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CRYPTOTANSHINONE
Cancer. Inhibits angiogenesis. Antibiotic. Anticancer



INGREDIENTS % Daily Value
Cryptotanshinone (Dan Shen Extract)


† Daily Value not established.




CONTRAINDICATION

Effects of Cryptotanshinone on Cytochrome P450 Isoforms. The cytochrome P450 enzymes are found primarily in the liver


Cryptotanshinone (CTS) significantly increased the activity of CYP1A2 in a dose-dependent manner. In CTS groups at the dosages of 20~ 540 mg/kg, the activity of CYP1A2 was 60 % ~ 430 % higher, CYP1A2 protein expression level was 130 % ~ 320 % higher, and CYP1A2 mRNA expression level was 10 % ~ 150 % higher than that of the negative control group. CTS had no effect on other kinds of CYP isoforms. CTS can induce hepatic microsome CYP1A2 expression significantly, which indicates potential drug-drug interaction might occurred when CTS is co-administrated with those drugs metabolised by CYP1A2 such as Capecitabine / Capecitabine (Xeloda), Lapatinib / Lapatinib (Tykerb)

Traditional Chinese Drug Research & Clinical Pharmacology 2009-04, DOI CNKI:SUN:ZYXY.0.2009-04-012



RESEARCH

Ginsenoside-Rg3
Objective To investigate the effects of Ginsenoside-Rg3 (GS-Rg3) on human fibroblasts’ proliferation and apoptosis.Methods After isolation and culture, fibroblasts were obtained from patients’s keloids.We collected and planted the fibroblasts in exponential growth phase on 96-hole plates, the added GS-Rg3 with final concentration of 25?50?100?200 μg/ml.Equal Volume medium was added in control groups as well.The plates were cultured in the same incubator with 37? and 5% CO2 for 24, 48 and 72 hours.Inhibition ratio was calculated ith MTT essay.Cells percentage in different phase of different cell cycle and AP region inferior hump was detected by fluorescence activated cell sorter.Results Inhibition ratio of GS-Rg3 on human extraorgan fibroblasts has positive correlation with concentration and time.The highest inhibition ration is 91.45% while the best concentration is 100 μg/ml.Conclusion GS-Rg3 can inhibit human fibroblasts from proliferation strongly, and induce them to apoptosis as well.
--LIU He-song, LAN Shan-shan, ZHAO Wei-gang, et al. Research on The effect of Ginsenoside-Rg3 on human fibroblasts proliferation and apoptosis. Zhong Guo Shi Yan Zhen Duan Xue. 2010; 14 (11): 1697-1700


Neo-tanshinlactone
Neo-tanshinlactone was isolated and synthesised for the first time and evaluated in vitro against several human cancer cell lines. Compound 1 showed significant inhibition against two ER+ human breast cancer cell lines and was 10-fold more potent and 20-fold more selective as compared to tamoxifen citrate. Compound 1 also potently inhibited an ER-, HER-2 over-expressing breast cancer cell line. Therefore, this novel compound merits further development as an anti-breast cancer drug candidate.

Xihong Wang, X., Bastow, K.F., Sun, C-M., Lin, Y-L., Yu, H-J., Don, M-J., Wu, T-S., Nakamura, S. & Lee, K-H. Antitumour Agents. Isolation, Structure Elucidation, Total Synthesis, and Anti-Breast Cancer Activity of Neo-tanshinlactone from Salvia miltiorrhizaJ. Med. Chem., 2004, 47 (23), Pp 5816–519. DOI: 10.1021/jm040112r

Cryptotanshinone & COX-2
Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes the biosynthesis of prostaglandins from arachidonic acid and plays a critical role in some pathologies including inflammation, neurodegenerative diseases and cancer. Cryptotanshinone is a major constituent of tanshinones, which are extracted from the medicinal herb Salvia miltiorrhiza Bunge, and has well-documented antioxidative and anti-inflammatory effects. This study confirmed the remarkable anti-inflammatory effect of cryptotanshinone in the carrageenan-induced rat paw edema model. Since the action of cryptotanshinone on COX-2 has not been previously described, in the present study, we further examined the effect of cryptotanshinone on cyclooxygenase activity in the exogenous arachidonic acid-stimulated insect sf-9 cells, which highly express human COX-2 or human COX-1, and on cyclooxygenases expression in human U937 promonocytes stimulated by lipopolysaccharide (LPS) plus phorbolmyristate acetate (PMA). Cryptotanshinone reduced prostaglandin E2 synthesis and reactive oxygen species generation catalyzed by COX-2, without influencing COX-1 activity in cloned sf-9 cells. In PMA plus LPS-stimulated U937 cells, cryptotanshinone had negligible effects on the expression of COX-1 and COX-2, at either a mRNA or protein level. These results demonstrate that the anti-inflammatory effect of cryptotanshinone is directed against enzymatic activity of COX-2, not against the transcription or translation of the enzyme.

Dao-Zhong Jin, D-Z., Yina, L-L., Jia, X-Q. & Zhu, X-Z. Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression. European Journal of Pharmacology. Volume 549, Issues 1-3, 7 November 2006, Pp. 166-72. doi:10.1016/j.ejphar.2006.07.055

Cryptotanshinone & STAT3
Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumours and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 µmol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

Shin, D-S., Kim, H-N., Shin, K.D., Yoon, J.Y., Kim, S-J., Han, D.C. & Kwon, B-M.Cryptotanshinone Inhibits Constitutive Signal Transducer and Activator of Transcription 3 Function through Blocking the Dimerization in DU145 Prostate Cancer Cells. Cancer Research 69, 193, January 1, 2009. doi: 10.1158/0008-5472.CAN-08-2575

Tanshinone I & Breast Cancer
The role of cell adhesion molecules has been studied extensively in the process of inflammation, and these molecules are critical components of carcinogenesis and cancer metastasis. This study investigated the effect of tanshinone I derived from the traditional herbal medicine, Salvia miltiorrhiza Bunge, on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha})-stimulated endothelial cells. Furthermore, this study investigated the effect of tanshinone I on cancer growth, invasion and angiogenesis on human breast cancer cells MDA-MB-231, both in vitro and in vivo. Tanshinone I dose dependently inhibited ICAM-1 and VCAM-1 expressions in human umbilical vein endothelial cells (HUVECs) that were stimulated with TNF-{alpha} for 6 h. Pretreatment with tanshinone I significantly reduced adhesion of either monocyte U937 or MDA-MB-231 cells to HUVECs. Interestingly, the inhibitory effect of tanshinone I on monocyte and cancer cell adhesion to HUVECs was mimicked by transfection with ICAM-1 and VCAM-1 small interfering RNA. In addition, tanshinone I effectively inhibited TNF-{alpha}-induced production of vascular endothelial growth factor (VEGF) and VEGF-mediated tube formation in HUVECs. Tanshinone I also inhibited TNF-{alpha}-induced VEGF production in MDA-MB-231 cells and migration of MDA-MB-231 cells through extracellular matrix.

Additionally, reduction of tumor mass volume and decrease of metastasis incidents by tanshinone I were observed in vivo. In conclusion, this study provides a potential mechanism for the anticancer effect of tanshinone I on breast cancer cells, suggesting that tanshinone I may serve as an effective drug for the treatment of breast cancer.

Nizamutdinova, I.T., Lee, G.W., Lee, J.S., Cho, M.K., Son, K.H., Jeon, S.J., Kang, S.S., Kim, Y.S., Lee, J.H., Seo, H.G., Ki Chang, C. & Kim, H.J. Tanshinone I suppresses growth and invasion of human breast cancer cells, MDA-MB-231, through regulation of adhesion molecules. Carcinogenesis 2008 29(10):1885-1892; doi:10.1093/carcin/bgn151


Cryptotanshinone Inhibits Constitutive Signal Transducer and Activator of Transcription 3 Function through Blocking the Dimerisation in DU145 Prostate Cancer Cells


Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumours and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy.
We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL.
To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analysed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 µmol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment.

Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalised with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modelling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

Dae-Seop Shin, Hye-Nan Kim, Ki Deok Shin, Young Ju Yoon, Seung-Jun Kim, Dong Cho Han and Byoung-Mog Kwon. Cancer Research 69, 193-202, January 1, 2009. doi: 10.1158/0008-5472.CAN-08-2575

Cryptotanshinone from Salvia miltiorrhiza BUNGE has an inhibitory effect on TNF-α-induced matrix metalloproteinase-9 production and HASMC migration via down-regulated NF-κB and AP-1.

Biochemical Pharmacology
Matrix metalloproteinases (MMP-9 and MMP-2) production and smooth muscle cell (SMC) migration may play key roles in the phathogenesis of neointima formation and atherosclerosis. Especially inducible MMP-9 expression was directly involved in the cancer cell invasion and SMC migration through vascular wall. In this study, we reveal that cryptotanshinone (CT) purified from Salvia miltiorrhiza BUNGE had an inhibitory effect on MMP-9 production and migration of human aortic smooth muscle cells treated with TNF-α in a dose-dependent manner. The down regulation of transcription of MMP-9 mRNA was evidenced by RT-PCR and MMP-9 promoter assay using luciferase reporter gene. Eletrophoretic mobility shift assay showed NF-κB and AP-1 nuclear translocations were suppressed. In addition, Western blot analysis indicated that extracellular signal regulated kinase 1 and 2, p38 and JNK MAP kinase signalling pathways were inhibited. From the results, it is suggested that CT has anti-atherosclerosis and anti-neointimal formation activity.
Volume 72, Issue 12, 15 December 2006, Pages 1680-1689
Cryptotanshinone inhibits angiogenesis in vitro

Abstract
In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone from Salvia miltiorrhiza ( dan shen ) was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydro-furan ring plays a crucial role in the activity.

Introduction
Angiogenesis is a physiological process of new blood vessel formation by endothelial cells, which is critical for normal physiology such as development and wound healing (Folkman, 1971; Carmeliet, 2003). In pathological states, angiogenesis is deregulated by numerous pro-angiogenic factors leading to induce several diseases such as diabetic retinopathy, rheumatoid arthritis and spreading of cancer (Folkman, 1985; Walsh, 1999; Martin et al., 2003). In particular, angiogenesis is crucially required for blood supply and metastasis of most types of solid tumours. Accordingly, abrogation of angiogenic process and enhancement of anti-angiogenic factors have been considered as potential targets for cancer therapy (Cao, 2001; Madhusudan and Harris, 2002; Tosetti et al., 2002; Kwon, 2003).
Based on this idea, a number of angiogenesis inhibitors have been developed from various sources, including endogenous protein fragments (O'Reilly et al., 1994; 1997; Kim et al., 2003), monoclonal anti-bodies (Brekken et al., 2000) and small molecules originated from natural products and organic synthesis (Ingber et al., 1990; Shim et al., 2003). As a result of our continuing search for new anti-angiogenic agents from chemical spheres, we found crypto-cryptotanshinone from the root of Salvia miltiorrhiza Bunge (Labiatae) exhibits a potent anti-angiogenic activity. Dried roots of S. miltiorrhiza have been used in traditional Chinese medicine for the treatment of several pathologies, including disorders caused by poor blood supply such as coronary artery disease and angina pectoris, hepatitis, menstrual disorder and miscarriage (Chang and But, 1986; Zhu, 1998). As major chemical constituents, more than 25 tanshinones which impart the reddish orange pigments, have been isolated from the plant (Ryu et al., 1997; Lin and Chang, 2000; Lin et al., 2001), and a variety of biological activities, including antioxidant, antibacterial, anti-inflammatory, neuroprotective activity have been reported (Lee et al., 1999; Ng et al., 2000; Kim et al., 2002; Lam et al., 2003). However, there has been no report related with their activity on angiogenesis. In this report, the isolation and anti-angiogenic activity of cryptotanshinone, one of tanshinones from S. miltiorrhiza, are described.

Materials and Methods

Active compound isolation
Dried roots of S. miltiorrhiza (11 kg) were extracted with CH2, Cl 2 at room temperature for 7 days and the solvent was evaporated to obtain crude extract (93.11g). CH 2 Cl 2 extract (90 g) was applied to the silica gel column chromatography eluted by n-Hexane-EtOAc mixture and CH 2 Cl 2 -EtOAc as mobile phases to obtain 6 fractions (SMC1-6), based on the TLC pattern.

Cell culture and growth assay
Early passages (4-8 passages) of bovine aortic endothelial cells (BAECs) were kindly provided by Dr. Jo at KNIH. BAECs were grown in MEM supplemented with 10% fetal bovine serum (FBS, Life Technology, Grand Island , NY ). CHANG (immortalized hepatocyte derived from normal human liver), HeLa (cervical carcinoma), and HT1080 (fibrosarcoma) cells were maintained in DMEM, and HT29 (colon carcinoma) cells in RPMI1640 containing 10% FBS. Cells were grown at 37 degrees C in a humidified atmosphere of 5% CO 2. Cell growth assay was carried out using MTT colorimetric assay. Cells were inoculated at a density of 5Â-10 3 cells per well in 96-well culture plates and incubated for 24 h for stabilization. Various concentrations of compounds were added to each well and the incubation was continued for 2 days. Fifty microliter of MTT (2 mg/ml stock solution, Sigma, St. Louis , MO ) was added and the plate was incubated for an additional 4 h. After removal of medium, DMSO (100 µl) was added. The plate was read at 540 nm by universal microplate reader (Bio-Tek Instruments, Inc., Winooski , VT ).

Chemoinvasion assay
The invasiveness of BAECs was examined in vitro using a Transwell chamber system with 8.0 µm pore- sized polycarbonate filter inserts (Corning Costar, Cambridge , MA ). The lower side of the filter was coated with 10 µl of gelatin (1 mg/ml), whereas the upper side was coated with 10 µl of Matrigel (3 mg/ ml). Cells (1 -10 5cells) were placed in the upper part of the filter and compounds were added in lower parts in the presence of bFGF (30 ng/ml, Upstate Biotechnology, Lake Placid, NY). The chamber was then incubated at 37 degrees C for 18 h. The cells were fixed with methanol and stained with hematoxylin/eosin. The cell invasion was determined by counting the whole cell numbers in a lower side of filter using optical microscopy at a  -100 magnifications.

Results and Discussion
Two tanshinones were obtained as active principles from the CH 2 Cl 2 extract of root of S. miltiorrhiza based on bioactivity-guided isolation of the inhibitory activity against the proliferation of BAECs. These active compounds were identified as cryptotanshinone and tanshinone IIA, which belong to abietane diterpenes from the comparison of spectral data with published values (Kang et al., 1997).

Anti-angiogenic activity of cryptotanshinone
We, next, conducted the cell invasion and tube formation assay using the BAECs to investigate the inhibitory effects of two tanshinones on angiogenesis in vitro. Basic fibroblast growth factor (bFGF) was used as a pro-angiogenic factor stimulating the spreading and migration of endothelial cell invasion. bFGF greatly increased cell invasion through the filter coated with Matrigel than that of the control. Cryptotanshinone dose-dependently blocked the invasion of BAECs into the filter induced by bFGF. Interestingly, tanshinone IIA did not inhibit bFGF -induced invasion of BAECs at the same concentration range. Moreover, cryptotanshinone dose-dependently inhibited the tube formation of BAECs induced by bFGF, whereas tanshinone IIA did not, either. The cytotoxicity was not observed at the concentration ranges up to 20 µM of the compounds examined by trypan blue staining. These results demonstrate that Cryptotanshin, one is a new small molecule angiogenesis inhibitor and can be used as a chemical probe for studying the regulatory mechanism of angiogenesis.

The mechanism of angiogenesis inhibition by cry- ptotanshinone is currently not understood. However, the present study provides a clue for structure-activity relationship of anti-angiogenic activity of cryptotanshinone. The only structural difference between two tanshinones is double bond at C-15 position of the dihydrofuran ring. This double bond of Cryptotanshin-one may play a critical role in angiogenesis inhibition by the compound. Moreover, the anti-angiogenic activity of cryptotanshinone may be due to a specific inhibition of angiogenic differentiation of endothelial cells rather than anti-proliferative activity against the cells, because tanshinone IIA also inhibits the proliferation of the endothelial cells. We currently investigate several mechanistic studies of anti-angiogenic activity of cryptotanshinone and attempt to identify the cellular target protein of the compound. The identification of the target protein of cryptotanshinone will help to elucidate the anti-angiogenic mechanism of the compound and provide a new therapeutic target for angiogenesis-related diseases.

Acknowledgement
The National Research Laboratory Grant from the Ministry of Science and Technology, Republic of Korea and the Brain Korea supported this work.

References
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Folkman J. Tumour angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182-6
Folkman J. Tumour angiogenesis. Adv Cancer Res 1985;43:175-203
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Kang HS, Chung HY, Jung JH, Kang SS, Choi JS. Antioxidant effect of Salvia miltiorrhiza. Arch Pharm Res 1997;20:496-500
Kim KS, Hong YK , Lee Y, Shin JY, Chang SI, Chung SI, Joe YA. Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain. Exp Mol Med 2003;35:578-85
Kim SY, Moon TC, Chang HW, Son KH, Kang SS, Kim HP.Effects of tanshinone I isolated from Salvia miltiorrhiza Bunge on arachidonic acid metabolism and in vivo inflammatory responses. Phytother Res 2002;16:616-20
Kwon HJ. Chemical genomics-based target identification and validation of anti-angiogenic agents. Curr Med Chem 2003; 10:717-36
Lam BY, Lo AC, Sun X, Luo HW, Chung SK, Sucher NJ . Neuroprotective effects of tanshinones in transient focal ce-rebral ischemia in mice. Phytomedicine 2003;10:286-91
Lee DS, Lee SH, Noh JG, Hong SD. Antibacterial activities of cryptotanshinone and dihydrotanshinone I from a medical herb, Salvia miltiorrhiza Bunge. Biosci Biotech Biochem 1999;63:2236-9
Lin HC, Chang WL. Diterpenoids from Salvia miltiorrhiza. Phytochemistry 2000;53:951-3
Lin HC, Ding HY, Chang WL. Two new fatty diterpenoids from Salvia miltiorrhiza. J Nat Prod 2001;64:648-50
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Pharmacokinetic interaction studies of tanshinones with tolbutamide, a model CYP2C11 probe substrate, using liver microsomes, primary hepatocytes and in vivo in the rat.
Wang X, Lee WYW, Or PMY & Yeung JHK. Phytomedicine: International Journal of Phytotherapy & Phytopharmacology. Mar 1, 2010. doi: 10.1016/j.phymed.2009.07.013  

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on tolbutamide 4-hydroxyIation was investigated in the rat. Danshen (0.125-2 mg/ ml) decreased 4-hydroxy-toIbutamide formation in vitro and in vivo. Enzyme kinetics studies showed that inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. The [K.sub.i] values of the tanshinones were: dihydrotanshinone (8.92 [micro]M), cryptotanshinone (24.5 [micro]M), tanshinone I (80.3 [micro]M) and tanshinone IIA (242.9 ). In freshly prepared primary rat hepatocytes, tanshinones inhibited tolbutamide 4-hydroxylation in a concentration-dependent manner, with EC40 values in the order: cryptotanshinone (15.8 [micro]M), tanshinone IIA (16.2 [micro]M), dihydrotanshinone (20.1 [micro]M) and tanshinone I (48.2 [micro].M). In whole animal studies, single dose Danshen treatment (50 or 200mg/kg, i.p.) increased tolbutamide clearance (17-26.9%), decreased AUC (14.4-20.9%) and increased the Vd (7.26%). Three-day Danshen treatment (200 mg/kg/day, i.p.) decreased the [C.sub.initial], increased [T.sub.[1/2]] and Vd but did not affect tolbutamide clearance and AUC. Tolbutamide-4-hydroxylation in vivo was decreased by Danshen after acute and after 3-day treatment, with decreases in the AUC of 4-hydroxy-tolbutamide (15-28%) over the time period studied. Despite competitive inhibition of rat CYP2C11 in vitro and in vivo, as shown by the decrease in tolbutamide 4-hydroxylation, only minor changes in tolbutamide pharmacokinetics was observed. This study illustrated that the herb-drug interaction potential should be monitored by both in vitro and in vivo biotransformation/ pharmacokinetic parameters.
Introduction
Danshen, (Salvia miltiorrhiza) has been widely used in China, and also in Japan, the United States of America and other European countries for the treatment of cardiovascular and cerebrovascular diseases (Zhou et al. 2005; Wang et al. 2007). However, Danshen has been associated with a number of clinically important herb-drug interactions leading to adverse outcome (Yu et al. 1997; Izzo et al. 2005; Holbrook et al. 2005). The Danshen-warfarin interaction may be mediated via both pharmacodynamic and pharmacokinetic mechanisms as Danshen exhibited both pharmacodynamic and pharmacokinetic interactions with warfarin (Lo et al. 1992). Danshen exaggerated the pharmacological effects of warfarin by prolonging the prothrombin time, an in vitro indicator of its anti-coagulation action and increased the bioavailability and decreased the elimination of warfarin in the rat (Chan et al. 1995). Recently, it has been reported that the major tanshinones of Danshen inhibited warfarin hydroxylation and increased the steady-state plasma warfarin concentration (Wu and Yeung 2009). The recent findings were in line with those from previous studies in which Danshen altered the metabolism of R-and S-warfarin (Chan et al. 1995), reactions widely accepted as being mediated through CYP isoforms such as 1A2, 2C9 and 3A4. Warfarin (R- and S-) is metabolised via CYP3A4 to 4-hydroxy- and 10-hydroxy-warfarin; CYP1A2 to form 6-hydroxy- and 8-hydroxy-warfarin; CYP2C9 (in man)/CYP2Cll (in rats) to form 6-hydroxy-and 7-hydroxy-warfarin. Given that warfarin can be metabolised through different CYP metabolic pathways, it is not an ideal model substrate to investigate the herb-drug interaction potential of Danshen for the individual CYP isoforms. It would be more appropriate to investigate the effects of Danshen on the disposition of specific model probe substrates of CYP isoforms such as caffeine (CYP1A2), tolbutamide (CYP2C9) and testosterone (CYP3A4) to investigate the effects of Danshen or other natural products on the metabolic activities of these individual CYP isoforms.

With advances in separation techniques, the active components of Danshen have been isolated and chemically characterised (Shi et al. 2005; Zhang et al. 2005; Liu et al. 2006; Ma et al. 2006), with extensive research currently undertaken to investigate the pharmacology and therapeutic potential of the individual components of the herb (Lam et al. 2006a, b). Tanshinone IIA, one of the major lipid soluble components of Danshen, has been reported to inhibit CYP1A2 activity in mouse, human and rat in vitro (Ueng et al. 2003; He et al. 2007; Wang et al. 2009). In this study, the effects of tanshinones (tanshinone I, tanshinone IIA, cryptotan-shinone and dihydrotanshinone) and Danshen on rat CYP2C11 activity was investigated in vitro and in freshly prepared rat hepatocytes to determine the potential of Danshen in affecting CYP2C mediated phase I metabolism in the rat, using tolbutamide as the probe substrate. Rat CYP2C11 is the equivalent of human CYP2C9 with common probe substrates specificity. The effects of Danshen on the metabolism of tolbutamide to 4-hydroxy tolbutamide and tolbutamide pharmacokinetics were also investigated in vivo. The combined in vitro and in vivo protocol may form the basis of a pharmacokinetic model for general screening to investigate herb-drug interactions with rat CYP2C11/human CYP2C9 substrates.

Materials and methods
Animals
Male Sprague-Dawley rats (250-300g) were supplied by the Laboratory Animal Service Center, The Chinese University of Hong Kong (CUHK) and housed in animal holding room under standard conditions with 12-hour light-dark cycle, with free access to rodent cubes (Glen Forrest Stockfeeders, Australia) and tap water. All the experimental procedures had been approved by the Animal Experimentation Ethics Committee (CUHK) in accordance to the Department of Health (HKSAR) guidelines in Care and Use of Animals.

Materials

Danshen was supplied by Winsor Health Products Ltd. (Hong Kong) where an initial screening test has been carried out that the batch of Danshen was free from other contaminants. Cryptotan-shinone, dihydrotanshinone, tanshinone I and tanshinone IIA were purchased from Chengdu Congon Bio-tech Co., Ltd. (China). Tolbutamide, 4-hydroxy-tolbutamide, chlorpropamide, B-nicoti-namide adenine dinucleotide phosphate (NADP), D-glucose 6-phosphate, glucose 6-phosphate dehydrogenase, heparin sodium, urethane, and phenacetin were from Sigma Chemical Co. (St. Louis, MO, USA). Sulfaphenazole was obtained from UFC Limited (Manchester, UK). Acetonitrile (HPLC Grade) was purchased from Labscan Analytical Sciences (Bangkok, Thailand). Methanol (HPLC Grade) was from BDH Laboratory Supplies (Poole, UK), ethyl acetate (HPLC grade) was from Fisher Chemicals (Leicester, UK). Acetic acid, glacial, (HPLC grade) was from Scharlau Chemie (Barcelona, Spain). Phenobarbitone sodium was obtained from Universal Pharmaceutical Lab. (Hong Kong). Carbon monoxide was supplied by Hong Kong Special Gas Co.

HPLC analysis of tanshinones

Individual tanshinones including cryptotanshinone, dihydrotanshinone, tanshinone I and tanshinone IIA were separated on an Agilent Zorbax Eclipse XDS-C8 5 [micro]m (4.6 x 150 mm) with Supelco Pelliguard[TM] LC-18 guard column in a gradient mobile phase consisting of acetonitrile and water. Typical conditions for elution were as follows: 55% acetonitrile (0-16 min); 55-80% acetonitrile (16-20 min); 80% acetonitrile (20-24 min), using a flow rate of l.0 ml/min. Detection was by UV absorbance at 245 nm. Standard curves for cryptotanshinone, dihydrotanshinone, tanshinone I and tanshinone IIA were linear between 5.0 and 100 [micro]M. The calibration curves had a minimum coefficient of determination (r2) 0.99. The intra-assay coefficients of variation of dihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone IIA were 2.04,1.49,2.53 and 3.17%, respectively. The inter-assay coefficients of variation of dihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone IIA were 1.91, 1.77, 2.39 and 2.54%, respectively. The accuracy of dihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone IIA were 109.8,108,103 and 91.3%, respectively. Fig. 1 showed the structures of the major tanshinones of Salvia miltiorrhiza present in the Danshen extract used in this study and a typical HPLC chromatogram of cryptotanshinone, dihydrotanshinone, tanshinone I and tanshinone IIA with retention times in the analysis of Danshen extract, after extraction with ethyl acetate.

Preparation of rat liver microsomes

Sprague-Dawley rats (male, 250-300g) were killed by exsanguinations. The liver was excised, rinsed with ice-cold 0.9% NaCl solution, weighed and homogenised in a 0.1 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 10,000g at 4 [degrees]C for 30 min. the supernatant was then centrifuged at 105,000g at 4 [degrees]C for 60 min. The pellet was reconstituted with 0.1 mM phosphate buffer (pH 7.4), and the protein concentration of the liver microsomes was determined by a protein assay (Lowry et al. 1951).

Analysis of rat tolbutamide 4-hydroxylase (CYP2CU) enzyme activities

Cytochrome P450 content of the rat liver microsomes was quantified and activity of P450 determined as described (Schenk-man and Jansson 1998). The interaction between Danshen and tolbutamide towards tolbutamide 4-hydroxylase (CYP2C11) activity in rat liver microsomes in vitro was evaluated by adding these substances at various concentrations: Danshen (0.125-2 mg/ml) and individual tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone; 6.25-100 [micro]M), tolbutamide 5[micro]M. Control incubations for tolbutamide 4-hydroxylation contained no Danshen or tanshinones. Liver microsomes (1 mg/ml) were incubated with 0.05 M Tris/HCl buffer (pH 7.4) with NADPH-regenerating system (10 mM NADP, 5mM G6P, 2 units/ml G6PDH, and 5 mM magnesium chloride). The incubation mixtures were incubated in an Eppendorf Thermomixer at 800 r.p.m. at 37 [degrees]G The reaction was initiated by addition of the rat liver microsomes. Termination of the enzyme reaction was by addition of 500 [micro]l of ice-cold acetonitrile. The incubation tubes were centrifuged and the supernatant extracted with 500 [micro]l ethyl acetate. The organic layer was dried under a gentle stream of nitrogen, resuspended in 120 [micro]l methanol, with 50 [micro]l used in HPLC analysis. Chlorpropamide (50[micro]g/ml, 10 [micro]l) was used as the internal standard for extraction and HPLC analysis.

Inhibition kinetics studies

Danshen (0.5-2 mg/ml), tanshinone I (12.5-75 [micro]M), tanshinone IIA (12.5-100 [micro]M), cryptotanshinone (12.5-100 [micro]M) and dihydrotanshinone (25-100 [micro]M) and tolbutamide (5 [micro]M-300 [micro]M) were used for inhibition kinetics studies. Sulfaphenazole (6.25-50 [micro]M), a selective human CYP2C9 inhibitor and rat inhibitor, was used as a positive control.

Preparation of freshly prepared rat hepatocytes

Male Sprague-Dawley rats (200-250 g) were used as liver cells donors. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (Alfasan, Woerden, Holland) (100mg/kg). Liver cells were isolated by the two-step collagenase perfusion technique previously described (Seglen 1976), with modification (Raman et al. 2004). The liver was perfused with sterile [Ca.sup.2+]-free perfusion buffer (8.3 g/1 NaCl, 0.5 g/1 KC1, 2.4g/1 HEPES, pH 7.4) at 37[degrees]C, gassed with 95% 02-5% C02. After 10min, a cannula (O.D.[approximately equal to] 2.08 mm) was inserted into the hepatic portal vein to serve as the outlet. Once the liver became pale yellow in colour, it was perfused with sterile collagenase (Type IV) (0.025%) containing-digestion buffer (3.9 g/1 NaCl, 0.5 g/1 KCl, 2.4 g/1 HEPES, 0.7 g/1 [CaCl.sub.2][H.sub.2]O, pH 7.6) at 37 [degrees]C, gassed with 95% [O.sub.2]-5% [CO.sub.2] for another 20 min. The Glisson's capsule was opened in ice-cold basal culture medium (DMEM) supplemented with 1% (w/v) bovine serum albumin (BSA). The resulting cell suspensions were filtered through a stainless steel mesh and a layer of sterile gauze and finally through a 100 [micro]m filter (Millipore, Billerica, MA.). Cells pellet after centrifugation was collected for determining cell viability by trypan blue exclusive assay. Hepatocytes viability was greater than 88%.

Effects of tanshinones on the metabolism of tolbutamide in freshly prepared rat hepatocytes

The freshly isolated hepatocytes was resuspended in Dulbec-co's Modified Eagle Medium (Gibco, Aucklang, NZ) supplemented with 0.1% Bovine Serum Albumin (Sigma), 100 nM dexamathaxone (Sigma) and 1% Antibiotics & Antimycotic (Gibco). Tanshinones, Danshen and sulfaphenazole were dissolved in 0.5% DMSO. Inhibitors of different concentrations were added to 1.5 ml centrifuge tubes. Rat hepatocytes cell suspension (5 x [10.sup.6] cells) were added to each centrifuge tube and preincubated for 10 min at 37 [degrees]C. A 500 [micro]l cell suspension was then added to 1.5 ml centrifuge tube containing tolbutamide (225 [micro]M) at 37 [deegres]C. The tubes were incubated on a thermomixer at 37 [degrees]C, 900 rpm for 30 min. After the incubation, the samples were immediately frozen in liquid nitrogen and kept at -20 [degrees]C until further analysis.

HPLC analysis of tolbutamide and 4-hydroxy-tolbutamide

Analysis of tolbutamide and 4-hydroxy-tolbutamide was a modified method by Veronese et al. (1990). The HPLC system consisted of a Hewlett Packard (HP) 1050 series pumping system and a multiple wavelength detector (seat at 313 nM). 4-hydroxytolbutamide and tolbutamide were separated on a Allsphere ODS2 5 urn (4.6 x 250 mm) (Alltech). The mobile phase is a mixture 10 mM sodium acetate, pH4.3 and acetonitrile in 60:40 ratio. The flow rate is 0.6 ml/min. The absorbance is 230 nm. Standard curves of tolbutamide and 4-hydroxy tolbutamide were linear between 0.1 and 5 [micro]g/ml. Precision tests gave good reproducibility of the HPLC analysis, the relative standard deviation was 3.7% at 50 [micro]g/ml and 2.3% at 100 [micro]g/ml. The accuracy of the standard curve is 96%, 98% and 99% at 25, 50 and 100 [micro]g/ml, respectively.

Effects of Danshen treatments on the pharmacokinetics of tolbutamide

For the acute treatments, rats were treated with Danshen (50-200 mg/kg, i.p.), or saline (control). Intraperitoneal administration of Danshen was selected in this study over the oral route of administration because previous report by Kuo et al. (2006) suggested possible induction of CYP3A by tanshinone IIA. Significant gastrointestinal absorption and first pass metabolism by CYP3A in the small intestine and CYP3A-linked drug transporter proteins (P-gp) may thus influence the interpretation of the CYP2C11 data in this study. The rats were anaesthetized with urethane (20% w/v, 6 ml/kg, i.p.) and the carotid artery was cannulated for collecting blood sample, jugular vein for injection of tolbutamide and replacing saline. A single dose of tolbutamide (10 mg/kg, i.v.) was given via jugular vein 30min after the treatment with Danshen. The dosage of tolbutamide used was based on similar herb-drug interaction study carried out previously (Yeung et al. 2006). Serial blood samples (0.3 ml) were collected via the carotid artery at 0, 10, 20, 40, 60, 90, 120, 180, 240, 300 and 360 min after tolbutamide administration. Saline was replaced via jugular vein each time after a blood sample collection. Plasma was separated and stored at -20 [degrees]C prior to analysis by High Performance Liquid Chromatography (HPLC). Cimetidine (60 mg/kg, i.p.) was used as positive controls for enzyme inhibition studies.

For the 3-day treatments, rats were pre-treated with Danshen (200 mg/kg/day, i.p.) or saline (control, i.p.) for 3 days. During the pre-treatment period, the rats were kept in a 12-h light/dark cycle animal room with controlled temperature and humidity. Free access to laboratory rodent diet and tap water was allowed. One day after the final pre-treatment, the rats were anaesthetized and experiments were performed as described in previous section. Phenobarbitone (40 mg/kg/day, i.p., 3 days) was used as positive controls for enzyme induction.

Data analysis

Statistical analysis of the data was carried out using ANOVA by a computer program (Statview 9.0, Abacus Concepts, USA). Enzyme kinetics data were fitted by non-linear regression analysis using GraphPad Prism 4 (GraphPad Software, CA, USA). [IC.sub.50] values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined by GraFit where appropriate using the following equation:

V = [Vo/[1+[(1/[IC.sub.50]).sup.s]]]

where Vo is uninhibited velocity, V is observed velocity, S is slope factor and I is inhibitor concentration. A Lineweaver-Burk Plot is a double reciprocal plot in which varying substrate concentrations are plotted against reaction velocities to obtain linear transformation. The enzyme parameter Michaelis constant ([K.sub.m]) and [V.sub.max] values were obtained from Lineweaver-Burk Plot. The inhibition constant ([K.sub.i]), the inhibitor concentration at which the reaction is half of the maximal rate, was obtained by a secondary plot using the slope of the primary Lineweaver-Burk Plot and fitted by GraphPad Prism 4. Pharmacokinetic parameter calculation was by standard non-compartmental methods by the PK Solutions 2.0 (Summit Research Services, USA). [C.sub.initial] is the initial tolbutamide concentration extrapolated to time zero. AUC from time 0 to 360 min was calculated by the trapezoidal rule.

Results

Effect of tanshinones and Danshen on tolbutamide 4-hydroxylase (CYP2C11) activities in rat liver microsomes in vitro

Danshen and the tanshinones inhibited the formation of 4-hydroxy-tobultamide concentration-dependently with decreases in the 4-hydroxy-tobultamide/tolbutamide ratios. Enzyme kinetics studies of rat tolbutamide 4-hydroxylase (CYP2C11) were determined with various tolbutamide concentrations, in the presence or absence of Danshen, tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and sulfaphenazole (positive control for rat CYP2C11 inhibition). The Lineweaver-Burke linear transformation of the enzyme velocities versus substrate concentration showed that Danshen act as a competitive inhibitor. Lineweaver-Burke plots of the Danshen extract (data not shown) and the subsequent secondary plots (Fig. 2a-f) confirmed that the inhibition by Danshen and the individual tanshinones were competitive in nature. Danshen had a [K.sub.i] value of 1.65 mg/ml while the [K.sub.i] values of the tanshinones were in the order: dihydrotanshinone (8.92 [micro]M), cryptotanshinone (24.5 [micro]M), tanshinone I (80.3 [micro]M) and tanshinone II (242.9 [micro]M). The inhibitory effects of dihydrotanshinone and cryptotanshinone on tolbutamide 4-hydroxylation were comparable to sulfaphenazole, a specific human CYP2C9 and rat CYP2C11 inhibitor which showed a [K.sub.i] value of 30.8 [micro]M.

Effect of tanshinones on rat tolbutamide 4-hydroxylase (CYP2C11) activities in freshly prepared rat hepatocytes

The tanshinones inhibited tolbutamide 4-hydroxylation in a concentration-dependent manner (Fig. 3). The [EC.sub.40] values for the individual tanshinones were in the order: cryptotanshinone (15.8 [micro]M), tanshinone IIA (16.2 [micro]M), dihydrotanshinone (20.1 [micro]M) and tanshinone I (48.2 [micro]M).

Effects of acute Danshen treatment on 4-hydroxylation and pharmacokinetics of tolbutamide

Fig. 4a and b showed the plasma concentration-time profiles of tolbutamide and 4-hydroxytolbutamide, respectively, after single dose treatment with Danshen (50mg/kg, i.p.). Fig. 4c and d showed the plasma concentration-time profiles of tolbutamide and 4-hydroxytolbutamide, respectively, after single dose treatment with Danshen (200 mg/kg, i.p.). Acute treatment of Danshen did not alter the clearance, [T.sub.[1/2]], and AUC of tolbutamide (Table 1) although the [C.sub.initial] was decreased by 7.4% and the Vd increased by a similar magnitude (7.4%). These may reflect changes induced by Danshen on the absorption/distribution of tolbutamide and may or may not be related to CYP2C11 inhibition. The formation of 4-hydroxytolbutamide was decreased, indicated by decreases in the AUC of 4-hydroxytolbutamide (AUC of 4-hydroxytolbutamide with no Danshen treatment was 559.4 [+ or -] 22.1 [micro]g/ml compared to 476.4 [+ or -] 23.5 [micro]g/ml after 50mg/ kg Danshen treatment and 451.7 [+ or -] 29.0 [micro]g/ml after 200 mg/kg Danshen treatment) representing decreases by 15% (P < 0.05) and 20% (P < 0.01), respectively. The decrease in the AUC of 4-hydroxytolbutamide confirmed the in vitro observation and confirmed that the metabolism of tolbutamide to 4-hydroxy tolbutamide through CYP2C11 was inhibited by tanshinones present in Danshen.

Table 1
Pharmacokinetics of tolbutamide (10mg/kg, i.v.) after acute treatment
of Danshen (50-200 mg/kg, i.p.).

                   Control (saline)     Danshen extract, 50 mg/kg

Cinital ([mu]g/ml)   57.3 [+ or -] 0.7      55.5 [+ or -] 1.1
AUC ([mu]gh/ml)     354.7 [+ or -] 12.7    280.4 [+ or -] 6.3 **
Vd (ml/kg)          170.7 [+ or -] 2.1     178.8 [+ or -] 3.5
CL (ml/min/kg)       0.47 [+ or -] 0.02     0.59 [+ or -] 0.01 ***
T 1/2 (min)         256.8 [+ or -] 7.7     210.2 [+ or -] 3.0 **

                   Danshen extract, 200 mg/kg  Cimetidine, 60 mg/kg

Cinital ([mu]g/ml)     54.2 [+ or -] 1.1 *       56.9 [+ or -] 1.4
AUC ([mu]gh/ml)       303.7 [+ or -] 6.8 *      431.6 [+ or -] 34.6 ***
Vd (ml/kg)            183.1 [+ or -] 4.1 **     174.7 [+ or -] 3.7
CL (ml/min/kg)         0.54 [+ or -] 0.01 ***     0.40 [+ or -] 0.0 **
T 1/2 (min)           234.0 [+ or -] 7.2 *      314.4 [+ or -] 22.2 ***

Results were mean[+ or -]S.E.M. of 8 animals. *p < 0.05, **p < 0.01
and
***p < 0.001 compared to corresponding controls (saline pretreatment
or cimetidine).a

Effects of 3-day Danshen treatment on 4-hydroxylation and pharmacokinetics of tolbutamide

The plasma concentration-time profiles of tolbutamide and 4-hydroxy-tolbutamide after 3-day Danshen pretreatment were shown in Fig. 5a and b, respectively. As shown in Table 2, pretreatment of Danshen (200 mg/kg/day, i.p.) for three days did not affect the overall clearance and AUC of tolbutamide. However, the [C.sub.initial] was decreased (9.4%), together with increases in the [T.sub.[1/2]] (21.4%) and Vd (9.2%). These may reflect changes induced by Danshen on the absorption/ distribution of tolbutamide and may be unrelated to CYP2C11 inhibition. Pretreatment of Danshen also decreased the AUC of 4-hydroxytolbutamide (AUC 4-hydroxytolbutamide = 1220 [+ or -] 48 [micro]g/ml compared to 879.8 [+ or -] 86.84 [micro]g/ml after 3-day danshen treatment) by 27.88% (P <0.05). The increases in [V.sub.d] after both acute and sub-chronic Danshen pretreatment indicated plasma distribution of tolbutamide was altered. The effects of cimetidine, a P450 inhibitor, and phenobarbitone, an enzyme inducer were as expected in terms of their ability to alter tolbutamide 4-hydroxylation and caused changes in the pharmacokinetics of tolbutamide, as previously reported (Yeung et al. 2006).

Table 2
Pharmacokinetics of tolbutamide (lOmg/kg, i.v.) after treatment of
Danshen (50-200 mg/kg/day, i.p.) for three days.

                           Control (saline)       Danshen
                                                 extract, 200
                                                  mg/kg/day,
                                                   3 days

[C.sub.initial] ([mu]g/ml)   69.9 [+ or -] 2.5    63.4 [+ or -] 1.1 *
AUC (ugh/ml)                428.2 [+ or -] 17.1  472.4 [+ or -] 24.3
[V.sub.d] (ml/kg)           141.8 [+ or -] 5.6   154.8 [+ or -] 2.8 *
CL (ml/min/kg)               0.38 [+ or -] 0.02   0.35 [+ or -] 0.02
T 1/2 (min)                 255.5 [+ or -] 11.1  310.1 [+ or -] 15.9 *

                           Phenobarbitone, 40 mg/kg/day, 3 days

[C.sub.initial] ([mu]g/ml)          40.0 [+ or -] 1.4 ***
AUC (ugh/ml)                       119.5 [+ or -] 1.8 ***
[V.sub.d] (ml/kg)                  250.5 [+ or -] 8.9 ***
CL (ml/min/kg)                      1.39 [+ or -] 0.0 ***
T 1/2 (min)                        124.8 [+ or -] 5.5 ***

Results were mean[+ or -]S.E.M. of 8 animals. *p<0.05, ***p < 0.001
compared to corresponding controls (saline pretreatment or
phenobarbitone).

Discussion

CYP2C9 is one of the major cytochrome P450 enzymes involved in the metabolism of a wide range of therapeutic agents (Rettie and Jones 2005) and metabolism of fatty acids, prostanoids and steroid hormones (Kirchheiner and Brockmoller 2005). Over 100 currently used drugs are known substrates of CYP2C9, corresponding to 10-20% of commonly prescribed drugs. The rat forms of the human CYP2C9 equivalent CYP2C isoforms include CYP2C6 and 2C11 where CYP2C11 being a homologue of the human 2C9 with 77% homology. In the rat, the CYP2C isoforms are more important since they are the more abundantly expressed CYP, in a way similar to CYP3A4 in man. It is clear from the in vitro inhibition kinetic studies in rat liver microsomes that Danshen can act as a competitive inhibitor to rat CYP2C11 which is mainly responsible for tolbutamide 4-hydroxylation. Although tolbutamide hydroxylation has been shown to be increased following phenobarbital (an inducer of CYP2B in rat liver) pretreatment (Veronese et al. 1990), tolbutamide 4-hydroxylation was observed in both human and non-induced rat liver microsomes (East-erbrook et al. 2001).

In this study, the [K.sub.i] values of cryptotanshinone and dihydro-tanshinone in inhibiting tolbutamide 4-hydroxylation in rat liver microsomes was in the same range to that of sulfaphenazole, a specific human CYP2C9/rat CYP2C11 inhibitor. It should be noted, however, that sulfaphenazole was much more effective in inhibiting tolbutamide metabolism in human than rat liver microsomes (Eagling et al. 1998). The effects of the tanshinones on the 4-hydroxy-tolbutamide/toIbutamide ratio showed that dihydrotanshinone and cryptotanshinone were more effective in altering the CYP2C11-mediated metabolic reaction, followed by tanshinone I and lastly, tanshinone IIA. Recent reports by Li et al (2006) and Sun et al (2007) have shown that tanshinones isolated from Danshen were metabolised mainly to hydroxy metabolites although the exact CYPs involved have not been elucidated. The changes in 4-hydroxylation of tolbutamide observed in this study may alternatively related to competition for the CYPs involved.

The effects of Danshen have also been carried out in vivo in this study, to investigate potential enzyme inducing effects on CYPs as well as to determine any pharmacokinetic changes after Danshen pretreatment. The dosages used in this study were in line with other studies previously reported (Lo et al. 1992; Kuo et al 2006; Song et al. 2007). In the pharmacokinetic studies, acute Danshen (50 or 200 mg/kg, i.p.) treatment increased tolbutamide clearance by 17-26.9%, but produced a decrease in the AUC (14.4-20.9%) and an increase in the Vd (7.26%) when a high dose was used. However, 3-day Danshen treatment (200 mg/kg/day) produced more pronounced changes in the pharmacokinetics of tolbutamide and 4-hydroxylation of tolbutamide. Although the formation of 4-hydroxytolbutamide was decreased, as indicated by the decreases in the AUC of 4-hydroxytolbutamide, the major pharmacokinetic parameter to be affected was the apparent volume of distribution (Vd) which suggested that Danshen, in addition to inhibiting P450-mediated reactions, may also affect the distribution of tolbutamide. The changes in the Vd thus led to an overall increase in plasma half-life of tolbutamide but with no effect on the clearance. This study thus confirmed that Danshen has no inducing effects on rat CYP2C11, although Danshen extracts has been suggested to exert an inducing effect of CYP1A2 in the mouse (Kuo et al. 2006). The amount of Danshen used in this study was similar in magnitude to studies carried out by other research groups, on mg/kg basis, in the laboratory animals and as such, the difference in the findings should not be due to difference in the dosage used. However, this study illustrated the importance of performing both in vitro and in vivo studies as well as combined pharmacodynamic-pharmacokinetic (PD-PK) to identify potential important drug-drug/herb-drug interactions. In vitro inhibition studies with enzymes have its limitation and must be supplemented with more thorough in vivo investigations.

Despite the inhibition of rat CYP2C11 activity by Danshen extract containing the major tanshinone in vitro and in vivo, the overall in vivo changes to tolbutamide was less that expected. Taken together the results from previous studies with Danshenwarfarin (Lo et al 1992; Wu and Yeung 2009), the in vitro and in vivo studies with rat CYP1A2 (Wang et al. 2009) and the current study with tolbutamide, it is clear that simple in vitro screening for potential CYP inhibitors may not reflect the in vivo effects in investigations for potential herb-drug interactions. In this case, the risk of Danshen-CYP2C11 effect would have been overestimated if the in vitro data was considered alone. Careful monitoring would still be essential for the concomitant use of Danshen with other drugs, particularly those metabolized by CYP1A2, CYP2C9 and CYP3A4. This study illustrated that the herb-drug interaction potential should be monitored by both in vitro and in vivo biotransformation/pharmacokinetic parameters. However, the significance of such metabolic interaction should be confirmed with in vivo studies, preferably combined PD-PK studies.



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Cryptotanshinione upregulates alpha-secretase by activation PI3K pathway in cortical neurons.
Mei Z, Situ B, Tan X, Zheng S, Zhang F, Yan P, Liu P. Brain Res. 2010 Aug 12;1348:165-73. Epub 2010 Jun 2.

The amyloid precursor protein (APP) is cleaved enzymatically by nonamyloidogenic and amyloidogenic pathways. alpha-Secretase cleaves APP within beta amyloid protein (Abeta) sequence, resulting in the release of a secreted fragment of APP (sAPPalpha) and precluding Abeta generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). We have shown previously that CTS modulated APP metabolism by elevation alpha-secretase activity. However, the molecular mechanisms involved were unclear. Here we reported that CTS increased alpha-secretase activity and sAPPalpha release. To gain insight into the molecular mechanism whereby CTS modulates alpha-secretase, we evaluated the involvement of three candidate alpha-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in CTS-induced non-amyloidogenic APP metabolism. Results showed that CTS treatment of cortical neurons overexpressing Swedish mutant human APP695 markedly elevated ADAM10 protein, and the inhibitor of ADAM10 inhibited the CTS-induced increase in alpha-secretase activity, suggesting CTS modulated alpha-secretase activity by upregulation ADAM10 protein. By using several specific protein kinase inhibitors, we showed that phosphatidylinositol 3-kinase (PI3K) mediated the CTS-induced alpha-secretase activation.

Cancer Lett. 2012 Mar;316(1):11-22. Epub 2011 Oct 10.
Cryptotanshinone suppresses androgen receptor-mediated growth in androgen dependent and castration resistant prostate cancer cells.
Xu D, Lin TH, Li S, Da J, Wen XQ, Ding J, Chang C, Yeh S.
Source
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Abstract
Androgen receptor (AR) is the major therapeutic target for the treatment of prostate cancer (PCa). Anti-androgens to reduce or prevent androgens binding to AR are widely used to suppress AR-mediated PCa growth; however, the androgen depletion therapy is only effective for a short period of time. Here we found a natural product/Chinese herbal medicine cryptotanshinone (CTS), with a structure similar to dihydrotestosterone (DHT), can effectively inhibit the DHT-induced AR transactivation and prostate cancer cell growth. Our results indicated that 0.5 μM CTS effectively suppresses the growth of AR-positive PCa cells, but has little effect on AR negative PC-3 cells and non-malignant prostate epithelial cells. Furthermore, our data indicated that CTS could modulate AR transactivation and suppress the DHT-mediated AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive PCa LNCaP cells and castration resistant CWR22rv1 cells. Importantly, CTS selectively inhibits AR without repressing the activities of other nuclear receptors, including ERα, GR, and PR. The mechanistic studies indicate that CTS functions as an AR inhibitor to suppress androgen/AR-mediated cell growth and PSA expression by blocking AR dimerization and the AR-coregulator complex formation. Furthermore, we showed that CTS effectively inhibits CWR22Rv1 cell growth and expressions of AR target genes in the xenograft animal model. The previously un-described mechanisms of CTS may explain how CTS inhibits the growth of PCa cells and help us to establish new therapeutic concepts for the treatment of PCa.




 
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