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Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen)

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ABSTRACT

1–45–1844444: Four novel compounds () as well as fourteen reported compounds () were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound demonstrated the best anti-inflammatory activity and was chosen for further research. Compound greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound in vitro. Compound was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

No MeSH data available.


The anti-inflammatory activities of the isolated compounds.(A) Cells were pretreated with compounds for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The nitrite production was determined by Griess assay. (B) The effect of compound 4 and Tan IIA on nitrite production was determined by Griess assay. *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS-treated group. (C) NO production was measured by flow cytometry with DAF-FM (3 μM). (D,E) Cells were pretreated with compound 4 for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The ELISA assay was employed to detect levels of TNF-α and IL-6 in the culture medium. *p < 0.05 versus the LPS-treated group. (F) Cells were pretreated with compound 4 as aforementioned and the expression of iNOS and COX-2 was determined by Western blotting. Tan IIA, tanshinone IIA.
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f3: The anti-inflammatory activities of the isolated compounds.(A) Cells were pretreated with compounds for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The nitrite production was determined by Griess assay. (B) The effect of compound 4 and Tan IIA on nitrite production was determined by Griess assay. *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS-treated group. (C) NO production was measured by flow cytometry with DAF-FM (3 μM). (D,E) Cells were pretreated with compound 4 for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The ELISA assay was employed to detect levels of TNF-α and IL-6 in the culture medium. *p < 0.05 versus the LPS-treated group. (F) Cells were pretreated with compound 4 as aforementioned and the expression of iNOS and COX-2 was determined by Western blotting. Tan IIA, tanshinone IIA.

Mentions: To date, there are around 60 tanshinones isolated and identified from Danshen23. However, only a few of them, such as tanshinone IIA, cryptotanshinone, were thoroughly investigated for their bioactivities. Although the anti-inflammatory activity of tanshinone IIA and cryptotanshinone has been well established2425, most of the known compounds isolated by our group such as compounds 5, 6, 7, 9, 10, 11, 12, 13 and 14 have not been reported. Synthesized endogenously from L-arginine by nitric oxide synthases (NOSs), NO becomes an inflammometer to modulate important cellular signaling involved in immunity and inflammation26. LPS-induced NO production in RAW264.7 macrophages has been considered as the convenient and credible method for anti-inflammation screening24252627. The Griess assay for the determination of nitrite resulted from the reaction of NO with O2 was used as effective and efficient method to quantitate NO production2728. Therefore, 18 compounds were primarily screened with a LPS stimulated RAW264.7 macrophage cell model for their anti-inflammatory activities evaluation. As shown in the results, most of compounds exhibited significant inhibition on nitrite production (Fig. 3A) suggesting the decreased production of NO. MTT results indicated that almost all of compounds exhibited no cytotoxicity except compound 10 (Figure S1) Among these compounds, the new compound 4 exhibited the highest inhibitory activity, far more than salvianolic acid B (Figure S2A), suggesting compound 4 was able to be a potential anti-inflammatory drug.


Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen)
The anti-inflammatory activities of the isolated compounds.(A) Cells were pretreated with compounds for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The nitrite production was determined by Griess assay. (B) The effect of compound 4 and Tan IIA on nitrite production was determined by Griess assay. *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS-treated group. (C) NO production was measured by flow cytometry with DAF-FM (3 μM). (D,E) Cells were pretreated with compound 4 for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The ELISA assay was employed to detect levels of TNF-α and IL-6 in the culture medium. *p < 0.05 versus the LPS-treated group. (F) Cells were pretreated with compound 4 as aforementioned and the expression of iNOS and COX-2 was determined by Western blotting. Tan IIA, tanshinone IIA.
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f3: The anti-inflammatory activities of the isolated compounds.(A) Cells were pretreated with compounds for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The nitrite production was determined by Griess assay. (B) The effect of compound 4 and Tan IIA on nitrite production was determined by Griess assay. *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS-treated group. (C) NO production was measured by flow cytometry with DAF-FM (3 μM). (D,E) Cells were pretreated with compound 4 for 1 h and then stimulated with/without LPS (1 μg/mL) for 24 h. The ELISA assay was employed to detect levels of TNF-α and IL-6 in the culture medium. *p < 0.05 versus the LPS-treated group. (F) Cells were pretreated with compound 4 as aforementioned and the expression of iNOS and COX-2 was determined by Western blotting. Tan IIA, tanshinone IIA.
Mentions: To date, there are around 60 tanshinones isolated and identified from Danshen23. However, only a few of them, such as tanshinone IIA, cryptotanshinone, were thoroughly investigated for their bioactivities. Although the anti-inflammatory activity of tanshinone IIA and cryptotanshinone has been well established2425, most of the known compounds isolated by our group such as compounds 5, 6, 7, 9, 10, 11, 12, 13 and 14 have not been reported. Synthesized endogenously from L-arginine by nitric oxide synthases (NOSs), NO becomes an inflammometer to modulate important cellular signaling involved in immunity and inflammation26. LPS-induced NO production in RAW264.7 macrophages has been considered as the convenient and credible method for anti-inflammation screening24252627. The Griess assay for the determination of nitrite resulted from the reaction of NO with O2 was used as effective and efficient method to quantitate NO production2728. Therefore, 18 compounds were primarily screened with a LPS stimulated RAW264.7 macrophage cell model for their anti-inflammatory activities evaluation. As shown in the results, most of compounds exhibited significant inhibition on nitrite production (Fig. 3A) suggesting the decreased production of NO. MTT results indicated that almost all of compounds exhibited no cytotoxicity except compound 10 (Figure S1) Among these compounds, the new compound 4 exhibited the highest inhibitory activity, far more than salvianolic acid B (Figure S2A), suggesting compound 4 was able to be a potential anti-inflammatory drug.

View Article: PubMed Central - PubMed

ABSTRACT

1–45–1844444: Four novel compounds () as well as fourteen reported compounds () were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound demonstrated the best anti-inflammatory activity and was chosen for further research. Compound greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-&alpha; and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-&kappa;B was attenuated after treatment with compound in vitro. Compound was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

No MeSH data available.