Comparative HPTLC analysis of stem and leaf of Achyranthes coynei with Achyranthes aspera

Leaf and stem materials of Achyranthes coynei and Achyranthes aspera were used for HPTLC analysis. HPTLC plates were developed on n-hexane: ethyl acetate (5:1 v/v) solvent system. The densitometric profiles were evaluated to elucidate differences within and among the species. The R f values and number of peaks obtained in densitrogram indicated chemical variation in the species. Although, both species had more or less equal number of peaks, their R f values, %height and %area varied. Thus HPTLC analysis in absence of external standards, proved to be an informative tool for evaluating differences between these species and their parts.


Introduction
Genus Achyranthes (Family: Amaranthaceae) comprises of about 15 species globally, including few medicinally and economically important species.Achyranthes coynei Sant. is an endemic, rare, endangered, threatened (RET) plant from India, recently been reported for its extended distribution from Karnataka (Pai et al., 2011).The plant is reported to possess antioxidant and antimicrobial properties (Upadhya et al., 2013;Ankad et al., 2013).On the other hand, Achyranthes aspera L. is a well known medicinal plant, reported as highly traded in India (Ved and Goraya, 2007).It is used in traditional medicinal systems in India to treat various disorders (Tandon, 2011;Upadhya et al., 2009).Additionally, identification and/or quantification of marker/ major compounds have been reported by Tondon (2011); Upadhya et al., (2014a); Pai and Joshi (2014) and Pai et al., (2014) from these species.
Various new and sophisticated analytical methods viz.HPTLC, HPLC, GC are being utilized for identifying compounds (Kokate et al., 2009;Patil et al., 2012;Upadhya et al., 2014b).Among these, HPTLC is the most popular, economical and reliable technique, used in differentiation and quality control analysis in pharmaceutical research (Hariprasad and Ramakrishnan, 2011).HPTLC technique offers better resolution of chemical constituents present in the plant extract, with reasonable accuracy in a shorter time (Sethi, 1996;Pawar et al., 2011).Generally, an identified compound serves as external standard for quality and quantity assurance of samples.In the absence of such reference compounds, the HPTLC fingerprints are compared with respect to number, sequence, position and colour of the separated bands (Mammen et al., 2011;Kamboj and Saluja, 2013).Furthermore, they are also employed in elucidating and comparing differences among species, their parts and/or herbal preparations (Kamboj and Saluja, 2013;Potawale et al., 2013).Such studies also provide an option of estimating differential patterns in samples.Thus, keeping in view of this aspect, present work was aimed towards finding out differentiation within and among leaf and stem extracts of A. coynei and A. aspera using HPTLC method.

Plant materials and chemicals
Leaf and stem materials of A. coynei and A. aspera were obtained from a single population from Madanabhavi, Belgaum district, Karnataka, India and specimens were authenticated and deposited at Herbarium, Regional Medical Research Centre, Belgaum (Voucher Number: A. coynei: RMRC 785; A. aspera: RMRC1250).All the solvents used during the study were of HPLC grade (Fischer Scientific, Mumbai, India).

Extraction
The plant materials (leaf and stem) of A. coynei and A. aspera, obtained from wild, were dried at room temperature and finely powdered.Previously employed method of Upadhya et al., (2014a) for total triterpenoid was used for extraction.A 5 g of powder was extracted with 25 mL (50% v/v) aqueous methanol by heating (2-3 min).To this, distilled water (75 mL) and concentrated H2SO4 (10 mL) was added with shaking.The mixture was refluxed for 5-6 hours over water bath at 95±2°C.The contents were cooled, filtered and transferred to separating funnel.To the above mixture chloroform (25 mL) was added and the layer was allowed to separate and the process was repeated twice.The chloroform layer obtained was washed using distilled water to get rid of acid.The acid free chloroform layer was dried to obtain residue.The residue was dissolved in methanol and the same was used for HPTLC analysis.

Instrument and chromatographic conditions
A CAMAG high performance thin layer chromatography (HPTLC) system was used for detection and separation.Analysis was performed on a pre-coated TLC silica gel G60 F254 plates (MERCK, Germany).Sample bands (6 mm) were applied using CAMAG Automated TLC Sampler (ATS-4) equipped with 25-µL syringe operated with settings: band length 6 mm, application rate 150 µL/s, distance from the bottom of the plate (Y) 10 mm, distance from edge of plate (X) 10 mm, distance between bands were auto set (6 mm).The plates were developed to a distance of 80 mm with hexane: ethyl acetate (5:1, v/v) as mobile phase in a CAMAG twin trough glass chamber previously saturated with the mobile phase at room temperature for 20 min.After the run, plates were dried in air current using drier and derivatised using anisaldehyde -sulphuric acid reagent followed by heating for 5 min at 110±2°C.Visualization of bands on plate in white light was recorded using CAMAG TLC visualizer.TLC plates were scanned at 540 nm and the data for the peaks were generated.

HPTLC analysis
During the present study a comparative analysis was carried out on two species of genus Achyranthes viz. A. coynei and A. aspera using HPTLC technique.Better band separation was obtained using hexane: ethyl acetate (5:1 v/v) as solvent system during the procedure.HPTLC plates were scanned at 540 nm in white lamp using automatic detector mode in CAMAG TLC scanner.The scan start position (Y axis) at 5.0 mm and scan end position (Y axis) at 85.0 mm with a slit dimension of 6.00 x 0.45 mm at a scan speed of 20 mm/ sec for each track was employed.The data resolution was set at 100 μm/ step.The densitogram and image for each track of A. coynei (leaf and stem) and A. aspera (leaf and stem) are presented in Fig. 1 a-h.The results (viz.peak number, Rf value, height, % height, area and % area) for the HPTLC run of both the species (leaves and stem) are presented in Table 1.It may be noted that the negative Rf values in the Table 1 indicate application position.

Comparison within the species
Variations in densitogram of leaf and stem extract of A. coynei and A. aspera were observed.Achyranthes coynei stem extracts showed 5 peaks different from that of leaf extracts while, in leaf extracts 3 peaks were different from stem extracts (Table 1).In contrary, 7 peaks observed in A. aspera leaf extracts were absent in stem extracts in comparison to 5 peaks not matching with leaf extracts.In general, stem extract was diverse in chemical nature over leaf extract.

Comparison of leaf extracts of A. coynei with A. aspera
Leaf extract of A. coynei showed 13 peaks whereas16 peaks were observed in leaf extract of A. aspera.This indicates the difference in the banding pattern and variations in the chemical constituents of both the species.Peak number seven in A. coynei and nine in A. aspera showed similar Rf value (0.42) indicating chemically identical constituents.Interestingly, height and area of the peak was higher A. aspera (Table 1, Fig. 1 a, c).Furthermore, other peaks showed different Rf values among the leaves of both species.Peak 13 in A. coynei had highest area and height at Rf 0.86 compared to the other species.

Comparison of stem extracts of A. coynei with A. aspera
A total of 15 peaks were observed in stem extracts of both the species.Peak with negative Rf values were not considered for analysis.Three peaks at Rf 0.21, 0.48 and 0.86 were coinciding in both the species of Achyranthes under study.However, these peaks in A. coynei were nearly double the area of those in A. aspera, indicating higher amount of those similar chemical constituents (Table 1,  Results of the present investigation indicate variations among parts and species.HPTLC analysis provided adequate information in differentiating these medicinal plants based on Rf values and number of peaks.Similar observations on HPTLC analysis have been worked and assessed by Hariprasad & Ramakrishnan (2011) in Rumex vesicarius, Sethi (1996) in pharmaceutical formulations and Mammen et al., (2011) in seasonal, geographical variation in Aerva lanata.Although, studies on specific triterpenoids viz., betulinic acid and oleanolic acid are reported in both the plant species using HPLC method (Pai et al., 2014;Upadhya et al., 2014a).However, these fingerprint patterns can be used to determine the chemical variations in and within the species.

Conclusion
A comparative account of leaf and stem extracts of A. coynei with that of A. aspera depicted large variation.HPTLC analysis reveals clear differentiation in leaves and stems, both within and among the species.Further, detailed qualitative and quantitative investigations are required for identification of these chemical entities.

Fig 1 .
Fig 1. HPTLC densitogram profile and plate image of a and b: A. coynei -Leaf extract; c and d: A. coynei -Leaf extract; e and f: A. aspera -Stem extract; g and h: A. aspera -Stem extract Fig. 1 e, g).Other peaks with different Rf Plant Science Today (2015) 2(1): 7-10 Horizon e-Publishing Group ISSN: 2348-1900 10 values among the stem extracts of both species indicated variation in chemical nature.

Table 1
Densitogram attributes of HPTLC run of leaf and stem extract of A. coynei and A.
aspera Pk -Peak; Rf -Retention factor; Ht -Height; AU -Area Under Curve; A -Area; the highlighted values in above table indicate application position of extracts on HPTLC plate, not considered for the analysis