Untitled Document
Salvia miltiorrhiza Bunge, a member of the Lamiaceae family, is valued in traditional Chinese Medicine. Its dried root (named Danshen) has been used for hundreds of years, primarily for the treatment of cardiovascular and cerebrovascular diseases. Tanshinones are the main active ingredients in S. miltiorrhiza and exhibit significant pharmacological activities, such as antioxidant activity, anti-inflammatory activity, cardiovascular effects, and antitumor activity. Danshen dripping pill of Tianshili is an effective drug widely used in the clinical treatment of cardiovascular diseases. With the increasing demand for clinical drugs, the traditional method for extracting and separating tanshinones from medicinal plants is insufficient. Therefore, in combination with synthetic biological methods and strategies, it is necessary to analyze the biosynthetic pathway of tanshinones and construct high-yield functional bacteria to obtain tanshinones. Moreover, the biosynthesis of tanshinones has been studied for more than two decades but remains to be completely elucidated. This review will systematically present the composition, extraction and separation, pharmacological activities and biosynthesis of tanshinones from S. miltiorrhiza, with the intent to provide references for studies on other terpenoid bioactive components of traditional Chinese medicines and to provide new research strategies for the sustainable development of traditional Chinese medicine resources.
Introduction
Salvia miltiorrhiza Bunge, an important Salvia species with terrific economic, social and medicinal benefits, was first recorded in the Shenlong Bencao Jing (200–300 AD, Han Dynasty), the oldest medicine monograph in China. Salviae miltiorrhizae Radix et Rhizoma, dry roots of Danshen, has been widely applied in the clinical treatment of cardiovascular diseases, dysmenorrhea, amenorrhea, and hypertension, hepatocirrhosis, chronic renal failure and other diseases. With the development of science and technology, the dosage forms containing Danshen have been gradually diversified. Tablet, injection solution, dripping pill, oral liquid, capsule, slow-release formulation, and soft gel are all dosage forms that have been prepared into medicines. Among these diverse preparations, composite Danshen droplet pills, used to treat angina pectoris and coronary heart disease, represent a star drug and, moreover, a demonstration of traditional Chinese medicine entering the international market. Currently, advanced clinical tests on this drug are ongoing in the United States. The active ingredients in S. miltiorrhiza can be mainly divided into two categories: fat-soluble tanshinone compounds and water-soluble salvianolic acids. Both ingredients have significant pharmacological activities and are also representative active ingredients in traditional Chinese medicine, especially tanshinones.
As secondary metabolites, tanshinones mainly accumulate in the roots, but the yield is very low. Currently, tanshinones are obtained from roots by chemical separation and purification. However, because traditional methods have low efficiency, high energy consumption and unfriendliness to the environment and plant resources, it is meaningful and necessary to find new methods. With the successful applications of synthetic biology technology in the study of taxol, ginsenoside, and artemisinin, this approach could also provide new developments and approaches for the modern study of tanshinones. Based on research on tanshinones at home and abroad, this paper will systematically review the research results of extraction, purification, pharmacological activity and biosynthesis of the original plant S. miltiorrhiza.
The Plant S. miltiorrhiza
Salvia miltiorrhiza Bunge belongs to the genus Salvia of the Lamiaceae family, which is a perennial erect herb. Its dry root is used as Danshen and has the effects of promoting blood circulation, relieving pain, regulating heat, and calming the nerves.
Wild S. miltiorrhiza generally grows in central and northeastern China. Environmental and soil conditions affect the quality of Danshen and the content of active ingredients. The Chinese Flora reports that there are two varieties in the S. miltiorrhiza species, namely, the original variety (S. miltiorrhiza var. miltiorrhiza) and S. miltiorrhiza Bunge var. charbonnelii. The original variety can be divided into two forms, namely, the original form (S. miltiorrhiza var. miltiorrhiza f. miltiorrhiza) and S. miltiorrhiza var. miltiorrhiza f. alba. These forms mainly differ in plant morphology and geographical distribution. For example, the flower color of S. miltiorrhiza var. miltiorrhiza f. alba is white, and it originates from Shandong, China.
In recent years, due to the excessive harvesting of wild S. miltiorrhiza, the resources are already on the verge of extinction. With the reduction in wild resources, artificial domestication cultivation of S. miltiorrhiza has been carried out since the 1970s. The main producing areas of the current cultivated S. miltiorrhiza are Linyi Shandong Province, Jiaozuo Henan Province, Wanrong Shanxi Province, Shangluo Shaanxi Province, Zhongjiang Sichuan Province and other places. To date, S. miltiorrhiza from Sichuan Province is still considered to have the best quality. Due to the low yield of secondary metabolites and the long growth cycle of cultivated plants, the production of tanshinones from cultivated S. miltiorrhiza can no longer meet the rapidly growing market demand. It is essential to use modern biotechnology methods to increase the yield. Therefore, various in vitro culture systems of S. miltiorrhiza, including suspension cell, callus, adventitious root and hairy root, as well as new techniques such as using endophytic fungi, can be used to accumulate tanshinone production.
Tanshinones of S. miltiorrhiza
In the 1930s, domestic and foreign scholars have begun to study the chemical constituents of S. miltiorrhiza. Japanese scholar Nakao first isolated tanshinone IIA from S. miltiorrhiza and identified its chemical structure in 1934. After the 1970s, with the development of clinical application of S. miltiorrhiza and the advancement of Chinese medicine extraction and separation technology, its chemical composition research has developed rapidly. Tanshinones, also known as total tanshinone, are a general term for a class of lipophilic phenanthrene compounds that are rich in the roots of S. miltiorrhiza. What these compounds have in common is that they possess an ortho- or fluorene structure and can be reduced to a diphenol derivative, which is converted to hydrazine after oxidation. In this process, these molecules facilitate electron transfer, and can promote or interfere with various reactions of the body; accordingly, these compounds exhibit significant pharmacological properties such as anti-cancer, antibacterial and anti-viral effects. To date, more than 40 tanshinones have been isolated from S. miltiorrhiza, including ortho-quinones (i.e., tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone) and para-quinones (i.e., isotanshinone I, isotanshinone IIA, and isocryptotanshinone). In addition, other diterpenoids and triterpenoids have been isolated from Danshen, such as danshenxinkun A/B/C/D and neo-tanshinlactone. With the increasing clinical demand for tanshinones, more research is currently being conducted to explore how to efficiently obtain tanshinones and enhance their pharmacological effects by exploring structure activity relationships.
Extraction and Separation Methods of Tanshinones
Due to the clinical application of tanshinones, the extraction, and purification processes of these compounds were developed early in tanshinone research, and the research on associated extraction and separation process technologies is relatively extensive. The main extraction methods include alcohol extraction, ultrasonic extraction, supercritical CO2 fluid extraction, pressurized liquid extraction (PLE), microwave-assisted extraction, high-speed countercurrent chromatography, column chromatography, and vacuum liquid phase extraction.
Because terpenoids generally have low polarity, they are often extracted with chloroform, ethyl acetate or petroleum ether. For some slightly more polar compounds, the plant roots are usually first degreased and then extracted with a polar solvent such as acetone and n-butanol. Although the alcohol extraction method is a relatively traditional extraction process, it is still widely used in the market for its simple and easy operation, and modern ideas have been incorporated to optimize the method. For example, by using orthogonal design, the best extraction and purification processes for total tanshinones were identified. The content of total tanshinones in crude extract increased from 18.23 to 57.29% by purification.
The ultrasonic extraction method does not require heating due to the short production cycle, thus avoiding the thermal degradation reaction of tanshinone IIA. Compared with traditional methods, such as methanol reflux extraction and organic solvents macerating at room temperature, ultrasonic-assisted extraction and microwave-assisted extraction of tanshinones have advantages such as extraction speed and high efficiency. Similarly, supercritical fluid extraction technology (SFE) has a short production cycle and does not require heating, which avoids the degradation reaction of tanshinone IIA. Compared with the traditional alcohol extraction method, SFE can retain more tanshinone IIA, but due to the expensive equipment and high production cost, it still needs further promotion to enter the market.
In addition, the extraction of tanshinones has many new methods and new technologies, such as non-ionic surfactant-assisted extraction, infrared-assisted extraction, ionic liquid-based ultrahigh pressure extraction, and selectively modified microfluidic chips. The combined application of methods can make extraction more efficient and productive, for example, ultrasound-assisted extraction combined with an amino-modified monolithic cartridge used as a solid-phase extraction sorbent to improve extraction efficiency; on-line continuous sampling combined with ionic liquid-based dynamic microwave-assisted extraction; a dynamic continuous ultrasound-assisted extraction with a high-intensity ultrasonic probe (CUAE-HIUP) combined with solid-phase extraction (SPE); and ionic liquid surfactant-mediated ultrasonic-assisted extraction combined with high performance liquid chromatography (HPLC).
Chromatography is commonly used for the separation and purification of tanshinone monomers. The semipreparative high-speed countercurrent chromatography (HSCCC) technique could successfully separate and purify the four major components of Danshen at high purities of over 95%. Furthermore, the high-efficiency separation of tanshinones could be achieved by the macroporous resin adsorption method and the countercurrent chromatography analog gradient elution method.
Pharmacological Activities of Tanshinones
Tanshinones have received extensive attention due to their remarkable activities in the clinical treatment of cardiovascular diseases. With deepening research, these compounds have been found to possess a wide range of pharmacological activities, such as antibacterial, antioxidant, anti-inflammatory, and anti-tumor properties. A brief introduction to the pharmacological activity of tanshinones will follow.
Anti-tumor Activity
There are many kinds of anti-tumor drugs, and their mechanisms of action are also different. For example, some drugs can interfere with the anabolism of nucleic acids, inhibit mitosis, and inhibit protein synthesis. Currently, most of the applied chemotherapy drugs are cytotoxic and cannot only kill cancer cells, but also cause side effects. The phenanthrene-quinone structure widely present in tanshinone compounds is a major cause of the cytotoxicity. During the antitumor reaction, the DNA molecule binds to the phenanthrene ring structure of the tanshinone, and the furan ring and steroid structure generate free radicals to hinder the synthesis of DNA in the tumor cells. Tanshinone IIA can regulate oxidative stress and reduce the mitochondrial membrane potential to induce apoptosis in cancer cells such as human lung cancer A549 cells; tanshinones inhibit the growth of human prostate cancer cell lines by arresting the cell cycle and inducing apoptosis in vitro. Among them, tanshinone I has the most significant activity with IC50 approximately 3–6 μM and its side effects on normal prostate epithelial cells are relatively small; cryptotanshinone induces tumor cell apoptosis by triggering cell cycle arrest, altering mitochondrial signaling pathways, and by other apoptosis-regulating proteins or death receptor pathways; the methanol extract of S. miltiorrhiza induced apoptosis in non-small cell lung cancer through phosphatase and tensin homolog (PTEN)-mediated inhibition of the PI3K/Alct pathway; S. miltiorrhiza exerted clear cytotoxic effects, and strongly inhibited the proliferation of HepG2 cells; and tanshinone I inhibits tumor angiogenesis by the phosphorylation of reducing signal transducers and activators of transcription (STAT3) at TYR705 and hypoxia-induced HIF-1a accumulation in both endothelial and tumor cells. In summary, tanshinones are expected to become a new type of anti-tumor drug with high efficiency and low toxicity.
Effects on the Cardiovascular System
Cardiovascular diseases refer to ischemic or hemorrhagic diseases in the heart, brain and whole body tissues caused by hyperlipidemia, blood viscous, atherosclerosis, and hypertension. S. miltiorrhiza and its compound preparations, such as composite Danshen droplet pills and Guanxin Danshen drop pills, have achieved good curative effects in the clinical treatment of cardiovascular diseases. Modern pharmacological research has found that S. miltiorrhiza has been widely used in the treatment of coronary artery, myocardial ischemia and myocardial infarction, improvement of microcirculation, and reduction of myocardial oxygen consumption.
Tanshinone IIA is a representative monomeric compound in S. miltiorrhiza. However, due to the poor water solubility, its water-soluble derivative, sodium tanshinone IIA silate (STS), is more widely used in the clinical treatment. Experimental results show that STS restrains the proliferation and migration of high glucose-induced vascular smooth muscle cells (VSMC), possibly through adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) activation. In addition, STS has protective effects on myocardial apoptosis induced by acute myocardial infarction (MI) in adult rats and inhibits cardiac fibrosis after pathological stimuli to the cardiovascular system. The clinical efficacy results also show that tanshinone IIA sodium sulfonate injection is effective in the treatment of acute cerebral infarction, and can also be used as an additional treatment for patients with unstable angina pectoris.
As the main lipophilic component of S. miltiorrhiza, tanshinone IIA also has cardiovascular protective effects. Its mechanisms of action mainly include the following: significantly reducing intimal thickening and restraining the proliferation and migration of VSMC, which is a significant event in the development of atherosclerosis (AS); by reducing the content of macrophages, reducing the uptake of oxidized low-density lipoprotein (ox-LDL), promoting cholesterol excretion, and reducing the formation of foam cells, thereby regulating blood lipids and reducing body weight; inhibiting the development of left ventricular hypertrophy and reducing the level of apoptosis; significantly reducing blood viscosity, inhibiting thrombin activation, promoting fibrin degradation and inhibiting the formation of thrombosis. In summary, tanshinone IIA has broad application prospects in the prevention and treatment of cardiovascular diseases, but its specific mechanism remains to be further studied. In addition to tanshinone IIA, cryptotanshinone, tanshinone I, dihydrotanshinone I, etc., have proved to be effective coronary dilators, which can improve microcirculation and enhance vascular blood flow.
Antioxidant Activity
Oxidative stress can cause DNA oxidative damage and abnormal expression of proteins puts the body in a vulnerable state, and is intently connected with the occurrence and development of various diseases. Previous studies have shown that tanshinone I, tanshinone IIB, cryptotanshinone, dihydrotanshinone I, methylenetanshinquinone, and miltiradiene were all found to act as antioxidants. The specific antioxidant activity of tanshinone IIA is that it can effectively restrain the interaction between DNA and the intracellular lipid peroxidation products and protect DNA by eliminating lipid free radicals and blocking the chain reaction of lipid peroxidation. In addition, tanshinone IIA inhibits atherosclerosis calcification in rats by inhibiting oxidative stress. Latter researches have indicated that it can also inhibit the activity and gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as the production of reactive oxygen species (ROS).
Untitled Document
Anti-bacterial Activity
The pharmacological effects of S. miltiorrhiza are very extensive, and in addition to the anti-tumor activity, which has been explored deeply, it has antibacterial and anti-inflammatory effects. The main lipophilic components that play the antibacterial roles in S. miltiorrhiza are cryptotanshinone, dihydrotanshinone I, hydroxyl tanshinone and tanshinone IIB. These compounds could significantly inhibit gram-positive bacteria, especially cryptotanshinone and dihydrotanshinone I exhibited strong antimicrobial activity. They could bring forth superoxide radicals, which are of great significance in the antibacterial action. In comparison, tanshinone IIA and tanshinone I were confirmed to have medium antimicrobial activity.
Effects on Neurodegenerative Disorders
Neurodegenerative diseases are caused by the loss of neurons or their myelin, which over time, in succession causes degeneration of the structure and function of the central nervous system. Neurodegenerative diseases could be divided into two categories according to their phenotype: one that affects body movement, like Parkinson’s disease, and one that affects memory and related dementia, like Alzheimer’s disease (AD). Due to the good lipophilicity and small molecular weight, tanshinones are able to penetrate the blood-brain barrier (BBB). For example, tanshinone I, tanshinone IIA, and cryptotanshinone have been reported to exhibit significant neuroprotective effects. Tanshinone I selectively suppressed pro-inflammatory gene expression in activated microglia and prevented nigrostriatal dopaminergic neurodegeneration in a mouse model of Parkinson’s disease. Tanshinone IIA protects neurons from the neurotoxicity of amyloid beta (Aβ) 25–35, increases neuronal viability and downregulates the expression of phosphorylated tau in AD. In addition, tanshinone IIA inhibits the transcription and expression of genes encoding inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-2, and human nuclear factor (NF-κBp65) pathway, thereby inhibiting the occurrence of neuroinflammation and reducing AD risk.
Other Bioactivities
In addition to the above mentioned pharmacological activities, tanshinone compounds exhibit anti-inflammatory effects, liver protection, protection against kidney damage, hormone-like activity, and so on. Methyl tanshinonate, hydroxytanshinone, tanshinone IIA, and tanshinone IIB show anti-platelet aggregation activity. Tanshinone IIA has the potential to ameliorate bone resorption diseases in vivo by reducing both the number and activity of osteoclasts. Cryptotanshinone and dihydrotanshinone I have been reported to have anti-cholinesterase activity. Due to their wide range of pharmacological activities, tanshinones are expected to contribute to the development of new drugs for clinical treatment.
Biosynthesis of Tanshinones in S. miltiorrhiza
Terpenoids are derived from methylpentahydroxy acid and have a molecular skeleton of an isoprene unit (C5 unit) as a basic structure unit. Terpenoids are widely found in nature and are also important class of compounds in Chinese herbal medicine. According to the number of C5 units, terpenoids can be divided into monoterpenes, sesquiterpenes, diterpenes, and triterpenes, in addition to the more specific structure of iridoids. The biosynthesis pathway of terpenoids has certain rules to follow. The common precursor iospentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are generated by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and/or the mevalonate (MVA) pathway. Under the action of prenyltransferase, IPP and DMAPP condense to form precursors of monoterpenes, sesquiterpenes, diterpenes, and similar compounds. These precursors form the basic skeleton of various terpenoids under the action of a quinone synthase, and then form a final product under the action of a modifying enzyme such as cytochrome P450 hydroxylase or glycosyltransferase. Tanshinones are a group of diterpenoids, and the biosynthesis pathway in S. miltiorrhiza also follows the above rules. Interference experiments have shown that the MVA pathway plays a more critical role in the cell growth of S. miltiorrhiza hairy roots, while the MEP pathway is more committed to the production of tanshinones. Of course, the interrelationship and crosstalk between the two pathways together promote cell growth and metabolite production.
Conclusion and Prospects
As a perennial cross-pollinating plant species, S. miltiorrhiza is strongly adaptive and widely distributed with generations having overlapping features and significant genetic diversity. Since ancient times, the single-component Danshen or its combination with other drugs has been used in the clinical treatment of various diseases exhibiting good effects. S. miltiorrhiza contains a variety of active ingredients, among which tanshinones have exhibited significant pharmacological properties, such as antibacterial, antioxidant, anti-inflammatory, and anti-tumor activities. In this paper, the extraction and purification methods of tanshinones are briefly introduced. Many new methods and new technologies have been applied, and it is worth mentioning that the combination of different methods can achieve high efficiency and low consumption and may have promising development prospects.
The traditional method of obtaining tanshinones has been unable to meet the clinical drug needs. In recent years by continuously investigating the functional genes in the tanshinone biosynthesis pathway, analyzing the complete pathway and reconstituting the biosynthetic pathway in heterologous hosts, the method of efficiently and heterologously producing tanshinones has become the focus of attention. To date, studies have shown that miltiradiene is an intermediate for the biosynthesis of tanshinones, and that it can be converted to tanshinones under the action of various modified enzymes. Therefore, more functional genes must be further discovered. Synthetic biology strategies and methods not only provide ideas for more efficient and convenient access to tanshinones, but also lay the foundation for the sustainable use of traditional Chinese medicine resources.
