Phytoconstituents Targeting the Serotonin 5-HT₃ Receptor: Promising Therapeutic Strategies for Neurological Disorders

Abstract

The 5-hydroxytryptamine-3 receptor (5-HT₃R), a subtype of serotonin receptor, is a ligand-gated ion channel crucial in mediating fast synaptic transmission in the central and peripheral nervous systems. This receptor significantly influences various neurological activities, encompassing neurotransmission, mood regulation, and cognitive processing; hence, it may serve as an innovative target for neurological disorders. Multiple studies have revealed promising results regarding the beneficial effects of these phytoconstituents and extracts on conditions such as nausea, vomiting, neuropathic pain depression, anxiety, Alzheimer’s disease, cognition, epilepsy, sleep, and dyskinesia via modulation of 5-HT₃R in the pathophysiology of neurological disorder. The review delves into a detailed exploration of in silico, in vitro, and in vivo studies and clinical studies that discussed phytoconstituents acting on 5-HT₃R and attenuates difficulties in neurological diseases. The diverse mechanisms by which plant-derived phytoconstituents influence 5-HT₃R activity offer exciting avenues for developing innovative therapeutic interventions. Besides producing an agonistic or antagonistic effect, some phytoconstituents exert modulatory effects on 5-HT₃R activity through multifaceted mechanisms. These include γ-aminobutyric acid and cholinergic neuronal pathways, interactions with neurokinin (NK)-1, NK2, serotonergic, and γ-aminobutyric acid(GABA)ergic systems, dopaminergic influences, and mediation of calcium ions release and inflammatory cascades. Notably, the phytoconstituent’s capacity to reduce oxidative stress has also emerged as a significant factor contributing to their modulatory role. Despite the promising implications, there is currently a dearth of exploration needed to understand the effect of phytochemicals on the 5-HT₃R. Comprehensive preclinical and clinical research is of the utmost importance to broaden our knowledge of the potential therapeutic benefits associated with these substances.

Serotonin, 5-hydroxytryptophan (5-HT), is a neurotransmitter derived from tryptophan and is involved in regulating mood, appetite, and sleep. It exerts its action through seven receptor subtypes (5-HT₁–5-HT₇ receptors).¹ Numerous preclinical and clinical investigations indicated the widespread distribution of 5-HT receptors in various brain areas like the hippocampus, amygdala, hypothalamus, olfactory bulb, choroid plexus, and islands of Calleja.²⁻⁹ They are involved in a variety of neuropsychological and neurodegenerative illnesses, including anxiety, depression, Alzheimer’s disease (AD), Parkinson’s disease (PD), and neuropathic pain, due to their widespread distribution in the brain.^10,11 Additionally, 5-HT plays an important role in gut health, and its stimulation can cause nausea and vomiting, especially in conditions such as cancer, inflammatory bowel disease, and colitis.¹²⁻¹⁴

5-HT and its receptors play a crucial role in developing gastric and central nervous system (CNS) illnesses and may be used as a therapeutic target. The agonists and antagonists of the receptor have gained significant market share for their therapeutic effects.^15,16 Except for the 5-hydroxytryptamine-3 receptor (5-HT₃R), all other 5-HT receptors are G-protein-coupled. The 5-HT₃R belongs to a ligand-gated cysteine-loop transmitter superfamily of ion channels, including the acetylcholine nicotinic receptor, the γ-aminobutyric acid A (GABA_A) receptor, and the glycine receptor. It operates as a selective cation ion channel (Figure 1).¹⁷ 5-HT₃Rs are located in both the CNS and the peripheral nervous system (PNS). A study conducted by Kilpatrick et al. reported the distribution of 5-HT₃R in the entorhinal cortex, retrosplenial cortex, hippocampus, and amygdala of the brain. Numerous autoradiographic labeling studies on rat brains showed the presence of 5-HT₃Rs in the subthalamic nucleus.^5,18In vitro autoradiographic assays and positron emission tomography imaging of mouse and rat brain slices suggested the highest 5-HT₃R in the hippocampus.¹⁹ The subtypes of 5-HT₃R are 5-HT_3A and 5-HT_3B. The immunostaining studies demonstrated a low occurrence of 5-HT_3AR in the limbic system, particularly in the hippocampus, amygdala, and entorhinal cortex, whereas the highest occurrence was observed in the hippocampus (CA1-CA3) regions of the forebrain region and brain stem, such as the nucleus tractus solitarius and trigeminal nerve.²⁰ The activation of 5-HT_3AR causes a rapid influx of Ca²⁺ ions at the nerve endings, essential in controlling the release of neurotransmitters such as 5-HT, GABA, acetylcholine (Ach), glutamate, and dopamine (DA). It can regulate the release and inhibition of these neurotransmitters, which can result in a variety of neurological disorders like nausea, vomiting, neuropathic pain, depression, anxiety, cognition, sleep AD, dyskinesia, and epilepsy (Figure 2).²¹

Figure 1.

Structure of 5-HT₃R (Created in Biorender.com)

Figure 2.

Mechanism of actions of phytochemicals on 5-HT₃R (Created in Biorender.com).

Herbs, plant extracts, phytochemicals, and other natural substances have been utilized extensively since time immemorial (Table 1). Traditional medicine has become widespread and increasingly popular globally due to its diverse pharmacological properties. Alkaloids, flavonoids, tannins, glycosides, and terpenoids are phytoconstituents with tremendous potential for treating various chronic disorders by modulating 5-HT₃R. Over the past 20 years, extensive research has been conducted on kampo medicine, a traditional Japanese remedy. Hoffman et al. conducted a study to screen the kampo remedies for their pharmacological effects. The effect of diluted ethanol tinctures of 121 kampo remedies on human 5-HT_3AR was studied through a voltage clamp method. Ligusticum striatum (rhizoma) activated 5-HT₃R, most strongly compared to other extracts, accounting for almost 30% of the 5-HT induced current. Further, the tinctures of different plant extracts such as Panax ginseng (radix), Syzygium aromaticum (flos), and Zingiber officinalis (rhizoma) were screened and found to have a potent 5-HT₃R antagonistic effect. In addition, the phytoconstituents such as boldine and leonurine present in Lindera aggregate (radix) and Leonurus japonicus (herba), respectively, were found to be potent 5-HT_3AR antagonists with IC₅₀ values of 0.53 ± 0.15 and 2.17 ± 0.15 μM, respectively, on the Xenopus laevis oocytes. In contrast, the other constituents of kampo medicine, such as tannic acid and schizandrin, also exhibited lower antagonistic effects with IC₅₀ 137 ± 22 and 48.2 ± 4.1 μM, respectively (Table 2).²² Extracts from Bolivian plants Xanthium spinosum and Senecio mathewsii demonstrated promising results obtained from the electrophysiology patch clamp technique. These extracts blocked 5-HT₃R and GABA_A receptors. However, their investigations on in vivo preclinical therapeutics remain untouched on neurological disorders.²³

Table 1. In Vivo Studies: Effects of Various Phytochemicals on 5-HT₃R.

S. No.	Plant Name	Chemical Constituent	Class	Doses	Disease	Animal Model	Effects	Reference
1.	Ganoderma lucidum (Ganodermatace)	Ganoderic acid A, Ganoderic acid C2, Ganodermanotriol	Triterpenoid	Varying doses (1,3,10 and 30 mg/kg, i.p.)	Nausea and vomiting	Cisplatin (3 mg/kg, i.p.) in rats	Improved food intake and Antiemetic effects	(39)
2.	Thymus persicus (Lamiaceae)	Carvacrol	Monoterpenoid	(50, 100, and 150 mg/kg, p.o.)	Neuropathic pain	Formalin induced paw licking in male mice	Antinociceptive and anti-inflammatory	(50)
3.	Cannabis Sativa (Cannabaceae)	CBD, THC	Alkaloids	CBD (2.5 mg/kg, i.p.) with THC (1 mg/kg, i.p.)	Vomiting and abdominal retching	Lithium-chloride-induced vomiting and abdominal retching	Antiemetic effects	(42)
4.	Salvia sclarea L. (Lamiaceae)	Sclareol	Diterpene	(15 mg/kg, p.o.)	Nausea and Vomiting	Copper sulfate-induced emesis in chicks	Decreased the number of retches and increased delay time	(43)
5.	Zingiber officinale (Zingiberaceae)	Gingerol, Shogaol	Polyphenol	Z. officinale aq. extracts (100, 300, and 500 mg/kg, p.o.)	Neuropathic pain	Oxaliplatin induced (6 mg/kg, i.p.)	Antiallodynic and antihyperalgesia effects	(51)
6.	Zingiber zerumbet (Zingiberaceae)	Zerumbone	Sesquiterpene	(10 mg/kg, i.p.)	Neuropathic pain	CCI-induced neuropathic pain in mice	Antiallodynic and antihyperalgesia effects	(52)
7.	Veratrum grandiflorum (Melanthiaceae)	Resveratrol	Stilbene	(2 mg/kg, i.v.)	Neuropathic pain	Electrical stimulation (jaw opening reflex) wistar rats	Antinociceptive effects	(55)
8.	Viola odorata (Violaceae)	Hydroxy flavoxone	Flavonoid	(1, 10, 30 mg/kg, i.p.)	Depression	FST and TST in mice	Reduce duration of immobility	(80)
9.	Spinacia oleracea (Amaranthaceae) and Brassica oleracea (Brassicaceae)	Alpha-lipoic acid	Organo sulfur	(50, 100, 200 mg/kg, p.o.)	Depression	FST and chronic stress induced depression in rats	Antidepressant and reduced anhedonia	(87)
10.	Terminalia catappa Linn (Combretaceae), Crocus sativus (Iridaceae), and Phyllanthus emblica L (Phyllanthaceae)	Gallic acid	Polyphenol	(30 and 60 mg/kg, p.o.)	Depression	FST and TST in mice	Antidepressant	(82)
11.	Hypericum perforatum (Clusiaceae)	Hyperforin	Terpene	(10 or 25 mg/kg, p.o.)	Depression	FST in rats	Antidepressant and increase % entries in open arm maze	(84)
12.	Gingko biloba (Ginkgoaceae)			(15, 30, and 60 mg/kg)	Cognition and memory	Scopolamine (3 mg/kg, i.p.) in male Swiss mice	Improve cognitive function through AchE Inhibition	(135)
13.	Zingiber officinale (Zingiberaceae)			(25, 50, and 100 mg/kg, i.p.)	Epilepsy	PTZ (30 mg/kg, i.v.) in mice	Increase seizures threshold	(136)
14.	Glycine max (Fabaceae)	Genistein	Flavonoid	(10 mg/kg, i.v.)	Epilepsy	PTZ (30 mg/kg, i.v.) in ovariectomized mice	Increase seizure threshold	(126)
15.	Matricaria chamomilla (Asteraceae)	Apigenin	Flavonoid	(50 mg/kg, p.o.)	Depression	FST, TST	Reduce duration of immobility	(90)
16.	Carpobrotus edulis (Aizoaceae)	Rutin	Flavonoid	(3 mg/kg, p.o.)	Depression	FST, TST	Reduce duration of immobility	(86, 137)

Table 2. In Vitro Studies: Effect of Various Phytochemicals on 5-HT₃R.

S. No.	Plant Name	Chemical Constituents/Extracts	Class	In Vitro Model	Concentration (IC50)	Binding Site	Reference
1.	Panax ginseng (radix) (Araliaceae)	Ginsenoside	Steroid glycoside	Xenopus laevis oocytes	27.6 ± 4.3 μM	Antagonism of 5-HT3AR	(22, 138)
2.	Syzygium aromaticum (flos) (Myrtaceae)	Tannic acid	Polyphenol	Xenopus laevis oocytes	48.2 ± 4.1 μM	Antagonism of 5-HT3AR	(22)
3.	Lindera aggregate (radix) (Lauraceae) and Peumus boldus (Monimiaceae)	Boldine	Aporphine alkaloid	Xenopus laevis oocytes	0.53 ± 0.15 μM	Antagonism of 5-HT3AR	(22, 139, 140)
4.	Leonurus japonicas (herba) (Lamiaceae)	Leonurine	Alkaloid	Xenopus laevis oocytes	2.17 ± 0.15 μM	Antagonism of 5-HT3AR	(22)
5.	Schisandra chinesis (fructus) (Schisandraceae)	Schizandrin	Dibenzocyclooctadiene	Xenopus laevis oocytes	48.2 ± 4.1 μM	Antagonism of 5-HT3AR	(22)
6.	Zingiber officinale Roscoe (Zingiberaceae)	Galanolactone	Diterpene	Guinea pig ileum	4.93 μM	Contractile inhibition of ileum via 5-HT3R	(27)
7.	Rosa damascene (Rosaceae)	Citronellol	Monoterpenes	Xenopus laevis oocyte	64 μM	Competitively inhibits 5-HT3AR	(40)
8.	Cannabis sativa (Cannabaceae)	Cannabinoid cannabidiol	Alkaloids	Xenopus laevis oocyte	121 nM to 29 μM	Noncompetitively inhibits 5-HT3AR	(41, 42)
9.	Syzygium aromaticum (Myrtaceae)	Eugenol vanillin	Benzaldehyde	Xenopus laevis oocytes	1159 μM eugenol and 4744 μM vanillin	Noncompetitive antagonist at 5-HT3AR	(40)
10.	Zingiber officinale (Zingiberaceae)	6-Shogaol	Polyphenol	Xenopus laevis oocytes	30.6 ± 0.54 μM	Noncompetitive antagonist at 5-HT3AR	(40)
11.	Zingiber officinale (Zingiberaceae)	6-Gingerol	Polyphenol	Xenopus laevis oocytes	46.6 ± 0.64 μM	Noncompetitive antagonist at 5-HT3AR	(40)
12.	Hypericum perforatum (Hypericaceae)	Hyperforin	Terpene	Peritoneal cells	3.4 μg/mL	Inhibiting serotonin uptake via 5-HT3R antagonism	(84)
13.	Uncaria hook (Rubiaceae)	Hirsutine	Indole alkaloid	Xenopus laevis oocytes	22.12 μM	Antagonism of 5-HT3R and 5-HT3ABR	(141)
14.	Eucalyptus globulus (Myrtaceae)	Eucalyptol	Monoterpene	Xenopus laevis oocytes	258 μM	Antagonism of 5-HT3R	(142)
15.	Lippia alba (Varbenaceae)	Citral	Monoterpene	Xenopus laevis oocytes	120 μM	Antagonism of 5-HT3R	(142)
16.	Gingko biloba (Ginkgoaceae)	Bilobalide, ginkgolides B	Sesquiterpenoid	Xenopus laevis oocytes	Bilobalide 470 μM, Ginkgolide B730 μM	Antagonism of 5-HT3R	(110)
17.	Cudrania tricuspidata (Moraceae)	Kaempferol	Flavonoid	Xenopus laevis oocytes	12.8 ± 2.0 μM	Antagonism of 5-HT3R	(103)
18.	Chili peppers (Solanaceae)	Capsaicin	Alkaloid	Xenopus laevis oocytes	62 μM	Antagonism of 5-HT3R	(143)
19.	Chili peppers (Solanaceae)	Capsaicin	Alkaloid	HEK-293 cells	54 μM	Antagonism of 5-HT3R	(143)
20.	Schisandra chinensis (Schisandraceae)	Schisandrin C	Lignan	Xenopus laevis oocytes	27.0 μM	Competitively antagonized 5-HT3R	(144)
21.	Xanthium spinosum (Asteraceae)			HEK-293 cells	4.6 ± 0.8 μg/mL	Antagonism of 5-HT3R	(23)
22.	Senecio mathewsii (Asteraceae)			HEK-293 cells	5.0 ± 1.0 μg/mL	Antagonism of 5-HT3R	(23)

Numerous chronic neurological diseases have shown resistance to available therapeutic options, leading to relapse and limited effectiveness. In light of these challenges, the potential benefits of phytoconstituents that target specific receptors and offer additional pharmacological effects have become a compelling area of exploration. The current review aims to explore and evaluate the potential of natural approaches in managing neurological disorders, focusing on modulation of 5-HT₃R by phytochemicals. The goal of the review is to present a thorough understanding of how phytoconstituents interact with the 5-HT₃R to mediate neurobehavioral activities, impacting neurological processes and perhaps having therapeutic advantages. Furthermore, to determine these natural chemicals’ potential as cutting-edge treatments for neurological disorders, the current review explores their modes of action, preclinical and clinical evidence, and safety profiles.

Nausea and Vomiting

5-HT₃R is involved in the physiology of nausea and vomiting.²¹ CNS and PNS are activated during a vomiting response, stimulating the enterochromaffin cells in the intestinal mucosa and causing the gastrointestinal tract to produce 5-HT. It activates the peripheral 5-HT₃R in the myenteric plexus, triggering synaptic and ganglionic transmission. This local 5-HT release induces vomiting by stimulating 5-HT₃R, inhibiting gastric motility.²⁴

The use of the Zingiber officinale Roscoe (Zingiberaceae) plant extract in ameliorating vomiting reflexes was widely studied. The administration of gingerol (1) in guinea pigs effectively inhibited upper gastric motility through its antiserotonergic actions and was proposed to be effective in emesis (Figure 3).²⁵ Investigations proved that gingerol strongly antagonizes 5-HT_3AR.^22,26 The other compounds of ginger, such as galanolactone (2), were also capable of suppressing contractile responses through antagonism of 5-HT_3AR with an IC₅₀ value of 4.93 μM against the effect of 2-methyl-5-HT, a 5-HT₃R agonist.²⁷ A clinical study regarding the effect of ginger root extract was conducted at St Bartholomew’s Hospital, London, on 60 women with major gynecological surgery. They were administered 0.5 g of ginger root extract (Zingiber officinale), which alleviated postoperative nausea and vomiting.²⁸

Figure 3.

Various natural products reported as 5-HT₃R antagonists.

In another randomized, double-blind, placebo-controlled, and crossover study, the patients with breast cancer were administered 500 mg of ginger-containing capsules; however, this was incapable of reducing chemotherapy-induced nausea and vomiting (CINV) in patients while the conventional medications such as ondansetron and domperidone reversed CINV.²⁹ Six previously published randomized controlled studies have assessed the effectiveness of oral ginger in preventing CINV in cancer patients. However, each study focused on a different cancer population, utilized varying chemotherapy regimens, administered different dosages of ginger, and employed different outcome assessment methods. Consequently, the findings from these studies are contradictory: four studies reported a positive impact of ginger, whereas two studies indicated no significant benefits.³⁰⁻³⁵

Most anticancer medications have nausea and vomiting as side effects, affecting the effectiveness of the therapy. The reduction of the CINV is a challenging task. Palonosetron (Aloxi), a potent 5-HT₃R antagonist, is effective and exhibits an antiemetic effect, but it induces headaches and constipation; therefore, it is less preferred.³⁶ Various phytoconstituents have been studied to alleviate these problems and enhance efficacy of anticancer drugs by lowering CINV. Menthol (3), a monoterpenoid obtained from Mentha piperita (Lamiaceae), antagonized human 5-HT₃R expressed in Xenopus laevis oocytes with an IC₅₀ of 160 μM.³⁷ Peppermint oil and menthol were evaluated for their binding affinity on 5-HT₃R using different in vitro paradigms where they have used [¹⁴C] Guanidinium influx into N1E-115 cells for 5-HT₃R expressions, isotonic contractions of the isolated rat ileum, and radioligand binding. Peppermint oil and menthol block 5-HT₃R in a concentration-dependent manner and also reduce the 5-HT-induced contraction of rat ileum.³⁸ The Ganoderma lucidum extract (GLE), a Chinese herb, was administered to rats at doses 1, 3, 10, and 30 mg/kg (i.p.) to evaluate its effect on cisplatin (3 mg/kg, i.p.) induced nausea. Results showed that GLE successfully increased food intake and minimized emesis.³⁹

The essential oil of Rosa damascene, a flowering plant, contains monoterpenes, viz., citronellol (4) and geraniol (5). These compounds competitively inhibit 5-HT_3AR with an IC₅₀ value of 175 μM and 64.3 μM, respectively, and are effective against nausea and vomiting.⁴⁰ Cannabidiol (6) (CBD), a nonpsychoactive substance, has shown inhibition in mice and human 5-HT_3AR expressed on Xenopus laevis oocytes. CBD (2.5 mg/kg i.p.) and Δ⁹-tetrahydrocannabinol (THC, 7) (1.0 mg/kg i.p.) diminished the effects of lithium-chloride-induced vomiting and abdominal retching in shrews.^41,42 Based on these findings, combining small doses of CBD with THC could enhance the antiemetic effect and mitigate the intoxicating effects associated with THC. Furthermore, research on eugenol (8) and vanillin (9) concluded that these have nonspecific inhibition of 5-HT₃R expressed on Xenopus laevis oocytes with IC₅₀ values of 1159 μM for eugenol and 4744 μM for vanillin.⁴⁰ The study on chicks was also demonstrated for the antiemetic effects of sclareol (10). Its 15 mg/kg oral dose attenuated the vomiting refluxes induced by copper sulfate, and sclareol decreased the number of retches and increased delay time. These results were supported through in silico findings suggesting that sclareol binds to 5-HT₃R and D₁ receptors more strongly than ondansetron and domperidone.⁴³

The FDA-approved drugs for CINV, such as granisetron, ondansetron, and palonosetron, are the best-selling medications in the market for several disorders associated with nausea and vomiting. Palonosetron has demonstrated its effects by selectively binding to 5-HT_3AR for CINV, as was previously mentioned. However, the lack of selectivity for 5-HT₃R subunits in the others may restrict their application in CINV.⁴⁴ On the other hand, the selectivity of most of the previously investigated natural 5-HT₃R antagonists supports their use in CINV, nausea, and vomiting.

Among these compounds, gingerol from Zingiber officinale, menthol from Mentha piperita, citronellol and geraniol from Rosa damascene, CBD, and eugenol have demonstrated effective inhibition of 5-HT₃R activity and reduction of nausea and vomiting symptoms. These natural compounds offer promising alternatives for managing nausea and vomiting, including CINV, with potentially fewer side effects compared to conventional medications like palonosetron.

Neuropathic Pain

Neuropathic pain is a chronic and debilitating condition marked by severe pain, heightened sensitivity to pain (hyperalgesia), and abnormal processing of sensory signals (nociception). There is a disruption in somatosensory signals between the brain and spinal cord. 5-HT₃R plays a significant role in transmitting pain in the central and peripheral nociceptive systems. The neurokinin (NK)-2 tachykinin receptor, substance P (SP), calcium calmodulin signaling, and protein kinase are also essential in transmitting central pain. SP is an 11-amino acid neuropeptide with a particular affinity for the neurokinin-2 receptor (NKR). Various types of non-neuronal cells, such as microglia and immune cells, express SP, which is essential to transmit nociceptive signals to second-order neurons in the brainstem and spinal cord via primary afferent fibers.

Additionally, the release of SP from primary afferent fibers increases the expression of the NK-2 receptor in dorsal horn neurons during neuropathic pain and inflammation.⁴⁵ The proper functioning of NK-2 tachykinin receptors relies on the ability of 5-HT₃R to facilitate the entry of Ca²⁺ ions. NK-2 receptors also play a role in the 5-HT₃R-mediated release of SP. Substantial evidence supports the interdependence of 5-HT₃R and the NK-2 tachykinin receptors, both crucial in transmitting pain signals within the CNS. Activation of NK-2 tachykinin receptors increases 5-HT release in the spinal cord, triggering 5-HT₃R. This activation may contribute to the development of pain hypersensitivity. Research indicates that the coactivation of NK-2 tachykinin receptor and 5-HT₃R enhances pain transmission in the spinal cord. When the 5-HT₃Rs are activated, there is an increase in the level of calcium release, forming a calcium-calmodulin complex. This complex upregulates the nitric oxide synthase (NOS) enzyme, producing nitric oxide (NO). NO presence can then modulate NK-2 tachykinin receptor activity. Overexpression of neurokinin or SP on the NK-2 tachykinin receptor intensifies pain sensitivity. The study in isolated nodose neurons from adult guinea pigs displayed that 5-HT, NO, and meta-chlorophenyl-biguanide (CPBG), a 5-HT₃R agonist, increased the response of SP (amplitude) in these neurons, which was absent with SP treatment alone. However, treatment with ICS205-930, a 5-HT₃R antagonist, completely abolished these effects, indicating the involvement of 5-HT₃R in pain stimuli.⁴⁶ Moore et al.⁴⁷ also showed that 5-HT promotes proinflammatory effects via 5-HT₃R.

Furthermore, this activation may increase the afferent vagal nerve sensitivity to SP on the tachykinin receptors. The administration of the 5-HT₃R antagonists, tropisetron and Y-25130 (at a concentration of 1 μM), did not result in noticeable alterations in the electrical characteristics of nodose neurons exposed to ovalbumin. These findings imply that 5-HT₃R antagonists might have pain-relieving abilities.⁴⁷ Several 5-HT₃ agonists have been used to investigate the involvement of 5-HT₃R in pain sensitivity. The intrathecal infusion of SR 57227, a 5-HT₃R agonist, stimulates glial cells and neuronal hyperexcitability, raising hypersensitivity to pain. It offered a rudimentary understanding of the spinal 5-HT₃R agonist as a pain mediator.⁴⁸

A polyherbal formulation of 11 extracts from plants, viz., as Commiphora mukul, Terminalia bellirica, Terminalia chebula, Emblica officinalis, Tinospora cordifolia, Piper longum, Withania somnifera, Alpinia galanga, Glycyrrhiza glabra, Curcuma longa, and Asparagus racemosus, was evaluated against acetic acid triggered writhing in mice with ondansetron (0.5 mg/kg, i.p.) as standard. The polyherbal formulation at doses of 390 and 585 mg/kg p.o. displayed significant antinociceptive effects in the writhing and tail immersion tests, possibly due to modulation of 5-HT₃R.⁴⁹ Iranian Avishan is the common name for the perennial herb Thymus persicus belonging to the Lamiaceae family. It has historically been used to treat back pain and toothaches. Previous studies have proved its antinociceptive effects in neuropathic pain against formalin-induced paw licking in male mice.⁵⁰ The essential oil Thymus persicus (TPEO) of aerial parts at doses of 50, 100, and 150 mg/kg p.o. significantly reduced allodynic effects and hyperalgesic stimuli, resulting in antinociceptive effects in mice. The study further elaborated on the impact of carvacrol (11) as a key compound in TPEO, which mediates the effect via the l-arginine/NO/cGMP/KATP signaling pathway and modulation of 5-HT₃R. Moreover, it was postulated that the analgesic effect against neuropathic pain was due to a reduction in proinflammatory mediators.⁵⁰

Further, certain studies explained the significance of Zingiber officinale Roscoe in analgesic effects at doses of 100, 300, and 500 mg/kg administered orally. One effect is against oxaliplatin-induced (2 mg/mL) cold and mechanical allodynia in mice.⁵¹ Zerumbone (12), a bioactive sesquiterpene derived from Zingiber zerumbet, has demonstrated antiallodynic and antihyperalgesic effects in a mouse model of neuropathic pain induced by chronic constriction injury. Another study revealed that zerumbone (10 mg/kg, i.p.) modulates the serotonergic system through 5-HT₃R, producing antiallodynic and antihyperalgesic effects.⁵²

For an extended period, tropisetron and ondansetron have been used to attenuate emesis. Still, their use in pain is also established by preclinical and clinical practices, which authenticate the crucial role of 5-HT₃R antagonism in pain relief. The activation of spinal 5-HT₃R regulates pain nociception, giving rise to allodynia. Further, 5-HT₃R antagonism contributes to comfort from mechanical allodynia in spinal cord injury following neuropathic pain in rats.⁵³ A double-blinded, placebo-controlled crossover study supported the finding from preclinical observations that 5-HT₃R antagonism alleviates symptoms related to neuropathic pain.⁵⁴

Resveratrol (2 mg/kg, i.v.) has been demonstrated to suppress the nociceptive, jaw-opening reflex in Wistar rats via 5-HT₃R-mediated GABAergic inhibition measured by digastric muscle electromyograms (dEMGs). Resveratrol activates 5-HT₃R, i.e., agonism, thereby inhibiting GABA receptor signaling and thus leading to inhibition of the Ca²⁺ channel, resulting in reduced pain as assessed by reduced amplification of dEMG.⁵⁵ Other investigations are underway to differentiate the role of the 5-HT receptor in neuropathic pain evaluated by paclitaxel (2 mg/kg i.p.)-induced neuropathic pain in male Sprague–Dawley rats. These rats were treated with the standard drug, 60 μg of WAY100635 (a 5-HT_1A receptor antagonist) or 30 μg of ketanserin (a 5-TH_2A/2C receptor antagonist) or 30 μg of ondansetron (a 5-HT₃R antagonist) or test drug Yokukansan, a Japanese medicine containing seven traditional mixture. Yokukansan attenuated PTX side effects by acting on the 5-HT₁ and 5HT₂ receptor and less via 5-HT₃R. It improved allodynia and increased the withdrawal threshold, which supports its therapeutic use in neuropathic pain via 5HT₁, 5-HT₂, and 5-HT₃R.⁵⁶ Grape seed extract (GSE) was evaluated for action by 5-HT₃ and GABA receptors in temporomandibular disorders characterized by dysfunctional jaw joints and muscles that control jaw movement. Animals were supplemented with 0.5% GSE dissolved in their water for 1 week, and nociception was induced by five injections of Freund adjuvant (each 100 μL per side) into the upper trapezius muscles. To investigate the mechanism of GSE, some animals were injected intracisternally with antagonists of 5-HT₃, 5-HT₇, GABA_A, or GABA_B receptors before jaw opening. The inhibitory effect of GSE is mediated via the activation of 5-HT_3/7 receptors and GABA_B to enhance central descending inhibitory pain pathways and suppress ongoing trigeminal nociception.⁵⁷

The existing studies investigating the use of phytochemicals to regulate 5-HT₃R for pain management, especially in neuropathic conditions, exhibit conflicting findings compared to similar research. This disparity complicates the comprehension of the impacts of 5-HT₃R agonists and antagonists on pain. Faerber et al. conducted a study that clarified the effects of 5-HT₃R agonists and antagonists. In their view, activating the spinal 5-HT₃R increases the GABAergic activity, a crucial mediator of analgesic effects. On the other hand, 5-HT₃R antagonists alleviate pain via NK1 and NK2 receptors by stopping the secretion of neurokinin, SP, and calcitonin gene-related peptides.^21,58

Among these, carvacrol from Thymus persicus, zerumbone from Zingiber zerumbet, and resveratrol have demonstrated significant antinociceptive effects through 5-HT₃R modulation. Carvacrol from Thymus persicus and zerumbone from Zingiber zerumbet exert their antinociceptive effects by modulating the l-arginine/NO/cGMP/KATP signaling pathway and through direct modulation of 5-HT₃R activity. Resveratrol suppresses nociception by activating 5-HT₃R-mediated GABAergic inhibition, leading to reduced pain sensitivity, and has demonstrated significant pain-relieving abilities by blocking 5-HT₃R-mediated pain transmission pathways.

Currently, there is a lack of research that supports the use of phytochemicals as 5-HT₃R agonists and antiagonists to treat central neuropathic pain pathways. Phytochemicals targeting 5-HT₃R inevitably hold promise as treatments for neuropathic pain. More research is necessary to identify specific phytoconstituents and assess their impact on experimental models of neuropathic pain.

Anxiety

Anxiety is a neuropsychiatric illness characterized by restlessness, fear, apprehension, difficulty concentrating, irritability, muscle tension, and sleep disturbances. In recent times, considerable evidence has substantiated the pivotal involvement of the serotonergic system in anxiety disorders. Preclinical research has been instrumental in formulating and supporting this hypothesis.^59,60

Antagonism of 5-HT₃R demonstrates encouraging anxiolytic effects observed across different behavioral paradigms, including the elevated X-maze, elevated T-maze, light/dark test, and potentiated acoustic startle.^61,62 Ondansetron, a 5-HT₃R antagonist, effectively treated anxiety in the early investigations, as evidenced by the elevated plus maze results in mice.⁶³Hallea ciliate and Datura stramonium were studied for their anxiolytic activity using a hole board test, increased head dips, sectional crossings, squares crossed, rearing in OFT, and decreased immobility time in force swim test (FST) in mice. This behavior pointed out that Datura has substantial anxiolytic and antidepressant-like activities through modulation of serotoninergic (5-HT₃R) and adrenergic (α₁) neurotransmissions.⁶⁴ In contrast, Hallea ciliate mediates its anxiolytic effects via GABA and 5-HT₃R antagonism.⁶⁵ The anxiolytic effect of petroleum ether extract of Zingiber officinale (5–300 mg/kg p.o.) in mice has demonstrated a decreased preference for a closed arm of an elevated plus maze.⁶⁶

Similarly, a combination of Ginkgo biloba and Zingiber officinale (0.5, 1, 10, and 100 mg/kg) via intragastric administration to male rats exhibited an anxiolytic effect without any amnesiac symptoms, as observed with benzodiazepines. Moreover, the combination displayed nootropic activity. Gingerol and galanolactone are the constituents of Zingiber officinale, both having antagonistic activity at the 5-HT₃R and are considered to have the potential to treat fear and anxiety-related disorders.⁶⁷

There has been little research on the anxiolytic effects of herbs, extracts, and phytoconstituents; molecular and clinical evidence is lacking, and behavioral assessments are the primary focus. Although some combinatorial effects like those of stramonium have been explored, little is known about particular phytoconstituents targeting anxiolytic effects via 5-HT and adrenergic receptors. More thorough research is necessary to investigate possible phytoconstituents acting on 5-HT3R and their molecular pathways for the anxiolytic effect.

Depression

The involvement of 5-HT₃R in the progression of depression-like behavior is widely accepted; however, the role of 5-HT₃R agonists and antagonists in the treatment of depression is still unclear and under investigation due to safety concerns.⁶⁸ The treatment of depression has traditionally emphasized the role of 5-HT₁ and 5-HT₂ receptors. Their agonists and antagonists have an impressive market presence and are in high demand as therapeutics among physicians; FDA-approved medications for treating depression and anxiety include buspirone, a 5-HT_1A agonist, methyl-5-HT, a 5-HT_2AB agonist, and mirtazapine, a 5-HT_2C receptor antagonist,⁶⁹ in addition to selective serotonin reuptake inhibitors like fluoxetine, duloxetine, and escitalopram.⁷⁰ The multifactorial pathophysiology of depression makes it challenging for researchers to identify compounds with multiple therapeutic effects with safety and efficacy. Buspirone, vortioxetine, mirtazapine, and quetiapine are some of the drugs utilized to alleviate neuropsychiatric symptoms that act through 5-HT₃R modulation.⁷¹⁻⁷⁵

Moreover, preclinical studies on phytochemicals and extracts have produced promising results in alleviating depressive symptoms through the regulation of 5-HT₃R. In rodents, 5-HT₃R agonism causes depressive symptoms; on the other hand, its antagonistic effects restore depressive-like behavior.^75,76HTR3a knock out mice displayed antidepressant effects evaluated by FST and elevated plus maze in a chronic social defeat stress model.⁷⁷ 5-HT₃R antagonism suppresses 5-HT binding to postsynaptic 5-HT₃R, thereby enhancing its accessibility to other receptors such as 5-HT_1A, 5-HT_1B, and 5-HT_1D, along with 5-HT₂ receptors. This mechanism elicits antidepressant-like effects.⁷⁸ In rodents, antidepressant effects were investigated using behavioral paradigms such as FST and the tail suspension test (TST). In FST, the antidepressant effects were reflected by an increase in serotonergic neurotransmission, which led to longer swimming durations (mobility), whereas those that were found to increase catecholaminergic neurotransmission led to longer struggling durations.⁷⁹ The Sweet violet (Viola odorata L) extract consisting of compound 1, 5,7-dihydroxy-3,6-dimethoxyflavone, compound 2, 5,7,4′-trihydroxy-3′,5′dimethoxyflavone, and compound 3, 5,7,4′-trihydroxy-3′-methoxyflavone, was critically investigated for their antidepressant activity via 5-HT₁, 5-HT₂, and 5-HT₃ receptors using WAY100635 (5-HT_1A antagonist; 0.1 mg/kg i.p.), ketanserin (5-HT_2A antagonist; 1 mg/kg, i.p.), or ondansetron (a 5-HT₃R antagonist; 1 mg/kg i.p.) in mice. The extract (1, 10, and 30 mg/kg, i.p.) effectively reduced immobility duration in mice comparable to ketanserin and ondansetron in TST and FST as well as raised 5-HT levels in the brain. Molecular docking studies demonstrated that these compounds showed a strong interaction with 5-HT₁ and 5-HT₂ receptors with greater affinity and higher binding energy for 5-HT₃R resulting in an antidepressant effect.⁸⁰ Other in silico studies are also presented in Table 3. Another study along these lines evaluated gallic acid (GA, 13), which was derived from Phyllanthus emblica, Terminalia catappa Linn, and Crocus sativus, among other plant sources.⁸¹ It was discovered that GA was useful in controlling dopaminergic, serotonin, and adrenergic receptors. The mechanistic effects of GA (30 and 60 mg/kg, p.o.) in mice were examined using several pharmacological contexts involving certain 5-HT_1A, 5-HT_2A/2C, and 5-HT₃R antagonists, such as NAN 190, ketanserin, and ondansetron, respectively. The higher dose 60 mg/kg GA was found to be effective to reduce the immobility time in TST and FST against above modulators which suggested that GA increased the 5-HT release in the synaptic cleft by interaction with 5-HT₂R and 5HT₃R.⁸²

Table 3. In Silico Studies of Phytochemical Binding on 5-HT3R.

S. No.	Constituents	Class	PDB ID	Binding Energy(kcal/mol)	Reference
1.	Gingerols	Polyphenol	4PIR	−8.7	(145)
2.	Shogaols	Polyphenol	4PIR	− 8.31	(145)
3.	α-Lipoic acid	Organo sulfur	6Y1Z	−5.4	(87)
4.	5,7-Dihydroxy-3,6-dimethoxyflavone	Flavonoid	4PIR	−8.15	(80)
5.	5,7,4′-Trihydroxy-3′,5′dimethoxyflavone	Flavonoid	4PIR	−7.90	(80)
6.	5,7,4′-Trihydroxy-3′-methoxyflavone	Flavonoid	4PIR	−7.39	(80)
7.	Kaempferol	Flavonoid	6Y1Z		(103)
8.	Capsaicin	Alkaloid		−7.8	(143)
9.	Psychotriasine	Alkaloid	5AIN	−5.811	(146)
10.	Schisandrin C	Lignan	VHH15	−5.62	(144)
11.	Meranzin hydrate		6Y1Z	−8.3	(88)
12.	Hesperidin	Flavonoid	6Y1Z	−9.4	(88)

5-HT₃R is widely distributed in the vagus nerve, nucleus tractus solitarius, guinea pig ileum, and peritoneal cells, among other places. Exposure to 5-HT activates these receptors.⁸³ The antidepressant activity of Hypericum perforatum extract and its principal constituent, Hyperforin (14), was also explored using in vivo and in vitro models. In a research study employing a hypericum extract (CO₂ extract) obtained through supercritical carbon dioxide extraction and hyperforin, notable spasmolytic effects were observed on the guinea pig ileum. The CO₂ extract hindered 5-HT uptake in rat peritoneal cells. Specifically, hyperforin, the CO₂ extract, and the ethanolic extract in peritoneal cells exhibited IC₅₀ values of 3.4, 4.5, and 47.5 μg/mL, respectively, for inhibiting 5-HT uptake. Further, hyperforin exhibited superior inhibitory activity on guinea pig ileum contraction at concentrations of 0.1, 0.3, and 1 μg/mL, with inhibition constants of 10%, 80%, and 97%, respectively. Bezold-Jarisch (B-J) reflex is characterized as 5-HT-induced transient bradycardia resulting from the stimulation of 5-HT₃R on the vagus nerve. Continuing the exploration of the B-J reflex, it was revealed that rats receiving only 5-HT bolus injections experienced a dose-dependent decrease in heart rates. Conversely, rats treated with hyperforin displayed less reduction in heart rate, possibly due to hyperforin’s 5-HT₃R antagonist activity. The above in vitro investigation suggested that hyperforin can inhibit 5-HT reuptake with an IC₅₀ value of 3.4 μg/mL. It also possesses 97% inhibitory activity on guinea pig ileum contraction at 1 μg/mL. It also reduced the 5-HT-induced bradycardia via 5-HT₃R antagonism. These noteworthy findings regarding the in vitro 5-HT₃R antagonism have set a promising direction for further research on hyperforin. Hyperforin (10 mg/kg) in rats significantly reduced immobility and increased % entries in the open arm in the behavioral despair test and elevated plus maze, which reflected its antidepressant and anxiolytic effects.⁸⁴ The aforementioned research on antidepressant action has been thoroughly investigated and demonstrates that medicinal plants are useful not only for providing complicated or unique compounds but also for uncovering novel and therapeutically significant pharmacological pathways. The pathophysiological implications of 5-HT₃R in depression have not been extensively investigated compared to those of 5-HT_1A and 5-HT₂ receptors. Nevertheless, literature data indicate that various types of antidepressants act as functional antagonists on 5-HT₃R, suggesting that reducing 5-HT₃R activity might play a role in the antidepressant mechanism of action.⁸⁵ An extract of Schinus molle L. (Anacardiaceae) and its constituent rutin (15) (3 mg/kg p.o.) reduced immobility in FST and TST as well as the number of crossings in OFT. The suggested mechanism of action was due to the modulation of serotonergic, adrenergic, and dopaminergic supply. They further postulated that the effects of 5-HT₁, 5HT₂, and 5-HT₃R primarily contribute to antidepressant action. They emphasized that 5-HT₃R antagonism may be a crucial mediator for mitigating depression-like behavior in mice.⁸⁶ α-Lipoic acid (14, ALA), a potent antioxidant commonly found in vegetables like spinach and cabbage, was found to increase % sucrose preference, decrease immobility duration, and restore levels of monoamines in the hippocampus. The docking study also revealed that ALA has a potent antagonistic effect on 5-HT₃R, perhaps mitigating symptoms of depression. Furthermore, the fact that ALA’s nutraceutical qualities prevent stress-induced depression-like behavior is also highlighted.⁸⁷Fructus Aurantii (FA) is the dried immature fruit of Citrus aurantium L. (Rutaceae). It contains multiple phytoconstituents that have neuroprotective effects. Meranzin hydrate (17, MH) and hesperidin (18) of FA demonstrated antidepressant action in behavioral and neurochemical paradigms and improvement in various biochemical indexes, including immobility time, gastric emptying, intestinal transit, corticotropin-releasing hormone CRH, ghrelin, ACTH, DA, noradrenaline NA, 5-HT, cortisol, and 5-HT₃ receptor. These effects were achieved through regulation of the 5-HT₃/growth hormone secretagogue receptor (GHSR) pathway. The study also validated the above-mentioned results using receptor antagonists and molecular docking, confirming the potential therapeutic mechanisms of MH in regulating the plasma biochemical indexes in rats with acute stress. The study also involved functional magnetic resonance imaging (fMRI) in providing anatomical and functional information about the active neural circuit regulating mood and gastroenteric motility. The study revealed the activation of excessive blood oxygen level-dependent (BOLD) signals in specific brain regions, including the left accumbens nucleus, left corpus callosum, and hypothalamus preoptic region, following acute stress stimuli. Additionally, it was found that FA and MH treatment reversed these BOLD signal changes to varying degrees.⁸⁸Bacopa monnieri was tested for its effects on 5-HT receptors using antagonists of 5-HT₁, 5-HT₂, and 5-HT₃ receptors. Furthermore, Bacopa monnieri (80 mg/kg p.o.) decreases immobility in FST and TST, which is indicative of antidepressant-like effects in albino mice. They proposed that 5-HT1 and 5-HT2, rather than 5-HT3R, receptors were responsible for Bacopa monnieri effects.⁸⁹ Recently, using antagonists, apigenin (19, APG) was assessed for its effects on catecholaminergic and serotonergic systems. APG in mice at the dose of 50 mg/kg p.o significantly reduced immobility time in FST and TST as compared to PCPA and ondansetron. APG reduced immobility and increased swimming and climbing time better than fluoxetine (30 mg/kg).⁹⁰

The phytoconstituents reported for antidepressant activity indicated that 5-HT₃R antagonism is effective in treating depression like behavior. GA has demonstrated antidepressant effects by interacting with multiple 5-HT and dopamine receptors, including 5-HT₃R, leading to increased 5-HT and dopamine release in the synaptic cleft. Hyperforin, derived, acts as a 5-HT₃R antagonist, contributing to its antidepressant and anxiolytic effects. Similarly, ALA exerts its antidepressant activity by acting as a strong antagonist on 5-HT₃R, thereby alleviating depression-like symptoms. Additionally, Meranzin hydrate + hesperidin (MH) demonstrated antidepressant effects by regulating the 5-HT₃/growth hormone secretagogue receptor (GHSR) pathway. Among these molecules, GA and MH have been particularly well-investigated, showing promising results in preclinical studies.

Cognition

Cognitive decline encompasses a range of mental disorders involving impairments in learning, memory, attention, and challenges in language and motor skills. Evidence suggests the involvement of the cholinergic system is necessary for cognition and may be under the inhibitory control of 5-HT₃R.^91,92 Since 1990, it has been firmly established that 5-HT₃R is implicated in cognitive processes. Studies have also been conducted on animal models of scopolamine-induced cognitive deficit in mice, rats, and marmosets, suggesting that 5-HT₃R antagonism may reverse cognitive dysfunctions.⁹³ Age-related decline in mental ability is also associated with decreased cholinergic neurotransmission in the CNS. Evidence suggests that inhibitory tone on the 5-HT receptor increases Ach release.⁹⁴In vitro experiments have shown that 5-HT agonists decrease Ach release, whereas blocking 5-HT₃R may increase Ach release and turnover in some brain regions. Ondansetron causing 5-HT₃R blockade improves novel object recognition (NOR), reversal learning tasks, and cholinergic release. The action of 5-HT₃R antagonists at such sites is suggested to facilitate cholinergic function and enhance the performance in cognition tests. The data strongly indicate that inhibiting the 5-HT₃R pathway in the hippocampus, which affects cholinergic function, holds promise as a viable approach for enhancing cognitive function through various strategies.⁹⁵

One more study conducted on rhesus monkeys using behavioral paradigms revealed that a low dose of ondansetron (0.00001–0.5 mg/kg, p.o.) improved the acquisition of visual object discrimination performance. This suggests that 5-HT₃R antagonists might be beneficial in cognitive illnesses. In rats, another research study showed that azasetron injected bilaterally at doses of 0.32 and 1.0 μg/side significantly reduced working memory errors caused by intrahippocampal scopolamine administration at a 3.2 μg/side dose. Furthermore, cognitive dysfunction associated with depression was investigated in rats using autoradiography and NORT. Vortioxetine effectively binds to 5-HT₃R with 95% occupancy and improved NORT. It was elucidated that these cognitive-enhancing effects of their 5-HT₃R antagonist are likely mediated through cholinergic mechanisms. However, despite consistent findings in preclinical literature regarding the positive impact of 5-HT₃R antagonists on memory function, it is crucial to note that ondansetron is considered to have limited clinical efficacy for improving cognitive function.⁹⁶⁻⁹⁸

Chronic administration of Zingiber officinale (Zingiberaceae) extract (50, 100, and 200 mg/kg, p.o.) improved memory function altered by morphine (5 mg/kg, i.p.) in male Wistar rats. The study suggested that these beneficial effects are attributed to the active constituents found in ginger, such as 6-Gingerol and Zingerone, which act on the 5-HT₃R as an antagonist and inhibit cholinesterase activity.⁹⁹ It was reported previously that scopolamine negatively affects cognitive functions in rats via modulation of 5-HT₃R associated with α7 nAChR, leading to cholinergic manipulation. Behavioral and gene expression investigations were conducted to assess the impact of the rosmarinic acid (20)-rich extract of Ocimum basilicum L (Lamiaceae) on 5-HT₃R.¹⁰⁰ Scopolamine (0.5 mg/kg, i.v.) was injected into male Wistar rats and showed memory deficits in novel object discrimination in support of these elevated gene expressions of 5-HT_3A R, NA7, M1, and nNOS leading to cognitive decline in the hippocampus of rats. At the same time, treatment with Ocimum basilicum L extract (200 and 400 mg/kg p.o.) for 14 days improved functions in the novel object recognition (NOR) test and restored cognitive functions by reducing expressions of 5-HT_3AR, NA7, M1, and NOS levels. These results further corroborated the previously reported studies and supported the involvement of 5-HT₃R in memory and cognitive processes. 5-HT₃R has been observed to coexpress and mutually regulate with NA7 (Neuronal acetylcholine receptor subunit alpha-7, also known as nAChRα7). Notably, 5-HT₃R shows significant expression in the hippocampus, prompting investigations into its role in learning and memory.¹⁰¹

Kaemferol (21), extracted from Cudriania tricuspidata leaves, exhibits promising cognitive enhancement effects in various experimental models-galactose/AlCl₃-induced cognitive dysfunctions and chlorpyrifos-induced dementia. In mice, kaemferol (60 mg/kg i.p) demonstrated good blood–brain barrier permeability and improved cognitive function, escape latency, and distance of target quadrants in MWM via increasing monoamine levels (5-HT, DA, NE) and antioxidant effects, and reduced neuroinflammation in in vivo settings.¹⁰² Further studies on kaempferol using in vitro electrophysiological investigations were performed with Xenopus oocytes expressing the human 5-HT_3AR to investigate the molecular relationship with IC₅₀ 12.8 ± 2.0 μM. The study confirmed a molecular-level interaction between kaempferol and 5-HT_3AR. Notably, kaemferol exhibited concentration-dependent inhibition of 5-HT_3AR, independent of voltage variations.¹⁰³

Peppermint often contains high amounts of the monoterpenes menthone (22) and menthol. Essential oils and menthol independently affect GABA_A, glycine, and 5-HT₃R receptors in the in vitro study. They also showed cholinesterase inhibitory properties. These components of monoterpenes may have a variety of impacts that are directly related to brain function. Among these are the effects of essential oils on cholinesterase inhibition;¹⁰⁴ menthol also affects GABA_A and 5-HT₃R receptors and negative allosteric modulation of GABA_A receptors by carvone (23). Research has indicated that they can improve cognitive function and mitigate the increase in mental exhaustion linked to extended cognitive assessments.¹⁰⁵

Ginkgo biloba has proven to be effective in various neurological illnesses, and it is a well-identified herb for interaction with the serotonergic system in memory-enhancing effects in the passive avoidance test.¹⁰⁶ Bilobalide, a sesquiterpenoid lactone of Ginkgo biloba, is effective in C. elegans for 5-HT-mediated cholinergic modulation.¹⁰⁷Ginkgo biloba was investigated in bisphenol A-induced cognitive deficit, where it demonstrated its effects via increasing hippocampal levels of DA, NE, and 5-HT and improved antioxidant potential, further protecting CA3 neurons.¹⁰⁸ Its constituents, such as bilobalide (24) and Ginkgolide B (25), block 5-HT_3AR expressed on Xenopus oocytes with an IC₅₀ value for bilobalide of 470 μM and for Ginkgolide B of 730 μM. However, the binding sites of these molecules on 5-HT₃R differ from that of granisetron. They were found to block the receptor pores.^109,110 Radioligand binding studies have revealed the presence of 5-HT₃R in the cholinergic-rich entorhinal cortex of the hippocampus. In vitro investigations have demonstrated that the activation of these 5-HT₃Rs leads to a decrease in Ach release in the cerebral cortex, while using a 5-HT₃R antagonist enhances the release of Ach.¹¹¹ This explanation correlates with the fact that 5-HT_3AR antagonism by Ginkgo biloba supports their correlation with cognition improvement and cholinergic enhancement. 5-HT₃R blockade plays a pivotal role in cognitive function through cholinergic mediation. Experimental evidence suggests that inhibiting 5-HT₃R enhances Ach release, highlighting the potential therapeutic significance of targeting this receptor in the modulation of cognitive processes and potential treatment avenues for cognitive disorders.

Various molecules have shown potential as cognitive enhancers through their interactions with 5-HT₃R and modulation of cholinergic function. Notable candidates include Zingiber officinale extracts (containing 6-Gingerol and Zingerone), rosmarinic acid-rich extract of Ocimum basilicum L, kaemferol from Cudriania tricuspidata leaves, and compounds found in peppermint, such as menthol and menthone. Ginkgo biloba constituents like bilobalide and Ginkgolide B have also demonstrated 5-HT₃R blockade activity. Among these, kaempferol and Ginkgo biloba constituents bilobalide and Ginkgolide B stand out as promising cognitive enhancers due to their ability to block 5-HT₃R and their potential to enhance cholinergic function, which is crucial for cognitive processes. More studies are required in this direction.

Sleep

CNS is crucial in regulating sleep and is associated with neurotransmitters, including 5-HT and melatonin. An appropriate serotonergic supply ensures the adequate formation of melatonin, which is responsible for sound sleep. Sleep is regulated by two process rapid eye movement (REM) and non rapid eye movement (NREM). REM is define as rapid movement of the eyes, vivid dreams, increased brain activity and NREM is a sleep phase that consists of three stages, ranging from light to deep sleep. It is important for physical restoration, energy conservation, and memory consolidation. NREM sleep accounts for about 75−80% of total sleep time.¹¹² Previously conducted studies in relation to 5-HT₃R and sleep were discussed. It was proven that activation of 5-HT₃R by the selective agonist m-CPBG has been shown to decrease the REM in rats. This proposed effect is related to the activation of glutamatergic interneurons expressing 5-HT₃R, leading to an increase in 5-HT release at postsynaptic sites involved in the induction of REM.¹¹³ Another study explained the effect of zacopride, a 5-HT₃R antagonist, effective in treating sleep apnea.¹¹⁴ Moreover, antidepressants such as vortioxetine and paroxetine increase NREM;¹¹⁵ a randomized clinical trial was conducted on Rosmarinus officinalis (RO) leaves for patients with withdrawal syndrome from opium abuse and sleep disturbances. The study demonstrated that patients administered RO capsules (16 mg) improved sleep and reduced insomnia via serotonergic interaction.^116,117 Suanzaorentang, a traditional Chinese herbal remedy, consists of Anemarrhenae rhizoma, Poria cocos, Zizyphi spinosi semen, Ligusticum wallichii, and glycyrrhizae in a ratio of 4:2:2:2:1. This herbal remedy is believed to regulate sleep by exerting an antagonistic effect on 5-HT₃R, which is involved in sleep regulation.¹¹⁸

Numerous studies have demonstrated the efficacy of synthetic drugs on 5-HT₃R antagonism in addressing sleep-related difficulties. A couple of studies were conducted using phytoconstituents; however, there remains a paucity of research investigating the role of phytoconstituents in treating sleep disorders through modulation of 5-HT₃R. Considering the side effects and habituation involved with synthetic drugs, the need for further exploration and attention toward phytochemicals in the context of sleep regulation via 5-HT₃R is highly desired.

Alzheimer’s Disease (AD)

It has been found that 5-HT interacts with 5-HT₃R in the hippocampus, a brain region involved in memory and learning. Activation of 5-HT₃R receptors can decrease the brain’s cholinergic tone, exacerbating cognitive deficits in AD. The recent development of a novel molecule (R3487/MEM3454) with dual properties of 5-HT₃R blocking and cholinesterase inhibitory activity holds promise as a potential therapeutic approach for AD. Acetylcholinesterase inhibitors (AChEIs) are currently the mainstay of treatment for AD, as they help increase the levels of Ach in the brain by blocking its degradation. They have shown improved attentional performance in rats with AD-like conditions.¹¹⁹

Moreover, there is evidence that ondansetron, a known 5-HT₃R antagonist, can potentiate the effects of donepezil, an AChEI.¹²⁰ Furthermore, RG3487, an α7-nAChR partial agonist/5-HT₃ antagonist, demonstrated a noteworthy increase in cortical and hippocampal DA and ACh effluxes upon acute administration.¹²¹ This finding suggests that combining 5-HT₃R antagonism with AChEIs might synergistically improve cognitive function in AD patients. Elevated HTR_3A positive interneurons levels were observed in the brains of transgenic AD (Tg AD) mice and post-mortem AD patients, preceding Aβ plaque appearance. 5-HT₃R is a Ca²⁺ permeable ionotropic receptor with heightened presynaptic activity in the hippocampus, leading to increased basal Ca²⁺ levels in the hippocampus and cortex. Chronic tropisetron administration mitigated cognitive deficits and reduced Aβ plaque levels in Tg AD mice, likely via an HTR₃-dependent mechanism and by attenuating Ca²⁺ and calcineurin signaling.¹²²

The presence of the β-amyloid protein significantly influences the pathogenesis of AD, which exacerbates several neuronal malfunctions. The in vitro study has shown β-amyloid 25–35 protein (10 μM) induced glutamate excitotoxicity and elevated calcium release, ROS, and caspase-3 leading to neuronal death. Such effects were observed to be reduced by 5-HT₃R antagonists tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1–10 μM). The study also suggested that 100 μM m-CPBG, an agonist of 5-HT₃R, resists the neuroprotective effects of MDL72222.¹²³ This study notifies the crucial role of 5-HT₃R antagonists in AD, attenuating neurotoxic effects, and provides neuroprotection.

Many synthetic substances have been examined in rodents and in patients for their potential to alleviate AD by interacting with 5-HT₃R. These substances have helped to reduce AD symptoms induced by scopolamine and amyloid beta. Although the advantages of 5-HT₃R antagonist therapy for AD are widely known, no studies on phytoconstituents and 5-HT₃R interactions have yet been documented. Consequently, future studies should investigate whether or not phytoconstituents can reduce AD by interacting with 5-HT3R in animal models.

Epilepsy

Epilepsy involves sudden abnormal neuronal discharges and is characterized by neuroinflammation and oxidative stress. An imbalance between glutamate and GABA plays a role in its pathogenesis. Past research data also pointed to the serotonergic supply, specifically 5-HT₃R, which could be correlated with epilepsy.¹²⁴ A study described the potential neuroprotective and antiepileptic benefits of hydroethanolic extract of Zingiber officinale (Zingiberaceae) (25, 50, 100 mg/kg, i.p.) in PTZ-induced tonic-clonic seizures in mice. The study discussed the antagonism of 5-HT₃R involved in releasing various neurotransmitters, including GABA. It also inhibited calcium release, contributing to fast excitatory synaptic transmission in the CNS.

Ginger extract has a potency to diminish the PTZ-induced seizures via modulation of 5-HT₃R.¹²⁵ Some other natural 5-HT₃R antagonists are effective in PTZ-induced seizures in ovariectomized mice. Genistein (10 mg/kg, i.p.), a flavonoid obtained from soya or soya products, provides phytoestrogen and exhibits estrogenic activity. Administering estrogen to ovariectomized female rats demonstrates neuroprotective effects against hippocampal damage induced by status epilepticus and was found to increase the seizure threshold, thereby reducing the chances of epileptic attacks. Similarly, administration of m-CPBG, a 5-HT₃R agonist, supports the anticonvulsant effects of genistein by increasing the seizure threshold. In contrast, the tropisetron (1 mg/kg, i.p.) 5-HT₃R antagonist reversed the anticonvulsant effects of genistein. Such results indicated that genistein provided potential antiepileptic effects in the PTZ-induced ovariectomized mice model via serotonergic pathways and 5-HT₃R modulation.¹²⁶ Traditional Chinese medicine reports pearl powder treatment for convulsion and tranquilization effects. Zhang et al. reported prevention of PTZ-induced generalized myoclonic seizures, reduced locomotor activity, and increased convulsion latency observed in mice in the pearl treatment group. These effects were further corroborated by receptor expression, measured by immunohistochemistry. It showed decreased 5-HT₃R and enhanced GABA release, supporting the anticonvulsant properties of conventional pearl powder.¹²⁷

Molecules targeting 5-HT₃R have shown potential in treating epilepsy by modulating neurotransmitter release and inhibiting calcium influx, thus reducing seizure activity.

Dyskinesia

Levodopa is the standard treatment for PD. However, its chronic use leads to dyskinesia.¹²⁸ The strategy to treat levodopa-induced dyskinesia (LID) involves modulation of NMDA and 5-HT receptors. AV101 (L-4-Chlorokinurenine) is an NMDA antagonist that reduced dyskinesia in primates, and proof of concept for the phase II trial in 20 PD patients continues.^128,129 The growing evidence suggests that 5-HT₃R might be involved in the development of dyskinesia.¹³⁰ 5-HT₃R is present in the basal ganglia, and its stimulation shows DA release.^131,132 Many studies have demonstrated that selective blockade of 5-HT₃R with ondansetron and granisetron reduces the LID without affecting the antiparkinsonian activity of levodopa. Blockade of 5-HT₃R reduces the DA release within the basal ganglia, suggesting that it could mitigate the aberrant DA release that characterizes the dyskinetic state.^133,134 Selective blockade of 5-HT₃R with medications like ondansetron and granisetron has been shown to reduce LID without affecting the antiparkinsonian activity of levodopa. This suggests that targeting 5-HT₃R could potentially mitigate the aberrant DA release associated with dyskinesia, making it a molecule with significant potential for managing LID in PD patients.

It is noteworthy that several pharmacological agents that target 5-HT₃R have demonstrated potential in reducing LID in PD. There is a dearth of research examining the potential effects of phytochemicals on dyskinesia through 5-HT₃R modulation.

Conclusion and Future Perspectives

Exploring natural approaches for treating neurological disorders, particularly by targeting 5-HT₃R modulation through phytochemicals, holds significant promise for advancing our understanding of and managing these complex conditions. Phytochemicals, owing to their safety and cost-effectiveness and, additionally, higher lipophilic balance, readily traverse the BBB, influencing 5-HT₃R and impacting vital neurotransmitter systems such as GABA, α7-nAChR, NK-R, 5-HT_1A, and D₂. This broad influence implicates them in alleviating a spectrum of symptoms ranging from nausea, vomiting, and cognitive impairment to conditions such as AD, sleep disorders, depression, anxiety, and epilepsy. The antagonistic action of phytochemicals on 5-HT₃R plays a pivotal role in regulating calcium release, inhibiting SP action, and mitigating inflammatory responses and oxidative stress. Consequently, these mechanisms contribute to a reduction in mitochondrial damage and caspase-3 activity, offering potential avenues for therapeutic intervention. Many constituents, such as gingerol, galanolactone, citronellol, geraniol, CBD, THC, eugenol, and vanillin, are reported to inhibit nausea and vomiting due to their 5-HT₃R antagonism. Similarly, zerumbone, resveratrol, carvacrol, gallic acid, genistein, resveratrol, and alpha-lipoic acid are found to be neuroprotective.

However, despite the promising implications, the current body of evidence exploring the effects of phytochemicals on 5-HT₃R is limited. Urgent attention is warranted for comprehensive preclinical and clinical studies to deepen our understanding of the therapeutic benefits these compounds may offer. The challenges of identifying optimal dosages, potential side effects associated with these molecules, and the development of standardized extracts may hamper the translation of these molecules into effective therapeutic strategies. Also, safety pharmacology studies and toxicity testing of these phytoconstituents are needed. Such investigations can shed light on their potential efficacy in treating not only common symptoms like nausea and cognitive impairment but also addressing more complex disorders like AD, dyskinesia, and epilepsy. Future perspectives should elucidate the specific molecular pathways and signaling cascades involved in the interaction between phytochemicals and 5-HT₃R.

Glossary

Abbreviations

5-HT_3AR

5-Hydroxytryptamine receptor 3A

5-HT

5-Hydroxytryptophan

ALA

Alpha-lipoic acid

AChEIs

Cholinesterase inhibitors

α7-nAChR

α7-nicotinic acetylcholine receptor

AD

Alzheimer’s disease

B-J

Bezold-Jarisch

CBD

Cannabidiol

CNS

Central nervous system

CINV

Chemotherapy-induced nausea vomiting

CA1

Cornu Ammonis 1

CA2

Cornu Ammonis 2

dEMG

Digastric muscle electromyograms

cGMP

Cyclic guanosine 3′,5′-monophosphate

FDA

The Food and Drug Administration

GABA

γ-Aminobutyric acid

GA

Gallic acid

GLE

Ganoderma lucidum extract

GIT

Gastrointestinal tract

FST

Force swim test

KATP

ATP-sensitive potassium channels

LID

Levodopa induced dyskinesia

m-CPBG

Meta-chlorophenyl-biguanide

NKR

Neurokinin-2 receptor

NOS

Nitric oxide synthase

NO

Nitric oxide

NMDA

N-Methyl-d-aspartate

NA7

Neuronal acetylcholine receptor subunit alpha 7

OFT

Open field test

PD

Parkinson disease

PET

Positron emission tomography

PNS

Peripheral nervous system

PTZ

Pentylenetetrazole

SP

Substance P

RO

Rosmarinus officinalis

TPEO

Thymus persicus aerial parts essential oil

THC

Tetrahydrocannabinol

TST

Tail suspension test

Author Contributions

Likhit Akotkar: Data curation, formal analysis, investigation, writing-original draft; Urmila Aswar: Conceptualization, validation, writing-review and editing and supervision; Ankit Ganeshpurkar: Data curation, formal analysis, reviewing, editing, drawing, and drafting of MS; Kundlik Rathod: Compilation of data, writing-original draft; Pradnya Bagad: Data curation, formal analysis, editing, drawing, and drafting of manuscript; Shailendra Gurav: Conceptualization and design of the study, validation, writing-review and editing.

The authors declare no competing financial interest.