Protective effect of Salvia miltiorrhiza extract against renal ischemia-reperfusion-induced injury in rats

The present study investigates the effect of pre-treatment with Salvia miltiorrhiza ethanol extracts (SMEE) on renal function markers, immunity and antioxidant activities in renal ischemia and reperfusion (IR) rats. Wistar rat kidneys were subjected to 60 min of global ischemia at 37 °C followed by 30 min of reperfusion, and were randomly assigned into the sham, IR model and three SMEE-treated groups (n = 8 per group). Results showed that high serum creatinin (Scr), blood urea nitrogen (BUN), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) and malondialhehyde (MDA) levels, and low antioxidant enzyme activities were observed in IR rats compared to the sham rats. Pre-treatment of Salvia miltiorrhiza ethanol extracts for 20 days prior to IR operation improved renal function, reduced IR induced renal inflammatory and oxidative injury. It is concluded that Salvia miltiorrhiza ethanol extracts could be beneficial in the treatment of renal ischemic injury.     

Effects of Lycium barbarum aqueous and ethanol extracts on high-fat-diet induced oxidative stress in rat liver tissue

This study evaluated the protective effects of aqueous extract of Lycium barbarum (LBAE) and ethanol extract of Lycium barbarum (LBEE) on blood lipid levels, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities and liver tissue antioxidant enzyme activities in rats fed a high fat diet (HF). The rats were randomly divided into seven groups of ten rats each and fed a different diet for eight weeks as follows: One group (NC group) was fed a standard diet, one group was fed a high-fat diet (HF group), one group was fed a high-fat diet and orally fed with 20 mg/kg b.w. simvastatin (HF + simvastatin group), and the other group was fed the high fat diet and orally fed with 50 mg/kg b.w. or 100 mg/kg b.w. LBAE (HF + LBAE), or 50 mg/kg b.w. or 100 mg/kg b.w. LBEE (HF + LBEE), respectively. After eight weeks, the HF diet caused deleterious metabolic effects. Rats fed the HF diet alone showed increased hepatocellular enzyme activities in plasma, a significant decline in antioxidant enzyme activities, and elevated liver lipid peroxidation indices. LBAE and LBEE administration significantly reduced liver damage and oxidative changes, and brought back the antioxidants and lipids towards normal levels. These data suggest that these antioxidants protect against toxicity parameters in HF rats.     

Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of six diterpenoids. dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude diterpenoids were obtained by extraction with ethanol-n-hexane (1:1, v/v) from S. miltiorrhiza Bunge. Preparative HSCCC with the two-phase solvent systems A composed of n-hexane-ethanol-water (10:5.5:4.5, v/v) and B composed of n-hexane-ethanol-water (10:7:3, v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The purities of dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B were 88.1, 98.8, 97.6, 93.5, 96.8 and 94.3%, respectively.     

Effect of Ginkgo biloba extract 50 on immunity and antioxidant enzyme activities in ischemia reperfusion rats

The aim of the study was to investigate the effect of Ginkgo biloba extract 50 (GBE50), a well-known natural antioxidant, against immunity and antioxidant enzyme activities in ischemia reperfusion (IR) rats. Rats were then divided into six groups fed for 15 days with the same diet: three groups (IV, V, VI) were treated by different doses of GBE50 suspension [20, 40, or 60 mg/kg body weight by oral gavage every day at a fixed time (10.00 a.m.)] (equal to 5, 10 and 20 times, respectively, the maximum recommended human dose), and three groups (I, II, III) were untreated. At the end of the experiment, rats' hearts were subjected to 30 min of ischemia followed by 90 min of reperfusion. Results showed that IR significantly enhanced heart rate, S-T height, myocardium (myeloperoxidase) MPO activity and blood interleukin-8 (IL-8), tumor necrosis factor Alpha (TNF-a), interleukin-1β (IL-1β) levels, blood aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine kinase (CK) activities, reduced myocardium sodium-potassium adenosine triphosphatase (Na(+)-K(+)-ATPase), calcium-magnesium adenosine triphosphatase (Ca(2+)-Mg(2+)-ATPase) activities and antioxidant enzyme activities in IR group (III) compared to sham control group (II). Pretreatment of GBE50 markedly significantly reduced heart rate, S-T height, myocardium MPO activity and blood IL-8, TNF-a, IL-1β levels, blood AST, LDH, and CK activities, enhanced myocardium Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase activities and antioxidant enzyme activities in IR group (II) compared to IR group (III). The results suggested that the GBE50 may reduce the oxidative stress in the reperfused myocardium, and increased immunity and antioxidant activities in IR rats.     

Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.     

Evaluation of pressurized liquid extraction and pressurized hot water extraction for tanshinone I and IIA in Salvia miltiorrhiza using LC and LC-ESI-MS

Pressurized liquid extraction (PLE) and pressurized hot water extraction (PHWE) using a laboratory-made system are applied for the extraction of thermally labile components such as tanshinone I and IIA in Salvia miltiorrhiza. PLE and PHWE are carried out dynamically at a flow of 1 mL/min, temperature between 95-140 degrees C, applied pressure of 10-20 bars, and extraction times of 20 and 40 min, respectively. Effects of ethanol added into the water used in PHWE are explored. PLE is found to give comparable or higher extraction efficiencies compared with PHWE with reference to Soxhlet extraction for tanshinone I and IIA in Salvia miltiorrhiza. The tanshinone I and IIA present in the various medicinal plant extracts are determined by liquid chromatography and liquid chromatography-mass spectrometry.     

Simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study of a standardized fraction of Salvia miltiorrhiza, PF2401-SF

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone, the active components of Salvia miltiorrhiza in rat plasma, was developed. After liquid-liquid extraction with tariquidar as an internal standard, tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone were eluted from an Atlantis dC18 column within 5 min with a mixture of methanol and ammonium formate (10 mm, pH 6.5; 85:15, v/v). The analytes were detected by an electrospray ionization tandem mass spectrometry in the selected reaction monitoring (SRM) mode. The standard curves were linear (r=0.999) over the concentration range of 0.25-80 ng/mL for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma. The coefficients of variation and the relative errors of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone for intra- and inter-assay at four quality control (QC) concentrations were 1.1-5.1% and -4.0-6.0%, respectively. The lower limit of quantification for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone was 0.25 ng/mL from 100 microL of plasma. This method was successfully applied to the pharmacokinetic study of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone after oral administration of PF2401-SF, the standardized fraction of Salvia miltiorrhiza enriched with tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone to male Sprague-Dawley rats.     

The effect of Lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats

The aim of this study was to investigate the effects of Lycium barbarum polysaccharide (LBP) on alcohol-induced liver damage in rats. A total of 36 rats were divided into control, ethanol and ethanol + LBP groups. Rats in the ethanol group were fed 7 g ethanol/kg body weight by gastric infusion, three times a day, for 30 consecutive days, while rats in the control group received the same volume of physiological saline instead of ethanol, and rats in ethanol + LBP group were fed both ethanol (7 g/kg body weight) and LBP (300 mg/kg body weight/day). Alcoholic liver injury was examined by serum ALT and AST activities, alcoholic fatty liver was assessed by lipid levels, and oxidative stress was evaluated by SOD, CAT, GSH-Px, GSH and MDA assays. In the ethanol group, a significant elevation of enzymes and lipid in serum, increased MDA level and depletion of SOD, CAT, GSH-Px and GSH in liver were observed. LBP administration significantly ameliorated liver injury, prevented the progression of alcohol-induced fatty liver, and improved the antioxidant functions when compared with the ethanol group. Histopathological examination of rat liver revealed that LBP administration protected liver cells from the damage induced by ethanol. The results suggest that LBP is a promising agent to protect the liver from hepatotoxicity and fatty liver induced by ethanol intake.     

Preparative isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

High-speed counter-current chromatography was applied to the isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude salvianolic acid B was obtained by extraction with ethanol-water from S. miltiorrhiza Bunge. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (3:7:1:9, v/v) was successfully performed yielding 342 mg salvianolic acid B at 98% purity from 500 mg of the crude extract in a one-step separation.     

Diterpenoid tanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhiza Bunge and their antimicrobial activities

Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7). These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.     

Modeling gradient elution in countercurrent chromatography: efficient separation of tanshinones from Salvia miltiorrhiza Bunge

Countercurrent chromatography (CCC) is a support-free liquid-liquid chromatography using centrifugal fields to hold the liquid stationary phase. CCC has been widely applied in the separation of various natural and synthetic components using a variety of biphasic liquid systems. The related hexane or heptane/ethyl acetate/methanol or ethanol/water biphasic liquid systems demonstrated their significance in CCC. Gradient is difficult in CCC since any composition change in one phase induces a composition change of the other phase to maintain phase equilibrium. This work provides a new insight into linear gradient elution in CCC that is feasible with some biphasic liquid systems such as selected compositions of the hexane/ethyl acetate/ethanol/water systems. The equations modeling solute motion inside the CCC column are proposed. Particular compositions of the liquid system, namely the hexane/ethyl acetate/ethanol/water 8:2:E:W compositions with E + W = 10, were studied from W = 1 to 9. They showed moderate changes in the upper organic phase compositions. The model is tested with the separation of tanshinones from the rhizome of Salvia miltiorrhiza Bunge. Different linear solvent gradient profiles were experimentally performed between 8:2:5:5 and 8:2:3:7 compositions and the results were evaluated using the proposed model. Five tanshinones including dihydrotanshinone I, cryptotanshinone, tanshinone I, 1,2-dihydrotanshinquinone, and tanshinone IIA have been successfully separated (>95% purities) using a gradient profile optimized by the developed model. The gradient model can be used only with biphasic liquid systems in which one phase shows minimum composition changes when the other phase composition changes notably. This case is not the general case for biphasic liquid systems but can be applied with specific compositions of the quaternary hexane or heptane/ethyl acetate/methanol or ethanol/water most useful CCC liquid systems.     

Microwave-assisted extraction of tanshinones from Salvia miltiorrhiza bunge with analysis by high-performance liquid chromatography

A novel microwave-assisted extraction (MAE) method has been developed for the extraction and determination of tanshinones (tanshinone IIA, cryptotanshinone and tanshinone I) from the root of Salvia miltiorrhiza bunge with analysis by HPLC. Various experimental conditions were investigated to optimize the percentage extraction. Under appropriate MAE conditions, such as ethanol concentrations of 95% (v/v), MAE for 2 min, liquid/solid ratio of 10:1 (ml/g), the percentage extraction can reach high in a short time. The percentage extraction (tanshinone IIA: 0.29%; cryptotanshinone: 0.23%; tanshinone I: 0.11%) by MAE was the same or even higher than conventional extraction methods. MAE only needs 2 min, but extraction at room temperature, heat reflux extraction, ultrasonic extraction and Soxhlet extraction need 24 h, 45 min, 75 min and 90 min, respectively. MAE was also available in pilot plant form for larger scale extraction.     

Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC-UV method

A sensitive and specific HPLC-UV method was developed for the simultaneous determination of major active components of danshen in rat plasma. Both water-soluble and lipid-soluble compounds were included, i.e. danshensu, salvianolic acid B and tanshinone IIA. Protocatechuic aldehyde and diazepam were used as internal standards. The chromatographic separation was achieved on a reversed-phase C(18) column by gradient elution using acetonitrile and 0.025% (v/v) phosphoric acid solution as mobile phase, at a flow rate of 1.0 mL/min. Salvianolic acid B, danshensu and internal standards were detected at 281 nm, while the detection of tanshinone IIA was carried out at 272 nm. All calibration curves showed good linearity (r(2) > 0.999) within test ranges. The limit of detection and the limit of quantification for danshensu, salvianolic acid B and tanshinone IIA in plasma were 0.065, 0.043, 0.022, 0.131, 0.085 and 0.044 microg/mL, respectively. This is the first report on the determination and pharmacokinetic study of danshensu, salvianolic acid B and tanshinone IIA in rat plasma and the results indicated that this method was reliable for the determination of the major active components of danshen in rat plasma.     

Separation of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of tanshinones from the roots of Salvia miltiorrhiza Bunge by stepwise elution. A set of three solvent systems and other experimental conditions were determined by analytical HSCCC. Using the optimized conditions, the preparative HSCCC separation was performed on 50 mg of crude light petroleum extract yielding pure tanshinones of tanshinone HA (7 mg), tanshinone I (3 mg) and cryptotanshinone (4 mg) all at purities of over 95% in a single run.     

One-step purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde from Salvia miltiorrhiza Bunge by isocratic stepwise hydrogen bond adsorption chromatography on cross-linked 12% agarose

Three major active components of the traditional Chinese medicinal herb Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde, are separated and purified from a crude water extract in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose 12 HR 10/30). Separation is achieved by stepwise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0-50 mL, 5% ethanol, 5% acetic acid; 50-100 mL, 20% ethanol, 20% acetic acid; and 100-200 mL, 30% ethanol, 30% acetic acid. The 3,4-dihydroxyphenyllactic acid is obtained with a purity of 97.3% and with a recovery of 88.1%. The corresponding figures for protocatechualdehyde are a purity of 99.4% with a recovery of 90.7%, and for salvianolic acid B a purity of 90.4% with a recovery of 50.3%, respectively. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.6 mg/mL gel), a 3,4-dihydroxyphenyllactic acid purity of approximately 94% with a recovery of 80.2% is obtained.     

Ipomoea aquatica extract shows protective action against thioacetamide-induced hepatotoxicity

In the Indian system of traditional medicine (Ayurveda) it is recommended to consume Ipomoea aquatica to mitigate disorders like jaundice. In this study, the protective effects of ethanol extract of I. aquatica against liver damage were evaluated in thioacetamide (TAA)-induced chronic hepatotoxicity in rats. There was no sign of toxicity in the acute toxicity study, in which Sprague-Dawley (SD) rats were orally fed with I. aquatica (250 and 500 mg/kg) for two months along with administration of TAA (i.p injection 200 mg/kg three times a week for two months). The results showed that the treatment of I. aquatica significantly lowered the TAA-induced serum levels of hepatic enzyme markers (ALP, ALT, AST, protein, albumin, bilirubin and prothrombin time). The hepatic content of activities and expressions SOD and CAT that were reduced by TAA were brought back to control levels by the plant extract supplement. Meanwhile, the rise in MDA level in the TAA receiving groups also were significantly reduced by I. aquatica treatment. Histopathology of hepatic tissues by H&E and Masson trichrome stains displayed that I. aquatica has reduced the incidence of liver lesions, including hepatic cells cloudy swelling, infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by TAA in rats. Therefore, the results of this study show that the protective effect of I. aquatica in TAA-induced liver damage might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects. Moreover, it confirms a scientific basis for the traditional use of I. aquatica for the treatment of liver disorders.     

Effects of Pithecellobium jiringa ethanol extract against ethanol-induced gastric mucosal injuries in Sprague-Dawley rats

Current anti-gastric ulcer agents have side effects, despite the progression and expansion of advances in treatment. This study aimed to investigate the gastroprotective mechanisms of Pithecellobium jiringa ethanol extract against ethanol-induced gastric mucosal ulcers in rats. For this purpose, Sprague Dawley rats were randomly divided into five groups: Group 1 (normal control) rats were orally administered with vehicle (carboxymethyl cellulose), Group 2 (ulcer control) rats were also orally administered with vehicle. Group 3 (positive control) rats were orally administered with 20 mg/kg omeprazole, Groups 4 and 5 (experimental groups) received ethanol extract of Pithecellobium jiringa ethanol extract at a concentration of 250 and 500 mg/kg, respectively. Sixty minutes later, vehicle was given orally to the normal control group, and absolute ethanol was given orally to the ulcer control, positive control and experimental groups to generate gastric mucosal injury. The rats were sacrificed an hour later. The effect of oral administration of plant extract on ethanol-induced gastric mucosal injury was studied grossly and histology. The level of lipid peroxidation (malondialdehyde-MDA), superoxide dismutase (SOD) and gastric wall mucus were measured from gastric mucosal homogenate. The ulcer control group exhibited severe gastric mucosal injury, and this finding was also confirmed by histology of gastric mucosa which showed severe damage to the gastric mucosa with edema and leucocyte infiltration of the submucosal layer. Pre-treatment with plant extract significantly reduced the formation of ethanol-induced gastric lesions, and gastric wall mucus was significantly preserved. The study also indicated a significant increase in SOD activity in gastric mucosal homogenate, whereas a significant decrease in MDA was observed. Acute toxicity tests did not show any signs of toxicity and mortality up to 5 g/kg. The ulcer protective effect of this plant may possibly be due to its preservation of gastric wall mucus along with increased SOD activity and reduction of oxidative stress (MDA). The extract is non-toxic, even at relatively high concentrations.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

Profiling the metabolic difference of seven tanshinones using high-performance liquid chromatography/multi-stage mass spectrometry with data-dependent acquisition

Tanshinones are a class of bioactive constituents in the roots of Salvia miltiorrhiza named Dan-Shen in Chinese, which possess diverse pharmacological activities. In this study, we employed a sensitive high-performance liquid chromatography/multi-stage mass spectrometry (HPLC/MS(n)) method with data-dependent acquisition and a dynamic exclusion program for the identification of phase I metabolites of seven tanshinones in rat bile after intravenous administration. These seven tanshinones are tanshinone IIA, sodium tanshinone IIA sulfonate (abbreviated as STS, a water-soluble derivate of tanshinone IIA), cryptotanshinone, 15,16-dihydrotanshinone I, tanshinone IIB, przewaquinone A and tanshinone I. Altogether 33 metabolites underwent monohydroxylation, dihydroxylation, dehydrogenation, D-ring hydrolysis or oxidation reactions in the C-4 or C-15 side chain which were characterized by analyzing the LC/MS(n) data. Different metabolic reactions for tanshinones were dependent on the degree of saturation and the substituent group in the skeleton. Dehydrogenation was the major metabolic modification for cryptotanshinone with saturated A and D rings. 15,16-Dihydrotanshinone I containing a saturated D ring was mainly metabolized through D-ring hydrolysis. For tanshinone IIA, possessing a saturated A ring, hydroxylation was the major metabolic pathway. When there was hydroxyl group substitution in the C-17 or C-18 position, such as przewaquinone A and tanshinone IIB, or sulfonic group substitution in the C-16 position, such as STS, higher metabolic stability than that of tanshinone IIA was shown and only trace metabolites were generated. Oxidation in the C-4 or C-15 side chain was a characteristic reaction for tanshinone IIA and hydroxylated tanshinone IIA. For tanshinone I, bearing unsaturated A and D rings simultaneously, no metabolites were detected.