stem is the most popular part Unlike a regular

fungal body, the fruiting body of G lucidum is relatively hardened, sharing

similarities with a wooden structure rather than a soft and limp body This part protrudes out front and accounts for most of the full proportion of an individual That is why it is easier to harvest and also offers more benefits

Figure 1: A Ganoderma lucidum’s fruiting body Credit: BGN Kingdom: Fungi

Phylum: Basidiomycota Class: Aragicomycetes Order: Polyporales Family: Ganodermaceae

Genus: Ganoderma Species: lucidum

As recorded in the Medicinal Journal of Wachtel-Galor et al (2011),

Ganoderma lucidum grows wildly in mainly China, Japan, Korea, some Southeastern

and Eastern Asian countries like Vietnam, Laos, Nepal, and a small part of Eastern Europe This is also observed in other studies regarding the natural distribution of the

fungus ( Jin et al., 2012; Du et al., 2019) That is likely the reason why G lucidum

appears more popular in these regions instead of others The study of Loyd et al

(2018) also found wild G lucidum in North America However, it was possibly the

product of migration

2.1.2 Chemical composition and bioactivity

2.1.2.1 Triterpenoids

The group of compounds that makes Ganoderma lucidum original is the

triterpenoids within the fruiting body According to Lee et al (2010), the most prominent names are lanostane triterpenoids, which are virtually not present in other

family than Ganoderma This study had also identified 18 different lanostane with 4

new compounds of butyl ganoderate A, butyl ganoderate B, butyl lucidenate N, and butyl lucidenate A Another study of Lin et al (2022) on the triterpenoid profile of GL’s fruiting body also revealed 17 different lanostanes as well as 4 new triterpenoids with various health effects The difference between the two studies proved that the

triterpenoids within Ganoderma lucidum can vary according to the origin and the

beta-(mono-, di-, tri-) The study of L F Li et al (2018) even compared the polysaccharide

of Ganoderma lucidum and Ganoderma sinense This direction had achieved

significance in terms of revealing the vast profile of polysaccharides within the fungus From that, it was possible to systematically speculate various health effects ranging from macrophage effects, tumor-suppressive effects, anti-inflammation, etc

2.1.2.3 Microelements

There are also other nutrients and constituents that contribute to the bioactivity

of Ganoderma lucidum Minerals, for example, are extremely abundant in a fruiting

body According to Wachtel-Galor et al (2011), the fungus is rich in potassium, magnesium, calcium, sulfur, and phosphorus There are also minor minerals like iron,

copper, sodium, and zinc The vitamins in G lucidum are also notable with original

types like riboflavin, niacin, and thiamin (El Sheikha, 2022)

2.1.3 Pharmacological effects on human health

With a diverse and plentiful profile of beneficial constituents and bioactive

compounds, Ganoderma lucidum offers numerous health benefits for the consumer

In terms of regular uses, the fungus was reported with abilities to enhance the immune

system The study of Zhao et al (2018) on the interaction between G lucidum extract

and mice’s immunological functions indicated that the administration of GL extract created impacts on several signaling pathways and receptors, thus enhancing the reflexes of the body while regulating the inner functions In 2020, Lei et al (2020) did not just find the same effects but also another mechanism, which referred to the effects of GL’s polysaccharides on the expression of the genes for white blood cells

The G lucidum extract also takes part in health enhancement and regulation The

considerable amount of micro-elements in the fungus also contributes to this ability

Anti-cancer is one of the most promising and prominent effect that G lucidum can offer The studies of Gao et al (2005) showed that a group of Gl polysaccharides

called Ganopoly could effectively inhibit and eliminate various cell-lines of lung

cancer like IL-2 and IL-6 Thyagarajan et al (2010) demonstrated that the

triterpenoids found in G lucidum were capable of regulating the expression of 1 and LC-3, which are the proteins in colon cancer The Gl’s triterpenoids can even

Beclin-inhibit certain protein kinases that take part in the growth of cancer cells Many other

studies have also found various positive results of G lucidum extract against breast

cancer (Bao et al., 2012), liver cancer (A Li et al., 2015), and prostate cancer (Wang

et al., 2020) In addition, the extract of Ganoderma lucidum was also proved to

contribute to the treatment of blood cancer in the study of Rossi et al (2014)

There are also other benefits that Ganoderma lucidum can offer The report of Klupp et al (2015) indicated that the G lucidum extract when treated on human

bodies had various effects of lowering blood cholesterol and reducing the risk of cardiovascular The effects on white blood cell regulation and stimulation also play a contributory role to anti-inflammation and lymphocyte function In addition, the rich nutrients within the fungus can be positive against fatigue and depression (Pazzi et al., 2020)

2.2 Usage on the global scale

2.2.1 Oriental herbal therapies

The most popular application of Ganoderma lucidum is definitely as an

ingredient for daily diet and herbal therapies It is considered the treat for longevity and health enhancement in China, Korea, Japan, and some other Asian countries In terms of daily use, the material is often infused in water to consume every day According to Bao et al (2012) and Loyd et al (2018), Chinese and Korean oriental

therapists often prescribe G lucidum as a supplement for their patients to simply

enhance perception and regulate respiration, digestion, and other essential activities

within the body For the elderly, drinking G lucidum infusion daily helps in reducing

depression, stabilizing sleep, and increasing nervous system regulation Furthermore, the bioactive compounds in the fungus can even stimulate appetite and thus promote nutrient consumption This is also recorded in other countries like Thailand and

Vietnam (Tsivileva et al., 2016; Luangharn et al., 2019) The use of Ganoderma lucidum has also been recently introduced in India The study of Bijalwan et al (2020)

clearly showed acknowledgement of the Indian market toward this fungus as well as

cultivation techniques that were presently used for propagating G lucidum The

grown fungus was then used for tea brewing and daily consumption

In herbal therapies, Ganoderma lucidum is often combined with other

ingredients, mostly other fungi and pharmaceutical herbs to produce a treatment for a particular symptom or disease Specifically, the fungus can be combined with

different types of ginseng (Panax, Myxopyrum smilacifolium, etc.) (Hijikata et al., 2005) Shanshoury et al (2021) integrated the use of G lucidum with Spirulina platensis (spiral algae), Silybum marianum (milk thistle), and Camelia sinensis (green

tea) to administer daily to human subjects, resulting in organ stabilization, enhanced kidney functions, and increased immunology Recently, the mushroom is shown to

even work with Cordyceps species and provide excellent qualities (Nie et al., 2021)

2.2.2 Modern medicine and pharmacy

Modern medicine and pharmacy often focuses on the internal constituents, which offer pharmacological effects when consumed In China, there are several

biotechnology companies that have developed specialized products from Ganoderma lucidum Most of them take form as extract in capsules Wicks et al in 2007 had researched and produce a capsules from G lucidum extract Blood tests with this

capsule showed the changes in speed, regulation, and even tolerability of the product

toward practical use In reality, the products of Ganoderma lucidum calsules in the

Chinese market are relatively diverse in designs and purposes

The use of Ganoderma lucidum has even been integrated with other herbs or pharmaceutical ingredients For example, Kim et al (2008) combined G lucidum with Duchesnea chrysantha, a local flower in China and Korea The extract provided

potent effects of inhibiting blood cancer as it blocked the metabolism of cell line

HL-60 in vitro In 2014, Rossi et al examined the combined effects of G lucidum and Ophiocordyceps sinensis, which is type of rare a parasitic fungus Tested on

professional cyclists, this combination was shown capable of preventing the decrease of the testosterone/cortisol ratio in saliva, which contributes to nonfunctional reaching and overtraining syndrome The anti-oxidation capability of these combined fungi

was also proved well The fungus can even be combined with red ginseng (Panax ginseng) to produce a condensed extract with the effects of enhancing insulin uptake

and inhibiting breast cancers (Bao et al., 2012; Hsu et al., 2021)

It is obvious that there have been significant pieces of evidence showing how

applicable Ganoderma lucidum can be in modern medicine and pharmacy However,

research and development of this material are only focused in certain countries like China and Korea The Western medical systems remain a potential market for

developing G lucidum, while surrounding areas like Russia, Bangladesh, and

Vietnam should also be accounted

2.3 Extraction and compound isolation techniques

2.3.1 Solid-Liquid extraction

Solid-liquid extraction is the based name for using a liquid solvent, which is capable of diluting the internal constituents of a solid subject to extract them The mechanism is quite simple yet efficient Particularly, it employs the affinity that the solvent has with the internal constituents and the osmotic pressure between the inner environment and the solvent environment (Priego-Capote, 2021) However, such a mechanism creates certain drawbacks like the time needed increases as the osmotic pressure decreases, or the affinity of the solvent is inadequate to extract the required constituents (Haan, 2006; W Li et al., 2021) Moreover, most solid-liquid extraction processes demand high temperatures As a result, there is quite a good chance that the internal constituents will be damaged or denatured during extraction (Ghaebi Panah et al., 2022)

Nevertheless, the advantages offered by solid-liquid extraction out-maneuvered the drawbacks as they are the reason why this is either the most popular technique of extraction or the basis for other types First, the operation is simple, leading to an insignificant likelihood that the process would go wrong Secondly, the technique is extremely versatile and adaptable with countless types of solvent that can be used based on different purposes and price This is also perceptible in the extraction of

Ganoderma lucidum The study of Choong et al (2018) extracted this fungus using

water and ethanol as the solvent and resulted in a referable fractionation profile in the retrieved polysaccharides Darsih et al (2019) tested methanol on the anti-oxidation

capability of the retrieved extract from G lucidum and reached a positive result

Specifically, the highest records of total polyphenols and the total triterpenoids extracted from the fungus were 13.152 garlic acid equivalent mg/g and 35.085 mg/g

As a result, the DPPH scavenging activity of the G lucidum extract was

approximately 23%, which is relatively high Other studies even used solvents like acetone, hexane (Cor et al., 2017), diethyl ether, butanol, and chloroform (Uddin Pk et al., 2019)

2.3.2 Advanced extraction techniques

Most of the more advanced techniques for extracting G lucidum were based on

solid-liquid extraction However, they employ additional stages, mechanisms, or instruments to carry out the process, thus minimize the drawbacks of the original technique One of the most used advanced extraction technique for this subject is super-critical fluid extraction Instead of using a regular solvent, this method replaces it with a fluid which is pushed to a super-critical state The solvent at this state possesses the traits of both a gas and a liquid, thus really flexible in extracting internal constituents (Sánchez-Camargo et al., 2014) Many studies have successfully applied

the super-critical fluid extraction to Ganoderma lucidum The study of Askin Uzel et

al (2007) extracted the fungus using supercritical CO2 and resulted in a desirable level of triterpenoids within the extract Recently, Karimi et al (2022) used this method to

extract nanoparticles from G lucidum and evaluated anti-oxidation capability The

IC50 value of the extract was ranging from 580 – 724 ppm

Another form that is also popular in extracting Ganoderma lucidum is

submerged extraction with assistance from media that are capable of breaking down cell membranes Regularly, researchers often use microwave assistance or ultrasound assistance Huang & Ning (2010) and Smiderle et al (2017) are two remarkable

studies that utilized microwave to facilitate the extraction of G lucidum On the other hand, many other studies have employed ultrasound in extracting G lucidum and

refined a particular profile of polysaccharides from the fungus (Alzorqi et al., 2017; Zheng et al., 2020) Exceptionally, the study of Chen et al (2014) even pushed the ultrasound assistance to another level when they circulated ultrasound while extracting, meaning the ultrasound was running through the whole process, not just the beginning

There are also some other notable techniques The study of Salvatore et al (2020) employed a Naviglio extractor, which generates static phase and a dynamic phase, accelerating molecular mass transfer and thus the extraction efficiency The new process was called Rapid Solid Liquid Dynamic Extraction (RSLDE) Other studies like those of Smiderle et al (2017) and Yeung et al (2022) used pressurized

solvents to hasten the extraction process for G lucidum, reducing the rate of extraction

by 20 – 30%

III METHODOLOGY 3.1 Materials

3.1.1 Material preparation

The primary material of this study if the fruiting body of Ganoderma lucidum

(Reishi, Linh Chi Do), which was purchased from Ha Giang Province, Vietnam The fruiting body of this fungus is mainly composed of a hardened cap The caps were cut into thin slices (0.5 cm thick) and then stored in plastic zip bag under a low temperature

3.1.2 Laboratory instruments

The instruments used in this study are exhibited in the table below:

Table 3.1 Laboratory instruments

Fundamental laboratory instruments like test tubes, glass stick, petri dishes, measuring cylinders, filter paper, etc, were also used during experimenting

3.1.3 Chemicals

The study employed the following chemicals:

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Chemicals involved in the process

There are 3 primary contents of this study:

Content 1: Determine the input quality of input Ganoderma lucidum This

Content 3: Optimize the extraction process for Ganoderma lucidum

3.3 Experiment design and methods

3.3.1 The quality of input materials

3.3.1.1 Moisture content

Moisture content is present in every natural material, including Ganoderma lucidum The primary mechanism of this method is to use heat and dehydrate all the

water Afterward, based on the difference in terms of weight between the input material and the output, the moisture content of the material will be determined This study employed a universal oven UN 100 Plus from Germany, which is capable of

generating natural convection heat to dry out the material within The Ganoderma lucidum was weighed exactly 10 grams and put in the oven operating at 105oC After 4 hours and every next 1 hour, the weight of the remaining was recorded The process continued until there was no significant difference the two records Then, by comparing the final weight and the initial weight, moisture content was deduced via the following equation:

%𝐻2𝑂 =𝑚𝑖𝑛𝑖𝑡𝑖𝑎𝑙 − 𝑚𝑓𝑖𝑛𝑎𝑙

𝑚𝑖𝑛𝑖𝑡𝑖𝑎𝑙 ∗ 100%

In the equation, %H2O represents the moisture content (%); minitial stands for 10

grams of the initial G lucidum; mfinal is the weight of the sample after drying The experiment was conducted in triplicate to further assert the integrity of the result

3.3.1.2 Ash content

The ash content, which refers to the percentage of mineral constituents and

inorganic salts, was determined for Ganoderma lucidum by the pyrolysis method The

mechanism of it is to break down organic bonds and disintegrate every organic

constituent, so only the inorganics remain In this study, the G lucidum was dried at

55oC until there was no weigh loss to obtain the dry sample Subsequently, a sample

of 3 grams G lucidum was installed in a crucible Afterward, the crucible was put in

a pyrolysis furnace operating at approximately 600oC in about 6 hours until all the remaining is white in color (no dark/black matter detected) After the process, the crucible was moved to a desiccation chamber to rest, preparing for weighing the ash Similar to the calculation of the moisture content, the difference between the final and the initial weight of the material will determine the ash content, particularly via the following equation:

%𝐴𝑠ℎ = 𝑚𝑓𝑖𝑛𝑎𝑙

𝑚𝑖𝑛𝑖𝑡𝑖𝑎𝑙 ∗ 100%

In the equation, %Ash represents the ash content (%); minitial stands for 3 grams

of the initial G lucidum; mfinal is the weight of the sample after pyrolysis The experiment was conducted in triplicate to further assert the integrity of the result

3.3.2 The extraction of Ganoderma lucidum

3.3.2.1 Ultrasonically-assisted Solid-liquid extraction

To extract Ganoderma lucidum, this study employed the standard method of

solid-liquid extraction with the assistance of ultrasound In the method, each extraction process requires a combination of various environmental factors, particularly the type of solvent, the solvent concentration, extraction time, temperature, and the ratio between the solvent and the material With the addition of ultrasound as a support in breaking down the cell membrane, facilitating the extraction rate, there are 6 environmental parameters in total This means there were also corresponding 6 experiments to determine the suitable and the best range of operation

for extracting G lucidum

The extraction process was conducted in a small Erlenmeyer flask with a volume

of 100 ml Each flask had 3 grams of the G lucidum slices and the solvent added in

accordance with the ratio set for the experiment Then, the flask was installed in the ultrasound water bath to initiate the breakdown of cell membranes Afterward, extraction was commenced in a temperature-controlled water bath, which was set in a certain time The time running out is when the extract was obtainable However, it needed to be filtered using a filtering membrane to remove the solid matters

For the investigation of the best range of operation for extracting Ganoderma lucidum, the designs of the 6 experiments were as follows:

Parameter 1: Time of ultrasonically assistance

Based on the principle of Ultrasonically-Assisted Solid-liquid extraction,

ultrasound was employed to break down the membrane of G lucidum cells, from that

facilitating the process of extraction The experiment to identify the most appropriate time for ultrasound assistance was conducted with flasks of sample undergoing the impact of Elma S100H, which generates 37 kHz soundwaves After finishing the process, the extract within the flasks was retrieved to measure the content of total polysaccharides to deduce the conclusion The tested range as well as other conditions for the experiment were as follows:

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Investigated range for ultrasound

Formula code Ultrasonically processing

time Other extracting conditions

Solvent concentration: 96%

Material – Solvent ratio: 1:15 g/ml

Parameter 2: The solvent

From this experiment, the flasks of sample were set through the whole process of extraction, unlike the previous After undergoing ultrasound processing, the flasks were installed in a temperature-controlled waterbath in a set amount of time Afterward, the extract was retrieved to measure the total polysaccharide content to

find out the best solvent for extracting G ludicum There were 3 solvents tested in this

experiments as the table below:

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Investigated types of solvent

Formula code Type of solvent Other extracting conditions

Time: 90 min / Temp: 60oC

Material – Solvent ratio: 1:15 g/ml

Parameter 3: The solvent concentration

There are 4 levels of solvent dilution like the table below The total polysaccharide extracted from these formulas were analyzed to assess the best solvent concentration

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Investigated concentrations of solvent

Formula code Solvent concentration Other extracting conditions

Ultrasound: Previous result Solvent: Previous result Time: 90 min / Temp: 60oC

Material – Solvent ratio: 1:15 g/ml

Parameter 4: The extraction temperature

There were 3 temperatures investigated in this study, including 60oC, 70oC, and 80oC The specific design is shown below

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Investigated temperature levels

Formula code Temperature Other extracting conditions

Parameter 5: The extraction time

Extraction time is one of the most important factors in herbal extraction as it considerably affects extraction efficiency, thus the cost of the process The range of examination was 120 minutes with records started from the 45th minute (Table 7)

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Investigated range of extraction time

Formula code Time Other extracting conditions

Solvent: Previous result

Solvent concentration: Previous result Temperature: 60oC

Material – Solvent ratio: 1:15 g/ml

Parameter 6: The ratio between material and solvent

There were 4 ratios investigated in this experiment The design was as follows:

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Investigated solid-liquid ratios

Formula code Solid-liquid ratio Other extracting conditions

3.3.2.2 Spectrometric quantification of total polysaccharides

In order to quantify the total polysaccharides extracted from Ganoderma lucidum after each experiment, the study employed the Phenol-Sulfuric Acid

carbohydrate estimation method, which was primarily based on the study of Nielsen (2010) This method was also used in many other studies with different subjects but the same purpose of quantifying total polysaccharides (Ye et al., 2008; Liu et al., 2022) In the operation, it is required to prepare a standard curve, which shows the linear interaction between the concentration and the spectrum absorbance of the standard solution In this case, the standard solution is glucose diluted in different concentration First, 100 mg of glucose was mixed with 1000 ml of deionized water to achieve the first solution with a concentration of 100 mg/l Subsequently, using the

first solution, the experiment continuingly diluted it into 6 dilution samples, corresponding to 6 concentrations as follows:

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Dilution formulas of the control solution

The first

solution 0 ml 0.5 ml 1 ml 2 ml 5 ml 10 ml

Concentration 0 mg/l 5 mg/l 10 mg/l 20 mg/l 50 mg/l 100 mg/l

Then, the diluted solutions were installed in small tubes, each had a volume of 10 ml Afterward, the colorization reaction was initiated with the addition of 1 ml Phenol 5% and 5 ml H2SO4 The tubes were let still in 30 minutes in order to finalize the reaction The result of this stage was 6 tubes of yellow-colored solution, which were processed in a spectrometer to determine the absorbance rate of each concentration to 490 nm wavelength Fitting the absorbance indexes in a linear graph, it was then possible to synthesize the following graph

Figure 3.1 Standard curve of diluted glucose

The correlation efficiency (R2) of the linear relationship was relatively high (0.99), which proves the adequate integrity of fitting The equation of the graph is 0.01146x + 0.05813 = y In this equation, y represents the absorbance rate (OD) from

the spectrometer after measuring a sample; x is the concentration of glucose, which was used as the equivalent for total polysaccharides From the equation, fitting the absorbance of the extract would result in the relative total polysaccharide content To convert the relative concentration of polysaccharides to percentage, the following equation was used:

3.3.2 Process optimization

Unlike the single-factor experiments, which focuses on the activity of only one environmental parameter, the optimization experiment was to determine the most accurate range of conditions when they function together in the extraction of

Ganoderma lucidum This study employed the Response Surface Methodology to

conduct optimization with the 3 selected variables including time, temperature, and solid-liquid ratio In this method, the input parameters are set in 3 levels Level 0 is the exact result obtained from the single-factor examination, Level -1 is the lower range, and Level +1 is the upper range For the 3 selected parameters in this experiment, the lower and upper ranges are set as the table bellows:

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Response surface method’s design and investigated parameters

Solid-liquid ratio (g/ml) 1/(C – 5) C 1/(C + 5) The parameters were then processed by the Box – Behnken matrix, which randomize the combinations among all the variables Subsequently, the matrix resulted in 17 extraction experiment with different conditions of extraction time, temperature, and material – solvent ratio The designs and corresponding variables of those experiments are as follows:

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Experiment design for optimization experiments

Experiment number

16 0 0 0

After gathering the total polysaccharides extracted under these 17 combinations, the Box – Behnken equation would base on the interaction between each two environmental components and infer the optimal ranges for the extraction of

Ganoderma lucidum

3.4 Data analysis

The data in this experiment was analyzed by Origin 2019 and PASW, which processed the ANOVA tests to scan over the credibility as well as the confidence range of the values Origin 2019 was also responsible for graphical design and exhibition

IV RESULTS AND DISSCUSION

4.1 Input indexes of Ganoderma lucidum

Table 4.1 shows the results obtain from experiments that measure the moisture

content and the ash content of the input Ganoderma lucidum’s fruiting body

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Moisture content and ash content of G lucidum

The moisture content of this study’s Ganoderma lucidum was 9.51% This result is relatively lower than the average moisture content of commercial Ganoderma lucidum often ranges from 9.7% to 11.2% (Solomon, 2018) According to the study of Kien et al (2019) on Ganoderma lucidum in Southern Vietnam, the moisture

content of a fruiting body can be stabilized at approximately 20% The moisture content of a fresh individual as well as the moisture needed for preservation vary depending on many different factors, including the cultivation conditions, nurturing processes, and the regional climate Therefore, the difference between the result of this studies and others is understandable

The ash content experiment of this study resulted in 2.06% of ash per dry weight Others like the study of Solomon (2018) showed that the ash content often ranges between 2.02% to 2.56%, which indicates that the ash of this study’s material is

regular In a study on the chemical composition of G lucidum, Veljović et al (2017)

identified the presence of Magnesium, Potassium, Calcium, and Phosphorus Other than those, there was insignificant amount of other microelements, which explains the relatively low ash content of this fungus

4.2 The effects of environmental factors on extracting G lucidum

4.2.1 Ultrasonically processing time

Ultrasound was proved extremely potent in facilitating herbal extraction in efficiency through creating cavitation bubbles in the slurry (mixture of solvent and the material) and exploding those bubbles, disrupting the cell membrane and consequently supporting extraction (Weggler et al., 2020) However, such a mechanism can affect the content of inner constituents in both quantity and quality That is why in this study, the application of ultrasound was used in the beginning instead of the entire extraction process, which was also successfully utilized in the studies of S Chen et al (2011) and Song et al (2020) on extracting polysaccharides

from Ginko bibola and Lycium barbarum, respectively The appropriate amount of time needed from 37 kHz ultrasound for assisting the extraction of Ganoderma lucidum was determined based on the results in Table 4.2 and Figure 4.1 below:

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Total polysaccharides from different ultrasonically processing time

*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05