Quantification and holistic quality evaluation of Wulingzhi extract by UHPLC-Q-Orbitrap-HRMS coupled with chemometric approaches.

The excreta of Trogopterus xanthipes ("Wulingzhi" in Chinese, WLZ) is a well-known traditional Chinese medicine. It has been used for centuries to treat amenorrhea, menstruation and postpartum abdominal pain. However, a systematic quality study on WLZ chemical markers has yet to be conducted. This study aimed to establish an ultra-high-performance liquid chromatography coupled with a hybrid quadruple extraction Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) method for the simultaneous quantitative determination of 20 compounds in 53 batches of WLZ; the method rapidly and sensitively determined the 20 plant- or animal-derived compounds. Firstly, the proposed approach was validated to satisfy the method's linearity, detection limits, precision, repeatability, stability and accuracy. Subsequently, multivariate analysis was used to identify correlations between the samples and feed, processing and regions. Finally, this method was used to further identify chemical markers for quality control in combination with chemometrics. This is the first report on pinusolide, betaine, hippuric acid, 4-oxorentinoic acid, 15-methoxypinusolidic acid and 4-oxoisotrentinoin in WLZ; the quality of WLZ became homogeneous after processing with vinegar (V-WLZ). Moreover, we screened for potential component markers, including uridine, allantoin, amentoflavone, hippuric acid, 3,4-dihydroxybenzoic acid, pinusolide, quercetin and kaempferol. These results were practical and efficient for the chemical clarification of WLZ and V-WLZ.

Total saponins from stems and leaves of Panax quinquefolius L. ameliorate podophyllotoxin-induced myelosuppression and gastrointestinal toxicity.

Podophyllotoxin (POD), a natural lignan distributed in podophyllum species, possesses significant antitumor and antiviral activities. But POD often causes serious side effects, such as myelosuppression, gastrointestinal toxicity, neurotoxicity, hepatic and renal dysfunction, and even death, which not only hinder its clinical application but also threaten the patient's health. Therefore, an effective treatment against POD-induced toxicity is important. Our preliminary study found that the total saponins from the stems and leaves of Panax quinquefolius L. (PQS) could significantly reduce the death of mice caused by POD. To reveal how PQS can alleviate POD-induced toxicity, further study was needed. Peripheral blood cell analysis, diarrhea score, and histological examination demonstrated that PQS could relieve myelosuppression and gastrointestinal side effects induced by POD. Then, metabolomics was performed to investigate the possible protective mechanism of PQS on POD-induced myelosuppression and gastrointestinal toxicity. Metabolomics analysis showed that metabolic changes caused by POD could be reversed by PQS to some extent; 23 metabolites altered significantly after POD exposure, and 11 metabolites significantly reversed by PQS pretreatment. Metabolic pathway analysis suggested that PQS might exhibit its protective effects by rebalancing disordered arginine, glutamine, and unsaturated fatty acid metabolism.

[Simultaneous determination of sixteen mycotoxins contaminants in cogon rootstalk by QuEChERS-UPLC-QqQ mass spectrometry].

A method for the simultaneous determination of sixteen mycotoxins in cogon rootstalk was developed using ultra-performance liquid chromatography coupled with triple quadropole mass spectrometry(UPLC-QqQ-MS/MS). The samples were extracted with acetonitrile contained 1% acetic acid and purified by QuEChERS method. The separation was performed on an Agilent Eclipse Plus C18column by gradient elution using methanol and 0.01% aqueous formic acid as mobile phase. The targeted compounds were detected in MRM mode by mass spectrometry with electrospray ionization(ESI)source operated in positive ionization mode. The linear relationships of the sixteen mycotoxins were good in their respective linear ranges. The correlation coefficients(r)ranged among 0.996 2-1.000. The LOQs of the sixteen mycotoxins were between 0.03 and 186.68 µg•kg ⁻¹. The average recoveries ranged from 60.28% to 129.2% with relative standard deviations(RSDs)within 0.29%-11%. The results demonstrated that the proposed method was sensitive and accurate, and suitable for the mycotoxins quantification in cogon rootstalk.

Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis.

BACKGROUND: T helper (Th) 17 and regulatory T (Treg) cells play a critical role in the pathogenesis of multiple sclerosis (MS) disease. Bu Shen Yi Sui Capsule (BSYSC), a traditional Chinese medicine formula, has been used clinically for the treatment of MS patients in China. METHODS: To evaluate the neuroprotective effects and the underlying mechanisms of BSYSC on MS, experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice was induced with myelin oligodendrocyte glycoprotein (MOG) 35-55. Th17 and Treg cells and the related cytokines were detected by flow cytometry, ELISA, real-time quantitative reverse transcription PCR, western blot and immunohistochemistry. RESULTS: We found that BSYSC improved neurological function, reduced inflammatory cell infiltration and damage to the axons and myelin in the brain and spinal cord. BSYSC down-regulated markedly the ratio of CD4 + IL-17+/CD4 + CD25 + FoxP3+ T cells in the spleen, decreased the cytokines of IL-17A, IL-6, IL-23, TGF-beta1 in the brain, and dropped the ratio of IL-17A and FoxP3 mRNA and protein in the brain or spinal cord at different stages. CONCLUSIONS: The study demonstrated that BSYSC had a strong neuroprotective effect on EAE mice. The protective mechanisms of BSYSC might be associated with mediating the regulation of Th17/Treg cells.

[Determination of 7 flavonol glycosides in Ginkgo biloba reference extract].

Six flavonol glycosides were isolated and calibrated from Ginkgo biloba extract, and then used to calibrate the content in 2 baiches of G. biloba reference extract, so was rutin. RSD values of rutin, kaempferol-3-O-rutinoside, kaempferol-3-O-rhamnoside-2-glu- coside, quercetin-3-O-rhamnop-yranosyl-2-O-(6-O-p-coumaroyl)-glucoside, kaempferol-3-O-rhamnopyranosyl-2-O-(6-O-p-coum-aroyl) - glucoside were around 1.1%-4.6%, nevertheless, RSD values of quercetin-3-O-glucoside and isorhamnetin-3-O-rutinoside were more than 5%. According to the results, the reference extract of G. biloba can be used as the substitute to determine rutin, kaempferol-3-O- rutinoside, kaempferol-3-O-rhamnoside-2-glucoside, quercetin-3-O-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside and kaempferol-3-0-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside instead of corresponding reference substances. So reference extract in place of single component reference in assay is feasible.

New oleanane-type triterpenoid saponins isolated from the seeds of Celosia argentea.

Three new oleanane-type triterpenoid saponins named celosins H, I, and J were isolated from the seeds of Celosia argentea L. Their structures were characterized as 3-O-ß-D-xylopyranosyl-(1 → 3)-ß-D-glucuronopyranosyl-polygalagenin 28-O-ß-D-glucopyranosyl ester, 3-O-ß-D-glucuronopyranosyl-medicagenic acid 28-O-ß-D-xylcopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl ester, and 3-O-ß-D-glucuronopyranosyl-medicagenic acid 28-O-α-L-arabinopyranosyl-(1 → 3)-[ß-D-xylcopyranosyl-(1 → 4)]-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl ester by NMR, MS, and chemical evidences, respectively. In our opinion, celosins H-J could be used as chemical markers for the quality control of C. argentea seeds.

[Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi].

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

Qualitative and quantitative analysis of major components of Qiye Shen'an tablet by UPLC Q-TOF/MS and UPLC-TQS-MS/MS.

The Qiye Shen'an tablet is formulated using total saponins extracted from Notoginseng stems and leaves. At present, the study on its chemical composition remains scarce and the quality control indicators are limited, which seriously hindering the effective quality control and clinical research. Hence, this study aims to comprehensively identify and characterize the Qiye Shen'an tablet while controlling its main component contents. To achieve a comprehensive understanding of this tablet, an ultra-high performance liquid coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method was employed for its separation and characterization. Through the analysis of 99 batches of Qiye Shen'an tablet produced by 9 enterprises, the characteristic quantitative components were further obtained. A total of 113 compounds were characterized and identified, among which 17 representative compounds were selected, and the ultra-high performance liquid-triple quadrupole tandem mass spectrometry (UPLC-TQS-MS/MS) method was established for further quantitative determination. It has been successfully applied to the content determination of 99 batches of Qiye Shen'an tablet, and a new quality control method is being formed. This study provides a new method for chemical spectrum analysis and determination of labeled compounds of Qiye Shen'an tablet, and lays a solid foundation for further study of potential active ingredients and comprehensive quality evaluation.

Simultaneous determination of eleven flavonoid glycosides in ginkgo biloba leaves collected in different seasons by UPLC PDA method.

A new UPLC method was developed for the simultaneous determination of eleven characteristic flavonoid glycosides in Ginkgo biloba leaves. The natural occurrence of flavonoid glycosides in Ginkgo biloba leaves within one vegetative season was investigated for the first time. The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 column (50 mm x 4.6 mm, 1.8 microm), the mobile phase A was acetonitrile, the mobile phase B was 0.4% phosphate aqueous solution in a gradient elution at a flow rate of 0.6 mL x min(-1), the detection was carried out at 360 nm. The result showed that eleven flavonoid glycosides had good linearity with good average recovery, separately. The method was proved to be accurate, rapid and good reproducible for the quality evaluation of Ginkgo biloba leaves, and provide an easy and rapid means for the quantitative analysis of flavonoid glycosides and their content fluctuation with seasons.

Identification and Quantification of Bu Shen Yi Sui Capsule by UPLC-LTQ-Orbitrap-MSn and UPLC-QTOF-MS/MS.

Traditional Chinese medicines (TCMs) have been considered as important alternative therapeutics because of their significant medicinal benefits in specific diseases. Chinese herb formula is characterized by a vast molecule that differs in routine medicines. Due to TCMs chemical complexity, proper quality control has been a great challenge. Choosing the appropriate method to identify and qualify these compounds is an important work to ensure its safety, efficacy and quality control. Thus, this study aimed at providing novel information on high-resolution LTQ-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap-MSn) based identification of Bu Shen Yi Sui capsule (BSYSC), which is used in treating multiple sclerosis as a kind of TCMs. Under the proposed chromatographic conditions, 80 chemical components classified as anthraquinone, phenolic acid and phenylethanoid glycosides were separated and identified from BSYSC. Coupled with the high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) method, eight of them were regarded as marker compounds for the quantitative evaluation of BSYSC. The identification and quantification with precision of UPLC-LTQ-Orbitrap-MSn and UPLC-QTOF-MS/MS could facilitate essential data for further pharmacokinetic studies of BSYSC.

[Metabolic pathway and metabolites of pseudolaric acid B].

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.

Characterization of diterpenoids in the bark of Pseudolarix kaempferi by HPLC-ESI/MSn.

Fragmentation behavior of diterpenoids was investigated by ESI/MSn and the qualitative analysis of diterpenoids in the bark of Pseudolarix kaempferi was performed using high-performance liquid chromatography/ multi-stage mass spectrometry (HPLC-ESI/MSn). The characteristic fragmentation behaviors of the diterpenoids are the cleavages of the lactone ring and C4-O bond. Furthermore, the eliminations of substituent groups at C-18, C-7 and C-8 can also be observed in the MS" (n = 3-4) spectra. For C-4 acetoxy subsititued diterpenoids, [M+Na-60]+ and [M-H-104] are the base peaks of MS2 spectra in the positive and negative ionization modes, respectively. For C-4 hydroxyl subsititued diterpenoids, [M+Na-44]+ and [M-H-62] are the base peaks of MS2 in the positive and negative ionization modes, respectively. For C-18 glucosylated or esterized diterpenoids, [M+Na-44]+ is the base peak of MS2 spectra in positive ionization mode. These fragmentation rules were successfully exploited in the identification of diterpenoids in methanol/water (6:4) extract of P. kaempferi by LC-MS in positive ionization mode. A total of 9 diterpenoids were identified or tentatively characterized, and one of them is reported here for the first time. The described method could be utilized for the sensitive and rapid qualitative analysis of P. kaempferi.

[Determination of main free amino acids in Banlangen Keli by UPLC].

OBJECTIVE: To establish a quantitative method with precolumn derivatization for determining the contents of six common amino acids in Banlangen Keli by UPLC. METHOD: Using 6-acetamido-4-hydroxy-2-methyl quinoline as the derivating agent, we determined the contents of arginine, threonine , alanine, gamma aminobutyric acid, proline, and valine. The UPLC analysis was performed on a Waters AccQ Tag TM Ultra C18 column (2.1 mm x 100 mm, 5 microm) with mobile phase AccQ Tag Ultra Eluent A and AccQ Tag Ultra Eluent B gradient elution at a flow rate of 0.7 mL x min(-1). The column temperature was 55 degrees C and detection wavelength was 260 nm. RESULT: The linear ranges of arginine, thremine, alanine, gamma aminobutyric acid, proline, and valine were 4. 15549.86 microg (r = 0.999 9), 0.595-5.95 microg (r = 0.999 8), 0.445-4.45 microg (r = 0. 999 9), 0.515-5. 15 pg (r = 0.999 9), 8.858-106.3 microg (r = 0.999 9) , 0.585-5. 85 microg (r = 0.999 8). Their average recoveries were 100.6%, 98.35%, 100.2%, 98.44%, 98.34%, 98.18% with RSD 1.8%,1.9%, 2.0%, 2.4%, 1.5% and 2.0%, respectively (n = 6). The contents of amino acids were different in samples from five productive enterprises. CONCLUSION: The method is efficient, good reproducible, sensitive, and accurate.

Consistency evaluation of Chaihuang granules based on pharmacokinetics and metabolomics.

Consistency evaluation of Traditional Chinese Medicinal preparations (TCMPs) with complex chemical composition is challenging. Chaihuang granules (CHG), as a well-known TCMP, consists of Chaihu (Bupleuri Radix) and Huangqin (Scutellariae Radix) extract. In this work, we used pharmacokinetics and metabolomics to evaluate consistency of CHG products from two different manufacturers. In the pharmacokinetic study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was applied to determine the plasma concentration-time profiles of baicalin in rat plasma. Pharmacokinetic parameters, including the maximum concentration in blood (Cmax), area under the curve (AUC), the time to reach Cmax (Tmax), and half-life (T1/2), were calculated to assess the consistency preliminarily. And there was no significant difference in these pharmacokinetic parameters between the two CHG. In LC-MS-based metabolomics, the metabolic response profiles changes based on relative distance values (RDV) to different CHG products were compared. Meanwhile, the kinetic process of 31 differential endogenous metabolites that altered by CHG were determined. Metabolomics data showed the similar metabolic regulation effects to rats of the two formulations. Both pharmacokinetic and metabolomics results indicated there was no significant difference between CHG products. Furthermore, metabolic pathways significantly altered by CHG were elucidated, including phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, phenylalanine metabolism, and sphingolipid metabolism. Pharmacokinetics combined with metabolomics could provide a comprehensive perspective for consistency evaluation of CHG.

Simultaneous Determination of Prostaglandin and Hormones in Excreta of Trogopterus xanthipes.

The excreta of Trogopterus xanthipes (also called Wulingzhi in Chinese, WLZ) is a well-known traditional Chinese medicine used for the treatment of irregular menstruation in clinic. Few reports are available on the chemical profiling of WLZ. In this work, qualitative and quantitative analyses of endogenous prostaglandin and hormones in WLZ were performed using UHPLC-orbitrap-MSn. In total, 48 compounds were identified in urine of T. xanthipes. Furthermore, the contents of four target compounds were simultaneously quantitated in 20 batches of samples by UPLC-MS/MS. The quantitative method showed a good linear correlation (R > 0.995) in a wide range for each compound. The method had a high sensitivity with LOD (0.5-1.0 ng/mL) and LOQ (1.0-2.5 ng/mL). The intra- and inter-day precisions were < 9.17 (RSD %), and repeatability and stability were < 6.14 (RSD %). The recovery of the analytes varied between 85.8% and 97.3% at three different concentrations. The present integrated qualitative and quantitative assessment of WLZ provides an evaluation strategy to assess the constituent in traditional Chinese medicine.

An Updated Review of Randomized Clinical Trials Testing the Improvement of Cognitive Function of Ginkgo biloba Extract in Healthy People and Alzheimer's Patients.

Alzheimer's disease (AD) is a common neurodegenerative disease, mainly manifested by cognitive dysfunction. It seriously reduces the quality of life, and there is no ideal treatment strategy in clinical practice. Clinical studies on the treatment of AD with Ginkgo biloba L. leaf extract (EGb) have been reported since 1980s, and many clinical studies have been carried out during the following 30 years. However, the benefits of EGb on the treatment of AD are still controversial. In this review, we collected the clinical trial reports of EGb on cognitive function from Pubmed, Elsevier, Europe PMC, and other database since the 1980s. Through analysis and comparison, we consider that EGb may be able to improve the cognitive function in patients who suffered from mild dementia during long-term administration (more than 24 weeks) and appropriate dosage (240 mg per day). The main evidences and existing problems of the negative and positive experimental results were summarized.

Antidepressant-like effect of liquiritin from Glycyrrhiza uralensis in chronic variable stress induced depression model rats.

Many flavonoids extracted from nature plants have been reported to exert antidepressant-like effect in animal studies. The present study was designed to observe the effects of liquiritin, a flavone compound derived from Glycyrrhiza uralensis, on the behaviors of chronic variable stress induced depression model rats and to explore the possible association between its antidepressant-like effect and antioxidative activity by measuring erythrocyte superoxide dismutase (SOD) activity and plasma malondialdehyde (MDA) level of the experimental animals. With the exposure to stressor once daily for consecutive 5 weeks, liquiritin and a positive control drug fluoxetine were administered via gastric intubation to rats once daily for consecutive 3 weeks from the 3rd week. The results showed that CVS reduced open-field activity and sucrose consumption significantly, but increased immobility time in forced swimming test. Treatment of liquiritin could effectively reverse alteration in immobility time and sucrose consumption but did not show significant effect on open-field activity. Moreover, liquiritin could increase SOD activity, inhibit lipid peroxidation, and lessen production of MDA, while fluoxetine did not. In conclusion, the present study demonstrated a potential antidepressant-like effect of liquiritin treatment on chronic variable stress induced depression model rats, which might be related to defense of liquiritin against oxidative stress.

Antidepressant-like effects of liquiritin and isoliquiritin from Glycyrrhiza uralensis in the forced swimming test and tail suspension test in mice.

Two classic animal behavior despair tests--the Forced Swimming Test (FST) and the Tail Suspension Test (TST) were used to evaluate the antidepressant activity of liquiritin and isoliquiritin from Glycyrrhiza uralensis in mice. It was observed that both liquiritin and isoliquiritin at doses of 10, 20 and 40 mg/kg significantly reduced the immobility time in the FST and TST in mice 30 min after treatment. Measurement of locomotor activity indicated that liquiritin and isoliquiritin had no central nervous system (CNS)-stimulating effects. The main monoamine neurotransmitters and their metabolites in mouse brain regions were also simultaneously determined by HPLC-ECD. It was found that these two compounds significantly increased the concentrations of the main neurotransmitters 5-HT and NE in the hippocampus, hypothalamus and cortex. Liquiritin and isoliquiritin also significantly reduced the ratio of 5-HIAA/5-HT in the hippocampus, hypothalamus and cortex, slowing down 5-HT metabolism compared with mice treated with vehicle+stress. In conclusion, liquiritin and isoliquiritin produced significant antidepressant-like effects, and their mechanism of action may be due to increased 5-HT and NE in the mouse hippocampus, hypothalamus and cortex.

[Fingerprint analysis of Radix Glycyrrhizae by fast HPLC].

The objective of this paper is to develop a fast analysis method to determine fingerprints of Radix Glycyrrhizae from different areas of China for identification and quality control. The experiments were carried out under following conditions: Agilent Eclipse Plus C18 (4.6 mm x 50 mm, 1.8 microm) column, acetonitrile and 0. 05% phosphoric acid solution as the mobile phases with gradient elution, flow rate 1.0 mL x min(-1), analysis time 11 min. The run time of the method was obviously decreased from 36 minutes to 11 minutes compared with routine HPLC method. The cluster analyses of the fingerprints of the 70 samples were performed by SPSS. The results showed that all samples were classified into 2 groups, 59 Glycyrrhiza uralensis as well as 11 G. inflata. Three compounds, liquiritin apioside, liquiritin and glycyrrhiza acid should be considered as effective references for quality control of Radix Glycyrrhizae. This method can be used widely for identification and quality control of Radix Glycyrrhizae.

Steroidal saponins from Smilax china and their anti-inflammatory activities.

Steroidal saponins, 1, 2, 3 and 4, were isolated from the BuOH extract of Smilax china L., along with 13 known compounds, 5-17. Their structures were elucidated on the basis of MS, 1D and 2D NMR spectroscopic analyses and chemical evidence. In the bioassay tests, all compounds showed inhibitory effects on cyclooxygenase-2 enzyme (COX-2) activities at final concentration of 10(-5) M, and only compound 5 showed an inhibitory effect on production of TNFalpha (tumor necrosis factor alpha) in murine peritoneal macrophages at the same concentration.