[Optimization of induction and culture conditions for hairy roots of Salvia miltiorrhiza].

To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.

Algal oil rich in n-3 polyunsaturated fatty acids suppresses B16F10 melanoma lung metastasis by autophagy induction.

Melanoma is a malignant tumor that arises from epidermal melanocytes with high morbidity and mortality, and currently, there are no effective conventional genotoxic treatments or systematic treatment. Increasing evidence shows that n-3 polyunsaturated fatty acids (PUFAs) exhibit anti-melanoma activity, but their anti-melanoma mechanism remains elusive. Here, C57BL/6 mice were injected with B16F10 melanoma cells via a tail vein to establish a lung metastasis model. n-3 PUFAs were significantly increased in lung metastatic tissues from mice treated with algal oil, especially rich in docosahexaenoic acid (DHA). Algal oil treatment significantly suppressed pulmonary metastases and outgrowth of melanoma cells, which was associated with autophagy induction, as evidenced by an increase in LC3-II levels. In addition, algae oil-triggered autophagy was mediated by inactivation of the mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein (MAP) kinase, and activation of c-Jun N-terminal kinases (JNKs), which led to a decrease in p62 accumulation and decreased secretion of proinflammatory cytokine interleukin-1ß (IL-1ß). These results suggest that algal oil exerts its antitumourigenic activities via autophagy-mediated p62 elimination and anti-inflammatory properties.