Chemical Constituents, Pharmacologic Properties, and Clinical Applications of Bletilla striata

Bletilla striata is a plant from the Orchidaceae family that has been employed as a traditional Chinese medicine (TCM) for thousands of years in China. Here, we briefly review the published studies of the last 30 years that were related to chemical constituents, pharmacologic activities, and clinical applications of B. striata. Approximately 158 compounds have been extracted from B. striata tubers with clarified molecular structures that were classified as glucosides, bibenzyls, phenanthrenes, quinones, biphenanthrenes, dihydrophenanthrenes, anthocyanins, steroids, triterpenoids, and phenolic acids. These chemicals support the pharmacological properties of hemostasis and wound healing, and also exhibit anti-oxidation, anti-cancer, anti-viral, and anti-bacterial activities. Additionally, various clinical trials conducted on B. striata have demonstrated its marked activities as an embolizing and mucosa-protective agent, and its application for use in novel biomaterials, quality control, and toxicology. It also has been widely used as a constituent of many preparations in TCM formulations, but because there are insufficient studies on its clinical properties, its efficacy and safety cannot be established from a scientific point of view. We hope that this review will provide reference for further research and development of this unique plant.


INTRODUCTION Dihydrophenanthrenes
Dihydrophenanthrenes, phenanthrenes, and benzyls are stilbenoids with an identical 1,2-distyrene skeleton. The substituents on their aromatic rings are mainly hydroxyl, methoxy, and p-hydroxybenzyl groups (Lin et al., 2005). In the ultraviolet spectrum, dihydrophenanthrene compounds, in general, have an α band at approximately 310 nm, a β band at approximately 250 nm, and a ρ band at approximately 280 nm. The skeleton of dihydrophenanthrenes can be synthesized in the phenylpropane metabolic pathway using phenolic compounds (Gu et al., 2013). The structures of dihydrophenanthrenes are shown in Figure 6.

Anthocyanins
Anthocyanins are the main pigments that give flowers their color. The flowers of B. striata are not commonly used in TCM, and therefore, few studies have been conducted on these flowers. Only five compounds (113-117) have been isolated from the flowers of B. striata. However, recent studies have shown that the anthocyanins in plants may affect human health (Stintzing and Carle, 2004), and have provided new material to study the medicinal uses of B. striata. The structures of anthocyanins are shown in Figure 7.

Steroids
All steroids are biosynthesized from mevalonic acid (Debieu et al., 1992). The steroids present in B. striata are complex, with diverse structures and many derivatives. Eleven steroids and their derivatives (118-128) have been isolated from B. striata, and their structures are shown in Figure 8.

PHARMACOLOGIC ACTIvITIeS
The basic research of TCM focuses mainly on chemical composition and pharmacology, of which the latter is the most important. Pharmacologic studies on B. striata have mainly focused on pharmacokinetics. The latter advances our understanding of the mechanism of action of B. striata components. In recent years, pharmacologic studies on B. striata have focused mainly on its hemostatic, wound-healing, antioxidative, anti-cancer, antiviral, and antibacterial activities.

Hemostasis
Hemostasis is a process that causes bleeding to stop (i.e., blood is retained within a damaged blood vessel). The hemostatic effect is one of the main pharmacologic effects of B. striata. Bensky and colleagues showed that when the dried powder of B. striata tubers was mixed with water, it could achieve a good hemostatic effect after it was spread onto a wound (Venkatraja et al., 2012). Interestingly, recent studies have shown that the water-soluble portion of B. striata has an active role in hemostasis, and its function is believed to be related to adenosine diphosphate, which promotes and accelerates platelet aggregation (Lu et al., 2005;Gachet, 2006). Hung and Wu found that BSP in this watersoluble component played a key role in hemostatic activity (Hung and Wu, 2016). Indeed, there has recently been intensive research on the procoagulant function of BSP. Blood coagulation is a process in which a series of coagulation factors is successively activated by enzymatic action to produce thrombin and fibrinogen clots. Animal experiments have shown that BSP can participate in multiple hemostasis processes, such as platelet adherence to the subendothelial matrix to block vessels (primary hemostasis), and the formation of fibrin clots (secondary hemostasis) by activation of various coagulation factors and promotion of thromboxane-A2 synthesis . Because of its obvious and non-toxic hemostatic effect, BSP has been developed as a new type of hemostatic agent that can be used as a drug delivery vehicle and wound dressing (Zhang et al., 2017a). BSP can be combined with other materials to develop new biomedical materials, such as hemostatic materials for surgical treatments Chen et al., 2019).
In addition to BSP, the steroids (123, 125-128) in B. striata exhibit obvious hemostatic activity and can significantly reduce clotting time . The hemostatic effect of this type of composition may be related to platelets, blood clotting, and fibrinolysis . wound Healing Wound healing has three main phases: inflammation, proliferation of granulation tissue, and repair (Kasuya and Tokura, 2014). Antioxidants in plants, such as polysaccharides and phenolic compounds, have important roles in these three phases (Süntar et al., 2012).
In the inflammation phase, BSP promotes the expression of cytokines such as tumor necrosis factor (TNF)-α, interleukin  (Cunha et al., 2007). Also, IFN-γ enhances the expression of major histocompatibility complex class-II molecules on macrophage surfaces to improve their ability to present antigens (Baldridge et al., 2010). Additionally, it was found that a BSP solution (11 μM) increased the nitric oxide (NO) concentration in a wound, which would promote the chemotaxis of neutrophils, monocytes, and macrophages, thereby providing adequate conditions for wound repair (Diao et al., 2008). NO can also regulate the diameter of and flow within blood vessels so that the wound obtains a more generous blood supply, which helps to repair the wound and advance the growth of new blood vessels. Studies have indicated that BSP can promote the cleanliness of necrotic tissue and provide conditions for subsequent tissue regeneration in a wound (Huang et al., 2019).
In the phases based on the proliferation of granulation tissue and repair, BSP can control the expression of pro-inflammatory factors (e.g., TNF-α) at an appropriate level, reduce the inflammatory reaction in the wound, and prevent damage to remaining cells (Lou et al., 2010). In addition, BSP promotes an increase in the expression of the vascular endothelial growth factor (VEGF), as well as the synthesis and release of hydroxyproline. These actions promote the growth of epithelial cells, accelerate fibroblast proliferation, and further promote healing by wound contraction Yu et al., 2011).
Apart from BSP, several phenolic acids (137-138, 140, 146-148) in B. striata are thought to promote wound healing (Song et al., 2017). Yang and colleagues reported that the esterification products of caffeic acid (140) and phenylethyl alcohol can inhibit the transforming growth factor-β1/Mothers against the decapentaplegic homolog 3 (TGF-β1/Smad3) signaling pathway . There is evidence that Smad3 can bind the DNA sequences of target genes at the transcriptional level, and, for pathologic skin conditions, assumes important roles in tissue repair and fibrosis (Ashcroft et al., 1999). Also, the degradation product of protocatechuic acid (138) can regulate high mobility group box (HMGB)1 expression by modulating the HMGB1/receptor for the advanced glycation end products (RAGE) pathway . During wound healing, classically activated macrophages can utilize HMGB1 to attract vessel-associated stem cells (e.g., endothelial progenitor cells, vascular progenitor cells, smooth muscle progenitor cells), which contribute to skin healing and angiogenesis (Lolmede et al., 2009).

Anti-Oxidation
Reactive oxygen species (ROS) have dual biologic effects. These are necessary to maintain cellular homeostasis during normal activities, but they also damage macromolecular matter. ROS over-accumulation results in cell death, can lead to multiple diseases, and accelerates the aging of the human body (Mittler, 2002). It is advantageous that various chemical constituents in plants can be used as natural antioxidants to remove free radicals in the body without the need for catalase, peroxidase, or superoxide dismutase (Grant and Loake, 2000).
Assays based on the scavenging of 2,2-diphenyl-1picrylhydrazyl (DPPH) radicals have shown that the fibrous root parts and pseudobulb parts of B. striata exhibit strong free radical-scavenging activity. Indeed, the partial antioxidant capacity of chloroform subfractions from ethanol extracts of the fibrous roots of B. striata was the strongest (half-maximal inhibitory concentration (IC 50 ) = 0.848 mg/L). Simultaneously, because the fibrous root parts contain more total phenols, they showed stronger reducing ability (reducing power RP 0.5AU = 83.68 mg/L) (Jiang et al., 2013).
BSP has been shown to be a natural antioxidant through various antioxidant test systems (DPPH, 2,2'-azino-bis, ferricreducing antioxidant power). The antioxidative effects of BSP are mediated through the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)/p22 phox signaling pathway (Qu et al., 2016). Through this signaling pathway, BSP can inhibit expression of NOX4 and p22 phox , thereby blocking angiotensin II-induced ROS generation (Yue et al., 2016). Furthermore, ferulic acid (147) in B. striata shows favorable free radicalscavenging ability, and its reducing ability is even stronger than that of vitamin C (Zhao et al., 2010). This antioxidant ability can protect cells from oxygen species-mediated DNA damage and radiation-induced free radicals (Srinivasan et al., 2006). The NOX4/ROS-mitogen-activated protein kinase pathway is the one through which ferulic acid (147) can decrease ethanolinduced ROS accumulation to improve anti-apoptotic responses .
In some cases, antioxidants can worsen oxidative stress (Miller et al., 2005). If an antioxidant effect is observed, trace ROS are important cellular messengers of molecular signaling. Excessive clearance of ROS may lead to redox imbalance and induce cell-signaling disturbances, thereby triggering oxidative systems in the body.
Woo and colleagues revealed that compound (100) exhibited growth inhibition against a human colon cancer cell line (HCT-15), with an IC 50 of 2.16 μM . Compounds (90, 98) isolated from B. striata tubers showed potent inhibition of proliferation of human hepatoma cells (HepG2), with an IC 50 of 29.1 μM and 25.5 μM, respectively. In HepG2 cells, these two dihydrophenanthrenes induced apoptosis by downregulating the expression of cyclin B1 and blocking the cell cycle in the G2/M phase . Compounds (40, 57, 94) were shown to sensitize K562/breast cancer resistance protein (BCRP) cells to the metabolically active product camptothecin, SN-38, by inhibiting the function of BCRP cells, thereby demonstrating that they could alter multidrug resistance in cancer treatment (Morita et al., 2005). Compounds (39, 40) inhibited tubulin polymerization at an IC 50 of 10 μM, and they limited cell growth by interfering with the mitosis of tumor cells (Morita et al., 2005). Compounds (70-71) exhibited significant cytotoxic effects against the MCF-7, HT-29, and A549 lines. After MCF-7 cells underwent treatment, the IC 50 for compounds (70-71) was 18.49 μg/ml and 12.64 μg/ml, which are greater values than that for cisplatin. Interestingly, their anti-cancer activity is related to their pro-oxidative activity.
In addition to the monomeric compounds stated above, BSP is also considered to have anti-cancer activity (Zhang et al., 2019b). Qian and colleagues showed that, compared with transcatheter arterial chemoembolization (TACE) alone, better results were obtained when the hepatocellular carcinoma in ACI rats was treated with a combination of TACE and arterial administration of BSP (Qian et al., 2003). BSP also inhibited the proliferation of HepG2 cells in a weak manner, and the division of HepG2 cells was not inhibited at <1.5 mg/ml. BSP can also induce apoptosis through caspase-3 expression (Liu et al., 2018). Using BSP as a drug carrier or mixing chemotherapeutic drugs with BSP is another anti-cancer strategy. In this manner, the drug concentration in the target organ can be maintained at a particular level. Based on this idea, copolymer micelles have been used in cancer chemotherapy . A macromolecular substance consisting of stearic acid-modified BSP as a carrier of docetaxel was created, and had a more pronounced effect on inhibiting the growth of HepG2 and HeLa cancer cells compared with that of docetaxel injection alone (Guan et al., 2017).

Antiviral
The efficacy of several first-line antiviral agents appears to be diminishing (Ruiz and Russell, 2012). The antiviral effect of some traditional Chinese herbs (e.g., Isatis tinctoria) has been demonstrated by experimental and clinical research (Li and Peng, 2013). The active pharmaceutical ingredient in these plants can be used as the lead compound for further structural optimization to develop new antiviral drugs that can strengthen the immune system to fight and eliminate viral infections.
B. striata can exert antiviral activity by (i) interfering with the surface proteins of viruses to reduce cell infection and inhibit virus invasion into tissue; (ii) interfering with the RNA replication of the invading viruses to inhibit viral proliferation in the body; and (iii) preventing the virus from being released from host cells, thereby reducing viral spread Zhang et al., 2017b). The water and ethanol extracts of B. striata avert invasion by the influenza virus by interfering with the hemagglutinin receptor on Madin-Darby canine kidney (MDCK) cells, and viral inhibition increases with the extract concentration, with an IC 50 of 18.3 mg/ml and 235.7 μg/ml, respectively, being recorded (Zhang et al., 2017b). Various compounds (53, 84-86, 88, 90, 97-98) in B. striata also show different levels of antiviral activity. Most of them have been shown to have significant antiviral activity against the H3N2 virus in an embryonated hen-egg model, with inhibition ranging from 17.2% to 79.3% .
B. striata can also assist the immune system of the human body to indirectly exert antiviral activity. Peng and colleagues investigated the immunomodulatory activity of BSPF2, which is a new polysaccharide identified from B. striata. BSPF2 significantly induced spleen cell proliferation in a dosedependent manner . The spleen can produce lymphocytes, macrophages, and various cytokines, and has an important role in the immune system. Animal experiments have demonstrated that B. striata can promote T-cell and B-cell immunity in immunocompromised mice (Qiu et al., 2011).

CLINICAL APPLICATION TCM Formulations
Based on classical theories of TCM, Chinese medicinal formulations are prepared by pooling different types of medicinal herbs together (Li and Peng, 2013). During the development of TCM theory, many formulations have been recorded in pharmacopeias or by folklore based on clinical experiences. Considering the different causes and symptoms of diseases, B. striata is often used with other drugs to offset the toxicity of one drug or enhance the bioavailability of another drug, which is known as the "correspondence of prescription and syndrome" in TCM.
For example, Bai Ji San (B. striata liniment) is frequently used as an astringent hemostatic medicine. It is composed of B. striata (Bai Ji  embolizing Agent TACE is a first-line treatment for most inoperable tumors (Tsurusaki and Murakami, 2015). An embolizing agent based on B. striata and TACE has been used in clinical applications. The B. striata embolizing agent can, in general, be classified into three types according to its formulation: liquid, compound, or microspheres. The B. striata liquid embolizing agent is composed of BSP, cellulose diacetate (solute), and dimethyl sulfoxide (solvent). It has the characteristics of easy flow, good biocompatibility, and no fixed morphology. It can be used for the embolization of irregular tumor cavities and reduces damage to vascular walls (Sun et al., 2005).
The B. striata compound embolizing agent is a synthetic moiety that can be used as an anti-carcinogen and coagulant. In this compound, B. striata can have a pharmacologic role with other medicinal ingredients. It can inhibit the proliferation and spread of tumor cells, and reduce the risk of adverse reactions (Chen et al., 2006).
B. striata polysaccharide microspheres (BSPMs) could be promising transarterial chemoembolization carriers for cancer treatment. BSPMs show favorable drug-loading, swelling, suspension, drug-entrapment, and release characteristics in vitro, which are conducive to long-term targeted chemotherapy (Li et al., 2018b). Studies have shown that BSPMs can embolize the blood supply of hepatic arteries and the hepatic portal vein, and completely inhibit the growth of tumors and surrounding microvessels by stopping the binding of VEGF to its receptor (Zhao et al., 2004).
The B. striata embolizing agent has also performed well in TACE of hepatic cirrhosis with portal hypertension and secondary hypersplenism. In a study by Liu and coworkers, long-term follow-up of surgical patients revealed a mean survival duration of 61.5 ± 9.1 (median, 60; range, 1-157) months in the control group and 63.4 ± 9.9 (52, 0-161) months in the B. striata group. The spleen thickness of treated patients was reduced compared with that before TACE, and the counts of white blood cells, platelets, and red blood cells returned to within normal ranges (Liu et al., 2011). Therefore, TACE using B. striata as an embolizing agent is a safe and efficacious treatment for patients with hepatic cirrhosis with portal hypertension and secondary hypersplenism.

Mucosa-Protective Agent
The imbalance between proteases and mucosal defense factors is an important cause of several digestive-system diseases (e.g., gastric ulcers, duodenal ulcers, and ulcerative colitis). In recent years, strengthening mucosal defenses has become a new strategy to treat peptic ulcers (Al-Jiboury and Kaunitz, 2012). B. striata can be used as a mucosa-protective agent alone or in combination with other Chinese herbal medicines (Zhang et al., 2019a). It can be administered by gastroscopic spraying, injection through a gastric tube, oral administration, or enemas (Zhou et al., 2019).
In the treatment of diseases of the upper gastrointestinal tract, B. striata forms a protective film on the damaged mucosal surface to protect it from erosion (by gastric acid) and digestion (by pepsin), thus enhancing the defensive function of the gastric mucosa (He et al., 2016). A preliminary clinical trial showed that B. striata combined with Panax notoginseng, omeprazole, and amoxicillin could reduce the inflammatory response of gastric ulcers, protect the ulcer surface, and promote the regeneration and repair of ulcerated tissue (Bai and Zhang, 2010). In the treatment of ulcerative colitis, B. striata can be added to an enema to act as a mucosa-protective agent. After B. striata powder enters the intestine, it can form a gel on the surface of the intestinal mucosa to protect it. It can also enhance adhesion of drugs to the intestinal wall, thereby increasing the drug concentration in the intestinal tract.
B. striata has a hemostatic function and can promote ulcer healing, which can aid regeneration of the intestinal mucosa and, thus, promote the healing of ulcerative colitis (Lan and Chang, 2014). BSP can alleviate the pathologic damage and symptoms of ulcerative colitis. It can inhibit the expression of TNF-α and nuclear factor-kappa B, upregulate the expression of IL-10, prevent the abnormal release of proinflammatory factors, promote repair of the intestinal mucosa, and inhibit inflammation (Ke and Zhao, 2011). BSP can also reduce the expression of Th2 cytokines in the colon by inhibiting macrophage activation, and relieve inflammation in the intestinal tract. Furthermore, enemas containing B. striata can reduce the risk of postoperative complications of patients with an artificial anus in the colon, reduce the possibility of inflammatory reactions in the surrounding skin, reduce erosion and hemorrhage in the artificial anal mucosa, and improve the quality of life for patients (Lan and Chang, 2014).

Novel Biomaterials
Recently, novel "dissolving microneedles" have been created using BSP. Microneedles can carry drugs into the skin for drug delivery and release encapsulated drugs over time. Compared with conventional transdermal patches and subcutaneous injections, B. striata polysaccharide microneedles are a minimally invasive method of drug delivery that prevents skin retention of biohazardous sharp waste. The pharmacologic activity of BSP contributes to the recovery from the micro-trauma caused by microneedles, such as prevention of bacterial infection and reduction of inflammation (Hu et al., 2018).
Mixing B. striata and polyvinyl alcohol to form a dressing matrix can result in a novel, high-quality biologic dressing. This material makes full use of the pharmacologic activity of B. striata for hemostasis and wound healing, and has been shown to have good mechanical properties and biocompatibility. Hence, it can absorb oozing blood and tissue fluid, which accelerates the healing of burns, surgical wounds, and acute wounds (Lin et al., 2012).
A novel, readily stripped bilayer composite was designed as a wound-dressing material, and exhibited excellent biocompatibility and mechanical properties. This composite comprised an upper layer of soybean protein nonwoven fabric coated with a lower layer of genipin-crosslinked chitosan and B. striata herbal extract. The extract in the wound-dressing material was non-toxic, but also promoted the growth of L929 fibroblasts, which are beneficial for wound healing (Liu and Huang, 2010).

Quality Control
The China Food and Drug Administration defines "geoauthentic" herbs as traditional Chinese crude drugs grown in unadulterated environments and subjected to natural conditions, or with specific cultivation techniques and processing methods (Brinckmann, 2013). Zheng'an County in Guizhou Province is accepted as the most optimal location to produce the crude tubers of B. striata in China. The B. striata produced here is called "Zheng'an Bai Ji". However, the material basis and potential mechanisms for producing geo-authentic herbs are not completely clear. The Chinese Pharmacopoeia recommends identifying the geo-genuine properties of B. striata according to morphologic, microscopic, and thin-layer chromatography approaches, and by ensuring that the residual inorganic components after ashing are ≤15.0% (He et al., 2016).
The standards mentioned above are accepted in formularies and pharmacopoeias, but may not be sufficient to evaluate the quality of all B. striata tubers. The latter contain various medicinally active ingredients (e.g., glycosides, bibenzyls, phenanthrenes, biphenanthrenes, and dihydrophenanthrenes) which should also be considered in quality control. For example, assessment of amounts of dactylorhin A (1), gymnoside V (5), gymnoside IX (6), militarine (8), and bletilnoside A (9) by ultra-high-performance liquid chromatography using photo-diode array detection showed obvious variation in response to the different origins of B. striata. The mass fraction of each chemical marker was 0.341-1. 110, 2.840-6.990, 5.790-34.400, 0.191-3.890, and 0.184-5.050 mg/g, respectively. Among them, a wild strain of B. striata produced in Zheng'an County contained relatively high levels of active ingredients at 1.110, 6.500, 31.400, 3.890, and 5.05 mg/g, respectively . Furthermore, Liu et al. used a parallel-line assay based on quantitative responses to evaluate the hemostatic potency of B. striata obtained from different habitats, which provided monitoring indicators for the clinical efficacy of B. striata . These preliminary results may provide evidence for the geographic specificity and quality control of geo-authentic herbs.

Toxicology
Toxic side effects and adverse reactions from B. striata have rarely been reported. If B. striata is used with Aconitum carmichaeli, it enhances the content of hypaconitine in the decoction (Weng et al., 2004). However, hypaconitine can increase the expression of RyR 2 (a regulatory protein related to the function of calcium channels) in cardiac muscle, which may result in an abnormal heart rate (Liu et al., 2016). Therefore, B. striata cannot be used with A. carmichaeli in TCM (He et al., 2016).
Tests to determine the acute toxicity of B. striata have shown that the mean mortality of mice is <20% if a single intragastric dose is increased to 80 g/kg body weight, and the median lethal dose of B. striata was not detected. Subsequent experiments using a maximum dose of B. striata that did not cause the death of experimental animals was determined to be 180 g/kg body weight (Zhang et al., 2013).
A series of toxicology experiments showed that BSP did not elicit allergic reactions, phototoxicity reactions and, most importantly, no obvious adverse reactions to human skin. These experiments included tests of acute oral toxicity (in mice), skin stimulation (rabbits), skin allergy (guinea pigs), skin phototoxicity (guinea pigs), and skin patches (humans) . Yue and colleagues showed that the acute toxicity of BSP was very low. Mice were given BSP (4 g/kg body weight, i.g.) twice during an interval of 6 h. None of the mice died, and no significant changes were detected in their activity, food intake, fur, or weight (Yue et al., 2003).

CONCLUSIONS AND PeRSPeCTIveS
B. striata has been used as a medicinal herb in China for thousands of years. However, due to the high degree of personalization of the diagnosis and treatment of TCM, its clinical efficacy cannot be comprehensively evaluated by evidence-based medicine. In this review, we categorized research on B. striata based on its chemical constituents, pharmacologic activities, and clinical applications. We also tried to establish connections between the conclusions of many studies carried out on B. striata.
More than 150 compounds have been isolated from various parts of B. striata, and they exhibit a wide range of biologic and pharmacologic properties. Efficacious use of B. striata is dependent upon the connection between these chemical components and their specific bioactivities. Based on extensive biologic testing, numerous phytochemicals identified in B. striata are efficacious against one or more diseases. Developing and applying monomer compounds isolated from this herb is another development direction. Therefore, B. striata has great potential to be further mined for its pharmacological effects.
These studies on component detection, pharmacological probing, and clinical exploration conducted on B. striata have provided us with additional data for this valuable herb. Additional systematic studies will offer sufficient proof for establishing the efficacy and safety of this herb so it can be used as a medicine from a scientific point of view. We hope that this review will allow further research and development of this unique plant.