{"paper_id":"f0c68708-aa71-4415-807c-da3ee1d26f1e","body_text":"Stephania\nrotunda  Lour. is a common\nmedicinal plant within the genus  Stephania , which\nis part of the family Menispermaceae. This genus encompasses approximately\n60 species, predominantly found across Southeast Asia, with around\n37 species in China, 15 species in Thailand, and about 16 species\nin Vietnam.  It has been employed in medical\npractice to treat various ailments, such as dysentery, fever, abdominal\npain, wounds, urinary diseases, malaria, headaches, asthma, and diarrhea. \n ,\nA total of 40 alkaloids isolated from this medicinal plant\n(including\ntetrahydropalmatine (THP), cepharanthine, and xylopinine) have shown\nto present analgesic, anti-inflammatory, gastric contractility, renal\nsystem modulation, antiplasmodial, and acetylcholinesterase inhibitory\nactivity. \n , \n  Tetrahydropalmatine (THP), also known as\nrotundine, is the principal active compound in  S. rotunda , and it plays a key role in traditional Asian medicine. THP has\nattracted significant interest due to its broad spectrum of pharmacological\neffects, including antiaddiction, anti-inflammatory, analgesic, neuroprotective,\nand antitumor activity. \n , \n  In antiaddiction studies, THP\nshowed a strong inhibitory effect on methamphetamine-induced responses\nin all phases of the conditioned place preference (CPP) paradigm and\nTHP also reduced morphine-induced CPP in a dose-dependent manner. \n , \n  Moreover, THP reduced ethanol intake through the modulation of D2\nreceptor-mediated PKA signaling in the caudate-putamen.  As an analgesic, THP alleviates acidosis-induced\npain by reducing proton currents in acid-sensing ion channels (ASICs)\nin dorsal root ganglion neurons.  It also\nalleviates neuropathic pain, formalin-induced pain, and bone-cancer-related\npain by inhibiting microglial activation and reducing proinflammatory\ncytokines. Additionally, THP reduced pain in endometriosis and dysmenorrhea\nvia oxidative stress reduction and anti-inflammatory effects.  THP reduces proinflammatory cytokines such as\nTNF-α, IL-1β, and IL-6 and mitigates apoptosis and autophagy\nthrough pathways like TRAF6/JNK and ERK/NF-κB. \n , \n  It exhibited protective effects in disease models such as acute\nlung injury and myocardial ischemia-reperfusion injury, highlighting\nits potential for treating inflammation-related diseases.  Neuroprotective effects of THP include reducing\nneuronal apoptosis in ischemia-reperfusion injury and oxidative stress\nin viral encephalitis. \n , \n  THP also modulates inflammatory\npathways and neurotransmitter systems, offering potential therapeutic\nbenefits for memory impairment, depression, and anxiety.  THP has shown anticancer potential, inhibiting\nglioblastoma progression via suppression of the ERK/NF-κB pathway\nand targeting melanoma by downregulating CDK2 activity. \n , \n  In ovarian cancer, it enhances cisplatin sensitivity through the\nmiR-93/PTEN/AKT pathway and reduces nephrotoxicity without affecting\nanticancer efficacy. \n , \n  Additionally, THP increases the\neffectiveness of tamoxifen and fulvestrant in ERα-positive breast\ncancer cells.  Beyond cancer, THP exhibits\nhypotensive properties, induces vascular relaxation, inhibits osteoclastogenesis,\nand has antifibrotic effects. It promotes muscle regeneration, reduces\nliver fibrosis, and demonstrates resistance to pathogens, such as\nplasmodium species, parasites, and pathogenic fungi. These findings\nunderscore THP’s potential as a multifaceted therapeutic agent.  Therefore, the successful extraction and quantification\nof THP from medicinal plants are essential for fully exploiting its\npharmacological potential.\nThe conventional extraction methods\nfor THP present several limitations.\nThese techniques often involve toxic and flammable solvents and harmful\ninorganic acids, thus causing true environmental and safety hazards.\nMoreover, they may lead to poor selectivity, low yield, and the degradation\nof bioactive compounds due to harsh extraction conditions. \n , \n  These drawbacks underscore the need for “greener”\nand more efficient extraction alternatives. The development of environmentally\nfriendly and sustainable techniques and solvents for extracting natural\nproducts is essential to protect both the environment and public health.\nAdditionally, such advancements play a critical role in enhancing\nthe ecological balance, economic performance, and innovative capacity\nof industries.\n“Green”\nsolvents stand out for their eco-friendly\ncharacteristics, such as low toxicity, thermal and chemical stability,\nnonflammability, and minimal volatility. These attributes position\nthem as an attractive alternative to traditional organic solvents,\nparticularly in light of growing environmental concerns. The researchers\nincreasingly favor “green” solvents due to their ability\nto efficiently extract bioactive compounds while ensuring safety and\nsustainability. By reducing environmental impact and producing high-quality\nextracts, they address both ecological and industrial demands. The\ndevelopment of these solvents marks a significant leap forward in\nestablishing safer, more sustainable extraction techniques. \n ,\nWhen focusing on the use of environmentally friendly (“green”)\nsolvents and for enhancing the extraction efficiency of THP from  S. rotunda  tubers, there is also a critical need\nfor a direct and reliable quantification method for THP in the resulting\nextracts. The quantification methods reported in the literature are\nnot suitable for this purpose due to their complex sample preparation\nprotocols, which involve multiple stages and the extensive use of\norganic solvents and inorganic acids. \n , \n  Previous studies\non the quantification of THP utilized a detection wavelength of 282\nnm. \n , \n  Although this approach proved effective,\nits sensitivity was limited, thus, highlighting an urgent need for\nthe development of enhanced analytical methods to improve detection\naccuracy and reliability.\nIn the present study, we developed\nan eco-friendly ultrasonic-assisted\nextraction method for THP and explored various types of solvents to\nextract THP from  S. rotunda  Lour. including\nvolatile organic compounds, inorganic acid solutions (used as controls),\nionic liquids, polyalcohols, and aqueous carboxylic acid solutions.\nWe also developed and validated a rapid HPLC-PDA method for the quantification\nof THP in  S. rotunda  medicinal plants\naccompanied by a straightforward sample preparation procedure. One\nkey goal of the study was also to identify and find potential “green”\nsolvents which could provide an improved extraction yield in an ultrasonic-assisted\nextraction over the traditional solvent-based extraction methods,\nwhile ensuring safety, cost-effectiveness, biodegradability, and regulatory\napproval for the use of such solvents in pharmaceutical, food, and\ncosmetic industries.\n\nTubers of  S. rotunda  were collected in Quang Tri Province,\nVietnam (geographical coordinates: 16°65′93.1″N;\n106°93′43.6″E). The plant material was identified\nby Le Tuan Anh from the Mientrung Institute for Scientific Research,\nVietnam. The voucher specimens (SR-01) were deposited at the Faculty\nof Pharmacy, University of Medicine and Pharmacy, Hue University,\nVietnam. The samples were dried, pulverized into a fine powder (1.12\nkg), and passed through a 0.71 mm mesh sieve. The resulting powder\nwas stored in a dry, light-protected environment for subsequent experimental\nuse.\nThe standard of THP was obtained from the National Institute of Drug\nQuality Control, Vietnam, and registered in the control logbook under\ncode C0420141.02. A stock solution with a concentration of 1000 μg/mL\nwas prepared by dissolving 10 mg of the THP standard in methanol (MeOH)\nwithin a 10 mL class-A volumetric flask. The calibration standards\nat different concentrations were subsequently obtained by diluting\nthe stock solution with MeOH to the desired levels.\nAcetone\n(Ace), ethanol (EtOH), MeOH, sulfuric acid (Sul), acetic acid (AA),\ncitric acid (CA), formic acid (FA), glycolic acid (GA), lactic acid\n(LA), malic acid (MA), malonic acid (MLA), oxalic acid (OXA), propionic\nacid (PPA), pyruvic acid (PA), tartaric acid (TA), 1,2-pentanediol\n(C5), 1,2-hexanediol (C6), 1,2-butanediol (C4), 1,2-propanediol (PP),\nethylene glycol (EG), glycerol (GL), Brij-35, Triton X-100 (TX100),\nTriton X-114 (TX114), Tween 40 (T40), Tween 60 (T60), Tween 65 (T65),\nTween 80 (T80), and Tween 85 (T85) were purchased from Macklin Inc.\n(Shanghai, China). The preparation of the solvents is described in  Table S1 . Various resins, including DM-301, HPD-300,\nHPD-400, AB-8, XAD-8, and LSA-40, were provided by Tianjin Haoju Resin\nTechnology Co., Ltd. (Tianjin, China).\nThe high-performance\nliquid chromatography (HPLC) analyses were performed using a Shimadzu\nLC-20A HPLC-PDA (Shimadzu Corporation, Kyoto, Japan) and an InertSustain\nC 18  analytical column (5 μm, 4.6 × 250 mm 2 ), protected by an InertSustain C 18  GL-Cart guard\ncartridge (5 μm, 5 × 4.6 mm 2 ) (MZ-Analysentechnik\nGmbH Barcelona, Spain). Sample preparation involved accurate weighing\nusing a Mettler Toledo analytical balance ( d  = 0.1\nmg, Mettler Toledo, Greifensee, Switzerland). UV–vis absorption\nspectra were recorded on a Jasco V-730 spectrophotometer (Jasco Corporation,\nTokyo, Japan). Homogenization and mixing were conducted by using a\nLabnet VX-200 vortex shaker (Labnet International, Edison) and a Julabo\nSW22 thermostatic shaker (Julabo GmbH, Seelbach, Germany). Ultrasonic\nextraction was carried out with an Elma ultrasonic cleaner (Elma Schmidbauer\nGmbH, Singen, Germany), followed by sample separation through centrifugation\nwith a Z326 K HermLe Labortechnik centrifuge (HermLe Labortechnik,\nWehingen, Germany). Filtration was performed under a vacuum using\na Gast DOA-P504-BN filtration system (Gast Manufacturing, Benton Harbor).\nPrecise liquid handling was ensured using BioPette micropipettes (Eppendorf\nAG, Hamburg, Germany) with volumes of 10, 100, 200, and 1000 μL.\nStandard laboratory glassware and consumables were employed as required\nthroughout the experimental procedures.\nQuantitative\nanalysis of all samples was conducted using reversed-phase HPLC-PDA.\nChromatographic separation was achieved on an InertSustain C 18  analytical column with a compatible guard column, as previously\ndescribed. The mobile phase comprised a mixture of acetonitrile (solvent  A ) and an aqueous solution containing 0.3% phosphoric acid\nand 0.2% trimethylamine (solvent  B ) at a volumetric\nratio of 10:90 (v/v). Analyses were performed at room temperature,\nemploying a flow rate of 0.70 mL/min and an injection volume of 5\nμL. Detection was set at 205 nm, complemented by full-spectrum\nscanning to ensure detailed peak characterization.\nThe analytical\nmethod was validated in accordance with the AOAC “Guidelines\nfor the Single Laboratory Validation of Chemical Methods for Dietary\nSupplements and Botanicals” to ensure its reliability and suitability\nfor routine application. Validation was conducted prior to routine\nuse to confirm that the method met the predefined performance criteria.\nThe validation process included evaluation of system suitability,\nspecificity, linearity, precision, accuracy, limit of detection (LOD),\nand limit of quantification (LOQ).\nTo evaluate suitable extraction\nsolvents and methods, 200 mg of  S. rotunda  dried tuber powder was added with 4 mL of solvents in centrifuge\ntubes, and the mixture was subjected to vortex agitation. The mixtures\nwere processed in an ultrasonic bath at 50 °C for 30 min. Following\nthis, the samples were centrifuged at 4000 rpm for 10 min to separate\nthe solid residues. The resulting supernatants were diluted and passed\nthrough 0.45 μm membranes for filtration. HPLC analysis was\nperformed on the prepared extracts, with each procedure repeated three\ntimes to ensure accuracy and consistency.\nTo comprehensively evaluate the\neffects of operational parameters\nand their interactions on the extraction yield, response surface methodology\n(RSM) was applied to optimize the extraction conditions. A Box-Behnken\ndesign was constructed using Design-Expert software version 13 (Stat-Ease\nInc., Minnesota) to optimize the extraction of THP from  S. rotunda  tubers. Statistical significance was determined\nat a p-value of less than 0.05. Four independent variables were examined:\nsolvent concentration ( A ), solvent-to-material ratio\n( B ), extraction time (C), and extraction temperature\n( D ), as presented in  Table  \n . The experimental design comprised 29 runs,\nincluding five center point replicates to evaluate the repeatability\nand reliability of the model.\nSix types\nof macroporous resins (HPD-300, HPD-400, DM-301, LSA-40,\nXAD-8, and AB-8) were employed in a solid–liquid extraction\nprocess to recover THP from the aqueous extract. Initially, the resin\ncolumns were preconditioned by sequentially treating them with absolute\nEtOH, 5% NaOH, and 5% HCl, followed by thorough rinsing with deionized\nwater until a neutral pH was achieved. A 5.0 mL THP-containing solution\nwas then passed through the prepared columns. THP adsorbed on the\nresin was eluted using absolute EtOH, followed by solvent evaporation\nto obtain a THP-enriched product. Recovery efficiency was calculated\nusing the formula: RE alka  = ( w / w \n o ) × 100%, where “ w ” and “ w \n o ” represent\nthe final and initial THP quantities, respectively.\nThe data were analyzed\nusing analysis of variance (ANOVA) conducted through SPSS software\n(version 26, IBM, Armonk, New York). Mean comparisons were performed\nusing the least significant difference test with a significance level\nset at  p  ≤ 0.05. Optimization of experimental\nconditions was carried out with the assistance of Design-Expert software\n(version 13.0, Stat-Ease Inc., Minnesota).\n\nThe chromatographic column, mobile phase composition,\nand flow\nrate were systematically adjusted by evaluating parameters, such as\npeak height, peak area, tailing factor, theoretical plate number,\ncapacity factor, and resolution, to achieve optimal separation. The\nfinalized chromatographic conditions are presented in  Sections  \n  and  2.4.1 . Subsequently, the quantification method was validated\nfollowing AOAC guidelines.  Key validation\nparameters assessed included specificity, system suitability, linearity,\nprecision, accuracy, LOD, and LOQ.\nSpecificity was assessed\nby injecting the blank solution, standard solution, test sample, and\ntest sample spiked with the standard into the chromatographic system.\nThe resulting chromatograms were analyzed and compared to confirm\nthat the method can effectively distinguish the THP compound from\nthe other components. The results are presented in  Figure  \n .\nChromatograms of the\nblank solution (a), standard solution (b),\nsample solution (c), sample solution spiked with the standard (d),\nthe chemical structure of tetrahydropalmatine, THP (e), and superimposed\nUV spectra of THP in standard and sample solutions (f).\nThe HPLC method demonstrated high specificity as the retention\ntime of the THP peak in the sample solution corresponded closely to\nthat in the standard solution. No THP signal was detected in the chromatogram\nof the blank solution. Additionally, the UV spectrum of THP in the\nsample solution matched perfectly with that of THP in the standard\nsolution.\nWith a higher proportion of acetonitrile (∼25\nto 30%) in\nthe mobile phase and the use of a C 18  column, chromatograms\nexhibited a well-defined, sharp THP peak with a short retention time\nof approximately 7–10 min. The UV spectrum of this peak corresponded\nto that of the THP standard. However, it is noteworthy that another\ncompound coeluted with the THP peak. According to Sothavireak,  the retention time of THP was 17.386 min, showing\nincomplete separation from an adjacent minor peak. Under our optimized\nconditions, the retention time of THP was 20.691 min; an additional\ncompound with a retention time of 22.220 min was detected, which was\nfully resolved from THP ( Figure  \n c).\nThe standard\nsolution at a specified concentration was injected six times consecutively.\nRetention time ( t \n R ), peak area ( S \n peak ), theoretical plate number ( N ), and tailing factor ( T \n f ) were recorded\nand evaluated. The results of the system suitability assessment are\nlisted in  Table  \n .\nNote:  t \n R : retention time;  S \n peak : peak area;  N : number of theoretical plates;  T \n f : tailing factor.\nThe results of the evaluation indicated that the relative\nstandard\ndeviations (RSD) for the retention time and peak area were 0.570 and\n1.804%, respectively, both within the acceptable limit of 2%. The\ntailing factor ( T \n f ) ranged between 0.8\nand 1.5, while the theoretical plate number ( N ) exceeded\n2000. These data confirm that the chromatographic system is suitable\nand adequate for the quantitative determination of the THP.\nLinearity was evaluated\nby measuring the responses of standard solutions at different concentrations\nto examine the relationship between the signal intensity and concentration.\nA calibration curve was plotted based on the concentration versus\npeak area of quinine standards, and the correlation coefficient ( r ) was calculated.\nThe results, presented in  Table  \n , demonstrated a strong\nlinear correlation between peak area and concentration for the THP\nstandard over the range of 16–160 μg/mL. The calibration\ncurve is described by the regression equation  y  =\n72,735 x  – 13,758, with a coefficient of determination\n( R \n 2 ) of 0.9999.\nPrecision\nwas assessed\nby injecting a test sample (diluted 10-fold from the extract) six\ntimes on the same day under consistent analytical conditions. The\nprocedure was repeated on a different day using the same sample to\nevaluate the intermediate precision. The results are summarized in  Table  \n .\nThe analysis\nindicated that the HPLC method demonstrated intraday repeatability\nwith RSD values of 0.601 and 0.429% ( n  = 6), and\ninterday precision with an RSD of 0.498% ( n  = 12).\nAccording to AOAC guidelines, for quantitative procedures with active\ningredient content ≤10%, the acceptable RSD is less than 1.5%.\nThus, the method complies with the AOAC precision criteria.\nThe\nTHP standard was spiked\ninto (10-fold-diluted) test samples to obtain three concentration\nlevels of the analyte for recovery evaluation. Each spiked sample\nwas injected into the HPLC system under the established analytical\nconditions with three replicates conducted at each concentration level.\nThe results of the accuracy assessment are shown in  Table  \n . The method met the accuracy\ncriteria, with recovery percentages ranging from 98.59% to 101.57%,\nin accordance with AOAC requirements.\nStandard solutions\nwere diluted to concentrations corresponding\nto signal-to-noise ratios (S/N) of 3 and 10, respectively, based on\nHPLC analysis results. These ratios were used to determine the LOD\nand LOQ, which were found to be 0.07 and 0.2 μg/mL for THP,\nrespectively.\nOverall, the developed HPLC method demonstrated\nacceptable system suitability and high specificity, thus ensuring\naccurate quantification of THP in complex sample matrices without\ninterference from impurities. The method also exhibited a wide linear\nrange, allowing for flexible application across various concentration\nlevels, thereby supporting diverse analytical objectives. Evaluation\nof precision and accuracy parameters showed consistently high performance,\nconfirming the method’s reliability. The analytical procedure\nsatisfied all validation criteria recommended by AOAC guidelines and\nwas suitable for the quantification of THP in  S. rotunda  tubers.\nBased on previous studies, the determination of THP\nfrom  S. rotunda  tubers typically involves\na multistep\nextraction procedure. In one approach, the powdered material is premoistened\nwith hydrochloric acid–acidified water for several hours, followed\nby extraction with dichloromethane and subsequent evaporation to dryness.\nThe dried residue is then reconstituted in an internal standard solution\nprior to the addition of the mobile phase for analysis.  Alternatively, another reported method involves\nextraction with chloroform, evaporation to dryness using a water bath,\ndissolution in sulfuric acid, basification with concentrated ammonia,\nand re-extraction with chloroform, followed by evaporation to dryness\nusing a water bath. The residue is then dissolved in the mobile phase\nand adjusted to the desired volume.  These\nmethods effectively reduce impurities and facilitate quantification\nof the target compound. However, they are not suitable for studies\naiming to identify alternative extraction solvents (e.g., “green”\nsolvents) to optimize the efficiency of THP extraction. The chemical\ncomposition of crude extracts analyzed directly differs from that\nof purified extracts obtained through extensive purification steps.\nTherefore, it is essential to develop a direct quantification method\nfor THP in crude extracts that incorporates a minimal and straightforward\nsample preparation procedure in order to support extraction studies\nand related applications.\nFurthermore, to the best of our knowledge,\nno validated analytical\nmethod has been reported for the quantification of THP in  S. rotunda  tubers using a detection wavelength of\n205 nm. This highlights the importance of developing and validating\nsuch a method in the present study. Notably, the use of a detection\nwavelength of 205 nmwhere THP exhibits significantly stronger\nabsorbance compared to the previously reported 282 nm (as shown in  Figure  \n f)resulted\nin substantial improvements in both the limits of detection (LOD)\nand quantification (LOQ). According to the study by Bory et al.,  the LOD and LOQ at 282 nm were 0.75 and 2.53\nμg/mL, respectively. In contrast, the values obtained in this\nstudy were approximately one-tenth of those previously reported, indicating\na marked enhancement in sensitivity. This improvement is particularly\nadvantageous for the quantification of THP in samples containing low\nanalyte concentrations.\nInorganic acids, bases,\nand organic solvents were initially evaluated as the control solvents.\nSubsequently, eco-friendly solvents were investigated to identify\nthe most feasible solvent for THP extraction. Standardized extraction\nconditions, including a solvent-to-material ratio of 20:1 (mL/g),\nultrasonic extraction for 30 min, and a temperature of 50 °C,\nwere applied to ensure consistency and comparability of results.\nFor the extraction of THP from  S. rotunda  tubers, limewater, water, a sulfuric acid\nsolution, and volatile organic solvents, including MeOH, EtOH, and\nAce, were prepared at concentrations of approximately 100 and 50%,\nas detailed in  Table S1 . These solvents\nserved as controls for comparative evaluation. The extraction yield\nof THP, presented in  Figure  \n  and  Table S2 , ranged from 2.15\nto 18.06 mg/g. Remarkably, 50% MeOH yielded the highest amount of\nTHP (18.06 mg/g), with no statistically significant difference compared\nto other solvents ( p  > 0.05).\nExtraction yields of\ntetrahydropalmatine (THP) from  S. rotunda  using solvents from the initial screening.\nColumns marked with different letters represent statistically significant\ndifferences ( p  < 0.05).\nTo identify\neffective alternative solvents for extracting THP from  S. rotunda , various polyalcohol solutions were prepared\nand tested ( Table S1 ). The results presented\nin  Figure  \n  and  Table S2  show that the 12 polyalcohol solutions\ntested for their ability to extract THP yielded the extraction values\nranging from 13.34 ± 0.11 to 18.06 ± 0.12 mg/g. Among these,\nthe 50% C6 and 50% C5 solvents exhibited the highest extraction efficiency\nwith the yields of 18.06 ± 0.12 and 17.90 ± 0.15 mg/g, respectively\n(the difference between these two values was not statistically significant).\nHowever, these values did not exceed the extraction yield obtained\nwith 50% MeOH solvent.\nThe subsequent investigation focused on the extraction\nof THP using\nsurfactants at a concentration of 5 mM. As shown in  Figure  \n  and  Table S2 , the extraction yields from the eight surfactant solutions\nstudied were significantly lower than those obtained with organic\nsolvents. The THP extraction yields ranged from 5.06 ± 0.03 to\n7.84 ± 0.02 mg/g, and Brij-35 exhibited the highest extraction\nefficiency among the surfactants tested.\nThe extraction yield of\nTHP from  S. rotunda  using 16 carboxylic\nacid solutions is presented in  Figure  \n  and  Table S2 . The results show that most carboxylic acid solutions produced\nhigher extraction yields compared to the 50% MeOH control solvent,\nand the THP concentrations ranged from 16.64 ± 0.09 to 19.60\n± 0.36 mg/g. Among five different solvent groups studied, including\ninorganic acids and bases, organic solvents, polyalcohols, surfactant\nsolutions, and carboxylic acid solutions, the carboxylic acid group\nconsistently exhibited a superior extraction efficiency. Markedly,\nthe 90% lactic acid solution yielded the highest THP content (19.60 ±\n0.36 mg/g).\nTHP is an alkaloid containing a basic amine group\nthat readily forms salts with carboxylic acids. These salt forms exhibit\nhigh solubility in polar solvents, which enhances the extraction efficiency\ncompared to the free base form in organic solvents. Carboxylic acids\ncan soften or disrupt the cellular structure of  S.\nrotunda  tuber powder, thereby facilitating the efficient\nrelease of THP. Additionally, carboxylic acids create a mild acidic\nenvironment (typically pH 3–5), which is optimal for protonating\nTHP without causing degradation of the active compound, as can occur\nunder strongly acidic conditions or elevated temperatures.  Compared with organic solvents, carboxylic acids\nallow for more selective extraction of THP, thus reducing impurities\nand thereby improving purification efficiency.\nAmong the five\nsolvent groups studied (comprising a total of 45\nsolvents), 90% lactic acid solution was found to provide the highest\nextraction yield ( Figure  \n  and  Table S2 ). The superior extraction\nefficiency of lactic acid compared to other carboxylic acids and solvent\ngroups can be attributed to several factors. First, the hydroxyl (−OH)\ngroup in lactic acid increases its polarity and hydrogen-bonding capacity\nrelative to simple carboxylic acids such as acetic, formic, and propionic\nacids, thus facilitating the softening of plant tissues and enhancing\nTHP release.  Second, lactic acid exhibits\nlower viscosity than poly­(carboxylic acid)­s like citric, malic, and\ntartaric acids, and therefore enabling easier penetration into plant\ntissues.  Finally, lactic acid provides\na mild pH environment that protects THP from degradation, which is\nsuperior to stronger acids such as formic, oxalic, or pyruvic acids\nthat may cause THP decomposition. \n ,\nNoticeably,\nlactic acid is widely applied in the food, cosmetics,\nand pharmaceutical industry. In the food industry, it serves primarily\nas an acidulant and preservative for regulating pH and extending the\nproduct shelf life. Within the cosmetics industry, lactic acid is\nvalued for its hydrating, antimicrobial, and skin-renewing properties,\nthus making it a common ingredient in skincare formulations as well\nas oral hygiene products. The pharmaceutical industry also benefits\nfrom lactic acid, which is incorporated into a range of medical devices\nand formulations, such as surgical implants, tablets, dialysis solutions,\nsutures, and controlled drug delivery systems. Beyond these established\nuses, recent advancements have expanded the role of lactic acid into\nthe synthesis of biodegradable and biocompatible polymers like polylactic\nacid, and “green” solvents. \n ,\nIn our current study, the 90% lactic acid solution was selected\nas the most feasible solvent for the further optimization of ultrasonic-assisted\nextraction. This choice was driven by the exceptional extraction efficiency\nof 90% lactic acid solution for THP. The results also suggest the\npotential of 90% lactic acid solution to yield even higher THP concentrations\nunder the optimized ultrasonic-assisted extraction process conditions.\nTherefore, in the next step, we focused on fine-tuning the critical\nprocess parameters of the extraction to further enhance both the yield\nand process efficiency (reference is also made to  Table  \n ).\nFor investigating the effects\nof process factors and their interactions\non the extraction yield, we used the RSM and Box-Behnken experimental\ndesign to optimize the ultrasonic-assisted extraction process of THP\nfrom  S. rotunda  tubers.\nThe extraction\nof natural compounds from medicinal herbs involves several key stages:\n(1) solvent penetration into the cellular matrix of the raw material;\n(2) dissolution of intracellular compounds by the solvent; (3) diffusion\nof the dissolved compounds from the cells into the bulk extraction\nsolvent; and (4) recovery of the extracted compounds. Enhancing the\nfactors that promote the diffusion and solubility of the compounds\nduring these stages can significantly improve the extraction efficiency.\nCritical parameters affecting the extraction performance include solvent\ncharacteristics, particle size of the raw material, solvent-to-material\nratio, extraction temperature, and extraction time.  In the present study, the particle size of the herbal material\nwas standardized by sieving through a 0.71 mm mesh. The effects of\nthe following four independent extraction process factors on the THP\nyield were investigated: lactic acid concentration ( A ), solvent-to-material ratio ( B ), extraction time\n( C ), and extraction temperature ( D ) ( Table  \n ). The experimental\ndesign comprised a total of 29 runs including five replicates at the\ncenter points to ensure model repeatability and reliability. The outcomes\nare presented in  Table S3 .\nThe results\nof the reliability and variance analyses are summarized\nin  Table S4 . The model demonstrated statistical\nsignificance with an  F -value of 49.44 and a corresponding  p -value <0.0001. The coefficient of determination ( R \n 2  = 0.9802) alongside a lack-of-fit test value\nof 0.7832 ( p  > 0.05) indicated a good fit between\nthe model and the experimental data. Furthermore, the adjusted  R \n 2  ( R \n 2 _adj = 0.9603)\nand predicted  R \n 2  ( R \n 2 _pred = 0.9200) values differ by less than 0.2, thus confirming\nthe stability of the model and a strong predictive capability. The\nregression equation describing the relationship between THP extraction\nyield and the independent variables based on the second-order Box-Behnken\nRSM is presented in  eq  \n  ( R \n 2  = 0.9802). \n 1.1 \n Y = 11.15469 − 0.064576 A + 0.259528 B − 0.000076 C + 0.054389 D + 0.000174 AB − 0.000385 AC + 0.000498 AD + 0.000548 BC − 0.000599 BD + 0.000014 CD + 0.001051 A 2 − 0.002942 B 2 + 0.000110 C 2 − 0.000587 D 2 \n where  Y  represents the THP\nextraction yield (mg/g),  A  denotes the lactic acid\nconcentration (%),  B  is the solvent-to-material ratio\n(mL/g),  C  corresponds to the extraction time (minutes),\nand  D  indicates the extraction temperature (°C).\nResponse surface plots visualize the interactions among independent\nvariables and their relationship to the dependent variables.  Figure  \n  shows the effects\nof the process factors studied here on the extraction yield of THP\nfrom  S. rotunda . Except for the interaction\nbetween temperature and extraction time, the response surfaces exhibited\nsteep slopes, indicating a strong impact of these factors on the extraction\nprocess. These findings are supported by the analysis of variance\nresults summarized in  Table S4 .\nResponse surface\ngraphs illustrating the optimization of the tetrahydropalmatine\n(THP) extraction.\nBased on the results\nobtained with RSM, the extraction conditions\nof choice were as follows: lactic acid concentration of 90.0% in the\nsolution, solvent-to-material ratio of 39.997 mL/g, extraction time\nof 10.002 min, and extraction temperature of 64.004 °C. Under\nthese conditions, the predicted THP extraction yield was 22.455 mg/g.\nTo align with the laboratory equipment capabilities, the practical\nexperimental conditions were slightly adjusted to lactic acid concentration\nof 90%, solvent-to-material ratio of 40 mL/g, extraction time of 10\nmin, and extraction temperature of 65 °C. Under these optimized\nexperimental conditions, the extracted THP content was 22.450 ±\n0.029 mg/g. The difference between the experimental and predicted\nresults was not statistically significant ( p  >\n0.05).\nMacroporous resins are polymeric materials characterized by highly\nporous structure. These materials are widely utilized for the separation\nand purification of bioactive compounds (including alkaloids from\nherbal extracts). The adsorption performance of these resins is influenced\nby both the physicochemical properties of the target molecules and\nthe intrinsic structural features of the resin beads such as particle\nsize, surface area, pore diameter, and polarity. This interaction\nadheres to the principle of “like dissolves like,” where\npolar compounds preferentially adsorb onto polar resins, while nonpolar\ncompounds exhibit greater affinity for nonpolar resins.  Adsorption mechanisms primarily involve hydrogen\nbonding, π-π interactions, and van der Waals forces, which\nare further modulated by the resin’s physical characteristics\nand operational parameters. \n , \n  However, excessive\nsimilarity in polarity between the macroporous resin and the target\ncompound can lead to overly strong adsorption forces, which hinders\nefficient desorption and thereby have a negative impact on the recovery\nefficiency and reusability of the resin.\nIn this study, six types of macroporous resins (XAD-8, LSA-40,\nAB-8, HPD-400, DM-301, and HPD-300) were evaluated for the recovery\nof THP from the  S. rotunda  extract.\nThe results showed that the XAD-8 resin presented the highest recovery\nefficiency (92.02 ± 0.60%), while the DM-301 resin exhibited\nthe lowest efficiency (86.46 ± 0.54%) ( Figure  \n  and  Table S5 ).\nSince THP possesses moderate polarity, the moderately polar XAD-8\nresin facilitates optimal van der Waals and hydrogen bonding interactions,\nthus leading to superior adsorption efficiency compared to nonpolar\nresins (such as HPD-300) and weakly polar resins (like AB-8). Although\nDM-301, HPD-400, and LSA-40 share similar moderate polarity, their\nrelatively smaller pore diameters compared to XAD-8 (22.5 nm) may\nlimit the adsorption capacity. Furthermore, the larger pore size of\nXAD-8 enhances desorption efficiency and minimizes the risk of pore\nblockage or undesired retention of adsorbates.\nTetrahydropalmatine (THP) recovery capacity was determined using\nvarious macroporous resins. Recovery efficiency (%) denotes the proportion\nof tetrahydropalmatine extracted from the solution via adsorption\nonto the resins. The THP content indicates the amount of tetrahydropalmatine\nsubsequently recovered after desorption from the resin using ethanol\n(EtOH) as the eluent. Columns marked with different letters represent\nstatistically significant differences ( p  < 0.05).\nIn general, the results of this study have significant\npractical\nimplications for quality control, extraction, and related applications.\nTraditional extraction methods involve soaking  S. rotunda  powder in diluted H 2 SO 4  solution for 24 h\nto obtain THP in its salt form, or alternatively extracting it with\nchloroform. The extract is subsequently dissolved in a H 2 SO 4  solution, alkalized with ammonia or NaOH to a pH of\n9–10, and subjected to chloroform extraction. This extraction\nstep is repeated three times. The combined chloroform extracts are\nthen concentrated by rotary evaporation and purified through crystallization\nin 96% EtOH. \n , , \n  Evidently, these conventional methods consume large amounts of organic\nsolvents and energy and require extended processing times. This study\nmakes a practical contribution by developing an efficient ultrasonic-assisted\nextraction method for THP (from  S. rotunda  medicinal plant) using “green” solvents as alternatives\nto conventional organic solvents. The ability of ultrasonic-assisted\nextraction to improve alkaloid extraction efficiency compared to nonultrasonic\nmethods has been well demonstrated. Aguilar-Hernández et al.\nreported that ultrasonic-assisted extraction increased the total alkaloid\ncontent in different tissues of  Annona muricata  by 2.96–56.31 times compared to maceration, with much shorter\nextraction times (5–15 min) depending on the matrix.  Rathod and Rathod found that ultrasonic-assisted\nextraction yielded 5.80 mg/g piperine from  Piper longum  in only 18 min, whereas Soxhlet extraction yielded 1.67 mg/g in\n4 h and batch maceration 0.98 mg/g in 8 h, corresponding to a 3.5–5.9-fold\nimprovement.  In another study, ultrasonic-assisted\nextraction of berberine from  Chromolaena odorata  reached 2.72% yield in 40 min, compared to only 0.22% for maceration\nin 60 min, representing approximately a 12.4-fold increase.  Furthermore, employing a 90% lactic acid solution\nas the solvent system resulted in a 1.2- to 1.4-fold increase in THP\nyield compared to conventional solvent-based methods. Lactic acid\nis an environmentally friendly water-soluble material; thus, the aqueous\nsolutions of it offer distinct advantages over potentially harmful\norganic solvents. The biodegradability and low toxicity of lactic\nacid contribute to reduced environmental impact and enhanced safety\nfor human exposure. Lactic acid is also produced from renewable biomass,\nsuch as corn starch and sugar cane, thus decreasing reliance on fossil-based\nresources and mitigating greenhouse gas emissions. Moreover, it possesses\nstrong solvent properties, enabling it to dissolve in a wide variety\nof media. This, in turn, broadens the utility of lactic acid in numerous\nindustrial applications. The low vapor pressure of lactic acid reduces\nthe level of emission of volatile organic compounds, thereby enhancing\nworkplace safety and facilitating environmentally sustainable manufacturing\nprocesses. Taken together, these characteristics position lactic acid\nand its aqueous solutions as embodiments of the principles of sustainable\nand safe chemical practices.  In the final\nstep, the THP compound was recovered from a 90% lactic acid solution\nusing a macroporous resin, which proved to be a convenient and efficient\npurification method. Conventional purification techniques, such as\nliquid–liquid extraction and silica gel column chromatography,\npresent several limitations, including excessive solvent consumption,\npotential retention of organic solvent residues, and the generation\nof environmental pollutants. In contrast, macroporous resins provide\nnotable advantages including operational simplicity, high adsorption\nefficiency, low operating costs, reduced solvent usage, improved product\nsafety, and ease of regeneration. These resins have been extensively\napplied for the isolation and concentration of bioactive constituents\nfrom various natural sources.\n\nA novel ultrasonic-assisted extraction\nmethod for the sustainable\nextraction of the tetrahydropalmatine alkaloid from  S. rotunda  Lour. tubers was developed and optimized.\nIn addition, a rapid and validated HPLC-PDA method using a C 18  column (4.6 × 250 mm 2 , 5 μm) was established\nfor quantifying THP in  S. rotunda  tubers\nat 205 nm. The present HPLC method requires only the dilution of the\nextract, followed by filtration through a 0.45 μm membrane filter,\nwithout any further sample preparation steps. The quantitative method\nmet the AOAC validation criteria. Various eco-friendly solvents were\nevaluated for ultrasonic-assisted THP extraction, and 90% lactic acid\nsolution showed the highest efficiency under optimized extraction\nconditions (90% lactic acid, 40 mL/g solvent-to-material ratio, 10\nmin extraction, 64 °C), yielding 22.450 ± 0.029 mg/g. This\nmethod improved the THP yield by 1.2 to 1.4 times compared to conventional\nsolvents. The recovery of THP using macroporous resin XAD-8 was 92.02\n± 0.60%. To the best of our knowledge, this work presents the\nfirst comprehensive report integrating a validated HPLC-PDA quantification\nmethod, ultrasonic-assisted extraction with “green”\nsolvents, and resin-based recovery for THP from  S.\nrotunda  tubers.\nThe improved extraction efficiency\nusing lactic acid solvent combined\nwith resin-based recovery methods can be applied to large-scale THP\nextraction, thereby promoting more sustainable and environmentally\nfriendly practices in industrial processes.","source_license":"CC-BY-4.0","license_restricted":false}