Optimal extraction, purification and antioxidant activity of total flavonoids from endophytic fungi of Conyza blinii H. Lév

Preparation of raw mater

The flavonoid-producing endophytic fungus CBL9 was inoculated into fresh PDA medium and cultured with shaking at 28 °C until the mycelial pellets grew to a certain condition (Bacteria is not growing and the concentration of flavonoids in the fermentation broth reaches 10 mg/L), then fermentation broth was rotary evaporation at 50 °C and raw material (L9F) was obtained after freeze-drying.

Ethyl acetate extraction

The extraction was conducted by following the method of Saraswaty with slight modifications (Saraswaty et al., 2013). A total of 200 mL concentrated fermentation broth was mixed with two times the volume of ethyl acetate to extract the fermentation broth, and repeat extraction three times. Crude flavonoid (L9F-E) was obtained by concentrating by evaporation under freeze-drying.

Preparation of standard curve

Sodium nitrite-aluminum nitrate colorimetric method was used to draw rutin standard song by following the method of Tang et al. (2020). The standard curve equation: A = 0.4164C-0.0003 (R² = 0.999 4).

Single factor test

The single factor test method was conducted with slight modifications (Bi & Tan, 2012) and the Macroporous resin D101 was used (Zhang et al., 2018). First, 1.0 g wet resin pretreated was put into 15 conical flasks and mixed with certain L9F. Then the mixture was incubated at different temperature, pH for different time. The adsorption ratio of flavonoid was measured.

Response surface analysis

According to the single-factor study, the Box-Benhnken optimization study was designed (Yu et al., 2019) with three levels (adsorption time, temperature, and pH). The adsorption ratio was chosen as inspection index. Design-Expert was used to analyze based on Box-Benhnken data. The possible mathematical model is: ${\displaystyle Y={\beta }_0+\Sigma {\beta }_{\mathrm{i}}{x}_{\mathrm{i}}+\Sigma {\beta }_{\mathrm{ij}}{x}_{\mathrm{i}}{x}_{\mathrm{j}}+\Sigma {\beta }_{\mathrm{ij}}{x}_{\mathrm{i}}^2}$ Y is the predicted response value; β⋅0, β_i, β_ii and β_ij is the regression coefficient representing the interaction of intercept, linear, squared and two factors; x_i and x_j is the independent factor of encoding (i ≠ j).

DPPH radical scavenging assay

The DPPH radical scavenging activities was collected as previously described in Kaur et al. (2020) and Tsimogiannis & Oreopoulou (2006). Vc was used as the standard antioxidant. L9F was separately dissolved in distilled water to prepare flavonoid solutions of different concentrations (0.01, 0.02, 0.03... 0.09, and 0.10 mg/mL). Equal volume of DPPH solution was mixed with different concentrations of flavonoid solution. The mixture was shaken and then put in a dark place for 30 min. Finally, the absorbance was measured at 517 nm by a spectrophotometer and each experimental group had three parallel controls. ${\displaystyle Y\left(\text{\%}\right)=\left[1-\left(A_1-A_2\right)∕A_3\right]\times 100\text{\%}}$ Y(%) is the DPPH radical scavenging activity A₁ is the absorbance of the sample with DPPH, A₂ is the absorbance of the sample without DPPH, and A₃ is the absorbance of DPPH without the sample.

ABTS radical scavenging assay

The ABTS radical scavenging activities was collected as previously described in Kaur and Zhang (Kaur et al., 2020; Zhang, Yang & Zhou, 2018; Zhang et al., 2018). Vc was used as the standard antioxidant. L9F was separately dissolved in distilled water to prepare flavonoid solutions of different concentrations (0.01, 0.02, 0.03 ... 0.09, and 0.10 mg/mL). Two milliliters of ABTS solution was mixed with one hundred Microliters of different flavonoid solution. The mixture incubated in a dark place at room temperature for 6 min. Finally, the absorbance was measured at 734 nm by a spectrophotometer and each experimental group had three parallel controls. ${\displaystyle Y\left(\text{\%}\right)=\left[1-\left(A_1-A_2\right)∕A_3\right]\times 100\text{\%},}$ Y(%) is the ABTS radical scavenging activity, A₁ is the absorbance of the sample with ABTS, A₂ is the absorbance of the sample without ABTS, and A₃ is the absorbance of ABTS without the sample.

Hydroxyl radical (•OH) scavenging assay

The hydroxyl radical scavenging activities of sample was collected as previously described in Chobot (Chobot & Hadacek, 2011), and Vc was used as the standard antioxidant. L9F was separately dissolved in distilled water to prepare flavonoid solutions of different concentrations (0.1, 0.2, 0.3 ... 0.9, and 1.0 mg/mL). A 0.5 mL flavonoid solutions of each concentration was mixed with 1 mL of Salicylic acid (6 mM), 1.5 mL of phosphate buffer solution (PBS, 0.15 M, pH 7.4), 1 mL of ferrous sulfate (6 mM) and 0.5 mL of H₂O₂ solution (0.01%). Then, the mixtures were incubated at 37 °C for 30 min. Finally, the absorbance was measured at 510 nm by a spectrophotometer and each experimental group had three parallel controls. ${\displaystyle Y\left(\text{\%}\right)=\left[\left(A_1-A_2\right)∕\left(A_3-A_2\right)\right]\times 100\text{\%}}$ Y(%) is the •OH radical scavenging activity (%), A₁ is the absorbance of the sample after reaction with hydroxyl radicals, A₂ is the absorbance of the sample, and A₃ is the absorbance without H₂O₂.

Superoxide radical (•O²⁻) scavenging assay

The superoxide radical scavenging activities of sample was collected as previously described in Zhishen and Leong (Zhishen, Mengcheng & Jianming, 1999; Leong et al., 2008). Similarly, Vc was used as the standard antioxidant. L9F were separately dissolved in distilled water to prepare polysaccharide solutions of different concentrations 0.01, 0.02, 0.03 ... 0.09, and 0.10 mg/mL. A 1.0 mL sample of each concentration was mixed with 3.0 mL of Tris-Hcl buffer (pH 8.2), 0.8 mL of Pyrogallol (0.05 M). Then, the mixtures were bathed at 25 °C for 5 min and 1.0 mL HCl (8.00 M) was mixed after that. Finally, the absorbance was measured at 325 nm using a spectrophotometer. ${\displaystyle Y\left(\text{\%}\right)=\left[1-\left(A_1∕A_2\right)\right]\times 100\text{\%}}$ Y is the •O²⁻ radical scavenging activity (%), A₁ is the absorbance of the sample and A₂ isthe absorbance of the control.

The influence of adsorption time

Figure 1 shows that the adsorption ratio increases first and then stabilizes with the increase of adsorption time. The adsorption ratio reached to the maximum first at 1 h,which was 59.06 ± 0.03%.

The influence of pH

Figure 2 shows that the adsorption ratio increases first, then goes down with the increase of pH. The adsorption ratio reach to the maximum at pH 3.5, which was (31.65 ± 0.03)%. There was no adsorption when pH was more than 5, which may be caused by the change of the flavonoid structure and the deactivation (Wu et al., 2017) or the weakening of the adsorption capacity of the macroporous resin (Li et al., 2007) under this pH condition.

The effect of temperature

Figure 3 shows that the adsorption ratio increases first and then goes down with the increase of temperature. The adsorption ratio reached to the maximum first at 30 °C, which was 51.26 ± 0.02%.

Selection of analysis factor level

According to Box-Benhnken’s central combination test design principle and single-factor test results, three factors (temperature, pH, and time) that have significant effects on the extraction of total flavonoids were selected, and the three-factor three-level response is adopted, which is shown in Table 1.

Response surface analysis experiment design scheme

A (temperature), B (pH), and C (adsorption time) was taken as independent variables, and Y (total flavonoid adsorption ratio) was taken as the response value. The test plan and results are shown in Table 2.

Establishment and analysis of multivariate quadratic response surface regression model

Quadratic regression response surface analysis Performs in Table 2, and a multiple quadratic response surface regression model was established: Y = 0.47-0.056A-0.042B+0.031C+0.088AB-0.043AC+ 4 × 10⁻⁴BC-0.064A²-0.047B²+0.056C². The variance analysis of each factor is shown in Table 3.

Table 3 shows that the model is significant (p < 0.05) and the Prob>F value of the decisive factor coefficient such as A (temperature), B (pH), C (adsorption time), AB (interaction between temperature and pH), AC (interaction between temperature and adsorption time) are 0.0008, 0.0039, 0.0161, 0.0004, 0.0192 (p < 0.05), indicating that the model has a good fit. In addition, the factors affecting the extraction ratio of total flavonoid were A (temperature), B (pH), and C (adsorption time) in order of magnitude, and the temperature reached a significant level (p < 0.001).

In this experiment, the interaction between AB and AC has significant effect. The results are shown in Figs. 4 and 5, and the interaction between BC is shown in Fig. 6. It can be seen from the response surface diagram that the extraction ratio of total flavonoids first increases and then decreases and increases in the end with the increase of A (temperature). B is in the range of −1.0 to 0. Due to the interaction of A, the extraction ratio of total flavonoids is higher. C is in the range of 0 to 1.0, due to the interaction of A, the extraction ratio of total flavonoids is higher.

Model verification

It is obtained that the supreme extraction ratio predicted is 64.2% under the process conditions of 25.02 °C, pH 2.80 and 1.85 h by solving the inverse matrix of the quadratic polynomial mathematical model of the total flavonoid yield.

The optimal conditions were revised to 25 °C, pH 2.80, and 1.85 h to extract the total flavonoids by the above to check the validity. The actual adsorption ratio was (64.14 ± 0.04)%, which is close to the theoretical value.

DPPH radical scavenging assay

DPPH has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants (Khled Khoudja, Boulekbache-Makhlouf & Madani, 2014). Figure 8 shows that the DPPH clearance ratio goes up as the concentration of each sample increases and it tends to be flat when the sample mass concentration is greater than 0.05 mg/mL. When the concentration reaches the maximum (0.1 mg/mL), the clearance ratio of total flavonoids on DPPH increases from (95.33 ± 0.01)% to (96.44 ± 0.04)% after purification, and Vc’s clearance ratio is (94.18 ± 0.002)%. It can be seen that the clearance effect of flavonoids on DPPH slightly better than that before purification, and the clearance effect of flavonoids on DPPH is close to Vc.

ABTS radical scavenging assay

ABTS has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants (Thaipong et al., 2012). Figure 9 shows that the ABTS clearance ratio goes up as the mass concentration of each sample increases. When the concentration reaches the maximum (0.1 mg/mL), the clearance ratio of total flavonoids on ABTS increases from (74.06 ± 0.04)% to (75.33 ± 0.03)% after purification, and Vc’s clearance ratio is (71.74 ± 0.05)%. It can be seen that the clearance effect of flavonoid on ABTS slightly better than that before purification, and the clearance effect of flavonoid on ABTS is better than Vc.

Hydroxyl radical (•OH) scavenging assay

Hydroxyl radicals are very active and have been associated with cancer risk when accumulated in the body excessively (Sakanaka, Tachibana & Okada, 2005). Figure 10 shows that the •OH clearance ratio goes up as the mass concentration of each sample increases and it tends to be flat when the sample mass concentration is greater than 0.3 mg/mL, but the clearance ratio of the sample before purification shows a downward trend, which may be the presence of oxidized and discolored impurities in the original fermentation broth. When the sample concentration reaches the maximum (1 mg/mL), the clearance ratio of total flavonoids on •OH increases from (3.98 ± 0.02)% to (73.79 ± 0.02)% after purification, and Vc’s clearance ratio is (93.72 ± 0.01)%. It can be seen that the clearance effect of flavonoids on •OH better than that before purification.

Superoxide radical (•O²⁻) scavenging assay

The superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxygen species, contributing to tissue damage and various diseases (Ozsoy et al., 2008; Aruoma, Grootveld & Bahorun, 2006). Figure 11 shows that the •O²⁻ clearance ratio goes up as the mass concentration of each sample increases and it tends to be flat when the sample mass concentration is greater than 0.05 mg/mL, but the clearance ratio of the sample before purification shows a downward trend, which may be the presence of oxidized and discolored impurities in the original fermentation broth. When the sample concentration reaches the maximum (0.1 mg/mL), the clearance ratio of total flavonoids on •O²⁻ increases from (0.15 ± 0.016)% to (28.11 ± 0.01)% after purification, and Vc’s clearance ratio is (31.14 ± 0.01)%. It can be seen that the clearance effect of flavonoids on •O²⁻ better than that before purification.