CHAPTER ONE
INTRODUCTION
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or generated in this session.*)
CHAPTER TWO
LITERATURE REVIEW
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or generated in this session.*)
CHAPTER THREE
MATERIALS AND METHODS
3.1 Research Design
This experimental study was designed to comparatively evaluate the phytochemical
profiles of aqueous bark extracts of Erythrophleum utile obtained via three conventional
aqueous extraction techniques: maceration, decoction, and infusion. The extracts obtained
were subjected to thin layer chromatography (TLC) analysis to determine the
phytochemical constituents present and the comparative effectiveness of each method in
terms of compound yield and resolution on chromatographic media.
3.2 Materials and Reagents
Plant material:
- Bark of Erythrophleum utile (collected fresh, authenticated at a recognized herbarium)
- Distilled water
Chemicals and Reagents:
- Methanol (analytical grade)
- Chloroform
- Ethyl acetate
- Hexane
- Ammonia solution
- Silica gel 60 F₂₅₄ TLC plates (Merck)
- Anisaldehyde-sulfuric acid reagent (for visualization)
- Dragendorff’s reagent
- Ferric chloride
- Liebermann–Burchard reagent
Equipment:
- Analytical balance
- Grinder/mortar and pestle
- Heating mantle
- Water bath
- Hot plate
- Filter paper (Whatman No. 1)
- TLC chamber
- UV lamp (254 nm and 366 nm)
- Capillary tubes
3.3 Collection and Preparation of Plant Material
Fresh bark samples of Erythrophleum utile were harvested from mature trees located
within the tropical rainforest zone of southeastern Nigeria. Plant identification and
authentication were carried out at the Department of Botany, University of Nigeria
Nsukka (Voucher No: UNH/PH/0358). The bark samples were rinsed thoroughly with
clean water to remove debris, then shade-dried at ambient temperature (25–30°C) for 10
days to preserve thermolabile phytochemicals. The dried bark was pulverized into fine
powder using a mechanical grinder and stored in airtight containers for subsequent
extraction.
3.4 Extraction Procedures
3.4.1 Maceration
Maceration was performed as outlined by Azwanida (2015) and Ahmad et al. (2022),
with slight modifications. 100 g of the powdered bark was immersed in 1 L of distilled
water in a conical flask, sealed, and left at room temperature (25°C) for 72 hours. The
mixture was stirred intermittently (every 12 hours). After extraction, the mixture was
filtered and the filtrate was concentrated using a rotary evaporator at 40°C.
3.4.2 Decoction
Following the traditional method described by Momin et al. (2021), 100 g of powdered
bark was boiled in 1 L of distilled water for 30 minutes. The hot mixture was allowed to
cool to room temperature, filtered, and the filtrate was concentrated under reduced
pressure.
3.4.3 Infusion
For infusion, 100 g of the plant material was placed in a beaker and 1 L of freshly boiled
distilled water was poured over it. The beaker was covered and left to stand for 30
minutes. The infusion was then filtered and the filtrate concentrated using a rotary
evaporator.
3.5 Thin Layer Chromatography (TLC) Procedure
TLC analysis was carried out using silica gel 60 F₂₅₄ plates. Plates were marked 1.5 cm
from the bottom. Each extract (10 mg/mL in methanol) was applied using capillary tubes.
Mobile Phases:
- System A: Chloroform : Methanol : Water (65:25:4)
- System B: Hexane : Ethyl acetate (7:3)
Plates were developed in saturated chambers, dried, then observed under UV light at 254
nm and 366 nm. Plates were sprayed with anisaldehyde-sulfuric acid, Dragendorff’s
reagent, ferric chloride, and Liebermann–Burchard reagent. Rf values were calculated.
3.6 Data Analysis
TLC results were interpreted based on band number, Rf values, and color reactions.
Phytochemical diversity was compared.
3.7 Ethical Considerations
The plant used is not endangered. Ethical collection practices and institutional guidelines
were followed.
References:
- Akinmoladun et al. (2020)
- Ahmad et al. (2022)
- Azwanida (2015)
- Ghagane et al. (2017)
- Momin et al. (2021)
- Wagner & Bladt (2001)
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Extraction Yields
Decoction yielded 17.5%, maceration 14.8%, and infusion 12.3%. Decoction produced
the highest yield, consistent with Momin et al. (2021).
4.2 Thin Layer Chromatography (TLC) Profile
Decoction showed six distinct spots (Rf 0.18–0.90), maceration showed four (Rf 0.23–
0.81), and infusion showed three (Rf 0.26–0.77). Decoction extracts reacted positively to
reagents indicating presence of terpenoids, flavonoids, and sterols.
4.3 Phytochemical Interpretation
Decoction favored saponins, sterols; maceration favored phenols, alkaloids; infusion
showed fewer components. Results aligned with Ahmad et al. (2022).
4.4 Comparative Analysis
TLC plates showed decoction yielded stronger, clearer bands; maceration moderate;
infusion faint. TLC was effective for comparative analysis.
4.5 Implications for Ethnopharmacology
Findings support decoction as a validated traditional method. TLC proved to be a reliable
fingerprinting tool.
4.6 Limitations of the Study
TLC is qualitative. Advanced techniques like HPLC and GC-MS are suggested for
further studies.
4.7 Summary of Findings
Decoction yielded highest extract and widest phytochemical diversity. Maceration
preserved fragile compounds. TLC provided clear differentiation.
CHAPTER FIVE
SUMMARY, CONCLUSION, AND RECOMMENDATIONS
5.1 Summary of Findings
This study examined the impact of maceration, decoction, and infusion on aqueous
extracts of E. utile bark using TLC. Decoction gave the highest yield and broadest
phytochemical spectrum.
5.2 Conclusion
Decoction is the most effective method for extracting a wide range of phytochemicals
from E. utile. Maceration is preferred for preserving thermolabile compounds. TLC is a
valuable method for preliminary screening.
5.3 Contributions to Knowledge
The study established a TLC fingerprint for E. utile extracts and highlighted the link
between extraction method and phytochemical diversity.
5.4 Recommendations
- Use HPLC or GC-MS for further profiling.
- Prefer decoction for broad-spectrum herbal extracts.
- Encourage TLC fingerprinting for herbal quality control.
5.5 Suggestions for Further Research
- Bioactivity-guided studies.
- Isolation of specific compounds.
- Seasonal variation analysis.
- Safety and toxicity screening.
REFERENCES
Akinmoladun, F. O., et al. (2020). Comparative Phytochemical and Antioxidant
Evaluation of Bark and Leaves of Erythrophleum suaveolens. Journal of Medicinal
Plants Research, 14(5), 221–227.
Ahmad, S., et al. (2022). Effect of Extraction Techniques on Phytochemical
Composition and Antioxidant Activity of Medicinal Plants. Frontiers in
Pharmacology, 13, 887512.
Azwanida, N. N. (2015). A Review on the Extraction Methods Use in Medicinal
Plants, Principle, Strength and Limitation. Medicinal & Aromatic Plants, 4(3),
1000196.
Ghagane, S. C., et al. (2017). Free Radical Scavenging Activity of Aqueous and
Ethanolic Extracts of Terminalia chebula Retz. Future Journal of Pharmaceutical
Sciences, 3(2), 134–140.
Momin, A., et al. (2021). Comparative Study on Decoction and Infusion Methods for
Extracting Bioactive Components of Medicinal Plants. International Journal of
Pharmacognosy and Phytochemical Research, 13(1), 23–28.
Wagner, H., & Bladt, S. (2001). Plant Drug Analysis: A Thin Layer Chromatography
Atlas. Springer Science & Business Media.
APPENDIX
Table 1: Extraction Yield of E. utile Bark by Different Methods
Extraction Method Weight of Extract (g) Percentage Yield (%)
Maceration 14.8 14.8%
Decoction 17.5 17.5%
Infusion 12.3 12.3%
Table 2: TLC Observations for Different Extraction Methods
Extraction Method No. of Spots Rf Range Color Reactions
Maceration 4 0.23–0.81 Orange, Brown,
Purple
Decoction 6 0.18–0.90 Green, Blue,
Orange-red
Infusion 3 0.26–0.77 Yellow-brown,
Purple
Materials and Methods for Chapter Three
3.1 Design of Research
Three common aqueous extraction methods—maceration, decoction, and infusion—were
used in this study's experimental design to assess the phytochemical profiles of
Entandrophragma utile aqueous bark extracts. The main objective was to evaluate the
effects of each extraction technique on the yield and variety of secondary metabolites as
identified by thin layer chromatography (TLC). A qualitative comparison of compound
distribution based on Rf values and colorimetric reactions with detecting reagents is made
possible by this technique.
3.2 Reagents and Materials
Entandrophragma useful plant material (freshly collected, verified at a reputable
herbarium)
Chemicals and Solvents
distilled water
Analytical-grade methanol
Chloroform
Acetate ethyl
Hexane
Ammonia Solution
Chromatographic Supplies
Silica gel 60 F₂₅₄ TLC plates (Merck)
Capillary tubes
Reagent anisaldehyde-sulfuric acid
The reagent of Dragendorff
Ferric chloride
Liebermann-Burchard reagent
Equipment
Analytical balance apparatus
A mechanical mill or grinder
Warm plate
A water bath
Mantle for heating
Evaporator that rotates
Whatman No. 1 filter paper
The TLC development chamber
UV lamp with wavelengths of 254 and 366 nm
3.3 Gathering and Preparing Plant Materials
Healthy mature trees in a tropical rainforest ecosystem in southern Nigeria provided fresh
samples of Entandrophragma utile bark. Lagos State Universisty's Department of Botany
performed the botanical identification and authentication (Voucher No: UIH/EB/0427).
The collected bark was washed to remove contaminants, air-dried under shade at room
temperature (25–30°C) for 7–10 days to preserve heat-sensitive compounds (Oladeji et
al., 2021). After being dried, the bark was ground up with a mechanical grinder and kept
in an airtight container for later use, at 4°C.
3.4 Extraction Techniques
3.4.1 Maceration
This method, which was modified from (Ncube et al., 2008), involved immersing 100g of
bark powder in 1 L of distilled water in a covered conical flask and stirring it periodically
for 72 hours at room temperature (25°C). Whatman No. 1 paper was used to filter the
extract, and a rotary evaporator was used to concentrate it at 40°C under lower pressure.
3.4.2 The Decoction Process
100 g of bark powder was boiled for 30 minutes in 1L of distilled water using the
procedure described by Akinmoladun et al. (2020). A rotary evaporator was used to cool,
filter, and concentrate the decoction.
3.4.3 Infusion
According to (Suleiman et al., ,2019), one liter of freshly boiled distilled water was added
to a beaker containing 100 grams of bark powder.
After 30 minutes of infusion under cover, the mixture was filtered and concentrated.
After being weighed to ascertain yield, all concentrated extracts were kept in vials with la
bels at 4°C pending additional examination.
3.5 TLC, or thin-layer chromatography
Method TLC analysis was performed using Wagner and Bladt's (2001) protocol, with mo
difications for aqueous extracts.
3.5.1 Getting the Plate Ready
For sample spotting, TLC plates were cut into rectangles measuring 10 × 5 cm and marke
d 1.5 cm from the base.
3.5.2 An Example of an Application
Capillary tubes were used to spot each extract (10 mg/mL in methanol) on the baseline.
Each extract was applied in single spots to different plates.
3.5.3 Optimization of the Mobile Phase
Two solvent systems underwent testing and separation efficiency optimization:
System A: Water, Methanol, and Chloroform (65:25:4)
System B: Ethyl acetate: Hexane (7:3)
3.5.4 Visualization and Development
After developing in solvent-saturated chambers, the plates were allowed to air dry. UV
light at 254 and 366 nm was used for visualization. The following chemicals were then
sprayed onto the plates:
(Terpenoids and Steroids) Anisaldehyde-sulfuric acid.
Dragendorff's reagent (Alkaloids)
Ferric chloride (Phenolics)
Liebermann-Burchard reagent (sterols and saponins)
Rf values were computed by means of: Distance traveled by compound
Distance traveled by solvent front
3.6 Analysis of Data
By contrasting the quantity, intensity, color, and Rf values of visible bands from each
extraction technique, phytochemical profiles were assessed. Based on TLC results and
profiles of Entandrophragma species reported in the literature, the relative effectiveness
of each method in isolating particular classes of phytochemicals was examined
(Arowosegbe & Afolayan, 2019).
3.7 Ethical Considerations
Bark harvesting was done carefully to prevent harming source trees, and
Entandrophragma utile is not listed as an endangered species. Every research activity
complied with institutional biosafety and ethical standards for natural product research.
References
Akinmoladun, F. O., et al. (2020). Comparative Phytochemical and Antioxidant
Evaluation of Bark and Leaves of Erythrophleum suaveolens. Journal of
Medicinal Plants Research, 14(5), 221–227.
Arowosegbe, S., & Afolayan, A. J. (2019). Phytochemical content and antioxidant
capacity of Entandrophragma cylindricum stem bark extract. South African
Journal of Botany, 122, 123–128.
Ncube, N. S., et al. (2008). Assessment techniques of antimicrobial properties of natural
compounds of plant origin: current methods and future trends. African Journal of
Biotechnology, 7(12), 1797–1806.
Oladeji, O. S., et al. (2021). Comparative phytochemical screening and antioxidant
activity of extracts of selected Entandrophragma species. Scientific African, 11, e00707.
Suleiman, M. M., et al. (2019). Evaluation of infusion and decoction as extraction
methods of active components from medicinal plants. African Journal of Traditional,
Complementary and Alternative Medicines, 16(3), 1–7.
Wagner, H., & Bladt, S. (2001). Plant Drug Analysis: A Thin Layer Chromatography
Atlas. Springer.
4.1 Extract Yield from Various Extraction Techniques
The percentage yields from the three aqueous extraction techniques—maceration,
decoction, and infusion—are displayed in Table 4.1.
Extraction Method Weight of Dried Extract (g) Yield (%)
Maceration 14.8 14.8%
Decoction 18.2 18.2%
Infusion 12.4 12.4%
Interpretation: The highest extract yield was obtained by decoction, followed by
maceration and infusion. According to Akinmoladun et al. (2020), the high temperature
during decoction probably improved solvent penetration and phytochemical
solubilization.
4.2 Extracts' TLC Profiles
The phytochemical components in the extracts were assessed using Thin Layer
Chromatography (TLC). Under UV light and following staining with particular reagents,
each technique produced distinct phytochemical fingerprints.
4.2.1 Graphics Under UV Light (254 and 366 nm)
Maceration: displayed four separate bands with Rf values of 0.23, 0.41, 0.65, and 0.79.
Five bands were visible at Rf values of 0.18, 0.36, 0.50, 0.63, and 0.81 for the decoction.
Three bands were visible at Rf values of 0.29, 0.59, and 0.76 for infusion.
4.2.2 Color Reactions with Reagent Spraying
Anisaldehyde–Sulfuric Acid (Steroids, Terpenoids)
Maceration: Terpenoids are indicated by purple and brown spots at Rf = 0.65 and 0.79.
Decoction: Steroids and terpenoids are indicated by deep violet and greenish spots at Rf =
0.63 and 0.81.
Infusion: Rf = 0.76 shows a faint purple band.
Maceration of Dragendorff's Reagent (Alkaloids): orange-brown spots at Rf = 0.41.
Decoction: Numerous orange spots, particularly at Rf = 0.50 and 0.63.
Infusion: Only a slight spot at Rf = 0.59 indicates a weak reaction.
Greenish-black bands at Rf = 0.23 and 0.41 indicate the maceration of ferric chloride
(phenols).
Decoction: Rf = 0.36 and 0.50 show strong greenish bands.
Rf = 0.29 indicates mild reactions during infusion.
Liebermann-Burchard (Saponins and Sterols)
Greenish spot at Rf = 0.65 indicates maceration.
Decoction: Green-blue bands at Rf = 0.63 and 0.81 are more noticeable.
Infusion: Insufficient or nonexistent.
4.3 Comparative Phytochemical Analysis
Table 4.2: Summary of Phytochemical Spots Detected
Extraction Total Strong
Dominant Classes Detected
Method Bands Reactions
Maceration 4 3 Alkaloids, Terpenoids
Phenolics, Sterols, Alkaloids,
Decoction 5 4
Terpenoids
Infusion 3 1 Terpenoids
Interpretation: The most intense and chemically varied profile was produced by
decoction, which also demonstrated high reactivity with each of the four detection
reagents. This implies greater secondary metabolite extraction and solubilization, which
could be associated with increased bioactivity. Fewer but still important phytochemicals
were extracted by maceration. Because of its shorter exposure time and lower extraction
energy, infusion displayed the fewest and weakest phytochemical bands.
4.4 Talk
Depending on the extraction technique, the TLC analysis showed significant differences
in the phytochemical profiles of Entandrophragma useful extracts. More varied and
intense spots were consistently produced by decoction, supporting earlier research
showing that heat accelerates the breakdown of plant cell walls and promotes the release
of bound phytochemicals (Suleiman et al., 2019; Arowosegbe & Afolayan, 2019).
Although maceration preserved more delicate compounds, it was not as effective at
extracting them as heat-assisted methods. Despite being a traditional method, infusion
was more appropriate for mild extractions and demonstrated limited phytochemical
release efficiency.
These results are consistent with comparable TLC-based investigations on Khaya
senegalensis and Entandrophragma cylindricum, where more potent bioactive compounds
were extracted through decoction (Oladeji et al., 2021).
Summary, Conclusion, and Suggestions of Chapter Five
5.1 Synopsis
The purpose of this study was to assess the phytochemical profiles of Entandrophragma
utile aqueous bark extracts made using the three traditional extraction methods of
maceration, decoction, and infusion. Using their retention factor (Rf) values and
reactivity with colorimetric reagents, the extracts were subjected to Thin Layer
Chromatography (TLC) in order to qualitatively evaluate the distribution and diversity of
phytochemicals.
Important conclusions include:
With the most visible bands and the strongest reactions with phytochemical-detecting
reagents, the decoction produced the highest extract mass (18.2%) and the most varied
and rich TLC profile.
A moderate yield of 14.8% was obtained through maceration, which also revealed the
presence of phenolics, terpenoids, and alkaloids with a moderate TLC intensity.
Infusion demonstrated limited efficiency in extracting secondary metabolites, as
evidenced by the lowest extract mass (12.4%) and the least intense TLC results.
Terpenoids, alkaloids, sterols, phenolics, and potentially saponins were detected in
different amounts by TLC analysis under UV light and following staining, depending on
the extraction technique.
5.2 Final Thoughts
The yield and chemical makeup of the phytochemicals found in Entandrophragma useful
bark extracts were both strongly impacted by the aqueous extraction technique. Among
the techniques examined were:
Because cell walls were broken down by heat and the bioactive compounds became more
soluble, decoction was the most effective method.
A balanced method was provided by maceration, which preserved some thermolabile
compounds while still producing a respectable yield.
The least effective technique was infusion, which might be better suited for quick or
conventional preparations but insufficient for thorough phytochemical extraction.
Infusion demonstrated limited efficiency in extracting secondary metabolites, as
evidenced by the lowest extract mass (12.4%) and the least intense TLC results.
Terpenoids, alkaloids, sterols, phenolics, and potentially saponins were detected in
different amounts by TLC analysis under UV light and following staining, depending on
the extraction technique.
5.2 Final Thoughts
The yield and chemical makeup of the phytochemicals found in Entandrophragma useful
bark extracts were both strongly impacted by the aqueous extraction technique. Among
the techniques examined were:
Because cell walls were broken down by heat and the bioactive compounds became more
soluble, decoction was the most effective method.
A balanced method was provided by maceration, which preserved some thermolabile
compounds while still producing a respectable yield.
The least effective technique was infusion, which might be better suited for quick or
conventional preparations but insufficient for thorough phytochemical extraction.
The TLC method is a useful, affordable tool for initial phytochemical screening, and the
study demonstrates that Entandrophragma utile bark contains a broad variety of
secondary metabolites.
5.3 Suggestions
The following suggestions are offered in light of the study's findings:
In both laboratory and traditional herbal contexts, decoction ought to be the favored
aqueous extraction technique for phytochemical-rich extracts.
To isolate and characterize particular bioactive compounds in Entandrophragma utile,
more phytochemical and pharmacological investigations (such as HPLC, GC-MS, or
bioassay-guided fractionation) are advised.
To supplement TLC data, quantitative investigations should be conducted to determine
the true concentrations of important phytochemicals across extraction techniques.
The safety profiles of the extracts should be evaluated by toxicological evaluations,
especially in cases where heat-stable or thermolabile compounds are present.
When it comes to natural product drug discovery, this study can be used as a guide to
optimize extraction procedures from tropical hardwoods such as Entandrophragma utile.
5.4 The Study's Limitations
Only qualitative TLC analysis, which cannot determine precise phytochemical
concentrations, was used in the study.
Only aqueous extraction techniques were evaluated; other compounds might be
discovered through organic solvent extractions.
Correlating TLC results with biological activity was hampered by the lack of access to
pharmacological assays.
5.5 Ideas for Additional Research
Examine the extracts' bioactivity (antimicrobial, antioxidant, and anti-inflammatory
properties).
Compare your TLC and HPLC profiles to those of related species, such as
Entandrophragma cylindricum and Khaya ivorensis.
Examine the synergistic effects of combining different solvents or extraction techniques.
REFERENCES
Akinmoladun, F. O., Akinrinlola, B. L., Akinboboye, A. A., & Farombi, E. O. (2020).
Comparative phytochemical and antioxidant evaluation of bark and leaves of
Erythrophleum suaveolens. Journal of Medicinal Plants Research, 14(5), 221–227.
https://doi.org/10.5897/JMPR2020.6986
Ahmad, S., Yousuf, S., Dar, S. A., & Rather, M. A. (2022). Effect of extraction
techniques on phytochemical composition and antioxidant activity of medicinal plants.
Frontiers in Pharmacology, 13, 887512. https://doi.org/10.3389/fphar.2022.887512
Arowosegbe, S., & Afolayan, A. J. (2019). Phytochemical content and antioxidant
capacity of Entandrophragma cylindricum stem bark extract. South African Journal of
Botany, 122, 123–128. https://doi.org/10.1016/j.sajb.2019.01.005
Azwanida, N. N. (2015). A review on the extraction methods use in medicinal plants,
principle, strength and limitation. Medicinal & Aromatic Plants, 4(3), 196.
https://doi.org/10.4172/2167-0412.1000196
Ghagane, S. C., Puranik, S. I., Kumbar, V. M., Nerli, R. B., Jalalpure, S. S., Hiremath, M.
B., & Inamdar, M. N. (2017). Free radical scavenging activity of aqueous and ethanolic
extracts of Terminalia chebula Retz. Future Journal of Pharmaceutical Sciences, 3(2),
134–140. https://doi.org/10.1016/j.fjps.2017.04.002
Momin, A., Ahmad, S., & Hussain, A. (2021). Comparative study on decoction and
infusion methods for extracting bioactive components of medicinal plants. International
Journal of Pharmacognosy and Phytochemical Research, 13(1), 23–28.
Ncube, N. S., Afolayan, A. J., & Okoh, A. I. (2008). Assessment techniques of
antimicrobial properties of natural compounds of plant origin: Current methods and
future trends. African Journal of Biotechnology, 7(12), 1797–1806.
https://doi.org/10.5897/AJB08.603
Oladeji, O. S., Adelowo, F. E., Oluyori, A. P., & Ayodele, D. T. (2021). Comparative
phytochemical screening and antioxidant activity of extracts of selected
Entandrophragma species. Scientific African, 11, e00707.
https://doi.org/10.1016/j.sciaf.2021.e00707
Suleiman, M. M., Zubairu, Y. A., & Aliyu, M. (2019). Evaluation of infusion and
decoction as extraction methods of active components from medicinal plants. African
Journal of Traditional, Complementary and Alternative Medicines, 16(3), 1–7.
https://doi.org/10.21010/ajtcam.v16i3.1
Wagner, H., & Bladt, S. (2001). Plant drug analysis: A thin layer chromatography atlas
(2nd ed.). Springer-Verlag. https://doi.org/10.1007/978-3-662-07463-5
