HPTLC Analysis and Antiproliferative Effect of Various Extracts of Swertia alata on Growth of Leishmania donovani Promastigotes in vitro

The present study was carried out to evaluate the quality control analysis and antiproliferative effects of petroleum ether, chloroform, ethanol and aqueous extracts of Swertia alata (family Gentianaceae) on Leishmania donovani. A basic, exact, quick and reproducible high performance thin layer chromatography (HPTLC) has been created for synchronous analysis of Oleanolic acid and Swertiamarin from S. alata. The method was validated for linearity, precision, specificity, accuracy and robustness. Densitometric scanning of the plates directly at 254nm was used for analysis of Swertiamarin, while as for the analysis of Oleanolic acid; plates were scanned for 520 nm after spraying with anis aldehyde-sulphuric acid reagent. For the extract of plants and Amphotericin B, the concentration response curve was plotted and IC50 values were determined and also MTT assay was carried out for comparative study. Compact bands of Oleanolic acid and Swertiamarin were obtained at Rf 0.62±0.001 and 0.66±0.002, linearity was satisfactory for oleanolic acid and swertiamarin. The developed and validated HPTLC methods were having Linearity range 200-2000 and 100-2000 ng/spot for oleanolic acid and swertiamarin respectively with r2 value 0.989±0.003 and 0.999±0.03, limit of detection (11.25 and 6.19 ng/spot), limit of quantification (37.51 and 20.64 ng/spot), recovery (98.4-101.0 % and 98.2-100.8%) and precision with % RSD < 2%. Various dried extracts were found to contain oleanolic acid in the range of 0.009%-0.012% and swertiamarin in the range of 0.113-0.135% (w/w). Cytotoxicity assay of extracts over promastigotes showed that ethanolic extract was found to be most cytotoxic (IC5031.43±1.90 μg/mL) after 96 h in comparison to other extracts. The ethanolic extract showed significant reduction in promastigotes which may be due to presence of oleanolic acid as compared with the other groups. The present HPTLC method is being reported for the first time and can be used for routine quality control.


INTRODUCTION
The genus Swertia belongs to family gentianaceae which is known to have about 700 species and 80 genera. 1 Plants belonging to this family are found in all parts of world.They have been widely used in folk medicine.Xanthones derivatives, flavonoids, irridoid glycosides, triterpenoids and dimeric xanthones have been isolated from the genus Swertia. 2,3The genus are well known bitter tonic used against loss of appetite and fewer their bitter taste can be attributed to their content of xanthones and irridoids. 4The most potential specie Swertia genus (S. chirata) is now nearly extinct from India.S. alata is being used as its replacement, and it has been reported to have Oleanolic acid and Swertiamarin. 5leanolic acid (3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid derived from the isopentenyl pyrophosphate derived from the mevalonate pathway. 6It is widely spreaded throughout the plant kingdom.This compound and its derivatives possess several interesting pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, and hepatoprotective effects. 7Swertiamarin is a dominant secoiridoid glycoside derived from loganic acid through mevalonic acid pathway.It is distributed predominantly among the members of Gentianaceae, mainly S. chirata and E. littorale.Some specific activities have been reported for swertiamarin such as anticholinergic, 8 antihyperlipidemic, 9,10 insulinotropic, 11,12 antinociceptive, 13 antioxidant and hepatoprotective. 14Oleanolic acid and swertiamarin are very important bioactive constituents of S. alata; therefore, there is an urgent need to develop a simple method for estimation of two chemotherapeutic compound using sophisticated instruments like high performance liquid chromatography (HPTLC).Till date, several analytical methods have been reported for analysis of these both the compounds.However, literature survey shows no single quantitative method available for estimation of both the compounds in the plant S. alata.
well closed containers under refrigeration until used for the biological testing.

Preparation of standard and quality control (QC) samples
Stock solutions of Oleanolic acid and Swertiamarin (10mg/mL) were prepared in HPLC grade methanol and by appropriate dilution standard solutions were prepared in the concentration range of 0.1 to 1.0 mg/mL.Peak area and amounts applied were treated by linear least-squares regression.Each amount was applied six times.Quality control samples at three different quantitative levels of 400,600 and 800 ng/band were prepared independently of the calibration standards to carry out validation of the method.Each extracts (100mg) was dissolved in 10mL of HPLC grade methanol in a 10-mL volumetric flask to get 10mg/mL solution.These solutions were sonicated and filtered through 0.22µm syringe filter and then used as test solution for HPTLC analysis.

HPTLC instrumentation and experimental conditions
Analysis was done on a Camag (Muttenz, Switzerland) HPTLC system, comprising a Linomat V automatic applicator and TLC scanner III.Chromatography was performed using precoated silica gel TLC (E.Merck, Germany) plated 60F 254 (20cmx20cm with 0.2mm thickness).The plates were activated at 60 o C for 5 min prior to chromatography.TLC sampler, fitted with a 100µL syringe and connected to nitrogen gas, was used for sample application and controlled by win CATS software version 1.2.0,CAMAG.The samples were applied in triplicate (8.0µL each) and were spotted in the form of bands having band width of 4.0mm.The plates were developed vertically ascending in a twin trough glass chamber (Camag, Switzerland) saturated with respective mobile phase.The optimized chamber saturation time for the mobile phase was 30 min at room temperature (25±2 º C).The chromatogram was developed up to 85% of total TLC plate height.After the development, HPTLC plates were dried in an oven for 5 min at 60 o C for complete removal of mobile phase.The wavelengths for fingerprinting were selected, by multiwavelength scanning, showing the highest number of peaks.The quantification of oleanolic acid and swertiamarin in different extracts were carried out for quality control of crude drugs.The Densitometric quantification was done at UV mode at 246 and 540nm with the help of tungsten lamp and deuterium lamps as a radiation source in reflectance mode.The plate was kept in photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at 254 and 520 nm.The peak numbers with its height and area, peak display, and peak densitogram were recorded.The quantification of oleanolic acid and Swertiamarin was carried out in two different sets of chromatography for quality control of extracts of S. alata.In the first chromatography set, the stock solutions of oleanolic acid was applied in triplicate in different volumes (1-10µL) on HPTLC plate and eluted using toluene: ethyl acetate: formic acid, 7.3:1:0.5,v/v/v as solvent system.In the second set of chromatography, swertiamarin was applied in triplicate in different volumes (1-10 µL) on HPTLC plate and eluted using n-butanol: acetic acid: water, 7.5:1:1.5,v/v/v as solvent system.The quantification was done by scanning the developed chromatograms at 254nm for swertiamarin and at 540nm for oleanolic acid after derivatisation with anis aldehyde sulphuric acid reagent.

Validation of the method developed
The newly developed HPTLC method was optimized and validated as per the ICH guidelines Q2 (R 1 ) for different parameters like calibration, linearity, precision, accuracy, robustness, specificity, LOD and LOQ. 21,22libration curve and linearity Different volumes (0.1 1 µL/spot) of the standard solutions (oleanolic acid and swertiamarin) were separately spotted on TLC plates (in triplicate) to obtain different concentrations of oleanolic acid (200-2000 ng/spot) Leishmaniasis is a wide spread life-threatening disease which is caused by obligate intracellular, kinetoplastid protozoa of the genus Leishmania. 15t is considered one of the serious tropical diseases in many countries.The drugs available for the treatment of leishmaniasis are, in general, toxic, expensive and require long term treatment.There is an inclusive lack of effective and inexpensive chemotherapeutic agents for treatment of Leishmaniasis.Pentamidine is very toxic but still is being used as first choice for the treatment of leishmaniasis because other drugs have shown varying degrees of resistance and efficacy against the Protozoa parasitic. 16These drugs are unsatisfactory because of their limited efficacy, frequent side effects and increasing drug resistance therefore, new, safer and more efficacious drugs are urgently required. 17Moreover, there is no effective vaccine against leishmaniasis. 18In this regard, the selection of bioactive molecules is initiated by a specific screening of plant extracts, isolation by activity directed fractionation and identification of the active compounds.Swertia alata Royle (Gentianaceae) is one of the common adulterants of S. chirata, which is widely used in indigenous medicine as a bitter tonic, febrifuge, laxative, antimalarial, anti-inflammatory, antioxidant and anthelmintic. 19,20Several species of Swertia are being used as substitutes or adulterants of S. chirata and are known for their therapeutic potential against fever, hepatitis, cholecystitis, pneumonia, osteomyelitis, dysentery, scabies, spasm, pain and malaria. 21he present investigation was designed to carry out with an objective of developing a validated sensitive and fast HPTLC method for simultaneous determination and quantification of oleanolic acid and swertiamarin and the extracts were further evaluated to investigate the antiproliferative effect of polar [ethanol (ETSA) and aqueous (AQSA)] and nonpolar compound enriched fractions [petroleum ether (PESA) (fat-rich fraction), chloroform (CHSA) terpenoid and flavonoid-rich fraction] against L. donovani.Analysis of these compounds was carried out using newly developed HPTLC method.The proposed method was developed as per the International Conference on Harmonization ICH guidelines. 22,23

Plant material and chemicals
The dried plant material was supplied by Almas Pharmaceutical Ltd, Uttar Pradesh and identified by Dr. H.B Singh, Scientist F and Head, Raw Material Herbarium and Museum, NISCAIR (National Institute of Science Communication and Information Resources) Pusa Gate, New Delhi.The voucher specimen (NISCAIR/RHMD/2013/2185/191) of the test drug has been deposited in the herbarium of NISCAIR for future reference.Standard Swertiamarin (Purity: 95.4% w/w) and Oleanolic acid (Purity: 98.8% w/w) were purchased from Natural remedies Pvt.Ltd, Bangalore, India.3-(4, 5 -dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) was procured from Sigma-Aldrich, USA.Tobie medium, amphotericin B and dimethyl sulfoxide (DMSO) were of molecular biology grade.Precoated silica gel 60 F 254 HPTLC plates were purchased from E. Merck, Germany.All the solvents used were of chromatography grade and other chemicals used were of analytical reagent (AR) grade.

Extraction
The aerial parts of the plant were air dried under shade and then ground to a coarse powder using a mixer.Ground plants material (500 gms) was extracted successively by petroleum ether, chloroform, ethanol and distilled water in soxhlet apparatus.After filtration, the solvent was evaporated to dryness under reduced pressure at a temperature lower than 40°C in rota evaporator (Heidolph, Germany).Each time before extraction with next solvents, the coarse powdered material was dried in hot air oven below 50ºC.All the dried extracts were kept in and swertiamarin (100-2000 ng/spot) for calibration plots.The data of peak area versus drug concentration were treated by linear least square regression and the concentration range showing best regressions was considered for linearity. 22,23ecision Precision of the proposed method was obtained by repeatability and intermediate precision.Inter-day and intra day precisions were done by preparing and applying three different concentrations of standards (in triplicate) on the same day and on three different consecutive days, respectively. 23The inter instrumental and inter-analyst precision was carried out by repeating the same procedure using different systems of the same make and by different analysts, respectively.Precision studies were done at three different concentration levels.The results were determined and reported in terms of % RSD. 24bustness of the method Robustness of the analytical procedure is a measure of its capacity to remain ineffective by small, but deliberate, variations in the method parameters and provide an indication of its reliability during normal usage.Robustness of the method was achieved by introducing small changes in the compositions of mobile phase and detection wavelength.The effect on the results was reported as % RSD.23

Specificity
The specificity of the method was ascertained by analysing standard drug and sample.The detection of spots for oleanolic acid and swertiamarin were confirmed by comparing R f and spectra of spots with those of the standards.The peak purity was assessed by comparing the spectra at three different levels, i.e. peak start, peak apex and peak end positions of the spot. 23

Limit of detection (LOD) and limit of quantification (LOQ)
The LOD was expressed as LOD = 3.3 σ /slope, whereas LOQ was expressed as LOQ = 10σ/slope of calibration curve, where σ is the standard deviation of the response at low concentrations.Slope = the slope of the calibration curve. 22

Accuracy as recovery
In analytical methods, the closeness of test results obtained by that method to the theoretical value is called the accuracy.The standard addition method was used by spiking at four different concentration levels, that is, 0, 50, 100 and 150%, of analyte in pre-analysed samples. 22

Estimation of oleanolic acid and swertiamarin in extracts
The newly developed and validated HPTLC method was applied for the analysis of oleanolic acid and swertiamarin in extracts of S. alata.Oleanolic acid and Swertiamarin were estimated in PESA, CHSA, ETSA and AQSA extract.The samples were applied in triplicate on HPTLC plates with standard and the contents of metabolites were analysed, using regression equations obtained from calibration plots, and expressed as %w/w.All the extracts showed number of UV active compounds and thus detected at 254nm and 540nm after visualization with or without derivatisation.

Antiproliferative Assay
Leishmania culture L. donovani used in this work was cultured promastigote forms which were obtained from the laboratory of National Communicable Disease Control programme (NCDC), New Delhi.Promastigotes were grown in modified Tobie's medium, 25 which consists of two phases blood agar (solid phase) and Locke's solution (liquid phase), 5 mL of solid phase was dispensed in to 25mL flask, and just before use, 2mL of Locke's solution was added to the flask.Then the flask was incubated at 24 o C for 72h.
After three days, the mortality was recorded by counting the live promastigotes in Neubauer counting chamber. 26

Antiproliferative assay of extracts
The antiproliferative assay of extracts was carried out to find out the best active extract.The stock solutions were prepared by dissolving 500mg of each extract in 10 mL DMSO.These solutions were passed through 0.45 μM membrane filter and stored at 4 0 C until used.These were diluted Tobie's medium to get a concentration of 1000µg/mL of every extract. 27urther, these solutions were passed through 0.22µM filter under sterile conditions in laminar flow hood before using for in vitro activity on promastigotes.Similarly, DMSO control was also prepared 27 while amphotericin B was used as the reference standard drug. 28omastigote forms from a logarithmic phase culture were suspended to yield 1 million of cells /mL after Neubauer chamber counting.Each well of 96 well plate was filled with 100 μL of the parasite suspension, and the plates were incubated at 24 0 C for 1h before drug addiction. 29

Promastigotes extracts susceptibility assay
The antileishmanial activity was performed according to a previously described method. 30Briefly Promastigotes of L. donovani were cultured in Tobie's medium.The viability of promastigotes was assessed by tetrazolium-dye (MTT) colorimetric method.MTT assay was performed on L. donovani promastigotes.1×10 6parasite/mL promastigotes were seeded in 96 well plastic tissue cultures flat plates supplemented with 100µL of the respective culture medium for a period of 24h maintained at 24 o C.It was then substituted by 100µL of fresh media containing varying concentrations of the extract (1000, 500, 250, 125, 62.5 and 31.2µg/mL).The plates were again incubated for 24, 48, 72 and 96h, separately at 37 o C, by changing fresh media containing extracts every 24h. 31After incubation period media were removed and fresh media added; 20µL of MTT reagent prepared in PBS (Phosphate buffer saline) was then added to all the wells and the plate was re-incubated for 4h.After seeing purple colour precipitation which was very well visible under microscope, media were carefully discarded for solubilisation of formazan crystals (MTT formazan).Further, 100µL of DMSO was added to each well and cells were incubated in dark at room temperature for 1h.The purple colour developed and the absorbance was read at 490 nm by a microplate reader (Bio-Rad, USA).Percentage of inhibition was calculated by comparing the percent viability with untreated control 32 and percentage promastigote viability was calculated using the formula of Mossman, 1983 33 at each concentration.

Statistical analysis
Data represented as the mean ± SD of triplicate samples from three independent assays.The IC 50 values were calculated using dose-response curves in graph pad prism 5.0 software (California, USA).Data with p<0.05 were accepted as not significantly different.

RESULTS AND DISCUSSION
The plant material was extracted using successive hot extraction using different solvents (Petroleum ether, chloroform, ethanol and distilled water) separately.The hot extraction was selected for the present study due to its high yields and the yield obtained were PESA (1.968 %), CHSA (1.613%), ETSA (7.963%) and AQSA (9.012%).

HPTLC Analysis
The various extracts were subjected to HPTLC analysis by specific solvent system.The composition of the mobile phase for development of chromatographic method was optimized by testing different solvent mixtures of varying polarity.The mobile phase, toluene: Ethyl acetate: formic acid (7.3:1:0.5)was optimized for separation and quantification of Oleanolic acid in PESA, CHSA, ETSA, AQSA.All the extracts showed good separation with compact spot Figure1.Oleanolic acid was well separated, using the same solvent as indicated above but visualized after derivatization with anis aldehyde sulphuric acid.It was scanned at 520nm wavelength, which showed compact peak at R f 0.66± 0.02 Figure 1 and 2(a) -2(e).The mobile phase n-butanol: acetic acid: Water, 7.5: 1: 1.5v/v/v was optimized for separation and quantification of Swertiamarin in PESA, CHSA, ETSA and AQSA.All the extracts showed good separation with compact spot Figure 3. Swertiamarin was well separated, using the same solvent as indicated without derivatization.It was scanned at 254 nm wavelength, which showed compact peak at R f 0.62± 0.01 Figure 3 and 4 (a) -4(e).

Validation of the method developed Calibration curve and linearity
The proposed method for simultaneous estimation of oleanolic acid and swertiamarin were found linear for a wide range of concentration with good regression coefficient (>0.99).The linearity was evaluated by data of peak area versus drug concentration and was treated by using linear least-squares regression and the concentration range showing best regressions was considered for its linearity.The regression equation with slope, intercept, and coefficient of correlation (R 2 ) was calculated and are given in Table 1.
Pharmacognosy Journal, Vol 9, Issue 6 (Suppl), Nov-Dec, 2017 Precision Table 2 represents inter-day and intra-day precision.Precision was checked by preparing and applying inter-day and intra day at three different concentration of standards (in triplicate) i.e. 200, 400 and 600 ng/spot on the same day and on three different days, respectively.The inter-analyst precision was done by repeating the same procedure using different systems of the same make and by different analyst.Low RSD (%) values obtained indicated that the method is precise.

Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ of different markers were calculated as per the standard protocol and reported in Table 1.This indicated that the proposed (d) (e) method exhibits a good sensitivity for the quantification of above compounds.

Accuracy (recovery)
The accuracy of the method was assessed by spiking 0, 50, 100 and 150% of analyte in prenasalized samples with known amounts of standard and then re-analysed by the HPTLC method.The %RSD values after spiking with 50, 100 and 150 % of drug was lying in the range of 98.7-101.0%.The experiment was conducted in triplicate Table 3.

Robustness
The low values of %RSD (< 2.0) obtained after introducing small deliberate changes in experimental conditions like change in wavelength at concentrations of 400, 600 and 800ng/spot in triplicate indicated the robustness of the method.The results were expressed as % RSD Table 4.

Estimation of oleanolic acid, swertiamarin in various extracts of S. alata
The newly developed and validated HPTLC method was applied for the simultaneous estimation of oleanolic acid and swertiamarin in various extracts of S.alata.The amount of oleanolic acid in various extract of drug i.e.PESA, ETSA, CHSA, and AQSA was found to be 0.02 % w/w, 0.05%w/w, 0.34%w/w and 0.32%w/w and swertiamarin found to be 0.14%w/w, 2.44%w/w, 1.24% w/w and 3.14% w/w respectively Table 5.

Antiproliferative assay of S. alata
Determining the absence of toxic effects on host cells is an important criterion that must be evaluated when searching for active compounds   with therapeutic potential against L. donovani.Up to 1% (v/v), DMSO had no effect on parasite growth rate and mobility morphology. 29The extracts of plants and drug inhibited the growth of promastigote forms of L. donovani in vitro after 24, 48, 72 and 96h of incubation and had a 50% inhibitory concentration (IC 50 ) that were shown in Figure 4.The IC 50 values were computed by entering the optical density readings in a preset MS excel template.The IC 50 values for the PESA, CHSA and AQSA extracts for L. donovani were significantly different (p<0.001) while in case of ETSA extract it was not significant statistically as compared to the standard Amphotericin B (p>0.05).From the determined IC 50 values and percentage of inhibition of promastigote number with the increase of concentration through different growth periods 24, 48, 72 and 96h, the concentration dependent effect of extract was shown in Figure 5.
The results of antileshmaniac activity exhibited that extracts of S. alata are having antileshmaniac activity.In the preliminary study, the extracts exhibiting >40% inhibition at the tested concentrations, were considered to have high antileishmanial potential, and are tested further on the L. donovani macrophage infection assay. 34The ETSA exhibited the highest inhibition percentage of L. donovani promastigote (94.17 ± 1.54 %) at 1000 µg/mL and (82.12 ± 0.45 %) at half of this concentration at 96h.Additionally, CHSA, PESA and AQSA extracts showed inhibition of approximately 50% at 500 µg/mL, and there was 5-10% reduction of the inhibition percentage at half of this concentration at 96h Figure 6.All the selected plants extracts were shown active or significant active at the lowest tested concentration (50 µg/mL).The L. donovani promastigotes treated with ETSA extract had the highest viability at all concentrations followed by CHSA.The gradual decrease in the number of promastigotes indicates that there is a clear toxic effect of the ethanolic extract as compared with all other extracts.There is a proportional relation between the concentration of the extract and its inhibitory effect.The genus Swertia has a wide range of uses in traditional medicine.To the best of our knowledge, no previous report was available regarding the evaluation of S. alata as antileishmanial activity against L. donovani.
In the present study, it was found that the antileishmanic activity of the ETSA extract of S. alata was very effective at 96h and showed similar results like the control i.e. amphotericin B. The opinion to the strong effect of S. alata on L. donovani promastigote may be due to presence of secoiridoid glycoside and oleanolic acid in this plant, which was reported previously with the same genus and different species. 35,36

CONCLUSION
Our results suggest that ethanolic fraction of S. alata might be a good candidate for development as antileishmanic drug and may come out as a new future phytopharmaceutical drug.In addition, a validated method was developed for quantification of oleanolic acid and swertiamarin in various fractions of extracts that can be used for its quality control as well as for that of other drugs containing them as ingredient.The analysis of two marker compounds for the quality control of various fraction using HPTLC methods in present investigation is unique and being reported for the first time.
Based on this study, S. alata could be considered as a potential candidate of leishmanicidal drug against L. donovani.Thus, more investigations should be done, first In vitro assay for finding anti-leishmanial effect by using Leishmania amastigote and then to investigate In vivo activity in laboratorial infected animals.This would help in obtaining a novel drug that could potentially be less toxic and more cost effective against the leishmania parasite.This is a first report of work carried out on the antileishmanial effects of this plant so far.Further urgent attention and research is needed for making this drug available soon before the epidemic of leishmaniasis rises further, however no work has been reported till date for antileishmanial activity of S. alata.