Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Shizhong Zheng; Weirui Zhang; Shengrong Liu

doi:10.1371/journal.pone.0244749

. 2020 Dec 31;15(12):e0244749. doi: 10.1371/journal.pone.0244749

Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Shizhong Zheng ¹, Weirui Zhang ^1,², Shengrong Liu ^1,^*

Editor: Branislav T Šiler³

PMCID: PMC7774858 PMID: 33382761

Abstract

Ganoderma lucidum (Fr.) Krast, commonly known as “Lingzhi” in Chinese, is a medicinal mushroom that is rich in biologically active substances. Polysaccharides and triterpenoids are the two major components responsible for the bioactivity of this fungus. In the present study, the ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids from G. lucidum was optimized using response surface methodology with a desirability function, with the equal importance for the two components. Following single factor experiments, the optimal conditions were determine as ultrasonic power of 210 W, extraction temperature of 80C, ratio of liquid to solid of 50 mL/g, and 100 min extraction time, using aqueous ethanol (50%, v/v) as the extracting solvent. Under the optimal conditions, the extraction yields of polysaccharides and triterpenoids reached 0.63% and 0.38%, respectively. On the basis of the scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl and evaluation of reducing power, the antioxidant capacities of the polysaccharides obtained by optimal UACE process were higher than those of polysaccharides extracted using traditional hot water extraction, whereas the triterpenoid-rich extracts showed antioxidant activities similar to those obtained using the ethanol maceration method. The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum. The developed UACE process could be useful in preparation of a polysaccharide- and triterpenoid-rich ingredient that holds great promise for application in the Ganoderma industry.

Introduction

Ganoderma lucidum (Fr.) Krast belonging to the family of Polyporaceae, commonly known as “Lingzhi” in Chinese and “Reishi” in Japanese, is one of the most highly prized medicinal mushrooms. Its fruiting bodies have been used as a folklore remedy to treat various illnesses and promote longevity for thousands of years in many oriental countries like China and Korea [1]. In recent years, there has been a growing worldwide interest in utilizing G. lucidum in the biomedical industry due to its pharmacological properties. Currently, a variety of G. lucidum products such as slices, tea, capsules, tablets, and drinks are available on the market, and commonly consumed in many parts of the world [2].

G. lucidum in the form of fruiting bodies, mycelium, and spores contains a broad range of bioactive compounds, including polysaccharides, triterpenoids, phenols, steroids, lectin, amino acids, nucleosides, and nucleotides [3]. Among these, the polysaccharides and triterpenoids are receiving special attention from researchers in the field of biomedicine. Several polysaccharides such as β-d-glucans and glycoprotein are generally recognized as having the most noticeable biological activity such as antitumor, antioxidant and immunomodulatory effects among various Ganoderma polysaccharides [4, 5]. The triterpenoids from this fungus exhibit structural diversity, and many of them such as ganoderic acids Me and T (a kind of highly oxygenated lanostane-type triterpenoids) have been proven to have potent antitumor activity [6]. In this context, much effort has been paid to the polysaccharides and triterpenoids of G. lucidum, including extraction techniques [7, 8], their pharmacological activities and mechanisms of action [9, 10], and fermentative production [11, 12].

Polysaccharides are generally prepared by traditional hot water extraction due to its easy operation and low instrument input [13]. However, it also has many shortcomings such as low extraction yield and long extraction duration, as well as requirement of high temperature [14]. To overcome these problems, a variety of novel techniques, including pressurized liquid extraction [15], ultrasound-assisted extraction (UAE) [16], and hydrothermal extraction [17], have been developed in extracting G. lucidum polysaccharides. For the extraction of triterpenoids, maceration as a conventional method is adopted widely using organic solvents as an extraction solvent [18]. A novel technique of microwave irradiation was shown to be effective for the extraction of triterpenoids from this fungus [7].

In recent years, the simultaneous extraction of multiple biologically active components from plants has become increasingly popular in order to improve extraction efficacy and reduce the number of extraction steps [19, 20]. However, there have been no available reports on the simultaneous extraction of multiple objective constituents from mushrooms to date. Most of previous studies pertaining to the extraction of polysaccharides and triterpenoids from G. lucidum have mainly focused on the extraction yield of one or the other [7, 8]. Hence, the significant loss of either polysaccharides or triterpenoids occurred during the extraction, and this would lead to a waste of resources. To our knowledge, the simultaneous extraction of polysaccharides and triterpenoids in G. lucidum has not yet been reported to date. In the present study, to better utilize G. lucidum, ultrasound technology was examined for the co-extraction of polysaccharides and triterpenoids, and an optimization study is essential.

Response surface methodology (RSM), a collection of mathematical and statistical techniques, is widely used for optimizing complex processes [21, 22]. The major advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions. RSM has been employed for optimizing the extraction of G. lucidum polysaccharides and ganoderic acids [8, 23] and triterpenoids from the medicinal fungus Sanghuangporus sanghuang [24]. In some optimization studies, multiple desirable response variables are commonly required. As desirability functions can transform two or more responses with different relative importances into a single objective function; thus, it is a desirable approach for the optimization of a multiple-response process [25]. It was used in tandem with statistical experimental designs for optimization of fermentation conditions [26], and extraction processes [27].

In the present study, the ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids was evaluated using ultrasound technology as an attempt to better utilize G. lucidum. The extraction parameters were optimized using RSM with a desirability function by setting the equal importance for the two components. Furthermore, to provide a scientific basis for better utilization of polysaccharides and triterpenoids from the optimal UACE, their in vitro antioxidant activities were investigated and compared with those of polysaccharides and triterpenoids obtained using conventional extraction approaches. The UACE process developed here could be economically useful and convenient in the preparation of a polysaccharide- and triterpenoid-rich ingredient that holds great promise for the Ganoderma industry.

Materials and methods

Materials and reagents

The dried G. lucidum was obtained from a local pharmacy (Kang-Bai-Jia Medical Chain Store, Fujian, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH), ursolic acid, ferric chloride, and trichloroacetic acid were products of Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemical reagents used were of analytical grade.

Hot water extraction of polysaccharides

The dried G. lucidum was grounded in a grinder (DFY-500, Zhejiang Linda Co. Ltd., China) to a fine powder (40 mesh). The fine powder sample was pretreated with 80% ethanol to remove some colored materials, oligosaccharides, and some small molecule materials [28], and then dried at 40C in an oven. The treated powder (1 g) was mixed with 40 mL of deionized water in a 150 mL flask, and extracted for 2 h at 95C. The extraction procedure was repeated once. The extract was centrifuged at 8000 ×g for 10 min. The combined supernatants were used for the estimation of crude polysaccharides or for further purification by ethanol precipitation when used in the assay of antioxidant activity.

Ethanol maceration for extraction of triterpenoids

A fine powder (1 g) of G. lucidum was extracted with 40 mL of 95% ethanol for 6 h at 30C with gentle shaking in a 150 mL flask. The flask was sealed with aluminum foil during the extraction. The extraction procedure was repeated once. The extraction solution was centrifuged at 8000 ×g for 10 min. The supernatants were combined, and used for measurement of triterpenoids and the assay of antioxidant activity.

UACE of polysaccharides and triterpenoids

The UACE was performed in an ultrasonic cleaning bath with a 3 L usable capacity (KQ-800KDE, Kun Shan Ultrasonic Instruments Co. Ltd., Jiangsu, China). The ultrasonic frequency was 40 kHz. Each of 1 g of G. lucidum fine powder was extracted with an aqueous ethanol in a 150 mL flask, and the other conditions were described elsewhere. The content of the flask was centrifuged at 8000 ×g for 10 min. The polysaccharides and triterpenoids in the resulting supernatant were determined. The yield for polysaccharides or triterpenoids was calculated according to the following equation:

E x t r a c t i o n y i e l d (%) = \frac{W e i g h t o f p o l y s a c c h a r i d e s o r t r i t e r p e n o i d s}{W e i g h t o f G . l u c i d u m p o w d e r} \times 100

(1)

Desirability function

On the basis of the desirability function [25], the extraction yields of polysaccharides and triterpenoids were compiled into one index (D value) according to Eqs (2) and (3):

d_{i} = {\begin{matrix} 0 \\ \frac{Y_{i} - Y_{\min}}{Y_{\max} - Y_{\min}} \\ 1 \end{matrix} \begin{matrix} Y_{i} \leq Y_{\min} \\ Y_{\min} < Y_{i} < Y_{\max} \\ Y_{i} \geq Y_{\max} \end{matrix}

(2)

where Y_i is the response value of an i-analyzed factor.

D = d_{1}^{w_{1}} \times d_{2}^{w_{2}} \times \dots \times d_{m}^{w_{m}}

(3)

where d_i represents individual desirability value and w_i is relative importance, which indicates the difference in the importance ascribed to different response variables. In Eq (3), w_i should be in the range of 0−1, and w₁ + w₂ + … + w₃ is 1.

According to the preliminary study, the parameters of the desirability function in this study were set as Y_min = 0.1%, Y_max = 2.0%, importance w = 0.5 for polysaccharides, and for triterpenoids, they were as Y_min = 0.05%, Y_max = 1.0%, and importance w = 0.5. The individual desirability value d₁ for polysaccharide was calculated based on Eq (4), and the individual desirability value d₂ for triterpenoid was calculated according to Eq (5). The overall desirability (D value) of the optimization was calculated using the individual desirability values according to Eq (6).

d_{1} = {\begin{matrix} 0 \\ \frac{Y_{i} - {0.1 %}_{}}{{2 %}_{} - {0.1 %}_{}} \\ 1 \end{matrix} \begin{matrix} Y_{i} \leq 0.1 % \\ 0.1 % < Y_{i} < \\ Y_{i} \geq 2 % \end{matrix} 2 %

(4)

d_{2} = {\begin{matrix} 0 \\ \frac{Y_{i} - 0.05 %}{{1 %}_{} - {0.05 %}_{}} \\ 1 \end{matrix} \begin{matrix} Y_{i} \leq 0.05 % \\ 0.05 % < Y_{i} < \\ Y_{i} \geq 1 % \end{matrix} 2 %

(5)

D = \sqrt{d_{1} d_{2}}

(6)

Single-factor experiments

The single-factor experiment was carried out to determine the preliminary range of the extraction variables. The G. lucidum fine powder (1 g) was extracted in 150 mL flasks with aqueous ethanol. The optimum extraction temperature was first determined by varying temperature from 40 to 90C under other conditions: 40% ethanol, ultrasonic power 140 W, liquid/solid ratio 40 mL/g, extraction time 60 min, and number of extractions 1. Then, the optimum values of ethanol concentration (20%−80%), ultrasonic power (70−245 W), ratios of liquid to solid (10−60 mL/g), extraction time (30−180 min), and number of extractions (1−4) were determined sequently using determined optimal values in the extraction. Each experiment was conducted in triplicate. The extraction yields of polysaccharides and triterpenoids were determined, and the D values were calculated.

Box-Behnken Design (BBD) of RSM

After the single factor experiments, four significant variables including X₁ (ultrasonic power), X₂ (extraction temperature), X₃ (liquid/solid ratio), and X₄ (extraction time) were selected and further optimized by RSM with BBD [29]. The coded and actual values of the four selected variables are listed in Table 1. A BBD matrix comprising 29 trials was formulated with Design-Expert software (version 8.0), and was shown in Table 2. A total of five replicates (runs 6, 7, 10, 16, and 29) were performed at the center point of the design to allow for estimation of a pure error sum of squares for statistical analysis. To simplify the study, only one extraction number was tested for all experiments in this design. Each experiment was performed in triplicate and average values were shown.

Table 1. Levels and codes of extraction variables used in BBD.

Extraction variables	Symbol		Level^a
	Coded	Uncoded	-1	0	+1
Ultrasonic power (W)	X₁	x₁	140	175	210
Extraction temperature (°C)	X₂	x₂	70	80	90
Liquid/solid ratio (mL/g)	X₃	x₃	30	40	50
Extraction time (min)	X₄	x₄	60	90	120

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^a X₁ = (x₁‒175)/35; X₂ = (x₂‒80)/10; X₃ = (x₃‒40)/10; X₄ = (x₄‒90)/30

Table 2. BBD matrix and its results for the optimization of UACE process.

Run	Coded levels of variables				Extraction yield (%)		D value
	X₁	X₂	X₃	X₄	Polysaccharides	Triterpenoids
1	-1	-1	0	0	0.45	0.35	0.24
2	0	0	1	1	0.63	0.41	0.32
3	1	1	0	0	0.41	0.26	0.17
4	0	0	1	-1	0.27	0.33	0.16
5	0	-1	1	0	0.30	0.44	0.21
6	0	0	0	0	0.58	0.31	0.26
7	0	0	0	0	0.72	0.34	0.31
8	-1	0	0	-1	0.54	0.23	0.21
9	0	1	0	1	0.39	0.25	0.18
10	0	0	0	0	0.68	0.32	0.29
11	1	0	0	1	0.48	0.35	0.25
12	0	0	-1	1	0.64	0.28	0.26
13	0	1	-1	0	0.26	0.11	0.07
14	0	-1	0	-1	0.27	0.24	0.13
15	-1	0	1	0	0.51	0.35	0.26
16	0	0	0	0	0.66	0.31	0.28
17	0	1	0	-1	0.33	0.24	0.15
18	-1	0	-1	0	0.41	0.38	0.24
19	1	0	0	-1	0.50	0.36	0.26
20	0	-1	0	1	0.26	0.37	0.17
21	-1	1	0	0	0.55	0.16	0.17
22	0	1	1	0	0.29	0.30	0.16
23	0	0	-1	-1	0.44	0.13	0.12
24	1	0	1	0	0.54	0.48	0.32
25	1	-1	0	0	0.22	0.29	0.13
26	1	0	-1	0	0.39	0.32	0.21
27	-1	0	0	1	0.50	0.27	0.22
28	0	-1	-1	0	0.14	0.28	0.07
29	0	0	0	0	0.56	0.32	0.26

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To correlate the relationship between response variables (D value) with the four independent variables of the UACE process, the D values were fitted to the following second-order quadratic polynomial equation:

Y = β_{0} + \sum_{i = 1}^{4} β_{i} X_{i} + \sum_{i = 1}^{4} β_{i i} X_{i}^{2} + \sum_{i = 1}^{3} \sum_{i < j}^{4} β_{i j} X_{i} X_{j}

(7)

where Y is the predicted D value, β₀ is an intercept of the equation, β_i, is linear coefficient, β_ii, is quadratic coefficient, β_ij is cross product term coefficient, and X_i and X_j are the coded independent variables. The significance of the polynomial equation was determined by an F test. The P-value was used as a tool to check the statistical significance of each coefficient in the polynomial equation [30]. The fit of the model was expressed by the coefficient of determination (R²). CV (%) was used as a measure of the accuracy and reliability of the experiments.

Quantitation of polysaccharides and triterpenoids

The supernatants obtained from the UACE processes were subjected to separation for polysaccharides and triterpenoids before being used for the assay. Appropriate amounts of 95% ethanol were added to the extract supernatants to a final ethanol concentration of 75% and kept for 12 h at 4C for polysaccharide precipitation. After centrifugation, the resulting supernatant was used to determine the concentration of triterpenoids, and the precipitate was washed twice with 95% ethanol, dissolved in distilled water, and used for assay of the polysaccharides. The vanillin-glacial acetic acid method was used for analysis of the triterpenoid with ursolic acid as the standard [24], and expressed as the equivalent amount of ursolic acid. The polysaccharides were measured by the phenol-sulfuric acid method using d-glucose as the standard [31]. The standard curves for estimation of both components are shown in S1 Fig.

DPPH radical scavenging activity assay

DPPH radical scavenging activity was determined using a method as described previously by Blois [32] with some modifications. Two milliliters of 0.2 mM DPPH in ethanol was added to 2 mL sample at different polysaccharide or triterpenoid concentrations in the range of 0.1−0.5 mg/mL. After vortexing, the mixture was kept for 15 min in the dark at 30C. The absorbance was measured at 517 nm using a UV-Vis spectrophotometer (TU-1810, Beijing Puxi General Instrument Co. Ltd., China). Ethanol was used as the negative control instead of DPPH, as well as the blank. Butylated hydroxytoluene (BHT) was used as a positive control. DPPH scavenging rate was calculated according to the following equation:

DPPH scavenging rate (%) = (1 - \frac{A_{s} - A_{c}}{A}) \times 100

(8)

where A_s is the absorbance determined for the sample, A_c is the absorbance for the ethanol control, and A is the absorbance for the DPPH solution (2 mL DPPH plus 2 mL ethanol).

Reducing power assay

The reducing capacity of a natural component can employ as a significant indicator of its potential antioxidant activity [33]. A higher absorbance of the reaction mixture at 700 nm shows a higher reducing power [34]. The reducing power of the polysaccharides and triterpenoid-rich extracts was determined following the method described by Ahmadi et al [35], with minor modifications. Briefly, 2 mL sample at varying concentrations (0.1−0.5 mg/mL) was mixed with 2 mL potassium phosphate buffer (0.2 M, pH 6.6), then incubated at 50C for 20 min. The reaction was terminated by adding 2 mL trichloroacetic acid solution (10%, w/v). Then, the reaction solution (2 mL) was mixed with 0.4 mL ferric chloride (0.1%, w/v) and distilled water (2 mL), and the absorbance was measured at 700 nm against a blank after 10 min.

Ethical statement

This study did not involve any protected species or animals. So any specific permit was not required for the present study.

Statistical analysis

The experimental data of the BBD were shown as the average of three repetitions, while all other data were represented as the mean ± SD (n = 3). Values of P < 0.05 were considered statistically significant. All the graphs were made either with Origin 8.0 (Origin Lab Inc., USA) or Design Expert (version 8.0, Stat-Ease Inc., USA).

Results

Extraction yields of polysaccharides and triterpenoids by conventional approaches

The polysaccharides yield of G. lucidum obtained by conventional HWE (two numbers of extraction) was 1.52%. For triterpenoids extraction using the conventional maceration method, an extraction yield of 0.59% was obtained using two extraction times.

Preliminary optimization by one-factor-at-a-time approach

Effect of extraction temperature on the yields of polysaccharides and triterpenoids

As presented in Fig 1A, the yield of polysaccharides slowly increased with increasing extraction temperature within the tested range from 40 to 90C. The highest polysaccharide yield was 0.41% at 90C. The triterpenoid yield increased from 0.11% to 0.15% as extraction temperature changed from 40 to 80C, and then decreased above 80C. As to the combined performance, the D value increased with the increase of extraction temperature and reached the maximum value of 0.13 when extraction temperature was 80C, and then it started to decline. Thus, an extraction temperature of 70−90C was considered the optimal range in subsequent RSM optimization studies.

Effect of ethanol concentration on the yields of polysaccharides and triterpenoids

Fig 1B shows that the polysaccharide yield decreased in a rapid manner with increasing ethanol concentration in the scope of test, with the maximum yield of 0.46% observed at the lowest concentration of 20%. Conversely, the highest triterpenoid yield of 0.34% was obtained at the highest investigated ethanol concentration of 80%. The D value increased to 0.15 from 0.04 as ethanol increased from 20% to 50%, then slightly decreased when it increased from 50% to 80%. The optimal ethanol concentration was therefore around 50%. As ethanol concentrations between 40%−60% gave little variation in the D value, and the extraction was poor outside of this range, the ethanol concentration of 50% was selected, and not included in BBD for further optimization.

Effect of ultrasonic power input on the yields of polysaccharides and triterpenoids

Fig 1C shows that the optimal ultrasonic power inputs for extraction of polysaccharides versus triterpenoids were opposite: maximum yield for polysaccharides was 0.54% at the lowest studied ultrasonic power (70 W), whereas the highest yield of triterpenoids, reaching 0.33%, was obtained when ultrasonic power applied was the highest at 245 W. As the ultrasonic power increased, the D value rose and reached a peak value of 0.17 at 175 W. An ultrasonic power of 175 W was therefore desirable in the extraction.

Effect of ratios of liquid to solid on the yields of polysaccharides and triterpenoids

As presented in Fig 1D, the yield of polysaccharides increased from 0.21% to 0.3% as the liquid/solid ratio rose from 10 to 20 mL/g but decreased as the ratio further increased to 30 mL/g, while a yield increase from 0.28% to 0.32% was observed when the liquid/solid ratio ascended from 30 to 40 mg/L. Afterward, the polysaccharide yield decreased gradually. The maximum yield of polysaccharides (0.32%) was obtained at a liquid/solid ratio of 40 mL/g. The yield of triterpenoids increased with the increasing liquid/solid ratio within the tested range except when the liquid/solid ratio was 20 mL/g, with a maximum yield of 0.34% at a ratio of 60 mL/g. As to the combined extraction efficacy, the maximum D value (0.17) was shown to be at a liquid/solid ratio of 40 mL/g, hence, this ratio was considered as the center point in the subsequent BBD.

Effect of extraction time on the yields of polysaccharides and triterpenoids

From Fig 1E, the polysaccharide yield increased with extraction time, until it reached the maximum value (0.44%) when extraction time was 90 min. With further extended time, the yield decreased. Triterpenoid yield increased from 0.11% to 0.35% as extraction time was increased from 30 to 120 min, and then it started to decrease. Regarding the combined performance, the D value increased first and reached a maximum value (0.23) when the extraction time was 90 min, and then decreased with time. An extraction time of 90 min was therefore considered desirable in subsequent studies.

Effect of number of extractions on the yields of polysaccharides and triterpenoids

As can be seen from Fig 1F, the yields of both polysaccharides (0.44%) and triterpenoids (0.32%) were high after the first extraction, and only small amounts of both components were extracted from the residue after an additional extraction. After extraction number two, almost no additional polysaccharides and triterpenoids could be extracted. The changing trend of D values against extraction number was the same for extraction yields of both polysaccharides and triterpenoids.

Optimization of the UACE process by RSM

Model fitting and statistical analysis

The matrix of BBD and experimental results are shown in Table 2. By applying multiple regression analysis on the D values, the following second-order quadratic polynomial equation in coded values was obtained:

\begin{array}{l} Y = 0.28 + 0.000825 X_{1} - 0.003217 X_{2} + 0.039 X_{3} + 0.03 X_{4} + 0.031 X_{1} X_{2} + 0.023 X_{1} X_{3} - \\ 0.005175 X_{1} X_{4} - 0.012 X_{2} X_{3} - 0.00245 X_{2} X_{4} + 0.0058 X_{3} X_{4} - 0.002946 X_{1}^{2} - 0.11 X_{2}^{2} - \\ 0.036 X_{3}^{2} - 0.031 X_{4}^{2} \end{array}

(9)

where Y is the predicted D value; X₁, X₂, X₃, and X₄ are the coded independent variables for ultrasonic power input, extraction temperature, ratio of liquid/solid, and extraction time, respectively.

The data of statistical analysis on the fitted response surface quadratic model are listed in Table 3. The high model F-value (4.46) and a low probability value (0.0042) indicated that the model was statistically significant. The low lack-of-fit F-value (4.63) and its corresponding high P-value (0.0765) indicated that the lack of fit was insignificant relative to the pure error and the model was reliable. The determination coefficient (R²) of the regression model was 0.817, indicating that more than 81% of total variability in the response could be explained using this model. The signal-to-noise ratio was as high as 6.952, and therefore the model was considered fit and could be used to navigate the design space. P-values of linear variables X₃ and X₄, and quadratic variable X₂² were lower than 0.05, thus, they were significant model terms, and had significant influences on the extraction.

Table 3. Analysis of variance for the fitted response surface quadratic model.

Source	Sum of squares	DF	Mean square	F-value	P-value
Model	0.11	14	8.105E ‒ 03	4.46	0.0042
X₁: Ultrasonic power	8.167E ‒ 06	1	8.167E ‒ 06	4.498E ‒ 03	0.9475
X₂: Extraction temperature	1.242E ‒ 04	1	1.242E ‒ 04	0.068	0.7975
X₃: Liquid/solid ratio	0.018	1	0.018	9.95	0.0070
X₄: Ultrasonic time	0.011	1	0.011	5.96	0.0285
X₁ × X₂	3.788E ‒ 03	1	3.788E ‒ 03	2.09	0.1706
X₁ × X₃	2.186E ‒ 03	1	2.186E ‒ 03	1.20	0.2911
X₁ × X₄	1.071E ‒ 04	1	1.071E ‒ 04	0.059	0.8116
X₂ × X₃	5.546E ‒ 04	1	5.546E ‒ 04	0.31	0.5892
X₂ × X₄	2.401E ‒ 05	1	2.401E ‒ 05	0.013	0.9101
X₃ × X₄	1.346E ‒ 04	1	1.346E ‒ 04	0.074	0.7894
X₁²	5.629E ‒ 05	1	5.629E ‒ 05	0.031	0.8628
X₂²	0.072	1	0.072	39.88	< 0.0001
X₃²	8.317E ‒ 03	1	8.317E ‒ 03	4.58	0.0504
X₄²	6.343E ‒ 03	1	6.343E ‒ 03	3.49	0.0827
Residual	0.025	14	1.816E ‒ 03
Lack of fit	0.023	10	2.340E ‒ 03	4.63	0.0765
Pure error	2.023E ‒ 03	4	5.057E ‒ 04
Cor total	0.14	28
R² = 0.8170
Adj R² = 0.6339
Adeq precision = 6.952
CV(%) = 20.19

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Response surface analysis

The graphical representation of response surface for Eq (9) is shown in Fig 2. The effect of ultrasonic power and extraction temperature on the D value and their interactions are depicted in Fig 2A. The D value increased as extraction temperature increased from 70 to approximately 80C, and then it started to decrease, but there was only a little change when varying ultrasonic power in the investigated range. The projection of the response surface indicated that there was no significant interaction between these two extraction variables. From Fig 2B, a high D value could be obtained by applying high levels of both ultrasonic power input and ratio of liquid to solid. When the liquid/solid ratio was low but ultrasonic power input was high, low D values were readily observed, implying the poor performance of the extraction.

From Fig 2C, a longer ultrasonic time helped to extract polysaccharides and triterpenoids, whereas the change in ultrasonic power input had little effect on the D value. The surface plot presents a flat plain, indicating that they had little interaction. Fig 2D shows that a small change in either the liquid/solid ratio or extraction temperature would lead to a considerable variation in the D value, and higher D values would be attained when a high liquid/solid ratio along with moderate extraction temperature were simultaneously applied in the extraction.

As is evident from Fig 2E, the D value increased first and then declined with time. Extraction temperature had an effect on the D value similar to extraction time. The maximum D value was attained at around intermediate levels (extraction temperature of 80C and 90 min extraction time) for both variables. As observed in Fig 2F, the D value first increased with extraction time, then decreased when extraction time reached a high level (around 105 min). At all investigated extraction times, the D value increased first, then decreased when the liquid/solid ratio was greater than 50 mL/g.

Optimization of extraction parameters of UACE

Through the numerical optimization technique of the Design-Expert software, the optimal conditions of UACE for the extraction of G. lucidum polysaccharides and triterpenoids, with the D value as the response variable, were determined to be the following: ultrasonic power 210 W, extraction temperature 80.77C, ratio of liquid/solid 49.85 mL/g, and extraction time 103.56 min. In consideration of actual experimental conditions, optimal extraction conditions were modified as ultrasonic power 210 W, extraction temperature 80C, ratio of liquid/solid 50 mL/g, and extraction time 100 min.

Validation of predictive model

The adequacy and reliability of the regression model was verified using three independent experiments in duplicate under the modified conditions. The experimental yield of polysaccharides was 0.63% and the triterpenoid yield reached 0.38%. The D value was calculated to be 0.31, which was generally consistent with the predicted value of 0.33 given by the model. The verification experiment confirmed the precision and reliability of the model.

Comparison of DPPH radical scavenging activity of the polysaccharides and triterpenoid-rich extracts between optimized UACE and conventional processes

Fig 3A shows that the DPPH scavenging activity of polysaccharides obtained by optimized UACE and HWE showed a dosage-dependent manner, and the activity of the polysaccharides obtained by optimal UACE was higher than that of HWE in the tested concentration range. At 0.2 mg/mL, the scavenging rate of the polysaccharides from the optimized UACE was 22.16%, higher than that (16.24%) of the HWE polysaccharides. Regardless of extraction methods, the DPPH scavenging rates of polysaccharides obtained were considerably lower than that of BHT at the same concentration. The results in Fig 3B indicated that there were no significant differences in the DDPH radical scavenging activity between the triterpenoid-rich extracts obtained by the optimized UACE process and the maceration method at all tested concentrations, and the activity of the two triterpenoid-rich extracts was strongly dependent on their concentration.

Comparison of reducing power of the polysaccharides and triterpenoid-rich extracts between optimized UACE and conventional processes

As shown in Fig 4A, the reducing power of polysaccharides obtained by optimal UACE and HWE methods exhibited a dosage-dependent pattern, and the polysaccharides obtained by optimal UACE showed a stronger reducing power than those extracted by the HWE method in the tested concentration range. At 0.2 mg/mL, the absorbance at 700 nm was 0.34 for the polysaccharides from optimal UACE, while it was only 0.24 for the HWE polysaccharides. The reducing power for both extracted polysaccharides was lower than that of BHT at the same concentration. Fig 4B shows that the reducing power of the triterpenoid-rich extracts obtained by the optimized UACE process and the ethanol maceration method was strongly dependent on their concentration, and there were no significant differences between the two extracts at all tested concentrations. The reducing power for both triterpenoid-rich extracts was considerably lower than that of the positive standard BHT.

Discussion

The efficient extraction of polysaccharides and triterpenoids from G. lucidum is a prerequisite for their further research and industrial application. Hence, studying the extraction of this medicinal mushroom for these two active ingredients is of particular importance. In the present investigation, the environmentally friendly ultrasonic-assisted extraction technology was employed for the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum in a simple process (S2 Fig), with a resulting satisfactory performance. The success of synchronously extracting these two contrasting components (in terms of water solubility) by the developed UACE process could be attributed to the synergistic action of an appropriate extraction solvent, a relatively high extraction temperature, and acoustic cavitations generated by ultrasound. In this process, the polysaccharides and triterpenoids might not dissolve well in the extraction solvent applied, while a relatively high temperature increases their solubility, and finally ultrasound accelerates the extraction. To the best of our knowledge, the present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum in a simple extraction process.

In the present study, the extraction yields of G. lucidum polysaccharides and triterpenoids using the optimized UACE process were 0.63% and 0.38%, respectively. These two yields were lower than those obtained with conventional processes (polysaccharide yield of 1.52% by HWE and triterpenoid yield of 0.59% by ethanol maceration). The obtained polysaccharide yield was also lower than that observed in the ultrasonic study done by Ma et al [8], where a yield of 2.87% was reported, and that of Kan et al [36], which showed 2.44% of polysaccharide yield. The low polysaccharide yield in this study may be largely due to its low solubility in aqueous ethanol, despite the use of ultrasound in the extraction. Regarding triterpenoids, the extraction yield was lower than that of the reported yield (0.97%) using microwave irradiation [7]. A reasonable explanation is that the solubility of some low polarity or non-polar triterpenoid compounds in this mushroom may be low in the extracting solvent.

Extraction processes influencing the bioactivity of extracts have been reported widely [37, 38]. For fungal polysaccharides, a large number of factors, including their chemical components, molecular mass, structure, and conformation, can remarkably affect their antioxidant activities [8, 39]. In the present study, the polysaccharides obtained by optimized UACE showed a higher antioxidant activity than that of polysaccharides obtained by the HWE method. Ultrasonic treatment in the extraction leading to the increase in antioxidant activity of the polysaccharides of longan fruit pericarp was also demonstrated, and the underlying mechanism has been attributed to the degradation of high molecular weight polysaccharides and further changes in chemical structure caused by acoustic cavitation effects [40]. The antioxidant activity of the polysaccharides from common mullein (Verbascum thapsus L.) flowers was also shown to be enhanced by ultrasound in the extraction [41]. We therefore postulated that the degradation of polysaccharides by the action of ultrasound and a higher portion of low-molecular-weight polysaccharides extracted from the matrice may be responsible for the higher antioxidant activity. The difference in the polysaccharide purity between the two extracted polysaccharides may also be a factor.

The triterpenoids of G. lucidum are related to its many important biological activities. In the present study, based on the DPPH radical scavenging activity and evaluation of reducing power, the antioxidant activity of triterpenoid-rich extracts of G. lucidum obtained under optimized UACE process did not alter by ultrasonic treatment in the process when compared with those obtained using the conventional ethanol maceration method. This may be because the triterpenoids have a relatively simple and stable chemical structure compared with the high-molecular-weight polysaccharides. Therefore, the ultrasonic conditions applied in this work did not lead to a significant detrimental effect on their chemical properties and therefore bioactivity. However, further studies would be needed to investigate the change in patterns of extracted triterpenoids of G. lucidum during the extraction by ultrasound.

Conclusion

In the present study, the simultaneous and efficient extraction of polysaccharides and triterpenoids from the medicinal fungus G. lucidum was achieved with ultrasonic extraction technology. The optimized extraction conditions by RSM with a desirability function were determined as: ultrasonic power of 210 W, extraction temperature of 80C, liquid/solid ratio of 50 mL/g, and extraction time of 100 min. On the basis of the scavenging capacity of DPPH and evaluation of reducing power, the antioxidant activity of the extracted polysaccharides by ultrasonic action in optimal UACE process was significantly enhanced, while the triterpenoid-rich extracts demonstrated unchanged activity. The present study is the first report on the application of ultrasound for the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum in a convenient extraction process.

Supporting information

S1 Fig

Standard curves for estimation of polysaccharides (A) and triterpenoids (B) used in this work. A, with d-glucose as the standard, and B, with ursolic acid as the standard.

(TIF)

Click here for additional data file.^{(926.4KB, tif)}

S2 Fig. Flow chart of the UACE process.

(TIF)

Click here for additional data file.^{(866.4KB, tif)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The research was supported by the Key Cultivating Program (No. 2018ZDK01) of Ningde Normal University and Ningde Science and Technology Plan Project (20190013).

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PLoS One. doi: 10.1371/journal.pone.0244749.r001

Decision Letter 0

Branislav T Šiler

29 Sep 2020

PONE-D-20-24854

Desirability function approach for optimization of ultrasound-assisted extraction of polysaccharides and triterpenoids from Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

PLOS ONE

Dear Dr. Liu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

It is rather out of the point to determine the antioxidant activity of a mix of compounds, particularly using only DPPH assay as noted by Reviewer #3. I can suggest the authors to isolate dominant polysaccharides and triterpenes and to further test their antioxidant activities employing multiple common assays in order to match the theme of the main manuscript title. One of the possibilities can be to shift the manuscript theme to the extraction methodology solely (as proposed by Reviewer #3), which, in the other hand, would decrease the manuscript informativeness. The Discussion section must deepened as per reviewers' suggestions. Language should be considerably revised in order to increase readability and overall presentation clarity.

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The manuscript describes the antioxidant capacity of polysaccharides and triterpenoids obtained from Ganoderma lucidum, using ultrasonic-assisted co-extraction (UACE) and response surface methodology (RSM). The paper is interesting but need major revision.

� The MS title is not appropriate and need revision.

� The abstract is generalized. Incorporate your significant data into the abstract.

� In introduction, the extraction procedures are focused rather than the importance of Ganoderma constituents and their ability to cure various ailments.

� In materials and method section, references are missing in subsections. Add pertinent references to the methods adopted.

� In results section, the authors have added methods too, which needs to be shifted to the relevant section. For instance line 265-268.

� The discussion is very shallow and needs major input.

� The linguistic quality of the MS is very poor (for instance line 237). Edit the MS drastically for grammatical mistakes and typos.

� I would suggest the authors to make a graphical model incorporating all information presented in the paper.

� The authors may add the recent relevant reference, Ryua DH, JY Cho, NB Sadiq, JC Kim, BY Lee, M Hamayun et al. (2020) Optimization of antioxidant, anti-diabetic, and anti-inflammatory activities and ganoderic acid content of differentially dried Ganoderma lucidum using response surface methodology. Food Chemistry, 335: 127645

Reviewer #2: The simultaneous recovery of polysaccharides and triterpenoids were reported by the authors using ultrasound-assisted extraction. The authors first performed one factor at a time experiment in order to determine the domain of the experimental design and optimised the influence of four significant variables (ultrasonic power, temperature, liquid/solid ratio, and extraction time). The introduction of the manuscript is well prepared and follows the theme of the article. A good methodology was established, the authors provide a clear rationale for each experiment conducted and the results are discussed thoroughly. Discussions are very well reported, and the authors focused on comparing their finding with previous reports. The figures are self-explanatory, clear, and easy to understand. However, I provided a few suggestions and recommend the acceptance of this paper in PLOSONE.

1. Please rephrase line 30-33 in the abstract for more clarity

2. Line 45: Include the classificator of Ganoderma lucidum mushroom for the first time (Karst.)

3. Line 37-38: "the triterpenoids showed antioxidant activities similar to those isolated using the ethanol maceration" This sentence is not correct because the authors performed only extraction. Hence, it should be corrected as "the triterpenoid rich extract showed antioxidant activities similar to those obtained using ethanol maceration.

4. Rephrase line 76

5. Line 208-210: The quantification of triterpenes should be better explained. Dis the authors used a standard (Ursolic acid for example). If yes, then the results should be expressed in the equivalent of the standard

6. In its current state, the manuscript has some grammatical errors and instances of badly constructed sentences. Please check the manuscript and refine the language carefully.

Reviewer #3: This study describes a methodology for simultaneously extracting polysaccharides and triterpenes.

However, the goals of bioactivity differ greatly between polysaccharides and triterpenes. For example, there are many types of triterpenes, and each triterpene has a unique bioactivity. Isn't each activity weakened by simultaneous extraction?

In this study, the author describes the usefulness of simultaneous extraction, but I think it is not enough to confirm with DPPH alone.If you have a reason, please state why you expressed the bioactivity only with the DPPH result. In some cases, I suggest removing the DPPH results and changing to a paper with only extraction methods.

P3L62: Please add a description of GA-MT and GA-T

P5 Materials and Methods: Particle size after sample grinding should be specified

P6L119-L147: The description of the extraction method is redundant. Please correct it to a concise description.

Figures: I can't consider the data because the figure doesn't have a legend.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2020 Dec 31;15(12):e0244749. doi: 10.1371/journal.pone.0244749.r002

Author response to Decision Letter 0

25 Nov 2020

Responses to the reviewer’s comments

Manuscript number: PONE-D-20-24854

Title: Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Dear Editors,

We appreciate your comments and suggestions on our manuscript submitted previously. The manuscript has been carefully revised according to the suggestions and recommendation by the reviewers, and now all problems have been addressed in revised manuscript. Revised portion are marked in red in the paper. Point-by-point responses to these comments are showed below in this letter.

Sincerely yours,

Shi-Zhong Zheng, Wei-Rui Zhang, Sheng-Rong Liu

Reviewer 1#: Some doubts, suggestions and recommendation

Comment 1: The manuscript describes the antioxidant capacity of polysaccharides and triterpenoids obtained from Ganoderma lucidum, using ultrasonic-assisted co-extraction (UACE) and response surface methodology (RSM). The paper is interesting but need major revision.

Response: Thanks. We have carefully and extensively revised the manuscript.

Comment 2: The manuscript title is not appropriate and need revision.

Response: Accept. It has been revised.

Comment 3: The abstract is generalized. Incorporate your significant data into the abstract.

Response: Thanks. The abstract has been carefully revised as suggested, and the quality was greatly improved. See in the revision.

Comment 4: In introduction, the extraction procedures are focused rather than the importance of Ganoderma constituents and their ability to cure various ailments.

Response: Thanks. Some abundant description has been deleted, and the focus has been paid to the extraction techniques and optimization. See in the revision.

Comment 5: In materials and method section, references are missing in subsections. Add pertinent references to the methods adopted.

Response: Accept. Several related references have been cited in the revised manuscript.

Comment 6: In results section, the authors have added methods too, which needs to be shifted to the relevant section. For instance line 265-268.

Response: Accept. The extraction conditions were deleted in Results section, and described in Materials and methods section. See in the revision.

Comment 7: The discussion is very shallow and needs major input.

Response: Thanks. We have carefully revised this section, and more in-depth explanation has been provided. See in the revision.

Comment 8: The linguistic quality of the MS is very poor (for instance line 237). Edit the manuscript drastically for grammatical mistakes and typos.

Response: Thanks. We have carefully checked the whole manuscript.

Comment 9: I would suggest the authors to make a graphical model incorporating all information presented in the paper.

Response: Accept. A graphical model was made and provided.

Comment 10: The authors may add the recent relevant reference, Ryua DH, JY Cho, NB Sadiq, JC Kim, BY Lee, M Hamayun et al. (2020) Optimization of antioxidant, anti-diabetic, and anti-inflammatory activities and ganoderic acid content of differentially dried Ganoderma lucidum using response surface methodology. Food Chemistry, 335: 127645

Response: Accept. It has been cited in the manuscript. See in the revision.

Reviewer 2#: Some doubts, suggestions and recommendation

Comment 1: Please rephrase line 30-33 in the abstract for more clarity.

Response: Accept. We has revised it for clarity.

Comment 2: Line 45: Include the classificatory of Ganoderma lucidum mushroom for the first time (Karst.)

Response: Accept. It has been described. See in the revision.

Comment 3: Line 37-38: “the triterpenoids showed antioxidant activities similar to those isolated using the ethanol maceration" This sentence is not correct because the authors performed only extraction. Hence, it should be corrected as” the triterpenoid-rich extract showed antioxidant activities similar to those obtained using ethanol maceration.

Response: Thanks. It has been revised as suggested.

Comment 4: Rephrase line 76: “For extraction of triterpenoids of G. lucidum, maceration using organic extracting solvents such as ethanol is often used”.

Response: Accept. It has been rephrased. See in the revision.

Comment 5: Line 208-210: The quantification of triterpenes should be better explained. Did the authors use a standard (ursolic acid for example). If yes, then the results should be expressed in the equivalent of the standard.

Response: Accept. We used D-glucose as the standard for polysaccharides measurement, and ursolic acid as the standard for the measurement of triterpenoids. The results were expressed in the equivalent of the standard. There information have been described in the manuscript.

Comment 6: In its current state, the manuscript has some grammatical errors and instances of badly constructed sentences. Please check the manuscript and refine the language carefully.

Response: Thanks. We have carefully checked the manuscript.

Reviewer 3#: Some doubts, suggestions and recommendation

Comment 1: This study describes a methodology for simultaneously extracting polysaccharides and triterpenes. However, the goals of bioactivity differ greatly between polysaccharides and triterpenes. For example, there are many types of triterpenes, and each triterpene has a unique bioactivity. Isn’t each activity weakened by simultaneous extraction?

Response: Thanks. We are very sorry that the separation of individual triterpenoids was not carried out, and the activity of each triterpene was not investigated in this study.

Comment 2: In this study, the author describes the usefulness of simultaneous extraction, but I think it is not enough to confirm with DPPH alone. If you have a reason, please state why you expressed the bioactivity only with the DPPH result.

Response: Thanks. For a systematic comparison, we have performed additional experiments on the reducing power of the extracts, and the results have been provided in the manuscript. See in the revision.

Comment 3: In some cases, I suggest removing the DPPH results and changing to a paper with only extraction methods.

Response: Thanks. We provided additional results on the antioxidant assay, and the results on the antioxidant assay were present in the manuscript. See in the revision.

Comment 4: Page 3, Line 62: Please add a description of GA-MT and GA-T

Response: Accept. It has been added.

Comment 5: Page 5 Materials and Methods: Particle size after sample grinding should be specified.

Response: Accept. The particle size was specified in the revision.

Comment 6: Page 6, L119-L147: The description of the extraction method is redundant. Please correct it to a concise description.

Response: Accept. We have shortened it. See in the revision.

Comment 7: Figures: I can’t consider the data because the figure doesn’t have a legend.

Response: Accept. Legends have been provided. See in the revision.

List of the changes in revision

Title

1. Line 1-3 of page 1, “Desirability function approach for optimization of ultrasound-assisted extraction of polysaccharides and triterpenoids from Ganoderma lucidum and evaluation of their in vitro antioxidant capacities” was revised as “Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities”.

Abstract

1. Line 24-25 of page 2, “Ganoderma lucidum is a medicinal mushroom that is rich in biologically active substances” was revised as “Ganoderma lucidum (Fr.) Krast, commonly known as “Lingzhi” in Chinese, is a medicinal mushroom that is rich in biologically active substances”.

2. Line 25 of page 2, “and widely used as a traditional Chinese herbal medicine in Asian countries” was deleted.

3. Line 26-27 of page 2, “Polysaccharides and triterpenoids are the two major bioactive ingredients of this mushroom” was changed as “Polysaccharides and triterpenoids are the two major components responsible for the bioactivity of this fungus”.

4. Line 28 of page 2, “of” was revised as “from”.

5. Line 29 of page 2, “with the equal importance for the two components” was inserted.

6. Line 29 of page 2, “single factor tests” was revised as “Following single factor experiments”.

7. Line 30-31 of page 2, “by RSM with the associated D value as the response variable” was deleted.

8. Line 33 of page 2, “With” was changed as “Under”.

9. Line 35 of page 2, “and evaluation of reducing power” was inserted.

10. Line 36 of page 2, “crude” was inserted.

11. Line 37 of page 2, “crude” was inserted.

12. Line 37-38 of page 2, “while the triterpenoids showed” was revised as “whereas the triterpenoid-rich extracts”.

13. Line38-39 of page 2, “using the ethanol maceration” was changed as “using the ethanol maceration method”.

14. Line 39 of page 2, “The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from G. lucidum” was inserted.

Keywords

1. Line 42-43 of page 2, “ultrasonic extraction” was inserted.

Introduction

1. Line 45 of page 3, “Ganoderma lucidum is the most famous medicinal mushroom in the world” was revised as “Ganoderma lucidum (Fr.) Krast belonging to the family of Polyporaceae, commonly known as “Lingzhi” in Chinese and “Reishi” in Japanese, is one of the most famous medicinal mushrooms”.

2. Line 47 of page 3, “in oriental countries” was revised as “in many oriental countries”.

3. Line 48-51 of page 3, “Nowadays, this mushroom is widely used to prevent and treat many challenging human diseases, such as hepatitis, hyperglycemia, high blood pressure, and cancers, and as a nutraceutical to improve health, and promote spiritual growth for human in many countries” was deleted.

4. Line 51-53 of page 3, “Owing to its medicinal properties and perceived health-promoting effects, there has been a growing worldwide interest in utilizing G. lucidum in the biomedical industry in recent years” was revised as “In recent years, there has been a growing worldwide interest in utilizing G. lucidum in the biomedical industry due to its pharmacological properties”.

5. Line 53 of page 3, “many G. lucidum products” was revised as “a variety of G. lucidum products”.

6. Line 54 of page 3, “in many parts of the world” was inserted.

7. Line 55 of page 3, “G. lucidum contains” was revised as “G. lucidum in the form of fruiting bodies, mycelium, and spores contains”.

8. Line 57-59 of page 3, “the two key bioactive components namely polysaccharides and triterpenoids are receiving special attention from researchers in the field of biomedicine” was revised as “the polysaccharides and triterpenoids are receiving special attention from researchers in the field of biomedicine”.

9. Line 59 of page 3, “Several kinds of polysaccharides” was revised as “Several polysaccharides”.

10. Line 60 of page 3, “are recognized” was revised as “generally recognized”.

11. Line 60 of page 3, “such as antitumor, antioxidant and immunomodulatory effects” was inserted.

12. Line 61-63 of page 3, “Regarding various chemically diverse triterpenoids, ganoderic acids such as GA-Me and GA-T have been proven to have potent pharmacological activity” was revised as “The triterpenoids from this fungus exhibit structural diversity, and many of them such as ganoderic acids Me and T (a kind of highly oxygenated lanostane-type triterpenoids) have been proven to have potent antitumor activity”.

13. Line 63-65 of page 3, “To date, a wide variety of important bioactivities, including antitumor, antioxidant, antimicrobial, and immunomodulatory effects, have been shown for both the polysaccharides and triterpenoids” was deleted.

14. Line 65-66 of page 3, “much attention” was revised as “much effort”.

15. Line 69-71 of page 4, “Hot water extraction (HWE) is a conventional method, and widely used in extraction of polysaccharides from mushrooms due to the fact that no special equipments are required in the extraction” was revised as “Polysaccharides are generally prepared by traditional hot water extraction due to its easy operation and low instrument input”.

16. Line 73 of page 4, “a variety of new techniques” was revised as “a variety of novel techniques”.

17. Line 75-76 of page 4, “for a higher yield and efficacy” was deleted.

18. Line 76-77 of page 4, “For extraction of triterpenoids of G. lucidum, maceration using organic extracting solvents such as ethanol is often used” was revised as “For the extraction of triterpenoids, maceration as a conventional method is adopted widely using organic solvents as an extraction solvent”.

19. Line 77-78 of page 4, “and microwave irradiation was shown to be effective for the promotion of extraction of triterpenoids from G. lucidum” was revised as “A novel technique of microwave irradiation was shown to be effective for the extraction of triterpenoids from this fungus”.

20. Line 80-82 of page 4, “To improve the extraction efficacy, the simultaneous extraction of multiple biologically active components from plant materials has become increasingly popular in recent years” was changed as “In recent years, the simultaneous extraction of multiple biologically active components from plants has become increasingly popular in order to improve extraction efficacy and reduce the number of extraction steps”.

21. Line 82 of page 4, “there have been no available reports on the simultaneous extraction of multiple objective constituents from mushrooms to date” was inserted.

22. Line 82 of page 4, “most studies” was revised as “most of previous studies”.

23. Line 84-85 of page 4, “The application of these available processes may result in the loss of either polysaccharides or triterpenoids during the extraction” was revised as “Hence, the significant loss of either polysaccharides or triterpenoids occurred during the extraction, and this would lead to a waste of resources”.

24. Line 86 of page 4, “a process for” was deleted.

25. Line 88-89 of page 4, “ultrasound technology was examined for the simultaneous extraction of polysaccharides and triterpenoids” was revised as “ultrasound technology was examined for the co-extraction of polysaccharides and triterpenoids”.

26. Line 89 of page 4, “and an optimization study is essential” was inserted.

27. Line 90-91 of page 4, “Response surface methodology (RSM) is a collection of mathematical and statistical techniques widely used for optimizing complex processes” was revised as “Response surface methodology (RSM), a collection of mathematical and statistical techniques, is widely used for optimizing complex processes”.

28. Line 93-94 of page 4-5, “RSM has been used extensively in optimizing the extraction processes of bioactive components” was changed as “RSM has been employed for optimizing the extraction of G. lucidum polysaccharides and ganoderic acids and triterpenoids from the medicinal fungus Sanghuangporus sanghuang”.

29. Line 94 of page 5, “On the other hand” was deleted.

30. Line 96 of page 5, “with different relative importances” was inserted.

31. Line 101-103 of page 5, “In an attempt to better utilize G. lucidum resources, the simultaneous extraction of polysaccharides and triterpenoids was examined using ultrasound technology in this work” was revised as “In the present study, the ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids was evaluated using ultrasound technology as an attempt to better utilize G. lucidum”.

32. Line 103-104 of page 5, “The parameters of ultrasonic-assisted co-extraction (UACE) of polysaccharides and triterpenoids were optimized using RSM integrated with a desirability function” was changed as “The extraction parameters were optimized using RSM with a desirability function by setting the equal importance for the two components”.

33. Line 104 of page 5, “Furthermore” was inserted.

34. Line 107-108 of page 5, “using conventional approaches” was revised as “using conventional extraction approaches”.

35. Line 110-112 of page 5, “The present study is the first report on the simultaneous extraction of polysaccharides and triterpenoids from the medicinal mushroom G. lucidum” was deleted.

Materials and methods

1. Line 115 of page 5, “The dried G. lucidum fruiting bodies were” was revised as “The dried G. lucidum was”.

2. Line 117 of page 5, “ferric chloride, and trichloroacetic acid” was inserted.

3. Line 119 of page 6, “Conventional hot water extraction of polysaccharides” was changed as “hot water extraction of polysaccharides”.

4. Line 120 of page 6, “The G. lucidum sample was processed into a fine powder in a grinder” was revised as “The dried G. lucidum was grounded in a grinder to fine powder (40 mesh)”.

5. Line 121 of page 6, “treated” was revised as “pretreated”.

6. Line 123 of page 6, “in the matrix” was deleted.

7. Line 123-125 of page 6, “The obtained powder (1 g) was placed into a 150 mL flask containing 40 mL deionized water, and extracted at 95�C in a water bath for 2 h” was revised as “The treated powder (1 g) was mixed with 40 mL of deionized water in a 150 mL flask, and extracted for 2 h at 95�C”.

8. Line 125 of page 6, “The extraction procedure was repeated once” was inserted.

9. Line 125 of page 6, “The whole extract” was revised as “The extract”.

10. Line 126 of page 6, “The supernatant was collected, and the residue was extracted again” was deleted.

11. Line 127-128 of page 6, “The supernatants were combined, and used for the estimation of polysaccharides or for further purification before the assay of antioxidant activity” was revised as “The combined supernatants were used for the estimation of crude polysaccharides or for further purification by ethanol precipitation when used in the assay of antioxidant activity”.

12. Line 129 of page 6, “Conventional method for extraction of triterpenoids” was revised as “Ethanol maceration for extraction of triterpenoids”.

13. Line 131-132 of page 6, “The mouth of the flask was sealed with aluminum foil to avoid solvent evaporation during the extraction” was revised as “The flask was sealed with aluminum foil during the extraction”.

14. Line 132-134 of page 6, “The residue was collected after centrifugation at 8000 ×g for 10 min. The supernatant was harvested, and the residue was extracted again” was revised as “The extraction procedure was repeated once. The extraction solution was centrifuged at 8000 ×g for 10 min”.

15. Line 136-137 of page 6, “Ultrasonic-assisted co-extraction of polysaccharides and triterpenoids” was revised as “UACE of polysaccharides and triterpenoids”.

16. Line 138-139 of page 6, “An ultrasonic bath having a 3 L usable capacity was used” was revised as “The UACE was performed in an ultrasonic cleaning bath with a 3 L usable capacity”.

17. Line 139-140 of page 6, “and ultrasonic power can be set between 35 and 310 W” was deleted.

18. Line 140-141 of page 6, “The G. lucidum fine powder (1 g) was extracted with an ethanol-water solution in a 150 mL flask” was revised as “Each of 1 g of the fine powder was extracted with an aqueous ethanol in a 150 mL flask, and the other conditions were described elsewhere”.

19. Line 141-142 of page 6-7, “The mouth of the flask was sealed with aluminum foil during the extraction” was deleted.

20. Line 143-144 of page 7, “and the resulting supernatant was used for measurements of polysaccharides and triterpenoids” was revised as “The polysaccharides and triterpenoids in the resulting supernatant were determined”.

21. Line 144-145 of page 7, “The number of extractions varied with experiments” was deleted.

22. Line 168 of page 8, “Single factor test” was revised as “Single-factor experiments”.

23. Line 169-170 of page 8, “Prior to RSM optimization, the preliminary range of process variables for the extraction was determined using one-factor-at-a-time approach with the desirability function” was revised as “The single-factor experiment was carried out to determine the preliminary range of the extraction variables”.

24. Line 170-174 of page 8, “The G. lucidum fine powder (1 g) was extracted in 150 mL flasks with varying temperatures (40 to 90�C), ethanol concentrations (20% to 80%), ultrasonic power (70 to 245 W), ratios of liquid to solid (10 to 60 mL/g), extraction times (30 to 180 min), and number of extractions (1 to 4)” was revised as “The G. lucidum fine powder (1 g) was extracted in 150 mL flasks with aqueous ethanol. The optimum extraction temperature was first determined by varying temperature from 40 to 90�C under other conditions: 40% ethanol, ultrasonic power 140 W, liquid/solid ratio 40 mL/g, extraction time 60 min, and number of extractions 1. Then, the optimum values of ethanol concentration (20%−80%), ultrasonic power (70−245 W), ratios of liquid to solid (10−60 mL/g), extraction time (30−180 min), and number of extractions (1−4) were determined sequently using determined optimal values in the extraction”.

25. Line 174 of page 8, “Each experiment was conducted in triplicate” was inserted.

26. Line 175 of page 8, “and the D values were calculated” was inserted.

27. Line 176 of page 8, “Box-Behnken design (BBD)” was changed as “Box-Behnken design (BBD) of RSM”.

28. Line 177 of page 8, “On the basis of the results of the single-factor experiments” was revised as “After the single factor experiments”.

29. Line 184-185 of page 8, “To simplify the research, one extraction number was used in all experiments” was revised as “To simplify the study, only one extraction number was tested for all experiments in this design”.

30. Line 185 of page 8, “Each trial” was revised as “Each experiment”.

31. Line 187 of page 9, “To determine the relationship” was revised as “To correlate the relationship”.

32. Line 201 of page 9, “Assay of polysaccharides and triterpenoids” was revised as “Quantitation of polysaccharides and triterpenoids”.

33. Line 206 of page 9, “at 8000 ×g for 10 min” was deleted.

34. Line 208-209 of page 10, “The vanillin-glacial acetic acid method was used for analysis of the triterpenoid amount in each sample” was revised as “The vanillin-glacial acetic acid method was used for analysis of the triterpenoid with ursolic acid as the standard”.

35. Line 210 of page 10, “and expressed as the equivalent amount of ursolic acid” was inserted.

36. Line 211 of page 10, “using D-glucose as the standard” was inserted.

37. Line 224 of page 10, “and A is the absorbance for the solution” was revised as “and A is the absorbance for the DPPH solution”.

38. Line 226 of page 10, “Reducing power assay” was inserted.

39. Line 226 of page 10, “The reducing capacity of a natural component can employ as a significant indicator of its potential antioxidant activity” was inserted.

40. Line 226 of page 10, “A higher absorbance of the reaction mixture at 700 nm shows a higher reducing power” was inserted.

41. Line 226 of page 10, “The reducing power of the polysaccharides and triterpenoids was determined following the method described by Ahmadi et al [35], with minor modifications” was inserted.

42. Line 226 of page 10, “Briefly, 2 mL sample at varying concentrations (0.1−0.5 mg/mL) was mixed with 2 mL potassium phosphate buffer (0.2 M, pH 6.6), then incubated at 50�C for 20 min. The reaction was terminated by adding 2 mL trichloroacetic acid solution (10%, w/v). Then, the reaction solution (2 mL) was mixed with 0.4 mL ferric chloride (0.1%, w/v) and distilled water (2 mL), and the absorbance was measured at 700 nm against a blank after 10 min” was inserted.

Results

1. Line 237 of page 11, “by HWE” was revised as “conventional HWE (two numbers of extraction)”.

2. Line 237-238 of page 11, “and 0.59% was obtained for the triterpenoids using the ethanol maceration” was revised as “For triterpenoids extraction using the conventional maceration method, an extraction yield of 0.59% was obtained using two extraction times”.

3. Line 240 of page 11, “on the yields of polysaccharides and triterpenoids” was added.

4. Line 241-243 of page 11, “Extraction temperature was tested between 40 and 90�C, while other variables were constant (ethanol concentration, 40%; ultrasonic power, 140 W; ratio of liquid/solid, 40 mL/g; extraction time, 60 min; and number of extractions, 1)” was deleted.

5. Line 243 of page 11, “As shown in Fig 1A” was revised as “As presented in Fig 1A”.

6. Line 244 of page 11, “within the tested range from 40 to 90�C” was inserted.

7. Line 244-246 of page 11, “The highest polysaccharide yield (0.41%) was achieved at the highest extraction temperature (90�C)” was revised as “The highest polysaccharide yield was 0.41% at 90�C”.

8. Line 246-248 of page 11, “The triterpenoid yield increased from 0.11% to 0.15% as extraction temperature changed from 40 to 80�C. When extraction temperature increased beyond 80�C, the triterpenoid yield decreased” was revised as “The triterpenoid yield increased from 0.11% to 0.15% as extraction temperature changed from 40 to 80�C, and then decreased above 80�C.”.

9. Line 248 of page 11, “As to the combined performance” was inserted.

10. Line 250 of page 11, “Above this, it started to decline” was revised as “Above this, it started to decline”.

11. Line 252 of page 12, “on the yields of polysaccharides and triterpenoids” was added.

12. Line 253-255 of page 12, “Ethanol concentrations ranging between 20%−80% were tested. Other conditions were as follows: temperature, 80�C; ultrasonic power, 140 W; ratio of liquid/solid, 40 mL/g; extraction time, 60 min; and number of extractions, 1” was deleted.

13. Line 255-257 of page 12, “Polysaccharide yield decreased in a rapid manner with increasing ethanol concentration (Fig 1B), with the maximum yield of 0.46% observed at the lowest studied concentration (20%)” was revised as “Fig 1B shows that the polysaccharide yield decreased in a rapid manner with increasing ethanol concentration in the scope of test, with the maximum yield of 0.46% observed at the lowest concentration of 20%”.

14. Line 257-258 of page 12, “the highest triterpenoid yield (0.34%)” was revised as “the highest triterpenoid yield of 0.34%”.

15. Line 260-261 of page 12, “The optimal level of ethanol was therefore around 50%” was revised as “The optimal ethanol concentration was therefore around 50%”.

16. Line 263 of page 12, “50% ethanol was used and not included in a further optimization” was revised as “the ethanol concentration of 50% was selected, and not included in BBD for further optimization”.

17. Line 264 of page 12, “on the yields of polysaccharides and triterpenoids” was inserted.

18. Line 265-268 of page 12, “The effects of ultrasonic power varying between 70 and 245 W were investigated, while other operational parameters were: extraction temperature, 80�C; ethanol concentration, 50%; ratio of liquid to solid, 40 mL/g; extraction time, 60 min; and number of extractions, 1” was deleted.

19. Line 268 of page 12, “As observed in Fig 1C” was revised as “As observed in Fig 1C”.

20. Line 271 of page 12, “applied” was inserted.

21. Line 273-274 of page 12, “An ultrasonic power of 175 W was therefore appropriate in the subsequent optimization” was changed as “An ultrasonic power of 175 W was therefore desirable in the extraction”.

22. Line 275 of page 12, “on the yields of polysaccharides and triterpenoids” was inserted.

23. Line 276-278 of page 13, “Extraction was conducted at different liquid/solid ratios from 10 to 60 mg/L under the following conditions: extraction temperature, 80�C; ethanol concentration, 50%; ultrasonic power, 175 W; extraction time, 60 min; and number of extractions, 1” was deleted.

24. Line 278-279 of page 13, “As illustrated in Fig 1D” was revised as “As presented in Fig 1D”.

25. Line 280 of page 13, “increased from” was revised as “rose from”.

26. Line286 of page 13, “As to the combin

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PLoS One. doi: 10.1371/journal.pone.0244749.r003

Decision Letter 1

Branislav T Šiler

1 Dec 2020

PONE-D-20-24854R1

Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

PLOS ONE

Dear Dr. Liu,

==============================

Authors have addressed all the major concerns raised in the reviewers' reports. However, one sentence in the conclusion section fails to present the verity of the study: "The antioxidant activity of the polysaccharides was enhanced by ultrasound."(L480), which does not hold the true since it means that you have somehow extracted polysaccharides, measured their antiox activity, let out the ultrasound through them and, measured their antiox activity once again and, finally, compared the two values. Please elaborate this.

Additionally, more attention should be paid to the Conclusion section .

==============================

Please submit your revised manuscript by Jan 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

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PLoS One. 2020 Dec 31;15(12):e0244749. doi: 10.1371/journal.pone.0244749.r004

Author response to Decision Letter 1

13 Dec 2020

Responses to the reviewer’s comments

Manuscript number: PONE-D-20-24854

Title: Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Dear Editors,

Sincerely yours,

Shi-Zhong Zheng, Wei-Rui Zhang, Sheng-Rong Liu

Reviewer 1#: Some doubts, suggestions and recommendation

Comment 1: Authors have addressed all the major concerns raised in the reviewers’ reports. However, one sentence in the conclusion section fails to present the verity of the study: “The antioxidant activity of the polysaccharides was enhanced by ultrasound”(L480), which does not hold the true since it means that you have somehow extracted polysaccharides, measured their antioxidant activity, let out the ultrasound through them and, measured their antioxidant activity once again and, finally, compared the two values. Please elaborate this.

Response: Accept. We have carefully revised this sentence. See in the revision.

Comment 2: Additionally, more attention should be paid to the Conclusion section.

Response: Accept. We have carefully revised this section, and we believe that the quality was greatly improved. See in the revision.

Comment 3: If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Response: Thanks. We have updated statement on the financial disclosure in our cover letter.

List of the changes in revision

Abstract

1. Line 34 of page 2, “crude” was deleted.

2. Line 35 of page 2, “crude” was deleted.

Introduction

1. Line 46 of page 3, “The most famous” was revised as “the most highly prized”.

Results

1. Line 322 of page 15, “the model was statistically very significant” was revised as “the model was statistically significant”.

2. Line 396 of page 19, “triterpenoids” was revised as “triterpenoid-rich extracts”.

Discussion

1. Line 435 of page 20, “in a convenient extraction process” was revised as “in a simple extraction process”.

2. Line 448 of page 21, “have been reported” was revised as “have been reported widely”.

3. Line 458 of page 21, “was shown” was revised as “was also shown”.

4. Line 461 of page 21, “were” was revised as “may be”.

Conclusion

1. Line 476 of page 22, “the simultaneous extraction” was revised as “the simultaneous and efficient extraction”.

2. Line 477 of page 22, “from G. lucidum” was revised as “from the medicinal fungus G. lucidum”.

3. Line 477 of page 22, “with ultrasonic technology” was revised as “with ultrasonic extraction technology”.

4. Line 480 of page 22, “On the basis of the scavenging capacity of DPPH and evaluation of reducing power” was inserted.

5. Line 480 of page 22, “The antioxidant activity of the polysaccharides was enhanced by ultrasound” was revised as “the antioxidant activity of the extracted polysaccharides by ultrasonic action in optimal UACE process was significantly enhanced”.

6. Line 480 of page 22, “while the triterpenoid-rich extracts demonstrated unchanged activity” was inserted.

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PLoS One. doi: 10.1371/journal.pone.0244749.r005

Decision Letter 2

Branislav T Šiler

16 Dec 2020

Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

PONE-D-20-24854R2

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0244749.r006

Acceptance letter

Branislav T Šiler

18 Dec 2020

PONE-D-20-24854R2

Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Dear Dr. Liu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Branislav T. Šiler

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Supplementary Materials

S1 Fig

Standard curves for estimation of polysaccharides (A) and triterpenoids (B) used in this work. A, with d-glucose as the standard, and B, with ursolic acid as the standard.

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S2 Fig. Flow chart of the UACE process.

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Optimization of ultrasonic-assisted extraction of polysaccharides and triterpenoids from the medicinal mushroom Ganoderma lucidum and evaluation of their in vitro antioxidant capacities

Shizhong Zheng

Weirui Zhang

Shengrong Liu

Roles

Abstract

Introduction

Materials and methods

Materials and reagents

Hot water extraction of polysaccharides

Ethanol maceration for extraction of triterpenoids

UACE of polysaccharides and triterpenoids

Desirability function

Single-factor experiments

Box-Behnken Design (BBD) of RSM

Table 1. Levels and codes of extraction variables used in BBD.

Table 2. BBD matrix and its results for the optimization of UACE process.

Quantitation of polysaccharides and triterpenoids

DPPH radical scavenging activity assay

Reducing power assay

Ethical statement

Statistical analysis

Results

Extraction yields of polysaccharides and triterpenoids by conventional approaches

Preliminary optimization by one-factor-at-a-time approach

Effect of extraction temperature on the yields of polysaccharides and triterpenoids

Fig 1. Effects of different extraction parameters on the yield of polysaccharides and triterpenoids of G. lucidum, as well as on the D value.

Effect of ethanol concentration on the yields of polysaccharides and triterpenoids

Effect of ultrasonic power input on the yields of polysaccharides and triterpenoids

Effect of ratios of liquid to solid on the yields of polysaccharides and triterpenoids

Effect of extraction time on the yields of polysaccharides and triterpenoids

Effect of number of extractions on the yields of polysaccharides and triterpenoids

Optimization of the UACE process by RSM

Model fitting and statistical analysis

Table 3. Analysis of variance for the fitted response surface quadratic model.

Response surface analysis

Fig 2. Response surface plots showing the effects of extraction parameters on the D values in the extraction of polysaccharides and triterpenoids from G. lucidum.

Optimization of extraction parameters of UACE

Validation of predictive model

Comparison of DPPH radical scavenging activity of the polysaccharides and triterpenoid-rich extracts between optimized UACE and conventional processes

Fig 3.

Comparison of reducing power of the polysaccharides and triterpenoid-rich extracts between optimized UACE and conventional processes

Fig 4.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Branislav T Šiler

Roles

Author response to Decision Letter 0

Decision Letter 1

Branislav T Šiler

Roles

Author response to Decision Letter 1

Decision Letter 2

Branislav T Šiler

Roles

Acceptance letter

Branislav T Šiler

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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