WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Maheep Kumar et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 3, 1075-1085. Research Article ISSN 2278 – 4357

USE OF TLC, UV, NMR, IR AND LC-MS/MS FOR PURIFICATION

AND IDENTIFICATION OF ANTIBACTERIAL COMPOUNDS

Maheep Kumar*1, Alok Kumar Ravi 2, Ravi Kumar Asthana3

1Department of Botany, Guru Ghasidas Vishwavidyalaya, Bilaspur-495009, India

2
Department of Ocular Pharmacology and Pharmacy, Dr. R.P. Centre for Ophthalmic
Sciences, All India Institute of Medical Sciences, New Delhi 110 029, India.
3
Centre of advanced study in Botany, Faculty of Science, Banaras Hindu University,
Varanasi-221005, India.

Article Received on
ABSTRACT
02 January 2014 Cyanobacteria are known to produce array of compounds. In an earlier
Revised on 26 January 2014, report, we reported antibacterial activity in methanolic crude extracts
Accepted on 18 February
2014 of laboratory grown Lyngbya aestuarii and Aphanothece bullosa
isolated from Chilka Lake and local paddy field respectively. In this
*Correspondence for Author report the same methanolic crude extracts were subjected to TLC
Dr. Maheep Kumar purification twice by altering the solvents and UV-illuminated bands
Department of Botany,
bioassayed. Such UV illuminated potent bands obtained after 2nd TLC
Guru Ghasidas
was subjected to spectroscopic analysis (UV, IR, 1H NMR and
Vishwavidyalaya, Bilaspur,
India. LCMS/MS). We have screened malyngolide from L. aestuarii
however, a diterpenoid from A. bullosa.
Keywords Lyngbya aestuarii∙ Aphanothece bullosa ∙TLC purification
spectroscopic analysis∙ antimicrobial.
INTRODUCTION
Drug resistance in bacterial pathogens such as Staphylococcus aureus, Streptococcus
pneumoniae and Pseudomonas aeruginosa indicated the ineffectivity of antibiotics1-3.
Therefore, search of new drug/ biomolecule/ lead molecule, is a major thrust area at the
moment. Modern approaches to drug discovery could not accelerate the pace of newer drug
development because of little exploration of natural resources especially microbial.
Metabolites produced by actinomycetes, fungi and unicellular bacteria along with
cyanobacteria contributed 45, 38 and 17% respectively4. However, secondary metabolites

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produced from the cyanobacteria are found to be antibacterial, antifungal, antialgal,

anticancerous, antimalarial with many more activities5-7.

Among the cyanobacterial genera screened, Lyngbya is distributed throughout tropical and
subtropical regions and proved as prodigious producer of secondary metabolites8,9.
Aphanothece sp. is an unicellular fresh water cyanobacteria reported to have antitumor and
antiproliferative properties10. Therefore, we have chosen L. aestuarii (brackish water) and
Aphanothece bullosa (fresh water) isolates as a target strains to screen antibacterial and
antifungal biomolecules after bioassay of crude extracts as well as TLC purified ones and
subjected to spectroscopic analysis for identification of biomolecules.

MATERIALS AND METHODS

Cyanobacterial strains and growth conditions
Lyngbya aestuarii (a brackish water strain isolated from Chilka lake, Orissa, India) and
Aphanothece bullosa (a fresh water strain from paddy field, around Banaras Hindu
University, Varanasi, India), were grown in ASN III and BG 11 medium, respectively11 for
screening of biological activity. Cultures were maintained at 28±2°C, with a light intensity of
14.40 W/m2, provided by cool white fluorescent tubes with a light/dark cycle of 18/ 6 h.

Preparation of crude extracts

Biomass of L. aestuarii and A. bullosa were harvested after 40 and 60 d respectively. The
harvested biomass was centrifuged at 10,000 rpm for 15 min (remi, India) and lyophilized
(christ-alpha 1-2, Germany). Lyophilized cyanobacterial biomass (5g) was extracted twice in
300 ml methanol (100%) by keeping it on shaker (150 rpm for 48 h) and centrifuged at
15,000 rpm for 15 min. Supernatants were dried in a rotary evaporator (prefit, India) at 40 °C
redissolved in 3 ml methanol (25 %) to be used for purification12.

Purification of crude extracts

Dried methanolic extracts (100 mg) of L. aestuarii and A. bullosa dissolved in 3ml methanol
(25%) for purification using TLC silica plates (TLC 60 Merck, Germany). Separations of
crude extracts were done using carbon tetrachloride and methanol (9: 1) as mobile phase.
Spots developed on such TLC plates were observed under UV illumination. The illuminated
orange spots were eluted separately and dissolved in 3 ml methanol (25%). Each elutes again
subjected to TLC purification using hexane: ethyl acetate (1: 1). Now all spots obtained at
second stage were bioassayed for antibacterial and antifungal activities.

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Antibacterial Bioassay
The designate spots in 2nd TLC were concentrated in rotary evaporator. These spots were
redissolved in 25%. Antibacterial bioassays of such samples were performed. Antibacterial
bioassay was performed using non-pathogenic Enterobacter aerogenes MTCC 2822
(IMTECH, India), Escherichia coli and Bacillus sp. This was carried out on 3.8 % Mullter
Hilton (MH) agar (Sigma-Aldrich) plates. The target bacteria were suspended in 0.91% NaCl
and turbidity adjusted to 107-108 CFU ml-1, corresponding to 0.5 McFarland standards
according to CLSI guidelines (Clinical and Laboratory Standards Institutes)13 . The wells of 4
mm diameter were made with sealing off bottom by soft agar (0.8%). Each MH plates were
swabbed with target bacterium aseptically. The spots elutes obtained after second TLC were
dissolved in 25% methanol and filled in well along with respective solvents (25% methanol)
and Rifampicin (50 µgml-1) as controls, and incubated at 37 °C for 24 h thereafter. The
diameters of the zones of inhibition were measured in mm.

Screening and identification of prospective compounds

UV spectra of antibacterial purified bands were recorded in methanol over scanning range of
195-500 nm using double beam spectrophotometer (U-2910, Hitachi, Japan). IR (infra red)
spectra of the same spots were obtained using Perkin Elmer spectrum version 10. 03. 05 as
film in KBr disc. 1HNMR (proton nuclear magnetic resonance) spectra of the same spots
were recorded on JEOL AL300 FTNMR spectrophotometers in downfield from TMS (tetra
methyl silane) in DMSO. D6 (dimethyl sulfoxide-d6) at 300 MHz.

Liquid chromatography mass spectrometry/ mass spectrometry

Preparation of unknown samples
1 mgml-1 stock solution of each samples were prepared in 50% MeOH containing 0.1%
formic acid and then further it was diluted in 50% MeOH containing 0.1% formic acid to
reach the final strength of 100 ngml-1, which was used for LC-MS/MS. For blank run, 50%
MeOH containing 0.1% formic acid was used. Mass grade formic acid, acetonitrile and
methanol were purchased from Merck, Germany. Water was purified using a Milli-Q
purification system (Millipore Corp., Bedford, MA, USA). All other chemicals and solvent
were one of the highest analytical grades available.

Information-Dependent Acquisition (IDA) Mode

Chromatography separation was achieved using Thermo Accela UHPLC system (Thermo
Electron Corp, Waltham, MA, USA) with a quaternary pump connected to an online

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degasser, autosampler and photodiode array detector (PDA). Chromquest software version
4.1 was used to control all parameters of UHPLC system. Analytical separation of the
unknown compound was achieved on a Purospher STAR RP-18 endcapped (3 µm particles,
55 x 4 mm size) column from Merck (Darmstadt, Germany). Column chamber was at
ambient temperature. The mobile phase was consisted of acetonitrile containing 0.1% formic
acid (A) and water containing 0.1% formic acid (B) with the following linear gradient
program: 0 min 100% B, 30 min 0% B. Mobile phase was pumped at the rate of 0.5 mlmin-1.
The autosampler tray was kept at ambient temperature. Twenty micro liter of the target
sample was injected into the UHPLC with a run time of total 30 min. To identify compound
in unknown samples by LC-MS/MS, an IDA method consisting of enhanced mass scan
(EMS), enhanced resolution (ER), and enhanced product ion (EPI) scan mode were
developed. LC-MS/MS IDA experiment for unknown compound was carried out on Linear
Ion Trap Quadrupole LC/MS/MS Mass Spectrometer 4000 Q TRAP AB Sciex instrument
(ABS, Foster City CA, USA) equipped with a Turbo Ion Spray (TIS) source operated in the
positive ion mode. Following IDA mass parameters were set for all experiments: Curtain gas
= 20; Collisional activated dissociation (CAD) = High; Ion Spary voltage = 5.5 kV; Ion
source Temperature = 450oC; Ion source gas-1 = 30; and Ion source gas-2 = 60. The
differential potential (DP) was set at 50. The Collision Energy (CE) was set at 10. Internal
heat was kept on. The EMS and EPI was performed with a scan rate of 4000 Da/s. For ER
scan, a scan rate of 250 Da/s was performed. All IDA experiments were performed in the
range of m/z 100 to m/z 1400 with an Q3 entry barrier of 8 V and disabled Q0-trapping.
Linear Ion Trap (LIT) fill time was set at 20 msec. Dynamic fill time was kept on. Data
acquisition and integration was performed by Analyst 1.4.2. software (ABS, Foster City CA,
USA)

RESULTS
TLC spots and Antibacterial activity
7 bands observed from L. aestuarii and 5 form A. bullosa. These potent bands were subjected
to 2nd TLC and UV illuminated designate bands were bioassayed against bacterial stains
(Table 1). The bioassay revealed that band F1 from L. aestuarii and L3 form A. bullosa were
potent antibacterial at 25µgml-1. The spot F1 form 3.3±0.321, 2.4±0.148, 2.1±0.048 inhibition
zone respectively against target bacterial strains. And L3 form 2.5±0.288, 1.2±0.32,
0.5±0.311 mm inhibition zone.

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Table 1: Bioassay of Second TLC bands against Enterobacter aerogens (1), Escherichia
coli (2) and Bacillus sp (3)
TLC spots 1st 2nd Rf Inhibition zone (mm)
TLC TLC values 1 2 3
Lyngbya A, B, F1 0.841 3.3±0.321 2.4±0.148 2.1±0.048
aestuarii C, D, G1 0.841 0.0 0.0 0.0
E, F,
G
Aphanothece H, I , L1 0.428 0.0 0.0 0.0
bullosa J, K, L L2 0.560 0.0 0.0 0.0
L3 0.645 2.5±0.288 1.2±0.321 0.5±0.311
L4 0.784 0.0 0.0 0.0
L5 0.831 0.0 0.0 0.0
Riffampicin - - - 11.0±0.572 8.5±0.511 9.2±0.533

The spots F1 and L3 produced 3.36±0.185 and 2.5±0.288 mm inhibition zones against E.
aerogenes. Rifampicin (50 µgml-1) was used as positive controls with inhibition zone sizes of
11.0±0.564. Therefore, we have spectroscopically analysed the potent bands obtained after
2nd TLC.

Spectroscopic analysis
UV absorption of F1 spot showed λmax at 203.5 nm indicated the presence of carboxylic acid/
acetic acid (Fig. 1a), IR absorption spectrum of target spots having 3419.06 cm-1 implied
presence of hydroxyl moiety, however, 1634 (carbonyl, C=O), 2922.20 (CH3), 2851.83 (C-H
or methoxy), 1414.90 (C=C) and 1106.58 (C-O-C) for other groups (Fig. 1b). The signal for
proton in 1H NMR spectrum revealed shift as: δ 3.393 (2H, d, J= 12 Hz) for isolated oxygen
bearing methylene (CH3-O), δ 2.490 (1H, s, J=30Hz) for carbonyl (C=O), δ 5.306 (1H, s) and
δ 1.971 (1H, s) for OH moiety. The other signals as: δ 1.218 (2H, d), δ 0.838 (2H, d), δ 0.817
(3H, t) led towards methyl moiety of the aliphatic chain. The chemical shift δ 8.467 revealed
the benzene ring (Fig. 1c). The m/z values of such target spots were as: 958.9, 537.7, 433.5,
415.4, 313.4 and 149.0. The m/z value, 541.44 coincided with described compound
malyngolide from cyanobacteria as screened from the data bank available on web (Fig. 1d).

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UV absorption spectrum of L3 spot had λmax at 219.5, 222.5 and 231.5 nm predicting the
carboxylic acid and benzene moiety in the compound (Fig. 2a). The IR absorption spectrum
revealed moiety as: 3436.88 (O-H, hydroxyl), 1638.18 (C=O, carbonyl) and 1605.19 cm-1
(aromatic ring) stretching. The other signals as 2925.93 (C-H stretching), 2852.41 (C=C
stretching) and 1384.65 showed (C-O-C) stretching (Fig. 2b).

Fig. 1. The spot F1 eluate derived from L. aestuarii was subjected to spectroscopic
analysis. UV Absorption spectrum (a); IR spectrum (b); 1H NMR spectrum (c); and LC
MS/MS (d).

The 1H NMR spectrum suggested the proton environment as chemical shifts, δ 0.736, 0.835,
0.853 and 1.233 indicated methyl (tertiary), 1.653 and 1.673 for methyl (secondary) group, δ
6.375 (1H, s, J=8.4), δ 6.485 (1H, s, J=0.715), δ 7.385 (1H, s, J=1.9), δ 7.445 (1H, s, J=2.4)
and δ 7.586 (1H, s, J=6.6) for aromatic ring moiety. Other chemical shifts δ 3.369 (1H, m,
J=6.2) and δ 3.571 (1H, s, J=1.3) pointed towards one proton attached to oxygenated carbon
(Fig. 2c). The m/z values were found as 758.4, 689.8, 610.3, 443.5, 437.5, 433.3, 415.4,
371.3, 355.5, 288.3, 149.2 and 149.0. The peaks 415.4, 443.5, 437.5 and 433.3 led the
possibility of a diterpenoid in the sample. Thus data from spectroscopic analysis confirmed
presence of a diterpenoid type of compound in A. bullosa (Fig. 2d).

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Fig. 2. The spot L3 eluate derived from A. bullosa was subjected to spectroscopic
analysis. UV Absorption spectrum (a); IR spectrum (b); 1H NMR spectrum (c); and LC
MS/MS (d).

DISCUSSION
Cyanobacteria posses various secondary metabolites specified through functional groups.
These functional groups are carbonyl, nitromethane, alcoholic, phenolic, etc. they show
illumination at a particular wavelength. We observed 20 illuminated band under ultraviolet
radiation. Spot F1 and L3 show antibacterial activity as methanolic crude extracts (100 µgml-
1
) of test cyanobacteria against 20 bacterial strains (17 gram-negative and 3 gram-positive)
possessed broad spectrum antibiotic activity, keeping rifampicin (100 µgml-1) as control14.

Through spectroscopic analysis of spot F1 showed the structure close to malyngolide

compound. However, presence of UV λmax, 203.5 nm indicated the carboxylic /acetic acid
moiety. The same compound malyngolide showed UV absorption at 208 nm15. IR absorption
at 3419.06 cm-1 was in tune with report of Cardllina and Moore16 where 3410 cm-1 as
absorption maxima was for hydroxyl group in malyngolide. Similarly IR spectra at 1643 cm-1
showed C=O stretching confirming the lactone carbonyl group17. Likewise, 1H NMR showed

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presence of carbonyl and hydroxyl moiety of malyngolide. The characteristic oxygen bearing
methylene group was found at δ 1.218 and carbonyl group at δ 2.490 as reported previously
by Cardllina and Moore16. The other methyl signal revealed at δ 0.832 and δ 0.817
corresponding to the aliphatic chain in tune with report of Gutierrez et al.15. Malyngolide is
an antibiotics naturally occurring from marine strain L. majuscula. Its molecular weight was
563.428818. The LCMS/MS value indicating Mr= 541.44 was close with the known
metabolite malyngolide. Malyngolide was reported as antibacterial against Bacillus subtilis,
S. aureus, Escherichia coli and P. aeruginosa19 as our previous report coincident with the
same property of malyngolide.

UV λmax of L3 spot at 219.5, 225.5, 231.5 and 400 nm suggested the conjugated and isolated
carbonyl, aromatic and hydroxyl group which create the base of diterpenoid20. The IR
absorption at 3436.88 cm-1, 1638.18 cm-1 and 1605.19 cm-1 confirm the hydroxyl, carbonyl
and aromatic moiety in the sample L3 similar to diterpenoid21. IR of diterpenoid also revealed
the similar results as hydroxyl at 3300 cm-1, carboxyl at 1710 cm-1 and aromatic at 1604 and
1520 cm-1 22
. Similarly, Norabictane a diterpenoid showed the IR absorption for aromatic
rings at 1605, 1580 and 1520 cm-1, the carbonyl moiety at 1723 and hydroxyl group at 3388
cm-1 17. The 1H NMR of spot L3 support the methyl moiety at chemical shifts δ 0.736, 0.835,
0.853, 1.233, 1.653, 1.673 and aromatic moiety at δ 6.375, 6.485, 7.385, 7.445 and at 7.586.
Four tertiary groups of methyl were also observed at δ 0.67, 0.88, 0.89, 1.11 and secondary at
δ 6.74, 7.70 and at 7.80 by Jaki et al.21. We could identify a proton attached with oxygenated
carbon as observed at δ 3.369 and at δ 3.571 of spot L3. Such oxygenated carbon was
observed in noscomin at δ 3.1921. Tertiary and secondary methyl groups were also observed
in commonstin A at similar chemical shifts as 0.86, 0.94, 0.99, 1.00 and 0.98 while aromatic
ring was also observed at similar shift at δ 6.93, 7.75 and 7.89 22. 1H NMR of norbietane, a
diterpenoid from Microcoleus lacustris, revealed methyl groups at chemical shifts at δ 1.14,
0.93 and at 0.9818. Diterpenoid of similar molecular weight were also reported as, tolypodiol
[M+H+ 456.56] a diterpenoid from Tolypothrix nodosa23, Norabietane [M+H+ 326.4091]18
and Noscomin [M-H- 425.2]21. Comnostin from Nostoc commune, showed various [M-H]-
values as 427 (comnostin A), 425 (comnostin B), 441 (comnostin C), 471 (comnostin D) and
425 (comnostin E)22. Similar 415.4, 433.3, 437.5 and 443.5 m/z values showed the molecular
weight of diterpenoid compound in spot L3.

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CONCLUSION
Cyanobacteria belonging to Chroococales, Stigonematales, Nostocales, and Oscillatoriales
produces antifungal compounds include fisherellin A, hapalindole, carazostatin, phytoalexin,
tolytoxin, scytophycin, toyocamycin, tjipanazole, nostocyclamide and nostodione. Fresh
water strains Stigonema, Fischerella, Anabaena, Nostoc, Synechococcus, Oscillatoria,
Schizothrix sp. found as source of antibacterial compounds. Secondary metabolites with
antibacterial activity are widely produced by cyanobacteria. These compounds are effective
against Grampositive and ⁄ or Gram-negative bacteria. Both toxic and nontoxic strains of
cyanobacteria are producers of antibacterialcompounds that are distinct from cyanotoxins.
The fresh water cyanobacterium Phormidium sp. has been also reported to inhibit growth of
different Gram-positive and Gram-negative bacterial strains, yeasts, and fungi. Thus selected
cyanobacterium belongs to Oscillatoriales and Chroococales order (from brackish and fresh
water bodies) and they produces antibacterial activity against gram negative bacteria. The
earlier report on methanolic crude extract also indicates that they have good potency to
produce antibacterial effect. Through purification of methanolic extract, they also presented
antibacterial activity. Specroscopic analysis indicates the presence of amine and terpenoid
like structure which is responsible for the antibacterial activity.Thus selected cyanobacteria
have potency to produce two compounds malyngolide and a diterpenoid.

ACKNOWLEDGEMENTS
We are grateful to Head and Programme Coordinator of Centre of Advanced Study in
Botany, for laboratory facilities, Department of Chemistry, for NMR. We are thankful to Dr.
M. S. Singh (Deaprtment of Chemistry) for consultant and suggestion in spectroscopic
analysis, Dr. U. S. Rai (Deaprtment of Chemistry) for IR spectroscopy. Financial support
from UGC-RGNFs [F-14-2 (SC)/2008/SA-III] is gratefully acknowledged.

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Abbreviation:
TLC Thin Layer Chromatography
UV Ultraviolet
NMR Nuclear magnetic resonance
LC-MS/MS Liquid Chromatography Mass Spectrometry Mass Spectrometry
LB Luria Broth
ASN Artificial Sea water Nutrient
BG Blue Green algae
MH Mullter Hilton

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