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The objective of current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for diclofenac sodium. The poloxamer 407, methyl cellulose, hydroxypropyl methyl cellulose and polyethylene glycol were used alone and in combination in different ratios to design the delivery system. The physical properties like Tsol-gel, viscosity, clarity of solution and gel were evaluated. The in vitro release of the drug delivery system was evaluated using membrane less method and the drug release kinetics and mechanism was predicted by applying various mathematical models to the in vitro dissolution data. Rabbits were used as in vivo model following subcutaneous injection to predict various pharmacokinetics parameters by applying Pk-Summit software. The in vitro and in vivo data revealed that the system consisting of the poloxamer 407 in concentration of 20% (DP20) was the most capable formulation for extending the drug release and maintaining therapeutic blood level of DS for longer duration (144 h). The data obtained for drug content after autoclaving the solutions indicate that autoclaving results in 6% degradation of DS. The data also suggested that the studied polymers poloxamer, MC and PG are good candidate to extend the drug release possessing a unique thermoreversible property.

The present study was conducted on fifteen medicinal plants and their respective branded formulations, commonly used in Khyber Pakhtunkhwa, for the evaluation of toxic heavy metals. The purpose of the study was to assess the toxic profile of the crude medicinal plants with respect to the worldwide permissible limits of metal concentrations and to correlate it with their respective herbal formulations available on the market. Chromium (Cr), Copper (Cu), Lead (Pb), Manganese (Mn) and Nickel (Ni) content were evaluated using wet digestion and Atomic Absorption Spectrophotometry technique. The results exhibited that in 100% of the analyzed medicinal plants Cr and Ni are present in excess of the maximum limits, Cu and Pb in 73% and 60% respectively, while Mn is in the normal range. Likewise in the respective branded formulations Cr and Ni exceed the normal limit in 100% of the products, Cu and Pb in 27% and 20% of the products respectively, while Mn is in the normal range. It indicates that majority of people in Pakistan who frequently use herbal drugs in various forms are exposed to the hazardous elements, which may pose serious health effects. Regulatory measures should therefore be taken to protect the general public from their hazardous health effects.

Alcoholic extract and various fractions of Achyranthes aspera leaves, traditionally used in Pakistan for treatment of infectious diseases was screened for in vitro antibacterial and antifungal activity. The chloroform and butanol fractions were found to be the most active among the fractions, showing considerable antibacterial activity against Shigella flexneri and Escherichia coli. The highest activity was found in the ethylacetate fraction (17 mm zone of inhibition) against gram-negative (Salmonella typhi) bacteria, with MIC value as 0.29 mg/mL. In antifungal screening, moderate activity was shown by the chloroform fraction (50 % inhibition) against Microsporum canis, with MIC value as 0.25mg/mL. Considerable level of antifungal activity was depicted by crude extract, hexane and butanol fractions against Aspergillus flavus and Microsporum canis. The ability of various extracts of Achyranthes aspera to inhibit different strains of fungi and bacteria indicates its potential use for the treatment of microbial infections.

The purpose of this study was to evaluate associations of the CYP1A1 gene variant rs4646903 polymorphism with the risk of developing esophageal cancer (EC). A case-control study was carried out in Pashtun population of Khyber Pakhtunkhwa province of Pakistan in which 140 hospital based EC cases and 196 population based healthy controls exposed to similar environmental conditions were included. A specific method based on the real time polymerase chain reaction (RT-PCR) was used to detect genotypes in case and control groups and results were then analyzed with SPSS version 20. In our population, individuals with CC and TC genotypes of the CYP1A1 rs4646903 polymorphism had significantly higher risk of EC (adjusted odds (OR): 15.709, 95%CI: 6.065-40.686, OR: 3.256 95%CI: 1.902-5.574 respectively). The 'C' allele was strongly associated with the disease (p0.0001). Adjusted OR was higher (1.5 times in C/C) in case of variant alleles that show the contribution of environmental and nutritional factors towards the development of EC. Our findings suggest that presence of the 'C' allele of rs4646903 (TC) may be one of the risk alleles for EC susceptibility in Pashtun population.

In present study four medicinal plants namely Valeriana wallichii, Xanthium strumarium, Achyranthes aspera and Duchesnea indica belonging to different families were collected in Khyber Pakhtunkhwa province and crude extract and subsequent fractions were analyzed for their inhibitory potential against acetylcholinesterase, butyrylcholinesterase and α-glucosidase enzymes. Valeriana wallichii, Xanthium strumarium and Achyranthes aspera were significantly active against cholinesterases. Chloroform and ethylacetate fractions of Valeriana wallichii exhibited significant activity against acetylcholinesterase (IC50: 61μg/ml) and butyrylcholinesterase enzymes (IC50: 58μg/ml), respectively. Similarly ethylacetate fraction of Achyranthes aspera showed significant activity against acetylcholinesterase (IC50: 61 μg/ml) and butyrylcholinesterase enzymes (IC50: 61 μg/ml), respectively. In case of α-glucosidase enzyme, the chloroform fraction of Xanthium strumarium exhibited significant inhibitory activity (IC50: 72 μg/ml) as compared to the standard compound acarbose (IC50: 483 μg/ml). Duchesnea indica showed no such activities.

In present study, the anti-inflammatory potential of three medicinal plants, Xanthium strumarium, Achyranthes aspera and Duchesnea indica were evaluated, using both in vitro and in vivo assays. Carrageenan induced hind paw edema model was used to carry out the in vivo anti-inflammatory activity, while for in vitro screening lipoxygenase inhibition assay was used. Crude extract of all the selected plants depicted significant (plt;0.001) anti-inflammatory activity, at late phase of inflammation. Achyranthes aspera also showed considerable anti-inflammatory activity (47%) at relatively lower concentration (200 mg/ml), at the initial phase of inflammation. Similarly the ethyl acetate fraction of all the selected plants showed significant lipoxygenase inhibition activity when compared with the standard drug (Baicalein). The results obtained from both in vitro and in vivo anti-inflammatory activity suggest that the ethyl acetate fraction of the crude extract of all the selected plants can be used for the isolation of new lead compounds with better anti-inflammatory activity.

Smilax china Linn. is extensively used in traditional Chinese medicine (TCM) as well as in Pakistan for several medicinal purposes including their use in inflammatory disorders.The aims of the current study were to validate and assess the folk use of Smilax china Linn. on pharmacological grounds using the isolated compound at molecular, in vivo and computational levels.Seiboldogenin was isolated from ethyl acetate fraction of the plant crude extract. In vitro lipoxygenase and in vivo carrageenan-induced hind paw oedema models were used in experimental studies while molecular docking technique was used to conduct computational study.Sieboldogenin showed significant lipoxygenase inhibition (IC50: 38 microM). It also exhibited significant inhibition (p0.05) of carrageenan-induced hind paw oedema at the doses of 10 and 50mg/kg. Computational molecular docking showed its molecular interaction with important amino acid residues in the catalytic site of lipoxygenase, revealing its potential binding mode at molecular level.Sieboldogenin seems to be a potential new anti-inflammatory compound responsible for anti-inflammatory activities of Smilax china Linn. Its in vitro and in vivo inflammatory activities are in good agreement with the folk medicinal use of Smilax china Linn. in inflammatory disorders.

The objective of the current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for Insulin. Thermoreversible in-situ gel system was developed and evaluated both in-vitro and in-vivo comprising of pluronic F-127 alone or in combination with methylcellulose in different ratios. The drug release kinetics and mechanism was predicted by applying various mathematical models to the in-vitro dissolution data. Rabbits were used as animal model following subcutaneous injection to predict various pharmacokinetic parameters by applying Pk-Summit® software. The in-vitro and in-vivo data revealed that the formulation IPM 15/3 consisting of the pluronic F-127 (15% w/v) and methylcellulose (3% w/v) was the most robust and capable formulation for extending the drug release and maintaining basal plasma insulin level between 10 and 40 µU/ml for 240 h (10 d).

A simple, specific, precise and rapid RP-HPLC-UV method was developed for simultaneous determination of lumefantrine and its metabolite desbutyl lumefantrine in human plasma. Experimental parameters were optimized and the method was validated according to standard guidelines. The method showed adequate separation for lumefantrine and desbutyl lumefantrine and best resolution was achieved with Supelco Discovery HS C18 RP (150mm×4.6mm, 5μm) column using acetonitrile and 0.05% trifluroacetic acid (70:30, v/v) as a mobile phase pumped at a flow rate of 1.0ml/min and wavelength of 335nm. The method was linear over the concentration range of 10-12,000ng/ml. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for lumefantrine were 10.0 and 18.0ng/ml, while for desbutyl lumefantrine were 7.5 and 15.0ng/ml, respectively. The proposed method was efficiently applied for determination of lumefantrine and desbutyl lumefantrine concentrations in plasma samples for pharmacokinetic studies.

In vivo and in vitro screening of anti inflammatory activity of Valeriana wallichii and Achyranthes aspera leaves crude extract was performed, using standardized procedures. Methanolic crude extract topical formulation (cream) of Valeriana wallichii and Achyranthes aspera leaves (Family Valerianaceae and Amaranthaceae respectively), were screened for their anti-inflammatory activity, through "Carrageenan induced hind paw edema" test, for their effect on the acute and chronic phase inflammation models in male Wistar rats. Methanolic extract and its fractions were also evaluated for their in vitro anti-inflammatory activity using lipoxygenase inhibition assay. Leaves of Valeriana wallichii showed significant (P0.001), dose dependant anti inflammatory activity, comparable with that of the standard, in animal model. The ethyl acetate fraction of Valeriana wallichii also showed considerable (IC 50=73 ± 0.36) in vitro anti-inflammatory activity as compared to standard (6.11 ± 0.02). Similarly Achyranthes aspera leaves showed relatively weak (p0.05) in vivo anti-inflammatory activity. However, its activity was comparable with that of standard at 10% concentration after 5 hrs of carrageenan injection. This activity was present in ethyl acetate fraction during in vitro screening (IC 50=76 ± 0.14) as compared to that of standard (IC 50=6.11 ± 0.02). The combined in vitro and in vivo Anti-inflammatory screening shows that the ethyl acetate fraction of the crude extract of Valeriana wallichii and Achyranthes aspera can be used for the isolation of new Anti-inflammatory lead compounds.

A highly sensitive and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of artemether and its metabolite dihydroartemisinin in human plasma using artemisinin as an internal standard. Chromatographic separation was achieved on a Supelco Discovery HS C18 RP column (150 mm × 4.6 mm, 5 µm; Bellefonte, USA) using acetonitrile and water with 0.1 % formic acid (80:20, v/v) as a mobile phase in isocratic mode. A high-resolution Thermo Electron Corporation LTQ-Orbitrap mass spectrometer (San Jose, USA) was used in single ion monitoring (SIM) mode using atmospheric pressure chemical ionization (APCI) as an interface. The following extracted ion ranges ([M + H]+) were monitored: m/z 267.14–267.16 for artemether, m/z 221.16–221.18 for dihydroartemisinin and m/z 283.14–283.16 for internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for artemether were 0.3 and 0.8 ng mL−1, while for dihydroartemisinin were 0.2 and 0.6 ng mL−1, respectively. The validated method was successfully applied to the quantification of artemether and dihydroartemisinin in plasma samples of healthy volunteers participating in pharmacokinetic drug–food interaction studies.

Valeriana wallichii DC (Valerianaceae) is one of the most widely used traditional remedies for various complications associated with nervous system and digestion. No antimicrobial and anti-inflammatory studies have so far been carried out on the aerial parts of the plant. The present work was focused to evaluate the antimicrobial (antifungal and antibacterial) and anti-inflammatory properties of V. wallichii using reported methods. Chloroform fraction (VW-2) and hexane fraction (VW-3) exhibited significant activity against S. aureus and B. subtilus, respectively. The chloroform fraction (VW-2) showed significant activity against S. aureus with 0.27 mg/ml MIC, where 0.31 mg/ml MIC was deduced for VW-3 fraction against B. subtilus. VW-3 fraction was also found to be the most potent inhibitor of M. canis, showing 70% inhibition with an MIC value of 0.19 mg/ml. Considerable inhibitory activity was also observed for VW-2 and water fraction (VW-6) against M. canis and A. flavus. A remarkable anti-inflammatory like activity was observed for the crude extract at a dose of 200 mg/kg at all observed durations. Other doses of the sample also showed excellent activity. Looking to these results it may be concluded that V. wallichii may be a potential source for activity guided isolation of natural products with antimicrobial and anti-inflammatory-like properties.

Background ‘Naswar’ is a smokeless tobacco product (STP) widely used in Pakistan. It has been correlated with oral and oesophageal cancer in recent clinical studies. The toxic effects associated with STPs have been associated with trace level contaminants present in these products. The toxin levels of Pakistani naswar are reported for the first time in this study. Methods A total of 30 Pakistani brands of naswar were tested for a variety of toxic constituents and carcinogens such as cadmium, arsenic, lead and other carcinogenic metals, nitrite and nitrate, and nicotine and pH. Results The average values of all the toxins studied were well above their allowable limits, making the product a health risk for consumers. Calculated lifetime cancer risk from cadmium and lead was 1 lac (100000) to 10 lac (1000000) times higher than the minimum 10E _ 4 (0.00001) to 10E _ 6 (0.000001), which is the ‘target range’ for potentially hazardous substances, according to the US Environmental Protection Agency. Similarly, the level of arsenic was in the range of 0.15 to 14.04 mg/g, the average being 1.25 mg/g. The estimated average bioavailable concentration of arsenic is 0.125e0.25 mg/g, which is higher than the allowable standard of 0.01 mg/g. Similarly, the average minimum daily intake of chromium and nickel was 126.97 mg and 122.01 mg, as compared to allowable 30e35 mg and 35 mg, respectively; a 4e5 times higher exposure. However, beryllium was not detected in any of the brands studied. The pH was highly basic, averaging 8.56, which favours the formation of tobacco specific amines thus making the product potentially toxic. This study validates clinical studies correlating incidence of cancer with naswar use in Pakistan. Conclusions This study shows that the production, packaging, sale and consumption of naswar should be regulated so as to protect the public from the health hazards associated with its consumption.