phytochemicalanalysis of the leaf, stem and seed extracts of cajanus

www.wjpps.com Vol 3, Issue 8, 2014.

694

Harris et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOCHEMICALANALYSIS OF THE LEAF, STEM AND SEED

EXTRACTS OF CAJANUS CAJAN L(DICOTYLEDONEAE:

FABACEAE)

Meena Sahu, Devshree Vermaand, K.K. Harris*

Department of Zoology, Government DB Girls’ PG College, Raipur (C.G)

ABSTRACT

Cajanus cajan L. is an important legume used as nutrient and rich

source of vitamin and protein. It is used as traditional medicine since

ancient times. Cajanus cajan L. is used for the treatment of hepatitis,

dysentery, diabetes and measles. Its leaves used to treat wounds,

malaria, bedsores, treat against hypoxic-ischemic brain damage and

alcohol-induced liver damage and also used as antioxidant, anti-

cancerous and antibacterial. Their seeds have been found to have anti-

sickling activity. This study aims to investigate the qualitative and

quantitative phytochemical analysis of leaf seed and stem extracts of

Cajanus cajan L.. Ethanol, methanol, chloroform and petroleumether

extract have been prepared using Soxhlet apparatus for the phytochemical analysis of leaves,

seeds and stems ofCajanus cajan L. Methods of Trease and Evans (1989) and Sofowora

(1993) were followed to revealed phytochemicals. Phytochemical analysis showed the

presence of saponins, tannins, alkaloids flavonoids, anthraquinones and reducing sugars,

however, terpenoids and cardiac glycosides were absent in some of the components of

Cajanus cajan L.. Quantitative phytochemical analysis of leaf, seed and stems for total

phenolic compounds were found with Folin-Ciocalteu’s reagent and revealed 16.61%, 3.82%

and 14.19% ; saponins 5.97%, 6.35% and 4.98%; tannins 0.49%, 0.23% and 0.22%;

alkaloids 2.65%, 2.65% and 2.51%; flavonoids 4.77%, 2.11% and 5.44% . The presence of

these bioactive compounds like phenol, alkaloids, flavonoids, saponins and tannins in

Cajanus cajan L. probably contributes to its medicinal properties making it widely used in

traditional medicines. Presence of these compounds is also directly linked by various

authorities to the valuable anti-sickling properties possessed by this plant.

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Article Received on 12 May 2014, Revised on 16 June 2014, Accepted on 13 July 2014

*Correspondence for Author

Dr. K.k. Harris,

Assistant Professor,

Department of Zoology,

Government DB Girls’ PG

College, Raipur (C.G)


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Keywords- Cajanus cajan L,Anti-sickling properties, Phytochemical analysis, Proximate

analysis

INTRODUCTION

Plant extract can be obtained by extraction methods involving various solvents such as water,

acetone, ethanol and methanol etc. Extracts can also be obtained from specific raw materials

such as fruit, leaf, seeds etc. These extracts have been used from times immemorial as phyto

remedy, traditionally all over the world. Many plant extracts have been used as herbal

medicines for the treatment of many diseases. Extracts are administered as syrup as well as

in the form of essential oils and creams. [1-4]Some of the plants which have shown to possess

antisickling properties are HymenocardiaacidaTul [5],Zanthoxylummacrophylla[6] and Carica

papaya. [7]Aloe vera and Pterocarpussantalinoides were found to be helpful in crisis

management. [8] Phytochemicals found in extracts of Piper guineensis, Pterocarpusosun,

Sorghum bicolor, Caryophyllala and Eugenia works as potential antisickling

agents.[9]Cajanus cajan seed[10], and HymenocardiaacidaTul[11]also shows antisickling

properties.Eucalyptuscamaldulensis, Rhoicissusdigitata, Sacoglottisgabonensis,

Mallotusoppositifolius, Adansoniadigitata, Bombaxcostatum, Bosciasenegalensis,

Entadraafricana, Hypaen thebaica and Leptadeniahastata shows antioxidant

activities.[12]Extracts of terminaliacatappaand Fagarazanthoxyloides extracts were reported to

possess potential sickling reversal properties.[13] Bruceaantidysenterica, Tabebulaspp and

Heliotropiumindicum were used in the treatment of Cancer disease.[14]Hymenocardia acida

shows antisickling property.[15, 16] Acalyphaindica, Calotropis gigantean, Crows sativus L.,

Euphorbia hirta, Ocimum sanctum L., Solanumxanthocarpum, Strychnos potatorum L.,

TerminaliabelliricaRoxb, TylophoraindicaMerrill, AdhatodavasicaNees, Apiumgraveolens

L., Boerhaviadiffusa L. Piper longum L., Piper nigrum L., Solanumnigrum, and

Solanumtrilobatum L. were used to treat respiratory diseases like asthma and chronic

obstructive pulmonary diseases.[17]Mangiferaindica, Xylopiaaethiopica, Centellaasiatica,

Ageratumconyzoides L., Carica papaya L., Garcinia kola Heckel, Ocimumgratissimum L.,

Zingiberofficinale, Thymus vulgaris L., Allium sativum L., Allium cepa L. and Piper

umbellatum were used for the treatment of cough. Carica papaya,

BryophyllumpinnatumLamOken and Aloe barbadensis L. were used for treating Asthma

.[18]Cinchona spp and Artemisia annua were shown to antimalarial effects.[19]

Terminaliacatappa, Terminaliasuperba, OlaxsubscorponoideaOliv. Cassia

fistulaandTetrapleura teraptera have shown antisickling properties. [20]Carica papaya[21],


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Plumbago zeylanica, Uvariachamae and Garcinia kola[22],Cajanus cajan

seed.[23]AsteracanthalongifoliaNees, Daemiaextensa, Euphorbia hirtaL., Euphorbiatirucalli L.,

Euphorbia neriifolia, Heliotropiumindicum L., Morus Alba L., Pitheacellobiumdulce,

Trichodesmaindicum, Curcuma amada L. and Curcuma longa L. were shos antimicrobial

activity against skin diseases caused by bacteria and fungi.[24] Nigella sativa seeds and oil

extracts were used to treat interruptions in kidney, liver, stomach, circulatory system,

respiratory system, immune system and also Cancer. Allium sativum L. and Allium cepa

shows protective effects on cancer. Punicagranatum L pomegranate fruits has been used as

antioxidant and is also used several diseases like cancer, inflammation, cardiovascular

disease, and diabetes etc. Triticumaestivumbread wheatis used to treat Cancer.[25] Extracts

from Capparisspinosa L., Artemisia herbaalbaAsso, Euphorbia macroclada L., Hibiscus and

Sabdariffa L. were used as antibiotics for treating infections caused by E.Coli. [26]Aloe

barbidensis[27],Justiciasecundavahl[28],Antandrophragmautil, Chenopodiumambrosioide,

Parquetinanigrescens and Petiveriaalliacea[29], Antandrophragmautil[30], Tremaorientalis[31],

Raphia hookeri[32],Enantiachlorantha[33], Chyrysophyllumalbidum.[34]Peumusboldus,

Agathosmabetulina, Echinacea angustifolia, Humuluslupulusand Mahoniaaquifolium extracts

were used for treating bacterial diseases.[35]Allium cepa, Allium sativum,

Amaranthuscaudatus, Althaeaofficinalis, Brassica rapa, Cichoriumintybus, Cannabis sativa

and Daturastramonium extracts have shown immense potential for the management and

treatment of various diseases. [36] Moringaoleifera seed shows in vitro cytotoxicity against

different cancer cell lines such as lung, colon and neuroblastoma. [37]MoringaoleiferaLam also

reported to posses antisickling properties.[38]Some of the plants which have shown to possess

antisickling properties are Aframomumalboviolaceum, Brideliaferruginea, Morindalucida,

Vignaunguiculata, Alchorneacordifolia, Annonasenegalensis, Cymbopogondensiflorus,

Ceibapentandra, Hymenocardiaacida, Coleus kilimandscharo, Dacryodesedulis,

Vignaunguiculata and Adansoniadigitata L. [39]

There are many studies pertaining to the phytochemical screening of plants in our country

and abroad. Studies from India include the phytochemical analysis of Momordica balsamina [40], Mentha pulegium, Ajuga reptans,Mentha longifolia and Mentha piperita [41], Carica

papaya, [42]Cassia siamea [43], Fusaea longifolia [44], Cleome nutidosperma, Emilia coccinea,

Euphorbia heterophylla, Physalis angulata, Richardia bransitensis, Scopania dulcis, Sida

acuta, Spigelia anthelmia, Stachytarpheta cayennensis and Tridax procumbens[45], Calotropis

procera [46], Jatropha curcas[47], Mombin [48], Senna alata L.[49],Aspilia africana and


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Bryophyllum pinnatum[50],Achyranthes Bidentata Blume[51], Pergularia tomentosa L.[52],

Chromolaena odorata[53], Morinda Citrifolia[54], Acalypha Linn.[55],

AspiliaMossambicensis[56], Cissus cornifolia [57], Anisopus mannii, Pavetta crassipes,

Anchomanes difformis, Vernonia blumeoides and Stachytarpheta angustifolia from Northern

Nigeria[58],Carica papaya Linn., Magnifera indica Linn., Psidium guajava Linn and Vernonia

amygdalina[59], Azadiracta indica, Colquhounia coccinea, Curcuma longa, Elsholtzia

fructicosa, Eucalyptus globules, Ocimum santrum, Rhodendron setosum and Zanthoxylum

aromatu [60], Tetracarpidium Conophorum,[61]Tigernut Cyperus esculentus[62], Vernonia

amygdalina and Talinum triangulare [63], Voacanga Africana [64],Aframomum Melegueta[65],

Sudenese medicinal plants like Acacia nilotica L. and Cassia obtusifolia L. etc.[66], Malva

Parviflora[67], Carica papaya and Parquetina nigrescens [68],Chromolaena odorata and Citrus

sinensis.[69] Asparagus racemosus, Clutia abbysinica,Clerodendrum myricoides, Ehretia

cymosia, Leucas calostachys, Toddalia asiatica, Rubia cordifolia, Spermacoce princeae,

Carrisa edulis and Ajuga remota [70], Moringa oleifera [71],Stephania glandulifera, Cuscuta

reflexa, Bergenia ciliata, Melia azadirachta, Drymaria diandra, Jasminum humile, Astilbe

rivularis, Oxalis corniculata, and Viola serpens[72],Phyllanthus Amarus[73], Silybum

Marianum [74], Hymenocardia ulmoides and Vitex ferruginea [7],Labisa pumila Benth[76],

Entandrophragma utile, Chenopodium ambrosioidesand Petiveria alliacea [77], Voacanga

globosaBlanco Merr. [78], Woodfordia fruiticosa, Adhatoda vasica, Chenopodium

ambrosoides, Viburnum cotinifolium, Euphorbia hirta, Vitex negundo, Peganum harmala,

Broussonetia papyrifera, Taraxacum officinale, Urtica dioica, Verbascum thapsus,

Caryopteris grata and Mimosa rubicaulis [79], Solanum Melongena [80], Cissus Populnea Guill [81], Piliostigma Thonningii [82],Telosma Africanum [83], Acacia Senegal [84], Euphorbia

Guyoniana [85], Moringa Oleifera [86], Trema Cannabina Lour.[87] ,AspiliaAfricana and

Tithonia Diversifolia [88], Oxytenanthera Abyssinica [89], Taraxacum Officinale [90],

Oxytenanthera Abyssinica [91], Lasia Spinosa [92], Ephedra Sinica [93], Persea Americana [94],

Lonchocarpus Cyanescens Benth[95]andMoringa Oleifera [96]were done abroad.

In our country studies pertaining to the phytochemical extracts of plantsStevia rebaudiana[97],

Helicteres Isora L[98], Dioscorea belophylla [99], Anethum graveolens, Foeniculum vulgare

and Trachyspermum ammi[100], Hygrophila spinosa [101], Corallocarpus epigaeus [102],

Mirabilis jalapa [103], Pergularia daemia [104], Pothos scandenL. [105], Urena lobata[106],

Ipomoea carnea [107],Lagenaria siceraria [108], Adhatoda vasica Nees [109], Corallocarpus

epigaeus,[110]Tephrosia purpureaLinn.[111], Celosia argentea[112], Pedalium Murex[113], Aegle


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marmelos [114], Juglans Regia [115], Ocimum sanctum L., Hyptis suaveolens L Poit., Croton

tiglium L., Tephrosia villosa L Pers., Malachra capitata L., Physalis minima L., Cleome

viscosa L., and Galphimia glauca Cav.[116], Ipomoea obscura[117], Aswagandha Withania

somnifera, Liquorice Glycyrhiza glabra, Shatavari Asparagus racemosus, Amla Emblica

officinalis, Gokhru Tribulus terrestris, Baidanka Mucuna pruriens, Koilikhia Arygyreia

nervosa, Talmuli Curculigo orchiolides[118], Stelleria media [119], Boswellia dalzielii [120],

Pterocarpus Marsupium [121], Bryophyllum pinnatum, Terminalia bellerica, Ipomea aquatica,

Tinospora cordifolia, Oldenlandia corymbosa, Ricinus communis, and Xanthium strumarium [122], Cajanus cajan Linn[123], Capparis Zeylanica Linn.[124], Woodfordia fruticosa L[125],

Juglans regia L.[126], Psidium [127], Podophyllum HexandruAnd Rheum Emodi [128], Wattakaka

Volubilis[129], Ocimum Gratissimum[130], Ficus racemosa[131], Rheum Emodi [132],

Achyranthus aspera, Parthenium hysterophorus Acalypha indica, Lindenbergia indica,

Euphorbia hirta,and Peristrophe bicalyculata[133],Aegle marmelos Bael, Annona squamosa

Sitaphal, Ficus racemosa Gular, Hibiscus rosa sinenses Jaswand and Psidium guajava

Guava[134], Indigofera aspalathoides[135], Solanum nigrum L and S.myriacanthus [136], Psoralea

corylifolia[137], Imperata Cylindrica [138], Costus Igneus [139], Cucurbita Maxima Duchense [140], Mesua ferrea Linn [141], Bt and Non-Bt Varieties of Cotton[142], Piper Nigrum [143],

Dodonaea viscose [144], MadderRubia cordifolia[145], Artemisia absinthium and Artemisia

annua.[146], Salvadora Oleoides[147], Pisonea aculeate [148], Cola nitida and Cola acuminate [149], Trewia nudiflora [150],Zanthoxylum Rhetsa[151], Murraya koenigii andCamellia Sinensisk [152], Bauhinia purpurea [153], Enhydra fluctuans, Lecuas aspera and Dillinia indica [154]

Limonia Acidissima L[155], Tridaxprocumbens Linn[156], Cyclea peltata [157], Marsilea Minuta

Linn[158],Pteris argyreae, Pteris vittata L, Pteris biaurita L, Pteris confusa and Pteris

multiaurita [159], Jasminum Multiflorum[160], Gloriossa Superba Linn., Costus Specious, and

Rauvolfia Serpentine Linn[161],Ziziphus oenoplia[162], Limonium Brasiliense[163], Curcuma

[164], Ecbolium viride and Justicia gendarussa [165], Propolis[166], Mangifera Indica [167],

Curcuma Zedoaria [168], Neoalsomitra clavigera [169], Genus Hedychium [170],Tridax

Procumbens[171], Bauhinia tomentosa L.[172]were analyzed in India.

Apart from these studies, works relating to photochemistry of plants with antisickling

properties is meager in our country. The present study aims to analyze the phytochemical

components present in the leaves, stem and seeds of a common plant Cajanus cajan L. which

has been found to possess potential antisickling properties (reversal of sickled erythrocytes in

our preliminary laboratory studies).


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MATERIALS AND METHODS

I.Collection of Plant Samples

The fresh leaves stems and seeds ofCajanus cajan L. were simultaneously collected from

cultivated farms and the open fields of Raipur district. Fresh parts of the plants were

identified and authenticated prior to phytochemical analysis. The leaves, stems and seeds

were separately cut into small bits, and air dried on shadow for two weeks. After dry they

were grinded into powdered with 1 mm size by using a Grinder machine before being

subjected to phytochemical screening.

II. Preparation of Extracts

Four solvents are used for the extraction of different parts of the plants based on their

increasing polarity. These are ethanol, methanol and chloroform and petroleum ether. 30g of

the powdered leaves, seeds and stems of Cajanus cajan L. were extracted with different

solvents in Soxhlet apparatus in 250 ml of each solvents separately for 48 hours and they

were concentrated by slow evaporation process[173]. The obtained crude extracts were kept in

closed container for preliminary qualitative phytochemical analysis.

III. Phytochemical Screening

The extract of each powdered parts of plants were used for phytochemical tests and to

identify the constituents, standard procedures were carried out as described by Trease and

Evans1989 and Sofowora 1993. Tannins, saponins, reducing sugars, alkaloids, terpenoides,

flavonoids, cardiac glycosides and anthraquinones were estimated following standard

methods.[174, 175]

A. Qualitative Analysis

a) Tannins

0.5 g of the extract was dissolved in 10 ml of distilled water, then a few drops of 1% ferric

chloride solution was added to obtain a brownish green or blue black precipitate, which

confirms the presence of tannin.

b) Saponins

0.5 g of the extract was dissolved in 5 ml distilled water. The mixture was shaken

vigorously. Formation of stable persistent froth shows the presence of saponins. A further

addition of 6 drops of olive oil while shaking forms an emulsion, confirming the presence of

saponins.


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c) Reducing sugars

1 gm of the extract was dissolved in 10 ml of distilled water. This extract was boiled with

Fehling solution A and B in test tube and colour changes were observed. Presence of brick

red colour indicated the presence of reducing sugar.

d) Alkaloids

6 ml of extract was mixed with 6 ml of 1% HCl in steam bath, then it was filtered. 1 ml of

Mayer’s reagent was added. Presence of turbidity shows presence of alkaloids. Further

addition of a few drops of olive oil to form an emulsion confirmed the presence of alkaloids.

e) Terpenoids

0.5 gm extract was dissolved in 2 ml of chloroform then 3 ml concentrated sulfuric acid was

added, a reddish brown colour in interphase indicates the presence of terpenoids.

f) Flavonoids

5 ml dilute ammonia was added to 5 ml extract and then 5 ml concentrated sulfuric acid was

added. Formation of yellow colour shows the presence of flavonoids.

g) Cardiac glycosides

2.5 g of extract was added to 2.5 ml distilled water. 1 ml glacial acetic acid containing a few

drops of ferric chloride was added then 0.5 ml of concentrated sulfuric acid was added.

Presence of brown ring at the interphase indicates the presence of deoxy sugar. A violet ring

below the brown ring was observed, while a greenish ring also appears above the brown ring,

confirming the presence of Cardiac Glycosides.

h) Anthraquinones

2.5 g extract was dissolved in 5 ml of conc. Sulfuric acid and filtered. The filtrate was

dissolved in 2.5 ml of chloroform. Chloroform layer was pipetted into a tube and 0.5 ml of

10% diluted ammonia was added. Formation of pink red or violet colour shows the presence

of anthraquinones.

i) Phenols

2 ml of extract was dissolved in 4 ml of distilled water and added few drops of 10% FeCl3.

Appearance of blue or green colour indicates presence of phenols.


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B. Quantitative Analysis

Quantitative analysis of phytochemical (alkaloids, saponins, flavonoids, phenols and tannins)

was done using standard methods. [176-183]

C. Thin Layer Chromatography

TLC was used for the conformation of the different secondary metabolites on analytical

plates. The prepared extract of leaves seeds and stems were dissolved in their respective

solvents with 1 mg/ml concentration. 10 µml of the extract were loaded on the analytical

plate (2.5 cm above from the bottom) and dried on air for thirty minutes. The spotted plates

were kept in a previously saturated developing chambers containing mobile phase and

allowed to run 3/4th of the height of the prepared plates. [184] There solvent system contains

petroleum ether: benzene: methanol (16:3:2) as mobile phase. The different bands of

chromatograms were observed under visible light and photographed. Different spraying

reagents were used for the detection of different bioactive compounds like Dragendorffs

reagent for alkaloids, Con. HCl for saponins, Ammonia solution for flavonoids, FeCl3 for

phenol, CHCl3 for Cardiac glycosides and 1 mg/ml KOH in CH3OH for anthraquinone. The

Rf values were calculated.

D. Proximate Analysis

The moisture, dry matter, total ash, total carbohydrate, total fat and protein were determined

by the following standardmethods.[185, 186] Apart from this, methods used in the proximate

analysis of plants likeAdansonia Digitata[187], Castor Seeds[188], Momordica

charantia[189], Argemone subfusiformisPapaveraceae and Urtica urens[190] , Vigna

Unguiculata [191] , fruit juice (Orange, Cashew, and mango, pineapple , pawpaw[192] ,

Telfairia occidentalis[193], Sida rhombifolia [194], Cajanus Cajan [195] , Star Apple Peel,

Pulp and Seed [196] , Fagonia cretica L., Peganum harmala L., Tribulus terrestris L.,

Chrozophora tinctoria and Ricinus communis L. [197], Eclipta alba,[198] Cajanus cajan

[199] , Annona sengalensis, [200] Jatropha CurcasArpb [201], Stevia Rebaudiana [202, 203]

Beta vulgaris, Mammillaria gracilis and Sempervivum tectorum [204], Macrotyloma

uniflorum [205], Pisum sativum [206] , Glycine Max [207] and Piper sarmentasum [208]

were also consulted and followed wherever necessary.

RESULTS

The results of present investigations on the leaves, seeds and stems of Cajanus cajan L. are

summarized below:


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I.QUALITATIVE PHYTOCHEMICAL ANALYSIS

The results of the qualitative analysis are presented in Table-01.

Tannins were present in the ethanolic, methanolic, chloroform and petroleum ether extracts of

leaves, seeds and stem, whereas the chloroform extract of leaves and seeds and petroleum

ether extract of seeds showed negative results; Saponins were absent in chloform extract of

leaves and petroleum ether extract of seeds; Reducing sugars were present in all the four

extracts of leaves, seeds and stem of Cajanus cajan; except the ethanol and methanol extract

of seeds and petroleum ether extract of leaves and stems; Alkaloids were absent only in

chloroform extract of leaves; Terpenoids were absent in the ethanolic extract of seeds,

methanolic extracts of seeds and methanolic, chloroform and petroleum ether extracts of

leaves; Cardiac glycoside were absent in ethanolic, methanolic and chloroform extracts of

seed and chloroform and petroleum ether extracts of leaves; Anthraquinones were absent in

all the four extracts of leaves, while the ethanolic and methanolic extracts of seeds and

chloroform extract of stem of Cajanus cajan L. Flavonoids and phenols were present in all

extracts of the plant.

II. QUANTITATIVE PHYTOCHEMICAL ANALYSIS

The results of the quantitative analysis are depicted in Table-16.

a) Cajanus cajan leaves: The secondary metabolites were found to be phenol 16.61%; tannins

0.49%; alkaloids, 2.65, %; flavonoids 4.77% and saponins 5.97%.

b) Cajanus cajan seeds: The secondary metabolites were found to be phenol 3.82%; tannins

0.23%; alkaloids 2.65%; flavonoids 2.11% and saponins 6.23%.

c) Cajanus cajan stem: The secondary metabolites were found to be phenol 14.19%; tannins

0.22%; alkaloid 2.51%; flavonoids 5.44% and saponins 4.98%.

Quantitative phytochemical estimation of phenol was summarized in table 16. In which the

total phenolics was determined with Folin-Ciocalteu reagent. Gallic acid was used as

standard compounds and were expressed as mg/g gallic acid equivalent using the standard

curve equation y = 0.0061x + 0.0396, R2 =0.9991, where y is absorbance at 760 nm and x is

total phenolic content in different parts of the plants. Maximum phenolic content was found

in leaves (166.16 ± 0.23 mg/g) than stems (141.93 ± 0.36 mg/g) and seeds (38.26 ± 1.53

mg/g).

For the determination of tannin standard procedure was followed by using Folin – Denis

method, the tannin concentration was determined by the standard graph of tannic acid


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solution and were expressed as mg/g tannic acid equivalent using standard curve equation y =

0.027x + 0.036, R2 =0.998, where y is absorbance at 700 nm and x is tannin content.

III. THIN LAYER CHROMATOGRAPHY (TLC)

TLC plates of Cajanus cajan leaves, seeds and stem are shown in figure 1-3. The method of

TLC involved solvent system of ethanol, methanol, Chloroform and petroleum ether extract,

in mobile phase petroleum ether: benzene: methanol (16:3:2). Number of spots and Rf values

with their detecting reagents are shown in Table no 2-13. IV. THE PROXIMATE ANALYSIS

The results of the proximate composition are presented in Table-17.

a) Cajanus cajan leaves: The proximate composition were found to be dry matter 93.68 ±

0.284; moisture 06.31 ± 0.284; ash 20.60 ± 0.114; fiber 21.82 ± 0.238; fat 13.00 ± 0.090;

protein 31.99 ± 0.070; carbohydrate 6.269 ± 0.153 and Nutritive value 236.72 ± 0.591.

b) Cajanus cajan seeds: The proximate composition were found to be dry matter 91.80 ±


protein 08.62 ± 0.035; carbohydrate 40.95 ± 0.244 and Nutritive value 333.73 ± 1.500.

c) Cajanus cajan stem: The proximate composition were found to be dry matter 93.88 ±


protein 21.34 ± 0.562; carbohydrate 8.131 ± 0.389 and Nutritive value 242.61 ± 1.569. Table 1: Phytochemical constituents of the extracts of Cajanus cajan L. the leaves,

stems and seeds.

CO

MPO

NEN

TS

ETH

AN

OL

EX

TRA

CT

MET

HA

NO

L

EXTR

AC

T

CH

LOR

OFO

RM

EX

TRA

CT

PETR

OLE

UM

ET

HER

EX

TRA

CT

1* 2 3 1 2 3 1 2 3 1 2 3 Tanins + + + + + + - - + + - + Saponins + + + + + + - + + + - + Reducing SUGARS + - + + - + + + + - + - Alkaloids + + + + + + - + + + + + Terpinoids + - + - - + - + + - + + Flavonoids + + + + + + + + + + + + Cardiac Glycosides + - + + - + - - + - + + Anthroquinon - - + - - + - + - - + + Phenols + + + + + + + + + + + +

*1= Leaves; 2= Seeds; 3= Stem; + (Positive); - (Negative)


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Thin layer chromatography of Cajanus cajan

Figure 1:TLC profiles of Cajanus cajan Leaves: A=Ethanol extract (Table-2);

B=Methanol extract (Table-3); C=Chloroform extract (Table-4); D= Petroleum ether

extract (Table-5).

Table 2: TLC of Cajanus cajan leaves ethanol extract in mobile phase petroleum ether:

benzene: methanol (16:3:2).

No.Of Bands

Rf Value Colour Of Bands In Visible Light

Spraying Reagents

Colour Of Bands Appeared

Phytoche Micals Detected

1 0.07 Dark green Con. HCl Dark brown Saponins 2 0.16 Yellow Ammonia

solution Yellow Flavonoid

3 0.51 Yellow Ammonia solution

Yellow Flavonoid

4 0.58 Light green - - 5 0.78 Dark grey Ammonia

solution Dark grey Flavonoid

6 0.84 Light grey FeCl3 Intense red Phenol 7 0.95 Light grey FeCl3 Green Phenol 8 0.99 Orange Dragendorffs

reagent Orange Alkaloid


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Table 3: TLC of Cajanus cajan leaves methanol extract in mobile phase petroleum

ether: benzene: methanol (16:3:2).

No.Of Bands

Rf Value

Colour Of Bands In Visible Light

Spraying Reagents

Colour Ofbands Appeared


1 0.10 Yellowish brown CHCl3 Orange Cardiac

glycosides 2 0.18 Dark green Con. HCl Deep brown Saponins

3 0.22 Dark grey Ammonia solution Grey Flavonoid

4 0.40 Light grey Ammonia solution Grey Flavonoid

5 0.82 - FeCl3 Intense red Phenol

6 0.99 Yellow Dragendorffs reagent Orange Alkaloids

Table 4: TLC of Cajanus cajan leaves chloroform extract in mobile phase petroleum


No.Of Bands

Rf Value


Spraying Reagents


Phytochemicais Detected

1 0.93 Yellow - - - 2 0.95 Light green FeCl3 Green Phenol

3 0.97 Grey Ammonia solution Dark grey Flavonoid

4 0.99 Yellow FeCl3 Intense red Phenol

No.Of Bands

Rf Value


Spraying Reagents


Phytochemicais Detected

1 0.93 Yellow - - - 2 0.95 Light green FeCl3 Green Phenol

3 0.97 Grey Ammonia solution Dark grey Flavonoid

4 0.99 Yellow FeCl3 Intense red Phenol Table 5: TLC of Cajanus cajan leaves petroleum ether extract in mobile phase petroleum ether: benzene: methanol (16:3:2). No. Of Bands

Rf Value


Spraying Reagents


Phytochemicals Detected

1 0.18 Light yellow Con. HCl Dark brown Saponins 2 0.34 Dark grey FeCl3 Intense red Phenol 3 0.62 Light grey FeCl3 Green Phenol 4 0.94 Dark yellow Dragendorffs



706


Figure 2:TLC profiles of Cajanus cajan Seeds: A=Ethanol extracts (Table-6); B=Methanol extracts (Table-7); C=Chloroform extracts (Table-8); D= Petroleum ether extract (Table-9). Table 6:TLC of Cajanus cajan seeds ethanol extract in mobile phase petroleum ether:

benzene: methanol (16:3:2).

No.Of Bands


Spraying Reagents



1 0.15 Dark yellow Ammonia solution

Yellow Flavonoid

2 0.24 Light yellow - - - 3 0.59 Light yellow - - - 4 0.93 Light yellow Ammonia


5 0.99 Orange Dragendorffs reagent

Orange Alkaloids

Table 7: TLC of Cajanus cajan seeds methanol extract in mobile phase petroleum


No.Of Bands

Rf Value


Spraying Reagents



1 0.05 Light brown Con. HCl Dark brown Saponins 2 0.08 Yellow Ammonia


3 0.10 Light grey Ammonia solution

Dark grey Flavonoid


707


Table 8: TLC of Cajanus cajan seeds chloroform extract in mobile phase petroleum


No.Ofbands

Rf Value


Spraying Reagents



1 0.06 Light brown Con. HCl Dark brown Saponins 2 0.11 Light blue FeCl3 Green Phenol 3 0.15 Light yellow Ammonia

solution yellow Flavonoid

4 0.19 Light blue Ammonia solution

Grey Flavonoid

5 0.29 Purple 1 mg/ml KOH in CH3OH

Purple Anthraquinone

6 0.66 Yellow - - - 7 0.83 Yellow Dragendorffs


Figure 3:TLC profiles of Cajanus cajan Stem: A=Ethanol extracts (Table-10); B=Methanol extracts (Table-11); C=Chloroform extracts (Table-12); D= Petroleum ether extract (Table-13). Table 9: TLC of Cajanus cajan seeds petroleum ether extract in mobile phase

petroleum ether: benzene: methanol (16:3:2).

No.Of Bands

Rf Value


Spraying Reagents


Phytochemicals Detected

1 0.17 Yellow - - - 2 0.53 Yellow Ammonia

solution yellow Flavonoid

3 0.99 Yellow Dragendorffs reagent

Light orange Alkaloid


708


Table 10: TLC of Cajanus cajan stems; ethanol extract in mobile phase-petroleum


No.Of Bands

Rf Value


Spraying Reagents



1 0.09 Light grey - - - 2 0.12 Yellow Ammonia


3 0.35 Light green - - - 4 0.42 Light grey Ammonia


5 0.53 Light grey Ammonia solution

Dark grey Flavonoid


Orange Alkaloid

Table 11: TLC of Cajanus cajan stems; methanol extract in mobile phase-petroleum


No.Of Bands

Rf Value


Spraying Reagents



1 0.06 Light grey Con. HCl Dark brown Saponins 2 0.10 Yellow - - - 3 0.21 Light green FeCl3 Green Phenol 4 0.33 Bluish green CHCl3 Purple Anthra

Quinine 5 0.52 Light green FeCl3 Intense red Phenol 6 0.64 Dark grey Ammonia

solution Grey Flavonoid


Alkaloid Alkaloid

Table 12: TLC of Cajanus cajan stems; chloroform extract in mobile phase-petroleum


No.of bands


Spraying Reagents



1 0.09 Dark yellow Con. HCl Dark brown Saponins 2 0.16 Light green FeCl3 Green Phenol 3 0.34 Light blue CHCl3 Purple Phenol 4 0.44 Dark green FeCl3 Green Phenol 5 0.58 Dark grey Ammonia


6 0.90 Light yellow - - - 7 0.97 Orange Dragendorffs



709


Table 13: TLC of Cajanus cajan stems petroleum ether extract in mobile phase

petroleum ether: benzene: methanol (16:3:2).

No. Of Bands

Rf Value


Spraying Reagents



1 0.29 Dark yellow 1 mg/ml KOH in CH3OH

Orange Cardiac glycoside

2 0.34 Green Con. HCl Brown Saponins 3 0.36 Green FeCl3 Green Phenol 4 0.47 Green CHCl3 Purple Anthraquinone 5 0.53 Green FeCl3 Green Phenol 6 0.64 Dark grey Ammonia

solution Grey Flavonoid

7 0.68 Light green CHCl3 Purple Anthraquinone 8 0.92 Light grey - - - 9 0.98 Orange Dragendorffs


Figure 4:Standard Curve for Phenol (Standard Concentration Curve for Tannic acid)

y = 0.027x + 0.036R² = 0.998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20 25 30

ABSO

RBAN

CE a

t 700

nm

CONCENTRATION µg/ml


710


Table 14: Absorbance of Standard compound (Gallic Acid) concentration (µg/ml)

Absorbance (mean) λ max = 760 nm.

Figure 5:Standard Curve for Phenol (Standard concentration curve of Gallic acid)

Table 15:Total phenolic contents in different parts of Hydro-alcoholic extracts of

Cajanus cajan L. Sample Concentration (µg/ml) Mean±SD.

Table 16:Percentage composition of the phytochemical constituents of leaves, seeds and

stems of Cajanus cajan L.

Concentration (µg/ml)

Absorbance (mean) λ max = 760 nm

0.8 0.0456 1.6 0.0505 3.12 0.0572 6.25 0.0786 12.5 0.1133 25 0.1937

Plant extract Concentration (µg/ml) Mean ± SD

LEAF 1000 166.16±0.23

SEED 1000 38.26±1.53

STEM 1000 141.93±0.36

CONSTITUENTS LEAF SEED STEM Alkaloid 2.65 2.65 2.51 Phenol 16.61 3.82 14.19 Flavonoid 4.77 2.11 5.44 Saponins 5.97 6.35 4.98 Tannin 0.49 0.23 0.22

y = 0.0061x + 0.0396R² = 0.9991

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 30

Abso

rban

ce 7

60 n

m

Concentration µg/ml


711


Figure 6: Graphical representation of percentage composition of phytochemicals found

in Cajanus cajan L.

Table 17:Quantitative phytochemical estimation of Cajanus cajan L. results are given as

percentage.

CONSTITUENTS ALKALOIDS PHENOL FLAVONOID SAPONIN TANNIN Leaf 2.65±0.1 16.61±0.02 4.77±0.06 5.97±0.04 0.49±0.02 Seed 2.65±0.53 3.82±0.15 2.11±0.03 6.35±0.96 0.23±0.01 Stem 2.51±0.19 14.19±0.03 5.44±0.72 4.98±0.14 0.22±0.01

The results are the mean of the percentage of triplicate estimation ± standard error.

Figure 7: Graphical representations of Proximate analysis of Cajanus cajan L. leaves, seeds

and stem.

02468

1012141618

Alkaloids Phenol Flavonoid Saponin Tannin

Leaf

Seed

Stem

0102030405060708090

100

Leaf

Seed

Stem


712


Table 18: Results of Proximate analysis of Cajanus cajan L. is given as percentage. The

results are the mean of the percentage of triplicate estimation ± SD.

% COMPOSITION LEAVES SEEDS STEM

Dry matter 93.68 ± 0.284 91.80 ± 0.229 93.88 ± 0.125 Moisture 06.31 ± 0.284 8.20 ± 0.229 06.11 ± 0.125 Ash 20.60 ± 0.114 22.11 ± 0.112 23.00 ± 0.222 Fiber 21.82 ± 0.238 05.09 ± 0.086 27.70 ± 0.360 Fat 13.00 ± 0.090 15.00 ± 0.090 14.19 ± 0.268 Protein 31.99 ± 0.070 08.62 ± 0.035 21.34 ± 0.562 Carbohydrate 6.269 ± 0.153 40.95 ± 0.244 8.131 ± 0.389 Nutritive value 236.72 ± 0.591 333.73 ± 1.500 242.61 ± 1.569

DISCUSSION

The medicinal properties possessed by plants extracts have been exploited by native people

from ancient times.[209] Phytochemicals from medicinal plants generally includes,saponins,

tannins anthraquinones, flavonoids, glycosides, etc. Some other examples of disease treating

components of plants include morphine, atropine, codeine, steroids, lactones and volatile oils.

In recent years these bioactive components are used in different forms such as infusions,

syrups, concoctions, decoctions, essential oils, ointments and creams. Many plants have been

investigated in vitro and have shown potential to cure SCD. The common examples are

Fagarazanthoxyloide[210], Cajanus cajan[9, 211, 212, 213]and Khayasenegalensis[214] in the

developing world phytomedicines could be important in the management of SCD, Some of

these plants reported are M. charantia[215], Cymbopogon citratesand Camellia sinensis[216],

Scopariadulcis[217], Aged garlic[218] and Picrorhizakurroa.[219]studied that crude aqueous

extract of Zanthoxylummacrophylla roots possessed anti-sickling properties [220, 221] showed

that Scopariadulcis can be used to cure sickle cell disease. Twelve plants were screened to

possess anti-sickle cell anemiaproperties [222] someof these plants are,

Cymbopogondensiflorus, Ceibapentandra, Dacryodesedulis, Brideliaferruginea,

Caloncobawelwitschii, and Vignaunguiculata. Khayasenegalensis contains potent

phytochemicals that have antisickling activities.Garcinia kola extracts is more effective in

membrane stabilization and used by the locals in Nigeria in the management of sickle cell

disease.[223]Phytochemical examination of the roots extract of Cissus populnea contained

steroidal glycosides and cardiac glycosides and was used for the treatment of inflammation

related diseases.[224]Pterocarpusosun, Eugenia caryophyllata and Sorghum bicolor extracts

can be used in the treatment of sickle cell disease.[225] The extract of Pterocarpussantalinoides

and Aloevera was reported in the management of sickle cell disorder.[226]It has been studied


713


that root extracts of Fagarazanthoxyloides has anti-sickling potential.[227] Doses of

Terminaliacatappa are of most importance in inducing hemolysis of human erythrocytes [228,

229, 230] studied that Scopariadulcis has a good effect on various diseases.

In the present study, results of phytochemical analysis shows the presence of alkaloids,

tannins, flavonoids and saponins in various extracts derived from leaves, stem and seeds of

Cajanus cajanL. All these phytochemicals have been shown to possess antisickling properties

by various authors, many authors have showed that saponins, carboxylic acids and flavonoids

were the active components behind the antisickling activity possessed by Hymenocardiaacida

leaves[15] . Phytochemicals detected in the extracts from various parts ofCajanus cajanL.

(Table 1) correspond to the earlier studies carried out in other plants by [9, 231, 232, 233, 234, 235,

236]also reported the presence of phenolic compounds, tannins, saponins and globulins.

CONCLUSIONS

In the light of the results of photochemical analysis of the leaves, stem and seeds of Cajanus

cajan L. we may conclude that theantisickling properties possessed by the plant is due to the

analyzed underlying phytochemicals present in the plant.

Further research towards formulation of an effective remedy, containing phytochemicals

from plants possessing antisickling properties, in various concentrations, for the relief of over

270 million Global Sickle Cell Disease patients during the “Crisis Stage” needs to be

initiated.

AKNOWLEDGEMENTS

We are thankful to Dr. Arvind Girolkar, Principal, Government DB Girls’ PG College Raipur

for providing necessary research facilities and encouragement.

Further, we sincerely thank the University Grants Commission, UGC-CRO, Bhopal, for

financial assistance in the form of various minor research projects sanctioned to the Zoology

Department of Government DB Girls’ PG College Raipur.

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