Optimization of the phenolic and antioxidants extraction from Ganoderma Lucidum using maceration method

Medicinal plants are rich in secondary metabolites and contain the active ingredients of many drugs(Davari et al., 2018). Over the years, the secondary metabolites in plants have been used to treat most diseases due to their ease of access and use and fewer side effects compared to chemical products(fazelinasb et al., 2017). Fungi are one of the types of medicinal plants that have attracted the interest of researchers in recent years. Ganoderma Lucidum is an annual fungus of the genus Basidiomycetes belonging to the class Agaricomycete, order Ployporaceae and family Ganodermataceae(chen et al., 2017). This effective medicinal fungus has been khown in East Asian countries for about 2000 years, such as Reishi or Mannetake in Japan(ten thousand years old fungus) and Ling Chih, Ling Chu and Ling Zhi(immortal fungus) in China and Korea is famous(Robertos 2004). Medicinal properties of Ganoderma Lucidum are due to the presence of biologically active compounds such as polysaccharides, sterols, amino acids, triterpenoids, alkaloids, proteins and peptides(Peterson 2006). As triterpenes and polysaccharids are the most important chemical compounds in Ganoderma that most scientific research has been done on them(stemets 2000).One of the most important polysaccharides in Ganoderma lucidum is Glucan, which strengthens the immune system(wang et al., 2002). The polysaccharides in Ganoderma lucidum also have antioxidant, antibacterial, antiviral, and radiation protection properties, and the protein in it has anti-tumor and anti-diabetic activity(Gao et al., 2004). Ganoderma lucidum contains a variety of compounds, including ketones, esters, lactones, alcohols, ethers, and hydroxybenzene (Taskin et al., 2013).The polyphenolic portion of Ganoderma lucidum consists mainly of flavonoids such as Quercetin, Routine, Myersitin, Morin, Hesperetin, and Naringenin (Saltarelli et al., 2015 , Veljovic et al., 2017).Different methods are used to extraction plant extracts such as ultrasound, microwave, supercritical fluid and maceration. Each of the obove methods has a differents ability in extracting plant compounds, depending on the sample, type of solvent used and extraction conditios.In the maceration method, the mass of the desired compound is transferred from the sample to the solvent used while the solution is in contact with the sample. Extraction also depends on the particle size and properties of the plant tissue. Extraction efficiency in maceration method is generally higer than new extraction methods and the probability of heat damage in this method is lower(wang et al 2002).

Ganoderma Lucidum was obtained from the Department of Medicinal Plants of the University of Tehran. The materials used for the tests, including ethanol and methanol solvents, were purchased from Pars Shimi Company, Iran. Folin ciocalteu reagent was purchased from Merk Company, Germany. Galic Acid, DPPH reagent, and sodium carbonate were purchased from Sigma Company, USA.Ganoderma lucidum was extracted by using the Shon method with modifications.The total amount of phenolic compounds in the Ganoderma lucidum extraction was measured by the Folin-Ciocalteu method. In this method, Folin reagent is reduced in the presence of phenolic compounds in alkaline solution and blue color is produced in the solution. Color intensity can be determined at a wavelength of 765 nm by the optical spectrometer.Evaluation of free radical inhibition activity with Diphenyl Picrylhydrazyl was measured. The IC50 factor, which represents the amount of required sample to inhibit 50 percent of free radicals, was used for better assessment of antiradical activity. The lower the IC50, the higher would be antioxidant potential (low concentrations of the sample can prevent large amounts of free radicals [ Khalili and Ebrahimzadeh 2015]. Minitab software version 16 was used to design the treatments from the response level method, the Box- Behnken model, and to assess the optimal conditions for extraction from Ganoderma Lucidum by the statistical method of the response level.

different extraction conditions (tempreature, time, and type of solvent)had significant effects on the total amount of phenolic compounds and IC 50 of Ganoderma lucidum extract, so that the total amount of phenolic compounds of produced extract from Ganoderma lucidum ranged from 7.244 to 21.089 mg100g-1, and IC 50 ranged from 2.409 to 8.369 percent. The results show that increasing temperature (20 to 40 C°), extraction time (12 to 48 h), and application of methanol solvent, compared to the use of water solvent and a mixture of 50/50 water and methanol, significantly (P ≤ 0.05) increases the total extraction of phenolic compounds and decreases the amount of IC 50 from Ganoderma Lucidum.The linear effects of all three variables of temperature, time and type of solvent, and square effect of solvent type (C2) on the total extraction of phenolic compounds and IC 50, were significant (P≤ 0.05). Meanwhile, the square effects of temperature, extraction time, interactional effects of temperature, extraction time, solvent type on the total extraction of phenolic compounds, and IC 50 from Ganoderma lucidum were not significant (P <0.05).According to Single optimization the maximum amount of phenolic composition of the produced extract from 20.9944 mg100g-1 Ganoderma lucidum with 99.32 percent desirability was achieved in the following condition: 48 h of extraction time, 40 C° of temperature, and the use of methanol solvents and the maximum antioxidant composition of the produced extract from Ganoderma lucidum which can inhibit free radicals was predicted to be 2.1128 percent with 100 percent utility belonging to 48 h extraction time, 40 C° temperature, and use of methanol solvent. According to Multiple optimizations total phenol optimum and IC 50 of the produced extract from Ganoderma lucidum. It was predicted that the optimal conditions for the produced extract from Ganoderma lucidum by macration method to achieve the maximum total phenolic composition and the minimum amount of IC 50 multiple with 99.662 percent of desirability on 48 h of extraction time, 40 C° of temperature and the using of methanol solvent that total phenol content and the IC50 were 20.9944 mg100g-1 and 2.1128 percent, respectively.

The results of this study proved that by optimizing the extraction conditions of Ganoderma Lucidum, a higer amount of its effective compounds can be achieved. Also, the response surface method can be an effective way to optimize the extraction conditions of effective compounds from Ganoderma Lucidum.