Multiphase Extraction And Separation Of Lithospermic Acid B And Tanshinone ⅡA From Salvia Miltiorrhiza Bunge

Lithospermic acid B（LAB）and tanshinone ⅡA（Tan ⅡA）are main bioactive components in Salvia miltiorrhiza Bunge which has anti-atherosclerosis,anti-neuritis,anti-tumor and other pharmacological effects.Up to now,solvent extraction or other auxiliary methods are used to extract and separate these two compounds.Traditional industrial methods for extracting LAB and Tan ⅡA have the problems of high costs,low efficiency and cumbersome operation.In this subject,three liquid phase system（TLPS）combined two phase system was used to extract and separate LAB and Tan ⅡA from Salvia miltiorrhiza Bunge at the same time,and the following research was conducted.（1）The n-hexane-ethanol-NaH₂PO₄-H₂O TLPS was used to extract LAB and Tan ⅡA.LAB was mainly distributed in the middle phase（ethanol-rich phase）,and Tan ⅡA was mainly distributed in the upper phase（n-hexane-rich phase）.Combining single factor experiment and response surface methodology（RSM）,TLPS extraction was optimized to achieve optimal condition:aqueous NaH₂PO₄ solution（3.75mol/L,p H2.55）:ethanol:n-hexane=384:376:240（v/v/v）,temperature 46.9℃ and time 6.65 h.Under the optimal conditions,the total yield of LAB was 142±8.8 mg/g,and the total yield of Tan ⅡA was 0.548±0.041 mg/g.（2）After extraction under optimal condition,the herb residue and bottom phase were removed.LAB and Tan ⅡA were separated by re-partitioning into different phases via addition of n-hexane and H₂O to the upper and middle phases.When the volume fractions of n-hexane and H₂O added were 16.4%and 20.5%,the partition coefficient of LAB was 22.4±1.2,95.8%of LAB was enriched in the middle phase.The partition coefficient of Tan ⅡA was 10.5±0.30,and 87.6%of Tan ⅡA was enriched in the upper phase.（3）The thermodynamic behaviors of LAB and Tan ⅡA at different temperatures in the process of n-hexane-ethanol-NaH₂PO₄-H₂O TLPS and re-partition were investigated.It was concluded that the phase separation of LAB and Tan ⅡA were spontaneous,irreversible and endothermic.（4）The n-hexane-acetone-ethanol-NaH₂PO₄-H₂O TLPSs were constructed,and finally the extraction was optimized to achieve optimal condition:aqueous NaH₂PO₄solution（3.75mol/L,p H 2.55）:acetone:ethanol:n-hexane=384:263:113:240（v/v/v/v）,temperature 46.0℃and extraction time 7.60 h.Under the optimal conditions,the total yield of LAB was 160±12 mg/g,and the total yield of Tan ⅡA was 0.582±0.038 mg/g.（5）Re-partition procedure was performed with the TLPS containing acetone.When the volume fractions of additional n-hexane and H₂O were 18.9%and 37.7%,respectively,the LAB partition coefficient was 14.0±0.91,and 93.3%of the LAB was enriched in the middle phase;The partition coefficient of Tan ⅡA was 11.2±0.73,and89.4%of Tan ⅡA was enriched in the upper phase.（6）The pseudo-first-order kinetic model was used to characterize the kinetic behaviors of the extraction of LAB and Tan ⅡA with n-hexane-ethanol-NaH₂PO₄-H₂O and n-hexane-acetone-ethanol-NaH₂PO₄-H₂O TLPS.The results showed that LAB desorbed from the herb faster than Tan ⅡA and reached distribution equilibrium,and the yields when LAB and Tan ⅡA reached equilibrium in the TLPS containing acetone was higher.（7）The methods for single-phase extractions for LAB and Tan ⅡA were referred from official Chinese pharmacopoeia.The yield of LAB extracted with 80.0%（v/v）methanol was 64.0±3.0 mg/g（extraction efficiency was 37.2%）,and the yield of Tan ⅡA extracted with absolute methanol was 0.352±0.014 mg/g（extraction efficiency was56.7%）.The yields with n-hexane-acetone-ethanol-NaH₂PO₄-H₂O TLPS extraction followed by optimized re-partition were 149±8.2 mg/g for LAB（extraction efficiency was 93.0%）,and 0.521±0.043 mg/g for Tan ⅡA（extraction efficiency was 93.7%）,which were 133%and 48.0%higher than the single-phase methods in the pharmacopoeia,respectively.