Animal model-based adjunctive herbal therapy in autism spectrum disorder: Therapeutic advances and prospects

Autism spectrum disorder (ASD) is a neurodevelopmental condition whose symptoms are noticeable in early postnatal life, affecting how children interact with their social environment. The global prevalence of this complex disorder is estimated to be 1% among children, but regional estimates vary substantially across countries depending on a panoply of genetic and environmental factors [1]. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, Text Revision (DSM-5-TR), autistic patients are diagnosed based on their difficulty communicating with people around them, their repetitive (stereotyped) behavior and restricted interests, and their inability to fulfill focus-demanding tasks at home and school. These clinical hallmarks of ASD are among the most difficult issues to deal with by both parents (or caregivers) and healthcare practitioners, thus posing a great socioeconomic burden worldwide [2].

Individuals with ASD are recognized to need particular care, compared to those with well-defined neuropsychiatric diseases, because the core communicative disabilities are mostly linked with a wide range of comorbidities, such as attention-deficit hyperactivity disorder (ADHD), anxiety, depression, irritability, and epilepsy [3]. Due to this multi-aspect condition, there is a consensus that ASD cannot be completely cured. Current treatments seek to attenuate symptoms while improving social interaction capabilities in autistic subjects of all age ranges. However, though these treatments are basically behavioral, cognitive, and/or physical, many autistic patients are prescribed medications to reduce anxiety, depression, and hyperactivity or to help manage some difficult traits such as aggression and epileptic seizures [4]. Even though these approved drugs are effective in targeting each comorbidity apart, there is strong evidence that their use in the context of ASD may be negatively impactful in the long term, interfering with therapeutic efforts that aim at enhancing social abilities, mainly due to the poor understanding of pathophysiological mechanisms underlying ASD, which in turn makes difficult the process of predicting the outcomes in later life.

Psychotropic drugs used to treat mental health disorders, namely antidepressants, anti-anxiety medications, psychostimulants, antipsychotics, and mood stabilizers, are known for their acute and chronic toxicity, and their severe side effects can significantly alter the prognostic course in ASD patients, for whom physicians in charge should examine the best therapeutic option before a decision of giving them such drugs can be made [5]. Interestingly, there are important studies whose authors attempted to take advantage of herbs as a natural source of medications for treating various health conditions such as kidney injuries [6] and Alzheimer’s disease [7], or even for low-toxicity contraception [8], reflecting that herbal therapy is emerging as an alternative to many synthetic drugs including psychotropic ones [9].

While not limited in practice, as about 80% of the world’s population still relies on traditional medicines for their primary health care [10], herbal therapy for ASD is considered a recent approach, as shown by the relative novelty of studies. Its objective is to test plant extracts of sufficient efficacy and lower toxicity to be administered to autistic patients, alone or in conjunction with standard medical therapies. A larger consideration of herbal therapy that includes isolated plant molecules was applied in many experiments and trials on autism pathology; however, this usage does not reflect the whole benefit of medicinal herbs as usually accounted for in alternative medicine. The main candidate molecules that gave promising results are resveratrol [11] and sulforaphane [12], which are widely distributed in fruits, vegetables, and herbs and whose benefits are usually attained when eaten or decocted as a whole.

Therefore, the current report reviews animal studies relating to the use of plant extracts in autism therapy research and proposes a timeline chart for herbal therapy to aid in guiding future supportive care of autistic patients.

In order to collect research articles relevant to herbal remedies applied to autistic patients and/or tested on animals made autistic-like experimentally, the authors conducted a literature search using the Mendeley reference management software and online system, targeting a variety of databases. The keywords utilized were “autism”, “herbal”, “therapy”, “animal model”, “autistic-like”, “medicinal plant”, “supportive care”, and “alternative treatment”. A reference script of relevant studies was obtained, organized, and then exported as a BibTeX file to the JabRef software that allowed the creation of a detailed HTML table file showing the author(s), title, abstract, year of publication, journal title, and DOI for each study. Further screening of the obtained studies prompted the authors to exclude those concerned with cannabis extracts and cannabinoids because the application of these treatments is beyond the scope of this review.

ASD is a complex brain dysfunction of developmental origin, meaning that many genetic and environmental factors combine in utero (and/or at the perinatal stage) to yield an unknown degree of damage to the brain structure and function, leading to a range of symptoms that generally appear in the first two years of life [13]. Clinically, this disorder is not looked at as a single disease but, rather, as a group of disabilities that assembles mild-to-severe communicative, behavioral, and intellectual abnormalities in the affected individuals, making almost every autistic person unique from a clinical point of view [14]. Therefore, although autistic patients share similarities, the clinical setting of follow-up and treatment can significantly differ between them, raising a major socioeconomic burden in modern societies. Patients with ASD are characterized by three coexisting neurobehavioral issues: deficient social communication and interaction skills, atypical repetitive behavior, and restricted and obsessive interests [15]. More or less related characteristics were also reported in the literature but are not specific to ASD, making the whole condition challenging for patients themselves and their caregivers (Figure 1).

Three levels of severity are described in the DSM-5-TR, correlating with the increasing support that autistic patients need. However, there is no clear-cut picture of which therapies should be assigned to patients diagnosed with a certain level. Evidence-based educational and behavioral therapies are the most resorted to, attempting to effectively relay social information to ASD subjects, who are known to inadequately perceive and process social cues from their adjacent environment. Given the developmental character of this disorder, non-pharmacological approaches are meant to be applied to children but are frequently extended to be used in adolescents and adults. Table 1 summarizes the main conventional therapies and their most pertinent approaches that aim to reduce maladaptive behaviors while enhancing socialization and personal independence.

Table 1. Main conventional therapies and approaches catering to patients with ASD.

Of interest, conventional therapies are not optional in the context of ASD but, rather, mandatory methodologies of chronic management of this lifelong condition. When necessary, pharmacological interventions are joined to the approach that appears to best fit the patient’s needs. This is often the case because ASD subjects are prone to distress and irritability due to their inability to fulfill correct reciprocal conversations, thus feeling that their needs and expressions are neglected or misunderstood by family members and peers. Accordingly, anxiety, depression, and aggression are among the most frequent comorbidities found [16], prompting physicians to prescribe psychotropic drugs to help accomplish therapy goals, improve patients’ quality of life, and minimize family suffering. In this respect, while the use of such drugs in neuropsychiatric diseases is regarded as primary care, many reports claim that their administration to ASD patients can negatively interfere with educational and behavioral efforts to alleviate the causal core symptoms, prejudicing the desired rehabilitation [17]. In addition, only two drugs have been approved by the Food and Drug Administration (FDA) for indication in autistic individuals, namely aripiprazole and risperidone, with the other psychoactive drugs being prescribed off-label to target certain symptoms due to the beneficial effects observed in clinical studies, thus limiting the range of medication choices and increasing the uncertainty associated with unapproved drugs in this context. Furthermore, there is currently no evidence regarding the efficacy of these drugs in ameliorating the core features of ASD. Yet, the overall toxicity and side effects of psychotropic drugs are widely reported [18], making it possible to exacerbate the autistic condition through somatic ailments. For these reasons, some clinical studies have been undertaken to administer plant extracts to autistic patients, medicated or not with an approved drug.

An adjunct therapy with Ginkgo biloba (EGb 761®, 100 mg twice a day) for at least 4 weeks was carried out on adult male patients (19.4-22.4 years old) already diagnosed with autistic disorder. Obvious improvement, though non-significant, was reported for scores relating to irritability, hyperactivity, inadequate eye contact, and inappropriate speech, suggesting a modest therapeutic effect [19]. In a similar study conducted by the same author, using Hypericum perforatum (commonly known as St. John's wort) as an adjunct treatment (20 mg daily), those autistic symptoms were significantly attenuated [20]. Moreover, when Ginkgo biloba (Ginko TD® tablets, 40 mg or 60 mg twice a day, depending on body weight) was administered daily to autistic children (4-10 years old) under risperidone treatment for 10 weeks, no significant behavioral changes were found in comparison to counterparts receiving risperidone alone, suggesting that the adjunction of this plant extract to risperidone did not affect the treatment outcome [21]. The effect of a 4-week adjunctive Panax ginseng treatment (pure extract tablets, 250 mg once a day) was also observed in three male autistic patients (18.4-22.2 years old) undergoing educational and behavioral interventions. This herb, which is comparable with the neuroprotective and anticonvulsant drug Piracetam [22], led to slight, non-significant improvement in the outcomes of interest (i.e., irritability, hyperactivity, inadequate eye contact, and inappropriate speech) [23]. In another study, capsules of sulforaphane-rich broccoli sprout extract were administered daily to young male ASD patients (13-27 years old) for 18 weeks without any psychotropic medication. The authors reported a substantial improvement of behavior in these patients, with a significantly greater number of them showing better social interaction and verbal communication compared to placebo-treated counterparts, proposing that this low-toxicity compound can be addressed for the prenatal prevention of ASD as well as for the early treatment of young children with this disorder [24]. Based on traditional Chinese medicine, clinical trials were also devoted to evaluating whether herbal preparations (as pills, capsules, or decoctions) help achieve better behavioral outcomes in ASD patients. Overall, when such herbal medicines were combined with conventional therapy, most findings indicated a significant improvement in the assessment scale scores (reviewed in [25]). Taken together, these studies shed light on herbal therapy as a valuable supportive approach, making use of the beneficial effects herbs exert in various pathological conditions to treat ASD symptoms.

Animal models play a major role in understanding the etiological mechanisms of diseases and discovering new drugs to efficiently prevent or treat them. Prior clinical observations and findings allowed researchers to create autistic-like animal models that fulfill the main features of ASD, particularly the impaired sociability of the animal toward its conspecific mates. Rodents are particularly preferred because of their similarity to human core autism phenotypes and their high suitability for drug screening and preclinical trials [26]. Autistic-like rodents are generally produced by injection of chemicals, especially valproic acid (VPA), either to pregnant dams (i.e., prenatal approach) or to their newborn pups (i.e., postnatal approach). An off-gestational approach was also adopted, whereby induction of autism is performed in adult, non-pregnant animals. To our knowledge, only a few published studies explored the herbal extract effects in autistic animal models, meaning that herbal therapy in autism basic research is still in its embryonic stage but develops rapidly through clinical efforts to apply alternative medicines.

Camellia sinensis, commonly known as green tea, was among the first herbs applied in animal studies seeking anti-autistic remedies. Using postnatal induction of autism-like condition, through subcutaneous (SC) injection of VPA to newborn albino mice aged 14 days, Banji et al. [27] administered oral doses of Camellia sinensis extract (75 and 300 mg/kg/day) to both male and female offspring from postnatal day (PND) 13 to 40. This chronic treatment, particularly at the high dose, significantly attenuated the proprioceptive, motor, nociceptive, and spatial memory anomalies, thus preventing hyperactivity, high anxiety, and disorientation in a spatial recognition context. Evaluation of cerebellar areas in autistic-like animals further revealed damage to the Purkinje cell layer, which was mostly avoided by Camellia sinensis extract. It was concluded that this plant is capable of attenuating autism-related changes in motor coordination, cognition, and anxiety, thereby exerting a neuroprotective effect, which is partly explained by the antioxidant action revealed by the significant decrease in plasma malondialdehyde (MDA), a marker of lipid peroxidation. These preliminary findings were recently corroborated by another study centered on the neurochemistry of ASD. Male autistic-like rats were obtained through intraperitoneal (IP) administration of VPA to pregnant females on gestational day (GD) 12.5. Beginning on PND 15, oral treatment with Camellia sinensis extract (300 mg/kg/day) was provided for 20 days. Post-experiment neurochemical assays in untreated animals showed a significant decrease in the cerebellar and cerebral (cortical) levels of serotonin (5-HT), dopamine (DA), norepinephrine (NE), γ-aminobutyric acid (GABA), serine, and taurine, with a concomitant decrease in whole brain rates of cholesterol and antioxidant markers (GSH, SOD, and CAT). On the contrary, a significant increase in MAO, AChE, glutamate, aspartate, glycine, oxidative stress markers (MDA and NO), and proinflammatory cytokines (TNF-α and IL-6) was registered. These disturbances in VPA-exposed rats were significantly mitigated in those receiving Camellia sinensis extract, except for cortical 5-HT, cerebellar serine, and cortical/cerebellar NE, DA, glycine, and aspartate [28].

Bacopa monniera, a prominent Ayurvedic plant largely employed as neuroprotective, was also tested and retested in autistic-like animals. Intraperitoneal injection of VPA to pregnant rats (GD 12.5) led to offspring manifesting impaired olfactory discrimination on PND 9, delayed eye opening on PND 13 and 14, and impaired motor development on PND 8-12 as early signs of abnormal neurodevelopment. Upon weaning (PND 21), autistic-like male pups were divided into saline-treated and Bacopa monniera (L.)-treated groups. The aqueous extract of this plant, at a dose of 300 mg/kg/day, was administered orally from PND 21 to 35. When submitted to various tests during adolescence (PND 30-40) and adulthood (PND 90-110), autistic animals showed pain hyposensitivity, hyperactivity, low exploratory/social behaviors, and high anxiety attitudes. The authors reported cytoarchitectural alterations of the cerebellum along with a significant increase in hippocampal serotonin levels, among other neurochemical changes. Treatment with Bacopa monniera was found to significantly improve behavioral alterations, ameliorate neurochemical markers, and preserve cerebellum tissue integrity [29]. The neuroprotective features of Bacopa monniera were recently recalled through in-depth analyses. Using a standardized methanolic extract marketed under the name of BacoMind®, various aspects of the autism-like condition were targeted in male Wistar rats prenatally exposed to VPA (on GD 12.5). Developmental assessment of newborn rats on PND 7-23 revealed a delay in eye-opening, lower body weight, and deficient vestibule-related sensorimotor reflex in a tilting plane test. Hallmarks of neurobehavioral dysfunction determined between PND 44 and 53, were social deficits, anxiety-like and repetitive behaviors, learning and memory impairments, and poor motor coordination. Inherent in these visible disabilities are intense oxidative stress, proinflammatory cytokines rise (IL-1β, IL-6, and TNF-α), anti-inflammatory cytokines decline (IL-10), high neuronal injury scores, and glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor overexpression in the hippocampus and prefrontal cortex. Post-weaning oral treatment with the standardized extract (20, 40, and 80 mg/kg), which was carried on from PND 23 to 43, resulted in a significant improvement in almost all parameters at the dose of 80 mg/kg. It is worth noting that while a much lesser extent of prevention was registered at 40 mg/kg, both extract doses proved efficient when compared to risperidone as a reference drug, which failed to significantly relieve the autistic status [30].

The advantage of reference drugs in such experiments was revealed by another recent study about the anti-autistic potential of Asparagus racemosus. Intraperitoneal injection of VPA to pregnant Wistar rats (GD 13) was shown to produce metabolic and enzymatic disturbances in the brains of their offspring. Autistic symptoms were manifested as a decrease in body weight (PND 7, 14, 21, 28, and 35), delayed eye-opening (observed once daily), and impaired olfactory discrimination (PND 9). Asparagus racemosus extract (50, 100, and 200 mg/kg/day) was introduced upon weaning (PND 21-35) by oral route. This two-week treatment, only at 100 and 200 mg/kg/day, was significantly effective in ameliorating the high plasma nitrite levels, low brain GSH and catalase activity, and high monoamine oxidase A (MAO-A) and acetylcholinesterase (AchE) activities. The use of fluoxetine (selective serotonin reuptake inhibitor) and donepezil (acetylcholinesterase inhibitor) as reference drugs allowed to demonstrate that Asparagus racemosus extract at the higher dose (300 mg/kg/day) is powerful at preventing the biochemical deficits measured in autistic-like preadolescent animals [31].

Korean Red Ginseng (KRG), a popular herbal remedy in South Korea, is reportedly shown to enhance central nervous system (CNS) functions. Pregnant ICR mice, subcutaneously injected with VPA on GD 10, delivered male offspring with a socially impaired phenotype, as indicated by lower sociability indices and sniffing scores, reflecting a poor social interaction of autistic-like animals with stranger mice. VPA-exposed animals also showed increased repetitive behavior and locomotor activity and decreased working memory scores and electroshock seizure threshold, while their motor coordination and balance measures did not differ from their control counterparts. These altered behavioral patterns, as assessed from PND 28 through 38, were normalized (except motor coordination ability) when autistic-like mice were given daily oral KRG solution (100 and 200 mg/kg) upon weaning (PND 21), revealing the potential therapeutic effects of KRG when administered post-weaning in animal models of ASD [32]. Using the same traditional remedy, Kim et al. [33] attempted to counteract VPA-induced autistic signs via concomitant administration of VPA and KRG during pregnancy. Pregnant Sprague-Dawley rats were subcutaneously injected with PVA on GD 12 while receiving daily oral doses of diluted KRG (20, 50, 100, and 200 mg/kg) from GD 10 to 15. Male autistic-like offspring showed hyperactivity (PND 28), social impairments (PND 30), and a lower electrical seizure threshold (PND 42) compared to their control counterparts. Poor social interaction was so evident that VPA-exposed pups significantly avoided conspecific strangers and preferred, thereafter, familial over novel rats. Depending on the dosage and variables measured, KRG treatment considerably improved the abnormal locomotor activity and sociability indices and prevented hypersensitivity to electric shock. Furthermore, KRG acted as an anti-teratogenic agent, counteracting the VPA-induced tail deformation in these animals. Those findings suggest that KRG may have positive therapeutic applications in ASD and serve as a preventive supplement against mild neural tube defects (NTD) in many contexts.

Oryza sativa, known within the Thai community as purple rice, was also explored for potential efficacy against autism symptoms. VPA was subcutaneously injected into newborn male and female rats on PND 14, followed by oral administration of the herbal extract (combined with pupae of the silkworm Bombyx mori) at doses of 50, 100, or 200 mg/kg between PND 14 and 40. A battery of repeated behavioral tests was applied throughout the treatment duration, with the cerebellum being isolated on PND 41 to assess biochemical markers and histomorphology. The extract was found to significantly reduce sensorimotor (reflex) deficits, anxiety-like behavior, social avoidance, and spatial memory deficiency shown in VPA-exposed offspring. However, this extract failed to counteract pain induced by thermal stimuli as a sign of nociceptive sensitivity following VPA exposure. Signs of severe oxidative stress, as well as a significant decrease in Purkinje cell density, were also unveiled in the cerebellum of autistic-like rats, whose treated counterparts had much less damage and even no apparent damage (at the dose of 200 mg/kg) at the end of the experiment [34].

Traditionally used for the treatment of many diseases, the leaf extract of Morus alba was recently shown to mitigate spatial memory deficit and hippocampal oxidative stress-related damage in Wistar rats. Subcutaneous VPA administration to 14-day-old male and female rats was later associated with pain hyposensitivity (PND 37-39), anxiety-like behavior, social avoidance, and spatial disorientation (PND 40). Neuronal density in hippocampal subregions (CA1, CA2, CA3, and dentate gyrus) was significantly decreased (PND 41), a finding that is linked with substantial oxidative stress. Applied by oral route (PND 14-40), daily treatment with Morus alba extract (25, 50, and 100 mg/kg) was unsuccessful in relieving social avoidance. While only low and medium doses differently affected nociceptive, oxidative, and cell density measures, all three doses resulted in a significant improvement in spatial memory performance [35].

Prangos ferulacea (L.) is a medicinal herb available in the Mediterranean and Middle East regions. Its various benefits in folk medicine prompted Saadat et al. [36] to evaluate its efficiency in male Wistar rats made autistic by exposing them to VPA in utero. Dams receiving this drug on GD 12.5 (by IP route) were allowed to raise their pups until PND 30, from which Prangos ferulacea extract (100 and 200 mg/kg) was intraperitoneally administered to the male offspring until PND 58. Several behavioral tasks were used at the start and end points of treatment (PND 30 and 58), then the animals were euthanized on PND 65 for histopathological and biochemical assays in the brain. Signs of increased anxiety, decreased motor coordination, pain hypersensitivity, and hippocampal oxidative stress were found in the VPA-offspring-vehicle group compared to control groups. Interestingly, hippocampal levels of the apoptosis regulator proteins BAX and BCL-2 were significantly changed in autistic-like animals, with the BAX/BCL-2 ratio being increased, pointing to apoptotic damage within the brain. Indeed, the hippocampal CA1, CA3, and DG subregions manifested a high percentage of neuronal death. Prangos ferulacea extract showed mild anxiolytic and high antioxidant effects. With no significant change in BAX and BCL-2 levels, this extract largely prevented neuronal cell death in the hippocampus, particularly at the dose of 200 mg/kg.

Moreover, the hydroalcoholic extract of Passiflora incarnata was also explored for possible anti-autistic effects in a Wistar rat model. This plant extract, traditionally used to treat patients with anxiety and sleep disorders, was dissolved in drinking water and then given ad libitum to adolescent male offspring (PND 35-81) prenatally exposed to VPA. Untreated autistic-like rats showed anxiety-like and repetitive (stereotypic) behaviors, learning and recognition memory impairments, and low-score sociability measures. Serum oxidative stress markers were significantly changed, while damage to hippocampal CA1 and prefrontal neurons was considerable. Passiflora incarnata extract was found to alleviate some autistic-like behaviors, relating this finding to the neuroprotective and antioxidant action of its chemical constituents [37].

Postnatal induction of autism-like phenotype through administration of VPA to newborn mice was further performed by Tejano et al. [38], who evaluated the anti-autistic potential of the ethanolic leaf extract of Balakat tree (Ziziphus talanai (Blanco) Merr.). On PND 14-16, mice pups were orally treated with VPA syrup followed by the plant extract (300 and 400 mg/kg/day). Autistic-like pups submitted to motor behavioral tests and cerebellar histological analyses, meanwhile, exhibited a significant increase in geotactic latency scores and a reduction in Purkinje cell layer density, both of which are signs of inappropriate proprioceptive and motor processing. Post-VPA treatment with Ziziphus talanai extract was successful in keeping most behavioral scores at control-group levels while preserving the number and size of Purkinje cells in the cerebellar cortex, suggesting that Ziziphus talanai possesses ameliorative effects against behavioral aberrations and altered cerebellar histology in murine models of ASD.

Notably, in contrast to earlier moderate results reported in the clinical setting [19], daily treatment of newborn albino male mice with Ginkgo biloba extract (100 mg/kg, IP route, PND 13-40) was very favorable. Post-VPA (PND 14) behavioral, biochemical, histological, and immunohistochemical analyses provided insight into the complexity of ASD. Signs of high anxiety, poor social interaction with stranger conspecifics, and deficient working memory were observed in VPA-exposed animals not receiving the plant extract. This autistic-like condition was accompanied by significant neurochemical changes (increase in MDA, IL-6, and IL-17, and decrease in GSH and TGF-β1), damage in the Purkinje cell layer, and low expression of myelin basic protein (MBP) and serotonin in the molecular and granular layers of the cerebellum. The intensity of these neurobehavioral aberrations was significantly reduced by the chronic Ginkgo biloba treatment, reflecting its therapeutic potential mainly through anti-inflammatory and antioxidant effects [39].

Interestingly, using an off-gestational approach whereby the chemical inducer propionic acid (PPA) was administered through intracerebroventricular (ICV) infusion, Jiji and Muralidharan [40] evaluated the ability of Clitoria ternatea L. root extract to counteract memory deficit and behavioral impairments seen in adult Wistar male rats (aged 4-8 weeks at the beginning of the study). Vehicle (1% Tween-80) or extract (250 and 500 mg/kg) solutions were provided orally for 28 consecutive days, with PPA being injected on days 22-28 in parallel to oral treatment. Altered learning and memory performances, along with a significant increase in serotonin and glutamate brain levels, were registered in PPA-exposed rats. Relating particularly to dampened object recognition and working memory, this animal model may mimic cognitive disabilities encountered in severe autism cases. Treatment with Clitoria ternatea L. extract significantly prevented the behavioral and neurochemical changes in a dose-dependent manner, highlighting that these significant nootropic effects are worthy of consideration in favor of patients with ASD.

Table 2 presents a summary underlining the main variables and findings of the reviewed animal studies. While we only mentioned positive results, it is noteworthy that half the reviewed studies enclose negative outcomes. Camellia sinensis did not alter certain neurotransmitters in the cerebral cortex (5-HT, aspartate, and glycine) and cerebellum (DA, NE, aspartate, glycine, and serine) [28]. Bacopa monniera failed to counteract thermal nociception in adolescent rats [29]. Treatment with Korean red ginseng did not reverse the impaired motor coordination and balance [32]. Treatment with Oryza sativa failed to significantly reduce thermal nociception and stereotypical behavior [34]. Morus alba was ineffective in improving anxiety, neuronal density, and SOD activity [35]. Prangos ferulacea (L.) did not affect the BCL-2/BAX apoptotic pathway, motor imbalance, anxiety, and aberrant responses to painful stimuli [36]. Moreover, treatment with Ziziphus talanai was unsuccessful in improving muscle strength in mouse pups [38]. Consequently, a more realistic and balanced view of the potential of herbal therapies for ASD is gained by taking into account the negative findings of the reviewed studies.

Table 2. Summary of the animal studies relating to using herbal extracts in autistic-like rodents.

Overall, the reviewed animal studies tried to replicate the complexities of human ASD by targeting the main pathophysiological factors thought to play a pivotal role in the occurrence and lifelong manifestations of this disease [41]. The autistic-like behaviors, which are relevant to most clinical observations in people with ASD, were associated with maternal use of medications during pregnancy (represented by valproic acid and propionic acid), as well as with neuroinflammation, brain oxidative stress, and neuronal cell damage at key cerebral regions in the offspring. Though describing a chemically induced autism-like condition, these studies provide interesting findings indicating that herbal therapy merits consideration as an adjunct treatment to conventional therapies in patients with ASD. Table 3 summarizes the relevant mechanisms of action afforded by herbal extracts and the expected effects in patients with ASD.

To encourage the widespread use of herbal therapy in this context, we propose a timeline chart that relies on the animal developmental stage equivalent to a certain human age range (Figure 2). This equivalence, yet approximative, is based on studies that compare rodents’ and humans’ age at different phases of their lives [42]. As shown, herbal extract supplementation covers both prenatal and postnatal phases, with prenatal and/or perinatal utilization being considered preventive as no autism diagnostic procedures exist during pregnancy and childbirth. Caregivers should, therefore, rely on the medical history of pregnant women and any known risk factors to justify preventive herbal therapy. Accounting for the global use of herbal remedies and the fact that many herbal remedies used traditionally have become modern medicines through drug development [43], plant extracts reviewed herein are worthy of testing in ASD to aid in ameliorating psychological and educational program outcomes in persons already diagnosed.

Table 3. Protective mechanisms of herbal extracts in patients with ASD.

Nevertheless, the proposed timeline chart is not meant to be blindly applied to human subjects due to ethical considerations and toxicological measures regarding the dosage, route of administration, and duration of treatment. Rather, it can be referred to as setting appropriate clinical trials within the framework of ethical practice. The context of herbal therapy in ASD has, therefore, some limitations and practical issues that need to be thoroughly addressed in future research.

Certainly, the most significant issue to solve is the animal-human correlation in herbal efficacy. As stated above, herbal therapy in ASD is a recent approach worldwide, creating a big challenge to the extrapolation from rodents to humans. Even though the few clinical trials made so far are advantageous in this regard, considering prenatal and/or early postnatal phases as the main therapeutic window in most animal studies would add complexity to that challenge. Another limitation lies in the fact that herbal extracts are chemically heterogeneous, containing a wide range of active constituents that may interact with each other or with other metabolites inside the organism. While this property might allow patients to gain the whole benefit (in contrast to isolated molecules), heterogeneity means that even if improvement in autistic symptoms is observed, strict medical surveillance of patients under therapy would be mandatory to prevent potential side effects. Added to this is the likelihood that any prescribed herbal extract may alleviate some symptoms while exacerbating others. Thus, the appropriate use of herbal therapy for the sake of autistic people's well-being would remain speculative unless the extract heterogeneity is neatly explored to understand the corresponding pharmacokinetics and drug interactions. Furthermore, a substantial issue resides in the route of administration and doses applied, which are manifold among the reviewed animal studies. Accounting for the interspecies variation in toxicity, researchers should determine and put into practice a safety factor to achieve an acceptable oral dosage for humans. However, there are two studies [36, 39] whose authors opted for the intraperitoneal route of administration, a choice that is inconsistent with the common human consumption of herbal extracts. So, it is highly desirable to reproduce the same experimental protocols using the oral route instead.

Otherwise, clinical attempts to implement herbal treatments for ASD should give particular attention to the poor communication skills of patients, who could be unable to express eventual discomfort (notably gastrointestinal). Their atypical behavior and sensitivity can make compliance with herbal ingestion a difficult task, particularly if the extract's taste or aroma is unpleasant. In these conditions, patient education is an integral part of herbal therapy, aiming at introducing it as a complementary and helpful care program that improves the quality of life.

On the way to overcoming these limitations, researchers should always bear in mind that autistic profiles are highly various, claiming the need for personalized schedules of herbal therapy in conjunction with conventional care. In this sense, biotechnology tools can be devoted to establishing patient clusters that share the same range of symptoms and severity, for whom standardized extract fractions fulfilling their therapeutic requirements are developed and manufactured. Refinement of the organoleptic properties of these extracts to facilitate ingestion and compliance should take place in the development of new biotechnological tools for ASD management.

Patients with ASD are currently handled by conventional therapies that aim to reduce their maladaptive behavior and enhance their communication skills. When needed, pharmacological interventions are called out by caregivers to control some comorbidities such as anxiety, depression, and aggressive behavior, but alternatives to psychoactive drugs are paving the way for herbal treatment of neuropsychiatric diseases. For instance, Ginkgo biloba, Hypericum perforatum, and Panax ginseng have shown promise in helping people with ASD. The emergence of animal studies focusing on the exploration of the anti-autistic effects of herbal extracts has further highlighted the forthcoming integration of herbal therapy into conventional healthcare programs, but this perspective presently confronts certain issues and limitations. In this review, we underlined the animal studies devoted to testing medicinal plants on autistic-like rodents, targeting the behavioral, histopathological, and molecular aspects of human ASD. A visual conclusion illustrating the key elements of this review is presented in Figure 3. To emphasize the developmental feature of these herbal remedy-based experiments, we deduced a timeline chart modeling the adjunctive herbal therapies in patients with ASD (Figure 2). Future research should be directed to analytical studies, in which predictive models of the relationships between herbal treatment and outcomes of interest are produced. Such statistical models would require a sufficiently large sample size, which is relatively small in the reviewed studies, and the comparison of herbal efficacy in males and females for better inference and precision. In this respect, extensive experiments covering the core symptoms and comorbidities of ASD should apply a variety of herbal fractions and therapeutic windows, allowing clinicians to systematically identify the best options for conducting cohort studies. Standardization of dosages and treatment durations among distinct experimental protocols is crucial in defining pharmacokinetics for clinical use. Taken together, both clinical and animal studies performed thus far should be endorsed, minimizing the time gap in the translation of their findings into approved herbal medications for patients with ASD.

This work is a part of the project PRFU–Biotechnology Doctoral Training at Chadli Bendjedid University – El Tarf (Project Number: D00L05UN360120230003).

MLT conceived the core idea, structured the review layout, performed the literature search and data extraction, and prepared the manuscript draft. SM prepared the manuscript draft, created the figures, and validated the final version. GS revised the draft and validated the final version. The authors read and approved the submitted version of the manuscript.

There is no conflict of interest among the authors.