When selecting a cultivation site for ALD, it is imperative to ensure that the slope faces east or north and the slope is maintained at 30°–35°. A shaded slope is preferable. Additionally, the soil should be loose, fertile and possess a deep profile, with loam or sandy loam enriched with humus being optimal, as this composition facilitates effective drainage. Low-lying areas, which are susceptible to flooding and consequently to root rot, should be avoided. For newly developed land, it is essential to thoroughly clear all the weeds and existing vegetation from the wasteland, incorporating these materials into the soil through deep plowing. Deep plowing should be carried out again in the following spring. In addition, cultivators may opt to grow wood-scented plants on land that has previously supported crops such as potatoes and corn (Chen, 2025). Given that ALD can accumulate harmful metals from its native soil, consumption of low-quality ALD may lead to the accumulation of toxic metal elements in the human body (Meng et al., 2021). Inductively coupled plasma mass spectrometry (ICPMS) provides a precise and reliable method for monitoring and controlling contamination levels in the extract, such as Cu, Pb, As, Cd, and Hg in ALD (Chen et al., 2023). The soil used for planting should effectively control the pollution of related metals. The arbuscular mycorrhizal fungi strains (Gigaspora decipiens, Scutellospora calospora, Racocetra coralloidea, Septoglomus deserticola, Entrophospora colombiana, Paraglomus brasilianum), which had a good symbiotic relationship with ALD, are the potential strains to inoculate ALD seedlings under artificial cultivation conditions (Yang et al., 2020). Symbiotic bacteria can significantly increase terpenoids accumulation (costunolide, dehydrocostus lactone, etc.), soluble protein, soluble sugar, antioxidant enzyme activity in the leaves.

Seed propagation serves as the primary method for the cultivation of ALD. In Yunnan Province, the cultivation of ALD predominantly involves seeds sowing, which can be conducted during the winter, autumn, and spring. Typically, spring sowing occurs from mid-March to early April, autumn sowing from late August to mid-September, and winter sowing in early November. Post-harvest, the seeds must be sun-dried and subsequently cleaned of impurities to prepare them for subsequent processes. Prior to sowing, it is essential to prepare the seeds by soaking them in lukewarm water with continuous stirring. As the water cools, impurities and non-viable seeds are removed, leaving only the viable seeds that settle at the bottom. These viable seeds are then soaked for 24 h before being partially dried in preparation for sowing.

If the seeds quantity is insufficient for planting requirements, asexual reproduction can be considered. It is crucial to avoid using fine roots with medicinal value as breeding material. During planting, the layout should be carried out according to the prescribed spacing. And when covering the soil, it is necessary to ensure that the root systems are completely and tightly buried. ALD propagation via cuttings requires a healthy selection of semi-hardened stems with 2–3 nodes. Stems are trimmed to retain 2–3 leaves for photosynthesis, followed by treatment with rooting hormones or basal soaking in diluted rooting agent for 30 min prior to air-drying. A sterile, well-draining substrate (e.g., a mixture of leaf mould and perlite) is prepared and sterilized to minimize pathogen risk. Processed cuttings are inserted into the substrate at a depth of 1/3–1/2 the stem length. Post-insertion, the medium is moderately watered to maintain moisture without waterlogging. Environmental conditions are maintained at 20 °C–25 °C with indirect light and adequate airflow to avoid direct sunlight exposure. Rooting initiates within 3–4 weeks, accompanied by new shoot development.

Large-scale planting has been carried out based on the growth characteristics of ALD in Yunnan and Guizhou Province and Chongqing of China. This large-scale planting is still mainly based on traditional agricultural cultivation methods. In the future, the yield, quality and production efficiency of ALD can be improved through multi-disciplinary means such as micropropagation and tissue culture technology, molecular biology, intelligent equipment and environmental control. Micropropagation and tissue culture technology represent a fundamental advancement in contemporary crop cultivation (Alanagh et al., 2014; Lu et al., 2019). By employing asexual reproduction and cellular engineering techniques, these methods effectively address the challenges of low efficiency and genetic instability inherent in traditional seed propagation (Pupilli and Barcaccia, 2012). The root tips of ALD serve as highly differentiated potential explants suitable for plant tissue culture. By precisely regulating the growth system through genetic modification and improving stress resistance and the targeted accumulation of metabolites, the production efficiency and product value of ALD can be significantly enhanced. The precise regulation of nutrient solutions during the cultivation process can now be easily implemented (Lu et al., 2022; Wang et al., 2023). The demand for elements such as nitrogen, phosphorus, potassium, and calcium varies significantly at different stages of plant growth. Through dynamic monitoring and intelligent intervention, the accumulation of secondary metabolites, stress resistance, and growth consistency of medicinal plants like ALD may be significantly improved. Furthermore, based on historical data, a growth prediction model for ALD can be constructed, integrating environmental variables (temperature, precipitation, soil EC value) to output the best irrigation and fertilization decisions. If synthetic biology and intelligent equipment can be effectively combined, the ALD industry is expected to become a benchmark model in the global medicinal plant field.

Leaf spot disease and root rot represent significant threats to the growth cycle of ALD, particularly during the rainy season when the prevalence of these diseases increases markedly, with July and August identified as peak periods. To mitigate these challenges, growers should prioritize land with superior drainage and a lower groundwater table for cultivating ALD. During field management, it is crucial to handle the plants carefully to prevent root injury and to rigorously implement quarantine measures to ensure that seeds are free from pathogens. Once infected plants are identified, they should be immediately removed, and the soil should be disinfected with quicklime to curb the further spread of root rot (Tan et al., 2023). In cases where ALD seedlings exhibit symptoms of root rot, growers need to take prompt action by precisely spraying an appropriate amount of thiophanate-methyl or carbendazim on the roots of the affected plants (Gaitnieks et al., 2016). Additionally, during the rainy season, the application of Bordeaux mixture on clear days is advised to effectively prevent leaf spot disease. Upon noticing symptoms of disease on ALD seedlings, an appropriate amount of bactericides (tebuconazole or chlorothalonil) should be evenly sprayed on the leaves, with attention to rotating different pesticides to enhance control effectiveness.

The primary pests impacting ALD include grasshoppers, aphids, cutworms, and grubs. During their nymphal stage, grasshoppers can be effectively managed by spraying with a solution of 90% crystalline trichlorfon diluted to 800 times (Zhang et al., 2019). Aphids can be controlled by spraying with a solution of 40% dimethoate emulsifiable concentrate diluted 800 to 1,500 times (Chandrasena et al., 2011). Cutworms and grubs, which damage seedlings, roots, and leaves, can be effectively trapped and eradicated using bait made by combining crystalline trichlorfon with wheat bran (Kumar and Pandey, 2022; Smirle et al., 2013).

AR originates from the dried root of ALD, which is collected during the autumn and winter seasons. Once dried, the coarse outer skin is removed. The final product is typically cylindrical or semi-cylindrical, measuring between 5 and 10 cm in length and 0.5–5 cm in diameter. Its surface boasts a yellowish-brown to grayish-brown coloration, adorned with prominent wrinkles, longitudinal grooves, and lateral root marks. The texture is notably firm, making it resistant to breaking. Upon sectioning, the interior reveals a grayish-brown to dark-brown coloration, with a grayish-yellow or light brownish-yellow periphery. A distinct brown cambium ring is evident, accompanied by a radial texture. It possesses a unique aroma and a slightly bitter taste (China Pharmacopoeia Committee, 2020a).

Four traditional AR-based prescriptions have been recorded in the Chinese Pharmacopoeia (2020 edition): Muxiang Fenqi Wan (China Pharmacopoeia Committee, 2020e), Muxiang Shunqi Wan (Bai, 2023; China Pharmacopoeia Committee, 2020f), Muxiang Binglang Wan (China Pharmacopoeia Committee, 2020d; Gao et al., 2024), and Liuwei Muxiang San (China Pharmacopoeia Committee, 2020c). These traditional medicines primarily treat gastrointestinal stagnation, abdominal distension pain, and bowel obstruction.

Additionally, other traditional prescriptions that were not included in the Chinese Pharmacopoeia have also been studied (Table 1). Simo Decoction has been shown to enhance gastrointestinal motility by influencing the contractions of antral circular smooth muscle strips (Dai et al., 2012). JianPi’I, which originates from the classical Chinese medicine formula Xiangshaliujunzi decoction, is known to alleviate symptoms associated with postprandial distress syndrome (Wang et al., 2016). The Xian-He-Cao-Chang-Yan formula has been demonstrated to ameliorate DSS-induced colitis in mice (Li J. et al., 2021). Weichang’an Pill has been widely used for decades in the treatment of irritable bowel syndrome and functional dyspepsia (Zhang et al., 2013). Kangen-karyu (GuanYuan-Ke-Li) is considered a promising therapeutic agent for Alzheimer’s disease (Paudel et al., 2020). Xianglian Pill, composed of Rhizoma coptidis and AR, has long been employed in the management of gastrointestinal disorders (Lu et al., 2020). Clinically, Wuwei Shexiang Pill is used in China to reduce joint pain and swelling, as well as to dispel wind and alleviate pain (Bai et al., 2023). Additionally, treatment with Muxiang gel plaster has been found to effectively prevent and mitigate mammary hyperplasia (Wu Y. et al., 2024).

With the development of TCM, the active metabolites in medicinal substances can be concentrated, extracted and combined to enhance their therapeutic efficacy. A metabolite formulation, consisting of three herbal extracts (CO₂ supercritical fluid extract of ginger, ethanol reflux extract of AR, and pogostemonis herba essential oil) has been developed as a promising anti-motion sickness treatment (Zhang et al., 2015).

The traditional processing techniques for ALD involves several methods aimed at enhancing its efficacy and adaptability for various Chinese medicinal formulations (Li X. et al., 2021; Song et al., 2022a). These methods include the use of raw AR, stir-fried AR, baked (or grilled) AR, and wine-processed AR. Raw AR denotes the cleaned and dried form of the original medicinal material, which undergoes impurity removal, washing, slicing, and drying. Stir-fried AR is prepared by lightly frying the slices with bran before they are used in formulations. Baked AR, also known as grilled AR, involves stir-frying the pieces with bran until they turn yellow, after which they are cooled and incorporated into medicinal applications (Wu M.-l. et al., 2024). Wine-processed AR is made by moistening the botanical drug with rice wine, followed by slicing and drying for medicinal use.

These traditional processing techniques for the botanical drug AR significantly influence the quality and therapeutic direction of the final product by carefully controlling the heating intensity and the use of auxiliary materials: raw AR retains abundant volatile oils, offering strong medicinal properties but also greater side effects due to alkaloids and other metabolites; roasting gently heats the botanical drug at low temperatures, promoting the release or transformation of some volatile oils, thereby reducing side effects and mildly enhancing its gastrointestinal therapeutic functions; plain stir-frying and bran-frying reduce toxicity and adverse effects while strengthening its therapeutic efficacy; wine-processing using rice wine facilitates the extraction of active metabolites, increasing pharmacological activity. Essentially, these methods work by regulating the content and transformation of volatile oils and other metabolites to achieve reduced toxicity, preserved efficacy, enhanced potency, or altered therapeutic targeting (Feng et al., 2023; Li R.-l. et al., 2021).

Although AR has good medicinal value, excessive use may lead to adverse effects. Skin allergic reaction is a common side effect in the use of AR, due to various irritant alkaloids (saussureanine, costunolide, etc.), for people who are sensitive or have existing skin inflammation, contact with these substances is easy to cause allergic reactions. Acute generalized exanthematous pustulosis also could induced by AR (Chu et al., 2019). Additionally, high doses of TCM usually have liver toxicity, and AR is no exception. Oxidative stress should be the primary mechanism for the high-dose AR-induced hepatotoxicity, and Nrf2, HO-1 and NQO1 were the main targets (Song et al., 2024). On the contrary, the appropriate dosage of ethanol extract of AR had a protective effect on liver injury induced by lipopolysaccharide in rats (Song et al., 2023). Reasonable control of the dosage of AR is important for patients to avoid side effects, which can effectively ensure the safety and effectiveness of Chinese herbal medicine.

In the field of traditional botanical drugs, species within the AR, Aristolochiae Radix (ARR), Vladimiriae Radix (VR), and Inulae Radix (IR), present persistent identification challenges due to morphological convergence and historical misclassification (Zhuang et al., 2021). These taxonomically related species exhibit overlapping botanical characteristics, particularly in root morphology and histological features, resulting in frequent substitution errors in herbal commerce. Current pharmacognostic authentication relies on UHPLC-QTOF-MS as the gold-standard methodology for precise differentiation (Guccione et al., 2017; Shum et al., 2007; Wang et al., 2024). This analytical approach enables simultaneous detection of multiple marker metabolites, achieving clear discrimination between structurally analogous aristolochic acid derivatives and sesquiterpene lactone.

VR (“Chuanmuxiang” in China), primarily derived from the dried roots of Vladimiria souliei (Franch.) Ling or V. souliei (Franch.) Lingvar. cinerea Ling, predominantly cultivated in western Sichuan, China, exhibits medicinal properties akin to those of Muxiang, albeit with distinct emphases (China Pharmacopoeia Committee, 2020b). Chemical profiling differentiates AR from VR based on significant differences in their quantitative composition (dihydrodehydrocostus lactone, mokkolactone, α-costol, etc.) (Chen et al., 2020; Liang et al., 2024; Yan et al., 2020). The total lactone extract of VR has the similar protective effects on cholestatic liver injury as AR, even better in terms of anti-inflammatory properties (Chen Z. et al., 2022).

IR (“Tumuxiang” in China) refers to the dried roots of Inula helenium L. or Inula racemosa Hook.f. (China Pharmacopoeia Committee, 2020g). Historically utilized as a substitute for AR, IR exhibits significant chemical distinctions from AR, with its primary differential metabolites being alantolactone and isoalantolactone (Cai et al., 2021; Rasul et al., 2013). These metabolites have been used to emesis and diarrhoea, and to eliminate parasites (Xu et al., 2015). Alantolactone and isoalantolactone have been confirmed to possess potential hepatotoxicity, nephrotoxicity, and allergenic effects. Long-term or excessive intake may lead to symptoms such as loss of appetite, fatigue, nausea, vomiting, and pain in the liver area. In severe cases, it can cause irreversible liver damage. Due to their toxicity, IR has been rarely used in modern clinical practice of TCM.

ARR (“Qingmuxiang” in China) is the dried roots of Aristolochia debilis Sieb. et Zucc. or Aristolochia contorta Bge. ARR originally appeared as a substitute for AR, but contemporary researches have revealed the presence of aristolochic acid in Aristolochiae species, which can cause nephrotoxic adverse reactions (Cheng et al., 2006; Wang X. et al., 2020; Zhang et al., 2016). Consequently, ARR has been banned from use in TCM formulations (Luo et al., 2024).

In summary, careful identification of AR’s species authenticity and quality is essential during procurement and use to prevent confusion with commonly confused botanical drugs. At the same time, it is recommended to use AR and its related botanical drugs under the guidance of a professional physician or pharmacist.

The commercially available herbal medicines exhibiting comparable qi-regulating, analgesic, spleen-strengthening, and digestion-promoting activities to AR primarily include Pericarpium Citri Reticulatae, Aurantii Fructus, Cyperi Rhizoma, and Amomi Fructus. However, their therapeutic emphases and clinical applications exhibit notably divergent profiles. Additionally, AR demonstrates multidimensional industrial advantages over other botanical drugs in its category.

Specifically, the flavonoid metabolites (hesperidin, nobiletin, tangeretin, etc.) in Pericarpium Citri Reticulatae are susceptible to rapid degradation under UV light, resulting in poor stability (Ho and Kuo, 2014; Li Y. et al., 2024). Aurantii Fructus contains alkaloids such as p-tyramine, N-methyltyramine, and tyramine that may cause cardiovascular adverse reactions including hypertension and arrhythmia, thus requiring careful evaluation of the patient’s baseline condition in clinical use (Gao et al., 2020). Cyperus volatile oils, rich in α-cyperone, cyperotundone, and limonene, face industrial scalability challenges due to low extraction yields (Bezerra et al., 2025). Amomi Fructus is constrained by its reliance on specific cultivation conditions and high production costs (Suo et al., 2018). In contrast, AR not only has a mild nature and comprehensive effects, but also stands out in terms of the stability of its metabolites, safety, and the maturity of industrialization. Therefore, it is more suitable for large-scale pharmaceutical production, food processing, and the development of health products.

The sesquiterpene lactones are the primary and characteristic metabolites of ALD, exhibiting a diverse and abundant range exceeding 130 species (Yuan et al., 2024; Zheng et al., 2022). Sesquiterpene lactones are a class of natural metabolites primarily found in plants of the Asteraceae family, characterized by a 15-carbon sesquiterpenoid backbone coupled with a lactone ring (Amen et al., 2025; Liu et al., 2021). They often serve as defensive metabolites in plants and are largely responsible for their characteristic bitter taste. These metabolites are renowned for their diverse and potent biological activities, including anti-inflammatory, anti-tumor, antimicrobial, and immunomodulatory effects (Amen et al., 2025). As a result, they represent not only key active metabolites in traditional herbal medicine but also important lead metabolites in modern drug development. However, their bioactivity is dual-edged: some members are strong allergens capable of inducing contact dermatitis and may exhibit cytotoxicity at higher concentrations (da Silva et al., 2021).

Among the sesquiterpene lactones, costunolide and dehydrocostus lactone are the key substances for quality control of AR (Figure 2) (Dong et al., 2018). The total content of costunolide and dehydrocostus lactone in AR must not be less than 1.8%, as stipulated by the Chinese Pharmacopoeia (2020 Edition). They are also the most important active substances in AR and could be quantified synchronously using high-performance liquid chromatography coupled with mass spectrometry (Seo and Shin, 2015; Zhang et al., 2014). Currently, costunolide is already available in substantial quantities through genetic engineering techniques. The biosynthetic pathway for costunolide (Figure 3) has been successfully built in Escherichia coli by the co-expression of three genes (GAS, GAO, COS) involved in costunolide biosynthesis, along with eight genes responsible for converting acetyl-CoA into farnesyl diphosphate via the mevalonate pathway. And costunolide yield was up to 100 mg L⁻¹ in E. coli (Yin et al., 2015). Meanwhile, the co-expression of GAS, GAO, and COS in yeast and Nicotiana benthamiana leaves has also facilitated costunolide production (Blazquez et al., 2011). Revealing the synthetic pathway of costunolide indicates that there are no obstacles at all in constructing transgenic plants of ALD that produce high yields of costunolide. However, despite the structural similarities between dehydrocostus lactone and costunolide, and their concurrent presence in plants, the biosynthetic pathway for dehydrocostus lactone in plants remains unidentified. Consequently, the production of dehydrocostus lactone still relies on the extraction from plant raw materials.

Monoterpenoids and triterpenoids are two important classes of natural terpenoid metabolites, each with distinct characteristics in structure, distribution, and function (Yang et al., 2025). Monoterpenoids, composed of two isoprene units (C10), are major metabolites of plant essential oils (Bhatti et al., 2014; Jiang and Wang, 2023). In contrast, triterpenoids consist of six isoprene units (C30), featuring higher molecular weight and greater structural complexity (Huang et al., 2024; Zeng et al., 2024). Monoterpenoids and triterpenoids are widely used in the flavor, food, and cosmetics industries. Their biological effects tend to be more profound and systemic, including notable anti-inflammatory, antitumor, immunomodulatory, and cholesterol-lowering activities (Yang et al., 2025). Representative metabolites such as α-amyrin and betulinic acid are core active metabolites in many traditional Chinese medicines. However, some triterpenoids may exhibit hepatotoxicity at high doses.

Phenylpropanoids are an important class of plant secondary metabolites characterized by a fundamental C6–C3 skeleton, which consists of a benzene ring (C6) attached to a propene group (C3), also known as the phenylpropane backbone (Vogt, 2010). These metabolites are primarily synthesized through the shikimate pathway and play crucial roles in plant defense, growth development, and signal transduction (Cao et al., 2025; Zhang et al., 2025). Additionally, they serve as significant sources for numerous pharmaceuticals, flavoring agents, and industrial raw materials.

Steroids represent a significant class of bioactive metabolites in TCM. Phytosterols (β-sitosterol, lappalanasterol, pregnenolone, etc.) are the predominant type in ALD. Structurally, phytosterols resemble cholesterol in animals, sharing the same cyclopentanoperhydrophenanthrene core skeleton, but differ in their side chain configurations. These steroidal metabolites exhibit diverse structures and broad biological functions, serving not only as a fundamental chemical basis for explaining the efficacy and mechanisms of TCM, but also as key resources for modern drug (steroid hormones) development (Ferreira-Guerra et al., 2020).

Flavonoids are ubiquitous in traditional Chinese medicine and are one of the key metabolites that enable many medicinal materials to exert their therapeutic effects. Flavonoids are distinguished by their broad spectrum of biological activities, including antioxidant effects, cardiovascular protection, and antiplatelet aggregation, which collectively contribute to reducing the risk of cardiovascular diseases and enhancing blood circulation (Jomova et al., 2025; Xu et al., 2025).

In addition to the previously mentioned metabolites, ALD has been identified to contain polysaccharides, higher fatty acids, small aliphatic alcohols, aldehydes, acids, amino acids, and cholamine, among other metabolites (Peng et al., 2025; Zhuang et al., 2021). The presence of these metabolites in AR may provide a pharmacological basis for the utilization of this herbal medicine in the treatment of constipation, intestinal infections, and associated inflammatory diseases. Higher fatty acids, amino acids, and cholamine are essential for maintaining normal physiological functions of the body (Chen and Fang, 2025; Kelling et al., 2024; Wu et al., 2022). Meanwhile, although the specific mechanisms of action of aliphatic alcohols, aldehydes, and acids require further investigation, they may play significant roles in regulating metabolism and participating in immune responses.

The antioxidant profile of ALD multifaceted, stemming from its rich and diverse phytochemical composition. The high values for total phenolic (188.2 ± 2.1 mg GAE/g DM) and flavonoid (129 ± 2.6 mg QE/g DM) content are paramount (Binobead et al., 2024). Phenolics and flavonoids are renowned antioxidants due to their hydrogen-donating ability, which neutralizes free radicals by stabilizing them. The in vitro assays (DPPH and ABTS) confirm this radical-scavenging capability. The IC₅₀ values (137.15 μg/mL for ABTS, 175.5 μg/mL for DPPH) represent the concentration required to scavenge 50% of the radicals (Binobead et al., 2024). Lower IC₅₀ values indicate higher potency. The extract of AR have comparable antioxidant activity to ascorbic acid (Adel et al., 2025). α,β-Unsaturated carbonyl metabolites (costunolide, dehydrocostus lactone, artemisitene, santamarine, isoalantolactone) were mainly featured as the antioxidant active metabolites of AR (Wu et al., 2025). This molecular structure is a key pharmacophore for antioxidant activity. It can quench free radicals through direct single-electron transfer. Ultimately, the most significant evidence of its antioxidant power is its in vivo protective effect against NaNO₂-induced toxicity. NaNO₂ is a potent oxidant that causes methemoglobinemia and oxidative damage to organs. The extract’s ability to improve hematological parameters and protect liver and kidney function biomarkers demonstrates that its antioxidants are bioavailable and active within a living system, effectively mitigating systemic oxidative stress (Elshaer et al., 2022). This bridges the gap from laboratory findings to potential therapeutic applications, suggesting ALD could be developed into a natural remedy for conditions where oxidative stress is a key pathological factor.

The extract of ALD and its isolated bioactive metabolites, particularly sesquiterpene lactones such as dehydrocostus lactone, alantolactone, and epoxymicheliolide, demonstrate significant anti-inflammatory activity across multiple experimental models. In LPS-stimulated RAW 264.7 macrophages and peritoneal macrophages, ALD and these metabolites consistently suppress the production of key proinflammatory mediators, including NO, PGE2, iNOS, COX-2, and the cytokines IL-6, IL-1β, and TNF-α (He et al., 2022; Lim et al., 2020). This broad inhibition of inflammatory outputs is not limited to macrophage models but extends to in vivo conditions such as osteoarthritis (Jo et al., 2021), where ALD reduces pain and serum IL-1β, and to inflammatory bowel disease (IBD) models (Lee et al., 2020), including dextran sulfate sodium (DSS)-induced colitis (Yuan et al., 2022), where it ameliorates symptoms, protects the colonic barrier, and lowers inflammatory cytokines and enzymes like myeloperoxidase (MPO).

The fundamental mechanism underlying this robust anti-inflammatory effect involves the dual suppression of the NF-κB and MAPK signaling pathways (Figure 4) (Lim et al., 2020). ALD and its metabolites inhibit the LPS-induced phosphorylation and degradation of IκBα, thereby preventing the nuclear translocation of the NF-κB subunits p65 and p50 (Chun et al., 2012; Lim et al., 2020). Concurrently, these metabolites inhibit the phosphorylation of MAPKs, including JNK, ERK, and p38 (Chun et al., 2012; Hsu et al., 2009; Lim et al., 2020). Further upstream, alantolactone was shown to suppress the expression of adaptor proteins MyD88 and TIRAP, which are critical for initiating these cascades (Chun et al., 2012). Importantly, this anti-inflammatory activity is complemented by a potent antioxidative effect mediated through the activation of the Nrf2/HO-1 pathway. Metabolites like dehydrocostus lactone and epoxymicheliolide enhance the nuclear accumulation of Nrf2, leading to the upregulation of antioxidant genes and a reduction in intracellular ROS (Yuan et al., 2022). Mechanistic studies reveal that many of these actions depend on the presence of an α,β-unsaturated carbonyl group, which allows the metabolites to form covalent bonds with key cysteine residues on target proteins like IKKα/β, Keap1, or TAK1 (He et al., 2022; Wu et al., 2025; Yuan et al., 2022).

The therapeutic implications of these mechanisms are vast, as evidenced by efficacy in diverse inflammatory disease models. Beyond colitis and osteoarthritis, ALD and dehydrocostus lactone alleviate atherosclerosis by promoting cholesterol efflux in macrophage-derived foam cells and modulating the TLR2/PPAR-γ/NF-κB pathway (Hong et al., 2025). They also show protective effects against irinotecan-induced intestinal mucositis by inhibiting the TLR4/NF-κB/NLRP3 axis and against benign prostatic hyperplasia by restoring apoptosis balance (Choi et al., 2021; Sun et al., 2024). Furthermore, a sesquiterpene lactone-rich fraction of ALD was significantly more effective than an aqueous extract in treating ulcerative colitis (Chen Y. et al., 2022), underscoring that these specific metabolites are the primary active anti-inflammatory agents. This multifaceted, multitargeted action, targeting both inflammation and oxidative stress, positions ALD and its metabolites as promising candidates for treating complex chronic inflammatory disorders.

The supercritical fluid extraction of oils from ALD demonstrates remarkable and concentration-dependent cytotoxic efficacy against various cancer cell lines. Specifically, the extract obtained at 10 MPa exhibited potent antitumor activity with IC₅₀ values of approximately 0.44, 0.46, and 0.74 μg/mL against HCT-116, MCF-7, and HepG-2 cells, respectively, whereas extracts obtained at higher pressures (20 MPa and 48 MPa) showed significantly reduced potency, highlighting the critical influence of extraction parameters on bioactive metabolite efficacy (Ahmed et al., 2022). Furthermore, specific sesquiterpene lactones like costunolide, dehydrocostus lactone, alantolactone, and isoalantolactone, isolated from the roots, have demonstrated significant activity against diverse cancer types including lung (A549), glioma (C-6), and prostate (PC-3) cancer cells, with hexane and chloroform fractions of ALD showing particularly low IC₅₀ values, such as 3.37 ± 0.14 and 7.53 ± 0.18 μg/mL against PC-3 cells, respectively (Bhushan et al., 2023b; Kumar et al., 2014).

The anticancer mechanisms of these extracts and metabolites are primarily mediated through the induction of apoptosis via both intrinsic and extrinsic pathways. Lyophilized ALD significantly suppressed the growth and proliferation of T-47D and HeLa cells by inducing apoptosis, as evidenced by suppressed LDH release, reduced NO production, and activation of death receptors in a dose-dependent manner (Hasson et al., 2018). Similarly, ALD extract promoted apoptosis in MCF-7 cells by activating caspase-3/7 and annexin V/PI pathways (Kumar et al., 2024b). In-depth mechanistic studies on dehydrocostus lactone have revealed its ability to inhibit angiogenesis by inducing G0/G1 cell cycle arrest in human umbilical vein endothelial cells (HUVECs) through the abrogation of the Akt/GSK-3β/cyclin D1 and mTOR signaling pathways (Ahmad et al., 2012). Moreover, in laryngeal carcinoma cells, dehydrocostus lactone induced mitochondrial apoptosis by inhibiting the PI3K/Akt/Bad pathway and stimulating endoplasmic reticulum stress-mediated apoptosis, accompanied by the upregulation of p53 and P21 (Zhang et al., 2020).

In vivo studies substantiate the anticancer potential and reduced systemic toxicity of these natural metabolites. In a 12-dimethylbenz (a) anthracene (DMBA)-induced mammary tumour model in rats, treatment with ALD root extract at 500 mg/kg body weight resulted in significant chemopreventive effects, demonstrated by inhibition of tumour parameters, minimal alterations in liver and kidney enzymes, reduction in oxidative stress, decreased pro-inflammatory cytokines (TNF-α and NF-κB), and downregulation of proliferation (Ki-67), metastasis (MMP-9), and angiogenesis (VEGF) markers (Kumar et al., 2024a). Another study in mice induced with cancer using Polyvinyl pyrrolidone K-30 (PVP) showed that ethanolic extract of ALD reduced cancer cell proliferation and mitigated histopathological damage in the liver and kidneys in a dose-dependent manner, contrasting with the severe damage observed in chemotherapy-treated groups (Al-Zayadi et al., 2023). Furthermore, molecular docking and virtual screening studies identified bioactive metabolites in ALD, such as stigmasterol, isoboldine, and beta-sitosterol, with favourable ADME (Absorption, Distribution, Metabolism, and Excretion) and toxicity profiles, showing strong binding affinities to hub genes like SRC, FGFR1, and HSP90AA1 involved in prostate cancer pathways, particularly PI3K/AKT signaling (Figure 5) (Kosanam et al., 2024). This multi-targeted approach, combined with a favourable safety profile, positions ALD and its bioactive metabolites as promising candidates for further development as anticancer therapeutics.

AR extract demonstrates significant organ-protective effects, particularly against acute lung injury, through a multi-target and multi-pathway mechanism. The study revealed that AR extract ameliorates cyclophosphamide-induced histological damage in the lung by concurrently modulating inflammatory, oxidative, and apoptotic pathways (Attallah et al., 2023). Specifically, its protective action is mediated through the reduction of iNOS and the caspase-3, which attenuates excessive inflammation and inhibits programmed cell death, respectively. Furthermore, AR extract alleviates oxidative stress by significantly decreasing the level of MDA, a marker of lipid peroxidation, while upregulating the gene expression of the HO-1. The accompanying downregulation of microRNA-let-7a suggests a potential involvement in epigenetic regulation, though its precise role requires further elucidation. This evidence collectively indicates that AR extract’s organoprotective efficacy is achieved via a synergistic combination of anti-inflammatory, antioxidant, and anti-apoptotic activities.

ALD exhibits multi-faceted anti-diabetic properties, demonstrated through both in vitro and in vivo studies. Two novel non-competitive proteinaceous inhibitors, ScAI-R (IC₅₀ = 23 μg/mL, Kᵢ = 0.38 µM) and ScAI-L (IC₅₀ = 28 μg/mL, Kᵢ = 0.32 µM), purified from the roots and leaves, showed high affinity towards human salivary and pancreatic α-amylases, achieving up to 90% inhibitory activity (Ben Abdelmalek et al., 2025). Further supporting these findings, the casein-encapsulated extract of bioactive metabolites from ALD significantly inhibited α-amylase and α-glucosidase activities and enhanced glucose uptake in HepG2 cells (Mushtaq et al., 2025). In diabetic rat models induced by Streptozotocin, oral administration of ALD extracts—containing high concentrations of phenolic metabolites such as dehydrocostus lactone, azulene, eicosapentaenoic acid, linoelaidic acid, isochlorogenic acid A, and chlorogenic acid resulted markedly reduced blood glucose, total cholesterol, and triglyceride levels, while improving HDL-cholesterol and body weight (Abouelwafa et al., 2024; Gomaa et al., 2021). These results collectively highlight the potential of ALD a source of effective anti-diabetic agents through enzyme inhibition and metabolic regulation.

Based on multiple studies, the extract of ALD and its specific bioactive metabolites, such as costunolide and dehydrocostuslactone, demonstrate broad-spectrum antiparasitic properties by exhibiting both direct lethal effects and indirect host-protective mechanisms. The ethyl acetate extract of AR (including arbusculin B, α-cyclocostunolide, costunolide, and dehydrocostuslactone) potently inhibited the growth of Trypanosoma brucei rhodesiense, with ICsos between 0.8 and 22 μM (Julianti et al., 2011). In vitro, extract of AR showed a significant anthelmintic effects on live adult Ascaridia.galli worms in terms of inhibition of worm motility, with worm motility inhibition of 100% at 24 h post-exposure at the 100 mg/mL (Mir et al., 2024). Beyond direct parasite killing, research reveals a complementary protective role; in a Trichinella spiralis-infected rat model, the extract significantly modulated infection-induced DNA damage and oxidative stress in host tissues (Alghabban et al., 2024). This collective evidence positions ALD as a highly promising source for novel antiparasitic agents, offering a dual mechanism of action that combines direct potency with alleviation of the pathological damage caused by parasitic infections.

The essential oil, hexane-chloroform, methanolic, and aqueous extracts of AR have been proven to have certain antimicrobial effects on Acinetobacter baumannii, Aspergillus flavus, Alternaria alternata, Bacillus cereus, Blumeria graminis, Botrytis cinerea, Candida tropicalis, Colletotrichum gloeosporioides, Candida albicans, Didymella glomerata, Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, Fusarium oxysporum, Fusarium graminearum, Fusarium lateritium, Klebsiella pneumonia, Pseudomonas aeruginosa, Pythium aphanidermatum, Phytophthora infestans, Sclerotinia sclerotiorum, Salmonella enterica, Staphylococcus aureus, and Staphylococcus epidermidis (Ahmed and Coskun, 2023; Ahmed et al., 2022; Cai et al., 2022; Idriss et al., 2023). Meanwhile, the smoke of ALD successfully inhibited Penicillium crustosum growth of fresh walnuts (Qiao et al., 2023). The antimicrobial mechanism of the extract of AR is multi-targeted, mainly including: damaging the structure of the cell membrane, inhibiting the formation of biofilms, inhibiting quorum sensing and inducing the production of ROS. The composites of ALD demonstrated significantly stronger antimicrobial activity compared to its individual metabolites, causing clear structural damage to resistant strains, such as chitosan-AR nanoconjugates (Alshubaily, 2019), iron oxide nanoparticles of AR (Al-Shaeri and Al-brahim, 2023), and AR-MgO nanoparticles (Mishra et al., 2020). AR extract is undoubtedly a natural antibacterial agent with great research and development value. Its broad-spectrum antibacterial activity, especially its effective effect on drug-resistant bacteria and biofilms, makes it a promising candidate for addressing the global antibiotic resistance crisis.

Studies have demonstrated the broad-spectrum antiviral potential of extracts and metabolites from ALD and related botanicals. Some lappanolides from ALD exhibits excellent anti-HBV activity (Li H.-B. et al., 2024). The value of the in vitro IC50 of AR extract against low pathogenic human coronavirus (HCoV-229E) and human influenza virus (H1N1) influenza virus were 23.21 ± 1.1 and 47.6 ± 2.3 μg/mL, respectively. (Attallah et al., 2023). The antiviral activity of ALD acetic acid extract had a significant positive influence against HSV-1 (EC50 = 82.6 g/mL; CC50 = 162.9 g/mL; selectivity index = 1.9) (Idriss et al., 2023).

The bioactive molecules from ALD can be as SARS-CoV-2 main protease inhibitors by computational approach investigation (Hajji et al., 2022). The ALD phytoconstituents have inhibitory potential against the receptor-binding domain of the spike glycoprotein and the main protease of the SARS-CoV-2 Delta (B.1.617.2) variant of the novel coronavirus using molecular docking, DFT, and ADME/Tox studies (Houchi and Messasma, 2022). However, in the actual experiment, no effect was still detected in terms of the inhibition of SARS-CoV2 entry (Idriss et al., 2023).

Currently, the vast majority of research on the antiviral effects of ALD (especially against SARS-CoV-2) remains at the stage of computer simulations (e.g., molecular docking) and in vitro studies. While these findings provide a solid theoretical foundation and valuable guidance for further investigation, there is still a long way to go before clinical application can be realized. Validation through animal studies and human clinical trials is still needed. It is also important to note that ALD a complex multi-metabolite system, and its antiviral activity likely results from the synergistic effects of various metabolites rather than a single metabolite. From a modern scientific perspective, its antiviral properties may be associated with holistic regulatory functions, such as anti-inflammatory and immunomodulatory effects.

Although ALD demonstrates significant medicinal potential, particularly its notable anticancer activity, there remain major limitations in current research that severely hinder its transition from a traditional remedy to a modern, internationally recognized drug.

First, the standardization of botanical drug sources and quality is the primary obstacle. The origin of ALD is complex, and variations in harvesting seasons and processing methods lead to significant differences in the types and concentrations of its internal chemical metabolites. Existing research has predominantly focused on exploring the activity of specific extracts or metabolites but lacks a comprehensive and systematic quality evaluation system for the botanical drug itself. Most studies rely only on individual active metabolites (such as costunolide and dehydrocostus lactone) as quality control indicators, which fails to comprehensively reflect the holistic nature of the botanical drug and its “multi-metabolite, multi-target” mechanism of action. This lack of standardization makes it difficult to reproduce, compare, and integrate research results from different laboratories, resulting in fragmented data that cannot provide a consistent and reliable material basis for clinical medication.

Secondly, research on the active metabolites and their mechanisms of action remains insufficient. Although numerous bioactive metabolites (costunolide, dehydrocostus lactone, alantolactone, etc.), have been isolated and identified, and their functions in inducing apoptosis and inhibiting angiogenesis have been confirmed, most studies remain at the stage of phenomenological observation and preliminary mechanistic exploration. The majority of mechanistic research relies on in vitro cell models, where the drug concentrations used are often significantly higher than the achievable levels in vivo, raising doubts about the extrapolation of the findings. There is a notable lack of research on how the multiple metabolites in ALD interact synergistically or antagonistically to produce therapeutic effects, which is an aspect central to the philosophy of traditional Chinese medicine. Furthermore, current studies focus predominantly on its anticancer properties, while modern scientific explanations of its traditional efficacy are severely lacking. Little effort has been made to integrate newly discovered functions (e.g., anti-inflammatory effects and regulation of gastrointestinal motility) with traditional knowledge.

Thirdly, the pharmacokinetic and safety evaluation systems require significant improvement. The processes of oral absorption, distribution, metabolism, and excretion of the main active metabolites in ALD, such as lactones, remain largely unclear. Key questions persist: What is their bioavailability? In what form do they exert effects in vivo, as parent metabolites or metabolites? What kind of pharmacokinetic interactions exist between these metabolites and commonly used chemotherapy drugs? All these questions remain unanswered. Although some studies suggest that its extracts exhibit low toxicity to normal cells, there is a lack of systematic toxicological studies that meet modern drug approval standards, including investigations into long-term toxicity, reproductive toxicity, and genotoxicity. The potential risk of “nephrotoxicity” has also been frequently mentioned, but solid experimental data to either confirm or refute this claim are still lacking. This uncertainty represents a major concern for its clinical adoption.

Finally, there is a significant lack of clinical research. Almost all encouraging data currently available come from preclinical studies (in vitro cell and animal experiments). There is a shortage of rigorously designed and standardized clinical trials to verify the actual efficacy and safety of these treatments in humans. In traditional Chinese clinical practice, ALD is most commonly used in formulations. However, modern research has rarely attempted to simulate such complex environments to explore its role and effects within these formulations. Moreover, the considerable gap between discovering highly active extracts or metabolites in the laboratory and developing them into quality-controlled, standardized preparations suitable for patient use remains largely unaddressed.

To promote the in-depth development of ALD research and facilitate its clinical translation, future work should focus on the following aspects to meet the needs of a comprehensive research pipeline from basic to applied studies.

Firstly, future research needs to focus on establishing a quality control methodology based on a “holistic perspective”. Modern chromatographic, spectroscopic, and coupling techniques (such as HPLC-MS and GC-MS) should be utilized, in combination with chemometric methods, to conduct systematic analysis of ALD from different sources. This will help establish a “fingerprint profiling” standard that covers multiple major active metabolites and characteristic metabolites. At the same time, active research on spectrum-effect relationships should be carried out to correlate chemical fingerprints with pharmacological efficacy data. This will help identify which groups of metabolites are the key material basis for specific pharmacological effects (such as anti-cancer, anti-inflammatory, and anti-ulcer activities).

Secondly, it is essential to employ multi-omics technologies (genomics, transcriptomics, proteomics, metabolomics) and systemic biological approaches to comprehensively and unbiasedly uncover the target sites and network pathways of ALD and its active metabolites. This represents a core requirement. Comprehensive clinical studies in line with international standards must be initiated. It is imperative to systematically complete pharmacokinetic studies on the main active metabolites and optimized extracts of ALD to clarify their ADME properties. Additionally, toxicological evaluations should be conducted to thoroughly assess the safety of long-term usage and fully elucidate potential risks such as nephrotoxicity.

Thirdly, innovative formulation technologies and translational research should be a top priority for future studies. Given that many lactone metabolites exhibit poor water solubility and low bioavailability, there is a need to develop novel drug delivery systems such as nanoparticles, liposomes, and phospholipid complexes, to enhance their targeting capability and therapeutic efficacy. Furthermore, these advanced systems can help mitigate systemic toxicity associated with non-selective drug exposure.

Faced with numerous research demands, it is crucial to concentrate resources on addressing key issues. In the short term, priority should be given to foundational work on quality standardization: immediately launching a large-scale chemical composition survey of mainstream commercial ALD, integrating genomics for origin identification, establishing rapid and accurate identification methods based on multi-metabolite quantitative analysis and DNA barcoding technology, and formulating unified and feasible standards for raw medicinal materials. The most promising core active metabolites should be prioritized, with resources focused on completing systematic preclinical pharmacokinetic and pharmacodynamic studies, as well as preliminary acute toxicity and repeated-dose toxicity tests, to quickly obtain an initial assessment of their safety risks. Meanwhile, advanced technologies such as CRISPR screening, molecular docking, and metabolite probes should be employed to precisely identify their direct target sites.

Research on the mechanisms of compatibility in TCM formulations is also one of the priorities. Conducting studies on the synergistic principles and toxicity-reducing effects of pairing ALD with other botanical drugs is essential to provide a scientific basis for the modern application of TCM formulas. For compounds that have been identified as highly active but with low bioavailability, it is necessary to initiate the development of novel drug delivery systems, such as preparing their nanocrystals or phospholipid complexes, and to validate their efficacy enhancement and toxicity reduction in animal models. Additionally, launching small-scale exploratory clinical studies on TCM will help accumulate preliminary data and experience for larger trials.

The establishment of a complete industrial chain can maximize the internationalization of the ALD industry. Facilitating the formation of an integrated industrial chain, which is from large-scale cultivation of ALD bases to standardized extraction and production, and further to the development of final formulations, will help translate research achievements into real industrial applications and build a modern traditional Chinese medicine brand. Meanwhile, establishing an ALD research database to integrate chemical, pharmacological, toxicological, and clinical data will promote global collaboration and knowledge sharing among researchers.