4.1 Alzheimer’s disease

AD is a classic neurodegenerative disease. Various theories that suggest that cholinergic, Aβ cascade, oxidative stress or inflammatory pathways are involved have been proposed to explain the pathogenesis of AD, but there is no unified definitive conclusion regarding its pathogenesis. The hypothesis that AZB might serve as an effective treatment for AD was first derived from Satoh's experiment. It was found that a traditional prescription, Daikenchuto, which is also known as “Da-Jian-Zhong-Tang” in China and contains ZBM, improves gastrointestinal transit by affecting acetylcholine (ACh) release (Satoh et al., 2001). ACh, an important neurotransmitter that is part of the central cholinergic system, is considered to act as a key mediator of learning and memory by regulating the cholinergic system, and reduced ACh release often causes the development of AD. Therefore, Nakamura et al. investigated whether ZBM could improve learning and memory by affecting ACh release in the central nervous system. In those experiments, the Morris water maze test was used to evaluate learning and memory ability. Administration of ZBM extract significantly decreased the platform finding time in scopolamine-induced mice. In addition, the effect of HAS on learning and memory ability was stronger under the same dose conditions (Nakamura et al., 2006). Unfortunately, this experiment did not address in depth whether ZBM and HAS improve learning and memory ability through ACh and or determine the underlying mechanism of action. Since then, numerous researchers have begun experimental studies of AZB.

As previously mentioned, HAS is an important active component of ZBM, and it is very abundant in AZB. In the experiments of Zhang et al., scopolamine was used to construct AD models, followed by oral administration of HAS. Based on the results of their experiments, these investigators reached a conclusion similar to that of Nakamura et al., namely, that HAS significantly alleviated learning and memory impairment and inhibited apoptosis of hippocampal neurons. To further explore the mechanism of action of HAS, Zhang et al. measured ACh and acetylcholinesterase (AChE) content in the brains of mice. AChE is an enzyme that rapidly hydrolyses ACh and is considered an important component of the pathway that regulates ACh content. Donepezil, a drug that is widely used in the treatment of AD, is a typical AChE inhibitor. Zhang et al.’s results showed that HAS inhibited the activity of AChE, thereby increasing ACh content in the brains of the model mice (Zhang et al., 2019). Since brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that is closely related to neuronal survival, its deletion in the hippocampus leads directly to the development of learning and memory impairment. In addition, cAMP response element-binding protein (CREB), an intranuclear factor that regulates gene transcription, is regulated by its own phosphorylation. Previous studies have shown that CREB is closely related to learning and memory function and that BDNF is an important target (Xue et al., 2015). Therefore, Zhang et al. continued to examine the expression of BDNF and CREB. HAS significantly upregulated the expression of BDNF and p-CREB in the hippocampi of model mice. This led to the conclusion that HAS exerts a therapeutic effect on AD by activating the cholinergic system and the CREB/BDNF signalling pathway (Zhang et al., 2019). Subsequently, Li and Zhang et al. continued to explore the mechanism of action of HAS using H₂O₂-induced PC12 cells as an in vitro model. They found that HAS significantly inhibited oxidative stress in the cells, as evidenced by the fact that it reduced intracellular ROS and malondialdehyde (MDA) content and increased mitochondrial membrane potential (MMP) and the levels of intracellular antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Through these effects, HAS maintained redox homeostasis in PC12 cells, which in turn inhibited apoptosis induced by H₂O₂. Similar results were obtained when the investigators identified the PI3K/AKT signalling pathway as the signalling pathway through which HAS inhibits cellular oxidative stress. The PI3K/AKT signalling pathway produces extensive antioxidant activity in both central and peripheral neurons and is considered a potential target in the treatment of neurodegenerative diseases. PI3K phosphorylates and activates AKT, a serine/threonine kinase that is activated by recruitment to the plasma membrane. Activation of the PI3K/AKT signalling pathway further affects the expression of downstream apoptosis-related proteins. In the experiments of Li et al., HAS activated the PI3K/AKT signalling pathway, thereby reducing the expression of the proapoptotic proteins cleaved caspase-3 and BAX and increasing the expression of the antiapoptotic protein Bcl-2 (Li et al., 2020). Liu et al. used mice to which D-galactose (D-gal) and AlCl3 had been administered as in vivo models to explore the mechanism of action of HAS in experiments that continued to focus on apoptosis induced by oxidative stress. The results showed that orally administered HAS inhibited oxidative stress and apoptosis in mice via the Nrf2/HO-1 signalling pathway and that it exerted a protective effect on hippocampal neurons and improved the learning and memory ability of the mice (Liu et al., 2022). To avoid problems associated with the structural instability of HAS, HAS-liposomes (HAS-LPs) were prepared and used to treat AD mice by nasal administration. This not only improved the stability of HAS but also resulted in a certain degree of brain targeting, thereby improving the therapeutic effect of HAS (Tang et al., 2013). Additional details of this work are shown in Table 1 and Fig. 2.

Table 1 Amides from Zanthoxylum bungeanum Maxim. (Rutaceae) are promising natural agents with neuroprotective activities.

Compounds	Compound’s structure	Doses/Con.	Experimental models	Detail effects and mechanisms	Related targets	References
Alzheimer’s disease
HAS		5 mg/kg, p.o., for 3 days	Scopolamine Induced mice	Enhancing the memory and learning of ddY mice		(Nakamura et al., 2006)
HAS		1.25, 2.5, 5 mg/kg, p.o.	Scopolamine Induced mice	Inhibiting scopolamine-induced cognitive impairments in mice; Inhibiting morphology changes and apoptosis in hippocampal neuron; Decreasing AChE activity and increasing ACh content; Up-regulating BDNF and CREB	BDNF, CREB,AChE	(Zhang et al., 2019)
HAS		15,30,60 μM	H2O2 induced PC12 cells	Protecting PC12 cells from H2O2 induced injury; Reducing H2O2-induced apoptosis in PC12 cells via reduction of intracellular ROS and increase of MMOP; Iincreasing SOD, CAT, GSH-Px, and decreasing MDA. Upregulating p-PI3k, Akt, p-Akt, Bcl-2, and down-regulating caspase-3, Bax in H2O2 induced PC12 cells.	SOD, CAT, GSH-Px, PI3K, Akt, Bcl-2, MDA, Bax, Caspase-3	(Li et al., 2020)
HAS		1.25, 2.5, 5 mg/kg, p.o.	D-gal/AlCl3 induced mice	Ameliorating spontaneous locomotion deficit of D-gal/AlCl3 induced mice; Improving the spatial learning and memory; Alleviating morphological changes and increasing Nissl neurons in hippocampus; Reducing MDA and increasing SOD, GSH-Px and CAT; Increasing HO-1 and Nrf2 in the hippocampus; Iinhibiting neuronal apoptosis by decreasing Cyt-c, Bax and Caspase-3, and increasing Bcl-2 in hippocampus.	SOD,GSH-Px, CAT,HO-1,Nrf2, Bcl-2,Caspase-3	(Liu et al., 2022)
HAS-LPs		1.25,2.5,5mg/kg, Intranasal	D-gal/AlCl3 induced mice	Exhibiting slow drug release and low toxic to the nasal mucosa; Alleviating D-galactose-induced learning memory deficits and protecting hippocampal neuronal cells. Being enriched in plasma and brain tissue via intranasal aministration.		(Li et al., 2022)
Gx-50		1 mg/kg, i.p.	Aβ transgenic mouse AβPP-Tg mice	Disassembling Aβ oligomers; Improve cognitive performance; Inhibiting Ca2+ inward flow.		(Tang et al., 2013)
		1 μM	Aβ42 induced primary cortical neurons of SD rats	Elevating the Akt phosphorylation and inhibiting GSK-3 to restoring the CREB’s transcriptional activity, and finally upregulating BDNF. Inhibiting Aβ-induced neuronal apoptosis and apoptotic gene expression, and Reducing neuronal calcium toxicity.	Akt, CREB, BDNF, GSK-3	(Tang et al., 2014)
Gx-50			Aβ-induced primary microglia	Inhibiting the chemotactic migration of microglia and CCL5 induced by Aβ. Upregulating TGF-β1 and enhancing phosphorylation of GSK-3β in Aβ-induced microglia.	TGF-β1,GSK-3β, CCL5	(Guo et al., 2014)
Gx-50		2.5,5, 10,20 μM	Aβ-induced microglia	Activating α7 nAChR; upregulating JAK2/STAT3 and PI3K/Akt signaling pathways to inhibit IL-1β release.	α7nAChR, JAK2, STAT3, PI3K, Akt, IL-1β	(Shi et al., 2016)
Qinbunamide B		20 μM	PC12 cells	Potentiating activity of NGF to stimulate neurite outgrowth from PC12 cells.	NGF	(Tian et al., 2016)
ZP-amide C
ZP-amide D
Hydroxy-γ-isosanshool		IC50 = 4.08 μM	Erastin induced HT-22 cells	Inhitibitng the erastin induced feroptosis of hippocampal neuron cell HT22 cell with the IC50 of 4.08 μM.		(Zhang et al., 2020)
Parkinson's disease
HAS		1 mM	HEK293T cells, Primary TGN, primary DRGN	Causing Ca2+ influx in cells transfected with TRPV1 or TRPA1 and evoking robust inward currents in cells transfected with TRPV1 or TRPA1; Inducing inward currents and Ca2+ influx which could be diminished in TRPV1–/– mice.	TRPV1, TRPA1	(Riera et al., 2009)
Depression
(−)-(6R)-ZP-amide A		3.125–100 μM	CORT induced PC 12 cells	Protecting against CORT-induced PC12 cells damage		(Chen et al., 2018)
(−)-(11R)-ZP-amide B		3.125–100 μM
(±)-ZP-amide G		100 μM
(±)-ZP-amide H		50–100 μM
ZP-amide I		25–54 μM
Zanthoamide		12.5–100 μM
HMDTD		25–100 μM

α7 nAChR, α7 nicotinic acetylcholine receptor; Ach, acetylcholine; AchE, acetylcholinesterase; AD, Alzheimer’s disease; Aβ, amyloid-beta; BDNF, brain-derived neurotrophic factor; cAMP response element-binding protein; CAT, catalase; CREB, cDNA, complementary DNA; CHO, Chinese hamster ovary; Con., concentration; CORT, corticosterone; D-gal, D-galactose; DRGN, dorsal root ganglia neurons; GSH-Px, glutathione peroxidase; GSK-3, glycogen synthase kinase; GX-50, N-[2-(3,4-Dimethoxyphenyl)ethyl]-3-phenylacrylamide; HAS, Hydroxy-α-sanshool; HAS-LPs, hydroxy-α-sanshool liposomes; HBS, Hydroxy-β-sanshool; HMDTD, (2E,7E,9E)-N-(2-hydroxy-2-methylpropyl)-6,11-dioxo-2,7,9-dodecatrienamide; MDA, malondialdehyde; MMOP, mitochondrial membrane potential; MWM, Morris water maze test; NGF, Nerve growth factor; PCN, Primary cortical neurons; SOD, superoxide dismutase; TDN, Tetrahydrobungeanool; TGN, trigeminal ganglion neurons.

Close

Fig. 2

Amides from Zanthoxylum bungeanum Maxim. (Rutaceae) are promising natural agents for the treatment of Alzheimer’s disease.

Export to PPT

AZBs other than HAS that have therapeutic potential for AD are also present in ZBM. N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenyl-acrylamide, also known as gx-50, penetrates the blood–brain barrier and acts directly on the brain to exert therapeutic effects. gx-50 has shown great therapeutic potential in a number of recent studies. It was initially found to reduce the level of amyloid-beta (Aβ) oligomers in the brains of amyloid-β protein precursor transgenic (AβPP-Tg) mice and to improve cognitive performance in mice. In in vitro experiments, gx-50 showed superior ability to break down Aβ oligomers, and this resulted in reduced apoptosis of primary cortical neurons induced by Aβ and inhibition of inward Ca²⁺ flow (Li et al., 2022). Gene microarray technology was subsequently used to explore the mechanism of this effect. After pathway analysis of changes in gene expression, immunoblotting, immunohistochemistry and real-time fluorescence quantitative PCR, it was concluded that gx-50 inhibits glycogen synthase kinase-3 (GSK-3) activity by promoting p-Akt phosphorylation and that this, in turn, restores the transcriptional activity of CREB and ultimately promotes BDNF expression (Tang et al., 2014). In studies using Aβ-induced primary microglia, gx-50 was found to downregulate the candidate chemokine CCL5 by activating the transforming growth factor β1 (GF-β1)/Smad2 signalling pathway, thereby inhibiting microglial migration to Aβ. In addition, gx-50 was found to inactivate GSK-3β by enhancing the phosphorylation of GSK-3β, a change that synergistically helped gx-50 inhibit microglial migration (Guo et al., 2014). In Shi et al.'s subsequent study, the effects of gx-50 on inflammation were investigated using Aβ-induced microglia as a model. The researchers found that gx-50 acts as a specific ligand to activate the α7 nicotinic acetylcholine receptor (α7nAChR) (Shi et al., 2016). The α7nAChR is a nicotinic receptor that occurs widely in the nervous system. It impairs the inflammatory response in AD via the cholinergic anti-inflammatory pathway and has both neuroprotective and anti-inflammatory effects that may reduce cognitive impairment (Conejero-Goldberg et al., 2008). During the pathogenesis of AD, Aβ binds not only to the translocase of the outer mitochondrial membrane (TOMM), thereby inducing oxidative stress, but also to the α7nAChR, thereby blocking neuroprotection. Surprisingly, gx-50 binds competitively to α7nAChR. Upon activation of α7nAChR by gx-50, JAK2, a downstream signalling partner of the α7nAChR, phosphorylates the transcription factor STAT3 and activates the PI3K/AKT signalling pathway, and this in turn inhibits the release of proinflammatory factors (Shi et al., 2016).

Qinbunamides B, ZP-amide C and ZP-amide D are isobutyl hydroxylamines that have been isolated from ZBM and identified; qinbunamide B is the first ethyl-containing isobutyl hydroxylamine to be isolated (Tian et al., 2016). Nerve growth factor (NGF), as a neurotrophic factor, promotes the growth and development of neurons. It is also an essential part of the mechanism that repairs the nervous system after damage. However, because NGF is a high-molecular-weight peptide, it is difficult for exogenous NGF to cross the blood–brain barrier (Allen et al., 2013). Therefore, promotion of endogenous NGF activity has been a popular topic in the treatment of AD in recent years. In experiments conducted by Tian et al., all three compounds (qinbunamides B, ZP-amide C and ZP-amide D) enhanced NGF activity and thereby stimulated the neuronal differentiation of PC12 cells, with potential neuroprotective effects (Tian et al., 2016).

Hydroxy-γ-isosanshool is another isobutyl amide compound. In a series of experiments, erastin was used to induce iron-related death of mouse hippocampal neuronal HT22 cells. After treatment with hydroxy-γ-isosanshool, cell morphology and cell survival improved. The ability of hydroxy-γ-isosanshool to inhibit iron-related death of neurons suggests that it may potentially be useful in the treatment of AD (Zhang et al., 2020). Additional details of this work are presented in Table 1.

4.2 Parkinson's disease

PD is the second most common neurodegenerative disease, and its symptoms manifest as nonmotor symptoms and motor deficits. The pathology of PD is mainly characterized by delayed development of dopaminergic neurons in the dense region of the substantia nigra and aggregation of Lewy vesicles containing misfolded α-synuclein (α-syn) (Li et al., 2022). Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential anchoring protein 1 (TRPA1), both of which are members of the transient receptor potential channel protein family (Khan et al., 2019), are abundantly expressed in sensory nerves and jointly regulate Ca²⁺ influx. In previous studies, cannabinoids attenuated seizures, but their alleviating effects could be reversed by TRPV1 antagonists (Gaston and Szaflarski, 2018). Activation of TRPV1 promotes the inward flow of Ca²⁺ and increases glutamate release and neural activity and therefore has some therapeutic potential for the treatment of PD (Patricio et al., 2020). HAS induces an inward current and inward flow of Ca²⁺ in HEK cells transfected with TRPV1 or TRPA1, whereas HBS does not. A similar conclusion was reached when primary cultured sensory nerves were used to further explore the role of HAS. In an in vivo experiment, TRPV1^-⁄- mice were used to explore whether the effects of HAS are mediated by TRPV1. The results were surprising in that inhibition of TRPV1 expression resulted in a significant reduction in both Ca²⁺ inward flow and current, even after HAS treatment (Koo et al., 2007; Riera et al., 2009).

4.3 Depression

Depression is an extremely common psychiatric disorder in which patients often suffer from cognitive impairment, fatigue, feelings of worthlessness, and even suicidal tendencies. The pathogenesis of depression is still being explored, and corticosterone-induced PC12 cells are often used as a model to screen drugs for the treatment of depression. In experiments conducted by Chen et al., a 95% ethanol extract of ZBM was prepared, and 21 isobutylamides were isolated from the extract, identified, and applied to corticosterone-induced PC12 cells as a way to screen for possible active compounds. In those experiments, seven compounds, (−)-(6R)-ZP-amide A, (−)-(11R)-ZP-amide B, (±)-ZP-amide G, (±)-ZP-amide H, ZP-amide I, zanthoamide and (2E,7E,9E)-N-(2-hydroxy-2-methylpropyl)-6,11-dioxo-2,7,9-dodecatrienamide, were found to effectively improve the survival rate of corticosterone-induced PC12 cells. In addition, the protective effects of these compounds were found to be closely related to their conformations. Isobutyl hydroxylamides with the R-configuration exhibited protective activity, while amides with the S-configuration showed no protective ability. The protective ability of isobutyl amides was also found to decrease with increasing unsaturated fatty chain length (Chen et al., 2018).