Separation of berberine and palmatine from Phellod

2022-10-19
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Separation of berberine and palmatine in Phellodendron amurense by high speed countercurrent chromatography

hsccc2tbe300 high speed countercurrent chromatograph, Shenzhen Tongtian Biochemical Co., Ltd

hd2212c2b nucleic acid protein detector, Shanghai Kanghua biochemical instrument factory

r2201 rotary evaporator, Shanghai Shensheng Biotechnology Co., Ltd

lc210a t high performance liquid chromatograph, Shimadzu chromatographic instrument company, Japan

chloroform, methanol, n-butanol and acetic acid are all domestic analytical pure, and the water is re distilled water. Phellodendron amurense was purchased from Huqingyutang, Hangzhou, and is Phellodendron amurense

1. 2 sample treatment

crush 1000 g of Phellodendron amurense into a 20 mesh sieve, soak it in 5 times the amount of industrial alcohol for 24 hours, and filter it for 3 consecutive times. After the combined filtrate is concentrated, add an appropriate amount of hydrochloric acid to acidify it to pH 2, place it overnight, and filter it to obtain the filtrate, which can be "self hydrolyzed" materials: it is expected to alleviate the problem of plastic and electronic waste, then alkalize it to pH 10 with NaOH, and then extract it for 3 times with the same amount of chloroform, and concentrate it to obtain extract 3 2 g, the extract is used for samples separated by high-speed countercurrent chromatography

1. 3. HSCCC solvent system selection

solvent system reference [4, 5] Select chloroform: methanol: 0 The solvent system is composed of 2 moles of dilute hydrochloric acid, mixed in various proportions, with a total volume of 4 ml. 10 mg of sample is taken each time and dissolved in the solvent system. After full dissolution, it is layered. Take the upper and lower phases and dot the plates respectively (silica gel plate, developing agent: n-butanol ∶ acetic acid ∶ water = 7:1:2). Look at the size of the spots in the two phases. Finally, it is found that when chloroform ∶ methanol ∶ 0 When the volume ratio of 2 mol/L dilute hydrochloric acid is 2 ∶ 1 ∶ 1, the spot size is about the same, so chloroform ∶ methanol ∶ 0 2 Moll dilute hydrochloric acid = 2:1:1 is the solvent system

2 results and discussion

2 1. The separation result

solvent system is: chloroform ∶ methanol: 0 2 mol/L dilute hydrochloric acid = 2:1:1, mix it fully in the separating funnel at room temperature, and then stand for layering (total volume 1000 ml), and then take the upper phase as the stationary phase and the lower phase as the mobile phase. 200 mg of sample is dissolved in 20 ml of solvent system each time, and the sample is injected once with a syringe. The flow rate of mobile phase is 2 mL min, the rotation speed of the instrument is 800 r min, and the wavelength of the detector is 254nm. After 4 hours of sample injection, stop the rotation of the host, and push out the stationary phase with nitrogen. The retention ratio is 91 4%。

Figure 1 high speed countercurrent chromatography separation of alkaloids in Phellodendron amurense figure

after high speed countercurrent chromatography separation, there are three main peaks (see Figure 1). We collected each peak with a partial collector, with an interval of 6m in each test tube, and monitored it with TLC. After removing the test tubes with overlapping components, the result was 20 4m g component, 64 The purity of 8 mg component and by HPLC was 99 6% and 99 3%, component 16 The technical problem of the conversion of 9m durometer G is a mixture

2. 2 structure identification

component I: yellow needle crystal (ethanol), melting point 198 ~ 200 ℃ (decomposition). Irtmax (KB 5g complex is really coming R) cm - 1:2923, 1605, 1510, 1364 (aromatic core), 1h2nmr (DM so 2d6) d: 3 25 (2H, t r, J = 6. 0Hz) , 4. 98 (2H, t r, J = 6. 0Hz) , 3. 88, 3. 94, 4. 08 and 4 11 (12h, 4 seven tips for selecting universal tension machine OCH3, s), 7 10 (1H, s) , 7. 73 (1H, s) , 8. 06 (1H, d, J = 910Hz) , 8. 21 (1H, d, J= 9. 0Hz) , 9. 07 (1H, s) , 9. 89 (1H, s) ; M s m z: 352, 337, 322, 308, palmatine hydrochloride

component II: yellow needle crystal, melting point 201 ~ 203 ℃ (decomposition). Irtmax (KB R) cm 21: 1600, 1505, 1364 (aromatic core), 1h2nmr (DM so 2d6) d: 3 22 (2H, t r, J = 5. 6Hz) , 4. 95 (2H, t r, J = 5. 6Hz) , 4. 07 and 4 09 (6H, s ) , 6. 17 (2H, s) , 7. 08 (1H, s) , 7. 78 (1H, s) , 8. 02 (1H, d, J = 9. 0Hz) , 8. 20 (1H, d, J = 9. 0Hz) , 8. 94 (1H, s) , 9. 89 (1H, s) ; M s m z: 337, 321, 306, 292, 278, is berberine hydrochloride

3 conclusion

in this paper, the alkaloids in Phellodendron amurense can be separated by semi preparative high-speed countercurrent chromatography with an injection amount of 200 mg, which shows that this method, especially the high-speed countercurrent chromatography with large preparation amount, is a good method for separating natural products

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