Antinociceptive Potential of Croton Genus: A Systematic Review

Abstract

The genus Croton (Euphorbiaceae) encompasses numerous species recognized for their diverse medicinal applications, particularly in pain management. This systematic review aims to compile and analyze the scientific evidence on the antinociceptive properties of Croton species. The review protocol was registered in the Open Science Framework (OSF) associated project: osf.io/z4juf. Using the PRISMA methodology, an exhaustive search was conducted in databases such as PubMed, Scopus, and Web of Science to identify relevant studies published to date. The review includes preclinical studies in animal models that investigate the mechanisms of action and efficacy of Croton extracts and isolated compounds in pain inhibition. The results indicate that 28 Croton species exhibit significant antinociceptive effects, attributed to bioactive compounds such as diterpenes, alkaloids, and flavonoids. These compounds interact with multiple biological pathways, including ion channels, such as TRPV1, K/ATP, and ASIC channels, as well as the inhibition of the synthesis of inflammatory mediators, particularly prostaglandins. This review highlights the potential of Croton as a source of novel analgesic agents and emphasizes the necessity for further clinical studies. Additionally, integrating ethnobotanical and pharmacological knowledge is suggested to develop innovative and effective treatments for pain management.

1. Introduction

Pain is an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage [1]. Subsequently, Williams and Craig [2] added the emotional and social component in a proposed update of the definition of pain, considering it as: ‘A distressing experience associated with actual or potential tissue damage, with sensory, emotional, cognitive, and social components’. From a clinical perspective, it is ‘an unpleasant sensory experience, accompanied by affective, motor, vegetative, and even personality responses’, thus establishing that pain is a subjective experience, differing from one individual to another, and there does not necessarily need to be a morphological lesion to justify a patient’s pain [3]. One of the most widely accepted pain classifications today is the one established by Woolf, as it encompasses the types of pain, resulting in four categories, based on its somatic origin and physiological mechanism: nociceptive, inflammatory, neuropathic, and functional. Pain typically occurs in response to nociceptor activation (nociceptive), alerting the presence of potentially harmful stimuli, which serves a protective role. However, clinical pain can arise from chronic inflammatory states (inflammatory pain), as secondary to nervous system damage (neuropathic pain), or even when there are no signs of damage that could cause it (functional pain) [4,5].

The treatment of pain often involves drugs that, although effective, mostly have side effects such as gastrointestinal, renal, cardiovascular, hematological, hepatic, and dependency, among others [6,7,8]. That is why it is important to look for natural alternatives that help to alleviate pain, with the least number of adverse effects [9]. Given the limitations and risks associated with conventional pharmacological treatments, there has been a growing interest in complementary and alternative therapies [10]. Among these, phytotherapy has gained significant attention; herbal medicine offers a range of natural compounds with analgesic properties that may provide effective pain relief with fewer side effects, as has been proven in a large number of species [11,12]. These natural remedies, rooted in traditional practices but increasingly supported by scientific research, represent a promising avenue for those seeking holistic approaches to pain management [12].

In this context, a large number of studies have been identified in species of the Croton genus, which show antinociceptive or analgesic effects. The genus name Croton is derived from the Greek word “kroton”, meaning “tick”, in reference to the thick, smooth seeds characteristic of most species within this genus, which belongs to the family Euphorbiaceae [13]. The Croton genus comprises around 1300 species distributed across tropical and subtropical regions worldwide, with the greatest diversity found in South America, the Caribbean, and parts of Africa [14]. These species inhabit a wide range of habitats and can be found in various types of vegetation, although they are most commonly associated with dry or open environments [15]. Several Croton species have been extensively studied and shown to exhibit multiple biological activities. Globally, plants of the Croton genus have a long history of use in traditional medicine, reflecting a consistent profile of biological activities and folk remedies. For example, in South Africa, Croton species are reliable traditional medicines for the treatment of microbial infections and malaria [16].

On the other hand, among the scientifically proven effects are treatments for cancer, constipation, diabetes, digestive disorders, external wounds, fever, hypercholesterolemia, hypertension, inflammation, gastric ulcers, and weight loss [14,16,17]. Additionally, various bioactivities of the essential oils from this genus, including anti-inflammatory, antinociceptive, gastroprotective, wound-healing, and cardiovascular effects, have been reported [17,18,19,20].

In a relatively recent review, a group of authors set out to report the chemical constituents of plants from the genus Croton documented over a 12-year period [14]. They found a total of 399 reported compounds, including 339 diterpenes, seven sesquiterpenoids, one triterpenoid, 21 glycosides, eight alkaloids, and two limonoids, among others. As can be seen, diterpenes are characteristic and predominant components of Croton species. Clerodane, tigliane, kaurane, crotofolane, labdane, cembrane, and abietane are some of the relevant diterpenes found in Croton.

Despite finding review articles on the health benefits of the Croton genus, no review was found that focuses on antinociceptive effects, nor one that delves into their mechanisms of action. Therefore, the objective of this work was to compile and analyze the scientific evidence on the antinociceptive properties of Croton species.

2. Materials and Methods

2.1. Study Protocol

The review protocol was registered in the Open Science Framework (OSF) platform (associated project: osf.io/z4juf). The PRISMA methodology [21] for systematic reviews was employed in this review (see Supplementary Materials PRISMA 2020 Checklist). Each author (seven) independently conducted searches without automation tools on the topic “Antinociceptive effect of Croton species” across various databases including Scopus, PubMed, Embase, Google Scholar, Springer, SciELO, Worldwide Science, Dialnet, and Web of Science. Researchers stored files in an EndNote folder (Clarivate Analytics), ensuring the removal of duplicate bibliographic records. The search was conducted up until September 2024. Authors were contacted for articles not available online. Searches were conducted in English, except on Dialnet and SciELO, where searches were done in Spanish and Portuguese. Terms included both Medical Subject Headings (MESH) and non-MESH terms, with Boolean operators “AND”, “NOT”, and “OR”. Specific terms searched were: (Croton OR Croton genus OR Euphorbiaceae OR “Croton extract” OR “Croton” + “anthocyanins”, “flavonoids”, “terpenes”, “alkaloids”, “phenolic compounds”, “lignans”, “fatty acids”) AND (“pain” OR “antinociceptive” OR “analgesic” OR “chemical composition”) NOT (anti-inflammatory). During the search process, no date restrictions and filters by item type were applied.

2.2. Eligibility Criteria

The eligibility criteria were established by the first author and the corresponding author. During the selection process, all authors independently searched databases using keywords and reviewed the titles and abstracts of retrieved references to identify relevant studies. Inclusion criteria included original or review articles on in vitro, preclinical (non-human species), and clinical models. Excluded were books and grey literature (theses, conference abstracts, etc.). We prioritized articles published in the last 5 years; however, we decided to include all those that provided relevant information (thematic relevance and contribution to knowledge), regardless of the year of publication. Studies on anti-inflammatory and other health effects were excluded (studies in which antinociception was evaluated, and other biological effects were also studied, were considered). The selected studies were validated by all authors. In case of disagreement, a meeting was held to hear everyone’s points of view and reach a consensus on whether or not to include the article, with the first author and corresponding author casting the final vote.

2.3. Study Quality Assessment

The quality of the studies was qualitatively evaluated using the quality criteria for conducting systematic reviews of preclinical evidence from the CAMARADES Preclinical Systematic Review & Meta-Analysis Wiki [22]. Special emphasis was placed on study design (included randomized controlled trials as well as laboratory studies conducted under controlled conditions); sample size (studies that included a sample size greater than 4 and preferably 6+ animals per group); methodology (whether studies detail the methods used to induce pain in experimental models, for example, chemical, thermal, or mechanical methods; whether they describe how treatments were administered and if standardized methods were used); presence of positive and negative controls (studies that included positive and negative controls to validate the results); statistical analysis (confidence intervals, p-values); and ethical considerations (studies that comply with ethical standards and have approval from an ethics committee).

2.4. Data Extraction

Data extraction utilized a checklist including title, year, country, Croton species, plant part (aerial parts, leaf, flower), pain induction model, animal species and sample, dosage, administration route, reference drug with dosage, findings, and mechanisms of action. We also identify mean differences for all pain tests in the studies.

Figure 1 provides details of the search process.

3. Results

3.1. Study Selection and Characteristics

A thorough search across various databases identified 2608 initial records. After eliminating 628 duplicates, 2608 unique studies were screened based on their titles and abstracts. From these, 88 studies were chosen for full-text review.

Following the application of predetermined inclusion and exclusion criteria, 59 studies were excluded for the following reasons: 36 did not involve antinociceptive measurements, 18 focused on anti-inflammatory evaluations, and 6 were deemed irrelevant to the research question.

Twenty-nine articles met the inclusion criteria. None of them evaluated the effect of any plant from the genus Croton in clinical models; 100% were conducted on experimental animals. Of these, only 13.8% were articles from the last 5 years. A total of 19 varieties of Croton were investigated (see

Table 1. Varieties of Croton in which studies on antinociceptive effects have been conducted.

Croton Variety	Number of Studies
Croton adamantinus Müll. Arg.	1
Croton antisyphiliticus Mart	1
Croton blanchetianus	2
Croton cajucara Benth	3
Croton celtidifolius	3
Croton conduplicatus Kunth	2
Croton cordiifolius Baill.	1
Croton crassifolius	1
Croton cuneatus Klotzsch	1
Croton guatemalensis Lotsy	1
Croton macrostachyus	1
Croton malambo	1
Croton nepetaefolius Baill	1
Croton pullei var. glabrior Lanj.	1
Croton sonderianus	1
Croton tiglium L.	1
Croton urucurana Baill	2
Croton zambesicus	1
Croton zehntneri	3
Total	28

). The most studied varieties were Croton cajucara (four articles), Croton celtidifolius and Croton zehntneri, with three articles, respectively.

The only pharmaceutical forms evaluated were extracts (mainly aqueous, ethanolic, and methanolic) and essential oils. The parts of the plant from which the extracts and essential oils were obtained included roots, leaves, bark, seeds, stems, and one that encompassed all aerial parts. On the other hand, the most commonly used pain induction model was the acetic acid-induced writhing test (21 times), followed by the formalin test (19 times). Other common models include the hot-plate test (12 times) and the tail-flick test (6 times). Additionally, there are several less frequent models, such as capsaicin-induced nociception and glutamate-induced nociception, among others.

The most frequently used animal species were Swiss mice and Wistar rats, although other mouse strains such as ICR or KM, as well as Sprague Dawley for rats, were also utilized. A zebrafish model was also used. Considering all the doses used in the articles, we found that the lowest oral dose was 3 mg/kg body weight and the highest was 1500 mg/kg. For the intraperitoneal route, the lowest dose was 0.1 mg/kg and the highest was 2000 mg/kg body weight. The average dose for oral administration was approximately 245.0 mg/kg, while for the intraperitoneal route, it was about 133.7 mg/kg. The most common route of administration was oral, with a predominant use compared to the intraperitoneal route. Several reference drugs were identified in the analyzed studies. The most common was morphine, used in 14 studies with an average dose of 6.78 mg/kg (lowest dose 0.1 mg/kg and highest 10 mg/kg). The second was indomethacin, reported in 12 studies with an average dose of 15.83 mg/kg (lowest 5 mg/kg and highest 20 mg/kg). In smaller proportions, acetylsalicylic acid (200 mg/kg), dipyrone (225 mg/kg), and gabapentin (70 mg/kg) were used. Additionally, other drugs such as fentanyl, N-acetylcysteine, and clonidine were used.

3.2. Main Findings on Antinociceptive Effects of Different Species of the Genus Croton

Essential oils and extracts from the genus Croton have shown significant antinociceptive effects in various pain models, such as acetic acid and formalin-induced tests. Some of these extracts exhibit antinociceptive activity comparable to that of morphine, indicating their potential action through opioid and cholinergic systems. A specific extract demonstrated efficacy in reducing hyperalgesia induced by sciatic nerve ligation, showcasing its ability to alleviate neuropathic pain symptoms over an extended period. Furthermore, it was found that an essential oil inhibits neuronal excitability, suggesting that the compounds present in the extract may be related to modifications in neuronal excitability. Most of the analyzed extracts and essential oils were effective in reducing the number of writhing movements induced by acetic acid and in both phases of the formalin test. Additionally, these extracts not only exhibited antinociceptive activity but also presented significant anti-inflammatory properties by reducing paw licking time in the formalin test. Antinociceptive activity was evident across all tested doses in various models. It is important to note that in studies evaluating more than one dose, a consistent dose-dependent effect was observed throughout the conducted tests (see

Table 2. Preclinical studies examining the antinociceptive properties of Croton species.

Title	Year	Croton Species	Plant Part (Extract, Essential Oil, or Resin)	Pain Induction Model	Animal Species and Sample	Dosage and Administration Route	Reference Drug and Dosage	Findings	Ref.
Antinociceptive effect of essential oil from Croton blanchetianus leaves	2024	Croton blanchetianus Baill	Essential oil from leaves	Acetic-acid writhing test, formalin test, and tail-flick test	Male and female Swiss mice (6 animals per group)	25, 50, and 100 mg/kg p.o.	Morphine 5 or 10 mg/kg i.p. Indomethacin 20 mg/kg i.p.	The essential oil demonstrated significant antinociceptive effects in both the acetic acid-induced writhing test and the formalin test (both p < 0.001). The results indicated a significant reduction in pain responses compared to control groups, suggesting the potential of this essential oil as an effective analgesic.	[23]
Dereplication of polar extracts of Croton antisyphiliticus Mart roots and its anti-inflammatory and antinociceptive potential	2024	Croton antisyphiliticus Mart	Ethanolic extract (EE) and Aqueous extract (AE) from Roots	Acetic-acid writhing test, tail-flick test, formalin test, and carrageenan-induced hyperalgesia	Female Albino Swiss mice (8 animals per group)	EE (125, 250, and 500 mg/kg) p.o. AE (300, 600, 900, 1200, and 1500 mg/kg) p.o.	Indomethacin 10 mg/kg p.o. Morphine 5 mg/kg s.c.	Both extracts significantly reduced the number of writhes (p ≤ 0.005 and ≤0.001) in the acetic acid-induced test and decreased pain induced by formalin (p < 0.05). They also reduced hyperalgesia induced by carrageenan and exhibited effects similar to those of indomethacin (p ≤ 0.001 versus control), indicating their potential as antinociceptive agents.	[24]
Flavonoid-Rich Fraction from Croton blanchetianus (Euphorbiaceae) Leaves Exerts Peripheral and Central Analgesic Effects by Acting via the Opioid and Cholinergic Systems	2022	Croton blanchetianus	Leaves extract Ethyl acetate fraction (EAF)	Acetic-acid writhing test, formalin test, hot-plate test, and tail immersion test	Male Swiss mice (6 animals per group)	EAF (12.5, 25, and 50 mg/kg p.o.	Morphine 10 mg/kg i.p. Indomethacin 20 mg/kg i.p.	The extract exhibited significant (p < 0.05 in all tests) antinociceptive activity comparable to morphine, with maximum inhibition values observed at the highest dose (50 mg/kg) in both the hot plate and acetic acid-induced writhing tests. The study suggests that the analgesic effects are mediated through the opioid and cholinergic systems.	[25]
Antinociceptive effect of triterpene acetyl aleuritolic acid isolated from Croton zehntneri in adult zebrafish (Danio rerio)	2020	Croton zehntneri	Stem bark triterpene acetyl aleuritolic acid (AAA)	Formalin test, Cinnamaldehyde, capsaicin, acid saline solution, glutamate, and hypertonic saline pain induction	Zebrafish Danio rerio (6 animals per group)	AAA (0.1, 0.3, and 1.0 mg/mL) i.p.	Morphine 0.1, 1, 0, and 2.5 mg/mL i.p.	AAA significantly (p < 0.05 in all tests) reduced nociceptive behavior induced by acid saline and capsaicin without altering locomotor activity and showed no toxicity. The analgesic effects of AAA were comparable to those of morphine, indicating its potential as an antinociceptive agent.	[26]
Neuropharmacological effects of essential oil from the leaves of Croton conduplicatus Kunth and possible mechanisms of action involved.	2018	Croton conduplicatus Kunth	Essential oil from leaves	Formalin test, acetic-acid writhing, and hot-plate test	Male Swiss mice (6 animals per group)	25, 50, and 100 mg/kg i.p.	Morphine 10 mg/kg i.p. Indomethacin 20 mg/kg i.p.	The oil demonstrated significant (p < 0.05 in all tests) antinociceptive activity in all pain models: In the acetic acid-induced nociception test, the oil reduced the number of writhes, indicating a decrease in pain sensitivity. In the formalin test, it significantly reduced the time spent licking the paw in both the early and late phases. In the hot plate test, it increased the latency to respond to thermal stimuli, further confirming its antinociceptive properties	[27]
Antinociceptive Effect of the Essential Oil from Croton conduplicatus Kunth (Euphorbiaceae).	2017	Croton conduplicatus Kunth	Stem bark Essential oil	Acetic-acid writhing, formalin test, and hot-plate test	Male Swiss mice (6 animals per group)	(25, 50, and 100 mg/kg i.p.	Morphine (10 mg/kg) i.p. Indomethacin (20 mg/kg) i.p.	The oil reduced nociceptive behavior at all tested doses in the acetic acid-induced nociception test. It also showed significant effects (p < 0.05) in both phases of the formalin test and extended latency time in the hot plate test.	[28]
Antinociceptive effect of aqueous extracts from the bark of Croton guatemalensis Lotsy in mice	2016	Croton guatemalensis Lotsy	Aqueous extract from bark	Acetic acid-induced writhing test and hot-plate test	Male ICR mice (5 mice per group)	100, 200, and 400 mg/kg i.p.	Dipyrone (250 mg/kg) Morphine 10 mg/kg	The study found that the aqueous extract of Croton guatemalensis exhibited significant dose-dependent antinociceptive effects in the acetic acid-induced writhing test (p < 0.05 all doses), with a maximum reduction of 85.5% compared to the control at the highest dose. The extract (25 mg/kg) also showed a significant difference (p < 0.05) in both phases of the formalin test when compared to the control group. However, no effects were observed in the hot plate model. The antinociceptive effects were not reversed by naloxone, indicating that the endogenous opioid system does not mediate these effects.	[29]
Anti-inflammatory and antinociceptive activities of Croton urucarana Baillon bark	2016	Croton urucarana Baillon	Methanol extract from bark	Formalin test, Acetic acid-induced writhing response	Male albino Swiss mice (7–8 animals per group)	25 mg/kg, 100 mg/kg, 400 mg/kg i.p.	Indomethacin (15 mg/kg) and Morphine (5 mg/kg)	100 and 400 mg/kg of the extract exhibited significant (p < 0.05) antinociceptive activities in both phases of the formalin test, as evidenced by reduced paw-licking time in the formalin test. 100 and 400 mg/kg of the extract also significantly reduced the number of abdominal writhings induced by acetic acid in a dose-dependent manner (p < 0.05).	[30]
TRPV1 channel inhibition contributes to the antinociceptive effects of Croton macrostachyus extract in mice	2015	Croton macrostachyus	Methanol/methylene chloride stem bark extract	Complete Freund’s Adjuvant (CFA) injection model Neuropathic pain induced by sciatic nerve partial ligation	Male and female Swiss mice (4–6 animals per group)	250 or 500 mg/kg per os	Gabapentin 70 mg/kg	The extract significantly reduced mechanical hyperalgesia induced by CFA injection, with antinociceptive effects maintained for up to 5 days after two administrations within the first 24 h (p < 0.001). At a dose of 500 mg/kg, it significantly reduced hyperalgesia (p < 0.05) in the mice subjected to sciatic nerve ligation, indicating that the extract effectively alleviated neuropathic pain symptoms.	[31]
Essential Oil of Croton zehntneri and its Main Constituent Anethole Block Excitability of Rat Peripheral Nerve	2015	Croton zehntneri	Essential oil from aerial parts (leaves)	Not specified in the text; the study focuses on excitability rather than a specific pain model.	Male and female Wistar rats (Not applicable)	0.1–1 mg/mL of essential oil and anethole In vitro exposure (not specified for in vivo administration)	Not applicable	The study showed that essential oil inhibits neuronal excitability. This is particularly significant (p < 0.05) given the pharmacological actions of these compounds, which are likely connected to changes in excitability.	[32]
Antinociceptive Effect of the Essential Oil Obtained from the Leaves of Croton cordiifolius Baill. (Euphorbiaceae) in Mice	2015	Croton cordiifolius Baill.	Essential oil from leaves	Acetic acid-induced writhing test Formalin test Capsaicin Induced Nociception Glutamate Induced Nociception	Male Swiss mice (7 animals per group)	50 and 100 mg/kg, i.p.	Indomethacin 10 mg/kg, i.p. Morphine 7.5 mg/kg, i.p.	The essential oil (both doses) showed significant (p < 0.05) antinociceptive effects in both the neurogenic and inflammatory phases of the formalin test, and significantly (p < 0.05) reduced the number of writhings induced by acetic acid. High doses reduced significantly (p < 0.05) the nociception induced by intraplantar glutamate	[33]
Antinociceptive and wound healing activities of Croton adamantinus Müll. Arg. essential oil	2013	Croton adamantinus Müll. Arg.	Essential oil from leaves	Acetic acid-induced writhing test and formalin test	Male Swiss mice (Animals per group not specified)	50 and 100 mg/kg, i.p.	Morphine 7.5 mg/kg, i.p. Indomethacin 10 mg/kg, i.p.	The study demonstrated that essential oil exhibited significant (p < 0.05) antinociceptive activity in both writhing and formalin tests at 50 and 100 mg/kg.	[34]
Antinociceptive and Smooth Muscle Relaxant Activity of Croton tiglium L. Seed: An In-vitro and In-vivo Study	2012	Croton tiglium L.	Methanol extract, petroleum ether parts, and ethyl acetate parts from seeds	Acetic acid-induced writhing test	KM male mice (7 animals per group)	20, 25, 50, 100, 200, 250, and 300 mg/kg p.o.	Aspirin 100 mg/kg p.o.	The study found that methanol extract at doses of 25, 50, and 100 mg/kg significantly (p < 0.05 and p < 0.01) inhibited abdominal writhing in a dose-dependent manner, with a maximum inhibition of 45.7% at 100 mg/kg.	[35]
Anti-nociceptive and anti-inflammatory effects of Croton crassifolius ethanol extract	2012	Croton crassifolius	Ethanol extract from root	Acetic acid-induced writhing test, hot-plate test, and formalin test	Male and female Kunming mice (10 animals per group)	45 mg/kg, 90 mg/kg, and 180 mg/kg per os	Indomethacin 10 mg/kg per os Pentazocine 10 mg/kg per os	The ethanol extract exhibited significant antinociceptive effects in the acetic acid-induced writhing test, with percentage inhibition of 28.89%, 38.37%, and 56.53% in the three doses (p < 0.05, p < 0.001, and p < 0.001, respectively). In the formalin test, the extract significantly reduced the time spent licking and biting the injected paws at higher doses (45 mg/kg, p < 0.05; 90 and 180 mg/kg, p < 0.001). However, it did not show significant effects in the hot-plate test compared to the control group.	[36]
Anti-inflammatory, analgesic, and antipyretic activities of ethanol root extract of Croton zambesicus	2010	Croton zambesicus	Ethanol extract from root	Formalin test, Acetic acid-induced writhing test, and hot plate test	Male and female mice (6 animals per group)	27 mg/kg, 54 mg/kg, and 81 mg/kg i.p.	Acetylsalicylic acid 100 mg/kg i.p.	The ethanol root extract of Croton zambesicus exhibited significant analgesic activities. The extract effectively reduced pain responses in the formalin (p < 0.001), acetic acid-induced writhing (p < 0.05), and hot plate tests (p < 0.05 and p < 0.001), indicating its potential use in managing pain and fever.	[37]
Evaluation of antinociceptive and anti-inflammatory effects of Croton pullei var. glabrior Lanj. (Euphorbiaceae)	2008	Croton pullei var. glabrior Lanj.	Methanol extract from leaves	Acetic acid-induced abdominal writhing, hot-plate, and formalin test	Swiss mice (6 to 9 per group)	0.1, 0.3, and 1.0 g/kg per os	Indomethacin 10 mg/kg per os Fentanyl 200 µg/kg	The methanol extract exhibited dose-dependent antinociceptive activity by reducing the number of acetic acid-induced abdominal writhings (0.3 g/kg, p < 0.05 and 1 g/kg, p< 0.01). It did not significantly alter thermal reactivity in the hot-plate test or the first phase of the formalin test, (second phase p < 0.01) indicating a non-opioid mechanism.	[38]
Comparative anti-inflammatory and antinociceptive effects of terpenoids and an aqueous extract obtained from Croton cajucara Benth	2007	Croton cajucara Benth	Aqueous extract of stem bark	Acetic acid-induced writhing test	Male Swiss mice	100, 300, and 1000 mg/kg per os	Indomethacin 10 mg/kg per os	The administration of the aqueous extract at all doses (100, 300, and 1000 mg/kg) resulted in a significant reduction (p < 0.05) in the number of abdominal contractions compared to the control group in mice. These results confirm the anti-nociceptive effect of the aqueous extract.	[39]
Dragon’s blood from Croton urucurana (Baill.) attenuates visceral nociception in mice	2007	Croton urucurana Baill	Red sap	Capsaicin- and cyclophosphamide-induced visceral nociception	Male Swiss mice (8 per group)	Red sap extract 200 mg/kg and 400 mg/kg (p.o.). Intraperitoneal (i.p.) for cyclophosphamide	N-acetylcysteine (750 mg/kg, i.p.) and morphine (7.5 mg/kg, s.c.)	The study found that dragon’s blood significantly suppressed spontaneous nociceptive behaviors induced by capsaicin and cyclophosphamide (p < 0.001 in both tests) in mice, suggesting its potential as a treatment for visceral pain associated with gastrointestinal or urinary tract pathologies.	[40]
Antinociceptive effect of Croton celtidifolius Baill (Euphorbiaceae)	2006	Croton celtidifolius Baill	Ethanolic extract and various fractions (ethyl acetate, n-butanol, aqueous) of bark	Formalin test and acetic acid-induced writhing test	Male Swiss mice (6–10 animals per group)	Ranged from 3 to 300 mg/kg (specific doses varied by extract and fraction) p.o. and i.p.	Indomethacin 10 mg/kg i.p., and acetylsalicylic acid 100 mg/kg p.o.	The study found that the ethanolic extract and fractions of Croton celtidifolius significantly reduced nociceptive responses in both phases of the formalin test (p < 0.05 and p < 0.01 in the second phase) and in the acetic acid-induced writhing test (p < 0.01). The ethyl acetate fraction was particularly effective, showing marked and dose-related inhibition of pain responses.	[41]
Anti-inflammatory activity of Croton cuneatus aqueous extract	2006	Croton cuneatus Klotzsch	Aqueous extract from the stem barks and leaves	Writhing test Tail flick test	Male albino mice (6 animals per group)	7 mg/kg i.p.	Morphine hydrochloride (3 mg/kg i.p.) Acetylsalicylic acid (200 mg/kg p.o.)	The results indicate that Croton cuneatus has significant antinociceptive properties (p < 0.05) in both tail-flick test and writhing tests, supporting its traditional use for pain relief.	[42]
Analysis of the antinociceptive effect of the proanthocyanidin-rich fraction obtained from Croton celtidifolius barks: Evidence for a role of the dopaminergic system	2006	Croton celtidifolius	Bark extract (proanthocyanidin-rich fraction)	Formalin test	Male Swiss mice (6–10 animals per group)	11 mg/kg (for 63SF), with other doses for antagonist drugs (e.g., L-ARG 600 mg/kg, L-NOARG 75 mg/kg)	Apomorphine 5 mg/kg i.p.	The study found that the proanthocyanidin-rich fraction (63SF) exhibited significant (p < 0.05) antinociceptive effects in the formalin test (nociceptive and inflammatory pain)	[43]
Antinociceptive effect of proanthocyanidins from Croton celtidifolius bark	2005	Croton celtidifolius	Hydroalcoholic extract of the tree bark followed by extraction with ethyl acetate and n-butanol fractions.	Formalin test, capsaicin test, glutamate test, tail-flick test, and hot-plate test	Male Swiss mice (6–10 animals per group)	100 mg/kg and 300 mg/kg i.p.	Morphine 5 mg/kg i.p.	The study demonstrated significant antinociceptive activity of the 63SF bark sub-fraction in both phases of the formalin test (p < 0.01), and in chemical and thermal tests, indicating its potential as an analgesic agent.	[44]
Antinociceptive effect of leaf essential oil from Croton sonderianus in mice	2005	Croton sonderianus	Essential oil of leaf	Acetic acid-induced writhing, hot-plate test, formalin test, and capsaicin-induced hind paw-licking	Male Swiss mice (8 per group)	50, 100, and 200 mg/kg per os	Morphine hydrochloride (7.5 mg/kg, s.c.) Acetylsalicylic acid (250 mg/kg)	The essential oil of Croton sonderianus produced significant antinociceptive effects against chemical nociception, particularly in acetic acid-induced writhing (p < 0.001) and capsaicin-induced paw-licking (p < 0.001) at doses of 100 and 200 mg/kg. Significant differences were also found in both phases (neurogenic and inflammatory) of the formalin test (p< 0.001).	[45]
Antinociceptive and anti-inflammatory effects of Croton malambo bark aqueous extract	2003	Croton malambo	Bark aqueous extract	Thermal and chemical methods (tail-flick test and acetic acid-induced writhing)	Male Albino mice (6 mice per group)	6 and 6.15 mg/kg i.p.	Acetylsalicylic acid 200 mg/kg p.o. Morphine hydrochloride 3 mg/kg i.p.	The extract exhibits a strong antinociceptive effect. In the tail-flick test, the extract significantly (p < 0.05 and p < 0.01) increased reaction time, indicating effective pain reduction. In the writhing syndrome test, the extract reduced the number of writhing episodes by 50% compared to the control group (p not shown). The antinociceptive effect of Croton malambo was greater than that of acetylsalicylic acid but less than morphine.	[46]
Investigations on the antinociceptive activity of crude extracts from Croton cajucara leaves in mice	2002	Croton cajucara Benth	Hexane, chloroform, and methanol leave extracts	Acetic acid-induced writhing test, hot-plate test, and formalin test	Male Swiss mice (6 per group)	100 and 200 mg/kg p.o.	Indomethacin 5 mg/kg p.o. Morphine 10 mg/kg s.c.	The study found that all tested extracts significantly (p < 0.05) reduced the number of writhing movements induced by acetic acid and effectively blocked the second phase of nociception in the formalin test (p < 0.05, 100 mg/kg; p < 0.01, 200 mg/kg). However, the extracts did not show significant efficacy in the hot-plate test.	[47]
Antinociceptive effects of the essential oil of Croton nepetaefolius on mice	2002	Croton nepetaefolius Baill	Essential oil from leaves	Acetic acid-induced writhing test, hot-plate test, and formalin test	Male Swiss mice (11 to 12 per group)	3, 30, and 300 mg/kg p.o.	Morphine 5 and 10 mg/kg s.c. Naloxone 5 mg/kg i.p.	The essential oil exhibited a dose-dependent antinociceptive effect, significantly reducing pain in the acetic acid-induced writhing test (p < 0.05) and both phases of the formalin test at higher doses (p < 0.05). The hot-plate test showed increased latency to pain response at certain doses (p < 0.05).	[48]
Antinociceptive effects of the essential oil of Croton zehntneri in mice	2001	Croton zehntneri	Essential oil from leaves	Formalin test, acetic acid-induced writhing test, hot-plate test	Male Swiss mice (from 6 to 10 animals per group)	100 mg/kg and 300 mg/kg per os	No reference drug	The essential oil significantly reduced paw-licking time in the second phase of the formalin test (p < 0.05). In the first phase of the formalin test, only the 300 mg/kg dose showed a significant reduction (p < 0.05). The number of contortions in the acetic acid-induced writhing test did not significantly differ from controls. In the hot-plate test, both doses significantly increased response latency compared to controls (p < 0.05).	[49]
Anti-inflammatory and Antinociceptive Effects in Rodents of the Essential Oil of Croton cajucara Benth	1999	Croton cajucara Benth	Essential oil from bark	Acetic acid-induced abdominal constriction and hot-plate	Male Albino Swiss mice	100, 500, and 1000 mg/kg per os	Morphine 10 mg/kg and Indomethacin 20 mg/kg Dipyrone 200 mg/kg p.o.	The essential oil exhibited significant antinociceptive effects, reducing abdominal constrictions at doses of 1000 mg/kg (p < 0.001). The oil showed a dose-dependent response, with higher doses leading to greater inhibition of pain. The oral administration of the essential oil at a dose of 1000 mg/kg significantly increased the latency compared to the control in the hot-plate test (p < 0.05).	[50]

Abbreviations: Per os (p.o), intraperitoneally (i.p.), ethanolic extract (EE), aqueous extract (AE), subcutaneous (s.c), leaves extract ethyl acetate fraction (EAF), stem bark triterpene acetyl aleuritolic acid (AAA), complete Freund’s adjuvant (CFA), trans-dehydrocrotonin (DCTN), trans-crotonin (CTN).

).

4. Discussion

The aim of this study was to compile and analyze the scientific evidence on the antinociceptive properties of various Croton species. Due to the decline in the publication of articles on this topic, we hypothesize that interest in exploring the analgesic potential of the Croton genus has decreased over the years. For this reason, we believe that this systematic review could serve as a reference point or turning point for the scientific community to revisit the study of the various Croton species, which, as mentioned earlier, total around 1300 species worldwide. Based on our inclusion criteria, we found 28 articles that evaluated 21 Croton species, suggesting that there is still much to be studied about this vast genus.

All the reviewed articles reported antinociceptive effects in different preclinical models of nociceptive, inflammatory, and neuropathic pain. The studies employed a range of pain models, including chemical (e.g., formalin-induced pain), thermal (e.g., hot-plate test), and mechanical (e.g., von Frey test) methods. This diversity in methodologies complicates direct comparisons of efficacy across species. For instance, while Croton cajucara demonstrated strong effects in the formalin test, its efficacy in thermal pain models remains less clear. Additionally, the sample sizes varied significantly across studies, with some utilizing small cohorts that may limit the generalizability of the findings.

Natural products derived from these plants serve as a significant source for the development and synthesis of new drugs due to their pharmacological effects, which are similar to those of conventional medications [51]. In this context, the authors attribute the antinociceptive effects to a variety of bioactive compounds found in these plants, primarily terpenoids, with flavonoids, alkaloids, and proanthocyanidins also playing a lesser but significant role [20,30,41,44,52]. Among the articles, the most frequently mentioned compounds include terpenoids such as acetyl aleuritolic acid and trans-crotonin [39]. Additionally, proanthocyanidins (polyphenolic compounds) have been highlighted for their ability to activate capsaicin-sensitive C fibers, contributing to pain reduction in the early stages of experimental models such as the formalin test [43]. The involvement of certain alkaloids and flavonoids present in the extracts has also been identified, as they appear to interact with inflammatory pathways and pain modulation mechanisms [20]. Other relevant compounds include 1,8-cineole, p-cymene, spathulenol, and caryophyllene oxide, which are found in the essential oil of certain species [27].

4.1. Mechanism of Action

We conducted an analysis of the proposed mechanisms of action for the antinociceptive effects of species within the genus Croton. Generally, these mechanisms include the modulation of ion channels, such as TRPV1, K/ATP, and ASIC channels, as well as the inhibition of the synthesis of inflammatory mediators, particularly prostaglandins [26,33,50,53,54]. The ability to interfere with the signaling of inflammatory mediators, such as cytokines (TNF-alpha, IL-1, IL-6) and prostaglandins, appears to be a central mechanism, akin to the effects of non-steroidal anti-inflammatory drugs (NSAIDs) [46,55,56]. Furthermore, the action on the dopaminergic and cholinergic systems, along with the inhibition of the glutamatergic system, has been highlighted in several studies, suggesting an effect at both central and peripheral levels [26,33,43]. Few articles reported an effect on the opioid system, and although it is not involved in all cases, some species exhibit interactions with mu, delta, and kappa opioid receptors, which also contribute to their analgesic effects [23,25,46,56]. In general, the mechanisms of action of Croton encompass both the modulation of central and peripheral nerve pathways and the inhibition of inflammatory mediators involved in pain perception. Additionally, effects on neuropathic pain or actions on the nervous system were also observed; Croton macrostachyus is one of the species that has demonstrated a clear effect on neuropathic pain by inhibiting TRPV1 receptors, which are directly involved in the sensitization and maintenance of mechanical hyperalgesia in neuropathic and inflammatory pain [18,31]. It has also been suggested that Croton urucurana and Croton cajucara may act on the central nervous system by modulating the nociceptive response at the spinal level, contributing to more profound analgesia [20,30,39]. The diversity of proposed mechanisms of action, including the modulation of ion channels and the inhibition of inflammatory mediators, suggests that Croton extracts could be effective in treating different types of pain, including nociceptive, inflammatory, and neuropathic pain. This is particularly relevant in the management of chronic pain, where current treatments are often inadequate or have undesirable side effects. Not all studies aimed to investigate the mechanisms of action or antinociceptive pathways.

4.2. Safety

Some of the reviewed studies included toxicity analyses to determine the safety of administration in murine models. For example, in the study by Suárez et al. [42], the acute toxicity of an aqueous extract of Croton cuneatus was evaluated in mice. Doses of 100, 300, and 1000 mg/kg were used. No significant toxic effects were observed at the lower doses; however, at 1000 mg/kg, a slight decrease in motor activity was noted. Similarly, in an antinociceptive study of proanthocyanidins from the bark of Croton celtidifolius [43], it was reported that Croton celtidifolius extracts showed an acceptable safety profile in the tests performed. Doses of 50, 100, and 200 mg/kg were used, and no significant adverse effects were reported. Oliveira et al. [49] assessed the toxicity of the essential oil of Croton zehntneri. Doses of 50, 100, and 200 mg/kg were used. No toxic effects were observed at therapeutic doses, although the authors noted that some compounds in the essential oil could have adverse effects at higher doses.

We found a couple of studies where antinociception was not assessed, but the toxicity of Croton was evaluated. For Croton gratissimus, a toxicity test was conducted in mice. The median lethal dose (LD50) of the extract was determined to be 1400 ± 148 mg/kg. This study revealed that the extract caused a reduction in respiratory rate and in the body and limb tone of the mice, leading to death in some cases [57]. In another study, the toxicity of extracts from four Croton species was evaluated using the murine macrophage cell line RAW 264.7. Although the concentrations used in the test were excessive compared to reasonable pharmacological doses, their use was justified to enhance the ability to detect subtle morphological changes that could correlate with drug-induced hepatotoxicity. Concentrations were evaluated at 30 to 100 times the maximum effective concentration (Cmax), implying that doses significantly higher than those expected in a normal therapeutic context were used. The findings indicated that, although all extracts showed toxicity towards Vero cells, only the Croton gratissimus extract presented a dose-dependent cytotoxic effect, with toxicities below the 55% threshold [58]. This suggests that, despite the observed toxicity, the C. gratissimus extract could have a relatively favorable safety profile compared to the other extracts evaluated.

Generally, extracts and essential oils have an acceptable safety profile at therapeutic doses; however, additional studies are recommended to evaluate chronic toxicity and long-term effects. A cautious approach to the use of essential oils in traditional medicine is advised. Nonetheless, it is essential to consider various factors that may influence toxicity, such as dosage, extraction method, and route of administration. The discussion in the articles suggests that, although Croton extracts may be safe in traditional contexts, further research is needed to fully understand their toxicity and the mechanisms of action of their compounds. Additionally, it is mentioned that variability in the chemical composition of plants, depending on factors such as climate and soil, can affect their safety and efficacy. Therefore, broader and more systematic toxicity studies are recommended to establish a clear safety profile before considering their use in clinical treatments.

Based on current evidence, it is suggested that preclinical studies be conducted to examine the effects of isolated bioactive compounds in order to identify those with the highest antinociceptive effects or interactions that enhance such effects. Following thorough safety studies, this could lead to clinical trials assessing the efficacy and safety of Croton extracts or other pharmaceutical forms in humans. Such studies would not only validate preclinical findings but also provide data on dosing and potential side effects.

Additionally, comparative studies between different Croton species are desirable, as current evidence makes it difficult to discern which species possesses greater potency and efficacy. Finally, fostering interdisciplinary collaboration among pharmacologists, clinical physicians, traditional healers, and botanists is essential to integrate knowledge and techniques that could enrich research on Croton and its application in allopathic treatment. This approach could promote education about the use of medicinal plants like Croton in pain management, both within the scientific community and among the general public, potentially increasing their acceptance and use in clinical practice.

5. Conclusions

This systematic study has compiled and analyzed scientific evidence on the antinociceptive effects of various species of the Croton genus. The findings indicate that certain species, such as Croton cajucara, have significant potential as sources of bioactive compounds with analgesic properties. In particular, the reviewed studies suggest that extracts of Croton cajucara can effectively reduce pain responses in experimental models, highlighting its viability as a natural alternative for pain management.

However, variability was observed in the results among different Croton species. For instance, while Croton cajucara showed consistent results in chemical pain tests, other species, such as Croton celtidifolius and Croton zehntneri, presented less clear antinociceptive effects, suggesting that not all species in the genus have the same therapeutic potential. This variability underscores the need for more comprehensive comparative studies to identify the most effective species and their specific mechanisms of action. These plants hold significant potential as sources of bioactive compounds with analgesic properties, suggesting their viability as natural alternatives in pain management. The identified mechanisms of action highlight the complexity and diversity of the biological interactions of Croton extracts and essential oils. With appropriate research, the Croton genus could significantly contribute to the development of new pain treatments, offering safer and more effective options compared to conventional analgesics.