Chromatographic fingerprint development for quality assessment of “ayurved Siriraj prasachandaeng” antipyretic drug.

Quality assessment of traditional herbal medicines is of benefit not only in research but also in practice. The method of quality assessment of the Thai traditional medicine, Ayurved Siriraj Prasachandaeng, was established by using High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography (HPLC). In HPTLC, the chromatographic fingerprints were developed; the color and the relative retardation factor (rRf) of bands were compared with those of reference markers. Likewise, relative retention time (rRt), and applied information content (f) were evaluated in HPLC fingerprints. Reference markers, gallic acid, caffeic acid, p-coumaric acid, ferulic acid, vanillic acid and kojic acid were used as qualitative markers in HPTLC whereas gallic acid, caffeic acid and vanillic acid were used as qualitative and quantitative markers in HPLC. Similarity of the chromatographic pattern among batches was determined by the presence of stated mathematic parameters in the range of 80 to 125 percent of the average. The HPTLC and HPLC fingerprints of three batches of Ayurved Siriraj Prasachandaeng showed similar chromatographic patterns. Such similarity showed that the productions of different batches in the recipe were consistent. Moreover, it revealed that some markers found in the recipe certainly came from various medicinal herbal components of their own recipes. In conclusion, the combination of rRf from HPTLC, and rRt and f from HPLC is the suitable method for identification and quality control of different batches of Ayurved Siriraj Prasachandaeng.

Cholangiocarcinoma cell induced platelet aggregation via activation of thrombin and cyclooxygenase.

Objective: To investigate whether Cholangiocarcinoma (CCA) cells affected platelet aggregation via activation of thrombin and cyclooxygenase. Methods: Human Cholangiocarcinoma (HuCCA) cells were cultured in T-75 Flasks with a standard technique. Cells were grown to confluence until used, after which, cells were detached and then resuspended in DMEM. Resuspended HuCCA cells were assessed for their effects on inducing platelet aggregation of normal volunteers using Born’s technique. Hirudin, apyrase, EDTA and indomethacin were used as pharmacological tools to investigate signaling mechanisms. Results: HuCCA cells can initiate aggregation of platelets in heparinized platelet rich plasma (hPRP). The potency of HuCCA cell induced platelet aggregation depends on the concentration of cell suspension. Interestingly, HuCCA cell induced platelet aggregation was inhibited by hirudin (10, 20 and 40 unit), EDTA (2, 3 and 4 mM) and indomethacin (0.1, 1 and 2 mM) in a dose dependent manner but not by apyrase (5, 10 and 20 units). Conclusion: HuCCA cells can induce platelet aggregation possibly via activation of thrombin and the cyclooxygenase signaling pathway but not the ADP pathway. Calcium may be an important factor required for this reaction.

Expression of cyclooxygenase-1 and -2 in cultured human glioma cells of various stages.

Cyclooxygenase-1 (COX-1) and -2 (COX-2) are two isoforms of enzymes responsible for the biosynthesis of several forms of prostaglandins (PGs) from arachidonic acid. COX-1 is constitutively expressed in most normal tissues while COX-2 expression is regulated by certain stimuli such as cytokines and growth factors. The expression of COX-2 has been associated with many pathological conditions such as atherosclerosis, inflammation, and cancers (e.g. colon, breast, and lung cancers). COX-2 expression may be associated with the progression of cancers since PGE2, a major product of both isoforms was identified as a tumor-derived immune suppressor. Therefore, the suppression of COX-2 activity using specific COX-2 inhibitors (recently classified non-steroidal anti-inflammatory drugs) may delay the progression of tumors harboring COX-2. The search for tumors that highly express both isoforms of COX serves as a guide to target tumors that are most likely to be susceptible to treatment with specific COX-2 inhibitors. To study a homogeneous population of tumor cells, fifteen samples of primary culture cells were prepared from different human brain tissues and tumors collected from subjects operated on at Siriraj Hospital. Cells were grown to confluent in 75-cm2 tissue culture flask for about 4 weeks. After which time, cells were extracted to evaluate COX-1 and COX-2 protein expressions by immunoblot using specific antibodies. Four out of seven samples of glioblastoma multiforme cells strongly expressed COX-2, while all samples of examined cultured cells expressed COX-1. Cultured cells from astrocytomas had only faint staining for COX-2, while maintaining strong COX-1 expression. Thus COX-2 expression was limited to samples of glioblastoma multiforme, the most advanced stage of astrocytoma. Further study for the suppression of COX-2 activity in inducing tumor apoptosis and in improving cellular immunity against this advanced cancer should be elucidated.

The side effects of non-steroidal anti-inflammatory drugs: Are they attributed to the selective inhibition of different isoforms of cyclo-oxygenase.

Cyclooxygenase (COX)exists as two isoforms. Cox-1 in present under physiological conditions and COX-2 is induced by inflammatory stimuli. Previous studies in vitro have suggested that the side-effects of non-steroidal anti-inflammatory drugs (NSAIDs) correlate with their ability to inhibit COX-1, while the anti-inflammatory effects are due to their ability to inhibit COX-2 In order to strengthen this hypothesis, we examined the correlation between the inhibitory effects of eight NSAIDs (ibuprofen, aspirin, diclofenac, sulindac, naproxen, indomethacin, tolmetin and piroxicam) on the activity of COX isoforms in vitro and the range of relative risks of gastrointestinal (GI) complications that have been reported with individual NSAID in a meta-analysis. Analysis by Spearman rank correlation test demonstrated that IC50 complications (rs = 0.74, p < 0.05). A similar analysis using the IC50 of NSAIDs on the activity of COX-2 did not demonstrate any significant correlation (rs =- 0.59, p = 0.12). The ratio of COX-2/COX-1 did not appear to have any significant correlation with the risks of clinical GI side-effects (rs = 0.5, p = 0.21). Further analyses of in vitro data from other sources are encouraged to validate the usefulness of this model in predicting the GI side effects of each particular NSAID.

Platelet serotonin in headache patients treated by new trend acupuncture.

Headache can be treated with acupuncture but its mechanism is virtually unknown. Since serotonin is considered to be an important factor as well as hyperactive platelets in the causation of migraine headache, a before and after study design was employed to evaluate the effects of acupuncture on the platelet serotonin and platelet aggregation in headache patients. Thirty-three patients, 18 suffering migraine headaches and 15 tension headaches were treated with dry needle acupuncture accompanying breathing exercise. Platelet serotonin was measured by spectrophotofluorometric method before and after treatment and found to be increased in 25 patients and decreased in 8 patients. The former groups show a statistically significant increase of p<0.01 (mean difference 0.15 + 0.26). Platelet aggregation induced by aggregating agents was also calibrated as to maximum response. The aggregations induced by collagen 1 ug, ADP 3 Um, adrenaline 1 or 25 uM, were decreased after acupuncture with significant collagen-induced aggregation (p<0.05). This suggests platelet serotonin and aggregation play a significant role in the treatment of headache by both inhibiting aggregation and by serotonin release. Thus the platelet serotonin is increased. Some interesting pints have been discussed in this report.