mTOR

Salidroside inhibits the growth of various human cancer cell lines in concentration- and time-dependent manners, and the sensitivity to salidroside is different in those cancer cell lines. Salidroside has been shown to inhibit CDK4, cyclin D1, cyclin B1, and Cdc2, while upregulating the levels of p27(Kip1) and p21(Cip1) and causing G1- or G2-phase arrest in various cancer cell lines. Salidroside also reduces the loss of cell viability and apoptotic cell death brought on by H(2)O(2) stimulation in cultured PC12 cells that have undergone NGF differentiation by activating the ERK1/2 pathway and inhibiting caspase-3 activation. [2] Salidroside induces PI3K/Akt pathway activation, which protects PC12 cells from MPP(+)-induced apoptosis and may be used to treat Parkinson's disease (PD).[3]

---

Salidroside (p-hydroxyphenethyl-beta-d-glucoside), which is present in all species of the genus Rhodiola, has been reported to have a broad spectrum of pharmacological properties. The present study, for the first time, focused on evaluating the effects of the purified salidroside on the proliferation of various human cancer cell lines derived from different tissues, and further investigating its possible molecular mechanisms. Cell viability assay and [(3)H] thymidine incorporation were used to evaluate the cytotoxic effects of salidroside on cancer cell lines, and flow cytometry analyzed the change of cell cycle distribution induced by salidroside. Western immunoblotting further studied the expression changes of cyclins (cyclin D1 and cyclin B1), cyclin-dependent kinases (CDK4 and Cdc2), and cyclin-dependent kinase inhibitors (p21(Cip1) and p27(Kip1)). The results showed that salidroside inhibited the growth of various human cancer cell lines in concentration- and time-dependent manners, and the sensitivity to salidroside was different in those cancer cell lines. Salidroside could cause G1-phase or G2-phase arrest in different cancer cell lines, meanwhile, salidroside resulted in a decrease of CDK4, cyclin D1, cyclin B1 and Cdc2, and upregulated the levels of p27(Kip1) and p21(Cip1). Taken together, salidroside could inhibit the growth of cancer cells by modulating CDK4-cyclin D1 pathway for G1-phase arrest and/or modulating the Cdc2-cyclin B1 pathway for G2-phase arrest. [1]

---

Salidroside is isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has a potent antioxidant property. The aim of this study was to investigate the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced cell apoptosis in nerve growth factor (NGF)-differentiated PC12 cells and the possible involvement of the extracellular signal-related protein kinase 1/2 (ERK1/2) signaling pathway. MTT assay, Hoechst 33342 staining, and TdT-mediated dUTP-biotin nick end labeling assay collectively showed that pretreatment with salidroside alleviated, in a dose-dependent manner, cell viability loss and apoptotic cell death induced by H(2)O(2) stimulation in cultured NGF-differentiated PC12 cells. According to Western blot analysis, pretreatment with salidroside transiently caused the activation of ERK1/2 pathway; a selective inhibitor of the mitogen-activated protein kinase kinase (MAPKK, MEK) blocked salidroside-activated ERK pathway and thus attenuated the influences of salidroside on H(2)O(2)-induced increase in the level of cleaved caspase-3, a chief executant of apoptosis cascades. Morphological analysis further indicated that in the presence of the MEK inhibitor, the neuroprotective effect of salidroside against H(2)O(2)-evoked cell apoptosis was significantly abrogated. Taken together, the results suggest that the neuroprotective effects of salidroside might be modulated by ERK signaling pathway, especially at the level or upstream of the caspase-3 activation. [2]

---

Oxidative stress plays an important role in the pathogenesis of Parkinson's disease (PD). Salidroside (SAL), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can exert potent antioxidant properties. In this study, we investigated the protective effects, and the possible mechanism of action, of SAL against 1-methyl-4-phenylpyridinium (MPP(+))-induced cell damage in rat adrenal pheochromocytoma PC12 cells. Pretreatment of PC12 cells with SAL significantly reduced the ability of MPP(+) to induce apoptosis in a dose and time-dependent manner. SAL significantly and dose-dependently inhibited MPP(+)-induced chromatin condensation and MPP(+)-induced release of lactate dehydrogenase by PC12 cells. SAL enhanced Akt phosphorylation in PC12 cells, and the protective effects of SAL against MPP(+)-induced apoptosis were abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. These findings suggest that SAL prevents MPP(+)-induced apoptosis in PC12 cells, at least in part through activation of the PI3K/Akt pathway. [3]

Salidroside (20, 50, and 100 mg/kg) guards against oxidative stress caused by D-galactosamine and lipopolysaccharide in the liver tissue. Salidroside attenuated the induced acute increase in serum aspartate aminotransferase and alanine aminotransferase activities, and levels of tumour necrosis factor-alpha levels and serum nitric oxide. It restored depleted hepatic glutathione, superoxide dismutase, catalase and glutathione peroxidase activities, decreased malondialdehyde levels and considerably reduced histopathological changes. Histopathological, immunohistochemical and Western blot analyses also demonstrated that salidroside could reduce the appearance of necrotic regions and expression of caspase-3 and hypoxia-inducible factor-1alpha in liver tissue. Conclusions: Salidroside protected liver tissue from the oxidative stress elicited by D-galactosamine and lipopolysaccharide. The hepatoprotective mechanism of salidroside appear to be related to antioxidant activity and inhibition of hypoxia-inducible factor-1alpha.[4]

---

Antioxidation effect of Salidroside [4]
Mice treated with D-galactosamine/LPS alone (Table 1) showed significant decreases in liver levels of enzymatic (SOD, CAT, GSH-Px) and non-enzymatic antioxidants (GSH) compared with the normal group. There was no difference between the normal group and Salidroside 100 group. The antioxidant defence system was protected by pretreatment with salidroside or NAC. The activities of enzymatic antioxidants in the mice pretreated with 20 mg/kg salidroside increased significantly compared with the D-galactosamine/LPS group, whereas changes in GSH were not significant. MDA produces oxygen free radicals through enzymatic antioxidant and non-enzymatic antioxidants. Liver MDA content, an end-product of lipid peroxidation, was increased in the D-galactosamine/LPS group compared with the normal group. The MDA levels were significantly suppressed in the groups pretreated with 50 or 100 mg/kg salidroside or NAC compared with the D-galactosamine/LPS group, but not in mice pretreated with 20 mg/kg salidroside (Table 1).

---

Effect of Salidroside on TNF-α production [4]
TNF-α is a critical mediator of liver injury induced by D-galactosamine/LPS. We postulated that salidroside protected against D-galactosamine/LPS-induced liver injury through inhibiting elevation of TNF-α levels. Two hours after D-galactosamine/LPS administration, the level of TNF-α was markedly induced compared with the normal group (764.72 ± 89.73 vs 21.25 ± 5.14 pg/ml; P < 0.001). Levels in the salidroside 100 group were 25.73 ± 4.16 pg/ml. Salidroside-pretreated mice showed significant and dose-dependent lower TNF-α levels than D-galactosamine/LPS-treated mice: 327.35 ± 58.36, 143.75 ± 27.48 and 84.36 ± 18.04 pg/ml in the groups treated with 20, 50 and 100 mg/kg salidroside, respectively, and 69.71 ± 13.89 pg/ml in the mice pretreated with NAC (P < 0.001 vs D-galactosamine/LPS group; Figure 2b).

---

Effect of Salidroside on the production of NO, and expression of tNOS and iNOS [4]
NO production was investigated by measuring nitrite accumulation in serum. Six hours after treatment with D-galactosamine/LPS, nitrite concentration in serum reached 59.31 ± 12.15 μmol/ml, which was significantly higher than that of the normal group (20.43 ± 5.74 μmol/ml, P < 0.001). Administration of salidroside or NAC 1 h before D-galactosamine/LPS injection produced a significant dose-dependent inhibition of NO. The NO contents were 50.39 ± 11.35, 47.45 ± 8.63 and 30.10 ± 4.45 μmol/ml at Salidroside doses of 20, 50 and 100 mg/kg, and 23.24 ± 2.70 μmol/ml with NAC. NO production with 50 and 100 mg/kg salidroside and NAC were significantly lower than in the D-galactosamine/LPS group. To investigate whether the inhibition of NO production is due to the reduction of iNOS and tNOS activities, we assessed the effect of salidroside on expression of these enzymes. Results were similar for tNOS and iNOS with salidroside 50 and 100 mg/kg and NAC. Levels of tNOS were 30.08 ± 6.55 and 26.26 ± 5.66 U/ml with salidroside 50 and 100 mg/kg, respectively, and 24.19 ± 4.13 U/ml with NAC. Levels of iNOS were 18.46 ± 3.86, 18.02 ± 5.82 and 16.57 ± 4.87 U/ml, respectively. The expressions of NO, tNOS and iNOS in the salidroside 100 group were 21.47 ± 4.35, 19.51 ± 7.26 and 16.16 ± 2.96 U/ml respectively, where were not significantly difference from the normal group (Figure 3).

---

Effect of Salidroside on histopathology, immunohistochemistry and Western blotting [4]
All mice treated with D-galactosamine/LPS showed histopathological signs of hepatotoxicity. Liver sections from these mice showed severe confluent and focal necrosis, apoptosis, inflammatory cell infiltrate around the central zone, periportal vacuolation and haemorrhage (Figure 4a). Many apoptotic bodies and nuclei with condensed chromatin appeared in these mice. The normal group did not show any abnormal changes in liver architecture (Figure 4b). The degree of tissue damage was obviously less in the mice pretreated with salidroside 20 and 50 mg/kg than in the D-galactosamine/LPS group: only central necrosis, cell infiltration and periportal vacuolation were observed, and apoptotic cell death was significantly reduced (Figures 4c and d). Prereatment with salidroside 100 mg/kg reduced degeneration: regular morphology of the liver parenchyma with well-defined hepatic cells and sinusoids was observed (Figure 4e). Mice treated with NAC also showed well-preserved hepatocytes and tissue architecture, with less necrosis and inflammatory cell infiltration (Figure 4f). [4]

---

Immunohistochemistry staining of caspase-3 was used to depict apoptosis degree in liver sections taken 6 h after treatment with D-galactosamine/LPS (Figure 5). Positive caspase-3 showed pale brown or brown in the endochylema after DAB coloration and was clearly identified. The D-galactosamine/LPS group presented large numbers of chocolate-brown areas (Figure 5a), with fewer in the normal group (Figure 5b). In livers from mice treated with Salidroside (20 and 50 mg/kg), the positive staining appeared as mild centrilobular staining, the positive cell population decreased and staining intensity gradually decreased with higher doses of salidroside −50 mg/kg of salidroside showed less positive staining than 20 mg/kg (Figure 5c and d). There was virtually no positive staining in the mice pretreated with 100 mg/kg Salidroside, similar to that with NAC (Figure 5e and f). Similar results were seen with Western blotting: degradation of 32 kDa procaspase-3 which generated the 17 kDa active fragment was observed. It is clear that most of the caspase-3 pro-form changed into active form in mice treated with D-galactosamine/LPS and there was less change in the salidroside-pretreated groups, and this change showed dose dependence. Salidroside administration resulted in further downregulation of activated caspase-3 levels induced by D-galactosamine/LPS.

SH-SY5Y cells are seeded in 96-well plates at 1×104 cells per well. After the treatment with Salidroside (25-100 μM) and MPP+, cell viability is measured by MTT assay. Briefly, cells are incubated with 500 μg/mL MTT at 37°C for 4 h. The medium is then taken out, 150 L of DMSO is added, and shaking is carried out for 10 minutes. In a microplate reader, absorbance is measured at a wavelength of 570 nm, and the results are presented as folds of control.

---

Cell viability assay [1]
Cells were seeded into sterile 96-well plates at a density of 1 × 104 cells/well and allowed to adhere. Then cells were treated with the appropriate salidroside solution in the concentration range of 0-32 µg/ml for 48 h. For time-course studies, cells were treated with salidroside (2 µg/ml) for 12, 24, 48, and 72 h. The cell proliferation Kit I (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; MTT) was used to measure cell viability. Briefly, 10 μl of the labeling solution was added into each well of the 96-well plates. After 2 h incubation in the CO2 incubator, 100 μl of the solubilization solution was added to dissolve purple crystals, which are the products of MTT substrates. The absorbance was measured at 570 nm on a plate reader. Absorbance measured in MTT assays was expressed as percentage of the control (defined as 100%).

---

[3H] thymidine incorporation [1]
Cell suspensions were seeded in 24-well plates (1 × 105 cells/well). Then cells were incubated with increasing concentrations of salidroside for 48 h. [3H]-thymidine (1 μCi/ml) next was added for 6 h at 37°C. Then cells were harvested by trypsinization, washed with cold PBS and centrifuged for 10 min at 1,500 rpm/min several times until the dpm in the washes were similar to the reagent control. Radioactivity was determined by liquid scintillation counting. The results were expressed according to maximal incorporation obtained for the untreated group. Each value was corrected for the quantity of proteins in each well. The data were expressed as percentage of the control (defined as 100%).

---

Cell cycle analysis [1]
Cells were seeded at 3 × 105 /well in 6-well plates and incubated overnight to allow cells to attach to the plate. After cells were treated with salidroside (2 and 4 µg/ml) for 6, 12, and 24 h, trypsin was used to release the cells from attaching to the plates. Cells then were harvested in cold PBS, fixed in 70% ethanol, and stored at 4°C for subsequent cell cycle analysis. Fixed cells were incubated with propidium iodide (PI), and RNAse A. The distributions of cells in the cell cycle were measured by flow cytometry.

---

Cell Treatment [2]
The cell culture was pretreated with normal medium containing different concentrations (0.5, 2, 8, 32, 128 μM) of salidroside, or containing 50 μM of D-α-tocopherol succinate (vitamin E, from Sigma) for 24 h. The cells were then exposed to the 1.0 mM H2O2 solution, which had been prepared by diluting the commercial available H2O2 with normal medium, for additional 90 min. We designated the cells as a control that underwent neither pretreatment with salidroside or vitamin E nor H2O2 stimulation. In separate experiments, 10 μM of PD98059, the MEK inhibitor, and 128 μM salidroside were sequentially added to the cell culture for pretreatment, followed by H2O2 stimulation as described above.

Mice: The 4-week-old male C57BL/6 mice are fed either a regular chow diet (n=8) or a high-fat diet (HFD) (n=16). salidroside intervention (100 mg/kg/day) is started by gavage once a day for five weeks after the HFD has been administered for ten weeks. Vehicle (saline) is administered to the control groups. Male C57Bl/KsJ (BKS) mice that are 4 weeks old and BKS.Cg-Dock7m +/+ Leprdb/J (db/db) mice that are 16 weeks old are used. For five weeks, salidroside is administered orally by gavage at a dose of 100 mg/kg/day. Vehicle (saline) is administered to the control groups. Every five days, mice's body weight and fasting blood glucose levels are checked. Using a Glucometer, glucose readings are taken from blood drawn from the tail vein.

---

C57BL/6 mice were fasted overnight (16–18 h) prior to administration of a single intraperitoneal dose of D-galactosamine (700 mg/kg) and LPS (10 μg/kg) dissolved in sterile phosphate-buffered saline (pH 7.4). Mice were randomly assigned to six experimental groups of 10 mice. The normal group was given sterile saline only. The salidroside100 group was given salidroside (100 mg/kg) only. The remaining four groups were all given D-galactosamine/LPS to induce liver failure and were treated with salidroside 20, 50 or 100 mg/kg or NAC (300 mg/kg). Dosages were determined from our previous work. Mice were injected with D-galactosamine/LPS 1 h after salidroside or NAC. Blood was collected 2 h after D-galactosamine/LPS injection for measurement of TNF-α levels, and after 6 h for measurement of serum ALT, AST and NOS activities and NO levels. Blood samples were allowed to coagulate at 4 °C for 30 min. Serum was then separated by centrifugation at 2000g at 4 °C. Mice were killed 6 h after D-galactosamine/LPS injection and the same liver lobe excised from each animal. Tissue samples were immersed in neutral buffered formaldehyde for histopathological and immunohistochemical examinations. The remainder was kept at −80 °C for subsequent analysis of MDA level and GSH, SOD, CAT and GSH-Px activities.[4]

mouse LD50 subcutaneous 28600 uL/kg Zhongcaoyao. Chinese Traditional and Herbal Medicine., 19(229), 1988

[1]. Cell Biol Toxicol. 2010 Dec;26(6):499-507.

[2]. J Mol Neurosci. 2010 Mar;40(3):321-31.

[3]. Food Chem Toxicol. 2012 Aug;50(8):2591-7.

[4]. J Pharm Pharmacol. 2009 Oct;61(10):1375-82.

Salidroside is a glycoside.
Salidroside has been reported in Hypericum erectum, Fraxinus formosana, and other organisms with data available.
See also: Sedum roseum root (part of); Rhodiola crenulata root (part of).

---

In summary, salidroside-caused growth inhibitions in several human cancer cells are associated with the induction of G1-phase and/or G2-phase arrest. Mechanistic investigations suggest that G1-phase arrest appear to be mediated via decrease in CDK4 and cyclin D1. Similarly, G2-phase arrest via decrease in Cdc2 and cyclin B1. The increased levels of p21Cip1 and p27Kip1 and this effect could be one of the mechanisms to inhibit the kinase activity of G1-phase and G2-phase. Since cell apoptosis is one of the consequences of cell cycle arrest, next, we will continue this part of our research work.[1]

---

To conclude, this study indicates that salidroside protects cultured NGF-differentiated PC12 cells from H2O2-evoked cell apoptosis by upregulating the antioxidant defense system. The neuroprotective effects of salidroside shown in this study might be associated with modulation of ERK signaling pathway.[2]

---

► MPP+-induced apoptosis in PC12 cells is a classic cellular model of PD. ► We investigated the effects of salidroside in PC12 cells. ► Salidroside reduced MPP+-induced apoptosis, chromatin condensation and LDH release. ► The cytoprotective effect of salidroside is associated with PI3K/Akt activation. ► Salidroside have therapeutic potential for preventing the development of PD.[3]

---

The aim was to investigate the protective effect of salidroside isolated from Rhodiola sachalinensis A. Bor. (Crassulaceae) on D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure. Methods: Hepatotoxicity was induced by an intraperitoneal injection of D-galactosamine (700 mg/kg) and lipopolysaccharide (10 mug/kg); salidroside (20, 50 and 100 mg/kg) was administered intraperitoneally 1 h before induction of hepatoxicity. Liver injury was assessed biochemically and histologically. Our research indicates that salidroside prevents oxidative stress induced by D-galactosamine/LPS and simultaneously attenuates HIF-1α expression in liver tissue. It is clear that D-galactosamine/LPS may increase HIF-1α via oxidative stress, and regulation of NO and TNF-α activity would affect hepatic injury induced by D-galactosamine/LPS. Further studies are needed to clarify these mechanisms. [4]

C14H20O7

300.3

300.12

10338-51-9

159278

White to off-white solid

1.5±0.1 g/cm3

549.5±50.0 °C at 760 mmHg

286.2±30.1 °C

0.0±1.6 mmHg at 25°C

1.629

-1.23

5

7

5

21

306

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H]

ILRCGYURZSFMEG-RKQHYHRCSA-N

InChI=1S/C14H20O7/c15-7-10-11(17)12(18)13(19)14(21-10)20-6-5-8-1-3-9(16)4-2-8/h1-4,10-19H,5-7H2/t10-,11-,12+,13-,14-/m1/s1

(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[2-(4-hydroxyphenyl)ethoxy]oxane-3,4,5-triol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 100 mg/mL (333.00 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)