Column Preparative Chromatography Research Articles

Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway through covalent modification of the 2-alkenal group of aliphatic electrophiles in Coriandrum sativum L.

Phytochemicals able to activate the transcription factor NF-E2-related factor 2 (Nrf2) were isolated from an extract of Coriandrum sativum L. (C. sativum) leaves by preparative octadecyl silica column chromatography. Ultraperformance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis of the isolated components after derivatization with 2-diphenylacetyl-1,3-inandione-1-hydrazone and experiments with HepG2 cells revealed that (E)-2-alkenals with different carbon numbers play a role in Nrf2 activation in these cells. Such Nrf2 activation appears to be attributable to S-alkylation of Kelch-like ECH-associated protein 1 (Keap1), the negative regulator for Nrf2, as determined by a biotin-PEAC5-maleimide assay. Interestingly, (E)-2-butenal caused Keap1 modification and Nrf2 activation, whereas butanal did not. These results suggest that (E)-2-alkenals with an α,β-unsaturated aldehyde moiety, which is a common substituent in phytochemicals isolated from C. sativum leaves, activate the Keap1/Nrf2 pathway associated with cellular protection.

Immunostimulating activity of maysin isolated from corn silk in murine RAW 264.7 macrophages

Corn silk (CS) has long been consumed as a traditional herb in Korea. Maysin is a major flavonoid of CS. The effects of maysin on macrophage activation were evaluated, using the murine macrophage RAW 264.7 cells. Maysin was isolated from CS by methanol extraction, and preparative C18 reverse phase column chromatography. Maysin was nontoxic up to 100 μg/ml, and dose-dependently increased TNF-α secretion and iNOS production by 11.2- and 4.2-fold, respectively, compared to untreated control. The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with maysin (1-100 μg/ml). Maysin also stimulated the phosphorylation of Akt and MAPKs (ERK, JNK). These results indicated that maysin activates macrophages to secrete TNF-α and induce iNOS expression, via the activation of the Akt, NF-κB and MAPKs signaling pathways. These results suggest for the first time that maysin can be a new immunomodulator, enhancing the early innate immunity. [BMB Reports 2014; 47(7): 382-387]

Flavonoid content and radical scavenging potential of extracts prepared from tobacco cultivars and waste

The flavonoid-rich natural products exert a wide range of pharmacological properties. This investigation aimed at obtaining extracts from tobacco cultivars and waste enriched with flavonoids and purified from nicotine and phenolic acids, and evaluating their radical scavenging potential. Extraction with a mixture of ethyl acetate–methanol (1:1, v/v) was employed resulting in 100% yield for flavonoids and 36% yield for phenolic acids. The crude extracts were purified using preparative column chromatography on silica gel. The content of flavonoids in the purified extracts varied from 8.8 ± 1.1% to 14.3 ± 1.8%. Nicotine was not detected in amounts higher than 0.3 μg mL− 1. The content of phenolic acids was lower than 1.0%. The radical scavenging potential of extracts from tobacco cultivars exhibited values from IC50 = 35.0 ± 3.1 to 64.6 ± 7.5 μg mL− 1. The extracts obtained by the proposed procedure are enriched with flavonoids with high radical scavenging potential and are purified from nicotine and phenolic acids. They can be regarded as potential biopharmaceuticals.

Studies of n-alkanes in the sediments of Colony Z3 (Pelotas - RS - Brazil)

This paper investigates the presence and concentration levels, of n-alkanes and also the potential sources of contamination of surface sediments in the fishing village Z-3 in the city of Pelotas, Brazil. The extraction of the sediments was performed according to EPA method 3540 and the separation of aliphatic hydrocarbons was done with preparative column chromatography. Gas chromatographic analysis with flame ionization detector was employed for the identification and quantification of compounds The level of aliphatic hydrocarbons found, was due mainly to n-alkanes originated from weathering of terrestrial plants and algae production. It is also possible to observe the dilution effect of the lagoon about analyzed compounds, comparing the results obtained from different sampling points. In general, the sediments sampled in the investigated region presented small levels of contamination by hydrocarbons

Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D

To improve biosurfactant production economics by the utilization of potential low-cost materials. In an attempt to utilize cost-effective carbon sources in the fermentative production of biosurfactants, various pure and waste frying oils were screened by a standard biosurfactant producing strain. Considering the regional significance, easy availability and the economical advantages, waste frying coconut oil was selected as the substrate for further studies. On isolation of more competent strains that could use waste frying coconut oil efficiently as a carbon source, six bacterial strains were isolated on cetyltrimethyl ammonium bromide-methylene blue agar plate, from a soil sample collected from the premises of a coconut oil mill. Among these, Pseudomonas aeruginosa D was selected as the potential producer of rhamnolipid. Spectrophotometric method, TLC, methylene blue active substance assay, drop collapse technique, surface tension measurement by Du Nouy ring method and emulsifying test confirmed the rhamnolipid producing ability of the selected strain and various process parameters were optimized for the production of maximum amount of biosurfactant. Rhamnolipid components purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, HPLC and TLC were characterized by fast atom bombardment mass spectrometry as a mixture of dirhamnolipids and monorhamnolipids. The rhamnolipid homologues detected were Rha-Rha-C(10) -C(10) , Rha-C(12) -C(10) and Rha-C(10) -C(8) /Rha-C(8) -C(10) . These results indicated the possibility of waste frying coconut oil to be used as a very effective alternate substrate for the economic production of rhamnolipid by a newly isolated Ps.aeruginosa D. Results of this study throws light on the alternate use of already used cooking oil as high-energy source for producing a high value product like rhamnolipid. This would provide options for the food industry other than the recycling and reuse of waste frying oils in cooking and also furthering the value of oil nuts.

Antimycobacterial Effect of Extract and Its Components from Rheum rhaponticum

The global threat of tuberculosis demands the search for alternative antimycobacterial drugs. The present study examined roots and petioles from Rheum rhaponticum for antimycobacterial activity. Crude methanol extracts and eight phenolic compounds isolated by preparative column chromatography were tested against Mycobacterium tuberculosis H37Ra and M. bovis using the broth dilution method. The extract from roots and its components, such as rhaponticin, deoxyrhaponticin, resveratrol, barbaloin, aloe-emodin, and chrysophanol were found to have antimycobacterial activity against both microorganisms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of all the investigated samples ranged from 32 to 512 microg/mL. The anthracene derivatives were the most active; their MICs were 32, 64, and 64 microg/mL (M. tuberculosis H37Ra) and 128, 64, and 64 microg/mL (M. bovis), respectively. The microorganisms were resistant to stimulation with extract from petioles, as were quercetin and rutin. The results showed that anthracene and stilbene derivatives play a prominent role in the antimycobacterial properties of R. rhaponticum.

Phytochemical, antimicrobial investigation and formulations development of extract of Semicarpus anacardium seed for their antidandruff potential

Background: Dandruff is one of the most commercially exploited skin disease by the personal care industry all over the world, which affects 5% of the global population. Plant kingdom is a gold mine for novel and affordable skin care acting through novel mechanisms against skin pathogens. Identification of these remedies for such skin care is an important study for the preservation of traditional knowledge for the antimicrobial treatments. Semicarpus anacardium Linn. (Anacardiaceae) has been used for the skin care since ancient times. Aim of the Study: The aim of the present study was to investigate the antidandruff potential of S. anacardium seed extract and development of phytochemical, antimicrobial, and formulations. Materials and Methods: Antimicrobial activity of petroleum ether extract of S. anacardium seed was studied by agar well diffusion method using Escherichia coli, Staphylococcus aureus, and Candida albicans strains. Two herbal antidandruff shampoo formulations were developed and evaluated using parameters, physical evaluation, that is, color and consistency, pH, washability, homogeneity, spreadability, and skin irritation tests. The developed herbal shampoo formulations were assessed for their antidandruff potential manually on the human volunteers, respectively. One phenolic compound was isolated using preparative column chromatography, followed by thin layer chromatography. Results: Results of present studies revealed that there was a concentration-dependent antimicrobial effect compared with standard antibiotic disc, that is, gentamicin. S. aureus showed more zone of inhibition than E. coli. The inhibitory zone of all isolates was determined as 0-23 mm. The observed antimicrobial activity was due to the potent bioactive phytoconstituents present in the extract. Our formulations were found to be semisolid consistency, brownish black color with very slightly alkaline pH, that is, 7.3 and 7.1, which is compatible with normal skin physiology, easily washable, homogenous aggregations, good spreadabilty along with free from skin irritation. The developed antidandruff herbal shampoo formulations were manually checked for its safety and efficacy on human volunteers for a week and were found that complete irradication of dandruff within a time period of 7 days. These formulations were also softening the hairs. The preliminary phytochemical analysis of the petroleum ether extract of S. anacardium was showed the presence of phenolic compounds, tannins, saponins, flavonoids, steroids, glycosides, carbohydrates, and alkaloids. Further, two compounds were detected in the thin layer chromatographic analysis of extract of S. anacardium which having their R f values 0.9 and 0.6. One phenolic compound was isolated which has R f value 0.85. Conclusion: The petroleum ether extract of S. anacardium seed possessed a broad spectrum of activity against a panel of bacteria and fungi responsible for the most common microbial diseases. This plant can be used as a cheap source of active therapeutics. Further, exploration of plant derived antimicrobials as well as their identity is needed. However, further series of scientific studies are required to prove its clinical reliability, safety, and efficacy for the beneficial of mankind.

Evaluation of Traditional Medicines I: Identification of Phela Using Different Chromatographic Techniques

PHELA is a herbal mixture of four African traditional medicinal plants that has been used for decades in wasting conditions and is now being developed by the Medical Research Council (MRC) as an immune booster for patients with compromised immune system. A chromatographic fingerprint of PHELA was needed for quality control purposes. Here, a comprehensive method for fingerprinting of PHELA using different chromatographic techniques is described. It involved extraction of the PHELA by either acidic or a simple 'salting-out' method, followed by Thin Layer Chromatography (TLC) analysis and/or preparative Column Chromatography (CC). The products were thereafter analyzed by High Performance Liquid Chromatography with UV-detector (HPLC-UV), HPLC with fluorescence-detector (HPLC-FL) and Gas-Chromatography with a Mass Selective Detector spectrometer (GC-MSD). The fingerprints were successfully used to differentiate PHELA from another common herbal product made from Hypericum perforatum (St. John's Wort), thereby illustrating its high potential for use in fingerprinting of PHELA and in differentiating it from other herbal medicines. By validating the different chromatographic techniques on the standardized extraction methods, this approach will enable wide application in quality control of PHELA using acceptable procedures, thereby promoting effective monitoring of the finished product in all countries where it will be used.

Evaluation of lipid removal from animal fats for the determination of organochlorine, pesticides, and polychlorinated biphenyl indicators by gas chromatography with electron capture detector

For cleanup of animal fat before GC analysis of organochlorine pesticides and polychlorinated biphenyls, freezing-lipid filtration with solid-phase extraction and matrix solid-phase dispersion were evaluated to replace the official Brazilian methodology that uses preparative alumina column chromatography. General drawbacks associated with this last technique, such as the use of large amounts of solvent, laborious and time-consuming procedure could be avoided by using these alternative approaches. Experiments were carried out to study the performance by using different combinations of sorbents and elution solvents. Efficiency of alternative extraction methods in terms of fat removal and recovery capability was monitored by gravimetry, TLC, and GC with electron capture detection. Freezing-lipid filtration with solid-phase extraction afforded better clean up efficiency with recoveries in a range of 54.5 to 103.6% with the relative standard deviation of less than 10% for all compounds under investigation.

Determination of carotenoids in Taraxacum formosanum by HPLC–DAD–APCI-MS and preparation by column chromatography

The objectives of this study were to determine the variety and content of carotenoids in Taraxacum formosanum, a traditional Chinese herb possessing vital biological activities, by developing an HPLC-DAD-APCI-MS method and a preparative column chromatographic method for carotenoid isolation. A total of 25 carotenoids were resolved within 66 min by employing a YMC C30 column and a gradient mobile phase of methanol-acetonitrile-water (79:14:7, v/v/v) and methylene chloride (100%) with flow rate at 1.0 mL/min and detection at 450 nm. All-trans-canthaxanthin was shown to be an appropriate internal standard for quantitation, with all-trans-β-carotene and its cis isomers present in largest amount (413.6 μg/g), followed by all-trans-violoxanthin and its cis isomers (209.5 μg/g), all-trans-lutein and its cis isomers (212.4 μg/g), all-trans-neoxanthin and its cis isomers (134.6 μg/g), antheraxanthin (16.5 μg/g), all-trans-β-cryptoxanthin and its cis isomers (5.8 μg/g), all-trans-zeaxanthin (3.6 μg/g) and neochrome (0.1 μg/g). For preparative chromatography, with a glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) as adsorbent, the carotenoid fraction was eluted with 300 mL of ethyl acetate with flow rate at 10 mL/min. Some more epoxides and cis isomers of carotenoids were generated during preparative column chromatography. Nevertheless, the carotenoids isolated from T. formosanum may be used as raw material for possible production of health food in the future.

Irresolvable complex mixture of hydrocarbons in soybean oil deodorizer distillate

Aliphatic hydrocarbons (HCs) can be used as a fingerprint of a given seed oil. Only by characterization of aliphatic HCs could contamination by mineral oil in that seed oil be confirmed. During the isolation of squalene from soybean oil deodorizer distillate, a significant amount of unknown HCs, ca. 44 wt%, was obtained. These seemingly-easy-to-identify HCs turned out to be much more difficult to elucidate due to the presence of an irresolvable complex mixture (ICM). The objective of this study was to purify and identify the unknown ICM of aliphatic HCs from soybean oil deodorizer distillate. Purification of the ICM was successfully achieved by using modified Soxhlet extraction, followed by modified preparative column chromatography, and finally by classical preparative column chromatography. FT-IR, TLC, elemental analysis, GC/FID, NMR and GC-MS analyses were then performed on the purified HCs. The GC chromatogram detected the presence of ICM peaks comprising two major peaks and a number of minor peaks. Validation methods such as IR and NMR justified that the unknowns are saturated HCs. This work succeeded in tentatively identifying the two major peaks in the ICM as cycloalkane derivatives.

Characterization of two new potential impurities of Valsartan obtained under photodegradation stress condition

A photostability study of Valsartan (VAL) is reported. Exposure of the drug to UV–vis radiation ( λ > 320 nm) yielded two previously unknown compounds, which were detected by HPLC. Preparative amounts of the new potential degradation products (DP-1 and DP-2) were obtained by submitting VAL bulk drug to extensive photodegradation. The impurities were isolated by preparative normal phase column chromatography. Analytical information from the infrared, nuclear magnetic resonance and mass spectral data of the degradation products revealed their structures as N-[2′-(1 H-tetrazol-5-yl)-biphenyl-4-ylmethyl]- N-isobutylpentanamide (DP-1) and N-(diazirino[1,3- f]phenanthridin-4-ylmethyl)- N-isobutylpentanamide (DP-2). DP-1 arose from decarboxylation of VAL, while DP-2 results from further loss of nitrogen from the tetrazole motif of DP-1, with concomitant cyclization to yield a tetracyclic diazacyclopropene derivative.

Anthocyanins from fruit of two species of the genus Rosa

We investigated the composition of anthocyanins from fruit of Rosa spinosissima L. and R. hracziana Tamam. (Rosaceae Juss.) growing near Bata-Bat, Shakhbuz Region, Republic of Azerbaidzhan. Fruit was collected on Sept. 16, 2009, during ripening. Freshly collected fruit (100 g each) was frozen with liquid N2 and pulverized. Pulp was separated from stones. Pulp (50 g each) was ground and extracted at room temperature for 2 d twice with EtOH (95%) and once with MeOH containing HCl (1%). The extracts were combined, filtered, and evaporated to dryness in vacuo at 30°C. The dry solid was treated with CHCl3 and then dissolved in MeOH (50 mL) containing HCl (0.5%). A part of the extract was hydrolyzed by HCl (2 N, 1:1 ratio) on a boiling-water bath for 60 min under reflux [1]. The hydrolysate was cooled. Aglycones were extracted by isoamyl alcohol for 20 h (shaking periodically). The isoamyl extract was studied by 2D chromatography on FN16 paper using Formic and Forestal systems [2]. Two aglycones were detected in the chromatogram. Preparative column chromatography using the Formic system isolated two pure aglycones. Aglycone-1 with Rf 0.32 (Formic) and 0.48 (Forestal) gave absorption maxima with fuchsine dye at 276 and 535 nm (MeOH containing 0.01% HCl). Adding AlCl3 produced a bathochromic shift of 20 nm. Aglycone-2 with Rf 0.12 and 0.31 (in the same systems, respectively) gave absorption maxima with purple dye at 277 and 546 nm. Addition of AlCl3 produced a bathochromic shift of 22 nm. Based on the chromatographic and spectral data and a comparison with authentic samples and the literature [2, 3], the first aglycone was identified as cyanidin; the second, delphinidin. Preparative 2D paper chromatography using n-BuOH:AcOH:H2O (4:1:2) (first direction) and AcOH:HCl (conc.):H2O (15:3:82) (second direction) showed that pulp of R. spinosissima fruit contained three; of R. hracziana, four anthocyanin glycosides. The first three components were identical in both species. Therefore, total anthocyanins from fruit pulp of R. hracziana was analyzed in detail. Preparative column chromatography (acidic cellulose powder) [4] and rechromatography on paper produced four pure anthocyanins. The pure anthocyanins were studied by chromatography and spectroscopy [2, 3, 5]. The anthocyanins were hydrolyzed as described above by neutralizing the hydrolysate with ion-exchange resin. Paper chromatography with authentic sugar samples identified D-glucose in hydrolysates of all glycosides. The chromatographic mobilities of anthocyanins 1 and 2 suggested that they were monoglucosides; 3 and 4, diglucosides. This agreed with results of stepwise acid analysis. The aglycone and sugar were cleaved after stepwise hydrolysis of anthocyanins 1 and 2 for 20 min. However, anthocyanins 3 and 4 formed four components. The first component corresponded to the starting glucoside; the second and third, to a monoglucoside; the fourth, the aglycone. The second spot did not fluoresce. Its Rf value indicated that it was the 3-glucoside. The third spot did fluoresce in UV light with emission analogous to the starting glucoside. It corresponded to the 5-glucoside according to fluorescence and chromatographic mobility. Oxidation of the anthocyanins with H2O2 produced glucose and confirmed that it was bonded to C3 in 1 and 2 and to C3 and C5 in 3 and 4 [1–3, 5, 6]. Thus, cyanidin-3-glucoside, cyanidin-3,5-diglucoside, delphinidin-3-glucoside, and delphinidin-3,5-diglucoside were identified for the first time in total anthocyanins from fruit pulp of R. hracziana using chromatography, spectroscopy, total and stepwise hydrolysis, oxidation by H2O2, and comparison with authentic samples. All components with the exception of delphinidin-3,5-diglucoside were found in R. spinosissima anthocyanidins. The monoglucosides of cyanidin and delphinidin dominated quantitatively the anthocyanin complex from fruit of both Rosa species.

Pectins from Canna edulis Ker residue and their physicochemical characterization

Water-soluble pectin (WSP), chelated-soluble pectin (CSP) and acid-soluble pectin (ASP) were sequentially extracted from Canna edulis Ker residue, a by-product after the extraction of starch, and their physicochemical properties were studied. ASP was further separated through preparative column chromatography, leading to isolation of two fractions, ASPI and ASPII, which chemical features were analyzed by GC, GC–MS and NMR. Moreover, their behaviors in the simulated gastric and intestinal fluid were monitored. The results exhibit that C. edulis residue contains high amount of ASP derived from the heteropolysaccharide with rhamnogalacturonan as main chain. Furthermore, three pectins show gel formation and dissolution behaviors. It well suggests that C. edulis residue can be exploited for a good source of pectin for functional materials.

SCREENING AND IDENTIFICATION OF RADICAL SCAVENGERS FROM NEO-TARAXACUM SIPHONANTHUM BY ONLINE RAPID SCREENING METHOD AND NUCLEAR MAGNETIC RESONANCE EXPERIMENTS

An online rapid screening method, the high-performance liquid chromatography (HPLC)–diode array detector (DAD)–radical scavenging detection (RSD)–electrospray ionization (ESI)–mass spectroscopy (MS)/MS system, was developed for the screening and identification of radical scavengers from Neo-Taraxacum siphonanthum, a new species found in China in 1989. For further characterization, the target compounds were isolated by silica column chromatography, preparative high-performance liquid chromatography (HPLC), HSCCC, and Sephadex LH-20 column chromatography and elucidated on the basis of ultraviolet (UV), ESI-MS/MS, and nuclear magnetic resonance (NMR) spectroscopy, as well as the chemical analysis. Eighteen antioxidative polyphenols (5 caffeic acid derivatives and 13 flavonoid derivatives) were characterized from Neo-T. siphonanthum. The distribution of all compounds was discussed in a chemosystematic context, which suggested that the genera Neo-Taraxacum and Taraxacum might relate chemosystematically.

Determination of total polar material in frying oil using accelerated solvent extraction

AbstractDuring use, frying fats undergo chemical deterioration. This leads to the formation of compounds that are more polar than the triacylglycerols of the fat. Collectively these are called total polar material (TPM), and the mass concentration of TPM is used as an indicator of the quality of frying fats. According to official methods, the fat or oil is separated into polar and non‐polar fractions by preparative column chromatography (PCC). The chromatographic step is time‐consuming and needs large amounts of solvents. Here we show that the chromatographic step can be replaced by accelerated solvent extraction (ASE). This was demonstrated for high‐oleic sunflower oil in which potatoes, carrots and parsnips were fried. The materials separated by these two procedures are shown to be identical as judged by HPLC of the TPM. The two procedures show good agreement.

Isolation of carotenoids, flavonoids and polysaccharides from Lycium barbarum L. and evaluation of antioxidant activity

A preparative column chromatography method was developed to isolate carotenoids, flavonoids and polysaccharides, from Lycium barbarum L., possessing vital biological activity, and their antioxidant activity was evaluated. Carotenoids were isolated by a column containing magnesium oxide and diatomaceous earth (1.5:1, w/w), and β-carotene was eluted with hexane, β-cryptoxanthin and neoxanthin with ethyl acetate and zeaxanthin with ethyl acetate–ethanol (80:20, v/v). Flavonoids and phenolic acids were separated using a Cosmosil 140 C18-OPN column, with phenolic acids being eluted with deionized water and neutral flavonoids with methanol. Polysaccharides were fractionated using a DEAE-Sepharose CL-6B column; neutral polysaccharides were eluted with water and acidic polysaccharides with different concentrations of NaCl. For antioxidant activity, the flavonoid fraction was the most effective in scavenging DPPH · and ABTS + free radicals, chelating metal ions and reducing power, while the zeaxanthin fraction and polysaccharides showed the most pronounced effect in scavenging hydroxy free radicals and superoxide anions, respectively.

Regioselective Addition of N‐(4‐Thiocyanatophenyl)pyrrolidine Addends to Fullerenes

AbstractThe regioselectivity of 1,3‐dipolar cycloaddition reactions between N‐(4‐thiocyanatophenyl)glycine and tetrakis[bis(ethoxycarbonyl)methylene]‐C60 (7) was studied. The mono‐addition resulted in two penta‐adduct isomers (8 and 9) in a 1:1 ratio. A second addition to each compound led to, in the case of 8, two hexa‐adduct isomer products (10 and 11), whereas addition to 9 led to 11 and 12. Finally, reaction of 11 and the glycine afforded a third addition, giving rise to hepta‐adduct isomers 13 and 14. Preparative TLC and column chromatography were used to separate and purify these compounds. The structures were assigned based on their molecular symmetries as analyzed by 1H, 13C, and 2D NMR spectra and MALDI‐TOF MS.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Synthesis and in vitro activities of a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue

To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octadecylglycerol residue we condensed 1- O-4-monomethoxytrityl-3- O-octadecyl- sn-glycerol-2-hydrogenphosphonate obtained from 3- O-octadecyl- sn-glycerol with AZT by the phosphonate method. The purified condensation product was de-tritylated resulting in 3′-azido-3′-deoxythymidylyl-(5′ → 2- O)-3- O-octadecyl- sn-glycerol, followed by treatment with (ethoxycarbonyl)phosphoric dichloride. The resulting 3′-azido-3′-deoxy-thymidylyl-(5′ → 2)-3- O-octadecyl- sn-glycerol-1- O-(ethoxycarbonyl)phosphonate was purified by preparative RP-18 column chromatography. The antiviral duplex drug 3′-azido-3′-deoxythymidylyl-(5′ → 2- O)-3- O-octadecyl- sn-glycerol-1- O-phosphonoformate trisodium salt (AZT–lipid–PFA) was obtained after alkaline cleavage of the phosphonoformate ethylester residue. The overall yield of the five step synthesis performed at gram scale was about 30%. According to a supposed pathway AZT–lipid–PFA could be cleaved to yield a mixture of different antiviral compounds such as AZT, AZT-5′-monophosphate, octadecylglycerol–AZT, PFA and octadecylglycerol–PFA, possibly producing additive and/or synergistic antiviral effects. In vitro studies showed that the duplex drug exhibits antiviral activities against HIV and especially against drug-resistant strains and clinical isolates of HSV and HCMV. The E 50 values of AZT–lipid–PFA against HIV ranged between 170 and 200 nM. The half-maximal inhibitory doses (IC 50) against highly acyclovir (ACV)-resistant HSV isolates determined by a plaque reduction assay ranged between 1.87 and 4.59 μM. Using ganciclovir (GCV)-sensitive, GCV resistant and drug cross-resistant HCMV strains the IC 50-values of AZT–lipid–PFA were between 2.78 and 1.18 μM. With regard to PFA, the IC 50-value of AZT–lipid–PFA determined on a multi-drug-resistant HCMV strain was about 90-fold lower than that of PFA, demonstrating the superior antiviral effect of the duplex-drug.

Analysis of Rhamnolipid Biosurfactants Produced Through Submerged Fermentation Using Orange Fruit Peelings as Sole Carbon Source

The fermentative production of rhamnolipid biosurfactant from Pseudomonas aeruginosa MTCC 2297 was carried out by submerged fermentation using various cost-effective waste materials such as orange peelings, carrot peel waste, lime peelings, coconut oil cake, and banana waste. The orange peel was found to be the best substrate generating 9.18 g/l of rhamnolipid biosurfactant with a surface tension reduction up to 31.3 mN/m. The production was growth independent, and optimum conditions were standardized. The emulsifying activity was highest against kerosene (73.3%). Rhamnolipid components were purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, high-performance liquid chromatography and thin-layer chromatography. The major rhamnolipid components were characterized, by fast atom bombardment mass spectrometry, as a mixture of dirhamnolipids and monorhamnolipids.