Phytochemical, Bioactive, and Toxicological Aspects of Ageratina adenophora (Spreng.) R.M.King & H.Rob.: A Narrative Review

Abstract

Due to limitations in the use of antibiotics as feed additives to promote growth, the development of safe, effective, and environmentally friendly antibiotic alternatives is a critical and urgent research priority in animal nutrition and veterinary medicine. Plants contain abundant active substances, which are important sources for developing new feed additives or discovering new active substances. Ageratina adenophora (Spreng.) R.M.King & H.Rob. (A. adenophora) is an herb native to Central America and a globally invasive species that has spread across Asia, Australia, Africa, and other continents. Studies indicated that it possesses a complex chemical composition, with over 100 constituents identified, including terpenoids, flavonoids, and phenolic acids, which exhibit diverse biological activities, including antibacterial, antioxidant, insecticidal, acaricidal, anti-inflammatory, neuroprotective, antidiabetic, antitumor, and gut health regulation effects. However, it also possesses distinct toxicity, primarily targeting organs such as the intestines, liver, and spleen, causing tissue damage by activating specific signaling pathways. This may limit its utilization as a feed additive or in medical drug development. Therefore, this review systematically summarizes the botanical characteristics, geographic distribution, phytochemical composition, biological activities, and toxicological effects of A. adenophora. We also discussed its potential application and current challenges. We hope this review can promote its development and utilization as potential feed additives or medicinal resources.

1. Introduction

In modern animal husbandry, feed additives played an indispensable role in enhancing growth performance, preventing diseases, and improving feed conversion efficiency [1]. Historically, antibiotics served as a major class of feed additives owing to the potent antimicrobial activity and significant growth-promoting effects. However, the widespread and even indiscriminate use of antibiotics in feed over several decades has precipitated a severe global health crisis [1]. Among the most pressing issues is the widespread proliferation and intensification of antimicrobial resistance (AMR). The emergence and spread of drug-resistant bacterial strains not only lead to therapeutic failures in veterinary medicine but also threaten human medicine by transferring through the food chain and the environment, severely jeopardizing global public health security [2]. Beyond fostering “superbugs,” the use of in-feed antibiotics presents other significant negative impacts. Drug residues can accumulate in animal-derived food products, raising food safety concerns and potentially disrupting the human intestinal microbiota [3]. Furthermore, the non-targeted effects of antibiotics can disrupt the diversity of the animal’s gut microbiota, impair intestinal barrier function and immune homeostasis, thereby affecting nutrient absorption and utilization [3]. In light of these escalating risks, regulatory agencies worldwide have implemented stringent restrictions, exemplified by the European Union’s 2006 and China’s 2020 bans on the use of antibiotics as growth promoters in animal feed [1,4]. This trend signals the dawn of a post-antibiotic era, making the search for safe, effective, and environmentally friendly antibiotic alternatives a critical and urgent research priority in animal nutrition and veterinary medicine.

Plant extracts are rich in diverse secondary metabolites such as polyphenols, alkaloids, and essential oils [5]. These active ingredients exert multi-targeted biological activities, including antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory effects, through synergistic interactions. More importantly, the natural attributes of plant-derived additives confer excellent ecological compatibility, i.e., their residues are readily biodegradable, minimizing environmental persistence and thus reducing the risk of contaminating the food chain. Studies have shown that specific plant extracts can maintain gut health by modulating the composition of the gut microbial community and enhancing intestinal epithelial barrier function [1]. Therefore, the development of feed additives based on natural products not only aligns with the global advocacy for “green” and sustainable animal farming practices but also represents a crucial strategic direction for combating the AMR crisis and ensuring the safety of animal-derived foods.

Ageratina adenophora (Spreng.) R.M.King & H.Rob. (A. adenophora) is a perennial invasive herbaceous plant of the Asteraceae family, and is native to Mexico and Costa Rica in Central America. It has now spread widely across multiple continents including Asia, Australia, and Africa [6]. Currently, its invasion covers over 30 million hectares in Southwest China and parts of South China, posing a serious threat to ecosystem stability [7,8,9]. This plant exhibits a complex and diverse chemical compositions, including terpenoids (sesquiterpenes, monoterpenes), flavonoids, phenolics, and cyclodipeptides. Among these, terpenoids, especially sesquiterpenes, constitute the major compound class of A. adenophora, and cadinene-type sesquiterpenes are regarded as its characteristic core constituents [10,11]. Based on this complex phytochemical foundation, A. adenophora exhibits diverse biological activities, including antimicrobial effects against microorganisms, insecticidal and acaricidal actions [11,12], and significant antioxidant and anti-inflammatory effects [11,13]. Additionally, its metabolites have demonstrated potential antitumor activity [14]. For example, the essential oil of A. adenophora displayed bactericidal activity against multiple bacteria in antimicrobial research [15]. Endophytic fungi isolated from A. adenophora also showed antibacterial potential against methicillin-resistant Staphylococcus aureus [9]. Moreover, dried A. adenophora biomass has been evaluated as a bio-fumigant to suppress Pythium aphanidermatum, and leaf extracts exhibited antioxidant and anti-inflammatory effects in oxidative-injury models of Caco-2 cells [16]. However, its exploration for applications in feed has been constrained due to concurrent reports of hepatotoxicity and other safety risks.

This article was designed as a narrative review rather than a formal systematic review. Relevant literature published up to July 2026 was identified through searches of the Web of Science Core Collection, CNKI, and PubMed using combinations of terms related to Ageratina adenophora, phytochemistry, biological activity, antimicrobial activity, toxicity, toxicology, detoxification, and feed utilization. Additional publications were identified by screening the reference lists of relevant studies and reviews. Peer-reviewed studies directly addressing the botanical characteristics, geographic distribution, chemical constituents, biological activities, toxicological effects, detoxification strategies, or feed applications of A. adenophora were considered, whereas duplicate records, conference abstracts, studies lacking sufficient methodological information, and publications without direct relevance to the scope of this review were excluded. Owing to the substantial heterogeneity in experimental models, preparations, exposure regimens, and outcome measures, no formal risk-of-bias assessment or meta-analysis was conducted. Instead, the evidence was synthesized qualitatively, with greater emphasis placed on studies providing clearly defined experimental conditions, quantitative activity or toxicity data, and mechanistic validation. On this basis, the present review critically integrates current knowledge of the phytochemistry, bioactivities, toxicological risks, detoxification approaches, and potential feed applications of A. adenophora, with the aim of identifying both its resource value and the evidence gaps that must be addressed before practical utilization.

2. Botanical Characteristics and Geographic Distribution

A. adenophora is classified within the genus Ageratina within the Asteraceae family. It is a perennial herb or subshrub with a chromosome number of 2n = 51. It is also called Eupatorium adenophorum Spreng., and it has several synonyms [17]. Plants attain a height of 1–3 m, with stems that are erect and clustered, and that are dark purple or green, covered in glandular hairs, and bearing opposite, ascending branches [18]. The leaves are categorized as either opposite, triangular or rhombic-ovate in shape, 4–15 cm long, with irregularly serrated margins, three basal veins, and petioles 1–6 cm long [19]. Capitula densely corymbose, the involucre broadly campanulate, with 3–4 layers of involucre bracts, tubular flowers white or pale purple, achenes pentagonal, apex with white bristly pappus, wind-dispersed [17].

A. adenophora, native to Mexico and Costa Rica in Central America, was introduced to Lincang, Yunnan Province, China from Myanmar in the 1940s [20]. It subsequently spread northeastward at an average annual rate of 20 km and is now widely distributed across southwestern and southern Chinese provinces including Yunnan, Guizhou, Sichuan, Guangxi, and Chongqing, with an invasion area exceeding 30 million hectares [19,21]. Globally, it has invaded parts of Asia, Africa, Oceania, and the Americas, including the fog belt of KwaZulu-Natal in South Africa, New South Wales in Australia, and Oahu in the Hawaiian Islands [20]. This species shows a marked preference for fertile, moist, and frequently disturbed habitats, such as stream banks, roadside verges, and forest clearings. It occurs across a broad elevational range of 500–2500 m and is particularly competitive in humid, high-rainfall environments, where it can rapidly establish dense, dominant stands [17,22].

3. Phytochemistry

Phytochemical studies have identified over 100 chemical constituents in A. adenophora (some of which are shown in Table 1), primarily isolated from the whole plant, leaves, and endophytic fungal tissues [23,24]. Notably, terpenoids (especially sesquiterpenes) constitute the major compound class, with cadinene-type sesquiterpenes representing core defence-related components [25,26]. Another significant group of active components is flavonoids, with the total flavonoid content in some populations of whole plants reaching 12.8 mg/g [24,27]. The total phenolic content of dried plant samples is 98.6 μg/g, notably including higher levels of caffeic acid and p-coumaric acid [20,24]. Among the terpenoids, geraniol and geraniol D have been shown to exhibit antioxidant, antibacterial, and insecticidal activities [9,23,27]. Additionally, amorpha-4,7(11)-diene (a sesquiterpene biosynthetic intermediate) [28], cyclo(L-proline-L-valine) (a novel cyclodipeptide) [10], and phorbol A (a novel terpene from endophytes) represent novel compounds first isolated from A. adenophora and its endophytes [13]. Other constituents include volatile oils, alkaloids, fatty acid esters, sterols, sugars, and other specialized metabolites [9,23,29].

Table 1.

A representative part of the isolated chemical constituents from A. adenophora.

No.	Compound	Molecular Formula	Structure	Reference
Terpenoids
1	9-Oxo-10,11-dehydroageraphorone (named euptox A)	C15H20O2		[28]
2	9-Oxo-ageraphorone	C15H22O2		[28]
3	9β-Hydroxyageraphorone	C15H24O2		[28]
4	β-Bisabolene	C15H24		[23]
5	p-cymene	C10H14		[23]
6	Bornyl acetate	C12H20O2		[23]
7	γ-Curcumene	C15H24		[23]
8	β-Caryophyllene	C15H24		[23]
9	Epi-α-cadinol	C15H26O2		[23]
10	Geraniol	C10H18O		[23]
11	Germacrene D	C15H24		[23]
12	β-Farnesene	C15H24		[23]
13	Nerolidol	C15H26O		[23]
14	2-Deoxy-2-(acetyloxy)-9-oxoageraphorone (named DAOA)	C17H22O4		[29]
15	Amorpha-4,7(11)-diene	C15H24		[28]
16	(–)-Amorph-4-en-7-ol	C15H26O		[28]
17	α-Phellandrene	C10H16		[23]
18	δ-2-Carene	C10H16		[23]
19	Safranal	C10H14O		[9]
Flavonoids
20	Isorhamnetin	C16H12O7		[9]
21	Quercetin	C15H10O7		[30]
22	Genistein	C15H10O5		[9]
23	Quercetin-3-O-β-D-glucoside	C21H20O12		[31]
24	Kaempferol-3-O-β-D-glucopyranoside	C21H20O11		[31]
25	Fortunellin	C28H32O14		[31]
26	Kaempferol	C15H10O6		[31]
Phenolic acids
27	Caffeic acid	C9H8O4		[9]
28	3-Coumaric acid	C9H8O3		[9]
29	2,3-Dihydroxybenzoic acid	C7H6O4		[9]
30	Salicylic acid	C7H6O3		[9]
Cyclic dipeptides
31	Cyclo (L-Pro-L-Val)	C10H16N2O2		[10]
32	Cyclo (L-Leu-L-Pro)	C11H18N2O2		[10]
33	Cyclo (L-Phe-L-Pro)	C14H16N2O2		[10]
Other Active Components
34	Phenyllactic acid	C9H10O3		[9]
35	Citric acid	C6H8O7		[9]
36	Glutaconic acid	C5H6O4		[9]
37	Dihydrocoumarin	C9H8O2		[9]

3.1. Sesquiterpenes

Sesquiterpenes are representative compounds of A. adenophora, featuring a 15-carbon (C₁₅) terpenoid backbone. These compounds are classified into several subclasses, including cadinene, and farnesene-type sesquiterpenes. The structural differences among these subclasses significantly influence their biological activities. The insecticidal and antibacterial activities of sesquiterpenes depend on the number and position of oxygen-containing functional groups in their structures, as well as the configuration of double bonds present [32]. For example, the cadinene sesquiterpene (9-oxo-10,11-dehydrodehydropangotenone, also named as euptox A) contains a carbonyl substituent at the 9-position of its skeleton. This structural feature is closely associated with its pronounced insect feeding deterrent activity and toxicity, which may function by disrupting insect energy metabolism [33]. The linear skeleton of the sesquiterpene biosynthetic intermediate (amorpha-4,7(11)-diene) serves as a key precursor for the subsequent formation of oxygen-containing active sesquiterpenes, and its structural transformation directly influences the chemical defense capacity of A. adenophora [28]. Liu et al. identified 9β-hydroxyageraphorone, which activates the salicylic acid signaling pathway by upregulating MYB (v-myb avian myeloblastosis viral oncogene homolog) and DOF (DNA binding with One Finger) genes, thereby enhancing tobacco resistance to Tobacco Mosaic Virus [34]. These findings highlight the importance of structure–activity relationships in shaping the biological functions of sesquiterpenes. In A. adenophora, sesquiterpenes accumulate predominantly in the leaves, with concentrations substantially higher than those in the roots and stems and peaking during the vigorous summer growth period, potentially reflecting increased defensive demands against insect herbivores [22].

3.2. Flavonoids

Flavonoids constitute major bioactive components in A. adenophora, featuring a 2-phenylchromenone backbone. These compounds are classified into several subclasses, including flavonoid aglycones and flavonoid glycosides, as well as other related compounds [31].

The structural differences among these subclasses exert a substantial influence on their biological activities. The antioxidant and anti-inflammatory properties of flavonoids depend on the number and position of hydroxyl groups in their structure. Hydroxylation at positions 3, 5, and 7 is crucial for enhancing antioxidant activity, as it neutralizes free radicals by providing electrons [30]. For example, quercetin has hydroxyl substitutions at positions 3, 5, 7, 3′, and 4′ of the flavonoid skeleton. This structural feature endows it with strong DPPH free radical scavenging ability while also exerting anti-inflammatory effects by inhibiting the release of inflammatory factors [9]. Additionally, the phenolic hydroxyl groups of flavonoid molecules bind to the active center of acetylcholinesterase while simultaneously acting on the peripheral site of the enzyme, with inhibition efficiency positively correlated with the number of phenolic hydroxyl groups [31].

3.3. Cyclodipeptides

Cyclodipeptides are distinctive bioactive compounds produced by endophytic bacteria within the A. adenophora. They feature a fundamental backbone formed by two amino acids cyclized via a peptide bond. They are classified into multiple subclasses, including cyclic (L-proline-L-valine) types, cyclic (L-leucine-L-proline) types, and cyclic (L-alanine-L-proline) types [10].

Cyclic dipeptides containing proline contribute to antibacterial activity, and their efficacy appears to depend on amino acid composition and stereochemical configuration. In the study by Ren et al., cyclo (L-Pro-L-Val) and cyclo (L-Leu-L-Pro) showed mild inhibitory activity against Staphylococcus aureus and Escherichia coli [10]. The cyclic structure of cyclodipeptides confers stability, enabling them to function long-term as signaling molecules or defense compounds in plant-microbe interactions [35].

4. Biological Activities of A. adenophora

Based on the various bioactive components, such as sesquiterpenes, flavonoids, and cyclic dipeptides, A. adenophora exhibited multiple biological activities, including antimicrobial, antioxidant, neuroprotective, antidiabetic, antitumor, and gut health regulation effects. In the following sections, we will discuss them in detail.

4.1. Antibacterial Activity of Extracts

Studies have shown that solvent extracts, essential oils, and endophyte-derived metabolites from A. adenophora exhibit notable antibacterial activity (Table 2), characterized by marked species selectivity and clear concentration dependence. For example, Zhang et al. prepared an A. adenophora extract using Soxhlet extraction with 75% ethanol and reported that the MIC values antibacterial activity against Escherichia coli and Staphylococcus aureus (S. aureus) were 0.075 g/mL and 0.05 g/mL, respectively [36]. Thapa et al. reported that the chloroform leaf extract of A. adenophora exhibited the most pronounced activity against E. coli and Proteus vulgaris, also showed consistent antibacterial effects against Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, and Salmonella typhi, all yielding inhibition zones of 8 mm [37]. In a subsequent study, Thapa et al. demonstrated that the essential oil of A. adenophora produced an inhibition zone of ~12 mm against Staphylococcus aureus and exerted inhibitory effects on Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus, with inhibition zones of 8 mm, 7 mm, and 7 mm, respectively [38]. Liu et al. isolated 9-oxo-10,11-dehydroageraphorone (ODA) from A. adenophora, this compound showed potent antibacterial activity against Ralstonia solanacearum, with MICs ranging from 0.25 to 1 mg/mL and MBCs of 2 mg/mL for all tested strains [39]. Zhang et al. further isolated eight thymol derivatives from the roots of A. adenophora. Among these compounds, 7-formyl-9-isobutyryloxy-8-hydroxythymol exhibited potent antibacterial activity, with a minimum inhibitory concentration (MIC) of 3.9 μg/mL, supporting its potential as a natural lead compound for antibacterial drug development [26].

4.1.1. Antibacterial Activity of Endophytic Microorganisms

In recent years, Ren et al. isolated an endophytic Bacillus velezensis (B. velezensis) strain Ea73 from A. adenophora. The fermentation broth exhibited broad-spectrum antibacterial activity against five pathogenic bacteria, namely Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and S. aureus, the inhibition zone against S. aureus reached 32.2 mm [10]. Two cyclic dipeptides, cyclo(L-Pro-L-Val) and cyclo(L-Leu-L-Pro), were further purified from the Ea73 fermentation broth. Both compounds exhibited moderate inhibitory activity against S. aureus and E. coli [10]. Tang et al. further demonstrated that the cell-free supernatant of B. velezensis EA73 had a minimum biofilm eradication concentration (MBEC) of 1.3 mg/mL against S. aureus biofilms. This antibiofilm effect was associated with the downregulation of biofilm-related genes (e.g., icaA, icaD, and sarA), accompanied by reduced levels of extracellular polysaccharides, extracellular proteins, and extracellular DNA (eDNA), providing a theoretical basis for developing endophyte-derived antimicrobial agents from A. adenophora [40].

4.1.2. Antibacterial Enhancement by Nanomaterials

Dua et al. employed an aqueous leaf extract of A. adenophora as both a bioreducing agent and a stabilizer to synthesize silver nanoparticles (AgNPs) via a green route. Mechanistic analyses indicated in antibacterial assays that AgNPs adhere to the bacterial cell surface and disrupt membrane integrity, resulting in leakage of intracellular components, concurrently, they induce the generation of reactive oxygen species, increasing the DNA strand-break rate by ~40% [41].

Table 2.

An overview of antibacterial activity of extracts of A. adenophora and related products.

Source Category	Key Active Component/Material	Target Microorganism(s)	MIC or MBC or ZOI or EC50	Reference
Crude extracts	70% ethanol extract	Escherichia coli	MIC = 0.075 g/mL, ZOI = 13.3 mm	[36]
		Staphylococcus aureus	MIC = 0.05 g/mL, ZOI = 14.2 mm
	Chloroform extract	Escherichia coli ATCC 8739	ZOI = 12 mm	[37]
		Proteus vulgaris ATCC 6380	ZOI = 12 mm
	Essential oil	Staphylococcus aureus KCTC 1916	ZOI = 12 mm	[38]
		Klebsiella pneumoniae KCTC 2242	ZOI = 8 mm
		Bacillus subtilis KACC 17047	ZOI = 7 mm
		Micrococcus luteus KACC 13377	ZOI = 7 mm
	ODA	Ralstonia solanacearum	MIC = 0.25–1 mg/mL, MBC = 2 mg/mL	[11,39]
		Fusarium oxysporum	EC50 = 0.3 mg/mL
	7-formyl-9-isobutyryloxy-8-hydroxythymol	Staphylococcus aureus, Bacillus thuringiensis	MIC = 7.8 μg/mL	[39,42]
		Bacillus cereus	MIC = 3.9 μg/mL
		Escherichia coli, Salmonella enterica	MIC = 15.6 μg/mL
	7-hydroxy-dehydrotremetone	Colletotrichum gloeosporioides	ZOI = 17.3 mm	[43]
		Colletotrichum musae	ZOI = 17.2 mm
		Rhizoctonia solani	ZOI = 16.4 mm
		Fusarium oxysporum f. sp. niveum	ZOI = 13.9 mm
		Alternaria alternata	ZOI = 14.4 mm
Endophyte-derived products	Fermentation broth of Bacillus velezensis Ea73	Staphylococcus aureus	ZOI = 32.2 mm	[10]
	Cyclo(L-Pro-L-Val)	Staphylococcus aureus	MIC = 256 μg/mL
	Cyclo(L-Pro-L-Val)	Escherichia coli	MIC = 512 μg/mL
	Ethyl acetate extract of endophytic fungus Penicillium sclerotigenum DCL06	Methicillin-resistant Staphylococcus aureus	MIC = 0.5–1 mg/mL, MBC = 1–2 mg/mL	[24]
	Ethyl acetate extract of endophytic fungus Diaporthe kochmanii DCL09	Escherichia coli (SMU1710)	MIC = 0.5–1 mg/mL, MBC = 1–2 mg/mL
Nanomaterials	Silver nanoparticles (AgNPs)	Staphylococcus aureus	MIC = 64.5 μg/mL	[41]
	Silver nanoparticles (AgNPs)	Escherichia coli	MIC = 82.5 μg/mL

Note: ZOI, zone of inhibition; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; ODA, 9-oxo-10,11-dehydroageraphorone; EC₅₀, half maximal effective concentration; MBEC, minimum biofilm eradication concentration.

Overall, the reported antibacterial activity of A. adenophora varies markedly among different studies, depending on the type of preparation, target microorganism, and evaluation index. Crude extracts and essential oils generally showed inhibitory effects at relatively high concentrations, whereas some purified compounds, such as thymol derivatives, exhibited lower MIC values at the μg/mL level. Endophyte-derived products and green-synthesized nanomaterials also displayed antibacterial potential, but their activity and mechanisms differed from those of plant extracts and isolated phytochemicals. Therefore, the current evidence indicates that A. adenophora possesses antibacterial potential, but these findings are mainly based on in vitro assays and are not yet sufficient to support its direct use as a mature antimicrobial alternative in animal production. At present, A. adenophora is more appropriately regarded as a source of antibacterial lead compounds or bioactive materials that require further purification, standardization, safety evaluation, and in vivo validation.

4.2. Inhibitory Effects Against Fungi

Chaiwaree et al. reported that the ethanol extract of A. adenophora exhibited pronounced antidermatophytic activity, with MIC of 0.04 mg/mL against Trichophyton mentagrophytes and <0.0025 mg/mL against Trichophyton rubrum [11]. Liu et al. isolated cadinene-type sesquiterpenes from the methanolic extract of A. adenophora, their antifungal activities were evaluated for the first time against wood-decay fungi. At 100 μg/mL, cadinene-type sesquiterpenes showed the highest inhibition against Inonotus cuticularis M1308 strain and Inonotus cuticularis M1239 strain, reaching 59.7% and 44.4% [44]. Liu et al. reported that ODA isolated from A. adenophora had an EC50 of 0.325 mg/mL (48 h) against Fusarium oxysporum [39]. Zheng et al. further found that the benzofuran derivative 7-hydroxy-dehydrotremetone, isolated from the roots of A. adenophora, exhibited broad-spectrum inhibitory activity. At a dose of 50 mg per paper disc, the inhibition zone diameters against Colletotrichum gloeosporioides, Colletotrichum musae, Rhizoctonia solani, Fusarium oxysporum f. sp. niveum, and Alternaria alternata were 17.3 mm, 17.2 mm, 16.4 mm, 13.9 mm, and 14.4 mm, respectively [43]. These results suggest that plant-derived natural products with activity against multiple agricultural pathogenic fungi may offer useful leads for the development of green fungicides.

4.3. Antioxidant Activity

In in vitro evaluations of the antioxidant activity of A. adenophora, the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay is the most commonly used. The median inhibition concentration (IC₅₀) values of its chloroform extract, 70% ethanol extract, ethyl acetate extract, petroleum ether extract, and aqueous extract were 1460 μg/mL, 74 μg/mL, 123 μg/mL, 232 μg/mL, and 55 μg/mL, respectively [37,45,46]. In the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, the IC₅₀ values of its aqueous extract, 95% ethanol extract, ethyl acetate extract, n-butanol extract, and petroleum ether extract were 13.2 μg/mL, 22.9 μg/mL, 29.7 μg/mL, 29.6 μg/mL, and 30.9 μg/mL, respectively [37,45,46]. Dua et al. synthesized AgNPs via a green approach using an aqueous leaf extract of A. adenophora, and the resulting nanoparticles exhibited a DPPH-scavenging IC₅₀ of 49.0 μg/mL [41]. In a t-BHP-induced oxidative-injury model in Caco-2 cells, Li et al. reported that pretreatment with the aqueous extract at 1 and 5 mg/mL significantly increased the cell viabilities to 69.0%, 78.9% and reduced intracellular ROS levels of 43.9% and 82.8%, respectively [16]. Okyere et al. isolated and identified an endophytic Bacillus toyonensis strain (i.e., SAU-19) from A. adenophora. In a high-fat diet mouse model, SAU-19 significantly attenuated the elevation of malondialdehyde (MDA) in serum and intestinal tissues and effectively reversed the decline in key antioxidant indices, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), highlighting endophyte-derived resources as a promising direction for the development and utilization of the antioxidant potential of A. adenophora [47].

Additionally, the antioxidant potential of A. adenophora can also be interpreted from its phytochemical composition. As abovementioned, A. adenophora contains multiple flavonoids and phenolic acids, including quercetin, kaempferol, isorhamnetin, caffeic acid, and p-coumaric acid, as well as their glycosidic derivatives, which are all chemical classes commonly associated with redox regulation [9,30]. Flavonoids rich in phenolic hydroxyl groups can directly donate hydrogen atoms or electrons to neutralize reactive radicals, and can also enhance endogenous antioxidant defense systems [48]. For instance, quercetin can trigger Nrf2 nuclear translocation and elevate GSH to defend cells against oxidative stress, kaempferol and isorhamnetin mitigate oxidative damage partially via the Nrf2/HO-1 pathway [49,50,51]. In parallel, phenolic acids such as caffeic acid and p-coumaric acid exhibit well-documented radical-scavenging and anti-lipid-peroxidation activities [52]. Therefore, the antioxidant activity of A. adenophora extracts is mainly attributed to the flavonoids and phenolic acids.

4.4. Anti-Inflammatory Activity

Okyere et al. identified the endophytic strain Bacillus toyonensis SAU-19 from A. adenophora and conducted a 35-day oral gavage study in high-fat diet mice. Compared with the high-fat diet group, SAU-19 significantly reduced the production of the pro-inflammatory cytokines IL-1β and TNF-α in small intestinal tissue, while maintaining the mRNA and protein levels of the anti-inflammatory cytokines IL-4 and IL-10. In addition, SAU-19 decreased the abundance of pathogenic bacteria such as Escherichia coli, Salmonella, and Staphylococcus and increased beneficial taxa including Lactobacillus in the small intestine and cecum [47]. Shi et al. reported that isolated essential oil from A. adenophora at concentrations ≤20 mg/mL showed no cytotoxicity toward RAW264.7 macrophages and significantly suppressed lipopolysaccharide (LPS)-induced the expression of Toll-like receptor 4 (TLR4) and IL-6 mRNAs, and upregulated the expression of IL-10 mRNA. These results indicated that isolated essential oil from A. adenophora can inhibit the inflammatory response via blocking the TLR4 pathway [53].

4.5. Insecticidal and Acaricidal Activity

Sun et al. reported that an ethanolic extract of A. adenophora exhibited strong nematicidal activity against Bursaphelenchus xylophilus. The same study further demonstrated pronounced fumigant toxicity against adult stored-product insects, including Sitophilus oryzae and Sitophilus zeamais [14]. Li et al. found that an aqueous extract of A. adenophora exhibited pronounced molluscicidal activity across developmental stages of Limax maximus [54]. Mechanistic analyses indicated that the extract disrupted redox homeostasis and detoxification metabolism, as evidenced by decreased activities of antioxidant enzymes, including SOD, peroxidase, and CAT, and detoxification enzymes such as cytochrome P450 and glutathione S-transferase. Changes in acetylcholinesterase activity were also observed, suggesting that interference with neural and metabolic functions may contribute to its molluscicidal effects [54].

Rajeswary et al. reported that the methanolic leaf extract of A. adenophora exerted concentration-dependent larvicidal activity against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi [55]. Samuel et al. further showed that petroleum ether and chloroform extracts displayed pronounced larvicidal activity against C. quinquefasciatus [12]. In addition, constituents such as safranal and dihydrocoumarin exhibited toxic effects against Solenopsis invicta, further supporting the potential of A. adenophora-derived compounds as sources of botanical insecticidal agents [9].

Collectively, these findings indicate that A. adenophora possesses insecticidal, nematicidal, and molluscicidal activities against several agriculturally and medically important pests. Its extracts and isolated constituents may therefore provide candidate materials for botanical pesticide development and integrated pest management. However, most available evidence is derived from laboratory bioassays. Further studies are required to establish standardized preparations, clarify target-specific mechanisms, assess environmental persistence and non-target toxicity, and verify efficacy under field conditions.

4.6. Others

It was reported that A. adenophora exerts significant regulatory effects on mammalian intestinal and rumen microbial communities [56]. Okyere et al. isolated and identified two strains of Bacillus toyonensis (SAU-19 and SAU-20) from A. adenophora. Studies revealed that in a high-fat diet-induced dysbiosis mouse model, oral administration of SAU-19 and SAU-20 significantly reduced body weight gain, improved intestinal structural integrity, increased ileal villus height and crypt depth, and upregulated mRNA expression levels of tight junction proteins (i.e., occludin and ZO-1). This restored gut microbiota balance, reduced levels of pathogenic bacteria such as Escherichia coli, Salmonella, and Staphylococcus aureus in the ileal and cecal contents, while increasing the abundance of beneficial Lactobacillus spp. [47].

In summary, A. adenophora contains a variety of bioactive constituents, including terpenoids, flavonoids, and polyphenols, which exhibit multiple biological activities, including antimicrobial, antioxidant, anti-inflammation, and gut health regulation effects. Notably, some components can scavenge ROS, alleviate oxidative stress, and suppress inflammation-related signaling pathways, which together contributed to the beneficial effects observed in mammalian cells (Figure 1). However, Current research has not conducted further comparative studies to determine which compound is primarily responsible for anti-inflammatory activity. Further research is still needed.

Figure 1.

Comprehensive biological activities of A. adenophora containing bioactive compounds in mammalian cells.

5. Toxicological Profiles of A. adenophora

Among the toxic constituents identified in A. adenophora, cadinene-type sesquiterpenes [e.g., 2-deoxo-2-(acetyloxy)-9-oxoageraphorone (DAOA), 9-oxoageraphorone (OA), and ODA] are prominent [57]. The following sections summarize toxic effects and mechanisms of A. adenophora in the intestine, liver, spleen, and rumen epithelium of animals, and are summarized in Table 3. Importantly, current evidence suggests that the toxic effects of A. adenophora are not uniform, but are strongly influenced by exposure dose and animal species. Therefore, the toxicity evaluation of A. adenophora should be interpreted from both dose–response and species-specific perspectives, especially when considering its potential utilization in animal production.

Table 3.

A summary of toxic effects and mechanisms of A. adenophora on major target organs in animals.

Toxicity Category	Animal Model	Key Toxic Constituents	Pathological Characteristics	Mechanisms of Action	References
Intestinal toxicity	Sprague–Dawley rats	Euptox A	Villus desquamation, inflammatory-cell infiltration, mucosal hemorrhage, decreased villus height, and reduced barrier integrity.	Activation of MLCK/ROCK signaling and downregulation of occludin, claudin-1, and ZO-1, following to increase intestinal permeability.	[58]
	Black goats	Euptox A and sesquiterpene derivatives	Necrosis and sloughing of intestinal epithelial cells, lamina propria edema, ileal villus disruption, and crypt hyperplasia.	MLCK/ROCK activation and membrane damage by sesquiterpene derivatives, then impair intestinal epithelial integrity.	[59]
Hepatotoxicity	Rats	Methanolic leaf extract of A. adenophora and a partially purified terpenoid toxic fraction	Elevated bilirubin, ALP, 5′-nucleotidase, ALT, and AST; bile-duct dilatation/hyperplasia; periductal hepatocellular necrosis.	Selective injury to the bile duct epithelium leads to cholestasis and secondary hepatocellular necrosis.	[60]
	Kunming mice	DAOA, OA, ODA	Disrupted hepatic lobules, focal/bridging necrosis, nuclear pyknosis, fibrosis, and increased ALT, AST, and ALP; DAOA showed the lowest LD50.	The exposure to high doses can cause cholestasis and hepatocellular necrosis through biliary and hepatocellular injury. Concurrent damage to immune organs (thymus and spleen) may then amplify hepatic inflammation.	[57]
	Chengdu Ma goats	2-deoxo-2-acetoxy-9-oxoageraphorone and nerolidol	Focal steatosis, ballooning degeneration, hepatocellular swelling, inflammatory infiltration, and increased ALT, AST, ALP, GGT, TC and total bilirubin (TBiL).	Imbalance of hepatic cytokine networks, i.e., pro-inflammatory mediators (TNF-α, IL-1β, IL-6, IL-9, IL-17, and CRP) were significantly upregulated at the mRNA level, whereas anti-inflammatory mediators (IL-4, IL-10, and TGF-β1) were significantly downregulated. Disruption of hepatic metabolic homeostasis, with perturbations in neuroactive ligand–receptor interaction and pyrimidine metabolism pathways.	[61]
Splenic toxicity	Rats	No specific single compound identified	Increased spleen index, white-pulp atrophy, red-pulp congestion/hemorrhage, and impaired splenic immune architecture.	Disruption of the splenic FRC network with downregulation of chemokines CCL21 and CCL19, resulting in reduced homing of CD3+ T cells to the spleen, concomitant skewing of the splenic Th1/Th2 balance toward a Th1 phenotype, ultimately suppressing splenic immune function and causing immune injury.	[62]
Splenic toxicity	Goats	No specific single compound identified	Dose-dependently increases apoptosis in splenocyte. Caspase-9 and caspase-3 activity and expression were significantly increased, Bax protein was upregulated and Bcl-2 was downregulated. Typical splenic lesions included splenic corpuscle atrophy and disordered lymphocyte arrangement.	Activation of the mitochondrial apoptotic pathway: upregulation of Bax and downregulation of Bcl-2, leading to collapse of mitochondrial membrane potential and cytochrome c release, thereby triggering the caspase-9/caspase-3 cascade, induction of G0/G1 cell-cycle arrest in splenocytes.	[63]

Note: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LD₅₀, median lethal dose; GGT, gamma-glutamyl transferase; TBiL, total bilirubin; TC, total cholesterol; CRP, C-reactive protein; FRC, fibroblastic reticular cell; CCL19, C-C motif chemokine ligand 19; CCL21, C-C motif chemokine ligand 21; Th1/Th2, T helper 1/T helper 2.

5.1. Hepatotoxicity

Ouyang et al. conducted acute and subacute toxicity assays in Kunming mice, thereby elucidating the hepatotoxicity of three cadinene-type sesquiterpenes from A. adenophora. In a single-dose oral acute toxicity test, the median lethal dose (LD₅₀) of DAOA in male mice was 926 mg/kg, whereas the LD₅₀ values of OA and ODA were both 1470 mg/kg, indicating that DAOA is more hepatotoxic than OA and ODA. In a 7-day oral gavage subacute study, DAOA treatment at 150 mg/kg significantly increased serum total bilirubin (TB) as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels, whereas OA induced significant elevations in these liver-injury indices only at 300 mg/kg, supporting the view that these sesquiterpenes can induce dose-dependent cholestasis and liver injury and may exert immunotoxic effects [57]. These findings provide relatively direct evidence for dose-dependent hepatotoxicity, as lower doses of DAOA were sufficient to induce significant increases in liver injury biomarkers, whereas higher doses were required for OA to produce comparable effects. Shao et al. fed goats with 40% A. adenophora powder for 90 days. Marked inflammatory response, accompanied by focal steatosis, ballooning degeneration, hepatocellular swelling, and inflammatory cell infiltration, was detected in the liver tissue, it also significantly upregulated the expression of IL-9, IL-17, TNF-α, C-reactive protein (CRP), IL-1β, and IL-6 mRNAs and significantly downregulated the expression of IL-4, IL-10, and TGF-β1 mRNAs. Untargeted metabolomics results showed that A. adenophora exposure also dysregulated hepatic energy and lipid metabolism [64]. In a 60-day feeding study conducted on rats, the subjects were provided with a diet consisting of 3:7 A. adenophora powder and standard feed, Ren et al. noted that DAOA, OA, and ODA identified from A. adenophora have been established as key toxic constituents responsible for animal liver injury, providing additional evidence for the hepatotoxic risk associated with this plant [62]. Euptox A is a major hepatotoxic component of A. adenophora, and it can induce hepatic lesions such as focal hepatocellular necrosis and degenerative desquamation of bile duct epithelium and cell cycle arrest and cell apoptosis in hepatocyte of experimental animals [65]. Kaushal et al. reported that hepatotoxic constituents of A. adenophora can be extracted with methanol and partially purified, following 48 h of exposure in rats. Serum total bilirubin, conjugated bilirubin, ALP, 5′-nucleotidase, ALT, and AST levels were markedly elevated. The histopathological alterations included bile duct dilatation and hyperplasia, periductal hepatocellular necrosis, and mononuclear cell infiltration. The toxic principles were mainly terpenoid compounds, which can induce cholestasis and hepatic parenchymal injury in rats [60].

Network toxicology predictions indicate that the primary toxic components of A. adenophora are 2-deoxy-2-acetoxy-9-oxo-cinnamaldehyde, 10Hβ-9-oxo-cinnamaldehyde, 10Hα-9-oxo-cinnamaldehyde, nerolidol, and 9-oxo-10,11-dehydro-phellandrene, all of which comply with Lipinski’s rules and exhibit good oral bioavailability [29]. A comprehensive metabolomic analysis was conducted on sheep liver tissue samples that had been fed a diet containing A. adenophora for a period of 90 days. The analysis revealed a significant enrichment of metabolites associated with histidine metabolism, arachidonic acid metabolism, and neuroactive ligand-receptor interactions. Transcriptomic analysis further identified epidermal growth factor receptor (EGFR), G protein-coupled bile acid receptor 1 (GPBAR1), and peroxisome proliferator-activated receptor alpha (PPARα) identified as core genes [29].

After 6 weeks of feeding mice a diet containing 300 g/kg A. adenophora, a marked increase in ROS levels, and MDA contents and decreases in the activities of SOD and GSH-Px were detected in the liver tissues [66]. Concurrently, mitochondrial ultrastructure was disrupted with increased swelling, while Na⁺K⁺-ATPase and Ca²⁺Mg²⁺-ATPase activities and mitochondrial DNA copy number were markedly decreased [66]. Furthermore, it has been demonstrated that this process results in the upregulation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome expression, which subsequently leads to elevated levels of caspase-1 activity and the secretion of IL-1β and IL-18. Ultimately, this culminates in the occurrence of hepatocyte pyroptosis [66]. This evidence indicates that A. adenophora-induced liver injury involves the oxidative stress, mitochondrial, pyroptosis pathways. The detailed mechanism of A. adenophora-induced liver injury presented in Figure 2.

Figure 2.

Mechanism of A. adenophora-induced hepatotoxicity via ROS-mitochondrial axis drives NLRP3-mediated pyroptosis.

5.2. Gastrointestinal Toxicity

After 60 days of feeding rats with a A. adenophora powder and basal diet in a 3:7 ratio, the small intestine exhibited villus shedding, inflammatory cell infiltration, and hemorrhage. The duodenal villus height decreased, the crypt depth increased, and the villus height/crypt depth (V/C) ratio significantly decreased. Concurrently, serum intestinal injury markers diamine oxidase and D-lactic acid were significantly increased and the expressions of several intestinal tight junction proteins, including occludin, claudin-1, and ZO-1 mRNA were significantly downregulated [58]. Similarly, Wang et al. observed marked pathological intestinal damage in goats fed 40% A. adenophora powder for 90 days, with significantly more severe injury in the small intestine than in the large intestine [59].

The primary toxic mechanism involves the activation of the myosin light-chain kinase (MLCK) and Rho-associated coiled-coil-containing protein kinase (ROCK) signaling pathway. It has been reported that euptox A has the capacity to induce the upregulation of MLCK and ROCK1/2 genes and protein expressions in ileal tissue of goats [59]. Concurrently, MLCK mediates myosin light chain (MLC2) phosphorylation, while ROCK inhibits myosin light chain phosphatase (MLCP) activity by phosphorylating myosin phosphatase target subunit 1 (MYPT1). Together, these processes induce centripetal contraction of the cytoskeleton, which ultimately leading to increased intestinal permeability [59].

Additionally, it was also reported that A. adenophora can specifically damage the ruminal epithelium of goats. After feeding a diet containing 40% powdered plant material for 90 days, the epithelium of both the dorsal and ventral rumen sacs became darker (with black–green adherent deposits), and ruminal papillae were sparse. The mRNA and protein expression levels of the tight-junction proteins occludin, claudin-1, and ZO-1 in epithelial cells decreased by 30% to 50% [67].

5.3. Spleen Toxicity

Ren et al. conducted an in vivo study in 7-week-old male rats fed a diet containing A. adenophora powder for 60 days. It significantly increased the spleen index and marked histopathology injury (i.e., white pulp atrophy and congestion/hemorrhage of the red pulp) in the spleen tissues of rats were observed [62]. A. adenophora exposure can also disrupt the splenic fibroblastic reticular cell (FRC) network, which was evidenced by a disruption of the Th1/Th2 immune balance in rats [62,68]. He et al. performed a 3-month feeding experiment in Saanen dairy goats receiving diets containing 40%, 60%, or 80% A. adenophora, respectively. They found that A. adenophora induced dose-dependent apoptosis in goat splenocytes [63]. In a 7-day subacute toxicity study, Ouyang et al. orally administered the cadinane-type sesquiterpene DAOA (at 300 mg/kg body weight) derived from A. adenophora, to Kunming mice. It significantly increased the spleen index and was accompanied by thymic atrophy, demonstrating a direct toxic effect of A. adenophora sesquiterpenes on immune organs such as the spleen. This phenomenon has been linked to the process of splenocyte apoptosis [57]. Mechanistically, this pro-apoptotic effect depended on the mitochondrial pathway, characterized by upregulation of Bax and downregulation of Bcl-2, release of cytochrome c (Cyt c) from mitochondria into the cytosol, and activation of the caspase-9/caspase-3 apoptotic cascade. In addition, A. adenophora exposure caused G0/G1 cell-cycle arrest in splenocytes, which ultimately resulted in splenocyte apoptosis and impaired splenic immune function [63].

Collectively, the above findings suggest that the toxicological effects of A. adenophora are jointly governed by exposure dose, duration of intoxication, and species-specific susceptibility. The severity of damage not only progressively increases with higher exposure levels and longer intoxication periods, but also differs markedly across species in terms of target organs and overall sensitivity. Ouyang et al. and Sun et al. showed that sesquiterpene toxins induced dose-dependent oxidative stress and pathological injury in the liver and spleen of mice, as evidenced by progressively increased ROS and MDA levels as well as aggravated histopathological lesions at higher doses [57,68]. Liao et al. and He et al. further demonstrated that A. adenophora extracts and euptox A significantly increased apoptosis and cellular injury in HeLa cells and goat splenocytes in a concentration-dependent manner; at higher concentrations, substantial apoptosis was induced through the mitochondrial and caspase-dependent pathways, whereas little or no obvious toxicity was observed at lower concentrations [13,63]. In an in vivo goat study [63,69], Wang et al. found that intestinal barrier injury progressively worsened with prolonged exposure to A. adenophora (30–60 d), and serum DAO and D-lactic acid levels increased in a time-dependent manner [67]. With regard to species-related variation, available studies suggest that horses are particularly susceptible to pulmonary toxicity, whereas cattle may exhibit anorexia, cessation of rumination, and photosensitization. In contrast, mice and rats mainly develop hepatotoxicity and cholestasis, while goats may display relatively different tolerance or altered feeding behavior depending on exposure level and plant source. Notably, even in rodent studies, toxic outcomes vary according to the geographic origin of the plant material and the method of preparation, suggesting that apparent species-related differences may also interact with the phytochemical heterogeneity of A. adenophora.

Accumulating evidence suggests that different livestock species exhibit significant differences in susceptibility to non-detoxified A. adenophora, while the safe dose thresholds have not yet been systematically established for most species. Current evidence predominantly demonstrates that goats develop significant hepatic and rumen damage after long-term feeding of a diet containing 40% A. adenophora meal [64], and no clear safe feeding window has been identified for this group to date. In rodent studies, dietary administration of A. adenophora at doses ≥100 g/kg diet in mice resulted in dose-dependent exacerbation of hepatic inflammatory injury, oxidative stress and mitochondrial damage [68]. For purified cadinene-type sesquiterpenes, the 7-day subacute safe dose in mice was determined to be 75 mg/kg body weight [57].

Therefore, from the perspective of practical application, subsequent studies have gradually shifted from solely evaluating the toxicity of the raw plant to the research and development of detoxification strategies and detoxified products, with the aim of reducing sesquiterpene-related toxicity while retaining its valuable bioactive components for potential development in feed or veterinary medicine.

6. A. adenophora as a Functional Feed Additive and Its Detoxification

A. adenophora exhibits enormous biomass and well-balanced crude protein and amino acid profiles, conferring it high potential as an unconventional feed resource. The core toxic constituents of A. adenophora are cadinene-type sesquiterpenoids, among which euptox A is the key mediator of hepatotoxicity, pulmonary toxicity and immunotoxicity in livestock and poultry [70,71]. Secondary metabolites including tannins, coumarins and essential oils can also reduce feed palatability and exert synergistic toxic effects [72]. Therefore, all existing detoxification technology systems are centered on the degradation or removal of sesquiterpenoid toxins, while maximally preserving the nutritional and functional active components of the plant such as crude protein, amino acids and flavonoids, which eliminates the core barriers to its feed resource utilization [72]. This section systematically sorts out the principles and research advances of mainstream detoxification technologies for A. adenophora, and comprehensively evaluates the feeding efficacy of detoxified A. adenophora in pigs, broilers, laboratory animals and ruminants in terms of growth performance, product quality and multi-faceted toxicological safety, offering theoretical basis and practical reference for its further development as functional feed.

6.1. Various Detoxification Technologies

Among various detoxification technologies, microbial fermentation detoxification is the core technology with the most in-depth research and mature application at present, and also the cornerstone for realizing the large-scale feed resource utilization of A. adenophora [1,72]. Early studies have screened fungal strains capable of efficiently degrading the toxic components of A. adenophora. Among them, Penicillium chrysogenum and Aspergillus ustus achieved a tannin degradation rate of over 70.0%, a coumarin degradation rate of 67.0%, and a total essential oil degradation rate of 91.2%, without damaging the original nutritional components of the plant during fermentation. This work was the first to confirm the feasibility of microbial detoxification for feed production [72]. Subsequent studies further screened functional bacterial strains capable of specifically degrading euptox A from A. adenophora plants, contaminated soil, and the rumen of migratory goats, including Klebsiella sp., Stenotrophomonas sp., and Pseudomonas sp. [70,71]. Among them, the Klebsiella pneumoniae XC-08 strain isolated from goat rumen achieved a 94.3% degradation rate of 45.0 mg/L euptox A within 24 h [71]. Furthermore, strains such as Klebsiella variicola PLP G-17 LC and Klebsiella pneumoniae PLP G-17 SC can simultaneously degrade hydrolyzable tannins and euptox A in feed, providing core bacterial resources for the development of ruminant-specific detoxification bacterial inoculants [70]. The aerobic fermentation technology with composite bacterial strains optimized for practical production scenarios has also been systematically verified. After treatment with composite bacteria, the crude fiber content of A. adenophora decreased from 27.8% to 20.2%, while the crude protein content was stably maintained above 22.0%. While achieving complete degradation of toxins, this technology also optimized the fiber structure and nutritional ratio of the feed, enabling it to replace conventional energy feeds such as wheat bran, which lays a solid foundation for its industrial production [73]. In addition to microbial fermentation, detoxification technology based on the targeted enrichment and purification of active components provides a new avenue for the development of functional feed additives derived from A. adenophora [74]. Through processes including macroporous resin enrichment and recrystallization, chlorogenic acid products with varying purities can be prepared from the leaves of A. adenophora. While directionally enriching anti-inflammatory and antioxidant active components such as chlorogenic acid and flavonoids, this process almost completely eliminates sesquiterpenoid toxins [74]. In addition, processes including chemical pretreatment and ensiling have been verified to auxiliary enhance detoxification efficiency, and are compatible with the feed production requirements of varying scales [75].

Consistently, detoxified A. adenophora exhibited high safety as a feed additive. For example, Xie et al. used Kunming mice to evaluate the subchronic toxicity of detoxified A. adenophora powder. In this study, detoxified A. adenophora powder was added to the basal diet at 2%, 5%, and 10%, and the mice were continuously fed for 90 days [76]. The results showed that throughout the experimental period, the behavior, coat condition, fecal characteristics, water intake, feed intake, and body weight of mice in all treatment groups remained normal, and no mortality was observed. Serum biochemical indicators measured at 30, 60, and 90 days, including AST, ALT, BUN, total protein, and albumin, showed no significant differences from the control group. After 90 days, body weight in the 2%, 5%, and 10% detoxified A. adenophora powder groups continued to increase normally over time. Organ coefficients of the spleen, kidney, and liver showed no obvious differences from those of the control group, and histopathological sections of the liver, kidney, and spleen showed no abnormal microscopic lesions [76]. Li et al. used a combined toxicological evaluation scheme consisting of the Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test, and chromosome aberration test of primary spermatocytes to evaluate the mutagenicity of biologically fermented detoxified A. adenophora powder. The results were all negative, with no dose–response relationship observed, indicating no mutagenic effect [77]. Liu et al. reported that detoxified A. adenophora powder had no obvious effects on feed intake, activity, body weight gain, implantation number, live fetuses, dead fetuses, or resorbed fetuses in pregnant mice. Fetal body weight, body length, and tail length showed no obvious differences from those of the negative control group, and no malformations were observed in external appearance, skeletal structure, or internal organs, indicated that detoxified A. adenophora powder had no maternal or embryotoxic effects [78]. Li et al. incorporated biologically fermented detoxified A. adenophora powder into the diet at doses of 10 g/kg and 20 g/kg and continuously fed New Zealand rabbits for 90 days [79]. The results showed that rabbits in all groups remained in good general condition throughout the experimental period, with no mortality, feed refusal, or abnormal behavior observed. During the trial, behavior, activity, feeding, and defecation were normal in all groups, and body weight gain, daily feed intake, and feed utilization showed no statistically significant differences compared with the control group [79]. Hematological parameters, serum biochemical indicators, organ coefficients, and histopathological examination showed that continuous feeding with 10 g/kg and 20 g/kg detoxified A. adenophora powder for 90 days caused no obvious adverse effects in rabbits [79]. The study also reported that the oral LD₅₀ of fermented detoxified A. adenophora powder in rats, mice, and rabbits was greater than 10,000 mg/kg, indicating that it could be classified as practically non-toxic [79].

6.2. Application Effects and Safety Evaluation of Detoxified A. Adenophora as Feed Additive in Animals

6.2.1. Swine

Yang et al. replaced wheat bran in swine diets with detoxified A. adenophora at proportions of 10%, 15%, and 20%, respectively, and formulated compound feeds for a 120-day feeding trial [73]. The results showed that pigs remained healthy throughout the experimental period, with no disease or adverse reactions observed [73]. In terms of production performance, the average daily gains of the control group and the 10%, 15%, and 20% detoxified A. adenophora groups were 558, 546, 527, and 508 g/head, respectively, all reaching the general domestic level of approximately 500 g/day, with no significant differences between the experimental groups and the control group [73]. The feed-to-gain ratios of the control group and the 10%, 15%, and 20% detoxified A. adenophora groups were 3.99:1, 4.05:1, 4.11:1, and 4.30:1, respectively, indicating an increasing trend in feed-to-gain ratio as the inclusion level increased [73]. Economic analysis showed that the cost per kilogram of weight gain and gross profit per pig in the 10% and 15% detoxified A. adenophora groups were not markedly different from those in the control group, whereas gross profit per pig decreased markedly when the inclusion level reached 20%. Therefore, the authors suggested that 15% may be a suitable inclusion level of detoxified A. adenophora in swine diets [73].

Song et al. further evaluated the effects of dietary detoxified A. adenophora powder on pork quality. In this study, Landrace crossbred pigs with body weights of 30–40 kg were fed complete diets supplemented with 10%, 15%, or 20% detoxified A. adenophora powder for 90 consecutive days, after which loin muscle samples were collected for analysis [80]. The results showed that replacement of part of the wheat bran with 10–20% detoxified A. adenophora powder did not adversely affect pork appearance, tissue condition, odor, taste, or boiled broth characteristics, all of which met the requirements of the NY/T843–2009 Green Food-Meat and Meat Products standard for fresh livestock meat [80]. In terms of physicochemical indicators, both moisture content and total volatile basic nitrogen were below the standard limits. With regard to hygienic indicators, inorganic arsenic, mercury, copper, lead, cadmium, chromium, tetracycline, oxytetracycline, chlortetracycline, chloramphenicol, and furazolidone were either not detected or were within the permitted limits. Microbiological indicators showed that the total bacterial count was less than 10 CFU/g, coliforms were less than 300 CFU/kg, and neither Salmonella nor diarrheagenic Escherichia coli was detected [80].

6.2.2. Broilers

Broilers are relatively sensitive to feed safety and can therefore be used to verify the safety and feasibility of the detoxification process for A. adenophora. Zhang et al. used broilers as experimental animals and supplemented diets with up to 6% detoxified A. adenophora powder in an 8-week feeding trial [75]. The results showed that the survival rates of the experimental groups ranged from 93.3% to 96.7%, with no obvious difference from the control group. The weight gain rate and feed-to-meat ratio of broilers were within the normal range, and no growth inhibition was observed [75]. These findings indicate that, under nutritionally comparable diet formulations, the addition of detoxified A. adenophora feed did not adversely affect broiler growth [75]. After slaughter, the appearance and tissue morphology of the heart, liver, spleen, lung, kidney, and other organs were normal, the meat was tender and free from abnormal odor, and the heavy metal contents in the liver met food hygiene standards.

6.2.3. Ruminants

Ruminants, particularly goats, are important target animals for evaluating the feed utilization potential of A. adenophora, because they are commonly exposed to this plant in invaded grazing areas. Direct feeding of non-detoxified A. adenophora to goats may cause damage to hepatocytes, renal tissues, myocardium, and lymphoid tissues, and may also affect immune function. In contrast, detoxified A. adenophora not only showed no obvious toxicity but also exhibited improved palatability [81]. Zhou et al. evaluated A. adenophora as a goat feed by incorporating detoxified A. adenophora into compound diets at 20%, 30%, and 40%, and compared these treatments with a non-detoxified group [82]. The results showed that feed intake in the 20%, 30%, and 40% detoxified A. adenophora groups increased by 31.7%, 30.2%, and 14.3%, respectively, with the 20% inclusion level showing better performance. In terms of weight gain, the 20% detoxified A. adenophora group showed a 136.8% increase in average weight gain, while the 30% and 40% groups also showed certain growth-promoting effects. In contrast, the non-detoxified A. adenophora group showed a negative weight-gain trend [82].

In summary, the feed utilization of A. adenophora has currently formed a detoxification technology system with microbial fermentation as the core and multiple technologies in synergy [72]. Multiple animal feeding trials and systematic toxicological assessments have verified that detoxified A. adenophora at appropriate inclusion levels causes no acute toxicity, subchronic toxicity or genotoxicity in pigs, broilers, rabbits, goats and other animals, nor does it exert negative impacts on animal growth and development, organ function or meat quality. It can effectively replace conventional energy feeds such as wheat bran, with both remarkable ecological benefits and economic value [77,78,79,81,83,84]. Nevertheless, existing studies are still focused on basic safety validation and growth performance observation; the regulatory effects and mechanisms of detoxified A. adenophora on animal gut microbiota structure, immune function, antioxidant status and pathogen resistance remain poorly understood. Future research should further carry out systematic investigations focusing on the optimization of appropriate inclusion ratios, long-term feeding safety, elucidation of functional mechanisms and product quality improvement, so as to establish standardized feeding application specifications and advance the industrial application process of A. adenophora feed resource utilization [78].

6.3. Advantages and Limitations of A. adenophora as a Feed Additive

A. adenophora has multiple unique advantages as a feed additive. First, as an invasive weed, A. adenophora features tremendous biomass and extensive distribution in southwestern China. Its raw material cost is much lower than that of artificially cultivated medicinal plants, making its feed products cost-competitive in the market [72]. Second, A. adenophora contains a wide variety of active components, including functional substances such as flavonoids, phenolic acids and polysaccharides, which exert multiple biological functions including antioxidant, anti-inflammatory, antibacterial and immunomodulatory activities [72,78]. Third, developing A. adenophora into a feed additive has certain ecological value: it can not only alleviate the ecological damage caused by its invasion, but also create economic value, realizing the resource utilization of harmful organisms [84]. Fourth, A. adenophora possesses a unique antiparasitic function, which is rare among conventional plant-derived feed additives and provides it with distinctive application value in the prevention and control of parasitic diseases [85].

However, A. adenophora also has obvious limitations when used as a feed additive. The sesquiterpenoid toxins in A. adenophora, especially euptox A, are the fundamental constraint on its direct feed utilization [72,85]. The LD₅₀ of euptox A in mice is 1470 mg/kg body weight [57]. Undetoxified A. adenophora (i.e., including euptox A) at high dose level can cause damage to the liver, lungs, intestinal tract and immune system in animals [67,71]. The main toxic pathogenesis of A. adenophora is oxidative stress and inflammatory response, which can induce hepatotoxicity, splenotoxicity and other related diseases in animals [70,71]. Therefore, all utilization schemes must take prior detoxification as a prerequisite, which increases the process complexity and production cost [78,86]. Furthermore, the detoxification process is not yet fully mature. Current research on A. adenophora detoxification is still in the developmental stage. Systematic studies are still needed in aspects including strain screening for microbial fermentation detoxification, process parameter optimization and large-scale production, and there is still no unified standardized evaluation system for the efficiency and safety of different detoxification methods [77]. Third, the application evidence is not sufficiently systematic. Compared to conventional additives such as astragalus polysaccharides and allicin, which have abundant long-term feeding and industrial application data, existing feeding studies on A. adenophora mainly focus on short-term safety evaluation and small-scale trials, lacking large-scale, multi-center and long-term production performance data [3,9]. Fourth, there are large species-specific differences. Different animal species show significantly different sensitivity to A. adenophora toxins. In goats, feeding diets containing undetoxified A. adenophora significantly upregulates the mRNA expression of inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-2, IL-6 and IL-10 in the ruminal epithelium, and downregulates the expression of tight junction proteins such as occludin and claudin-1 [59]. Poultry are highly sensitive to the toxins, while ruminants have relatively higher tolerance due to the detoxification effect of rumen microorganisms [76]. Such species differences require the development of differentiated detoxification standards and inclusion level schemes for different animals, which further increases the complexity of application and popularization.

7. Conclusions and Prospects

A. adenophora should be regarded as a chemically rich but intrinsically hazardous biomass rather than as a ready-to-use feed additive. Its terpenoids, flavonoids, phenolic acids, and endophyte-derived metabolites exhibit a broad range of reported biological activities, particularly antimicrobial, antioxidant, anti-inflammatory, and gut-regulatory effects. However, most of these effects have been demonstrated in chemical assays, cell models, or small-scale animal experiments. More importantly, the same plant contains cadinene-type sesquiterpenes and other constituents capable of inducing hepatobiliary injury, intestinal and ruminal barrier disruption, splenic immune dysfunction, oxidative stress, inflammation, and programmed cell death. Therefore, the presence of potentially beneficial activities cannot, by itself, justify the direct inclusion of raw or insufficiently characterized A. adenophora in animal diets.

Based on the available evidence, the most feasible feed-related application is not the untreated plant, but a standardized and adequately detoxified feed ingredient produced through validated microbial fermentation, selective extraction, or fractionation. Such products may retain useful nutrients and selected antioxidant or antimicrobial constituents while reducing the concentrations of euptox A and other toxic sesquiterpenes. Nevertheless, current feeding studies demonstrate preliminary feasibility rather than commercial readiness. Many studies were conducted on a limited number of animals, used relatively short exposure periods, or lacked standardized characterization of the starting material, detoxification efficiency, and residual toxin concentrations. Species-specific safe inclusion levels also remain uncertain, and findings obtained in rodents or individual livestock species cannot be directly generalized across animal-production systems.

Purified phytochemicals and endophyte-derived products may represent more controllable application routes than whole-plant preparations because their composition, potency, and toxicological profiles can be characterized more precisely. Some isolated thymol derivatives and microbial metabolites may provide leads for antimicrobial development, whereas the insecticidal, nematocidal, and molluscicidal properties of A. adenophora may support separate applications in botanical pest control. These pesticidal effects, however, should be interpreted primarily as evidence of biological potency and possible toxicity rather than as support for feed use. Their development will require independent evaluation of mammalian toxicity, environmental persistence, non-target effects, and field efficacy.

Future research should move beyond descriptive screening of biological activity toward a risk-controlled product-development framework. Priorities include: (i) characterization of geographic, seasonal, and plant-part variation in both beneficial and toxic constituents; (ii) establishment of validated marker compounds and quantitative limits for residual sesquiterpene toxins; (iii) standardization and scale-up of detoxification processes, including batch-to-batch quality control and stability assessment; (iv) long-term, dose–response feeding studies in target livestock species that assess growth performance, feed efficiency, organ pathology, immune and reproductive outcomes, product quality, and tissue residues; and (v) direct comparison with established feed additives to determine whether detoxified A. adenophora provides meaningful biological or economic advantages. The environmental consequences of harvesting, processing, and transporting this invasive plant should also be evaluated to avoid unintentionally facilitating its further spread.

In conclusion, the practical value of A. adenophora lies not in the direct use of the invasive plant, but in its controlled conversion into chemically defined, detoxified, and application-specific products. Until reproducible detoxification standards, species-specific safety thresholds, and long-term efficacy data are established, its use in animal nutrition should remain cautious and experimental. A safety-first strategy integrating phytochemical standardization, toxicological validation, and production-scale assessment is essential for determining whether this invasive and toxic plant can be transformed into a genuinely useful feed or veterinary resource.

Author Contributions

Writing—original draft preparation, M.W., H.X. and C.D.; methodology, C.D., H.X., Z.H. and J.S.; writing—review and editing, H.J., Z.H., C.D. and J.S.; funding acquisition, C.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

The research was supported by the Beijing Natural Science Foundation (L252139) and the National Natural Science Foundation of China (32573432).