Ultrasound Enhanced Subcritical Water Extraction Of Active Ingredients Of Different Polarity From Lithospermum Erythrorhizon

Subcritical water extraction is a technique based on the use of water as extractant at temperatures between 100℃and 374℃and a pressure high enough to maintain the liquid state. The polarity of water at ambient temperature and pressure is high, while the polarity of subcritical water decreases with increasing temperature and the nature is similar to the organic solvent which can be used for the sequential extraction of water-soluble and fat-soluble components from natural products. This technique has become a hotspot of separation and purufication study because of the advantages of low cost and environment protection. However, some heat-sensitive components are prone to occur irreversible changes at high temperatures keeping for a long time, and the extraction conditions of some nonpolar and macromolecules materials are relatively harsh.In order to overcome these problems, the weak polar naphthoquinone pigment and non-polar volatile oil from Lithospermum erythrorhizon were used as the studied obfects. A physical technique was applied to enhance the extraction procedure of subcritical water by means of introducing an ultrasound probe into the extraction kettle to study the impact of ultrasound field parameters such as output power, frequency, irradiation method and irradiation time on extraction efficiency of subcritical water. Effect of ultrasound enhanced subcritical water, supercritical CO₂, traditional solvent and steam distillation extraction methods on extract was studied. The mechanism of ultrasound enhanced subcritical water extraction was studied from the perspectives of cavitation effect, mechanical effect, impact on micro-structurre of plant and mass transfer rate. A new green efficient method of separating natural products was proposed in this paper.The results show that the ultrasound can significantly improve efficiency and shorten mass transfer equilibrium time. The low-frequency ultrasound irradiation with high output power has a better enhancement effect. The extraction yield of naphthoquinone pigments from Lithospermum erythrorhizon by subcritical water extraction was increased from 0.22 % to 0.31 % by ultrasound of 250 W and 20 kHz intermittent radiating(6 s/4 s) for 20 min at conditions of 120℃and 5 MPa, and the extraction yield of volatile oil was increased from 1.87 % to 2.39 % at conditions of 160℃and 5 MPa keeping for 25 min.Ultrasound enhanced subcritical water extraction(USWE), supercritical CO₂ extraction(SCE) and Soxhlet extraction(SE) of naphthoquinone from Lithospermum erythrorhizon were compared, and the results showed the yield of USWE was maximum being 0.31 %, followed by SE and SCE being 0.21 % and 0.19 % respectively. The extracts by these three methods all contained shikonin, acetylshikonin, isobutylshikonin,β,β-dimethylacrylshikonin, 2-methyl-n-butyrylshikonin and isovalerylshikonin. Besides the above six main components, the SE and SCE extracts were detected to contain more impurities. The total bioactivity of naphthoquinone was stronger than volatile oil and showed differences depending on extraction methods. The DPPH scanvenging and reducing activities of SE extract were the strongest, followed by SE and SCE. The inhibitory effect of Escherichia coli and Staphylococcus aureus of USWE was the strongest, followed by SE and SCE. USWE, steam distillation extraction(SD) and ultrasound enhanced solvent extraction(USE) of volatile oil from Lithospermum erythrorhizon were compared and the results showed the yield of USWE was the highest being 2.39 %, followed by USE and SD, being 1.93 % and 0.62 % respectively. The extracts by these three mothods were all detected to contain 8, 11-18 carbon diene acid methyl ester, 7-18 carbon olefin acid methyl ester, palm methyl ester, 15 alkanes, 18 carbon-triene methyl ester and 9-18 carbon olefin acid methyl ester. SD and USE extracts were additionally detected to contain more volatile active components with low molecular weight. The volatile oil didn't possess DPPH scavenging capacity but showed certain reducing and antibacteria ability. The bioactivity of SD extract was the strongest, followed by USE and USWE.The result of solubility experiment showed that the maximum solubility of stearic acid in subcritical water was 0.136 at charge amount of 0.8 g, temperature of 180℃, pressure of 15 MPa and equilibrium time of 30 min. The maximum solubility of palmitic acid in subcritical water was 0.178 at charge amount of 1.0 g, temperature of 160℃, pressure of 15 MPa and equilibrium time of 30 min. The dissolution equilibrium time of fatty acid was shortened to 20 min by the ultrasound irradiation of 250 W and 20 kHz.The study of ultrasound enhanced mechanism showed that the cavitation yield increased with temperature, irradiation time, output power, decreasing prressure and frequency. The dramatic oscillation of mass point within limited space in subcritical water under ultrasonic function which led to a huge speed and accelerated velocity. Many irregular grooves appeared on surface of granules which was initially regular and plane after being dealt with ultrasonic oscillation. The compacted structure became unconsolidated which increased the permeability and swelling capacity of granules and reducing the resistance of mass transfer procedure effectively.