Physicochemical and antioxidant properties of extruded Rhodiola as affected by twin-screw extrusion

ABSTRACT In order to explore the effect of extrusion operating parameters on the physicochemical and antioxidation properties of the extruded Rhodiola, Rhodiola was extruded as raw material under different moisture content (24, 26, 28%), barrel temperature (120, 130, 140°C) and screw speed (200, 250, 300 rpm) conditions. After extrusion, the water solubility index of Rhodiola was significantly higher than that of raw material, while the water absorption index was significantly lower than that of raw material. In addition, due to Maillard reaction, L* and a* values of color decreased significantly. The extraction rate of soluble substances, rhodioloside, total flavonoids and total phenols of Rhodiola rosea after extrusion were significantly higher (P < .05) than those of the raw material. Both moisture content and barrel temperature had important effects on the total phenolic content and antioxidant activity. At the same time, moisture content also significantly affected the rhodioloside content when the barrel temperature significantly affected the total flavonoid content. Whereas, the screw speed significantly affected the total flavonoid content. In conclusion, the extrusion process facilitated the increase of active ingredients of Rhodiola and significantly enhanced its antioxidant capacity. Graphical Abstract


Introduction
Rhodiola rosea is a perennial herb belonging to Rhodiola genus of the Crassulaceae family. [1]It is mainly distributed in the west and north of Asia, Europe, north America and other places, under the hillside forest, gravel hillside and alpine tundra at an altitude of 1600-2500 meters. [2,3][6] In addition, Rhodiola rosea exhibits a wide range of biological and medicinal activities such as antifatigue, [7] anti-oxidation, [8] anti-altitude reaction, [9] anti-inflammatory, [5] anti-depressant, [10] anti-aging [11] activities.Currently, antioxidant is an important direction in the study of Rhodiola rosea and there are many reports.For instance, [12] investigated the antioxidant activity and antibacterial activity of Rhodiola rhizome and its aqueous and ethanolic extracts, and the test results showed that the ethanolic extract showed the strongest antioxidant and antibacterial activity compared to the aqueous and dried raw materials.The hypoglycemic effect of irradiated Rhodiola ethanol extract was studied by. [13]The results showed that Rhodiola ethanol extract had inhibitory effect on α-Glucosidase activity after irradiated by 20 kGy 60Co-γ ray when compared, the inhibitory effect of α-Glucosidase activity was significantly better than that of other irradiation doses (P < .05). [14]reported the in vitro antioxidant effects of four purified components of Rhodiola rhizome.Salidroside, Arbutin, Rhodiolinin and Kaempferol showed various antioxidant effects in free radical scavenging assay and ferric-reducing antioxidant power assay, with kaempferol showing the most significant antioxidant effect in vitro.
Currently, most of the studies on Rhodiola rosea have been conducted by ethanol extraction, and studies on twin-screw extrusion of Rhodiola have not been reported.Twin screw extrusion technology is a continuous operation process that shows higher processing capacity when compares to that of batch system. [15,16]The process integrates mixing, stirring, crushing, heating, cooking, sterilization, extrusion and molding steps, and the twin-screw extruder is capable of carrying out the abovementioned multiple operating units simultaneously and continuously. [17]Due to the pushing effect of the extruder screw, the material is continuously squeezed forward in the barrel, and is mixed and stirred in the process.At the same time, under the action of high temperature and high shear force, the internal molecules of the material are disintegrated, denatured and rearranged, therefore, the process changes the structure of some components, content and type.At the die head, the material is extruded quickly from the tiny die hole, instantly from the high temperature and high-pressure state down to normal temperature and pressure state, at this time, water that containing in the material undergoes rapid vaporization due to the huge temperature and pressure differences, hence, leading the material to be puffed. [18]In addition, the cell wall of the plant treated by the twin-screw extruder is destroyed, which helps the overflow of soluble components inside the cell, and the extraction rate of soluble substances increases dramatically.The process is not only enables the full releasing of active ingredients, but also allows the structure of some active substances to be changed and better perform the functional role of the plant itself. [19]The results of the experiment by [20] showed that the crude saponin and acidic polysaccharide contents of white ginseng root extrudate were significantly higher than those of raw material.In addition, antioxidant properties of extrudate were also increased, while the reducing sugar content was markedly lower than that of raw material.Another report indicated that the antioxidant properties of ginseng extrudates were significantly increased after ginseng powder was extruded by different configurations of screws, and the content of total saponins in ginseng extrudates was up to 14.31%, and the content of rare ginsenoside Rg3, which is almost absent in raw ginseng powder, was increased by 73.33%. [21]Extrusion operation parameters (moisture content, barrel temperature, screw speed, etc.) are very important factors affecting the physical and chemical properties of extrusion.The effects of moisture content, barrel temperature and screw speed on the physicochemical properties of red ginseng extrusion were verified in an experiment by Gui. [22]It was reported that the physicochemical properties of the extrudate were mainly dependent on the feed moisture content and barrel temperature, while the screw speed had less effect.
In order to investigate the effects of operation parameters on the physicochemical (soluble substances, Rhodiolosides content, Total flavonoids and Total phenolic content) and antioxidant (DPPH and ABTS free radical scavenging capacity) properties of Rhodiola rosea, this experiment was conducted using Rhodiola as the raw material under different moisture content (24, 26, 28%), barrel temperature (120, 130, 140°C) and screw speed (200, 250,  300rpm).

Material
Rhodiola dried root from Changbai Mountain was purchased from Weiye Pharmacy (Yanji City, Jilin Province, China), DSE-30 twin-screw extruder (Shandong Shengrun Machinery Co. Shandong Province, China).All reagents used in this experiment were analytically pure except methanol and acetonitrile, which were chromatographically pure.

Extrusion process
The dried Rhodiola roots were crushed and passed through a 40-mesh sieve.Rhodiola powder was extruded by a DSE-30 twin-screw extruder with a feeding rate of 100 g/min, a die hole diameter of 3.00 mm, a screw diameter of 32 mm, and a screw length of 742 mm.A three-factor, three-level (moisture content: 24, 26 and 28%, barrel temperature: 120, 130 and 140°C, screw speed: 200, 250 and 300 rpm) response surface analysis experimental design was used for this experiment.The conditions of moisture content, barrel temperature, and screw speed are shown in Table 1.The extruded Rhodiola sample is dried in an oven set at 80°C for 5 h, then crushed with a pulverizer and passed through 60mesh sieve.The final powder samples were bagged and stored sealed.The configuration of the extruder screw used in the experiments is shown in Figure 1.
Water solubility index and water absorption index The water solubility index and water absorption index were determined with reference to the determination method of Anderson et al. [23] The powder sample with M0 = 1 g (dry weight) and 20 mL of distilled water were added to a centrifuge tube with weight M1, shaken vigorously with an oscillator for 1 min and then heated in a water bath at 30°C for 30 min.And then centrifuged at 4000 r/ min for 15 min.After centrifugation, the supernatant was poured into a clean evaporating dish with known weight M2, marked and then the evaporating dish was put into a drying oven at 105°C for drying.The weight after drying was recorded as M3.The weight of the centrifuge tube after pouring off the supernatant was recorded as M4.Three groups of each sample were taken for parallel experiments, and the average value was finally taken.The results were compared with the measurement of unextruded Rhodiola.The formula was calculated as follows: Water Absorption Index WAI= % Where: M 0 : sample weight (g), M 1 : net weight of centrifuge tube (g), M 2 : net weight of evaporation dish (g), M 3 : evaporation dish weight after drying (g), M 4 : weight of the centrifuge tube after removing the supernatant (g).

Color
Using spectrophotometer to analyze the color of the sample, the L*, a*, b* values of the powder sample and raw material were measured, data were recorded, and the average value was calculated.

Extraction yield of soluble substances
The extraction yield of soluble substances was determined following the method of Jung et al. [24] Briefly, Rhodiola powder sample (M 0 = 2 g (dry weight)) was extracted with 40 mL distilled water at 80°C for 3 h.After centrifugation at 4000 rpm for 15 min, the supernatant was collected, but, solid residue was two times again subjected to the extraction process.The supernatant solutions collected three times were combined and poured into an evaporating dish (M 1 ) prior to dry in a drying oven at 105°C until the constant weight of sample (M 2 ) was reached.The extraction yield of soluble substances was then calculated using an equation 1.

Rhodiolosides content
A high performance liquid chromatography (1260 Infinity, Agilent, Germany) was used to investigate the Rhodiolosides content containing in Rhodiola sample.Firstly, Rhodiola powder (0.5 g) was dissolved in 10 mL of methanol (70% v/v) prior to sonicate (KQ-500DE, Kunshan Ultrasonic Instrument Co., Kunshan, China) at 40°C, 400 W for 75 min.The sample was then cooled to room temperature and centrifuged at 4000 rpm for 15 min.The supernatant was collected and filtered through a 0.45 μm membrane prior to subject to the HPLC system.In this study, a C18 column (4.6 mm × 250.0 mm, 5 μm) was used as a separating column and an elution mobile phase was acetonitrile (A)-water (B).The HPLC conditions were as follow: 0-7 min, 8%A; 7-10 min, 8%A-12% A; 10-15 min, 12%A.The flow rate was 1.0 mL/min, wavelength 276 nm, column temperature 25°C, and injection volume 20 μL. [25]The concentration of rhodiolosides containing in Rhodiola samples was reported as an average value from triplicate experiments.

Preparation of Rhodiola extrudate extract solution
The Rhodiola extrudate extract was prepared using the method described by Zhang et al. [21] with slight modifications.Firstly, Rhodiola extrudate powder (2.0 g) was mixed with 30 mL of 70% (v/v) ethanol and heated in a water bath at 50°C for 1 h.Sample was then, ultrasonicated at 50°C, 40 W for 1 h, prior to centrifuge at 4000 rpm for 15 min.The obtained supernatant was referred to as Rhodiola extrudate extract solution, which was collected and stored at 4°C until used.

Total flavonoids content
According to the method of Pazilat et al., [26] the total flavonoid content of Rhodiola extract was determined by adding 0.4 mL of 10% aluminum nitrate to 1 mL of Rhodiola extrudate extract solution mixed with 0.4 mL of 5% sodium nitrite after 6 min of standing.Then, 4 mL of 1 mol/L sodium hydroxide was added to the solution after another 6 min of standing.The final volume of solution was set to 10 mL by adding 3.6 mL of distilled water.After 15 min of standing, the absorbance value of solution was measured under a spectrophotometer at 510 nm.Total flavonoids content of sample was then calculated using an equation (2).This experiment was carried out in triplicate.
Total flavonoid content mg=g where: D is dilution times, Rut is rutin concentration (mg/mL), RV is Rhodiola extract volume (mL), and M is weight of Rhodiola sample (g)

Total phenolic content
The total phenolic content of Rhodiola extrudate was determined by the forinol method. [27]0.01 mL of Rhodiola extrudate extract was diluted to 1 ml with distilled water and then mixed with 0.5 mL forinol and 1.5 mL Na 2 CO 3 (m/v = 20%).The final volume of sample solution was then set to 10 mL using distilled water.After heating the solution in a water bath set at 75°C for 10 min, the solution was cooled down to room temperature before determining its absorbance value in the spectrophotometer (model and company) at 760 nm.Total phenolic content of sample was then calculated using an equation (3).
where GAE is gallic acid concentration (mg/mL), RV is Rhodiola extract volume (mL), DF is dilution times, and M is weight of Rhodiola sample (g)

DPPH free radical scavenging capacity
The DPPH and ABTS free radical scavenging rates were determined based on the method of Kosakowska et al. [12] with slight modifications.For the experiment, 2 mL of Rhodiola extract was mixed with 2 mL of 0.04 mg/mL of DPPH solution.The mixture was kept for 30 min in the dark at room temperature.The absorbance (A i ) at 517 nm was then measured using a spectrophotometer.In the present study, the absorbance (A j ) of 2 mL of Rhodiola extract mixed with 2 mL of anhydrous ethanol and the absorbance (A 0 ) of 2 mL of DPPH solution mixed with 2 mL of anhydrous ethanol were also measured as the absorbance of blank and control, respectively, Equation (3) was used to calculate the DPPH free radical scavenging rate (%) where: A i is the absorbance value of Rhodiola rosea extract, A j is the absorbance value of blank Rhodiola rosea extract, and A 0 is the absorbance value of control.

ABTS Free radical scavenging capacity
Following the method of Kosakowska et al. [12] with slightly modify, the ABTS powder and potassium persulfate were added to a volumetric flask and the volume was fixed to 100 ml so that the concentration of ABTS was 7 mmol/ml and the concentration of potassium persulfate was 2.45 mmol/ml.The prepared solutions were allowed to stand at 25°C for 14 h in the dark condition.The ABTS radical solution was diluted with phosphate buffer (PBS, 0.01 mol/L, pH = 7.4) until its absorbance at 734 nm was equal to 0.70 to obtain the ABTS reaction solution.20 μL of Rhodiola extrudate extract was mixed with 2 mL of ABTS reaction solution and then shaken thoroughly, and the absorbance value at 734 nm was measured after standing for 10 min.The absorbance value of the sample was determined three times, and the ABTS reaction solution without the addition of extruded rhodiola was used as the blank control.

ABTS free radical scavenging rate %
where A S is an Absorbance value of the sample tube, and A 0 is an Absorbance value of the control tube

Statistical analysis
The differences between sample groups were analyzed by Duncan's analysis (SPSS 17.0, P < .05),and the selection of the best combination of operating parameters for twin-screw extrusion of Rhodiola was performed by response surface analysis (Design Expert 8.0.6).

Water solubility index and water absorption index
As can be seen from Table 1, the water solubility index of all samples after extrusion were all significantly higher (P < .05)than the original Rhodiola sample, while the water absorption index of all samples were significantly lower (P < .05)than the original Rhodiola sample.The reason for this is that during the extrusion process the material was subjected to high temperature, mechanical energy was converted into intermolecular thermal energy, large molecules were transformed into small molecules, most of the branched chains in the starch were decomposed, therefore the content of soluble components increased and the water solubility index of Rhodiola rosea increased after extrusion. [28]Hagenimana et al. [29] concluded that extrusion cooking increased the degree of starch pasting and dextrinisation, which increased the content of soluble components in the samples, resulting in The water solubility index values increased.In addition, as the high temperature caused the Rhodiola powder to paste, the distance between starch molecules increased, weakening the hydrogen bonding between starch molecules and resulting in the exposure of a large number of hydrophilic groups within the starch molecules. [30]The greater shearing effect on the Rhodiola extrudate leads to partial cleavage of some of the material, an increase in water-soluble material and a decrease in water-absorbent material.

Color
The color of the product is an important quality index.L* value is called the brightness index, which reflects the combined value of whiteness and brightness.a* and b* values are called the chromaticity index, which together determine the hue. [31]From Table 1, it can be learned that the L* and a* values of Rhodiola extrudates all decreased and were significantly lower (P < .05)than those of raw material after extrusion, and the b* values all increased but were not significantly (P > .05)different from those of raw material powder.It is possible that the non-enzymatic browning of amino acids and reducing sugars in Rhodiola powder occurred under the action of high temperature, high pressure and high shear force, which produced black essence-like substances resulting in the decrease of brightness of Rhodiola extrudates.Zhang et al. [32] concluded that the stronger the degree of browning, the chromaticity showed an increase in a* and b* values and a decrease in L* value.

Extraction yield of soluble substances
It can be seen from Table 1 that after extrusion, the extraction yield of soluble substances of all samples increased and was significantly greater than that of the original Rhodiola (P < .05),suggesting that the twin-screw extrusion process was beneficial to the precipitation of soluble substances in Rhodiola.
During the extrusion process, Rhodiola rosea cell walls were destroyed by extrusion, friction, multiple shear forces as well as high temperature and pressure, therefore, soluble substances were more easily dissolved and released.During the extrusion process, due to friction, shear and high temperature and high pressure steam, the lignin between the cells and between the layers within the cell wall can be melted and some of the hydrogen bonds are broken.Lignin, cellulose and hemicellulose undergo hightemperature hydrolysis, which causes the cell wall to be broken and loosened, and the functional effective material is exposed. [19]When the extrusion conditions were 24% moisture content, 130°C barrel temperature, and 300 rpm screw speed, the maximum yield of soluble substances extraction of Rhodiola samples was 35.91%, which was 17.25% higher than that before extrusion.This is similar to the result obtained by, [33] where the extraction yield of soluble substances of ginseng extrudates was observed increasing from 24.5% to 73.2% after extrusion.

Rhodiolosides content
Rhodiolosides is thought to be one of the most critical plant constituents needed for the therapeutic activity. [2]As can be seen from Table 2, the rhodiolosides content of samples 1, 2, 3, and 4 were significantly (P < .05)smaller than those of samples 14, 15, 16, and 17, which indicated that the content of rhodiolosides increased as the moisture content increased.The effect of barrel temperature on the increasing of rhodiolosides content were also seen in this study when that content in samples 1, 5, and 6 were compared with ones in samples 4, 12, and 13, respectively.However, the higher rhodiolosides content in sample 14 than that of sample 17 could imply to the degradation of rhodiolosides molecules that might occur at too high temperature level.In order to determine the influence of screw speed, the rhodiolosides content in samples 2, 5, and 12 were compared to those of samples 3, 6, and 13, respectively.Since samples in the former group showed smaller rhodiolosides content when compared to ones containing in samples in the latter group, this result also indicated that rhodiolosides content increased along with the increasing of screw speed.However, the rhodiolosides content remained constant once the screw speed was increased to the certain level.The increasing in rhodiolosides content when increasing the level of extrusion parameters could be attributed to the impact of high temperature, pressure and shear force in the extrusion process to weakening the molecular bonds and increasing water absorption. [22,34]These results are in line with those of, [33] who found the increasing of ginsenoside content of white ginseng after extrusion.Data in Table 2 revealed that moisture content, barrel temperature, screw speed and the interaction between moisture content and barrel temperature had significant (P < .05)impact on the content of rhodiolosides, in which the effect those factors from highest to lowest was moisture content > screw speed > barrel temperature.Sample 11 (26%, 130°C, 250rpm) had the highest content of rhodiolosides.

Total flavonoid
The total flavonoid contents of Rhodiola raw material and Rhodiola extrudates were shown in Table 2.The total flavonoid content of raw material was 6.42 mg/g, whereas those content of all samples after extrusion were observed significantly (P < .05)higher.This result indicated that the extrusion process had significantly impact on the increasing of total flavonoid content of Rhodiola.According to Table 3, the total flavonoid content of Rhodiola was positively correlated with the barrel temperature, but negatively correlated with the screw speed.However, there was no significant (P > .05)pattern observed for the effect of moisture content on the total flavonoid content.The reasons could be due to the influence of high temperature, high pressure, and high shear force that generate the destructive force to destroy the cell wall and fiber structure of Rhodiola, and led to the release of some flavonoid compounds, hence, the total flavonoid content increases. [35]In addition, the increase in temperature can accelerate the melting of the material, and the particles in the material can be easily destroyed by the mixing and shearing action of the screw, and the mechanical strength is reduced and easily expanded. [19]The effect of screw speed on the extrudate of Rhodiola was mainly due to the fact that as the screw speed increases, the residence time of Rhodiola in the barrel becomes shorter and the cell wall was less damaged and the biochemical reaction was not complete. [36]Among the three parameters used, screw speed was influence the most to the flavonoid content, follow by barrel temperature, and moisture content, respectively.

Total phenol content
The total phenolic content of Rhodiola raw material was 18.32 mg/g, which was significantly lower (P < .05)than those content containing in all samples after extrusion (Table 2), in which the phenolic content was observed maximum at 90.63 mg/g in the condition using 24% moisture content, 130°C barrel temperature, and 300rpm screw speed.This result was in consistence with the results of, [35] who demonstrated that the extraction of polyphenols by extrusion-treated mulberry powder was increased by 73.4% compared to normal crushing.However, the total phenolic content of Rhodiola decreased during the extrusion when increasing the moisture content.Similar results have also been reported by [22] and, [20] where the reason behind this effect was explained due to the polymerization of phenolic compounds in the presence of moisture content.In the extrusion process, the proper water content can make the material fully wetted and swollen, which helps to open, break and reorganize the macromolecular entanglement structure in the material under the action of external force.When the material is extruded, the water is heated and vaporized sharply in the reaction chamber of the extruder, producing a strong impact and cutting and mixing the material molecules, thus changing the chemical composition of the material. [19]The barrel temperature was also negatively correlated with the total phenolic content. [37]also reported a negative effect of barrel temperature on total phenolic content during chestnut extrusion.As seen in Table 3, the order of influence of the total phenol content of each factor was barrel temperature > moisture content > screw speed.

Antioxidant properties
The DPPH and ABTS free radical scavenging results of raw and extruded Rhodiola are presented in Table 2.The DPPH radical scavenging rate of all samples and the ABTS radical scavenging rate of most samples were significantly (P < .05)higher than the raw material.This result is in consistent with those previously reported in the literature. [21,38]The scavenging capacity against DPPH and ABTS free radicals in Rhodiola extrudates could be due to the increase in rhodiolosides and phenolic acids. [39]his is consistent with the results of the present experiment in which rhodioloside and total phenol content were increased after being extruded.
The DPPH radical scavenging rate of Rhodiola decreased with increasing moisture content during extrusion, while barrel temperature had a significant (P < .05)but irregular effect on it.Screw speed had a non-significant (P > .05)effect on the DPPH radical scavenging rate. [40]found extrusion processing of germinated brown rice at lower feed moisture content led to higher DPPH and ferric reducing antioxidant power values.From the results of the response surface analysis in Table 3, the effect of each factor on the scavenging rate of DPPH radicals was in the order of moisture content > barrel temperature > screw speed.Figure 2(A) shows the 3D plot of the effect of extrusion operation parameters on the DPPH radical scavenging rate of Rhodiola.The DPPH radical scavenging rate was negatively correlated with moisture content-barrel temperature and moisture content-screw speed, which increased and then decreased with the increase of barrel temperature-screw speed.The best extrusion conditions for the DPPH radical scavenging rate of Rhodiola were determined as follows: moisture content 24%, barrel temperature 135°C, and screw speed 260 rpm.According to Table 3, it was found that moisture content, barrel temperature, and screw speed had significant (P < .05)effects on the ABTS radical scavenging rate of Rhodiola, in the order of moisture content > screw speed > barrel temperature.In addition, ABTS radical scavenging rate was positively correlated with moisture content.Figure 2(B) showed a 3D plot of the effect of extrusion operating parameters on the ABTS radical scavenging rate of Rhodiola.The relationship between ABTS radical scavenging rate of Rhodiola and moisture content-barrel temperature, moisture content-screw speed, barrel temperature-screw speed was similar to ones observed with the DPPH scavenging rate experiment.However, the optimal extrusion conditions for the ABTS radical scavenging rate of Rhodiola rosea were moisture content 24%, barrel temperature 127°C, and screw speed 200 rpm.

Figure 1 .
Figure 1.The screw configuration of twin-screw extruder.

Figure 2 .
Figure 2. Effect of extrusion operating parameter on DPPH radical scavenging rate (A) and ABTS radical scavenging rate(B) of extruded Rhodiola.

Table 1 .
Effects of extrusion operation parameters on physical properties of extruded Rhodiola.
Note: Different letters marked in the table indicate significant differences between means (P < 0.05)

Table 2 .
Effects of extrusion operation parameters on chemical and antioxidant properties of extruded Rhodiola.Note: Completely different letters marked in the table indicate significant differences between each other (P < 0.05)

Table 3 .
Variance analysis of regression model for chemical and antioxidant properties in extruded Rhodiola.