Review of traditional uses, botany, chemistry, pharmacology, pharmacokinetics, and toxicology of Radix Cyathulae

Cyathulae Radix (CR), also known as “Chuanniux” is a well-known traditional Chinese herbal medicine that has been used in China for thousands of years. The present work reviewed advances in traditional uses, origin, chemical constituents, pharmacology, pharmacokinetics, and toxicity studies of CR. This work aims to provide more up-to-date references for modern study and application of this plant. Furthermore, the possible trends and perspectives for future research of this plant are also discussed. In China, the roots of CR have been widely used in clinical practice to treat orthopedic, gynecological, and urologic diseases. Currently, over 59 compounds have been isolated and identified from CR, including alkaloids and flavonoids. The extracts and compounds from CR have many pharmacological activities both in vivo and in vitro. They provide beneficial effects on the hematological system and anti-inflammatory properties. However, few studies have investigated the pharmacokinetics and toxicity of CR. Further studies should be undertaken to investigate the clinical effects, toxic constituents, and pharmacokinetics of CR; perform quality evaluation; and establish quality criteria for processed C. officinalis. Furthermore, studying the changes of raw and processed CR and the variety of this plant between different cultivated areas and cultivars will be interesting.


Introduction
Cyathulae Radix is a perennial herbaceous plant distributed in Sichuan, Guizhou, and Yunnan provinces in China. Its root is commonly used as a traditional Chinese herbal medicine called "Chuanniux. " It is traditionally believed to have the effects of removing "blood stasis, " antifertility, anti-inflammation, stimulating menstruation, and curing orthopedic diseases including bone injury [1].
Cyathulae Radix has been listed in the Pharmacopoeia of the People's Republic of China. Since 1953, over 100 prescriptions containing CR have been utilized to treat gynecological diseases, orthopedic diseases, blood stasis Origin Cyathulae Radix is a herbaceous perennial plant. It reaches approximately 50-100 cm in height and contains many branches. The stems of this plant are erect and four prismatic but circular at the bottom. They exhibit green color, which is sometimes mixed with purple. The stems are covered with sparse coarse hairs. The leaves are elliptic to narrowly elliptic, opposite, and petiolate. The leaf margins are entire. The leaves are 3-13 cm long and 1.5-5 cm wide and covered with thick lodging coarse hairs on the pressure side and long pubescence hairs on the reverse side. The flowers have a green-white color and are clustered in inflorescences. The compound cymes gather into flower pellets with a diameter 1-1.5 cm. The flower pellets interactive opposite on rachis. The bisexual flowers are located in the center, whereas sterile flowers are located on both sides. The bracts are ovoid and approximately 4-5 mm in length. The bisexual flowers are 3-5 mm in length with lanceolate sepals. The flower has five stamens and five staminodes. The base of the stamen is densely villous. The staminode is rectangular in shape, 0.3-0.4 mm long, and 0.1-0.2 mm wide. The pistil is ovary superior and 1-loculed and has a fine style. The utricles are long elliptical, obovate, and 2-5 mm long. The seeds are ovoid, lentoid, and 1.5-2 mm long. CR flowers from June to July and sets fruit from August to September. The roots of CR are nearly cylindrical, slightly twisted, and 30-60 cm long and have a diameter of 0.5-3 cm. The underpart of the root is slightly slender and has few branches. The surface root is yellowish brown or grayish brown with longitudinal wrinkles, branch marks, and many horizontal long leather-like protuberances. The roots are pliable but strong; they have a light yellow or brown section. The vascular bundles are point-like and arranged in several rounds of concentric rings. The stele is large in the transverse section. The outer tough vascular bundles are intermittently arranged in 4-11 rounds, and the inner stratification layer is visible in the inner vascular bundles. The xylem catheter normally has a single radial arrangement and is ligneous. Wood fiber is developed, and parenchyma cells contain calcium oxalate crystal [1]. Different from other plants of the same genus, the cylinder number of tertiary vascular bundles in 1-year-old CR roots can reach up to six laps, and that in 2-year-old CR roots can reach up to eight laps [10].
This plant is native to South and Southwest China. At present, it is mainly cultivated in Sichuan and Chongqing. The plant is also distributed in some provinces, including Guizhou, Hubei, Hunan, Shanxi, Gansu, Jiangxi, and Zhejiang. The altitude in the cultivation areas ranges from 1150 to 2680 m [11,12]. The plant has two close relatives, C. capitata (Wall.) Moq. and the hybrid of C. officinalis and C. capitata (Wall.) Moq., which are common in origin and widely found in markets. Their roots are identified as adulterant of Chuanniuxi [13]. The main differences between CR and its adulterants are the shape of inflorescence, roots, and taste of the root. Compared with CR, Cyathula capitata (Wall.) Moq. has larger and longer inflorescence and exhibits a taupe color after being dried. The roots are thick, large, and always lignified and have an acrid taste. The hybrid of these two species also has a large inflorescence with a black gray color after being dried. The roots taste bitter and cause numb tongue, and they have multiple branches at the bottom. In addition, the duramen of the root is hard [14]. The leaf morphology and microscopic features of these three plants are different [15]. Genetic diversity analysis shows large genetic differences between CR and its adulterants. However, molecular marker technology can easily distinguish them. In 2010, CR and its adulterants were identified by SCAR markers. The primer SC-495 is effective for distinguishing CR and C. capitata (Wall.) Moq. [16].

Phytochemistry
Many chemical compounds have been isolated from CR. The chemical components in CR can be roughly divided into three categories: triterpenoid saponins, steroid ketones, and polysaccharide and other compounds ( Fig. 1).

Triterpenoid saponins
Triterpenoid saponins are composed of three saponins and sugar molecules. Tetracyclic triterpenoids and pentacyclic triterpene are common. Sugar that is connected to the glycosides usually contains glucose, rhamnose, glucuronic acid, and arabia sugar. Saponins found in CR are pentacyclic triterpene saponins. Three terpenoid saponins, including oleanolic acid type three terpenoid saponins, ivy saponins, and bamboo saponins, have been reported in CR (Table 1) [17].

Steroid ketones
Steroid compounds are another important kind of active compounds in CR (Table 2).

Pharmacology
Cyathulae Radix possesses extensive pharmacological effects, including effects on the hematological system, urogenital system, and immune system; antifertility effects; anti-inflammatory effects; antitumor effects; senility-delaying effects; hypolipidemic effects; antihypertensive effects; antioxidation effects; and effects on climacteric syndrome, material metabolism, growth and development, and articulation [20]. In the following sections, the primary pharmacological abilities of CR are summarized and analyzed, as listed in Table 3.

Effects on the hematological system
The characteristic pharmacological ability of CR on the hematological system has been researched on several levels, including the improvement of mesenteric microcirculation, enhancing the deformability of red blood cells (RBCs), reduction of platelet aggregation, decreasing the level of fibrinogen, promotion of wound healing, and lowering blood pressure and blood fat. From the point of view of TCM, CR has the function of activating blood and removing blood stasis. Cheng et al. [25] revealed that the water extracts of RC (WERC) at a dose of 5 g/kg could improve mesenteric microcirculation, decrease whole blood viscosity, and strengthen the deformation ability of RBC in acute blood stasis model rats. Wang et al. [26] reported that the addition of CR in Sijunzitang (Decoction of Four Mild Drugs, DFMD) accelerated the healing time and improved the healing rate for refractory wounds in a rat model of lower extremity. The mechanism of these effects is that DFMD increased the expression of vascular endothelial growth factor and the number of microvessels in the wound, which improved the anoxia and ischemia condition, accelerated the healing speed, and shortened the healing time of the wound [26]. After the addition of CR, the effects were stronger than those of DFMD alone. Further investigation revealed that DFMD and CR upregulated the synthesis and secretion of TGFβ 1 and bFGF, accelerated fibroblast proliferation from the G0/G1 phase to the S phase, promoted the proliferation of fibroblasts and granulation tissues, and filled tissue defects [27]. Moreover, CR increased the proportion of fibroblasts in the S phase. Guo et al. suggested that CR has significant effects on hyperlipidemia and hepatic steatosis. WERC, components of 20% ethanol elution parts, and steroid components of CR could reduce the serum TC, TG, LDL-C, ALT, and AST values of hyperlipidemia rats; increase HDL-C value; reduce MDA levels in liver tissue; and increase SOD levels.
Moreover, Qi et al. [28,29] revealed that the ethanol extracts of CR could reduce the blood pressure, decrease the activity of angiotensin-converting enzyme (ACE) and the apoptosis of cardiac cells, and block the effect of ACE in spontaneously hypertensive rats. In addition, a previous study [30] has also demonstrated that WERC could effectively alleviate left ventricular hypertrophy and improve myocardial remodeling in a dose-dependent manner.

Effects on climacteric syndrome
Climacteric syndrome in women is due to the decline of ovarian function. Estrogen levels are significantly decreased by cardiovascular, nervous, and endocrine system disorders, leading to dysfunctional uterine bleeding, paroxysmal excess heat and sweating, vulvar and vaginal atrophy, osteoporosis, and other symptoms of the autonomic nervous system. Additionally, estrogen deficiency is the main cause of postmenopausal osteoporosis. Phytoestrogens, an alternative to estrogen, have attracted increasing attention in terms of their safety and efficacy [31].
Wang et al. [32] revealed that CR has a weak estrogenlike effect in ovariectomized rats. It could reduce the atrophy of the reproductive system induced by estrogen deficiency, improve lipid metabolism, and reduce body weight. It may be effective in treating climacteric syndrome. CR could increase uterine weight and improve Zhou et al. [18,19] Zhou et al. [18,19] 10   endometrial atrophy in ovariectomized rats, indicating that it has the same function with endogenous estrogen. However, its stimulating effect on the endometrium is far less than that of estrogen. So the treatment of postmenopausal estrogen deficiency by using RC instead of estrogen can avoid the risk of inducing endometrial and breast cancer. Furthermore, CR shows a notable inhibitory  effect on bone loss, improving bone biomechanical properties and preventing the occurrence of osteoporosis in ovariectomized rats. In addition, CR reduces the degree of joint edema in rats with acute ventilated arthritis. The mechanism of the effect may be associated with downregulating the expression of NF-kB P65 and inhibiting the activity of NF-kB, thereby reducing inflammation [33].
PCRs have attracted increasing attention from researchers. Li et al. [35] found that PCRs (2.5, 5, 10 g/kg) played a key role in enhancing the PFC response ability of mice immunized with SRBC, strengthening the cytotoxic activity of NK cells. However, the lymphocyte transformation rate did not change significantly. This finding showed that PRC could enhance the role of humoral immunity mediated by B cells, had no significant effect on T cell-mediated immunity, and could enhance the cytotoxic activity of NK cells. Wang et al. [36,37] discussed the immune function of Achyranthes bidentata polysaccharides in vivo and in vitro. In vivo, PCR (25-100 mg/ kg) could enhance delayed hypersensitivity and NK cell activity in normal mice and increase the carbon clearance rate in mice, number of antibody-producing cells, and macrophage phagocytosis of chicken RBCs with increasing polysaccharide concentrations. PCR could also significantly improve the number of white blood cells caused by cyclophosphamide (Cy) reduction. However, it had no effect on the transformation rate of spleen lymphocytes. In vitro, PCR in the concentration range of 10-300 μg/ mL did not have a direct toxic effect on cells. It could promote the proliferation of B lymphocytes (P < 0.01) and enhance NK cell activity in mice peritoneal macrophages (P < 0.05) and neutral RBC activity (P < 0.01) with increasing polysaccharide concentration. However, it had no effect on the proliferation of T lymphocytes (P > 0.05). In the theory of TCM, CR possesses a tonic effect, which may be related to its polysaccharides.

Antitumor effect
Song et al. [38] revealed that PCR has a certain inhibitory effect on mouse hepatoma cells. Ding [39] reported that PCR at doses of 1, 2.5, and 5 g/kg could effectively suppress mouse H22 hepatoma cells with inhibition rate of 21.99-42.21%. In S180 tumor-bearing mice, the inhibition rate was 10.00-48.08% with PCR dose of 1 and 5 g/ kg. PCR not only had strong antitumor effects but also played a significant role in the recovery of peripheral leukocytes from mice (normal and tumor-bearing mice) induced by Cy. Zong et al. [40] revealed that 100, 200, and 400 μg/kg of PRC inhibited the growth of BEL-7402 cells and promoted cell apoptosis. The mechanism might be related to the upregulation of caspase-3 protein expression. Cao et al. [41] studied the effects of PCR on the tumor microenvironment of mice bearing HepG-2 cells. They administered 0.825 g/kg per day (low dose), 1.65 g/ kg per day (middle dose), and 3.3 g/kg per day (high dose) of PCR in mice by gavage for 2 weeks. The results showed that PCR improved tumor immune inhibition, and high dose of PCR inhibited the expression of immunosuppressive factors in local tumor tissues and delayed tumor progression.

Antioxidation and antiaging effects
Ding et al. [42] established a rat model of hepatocyte injury induced by H 2 O 2 in vitro. They treated rats with 10 mg/mL of PCR with 73% purity. The results showed that PCR could reduce the increased levels of aminotransferase induced by H 2 O 2 in rat stem cells, which might be related to the antioxidant effect of PCR. In a senescent mouse model, 0.25 mL of 55.6% pure Achyranthes bidentata polysaccharide was intragastrically administered, and 0.2 mL of d-galactose was injected subcutaneously [43]. The results showed that A. bidentata polysaccharides had stronger antioxidant activity than vitamin C, which is an excellent natural oxidant.

Effects on material metabolism and growth
Cheng et al. [44,45] revealed that CR contains molting sterones. Molting steroids increase glucose consumption within a certain concentration range, and the hypoglycemic effect of molting sterones decreases with increasing glucose concentration in the culture medium. The hypoglycemic effect of the molt is associated with hepatocytes and is non-insulin dependent. In a cell model of insulin resistance, ecdysterone can increase insulin sensitivity and improve glucose metabolism. As a reverse inhibitor, ecdysterone can improve insulin resistance caused by hyperglycemia, and its toxicity is very negligible.

Effects on promoting angiogenesis
Cyathulae Radix is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. Zhou et al. reported that the whole extract of CR evidently increased cell migration. The open wound area was smaller than that of the control group by 14.9% after an 8 h treatment. The whole extracts of CR promoted angiogenesis mainly via stimulating the migration of endothelial cells in human umbilical vein endothelial cells in vitro. The authors found that several migration-related genes in zebrafish appeared to play pro-angiogenic roles of the CR extract in vivo. The results in human ECs are quite comparable to those in zebrafish. The mechanism of these effects is related to the enhanced gene expression of MMP9 and β-catenin by CR extract, revealing that matrix degradation and cell-cell adhesion are simultaneously triggered by the CR extract for stimulating angiogenesis. β-Catenin is a critical member of the canonical Wnt signaling pathway that is responsible for vascular sprouting, particularly for cell-cell adhesion. Moreover, it can trigger signal transduction for vascular remodeling and differentiation [46].

Pharmacokinetics
To date, pharmacokinetic studies of CR extracts are few. Previous studies mainly focused on its ketosteroids such as cyasterone. In 2010, a study [47] reported that cyasterone is abundant in CR and can be used as a representative ingredient in pharmacokinetic studies. A rapid and sensitive method using reverse phase high-performance liquid chromatography (HPLC) was established to determine the cyasterone concentrations in rabbit plasma. The method provided bases for the study on CR pharmacokinetics. CR was traditionally considered as a guiding herb in TCM composite preparations and deeply valued by tens of millions of Chinese medicine practitioners. Tang et al. studied the effects of CR on paeoniflorin pharmacokinetics of Xuefu Zhuyu Tang in normal and blood-stasis model rats. After oral administration of solution with or without CR at a dose of 5 mL/kg, the peak plasma concentration (Cmax), peak time (Tmax), and half-life values of paeoniflorin were determined. The results indicated that the presence of CR in Xuefu Zhuyu Tang increased the Cmax of paeoniflorin from 0.099 to 0.363 mg/L and 0.228 to 0.315 mg/L in the normal and blood-stasis model group, respectively. Moreover, CR decreased the Tmax from 0.555 to 0.276 h in the normal group and the t1/2 from 0.853 to 0.501 h and 0.708 to 0.408 h in the two groups. Furthermore, the area under the concentration-time curve (AUC) was also determined. AUC0-∞ increased from 0.166 to 0.381 mg/L h in the normal group only. As a guiding herb, RC could significantly increase the absorption amount and rate of paeoniflorin in decoction and accelerated its elimination from the blood [48].

Toxicity
Throughout its long history, CR has been generally considered to be a safe TCM in China [49]. No reports of poisoning have been reported in clinics. In a previous study, the toxicity of its ingredients such as ecdysone was found to be very low. The embryotoxicity of CR was also mentioned in the literature; however, a study on mice showed that its water decoction had no obvious induction effect of chromosomal aberration and embryonic micronucleus [50]. Recently, the acute toxicity of CR was reported in a dissertation [51]. The experimental results showed that after high-dose administration of water decoction, the mice exhibited neurotoxicity and gastrointestinal toxic reaction. Obvious toxic reactions were observed. Moreover, the maximum tolerance dose of samples from different origin ranged from 56.8 to 34.9 g/ kg. This experimental result was different from the traditional cognition of safety of CR. However, the toxicity has not been clarified to date. Thus, further studies should be conducted to confirm the clinical innocuity of CR.

Future perspectives and conclusions
CR has been used in China as an effective TCM for many years. Recently, it has been the subject of increasing interest. Many kinds of chemical constituents have been isolated and identified from this plant, and the pharmacological activity of its main chemical constituents such as alkaloids, steroids, and polysaccharides has been verified. Meanwhile, many traditional uses of this plant have been validated by pharmacological studies. In addition, previous animal investigations and in vitro studies have revealed that CR possesses significant effects on the hematological, urogenital, and immune systems. There is no doubt that continued progress has been made on various aspects of this plant in the past decade. However, further preliminary studies are needed to fully elucidate the mechanisms and constituents of this plant.
First, due to the complex introduction process, the mixture of CR and its adulterants has been widely used. This process has led to the common occurrence of adulterants in the herbal medicine market and misapplications in clinics, thus significantly affecting the value of CR. Therefore, cultivation management should be strengthened to remove the adulterants from the cultivation base. In addition, a unified identification is needed to control the variety and quality of this herbal medicine, and establishing a standardized finger print of this plant might be indispensable. Second, the current pharmacological studies of this plant have primarily focused on several chemicals such as cyasterone and achyranthan. Evidence that can explain the special mechanism of action for the pharmacological activity of this plant is lacking. Thus, future studies should investigate more bioactive compounds and their mechanism of action and structure-function relationship. In addition, toxicity studies of aqueous extract are few, and no evaluations of target-organ toxicity have been documented. Furthermore, the experimental results about neurotoxicity and gastrointestinal toxicity contradict traditional understanding of CR. Therefore, further studies should devote more effort to investigate the compounds of CR and their toxicity.
Third, studies regarding the pharmacokinetics of CR are lacking. According to the only pharmacokinetic report of this plant, the marker compound cyasterone could be determined accurately in rabbit plasma via HPLC. Additionally, as a guiding-herb, CR could affect the pharmacokinetics of other medicines in Chinese compound formula. Therefore, future studies should investigate the pharmacokinetics of its main compounds.
Fourth, to date, studies on the parts of CR other than its roots are few. Other parts of this plant are worthy of multi-aspect research in the future. Thus, the chemical constituents and pharmacological effects of the aboveground parts should be investigated to reveal their possible medicinal potential.
Fifth, the traditional uses of CR include several processed products including the roots stir-fried with wine and salt. The traditional theory considered that the processing techniques with wine and salt could enhance its function of promoting menstruation and blood circulation and strengthening the muscles and bones [52]. Current investigations have found that the two processing techniques have significant effect on herbal chemical profiles, and several new compounds are generated in the process [53]. However, there is still not enough research to confirm the change of efficacy in the process. Moreover, special quality standards for processed CR are still lacking. Therefore, studies should investigate the chemical and pharmacological characteristics of processed CR systematically. Lastly, although CR is currently widely used and cultivated in many provinces of China, managing the variety of this plant and enhancing fundamental research are also important.
In addition, a similar Chinese medicine, Achyranthis Bidentatae Radix which is named Niuxi or Huainiuxi is used in the clinic. The original plants of Achyranthis Bidentatae Radix (Niuxi) and Cyathulae Radix (Chuanniuxi) looked similar and both of them are used to invigorate the circulation of blood. However, the main difference between these two herbs is efficacy. Achyranthis Bidentatae Radix is more suitable for tonifying the kidney and strengthen the muscles and bones, while Cyathulae Radix prefers promoting blood circulation and removing blood stasis, according to Chinese Pharmacopoeia 2015 Edition and teaching materials. Although this distinction is controversial now [54], it is used clinically according to this distinction.
Our present paper has systematically reviewed the traditional uses, origin and variety, phytochemistry, pharmacology, and toxicology of CR and provided comprehensive information on this plant. We hope that this review highlights the importance of CR and provides some directions for the future development of this plant.