Extraction, preparation and an assessment of the activity of carboxymethyl polysaccharide from Panax japonicus

A response surface method (RSM) was employed to optimize polysaccharide extraction from Panax japonicus (PJPS). Also, carboxymethyl substitution of PJPS (CM-PJPS) was studied using a response surface methodology. A three-variable Box– Behnken design (BBD) methodology was applied. RSM analyses revealed that conditions that maximized polysaccharide yield were as follows: a liquid-to-material ratio 22:1, an extraction temperature of 83.99 °C and an extraction duration of 2.32 h. Under optimum extraction conditions, the average absorbance value of PJPS was 0.6001, and the purity of PJPS was 98.2%. A quadratic regression model for CM-PJPS was obtained via BBD. The optimum extraction conditions for the process were determined to include a 1.38 h reaction time, 1.24 g monochloroacetic acid (MCA) and a reaction temperature of 52.85 °C. Under optimum extraction conditions, the average degree of CM-PJPS substitution was 0.9733. The structure of the PJPS was examined by means of Fourier transform infrared spectroscopy (FTIR). CM-PJPS exhibited the strongest anti-oxidant activity in vitro . In addition, CM-PJPS inhibited Skov3 and A2780 cancer cell proliferation. Our data revealed that CM-PJPS is a promising natural antioxidant with potential value as a food supplement and for treatment of cancer.


Introduction
Panax japonicus C. A. Mey is a member of the Araliaceae.It is named for its rhizome-like bamboo knot.The plant is distributed mainly within Western Hubei.It is a variety included in the Chinese Pharmacopoeia and has widely been used in Chinese medicine (Tang & Eisenbrand, 1992).P. japonicus is of special importance to Miao medicine as a result of its capacity to nourish and strengthen, stop bleeding, dredge meridians, relax tendons, relieve pain, enhance circulation, remove blood stasis, and invigorate the spleen and stomach (CP-Chinese Pharmacopoeia, 2015).Polysaccharides from P. japonicus are important active ingredients of the plant and have strong immune regulatory and anti-fatigue effects (Shu et al., 2018).Domestic research on P. japonicus has mainly been focused on saponins, and has rarely involved the assessment of polysaccharides (Shin et al., 2015).Polysaccharides of P. japonicus are of global interest.These mainly consist of acidic heteropolysaccharides and dextran with complex structures consisting of galacturonic acid, galactose, rhamnose, arabinose and arabinose.After saponins, polysaccharides are believed to be the compounds that most greatly influence bioactivity of P. japonicus (Wang et al., 2012;Yang et al., 2014).
Polysaccharides are type of natural macromolecule in which aldose or ketose molecules are linked through a glycosidic bond.
Polysaccharides are essential components of all living organisms, and are needed to maintain biological functioning (Liu et al., 2015;Xu, 2017).A large number of studies over the past decade have indicated that polysaccharides have a wide range of biological functions that include anti-cancer, anti-infection, anti-coagulation, anti-virus, and anti-radiation activities (Hetland 2003;Liu et al., 2015;Sohretoglu & Huang, 2018).The compounds also have been determined to regulate immune function, hypoglycemia, hypolipidemia, cell division and differentiation, cell growth and aging (Shu et al., 2018;Han et al., 2006;Willats et al., 1999;Ye et al., 2012).Uses of polysaccharides as drugs has very little toxicity.As a result of these findings, polysaccharide research has attracted great interest.
The pharmacological activities of polysaccharides are closely related to their structures.In order to identify polysaccharides with new or improved biological activities, researchers have altered the structures of polysaccharides.Modifications generally include adding hydroxyl, carboxyl and/or amino groups to polysaccharide residues and the formation of polysaccharide complexes (Fry et al., 2001;Charvet et al., 2011;Kadokawa et al., 1998).Each modification has a unique effect on the activity of a polysaccharide.

Extraction, preparation and an assessment of the activity of carboxymethyl polysaccharide from Panax japonicus
Qi-Yang YU 1,2 , Shuai YUAN 1,2 , You-Yu YAN 2 , Xi-Feng ZHANG 1 *  a In this study, water-soluble polysaccharides of P. japonicus from Enshi, Hubei Province, were extracted using water, precipitated using ethanol and carboxymethylated.Based on previous studies that assessed the feasibility of separation and purification of polysaccharides from Panax japonicus, carboxymethylation of the polysaccharides was optimized using a response surface methodology (RSM).Antioxidant and antineoplastic activities of carboxymethylated polysaccharides were preliminarily explored, and the effects of carboxymethylation modification of P. japonicus polysaccharide activity were investigated.

Materials
P. japonicus plants were purchased from the Hubei Province of China.All chemicals and solvents were of analytical grade.

Preparation of P. japonicus polysaccharide (PJPS) extraction
The polysaccharides were extracted by water extraction and alcohol precipitation.The polysaccharide content was determined using sulfuric acid-phenol method with glucose functioning as a standard (Yao et al., 2020).Three factors affecting extraction conditions were categorized into three different levels, as specified in Supplemental Table 1.Single factor experiments were conducted to assess the effects of different extraction conditions on PJPS extraction efficiency, as follows: X 1 (liquid-material ratio), X 2 (extraction time) and X 3 (extraction temperature).On the basis of single factor analysis, optimization of the three factors were determined, and a multi-factor response surface optimization test was carried out.Further optimization of the polysaccharide extraction process using a Box-Behnken central combination test was accomplished (Yang et al., 2016).After decompression and concentration, aqueous solutions were precipitated with 95% ethanol three times, and precipitates were re-dissolved, alcohol precipitated, and freeze dried to obtain crude PJPS.
Crude PJPS (about 80 mg) was re-dissolved in ultrapure water (4 mL), and a Cellulose DE-52 (DEAE) column (1.0 × 40 cm) was used to separate compounds.Gradient elution was carried out using 0, 0.05, 0.1, 0.2, and 0.3 mol/L NaC1 solution.Sugars were assessed using phenol sulfuric acid method and the ultraviolet absorbance was assessed at 490 nm.The major peak of polysaccharide corresponding to PJPS was concentrated and further purified using a 1.0 × 40 cm Sephadex G-100 column at a flow rate of 0.5 mL/min.PJPS fractions from the purified polysaccharide were analysed, concentrated and freeze-dried.

Carboxymethyl substitution of PJPS (CM-PJPS)
CM-PJPS was accomplished using a method described in Jiang et al. (Jiang et al., 2013).A proper amount of PJPS was dissolved in sodium hydroxide solution (2 Mol/L), stirred for 30 minutes, followed by the addition of 2g monochloroacetic acid.Then the mixture was stirred and refluxed at a certain temperature for a certain time.After the reaction was completed, it was cooled to room temperature and adjusted to pH 7 using hydrochloric acid.Further the reaction was placed in the dialysis bag and dialyzed for 48 h.After decompression and concentration, the dialysate was precipitated three times with 95% ethanol and dried to obtain CM-PJPS.
In Table 1, reaction time, MCA, and temperature were assigned to three different levels; A, B and C. The effects of A (reaction time), B (MCA) and C (reaction temperature) on the degree of polysaccharide carboxymethylation achieved were assessed using a single factor experiment.Based on single factor analysis, a response surface optimization value for each factor was determined.Carboxymethylation of polysaccharides was further optimized using the Box-Behnken central combination test.

Determination carboxymethyl substitution degree
The degree of carboxymethyl substitution achieved was determined as described by Zhao et al. (Zhao & Zhang 2015).Briefly, 10 mg CM-PJPS was added to 3 mL 70% ethanol, mixed and incubated 5 min.Next 10 mL distilled water and 50 mL 0.5 mol/L sodium hydroxide solution were mixed until all the samples had dissolved.The solution was then titrated using 0.1 mol/L hydrochloric acid solution and phenolphthalein served as a chromogenic agent.Degree of substitution (DS) was calculated using the following formulae 1 and 2: ( ) (1) and ( ) Where V 0 is the volume sodium hydroxide (mL); V 1 is the volume of hydrochloric acid consumed by blank determination (mL); V 2 is the volume of hydrochloric acid consumed by sample determination (mL); M 0 is the concentration of sodium hydroxide; M is the concentration of hydrochloric acid; and m is the quantity of the samples (g).

Ultraviolet spectrum
To determine the absorbance of PJPS and CM-PJPS, 0.1mg/ ml solutions were scanned using an ultraviolet spectrophotometer at wavelengths that ranged from 200 to 600 nm.

FTIR analysis
The composition of dried PJPS and CM-PJPS was characterized via Fourier transform infrared spectroscopy (FTIR) from 400-4,000 cm -1 .

Scanning electron microscopy (SEM)
Conductive film was bonded to the sample base, and evenly sprinkled with a small amount of sample, then the sample that did not stick was blown lightly with ear wash balls, further, coated with a conductive film.The morphologies of samples were observed using 500, 1,000 and 2,000× magnification using field emission SEM (Yin et al., 2019).

Monosaccharide composition analysis and Determination of molecular weight
Monosaccharide composition of purified PJPS was analyzed using a Promosil C18 column (250mm × 4.6mm × 5μm).Mobile phase A consisted of 0.1 mol/L phosphate buffer saline (PBS) with pH 6.7, and phase B consisted of acetonitrile.The flow rate was 1 mL/min.The column temperature was 30 °C, the detection wavelength was 250 nm, and the injection volume was 20 μL.Monosaccharides were identified based on comparing their mass spectra with those in the Wiley mass spectral library, and the relative abundance of each monosaccharide was calculated using the peak area normalization method (Yao et al., 2020).The molecular weights of PJPS were measured using HPSEC-MALLS-RID according to previous reported methods (Wu et al., 2016;Cheong et al., 2015).

Hydroxyl radical scavenging activity
To determine hydroxyl radical scavenging activity, 500 μL of 2, 4, 6, 8, and 10 mg/mL samples were mixed with 250 μL FeSO 4 (9 mmol/L) and 500 μL salicylic acid-ethanol solution (9 mmol/L).Then 500 μL H 2 O 2 was added and the solution was incubated at room temperature for 60 min.Ascorbic acid was used as positive control, while distilled water was used as a negative control.Finally, the absorbance was measured at 510 nm.Hydroxyl radical scavenging activity was calculated using the following Equation 3: ( ) Where A 0 is the absorbance of the control (water).A 1 is the absorbance of the tested samples, and A 2 is the absorbance of the samples to which water was added, rather than H 2 O 2 solution (Yang et al., 2016).

Fe 2+ chelating activity
Fe 2+ chelating activity was determined by mixing 400 μL of different sample concentrations (2, 4, 6, 8, and 10 mg/mL) respectively with 40 μL FeCl 2 (5 mmol/L), 80 μL ferrozine solution (9 mmol/L) and 1,080 μL water.Ascorbic acid was used as a positive control, while distilled water was used as a negative control.The absorbance of the mixtures was measured at 562 nm.Fe 2+ chelating activity was calculated using the following Equation 4: ( ) Where A 0 is the absorbance of the control group (water).A 1 is the absorbance of the test group, and A 2 is the absorbance of the samples mixed with water instead of FeCl 2 solution.

2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity
To determine ABTS radical scavenging activity, 2.5 mL 7 mmol/L ABTS diammonium salt was mixed with 0.5 mL 15 mmol/L potassium persulfate.After storage in the dark for 24 h, the solution was diluted with distilled water to yield a substance with an absorbance of 0.700 at 734 nm.The diluted solution (1,600 μL) was added to 400 μL of sample solutions (2, 4, 6, 8, and 10 mg/mL) respectively.Absorbance of each solution was measured at 734 nm after they were allowed to react for 10 min at room temperature.Ascorbic acid was used as positive control, while distilled water was used as the blank.The scavenging activity of the samples was calculated according to the following Equation 5: ( ) Where A 0 is the absorbance of the control group (water was added instead of sample).A 1 is the absorbance of the test group, and A 2 is the absorbance of the samples mixed with water instead of ABTS solution.

Superoxide anion scavenging activity
To determine superoxide anion scavenging activity, 300 μL of different sample concentrations (2, 4, 6, 8, and 10 mg/mL) were mixed respectively with 900 μL Tris-HCl buffer (pH 8.0) and 90μL pyrogallol, and then to which 300 μL HCl (10mol/L) was added, further, the solutions were incubated at room temperature for 20 min.Ascorbic acid was used as positive control, and distilled water was used as a blank.The absorbance of the mixtures was measured at 420 nm.Hydroxyl radical scavenging activity was calculated using the following Equation 6: ( ) Where A 0 indicates the absorbance of the control group (water).
A 1 is the absorbance of the experimental samples, and A 2 is the absorbance of the samples mixed with absolute ethanol instead of pyrogallol.

Cytotoxicity of PJPS and CM-PJPS
The cytotoxicity of PJPS was calculated according to a method reported previously (Xing et al., 2018).Human ovarian carcinoma cells (Skov3 and A2780) were cultured in DME/F12 medium (Hyclone, Logan, UT, USA), together with supplemented with fetal bovine serum (10%) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37°C as well as 5% CO 2 in a humidified incubator.Cells were treated respectively with different concentrations of PJPS and CM-PJPS (0, 200, 400, and 800 μg/mL), and then incubated for 24 h.An MTT assay was used to evaluate cell proliferation as well as the cytotoxicity of PJPS and CM-PJPS.The experiment was repeated three times.

Optimization of polysaccharide extraction conditions
Using the absorbance as an index for detection, the influence of each factor on extraction of PJPS was investigated.Specific experimental results determined using the (Box-Behnken design) BBD were shown in Supplemental Design-Expert 8.0.6 software was used to further analyze the experimental results determined via BBD shown in Supplemental Table 3. From Supplemental Table 3, X 1 , X 2 , X 3 , X 2 X 3 , X 1 2 , X 2 2 , X 3 2 significantly impacted the response value (P < 0.01), and X 1 X 3 significantly impacted the response value (P < 0.05).The judgment coefficient value (R 2 = 0.9932) indicated that data correlated with the model and identified experimental factors that most greatly influenced the response value.The calibration coefficient of R 2 Adj = 0.9845 indicates that 98.45% of observed experimental data variability can be explained by this regression model.Coefficient of variation (CV) = 0.89%, which shows that the reliability and accuracy of the experiment was good, and precision was determined to be 28.098.The F value of the model was 113.64, which indicates that the model reached a significance level of P < 0.0001.In addition, lack of fit was 0.36 (P = 0.7878 > 0.05), which indicates that there is no significant relationship between the missing values and pure error.The regression model shown was used to guide further experiments, including determining the two-factor effect of the model.Results of these analyses were shown in Figure 1.
Analysis using Design Expert 8.0.6 showed that the optimum conditions for PSPF extraction included a ratio of liquid to material, 22:1, an extraction temperature of 83.99°C, and an extraction time of 2.32 h.Under these conditions, the predicted absorbance of the experimental sample was 0.6208.In order to verify the feasibility of the response surface results, optimal conditions were further optimized and validated.The optimal extraction conditions were applied to confirmative the predictive capacity of the model.An average value 0.6001was produced as a result of five independent laboratory experiments, and the standard deviation was determined to be 0.017.The relative error between experimental and predicted values was determined to be 3.4%, which indicated that the response surface results were reliable.Additionally, polysaccharide purity was 90%.

Optimization experiment of CM-PJPS
The optimum conditions for single factor test were determined to require a reaction time of 2 h, 1.5 g monochloroacetic acid and a reaction temperature of 50 °C.Using substitution degree as a detection index, the influence of the three factors on levels of P. japonicus polysaccharide carboxymethyl substitution was investigated.BBD factor level results were shown in Table 2 Experimental results were further analyzed using Design-Expert 8.0.6 software.The results were shown in Table 3. 3 indicated that A, B, C, AB, AC, BC, A 2 , B 2 , C 2 significantly impacted the response value (P < 0.01).The judgment coefficient (R 2 = 0.9992) showed that the model was reliable and experimental factors influenced the response value.The calibration coefficient of R 2 Adj = 0.9982 indicated that 99.82% of experimental data variability could be explained by this regression model.CV = 1.33% indicated that the reliability and accuracy of the model was good, and precision was determined to be 77.180.

Results of Table
The F value of the model was 968.53, which indicated that the model reached a significant level (P < 0.0001).In addition, the Lack of fit was determined to be 6.13 (P = 0.0562 > 0.05), which showed that there was no significant relationship between the missing value and pure error.Since the regression model was determined to be able to guide the experiment, further analysis of the two-factor effect of the model was carried out, and results were shown in Figure 2. Design Expert 8.0.6 analysis revealed that the optimal conditions of polysaccharide extraction included a reaction duration of 1.38 h, 1.24 g monochloroacetic acid and a reaction temperature of 52.85 °C.Under these conditions, the predicted degree of substitution was determined to be 0.9723.In order to verify the feasibility of response surface results, the optimal conditions were optimized and validated.The average value determined after six repeated experiments was 0.9733 and the standard deviation was 0.0073.These results revealed that the experimental results were stable under conditions, and the relative error with the predicted value was 0.10%, indicating that the results of the response surface were reliable.
The activity of polysaccharides is directly or indirectly affected by their molecular structure.The structure of polysaccharides includes primary structure, secondary structure, tertiary structure and quaternary structure.The activity of polysaccharides can be changed by modifying its molecular structure.Molecular modification can affect the activity of polysaccharides by changing their spatial structure, molecular weight, substituent type, number and position (Getachew & Chun, 2017;Xie et al., 2020).There are many methods for the modification of polysaccharides, including physical method, chemical method and biological method (Liu et al., 2018;Xie et al., 2020;Zhu et al., 2019).In addition to the modification of the main chain, the branched chain of polysaccharides can also be modified (Lee et al., 2017;Xiao et al., 2020).The common methods of modification of polysaccharides or oligosaccharides include acidification, phosphorylation, acetylation, alkylation, sulfonyl formation, carboxymethylation, etc (Cai et al., 2018;Xu et al., 2019).After molecular modification, the bioactivity of polysaccharides has been improved, and even new functions have been produced (Huang & Huang 2017).

Separation and purification of PJPS
As shown in Figure 3A and B, crude PJPS was purified with DE-52, revealing five peaks.The major peak (neutral polysaccharide) was corresponded to PJPS (Figure 3A).Then, PJPS was further purified using SephadexG-100 column chromatography and eluted using deionised water.PJPS was

FTIR spectrophotometric analysis
As shown in Figure 4B, polysaccharide peaks occurred at 3407 cm -1 (O-H stretching vibration) and 2930 cm -1 (C-H stretching vibration).Peaks at 1605 cm -1 and 1418 cm -1 , which were characteristic of asymmetric and symmetric stretching vibrations of -COO-, respectively, indicated that the carboxymethyl had been successfully attached to the polysaccharide chain.

Scanning electron microscopy
Scanning electron microscopy (SEM) is an effective method for assessing polysaccharide morphology.The method involves assessing interactions between an electron beam and sample, producing a high-resolution, three-dimensional image of a sample.Generally, it is used to identify the surface structure of samples.Figure 5 shows that PJPS is porous, middle degree of substitution of CM-PJPS(M-CM-PJPS) is loosely porous, and high degree of substitution of CM-PJPS(H-CM-PJPS) forms flaky aggregation, which appears even more loosely packed than M-CM-PJPS.

Antioxidant analysis
As shown in Figure 6, CM-PJPS has hydroxyl radical, metal ion radical, ABTS radical and superoxide radical ion scavenging activity.After carboxymethyl modification, the antioxidant capacity of PJPS significantly improved.However, the hydroxyl radical scavenging activity of unmodified polysaccharide was also strong.The removal rate of metal ions by polysaccharides of H-CM-PJPS was significantly higher than those of M-CM-PJPS and PJPS, and the maximum removal rate was determined to be 56.90%,while the positive ascorbic acid control possessed no significant removal rate.The free radical scavenging rate of modified PJPS was significantly higher than that of unmodified PJPS, and the maximum scavenging rate was 73.26%, which was lower than that of the ascorbic acid positive control.Modified polysaccharide superoxide radical ion scavenging rates were significantly higher than those of unmodified polysaccharides.The maximum scavenging rate was determined to be 72.73%,which was less than that of ascorbic acid (positive control).
Polysaccharides are a kind of important natural product resources.Extensive studies have shown that active polysaccharides have a variety of biological activities, including antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic and so on.Researchers at home and abroad are very concerned about the antioxidant capacity of active polysaccharides, especially in vitro antioxidant activity, and have published a large number of studied results (Chen et al., 2009;Zhao et al., 2014;Pereira et al., 2012).The antioxidant mechanism of antioxidants can be summarized as: elimination of active free radicals, elimination of inactive oxidation factors, combination of metal ions and so on.There are many factors affecting the antioxidant activity of polysaccharides, which may include the source, chemical structure, molecular weight, purity and spatial structure of polysaccharides (Huang et al., 2017;Mei et al., 2017;Mirzadeh et al., 2020).
unimodal, and peaks were symmetrical, which indicated that the sample was relatively uniform (Figure 3B).

UV absorption
As shown in the Figure 4A, UV absorption of PJPS and CM-PJPS (DS of H-CM-PJPS and M-CM-PJPS were 0.97 and 0.78, respectively) were observed from 200-600 nm, which indicated that modification did not significantly affect polysaccharide absorption.apoptosis of tumor cells and so on.Among which the research on the antitumor activity of polysaccharides has attracted the most attention, and polysaccharides has been applied in clinical practice, such as lentinan, Polyporus umbellatus polysaccharides, Ganoderma lucidum polysaccharides, Coriolus versicolor polysaccharides, astragalus polysaccharides, etc (Li et al., 2020;Sohretoglu & Huang 2018;Zhang & Zhao 2019).Polysaccharides can directly kill tumor cells by inducing differentiation or apoptosis of tumor cells and affecting oncogene expression, or improve host anti-tumor immune function by activating macrophages, activating lymphocytes, promoting cytokine secretion and activating complement, so as to inhibit tumor and play a better anti-tumor effect (Fu et al., 2014;Liu et al., 2019).

Analysis of anticancer results
Data from Figure 7 indicated that PJPS did not inhibit A2780 and Skov3 tumor cell survival after modification, the inhibitory effect of polysaccharides on tumor cell survival was significantly enhanced.As concentration increased the inhibitory effect of H-CM-PJPS was enhanced relative to that of M-CM-PJPS.The A2780 tumor cell survival was 66.15%.The lowest survival rate of Skov 3 cancer cells was 76.08%.
Polysaccharide has a wide range of biological functions.It can not only be used as energy supply material and basic components of some substances in the body, but also participates in the process of intercellular recognition, regulation of immune energy supply, transportation of intercellular substances, cell transformation,

Conclusion
A single factor experiment revealed that optimal PJPS extraction conditions established using RSM contained a liquid to material ratio, 22:1, an extraction temperature of 83.99 °C, and an extraction duration of 2.32 h.Under these conditions, the purity of polysaccharide reached 90%.Single factor experiments with RSM also were used to determine optimal conditions for CM-PJPS.To produce optimal levels of CM-PJPS, a reaction duration of 1.38 h, 1.24 g MCA and a temperature of 52.85°C was determined.Under these conditions, the degree of substitution of polysaccharides reached 97%.After CM-PJPS, antioxidant capacity and anticancer activity of polysaccharides were significantly improved.

Figure 1 .
Figure 1.Response surface plots showing effect of variables on PJPS yield.Effects of (X 1 ) liquid-material ratio, (X 2 ) extraction time, h, and (X 3 ) extraction temperature (°C) on PJPS extraction yield are shown.

Figure 2 .
Figure 2. Response surface plots showing the effect on CM-PJPS.Effects of (A) reaction time; (B) MCA (g) and (C) reaction temperature (°C) on the degree of substitution of CM-PJPS.

Figure 3 .
Figure 3. Isolation and analysis of PJPS.(A) Elution curve of PJPS using the DEAE-52 ion-exchange column.The eluent was H 2 O and NaCl (0.1, 0.2, 0.3 and 0.5 mol/L) with a flow rate of 0.5 mL/min (10 min/tube); (B) Elution curves of PJPS using the Sephadex column.The eluent was H 2 O with a flow rate of 0.4 mL/min (20 min/tube); (C) Monosaccharide composition of PJPS; (D) GPC chromatogram of PJPS for molecular weight determination with HPSEC-MALLS-RID system.

Figure 5 .
Figure 5. Morphology of PJPS and CM-PJPS observed by SEM at different magnifications.

Table 1 .
Levels and code of extraction variables used in Box-Behnken design.

Table 2 .
Using Design-Expert 8.0.6 software, results of Supplemental Table2were analyzed and the following quadratic regression Equation 7 was obtained: .

Table 2 .
Experimental Design and Results of CM-PJPS Box-Behnken.

Table 3 .
Analysis of variance of the experimental results of the CM-PJPS.
p:Significance test; CV: Coefficient of variation; F: F test; df: Degree of Freedom.