Response surface methodology-based optimization of ultrasound-assisted extraction of β-sitosterol and lupeol from Astragalus atropilosus(roots) and validation by HPTLC method

Objective:To optimize the ultrasonication method for efficient extraction ofβ-sitosterol and lupeol from the roots of Astragalus atropilosus using Box-Behnken design of response surface methodology(RSM),and its validation by high performance thin layer chromatography(HPTLC)method.Methods:Ultrasonication method was used to extractβ-sitosterol and lupeol from Astragalus atropilosus(roots).RSM was used to optimize the different extraction parameters viz.liquid to solid ratio(10–14 m L/g),temperature(60-80℃)and time(40–60 min)to maximize the yield ofβ-sitosterol and lupeol.The quantitative estimation ofβ-sitosterol and lupeol was done in chloroform extract of Astragalus atropilosus by validated HPTLC method on 10 cm×20 cm glass-backed silica gel 60 F254 plate using hexane and ethyl acetate(8:2,v/v)as mobile phase.Results:A quadratic polynomial model was found to be most appropriate with regard to R1(yield of total extraction;R2/%CV=0.9948/0.28),R2(β-sitosterol yield;R2/%CV=0.9923/0.39)and R3(lupeol yield;R2/%CV=0.9942/0.97).The values of adjusted R2/predicted R2/signal to noise ratio for R1,R2,and R3 were 0.9782/0.9551/48.77,0.9904/0.9110/31.33,and 0.9927/0.9401/36.08,respectively,indicating a high degree of correlation and adequate signal.The linear correlation plot between the predicted and experimental values for R1,R2,and R3 showed high values of R2 ranging from 0.9905-0.9973.β-sitosterol and lupeol in chloroform extract of Astragalus atropilosus were detected at Rf values of 0.22 and 0.34,respectively,atλmax=518 nm.The optimized ultrasonic extraction produced 8.462%w/w of R1,0.451%w/w of R2 and 0.172%w/w of R3 at 13.5 m L/g liquid to solid ratio,78℃of temperature and 60 min of time.Conclusions:The experimental findings of RSM optimized extraction and HPTLC analysis can be further applied for the efficient extraction ofβ-sitosterol and lupeol in other species of Astragalus.

Theoretical and experimental study on lipophilicity and wound healing activity of ginger compounds

Objective:To correlate the chromatographic and computational method to calculate lipophilicity of selected ginger compounds and to observe the effects of log P on wound healing.Methods:Mixtures of acetonitrile and water with acetonitrile content between 95%and 50%v/v in 5%increments were kept separately in 10 different chromatographic chambers,saturated with solvent for 2 h.Spots were observed under UV light atλ=254 nm p-anisaldehyde used as a spraying reagent Theoretical calculation was done using the Alogps 2.1 online program at www.vcclab.org/lab/alogps.For percentage wound contraction,five groups of animal(mice)(25-30 g)of either sex were selected.Wound were created on dorsal surface of animals using toothed forceps,scalpel and pointed scissors.The wound areas were calculated using vernier caliper.After making wound mice were orally administered 35 mg/kg 6-shogoal,6-gingerol,8-gingerol and 10-gingerol respectively.Croup E as the control group received tap water.Results:The Iipophilicity values determined in thin layer chromatography were correlated with the theoretically calculated various log P by linear regression analysis.Significant correlations were found between log P values calculated by software program and the experimental reversedphase thin-layer chromatography data.Order of wound healing property of ginger compounds is directly dependent on lipophilieity i.e.more lipophilic compound has highest activity.Conclusions:Experimentally determined lipophilieity(R_(MO))values were correlated with log P determined by software's and found satisfactory.Lipophilieity(R_(MO)is a useful parameter for the determination and prediction of biological activity of ginger compounds.

Corchorus olitorius aqueous extract attenuates quorum sensing-regulated virulence factor production and biofilm formation

Objective:To investigate the effect of Corchorus olitorius aqueous fraction(COAF)on quorum sensing(QS)-regulated virulence factors and biofilm formation in Pseudomonas aeruginosa(PAO1).Methods:The preliminary screening of the anti-QS effect of COAF was performed by evaluating the anti-pathogenic activity against Chromobacterium violaceum CV026 biosensor strain.Next,the inhibitory effects of COAF on QS-regulated pyocyanin production,proteolytic and elastolytic activities,swarming motility,and biofilm formation were evaluated in PAO1.Results:The results showed that the treatment of COAF significantly decreased the biofilm biomass,attenuated virulence factors,and inhibited swarming motility of PAO1 without affecting the growth of the bacteria in a dose-dependent manner.COAF at 2000μg/mL significantly decreased Las B elastase activity in PAO1 culture,exopolysaccharide production,swarming motility,pyocyanin level,and biomass of PAO1 by 55%(P<0.05),60%(P<0.01),61%(P<0.01),65%(P<0.01)and 73%(P<0.01),respectively.In addition,the production of violacein was decreased by 62%(P<0.01)with the treatment of a high dose of COAF.Conclusions:These findings indicate that COAF can be a potential source of anti-QS agents.

Anti-inflammatory activity and qualitative analysis of different extracts of Maytenus obscura(A.Rich.) Cuf.by high performance thin layer chromatography method

Objective:To perform aqueous elhanol soluble fraction(AESK) and dichloromethane extract of aerial parts of Maytenus obscura(A.Rich.) Cuf.using high performance thin layer chromatography(HPTLC) and to test anti-inflammatory activity of these extracts.Methods:HPTLC studies were carried out using CAM At;HPTLC system pquipped with Linomat IV applicator,TLC scanner 3.Reprostar 3.CAMAG ADC 2 and WIN CAT5-4 software were used.The anti-inflammatory activity was tested by injecting different groups of rats(6 each) with formalin in hind paw and measuring the edema volume before and 1h later formalin injection.Control group received saline i.p.The extracts treatment was injected i.p.in doses of 100 and 200 mg/kg 1h before formalin administration.Indomethaein(30 mg/kg) was used as standard.Results:The results of preliminary phytochemical studies confirmed the presence of protein,lipid,carbohydrate,phenol,flavonoid.saponin,triterpenoid,alkaloid and anthraquinonc in both extracts.Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents toluene:ethyacetate:glacial acetic acid(5:3:0.2,v/v/v) as mobile phase.HPTLC.finger printing of AESF revealed major eight peaks with R_f values in the range of 0.28 to 0.80 and the dichloromethane revealed major 11 peaks with R_f values in the range of 0.12 to 0.76.The purity of sample was confirmed by comparing the absorption spectra at start,middle and end position of the hand.Treatment of rats(i.p.) with AESF and dirhloromethane in doses of 100 and 200 mg/kg inhibited singnificantly(P<0.05.n=6) formalin-induced inflammation by 50%.55.9%,45.5%,and51.4%.respectively.Conclusions:HPTLC finger printing of AESF and dichloromethane of Maytenus obscura revealed eight major spots for alcoholic extracts and nine major spots for dichloromethane extracts.These HPTLC profiles may be of great usefulness in the quality control of herbal products containing these extracts.The anti-inflammatory activity of both extracts also revealed the medicinal importance of these extracts.The plant can be further explored for the isolation of phyloconstiluents having anti-inflammatory activity.

Chromatographic finger print analysis of anti-inflammatory active extract fractions of aerial parts of Tribulus terrestris by HPTLC technique

Objective:To develop HPTLC fingerprint profile of anti-inflammatory active extract fractions of Tribulus terrestris(family Zygophyllaceae).Methods:The anti-inflammatory activity was tested for the methanol and its fractions(chloroform,ethyl acetate,n-butanol and aqueous)and chloroform extract of Tribulus terrestris(aerial parts)by injecting different groups of rats(6 each)with carrageenan in hind paw and measuring the edema volume before and 1,2 and 3 h after carrageenan injection.Control group received saline i.p.The extracts treatment was injected i.p.in doses of 200 mg/kg 1 h before carrageenan administration.Indomethacin(30 mg/kg)was used as standard.HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator.TLC scanner 3.Reprostar 3,CAMAG ADC 2 and WIN CATS-4 software for the active fractions of chloroform fraction of methanol extract.Results:The methanol extract showed good antiedematous effect with percentage of inhibition more than 72%,indicating its ability to inhibit the inflammatory mediators.The methanol extract was re-dissolved in 100 mL of distilled water and fractionated with chloroform,ethyl acetate and n-butanol.The four fractions(chloroform,ethyl acetate,n-butanol and aqueous)were subjected to anti-inflammatory activity.Chloroform fraction showed good anti-inflammatory activity at dose of 200 mg/kg.Chloroform fraction was then subjected to normal phase silica gel column chromatography and eluted with petroleum ether-chloroform,chloroform-ethyl acetate mixtures of increasing polarity which produced 15 fractions(F1-F15).Only fractions F1,F2,F4,F5,F7,F9,F11 and F14 were found to be active,hence these were analyzed with HPTLC to develop their finger print profile.These fractions showed different spots with different R_f values.Conclusions:The different chloroform fractions F1,F2,F4,F5,F7,F9,F11 and F14 revealed 4,7,7,8,9,7,7 and 6 major spots,respectively.The results obtained in this experiment strongly support and validate the traditional uses of this Sudanese medicinal plant.

Determination of bioactive marker glycyrrhizin in Glycyrrhiza glabra root and commercial formulation by validated HPTLC-densitometric method

Objective:To develop a simple sensitive high performance thin layer chromatography(HPTLC)-densitometric method for quantification of glycyrrhizin in the Glycyrrhiza glabra root methanol extract and licorice root capsule methanol extract.Methods:Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents ethyl acetate:glacial acetic acid:methanol:water in proportion of 6:2:1:0.5,v/v/v/v as mobile phase.Scanning and quantification of developed plate was done densitometrically at 254 nm.The method was validated for detection and quantification limits,precision,recovery and robustness according to International Conference on Harmonization guidelines.Results:The system gave compact spot for glycyrrhizin(R_(f)=0.280±0.001).The regression curve of standard was found to be Y=4.213x+22.078.The limit of detection(15.7 ng per band),limit of quantification(47.1 ng per band),recovery(99.4%-99.8%)and precision(≤1.62%and≤1.84%;intraday and interday)were satisfactory for glycyrrhizin.Linearity range for glycyrrhizin was 20-200 ng(r^(2)=0.996).The content of glycyrrhizin was estimated as 5.9%and 11.2%w/w in glycyrrhizin in the Glycyrrhiza glabra root methanol extract and licorice root capsule methanol extract,respectively.Conclusions:This estimation technique is very much useful for the estimation of glycyrrhizin present in various formulations as well as for quality control of crude drugs containing glycyrrhizin.