Hydroxysafflor Yellow A: A Systematical Review on Botanical Resources, Physicochemical Properties, Drug Delivery System, Pharmacokinetics, and Pharmacological Effects

Hydroxysafflower yellow A (HSYA), as a principal natural ingredient extracted from safflower (Carthamus tinctorius L.), has significant pharmacological activities, such as antioxidant, anti-inflammatory, anticoagulant, and anticancer effects. However, chemical instability and low bioavailability have been severely hampering the clinical applications of HSYA during the treatment of cardiovascular and cerebrovascular disease. Therefore, this present review systematically summarized the materials about HSYA, including acquisition methods, extraction and detection methods, pharmacokinetics, pharmacological effects and molecular mechanism, especially focus on the possible causes and resolutions about the chemical instability and low bioavailability of HSYA, in order to provide relatively comprehensive basic data for the related research of HSYA.


INTRODUCTION
Cardiovascular and cerebrovascular disease (CCD), as one of the leading causes of mortality worldwide, has been increased rapidly and presented younger trend, with high mortality all of the world (Collins et al., 2017;Donahue and Hendrikse, 2017). Although many types of therapeutic strategies were used to treat patients with CCD, such as angiotensin converting enzyme inhibitor (ACEI), ß-receptor blocker, and statins, the outcome remains not satisfactory due to the inevitable side effects and high treatment expenditure. To address the problem, more and more studies are trying to seek treatment strategy from Traditional Chinese Medicine (TCM). Characterized by minor side effects, TCM has become an important source of natural product, such as Aspirin, Digoxin, Hydroxysafflor yellow A, which exhibit substantially protective effects against CCD (Eichhorn and Gheorghiade, 2002;Dai and Ge, 2012;Desborough and Keeling 2017;Hu et al., 2020).
Hydroxysafflor yellow A (HSYA) is a primary active product derived from safflower (Carthamus tinctorius L.), a plant of the Compositae (Asteraceae) family, which was used to improve blood circulation, eliminate blood stasis, and relieve menstrual pain as early as recorded in Kaibao Bencao. HSYA, as an indicator component to characterize the medical value of safflower recorded in The Pharmacopoeia of the People's Republic of China from 2005 edition, possesses a broad spectrum of pharmacological activities, such as antioxidant, anti-inflammatory and anticoagulant effects, which play an important role acting on cardiovascular and cerebrovascular disease Wu et al., 2012;Ma et al., 2019a;Zhou et al., 2019;Bacchetti et al., 2020). However, chemical instability and low bioavailability of HSYA severely hamper the clinical applications. It can be easily oxidized, hydrolyzed, polymerized by light, high temperature, and alkaline conditions due to its structural characteristics. The high polarity directly leads to difficulty of transmembrane transport, resulting in low bioavailability. To address these problems, new drug delivery systems were developed to improve the therapeutic efficacies of HSYA based on lipid-based carriers, such as microemulsions, selfemulsifying systems, nanoparticles, chitosan, and the combination of HSYA with other drugs, which may have a good application prospect.
The present review systematically summarized the literatures about HSYA, including botanical resources, extraction and detection methods, pharmacokinetics, pharmacological effects and molecular mechanism, especially focus on the possible causes and resolutions about the chemical instability and low bioavailability of HSYA, in order to provide relatively comprehensive basic data for the related research of HSYA.

ACQUISITION OF HYDROXYSAFFLOWER YELLOW A
HSYA is mainly extracted from safflower, but the amount is not enough to support current clinical applications. Therefore, it is urgent find other ways to obtain HSYA. At present, chemical synthesis and biosynthesis are two promising ways to obtain HSYA. The information was detailed as follows.

Acquisition From Safflower
Safflower, as the natural source of HSYA, is widely planted worldwide. It is said that safflower originated in West Asia (Iran, Nicaragua and Turkey), and were later introduced for cultivation on almost every continent except Antarctica, such as America, Australia, China, Ethiopia, India, Italy, Mexico, Spain, and so on (Figure 1). India has developed into the most productive country with the planting areas of over 760,000 hm 2 , and the yield of about 460,000 t, accounting for 50% of the total area and yield in the world (Liang et al., 2015).
Safflower is also widely cultivated in China with planting area of about 30,000-58,000 hm 2 , and dried flowers yield of about 1,500-2000 t every year. Xinjiang province has become the major safflower production area, covering an area of 16,700-40,000 hm 2 , which accounts for more than 3/4 of the total planting areas and provides more than 80% dried flowers and seeds in China. Henan province (Weihonghua, 卫红花), Zhejiang province (Duhonghua, 杜 红花) and Sichuan province (Chuanhonghua, 川红花) are described as the authentic product areas of safflower in the history of China.
The content of HSYA containing in safflower were affected by many factors, including geographical origins, color and harvest time. The content of HSYA ranged from 0.05 to 14.99 mg/g by comparing 80 safflower cultivars collected from Africa, America, Asia, and Europe. HSYA in Africa cultivars was higher than that in Asia and Europe, and China cultivars is higher than that in Turkey, India and Kenya. Moreover, color is another factor to influence the content of HSYA. The darker the color of safflower, the higher the content of HSYA (red > orange > yellow > white according to PANEONE) (Tong et al., 2011;Xu et al., 2018). For example, Hebei safflower (red) was richer in HSYA (26.943 mg/g) than that in Wei safflower (white flowers, 0.472 mg/g) in China (Zhao, 2015). The most appropriate time to pick safflower is the morning of the third or fourth day after flowering (Tian et al., 2007).

Oxidation Synthesis Pathway
Chemical synthesis is an efficient way to obtain the natural or unnatural products within a short time period. Oxidation synthesis for HSYA is a rapid and highly efficient chemical synthesis method, and the synthetic pathway was shown in Figure 2A.

Biosynthetic Pathway
Biosynthesis is a multi-step enzymatic process, in which a simple product is converted into a more complex desired product in a living organism. Biosynthesis is characterized by continuous and effective production, low-carbon and friendly environment, which provides a great support to the development of natural products (Pang et al., 2015). This production model will be the main source of HSYA in the future. However, the biosynthetic pathway of HSYA in plant remains unclear. According to the vital reaction of chalcone biosynthetic pathway, one molecular of 4-coumaroyl-CoA and three molecules of malonyl-CoA are converted into naringenin chalcone (4,2′,4′,6′-tetrahydroxychalcone) via the intervention of chalcone synthase, and it was transformed into HSYA after glycosylated (Heller and Hahlbrock, 1980;Knogge et al., 1986). The possible biosynthetic pathway of HSYA in phytosomal was shown in Figure 2B.
The content of HSYA is controlled by a nuclear gene with two alleles, HSya and hsya gene. HSya gene dominates completely over hsya gene to promote HSYA biosynthesis (Zhang et al., 2009;Yang et al., 2011). sHSP is a small heat shock protein and encoded by CTL-hsyapr, which might be directly or indirectly disturb HSYA biosynthetic pathway (Tang et al., 2010). When safflower is under external pressure, CT-wpr (TDF-11) was activated, which might arouse sHSP and inhibit the expression of HSya to some extent, finally leading to the inhibition of biosynthetic pathway of HSYA (Li et al., 2010b).

EXTRACTION AND DETECTION Extraction Methods
HSYA is highly soluble in water, while hardly dissolve in lipophilic solvents such as ethyl-acetate, ether, benzene, and   chloroform. So, the most general and traditional extraction method to get HSYA is water immersion. However, water immersion has the characteristic of low yield and high consumption of raw materials (e.g., the yield of 0.023%, 800 g of safflower; the yield of 0.066%, 2000 g of safflower) (Bai et al., 2012;Li et al., 2013a). Simultaneously, high temperature, alkaline conditions, and illumination all accelerate the degradation of HSYA in the traditional procedure. As shown in Table1, many other extraction systems were developed. Smashing tissue extraction holds an absolute advantage in time consumption compared with other extraction systems, with a yield of 1.359% in two minutes . MAS-I microwave extraction system with a solid and liquid ratio of 100 maintained at 70°C for 3 cycles in 20 min could obtain HSYA with higher yield of 6.96% . Hong et al. (2015) compared the effectiveness of MSPD, ultrasound extraction, and Soxhlet extraction methods. MSPD system obtained the highest yield at 14.89% compared with ultrasonic (12.25%) and Soxhlet extraction (13.09%), and also achieved the lowest consumption of raw materials.
Solvents also affect the extraction efficiency. Li et al. (2016) developed the DMSO extraction method as follows: Soak 2.0 g safflower with 14 volumes DMSO and stir for 30 min to remove impurities at room temperature in dark environment. Then soak the filter residue with 14 volumes of DMSO at 80°C for 1 h before heating for 50 min in the same environment. Soak the filter residue with another 12 volumes DMSO and repeat the steps above. Then add 3 volumes butyl acetate to the filtrate and centrifuge to obtain crimson precipitates. Finally, wash the precipitates with an appropriate amount of ethanol before drying to obtain light yellow powder with yield at 14.56%. DMSO is a "universal solvent" in areas of pharmaceutical sciences and cell biology, so it is inevitable to dissolve a lot of impurities during the extraction process, and the toxicity of DMSO is difficult to remove. Therefore, this method is not recommended to be extensively promoted.

Detection Methods
Several detectors coupled with a liquid chromatography (LC) system have already been used for HSYA detection, including diode (DAD) (Qi et al., 2007), electrochemical (ECD), mass spectrometer (MS), and ultraviolet (UV). In addition, the novel multilayer porous silicon-based immunosensor has also been applied for HSYA detection. Detailed information is provided in Table 2.
MS is the most effective detector for qualitative analysis of HSYA with a high detection sensitivity, selectivity and lowinterference. HSYA and its metabolites can be accurately identified, based on mass-to-charge ratio (m/z) and fragmentation patterns.
The content of HSYA in human plasma was determined after oral administration of safflower. LC-MS/MS method with isocratic elution system composing of methanol and 5 mM ammonium acetate (80:20, v/v) was proved with a linear range of 1-1,000 ng/ml, a correlation coefficient ≥0.999 (Wen et al., 2008).  optimized the sensitivity and selectivity of this method to be more simplified and effective. The researchers replaced the Thermo syncronis C8 with Agilent ZORBAXSB C18, added ammonium acetate to the mobile phase and increased concentration dilution ratio. The modified method reduced the injection volume, improved the response intensity and peak shape, and shortened the retention time.
UPLC-DAD-MS method had the advantages of fast detection speed, good peak shape and less solvent consumption. Twentyeight compounds were identified only in 30 min, including HSYA from Xue Fu Zhu Yu decoction, a classic prescription of TCM .
UPLC-TOF-MS is the most powerful tool with accurate activity measurement and full spectral sensitivity to determine HSYA using gradient elution with acetonitrile and 0.1% (v/v) formic acid aqueous solution in ESI + . This method showed good linearity (r 2 ≥ 0.9992) and precision (RSD ≤3.4%) with the limits of detection (LOD) at 35.2 ng/ml (Hong et al., 2015). Jin et al. (2016b) successfully developed a UFLC-Q-TOF-MS method to detect HSYA and its multiple metabolites in the plasma, bile, urine and feces of SD rats after oral administration with HSYA using the mobile phase consisting of methanol and 0.5‰ acetic acid in water.
The analysis speed of UV detector for HSYA is very fast, but its sensitivity and selectivity are slightly weaker than that of MS system (Chen et al., 2010). Novel multilayer porous silicon-based immunosensor is an easy way to alter the etching current periodically, which fabricated of pSi photonic crystal. The linear relationship was ranging from 1 to 3 g/ml and detection limit at 0.78 ng/ml for HSYA detection (Lv et al., 2011).

Physical Properties
HSYA, a C-glucosyl quinochalcone, is a yellow amorphous powder with a molecular formula of C 27 H 32 O 16 (Meselhy et al., 1993). It is usually used as a dye owing to its attractive color. It shows maximum absorption at 403 nm due to p-conjugated system coupled with several hydroxyl groups (Ma et al., 2014). The C-glycoside bond, located between the 1,3-diketone on the ring A, is unstable in HSYA. The hydroxyl group located at the C-2 position in the glycoside is easily condensed with the adjacent enol due to the strain effect. The pyranose ring is opened, and then forms an oxyfuran [3,2 days] benzofuran ring by a cyclization reaction (Suzuki et al., 2017). HSYA is easily degraded by light, high temperature, and alkaline conditions. HSYA emits weak fluorescence at 450 nm in aqueous solution owing to the lack of rigid planar configuration in the molecular structure, and borax can significantly increase the HSYA fluorescence intensity by 20 times (Cao et al., 2020). Due to the existence of phenolic hydroxyl groups, HSYA exists in protonated form in natural or alkaline aqueous solutions, which seriously affects its transmembrane ability and leads to low bioavailability.

Chemical Stability
Effect of pH HSYA is easy to degrade under alkaline conditions. Pu et al. (2017) had illustrated the pH profile (1-14) of HSYA stability in aqueous solution follows an inverted V curve, and it was most unstable at pH 9. When HSYA was transferred from the aqueous solution to the buffer solution at pH 9.16, the UV absorbance was red-shifted from 404 nm to 426 nm, with the gradually increased absorbance of degradation products at 300 and 380 nm, which indicated that the electron cloud density of the conjugate system increased after rapid ionization under alkaline conditions. As shown in Figure 3, there were two degradation products proposed in HSYA aqueous solution. The hydroxyl group at C-2ʹ was ionized under the moderate solution (pH 7-9). Intramolecular nucleophilic attacks Cβ and add O 2 to the α, ßunsaturated double bond. The two products are isomers, which obtained after hydrogen migration. There were chalcone, flavones, and carbanion intermediates detected in HSYA acidic neutral and aqueous solution. However, only flavones were found under strong alkaline conditions (pH 13).

Effect of Temperature
When HSYA was incubated in boiling water under dark conditions for 0, 0.5, 1, 2, and 4 h, the degradation rate of HSYA was increased gradually detected by HPLC system. The possible mechanism of HSYA degradation was that the colorless glycoside of HSYA bonded with H 2 O to form conjugate system, which reacted with the adjacent enol to transform the chromophore structure under the high temperature (Yue et al., 2003;Li et al., 2009). HSYA can be directly hydrolyzed and transformed into p-coumaric acid. And the hydroxyl group at the 2-position of C-glycoside of HSYA can be also condensed with the adjacent enol on A ring, and oxidized by O 2 at pH 8 under the high-temperature condition, then obtained the degradations ( Figure 4). Due to the instability of the degradations as enols, a mixture of the two degradations were obtained (Fan et al., 2011).

Effect of Light
HSYA degrades when exposed to light, so it is generally stored in dark conditions. When distilled water solution of HSYA was exposed under sunlight, ultraviolet, incandescent light, and the dark environment respectively, the content of HSYA decreased in turn analyzed by HPLC . The content of HSYA in aqueous solution decreased when stored under natural light at room temperature for 20 days (Wang, 2017).

DRUG DELIVERY SYSTEM
A drug delivery system is responsible to control the release rate, extend the duration of drug action, and eliminate side effects, which can be divided into 4 types, including lipid-based carriers, polymer nanoparticles, inclusion complexes, and capsulations. At present, the studies on the HYSA drug delivery systems mainly focuses on lipid-based carriers, which can improve the bioavailability by reducing the high-water solubility of HSYA. Moreover, microemulsions, self-emulsifying systems, and nanoparticles can also enhance the transmembrane capacity of HSYA. In this review, we also summarized other drug deliveries, such as chitosan, and the combination of HSYA with other drugs. A detailed description is provided in Ahmed and Aljaeid, 2016, Figure 5.

Microemulsions
Microemulsions are stable liquid solution consisted of water, oil, surfactant, and co-surfactant, with the characteristic of isotropic and thermodynamics. They are transparent or translucent with small droplet size, typically up to 150 nm (Lopes et al., 2014;Lv et al., 2018). Microemulsions are used to increase the permeability of hydrophilic peptides by enhancing the fluidity of cell membranes and opening tight connections between cells, which is a potential tool for hydrophilic drug molecules. Qi et al. (2011) investigated the bioavailability of HSYA microemulsion by intraduodenally administration.   Frontiers in Pharmacology | www.frontiersin.org December 2020 | Volume 11 | Article 579332 8 Self-emulsifying drug delivery system (SDEDDS) is a kind of the microemulsions. It could rapidly and spontaneously form a microemulsion in the gastrointestinal tract, where the peristalsis of the gastrointestinal tract and small intestine provides the necessary agitation for emulsification. SDEDDS have multiple advantages, including 100% drug entrapment capacity, physically stable formulations, no dissolution required, and submicron droplet sizes (Ghasemiyeh and Mohammadi, 2018). SDEDDS of HSYA were synthesized by inner water phase of 0.5% gelatin solution, and the external oil phase of bean phospholipids, medium chain triglycerides, Tween 80, oleic acid, and labrasol (20/65/7.4/2.5/0.1, in wt%). The study demonstrated that SDEDDS significantly improved the permeability of HSYA through Caco-2 cells monolayers, and plasma concentration increased by 2.17 times. The apparent permeability coefficient (Papp) of HSYA and HSYA-SDEDDS were (3.52 ± 1.41) × 10 −6 cm/s and (6.62 ± 2.61) × 10 −6 cm/s at the same concentrate (0.4 mg/ml), which improved to 1.88-fold by SDEDDS (Lv et al., 2012).
Labrafac lipophilic WL1349 (WL1349) is a medium-chain triglyceride that can be digested, absorbed, and hydrolyzed by pancreatic lipase after being emulsified by an endogenous emulsifier (such as bile). Preparation of HSYA-phospholipid complex increased lipophilicity, and dissolved in WL1349 to form a stable oil solution (a lipid-based preparation). Compared with HSYA aqueous solution, the oral bioavailability of HSYA-phospholipid complex WL 1349 oil solution in rats increased by about 37 times (Cmax of 2.79 μg/ml vs. 0.08 μg/ml within 24 h) and reduced the excretion of the drugs (8.80 ± 2.30% vs. 44.66 ± 8.00% in feces within 24 h) (Wang et al., 2008;Li et al., 2010a).

Nanoparticles
Solid lipid nanoparticles (SLN) is the earliest lipid-based nanocarriers formulated from lipids with a submicron size less than 1,000 nm, which are solid at body temperature and stabilized by emulsifiers (Koga et al., 2010). They can protect drugs against harsh environmental and are easy to mass-produce. However, owing to the crystalline structure existed in SLN, the drug-loading efficiency is poor. Some of the main lipids that have been used so far are monostearic acid, stearyl alcohol, stearic acid, glyceryl monostearate, cetyl palmitate, poloxamer 188, Tween 80, and dimethyl octadecyl ammonium bromide (DDAB) (Lee et al., 2016;Tapeinos et al., 2017;Ghasemiyeh and Mohammadi, 2018). As reported by Zhao et al. (2017), 1% Tween 80 was used as an emulsifier. HSYA-SLN with w/o/w structure prepared by micro emulsification procedure significantly improved oral bioavailability. HSYA SLNs is spherical with an average diameter of 174 ± 20 nm, zeta potential of −12.4 ± 1.2 mV, and the encapsulation efficiency is 55%. The SLNs of HSYA were stable within ten days at 4 or 30°C. SLNs of HSYA increased the oral bioavailability of HSYA in rats about 3.97-fold. It also significantly enhanced the C max and AUC by 7.76 and 3.99 folds. The pharmacodynamic evaluation showed that HSYA-SLNs had a better therapeutic effect on the cerebral ischemia rats compared to HSYA aqueous solutions (Zhao et al., 2018).

Others Delivery System
The combination of Ligusticum chuanxiong volatile oil (CVO) and HSYA also improved the bioavailability in rats. When HSYA co-administered with CVO of 0.02 mg/ml, the bioavailability of HSYA in rats was increased by 6.48 folds. The emulsification of CVO increased Papp of HSYA and the paracellular transport by opening the integral tight junction of Caco-2 cells. Chitosan, a kind of biological polysaccharide, is a molecule usually obtained by deacetylation of chitin with the carbohydrate backbone structure similar to cellulose, which is composed of two kinds of repeating units, N-acetyl-D-glucosamine and D-glucosamine, combined with (1-4)-β-glucoside linkage. It is characterized by the presence of a lot of amino groups on the chain (Ahmed and Aljaeid, 2016;Hong et al., 2017). HSYA-Chitosan complex effectively improved the oral absorption of HSYA, and the bioavailability increased to 476% (Ma et al., 2015).
Drug delivery system has great potential to improve the bioavailability and chemical instability of HSYA. However, there are also some several challenges, such as high preparation cost and poor promotional effect, which need further improvement.

PHARMACOKINETICS
HSYA is one of the representative chemical compounds of biopharmaceutics classification system (BCS) class III drugs.
Caco-2 cell monolayer model was usually used to study the transmembrane characteristic of HSYA, and the result prompted that the absorption of HSYA is basically in line with the passive diffusion. P-gp inhibitors (verapamil) and energy metabolism inhibitors (sodium azide) failed to block the intake of HSYA, which demonstrated that the absorption of HSYA is irrelevant to the P-gp protein (Zhou et al., 2014). However, this conclusion needed more evidence to verify owing to the opposite result in another study (Wang et al., 2009). The peak concentration of HSYA generally appeared at 10 min after oral administration (Li et al., 2007). A study has shown that healthy volunteers received intravenous injections of 35, 70, and 140 mg of HSYA, the elimination half-life values (t 1/2 ) of HSYA was 3.32 h, and the C max was (2.02 ± 0.18), (7.47 ± 0.67), (14.48 ± 4.70) mg L −1 , respectively, (Qiao et al., 2009). HSYA has a low plasma protein binding rate (48%-54.6%, 72 h, i. v.), and it has no competitive binding to other drugs. So, HSYA is highly safe in vivo (Chu et al., 2006). Yang et al. (2009) suggested that the therapeutic effect of the single-dose HSYA indicated proportional to the dose ranging from 35 to 140 mg/kg, which conformed to first-order kinetics in    HSYA and ginsenoside Rb1 Luo et al. (2020) Frontiers in Pharmacology | www.frontiersin.org December 2020 | Volume 11 | Article 579332 healthy Chinese female volunteer. The t 1/2 corresponded to the median of the range from 2.23 to 4.63 h (the average value of 3.17 h) at each dose. But this research had some potential limitations, such as a low-volume sample and only female volunteers, which lack of universality. Moreover, the combination therapy of HSYA and protocatechuic aldehyde greatly promoted the intake of HSYA (Ao et al., 2018). HYSA is reduced and hydrolyzed, such as hydroxylation, hydroxylation and methylation, dehydration, hydrogenation and hydration, to obtain phase I metabolites under the action of hepatic microsomal drug-metabolizing enzyme in the liver. Phase II metabolism included acetylation and glucuronidation, dehydration, hydrogenation, hydration, hydroxylation with glucuronidation, deglycosylation, methylation and glucuronic acid conjugation reactions (Jin et al., 2016b;Wu et al., 2018). The possible metabolic pathways were summarized in Ahmed and Aljaeid, 2016, Figure 7. HSYA and its metabolites are distributed in the heart, liver, spleen, lung, kidney, brain and intestines. In addition, it is almost impossible to cross the healthy blood-brain-barrier (BBB), but easily to penetrate damaged BBB (Sheng et al., 2019).
The low bioavailability of HSYA directly blocks its therapeutic efficiency. It is difficult for HSYA to pass through phospholipid bilayer because of its strong polarity and poor membrane permeability, which is also an important reason for its low bioavailability. Therefore, the design of HSYA delivery systems mainly focused on improving its lipsolubility. Despite, HSYA exists as an undivided molecule in the strong acid environment of the stomach, it cannot be absorbed by the stomach due to its high molecular weight and strong hydrophilicity (Zhang, 2006). Besides, the small intestine actually is the main absorption site of HSYA. However, gastrointestinal metabolism in a weak alkaline environment is not conducive to the absorption of HSYA, on the contrary, it promotes degradation (Wu et al., 2018).

PHARMACOLOGICAL EFFECTS AND MOLECULAR MECHANISM
HSYA shows excellent therapeutic effects on various diseases, such as cardiovascular and cerebrovascular diseases, cancer, and so on. This part provided the biological activity of HSYA in detail (Table 3, Figure 8).

Cardioprotective Effects
HSYA has been proved to be a superior agent on the cardioprotective system in vivo and in vitro. HSYA (5 mg/kg, 30 min before ischemia, i. p.) was found to improve ischemia/ reperfusion (I/R) injury by reducing the releases of cTnI, IL-6. LDH and the myocardial infarction size . Similarly, HSYA (4 or 8 mg/kg) could reduce the expression of MIS, CK-MB, and MDA in experimental acute myocardial ischemic model, which induced by left anterior descending coronary artery (LAD) ligation (Tu et al., 2009). Furthermore, HSYA combined with nitroglycerin showed a better therapeutic action on acute myocardial infarction than HSYA alone, which produced a marked increase in SOD, eNOS, and NO content.
The mechanisms of HSYA on cardioprotective effects are related to antioxidant, free radical scavenging abilities, and antiinflammatory activity. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for the regulation of cellular redox balance and protective antioxidant and phase II detoxification responses in mammals. On basal condition, Nrf2 binding with the chaperon protein Kelch like-ECH-associated FIGURE 8 | Schematic showing multiple signaling pathways of HSYA. The four crucial signaling pathways were displayed above, including NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway (Erk1/2, JNK, p38 signaling pathway), and Nrf2/HO-1 signaling pathway.

Neuroprotective Effect
HSYA showed excellent neuroprotective effect. HSYA injection (50 mg/d and 75 mg/d) in patients with acute ischemic stroke of blood stasis syndrome might be to undergo for a phase III clinical trial (Zhu et al., 2020). HSYA (2 mg/kg, tail vein injection) protected the C57BL/6J mice against middle cerebral artery occlusion (MCAO) by decreasing the expression of inflammatory genes factors, including TNF-α, IL-1β, and NO (Lv et al., 2015). HSYA (0.6 mg/100 g, tail vein injection) could also improve spatial learning and memory ability of vascular dementia model rat by promoting angiogenesis and increasing synaptic plasticity . Moreover, HSYA (6 mg/kg per day, 2 weeks, i. v.) could reduce the accumulation of amyloid precursor protein, improve synaptic function and reverse homocysteine (Hcy) induced cognitive impairment in Alzheimer's disease mice (Lu et al., 2013). The neuroprotective effect of HSYA might be related to the regulation of TLR4, NF-κB, p-p65, MAPK, PI3K/Akt and Nrf2/ HO-1 signaling pathways (Wei et al., 2005;Lv et al., 2015;Deng et al., 2018). NF-κB is a family of dimeric transcription factors central to regulate immune development, immune responses, inflammation, cancer, and other diseases. HSYA inhibited NF-κB to exert neuroprotective effect (Yu et al., 2020). Similarly, HSYA (20 mg/kg for 28 days) could improve motor dysfunction in C57/ BL6 mice model of Parkinson by promoting the expression of BDNF, p-TrkB/TrkB, DRD3, p-PI3K/PI3K and p-AKT/AKT .

Anticancer Effect
Some studies have found that HSYA exerted anticancer activity in several cancer cells, such as human umbilical vein endothelial cells (HUVEC), HepG2 cells, Skov3 cells, as well as some in vivo studies. HSYA could eliminate reactive oxygen species (ROS), prevent apoptosis from membrane permeability, and inhibit proliferation and migration (Ma et al., 2019a;Chen et al., 2019;Zhang et al., 2019). HSYA also increased the thymus index, and effectively down-regulate the mRNA levels of forkhead box P3-positive (Foxp3) and retinoic acid receptorrelated orphan receptor-gamma-t (RORγt), which contributed to improve the tumor immune microenvironment-the key points of tumor cell proliferation and invasion (Ma et al., 2019b). What is particularly noteworthy is the anticancer abilities of HSYA might related to the excellent antioxidant effect.

Anticoagulant Effect
PAF, as the most effective platelet activator known so far, has a wide range of biological activities, and can be synthesized by a variety of cells such as platelets, leukocytes, endothelial cells. Anticoagulant effect of HSYA plays a significant mediating role in many pathological processes, such as tissue IR injury, coronary heart disease, atherosclerosis, cerebrovascular disease and many other cardiovascular diseases. HSYA dosedependently inhibited the specific binding between [ 3 H] PAF and washed rabbit platelet, which is considered as a new generation PAF receptor antagonist in the future (Zang et al., 2002). PAF-activated human small airway epithelial cells model was pretreated with HSYA. Guo et al. (2018) proposed that HSYA could attenuate the PAF-induced inflammatory factors, destruct cell-barrier function, and inhibit the expression of protein kinase C, MAPK, activator protein-1, and NF-κB activation to show anticoagulant effect.

Other Pharmacological Effects
In addition to the biological activities discussed above, HSYA also has other diverse pharmacological effects, such as immunodeficiency, anaphylactoid, hepatic fibrosis, pulmonary arterial hypertension, skin photosensitivity, Type 1 diabetes, vascular dementia, and so on.
HSYA is an immunomodulator that regulates the inflammatory response induced by lipopolysaccharide in various cells, including human non-small lung cancer cell line (A549 cells), H1299 and Eahy926 human endothelium cells. It could inhibit many kinase activities related to inflammatory factors, such as TNFα, IL-1β, and IL-6. HSYA could also inhibit the pro-inflammatory proteins expression, such as NF-κB p65, PI3K/Akt/mTOR, MAPK TLR-4, Myd88 and ICAM-1 (Han and Zhao, 2010;Jin et al., 2016;Jiang et al., 2019. HSYA was also proved to relieve certain respiratory conditions by decreasing mRNA levels of ICAM-1 and E-selectin elevation (Liu et al., 2014;Zhang et al., 2017). Moreover, another study implemented by Zheng et al. (2019) showed that HSYA could attenuate ovalbumin-induced allergic asthma in guinea pigs.
HSYA possesses a strong vascular relaxation effect on pulmonary arterial hypertension (PAH). It reduced the vascular tension by activating the Kv channel in pulmonary vascular smooth muscle cells (PVSMCs) (Bai et al., 2012). HSYA (6 mg/kg for 11 days) promoted angiogenesis in ischemic mice. The therapeutic mechanisms of HSYA might be associated to significantly increase the capillary-like tube formation and migration of HUVEC, enhance the expression of angiopoietin 1 and Tie-2, phosphorylations of Tie2, Akt and extracellular signal-regulated kinase 1/2 (Chen T. et al., 2016).
Frontiers in Pharmacology | www.frontiersin.org December 2020 | Volume 11 | Article 579332 It is also reported that HSYA could exert protection on macroscopic skin lesions induced by ultraviolet rays (UV). It could promote the skin to regain its initial shape, elevate the activities of skin anti-oxidant enzymes, increase skin collagen content and maintain the structural integrity of the skin (Kong et al., 2013).
The combination of HSYA and deferoxamine (DFO) was discovered to improve type 1 diabetes by accelerating diabetic wound healing, promoting angiogenesis, reducing the inflammatory response, and up-regulating the expression of hypoxia-inducible factor-1 a (HIF-1 α) (Gao et al., 2018).

DISCUSSION AND CONCLUSION
In the present review, we systematically summarized the materials about HSYA, including acquisition methods, extraction and detection methods, pharmacokinetics, pharmacological effects and molecular mechanism. HSYA is proved to be an excellent antioxidant, anti-inflammatory and anticoagulant agent, so it plays an exceptional role in the treatment of cardiovascular and cerebrovascular diseases by down-regulating NF-κB signaling pathways, inhibiting MAPK signaling pathways, and attenuating the activation of Nrf-2/HO-1 signaling pathways. However, it is easily to degrade in the process of storage, extraction and separation procedure due to its chemical instability, which brings great challenges to the application of HSYA. Another major challenge is the low bioavailability caused by strong polarity. A large number of studies have been carried out to improve the chemical instability and bioavailability. Microemulsions, self-emulsifying systems, nanoparticles and other drug delivery systems have gradually improved the bioavailability, chemical stability, cellular uptake and biological activity of HSYA, which will be evaluated in clinical trials later.
Although great progress has been made in the research and application of HSYA in the past few decades, there are still many problems and challenges. First of all, chemical instability and low bioavailability of HSYA are still far from being resolved, which is still a key issue of the future research. It is important to continue the development of viable drug delivery systems for HSYA. The crucial aspects will involve in the enhancement of the solubility and bioavailability, as well as methods for selectively targeting these delivery systems to disease sites. Secondly, HSYA has been used to treat cardiovascular and cerebrovascular diseases clinically in China, but the drug target has not been completely revealed at present, which needs in-depth study later. Finally, the approach of obtaining HSYA from plants is cumbersome and complicated, and the yield is unsatisfactory. Chemical synthesis might pollute the environment, so it does not recommend to popularize. Biosynthesis is characterized by high efficiency, energy saving, and environmental protection, and it will transform into the major source of HSYA in the near future.
In short, it is the first time to systematically summarize the basic information about HSYA, which might provide relatively comprehensive basic data for the related research of HSYA.

AUTHOR CONTRIBUTIONS
HX, FZ and PW performed the frame design of the manuscript. FZ collected the data and drafted the manuscript. YJ, XZ and DC helped to organize the data. PW revised the manuscript. All authors read and approved the final manuscript. The funding agencies had no role in the study design, the collection, analysis, or interpretation of data, the writing of the report, or the decision to submit the article for publication.