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446 © 2015 Indian Journal of Pharmaceutical Sciences | Published
by Wolters Kluwer - Medknow
*Address for correspondence E-mail: [email protected]
Variation in Pharmacognostic Characters and Polyphenolic
Contents among Four Species of Medicinal Plants from the Genus
Spermacoce (Rubiaceae)G. M. ANKAD, R. T. KONAKERI1, H. V. HEGDE*
AND S. ROYRegional Medical Research Centre, Nehru Nagar,
Belgaum-590 010, 1Department of Dravya Guna, Shri C. B. Guttal
Ayurvedic Medical College, Dharwad-580 008, India
Ankad, et al.: Pharmacognostic Analysis of Spermacoce
Spermacoce hispida L. is one of the important medicinal plants
used in traditional systems of medicine. It is observed that,
several times it is difficult to differentiate the plant from the
other allied species from the same genus, Spermacoce, especially,
when they are in drug form. Therefore, the present study aims to
document the differences in the pharmacognostic characters,
preliminary phytochemical analysis and polyphenolic contents from
the leaves of four species belonging to the genus Spermacoce, viz.
S. hispida L., S. mauritiana O. Gideon, S. stricta L. and S.
ocymoides Burm. Transverse section passing through the midrib with
lamina on either sides, epidermal characters, leaf constants,
organoleptic characters, physicochemical analysis, extractive
values and preliminary phytochemical analysis were carried out for
all these species. Total phenolic content by Folin‑Ciocalteu method
and total flavonoids by AlCl
3 method were also estimated from the leaves of all these
species. The results indicated that
S. hispida can be clearly differentiated from the other selected
species on the basis of size and number of epidermal cells, size of
trichomes, leaf constants, physicochemical analysis and extractive
values. However, it is also found that S. hispida possess total
phenolic content at 6.88±0.34 mg CAE/g and 9.17±0.46 mg TAE/g.
Total flavonoids was at 5.98±0.30 mg QE/g. The study will provide
information with respect to identification and differentiation
amongst selected species of genus Spermacoce.
Key words: Traditional medicine, Spermacoce, pharmacognostic
study, phytochemical, total phenolic content, total flavonoids
Plants areutilized extensively as rawdrugs formanyformulations
in traditional systems of medicine.Often, pharmacognosy is employed
as a tool tocheck thegenuinenessof the rawdrugs and
todetectadulteration/substitution of these plant materials.Despite
of several modern techniques, the QC ofthe raw drugs still rely
upon the pharmacognosticstudies by and large.According to
theWorldHealthOrganization, the macroscopic and
microscopicdescription of a medicinal plant is the first
steptowards establishing the identity and the degreeof purity of
suchmaterials and should be carriedout before any tests are
undertaken[1]. Usually,the identification of the medicinal plants
in thefield is based onmorphological features or othertraditionally
known characteristics. In several suchinstances, there is a
chanceof selecting incorrect rawdrugs/adulterants, especially
because of their similar
morphological featureswith other plants.Therefore,an extensive
microscopical and phytochemicalscreening is needed for raw drug to
avoid anyambiguity and such a study will serve also as areference
for further studies[2].
Traditionally, leavesof several speciesofSpermacoce,belonging to
family Rubiaceae, are being used invarious Indian systems of
medicine. The majorspecies among them is Spermacoce hispida
L.,which is locally known as ‘Madanaghanti’[3]. The
Research Paper
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For reprints contact: [email protected]
Accepted 03 August 2015Revised 24 January 2015Received 17 March
2014
Indian J Pharm Sci 2015;77(4):446-452
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leavesS. hispida are reported to be used in
treatingconjunctivitis, haemorrhoids, gallstones and to
relieveheadache[4]. Generally,most of these activities
areattributed to the secondarymetabolites in the plant[5].In recent
past, it is understood that, the phenoliccompounds present in
plants have a great deal ofbiologically active constituents and
therefore havebeen studied extensively. One of the
prominentproperties of the phenolics is their excellent
radicalscavenging activity[6], whereas flavonoids, a groupof
polyphenolic compounds, are known for series ofproperties such as
free radical scavenging activity,inhibition of hydrolytic and
oxidative enzyme andantiinflammatory action[7].However, no
information isavailableon the totalphenolic contents
andflavonoidsin thegenusSpermacoce.
Meantime, it was observed that, it is difficult todifferentiate
theplants and the leavesmorphologically,both in the field and in
raw form, due to itssimilaritywith other allied species of same
genusviz. S. mauritiana O. Gideon., S. stricta L. andS.
ocymoidesBurm.[3,8].Therefore, thepresent study isdesigned
toprovide informationonpharmacognosticalcharacters, preliminary
phytochemical analysis andpolyphenolic contents from the leaves of
selectedspecies from the genusSpermacoce, in the processof
standardizing parameters for correct identificationofS. hispida
leaves and for their differentiation fromother allied species of
the samegenus.
MATERIALS AND METHODS
Fresh leaves ofS. hispida,S. mauritiana,S. strictaand S.
ocymoides were collected from Belgaum,India (N 15.71369o, E
074.37958o) and wereauthenticated.Herbariawere prepared and
depositedin herbarium repository (VoucherNo.
RMRC-530,RMRC-531,RMRC-532,RMRC-533, respectively) atRMRCBelgaum
for future reference.
Transverse section of midrib and epidermal characters:Fresh
leaves of selected species ofSpermacocewereused formicroscopic
sectioning. Sharp razor bladeswere used to take transverse section
passing throughmidribwith lateral extensions of lamina on
eitherside. Phloroglucinol and conc. HCl were used toobserve
lignified tissues.Upper and lower epidermallayerswere peeled to
study the epidermal characters.
Numerous temporary and permanentmounts of thesectionsweremade
and examined[9,10].
Determination of leaf constants:Leaf constants were determined
according tostandardmethods with the help of camera lucidaafter
calibrating the microscope using stage andocularmicrometers. Leaf
constants were observedfrom leaves collected fromdifferent plants
to ensurethe variations of characters and average result
ofcharacterswerementioned[10].
Powder characteristics:The collected leaveswerewashed, shade
dried andpulverized.The powderwas then passed through
#85meshandcollectedfinepowder isused forpowdermicroscopy. Fragments
of powderwere clearedwithchloral hydrate. Phloroglucinol and
conc.HClwereused to observe lignified tissues, iodine to
observestarch grains, acetic acid and dil. H2SO4 to detectcalcium
oxalate crystals and glycerinewas used asmountant.The diagrams of
different structuresweredrawnwith thehelpof camera lucidamentioning
thescale[10].
Microphotographs:Microscopicdescriptionsof tissueswere
justifiedwithmicrophotographsmentionedwith appropriate scalebars,
photographswere takenwithOlympusBX-41microscope.
Organoleptic characters, physicochemical analysis and extractive
values:Fine powder obtained by pulverizationwas used
fororganoleptic characters to evaluate color, texture,odour and
taste. Physicochemical parametersweredone to evaluate the
percentage of loss on drying,total ashcontent, acid insoluble
ashandwater solubleash.Extracts of powdered leaveswere
preparedwithdifferent solvents for the studyof
extractivevalues[11].
Preliminary phytochemical analysis:Aqueous and ethanol
extractswere prepared to findout thepresenceof secondarymetabolites
performingvarious tests[11].
Total phenolic content:Total phenolic content (TPC)was
quantified
usingmodifiedFolin–Ciocalteumethod[6].Theassaymixturewaspreparedusing0.5mlofdistilledwater,0.125ml
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448 Indian Journal of Pharmaceutical Sciences July - August
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different concentrationsof standard tannic acid and/orcaffeic
acidwith0.125mlofFolin–Ciocalteu reagent,incubated for 10min in
dark.After 10min 1.25ml7%aq. sodiumcarbonate and1ml of
distilledwaterwas added and the reactionmixturewas incubatedin dark
for 90min at 37o. The absorbance of bluecolour was read at 760 nm
using distilled waterinstead of standards in the reactionmixture as
blankondoublebeamspectrophotometer.Similarly, extractsprepared
(10%w/v inmethanol)were alsoquantifiedand the resultswere compared
to the standard curvesand expressed asmg/tannicor caffeic acid
equivalentper gramdrypowder for the samples.
Total flavonoids:Total flavonoid (TF) contentswere quantified
usingmethod given by Luxmon-Ramma et al.[12]. Onemilliliter of
2%methanolicAlCl3was reactedwith1mlof different concentrationsof
standardquercetinfor 10 min in dark. Absorbance was measuredat 367
nm on double beam spectrophotometerusing 2% methanolic AlCl3 as
blank. Standardwas replacedwith extracts prepared (10%w/v
inmethanol) and resultswere compared to the standardcurves
obtained. The results were expressed asmg/quercetin equivalent per
gramdrypowder for thesamples and the datawas represented as showed
bySandeepet al[13].
RESULTS
Transverse section of midrib:The characters of transverse
section (TS) ofmidribwith lateral extensions of the lamina on its
eithersides are shown in fig. 1a-d.All the speciesweredorsiventral
in nature, plano convex shaped witha depression on middle of dorsal
side. DetailedTS showed rectangular shaped upper and
lowerepidermis. Lamina showed two layers of palisadeparenchyma
underneath the upper epidermis and3 to 5 rows of spongy parenchyma
were foundbeneath palisade layer. The rest of the midribwas
occupied by the cortical parenchyma withcollateral vascular bundle
embedded in themiddle.Xylem was towards center and phloem
towardsperiphery. Parenchymatous tissuewas thinwalledwith prominent
intercellular spaces.
Epidermal characters:The comparative results of upper epidermis
arementioned inTable 1 (fig. 2) and lower epidermis
are mentioned in Table 2 (fig. 3). The resultsrevealed size and
type of epidermal cells, size andtype of trichomes, type of stomata
and numberof epidermal cells. The upper epidermal cells ofS.
hispida were rectangular to polygonal shapedwhichwere similar in S.
stricta and S. ocymoides whereas S. mauritiana possessed irregular
shapewith papillae. S. hispida has shown variation insize of
epidermal cells and number of epidermalcells as compared to other
species.Trichomeswereabsent in S. hispida and were present in
otherspecies. The lower epidermal cells were
irregularshapedwithwavymargin in all species,S. hispidahas shown
variation in size of epidermal cells andnumber of epidermal cells
as compared to otherspecies. Trichomeswere present in S. hispida
andS. mauritiana andwere absent in other two species.All species
possessedRubiaceous (paracytic) stomatain both upper and lower
epidermal layer.
Leaf constants:The comparative results of leaf constants
arementioned inTable3.The leaf constantsofS.
hispidashoweddifferenceswith respect to stomatal number,stomatal
index, vein islet number, vein terminationnumber and palisade ratio
comparing with otherspecies.
Powder characteristics:Powders of all the species revealed the
presence offragmentsofupper and lower epidermal cells,
lamina,mesophyll tissue, palisade parenchyma, trichomes,annular and
spiral vessels and prismatic and acicularcrystals of calciumoxalate
(fig. 4).
Fig 1: Transverse section of midrib.Transverse section of midrib
of S. hispida (a), S. mauritiana (b), S. stricta (c), S. ocymoides
(d). UE–Upper epidermis, Pal–palisade parenchyma, Spar–spongy
parenchyma, LE–lower epidermis, Xy–xylem, Phl-phloem. Scale bar-200
μm.
ba
c d
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Organoleptic characters, physicochemical analysis and extractive
values:The results of organoleptic characters arementioned in Table
4, they were similar in allspecies. Physicochemical analysis and
extractivevalues results are mentioned in Table 5. Theresults of S.
hispida shown difference in termsof loss on drying (5.7263% w/w),
totalash content (9.6457% w/w), acid insolubleash (0.0794% w/w) and
water solubleash (7.0412% w/w) with other selected species.The
results of extractive values revealed,S. hispidashown difference in
terms of water solubleextractive (21.9649% w/w) and ethanol
solubleextractive (6.9464%w/w)withother selected species.
Preliminary phytochemical analysis:The results of tests for the
detection ofphytochemicalsarementioned inTable6.Ethanol andaqueous
extractswere treatedwith various reagentsto detect the
phytochemicals.All the species showedthe presence of alkaloids and
glycosides, tanninsand phenolic compounds, carbohydrates and
aminoacids.S. hispida andS. mauritiana showedpresenceofflavonoids
and in other two species itwas absent.Steroids andproteinswere
absent in all species.
Total phenolic content:The TPC of selected Spermacoce
specieswere expressed in terms of caffeic acid/tannicacid
equivalents using the standard curve
TABLE 1: EPIDERMAL CHARACTERS FOR UECharacters Spermacoce
hispida Spermacoce mauritiana Spermacoce stricta Spermacoce
ocymoidesType of epidermal cells Rectangle to
polygonal shapedIrregular shaped with papillae
Rectangle to polygonal shaped
Rectangle to polygonal shaped
Size of epidermal cells (µ) 149.86–368.42 200.67–367.17
164.11–228.22 98.54–213.63Type of stomata Rubiaceous Rubiaceous
Rubiaceous RubiaceousType of trichome - Uniseriate Thick walled,
uniseriate Thick walled, uniseriateSize of trichome (µ) -
50.99–202.62 59.09–82.25 85.90–110.28Epidermal cells number 175–208
192–218 325–349 400–430UE: Upper epidermis
TABLE 2: EPIDERMAL CHARACTERS FOR LECharacters Spermacoce
hispida Spermacoce mauritiana Spermacoce stricta Spermacoce
ocymoidesType of epidermal cells Irregular shaped, wavy Irregular
shaped wavy Irregular shaped, wavy Irregular shaped wavySize of
epidermal cells (µ) 133.10‑369.89 165.33‑391.64 93.50‑280.59
100.25‑209.11Type of stomata Rubiaceous Rubiaceous Rubiaceous
RubiaceousType of trichome Uniseriate Uniseriate - -Size of
trichome (µ) 200.39‑479.60 64.55‑355.50 - -Epidermal cells number
236-252 350-380 496‑520 530-560LE: Lower epidermis
Fig 2: Epidermal characters for upper epidermis.Epidermal
characters for upper epidermis of S. hispida (a), S. mauritiana
(b), S. stricta (c), S. ocymoides (d). S–Stomata, E–epidermal cell,
T–trichome. Scale bar-200 μm.
ba
c dFig 3: Epidermal characters for lower epidermis.Epidermal
characters for lower epidermis of S. hispida (a), S. mauritiana
(b), S. stricta (c), S. ocymoides (d). S–Stomata, E–epidermal cell,
T–trichome. Scale bar-200 μm.
ba
c d
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equation as shown in Table 7. Results of TPCare presented in
Table 8. TPC of S. hispidawas 6.88±0.34 mg CAE/g and 9.17±0.46
mgTAE/g. The selected Spermacoce species canbe arranged on basis of
TPC from lowest as inS.mauritiana
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to be useful in reducing obesity, to control
bloodydiarrhea,urinary infections,oliguria,bonediseaseandfracture
healing[14].Themethanol extract of the plantwas also reported for
its antibacterial activity[15] andhydroxyl radical scavenging and
nitric oxide
radicalscavengingactivities[16].Effortswerealsomadeearlierto
identify the chemical constituents ofS. hispida[17], S.
ocymoides[18] andothernon-Indian species[19] of thegenus.
In spite of its wider and known utility, thepharmacognostic
information on S. hispida incomparison with other allied species is
notavailable. Hence, the present study forms thefirst report on
comparative pharmacognostic and
phytochemical analysis of leaves from the genusSpermacoce.
The results of pharmacognostic studies revealed that,TS
ofmidrib, leaf constants (Table 3) organolepticcharacters (Table 4)
and preliminary phytochemicalanalysis (Table 6) remains almost same
for all theinvestigated species ofSpermacoce.The results
arecomparablewith theearlier reports forS. hispida[20] and S.
ocymoides[21].However, significantdifferenceswereobserved in
theepidermal characters
(Tables1and2),physicochemicalanalysisandextractivevalues(Table5)andpolyphenoliccontents
(Table8).
ThehighestTPCandTFwereobserved in the leavesofS.
ocymoides.Thepresent studygains importance,as the TF content was
linked to the antioxidantactivity inS. hispida[22].
The present study is an effort to fill the gaps in
thepharmacognostic studies of the genusSpermacoce,along with
providing the differentiating featuresbetween the allied species
for their correctidentification. These parameters, hopefully,
shallserve as quality control parameters and also help infurther
studies on better and correct utility of the
TABLE 5: PHYSICOCHEMICAL ANALYSIS AND EXTRACTIVE VALUESName of
the test (% w/w)
Spermacoce hispida
Spermacoce mauritiana
Spermacoce stricta
Spermacoce ocymoides
Loss on drying 5.7263 4.1420 6.7941 4.8378Total ash content
9.6457 9.0938 10.6420 11.3207Acid insoluble ash 0.0794 1.7031
2.7908 2.0841Water soluble ash 7.0412 2.6844 2.4599 4.2453Water
soluble extractive
21.9649 27.5432 13.5298 15.0250
Ethanol soluble extractive
6.9464 9.0976 10.2009 12.8647
TABLE 6: PRELIMINARY PHYTOCHEMICAL ANALYSISSecondary
metabolite
Tests Spermacoce hispida
Spermacoce mauritiana
Spermacoce stricta
Spermacoce ocymoides
Aqueous extract
Ethanol extract
Aqueous extract
Ethanol extract
Aqueous extract
Ethanol extract
Aqueous extract
Ethanol extract
Alkaloids Dragendorff’s −ve −ve −ve −ve −ve −ve −ve −veHager’s
+ve +ve +ve +ve +ve +ve +ve −veWagner’ +ve −ve +ve −ve +ve −ve +ve
−ve
Glycosides Legal’s +ve +ve +ve −ve +ve −ve +ve −veDeoxysugars
+ve +ve +ve +ve −ve +ve +ve +ve
Flavonoids Shinoda’s −ve +ve −ve +ve −ve +ve −ve +veTannins and
phenolic compounds
5% FeCl3 −ve +ve −ve +ve −ve +ve −ve +veLead acetate −ve +ve −ve
+ve −ve +ve −ve +veBromine water −ve +ve −ve +ve −ve +ve −ve
+veDilute HNO3 −ve +ve −ve +ve −ve +ve −ve +ve
Steroids Salkowski reaction −ve −ve −ve −ve −ve −ve −ve
−veLiebermann‑Burchard +ve −ve +ve −ve +ve −ve +ve −ve
Carbohydrates Molisch’s −ve +ve +ve +ve −ve +ve −ve +veFehling’s
−ve +ve +ve +ve −ve +ve −ve +veBenedict’s −ve +ve +ve +ve −ve +ve
−ve +veBarfoed’s +ve +ve +ve −ve −ve +ve −ve +veSelwinoff’s −ve +ve
−ve +ve −ve +ve −ve +ve
Proteins Biuret’s −ve −ve −ve −ve −ve −ve −ve −veMillion’s −ve
−ve −ve −ve −ve −ve −ve −ve
Amino acids Ninhydrin −ve +ve −ve +ve −ve +ve −ve +veTest for
tryptophan −ve +ve −ve +ve −ve +ve −ve -ve
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intendedplant from the genusSpermacoce accordingto traditional
systemofmedicine.
Acknowledgements:Authorsare indebted to the
IndianCouncilofMedicalResearch (ICMR) for funding the study
throughthe intramural funds
ofRegionalMedicalResearchCentre,Belgaum.Authorsare thankful
toMr.BhoopalTalawar, LabAttendant, RMRCBelgaum, for
hisassistance.
Financial support and sponsorship:The IndianCouncil
ofMedicalResearch for fundingthe study through the intramural funds
ofRegionalMedicalResearchCentre,Belgaum.
Conflicts of interest:There are no conflicts of interest.
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TABLE 7: STANDARD CURVE EQUATION OF TPC AND TFActivity
Concentration
range (μg/ml)Regression equation
Correlation coefficient (R2)
TPCCaffeic acid 10–800 y=0.0029x−0.0458 0.9940Tannic acid 10–400
y=0.0022x+0.0225 0.9977
TFQuercetin 10–400 y=0.0059x−0.0506 0.9914
TPC: Total phenolic content, TF: total flavonoids
TABLE 8: TPC AND TF FROM LEAVESActivity Spermacoce
hispidaSpermacoce mauritiana
Spermacoce stricta
Spermacoce ocymoides
TPCCAE (mg/g) 6.88±0.34 6.67±0.33 7.08±0.35 8.17±0.41TAE (mg/g)
9.17±0.46 8.90±0.45 9.44±0.47 10.88±0.54
TFQE (mg/g) 5.98±0.30 5.62±0.28 6.18±0.31 6.31±0.32
Figures in tables are represented as mean of three readings±SD.
TPC: Total phenolic content, TF: total flavonoids, TAE: tannic acid
equivalent, CAE: caffeic acid equivalent, QE: quercetin equivalent,
SD: standard deviation
