1. INTRODUCTION
The herb Zeqi (also called Euphorbia helioscopia L.), belongs to the genus Euphorbiaceae in the family Euphorbiaceae. In China, it is also called Wu Duo Yun, cat’s eye grass, and Wu Feng Ling zhi. Zeqi is widely distributed worldwide and is very common in Eurasia and North Africa. As a widely used ingredient of traditional Chinese medicine, Zeqi has multiple functions including as a febrifuge, expectorant, and diuretic for edema. Zeqi was first recorded in Shennong’s Classic of the Materia Medica (Eastern Han Dynasty) [1], and is described in the Dictionary of Traditional Chinese Medicine as slightly cold in nature; pungent and bitter in flavor; poisonous; belonging to the lungs, the small intestine, and the large intestine meridians; and having main effects of eliminating phlegm, killing pathogens, and relieving itching [2]. Before the Song Dynasty, all materia medica works considered Euphorbia plants as Zeqi, until The Compendium of Materia Medica (1552–1578) [3], in which Li Shizhen, a physician in the Ming dynasty, suggested that cat’s eye grass was the authentic source of Zeqi. Later, the Illustrated Book on Plants (1841–1846) [4] illustrated Zeqi as cat’s eye grass, thus indicating that in the Qing Dynasty, cat’s eye grass was used as the aboveground part of Zeqi [5]. Currently, the medicinal parts of Zeqi are primarily the whole grass, including stems and leaves. This herb has been widely used for decades in the treatment of various diseases, including tuberculosis, phlegm and cough, dysentery, malaria, warts, and lung cancer [6–8].
In recent years, researchers have extracted various chemical components from Zeqi, including diterpenes, triterpenoids, and total flavonoid components (TFC), and investigated their biological properties. For example, Zeqi diterpenes have been found to have antitumor activity and anti-inflammatory activity. On the basis of this activity, Zeqi has been used to treat cancer and inflammatory diseases, such as psoriasis and endotoxic shock. However, because of the limited scope of existing experiments, particularly human and mechanistic studies, further clinical trials are necessary to demonstrate the therapeutic potential of Zeqi.
This review summarizes recent pharmacological studies on Zeqi and its extracted chemical components (for example, jatrophane, lathyrane, and ingenane diterpenoids of Euphorbia helioscopia L.) to provide a reference for their clinical application in traditional Chinese medicine.
2. STUDIES ON ZEQI’S PHARMACOLOGICAL ACTIVITY
Terpenes are the most abundant chemical constituents of Zeqi. Other major components of Zeqi include steroids, tannins, flavonoids, phenylpropanoids, and cerebrosides [9, 10]. The terpenes of Zeqi include monoterpenes, sesquiterpenes, desmethyl sesquiterpenes, diterpenes, and triterpenes, among which diterpenes show high structural diversity and are the most abundant. The structural diversity of these compounds arises from a variety of backbone types, such as lathyrane, jatrophane, ingenane, ent-abietane, ent-kaurene, and tigliane [11, 12]. In contrast, relatively few triterpene compounds are found in Zeqi are and are classified primarily into lupane and cycloalkanes [13]. The flavonoids of Zeqi are also abundant and important in reducing diabetes mellitus, including chalcones [14] and isoflavones [14, 15]. Figure 1 shows the structures of some compounds in Zeqi. Zeqi’s pharmacological activity has been extensively investigated in recent years. This herb has been found to possess various beneficial properties, including antitumor, antiviral, anti-inflammatory, anthelmintic, antimicrobial, and antioxidant activity.
2.1 Extracts of Zeqi
Many studies have experimentally examined Zeqi crude extract alone or in combination with other herbs; examples include ethyl acetate extract (EAE), Zeqi root aqueous extract (EWE), and Zeqi methanol extract. Various crude extracts have shown significant inhibitory effects on a variety of cancer cells ( Figure 2 ).
2.1.1 Ethyl acetate extract
In vivo experiments using a nude mouse xenograft model of hepatocellular carcinoma have shown that EAE at concentrations of 100–200 μg/ml significantly inhibits SMMC-7721 human hepatocellular carcinoma cells. The mechanism involves inhibiting tumor cell invasion and decreasing expression of MMP-9 in a time-and dose-dependent manner, thereby blocking the cell cycle in G1 phase [8]. In addition, dose-dependent inhibitory effects of EAE on tumor cell invasion and MMP-9 expression have been observed in vitro experiments [16].
2.1.2. Methanol extract of Zeqi
Studies have reported that methanol extract of Zeqi has antioxidant, antimicrobial, and anthelmintic activity.
Crude methanol extract of Zeqi has been found to have antidiabetic and antioxidant effects in a rat model of type 2 diabetes. In that study, diabetes was induced in male Wistar rats by administration of 5% sucrose, nicotinamide, and streptozotocin for 8 consecutive weeks. The diabetic rats were divided into positive control (no treatment), standard control (metformin 10 mg/kg bw), treatment 1 (methanol extract 200 mg/kg bw), and treatment 2 (methanol extract 400 mg/kg bw) groups. After 21 days of treatment, the rats were decapitated, and blood was collected to assess antidiabetic potential, antioxidant and lipid profiles, thyroid hormones, amylin, leptin, and carbohydrate metabolic enzymes. The serum levels of glucagon, glucose, and c-peptide were significantly (P ≤ 0.05) lower in the treatment 1 and treatment 2 groups than the standard control group. The serum insulin levels were lower in the treatment 2 group as compared to the treatment 1, and it may be related to the concentration of methanol extract in treatment 2 group being higher than that in treatment 1 group. Therefore, the methanolic extract of Zeqi effectively decreased the total oxidative state and MDA levels; increased the total antioxidant capacity in diabetic rats; and increased the levels of SOD, catalase, paraoxonase, and arylesterase. In addition, the treatment had anti-hyperlipidemic activity, and normalized the levels of thyroid hormones, glucagon, and leptin. Methanolic extract of Zeqi was found to be rich in chlorogenic acid, ferulic acid, caffeic acid, catechin, rutin, and quercetin, and these phenolics and flavonoids positively correlated with antioxidant and antidiabetic activity. The authors hypothesized that the antioxidant activity of Zeqi might be associated with the phenolic constituents present in its methanolic extract [17].
Other studies demonstrated that the methanolic extract of Zeqi can achieve antioxidant effects through the elimination of free radicals, mainly related to free-radical scavenging activity of (2S)-1,2-O-di-linolenoyl-glyceryl-6-O-(α-d-galactopyranosyl)-β-d-galactopyranoside in the complex mixture of seven cerebrosides together with glucoclionasterol, a digalactosyldiacylglycerol and a diacylmonogalactosylglycerol [6]. In a study that fed two groups of mice Zeqi latex or Zeqi methanol extract (600 or 1200 mg/kg, respectively) for 2 weeks, the methanol extract, compared with the latex, showed significant in vivo antioxidant effects. The levels of antioxidant enzymes (catalase, superoxide dismutase, and glutathione) significantly increased after administration of a dose of 1200 mg/kg. Thus, the methanolic extract eliminated free radicals by increasing the levels of antioxidant enzymes. Moreover, a 1200 mg/kg dose methanolic extract significantly decreased malondialdehyde levels, thus suggesting therapeutic effects on liver and kidney damage, hypolipidemic effects, and hemostatic potential, which may help biological systems defend against stress-induced pathological conditions [18].
In traditional Chinese Medicine, Zeqi is frequently used to treat various gastrointestinal disorders. Current research has confirmed the antimicrobial activity and anthelmintic activity of Zeqi methanol extract, thus providing a basis for the treatment of gastrointestinal disorders. The methanolic extract has shown dose-dependent antibacterial activity against four bacteria (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas multocida, and Escherichia coli) and two fungi (Aspergillus flavus and Candida albicans) in the range of 100–500 mg ml−1. The extract showed better inhibitory effects on Staphylococcus aureus and Escherichia coli, whereas the lowest inhibitory effect was observed for Aspergillus flavus.
In the anthelmintic experiments, worm motility inhibition and egg hatch studies were used for in vitro assessment, and fecal egg count reduction was assessed in an in vivo study. The methanolic extract of Zeqi at various concentrations (12.5 mg ml−1, 25 mg ml−1, and 50 mg ml−1) showed dose-dependent anthelmintic effects toward H. contortus. These findings suggest that Zeqi might serve as a potential alternative drug for the treatment of helminth infections in ruminants [19].
In addition, silver nanoparticles (AgNPs) synthesized from Zeqi methanol extract have been investigated. AgNPs and AgNP-loaded chitosan-alginate constructs have shown antimicrobial activity against six species of bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Morganella morganii, and Haemophilus influenzae. Almost all constructs showed cytotoxicity toward epithelial cancer cells, thus suggesting the biomedical potential of the newly designed AgNPs and AgNP-loaded chitosan-alginate constructs [20].
2.1.3 Zeqi root aqueous extract
EWE has shown good antitumor activity and antimicrobial activity [21, 22]. Its preparation method is as follows: First, 5 g Zeqi root is soaked in distilled water, boiled for 6 hours at 100°C, then filtered and concentrated to 1 g/ml. The extract is filter sterilized, diluted in RPMI 1640 + 10% FCS, and finally refrigerated at 4°C.
EWE has also shown favorable antitumor activity in vitro. Antitumor activity has been evaluated by live cell counting, MTT staining, and colony formation of three cancer cell lines. The IC50 values of EWE against 7721, HeLa, and MKN-45 cells have been reported to be 1.26, 1.98, and 1.72 mg/ml (72 h), respectively. However, MTT staining of EWE-treated 7721, HeLa, and MKN-45 cells showed IC50 values of 1.43, 1.67, and 0.97 mg/ml, respectively. Treatment with EWE at 4 mg/ml showed 59.8%, 66.4%, and 70.5% inhibition of 7721, HeLa, and MKN-45 cells, respectively. EWE therefore has efficient antitumor activity ( Figure 3 ) [21].
Antitumor experimental data for Zeqi root aqueous extract (Adapted with permission from ref. [21]. Copyright Journal of Chinese Medicinal Materials).
EWE also has antimicrobial activity and has been found to inhibit the growth of the bacteria Bacillus subtilis and Klebsiella pneumoniae, and the fungus Candida albicans. Among Gram-positive bacteria, Staphylococcus aureus is most sensitive, and Bacillus subtilis is most resistant. Among Gram-negative bacteria, Pseudomonas aeruginosa is most sensitive, and Klebsiella pneumoniae is most resistant [22].
2.1.4 Dichloromethane extract of Zeqi
The dichloromethane extract of Zeqi has favorable antimicrobial activity in bacteriostatic tests on fungi such as Aspergillus longum, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solanacearum, and Candida glabrata. The inhibition rate of Fusarium solanacearum has been found to be 90%, whereas no significant inhibition of bacteria such as Salmonella typhimurium and Bacillus subtilis has been observed [23, 24].
2.1.5 Ethanol extract of Zeqi
Both ethanol and methanol extracts of Zeqi show significant antioxidant activity. In experiments investigating the effects of Zeqi extract on oleic-acid-induced fat accumulation in HepG2 cells, 50% ethanol extract has shown the best antioxidant effect. This treatment inhibits oleic-acid-induced fat accumulation and enhances the endogenous antioxidant capacity of HepG2 cells. These findings have provided a theoretical basis for understanding antioxidant properties and treating NAFLD [25]. The preparation method of methanol and methanol extracts of Zeqi is as follows: First, 5.0 g Zeqi powder is divided into six conical flasks, pre-soaked in 100 ml methanol and ethanol at various concentrations (25%, 50%, 75%) for 30 min, then ultrasonicated at 30°C and 300 W for 30 min. Finally, the extracts are filtered and dried at 80°C.
2.1.6 Zeqi lectin
Lectins are proteins that specifically bind and cross-link to carbohydrates, and are involved in activation of the lectin pathway, which is believed to have roles in innate and adaptive immunity. Experiments have purified a lectin (EHL) from Zeqi leaves through a combination of ion-exchange and gel filtration chromatography. The lectin agglutinates only chicken erythrocytes, but shows no agglutination toward all human blood group erythrocytes. Fructose has been found to inhibit EHL-induced hemagglutination. In addition, in antimicrobial experiments, EHL has shown favorable antibacterial activity, including inhibition of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli growth [26].
2.1.7 Other extracts of Zeqi
The ethyl acetate extract, n-butanol extract, hexane extract, and chloroform extract of Zeqi have favorable antimicrobial and anthelmintic activity. In one study, n-hexane extract inhibited all tested microbial species except Pseudomonas aeruginosa and Escherichia coli, such as S. aureus (Gram positive), B. subtilis (Gram positive) [22]. The chloroform extract of Zeqi has shown cytotoxic effects toward Cimex lectularius, with maximum mortality rates of 8.67% (after 12 h), 10.34% (after 24 h), and 14.67% (after 48 h), respectively [27].
In addition, Zeqi extracts have been reported to promote wound healing. Extracts of the above-ground parts of Zeqi have been prepared with n-hexane, ethyl acetate, and methanol as solvents, and the wound healing activity has been evaluated with linear incision, a circular excision wound model, and the hydroxyproline method. The tensile strength of the linear incision wound model was 43.03%, and the wound area of the circular incision wound model decreased by 65.24% on the 10th day of applying extracts of the above-ground parts of Zeqi. The hydroxyproline content in tissues treated with the extracts was 35.47 μg/mg. These findings indicate that the above-ground part of Zeqi promotes wound healing in various models [28].
2.2 Compounds of Zeqi
2.2.1 Terpenes
Among the many types of secondary metabolites in Zeqi, terpenes are a major chemical component. The most important biologically active substances of Zeqi are diterpenes and triterpenoids.
Diterpenes, the most abundant terpenes in Zeqi, have many pharmacological activities, including antitumor, anti-inflammatory, anti-microbial, antioxidant, antilipidemic, and antiviral activity ( Figure 4 ).
The diterpenes of Zeqi have significant antitumor potential. Zeqi decoction, as recorded in the Synopsis of the Golden Chamber, is an ancient formula for lung cancer treatment. Zeqi, the monarch drug in Zeqi decoction, has been hypothesized to play an important role in this treatment. Modern pharmacological studies have confirmed that the most abundant diterpene compounds in Zeqi, at a concentration of 2.5 μM, can be used to treat lung cancer by enhancing NK cell killing of H1299-luci and A549-luci cells [29].
Euphornin, a diterpene isolated from Zeqi, is the most abundant component and the major bioactive component in Zeqi. Research on sulforhodamine B has demonstrated inhibition of HeLa cell proliferation by euphornin in a dose-dependent and time-dependent manner. Apoptosis is also induced by euphornin in a concentration-dependent manner, with apoptosis rates ranging from 25.3% to 52.6%. The underlying mechanism is that high concentrations of euphornin block HeLa cells at the G2/M stage, alter the ratio of Bax/Bcl-2 in HeLa cells, and lead to the release of cytochrome complexes, thus inducing cell apoptosis. In addition, euphornin significantly increases the levels of cleaved caspase-3, caspase-8, caspase-9, and caspase-10. Various studies have demonstrated that euphornin significantly suppresses cell growth—a response accompanied by G2/M cell cycle arrest and increased apoptosis rates via mitochondrial and caspase pathways ( Figure 5A ) [30].
Furthermore, the macrocyclic diterpenes euphornin L (1) and euphoscopin F (2) isolated from Zeqi exhibit significant cytotoxicity against the HL-60 cell line, with IC50 values of 2.7 μM and 9.0 μM, respectively [31]. Two jatrophane-derived diterpenoids with a 5/10 fused-ring skeleton from Zeqi, heliojatrone A(1) and particularly heliojatrone B(2), exhibit p-glycoprotein inhibitory activity against MCF-7/ADR, thus providing a new structural template for the development of MDR-reversing drugs from natural products [32]. Another bioactive jatrophane diterpene ester extracted from Zeqi has been evaluated for inhibitory effects on P-glycoprotein inadriamycin-resistant MCF-7/ADR; the compounds are also neuroprotective against damage induced by serum deprivation and rotenone in PC12 cells [33]. The diterpene lactones F and L isolated from Zeqi show significant cytotoxicity toward the MCF-7 breast cancer cell line as well as the PANC-1 pancreatic cancer cell line, thus suggesting Zeqi’s potential for the treatment of pancreatic adenocarcinoma [34]. However, the antitumor mechanism remains unclear, and further studies are required to confirm the effects and related mechanisms.
The inhibition of Kv1.3 channels is another antitumor mechanism. Kv1.3 promotes calcium signaling, and Kv1.3 is involved primarily in the activation of effector T cells. The sensitivity of various subsets of T cells to Kv1.3 inhibitors differs. In addition, Kv1.3 shows neurotoxic effects in organisms and in cancer development. Abnormal expression of Kv1.3 has been detected in a variety of cancer types, including breast cancer, prostate cancer, ovarian cancer, and microglioblastoma. Inhibition of Kv1.3 activity has been found to induce apoptosis in orthotopic melanoma cells [35]. In a human embryonic kidney 293 cell model transfected with a plasmid encoding Kv1.3, the inhibitory effects of isolated Zeqi diterpenoids on Kv1.3 ion channels have been found to be significant ( Figure 5A ) [36].
In general, the diterpene compounds of Zeqi demonstrate antitumor efficacy. Euphornin inhibits the growth of HeLa cells, and euphornin L (1) and euphoscopin F (2) exhibit significant cytotoxicity against the HL-60 cell line. However, many studies to date have provided only preliminary information on the in vitro cytotoxic activity of the isolated compounds.
Inflammation is a fundamental pathological process that can be triggered by any factor causing tissue damage. LPS is a major contributor to the pathogenesis of Gram-negative bacilli and induces various inflammatory reactions in the body. Modern pharmacological studies have shown that the diterpenes in Zeqi exhibit favorable anti-inflammatory activity ( Figure 5B ). Specifically, the ent-atisane diterpenoids from Zeqi have been found to dose-dependently inhibit LPS-induced NO production, thus indicating their potential anti-inflammatory effects [37]. Macrocyclic diterpenes (1–11) isolated from the above-ground parts of Zeqi have also shown significant inhibitory effects on LPS-stimulated NO production in RAW264.7 cells. Among the isolated compounds, diterpene 1 has an IC50 value of 7.4±0.6 μM; this compound might be associated with regulation of the NF-kB signaling pathway, through suppressing translocation of the p65 subunit, and consequently decreasing IL-6 and TNF-α secretion [38]. NF-kB regulates apoptosis, stress responses, NF-kB over-activation, and many human diseases such as those involving inflammatory changes. Therefore, blocking the NF-kB signaling pathway inhibits inflammation. Another study has indicated that euphohelides A–C and ent-abietane-type norditerpene lactones from Zeqi significantly alleviate LPS-induced NO release, with IC50 values of 32.98±1.13 μM and 33.82±3.25 μM, respectively; this activity might be associated with regulation of the NF-kB signaling pathway [39]. However, because this study did not identify the mechanism underlying how these components affect the NF-kB signaling pathway, the findings remain speculative and will require further verification in the future. Another study has found that two other diterpenes isolated from Zeqi, 15-o-acetyl-3-o-benzoylcharaciol and helioscopinolide A, inhibit the production of PGE2, ROS, and proinflammatory cytokines, as well as the expression of iNOS and COX-2, in LPS-stimulated BV2 microglia cells, thus leading to anti-inflammatory effects [11].
NLRP3 is an inflammatory vesicle sensor protein with key roles in many inflammatory diseases. Several studies have indicated that Zeqi diterpenes have strong inhibitory effects on NLRP3 inflammasomes, with IC50 values of 3.34–14.92 μM in vitro. Four new diterpenoids (1–4) and four known diterpenoids (5–8) have been purified from whole Zeqi plants. Anti-inflammatory activity tests on the separated compounds have indicated that diterpenoid 4 has a significant inhibitory effect on NLRP3 inflammasomes, with an IC50 value of 7.75 μM [40, 41]. Euphopia D inhibits the maturation of caspase-1, thereby regulating the secretion of the cytokine interleukin-1β (IL-1β) and indirectly blocking the pyroptosis mediated by GSDMD cleavage.
Zeqi can be used to treat psoriasis, on the basis of its anti-inflammatory activity. Psoriasis is a common clinically refractory, systemic, and chronic autoimmune skin disease [42]. Increased expression of voltage-gated Kv1.3 ion channels, as well as the activation and proliferation of effector memory T cells, leads to the secretion and release of many inflammatory factors, such as interleukin 12 (IL-12) and interleukin 17 (IL-17), and eventually causes inflammation [43–45]. Therefore, inhibiting the function of Kv1.3 ion channels, and blocking the proliferation and cytokine production of effector memory T cells have shown promise in the treatment of psoriasis. Researchers have isolated 43 diterpenoid constituents from the petroleum ether site of the methanolic extract of Zeqi. The production process is as follows: dry and crush the whole Zeqi grass. Then, soak it in methanol four times at room temperature. After that, concentrate the extract under reduced pressure to obtain the total paste. Finally, extract the total paste with petroleum ether to obtain the petroleum ether part. All these constituents were jatrophanes and their analogs, including 17 new compounds (heliojatone D and helioscopids A–P). Among these new compounds, heliojatone D (1), the fourth naturally occurring diterpene discovered to date, has a rare bicyclo[8.3.0] tridecane skeleton. The ability of compounds to inhibit Kv1.3 ion channels has been tested in a model of HEK293 cells transfected with, and stably expressing, Kv1.3 [46]. Psoriasis can therefore be treated by inhibiting Kv1.3 channels to achieve anti-inflammatory effects.
Diterpene compounds of Zeqi also have anti-endotoxin shock activity. Endotoxin, also known as LPS, is the main healing component responsible for endotoxic shock. However, bacterial toxins, TNF-alpha, IL-1β, IL-6, and nitric oxide are the main mediators involved in endotoxic shock [47, 48]. NO is closely associated with LPS-induced endotoxic shock; therefore, methods for inhibiting NO production can be used to treat endotoxic shock. However, many experiments in recent years have shown that the use of NO synthase inhibitors in endotoxic shock is sometimes harmful. Therefore, studying the molecular mechanism of LPS-induced NO production, and searching for substances that inhibit NO production, might provide new ideas for the treatment of endotoxic shock [49]. Three new jatrophane diterpenes (1–3), a jatrophane diterpene (4) with unreported spectroscopic data, and nine known analogues (5–13) have been isolated from Zeqi. Their structures have been determined through detailed spectroscopic analysis (IR, ESIMS, HR-ESIMS, and 1D and 2D NMR) and X-ray crystallography of jatrophane diterpene 1. The diterpenes have shown inhibitory effects on LPS-induced NO production in mouse microglial BV-2 cells [7]. Three previously unreported ent-abietane-type norditerpene lactones, euphohelides A–C (1–3) and 11 known analogs (4–14) have been isolated from whole Zeqi plants. Euphohelide A and analog 5 have been found to significantly alleviate LPS-induced NO release, with IC50 values of 32.98±1.13 μM and 33.82±3.25 μM, respectively [39]. The above studies suggest that Zeqi might potentially open new avenues for the treatment of endotoxic shock.
The diterpene compounds of Zeqi also have anti-microbial activity directed primarily against bacteria. The new diterpenoid 1 extracted from Zeqi has been screened against the Gram-positive bacteria ATCC 25175 and ATCC 27044, and the antibacterial activity may be associated with the macrocyclic structure and ester group of the compound [50]. Moreover, a new jatrophone-type diterpenoid named ephhoheliosnoid E (1) extracted from Zeqi has shown significant antibacterial activity against Gram-positive bacteria, with an MIC toward ATCC 25175 and ATCC 27044 of 3.9 μg/ml [9]. The antimicrobial activity of Zeqi diterpenoids suggests that they might provide a new avenue for the development of highly antimicrobial agents. However, research gaps exist regarding whether the diterpenoids have comparable antimicrobial activity against fungi. This topic may be a future research direction.
Biologist Dr. Denham Harman has found [51] that the aging and degeneration of the human body are due primarily to the accumulation of free radicals produced during cellular metabolism. Free radicals impair normal functioning of the body by oxidizing other substances. Therefore, the elimination of free radicals is important in combatting oxidation and protecting organisms. Euphelionolide A, a natural oxidized diterpene in Zeqi, eliminates free radicals and repairs DNA through the HAT and RAF mechanisms at multiple reaction sites, and shows good antioxidant activity [52].
According to China’s lipid management guidelines, cardiovascular disease, particularly atherosclerotic cardiovascular disease, is the main cause of death among both urban and rural residents; moreover, LDL-C is a causative risk factor for atherosclerotic cardiovascular disease [53]. Therefore, effective management of lipids is critical. Zeqi diterpenoid compounds have shown significant lipid-lowering activity. Screening results for LDL uptake have revealed that most Zeqi diterpenoid compounds increase the LDL-uptake rate of HepG2 cells. Additionally, these compounds have been found to have significant lipid-lowering activity in vivo, thus marking a crucial step toward developing new lipid-lowering agents [54]. One study has found that Zeqi diterpenes A and B, containing a bicyclo[4.3.0]nonane core, exhibit triglyceride-lowering effects in oleic-acid-stimulated HuH7 cells at concentrations of 1–50 μM; these effects were comparable to those of the lipid-lowering drug rosiglitazone in the positive control group [55]. However, whether diterpenes lower only LDL cholesterol and what their specific lipid-lowering mechanisms might be remain to be studied in the future.
Zeqi G–J macrocyclic diterpenes extracted from Zeqi show anti-Zika virus activity. Zika virus infection may lead to several serious neurological complications such as Greene-Barrow syndrome and congenital Zika syndrome. To date, no vaccines (prophylactics) or antiviral drugs (therapeutics) have been approved by the FDA for the treatment of Zika virus infection. Developing natural products is an important method to treat Zika virus infection. Four newly identified diterpenes (1–4) and 16 known diterpenes (5–20) have been isolated from whole Zeqi plants, among which diterpenes 1 and 2 are rhamofolane diterpenoids with a 5/7/6 tricyclic system, diterpene 3 is a lathyrane diterpene, and diterpene 4 is a jathophane diterpene. Diterpenes 9 and 15 have EC50 values of 2.63 μM and 5.94 μM, respectively, low cytotoxicity, and strong anti-Zika virus activity [56].
Among secoheliospholanes A (1) and B (2), and A (3) isolated from Zeqi, which have an unusual 7,8-seco-jatrophane skeleton and unprecedented 9,10-seco-7,10-epoxyjatropholane skeleton, secoheliospholane B (2) exhibits inhibitory activity toward HSV-1, with an IC50 value of 6.41 μM [57].
The amounts of triterpene compounds in Zeqi are low, and the related activities of triterpene has not been adequately studied. However, compounds 5 and 6 of the (19αH)-lupane and (9βH)-lanostane triterpenoids extracted from Zeqi have been found to induce apoptosis in HeLa cells, with EC50 values of 1.59±0.25 and 26.48±0.78 μM, respectively, thus suggesting that triterpenoids from Zeqi trigger apoptosis in HeLa cells [13]. However, whether triterpene compounds might have the same effects on other cancer cells or have other pharmacological effects remain to be discovered in further research.
2.2.2 Total flavonoid components and total phenolic components
The phenolic compounds and flavonoids of Zeqi are relatively low in abundance but show remarkable activity against diabetes mellitus. Experiments have been conducted to obtain the TFC and total phenolic components (TPC) of Zeqi in each extract type. According to statistical analysis, the methanolic extract of Zeqi had the highest content of TFC and TPC, whereas the EWE had the lowest content of TFC and TPC. In vitro results indicated that the methanolic extract of Zeqi had the strongest scavenging ability for DPPH (IC50 = 0.06±0.02 mg/ml), FRAP (758.9±25.1 μM Fe+2/g) and ABTS (689±25.94 μMTEq/g), because its TPC (24.77±0.35 mgGAEq/g) and TFC (17.95±0.32 mgQEq/g) were highest. The methanolic extract showed the highest inhibition of α-amylase and α-glucosidase, and therefore the highest antidiabetic activity. A close association between TFC and TPC in decreasing diabetes mellitus has been suggested ( Figure 6 ) [58].
(A) The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging activity of five concentrations of various Zeqi extracts. (B) IC50 values of DPPH• radical scavenging activity of various Zeqi extracts. Results are mean ± standard deviation of three replicates per group. Different lowercase letters (a to c) above the bars indicate significant differences between groups (P ≤ 0.05). MthEh: methanolic extract of Euphorbia helioscopia; EthEh: ethanolic extract of Euphorbia helioscopia; AqEh: aqueous extract of Euphorbia helioscopia (Adapted with permission from ref. [58]. Copyright the Authors).
2.2.3 Tannins
Beyond terpenes, flavonoids and phenolic compounds, tannins have also been isolated from Zeqi, including more than 20 types, such as corilagin, euphorscopin, euphorhelin, furosin, helioscopinin A, helioscopinin B, helioscopin A, helioscopin B, punicafolin, and terchebin [59–61]. However, research on the pharmacological activity of tannins is limited, and further exploration is necessary.
2.3 Structural parts of Zeqi
In addition to the compounds of the Zeqi, studies also found that the whole seeds in Zeqi showed great pharmacological activity.
The seed of Zeqi (also known as EHS) and has been found to have potential in treating lung diseases such as pulmonary fibrosis. Experiments have explored the mechanism of action of EHS in treating pulmonary fibrosis. Widely targeted metabolomics has identified 231 compounds in 12 categories. Furthermore, network pharmacology and molecular docking analyses using a multi-ingredient, multi-target, and multi-pathway approach have indicated that EHS has anti-pulmonary fibrosis medicinal value. Molecular dynamics simulations have indicated that HSP90AA1 and 9-hydroxy-12-oxo-15(Z)-octadecenoic complex (with the highest docking score) have stable combination effects. And the binding free energy is about −82.5 kcal/mol, which proves that the complex has high stability [62]. EHS also contains major active ingredients such as terpenoids, lipids, flavonoids, and phenolic acids, although the content of the active ingredients differs from that in Zeqi plants. Therefore, EHS and Zeqi may have similar antitumor activity. Bioinformatics and computer-assisted drug design have been used to investigate the potential anti-pulmonary fibrosis effects of EHS. Network pharmacology and molecular docking results have shown that unsaturated fatty acids (EHS1) and flavonoids (EHS8) are primarily responsible for the antifibrotic effects of EHS in the lungs. EHS may exert antifibrotic effects on lung fibrosis by inhibiting HSP90AA1, EGFR, AKT1, and EP300, and modulating the AGE-RAGE signaling pathway. Moreover, 9-hydroxy-12-oxo-15 (Z)-octadecenoic acid, nordihydrocapsiate, 1-O-salicyl-d-glucose,9-(arabinosyl) hypoxanthine, xanthosine, and galangin-7-O-glucoside might potentially be the pharmacological components responsible for the antifibrotic effects of EHS in lung fibrosis [62]. However, to promote the clinical effectiveness of EHS, additional basic medical research must be performed.
3. DISCUSSION
Zeqi is a plant with considerable medicinal value and a documented history in ancient Chinese medicine texts. The clinical application of Zeqi has been increasing in recent years, and its antitumor, anti-inflammatory, anti-bacterial, and anthelmintic activity have attracted substantial research attention.
Beyond studies of antitumor activity, and the reported treatment of cancers including liver cancer, lung cancer, and breast cancer, Zeqi has been used in folk medicine to treat esophageal cancer [63]. However, in most current studies, only the components of Zeqi have been found to have killing effects on cancer cells, but the intrinsic antitumor mechanisms and the apoptotic pathways involved remain unclear. Therefore, research on the antitumor activity of Zeqi is insufficient. Interestingly, however, few studies have reported the pro-tumor activity of Zeqi, and the Zeqi factors tigliane and the ingenane diterpene ester type have been considered to be active tumor promoters. These factors exhibit moderate (H1) to low (H8) relative tumor promoting potency with respect to the ingenane prototype DTE tumor promoter 3-TI. However, current reports are insufficient, and, in general, the antitumor activity of Zeqi is more significant than the anti-inflammatory activity, antibacterial activity and other activities [64].
Crude extracts of Zeqi have shown significant potential in many studies, but most prior studies have examined crude extracts only, and no in-depth experiments have been performed to study specific components of crude extracts. For example, a study on the anthelmintic activity of Zeqi methanol extract has indicated favorable anthelmintic activity in vitro and in vivo, and the methanol extract has been speculated to contain oocytocidal compounds. However, no experiments have been designed to investigate this possibility, and the mechanism remains unclear. In the future, breakthroughs in this area may improve mechanistic understanding, and relevant clinical experiments may be performed for verification. Notably, in several studies, the antioxidant activity of the methanol and ethanol extracts of Zeqi has been hypothesized to be due to the presence of TPC and TFC in the extracts. Moreover, experiments have shown that higher TPC and TFC in the extracts is associated with higher extract antioxidant activity. Unfortunately, the specific mechanisms of TPC and TFC remain unclear. Breakthroughs in this topic are anticipated in the future.
Zeqi has significant anti-inflammatory activity, but some studies have found that the milky white sap produced by Zeqi is toxic and elicits toxic reactions in the skin and mucous membranes, primarily herpetic phototoxic dermatitis caused by psoralens [65]. Some studies have extracted 12-deoxyphosphorothioate-13-phenylacetate-20-acetate from the sap and found that it is the main component of the toxic locus of Zeqi and the most irritating substance isolated. Therefore, this compound is considered the root cause of toxicity [66]. However, another study extracting components from Zeqi sap has found that the main diterpenes in the sap are of the jatrophane and lathyrane types; therefore, these two types of diterpenes might be the main toxic components in the sap [67]. Although Zeqi is toxic, many studies have traced the toxicity to the sap, and the overall medicinal value of Zeqi cannot be underestimated. In the future, more in-depth research on the toxicity of Zeqi is necessary to prevent toxic harm.
In summary, many effective chemical constituents of Zeqi, including terpenes, flavonoids, and tannins, have been studied through pharmaceutical analysis. However, systematic phytochemistry research remains to be performed. In the field of pharmacology, studies on the bioactivity of extracts, terpenes, and flavonoids of Zeqi have focused on antitumor, anti-inflammatory, and anti-microbial activity, whereas fewer studies have examined tannins as well as structural parts of Zeqi. Although many pharmacological studies have used crude extracts of Zeqi, the relationship between the chemical constituents and activity remains unclear.
Zeqi has shown encouraging outcomes in several clinical studies; however, further well-designed multicenter, large cohort trials, as well as randomized controlled trials, are necessary to validate its efficacy as a single agent. Moreover, challenges pertaining to drug absorption, distribution, metabolism, excretion, and toxicity must be addressed. Hence, future research may pursue pharmacochemical modifications through chemical structure refinement, optimizing the pharmacokinetic and pharmacodynamic properties of related active ingredients, increasing their therapeutic potential, and pursuing clinical trials and successful drug development. Additionally, more studies on pharmacokinetics and bioavailability are necessary, including the design of experiments on combinations of Zeqi with other drugs in the treatment of diseases. To enhance the clinical effectiveness of Zeqi, expand its clinical applications, and make significant contributions to human health, a comprehensive understanding of Zeqi’s potential value is required.
ABBREVIATIONS
HL-60, human promyelocytic leukemia cell; MCF-7/ADR, human breast adenocarcinoma cell line; PANC-1, human pancreatic cancer cells; LPS, bacterial lipopolysaccharide; RAW264.7, mouse monocyte macrophage leukemia cells; HEK293, human embryonic kidney cells 293; MDR, multidrug resistance; NAFLD, nonalcoholic fatty liver disease; DPPH, DPPH research; FRAP, ferric ion reducing antioxidant power; ABTS, Total Antioxidant Capacity Research Kit with ABTS method; HSP90AA1, heat shock protein; EGFR, epidermal growth factor receptor.