Ethanolic Extracts of Cupressaceae Species Conifers Provide Rapid Protection against Barium Chloride-Induced Cardiac Arrhythmia

Sudden cardiac death (SCD) is responsible for a high percentage of cardiovascular fatalities, with ventricular arrhythmias being the most common cause. Despite numerous clinically available antiarrhythmic drugs (AADs), AADs retain some undesirable arrhythmic effects, and their inappropriate use can lead to severe adverse reactions. The exploration of new therapeutic options against arrhythmias with fewer unreceptive effects is of utmost importance. The ethanolic extracts of seven Cupressaceae species, namely, Chamaecyparis obtusa, Juniperus chinensis (L.) Ant., Sabina chinensis (L.) Ant. cv. Kaizuca, Platycladus orientalis (L.) Franco, Juniperus sabina L., Fokienia hodginsii, and Juniperus chinensis ‘Pyramidalis’ were investigated for their pharmacological effects on barium chloride (BaCl2)-induced arrhythmia using normal II lead electrocardiogram (ECG) measurements in a mouse model. According to the ECG profiles, pretreatment with C. obtusa, P. orientalis, and J. sabina extracts provoked dose-dependent protection against BaCl2-induced arrhythmia, while pretreatment with the other four species and amiodarone did not exert cardioprotective effects. The treatment effects were confirmed using a rat model. The therapeutic effects of C. obtusa, P. orientalis, and J. sabina extracts on the M2 and M3 receptors but not the M1 receptor were mediated by the inhibition of the M2 receptor blocker (methoctramine tetrahydrochloride), M3 antagonist (4-DAMP), or M1 receptor blocker (pirenzepine dihydrochloride). This first-line evidence illustrates that certain Cupressaceae species possess active antiarrhythmic components. The first line of key findings revealed that active components of certain Cupressaceae species have cardioprotective effects, suggesting that these innovative phytochemicals have promising potential for preventing the occurrence of cardiac arrhythmia and reducing sudden cardiac death.


Introduction
Cardiovascular diseases (CVDs) are major causes of health problems and death, representing prominent causes of morbidity and mortality worldwide and a total health and economic burden.Numerous lines of evidence suggest that CVDs include complex remodeling responses, such as hypertension, myocardial ischemia, and valve disease, which lead to poor clinical outcomes, including arrhythmia, heart failure (HF), and sudden cardiac death (SCD).These deleterious effects not only aggravate intrinsic heart disease and influence the eminence of life of patients but also induce SCD, which is life-threatening [1].As the utmost populous country worldwide, China has ≈290 million patients with CVDs, and thus, CVDs represent the leading cause of death in the Chinese population [2].In most cases, SCD leads to a sudden and unpredictable death due to an alteration in heart rhythm (sudden cardiac arrest).It estimates about 300,000 to 400,000 deaths annually in the United States and approximately 600,000 deaths annually in China [3,4].
Notably, the pathogenesis of arrhythmia is complicated and unpredictable.Intervention alternatives for arrhythmias include medications, radiofrequency ablation, cardiac resynchronization, implantable cardioverter-defibrillators, artificial pacemakers, and heart transplants [5][6][7][8][9].Notwithstanding technological advances in catheter ablation therapy, antiarrhythmic drugs (AADs) are a basis for the management of cardiac arrhythmias.Experimental and translational data have demonstrated that regularly used AADs exert numerous effects on the heart, and the manifestation of these effects intensely depends on the specific experimental or clinical settings [10].In the clinic, the treatment of arrhythmia is mainly based on Western medicine, i.e., propafenone (calcium channel blocker) and propranolol (β receptor blocker), but medical research has shown that AADs also have undesirable arrhythmic effects and that their improper usage can frequently cause more severe adverse reactions.Amiodarone [2-butyl-3-(3 ′ ,5 ′ -diiodo-4 ′ α-diethylaminoethoxybenzoyl)benzofuran] (AMD), a class III antiarrhythmic drug that is an antagonism of Na + , Ca 2+ , and K + channels, and the β-adrenergic receptors as well, is a highly efficient AAD with potentially serious side effects in humans, such as idiosyncratic hepatotoxic reactions [11] and severe pulmonary toxicity (interstitial pneumonia and lung fibrosis) [12].Despite numerous clinical options for AADs, pharmacotherapy is still ineffectual for the majority of patients.Additionally, all antiarrhythmic agents that act via diverse ion channels retain lifethreatening proarrhythmic potential.Though the long-term consumption of these drugs can release symptoms, they can lead to an inadequate prognosis and may even upsurge mortality; hence, there is an urgent necessity to discover new antiarrhythmic treatment strategies involving natural products [13,14].
New phytoconstituent drugs and their physicochemical properties can be used for the treatment of various diseases because the secondary metabolites of plants include important bioactive compounds that are selected and propagated naturally for application as remedies against various human diseases and health disorders [15].Remarkably, Juniperus sabina L. and Sonchus oleraceus (L.) L. are broadly utilized as traditional medicinal plants in China, and their aqueous extracts have been taken as a folk medicine to treat infections, inflammatory diseases, and tumors [16].Several studies have shown that J. sabina L. significantly improves hepato-protective activity, antioxidant activity, antidiabetic activity, and antitumor effects [17][18][19].J. chinensis has demonstrated noteworthy improvements in anti-proliferative and antifungal activities [20][21][22].In addition, J. formosana Hayata has been shown to have an inhibitory effect on the progression of kidney disease, urinary disorders, gynecological diseases, and lung cancer [23][24][25].The fruit and leaves of junipers are usually used as tea, and minced fruits are applied to lower blood sugar contents in Anatolia [19].Chamaecyparis obtusa was found to be effective in preventing bleeding, possessing antibacterial activity, and exerting antitussive effects [26].Whether Cupressaceae plants possess antiarrhythmia activity remains to be investigated.
In this study, we attempted to evaluate the efficacy of pretreatment with Cupressaceae in a murine model of barium chloride (BaCl 2 )-induced arrhythmia.To achieve this goal, seven Cupressaceae species, including Chamaecyparis obtusa, Juniperus chinensis (L.) Ant., Sabina chinensis (L.) Ant.cv.Kaizuca, Platycladus orientalis (L.) Franco, Juniperus sabina L., Fokienia hodginsii, and Juniperus chinensis 'Pyramidalis', were collected and extracted with ethanol.The biological function (protection) of the ethanolic extracts against BaCl 2 -induced arrhythmia was examined using normal II lead electrocardiogram (ECG) measurements in mouse models.

A Survey of the Medicinal Value of Various Conifer Plants
The literature search of electronic databases, including PubMed, MEDLINE, ScienceDirect, Google Scholar, the Cochrane Library, and Web of Science, was performed to iden-tify articles published from inception until May 2023, illustrating that various conifer plant extracts composed of a variety of active components have great medicinal value (Supplemental Table S1 in Supplementary File).For example, F. hodginsii, a rich source of structurally diverse diterpenoids, has been used for the treatment of stomach aches, nausea, vomiting, and anti-proliferative activity [27][28][29][30], which are dependent on various components.One report shows that the volatile oil (100 mg/kg body weight, p.o.) of Cupressus funebris Endl can significantly enhance its anti-inflammatory activity [31].The volatile oil of J. chinensis 'Pyramidalis' has significant antitumor and antibacterial effects [32].In addition to its blood circulation, diuretic activity, and antibacterial activity, S. chinensis L. exerts antidiabetic effects via its α-amylase inhibitory activity [33].The compounds β-phellandrene, terpinen-4-ol, and bornyl acetate are major constituents of essential oils in S. chinensis and have been found to possess effective antifungal activity [22,34].Three phenolic compounds, cosmosiin, caffeic acid, and p-coumaric acid, were firstly isolated from the leaves of Cupressus sempervirens L.; together with cupressuflavone, amentoflavone, rutin, quercitrin, quercetin, and myricitrin, these compounds have been found to have high antioxidant activity, and quercetin, rutin, caffeic acid, and p-coumaric acid are further used for hepatoprotective activity [35].The previous study affords a promising complementary alternative for the proper use of a selection of essential oil combinations for use in the respiratory tract [36].In total, 33, 37, and 37 compounds were found in the oils from the leaves of S chinensis (L.) Ant, C. lusitanica 'Zhongshan' Mill, and S. chinensis (L.) Ant.Cv.Kaizuca, respectively.Fourteen compounds were commonly identified, such as thujene, alpha-pinene, camphene, sabinene, beta-myrcene, alpha-terpinene, gammaterpinene, alpha-terpinolene, bornyl acetate, beta-elemene, alpha-amorphene, germacrene D, delta-cadinene, and elemol.Moreover, each species contained its own particular compounds.Remarkably, the main components are sabinene (20.99%), limonene (19.78%), and bornyl acetate (11.68%) for S. chinensis (L.) Ant; alpha-pinene (10.39%), sabinene (11.19%), and delta-3-carene (8.88%) for C. lusitanica 'Zhongshan' Mill; and limonene (24.56%) and beta-myrcene (8.04%) for S. chinensis (L.) Ant.Cv.Kaizuca [37].Because of the various associated phytochemicals with numerous medicinal properties, diverse conifer plants might represent a new source of materials with pharmacological properties involved in protection against cardiac arrhythmia that is worth investigating.

Screening of Various Conifer Plant Extracts in Mice
To investigate the antiarrhythmic effects on conifer plants, mice were gavaged with the crude extracts of F. hodginsii, C. obtusa, J. chinensis (L.) Ant., S. chinensis (L.) Ant.cv.Kaizuca, P. orientalis (L.) Franco, J. sabina L., or J. chinensis (0.1 mL/10 g Bwt) for 10 min and injected (i.p.) with 0.8% BaCl 2 solution (0.1 mL/10 g Bwt).First, mice were orally administered olive oil alone for 5 or 10 min before BaCl 2 injection, and the mice with an ECG of BaCl 2 -induced arrhythmia failed to show any effect of the addition of olive oil (Figure 1A,C).Olive oil was used to dissolve all the extracts in subsequent experiments.Next, to evaluate time points after administration of the extracts, mice were orally administered the C. obtusa, P. orientalis, and J. sabina extracts for 5 or 10 min followed by a subsequent BaCl 2 injection.The results revealed no protective effect at 5 min after administration (Figure 1B), while 10 min prior to BaCl 2 injection was found to have a protective effect (Figure 1D).To investigate the protective effect of amiodarone (clinical medicine as a reference), mice were i.p. injected with 0.6% amiodarone (15 mg/kg Bwt) for 10 min before BaCl 2 injection, and the ECG signals of the mice appeared normal (Figure 1E).The results further illustrated that BaCl 2 -induced arrhythmia in mice is remediable via clinical drugs.Subsequently, considering the intestinal absorption of the extracts, the conifer plant extract was intragastrically administered for 10 min, followed by an injection of the BaCl 2 solution.After seven conifer plants were screened, F. hodginsii, J. chinensis (L.) Ant., S. chinensis (L.) Ant.cv.Kaizuca, and J. chinensis showed no protective effects against BaCl 2 -induced arrhythmia in mice.Surprisingly, the ECG data revealed protective effects of C. obtusa, P. orientalis, and J. sabina against BaCl 2 -induced arrhythmia in mice (Figures 2-4).

Dose-Dependent Protective Effects of C. obtusa, P. orientalis, and J. sabina Extracts on BaCl2-Induced Arrhythmia in Mice
To determine the antiarrhythmic effects of C. obtusa, P. orientalis, and J. sabina on BaCl2-induced arrhythmia in mice, various doses of C. obtusa, P. orientalis, and J. sabina extracts were tested.The data revealed dose-dependent protective effects of the C. obtusa, P. orientalis, and J. sabina extracts, ranging from 0.075 mL/10 g Bwt to 0.15 mL/10 g Bwt or 0.2 mL/10 g Bwt, while 0.05 mL/10 g Bwt had no significant effect (Figures 2-4).

Efficacy of C. obtusa, P. orientalis, and J. sabina Extracts in Protection against BaCl2-Induced Arrhythmia in Mice
To investigate the protective potential (duration) of BaCl2-induced cardiac arrhythmia, mice were orally administered various doses of C. obtusa, P. orientalis, and J. sabina extracts for 10 min, followed by an injection of the BaCl2 solution.The results showed that with increasing doses of P. orientalis, and J. sabina extracts, the duration of protection increased with increasing dosage.However, due to the toxicity of C. obtusa, the duration of  To determine the antiarrhythmic effects of C. obtusa, P. orientalis, and J. sabina on BaCl 2 -induced arrhythmia in mice, various doses of C. obtusa, P. orientalis, and J. sabina extracts were tested.The data revealed dose-dependent protective effects of the C. obtusa, P. orientalis, and J. sabina extracts, ranging from 0.075 mL/10 g Bwt to 0.15 mL/10 g Bwt or 0.2 mL/10 g Bwt, while 0.05 mL/10 g Bwt had no significant effect (Figures 2-4).

Efficacy of C. obtusa, P. orientalis, and J. sabina Extracts in Protection against BaCl 2 -Induced Arrhythmia in Mice
To investigate the protective potential (duration) of BaCl 2 -induced cardiac arrhythmia, mice were orally administered various doses of C. obtusa, P. orientalis, and J. sabina extracts for 10 min, followed by an injection of the BaCl 2 solution.The results showed that with increasing doses of P. orientalis, and J. sabina extracts, the duration of protection increased with increasing dosage.However, due to the toxicity of C. obtusa, the duration of protection increased to 0.15 mL/10 g Bwt (Table 1).These results revealed that C. obtusa, P. orientalis, and J. sabina extracts contain antiarrhythmic characters.One report indicated that the ethanol extract of Sophora flavescens Ait. has been shown to have antiarrhythmic activity via investigations of cardiac arrhythmias induced by aconitine infusion in mice and by coronary artery ligation in rats [38].To confirm the potential of BaCl 2 -induced cardiac arrhythmia treatment with C. obtusa, P. orientalis, and J. sabina extracts, rats were orally injected with 0.4 mL/100 g Bwt of C. obtusa, P. orientalis, and J. sabina extracts, respectively, of the BaCl 2 solution.The results revealed the antiarrhythmic effects of C. obtusa, P. orientalis, and J. sabina extracts against BaCl 2 -induced cardiac arrhythmia in rats (Figure 5).These results were consistent with the results obtained in mice, illustrating that C. obtusa, P. orientalis, and J. sabina extracts possessed antiarrhythmic effects.

Treated Efficacy of C. obtusa, P. orientalis, and J. sabina Extracts in BaCl2-Induced Arrhythmia in Rats
One report indicated that the ethanol extract of Sophora flavescens Ait. has been shown to have antiarrhythmic activity via investigations of cardiac arrhythmias induced by aconitine infusion in mice and by coronary artery ligation in rats [38].To confirm the potential of BaCl2-induced cardiac arrhythmia treatment with C. obtusa, P. orientalis, and J. sabina extracts, rats were orally injected with 0.4 mL/100 g Bwt of C. obtusa, P. orientalis, and J. sabina extracts, respectively, of the BaCl2 solution.The results revealed the antiarrhythmic effects of C. obtusa, P. orientalis, and J. sabina extracts against BaCl2-induced cardiac arrhythmia in rats (Figure 5).These results were consistent with the results obtained in mice, illustrating that C. obtusa, P. orientalis, and J. sabina extracts possessed antiarrhythmic effects.To further understand which M-receptor type was involved in the antiarrhythmic effects of the C. obtusa, P. orientalis, or J. sabina extracts compared with the reference drug amiodarone, mice were first administered BaCl 2 ; once arrhythmia developed, the mice received pirenzepine dihydrochloride (M1 antagonist, 0.3 mg/kg Bwt), methoctramine tetrahydrochloride (M2 antagonist, 0.3 mg/kg Bwt), or 4-DAMP (M3 antagonist, 1 mg/kg Bwt) for 2 min and then amiodarone, C. obtusa, P. orientalis, or J. sabina extracts (0.1 mL/10 g Bwt) to monitor alterations in the ECG.The results demonstrated that the antiarrhythmic effects of the C. obtusa, P. orientalis, and J. sabina extracts could occur through the M2 (Figure 6) and M3 receptors (Figure 7) but not the M1 receptor (Figure 8).

The Involvement of the M Receptor in the Antiarrhythmic Effects of C. obtusa, P. orientalis, and J. sabina Extracts Compared with That of Amiodarone in Mice
To further understand which M-receptor type was involved in the antiarrhythmic effects of the C. obtusa, P. orientalis, or J. sabina extracts compared with the reference drug amiodarone, mice were first administered BaCl2; once arrhythmia developed, the mice received pirenzepine dihydrochloride (M1 antagonist, 0.3 mg/kg Bwt), methoctramine tetrahydrochloride (M2 antagonist, 0.3 mg/kg Bwt), or 4-DAMP (M3 antagonist, 1 mg/kg Bwt) for 2 min and then amiodarone, C. obtusa, P. orientalis, or J. sabina extracts (0.1 mL/10 g Bwt) to monitor alterations in the ECG.The results demonstrated that the antiarrhythmic effects of the C. obtusa, P. orientalis, and J. sabina extracts could occur through the M2 (Figure 7) and M3 receptors (Figure 8) but not the M1 receptor (Figure 6).(A) M2/Amiodarone

Discussion
In the present study, we first demonstrated the pharmacological effects of C. obtusa, P. orientalis, and J. sabina extracts in a concentration-dependent manner as the cardioprotective agents against BaCl 2 -induced arrhythmia.As we know, traditional Chinese medicine (TCM) has played a critical role in ameliorating symptoms, thwarting disease recurrence, reducing toxic side effects, and improving the quality of life.The use of Western medicine for the management of AF is limited, and several studies have revealed that traditional herbs comprise a variety of pharmacologically active constituents that have great efficacy and prospective for the hindrance and treatment of cardiac arrhythmia [39][40][41][42] with distinctive advantages, such as few side and adverse effects, low toxicity, a long effect duration, and high compliance.In terms of clinical usage, TCMs with alkaloids, flavonoids, and saponins as the leading effective constituents have a positive effect on the treatment of CVDs such as angina pectoris, arrhythmia, myocardial ischemia, and myocardial infarction (MI) [43].Another report revealed that Fuzi, Aconiti lateralis Radix Praeparata, has been broadly used for 2000 years in TCM for the treatment of acute HF.Notably, the results revealed that the longterm use of Fuzi has a main benefit in averting cardiovascular problems [44].Calycosin and its derivatives, the major bioactive flavonoids in Astragalus membranaceus, have promising potential for the cardiovascular protection of cardiac myocytes and vascular endothelial cells [43].Wenxin Keli (WXKL), a typical Chinese patent medicine with apparent effectiveness and promising safety, has played a prominent role in the treatment of CVD patients.Accumulating evidence from various cell and animal studies has shown that WXKL plays cardioprotective roles by impeding inflammation, diminishing oxidative stress, mediating vasomotor disorders, decreasing cellular apoptosis, and protecting against endothelial injury, myocardial ischemia, cardiac fibrosis, and cardiac hypertrophy [45].Furthermore, the action of WXKL may reduce the QT interval and dawdle the heart rate by downregulating sodium channel protein type 5 subunit alpha (SCN5A) and the beta-2 adrenergic receptor (ADRB2) and upregulating muscarinic acetylcholine receptor M2 (CHRM2) during myocardial ischemia.These findings afford novel insight into the molecular mechanisms by which WXKL reduces the prevalence of ventricular arrhythmia [46].
The results of this study disclose that the pharmacological effects of C. obtusa, P. orientalis, and J. sabina extracts exert cardioprotection against BaCl 2 -induced (inhibiting IK1) arrhythmia.Basal and acetylcholine-gated inward-rectifier K + -currents (IK1 and IKACh, respectively) play vital roles in cardiac excitability by mediating heart rate variability and susceptibility to atrial arrhythmias and AF.Recent studies have indicated the coexistence of multiple muscarinic acetylcholine receptor (mAChR) subtypes that regulate several distinct K + currents in the heart, namely, the inward rectifier K + current (IKACh) by M2 and two delayed rectifier K + currents, IKM3 and IK4AP, by the M3 and M4 receptors, respectively.Gi-protein-coupled muscarinic receptor M2 is considered the predominant receptor that activates IKACh [47].Calcium/calmodulin-dependent protein kinase II (CaMKII) is a vital ion channel mediator that participates in excitation-contraction coupling to regulate its electrophysiological function.These effects can be largely abolished by the co-application of the IK1 blocker BaCl 2 .IK1-dependent suppression of CaMKII activity is a crucial cardiac salvage signaling pathway during Ca 2+ dyshomeostasis and oxidative stress (reactive oxygen species, ROS).IK1 might be a unique target for the pharmacological conditioning of reperfusion arrhythmia, especially during intervention after unpredictable ischemia [48].
Cardiac autonomic nerve dysfunctions, such as the excitement of the Vagal nerve and inhibition of sympathetic nerves, have been exposed by molecular biology studies [49,50].Accordingly, arrhythmia can be triggered by the abnormal structure and function of ion channels [51].Accumulating studies have demonstrated a role for α1-adrenolytics in the management of arrhythmias.The stimulation of α1-adrenoceptor facilitates inositol trisphosphate (IP 3 ) production and subsequent Ca 2+ release from the sarcoplasmic reticulum (SR) [52].Therefore, the blockade of α1-adrenoceptors may result in the stabilization of Ca 2+ levels, generating antiarrhythmic effects in catecholamine-induced arrhythmias, e.g., catecholaminergic polymorphic ventricular tachycardia.One study has reported that prazosin not only reduced the norepinephrine-induced elongation of AF in mice but also mitigated norepinephrine-induced SR Ca 2+ leakage and spontaneous SR Ca 2+ release in cultured atrial cardiomyocytes.These findings confirm that α1-adrenoceptors may have a role in preventing cardiac arrhythmias [53] and have been confirmed in numerous animal studies, thus validating the antiarrhythmic properties of α1-adrenolytics [54][55][56].In earlier experiments, 2-methoxyphenylpiperazine derivatives were shown to have a high affinity for α1-adrenoceptors [57], and the activities of these compounds were compared with those of carvedilol, which is a β1and α1-adrenoceptor blocker with antioxidant properties [58].Nevertheless, the mechanism by which C. obtusa, P. orientalis, and J. sabina extracts protect against BaCl 2 -induced arrhythmia requires verification.
The experiments also depict the involvement of the M receptor in the antiarrhythmic effects of C. obtusa, P. orientalis, and J. sabina extracts compared with that of amiodarone in mice.In primary tissues, at least four pharmacological M receptors (M1, M2, M3, M4) are defined, and five muscarinic receptors have been cloned (m1, m2, m3, m4, m5).There are few selective agonists for M-receptor subtypes, and all classical agonists (acetylcholine, carbachol, etc.) are evidently nonselective.Several selective antagonists for M1 (pirenzepine) and M2 receptors (AF-DX 116) have been critically studied [59].A comparative study of the ability of selective M-cholinoblockers to prevent arrhythmias induced by phosphacol and potassium cyanide showed that the activity of the M1 antagonist pirenzepine is greater than that of the M2 antagonist AF-DX-116; simultaneously, both drugs revealed the equivalent activity with regard to arrhythmias induced by aconitine, calcium chloride, and carbachol [60].The present results demonstrated that the antiarrhythmia effects of C. obtusa, P. orientalis, and J. sabina extracts could occur through the M2 (Figure 7) and M3 receptors (Figure 8) but not the M1 receptor (Figure 6).One study has reported that (1) the stimulation of the M1 mAChR subtype on sympathetic postganglionic cells results in catecholamine-mediated cardiac stimulation, (2) M1 mAChR is not expressed in the mouse heart, and (3) the administration of ACh to mice induces arrhythmia [61].The IKACh plays a vital role in cardiac excitability by mediating heart rate variability and vulnerability to atrial arrhythmias.Both inward rectification mechanisms are extrinsic to the KACh channel; from our understanding, this is the first depiction of an extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of the G protein-coupled receptor M2 [62].Recent studies have indicated the presence of multiple mAChR subtypes that regulate several distinct K+ currents in the heart, namely, the IKACh by M2 and the two delayed rectifier K+ currents IKM3 and IK4AP by the M3 and M4 receptors, respectively [63].This is the first report to demonstrate the downregulation of three types of mAChR-coupled K + currents (IKM2, IKM3, and IKM4) and of M2, M3, and M4 mAChR subtype proteins in a canine model of atrial tachycardia (AT)-induced remodeling [64].Both D,L-sotalol and MS-551 interact with cardiac M2 and peripheral M3 receptors, and they exert anticholinergic activity in cardiac and peripheral tissues at high concentrations [65].Activation of M3 has been previously shown to promote shortterm cardioprotection against ischemic injury with the M3 agonist choline, the antagonist 4-DAMP, or the M2-mAChR antagonist methoctramine followed by the administration of choline.M3-mAChRs denote a promising target for interpreting cardiomyocytes tolerant to ischemic injury [66].The prevention of ischemia-induced changes in Gi-mediated signal transduction and/or (with certain limitations discussed below) the selective activation of cardiac muscarinic M2 receptors could hence be an alternative pharmacological treatment for acute myocardial ischemia [67].Atrial G protein-gated inwardly rectifying K+ (GIRK) channels are critical mediators of parasympathetic effects on cardiac physiology.The mouse ventricular GIRK channel is a GIRK channel subunit (GIRK1, GIRK4), a GIRK1/GIRK4 heteromer, and although it contributes to the cholinergic suppression of ventricular myocyte excitability, this impact does not substantively influence cardiac physiology or ventricular arrhythmogenesis in mice [68].Accumulating evidence indicates the presence of functional M3-mAChRs, in addition to the well-recognized M2-mAChRs, in the hearts of various species comprising humans.Choline improves cardiac function and moderates ischemic myocardial injury by stimulating cardiac M3-mAChRs, which in turn results in changes in the multiple signaling pathways, leading to cytoprotection.These findings suggest that M3-mAChR is a new target for improving cardiac function and preventing cardiac injury during ischemia/reperfusion [69].
Remarkably, bioactive compounds such as phenolics, flavonoids, terpenoids, glycosidic derivatives, alkaloids, iridoids, and saponins from various parts of plants including Terminalia arjuna [70] and Emblica officinalis fruit [71] have gained important applications in exerting significantly cardioprotective effects.Furthermore, many lines of medicinal values have been discovered from Cupressus sempervirens L. (Cupressaceae) including headache, toothaches [72], sneeze bronchitis, obesity, and laryngitis along with biological properties such as anti-inflammatory, anti-microbial, and insecticidal actions [73].In addition, nineteen various polyphenolic molecules comprising gallic acid, chlorogenic acid, catechin, methyl gallate, coffeic acid, syringic acid, pyrocatechol, rutin, ellagic acid, coumaric acid, vanillin, ferulic acid, naringenin, rosmarinic acid, daidzein, querectin, cinnamic acid, kaempferol, and hesperetin upon extraction of C. sempervirens using supercritical fluid extraction have recently been identified and further been investigated for their antibacterial and anti-biofilm activities [74,75].To the best of our knowledge, this study is the first investigation to explore the potential benefit of Cupressaceae in the treatment of arrhythmia, making our findings particularly valuable.In the future, we will be treating large animal arrhythmia with the extracts to confirm the efficacy of ethanolic extracts.And the extracts are isolated and purified to obtain the active components via HPLC and NMR.The experiments will further be conducted to investigate the purified components in in vivo and in vitro assays in the treatment of animal arrhythmia to validate the efficacy, acting mechanisms, and toxicity for the completion of preclinical tests.

Extraction of Cypress Leaves
First, all the dried cypress leaves were cut into small pieces.Second, all the cut leaves were soaked in 75% ethanol (1:10, w/v) in an ultrasonic bath (KQ-5200 type DE, Kun Shan Ultrasonic Instruments Co., Ltd., Kunshan, Jiangsu, China) to obtain the extraction solutions.Finally, the extraction solutions were dried in an evaporator (DHG-9070A, Shanghai Heheng Instruments and Equipment Co., Ltd., Shanghai, China) at 42 • C to obtain the products as dark green solids.The extracts were then dissolved in olive oil for subsequent experiments.The yield of crude extract was 8.6 ± 1.2%.The experimental concentration was 40 mg/mL.

Animal Care
All experimental procedures were executed according to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals, 8th edition" published by the National Institutes of Health, and they fulfilled the ARRIVE guidelines.The animal experiments were approved by the Animal Ethics Committee of Medical College according to the "Guide for the Care and Use of Laboratory Animals" of Xiamen Medical College (approved protocol ID SYXK 2018-0010).Evaluations of experimental animal care were periodically performed in accordance with the Laboratory Animal Guidelines for Ethical Review of Animal Welfare (GB/T 35892-2018, China).
Eighty male ICR mice (6 weeks old, 22 ± 3 g Bwt) and male Sprague Dawley rats (150 ± 30 g Bwt) were obtained from Hangzhou Medical College (Zhejiang, China) and kept at RT (22 ± 2 • C) and a specific humidity (50 ± 10%).A 12/12 h light/dark (6 a.m.-6 p.m.) cycle was maintained throughout the entire study.The mice had free access to a standard laboratory diet (Rodent Feed 1022, Beijing HFK Bioscience Co., Ltd., Beijing, China) and tap water ad libitum.Sprague Dawley rats and ICR mice were anesthetized with 5% isoflurane gas in an inhalation chamber with a vaporizer (R583S rodent gas anesthesia machine, RWD Life Science Co., Ltd., Shenzhen, China), and 2% isoflurane was administered during the entire experimental procedure.The mice were anesthetized according to the protocol supplied in the McGill Standard Operating Procedure (SOP) (#110 for mice and #111 for rats), which describes methods used for mouse and rat anesthesia.Mice were randomly divided into 9 groups: an NS group, a positive control group (amiodarone), and 7 test groups of Cupressaceae leaves.First, the mice were generally anesthetized with 5% inhaled isoflurane in a rodent gas anesthesia machine (RWD Life Science Co., Ltd.Shenzhen, China) and fixed on a plank.Second, acupuncture needles were subcutaneously inserted into the limbs of the mice to monitor and record normal II lead electrocardiograms (ECGs) via the BL-420I biological function experiment system (Techman Inc., Chengdu, Sichuan, China) under 2% isoflurane anesthesia.Then, the anesthetized mice were orally administered different treatments at the test dose.Ten minutes later, a 0.8% barium chloride solution was i.p. injected into each mouse (0.1 mL/10 g Bwt) to induce arrhythmia.ECG signals were individually monitored and recorded during the experiments.The number of mice that maintained a normal rhythm and the duration (5, 10, and 30 min) of normal rhythm mice were recorded to calculate the efficacy of the tested samples in each group.At the end of the experiments, all rats were sacrificed by CO 2 euthanasia.Additional experiments were performed to explore the effect of the muscarinic acetylcholine receptor type on the antiarrhythmic effects of the C. obtusa, P. orientalis, and J. sabina extracts (5 mg/mL of olive oil).Mice were treated with pirenzepine dihydrochloride (an M1 antagonist, 0.3 mg/kg Bwt), methoctramine tetrahydrochloride (an M2 antagonist, 0.3 mg/kg Bwt), or 4-DAMP (an M3 antagonist, 1 mg/kg Bwt) for 2 min and administered amiodarone, C. obtusa, P. orientalis, or J. sabina extracts (0.1 mL/10 g Bwt) to detect alterations in the ECG profile.

Statistical Analysis
The in vivo data are expressed as means ± SEMs.The results were carried out by using a one-way analysis of variance (ANOVA) for statistical comparisons among treatments.The means within each column followed by different letters are significantly different at p < 0.05 according to the post hoc Tukey's test.

Conclusions
CVDs are the leading cause of global mortality and impose a considerable economic burden on both governments and individuals.Ethanolic extracts of seven species of Cupressaceae, namely, Chamaecyparis obtusa, Juniperus chinensis (L.) Ant., Sabina chinensis (L.) Ant.cv.Kaizuca, Platycladus orientalis (L.) Franco, Juniperus sabina L., Fokienia hodginsii, and Juniperus chinensis 'Pyramidalis' were screened for their cardioprotective effects against BaCl 2 -induced arrhythmia in a mouse model, and the results of the ECG profiles revealed that pretreatment with C. obtusa, P. orientalis, and J. sabina extracts exerted dose-dependent cardioprotective effects.The cardioprotection of the C. obtusa, P. orientalis, and J. sabina extracts was exerted through the M2-and M3-mAChRs.These treatment effects were also confirmed in a rat model.These first-line key findings reveal that certain Cupressaceae species possess active antiarrhythmic components, suggesting that these innovative phytocompounds might have promising potential for preventing the occurrence of cardiac arrhythmia and reducing SCD.The identification of active phytochemicals and their antiarrhythmic implications in the clinic requires further study.

2. 3 .
Dose-Dependent Protective Effects of C. obtusa, P. orientalis, and J. sabina Extracts on BaCl 2 -Induced Arrhythmia in Mice 2.3 min *** Mice was orally pretreated with various concentrations of ethanol extracts of C. obtusa, P.

Figure 5 .
Figure 5. Treatment with 0.4 mL/100 g Bwt of (A) Chamaecyparis obtusa, (B) Platycladus orientalis, and (C) Juniperus sabina in rats after BaCl2-induced arrhythmia.2.6.The Involvement of the M Receptor in the Antiarrhythmic Effects of C. obtusa, P. orientalis, and J. sabina Extracts Compared with That of Amiodarone in MiceTo further understand which M-receptor type was involved in the antiarrhythmic effects of the C. obtusa, P. orientalis, or J. sabina extracts compared with the reference drug amiodarone, mice were first administered BaCl2; once arrhythmia developed, the mice received pirenzepine dihydrochloride (M1 antagonist, 0.3 mg/kg Bwt), methoctramine tet-

2. 6 .
The Involvement of the M Receptor in the Antiarrhythmic Effects of C. obtusa, P. orientalis, and J. sabina Extracts Compared with That of Amiodarone in Mice

4. 4 .
Antiarrhythmic Activity of the Extract in a Mouse Model of BaCl 2 -Induced Arrhythmia Prior to (Protection) BaCl 2 Induction

Table 1 .
The protection duration (min) of barium chloride-induced cardiac arrhythmia pretreated with the ethanol extracts of Chamaecyparis obtusa, Platycladus orientalis, and Juniperus sabina leaves.

Table 1 .
The protection duration (min) of barium chloride-induced cardiac arrhythmia pretreated with the ethanol extracts of Chamaecyparis obtusa, Platycladus orientalis, and Juniperus sabina leaves.