Chemicals involved in the process There are 3 primary contents of this study: Content 1: Determine the input quality of input Ganoderma lucidum.. Ultrasonically-assisted Solid-liquid ext
INTRODUCTION
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
2 in terms of the total polysaccharides as well as the proper extraction method in order to optimize both the operation and the cost.
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 infusion
3 Optimization is required to produce the most efficient extraction process while saving the cost
From those hypotheses, questions were made and information was required to answer the practicality, novelty, and topicality of this study The objectives and the work frame of this study were also inferred during the process of gathering literature bases.
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.
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 a certain extent and thwart achievements at higher levels
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
LITERATURE REVIEW
The research subject – 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
Class: Aragicomycetes Order: Polyporales Family: Ganodermaceae
5 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
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 cultivation environment
Polysaccharides in Ganoderma lucidum are also renowned for their diversity and potentials The profile of polysaccharides in Ganoderma lucidum is vast and even remains with undiscovered groups of compounds Nevertheless, there are beta- glucans, which are the representative for GL’s polysaccharides (Benkeblia, 2016)
The components of this group are often mannose, xylose, glucose, galactose, and arabinose They form a backbone of beta-1-3-D-glucopyranosyl and various branches
6 (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
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
7 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 Beclin- 1 and LC-3, which are the proteins in colon cancer The Gl’s triterpenoids can even 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).
Usage on the global scale
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
8 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)
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-
9 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.
Extraction and compound isolation techniques
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)
10 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)
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
11 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%
METHODOLOGY
Materials
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
The instruments used in this study are exhibited in the table below:
1 Drying oven UN 110 plus Germany
9 Temperature-controlled water bath China
Fundamental laboratory instruments like test tubes, glass stick, petri dishes, measuring cylinders, filter paper, etc, were also used during experimenting
The study employed the following chemicals:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Chemicals involved in the process
Research contents
There are 3 primary contents of this study:
Content 1: Determine the input quality of input Ganoderma lucidum This includes
• The initial moisture content of the material
• The initial ash content of the material
Content 2: Determine the single – factors as the conditions for extraction There were 6 factors that required examination to deduce the appropriate level for each The factors included ultrasound time, solvent, solvent concentration, extraction time, temperature, and material – solvent ratio
Content 3: Optimize the extraction process for Ganoderma lucidum.
Experiment design and methods
3.3.1 The quality of input materials
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
14 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 105 o C 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:
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
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 55 o C 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 600 o C 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:
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
16 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:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Formula code Ultrasonically processing time Other extracting conditions
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:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Formula code Type of solvent Other extracting conditions
Time: 90 min / Temp: 60 o C Material – Solvent ratio: 1:15 g/ml
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
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Formula code Solvent concentration Other extracting conditions
Ultrasound: Previous result Solvent: Previous result Time: 90 min / Temp: 60 o C Material – Solvent ratio: 1:15 g/ml
There were 3 temperatures investigated in this study, including 60 o C, 70 o C, and 80 o C The specific design is shown below
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Formula code Temperature Other extracting conditions
Material – Solvent ratio: 1:15 g/ml Other conditions: Previous results
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 45 th minute (Table 7)
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Investigated range of extraction time
Formula code Time Other extracting conditions
Solvent: Previous result Solvent concentration: Previous result Temperature: 60 o C
Parameter 6: The ratio between material and solvent
There were 4 ratios investigated in this experiment The design was as follows:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
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
19 first solution, the experiment continuingly diluted it into 6 dilution samples, corresponding to 6 concentrations as follows:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Dilution formulas of the control solution
The first solution 0 ml 0.5 ml 1 ml 2 ml 5 ml 10 ml
Deionized water 10 ml 9.5 ml 9 ml 8 ml 5 ml 0 ml
Concentration 0 mg/l 5 mg/l 10 mg/l 20 mg/l 50 mg/l 100 mg/l
20 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 (R 2 ) 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
21 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:
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
RESULTS AND DISSCUSION
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
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Moisture content and ash content of G lucidum
Criteria Moisture content Ash content
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
The effects of environmental factors on extracting G lucidum
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:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Total polysaccharides from different ultrasonically processing time
Ultrasound time 10 min 15 min 20 min
*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05
Figure 4.1 Polysaccharides from ultrasonically-processing time
Compared to 0.27% of polysaccharides extracted from G lucidum with 10- minute ultrasound processing, the efficiency of 15 minutes increased substantially by approximately 84% to 0.53% of polysaccharides However, the use of 20 minutes resulted in a reduced number of only 0.50% with significant difference at a confidence level of 95% The use of ultrasound assistance can be adjusted depending on the model of the machine, the power input, the mode of operation, and operator’s requirements
As the matter of fact, many studies extracting Ganoderma lucidum utilized different range of ultrasound assistance The study of Shen et al (2020) only required 5.4 minutes However, it employed a great amount of solvent and a temperature of 90 o C
27 with a reflux condition Alzorqi et al (2017), on the other hand, used 45-65 minutes but for the extraction of only β-D-glucan polysaccharides without any concerns toward other substances The power of ultrasound was also only 20 kHz In this study, 15 minutes was concluded the optimal range for ultrasound application
Solvent is perhaps present in every herb or fungal extraction process The solvent can vary substantially in types, from water to complex compounds like ether, alkaline, aldehydes, etc This study investigated ethanol, methanol, and water for extracting Ganoderma lucidum Using the total polysaccharides measured from the extract, the results of this experiment were exhibited as follows:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Total polysaccharides from different types of solvent
*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05
Figure 4.2 Polysaccharides from types of solvent
Overall, the performance of ethanol was higher than that of methanol with 0.90% polysaccharides extracted compared to 0.77 In many aspects, these two alcohols share similarity as solvent for herbal extraction However, in this case, methanol has some drawbacks Prominently, its affinity toward non-polar is lower compared to ethanol (Zarrinmehr et al., 2022) The study L F Li et al (2018) identified various polysaccharides in Ganoderma lucidum and showed the content of non-polar, consequently explaining the effect of methanol’s drawback in dissolving non-polar contents Furthermore, the boiling point of methanol is only about 65 o C At 60 o C in a non-reflux condition of this study, the solvent evaporates and leads to degradation
29 Water extraction performed as anticipated when its result was 0.42 % of polysaccharides (w/w), much lower than the other two alcohol solvents This result was in good alignment with the study of Ma et al (2013), which also used water extraction for G lucidum and obtained approximately 0.56%
Therefore, Ethanol was selected as for its superior performance as well as affordability and accessibility
There were 5 different ethanol concentrations tested in this study, including 50, 60, 70, 80, and 90% The results were shown in Table 4.4 and Figure 4.3 as follows:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Total polysaccharides from different solvent concentrations
*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05
Figure 4.3 Polysaccharides from solvent concentration
Statistically, the extraction efficiency of ethanol 80% and 90% did not have any significant difference at a confidence level of 95% This also means concentration higher than 80% did not make extraction more efficient The commercially-pure ethanol (96%) even performed more poorly with only 0.91% polysaccharides extracted as for its lack of water, which has affinity toward certain bioactive constituents (Zarrinmehr et al., 2022) The maximum polysaccharide extraction efficiency in this experiment was 0.99%, achieved at the concentration of 80% Lower concentrations of 60% and 70% produced less efficiency with 0.92% and 0.83%, respectively Therefore, 80% was selected as the primary solvent concentration for
31 extracting Ganoderma lucidum The study of Alzorqi et al (2017) also employed 80% ethanol as the primary solvent for extracting β-D-glucan polysaccharides from G lucidum Aligning the studies of Zhu et al (1999) on fungal triterpene and Choong et al (2018) on various species fungal extraction, 80% is preferable concentration for extracting fungi thanks to the balance between solubility and selectivity for complex organic forms
Different temperatures led to different extraction rate In this study, after examining 3 levels including 60 o C, 70 o C, and 80 o C, the results were demonstrated on Figure 4.4 and Table 4.5 as follows:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Total polysaccharides from different temperatures
*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05
From 60 o C to 70 o C, it was noticed that the content of polysaccharides rose drastically from 1.01% to 1.19%, which is the maximum level in this experiment
Afterward, at 80 o C, it decreased considerably to 0.96% According to Jawade &
Chavan (2013), the higher the temperature, the more kinetic-active matters are
Consequently, diffusion between the Ganoderma lucidumi’s internal environment and the solvent increased, which explains the rise from 60 o C to 70 o C This trend of increase is also similar to the study of Ma et al (2013) on extracting Ganoderma lucidum from 50 o C to 70 o C
33 Among many studies on the extraction of herbs and fungi, the level of suitable temperatures for extracting polysaccharides or carbohydrates was relatively consistent Particularly, most resulted in the temperature of 80 o C, at which polysaccharides of herbs and fungi (e.g Panax ginseng, shiitake mushroom) would remain stable (Galoburda et al., 2015; H M Kim et al., 2020) However, when the temperature was pushed up to 100 o C, the polysaccharide contents degraded significantly Despite that, the suitable temperature in this study for extracting
Ganoderma lucidum could only be 70 o C due to the non-reflux experimental condition, which could not prevent solvent evaporation and subsequently degradation
Observation over the 80 o C flasks showed the amount of solution was lower than that of others, proving evaporation played a non-negligible role in extraction of this study
Furthermore, the final result obtained from 70 o C was relatively comparable to the study of Kan et al (2015), which chose 66 o C as the primary input temperature for extracting G lucidum and the study of Ma et al (2013), which also used 70 o C to yield approximately 2% of Gl’s polysaccharides
Extraction time is a key factor in any extraction process as it does not only affect efficiency but also the economic prospects The total polysaccharide contents obtained from various extraction time were shown in Table 4.6 and Figure 4.5 below:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Total polysaccharides from different time
*Note: Values in the same row with different lowercase letters indicate significant differences at α = 0.05
Figure 4.5 Polysaccharides from extraction time
In general, the total polysaccharides extracted from G lucidum increased through time This tendency is ordinary as in almost every extraction process when the osmotic pressure between the inner environment and the solvent was unbalanced, causing matters including polysaccharides diffusing to the extract (Raghav & Kumar, 2018) However, as matters diffused to the solvent, the osmotic pressure reduced and it led to the decrease in terms of the extraction rate like in this experiment Particularly, in 60 minutes from the 60 th min to the 120 th min, the total polysaccharide content rose by 0.33 g/100g (from 0.83 to 1.16), corresponding to a rate of 109.4% However, in the next 60 minutes, the rate of increase was only 11.3%, corresponding to an increase
Optimal extraction conditions for G lucidum
From the experiments on single-factors, the optimal conditions for extracting
Ganoderma lucidum were an ultrasonically-processing time of 15 minutes, a temperature of 70 o C, extraction time of 150 minutes, and a solid-liquid ratio of 1:25 with ethanol 80% as the primary solvent The optimization experiment was conducted on temperature, time, and solid-liquid ratio with the following design in terms of input values:
Table Error! Use the Home tab to apply 0 to the text that you want to appear here
Input values of optimization matrix
The analysis of Box-Behnken required 17 experiments using various combinations of the 3 investigated parameters After obtaining the results of the 17 experiment, response surface was conducted and proposed Figure 4.7 as follows:
According to Figure 4.7., overall, the interactions between time – temperature, solid-liquid ratio, and time – liquid-solid ratio share prominent features in order to maximize the output polysaccharides extracted Particularly, interactions with the parameter temperature often gave the highest result in the region of 69 o C In other words, the optimized process needs to slightly reduce in temparature For time, the maximum polysaccharides reside the upper limits Finally, the Box-Behnken matrix suggested that solid-liquid ratio should be between 1:26 g/ml – 1:28 g/ml The lack of fit was not significant while the pure error was inconsiderable (appendix 1) Taking a solution from the set (Figure 4.8.), the optimized extraction having a temperature of
39 68 o C, extraction time of 160 minutes, a solid-liquid ratio of 1:28 g/ml would result in 1.55% polysaccharides extracted from Ganoderma lucidum
CONCLUSION
Conclusion
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 68 o C, 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
Compared to other studies, this range is relatively good, providing an insight toward a production process using UAE extraction to specialize Ganoderma lucidum.
Recommendation
In general, the extraction process was relatively successful, there remains room for improvement Particularly, the phenol-sulfuric acid method of quantifying polysaccharides, no matter how precise it is, could be further fortified with a step of polysaccharide isolation from the G lucidum extract From that, the absorbance of the tested sample would be more reliable
A reflux condition would definitely have contributed to the examination of temperature, providing more insight toward the interaction between the extraction efficiency and temperature, particularly
APPENDIX
Data analysis
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Dependent Variable:Polysaccharides (I) Ultrasound (J) Ultrasound Mean
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Dependent Variable:Polysaccharides (I) SolventType (J) SolventType Mean Difference
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Dependent Variable:Polysaccharides (I) SolventConcentration (J) SolventConcentration Mean
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Dependent Variable:Polysaccharides (I) Temperature (J) Temperature Mean
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Dependent Variable:Polysaccharides (I) Time (J) Time Mean
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Test of Homogeneity of Variances
Levene Statistic df1 df2 Sig
Sum of Squares df Mean Square F Sig
Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3.000
Dependent Variable:Polysaccharides (I) LiquidSolid (J) LiquidSolid Mean
95% Confidence Interval Lower Bound Upper Bound L
* The mean difference is significant at the 0.05 level