# Preparative Purification of Total Flavonoids from Sophora tonkinensis Gagnep. by Macroporous Resin Column Chromatography and Comparative Analysis of Flavonoid Profiles by HPLC-PAD

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## Abstract

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## 1. Introduction

## 2. Results and Discussion

#### 2.1. Resin Selection

#### 2.2. Effect of Sample pH on Adsorption Capacity

#### 2.3. Adsorption Isotherms

_{e}–q

_{e}plots decreased with the increasing adsorption temperature, which indicated that the increase in the adsorption temperature was favorable to the adsorption of the total flavonoids onto the AB-8 resin.

_{e}/q

_{e}versus C

_{e}(Figure 3b), lnq

_{e}versus lnC

_{e}(Figure 3c), and q

_{e}versus lnC

_{e}(Figure 3d) would give linear regression lines, respectively, and the parameters of the isotherm models for the adsorption of the total flavonoids on the AB-8 resin are listed in Table 1. K

_{L}represents a tendency, where the adsorbate is attached to an adsorbent, and the larger the K

_{L}value the higher the adsorption energy, and in this study, the values of K

_{L}were decreased with the temperature increasing, which indicated that raising the temperature was adverse to the adsorption. K

_{F}for the Freudlich model is related to the adhesion ability. The values of K

_{F}decreased from 3.3953 to 2.9300 mg/g(L/mg)

^{1/n}when the adsorption temperature increased from 298.15 to 318.15 K. This indicated that the increase in temperature would decrease the adhesion ability of the total flavonoids onto the AB-8 resin. An exponent, n, is a heterogeneity factor, and is also an indicator of the non-linearity degree of adsorption isotherms. As shown in Table 1, all of the values of n were between 2 and 10, which indicated that the adsorption was a physical process [34]. Likewise, the Temkin model was also employed to describe the experimental data, and K

_{T}and B

_{T}represent the binding energy and adsorption heat, respectively. The values of K

_{T}decreased from 0.5656 to 0.4865 as the temperature increased from 298.15 to 318.15 K, which indicated that the higher temperature could weaken the binding capability. In addition, compared to the Freudlich and Temkin models, the Langmuir model fitted the experimental data best because of the highest values of the regression coefficient R

^{2}(0.9977–0.9986) within the range of the temperature tested. This revealed that a monolayer adsorption behavior of the total flavonoids from S. tonkinensis happened to the AB-8 resin.

#### 2.4. Adsorption Kinetics

_{e}-q

_{t}) versus t (Figure 4b), t/q

_{t}versus t (Figure 4c), and q

_{t}versus t

^{1/2}(Figure 4d)), the kinetic equations and relevant parameters for the adsorption of the total flavonoids from the S. tonkinensis on the AB-8 resin were calculated and are listed in Table 2. It was found that the theoretical maximum adsorption capacity (20.92 mg/g) calculated from the pseudo-second-order model was fairly close to the experimental value (20.46 mg/g), whereas the theoretical maximum adsorption capacity (7.70 mg/g) calculated from the pseudo-first-order model was much less than the experimental value. Moreover, the pseudo-second-order model yielded relatively higher R

^{2}values (0.9999) than the pseudo-first-order model (0.9696). Taken together, this adsorption process was in well agreement with the pseudo-second-order model. In the case of the intra-particle diffusion model, three consecutive steps were involved, more specifically, a sharper line segment represented the diffusion of the total flavonoids through the solution to the external surface of the AB-8 resin, or the boundary layer diffusion of the flavonoids. Then, a line segment that sloped slightly represented the movement of the flavonoids into the interior part of the AB-8 resin. The final equilibrium stage was the adsorption of flavonoids onto the interior surface of the AB-8 resin. It is worth mentioning that the value of I, the y-intercept, can indicate the thickness of the boundary layer; the larger the y-intercept, the smaller the contribution of the intra-particle diffusion. When I = 0, there was no boundary layer thickness, and the intra-particle diffusion model is the only rate-limiting step [36]. As seen from Figure 4d, the plots did not pass through the origin, which implied that the rate-limiting step was not only the intra-particle diffusion, but also the adsorption or boundary layer diffusion involved in the adsorption process.

#### 2.5. Column Chromatography

#### 2.5.1. Dynamic Breakthrough Curves

#### 2.5.2. Effect of Ethanol Concentration on Desorption Ratio

#### 2.5.3. Dynamic Desorption Curves

#### 2.6. Preparative Purification of Total Flavonoids Under Optimized Conditions

#### 2.7. Comparative Analysis of Flavonoid Profiles by HPLC-PAD

## 3. Materials and Methods

#### 3.1. Chemicals and Reagents

#### 3.2. Preparation of Sample Solutions

#### 3.3. Determination of Total Flavonoids Content

_{2}was added and mixed. Six minutes later, 150 μL of 10% (w/v) Al(NO

_{3})

_{3}was added, and the mixture was mixed for 6 min, followed by the addition of 1 mL of 4% (w/v) NaOH. Afterwards, the solution was supplemented to 5 mL with 30% (v/v) aqueous ethanol, and then rested for 15 min. The absorbance value was detected at 510 nm using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). The calibration curve was obtained based on the absorbance values of a series of rutin standard solutions, which showed a good linearity ($A=11.1353C+0.0078$, R

^{2}= 0.9998) in the range of 18.12–63.42 μg/mL, where A was the absorbance value and C was the total flavonoids content (mg/mL).

#### 3.4. Comparison of Adsorption Capacity, Desorption Capacity and Ratio

_{e}(mg/g) is the equilibrium adsorption capacity, C

_{0}(mg/mL) is the initial concentration of total flavonoids, C

_{e}(mg/mL) is the equilibrium concentration of the total flavonoids, V

_{i}is the initial volume of sample solution, and m (g) is the dry weight of resin.

_{d}(mg/g) represents the desorption capacity, C

_{d}(mg/mL) represents the concentration of the total flavonoids in the desorption solution, D (%) represents the desorption ratio, and V

_{d}represents the volume of the desorption solution.

#### 3.5. Adsorption Isotherms

_{m}(mg/g) represents the theoretical maximum adsorption capacity, K

_{L}(L/mg) is the Langmuir adsorption constant, n and K

_{F}[mg/g(L/mg)

^{1/n}] are the Freundlich constants, and K

_{T}(L/mg) and B

_{T}(J/mol) are Temkin constants.

#### 3.6. Adsorption Kinetics

_{1}and k

_{2}represent the rate constants of the pseudo-first-order kinetic model and pseudo-first-order kinetic model, respectively. k

_{i}and I are the constants of the Weber–Morris intra-particle diffusion model.

#### 3.7. Optimization of Resin Column Chromatography Conditions

#### 3.8. HPLC-PAD Analysis of S. tonkinensis Extract Before and After Purification

## 4. Conclusions

## Author Contributions

## Funding

## Conflicts of Interest

## References

- Yoo, H.; Ryu, K.H.; Bae, S.K.; Kim, J. Simultaneous determination of trifolirhizin, (-)-maackiain, (-)-sophoranone, and 2-(2,4-dihydroxyphenyl)-5,6-methylenedioxy benzofuran from Sophora tonkinensis in rat plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study. J. Sep. Sci.
**2014**, 37, 3235–3244. [Google Scholar] [PubMed] - Tang, L.; Dong, L.N.; Peng, X.J.; Li, Y.; Shi, J.; Zhou, F.Y.; Liu, Z.Q. Pharmacokinetic characterization of oxymatrine and matrine in rats after oral administration of radix Sophorae tonkinensis extract and oxymatrine by sensitive and robust UPLC-MS/MS method. J. Pharm. Biomed. Anal.
**2013**, 83, 179–185. [Google Scholar] [CrossRef] [PubMed] - Ahn, J.; Kim, Y.M.; Chae, H.S.; Choi, Y.H.; Ahn, H.C.; Yoo, H.; Kang, M.; Kim, J.; Chin, Y.W. Prenylated flavonoids from the roots and rhizomes of Sophora tonkinensis and their effects on the expression of inflammatory mediators and proprotein convertase subtilisin/kexin type 9. J. Nat. Prod.
**2019**, 82, 309–317. [Google Scholar] [CrossRef] [PubMed] - Yang, X.Z.; Deng, S.H.; Huang, M.; Wang, J.L.; Chen, L.; Xiong, M.R.; Yang, J.; Zheng, S.J.; Ma, X.H.; Zhao, P.; et al. Chemical constituents from Sophora tonkinensis and their glucose transporter 4 translocation activities. Bioorg. Med. Chem. Lett.
**2017**, 27, 1463–1466. [Google Scholar] [CrossRef] [PubMed] - Yang, R.Y.; Lan, Y.S.; Huang, Z.J.; Shao, C.L.; Liang, H.; Chen, Z.F.; Li, J. Isoflavonoids from Sophora tonkinensis. Chem. Nat. Compd.
**2014**, 48, 674–676. [Google Scholar] [CrossRef] - Li, X.N.; Sha, N.; Yan, H.X.; Pang, X.Y.; Guan, S.H.; Yang, M.; Hua, H.M.; Wu, L.J.; Guo, D.A. Isoprenylated flavonoids from the roots of Sophora tonkinensis. Phytochem. Lett.
**2008**, 1, 163–167. [Google Scholar] [CrossRef] - Ding, P.; Chen, D. Three cyclized isoprenylated flavonoids from the roots and rhizomes of Sophora tonkinensis. Helv. Chim. Acta
**2007**, 90, 2236–2244. [Google Scholar] [CrossRef] - Deng, Y.H.; Xu, K.P.; Zhou, Y.J.; Li, F.S.; Zeng, G.Y.; Tan, G.S. A new flavonol from Sophora tonkinensis. J. Asian Nat. Prod. Res.
**2007**, 9, 45–48. [Google Scholar] [CrossRef] - Wu, C.; He, L.; Yi, X.; Qin, J.; Li, Y.; Zhang, Y.; Wang, G. Three new alkaloids from the roots of Sophora tonkinensis. J. Nat. Med.
**2019**, 73, 667–671. [Google Scholar] [CrossRef] - Pan, Q.M.; Zhang, G.J.; Huang, R.Z.; Pan, Y.M.; Wang, H.S.; Liang, D. Cytisine-type alkaloids and flavonoids from the rhizomes of Sophora tonkinensis. J. Asian Nat. Prod. Res.
**2016**, 18, 429–435. [Google Scholar] [CrossRef] - Pan, Q.M.; Li, Y.H.; Hua, J.; Huang, F.P.; Wang, H.S.; Liang, D. Antiviral matrine-type alkaloids from the rhizomes of Sophora tonkinensis. J. Nat. Prod.
**2015**, 78, 1683–1688. [Google Scholar] [CrossRef] [PubMed] - Yoo, H.; Chae, H.S.; Kim, Y.M.; Kang, M.; Ryu, K.H.; Ahn, H.C.; Yoon, K.D.; Chin, Y.W.; Kim, J. Flavonoids and arylbenzofurans from the rhizomes and roots of Sophora tonkinensis with IL-6 production inhibitory activity. Bioorg. Med. Chem. Lett.
**2014**, 24, 5644–5647. [Google Scholar] [CrossRef] [PubMed] - Luo, G.Y.; Yang, Y.; Zhou, M.; Ye, Q.; Liu, Y.; Gu, J.; Zhang, G.L.; Luo, Y.G. Novel 2-arylbenzofuran dimers and polyisoprenylated flavanones from Sophora tonkinensis. Fitoterapia
**2014**, 99, 21–27. [Google Scholar] [CrossRef] [PubMed] - Wang, W.; Nakashima, K.I.; Hirai, T.; Inoue, M. Anti-inflammatory effects of naturally occurring retinoid X receptor agonists isolated from Sophora tonkinensis Gagnep. via retinoid X receptor/liver X receptor heterodimers. J. Nat. Med.
**2019**, 73, 419–430. [Google Scholar] [CrossRef] [PubMed] - Chae, H.S.; Yoo, H.; Kim, Y.M.; Choi, Y.H.; Lee, C.H.; Chin, Y.W. Anti-inflammatory effects of 6, 8-diprenyl-7,4′-dihydroxyflavanone from Sophora tonkinensis on lipopolysaccharide-stimulated RAW 264.7 cells. Molecules
**2016**, 21, 1049. [Google Scholar] [CrossRef] [PubMed] - Lee, J.W.; Lee, J.H.; Lee, C.; Jin, Q.; Lee, D.; Kim, Y.; Hong, J.T.; Lee, M.K.; Hwang, B.Y. Inhibitory constituents of Sophora tonkinensis on nitric oxide production in RAW 264.7 macrophages. Bioorg. Med. Chem. Lett.
**2015**, 25, 960–962. [Google Scholar] [CrossRef] [PubMed] - Wang, W.; Nakashima, K.I.; Hirai, T.; Inoue, M. Neuroprotective effect of naturally occurring RXR agonists isolated from Sophora tonkinensis Gagnep. on amyloid-β-induced cytotoxicity in PC12 cells. J. Nat. Med.
**2019**, 73, 154–162. [Google Scholar] [CrossRef] [PubMed] - Huang, M.; Deng, S.; Han, Q.; Zhao, P.; Zhou, Q.; Zheng, S.; Ma, X.; Xu, C.; Yang, J.; Yang, X. Hypoglycemic activity and the potential mechanism of the flavonoid rich extract from Sophora tonkinensis Gagnep. in KK-Ay mice. Front. Pharmacol.
**2016**, 7, 288. [Google Scholar] [CrossRef] - Ding, P.L.; He, C.M.; Cheng, Z.H.; Chen, D.F. Flavonoids rather than alkaloids as the diagnostic constituents to distinguish Sophorae Flavescentis Radix from Sophorae Tonkinensis Radix et Rhizoma: An HPLC fingerprint study. Chin. J. Nat. Med.
**2018**, 16, 951–960. [Google Scholar] [CrossRef] - He, C.M.; Cheng, Z.H.; Chen, D.F. Qualitative and quantitative analysis of flavonoids in Sophora tonkinensis by LC/MS and HPLC. Chin. J. Nat. Med.
**2013**, 11, 690–698. [Google Scholar] [CrossRef] - Liu, E.H.; Qi, L.W.; Cao, J.; Li, P.; Li, C.Y.; Peng, Y.B. Advances of modern chromatographic and electrophoretic methods in separation and analysis of flavonoids. Molecules
**2008**, 13, 2521–2544. [Google Scholar] [CrossRef] [PubMed] - Sun, Y.; Sun, Y.; Chen, H.; Hao, Z.; Wang, J.; Guan, Y.; Zhang, Y.; Feng, W.; Zheng, X. Isolation of two new prenylated flavonoids from Sinopodophyllum emodi fruit by silica gel column and high-speed counter-current chromatography. J. Chromatogr. B
**2014**, 969, 190–198. [Google Scholar] [CrossRef] [PubMed] - Zhu, Y.; Liu, Y.; Zhan, Y.; Liu, L.; Xu, Y.; Xu, T.; Liu, T. Preparative isolation and purification of five flavonoid glycosides and one benzophenone galloyl glycoside from Psidium guajava by high-speed counter-current chromatography (HSCCC). Molecules
**2013**, 18, 15648–15661. [Google Scholar] [CrossRef] [PubMed] - He, J.Z.; Shao, P.; Liu, J.H.; Ru, Q.M. Supercritical carbon dioxide extraction of flavonoids from Pomelo (Citrus grandis (L.) Osbeck) peel and their antioxidant activity. Int. J. Mol. Sci.
**2012**, 13, 13065–13078. [Google Scholar] [CrossRef] - Wen, L.; Lin, Y.; Lv, R.; Yan, H.; Yu, J.; Zhao, H.; Wang, X.; Wang, D. An efficient method for the preparative isolation and purification of flavonoids from leaves of Crataegus pinnatifida by HSCCC and Pre-HPLC. Molecules
**2017**, 22, 767. [Google Scholar] [CrossRef] [PubMed] - Xie, Y.; Guo, Q.-S.; Wang, G.-S. Preparative separation and purification of the total flavonoids in Scorzonera austriaca with macroporous resins. Molecules
**2016**, 21, 768. [Google Scholar] [CrossRef] [PubMed] - Yang, Z.; Tang, H.; Shao, Q.; Bilia, A.R.; Wang, Y.; Zhao, X. Enrichment and purification of the bioactive flavonoids from flower of Abelmoschus manihot (L.) Medic using macroporous resins. Molecules
**2018**, 23, 2649. [Google Scholar] [CrossRef] - Ren, J.Y.; Zheng, Y.M.; Lin, Z.H.; Han, X.; Liao, W.Z. Macroporous resin purification and characterization of flavonoids from Platycladus orientalis (L.) Franco and their effects on macrophage inflammatory response. Food Funct.
**2017**, 8, 86–95. [Google Scholar] [CrossRef] - Cao, Q.; Wang, L.; Ur Rashid, H.; Liang, H.; Liu, X.; Xie, P. Ultrasonic-assisted reductive extraction of matrine from sophorae tonkinesis and its purification by macroporous resin column chromatography. Sep. Sci. Technol.
**2018**, 53, 745–755. [Google Scholar] [CrossRef] - Li, J.; Chase, H.A. Development of adsorptive (non-ionic) macroporous resins and their uses in the purification of pharmacologically-active natural products from plant sources. Nat. Prod. Rep.
**2010**, 27, 1493–1510. [Google Scholar] [CrossRef] - Aksu, Z.; Kabasakal, E. Batch adsorption of 2, 4-dichlorophenoxy-acetic acid (2, 4-D) from aqueous solution by granular activated carbon. Sep. Purif. Technol.
**2004**, 35, 223–240. [Google Scholar] [CrossRef] - Huang, J.H.; Liu, Y.F.; Wang, X.G. Selective sorption of tannin from flavonoids by organically modified attapulgite clay. J. Hazard. Mater.
**2008**, 160, 382–387. [Google Scholar] [CrossRef] [PubMed] - Foo, K.Y.; Hameed, B.H. Insights into the modeling of adsorption isotherm systems. Chem. Eng. J.
**2010**, 156, 2–10. [Google Scholar] [CrossRef] - Vargas, A.M.M.; Cazetta, A.L.; Martins, A.C.; Moraes, J.C.G.; Garcia, E.E.; Gauze, G.F.; Costa, W.F.; Almeida, V.C. Kinetic and equilibrium studies: adsorption of food dyes acid yellow 6, acid yellow 23, and acid red 18 on activated carbon from flamboyant pods. Chem. Eng. J.
**2012**, 181–182, 243–250. [Google Scholar] [CrossRef] - Siddiqui, S.I.; Rathi, G.; Chaudhry, S.A. Acid washed black cumin seed powder preparation for adsorption of methylene blue dye from aqueous solution: Thermodynamic, kinetic and isotherm studies. J. Mol. Liq.
**2018**, 264, 275–284. [Google Scholar] [CrossRef] - Shin, H.S.; Kim, J.H. Isotherm, kinetic and thermodynamic characteristics of adsorption of paclitaxel onto Diaion HP-20. Process Biochem.
**2016**, 51, 917–924. [Google Scholar] [CrossRef] - Li, W.; Zhang, S.; Zu, Y.G.; Fu, Y.J.; Ma, W.; Zhang, D.Y.; Kong, Y.; Li, X.J. Preliminary purification and separation of genistein and apigenin from extracts of pigeon pea roots by macroporous resins. Bioresour. Technol.
**2010**, 101, 4667–4675. [Google Scholar] [CrossRef] - Jayaprakasha, G.K.; Singh, R.P.; Sakariah, K.K. Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chem.
**2001**, 73, 285–290. [Google Scholar] [CrossRef]

Sample Availability:Sophora tonkinensis Gagnep. and flavonoids standards are available from the authors. |

**Figure 1.**Static adsorption/desorption capacity and desorption ratio of the total flavonoids from S. tonkinensis on different resins.

**Figure 3.**Adsorption isotherms (

**a**) and linear correlations on the basis of the Langmuir (

**b**), Freundlich (

**c**), and Temkin (

**d**) models for the total flavonoids from S. tonkinensis on the AB-8 resin at 298.15, 308.15, and 318.15 K.

**Figure 4.**Adsorption kinetic curve (

**a**) and linear correlations on the basis of the pseudo-first-order (

**b**), pseudo-second-order (

**c**), and intra-particle diffusion (

**d**) models for the total flavonoids from S. tonkinensis on the AB-8 resin at 298.15 K.

**Figure 5.**Dynamic breakthrough curves of the total flavonoids from S. tonkinensis on the column packed with the AB-8 resin at different flow rates.

**Figure 7.**Dynamic desorption curves of the total flavonoids from S. tonkinensis on the column packed with the AB-8 resin at different flow rates.

**Figure 8.**High-performance liquid chromatography coupled with photodiode-array detection (HPLC-PAD) chromatograms of the flavonoid extracts from S. tonkinensis before (

**a**) and after (

**b**) purification at 254 nm. Peaks 1, 2, 3, 4, 5, and 6 represent rutin, trifolirhizin, quercitrin, quercetin, maackiain, and formononetin, respectively.

**Table 1.**Adsorption isotherm equations and parameters of the total flavonoids from the S. tonkinensis on the AB-8 resin.

Model | T (K) | Equations | Parameters | ||
---|---|---|---|---|---|

K_{L} (L/mg) | q_{m} (mg/g) | R^{2} | |||

Langmuir | 298.15 | $\frac{{C}_{e}}{{q}_{e}}=0.0411{C}_{e}+0.7806$ | 0.0527 | 24.33 | 0.9986 |

308.15 | $\frac{{C}_{e}}{{q}_{e}}=0.0443{C}_{e}+0.9388$ | 0.0472 | 22.57 | 0.9981 | |

318.15 | $\frac{{C}_{e}}{{q}_{e}}=0.0477{C}_{e}+1.1071$ | 0.0431 | 20.96 | 0.9977 | |

K_{F} [mg/g(L/mg)^{1/n}] | n | R^{2} | |||

Freundlich | 298.15 | $\mathrm{ln}{q}_{e}=0.3882\mathrm{ln}{C}_{e}+1.2224$ | 3.3953 | 2.5760 | 0.9215 |

308.15 | $\mathrm{ln}{q}_{e}=0.3767\mathrm{ln}{C}_{e}+1.1559$ | 3.1769 | 2.6546 | 0.9432 | |

318.15 | $\mathrm{ln}{q}_{e}=0.3710\mathrm{ln}{C}_{e}+1.0750$ | 2.9300 | 2.6954 | 0.9439 | |

K_{T} (L/mg) | B_{T} (J/mol) | R^{2} | |||

Temkin | 298.15 | ${q}_{e}=5.0133\mathrm{ln}{C}_{e}-2.8570$ | 0.5656 | 5.0133 | 0.9696 |

308.15 | ${q}_{e}=4.5858\mathrm{ln}{C}_{e}-2.8858$ | 0.5330 | 4.5858 | 0.9736 | |

318.15 | ${q}_{e}=4.2617\mathrm{ln}{C}_{e}-3.0703$ | 0.4865 | 4.2617 | 0.9704 |

**Table 2.**Thermodynamic parameters for the adsorption of the total flavonoids from S. tonkinensis on the AB-8 resin.

Kinetics Model | Regression Equations | Parameters |
---|---|---|

Pseudo-first-order | $\mathrm{ln}({q}_{e}-{q}_{t})=-0.0186t+2.0411$ | k_{1} = 0.0186 min^{−1}Q _{e} = 7.70 mg/gR ^{2} = 0.9696 |

Pseudo-second-order | $\frac{t}{{q}_{t}}=0.0478t+0.3289$ | k_{2} = 6.9469 × 10^{−3} g/(mg·min)Q _{e} = 20.92 mg/gR ^{2}= 0.9999 |

Intra-particle diffusion (first stage) | ${q}_{t}=3.3432{t}^{1/2}+2.3129$ | k_{i} = 3.3432 mg/(g·min^{1/2})I = 2.3129 mg/g R ^{2} = 0.9721 |

(second stage) | ${q}_{t}=0.7191{t}^{1/2}+12.3295$ | K_{i} = 0.7191 mg/(g·min^{1/2})I = 12.3295 mg/g R ^{2} = 0.9843 |

(third stage) | ${q}_{t}=0.0797{t}^{1/2}+19.0885$ | K_{i} = 0.0797 mg/(g·min^{1/2})I = 19.0885 mg/g R ^{2} = 0.9966 |

**Table 3.**Comparison of the flavonoid contents in the extracts from S. tonkinensis before and after purification.

Compounds | Retention Time (min) | Content (%) | Recovery | |
---|---|---|---|---|

Before Purification | After Purification | |||

Rutin | 28.362 | 0.014% | 0.036% | 83.47% |

Trifolirhizin | 37.825 | 0.756% | 3.198% | 86.51% |

Quercitrin | 40.641 | 0.026% | 0.032% | 84.22% |

Quercetin | 42.103 | 0.007% | 0.067% | 91.53% |

Maackiain | 48.759 | 1.617% | 7.842% | 87.16% |

Formononetin | 49.537 | 0.067% | 0.842% | 85.90% |

Resins | Particle Size (mm) | Surface Area (m^{2}/g) | Average Pore Diameter (nm) | Polarity |
---|---|---|---|---|

NKA-9 | 0.3~1.25 | 170~250 | 15.5~16.5 | Polar |

NKA-II | 0.3~1.25 | 160~200 | 14.5~15.5 | Polar |

HPD-600 | 0.3~1.25 | 550~600 | 8.0~9.0 | Polar |

DM301 | 0.3~1.25 | 330~380 | 9.0~11.0 | Middle polar |

HPD-400 | 0.3~1.25 | 500~550 | 7.5~8.0 | Middle polar |

HPD-750 | 0.3~1.25 | 650~700 | 8.5~9.0 | Middle polar |

AB-8 | 0.3~1.25 | 450~530 | 13.0~14.0 | Weak polar |

HPD-100 | 0.3~1.25 | 650~700 | 8.5~9.0 | Non-polar |

D101 | 0.3~1.25 | 600~700 | 10.0~12.0 | Non-polar |

X-5 | 0.3~1.25 | 500~600 | 21.0~23.0 | Non-polar |

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**MDPI and ACS Style**

Hou, M.; Hu, W.; Xiu, Z.; Jiang, A.; Men, L.; Hao, K.; Sun, X.; Cao, D.
Preparative Purification of Total Flavonoids from *Sophora tonkinensis* Gagnep. by Macroporous Resin Column Chromatography and Comparative Analysis of Flavonoid Profiles by HPLC-PAD. *Molecules* **2019**, *24*, 3200.
https://doi.org/10.3390/molecules24173200

**AMA Style**

Hou M, Hu W, Xiu Z, Jiang A, Men L, Hao K, Sun X, Cao D.
Preparative Purification of Total Flavonoids from *Sophora tonkinensis* Gagnep. by Macroporous Resin Column Chromatography and Comparative Analysis of Flavonoid Profiles by HPLC-PAD. *Molecules*. 2019; 24(17):3200.
https://doi.org/10.3390/molecules24173200

**Chicago/Turabian Style**

Hou, Mengyang, Wenzhong Hu, Zhilong Xiu, Aili Jiang, Lei Men, Kexin Hao, Xingsheng Sun, and Duo Cao.
2019. "Preparative Purification of Total Flavonoids from *Sophora tonkinensis* Gagnep. by Macroporous Resin Column Chromatography and Comparative Analysis of Flavonoid Profiles by HPLC-PAD" *Molecules* 24, no. 17: 3200.
https://doi.org/10.3390/molecules24173200