IT202200000581A1 - Quinic acid for use in the treatment of mood disorders - Google Patents

Description

Quinic acid for use in the treatment of mood disorders

The present invention concerns quinic acid for use in the treatment of mood disorders. In particular, the present invention concerns quinic acid, a derivative thereof or a precursor thereof or a pharmaceutical composition comprising quinic acid, a derivative thereof or a precursor thereof for use in the treatment of mood disorders, in which the quinic acid can? be obtained from plants such as kiwi.

The Depression ? A common and heterogeneous mental health disorder, it contributes significantly to the global burden of disease and affects people in all communities. of the world. To date, it is estimated that depression affects 322 million people (4.4% of the world population), approximately half of them. of these people live in the Southeast Asia region and the Western Pacific region, reflecting the relatively large populations of these two regions (which, for example, include India and China) [1].

Depressive disorders are the most common mental illnesses? common and the main source of disability? worldwide, affecting over 10-15% of the population at some point in their lives.

Only since the last four decades have there been precise criteria for diagnosing depression. Mood disorders are currently classified on the basis of symptoms, clinical history (including age of onset, course and outcome), family inheritance patterns and response to treatment. Nowadays, diseases and therefore also depression are classified with two diagnostic systems: the Diagnostic Statistical Manual of Mental Disorders - (DSM-V), of the American Psychiatric Association (APA) and the International Classification of Diseases (ICD -11) of the World Health Organization? (WHO) although the former covers mental disorders in detail. In the DSM-V, depressive disorders were separated from bipolar and related disorders, making the classification into unipolar and bipolar depressive disorders (people suffering from both depression and manic symptoms) obsolete.

The two most depressive disorders? DCS and dysthymia are frequent. Dysthymia? a chronic form of mild depression, while MDD is the most shape? severe depression. Symptoms vary from one type of mental illness to another, but are primarily characterized by problems with emotions, ways of thinking, and the way people behave and relate to each other in society. Are some mental health problems also comorbid? with one or more? mental health conditions such as depression and anxiety. Mood disorders and anxiety are generally discussed together because? both involve a negative emotional state. Depending on the gravity? of the symptoms, the episode can? be further classified as mild, moderate or severe:

- Mild depression: there are few, if any, symptoms in excess of those necessary to make the diagnosis, the intensity? any symptoms? distressing but manageable, and the symptoms cause mild impairment in social or professional functioning.

- Moderate depression: the number of symptoms, the intensity? symptoms, functional impairment, or all of these variables are among those specified for “mild” and “severe.”

- Severe depression: the number of symptoms? substantially higher than that required to make the diagnosis, the intensity? any symptoms? severely distressing and unmanageable, and the symptoms markedly interfere with social and professional functioning.

Treatments for depression can be divided into three; those involving the use of medications (referred to as pharmacotherapy), those using alternative biological treatments, and those using psychotherapy. Treatments classified as part of pharmacotherapy include antidepressant medications, mood stabilizers, and antipsychotics. Alternative biological treatments can be electroconvulsive therapy (ECT), transcranial magnetic stimulation (TMS), deep brain stimulation, bright light therapy, while psychotherapy includes cognitive behavioral therapy (CBT), behavioral activation treatment , interpersonal therapy (IP) and family and marital therapy.

Several antidepressants have proven effective in treating depression. However, antidepressant drugs have slow clinical efficacy, which is fundamental for the therapeutic outcome and patient compliance, in fact current pharmacotherapies take weeks or months to obtain their effects with an increased risk of suicidal behavior.

During this "therapeutic delay", 15% of patients suffering from MDD commit suicide [1,2]. This ? the reason why electroconvulsive therapy can? be the treatment of choice for agitated depressed patients with a high risk of suicide. Further problems arise from the limited effectiveness of all antidepressant drugs, since at least 20% of all depressed patients are refractory to pi? different antidepressants at adequate doses. In general, if a patient does not respond to a certain antidepressant after an 8-week study on an adequate dose, switching to another antidepressant with a different mechanism of action is ? the reasonable next step (for example, from SSRI to SNRI). All these problems increase the urgency of finding more effective drugs? active and with fewer side effects.

The most drugs Commonly used, often referred to as second-generation antidepressants, are selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), which have greater efficacy and safety than more common drugs. dated (i.e. first generation antidepressants). Relatively selective norepinephrine reuptake inhibitors have also been developed as antidepressants (e.g., maprotiline, reboxetine). In monoaminergic systems, transmitter reuptake is ? the main mechanism by which neurotransmission is terminated; therefore, the inhibition of reuptake can? improve neurotransmission, presumably by slowing transmitter clearance and prolonging its residence time in the synapse. Reuptake inhibitors inhibit SERT, NET, the neuronal norepinephrine (NE) transporter; or both. Similarly, first-generation drugs, which include monoamine oxidase inhibitors (MAOIs) and tricyclic antidepressants (ATDs), also improve monoamine neurotransmission: MAOIs inhibit monoamine metabolism and thus improve the storage of neurotransmitters in secretory granules, ATDs inhibiting serotonin or 5-hydroxytryptamine (5-HT) and the reuptake of norepinephrine. Although effective, these first-generation agents exhibit side effects and drug and food interactions that limit their use compared to more advanced drugs. recent. Is the hypertensive crisis resulting from food or drug interactions? one of the toxicities? potentially lethal conditions associated with the use of MAOIs. Foods containing tyramine are a causal factor. MAO-A within the intestinal wall and MAO-A and MAO-B in the liver normally degrade dietary tyramine. However, when is MAO-A? inhibited, the ingestion of some mature cheeses, red wines, sauerkraut, broad beans and a variety? of other tyramine-containing foods leads to the accumulation of tyramine in adrenergic nerve endings and neurotransmitter vesicles inducing the release of norepinephrine and epinephrine. The released catecholamines stimulate peripheral postsynaptic receptors, increasing blood pressure to dangerous levels (cheese reaction). These episodes can be reversed by antihypertensive drugs. Even when the patient is very careful, dietary inadvertence or the use of prescription or over-the-counter drugs that contain sympathomimetic compounds may occur, resulting in a potentially lethal increase in blood pressure. Another serious and life-threatening problem with chronic MAOI administration? hepatotoxicity.

In light of the above, ? therefore the need is evident? to provide new therapeutic agents for mood disorders capable of overcoming the known disadvantages of mood disorder therapy.

The beneficial effects of a diet rich in fruits, vegetables, nuts and plant-based ingredients (whole grains, olive oil, especially on the prevention of cardiovascular disease are well known and generally recognized [3]. In addition, poor nutrition correlates with high blood cholesterol levels, obesity, heart disease, diabetes and some types of cancer.

In recent years, however, evidence is emerging of the possible effects of a diet rich in fruit, vegetables, dried fruit, whole grains and olive oil on various aspects of mental health.

In the last ten years, nutritional psychiatry has become developed as a promising research area, in a field with large unmet needs. Nutrition and mental health are interconnected and influence each other. Yes ? given that healthy nutrition plays an important role in the onset and severity of the disease. of several mental health disorders, and in fact people with mental health disorders often have nutritional deficiencies. This link between nutrition and mental health? was first noted with depression [4].

The first observation studies and small intervention studies have mainly focused on the possible effects of a diet rich in the food products indicated above or on the entire dietary pattern that contains them. An example ? the so-called "Mediterranean diet", the plant-based diet typical of the regions around the Mediterranean Sea dedicated to olive cultivation. These reports have focused more on cognitive performance [6], on the prevention of depression [7] and, generally, on psychological well-being [8]. The available evidence suggests that an increased consumption of fruit and vegetables? associated with both cognitive benefits and psychological well-being. Meanwhile, in the field of intervention studies, registered, randomized and controlled trials have begun to appear [9]. Observation and intervention studies on the effects of entire dietary patterns on mental health, while being very powerful tools, cannot explain which specific fruits and vegetables? which molecules are responsible for the observed effects. Intervention studies with individual fruits or vegetables are needed to evaluate the possible effectiveness of individual types of fruits or vegetables on brain health. So far, various in vitro and in vivo studies (the latter on animal models and humans) have suggested various possible effects on the brain of various fresh or processed fruits and vegetables, such as yellow kiwi (Actinidia chinensis, Planch.) [10], blackcurrant, blueberry, cherry, cranberry, grapes, apples [11], orange juice [12], onion and green kiwi (Actinidia deliciosa) [13]. In vivo experiments allow testing fruits/vegetables or their molecules (single or in combination) within the complex context of a mammal or even a human but, on the other hand, they are generally not able to deal with the molecular targets of treatment. In vitro experiments can provide precise indications on specific molecular targets but do not provide information on the actual efficacy in the entire organism.

Approximately 25% of patients with neuropsychiatric conditions, including mood disorders and schizophrenia, show high levels of inflammation [14] and oxidative stress [15]. AND? It is widely reported that a healthy diet has properties? anti-inflammatory, and could improve oxidative stress by providing compounds with well-known properties? anti-inflammatory and antioxidant. For example, vitamins such as ascorbic acid (vitamin C) and alpha tocopherol (vitamin E) have beneficial properties. of direct scavenging of free radicals [17]. Nutrients such as selenium, zinc and cysteine are co-factors for antioxidant systems such as glutathione peroxidase and superoxide dismutase. Therefore, fruits/vegetables can act on more processes? general which are also responsible for neuropsychiatric disorders. As regards the possible molecules responsible for the activities? observed, very few reports reveal the precise molecular components that are responsible for the activity? of fruits/vegetables (an example is the identification of two phytochemical compounds in a Concorde grape juice preparation responsible for the reduction of stress and peripheral inflammation [18]).

The identification of the precise active molecules of fruits/vegetables? complicated by the fact that some biological effects of fruits and vegetables may depend on additive, synergistic and antagonistic effects between components of the phyto-complex, more? than from individual molecules, and this makes the matter more difficult [19].

Actinidia fruits can be described as berries with many seeds enclosed by a juicy pericarp. The biggest review? recent history of the genus recognizes 55 species and 20 varieties. Traditionally, the composition of the fruit is been considered primarily in terms of nutritional components (proteins, lipids, carbohydrates, vitamins and minerals). The kiwi? often regarded as a fruit with high fiber content; however, when compared to other fruits, its fiber content is not ? particularly tall; with its 2-3% fiber on fresh weight? similar to that of apples, oranges and bananas. The fruit ? also rich in specialized secondary metabolites belonging to the chemical classes of carotenoids, phenolic compounds, flavonoids, vitamins, chlorophylls and organic acids. Secondary metabolites play a role in the interaction of the cell with its environment, represented by other cells in the organism, or by external organisms or abiotic factors, for example, in the case of the entire plant, they can attract pollinators or defend it from pests and diseases[20].

As main secondary metabolites, the fruit of Actinidia deliciosa contains phenolic compounds and indolealkaloids (tryptamine and serotonin). Phenols are a large group of compounds characterized by a chemical structure containing at least one phenolic group. Phenolic compounds have been associated with active antioxidant and have been intensely studied why? considered healthy for consumers. In plant-derived foods, phenolic compounds are divided into two large groups known as phenolic acids and flavonoids. Tryptamine and 5-hydroxy tryptamine are secondary metabolites belonging to the class of indolealkaloids, and more? in particular they are indoleamines formed by an indole skeleton with a side chain of the ethylamine type, both derived from the amino acid tryptophan. These compounds have been seen to be widely distributed in the living kingdoms, from bacteria to higher eukaryotes, with very different distribution and functions [21].

The kiwi fruit? also recognized as a huge dietary source of vitamin C, potassium and folic acid, it also contains relatively high levels of vitamin E which are associated with anti-inflammatory effects. For many years yes? assumed that kiwi vitamin E was predominantly contained in the seeds in association with the oil content, and was not bioavailable given that the seeds generally exist upon digestion. However, more analysis? recent studies have shown that the main form of vitamin E, a-tocopherol, is in the pulp, probably associated with the cell membranes, and therefore? potentially bio-available [22]. Proteins represent a minor but significant component of the fruit. Raw proteins typically represent about 1% of fresh weight; however, soluble proteins are much less, around 0.3%. In green kiwi, actinidin is the predominant soluble protein, accounting for up to 40% of soluble proteins.

Despite its components that are beneficial to health and nutrition, kiwi contains a number of elements that can be harmful to health, particularly allergens and oxalates.

? It is well known that animal research is has been an integral part of scientific discovery and the development of therapeutic drugs for decades also because? many responses to stimuli are well conserved across mammalian species. For this reason, animal models, particularly rodents, are potentially relevant to human disorders. The results that have been facilitated through animal modeling of the pathology are invaluable, demonstrating their necessity? in promoting understanding of human pathophysiology and treatment.

To test neurobiological theories of common human diseases and to develop new targets for drug discovery, good animal models are needed. There have been only a few criteria proposed for this? which constitutes an ideal animal model, and small adaptations have developed these criteria to include more evaluations and interpretations? complex, in fact animal models are expected to have validity? form, construct and predictive. The question of validity criteria? ? was raised by [23], the authors proposed that: - an animal model should recapitulate most of the main symptoms of the disorder to be modeled (form validity);

- the symptoms induced by the model should be malleable to pharmacological intervention (predictive validity) and should be similar, based on this? That ? known about the human disorder, the condition modeled, the etiology of the disease (construct validity).

Many models satisfy more? of a validity criterion? and, as understanding of the underlying biology of disorders advances, ? It is possible to extend the criteria to include "biological validity". Recently there? was a push for biologically more animal models? relevant whose main requirement is? that of imitating the neurobiological correlates of diseases (pathological validity); such models require complex tools, and this is? a rapidly growing area of research [24].

Regarding animal models of depression, to date many different types of stress have been found to induce various depression-like behaviors in rodents. One of the earliest forms of stress-based models? It was the application of electric shocks to the paw that led to a behavioral response called ?learned helplessness?. This behavioral phenomenon means acquisition of a passive approach to coping with stress, which occurs after several exposures to unavoidable conditions and manifests as a failure to escape the shock when provided with an escape route.

Passive coping? became a focus of attention in another model, the Porsolt swim test [25], commonly referred to as the forced swim test (FST). Porsolt found that rats placed in a cylinder with water in an inescapable condition first show active escape attempts and swim vigorously around the cylinder, however within a few minutes they become less active and finally resort to passive floating, with just enough movement to keep your head above water. This answer? It has been nicknamed "behavioral despair" as it seems to reflect animals "giving up" on their chance. to escape the water. Behavioral desperation can It can also be triggered in a tail suspension test (TST), in which mice suspended by the tail progress from initial attempts at fighting and climbing to immobility. passive [26]. The validity? of these models is mainly based on their sensitivity? to antidepressant treatment. Indeed, antidepressants from most classes administered acutely and chronically have been shown to delay the onset of acquired helplessness and behavioral hopelessness in the FST and TST [27,28]. The main strong point of these models? their ability? to quickly select new compounds and for this reason they have become a gold standard for the early screening of new molecules with putative antidepressant effects.

According to the present invention? It has now been discovered that quinic acid (QA), a specialized secondary metabolite of kiwifruit, taken alone or in combination with kiwifruit caffeic acid, exhibits beneficial activity. antidepressant in two reference animal models for depressive behavior in mice.

Quinic acid [(3R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylic acid] and its derivatives are compounds containing the molecular structure of quinic acid, formed by a cyclohexane ring bearing a carboxylic acid residue in position 1 and four hydroxyl groups at positions 1,3,4, and 5 as illustrated in Figure 1a. The first discovery of quinic acid is due to the French chemist who described it for the first time in the medicinal plant Chincona officinalis L.. Subsequently, quinic acid was widely studied until in 1932 Fischer and Dangschat elucidated its structure and properties? stereochemical, also studying the relationship between the biogenetic origin of quinic, gallic and shikimic acids (Figure 1).

They discovered that these three acids have a common biogenetic origin and that the presence of quinic acid can be correlated as a precursor to the shikimate pathway, an important biochemical process that is divided into several passages inside the cell. The way of shikimato? used as a biosynthetic pathway by bacteria, plants, archaea and other organisms and provides precursors for the synthesis of aromatic amino acids (Figure 2).

? interesting to note that quinic acid can? constitute a sole or alternative carbon source in various microorganisms (for example Aerobacter aerogenes and Klebsiella pneumonia). In nature, quinic acid is widely spread within plants, such as in coffee beans, in the bark of Cinchona (L.), in the bark of Eucalyptus globulus (Labill.), in tobacco leaves or of Urtica dioica L. [29]. However, quinic acid is rarely found in plants in free form and is mainly and more? abundantly present in ansterified form together with other secondary metabolites, in particular hydroxycinnamic acids, such as caffeic acid, producing in this case chlorogenic acid (CGA) (Figure 2). In this form? widespread within the plant kingdom with an activity? protective against microbial attacks [30].

In nature, quinic acid can? be produced from D-glucose through four enzymatic reactions, some in common with the shikimate pathway. The biosynthetic pathway begins by converting D-glucose into D-erythrose-4-phosphate via the enzyme transketolase. Phosphoenolpyruvate is added to it forming 3-deoxyheptulosonic acid (DAHP). The enzyme DHQ synthase converts DAHP to 3-dehydroquinic acid (DHQ), which is in turn converted to quinic acid by the enzyme DHQ dehydrogenase. Can quinic acid? can also be produced from shikimic acid by the action of the quinate hydrolyase enzyme, as shown in Figure 2. Quinic acid derivatives, in particular those derived from phenolic acids, have shown a broad spectrum of anticancer, antioxidant, anti-inflammatory, neuroprotective actions and hepatoprotective in the treatment of various cancers and central nervous system-related diseases [31]. For example, (-)-4-O-(4-O-?-D-glucopyranosylcaffeoyl)quinic acid has shown active activity. anticancer against human colon cancer [32].

Caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid) (Figure 3) is a natural phenolic compound belonging to the class of hydroxycinnamic acids (coumaric acid, caffeic acid, ferulic and sinapic acid and their derivatives), a class of aromatic acids that has a phenylpropanoid structure (C6-C3) with a 3,4-dihydroxylated aromatic ring bearing a carboxylic acid residue at the end of a transethylene chain. These compounds are hydroxy derivatives of cinnamic acid produced by the secondary metabolism of plants, in which it is widely distributed [33].

Caffeic acid is found in simple (monomeric) form as esterased organic acids, esters of sugars, amides and glycosides (for example caffeic acid 3-?-D-glucoside), or in more complex forms? complex such as dimers, trimers and derivatives of flavonoids, or they can also be linked to proteins and other polymers in the cell wall of plants. CA participates in the defense mechanism of plants against predators, parasites and infections, as it has an inhibitory effect on the growth of insects, fungi and bacteria, and also promotes the protection of plant leaves against ultraviolet B (UV-B) radiation [33 ].

The biosynthesis of this compound in plants occurs through the endogenous shikimate pathway, responsible for the production of aromatic amino acids from glucose. Biosynthesis begins with shikimic acid which undergoes three enzymatic reactions: the first? a phosphorylation mediated by the enzyme shikimate kinase, followed by the conjugation of a molecule of phosphoenolpyruvate, mediated by 5-enolpyruvilshikimate-3-phosphate (EPSP) synthase and finally the synthesis of chorismic acid mediated by the enzyme chorismate synthetase, one of the metabolic intermediates more? important in this way. This is transformed into prephenic acid via the enzyme chorismate mutase (a precursor of L-phenylalanine). The formation of L-phenylalanine? mediated by pyridoxal phosphate (PLP) as a coenzyme in the deamination process and by nicotinamide adenine dinucleotide (NAD) as an electron exchanger. Deamination of L-phenylalanine by the enzyme phenylalanine ammonium lyase (PAL) forms cinnamic acid. This is then converted to p-coumaric acid by cinamate 4-hydroxylase (C4H) and finally to caffeic acid via the enzyme 4-coumarate 3-hydroxylase (C3H) [33] (Figure 3).

Caffeic acid shows a wide range of beneficial health effects including anti-inflammatory, antitumor, antithrombotic, antihypertensive, antifibrotic, antiviral, antioxidant and anticancer [33]. The activity antioxidant probably comes from the properties? of scavenging against free radicals. Furthermore, it inhibits lipid peroxidation and protects DNA from oxidative damage. Caffeic acid has also been shown to have promising beneficial effects in animal models for Alzheimer's disease [34].

Additionally, caffeic acid has been shown to prevent neurotoxicity? induced by acrolein in vitro. The toxic action of acrolein mimics many mechanisms that underlie neurodegenerative pathways, such as those activated in AD. Pretreatment of cells with caffeic acid reduces their neurotoxicity, reduces the level of ROS, and also exerts a beneficial activity. indirect antioxidant by protecting glutathione, an essential endogenous antioxidant[35].

According to the present invention, ? Was the capacity first assessed and then confirmed? of the kiwi to reduce the time of immobility? both in the TST and in the FST, and therefore to carry out an activity? antidepressant, on mice treated chronically for 10 days with fruit juices through the administration of intragastric gavage (IG). After completing the behavioral tests, the mice were sacrificed to collect serum and brain samples for metabolomic analysis, and the behavioral data were then compared to the analytical data. Furthermore, pharmacokinetic (PK) experiments were conducted to estimate the absorption and clearance of selected kiwi compounds.

Following the execution of in vivo experiments in which carried out the chronic administration of kiwi juice in animal models, the proponent discovered that only very few new metabolites can be found both in mouse serum and in the brain after administration of the juice. Among them, the proponent recognized quinic acid (QA), a specialized secondary metabolite of kiwi, and caffeic acid sulfate (CA-sulfate), a mouse metabolite produced from kiwi caffeic acid. The latter ? abundantly present in the kiwi juice metabolome in the form of various glucosides and esters. Therefore, according to the present invention, the possible effects of these two molecules have been analyzed more? in detail. In particular, according to the present invention, the kiwi (i.e. the fruit of Actinidia deliciosa, the "green" kiwi) is Was it originally chosen on the basis of previous sources reporting the activity? antidepressant of the fruit and its close relative Actinidia chinensis (the yellow kiwi) [10,13]. Furthermore, other investigations have suggested the ability of the green kiwi to carry out activities? at a cerebral level, such as improving the quality? of sleep in young people and adults with sleep problems, and to show activity? antidepressant in pre-clinical models (36,13). Furthermore, green kiwi contains a quantity? relevant serotonin and its plant precursor, tryptamine (data not shown). In principle, both serotonin and its derivative, melatonin, could be involved in improving sleep, even if melatonin is not ? never detected in the present analysis. Even the plant precursor of serotonin, indoleamine tryptamine, could act in the same context, since? can? behave as a serotonin agonist [37].

Serotonin and tryptamine ingested through diet are generally not bioavailable because? become substrates of the intestinal MAO-A and B enzymes, but not? Is it possible to exclude their possible role when consuming a phytocomplex such as the one present in fruit and vegetables and considering the activity? synergistic between its different components or between a combination of them. For example, the shamanic ayahuasca beer retains the properties hallucinogen of dimethyltryptamine (DMT) contained in the herb Psychotria viridis, Ruiz & Pav thanks to the activity? MAO-inhibitory of ?-carbolines (harmine, harmaline and tetrahydroharma) of Banisteriopsis caapi, (Griseb.) Morton [38]. Therefore, in principle, possible MAO-A MAO-B inhibitory components present in kiwifruit could change the fate of the fruit's serotonin and tryptamine, making them more bioavailable once consumed.

As mentioned previously, the experimental results carried out by the proponent and described in the example indicate that the oral administration of kiwi has an antidepressant effect. Furthermore, on the basis of the present invention, the kiwi metabolites that may be candidates for this activity have been identified. According to the present invention, in behavioral experiments the data of different treatment groups were compared with the negative control, which was a vehicle solution, a mix of the most common primary metabolites. abundant kiwi without any activity.

TST and FST have been adopted as a behavioral paradigm to evaluate depressive-like behavior although there are many other tests commonly used to evaluate this topic since? these tests are highly sensitive and are regularly used to evaluate the potency of antidepressant drugs. Furthermore, intragastric (IG) administration is was chosen why? allows you to precisely control the dose administered, and why? imitates the actual intake of the phytocomplex compared to other types of administration.

Kiwi extract significantly reduces immobility time. of mice in both the TST and FST also compared to the positive control, a drug commonly used in the clinical treatment of depression, without causing locomotor impairment, as reported by the OFT data.

In the example, fluoxetine (FLX) at 20 mg/kg? was used as a positive control, since? this dose? comparable to the plasma levels of patients treated with it according to Dulawa and colleagues [39] both in acute and chronic regimes.

However, in the FST the positive control did not work as reported in the literature with this mouse strain [40]. There? ? probably due to the so-called serotonin syndrome, a serious disease that commonly appears in humans after overdose of antidepressants or after combining several psychotropic drugs. This toxicity? acute due to 5-HT? has been characterized in mice by the expression of some behavioral and physiological responses such as: hind limb abduction, forepaw treading, backward movement, straub tail, head weaving, tremor and low flat posture [41]. Not ? no locomotor impairment was observed in the OFT test carried out two days before the end of chronic treatment (Day 8) although ? reported in the literature that motor impairment, if it occurs, could appear after 3-4 days of chronic treatment [39]. For this reason, in the following experiments the positive control is been changed to another SSRI drug, Escitalopram.

According to the present invention, UPLC-ESI-MS analytical data reveal two potential kiwifruit molecules (quinic acid and caffeic acid) candidates for the activity behavioral observed, present in the serum and brain of mice administered with kiwi. These two molecules show serum and brain levels that decrease linearly with increasing dilutions of kiwi administered (kiwi1>kiwi2>kiwi3).

Furthermore, the "pharmacokinetics" of these kiwi metabolites? was performed in mice treated with pi? high dilution of kiwi (kiwi1) and relative quantification in brain and serum samples ? was evaluated with the UPLC-ESI-MS technique over a 24-hour period. The accumulation? was very quick, since? levels of both metabolites decrease to baseline levels between 8 and 24 hours.

The approach to study bioavailability? of single molecules after administration/consumption of a whole fruit as a tool to identify possible neuroactivity candidates? ? already? been used positively by Wang and collaborators [22] thanks to which they were able to identify two active molecules, dihydrocaffeic acid (DHCA) and malvidin-3?-O-glucoside (Mal-gluc), after the administration of the Concord grape juice solution.

Therefore, ? a group of animals treated with the same dilution of kiwi (kiwi1) subjected to a perfusion approach was analyzed to evaluate the possible cerebral penetration of the two metabolites of interest; brain levels after perfusion were about half of the level of non-perfused animals, confirming that part of these molecules is able to pass the BBB. These data confirm the passage of both molecules in the brain parenchyma.

The cold saline perfusion technique? Widely used to remove blood from the brain or other target tissues in various brain investigations, mainly because? in many cases the blood could interfere with the subsequent analysis [18, 42] or, in the case of homogenized brain, the molecules present in the cerebral vessels could influence the analysis.

As in this work, Wang and collaborators [43] used this technique to evaluate the ability of various polyphenolic metabolites to pass the BBB.

As described in the example of the present invention, in a 10-day chronic treatment with different dilutions of kiwi on TST and FST in mice, the kiwi at the maximum dilution, the so-called Kiwi 1, strongly reduced the immobility time. in the above-mentioned tests without causing any locomotor alterations. Furthermore, few kiwi metabolites were found in the serum and brains of kiwi-treated mice compared to controls, and two of these were identified: one, quinic acid, ? a metabolite of kiwi, while the other, sulphated caffeic acid, is an organic metabolite of the same. N? serotonin or tryptamine were different in the serum or brain of kiwi-treated animals compared to the control group.

Quinic acid and caffeic acid sulfate levels were assessed by mass spectrometry. Furthermore, PK experiments were conducted on animals fed kiwifruit to evaluate the absorption and elimination of the two compounds. Furthermore, cerebral penetration? was evaluated in animals treated with the two metabolites after transcardiac perfusion technique. The post-perfusion brain level of QA and CA-sulfate was lower than in non-perfused samples (about half), but still detectable, demonstrating that the two molecules overcome the blood-brain barrier and are able to enter the brain parenchyma. .

Subsequently, the two individual specialized kiwi metabolites were evaluated in the same behavioral paradigms after a 10-day regimen in chronic treatment.

To this end, ? Was the quantity carefully determined first? absolute of these metabolites in kiwi juice and then these metabolites were administered, alone or in combination, to mice at the same concentration as undiluted kiwi extract (kiwi 1).

The level of quinic acid in the kiwi juice used in the example was ~492 mg/100 g of fresh fruit, in line with that reported in the literature (390 mg/100 g of fresh weight) [44], while the levels of three caffeic acid hexosides detected in kiwi were ~6.5 mg/100 g of fresh fruit. As far as is known to the proposing party, this is? the first report to certify a quantification of caffeic acid 3-?-D-glucoside since? in literature ? already? been detected but never quantified [45].

When QA and CA-glucoside were administered to mice, alone and in combination, at the same concentration as undiluted kiwifruit extract, QA ? was able to partially mimic the effect of kiwi 1 showing a marked effect, although not so? as high as that of kiwi 1, in reducing the time of immobility? in both behavioral tests. Instead, CA-glucoside administered alone showed no effect, while the combination of the two molecules showed a synergistic effect in TST. Since? in previous behavioral experiments there were some problems with the positive control FLX, in this experiment the control was ? been replaced with escitalopram 10 mg/kg, a more SSRI drug? selective that worked on both tests. All treated groups were also assessed to determine the degree of motor impairment in OFT on day 8 and the fact that no impairment was detected suggested that the reduction in immobility was due to the antidepressant effect.

Therefore, the kiwi has shown to have an activity? rather strong antidepressant, although the fruit's serotonin and tryptamine were unable to reach the blood or brain while a previously unaccounted for metabolite, quinic acid, is ? turned out to be both able to reach the blood and the brain, passing the blood-brain barrier, and to be at least partially responsible for the activity? kiwi antidepressant.

In free form, free quinic acid is found at low levels (<100 mg/100 g fresh weight) in fruits and vegetables [46]. Ripe fleshy fruits generally accumulate citric and malic acids as the main organic acids instead of quinic acid, and edible fruits with high levels of quinic acid are rare. Exceptions include black chokeberry (Aronia melanocarpa, Michx., Elliot) [47] and kiwi [44]. Quinic acid in free form is also found in some processed foods due to the hydrolysis of esters that occurs, for example, during the roasting of coffee beans.

In recent years, the literature has largely focused attention on CGA derivatives and their metabolism with both in vitro and in vivo studies, while the metabolic fate of quinic acid? been neglected so far. The bioavailability? of pure QA isn't it? reported in the literature, but its detection in human serum after the consumption of coffee? suggests that it can? reach the blood [48]. Nowadays, in literature? There is only one article that quantifies the recovery of quinic acid after the consumption of apple smoothies in humans [49].

Other work has focused on the actions of this metabolite via in vitro and in vivo approaches. ? was it like this? discovered that QA ? active, for example, in the release of insulin from cells ? pancreatic cancer, making it a possible therapeutic tool against diabetes, in reducing the activation of TNF-mediated expression? of the adhesion molecule, thus reducing vascular inflammation and also having activity? radioprotective against X-rays [50,51,52]. Furthermore, the QA ? already? used in the creation of nanoparticles in which it carries out the task of conveying drugs to solid tumors being able to interact with endothelial selectins mimicking their ligands [53].

? Interestingly, when QA and CA-sulfate were quantified (relative quantification) in the serum and brain of mice, their levels in the single or combined treatment (both molecules) were lower than those detected for the kiwi dilution 1, even though the concentrations of the metabolites administered to the animals were the same. There? suggests that other molecules present in the whole fruit are necessary for stability? and/or the "correct" intestinal absorption of the two metabolites. However, perfusion experiments demonstrated that quinic acid administered alone was able to reach the brain parenchyma, but showed less penetration into the brain than the entire kiwi treatment.

Furthermore, the ?pharmacokinetic? analyzes conducted in a 24-hour window on serum and brains of mice treated with QA reveal that its accumulation? was very quick, since? levels of this metabolite decline to baseline between 8 and 24 hours. These data are in line with previous data obtained on kiwi-fed mice although the relative quantifications are lower.

The effects of secondary metabolites present in fresh fruits and vegetables are difficult to reveal not only because of the large number of different molecules, but also because? their business? could it depend on bioavailability? of each individual metabolite and the additive, synergistic and antagonistic effects between them [54]. Are there a number of factors that influence bioavailability? of metabolites in an individual; as regards polyphenols, for example, this can? be influenced by many factors including the microbial composition of the colon, the dose consumed and the presence of other polyphenols and macronutrients within the food matrix. Furthermore, are studies on the interferences that occur in bioaccessibility limited? and bioavailability? of phytochemicals and the impact these have on their bioactivity.

Synergy and antagonism are notoriously difficult to study rigorously because the research methodology applied to the chemistry of natural products? typically dedicated to a reduction in complexity? and the identification of individual active constituents for drug development [55].

The synergy in the activities? biological can? derive from phytochemical combinations that can promote solubility, safety, absorption, stability or bioavailability? of the main active compounds.

A less discussed phenomenon? antagonism, in which the effects of the active constituents are masked by other compounds in a complex mixture.

This explains, in general, why? no single compound can replace the combination of natural phytochemicals present in fruit and vegetables to obtain beneficial health effects.

In this case, the data demonstrate that QA administered alone has a marked effect consistent with undiluted kiwifruit extract, but the activity minor? well correlated with the higher levels? low in both serum and brain samples. There? confirms that the entire phytocomplex could be important in stability? and in absorption but not necessarily in activity.

Only QA but not CA-glucoside was active in reducing immobility time? in both tests, although it was less efficient than the entire kiwi phytocomplex. Furthermore, when administered in combination, the two molecules appeared to have a synergistic effect that manifests itself only in the TST. PK and perfusion experiments demonstrated that QA administered alone had lower absorption and brain penetration than the whole fruit. Therefore, QA administered alone at the same concentration found in the whole fruit can partially mimic the activity? kiwi antidepressant in mice, indicating that QA is one of the active antidepressant molecules although its activity? is lower than the kiwi.

The complete activity, the bioavailability? and the penetration of the two kiwi metabolites into the brain could require other molecules present in the fruit whose presence could be fundamental for correct intestinal absorption and/or stability? in mice.

Furthermore, a combination of additional molecules, which were believed to be present in trace amounts because not detected by the extremely sensitive UPLC-ESI-MS platform, could be responsible for some of the activity? antidepressant of the whole fruit, probably acting in synergy with each other, thus clarifying the minor behavioral and analytical effects observed in single-molecule treatments. The activity? synergistic of secondary metabolites? well known in the literature and could explain the current data obtained by administering QA and CA-glucoside together.

Based on the effective dose of kiwi juice and the exact quantification of QA and CA-sulfate obtained by mass spectrometry, can it be possible? hypothesize to translate this antidepressant effect in humans to approximately 700 grams of kiwi/day.

One of the main limitations in the use of plant secondary metabolites in therapy? the possible low stability? and bioavailability? of these molecules in animals and humans, in fact quinic acid, caffeic acid and CGA are poorly absorbed through the digestive system in humans and animals. In particular, regarding quinic acid, the intestinal flora plays a crucial role in the conversion of quinic acid into benzoic and hippuric acids, probably significantly decreasing its bioavailability. Thus, the development of new delivery systems for fruit metabolites, such as the use of nanoparticles as food supplements, could overcome this problem.

Furthermore, since? the above nutraceutical products show a long history of safe consumption, they could be readily used as adjuvants in the treatment of depression, since? they do not require oversight by regulatory agencies such as FDA and EMA.

Therefore, ? a specific object of the present invention a product consisting of

a) quinic acid, a derivative thereof or a precursor thereof, or

b) a pharmaceutical composition comprising or consisting of quinic acid, a derivative or precursor thereof, as an active ingredient, together with one or more? pharmaceutically acceptable excipients and/or adjuvants for use in the treatment of a mood disorder.

According to the present invention, the product consisting of a) or b) is not? a kiwi or a grapefruit in itself, for example in the form of a concentrated plant extract, however, called quinic acid, a derivative or its precursor can be isolated from kiwi or grapefruit as well as? from other plants comprising said compounds.

According to the present invention, the mood disorder, which can? be treated with the product of the present invention, can it? be chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder, and depressive disorder due to another medical condition, premenstrual dysphoric disorder, mental dysregulation disorder disruptive mood.

In accordance with the present invention, such derivatives may be selected from the group consisting of an ester or ether of quinic acid with a sugar or of an ester of quinic acid with an organic acid, of an amide of quinic acid with a nitrogen-containing metabolite, such as an amino acid, of a pharmacologically acceptable quinic acid salt.

According to the present invention, sugar can be a monosaccharide, such as a hexose, a pentose, a deoxyhexose, or an oligosaccharide.

As for the ester of quinic acid with a sugar, according to the present invention, it can? be chosen from the group consisting of quinoyl glucose, quinoyl galactose, while the quinic acid ether with a sugar is quinic acid glycoside.

According to the present invention, organic acid can? be selected from the group consisting of pharmaceutically acceptable aromatic organic acids such as cinnamic acid, hydroxycinnamic acids, benzoic and hydroxybenzoic acids, or aliphatic organic acids such as malic acid, citric acid, isocitric acid, fumaric acid, lactic acid, malonic acid, succinic acid, tartaric acid, provided that caffeic acid is excluded.

Therefore, according to the present invention, the ester of quinic acid with an organic acid can? be for example an ester of quininic acid with cinnamic acid, with a hydroxycinnamic acid, with a benzoic acid, with a hydroxybenzoic acid, with malic acid, with citric acid, with isocitric acid, with fumaric acid, with lactic acid, with acid malonic acid, with succininic acid or with tartaric acid, provided that the ester of quinic acid with caffeic acid is excluded.

According to the present invention, the quinic acid salt can be a quinic acid salt with a pharmacologically acceptable alkali metal or alkaline earth metal.

As regards the precursor of quinic acid, according to the present invention, it can? be shikimic acid.

According to the present invention, the pharmaceutical composition can further understand caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids.

According to an expression of the present invention, the product can? be administered in a quantity? such that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably 2 g to 6.5 g, optimally 3.5 g to 4.5 g.

In particular, the product, according to the present invention, can be administered orally.

Therefore, the product or pharmaceutical composition according to the present invention can? be in a form suitable for oral administration, such as nanoparticles, tablets, capsules, syrup.

Furthermore, the present invention relates to a plant product comprising quinic acid, a derivative or a precursor thereof for use in the treatment of a mood disorder, provided that the plant product is not kiwi or grape. For example, the plant product can? be raspberry, blueberry, blackberry, gooseberry, a drupe such as cherry, peach, apricot, an apple such as apple and pear, a pepo such as melon and watermelon, a hesperidium such as orange, lemon, kumquat, clementine.

A further object of the present invention? a herbal product comprising quinic acid, a derivative or precursor thereof for use in the treatment of a mood disorder, wherein the herbal product is administered in quantity? such that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably 2 g to 6.5 g, optimally 3.5 g to 4.5 g.

According to the present invention the term vegetable product includes vegetables and fruits that contain quinic acid in the form of a powder of dehydrated plant material or in a form of concentrated plant extract. Dehydrated plant material can be obtained, for example, by drying, freeze-drying or spray-drying, while the concentrated plant extract can? be obtained by concentrating or dehydrating a plant extract. According to a preferred embodiment, the plant material to be administered according to the dosage mentioned above is the kiwi.

As mentioned above, according to the present invention, the mood disorder that can can be treated? be chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder, and depressive disorder due to another medical condition, premenstrual dysphoric disorder, from disruptive mood dysregulation.

According to the present invention, the precursor can be shikimic acid.

According to one embodiment, the plant product can? further include caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids.

According to the present invention, the herbal product to be administered according to the above-mentioned dosage can be a fleshy fruit such as kiwi or other berries such as grapes, raspberries, blueberries, blackberries, gooseberries, stone fruits such as cherries, peaches, apricots, apples such as apples and pears, peppers such as mellons and watermelon, hesperides such as oranges, lemons, grapefruits , kumquat, clementine, preferably kiwi.

According to the present invention, the plant product can? be administered orally.

The present invention also relates to a combination of quinic acid, a derivative or a precursor thereof with caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids for separate or sequential use in the treatment of a mood disorder.

According to the present invention, the term "separate use" is understood as the administration, at the same time, of the two compounds of the combination according to the invention in distinct pharmaceutical forms. The term "sequential use"? understood as the subsequent administration of the two compounds of the combination according to the invention, each in a distinct pharmaceutical form.

The mood disorder that can be treated with the combination of the present invention can? be chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder, and depressive disorder due to another medical condition, premenstrual dysphoric disorder, mental dysregulation disorder disruptive mood.

In particular, this derivative can be selected from the group consisting of an ester or ether of quinic acid with a sugar or an ester of quinic acid with an organic acid, an amide of quinic acid with a nitrogen-containing metabolite, such as an amino acid, a pharmaceutically acceptable than quinic acid.

According to the present invention, sugar can be a monosaccharide, for example hexose, pentose, deoxyhexose, or an oligosaccharide.

According to the present invention, the quinic acid ester with a sugar to be used in combination can be chosen from the group consisting of quinoyl glucose, quinoyl galactose, while the ether of quinic acid with a sugar can? be quinic acid glycoside.

As mentioned previously, organic acid can be selected from the group consisting of pharmaceutically acceptable organic acids such as cinnamic acid, hydroxycinnamic acids, benzoic and hydroxybenzoic acids, or aliphatic organic acids such as malic acid, citric acid, isocitric acid, fumaric acid, lactic acid, malonic acid, succinic acid, tartaric acid, provided that caffeic acid is excluded.

Therefore, according to the present invention, the ester of quinic acid with an organic acid can be for example an ester of quinic acid with cinnamic acid, with a hydroxycinnamic acid, with a benzoic acid, with a hydroxybenzoic acid, with malic acid, with citric acid, with isocitric acid, with Fumaric acid, with lactic acid, with malonic acid, with succinic acid or with tartaric acid, provided that the ester of quinic acid with caffeic acid is excluded.

According to the present invention, the quinine acid salt which can can be used in combination? be a quinic acid salt with a pharmaceutically acceptable alkali metal or alkaline earth metal.

As mentioned above, the precursor can? be shikimic acid.

According to one embodiment of the present invention, the combination? administered so that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably from 2 g to 6.5 g, ideally from 3.5 g to 4.5 g.

According to the present invention, the combination can? be administered orally. Therefore, the combination can? be in a form suitable for oral administration such as nanoparticles, tablets, capsules, syrup.

The present invention will now come? described in an illustrative, but not restrictive, manner based on the preferred embodiments, with particular reference to the examples and attached drawings, such as:

Figure 1. Natural products with common biogenetic origin. Chemical structure of quinic acid (a), gallic acid (b) and shikimic acid (c).

Figure 2. Quinic acid biotransformation and shikimate pathway in plants. Bold arrows indicate reactions of the main branch of the shikimate pathway leading to the production of chorismate (not shown in the figure, see figure 3). Quinic acid? the precursor of CGA via the addition of caffeoyl-CoA or via pcoumaroyl quinic acid (not shown in the figure).

Figure 3. Biotransformation of caffeic acid. Bold arrows indicate reactions of the main branch of the shikimate pathway leading to phenylalanine production.

Figure 4. Bar graph representing the effect of Kiwi extract at different dilutions on immobility time? in the TST and FST. The times of immobility? of TST (a) and FST (b) were evaluated 90 minutes after the administration of Vehicle, Neutral juice, Kiwi 1, Kiwi 2, Kiwi 3 at a volume of 10 ml/kg and 30 minutes after the administration of 20 mg /kg of fluoxetine (FLX) at a volume of 10 ml/kg. The values were evaluated through a one-way ANOVA followed by a Tukey's post hoc test and expressed as mean ? SD (n=12/16 per group). **p?0.01, ****p ?0.0001 vs. group treated with vehicle; #p?0.05, ###p?0.001, ####p?0.0001 vs. group treated with neutral.

Figure 5. Bar graph representing the effect of Kiwi extract at different dilutions on distance traveled and speed? average of mice in the OFT. Distance (a) and speed? average (b) were evaluated in a 5-minute open field test 90 minutes after the administration of Vehicle, Neutral, Kiwi 1, Kiwi 2, Kiwi 3 at a volume of 10 ml/kg and 30 minutes after the administration of 20 mg /kg of fluoxetine (FLX) at a volume of 10 ml/kg. No difference ? was found among the different treatment groups. The values were evaluated through a one-way ANOVA and expressed as mean ? SD (n=12/16 per group).

Figure 6. O2PLS-DA score plot and S-loading plot of serum samples analyzed in of negative ionization with C18 (a) and HILIC (b) columns. In the score plot each dot represents a sample, in the S-loading plot each dot represents a metabolite.

Figure 7. Relative quantification of quinic acid and caffeic acid sulfate. Relative quantification of quinic acid (a, c) under C18 chromatography conditions in serum and brain respectively, and of caffeic acid sulphate (b) in serum under HILIC conditions. The values were evaluated through a one-way ANOVA followed by a Dunnett's post hoc test and expressed as mean ? SD (n=12/16 per group). *p?0.05 **p?0.01, ***p?0.001 ****p ?0.0001 vs. group treated with Kiwi 1. QA=quinic acid, CAS=caffeic acid sulphate.

Figure 8. Relative quantification of quinic acid and caffeic acid sulfate in the serum and brain of mice after administration of kiwi juice. Relative quantification of quinic acid (a,c) under C18 chromatography conditions in serum and brain and of caffeic acid sulfate (b,d) under HILIC chromatography conditions in serum and brain. Does each timing represent the average? SD of n=4 per group. QA=quinic acid, CAS=sulphated caffeic acid.

Figure 9. Relative quantification of quinic acid and caffeic acid sulfate in PK experiments after perfusion. Relative PK quantification in the brain of quinic acid under C18 chromatography conditions (a) and of caffeic acid sulfate under HILIC chromatography conditions (b). The values were compared using unpaired t-test and expressed as mean? SD (n=6 per group). **p?0.01.

Figure 10. UPLC-ESI-MS chromatograms of specialized kiwifruit metabolites and their corresponding commercial standards. Peaks outlined with black dashes represent the authentic standards while those not contoured represent specialized metabolites found in kiwifruit. Both molecules are visible only in mode of negative ionization. In panel (a) ? quinic acid is shown while in panel (b) it is caffeic acid 3-?-D-glucoside shown. The retention time and l?m/z (-) are reported above each peak.

Figure 11. Bar graph representing the effect of kiwi and its metabolites on immobility time? in the TST and FST. The time of immobility? of TST (a) and FST (b) were measured 90 minutes after the administration of Vehicle (negative control), Kiwi 1, Quinic Acid, Caffeic Acid and a solution of Caffeic Quinic Acids at a volume of 10 ml/kg, and 30 minutes after administration of 10 mg/kg escitalopram (ESC, positive control) at a volume of 10 ml/kg. The values were evaluated through a one-way ANOVA followed by a Tukey's post hoc test and expressed as mean ? SD (n=12 per group). *p ?0.05, **p ?0.01,****p ?0.0001 vs. group treated with vehicle; #p?0.05, ####p?0.0001 vs. group treated with kiwi 1.

Figure 12. Bar graph representing the effect of kiwi and its metabolites on distance traveled and speed? average of mice in the OFT. The distance (a) and the speed? average (b) were measured in a 5 minute open field test, 90 minutes after administration of Vehicle (negative control), Kiwi 1, Caffeic Acid, Quinic Acid and a Caffeic Quinic Acid solution at a volume of 10 ml /kg, and 30 minutes after administration of 10 mg/kg escitalopram (ESC, positive control) at a volume of 10 ml/kg. No difference ? was detected between treatment groups. The values were evaluated through a one-way ANOVA and expressed as mean ? SD (n=12 per group).

Figure 13. Relative quantification of quinic acid and caffeic acid sulfate. Relative quantification of quinic acid (a,c) under C18 chromatography conditions in serum and brain respectively, and of caffeic acid sulphate (b) in serum under HILIC chromatography conditions. The values were evaluated through a one-way ANOVA followed by a Dunnett's post hoc test and expressed as mean ? SD (n=12 per group). **p?0.01, ****p ?0.0001 vs. group treated with Kiwi 1. QA=quinic acid, CAS=caffeic acid sulphate.

Figure 14. Relative quantification of quinic acid in mouse serum and brain after single quinic acid treatment. Relative pharmacokinetic quantification of quinic acid in serum (a) and brain (b) under C18 chromatography conditions. Does each timing represent the average? SD of n=4 per time point.

Figure 15. Relative quantification of quinic acid in PK experiments after perfusion. Relative PK quantification of quinic acid in the brain under C18 chromatography conditions, values were evaluated with unpaired t-test and expressed as mean ? SD (n=6 per group). ****p?0.0001 (a).

Comparison between perfused animals treated with kiwi and quinic acid 30 minutes before sacrifice. The values were evaluated through a two-way ANOVA followed by a Newman-Keuls post hoc comparison and expressed as mean ? SD (n=6 per group). *p?0.05, **p?0.01 (b).

EXAMPLE 1: Study of the effects of kiwi and its metabolites on depression

Animal materials and essays

Animals

5-week-old na?ve male C57BL/6JOlaHsd mice (Envigo RMS Srl, San Pietro al Natisone, Udine, Italy) were used (n=12/16 for behavioral experiment, n=4 for PK experiment and n=6 for perfusion). Upon arrival the mice, weighing 20-25 g, were housed six per cage in Optimice? (36.3x29.2x15.5) with sawdust as litter at constant room temperature (21? 1?C) and humidity? relative (60%) with a 12-hour light cycle (7am-7pm light) with food (Mucedola NFM18) and water accessible ad libitum. The animals were left to adapt to laboratory conditions for at least two weeks before starting the experimental procedures. Were the procedures involving animals conducted at the University? of the Studies of Verona which adheres to the principles set out in the following laws, regulations and policies governing the care and use of laboratory animals: Law (Legislative Decree 26/2014; Authorization n.19/2008-A issued on 6 March 2008 by the Ministry of Health); the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/EU).

Reagents

Fluoxetine HCl (FLX) ? was obtained from Alomone laboratories (Jerusalem, Israel); acetonitrile (ACN), methanol (MeOH), and water were purchased from Honeywell (Seezle, Germany); formic acid (HCOOH) from Biosolve (Dieuze, France) and leucine-enkephalin solution from Waters (Milan, Italy). All solvents were LC-MS grade. Escitalopram? was obtained from MedChemExpress (Monmouth Junction, NJ, USA); D-(?)-quinic acid and caffeic acid 3-?-D-glucoside were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). All chromatographic solvents (ACN, MeOH, Water, and HCOOH) were LC-MS grade and purchased from Honeywell (Seezle, Germany); leucine-enkephalin solution from Waters (Milan, Italy).

Fruit sampling and preparation of fresh juices

Kiwis (Actinidia deliciosa cv. Hayward) were obtained from local producers (Verona, Italy), prepared by cutting peeled slices of each sample, freezing them immediately in liquid nitrogen and storing them at ? 80?C. The frozen material? was pulverized using an A11 analytical mill (IKA-Werke, Staufen, Germany) and the powder ? been preserved at ? 80?C. Fresh juice? was prepared by weighing and thawing 27 g of frozen homogenized powder and centrifuging at 3650? g for 15 minutes at 4 ?C. The supernatant? was then transferred to a new tube and centrifuged at 21,000? g for 15 minutes at 4 ?C. The supernatant? was then passed through a 0.22 ?m Millex PES filter (MilliporeSigma) to remove the high fiber content of the phytocomplex.

The kiwi? was also divided into two other dilutions, starting from the pi? concentrated and undiluted called Kiwi 1 and with a 1:2 and 1:3 dilution in MilliQ water? (MilliporeSigma) for Kiwi 2 and Kiwi 3 respectively and administered to animals at a volume of 10 ml/kg.

NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy? was conducted in collaboration with the organic chemistry group of the University? of Verona led by Professor Michael Assfalg. Kiwi juice samples were prepared by thawing 2?3 g of homogenized kiwi pulp powder as described previously (Section: Fruit Sampling and Preparation of Fresh Juice). The thawed fruit? was first carefully mixed and then sonicated for 15 minutes in a cold water bath. The suspension ? was then centrifuged at 15000 ? g for 10 minutes at 4 ?C and the supernatant ? was transferred to a new tube and centrifuged at 18000 ? g for 20 minutes at 4 ?C to remove remaining insoluble debris. Then 0.56 ml of the soluble aqueous extracts were diluted to a final volume of 0.7 ml in 0.15 M potassium phosphate buffer (pH 6.0), 0.02% (w/v) sodium azide, 5% (v/v) D2O (Cambridge Isotope Laboratories, Cambridge, UK) and 1 mM 4,4-dimethyl-4-silapentan-1-sulfonic acid-d6 (DSS-d6) (MilliporeSigma). NMR spectra were recorded at 298 K using a Bruker Avance III instrument (Bruker, Karlsruhe, Germany) equipped with a triple-resonance TCI cryogenic probe and operating at a <1>H Larmor frequency of 600.13 MHz. Spectra were acquired <1>H-NOESY with a mixing time of 100 ms, a recycling time of 10 s, 64 transients (FID), 64000 points and a spectral width of 20 ppm. Spectra were processed with Topspin v3.2 (Bruker) by multiplying the FIDs with an exponential function with a line broadening of 0.3 Hz before Fourier transformation, phasing, and baseline correction. The spectra were then calibrated to the DSS-d6 singlet signal and analyzed using Chenomx NMR Suite v8.0 (Chenomx, Alberta, Canada) and compared to the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/) . The identified metabolites were quantified by integrating NMR signals using DSS-d6 as an internal standard. The Chenomx software? It has also been used to quantify metabolites having overlapping signals, such as fructose. The resulting values were then corrected with the dilution factor and converted into milligrams per 100 grams of fresh fruit based on the initial weights and volumes.

Vehicle Preparation

On the basis of the NMR analyzes of the kiwi and to exclude the possibility? that the main primary metabolites (sugars, organic acids and ascorbic acid) could have some effect on behavioral tests, ? a vehicle solution was created that replicated the concentrations of these main components. The complete composition of the vehicle solution was: 10.29 g/L sucrose, 33.5 g/L fructose, 35.94 g/L glucose, 0.7 g/L ascorbic acid, 9 g/L citric acid and 9 g/L malic acid. In order to limit the number of candidate kiwi molecules, undiluted kiwi juice (kiwi 1) ? been passed through cartridges in mode? mixed with cation exchange (MilliporeSigma) following the manufacturer's instructions to remove a fraction of the secondary metabolites. Briefly, the columns were activated with 15 ml of ethanol for 15 minutes, then equilibrated with 25 ml of an acidic aqueous solution containing 1 mM malic acid (pH 3.3), and finally the samples were loaded and eluted. The composition? of juice and neutralized kiwi juice, determined by untargeted metabolomics (see below) based on LC-MS ? shown in Table 1. M/Z, retention time, and relative quantification are reported for both identified and unidentified metabolites. The vehicle and neutralized juice were administered to the animals at a volume of 10 mg/kg.

Table 1 contains the untargeted metabolomics data matrix. The molecules are listed starting from the kiwi/neutral ratio pi? high. ui=unidentified compounds.

Table 1

Forced swim test

Mice were placed in a transparent plexiglass cylinder (height 46 cm x 20 cm diameter) filled with 30 cm of water at a temperature of 25?1 ?C for 6 minutes. The dimensions of the cylinders were selected to ensure that the mice were not able to touch the bottom of the tank during the test, n? with paws n? with the tail. A rectangular wooden divider? was used between cylinders to prevent mice from seeing each other during the test and potentially alter their behaviors. A white background? was used to increase the contrast in the video recorded between mice and the wall. The test consists of 6 minutes, however only the last four minutes of the test are analyzed due to the fact that most mice are very active at the beginning of the FST [56]. The time of immobility? ? was scored manually by two trained observers.

Tail suspension test

Similar to the forced swim test, the tail suspension test involves recording immobility? of mice exposed to an unavoidable stressful situation. In this case the mice were suspended from the ground by their tails through a 17 cm ribbon connecting the tail to a grid suspended 60 cm from the counter [26]. A rectangular wooden divider is used to prevent mice from seeing each other during the test and potentially alter their behaviors. A white background? was used to increase the contrast in the video recorded between mice and the wall. This test? has been widely validated pharmacologically [27] and can? be used together with or as an alternative to the FST. Another problem with this test? the curious ability? of mice of the C57Bl/6J strain to climb on their own tails, thus altering the evaluation of the immobility? [40]. Mice displaying this acrobatic escape attempt are usually excluded from further analysis, which can lead to errors in the analysis. For this reason, a climbstopper (4 cm long, 1.6 cm external diameter, 1.3 cm internal diameter, 1.5 grams) is ? been used to prevent this behavior according to Can and colleagues

[57]. The time of immobility? ? was scored manually by two trained observers.

The behavioral tests were repeated twice and the sum of the two experiments was ? been reported.

Open field tests

Since? Both FST and TST are basically tests involving locomotion, should independent assessments of activity be performed? locomotive to ensure selectivity? of any changes in immobility? data from the treatments. The mice were tested for activity. in an open 40x40cm square arena for 5 minutes. During the test the arena was evenly lit with low lighting (~40 Lux). Every mouse? was taken from his cage and placed in the pre-room for acclimatization to the new condition at least 1 hour before the test. The total distance traveled and speed? average of each mouse were recorded and analyzed using a MATLAB tool<?>.

Sample preparation, extraction and analysis After completing the behavioral test, mice were sacrificed to collect serum and brain samples. Blood samples were collected from the retro-orbital sinus of animals under deep isoflurane-induced anesthesia, through a non-heparinized glass capillary. The blood ? was left to coagulate at room temperature for 20 minutes and then centrifuged at 6800? g for 5 minutes at 4?C. Supernatants were collected, frozen in liquid nitrogen, and then stored at ?80?C. Brain samples were extracted from the skull, washed in 0.9% saline, immediately frozen in liquid nitrogen, and then stored at -80?C, until further analysis.

Metabolite extraction for targeted and non-targeted UPLC-MS analyses

Serum samples were thawed at room temperature and, once thawed, 10 volumes of cold (?20?C), LC-MS grade methanol was added to 1 ml of serum. The mixture ? was gently mixed using a benchtop "vortex" for 30 s and then centrifuged at 3650 ? g for 15 minutes at 4?C. The supernatant? was then transferred to a new tube and centrifuged at 21000 g for 20 minutes at 4?C. The supernatant? was then passed through an Oasis? PRiME HLB 1 cc Vac, with 30 mg of adsorbent material per cartridge

connected to a Waters brand 20-position extraction manifold (Waters, Milan, Italy), to remove common matrix interferences, such as proteins and phospholipids, according to the manufacturer's instructions. In short, these cartridges do not need to be activated or equilibrated, simply the samples are loaded and collected immediately with a so-called ?pass-through? method.

For metabolomic analysis, methanol extracts were diluted 1:2 (v/v) with LC-MS grade water (Honeywell) for C18 analysis and 1:2 with LC-MS grade methanol for C18 analysis. HILIC analysis. The extracts were then passed through 0.2 ?m pore size Minisart RC4 filters and 3 ?L were injected into the UPLC device.

Brain samples were weighed and subsequently 10 volumes of cold (?20?C), LC-MS grade methanol were added. The mixture ? been homogenized with a Precellys cryolys? evolution at 4?C, then sonicated at 40 kHz in an ultrasonic bath (Sonica Ultrasonic Cleaner; SOLTEC, Milan, Italy) for 20 minutes, followed by two centrifugation cycles both conducted at 4?C, the first at 3650 ? g for 15 min and the second at 21000 ? g for 15 min. The supernatant? been run through an Oasis cartridge? PRiME HLB 1 cc Vac, with 30 mg of adsorbent material as described above, diluted and filtered as before, for injection into the UPLC-ESI-MS.

For kiwi extract and neutralized juice, samples were prepared as described in the sections ?Fruit sampling and preparation of fresh juice? and ?Vehicle Juice Preparation? and 100 ?L of sample was added to 900 ?L of cold (?20?C), LC-MS grade methanol. The mixture ? was gently mixed and centrifuged at 21000 ? g for 10 minutes at 4?C. The resulting supernatant? was collected, diluted, and filtered as previously described, for injection into the UPLC-ESI-MS.

UPLC-MS analysis of untargeted metabolomics

The analysis system included an ACQUITY I CLASS UPLC (Waters, Milford, MA, USA), equipped with a refrigerated autosampler, to which? A Xevo G2-XS qTOF mass spectrometer was connected

equipped with an electrospray ionization (ESI) source, operating in the modes? positive or negative and controlled by MassLynx v4.1. All extracts were injected into a Waters ACQUITY UPLC BEH C18 and HILIC column (2.1 mm ? 100 mm, 1.7 ?m), both maintained at 30?C. The mobile phases used for the separation of the metabolites with the C18 column consisted of water acidified with formic acid (final concentration of 0.1%, v/v) (A) and acetonitrile (B). The initial conditions of the analysis were 99% A and 1% B and ? the following elution profile was applied: 0-1 min, 1% B; 1-10 minutes, 1-40% B; 10-13.50 min, 40-70% B; 1.50-15 min, 70-90% B; 15-16.50 min, 90-100% B, 16.50-20 min, 100% B, 20-20.1 min, 100-1% B (initial conditions). Subsequently, the system ? been equilibrated at 99% A and the elution ? completed after 25 min. The HILIC mobile phases consisted of water with ammonium formate at the final concentration of 20 mM (A) and 95% acetonitrile with 5% water and 10 mM ammonium formate (B). The initial conditions were 0% A and 100% B and ? the following elution profile was applied: 0?3 min, 100% B; 3?7 minutes, 100?85% B; 7?10 minutes, 85% B; 10? 15 min, 85?50% B; 15?20 minutes, 50% B; 20?20,10 min, 50? 100% B (initial conditions).

Subsequently, the system ? been equilibrated to 100% B and the elution ? completed after 30 min. The flow ? was set to 0.350 mL/min for both columns. The samples were kept at 8?C and the sample sequence ? was randomized. A sample, defined as quality control? (QC), ? was prepared by mixing equal parts of all samples, in order to verify the UPLC-qTOF performance throughout the entire experiment. The QC? was injected after every nine samples analyzed. The ion source parameters were: capillary voltage 0.8 kV, sampling cone voltage 40 V, source offset voltage 80 V, source temperature 120?C, desolvation temperature 500?C, ionization gas flow rate cone 50 L/h and desolvation gas flow rate 1000 L/h. Nitrogen gas? was used for nebulization and desolvation, while argon was used was employed to conduct collision-induced dissociation. ? Was an MS method created to acquire data in mode? ?profile? using a fixed collision energy in two scan functions. In function 1, the low collision energy ? been disabled, while in function 2 the high energy is was set to 35 V. For some samples, high energy was was increased to 45 V to obtain better fragmentation of some metabolites. In both functions, the Xevo G2-XS ? been set to perform analyzes in mode? of sensitivity, making scans in the range of 50?2000 m/z and with a scan time of 0.3 s. The lockmass solution used as a ?calibrator?, to verify the accuracy of the mass spectrometer, consisted of a leucine-enkephalin solution (Waters) of 100 pg/?L, injected with a flow of 10 ?L/min and generating a signal of 556.2771 in mode? positive and 554.2615 in mode? negative.

Data processing and metabolite identification

The statistical analysis of the behavioral experiment? was performed using the Prism v9.0 program (GraphPad Software, San Diego, CA, USA). Significant differences between samples were determined by one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. Raw data generated during untargeted metabolomics analysis were processed using Progenesis QI software (Waters). An intensity? absolute ion for peak selection? was set to 300 and a minimum chromatographic peak width to 0.03 min. An automatic online search on public databases (MassBank, PlantCyc, Plant Metabolic Network and Human Metabolome Database) ? was used for the provisional identification of metabolites by comparing m/z ratios, isotopic similarities and fragmentation patterns, with the fragmentation profiles, extrapolated by the Progenesis QI software, of the individual compounds. Further identifications were achieved using Metlin (https://metlin.scripps.edu) with a tolerance of 0.003 Da and an internal library of authentic commercial standards. Finally, the data reported in the literature were used to support the alleged annotations. Since? internal standards were not used, relative quantification (i.e. comparison between samples) ? was based on the area of each of the signals extracted from the chromatograms and expressed in units? of intensity? arbitrary.

Quantification of quinic and caffeic acids in kiwifruit

Absolute quantification of metabolites by LC-MS requires careful evaluation of possible matrix effects that may result in suppression or enhancement of the ion signal. Using the dilution method shown by Cavaliere et al. [58], ? The dilution at which matrix effects disappeared for the metabolite of interest was determined.

Kiwi juice (kiwi 1) ? been diluted in sequence and yes? found that at a dilution of 1:1600 for quinic acid and 1:100 for caffeic acid, the matrix effect was no longer present, although the two metabolites were still well detectable.

Subsequently, the absence of interference resulting from the matrix effect? was further verified, for both metabolites in kiwi juice, using the addition method, as reported by Toffali and collaborators [59]. Chromatographic peak areas were compared in three groups of samples: (1) diluted kiwi juice only; (2) the same kiwi juice as group 1 added with 1.5 ng/?L of D-(-)-quinic acid and 125 pg/?L of caffeic acid 3-?-D-glucoside; (3) solution of pure standard compounds, using the same compounds as group 2 at the same concentrations. 1 ?L of each diluted solution ? been analyzed three times by UPLC-ESI-MS and was not? No matrix effect was ever found for both metabolites. The peak area of the metabolites of interest? was normalized by the dilution factor and compared to a calibration curve obtained using authentic commercial standards.

Quantification of quinic acid in serum Quinic acid? was also quantified in the serum of perfused and non-perfused animals treated with kiwi and sacrificed after 30 minutes. ? a final dilution of 1:80 was used. The matrix effect? was determined through the addition method, as described by Toffali and collaborators [59], and using the following three groups of samples: (1) serum-only sample; (2) the same serum sample as group 1 spiked with 18 pg/?L of D-(-)-quinic acid; (3) pure quinic acid solution, using the same concentrations as in group 2. 1 ?L of each dilution ? was analyzed three times by UPLC-ESI-MS and a small matrix effect ? was detected, determined and used to obtain correct absolute quantification of the metabolite.

Data analysis

Statistical analysis? was performed using Prism v9.0

Significant differences between samples were determined by one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. UPLC-ESI-MS data processing ? already? described in the ?Materials and Methods? section. (see section ?Data processing and metabolite identification?).

Results

In vivo experiments on well-validated behavioral models combined with metabolomic approaches reveal unique activity. kiwi antidepressant

Chronic kiwi treatment reduces immobility time? of the mouse in TST and FST

The possible activity? antidepressant of different dilutions of kiwi extracts (kiwi 1, kiwi 2 and kiwi 3) ? was studied with both TST and FST through the evaluation of the capacity? to reduce the time of immobility. Once the animals arrived, they were left to adapt to laboratory conditions for at least 2 weeks. After this period the animals were weighed and handled every day to get them used to it and therefore to reduce the stress of human handling, in fact the intra gastric administration (IG)? very stressful because? causes immediate changes in physiological parameters indicative of stress (increased blood corticosterone, glucose, growth hormone and increased heart rate and blood pressure). For this reason, 1 week before starting the procedure on the animal, the mice were left to adapt to the administration of IG (IG of water twice a day) because ? reported in the literature that after 4 days there is? a decrease in all the parameters mentioned above. For kiwi extracts, vehicle and neutralized juice, mice were treated chronically for 10 days through the administration of IG and tested after 90 minutes, while fluoxetine (Prozac?) ? was used as a positive control drug and administered intraperitoneally (IP) 30 minutes before the test at a concentration of 20 mg/kg [60]. All groups were compared to the negative control, vehicle-treated group. Kiwi 1 strongly reduces immobility time? in both tests compared to the vehicle-treated group [F(5,81)=16.00, p<0.0001; F(5,81)=15.29, p<0.0001 for TST and FST respectively] while kiwi 2 and kiwi 3 were less active, with activity? of kiwi 3 which disappears completely in FST (Figure 4). Fluoxetine, the positive control did not work in the FST paradigm, but this problem? examined below in the discussion section. The presence of possible locomotor impairment? was assessed on day 8 in an OFT, as previously described. Not ? No motor impairment was found between the different groups of animals (Figure 5).

The results of the comparison of the samples treated with kiwi juice and the vehicle showed that only a few fruit-specific metabolites were present in the serum and brain of the mouse. ? A multivariate statistical analysis of the data on the metabolite quantification matrix was conducted to discriminate the differences between these two groups. The metabolite quantification matrix, obtained using Progenesis QI and containing the m/z values and their relative abundances in each of the samples, is ? was subjected to orthogonal and bidirectional partial least squares discriminant analysis (O2PLS-DA), using SIMCA 13.0 (Umetrics), after Pareto scaling and data centering operations. Even following the use of both types of chromatographic analyzes (based on C18 and HILIC columns, to include the greatest possible number of metabolites, from highly polar ones to those with medium and low polarity), the kiwi metabolites found in There were few serum samples and, notably, only detectable metabolites in the kiwifruit-treated group 1 compared to vehicle-fed mice (Figure 6). The majority of them ? was presumably identified and two metabolites were found to be quinic acid, a secondary metabolite abundantly present in kiwis, and caffeic acid sulfate, which is probably the murine catabolite of caffeic acid-based molecules present in kiwi fruit (glucosides and esters of caffeic acid, see Table 1). The parameters used for the identification of these two metabolites are discussed in detail below.

Two other metabolites found only in kiwi-treated mice, presumably identified as indoxyl sulfate and hydroxybenzene sulfate, are not kiwi metabolites: they could be catabolites of some metabolites in the fruit or endogenous mouse metabolites that increase following treatment.

The same two metabolites, quinic acid and caffeic acid sulfate, were also found in brain samples. The relative quantification, obtained from the above UPLC-ESI-MS analysis, of quinic acid and caffeic acid sulfate in serum and brain tissue of mice treated with all kiwi juice dilutions and with kiwi juice "neutralized", ? shown in Figure 7. Quinic acid? was detected only with the C18 chromatographic condition, while caffeic acid sulfate is better detected with the HILIC condition. Since? Caffeic acid sulfate levels detected in the brain using the HILIC technique were below the Limit of Detection (LOD, the lowest amount of a substance that can be determined with a level at least 5 times above background), they did not have been evaluated.

As expected, both molecule levels decreased linearly with increasing dilutions of kiwifruit extracts [F(3,20)=46.35, p<0.0001; F(3,20)=9.644, p<0.0004 of quinic acid, for serum and brain respectively, and of caffeic acid sulfate only for serum F(3,20)=16.34, p<0.0001] . Furthermore, by comparing the metabolomes, already? previously characterized, of the kiwi with that of the "neutralized" kiwi, it was possible to see that caffeic acid derivatives were almost absent in the "neutralized" kiwi juice and the abundance of quinic acid was reduced (Table 1). These observations are in line with the very low recovery of these metabolites in mice treated with "neutralized" juice. Furthermore, the low level of these metabolites in "neutralized" juice, which was inactive in the TST and FST behavioral tests, reinforces the hypothesis that one or both of these molecules may be at least partially responsible for the behavioral activities of kiwi juice in mice.

The kinetics of appearance and elimination of quinic acid and caffeic acid sulphate in the serum and brain of mice, following the administration of kiwi, were followed using a pharmacokinetic (PK) approach on different groups of mice treated only with undiluted kiwi (1 kiwi) and sacrificed at different times. The time points used in this experiment are as follows: 15min, 30min, 45min, 1h, 1h15 min, 1h30min, 2h, 4h, 8h and 24h. Figure 8 shows the relative quantification, in units? arbitrary, of quinic acid and caffeic acid sulfate in serum and brain samples. Not surprisingly, the relative levels of the two molecules decrease as time passes.

The presence of quinic acid and caffeic acid sulfate in the whole brain tissue homogenate is not ? necessarily indicative of the capacity? of the molecules to overcome the blood-brain barrier (BBB) and, consequently, to reach the brain parenchyma, as their presence can? be due to the blood vessels that supply the brain. To verify the real capacity? of crossing of the BBB by quinic acid and caffeic acid sulphate? the perfusion technique was used. This allowed blood to be taken from the brain and therefore the real presence of quinic acid and caffeic acid sulphate in the tissue to be assessed. In practice, the animals after treatment (administration of kiwi juice) were anesthetized and the entire blood volume was was flushed through cold 0.9% saline, and after 5 minutes of perfusion, brain samples were collected and frozen in liquid nitrogen. The samples were stored at ? 80?C until subsequent LC-MS analyses. Figure 9 shows the relative quantification in units? arbitrary concentrations of quinic acid and caffeic acid sulfate in the brain after perfusion: the post-perfusion levels of both metabolites are lower than in non-perfused samples (about half), but still detectable, proving that the two molecules cross the blood-brain barrier and are able to enter the brain parenchyma [F(5,5)=6.112, p=0.0020 and F(5,5)=18.61, p=0.0014 for brain quinic acid and caffeic acid sulfate respectively].

Bioavailability? of kiwi indolealkaloids. The bioavailability? of serotonin and tryptamine? was evaluated, after chronic treatment, in serum and brain samples of mice through UPLC-ESI-MS analysis.

In general, serotonin and tryptamine in the diet are usually degraded by MAO isoenzymes present in the intestine and for this reason they are not bioavailable, but you can't? exclude a role for these molecules when eaten within a composite phytocomplex, due to the activity? synergy between the different components. Therefore, in principle, any kiwi inhibitors of MAO-A and B could change the fate of serotonin and tryptamine taken with the fruit, making them more bioavailable.

In the data contained in the present invention, not? Tryptamine was found in both samples analyzed, while for serotonin no differences were found between mice fed kiwifruit 1 and the control group. To definitively exclude bioavailability? of kiwi serotonin, ? PK analysis was performed on mice treated with a dilution of kiwifruit 1 added with a quantity? detectable deuterated serotonin (Serotonin-d4).

In the pharmacokinetic analysis, no differences in administered serotonin-d4 were found (data not shown).

Quinic acid? capable of mimicking the effects of kiwi juice on mouse models of depression Identification and quantification of putative active kiwi metabolites

In previous experiments (Materials and Methods) only a few kiwi fruit metabolites were found in the sera and brains of kiwi-treated mice and presumably identified, namely quinic acid and caffeic acid sulfate, a putative murine metabolite of caffeic acids. present in the kiwi fruit.

Quinic acid, abundantly present in kiwi fruits in free form, is was identified using commercially available authentic quinic acid, based on retention time (0.81 min) and mass-to-charge ratio (m/z) (expected m/z: 191.0555, detected m/z: 191, 0550, mass error: 2.6 ppm) (Figure 10).

Caffeic acid sulfate, a putative murine metabolite derived from kiwifruit caffeic acids, is was identified on sera and brains based on mass-to-charge ratio (m/z) (predicted m/z: 258.991, detected m/z: 258.990, error mass: 3.8 ppm) and fragmentation pattern (ms/ms) , as the commercial standard was not available: after fragmentation, the metabolite generated a fragment of m/z 179.0330, corresponding to caffeic acid (predicted m/z: 179.0344) and a fragment of m/z 135 .0423 (expected m/z 135.0446), which is ? a typical and diagnostic fragment of caffeic acid, generated by its decarboxylation (caffeic acid-COOH-H<+>).

As shown in Table 1, the kiwi juice used in the behavioral tests contained four caffeic acid hexoses, one caffeic acid dihexose, one caffeic acid glucuronide, and two unidentified caffeic acid derivatives. Caffeic acid hexoses were identified based on their mass-to-charge ratio (m/z) (predicted m/z: 341.0872, detected m/z: 341.0873, mass error 0.29 ppm) and to the fragmentation patterns (ms/ms, all generated fragments with m/z 179.0344 /-0.005, corresponding to caffeic acid, and 135.0446 /-0.005, corresponding to caffeic acid ?COOH-H<+>) . Three hexoses of caffeic acid showed a quantity interesting in kiwi juice and one of these, the caffeic acid hexose pi? abundant, ? was precisely identified as caffeic acid 3-?-D-glucoside using a commercially available authentic standard and by comparison with retention time (4.6 min), mass-to-charge ratio (m/z), and fragmentation pattern (figure 10). The other two peaks showed a retention time of 3.98 min and 5.29 min. Based on their mass-to-charge ratio (m/z) and fragmentation profiles, these were presumably identified as structural isomers of caffeic acid 3-?-D-glucoside. On the other hand, the detection of one of the other two peaks in the authentic commercial standard of caffeic acid 3-?-D-glucoside suggests that the peak at 4.6 minutes and the other peak at 5.29 minutes are isoforms which easily convert to each other (Figure 10).

In order to verify whether the activity? kiwi antidepressant, studied in ?Materials and Methods?, could derive from quinic acid and caffeic acid glucosides, i.e. those kiwi molecules that managed to reach both the sera and the brains of the treated animals, the tests were carried out behavioral with pure molecules, at the same concentration as the fruit.

Therefore, quinic acid and the two putative isoforms of caffeic acid 3-?-D-glucoside in the fruit had to be quantified exactly.

For the quantification of quinic acid in kiwi juice? a calibration curve was constructed with concentrations of the commercial standard between 0.5 and 5 ?g/?L with an R2 of 0.9993. Three measurements were performed with a 1:1600 dilution of kiwi juice and the data were compared to the calibration curve, resulting in an exact quantification of 491.26 ? 1.80 mg/100 g fresh weight of fruit.

The calibration curve of caffeic acid 3-?-D-glucoside ranged from 31.25 to 312.5 pg/?L with an R2 of 0.9995. Three different measurements of a 1:100 dilution of kiwi juice were compared to the calibration curve to obtain an exact quantification of 6.475 ? 0.026 mg/100 g fresh weight of fruit.

Quinic acid? able to partially mimic the activity? kiwi antidepressant in chronic treatment

The capacity? of the aforementioned specialized kiwi metabolites to reduce the time of immobility? ? was evaluated in both the TST and FST paradigms using the exact concentration of the metabolites present in the fruit. Kiwi extract at kiwi dilution, vehicle (negative control), quinic acid and caffeic acid glucoside at the same concentration as kiwi 1 and a combination of the two metabolites were used to chronically treat mice for 10 days via IG administration. The TST and FST tests were performed on day 10, 90 minutes after the administration of juice/metabolite, while Escitalopram (Cipralex?) ? was used as a positive control drug and administered intraperitoneally (IP) 30 minutes before testing, at a concentration of 10 mg/kg. As in the previous experiment (see section ?Chronic treatment of kiwi reduces mouse immobility time in TST and FST? and figure 4), kiwi 1 confirmed its ability to to greatly reduce the time of immobility? in both tests compared to the vehicle-treated group. Quinic acid administered alone and in combination with caffeic acid also had a strong to reduce the time of immobility? while caffeic acid alone was not active, although in this group it was was a high variability observed? inter-subjective [F(5,66)=26.33; p<0.0001; F (5,66)=11.54, p<0.0001 for TST and FST respectively] (Figure 11). Escitalopram (ESC), in this case, ? was a good positive control for both tests compared to fluoxetine, which works only on the TST (see Figure 4). The activity? locomotive? was evaluated on day 8 in OFT, to check for possible locomotor impairment, as previously described (see ?Open field testing? section). Not ? No motor impairment was found among different groups of animals (Figure 12). Therefore, the administration of quinic acid at the same concentration found in kiwi can partially mimic the activity? kiwi antidepressant on mice, indicating that quinic acid is one of the fruit's active antidepressant molecules.

Following the behavioral experiments, serum and brain samples were collected and analyzed by UPLC-ESI-MS. Figure 13 shows the relative quantification, in units? arbitrary, of caffeic acid sulfate and quinic acid in both serum and brain samples of mice treated with kiwi 1, quinic acid, and a combination of quinic acid and caffeic acid glucoside, which were the effective treatments in the TST and FST. Both results obtained with the two chromatographic conditions (C18 and HILIC) are shown [F(2,33)=63.46, p<0.0001; F(2,33)=35.96, p<0.0001 of quinic acid for serum and brain respectively and of caffeic acid sulfate for serum only F(2,33)=18.86, p<0.0001] .

As previously reported in ?Materials and Methods?, quinic acid was detectable only under C18 chromatographic conditions, while caffeic acid sulfate was ? best detected with HILIC chromatography. However, the levels of caffeic acid sulphate in the brain were not evaluated because, although still detectable, they were below the LOD (the lowest amount of a substance that can be determined with a level at least 5 times higher at the bottom) of the instrument.

? Interestingly, the levels of quinic acid and caffeic acid sulfate in sera and brain were lower in all treatments with pure metabolites (QA, CA or QA+CA) compared to the levels of the same molecules in samples treated with untreated kiwifruit. diluted (kiwi 1). Since? quinic acid and caffeic acid glucosides were administered at exactly the same concentration as the phytocomplex, so suggests that other molecules or components are present in the fruit that could increase adsorption and/or stability? of metabolites in the living organism. Therefore, the lower activity? of pure quinic acid and of the quinic acid-glucoside caffeic acid combination compared to the treatment with kiwi 1 can? be due to the activity? of other low-level and undetectable kiwi molecules or with limited absorption and/or stability? of quinic and caffeic acid when taken as pure molecules.

Kinetic analysis and quantification of serum in mice treated with quinic acid

PK experiments were performed on different groups of mice treated with quinic acid and sacrificed at different timepoints. The timepoints used in this experiment are as follows: 15min, 30min, 45min, 1h, 1h15 min, 1h30min, 2h, 4h, 8h and 24h. Figure 14 shows the relative quantification in units? arbitrary levels of quinic acid in both serum and brain samples. Obviously and as seen with kiwifruit treatment, the relative levels of quinic acid decrease over time.

Also in this case, as for the entire kiwi phytocomplex, is the actual cerebral penetration? was evaluated using the perfusion technique in cold 0.9% saline buffer for 5 minutes (more details are presented in Materials and Methods). Figure 15 shows the relative quantification in units? arbitrary quantities of cerebral quinic acid under C18 chromatographic conditions. Brain levels after perfusion were, as expected, higher low compared to non-perfused samples (see also figure 9). Furthermore, the brain levels of QA- and kiwi-treated mice after the perfusion technique were compared. The levels of mice treated with QA alone were much lower. low compared to the other group [F(5,5)=6.189, p<0.0001 for brain quinic acid (a) and Finteraction(1,20)= 1.147, p=0.2970; Ftreatment(1,20)= 18.03, p=0.0004; Fperfusion(1,20)= 43.25, p<0.0001 (b)].

Furthermore, ? The exact quantification of quinic acid in the serum and brain of animals treated with an acute administration of quinic acid at the same concentration as kiwi 1 (undiluted kiwi) was also proven. ? A calibration curve was created with authentic quinic acid standard concentrations ranging from 6 to 60 pg/?l with an R2 of 0.9969. Eight different serum samples of 1:80 dilutions in three replicates were plotted against the calibration curve. This dilution? was chosen why? was it the best shape? diluted which allows us to clearly detect quinic acid in the serum samples even if a small matrix effect was still present. For this reason, the matrix effect is was determined through spiking experiments and the data were corrected to obtain the exact quantification which was 1.451 ? 0.318 ng of quinic acid/ul of serum.

Quantification in the brain is not? was it possible why? during the dilution of brain extracts the signal due to quinic acid? disappeared at dilutions where the matrix effect was still too high to allow accurate quantification. This isn't it? surprising, given that phospholipids are among the main responsible for the matrix effect and given that the brain contains quantities very relevant of phospholipids. For caffeic acid, accurate absolute quantification was not possible in both sera and brains.

Claims (28)

1) Product consisting of a) quinic acid, a derivative thereof or a precursor thereof, or b) a pharmaceutical composition comprising or consisting of quinic acid, a derivative or precursor thereof, as an active ingredient, together with one or more? excipients and/or adjuvants, for use in the treatment of a mood disorder. 2) Product according to claim 1, for use according to claim 1, wherein said mood disorder is? chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder and depressive disorder due to other medical condition, premenstrual dysphoric disorder, mood dysregulation disorder disruptive. 3) Product according to claim 1 for use according to any of indications 1-2, wherein said derivative is? selected from the group consisting of an ester or ether of quinic acid with a sugar or an ester of quinic acid with an organic acid, an amide of quinic acid with a nitrogen-containing metabolite, such as an amino acid, a pharmaceutically acceptable salt of quinic acid . 4) Product according to claim 3 for use according to any of claims 1-2, wherein the quinic acid ester with a sugar is ? chosen from the group consisting of quinoyl glucose, quinoyl galactose, while the quinic acid ether with a sugar is the glycoside of quinic acid. 5) Product according to claim 3 for use according to any of claims 1-2, wherein the organic acid is selected from the group consisting of pharmaceutically acceptable aromatic organic acids such as cinnamic acid, hydroxycinnamic acids, benzoic and hydroxybenzoic acids or acids aliphatic organic acids such as malic acid, citric acid, isocitric acid, fumaric acid, lactic acid, malonic acid, succinic acid, tartaric acid, provided that caffeic acid is excluded. 6) Product according to claim 3 for use according to any of claims 1-2, wherein the quinic acid salt is? a salt of quinic acid with a pharmaceutically acceptable alkali or alkaline earth metal. 7) Product according to claim 3 for use according to any of claims 1-2, wherein the precursor is? shikimic acid. 8) Product according to any one of claims 1-7, for use according to claim 1-7, wherein the pharmaceutical composition further comprises caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids. 9) Product according to any of claims 1-8, for use according to claim 1-8, wherein the product is administered in a quantity such that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably 2 g to 6.5 g, optimally 3.5 g to 4.5 g. 10) Product according to claim 9 for use according to any of claims 1-8, wherein the product is administered orally. 11) Product according to any of claims 1-10, for use according to claim 1-10, wherein the pharmaceutical composition is? in a form suitable for oral administration, such as nanoparticles, tablet, capsule, syrup. 12) Plant product composed of quinic acid, a derivative or a precursor thereof for use in the treatment of a mood disorder, provided that the plant product is not kiwi or grape. 13) Herbal product comprising quinic acid, a derivative or a precursor thereof for use in the treatment of a mood disorder, wherein the herbal product is administered in a quantity such that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably 2 g to 6.5 g, optimally 3.5 g to 4.5 g. 14) Herbal product according to any of claims 12-13 for use according to any of claims 12-13, wherein said mood disorder is? chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder, and depressive disorder due to another medical condition, premenstrual dysphoric disorder, mental dysregulation disorder disruptive mood. 15) Plant product according to claim 12, for use according to any of claims 12-13, wherein the precursor is? shikimic acid. 16) Herbal product according to any one of claims 12-13 for use according to any one of claims 12-13, wherein the herbal product further comprises caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids. 17) Plant product according to any of claims 13-16, for use according to claims 13-16, wherein the plant product is? a fleshy fruit such as kiwi or other berries such as grapes, raspberries, blueberries, blackberries, gooseberries, stone fruits such as cherries, peaches, apricots, apples such as apples and pears, peppers such as melons and watermelons, hesperides such as oranges, lemons, grapefruits, kumquats, clementine, preferably kiwi. 18) Herbal product according to any of claims 12-17, for use according to claims 12-17, wherein the herbal product is administered orally. 19) Combination of quinic acid, a derivative or a precursor thereof with caffeic acid or other pharmaceutically acceptable hydroxycinnamic acids for separate or sequential use in the treatment of a mood disorder. 20) Combination according to claim 19 for use according to claim 19, wherein said mood disorder is? chosen from the group consisting of depressive disorder, such as major depressive disorder (MDD), persistent depressive disorder (dysthymia), substance/medication-induced depressive disorder and depressive disorder due to other medical condition, premenstrual dysphoric disorder, mood dysregulation disorder disruptive. 21) Combination according to claim 19 for use according to any of claims 19-20, wherein said derivative is? selected from the group consisting of an ester or ether of quinic acid with a sugar or an ester of quinic acid with an organic acid, an amide of quinic acid with a nitrogen-containing metabolite, such as an amino acid, a pharmaceutically acceptable salt of quinic acid . 22) Combination according to claim 19 for use according to any of claims 19-20, wherein the quinic acid ester with a sugar is chosen from the group consisting of quinoyl glucose, quinoyl galactose, while the quinic acid ether with a sugar is a glycoside of quinic acid. 23) Combination according to claim 21 for use according to any of claims 19-20, wherein the organic acid is? selected from the group consisting of pharmaceutically acceptable aromatic organic acids such as cinnamic acid, hydroxycinnamic acids, benzoic and hydroxybenzoic acids or aliphatic organic acids such as malic acid, citric acid, isocitric acid, fumaric acid, lactic acid, malonic acid, succinic acid , tartaric acid, provided that caffeic acid is excluded. 24) Combination according to claim 21 for use according to any of claims 19-20, wherein the quininic acid salt is? a salt of quinic acid with a pharmaceutically acceptable alkali or alkaline earth metal. 25) Combination according to claim 21 for use according to any of claims 19-20, wherein the precursor is? shikimic acid. 26) Combination according to any one of claims 19-25, for use according to claim 19-25, wherein quinic acid, a derivative or a precursor thereof is administered in a quantity such that the daily dosage of quinic acid varies from 10 mg to 16 g, preferably from 0.5 g to 8.5 g, more? preferably from 1 g to 8 g, even more? preferably 2 g to 6.5 g, optimally 3.5 g to 4.5 g. 27) Combination according to any of claims 19-26, for use according to claim 19-26, wherein the combination is? administered orally. 28) Combination according to any of claims 19-27, for use according to claim 19-27, wherein the combination is? in a form suitable for oral administration, such as nanoparticles, tablet, capsule, syrup.