A sensitive method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (MS) was optimized for the separation and identification of phenolic acids, flavonoid glycosides and flavonoid aglycones in the extract of burr parsley (Caucalis platycarpos L.). Fragmentation behavior of flavonoid glycosides and phenolic acids were investigated using ion trap mass spectrometry in negative electrospray ionization. The MS, MSn and UV data together with HPLC retention time (TR) of phenolic acids and flavonoids allowed structural characterization of these compounds. Caffeoylquinic acid (CQA) isomers, p-coumaroyl-quinic acids (p-CoQA), feruloylquinic acids (FQA), dicaffeoylquinic acids (diCQA), luteolin-7-O-rutinoside, apigenin-7-O-rutinoside as well as isolated chrysoeriol-7-O-rutinoside have been identified as constituents of C. platycarpos for the first time. An accurate, precise and sensitive LC-DAD method for quantification of four phenolic acids (3-O-caffeoylquinic, caffeic, p-coumaric, o-coumaric acid), four flavonoid glycosides (luteolin-7-O-glucoside, apigenin-7-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-rhamnoside), and three flavonoid aglycones (luteolin, apigenin, chrysoeriol) in C. platycarpos extract was validated in terms of linearity, limit of detection, limit of quantification, precision and accuracy. 3-O-caffeoylquinic acid was the predominant phenolic acid and luteolin-7-O-glucoside was the predominant flavonoid glycoside.

The presence of peritoneal carcinomatosis arising from gastrointestinal and gynecologic tumors is associated with a poor prognosis. Animal models of peritoneal carcinomatosis are important in the evaluation of new treatment modalities. The purpose of this study was to investigate the effect of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in an animal model of induced peritoneal carcinomatosis in the mouse. For induction of peritoneal carcinomatosis, cells from transplantable mammary carcinoma (MCa) were implanted intraperitoneally in CBA mice. Seven or 3 days before implantation of MCa cells (5 x 10 (3)) the mice were injected with lyophilized water extract from CAUCALIS PLATYCARPOS L. (CPL; 200 mg . kg (-1)) into the abdominal cavity. Immediately after implantation of MCa cells in the abdominal cavity, mice were treated two times with 2 mL of saline that was heated either at 37 degrees C or 43 degrees C (hyperthermal treatment) and cytostatics (doxorubicin 20 mg . kg (-1), cisplatin 10 mg . kg (-1), mitomycin 5 mg . kg (-1), 5-FU 150 mg . kg (-1)). We followed the survival of animals and the side effects appearing with different forms of treatment. CPL increased the life span of mice with peritoneal carcinomatosis without hyperthermal treatment (ILS% = 32.55 %) but showed no effect on the life span of mice with hyperthermal treatment (ILS% = 1.44). Combined treatment with CPL and cytostatics (CIS, DOX, and MIT) significantly affected the development of peritoneal carcinomatosis and increased the survival of mice (ILS% - 37 degrees C = 144.17, 415.46, and 124.13, ILS% - 43 degrees C = 311.42, 200.74, and 138.33, respectively). However, intraperitoneal chemotherapy with 5-FU alone resulted in greater survival time of mice than the treatment with 5-FU + CPL. Results suggest the synergistic effect of hyperthermia, chemotherapy, and immunotherapy. CPL significantly increases the antitumor activity of the hyperthermic chemotherapy and the survival rate of mice with peritoneal carcinomatosis. The stimulative effect of CPL on immunomodulation may be a possible mechanism which protects mice from developing peritoneal carcinomatosis and reduces the side effects of chemotherapy, increasing the life span of mice.

The water extract of burr parsley (Caucalis platycarpos L.) showed remarkable antitumor activity in rats and mice. Phenolic compounds, including phenolic acids and flavonoids, are considered to be the major bioactive compounds. The aim of this work was to develop a reverse phase HPLC-DAD method for the simultaneous quantification of flavonoid aglycones and phenolic acids, and an HPLC-DAD-MS/MS method for structural characterization of phenolic compounds, obtained after hydrolysis of C. platycarpos methanolic extract. Caffeic acid was the predominant phenolic acid, and luteolin was the predominant flavonoid aglycone. The optimized and validated method for the determination of the five phenolic acids and four flavonoid aglycones ensured reliable results and could be used for the quality control of raw plant material.Naturally occurring immunological response modifiers in combination with cytostatics are widely used in cancer treatment [1,2]. Burr parsley (Caucalis platycarpos L., Caucalis daucoides L., Apiaceae), grows in Mediterranean and Central Europe. It has been shown that the water decoction of C. platycarpos after intraperitoneal injection had antitumor activity on the rat model of liver metastasis of colorectal cancer. The antitumor effect results from stimulation of the immunological system of the host, by involving the spleen as a lymphatic organ to produce antitumor factors [3]. Flavonoids and phenolic acids as ubiquitous secondary plant metabolites are distributed in nature in their free and bound forms [4]. Luteolin-7-Oglucoside was found to be the predominant flavonoid and 3-O-caffeoylquinic acid was the predominant phenolic acid in C. platycarpos methanolic extract, where 31 compounds were identified [5]. The goal of this work was to develop an HPLC-DAD-MS/MS method for structural characterization and identification of phenolic compounds obtained after hydrolysis of methanolic extract and to develop and validate the HPLC-DAD method for the determination of aglycones and phenolic acids.The hydrolysis conditions of phenolic acid esters and flavonoid glycosides were optimized in terms of duration of hydrolysis and temperature and concentration of hydrochloric acid [6]. Different conditions were examined by recording the total ion chromatogram and extracted ion chromatogram at m/z 447, which is the [M -H] -for the main flavonoid glycoside present in C. platycarpos, namely luteolin-7-O-glucoside. Hydrolysis was considered to be completed when there was no peak assigned with m/z 447 in the hydrolyzed methanolic extract. BHT at concentration 1.5 g/L was added as antioxidant. It was found that optimum hydrolysis conditions were in 1.5 M HCl for 3 h on boiling water bath, protected from light. Under these conditions, small amounts of 3-O-caffeoylquinic acid and 5-O-feruloylquinic acid are not completely hydrolyzed. By increasing the concentration of hydrochloric acid and duration of hydrolysis, degradation of phenolic components of interest and formation of quinones occur.

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Since C. platycarpos contains a wide variety of antioxidants, in the present study total flavonoid and phenolic acid content as well as antioxidative activity of various C. platycarpos extracts were investigated. The results obtained show a significant polyphenol content and antioxidant activity of the investigated plant. Moreover, a positive correlation between antioxidant activity and content of flavonoids and phenolic acids was found, indicating the responsibility of these compounds for the antioxidant effectiveness of C. platycarpos extracts and making C. platycarpos a good potential source of natural antioxidants.