Research Article HPTLC Fingerprint Profile (Phenolics) of...
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Research Article HPTLC Fingerprint Profile (Phenolics) of Selected Cyathea Species from Western Ghats, South India Janakiraman Narayanan and Johnson Marimuthu alias Antonysamy Centre for Plant Biotechnology, Department of Botany, St. Xavier’s College (Autonomous), Palayamkottai, Tamil Nadu 627 002, India Correspondence should be addressed to Johnson Marimuthu alias Antonysamy; [email protected] Received 25 August 2015; Revised 2 January 2016; Accepted 4 January 2016 Academic Editor: Vivian Y. Shin Copyright © 2016 J. Narayanan and J. Marimuthu alias Antonysamy. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HPTLC analysis was performed to study the phenolics, flavonoids, and tannins profile of Cyathea nilgirensis Holttum, Cyathea gigantea (Wall. ex. Hook.) Holttum, and Cyathea crinita (Hook.) Copel. e maximum number of bands was observed in the ethanolic extracts of C. crinita. e phenolic bands with values 0.05, 0.32, 0.43, 0.75 (quercetin), and 0.83 demonstrated their occurrence in all the three studied species of Cyathea. Flavonoids with values 0.05, 0.23, 0.30, 0.43, and 0.73 showed their presence in the three studied species of Cyathea. Tannins with the values 0.05, 0.09, and 0.82 determined their existence in all the three studied Cyathea species. Gallic acid was present only in C. crinita with the value 0.49. e developed HPTLC profiles can be used for identification and quantitative determination of phenolics, flavonoids, and tannins. is method is also suitable for rapid screening of Cyathea species for chemotypic assessment and also for quality control purposes. 1. Introduction Plants synthesize a wide variety of chemical compounds which can be sorted by their chemical class, biosynthetic origin, and functional groups. e medicinal value of plants lies in chemical substances or group of compounds that produce a definite physiological action in the human body [1]. e active ingredients present in medicinal plants can be used for therapeutic purposes and are precursors of chemotherapeutical semisynthesis [2]. e beneficial effects of plants are usually due to the secondary metabolites which provide temporary relief to symptomatic problems, health promoting characteristics, and curative properties. Plant phenolic compounds include flavonoids, tannins, gly- cosides, coumarins, anthraquinones, lignans, and lignins. ey may act as phytoalexins, antifeedants, and attractants for pollinators. In addition, they act as contributors to the plant pigmentation [3]. With the advent of modern scientific methods, medicinal plants came under chemical scrutiny, leading to the isolation of the active principles. Soon aſter their isolation and characterization, these compounds either in pure state or in the form of well-characterized extracts became part of pharmacopoeias of several countries. According to draſt guidelines stated by the USFDA, a marker compound is a chemical constituent of a botanical raw material that is used for identification or quality con- trol purposes, especially when the active constituents are not identified. e active constituent is responsible for the intended pharmacological activity or therapeutic effects. Chemical standardization oſten involves chemical identifi- cation by spectroscopic or chromatographic fingerprint and chemical assay for active constituents or marker compounds if available. e analytical methods developed can be used for chemical fingerprinting and assaying of marker or active compounds [4]. Chemical fingerprints obtained by chro- matographic techniques are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the chemical integrity of the herbal medicines and its products are therefore used for authentication and identification of the plant [5]. Tree ferns are usually considered under a single family Cyatheaceae except Holttum [6] who suggested a poly- phyletic derivation with four families. It is one of the most interesting families among the pteridophytes due to their striking morphology and wide geographical distribution with diversity centers in the tropics, subtropics, and southern Hindawi Publishing Corporation Chinese Journal of Biology Volume 2016, Article ID 6420371, 7 pages http://dx.doi.org/10.1155/2016/6420371
Transcript of Research Article HPTLC Fingerprint Profile (Phenolics) of...
Research ArticleHPTLC Fingerprint Profile (Phenolics) of SelectedCyathea Species from Western Ghats South India
Janakiraman Narayanan and Johnson Marimuthu alias Antonysamy
Centre for Plant Biotechnology Department of Botany St Xavierrsquos College (Autonomous) Palayamkottai Tamil Nadu 627 002 India
Correspondence should be addressed to Johnson Marimuthu alias Antonysamy ptcjohnsongmailcom
Received 25 August 2015 Revised 2 January 2016 Accepted 4 January 2016
Academic Editor Vivian Y Shin
Copyright copy 2016 J Narayanan and J Marimuthu alias Antonysamy This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited
HPTLC analysis was performed to study the phenolics flavonoids and tannins profile of Cyathea nilgirensis Holttum Cyatheagigantea (Wall ex Hook) Holttum and Cyathea crinita (Hook) Copel The maximum number of bands was observed in theethanolic extracts of C crinita The phenolic bands with 119877119891 values 005 032 043 075 (quercetin) and 083 demonstrated theiroccurrence in all the three studied species of Cyathea Flavonoids with 119877119891 values 005 023 030 043 and 073 showed theirpresence in the three studied species of Cyathea Tannins with the 119877119891 values 005 009 and 082 determined their existence in allthe three studied Cyathea species Gallic acid was present only in C crinita with the 119877119891 value 049 The developed HPTLC profilescan be used for identification and quantitative determination of phenolics flavonoids and tanninsThis method is also suitable forrapid screening of Cyathea species for chemotypic assessment and also for quality control purposes
1 Introduction
Plants synthesize a wide variety of chemical compoundswhich can be sorted by their chemical class biosyntheticorigin and functional groups The medicinal value of plantslies in chemical substances or group of compounds thatproduce a definite physiological action in the human body[1] The active ingredients present in medicinal plants canbe used for therapeutic purposes and are precursors ofchemotherapeutical semisynthesis [2] The beneficial effectsof plants are usually due to the secondary metaboliteswhich provide temporary relief to symptomatic problemshealth promoting characteristics and curative propertiesPlant phenolic compounds include flavonoids tannins gly-cosides coumarins anthraquinones lignans and ligninsThey may act as phytoalexins antifeedants and attractantsfor pollinators In addition they act as contributors to theplant pigmentation [3] With the advent of modern scientificmethods medicinal plants came under chemical scrutinyleading to the isolation of the active principles Soon aftertheir isolation and characterization these compounds eitherin pure state or in the form of well-characterized extractsbecame part of pharmacopoeias of several countries
According to draft guidelines stated by the USFDA amarker compound is a chemical constituent of a botanicalraw material that is used for identification or quality con-trol purposes especially when the active constituents arenot identified The active constituent is responsible for theintended pharmacological activity or therapeutic effectsChemical standardization often involves chemical identifi-cation by spectroscopic or chromatographic fingerprint andchemical assay for active constituents or marker compoundsif available The analytical methods developed can be usedfor chemical fingerprinting and assaying of marker or activecompounds [4] Chemical fingerprints obtained by chro-matographic techniques are strongly recommended for thepurpose of quality control of herbal medicines since theymight represent appropriately the chemical integrity of theherbal medicines and its products are therefore used forauthentication and identification of the plant [5]
Tree ferns are usually considered under a single familyCyatheaceae except Holttum [6] who suggested a poly-phyletic derivation with four families It is one of the mostinteresting families among the pteridophytes due to theirstrikingmorphology andwide geographical distributionwithdiversity centers in the tropics subtropics and southern
Hindawi Publishing CorporationChinese Journal of BiologyVolume 2016 Article ID 6420371 7 pageshttpdxdoiorg10115520166420371
2 Chinese Journal of Biology
temperate regions They are considered as primitive thoughthey represent different lines of evolutionThese ferns displaygreat ecological conservatism as most species are terrestrialplants of moist forests and are intolerant to longer peri-ods of drought or frost Furthermore they show a greaterprovincialism and endemism thanmost fern groups [7]Withthe traditional healing system that is actively searching andexpanding its pharmacopoeia in order to treat a large numberof complaints an environment with great floral diversity isslipping away unlearned by a new generation of healersThe scientific and traditional communities need a resourcewhere data on the phytochemical aspects of these ferns arecollated Hence the present study was intended to studythe HPTLC fingerprint profile (phenolics flavonoids andtannins) of Cyathea nilgirensis Holttum Cyathea gigantea(Wall ex Hook) Holttum and Cyathea crinita (Hook)Copel
2 Materials and Methods
21 Collection of Plant Materials Specimens for the presentstudy were collected from various natural habitats of TamilNadu C nilgirensis were harvested in and around Kakkachistream (1725m) Kothayar Tirunelveli Hills (8∘441015840N and77∘441015840E) C gigantea from the roadsides near Nadugani(2637m) Nilgiris Hills (11∘241015840N and 76∘441015840E) and C crinitafrom the Anglade Institute of Natural History ShenbaganurKodaikanal (2195m) Palni Hills (10∘131015840N and 77∘321015840E)Western Ghats South IndiaThe plants were identified basedon the Pteridophyte Flora of the Western Ghats South IndiabyManickam and Irudayaraj [8] Herbarium specimens wereprepared and the voucher specimens were deposited in theSt Xavierrsquos College Herbarium (XCH) Palayamkottai TamilNadu India for further reference (C nilgirensis XCH 25423C gigantea XCH 25422 and C crinita XCH 25424)
22 Preparation of Extracts The collected species of Cyatheawere thoroughly washed with tap water followed by distilledwater They were blotted on the blotting paper and shadedried at room temperature in the dark The shade driedplant samples were ground to fine powder using mechanicalgrinder 30 g powdered samples were extracted successivelywith 180mL of ethanol using Soxhlet extractor for 8ndash12 h ata temperature not exceeding the boiling point The extractswere concentrated in a vacuum at 40∘C using rotary evapo-rator
23 HPTLC Analysis HPTLC studies were carried out usingthe standard method described by Wagner et al [9] 25mgof ethanolic extracts of selected Cyathea species was weighedaccurately in an electronic balance (Shimadzu) It was dis-solved in 05mL of ethanol and centrifuged at 3000 rpmfor 5min These solutions were used as test solution forphenolics flavonoids and tannins 2120583L of test solutionsand 2 120583L of standard solution were loaded as 5mm bandlength in the silica gel 60F254 TLC plate using Hamiltonsyringe and CAMAG Linomat 5 instrument The samplesloaded plate was kept in TLC twin trough developing
Table 1 HPTLC phenolics profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Phenolic 2026 + + Phenolic 4028 + Catechin032 + + + Unknown043 + + + Phenolic 5049 + Phenolic 6055 + + Unknown059 + Phenolic 7066 + Unknown072 + Phenolic 8075 + + + Quercetin080 + Unknown083 + + + Unknown092 + + Unknown
chamber (after being saturated with solvent vapour) withrespective mobile phases namely toluene-acetone-formicacid (45 45 1) for phenolics toluene-acetone-formic acid(45 45 1) for flavonoids and toluene-ethyl acetate-formicacid-methanol (3 3 08 02) for tannins The plate wasdeveloped up to 90mm
The developed plate was dried by hot air to evaporatesolvents from the plateThe plate was kept in photodocumen-tation chamber and the images were captured under visiblelight UV 254 nm and UV 366 nm The developed plate wassprayed with respective spraying reagents namely Folin-Ciocalteu reagent for phenolics 1 ethanolic aluminiumchloride reagent for flavonoids and 5 ferric chloride reagentfor tannins The plates were dried at 100∘C in hot air ovenThe plate was photodocumented in visible light and UV366 nm mode using photodocumentation chamber Beforederivatization the plate was fixed in scanner stage andscanned at UV 254 nm and UV 366 nm After derivatizationthe plate was fixed in scanner stage and scanned at UV366 nm The peak table peak display and peak densitogramwere noted The software used was winCATS 134 version
3 Results
31 Phenolics Profile HPTLC separation of phenolics deter-mined high resolution and reproducible peaks in the studiedCyathea species The results determined the presence of 27different types of phenolics bands and validated 14 different119877119891 values ranged from 005 to 092 (Figure 1 Table 1) Themaximum number (11) of phenolics has been observed inthe ethanolic extract of C crinita Among the different typesof phenolics the bands with 119877119891 values 005 032 043 075and 083 demonstrated their presence in all the three studiedspecies of Cyathea The phenolic band with 119877119891 value 075confirmed the presence of quercetin in the ethanolic extractof all the three studied Cyathea species The percentage ofquercetin presence was as follows C nilgirensis (019) C
Chinese Journal of Biology 3
(a) (b) (c) (d) (e)
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0
(AU
)
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6 78
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5
67
8
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(AU
)
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(g)
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56 7
89
1011
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(AU
)
Rf
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(h)
1000
900
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0
(AU
)
Rf
Phenolic standard
000 020 040 060 080
(i)
8000
6000
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00
(AU
)
(AU
)
000
00
020
200
1000
040
060080
100
Rf
400600
800
(mm)
8000
6000
4000
2000
00
All tracks 254nm
020
20 0
040
0600 80
Rf
(j)
Figure 1 HPTLC (chromatogram) Phenolics profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard phenolic-quercetin (j) 3D display of all tracks
4 Chinese Journal of Biology
gigantea (032) andC crinita (030) HPLC analysis validatedthe quercetin occurrence in the ethanolic extracts of Cyatheaspecies with the retention times namely RT 1739min in Cgigantea RT 1809min in C nilgirensis and RT 1606min inC crinitaTheband with119877119891 value 028 validated the presenceof catechin in C nilgirensis The phenolic bands with 119877119891values 028 and 080 showed their unique presence in Cnilgirensis whereas the bands 026 and 066 displayed theiroccurrence only inC giganteaThebandswith119877119891 values 049059 and 072 expressed their existence only in C crinita
32 Flavonoids Profile Ethanolic extracts of studied Cyatheaspecies represented the presence of 28 bands and substan-tiated 13 types of flavonoids with 119877119891 values ranged from005 to 082 (Figure 2 Table 2) The maximum number (10)of flavonoids was illustrated in C crinita followed by Cnilgirensis andC gigantea (9) Flavonoidswith119877119891 values 005023 030 043 and 073 showed their presence in the threestudied species of Cyathea C nilgirensis showed the presenceof distinct band with 119877119891 value 077 The flavonoid bandswith 119877119891 values 014 and 071 displayed their unique presencein C crinita The flavonoid with 119877119891 value 018 confirmedthe presence of rutin in C gigantea The HPLC analysisconfirmed the rutin existence in the ethanolic extract of Cgiganteawith RT 1924min Rutin was failed to observe in theother two studied Cyathea species
33 Tannins Profile Mobile phases of different compositionswere tested in Cyathea species in order to obtain highresolution and reproducible peaks The results showed thepresence of 30 bands and validated 17 diverse types of tanninswith 119877119891 values ranged from 005 to 093 (Figure 3 Table 3)Themaximumnumber (13) of tannins was found inC crinitawhen compared to the other studied species C nilgirensisshowed 10 different tannins andC gigantea demonstrated thepresence of 8 different tannins Among the different typestannin with the 119877119891 values 005 009 and 082 showed theirpresence in all the three studied Cyathea species The tanninbands 024 034 066 and 073 showed their unique presencein C nilgirensis C crinita expressed three distinct bandsnamely 049 056 and 093 Gallic acid was present only inC crinita (009) with the 119877119891 value 049
4 Discussion
Chromatography is the lynchpin of phytochemistry and is thekey to obtain pure compounds for development into ther-apeuticals Separation identification and structure elucida-tion of biologically active compounds have been facilitated bycontinual development of chromatographic methods Theyalso play a fundamental role as an analytical technique forquality control and standardization of phytotherapeuticalsGenerally two approaches being used for standardizationare fingerprint analysis by HPTLCHPLC and quantificationof individual chemical markers [10] It ensures reproduciblepharmaceutical quality of herbal products CharacteristicHPTLC fingerprinting of particular plant species will not
Table 2 HPTLC flavonoids profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Unknown014 + Flavonoid 1018 + Rutin023 + + + Flavonoid 2030 + + + Flavonoid 3036 + + Flavonoid 4043 + + + Unknown049 + + Unknown060 + + Flavonoid 5068 + + Flavonoid 6071 + Unknown073 + + + Unknown077 + Unknown082 + + Unknown
only help in identification of that species but also pro-vide basic information useful for the isolation purificationand characterization of marker chemical compounds of thespecies [11] It is useful as a phytochemical marker and also agood estimator of genetic variability in plant populationsThepresence or absence of chemical constituent has been founduseful in the placement of the plant in taxonomic categoriesHPTLC profile differentiation is an important procedure [12]which produces visible chromatograms and complex infor-mation about the entire sample It also provides visualizationof the separated constituents and online identification ofthe analyte by in situ spectrum scanning and postchromato-graphic derivatization along with 119877119891 comparison with thestandard [13] HPTLCmethod can be used for phytochemicalprofiling and quantification of compounds present in plantsamples
With the increasing demand for natural products asmedicines there is an urgent need for standardization of plantproducts Chromatographic fingerprint is a rational optionto meet the need for more effective and powerful qualityassessment to traditional system of medicine throughoutthe world [14] The optimized chromatographic fingerprintis not only an alternative analytical tool for authenticationbut also an approach to express the various patterns ofchemical ingredients distributed in the plant material andto preserve such ldquodatabaserdquo for further sustainable studies[15] HPTLC results on ethanolic extracts of C nilgirensisC gigantea and C crinita provided an impressive resultdirected towards the presence of diverse type of phytochem-icals (phenolics flavonoids and tannins) The selection ofappropriate solvent system for a particular plant extract canbe achieved only by analyzing the 119877119891 values of compoundsin different solvent system The variation in 119877119891 values of thephytochemicals provides an important clue about selection ofappropriate solvent system for separation of pure compoundsby column chromatographyMixture of solvents with variablepolarity in different ratio can be used for separation of
Chinese Journal of Biology 5
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Rf
00
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(AU
)
(mm)
All tracks 366nm
(j)
Figure 2 HPTLC (chromatogram) Flavonoids profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard flavonoid-rutin (j) 3D display of all tracks
6 Chinese Journal of Biology
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Tannin standard
000 020 040 060 080
(h)
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00
200400
600800
1000
(mm)
(AU
)
8000
10000
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00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
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2 Chinese Journal of Biology
temperate regions They are considered as primitive thoughthey represent different lines of evolutionThese ferns displaygreat ecological conservatism as most species are terrestrialplants of moist forests and are intolerant to longer peri-ods of drought or frost Furthermore they show a greaterprovincialism and endemism thanmost fern groups [7]Withthe traditional healing system that is actively searching andexpanding its pharmacopoeia in order to treat a large numberof complaints an environment with great floral diversity isslipping away unlearned by a new generation of healersThe scientific and traditional communities need a resourcewhere data on the phytochemical aspects of these ferns arecollated Hence the present study was intended to studythe HPTLC fingerprint profile (phenolics flavonoids andtannins) of Cyathea nilgirensis Holttum Cyathea gigantea(Wall ex Hook) Holttum and Cyathea crinita (Hook)Copel
2 Materials and Methods
21 Collection of Plant Materials Specimens for the presentstudy were collected from various natural habitats of TamilNadu C nilgirensis were harvested in and around Kakkachistream (1725m) Kothayar Tirunelveli Hills (8∘441015840N and77∘441015840E) C gigantea from the roadsides near Nadugani(2637m) Nilgiris Hills (11∘241015840N and 76∘441015840E) and C crinitafrom the Anglade Institute of Natural History ShenbaganurKodaikanal (2195m) Palni Hills (10∘131015840N and 77∘321015840E)Western Ghats South IndiaThe plants were identified basedon the Pteridophyte Flora of the Western Ghats South IndiabyManickam and Irudayaraj [8] Herbarium specimens wereprepared and the voucher specimens were deposited in theSt Xavierrsquos College Herbarium (XCH) Palayamkottai TamilNadu India for further reference (C nilgirensis XCH 25423C gigantea XCH 25422 and C crinita XCH 25424)
22 Preparation of Extracts The collected species of Cyatheawere thoroughly washed with tap water followed by distilledwater They were blotted on the blotting paper and shadedried at room temperature in the dark The shade driedplant samples were ground to fine powder using mechanicalgrinder 30 g powdered samples were extracted successivelywith 180mL of ethanol using Soxhlet extractor for 8ndash12 h ata temperature not exceeding the boiling point The extractswere concentrated in a vacuum at 40∘C using rotary evapo-rator
23 HPTLC Analysis HPTLC studies were carried out usingthe standard method described by Wagner et al [9] 25mgof ethanolic extracts of selected Cyathea species was weighedaccurately in an electronic balance (Shimadzu) It was dis-solved in 05mL of ethanol and centrifuged at 3000 rpmfor 5min These solutions were used as test solution forphenolics flavonoids and tannins 2120583L of test solutionsand 2 120583L of standard solution were loaded as 5mm bandlength in the silica gel 60F254 TLC plate using Hamiltonsyringe and CAMAG Linomat 5 instrument The samplesloaded plate was kept in TLC twin trough developing
Table 1 HPTLC phenolics profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Phenolic 2026 + + Phenolic 4028 + Catechin032 + + + Unknown043 + + + Phenolic 5049 + Phenolic 6055 + + Unknown059 + Phenolic 7066 + Unknown072 + Phenolic 8075 + + + Quercetin080 + Unknown083 + + + Unknown092 + + Unknown
chamber (after being saturated with solvent vapour) withrespective mobile phases namely toluene-acetone-formicacid (45 45 1) for phenolics toluene-acetone-formic acid(45 45 1) for flavonoids and toluene-ethyl acetate-formicacid-methanol (3 3 08 02) for tannins The plate wasdeveloped up to 90mm
The developed plate was dried by hot air to evaporatesolvents from the plateThe plate was kept in photodocumen-tation chamber and the images were captured under visiblelight UV 254 nm and UV 366 nm The developed plate wassprayed with respective spraying reagents namely Folin-Ciocalteu reagent for phenolics 1 ethanolic aluminiumchloride reagent for flavonoids and 5 ferric chloride reagentfor tannins The plates were dried at 100∘C in hot air ovenThe plate was photodocumented in visible light and UV366 nm mode using photodocumentation chamber Beforederivatization the plate was fixed in scanner stage andscanned at UV 254 nm and UV 366 nm After derivatizationthe plate was fixed in scanner stage and scanned at UV366 nm The peak table peak display and peak densitogramwere noted The software used was winCATS 134 version
3 Results
31 Phenolics Profile HPTLC separation of phenolics deter-mined high resolution and reproducible peaks in the studiedCyathea species The results determined the presence of 27different types of phenolics bands and validated 14 different119877119891 values ranged from 005 to 092 (Figure 1 Table 1) Themaximum number (11) of phenolics has been observed inthe ethanolic extract of C crinita Among the different typesof phenolics the bands with 119877119891 values 005 032 043 075and 083 demonstrated their presence in all the three studiedspecies of Cyathea The phenolic band with 119877119891 value 075confirmed the presence of quercetin in the ethanolic extractof all the three studied Cyathea species The percentage ofquercetin presence was as follows C nilgirensis (019) C
Chinese Journal of Biology 3
(a) (b) (c) (d) (e)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
Rf
1
2
3 4
5
6 78
(f)
1
2
3
4
5
67
8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(g)
1
2
3
4
56 7
89
1011
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(h)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Phenolic standard
000 020 040 060 080
(i)
8000
6000
4000
2000
00
(AU
)
(AU
)
000
00
020
200
1000
040
060080
100
Rf
400600
800
(mm)
8000
6000
4000
2000
00
All tracks 254nm
020
20 0
040
0600 80
Rf
(j)
Figure 1 HPTLC (chromatogram) Phenolics profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard phenolic-quercetin (j) 3D display of all tracks
4 Chinese Journal of Biology
gigantea (032) andC crinita (030) HPLC analysis validatedthe quercetin occurrence in the ethanolic extracts of Cyatheaspecies with the retention times namely RT 1739min in Cgigantea RT 1809min in C nilgirensis and RT 1606min inC crinitaTheband with119877119891 value 028 validated the presenceof catechin in C nilgirensis The phenolic bands with 119877119891values 028 and 080 showed their unique presence in Cnilgirensis whereas the bands 026 and 066 displayed theiroccurrence only inC giganteaThebandswith119877119891 values 049059 and 072 expressed their existence only in C crinita
32 Flavonoids Profile Ethanolic extracts of studied Cyatheaspecies represented the presence of 28 bands and substan-tiated 13 types of flavonoids with 119877119891 values ranged from005 to 082 (Figure 2 Table 2) The maximum number (10)of flavonoids was illustrated in C crinita followed by Cnilgirensis andC gigantea (9) Flavonoidswith119877119891 values 005023 030 043 and 073 showed their presence in the threestudied species of Cyathea C nilgirensis showed the presenceof distinct band with 119877119891 value 077 The flavonoid bandswith 119877119891 values 014 and 071 displayed their unique presencein C crinita The flavonoid with 119877119891 value 018 confirmedthe presence of rutin in C gigantea The HPLC analysisconfirmed the rutin existence in the ethanolic extract of Cgiganteawith RT 1924min Rutin was failed to observe in theother two studied Cyathea species
33 Tannins Profile Mobile phases of different compositionswere tested in Cyathea species in order to obtain highresolution and reproducible peaks The results showed thepresence of 30 bands and validated 17 diverse types of tanninswith 119877119891 values ranged from 005 to 093 (Figure 3 Table 3)Themaximumnumber (13) of tannins was found inC crinitawhen compared to the other studied species C nilgirensisshowed 10 different tannins andC gigantea demonstrated thepresence of 8 different tannins Among the different typestannin with the 119877119891 values 005 009 and 082 showed theirpresence in all the three studied Cyathea species The tanninbands 024 034 066 and 073 showed their unique presencein C nilgirensis C crinita expressed three distinct bandsnamely 049 056 and 093 Gallic acid was present only inC crinita (009) with the 119877119891 value 049
4 Discussion
Chromatography is the lynchpin of phytochemistry and is thekey to obtain pure compounds for development into ther-apeuticals Separation identification and structure elucida-tion of biologically active compounds have been facilitated bycontinual development of chromatographic methods Theyalso play a fundamental role as an analytical technique forquality control and standardization of phytotherapeuticalsGenerally two approaches being used for standardizationare fingerprint analysis by HPTLCHPLC and quantificationof individual chemical markers [10] It ensures reproduciblepharmaceutical quality of herbal products CharacteristicHPTLC fingerprinting of particular plant species will not
Table 2 HPTLC flavonoids profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Unknown014 + Flavonoid 1018 + Rutin023 + + + Flavonoid 2030 + + + Flavonoid 3036 + + Flavonoid 4043 + + + Unknown049 + + Unknown060 + + Flavonoid 5068 + + Flavonoid 6071 + Unknown073 + + + Unknown077 + Unknown082 + + Unknown
only help in identification of that species but also pro-vide basic information useful for the isolation purificationand characterization of marker chemical compounds of thespecies [11] It is useful as a phytochemical marker and also agood estimator of genetic variability in plant populationsThepresence or absence of chemical constituent has been founduseful in the placement of the plant in taxonomic categoriesHPTLC profile differentiation is an important procedure [12]which produces visible chromatograms and complex infor-mation about the entire sample It also provides visualizationof the separated constituents and online identification ofthe analyte by in situ spectrum scanning and postchromato-graphic derivatization along with 119877119891 comparison with thestandard [13] HPTLCmethod can be used for phytochemicalprofiling and quantification of compounds present in plantsamples
With the increasing demand for natural products asmedicines there is an urgent need for standardization of plantproducts Chromatographic fingerprint is a rational optionto meet the need for more effective and powerful qualityassessment to traditional system of medicine throughoutthe world [14] The optimized chromatographic fingerprintis not only an alternative analytical tool for authenticationbut also an approach to express the various patterns ofchemical ingredients distributed in the plant material andto preserve such ldquodatabaserdquo for further sustainable studies[15] HPTLC results on ethanolic extracts of C nilgirensisC gigantea and C crinita provided an impressive resultdirected towards the presence of diverse type of phytochem-icals (phenolics flavonoids and tannins) The selection ofappropriate solvent system for a particular plant extract canbe achieved only by analyzing the 119877119891 values of compoundsin different solvent system The variation in 119877119891 values of thephytochemicals provides an important clue about selection ofappropriate solvent system for separation of pure compoundsby column chromatographyMixture of solvents with variablepolarity in different ratio can be used for separation of
Chinese Journal of Biology 5
(a) (b) (c) (d) (e)
1
2
3 4 5
6
78
9
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)
1
2
3 4
5
6 7 8
9
Rf
600
700
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(g)
1
2
3
45
67
8
9
10
Rf
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(h)
Rf
800
700
600
500
400
300
200
100
0
(AU
)
Flavonoid standard
000 020 040 060 080
(i)
(AU
)
8000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200
1000
400
600
800
8000
6000
4000
2000
00
(AU
)
(mm)
All tracks 366nm
(j)
Figure 2 HPTLC (chromatogram) Flavonoids profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard flavonoid-rutin (j) 3D display of all tracks
6 Chinese Journal of Biology
(a) (b) (c) (d)
1
2
3
45
6
78 910
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(e)
1
2
3
4
5 67 8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
1
2
3
4
5 6 7
8
91011
1213
000 020 040 060 080
(g)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Tannin standard
000 020 040 060 080
(h)
(AU
)
8000
10000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200400
600800
1000
(mm)
(AU
)
8000
10000
6000
4000
2000
00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Microbiology
Chinese Journal of Biology 3
(a) (b) (c) (d) (e)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
Rf
1
2
3 4
5
6 78
(f)
1
2
3
4
5
67
8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(g)
1
2
3
4
56 7
89
1011
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(h)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Phenolic standard
000 020 040 060 080
(i)
8000
6000
4000
2000
00
(AU
)
(AU
)
000
00
020
200
1000
040
060080
100
Rf
400600
800
(mm)
8000
6000
4000
2000
00
All tracks 254nm
020
20 0
040
0600 80
Rf
(j)
Figure 1 HPTLC (chromatogram) Phenolics profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard phenolic-quercetin (j) 3D display of all tracks
4 Chinese Journal of Biology
gigantea (032) andC crinita (030) HPLC analysis validatedthe quercetin occurrence in the ethanolic extracts of Cyatheaspecies with the retention times namely RT 1739min in Cgigantea RT 1809min in C nilgirensis and RT 1606min inC crinitaTheband with119877119891 value 028 validated the presenceof catechin in C nilgirensis The phenolic bands with 119877119891values 028 and 080 showed their unique presence in Cnilgirensis whereas the bands 026 and 066 displayed theiroccurrence only inC giganteaThebandswith119877119891 values 049059 and 072 expressed their existence only in C crinita
32 Flavonoids Profile Ethanolic extracts of studied Cyatheaspecies represented the presence of 28 bands and substan-tiated 13 types of flavonoids with 119877119891 values ranged from005 to 082 (Figure 2 Table 2) The maximum number (10)of flavonoids was illustrated in C crinita followed by Cnilgirensis andC gigantea (9) Flavonoidswith119877119891 values 005023 030 043 and 073 showed their presence in the threestudied species of Cyathea C nilgirensis showed the presenceof distinct band with 119877119891 value 077 The flavonoid bandswith 119877119891 values 014 and 071 displayed their unique presencein C crinita The flavonoid with 119877119891 value 018 confirmedthe presence of rutin in C gigantea The HPLC analysisconfirmed the rutin existence in the ethanolic extract of Cgiganteawith RT 1924min Rutin was failed to observe in theother two studied Cyathea species
33 Tannins Profile Mobile phases of different compositionswere tested in Cyathea species in order to obtain highresolution and reproducible peaks The results showed thepresence of 30 bands and validated 17 diverse types of tanninswith 119877119891 values ranged from 005 to 093 (Figure 3 Table 3)Themaximumnumber (13) of tannins was found inC crinitawhen compared to the other studied species C nilgirensisshowed 10 different tannins andC gigantea demonstrated thepresence of 8 different tannins Among the different typestannin with the 119877119891 values 005 009 and 082 showed theirpresence in all the three studied Cyathea species The tanninbands 024 034 066 and 073 showed their unique presencein C nilgirensis C crinita expressed three distinct bandsnamely 049 056 and 093 Gallic acid was present only inC crinita (009) with the 119877119891 value 049
4 Discussion
Chromatography is the lynchpin of phytochemistry and is thekey to obtain pure compounds for development into ther-apeuticals Separation identification and structure elucida-tion of biologically active compounds have been facilitated bycontinual development of chromatographic methods Theyalso play a fundamental role as an analytical technique forquality control and standardization of phytotherapeuticalsGenerally two approaches being used for standardizationare fingerprint analysis by HPTLCHPLC and quantificationof individual chemical markers [10] It ensures reproduciblepharmaceutical quality of herbal products CharacteristicHPTLC fingerprinting of particular plant species will not
Table 2 HPTLC flavonoids profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Unknown014 + Flavonoid 1018 + Rutin023 + + + Flavonoid 2030 + + + Flavonoid 3036 + + Flavonoid 4043 + + + Unknown049 + + Unknown060 + + Flavonoid 5068 + + Flavonoid 6071 + Unknown073 + + + Unknown077 + Unknown082 + + Unknown
only help in identification of that species but also pro-vide basic information useful for the isolation purificationand characterization of marker chemical compounds of thespecies [11] It is useful as a phytochemical marker and also agood estimator of genetic variability in plant populationsThepresence or absence of chemical constituent has been founduseful in the placement of the plant in taxonomic categoriesHPTLC profile differentiation is an important procedure [12]which produces visible chromatograms and complex infor-mation about the entire sample It also provides visualizationof the separated constituents and online identification ofthe analyte by in situ spectrum scanning and postchromato-graphic derivatization along with 119877119891 comparison with thestandard [13] HPTLCmethod can be used for phytochemicalprofiling and quantification of compounds present in plantsamples
With the increasing demand for natural products asmedicines there is an urgent need for standardization of plantproducts Chromatographic fingerprint is a rational optionto meet the need for more effective and powerful qualityassessment to traditional system of medicine throughoutthe world [14] The optimized chromatographic fingerprintis not only an alternative analytical tool for authenticationbut also an approach to express the various patterns ofchemical ingredients distributed in the plant material andto preserve such ldquodatabaserdquo for further sustainable studies[15] HPTLC results on ethanolic extracts of C nilgirensisC gigantea and C crinita provided an impressive resultdirected towards the presence of diverse type of phytochem-icals (phenolics flavonoids and tannins) The selection ofappropriate solvent system for a particular plant extract canbe achieved only by analyzing the 119877119891 values of compoundsin different solvent system The variation in 119877119891 values of thephytochemicals provides an important clue about selection ofappropriate solvent system for separation of pure compoundsby column chromatographyMixture of solvents with variablepolarity in different ratio can be used for separation of
Chinese Journal of Biology 5
(a) (b) (c) (d) (e)
1
2
3 4 5
6
78
9
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)
1
2
3 4
5
6 7 8
9
Rf
600
700
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(g)
1
2
3
45
67
8
9
10
Rf
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(h)
Rf
800
700
600
500
400
300
200
100
0
(AU
)
Flavonoid standard
000 020 040 060 080
(i)
(AU
)
8000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200
1000
400
600
800
8000
6000
4000
2000
00
(AU
)
(mm)
All tracks 366nm
(j)
Figure 2 HPTLC (chromatogram) Flavonoids profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard flavonoid-rutin (j) 3D display of all tracks
6 Chinese Journal of Biology
(a) (b) (c) (d)
1
2
3
45
6
78 910
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(e)
1
2
3
4
5 67 8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
1
2
3
4
5 6 7
8
91011
1213
000 020 040 060 080
(g)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Tannin standard
000 020 040 060 080
(h)
(AU
)
8000
10000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200400
600800
1000
(mm)
(AU
)
8000
10000
6000
4000
2000
00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 Chinese Journal of Biology
gigantea (032) andC crinita (030) HPLC analysis validatedthe quercetin occurrence in the ethanolic extracts of Cyatheaspecies with the retention times namely RT 1739min in Cgigantea RT 1809min in C nilgirensis and RT 1606min inC crinitaTheband with119877119891 value 028 validated the presenceof catechin in C nilgirensis The phenolic bands with 119877119891values 028 and 080 showed their unique presence in Cnilgirensis whereas the bands 026 and 066 displayed theiroccurrence only inC giganteaThebandswith119877119891 values 049059 and 072 expressed their existence only in C crinita
32 Flavonoids Profile Ethanolic extracts of studied Cyatheaspecies represented the presence of 28 bands and substan-tiated 13 types of flavonoids with 119877119891 values ranged from005 to 082 (Figure 2 Table 2) The maximum number (10)of flavonoids was illustrated in C crinita followed by Cnilgirensis andC gigantea (9) Flavonoidswith119877119891 values 005023 030 043 and 073 showed their presence in the threestudied species of Cyathea C nilgirensis showed the presenceof distinct band with 119877119891 value 077 The flavonoid bandswith 119877119891 values 014 and 071 displayed their unique presencein C crinita The flavonoid with 119877119891 value 018 confirmedthe presence of rutin in C gigantea The HPLC analysisconfirmed the rutin existence in the ethanolic extract of Cgiganteawith RT 1924min Rutin was failed to observe in theother two studied Cyathea species
33 Tannins Profile Mobile phases of different compositionswere tested in Cyathea species in order to obtain highresolution and reproducible peaks The results showed thepresence of 30 bands and validated 17 diverse types of tanninswith 119877119891 values ranged from 005 to 093 (Figure 3 Table 3)Themaximumnumber (13) of tannins was found inC crinitawhen compared to the other studied species C nilgirensisshowed 10 different tannins andC gigantea demonstrated thepresence of 8 different tannins Among the different typestannin with the 119877119891 values 005 009 and 082 showed theirpresence in all the three studied Cyathea species The tanninbands 024 034 066 and 073 showed their unique presencein C nilgirensis C crinita expressed three distinct bandsnamely 049 056 and 093 Gallic acid was present only inC crinita (009) with the 119877119891 value 049
4 Discussion
Chromatography is the lynchpin of phytochemistry and is thekey to obtain pure compounds for development into ther-apeuticals Separation identification and structure elucida-tion of biologically active compounds have been facilitated bycontinual development of chromatographic methods Theyalso play a fundamental role as an analytical technique forquality control and standardization of phytotherapeuticalsGenerally two approaches being used for standardizationare fingerprint analysis by HPTLCHPLC and quantificationof individual chemical markers [10] It ensures reproduciblepharmaceutical quality of herbal products CharacteristicHPTLC fingerprinting of particular plant species will not
Table 2 HPTLC flavonoids profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Unknown014 + Flavonoid 1018 + Rutin023 + + + Flavonoid 2030 + + + Flavonoid 3036 + + Flavonoid 4043 + + + Unknown049 + + Unknown060 + + Flavonoid 5068 + + Flavonoid 6071 + Unknown073 + + + Unknown077 + Unknown082 + + Unknown
only help in identification of that species but also pro-vide basic information useful for the isolation purificationand characterization of marker chemical compounds of thespecies [11] It is useful as a phytochemical marker and also agood estimator of genetic variability in plant populationsThepresence or absence of chemical constituent has been founduseful in the placement of the plant in taxonomic categoriesHPTLC profile differentiation is an important procedure [12]which produces visible chromatograms and complex infor-mation about the entire sample It also provides visualizationof the separated constituents and online identification ofthe analyte by in situ spectrum scanning and postchromato-graphic derivatization along with 119877119891 comparison with thestandard [13] HPTLCmethod can be used for phytochemicalprofiling and quantification of compounds present in plantsamples
With the increasing demand for natural products asmedicines there is an urgent need for standardization of plantproducts Chromatographic fingerprint is a rational optionto meet the need for more effective and powerful qualityassessment to traditional system of medicine throughoutthe world [14] The optimized chromatographic fingerprintis not only an alternative analytical tool for authenticationbut also an approach to express the various patterns ofchemical ingredients distributed in the plant material andto preserve such ldquodatabaserdquo for further sustainable studies[15] HPTLC results on ethanolic extracts of C nilgirensisC gigantea and C crinita provided an impressive resultdirected towards the presence of diverse type of phytochem-icals (phenolics flavonoids and tannins) The selection ofappropriate solvent system for a particular plant extract canbe achieved only by analyzing the 119877119891 values of compoundsin different solvent system The variation in 119877119891 values of thephytochemicals provides an important clue about selection ofappropriate solvent system for separation of pure compoundsby column chromatographyMixture of solvents with variablepolarity in different ratio can be used for separation of
Chinese Journal of Biology 5
(a) (b) (c) (d) (e)
1
2
3 4 5
6
78
9
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)
1
2
3 4
5
6 7 8
9
Rf
600
700
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(g)
1
2
3
45
67
8
9
10
Rf
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(h)
Rf
800
700
600
500
400
300
200
100
0
(AU
)
Flavonoid standard
000 020 040 060 080
(i)
(AU
)
8000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200
1000
400
600
800
8000
6000
4000
2000
00
(AU
)
(mm)
All tracks 366nm
(j)
Figure 2 HPTLC (chromatogram) Flavonoids profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard flavonoid-rutin (j) 3D display of all tracks
6 Chinese Journal of Biology
(a) (b) (c) (d)
1
2
3
45
6
78 910
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(e)
1
2
3
4
5 67 8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
1
2
3
4
5 6 7
8
91011
1213
000 020 040 060 080
(g)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Tannin standard
000 020 040 060 080
(h)
(AU
)
8000
10000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200400
600800
1000
(mm)
(AU
)
8000
10000
6000
4000
2000
00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Chinese Journal of Biology 5
(a) (b) (c) (d) (e)
1
2
3 4 5
6
78
9
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)
1
2
3 4
5
6 7 8
9
Rf
600
700
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(g)
1
2
3
45
67
8
9
10
Rf
600
500
400
300
200
100
0
(AU
)
000 020 040 060 080
(h)
Rf
800
700
600
500
400
300
200
100
0
(AU
)
Flavonoid standard
000 020 040 060 080
(i)
(AU
)
8000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200
1000
400
600
800
8000
6000
4000
2000
00
(AU
)
(mm)
All tracks 366nm
(j)
Figure 2 HPTLC (chromatogram) Flavonoids profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d and e) After derivatization ((d) visible light (e) UV light 366 nm) (f) peak densitogram of C nilgirensis (g) peakdensitogram of C gigantea (h) peak densitogram of C crinita (i) standard flavonoid-rutin (j) 3D display of all tracks
6 Chinese Journal of Biology
(a) (b) (c) (d)
1
2
3
45
6
78 910
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(e)
1
2
3
4
5 67 8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
1
2
3
4
5 6 7
8
91011
1213
000 020 040 060 080
(g)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Tannin standard
000 020 040 060 080
(h)
(AU
)
8000
10000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200400
600800
1000
(mm)
(AU
)
8000
10000
6000
4000
2000
00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 Chinese Journal of Biology
(a) (b) (c) (d)
1
2
3
45
6
78 910
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(e)
1
2
3
4
5 67 8
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
000 020 040 060 080
(f)1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
1
2
3
4
5 6 7
8
91011
1213
000 020 040 060 080
(g)
1000
900
800
700
600
500
400
300
200
100
0
(AU
)
Rf
Tannin standard
000 020 040 060 080
(h)
(AU
)
8000
10000
6000
4000
2000
00000
020
040
060
080
100
Rf
00
200400
600800
1000
(mm)
(AU
)
8000
10000
6000
4000
2000
00
All tracks 254nm
(i)
Figure 3 HPTLC (chromatogram) Tannins profile of Cyathea species (andashc) Before derivatization ((a) visible light (b) UV light 366 nm(c) UV light 254 nm) (d) After derivatization (visible light) (e) peak densitogram of C nilgirensis (f) peak densitogram of C gigantea (g)peak densitogram of C crinita (h) standard tannin-gallic acid (i) 3D display of all tracks
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Chinese Journal of Biology 7
Table 3 HPTLC tannins profile of studied Cyathea species
119877119891
values C nilgirensis C gigantea C crinita Assigned substance
005 + + + Tannin 1009 + + + Tannin 2015 + + Unknown022 + + Tannin 3024 + Unknown034 + Unknown040 + + Unknown049 + Gallic acid056 + Tannin 4060 + + Unknown063 + + Unknown066 + Unknown069 + + Unknown073 + Unknown082 + + + Unknown088 + + Unknown093 + Unknown
pure compound from plant extracts The HPLC analysisconfirmed the presence of quercetin and rutin in the ethano-lic extracts of Cyathea species The results of the presentstudy directly coincided with the previous observations [1617] The developed HPTLC method will provide sufficientinformation about therapeutic efficacy of the drug and alsoin the identification standardization and quality control ofstudied Cyathea species
5 Conclusion
The results of the present study revealed a better separationof individual secondary metabolites and further facilitatetheir quantitative estimation and qualitative separation ofpharmacologically active chemical compounds
Conflict of Interests
The authors declare that they have no conflict of interests
References
[1] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005
[2] WHO The Selection of Essential Drugs Second Report of theWHO Expert Committee vol 641 of WHO Technical ReportSeries World Health Organization Geneva Switzerland 1979
[3] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsSources Applications andHealth Effects CRCPress Boca RatonFla USA 2004
[4] E S Ong ldquoExtractionmethods and chemical standardization ofbotanicals and herbal preparationsrdquo Journal of ChromatographyB vol 812 no 1-2 pp 23ndash33 2004
[5] N A Farooqui A Dey G N Singh T S Easwari and M KPandey ldquoAnalytical techniques in quality evaluation of herbaldrugsrdquo Asian Journal of Pharmaceutical Research vol 4 no 3pp 112ndash117 2014
[6] R E Holttum ldquoPosing the problemsrdquo in The Phylogeny andClassification of the Ferns A C Jermy J A Crabbe and B AThomas Eds Supplement no 1 to the Botanical Journal of theLinnean Society vol 67 pp 1ndash284 Linnean Society of LondonLondon UK 1973
[7] RM Tryon and G J Gastony ldquoThe biogeography of endemismin the Cyatheaceaerdquo Fern Gazette vol 11 pp 73ndash79 1975
[8] V S Manickam and V Irudayaraj Pteridophyte Flora of theWestern Ghats South India BI Publications Private LimitedNew Delhi India 1992
[9] H Wagner S Baldt and E M Zgainski Plant Drug AnalysisSpringer Berlin Germany 1996
[10] M B Patel and S H Mishra ldquoQuantitative analysis of markerconstituent swertisin in Enicostemma hyssopifolium verdoon byRP-HPLC and HPTLCrdquo Acta Chromatographica vol 24 no 1pp 85ndash95 2012
[11] D Yadav N Tiwari and M M Gupta ldquoSimultaneous quantifi-cation of diterpenoids in Premna integrifolia using a validatedHPTLC methodrdquo Journal of Separation Science vol 34 no 3pp 286ndash291 2011
[12] D S S Kpoviessi F Gbaguidi J Gbenou et al ldquoValidation of amethod for the determination of sterols and triterpenes in theaerial part of Justicia anselliana (Nees) T Anders by capillarygas chromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 4 pp 1127ndash1135 2008
[13] Md Faiyazuddin A Rauf N Ahmad et al ldquoA validatedHPTLCmethod for determination of terbutaline sulfate in biologicalsamples application to pharmacokinetic studyrdquo Saudi Pharma-ceutical Journal vol 19 no 3 pp 185ndash191 2011
[14] L P Halinski J Szafranek B M Szafranek M Gołebiowskiand P Stepnowski ldquoChromatographic fractionation and analy-sis of themain components of eggplant (Solanummelongena L)leaf cuticular waxesrdquo Acta Chromatographica vol 21 no 1 pp127ndash137 2009
[15] S Tiwari U Bhadoriya L Saini A Gupta and S SolankildquoQuantitative analysis of glycyrrhizic acid by HPTLC in herbalformulationrdquo Asian Journal of Pharmacy amp Life Science vol 1no 2 pp 124ndash127 2011
[16] G Toker S Turkoz and N Erdemoglu ldquoHigh performanceliquid chromatographic analysis of rutin in some Turkish plantsIIrdquo Journal of Chemical Society of Pakistan vol 20 no 4 pp240ndash243 1998
[17] G Mradu S Saumyakanti M Sohini and M Arup ldquoHPLCprofiles of standard phenolics compounds present in medicinalplantsrdquo International Journal of Pharmacognosy and Phytochem-ical Research vol 4 no 3 pp 162ndash167 2012
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
