Pharmacological Activities and Progress in Structure Modification of Salidroside

Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal (TCM) plant, and has displayed a broad spectrum of pharmacological properties, such as resisting anoxia, antiradiation and antifatigue. In addition, it has low toxicity as traditional Chinese medicinal plant. However, the bioavailability of salidroside is low because of its instability for hydrolase. Lots of researchers modified the structure of salidroside to enhance its bioactivity. This paper summarizes the structure modification and activities study of salidroside in recent ten years, and preliminary discuss the structure-activity relationship of salidroside analogues, which hope to provide new ideas of modification in future.


Introduction
Salidroside is the active ingredient isolated from tuber and root tuber of Rhodiola rosea L., a traditional Chinese medicinal (TCM) plant. In Shennong Classic of Materia Medica, the ancient people regarded R. rosea as the best supplement which can tonify the kidney, promote blood circulation to stop bleeding and clear lung to stop coughing. The Compendium of Materia Medica recorded R. rosea as the rare tonic and had been used for the treatment of chest congestion, nausea, weakness, etc. Modern pharmacological studies have demonstrated that salidroside from R. rosea is the main bioactive compound with many biological activities, such as resisting anoxia, anti-radiation, antifatigue, improving oxygen lack, anti-tumor, preventing cardiovascular and postponing aging [1].
Due to obvious inhibitory activities of salidroside on many kinds of tumor cells, the mechanisms of salidroside were summarized according to the corresponding tumor cell lines.
After being compatible with cyclophosphamide (CTX) at small dose (10 mg/kg), the inhibitory rates of salidroside for H22 solid tumor in different doses were 35.2%, 48.0%, 55.9%, which were better than the inhibitory rates of CTX (34.1%). In addition, the spleen and thymus weights of each dose group were obviously increased. This suggests that salidroside has strong anti-hepatoma effect in vivo and in vitro, and when it has been compatible with CTX, it can increase the antihepatoma effect of CTX with decreasing toxicity.
Study on the related mechanism: 1. Salidroside has immunopotentiation effect (improving the mice spleen index and thymus index; improving the activation capacity for T lymphocytes and NK cells; improving secretion level of IL-2, IL-12 in tumor-bearing mice's serum [13]). 2. Salidroside can inhibit DNA Topo II activity and present a certain dose-effect relationship [12].
SW1116 cells: Sun found that high concentrations of salidroside (10, 20, 50 μg/ml) can significantly inhibited proliferation of SW1116 cells in a parallelly [16]. Through western blot they found that salidroside decreased proteins expression of phosphorylation levels in JAK2/STAT3 signaling, while MMP-2 and MMP-9 proteins levels were decreased and protein expression of VEGF and VEGFR-2 were downregulated.
Human Skin Melanoma A375 cells: Yang found that saldroside can inhibit invasion and metastasis of human skin melanoma A375 cells with the dose-effect dependence [13]. The inhibition rate reached 74.2% when the concentration of salisroside is 100 μg/ml. This suggested that the mechanism may be related to salidroside's inhibiting adhesion for tumor cells to the base membrane. content of sodium, calcium ions. 4. Scavenge oxygen free radicals and inhibit lipid peroxidation.
Hepatoprotective effect: Salidroside can attenuate the induced acute increase in serum aspartate aminotransferase and alanine aminotransferase activities, and lessen the levels of tumour necrosis factoralpha and serum nitric oxide. Wu found that salidroside could reduce the appearance of necrotic regions and expression of caspase-3 and hypoxia-inducible factor-1a in liver tissue. And salidroside perhaps worked as a precursor of GSH and free-radical scavenger to reduce cellular GSH depletion and balance ROS to ameliorate peroxide and superoxide activities [23].
Nerve cell protection: Aβ (β-amyloid) stimulates the activation of microglia cells, NADPH (nicotinamide adenine dinucleotide phosphate), iNOS (inducible Nitric Oxide Synthase), NF-κB, and releases the TNF, an inflammatory factor which can directly or indirectly cause nervous system damage and nerve cells dysfunction.
Zhang used AD mice models induced by Aβ1-40 to examine the intervention effect for AD of salidroside [24]. The study found that salidroside may inhibit the NF-κB-iNOS-NO pathway. Salidroside can inhibit the expression of NF-κB and reduce the expression of iNOS to suppress the generation of NO, which can finally alleviate neuron cell damage induced by oxidative stress. At the same time, salidroside can significantly reduce the amount of NO in cells and reduce excessive expression of iNOS and nNOS, thus it can inhibit the NO pathway and protect nerve cells.
The structural modification for salidroside and biological activities Salidroside have broad application prospects and high medicinal values. However, the basic structure of salidroside is the oxygen glycoside, which is easily hydrolyzed by enzyme in vivo. This makes the bioavailability of salidroside low and cannot reach the effective blood drug concentration. In addition, on the base of pharmacokinetic research [28], salidroside only distributes over liver, kidney and heart which have the adequate blood supply. Due to its low fat soluble, salidroside cannot pass the blood brain barrier.
In order to enhance the bioavailability and bioactivity of salidroside, a lot of researchers have modified its structure. The structural modification is mainly focused on two aspects: the structural modification of tyrosol and glucoside.
Comparing the IC 50 values of FRAP, DPPH and structures of the synthesized compounds, we found that the adjacent hydroxyl on benzene ring ( Figure 2) can enhance anti-radiation activity. It enhances the stability of oxygen free radicals through hydrogen bonding interaction.
To sum up, salidroside can inhibit the growth of various tumor cells. When it is compatible with the chemotherapy drugs, it can increase their anti-tumor effects and decrease toxicities. Such characteristics mainly associated with salidroside's good improvement of the immune system function.

Antioxidant activity
Promote the biosynthesis of mitochondria: Mitochondria is the main place to produce ROS. When excessive ROS produced, mitochondria exposed to high concentration active oxygen environment which led to oxidative damage and affected the normal physiological function of cells. Therefore, promoting the biosynthesis of mitochondrial function and increasing the activity of antioxidant enzymes in mitochondria can decrease levels of ROS and reduce the oxidative damage of mitochondria [14].
Salidroside can promote the biosynthesis of mitochondria and improve numbers of mitochondria by activating the signal pathway controlled by PGC-1α through raising AMPK phosphorylation [15,16].

Scavenge free radicals in cells:
Free radicals, including superoxide anion, hydroxyl radical, NO free radical, alcoxyl radical and lipid peroxide (LPO), are widely-existing in human body. The oxygen free radical can induce starting the free radical chain reaction with its unstable property, and can continuously generate new active free radicals.
Investigation shows that salidroside can increase the activities of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase), GSH (glutathione), and reduce the content of MDA (malondialdehyde) at the same time, which can speed scavenging free radicals [17,18].

Anti-Aging Effect
When numbers of ROS exceeded in mitochondria, it would cause the DNA oxidative damage. Due to the lacking of integrated DNA damage repair system in the mitochondria, it causes oxidative damage accumulation, and gradually leads to mitochondrial dysfunction. Then it causes changes of physiological function and leads to the age-related diseases [19].
One of the main causes for aging is the mitochondrial dysfunction, salidroside can inhibit cell oxidative damage, maintain the numbers and function of mitochondria. Researches showed that salidroside could reduce the content of MDA and increase the activity of SOD and GSH-Px in the natural aging mice [20].
In addition, salidroside can enhance sports abilities and extend the memory time of aging mice induced by D-galactose. It also shows that salidroside can reverse aging by promoting mitosis of lymphatic cells and synthesis of IL-2 [21].

Anti-myocardial ischemia and hypoxia activities:
Salidroside is an oxygen glycoside molecule with the basic pharmacophore to treat coronary atherosclerotic heart disease, and the activity is affected by the appropriate length of the connection chain between glycosyl and the polysubstituted aromatic ring.
Studies have shown the mechanisms of salidroside's antimyocardial ischemia and hypoxia activities [22]: 1. Reduce the synthesis of angiotensin converting enzyme, and obviously inhibit the proliferation and contraction of vascular smooth muscle cells. 2. Reduce the myocardial oxygen consumption, and strengthen the pumping force of heart to improve the symptoms of heart failure. 3. Improve the activity of Na + -K + -ATP enzyme in cells, and reduce the change activities of compounds but also can enhance the stability for acid, alkali and enzyme [31,32]. In addition, the introduction of amine structures can improve LogP of compounds, thus it can enhance the stability and bioavailability.
In the test of DPPH EC 50 , the activities of compounds 6, 7, 8 and 9 ( Figure 5) were stronger than salidroside and Vc (Vc DPPH EC 50 =14.5; salidroside DPPH EC 50 =17.1; compound 6 DPPH EC 50 = 12.5; compound 7 DPPH EC 50 =10.4; compound 8 DPPH EC 50 =10.0; compound 9 DPPH EC 50 =11.0). It is speculated that the introduction of nitro on the benzene ring can enhance the ability of scavenging free radicals. When the nitro is at 2 or 3 site, the ability of scavenging free radicals is better than others. In addition, the ability of scavenging free radicals for S-glycosides and N-glycosides are better than O-glycosides.
To sum up, the properties and positions of substituent on the benzene ring, the type of glycosidic bond and glycosyl can influence the activity ofsalidroside. The introduction of methylenedioxy, N-glycoside, S-glycoside and C-glycosides can increase the stability and pharmacological activities.
Peng designed and synthesised a series of C-glycosides and tested myocardial protection [33].
The main modification sites were: 1. Change phenol hydroxyl groups into electron-donating groups or electron-withdrawing groups. 2. Introduce amine structures on the connection chain. 3. Change O-glycoside into C-glycoside.
In the test of LDH inhibition rate, compounds 10, 11, 12, 13 and 14 in different doses were better than salidroside with obvious concentration-response relationship (Table 1).
Compound 11 is the best in different dosage group. It is speculated that the introduction of halogen atoms or methoxyl to o-, m-, pposition on the benzene ring can improve myocardial protection, and the introduction of fluorine atoms had the best protection. C-glycosides. had stronger protection than O-glycosides.
Zhang [34] modified structure of salidroside from following The experimental results show that increasing the numbers of hydroxyl on benzene ring can increase activities of anti-radiation and anti-anoxia. However, the number of hydrogen bond donor (hydroxyl, amino, etc.) must be less than five according to "drugs 5 rule".

The Modification of Glucoside
Most researchers usually choose four main points to modify the glucoside of salidroside: 1. The groups on the benzene ring; 2. The length and saturability of carbon chain between benzene ring and glycosyl donor; 3. The type of glycosidic bond; 4. The type and configuration of glycosyl donor. Most modification focused not only on a single site, but usually on multiple sites. In the test of anti-anoxia activity under normal pressure, compounds 3 and 4 ( Figure 3) had the quite activities in different doses (16.6, 50, 150 mg/kg/day) compared with salidroside (salidroside, 50 mg/kg/ day, the survival time 42.8 ± 5.1 min; compound 3, 50 mg/kg/day, the survival time 40.1 ± 6.4 min; compound 4, 50 mg/kg/day, the survival time 44.1 ± 6.0 min). It is speculated that S-glycosides and C-glycosides have better stability to enhance anti-anoxia activity.
In the test of OD value in endotheliocyte, the activity of compound 5 (Figure 4) was better than salidorside (OD values of model, salidroside and compound 5 were 0.301 ± 0.018, 0.473 ± 0.010 and 0.533 ± 0.012, respectively). It could speculate that the introduction of methylenedioxy can enhance the stability for acid, alkali and enzyme. Methylenedioxy is the isosteres for oxygen, which not only can largely aspects: 1. Remove phenolic hydroxyl groups; 2. Change carbon numbers of connection chain; 3. Introduce alkenes structure to connection chain; 4. Change glycosyl into galactose. They got a series of novel compounds and test anti-fatigue activity. The anti-fatigue activities of novel compounds were weaker than salidroside. The result showed that phenolic hydroxy is the active site for anti-fatigue activity. The anti-fatigue activities of galactose groups were better than glucose groups, which showed that galactose can enhance anti-fatigue activity. The three-dimensional structures of carbohydrate are different, and biological activities of polysaccharides are determined by glycosyls. So different types of glycosyl have an influence on biological activities of salidroside.
Many studies have shown that compounds with glucosamine have nerve cells protection [35]. Guo changed glycosyl donors into N-acetylglucosamine, glucosamine and synthesized compounds 15, 16 ( Figure 6) [36]. They studied the mechanism of protecting PC12 nerve cells.
Pretreatment with compounds 15, 16 and salidroside at different concentrations (80, 160, 320 mg/ml) significantly attenuated the cell viability loss evoked by hypoglycemia and serum limitation, and the attenuating effect on the survival of cultured PC12 cells displayed a dose dependent pattern. The changes in the percentage of apoptotic cells were also analyzed by Hoechst 33342 staining. In comparison to control group, exposure to hypoglycemia and serum limitation alone produced significantly apoptosis. Pre-incubation with 80, 160, and 320 mg/ml of compounds 15, 16 and salidroside, the apoptotic percentages were significantly lower than that produced by exposure to hypoglycemia and serum limitation alone. Western blot analysis showed that compounds 15 and 16 against hypoglycemia and serum limitationinduced cell apoptosis in the PC12 cells partly attributed to their evoked modulation of apoptosis-related gene expression(especially Bcl-2), which was in accordance with salidroside. Therefore the introduction of N-acetylglucosamine can enhance the protection of PC12 cells by inhibiting apoptosis gene (Bcl-2) expression, inhibiting cell apoptosis and enhancing cell viability.
Zheng modified structure of salidroside from following aspects [37]: 1. Introduce more phenolic hydroxyl or methoxyl groups on the benzene ring    Compound 17 have exhibited the best anti-anoxia activity. Structure and activity relationships (SARs) revealed that: 1. Introduction of more phenol hydroxyl groups on the benzene ring can improve antioxidant activity; 2. The potency of β-glucoside is better than α-glucoside; 3. when containing two carbons in linker manifested best activity.
Compound 17 and 18 then were investigated for their protective effect on human normal liver cells and the mechanism. Noticeably, compounds 17 and 18 comparing with salidroside showed better cell protection (CCl 4 mediated apoptosis). And salidroside showed good cell protection merely in the concentration for 100 mM compounds 18 (50, 100 mM) and compounds 17 (10, 50, 100 mM) in low doses showed better cell protective effect.
In an effort to detect the activities of SOD, GSH-Px and the content of MDA in human liver apoptotic cells, we further decided to add 10 mM CCl 4 into LO2 cells, we found the activity of GSH-Px was reduced by 51%, SOD was reduced by 53%, and content of MDA was raised by 70%. Through treated with compounds 17, 18 and salidroside, the activities of GSH-Px and SOD activities in LO2 cells were enhanced. The toxicity caused by CCl 4 was accompanied by increased lipid peroxides. And the activities of compounds 17, 18 were better than salidroside (salidroside 100 mM, SOD 61%, GSH-Px 61%, MDA 157%; compound 17 100 mM, SOD 89%, GSH-Px 86%, MDA 110%; compound 18 100 mM, SOD 83%, GSH-Px 80%, MDA 117%). It can be speculated that compounds 17 and 18 can inhibit the production of peroxide, scavenge free radicals, and inhibit apoptosis mediated by CCl 4 .
Preliminary discussion of structure-activity relationship for salidroside analogues To sum up, aglycone, chain between aglycone and glycosyl, the type of glucosidic bond and the configuration of glycosyl can affect the biological activities of salidroside analogues.
The preliminary structure-activity relationship studies can be concluded as follows: 1. Substituents on the benzene ring of aglycone. Phenolic hydroxy is the active site for anti-fatigue and anti-oxidant activities [34]. When no phenol hydroxyl was introduced, the antioxidant activity could be reduced. And when the length of phenolic hydroxyl increases, the antioxidant activity improved [29,37]; The positions of phenol hydroxyl also had the had certain differences to antioxidant activity. When phenol hydroxyl is at position C1, the antioxidant activity has no influence. When phenol hydroxyl is at position C2 or C6, the antioxidant activity is reduced. When phenol hydroxyl is replaced by the electron withdrawing group, the hydrogen on the benzyl carbon is tend to leave, which can enhance the ability of catching DPPH and show better antioxidant activity [30]; When introduces electron-donating groups to position C 2 or C 6 on the benzene ring, the antioxidant ability is enhanced. It also can improve the protection for myocardial cells [33]; whereas replacing methoxyl to position C 2 or C 6 , the antioxidant activity is reduced [30].
2. The influence of the link chain. Various length of chain have differences to antioxidant activity, when the carbon is only one, the anti-hypoxia activity is shinking and antioxidant activity is reduced [30,37].
3. The type of glucosidic bond. S-glycosides, C-glycosides and N-glycosides exbited better antioxidant activity than O-glycosides. It can be speculated that S-glycosides, C-glycosides and N-glycosides have better stability in vivo [30]. And N-glycosides have better water-soluble. It can enhance the combination of compounds with receptors; When there exits amine groups on benzene ring, the electron cloud on the benzyl carbon redistributes due to strong electron-withdrawing effect. It tends to form more stable C-N double bond with amino, which lead to increased numbers of free oxygen. However, oxygen atoms cannot combine with carbon atoms to form a more stable structure, so the number of free oxygen is less. Therefore the ability of scavenging free radicals of N-glycosides is better than O-glycosides [30,33].  4. The type and configuration of glycosyl. The anti-fatigue activities of galactose groups were better than glucose groups [34], and the introduction of N-acetylglucosamine can enhance the protection of PC12 cells [36]. The antioxidant activity of β-glucoside is better than α-glucoside.

Conclusion
Salidroside is a phenylpropanoid glycoside isolated from Rhodiola L. which which have been extensively used for the treatment of resisting anoxia, anti-radiation and antifatigue in Traditional Chinese Medicines. In addition, it has anti-tumor, radioresistance, antiviral and cytoprotection effects. Due to its low toxicity, salidroside has been promising used in clinic. However, drugs about salidroside on the market at present mainly tend to cosmetics. The main reason is that the structure of salidroside is O-glycosides, which have low metabolism stability. This paper review on the structural modifications and activities study of salidroside in recent ten years, and the preliminary structureactivity-relationship of salidroside analogues, which intend to provide perspectives for rational modification of salidroside for antioxidant and acetylcholinesterase inhibitory activities, along with the other potential pharmacological activities.