Evaluation of antioxidant and anticancer activities of naphthoquinones‐enriched ethanol extracts from the roots of Onosma hookeri Clarke. var. longiforum Duthie

Abstract In this study, the optimal naphthoquinones‐enriched ethanol extract from the roots of Onosma hookeri Clarke. var. longiforum Duthie (OHC‐LD) was obtained under an optimal condition (69% ethanol, material to solution ratio of 27:1 at 60℃ for 59 min) by the ultrasound‐assisted extraction, according to four‐variable three‐level Box–Behnken design‐response surface methodology. The experimental yield of ethanol extract was 42.08 ± 0.65%, and the contents of naphthoquinones reached to 1.07 ± 0.004%. The optimal extract exhibited similar scavenging activity against ABTS (2,2'‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulfonic acid) radical as BHT(butylated hydroxytoluene) at 1,250 µg/ml, and better DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) scavenging activity than BHT at 250 µg/ml. However, the optimal ethanol extract was not sensitive to MCF‐7 cell line ( IC50 of 321.849 µg/ml). The results revealed the naphthoquinones‐enriched ethanol extract from the roots of OHC‐LD had could be used as a potential natural antioxidant.

Therefore, in this study, an naphthoquinones-enriched ethanol extracts from the roots of OHC-LD was conducted by the ultrasonic-assisted technology (Hao, Li, & Lin, 2017;Jovanović et al., 2017)

| Plant materials
The roots of OHC-LD were collected from Shigatse, Tibet and authenticated by Aga Er-bu of Tibet University. The voucher specimen (No. 20171216-1) has been deposited at Pharmacy Department, Sichuan Agricultural University.

| Cell lines and culture medium
MCF-7 cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. MCF-7 cells were cultured in 5% CO 2 , for 24 hr at 37℃ in an incubator (Thermo). The growing medium consisted of the following: Roswell park memorial institute (RPMI) 1640 medium (HyClone), 10% Fetal bovine serum (Gibco), 2% antibiotics [penicillin in a concentration of 100 U/mL and streptomycin in a concentration of 100 U/mL (Gibco)], and 1% insulin (Gibco).

| Preparation of the naphthoquinones-enriched ethanol extract by Ultrasonic-assisted extraction according BBD-RSM
The roots of OHC-LD (2 g) were added ethanol solution (55%-95%) with the ratio of material to solution of 1:10 to 1:30 g/ml into a 100 ml conical flask. The ethanol extract was extracted by ultrasonic-assisted extraction at 20℃ to 70℃ for 20 to 70 min. After being centrifuged at 3,500 rpm for 5 min, the supernatant was collected and made up to 50 ml, 10 ml of which was concentrated by a rotary evaporator under vacuum at 50℃ and dried to constant weight for the measurement of its total weight. The content of naphthoquinones was calculated by the UV absorption value of L-shikonin at 516 nm. A standard curve was prepared using L-shikonin (Y = 18.495X + 0.0257 R 2 = 0.9991, Linearity range, 4 μg/ml to 44 μg/ml). The contents of naphthoquinones were expressed as micrograms of L-shikonin per gram of sample.

| Single-factor experiment
During ultrasonic-assisted extraction of the roots from OHC-LD, the effects of extraction temperature and time, the ratio of material to solution, and ethanol concentrations were investigated by a singlefactor design. Each experiment was done when one factor was changed, while the others were remained (Chen et al., 2017). The effects of each factor were analyzed by the total weight of ethanol extract and its content of naphthoquinones as Equation (1): where X max represents the highest extraction rate of naphthoquinones in all experimental groups, X represents the extraction rate of naphthoquinones for each experiment; Where Y max represents the highest weight of ethanol extract in all experimental groups, Y represents the weight of ethanol extract for each experiment.

| BBD-RSM experimental design
A BBD-RSM was designed by a commercial statistical package, Design-Expert version 8.0.6, to estimate the effect of each independent variables (extraction temperature and time, the ratio of material to solution, and ethanol concentration) basing on both the extraction yields and the contents of naphthoquinones. According to the results of single-factor experiments, each parameter experiment was performed on three different levels, see in Then, the variables were calculated by the Equation (2): where X is a coded value for the variable, X i is the corresponding actual value, X 0 is the actual value in the center of the domain, and ΔX is the step change value.
The scoring of extraction yields and the contents of naphthoquinones were calculated via second-order polynomial equation as Equation (3): where Y represents the dependent variable (scores of yields of ethanol extract and the contents of naphthoquinones); A 0 is the constant coefficient; A i , A ii, and A ij are the coefficients estimated by the model; X i and X j are the coded independent variables. They represent the linear, quadratic, and interaction effects of the X 1 , X 2 , X 3, X 4 factors, respectively; (i ≠ j) (Noshad et al., 2012).

| Antioxidant activity
The antioxidant capacity of the crude extract, which was obtained under the optimal conditions, was investigated by DPPH method, ABTS method, and reduction powder method.

| The radical scavenging activity against DPPH
The radical scavenging activity of the ethanol extract against DPPH was according to the literature (Li, Hao, Wang, Huang, & Li, 2009). In brief, the mixture of DPPH solution (1 ml, 0.048 mg/ml in 75% ethanol) and the ethanol extract solution (1 ml, 3.91-1000 µg/ml) was incubated at 20℃ in the dark for 30 min. Then, the absorbance was measured at 517 nm. The butylated hydroytoluene (BHT) was used as a positive control. Equation (4): where A i represents the absorbance of the DPPH/sample mixture, A 0 represents the absorbance of the DPPH without sample (the sample was replaced by 75% ethanol), and A j represents the absorbance of the sample without DPPH (the DPPH was replaced by 75% ethanol).

| The radical scavenging activity against ABTS
The radical scavenging activity against ABTS was measured as followed: the 1:1 (v/v) mixture of K 2 S 2 O 2 solution (2.6 mmol/L) with ABTS (7.4 mmol/L) solution was kept in the dark at room temperature for 12-16 hr. The ABTS working solution (0.8 ml), which was obtained by diluting the mixture above with absolute ethanol, was mixed with ethanol extract solution (0.2 ml, 19.53-5000 µg/ml).
Taking BHT as a positive control, the absorbance of the sample was determined at 734 nm (Li, Lin, Gao, Han, & Chen, 2012). Equation (5): where A i represents the absorbance of the ABTS/sample mixture, A 0 represents the absorbance of the ABTS without sample (the sample was replaced by 75% ethanol), and A j represents the absorbance of the sample without ABTS (the ABTS was replaced by 75% ethanol).

| Reducing power
The Reducing power was measured according to the literature (Oyaizu, 1986). The mixture of the ethanol extract solution (1 ml, 19.53-10000 µg/ml), phosphate buffer (2.5 ml, 0.2 mol/L, pH = 6.6), and potassium ferricyanide solution (2.5 ml, 10 g/L) was incubated at 50℃ under water bath for 30 min and then added trichloroacetic acid (2.5 ml, 100 g/L), centrifuged at 3,000 r/min for 10 min. The supernatant (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl 3 (0.5 ml, 0.1% w/v). After incubating at room temperature for 10 min, the absorbances of the sample mixture and BHT were measured at 700 nm.

| Anticancer activity
The cytotoxic activity of the optimal ethanol extract was measured in  were seeded in each well of the 96-well plates with 1 × 10 5 cells/ well. After incubation with the optimal ethanol extract with the appropriate concentration ranges of drugs (150, 75, 37.5, 18.75, and 9.375 µg/ml) for 48 hr, MTS solution (2.5 mg/ml in PBS) was added (20 μl/well), and the plates were incubated for an additional 2-4 hr at 37℃. The optical density of the solution was measured at 490 nm using a microplate reader (TECAN, Austria). Growth inhibition was estimated from the optical density of the solution. The percentage of cell survival was calculated as follows. Equation (6):

TA B L E 1 Independent factors and their levels used in ultrasonic extraction
The relation between surviving fraction and compound concentration was plotted, and IC 50 (the concentration required for 50% inhibition of cell viability) was calculated for each test compound.

| Statistical and analysis
All experiments were performed in triplicate, all data were expressed at least 3 independent evaluations, and standard deviation (SD) were also calculated using SPSS 23.0.

| Preparation of the ethanol extract
In order to achieve the highest extraction yields as well as the better contents of naphthoquinones, the effects of extraction temperature and time, the ratio of material to solution and ethanol concentration were investigated.

| Optimization of extraction process
As shown in Figure 1a, the comprehensive score increased with temperature increasing from 20℃ to 50℃, reached maximum at 50℃, and decreased from that point. Thus, the favorable extraction temperature was 50℃.
As shown in Figure 1b, the highest comprehensive score was obtained when the extraction time was 60 min. After that point, the scores decreased. It may due to the degradation of naphthoquinones for over-time extraction. Consequently, 60 min was the best extraction time.
As shown in Figure 1c, with the increase of the ratio of material to solution, the comprehensive scores were also increased. However, As shown in Figure 1d, the comprehensive scores increased along with ethanol concentration increase, and the highest score was obtained when the ethanol concentration was 65%. Accordingly, the favor the ethanol concentration ratio was 65%.

| Statistical analysis and model fitting using response surface methodology (RSM)
Compared with traditional single parameter optimization, RSM is more advantageous for saving time, space, and raw material. For optimizing the four independent parameters, a total of 29 runs were in the current Box-Behnken design (see in Table 2). The fitting equation used to predict the extraction yield was as follows Equation (7): where Y represents the scores of naphthoquinones extract and alcohol extract, and A, B, C, and D are the coded variables for the extraction temperature, extraction time, the ratio of material to solution, and ethanol concentration, respectively.
The experimental data were analyzed by the Analysis of Variance, and the significance of the regression coefficients was evaluated by their corresponding P-values (Ahmad, Alkharfy, Wani, & Raish, 2015). As shown in TABLE 3, the model difference was extremely significant (p-value < .0001), and the Lack of Fit was not significant (p-value = .1139 > .05). The R 2 (0.9568) proved a reasonable fit between the model and the experimental data. The R 2 adj (0.9136) also indicated the accuracy of the model. At the same time, three linear coefficients (A, C, and D), one interactive coefficients (BD), and three quadratic coefficients (B 2 , C 2 , and D 2 ) were extremely significant (p < .01), and others were not significant (p > .05), indicating that the results were statistically significant.
The results also indicated that the four factors of the test were not a simple linear relationship with the response value, but a result of multiple factors.
In the four-factor three-level response surface experiment, the response graph was a three-dimensional graph (Figure 2), which fixed two factors and drew the influence of two other factors on the response value according to the fitting equation. The three-dimensional map could intuitively reflect the influence of the interaction of these two factors on the response value. After the fitting equation of all the experimental data by Equation (7), the best process

| Verification of analytical method and the content of naphthoquinones in the optimal extraction
Three repeated verification tests were conducted under the optimal conditions, in which the sample was extracted with 69% ethanol with the ratio of material to solution of 27:1 at 60℃ for 59 min.
Under these conditions, the extraction yield of ethanol extract was 42.08 ± 0.65% and the content of naphthoquinones reached to 1.07 ± 0.004%. The comprehensive score was 98.04 ± 0.89, which is close to the predicted value (100.467). The agreement of the results indicated that the experimental model had a good reliability, and the experimental value has little error. Therefore, an effective ultrasonic-assisted extraction technique was optimized.

| The radical scavenging activity against DPPH
As a free radical with an unpaired electron, DPPH could decrease significantly when it is exposure to proton radical scavengers (Rostami & Gharibzahedi, 2017). The DPPH scavenging activities of the ethanol extract were shown in Figure 3a, which exhibited dose-dependent scavenging activities in the studied concentration range, with IC 50 value of 51.30 µg/ml, higher than that of BHT (IC 50 = 4.90 µg/ml). The BHT achieved maximum scavenging value (73.8 ± 0.56%) at 125 μg/ mL. However, when the concentration was 250 μg/mL, the DPPH radical scavenging activity of ethanol extract was higher than that of the BHT. The results indicated that the DPPH radical scavenging activity of BHT was higher than that of alcohol extract at low concentration, but the latter was better than BHT at high concentration. Compared with the previous studies, the DPPH radical scavenging activity of the alcohol extract (IC 50 = 80 µg/ml) from L. erythrorhizon  was lower than that of OHC-LD. Meanwhile, the naphthoquinone compounds have been proven to exhibit excellent antioxidant capacity (Ordoudi et al., 2011). However, in the early report, the contents of naphthoquinones of L. erythrorhizon are higher than that of OHC-LD (Hu, Jiang, Leung, & Zhao, 2006). It is referred that the increase of naphthoquinone content in alcohol extract under the optimal condition leads to the enhancement of DPPH radical scavenging ability.

| The radical scavenging activity against ABTS
As an excellent substrate for peroxidases, ABTS is frequently used to study the antioxidant properties of natural compounds (Zaidi, Shah, Parmar, & Thawani, 2018). From the Figure 3b, although the ABTS radical scavenging activity of the ethanol extract (IC 50 = 295.81 µg/ml) TA B L E 3 Analysis of variance of response surface quadratic model analysis for the extraction yield was lower than that of BHT (IC 50 = 32.423 µg/ml), it also could reach to nearly 100 ± 0.79%, when the concentration higher than 1,250 µg/ml.
The good ABTS radical scavenging activity may derive from the large number of hydroxyl groups in naphthoquinone molecules (Zaidi et al., 2018), which can provide electrons to decrease ABTS free radical.

| Reducing power
The reducing power is another useful method for assessing antioxidant activity. It is indicated by the absorbance quantity for the reaction mixture at 700 nm (Gharibzahedi, Razavi, & Mousavi, 2013). Figure 3c indicated the reducing power values of the ethanol extract increased in a concentration-dependent manner, but lower than that of BHT. The reducing power value of the ethanol extract reached 1.17 ± 0.03 at 10 mg/ ml, while the BHT reached the maximum value (1.2 ± 0.01) at 5 mg/ml.
In general, the optimal extract exhibited similar scavenging activity against ABTS radical as BHT at 1,250 µg/ml, better DPPH scavenging activity than BHT at 250 µg/ml, and similar reducing power as BHT after 10 mg/ml. The existence of numerous hydroxy groups in naphthoquinone molecules could be attributed to antioxidant capacity (Belhaj et al., 2017).

| Evaluation of the anticancer activity of the ethanol extract in vitro
The optimal ethanol extract from OHC-LD was evaluated the cy-

| CON CLUS ION
In this study, an efficient and economical ultrasonic-assisted extraction of ethanol extract from the roots of OHC-LD was established by BBD-RSM. The optimal conditions were obtained (69% ethanol, material to solution ratio of 27:1 at 60℃ for 59 min). The yield of ethanol extract was 42.08 ± 0.65%, and the content of naphthoquinones reached to 1.07 ± 0.004%. Furthermore, the naphthoquinonesenriched extract obtained under the optimal condition exhibited better DPPH scavenging activity, similar ABTS radical scavenging effects and reducing power as BHT at high concentrations (250 µg/ ml, 1,250 µg/ml, 10 mg/ml, respectively), indicating its good antioxidant activity, comparable to or better than BHT. However, the op-