European Journal of Biotechnology and Bioscience

European Journal of Biotechnology and Bioscience ISSN: 2321-9122; Impact Factor: RJIF 5.44 Received: 24-04-2019; Accepted: 27-05-2019 www.biosciencejournals.com Volume 7; Issue 4; July 2019; Page No. 50-59

Chemical profiling of Genus Cassia L. sensu lato using High Performance thin Layer Chromatography (HPTLC) and its taxonomic implications.

Usha Veerachari1*, Jyothi SG2, Senthilkumar U3, Mithra Madappa4, Ravikanth G5, Somashekariah BV6 1Department of Life Sciences, Jain University, Bengaluru, Karnataka, 2 Department of Biochemistry, BMS College for Women, Bengaluru, Karnataka, India 5, 3 Ashoka Trust for Research in Ecology and Environment, Srirampura, Jakkur Post, Bengaluru, Karnataka, India 4 Department of Biotechnology, St. Joseph’s College PG and Research Centre, Bengaluru, Karnataka, India 5 School of Ecology and Conservation, University of Agricultural Sciences, GKVK, Bengaluru, Karnataka, India 6 Department of Biochemistry, St. Joseph’s College PG and Research Centre, Bengaluru, Karnataka, India

Abstract Objective: To establish the phenolic (flavonoids) fingerprint profile for identification and making a comparative analysis of the economically important Cassia L. at level. Fingerprint profiles inturn act as biochemical markers and are useful in differetiating the species thus supplementing chemotaxonomic studies. Method: Methanolic extract of the were screened for Quercetin, Gallic acid, Cinnamic acid, Caffeine, Curcumin and Ferulic acid using standard procedure and densitometric evaluation was performed at 254 nm in CAMAG TLC scanner 3 linked to WINCATS software. The image of the plate was captured at 366nm, UV chamber. Results: The contour graph obtained from the densitometric evaluation indicates that the percentage of each compound varies within the species. Quercetin and Curcumin being present in higher concentrations than Gallic acid, Ferulic acid, Cinnamic acid and Caffeine among all the species of Cassia L. Conclusion: Chromatographic fingerprinting is useful in differentiating the species and acts as an important biochemical marker in plant systematic studies and also in authentication of the species.

Keywords: Cassia sensu lato, chromatography, chemotaxonomy, phenolic compounds

1. Introduction compounds in a series of related . Increase in the The genus Cassia sensu lato., erected by Linnaeus [15], knowledge of these natural products and their occurrence in includes , shrubs and herbs which have a pan tropical the potentialities of chemotaxonomy has now become distribution [10, 32] with 62 species being present in India. increasingly obvious [6]. HPTLC method is able to generate Bentham [3] in his revision of the genus indicated that it is a rapid low cost and easily understandable chromatographic comprised of three distinct groups and suggested that there fingerprint in the form of a unique sequence of peaks, are three subgenera in this genus; viz., Cassia, and corresponding to the analyzed samples in its chemical Lasiorhegma [26]. These three genera belong to the tribe fullness [22]. Classification of plants based on the phenolic Cassieae Bronn ex., of Caesalpiniaceae [10, 17, 14, 19]. However chemotypes can be used as a powerful chemotaxonomic tool systematics and biochemical analysis on this particular that provides a detailed view of the differences and group is limited with sparse literature and reports on similarities between species [16]. cytological studies contradicting each other [29]. These Phenolics have proved to be of significance to genera have significant importance in Indian traditional chemotaxonomists in providing much taxonomic data and medicines to treat various diseases such as skin diseases, flavonoids are taxonomically the most important phenolics. inflammatory diseases, rheumatism, jaundice, anorexia, etc. Thus analysis for some phenolics by chromatographic [1]. techniques provides a strong base for further quantification. The study on chemical variation at inter-specific and intra- Quercetin, a flavonol, is a plant pigment found in , specific level of the taxa helps in distinguishing the species vegetables, leaves and grains. It can be used as an ingredient and also highlights the patterns of chemical variation across in supplements, beverages or foods. Preliminary research genera that may ultimately demonstrate the diversity of work has shown that quercetin can inhibit reverse phyto-constituents among them. Hence in cases where transcriptase [27], is an effective bronchodilator [24] and helps fundamental disagreements on classification of taxa exist, reduce the release of histamine and other allergic or phytochemical investigations would either resolve the inflammatory chemicals in the body. It is widely used for its taxonomic complexities [5] or improve the better activity of eliminating phlegm and cough relieve. It also understanding on the history of chemical evolutions among shows activity like Vitamin P. It is also an antinflammatory them [8] and also leads to the discovery of novel metabolites. and used in anticancer application [31]. Cinnamic acid is a Relationship between plant constituents and plant white crystalline organic compound with honey like odour classification is a significant need in chemical ; it [11]. It is used in the preparation of flavours, synthetic indigo, consists of the investigation of the distribution of related certain pharmaceuticals and also in perfumeries [30].

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Cinnamic acid and its associated compounds are present in The residue of the tender leaves from 10 individuals of each coffee, apples, citrus fruits, vegetable oils, propolis and species, collected during the same season was dissolved in wine. It has been shown to possess antitumour and 1mL ethanol and used for chromatographic studies. chemopreventive properties [16]. Caffeine is a bitter, white crystalline alkaloid found in varying quantities in seeds, 2.3 Thin Layer Chromatography leaves and fruits of some plants. It acts as a natural pesticide The chromatographic plates were prepared using Cellulose that paralyzes and kills certain insects feeding on the plants. microcrystalline of ‘Merck’ grade. A combination of 15g It is commonly consumed by humans in infusions extracted cellulose powder and 90mL distilled water was blended in a from the seed of coffee plant and leaves of tea bush and also mixer for 1min; the slurry was poured into the applicator, products derived from kola nut. It acts as a stimulant on the with thickness adjusted to 0.3mm. Glass plates (20×20cm of central nervous system (CNS), temporarily warding off 3mm thickness) wiped with acetone were used as supporting drowsiness and resort alertness. Low consumption has less material on which the slurry was uniformly poured using the health risks and a modest protective effect against spreading tube. The chromatographic plates were allowed to Parkinson’s disease and certain types of cancer [18]. Gallic dry at room temperature and activated at 120 ͦ C for 1hr acid is a commonly occurring phenolic acid found in before development. nuts, , witch hazel, tea leaves, and other The spotting guide was used to assure the uniformity of the plants. It has antifungal, antioxidant and anticancerous sample spot on the TLC plate. 3µL of the extract was properties due to its cytotoxic effect. Gallic acid is used in applied as the starting point on the cellulose TLC plates and the treatment of internal haemorrhage, albuminuria, diabetes placed in the developing chamber saturated with the mobile and used in the preparation of ointments to treat psoriasis phase n-Hexane and ethyl acetate, in the ratio of 1:1 in the and external haemorrhoids) [25]. Ferulic acid is an abundant first run and glacial acetic acid in the second. The phenolic compound found in the plant cell wall components, developing chamber was kept covered with the glass plate to apple, peanut, orange, pineapple, seeds of coffee, rice, maintain constant saturation. wheat, barley and oats [7, 2, 34]. It exhibits antioxidant, After two dimensional development and total drying, the antibacterial and antitumour activity [4, 7]. Curcuminoids are plates were exposed to ammonia vapours in order to natural phenols that are responsible for the yellow colour of intensify the fluorescence of the phenolic spots and were turmeric. The bioactivity of Curcumin ingested in foods viewed under long UV light of wave length 360nm. The may be increased as a result of cooking or dissolution in oil patterns were photographed and the boundaries of flavonoid [13, 20]. Curcumin is used against cancer, diabetes, allergies, spots were marked on the tracing sheets after noting the [21] arthritis, Alzheimer's disease and other chronic illnesses . colours of the spots. Each distinct colour at any particular Rf The chemical content present in plants has high therapeutic value was regarded as a separate character and the total values which are usually variable depending on many number of characters of particular taxon, were the total [33] factors , can act as biochemical markers in plant number of spots present on its TLC profile. Rf value of each systematics. Hence the present investigation on the spot was calculated with reference to both the directions. A distribution of phenolics was performed among 12 species minimum of 10 TLC plates was used for each species for of Cassia L. to examine their relative distance, as evident averaging the Rf values of flavonoids spots of single plant. from the chromatographic analysis. On the basis of the Rf values and the colour under UV light, spots presumed to be identical on the chromatogram of two 2. Materials and Methods or more taxa have been assigned the same number. Total 2.1 Sampling spots obtained on the chromatographic profiles of all the Twelve Cassia L. species viz, Senna alata (L.) Roxb., taxa were traced on a tracing sheet, to prepare a ‘Master Senna auriculata (L.) Roxb., Cassia fistula L., Senna key’ or ‘Master chromatogram’. The spot areas on the hirusta (L.) H.S. Irwin & Barneby., Senna occidentalis (L.) chromatogram of each species were also recorded with the Link., Chamaecrista Kleinii (Wt. & Arn.) V.Singh., Senna help of a planimeter for numerical assessment, which are polyphylla (Jacq.) H.S.Irwin and Barneby., Senna uniflora represented graphically. The above procedure was followed (Mill.) H.S.Irwin and Barneby., Senna siamea (Lam.)., to extract mainly the different flavonoid contents in the H.S.Irwin and Barneby., (DC.) H.S.Irwin individual species. and Barneby., Senna surattensis (Burm.f) H.S.Irwin and Barneby., Senna tora (L.) Roxb., were collected from 2.4 Numerical assessment different regions of Bangalore for the present study. These In the present study, three similarity measures have been species were authenticated using morphological characters used to interpret the data available on TLC profiles to by the Centre for Ecological studies, Indian Institute of determine the homology of phenolic compounds amongst Science, Bangalore by the taxonomist using different species studied belonging to the genera, Cassia s.l. literature. Voucher specimens were prepared for all species The similarity measures like ‘Matching Co-efficient’ and’ and deposited at the Herbaria, National Ayurveda Dietetics Co-efficient of Similarity or Jaccard Co-efficient’ relate to Research Institute, Bangalore the qualitative variations with respect to the presence or absence of spots. The ‘Biochemical Distance’ relates to 2.2 Acid hydrolysis quantitative variations according to the differences in the sample (ca. 5g) from 12 species of Cassia were intensity of spots, i.e., spot area. Expression for Matching immersed in 2N HCl in different test tubes and heated for 45 Co-efficient and Co-efficient of Similarity are min in hot water bath. After cooling, the solution was filtered and the filtrate was collected twice each time with 5mL ethyl acetate. The combined ethyl acetate portion was left overnight for evaporation and concentrated to dryness.

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value 0.9 is seen in almost all species except in S. siamea and S.spectabilis and can be considered as the generic

criteria as “marker spots”. where, m=number of positive matches (substances in All species in the present study could be identified by their question occurring in both the taxonomic units), n=number characteristic chromatographic units (Table 1). In addition, of negative matches (substances in question missing in both they were observed to have specific spots which assist in the the taxonomic units), d=number of differences (substances identification of species. Among 12 species of the genus occurring only in one of the taxonomic units) Cassia studied, seven species namely, S. alata, S. hirsuta S. The differences between the above two coefficients is occidentalis, S. uniflora, S. surattensis, S.siamea and S. tora simply that, in the Matching Coefficient, the negative were found to have ‘species specific spots’ or ‘species matches are regarded as true similarities while for markers’. The qualitative TLC data so obtained were calculating coefficient of similarity, they are not used. numerically analyzed and assessed linking the proximity Expression for Biochemical Distance is existing amongst the selected Cassia species in terms of (Biochemical Distance)⁄(Taxonomic Distance) (d)=√(1-S sm matching co-efficient (Cm) and Jaccard co-efficient or co- where, S = Simple Matching Co-efficient. sm efficient of similarity (Cs).

Cm and Cs values belonging to different species show the 2.5 Cladistic analysis degree of the species relativity. A concordance between Unweighted pair group method with arithmetic mean classical taxonomy and chromatographical data was derived (UPGMA) was used to construct the dendrogram for the for the phenolic spots based on correlations of Cm and Cs total phenolic spots obtained from chromatographic profiles. (Figure 1 and 2).

Biochemical distance is calculated based on the 2.6 High performance thin layer chromatography chromatographic patterns across all the species and the (HPTLC) quantitative characters, like the amount of various HPTLC was performed on HPTLC plate pre-coated with compounds present in the different taxonomical units are silica gel 60 F of thickness 0.2mm (E. Merck) of size 254 measured using correlation co-efficient and Taxonomic 10×10 cm. Quercetin, Cinnamic acid, Ferulic acid, Caffeine, distance/Biochemical distance. The degrees of their Curcumin and Gallic acid (4.0µL each) and samples (4.0 µL relativity (species) were established according to the each) were applied on the plate as band of 8mm width using Taxonomical distance/Biochemical distance (Table 2). Hamilton syringe and CAMAG Linomat V sample Systematic position of Cassia sensu lato was studied applicator. The plate was developed to a distance of 8cm in chemotaxonomically with respect to presence and absence a CAMAG twin trough chamber previously saturated with of the phenolic spots and was rearranged on the basis of mobile phase [12] for 30 min. After development, the plate chemical findings based on the result of numerical analysis. was dried at room temperature and densitometric evaluation Biochemical overlapping is observed across the species with was performed at 254nm in CAMAG TLC scanner 3 linked respect to phenolic spots obtained from TLC studies. This to WINCATS software. The image of the plate was captured indicates the chemical linkage wherein similarity and at 366nm UV chamber. dissimilarity of spots are shown across the different species, The quantification of the phytochemicals in all the samples which is very significant in S. alata with a total of 9 spots. were determined by comparing the total peak area observed This suggests that S. alata has some unique chemical spots for standard bands with that in sample solutions and not found in the remaining species of Cassia. All the other calculated using the formula species of Cassia possess more or less similar number of

spots ranging from 4 to 7 which indicates some similarity

existing amongst them. The preliminary qualitative TLC analysis expressed the presence of nine spots in S. alata, out of which four spots (spot number 1b with Brown colour and Rf of 49, 2b with

fluorescent orange and Rf of 23, 7b with light fluorescent 3. Results orange and Rf of 53, 7b1 with light fluorescent orange and The number of the spots, their Rf values in two directions, Rf of 57) are species specific and the numerical evaluation colours in UV light and spot areas were tabulated (Table 1). also articulated the occurrence of these unique spots not The species can be distinguished by their characteristic observed in other species of Cassia considered. chromatographic profiles using different spot types. In addition, the specific spots have further aided in identifying 3.1 High Performance Thin Layer Chromatography their individuality. The details of spot pattern observed for (HPTLC) each species were noted where a total of 27 spot types were The chromatograms when observed under UV light, showed visualized on the chromatographic profiles of the 12 taxa. well defined spots against respetive standards and the This study on the phenolic spot patterns of the genus Cassia densitograms were obtained for the same (Supplementary L. compares the similarity and dissimilarity among the Figures S1 to S5). HPTLC technique provides an authentic species. The following criteria such as the total number of data that ascertains the presence of specific spots observed, their presence or absence and relative 2 phytoconstituents which decides the biological nature of the concentration as spot area in mm were taken for the species thereby accomplishing their correlation [28]. This comparison. analysis gives clarity of the quantified spots rather than the The total number of spot types on the chromatogram of qualitative biochemical spots obtained from TLC studies. individual species varied from 4 to 9. The constancy of the The HPTLC analysis was performed with respect to some occurrence of the spot type 1b1 of brown colour with Rf well known phenolics and flavonoids like Quercetin,

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Cinnamic acid, Caffeine, Gallic acid, Ferulic acid and C. fistula and C. kleinii have shown to possess higher Curcumin. percentage of curcumin followed by S. hirsuta and S. auriculata. Curcumin content in the remaining species was Quercetin found to be very less while the other leaf extracts did not The study species was chromatographically analyzed for the show the phenolic spots (Table 8). presence of the familiar flavonoid quercetin and it is found The presence of quercetin in Senna alata, Senna hirsuta, to be present in all (Table 3). Chamaecrista kleini, Senna polyphylla, Senna uniflora, Senna spectabilis, Senna surattensis and Senna tora; Cinnamic acid cinnamic acid in all the 12 species taken for study; caffeine S. occidentalis exhibited maximum cinnamic acid content in Senna alata, Senna hirsuta, Chamaecrista kleinii, Senna along with S. auriculata and S.alata, while S.hirsuta and S. occidentalis, Senna surattensis and Senna tora; gallic acid polyphylla showed minimum concentration (Table 4). in Senna uniflora, Senna siamea, Senna surattensis and Senna tora; curcumin in Senna alata, Senna auriculata, Caffeine Cassia fistula, Senna hirsuta, Chamaecrista kleinii, and The leaf extracts of Senna alata, Senna tora and Senna Senna surattensis, ferulic acid in Senna alata, Senna surattensis contains almost similar content of caffeine in auriculata, Senna occidentalis, Senna siamea, Senna comparison to Cassia fistula, Senna auriculata and spectabilis, Senna surattensis and Senna tora have been Chamaecrista kleinii. Caffeine was found to be present in reported for the first time in the present investigation. These lowest amount in Senna occidentalis and could not be compounds were quantified by HPTLC analysis, where the detected in other species of Cassia under the performed percentage concentration is represented pictorially by the experimental conditions (Table 5). densitograms. The presence of these phenolics in the evaluated taxa tends to comprehend the relative association Gallic acid existing between the Cassia species (Figure 3). HPTLC analysis revealed that S. hirsuta leaf extract The contour graph shows the percentage of each compound contains maximum amount of gallic acid when compared from low to high in Cassia sensu lato where, Quercetin and with other Senna species of Peninsular India (Table 6). Curcumin are in higher concentration than Gallic, Ferulic and Cinnamic acids along with Caffeine among all the Ferulic acid species (Supplementary Figure S6). Based on the whisker Higher percentage of ferulic acid was observed in S. plot (Figure 4), though the deviation (SD) is defined, SE surattensis leaf extract compared to low percentage found in errors indicate that Quercetin and Ferulic acid have higher S. spectabilis and S. occidentalis. Comparable ferulic acid variation among the species; perhaps both of them would be content was shown by Senna alata and Senna tora followed the better indicators to distinguish a species (Supplementary by Senna siamea and Senna auriculata (Table 7). Figure S6). Also Caffeine and Gallic acid contribute significantly to the systematics of Cassia sensu lato. Curcumin

cm

Fig 1: Dendrogram constructed for the Matching Coefficient

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cs

Fig 2: Dendrogram constructed for the Coefficient of Similarity

Fig 3: Dendrogram for the contour graph derived from HPTLC

Fig 4: Graphical representation of SD values with respect to total mean

Fig 5: Supplementary Figure S1 HPTLC graph showing querctin at all tracks observed @254nm.

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Fig 6: Supplementary Figure S2 HPTLC graph showing cinnamic acid at all tracks observed @254nm.

Fig 7: Supplementary Figure S3 HPTLC graph showing caffeine at all tracks observed @254nm.

Fig 8: Supplementary Figure S4 HPTLC graph showing gallic acid at all tracks observed @254nm.

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Fig 9: Supplementary Figure S5 HPTLC graph showing curcumin and ferulic acid at all tracks observed @254nm.

Fig 10: Supplementary Figure S6 Contour Graph for specific metabolites identified by HPTLC analysis.

Table 1: Rf values, Fluorescent colour and spot area in mm2 of the phenolic compounds in leaves of different species of the genus Cassia after spraying with 1% AlCl3 + NaOH.

Colour of Sl No Spot No Rf

S.tora S.alata the spot

C.fistula C.kleinii

S.siamea

S.hirsuta

S.uniflora

S.auriculata S.polyphylla S.spectabilis

S.surattensis

S.occidentalis Area of the spot in mm2 1 1b 49 ------Br 0.03 2 1b1 77 124 37 200 170 36 220 150 - - 156 261 Br 0.9 3 2b 23 ------Fl O 0.2 4 2b1 - 144 - - 50 - - 70 - 53 - - Fl O 0.05 5 2b2 ------77 - Fl O 0.01 6 3b 232 77 - - - 175 ------Fl Y 0.6 7 4b 117 92 97 - 55 75 - 168 32 80 136 - Fl B 0.9 8 4b1 - - - - - 187 ------Fl B 0.9 9 5b 123 - - - - 119 - - - 157 - - Fl G 0.8 10 5b1 183 - - - 352 ------Fl G 0.3 11 6b - 43 60 - - - - - 80 - - - L Fl Br 0.8 12 6b1 - - - 107 ------L Fl Br 0.07 13 6b2 ------65 - - - L Fl Br 0.4 14 7b 53 ------L Fl O 0.6 15 7b1 57 ------L Fl O 0.3 16 7b2 - - 67 55 ------L Fl O 0.9 17 7b2 - - - - - 40 ------L Fl O 0.01 18 7b2 ------49 - - - L Fl O 0.06 19 8b1 - - - 150 ------24 - L Fl Y 0.8 20 8b1 - - 29 67 - - 28 - - 27 19 - L Fl Y 0.7

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21 8b2 ------13 L Fl Y 0.02 22 9b - - 26 - 52 - 105 62 90 - - 58 L Fl B 0.9 23 9b1 - - - 31 ------L Fl B 0.8 24 9b2 - - - 374 ------L Fl B 0.4 25 10b - 26 56 - 75 - 228 95 108 - 13 108 L Fl G 0.8 26 10b1 ------68 - L Fl G 0.9 27 11b ------125 - - - - L Fl Y Br 0.8 Note: Br-Brown, Fl.O-Fluorescent orange, Fl.Y-Fluorescent yellow, Fl.B-Fluorescent blue, Fl.G-Fluorescent green, L.Fl.Br- Light fluorescent brown, L.Fl.O-Light fluorescent orange, L.Fl.Y-Light fluorescent yellow, L.Fl.B-Light fluorescent blue, L.Fl.G-Light fluorescent green, L.Fl.Y.Br-Light fluorescent yellow brown).

Table 2: Taxonomic distance / biochemical distance

2 3 4 5 6 7 8 9 10 11 12 1 0.61 0.64 0.77 0.54 0.58 0.64 0.64 0.7 0.91 0.64 0.64 2 0.47 0.64 0.50 0.50 0.50 0.43 0.50 0.50 0.54 0.47 3 0.58 0.38 0.91 0.28 0.38 0.38 0.47 0.43 0.38 4 0.64 0.67 0.50 0.64 0 0.58 0.54 0.91 5 0.54 0.38 0.28 0.47 0.50 0.50 0.38 6 0.54 0.54 0.67 0.47 0.58 0.61 7 0.38 0.50 0.47 0.58 0.61 8 0.47 0.54 0.50 0.38 9 0.54 0.54 0.43 10 0.50 0 11 0.50

Table 3: HPTLC results showing % concentration of quercetin in Table 5: HPTLC results showing % concentration of Caffeine in Cassia species. Cassia species.

Area of the Sample RF values Area of the peak % of caffeine Sample Rf values % of quercetin peak S.alata 0.52 4260.17 0.5408 S.alata 0.94 343.50 0.1112 S.auriculata 0.48 901.47 0.1144 S.auriculata 0.88 4423.80 1.4321 C.fistula 0.53 2168.65 0.2752 C.fistula 0.89 3600.34 1.1655 C.kleinii 0.55 974.05 0.1236 S.hirsuta 0.91 4875.80 1.5784 S.occidentalis 0.45 345.61 0.0438 C.kleinii 0.90 3833.25 1.2409 S.surattensis 0.51 2745.60 0.3485 S.occidentalis 0.90 3213.14 1.040 S.tora 0.53 3555.54 0.4513 S.polyphylla 0.91 1914.50 0.6197 S.uniflora 0.89 210.66 0.0681 Table 6: HPTLC results showing % concentration of Gallic acid in S.siamea 0.90 718.91 0.2327 Cassia species. S.spectabilis 0.90 437.23 0.1415 Area of the % of gallic S.surattensis 0.91 1460.83 0.4729 Sample Rf values peak acid S.tora 0.91 2702.64 0.8749 S.auriculata 0.40 262.08 0.0366

S.hirsuta 0.44 4923.06 0.6875 Table 4: HPTLC results showing % concentration of Cinnamic S.uniflora 0.40 1365.48 0.1906 acid in Cassia species. S.siamea 0.43 623.51 0.0870 Rf Area of the % of cinnamic S.surattensis 0.39 2703.91 0.3776 Sample values peak acid S.tora 0.40 458.13 0.0639 S.alata 0.48 2759.36 0.5111 S.auriculata 0.47 2725.97 0.5049 Table 7: HPTLC results showing % concentration of Ferulic acid C.fistula 0.45 1477.29 0.2736 in Cassia species. S.hirsuta 0.46 1581.47 0.2929 C.kleinii 0.46 1842.09 0.3411 Sample Rf values Peak area % of ferulic acid S.occidentalis 0.46 3011.41 0.5576 S.alata 0.52 4935.46 1.1663 S.polyphylla 0.47 1401.21 0.2595 S.auriculata 0.49 578.24 0.1366 S.uniflora 0.47 2218.48 0.4109 S.occidentalis 0.52 374.69 0.0885 S.siamea 0.48 1727.74 0.3200 S.siamea 0.49 785.11 0.1855 S.spectabilis 0.48 1992.72 0.3690 S.spectabilis 0.47 370.64 0.0875 S.surattensis 0.49 2526.20 0.4679 S.surattensis 0.48 6015.37 1.4214 S.tora 0.49 2332.43 0.4320 S.tora 0.47 4323.91 1.0217

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Table 8: HPTLC results showing % concentration of Curcumin in Ecology and Conservation, UAS, GKVK, Bangalore, Prof. Cassia species. K. M. Diwakar, and Prof. K. B. Purushothama, Department

Area of the % of of Botany, St. Joseph’s PG and Research Centre, Bengaluru, Sample Rf values peak curcumin for their kind support and valuable suggestions. S. alata 0.41 183.42 0.0324 S. auriculata 0.44 1167.18 0.2061 7. References C. fistula 0.45 3791.16 0.6696 1. Bhakta T, Banerjee S, Mandal SC, Maity TK, Saha BP, S. hirsuta 0.45 2001.16 0.3534 Pal M. Hepatoprotective activity of Cassia fistula leaf C. kleinii 0.45 3769.07 0.6657 extract. Phytomedicine. 2001; 8:220-4. [PubMed] S. surattensis 0.61 407.02 0.0718 2. Beejmohun V, Fliniaux O, Grand E, Lamblin F, Bensadde L, Christen P, et al. Microwave-assisted 4. Discussion extraction of the main phenolic compounds in flaxseed. Chemosystematics is the attempt to classify and identify Phytochemical Analysis. 2007; 18:275-282. plants, according to demonstrable differences and 3. Bentham G. Revision of the genus Cassia Linn. Trans. similarities in their biochemical composition [23]. Flavonoids Linn. Soc.London. 1871; 27:503-591. the most important phenolics are found to be present in all 4. Bouftira I, Chedly A, Souad S. Antioxidant and parts of the plant. The present investigation confirms the antibacterial properties of Mesembryanthemum occurrence of flavonoids in the methanolic leaf extracts of crystallinum and Carpobrotus edulis extracts. Advances all the species of Cassia L. along with the comparative in Chemical Engineering and Science. 2012; 2:359-365. account of it. 5. Cronquist A. Bull. Jardin Botan. Bruxelles. 1999; Morphological studies play a vital role in the study of plant 27:13. systematics and are a useful tool in the classification of a 6. Erdtman H. Some aspects of Chemotaxonomy. taxon. Along with morphological markers, biochemical Institution for Organisk Kemi, KTH, Stockholm 70, markers are also used to classify organisms. A multitude of Sverige, 1963, 679-708. phytochemical patterns gives an input to judge the overall 7. Gelinas P, Mckinnon CM. Effect of wheat variety, taxonomic implications. The presence or absence of farming site and bread baking on total phenolics. chemical constituents has been found useful in the International journal of food Science and Technology. placement of the plant in taxonomic categories. 2006; 42:329-332. Chromatographic finger printing of the phytoconstituents 8. Hegnauer R. Chemical characters in plant taxonomy: can be used efficiently for the identification and also for some possibilities and limitations, Laboratorium voor quality assessment of the herbal extracts. Six pure bioactive experimentele plantensystematiek, Leidea, Netherlands, compounds were used as chemotaxonomic markers to 2006, 173-187. distinguish the different Cassia samples. HPTLC 9. Hostettmann K, Marston A, Mailland M, Hamburger fingerprints thus obtained for the species not only help in M. Phytochemistry of plants used in traditional differentiating the species and to make a comparative medicine. 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