HPTLC Analysis of Bioactivity Guided Anticancer Enriched Fraction of Hydroalcoholic Extract of Picrorhiza kurroa

Objective. Hydroalcoholic extract of Picrorhiza kurroa and its fractions were subjected to in vitro screening for cytotoxicity; further best active fraction (BAF) obtained was tested against Ehrlich ascites carcinoma (EAC) model in Balb/c mice after its quality control analysis. Methods. Cytotoxicities of all the fractions and mother extract of P. kurroa were determined, using MTT assay on breast cancer (MCF-7, MDA-MB 231) and cervical cancer (HeLa, SiHa) cell lines. Metabolic fingerprinting was developed using HPTLC with quantification of biomarkers (cucurbitacins B and E; betulinic acid; picrosides 1 and 2; and apocynin) in BAF. The EAC tumor-bearing mice were used for in vivo anticancer activity after oral administration (50 mg Kg−1) for 10 days. Results. Cytotoxicity assay of mother extract and its fractions over breast cancer and cervix cancer cell lines showed that dichloromethane (DCM) fraction was most cytotoxic (IC50 36.0–51.0 µg mL−1 at 72 h). Oral administration of DCM fraction showed significant reduction in tumor regression parameters, viable tumor cell count and restoration of hematological parameters may be due to presence of cucurbitacins B and E; betulinic acid; picrosides 1 and 2; and apocynin, as compared to the untreated mice of the control group. Conclusion. The DCM fraction of P. kurroa displayed potent anticancer activity and can be further explored for the development of a potential candidate for cancer therapy.


Introduction
Cancer is a major public health problem in many parts of the world. It is currently the second leading cause of death and is expected to surpass heart diseases as the leading cause of death in the next few years [1]. Normal tissue homeostasis is maintained by the two counterparts, namely, cell proliferation and apoptosis [2]. Failure of apoptosis mechanism may result in limitless growth and division of cells. The conventional therapies for cancer include chemo-and radiotherapies mediated by inducing apoptosis or inhibiting proliferation in neoplastic cells [3]. These therapies cause damage to healthy tissues around the tumors [4,5] and also develop resistance by numerous tumors [6]. Researchers have been studying alternatives of cancer therapy by applying potential biological molecules to target neoplastic tumors [7].
Plant-based immunomodulators are nowadays receiving adequate attention and have been evaluated for their active potential to modulate immune responses [8,9]. Many of the natural products are in clinical use [10]. Identification of the active components and molecular basis for the action of a traditional medicine is likely to make natural products more acceptable for humans, an approach sometimes referred to as Reverse Pharmacology [11].
The most cytotoxic fraction, that is, best active fraction (BAF), was further evaluated for in vivo anticancer potential after its quality control analysis, using HPTLC. The contents of picrosides 1 and 2, betulinic acid, cucurbitacins, and apocynin were quantified in BAF since it was terpenoid enriched fraction (DCM). The contents of one flavonoid and 6 terpenoid markers were quantified using newly developed and validated simultaneous HPTLC methods for the first time in any medicinal plant. The anticancer potential of these compounds have already been reported separately [18,20,[22][23][24][25][26][27][28][29][30][31][32][33]; however no work has been reported till date on terpenoid enriched fraction in totality for P. kurroa.

Hydroalcoholic Extract (Mother Extract) and
Its Fractionation. The 500 gm of powdered P. kurroa rhizome was extracted with 70% alcohol in Reflux extractor for five hours on water bath and filtered. The filtrate was evaporated to dryness under reduced pressure. The hydroalcoholic (mother) extract thus obtained was suspended in double distilled water (1 gm/10 mL) and sonicated for 15 min at 45 ∘ C. Prepared aqueous suspension was partitioned with equal proportions of hexane, DCM, and n-butanol (thrice each). The aqueous suspension left after partitioning was evaporated to dryness and the residue was sonicated further with acetone and methanol separately for 20 min, thrice each. The remaining residue and solvent fractions obtained were evaporated to dryness under reduced pressure. The extractive values and % yields of different fractions were calculated and stored at 4 ∘ C for bioactivity and quantitative analysis.

Cell Line and Cell
Culture. All cell lines (MCF-7, SiHa, Hela, and MDA-MB 231) used in the study were obtained from National Centre for Cell Science (NCCS) at Pune, India. The cell lines were grown as monolayer cultures in RPMI-1640 media with 10% foetal calf serum (FCS) and 1% PSA (penicillin, streptomycin, and amphotericin) in a humidified atmosphere of 5% CO 2 at 37 ∘ C.

Cytotoxicity Assay of Picrorhiza Extract and Its Fractions.
The cytotoxicity assays of mother extract and its hexane, DCM, n-butanol, acetone, methanol, and water fractions were carried out to find out the best active fraction (BAF). The stock solution was prepared by dissolving 500 mg of each extract/fraction in dimethyl sulfoxide (DMSO) and volume was made up to 10 mL in volumetric flask, separately. These solutions were passed through 0.45 m membrane filter and stored at 4 ∘ C until used. These were diluted fifty times using RPMI-1640 media (1 mL to 50 mL) to get a concentration of 1000 g mL −1 of every extract/fractions. Further, these solutions were passed through 0.22 m membrane filter in aseptic condition before using for in vitro activity on different cell lines. Similarly, DMSO control was also prepared and used for every cell line.
In brief, MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed on MCF-7, SiHa, HeLa, and MDA-MB 231 cell lines [34]. 1 × 10 4 cells were seeded on 96-well plates supplemented with 100 L of the respective culture media for a period of 24 h at 37 ∘ C. It was then substituted by 100 L of fresh media containing varying concentrations of the extract/fractions (3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 g mL −1 ). The plates were again incubated for 24, 48, and 72 h, separately at 37 ∘ C, by changing fresh media containing extracts/fractions every 24 h. After incubation period media were removed and fresh media added; 20 L of MTT reagent prepared in respective media (5 mg mL −1 ) was then added to all the wells. This was followed by incubation for 3 h. After seeing purple color precipitation which was very well visible under microscope, media were carefully discarded for solubilization of formazan crystals (MTT formazan). Further, 100 L of DMSO was added to each well and cells were incubated in dark at room temperature for 1 h. The purple color developed was measured at 570 nm by a microplate reader (Bio-Rad, USA). The percentage of cytotoxicity of these extracts was calculated by using the following formula: where is the absorbance of the control and is the absorbance of the sample.

Sample Preparation and Chromatographic Conditions.
The dried mother extract and fractions (100 mg each) of P. kurroa were reconstituted using HPLC grade methanol in a 10 mL volumetric flask to get 10 mg mL −1 solution. These were sonicated and filtered through 0.22 m syringe filter before being used for HPTLC analysis. The samples were applied in triplicate (8.0 L each) and the width of the track was kept to 4.0 mm on precoated silica gel 60 F 254 plates (E. Merck, 0.20 mm thickness), using Linomat V (HPTLC sample applicator). Linear ascending development was carried out in 10 × 20 cm twin trough-glass chamber (Camag, Muttenz, Switzerland). The optimized chamber saturation time for solvent system was 30 min at 25 ∘ C and relative humidity of 60%. The chromatogram was developed up to 85% of total TLC plate height. Developed chromatograms were scanned at 254 nm for DCM extract without derivatization but at 520 nm for other extracts after derivatization with anisaldehyde sulphuric acid. The wavelengths for fingerprinting were selected by multiwavelength scanning showing the highest number of peaks. The quantification of cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin was carried out in three different sets of chromatography for quality control of BAF.
Stock solutions of cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin were prepared in HPLC grade methanol to get a known concentration of 500 g mL −1 . In the first set, the stock solutions of cucurbitacins B, D, and E were mixed in equal volume to get cucurbitacin standard (STC, 166.66 g mL −1 each). It was applied in triplicate in different volumes (0.1-10 L) on HPTLC plate and eluted using toluene : ethyl acetate : formic acid, 60 : 40 : 0.5, v/v/vas solvent system. The second set of chromatography was done for quantification of picrosides 1 and 2 and apocynin; similarly equal volumes of all three    stock standards were mixed to get the picroside and apocynin standards (STPA, 166.66 g mL −1 each). This was also applied in triplicate in different volumes (0.1-10 L) on HPTLC plate and eluted and toluene : ethyl acetate : methanol : formic acid, 40 : 50 : 10 : 0.2, v/v/v/v, was used as solvent system. In the third set of chromatography, betulinic acid (STB) was applied as such (500 g mL −1 ) in triplicate in different volumes (0.1-10 L) and eluted, using same solvent as used for cucurbitacin. The sample (BAF, 8.0 L each) was applied in triplicate on every plate with the same chromatographic conditions as mentioned above. The quantification was done by scanning the developed chromatograms at 240 nm for cucurbitacins (without derivatization), at 595 nm for betulinic acid, and at 500 nm for picrosides and apocynin after derivatization with anisaldehyde sulphuric acid reagent.  2.11. Robustness 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 examined as % RSD [35].

2.12.
Specificity. The specificity of the method was ascertained by analysing standard drug and sample. The detection of spots for cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin in BAF was confirmed by comparing and spectra of spots with those of the standards. The peak purity was assessed by comparing the spectra at three different levels, that is, peak start, peak apex, and peak end positions of the spot [36]. (LOQ). The LOD was expressed as LOD = 3.3 /slope, whereas LOQ was expressed as LOQ = 10 /slope of calibration curve [36]. 2.14. 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 preanalyzed samples [36].

Analysis of Cucurbitacins B, D, and E; Betulinic Acid; Picrosides 1 and 2; and Apocynin in BAF.
The newly developed method was applied for simultaneous estimation of cucurbitacins B, D, and E and betulinic acid as well as picrosides 1 and 2 and apocynin in DCM fraction of P. kurroa. The samples were applied in triplicate on HPTLC plates with standard and the contents of metabolites were analyzed, using regression equations obtained from calibration plots, and expressed as % w/w.

Analysis of Tumor Regression and Hematological Parameters after Oral Administration of DCM Fraction.
The tumor regression parameters (tumor volume, packed cell volume, tumor weight, and viable and nonviable cell count) were analyzed after administration of last dose. The mice from each group were kept fasting for 18 h and blood samples were collected in ethylenediaminetetraacetic acid coated vials following anesthesia with ketamine-xylazine by cardiac puncture for the estimation of haematological toxicity. The animals were then sacrificed by cervical dislocation for the study of antitumor activity. Hematologic analysis was carried out using an automated hematologic analyzer (MS9 Differential Cell Counter 3 Part, HD Consortium, India). The mice were dissected and the peritoneal cavity was used to collect the ascetic fluid. The tumor volume was measured in a graduated centrifuge tube (in mL). The packed cell volume frequently occurring and rare cells correlating to basophils, monocytes, eosinophils, etc.) were determined using a blood automatic analyzer.

Statistical Analysis.
Values were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Dunnett's test (Graph Pad, San Diego, CA) was used for statistical analysis. All the treatment groups were compared with the toxic control group. values < 0.05 were considered as statistically significant.

Results and Discussion
The plant material was extracted using crude alcohol (70%) by maceration and Reflux extraction after optimization. The hot extraction was selected for study due to its high yields and called mother extract (25.6% w/w). This was further fractionated using hexane (9% w/w), DCM (31% w/w), nbutanol (23% w/w), acetone (11% w/w), methanol (16% w/w), and water (7% w/w). However, 5.2 g of mother extract (4% w/w) of the drug was lost during the processing (Figure 1).

Cytotoxicity Assay.
The cytotoxicity of hydroalcoholic (mother) extract and its fractions of P. kurroa on selected cancer cell lines was determined by MTT assay at 24, 48, and 72 h, which showed the best activity at 72 h. The results of cytotoxicity assay showed that hexane and water fractions did not produce any substantial cytotoxicity and were found safe in the tested concentration (500 g mL −1 ) in all cell lines. However, mother extract, DCM, n-butanol, acetone, and methanol fractions produced good cytotoxicity varying between 36 and 270 g mL −1 at 72 h among different cell lines (Figure 2). The DCM fraction (IC 50 ranging from 36 to 51 g mL −1 at 72 h) showed best cytotoxic activities towards all cancer cell lines (Figures 3 and 4(a)-4(d)). The best cytotoxic activity of DCM fraction from P. kurroa may be attributed to the presence or synergistic activities of phytochemical components including sterol, triterpenes, and polyphenols [18,20,[22][23][24][25][26][27][28][29][30][31][32][33][34]. However, betulinic acid may be attributed to this activity [41], since DCM fraction is rich in it, as proved by our analytical studies. MTT assay results of all the four cell lines proved that cytotoxicity was highest in DCM, followed by n-butanol, methanol fraction, and mother extract at 72 h (Figures 4(a)-4(d)). As the DCM fraction of hydroalcoholic extract of P. kurroa exhibited the  highest cytotoxicity towards the tested cell lines, and this may be due to vacuole formation, membrane blebbing, nuclear condensation, and detachment of cells from the substratum and shrinkage of cells as well as development of apoptotic bodies [42,43].

HPTLC Analysis.
The HPTLC fingerprinting of mother extract and different fractions was developed on silica gel. DCM fraction showed the maximum number of UV active compounds and was thus detected at 254 nm, whereas other fractions and mother extract were detected at 520 nm after visualization, using anisaldehyde sulphuric acid reagents (Figures 5(a)-5(f); Table 1).  (Figures 6(a)-6(c)) on scanning at 240 nm without derivatization. Betulinic acid was well separated, using the same solvent as indicated above for cucurbitacins, but visualized after derivatization using anisaldehyde sulphuric acid. It was scanned at 595 nm wavelength, which showed compact spot and sharp peak at 0.76 ± 0.01 (Figures 7(a)-7(c)). The toluene : ethyl acetate : methanol : formic acid (40 : 50 : 10 : 0.2, v/v/v/v) was optimized for separation and quantification of picrosides 1 and 2 and apocynin, which gave a good separation among components. The plate was scanned at 500 nm wavelengths after derivatization with anisaldehyde sulphuric acid, which Table 4: (a) Robustness of the HPTLC method for estimation of cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin by changing detecting of wavelengths. (b) Robustness of the HPTLC method for estimation of cucurbitacins B, D, and E and betulinic acid by changing detecting of mobile phase composition. (c) Robustness of the HPTLC method for estimation of picrosides 1 and 2 and apocynin by changing detecting of mobile phase composition. produce very well defined peaks of picrosides 1 and 2 and apocynin at values 0.23 ± 0.01, 0.11 ± 0.02, and 0.77 ± 0.01, respectively (Figures 8(a)-8(c)).

Validation of the Method Developed
3.4.1. Calibration Curve and Linearity. The newly developed methods for simultaneous estimation of cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin were found linear for a wide range of concentration with good regression coefficient (>0.99). The linearity data of all the biomarkers developed such as range linearity, regression equation, regression coefficient, slope, intercept, LOD, and LOQ are given in Table 2.

Precision.
The method precision and intermediate precisions were determined and reported in terms of % RSD. Precision of the proposed method was obtained by repeatability and intermediate precision at three different concentration levels. The % RSD of interday precision, intraday precision, and interanalyst precision was within the range of 1.61-2.12 for all compounds, as reported in Table 3.

Robustness of the Method.
The low values of % RSD obtained after introducing small but deliberate changes in mobile phase composition and wavelength indicated robustness of the methods (Tables 4(a)-4(c)) at 3 different concentration levels.

Specificity.
The specificity of the methods was ascertained by analysing standard drugs and samples. The detection of spot for cucurbitacins B, D, and E; betulinic acid; picrosides 1 and 2; and apocynin in DCM sample was confirmed by equating and spectra of spot with the standard. The peak purity was estimated by comparing the spectra at three different levels, that is, at peak start, peak apex, and peak end positions of the spot. (LOQ). The LOD and LOQ of different markers were calculated as per the standard protocol [36,37] and reported in Table 3. The LOD of markers lies in the range of 21.94-133.0 ng, indicating good sensitivity of methods for simultaneous quantification of compounds.

Limit of Detection (LOD) and Limit of Quantification
3.4.6. Accuracy as Recovery. The accuracy was calculated as recovery by standard addition method by spiking 0, 50, 100, and 150% of analyte in preanalyzed samples, showing good recovery of all biomarkers used and lying in the range of 99-101.4% (Table 5).  Figure 9: Showing tumor regression parameters as obtained on Balb/c mice after ten days of oral administration of DCM fraction of hydroalcoholic extract of P. kurroa ( * * showed < 0.01 followed by Dunnett's test in comparison to EAC control).
rhizome. The peak areas of triplicate samples were analyzed by regression equation obtained from the calibration plot. The content obtained for different markers is reported in Table 6. Cucurbitacin D was found absent in DCM fraction (BAF) of P. kurroa.

3.5.
In Vivo Anticancer Activity of DCM Fraction. DCM fraction of P. kurroa showed a significant effect on tumor regression parameters of EAC cell bearing mice. The DCM fraction significantly ( < 0.01) reduced the tumor volume, tumor weight, and % packed cell volume at a dose of 50 mg Kg −1 body weight, as compared with EAC (toxic) control group (Figure 9). The results were almost comparable to that of 5-FU, a standard marketed drug. There was a significant decrease in number of tumor cells on treatment with DCM fraction and 5-FU in tumor-bearing mice, as compared with EAC control. Similarly, a percentage of viable cells were decreased significantly in treatment groups, as compared with untreated EAC control (Table 7).
Haematological parameters of EAC tumor-bearing and treatment group mice were studied on day 14, which showed significant changes in the number of WBCs only, and that was reversed in treated groups as compared with untreated EAC control. Other parameters such as haemoglobin, RBC, lymphocytes, hematocrit (HCT), RDW, and PLT were found to be near normal and did not produce any significant alteration ( Table 8).
The well-founded criteria for assessing the value of any anticancer drug are the increase in life span, the loss of leukemic cells from the blood, and reduction of solid tumor volume. Transplantable tumor cells, such as EAC, are rapidly growing cancer cells with aggressive behavior [37][38][39]. The tumor implantation includes a local inflammatory reaction by increasing vascular permeability and results in an intense ascetic fluid accumulation [37,39]. Our results showed significant reversal of tumor regression parameters accompanied by a reduction in WBC count after treatment with DCM fraction of hydroalcoholic extract of P. kurroa. The best active/enriched fraction also inhibited the accumulation of

Conclusion
This study has indicated that hydroalcoholic (mother) extract and its medium polar fractions of P. kurroa exhibited cytotoxic potential, while water and hexane fractions did not produce cytotoxicity in cervical and breast cancer cell lines up to 500 g mL −1 and 72 h. The DCM fraction was found as best active fraction in in vitro testing with lowest IC 50 value (36-51 g mL −1 at 72 h) among the four tested cell lines. This might be due to the presence of cucurbitacins B and E; betulinic acid; picrosides 1 and 2; and apocynin, as obtained from analytical studies and supported by earlier reports [15,[18][19][20][21]. The analysis of seven markers (six terpenoid and one flavonoid) for quality control of DCM fraction using simultaneous HPTLC methods in present investigation is unique and being reported for the first time. The oral administration of DCM fraction (BAF) of hydroalcoholic (mother) extract of P. kurroa (50 mg Kg −1 ) in Balb/c mice reduced the tumor volume and weight and % packed cell volume as well as WBC reflecting antitumor activity of P. kurroa. Our results suggest that DCM fraction of hydroalcoholic extract of P. kurroa might be a good candidate for development as anticancer drug and may come out as a new future phytopharmaceutical drug since inclusion of phytopharmaceuticals/enriched fractions is already in the process in several pharmacopoeias. In addition, simultaneous methods developed and validated for quantification of cucurbitacin (B, D, and E), betulinic acid, picroside 1, picroside 2, and apocynin can be used for its quality control as well as for that of other drugs containing them as ingredient. EAC: Ehrlich ascites carcinoma, 5-FU: 5-fluorouracil, DPK: dichloromethane fraction of P. kurroa ( * * showed < 0.01 followed by Dunnett's test in comparison to EAC control). Table 8: Comparative haematological profile of EAC, control, standard, and DCM fraction treated groups of Balb/C mice after ten days of treatment.