Authentication of Senecio scandens and S. vulgaris based on the comprehensive secondary metabolic patterns gained by UPLC-DAD/ESI-MS

A secondary metabolic pattern using ultra-performance liquid chromatography (UPLC)-DAD/ESI-MS was constructed to gain chemical information for authentication of Senecio scandens (SS) and Senecio vulgaris (SV), the two representative species containing hepatotoxic pyrrolizidine alkaloids (HPAs). The metabolic pattern showed three groups of bioactive constituents: phenolic/aromatic acids, flavonoid glycosides and the HPAs. 47 peaks were identified including 19 phenolic/aromatic acids, 10 flavonoid glycosides and 18 PAs by direct comparison with the available reference compounds or deduced from the UV absorption and their ESI-MS fragmentation patterns.The two species could be authenticated diagnostically by their metabolic profiling of the three chromatographic fingerprints. Although both SS and SV contain PAs as the characteristic constituents, only 2 PAs, adonifoline and adonifoline N-oxide were detected in SS, while other 16 PAs were detected in SV, including the highly toxic senecionine, retrorsine, seneciphylline and their corresponding N-oxides. The concentration of PAs in SV is also higher than that in SS. The number and concentration of the phenolic compounds in SS were higher than in SV. Jacaranone derivatives were only detected in SS and jacaranone ethyl ester was detected as the predominant constituent.In the fingerprint of the n-butanol extracts, 10 quercetin and kaempferol glycosides derivatives were detected. 9 were found in SS and only 2 in SV. PAs, jacaranone derivatives and flavonoid glycosides can serve as the metabolic markers to distinguish the Senecio plants from each other, and provide evidence for their clinical application in the consideration of safety and efficacy.     

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC–DAD and HPLC–ESI-MS

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC–DAD–ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC–ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids.     

Characterization of flavonoids in the extract of Sophora flavescens Ait. by high-performance liquid chromatography coupled with diode-array detector and electrospray ionization mass spectrometry

A method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (ESI) was established for the separation and characterization of flavonoids in Sophora flavescens Ait. Based on the chromatographic separation of most flavonoids present in S. flavescens Ait., a total of 24 flavonoids were identified. Fourteen compounds were unambiguously identified comparing experimental data for retention time (t(R)), UV and MS spectra with those of the authentic compounds: 3',7-dihydroxy-4'-methoxy-isoflavone (13), trifolirhizin (14), kurarinol (18), formononetin (19), 7,4'-dihydroxy-5-methoxy-8-(gamma,gamma-dimethylallyl)-flavanone (22), maackiain (21), isoxanthohumol (23), kuraridine (26), kuraridinol (27), sophoraflavanone G (30), xanthohumol (31), isokurarinone (33), kurarinone (35) and kushenol D (38), and additional 10 compounds were tentatively identified as kushenol O (10), trifolirhizin-6'-malonate (15), sophoraisoflavanone A (20), norkurarinol/kosamol Q (24), kushenol I/N (25), kushenol C (28), 2'-methoxykurarinone (29), kosamol R (32), kushecarpin A (34) and kushenol A (37) by comparing experimental data for UV and MS spectra with those of literature. Furthermore, fragmentation pathways in positive ions mode of 24 flavonoid compounds of types of flavanone, flavanonol, flavonol, chalcone, isoflavone, isoflavanone and ptercocarpane were summarized. Some common features, such as CH(3)., H(2)O, CO, CO(2), C(3)O(2) and C(2)H(2)O losses, together with Retro-Diels-Alder fragmentations were observed in the prenylated flavonoids in S. flavescens Ait. The loss of the lanandulyl chain was their characteristic fragmentation, which might help deducing the structure of unknown flavonoid compounds. The present study provided an approach to rapidly characterize bioactive constituents in S. flavescens Ait.     

Metabolic profile of the bioactive compounds of burdock (Arctium lappa) seeds, roots and leaves

In this work the bioactive metabolic profile, the antioxidant activity and total phenolic content of burdock (Arctium lappa) seeds, leaves and roots were obtained. TEAC values and total phenolic content for hydro-alcoholic extracts of burdock ranged from 67.39 to 1.63 μmol Trolox equivalent/100 g dry weight (DW), and from 2.87 to 45 g of gallic acid equivalent/100 g DW, respectively. Phytochemical compounds were analyzed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS/MS) in negative mode. The main compounds of burdock extracts were caffeoylquinic acid derivatives, lignans (mainly arctiin) and various flavonoids.The occurrence of some phenolic acids (caffeic acid, chlorogenic acid and cynarin) in burdock seeds; arctiin, luteolin and quercetin rhamnoside in burdock roots; phenolic acids, quercetin, quercitrin and luteolin in burdock leaves was reported for the first time.     

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC-DAD-ESI-MS(n)

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography-diode array detection-electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MSⁿ) combined with liquid chromatography-time of flight-mass spectrometry (HPLC-TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MSⁿ fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC-DAD using a C₁₈ column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.     

HPLC-DAD-ESI-MS analysis of the constituents of aqueous preparations of verbena and lemon verbena and evaluation of the antioxidant activity

Verbena and lemon verbena aqueous preparations were investigated for their content of constituents, especially polyphenols by HPLC/DAD/ESI/MS analysis because they are used worldwide as herbal teas. The main class of compounds of these plants were phenylpropanoids (from 16 to 120 mg/g of dried extract), being verbascoside the most abundant in all the preparations up to 97% of the total phenylpropanoids. Also iridoids, hastatoside and verbenalin together with flavonoids, mono- and di-glucuronidic derivatives of luteolin and apigenin were found. These simple preparations, especially that obtained from infusion of lemon verbena, could be lyophilized to obtain a powder having interesting technological properties to be used as ingredients of cosmetics, food supplements and herbal medicinal products do to the many biological properties of verbascoside. In addition, the antioxidant property of the lemon verbena infusion was evaluated by the DPPH test using Trolox as the reference compound.     

Determination of phenolic compounds in fennel by HPLC and HPLC-MS using a monolithic reversed-phase column

A reversed-phase high-performance liquid chromatography (HPLC) method for analyzing phenolic compounds in fennel (Foeniculum vulgare) has been developed. The use of a monolithic column with short dimensions in combination with optimized chromatographic conditions allows over 100 samples per day to be analyzed. Chromatographic parameters such as column temperature and injection volume, were found to be crucial in obtaining adequate selectivity and resolution, consequently allowing short run times. The method was validated for the major phenolic compounds present in fennel plant material: 3-O-caffeoylquinic acid (3-CQA), chlorogenic acid, 4-O-caffeoylquinic acid (4-CQA), eriocitrin, rutin, miquelianin, 1,3-O-dicaffeoylquinic acid (1,3-diCQA), 1,5-O-dicaffeoylquinic acid (1,5-diCQA), 1,4-O-dicaffeoylquinic acid (1,4-diCQA) and rosmarinic acid. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.05 to 1.0 microg/mL and from 0.15 to 2.5 microg/mL, respectively. With some adaptation, the extraction procedure could be even less invasive, which is useful in screening work.     

Phenolic compounds, organic acids profiles and antioxidative properties of beefsteak fungus (Fistulina hepatica).

The phenolic compounds and the organic acids composition of the edible beefsteak fungus Fistulina hepatica was determined by HPLC/DAD and HPLC/UV, respectively. The results showed a profile composed by five phenolic compounds (caffeic, p-coumaric and ellagic acids, hyperoside and quercetin) and six organic acids (oxalic, aconitic, citric, malic, ascorbic and fumaric acids). The quantification of the identified compounds revealed that ellagic acid (ca. 49.7%) and malic acid (ca. 57.9%) are the main compounds in this species. In a general way the phenolic profile revealed to be more constant than the organic acids one and could be more useful for the quality control of the species. Beefsteak fungus was also investigated for its capacity to act as a scavenger of DPPH() radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid). Good results were obtained against DPPH in a concentration-dependent manner. Beefsteak fungus also displayed good activity against superoxide radical, achieved by its capacity to act as both scavenger and xanthine oxidase inhibitor. A prooxidant effect was noticed for hydroxyl radical, which may be due to its capacity for iron ions reduction. Little ability for iron chelation was also observed. Beefsteak fungus showed a weak protective effect against hypochlorous acid.     

Determination of phenolic compounds in Prunella L. by liquid chromatography-diode array detection

Four species of Prunella L. (Prunella vulgaris L., Prunella laciniata L., Prunella grandiflora L. and Prunella orientalis Bornm.) belong to the family of Lamiaceae and representing popular Western and Chinese herbal medicine were examined for the content of phenolic compounds. Phenolic acids (rosmarinic acid, caffeic acid, ferulic acid, chlorogenic acid, protocatechuic acid), flavonoids (rutin, quercetin) in different quantitative proportions depending on extracts were determined by the rapid, selective and accurate method combining solvent/acid hydrolysis extraction and high performance liquid chromatography-diode array detection (HPLC-DAD). Water, methanol, butanol, acetonitrile, ethyl acetate, hexane and their acidic solutions were used to examine the efficiency of different solvent systems for the extraction of phenolic compounds. Acid hydrolysis extraction was established as the most suitable extraction method for phenolic compounds.     

Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts

Mango is one of the important tropical fruits in the world. As it is a seasonal fruit, it is processed for various products. During its processing, peel is one of the major byproducts, which is being wasted. Bioactive conserves were extracted using 80% acetone from peels of raw and ripe mango fruits and subjected to acid hydrolysis. The prominent phenolic compounds identified by HPLC were protocatechuic acid, gentisic acid and gallic acid. The phenolic acid derivatives present in acetone extracts of raw and ripe peels were tentatively identified by LC–MS. Gallic acid, syringic acid, mangiferin, ellagic acid, gentisyl-protocatechuic acid, quercetin were the phenolic compounds identified in both raw and ripe peels, while raw peel showed the presence of glycosylated iriflophenone and maclurin derivatives also. β-Carotene was the major carotenoid followed by violaxanthin and lutein. Thus, both raw and ripe mango peel extracts have different phenolic compounds and carotenoids, which will have various pharmaceutical applications.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Phenolic compounds from Byrsonima crassifolia L. bark: phytochemical investigation and quantitative analysis by LC-ESI MS/MS

Phytochemical investigation of the methanolic extract of Byrsonima crassifolia's bark led to the isolation of 8 known phenolic compounds 5-O-galloylquinic acid, 3-O-galloylquinic acid, 3,4-di-O-galloylquinic acid, 3,5-di-O-galloylquinic acid, 3,4,5-tri-O-galloylquinic acid, (+)-epicatechin-3-gallate along with (+)-catechin and (+)-epicatechin.Due to their biological value, in the present study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, working in multiple reaction monitoring (MRM) mode, has been developed to quantify these compounds. B. crassifolia bark resulted in a rich source of phenolic compounds and particularly of galloyl derivates. The proposed analytical method is promising to be applied to other galloyl derivatives to quantify these bioactive compounds in raw material and final products.     

Hydroxycinnamic acid derivatives occurring in Cichorium endivia vegetables

The hydroxycinnamic acid derivatives found in Chicorium endivia var. crispum and var. latifolium polyphenolic extracts were detected and characterized by high-performance liquid chromatography (HPLC) combined with photodiode array detector (DAD) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The method provides data (molecular weight and diagnostic fragment ions) on the molecular structure of compounds. The combined approach enabled identification of four hydroxycinnamic derivatives in each chicory extract; three derivatives (5-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, and 5-O-feruloylquinic acid) were found in both chicories, while 3,5-di-O-caffeoylquinic acid was typical of var. crispum and cis-caftaric acid of var. latifolium.     

Simultaneous estimation of phenolic acids in sea buckthorn (Hippophaë rhamnoides) using RP-HPLC with DAD

A RP-HPLC-DAD method was developed and validated for the simultaneous analysis of nine phenolic acids including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, p-coumaric acid, cinnamic acid, caffiec acid and ferulic acid in sea buckthorn (SB) (Hippophaë rhamnoides) berries and leaves. The method was validated in terms of linearity, LOD, precision, accuracy and recovery and found to be satisfactory. Phenolic acid derivatives in anatomical parts of SB berries and leaves were separated into free phenolic acids, phenolic acids bound as esters and phenolic acids bound as glycosides and profiled in HPLC. Berry pulp contained a total of 1068 mg/kg phenolic acids, of which 58.8% was derived from phenolic glycosides. Free phenolic acids and phenolic acid esters constituted 20.0% and 21.2%, respectively, of total phenolic acids in SB berry pulp. The total phenolic acid content in seed kernel (5741 mg/kg) was higher than that in berry pulp and seed coat (Table 2). Phenolic acids liberated from soluble esters constituted the major fraction of phenolic acids (57.3% of total phenolic acids) in seed kernel. 8.4% and 34.3% of total phenolic acids in seed kernel were, respectively contributed by free and phenolic acids liberated from glycosidic bonds. The total soluble phenolic acids content in seed coat (448 mg/kg) was lower than that in seed kernel and pulp (Table 2). Proportion of free phenolic acids in total phenolic acids in seed coat was higher than that in seed kernel and pulp. Phenolic acids bound as esters and glycosides, respectively contributed 49.1% and 20.3% of total phenolic acids in seed coat. The major fraction (approximately 70%) of phenolic acids in SB berries was found to be concentrated in the seeds. Gallic acid was the predominant phenolic acid both in free and bound forms in SB berry parts and leaves.     

Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC–DAD–ESI-MS

A high-performance liquid chromatography–diode array detection–mass spectrometry method with electrospray ionization mode (HPLC–DAD–ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC–DAD–ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70 min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile–water (94:6, v/v) with flow rate at 1 mL/min, column temperature at 30 °C and detection wavelength at 280 nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6 μg/g), followed by quercetin-3-O-rutinoside (281.3 μg/g), dicaffeoylquinic acid isomers (250.1 μg/g), chlorogenic acid (237.0 μg/g), quercetin-di-(rhamnohexoside) (117.5 μg/g), quercetin-di-(rhamno)-hexoside (116.8 μg/g), kaempferol-3-O-rutinoside (97.7 μg/g), isorhamnetin-3-O-rutinoside (72.1 μg/g), p-coumaric acid (64.0 μg/g), caffeic acid (23.7 μg/g) and vanillic acid (22.8 μg/g).     

羊耳菊化学成分和药理活性研究进展

羊耳菊(Inula cappa)为汉族、傣族等多民族常用药,本文综述了其化学成分和药理活性的研究进展。近40年来,已从羊耳菊中分离得到了100余种化合物,主要为倍半萜类、肌醇类、三萜类、黄酮类和酚类化合物。羊耳菊药理活性的报道较少,目前仅发现有抗氧化作用和抑菌作用。     

Evaluation of Antioxidant Potential of Kaempferia rotunda Linn

The plant Kaempferia rotunda Linn. has been explored for its anti oxidant potential in the present study. The antioxidant property was assessed by lipid peroxidation markers such as malonaldehyde (MDA) and 4-hydroxyl-2-nonenal (4-HNE). The lipid peroxidation byproducts are highly toxic and responsible for various diseases like myocardial infarction, diabetes mellitus, hepatic injury, atherosclerosis, rheumatoid arthritis and cancer. The chemical constituents of the plant were critically and qualitatively analyzed to confirm the presence of flavonoids and phenolic derivatives. Hence our objective has been designed to evaluate the antioxidant effect of Kaempferia rotunda linn. and its contribution to control the lipid peroxidation.     

Phytochemical screening and evaluation of cardioprotective activity of ethanolic extract of Ocimum basilicum L. (basil) against isoproterenol induced myocardial infarction in rats

Background and the purpose of the study

The objectives of the present study were phytochemical screening and study of the effects of ethanolic extract of aerial parts of Ocimum basilicum (basil) on cardiac functions and histopathological changes in isoproterenol-induced myocardial infarction (MI).

Methods

The leaves of the plant were extracted with ethanol by maceration and subjected to colorimetry to determine flavonoids and phenolic compounds. High-performance TLC analysis and subsequent CAMAG''s TLC scanning were performed to quantify rosmarinic acid content. Wistar rats were assigned to 6 groups of normal control, sham, isoproterenol, and treatment with 10, 20, and 40 mg/kg of the extract two times per day concurrent with MI induction. A subcutaneous injection of isoproterenol (100 mg/kg/day) for 2 consecutive days was used to induce MI.

Results

Phytochemical screening indicated the presence of phenolic compounds (5.36%) and flavonoids (1.86%). Rosmarinic acid was the principal phenolic compound with a 15.74% existence. The ST-segment elevation induced by isoproterenol was significantly suppressed by all doses of the extract. A severe myocardial necrosis and fibrosis with a sharp reduction in left ventricular contractility and a marked increase in left ventricular end-diastolic pressure were seen in the isoproterenol group, all of which were significantly improved by the extract treatment. In addition to in-vitro antioxidant activity, the extract significantly suppressed the elevation of malondialdehyde levels both in the serum and the myocardium.

Conclusion

The results of the study demonstrate that Ocimum basilicum strongly protected the myocardium against isoproterenol-induced infarction and suggest that the cardioprotective effects could be related to antioxidative activities.     

Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris SW aqueous extract with anti-PLA2 activity

The Casearia sylvestris SW (Flacourtiaceae) is utilized in folk medicine (Brazil and all Latin American) to treat several pathologic processes as inflammation, cancer, microbial infection and snake bites. Studies showed that C. sylvestris aqueous extract can inhibit many toxic effects caused by snake venoms (or caused by phospholipase A₂ isolated) from different species, mainly of Bothrops genus. Inhibition of enzymatic and myotoxic activities, decrease of edema formation and increase of the survival rate of rats injected with lethal doses of bothropic venoms are some toxic effects inhibited by C. sylvestris. In this study, four ellagic acid derivatives from aqueous extracts of C. sylvestris were isolated, characterized, and tested against effects from both total venom and PLA₂ (Asp 49 BthTX-II) from the venom of Bothrops jararacussu. The isolated compounds were as follows: ellagic acid (A), 3′-O-methyl ellagic acid (B), 3,3′-di-O-methyl ellagic acid (C), 3-O-methyl-3′,4′-methylenedioxy ellagic acid (D). The inhibition constant values (Ki) for enzymatic activity, as well the IC₅₀ values found in the edematogenic and myotoxic activities, indicate that the ellagic acid is the best inhibitor of these activities, while compounds C and D are the substances with lowest capacity on inhibiting these same effects. Our results show that the presence of hydroxyls at position 3 or 3′ (compounds A and B) increases the capacity of these derivatives on inhibiting these toxic effects. However, the presence of methoxyl groups at position 3 or 3′ reduced, but did not completely inhibit the capacity of compounds C and D on inhibiting all the toxic effects studied.     

Effect of Ilex extracts and isolated compounds on peroxidase secretion of rat submandibulary glands.

Free radicals are involved in diverse disorders such as tumoral, central nervous system alterations, immunological and inflammatory pathologies. Peroxidase is an oral enzyme involved in the defense of the oral cavity. Ilex species such as Ilex paraguariensis St. Hil. and the commercial product made with it "Yerba Mate" are used traditionally as antirheumatics and for the treatment of gastrointestinal diseases among others and also as a beverage with nutritional and stimulant properties. The presence of polyphenolic derivatives and flavonoids in the aqueous extract has been determined by HPLC analysis. In this study, the activity of aqueous extracts of I. paraguariensis and "Yerba Mate" on peroxidase secretion in female rat submandibular glands was investigated. The contribution to this pharmacological activity by some major hydrocynnamic acid derivatives present in the crude extracts, such as chlorogenic acid and caffeic acid and the most abundant methylxanthine, caffeine, was also evaluated. Spectrophotometrical determination of peroxidase activity showed that both extracts produced a significant increase in both secreted peroxidase and total peroxidase activity, though "Yerba Mate" showed a higher activity (EC(50) "Yerba Mate": 148+/-10 microg/ml; EC(50)I. paraguariensis: 841+/-20 microg/ml). The HPLC/DAD analysis of the crude extracts was performed and chlorogenic acid, caffeic acid and caffeine were identified and quantified. The results (expressed as W/W percentage of dried material) were as follows: I. paraguariensis: chlorogenic acid: 2.80+/-0.30, caffeic acid: 0.023+/-0.004, caffeine: 1.06+/-0.06; "Yerba Mate": chlorogenic acid: 1.98+/-0.37; caffeic acid: 0.020+/-0.003, caffeine: 0.70+/-0.06. Caffeine and chlorogenic acid were proved to play an important role in the induction of peroxidase secretion induced by the extracts.