Electronic Supporting Information

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1 Electronic Supplementary Material (ESI) for Green Chemistry. This journal is The Royal Society of Chemistry 2018 Electronic Supporting Information Two-phase systems developed with hydrophilic and hydrophobic deep eutectic solvents for simultaneously extracting various bioactive compounds with different polarities Jun Cao a, b, Luyao Chen a, Mohan Li a, Fuliang Cao b, Linguo Zhao b, Erzheng Su a, b, c* a College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing , China. b Co-innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing , China. c State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing , China. * Corresponding author: Erzheng Su Tel Fax: address: ezhsu@njfu.edu.cn 1

2 1. Supplementary methods 1.1 Methodology validation for the determination methods The methodology validation for the determination of the bioactive compounds (procyanidine, flavonoids, terpene trilactones and polyprenyl acetates) were investigated. The equation of the regression line, correlation coefficient (R 2 ), linear range, limit of detection (LOD) and limit of quantification (LOQ) for each compound were presented in Table S1. LOD was established at a signalto-noise ratio (S/N) of 3. LOQ was established at signal-to-noise ratio (S/N) of 10. LOD and LOQ were experimentally verified by six injections of procyanidine, flavonoids, terpene trilactones and polyprenyl acetates at the LOD and LOQ concentration range. A standard working solution containing polyprenyl acetates of 600 μg/ml, procyanidine of 75 μg/ml, flavonoids of 44 μg/ml and terpene trilactones of 200 μg/ml was prepared. The precision of the method (within-day variations of replicate determinations) was checked by injecting six times of standard working solution. The precision of the method expressed as the RSD % was shown in Table S2. The accuracy of the determination method was also investigated. The accuracy, defined in terms of % deviation of the calculated concentration from the actual concentration was listed in Table S3. The results were obtained at the range of ±5%, which showed that the methods were deemed to be accurate. 1.2 Single-factor experiments about the effects of solid to solvent ratio, extraction temperature and time on the extraction yield. In order to choose appropriate variable range for optimization by experimental design based on the response surface methodology, single factor experiments were conducted. First of all, the solid to 2

3 solvent ratio was investigated at 1:5, 1:7.5, 1:10, 1:12.5, 1:15, 1:20 and 1:30 (w/v, g/ml). Next, under the optimum solid to solvent ratio, the extraction temperature was investigated from 45 C to 70 C at an interval of 5 C. Under the optimal solid to solvent ratio and extraction temperature, the effect of the extraction time was investigated from 25 min to 65 min at an interval of 5 min. 2. Supplementary tables Table S1. The regression equation, R 2, linear range, LOD and LOQ for polyprenyl acetates (PPAs), procyanidine (PAC), flavonoids and terpene trilactones (TTLs). Equation of regression line R 2 Linear range LOD LOQ (μg/ml) (μg/ml) (μg/ml) PPAs y = x PAC y = x flavonoids y= x TTLs y = x Table S2. Precision of the determination methods for PPAs, PAC, flavonoids and TTLs. The number of parallel experiments was six (n=6). Concentration of sample (μg/ml) Estimated value (μg/ml) Average value (μg/ml) RSD % PPAs PAC flavonoids TTLs

4 Table S3. Accuracy of the determination methods for PPAs, PAC, flavonoids and TTLs. Concentration Average Addition Estimated value Recovery of sample Recovery (μg/ml) (μg/ml) rate (%) (μg/ml) rate (%) RSD % PPAs PAC Flavonoids TTLs Table S4. Effect of solid to solvent ratio on the extraction yields of Ginkgo biloba leaves bioactive compounds. Error bars indicate the SEM (n = 3). Solid to Bioactive compounds in hydrophilic DES phase (mg/g) PPAs in MCO phase solvent ratio PAC Flavonoids TTLs (mg/g) 1: ± ± ± ±2.15 1: ± ± ± ±1.33 1: ± ± ± ±0.15 1: ± ± ± ±1.22 1: ± ± ± ±0.45 1: ± ± ± ±0.78 4

5 Table S5. Results of variance analysis for regression equantion of procyanidins Source Sun of Degree of Mean square freedom square F-value p-value significance Model < ** ** < ** ** * * < ** ** Residual Lack of fit Pure error Total Table S6. Results of variance analysis for regression equantion of flavonoids Source Sun of Degree of Mean square freedom square F-value p-value significance Model < ** ** < ** < ** < ** < ** < * * ** < ** Residual Lack of fit Pure error E-05 Total

6 Table S7. Results of variance analysis for regression equantion of terpene trilactones Source Sun of Degree of Mean square freedom square F-value p-value significance Model < ** < ** < ** ** ** * < ** < ** Residual Lack of fit Pure error Total Table S8. Results of variance analysis for regression equantion of polyprenyl acetates Source Sun of Degree of Mean square freedom square F-value p-value significance Model ** ** ** * * ** * Residual Lack of fit Pure error Total

7 3. Supplementary figures Extraction yield of PAC, flavonoids an TTLs (mg/g) PAC Flavonoids TTLs Extraction 55 temperature( ) Extraction yield of PPAs (mg/g) Figure S1. Effect of temperature on the extraction yields of Ginkgo biloba leaves bioactive compounds Extraction yield of PAC, flavonoids an TTLs (mg/g) PAC Flavonoids TTLs PPAs Extraction yield of PPAs (mg/g) Extraction time (min) Figure S2. Effect of extraction time on the extraction yields of Ginkgo biloba leaves bioactive compounds 7

8 Figure S3. Response surface plots of the model for extraction yield of PAC. (A) Varying extraction temperature and solid to solvent ratio, extraction time 40min; (B) Varying extraction temperature and time, solid to solvent ratio 1:20; (C) Varying solid to solvent ratio and extraction time, extraction temperature 60 C. 8

9 Figure S4. Response surface plots of the model for extraction yield of flavoindins. (A) Varying extraction temperature and solid to solvent ratio, extraction time 40min; (B) Varying extraction temperature and time, solid to solvent ratio 1:20; (C) Varying solid to solvent ratio and extraction time, extraction temperature 60 C. 9

10 Figure S5. Response surface plots of the model for extraction yield of TTLs. (A) Varying extraction temperature and solid to solvent ratio, extraction time 40min; (B) Varying extraction temperature and time, solid to solvent ratio 1:20; (C) Varying solid to solvent ratio and extraction time, extraction temperature 60 C. 10

11 Figure S6. Response surface plots of the model for extraction yield of PPAs. (A) Varying extraction temperature and solid to solvent ratio, extraction time 40min; (B) Varying extraction temperature and time, solid to solvent ratio 1:20; (C) Varying solid to solvent ratio and extraction time, extraction temperature 60 C. 11