Salvia miltiorrhiza and Salvia Species:identification,traditional use,application research of Radix Salvia miltiorrhiza and its useful fraction components.
- Basic Botanical Data of Radix Salvia miltiorrhiza.
- Salvia and the History of Microcirculation Research in China.
- Status of Salvia In Ancient Chinese Medicine:Radix Salvia miltiorrhiza as the heart-mind stimulator.
- Active Constituents and phytochemicals of Radix Salvia miltiorrhiza.
- What is Tanshinone IIA? Application and function of Tanshinone IIA.
- Application research of Radix Salvia miltiorrhiza and its useful fraction and components.
- More About Sage Family.:History,Key Actions,Key Components,Medicinal Parts,Remedies,Traditional Uses of Sage Family world wide.
- Dosage, Toxicity,And Adverse Effects of Danshen.
- Photo Gallery of Salvia miltiorrhiza.
What is Tanshinone IIA? Application and function of Tanshinone IIA.:
Tanshnone IIA is a diterpenoid naphthoquinone extracted and isolated from Radix Salvia miltiorrhiza(Danshen).
Figure: Molecular Structure of Tanshinoneiia
Physical Appearance:Orange Solid (m.p. 205-207 deg C)
Molecular Formula: C19H18O3 M.W.:294.3
Purity: 98%(TLC:30% Ethyl Acetate/Petroleum ether; Rf=0.47)
Solubility: Soluble in DMSO (25mg/ml) and ethanol (5mg/ml)
Tanshinone IIA show good function and inhibits AP-1 activity by suppressing junfos-DNA complex formation(IC50=0.22um),and displays anti-inflammatory activity and induces apoptosis in a variety of cell lines.
Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE induced apoptosis in HL60 human premyelocytic leukemia cell line.
Apoptosis is a new therapeutic target of cancer research. Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE, a traditional oriental medical herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Tanshinone IIA induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide (PI) staining. Tanshinone IIA-induced apoptosis is accompanied by the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-3, a major component in apoptotic cell death mechanism.
Apoptosis is cellular suicide or programmed cell death which is mediated by the activation of an evolutionary conserved intracellular pathway. Recently the relation of apoptosis and cancer has been emphasized and increasing evidence suggests that the processes of neoplastic transformation, progression and metastasis involve alteration of normal apoptotic pathways. Apoptosis also gives some clues about effective anticancer therapy, and many chemotherapeutic agents were reported to exert their anti-tumor effects by inducing apoptosis of cancer cells.
Salvia miltiorrhiza BUNGE is a traditional oriental medical herb which belongs to the family of Labiatae, and has been used for treatment of coronary heart disease, particularly angina pectoris and myocardial infarction. According to several phytochemical reports, major constituents of the root of Salvia miltiorrhiza BUNGE are composed of phenolics such as lithospermate B and diterpene quinone pigments such as tanshinones. Medical researchers have studied on the effect of magnesium lithospermate B on renal failure, and diterpene quinones have been reported for anti-platelet aggregation effect. Recently, it was found the growth inhibitory effects of various tanshinones on five tumor cell lines, and it was shown that HCTIS/CL02, a multidrug-resistant tumor cell line, did not exhibit resistance to tanshinones even though it has resistance to doxorubicin.
In this study, apoptosis-inducing effect of tanshinone IIA which is most abundant and structurally representative among the tanshinones of Salvia miltiorrhiza BUNGE was investigated in HL60 human premyelocytic leukemia cell line.
3. Results and Discusssion
Recently, the anti-tumour effect of tanshinones isolated from Salvia miltiorrhiza BUNGE was elucidated using human tumour cell lines. Since many anticancer drugs induce apoptosis of tumour cells, the apoptosis-inducing activity of the crude extract of Salvia miltiorrhiza BUNGE was investigated. Human premyelocytic leukemia cell line, HL60, was treated with various concentrations of the ether extract of Salvia miltiorrhiza BUNGE for 4 h, and it was found that it induced internucleosomal DNA fragmentation, one of the biochemical hall mark of apoptosis, at the concentration above 10 ug/ml. UV radiation was used as a positive control for the induction of apoptosis. Among the constituents of Salvia miltiorrhiza BUNGE, tanshinone IIA was focused on, because it is most abundant and structurally representative having a basic tanshinone skeleton. Using a similar phytochemical procedure with the previous report, tanshinone IIA was successfully purified and the structure of it was clearly identified compared with the previous report. When HL60 cells were treated with various concentrations of tanshinone IIA, internucleosomal DNA fragmentation was induced by the concentrations as low as 1 ug/ml. In time course experiment in which 3 ug/ml of tanshinone IIA was treated, 180 bp ladder was generated at 2 or 3 hours after the treatment. Microscopic observation showed that the crude ether extract of Salvia miltiorrhiza BUNGE and purified tanshinone IIA both induced cellular morphological changes characteristic of apoptosis including membrane blebbing and apoptotic body formation. Flow cytometry analysis of tanshinone IIA-treated HL60 cells showed the increase of hypodiploid apoptotic cells and the decrease of the cells at G1 phase of cell cycle, suggesting a possibility that tanshinone IIA induced apoptosis occurs at G1 phase of cell cycle. PARP is a nuclear enzyme which is involved in DNA repair process, and recently, it was found that 113 kDa PARP protein is cleaved into 89 and 24 kDa fragments by the action of CPP32, a protease recently named as caspase-3.
Since the specific proteolytic cleavage of PARP is considered to be a biochemical characteristic of apoptosis, the Western blotting experiment was carried out using the antibody against PARP. The results demonstarted that PARP is cleaved into 89 kDa fragment 4 hours after the addition of tanshinone IIA suggesting that caspase-3 was activated. To measure the caspase-3 activity directly and quantitatively, Ac-DEVD-pNA was used, a specific colorimetric substrate of caspase-3. The results demonstrated that caspase-3 is activated during tanshinone IIA-induced apoptotic process. Recently, many papers reported that internucleosomal DNA fragmentation is not essential in apoptotic cell death, and some necrotic cell death is accompanied by internucleosomal DNA fragmentation, suggesting the possibility that internucleosomal DNA fragmentation may be not enough as an indicator of apoptotic cell death. It is, however, clear that the central mechanism of apoptosis is evolutionary conserved and caspase activation is an essential step in this complex apoptotic pathways. The results of the study, therefore, give more important evidences that tanshinone IIA-induced cell death is apoptosis.
Note:Normal and Adoptotic Morphology of HL-60 Cells.:
A:Control HL-60 cells are majority premylocytes which usually appear in ovoid shape with large nucleus(arrow).H and E stain,at x 1000.
B:After incubation at 0.1mg of Danshen for 48hrs,HL-60 cells exhibit characteristic apoptotic features with condensed chromatin along the margin of the nucleus(arrow).H and E stain,at x 1000.
In this study the apoptosis inducing activity of tanshinone IIA, a main ingredient of Salvia miltiorrhiza BUNGE, was examined. Other tanshinone components structurally related to tanshinone IIA such as cryptotanshinone and tanshinone I also exist in the root of Salvia miltiorrhiza BUNGE, and the apoptosis-inducing activity of other tanshinones will be examined later especially focusing on caspase activity which is central component of apoptotic pathway.
- 1.Salvia miltiorrhiza and Salvia Species:identification,traditional use,application research of Radix Salvia miltiorrhiza and its useful fraction components.
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