Saussurea costus in nanomedicine: green-synthesized metal nanoparticles and advanced nanosystems for enhanced therapeutic efficacy

Abstract

Saussurea costus (S. costus), a medicinal plant widely utilized in traditional Ayurveda, Chinese, and Tibetan medicine, is rich in pharmacologically active compounds, including sesquiterpene lactones, flavonoids, phenolics, and essential oils. Despite its reported antimicrobial, anti-inflammatory, antioxidant, anticancer, and immunomodulatory activities, clinical translation of S. costus remains hindered by its low bioavailability and off-target effects. This review explores the use of nanosystems to address these limitations and enhance the biological performance of S. costus extracts. Metal-based nanoparticles (silver, copper, palladium, magnesium oxide) and other nano-formulations, including polymeric, lipid-based, and inorganic nanoparticles, detailing their synthesis, characterization techniques, and biomedical applications. The integration of S. costus into nanosystems is shown to improve cellular uptake and facilitate prolonged release and superior therapeutic outcomes as supported by several in vitro and in vivo studies. This review highlights the incorporation of Saussurea costus into different nanosystems towards the development of effective nanotherapeutics.

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1. Introduction

The genus Saussurea, a member of the Asteraceae family, comprises over 400 species globally.^1,2 These are widely distributed across temperate regions, such as south and central Asia, China, Himalayas, and Russia.^3,4 Saussurea species growing in alpine and subalpine regions has diverse phytochemicals as a mechanism for adaptation and survival in such unique environments.⁵ The botanical diversity of this plant, as well as the various morphologically distinct members ranging from low-growing herbs to taller perennial species, reflect the wide spectrum of pharmacological applications across different species.⁶ Several Saussurea species, including Saussurea costus (synonym: S. lappa), S. laniceps, S. obvallata, S. involucrate, and S. gossypiphora are traditionally used in different medicine systems such as Traditional Chinese Medicine (TCM), Ayurveda, Tibetan, and Korean medicine.^1,7–10 These species have been utilized in the treatment of various illnesses such as digestive disorders,¹¹ respiratory diseases,^12,13 inflammatory conditions,^10,14 gynecological issues,¹⁵ and infectious diseases.¹⁶ Among the genus, Saussurea costus (S. costus), known for its significant use in various traditional medicine in Pakistan and Himalayan region of India. For instance, In Ayurvedic medicine, S. costus was utilized to treat asthma, inflammatory diseases, ulcers, and stomach disorders.¹⁷ Within the Unani system, the root of S. costus is used to treat kidney, liver, and blood diseases. It is also known to be employed as a carminative, anthelmintic, and brain stimulant.¹⁸

The phytochemical analyses of S. costus revealed a diversity of secondary metabolites such as sesquiterpene lactones (costunolide and dehydrocostus lactone), phenolic acids (chlorogenic acid and caffeic acid), flavonoids (quercetin and kaempferol), and alkaloids, phytosterols, lignans, tannins, triterpenes, and essential oils.^19,20 While crude extracts from S. costus demonstrate valuable pharmacological activity, several challenges hinder their effectiveness to be integrated into mainstream therapeutics. For example, sesquiterpenes and lipophilic flavonoids have low water solubility, resulting in low plasma concentration and minimal therapeutic effect, even at high doses.^21–23 Even when absorbed, poor permeability through biological membranes and rapid hepatic metabolism may result in short half-life and low effectiveness.²⁴ Polyphenols and essential oils are sensitive to pH, light, temperature, and oxidation, resulting in loss of potency upon administration or during storage.^23,25–27 Additionally, non-selective targets throughout the body could reduce the efficacy in targeted disease sites like tumors.²⁸ These limitations necessitate advanced delivery strategies to the delivery of S. costus derived compounds.

To overcome these limitations, plant extracts were incorporated into different delivery systems to improve their biological performance.²⁹ These innovative systems include lipid-based nanocarriers such as niosomes, liposomes and solid lipid nanoparticles which enhance the solubility of hydrophobic compounds.^30,31 Polymeric systems, on the other hand, like chitosan and polycaprolactone (PCL), protect essential oils from degradation and enable controlled release of active constituents.^32,33 Additionally, metal-based nanosystems (including silver, gold, zinc, and copper oxides) are prepared using green approaches to carry natural compounds which add to their intrinsic antimicrobial or anticancer properties.^34,35

In this review, the natural distribution and traditional use of S. costus was examined to highlight its ethnopharmacological relevance. S. costus is known for its diverse phytochemicals which contribute to a wide range of biological activities. However, despite its therapeutic promise, the use of S. costus in clinical applications is limited by challenges such as poor water solubility, low bioavailability, and possible toxicity in its crude form. To address these issues, this review focuses on recent progress in nanoformulation strategies, including green synthesis of metal and metal oxide nanoparticles, as well as different nanocarriers. These nanosystems are evaluated based on synthesis, physicochemical features, and biological activities to provide a comprehensive understanding of their therapeutic potentials.

2. Discovery, taxonomy, and geographical distribution of Saussurea species

The genus Saussurea belongs to the kingdom Plantae and is classified under clade Asterids. Within the Asteraceae family, Saussurea genus is further assigned to the subfamily Carduoideae and belongs to the tribe Cardueae with over four hundred species of perennials.^1,36 Additionally, the taxonomy of different species is often challenging to resolve within this genus due to frequent hybridization, morphological plasticity, and convergent evolution in high-altitude environments. To resolve the complex taxonomy of Saussurea, many studies have turned to study the molecular phylogenetics of these species, where they analyzed the DNA sequences to understand the evolutionary relationships among species.^2,6,37,38 For example, Wu et al. found that genes, such as (CBF1, COR15A, COR47, and KIN1) are upregulated, aiding the adaptation of S. involucrate to cold and high-altitude environments.³⁹ The genus Saussurea was named in honor of the Swiss naturalists Horace-Bénédict de Saussure based on specimens collected during 18th century.^40,41 The species of Saussurea exhibit broad distribution across the Northern Hemisphere including North America, Europe, Asia, and North Africa. China exhibits the highest diversity of the of this plant with an estimation of 317 species, concentrated in alpine regions such as the Hengduan Mountains and Qinghai-Tibet Plateau.⁴² For example, S. involucrata is endemic to the Altai and Tianshan mountains,⁴³ while S. minutiloba,⁴² S. balangshanensis,⁴⁴ and S. medusa⁴⁵ are found in the Hengduan mountains. In the Indian Himalayan region, 62 species of Saussurea are documented to hold medicinal and cultural significance such as, S. costus and S. obvallata.⁴ In Mongolia around the Khangai mountains species such as S. salicifolia are found.⁴⁶ Additionally, in Japan and South Korea, endemic species like S. tanquetti and S. japonica are observed.⁴⁷ In Russia, particularly in Siberia, species like S. amara and S. daurica are commonly reported.⁴⁸ Beyond Asia, the genus also extends into Europe, with species like S. pygmaea, S. discolor, and S. alpina, inhabiting the Alps and Slovakia.^38,49 In North America, S. weberi and S. americana are distributed from Alaska through the Rocky mountains to northern California.³⁸ Additionally, a few Saussurea species were recorded in North Africa, particularly in the Atlas mountains of Algeria and Morrocco.^50,51

3. Traditional use of Saussurea species

The Saussurea genus has a long-standing ethnomedicinal history, particularly in Asia. Several Saussurea species were traditionally used in different medicine systems such as Traditional Chinese Medicine (TCM), Ayurveda, Tibetan, and Korean medicine.^1,7–10 They are often administered as powders, decoctions, and oils, either alone or in polyherbal formulations. In TCM, S. involucrate (known as Snow Lotus) was documented in the Chinese Compendium of Materia Medica, where it was described as a remedy for arthritis, menstrual pain, and altitude sickness, highlighting its traditional use in regions of Xinjiang and Tibet.⁵² Similarly, in Tibetan medicine, S. medusa and S. laniceps were traditionally used in polyherbal formulations as anti-inflammatory agent to treat cold disorders.⁷

Among the Saussurea genus, S. costus (also known as costus root) is extensively documented for its traditional use. In Ayurveda, it is referred to as Kushtha and is regarded as one of the most potent herbs for treating respiratory disorders (asthma and bronchitis), skin diseases, rheumatism, and ulcers due to its antimicrobial, anti-inflammatory, and expectorant properties.⁵³ Ayurvedic texts like Sushruta Samhita and Charaka Samhita described the importance of S. costus in formulations used for detoxification, digestive disorders, and respiratory diseases.⁵⁴ In Unani medicine, the root is valued for its ability stimulate brain activity, and improve liver and kidney functions, and is commonly prescribed for anemia, hyperglycemia, hyperlipidemia, ulcer and chronic cough.⁵⁵ Furthermore, in the Chinese Pharmacopoeia, it is recommended for managing abdominal pain, bloating, and dysentery by alleviating gastrointestinal stagnation.⁵⁶

Recently, S. costus has attracted growing scientific interest due to its strong medicinal potential. Studies on its root extract have revealed a range of important pharmacological properties.^1,57,58 However, overharvesting this plant has led to a significant decline in its wild populations. As a result, the species was classified under Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which includes species threatened with extinction.⁵⁹ This highlights the urgent need for conservation measures and the development of sustainable cultivation methods to protect this valuable medicinal plant.^57,60

4. Bioactive metabolites of S. costus

A wide range of extraction methods were employed to isolate the phytoconstituents of S. costus, each of which generated different yields and profiles of the bioactive compounds. Maceration,⁵⁷ Soxhlet extraction,⁶¹ and percolation⁶² were used to isolate key constituents using solvents like acetone, ethanol, methanol, hexane, and water. Additionally, techniques such as ultrasonic-assisted extraction,⁶² microwave-assisted extraction,⁶³ and supercritical fluid extraction⁶⁴ have enhanced extraction selectivity, particularly for thermolabile and non-polar compounds. The phytochemical profile of S. costus is rich in secondary metabolites that are pharmacologically active (Fig. 1). Among the secondary metabolites, sesquiterpene lactones such as costunolide, alantolactone, isoalantolactone, dehydrocostuslactone, and cynaropicrin are the most abundant and are primarily responsible for the antimicrobial, anti-inflammatory, antifungal, and immunomodulatory effects.⁶⁵ These are accompanied by a range of guaiane-, and germacrene-type lactones, such as saussureamines A–E, santamarine, lappalone, reynosin, epizaluzanins, arbusculin A & B, and colartin, further enhancing its therapeutic diversity.⁶⁵ The root of S. costus also contains notable flavonoids like luteolin, kaempferol, quercetin and its glycosides, which exhibit antioxidant and antimicrobial activities.^18,65 Other secondary constituents include phenolic acids (gallic acid, naringenin, chlorogenic acid, caffeic acid, and ferulic acid),⁶⁶ coumarins (umbelliferone),¹⁸ lignans (sesamin),¹⁸ alkaloids (saussurine, costine).¹⁸ The triterpenoid profile comprises (lupeol, daucosterin, α-amyrin betulinic acid) all of which are associated with anti-inflammatory, hepatoprotective, and anticancer effects.^62,65,67 Additionally, a range of phytosterols, including (β-sitosterol, lappasterol, daucosterol, pregnenolone) have been identified, which may contribute to lipid-lowering and immunomodulatory functions.^65,68 Furthermore, S. costus is rich in volatile essential oils including β-caryophyllene oxide, γ-bisabolene, anethole, 1,8-cyclopentadecadiyne, and patchoulene, which have been linked to anticancer activities.^62,67,69

Fig. 1 Chemical structures of bioactive compounds extracted from S. costus.

5. Pharmacological activities of S. costus and their mechanisms of action

Many of the broad therapeutic properties of S. costus, as reported in traditional medicine, are now supported by pharmacological evidence. Notably, S. costus exhibits anti-inflammatory,^70,71 anticancer,⁷² antioxidant,⁷³ antimicrobial,⁷⁴ antifungal,⁷⁴ hepatoprotective,⁷⁵ antidiabetic,⁷⁶ neuroprotective,⁷⁷ and immunomodulatory activities. For example, Choi et al. investigated the anti-inflammatory activity of S. costus extract in Wistar rats with benign prostatic hyperplasia (BPH), demonstrating that the S. costus significantly downregulated pro-inflammatory cytokines (TNF-α and IL-6) in inflamed prostate tissues. Additionally, it modulated apoptotic pathways by upregulating BAX (a pro-apoptotic protein) and downregulating BCL-2 (an anti-apoptotic protein), thereby restoring the apoptosis imbalance commonly associated with chronic inflammation in BPH.⁷⁰

Another group evaluated the anti-inflammatory effects of S. costus on TNF-α and IFN-γ-induced inflammation in human keratinocyte cells. Results showed that there was downregulation of inflammatory chemokines (RANTES, MDC, TARC, and IL-8) through the inhibition of STAT1 phosphorylation, particularly by alantolactone, thereby blocking the downstream signaling pathways responsible for pro-inflammatory cytokine production.⁷¹

In terms of anticancer activity, S. costus has shown cytotoxic effects against a range of cancer cell lines, including colon (HCT116), liver (HepG2), and breast (MCF-7) cancers. In their research, Shati et al. reported that the S. costus leaf extract treated cancer cells were arrested in the G1 phase of the cell cycle, where levels of pro-apoptotic proteins increased and anti-apoptotic proteins decreased, suggesting that S. costus induced cancer cell death by targeting the mitochondrial pathway of programmed cell death.⁷²

Elazab et al. investigated the protective effects of S. costus and Glycyrrhiza glabra extracts against arsenic-induced nephrotoxicity in chickens.⁷³ The administration of S. costus significantly ameliorated kidney damage caused by arsenic exposure through the activation of the Nrf2/HO-1 signaling pathway, which upregulates antioxidant enzymes such as superoxide dismutase and catalase. It also exerted anti-inflammatory effects by downregulating pro-inflammatory genes TNF-α and NF-κB, and decreased stress-related responses via reduced expression of heat shock proteins.⁷³

The antimicrobial activity of S. costus against SARS-CoV-2 and Candida albicans was also investigated. Molecular docking analysis revealed that 12 out of 69 identified compounds were triterpenoids, of which 70% showed strong binding affinities (−7.8 to −5.6 kcal mol⁻¹) against SARS-CoV-2 targets, suggesting promising antiviral potential. Additionally, S. costus extract demonstrated notable antifungal activity, with Candida albicans being the most susceptible organism.⁷⁴

El Gizawy et al. studied the hepatoprotective activity of S. costus extract against paracetamol-induced liver toxicity in male Wistar rats.⁷⁵ Paracetamol (acetaminophen), when administered in high doses, induces hepatotoxicity due to its metabolism into a toxic intermediate (NAPQI), which leads to liver cell apoptosis. Results showed that S. costus extract significantly attenuated paracetamol-induced liver injury in rats by increasing catalase activity and reducing oxidative stress markers like caspase 2 and CK18. Furthermore, S. costus modulated genetic markers involved in apoptosis and inflammation, including downregulation of miRNA-34a (less activation of cell death pathways) and miRNA-223 (suppress excessive inflammation), and upregulation of Hepatocyte Nuclear Factor 1 Alpha (restore metabolic homeostasis and enhance liver function), sirtuin-1 (reduce oxidative damage in liver), and (CCAAT/Enhancer-Binding Protein Alpha which supports cell regeneration and suppresses inflammatory damage).⁷⁵

Abouelwafa et al. investigated the antidiabetic activity of S. costus extracts in a streptozotocin induced diabetic rat model.⁷⁶ The S. costus extracts demonstrated inhibitory activity against digestive enzymes such as α-amylase, α-glucosidase, and lipase, which are involved in carbohydrate and lipid metabolism. S. costus extract significantly lowered blood sugar (in terms of glucose and HbA1c levels), supported gluconeogenesis (decreased F-1,6-BP, G-6-P), and enhanced glycolysis (increased glucokinase). It also improved the lipid profile by increasing HDL and decreasing triglycerides and total cholesterol levels.⁷⁶

Moreover, Abdel-Rahman et al. evaluated the neuroprotective and endocrine-modulating effects of S. costus root extract in male albino rats exposed to thorium-induced toxicity.⁷⁷ The cerebellum accumulated thorium caused significant oxidative damage and monoamine depletion. Pre-treatment of the albino rats with S. costus extract significantly enhanced the antioxidant defense system evidenced by reduced lipid peroxidation and nitric oxide levels and restored glutathione content.⁷⁷

6. Limitations of using S. costus extracts in therapeutic applications

Despite the reported pharmacological activities of S. costus, the direct use of its free extract in medicinal formulations is limited (Fig. 2). One of the primary challenges is hydrophobicity of its bioactive compounds such as sesquiterpene lactones and flavonoids.^78,79 These constituents exhibit low dissolution in physiological fluids (blood or gastric juice), resulting in suboptimal absorption and low systemic bioavailability.^78,79 For example, costunolide, a sesquiterpene lactone that is abundant in S. costus extract, exhibits low water solubility estimated at approximately 9.38 mg L⁻¹ at 25 °C.⁸⁰ Similarly, dehydrocostus lactone is insoluble in water, further limiting its bioavailability. ⁸¹ S. costus phytochemicals are also highly prone to environmental degradation. Vadaparthi et al. investigated on the stability of costunolide and dehydrocostus lactone in S. costus using liquid chromatography which exhibited significant degradation under alkaline conditions.⁸² Additionally, polyphenols and essential oils degrade rapidly upon exposure to heat, oxygen, or light, reducing the shelf life and the therapeutic efficacy.^83,84 Furthermore, these compounds undergo hepatic first-pass metabolism, leading to rapid elimination. A pharmacokinetic study conducted by Peng et al. on dehydrocostus lactone and costunolide showed that these compounds experience rapid hepatic metabolism in rats. This necessitates higher or more frequent dosing to achieve therapeutic levels, which may increase the risk of toxicity.⁸⁵ The application of S. costus sesquiterpene lactones in topical formulations is challenging due to potential skin irritation. These compounds can act as haptens, binding to skin proteins and triggering immune responses. A study by Cheminat et al. showed that sesquiterpene lactones, can be recognized by the immune system of sensitive individuals, causing allergic reactions.⁸⁶ This could be mitigated by encapsulating S. costus extracts to reduce direct skin exposure and irritation.⁸⁶ Another major limitation is the lack of specificity. When administered in free form, the active constituents of S. costus are distributed non-selectively throughout the body.⁸⁷ Furthermore, batch-to-batch variability due to differences in plant growth conditions, harvesting methods, and extraction techniques pose challenges for standardization and reproducibility in therapeutic applications.⁸⁸ These issues collectively underscore the need for advanced formulations to optimize the therapeutic potential of S. costus.

Fig. 2 Challenges limiting the therapeutic potential of free S. costus extract.

7. Advanced nanosystems for maximizing therapeutic potential of S. costus

Advanced delivery systems have emerged as effective tools to improve the delivery S. costus, with green synthesis and nanoformulation strategies showing considerable promise.^89–98 Plant-mediated green synthesis of metal and metal oxide nanoparticles offers a simple, sustainable route in which phytochemicals from the extract act as both reducing and capping agents, often conferring additional biological activity compared with chemically synthesized nanoparticles.^99,100 Moreover, nanoformulations such as polymeric,⁹⁴ lipid,⁹⁷ inorganic nanoparticles,⁹⁸ and nanofibers⁹⁶ can improve the bioavailability, stability and target-site accumulation of phytoconstituents while protecting them from rapid metabolism and degradation. This review presents nanosystems developed for S. costus and highlights how green synthesis and nanoencapsulation strategies helped overcome key limitations of the free extracts and consequently enhanced their biological activity in different biomedical applications. A structured search was conducted in PubMed (n = 19), Scopus (n = 33), and Web of Science (n = 47) to identify reports on S. costus/S. lappa formulated as metallic, polymeric, lipidic, or hybrid nanosystems. After removal of 47 duplicates, 52 records were screened. 32 were excluded (not nano, no S. costus, review), and 20 studies met the eligibility criteria and were included in this review. The selection process is summarized in Fig. 3.

Fig. 3 PRISMA-style flow diagram for selection of S. costus nanoformulation studies.

7.1 Green synthesis of metal-based nanoparticles using S. costus

Recent studies have demonstrated the use of S. costus extract as a natural reducing agent for the green synthesis of metal and metal oxide nanoparticles with enhanced biological activities (Table 2).

7.1.1 Silver nanoparticles (AgNPs). Bharathi et al. prepared AgPNs utilizing the aqueous root extract of S. costus as a reducing agent and incorporated AgNPs in a chitosan hydrogel matrix and evaluated their antimicrobial activities for wound healing application.⁹⁹ Characterization of the prepared nanoparticles using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) confirmed the spherical shape of the nanoparticles with a size in the range of 10–20 nm. Dynamic light scattering (DLS) showed that the diameter of the nanoparticles was 44 nm. X-ray chromatography (XRD) confirmed the crystallinity of silver (face-centered cubic), while Fourier transform infrared (FTIR) revealed functional groups from S. costus phytochemicals, possibly responsible for reduction of silver ions. Thermal analysis included thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA indicated a weight loss occurred between 200–500 °C with a total weight loss of 49.71%, indicating the presence of organic molecules of S. costus extract stabilizing AgNPs. The melting point was 966.4 °C. To evaluate the biocompatibility AgNPs, MTT assay was performed on human leukemia cell lines (THP-1) for 24 h, showing an inhibitory concentration (IC₅₀) of 151.10 µg mL⁻¹. The AgNPs were then loaded into a chitosan hydrogel, showing a swelling ratio of 41% and good spreadability, with sustained release for 3 h. Five bacterial strains were used to evaluate the antimicrobial activities including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Bacillus cereus (B. cereus) Staphylococcus aureus (S. aureus), and Klebsiella pneumoniae (K. pneumoniae), using agar well diffusion method. Among the tested strains, P. aeruginosa exhibited the highest susceptibility with an inhibition zone of 21.47 ± 0.50 mm, followed by S. aureus (19.97 ± 0.73 mm), E. coli (17.30 ± 0.03 mm), and K. pneumoniae (15.50 ± 0.51 mm), while B. cereus showed the lowest response at 11.52 ± 0.19 mm. For the in vivo study, P. aeruginosa-infected Wistar rats were treated with chitosan hydrogel-AgNPs for 15 days. This resulted in significant reduction in wound area from 518 mm² to 16 mm² with no evident bacterial load (Fig. 4). Histological analysis showed enhanced collagen and fibroblasts production in the wound. Additionally, tissue remodeling biomarkers showed a significant elevation in the treated group confirming tissue regeneration.⁹⁹

Fig. 4 Wound healing progression in P. aeruginosa-infected Wistar rats over a 15-day period under different treatment conditions. (A) Negative control (no treatment) exhibited delayed healing and persistent wound area. (B) Positive control group treated with silver sulfadiazine showed partial wound contraction. (C) Test I group treated with blank chitosan hydrogel displayed moderate healing. (D) Test II group treated with chitosan hydrogel-AgNPs synthesized using S. costus extract demonstrated significant healing, with complete wound closure (from 518 mm² to 16 mm²) and absence of bacterial infection. Reproduced with permission from,⁹⁹ Springer Nature, 2021.

Hijazi et al. utilized S. costus root aqueous extract to evaluate the antibacterial activity of AgNPs against different microbial strains, and their catalytic efficiency in degrading organic dyes.¹⁰¹ TEM showed spherical particles of 22 nm in size. The hydrodynamic diameter of AgNPs using DLS was 66.2 nm, with polydispersity index (PDI) of 0.249, and a zeta potential of −21.57 ± 2.72 mV, indicating monodispersity of the prepared AgNPs. TGA indicated a total weight loss of 68.6%, signifying stabilization of AgNPs by organic content of S. costus. Photoluminescence (PL) spectroscopy confirmed the successful formation of AgNPs with excitation wavelength at 400 nm and emission wavelength at 534 nm, indicative of surface plasmon resonance (SPR) with no spectral overlap observed with either S. costus or the silver nitrate solution. The antibacterial efficacy of the green-synthesized silver nanoparticles was assessed against Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), and Staphylococcus haemolyticus (S. haemolyticus) using the agar well diffusion method. Among the tested strains, S. haemolyticus exhibited an inhibition zone of 15.33 ± 0.57 mm (highest susceptibility), while E. faecalis showed inhibition zone of 12.0 ± 0.4 mm (lowest susceptibility). The minimum inhibitory concentration (MIC) values ranged from 0.0394 to 0.158 g L⁻¹, and the minimum bactericidal concentration (MBC) values ranged from 0.0788 to 0.315 g L⁻¹. Based on the MBC/MIC ratios, the nanoparticles exhibited bacteriostatic effect (ratio = 8) against K. pneumoniae, bactericidal activity (ratio ≤4) against S. aureus, S. haemolyticus, and E. faecalis. The nanoparticles also demonstrated catalytic activity in degrading toxic dyes like safranin, methyl orange, methylene blue, Congo red, and orange G. These results highlight the potent antibacterial and catalytic activity of silver nanoparticles green-synthesized using S. costus extract.¹⁰¹

Another group tested the antimicrobial and antioxidant activity of AgNPs synthesized using the aqueous S. costus root extract.¹⁰² SEM images revealed spherical shape of the prepared nanoparticles. XRD analysis confirmed the crystallinity of AgNPs, and the calculated crystallite size was 20.15 nm, with lattice constant of 4.086 Å. Surface plasmon resonance (SPR) peak was detected at 440 nm using UV-Vis spectroscopy, indicative of nanoparticle formation. FTIR analysis showed functional groups corresponding to flavonoids, phenols, amides, and amines, indicating successful bio-reduction of silver nitrate. The antimicrobial activity against P. aeruginosa and E. coli of AgNPs, methanol S. costus root extract, and aqueous S. costus root extract were evaluated and compared. The agar well diffusion method was utilized showing zones of inhibition of 11.0 mm against E. coli and 9.0 mm against P. aeruginosa in case of AgNPs. In comparison, the methanolic root extract alone showed moderate inhibition (9.0 mm for P. aeruginosa and 6.0 mm for E. coli), while the aqueous extract produced a 6.0 mm inhibition zone for both bacteria. Additionally, the antioxidant activity of methanolic extract was tested showing an IC₅₀ value of 0.814 µg mL⁻¹, attributable to its rich content of total flavonoids (145 mg rutin equivalent per g) and total phenolics (205 mg gallic acid equivalent per g).¹⁰²

Aqueous extracts from both S. costus leaves and roots were utilized to synthesize AgNPs.⁹² SPR peaks was evident at 420 nm for S. costus root-derived AgNPs (R-AgNPs) and at 407 nm for leaf-derived nanoparticles (L-AgNPs). TEM analysis revealed smaller particle size for R-AgNPs (26 nm), while L-AgNPs had a size of 55 nm. Monodispersity of the nanoparticles was examined by DLS in which R-AgNPs exhibited PDI of 0.2578 (diameter = 50.32 nm), and for L-AgNPs, PDI was 0.1448 (diameter = 148.8 nm). Energy-dispersive X-ray spectroscopy (EDX) confirmed the phytochemical surface capping evident by signals corresponding to carbon, oxygen, and potassium in addition to strong silver peaks. Three assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP), were employed to assess the antioxidant activity of R-AgNPs and L-AgNPs (Table 1). The scavenging activity was significantly higher in AgNPs than in their corresponding plant extracts. Notably, R-AgNPs exhibited superior antioxidant activity, which can be attributed to the higher content and diversity of phenolic and flavonoid compounds in the root extract. The study also evaluated anti-inflammatory activities through the inhibition of proinflammatory enzymes, such as 5-LOX, COX-1, and PLA₂s, achieving over 99% inhibition at a concentration of 120 µg mL⁻¹ for both R-AgNPs and L-AgNPs. MTT assay was utilized to evaluate the anticancer activity of the prepared nanoparticles and their free extracts against colorectal adenocarcinoma (LoVo), colorectal carcinoma (HCT-116), and triple-negative breast cancer (MDA-MB-231) cells. The results indicated enhanced antiproliferative effects of R-AgNPs and L-AgNPs compared to the free form of the extracts with IC₅₀ values ranging between 42 and 60 µg mL⁻¹ (Table 1). Finally, antimicrobial activity against bacteria (E. coli, S. aureus, Bacteroides fragilis (B. fragilis), and Enterococcus faecalis (E. faecalis)) and fungi (Aspergillu niger (A. niger), Penicillium digitatum (P. digitatum), and Aspergillus oryzae (A. oryzae)) was evaluated. Both AgNPs displayed a more significant antimicrobial effect than the corresponding S. costus aqueous extracts. L-AgNPs were the most effective, especially against E. coli and E. faecalis with an IC₅₀ of 14.3 µg mL⁻¹. For the antifungal activities, L-AgNPs and R-AgNPs showed 2–3 times more potency than their free extracts. R-AgNPs were particularly potent against A. oryzae with IC₅₀ of 2.50 µg mL⁻¹.⁹²

Table 1 Evaluation of antioxidant and anticancer activities of L-AgNPs and R-AgNPs and their corresponding free extracts.⁹²

	Leaf extract	Root extract	L-AgNPs	R-AgNPs	Ref.
DPPH (% scavenging activity)	81.0	83.5	95.3	97.7	92
ABTS (% scavenging activity)	84.7	81.0	97.7	95.3
FRAP (% Fe^3+ reduction)	74.7	67.2	91.9	95.6
FRAP (mg gallic acid equivalent per L)	59.5	39.2	43.5	65.1
MDA-MB-231 (IC50 µg mL^−1)	86	120	56	60
LoVo (IC50 µg mL^−1)	74	74	50	42
HCT-116 (IC50 µg mL^−1)	82	82	56	60

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Table 2 Characteristics and biological activities of S. costus-derived metal or metal oxide nanoparticles^a

Nanoparticle type	S. costus part/extract type	Characterization of nanoparticles	Reported bioactivity	Ref.
a Abbreviations: AChE: acetylcholinesterase; AgNPs: silver nanoparticles; AuNPs: gold nanoparticles; BChE: butyrylcholinesterase; CHO: Chinese hamster ovary; CuNPs: copper nanoparticles; CuO NPs: copper oxide nanoparticles; DLS: dynamic light scattering; DSC: differential scanning calorimetry; EDX: energy-dispersive X-ray spectroscopy; IC50: half-maximal inhibitory concentration; IONPs: iron oxide nanoparticles; MCF-7: Michigan Cancer Foundation-7 cell line; MgO NPs: magnesium oxide nanoparticles; MRSA: methicillin-resistant Staphylococcus aureus; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay; PDI: polydispersity index; PdNPs: palladium nanoparticles; PL: photoluminescence; ROS: reactive oxygen species; R-AgNPs: root-extract silver nanoparticles; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TGA: thermogravimetric analysis; UV-Vis: ultraviolet-visible spectroscopy; XRD: X-ray diffraction; ZnO NPs: zinc oxide nanoparticles.
Chitosan hydrogel loaded-AgNPs	Root aqueous crude extract	-UV-Vis peak at 419 nm	- Antibacterial with zone of inhibition from 11.52 to 21.47 mm (S. aureus, B. cereus, E. coli, K. pneumoniae) largest against P. aeruginosa (21.47 mm)	99
		- Size: 10–20 nm (TEM and SEM) 44 nm (DLS)	- Wound healing: P. aeruginosa-infected Wistar rat model showed complete bacterial clearance by day 15
		- Sustained release for 3 h	- No adverse effects on skin or systemic health
		Organic content: 49.71% (TGA)
		Melting point: ∼966 °C (DSC)
AgNPs	Root aqueous crude extract	UV-Vis peak at 460 nm	- Antibacterial effect against K. pneumoniae, S. aureus, S. haemolyticus, E. faecalis	101
		PL emission peak at 534 nm	- Lowest MIC: 0.0394 g L^−1 (K. pneumoniae)
		Size: 22 nm (TEM), 66.2 nm (DLS)	- Bactericidal effect (MBC/MIC ≤4) vs. 3 strains; bacteriostatic vs. K. pneumoniae
		PDI: 0.249	- Membrane disruption, ROS generation, Ag^+ ion release
		Zeta potential: −21.57 mV	- Catalytic degradation of dyes
		Crystalline peaks at (111), (200), (220), (311)
		Weight loss: 69% (TGA)
AgNPs	Root aqueous extract	- UV-Vis peak at 440 nm	- Antibacterial effect against E. coli (11.0 mm) and P. aeruginosa (9.0 mm)	102
		- Crystallite size: 20.15 nm and lattice constant: 4.086 Å (XRD)	- Antioxidant
		- Spherical morphology (SEM)
Polyvinyl alcohol (PVA) sheets loaded-AgNPs	Root methanolic (70%) crude extract	- UV-Vis peak at 422 nm	- Antibacterial effect against E. coli, S. aureus Acinetobacter baumannii, and MRSA at 1 µg mL^−1	105
		- Size 45 ± 15 nm (TEM/SEM)	- High catalytic activity of 4-nitrophenol with 97% reduction in 18 min
			- Reusable for 8 cycles with >80% catalytic efficiency
AgNPs	Root and leaves aqueous crude extracts	- UV-Vis peak at 407 nm L-AgNPs and 420 nm for R-AgNPs	- R-AgNPs exhibited superior antioxidant activity to L-AgNPs	92
		- L-AgNPs: 55 nm (TEM), 148.8 nm (DLS), PDI: 0.1448	- AgNPs showed >99% inhibition of pro-inflammatory enzymes
		- R-AgNPs 26 nm (TEM), 50.32 nm (DLS), PDI: 0.2578	- IC50 for cancer cell lines (HCT-116, LoVo, MDA-MB-231) ranged 42–60 µg mL^−1, showing most potency against LoVo (R-AgNPs)
		- EDX confirmed presence of Ag, O, C, K	- Antibacterial IC50 values: 14.3–20.65 µg mL^−1 most effective against E. coli and E. faecalis (L-AgNPs)
			- Antifungal effect against A. oryzae with IC50 of 2.5 µg mL^−1 (R-AgNPs)
CuNPs	Flower aqueous crude extract	- UV-Vis peak at 280–500 nm	- Antimicrobial effect (P. aeruginosa, E. coli, S. aureus, and K. pneumoniae). Inhibition zones of 17–19 mm	103
		Size: 98.03 nm (DLS)	- Anti-obesity: lipase inhibition reached 47.01% with IC50 of 60.01 µg mL^−1
		- Irregular morphology of CuNPs (SEM)	- Anti-diabetic: amylase inhibition reached 56.45% with IC50 of (45.31 µg mL^−1)
		- EDX confirmed presence of Cu and sulfur
Gold (AuNPs)	Root methanolic crude extract	- UV-Vis peaks at 500 nm	- Improved antioxidant capacity and stronger radical scavenging effects	106
		- XRD peaks at 38°, 43.8°, 65° (111, 200, 220 planes)	- Enhanced inhibition of digestive enzymes related to diabetes
		- Size:19 nm with spherical shape (TEM)	- Possible anti-Alzheimer's activity through acetylcholinesterase inhibition
			- Sustained anti-inflammatory potential via protein stabilization mechanisms
			- Increased cytotoxic effectiveness against liver (HepG2) and colon cancer (Caco2) cells
			- Induced cancer cell apoptosis and modulated gene expression favorably
IONPs	Root peel methanolic (50%) crude extract	- Size: 295 nm, zetapotential: +0.058 (DLS)	- Antimicrobial activity against P. aeruginosa, E. coli, Staphylococcus sp	104
		- Spherical, prismatic, elongated particles (SEM/TEM)	- Morphological damage to IONPs-treated E. coli via membrane disruption
Zinc oxide (ZnO NPs)	Root methanolic crude extract	- UV-Vis peak 430 nm	- Strong antibacterial activity: 3.7 mm inhibition zone for E. coli and 3.5 mm for P. aeruginosa	107
		- Hexagonal shape of ZnO NPs (SEM)	- Antifungal activity against Meyerozyma caribbica and Rhizopus oryzae (inhibition zone of 2.9 mm for both)
		- DLS: size: 123.5 nm, zetapotential of −99.9 mV	- Cytotoxicity (MTT assay) against CHO cell line with IC50 of 3.16 µg mL^−1
		- EDX confirmed presence of Zn and O
MgO NPs	Root methanolic crude extract (Qust al-Hindi and Qust al-Bahri)	- UV-Vis peaks at 250 and 320 nm	- Significant antibacterial effect (16 mm against P. aeruginosa)	93
		- Crystallite size (XRD): 30–34 nm	- Strong antifungal activity (20 mm against C. tropicalis)
		- Uniform cubic morphology (SEM/TEM)	- Dose-dependent cytotoxicity on MCF-7 (IC50 = 52.1–67.3%)
		- DLS: size: 20–100 nm, zeta potential: −20.5 mV	- 92% methylene blue dye degradation under UV by Qust al-Bahri-derived MgO NPs
PdNPs	Root aqueous crude extract	- UV-Vis peaks at 350 nm	- Enhanced antimicrobial activity compared to free extract	90
		- DLS: zeta potential: −10.6 Mv	- Antioxidant IC50 of 90 µg mL^−1
		- Size: 1.9–17.6 ± 1.2 nm and spherical shape (TEM)	- AChE/BChE inhibition 79.23%/76.13%
		- EDX confirmed presence of Pd, C, O	- NO inhibition in RAW 264.7 cells by 7.84%
			- Effective cytotoxicity against HCT-116 (IC50 7.8 µg mL^−1), HepG2 (11.8 µg mL^−1), and MCF-7 (26.7 µg mL^−1)
CuO NPs	Mixture of S. costus root (methnolic) and O. sanctum seed aqueous crude extracts	- UV-Vis peak at 27 (CuO NPs) and 500 nm (phytochemicals)	- Strong antibacterial activity (3.2 mm zone for S. aureus and B. subtilis)	100
		- Size: 40–60 nm with aggregated rough surfaces (SEM)	- Effective antifungal activity (2.8 mm zone for Meyerozyma guilliermondii)
		- Size: 103.4 nm and zetapotential: 63.2 mV (DLS)	- Cytotoxic effect (MTT assay) on CHO cells with IC50 of 4.14 µg mL^−1
		- EDX confirmed presence Cu and O	- Biological activity attributed to ROS generation, Cu^2+ release, and membrane damage
		- Crystallite size 38 nm
		- Weight loss: 22.5% (organic content), stable up to 800 °C (TGA/DSC)
Titanium oxide (TiO2)/ZnO nanocomposite coated by Cu(II) complex of di-Schiff bases	Root aqueous crude extract was used to produce TiO2	- XRD: anatase TiO2 and wurtzite ZnO, some ZnO peaks weakened after coating (amorphous organic shell), no lattice shift	- Antibacterial effect (agar diffusion, 5 µg mL^−1) after 3 days gave 30 mm vs. Serratia marcescens, 30 mm vs. E. coli, 34 mm vs. S. aureus (all much higher than bare TiO2/ZnO with ≤7 mm)	108
		- TEM/SEM: irregular/spherical agglomerates about 40–100 nm, coated sample shows thicker outer layer	- Antifungal activity with clear inhibition of C. albicans, A. flavus, Trichophyton rubrum
		- EDX: Ti, Zn, O plus C, N, Cu after coating	- S. costus phytochemicals create a reactive/organic surface on TiO2/ZnO, allowing uniform fixing of the copper(II) di-Schiff base complex, synergizing its antimicrobial activity
		- TGA: coated nanocomposite more thermally stable than the free complex
		- No EE% or release data reported

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7.1.2 Copper nanoparticles (CuNPs) and copper oxide nanoparticles (CuONPs). Kumar et al. synthesized CuNPs using two aqueous extracts of Bauhinia variegata (B. variegata) flower (6%) and S. costus flower (3%) to explore their anti-obesity, anti-diabetic, and antimicrobial activities.¹⁰³ The solution color change from blue to green indicated formation of CuNPs. The SPR peaks of CuNPs were detected using UV-Vis spectroscopy showing absorption from 280–500 nm range. DLS showed a particle size of 98.03 nm (S. costus) and 8.721 nm (B. variegata). SEM imaging of CuNPs revealed irregular shapes, surfaces, and edges of CuNPs. EDX confirmed Cu-rich composition. XRD confirmed high crystallinity of the synthesized CuNPs. Antimicrobial tests were conducted against P. aeruginosa, E. coli, S. aureus, and K. pneumoniae using agar well diffusion method. The zones of inhibitions ranged from 17 to 19 mm, comparable to conventional antibiotics like ampicillin. This antimicrobial action could be attributed to generation of reactive oxygen species (ROS) that disrupted the membrane integrity of bacteria, and the release of Cu²⁺ ions. Additionally, the presence of residual flavonoids and polyphenols from the B. variegata and S. costus extract provided a synergistic antibacterial effect. Pancreatic lipase inhibition assay was conducted to evaluate anti-obesity activity. S. costus derived-CuNPs showed 47.01% inhibition of pancreatic lipase (IC₅₀ of 60.01 µg mL⁻¹), while B. variegata-mediated CuNPs revealed 59.67% inhibition of pancreatic lipase at 100 µg mL⁻¹. In terms of anti-diabetic activity, results showed strong potential for reducing postprandial glucose levels evaluated by α-amylase inhibition assay, where the IC₅₀ of B. variegata-mediated CuNPs was 78.06 µg mL⁻¹ showing 89.02% inhibition of α-amylase and S. costus CuNPs had an IC₅₀ = of 45.31 µg mL⁻¹ with 56.45% inhibition.¹⁰³

Another study conducted by Kolahalam et al. synthesized CuONPs using a mixture of S. costus root extract and Ocimum sanctum (O. sanctum) seeds extract as reducing agents to explore their cytotoxic effect on Chinese Hamster Ovary (CHO) cells, antibacterial, and antifungal activities.¹⁰⁰ UV-Vis spectroscopy revealed peaks at 270 nm corresponding to CuONPs and 500 nm related to S. costus and O. sanctum-derived compounds. The size of the CuONPs was estimated by DLS to be 103.4 ± 27.4 nm with zetapotential of +63.2 mV suggesting high stability. XRD revealed a crystallite size of 38 ± 1 nm confirming crystallinity. SEM images showed cuboidal and round particles exhibiting a size range of 40–60 nm, while EDX showed copper and oxygen peaks. Functional groups related to plant extracts including N–H, O–H, and C≡C were detected in FTIR spectrum. Thermal analysis indicated a weight loss of 22.5% due to the decomposition of organic compounds form the extracts TGA, while DSC analysis showed a strong exothermic peak at 700 °C. To assess the antimicrobial activities, different bacterial strains isolated from drinking water were used, including Sphingobacterium thalpophilum, Ochrobactrum sp., Acinetobacter sp., Sphingobacterium sp., P. aeruginosa, E. coli, and Bacillus subtilis, Streptococcus aureus, Staphylococcus aureus. Staphylococcus aureus and Bacillus subtilis exhibited the highest sensitivity towards CuONPs (170 ppm), both showing zones of inhibition of 3.2 ± 0.03 and 3.2 ± 0.14 mm, respectively. P. aeruginosa and E. coli showed zones of inhibition of 2.9 ± 0.08 mm and 2.7 ± 0.05 mm, respectively. The least sensitive strains were Streptococcus aureus (2.0 ± 0.02 mm) and Sphingobacterium thalpophilum (2.2 ± 0.04 mm). Antifungal properties were tested against 8 fungal strains including Meyerozyma guilliermondii, Fusarium oxysporium, Rhizopus oryzae, Aspergillus flavus, Meyerozyma caribbica, Aspergillus oryzae, and Aspergillus niger, and Trichoderma asperellum. Best antifungal activity was induced by 170 ppm of CuONPs, with reported zones of inhibition of 2.8 ± 0.05 mm and 2.7 ± 0.12 mm for Meyerozyma guilliermondii and Meyerozyma caribbica, respectively. Fusarium oxysporium, Rhizopus oryzae, and Aspergillus flavus showed zones of 2.0–2.1 mm. The least sensitive strains were Aspergillus oryzae and Aspergillus niger. MTT assay was conducted on CHO cell line and revealed an IC₅₀ value of 4.14 µg mL⁻¹ following a dose-dependent decrease in cell viability. The mechanism of inhibition includes ROS generation, metal ion release, and membrane interaction.¹⁰⁰

7.1.3 Iron oxide nanoparticles (IONPs). Al-brahim biosynthesized IONPs using aqueous extract of S. costus root and iron salts FeCl₂ : FeCl₃ (2 : 1).¹⁰⁴ The solution color change from yellow to dark brown indicated the formation of IONPs. TEM and SEM revealed that IONPs exhibited spherical, elongated, or prismatic curved shapes. The average size of the prepared nanoparticles was 295 nm with zetapotential of +0.058 mV. For antimicrobial evaluation of S. costus-derived IONPs, agar well diffusion method was utilized against bacterial strains (Staphylococcus sp., B. subtilis, K. pneumoniae, Shigella sp., P. aeruginosa, and E. coli) and fungal strains (Aspergillus niger, Alternaria sp., and Rhizopus sp.). P. aeruginosa, Staphylococcus sp., E. coli, Shigella sp., and Aspergillus niger showed inhibition zones of 11, 9, 9, 6, and 5 mm, respectively. Comparative tests also showed that IONPs had higher inhibitory effects than either the S. costus free extract or Fe ions alone. SEM imaging revealed morphological differences between untreated and IONPs-treated E. coli (Fig. 5). While the untreated cells appeared smooth with typical rod-shaped morphology, the IONPs-treated E. coli showed structural damage, including membrane distortion, surface corrugation, and reduction in both cell length and diameter. These alterations suggest that the IONPs compromised the bacterial cell wall integrity, supporting membrane disruption as a primary mechanism of antimicrobial action of S. costus -derived IONPs.¹⁰⁴

Fig. 5 SEM imaging of untreated E. coli (left) vs. S. costus -IONPs-treated E. coli (right). (A) Untreated E. coli displaying intact rod-shaped structure with typical cell dimensions. (B) IONPs-treated E. coli showing clear signs of membrane damage and reduced cell diameter and length.¹⁰⁴ Copyright license CC BY 4.0.

7.1.4 Magnesium oxide nanoparticles (MgO NPs). Amina et al. biosynthesized MgO NPs using methanolic root of S. costus and evaluated their antimicrobial, antifungal, and anticancer activities.⁹³ Two varieties of S. costus were used one is Qust al-Hindi obtained from Indian woods (mountains) and the other is Qust al-Bahri retrieved from sea (coastal regions). The change of yellow colored aqueous solution of Mg (NO₃)₂ to dark brown confirmed the formation of MgO NPs. UV-Vis spectroscopy showed SPR peaks at 320 nm (indicating MgO NPs formation) and 250 nm (possibly due to phytochemical constituents). FTIR analysis indicated the presence of functional groups such as –OH, C=O, and Mg–O, while XRD revealed crystalline structure with particle sizes of approximately 30 nm and 34 nm for MgO NPs prepared using Qust al-Hindi and Qust al-Bahri, respectively, as confirmed by SEM and TEM imaging. DLS confirmed the colloidal stability of the nanoparticles (−20.5 mV). EDX confirmed the presence of Mg and oxygen.

Antimicrobial activity was performed using agar well diffusion method against B. subtilis, P. aeruginosa, S. aureus, and E. coli. MgO NPs synthesized from Qust al-Bahri (35 µg mL⁻¹), showed strong antibacterial activity, most effective against P. aeruginosa (zone of 16 mm), followed by E. coli (15 mm), S. aureus (14 mm) and B. subtilis (10 mm). In contrast, Qust al-Hindi -derived MgO NPs were only effective against B. subtilis (17 mm) and E. coli (8 mm), showing no activity against S. aureus and P. aeruginosa. Additionally, the MIC of Qust al-Bahri-MgO NPs for both E. coli and P. aeruginosa was 320 µg mL⁻¹, while the MBC was 320 µg mL⁻¹ for E. coli and 1280 µg mL⁻¹ for P. aeruginosa, suggesting strong bactericidal potential, particularly against E. coli. SEM imaging further confirmed bacterial cell wall disruption and morphological deformation in treated samples (Fig. 6).⁹³

Fig. 6 SEM images illustrating the morphological changes in E. coli and P. aeruginosa upon treatment with Qust al-Bahri -mediated MgO NPs. (a) Untreated E. coli (control) showing rod-shaped morphology, (b) E. coli treated with Qust al-Bahri extract exhibiting partial surface disruption, (c) E. coli treated with MgO NPs displaying significant morphological deformation and membrane damage. (d) Untreated P. aeruginosa (control), (e) P. aeruginosa treated with Qust al-Bahri extract showing moderate surface changes, (f) P. aeruginosa treated with Qust al-Bahri-MgO NPs revealing extensive structural disruption and cell wall lysis.⁹³ Copyright license CC BY 4.0.

In terms of antifungal activity, Qust al-Hindi -derived MgO NPs exhibited more potent effects than Qust al-Bahri. At 35 µg mL⁻¹, they produced inhibition zones of 20 mm and 19 mm against Candida tropicalis and Candida glabrata, respectively. Qust al-Bahri -MgO NPs were active only against C. tropicalis (20 mm), with no effect on C. glabrata. The antifungal mechanism likely involves ROS-mediated oxidative stress and disruption of fungal membrane integrity.⁹³

To assess the anticancer activity of MgO NPs, MTT assay was conducted on breast cancer cells (MCF-7). The IC₅₀ values were 67.3% for Qust al-Bahri -MgO NPs and 52.1% for Qust al-Hindi-MgO NPs. Microscopic examination showed morphological changes in treated cells, including shrinkage, chromatin condensation, and membrane blebbing. The morphological evidence was further confirmed by LDH assay, which demonstrated increased enzyme release confirming cytotoxic damage. DNA fragmentation assay revealed fragmentation patterns in Mg ONPs-treated cells, suggesting apoptosis induction. Moreover, intracellular ROS quantification via fluorescence microscopy demonstrated a significant rise in ROS levels in treated cells, supporting the hypothesis that oxidative stress was a primary driver of apoptosis. Additional confirmation came from Rhodamine 123 staining, which showed loss of mitochondrial membrane potential, further implicating the apoptotic pathway. Acridine orange/ethidium bromide (AO/EtBr) dual staining was employed to differentiate between viable, early apoptotic, late apoptotic, and necrotic cells. MgO NPs-treated cells showed an 84.3 ± 0.01% apoptotic population compared to only 8.3 ± 0.7% in the untreated group, and 65.4 ± 0.06% in paclitaxel-treated cells. Additionally, DAPI staining revealed nuclear condensation and fragmentation in treated cells, further validating apoptotic cell death. A scratch assay demonstrated that MgO NPs significantly impaired MCF-7 cell migration and delayed wound closure, indicating strong antiproliferative and antimigratory effects that could help limit tumor growth and metastasis. Finally, MgO NPs also exhibited strong photocatalytic activity against methylene blue dye, with degradation efficiencies of 92% (Qust al-Bahri) and 59% (Qust al-Hindi) after 1 h of UV exposure, demonstrating their potential in environmental and therapeutic applications.⁹³

7.1.5 Palladium nanoparticles (PdNPs). Al-Radadi utilized the aqueous root extract of S. costus to reduce PdCl₂ into palladium nanoparticles and explored their biomedical applications including antibacterial, antioxidant, anti-inflammatory, anti-Alzheimer's, and anticancer activities.⁹⁰ The successful formation of PdNPs was confirmed by the color change from yellow (PdCl₂) to dark brown (PdNPs). SPR peak at 350 nm indicated Pd⁰ formation. PdNPs had a diameter range of 1.9 nm to 17.6 ± 1.2 nm and spherical shape (TEM). Zeta potential analysis showed zeta potential of −10.6 mV, suggesting moderate colloidal stability. FTIR analysis identified functional group peaks at 554.30 cm⁻¹ (Pd–O), 3442 cm⁻¹ (hydroxyl), 2083 cm⁻¹ (alkyne), and 1635 cm⁻¹ (amide). Face-centered cubic crystalline structure was determined using XRD with a crystallite size of 2.88 nm. EDX confirmed the presence carbon, oxygen, and nitrogen from the plant extract as well as the Pd, while X-ray photoelectron spectroscopy (XPS) confirmed elemental palladium (Pd⁰) with Pd3d peaks at 340.89 and 335.63 eV. Antimicrobial assessment was conducted against S. aureus, B. subtilis, E. coli, and P. aeruginosa, using agar well diffusion method. Zones of inhibition for S. costus-derived PdNPs and the free extract (at 100 µg mL⁻¹) were 22 ± 0.5 mm and 16 ± 0.1 mm (S. aureus), 23 ± 0.1 mm and 18 ± 0.0 mm (B. subtilis), 22 ± 0.3 mm and 17 ± 0.0 mm (E. coli), and 19 ± 0.4 mm and 15 ± 0.0 mm (P. aeruginosa), respectively. Furthermore, the antioxidant effect of S. costus-mediated PdNPs, assessed by DPPH and ABTS, was superior to the free extract, showing IC₅₀ values of 90 µg mL⁻¹. This could be attributed to the high phenolic and flavonoid content in the S. costus and the increased surface area of the PdNPs, promoting efficient electron donation to neutralize free radicals. Furthermore, inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) implicated in Alzheimer's disease was also assessed. At 400 mg mL⁻¹, the inhibition rates were 79.23 ± 1.11% for AChE and 76.13 ± 0.43% for BChE, indicating the ability of S. costus-PdNPs to modulate cholinergic activity. The anti-inflammatory potential was evaluated using the Griess assay where PdNPs reduced nitric oxide production by 7.84% in LPS-stimulated murine macrophage (RAW 264.7) cells. Finally, anticancer activity against different cell lines HCT-116 (colon), HepG2 (liver), and MCF-7 (breast) was assessed using MTT assay. S. costus-PdNPs showed selective cytotoxicity towards tumor cells due to ROS production and apoptosis induction. The MTT assay on S. costus-PdNPs revealed IC₅₀ values of 7.8 µg mL⁻¹ (HCT-116), 11.8 µg mL⁻¹ (HepG2), and 26.7 µg mL⁻¹ (MCF-7), which are much lower than IC₅₀ values of the free S. costus extracts (82.5–114.6 µg mL⁻¹). S. costus-PdNPs showed enhanced biomedical activities compared to free extract.⁹⁰

7.2 Nanoformulations of S. costus extract

S. costus extract were incorporated into nanocarriers, such as polymeric, lipid-based nanoparticles, and inorganic nanocarriers as well as electrospun nanofibers to improve its therapeutic efficacy by enhancing bioavailability and increasing its overall stability. The following subsection covers different nanoformulations encapsulating S. costus extract and investigates their biological activities (Table 3).

Table 3 Characteristics and biological activities of S. costus polymeric nanosystems^a

Nanosystem	S. costus part/extract type	Characteristics	Reported bioactivity	Ref.
a Abbreviations: AchE: acetylcholinesterase; BAX: Bcl-2-associated X protein; BChE: butyrylcholinesterase; C3: complement component 3; C4: complement component 4; CS: chitosan; DLS: dynamic light scattering; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FCS: fungal chitosan; GM-CSF: granulocyte-macrophage colony-stimulating factor; HSF cells: human skin fibroblast cells; IFN-γ: interferon gamma; IL-1β: interleukin-1 beta; IL-6: interleukin-6; Ki-67: proliferation marker protein; MMP-3: matrix metalloproteinase-3; MMP-9: matrix metalloproteinase-9; MIC: minimum inhibitory concentration; PCL–GL: polycaprolactone–gelatin; PDI: polydispersity index; PMMA: poly(methyl methacrylate); SEM: scanning electron microscopy; TEM: transmission electron microscopy; TLC: total leukocyte count; TNF-α: tumor necrosis factor-alpha.
FCS	Root ethanolic (70%) crude extract	- Size: 48 nm (TEM, spherical)	- S. costus -loaded FCS outperformed the antimycotic activity of both free S. costus and FCS	89
		- Zeta potential: +3.28 mV (high stability)	- S. costus -loaded FCS showed the highest zone of inhibition (13.8 mm for standard C. albicans) and lowest MIC (1.75 mg mL^−1)
		- EE%: NR	- SEM imaging of treated Candida confirmed cell wall lysis after 6–12 h of exposure
CS	Root ethanolic (70%) crude extract	- Size: 924 ± 112.6 nm	- Immunostimulatory activity in dexamethasone-induced immunosuppressed rats	91
		Morphology: spherical (TEM)	- Significant increase in TLC, C3, C4, IFN-γ
		- Zeta potential: +39.4 ± 7.04 mV	- Decrease in TNF-α
		- EE%: 50.09 (direct method)	- Ki-67 upregulation
			- Normal liver and kidney function
			-No cytotoxicity up to 5 mg mL^−1
PMMA	Root essential oils	- DLS: size: 145 nm, PDI: 0.18, zeta potential: +45 mV	- Non-cytotoxic at ≤25 µg mL^−1 (A549 cells)	94
		- Spherical morphology (TEM)	- Anti-inflammatory effect: reduced TNF-α, GM-CSF, IL-1β and MMP-9 levels
		- EE%: NR	- Antidiabetic effect: IC50 for α-amylase was 22.9 µg mL^−1 and for α-glucosidase 75.8 µg mL^−1
			- Cholinergic modulation: IC50 for AchE and BChE were 25.0 and 14.9 µg mL^−1, respectively
Eudragit® RS100	Root ethanolic crude extract	- DLS: size (119.7 nm), PDI (0.182)	- Significant anti-inflammatory effect in a P. gingivalis periodontitis rat model	95
		- EE%: NR	- Day-2 changes were not significant
			- NF-κB and TNF-α reduced significantly by day 5
Eudragit® RS100	Root ethanolic crude extract	- Hydrodynamic size: 119.7 nm	- Anticancer & antiproliferative activity	109
		- EE%: NR	- Inhibition of HeLa (cervical cancer) showed a dose-dependent with 50–60% viability with IC50 of 81.80 µg mL^−1
			- Proliferation of SHED (human dental pulp stem cells) showed a significant decrease at 12.5 µg mL^−1 across 24/48/72 h
PCL-GL nanofibers	Root ethanolic (100%) crude extract	- Diameter: 253 nm (SEM)	- High antimicrobial activity (log reduction 7)	96
		- Water contact angle: 54.6° (hydrophilic)	- DPPH scavenging of 86.4%
		- Controlled release: 45.99% release over 7 days	- Biocompatible with HSF cells
		- EE%: NR	- In vivo enhanced wound closure in male albino rats by 82.2%
			- Significant collagen deposition, downregulation of IL-6, and upregulation of BAX and MMP-3
Bilosome	Costunolide	- DLS (size of 119.7 ± 3.63 nm)	- Costunolide-loaded bilosomes showed greater anticancer potency than free costunolide in LS174T cells and maintained a more favorable profile relative to the blank carrier in normal colonic cells, supporting improved selectivity	97
		- EE%: 63.4 ± 3.59%	- Nanoformulation drove apoptosis (sub-G1 accumulation) and produced a pro-apoptotic gene expression. It also triggered oxidative stress and mitochondrial pathway activation
Silica coated with methacrylic acid	Costunolide	- Pore size: 10 nm (TEM); – drug loading: 30.5 ± 2.45% (HPLC)	- Blank nanoparticles were biocompatible (LX-2 cells)	98
		- Acid shielding at pH 1.0, rapid release at pH 6.8	- Loaded + coated particles suppressed TGF-β1, MMP2, α-SMA, COL1A1 more than free drug in water
		- XRD/DSC confirmed the crystallinity of costunolide	- In vivo model showed that at ½ the dose, loaded + coated particles reduced necrosis and collagen, and improved ALT/AST more than free drug
		- EE%: NR

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7.2.1 Polymer-based nanoparticles. Alshubaily incorporated S. costus root ethanolic extract into fungal-derived chitosan (FCS) and tested their antifungal and antimicrobial activities.⁸⁹ FCS was isolated from Aspergillus niger mycelia using alkaline deacetylation and acidic solubilization methods. S. costus-loaded FCS was prepared via ionic gelation using sodium tripolyphosphate as a cross-linker. TEM imaging showed spherical morphology and a particle size of 48 nm. DLS analysis revealed a zeta potential of +3.28 mV, indicating good colloidal stability. FTIR analysis confirmed strong interactions between S. costus and FCS. To evaluate their antimycotic activity, disc diffusion and MIC assays were utilized against standard and drug-resistant Candida albicans and Candida glabrata. S. costus-loaded FCS exerted the highest antifungal activity against all strains in comparison to free S. costus and FCS. The zones of inhibition of S. costus-loaded FCS against all fungal strains ranged from 13.8 ± 0.7–11.1 ± 1.0 mm. Furthermore, S. costus-loaded FCS showed an MIC range of 1.75–3 mg mL⁻¹, lower than both free FCS and S. costus. Morphological changes in the treated yeast cells with their respective MICs were assessed using SEM imaging. Candida cells began showing membrane swelling and deformation after 6 h of treatment. By 12 h, most cells exhibited severe membrane rupture, loss of structural integrity, and lysis.⁸⁹

Milad et al. examined the immunological and anti-inflammatory activities of S. costus-loaded chitosan nanoparticles (S. costus-CS NPs) in dexamethasone immunosuppressed male rats.⁹¹ The study compared the immunomodulatory effects of S. costus, Moringa oleifera, Ziziphus spina-christi, and their CS nano-conjugates. Free CS nanoparticles exhibited small size (46.21 ± 28.75 nm), compared to S. costus-loaded CS (924 ± 112.6 nm), while maintaining a favorable positive zeta potential (+39.4 ± 7.04 mV). FTIR analysis revealed functional group interactions between S. costus and CS, with characteristic shifts in amide, carboxyl, and aromatic bands, confirming their successful conjugation. EE% for the S. costus-CS NPs system was determined by a direct method where the loaded nanoparticles were precipitated at pH 9, centrifuged, the pellet redissolved in 1% acetic acid, and EE% was calculated to be 50.09%. This value was comparable to that of Ziziphus-CS NPs (49.98%) and only slightly lower than that of Moringa-CS NPs (66.14%), demonstrating that S. costus can be efficiently incorporated into the chitosan nanocarrier. In vitro cytotoxicity of S. costus-CS NPs was evaluated using the water-soluble tetrazolium (WST-1) viability assay. S. costus-CS NPs exhibited no cytotoxic effects on human lung fibroblast (WI-38) and human embryonic kidney (HEK-293) cell lines at the tested concentration (100 µg mL⁻¹), similar to Moringa-CS and Ziziphus-CS formulations. Furthermore, S. costus-CS NPs were administered orally in dexamethasone-induced immunosuppressed male rats for 28 days. The S. costus-CS NPs-treated group showed elevation of total leukocyte count (at day 14 and day 28) and complement protein C3 compared to immunosuppressed rats, with a higher increase in C3 than that in Ziziphus-CS NPs, reflecting a robust activation of the complement cascade. In general, plant extract-CS conjugates induced higher TLC and C3 responses than the corresponding free extracts; accordingly, S. costus-CS NPs produced a more pronounced enhancement of leukocyte counts and C3 levels than free S. costus extract, particularly at earlier time points, indicating that nanoencapsulation amplifies the immunostimulatory effect of the traditional extract. C4 also increased at day 14 in the S. costus-CS group, indicating an early complement response, whereas the free S. costus group did not show a sustained time-dependent rise in C3. Importantly, tumor necrosis factor-alpha (TNF-α) was markedly suppressed in S. costus-CS NPs-treated rats. Both free S. costus extract and S. costus-CS NPs reduced TNF-α relative to the immunosuppressed group, but the nanoformulation achieved the most sustained and profound effect. S. costus-CS NPs were the only conjugate that significantly reduced TNF-α levels below those of the control group across the entire experimental period and displayed the lowest TNF-α values among all plant-CS nanoformulations, underlining the particularly strong anti-inflammatory potential of the nanoformulated S. costus extract compared with its free counterpart. In parallel, histopathological evaluations of the spleen demonstrated a clear reversal of dexamethasone-induced white pulp atrophy and restoration of normal follicular architecture in animals treated with S. costus-CS NPs, supported by a pronounced elevation in Ki-67 expression. Notably, the highest Ki-67 area percentage was recorded in both the S. costus extract and S. costus-CS NPs groups, indicating that nanoencapsulation preserves the intrinsic lymphoproliferative capacity of the traditional extract while enhancing its systemic immunomodulatory and anti-inflammatory profile relative to free S. costus. Liver (ALT, AST) and kidney (urea, creatinine) function markers remained within normal ranges, indicating no toxicity. Also, histology of liver and kidney of S. costus-CS NPs-treated animals showed normal hepatocyte architecture and no signs of glomerular or tubular damage. Thus, within this comparative study, the S. costus-CS nanoformula were superior not only against other plant-based chitosan nanoformulations but also relative to free S. costus extract, offering stronger and faster immunomodulatory effects with a comparable safety profile.⁹¹

Anti-inflammatory, anti-diabetic, and anti-Alzheimer activities of S. costus essential oil were evaluated after incorporation in polymethyl methacrylate (PMMA) nanocapsules (Fig. 7).⁹⁴ S. costus essential oil was extracted by supercritical CO₂ extraction and was rich in dehydrocostus lactone (55.39%) and costunolide (8.87%). Nanoprecipitation was used to prepare S. costus-PMMA nanocapsules with a mean size of 145 ± 1 nm, PDI of 0.18 ± 0.01, and zeta potential of +45 ± 2 mV. TEM images indicated spherical morphology, while FTIR confirmed successful encapsulation of the essential oil. Cytotoxicity was assessed on human lung epithelial (A549) cells using the LDH assay. S. costus-PMMA nanocapsules were non-cytotoxic at 12.5–25 µg mL⁻¹, similar to free S. costus essential oil, although the encapsulated form became more cytotoxic at higher concentrations (≥50 µg mL⁻¹), consistent with increased cellular uptake and bioavailability upon nanoencapsulation. Anti-inflammatory effects were investigated in LPS-stimulated A549 cells using qRT-PCR and gelatin zymography. S. costus-PMMA nanocapsules significantly reduced the expression of TNF-α, IL-1β and GM-CSF and suppressed MMP-9 activity, whereas blank PMMA nanoparticles showed no such effect, confirming that the anti-inflammatory activity arised from the S. costus oil and is potentiated by its nano-encapsulation.

Fig. 7 Therapeutic activities of S. lappa-loaded PMMA nanoparticles. The nanoformulation exhibited anti-diabetic activity by inhibiting α-amylase and α-glucosidase enzymes, thereby reducing glucose release. It also demonstrated cholinergic modulation activities, and consequently could delay progression of Alzheimer's disease, by inhibiting AChE and BChE. Additionally, it showed anti-inflammatory action by downregulating LPS-induced expression of pro-inflammatory cytokines. Reproduced with permission.⁹⁴ Elsevier, 2021.

Cholinesterase inhibition assays revealed that encapsulation markedly enhanced the anti-Alzheimer potential of S. costus. The IC₅₀ values against AChE (Acetylcholinesterase) decreased from 67.4 ± 3.7 µg mL⁻¹ for free oil to 25.0 ± 2.0 µg mL⁻¹ for S. costus-PMMA, and against BChE (butyrylcholinesterase) from 17.4 ± 1.1 µg mL⁻¹ to 14.9 ± 1.9 µg mL⁻¹, while both nanoformulated and free oil compared favorably with galantamine for BChE inhibition. This improvement is attributed to the α-methylene-γ-butyrolactone moiety of sesquiterpene lactones, which interacts with sulfhydryl groups on cholinesterases.

Inhibition of carbohydrate-digesting enzymes (α-amylase and α-glucosidase) was also evaluated to assess antidiabetic activity. For α-amylase, nanoencapsulation substantially increased potency, with IC₅₀ decreasing from 65.9 ± 1.7 µg mL⁻¹ (free S. costus oil) to 22.9 ± 0.3 µg mL⁻¹ (S. costus-PMMA), both outperforming acarbose (365.3 ± 10.7 µg mL⁻¹). For α-glucosidase, the free oil remained more potent (IC₅₀ 42.1 ± 3.0 µg mL⁻¹) than the encapsulated form (75.8 ± 4.4 µg mL⁻¹), an effect attributed to the slower release of the oil from the PMMA matrix. Nevertheless, both free and nanoencapsulated S. costus outperformed acarbose (271.7 ± 7.1 µg mL⁻¹).

Thus, encapsulation of S. costus essential oil in PMMA enhanced its solubility, stability, cellular uptake and sustained release, resulting in stronger anti-inflammatory, anti-cholinesterase and α-amylase inhibitory activities than the traditional free extract, while maintaining superior safety at non-cytotoxic concentrations.⁹⁴

Moelyanto et al. evaluated whether S. costus-loaded Eudragit® RS100 polymeric nanoparticles can attenuate Porphyromonas gingivalis-induced periodontal inflammation by quantifying changes in NF-κB and TNF-α at 2 and 5 days post-treatment, addressing the need for bioavailable, plant-based anti-inflammatories in periodontitis.⁹⁵ For this purpose, the nanoparticles were prepared using the nanoprecipitation method, having a mean size of 119.7 nm with PDI of 0.182. For biological testing (in vivo), male Wistar rats received P. gingivalis injections into the mandibular gingival sulcus every 3 days for 14 days until periodontitis was evident. Treated groups were dosed with the prepared S. costus-loaded nanoparticles once daily. The inflammatory signaling in alveolar bone was evaluated for NF-κB p65 (transcription factor that drives the expression of pro-inflammatory cytokines) and TNF-α (promotes inflammation, osteoclast activity, and alveolar bone resorption). Results have shown a significant reduction in the expression of both NF-κB (p = 0.001) and TNF-α (p = 0.002) by day 5 in the alveolar bone of the lower, indicating effective suppression of the periodontal inflammatory cascade. Furthermore, rats exhibited gingival redness, edema, recession, and bleeding on probing, and radiographs showed decreased bone density with radiolucent areas around the mandibular central incisors. Histology (H&E) demonstrated chronic inflammatory infiltrates composed of lymphocytes, plasma cells, and macrophages in the alveolar bone, together confirming active periodontitis prior to treatment.⁹⁵

7.2.2 Electrospun nanofibers. Lababidi et al. synthesized polycaprolactone–gelatin (PCL–GL) nanofibers loaded with 6% ethanolic extract of S. costus (S. costus -loaded PCL–GL) and assessed their antimicrobial, antioxidant, anti-inflammatory, and wound healing potency (Fig. 8).⁹⁶ Incorporation of the S. costus extract increased the nanofiber diameter to 253.07 ± 44.54 nm (SEM). Fast Fourier transform (FFT) confirmed the alignment of S. costus -loaded PCL–GL. FTIR analysis indicated bonding interactions, XRD confirmed crystallinity of PCL, and thermal stability was evaluated by TGA. Water contact angle was 54.6° ± 2.03, indicating enhanced hydrophilicity upon incorporation of S. costus into the nanofibers. The in vitro release profile of S. costus from nanofibers demonstrated a controlled, triphasic release behavior over 7 days. An initial burst release occurred during the first 12 hours, attributed to extract located near or on the fiber surface, with release value of 36.72%. This was followed by a slower, controlled diffusion phase, likely associated with gradual polymer swelling and limited matrix degradation, with cumulative release after 72 hours reaching 43.33 by day 7, the final bulk erosion phase was observed, achieving cumulative release value of 45.99%. Release kinetic modeling showed that the data best fitted the Higuchi model, indicating diffusion-controlled release through the polymer matrix. The addition of hydrophilic GL facilitated swelling and pore formation, enhancing extract diffusion compared to PCL alone. The Korsmeyer–Peppas release exponent further confirmed Fickian diffusion as the predominant release mechanism.⁹⁶ Antimicrobial activity of S. costus -loaded PCL–GL against P. aeruginosa, S. aureus, and E. coli achieved a log reduction of 7 (99.999% inhibition) in all strains. Antioxidant activity (DPPH assay) was 80.85%. MTT assay demonstrated biocompatibility with no significant cytotoxic effects on human skin fibroblasts. In vivo studies using male albino rats revealed that S. costus -loaded PCL–GL-treated wounds induced significant closure of the wound (82.22 ± 4.96%) vs. untreated wounds (68.88 ± 7.37%). Additionally, total leukocyte count and inflammatory markers were downregulated, while pro-apoptotic and angiogenic genes were upregulated. Histological and immunohistochemical analysis confirmed thick epithelial regeneration, dense collagen deposition, moderate VEGF and TGF-β1 expression, and high BAX levels in treated wounds. The findings suggest that S. costus -loaded PCL–GL nanofibers represent a safe intervention to accelerate wound healing.⁹⁶

Fig. 8 Evaluation of S. costus-loaded PCL–GL nanofibers for wound healing applications. Nanofibers were characterized using SEM, FTIR, XRD, and TGA to confirm successful nanofiber formation and incorporation of extract phytochemicals. In vitro studies demonstrated strong antioxidant (DPPH scavenging) and antimicrobial activities and non-cytotoxic properties. In vivo studies showed significantly enhanced rat wound closure and re-epithelialization compared to the untreated control.⁹⁶ Copyright license CC BY 3.0.

7.2.3 Lipid-based nanoparticles. Alamoudi et al. encapsulated costunolide (a major sesquiterpene lactone occurring predominantly in S. costus) into bilosome nanoparticles to overcome its poor solubility and permeability and thereby boost its anti-colon-cancer activity against LS174T colorectal adenocarcinoma cells.⁹⁷ Costunolide-loaded bilosomes were prepared by thin film hydration using cholesterol, Span-85 and bile salt. Higher cholesterol : Span ratios and bile salt levels increased EE%, with the optimum condition (cholesterol : Span 1 : 5 and bile salt 0.5 mM) yielding a vesicle size of 119.7 ± 3.63 nm and an entrapment efficiency of 63.4 ± 3.59%, indicating efficient drug loading in a nanoscale carrier. In vitro cytotoxicity quantified by the sulforhodamine B assay (48 h) showed that costunolide-loaded bilosomes (IC₅₀ = 6.20 µM) were more than twice as potent as free costunolide (IC₅₀ = 15.78 µM) against LS174T cells, while oxaliplatin (positive control) had an IC₅₀ of 0.70 µM and the blank bilosomes were much less toxic (IC₅₀ = 50.08 µM). Selectivity was further supported in normal human colonic epithelial cells (HCoEp), where IC₅₀ values were 65.11 µM for blank bilosomes, 55.75 µM for free costunolide, and 25.84 µM for costunolide-loaded bilosomes, indicating that nanoencapsulation preferentially enhances cytotoxicity in cancer cells while maintaining an acceptable safety margin in normal cells. Cell-cycle analysis revealed that both free costunolide and loaded-bilosomes increased the sub-G1 population, consistent with apoptotic DNA fragmentation, but the nanoformulation caused a more pronounced sub-G1 accumulation. Annexin-V/PI staining confirmed a stronger pro-apoptotic effect for costunolide-loaded bilosomes, with a 208% increase in late apoptosis vs. control and higher necrosis (+144.7% vs. control and +42.6% vs. free costunolide), showing that the nanoformulation drives programmed cell death more effectively than the free compound. Costunolide-loaded bilosomes induced greater up-regulation of CASP3, BAX and TP53 and more pronounced down-regulation of BCL2 compared with free costunolide, accompanied by a 173.2% increase in ROS vs. control (97.0% vs. free costunolide), loss of mitochondrial membrane potential and cytochrome-c release, indicating more efficient activation of the intrinsic mitochondrial apoptotic pathway. Therefore, bilosomal encapsulation of this S. lappa-derived component significantly enhanced its cytotoxic, pro-apoptotic and mitochondrial ROS-mediated effects relative to traditional (non-encapsulated) costunolide, while preserving selectivity toward malignant over normal colonic epithelial cells.⁹⁷

7.2.4 Inorganic nanoparticles. Niu et al. enhanced the aqueous solubility and gastric instability of costunolide, a key S. costus sesquiterpene lactone, by loading it into pH-responsive mesoporous silica nanoparticles coated with a methacrylic acid copolymer (Eudragit/MAC) to improve its dissolution in intestinal fluid and anti-liver-fibrosis efficacy.⁹⁸ The nanoparticles were synthesized using cetyltrimethylammonium bromide (CTAB) as a template, then costunolide was adsorptively loaded into the mesopores and finally MAC-coated. TEM imaging showed 10 nm channels in empty mesoporous silica nanoparticles that became less distinct after loading and coating, and N₂ sorption revealed marked decreases in surface area and pore volume, together confirming pore filling and encapsulation of costunolide. HPLC quantified drug loading at 30.5 ± 2.45%. The release of costunolide was evaluated in simulated stomach (pH 1.0) versus intestinal (pH 6.8) fluid. In acidic medium (pH 1.0), pure costunolide was highly unstable, with its remaining content dropping to 1.68% ± 0.47% within 2 h. In contrast, the coated nanoparticles released only a small initial surface fraction detectable within the first 10 min, after which costunolide became hardly detectable beyond 30 min, indicating that the MAC coating effectively acted as an acid shield and prevented further drug release and degradation. Under intestinal conditions (pH 6.8), both loaded and coated + loaded nanoparticles showed rapid and almost complete release within 45 min, whereas only 12.8% ± 2.39% of pure costunolide dissolved over the same period. Thus, MAC-coating protected costunolide in the stomach and then enabled fast release in the intestine, improving both its stability and dissolution profile for absorption.⁹⁸ In human hepatic stellate cells (LX-2), blank nanoparticles were biocompatible, and at an equal nominal dose, loaded + coated nanoparticles suppressed fibrogenic proteins (TGF-β1, MMP2, α-SMA, COL1A1) more strongly than free costunolide dispersed in water, indicating that improved dispersion and release translate into a stronger cellular antifibrotic response. Crucially, this superiority was confirmed in vivo in a bile duct ligation rat model. Loaded nanoparticles administered at half the costunolide dose produced greater antifibrotic benefit than free costunolide, with lower necrosis scores, smaller Sirius Red-positive collagen areas, more pronounced reductions in hepatic TGF-β1, MMP2 and α-SMA, and significant improvements in serum ALT/AST, whereas free costunolide produced weaker or limited changes. Therefore, these findings provide a clear comparison showing that, compared with traditional (non-encapsulated) costunolide, the nanoformulated S. costus constituent exhibits superior gastric stability, intestinal dissolution, antifibrotic potency and dose-sparing efficacy in preclinical models.⁹⁸

8. Discussion

The studies summarized in this review collectively demonstrate that nanoformulations enhance the therapeutic potential of S. costus extracts. Across different platforms, nanoencapsulation was shown to improve solubility, protect labile sesquiterpene lactones and essential oils from degradation, and increase cellular uptake, often leading to stronger biological effects than those observed with the corresponding free extracts or pure compounds.^89–98 Furthermore, these nanoformulations achieved comparable or superior activity to conventional therapies, positioning S. costus as a promising candidate for plant-based nanotherapeutics rather than simply a traditional herbal remedy.^90–99,101 Chitosan-based systems and polymeric nanocapsules amplify immunomodulatory, anti-inflammatory, antidiabetic and anti-Alzheimer activities, and in some cases reduce the effective dose required to achieve a therapeutic response.^89,91,94 For example, chitosan nanoparticles and PMMA nanocapsules improved complement activation, TNF-α suppression, cholinesterase inhibition and α-amylase inhibition relative to free extract or essential oil, while maintaining acceptable biocompatibility in the tested models.^91,94 Similarly, lipid-based bilosomes and pH-responsive inorganic carriers such as mesoporous silica coated with methacrylic copolymers illustrate how rational design can overcome the poor solubility and gastric instability of costunolide, resulting in enhanced antifibrotic or anticancer activity and dose-sparing effects in vivo.^97,98 These trends support that nanocarriers can be tailored to address specific biopharmaceutical limitations of S. costus and its constituents, thereby unlocking pharmacological effects that are not fully accessible with traditional formulations.^91,94,97,98

Topical and localized delivery systems represent another promising area for S. costus nanoformulations. Silver nanoparticles embedded in chitosan hydrogels, as well as S. costus-loaded PCL–GL electrospun nanofibers, have shown robust antibacterial, antioxidant and wound-healing properties in vitro and in vivo.^96,99 These platforms combine the intrinsic antimicrobial and anti-inflammatory properties of S. costus with the structural benefits of hydrogels or fibrous scaffolds, such as moisture retention, controlled release and physical support for tissue regeneration.^96,99 The demonstration of accelerated wound closure and improved collagen deposition suggests that S. costus nanomaterials could be used for cutaneous and mucosal indications where localized high concentrations (while minimizing systemic exposure) are desired.^96,99

Metal and metal oxide nanoparticles synthesized using S. costus extracts (Ag, Cu, ZnO, MgO, Pd, IONPs, CuO) also show impressive antimicrobial and, in some cases, anticancer activities, reflecting synergistic effects between the metallic core and phytochemical capping layers.^{90,92,93,99–104,107,108} These systems have very high reactivity and strong ROS-generating capacity underlining the need for careful balance between therapeutic benefit and potential toxicity, especially for systemic applications.^{92,93,98,100,106–108,110–112} Polymeric and lipid/inorganic carriers appear particularly suitable for systemic delivery and for overcoming solubility and stability barriers,^{89,91,94,95,97,98} whereas hydrogels and nanofibers are more appropriate for topical wound management,^96,99 and metal/metal oxide nanoparticles may be most useful when high local antimicrobial activity is required.^{90,92,93,99–104,107,108} This review aims to highlight the use of nanoformulations to maximize the value of S. costus as a medicinal plant and to identify biomedical applications where further preclinical and translational work are needed.

9. Challenges and limitations

Despite the reported biomedical application of S. costus nanoformulations, several challenges still need to be addressed. As for all plant extracts, differences in geographical origin and cultivation conditions cause batch variability in phytochemical composition of S. costus extract. In addition to yield percent variability and low stability of the extract, different extraction methods could also yield variable sets of phytochemicals. The reported chemical components in S. costus extracts fluctuate dramatically as reported by different studies, making it difficult to establish a reproducible therapeutic profile across different batches.⁷⁴

EE% values were generally in the range of 50–65%, a pattern that is consistent with the pronounced hydrophobicity of major S. costus constituents, such as sesquiterpene lactones and essential oils.^89,91,98 However, several studies did not report encapsulation parameters, EE% and loading capacity, making it difficult to assess incorporation efficiency or compare systems quantitatively. The key variables that are known to influence EE%, including carrier composition, medium pH, solvent removal conditions and nanoparticle size, were rarely explored.¹¹³ Lack of these parameters limits cross-study comparability to optimize S. costus-loaded nanoformulations for future therapeutic applications.

Although nanoencapsulation of S. costus compounds could protect them from light and thermal degradation, long-term stability studies are still needed.⁹² To date, most published work on S. costus-based nanoformulations consists of in vitro experiments or short-term in vivo studies. For the majority of nanosystems, available data are limited to physicochemical characterization and in vitro cytotoxicity/efficacy assays, with no pharmacokinetic analysis and only very limited, non-comprehensive in vivo toxicity profiling. There is, therefore, a clear need for long-term animal studies and clinical validation to define the therapeutic efficacy, pharmacodynamics and pharmacokinetics of nanoformulated S. costus extracts in humans. In addition, metal-based S. costus nanosystems raise specific safety and cytotoxicity concerns that are still insufficiently addressed.¹¹⁰ Most studies on silver, zinc oxide and other metal nanoparticles synthesized using S. costus extracts focused on antimicrobial or anticancer efficacy, while providing little or no information on systemic toxicity, hemocompatibility, genotoxicity, biodistribution, or elimination pathways.^92,93,99,106 Metal nanoparticles can generate reactive oxygen species, induce oxidative stress, interact with cellular membranes and proteins, and potentially accumulate in clearance organs such as the liver, spleen, and kidneys, particularly at higher doses or upon repeated administration.^111,112 In vitro work has indicated dose-dependent cytotoxicity and intracellular accumulation in mammalian cells for some S. costus-based formulations, but comprehensive dose–response and long-term toxicity studies are lacking.^92,93,99,106 Consequently, careful toxicological evaluation and dose optimization are required before metal-based S. costus nanosystems can be considered for translational or clinical use.^92,114 The available studies evaluating S. costus-based nanoformulations are still limited, and future research should prioritize systematic comparative studies in which S. costus nanoformulations are assessed alongside other medicinal plants. Such comparative work will be essential to clarify whether S. costus provides consistent advantages over other botanicals in terms of efficacy, safety and translational potential. Finally, large-scale production of these nanoformulations should be explored to address technical challenges, develop relevant quality control procedures, and meet regulatory guidelines.

The studies summarized in this review collectively demonstrate that nanoformulations enhance the therapeutic potential of S. costus extracts. Across different platforms, nanoencapsulation was shown to improve solubility, protect labile sesquiterpene lactones and essential oils from degradation, and increase cellular uptake, often leading to stronger biological effects than those observed with the corresponding free extracts or pure compounds.^89–98 Furthermore, these nanoformulations achieved comparable or superior activity to conventional therapies, positioning S. costus as a promising candidate for plant-based nanotherapeutics rather than simply a traditional herbal remedy.^90–99,101 Chitosan-based systems and polymeric nanocapsules amplify immunomodulatory, anti-inflammatory, antidiabetic and anti-Alzheimer activities, and in some cases reduce the effective dose required to achieve a therapeutic response.^89,91,94 For example, chitosan nanoparticles and PMMA nanocapsules improved complement activation, TNF-α suppression, cholinesterase inhibition and α-amylase inhibition relative to free extract or essential oil, while maintaining acceptable biocompatibility in the tested models.^91,94 Similarly, lipid-based bilosomes and pH-responsive inorganic carriers such as mesoporous silica coated with methacrylic copolymers illustrate how rational design can overcome the poor solubility and gastric instability of costunolide, resulting in enhanced antifibrotic or anticancer activity and dose-sparing effects in vivo.^97,98 These trends support that nanocarriers can be tailored to address specific biopharmaceutical limitations of S. costus and its constituents, thereby unlocking pharmacological effects that are not fully accessible with traditional formulations.^91,94,97,98

Topical and localized delivery systems represent another promising area for S. costus nanoformulations. Silver nanoparticles embedded in chitosan hydrogels, as well as S. costus-loaded PCL–GL electrospun nanofibers, have shown robust antibacterial, antioxidant and wound-healing properties in vitro and in vivo.^96,99 These platforms combine the intrinsic antimicrobial and anti-inflammatory properties of S. costus with the structural benefits of hydrogels or fibrous scaffolds, such as moisture retention, controlled release and physical support for tissue regeneration.^96,99 The demonstration of accelerated wound closure and improved collagen deposition suggests that S. costus nanomaterials could be used for cutaneous and mucosal indications where localized high concentrations (while minimizing systemic exposure) are desired.^96,99

10. Conclusions

S. costus has been known for decades for its therapeutic uses in multiple cultures including traditional Ayurveda, Chinese, and Tibetan medicine. It has been used in treating a broad spectrum of medical conditions such as respiratory, digestive, gynecological, and infectious diseases. The remarkable biological activities of S costus are attributed to its rich content of pharmacologically active phytochemicals including sesquiterpene lactones, costunolide, flavonoids, phenolics, and essential oils. However, the full potential of this medicinal plant remains largely untapped in clinical settings due to its low water solubility and bioavailability, lack of targetability, and environmental instability. Incorporation of S. costus-derived compounds into metallic nanoparticles, polymeric, lipid-based, inorganic nanocarriers and electrospun nanofibers was introduced to overcome these limitations. These nanoformulations were reported to exert significant antimicrobial, anticancer, antioxidant, anti-inflammatory, and wound healing activities. Additional nanoformulations would help transform S. costus from traditional medicine into a modern, evidence-based therapeutic for diverse biomedical applications.

Conflicts of interest

The authors declare no potential competing interests.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Acknowledgements

The current work was funded by a grant from the American University in Cairo to Prof. Hassan Azzazy.

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