Zanthoxylum bungeanum Maxim. (Rutaceae): A Systematic Review of Its Traditional Uses, Botany, Phytochemistry, Pharmacology, Pharmacokinetics, and Toxicology

Zanthoxylum bungeanum Maxim. (Rutaceae) is a popular food additive and traditional Chinese herbal medicine commonly named HuaJiao in China. This plant is widely distributed in Asian countries. The aim of this paper is to provide a systematic review on the traditional usages, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology of this plant. Furthermore, the possible development and perspectives for future research on this plant are also discussed. To date, over 140 compounds have been isolated and identified from Z. bungeanum, including alkaloids, terpenoids, flavonoids, and free fatty acids. The extracts and compounds have been shown to possess wide-ranging biological activity, such as anti-inflammatory and analgesic effects, antioxidant and anti-tumor effects, antibacterial and antifungal effects, as well as regulatory effects on the gastrointestinal system and nervous system, and other effects. As a traditional herbal medicine, Z. bungeanum has been widely used to treat many diseases, especially digestive disorders, toothache, stomach ache, and diarrhea. Many traditional usages of this plant have been validated by present investigations. However, further research elucidating the structure-function relationship among chemical compounds, understanding the mechanism of unique sensation, as well as exploring new clinical effects and establishing criteria for quality control for Z. bungeanum should be further studied.


Introduction
The Zanthoxylum genus (Rutaceae) consists of 250 species worldwide, including 45 species and 13 varieties in China. Z. bungeanum is a species of the genus Zanthoxylum, widely distributed in Asian countries including China, Japan, India, Korea, etc. The fruits of Z. bungeanum are the most popular commercial product in the genus Zanthoxylum, and are largely used as a popular condiment in cooking and medicine with a long history for both medicinal and economic uses in China. So far, multiple cultivars of HuaJiao have been cultivated during the process, such as Yuexigong Jiao, Da Hongpao, and Hanyuan HuaJiao [1,2]. The pericarps color of Z. bungeanum cultivars is bright red, and therefore these cultivars are commonly known as "HonghuaJiao" in Chinese [3,4].
Since 1977, the Z. bungeanum has been listed in the Pharmacopoeia of the People's Republic of China (Ch. P), and over 30 prescriptions containing Z. bungeanum have been applied for the treatment of abdominal pain, toothache, dyspepsia, vomiting, diarrhea, ascariasis, eczema, etc. [5][6][7].   The distribution of Z. bungeanum is very wide due to its wide niche breadth. It is native to China and widely distributed in the provinces of Sichuan, Shaanxi, Yunnan, Guizhou, Gansu, etc., among which Sichuan province contains the largest production areas, famous for their high production and quality. In addition, it is also widely cultivated in the Japanese islands, the Korean peninsula, India, and other regions [25,26].

Phytochemistry
To the best of our knowledge, many chemical compounds have been isolated and identified from Z. bungeanum between the later 1880s and the present day. Currently, more than 140 constituents have been identified from this plant; furthermore, alkaloids and terpenoids have been identified as the characteristic components. This section details phytochemical studies that have been conducted on many parts of Z. bungeanum, including the stem, the leaf, the seed, the pericarps, and the roots. The identified compounds are listed in the following tables and the corresponding structures are also comprehensively presented. Alkylamides, creating a strong numbing sensation in the mouth, are considered to be the main characteristic compounds. So far, more than 25 alkylamides have been isolated and identified from this plant. They are usually highly unsaturated with a unique taste because of the two or more conjugated double bonds. Hydroxy-α-sanshool (HAS), having four double bonds in the cis-configuration, is the active ingredient most responsible for the unique tingling sensation evoked by the pericarps of Z. bungeanum. HAS was first isolated from the pericarps of Z. bungeanum and identified by Yasuda et al. (1982) [27]. There are two different viewpoints on the unique sensation produced by HAS. In vitro, HAS has been shown to activate TRPV1 and TRPA1 in sensory neurons by influx of Ca 2+ in cells [28]. Subsequently, Bautista et al. (2008) reported the activation of somatosensory neurons including smalland large-diameter cells elicited through the unique ability of HAS to inhibit two-pore potassium channels (KCNK3, KCNK9, and KCNK18) [18]. However, Hydroxy-β-sanshool (HBS) with four double bonds in the all trans-configuration has no effect when applied to the human tongue [29]. Additionally, Galopin et al. (2004) provided the minimum structure unit for the tingling sensation elicited by alkylamides ( Figure 2). It is noteworthy that the all-trans alkylamides are tasteless, whereas the amides having a cis double bond are very pungent [30,31] (Table 2, Figure 3).

Other Alkaloids
Apart from the alkylamides, there are also other alkaloids isolated from Z. bungeanum. To date, eight alkaloids isolated from Z. bungeanum have been reported. In 1981, zanthobungeanine, des-N-methylchelerythrine, 11-methoxychelerythrine, L-N-acetylanonanine, arnothianamide, and skimmianine were isolated from the roots of Z. bungeanum [32]. Moreover, haplopine and kokusaginine were also found to be present in the pericarps of Z. bungeanum in 1984 [33]. The chemical constituents of alkaloids and their corresponding structures are exhibited in Table 2

Phytochemistry
To the best of our knowledge, many chemical compounds have been isolated and identified from Z. bungeanum between the later 1880s and the present day. Currently, more than 140 constituents have been identified from this plant; furthermore, alkaloids and terpenoids have been identified as the characteristic components. This section details phytochemical studies that have been conducted on many parts of Z. bungeanum, including the stem, the leaf, the seed, the pericarps, and the roots. The identified compounds are listed in the following tables and the corresponding structures are also comprehensively presented.  4.1.1. Alkylamides Alkylamides, creating a strong numbing sensation in the mouth, are considered to be the main characteristic compounds. So far, more than 25 alkylamides have been isolated and identified from this plant. They are usually highly unsaturated with a unique taste because of the two or more conjugated double bonds. Hydroxy-α-sanshool (HAS), having four double bonds in the cis-configuration, is the active ingredient most responsible for the unique tingling sensation evoked by the pericarps of Z. bungeanum. HAS was first isolated from the pericarps of Z. bungeanum and identified by Yasuda et al. (1982) [27]. There are two different viewpoints on the unique sensation produced by HAS. In vitro, HAS has been shown to activate TRPV1 and TRPA1 in sensory neurons by influx of Ca 2+ in cells [28]. Subsequently, Bautista et al. (2008) reported the activation of somatosensory neurons including small-and large-diameter cells elicited through the unique ability of HAS to inhibit two-pore potassium channels (KCNK3, KCNK9, and KCNK18) [18]. However, Hydroxy-β-sanshool (HBS) with four double bonds in the all trans-configuration has no effect when applied to the human tongue [29]. Additionally, Galopin et al. (2004) provided the minimum structure unit for the tingling sensation elicited by alkylamides ( Figure 2). It is noteworthy that the all-trans alkylamides are tasteless, whereas the amides having a cis double bond are very pungent [30,31] (Table 2, Figure 3).

Other Alkaloids
Apart from the alkylamides, there are also other alkaloids isolated from Z. bungeanum. To date, eight alkaloids isolated from Z. bungeanum have been reported. In 1981, zanthobungeanine, des-N-methylchelerythrine, 11-methoxychelerythrine, L-N-acetylanonanine, arnothianamide, and skimmianine were isolated from the roots of Z. bungeanum [32]. Moreover, haplopine and kokusaginine were also found to be present in the pericarps of Z. bungeanum in 1984 [33]. The chemical constituents of alkaloids and their corresponding structures are exhibited in Table 2 and Figure 3.    Kokusaginine Pericarps [33] Alkaloids isolated from Z. bungeanum.  Essential oils are the principle source of the special flavor in HuaJiao, and terpenoids are considered to be significant components due to their relatively high percentage among these compounds. To date, more than 65 constituents of terpenoids have been identified by gas chromatography coupled with mass spectrometry, which mainly consisted of high contents of monoterpenes and sesquiterpenoids. However, the chemical constituents and contents of terpenoids constituents are different in different studies, which can be explained by genetic characteristics, growth conditions, extraction methods, and other factors [39,40]. The terpenoids isolated from Z. bungeanum are presented in Table 3 and their corresponding structures are shown in Figure 4.      Terpinolene Pericarps [45] Terpenoids isolated from Z. bungeanum.

Flavonoids (104-129)
Flavonoids are common ingredients of numerous plants all over the world. Increasing the number of flavonoids isolated from Z. bungeanum has attracted much attention because of their broad range of pharmacological activities including antioxidant activity, antithrombotic activity, anti-aging activity, anti-tumor activity, etc. To date, more than 25 flavonoids, displayed in Table 4 and Figure 5, have been identified from this plant, such as quercetin, rutin, and quercetin 3-O-α-L-rhamnoside. Yang et al. (2013) and  provided significant data confirming that the leaves contain abundant flavonoids with prominent antioxidant abilities [10,23]. This could also explain the frequent addition of Z. bungeanum leaves to the Chinese diet for the promotion of human health. anti-aging activity, anti-tumor activity, etc. To date, more than 25 flavonoids, displayed in Table 4 and Figure 5, have been identified from this plant, such as quercetin, rutin, and quercetin 3-O-α-L-rhamnoside. Yang et al. (2013) and  provided significant data confirming that the leaves contain abundant flavonoids with prominent antioxidant abilities [10,23]. This could also explain the frequent addition of Z. bungeanum leaves to the Chinese diet for the promotion of human health.

Fatty Acids (130-139)
A few studies have been conducted investigating the fatty acids in Z. bungeanum. In 2007, palmitoleic acid was isolated from Z. bungeanum [47]; in addition, Xia (2011) reported that eicosoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanic acid, oleic acid, and stearic acid were also present in the seeds of Z. bungeanum [11]. Lately, linolenic acid, linoleic acid, and nonanoic acid have been isolated and identified in Z. bungeanum [48,49]. As presented in Table 5 and Figure 6, the fatty acids isolated and identified from Z. bungeanum are mainly long carbon chains with a terminal carboxyl group. Linoleic acid Seeds [48] Fatty acids isolated from Z. bungeanum.

Pharmacology
To the best of our knowledge, Z. bungeanum has been demonstrated to possess wide-reaching pharmacological effects, including effects on the digestive system, nervous system, and circulatory system, as well as anti-inflammatory and analgesic effects, antioxidant effects and anti-tumor effects, anti-fungal and antibacterial effects, insecticidal effects, and so on. In this section, the main pharmacology activities of Z. bungeanum are summarized and analyzed, as listed in Table 7.

Effect on the Digestive System
The characteristic pharmacological effect of Z. bungeanum on the digestive system has been comprehensively reviewed. The gastrointestinal smooth muscle in rabbits was stimulated by lower concentrations (4 mg/mL, intragastric (i.g.)) and depressed by higher concentrations (12 mg/mL, i.g.) of the water extracts of Z. bungeanum (WEZB), and this stimulating effect could be completely inhibited by atropine [51,52]. Furthermore, Zhang et al. (1991) reported that the WEZB (2.5, 5.0, and 10 g/kg, i.g., crude herb mass equivalent) showed significant inhibiting effects on experimental gastric ulcers in mice, including the pylorus ligation ulcer, water immersion stress ulcer, indomethacin-ethanol ulcer, and hydrochloric acid ulcer; additionally, they also found that the petroleum ether extracts of Z. bungeanum (PEEZB) could markedly inhibit diarrhea induced by castor oil (3.0 and 6.0 mL/kg, i.g.), while the WEZB (5 and 10 g/kg, i.g., crude herb mass equivalent) exhibited strong inhibition on diarrhea caused by senna leaf, which is different from the PEEZB [53]. Yuan et al. (2009) demonstrated that the essential oils of Z. bungeanum (EOZB) at doses of 0.1, 0.2, 0.4, and 0.8 mg/mL could dose-dependently inhibit the contraction of isolated duodenal smooth muscle of rabbits through a mechanism possibly associated with the blocking of the Ca 2+ channel, calcium inward current, and release of intracellular calcium [54]. Meanwhile, a study also reported that the EOZB strongly inhibits the contraction of the colon smooth muscle in rabbits [55]. In addition, the WEZB (0.5, 1.0, and 2 g/kg, i.g., for 14 days) was reported to show great improvement in colonic shortening and body weight loss in dextran sodium sulfate (DSS)-induced experimental colitis in mice, whereas it decreased Disease activity index (DAI), a clinical parameter reflecting the severity of weight loss [56]. The mechanism may be related to the reduction of pro-inflammatory cytokines such as TNF-α, IL-β, and IL-12; additionally, suppression of NF-κB p65, IκBα phosphorylation, and the TLR4 pathway was also involved [56].
In addition to this, Kono et al. (2011) suggested that HAS, an active ingredient of Z. bungeanum pericarps, at doses of 0.3, 3.0, and 30 µmol/L, showed notable effects on improving the release of adrenomedullin (ADM) from intestinal epithelial cells in a dose-dependent manner [57]; in addition, they also showed that HAS (0.3 mg/kg) could markedly enhance the colonic blood flow in colitis rats. Additionally, HAS could also significantly evoke long-distance contraction (LDC) in vitro, and the mechanism may be associated with the blockage of the KCNK9 channel in the rat proximal colon (3, 10 and 30 µM) [58].

Effect on the Nervous System
It was reported that the EOZB and WEZB could reversibly inhibit the sciatic nerve impulse conduction of toads, and the required time of 20% EOZB and 20% WEZB for blocking the nerve impulse was approximately equal to that of procaine [59,60]. In addition, the polyphenol extracts from Z. bungeanum (PEZB) showed significant anti-depressive effects on behavioral despair models (induced by forced swimming and tail suspension) (50, 100, and 200 mg/kg, i.g.) and the underlying mechanism might involve the central monoaminergic systems [61]. Furthermore, previous studies have also demonstrated that the PEZB (50, 100, and 200 mg/kg, i.g., for 21 days) could improve climacteric depression caused by chronic unpredictable stress behavior. The mechanism for this effect might be related to the reduction of depressive symptoms, which are elicited by the regulation of the nerve-endocrine system [62]. In addition, it has been reported that the PEZB can upregulate the level of NE and 5-HT in the brain tissue of rats with post-stroke depression, and the mechanism may be associated with an inhibitory effect on monoamineoxidase (MAO) activity (50, 100, and 200 mg/kg, i.g., for 21 days) [63]. Additionally, the PEZB (50, 100, 200 mg/kg, i.g., for 14 days) also showed anti-depressive effects in an unpredictable stress model of depression in rats and an ovariectomized model of depression in mice [64,65].
One interesting study indicated that HAS (5 mg/kg, per os (p.o.)) can significantly shorten the escape latency in mice via a Morris water maze test, and this also exhibited a tendency of HAS to reduce the effect of scopolamine-induced dementia, which is probably mediated by the facilitation of ACh release [66]. Additionally, it is worth noting that gx-50, an active ingredient isolated from Z. bungeanum, could significantly enhance the cross-platform times (1.0 mg/kg, i.p., for 2 months), inhibit the release of cytokine induced by Aβ in microglia cells (2.5, 5, 10, and 20 µM), penetrate the blood-brain barrier, improve the cognitive abilities of mice in vivo (1.0 mg/kg, i.p., for 2 months), disassemble Aβ oligomers (5 µM), inhibit Aβ-induced neuronal apoptosis, increase bax expression, and reduce neuronal Ca 2+ influx toxicity, which strongly suggests that gx-50 is a potential candidate drug for treating Alzheimer's Disease (AD) [37,67]. In one report of 2016, eight isobutylhydroxyamides were isolated and identified from the pericarps of Z. bungeanum, and three of them-including qinbunamide,

Effect on the Circulatory System
In 2005, the seed oil of Z. bungeanum (SOZB, 5, 10, and 20 mL/kg, i.g., for 4 weeks) was reported to have a notable effect on reducing cholesterol (CHOL) hyperlipidemia, triglyceride (TG), and low-density lipoprotein (LDL), as well as increasing high-density lipoprotein (HDL-C) [68]. Later, Liu et al. (2007) indicated that the SOZB (2.5 mL/kg, i.g., for 10 weeks) could decrease blood lipids and lower blood viscosity through reducing high blood viscosity (HBV), high low viscosity (HLV), CHOL, TG, and increasing HDL-C [69]. Furthermore, the SOZB at doses of 2.5, 5, and 10 g/kg also showed significant effect on reducing the serum levels of TG, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), malondialdehyde (MDA), and nitric oxide (NO) through the activation of PPARγ, which indicates that SOZB is a promising novel hypolipidemic health product [70]. In addition, previous studies have also demonstrated that the EOZB (2.0, 4.0, 6.0, 8.0, and 10.0 µL/mL) displays a dose-dependent relaxation of the contracted aortic muscle elicited by PE and KCl in rats, and the mechanism may decrease calcium influx and inhibit calcium channels [71].

Anti-Inflammatory and Analgesic Effects
Z. bungeanum has a long history of usage in China as an anti-itching agent as well as for the treatment of pruritus vulvae-related diseases. In accordance with the traditional usage of Z. bungeanum, a few studies have demonstrated that these plants possess anti-inflammatory and analgesic effects both in vitro and in vivo. In 1994, the anti-inflammatory and analgesic abilities were evaluated through many animal models. The WEZB (2.5, 5.0, and 10 g/kg, i.g., for 3 days, crude herb mass equivalent) and the ether extracts (EEZB) (1.5, 3.0, and 6.0 mL/kg, i.g.) of Z. bungeanum exhibited significant anti-inflammatory and analgesic effects in animal models of inflammation (dimethylbenzene-induced ear oedema test and carrageenan-induced rat paw oedema in mice) and pain (acetic acid-induced torsion test in mice) [73]. Later, in 2010, the EOZB was also reported to be effective against dimethylbenzene-induced ear oedema and acetic acid-induced pain at doses of 0.05, 0.1, and 0.2 g/kg (i.g., for 14 days). At a dose of 0.1 g/kg, the WEZB showed notable activity with an inhibition ratio of 65.76% for oedema weight and 51% inhibitory value for the writhing responses [74].
In another study of formalin-induced pain in rats, USV (ultrasonic vocalization) data was significantly lower than that of the control groups after the EEZB and the WEZB treatment, and the EEZB had better analgesic effect than WEZB. Additionally, the EEZB could relieve pain caused by warming similar to Lidocaine [77]. Additionally, the effects of HAS to quickly mediate pain were evaluated using recordings of cutaneous sensory fibres, whole-cell patch clamp, and calcium imaging. The results revealed that HAS (IC 50 = 70 ± 7 µM) could inhibit the excitability of Aδ mechanosensory nociceptors by blocking voltage-gated sodium channels to induce "fast pain" analgesia [78]. In addition, the seed oil of Z. bungeanum (SOZB) at doses of 0.5, 1.0, and 2.0 g/kg (i.g.) could markedly inhibit the dimethylbenzene-induced auricle edema with the inhibition rates of 68.52%, 70.43%, and 71.83% for the ear weight [48].
Apart from these, some compounds isolated from the pericarps of Z. bungeanum were found to exhibit notable anti-inflammatory effects by the suppression of nitric oxide (NO) production. Results revealed that the compounds ZP-amide D, ZP-amide E, ZP-amide F, and ZP-amide G showed inhibitory effects on nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages, with IC 50 values of 48.7 ± 0.32, 27.1 ± 1.15, 49.8 ± 0.38, and 39.4 ± 0.63 µM, respectively [38].
In the treatment with the EEZBL (0.015%, 0.030%, and 0.045%, for 8 days), the hexanal content, the thiobarbituric acid reactive substances (TBARS) value, and the lipoxygenase (LOX) activity were remarkably lower during processing, both in the dorsal and ventral muscles [25]. In addition, salted fish with the EEZBL (0.018%) and the polyphenols (0.01% chlorogenic acid, hyperoside and quercitrin) had higher endogenous antioxidant enzyme (catalase, superoxide dismutase, and glutathione peroxidase) activities, lower peroxide value (PV), and thiobarbituric acid-reactive substance (TBARS) values than those of the control [84]. These findings showed that EEZBL can be regarded as a source of natural antioxidants.

Insecticidal Effects
Bowers et al. (1993) reported that three active monoterpenes isolated from Z. bungeanumpiperitone, 4-terpineol, and linalool-exhibited high repellent activity against ants, with 80% of the ants repelled from feeding on the sucrose solution of 8.91, 14.13, and 20 µg/cm 2 [94]. It was reported that the EOZB obtained by supercritical CO 2 had high insecticidal activity against Sitophilus zeamais and Tribolium castaneum [95]. In addition, three fractions of the EOZB (petroleum ether fraction, dichloromethane fraction, and diethyl ether fraction) showed significant insecticidal effects towards T. castaneum with LD 50 values of 0.0713, 0.11699, and 0.12267 µL [95]. In addition, Kou (2015) suggested that the EOZB obtained by steam distillation displayed strong anti-insect activity against aedes albopictus at doses of 15, 25, 35, and 45 µg/mL [96]. Furthermore, T. castaneum could be killed by the MEZB at doses of 0.5, 1.0, and 1.5 mg/mL [96]. Additionally, two EOZB samples were obtained from the pericarps of Z. bungeanum with the methods of hydrodistillation (HD) and supercritical fluid CO 2 extraction (SFE), and their bioactivities against Lasioderma serricorne adults were evaluated. The results indicated that the SFE sample and HD sample showed significant anti-insect activity against Lasioderma serricorne adults with LC 50 values of 3.99 µg/mL and 12.54 µg/mL [41].

Other Pharmacological Effects
In addition to the pharmacological effects listed above, Z. bungeanum also exhibited other effects. The sanshools isolated from the pericarps of Z. bungeanum were regarded as a topical lifting agent for wrinkles based on their capacity to relax subcutaneous muscles [97]. Furthermore, the sanshools (0.8 mg/mL) of Z. bungeanum elicited protective effects on rice-seedling growth, chlorophyll content, and root activity in rice seedlings exposed to metolachlor; this activity was related to the upregulated expression of four Glutathione transferases (GST) genes, especially representative GST genes (OsGSTU3) [98].  evaluated the anti-asthma effects of the seeds of Z. bungeanum (SZB), and they found that the SZB at doses of 0.25, 0.5, and 1.0 g/kg could obviously prolong the latency period of induced asthma (LPIA) and reduce the frequency of citric acid-induced cough compared with that of vehicle treatment. Moreover, the SZB (0.5, 1.0, and 2.0 g/kg, i.g.) also showed anti-fatigue and anti-anoxia ability [48]. Additionally, Lan et al. (2014) demonstrated that the EOZB (3%) could significantly enhance the percutaneous absorption of drugs with different lipophilicities [46]. Meanwhile, limonene (3%) exhibited the highest permeation fluxes and cumulative amounts in comparison with terpinen-4-ol (3%) and 1,8-cineole (3%). The mechanism of these enhancers which promoted the skin permeation of drugs may be associated with the effect on skin stratum corneum (SC) lipids [45].
Apart from these, the n-butanol fraction isolated from the EEZB has effects on the cholesterol metabolism. In an in vivo study, it (50 and 200 mg/kg/day, i.g., for 4 weeks) can inhibit hyperlipidemia effects with decreased serum TC and TG levels in apoE-ko mice. In an in vitro study, with the n-butanol fraction of the EEZB treatment (0.05, 0.1, and 0.2 mg/mL), the TC, TG, FC (free cholesterol) levels and apolipoprotein B (apoB) secretion were significantly decreased in HepG2 cells exposed to sterols and 25-hydroxycholesterol, whereas apolipoprotein A1 (apoA1) secretion was increased [99]. The mechanism may be related to the increase in low density lipoprotein receptor (LDLR) protein and inhibition of the expression of hydroxy methylglutaryl coenzyme A reductase, HMGCR [99].

Summary of Pharmacologic Effects
In conclusion, Z. bungeanum has an extensive range of pharmacological effects which includes effects on the digestive system, effects on the nervous system, effects on the circulatory system, anti-inflammatory and analgesic effects, anti-bacterial and anti-fungal effects, as well as antioxidant effect and anti-tumor effects, etc. These pharmacological activities mainly have focused on the extraction or preparations of Z. bungeanum, which indicates that this plant has a promising potential for treating disease. However, there are few systemic investigations regarding the individual compounds and their corresponding pharmacological activities, as well as action mechanism. Therefore, future research into pharmacological effects, structure-function relationships, and mechanisms of the plant's bio-active components should be explored by in vivo and in vitro experiments.
After oral administration of the WEZB at a dose of 1.3 g/mL, the peak time (Tmax) and peak plasma concentration (Cmax) values were determined to be 30.0 min and 46.720 g/kg, respectively, and the t1/2 of the WEZB was 79.26 min. Furthermore, the area under the concentration-time curve (AUC) was also determined, and the AUC 0-t was 102.015 g/h/kg [100]. Furthermore, Fang et al. (2014) reported that the t1/2 values of alkylamides were determined to be 179.33, 118.03, 134.01, and 241.51 min of different intestinal segments of rats (including duodenum, jejunum, ileum, and colon), and the jejunum was regarded as the best absorption site of alkylamides [101].
In addition, a simple, rapid, and sensitive UHPLC-MS/MS method was developed for the determination of HAS, HBS, and HRS concentration in rat plasma. After the subcutaneous administration of the EEZB at a dose of 11.0 mg/kg (equivalent to 6.21 mg/kg of HAS, 1.36 mg/kg of HBS and 0.32 mg/kg of HRS), the peak times (Tmax) of HAS, HBS, and HRS were determined to be 36, 42, and 69 min, respectively, and the peak plasma concentration (Cmax) values were 1468, 432, and 41.49 ng/mL, respectively. Moreover, the area under the concentration-time curve (AUC) was also determined, and the AUC 0-t of HAS, HBS, and HRS were 3816, 819, and 147 ng/mL, respectively; additionally, the AUC 0-∞ were 3890, 839, and 160 ng/mL, respectively. Meanwhile, pharmacokinetics studies of EOZB (4.4 mg/kg) after intravenous injection were also conducted. The Cmax values of HAS, HBS, and HRS were 1215, 324, and 34.70 ng/mL, respectively, and the t1/2 were 65.4, 91.2, and 99.6 min, respectively, and the AUC 0-t were 1498, 385, and 65 ng/mL, respectively, and the AUC 0-∞ were 1551, 441, and 72 ng/mL, respectively. In addition, the subcutaneous absolute bioavailability were 100.2, 76.2, and 90.3% for HAS, HBS, and HRS, respectively [102].
Apart from these, the study of gx-50 (20 mg/kg, p.o.) on metabolism was also determined by LC-MS/MS. The results demonstrated that gx-50 could be absorbed into the blood and penetrate the blood-brain barrier (BBB) after oral administration, and it immediately distributed to brain tissue (5 min post-per os (PO)) and was finally excreted approximately 4 h post-PO [37].

Toxicology
For thousands of years, Z. bungeanum was commonly considered to be a traditional Chinese medicine with low toxicity [15]. To date, investigations regarding the toxicities of Z. bungeanum are scarce, and previous studies mainly focus on its extracts (Table 8). In 1995, Tong et al. (1995) demonstrated that the median lethal concentration (LD 50 ) value of the WEZB in mice was 45 g/kg (i.g., crude herbs mass equal) [16]. However, a report in 2010 suggested that the LD 50 value of the WEZB in mice was 51.14 g/kg (crude herbs mass equal, i.g.), and this can be explained by the toxicity of Z. bungeanum which varied with the genetic characteristics, growing conditions, and medicinal materials [100]. Furthermore, Zhao et al. (2003) studied the toxicity of the WEZB on the viscera of mice, and the WEZB (0.5, 1.0, 2.0, and 4 g/kg, i.g., crude herbs mass equal) showed low toxicity on liver including ballooning degeneration, cytoplasm rarefaction, and some spotty necrosis [103]. In a cell-based model, the WEZB was added to J774.1 cells, and the results showed that the WEZB had no toxicity at doses of 100, 200, and 400 µg/mL [56].
The toxicity of the EOZB has also been investigated in recent years. The LD 50 value of different approaches including intragastric administration (i.g.), intraperitoneal injection (i.p.), intramuscular injection (i.m.), and hypodermic injection (i.h.) of the EOZB was determined to be 2.27, 2.03, 4.64, and 5.32 g/kg, respectively [104]. After treatment with the lethal dose of EOZB, drowsiness, diarrhea, arrhythmia, twitchy limbs, and even death were observed in mice. Moreover, it has been reported that the EOZB resulted in low toxicity in both HaCaT cells and CCC-ESF-1 cells with a dose-dependent decrease in cell viability at IC 50 values (i.e.) of 2.435 and 3.649 mg/mL, respectively [45].
In conclusion, Z. bungeanum showed low toxicity potential for use as a flavoring and traditional Chinese medicine. The occurrence of adverse reaction to Z. bungeanum mainly resulted from the over-dosage or misuse of Z. bungeanum. Obviously, the over-dosage of these plants will cause severe adverse reactions or even death. Furthermore, certain people should take Z. bungeanum with caution, such as pregnant women and those with Yin deficiency [15]. Toxicities and side effects of Z. bungeanum.

Future Perspectives and Conclusions
In summary, Z. bungeanum Maxim. has been used in Asian countries for many years, and many kinds of chemical constituents have been isolated and identified from this plant. There is no doubt that Z. bungeanum is an important and effective traditional Chinese medicine and food additive with a long history. Significant breakthrough has been made on multiple aspects of this plant in the past decade. However, it is worthy to note that there are still several challenges tha require further investigation to satisfy clinical research.
First, pharmacological studies have mainly focused on crude extracts and preparation, and there is not sufficient evidence to explain the special action mechanism for the pharmacological activity of this plant. Therefore, further investigation should be carried out to study the bioactive compounds and its action mechanism and structure-function relationship. Second, there are not enough studies regarding the pharmacokinetics and clinical research of Z. bungeanum, and few evaluations of the toxicity on a cellular and molecular level have been explored. Thus, future study of Z. bungeanum should focus more on the pharmacokinetics study of other constituents besides alkylamides, and toxicity studies should be performed on its molecular and cellular level to investigate any side effects in clinical research. Third, according to the current in vivo and in vitro investigation, HAS is the main active compound responsible for the special taste and diverse pharmacological activity. However, HAS is not stable in normal storage conditions and it may be sensitive to oxygen due to the conjugated triene system [9]. Therefore, more stable derivatives of HAS and other sanshools should be synthesized by modification of the structure. Fourth, traditional usages of Z. bungeanum include few prescriptions, and the processed products of Z. bungeanum are becoming fewer and fewer. Therefore, new prescriptions and products need to be developed to meet the clinical requirement. Fifth, the study of leaves, seeds, stems, and roots in Z. bungeanum has been relatively slow in comparison with research of pericarps, and it is necessary to investigate the chemical compounds and pharmacological activity of every part of Z. bungeanum to ensure the full utilization of the possible medicinal usages of this plant. Sixth, because of its rich biodiversity and morphological similarity to the Zanthoxylum genus, it is difficult to identify Z. bungeanum and its related plants. Thus, it is important to find a potential strategy that can control the quality of this plant and establish a unified international quality evaluation system. Lastly, due to its wide distribution and cultivation in many areas of the world, it is very important to enhance the efficiency and quality of the picking process.
The current literature provides a full-scale review on the progress of traditional uses, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology of Z. bungeanum, and proposes some issues worth investigating in the future, which will greatly facilitate the comprehensive understanding and effective development of Z. bungeanum treaments and applications.