Bioassay-Guided Isolation and Partial Characterization of the

Bioassay-Guided Isolation and Partial
Characterization of the Antibacterial Components
from Jasminum sambac Extract
by
Maria Katrina Diana M. Cruz
A Thesis Submitted
to the School of Chemical Engineering and Chemistry
in Partial Fulfillment of the Requirements for the Degree
Bachelor of Science in Chemistry
Mapúa Institute of Technology
May 2006
APPROVAL SHEET
This is to certify that we have supervised the preparation of and read the thesis entitled
Bioassay-Guided Isolation and Partial Characterization of the Antibacterial
Components from Jasminum sambac Extract prepared by Maria Katrina Diana M. Cruz
and that the said thesis has been submitted for final examination by the Oral Examination
Committee.
Marilyn A. Miranda
Thesis Adviser
Rhoda B. Leron
Course Adviser
As members of the Oral Examination Committee, we certify that we have examined this
thesis, presented before the committee on March 2006, and hereby recommend that it be
accepted as fulfillment of the thesis requirement for the degree in Bachelor of Science in
Chemistry.
John Ysrael G. Baluyut
Member
Kevin B. Dagbay
Member
Joy G. Hofileña
Member
This thesis is hereby approved and accepted by the School of Chemical Engineering and
Chemistry as fulfillment of the thesis requirement for the degree in Bachelor of Science in
Chemistry.
Luz L. Lozano
Dean, School of CHE-Chm
ii
ABSTRACT
The crude extract was obtained from the leaves of Jasminum sambac by extraction
with petroleum benzene showed effective antimicrobial activity against Gram-positive
bacteria: Staphylococcus aureus, and against Gram-negative bacteria: Escherichia coli. It
was then run through normal phase column chromatography having petroleum benzene as the
eluting solvent for the isolation of the active compounds present. The fractions with
effective activity were further isolated using 3 % and 10 % ethyl acetate in petroleum
benzene as the eluting solvents. Activity was found to vary among microorganisms. It was
found out that the first compound has effective antimicrobial activity against S. aureus
(AI = 0.58) and the second compound has effective antimicrobial activity against E. coli
(AI = 0.61). The available data using IR spectra of the first compound revealed the presence
of aromatic, C=O, and C-O of aliphatic ethers and esters while the second compound
revealed the presence of C=O, and C-O of aliphatic ethers and esters and aromatic group.
The 1HNMR spectra of the first compound revealed the presence of a mono-substituted
aromatic ring, aliphatic double bonds with methyl groups, and an acetate ion. While the
1
HNMR spectra of the second compound revealed the presence of a mono-substituted
aromatic ring, an ethyl group attached to the carboxylate oxygen of the acetate group and a
secondary or tertiary alkyl group attached to the carbonyl carbon of an ester. However,
overlapping of peaks observed in the 1HNMR spectra of the two compounds suggest that
impurities might still be present and can be removed through further isolation.
Keywords:
extraction with petroleum benzene, antimicrobial
chromatography, IR spectra, 1HNMR spectra
iii
activity,
column
ACKNOWLEDGEMENT
iv
TABLE OF CONTENTS
TITLE PAGE
i
APPROVAL PAGE
ii
ACKNOWLEDGEMENT
iii
TABLE OF CONTENTS
iv
LIST OF TABLES
vi
LIST OF FIGURES
vii
ABSTRACT
viii
Chapter 1: INTRODUCTION
1
Chapter 2: REVIEW OF LITERATURE
4
Jasminum sambac
Isolation, Purification, and Characterization
of Major Bioactive Components of Different Plants
The Fungi Isolated from Sampaguita
Other Herbal Treatments for Pimples
Other Herbal Treatments for E. coli
Sampaguita as a Herbal Treatment for Pimples and Fever
Chapter 3: BIOASSAY-GUIDED ISOLATION AND
PARTIAL CHARACTERIZATION OF THE
ANTIBACTERIAL COMPONENTS FROM
Jasminum sambac EXTRACT
4
5
5
6
6
7
8
Abstract
8
Introduction
8
Methodology
9
Collection and identification of sampaguita leaves
Preparation of sampaguita leaves
Extraction of the crude extract
Selection of solvent system for the isolation
Isolation of the desired component
v
9
9
10
10
12
Antimicrobial assay of the isolated compounds
Structure elucidation of the isolated component
12
14
Results and Discussion
14
Identification of sampaguita leaves
Extraction of the crude extract
Selection of solvent system for the isolation
Isolation of the desired component
Antimicrobial assay of the isolated compounds
Infrared spectral analysis
H NMR spectral analysis
14
14
14
15
16
19
20
Conclusion
22
Recommendation
23
References
24
Chapter 4: CONCLUSION
25
Chapter 5: RECOMMENDATION
27
REFERENCES
28
APPENDICES
30
vi
LIST OF TABLES
TABLE 1: Percent Recovered of the Crude Extract
from the Fresh Poultice
TABLE 2: Rf Values of the Fractions
of the Second Column
TABLE 3: Rf Values of the Fractions
of the Third Column
TABLE 4: Antimicrobial Assay of the Crude
Expressed in Terms of AI
TABLE 5: Antimicrobial Assay of the Fractions
of the First Column
TABLE 6: Antimicrobial Assay of the
of the Second Column
TABLE 7: Antimicrobial Assay of the
of the Third Column
TABLE 8: Prominent Bands in the IR Spectra
of Compound C4b
TABLE 9: Prominent Bands in the IR Spectra
of Compound C4c
TABLE 10: Important Chemical Shifts in the 1HNMR Spectrum
of Compound C4b
TABLE 11: Important Chemical Shifts in the 1HNMR Spectrum
of Compound C4c
vii
14
16
16
17
17
18
18
19
20
20
21
LIST OF FIGURES
FIGURE 1:
FIGURE 2:
FIGURE 3:
FIGURE 4:
Poultice of Fresh Sampaguita Leaves
Diagram Showing the Procedural Flow of the Study
Diagram of Fractions Isolated
Separation of Compounds Using 3%, 10%, and 13% Ethyl acetate
in Petroleum benzene
FIGURE 5: 1HNMR Spectra of Compound C4b
FIGURE 6: 1HNMR Spectra of Compound C4c
FIGURE 7: IR Spectra of Compound C4b
FIGURE 8: IR Spectra of Compound C4c
FIGURE 5: Plant Identification Certificate
viii
10
11
13
15
31
32
33
33
34
Chapter 1
INTRODUCTION
Pimples is a common skin disease caused by Staphylococcus aureus. The common
treatment for pimples is the prescription of creams and gels. However, most creams produce
stinging effects and redness on the affected area. Also, most of the effective treatments are
expensive. Another common illness is fever caused by endotoxins. Endotoxin stimulates
host cells to release proteins called endogenous pyrogens, which affect the temperaturecontrolling center of the brain. Large doses of endotoxin can cause death, primarily through
hemorrhagic shock and tissue necrosis (Madigan et al., 2003). Escherichia coli are known to
produce these Endotoxins. The common treatment for fever is analgesic. Conventional
drugs usually provide effective therapy but there is an increasing problem of side-effects.
And therefore, there’s a continuing need for new solutions.
Most people prefer medicinal plants, because they are natural. Plants have long
provided mankind with a source of medicinal agents. Two different extracts of Thymine
leaves have been known to have antioxidant property, replacing synthetic BHT for use in
foods (Dapkevicius et al., 2002). Active components namely: alpha-terpinine-4-ol, and
Cineole compounds found in the plant extracts Matricaria recutita, Lavendula, and
Maleleuca alternifola have well proven anti-microbial, anti-viral, and anti-inflammatory
properties. However, these extracts are found in German chamomile (Matricaria recutita),
Lavender (Lavendula), and Tea tree (Maleleuca alternifola) which are rare. Other plants like
Putod, Pitogo, Malasambung, and Tabako are used to cure skin diseases (Brown, 1958). For
fever, the following are used as treatment: Kauayan, Palai, Niyog, Anonas, and Sampalok
(Brown, 1958).
1
Sampaguita (Jasminum sambac) is commonly grown here in the. Its leaves are used
as a poultice for treating skin diseases and wounds (Brown, 1958).
Endophytes culture (a plant host) found in Jasminum sambac do not show antimicrobial and anti-tumor activity. However, it is possible that these endophytes may produce
substances that may ward off microbial infections by stimulating the host’s immune system
rather than by antimicrobial activity. These substances present in the extract of Jasminum
sambac may stimulate the growth of the microorganisms, as was evident in several isolates
showing good bacterial growth forming wide zone of inhibition around the disk, thus
counteracting the effect of inhibitory substances (Radu and Kqueen, 2002). This study
concludes that there is a probability that Jasminum sambac can treat pimples and fever.
Though there had been a study on the isolation of fungi from Jasminum sambac, the
isolated sample is tested against Bacillus subtilis, Saccharomyces cerevisiae, and Salmonella
typhimurium and not for the microbes: Staphylococcus aureus and Escherichia coli. The
component/s of Jasminum sambac that have the property to treat pimples and fever are not
yet known.
The objectives of this study are to provide experimental results for the activity of
Jasminum sambac against Staphylococcus aureus and Escherichia coli, and to isolate and
partially characterize the compound responsible for these activity using Normal Phase
Column Chromatography, 1HNMR Spectroscopy and IR Spectroscopy, respectively.
The focus of this study was on the isolation and partial characterization of the active
component/s of the leaves of Jasminum sambac against S. aureus and E. coli. The solvent
for the isolation was selected through Thin Layer Chromatography (TLC). The following
solvents were used: petroleum benzene, and ethyl acetate in petroleum benzene. The desired
2
1
component of the sample was separated using Column Chromatography. Isolation was done
until appearance of a good spot on the TLC plate. The isolated compounds were subjected to
IR and 1HNMR for their partial characterization. And finally, the sample was tested against
S. aureus and E. coli to confirm the initial findings that it can treat pimples and fever. The
fractions extracted and isolated were all tested for its anti-microbial (S. aureus and E. coli)
activity. The number of fractions was reduced using TLC, before its anti-microbial activity.
The importance of this study was that the compounds isolated, were found effective,
and could provide a cheaper, and a natural alternative for the treatment of pimples and fever.
3
1
Chapter 2
REVIEW OF LITERATURE
Through the help of Natural Product discoveries, we are able to isolate and obtain
components of plants that have the ability to cure different diseases. The studies that were
done usually include isolation, purification, analysis, and characterization of a desired
component.
Jasminum sambac
The local names of Jasminum sambac are: Kampupot in Tagalog; Sampagita in Spanish
and Filipino; and Arabian Jasmine, sambac in English.
This plant is a spreading or sprawling, smooth, shrub usually less than 2 meters in
height. The leaves are glossy, ovate or rounded, and 6 to 12 cm long, with short stalks,
pointed or blunt tip, and pointed or rounded base. The flowers are white, very fragrant, and
borne singly or in threes on axillary or terminal inflorescences. The calyx-teeth are 8 to 10,
very slender, and 5 to 8 mm long. The corolla-tube is slender and 1 to 1.5 cm long; the limb
is usually double and 1.5 to 2 cm in diameter.
In China, the flowers are used for providing aroma to tea. In Malaya, women soak the
flowers in water to be used for washing the face. The flowers are applied as a poultice to the
breasts of women as a lactifuge. The leaves are given internally for fevers. They are used
for poulticing skin complaints and wounds (Brown, 1958).
4
1
Isolation, Purification and Characterization of Major Bioactive Components of
Different Plants
Although it has been reported that Sampaguita has possible anti-microbial activity
against Staphylococcus aureus and Escherichia coli, the active components have not been
isolated and identified. Isolation, purification and characterization of the major bioactive
components of several other plants have been reported.
In the isolation, purification and characterization of the major bioactive components of
the rhizomes of Ethlingeria elatior, the sample was prepared by air drying. The crude
extracts of the air dried samples were extracted using DCM as solvent and isolated using
column chromatography (Budoy et al., 2003).
The antioxidant compounds of avocado, was extracted by solvent extraction by using
organic and aqueous solvents. The crude extract from avocado was partitioned between
hexane and chloroform. Purification of the chloroform fraction was done by isocratic and
gradient elution column chromatography (De Asis and Espeso, 2003).
Adoption of the above mentioned extraction and isolation methods can be done to
sampaguita to successfully isolate, purify and partially characterize the components
responsible for its antibacterial activity against Staphylococcus aureus and Escherichia coli.
The Fungi Isolated from Sampaguita
Fungi can also be isolated from plants. The dried and sterilized leaves were divided
into 3 segments and placed on Potato Dextrose Agar (PDA) to suppress bacterial growth.
The plates were incubated at 27°C for 3 weeks. Emerging fungi were transferred to fresh
PDA plates, incubated for 1 week and periodically checked for purity.
5
1
Endophytes fungi have also been isolated in Jasminum sambac. The culture isolated did
not show anti-microbial activity against Bacillus subtilis, Saccharomyces cerevisiae, and
Salmonella typhimurium; and anti-tumor activity.
The Endophyte culture found in Jasminum sambac may (1) produce substances that may
ward off microbial infections by stimulating the host’s immune system rather than by antimicrobial activity. Or maybe, (2) these substances present in the extract can stimulate the
growth of the microorganisms, showing good bacterial growth forming wide zone of
inhibition around the disk, thus counteracting the effect of inhibitory substances (Radu and
Kqueen, 2002).
Other Herbal Treatments for Pimples
In China, the whole plant of Putod is boiled and the extract is used for wounds and other
skin disorders. In the Philippines, the whole seed of Pitogo is roasted, powdered into small
pieces, soaked into coconut oil, stirred, and applied to wounds, boils, itches, and other skin
diseases. Malasambung is used against skin diseases here in the Philippines. In India,
Tabako leaves are used for skin diseases (Brown, 1958).
Other Herbal Treatments for E. coli
The bud of the leaf of Kauayan is used to treat leprosy, fevers, and haemoptysis. In IndoChina, the decoction of the fruits of palai is used as an emollient in diarrhea. The flowers of
Niyog are useful in the treatment of urinary discharges. Malays use the powdered seeds of
Anonas as an astringent for diarrhea. Decoction of Sampalok leaves is used as a bath in
fevers (Quisumbing, 1978).
6
1
Sampaguita as a Herbal Treatment for Pimples and Fever
Sampaguita has been traditionally known for treating skin diseases. Its leaves are used
as poultice for skin diseases and wounds, and used in decoction for fever (Quisumbing,
1978). However, these methods are used with no tests done to determine their effectivity.
7
1
Chapter 3
BIOASSAY-GUIDED ISOLATION AND PARTIAL CHARACTERIZATION
OF THE ANTIBACTERIAL COMPONENTS FROM Jasminum sambac EXTRACT
Abstract
The crude extract was obtained from the leaves of Jasminum sambac by extraction
with petroleum benzene showed effective antimicrobial activity against Gram-positive
bacteria: Staphylococcus aureus, and against Gram-negative bacteria: Escherichia coli. It
was then run through normal phase column chromatography having petroleum benzene as the
eluting solvent for the isolation of the active compounds present. The fractions with
effective activity were further isolated using 3 % and 10 % ethyl acetate in petroleum
benzene as the eluting solvents. Activity was found to vary among microorganisms. It was
found out that the first compound has effective antimicrobial activity against S. aureus
(AI = 0.58) and the second compound has effective antimicrobial activity against E. coli
(AI = 0.61). The available data using IR spectra of the first compound revealed the presence
of aromatic, C=O, and C-O of aliphatic ethers and esters while the second compound
revealed the presence of C=O, and C-O of aliphatic ethers and esters and aromatic group.
The 1HNMR spectra of the first compound revealed the presence of a mono-substituted
aromatic ring, aliphatic double bonds with methyl groups, and an acetate ion. While the
1
HNMR spectra of the second compound revealed the presence of a mono-substituted
aromatic ring, an ethyl group attached to the carboxylate oxygen of the acetate group and a
secondary or tertiary alkyl group attached to the carbonyl carbon of an ester. However,
overlapping of peaks observed in the 1HNMR spectra of the two compounds suggest that
impurities might still be present and can be removed through further isolation.
Keywords:
extraction with petroleum benzene, antimicrobial
chromatography, IR spectra, 1HNMR spectra
activity,
column
Introduction
Sampaguita is known to have a lot of medicinal uses. Its flowers are applied as a
poultice to the breasts of women as a lactifuge. Its roots may be used with its leaves to make
an eye-lotion. It is given fresh for vereneal diseases. Its leaves are given internally for ever.
Its leaves are used to poulticing skin complaints and wounds (Quisumbing, 1978).
However, these statements are folkloric. The active components were not tested to the
microorganisms responsible for these diseases. Therefore, further study is needed.
8
1
The objectives of this study are to provide experimental results for the activity of
Jasminum sambac against Staphylococcus aureus and Escherichia coli, and to isolate and
partially characterize the compound responsible for these activity using Normal Phase
Column Chromatography, 1HNMR Spectrocopy and IR Spectroscopy, respectively.
This study reports the isolation of the active compounds from the sampaguita leaf
extracts by means of column chromatography and TLC. The eluting solvents were pure
petroleum benzene, 3% and 10 % ethyl acetate in petroleum benzene. It also provides the
antimicrobial activity of the fractions isolated against S. aureus and E. coli, the IR spectra
and 1HNMR Spectra of the two active compounds isolated, C4b and C4c.
Methodology
Collection and identification of sampaguita leaves
The leaves of Sampaguita were harvested from Tondo, Manila in December of 2005.
The plant species were identified at the Bureau of Plants as Jasminum sambac (see Appendix
A).
Preparation of sampaguita leaves
Fresh sampaguita leaves were made into a poultice as shown on Figure 1. It was
boiled in water and then, strained and squeezed to remove excess water. The resulting
product was evaluated for its antimicrobial activity against Staphylococcus aureus and
Escherichia coli using the agar diffusion method.
9
1
Figure 1 Poultice of fresh sampaguita leaves
Extraction of the crude extract
To facilitate the preliminary solvent selection, a small amount undergone maceration
with petroleum benzene, methanol, and a mixture of methanol/petroleum benzene (1:1) to
yield three different organic extracts. Each of the organic extract was again evaluated against
the selected microorganisms. Those that contain activity had undergone a series of isolation
and purification process using column chromatography with appropriate eluting solvents.
Figure 2 shows the procedural flow diagram.
Selection of solvent system for the isolation
The selection of the solvent system for isolation was performed based on the results
of the preliminary solvent selection that gave an activity against the S. aureus and E. coli. A
minute amount of the macerated poultice were transferred in the TLC plate coated with silica
gel (MERCK Silica Gel 60 F254) using a capillary tube. Several eluting solvents that exhibit a
wide range of polarity gradient like petroleum benzene, ethyl acetate in petroleum benzene,
methanol, and acetone were tested to give a better separation of compound present in the
sample.
10
1
Poultice
Fresh Sampaguita
Leaves
*Analysis
N
Stop
+
*Analysis: Anti-microbial
avtivity against
Staphylococcus aureus
Escherichia coli.
Y
Solvent Selection
Column Chromatography
**Reduction by
selecting those that
contain the same
retention factor.
Fractions
TLC
**Reduced Number
of Fractions
1
Disregard
the Fraction/s
that produced
(-) result/s
2
3
N
*Analysis
+
Y
Y
IR and
HNMR
1
N
TLC
Showed
good
separati
TLC
Y
*Analysis
+
N
Stop
Figure 2 Diagram showing the procedural flow of the study
11
1
The developed chromatogram in the TLC plates were sprayed with 1:1 mixture of 2%
vanillin in 95% ethanol (2 grams in 100 ml) and 95% ethanol and 10% sulfuric acid (in 9:1
ratio) and were heated to reveal the spot(s)/band(s) of the compounds present. The solvent
system that showed the best resolution of spots (more spots and less tailing) was selected as
the eluent for column chromatography.
Isolation of the desired component
The active extract were fractionated by column chromatography on silica gel using
the ratio 1:40 and were eluted with a step polarity gradient of the selected solvent. Fractions
were collected and pooled on the basis of their TLC profiles. The fractions were evaluated
for its antimicrobial activity against S. aureus and E. coli. Active fractions were further
fractionated by successive column chromatography followed by thin layer chromatography
until there is an appearance of a good spot on the TLC plate that also contain activity against
S aureus and E. coli. Figure 3 shows the fractions isolated diagram. Fraction C showed the
best activity against the two test microorganism, and therefore, it was further isolated.
Antimicrobial assay of the isolated compounds
The fractions obtained in the isolation process were gathered for the antimicrobial
assay of the compound (Qualitative screening) using S. aureus – Gram-positive bacteria
(common pur cells, common skin microflora that can infect wounds), and E. coli – Gramnegative bacteria (Endotosin-producing bacteria that cause fever and UTI).
Microbial suspensions were prepared from 1-day old culture of bacteria.
suspending medium used was 0.1 % peptone water.
12
1
The
Crude Extract
A
Fractions
A1-A9
B
*C
Fractions
B1-B6
C1
C2
C4a
C3
*C4
b
*C4
*C4
c
Figure 3 Diagram of the fractions isolated
The suspending medium, the nutrient agar, and all apparatus used were sterilized
using the autoclave for 20 minutes at 120°C.
One-tenths (0.1) mL of the bacterial suspension was aseptically transferred into each
plate. Nutrient Agar were melted and cooled to 45°C; 5 mL of the corresponding medium
were poured to the plate. The plate was gently rotated to disperse inocolum evenly.
Six millimeter (6 mm) filter paper disc were soaked in the fractions allowing two
discs per fraction per organism. The inverted plates were incubated at 35°-36°C for 24-48
hours.
The inhibition zone was measured with a ruler and antimicrobial index was calculated
as:
13
1
Structure elucidation of the isolated component
The isolated compounds were subjected to IR and 1HNMR for its characterization.
The IR Analysis was done to determine the functional groups present at the Analytical
Chemistry Laboratory of De La Salle University. The 1HNMR Analysis was done to further
characterize and verify the IR results, at the National Chemistry Instrumentation Center of
Ateneo de Manila. The solvent used is CDCl3.
Results and Discussion
Identification of sampaguita leaves
The scientific name of the plant harvested from Tondo, Manila was identified at the
Bureau of Plants as Jasminum sambac from the family Oleaceae.
Local names are
Sampagita, Hubar, Arabian Jasmine, sambac (see Appendix A).
Extraction of the crude extract
The crude extract was obtained from the poultice of fresh sampaguita leaves by
soaking in Petroleum benzene for three days gave a percent yield of 0.581%
(refer to Table 1).
Table 1 Percent Recovered of the Crude Extract from the Fresh Poultice
Mass
% Recovered
Poultice
504 g
0.581 %
Crude Extract
2.93 g
Selection of solvent system for the isolation
Several eluting solvents like Petroleum benzene, 3%, 5%, 7%, 10%, and 13 % Ethyl
acetate in petroleum benzene were tested to check the separation of compounds present in the
14
1
sample. The good separation was observed with: petroleum benzene, 3 %, and 10 % ethyl
acetate in petroleum benzene. Thus, these were used for the isolation of compounds (see
Figure 4).
Figure 4 Separation of compounds using 3 %, 10 %, and 13
% ethyl acetate in petroleum benzene.
Isolation of the desired component
The first isolation resulted to 54 fractions. These fractions have undergone TLC to
check if the fractions were already purified. Fractions that have the same Rf values were
combined together. The fractions collected were grouped into 7, depending on their Rf
values. Fraction 19-28 were Group A, fractions 29-35 were Group B, fractions 37-45 were
Group C, fractions 1-18 were Group D, fraction 36 was Group E, fractions 46-49 were Group
F, and fractions 50-54 were Group G. All the seven fractions were subjected to antimicrobial
analysis against S. aureus and E. coli. Fraction C gave the highest activity against the two
microorganisms, so second column chromatography was performed. The four compounds,
C1, C2, C3, and C4, were isolated from Fraction C with 3 % ethyl acetate in petroleum
benzene as the eluting solvent. Table 2 gives the retention factors of the four fractions. The
color of fraction C1 and C2 is yellow. C3 is pale yellow, and C4 is pale yellow green.
15
1
Table 2 Rf Values of the Fractions of the Second Column, Solvent is 3 % Ethyl
acetate in Petroleum benzene
Fraction
Rf Value
C1
1.00
C2
0.29
C3
0.50; 0.67; 0.83
C4
0.54; 0.63; 0.75
All four fractions were again subjected to antimicrobial analysis against S. aureus
and E. coli. Fraction C4 gave the good activity against the two microorganisms, so third
column chromatography was performed forming five more fractions - C4a, C4b, C4c, C4d,
and C4e. Table 3 gives the retention factors of the five fractions using 10% ethyl acetate in
petroleum benzene. Fraction C4b, C4c, C4d and C4e showed one spot in the TLC plate with
13% ethyl acetate in petroleum benzene as the eluting solvent. Combining fractions that has
the same Rf values reduced the number of fractions. Fraction C4b and C4c have the same Rf
so it was combined and reduced to C4b, as well as C4d and C4e, which was reduced to C4c.
Table 3 Rf Values of the Fractions of the Third Column, Solvent is 10 % Ethyl acetate
in Petroleum benzene
Fraction
Rf Value
C4a
0.44; 0.56
C4b
0.52
C4c
0.52
C4d
0.44
C4e
0.44
Antimicrobial assay of the isolated compounds
The results of the preliminary screening of the three extracts from three different
solvent is presented in Table 4. The antimicrobial activity of the three compounds against S.
aureus and E. coli was shown by the observed hindered growth of the microorganism around
the paper disc which can be designated as the inhibition zones. Higher AI of the samples
16
1
could be attributed to the greater diffusion rates of the sample as compared to
Chloramphenicol. The positive control contains 30 µg Chloramphenicol.
Table 4 Antimicrobial Assay of the Crude Extract Expressed in Terms of Activity Index
S. aureus
E. coli
Diameter of
Diameter of
AI
AI
Sample
Clearing zone
(mm)
Petroleum
benzene
Petroleum
benzene
And Methanol
Methanol
Chloramphenicol
Clearing zone
(mm)
>55
>4.5
17
1.75
>55
>4.5
n/a
n/a
18
23
0.8
2.8
n/a
8
n/a
0.33
Based from the antimicrobial assay, the extract having petroleum benzene as the
solvent, has the best antimicrobial activity due to its high AI and no bacterial growth at the
first trial. This extract had undergone isolation and the antimicrobial assay of its fractions is
shown in Table 5.
Table 5 Antimicrobial Assay of the Fractions from the First Column Expressed in Term of
Activity Index.
S. aureus
E. coli
Sample
Diameter of
Clearing zone
(mm)
AI
Diameter of
Clearing zone
(mm)
AI
A
B
C
D
E
F
G
9
11
11
10
10
10
0.5
0.75
0.75
0.67
0.67
0.67
9.5
14.5
12.5
7
12.5
7
0.58
1.42
1.08
0.17
1.08
0.17
Chloramphenicol
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1
Based from the antimicrobial assay, fraction C has the best antimicrobial activity due
to its high AI on both test microorganisms. This fraction had undergone second isolation and
the Antimicrobial assay of its fractions is shown in Table 6.
Table 6 Antimicrobial Assay of the Fractions from the Second Column Expressed in Terms
of Activity Index (AI).
S. aureus
E. coli
Diameter of
Diameter of
Sample
AI
AI
Clearing zone
(mm)
C1
C2
C3
C4
Chloramphenicol
10
10
Clearing zone
(mm)
0.67
0.67
14.5
9
9.5
12
7
1.42
0.50
0.58
1.00
0.17
Based from the antimicrobial assay, fractions C1, C2, and C3 have no activity against
S. aureus but have good activity against E. coli. Fraction C4 has the best antimicrobial
activity due to its high AI compared to Chloramphenicol, and it has an activity against the
two test microorganisms. This fraction undergone third isolation and the antimicrobial assay
of its fractions are shown in Table 7.
Table 7 Antimicrobial Assay of the Fractions from the Third Column Expressed in Terms
of Activity Index
S. aureus
E. coli
Sample
Diameter of
Clearing zone
(mm)
AI
Diameter of
Clearing zone
(mm)
AI
C4a
7
0.17
7.5
0.25
C4b
9.5
0.58
7
0.17
C4c
-
-
9.7
0.61
Chloramphenicol
10
0.67
7
0.17
18
1
Based from the antimicrobial assay, the fraction C4b has the best antimicrobial
activity against S. aureus and fraction C4c has the best antimicrobial activity against E. coli
due to their high AI to the corresponding test microorganisms.
As observed, as the number of isolation increases, the diffusion rate of its
corresponding fraction decreases. It is possible that this is due to, aside from the isolation
process, the crude extract contains more active compounds than the purified fraction.
Infrared (IR) spectral analysis
The IR spectra from fractions C4b and C4c are shown on Appendix B.
The
prominent bands and its corresponding functional group are shown on Tables 3.8 and 3.9,
respectively.
The two compounds have carbonyl groups. Probably, these carbonyl group is an ester
because the frequency of C=O of an ester ranges from 1735 cm-1 to 1800 cm-1 . Also, the
frequency at 1220 cm-1 to 1280 cm-1 is an indication of COC stretching.
Absorption
frequency at 1050 cm-1 to 1250 cm-1 is an indication of CO stretching. The IR spectra of
fractions C4b and C4c contain weak bands at 1601.65 cm-1 and 1590 cm-1 respectively which
indicate a C=C stretch of an aromatic group. Table 8 and Table 9 summarize the prominent
bands of compounds present in C4b and C4c.
Table 8 Prominent Bands in the IR Spectra of C4b
Group
Absorption Frequency
C=O
1739.31 cm-1
C=C aromatic stretching
1601.65 cm-1
C-O-C
1278.05 cm-1
C-O
1121.99 cm-1
C-H (alkane)
2955.49 cm-1
C-H (alkane)
2923.80 cm-1
Abbreviation: s = strong, m = medium, w = weak , v = variable
19
1
Intensity
s
w
m
m
s
very s
Table 9 Prominent bands in the IR Spectra of C4c
Group
Absorption Frequency
C=O
1734.97 cm-1
C-O-C
1259.00 cm-1
C-O
1092.39 cm-1
C=C aromatic stretching
1590 cm-1
C-H (alkane)
2954.67 cm-1
C-H (alkane)
2919.90 cm-1
-CH2-(alkane)
2850.93 cm-1
Intensity
m
m
m
m
s
very s
s
Abbreviation: s = strong, m = medium, w = weak , v = variable
1
H NMR spectral analysis
The 1H NMR spectra of compounds C4b and C4c are shown on Appendix C. The solvent
used is CDCl3 and the frequency used is 400 MHz. The chemical shifts were expressed in
ppm.
The chemical shift of compound C4b at 7.2363 ppm (5H, m, J = 6 Hz) revealed the
presence of a mono-substituted aromatic ring. The solvent CDCl3 causes the peak at 7.2314
ppm. The chemical shift at 4.3110 ppm (1H, dd, J = 2 Hz) revealed the presence of aliphatic
double bonds with methyl groups present. This peak overlapped with other peak due to
presence of impurities, which can be removed through further isolation. The chemical shift
at 2.3505 ppm (3H, s) revealed the presence of an acetate group. The prominent peaks of
compound C4b from the 1H NMR analysis is shown on Table 10.
Table 10 Important Chemical Shifts in the 1H NMR Spectrum of Compound C4b
Chemical
Number of
Multiplicity
J values (Hz)
Type of Proton
Shift
Proton
(ppm)
7.2363
Mono-substituted
5
m
6
aromatic ring
Aliphatic double
4.3110
1
dd
2
bonds with
methyl groups
2.3505
3
s
Acetate group
20
1
The chemical shift of compound C4c at 7.1681 ppm (5H, m, J = 4 Hz) revealed the
presence of a mono-substituted aromatic ring. The chemical shift at 4.1000 ppm (1H, q, J =
2 Hz) revealed the presence of an ethyl group. This peak has the same coupling constant
with the peak at 3.4500 ppm (1H, d/m, J = 2 Hz) which indicates that the proton with
chemical shift at 4.1000 ppm is adjacent to the proton on the methyl group at 3.4500 ppm.
The coupling of these two peaks might have caused the splitting. Also, the multiplicity of
peak at 3.4500 ppm was not clearly shown in the spectra and might have been caused by
impurities, which can be removed through further isolation. The chemical shift at 2.3358
(3H, s) revealed the presence of a carbonyl group of an ester. The chemical shift at 1.2338
ppm (1H, m, J =2Hz ) revealed the presence of a secondary or tertiary alkyl group. Since the
peak is on the upfield region of the spectrum, it must be attached to a carbon adjacent to an
electron withdrawing group such as the carbonyl carbon (C=O). However, the multiplicity of
this peak is not clear because of the impurities present that can be removed through further
isolation. The prominent peaks of compound C4c from the 1H NMR analysis is shown on
Table 11.
Table 11 Important Chemical Shifts in the 1H NMR Spectrum of Compound C4c
Chemical
Shift
(ppm)
Number
of Proton
Multiplicity
J Values
(Hz)
Type of Proton
7.1681
5
m
4
Mono-substituted aromatic ring
4.1000
1
q
2
Ethyl group
3.4500
1
d/m
2
Methyl group
2.3358
1.2338
3
1
s
m
2
Carbonyl group of an ester
Secondary or tertiary alkyl group
21
1
Conclusion
The crude extract from sampaguita leaves was obtained using petroleum benzene as
the solvent. From the antimicrobial assay, it is found out that non-polar compounds of the
poultice of sampaguita leaves has good antimicrobial activity against S. aureus and E. coli.
Column chromatography having petroleum benzene, 3 %, and 10 % ethyl acetate in
petroleum benzene as the eluting solvents isolated two possible new compounds – C4b and
C4c. The available data using IR spectra of the first compound revealed the presence of
aromatic, C=O, and C-O of aliphatic ethers and esters while the second compound revealed
the presence of C=O, and C-O of aliphatic ethers and esters and aromatic group. The
1
HNMR spectra of compound C4b revealed the presence of a mono-substituted aromatic
ring, aliphatic double bonds with methyl groups, and an acetate group. While the 1HNMR
spectra of compound C4c revealed the presence of a mono-substituted aromatic ring, an ethyl
group attached to the carboxylate oxygen of the acetate group and a secondary or tertiary
alkyl group attached to the carbonyl carbon of an ester. However, overlapping of peaks
observed in the 1HNMR spectra of the two compounds - C4b and C4c- suggest that
impurities might still be present and can be removed through further isolation.
This study was able to isolate only two compounds –C4b and C4c- with activity
against S. aureus and E. coli. However, there are some fractions that also showed good
activity against the two microorganisms but were not isolated. It is then recommended that
these fractions should further be isolated using the necessary solvents for column
chromatography and characterized using other spectroscopic technique to elucidate fully their
structures.
22
1
Also fraction F derived from the petroleum benzene extract showed good activity
against B. subtilis. It is recommended then that further study about the isolation of this active
compound against B. subtilis is done.
Also,
13
C Spectroscopy and HPLC-MS Spectroscopy may be performed to the active
compound isolated –C4b and C4c- to fully characterize and elucidate the structures.
Recommendation
The minor peaks present in the 1HNMR spectra of compounds C4b and C4c shows
that the two compounds have impurities. These impurities must be eliminated through
further isolation using column chromatography.
13
C NMR Spectroscopy and HPLC-Mass
Spectroscopy may be performed to the purified sample of C4b and C4c to elucidate fully
their structures.
This study was able to isolate only two compounds with activity against S. aureus and
E. coli. However, there are some fractions that also showed good activity against the two
microorganisms but were not isolated. It is then recommended that these fractions should
further be isolated using the necessary solvents for column chromatography and
characterized using other spectroscopic technique to elucidate fully their structures.
Fraction F derived from the petroleum benzene extract showed good activity against
B. subtilis. It is recommended then that further study about the isolation of this active
compound against B. subtilis be conducted.
23
1
References
De Asis, A. and E. Espeso (2003). Isolation and Chemical Characterization of Antioxidant
Compounds from the Leaves of Persea Americana (Avocado) in the Linoleic Acid
Peroxidation Reaction. B.S. Thesis, University of Santo Tomas.
Gutierrez, G. (2004) Isolation, Structure Elucidation and Antimicrobial Activity of a
Secondary Metabolite from Dichloromethane Leaf Extract of Kamias (Averrhoabilimbi L.).
B.S. Thesis, Mapua Institute of Technology.
Luis, K. (2005). Isolation, Purification and Structural Elucidation of the Biologically
Active Component of Cogon Roots. B.S. Thesis, Mapua Institute of Technology.
Quisumbing E. (1978). Medicinal Plants of the Philippines, 3rd Edition, Katha Publishing
Inc., Manila.
Radu, S. and C. Kqueen (2002). Preliminary Screening of Endophtic Fungi from Medicinal
Plants in Malaysia for Antimicrobial and Anti-tumor Activity. Malaysian Journal of Medical
Sciences, Volume 9, (2), 23-33.
24
1
Chapter 4
CONCLUSION
The crude extract from sampaguita leaves was obtained using petroleum benzene as
the solvent. From the antimicrobial assay, it is found out that non-polar compounds of the
poultice of sampaguita leaves has good antimicrobial activity against S. aureus and E. coli.
Column chromatography having petroleum benzene, 3 %, and 10 % ethyl acetate in
petroleum benzene as the eluting solvents isolated two possible new compounds – C4b and
C4c. The available data using IR spectra of the first compound revealed the presence of
aromatic, C=O, and C-O of aliphatic ethers and esters while the second compound revealed
the presence of C=O, and C-O of aliphatic ethers and esters and aromatic group. The
1
HNMR spectra of compound C4b revealed the presence of a mono-substituted aromatic
ring, aliphatic double bonds with methyl groups, and an acetate group. While the 1HNMR
spectra of compound C4c revealed the presence of a mono-substituted aromatic ring, an ethyl
group attached to the carboxylate oxygen of the acetate group and a secondary or tertiary
alkyl group attached to the carbonyl carbon of an ester. However, overlapping of peaks
observed in the 1HNMR spectra of the two compounds - C4b and C4c- suggest that
impurities might still be present and can be removed through further isolation.
This study was able to isolate only two compounds –C4b and C4c- with activity
against S. aureus and E. coli. However, there are some fractions that also showed good
activity against the two microorganisms but were not isolated. It is then recommended that
these fractions should further be isolated using the necessary solvents for column
chromatography and characterized using other spectroscopic technique to elucidate fully their
structures.
25
1
Also fraction F derived from the petroleum benzene extract showed good activity
against B. subtilis. It is recommended then that further study about the isolation of this active
compound against B. subtilis be conducted.
Also,
13
C Spectroscopy and HPLC-MS Spectroscopy may be performed to the active
compound isolated –C4b and C4c- to fully characterize and elucidate the structures.
26
1
Chapter 5
RECOMMENDATION
The minor peaks present in the 1HNMR spectra of compounds C4b and C4c shows
that the two compounds have impurities. These impurities must be eliminated through
further isolation using column chromatography.
13
C NMR Spectroscopy and HPLC-Mass
Spectroscopy may be performed to the purified sample of C4b and C4c to elucidate fully
their structures.
This study was able to isolate only two compounds with activity against S. aureus and
E. coli. However, there are some fractions that also showed good activity against the two
microorganisms but were not isolated. It is then recommended that these fractions should
further be isolated using the necessary solvents for column chromatography and
characterized using other spectroscopic technique to elucidate fully their structures.
Fraction F derived from the petroleum benzene extract showed good activity
against B. subtilis. It is recommended then that further study about the isolation of this active
compound against B. subtilis be conducted.
27
1
REFERENCES
Budoy, C., A. Aguinaldo, and S. Franzblau (2003). Isolation, Purification and
Characterization of Major Bioactive components from the Rhizomes of Etlingeria
elatior(jack) R.M. Smith (IK) against Mycobacterium tuberculosis H37Rv. B.S. Thesis,
University of Santo Tomas.
Brown, W. (1958). Useful Plants in the Philippines, Technical Bulletin 10, Bureau of
Printing, Manila. pp. 221
Cheng, Y., R. Teng, O. Zheng, N. Tan, C. Wang, and J. Zhou (2002). Isolation and
Characterization of Brachystemidines A-E, Novel Alkaloids from Brachystemma Calycinum.
Journal of Natural Products, Volume 65, (5), 750-752.
Chu, M., T. Chan, P. Das, J. Jenkins, R. Mierzwa, V. Gullo, M. Patel, and B. Pramanik
(2002). Isolation and Characterization of Novel Oligosaccharides Related to Ziracin. Journal
of Natural Products, Volume 65, (11), 1588-1593.
Dapkevicius, A., T. Van Beek, G. Lelyveld, A. de Groot, J. Linssen, A. Van Veldhuizen, and
R. Venskutonis (2002). Isolation and Structure Eelucidation of Radical Scavengers from
Tymus vulgaris leaves. Journal of Natural Products, Volume 65, (6), 892-896.
De Asis, A., and E. Espeso (2003). Isolation and Chemical Characterization of Antioxidant
Compounds from the Leaves of Persea Americana (Avocado) in the Linoleic Acid
Peroxidation Reaction. B.S. Thesis, University of Santo Tomas.
Gloer, J., B. Joshi, and D. Wicklow (2002). Bioactive Natural Products from a SclerotiumColonizing Isolate of Humicola fuscoatra. Journal of Natural Products, Volume 65, (11),
750-752.
Gutierrez, G. (2004) Isolation, Structure Elucidation and Antimicrobial Activity of a
Secondary Metabolite from Dichloromethane Leaf Extract of Kamias (Averrhoabilimbi L.).
B.S. Thesis, Mapua Institute of Technology.
Halinger, E. and S. Korhammer (1994). Isolation of Biologically Active Substance from
Rhizomes of Quackgrass. Journal of Agricultural Food Chemistry, Volume 42, (9), 20482050.
Luis, K. (2005). Isolation, Purification and Structural Elucidation of the Biologically
Active Component of Cogon Roots. B.S. Thesis, Mapua Institute of Technology.
Petersen K., P. Nielsen, G. Bertelsen, M. Lawther, M. Oleen, N. Nilsson, and G. Mortensen
G. (1999) Potential of Biobased Materials for Food Packaging. Trends in Food Science and
Technology, Volume 10, (2), 52-68.
28
1
Quisumbing E. (1978). Medicinal Plants of the Philippines, 3rd Edition, Katha Publishing
Inc., Manila.
Radu, S. and C. Kqueen (2002). Preliminary Screening of Endophtic Fungi from Medicinal
Plants in Malaysia for Antimicrobial and Anti-tumor Activity.” Malaysian Journal of
Medical Sciences, Volume 9, (2), 23-33.
Weber, C. (2000) Biobased Packaging Materials for the Food Industry. The Royal
Veterinary and Agricultural University. Denmark.
29
1
APPENDICES
30
1
APPENDIX A
31
1
APPENDIX B
IR Spectra of Compound C4b
IR Spectra of Compound C4c
32
1
APPENDIX C
H NMR Spectra of Compound C4b
33
1
H NMR Spectra of Compound C4c
34
1
APPENDIX D
A. % Yield
% Yield = (mass extract/mass poultice) x 100
% Yield = (2.93/504) x 100 = 0.581 %
B. Antimicrobial Index
AI = (Diameter of Clearing Zone – Diameter of Well)/Diameter of Well
AIC4b = (9.5 – 6.0)/6.0 = 0.58
AIC4c = (9.7 – 6.0)/6.0 = 0.61
C. Coupling Constant (J)
J = [(L-R) x 400 MHz] / 10
J = [(4.3110 - 4.2708) * 400] / 10
J=2
35
1