Biochemical Estimation and Antimicrobial Activities of the Extracts of Caesalpinia Sappan Linn.

Abstract

Caesalpinia sappan Linn., a traditional Indian medicinal plant used widely in oriental medicine. The plant extracts were found to be a good source of secondary metabolites, vitamins and metals. The extracts were further tested against certain human pathogenic microbes. The methanol and ethyl acetate extracts of the heartwood was found to be effective against certain pathogenic microbes. Key words: Caesalpinia sappan; Secondary metabolites; Vitamins; Antimicrobial activity DOI: http://dx.doi.org/10.3329/bjsir.v46i4.9586 BJSIR 2011; 46(4): 429-436

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Bangladesh J. Sci. Ind. Res. 46(4), 429-436, 2011
Biochemical Estimation and Antimicrobial Activities of the Extracts of
Caesalpinia Sappan Linn.
N. Senthilkumar*, S. Murugesan, N. Bhanu, S. Supriya and C. Rajeshkannan
Division of Bioprospecting, Institute of Forest Genetics and Tree Breeding, Coimbatore
Abstract
Caesalpinia sappan Linn., a traditional Indian medicinal plant used widely in oriental medicine. The plant extracts were found to be a good
source of secondary metabolites, vitamins and metals. The extracts were further tested against certain human pathogenic microbes. The
methanol and ethyl acetate extracts of the heartwood was found to be effective against certain pathogenic microbes.
Key words: Caesalpinia sappan, Secondary metabolites, Vitamins, Antimicrobial activity
Introduction
Plants have always been a common source of medicaments
due to their bioactive principles. Inadequate supply of drugs,
prohibitive cost of treatments, side effects of several allo-
pathic drugs and development of resistance to currently used
drugs for infectious diseases have led to increased emphasis
on the use of plant material as a source of medicines for a
wide variety of human ailments. Of the 2,50,0 00 higher
plant species, more than 80,000 are medicinal. India is one
of the world's 12 biodiversity centers with the presence of
over 45000 different plant species. Of these, about 15000-
20000 plants have good medicinal value. However, only
7000-7500 species are used for their medicinal values by tra-
ditional communities. In India, drugs of herbal origin have
been used in traditional systems of medicine such as Unani
and Ayurveda since ancient times. The Ayurveda system of
medicine uses about 700 species, Unani 700, Siddha 600,
Amchi 600 and modern medicine around 30 species. The
drugs are derived either from the whole plant or from differ-
ent organs, like leaves, stem, bark, root, flower, seed etc.
some drugs are prepared from exudates of plant product such
as gums, resins and latex. Caesalpinia sappan Linn. (Sappan
lignum) heartwood is being used in Kerala as herbal drink-
ing water for its antithirst, blood purifying, antidiabetic
(Moon et al., 1992; Kim et al., 1997), improvement of com-
plexion and several other properties. The plant is also being
used worldwide for a large number of traditional medicinal
purposes. Recent research confirms its anticancer, antitumor,
antimicrobial (Kim et al., 2004), antiviral, immunostimulant
(Moon et al, 1992; Mok et al., 1998), antifungal activity
(Reddy et al., 2003) and several other activities. In addition
to medicinal and aromatic properties trunk wood, stem and
leaves are also used as dyeing agent. Heartwood is also used
as a colouring agent for wine, meat, fabric and it is well
established as a safe natural colouring agent with good
medicinal value for food products, beverages and pharma-
ceuticals. The woody part contains brazilin and brasilein and
an essential oil consisting of D-a-phellandrene, ocimene,
tannin, gallic acid and saponin. Brazilin is found to be the
main constituent of the plant responsible for several of its
biological activities (Mar et al., 2003). In recent years, the
extract of Sappan Lignum (the dried heartwood of
Caesalpinia sappan L.) has been found to be a potential
immunosuppressive agent. The reported main phenolic com-
pounds in Sappan Lignum were divided into to four structur-
al sub-types: i.e. brazilin, chalcone, protosappanin and
homisoflavonoid. Among the protosappanin derivatives,
such as protosappanin B and isoprotosappanin B, 10-O-
methyprotosappanin B and 10-O-methylisoprotosappanin B,
as well as protosappanin E1 and protosappanin E2 occur as
pairs of epimers. Meanwhile, the homisoflavonoid epimers
sappanol and episappanol, 4- O-methylsappanol and 4-O-
methylepisappanol, 3'-O-methylsappanol and 3'-O-
methylepisappanol were successively isolated along with a
new compound, a 3-benzylchroman derivative 3'-deoxy-4-
O-methylepisappanol (Fu et al., 2008). Considering the
medicinal importance of C. sappan, a study on the efficacy
of C. sappan extracts on human pathogenic bacteria and fun-
gus has been made anticipating to develop antibiotic in
future.
BANGLADESH JOURNAL
OF SCIENTIFIC AND
INDUSTRIAL RESEARCH
E-mail: bjsir07@gmail.com
Corresponding author. E-mail: lings02@gmail.com
BCSIR

430 Biochemical Estimation and Antimicrobial Activities 46(4) 2011
Materials and Methods
Different parts of C. sappan such as leaves, pods, twigs and
heartwood were used for the analysis. All the plant parts
were sequentially extracted. Leaves were extracted with
water, methanol and ethyl acetate and all other tissues were
extracted in the order of water, ethyl acetate and methanol.
Estimation of secondary metabolites and vitamins
Plants secondary metabolites have recently been referred to
as phytochemicals, which are naturally occurring and bio-
logically active plant compounds that have potential disease
inhibiting capabilities (Akinmoladun et al., 2007). Hence in
this study the secondary metabolites and vitamins were esti-
mated using the following methods.
Total phenol
Principle
Phenol reacts with phosphomolybdic acid in follin-
Ciocalteau reagent in alkaline medium and produce blue
coloured complex (Molybdenum blue).
The method of Malick and Singh (1980) was followed. 0.5
ml of Folin-Coicalteau reagent, diluted with an equal volume
of distilled water before use was added to 1 ml of the alco-
hol extract in a test tube followed by 2 ml of 20% Na2CO3
and the mixture was heated on a boiling water bath for 1
minute. The blue colour was diluted to water and read at 650
nm. Reagent blank was maintained with 80% ethanol. Total
phenol was calculated keeping pyrogallol as the standard.
Estimation of Tannins
Principle
Tannin like compounds reduces phosphotungstic molybdic
acid in alkaline solution to produce a highly colored blue
solution, the intensity of which is proportional to the amount
of tannins. The intensity is measured in a spectrophotometer
at 700nm.
The method of Schanderl (1970) was followed to estimate
tannins. 0.1g of the sample was ground with 75ml of distilled
water. Heated for 30 minutes, centrifuged (2000rpm-20min).
The supernatant was used as sample after making up to
100ml. 1ml of the sample was added to 75ml water along
with 5ml of Folin-Denis reagent, 10 ml of sodium carbonate
solution and dilute to 100ml with water. A blank was pre-
pared with water instead of the sample. The absorbance was
read at 700nm after 30 minutes. A standard graph was pre-
pared by using 0-100µg tannic acid.
Quantification of Alkaloids
Principle
10mg of the sample were weighed into a 250 ml beaker and
200 ml of 20% acetic acid in ethanol was added and covered
to stand for 4 h. This was filtered and the extract was con-
centrated using a waterbath to one-quarter of the original
volume. Concentrated ammonium hydroxide was added
drop wise to the extract until the precipitation was complete.
The whole solution was allowed to settle and the precipitate
was collected by filtration and weighed (Harborne, 1973;
Obadoni and Ochuko, 2001).
Quantification of Flavonoids
Principle
10mg of the plant samples were extracted repeatedly with
100 ml of 80% aqueous methanol at room temperature. The
whole solution was filtered through Whatman filter paper
no. 42 (125 mm). The filtrate was later transferred into a cru-
cible and evaporated to dryness over a water bath and
weighed (Boham and Kocipai, 1994).
Vitamins
Determination of ascorbic acid (Vitamin C)
Ascorbic acid reduces the 2,6-dichlorophenol indophenol
dye to a colourless leuco-base. The ascorbic acid gets oxi-
dized to dehydroascorbic acid. The dehydroascorbic acid
alone reacts quantitatively and not the other reducing sub-
stances present in the sample extract (Sadasivam and
Manickam, 1996).
1g of sample was homogenized with 5ml of 4% oxalic acid.
Centrifuge and collect the liquid Transfer the supernatant in
one test tube, add 5 drops of bromine water and make up
the volume up to 10ml with 4% oxalic acid. 0.1 ml of bromi-
nated sample extract was taken and made up to 3 ml with
distilled water. To this 1 ml of DNPH (Dinitrophenyl
Hydrazine) reagent with 1-2 drops of thiourea was added.
The tubes were incubated at 37°C for 3 hrs. After incubation
7 ml of 80% sulphuric acid was added to dissolve the
orange-red osazone crystals. Measure the absorbance at
540nm.

Senthilkumar, Murugesan, Bhanu, Supriya and Rajeshkannan 431
Determination of riboflavin
Riboflavin is water soluble and photosensitive vitamin.10mg
of the sample was extracted with 10 ml of 50% ethanol solu-
tion and shaken for 1 hour. This was filtered into a 25 ml
flask; 2 ml of the extract was pipette into 25 ml volumetric
flask. 2 ml of 5% potassium permanganate and 2 ml of 30%
Hydrogen peroxide were added and allowed to stand over a
hot water bath for about 30 min. 0.4 ml of 40% sodium sul-
phate was added. This was made up to 10 ml mark and the
absorbance measured at 510 nm in a spectrophotometer
(Okwu and Josiah, 2006).
Determination of thiamin
Thiamin is one of the B-group vitamin whose deficiency
causes beri-beri. 10mg of the sample were homogenized
with ethanolic sodium hydroxide. It was filtered in to a 100
ml flask. 2 ml of the filtrate was pipette and the colour devel-
oped by addition of 2 ml of potassium dichromate and read
at 360 nm. A blank sample was prepared and the colour also
developed and read at the same (Okwu and Josiah, 2006).
Antibacterial and antifungal activity of the plant extracts
The extract of the dried heartwood samples were used for the
study. The extract of different concentrations such as 50,100
and 200 ppm was tested against many bacterial pathogens
such as Escherichia coli, Proteus vulgaris, Pseudomonas
aeruginosa, Staphylococcus epidermidis and Citrobacter
divergens causing diarrhea, urinary tract infection, intra-
venous liver infection, nosocomial blood stream infection
and neonatal meningitis respectively. The ethanolic and
aqueous extracts were tested against different fungal
pathogens such as Aspergillus flavus, Aspergillus niger and
Fusarium sps causing lung and ear disease for their antifun-
gal activity. It was demonstrated by well diffusion method
described by Bauer et al., (1966).
The disc diffusion method was used to screen the antimicro-
bial activity. In vitro antimicrobial activity was screened by
using Mueller Hinton Agar (MHA) obtained. The agar plates
were prepared and were allowed to solidify for 5 minutes
and 0.1 % inoculum suspension was swabbed uniformly and
the inoculum was allowed to dry for 5 minutes. The different
concentrations of extracts (1.25, 2.5 and 5 mg/disc) were
loaded on 4 mm sterile disc. The loaded discs were placed on
the surface of medium and the compound was allowed to dif-
fuse for 5 minutes and the plates were kept for incubation at
37°C for 24 hrs. The filter paper discs containing methanol
and ethyl acetate served as negative controls. Standards
broad spectrum antibiotics like ampicillin, chloramphenicol,
kanamycin, penicillin and Amphotericin B were used as pos-
itive controls.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) was deter-
mined, using a common broth micro dilution method in 96-
well microtiter plates (Camporese et al., 2003; NCCLS,
2001). Two fold dilutions of each extract were carried out,
starting from 16 to 0.062 mg/mL. 10 µL of the previously
prepared different methanol microbial suspensions (105
CFU/ mL) were added to each well. Plates were incubated
for 18 h at 37º C and then were examined with Elisa reader
(Awareness Tech, USA) at 620 nm and the lowest concentra-
tion of each extract showing no growth was taken as its min-
imum inhibition concentration (MIC). The solution DMSO
(100 µL/mL) served as the negative control. All the samples
were tested in triplicates to confirm the activity.
Results and Discussions
The plants form secondary metabolites, which are organic
compounds for the protection against pests and dysfunctions
in the human body, used as colouring agents, scents or attrac-
tants and as the plant's own hormones. A diet which is rich in
plant foods contain a variety of secondary metabolites and
contribute to protecting the body against cancer and cardio-
vascular illnesses (Crozier et al., 2006).
Secondary metabolites of C. sappan
Quantitative determination of secondary metabolites of C.
sappan has been made and the results are presented below in
figure 1. High quantities of alkaloids were found in various
tissues of C. sappan twig (14 mg/g) followed by the heart-
wood (10mg/g), flavonoid content was estimated to be 28
mg/g in heartwood sample followed by 21.3 mg/g in the pod.
Alkaloids and their derivatives are used for analgesic, anti-
spasmodic and antibacterial effects anticancer activity and
anti-inflammatory activity (Del-Rio et al., 1997; Okwu,
2004; Stray, 1998; Okwu and Okwu, 2004). The presence of
such high proportion of alkaloid and flavonoids seem to con-
tribute to the pharmacological and antimicrobial activity of
the plant, as reported by many researchers (Moon et al.,
1990 and 1992, Nagai et al., 1984, Hufford et al., 1975).
High values of phenolic compounds and tannins were esti-
mated in C. sappan heartwood 150mg/g and 171mg/g
respectively and thus it possesses potent anti-microbial
activity (Xu et al., 2005).

432 Biochemical Estimation and Antimicrobial Activities 46(4) 2011
Vitamins of C. sappan
All the tissues of C. sappan plant are good sources of ascor-
bic acids, riboflavin, thiamin and niacin (Figure 2). The
heartwood of C. sappan showed maximum amount of about
70.3 mg/g of ascorbic acid, 39.9 mg/g of niacin, 9.3 mg/g of
thiamine and 8.3 mg/g of riboflavin. Vitamins are required
for normal wound healing and can be efficiently used to
reverse the adverse effects of deficiencies by proper dose
administration of the extract (Okwu, 2003 and 2004). Intake
of the aqueous extracts of C.sappan heartwood may suffice
the daily recommended dose of vitamins required by man.
Metals of C. sappan
Some metals like copper, zinc, iron, magnesium, sodium,
potassium and calcium are essential trace nutrient to all high
plants and animals. In animals, including humans, metals are
found primarily in the bloodstream, as a co-factor in various
enzymes and in pigments like hemoglobin. They are needed
in trace amounts for the normal metabolism of the cell.
However in sufficient amounts; metals can be poisonous and
even fatal to organisms. C. sappan has a fair amount of cop-
per, mercury, zinc, iron, sodium and potassium of about 0.69
ppm, 1.16 ppm, 1.12 ppm, 2.02 ppm, 100 ppm and 1500 ppm
Fig. 1: Secondary metabolites of C. sappan
Fig. 2: Vitamins of C. sappan

Senthilkumar, Murugesan, Bhanu, Supriya and Rajeshkannan 433
respectively (Table I). Copper acts as a cofactor in various
enzymatic actions. The antimicrobial effects of the C.sappan
extracts may be attributed by the presence of copper ions in
it. Mercury as the main ingredient is being prescribed by the
physicians around the world for numerous ailments includ-
ing constipation, depression, child bearing and toothaches
(Mayell, 2001). Zinc is one of the most important minerals
used by the body for various functions Iron is often incorpo-
rated into the heme complex. Potassium is important in neu-
ron function and in influencing osmotic balance between
cells and the interstitial fluid. Sodium ions are necessary for
regulation of blood and body fluids, transmission of nerve
impulses, heart activity and certain metabolite functions.
Antimicrobial studies on C. sappan
Explant extracts derived from medicinal and aromatic plants
especially phenols, phenolics and polyphenolic compounds
have been claimed to have multiple biological activities
including antioxidant, anti-inflammatory, antiviral, anticar-
cinogenic, antibacterial, antifungal, antioxidant effects etc
(Halliwell et al., 1995; Halliwell, 1997; Bisignano et al.,
1999). Different extracts of heartwood of C. sappan were
examined for their antimicrobial property activity.
Methanolic and ethyl acetate extracts of the heartwood sam-
ple of C. sappan showed maximum zone of inhibition at 200
ppm against E. coli, Proteus vulgaris, Klebsiella pneumonia,
Citrobacter divergens and Pseudomonas sp. and inhibition
zone from 1.1 - 2.7cm was observed against the above men-
tioned organisms (Table II). Egwaikhide and Gimba, (2007)
evaluated the antibacterial activity of ethyl acetate extracts
of Plectranthus glandulosus and observed the zone of inhi-
bition ranging from 1.0-2.6 cm against Bacillus subtilis and
Staphylococcus aureus. The ethyl extracts of Parkia biglo-
bosa and Parkia bicolor were studied against Bacillus cercus
and Staphylococcus aureus in varying concentrations such as
50mg/ml and 100mg/ml and it was concluded that higher the
concentration of the plant extracts increased the antibacteri-
al activity Ajaiyeoba and Fadare, (2002). It is evident from
this study, that as the concentration of the various extracts of
heartwood sample were increased, there is increase in the
diameter of the inhibition zone irrespective of the organism
used. The methanol and ethyl acetate extracts were used in
this study as they were found to give better effect against the
microorganisms, a study by Lin et al., (1999) reported that
the methanol extracts of the plant provide more consistent
antimicrobial activity compared to those extracted by other
solvents. A study was performed by Chaudhary and Tariq
(2006), who indicated that Coriander sativum has inhibited
the growth of Micrococcus leuteus and Mycobacterium
smegmatus in their ethyl acetate extracts mainly due to the
presence of phenolic compounds. High values of phenolic
compounds and tannins were detected in the heartwood sam-
ple of C. sappan in our study which might have contributed
to the anti-microbial activity of the various extracts.
Table I: Metal composition of aqueous extract of C. sappan
Sl. No. Metal Amount
Detected (ppm)
1 Copper 0.69
2 Mercury 1.16
3 Zinc 1.12
4 Iron 2.02
5 Manganese ND
6 Sodium 100
7 Potassium 1500
Table II: Antibacterial activity of heartwood of C. sappan
S.No. Name of the Zone of inhibition (mm)
bacterial pathogen Control Methanolic Ethylacetate extract
extract (ppm/well) (ppm/well)
50 100 200 50 100 200
1Escherichia coli 20C06 08 11 07 10 13
2Proteus vulgaris 30A11 19 26 - - -
3Staphylococcus aureus 25P- -----
4Pseudomonas aeruginosa 22K---132025
5Klebsiella pneumonia 28K12 16 26 14 21 27
6Citrobacter divergens 27A17 18 25 09 13 19
Standards antibiotic discs of C-Chloramphenicol, A-Ampicillin, P- Penicillin and K-Kanamycin, Concentration 30 µg/disc.

434 Biochemical Estimation and Antimicrobial Activities 46(4) 2011
Table III shows the inhibitory zone profile of heartwood
sample against fungal pathogens. Fusarium sps. was found
to be resistant to the extracts of heartwood sample, it might
be susceptible to the extracts of the other tissues of C. sap-
pan. From this study it may be recorded that the methanolic
and ethyl acetate extracts of the heartwood of C. sappan
have least impact on the human pathogenic fungi.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was deter-
mined for the methanol and ethyl acetate extracts against
bacterial strains (Table IV). E. coli had an MIC of 50µg/ml
for both the methanol and ethyl acetate extracts. The
methanol extract was effective on P. vulgaris at 150µg/ml;
P. aeruginosa was sensitive to the ethyl acetate extract and
exhibited an MIC of 100µg/ml. K. pneumonia and C. diver-
gens were sensitive to both the extracts and had an MIC from
50-150µg/ml.
Conclusion
Caesalpinia sappan is one of the trees found to possess
many medicinal values. The present study therefore has pro-
vided some biochemical basis for the ethnomedicinal values
of extracts from C. sappan in the treatment and prevention of
infections like diahorea. As rich source of phytochemicals,
minerals and vitamins C. sappan could be a potential source
of useful drugs. HPLC analysis reveals the presence of
anthocyanin in C.sappan which is also a rich source of
polyphenols and hence it can be widely used in food indus
try as colouring agent. The antimicrobial studies revealed the
potential of C. sappan to be used as an efficient antibiotic
drug.
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Received : November 17, 2009;
Accepted : April 25, 2011