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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 Verma and, 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 ±
0.229; moisture 8.20 ± 0.229; ash 22.11 ± 0.112; fiber 05.09 ± 0.086; fat 15.00 ± 0.090;
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 ±
0.125; moisture 06.11 ± 0.125; ash 23.00 ± 0.222; fiber 27.70 ± 0.360; fat 14.19 ± 0.268;
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.
COMPONENTS
ETHANOL
EXTRACT
METHANOL
EXTRACT
CHLOROFO
RM
EXTRACT
PETROLEU
M ETHER
EXTRACT
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
Phytoche
Micals
Detected
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
ether: benzene: methanol (16:3:2).
No.Of
Bands Rf
Value Colour Of Bands
In Visible Light Spraying
Reagents
Colour Of
Bands
Appeared
Phytochemica
is 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 Colour Of Bands
In Visible Light Spraying
Reagents
Colour Of
Bands
Appeared
Phytochemica
is 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 Colour Of Bands
In Visible Light Spraying
Reagents Colour Of
Bands
Appeared
Phytochemic
als 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
reagent Orange Alkaloid
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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 Rf Value Colour Of
Bands In
Visible Light
Spraying
Reagents Colour Of
Bands
Appeared
Phytoche
Micals
Detected
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
solution Yellow Flavonoid
5 0.99 Orange Dragendorffs
reagent Orange Alkaloids
Table 7: TLC of Cajanus cajan seeds 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 Of
Bands
Appeared
Phytoche
Micals
Detected
1 0.05 Light brown Con. HCl Dark brown Saponins
2 0.08 Yellow Ammonia
solution Yellow Flavonoid
3 0.10 Light grey Ammonia
solution Dark grey Flavonoid
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Table 8: TLC of Cajanus cajan seeds chloroform extract in mobile phase petroleum
ether: benzene: methanol (16:3:2).
No.Ofba
nds Rf
Value Colour Of Bands
In
Visible Light
Spraying
Reagents Colour Of
Bands
Appeared
Phytoche
Micals
Detected
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
reagent Orange Alkaloid
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 Colour Of Bands
In Visible Light Spraying
Reagents Colour Of
Bands
Appeared
Phytochemic
als Detected
1 0.17 Yellow - - -
2 0.53 Yellow Ammonia
solution yellow Flavonoid
3 0.99 Yellow Dragendorffs
reagent Light orange Alkaloid
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Table 10: TLC of Cajanus cajan stems; 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 0.09 Light grey - - -
2 0.12 Yellow Ammonia
solution Yellow Flavonoid
3 0.35 Light green - - -
4 0.42 Light grey Ammonia
solution Dark grey Flavonoid
5 0.53 Light grey Ammonia
solution Dark grey Flavonoid
6 0.99 Yellow Dragendorffs
reagent Orange Alkaloid
Table 11: TLC of Cajanus cajan stems; 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 Of
Bands
Appeared
Phytoche
Micals
Detected
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
7 0.99 Yellow Dragendorffs
reagent Alkaloid Alkaloid
Table 12: TLC of Cajanus cajan stems; chloroform 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 0.09 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
solution Dark grey Flavonoid
6 0.90 Light yellow - - -
7 0.97 Orange Dragendorffs
reagent Orange Alkaloid
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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 Colour Of Bands In
Visible Light Spraying
Reagents Colour Of
Bands
Appeared
Phytoche
Micals
Detected
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
reagent Orange Alkaloid
Figure 4:Standard Curve for Phenol (Standard Concentration Curve for Tannic acid)
y = 0.027x + 0.036
R² = 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
ABSORBANCE at 700nm
CONCENTRATION µg/ml
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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 + 0.0396
R² = 0.9991
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30
Absorbance 760 nm
Concentration µg/ml
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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.
0
2
4
6
8
10
12
14
16
18
Alkaloids Phenol Flavonoid Saponin Tannin
Leaf
Seed
Stem
0
10
20
30
40
50
60
70
80
90
100
Leaf
Seed
Stem
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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
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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 phytochemical 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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