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International Journal of Natural Products Research

Universal Research Publications. All rights reserved

ISSN: 2249-0353
Original Article
Alkaloid from Jatropha curcas Linn and its oral pathogenic activity
Choudhari Chetan and Dr (Mrs) Sandhya Parameswaran
Assistant Professor in Pharmacognosy, Saraswathi Vidya Bhavan`s College of Pharmacy, Sankara nagar, Sonarpada,
Dombivili (E), Maharashtra, 421203, India.
E-mail - iamsandhyap@yahoo.com. Mobile No. 9324513963
Received 08 February 2013; Accepted 25 February 2013
ABSTRACT
The fresh stems of Jatropha curcas Linn. (Euphorbiaceae) are used as chewing sticks and toothbrushes to strengthen gums,
cure bleeding, spongy gums and gum boils. This study reports the isolation and preliminary characterization of a new
alkaloid from the chloroform extract of the stems of J.curcas and its antimicrobial activity against oral pathogens. The
isolated alkaloid was tested for its purity with HPLC and HPTLC and subjected to spectral analysis. The MIC value of the
alkaloid extract against oral pathogens ranged from 0.125mg/ml to 2.5mg/ml with Lactobacillus acidophilus and
Streptococcus mutans being more susceptible. The isolated alkaloid was found to have good antibacterial activity by TLC
bioautography method.
2013 Universal Research Publications. All rights reserved
KEYWORDS: Jatropha curcas stems, alkaloidal extract, antibacterial activity, oral pathogens

INTRODUCTION: alkaloids, tannins, glycosides, terpenes etc.(6-7) Earlier

Dental plaque is formed by the colonization and
accumulation of oral microorganisms in the insoluble
glucan layer that are synthesized by glucosyltransferase
(GTase) from Streptococcus mutans. Acid forming
microorganisms, including S. mutans, in the plaque elute
lactic acid by metabolizing fructose as its carbon source,
which finally result in dental caries [1-2]. Numerous
chemicals and antibiotics like sporamycin, vancomycin,
chlorhexidine, etc. are being used for treating dental caries.
However, these chemicals possess many adverse effects
such as microorganisms getting tolerance, vomiting,
diarrhea and teeth staining. These drawbacks justify further
research and development of natural antimicrobial agents
that are safe for the host or specific for the oral pathogens
[3]
Literature reports the use of stems of Jatropha curcas as
tooth brushes to strengthen the gums and to cure bleeding,
in spongy gums, gum boils and tooth ache [4]. The plant
Jatropha curcas Linn (Fam. Euphorbiaceae) is a glabrous
erect branched shrub growing to a height of 2-5 meters. It
has stout cylindrical green branches with a visical milky or
reddish sap. The viscid juice flowing from the stem is
employed to arrest bleeding or hemorrhage from wound,
ulcer, cuts, and abrasions due to its coagulant effect (5).
The plant contains various secondary metabolites like
phytochemical study revealed the presence of alkaloids in
the stems, but none of them were isolated and
characterized. Hence present study was aimed to isolate
the alkaloid from the chloroform extract (ChE) of the stems
of Jatropha curcas and to evaluate both the extract and the
alkaloid for its oral pathogenic activity.
MATERIALS AND METHODS
Plant materials :
Stems of Jatropha curcas Linn were collected in the month
of March from Kasbe-sukene, District- Nashik,
Maharashtra, India. The plant was authenticated at
Agharkar Research institute, Pune, India and a voucher
number (AHMA-24141) was assigned.
Isolation of alkaloid C1:
The dried and powdered stems of the Jatropha curcas was
first defatted using petroleum ether (60-80C). The defatted
marc was dried, soaked in ammonia and extracted with
chloroform to obtain chloroform extract (ChE). This was
subjected to TLC and HPTLC using Silica gel 60 GF254
plate as a stationary phase and chloroform : toluene :
ethanol (4:2:2) as a mobile phase. HPTLC fingerprint
(using Camag TLC Scanner 3) showed the presence of a
compound at Rf 0.47 with maximum area under the peak.
The alkaloid labeled as C1 was isolated using preparative
TLC. The recovery at each stage was monitored by HPTLC

International Journal of Natural Products Research 2013; 2(1): 25-28

25
Figure 1: HPTLC chromatogram and wave length scanning data of CHE extract (track 1), partially purified alkaloid
(Track 2) and isolated alkaloid (Track 3).
by verifying the Rf and max of the alkaloid C1. HPLC
was done to determine the purity of the isolated alkaloid. It
was performed on isocratic Shimadzu LC system equipped
with SPD-10 AVP/10AVvp UV-Visible detector and LC-
10 ATVP solvent delivery module. The column used was
C18 (Phenomenex Luna 5u C18, 250 x 4.60mm). The
mobile phase used was methanol: water (60:40). The flow
rate of the pump was maintained at 1ml/min, sample
volume injected was 20ul and absorption was monitored at
397 nm.
Spectral studies of alkaloid C1 :
The alkaloid C1 was dissolved in methanol (1500 ppm) and
UV-Visible spectrum was recorded using a UV-Visible
Spectrophotometer ( Elico SL 159 UV-VIS). The FTIR
spectrum of alkaloid C1 was recorded on a MAGNA 550
(Nicholet Instrument Corporation USA) FTIR by KBR
pellet method in the range of 400 to 4000 cm-1 region. LC-
MS spectrum of compound C1 was recorded on 410 Prostar
Binary LC with 500 MS IT PDA Detectors (Varian Inc,
USA) using Electrospray Ionisation method.
International Journal of Natural Products Research 2013; 2(1): 25-28
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Antibacterial activity.
Bacterial strains and culture conditions :
The antimicrobial activity of ChE was evaluated against
oral pathogens viz., facultative anaerobes namely
Streptococcus mutans (MTCC 890), Streptococcus mitis
(MTCC 2695), Streptococcus anginosus (MTCC 1929),
Actinomyces viscosus (MTCC 7345) maintained on Brain-
Heart-Infusion Agar slants and Lactobacillus acidophilus
(NCIM 2903), Lactobacillus fermentum (NCIM 2166)
maintained on MRS agar slants.[1]
Agar well diffusion method for CHE:
Bacteria from 24 hours slants were suspended in saline
solution (0.9% w/v NaCl) individually till the turbidity
matches with that of Mc farland 0.5 solution (mixture of
9.9 ml of 1% H2SO4 and 0.1 ml of 1% w/v solution of
BaCl2). Antimicrobial activity was carried out by the Agar
cup assay method with ChE extract dissolved in DMSO in
the concentrations of 10%, 5%, 2.5% and 1.25% w/v.
Solvent DMSO was used as negative control while standard
Chlorhexidine (0.01%w/v) was used as a positive control.
Table I : MIC and IZD values for Chloroform extract and alkaloid C1
Test Organism Zone of inhibition (mm)
Chloroform Extract (%w/w)
10 5 2.5 1.25
L.acidophilus 27 23 21 16
L.fermentum 20 15 12
S.mutans 20 16 13
S. mitis 13 11 10
S. anginosus 13 12 10
A.viscosus 12 11 9 8
Standard : Chlorhexidine (0.01%w/w)
80 l solution of each test solution was delivered to four
wells of each agar plate. The inhibition zone diameters
(IZD) were measured on plates incubated at 37 0 C for 18
hours.
Minimum Inhibitory Concentrations (MIC) of alkaloidal
extract :
Agar cup plate method was used to determine the MIC of
the ChE. The ChE was serially double diluted to lowest
concentration and tested for its IZD value against all
bacterial strains to get the MIC value. MIC value is the
lowest concentration of the extract at which IZD value was
more than or equal to 7mm (for well of 6 mm diameter).
TLC bioautography method :
Agar plates seeded with test bacteria were prepared. The
alkaloid C1 was dissolved in chloroform (0.15%w/v) and
applied over the silica gel 60 F254 TLC plates (Merck,
Germany). TLC plates were developed in the mobile phase
chloroform: toluene: ethanol (4:2:2) and carefully dried.
The spot of alkaloid was observed and that part of the plate
was cut out, sterilized under U.V. light for half an hour and
pressed on agar plate seeded with individual
microorganisms with the help of sterile forceps. Agar plates
were kept at a temperature of 10 to 15 0C to allow the
compound to diffuse in agar base. One TLC plate without
any sample spot was developed with the same solvent
system, dried and used as a negative control. TLC plates
were removed from agar plates after one hour with the help
of sterile forceps and the plates were incubated at 37 0 C for
24 hours. After incubation, plates were observed for
inhibition zone diameter.
RESULTS AND DISCUSSIONS:
The chloroform extract (ChE) from the dried stem powder
showed positive test for the dragendroffs reagent
indicating alkaloid. The alkaloid showed an Rf of 0.57 in
TLC and HPTLC studies in the solvent system chloroform:
toluene: ethanol (4:2:1). The alkaloid was isolated by
repeated preparative chromatography and its purity was
confirmed by HPTLC and HPLC data. HPTLC wavelength
scanning data indicated the max of the alkaloid to be
397nm (Fig.1). In HPLC studies, the isolated alkaloid (C1)
showed a retention time of 4.563 minutes (Fig. 2). The
alkaloid C1, a white solid showed an [M+] at m/z =324.2
with characteristic IR bands at 1735cm-1 (carbonyl
vibrations of ester group), 1643 (carbonyl vibrations of
amide group), 3433 (OH/NH stretching vibrations), 1023,
1114 (CN and CO stretching vibrations). The chloroform
extract and the alkaloid C1 were tested for their oral
pathogenic activity against Lactobacillus acidophilus,
International Journal of Natural Products Research 2013; 2(1): 25-28
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Std MIC Alkaloid C1
(%w/w) (%w/w)
27 0.0125 19
10 21 0.05 23
11 23 0.05 22
9 24 0.25 20
9 24 0.125 15
23 0.25 13
Lactobacillus fermentum, Streptococcus mutans,
Streptococcus mitis, Streptococcus anginosus and
Actinomyces viscosus. It was found that L.acidophilus and
S.mutans were highly susceptible to the chloroform extract.
A concentration dependent activity was observed for the
chloroform extract and the MIC was found to be as low as
0.01% for L.acidophilus and 0.05% w/v for L. fermentum
and S. mutans (Table 1). A TLC bioautography method was
used for studying the antimicrobial activity of alkaloid C1
against the oral pathogens. Maximum zone of inhibition
was seen for S. mutans and minimum with A. viscosus. The
isolated alkaloid C1 also showed superior antimicrobial
activity against oral pathogens.
DISCUSSIONS
Combination of LCMS and IR spectroscopic data
confirmed the compound as high molecular weight,
showing the presence of ester, amide, ether as well as
aromatic ring in the structure. Literature reviews shows the
presence of Jatrophine and four new alkaloids of the
imidazoline class in the latex of Jatropha gossypifolia. (9)
An alkaloid tetramethylpyrazine is also reported in the
stems of Jatropha podagrica (10). Alkaloids in the stems of
Jatropha curcas is reported for the first time here. A
concentration dependent oral pathogenic activity was found
for the alkaloid extract. The isolated alkaloid C1 when
tested for antimicrobial activity by the TLC bioautography
method showed superior antimicrobial activity with
maximum zones of inhibition with S. mutans.
Figure 2: HPLC chromatogram of alkaloid C1
CONCLUSION :
This research work gives a scientific insight towards the
tradition claim of the stems as tooth brush. It can also be
suggested that the oral hygiene produced by the stems may
be partly due to the alkaloids present. The large scale
isolation and identification of this alkaloid will be the next
step in searching for new bioactive natural compounds with
antimicrobial activity against oral pathogens.
References
(1) Samaranayke L. Essential Microbiology of dentistry.
3rd ed. Elsevier, Oxford (2000) 255-270
(2) Shafer WG, Hine MK, Levy BM. Textbook of Oral
Pathology. 4th ed. HardCourt India Pvt.Ltd, India
(2004) 409-415.
(7) 2,4dione isolated from Jatropha curcas .
Phytochemistry (1999), 50: 337- 338.
(8) Talapatra SK, Mandal K, Jatrocurcin a new tetracyclic
tritepene from Jatropha curcas, Journal of Indian
Chemical Society, (1993) 70(6): 543-548.
(9) Silverstein, RM, Spectroscopic Identification of
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(2005) 1:148.
(3) Leland JC, Peter B. Natural Products from Plants. 2nd
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(4) Nadkarni KM, Indian Materia Medica. 2nd ed. Popular
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(5) Jha NK, Pande KL, Jha AK. Jatropha curcas : Jangali
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(10) Ahmad MU, Islam MR, Alkaloids of Jatropha
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Source of support: Nil; Conflict of interest: None declared

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