These presentation slides are about extraction, identification and antioxidant activities of carotenoids from Ipomoea aquatica Forsk. It is commonly known as Morning water glory or water spinach which is rich source of nutrients,, wide array of carotenoids are extracted and identified by GC- MS techniques by researcher arond the Globe. Antioxidant activity of natural products can be evaluated by different assay; some them are discussed in these slides.

1. Extraction, identification and antioxidant activities of carotenoids from
Ipomoea aquatica Forsk
Name: Pragati Shah
Roll no: 63/072, Msc. 4th semester
Central Department of Chemistry
Tribhuvan University, Kirtipur
Kathmandu, Nepal
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2. CONTENTS
• INTRODUCTION
• OBJECTIVES
• MATERIALS AND METHODS
• RESULTS AND DISCUSSION
• CONCLUSIONS
• REFERENCES
• ACKNOWLEDGEMENT
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3. INTRODUCTION
Carotenoids are group of compounds found in plants, algae and various
microorganisms.
Chemical structure of carotenoids is based on a C40 tetraterpenoid structure having
extended conjugated double-bond system.
On the basis of their chemical structures, these compounds can be divided into 2
different types:
1.Hydrocarbon carotenoids, generally named carotenes.
2. Oxygenated carotenoids, commonly known as xanthophylls.
Carotenoids are found to be strong antioxidants being effective for scavenging the
reactive oxygen species (ROS).
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(Zhu et al., 2011 )

4. • Antioxidants are the molecules that inhibit oxidation of other molecules.
• Body requires a supplement of dietary antioxidants which can be obtained only by
the consumption of an antioxidant-rich diet.
• Among dietary supplements, green leafy vegetables are the one which can provide
necessary antioxidant to our body.
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5. Ipomoea aquatica, commonly called ‘Water spinach’.
Family: Convolvulaceae
Native to southeast Asia and is herbaceous vine
found in marshy places.
The study on Ipomoea aquatica has revealed
diverse array of compounds like vitamins, carotenoids, alkaloids,
flavonoids, chlorophylls, amino acids etc.
 β-carotene, β-cryptoxanthin,lutein, lutein epoxide,
violaxanthin and neoxanthin are the carotenoids
found in water spinach.
In traditional medicine system it has been used against various disorders like
nosebleed , nervous debility, liver malfunction ,heavy metal poisoning.
( Lawal et al., 2015 )
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6. OBJECTIVES
 To extract carotenoids from Ipomoea aquatica .
 To identify carotenoids compounds by LC-MS.
 To carry out ABTS radical cation-scavenging assay.
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7. MATERIALS AND METHODS
100 g of fresh and mature
leaves were purchased
from market, washed and
mixed with deionized
water.
Carotenoids were extracted
with mixture of methanol,
acetone and petroleum ether
(1: 1:1:) then saponified
with KOH in methanol.
Then extract was washed
with water, concentrated
by rotary evaporator,
resolved by acetone and
purified by TLC .
Then carotenoids were
prepared as stock solution of
1 g/l in acetone ,filled with N2
and stored at -20 ºC for
further experiments.
The total extract and purified
fractions were analysed by
TLC with β-carotene standard
and Lutein standard and their
Rf values were measured.
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8. Visible spectroscopic data for
purified carotenoid were measured
in different solvents by UV-visible
spectrophotometer.
Some test for particular
functional group in carotenoids
epoxide, aldehyde, ketone
group were carried out and
monitored for diagnostic
change in UV-spectrum.
Purity of fractions were
checked by reversed phase
HPLC and analysed by using
water HPLC system.
LC-MS was also used for the
identification of specific
carotenoids.
1. Identification of extracted carotenoids
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9. 2. ABTS radical cation-scavenging assay
Decrease in absorbance was measured exactly 1 min after mixing the solution then up to 6
min. The final absorption was noted. BHT was used as standard.
Different conc. of sample (1.6, 2.4, 4, 6 and 8 μg/mL) were prepared. 0.5 mL of sample
was mixed with 3 mL of ABTS working standard in a cuvette.
ABTS solution (7 mM) mixed with ammonium persulphate (2.45 mM) solution, kept for
12–16 h in the dark to produce colored solution of ABTS radical cations. This stock
solution was diluted with methanol and initial absorbance was measured at 734 nm.
% inhibition of radicals due to antioxidant property of the carotenoid was calculated as:
%inhibition = [(Acontrol - Asample)/Acontrol] × 100%
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10. 1.Identification of the isolated fractions
TLC of carotenoids showed 7 spots, and c1, c2 and c3 were the major spots, their Rf values
were 0.160, 0.247 and 0.960, respectively.
 Rf values of c2 and c3 matched the values of Lutein and of β-carotene standard in the TLC,
therefore c2 and c3 were identified as Lutein and β-carotene.
RESULTS AND DISCUSSIONS
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1. Total ion chromatogram and mass spectrum of C1
601
583

13. 13
2.Total ion chromatogram and mass spectrum of C2
569
533

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3.Total ion chromatogram and mass spectrum of C3
537
445

15. 4.1. ABTS-radical cation scavenging activity of individual carotenoid.
 Group bar chart indicate ABTS radical cation scavenging potential of 3 different
carotenoids at concentration range of 1.6, 2.4, 4, 6 and 8 μg/mL.
 Violaxanthin showed an inhibition of 88.21±1.03% at 8 μg/mL.
Fig 1:Percentage inhibition of ABTS radical cation by carotenoids and BHT
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16. S. N. Compounds IC50 value (μg/mL)
1. β-carotene 5.85
2. Lutein 3.54
3. Violaxanthin 3.38
4. BHT 10.93
Table 2: IC50 value for carotenoids and BHT
Fig 2: Concentration dependent inhibitory effect of carotenoid and BHT
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17. CONCLUSIONS
 The carotenoids extracted from water spinach were identified as β-Carotene,
Lutein and Violaxanthin.
 Violaxanthin, with double 5,6-epoxy groups, performed highest percentage
inhibition in ABTS radical cation scavenging assay.
 It was concluded that the individual carotenoid scavenges the free radical in a
concentration dependent manner.
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18. Statistical analysis
 All analyses were run in triplicate.
 Results were expressed with standard deviation of mean.
 Correlation and regression analyses were carried out to determine the
relationship between free radical scavenging activity and concentration of
carotenoids.
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19. REFERENCES
• El-Sawi, N., Gad, M. H., Al-Seeni, M. N., Younes, S., Soad, E. M. E. G., & Ali, S.
(2017). Evaluation of Antidiabetic Activity of Ipomoea Aquatica Fractions in
Streptozotocin Induced Diabetic in Male Rat Model. Sohag Journal of Science,
2(1), 9-17.
• Fu, H., Xie, B., Ma, S., Zhu, X., Fan, G., & Pan, S. (2011). Evaluation of
antioxidant activities of principal carotenoids available in Ipomoea aquatica.
Journal of Food Composition and Analysis, 24(2), 288-297.
• Hamid, K., Ullah, M. O., Sultana, S., Howlader, M. A., Basak, D., Nasrin, F., &
Rahman, M. M. (2011). Evaluation of the Leaves of Ipomoea aquatica for its
Hypoglycemic and Antioxidant Activity. Journal of Pharmaceutical Sciences and
Research, 3(7), 1330-1333.
• Lawal, U., Shaari, K., Ismail, I. S., Khatib, A., & Abas, F. (2016). Antioxidant and
[alpha]-Glucosidase Inhibitory Activities of Isolated Compounds from Ipomoea
aquatica. Records of Natural Products, 10(6), 701-707.
• Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. J., &
McLaughlin, J. L. (1982). Brine shrimp: a convenient general bioassay for active
plant constituents. Planta Medica, 45(05), 31-34.
• Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American journal of Enology and
Viticulture, 16(3), 144-158.
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20. ACKNOWLEDGEMENT
I want to express my sincere gratitude to
Prof. Dr. Ram Chandra Basnyat (HOD)
And my supervisors:
Prof. Dr. Niranjan Parajuli
Dr. Surya Kant Kalauni
Dr. Bimala Subba
Dr. Sushika Mulmi
Dr. Khaga Raj Sharma
And my all friends .
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21. THANK YOU
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22. 22
Mass spectrum of C2
569

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C: mass spectrum of fraction C1
A: HPLC profile of fraction C1
601
583

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HPLC profile of C2
Mass spectrum of C2
569
533

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HPLC profile of C3
Mass spectrum of C3
537
445

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1. HPLC profile of fraction C1
2. HPLC profile of fraction C2

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3. HPLC profile for fraction C3