Cavitation-driven extraction: how ultrasound-induced acoustic cavitation maximizes bioactive compound recovery from S. costus roots

Abstract

Saussurea costus (Falc.) Lipsch., an endangered Indo-Himalayan medicinal plant, is highly valued for its roots enriched with polyphenols and sesquiterpenes of therapeutic relevance. In this study, ultrasonic-assisted extraction (UAE) was evaluated as a sustainable and efficient alternative to conventional techniques for recovering bioactive metabolites from S. costus roots. Process optimization was carried out using response surface methodology (Box–Behnken design) with aqueous ethanol (50:50, v/v) as the extraction medium. The cavitation-driven process significantly (p ≤ 0.05) improved recovery efficiency, yielding 29.98 ± 0.23 % extract compared to Soxhlet (26.53 %) and maceration (24.67 %). UAE extracts exhibited elevated levels of phenolics (66.27 ± 0.02 mg GAE/g) and flavonoids (73.33 ± 1.23 mg QE/100 g), alongside superior antioxidant capacity (52.29 ± 2.20 % DPPH inhibition; 197.56 ± 1.10 µmol TEAC/g FRAP). Mechanistic insights suggest that acoustic cavitation enhances mass transfer and disrupts cellular structures, thereby facilitating the release of both polar and non-polar phytoconstituents more effectively than thermal approaches. HPLC profiling further confirmed selective enrichment of key phenolics, including gallic acid, chlorogenic acid, and naringenin. Collectively, these findings establish UAE as a scalable, eco-friendly, and high-performance extraction strategy, underscoring its potential for producing S. costus extracts with significant nutraceutical and pharmaceutical applications.

1. Introduction

Indian costus (S. costus), kuth, or putchuk, is a slender, ever-lasting plant native to the Western Himalayan regions of Pakistan and India, thriving at altitudes between 2600 and 4000 m. Typically growing to a height of 1 to 2 m, the plant is characterized by its erect, sturdy, fibrous stem and distinctive aromatic roots, which can reach up to 60 cm in length. Among the Saussurea genus, S.costus is particularly notable for its commercial value and extensive use in traditional medicine systems, including Ayurvedic, Chinese, and Tibetan practices. It is listed as ingredient in around 175 recipes in The Handbook of Traditional Tibetan Drugs. [1]. The roots, rich in essential oils, alkaloids, and other bioactive constituents, are the most utilized part of the plant and are employed in treating various ailments such as stomach aches, headaches, typhoid, and colds [2].

S. costus is valued for its diverse medicinal properties, which include anti-inflammatory, antioxidant, anticancer, hepatoprotective, and immunomodulatory effects. Major phtochemicals present in the shrub include sesquiterpenes, flavonoids, and essential oils [3]. Essential oil extracted from its roots contains sesquiterpene lactones like costunolide and dehydrocostus lactone, which are known for their anti-cancerous activities [4]. These compounds have demonstrated efficacy against various microbial infections and possess anticarcinogenic potential, especially in breast cancer [5]. Studies have also reported that compounds like elemol and dehydrocostus lactone inhibit microbial growth, and cell degradation [6], while dehydrocostus lactone influences apoptosis pathways in human leukemia cells [7]. Early research on S.costus included the characterization of basic fractions and the isolation of compounds such as Saussurea lactone (C₁₅H₂₀O₂) [8], costunolide and other bioactive compounds including costic acid, palmitic acid, linoleic acid, β-sitosterol, β-cyclocostunolide, alantolactone, α-cyclocostunolide, and isoalantolactone [9]. Subsequent studies identified additional compounds such as isodehydrocostus lactone, isozaluzanin-C, and guaianolides, 12-methoxydihydrodehydrocostus lactone and 4′-methoxydehydrocostus lactone contributing to the rich chemical profile of this medicinal plant [10].

Phytochemical analysis have revealed that sesquiterpene lactones are the major bioactive compounds in S. costus, with significant pharmacological activities [11]. However, alongside lactones, polyphenols in S. costus also contribute to its therapeutic potential [12]. Polyphenols are well-known for their antioxidative, inflammation suppressing and chemo preventive abilities, thus their extraction and subsequent research is needed. While lactones are significant, the increasing recognition of polyphenols in the plant’s bioactivity has led to a growing interest in optimizing their extraction. Polyphenols, in particular, are often highlighted due to their stability, widespread health benefits, and potential for incorporation into various medicinal formulations. This shift in focus reflects a broader trend in herbal medicine research, where the extraction of polyphenols is prioritized to fully harness the plant's therapeutic potential. Traditional extraction methods like solvent, cold extraction, seeping, boiling, and soxhelation though cost-effective often yield lower extraction efficiencies [13]. The increasing interest in phytonutrients from botanical origin has prompted advancement for faster, more cost-effective extraction methods. Solvant choice is crucial as studies indicate that organic solvents are effective in extracting phytochemicals from natural sources [14]. Traditional extraction methods are time-intensive, solvent- and energy-demanding, and often cause degradation of phytonutrients [15]. In response, novel extraction techniques, such as ultrasonic-assisted extraction (UAE), offers a viable choice. UAE ensures high replicability within a limited time frame, while minimizing solvent and energy use and operating at reduced temperatures. Sonic assisted nucleation rupture cell wall, promoting mass transfer through shearing stress while preserving the structural integrity of target compounds [16]. Moreover, UAE allows for the use of more economical and environmentally friendly solvents [17].

Given the need for efficient and sustainable methods to valorize endangered medicinal plants, this study presents the first comprehensive optimization and application of Ultrasonic-Assisted Extraction (UAE) for recovering polyphenols and flavonoids from the roots of Saussurea costus. The research aims to: (1) compare the efficiency of UAE against conventional Soxhlet and maceration techniques; (2) evaluate the impact of different solvents on extraction yield and bioactivity; and (3) identify the optimal parameters for maximizing the recovery of bioactive compounds with enhanced antioxidant potential. By achieving these objectives, the research aims to establish advanced and sustainable separation techniques for use in modern nutraceutical and pharmaceutical applications.

2. Materials and methods

2.1. Botanical source and compound recovery

Healthy, undamaged rhizome of S. costus acquired from Gangbal area, situated at 34°24′8.6″ North and 74°58′38″ East in the Sindh Valley, at an approximate altitude of 4,000 m (Voucher No. 574, Dated: 13/11/2022), was authenticated by the Forest Research Institute of Jammu and Kashmir. The roots were meticulously washed, shade-dried, and then powdered finely using a Lab Hammer Mill. Preliminary trials were conducted using various solvent concentrations (100:0, 75:25, 50:50, 25:75 ratios of ethanol, methanol, and petroleum ether to distilled water) to determine the optimal extraction yield and Total Phenolic Content (TPC). As summarized in Supplementary Table S1, the 50:50 ethanol-to-water ratio yielded the highest extraction efficiency (28.5 %) and a high concentration of bioactive compounds, as indicated by its TPC (58.2 mg GAE/g). Consequently, this solvent concentration was selected for use in both conventional and ultrasonic-assisted extraction methods.

2.2. Soxhlet extraction

Soxhlet extraction was carried out following the protocol of López-Bascón et al. [18] with modifications. Briefly, 5 g of the dried, ground S. costus powder was placed in an extraction thimble and refluxed with 50 mL of different organic solvents (50 % ethanol, 50 % methanol, and 50 % petroleum ether), using a solid-to-solvent ratio of 1:10 (w/v). The extraction was continued for 6 cycles (approximately 6 h) until the solvent in the siphon tube became colorless, with heat maintained at 80 °C. After the extraction was complete, the thimble was removed, and the solvent was recovered. The extract was obtained by evaporating the solvent using a vacuum oven set at 35 °C.

2.3. Maceration

The maceration procedure was adapted from Romero-Cascales et al. [19]. Five grams of the dried, ground S. costus powder was macerated with 100 mL of solvent, using an initial solid-to-solvent ratio of 1:20 (w/v). The mixture was maintained at 25 ± 2°C for 48 h with occasional shaking. After this period, the supernatant was decanted. The residual solid (retentate) was subjected to a second maceration by adding 20 mL of fresh solvent. This process was repeated six times (20 mL × 6) until the plant material was fully exhausted. All the supernatants were combined and filtered through filter paper to obtain the final macerate.

2.4. Ultrasonic assisted extraction (UAE)

UAE was proceeded according to method described by Shen et al. [20]. One gram of S.costus root powder was dissolved in 25 mL of extraction solvent in a 250 mL Erlenmeyer flask. UAE was performed using an ultrasonic probe system (Indosati, Model INDO/AB/008). A 13 mm titanium probe was immersed in the mixture operating at 50 % amplitude for 30 min. To control the temperature, the Erlenmeyer flask was placed in a circulating water bath maintained at 25 °C. The pulse duration was set to alternate between “on” (5 s) and “off” (3 s) modes throughout the extraction process. To prevent overheating from the ultrasound, water at room temperature (25 °C) was circulated in the ultrasonic bath. The supernatant was processed similarly to the conventional extraction method to obtain dried ultrasonic-assisted extracts of S. costus. The dehydrated samples were then preserved tightly in sealed vessel at 4 °C.

2.5. Yield Percentage

The yield was calculated using the formula;

$$Y\left(\%\right)=100\frac{M_{\mathit{ext}}}{M_{\mathit{ech}}}$$ (1)

where Y is extraction recovery in %, M_ext = extract mass after evaporating the solvent and M_ech = mass of fresh sample. All measurements were performed in triplicate (n = 3).

2.6. Total phenolic compounds (TPC) (mg GAE/g)

The TPC of S. costus root extract fraction was carried out as per Bhat et al. [21] after slight adaptation. Sodium bicarbonate (Na₂Co₃) (7.5 g) (7.5 % w/v) solution was prepared in 100 ml of distilled water. Gallic acid stock was formulated by solubilizing 100 mg in 100 mL of methanol. The phenols were extracted from sample, using methanol or ethanol. Folin-Ciocalteu (5 mL) was incorporated to each tube followed by addition of sodium carbonate solution (4 ml). Following mixing, the solutions were kept undisturbed in dark at room temperature for 30 min. Afterwards, the optical density of each solution was measured spectrophotometrically at 765 nm. All measurements were performed in triplicate (n = 3).

2.7. Total flavonoid content (TFC) (mg QE/100 g)

TFC of the S. costus root extracts was determined following Saboon et al. [22]. A stock solution was prepared by dissolving a 100 mg of quercetin in 100 mL of methanol or water. Following preparation, the stock solution was diluted to formulate multiple standard solutions. S. costus samples were dissolved in methanol. To it, 0.3 mL of NaNO₂ solution (5 %) was added and allowed to rest for 5 min. Following rest, 0.3 mL of aluminum chloride solution (10 %) was added and further allowed to stand for 5 min. Afterwards, 2 mL of NaOH (1 M) was incorporated in each tube, followed by addition of 2.4 mL of distilled water. The solution was mixed vigorously well to ensure complete reaction and flavonoid content was measured spectrophotometrically against water as blank by recording the optical density at 510 nm using a UV–Vis spectrophotometer (model). All measurements were performed in triplicate (n = 3).

2.8. Radical inhibition activity using DPPH method

The method proposed by Bhat et al. [21] was followed for estimation of DPPH radical scavenging activity of the S. costus root extracts. All measurements were performed in triplicate (n = 3).

2.9. Ferric ion reducing anti-oxidant array (FRAP)

FRAP of the S. costus root extract was accessed following procedure of Bhat et al. [21]. All measurements were performed in triplicate (n = 3).

2.10. HPLC analysis of polyphenols

The phenolic profile of the S. costus root extracts was analyzed and quantified using High-Performance Liquid Chromatography (HPLC) following methodologies outlined by El-Nashar et al. [23] with modifications. The analysis was performed on a chromatographic system (Agilent 1260 series) equipped with a multi-wavelength detector.

Chromatographic separation was achieved using a Kromasil C18 column (4.6 mm × 250 mm, 5 µm particle size) maintained at 35 °C. The mobile phase consisted of HPLC-grade water (Solvent A) and acetonitrile containing 0.05 % trifluoroacetic acid (TFA) (Solvent B). The solvents were delivered at a flow rate of 1.0 mL/min under the following gradient program: 0 min, 82 % A and 18 % B; 0–5 min, 80 % A and 20 % B; 5–8 min, 60 % A and 40 % B; 8–12 min, 60 % A and 40 % B (isocratic); 12–15 min, 85 % A and 15 % B; and 15–16 min, 82 % A and 18 % B for column re-equilibration. The detection wavelength was set at 280 nm, and the injection volume was 10 µL.

The analysis utilized sixteen phenolic standards: gallic acid, chlorogenic acid, catechin, methyl gallate, caffeic acid, syringic acid, pyrocatechol, rutin, ellagic acid, coumaric acid, vanillin, ferulic acid, naringenin, taxifolin, cinnamic acid, and kaempferol. Standard solutions were prepared in ethanol at defined concentrations for calibration. Compounds in the samples were identified by comparing their retention times and UV–Vis spectral characteristics with those of the authentic standards. Quantification was performed using peak area integration against the standard calibration curves. Data acquisition and peak integration were performed using Agilent OpenLAB CDS ChemStation Edition, version C.01.07.

2.11. Statistical analysis & optimization

Statistical analysis was performed using [Software Name, e.g., SPSS Statistics, version 28.0]. A two-way analysis of variance (ANOVA) was conducted with extraction method and solvent type as independent factors. Post-hoc comparisons were performed using Tukey's Honest Significant Difference (HSD) test. A p-value ≤ 0.05 was considered statistically significant. All measurements were performed in triplicate (n = 3).

2.12. Process optimization using response surface methodology

Response surface methodology (RSM) coupled with four factors three level Box Behnken Design (BBD) was employed to investigate the individual and interactive effects of independent variables on dependent variables via Design- Expert 12 (State-Ease Inc., Minneapolis, MN, USA) statistical package. Multiple regression models were used to analyse the data, and analysis of variance (ANOVA) was employed to establish the statistical significance of each result. Second order polynomial mathematical models were developed to show the relationship between dependent and independent variables. Analysis of variance (ANOVA) investigated the adequacy of developed mathematical models which were used to plot the prediction contour graphs to study the interactive effect of independent variables on the responses. Optimum conditions were determined by BBD’s desired function methodology. The optimum condition criteria applied for numerical optimization was to maximize yield percentage, total phenolic compounds (TPC) (mg GAE/g), total flavonoid content (TFC) (mg QE/100 g). and anti-oxidants.

3. Results and discussion

3.1. Recovery rate (yield)

The extraction yield is a critical parameter for evaluating the efficiency of a process, as a higher yield often indicates a more effective release of cellular constituents, including target bioactive compounds. In this study, the recovery rates from S. costus roots using three techniques—maceration, Soxhlet, and Ultrasonic-Assisted Extraction (UAE)—with various aqueous-organic solvents (1:1 ratio) were significantly different (p ≤ 0.05) (Fig. 1).

Fig. 1.

Various solvent extraction yields of Saussurea costus using different methods of Extraction.

UAE consistently achieved the highest extraction yield across all solvent systems. For instance, with 50 % aqueous ethanol, UAE yielded 29.98 %, significantly outperforming Soxhlet extraction (26.53 %) and maceration (24.67 %). This superior performance is directly attributable to the mechanism of acoustic cavitation. The collapse of cavitation bubbles generated by high-intensity ultrasound creates micro-jets and intense shear forces that physically disrupt cell walls and enhance solvent penetration into the plant matrix [24]. This phenomenon leads to faster extraction kinetics and more efficient mass transfer compared to the passive diffusion in maceration or the thermal-driven continuous washing in Soxhlet extraction [25].

Furthermore, the choice of solvent significantly influenced the yield, following the order: 50 % ethanol > 50 % methanol > 50 % petroleum ether > distilled water. Aqueous ethanol (50 %), being a medium-polarity solvent, proved most effective. The presence of water increases the solvent's polarity compared to absolute ethanol, enabling a wider spectrum of action [26]. This allows for the co-extraction of a broad range of metabolites, from hydrophilic compounds (e.g., carbohydrates, proteins) to medium-polarity phytochemicals, thereby enhancing the total extract mass [27,28]. The results align with findings for other plant materials, where aqueous-organic mixtures facilitate the extraction of both hydrophilic and organophilic compounds [29].

While the high yield from UAE is advantageous, it is important to note that it represents the total mass of extracted material, which includes target polyphenols as well as other constituents like sugars and proteins. Future research could focus on coupling UAE with subsequent purification steps to isolate specific bioactive fractions. Nevertheless, the efficiency, reduced extraction time, and lower operational temperature of UAE make it a highly promising and scalable method for the valorization of S. costus.

3.2. Total phenolic content (TPC)

Total Phenolic Content (TPC) is a vital metric for assessing the antioxidant potential of plant extracts, as the diverse structures of phenolic compounds contribute to their ability to scavenge free radicals [21]. The extraction method and solvent system significantly (p ≤ 0.05) influenced the TPC of the S. costus root extracts (Fig. 2).

Fig. 2.

Total phenolic content of Saussurea costus root extract by various solvents and extraction methods.

Among the solvents, 50 % aqueous ethanol consistently yielded the highest TPC, a finding that underscores the importance of solvent polarity. Phenolic compounds often exist as glycosides or possess varying numbers of hydroxyl groups, making them more soluble in polar solvents. Aqueous ethanol, with its balanced polarity, effectively solvates a wide range of these compounds. The water fraction disrupts hydrogen bonding and polar interactions between phenolics and the plant matrix (e.g., cell walls, proteins), while ethanol solubilizes the less polar aglycones [30]. This explains the superior performance of 50 % ethanol over pure solvents and the significantly lower TPC obtained with the non-polar petroleum ether. This principle is consistent with findings in other plants, where a moderate water content in ethanol enhances the extraction of polar metabolites like phenolics [31].

More importantly, the extraction technique itself had a profound impact. Ultrasonic-Assisted Extraction (UAE) resulted in the highest TPC values (66.27 mg GAE/g with 50 % ethanol). The mechanism behind this enhancement is the cavitation-driven physical disruption of cell walls. This process creates finer particles and increases the surface area for mass transfer, facilitating a more complete and rapid release of intracellular phenolic compounds compared to the slower diffusion in maceration or the potentially degrading thermal stress of Soxhlet extraction.

While a high TPC is desirable, it is a collective measure and does not distinguish between individual phenolic compounds. Future work should involve correlating the TPC with specific biological activities and using advanced techniques like LC-MS to fully characterize the phenolic profile released by UAE. Nonetheless, the high TPC achieved with the optimized UAE process confirms its efficacy in recovering potent antioxidant fractions from S. costus.

3.3. Total flavonoid content (TFC)

Flavonoids are a major class of phenolic secondary metabolites renowned for their antioxidant, anti-inflammatory, and antimicrobial properties, which underpin their role in preventing chronic diseases [32]. The extraction of these valuable compounds from S. costus roots was significantly influenced (p ≤ 0.05) by both the solvent system and the extraction technique (Fig. 3).

Fig. 3.

Total Flavonoid content of Saussurea costus root extract by various solvents and extraction methods.

The data confirmed that 50 % aqueous ethanol was the most effective solvent for flavonoid extraction. Flavonoids, often occurring as glycosides, are inherently polar, and the balanced polarity of aqueous ethanol optimally solvates this diverse group. The water component facilitates the swelling of plant tissues and disrupts hydrogen bonds, enhancing the solvent's access to and solubility of flavonoids, a mechanism supported by findings in other medicinal plants [15,27].

Crucially, the extraction technique was a dominant factor. Ultrasonic-Assisted Extraction (UAE) yielded the highest TFC, significantly outperforming both Soxhlet extraction and maceration. This superior performance is a direct consequence of the cavitation mechanism. The implosion of ultrasonic cavitation bubbles generates intense shear forces and micro-jets that cause severe physical disruption of the cell walls. This not only creates a greater surface area for mass transfer but also mechanically facilitates the release of bound and intracellular flavonoids, leading to a more complete and rapid extraction than the slow diffusion of maceration or the potentially degrading thermal environment of Soxhlet. This aligns with the established efficacy of UAE for extracting sensitive phytochemicals [33,34].

While the TFC assay confirms the high concentration of total flavonoids, it is a collective measure that does not reflect the specific composition of the flavonoid profile. Future research should employ LC-MS/MS to identify and quantify individual flavonoid aglycones and glycosides present in the UAE extracts. Furthermore, the correlation between the enhanced TFC and specific bioactivities, such as neuroprotective or cardioprotective effects, warrants investigation in cellular or animal models to fully validate the nutraceutical potential of the optimized extracts.

3.4. Oxidative inhibition action

3.4.1. DPPH free radical scavenging capacity

The DPPH assay revealed that the antioxidant capacity of S. costus root extracts was significantly (p ≤ 0.05) influenced by both the extraction technique and the solvent used (Fig. 4). A clear hierarchy was observed, with Ultrasonic-Assisted Extraction (UAE) consistently yielding extracts with the highest radical scavenging activity, followed by Soxhlet and then maceration.

Fig. 4.

DPPH (%) of Saussurea costus root extract by various solvents and extraction methods.

The superior performance of UAE can be directly attributed to its cavitation-driven mechanism. The violent collapse of acoustic bubbles generates micro-jets and shear forces that not only enhance the release of antioxidants from the plant matrix but may also preserve their integrity by operating at lower temperatures than Soxhlet extraction [35]. This efficient, non-thermal process results in a higher concentration of intact antioxidant compounds in the extract, leading to enhanced free radical quenching.

Furthermore, the solvent polarity played a critical role. Aqueous ethanol (50 %) produced extracts with the highest DPPH scavenging capacity. This is because the antioxidant potential is largely derived from phenolic compounds, whose efficient extraction is maximized by the balanced polarity of aqueous ethanol. The water component facilitates hydrogen atom transfer—the key mechanism in neutralizing DPPH radicals—by disrupting the internal hydrogen-bonding network of the phenolics, making their reactive hydroxyl groups more accessible [36,37]. This explains why pure water was less effective, as it lacks the solvation power for the full spectrum of antioxidants, and why non-polar solvents like petroleum ether yielded extracts with lower activity.

While the DPPH assay is a valuable initial screening tool, it is a simplified model conducted in an organic medium, which may not fully represent antioxidant behavior in complex biological systems. Future work should validate these findings using cell-based antioxidant assays (e.g., CAA − Cellular Antioxidant Activity) and in vivo models to better predict the physiological relevance of the antioxidant effects. Nevertheless, the strong DPPH activity of the UAE extracts underscores their potential as potent sources of natural antioxidants.

3.4.2. Ferric reducing antioxidant power (FRAP) assay

The Ferric Reducing Antioxidant Power (FRAP) assay provides a direct measure of an extract's electron-donating capacity, a key mechanism of antioxidant action. The results demonstrated a significant (p ≤ 0.05) influence of both extraction technique and solvent on the reducing power of S. costus root extracts (Fig. 5).

Fig. 5.

FRAP (µmol TEAC/g) of Saussurea costus root extract by various solvents and extraction methods.

A striking finding was the marked superiority of Ultrasonic-Assisted Extraction (UAE), which yielded a FRAP value nearly double that of Soxhlet extraction and maceration when using 50 % aqueous ethanol. This profound enhancement is attributed to the synergy of two key mechanisms. Firstly, the cavitation process of UAE ensures a more efficient and complete extraction of a broad spectrum of reducing agents, including phenolic acids and flavonoids. More importantly, the low-temperature, rapid nature of UAE is critical for preserving the integrity of thermolabile antioxidants. Many potent reducing compounds can degrade or polymerize under the prolonged heat of Soxhlet extraction, diminishing their electron-donating capacity. UAE effectively bypasses this thermal degradation, resulting in an extract with a higher concentration of functionally active antioxidants.

Regarding solvent effects, 50 % aqueous ethanol again yielded the extract with the highest reducing power. The FRAP mechanism relies on electron transfer from antioxidants to the Fe(III)-TPTZ complex. The balanced polarity of aqueous ethanol optimally extracts compounds rich in reducing functional groups (e.g., phenolic –OH). Furthermore, the ionization state of these compounds, which is crucial for their reducing potency, is favorably influenced by the solvent environment, enhancing their ability to act as electron donors [38]. The poor performance of distilled water underscores that while it can ionize compounds, its limited solvation power for the full profile of antioxidants restricts the overall reducing capacity of the extract.

While the FRAP assay is excellent for quantifying reducing capacity, it is important to note its limitation: it measures potential under controlled, non-physiological conditions. The exceptional FRAP value of the UAE extract, however, strongly indicates a high concentration of potent redox-active compounds. Future research should focus on correlating this high reducing power with specific health-relevant outcomes, such as the ability to mitigate oxidative stress in biological systems, to fully unlock the therapeutic potential of S. costus extracts produced via this green technology.

3.5. Phytochemical profiling of polyphenols by HPLC

HPLC analysis provided a detailed phytochemical profile, confirming the selective enrichment of specific phenolic compounds influenced by the extraction solvent. The chromatographic separation successfully identified and quantified key phenolics, with their concentrations detailed in Table 1.

Table 1.

HPLC analysis of the total polyphenols of different S. costus extracts.

Polyphenols	Concentration (μg/g)
	Standards	SCEE	SCME	SCAE
Gallic acid	16.50	7882.02 ± 20.01	5610.21 ± 15.23	3057.65 ± 10.23
Chlorogenic acid	27.75	3260.11 ± 11.10	3120.56 ± 11.25	1188.15 ± 23.20
Catechin	67.95	0.00	0.00	0.00
Methyl gallate	10.85	80.23 ± 1.23	27.96 ± 0.22	11.39 ± 0.12
Coffeic acid	18.32	60.23 ± 0.39	67.58 ± 0.35	48.85 ± 0.55
Syringic acid	17.01	50.21 ± 0.35	62.03 ± 0.34	0.00
Pyro catechol	28.02	0.00	0.00	0.00
Rutin	60.05	0.00	0.00	0.00
Ellagic acid	33.65	70.56 ± 0.85	80.45 ± 0.40	0.00
Coumaric acid	11.21	0.00	0.00	0.00
Vanillin	12.85	116.12 ± 1.03	120.30 ± 1.10	65.18 ± 0.59
Ferulic acid	12.02	190.14 ± 2.95	221.85 ± 2.23	119.66
Naringenin	14.25	1195.54 ± 15.23	1640.55 ± 21.01	449.25 ± 9.59
Taxifolin	14.23	98.36 ± 1.20	150.02 ± 1.55	0.00
Cinnamic acid	5.85	197.85 ± 2.53	15.25 ± 0.12	0.00
Kaempferol	11.12	0.00	0.00	0.00

S. costus aqueous extract SCAE S. costus aqueous methanol extract SCME S. costus aqueous ethanol extract SCEE

*Values are mean ± SD.

A clear solvent-dependent trend was observed. The 50 % aqueous ethanol extract (SCEE) demonstrated the most efficacious extraction for the majority of quantified polyphenols. It yielded the highest concentrations of prominent compounds such as gallic acid (7882.02 μg/g) and chlorogenic acid (3260.11 μg/g). This reaffirms the role of aqueous ethanol as a versatile solvent, capable of extracting a wide range of polar to moderately polar phenolics due to its ability to disrupt hydrogen bonds and solvate diverse molecular structures.

Notably, the extraction efficiency was compound-specific. For instance, while ethanol was superior for gallic acid, 50 % aqueous methanol (SCME) resulted in a higher yield of naringenin (1640.55 μg/g) compared to the ethanol extract. This selectivity can be attributed to the subtle differences in solvent polarity and the specific solubility of each compound, suggesting that the choice of solvent can be tuned to target specific bioactive phenolics.

The significantly lower concentrations obtained with the aqueous extract (SCAE) highlight the limitation of water alone in extracting the full spectrum of phenolics, particularly the less polar aglycones. This solvent-dependent variability is a common phenomenon in plant extraction, as supported by similar trends reported in related species like Costus speciosus [39]. The findings underscore that the enhanced extraction yield and TPC achieved with UAE using 50 % ethanol (as shown in previous sections) correspond to a tangible increase in the concentration of specific, high-value antioxidant compounds, thereby validating the overall efficiency of the optimized process.

Process optimization:

Optimization was carried out using Derringer’s desirability function approach, following analysis of the polynomial model that demonstrated the effects of independent and dependent variables on the extraction process. The best option was chosen based on its highest desire score of 0.70 which suggested the combination of aqueous ethanol (50:50) and ultrasonic-assisted extraction (UAE) yielded the best results. The predicted values for recovery rate, total polyphenolic content, total flavone content (TFC), and oxidative inhibition capacity closely matched the experimental results obtained under optimized extraction conditions, with deviations not exceeding 3.24 %.

Conclusion:

Saussurea costus is a therapeutically renowned plant whose roots are a rich source of bioactive compounds, including sesquiterpene lactones, polyphenols, and flavonoids. This study successfully establishes Ultrasonic-Assisted Extraction (UAE) as a superior, scalable, and eco-friendly alternative to conventional extraction methods for this endangered species. Our optimized UAE process, utilizing aqueous ethanol, demonstrated a significant (p ≤ 0.05) enhancement in extraction yield, total phenolic and flavonoid content, and in-vitro antioxidant capacity (DPPH and FRAP) compared to Soxhlet extraction and maceration.

The superiority of UAE is attributed to the mechanism of acoustic cavitation, which effectively disrupts cellular structures and enhances mass transfer, facilitating the efficient release of both polar and non-polar phytoconstituents. HPLC analysis further confirmed that UAE selectively enriches key phenolic compounds, such as gallic acid, chlorogenic acid, and naringenin. The method's efficiency, coupled with its lower operational temperature, reduced solvent consumption, and shorter processing time, makes it particularly suitable for producing high-quality, thermally-sensitive extracts for nutraceutical and pharmaceutical applications.

While this study comprehensively validates the advantages of UAE for S. costus, future work should focus on scaling up the process, conducting a detailed economic analysis, and investigating the extraction of specific, high-value sesquiterpene lactones. Furthermore, in-vivo studies to confirm the bioactivity of the UAE-derived extracts are recommended. In conclusion, UAE presents a robust and sustainable strategy for maximizing the valorization of S. costus, promoting its conservation through enhanced utilization efficiency.

CRediT authorship contribution statement

Madikha Mushtaq: Writing – review & editing, Writing – original draft. Quraazah Akeemu Amin: Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Towseef Ahmad Wani: Project administration. Syed Zameer Hussain: Formal analysis. Tashooq Ahmad Bhat: Formal analysis. Shahnaz Parveen: Formal analysis. Anis Ahmad Chaudhary: Formal analysis. Mohamed A.M.Ali: Software. Tahiya Qadri: Funding acquisition. Irtiqa Amin: Software.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: