1 Introduction
Polyphenols are a natural product in plants that are widely distributed. They can be obtained in large yields (Daglia, 2012). The structures of polyphenols are diverse and include phenolic substances with phenolic hydroxyl groups on a single benzene ring and highly polymerized phenolic substances (Cheynier, 2005). Despite the diversity, they have the same characteristic of potent biological activities, which include antioxidant and antibacterial activities (Crozier et al., 2009). Polyphenols can be classified according to their source, function, and chemical structure. In terms of chemical structure, polyphenols can be simply considered as substances with one or more hydroxyl groups on an aromatic compound. Furthermore, they can be divided into flavone (flavonoids, flavanols, anthocyanins, flavanols, etc.) and flavonoids (coumarin, caffeic acid, and ferulic acid). In addition, some polyphenols can also form complex high molecular weight polymers with polysaccharides, organic acids, proteins, and other substances in plants, such as tannins. Different extraction methods have been used to obtain the phenolic compounds from different kinds of plants. These include organic matter extraction and water extraction. In addition, several physically assisted extraction methods, such as high pressure assisted extraction (Putnik et al., 2016), microwave extraction (Dahmoune et al., 2015), ultrasonic extraction (Kayan et al., 2021), enzyme extraction, and supercritical carbon dioxide extraction (Essien et al., 2020) have been described. These methods have become widely used in the extraction of polyphenols.
Polyphenols can exhibit good antioxidant properties because of their easily reduced phenolic hydroxyl groups. Their antioxidant capacity is related to the group position of phenolic hydroxyl groups. Reflecting the good antioxidant capacity of polyphenols, phenols can reduce some chronic diseases caused by oxidative stress, such as cardiovascular disease and chronic inflammation (Tsao and Rong, 2010). In addition to their antioxidant capacity, many phenolic compounds can also exhibit antibacterial activity (Sikkema et al., 1995). The complete details of the antibacterial mechanism of phenolic compounds remain unclear. However, it is known that phenolic substances can interact with the cell membrane structure, resulting in irreversible damage that inhibits cell growth and reproduction (Chirico and Gramatica, 2011). Polyphenols can reportedly change the permeability of cell membranes and lead to changes in various cellular functions. Polyphenols can also combine with cell enzymes, resulting in altered cell wall stiffness (Gokhale and Kulkarni, 2000). Phenolic acids destroy the integrity of cell membranes, resulting in the leakage of intracellular substances (Borges et al., 2013). Flavonoids can inhibit energy metabolism in cells and inhibit essential enzymes required for DNA and RNA replication, resulting in cell death (Hiroyuki et al., 1998).
These substances are stored in roots, stems, leaves, fruits, and seeds of many plants, including sweet sorghum. This gluten-free grain is the fifth most-harvested crop globally. It is an economically important agricultural crop with highly efficient photosynthetic activity. It is the main food source in Asia and Africa. Sweet sorghum has a high biological yield and high fermented sugar content (Petr et al., 2003), excellent salt tolerance and drought resistance, and can be planted in semi-arid areas.
Sweet sorghum is planted worldwide as a bioenergy crop (Naeini et al., 2016). Therefore, as a non-food crop, sweet sorghum has great potential for development. However, sweet sorghum is mainly used as a raw material for a single purpose, and research has mainly focused on its sugar or cellulose components (Yu et al., 2012). The straw by-product is seldom used and is typically stripped-off and discarded. Sweet sorghum stalk anthocyanins, phenolic acids, and sterols are good sources of phytochemicals. It is also a potential source of antioxidants (Yang et al., 2015). The stems are rich in phenolic compounds, which may play a role in the prevention of some chronic diseases caused by oxidative stress (Soltani et al., 2021).
The roles of polyphenols as antioxidants, preservatives, and bacteriostatic agents has been widely studied in recent years, and microbial resistance to antibiotics has become an active research area. However, sweet sorghum, as a C4 energy compound widely planted all over the world, is rarely studied in this field. In this context, exploration of the potential value of polyphenol extract from fresh sweet sorghum stem as a natural antioxidant and antibacterial agent is warranted.
2 Materials and Methods
2.1 Chemicals and Materials
Folin phenol reagent, ethanol, ethyl acetate, hydrochloric acid, AlCl3, ZnCl2, beef extract, peptone, NaCl, agar, phosphate buffered saline (PBS), and Coomassie brilliant blue were obtained as analytically pure products from Beijing Solabao Biology Co., Ltd. Sweet sorghum (BJ0602) was grown in saline soil in Wuwei City (longitude: 102.634; Latitude: 37.929), Gansu Province, China, at a site at the Radiation Mutation Engineering Laboratory. It was planted in early May 2020 and harvested on October 15, 2020. The total fermented sugar content of sweet sorghum was relatively high, accounting for approximately 12–13% of the total sugar content. To make complete use of sweet sorghum, straw residue was used to extract phenolic acids.
2.2 Extraction of Polyphenols From Fresh Sweet Sorghum Stalks
Polyphenol extraction from fresh sweet sorghum stalks was performed as previously described (Chen et al., 2021) with some modifications. Briefly, the newly harvested sweet sorghum stems were collected from the sweet sorghum planting base, washed, and then cut into small sections of approximately 10 cm using a juicer. After juicing, the fresh sweet sorghum straw juice residue mixture was vacuum filtered with medium-speed qualitative filter paper to remove the residue. The sample served as the control for the determination of total phenol content and total antioxidant capacity. One hundred grams of fresh sweet sorghum stalk juice residue mixture was added to 500 ml of acid ethanol with an ethanol content of 60% (1.6 mol/L HCl, volume percentage 2%) and the pH was adjusted to 6.0 with NaOH buffer. The mixture was stirred and extracted in a 600 W ultrasonic water bath.
After extraction, the extract and residue were separated using medium-speed qualitative filter paper. The residue was collected and three cycles of washing and suction were performed to completely wash the contents. The dilute solution of polyphenol crude extract (1 L) received 50 g AlCl3 + ZnCl2 composite precipitant (mass ratio AlCl3: ZnCl2 = 1:2; mass ratio of precipitant: sweet sorghum stalk = 1:20) at room temperature to precipitate polyphenol at an adjusted pH of 6.0, using 1 mol/L NaHCO3. After allowing the sample to stand for 30 min, it was centrifuged at room temperature and 6,000 rpm for 15 min. The supernatant was discarded to obtain a complex of polyphenols and precipitate.
A total of 100 g of the complex of polyphenol and precipitant was washed by addition to 500 ml of 60% acid ethanol. After the extract and wash solution were fully mixed, they were centrifuged at 6,000 rpm for 15 min at room temperature. The supernatant was removed and washing was repeated. The procedure was repeated two more times. Following the final wash, 500 ml of 1 mol/L HCl solution was added to the precipitate to completely dissolve it. The same volume of ethyl acetate was added to the solution for static extraction. Each extraction was performed three times for 15 min each.
The extract (500 ml) was placed in a round-bottom flask. Ethyl acetate was recovered by rotary evaporation at 40°C. A polyphenol concentrate was obtained. The concentrate was dried in an aluminum box at room temperature to completely volatilize the ethyl acetate. Subsequently, a small amount of deionized water was added to redissolve it to obtain the polyphenol purification concentrate. The concentrated solution was frozen at-80°C for 18 h and then lyophilized under vacuum at -60°C for 36 h to obtain fresh sweet sorghum stem polyphenol lyophilized powder.
2.3 Optimization of Extraction Technology of Polyphenols From Sweet Sorghum Stalks
2.3.1 Standard Curve
The standard curve was determined using the classical and widely used Folin method (Li et al., 2008). The gallic acid standard was prepared at 0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml. One milliliter of each solution was fully reacted with 0.25 ml of Folin reagent for 5 min in the dark at 25°C. Further, 1 ml of Na2CO3 solution was added. After the mixture was fully mixed, it was heated in a water bath at 50°C for 10 min, cooled to 25°C, and the absorbance was measured at 760 nm.
2.3.2 Determination of Polyphenol Extraction Rate
The crude polyphenol extract was extracted, and the polyphenol content was determined using the standard curve described above. Three measurements were made and the average value was reported. The polyphenol content was calculated using the linear regression equation:
Extraction
rate
(
mg
.
g
-
1
)
=
(
C
×
V
×
n
)
/
m
,
Where C is the polyphenol concentration in mg/mL, V is the volume of crude extract (ml), n is the dilution ratio, and m is the weight of the raw material (g).
2.3.3 Single Factor Experiment
The effects on the extraction rate were determined for acidic ethanol (40, 50, 60, 70, and 80%; 1.6 mol/L HCl, volume percentage 2%), liquid-solid ratio (5:1, 10:1, 15:1, 20:1, and 25:1; mL: g), ultrasonic time (30, 60, 90, 120, and 150 min), and ultrasonic temperature (30, 40, 50, 60, and 70 °C).
2.3.4 Response Surface Optimization
In each single factor experiment, the top three high levels of extraction rate were selected. In the whole process of polyphenol extraction, the response surface design method of four factors and three levels was adopted to systematically optimize the above four conditions (Zhang et al., 2009).
2.4 Analysis of Polyphenol Extracts From Sweet Sorghum Stalks
The liquid chromatography-mass spectrometry (LC-MS) method used was slightly modified according to Wei et al.
(2013) and Garcia (2011). LC-MS was performed using an Ultimate 3000 LC device and Q Exactive HF (Thermo Fisher Scientific) equipped with a newly activated C18 column (Zorbax eclipse C18, 2.0 μm∗2.3∗120 mm), and the extract analysis platform of Qingdao Kechuang Quality Detection Co., Ltd. to determine the types and contents of the main phenolic compounds in sweet sorghum extract. The system operated with a column temperature of 30°C, flow velocity of 0.3 ml/min, mobile phase A of water and 0.1% formic acid, and mobile phase B of acetonitrile. Two microliters of sample were used for each determination. The sample injection temperature of the automatic sampler was set to 4°C, and the mobile phase was 0.1% formic acid and H2O (A) and acetonitrile (B). The gradient used was 95% solution A from 0 to 2 min, followed by the decrease of A from 95% at 2 min to 5% at 20 min, followed by the flow of 5% solution A at 300 μL/min for 5 min. The mass spectrometry conditions were as follows: heater temperature 325°C, sheath gas velocity 45 arb, auxiliary gas flow rate of 15 arb, purge gas flow rate of one arb, electrospray voltage of 3.5 KV, capillary temperature of 330°C, and S-Lens RF level of 55%. Scanning was done in the full scan mode from 100 to 1,500 m/z). Data-dependent secondary mass spectrometry (dd-MS2, TopN = 10) was performed at a resolution of 120,000 (primary MS) and 60,000 (secondary MS). The collision mode was high-energy collision dissociation.
2.5 Determination of Total Phenol Content and Total Antioxidant Capacity
Briefly, 10 μL of polyphenol sample solution was repeatedly mixed with the corresponding liquid in the total phenol content kit (Suzhou Keming Biotechnology Co., Ltd. Suzhou, China), measured at 760 nm, and the total phenol content of the sample was converted according to the standard curve. The results are expressed as mg/g sample. The antioxidant capacity of the samples was determined using the Total Antioxidant Capacity kit (Suzhou Keming Biotechnology Co., Ltd. Suzhou, China). After the sample was fully mixed with the reagent, the absorbance was measured at 593 nm. The T-AOC of the extract was expressed as μmol Trolox/mL.
2.6 Antibacterial Activity of Polyphenols From Sweet Sorghum Stalks
2.6.1 Microbial Cultivation
Staphylococcus aureus CMCC 26003, Listeria monocytogenes ATCC 19114, Escherichia coli CMCC 4410, and Salmonella Typhimurium ATCC 14028 were obtained from the China Medical Culture Center. The antibacterial activity of polyphenol extracts from sweet sorghum was studied. Each strain was inoculated in 25 ml nutrient broth medium (peptone 10 g/L, beef extract 3 g/L, sodium chloride 5.0 g/L), incubated at 37 °C for 8 h in a ZQLY-180 N rotary incubator (Shanghai Zhichu Co., Ltd.) as previously described (Jy et al., 2004). The optical density at 600 nm (OD600) of each strain was adjusted to 1.0. The bacterial suspension (100 μL) was centrifuged at 12,000 rpm for 15 min to obtain the precipitate. The cells of each strain were resuspended in 10 μL of different concentrations (20 g/L) of sweet sorghum phenolic compounds extract and treated for 10 min. Ten microliters of PBS buffer was added to the blank control group of each strain, fully suspended, and maintained for 10 min.
2.6.2 Flow Cytometry
Annexin V-FITC/PI staining (BD Pharmingen, San Diego, CA, United States) was used to assess the degree of apoptosis. The treated cells were fully mixed with 500 μL buffer and reacted with annexin V conjugated to fluorescein isothiocyanate (FITC) and with propidium iodide (PI) for 15 min at 25°C in the dark. Annexin V-positive cells were regarded as apoptotic cells (Xia et al., 2019).
2.6.3 Permeability of Bacterial Membrane
Conductivity was determined by a modification of a previously described method (Lee et al., 1998). After incubation at 37°C overnight, the bacteria were centrifuged at 10,000 rpm for 10 min at room temperature. The collected bacteria were washed with 10 mM PBS (pH 7.0) three times and diluted to approximately 105 colony forming units (CFU)/mL with PBS. The polyphenol extract of sweet sorghum was added to the bacterial cell suspension until the final concentration was 20 mg/ml. PBS buffer with the same volume as the polyphenol extract solution was added to the blank control group. The samples were mixed and incubated at 37°C with shaking (150 rpm at room temperature). The conductivity of the cell culture medium was measured at 0, 1, 2, 3, 4, 5, 6, and 7 h.
2.6.4 Bacterial Protein and Nucleic Acid Leakage
Protein leakage was checked following a previously described method with modifications (Zhao et al., 2017). Bacterial suspension (100 µL) was treated with sweet sorghum phenolic extract and then centrifuged at 12,000 rpm for 15 min at room temperature. The supernatant (20 μL) and 200 μL Coomassie Brilliant Blue G-250 solution diluted with deionized water was mixed at 25°C for 5 min. A fluorescent plate reader was used to measure the absorption of the mixture at 595 nm of the sweet sorghum phenolic compound extract (100 μL). The treated bacterial suspension was then centrifuged at 12,000 rpm for 15 min at room temperature. A 0.22 μM pore size sterile microporous membrane filter was used to filter the supernatant. The remaining liquid was diluted 100-fold and the nucleic acid concentration was measured as described above.
2.7 Statistical Analysis
All tests were repeated three times in parallel. All data are expressed as the mean ± standard deviation (SD; n = 3). The experimental results were evaluated by one-way ANOVA. Significant differences between the means were analyzed by Tukey’s test (p < 0.001).
4 Discussion
Vegetable food processing by-products are one of the main processing problems in the food industry. One of the main processing problems in this industry is the utilization of by-products. However, owing to their good technical or nutritional characteristics, they are also promising sources of compounds (Han et al., 2012). By-products in food processing, including waste, wastewater, seeds, peel, and rhizomes in the production and processing process, are good and effective sources of antioxidants (Patel, 2012).
The types and contents of phenolic acids are important indices to evaluate the utilization potential of plant polyphenol extracts (Scania and Chasani, 2021). However, the antibacterial activity of polyphenols may depend mainly on the concentration of polyphenols and the substitution position of phenolic hydroxyl groups on the aromatic ring. Phenolic acids in plants may eventually bind to proteins and polysaccharides through hydrogen bonds and hydrophobicity (Hammouda et al., 2012). Therefore, in the process of extracting phenolic acids, good solvents are needed to reflect high solubility and some metal ions are needed to destroy hydrogen bonds and increase the extraction rate. Generally, by-products in agricultural processing are the main sources of lignocellulose and phenolic acids (Monlau et al., 2015). For example, the grains after brewing beer are rich in ferulic acid and coumarin (Du et al., 2009). The by-products produced during onion and potato processing have anti-inflammatory effects (Albishi et al., 2013). The by-products of lettuce and chicory are rich in antioxidants and can be used in food (Kerdudo et al., 2014). There are a large number of phenolic acids in sugarcane and they have important effects on the color of fruit juice (Bucheli and Robinson, 1994). Furthermore, phenolic acids have good antioxidant capacity, inhibit the growth of bacteria, and kill tumor cells (Vaquero et al., 2007).
In the single factor experiment, when the concentration of ethanol in acid ethanol was 60%, the extraction rate of polyphenols from fresh sweet sorghum stems was the highest. This finding may be related to the solvent characteristics of ethanol as an organic solvent. Organic solvents have strong polarity, which can promote the dissolution of organic compounds, such as phenolic acids, to a certain extent. However, in the present study, with an increase in ethanol concentration in acid ethanol the extraction rate of polyphenols did not significantly improve. It is very likely that some substances with weak polarity, such as pigments and impurities, dissolve more in this process. This inhibits the complete dissolution of phenolic acid compounds (Gambuti et al., 2009). The optimal ethanol concentration determined in the present study was 60%.
When the solid-liquid ratio was 20:1 ml/g, the solubility of phenolic acids approached saturation. With a continuous increase in the solid-liquid ratio, the extraction rate of phenolic acids did not change significantly. This may be because the increase in ethanol solvent dissolves some impurities, such as pigment, and because of alcohol solubility (Zhang, 2010). Dissolution of effective components is prevented, so the ratio of liquid to material is 20:1 ml/g. The time of ultrasonic treatment in the extraction process was closely related to the dissolution of the solute. In this extraction process, the optimal ultrasonic treatment time was 60 min. A long treatment time may lead to the decomposition and transformation of polyphenols due to thermal effects. As well, if the ultrasonic time is too long, the polyphenols will be oxidized and decomposed, resulting in a decrease in the polyphenol extraction rate. Sixty minutes was determined to be the optimum ultrasonic time. The extraction rate of polyphenols from sweet sorghum reached a maximum at 40 °C. The increase in polyphenol extraction rate may be due to the strong diffusion and dissolution of polyphenols at a certain temperature (Wei, 1961). Higher temperatures may also destroy the structure of phenolic acids and aggravate the oxidative decomposition of polyphenols, resulting in a reduction in the extraction rate (Huo et al., 2017). The optimum ultrasonic temperature was determined to be 40°C.
The experimental data in Table 2 were fit and analyzed using the statistical analysis software Desig Expert 8. The quadratic multiple regression linear equation that was obtained was Y = 5.37 + 0.078A + 0.14B−0.005C−0.091D−0.21AB+0.20AC−0.15AD+0.14BC−0.068BD+0.26CD−0.58A2−0.67B2−0.79C2−0.71D2.
The analysis of the linear regression equation showed that F = 25.06, p < 0.0001. The mismatch difference was not significant (R2 = 0.962, R2 Adj = 0.923), indicating that the experimental model has a good fit and can effectively explain the changes in various values in the response surface test. The coefficient of variation was 3.82%, which is <10%, indicating that the experiment was highly accurate and reliable, with the same experimental results expected in repeat experiments.
The F value reflects the influence of each factor on the experiment. The sequence factors are treatment time, ethanol concentration, treatment temperature, and solid-liquid ratio. Among them, the primary term B, secondary term, and interactive term CD had significant effects on the extraction rate of polyphenols (p < 0.01). The interactive terms AB and AC were significantly different (p < 0.05).
Based on the optimization of the process of polyphenol extraction from fresh sweet sorghum stem by response surface methodology, it was found that the theoretical best extraction process was ethanol concentration 60.61%, liquid-solid ratio 20.51:1, ultrasonic time 60.02 min, and ultrasonic temperature 39.25°C. Under these conditions, the extraction rate of polyphenols from sweet sorghum was 5.39 mg/g. However, considering the convenience and efficiency in practical operation, the process parameters of polyphenol extraction were adjusted to an ethanol concentration of 61%, liquid-solid ratio of 21:1 ml/g, ultrasonic time of 60 min, and ultrasonic temperature of 40°C. The average extraction rate of polyphenols from sweet sorghum was 5.77 mg/g. The error was 1.5%. Therefore, the modified extraction process optimized by response surface methodology was stable and reliable.
To evaluate the bioactivity of polyphenols in fresh sweet sorghum stems, we measured the total phenol content and total antioxidant capacity before and after extraction. After the optimized extraction process, the total phenol content and total antioxidant capacity of fresh sweet sorghum stem extract were greater than those without extraction, which was also consistent with Qin et al. (2020) and Khan et al. (2018). The phenolic compounds extracted from green onion and chrysanthemum had higher 2,2-diphenyl-1-picrylhydrazyl scavenging ability and superoxide anion radical scavenging ability. The whole extraction process can enrich a large number of phenolic acids. Sweet sorghum, as a cheap cash crop, can also be an ideal material for the production of phenolic acids.
Fresh sweet sorghum juice is rich in phenolic acids, including polyphenols and flavonoids. These substances have good biological activities that include antioxidant, anti-inflammatory, and anti-tumor activities (Vanamal et al., 2017). In this study, sweet sorghum juice was rich in 31 polyphenols that included betaine, coumarin, guanosine, cinnamic acid, and naringenin. The polyphenol extract of sweet sorghum was rich in coumarin, vanillin, caffeic acid, coumaric aldehyde, cinnamic acid, and other 18 polyphenols. The main components of these extracts have a strong reducing ability. Zhao et al. (2015) described that extract from sugarcane was enriched in gallic acid, ferulic acid, coumarin, and chlorogenic acid, which have antioxidant and antibacterial properties. In addition, similar to the results of this study, Deseo et al. (2020) showed that the phenols in sugarcane molasses extract were closely related to antioxidant activity.
Escherichia coli, S. aureus, Listeria spp. and Salmonella spp. treated with 25 mg/ml sweet sorghum polyphenol extract were examined to determine the relationship between the antibacterial activity of the polyphenol extract and bacterial membrane permeability. The conductivity of the bacterial culture medium increased significantly in 1 h, but almost did not change between 1 and 7 h. At the same time, the electrical conductivity of the control group displayed almost no change within 7 h. This indicates that polyphenols extracted from sweet sorghum affect the integrity of bacterial membranes and cause electrolyte leakage. The test results of protein and nucleic acid content in the cell culture medium were consistent with the previous experimental results. Protein leakage and nucleic acid leakage were relatively low in the control group, and after treating four foodborne pathogenic bacteria with sweet sorghum polyphenol extract, it was found that the protein leakage and nucleic acid leakage in each treatment group increased significantly. The findings further indicate that the polyphenol extract from fresh sweet sorghum stem can increase the conductivity in bacterial culture medium in a short time, and its effect on gram-positive bacteria is significantly higher than the effect on gram-negative bacteria. The reason may be that polyphenols destroy the integrity of the bacterial cell membrane structure, resulting in the exudation of intracellular molecules, further resulting in an increase in the electrolyte content in the cell culture medium. This result is similar to that reported by Shen et al. (2015). The latter authors opined that cinnamaldehyde can destroy bacterial cell membranes. At the same time, with the increase in cinnamaldehyde concentration, the degree of damage of the cell membrane increased. Xu (2014) studied the effect of fennel seed extract on bacteria and found that an increase in treatment concentration and treatment time of polyphenol extract increased the permeability of the cell membrane. In the present study, the determination of cell apoptosis by flow cytometry confirmed the antibacterial effect of sweet sorghum polyphenol extract on foodborne pathogens. After treatment of the four foodborne pathogens with fresh sweet sorghum stem polyphenols, the number of apoptotic cells exceeded the numbers in the control group of each bacterium.
In general, the position of phenolic hydroxyl groups in phenols has a significant impact on the antioxidant and antibacterial capacities of polyphenols (Toshitsugu et al., 2004). For example, cinnamic acid can lead to irreversible changes in membrane structure, so as to release intracellular components out of cells (Borges et al., 2013). This may be because polyphenols change the hydrophobicity of the membrane structure, resulting in local rupture of membrane structure and formation of holes. The antibacterial effect of flavonoids is different from that of polyphenols (McRae et al., 2007). It may form a complex with the components of the bacterial cell wall, resulting in a gap in the cell wall. The antibacterial mechanism of some substances involves the inhibition of DNA helicase (Matja et al., 2010), resulting in the failure of membrane structure to inhibit bacterial growth. In this study, the main antibacterial mechanism of fresh sweet sorghum stem polyphenols appears to be polyphenol-mediated destruction of the integrity of the cell membrane, resulting in cell apoptosis. Polyphenols may also form a complex with essential substrates in bacterial metabolism, resulting in bacterial metabolic disorder and inactivation of protein variability. These mechanisms require further study.
In conclusion, fresh sweet sorghum stem extract is rich in phenolic compounds, has good antioxidant capacity, and has potent antibacterial action for common foodborne pathogenic bacteria. Its antibacterial mechanism involves the toxicity of polyphenols, particularly the destruction of the microbial membrane structure. Therefore, sweet sorghum polyphenol extract can be used as a natural bioactive material with high added value for antibacterial applications in the food industry.