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1.  HPLC-DAD analysis, antioxidant potential and anti-urease activity of Asparagus gracilis collected from District Islamabad 
Asparagus gracilis subspecie of Asparagus capitatus Baker, is described as food and medicine for various ailments. In this study we investigated, its phenolic constituents, in vitro antioxidant potential against various free radicals and anti-urease potential.
Asparagus gracilis aerial parts collected from District Islamabad, Pakistan were extracted with crude methanol which was further fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fraction. Total phenolic and flavonoid contents were estimated for extract and all the derived fractions. Diverse in vitro antioxidants assays such as DPPH, H2O2, •OH, ABTS, β-carotene bleaching assay, superoxide radical, lipid peroxidation, reducing power, and total antioxidant capacity were studied to assess scavenging potential. Antiurease activity of methanol extract and its derived fractions was also investigated. HPLC-DAD analysis of crude methanol extract was performed by using different phenolic standards.
Ethyl acetate fraction expressed maximum content of flavonoids (240.6 ± 6.1 mg RE/g dry sample), phenolics (615 ± 13 mg GAE/g dry sample) and best antioxidant potential among different fractions of crude methanol extract. Hydrogen peroxide assay and hydroxyl, supeoxide, nitric oxide free radicals antioxidant assays as well as beta carotene assay showed significant correlation with flavonoid content while hydrogen peroxide, ABTS and lipid peroxidation assay displayed significant correlation with phenolic content. HPLC analysis showed the presence of important phenolics i.e. catechin (4.04 ± 0.02 μg/mg sample), caffeic acid (0.89 ± 0.003 μg/mg sample), rutin (24.58 ± 0.1 μg/mg sample), myricetin (1.13 ± 0.07 μg/mg sample) and quercetin (14.91 ± 0.09 μg/mg sample). Ethyl acetate fraction expressed lowest IC50 in antiurease activity. Correlation analysis of antiurease activity expressed significant correlation with flavonoids (P < 0.004) and phenolics (P < 0.02) proposing multipotent activity of fractions.
These results revealed the presence of some bioactive compound in the ethyl acetate fraction having both antioxidant as well as antiurease potential.
PMCID: PMC4179820  PMID: 25245231
Antiurease; Phytochemistry; Antioxidant; Mutltipotent activity; Fractionation
2.  Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens 
PLoS ONE  2014;9(5):e96979.
Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.
PMCID: PMC4016209  PMID: 24817272
3.  Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene 
BMC Research Notes  2012;5:285.
Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants.
Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1 along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis.
Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.
PMCID: PMC3507645  PMID: 22691292
Genetic transformation; Yeast halotolerant gene (HAL2); Citrus jambhiri Lush and Agrobacterium tumefaciens
4.  Isolation of antibacterial compounds from Quercus dilatata L. through bioassay guided fractionation 
Four medicinal plants (Chrozophora hierosolymitana Spreng, Chrysanthemum leucanthemum L., Ephedra gerardiana Wall. ex Stapf, and Quercus dilatata L.) used by indigenous healers to treat various infectious diseases were selected for the present study. The major objective of the present study was isolation and characterization of antimicrobial components from the crude plant extracts using bioassay guided fractionation.
Seven methanolic extracts of the four plants were screened to identify any antimicrobial agents present in them. The active crude plant extract was fractionated first by solvent partitioning and then by HPLC. Characterization of the active fractions was done by using spectrophotometer.
All the seven methanolic extracts showed low antifungal activity, however, when these extracts were tested for antibacterial activity, significant activity was exhibited by two extracts. The extract of aerial parts of Q. dilatata was most active and therefore, was selected for further analysis. Initially fractionation was done by solvent-solvent partitioning and out of six partitioned fractions, ethanol fraction was selected on the basis of results of antibacterial activity and phytochemical analysis. Further, fractionation was carried out by RP- HPLC and purified active subfractions were characterized by comparing their absorption spectra with that of the known natural products isolated from the plants of Quercus genus.
Discussion and conclusion
The results suggest that this is the first report of the isolated antibacterial compounds from this genus.
PMCID: PMC3416653  PMID: 22554280
Absorption spectrum; Antibacterial activity; Phytochemical analysis; RP-HPLC analysis; Solvent partitioning
5.  Antioxidant and Cytotoxic Activities and Phytochemical Analysis of Euphorbia wallichii Root Extract and its Fractions 
Euphorbia wallichii a perennial herb growing mainly in Himalayas has been widely used in folk medicines for its medicinal properties. In the present study, the crude methanolic root extract (CME) and its fractions; n-Hexane Fraction (NHF), n-Butanol Fraction (NBF), Chloroform Fraction (CHF), Ethyl acetate Fraction (EAF) and Aqueous Fraction (AQF) of this plant specie were investigated for antioxidant and cytotoxic activities and phytochemical analysis. Antioxidant activity was determined by using 2,2-diphenyl-1-picryl-hydrazyl free radical (DPPH) and DNA protection assay performed on pBR322 plasmid DNA. In both these assays, promising results were obtained for CME as well as other fractions. The IC50 values for DPPH assay were in a range of 7.89 to 63.35 μg/ml in which EAF showed the best anti-oxidant potential and almost all the tested samples showed certain level of DNA protection. The cytotoxic activity was assessed by using Sulforhodamine B (SRB) assay on human cell lines; H157 (Lung Carcinoma) and HT144 (Malignant Melanoma). The IC50 values of the tested samples ranged from 0.18 to 1.4 mg/mL against H157 cell line whereas against HT144 cell line the IC50 values ranged from 0.46 to 17.88 mg/mL with NBF fraction showing maximum potential for both. Furthermore, the phytochemical analysis of CME and its fractions showed the presences of flavonoids, saponins, tannins, terpenoides and cardiac glycosides with varying concentrations.
PMCID: PMC3813110  PMID: 24250446
Antioxidant; Cytotoxicity; DNA protection; E. wallichii; Phytochemical analysis
6.  Genetic differentiation and geographical Relationship of Asian barley landraces using SSRs 
Genetics and Molecular Biology  2011;34(2):268-273.
Genetic diversity in 403 morphologically distinct landraces of barley (Hordeum vulgare L. subsp. vulgare) originating from seven geographical zones of Asia was studied using simple sequence repeat (SSR) markers from regions of medium to high recombination in the barley genome. The seven polymorphic SSR markers representing each of the chromosomes chosen for the study revealed a high level of allelic diversity among the landraces. Genetic richness was highest in those from India, followed by Pakistan while it was lowest for Uzbekistan and Turkmenistan. Out of the 50 alleles detected, 15 were unique to a geographic region. Genetic diversity was highest for landraces from Pakistan (0.70 ± 0.06) and lowest for those from Uzbekistan (0.18 ± 0.17). Likewise, polymorphic information content (PIC) was highest for Pakistan (0.67 ± 0.06) and lowest for Uzbekistan (0.15 ± 0.17). Diversity among groups was 40% compared to 60% within groups. Principal component analysis clustered the barley landraces into three groups to predict their domestication patterns. In total 51.58% of the variation was explained by the first two principal components of the barley germplasm. Pakistan landraces were clustered separately from those of India, Iran, Nepal and Iraq, whereas those from Turkmenistan and Uzbekistan were clustered together into a separate group.
PMCID: PMC3115321  PMID: 21734828
cluster analysis; genetic diversity; geographical differentiation; Hordeum vulgare; principal component analysis
7.  Survey of artemisinin production by diverse Artemisia species in northern Pakistan 
Malaria Journal  2010;9:310.
Artemisinin is the current drug of choice for treatment of malaria and a number of other diseases. It is obtained from the annual herb, Artemisia annua and some microbial sources by genetic engineering. There is a great concern that the artemisinin production at current rate will not meet the increasing demand by the pharmaceutical industry, so looking for additional sources is imperative.
In current study, artemisinin concentration was analysed and compared in the flowers, leaves, roots and stems of Artemisia annua and 14 other Artemisia species including two varieties each for Artemisia roxburghiana and Artemisia dracunculus using high performance liquid chromatography (HPLC).
The highest artemisinin concentration was detected in the leaves (0.44 ± 0.03%) and flowers (0.42 ± 0.03%) of A. annua, followed by the flowers (0.34 ± .02%) of A. bushriences and leaves (0.27 ± 0%) of A. dracunculus var dracunculus. The average concentration of artemisinin varied in the order of flowers > leaves > stems > roots.
This study identifies twelve novel plant sources of artemisinin, which may be helpful for pharmaceutical production of artemisinin. This is the first report of quantitative comparison of artemisinin among a large number of Artemisia species.
PMCID: PMC2989329  PMID: 21047440
8.  ATR-FTIR spectroscopy detects alterations induced by organotin(IV) carboxylates in MCF-7 cells at sub-cytotoxic/-genotoxic concentrations 
PMC Biophysics  2008;1:3.
The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 μg/ml, 0.1 μg/ml or 1.0 μg/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 – 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 μg/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 μg/ml, 1.0 μg/ml or 10.0 μg/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with ≥ 1.0 μg/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.
PACS codes: 87.15.-v, 87.17.-d, 87.18.-h
PMCID: PMC2666631  PMID: 19351425

Results 1-8 (8)