A review of frequently used Kampo prescriptions. Part 3. Yokukansan

The source of yokukansan (YKS) is thought to be Baoying‐Jinjiang (Hoeikinkyōroku in Japanese) by Xue Ji. YKS was originally designed for children and indicated for spasms, clenching of teeth, digestive dysfunction due to mental instability, and insomnia.


HISTORY OF YOKUKANSAN
The original source of yokukansan (YKS) is Xue Ji's Bao Ying Jin Jing Lu, but the best-known description is found in Bao Ying Cuo Yao [1,2].This formula was originally designed for children and is indicated for spasms, clenching of teeth, digestive dysfunction due to mental instability, and insomnia.Furthermore, simultaneous administration of YKS to both a child and mother is recommended.In the Edo period (1603-1867), with the stabilization of society, YKS was widely used for neuropsychiatric disorders in children according to original texts.Gradually, its use has been extended to adults and widely applied to neuropsychiatric disorders with an emphasis on muscle tension, mental agitation, and irascibility.Historically, YKS has attracted more attention in Japan than it has in China.Currently, YKS is widely used in children and adults.Yokukansankachimpihange (YKSCH) is developed in Japan, adding chimpi (Citrus peel) and hange (Pinellia tuber) to YKS.YKSCH has also been used as an independent formula.

Origin of YKS
The source of YKS is considered to be Bao Ying Jin Jing Lu (1550) by Xue Ji [1].The most well-known description is mentioned in Bao Ying Cuo Yao (1556), which described his and his father's experience [2].Both books were pediatric textbooks.The descriptions in other following books are almost identical to the original ones.Since YKS is not mentioned in any internal medicine, surgery, or gynecology book written by Xue Ji, it is clear that YKS was initially created as a prescription for children.
The description of YKS in Bao Ying Cuo Yao is as follows: "YKS is indicated for spasms due to deficiency heat in the liver meridian, teeth grinding due to phlegm heat, or palpitations due to fright, and also vomiting, drooling, abdominal distention, anorexia, and sleep anxiety due to mental instability (Figure 1)".After the description of the seven component crude drugs (Table 1), a characteristic administration method is mentioned, "The decoction is taken together by a child and his or her mother.If it is made into a rounded formulation with an excipient of honey, it is renamed as 'Yi-qing wan.'" From a modern medical standpoint, YKS might be indicated for febrile convulsion, breath-holding spells, epilepsy, night terrors, and other central nervous system (CNS)-related muscle tension conditions, with the expectation of antispasmodic and sedative effects.The formula was designated as a powder or decoction in water or as a pill, presumably due to the ability of children to ingest the drug and the fact that the main ingredient in Uncaria hook is volatile.The unique method by which mothers and children take their medications simultaneously will be described later.

Subsequent influence on pediatric Kampo medicine
A search for references to YKS in Chinese classical pediatric textbooks from the Qing dynasty after Xue Ji found few descriptions, suggesting that YKS has received little attention in China.Table 2 shows the information on YKS in representative pediatric medical textbooks in Japan.Karei-syoni-hou, the first pediatric book in Japan, did not mention YKS because of the strong influence of Li Zhu's medicine [3].During the Edo period, with society's stabilization, medical books from the Ming dynasty were imported, and the development of the letterpress and printing technology led to the publication of pediatric books [4].
Yoyo-kasoku [9] published in 1885, the early Meiji period, while respecting the original texts, expanded the range of applications to include growth fever, eye malnutrition, night blindness, and fetal toxin eye, as well as stuttering and slow speech.However, pediatric books during this period focused on treating acute and severe organic diseases, especially infectious diseases, and there was little mention of psychosomatic conditions in children.However, in recent years this formula is frequently used for neurodevelopmental, sleep, and adjustment disorders.Although the social background is largely reflected in the use of YKS in children, the concepts of the original texts are utilized.

Adult application of YKS
YKS was initially devised for children with seizures and catatonia; however, its applicability was extended to adults during the Edo period (Table 3).Fukui Futei (1725-1792) described its use for epilepsy in adults and children with deficiency [10].During the 19th century, descriptions of its use increased to include paralysis and other nervous disorders that caused muscle tonus, and its use was extended to mental symptoms such as overexcitement.Wada Tokaku (1743-1803) added peony root to the formula to treat irascibility, insomnia, and impatience [11].Meguro Dotaku (1739-1798) noted its efficacy for paraplegia and insomnia with upper abdominal blockage and anger [12].In addition, Asada Sohaku (1815-1894) emphasized the presence of anger and muscle cramps in the extremities [13].These symptoms, particularly the presence of irascibility, are still considered essential targets for the use of this formula.In addition, Seiken-shu, published just before the government policy of repressing Kampo medicine, contained various adult cases of this formula.Interestingly, this formulation is helpful for severe fatigue caused by persistent mental stress [14].We discuss the modern usage of YKS in a further section.

YKS-based formulations with additional crude drugs
Various crude drugs have been added to YKS, including anthelmintics, chrysanthemum flowers, fine spices, peony roots, yellow lotus roots, and gardenia.The most famous example is YKSCH, which is composed of YKS and Chimpi (Citrus peel, Chenbin) and Hange (Pinellia tuber, Hanxia), and is recognized as an independent formulation and is widely used currently.Its origin is unknown, but it is thought to have been developed by an experienced practitioner of this formula from the Edo period.Asai Nanmei (1734-1781) described the abdominal manifestation of this formula as palpitations from the left side of the navel to the epigastric region (Figure 2) [15].

Simultaneous dosing for a child and mother
As mentioned above, Xue Ji recommended that both a child and its mother take this formula simultaneously in Bao Ying Cuo Yao.This may be because infants have difficulty taking the drug and the drug could be transferred to the child through breast milk.The same idea was also proposed by Hoyogei [16].At the same time, Xue Ji recommended the usage of kamishoyosan for infant agitation and uncontrolled laughter, and for anger of a mother.This use is intended to calm both child and mother simultaneously, and this idea has influenced modern Kampo medicine's simultaneous therapy of mother and child for childcare anxiety.

Name of the book (year of publication) Author Indications considered important References
Hodoku-benkai (unknown) Fukui Futei (1725-1792) Epilepsy with deficiency pattern [10] Sousyo-hoi-kai (1813) Wada Tokaku Irascibility, Insomnia, Impatience [11] Saneikan-ryochi-zatsuwa (1847) Meguro Dotaku Hemiplegia, insomnia [12] Hutugo-yakusitu-hokan-kuketu (1878) Asada Sohaku Irascibility [13] Seiden-shu (1901) Yamada Narihiro Severe fatigue caused by persistent mental stress [14] extracts were classified as Kampo extract formulations for a medical prescription (ethical Kampo formulation) and over-the-counter (OTC) Kampo extract formulations for self-medication.The decoction includes the formulation by doctors' prescriptions and pharmacists dispensing with doctors' prescriptions (in-pharmacy formulation).These forms of YKS were approved by JP XVIII, the Guide Book of the Approval Standard for OTC Kampo Products (Revised Edition), and the sixth edition of the Guidelines for Pharmacy Preparation.The original formulation of YKS is a decoction prepared by boiling a mixture of crude drugs in water at room temperature.Boiling the mixture without the Uncaria hook under low heating in 500-600 mL of water reduced the volume to half.Before boiling, the Uncaria hook is usually boiled for a few minutes.After removing the crude drug residue, the extraction fluid was divided into two or three doses per day before or between meals.
Currently, YKS extract formulations are mainly distributed in Japan.The amount of crude drug in each formulation is shown in Table 4. Two pharmaceutical companies manufacture Kampo extract formulations for the medical prescription of YKSCH.The usual dose for adult patients consists of two or three divided doses for oral administration before or between meals.The dosage forms include granules, fine granules, and tablets.The OTC formulations of YKS usually contain smaller amounts of extract than those used in medical prescriptions.

Constituents
A high-performance liquid chromatography profile of YKS extract (Tsumura & Co, Tokyo, Japan) demonstrated various detectable constituents from eight crude drugs (Figure 3).The JP XVIII defined the amount of major constituents in YKS extract as follows: not less than 0.15 mg of total alkaloids (rhyncophylline and hirsutine), not less than 0.6 mg and not more than 2.4 mg of saikosaponin b 2 , and not less than 10 mg and not more than 30 mg of glycyrrhizic acid (GL), per extract prepared with the amount specified in the method of preparation [17].

Cnidium rhizome
Cnidium rhizome is the rhizome of Cnidium officinale Makino (Umbelliferae), usually passed through hot The high-performance liquid chromatography fingerprint of yokukansan extract formulation.

Bupleurum root
The Bupleurum root is the root of Bupleurum falcatum Linné (Umbelliferae).It contains not less than 0.35% of the total saponin (saikosaponin a and saikosaponin d), calculated based on dried material [17].More than 100 types of saikosaponins have been found only in Bupleurum plants and have multiple pharmacological activities, including antiinflammatory, immunomodulatory, antiviral, antipyretic, antitumor, sedative, anticonvulsant, and liver-and kidney-protective activities [39,40].

Pharmacokinetics of the components in YKS
Previous studies have investigated the pharmacokinetic properties of YKS.A pharmacokinetic study of healthy volunteers with YKS extract administration revealed a dose-dependent increase in the maximum plasma concentration (C max ) of GM and hirsuteine derived from the Uncaria hook.The time to maximum plasma concentration (t max ) of these alkaloids was within 1 h of oral administration, and the apparent elimination half-life (t 1/2 ) was approximately 2-3 h.In contrast, the t max of GA is approximately 8 h and t 1/2 is 9-12 h [43].
An experimental study in mice with orally administered YKS extract showed that GA absorption is almost completely inhibited upon gut microbiome ablation with ampicillin or vancomycin treatment.These results indicate that the use of some antibiotics and gut bacterial alternation may decrease GA absorption in YKS [44].
Several randomized clinical trials (RCTs) have demonstrated the efficacy of YKS for BPSD, as assessed using the Neuropsychiatric Inventory (NPI).However, individual clinical trials have not obtained consistent results regarding its effects on cognitive function, as  In an open-labeled RCT by Iwasaki et al. [57], which marked the beginning of clinical trials in this area, 52 patients (mean age ± standard deviation, 80.3 ± 9.0 years) with Alzheimer's disease, vascular dementia, and dementia with Lewy bodies diagnosed with an MMSE score less than 24 and BPSD with an NPI score higher than 6 were treated with YKS.Twentyseven patients were assigned to the YKS treatment group (7.5 g/day) and 25 to the control group (without Kampo medicine), using a random number table, and were followed for up for four weeks.If the effect was inadequate after one week of treatment, thiapride hydrochloride (25 mg/day) was administered.The NPI and BI were significantly improved in the YKS treatment group.Among the NPI subscales, significant improvement was noted in delirium, agitation, irritability, and abnormal behavior.Thiapride hydrochloride was prescribed to 11 patients in the control group, but not in the YKS treatment group.Six of 11 patients experienced dizziness and lightheadedness as adverse reactions.During the trial, two patients in the YKS treatment group experienced hypersedation, and the dose of YKS was reduced to 5.0 g/day.In one patient, the NPI, which decreased after YKS treatment, increased again one month after the YKS dose reduction.
In an open-label RCT crossover by Mizukami et al. [58], 106 patients with AD or dementia with Lewy bodies were randomly assigned to two groups and treated with YKS (7.5 mg/day) for four weeks, followed by a four-week discontinuation period, or in a reversed order.Symptoms were assessed using NPI, instrumental ADL (IADL), and MMSE scores.The intentionto-treat (ITT) analysis was performed on 57 outpatients (mean age, 78.7 ± 5.4 years) and 46 inpatients (mean age, 78.5 ± 6.7 years).The NPI score significantly improved in the YKS group; however, no changes were observed in the MMSE or IADL.Serious adverse effects were also not observed.
In an open-label RCT by Monji et al. [59], 15 patients (mean age, 80.2 ± 4.0 years) diagnosed with AD were randomly assigned to two groups, with 10 patients receiving YKS (7.5 mg/day), and five followed up for 12 weeks without Kampo medicine.Blinding was not performed.Both groups were treated with 50 mg/day of sulpiride with dosage adjustment according to the inhouse criteria.The YKS group showed a significant improvement in the total NPI score after 12 weeks compared to that before treatment.MMSE and BI scores did not change significantly.The YKS group tended to receive lower doses of sulpiride than the control group.Two patients in the YKS group had hypokalemia and one had to reduce the dose of sulpiride owing to extrapyramidal symptoms.One patient in the control group was excluded from the study because of acute edema.In an open-label RCT by Okahara et al. [60], 61 patients diagnosed with AD, who had an NPI score of 4 or more and had been taking donepezil hydrochloride for at least four weeks, were randomly assigned to two groups.A total of 30 patients (mean age, 76.1 ± 8.1 years) were treated with YKS (7.5 g/day), and 33 (mean age, 77.1 ± 6.8 years) were followed up for four weeks without additional medication.An ITT analysis was conducted, and the YKS group showed significant improvement in the total NPI score at four weeks compared with the control group, specifically in the two NPI subscales of excitement and irritability.The MMSE, DAD, Zarit Burden Interview, and self-rating depression scale scores were unchanged in betweenand within-group comparisons.None of the patients developed hypokalemia and no serious adverse effects were observed.
The effects of YKS on BPSD and ADL were demonstrated in the aforementioned RCTs and metaanalyses; however, an important limitation was that all RCTs were performed in an open-label manner.In 2015, Furukawa et al. conducted the first placebocontrolled double-blinded (DB)-RCT [62], in which 145 patients with AD were divided into 75 (mean age, 78.3 ± 5.4 years) and 70 (mean age, 78.5 ± 5.1 years) patients in the YKS and placebo groups, respectively, for 12 weeks.The NPI-Brief Questionnaire Form (NPI-Q) showed no significant differences between the two groups at four and 12 weeks.This is presumably due to the use of the NPI-Q score, which is simpler than that used in previous RCTs, the lower baseline NPI-Q score than that used in previous RCTs, and the fact that the placebo group also showed a significant improvement in BPSD.In the subgroup analysis, there was a significant improvement in patients with high irritability and delirium subscale scores in the YKS group, which is consistent with the results of a previous metaanalysis [61].
In an observer-single-blinded RCT by Teranishi et al. [63], the efficacy of YKS was compared headto-head with that of two active controls, risperidone and fluvoxamine.A total of 82 patients diagnosed with dementia according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria were randomly allocated to three groups: 27 patients (mean age, 83.5 ± 5.8 years) to the YKS group, 27 (mean age, 80.7 ± 8.8 years) to the risperidone group, and 28 (mean age, 83.2 ± 5.4 years) to the fluvoxamine group, and treated for eight weeks.An ITT analysis was performed, in which the total NPI scores were found to be significantly improved in all three groups compared to baseline values; however, there were no significant differences between the groups.MMSE and functional independence measures did not show significant differences between and within the groups.The extrapyramidal symptom score was significantly worse in the risperidone group.
In addition to dementia, YKS has been used to treat a variety of neuropsychiatric disorders.Most of them were single-arm case series; however, we found two RCTs and discuss them here.
In a DB-RCT by Miyaoka et al. [64], 120 inpatients with refractory schizophrenia were treated with YKS or a placebo for 4 weeks, and their symptom scores were evaluated using the Positive and Negative Symptom Scale.There was a trend toward lower positive and general scores in the YKS group; however, no significant difference was observed in the per-protocol or ITT analyses.However, in the per-protocol analysis, the YKS group showed significant improvements in conversational fluency, tension, and impulse control, among the subscale items.
In a DB-RCT by Huang et al. [65], 38 children and adolescents with Tourette syndrome were randomly allocated to the YKS group that received 5 g/day of YKS extract for four weeks or the control group that received a placebo.The Yale Global Tic Severity Scale scores were compared at weekly intervals after administration; however, a significant difference was found only in the first week after administration.
Starting with the RCT by Arai et al. [66], several RCTs have examined the effects of YKS on perioperative sedation, anxiety, and delirium prevention; however, they have yet to demonstrate constant efficacy.
An observer-single-blinded RCT by Arai et al.
[66] compared YKS with diazepam for preoperative anxiety.At 1.5 hours preoperatively, 34 patients in the YKS group (median age, 63, range 30-85 years) received 2.5 g/day of YKS extract, and 36 patients in the control group (median age, 66, range 35-85 years) received 5 mg of diazepam.The YKS group was significantly more sedated than the diazepam group, based on the modified Observer's Assessment of Alertness/Sedation Scale administered upon entering the operating room.There were no differences in the degree of anxiety or salivary α-amylase levels between groups.
An observer-single-blinded RCT by Tanaka et al. [67] examined the effects of YKS on preoperative anxiety and postoperative pain in breast cancer.A total of 35 patients (mean age, 49 ± 6.2 years) were allocated to the YKS group, which received 2.5 g YKS extract the night before surgery and before anesthesia; 42 patients (mean age, 48 ± 5.7 years) were assigned to the control group.According to the authors, The Hospital Anxiety and Depression Scale (HADS)-A values were better in the YKS group; however, no group differences were observed.Salivary α-amylase levels immediately before surgery in the YKS group were lower than the α-amylase levels the night before surgery and lower than the α-amylase levels in the control group at the same time.
The DB-RCT by Wada et al. [68] examined the effects of YKS with more dosages of YKS on preoperative anxiety and the frequency of postoperative delirium in patients with cancer.The YKS group (n = 84, mean age, 64.4 ± 11.9 years) received 7.5 g/day of extract for 4-8 days before surgery, whereas the control group (n = 76, mean age, 61.7 ± 13.5 years) received a placebo for the same period.Patients were enrolled and assigned in a 1:1 ratio.HADS-A before and after surgery and the frequency of postoperative delirium diagnosed according to the DSM-V criteria in the first five days after surgery were compared; however, no significant differences were found for either outcome.

Mechanisms of action
As described in the previous sections, recent clinical studies of YKS have drawn attention to its beneficial effects on adult psychiatric abnormalities, including BPSD and perioperative psychiatric symptoms; in particular, the effects of YKS are most remarkable against "positive symptoms" in these patients, that is, psychiatric symptoms, including delusions, hallucinations, agitation/aggression, anxiety, and irritability/lability.The mechanisms underlying the effects of YKS on these positive symptoms have been extensively examined in cellular, animal, and pharmacokinetic experiments.As reviewed previously [69][70][71][72], it has been revealed that YKS modulates many neuronal pathways including serotonergic, glutamatergic, GABAergic, and other neurotransmitter pathways in CNS.The most wellcharacterized targets of YKS in treating positive psychiatric symptoms are the serotonergic and glutamatergic pathways.Therefore, this section briefly summarizes previous and up-to-date findings regarding the mechanisms of action of YKS on these two neural pathways, as well as the relevant pharmacokinetic findings.The overview of the effects of YKS on serotonergic and glutamatergic pathways in CNS is shown in Figure 4.

Serotonergic pathways
Along with cognitive deficits, which are core symptoms of dementia, BPSD significantly decreases the quality of life in both patients with dementia and their caregivers.Although the pathogenic mechanism of BPSD is complicated and poorly understood, alteration of 5-hydroxytriptamine (5-HT, or serotonin) neurotransmitter and neuromodulator systems in brains with dementia is involved [73][74][75].To investigate the relationship between the effects of YKS and serotonergic pathways, researchers conducted several in-vitro assays and pharmacological experiments using animal models.In animals, Kanno et al. demonstrated that the aggressive behavior of rats injected with parachloroamphetamine (PCA), which causes depletion of cerebral 5-HT, was ameliorated by buspirone, a 5-HT 1A agonist, and ketanserin, a 5-HT 2A antagonist, whereas it was exacerbated by 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT 2A agonist [76], suggesting that aggression in this animal model is regulated by these 5-HT receptors.In this model, repeated treatment with YKS for 14 days decreased the PCA-induced aggressive behavior.In addition, the co-administration of WAY-100635, an antagonist of 5-HT 1A receptors, suppressed the ameliorative effect of YKS on aggression, indicating that the antiaggressive effect of YKS is mediated by 5-HT 1A agonism.Terawaki et al. used in-vitro experiments to demonstrate that YKS binds to 5-HT 1A , but not to 5-HT 2A receptors, and exerts partial agonistic activity on 5-HT 1A receptors [77].Notably, similar to YKS, Uncaria hook, one of the seven constituent drugs of YKS, showed partial 5-HT 1A agonistic activity when administered alone, whereas the other constituent drugs did not.Furthermore, deprivation of Uncaria hook from the YKS formula resulted in attenuation of the partial agonistic activity of 5-HT 1A receptors.These results indicate the presence of active ingredients in Uncaria hook that act on the 5-HT 1A receptors.To identify the active ingredients in Uncaria hook, Nishi et al. examined the binding and agonistic activity of the 5-HT 1A receptors of the main alkaloids contained in Uncaria hook [78].Among the seven compounds tested, only GM was found to potently bind to 5-HT 1A receptors and act as a partial agonist.The authors further evaluated the effects of YKS, Uncaria hook, and GM in socially isolated mice, which is another mouse model of aggression.They demonstrated that these drugs attenuated the aggressive behavior in these mice and that this effect was blocked by co-administration with WAY-100635, indicating the involvement of 5-HT 1A receptors.More recently, YKS was shown to inhibit aggressive behavior in mice that underwent cholinergic degeneration in the nucleus basalis of Meynert, which presumably contributes to the pathogenesis of cognitive deficits and emotional disturbances in dementia patients [79].The antiaggressive effect of YKS is also mediated by 5-HT 1A receptors.In addition to its antiaggressive effect, YKS exerted an anxiolytic-like effect in the contextual fear-conditioning test, which was reversed by WAY-100635 [80].Notably, the anxiolytic-like effect of YKS observed in the elevated plus maze test was not suppressed by WAY-100635, suggesting that YKS produces an anxiolytic effect via 5-HT 1A receptors against memory-dependent fear.In terms of the binding of YKS  F]MPPF), a radioligand for 5-HT 1A receptors, and demonstrated the 5-HT 1A receptor occupancy induced by oral administration of YKS to be 10%-17%, indicating that YKS affects 5-HT 1A receptors in vivo [81].These results suggest that YKS suppresses aggressive behavior and some kinds of anxiety through its partial agonistic activity on 5-HT receptors and that these effects are attributable to GM, an ingredient in Uncaria hook.In addition to its partial agonistic effect on 5-HT1A receptors, YKS also affects the density and affinity of 5-HT 1A receptors.Ueki et al. reported that chronic intake of YKS-containing food for six weeks significantly increased 5-HT 1A receptor density and decreased its affinity in the prefrontal cortex (PFC) without affecting its mRNA or protein expression levels [82].At the behavioral level, YKS treatment enhanced the 5-HT 1A agonist-induced anxiolytic effect, indicating that YKS potentiates 5-HT 1A signaling by increasing its number, although the underlying mechanism and active ingredients remain to be identified.Taken together, YKS exerted anti-aggressive effects through partial agonistic action on 5-HT 1A receptors, and this effect is attributed to GM, an ingredient in Uncaria hook.In addition, YKS can attenuate anxiety-like behavior by increasing 5-HT 1A receptor signaling due to 5-HT 1A agonistic activity and/or potentiation of receptor density in the PFC.
Moreover, the 5-HT 2A receptor is also involved in the psychiatric effects of YKS, despite the absence of a direct action of YKS on this receptor [77].Egashira et al. reported that injection of DOI, a psychedelic 5-HT 2A agonist, induced a head-twitch response in mice that was inhibited by repeated treatment with YKS for 14 days [83].Additionally, the protein expression of 5-HT 2A receptors decreased in the prefrontal cortex (PFC) following a two-week treatment with YKS, suggesting that the effect of YKS on the head-twitch response is mediated by a decrease in 5-HT 2A receptors.Similar results were obtained by Ueki et al. [84], who showed that social isolation stress increased the DOI-induced head-twitch response, indicative of hallucinations in mice, and a six-week intake of YKS-containing food counteracted this increase.Furthermore, social isolation increased the density of 5-HT 2A receptors in the PFC, and YKS treatment downregulated this increase without affecting the affinity or expression level of 5-HT 2A receptors.It should also be noted that each of the seven constituent drugs failed to downregulate 5-HT 2A receptor density when administered alone, indicating that the additive or synergistic effects of these drugs are required for YKS to exert its down-regulatory action.More recently, repeated treatment with YKS for six days was reported to enhance the anxiolytic-like effect of fluvoxamine, a selective serotonin reuptake inhibitor, in the contextual fearconditioning paradigm in mice, with decreased 5-HT 2A receptor expression in the PFC [85].
Nogami-Hara et al. also demonstrated that YKS reduced anxiety-like behavior and DOI-induced abnormal behavior in a rat model of cerebral ischemia combined with intracerebroventricular injection of amyloid-beta peptides [86].This anxiolytic-like effect of YKS was blocked by DOI, indicating that 5-HT 2A antagonism by YKS reduces anxiety.Furthermore, an antiaggressive effect of YKS was also shown to be involved with the altered 5-HT 2A receptor expression; a two-week treatment with YKS decreased aggression in socially isolated mice and it concomitantly suppressed the increase in 5-HT 2A receptors in the amygdala [87].These results suggest that YKS exerts ameliorative effects against hallucinations, anxiety, and aggression by decreasing the expression levels or density of 5-HT 2A receptors in some parts of the brain, including the PFC and amygdala.Although the mechanism underlying the decrease in 5-HT 2A receptors induced by YKS is unclear, it could be orchestrated by multiple constituent drugs in YKS.In addition to the reduction in 5-HT 2A receptors, Iba et al. showed a decline in 5-HT 3A receptor expression in the amygdala when YKS reduced aggressive behavior in socially isolated mice [87].Recent studies have suggested that the 5-HT 3 receptor, the sole ion-channel receptor among the 5-HT receptor subtypes, can be a promising therapeutic target for treating drug abuse and addiction, as well as anxiety, depression, and schizophrenia [88].YKS reduced ethanol preference in mice, but this reduction was not observed in 5-HT 3 receptor-deficient mice [89].In addition, the co-application of several alkaloids contained in Uncaria hook strongly inhibited 5-HT 3 receptor currents, although each applied alone exhibited only a weak inhibitory effect [90].These findings suggest the possible involvement of 5-HT 3 signaling inhibition in the effects of YKS against aggression and addiction.
A summary of the abovementioned basic studies examining the effects of YKS on serotonergic pathways is provided in Table 6.Collectively, the primary target receptors in the serotonergic system that are involved in the beneficial effects of YKS on BPSD-like behaviors are 5-HT 1A and 5-HT 2A receptors.YKS potentiates 5-HT 1A receptor signaling, which is primarily due to the partial agonistic effect of GM, an ingredient of Uncaria hook, on 5-HT 1A receptors.Meanwhile, YKS suppresses 5-HT 2A receptor signaling by suppressing its expression.The underlying mechanism cannot be explained by a single molecule or pathway but would be formed by the combinatorial effect of multiple drugs in YKS.In addition, reports on 5-HT 3 receptors have been increasing in recent years, and further research would clarify the characteristics of YKS's action on these receptors and their contribution to the overall effects of YKS.

Glutamatergic pathways
Excitotoxicity through glutamate receptors and changes in glutamatergic neurotransmission have been proposed to contribute to the pathological processes of many neurodegenerative disorders, including AD [91][92][93].Moreover, altered glutamate neurotransmission in patients with dementia is involved in the pathogenesis of BPSD, and its pharmacological modulation can be a promising therapeutic strategy for BPSD, as exemplified by the beneficial effects of memantine, a partial antagonist of N-methyl-D-aspartate (NMDA) receptors [94,95].To clarify the effects of YKS on glutamatergic neurotransmission, the action of YKS was examined in rats fed a zinc-deficient diet for four weeks, a neurological disease model with dysregulated glutamatergic neurotransmitter system [96].In this model, a 10-day treatment with YKS significantly suppressed the abnormal increase in extracellular concentrations of glutamate in the hippocampus after stimulation with 100 mM KCl, suggesting modulation of the glutamatergic neurotransmitter systems by YKS.In the same animal model, Takeda et al. further demonstrated that the suppressive effect of YKS on the excessive release of glutamate was coincident with a decline in increased exocytosis in mossy fiber boutons, suggesting that YKS decreases excessive increases in glutamate release [97].Concomitantly with the decrease in excessive glutamate release, YKS attenuated the zinc deficiency-induced increase in aggressive behavior in socially isolated mice [98], similar to MK-801, an NMDA receptor antagonist [99].Tamano et al. showed that GM and GA reduced excessive exocytosis in mossy fiber boutons in hippocampal slices from zinc-deficient rats, suggesting that these compounds may be responsible for the suppressive effect of excessive glutamate release [100].
Similar inhibitory effects of YKS on extracellular glutamate rise were confirmed in rats fed a thiaminedeficient diet, another animal model for an aberrant increase in extracellular glutamate, which also expresses cognitive deficits and BPSD-like abnormal  [101,102].Furthermore, histopathological analyses revealed that administration of YKS also ameliorated thiamine-deficiency-induced degeneration of neurons, astrocytes, oligodendroglia, and myelin sheaths [101,102].Notably, behavioral changes preceded neurological symptoms in thiamine-deficient rats.Furthermore, considering that the degeneration of astrocytes was most prominent compared to that of other brain cells at an earlier stage where neurological symptoms were absent, the effects of YKS on astrocytes may be closely related to its beneficial effects on BPSD-like behavior in thiamine-deficient rats.
Astrocytes play critical roles in the metabolism of synaptic and perisynaptic glutamate through two glutamate transporters, glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1), and the expression and/or function of these glutamate transporters is altered in many neurodegenerative disorders.Notably, the expression and function of these transporters are decreased in AD brains, leading to reduced uptake of extracellular glutamate [103,104].Considering the beneficial effects of YKS on astrocyte degeneration and excess extracellular glutamate [101,102], YKS was speculated to affect the expression and/or function of glutamate transporters in astrocytes.Indeed, Kawakami et al. demonstrated that YKS dose-dependently restored the reduced glutamate uptake ability in rat astrocytes cultured in thiamine-deficient conditions [105][106][107] and that this effect was attributed to an increase in mRNA and protein levels of GLAST, which were reduced by thiamine deficiency [107].The authors further identified Glycyrrhiza as the only constituent drug that significantly recovered the glutamate uptake ability among the seven drugs of YKS [106].Moreover, GL and GA ameliorated the thiaminedeficiency-induced decrease in glutamate uptake among the eight ingredients in Glycyrrhiza.Another finding of note was that these compounds inhibited protein kinase C (PKC) [106].In addition to GLAST, YKS also increased the expression of GLT-1, although glutamate uptake activity in astrocytes cultured in this thiamine-deficient condition was mediated predominantly through GLAST [105].As to the GLT-1-mediated transport of glutamate, Ueki et al. cultured astrocytes in the presence of tumor necrosis factor-α (TNF-α) and dibutyryl-cyclic adenosine monophosphate (dBcAMP) to enhance GLT-1 expression, and found that, in this condition, YKS significantly augmented glutamate transport through GLT-1 by increasing its expression [108], indicating that YKS can potentiate the expression of both GLAST and GLT-1.Although in vivo evidence on the effects of YKS on glutamate transporters are not adequate to date, it has been shown that YKS inhibits a chronic stress-induced decrease in excitatory amino acid transporter 2 (EAAT2) (alias GLT-1) in the hippocampus of stressed mice [109].
Thus, YKS is thought to alleviate the excess extracellular glutamate in two modalities, that is, inhibition of glutamate release and potentiation of glutamate uptake by astrocytes.The former may be mediated by GM and GA, although the receptors for these molecules underlying this effect are unknown.In contrast, the latter is attributable to the upregulation of glutamate transporters by Glycyrrhiza, GL, and GA.
In addition to the modulatory effects on extracellular glutamate concentration, some reports have suggested that the neuroprotective effects of YKS occur via direct action on neurons.Kawakami et al. showed that YKS inhibited glutamate-induced death of PC12 cells in a dose-dependent manner and that it bound to glutamate-recognition and glycine-recognition sites of NMDA receptors, which mediate excitotoxicity [105].In addition, the authors further demonstrated that YKS protects against glutamate-induced cell death in rat cortical neurons and that Glycyrrhiza, Uncaria hook, and Japanese Angelica root exert this neuroprotective activity [110].Moreover, isoliquiritigenin, an ingredient in Glycyrrhiza, exerted an antagonistic effect on NMDA receptors and exhibited a neuroprotective effect similar to YKS, although it remains to be determined whether the neuroprotective effects of YKS and isoliquiritigenin are caused by its antagonistic effect against NMDA receptors [110].In addition to its effects on NMDA receptors, Kanno et al. showed a cell-protective effect through augmentation of the cystine/glutamate antiporter system Xc À , of which inhibition by excessive extracellular glutamate causes intracellular deficiency of glutathione and consequent cell death [111].They showed the cell-protective effect of YKS on PC12 cells cultured in an excessive glutamate condition, along with an increase in the expression of the system Xc À subunits and concomitant recovery of the decreased glutathione levels.Moreover, experiments to clarify the active compounds revealed that GM, hirsuteine, hirsutine, and procyanidin B 1 in Uncaria hook had cell-protective effects and that these ingredients upregulated the system Xc À subunits and ameliorated glutathione levels, indicating their involvement in the cytoprotective effects of YKS.Taken together, in addition to the regulation of glutamate content, YKS could exert cytoprotective effects by affecting the NMDA receptor and cystine/glutamate antiporter.The aforementioned non-clinical reports that show the beneficial effects of YKS on glutamatergic pathways are summarized in Table 7.

Pharmacokinetic findings
YKS contains a myriad of compounds, both known and unknown, among which GM and GA, as aforementioned, play important roles in the effects of YKS in modulating the serotonergic and glutamatergic pathways.Several pharmacokinetic studies on YKS have evaluated the absorption of these compounds into blood circulation and their subsequent transferability into the brain (summarized in Table 8).The concentration-time curve of plasm GM measured with liquid chromatography with tandem mass spectrometry after a single administration of YKS (4.0 g/kg) in male rats was shown by Kushida et al., where the pharmacokinetic parameters were calculated to be as follows: AUC 0-∞ : 6.79 ± 1.31 (ngh/mL); C max : 1.98 ± 0.43 (ng/mL); t max : 0.67 ± 0.29 (h); t 1/2 : 1.6 ± 0.5 (h) [112].In addition, the pattern of the concentration-time curve of brain GM showed a similar shape to that of the plasma, with the following pharmacokinetic parameters: AUC 0-∞ : 2.90 ± 0.65 (ngh/g); C max : 1.18 ± 0.32 (ng/g); t max : 0.50 ± 0.00 (h); t 1/2 : 3.4 ± 2.0 (h).Furthermore, in an in-vitro model of the blood-brain barrier (BBB) composed of a co-culture of endothelial cells, pericytes, and astrocytes, GM was able to cross the BBB with a permeability rate of 27.3 ± 0.2 (%) [113].In-vitro autoradiographic binding assays with tritium-labeled GM revealed that specific binding sites for GM include the frontal cortex, hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus, and cerebellum.The binding density was higher in the frontal cortex, moderate in the dorsal raphe nucleus, and lower in the cerebellum [114].Furthermore, GM was suggested to bind to 5-HT receptors, such as 5-HT 1A , 5-HT 2A , 5-HT 2B , 5-HT 2C , or 5-HT 7 , and to α 2A and μ-opioid receptors from the findings obtained using competitive ligands for the respective receptors [114].More recently, Matsumoto et al. reported that, with the mass spectrometry imaging technique, GM diffusely distributes throughout the brain, including in the cerebral cortex, hippocampus, striatum, thalamus, amygdala, cerebellum, and cerebral ventricle, 0.25 h after intravenous injections [115].In addition to results obtained in animals, after a single administration of YKS at 2.5, 5.0, and 7.5 g in healthy humans, GM was detected in the plasma in a dose-dependent manner and the ranges of pharmacokinetic parameters were determined as follows: C max : 0.650-1.98(ng/mL), AUC 0-∞ : 1.18-4.81(ngh/mL), t 1/2 : 1.72-1.95(h), and t max : 0.500 (h) [43].These results indicate that GM is transferred into the blood after oral administration of YKS and then crosses the BBB to be distributed in some brain regions.Additionally, of interest, there is a gender difference in plasma pharmacokinetics of GM in rats; the plasma concentrations of GM in females are approximately four times higher than those in males after oral administration of YKS, and male-specific CYP2C11 and CYP3A2 in rats are involved in this gender difference, although such pharmacokinetic differences are not reported in humans [116].
Although GL, one of the main ingredients in Glycyrrhiza [117], is poorly absorbed from the gut, its hydrolyzed aglycon by intestinal bacteria, GA, is absorbed into the blood circulation [118].After single oral administration of YKS (1 g/kg) in rats, GA was detected in the plasma with t max of 8.7 ± 0.7 (h), C max of 839 ± 112 (ng/mL), and AUC 0-∞ of 8485 ± 773 ngh/mL [119].Besides, at eight hours after a single administration of YKS (0.5-2.0 g/kg), GA was also detected in the brain and CSF in a dose-dependent manner, with the concentrations ranging from 8.5 to 21.5 (ng/g) and 1.3-2.1 (ng/mL), respectively.Furthermore, the authors demonstrated, using the in-vitro BBB model, that GA can cross the BBB with a permeability rate of 13.3 ± 0.5 (%).In addition to the findings in animals, Kitagawa et al. showed a dose-dependent increase in the plasma concentrations of GA after oral administration of YKS at 2.5, 5.0, and 7.5 g in healthy humans, with C max in the range of 57.7-108 (ng/mL) [43].The values of t max , t 1/2 , and AUC were determined ranging from 8.00 to 8.01 (h), 11.0-12.3(h), and 690-1670 (ngh/mL), respectively.The in-vitro autoradiographic binding assays with tritiumlabeled GA by Mizoguchi et al. further revealed that there were specific binding sites for GA in rat brain slices, and the density of binding was higher in the hippocampus, moderate in the caudate putamen, nucleus accumbens, amygdala, olfactory bulb, cerebral cortex, thalamus, and midbrain, and lower in the brain stem and cerebellum [120].Moreover, subsequent microautoradiography showed that [ 3 H] GA signals in the hippocampus were detected in small non-neuronal cells similar to astrocytes.These lines of evidence suggest that GL contained in YKS is metabolized into GA by intestinal bacteria, and GA is absorbed into the blood circulation and then crosses the BBB to enter the brain, where it possibly binds to astrocytes in some regions, including the hippocampus.A recent update on the pharmacokinetics of GA has focused on the gut microbiota to identify the reason for the large inter-individual differences in GA pharmacokinetics [44].Ishida et al. showed that oral antibiotics, which change the proportion of gut microbiota and the number of microbes, affected the plasma levels of GA, and that there were correlations between plasma GA levels and the gene copies of bacteria, such as those belonging to the phylum Bacteroides and Firmicutes [44].Similar studies focusing on the interindividual differences in the effects and pharmacokinetics of YKS will help to further understand the mechanisms underlying the beneficial effects of YKS, given the basic idea that Kampo medicines should be carefully prescribed depending on the individual differences in patients' conditions and constitutions, and that, in some cases, patients are separated into drug responders and nonresponders even if those with the same disease are treated with the same Kampo medicine.The target cell of GM might be neurons [114] Healthy humans YKS: (1) 2.5 g, (2) 5.0 g, (3) 7.5 g, oral administration, single dose 0, 0.25, 0.

ADVERSE REACTIONS AND POSTMARKETING SURVEILLANCE OF YKS
In Japan, all drugs causing adverse events, including suspected causal drugs and drugs for which a causal relationship cannot be ruled out, should be reported to the Pharmaceutical and Medical Devices Agency (PMDA) by pharmaceutical companies, attending physicians, and pharmacists who identify them.The Japanese Adverse Drug Event Report database by PMDA showed 96 cases of YKS-related adverse drug reactions reported between 2005 and 2022 [121].Of these, 34 cases were reported with YKS as the suspected causal drug, and the remainder were reported with concomitant medications as the causal drug.Among the 34 cases, the most common adverse reaction was hypokalemia (13 cases), followed by pseudohypoaldosteronism (six cases).In addition, two cases of congestive heart failure and one case of rhabdomyolysis were reported, both of which were considered to result from licorice-induced pseudoaldosteronism (PA).Finally, 22 cases of YKS-related adverse events were recorded.Subsequently, four cases of interstitial lung disease, two cases of liver damage, one case of drug eruption, and six other cases were recorded.There was one fatal case each of loss of appetite and cerebral hemorrhage.Takamitsu et al. conducted an adverse drug reaction frequency survey for a representative dosage form of YKS, Tsumura YKS Extract Granules for ethical use, for a maximum observation period of 52 weeks to clarify the safety profile, particularly the frequency of adverse drug reactions [122].Of 3156 patients (mean age: 65.3 ± 21.9 years; 1172 men and 1984 women) included in the safety analysis, adverse reactions occurred in 136 patients after the start of treatment with this drug, with an incidence of 4.3%.The most common adverse reactions were metabolic and nutritional disorders (1.4%), mainly hypokalemia, general and systemic disorders, administration site conditions (1.1%), primarily edema and digestive disorders (0.6%), and diarrhea and nausea.The incidence of adverse drug reactions was higher in patients aged 65 years or older.
Arai et al. surveyed clinical trials of Kampo products from the Kampo Treatment Database to determine the number of patients treated with Kampo products, and the number and type of adverse events [123].The results showed that 12 of 232 (5.17%) patients had adverse events with YKS, including five patients with gastrointestinal disorders, four patients with metabolic and nutritional disorders, and three patients with psychiatric and nervous system disorders.
dementia (OR = 2.8; 95% CI: 1.6-4.9;p < 0.001), and low body weight (<50 kg; OR = 2.2; 95% CI: 1.1-3.5;p = 0.034) were risk factors for PA; although not significant, treatment with loop diuretics (OR = 1.8; 95% CI: 0.98-3.5;p = 0.059) tended to increase the risk of PA [124].Additionally, the concomitant use of thiazide diuretics, loop diuretics, and glucocorticoids may increase the risk of PA and should be treated with caution.Currently, YKS is being used more frequently in patients with dementia, and the presence of dementia may delay the detection of PA.Therefore, caregivers should pay careful attention to these issues.

CONCLUSION
YKS is useful for BPSD and perioperative mental instability.The modulation of the serotonergic and glutamatergic neural pathways is one of its mechanisms.Pseudoaldosteronism is one of the side effects.

T A B L E 2
Description of yokukansan in representative pediatric texts up to the early Meiji Era.Name of the book (year of publication) Author Indications of yokukansan ReferencesKarei-shoni-ho (1566

F I G U R E 2
The abdominal sign for the appropriate use of yokukansankachimpihange.T A B L E 4 Plant names and part of each ingredient of yokukansan.

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The binding density of [ 3 H]GA was higher in the hippocampus, moderate in the caudate putamen, nucleus accumbens, amygdala, olfactory bulb, cerebral cortex, thalamus, and mid brain, and lower in the brain stem and cerebellum The major target cells of GA were suggested to be astrocytes expressing 11βMice treated with amoxicillin and vancomycin had low plasma concentrations of GA, with altered composition pattern of gut microbiotaMice with low plasma levels of GA had lower levels of the phylum Bacteroides and Firmicutes - 18 Atractylodes lancea rhizome; dBcAMP, dibutyryl-cyclic adenosine monophosphate; EAAT2, excitatory amino acid transporter 2; ED, effective dose; GA, glycyrrhetinic acid; GLAST, glutamate aspartate Abbreviations: ALR, Lower plasma concentration of GM in male than in female was most likely attributed to male dependent CYP2C11 and CYP3A2GA crossed the brain endothelial cells with the permeability rate and permeability coefficient of 13.3 ± 0.5 (%) and 16.5 ± 0.7 (10 À6 cm/s), respectively GL, isoliquiritigenin, and liquiritigenin also crossed the in-vitro BBB, but glycyrrhetic acid 3-O-gluculonide, isoliquiritin, isoliquiritin apioside, liquiritin, and liquiritin apioside had difficulties crossing the BBB max : 1.98 ± 0.35 ng/mL; t max : 0.67 ± 0.24 h; t 1/2 : 1.63 ± 0.40 h H. Yamaguchi wrote "History of Yokukansan."R. Arita wrote "Pharmaceutical Information about YKS." T. Yoshino wrote "Clinical Studies of YKS." H. Oizumi wrote "Preclinical Studies on YKS." T. Nogami wrote "Adverse Reactions and Postmarketing Surveillance of YKS." S. Takayama designed the project and wrote the abstract, summary, and conclusion.