Effect of employing different extraction techniques and solvents on the total phenolic content and antioxidant properties of an Iranian endemic species Zeravschania khorasanica (Apiaceae).

Zeravschania khorasanica, a species endemic to the eastern part of Iran, possesses distinct characteristics that distinguish it from its two closely related species. This research employed five different extraction techniques to identify the active components, total phenolic content and in vitro antioxidant activity of the extract. Furthermore, hydro-distillation was utilized for GC/MS analysis to determine the composition of the essential oil. The total phenolic content was estimated using the Folin-Ciocalteu assay and the antioxidant capacity was evaluated using the DPPH radical scavenging test. The findings revealed that ethanolic Soxhlet extraction yielded the highest efficiency in extracting total phenolic content (88.19 ±1.99 gallic acid mg/100g). In contrast, water maceration extraction demonstrated the highest antioxidant activity (68.1 ±5.4%). Interestingly, the study uncovered that there is no significant positive correlation between the phenolic content and the antioxidant activity of the plant. Additionally, HPLC analysis identified three phenolic constituents in the extract. The Soxhlet extraction method yielded the highest levels of chlorogenic acid (5.8 ppm), caffeic acid (4.1 ppm) and salicylic acid (10.3 ppm). As per the GC/MS analysis, a total of eleven compounds were identified. The predominant compounds were elemicin at 58.19% and trans--bergamotene at 25.78%.

Identification and structural characterization of a novel (+)-catechin-caffeic acid adduct present in wines.

A new compound with a molecular ion mass of m/z 467 in the negative ion mode was found to occur in a white wine aged 30 months in bottle. In this latter, fragment ions compatible with the loss of a carboxylic acid (-44 a.m.u.), a caffeic acid unit (-178 a.m.u.), and a Retro-Diels Alder (-152 a.m.u.) were observed. The present work reports the synthesis of a (+)-catechin-caffeic acid adduct resulting from the condensation reaction between caffeic acid and (+)-catechin. The structural characterization by NMR showed that this adduct is formed by the linkage between carbon 8 at ring A from (+)-catechin and carbon 9 from caffeic acid. In addition, the similarity in the HPLC retention time and UV-Visible spectra of the synthesized compound with the one detected in white wine and the bottling experiments, confirms the presence of this novel (+)-catechin-derived compound in those matrices.

Chlorogenic acid as an indispensible partner of caffeic acid in coffee via selective regulation of prooxidative actions of caffeic acid.

Chlorogenic acid (CGA) and caffeic acid (CA) are two major phenolic acids in coffee. Though the International Agency for Research on Cancer has classified CA as a Group2B carcinogen, coffee consumption seems generally safe within the usual levels of intake and is more likely to benefit health than to harm it. We thus speculated that CGA may effectively suppress the carcinogenic potential of CA. In a molar ratio achievable in vivo, this study shows that CGA can inhibit (i) copper reduction caused by CA, (ii) CA oxidation caused by copper, (iii) the formation of hydroxyl radicals by CA and copper, and (iv) DNA damage induced by CA, quercetin or (-)-epigallocatechin-3-gallate in the presence of copper. CA tends to undergo autoxidation to produce hydrogen peroxide and quinone, which further reacts with proteins to form quinoproteins. This autoxidation at a tolerable level normally induces beneficial adaptive responses. This study shows that CGA is less efficient than CA in producing hydrogen peroxide and quinoprotein; however, together they synergistically produce hydrogen peroxide and quinoprotein in vitro at a molar ratio achievable in vivo. In conclusion, CGA can selectively regulate the prooxidant activities of CA depending on whether copper is involved or not. CGA could be viewed as an indispensable partner of CA in coffee, given its dual role in suppressing the carcinogenic potential of CA and boosting CA autoxidation which is beneficial for disease prevention.

Biotinylated caffeic acid covalent binding with myofibrillar proteins in alkaline conditions: Identification of protein-phenol adducts and alterations in protein properties.

In this study, the effects of covalent interactions between myofibrillar proteins (MP) and caffeic acid (CA) were investigated. Protein-phenol adducts were identified by biotinylated caffeic acid (BioC) used as a substitution of CA. The total sulfhydryls and free amines content were decreased (p < 0.05). The α-helix structure of MP increased (p < 0.05) and MP gel properties enhanced slightly at low dosages of CA (10 and 50 µM), and both were impaired significantly (p < 0.05) at high dosages of CA (250 and 1250 µM). Two prominent adducts of myosin heavy chain (MHC)-BioC and Actin-BioC were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which gradually increased at low concentrations of BioC (10 and 50 µM), and raised significantly at the concentration of 1250 µM. According to the correlation analysis, MHC-BioC and Actin-BioC adducts showed a significant negative correlation with gel properties, such as G', hardness, and water holding capacity (WHC) (p < 0.01), which indicated that the covalent interactions between MP and CA significantly affected the quality of meat products.

Influence of Caffeic and Caftaric Acid, Fructose, and Storage Temperature on Furan Derivatives in Base Wine.

The aim of this study was to determine the influence of caffeic and caftaric acid, fructose, and storage temperature on the formation of furan-derived compounds during storage of base wines. Base wines produced from Chardonnay grapes were stored at 15 and 30 °C for 90 days with additions of fructose, caffeic acid, and caftaric acid independently or in combinations. Wines were analyzed following 90 days of storage for: total hydroxycinnamic acids, degree of browning, caffeic acid and caftaric acid concentrations, and nine furan-derived compounds. Caffeic and caftaric acid additions increased homofuraneol concentration by 31% and 39%, respectively, at 15 °C (p < 0.05). Only the addition of caffeic acid increased furfural by 15% at 15 °C (p < 0.05). Results demonstrate that some furan derivatives over 90 days at 15 °C increased slightly with 5 mg/L additions of caffeic and caftaric acid. This is the first time the influence of hydroxycinnamic acids on furan-derived compounds has been reported during short-term aging of base wine at cellar temperature.

Fast Analysis of Caffeic Acid-Related Molecules in Instant Coffee by Reusable Sonogel-Carbon Electrodes.

Reusable Sonogel-Carbon electrodes containing carbon black (SNGC-CB) have been used for the electrochemical analysis of caffeic acid (CA) in real matrices. Measurements were firstly performed in standard solutions, in which SNGC-CB electrodes allowed the electrochemical determination of CA with high sensitivity and low limit of detection, equal to 0.76 µM. The presence of CB nanostructures in the formulation led to improved performances with respect to pristine SNGC electrodes. Then, measurements were performed in four instant coffees of different brands. A comparison between the results obtained by electrochemical, chromatographic and spectroscopic methods showed that SBGC-CB electrodes represent a simple and economic tool for the rapid assessment of caffeic acid-related molecules in instant coffees.

Haskap Berry Leaves (Lonicera caerulea L.)-The Favorable Potential of Medical Use.

The presented research evaluates the medical use potential of Lonicera caerulea leaves, which are waste plants in cultivating berries. The study's screening activity included the leaves of five varieties of Lonicera caerulea: Atut, Duet, Wojtek, Zojka, and Jugana. The microbiological analysis confirmed the safety of using Lonicera caerulea leaves without significant stabilization. Lonicera caerulea leaves standardization was carried out based on the results of the chromatographic analysis, and it showed differences in the contents of active compounds (loganic, chlorogenic and caffeic acids, and rutin), which are attributed to biological activity. For the Lonicera caerulea leaves varieties tested, the differences in the content of total polyphenol content, chlorophylls, and carotenoids were also confirmed. The screening of biological activity of five Lonicera caerulea leaf varieties was carried out concerning the possibility of inhibiting the activity of α-glucosidase, lipase, and hyaluronidase as well, and the antioxidant potential was determined. The defined profile of the biological activity of Lonicera caerulea leaves makes it possible to indicate this raw material as an essential material supporting the prevention and treatment of type II diabetes. However, this research showed that tested enzymes were strongly inhibited by the variety Jugana. The health-promoting potential of Lonicera caerulea leaves was correlated with the highest chlorogenic acid and rutin content in the variety Jugana.

Application of insecticides on peppermint (Mentha × piperita L.) induces lignin accumulation in leaves by consuming phenolic acids and thus potentially deteriorates quality.

Irrational use of pesticides may lead to physiological and metabolic disorders in different crops. However, there are limited investigations on impacts of insecticides on physiology and biochemistry, secondary metabolic pathways, and associated quality of medicinal plants such as peppermint (Mentha × piperita L.). In this study, target metabolites in peppermint were monitored following foliar spraying of five insecticides: imidacloprid, pyriproxyfen, acetamiprid, chlorantraniliprole, and chlorfenapyr. Compared with the control, all insecticide treatments caused a significant loss of soluble protein (decreased by 22.3-38.7%) in peppermint leaves. Insecticides induced an increase in the levels of phytohormones jasmonic acid and abscisic acid in response to these chemical stresses. Among them, imidacloprid increased jasmonic acid by 388.3%, and pyriproxyfen increased abscisic acid by 98.8%. The contents of phenylpropanoid metabolites, including rutin, quercetin, apigenin, caffeic acid, 4-hydroxybenzoic acid, ferulic acid, syringic acid, and sinapic acid showed a decreasing trend, with pyriproxyfen decreasing the levels of quercetin and 4-hydroxybenzoic acid by 78.8% and 72.6%, respectively. Combined with correlation analysis, the content of lignin in leaves shows different degrees of negative correlations with several phenolic acids. It could be inferred that insecticides may trigger plant defense mechanisms that accumulate lignin (increased by 24.6-49.1%) in leaves by consuming phenolic acids to barricade absorption of insecticides. Through constructing networks between phytohormones and secondary metabolites, peppermint may regulate the contents of caffeic acid, 4-hydroxybenzoic acid, and sinapic acid by the antagonistic effect between salicylic acid and abscisic acid in response to insecticidal stresses. Principal component analysis and systemic cluster analysis revealed that the most pronounced changes in physiological indexes and metabolites were caused by the pyriproxyfen treatment. In conclusion, this study improves our understanding of the mechanism by which insecticides affect plant physiological and metabolic processes, thus potentially altering the quality and therapeutic value of peppermint as an example.

Coffee simulated inhibition of pancreatic lipase and antioxidant activities: Effect of milk and decaffeination.

Noncommunicable diseases (NCDs) are the leading cause of mortality worldwide. Dietary intake of polyphenols may protect against the development of NCDs. Coffee is a rich source of phenolic acids in the Western diet that may prevent or treat hyperlipidemia, type 2 diabetes, chronic liver diseases, and obesity. These health effects are attributed, at least partially, to the antioxidant properties and inhibitory activity of phenolic acids on lipases. However, the effect of milk on these properties is not clear. Thus, we aimed to evaluate the antiradical properties and inhibitory activity on pancreatic lipase in vitro of phenolic acids from coffee. We obtained commercial traditional and decaffeinated espresso coffee capsules and prepared the beverages according to the manufacturer's instructions using a domestic coffee maker. Espresso prepared with traditional and decaffeinated coffee capsules were evaluated with and without the addition of milk following in vitro digestion.The total phenolic content ranged from 168.21 to 397.38 mg equivalent to chlorogenic acid/mL. All coffee-based beverages showed antioxidant activity, with emphasis on decaffeinated and milk-added beverages, respectively. Caffeic acid was the most abundant phenolic compound followed by 5-caffeoylquinic acid before digestion. In contrast after in vitro digestion, only caffeic acid was bioaccessible. The addition of milk improved the bioaccessibility of caffeic acid and caffeine. Overall, the half-maximal inhibitory concentration of the samples for pancreatic lipase varied between 222 and 3035.8 µg/mL. Decaffeinated coffee had a greater inhibitory effect than regular coffee regardless of milk addition. In conclusion, decaffeinated and milk-added coffee beverages have a greater effect on lipase inhibition. This may be related to the greater bioaccessibility of phenolic compounds in these samples. Further studies are needed to elucidate the mechanisms of enzymatic inhibition by phenolic acids.

Ferocactus herrerae Fruits: Nutritional Significance, Phytochemical Profiling, and Biological Potentials.

The current study reports for the first time the nutritional, fruit volatiles, phytochemical, and biological characteristics of Ferocactus herrerae J. G. Ortega fruits. The nutritional analysis revealed that carbohydrate (20.6%) was the most abundant nutrient followed by dietary fibers (11.8%), lipids (0.9%), and proteins (0.8%). It was rich in vitamins, minerals, essential, and non-essential amino acids. Gas chromatography-mass spectrometry (GC-MS) analysis of the headspace-extracted volatiles showed that 3-methyl octadecane (35.72 ± 2.38%) was the major constituent detected. Spectrophotometric determination of total phenolic and flavonoid contents of the fruit methanolic extract (ME) showed high total phenolic [9.17 ± 0.87 mg/g gallic acid equivalent (GAE)] and flavonoid [4.99 ± 0.23 mg/g quercetin equivalent (QE)] contents. The ME was analyzed using high-performance liquid chromatography with ultraviolet (HPLC-UV), which allowed for both qualitative and quantitative estimation of 16 phenolic compounds. Caffeic acid was the major phenolic acid identified [45.03 ± 0.45 mg/100 g dried powdered fruits (DW)] while quercitrin (52.65 ± 0.31 mg/100 g DW), was the major flavonoid detected. In-vitro assessment of the antioxidant capacities of the ME revealed pronounced activity using three comparative methods; 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (132.06 ± 2.1 µM Trolox equivalent (TE) /g), 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS), (241.1 ± 5.03 uM TE/g), and ferric reducing antioxidant power (FRAP) (258.9 ± 1.75 uM TE/g). Besides, remarkable anti-inflammatory [COX-1 (IC50 = 20.2 ± 1.1 µg/mL) and COX-2 (IC50 = 9.8 ± 0.64 µg/mL)] and acetylcholinesterase inhibitory (IC50 = 1.01 ± 0.39 mg/mL) activities were observed. Finally, our results revealed that these fruits could be used effectively as functional foods and nutraceuticals suggesting an increase in their propagation.

Antidepressant-like effect of caffeic acid: Involvement of the cellular signaling pathways

Abstract Caffeic acid is a phenolic compound widely distributed in plants and beverages such as coffee. Although its mechanism of action is poorly understood, caffeic acid reportedly induces antidepressant-like and neuroprotective effects. This study aimed to investigate the involvement of cellular signaling pathways in acute antidepressant-like effect induced by caffeic acid in mice. All procedures were approved by the Institutional Animal Ethics Committee of the UNIVALI n. 021/2013. Female Swiss mice were administered with vehicle, caffeic acid (5 mg/ kg, p.o.), inhibitor (H-89, U0126, chelerythrine, or PD9859, i.c.v.) or caffeic acid plus inhibitor. The behavioral effects were evaluated 1h after the administration of compounds to mice using tail suspension test (TST) and open field test (OFT). The results showed that the antidepressant- like effect of caffeic acid in mice was possibly mediated by the activation of PKA, MEK 1/2, PKC and MAPK (as assessed using TST), without compromising their locomotor activity (as assessed using OFT). Our results demonstrated, at least in part, the pathways involved in the neuroprotective and behavioral effects of caffeic acid.

Effect of Elicitation with (+)-Usnic Acid on Accumulation of Phenolic Acids and Flavonoids in Agitated Microshoots of Eryngium alpinum L.

The present work was aimed at studying the potential of elicitation on the accumulation of phenolic compounds in in vitro shoot cultures of Eryngium alpinum L., a protected plant from the Apiaceae family. The study examined the influence of (+)-usnic acid on the biomass growth as well as on the biosynthesis of the desired flavonoids and phenolic acids in the cultured microshoots. The phenolic compound content was determined by HPLC-DAD. The flavonoid of the highest concentration was isoquercetin, and the phenolic acids of the highest amount were rosmarinic acid, caffeic acid and 3,4-dihydroxyphenylacetic acid, both in the non-elicited and elicited biomass. Isoquercetin accumulation was efficiently increased by a longer elicitation with a lower concentration of lichenic compound (107.17 ± 4.67 mg/100 g DW) or a shorter elicitation with a higher concentration of acid (127.54 ± 11.34 and 108.37 ± 12.1 mg/100 g DW). Rosmarinic acid production generally remained high in all elicited and non-elicited microshoots. The highest content of this acid was recorded at 24 h of elicitation with 3.125 µM usnic acid (512.69 ± 4.89 mg/100 g DW). The process of elicitation with (+)-usnic acid, a well-known lichenic compound with allelopathic nature, may therefore be an effective technique of enhancing phenolic compound accumulation in alpine eryngo microshoot biomass.

Preclinical safety assessment of the crude extract from Sida rhombifolia L. aerial parts in experimental models of acute and repeated-dose 28 days toxicity in rats.

Sida rhombifolia (Malvaceae) is popularly used as a treatment for several pathological conditions; however, there is a lack of studies that identify its compounds and that evaluate comprehensively the safety of its consumption. Therefore, the aim of this study was to determinate the phytochemical constitution of the crude extract of Sida rhombifolia (CESR), and its safety in models of acute and repeated doses (28 days) toxicity. The tested dose for the model of acute toxicity was 2000 mg/kg doses for the repeated dose model were 150, 300 e 600 mg/kg. Hematological, biochemical, histopathological and oxidative markers were investigated. HPLC-DAD-MS analysis evidenced the presence of caffeic acid, coumarin, and rutin. In the acute toxicity model the only altered parameters were tissue ROS, and AST and BUN in serum. As for the repeated dose experiment both hematological and biochemical markers remained within the values of reference for the species. Obtained results demonstrate that the CESR did not present significant toxic effects when administrated orally to male and female rats in acute and repeated doses.

Use of non-volatile compounds for the classification of specialty and traditional Brazilian coffees using principal component analysis.

Coffee beans contain different volatile and non-volatile compounds that are responsible for their flavor and aroma. Herein, principal component analysis (PCA) was employed to correlate the non-volatile composition of specialty and traditional coffees with drink quality. The quantified non-volatile compounds included caffeine, chlorogenic acid, caffeic acid, and nicotinic acid in both types of coffee samples, while 5-hydroxymethylfurfural was only quantified in the specialty coffee samples. The most abundant compounds present in specialty coffees were associated with the aroma and flavor, affording a high drink quality. In traditional coffees, the most abundant compounds included nicotinic acid and caffeine, indicating a stronger roasting process, loss of sensory characteristics, and blended formulations. PCA showed a distinction between the traditional and specialty coffees such that a relationship between the contents of the compounds in each type of coffee, quality, and classification could be established.

A Rapid UPLC Method for the Simultaneous Quantitation of Caffeic Acid Derivatives in Dried Extracts of Echinacea Purpurea.

Echinacea purpurea is a traditional medicinal plant widely used as adjuvant for the treatment of respiratory and urinary infections. Caffeic acid derivatives are considered the main active markers, such as chicoric acid, caftaric acid and chlorogenic acid. An analytical method using ultra performance liquid chromatography (UPLC) and diode array detector was developed and validated, to quantify caffeic acid derivatives in commercial dried extracts of EP. UPLC method was developed using a C18 column (50 × 2.1 mm, 1.8 µm), at 30°C. Mobile phase was composed of acetonitrile and 0.05% (v/v) formic acid aqueous solution (10:90), flow rate 0.2 mL/min. Injection volume was 10 µL and detection was performed at 300 and 330 nm. The developed method complied with all required validation parameters, and showed to be linear, precise, accurate, selective and robust for all caffeic acid derivatives. Using the validated method, the levels of caftaric acid (0.110-0.507%w/w), chicoric acid (0.040-0.179%w/w) and chlorogenic acid (0.013-0.084%w/w) were determined in five commercial dried extracts of E. purpurea, with significant variation in the contents between different samples, indicating the need of standardization and control of individual caffeic acid derivatives in commercial extracts.

Paper-based Chemiluminescence Device with Co-Fe Nanocubes for Sensitive Detection of Caffeic Acid.

In this work, a new chemiluminescence (CL) system of Co-Fe prussian blue analogs nanocubes (Co-Fe PBA NCs) that can catalyze luminol to produce strong CL in the absence of H2O2 was established. Co-Fe PBA NCs have the property of oxidase-like activity, and it can catalyze the generation of active oxygen radicals in a dissolved oxygen system. Since caffeic acid (CA) can remove reactive oxygen species in the system, a sensitive detection method for CA on a paper-based chip was developed. Under the optimal conditions, this method showed a good linear response to CA in the range of 10 - 800 ng mL-1 with a limit of 3 ng mL-1. The proposed method had been used for the determination of CA in tea samples. The results may open a new avenue for the catalytic property on luminol CL system without extra oxidants.

Use of asparaginase for acrylamide mitigation in coffee and its influence on the content of caffeine, chlorogenic acid, and caffeic acid.

A factorial design with a duplicate in the central point was used to investigate the effect of treating arabica coffee beans with asparaginase. The investigated factors were enzymatic load (1000 and 5000 ASNU/Kg), water percentage (30 and 90%), and hydrolysis time (1 and 3 h). The acrylamide content was determined by UPLC-MS/MS, and the caffeic acid, chlorogenic acid and caffeine concentrations were determined by HPLC-DAD. The statistical analysis was carried out in the R platform using RStudio graphical interface. The results indicated the importance of coffee bean pretreatment with steam, and that the enzyme load reduced the acrylamide content to 65 mg/kg in coffee beans. The predicted reduction was obtained with hydrolysis time of 2 h, water content of 90%, and asparaginase load of 5000 ASNU/kg. The asparaginase treatment did not influence the major bioactive compounds in coffee.

Triacylglycerols compositions, soluble and bound phenolics of red sorghums, and their radical scavenging and anti-inflammatory activities.

Six commercial red sorghum varieties (Tong Za 117, 141, 142 and 143, Chi Za 109 and 101) were investigated for their triacylglycerol (TAG) profiles, soluble and bound phenolics, and radical scavenging and anti-inflammatory activities. A total of 21 TAGs were identified in red sorghum oils for the first time. Total phenolic (TPC) and flavonoid contents (TFC) in the soluble or bound phenolic fractions differed among red sorghums. Significant correlation among TPC, TFC and DPPH radical scavenging activities was observed in both fractions. Except for caffeic acid, most of phenolic acids in red sorghums are in the bound form. Soluble 3-deoxyanthocyanidins contents (2.12-57.14 µg/g) were significantly higher than those of bound forms (0.01-0.18 µg/g) regardless of sorghum varieties and types of 3-deoxyanthocyanidins. Moreover, the stronger anti-inflammatory capacity of soluble phenolic fraction in Tong Za 117 correlated with its higher TPC, TFC and radical scavenging activity than those of its bound counterpart.

Micronization increases the bioaccessibility of polyphenols from granulometrically separated olive pomace fractions.

The effect of micronization of granulometrically fractionated olive pomace (OP) on the bioaccessibility of polyphenols and the antioxidant capacity was investigated during sequential in vitro static digestion. Crude OP was fractionated in a 2-mm sieve (F1: > 2 mm; F2: < 2 mm) and then micronized (300 r min-1, 5 h) generating F1AG (17.8 µm) and F2AG (15.6 µm). Micronization increased the release of hydroxytyrosol, oleuropein, caffeic acid, and decarboxymethyl oleuropein aglycone (3,4-DHPEA-EDA) in the salivary and gastric phase, beyond luteolin in the gastric phase. Micronization also increased the intestinal bioaccessibility of hydroxytyrosol, 3,4-DHPEA-EDA, oleuropein, luteolin, and apigenin; it was more effective for F2AG than F1AG. Micronized samples increased antioxidant capacity in the gastric phase. F2AG exhibited the highest antioxidant capacity in the insoluble intestinal fraction. Thus, micronization can be further exploited to improve the nutraceutical properties of OP by increasing the bioaccessibility and antioxidant capacity of phenolic compounds.

Distributions of eight bioactive components in rat tissues administered Marsdenia tenacissima extract orally detected through UPLC-MS/MS.

Marsdenia tenacissima (Roxb.) Wight et Arn. (M. tenacissima) is considered an anticancer medicine in traditional Chinese medicine, which is extensively used in clinical application since it has great therapeutic effects. Currently, although a number of articles have examined M. tenacissima in terms of its pharmacology and quality control, few have investigated the in vivo mechanism of M. tenacissima active ingredients. Previously, we have studied the pharmacokinetics of eight active ingredients after oral administration of M. tenacissima extracts in rat plasma. This study constructed a new scientific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach to simultaneously quantify the contents of tenacissosides B, G, H and I, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid and caffeic acid in rats orally administered M. tenacissima extract. The proposed approach was successfully used for investigating the distributions of those eight analytes in rat tissues, with digoxin being used as an internal control. The Eclipse Plus C18 RRHD column was used for determination at a column temperature of 30°C. The mobile phase system consisted of acetonitrile and water (supplemented with 0.1% formic acid) under optimal gradient elution conditions. Afterwards, this approach was validated according to the requirements for the analysis of biological samples developed by the US Food and Drug Administration, including precision, accuracy, stability and matrix effects. Based on tissue distribution analysis, those eight analytes showed rapid distribution within all the tested tissues. With regard to organic acid distribution, it followed the order stomach > liver > kidney > small intestine > lung > spleen > heart, whereas the four steroids followed the order stomach > lung > spleen > small intestine > liver > kidney > heart. The present study lays the theoretical foundation for the use and development of M. tenacissima in clinical practice.