Introduction

A report published by World Health Organization (WHO) has documented that cancer is the second highest cause for death around the world and 70% of deaths because of different cancers have been reported in middle- and low-income countries. Cancer rates could increase by 50% to 15 million new cases a year by the year 2020 [1–3]. Cancer is mainly caused by various mutations in the genome, which can cause deregulation of diverse molecular signaling cascades [4–6]. The presence of constant growth signals, unresponsiveness to antigrowth signals, apoptosis resistant, heightened angiogenesis, tissue invasion in addition to metastasis, enhanced replicative potential, as well as genome variabilities are the major signs of cancerous cell growth [7]. Numerous therapeutic approaches against cancer have been established over the last few decades after acquiring a deeper understanding of several underlying signaling mechanisms that can lead to the enhanced survival and proliferation of neoplastic cells. They include adjuvant and neoadjuvant chemotherapy, targeted therapy, immunotherapy, surgery, and radiotherapy [8–11]. The prevalence of cancer and mortality remains high despite substantial improvements in treatment procedures [12]. This phenomenon can be generally attributed to the limited effects of existing anti-cancer therapies and the expensive cost of the treatment, in addition to substantial adverse reactions [13]. Additionally, modern cytotoxic agents commonly possess life-threatening toxicity [14, 15]. In the past six decades, there have been several cases of removal of pharmaceutical products from the market because of antagonistic drug reactions, with the most prominent adverse event being hepatotoxicity [16]. Additionally, some cancers such as breast cancer may often reappear after remaining inactive for a long time even after successful treatment [17], indicating that discovery of new and safe treatment methods is still needed.

Mother Nature is a reservoir of a significant number of plant-based natural products, which possess significant anti-cancer potential [18]. Several plant-based molecules can act as a chemosensitizer as well as overcome chemoresistance in different types of cancer [19–23]. In addition, 40% of the medicines approved by the FDA available in the market have been derived from products obtained from plants, 74% of which are anticancer drugs [24, 25]. The research in this field has emphasized on the utilization of undiscovered reservoirs of phytochemicals such as alkaloids, glycosides, terpenoids, phenolics, and saponins, in order to avoid the harmful side effects of medications used in chemotherapy, to prolong recovery time as well as to boost the quality of life in cancer patients [5, 19, 26, 27]. Salvia miltiorrhiza (S. miltiorrhiza, Danshen) belongs to the family Labiatae, is a well-known traditional Chinese herb. In addition, due to its excellent medicinal properties, it has been in use for thousands of years to treat many diseases and is regarded as “Super-grade drug” in Pen-Ts’ao of Shen-Nung [28]. Traditionally, Danshen has been extensively used to treat cardiovascular diseases, mental agitation, memory weakness, insomnia, cancer, and liver fibrosis [29–31]. It also delays the development of atherosclerosis [32]. Its role as an anti-hypertensive and anti-platelet aggregation agent also may result in the prevention of cerebral infarction [33]. Danshen may have a role in the elevated expression of certain antioxidant enzymes [34].

Pharmacologically, Danshen has two categories of compounds, i = lipophilic diterpenoid tanshinones, ii = water-soluble phenolic acids [35]. Tanshinones are important antioxidants, anti-cardiovascular, anti-inflammatory, and antitumor agents and are the main ingredient in S. miltiorrhiza [36]. The water-soluble phenolic acids possess various bioactivities including those of antioxidant, anticoagulant, anti-thrombotic, antitumor, and anti-HIV [37]. Among the water-soluble phenolic acids, salvianolic acid B (SAB) is the chief component as per official Chinese Pharmacopoeia. Many studies have reported the promising antitumor, neuroprotective and cardio-protective properties of SAB in different model systems [38–40]. This review briefly highlights the important cellular pathways involved in the antitumoral actions of SAB in different cancer cell lines and in animal models and strategies to utilize its potential for cancer therapy.

SAB chemistry

The basic chemical structure of different derivatives of SAB contain [(R)-3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoic acid] and it is also known as lithospermic acid B [41]. Molecular formula of SAB is C₃₆H₃₀O₁₆ and its molecular weight is 718.6138 g/mol. SAB is yellowish and an amorphous compound formed by three Danshensu (Salvianic acid A) molecules and one molecule of caffeic acid. Interestingly, phenolic groups in different compounds are responsible for inhibition of tumor invasion, induction of apoptosis, reversal of drug resistance, modulation of immune response to tumor cells, inhibition of metastasis of tumor cells, and reducing abnormal proliferation [42]. Blocking of lipid peroxidation can be executed by release of hydrogen from activated phenolic hydroxyl groups (9 in number) [43]. Interestingly, antioxidant activities demonstrated by SAB and Danshensu have been attributed to the functional phenolic groups present in their structures [44, 45]. Chemical structure of SAB were shown in Figure 1.

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Figure 1. 

Chemical structure of salvianolic acid B

Biosynthesis of SAB

Among different Salvianolic acids, SAB and Rosmarinic acid (RA) can be synthesized primarily through tyrosine derived phenolic acid and phenylpropanoid biosynthetic pathways (Figure 2) [46]. Biosynthesis of RA is initiated when L-phenylalanine and L-tyrosine are converted to two different intermediates, i.e. 4-Coumaroyl-CoA and 4-hydroxyphenyllactic acid respectively by two parallel but independent pathways. Several subsequent biochemical reactions initiated by covalently joining these two intermediates can form RA at the end. SAB is thought to be derived from RA (Figure 2). However, the detailed mechanism of SAB synthesis has not been studied until date [47]. The extent/quantification of biosynthesis of SAB is dependent on the yield recovered during its extraction process. In general, using conventional reflux-based extraction methods for extraction from roots requires high temperature for a longer time, which may contribute to a lower yield of SAB mainly due to its hydrolysis into tanshinol [48]. However, a higher extraction yield can be achieved over a shorter time and lower temperature when an ultrasound-assisted extraction method is used. Using the ultrasound-assisted extraction method, the yield of salvianolic acid B was 33.93 mg/g in S. miltiorrhiza roots higher than those with a conventional refluxing method (28.76 mg/g) [49].

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Figure 2. 

Biosynthetic pathways of SAB in S. miltiorrhiza. TAT: tyrosine amino transferase; HPPR: 4-hydroxyphenylpyruvate reductase; PAL: phenylalanine ammonia-lyase; C4H: cinnamic acid 4-hydroxylase; 4CL: 4-coumarate CoA ligase; RAS: rosmarinic acid synthase; CYP98A14: cytochrome P450-dependent monooxygenase

Various molecular targets affected by SAB

As cancer is a disease in which several signaling molecules may be deregulated causing the cells to multiply, invade, or metastasize [50]. SAB has been identified as a potential antitumor compound and has been observed to target multiple steps in the apoptotic pathway [51] (Figure 3). The studies on the mechanistic action of SAB showed up-regulation of caspase-9, pro-apoptotic proteins, i.e. B-cell lymphoma 2 (BCL-2)-associated X protein (BAX) and BAK, enhanced caspase-3 level with poly ADP-ribose polymerase (PARP) cleavage and down-regulation of anti-apoptotic proteins such as Bcl-2, which can promote apoptosis [52–55]. In SKOV3 ovarian cancer cells, SAB induced apoptosis by the activation of caspase-3 in a dose-dependent manner [56]. It was also found to cause cell cycle arrest at M/G2 phase by inhibiting the expression of cyclin E and cyclin D [57, 58].

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Figure 3. 

A diagram showing the induction of apoptosis and cell cycle arrest induced by SAB treatment. FADD: FAS-associated death domain protein; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; Chk: Checkpoints; ↑ increase; ↓ decrease

In addition, among several signal transduction pathways, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) has been found to be deregulated in most types of tumor cells. PI3K/AKT, a serine-threonine kinase protein binds to phosphatidylinositol triphosphate (PIP3) on the surface of a cell and its activity can be modulated by phosphoinositide dependent kinase-1 (PDK-1) after its binding to PIP3. Activated AKT can phosphorylate downstream target proteins, including forkhead box O (FOXO1), glycogen synthase kinase-3 (GSK3β), and mTOR, resulting in cancer cell survival, cell-cycle progression, ribosome biogenesis, and/or protein synthesis [59]. AKT and mTOR are the key proteins involved in regulation of apoptotic and autophagy pathways in different tumor cells. Therefore, it represents a potential antitumor target in therapeutic studies [60–62]. SAB has been found to induce apoptosis and autophagy by inhibiting AKT/PI3K mediated activation of mTOR pathway in colorectal [63] and hepatocellular carcinoma (HCC) cells [60]. Autophagy is an important physiological process, which has been reported to be involved in the maintenance of cellular homeostasis by degrading old proteins and damaged cellular organelles [64]. Autophagy plays a dual role in cancer. It may be involved in tumor suppression as well as in increased proliferation. Apoptosis and autophagy could be induced by the same stimulus, but the interaction between them was still unclear [65]. Most studies have shown effects of SAB on PI3K/AKT signaling pathway resulting in down-regulation of m-TOR. There is need to explore further molecular targets modulated by SAB within PI3K/AKT pathway, i.e. FOXOs and GSK3β as they are also involved in the process of cell survival and cell cycle progression in tumor cells [66].

Tumor cells exhibit elevated activation of constitutive nuclear factor kappa light chain enhancer of activated B cells (NF-κB), which may lead to increased cell growth, reduced apoptosis, metastasis of tumor cells, angiogenesis and alter cellular metabolism by regulating the expression of many genes such as cyclooxygenase-2 (Cox-2) [67–69]. NF-κB activation can cause tumor cells to become resistant to apoptosis and it can facilitate increased proliferation and metastasis [70–72]. NF-κB has been considered as an important target for modulation by different therapeutic approaches in many tumor types [73, 74]. NF-κB and MDM-2 (mouse double minute 2 homolog) were found to be down-regulated in SAB treated JHU-013 head and neck cancer cells in a dose-dependent sequential inhibition of LPS-stimulated Cox-2 and PGE-2 [57, 58]. The mechanism(s) by which SAB can exert its inhibitory effects on NF-κB pathway is still unclear and requires detailed studies. In 7,12-dimethylbenzanthracene (DMBA) treated hamster model, modifications of key metabolic pathways, including elevated glutaminolysis and glycolysis, and decreased cholesterol and myo-inositol metabolism were observed, which were attenuated by SAB exposure. SAB also inhibited important regulators of cellular proliferation and tumorigenesis, i.e. hypoxia induced factor (HIF)-1α, matrix metalloproteinase (MMP)-9, and tumor necrosis factor (TNF)α [75, 76]. The action of SAB in modulating Warburg effect and affecting tumor growth were summarized in Figure 4.

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Figure 4. 

Schematic diagram showing SAB inducing mechanism of action in cancer. PGH2: prostaglandin H2; PGE2: prostaglandin E2; VEGF: vascular endothelial growth factor; MMP: matrix metalloproteinases; IP3K: indole phosphatide 3 kinase; GLU-6PO4: glucose 6 phosphate; ↑ increase; ↓ decrease

A brief overview of anti-cancer potential of SAB in different tumor malignancies, i.e. breast cancer, HCC, leukemia, colorectal cancer, head and neck squamous cell carcinoma (HNSCC), glioma cells, cervical cancer, ovarian cancer, and retinoblastoma has been shown in tables 1 and 2. The detailed pharmacological impact of SAB against different cancers has been discussed briefly below:

Role of SAB in regulating epithelial-mesenchymal transition (EMT)

For tumor growth and spread, epithelial-mesenchymal transition (EMT) is an important process and enhanced expression of mesenchymal genes (Fibronectin, Vimentin, N-Cadherin), as well as reduced expression of epithelial genes (E-Cadherin), are the most important characteristics of EMT [126, 127]. Metastasis poses the biggest hurdle in effective cancer treatment and accounts for 90% mortality rate caused by different cancers [128–133]. SAB suppressed extracellular matrix modelling and cellular proliferation through inhibition of NF-κB associated activation of MMP-9 and MMP-2 in high glucose induced mesangial cells [134]. In addition, MMP-9 levels were also noted to be significantly downregulated in SAB treated breast cancer cells [83]. Moreover, studies have indicated that SAB may have a role in the inhibition of EMT by modulating the expression levels of different micro-RNAs. Yu et al. [135] showed that administration of SAB reversed liver fibrosis, repressed Hedgehog pathway and EMT by up-regulation of Patched-1, miR-152, and DNA methyl transferase 1 (DNMT1). In addition, miR-106b, miR-93 and miR-25 were significantly downregulated in TGF-β induced EMT [135].

In addition, another study showed a dose-dependent increase in the expression of miR-106b-25 cluster in SAB treated HK-2 human kidney cancer cells. Interestingly, miR-106b can reduce EMT by increasing the expression levels of E-cadherin and lowering expression levels of α-smooth muscle actin (α-SMA) [136]. In-vivo studies have also revealed inhibition of TGB-β1-induced EMT in SAB treated HK-2 cells by modulation of TGF-β/Smad signaling pathway [137]. Moreover, a reversal of TGF-β1 induced EMT in KH-2 cervical cancer cells by nano-formulation of SAB (HCA-Chi-Ca-SAB) has been reported [138]. These findings suggest the potential role of Salvianolic acids in the reversal of EMT in cancer. EMT reversal may also result in the regeneration of already disseminated cancerous cells within the body [139]. Further studies on the exploration of mechanistic pathways involved in EMT reversal by SAB will establish its role and usefulness as an anti-EMT drug.

Pharmacokinetics and bioavailability of SAB

A number of naturally derived biological compounds have deficiency in distribution, rapid metabolism, excretion, and poor absorption that may restrain their bioavailability [140]. Several experiments have been conducted to study the pharmacokinetics behavior of SAB in humans and animals for instance dogs, rabbits, and rats. The pharmacokinetic profile of SAB in rats was studied using two-compartment open models by subsequent oral course of 200 mg/kg. The absolute/standard SAB bioavailability is 0.022%. Several investigations suggested that after oral administration of SAB, 60% of given SAB remains in the gut for at least 180 min, thus contributing to its poor bioavailability in the body [141]. Oral bioavailability in dogs was noted to be 1.1% only, after the administration of SAB doses, i.e. 80 mg/kg orally, and 9 mg/kg [142]. The intravenously injected doses with 3, 6, and 12 mg/kg of magnesium SAB in beagle dogs have also been examined. It was evident from the previous studies that the elimination and distribution of SAB were fast enough [143]. The normal urinary excretion rate of SAB reported from the studies was 0.16%. However, the recovery of SAB from the gastrointestinal (GI) tract was 41.2% and 23.3% respectively when the oral course of 10 and 50 mg/kg doses were applied [143].

The intravenous dose of SAB in humans exhibited 0.29 h half-life of SAB’s elimination with the application of 100 mg/kg dose, although the concentration of SAB gradually and quickly increased in the bile. It attained the highest value within 30 min [144]. It was observed that the concentration of SAB is greater in the bile than in plasma at certain points, which suggested that the hepatobiliary eradication of SAB might imply an active transport [145]. Furthermore, the cumulative absorption concentration of salvianolic acid B was greater in rear jejunum segments than in middle and front segments. SAB has a low oral bioavailability of 4% due to the confined intestinal permeability [146]. Moreover, human epithelial colorectal adenocarcinoma cells (Caco-2) study revealed the lower concentration of SAB in cell membrane permeability. About 5% of SAB bioavailability was noted after oral administration [147]. One hundred mg/kg SAB was administered intravenously, and 500 mg/kg of SAB was administered orally in conscious and freely moving rats. The oral bioavailability of SAB was found to be 2.3% in freely moving rats [148]. Fluorescent poly (ethyl-cyanoacrylate) nanoparticles (300 nm size) were loaded with SAB and it enhanced the bioavailability and sustenance of SAB and allowed the nano formulation to cross blood-brain barrier [149].

Approaches to ameliorate the bioavailability of SAB

The low bioavailability of SAB can be correlated to the rapid metabolic clearance or its poor absorption in the body. However, two main strategies can be adopted to enhance the SAB absorption, i.e. preparation of fat-soluble complexes and the use of absorption enhancers. The sodium caprateused as co-administration of absorption enhancer can significantly increase the intestinal permeability with the in vivo bioavailability of SAB [147]. Borneol is a common Chinese herbal medicine, which can improve the intestinal absorption of SAB in a dose-dependent manner [150, 151]. The production of fat-soluble complexes such as phospholipids may possibly be another suitable preference that can enhance the absorption of SAB in GI tract. This can be attained through the phospholipid complex loaded with the nanoparticle formation. This formulated complex can lead to an increase of 2.9 folds in the relative bioavailability in comparison to the normal salvianolic acid B formulation [152]. Increased bioavailability of SAB has been reported using SAB phospholipid complex loaded pellets without causing any significant increase in C_max when compared to SAB alone [153, 154]. This finding supports the utilization of nanoparticle establishment that produces a greater bioavailability than the pellet formation.

The two major metabolites of SAB produced after it was injected intravenously in rats are, i.e. monomethyl-SAB (3-MMS) and Lithospermic acid (LSA). The metabolic pathway for SAB in rats included methylation by means of catechol-O-methyltransferase (COMT) in kidney and liver [152]. The blood concentration can greatly be increased in rats by co-administration of an intravenous dose of 50 mg/kg SAB and 0.5 mg/kg ferulic acid [155]. Additionally, the accumulative absorption of SAB in the rats was increased from 3% to 40% in the bile excretion, when used with L-DOPA and tolcapone. Likewise, with the single intravenous dose of SAB, the plasma concentration of 3-O-methyl dopa (3-OMD) was noted to be decreased. The current information related to CYP enzymes and SAB is debatable. Qui et al. [156] revealed that SAB has no obvious effect on CYP enzyme using human liver microsomes (HLMs).

In contrast to previous study, SAB was reported to exhibit effectual concentration-dependent inhibitory effect on CYP3A4 activity with IC₅₀ values of 1.44 mg/L on HLMs thus demonstrating the role of CYP3A4 substrate [157]. Ferulic acid nano particles (FA-NPs) loaded with SAB displayed significantly stronger antitumor effects via receptor-mediated targeted delivery in MCF-7 and MDA-MB-231 cells [158]. Two novel analogs of SAB were recently isolated from Salvianolic acid injection. 7′(Z)-(8″S,8‴S)-epi-salvianolic acid E (compound 1) is a ring opened product of SAB and (7′R,8′R,8″S,8‴S)-epi-salvianolic acid B (compound 2) is a non-enantiomer of SAB (Figure 5). Both these compounds were around 6% of total SAB injection. Compound 1 showed neuroprotective effects comparative to SAB while compound 2 exhibited potent antioxidants effects [159]. Pharmacokinetics study on both SAB analogs and SAB showed that at a dose of 6 mg/kg, compound 1 and compound 2 had a slow elimination rate than SAB. Interestingly, compound 1 and SAB had higher exposure at the same dosage than compound 2. These findings showed the potential therapeutic role of novel analogs of SAB [160].

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Figure 5. 

Chemical structures of Salvianolic acid B analogues

Biosafety profile of SAB

Biosafety and toxic profiling of SAB in rats showed significant adverse effects in lungs, liver, kidney, brain, and heart after administration of SAB in combination with ginsenoside Rg1 at a lethal dose (LD₅₀) of 1, 747 mg/kg. This dose is 100 times more than the effective dose of SAB [161]. Many clinical trial studies have cleared S. miltiorrhiza for the treatment of stroke, heart attack, and many other clinical pathologies [162]. As a non-toxic and bio-safe ingredient in a traditional medicine being used for a century, SAB has primarily emerged as a safe drug for clinical use. However, additional studies are required to explore and validate its safety for human use.

Conclusion and Future perspectives

The cancer has created havoc for the humanity and its treatment has baffled scientists and researchers for past many decades. The use of natural compounds to treat different types of cancers has a promising future, as these compounds can be effective at doses, which are likely to cause lesser adverse effects. SAB provides an alternative to already existing therapies for different typed of cancer due to its ability to target multiple cellular pathways, i.e. MAPK, PI3K/AKT, and NF-ĸB to induce apoptosis, inhibit invasion and proliferation of tumor cells. It can also sensitize tumor cells to other antitumor agents by reversing the multi-drug resistance mechanisms operational in tumor cells. The limitation of bioavailability of SAB is a major hurdle for its use in treatment, which can be overcome by using nanoparticles-based drug delivery systems to enhance its efficacy and retention in the living systems. Further pre-clinical and clinical studies are essential to cement the idea that SAB can be used effectively in therapy of different types of cancers.