Optimization of Ethanol Extraction Process of Antioxidant Components of Rosemary and Antioxidant

 Abstract: To optimize the extraction process of the antioxidant components of rosemary by ethanol extraction and to evaluate their antioxidant activities in vitro. The effects of ethanol concentration, liquid-liquid ratio and extraction time on the yield of antioxidant components (rhamnosus acid + rhamnol + rosmarinic acid) were investigated. Response surface test was used for the optimization of the extraction process and the in vitro antioxidant activity of the freeze-dried crude extract of rosemary was investigated. The optimal extraction protocol was obtained: ethanol concentration of 65%, liquid/feed ratio of 26:1 mL/g and extraction time of 4 days; the crude extract of rosemary had good Fe3+ reducing power, and its IC50 values against ABTS, DPPH and OH radicals were 0.081, 0.280 and 0.360 mg/mL, respectively; the total antioxidant yield of rosemary was 5.418% under these conditions. The results showed that the response surface model had good predictive ability and the crude extract of rosemary had certain antioxidant activity.

 

Rosemary (Rosmarinius officinalis L.) belongs to the dicotyledonous plant family, Labiatae, native to the European region and the Mediterranean coast of northern Africa [1], is a natural, safe, non-toxic, heat-resistant and highly efficient antioxidant, the active ingredients are mainly sage, sageol and rosemarinic acid [2-3], which makes up for the BHA, BHT and TBHQ and other artificial antioxidants with potential toxicity and side effects [4], becoming one of the hot spots in food, medicine and health care research [5-6]. It makes up for the defects of artificial antioxidants such as BHA, BHT and TBHQ, which have potential toxicity and side effects [4], and has become one of the hot spots of research in the fields of food, medicine and health care [5-6]. Therefore, it is of great significance to optimize the extraction process and activity of antioxidants in rosemary.

 

The extraction of antioxidant components of rosemary is mostly done by solvent extraction method, generally using ethanol as an organic solvent at 10:1 liquid/feed ratio (mL/g) for 2 h. JACOTET et al. [8] extracted the antioxidant components of rosemary by ultrasonic and microwave methods, and found that the use of ultrasonic waves to assist the extraction of rhamnosus and ursolic acid could increase the extraction rate of the method is short, but it may easily destroy the active components in the extract. In this paper, we used ethanol extraction to extract the antioxidant components from rosemary, but it is easy to destroy the active components in the extract. In this paper, ethanol extraction was used to optimize the extraction process of antioxidant components from rosemary by a simple extraction method without destroying the active components as much as possible, and then the in vitro antioxidant activity of the extracts was investigated to provide further theoretical references for the study of rosemary.

 

1 Test materials and equipment

1.1 Test materials

Rosemary leaves; acetonitrile, methanol and phosphoric acid were all chromatographically pure; standard of rhamnoside acid and rosemary acid (Dalian Meilun Biotechnology Co., Ltd., purity ≥ 98%); 2,2-iamino-bis(diammonium) salt (ABTS, Ruyoshi Bio-technology, purity ≥ 98%); 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH, Ruyoshi Bio-technology, purity ≥ 97%); anhydrous ethanol, ferrous sulfate, potassium per sulfate, sodium dihydrogen phosphate, potassium ferricyanide, ferric chloride and trichloroacetic acid were all analytically pure. The analytical purity of the products includes ferrous sulfate, potassium persulfate, sodium dihydrogen phosphate, potassium ferricyanide, ferric trichloride and trichloroacetic acid.

 

1.2 Instruments and equipment

VELOCITY 14R centrifuge (Dynamica Scientific); SHB-III circulating water multi-purpose vacuum pump (Gongyi Zhongtian Instrumentation Co.  Ltd.); SHB-III circulating water multi-purpose vacuum pump (Gongyi Zhongtian Instrument Technology Co., Ltd.); U3000 high-performance liquid chromatograph (Thermo Fisher Scientific); UV-1780 UV-visible spectrophotometer (SHIMADZU); SCIENTZ-18N freeze dryer (Ningbo Xinzhi Bio-technology Co., Ltd.); RE-5299 rotary evaporator (Gongyi Yuhua Instrument Co., Ltd.). Ltd.); RE-5299 rotary evaporator (Gongyi Iowa Instrument Co., Ltd.).

 

2 Test methodology

A certain amount of rosemary powder was weighed into a conical flask, and according to the conditions set in the one-way or response surface test, a certain concentration and liquid-liquid ratio of ethanol solution was added to the flask, and the extraction was carried out for a certain period of time at room temperature, and then the extract was centrifuged and filtered to obtain the extract of antioxidant components of rosemary, and the supernatant was concentrated by rotary evaporator, and freeze-dried to obtain the crude extract of antioxidant components of rosemary powder.

2.1 Univariate tests

The effects of ethanol concentration (20%, 40%, 60%, 80%, 100%), liquid/feed ratio (10:1, 15:1, 20:1, 25:1, 30:1 (mL/g)) and extraction time (2, 4, 6, 8, 10 days) on the yield of antioxidant constituents of rosemary were investigated by setting the ethanol concentration at 60%, liquid/feed ratio at 20:1, 25:1, 30:1 (mL/g) and the extraction time at 6 days. The effect of the ratio [10:15:20:1, 25:1, 30:1 (mL/g)] and extraction time (2, 4, 6, 8, 10 days) on the yield of antioxidant components of rosemary, each group of experiments was repeated three times, and the average was taken.

 

2.2 Response surface test

The optimal ethanol concentration, liquid/feed ratio and extraction time for each condition were determined by the above one-way tests, and the response surface optimization test was conducted. The factors and coding levels are shown in Table 1.

Table 1 Box-Behnken test factor table

 

3 Measurement methods

3.1 Determination of antioxidant components of rosemary

Referring to GB 1886.172-2016 "Food Safety National Standard Food Additives Rosemary Extract", high performance liquid chromatography (HPLC) was performed to determine the antioxidant constituents of rosemary (in terms of rhamnosus acid, rhamnol, and rosmarinic acid) by adopting the chromatographic conditions of Wang Ying et al [9].

Antioxidant yield = (C1 +C2 +C3 ) × V / M7" × 100 %

(1) where C1 - the concentration of rhamnosus acid in the sample solution, mg/mL; C2 - the concentration of rhamnol in the sample solution, mg/mL; C3 - the concentration of rosmarinic acid in the sample solution, mg/mL; V - the volume of sample solution in a constant volume; V - the volume of sample solution in a constant volume; V - the volume of sample solution in a constant volume C3 - concentration of rosmarinic acid in the sample solution, mg/mL; V - volume of sample solution, mL;

M - mass of the specimen, mg.

 

3.2 ABTS Free Radical Scavenging Rate Measurement

Referring to the method of BKHAIRIA et al. [10], 2.0 mL of different concentration gradient sample solution was added to 2.0 mL of ABTS working solution, and the reaction was kept at room temperature and protected from light for 30 min, and the absorbance value (ABS) was determined at 734 nm.

Clearance = 1 - (AS - AB ) / A0 7" x 100 % (2)

Where AS - absorbance value of sample reaction group; AB - absorbance value of negative control group; A0 - absorbance value of blank control group.

 

3.3 DPPH free radical scavenging rate measurement

Referring to the method of Mu Yuwen et al [11-12], 2.0 mL of sample solution with different concentration gradients were added with 2.0 mL of DPPH working solution, and the reaction was kept at room temperature and protected from light for 30 min, and the absorbance value was measured at 517 nm, which was calculated as shown in Equation (2).

 

3.4 OH radical scavenging rate measurement

Referring to the method of DONG et al [13], 1.0 mL of sample solution with different concentration gradients was taken, and 1.0 mL of FeSO4 solution, 1.0 mL of salicylic acid solution and 1.0 mL of H2O2 solution were added respectively, and the reaction was carried out in a water bath at 37 ℃ for 30 min, and the absorbance was measured at the wavelength of 510 nm, and the calculations were as shown in Equation (2).

 

3.5 Measurement of Fe3+ reducing power

Referring to the method of Lai Kecun et al. [14], 1.0 mL of sample solution with different concentration gradients was added 2.5 mL of sodium dihydrogen phosphate solution and potassium ferricyanide solution, and reacted at 50 ℃ for 20 min; 2.5 mL of trichloroacetic acid solution was added, and the supernatant was centrifuged for 10 min to obtain 2.5 mL of the sample solution. 2.5 mL of distilled water was added, 0.5 mL of ferric chloride solution, and the reaction was placed in the room temperature for 10 min. React for 10 min; determine the absorbance at 700 nm.

Absorbance value =AS -A0 (3)

Where AS - absorbance value of sample reaction group; A0 - absorbance value of blank control group.

 

3.6 Data processing

Response surface experiments were designed and data processed using Design Expert 10 and plotted via Excel.

 

4 Results and analysis

4.1 One-way tests

As shown in Figure 1, with the increasing of ethanol concentration, the rate of antioxidant components gradually increased; when the ethanol concentration was 60%, the rate of antioxidant components reached the maximum; then with the increasing of ethanol concentration, the rate of antioxidant components first showed a slightly decreasing trend, and then a large decline. This may be due to the increase in the amount of some weakly polar substances dissolved, resulting in the slow diffusion of phenolic substances into the ethanol solvent [15]. Therefore, the best ethanol concentration is 60%.

 

With the ethanol concentration and extraction time fixed, the antioxidant yield of rosemary decreased and then increased with the increase of the liquid-liquid ratio, until the liquid-liquid ratio of 25 mL/g, the antioxidant yield reached the maximum value, and then showed a significant decrease. This is due to the fact that the liquid-liquid ratio increases, the penetration of the solvent and the dissolution of the active ingredient proceed faster, and after reaching a certain amount, the higher liquid-liquid ratio will lead to an increase in the mass-transfer impetus [16], which will cause the yield of antioxidant components to decrease. Therefore, the best liquid to material ratio is 25 mL/g.

 

With the ethanol concentration and the liquid-liquid ratio fixed, the antioxidant yield showed a significant increase with the increase of the extraction time, until the maximum antioxidant yield was reached at 4 days, after which the antioxidant yield decreased with the increase of the extraction time, and then it showed a slight upward trend, and then leveled off. This may be due to the fact that the phenolic substances are easy to decompose and deplete after too long an extraction time [17], which reduces the antioxidant components and is not favorable for extraction. Therefore, an extraction time of 4 days is optimal.

 

4.2 Box-Behnken optimization test

4.2.1 Regression modeling and testing

The results of the response surface optimization of ethanol concentration, liquid/feed ratio and extraction time on the yield of antioxidant components of rosemary are shown in Table 2.

Multiple linear regression analysis and model fitting of the experimental results in Table 2, the relationship between the dependent variable and the respective variables can be expressed by the following quadratic polynomial equation:

Y=5.25+0.945 6A+0.171 6B+0.066 3C+0.110 3AB -0.079 5AC-0.332BC-0.723 3A2-0.765 3B2

-0.651C2

This regression equation was analyzed by ANOVA and the results are shown in Table 3.

The regression model was highly significant (p<0.01), the misfit term was not significant (p>0.05), the coefficient of determination was R2=0.992 3, and the corrected coefficient of determination was Adj-R2=0.982 4, which indicated that the model had a good predictive power. Based on the F-value, it can be inferred that the order of influence of each factor is ethanol concentration > liquid/feed ratio > extraction time.

 

4.2.2 Interaction analysis between the two factors

As shown in Table 3, the interaction between liquid ratio and extraction time had a significant effect (p<0.05) on the yield of antioxidant components. The response surface of the interaction from the regression equation is shown in Fig. 2, which shows that the effects of ethanol concentration and liquid-liquid ratio on the amount of antioxidants extracted were significant, while the effect of extraction time on the amount of antioxidants extracted was insignificant, which was consistent with the results of the analysis of variance (ANOVA).

 

4.2.3 Optimal conditions optimization and validation results

The optimum conditions for the extraction of antioxidant components from rosemary by ethanol extraction were obtained by response surface methodology: ethanol concentration of 66.688%, liquid/feed ratio of 25.839:1 (mL/g), and extraction time of 3.967 days, and the yield of antioxidant components was 5.584% under these conditions. According to the actual operation, the antioxidant yield was 5.418% under the conditions of 65% ethanol concentration, 26:1 liquid/feed ratio (mL/g) and 4 days of leaching time in 3 parallel tests, which was very close to the theoretical value.

 

4.3 Antioxidant activity of rosemary extracts

As can be seen in Fig. 3(a), both the crude extract of rosemary and the VC control group showed better scavenging of ABTS free radicals.

In a certain range, increasing the concentration of crude extract could significantly increase the radical scavenging rate of ABTS. At 0.02 mg/mL, the radical scavenging rate of ABTS was 15.07%; when the concentration increased to 0.20 mg/mL, the radical scavenging rate of ABTS increased to 85.56%, which was a significant increase in the range; after 0.20 mg/mL, there was no significant change in the radical scavenging rate of ABTS. After reaching 0.20 mg/mL, the ABTS radical scavenging rate increased to 85.56%, and the increase was very significant in this range. After data processing, the IC50 value of ABTS radical scavenging of rosemary crude extract was 0.081 mg/mL, and the IC50 value of ABTS radical scavenging of VC control group was 0.037 mg/mL. The scavenging ability of the antioxidant substances of rosemary was slightly weaker than that of the VC control group, which belongs to the group of substances with stronger antioxidant activity.

 

The scavenging rate of DPPH radicals by the crude extract of rosemary and the VC control group increased steadily with the increase of concentration, from 12.56% at 0.02 mg/mL to 56.11% at 0.40 mg/mL, which was a significant increase. By comparison, the trend line of VC was much higher than that of the crude extract of rosemary, and the half inhibitory concentration of the crude extract for scavenging DPPH radicals was calculated to be 0.280 mg/mL, while the half inhibitory concentration of the VC control group for scavenging DPPH radicals was 0.046 mg/mL, which showed that the crude extract of rosemary had better scavenging activity against DPPH radicals but weaker than that of the VC control group. The crude extract of Dieffenbachia officinalis had better scavenging activity against DPPH radicals, but weaker than the VC control group.

 

As shown in Figure 3(b), the OH radical scavenging rate was only 3.27% at a concentration of 0.02 mg/mL, and increased to 47.44% when the concentration of rosemary crude extract was increased to 0.40 mg/mL. The OH radical scavenging rate was only 3.27% when the concentration of the crude extract was increased to 0.40 mg/mL, and increased to 47.44% when the concentration of the crude extract was increased to 0.40 mg/mL. The IC50 values for OH radical scavenging were calculated to be 0.360 mg/mL for the crude extract of rosemary and 0.049 mg/mL for the VC control group. Due to the large amount of impurities in the crude extract of rosemary, the VC control group showed a significantly stronger scavenging ability for OH radicals than the crude extract of rosemary at the same concentration.

 

The determination of the reducing power of Fe3+ is actually a test of the electron supplying ability of the sample, and the larger the absorbance value, the stronger the reducing power. From the figure, it can be seen that both the crude extract of rosemary and the VC control group had certain reducing power for Fe3+ at different mass concentrations. The ABS value of rosemary crude extract increased from 0.065 to 0.915 when the concentration was increased from 0.02 mg/mL to 0.40 mg/mL. Although the reducing power of Fe3+ in the VC control group was stronger than that in the crude extract of rosemary at the same mass concentration, the crude extract of rosemary, which had not been purified and enriched, still showed that the reducing power of Fe3+ increased with the increase of mass concentration.

 

5 Conclusion

In this paper, the optimum process conditions for the extraction of antioxidant components from rosemary by ethanol extraction were established. Under the conditions of ethanol concentration of 65%, liquid/feed ratio of 26:1 (mL/g) and extraction time of 4 days, the antioxidant components were obtained at a rate of 5.418%, which showed a good predictive ability of the model. The antioxidant activity of ABTS, DPPH, OH radical scavenging capacity and Fe3O4 free radical scavenging capacity of the crude extract of rosemary were found to be good in the range of the determined mass concentrations,  In the range of mass concentration of rosemary crude extract, the scavenging ability of ABTS, DPPH, OH free radicals and the reducing power of Fe3+ increased with the increase of mass concentration, and the IC50 values of ABTS, DPPH and OH free radicals were 0.081, 0.280, and 0.360 mg/mL, respectively, which indicated that the antioxidant activity of the crude extract of rosemary could be a useful tool for the study. This study can provide a theoretical reference for the comprehensive development and utilization of the natural antioxidant components of rosemary.

 

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