RESUMEN
Whey protein isolates (WPI) are known to have mineral-binding capacity to promote iron absorption. The aim of this study was to investigate the effect of iron ratio on the conformational structure of iron-bound whey protein isolate (WPI-Fe) and its thermodynamic stability. It was shown that the iron to protein ratio affects both the iron binding capacity of WPI and the iron valence state on the surface of WPI-Fe complexes. As the iron content increases, aggregation between protein molecules occurs. In addition, WPI-Fe nanoparticles have thermodynamic stability and Fe2+ has a high affinity with WPI for spontaneous exothermic reactions. This study demonstrates that WPI-Fe complexes can be used to efficiently deliver high-quality iron source (Fe2+) for future iron supplements.
Asunto(s)
Hierro , Nanopartículas , Proteína de Suero de Leche/química , TermodinámicaRESUMEN
Resina Draconis (RD) is known as the "holy medicine for promoting blood circulation" and possesses antitumor properties against various types of cancer, including breast cancer (BC); however, the underlying mechanism is not well understood. To explore the potential mechanism of RD against BC using network pharmacology and experimental validation, data on bioactive compounds, potential targets of RD, and related genes of BC were obtained from multiple public databases. Gene Ontology (GO) and KEGG pathway analyses were performed via the DAVID database. Protein interactions were downloaded from the STRING database. The mRNA and protein expression levels and survival analysis of the hub targets were analyzed using the UALCAN, HPA, KaplanâMeier mapper, and cBioPortal databases. Subsequently, molecular docking was used to verify the selected key ingredients and hub targets. Finally, the predicted results of network pharmacology methods were verified by cell experiments. In total, 160 active ingredients were obtained, and 148 RD target genes for the treatment of BC were identified. KEGG pathway analysis indicated that RD exerted its therapeutic effects on BC by regulating multiple pathways. Of these, the PI3K-AKT pathway was indicated to play an important role. In addition, RD treatment of BC seemed to involve the regulation of hub targets that were identified based on PPI interaction network analysis. Validation in different databases showed that AKT1, ESR1, HSP90AA1, CASP3, SRC and MDM2 may be involved in the carcinogenesis and progression of BC and that ESR1, IGF1 and HSP90AA1 were correlated with worse overall survival (OS) in BC patients. Molecular docking results showed that 103 active compounds have good binding activity with the hub targets, among which flavonoid compounds were the most important active components. Therefore, the sanguis draconis flavones (SDF) were selected for subsequent cell experiments. The experimental results showed that SDF significantly inhibited the cell cycle and cell proliferation of MCF-7 cells through the PI3K/AKT pathway and induced MCF-7 cell apoptosis. This study has preliminarily reported on the active ingredients, potential targets, and molecular mechanism of RD against BC, and RD was shown to exert its therapeutic effects on BC by regulating the PI3K/AKT pathway and related gene targets. Importantly, our work could provide a theoretical basis for further study of the complex anti-BC mechanism of RD.
Asunto(s)
Neoplasias de la Mama , Extractos Vegetales , Femenino , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proliferación Celular , Células MCF-7 , Simulación del Acoplamiento Molecular , Farmacología en Red , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Extractos Vegetales/farmacologíaRESUMEN
Prostate cancer (PCa) is a common and fatal cancer for men and effective treatment options are still not enough for patients. Radix et Rhizoma Rhei had been applied to treat PCa long-termly and effectively when combined with surgical treatment and chemotherapy. However, its active components and target proteins are still not quite clear. As membrane receptors play a vital role in PCa, in this study, a novel strategy that combines comprehensive 2D 3-aminopropyltriethoxysilane-decorated prostate cancer cell (DU145) membrane chromatographic (CMC) system with network pharmacology approach was developed to characterize membrane binding active components proteins from Radix et Rhizoma Rhei and their targets. Thirteen active components were screened out by CMC system, among which emodin and rhapontigrnin with good membrane binding behaviors were validated to show ideal inhibitory effects on DU145 cells by cell viability and cell apoptosis assays. Five membrane proteins were predicted as the potential targets by the a specific network pharmacology approach, among which mast/stem cell growth factor receptor Kit (KIT) was identified as the most possible target by network data mining. Surface plasmon resonance analysis verified that the dissociation constant (KD) of rhapontigrnin and emodin with KIT was 6.06â¯×â¯10-5 M and 8.82â¯×â¯10-5 M, respectively. Our results showed that the combination of comprehensive 2D CMC system and network pharmacology based target identification could not only rapidly identify the membrane binding components but also find the potential membrane protein targets with high confidence, which could broaden the range of application scope of CMC, especially for the screening of active compounds from complex chemical samples using primary pathologic cell lines.
Asunto(s)
Membrana Celular/metabolismo , Medicamentos Herbarios Chinos/química , Neoplasias de la Próstata/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodos , Humanos , Masculino , Simulación del Acoplamiento Molecular , Propilaminas/química , Neoplasias de la Próstata/tratamiento farmacológico , Reproducibilidad de los Resultados , Silanos/química , Resonancia por Plasmón de SuperficieRESUMEN
Rhizoma corydalis and Radix Angelicae Dahurica (Yuanhu-Baizhi) herbal medicine pair has been used for thousands of years and has been reported to be potentially active in recent cancer therapy. But the exact active components or fractions remain unclear. In this study, a new comprehensive two-dimensional (2D) 3-aminopropyltriethoxysilane (APTES)-decorated MCF7-cell membrane chromatography (CMC)/capcell-C18 column/time-of-flight mass spectrometry system was established for screening potential active components and clarifying the active fraction of Yuanhu-Baizhi pair. APTES was modified on the surface of silica, which can provide an amino group to covalently link cell membrane fragments with the help of glutaraldehyde in order to improve the stability and column life span of the MCF7 CMC column. The comprehensive 2D MCF7-CMC system showed good separation and identification abilities. Our screen results showed that the retention components are mainly from the alkaloids in Yuanhu (12 compounds) and the coumarins (10 compounds) in Baizhi, revealing the active fractions of Yuanhu-Baizhi herbal medicine pair. Oxoglaucine, protopine, berberine, osthole, isopimpinellin and palmitic acid were selected as typical components to test the effects on cell proliferation and their IC50 were calculated as 38.17 µM, 29.45 µM, 45.42 µM, 132.7 µM, 156.8 µM and 90.5 µM respectively. Cell apoptosis assay showed that the drug efficacy was obtained mainly through inducing cell apoptosis. Furthermore, a synergistic assay results demonstrated that oxoglaucine (representative of alkaloids from Yuanhu) and isopimpinellin (representative of coumarins from Baizhi) showed significant synergistic efficacy with GFT, indicating that these components may act on other membrane receptors. The proposed 2D CMC system could also be equipped with other cells for further applications. Besides, the follow-up in-vitro experimental strategy using cell proliferation assay, cell apoptosis assay and synergistic assay proved to be a practical way to confirm the active fractions of herbal medicine.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Membrana Celular/efectos de los fármacos , Cromatografía/métodos , Corydalis/química , Medicamentos Herbarios Chinos/farmacología , Antineoplásicos Fitogénicos/química , Neoplasias de la Mama/fisiopatología , Membrana Celular/química , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Femenino , Humanos , Células MCF-7 , Espectrometría de Masas , Plantas Medicinales/química , Propilaminas/química , Rizoma/química , Silanos/químicaRESUMEN
The samples of Huangqi injection (HI) were analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-TOF-MS), and both positive and negative ion modes were employed to obtain the LC-TOF-MS analysis information of chemical compounds in HI. Then the mass defect filtering (MDF) approach, which was developed based on the previously published articles, was utilized to rapidly screen the astragalosides from the obtained LC-TOF-MS data. Each screened astragaloside was confirmed by the presence of no less than 2 quasi-molecular ions. All the screened astragalosides were then tentatively assigned according to the parent ion and daughter ion information. Finally, a total of 62 astragalosides were screened and characterized from the HI samples, including 15 new detected ones. The identification results indicated that acetylation, hydrogenation, dehydrogenation, methoxylation and hydration might be the major conversion reactions involved in the formation of the astragalosides. The LC-TOF-MS-based MDF approach was proved to be a feasible and efficient tool to screen the chemical constituents in complex matrices such as herbal medicines.
Asunto(s)
Medicamentos Herbarios Chinos/química , Saponinas/análisis , Astragalus propinquus , Cromatografía Liquida , Plantas Medicinales/química , Espectrometría de Masas en TándemRESUMEN
The side effects of cisplatin (CDDP), notably nephrotoxicity, greatly limited its use in clinical chemotherapy. HuangQi Injections (HI), a commonly used preparation of the well-known Chinese herbal medicine Astragali radix, appeared to be promising treatment for nephrotoxicity without compromising the anti-tumor activity of CDDP. In this study, the urinary metabolomics approach using liquid chromatography time of flight mass spectrometry (LC-TOF/MS) was developed to assess the toxicity-attenuation effects and corresponding mechanisms of HI on CDDP-exposed rats. As a result, successive administration of HI significantly recovered the decline of body weight and downregulated the abnormal increase of serum creatinine and urea. HI partly restored the CDDP-induced alteration of metabolic profiling back into normal condition. Totally 43 toxicity-attenuation potential biomarkers were screened and tentatively identified, which were involved in important metabolic pathways such as amino acid metabolism, TCA cycle, fatty acid metabolism, vitamin B6 metabolism and purine metabolism. The results clearly revealed that HI could alleviate CDDP-induced nephrotoxicity and improve the disturbed metabolic balance induced by repeated CDDP exposure. The present study provided reliable evidence for the protective effect of HI on CDDP-induced toxicity with the multi-target pharmacological characteristics.
Asunto(s)
Antineoplásicos/efectos adversos , Cisplatino/efectos adversos , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Metaboloma , Metabolómica , Animales , Biomarcadores/orina , Cromatografía Liquida , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/orina , Masculino , Redes y Vías Metabólicas , Metabolómica/métodos , Ratas , Espectrometría de Masas en TándemRESUMEN
Liquid chromatography with quadrupole time-of-flight mass spectrometry coupled with automated data analysis by Peakview software was employed to systematically screen and characterize the astragalosides in Radix Astragali, a Chinese medical preparation. The separation was performed on a poroshell 120 SB-C18 column equipped in a conventional liquid chromatography system. After being separated using a general gradient elution, the analytes were detected by the triple quadrupole time-of-flight mass spectrometer in both positive- and negative-ion modes. The mass defect filtering function built in the Peakview software was utilized to rapidly screen the potential ions of interest, while some functions of Peakview such as Formula Finder, XIC manager, and IDA Explorer were employed to facilitate the assignment or characterization of the screened astragalosides. A total of 42 astragalosides were screened and tentatively characterized or assigned, and 20 of them were firstly detected in Radix Astragali. According to the screened astragalosides, acetylation, glycosidation, hydrogenation, oxidation, and hydration were considered to be the major secondary metabolic pathways involved in the formation of the astragalosides. The combination of liquid chromatography with quadrupole time-of-flight mass spectrometry and automated Peakview analysis is a feasible and efficient tool to screen and identify the constituents in complex matrices of herbal medicines.
Asunto(s)
Planta del Astrágalo/química , Medicamentos Herbarios Chinos/análisis , Glucósidos/análisis , Plantas Medicinales/química , Programas Informáticos , Astragalus propinquus , Cromatografía Líquida de Alta Presión , Medicamentos Herbarios Chinos/administración & dosificación , Glucósidos/administración & dosificación , Espectrometría de Masas , Soluciones/química , Factores de TiempoRESUMEN
Cell membrane chromatography (CMC) is a bioaffinity chromatography technique for characterizing interactions between drugs and membrane receptors and has been widely used to screen active components from complex samples such as herbal medicines (HMs). However, it has never been applied in vivo due to its relatively high limit of detection (LOD) and the matrix interferences. In this study, a novel on-line comprehensive two-dimensional HepG2/CMC/enrich columns/high performance liquid chromatography/time-of-flight mass spectrometry system was developed to rapidly screen potential anti-hepatoma components from drug-containing serum of rats after oral administration of Radix scutellariae. A matrix interference deduction method with a home-written program in MATLAB was developed, which could successfully eliminate the interference of endogenous substances in serum. Baicalein, wogonin, chrysin, oroxylin A, neobaicalein and rivularin from Radix scutellariae extraction were significantly retained in the HepG2/CMC column. Three potential active components, wogonin, oroxylin A and neobaicalein were firstly screened from the drug-containing serum as well. The cell counting kit-8 assay demonstrated that wogonin, oroxylin A and chrysin showed high inhibitory activities in a dose-dependent manner on HepG2 cells at the concentration of 12.5-200 µM (p<0.05) and the IC50 values were 69.83, 16.66 and 51.6 µM, respectively. Wogonin and oroxylin A, which were screened both from Radix scutellariae extraction and the drug-containing serum, could be selected as lead compounds to obtain good anti-hepatoma effects. The proposed comprehensive 2D CMC system and matrix interference elimination strategy have significant advantages for in vivo screening of active components from complex biological samples and could be applied to other biochromatography models.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/sangre , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/análisis , Scutellaria baicalensis , Espectrometría de Masas en Tándem/métodos , Administración Oral , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC50 of 10.30 µM, 17.85 µM, and 2.53 µM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.
Asunto(s)
Membrana Celular/química , Extractos Vegetales/química , Propilaminas/química , Salvia miltiorrhiza/química , Silanos/química , Gel de Sílice/química , Abietanos/química , Abietanos/metabolismo , Abietanos/farmacología , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Células Hep G2 , Humanos , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fenantrenos/química , Fenantrenos/metabolismo , Fenantrenos/farmacología , Extractos Vegetales/metabolismo , Salvia miltiorrhiza/metabolismo , Trasplante Heterólogo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/químicaRESUMEN
Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well-developed biological chromatographic technique. In this study, we have developed combined SMMC-7721/CMC and HepG2/CMC with high-performance liquid chromatography and time-of-flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R(2) = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.
Asunto(s)
Membrana Celular , Técnicas de Química Sintética/métodos , Cromatografía , Quinazolinas/química , Quinazolinas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Técnicas de Química Sintética/instrumentación , Química Farmacéutica , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Humanos , Espectrometría de Masas , Estructura MolecularRESUMEN
To explore the effect and mechanism of action of different ω-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio on the expression of AKT and mTOR in mice bearing endometrial carcinoma. Once the human endometrial carcinoma xenograft models were successfully established, 40 BALB/C mice were randomized into five groups: group A (ω-6 PUFAs), group B (10:1 ω-6/ω-3 PUFAs), group C (control group), group D (1:1 ω-6/ω-3 PUFAs), and group E (ω-3 PUFAs). Six weeks post-treatment, mice were sacrificed and the xenograft tissues were harvested for immunohistochemical SP analysis of AKT and mTOR expression. AKT and mTOR mRNA expression was determined by reverse transcription polymerase chain reaction. Group A and group B had the highest positive expression of AKT and mTOR, with increased mRNA expression. Group D and group E had the lowest positive expression of AKT and mTOR, with decreased mRNA expression. There was a positive correlation between the expression of AKT and that of mTOR (r = 0.92). Thus, ω-6/ω-3 PUFAs in different proportions are associated with the mRNA expression of AKT and mTOR in the tissues of mouse xenograft model of human endometrial cancer.
Asunto(s)
Neoplasias Endometriales/patología , Ácidos Grasos Omega-3/fisiología , Ácidos Grasos Omega-6/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K(+) channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.
Asunto(s)
Antibióticos Antineoplásicos , Membrana Celular/metabolismo , Membrana Celular/patología , Doxorrubicina , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Miocardio/patología , Extractos Vegetales/farmacología , Ranunculaceae/química , Animales , Supervivencia Celular , Diterpenos , Medicamentos Herbarios Chinos , Ratas , Ratas Sprague-DawleyRESUMEN
The combined use of the state-of-the-art hybrid mass spectrometer (MS) together with high efficient liquid chromatography may prove to be a useful tool for neutral saccharide analysis. In the present work, we used hydrophilic interaction liquid chromatography (HILIC) to separate selected saccharides (fructose, glucose, galactose, sucrose, melibiose, raffinose, manninotriose, stachyose and verbascose). Influences of the column type, additive type, temperature, pH and other separation factors on analyte retention were evaluated. The new method did not require reduction, derivatization or postcolumn addition of reagents, which are commonly used in conventional saccharide analysis. Our results demonstrate the potential of HILIC-MS for sensitive and robust determination of saccharides in crude and processed Radix Rehmanniae, and may promote new perspectives in the research of other medicinal herbs.
Asunto(s)
Carbohidratos/química , Carbohidratos/aislamiento & purificación , Cromatografía Liquida/métodos , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Rehmannia/química , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/instrumentación , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The aim of this study was to compare the pharmacokinetics of timosaponin B-II and timosaponin A-III in rat plasma after oral administration of Zhimu-Baihe herb-pair, Zhimu extract, free timosaponin B-II and free timosaponin A-III. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a SHISEIDO CAPCELL PAK C18 column (100 mm × 3 mm i.d., 3.0 µm) with the mobile phase consisting of acetonitrile and 0.05% (v/v) formic acid by linear gradient elution. The detection was performed on an Agilent G1946D single quadrupole mass spectrometer with negative electrospray ionization (ESI) interface in select-ion-monitoring (SIM) mode. The following ions: m/z 919 for timosaponin B-II, m/z 739 for timosaponin A-III and m/z 683 for the IS were used for quantitative determination. Good linearity was achieved over the concentration ranged from 4.0-793.3 ng/mL to 3.9-781.3 ng/mL for the two saponins. The precision of the in vivo study was evaluated by intra- and inter-day assays and the percentages of relative standard deviation were all within 15%. The plasma concentrations of timosaponin B-II and timosaponin A-III in rats at designated time periods were successfully determined using this fully validated method, and statistically significant differences (p<0.5) in pharmacokinetic parameters including AUC0-t, AUC0-∞ and MRT (mean residence time) were obtained. Compared to these pharmacokinetic parameters after oral administration of Zhimu extract and monomer solution, higher peak concentration (Cmax is higher), slower elimination (MRT is longer) and larger AUC values could be observed after giving Zhimu-Baihe herb-pair in our study. Therefore, this result not only elucidated the steady and long-lasting pharmacological properties but also revealed the practical value of the compatibility of herb-pair remedy.