Characterization and application of red mangrove (Rhizophora racemosa) bark extract as an indicator

1. Characterization and Application of Red Mangrove (Rhizophora racemosa) Bark
Extract as an Indicator
Master’s Degree Research Presentation
By
Korfii Uebari
(PG.2017/01982)
(HND RIVPOLY, PGD RSU)
Department of Chemistry
Supervisors: Dr. N. Boisa
Prof. T. J. K. Ideriah
Rivers State University
Faculty of Science

2. Graphical Abstract
Introduction
Justification of the Study
Aim and Objectives
Literature Review
Materials and Methods
Results and Discussion
Conclusion
Recommendations
Contributions to Knowledge
References
Outline

3. 0
1
2
3
0 500 1000
Absorbance
wavelength nm
Characterization
Application
Collection of Sample
Graphical Abstract
Classical
Phytochemical Screening
Endpoint color changes
Titration
GC-FID Results
Preparation of the Plant Sample
Extraction
UV-Visible Spectrum
I.R Spectrum
1 2 3 4 5
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
water extract
Absorbance
% Concentration
Molar Absorptivity
Bark of the plant

4. 1
Introduction
An indicator is a substance that gives a visible sign, usually by a colour change, of the
presence or absence of a threshold concentration of a chemical species, such as an acid or an
alkali in a solution (Eze & Ogbuefi, 2014).
Dyes are conjugated molecules, generally consisting of aromatic and/or unsaturated
compounds that are either derived from natural sources or are made synthetically.
Natural dyes are derived from plants, invertebrates, or minerals. The majority of natural dyes
are dyes from plant sources (Purwar, 2016).
Dyes applications are feasible because of the presence of chromophores and auxochromes
(Luqman et al., 2017).

5. 2
Justification of the Study
Environmental issues from synthetic indicators (Pathade et al., 2009).
Apart from the environmental related issues of synthetic indicators, studies have shown that
some of these synthetic indicators have toxic effects on users (Okoduwa et al., 2015; Abugri
et al., 2012; Pathade et al., 2009).
Several researchers; Abugri et al., (2012), Izonfuo et al., (2006), Okoduwa et al., (2015), and
Trivedi et al., (2016) have reported a variety of local plants to have contained different types
of dyes which are used as indicators.

6. 3
Aim and Objectives
Aim
This study characterized and evaluated the potential of extracts from red mangrove
(Rhizophora racemosa) bark as an indicator.

7. 4
1. Extraction of dye from red mangrove plant (Rhizophora racemosa) bark
2. Identification of the components in the extracted plant using classical phytochemical screening
and GC-FID techniques
3. To determine the wavelength of maximum absorptions (λmax) of the extracted dyes using Ultra-
Violet/Visible spectroscopy to determine the optical properties of the extracts
4. Identification of functional groups present in the extracted dye from the plant using Fourier
Transform Infra-Red spectroscopy (FT-IR)
5. Application of the extracts as potential indicator through titration
Objectives

8. 5
Literature Review
Authors of other
Researches
Plants used Methods and Solvents used Application Instrumental
Characterization
Izonfuo et al., (2006) Hibiscus sabdariffa and
Basella alba
Traditional Method, Aqueous and
Ethanol
Acid-Base
Titration
UV/Visible spectroscopy
Abugri et al., (2012) Guinea corn Traditional Method, Ethanol Acid-Base
Titration
UV/Visible spectroscopy
Eze & Ogbuefi, (2014) Urena Lobata (Mgbo) Traditional Method
Ethanol, Cold Water, and Hot Water
Acid-Base
Titration
NA
Onwuachu et al., (2014) Hibiscus, Mango, Ginger
& Kolanut
Ethanol Acid-Base
Titration
NA
Okoduwa et al., (2015) Rose (Rosa setigera),
Allamanda (Allamanda
cathartica), Hibiscus
(Hibiscus rosa-sinensis)
Soxhlet Extraction Method
Cold Method
Methanol and Water
Acid-Base
Titration
UV/Visible spectroscopy
Trivedi et al., (2016) Euphorbia milii Traditional method, Methanol Acid-Base
Titration
NA
Present Research Red Mangrove Plant
(Rhizophora racemosa)
Traditional Method, Ethanol and
Water
Acid-Base Titration UV/Visible spectroscopy,
Infra-Red spectroscopy,
and GC-FID
NA = Not Applicable

9. 6
Chemical Properties of Dyes
The hue of dyes depend on the entire light absorbing system.
Direct Red 81
(James, 1997)
Indicators are able to change colors with pH changes by donating or
accepting protons

10. 7
Materials and Methods Application
Titration
Characterization
Collection
of Sample
Preparation of the Plant Sample
Extraction
Identification of the Plant
Traditional Method
Classical
Phytochemical Screening
(Onwuka, 2018)
GC-FID Results
UV-Visible Analysis
Molar Absorptivity I.R Analysis
Data Analysis
Data Analysis
Bark of the plant

11. 8
Results and Discussion
Table 1. Classical Phytochemical Screening
Sample Tannin Saponin Cardiac
Glycoside
Steroids Terpenoids Alkaloids Anthraquinone Flavonoids
Water
Extract
+ + + + - - + +
Ethanol
Extract
+ + + + + + + +
The same phytochemical compounds were reported by Udeozo et al., (2018), Edu et al.,
(2015), Poompozhil & Kumarasamy, (2014), Ukoima et al., (2013), Ganesh & Vennila
(2011) and Obi & Onuoha, (2000) in Rhizophora racemosa, and other mangrove plant
species.
+ Present, - Absent

12. GC-FID Results
Phytochemicals Water Extract ug/ml Ethanol Extract ug/ml
Alkaloids 2.5171 1.6344
Tannins 4.7026 4.6642
Flavonoids 26.084 28.859
Phenol 11.8429 6.9698
Saponins 4.2997 3.9587
Oxalate 2.3746 2.5587
Phytate 1.9860 0.6926
Steroids 11.9544 12.4892
Total 65.7613 61.8366
Table 2. GC-FID Results of Red Mangrove Plant (Rhizophora racemosa) Extracts
Amadi et al., (2017), Emejulu et al., (2017), Azubuike et al., (2016) and Njoku & Obi, (2009) have reported
that plants contain varying amounts of flavonoids, tannins, saponins, alkaloids, phenolic acid, oxalate, phytate
and steroids. 9

13. 0.472
0.183
1.748
2.183
2.483
0.288
0.184
0.544
0.099
0
0.5
1
1.5
2
2.5
3
0 200 400 600 800 1000
Absorbance
Wavenumber nm
Figure 1a. UV-Visible spectra of water and ethanol extracts
1.189
1.378
1.074
2.595
1.103
0.348
0.084 0.106 0.04
0
0.5
1
1.5
2
2.5
3
0 200 400 600 800 1000
Absorbance
Wavelength nm
Ethanol Extract
UV-Vis Spectroscopy Results
Immediately After Extraction
10
Water Extract

14. UV-Vis Spectroscopy Results
Water Extract
Ethanol Extract
72 hrs After Extraction
11
Figure 1b. UV-Visible spectra of water and ethanol extracts

15. Findings from the present study agree with the reports of Sudarshan et al., (2011) on
Thevetia thvetiodes and Thevetia peruvianei and Espinosa-Morales et al., (2012) on Justicia
spicigera that extracts from plants could absorb at 581 nm, 555nm and 537nm respectively.
Izonfuo et al., (2006) also obtained a λmax of 520 nm for the ethanol extracts of Hibiscus
rosasinensis.
These findings showed that the some plant extracts absorbed within the visible region (400 –
750 nm) of the electromagnetic spectrum justified by their colour productions.
12

16. Water Extract_001
Name
Water Extract
Description
4000 350
3500 3000 2500 2000 1500 1000 500
104
7
10
20
30
40
50
60
70
80
90
100
cm-1
%T
3440.91cm-1
436.82cm-1
529.92cm-1
1634.43cm-1
411.00cm-1
390.64cm-1
378.21cm-1
354.51cm-1
366.71cm-1
358.96cm-1
2059.32cm-1
1441
1111.7
1290.3
FT-IR Results
Figure 2a FT-IR Spectrum of the Water Extract
13

17. Frequency
Range (cm-1)
Absorption
(cm-1)
Appearanc
e
Functional
Group
Compound
Class
Comment
4000-3000 3440.91 Strong
Broad
O-H Stretching Alcohol, Phenol Intermolecular
bonded
1800-1600 1634.43 C=O Stretching Carboxylic acid
1600-1400 1441.0 Medium C-H Bending Alkane Methyl group
1400-1000 1290.3 Strong C-O Stretching Aromatic ester
1111.7 Strong C-O Stretching Secondary
alcohol
Table 3. Interpretation for Rhizophora racemosa Water Extract
Merck Life Science (Sigma-Aldrich) Standard for IR Spectrum Table and Chart
14

18. Ethanol Extract_1
Name
Ethanol Extract
Description
4000 350
3500 3000 2500 2000 1500 1000 500
161
15
20
30
40
50
60
70
80
90
100
110
120
130
140
150
cm-1
%T
3447.67cm-1
1636.77cm-1
638.37cm-1
366.80cm-1
396.05cm-1
1065.95cm-1
2062.11cm-1
377.09cm-1
359.08cm-1
1520.4
1445
1286.3
421.41
Figure 2b FT-IR Spectrum of the Ethanol Extract
15

19. Frequency
Range (cm-1)
Absorption
(cm-1)
Appearance Functional
Group
Compound
Class
Comment
4000-3000 3447.67 Strong
Broad
O-H Stretching Alcohol,
Phenol
Intermolecular
bonded
1800-1600 1636.77 C=O Stretching Carboxylic acid
1600-1400 1520.4 Strong N-O Stretching Nitro
compound
1445 Medium C-H Bending Alkane Methyl group
1400-1000 1256.3 Strong C-O Stretching Aromatic ester
Table 4. Interpretation for Rhizophora racemosa Ethanol Extract
Merck Life Science (Sigma-Aldrich) Standard for IR Spectrum Table and Chart
16

20. Findings from the present study agree with the FTIR reports by Al-Alwani, (2017) and
Nhapi, (2016) on their reports for functional group characterization of Strelitzia reginae
flowers and Eichhornia crassipes dyes.
The present findings also agree with the reports by Espinosa-Morales et al., (2012) in their
research on characterization of a natural dye by spectroscopic techniques.
Reports by Udeozo et al., (2018) on the efficacy of Rhizophora racemosa wood revealed the
presence of the functional groups C=O, O-H and C=N. Findings from the present study
agree with this report.
17

21. Titration (Acid-Base)
Indicators HCl / NaOH HAc / NaOH HCl / NH4OH HAc / NH4OH
Methyl orange 26.63±0.2 25.67±0.2 3.02±0.0 5.4±0.1
Methyl red 25.53±0.7 26.37±0.8 3.00±0.1 9.5±0.1
Phenolphthalein 23.53±0.4 25.17±0.2 2.02±0.0 3.02±0.0
Water Extract 26.70±0.2 28.1±0.3 4.17±0.2 3.3±0.1
Ethanol Extract 26.03±0.1 25.43±0.1 4.03±0.1 3.37±0.1
Table 5.Titration for 25.00ml of 0.1M of the Base was Titrated against 0.1M Solution (Acid)
*All values are mean ± S.D. for n=3
HCl: Hydrochloric acid, HAc: Acetic Acid, NaOH: Sodium Hydroxide, NH4OH:
Ammonium Hydroxide 18

22. Endpoint Color Change from the Titration
Acid
Yellow
Red
Base
This agree with other findings reported by Kapilraj et al., (2019), Nair et al., (2018), Trupti,
(2017), Byamukama et al., (2016), Eze & Ogbuefi, (2014), Abugri et al., (2012), Udachan et
al., (2012), Pathade et al., (2009), Patil et al., (2009), Bhagat et al., (2008), and Nwosu et
al., (2004) on the fact that extracts from plants could act as indicators in titration. 19
Figure 3. Endpoint Color Change from the Titration

23. 1 2 3 4 5
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
water extract
Absorbance
% Concentration
1 2 3 4 5
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ethanol extract
Absorbance
% Concentration
Figure 4. Molar Absorptivity of the Red Mangrove Plant (Rhizophora racemosa)
Extracts
Molar Absorptivity for Water Extract Molar Absorptivity for Ethanol Extract
From the present findings, it is revealed that molar absorptivity of extracts from plant can be derived.
This agree with the reports of Izonfuo et al., (2006) where they derived the molar absorptivity of H.
sabdariffa and B. Alba.
450 nm 400 nm
20

24. 21
Table 6. Comparative Properties of the Indicators
Indicators Functional
Groups
λmax Colour
changes
Solubility Color/Forms Presence of
Chromophore
Presence of
Auxochrome
Methyl Orange N=N
S=O +
420 nm + Red - Yellow Soluble in water,
practically
insoluble in
ethanol
˧
Orange-Yellow/Powder
crystalline +
Yes Yes
Methyl Red C=O
O-H
N=N +
410 nm ͯ Red - Yellow Soluble in
ethanol, partially
soluble in water ˧
Dark red crystalline
powder+
Yes Yes
Phenolphthalein O-H
C=O +
550nm + Colourless -
Pink
1 g dissolves in
12 ml alcohol.
In water, 400
mg/L at room
temperature
+
white or yellowish-white
to pale orange fine
crystalline powder
+
Yes Yes
Water Extract of
Rhizophora
racemosa
O-H, C=O, and
N-O
450 nm,
559 nm
Yellow – Red Soluble in water
and ethanol
Ox-blood/Liquid Yes Yes
Ethanol Extract
of Rhizophora
racemosa
O-H, C=O, and
N-O
400 nm,
572 nm
Yellow – Red Soluble in water
and ethanol
Ox-blood/Liquid Yes Yes
Sources: + PUBCHEM, ˧ CHEMSPIDER, ͯ Sigma-Adrich

25. Conclusion
The results of this study showed that the red mangrove plant (Rhizophora racemosa) extracts are suitable to
be used as indicators.
Classical phytochemical analysis and GC-FID revealed the presence of tannins, alkaloids, steroids,
cardiac glycosides, flavonoids, phenol, anthraquinone, saponins, terpenoids in the extracts.
UV-Vis spectroscopy showed the maximum absorptions of the extracts. The Wavelength of maximum
absorbance fall between 400 – 450nm and 559 – 572 for the fresh extracts.
I.R spectroscopy showed the presence of C-O, O-H, C=O, C-O and N-O groups in the extracts from the
plant.
Titration revealed that the plant changed colour from yellow in an acidic solution to red in the alkaline
solution at the endpoint.
The molar absorptivity of the extracts from the plant showed the intrinsic property of the chemical
species of the plant extracts. 22

26. It is
recommended
that:
Schools
Development of
Analytical
Methods
Development of
Indicator Papers
Research
Laboratories
Recommendations
23

27. Contributions to Knowledge
The research revealed that extracts from red mangrove (Rhizophora racemosa) bark has
the potential to serve as an indicator in titration.
The research also revealed that extracts from red mangrove (Rhizophora racemosa) bark
absorb within the visible region of the electromagnetic spectrum.
24

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