American Journal of Bio Science

2015; 3(2-1): 12-18

Published online February 26 2015 (http://www.sciencepublishinggroup.com/j/ajbio)
doi: 10.11648/j.ajbio.s.2015030201.13
ISSN: 2330-0159 (Print); ISSN: 2330-0167 (Online)

Investigation of the Medicinal Potentials of Syzygium

jambos (L.) Extract and Characterization of the Isolated
Compounds
Majidul Haque1, Mst. Marium Begum2, Moynul Hasan3, A. F. M. Towheedur Rahman4,
Md. Iftekhar Hussain5, Mohammad Mizanur Rahman5, Md. Hazrat Ali6, Md. Ashraful Islam7,
Md. Zakir Sultan1, Md. Reyad-ul- Ferdous4, Choudhury Mahmood Hasan1, *
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
2
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
3
Department of Pharmacy, Dhaka International University, Dhaka-1213, Bangladesh
4
Department of Pharmaceutical Sciences, North South University, Dhaka-1229, Bangladesh
5
Department of Pharmacy, Primeasia University, Dhaka-1213, Bangladesh
6
Department of Pharmacy, International Islamic University, Chittagong-4203, Bangladesh
7
Department of Biomedical Imaging, Faculty of Bioscience, Abo Akademi University, Turku, Finland

Email address:
cmhasan@gmail.com (C. M. Hasan)

To cite this article:

Majidul Haque, Mst. Marium Begum, Moynul Hasan, A. F. M. Towheedur Rahman, Md. Iftekhar Hussain, Mohammad Mizanur Rahman,
Md. Hazrat Ali, Md. Ashraful Islam, Md. Zakir Sultan, Md. Reyad ul Ferdous, Choudhury Mahmood Hasan. Investigation of the Medicinal
Potentials of Syzygium jambos (L.) Extract and Characterization of the Isolated Compounds. American Journal of Bio Science. Special Issue:
Pharmacological and Phytochemicals Investigation. Vol. 3, No. 2-1, 2015, pp. 12-18. doi: 10.11648/j.ajbio.s.2015030201.13

Abstract: Syzygium jambos (L.) Alstone belonging to the Myrtaceae family have been investigated for isolation of their
secondary metabolites and evaluation of their biological activities. A total of three compounds have been isolated. The isolated
compounds were identified by extensive analysis of their high resolution 1H-NMR spectroscopic data.The dried stem barks of
Syzygium jambos was extracted with methanol. The concentrated methanolic extract was then partitioned with vacuum liquid
chromatography. The first investigation was conducted with the VLC fraction-6 afforded one pentacyclic triterpenes (SJ-61),
VLC fraction-9 afforded a sitosterol (SJ-92) and VLC fraction-16 afforded a triterpene 3-nor-2,3-fridelan derivative (SJ-184).
In the brine shrimp lethality bioassay, The LC50 values were found to be 6.97, 18.07, 64.943, 247.596 and 12.75µg/ml for
crude methanolic extract, carbon tetrachloride, chloroform and n-hexane soluble fractions of methanolic extract of Syzygium
jambos respectively representing methanolic extract fraction as highly cytotoxic.

Keywords: Medicinal Plant, Extraction, Isolation, Characterization, Cytotoxicity, Biological Activity

gymnosperms) on this planet is estimated at 250,000[4], with

1. Introduction a lower level at 215,000[5] and an upper level as high as
In the plant kingdom there is a remedy for every disease[1] . 500,000[6,7]. Of these, only about 6% have been screened for
Two hundred and fifty years ago, there were few or no biologic activity, and a reported 15% have been evaluated
synthetic medicines. The plants were the main source of phytochemically[8]. Chemical diversity of secondary plant
drugs for the world's population. Today, 75% of the world's metabolites that results from plant evolution may be equal or
population, the poor 3/4ths, still relies on those plants and superior to that found in synthetic combinatorial chemical
other tools of traditional medicine[2] . Plants have provided a libraries. Success in natural products research is conditioned
source of inspiration for novel drug compounds, as plant by a careful plant selection, based on various criteria such as
derived medicines have made large contributions to human chemotaxonomic data, ethnomedical information, field
health and well-being[3] . observations or even random collection. One main strategy in
The number of higher plant species (angiosperms and the isolation of new leads consists of the so-called
 American Journal of Bio Science 2015; 3(2-1): 12-18 13

bioactivity-guided isolation, in which pharmacological or 3. Results and Discussion of the Test

biological assays are used to target the isolation of bioactive
compounds[9]. Samples of S. jambos
The work described in this dissertation is an attempt to Bioactive compounds are almost always toxic at higher
isolate and characterize the chemical constituents of dose. Thus, in vivo lethality in a simple zoological organism
medicinal plants, viz., Syzygium jambos (Fam. Myrtaceae) can be used as a convenient informant for screening and
and to evaluate the possible cytotoxic profiles of the crude fractionation in the discovery of new bioactive natural
extracts products.

2. Materials and Methods 3.1. Chemical Investigation of the Purified Crude Extract
of S. Jambos
Collection and preparation of the plant (Syzygium jambos)
material: Plant sample of Syzygium jambos were collected One steroid and two triterpenes were yielded from the
from Gazipur, Bangladesh and was identified by an expert methanolic extracts of stem barks of S. jambos. Through
taxonomist of Botany Department, University of Dhaka repeated chromatography and purification, extensive
(plant reference no. is DUSH7179). The stem barks were spectroscopic technique (proton NMR techniques) was
separated, cut into small pieces, sun dried for several days applied to determine the structure.
followed by oven dried for 24 hours at 40° C to facilitate 3.1.1. Characterization of SJ-61 as Friedelin
grinding and finally crushed into course powder.
Extraction and fractionation: About 200gram of powdered
stem barks in methanol for two weeks with occasional
shaking. The mixture was filtered, concentrated at 39˚C to
have a concentrated crude extract. 5gram of the extract was
subjected to solvent-solvent partition for investigation of the
biological activities and the remaining amount was used for
chemical investigation[10,11]. The concentrated methanol
extract (5 g) was fractionated by modified Kupchan
method[12] and the resultant partitionates i.e., petroleum ether,
carbon tetrachloride, chloroform and aqueous soluble
materials were used for different biological screenings.

2.1. Chemical Investigation of the Purified Crude Extract

of S. jambos
Figure 1. Compound SJ-61 (Friedelin)
The methanol extract was subjected to extensive
chromatographic separation. The isolated pure compounds Compound SJ-61 was obtained (Fig-1) from the VLC
were then characterized by extensive spectroscopic studies fraction 6 (15% ethyl acetate in hexane) of crude extract as
like proton NMR techniques, using CDCl3 as solvent. white crystals. It was evident as a yellow spot on TLC (Silica
gel PF254) when the developed plate was sprayed with
2.2. Brine Shrimp Lethality Assay of the Purified Crude
vanillin-sulfuric acid followed by heating at 110°C for 5-10
Extract of S. jambos
minutes. It was found to be soluble in petroleum ether,
This technique[13] was applied for the determination of hexane, ethyl acetate and chloroform.
general toxic property of the plant extractives indicating The 1H NMR spectrum (500 MHz, CDCl3) of SJ-61
bioactive materials using Artemia salina in a 1-day in vivo (Figure-2 & table-1) revealed signal for two double doublet
assay. Vincristine sulfate was used as positive control. Brine of proton intensity at δ 2.23 typical for H-2b and at δ 2.23
shrimp lethality bioassay[14] is a rapid and comprehensive typical for H-1a (1H, dd J=13.0, 7.5 Hz). A one proton
bioassay for the bioactive compounds of natural and multiplet was found for H-4 of pentacyclic triterpenoid and
synthetic origin and is considered a useful tool for one equatorial proton (Heq-2) at δ 2.37. The spectrum
preliminary assessment of toxicity. displayed another multiplate at δ 1.96 for H-1a, originated
Brine shrimp toxicity is closely correlated with 9KB from eight singlet signals in the regions 0.71 ppm, 0.86 ppm,
(human nasopharyngeal carcinoma) cytotoxicity (p=0.036 0.87 ppm, 0.94 ppm, 0.99 ppm, 1.00 ppm, 1.04 ppm and 1.17
and kappa = 0.56). ED50 values for cytotoxicities are ppm each integrating for three protons assignable to eight
generally about one-tenth the LC50 values found in the brine tertiary methyl groups at C-24, C-25, C-23, C-30, C-26, C-27,
shrimp test. Thus, it is possible to detect and then monitor the C-28 and C-29 respectively.
fractionation of cytotoxic, as well as 3PS (P388) (in vivo
murine leukaemia) active extracts using the brine shrimp
lethality bioassay[11].
14 Majidul Haque et al.: Investigation of the Medicinal Potentials of Syzygium jambos (L.) Extract and
Characterization of the Isolated Compounds

Table 1. Comparison between 1H NMR spectral data of SJ-61(500 MHz) and Friedelin in CDCl3 (Dhaka Univ. J. Sci. 59(2): 179-180, 2011 (July).

δH in ppm in CDCl3
Protons
SJ-61 Friedelin
H-1a 1.96 (1H, m) 1.97 (1H, m)
H-2b 2.29 (1H, dd, 13.0, 7.5 Hz) 2.28 (1H dd 13.2, 7.2 Hz)
H-4 2.37 (1H, m) 2.40 (1H, m)
H3-23 0.87 (3H, d 7.5Hz) 0.89 (3H, d, J=7.2 Hz)
H3-24 0.71 (3H, s) 0.74 (3H, s)
H3-25 0.86 (3H, s) 0.88 (3H, s)
H3-26 0.99 (3H, s) 1.01 (3H, s)
H3-27 1.00 (3H, s) 1.02 (3H, s)
H3-28 1.04 (3H, s) 1.06 (3H, s)
H3-29 1.17 (3H, s) 1.19 (3H, s)

Figure 2. ¹H NMR of spectrum of SJ-61 in CDCl3

 American Journal of Bio Science 2015; 3(2-1): 12-18 15

Table 2. 1H NMR spectral data of SJ-92 (500 MHz) and β-sitosterol (Dhaka Univ. J. Sci. 59(2): 179-180, 2011 (July) in CDCl3.

δH in ppm in CDCl3
Position
SJ-92 β-sitosterol
H-3 5.34 (1H d, J = 5.2 Hz) 5.33 (1H d, J = 7.0 Hz)
H-6 3.52 (1H, m) 3.51 (1H, m)
H3-18 0.67 (s) 0.67 (s)
H3-19 1.00 (s) 1.00 (s)
H3-21 0.91 (3H d, J = 6.4 Hz) 0.92 (3H d, J = 6.6 Hz)
H3-26 0.84 (3H d, J = 7.2 Hz) 0.84 (3H d, J = 7.0 Hz)
H3-27 0.82 (3H d, J = 7.2 Hz) 0.81 (3H d, J = 7.0 Hz)
H3-29 0.85 (3H t, J = 6.0 Hz) 0.85 (3H, t, J = 7.0 Hz)

Figure 3. ¹H NMR spectrum of SJ-92 in CDCl3

16 Majidul Haque et al.: Investigation of the Medicinal Potentials of Syzygium jambos (L.) Extract and
Characterization of the Isolated Compounds

3.1.2. Characterization of SJ-92 as β-Sitosterol

3.1.3. Characterization of SJ-184 as 3-nor-2,4-
29 Secofriedelan-4-oxo-2-oic Acid Derivative
28
21
26
18 25

27
19
1

3
HO 6

H
Figure 4. Compound SJ-92 (β-Sitosterol)

Compound SJ-92was obtained as needle shaped crystals

Figure 5. Compound SJ-184 (3-nor-2,4-Secofriedelan-4-oxo-2-oic Acid
(Fig-4). It was evident as a purple spot on TLC (Silica gel Derivative)
PF254) when the developed plate was sprayed with vanillin-
sulfuric acid followed by heating at 110°C for 5-10 minutes. Compound SJ-184 was obtained (Fig-5) from the VLC
It was found to be soluble in petroleum ether, hexane, ethyl fraction 16 (20% methanol in ethyl acetate) of crude extract
acetate and chloroform. as white crystals. It was evident as a purple spot on TLC
The 1H NMR spectrum (500 MHz, CDCl3) of SJ-92 (Silica gel PF254) when the developed plate was sprayed
(Figure-3 & Table-2) revealed the typical signal for the with vanillin-sulfuric acid followed by heating at 110°C for
olefinic H-3 of the steroidal skeleton was evident from a 5-10 minutes. It was found to be soluble in petroleum ether,
doublet at δ 5.34 integrating one proton. A one proton hexane, ethyl acetate and chloroform.
multiplet was found at δ 3.52. The position and multiplicity The 1H NMR spectrum (500 MHz, CDCl3) of SJ-184
of which was indicative of H-6 of the steroidal nucleus. The (Figure-6 & table-3) revealed signal for multiplet of proton
spectrum further revealed signals at δ 0.67 and δ 1.00 (3H intensity at δ 2.27 typical for H-1. Seven singlet signals at
each) assignable to two tertiary methyl groups at C-18 and C- 2.16ppm, 0.73ppm, 0.80ppm, 0.89ppm, 0.98ppm, 1.23ppm
19, respectively. The 1H NMR spectrum showed two and 0.94ppm, integrating for three protons assignable to
doublets centered at δ 0.83 (J = 7.2 Hz) and at δ 0.81 (J = 7.2 seven tertiary methyl groups at C-22, C-23, C-24, C-25, C-26,
Hz) which could be attributed to two methyl groups at C-25. C-28 and C-29 respectively.
The doublet at δ 0.91 (J = 6.4 Hz) was demonstrative of a Three proton singlet in the more deshielded region
methyl group at C-20. On the other hand, the triplet of three- (δ=2.16ppm) for methyl group at C-22. This high chemical
proton intensity at δ 0.85 could be assigned to the primary shift is due to the attachment to ketone group.
methyl group attached to C-28. From this above spectral features it is assumed that the
The above spectral features (Table-3) are in close compound have seven methyl protons, one of which is
agreement to those observed for β-sitosterol (Dhaka Univ. J. attached to a ketone group. So, it is proposed that the
Sci. 59(2): 179-180, 2011 (July). On this basis, the identity of compound SJ-184 may be 3-nor-2,3-secofriedelan derivative.
SJ-92 was confirmed as β-sitosterol. The identity of SJ-92
was further confirmed by CO-TLC with authentic sample,
previously isolated in Phytochemical Research Laboratory.
Table 3. ¹H NMRspectral data of SJ-184 as 3-nor-2,3-secofriedelan derivative (proposed) (J. Agric. Food Chem. 2006, 54, 35663571)

Protons Chemical shift

H-1 2.27 (1H, m)
H-22 2.16 (3H, s)
H-23 0.73 (3H, s)
H-24 0.80 (3H, s)
H-25 0.89 (3H, s)
H-26 0.98 (3H, s)
H-28 1.23 (3H, s)
H-29 0.94 (3H, s)
 American Journal of Bio Science 2015; 3(2-1): 12-18 17

Figure 6. ¹H NMR spectrum of SJ-184 in CDCl3

3.2. Brine Shrimp Lethality Assay o of the Purified Crude mortality of brine shrimp nauplii occurred) was determined
Extract of S. Jambos for the samples.
The methanolic extract of bark and the different
In the present bioactivity study all the crude extracts, partitionate of the methanolic extract i.e. hexane soluble
hexane, carbon tetrachloride, chloroform and aqueous soluble partitionate (HSF), carbon tetrachloride soluble partitionate
fractions of methanolic extract showed positive results (CTCSF), chloroform soluble partitionate (CSF) and aqueous
indicating that the test samples are biologically active. Each soluble partitionate (AQSF) of S.jambos were subjected to
of the test samples showed different mortality rates at brine shrimp lethality bioassay following the procedure of
different concentrations. Plotting of log of concentration Meyer et al., (1982). The lethality of the extractives to brine
versus percent mortality for all test samples showed an shrimp was determined and the results are given in Table-4.
approximate linear correlation. From the graphs, the median The lethal concentration LC50 of the test samples after 24
lethal concentration (LC50, the concentration at which 50% hr. was obtained by a plot of percentage of the shrimps died
18 Majidul Haque et al.: Investigation of the Medicinal Potentials of Syzygium jambos (L.) Extract and
Characterization of the Isolated Compounds

against the logarithm of the sample concentration (toxicant were compared to this positive control.
concentration) and the best-fit line was obtained from the The LC50 values of HSF, CTCSF, CLSF and MESSJ were
curve data by means of regression analysis. found to be 257.596µg/ml, 13.616µg/ml, 64.943 µg/ml, and
Vincristine sulfate (VS) was used as positive control and 6.97µg/ml respectively. MESF and CTCSF showed
the LC50 was found 0.451 µg/ml for VS. Compared with the significant lethality whereas CLSF showed moderate activity
negative control VS (positive control) gave significant and HSF showed least activity.
mortality and the LC50 values of the different extractives
Table 4. LC50 values of the test samples of S.jambos

Test samples Regression line R2 µg/ml)

LC50 (µ
VS y = 30.799x + 60.64 97.29 0.451
MESF y = 34.629x+20.803 0.9497 6.97
HSF y = 19.485x+3.3579 0.972 247.596
CTCSF y = 39.689x + 4.9914 0.9463 13.616
CLSF y = 35.233x-14.95 0.8681 69.734
AQSF y = 35.233x + 11.05 0.982 12.75

VS = Vincristine sulphate, MESF = Methanol soluble fraction, HSF = Hexane soluble fraction, CTCSF = Carbon tetrachloride soluble fraction, CLSF =
Chloroform soluble fraction

[4] Ayensu E.S., DeFilipps R. A., 1978, Endangered and

4. Conclusion Threatened Plants of the United States. Washington, DC:
Smithsonian Institution
Different partitionates of the methanolic extract of the
stem bark of S. jambos were investigated for isolation of the [5] Cronquist A., 1988, The Evolution and Classification of
Flowering Plants, Bronx, NY:New York Botanical Garden
potent secondary metabolites of this plant. Successive
chromatographic separation and purification of the VLC [6] Tippo O., Stern W. L., 1977, Humanistic Botany. New
partitionates of the crude methanolic extract yielded a total of York:W.W. Norton
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elucidated as β-sitosterol, friedelin and 3-nor-2,3- biodynamic compounds: Plants in the Development of
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[9] Burbaum J.J., Ohlmeyer M.H., Reader J.C., Henderson I,
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In conclusion, considering the potential bioactivity, this
[10] Md. Reyad-Ul-Ferdous, Sayma Akhter, Md. Zahirul Islam
plant can be studied extensively to find out their unexplored Khan, Md. Eshak Khan, Md. Atiqul Islam, Md. Sharif Ullah.
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[11] Reyad-ul-Ferdous M, Alam TT, Islam MA, Khan MZI,

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