DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF QUERCETIN, ELLAGIC ACID AND RUTIN IN HYDROALCOHOLIC EXTRACT OF TRIPHALA CHURNA

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of quercetin, ellagic acid and rutin in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3 with orthophosphoric acid) and methanol in the ratio 55:45, at flow rate of 1 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 254 nm with a UV-visible detector.Results: The retention time of quercetin, ellagic acid and rutin were found to be 7.52 min, 9.10 min and 12.47 min respectively. Linearity of quercetin, ellagic acid and rutin were found in the range of 8-12 ppm, 9-17 ppm and 7-11 ppm respectively. The correlation coefficient for quercetin, ellagic acid and rutin were 0.997, 0.999 and 0.999 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of quercetin, ellagic acid and rutin in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulation containing these three markers

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ELLAGIC ACID AND QUERCETIN IN AN AYURVEDIC FORMULATION

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines. Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and acetonitrile in the ratio 60:40, at flow rate of 1.2 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 255 nm with UV-visible detector. Results: The retention time of ellagic acid and quercetin were found to be 1.65 min and 2.94 min respectively. Linearity of ellagic acid and quercetin were tested in the range of 6-14 ppm and 3-11 ppm respectively. The correlation coefficient for ellagic acid and quercetin were 0.997 and 0.993 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise. Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulation containing these two markers

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF GABAPENTIN AND AMITRIPTYLINE HYDROCHLORIDE IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of gabapentin and amitriptyline hydrochloride in gabantip-at tablet and validate as per international conference on harmonization (ICH) guidelines and to perform the force degradation studies using the developed method.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and mobile phase consisting of 0.05 M potassium dihydrogen orthophosphate pH 2.1 adjusted with orthophosphoric acid and acetonitrile in the ratio (55:45), at flow rate 1 ml/min and column temperature (25 °C). The effluents obtained were monitored at 221 nm with the UV-visible detector.Results: The retention time of gabapentin and amitriptyline hydrochloride was found to be 1.959 min and 4.221 min respectively. The linearity of gabapentin was found in the range of 720-1680 ppm and that for amitriptyline hydrochloride was found to be 24-56 ppm. The correlation coefficient for gabapentin and amitriptyline hydrochloride were 0.999 and 0.9963 respectively. The high recovery values (98%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous estimation of gabapentin and amitriptyline hydrochloride tablet dosage form. Hence the proposed method may find practical applications as a quality-control tool in the simultaneous analysis of the two drugs in combined dosage forms in quality-control laboratories

A NOVEL RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF BERBERINE, QUERCETIN, AND PIPERINE IN AN AYURVEDIC FORMULATION

Objective: The objective of this study was to develop and validate a novel, simple, rapid, precise and accurate reversed-phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of berberine, quercetin, and piperine in Ayurvedic formulation.Methods: The chromatographic separation was achieved using a stationary phase C18 shim-pack (150 mm x 4.6 mm, 5µ) column and mobile phase consisted of acetonitrile: 0.04 M potassium dihydrogen phosphate buffer (pH 3.0 adjusted using orthophosphoric acid) in a ratio of 65:35 v/v, with a flow rate of 1 ml/min and UV detection at 255 nm.Results: The retention time of berberine, quercetin, and piperine were found to be 2.7, 3.0 and 6.3 min respectively. Linearity for berberine, quercetin, and piperine were found in the range of 12-28 µg/ml. All calibration curve showed good linear correlation coefficients (r2˃ 0.999) within the tested ranges. Mean percent recoveries for berberine, quercetin, and piperine were found to be within the acceptance limits (98-120%). The percent relative standard deviation (% RSD) for precision was found to be less than 2% which indicates method is precise.Conclusion: The developed method is novel, simple, precise, accurate and can be used for quantitative analysis and quality control of the raw material as well as other commercial formulations containing these three markers

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF CURCUMIN, PIPERINE AND CAMPHOR IN AN AYURVEDIC FORMULATION

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of curcumin, piperine and camphor in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and acetonitrile in the ratio 40:60, at flow rate of 1 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 255 nm with UV-visible detector.Results: The retention time of curcumin, piperine and camphor was found to be 6.57 min, 7.32 min and 8.57 min respectively. Linearity of curcumin and camphor were found in the range of 4-8 ppm and that of piperine was found to be 5-9 ppm. The correlation coefficient for curcumin, piperine and camphor were 0.998, 0.99 and 0.994 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of curcumin, piperine and camphor in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other species containing these three markers

Effectiveness of sustained maximal inspiration along with transcutaneous electrical nerve stimulation in patients with malignant pleural effusion with intercostal drainage tube: a randomized controlled trial

Background: Malignant pleural effusion (MPE) is one of the most common causes of an exudative pleural effusion. It is the most common cause of a unilateral massive pleural effusion. Most MPE s are secondary to metastases to the pleura, most often from lung or breast cancer.  Medical management includes aspiration of fluid with the insertion of a chest tube, which may be necessary to relieve dyspnea. Hypoventilation does occur in certain areas of the lungs because of pain and muscle guarding after intercostal drainage tube (ICD) tube placements in pleural effusion. Therefore, it is important to emphasize pain management and expansion of affected areas of the lungs and chest wall. SMI is the basic maneuver of incentive spirometry and is mainly given to prevent atelectasis and lung collapse in postoperative patients. TENS is a method of producing an electro-analgesic effect and is effective in providing post-operative pain control. Methods: The study design of the study was a randomized controlled trial. A total of 44 participants with a diagnosis of MPE with an intercostal drainage tube were included in this study. The study duration was 5 years with an intervention period of 2 weeks and the outcome measures were pulmonary functions and intercostal pain severity using the NRS Scale. Results: There was a significant improvement in pulmonary functions in the control group and a highly significant improvement in pulmonary functions in the experimental group after 2 weeks of interventions and pain severity (NRS) was significantly reduced in the experimental group only (p<0.001) Conclusions: This study concluded that sustained maximal inspiration (SMI) along with TENS significantly improves pulmonary functions and reduces pain at the site of ICD in patients with MPE

SIMULTANEOUS RP-HPLC ANALYSIS OF QUERCETIN AND KAEMPFEROL IN DIFFERENT PLANT PARTS OF CISSUS QUADRANGULARIS

Objective: To develop a simple, rapid and specific reversed phase High-Performance Liquid Chromatography (HPLC) method for comparative analysis of flavonoids, quercetin and kaempferol, in different plant parts (leaves, stem and roots) of Cissus quadrangularis Linn.Methods: The HPLC method which can be used effectively for separation of components from plants has been developed to perform a comparative analysis of flavonoids, quercetin and kaempferol, in different plant parts (leaves, stem and roots) of Cissus quadrangularis Linn. An endcapped C18 column at 370 nm, and water: acetonitrile (45:55) containing 0.1% o-phosphoric acid as mobile phase was used.Results: Quercetin and kaempferol were well resolved at about 5 min and 7 min respectively. Calibration curves for quercetin and kaempferol were linear in the range of 1-10 µg/ml (R2= 0.999) and 0.5-10 µg/ml (R2= 0.999) respectively. The sensitivity of the method was found to be higher, with a limit of detection (LOD) values of 0.42 μg/ml for kaempferol and 0.48 μg/ml for quercetin and limit of quantification (LOQ) values for kaempferol and quercetin 1.27 μg/ml and 1.47 μg/ml respectively. Percentage recovery values for kaempferol were found to be 99.18, 99.03 and 98.32 for the leaves, stem and root respectively, and for quercetin, they were 99.77, 100.12 and 100.54 for the leaves, stem and root respectively.Conclusion: The developed HPLC method for the analysis of flavonoids has enabled rapid, accurate and reproducible determination. The method can be applied successfully for analysis of quercetin and kaempferol in various plant parts of C. quadrangularis.Keywords: Cissus quadrangularis, HPLC, Kaempferol, Querceti

Stromal Expression of CD10 in Invasive Breast Carcinoma and Its Correlation with ER, PR, HER2-neu, and Ki67

CD10 is a cell surface zinc-dependent endopeptidase, which degrades many bioactive peptides. CD10 expression in tumour stroma is associated with biological aggressiveness of many epithelial malignancies. To date, only one study has correlated with expression of CD10 with well-known prognostic markers of breast, that is, ER, PR, HER2-neu, and tumour grade; however, its correlation with ki67 is still not studied. The aim of this study is to evaluate stromal CD10 expression in breast carcinoma and to examine its correlation with ER, PR, HER2-neu, and Ki67. Methods and Results. CD10 expression in fifty patients was assessed by immunohistochemistry and scored as negative, weak and strong. CD10 was found to be positive in stroma of 40/50 (80%) cases. Stromal CD10 showed positive correlation with tumour grade, HER2-neu (P = .000), and ki67 (P = .027), negative correlation with ER and PR. Conclusions. Hence CD10 expression correlated strongly with well-established negative prognostic markers, that is, HER2-neu and ki67 positivity, ER/PR negativity, and higher tumour grade, thus indicating that CD10 can be used as independent marker indicating poor prognosis and can be used as target for the development of novel therapies

SCREENING, CHARACTERIZATION, AND IN VITRO EVALUATION OF PROBIOTIC PROPERTIES OF LACTOBACILLUS STRAINS

  Objective: The aim of the present investigation was to isolate and identify Lactobacillus strains from dairy and cattle dung samples. Potent isolates were selected for screening by antimicrobial activity; selected lactobacilli were further tested for probiotic properties and adhesive attributes.Methods: Lactobacilli were isolated aseptically on specific de man, rogosa and sharpe medium from dairy and cattle dung samples. Isolates were identified by Gram-staining, motility, catalase, endospore, and carbohydrate fermentation tests. Further, the isolates were screened for antimicrobial activity by disk diffusion assay, and potent lactobacilli were observed for probiotic properties: Acid and bile salt tolerance, gelatinase activity, and autolytic activity. For analyzing the adhesive attributes, isolates were observed for autoaggregation, coaggregation and microbial adhesion to solvents assay.Results: About 12 Lactobacillus strains among 98 isolates exhibited maximum antimicrobial activity were further selected for identifying their probiotic and adhesive attributes. Among 12 selected isolates, cell-free supernatant (CFS) of buffalo milk BM10 and goat milk GM10 showed excellent antimicrobial activity, 20.34±0.02 mm against Staphylococcus aureus and 18.65±0.11 mm against Escherichia coli. Isolates showed survival at pH 2 and 3 and can tolerate 0.2-0.3% bile salt concentrations. The GM5 showed maximum autoaggregation (67.04±0.61%) and minimum coaggregation (11.51±0.50%) showed by GM3. The BM10 exhibited maximum adherent value 64.84±1.41% for n-hexadecane.Conclusion: The two lactobacilli, BM10 and GM10 identified as Lactobacillus fermentum and Lactobacillus pentosus on the basis of phenotypic and sugar utilization tests. The CFS of both lactobacilli can be used as antimicrobial agent. Both isolates showed significant results of probiotic and adhesive attributes, therefore, can be evaluated for clinical and therapeutic applications

A NOVEL REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF ALOE-EMODIN AND PIPERINE IN AYURVEDIC FORMULATION

Objective: The objective of this study was to develop and validate a novel, simple, rapid, accurate, and precise reverse-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of aloe-emodin and piperine in the ayurvedic formulation as per the International Conference on Harmonization guidelines. Methods: Chromatographic separation was achieved using a Prontosil C18 (250 × 4.6 mm, 5 μ), with a mobile phase consisting of 0.05% orthophosphoric acid and acetonitrile in the ratio of 50:50, at a flow rate of 1 ml/min and column temperature maintained at 28°C and ultraviolet (UV) detection at 225 nm. Results: The retention time of aloe-emodin and piperine was found to be 9.38±0.2 min and 13.45±0.2 min, respectively. The linearity of aloe-emodin and piperine was tested in the range of 1–20 μg/ml. The correlation coefficient for aloe-emodin and piperine was found to be 0.998 and 0.997, respectively. The recovery values (98–102%) indicate a satisfactory accuracy. The percentage relative standard deviation for precision was found to be <2% which indicates that the method is precise. Conclusion: A precise method for the simultaneous quantification of aloe-emodin and piperine was developed using high-performance liquid chromatography. Hence, the developed method can be used for quantitative and quality control analysis of formulations containing these phytoconstituents