Influence of Extraction Methods on the Antibacterial and Antioxidant Activities of Rosemary Processing Residue Extracts

 Abstract:Objective to provide reference for the high-value utilization of the residue after essential oil extraction from rosemary. Methods] The solid residue of rosemary after essential oil extraction was used as raw material, and the effective components were extracted by four methods, namely, solvent extraction (CSE), ultrasonic-assisted extraction (UAE), supercritical CO2 extraction (SC-CO2) and subcritical extraction (SE), and then analyzed by liquid chromatography - quadruple quadruple bar - high resolution tandem mass spectrometry (LC-QRT-MS/MS), and the antioxidant capacity of the obtained extracts were comprehensively evaluated by the scavenging capacity of three kinds of free radicals, including DPPH, ABTS and hydroxyl radical, The antioxidant capacity of the extracts was comprehensively evaluated by analyzing the scavenging ability of three kinds of free radicals, such as DPPH, ABTS, hydroxyl, etc., and the antimicrobial effect was evaluated by using three indexes, namely, the circle of inhibition, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC).

 

The main active substances in the four extracts were terpenoids such as salvinorin, oleanolic acid and ursolic acid, flavonoids such as lignans, hesperidin, geranylgeranylgeranyl and hesperidin, as well as phenolic compounds such as salvianic acid and chlorogenic acid. The extracts obtained by the four methods had significant antioxidant effects, and their antioxidant capacities were SC-CO2, CSE, UAE, and SE in descending order, and the extracts obtained by the four methods had significant inhibitory effects on Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), and the inhibitory effects on Staphylococcus aureus were SC-CO2, SE, CSE, UAE, SC-CO2, and UAE, and on Staphylococcus aureus, respectively, in descending order, The inhibitory effects on Staphylococcus aureus were SC-CO2, SE, CSE and UAE, and on Bacillus subtilis were SC-CO2, SE, UAE and CSE in descending order of strength, among which the extracts obtained by using SC-CO2 had obvious inhibitory effects on Bacillus cereus; the synthesized analyses of the circles of inhibition, MICs and MBCs of the five test strains showed that the inhibitory abilities were SC-CO2, UAE, SE and CSE in descending order of strength.

 

[Conclusion] SC-CO2 is the optimal way to recover antioxidant and bacteriostatic components from rosemary processing residues.

 

Rosemary Rosmarinus officinalis (synonym Salvia rosmarinus) is a perennial aromatic evergreen shrub of the Labiatae family, originating from the Mediterranean coastal region, which has been successfully introduced and widely promoted in China. Rosemary is rich in various natural bioactive substances with antioxidant, antibacterial, anti-inflammatory and anticancer effects, and is widely used in cosmetics, medicine, food additives and biocontrol. The traditional uses of rosemary are mainly edible and medicinal. The leaves are widely used as a spice and flavoring [1], a decoction of the leaves and branches is used to treat low blood pressure, abdominal pain, and diarrhea, and an infusion of the leaves is used as a tonic, antitussive, expectorant, anti-asthmatic, antipyretic, and antiparalytic [2]. Rosemary also has good ecological effects such as insect repellency, weed suppression, and soil and water nourishment, and has been used as a cash crop for oil tea and other economic forests [3].

 

As a traditional multi-purpose essential oil, rosemary has high antioxidant and antibacterial activity, which is beneficial to human health and food preservation. As research on the active constituents of rosemary essential oil has progressed, the use of rosemary essential oil in food processing, aromatherapy, medicine and other applications has increased. However, in the process of essential oil preparation, after the essential oil components are extracted from the plant by steam distillation, a large amount of solid residue remains, which is currently not exploited in a high value way. Landfill or incineration is the conventional treatment of essential oil residues, which is not only costly, but also causes environmental problems [4], and can be further processed to achieve high-value utilization and reduce industrial pollution [5].

 

In recent years, domestic and international studies on the development and utilization of solid residues of aromatic plants have mainly focused on composting, animal feed and biosorption [6]. The research results show that the solid residue of rosemary leaves after the extraction of essential oil still contains substances with antioxidant activity, such as sage acid, sage phenol, rosemarinic acid, rosemarinol, etc., of which sage acid has better antioxidant activity on oils and fats, and rosemarinic acid has obvious anti-inflammatory, antioxidant, immunosuppressive, etc. Pharmacological effects, and there is a huge potential for the application of these active substances in the fields of cosmetic, pharmaceutical and food production [7-8], and the traditional utilization of the solid residue of aromatic plants has mainly focused on composting, animal feed, and biosorption [6]. These active substances have great potential for application in the fields of cosmetics, pharmaceuticals and food production [7-8], but the traditional way of utilization does not give full play to the residual value of these essential oil residues. The use of suitable extraction methods to extract these active ingredients, and understand the antioxidant and antibacterial biological activities of the extracts can provide a scientific basis for their high value utilization [9].

 

The extraction method has an important influence on the recovery and utilization of active substances in the residue, and the contents and types of active components in the extracts obtained by different extraction methods are different. Understanding the commonalities and differences of the active components in the extracts obtained by different extraction methods is beneficial to the selection of suitable extraction methods according to the requirements of different applications, and can provide a theoretical basis for the efficient extraction and utilization of the active components in rosemary processing residues. Solvent extraction is the most commonly used method for extracting the natural antioxidant components of rosemary, which is low in cost and easy to scale up, but has the disadvantages of low selectivity and solvent residue.

Bi Liangwu et al. [10] used a two-step extraction method with polar and non-polar solvents to extract antioxidants from rosemary with an extraction rate of 16.08%, in which the contents of rhamnosus acid, rhamnol, and rosmarinic acid were 17.78%, 6.23%, and 3.37%, respectively. Ultrasonic-assisted extraction is to insert the probe emitting ultrasonic waves directly into the solvent mixed with raw materials, so that the raw materials are directly subjected to the action of ultrasonic waves, so as to promote the rapid dissolution of the active ingredients in the raw materials in the solvent, which has the advantages of simple operation, short process cycle, and the active ingredients will not be destroyed, and so on.

 

Luo Xiaofang et al. [11] used ultrasonic-assisted extraction to extract rhamnosus acid from rosemary leaves, and the extraction rate was as high as 93.6%, indicating that ultrasonic-assisted extraction can significantly improve the extraction efficiency of rhamnosus acid. Supercritical CO2 extraction has the advantages of ultra-low temperature, non-toxicity and recyclability [12], and does not destroy the activity of the active ingredients, and is often used to extract rosemary phenolic compounds [13]. Subcritical extraction also has the advantages of low temperature, non-toxicity, recyclability, etc., and is not easy to destroy the active ingredients, and the use of extraction solvents is small, which is suitable for industrial production, and it has been used in the extraction of essential oil of rosemary and the preparation of antioxidants [14].

 

In this experiment, the above four extraction methods were selected for the comparative study on the extraction of rosemary residues, in order to analyze the active components and their antioxidant and bacteriostatic activities under different extraction methods, and to screen the optimal extraction methods with promising prospects, so as to provide a reference for the development and utilization of antioxidant and bacteriostatic products from the residues of rosemary with high value.

 

1 Materials and Methods

1.1 Materials and Instruments

Samples: Sampling was carried out at the rosemary plantation of Yuzhou City Hefeng Agricultural Development Company Limited in Henan Province, and the sampling site was the mature leaves of the current year. The leaves of rosemary were picked and dried naturally. The residue of the dried rosemary leaves after extraction of essential oil by steam distillation is called rosemary processing residue.

 

Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella, Bacillus cereus were all provided by the Laboratory of Economic Forestry, Henan Agricultural University. Each species has been identified.

 

Instruments: XH-2008DE Intelligent Temperature-Controlled Dual-Frequency Ultrasonic Synthesizer/Extractor was purchased from Beijing Xiangbao Technology Development Co., Ltd, Subcritical Dual Solvent Extractor was purchased from Henan Subcritical Biotechnology Co., Ltd, SFE-2 Supercritical Fluid Extractor was purchased from Applied Separations, Inc, USA, Gen5 Enzyme Labeling Instrument was purchased from Biotek, Inc, USA, Heraeus Megafuge 8R High-Speed Freezer Centrifuge was purchased from Zhengzhou Jinyouning Instrument Co. The Heraeus Megafuge 8R high-speed refrigerated centrifuge was purchased from Zhengzhou Jin Youning Instrument Co., Ltd. and the Q-Exactive Plus System liquid chromatography - quadruple quadruple rod - high resolution tandem mass spectrometer was purchased from Thermo Fisher, USA.

 

1.2 Test methods

1.2.1 Ingredient extraction

Pre-treatment of samples: The rosemary processing residues were completely dried in an oven, then removed, crushed and sieved in a pulverizer (pore size 0.18 mm) to obtain rosemary processing residues powder.

 

1）Conventional solvent extraction (CSE). Weighing 15.0 g of rosemary processing residue powder, adding 300 mL of anhydrous ethanol, heating and refluxing for 2 h, filtering the extract, rotary evaporation of the filtered extract, concentrated to about 20.0 mL, put into the oven drying, collected and spare [15-16].

2）

(2) Ultrasoni-assisted extraction (UAE). 1.5 g of rosemary processing residue powder was weighed, 37.5 mL of anhydrous ethanol was added, and the extract was extracted for 2 h at an extraction temperature of 50 ℃, then the extract was filtered, and then rotary evaporated, and the extract was concentrated to 20.0 mL, then dried in an oven, and then collected for spare use [17].

 

(3) Supercritical CO2 fluid extraction (supercritical CO2 fluid extraction, SC-CO2). Weighing 8.0 g of rosemary processing residue powder, adding 95% volume fraction of ethanol as the entrainment agent, entrainment agent and rosemary powder mass ratio of 2:5, extraction pressure of 4 × 104 kPa, extraction for 3 h, collect the extracts drying spare [18].

(4) Subcritical extraction (SE). Weigh 100 g of rosemary processing residue, add 1.5 L of n-butane, extraction 0.5 h, to get a paste extract, drying spare.

 

1.2.2 Component analysis

The compositional analysis of rosemary processing residue extract was carried out by liquid chromatography coupled with quadruple quadruple rod-high resolution tandem mass spectrometry (LC-QRT-MS/MS).

Chromatographic conditions: Agilent Eclipse Plus C18 column (150 mm × 3 mm, 1.8 μm) was used; 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid acetonitrile was used as mobile phase B, and the linear gradient type was ramp; the temperature of the column was 30 ℃, and the temperature of the injection plate was 4 ℃, and the sample was injected into the sample automatically at a flow rate of 5 μL. 95% from 5% for 0~15 min, 95% for 15~18 min, 5% for 18~20 min, 5% for 20~23 min, and the flow rate was 0.3 mL/min.

 

Mass spectrometry conditions: Heated electrospray ionization source (HESI), positive ion scanning mode, spray voltage of 3.50 kV, capillary temperature of 350 ℃, auxiliary gas heating temperature of 200 ℃, atomized nitrogen pressure of 3.5 × 104 Pa, auxiliary gas pressure of 7.0 × 104 Pa, ion sweeping gas pressure of 3.5 × 103 Pa, sheath gas flow rate of 40 L/min, auxiliary gas 10 L/min. The full Ms/dd-MS2 scanning mode was adopted, with a full scanning resolution of 70 000 and an automatic gain control target ion number of 1×106; the scanning range was 80-1000 m/s, and the second scanning resolution was 17 500 and an automatic gain control target ion number of 2×105; the separation window was 1.0 m/z, and the collision energy was 20%, 40%, and 60%, 60%.

 

TraceFinder 3.3 software was used for the characterization of the target, combined with the spectral library, to establish the target screening method OTCML Screening, with a match score of not less than 80, and the quantitative analysis was carried out by the area normalization method to obtain the relative content.

 

1.2.3 Analysis of antioxidant capacity

(1) Determination of DPPH free radical scavenging ability. The extract was diluted with anhydrous ethanol into 2, 4, 8, 16, 32, 64, 128, 256, 512, 1 024 mg/L mass concentration gradient solution, and vitamin C was diluted with deionized water into the same mass concentration gradient solution as the positive control. 20 μL of the sample solution to be tested and 180 μL of the DPPH working solution were mixed in a 96-well plate, and the reaction was carried out for 30 min at room temperature, protected from light, and then the absorbance was measured by an enzyme counter at 517 nm. After the reaction for 30 min at room temperature and protected from light, the absorbance was measured at 517 nm with an enzyme meter, and the results were repeated three times for each sample and control.

 

R1=[1-(A1-A2)/A0] × 100%.

Where: R1 is the clearance of DPPH; A0 is the absorbance of the mixture of anhydrous ethanol and DPPH working solution; A1 is the absorbance of the mixture of DPPH working solution and different concentrations of rosemary extract or vitamin C; A2 is the absorbance of the mixture of different concentrations of the extract and anhydrous ethanol.

 

2) ABTS free radical scavenging capacity assay. The concentration of

7 mmol/L ABTS solution was mixed with 2.45 mmol/L potassium persulfate solution in equal volume, and then left at room temperature and protected from light for 16~24 h, i.e., the storage solution, which was diluted with anhydrous ethanol to obtain the working solution with absorbance at 734 nm of 0.70±0.02, and then set aside. Dilute the extract with anhydrous ethanol into 2, 4, 8, 16, 32, 64, 128, 256, 512, 1 024 mg/L by two-fold dilution method, and dilute vitamin C with deionized water into the same mass concentration gradient as the positive control. 20 μL of the sample to be tested and 180 μL of the ABTS working solution were mixed into a 96-well plate and reacted for 6 min, and then analyzed by ELISA. The reaction was carried out for 6 min, and the absorbance was measured at 734 nm with an enzyme counter, and the results were repeated three times for each sample and control [19-20].

R'=[1-(A1'-A2')/A0'] × 100%.

Where: R' is the free radical scavenging rate of ABTS; A0' is the absorbance of the mixture of anhydrous ethanol and ABTS working solution; A1' is the absorbance of the mixture of ABTS working solution and different concentrations of rosemary extract or vitamin C; A2' is the absorbance of the mixture of different concentrations of the extract and anhydrous ethanol.

 

3) Hydroxyl radical scavenging capacity assay.

The hydroxyl radical scavenging ability was determined by the salicylic acid method. A 96-well plate was prepared by adding 200 μL of 6 mol/L ferrous sulfate solution and 200 μL of 6 mmol/L salicylic acid-ethanol solution, and then the extracts were diluted with anhydrous ethanol to form a solution with a gradient of 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 mg/L, and 100 μL of the extracts were added (deionized water was used as the blank control instead of the extracts). Add 100 μL of the extract solution to be tested (deionized water was used to replace the extract solution for the blank control), and finally add 200 μL of 6 mmol/L hydrogen peroxide solution, shake well, and take a water bath at 35 ℃ for 30 min, and then measure the absorbance at 510 nm using an enzyme meter. Since the sample solution has its own absorbance, deionized water was used to replace the hydrogen peroxide in the measurement of its background value, and the hydroxyl radical scavenging rate (R'') was calculated according to the formula The hydroxyl radical scavenging rate (R'') was calculated according to the formula [21-22].

R''=[(A0''-A1''+A2'')/A0''] × 100%.

Where: A0'' is the absorbance of the blank control; A1'' is the absorbance after adding the sample solution; A2'' is the background absorbance.

 

1.2.4 Bactericidal effect analysis

Prepare MHA medium, inoculate each test bacterial species on MHA medium, put it into 37 ℃ constant temperature incubator for activation for 24 h. Each test bacterial species should be activated for 3 generations consecutively. Prepare MHB medium, pick single colonies onto MHB medium with an inoculating ring, put it into a shaker, and incubate it at 37 ℃ for 8-10 h. After incubation, take an appropriate amount of bacterial suspension, dilute it to the specified concentration with sterile water (the absorbance is 0.08～0.10, and the total number of bacterial colonies per ml of the sample is 1.5×108, as determined by an ultraviolet spectrophotometer at 625 nm), and dilute it to the specified concentration per ml of the sample. Dilute the suspension to a total of 1.0×106 bacteria per ml of sample.

 

1) Bacteriostatic circles. Use a perforator to make a number of 6 mm diameter circular pieces of filter paper, which were autoclaved and prepared for use. Pipette 100 μL of diluted bacterial solution into the MHA medium, evenly spread the bacterial solution, blow dry, use sterile tweezers to clip the filter paper sheet, each medium to paste 3 points, each point to paste 2 layers of filter paper sheet, drop 4 μL of the extract with a quality concentration of 20 g / L, each bacterial species to do 3 replicates, incubate at 37 ℃ for 24 hours, use vernier calipers to measure the diameter of the two vertical directions of each inhibition circle, and take the average value. Measure the diameter of each inhibition circle in two vertical directions with a vernier caliper and take the average value.

 

2）Minimum inhibitory concentration (MIC). The MIC of the extracts was determined by two-fold broth dilution method using a 96-well plate. The extracts were diluted with liquid medium to obtain the samples of 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039 g/L. 150 μL of the pathogenic bacteria suspension was added, and 50 μL of the samples of different concentrations were used to determine the MIC. Add 150 μL of bacterial suspension, 50 μL of different concentration samples, incubate at 37 ℃ for 24 h, observe the growth of the bacterial solution, and the minimum concentration corresponding to the transparency and clarification of the bacterial solution is the MIC, and repeat the determination for three times.

3）

(3) Minimum bactericidal concentration (MBC). According to the results of MIC, 2 μL of each bacterial solution of different concentrations were taken and planted on the culture medium, and then incubated at 37 ℃ for 24 h. The growth of bacteria was observed, and the minimum concentration of the extract corresponding to the absence of colonies was the MBC [23].

 

1.3 Data analysis

The data were statistically analyzed using SPSS 23 and Origin 2021 software.

 

2 Results and analysis

2.1 Effect of extraction methods on the composition of extracts

The total ion flow of the rosemary processing residue extract measured by liquid chromatography coupled with quadrupole-high resolution tandem mass spectrometry (LC-QRT-MS/MS) using four extraction methods is shown in Fig. 1.

 

The relative contents of the active components of the rosemary processing residue extracts obtained by different extraction methods are shown in Table 1. According to the results in Table 1, there were some differences in the composition and relative contents of the active substances extracted by the four extraction methods, of which 16 compounds were extracted by the CSE method, 17 compounds were extracted by the UAE method, 18 compounds were extracted by the SC-CO2 method and 8 compounds were extracted by the SE method. Among them, 16 compounds were extracted by CSE method, 17 compounds by UAE method, 18 compounds by SC-CO2 method, and 8 compounds by SE method. The active substances detected were terpenoids, such as salvianic acid, salvinorin, oleanolic acid and ursolic acid, flavonoids, such as quercetin, apigenin, lignans, geraniol and hesperidin, and phenolic acids, such as rosemarinic acid, caffeic acid, ferulic acid and chlorogenic acid, etc. The results showed that the extracts of these compounds were very similar to those of the UAE method.

 

The relative contents of the active compounds in the extracts obtained by the UAE and SC-CO2 methods were higher, 10.04% and 10.03%, respectively, while the relative contents of the active compounds in the extracts obtained by the CSE and SE methods were 7.67% and 5.01%, respectively. The relative contents of terpenoids in the extracts obtained by the CSE and SE methods were 7.67% and 5.01%, respectively. The highest relative content of terpenoids extracted by the SC-CO2 method was 9.35%, while the relative contents of terpenoids extracted by the CSE, UAE and SE methods were 6.76%, 8.06%, and 4.38% in the following order. The relative contents of flavonoids extracted by UAE method were higher at 1.70%, while the relative contents of flavonoids extracted by CSE, SC-CO2 and SE methods were 0.58%, 0.62% and 0.60%, respectively. In addition, phenolic acids were extracted from rosemary processing residues by the four extraction methods of CSE, UAE, SC-CO2 and SE, with the relative contents of 0.34%, 0.29%, 0.06% and 0.02%, respectively.

 

In summary, compared with CSE, the UAE method was able to recover terpenoids and flavonoids from rosemary processing residues more efficiently, probably due to the fact that ultrasonic cavitation increased the fragmentation of the plant cell wall, which was more conducive to the solubilization of the active ingredients; the advantage of the low-temperature extraction with SC-CO2 to a certain extent avoided the transformation of oleanolic acid to salvinorin at high temperatures, and the method was able to recover terpenoids from rosemary processing residues more effectively. In the SE method, n-butane was used as the solvent, and according to the principle of similar solubility, the extraction was carried out under low pressure and low temperature, which increased the solubility of oleanolic acid and rhamnolic acid, and the terpenes could be recovered more efficiently from the processing residues of rosemary compared with the other three extraction methods.

 

2.2 Effect of extraction method on antioxidant activity of extracts

2.2.1 DPPH free radical scavenging capacity

The DPPH radical scavenging ability of rosemary processing residue extracts obtained by different extraction methods is shown in Figure 2A. As shown in Figure 2A, the DPPH radical scavenging ability of the extracts obtained by the four methods increased with the increase of the extract concentration. When the mass concentration of the extract was higher than 1.28 g/L, the DPPH radical scavenging rate of the extract obtained by the SC-CO2 method slowed down; when the mass concentration of the extract was higher than 2.56 g/L, the DPPH radical scavenging rate of the extract obtained by the SE method slowed down, and the DPPH radical scavenging rate of the extract obtained by the four methods slowed down when the mass concentration was 2.56 g/L. The results of the DPPH radical scavenging rate of the extract obtained by the SC-CO2 method were shown in Fig. 2A. When the concentration of sample mass was 2.56 g/L, the difference of DPPH radical scavenging rate of the extracts obtained by the four extraction methods was significant (P < 0.05), 95.12%, 95.26% and 94.88%, respectively.

 

The IC50 (half inhibitory concentration) of the DPPH radical scavenging ability of the rosemary processing residue extracts obtained by different extraction methods is shown in Fig. 3. As shown in Figure 3, the DPPH radical scavenging abilities of the extracts were in the order of SC-CO2, CSE, SE and UAE, among which the DPPH radical scavenging ability of the rosemary processing residue extract obtained by SC-CO2 method was the strongest, with an IC50 of (327.43±2.48) mg/L. The weakest scavenging ability was observed in the extract obtained by UAE method, with an IC50 of (1 353.67±34.48) mg/L. The IC50 of rosemary processing residue extract obtained by UAE method was (1 353.67±34.48) mg/L. The weakest DPPH radical scavenging ability was found in the extract obtained by the UAE method, with an IC50 of (1,353.67±347.43) mg/L. Taken together with the results shown in Table 1, the relative contents of rhamnoside and oleanolic acid in the rosemary processed residue extract obtained by the SC-CO2 method were at a high level, and it can be seen that rhamnoside and oleanolic acid have a strong scavenging ability for DPPH free radicals.

 

2.2.2 ABTS free radical scavenging capacity

The ABTS radical scavenging ability of rosemary processing residue extracts obtained by different extraction methods is shown in Figure 2B. As shown in Figure 2B, the ABTS radical scavenging ability of the extracts obtained by the four extraction methods increased with the increase of the concentration of the extracts, and the difference of the ABTS radical scavenging ability of the extracts obtained by the four extraction methods was significant (P＜0.05) at the concentration of 0.32 g/L. The results were summarized as follows. The ABTS radical scavenging effect of the extracts of rosemary processing residues obtained by the four extraction methods was significant, and the ABTS radical scavenging rate of the extracts obtained by CSE, UAE, SC-CO2 and SE methods reached 98.18%, 98.48%, 99.34% and 99.99%, respectively, when the mass concentration of the extract was 10.24 g/L. The results of the four extraction methods were summarized in the table below.

The IC50 of ABTS radical scavenging of rosemary processing residue extract obtained by different extraction methods is shown in Fig. 3.

 

As shown in Fig. 3, the ABTS radical scavenging abilities of the extracts were UAE, SC-CO2, CSE and SE in descending order, among which the strongest ABTS radical scavenging ability was found in the rosemary processing residue extract obtained by the UAE method, with an IC50 of (152.13±6.82) mg/L, and the weakest ABTS radical scavenging ability was obtained by the SE method, with an IC50 of (367.30±6.92) mg/L. The results in Table 1 were summarized in the following table. The IC50 of the extract obtained by the SE method was the weakest, with an IC50 of (367.30±6.92) mg/L. The results in Table 1 showed that the relative content of rhamnetol was the highest, and the relative content of flavonoids, such as geranylgeranyl and hesperidin, were also at a high level in the extract obtained by the UAE method, so that it is known that the rhamnetol and the flavonoids have the strong scavenging ability of the free radicals in the ABTS process.

 

2.2.3 Hydroxyl radical scavenging capacity

The hydroxyl radical scavenging ability of the rosemary processing residue extracts obtained by different extraction methods is shown in Figure 2C. As shown in Figure 2C, the hydroxyl radical scavenging ability of the extracts obtained by the four extraction methods increased with the increase of the extract concentration, and the difference of the hydroxyl radical scavenging ability of the extracts obtained by the four extraction methods was significant (P＜0.05) when the sample concentration was 6 g/L. The results showed that the hydroxyl radical scavenging ability of the rosemary processing residue extract obtained by the four extraction methods was significantly lower than that of the rosemary processing residue extract obtained by the four extraction methods. The hydroxyl radical scavenging effect of the extracts of rosemary processing residues obtained by the four extraction methods was significant, and the hydroxyl radical scavenging rates of the extracts obtained by the CSE, UAE, SC-CO2 and SE methods reached 93.38%, 94.68%, 99.14% and 82.71%, respectively, at a concentration of 20 g/L. The results were summarized as follows.

 

The IC50s of hydroxyl radicals scavenging of the rosemary processing residue extracts obtained by different extraction methods are shown in Figure 3. As shown in Fig. 3, the hydroxyl radical scavenging ability of the extracts, in descending order, was as follows: SC-CO2, CSE, UAE, and SE, among which: the hydroxyl radical scavenging ability of the extract of rosemary processing residue obtained by SC-CO2 method was the strongest, with the IC50 of (3.59±0.09) g/L; and the hydroxyl radical scavenging ability of the extract obtained by SE method was the weakest, with the IC50 of ( 10.28±0.55) g/L. The results in Table 1 are summarized in the table. The results in Table 1 showed that the extracts obtained by SC-CO2, CSE and UAE had significant advantages in terms of the types and relative contents compared with those obtained by SE, and it can be concluded that the extracts obtained by these three extraction methods have significant hydroxyl radical scavenging ability.

 

The results showed that the main antioxidant components in rosemary were diterpene phenols, flavonoids and a small amount of triterpenes. According to Table 1, the compounds with strong antioxidant activity detected in the extracts of rosemary processing residues were rosmarinic acid, caffeic acid, salvia divinorum, and rhamnolic acid. The relative contents of antioxidant activities in the extracts obtained by CSE, UAE, SC-CO2 and SE methods were 6.77%, 7.74%, 7.30% and 2.65%, which were in agreement with the antioxidant results.

 

2.3 Effect of extraction methods on the antibacterial activity of the extracts

2.3.1 Circle of inhibition

The circles of inhibition of the extracts of rosemary processing residues extracted by the four methods are shown in Fig. 4, and the specific diameters are shown in Table 2. In general, the extracts obtained by the four extraction methods showed significant inhibitory effects mainly on Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Bacillus cereus), but not Gram-negative bacteria (Salmonella spp. and Escherichia coli), which is in line with the findings of Weerakkody et al [24] and Tornuk et al [25], who found that Gram-positive bacteria were more sensitive than Gram-negative ones. This result is in line with the findings of Weerakkody et al [24] and Tornuk et al [25] that Gram positive bacteria are more sensitive than Gram negative bacteria.

 

When the mass concentration of rosemary processing residue extract was 20 g/L, the extracts of rosemary processing residue obtained by the four extraction methods had obvious inhibitory effects on Staphylococcus aureus and Bacillus subtilis, among which the extract obtained by the SC-CO2 method had obvious inhibitory effects on Bacillus cereus and the extract obtained by the four extraction methods had no obvious inhibitory effects on Salmonella and Escherichia coli at this mass concentration. The extracts obtained by the SC-CO2 method had no significant inhibitory effect on Salmonella and E. coli at this mass concentration. In conclusion, the SC-CO2 method showed the strongest inhibitory effect on rosemary processing residues, with the circle diameters of S. aureus (13.88±0.44), B. subtilis (11.58±0.60) and B. cereus (10.71±0.78) mm, respectively.

 

2.3.2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The minimum inhibitory and bactericidal concentrations of the extracts of rosemary process residues obtained by the four extraction methods on the five test organisms are shown in Table 3. The results showed that the extracts obtained by the four extraction methods had obvious inhibitory effects on these five species, and the best inhibitory effect was obtained by the SC-CO2 method, with the minimum inhibitory mass concentration (MIC) of 0.63 g/L against S. aureus, the minimum inhibitory mass concentration (MIC) of 0.16 g/L against Bacillus subtilis and Bacillus cereus, the minimum inhibitory mass concentration (MIC) of 2.50 g/L against Salmonella, and the minimum inhibitory mass concentration (MIC) of 2.50 g/L against Escherichia coli. 2.50 g/L for Salmonella and 5.00 g/L for Escherichia coli. The extracts obtained by the four extraction methods had obvious bactericidal effects on these five bacteria, among which the extracts obtained by the SC-CO2 method had the best bactericidal effect, with a minimum bactericidal mass concentration of 1.25 g/L for Staphylococcus aureus, and 0.31 g/L for Bacillus subtilis and Bacillus cereus. The minimum bactericidal mass concentration was 1.25 g/L for Staphylococcus aureus, 0.31 g/L for Bacillus subtilis and Bacillus cereus, 5.00 g/L for Salmonella, and 10.00 g/L for Escherichia coli.

 

In addition, oleanolic acid and ursolic acid also had some inhibitory effects, which might be the reason for the significant inhibitory effect of the extracts obtained by the SE method; the extracts obtained by the UAE and CSE methods also had some inhibitory effects on Gram-positive bacteria. The extracts obtained by UAE and CSE methods also showed some inhibitory effects on Gram-positive bacteria, and the contents of the extracts were rich and relatively high.

 

3 Conclusion and discussion

The results of liquid chromatography-mass spectrometry (LC-MS/MS) were compared with those of four extraction methods, namely, CSE, UAE, SC-CO2 and SE, using the processing residue of rosemary as raw material. The results showed that the four extraction methods were effective in extracting a variety of active ingredients, which were mainly terpenoids, phenolic acids and flavonoids. The antioxidant and antibacterial activities of the extracts obtained by the SC-CO2 method were optimized, and this method is the best way to efficiently extract and recycle the constituents from rosemary residues.

 

By comparing the extracts obtained by the UAE and CSE methods, it can be found that the ultrasound-assisted extraction can better improve the extraction efficiency of the active substances. In addition, although the relative content of active ingredients and antioxidant effect of the extracts obtained by the SE method were not optimal, the extraction efficiency of oleanolic acid and ursolic acid, which are triterpenoids, was higher in this method, and it can be used as an effective route for the preparation of unique natural antioxidants and specialized bacteriostatic agents. The results of this study suggest that rosemary processing residues can be a potential source of natural antioxidants and bacteriostatic agents, which have great potential for application in the production of pharmaceuticals, cosmetics and food.

 

Since the beginning of the 21st century, countries around the world have been committed to the recycling of high-value bioactive substances from residual biomass in agriculture and industry. As research on the bioactivities of rosemary continues to expand, so do its applications, and the amount of rosemary waste generated worldwide has increased dramatically. The reuse of rosemary processing residues has been gradually emphasized in foreign countries: Sánchez-Vioque et al. [26] used Soxhlet extraction and ultrasound-assisted extraction to recover active compounds from the solid residue of rosemary steam distillation, and the results showed that the extracts obtained by these two extraction methods exhibited good antioxidant activity; Santana-Méridas et al. ] found that a large amount of phenolic compounds were present in the rosemary residue after the extraction of the essential oil, and by evaluating the antioxidant and biocidal activity of the residue extracts, it was found that the solid residue of the rosemary distillation could be a potential source of antioxidants and natural crop protection agents.

 

Currently, there are few domestic studies on the recycling of active ingredients from rosemary processing residues. Only Le Zhenqiao et al [28] used supercritical CO2 process to extract antioxidant components from rosemary processing residues, and Liao Xiali et al [29] extracted antioxidant components from rosemary processing residues by solvent method. In this experiment, the extraction method and solvent were not screened for a single type of active ingredient, which should be further investigated. The preparation of biochar from the extracted rosemary residue can also be investigated as an alternative for the development of a biochar.

Provide new ideas to realize the circular economy of rosemary essential oil industry.

 

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