OBJECTIVE: To evaluate the presence of any high value added compounds in the spent biomass of C. vulgaris To identify the biological activity of the extracted compounds To evaluate the structure and nature of the compounds using Nuclear Magnetic Resonance Spectroscopy and other analytical techniques. Development of economically viable methodologies for the simultaneous extraction of by-products from a single set of biomass. biological activities performed -Total antioxidant capacity, Anti bacterial activity, Anti-tuberculosis activity, Anti proliferative assay

1. R.Priyanka
M.Sc biotechnology
Marine Biotechnology department,
National Institute of Ocean Technology,
Chennai

2. With an idea to extract as many products as possible from a single
set of biomass, protein rich-extract was first separated from
C. vulgaris using hot water treatment. The spent biomass obtained
after the extraction, which is literally considered as a waste
material was further evaluated for high-value added products such
as lipid and carotenoids.

3. OBJECTIVE
• To evaluate the presence of any high value added compounds in the
spent biomass of C. vulgaris
• To identify the biological activity of the extracted compounds
• To evaluate the structure and nature of the compounds using Nuclear
Magnetic Resonance Spectroscopy and other analytical techniques.
• Development of an economically viable methodologies for
simultaneous extraction of by-products from a single set of biomass.

4. VISIBLE short UV Long UV Stained
Spent biomass characterization using TLC fractions

5. Column
chromatography
Collected fractions
Spent biomass
Extraction Concentrated in rotary
vacuum evaporator

6. spotted Under UV Stained

7. spotted Under UV Stained

8. spotted Under UV Stained

9. spotted Under UV Stained

10. spotted Under UV Stained

11. spotted Under UV Stained

12. spotted Under UV Stained

13. spotted Under UV

14. 33 35
34 36

15. spotted Under UV Stained

16. spotted Under UV Stained

17. spotted Under UV

18. spotted Under UV Stained

19. Purified
compound
TLC
Mini column for Further purification

20. VISUAL
Green pigment
Short UV Long UV Stained
Thin layer chromatography fractions of target
compound

21. Preparative
TLC under
long UV
radiation
Preparative TLC
under visible
light
Purified pigment

22. Purified target compound
VISUAL Long UV Stained

23. Isolation and characterization of pigments from
C. vulgaris
Nuclear Magnetic Resonance Spectroscopy (NMR)
The structure of the compound was elucidated using the technique of
NMR, Bruker 400 MHz, Narrow Bore, High Resolution Solution State NMR
Spectrometer. NMR spectra were recorded using Deuterated Chloroform (CDCl3).
Liquid Chromatography-Mass Spectroscopy (LC-MSMS)
The obtained lutein sample was analyzed using Liquid Chromatography-Mass
Spectroscopy (LC-MS-MS) Shimadzu LCMS 8040 triple quadrupole mass spectrometer
coupled with UHPLC NEXERA. To enhance separation in the column, 0.5mM Trifluoro
ammonium acetate in water and acetonitrile was used in mobile phase composition
and retention time was 4.110 min.

24. Isolation and structural elucidation of carotenoid
The 1HNMR spectrum of lutein was recorded in
deuterated chloroform at 400 MHz. The spectrum
shows peaks between 5.5 and 6.8ppm integrating
for 12 protons, confirming the presence of
conjugated olefinic chain in carotenoids. Singlets in
the region of 0.8-1.5 ppm indicate the presence of
methyls.

25. 1H NMR spectrum of lutein
The carbon NMR spectrum of lutein shows the presence of 40 carbons. Peaks in the region of
124-136 ppm are characteristic of olefinic carbons. Two peaks at 65.9 and 65.1ppm confirm
the presence of oxymethine carbons. 10 methyl carbons were observed in the upfield
region.This confirms that lutein is a carotenoids, without any carbonyl carbon and has
hydroxyl group attached to the β-ionone ring.

26. Liquid Chromatography-Mass
Spectroscopy/Mass Spectroscopy
The mobile phase used was acetonitrile: 0.5mM trifluoro
ammonium acetate in water (70:30) with the flow rate of
0.6mL/min at UV absorption of 254nm. The LC showed a major
peak at 14.110mins, whose molecular weight was 569 (M+H)
and the minor peak at 13.718 mins, showed a mass at 552. The
molecular weights correspond to the carotenoids are lutein and
cryptoxanthin, respectively. However, the compounds need to be
further purified for confirmation studies

28. Mass spectrum for lutein

29. High Resolution Mass spectrum of lutein
The high resolution mass spectrum of lutein shows the molecular weight of
the molecule as 568.4335 amu. Hence the molecular formula of lutein was
deduced as C40H56O2.

30. Structure of lutein
H3C OH
H3C CH3
CH3
HO
H3C CH3
H3C H3C
CH3
CH3
1
3
1'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
15'
4
5
7
8
9
10
11
12
13
14
15
The high resolution mass spectrum of lutein shows the molecular
weight of the molecule as 548.67amu. Hence the molecular
formula of lutein was deduced as C34H36N4O3.

31. NMR spectrum of green pigment
The NMR spectrum showed singlets at 9.5, 9.3 and 8.5 ppm respectively. This is characteristic of olefinic
protons of the porphyrin ring. A doublet of doublet at 8.0ppm and two doublets at 6.1 and 6.3ppm are
characteristic of exomethylene protons. Three singlets at 3.2, 3.4 and 3.6ppm indicate the presence of
methyls attached to the pyrrole ring. Based on these observations, the molecule was characterized as
methyl pyropheophorbide-a (Figure 13), a compound formed by the degradation of chlorophyll-a in higher
plants and algae. The central magnesium atom is absent.

32. Structure of methyl pyropheophorbide-a
N
NH N
HN
O
H3C
CH3
CH3
H3C
O
OCH3
This particular pigment derivative, Methyl pyropheophorbide finds major applications in
photodynamic therapy, where this derivative is accumulated in tumorous tissues, which acts
as a photosensitizer when a laser radiation is passed to target the tumor cells. It is also used
in photosynthetic studies.

33. Total antioxidant capacity
The diluted extract solution (0.5 mg/ml and 1mg/ml)
were added to a test tube containing 5 ml reagent
solution(0.6 M sulfuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate).
Various concentrations of Gallic acid were used as
reference standard. The reaction mixture was
incubated at 90C for 1 hour and then cooled to room
temperature. The absorbance was measured at 695
nm against distilled water as blank.

34. Result
0
10
20
30
40
50
60
70
Control CV5
Antioxidant
activity
(%)
Lutein
The total antioxidant capacity of the lutein shows good efficiency of about
63.9% when compared with control which is 33.5%.

35. Bacteria used- Bacillus subtilis, Bacillus cereus, Micrococcus luteus,
Staphylococcus aureus, Streptococcus pyogens, Staphylococcus epidermis ,
Escherichia coli , Klebsiella pneumonia, Proteus mirabilis, Pseudomonas
aeruginosa, Salmonella enterica, Serratia marcescens , Vibrio cholera (01),
Yersinia enterocolitica, Enterobacter aerogenes and Vibrio cholerae (0139)

36. Result
Studies on the anti-bacterial activity
against 16 human pathogenic bacterial
strains revealed that lutein exhibited a
small zone of inhibition. However, it
needs further confirmation studies. On
the control, Methyl pyropheophorbide-a
did not showed any activity.
Lutein
Methyl
pyropheophorbide a

37. Anti-tuberculosis activity
Screening of Compounds by Luciferase Reporter Phage (LRP) at a concentration of 100 µg/ml concentration against
a set of standard strains including M. tuberculosis H37Rv, sensitive clinical isolate and resistant clinical isolate.
Read with luminometer at 10S integration
% RLU Reduction = Control RLU – Test RLU X 100
----------------------
Control RLU
Result= 24.74%(lutein)

38. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. About 5 x 103 cells of HEK 293 and
MDA-MB-231S were seeded in 96-well plates and allowed to grow for 24 h.
Different concentrations of lutein and methyl pyropheophorbide a ranging
from 0.1-100 µg/mL (0.1, 0.5, 1.0, 2.5, 5, 10, 25, 50 and 100 µg/mL) were
tested. The cells were incubated with the extracts for 48 h followed by the
addition of MTT and further incubation for 4 h. The medium was removed
completely and the formazan crystals were dissolved in DMSO. The optical
density was measured using an ELISA reader (Molecular Devices, Spectra Max
190, USA) at 570 nm.
Anti proliferative assay

39. MDA MB 231S
The cell viability was calculated using the following
formula:
% Cell Viability = (A570 sample / A570 control) x 100.
0
20
40
60
80
100
120
Control Solvent
control
0.1 0.5 1 2.5 5 10 25 50 100
CV5 CV6

40. Result
The cell viability tests demonstrated that the lutein and methyl
pyropheophorbide-a exerts 50 % of inhibition of growth of breast cancer
cells, MDA-MB-231S at 0.1-0.5 µg/mL. Further confirmatory studies are
required to prove the efficacy of the compounds as anti-cancer drug.
However, it has been reported that methyl pyropheophorbide reduces the
tumour size by 14 % in experimental rats