Chromatographic analysis of Crataegus azarolus and possible psychiatric implications

Introduction

Pharmaceutical herbals and their use in psychiatry are common in different cultures and medical practices. Hawthorn (Crataegus) is an herb comprising over 280 species belonging to the rose family, with multiple medical applications dating back to the first century A.D., as described in De Materia Medica of Dioscorides [1], 2]. It was also used in traditional Chinese and Native American medicine [3]. Flowers, leaves, and berries of hawthorn are used separately for both medical and culinary purposes, including supplements, teas, jams, or wine [4]. Modern techniques have allowed researchers to identify available compounds in different hawthorn species. Consistent with findings across hawthorn species, studies examining Crataegus azarolus have identified phenolic compounds, flavonoids, tannins, terpenoids, resins, and diterpenes, with antioxidant activities [5], 6]. Leaves and berries exhibited distinct compositions with respect to polyphenolic compounds and mineral content, which also varied according to bioclimatic factors such as humidity [7], 8]. Phytochemicals in hawthorn have been widely suggested to exert beneficial properties, including anti-inflammatory, antihyperglycemic, neuroprotective, and cardioprotective effects [9], 10]. While clinical evidence in these areas remains largely absent, most clinical research on hawthorn has focused on heart failure [11]. Most hawthorn products in the U.S. are currently marketed as alternative treatments supporting cardiovascular health, including blood pressure regulation. According to the National Institutes of Health (NIH) Dietary Supplement Label Database, approximately 620 different products from 270 distinct brands in the U.S. market contain hawthorn as an ingredient [12].

Apart from its cardiovascular applications, the effects of hawthorn on the central nervous system are less studied in the literature, although folkloric reports mentioned benefits on nervousness and insomnia. Furthermore, there are multiple brand formulations, especially in Europe, that combine herbals, including Hawthorn subspecies, marketed for mild anxiety and insomnia (e.g., Sympathyl, Euphytose, Neurofast, Pasalix). Some preclinical studies have suggested anxiolytic and hypnotic effects, most likely attributable to its polyphenolic compounds [13]. A preclinical study indicated that Crataegus pinnatifida shows anxiolytic and antidepressant-like effects, comparable to escitalopram [14]. It also showed analgesic effects in an experimental mouse model, which were reversed by naloxone, suggesting the involvement of central opioidergic systems [15]. Interestingly, there are clinical trials assessing particular Crataegus species in the treatment of anxiety with reported benefits over placebo [16], 17]. However, these studies involved combinations with other herbal ingredients. There is a lack of literature analyzing and discussing the compounds in hawthorn and their potential psychiatric effects. Therefore, we aimed to investigate the organic volatile compounds and the anion and cation contents of C. azarolus, while discussing how these findings could inform its possible psychiatric effects.

Materials and methods

Results

The total-ion chromatogram (TIC) of the methanolic C. azarolus extract is shown in Figure 1, with the late baseline rise attributable to column bleed (>32 min) omitted for clarity. Peak areas were normalized and expressed as relative percentages. GC–MS library matching using the NIST and Wiley databases yielded 13 tentative bioactive compounds. The most abundant signals tentatively corresponded to oleic acid, followed by a broad cluster including fatty acids such as α-linolenic, linoleic, palmitic, and stearic acids, together forming the most abundant major chemical group in the extract. Quercetin (3,4′,7-O-trimethylquercetin) and lucenin-2 were tentatively predicted as flavonoids. Additional peaks were tentatively assigned to ethyl iso-allocholate, ergosta-5,22-dien-3-ol, 4-methyl-benzaldehyde, pregnane-3,11,21-triol, and 24,25-dihydroxy-vitamin D3. Digitoxin was also predicted as a cardiac glycoside. Tentatively identified compounds are presented with retention time (min), molecular formula, molecular weight, class name, and match scores in Table 1.

Figure 1:

The total-ion chromatogram (TIC) of the methanolic extract of Crataegus azarolus.

Anion and cation content (F⁻, Cl⁻, NO₂⁻, NO₃⁻, Na⁺, K⁺, Mg²⁺, Ca²⁺, Li⁺) through ion chromatography is shown in Table 2. Chromatographic quality, column, and wellness plots were appropriately ranged. Linear calibration was achieved for all quantified ions. Coefficients of determination (R²) were 0.996–0.999 for Na⁺, K⁺, Mg²⁺, Ca²⁺, and Li⁺, and 0.988–0.995 for F⁻, Cl⁻, NO₂⁻, and NO₃⁻. Potassium dominated the cation spectrum at 30.4 mg per 100 g dry plant material, higher than magnesium, which was 2.36 mg per 100 g. Sodium and calcium were minor constituents, contributing 0.62 and 0.21 mg per 100 g, respectively. Lithium was 0.001 mg per 100 g, which can be considered at trace levels. Among the anions, fluoride was prominent at 7.67 mg per 100 g, whereas chloride (0.94 mg), nitrite (0.21 mg), and nitrate (0.48 mg) remained below 1 mg per 100 g.

Discussion

Secondary metabolites, including flavonoids and other organic compounds from herbal sources, are typically considered beneficial and involved in treating various diseases. GC–MS and ion chromatographic analysis of C. azarolus tentatively suggested several bioactive compounds, such as flavonoids, fatty acids, sterols, and vitamin D derivatives. Although ionic components were detected, their low abundance limits the ability to hypothesize any impact on neuropsychiatric effects from the present data. It is critical that GC–MS identification cannot infer functional claims without additional experiments, while a brief review of the tentative compounds could provide a context for future studies and hypotheses.

Quercetin is a flavonoid and is widely recognized for its anti-inflammatory, antioxidant, antimicrobial, and cardiovascular effects in preclinical studies. Experimental studies suggests that quercetin may be beneficial in slowing neurodegenerative processes and exerting neuroprotective effects through multiple mechanisms [18]. Several in vivo studies have further reported antidepressant-like effects, while these findings remain limited to animal models [19], 20]. Proposed mechanisms may be mediated by regulating the hypothalamic-pituitary-adrenal (HPA) axis, serotoninergic and cholinergic neurotransmission [21], 22]. In a rodent study involving lipopolysaccharide-induced neuroinflammation, quercetin administration reduced inflammatory markers, increased brain-derived neurotrophic factor (BDNF) mRNA levels, and decreased inducible nitric oxide synthase (iNOS) mRNA levels, leading to improvements in anxiety-like symptoms [23]. Additionally, it has been suggested to reduce anxiety in rodents with traumatic brain injury by attenuating HPA axis activity, with effects comparable to diazepam [24]. Human studies with quercetin for various neuropsychiatric conditions are significantly limited and clinical efficacy cannot be inferred with current literature.

Another flavonoid, lucenin-2, was also tentatively detected in C. azarolus and has been reported to exhibit anti-inflammatory effects in experimental studies [25], 26]. Lucenin-2 (luteolin 6,8-di-C-glucoside) is a glycosylated form of luteolin (3′,4′,5,7-tetrahydroxyflavone), which was not directly identified in our analysis but is important for providing structural context. Luteolin has been more characterized in preclinical studies related to neuropsychiatric effects and receptor-level interactions, however, these findings should not be interpreted as evidence for similar effects of lucenin-2 [27], [28], [29]. The literature lacks experimental studies examining lucenin-2 and its potential biological effects.

Polyunsaturated fatty acids (PUFAs) are known to exert various psychiatric effects and contribute to mood regulation. Hawthorn was tentatively found to contain the omega-3 PUFA alpha-linolenic acid and the omega-6 PUFA linoleic acid. PUFAs are essential for synaptic plasticity, and both omega-3 and omega-6 fatty acids are considered necessary for healthy brain function [30]. Reduced omega-3 PUFA levels have been associated with neurodevelopmental, mood, and psychotic disorders, whereas findings related to omge-6 PUFAs and mood remain inconsistent [31], [32], [33]. Experimental studies hypothesized that PUFAs may involve in neuroinflammation, modulation of endocannabinoid system, and impact HPA axis [34].

The association between oleic acid and depressive symptoms is inconclusive, with studies reporting positive, negative, or no correlation [35], 36]. However, some evidence suggests that oleic acid may contribute to sleep regulation and could be deficient in individuals with depression-related sleep disturbances [37]. A cross-sectional study found that higher intake of oleic acid and alpha-linolenic acid was associated with lower anxiety levels [38]. Palmitic acid is typically considered proinflammatory and has been associated with increased anxiety-like behavior [39]. Interestingly, a postmortem study reported increased levels of palmitic acid and decreased levels of oleic acid in the amygdala of patients with major depressive disorder compared to healthy controls [40]. Another saturated fatty acid, stearic acid, was also tentatively identified in the GC–MS analysis. In preclinical studies, stearic acid has been suggested to exhibit anti-inflammatory and antidepressant-like effects [41], 42]. Furthermore, an in vitro study on cortical neurons reported neuroprotective effects of stearic acid against glutamate excitotoxicity and peroxidative injury [43]. Taken together, these predominantly preclinical data provide limited context regarding the relevance of the tentatively identified fatty acids to mood and central nervous system function, precluding direct interpretation of these findings.

Various phytosterols were also predicted in hawthorn. Ergosta-5,22-dien-3-ol has been described in experimental studies as involving in antioxidative and anti-inflammatory processes, and ergosterol derivatives have been proposed to exhibit antidepressant-like effects in preclinical models [44], 45]. Vitamin D derivatives represent a relatively well-studied area in psychiatry and may be beneficial for mood, anxiety, and sleep through mechanisms involving the regulation of serotonin and melatonin systems [46], 47]. Although, it is critical to highlight that these identifications might indicate structurally related compounds rather than directly reported chemicals. Pregnane-3,11,21-tetrol, cyclic 20,21-(butyl boronate), was also tentatively detected in C. azarolus and has also been reported in other plants, including Phlomis stewartii and Passiflora incarnata [48], 49]. It is a polyhydroxylated steroid that may be considered a sapogenin-type fragment. While this interpretation should be made with caution, sapogenins have been reported in hawthorn and have been investigated in preclinical models for its anti-inflammatory, anxiolytic, and antidepressant-like properties [50], 51]. Ethyl iso-allocholate was another steroid derivative that was tentatively identified, for which various in vitro studies suggesting anti-inflammatory and anti-oxidant activities [52], 53].

Tentative identification of C. azarolus included digitoxin, which may reflect the presence of structurally related phytochemicals, as the literature indicates that hawthorn species contain alkaloids structurally similar to cardiac glycosides [54]. Digitoxin is a phytosteroid and cardiac glycoside with well-established effects on the heart, but its role in mental health remains not well defined. An early study suggested that Na⁺/K⁺-ATPase inhibitors like digitoxin may counteract neuronal depression induced by monoamines such as serotonin and noradrenaline [55]. A recent study proposed that lithium and cardiac glycosides may share overlapping molecular mechanisms and transcriptional responses, raising the possibility that cardiotonic steroids in relation to mood impact [56]. These findings remain preliminary and do not permit direct interpretation. Moreover, there is a paucity of literature examining cardiac glycosides and their association with neuropsychiatric effects at clinically relevant doses.

The overall ionic profile of hawthorn reflected a generally low level of inorganic anions, while providing detectable amounts of fluoride and potassium. Although the effects of lithium and magnesium on mood are well studied [57], 58], the levels of these cations in the sample were far below the doses associated with direct neuropharmacological effects. On similar context, while nitrate and nitrite ions might have central effects [59], the levels in C. azarolus sample were low and it is unlikely provide any direct effects. This composition may be characteristic of C. azarolus compared to other Crataegus species or different varieties within azarolus. It is unclear if having trace inorganic ion content alone or in synergy with volatile polyphenolic compounds has any psychiatric effects, and this cannot be inferred from presented data. Overall, there is no direct preclinical or clinical evidence linking C. azarolus to psychiatric effects, and existing claims remain speculative, warranting further investigation.

Conclusions

Hawthorn (C. azarolus) was tentatively included various flavonoids with possible distinct benefits that may contribute to both general and mental health. Hypothetically, these compounds may influence mental health by reducing inflammation, enhancing antioxidant mechanisms, affecting monoaminergic systems, or modulating trophic factors such as brain-derived neurotrophic factor (BDNF). Although C. azarolus contains inorganic ions at levels unlikely to directly affect the central nervous system, additional studies are needed. Further investigation into hawthorn and its potential applications in psychiatry is warranted through preclinical studies and confirmatory analyses.