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Separation and determination of active components in Radix Salviae miltiorrhizae and its medicinal preparations by nonaqueous capillary electrophoresis.

A nonaqueous capillary electrophoresis (NACE) method was developed for simultaneous assay of three bioactive components (1: cryptotanshinone; 2: tanshinone IIA, and 3: tanshinone I) in Radix Salviae miltiorrhizae and in its herbal preparations for the first time. After optimization of separation conditions, a buffer of 250 mmol L(-1) ammonium acetate containing 30% acetonitrile and 1.0% acetic acid (V:V) in methanol was selected for separating the three analytes, but baseline separation of tanshinon I and tanshinone IIA was not obtained. Therefore second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.9943-0.9991) between peak heights in second-order derivative electropherograms and concentrations of the three analytes. The relative standard deviations (RSD) of the migration times and the peak height of the three constituents were in the range of 0.81 -0.88% and 0.34-1.13% (intra-day), 1.57-1.86% and 3.05-5.52% (inter-day), respectively. The recoveries of three constituents ranged from 90.2 to 108.5%. The results indicated that baseline separation of the analytes was sometimes hard to obtain and second-order derivative electropherograms were applicable for the resolving and analysis of overlapping peaks.

Acetic Acid↗

Complete 1H and 13C NMR assignments of clerodane diterpenoids of Salvia splendens.

Unambiguous and complete assignments of 1H and 13C NMR chemical shifts for five clerodane diterpenes, four of them isolated from Salvia splendens (salviarin, splendidin and splenolides A and B) and one obtained by acetylation of splenolide A, are presented. The assignments are based on 2D shift-correlated [1H,1H-COSY, 1H,13C-gHSQC-1J(C,H) and 1H,13C-gHMBC-nJ(C,H) (n=2 and 3)] and nuclear Overhauser effect (NOE) experiments. The conformation of the rings of these compounds is supported by the 3J(H,H) values and NOE results.

Acetylation↗

A direct liquid, non-equilibrium solid-phase micro-extraction application for analysing chemical variation of single peltate trichomes on leaves of Salvia officinalis.

A non-equilibrium solid phase micro-extraction application was tested for the rapid extraction of essential oil from single oil glands of sage and the oil components compared with those determined by solvent extract and distillation. Oil glands were directly ruptured with a polydimethylsiloxane-coated fused silica fibre and the essential oil was sorbed. Three insertion levels of an individual plant of Salvia officinalis involving an immature apical young leaf, an expanding and a fully developed leaf, respectively, were used to determine the applicability of this method. Thirty-eight components in the oil could be identified by GC-MS. The method only showed small semi-quantitative differences compared with conventional methods. Chemical variation of single oil glands within the immature and premature leaf was higher than within the homogeneous mature leaf. The intermediary, still-expanding leaf was used to carry out a detailed study of the glands. The basal region of the intermediary leaf contained compounds in high conformity with the young leaf trichomes. The remaining oil glands of this leaf showed inconsistent accumulation patterns.

Chromatography, High Pressure Liquid↗

Antinociceptive, antiinflammatory and acute toxicity effects of Salvia leriifolia Benth seed extract in mice and rats.

The antinociceptive and antiinflammatory effects as well as the acute toxicity of Salvia leriifolia aqueous seed extract were studied in mice and rats. Antinociceptive activity was assessed using the hot-plate and tail flick tests. The effect on acute inflammation was studied using vascular permeability increased by acetic acid and xylene-induced ear oedema in mice. The activity against chronic inflammation was assessed using the cotton pellet test in rats. The LD(50) of the extract was found to be 19.5 g/kg (i.p.) in mice. The aqueous seed extract showed significant and dose-dependent (1.25-10 g/kg) antinociceptive activity over 7 h, and was inhibited by naloxone pretreatment. Significant and dose-dependent (2.5-10 g/kg) activity was observed against acute inflammation induced by acetic acid and in the xylene ear oedema test. In the chronic inflammation test the extract (2.5-5 g/kg) showed significant and dose-dependent antiinflammatory activity. The aqueous seed extract of S. leriifolia may therefore have supraspinal antinociceptive effects which may be mediated by opioid receptors, and showed considerable effects against acute and chronic inflammation.

Acetic Acid↗

Inhibition of angiotensin converting enzyme by lithospermic acid B isolated from Radix Salviae miltiorrhiza Bunge.

The roots of Salviae miltiorrhiza (RSM) have been traditionally used for treatment of hypertensive disease in China, Korea, and Japan. Bioassay guided fractionation and purification as assayed by angiotensin converting enzyme (ACE) inhibitory assay resulted in the isolation of lithospermic acid B (LSB) as an active principle. The ACE plasma activities were significantly inhibited by the addition of LSB in a dose-dependent manner of which IC50 value was 86 microg/ml (120 microM). Moreover, angiotensin I-induced contraction was markedly attenuated by prior exposure of endothelium-intact aortic rings to LSB. These results suggest that RSM-induced antihypertensive effect may be, at least in part, due to ACE inhibitory effect of LSB.

Angiotensin-Converting Enzyme Inhibitors↗

Protonophoric and uncoupling activity of royleanones from Salvia officinalis and euvimals from Eucalyptus viminalis.

The mechanism of action of quinones from the roots of Salvia officinalis L. (royleanones) and terpenoid phenolaldehydes from the leaves of Eucalyptus viminalis Labill. (euvimals) was studied. Royleanones and euvimals displayed marked protonophoric activity on artificial bilayer lipid membranes in vitro, and exerted an uncoupling action on oxidative phosphorylation in isolated rat liver mitochondria. The results suggest that biological membranes are the primary targets of royleanones and euvimals, and the protonophoric activity may contribute to the cytotoxicity and antimicrobial properties of these compounds.

Abietanes↗

Butyryl- and acetyl-cholinesterase inhibitory activities in essential oils of Salvia species and their constituents.

Extracts of Salvia (sage) species have been reported to have cholinergic activities relevant to the treatment of Alzheimer's disease. A lack of information on the inhibition of the enzyme butyrylcholinesterase, also considered to be a target in the treatment of the disease, prompted this in vitro investigation of the essential oils of S. fruticosa, S. lavandulaefolia, S. of ficinalis and S. of ficinalis var. purpurea for anti-butyrylcholinesterase activity. Dose-dependent inhibition of human cholinesterases by the extracts and constituents was determined using the method of Ellman. A time dependent increase in the inhibition of butyrylcholinesterase by the oils of S. fruticosa and S. of ficinalis var. purpurea was evident. IC(50) values decreased from 0.15 +/- 0.007 and 0.14 +/- 0.007 mg/mL after 5 min to 0.035 +/- 0.016 and 0.06 +/- 0.018 mg/mL after 90 min incubation time respectively. The slow onset of inhibition of butyrylcholinesterase was also shown by individual constituents, such as 3-carene and beta-pinene. Analyses of the chemical composition of the oils and anti-butyrylcholinesterase activity of their constituents revealed that none of the compounds tested would account for the total activity of the oils and that synergy is likely.

Acetylcholinesterase↗

Salviae miltiorrhizae radix increases dopamine release of rat and pheochromocytoma PC12 cells.

The Radix of Salvia miltiorrhiza Bunge (Labiatae) (SMR), an eminent herb, is often included as an ingredient in various herbal remedies recommended for vascular circulation therapies. The present study investigated the effect of SMR on dopaminergic neurotransmission. Various extracts prepared from the stems of SMR were tested for cytotoxic activity on pheochromocytoma PC12 cells using the XTT assay method. The ethanol extract (IC50 > 100 microg/mL), water extract (IC50 > 100 microg/mL) and chloroform (IC50 = 90 microg/mL) fraction exhibited weak cytotoxic activity. However, the butanol (IC50 = 80 microg/mL) and ethyl acetate (EtOAc; IC50 = 70 microg/mL) fractions exhibited strong cytotoxic activity. Also, the extracts and fractions were investigated for dopamine release effects. The EtOAc fraction showed a stronger stimulatory effect on dopamine release activity than the other fractions. The effect of the crude EtOAc fraction (50 microg/mL) of SMR on K+ (20 mm)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10(-4) m) using high-performance liquid chromatography with electrochemical detection to measure endogenous DA. The EtOAc fraction significantly increased K+ -stimulated DA release (p < 0.001) from rat striatal slices when compared with K+ -stimulated alone. The EtOAc fraction potentiated the effect of amphetamine on K+ -stimulated DA release (p < 0.001) when compared with amphetamine alone. To examine whether in vitro the EtOAc fraction treatment induces DA release in PC12 cells, the role of protein kinases was investigated in the induction of the EtOAc fraction-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). The PKC inhibitors chelerythrine (50 nm and 100 nm) and Ro31-8220 (100 nm) and the MAP kinase kinase inhibitor, PD98059 (20 microm), inhibited the ability of the EtOAc fraction of SMR to elicit the EtOAc fraction-stimulated DA release. The PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nm) mimicked the ability of the EtOAc fraction of SMR to elicit DA release. In contrast, a selective PKA inhibitor, 50 microm Rp-8-Br-cAMP, blocked the development of EtOAc fraction-stimulated DA release. It was demonstrated that the EtOAc fraction of SMR stimulated DA release. Therefore the mechanism by which the EtOAc fraction of SMR induced the enhancement in EtOAc fraction-stimulated DA release is apparent.

8-Bromo Cyclic Adenosine Monophosphate↗

Effects of tanshinone I isolated from Salvia miltiorrhiza bunge on arachidonic acid metabolism and in vivo inflammatory responses.

Arachidonic acid (AA) mainly released from the cell membrane by phospholipase A(2) (PLA(2)) is converted to eicosanoids by the action of cyclooxygenase (COX) and lipoxygenase (LO). In order to find the specific inhibitors of AA metabolism especially PLA(2) and COX-2, 300 plant extracts were evaluated for their inhibitory activity on PGD(2) production from cytokine-induced mouse bone marrow-derived mast cells in vitro. From this screening procedure, the methanol extract of Salvia miltiorrhiza was found to inhibit PGD(2) production and the ethyl acetate subfraction gave the strongest inhibition of five subfractions tested. From this ethyl acetate subfraction, an activity-guided isolation finally gave tanshinone I as an active principle. This investigation deals with the effects of tanshinone I on AA metabolism from lipopolysaccharide (LPS)-induced RAW 264.7 cells and in vivo antiinflammatory activity. Tanshinone I inhibited PGE(2) formation from LPS-induced RAW macrophages (IC(50) = 38 microM). However, this compound did not affect COX-2 activity or COX-2 expression. Tanshinone I was found to be an inhibitor of type IIA human recombinant sPLA(2)(IC(50) = 11 microM) and rabbit recombinant cPLA(2) (IC(50) = 82 microM). In addition, tanshinone I showed in vivo antiinflammatory activity in rat carrageenan-induced paw oedema and adjuvant-induced arthritis.

Abietanes↗

Quantification of the plant-derived hallucinogen Salvinorin A in conventional and non-conventional biological fluids by gas chromatography/mass spectrometry after Salvia divinorum smoking.

A gas chromatography method with mass spectrometric detection is described for the determination of Salvinorin A, the main active ingredient of the hallucinogenic mint Salvia divinorum. The method was validated in plasma, urine, saliva and sweat using 17-alpha-methyltestosterone as internal standard. The analytes were extracted from biological matrices with chloroform/isopropanol (9:1, v/v). Chromatography was performed on a 5% phenyl methyl silicone capillary column and analytes were determined in the selected ion monitoring mode. The method was validated over the concentration range 0.015-5 microg/mL plasma, urine and saliva and 0.01-5 microg/patch in the case of sweat. Mean recoveries ranged between 77.1 and 92.7% for Salvinorin A in different biological matrices, with precision and accuracy always better than 15%. The method was applied to the analysis of urine, saliva and sweat from two consumers after smoking 75 mg plant leaves to verify the presence of the active ingredient of S. divinorum in human biological fluids as a biomarker of plant consumption. Salvinorin A was detected in urine (2.4 and 10.9 ng/mL) and saliva (11.1 and 25.0 ng/mL), but not in sweat patches from consumers.

Body Fluids↗

Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry.

The phenolic components from Radix Salvia miltiorrhizae Bunge, a well-known herbal medicine (Dan-Shen in Chinese), have been investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the ESI mass spectra a predominant [M-H]- ion was observed in negative mode and provided molecular mass information. ESI-MS/MS spectra of the [M-H]- ions were used for structural analysis, based on the spectra of standards. It was found that caffeic acid and its monomeric analogs containing a carboxyl group readily lost CO2, while dimers, trimers and tetramers of caffeic acid expelled successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in S. miltiorrhizae were characterized, of which eight compounds were positively identified by comparison with standards. The remaining twenty phenolics for which standards were not available were tentatively identified based on their UV spectra and MS/MS fragmentation characteristics.

Chromatography, High Pressure Liquid↗

Characterization of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen) by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry.

The qualitative analysis of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen in Chinese) was performed using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (ESI-MS(n)). Tanshinones are the major bioactive constituents of Dan-shen, which is used in China for the treatment of haematological abnormalities and cardiovascular diseases. The ESI-MS(n) fragmentation behavior of tanshinones was investigated. For tanshinones with the tanshinone I nucleus, the fragmentation was triggered by loss of a molecule of CO except bearing a substituent at C17 or C18, followed by sequential eliminations of CO. If C(15-16) was a saturated bond, the fragmentation was triggered by elimination of a molecule of H2O. For tanshinones with the tanshinone IIA nucleus, the fragmentation was triggered by loss of a molecule of H2O, followed by successive eliminations of CO. Ions corresponding to loss of a molecule of propylene (Delta m = 42) were also observed. Moreover, when C(15-16) was a saturated bond, ions corresponding to losses of CH3, H2O and propylene were more abundant. If no D-ring existed, the presence of isopropyl resulted in an elimination of a molecule of H2O with an adjacent CO or OH. In addition, the extension of the pi-conjugation in the A-ring (especially at C(1-2)) induced the fragmentation by loss of a molecule of CO. These fragmentation rules were applied to the identification of tanshinones in a chloroform/methanol (3:7) extract of Dan-shen, which was separated on a C18 column with gradient elution. A total of 27 tanshinones were identified, including five new constituents. The established method could be used for the sensitive and rapid identification of tanshinones in the Dan-shen drug and its pharmaceutical preparations.

Abietanes↗

Salvia miltiorrhiza inhibits biliary obstruction-induced hepatocyte apoptosis by cytoplasmic sequestration of p53.

Cholestatic liver injury is caused by hepatocellular apoptosis induced by toxic bile salts. We have studied the effects of a traditional Chinese herbal medicine, Salvia miltiorrhiza, on apoptotic cell death in bile duct-ligated (BDL) rats. We also attempted to clarify the molecular mechanisms of the hepatoprotective effects of S. miltiorrhiza in this animal model. A water extract of S. miltiorrhiza (Sm-X; 200 mg/kg; po) was administered to BDL rats for 10 days. Rats were euthanized and apoptosis was detected in liver tissue by TUNEL staining. Western blot analysis and immunostaining for alpha-smooth muscle actin (alpha-SMA), Bax, Bcl-2, and p53 were performed. Results show that the treatment of BDL rats with Sm-X significantly improved the liver function parameters, although the expression of alpha-SMA, a marker of hepatic stellate cell activation, was not affected. Treatment with Sm-X significantly reduced the number of apoptotic cells. A time-dependent decrease in Bax protein level and an increase in Bcl-2 protein level were observed in BDL rats treated with Sm-X. Immunohistochemical analysis demonstrated that p53 was strongly positive in hepatocyte nuclei of BDL rats but that treatment with Sm-X induced a cytoplasmic sequestration of p53. Taken together, hepatoprotective effects of Sm-X partially ascribe to the antiapoptotic property in hepatocytes. Treatment of Sm-X-induced cytoplasmic sequestration of p53, downregulation of Bax, and upregulation of Bcl-2 protein. This study identifies and delineates signaling factors involved in the antiapoptotic properties of Sm-X and suggests a potential application of S. miltiorrhiza in the clinical management of hepatic disease induced by toxic bile salts following biliary obstruction.

Actins↗

Experimental study on the preventive mechanism of salviae miltiorrhizae against atherosclerosis in rabbits models.

The preventive mechanism of salviae miltiorrhizae (SM) against experimental atherosclerosis (AS) in rabbits models was investigated. The experimental AS rabbit models were reproduced by feeding the high cholesterol diet. The changes of atherosclerotic plaques in normal group, model group and SM treated group were observed. The levels of serum TG, TC, HDL-C and LDL-C were determined. The immunohistochemistry was used to detect the expression of Bcl-2, Bax and IL-6 proteins in atherosclerotic plaques. The results showed that the level of serum TG in SM treated group was significantly lower than in model group (P<0.01). Immunohistochemistry revealed that the expression of Bcl-2, Bax and TL-6 in model group was significantly higher than in normal group. In the SM group, the expression of Bcl-2 protein was up-regulated and that of Bax was down-regulated. It was suggested that SM could inhibit formation of AS in experimental rabbits. To decrease the expression of Bax and increase the expression of Bcl-2 protein may be one of the mechanisms of SM against atherosclerosis.

Animals↗

Study on effects of extracts from Salvia Miltiorrhiza and Curcuma Longa in inhibiting phosphorylated extracellular signal regulated kinase expression in rat's hepatic stellate cells.

OBJECTIVE: To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it. METHODS: Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4, 5-dimthyl-2-2thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of alpha smooth actin (alpha-SMA), collagen type I, and ERK were determined by Western blot. RESULTS: SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level of alpha-SMA (P < 0.01). Curcumin significantly reduced the expression of collagen type I (P < 0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group (P < 0.01 and P < 0.05 respectively). CONCLUSION: SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type I collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.

Animals↗

The inhibitory effect of radix Salviae miltiorrhizae on hypoxic structural remodeling of intra-acinar pulmonary arteries.

The inhibitory effect of Radix Salviae Miltiorrhizae (RSM) on hypoxic structural remodeling of intra-acinar pulmonary arteries (IAPA) was observed by light and electron microscopy and morphometry. It was found that RSM can not only dilate IAPA and relieve the hypoxic injuries to endothelia cells, but also inhibit the active muscularization of IAPA in the hypoxic animals, suggesting that RSM plays a very important role in inhibiting structural remodeling of IAPA and pulmonary hypertension.

Animals↗

New butyrylcholinesterase inhibitory triterpenes from Salvia santolinifolia.

Slavins A (1) and B (2), the new amyrin type triterpenes, have been isolated from the chloroform soluble fraction of Salvia santolinifolia and assigned structures on the basis of spectral studies including 2D NMR. Both the compounds displayed inhibitory potential against the enzyme butyrylcholinesterase.

Butyrylcholinesterase↗

Inhibition of osteoclast differentiation by tanshinones from the root of Salvia miltiorrhiza bunge.

We screened natural products to find compounds with anti-osteoporotic potential using a coculture-based system by which osteoclast differentiation is effectively achieved. We found that methylene chloride soluble fraction of the root of Salvia miltiorrhiza Bunge (Labiatae) suppressed osteoclast differentiation. Five tanshinones, tanshinone IIA (1), tanshinone 1 (2), cryptotanshinone (3), 15,16-dihydrotanshinone I (4), and ferruginol (5) were subsequently isolated from fraction. Among the five compounds, compounds 1-4 reduced the formation of TRAP-positive multinuclear osteoclasts. These results suggest that the identified tanshinones may be useful candidates for development of therapeutic agents to treat osteoporosis and other bone-resorptive diseases.

Abietanes↗