Chromatography: An important tool for drug discovery.

Despite substantial developments of extraction and separation techniques, isolation of natural products from natural sources is still a challenging task. Undoubtedly hybrid methods like liquid chromatography with NMR spectroscopy or liquid chromatography coupled with mass spectrometry made on-line structure elucidation possible and provided impressive examples of natural product identification without prior isolation, however, in many cases the necessity to get the purified compounds in hand is still a fact. The process begins with the collection of desired plant material which is subjected to the suitable extraction process. The complex crude extracts are then monitored by various chromatographic procedures to separate and quantify the desired compounds. The active plant extracts are then fractionated to isolate the bioactive compounds in their pure form. The fully identified compound is used as a lead for the production of related analogues to modulate the biological activity and to carry out structure-activity relationship. The major isolated bioactive compound is used for semi-synthetic modification or total synthesis should be carried out such that it is relatively easy to modify the structure of the lead compound. This is a simple and cost-effective way to increase the chance to discover lead compounds. The biological activity in vitro and in vivo has to be done after purification.

The effect of an intraorifice barrier and base under coronal restorations on the healing of apical periodontitis: a randomized controlled trial.

AIM: To evaluate the effect of intraorifice barriers and bases on the healing of apical periodontitis following root canal treatment in mandibular molars. METHODOLOGY: A total of 120 permanent mandibular molars with necrotic pulps and periapical radiolucencies (PAI score ≥ 3) were recruited. Root canal treatment was performed in all teeth using a standard protocol, following which they were randomly allocated to one of the three treatment groups: intraorifice barrier group: coronal 3-mm gutta-percha was removed and replaced with glass-ionomer cement (GIC) barrier. The floor of the pulp chamber was then sealed with 2-mm-thick GIC base followed by final composite resin restoration; base group: received 2-mm-thick GIC base before placement of composite resin restoration; and control group: had pulp chamber entirely filled with composite resin only. Follow-up was done at 3, 6, 9 and 12 months. Combination of clinical and radiographic parameters were used to assess treatment outcome. The data were analysed using Kruskal-Wallis, chi-square and Wilcoxon signed-rank tests and logistic regression analysis. RESULTS: At the end of 12 months, the base group had the most favourable healing (97.1%), whilst the control group had the least favourable healing (83.8%). The intraorifice barrier group had healing of 92.1%. However, there was no significant difference in healing between groups at the end of the follow-up period (P > 0.05). Additional subgroup analysis revealed a nonsignificant effect of periodontal status and root filling level on periapical healing. CONCLUSION: The use of an additional barrier under permanent restorations did not significantly improve the outcome of primary root canal treatment in posterior teeth after 12 months. However, its influence in the long term requires further evaluation.

2D and 3D radiographic outcome assessment of the effect of guided tissue regeneration using resorbable collagen membrane in the healing of through-and-through periapical lesions - a randomized controlled trial.

AIM: To evaluate the effect of collagen membrane on the healing of through-and-through periapical lesions using 2-dimensional (2D) and 3-dimensional (3D) imaging techniques. METHODOLOGY: Thirty-two patients with periapical radiolucencies measuring at least 10 mm and with confirmed loss of buccal and lingual cortical plates were randomly divided into GTR and control groups. Periapical surgery was performed in both groups, using a resorbable collagen membrane in the GTR group only. 2D healing was evaluated according to Molven's criteria, while 3D healing was assessed using modified PENN 3D criteria, RAC indices and the B index, 12 months after surgery. Data were analysed using Chi-square, Mann-Whitney and Wilcoxon signed rank tests. RESULTS: Thirty patients were analysed at the 12 months follow-up. Both groups had a significant reduction in the size of lesions [92 ± 12% (control) and 86 ± 14% (GTR) in 2D and 85 ± 19% (Control) and 82 ± 13% (GTR) in 3D], with no significant difference between the groups (P > 0.05). A total of 29(14 control, 15 GTR) teeth and 26(13 control, 13 GTR) teeth were classified as success according to Molven's (2D) and modified PENN 3D criteria, respectively, with no significant difference in success between 2D and 3D assessments. RAC indices of 3D images revealed the greatest percentage of complete healing in R scores (55% for Control, 41% for GTR), whereas cortical plate had the lowest percentage of healing (30% for Control, 27% for GTR) (P > 0.05). Only 2 (13%) patients in each group had complete healing using the B index. CONCLUSION: Periapical surgery with or without GTR was a predictable and viable solution for through-and-through lesions. There was no benefit in using a collagen membrane with regard to the outcome of periapical surgery in through-and-through lesions. Both cone beam computed tomography (CBCT) and periapical radiographs (PA) allowed similar assessment of healing after periapical surgery.

Effect of continuous ultrasonic irrigation on postoperative pain in mandibular molars with nonvital pulps: a randomized clinical trial.

AIM: To evaluate via a randomized clinical trial the effect of continuous ultrasonic irrigation (CUI) on postoperative pain in mandibular molars with nonvital pulps. METHODOLOGY: Seventy mandibular molars with nonvital pulps and apical periodontitis were treated endodontically using two different irrigation techniques. The patients were randomly allocated to one of two groups, CUI (n = 35) and syringe irrigation (SI) (n = 35). The CUI group received irrigant activation using a Proultra Piezoflow ultrasonic needle as the final irrigation protocol, whilst in the SI group, the final irrigation was performed using 27-gauge needle. All the patients were prescribed ibuprofen 400 mg to be taken every 8 h, if required. Postoperative pain using a visual analog scale and analgesic intake were recorded everyday for 7 days. Data were analysed using chi-Square, Mann-Whitney and multiple linear regression tests. RESULTS: Mean postoperative pain was lower in the CUI as compared to SI group, but the difference was significant on the first day only (P = 0.032). The overall 24-h pain prevalence was 41.4%. CUI had a lower incidence of pain (31.4%) as compared to the SI group (51.4%), but the difference was not significant (P > 0.05). No significant difference was observed in analgesic consumption between the groups (P > 0.05). Regression analysis revealed a significant association of mean postoperative pain at 24 h with the irrigant protocol (P = 0.017) and preoperative pain (P = 0.000). CONCLUSION: A significant difference was observed between CUI and syringe irrigation on the first postoperative day following chemo-mechanical preparation. However, the benefit observed was not clinically relevant.

Effect of maintaining apical patency on endodontic pain in posterior teeth with pulp necrosis and apical periodontitis: a randomized controlled trial.

AIM: To evaluate the association between apical patency and post-operative pain in posterior teeth with pulp necrosis and apical periodontitis. METHODOLOGY: Sixty-eight patients requiring primary root canal treatment in mandibular first molars with necrotic pulps and apical periodontitis were included. The patients were randomly allocated to one of two groups: patency (n = 34) and nonpatency (n = 34). After administering local anaesthesia, root canal preparation was completed using ProTaper rotary instruments. A size 10 K-file was used as a patency file and carried 1 mm beyond the working length (WL) between each instrument change in the patency group, while it was carried up to WL in the nonpatency group. Patients were asked to record their pain experience on a pain chart daily for 7 days. Three patients (two in the patency group, one in the nonpatency group) did not return with completed pain charts on the subsequent visit, resulting in a total of 65 patients for the final analysis (patency, n = 32; nonpatency, n = 33). Data was analysed using Chi Square test, t-test, Mann-Whitney test and Wilcoxon Signed Ranks test. RESULTS: Overall, 43% of the patients experienced post-operative pain. The patency group had less incidence of pain (34%) as compared to the nonpatency group (52%), but the difference was not significant (P = 0.163). CONCLUSION: Maintenance of apical patency during chemomechanical preparation had no significant influence on post-operative pain in posterior teeth with necrotic pulps and apical periodontitis.

Pain following foraminal enlargement in mandibular molars with necrosis and apical periodontitis: A randomized controlled trial.

AIM: This randomized controlled trial was conducted to evaluate the effect of foraminal enlargement during chemomechanical root canal preparation on postoperative pain. METHODOLOGY: Seventy mandibular first molars with asymptomatic pulp necrosis and chronic apical periodontitis were randomized into two groups - foraminal enlargement (FE) and conventional nonforaminal enlargement (NFE). Canals were prepared according to the respective protocols, and the teeth were restored temporarily. Postoperatively, the patients were prescribed ibuprofen 400 mg, to be taken if required. Pain experience and analgesic intake were recorded for 7 days following chemomechanical preparation. The data were analysed using Mann-Whitney U-test and chi-square tests. RESULTS: Pain experience was higher with foraminal enlargement than when using a conventional technique. A significant difference was observed in postoperative pain during the first 4 days and the sixth day (P < 0.05), with greater pain in the FE as compared to the NFE group. There was no significant difference in prevalence of analgesic intake and number of doses between the experimental groups (P > 0.05). CONCLUSION: Enlargement of the apical foramen during root canal treatment increased the incidence and intensity of postoperative pain.

Tertiary dentinogenesis with calcium hydroxide: a review of proposed mechanisms.

Calcium hydroxide has been used extensively in dentistry for a century. Despite its widespread use as a pulp-capping agent, its mechanisms of action still remain ambiguous. Understanding its modes of action will lead to a broader understanding of the mechanisms associated with induced dentinogenesis and help in optimizing the currently available agents to target specific regenerative processes to obtain the best possible clinical outcomes. A literature search relating to mechanisms of dentinogenesis of calcium hydroxide up to December 2011 was carried out using pubmed and MEDLINE database searches as well as manual searching of cross-references from identified studies. Resulting suggestions regarding dentinogenic mechanisms of calcium hydroxide range from direct irritating action of the material to induction of release of biologically active molecules. The purpose of this article is to discuss various mechanisms through which calcium hydroxide may induce tertiary dentinogenesis in the light of observations made in included studies.

Comparative evaluation of antimicrobial efficacy of Syzygium aromaticum, Ocimum sanctum and Cinnamomum zeylanicum plant extracts against Enterococcus faecalis: a preliminary study.

AIM: To evaluate the antimicrobial efficacy of Ocimum sanctum, Cinnamomum zeylanicum, Syzygium aromaticum and 3% sodium hypochlorite (NaOCl) against Enterococcus faecalis in planktonic suspension and biofilm phenotypes. METHODOLOGY: The antibacterial efficacy of different concentrations of aqueous ethanolic extracts of O. sanctum, C. zeylanicum and S. aromaticum against E. faecalis at various time intervals was assessed using the agar well diffusion test, microdilution test and biofilm susceptibility assay (BSA) on cellulose nitrate membrane as well as in a tooth model. NaOCl was used as the positive control. Distilled water was used as negative control for agar diffusion and microdilution tests and phosphate-buffered saline for the BSA. The results of the agar diffusion test were analysed statistically using anova and Tukey's tests. RESULTS: Cinnamomum zeylanicum, S. aromaticum and O. sanctum exhibited minimum bactericidal concentration at 10%, 10% and 40%, respectively. Cinnamomum zeylanicum, S. aromaticum, O. sanctum and NaOCl showed complete bacterial inhibition in planktonic form after exposure of 30, 15, 35 and 1 min, respectively. In BSA on cellulose nitrate membrane, NaOCl was associated with complete bacterial inhibition after contact of 2 min, whilst 10% C. zeylanicum, 10% S. aromaticum and 40% O. sanctum showed cessation of growth after 12, 12 and 24 h, respectively. The results of BSA on tooth model were similar except for O. sanctum, which was not included in the model. CONCLUSION: Cinnamomum zeylanicum, S. aromaticum and O. sanctum demonstrated antimicrobial activity against planktonic and biofilm forms of E. faecalis with C. zeylanicum and S. aromaticum having better antimicrobial efficacy than O. sanctum. NaOCl had superior antimicrobial efficacy amongst all the groups.

4-epi-Pimaric acid: a phytomolecule as a potent antibacterial and anti-biofilm agent for oral cavity pathogens.

The present study focused on the antibacterial and biofilm inhibitory potential of 4-epi-pimaric acid isolated from aerial parts (stem and leaves) of Aralia cachemirica L. (Araliaceae) against oral cavity pathogens. 4-epi-Pimaric acid exhibited minimum inhibitory concentration (MIC) in the range of 4-16 µg/ml and minimum bactericidal concentration (MBC) two- to four-folds higher than MIC. There was significant inhibition in the biofilm formation by Streptococcus mutans on the saliva coated surface (P < 0.05), and confocal microscopy revealed that 4-epi-pimaric acid inhibited the clumping and attachment of S. mutans. At 8 × MIC concentration, it significantly prevented the pH drop and reduced S. mutans biofilms (P < 0.05). Increased propidium iodide staining and leakage of 260- and 280-nm absorbing material by 4-epi-pimaric acid treated cells of S. mutans suggested that it probably causes disruption of the cytoplasmic membrane structure. It also exhibited significant suppression of TNF-α expression in human neutrophils, suggestive of its anti-inflammatory activity. Furthermore, the compound was found to be significantly safe (IC(50) >100 µg/ml) in the MTT assay on AML-12 cell lines. In conclusion, 4-epi-pimaric acid showed promising antibacterial, anti-biofilm and anti-inflammatory potency and this compound can be exploited for therapeutic application in oral microbial infections.

Evaluation of the immunomodulatory and anti-inflammatory activity of Bakuchiol using RAW 264.7 macrophage cell lines and in animal models stimulated by lipopolysaccharide (LPS).

Bakuchiol (BAK) has been reported to have a diverse pharmacological property as an antibiotic, anti-cancer, anti-hypolipidemic, anti-inflammatory and anti-convulsant agent. This study aimed to elucidate the immunomodulation and anti-inflammatory mechanism of bakuchiol using lipopolysaccharide stimulated RAW 264.7 macrophages and various animal models. The present study has shown that BAK significantly suppressed the pro-inflammatory cytokine expression in a dose-dependent manner and its oral administration significantly decreased delayed hypersensitivity responses as compared to control group. The assessment of immunomodulatory activity was carried out by the testing Hemagglutinating antibody (HA) titer, delayed type hypersensitivity (DTH) responses and phagocytic index by carbon clearance test. On the other hand, it showed significant decrease in circulating antibody titer and carbon clearance assay in a concentration-dependent manner. BAK has significantly potentiated the cellular immunity as well as humoral immunity by facilitating the footpad thickness responses in sheep RBCs in sensitized mice by significantly decreasing circulating antibody titer. Molecular studies revealed that BAK inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. The responses were statistically significant as compared with the control (*p < 0.05, **p < 0.01).

Vermicomposting of sugar industry waste (press mud) mixed with cow dung employing an epigeic earthworm Eisenia fetida.

In India, millions of tons of press mud (PM) are generated by sugar mills every year. This paper reports the potential of vermitechnology to convert sugar industry waste PM mixed with cow dung (CD) into vermicompost, employing an epigeic earthworm Eisenia fetida. A total of six different reactors were established having different ratios of PM and CD including one control (CD only). The growth and fecundity of E. fetida was monitored for 13 weeks. Maximum growth was recorded in 100% CD, but earthworms grew and reproduced favourably up to 1:1 PM and CD feed composition. However, greater percentages of PM in different reactors significantly affected the growth and fecundity of worms. Vermicomposting resulted in a decrease in carbon concentration and an increase in nitrogen, phosphorus and calcium concentrations of the vermicompost. Investigations indicated that vermicomposting could be an alternative technology for the management of PM into useful fertilizing material, if mixed at maximum 50% with CD.

Anesthetic considerations in a parturient with renal tubular acidosis and hypothyroidism undergoing cesarean delivery.

Renal tubular acidosis, associated with hypothyroidism, is rare. We present the case of a woman with known renal tubular acidosis and treated hypothyroidism who underwent emergency cesarean delivery under uneventful combined spinal-epidural anesthesia. The rationale for choosing the anesthetic technique and the potential risks associated with anesthesia and renal tubular acidosis are discussed.

Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin.

Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3ß. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

Sub-acute toxicity evaluation of an aqueous extract of Labisia pumila, a Malaysian herb.

Labisia pumila (Myrsinaceae), is a popular herb among the women in Malaysia known locally as "Kacip Fatimah". Recently many nutraceutical products containing the powdered or extracted parts of the plant have become available for women's health care. However no evaluation of the effect of the repeated dosing of any herbal product of this plant had been undertaken prior to a 28-day sub-acute study presented in this report. The results showed that a dose of 50mg/kg of an aqueous extract of L. pumila corresponded to no-adverse-effect-level (NOAEL), whereas higher doses were associated with some toxicity concerns.