Preparation, Characterization, and Evaluation of Physcion Nanoparticles for Enhanced Oral Bioavailability: An Attempt to Improve Its Antioxidant and Anticancer Potential.

This study aims to enhance the dissolution rate of a poorly water-soluble drug physcion by producing its nanoparticles (NPs) using an antisolvent precipitation with a syringe pump (APSP) method and to assess its antioxidant and cytotoxic potential. The NPs were prepared using a simple and cost-effective APSP method and subsequently characterized by different analytical techniques including dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray powder diffractometry (XRD). They were also subjected to solubility and dissolution studies, and different parameters such as dissolution efficiency (DE), mean dissolution time (MDT), and difference (f1) and similarity factors (f2) were determined. Furthermore, physcion and its NPs were investigated for antioxidant and cytotoxic effects using various in vitro assays. SEM and DLS analysis indicated that the average size of physcion NPs was 110 and 195 ± 5.6 nm, respectively. The average ζ-potential and polydispersibility index (PDI) of the prepared NPs were -22.5 mV and 0.18, respectively, showing excellent dispersibility. XRD confirmed the amorphous nature of physcion NPs. The solubility and dissolution rates of NPs were significantly higher than those of the original powder. The antioxidant potential studied by the (DPPH), FRAP, and H2O2 assays was greater for physcion NPs than that for the raw powder. The IC50 values of physcion NPs against the aforementioned models were 57.56, 22.30, and 22.68 µg/mL, respectively. Likewise, the cytotoxic potential investigated through the MTT assay showed that physcion NPs were more cytotoxic to cancer cell lines A549 (IC50 4.12 µg/mL), HepG2 (IC50 2.84 µg/mL), and MDA-MB-231 (IC50 2.97 µg/mL), while it had less effect on HPAEpiC (IC50 8.68 µg/mL) and HRPTEpiC (IC50 10.71 µg/mL) normal human epithelial cells. These findings have proved that the APSP method successfully produced physcion NPs with enhanced solubility, dissolution rate, and antioxidant and cytotoxic activities.

Biosynthesized Silver Nanoparticles Using Alnus nitida Leaf Extract as a Potential Antioxidant and Anticancer Agent.

Biogenic synthesis of silver nanoparticles (AgNPs) using plant extracts is gaining attention as a substitute to the conventional physical and chemical synthesis methods. This study reports a facile, cost-effective, and ecofriendly synthesis of AgNPs using leaf extract of Alnus nitida (A. nitida) and their antioxidant and antiproliferative activities. The biosynthesized AgNPs were characterized using various analytical techniques including UV-visible spectroscopy, energy-dispersive spectrometry, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), and dynamic light scattering. The antioxidant and cytotoxic potential of the extract and AgNPs was evaluated using different in vitro models. The UV-vis analysis revealed a surface plasmon resonance peak of 400 nm corresponding to the synthesis of AgNPs. SEM analysis confirmed the formation of heterogeneously dispersed particles of nano size, while the XRD and FTIR spectra confirmed the crystallinity and existence of different functional groups that helped in capping and stability of AgNPs. The antioxidant activity of AgNPs and extract, studied by 1,1-diphenyl 2-picryl hydrazyl (DPPH), fluorescence recovery after photobleaching (FRAP), 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and H2O2 scavenging assays, showed a dose-dependent effect. The AgNPs at 1000 µg/mL significantly scavenged DPPH, FRAP, ABTS, and H2O2 by 66.45, 74.65, 78.81, and 72.56% with an average IC50 value of 33.31, 18.50, 16.46, and 15.65 µg/mL, respectively. The cytotoxic potential investigated by MTT assay revealed promising antiproliferative effects against different cancer cell lines. The IC50 values of AgNPs on MDA-MB-231, A549, and Hep-G2 cells were 14.88, 3.6, and 5.38 µg/mL, respectively. The results showed that AgNPs were more effective against lung and hepatocellular carcinoma. The selectivity index showed that AgNPs remained highly selective in retarding the growth of A549 and Hep-G2 cells as compared to normal cell lines HPAEpiC and HRPTEpiC. Overall, this study showed that biosynthesized AgNPs were associated with considerable antioxidant and cytotoxic effects. Our work suggests that A. nitida-mediated AgNPs should be evaluated further in order to develop safe and effective formulations for the treatment of different degenerative diseases.

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1 mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2 v/v) to 0.5 mL of plasma. The internal standard solution (artemisinin 500 ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 µL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 µL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Association between Aldosterone Synthase (CYP11B2) Gene Polymorphism and Hypertension in Pashtun Ethnic Population of Khyber Pakhtunkwha, Pakistan.

Genome-wide association studies significantly increased the number of hypertension risk variants; however, most of them focused on European societies. There is lack of such studies in developing countries, including Pakistan. The lack of research studies and the high prevalence of hypertension in the Pakistani community prompted us to design this study. Aldosterone synthase (CYP11B2) was thoroughly studied in different ethnic groups; however, no such study has been conducted in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. In essential hypertension, the aldosterone synthase gene (CYP11B2) plays a significant role. Aldosterone synthesis is affected by both hereditary and environmental factors. Aldosterone synthase (encoded by the CYP11B2 gene) controls the conversion of deoxycorticosterone to aldosterone and, thus, has genetic influences. Polymorphisms in the CYP11B2 gene are linked to an increased risk of hypertension. Previous research on the polymorphism of the aldosterone synthase (CYP11B2) gene and its relationship to hypertension produced inconclusive results. The present study investigates the relationship between CYP11B2 gene polymorphism and hypertension in Pakistan's Pashtun population. We used the nascent exome sequencing method to identify variants associated with hypertension. The research was divided into two phases. In phase one, DNA samples from 200 adult hypertension patients (of age ≥ 30 years) and 200 controls were pooled (n = 200/pool) and subjected to Exome Sequencing. In the second phase, the WES reported SNPs were genotyped using the Mass ARRAY technique to verify and confirm the association between WES-identified SNPs and hypertension. WES identified a total of eight genetic variants in the CYP11B2 gene. The chi-square test and logistic regression analysis were used to estimate the minor allele frequencies (MAFs) and chosen SNPs relationships with hypertension. The frequency of minor allele T was found to be higher in cases compared to the control (42% vs. 30%: p = 0.001) for rs1799998 of CYP11B2 gene, while no significant results (p > 0.05) were observed for the remaining SNPs; rs4536, rs4537, rs4545, rs4543, rs4539, rs4546 and rs6418 showed no positive association with HTN in the studied population (all p > 0.05). Our study findings suggest that rs1799998 increases susceptibly to HTN in the Pashtun population of KP, Pakistan.

Validation of Genome-Wide Association Studies (GWAS)-Identified Type 2 Diabetes Mellitus Risk Variants in Pakistani Pashtun Population.

Objective: Recent GWAS largely conducted in European populations have successfully identified multiple genetic risk variants associated with Type 2 Diabetes Mellitus (T2DM). However, the effects conferred by these variants in the Pakistani population have not yet been fully elucidated. The objective of this study was to examine European GWAS-identified T2DM risk variants in the Pakistani Pashtun population to better understand the shared genetic basis of T2DM in the European and Pakistani cohorts. Methodology: A total of 100 T2DM patients and 100 healthy volunteers of Pashtun ethnicity were enrolled in this study. Both groups were genotyped for 8 selected single nucleotide polymorphisms (SNPs) using the Sequenom MassARRAY® platform. The association between selected SNPs and T2DM was determined by using appropriate statistical tests. Results: Of the 8 studied SNPs, 5 SNPs, SLC30A8/ rs13266634 (p=0.031, OR=2.13), IGF2BP2/ rs4402960 (p=0.001, OR=3.01), KCNJ11/ rs5219 (p=0.042, OR=1.78), PPARG/ rs1801282 (p=0.042, OR=2.81) and TCF7L2/ rs7903146 (p=0.00006, 3.41) had a significant association with T2DM. SNP GLIS3/ rs7041847 (p=0.051, OR=2.01) showed no sufficient evidence of association. SNPs KCNQ1/ rs2237892 (p=0.140, OR=1.61) and HHEX/IDE/ s1111875 (p=0.112, OR=1.31) showed opposite allelic effects and were not validated for T2DM risk in the study population. Among the studied SNPs, TCF7L2/ rs7903146 showed the most significant association. Conclusion: Our study finding indicates that selected genome-wide significant T2DM risk variants previously identified in European descent also increase the risk of developing T2DM in the Pakistani Pashtun population.

Association of APOE (rs429358 and rs7412) and PON1 (Q192R and L55M) Variants with Myocardial Infarction in the Pashtun Ethnic Population of Khyber Pakhtunkhwa, Pakistan.

Coronary Artery Diseases (CAD) remains the top among Non-communicable Diseases (NCDs). Variations in Apolipoprotein E (APOE) and Paroxonase 1 (PON1) have been associated with Myocardial Infarction (MI) in several populations. However, despite the high prevalence of CAD, no such study has been reported in the Pashtun ethnic population of Pakistan. We have conducted a two-stage (i.e., screening and validation) case-control study in which 200 cases and 100 control subjects have been recruited. In the first stage, Whole Exome Sequencing (WES) was used to screen for pathogenic variants of Myocardial Infarction (MI). In the second stage, selected variants of both APOE and PON1 genes (rs7412, rs429358, rs854560, and rs662) were analyzed through MassARRAY genotyping. Risk Allele Frequencies (RAFs) distribution and association of the selected SNPs with MI were determined using the Chi-square test and logistic regression analysis. WES identified a total of 12 sequence variants in APOE and 16 in PON1. Genotyping results revealed that APOE variant rs429358 (ɛ4 allele and ɛ3/ɛ4 genotype) showed significant association in MI patients (OR = 2.11, p value = 0.03; 95% CI = 1.25-2.43); whereas no significant difference (p˃ 0.05) was observed for rs7412. Similarly, the R allele of PON1 Q192R (rs662) was significantly associated with cases (OR = 1.353, p value = 0.048; 95% CI = 0.959-1.91), with particular mention of RR genotype (OR = 1.523, p value = 0.006; 95% CI = 1.087-2.132). Multiple logistic regression analysis showed that rs429358 (C allele) and rs662 (R allele) have a significantly higher risk of MI after adjustment for the conventional risk factors. Our study findings suggested that the rs429358 variant of APOE and PON1 Q192R are associated with MI susceptibility in the Pashtun ethnic population of Pakistan.

Decoding type 2 diabetes mellitus genetic risk variants in Pakistani Pashtun ethnic population using the nascent whole exome sequencing and MassARRAY genotyping: A case-control association study.

Genome-wide association studies have greatly increased the number of T2DM associated risk variants but most of them have focused on populations of European origin. There is scarcity of such studies in developing countries including Pakistan. High prevalence of T2DM in Pakistani population prompted us to design this study. We have devised a two stage (the discovery stage and validation stage) case-control study in Pashtun ethnic population in which 500 T2DM cases and controls each have been recruited to investigate T2DM genetic risk variants. In discovery stage Whole Exome Sequencing (WES) was used to identify and suggest T2DM pathogenic SNPs, based on SIFT and Polyphen scores; whereas in validation stage the selected variants were confirmed for T2DM association using MassARRAY genotyping and appropriate statistical tests. Results of the study showed the target positive association of rs1801282/PPARG (OR = 1.24, 95%Cl = 1.20-1.46, P = 0.010), rs745975/HNF4A (OR = 1.30, 95%Cl = 1.06-1.38, P = 0.004), rs806052/GLIS3 (OR = 1.32, 95%Cl = 1.07-1.66, P = 0.016), rs8192552/MTNR1B (OR = 1.53, 95%Cl = 0.56-1.95, P = 0.012) and rs1805097/IRS-2 (OR = 1.27, 95%Cl = 1.36-1.92, P = 0.045), with T2DM; whereas rs6415788/GLIS3, rs61788900/NOTCH2, rs61788901/NOTCH2 and rs11810554/NOTCH2 (P>0.05) showed no significant association. Identification of genetic risk factors/variants can be used in defining high risk subjects assessment, and disease prevention.

Development and Evaluation of Self-Emulsifying Drug-Delivery System-Based Tablets for Simvastatin, a BCS Class II Drug.

Background: Self-emulsifying drug-delivery systems (SEDDSs) are designed to improve the oral bioavailability of poorly water-soluble drugs. This study aimed at formulating and characterization of SEDDS-based tablets for simvastatin using castor and olive oils as solvents and Tween 60 as surfactant. Methods: The liquids were adsorbed on microcrystalline cellulose, and all developed formulations were compressed using 10.5 mm shallow concave round punches. Results: The resulting tablets were evaluated for different quality-control parameters at pre- and postcompression levels. Simvastatin showed better solubility in a mixture of oils and Tween 60 (10:1). All the developed formulations showed lower self-emulsification time (˂200 seconds) and higher cloud point (˃60°C). They were free of physical defects and had drug content within the acceptable range (98.5%-101%). The crushing strength of all formulations was in the range of 58-96 N, and the results of the friability test were within the range of USP (≤1). Disintegration time was within the official limits (NMT 15 min), and complete drug release was achieved within 30 min. Conclusion: Using commonly available excipients and machinery, SEDDS-based tablets with better dissolution profile and bioavailability can be prepared by direct compression. These S-SEDDSs could be a better alternative to conventional tablets of simvastatin.

Factors and Predictors of Health Related Quality of Life of the General Population of Pakistan.

Background and Objective: The standards of living, improvement in public health, and medical care in Pakistan are increasing day by day, health-related quality of life (HRQoL) has been increasingly acknowledged in various patient's reported outcomes in Pakistan. However, a large-scale general population-based study on assessing HQRoL in Pakistan was not conducted. Therefore, this study aimed to evaluate HRQoL for the general Pakistani population. Material and Methods: A cross-sectional study with a population sample (n = 16,672) was selected from all Pakistan provinces using a stratified sampling approach. The EQ-5D-3L tool was used to measure the HRQoL of the general population of Pakistan. The descriptive and inferential statistics have been done by using SPSS version 20. Results: Overall, 121 health states were reported in this study. EQ-5D index and EQ-VAS scores were 0.74 ± 0.32 and 0.75 ± 0.25, respectively. The percentage of people responding to any problems increased with age. Males have better health as compared to females in all age groups. All demographics were significantly associated (P < 0.01) with the mean EQ5D index and VAS scores except residence (p > 0.05). The regression model reported that age was the best predictor of the EQ-5D index scores after adjusting for the covariates (beta = 0.19; p < 0.001). This study provides Pakistani population HRQoL data measured by the EQ-5D tool, based on a national representative sample. Conclusion: The current study concluded that Age, City, Gender, Education, Occupation, Residence, and House occupancy are significantly affecting HRQOL. The socioeconomically deprived groups and females have inferior health status than more advantaged. The trends detected in high-income nations were usually similar to Pakistan.

An Overview on Rumex dentatus L.: Its Functions as a Source of Nutrient and Health-Promoting Plant.

Rumex dentatus L. (Polygonaceae), also known as toothed dock or Aegean dock, is a medicinal plant with a high culinary value in addition to being used as an ethnomedicinal plant. This review focuses on the botanical, nutritional, phytochemical, and pharmacological activities of R. dentatus, as well as the future prospects for systematic investigations into these areas. R. dentatus has been subjected to scientific evaluation, which has confirmed its traditional uses and demonstrated a wide range of biological and pharmacological potentials, including antioxidant, anticancer, antifungal, antibacterial, anti-inflammatory, and other biological properties. Phytochemical analyses showed the presence of anthraquinones, chromones, flavonoids, and essential oils. As a result of this current review, the medicinal significance of R. dentatus has been confirmed, and future research on its unexplored aspects, such as the identification of pharmacologically active chemical constituents and related mechanisms and safety, may be stimulated, with the goal of developing it into a drug.

Assessment of Tocilizumab (Humanized Monoclonal Antibody) for Therapeutic Efficacy and Clinical Safety in Patients with Coronavirus Disease (COVID-19).

Background and objectives: COVID-19 patients exhibit a broad range of manifestations, presenting with a flu-like respiratory tract infection that can advance to a systemic and severe disease characterized by pneumonia, pulmonary edema, severe damage to the airways, and acute respiratory distress syndrome (ARDS, causing fatality in 70% of COVID-19 cases). A 'cytokine storm' profile is found in most severely influenced COVID-19 patients. The treatment protocol of the disease also includes tocilizumab, which is a humanized monoclonal antibody used to treat autoimmune and inflammatory conditions. This study was designed (1) to assess the role of tocilizumab in COVID-19 patients regarding therapeutic efficacy through evaluation of cytokine release syndrome (CRS) resolution and anticoagulant effect, analyzing clinical safety via monitoring of associated adverse effects profile; and (2) to compare the clinical safety and therapeutic efficacy of institutional treatment regimen (alone) versus tocilizumab added to an institutional treatment module in COVID-19 patients. Materials and Methods: In this study, the endpoints parametric assessment of severely diseased patients of COVID-19 was performed (total n = 172, control group (institutional protocol treatment provided), n = 101 and test group (tocilizumab provided), n = 71) at the Khyber Teaching Institution, MTI, Peshawar. The assessments were compared using non-parametric analyses at baseline and after a follow-up of 12−18 days until the patient discharged or expired. Results: Results of the study revealed an insignificant difference among the control vs. test group in resolving inflammatory parameters (C-reactive protein (CRP) 21.30 vs. 50.07; p = 0.470, ferritin 482.9 vs. 211.5; p = 0.612, lactate dehydrogenase (LDH) 29.12 vs.18.8; p = 0.0863, and D-dimer 464 vs.164.4; p = 0.131). However, a statistically significant difference was found between the control group and test group regarding coagulation parameters (international normalized ratio (INR) 0.12 vs. −0.07; p ≤ 0.001; activated partial thromboplastin time (aPTT) 0.42 vs. −1.16; p ≤ 0.001; prothrombin time (PT) 0.31 vs. −0.96; p ≤ 0.001; platelet count −12.34 vs. −1.47; p = 0.012) and clinical survival rate (89.10 vs. 90.14; p < 0.001). Furthermore, there was significantly higher infection rates and raised alanine aminotransferase (ALT) and alkaline phosphatase (ALP) associated with the tocilizumab group as compared to those receiving institutional treatment (bacterial infections: 0.99% vs. 15.49%; p ≤ 0.01, ALT: 3.96% vs. 28.16%; p ≤ 0.01, ALP: 1.98% vs. 22.53%; p ≤ 0.01). Conclusions: From this study, it was concluded that tocilizumab can be a better drug of choice in terms of efficacy, particularly in resolving coagulopathy in severe COVID-19 patients.

Efficacy Evaluation of 10-Hydroxy Chondrofoline and Tafenoquine against Leishmania tropica (HTD7).

Leishmaniasis is affirmed as a category one disease (most emerging and unmanageable) by the World Health Organization (WHO), affecting 98 countries with an annual global incidence of ~1.2 million cases. Options for chemotherapeutic treatment are limited due to drug resistance and cytotoxicity. Thus, the search for new chemical compounds is instantly desirable. In this study, we used two compounds, i.e., 10-hydroxy chondrofoline and tafenoquine, for their antileishmanial activity against L. tropica (HTD7). First, the cytotoxicity assay of the test compounds against THP-1 cells was carried out, and these compounds were found safe. Intra-THP-1 amastigote activity (in vitro) was performed, which was then followed by the in vivo activity of 10-hydroxy chondrofoline in the murine cutaneous leishmaniasis (CL) model. A total of three concentrations were used, i.e., 25, 50, and 100 µM, to check the in vitro activity of the test compounds against the amastigotes. 10-hydroxy chondrofoline was found to be the most potent compound in vitro (and thus was selected for in vivo studies) with an LD50 value of 43.80 µM after 48 h incubation, whilst tafenoquine had an LD50 value of 53.57 µM. In vivo activity was conducted by injecting 10-hydroxy chondrofoline in the left hind foot of the infected BALB/c mice, where it caused a statistically significant 58.3% (F = 14.18; p = 0.002) reduction in lesion size (0.70 ± 0.03 mm) when compared with negative control (1.2 ± 0.3 mm).

Screening of Rhamnus Purpurea (Edgew.) Leaves for Antimicrobial, Antioxidant, and Cytotoxic Potential.

Exploring new antimicrobial and cytotoxic drugs has been one of the most active areas of research. Rhamnus purpurea (Edgew.) buckthorn (Rhamnaceae) is a wild shrub traditionally used in Pakistan for the treatment of various ailments including cancer and infectious diseases. The aim of this study is to find novel antimicrobial and cytotoxic agents of plant origin. The crude methanol extract and full range of fractions of R. purpurea leaves were screened for the said activities using in vitro antimicrobial, antioxidant, and cytotoxic models following standard protocols. The antimicrobial activity was evaluated using the agar well diffusion method, while the antioxidant activity was assessed with 1,1-diphenyl-2-picryl hydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. The cytotoxic effect was investigated against the human cancer cell lines i.e. Caco-2 (gut), A549 (lung), HepG2 (liver), and MDA-MB-231 (breast) by MTS assay. In addition, toxicity studies were conducted on renal and alveolar primary epithelial cells (HRPTEpiC and HPAEpiC, respectively). Phytochemical investigation showed the presence of secondary metabolites such as alkaloids, saponins, tannins, glycosides, phenols, carbohydrates, proteins, and flavonoids. The n-hexane and chloroform fractions showed significant activity against Staphylococcus aureus (MIC 0.60 and 0.68 mg/mL, respectively), Salmonella typhi (MIC 0.48 and 0.45 mg/mL, respectively), and Bacillus subtilis (MIC 0.54 and 0.76 mg/mL, respectively). Among fungal strains, crude methanol and chloroform fractions exhibited significant activity against Fusarium solani (MIC 0.53 and 0.44 mg/mL, respectively) and Aspergillus niger (MIC 0.47 and 0.42 mg/mL, respectively). The crude methanol, n-hexane and chloroform fractions revealed the highest antioxidant activity at 1000 µg/mL, compared to that of ascorbic acid. The n-hexane fraction showed a significant cytotoxic effect against Caco-2, A549, and HepG2 cell lines with IC50 values of 5.65 ± 0.88, 5.50 ± 0.90, and 4.95 ± 1.0 µg/mL, respectively. Similarly, the chloroform fraction depicted significant activity against Caco-2, A549, and HepG2 cell lines with IC50 values of 4.55 ± 1.25, 4.65 ± 1.55, and 2.85 ± 0.98 µg/mL, respectively. The crude methanol extract and almost all fractions exhibited the highest selectivity index (>2.0) for Caco-2, A549, and HepG2 cancer cell lines, providing safety data for this study. The results showed that R. purpurea leaves have excellent antimicrobial, antioxidant, and cytotoxic potential and warrant further studies to search for novel compounds for the said activities.

Formulation Development of Mirtazapine Liquisolid Compacts: Optimization Using Central Composite Design.

Mirtazapine is a tetracyclic anti-depressant with poor water solubility. The aim of this study was to improve the dissolution rate of mirtazapine by delivering the drug as a liquisolid compact. Central composite design (CCD) was employed for the preparation of mirtazapine liquisolid compacts. In this, the impacts of two independent factors, i.e., excipient ratio (carrier:coating) and different drug concentration on the response of liquisolid system were optimized. Liquisolid compacts were prepared using propylene glycol as a solvent, microcrystalline cellulose as a carrier, and silicon dioxide (Aerosil) as the coating material. The crystallinity of the formulated drug and the interactions between the excipients were examined using X-ray powder diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR), respectively. The dissolution study for the liquisolid compact was carried out as per FDA guidelines. The results showed loss of crystallinity of the mirtazapine in the formulation and was completely solubilized in non-volatile solvent and equally dispersed throughout the powder system. Moreover, drug dissolution was found to be higher in liquisolid compacts than the direct compressed conventional tablets (of mirtazapine). The liquisolid technique appears to be a promising approach for improving the dissolution of poorly soluble drugs like mirtazapine.

Assessment of Contributing Factors and Treatment Practices for Therapeutic Efficacy and Drug-Related Problems in Suicidal Psychotic Patients.

Suicide, a deliberate act of self-harm with the intention to die, is an emerging health concern but, unfortunately, the most under-researched subject in Pakistan, especially in Khyber Pukhtunkhwa (KPK). In this study, we aimed to identify risk factors that can be associated with suicidal behavior (SB) and to evaluate the prevailing treatment practices for therapeutic efficacy and drug-related problems (DRPs) in psychotic patients among the local population of KPK. A prospective, multicenter study was conducted for suicidal cases admitted to the study centers by randomized sampling. Socio-demographics and data on suicidal behavior were assessed using the Columbia-Suicide Severity Rating Scale (C-SSRS), socioeconomic condition by Kuppuswamy socioeconomic scale (KSES) and treatment adherence by Morisky Medication-Taking Adherence Scale (MMAS-4). Drug-related problems and the therapeutic efficacy of prevailing treatment practices were assessed at baseline and follow-up after 3 months of treatment provided. Regarding suicidality (N = 128), females reported more ideations (63.1%), while males witnessed more suicidal behavior (66.6%, p < 0.001). Suicide attempters were mostly married (55.6%, p < 0.002); highly educated (53.9%, p = 0.004); dissatisfied with their life and had a previous history (p < 0.5) of suicide attempt (SA) (20.6%), self-injurious behavior (SIB) (39.7%) and interrupted (IA) or aborted attempts (AA) (22.2%). A greater improvement was observed in patients receiving combination therapy (p = 0.001) than pharmacotherapy (p = 0.006) or psychotherapy (p = 0.183), alone. DRPs were also detected, including drug-selection problems (17.88%), dose-related problems (20.64%), potential drug−drug interactions (24.31%), adverse drug reactions (11.46%) and other problems like inadequate education and counseling (21.55%). Furthermore, it was also found that psychotic patients with suicidal ideations (SI) were significantly (p = 0.01) more adherent to the treatment as compared to those with suicidal attempts. We concluded that suicide attempters differed significantly from patients with suicidal ideations in psychotic patients and presented with peculiar characteristics regarding socio-demographic factors. A combination of therapies and adherence to the treatment provided better outcomes, and targeted interventions are warranted to address drug-related problems.

Evaluation of in-vitro and in-vivo antimicrobial potential of Typha elephantina leaves extracts using Cyprinus carpio as a research model.

Present study investigate the in-vitro antibacterial and antifungal potential of Typha elephantina leaves aqueous extract (T. Eaq), ethanolic extract (T. Eeth) and methanolic extract (T. Emth) at different dosages against selected bacteria and fungi using dis diffusion method and Potato Dextrose Agar method. The study was also proceeded in- vivo against one strain of fungi (Aspergillus niger) using aqueous (T. Eaq) extract only. In-vitro study showed that Citrobacter freundii was highly sensitive while Salmonella typhimurium was the least among all. The antifungal activity was dose dependent and differs according to the fungal strain. Aspergillus niger was highly sensitive in order of aqueous extract (T. Eaq), ethanolic extract (T. Eeth) and methanolic extract (T.Emth), followed by Alterneria solani, Candida albicans and Aspergillus ustus. The in-vivo antifungal study was carried using Cyprinus carpio which were first infected with Aspergillus niger and then treated with (T. Eaq) at different doses. During in-vivo study various hematobiochemicl parameters and bio-accumulative stress of some heavy metals were assessed. Highly significant (P<0.05) remedial effects were observed at day 21st of treatment with extract at 100mg/ kg body weight. Differential accumulation was found i.e in skin the accumulation was highest followed by intestine gills and muscles tissues. Liver showed least accumulation.

Association of ATM, CDH1 and TP53 genes polymorphisms with familial breast cancer in patients of Khyber Pakhtunkhwa, Pakistan.

Background: Genetic studies play a significant role in understanding the underlying risk factors of breast cancer. Polymorphism in the tumor suppressor gene TP 53, CDH1 and ATM genes are found to increase susceptibility for breast cancer globally. Objective: This study aimed to identify/analyze the contribution of genetic polymorphisms in the breast cancer candidate genes ATM, TP53 and CDH1 that may be associated with familial breast cancer risk in the Khyber Pakhtunkhwa population. Subjects and Methods: In the present case-control study, Whole Exome Sequencing (WES) of the 100 breast cancer patients and 100 ethnic controls were performed for the selected genes in the target population. Results: Of the studied variants rs3743674 of the CDH1 gene (crude P=0.014 and adjusted p=0.000) evident significant association with breast cancer in Pakistani Pashtun population. Whereas TP53rs1042522 (crude P=0.251 and adjusted P=0.851) and ATM rs659243 (crude p=0.256 and adjusted p=0.975) showed no or negative association with breast cancer in study population. Conclusion: The present study demonstrates that CDH1rs3743674 polymorphism is associated with elevated breast cancer risk in the Pashtun ethic population of Khyber Pakhtunkhwa.

Clinical Investigation on the Impact of Cannabis Abuse on Thyroid Hormones and Associated Psychiatric Manifestations in the Male Population.

Cannabis abuse is a common public health issue and may lead to considerable adverse effects. Along with other effects, the dependence on cannabis consumption is a serious problem which has significant consequences on biochemical and clinical symptoms. This study intends to evaluate the harmful effects of the use of cannabis on thyroid hormonal levels, cardiovascular indicators, and psychotic symptoms in the included patients. This prospective multicenter study was conducted on cannabis-dependent patients with psychotic symptoms (n = 40) vs. healthy control subjects (n = 40). All participants were evaluated for psychiatric, biochemical, and cardiovascular physiological effects. Patients were selected through Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria and urine samples, exclusively for the evaluation of cannabis presence. Serum thyroid stimulating hormone (TSH), T3, and T4 levels were measured using the immunoassay technique. Patients were assessed for severity of depressive, schizophrenic, and manic symptoms using international ranking scales. Various quantifiable factors were also measured for the development of tolerance by cannabis. Among the patients of cannabis abuse, 47.5% were found with schizophrenia, 20% with schizoaffective symptoms, 10% with manic symptoms, and 22.5% with both manic and psychotic symptoms. In the group-group and within-group statistical analysis, the results of thyroid hormones and cardiovascular parameters were non-significant. The psychiatric assessment has shown highly significant (p < 0.001) difference of positive, negative, general psychopathology, and total scores [through Positive and Negative Syndrome Scale (PANSS) rating scales] in patients vs. the healthy control subjects. The study revealed that cannabis abuse did not significantly alter thyroid hormones and cardiovascular parameters due to the development of tolerance. However, the cannabis abuse might have a significant contributing role in the positive, negative, and manic symptoms in different psychiatric disorders.

Phytochemical and Biological Screening of Leaf, Bark and Fruit Extracts from Ilex dipyrena Wall.

The Aquifoliaceae is an important family and has been used traditionally for some time. One of the members of this family is the Ilex dipyrena wall, which itself possesses a potential medicinal importance. This plant is traditionally used for the treatment of various ailments including pain, swelling, burns, and fever. The current study was designed to screen out the antioxidant and analgesic potential of this plant and to verify its traditional uses, along with its phytochemical profile. Extracts were subjected to antioxidant, analgesic, and phytochemical analysis using DPPH, chemical-induced (acetic acid and formalin) nociception models and GC-MS analysis, respectively. The leaf, bark, and fruit extracts showed significant antioxidant activity compared to that of standard. Likewise, all the extracts demonstrated significant (p < 0.01) analgesic activity in a mice model. In acetic acid induced analgesia, the leaf, bark, and fruit extracts caused 51.64, 56.13 and 59.52% inhibition, respectively at a dose of 100 mg/kg while at 200 mg/kg it showed 83.01, 71.69 and 75.47% inhibition, respectively. In Formalin-induced paw-licking assay, fruit extract showed 59.42 and 64.19% inhibition at 200 mg/kg dose in the first and second phase, respectively. The GC-MS analysis revealed the presence of cathinone, phenylpropanolamine, dl-phenylephrine, amphetamine, myristic acid, and palmitic acid. Results of the study suggest that crude extracts from different parts of this plant may be a useful source for the development of novel analgesics. However, further investigation in terms of isolation of bioactive compounds and their toxicological evaluations are needed to validate the observed results.

Association of HLA-B Gene Polymorphisms with Type 2 Diabetes in Pashtun Ethnic Population of Khyber Pakhtunkhwa, Pakistan.

Human leukocyte antigen (HLA) system is the most polymorphic and gene dense region of human DNA that has shown many disease associations. It has been further divided into HLA classes I, II, and III. Polymorphism in HLA class II genes has been reported to play an important role in the pathogenesis of type 1 diabetes (T1D). It also showed association with T2D in different ethnic populations. However, a little is known about the relationship of HLA class I gene polymorphism and T2D. This study has evaluated the association of HLA-B (class I gene) variants with T2D in Pashtun ethnic population of Khyber Pakhtunkhwa. In the first phase of the study, whole exome sequencing (WES) of 2 pooled DNA samples was carried out, and DNA pools used were constructed from 100 diabetic cases and 100 control subjects. WES results identified a total of n = 17 SNPs in HLA-B gene. In the next phase, first 5 out of n = 17 reported SNPs were genotyped using MassARRAY® system in order to validate WES results and to confirm association of selected SNPs with T2D. Minor allele frequencies (MAFs) and selected SNPs×T2D association were determined using chi-square test and logistic regression analysis. The frequency of minor C allele was significantly higher in the T2D group as compared to control group (45.0% vs. 13.0%) (p = 0.006) for rs2308655 in HLA-B gene. No significant difference in MAF distribution between cases and controls was observed for rs1051488, rs1131500, rs1050341, and rs1131285 (p > 0.05). Binary logistic regression analyses showed significant results for SNP rs2308655 (OR = 2.233, CI (95%) = 1.223-4.077, and p = 0.009), while no considerable association was observed for the other 4 SNPs. However, when adjusted for these variants, the association of rs2308655 further strengthened significantly (adjusted OR = 7.485, CI (95%) = 2.353-23.812, and p = 0.001), except for rs1131500, which has no additive effect. In conclusion, the finding of this study suggests rs2308655 variant in HLA-B gene as risk variant for T2D susceptibility in Pashtun population.