Internship Report

Submitted to

Manonmaniam Sundaranar University, Tirunelveli

In partial fulfilment of the requirements for the award of the degree of

MASTER OF SCIENCE

IN

BIOTECHNOLOGY

Submitted by

A. Sheik Shahul Meeran

(Reg No: 23083126503011014)

DEPARTMENT OF BIOTECHNOLOGY

NOORUL ISLAM COLLEGE OF ARTS & SCIENCE

KUMARACOIL - 629 180

OCT 2024
EXTRACTION OF PHYTOPHENOLS
SI.no CONTENTS Page no

1 Introduction

2 Reagents Required

3 Materials Required

4 Methodology

5 Result

6 Conclusion

7 Reference
I.INTRODUCTION

Aerva lanata, a perennial herb belonging to the Amaranthaceae family, has gained
attention for its diverse medicinal properties and rich phytochemical profile Aerva lanata
is particularly valued for its high content of polyphenols, which are bioactive compounds
known for their antioxidant, anti-inflammatory and antimicrobial effects(Bitasta and
Madan 2016). This study focuses on the extraction of polyphenols from two distinct plant
parts leaves and stem using an innovative non-conventional method Microwave-Assisted
Extraction(Egbuna et al. 2019).

Polyphenols are naturally occurring compounds that contribute significantly to

human health by combating oxidative stress and reducing the risk of chronic diseases.
The extraction of these compounds is critical for utilizing the therapeutic potential of
Aerva lanata (Muscolo et al. 2024). Traditional extraction methods, while effective, can
be time-consuming and may lead to the degradation of sensitive compounds. In contrast,
MAE offers a rapid and efficient alternative that utilizes microwave energy to enhance
the extraction process. This technique is particularly advantageous as it reduces solvent
usage and extraction time while improving the yield and quality of the extracted
compounds(Polash et al. 2017).

The leaves, rich in flavonoids and other phenolic compounds, are expected to yield
a higher concentration of bioactive constituents(Santhosh et al. 2016). Meanwhile, the
stems, often overlooked, may also contribute valuable polyphenolic profiles that warrant
investigation. By analyzing these two samples, we aim to compare their polyphenol
content and evaluate the effectiveness of MAE as a sustainable extraction method(Vitale
et al. 2022).Optimizing extraction methods for plants like Aerva lanata can lead to
increased availability of polyphenol-rich extracts for use in functional foods,
nutraceuticals and pharmaceuticals. This study aims to provide insights into the potential
of Aerva lanata as a sustainable source of bioactive compounds, highlighting its role in
promoting health and wellness(Bitasta and Madan 2016).

II.WORKPLAN

PLANT EXTRACTION

EXTRACTION

MICROWAVE ASSISTED EXTRACTION(MAE)

100% H2O 50% ETHANOL: 50% H2O

TOTAL POLYPHENOL CONTENT

 RESULT

III.Reagents required;

• Folin- Ciocalteu Reagent (FCR)

• Sodium Carbonate (Na2CO3)

IV.Materials required

• Plant samples (Stem and Leaf)

• Microwave oven

• Borosilicate glass bottle

• UV Spectrophotometer

• Incubator

• Micropipette (20µl and 1000µl)

• Microtips

• Eppendorf tubes

• Cuvette

• Test tubes
 • Distilled water

• Cotton

• Tissue paper

V.METHODOLOGY

1) Selection of Plant

Aerva lanata., an abundant plant in the environment, was to quantify its

polyphenol content using a non-conventional extraction method known as maceration-
assisted extraction (MAE)(Kodhaiyolii et al. 2019). Healthy samples were collected from
a pristine habitat to ensure quality, and the plant material was dried using air drying or a
dehydrator at low temperatures to preserve the integrity of the polyphenolic
compounds(Alara et al. 2019). After drying, the material was ground into a fine powder
to enhance its extraction efficiency. Various solvents, including ethanol, methanol and
distilled water, were employed in conjunction with the MAE technique, optimizing
parameters such as temperature and extraction time to maximize yield(Bastola et al.
2017). Following extraction, the solution was filtered to remove solid residues, and the
extract was concentrated using methods like rotary evaporation or freeze-drying. The
extracted polyphenols were characterized using analytical techniques such as high-
performance liquid chromatography (HPLC) or spectrophotometry to assess their
composition and concentration. Finally, the extracts were stored in dark, airtight
containers at low temperatures to maintain stability, while strict adherence to safety
protocols and regulatory guidelines ensured reliable results in quantifying the polyphenol
content of Aerva lanata (Swamy et al. 2012).
2) Extraction

Extraction refers to the process of separating polyphenolic compounds from plant

materials, such as fruits, vegetables, leaves, seeds, or herbs, using a solvent or a
combination of solvents. Polyphenols are naturally occurring compounds with
antioxidant properties, commonly extracted for use in nutraceuticals, pharmaceuticals,
cosmetics and food products(Ballard et al. 2010). The extraction process typically begins
with the selection of a solvent, such as ethanol, methanol, water, or acetone, chosen based
on its ability to dissolve polyphenols while ensuring safety for the intended use(Filip et
al. 2017). Plant material is often ground or dried to increase the surface area for better
solvent contact. The solvent is then applied to the plant material using various techniques
like maceration, Soxhlet extraction, ultrasound-assisted extraction or microwave-assisted
extraction(Deng et al. 2022). During this process polyphenolic compounds dissolve into
the solvent. Finally, after extraction, the polyphenols are separated from the solid plant
residue, and the solvent is typically evaporated or removed to obtain a concentrated
extract, which can be further purified or analyzed(Pandey and Rajbhandari 2014).

View

3) Microwave Assisted Extraction

Microwave-Assisted Extraction (MAE) is a green extraction method that uses

microwave energy to enhance the release of polyphenolic compounds from plant
materials(Do et al. 2014). In this process, the plant material is ground or dried and mixed
with a solvent such as ethanol, methanol, or water. The mixture is then exposed to
microwave radiation, which rapidly heats both the solvent and the plant material,
increasing the temperature and pressure inside the plant cells. This heat causes the cells to
rupture, allowing polyphenols to be released into the solvent more efficiently(Genwali et
al. 2013). After extraction, the liquid extract is separated from solid residues and can be
purified or concentrated further. MAE offers several advantages over traditional methods,
including faster extraction, improved efficiency, reduced solvent use, and lower energy
consumption, making it more environmentally friendly. These benefits make MAE a
preferred choice for extracting bioactive compounds like polyphenols, as it provides
higher yields with less environmental impact(García-Mahecha et al. 2023).

4) Total Phenolic Content

Total Polyphenol Content (TPC) is a measure of all the polyphenols in a plant

extract, which are natural compounds in plants known for their health benefits,
particularly as antioxidants that protect cells from damage. TPC indicates how much
polyphenol is present in the extract and is typically measured using a color-changing test,
often the “Folin-Ciocalteu method”. The result is expressed in **mg of gallic acid
equivalents (GAE)** per gram of sample, where gallic acid serves as a standard for
comparison. A higher TPC means more polyphenols, which translates to stronger
antioxidant activity and potential health benefits. TPC is useful for comparing different
plant extracts, helping to identify those rich in polyphenols. Overall, TPC provides
insight into the polyphenolic content of an extract and its potential health-promoting
properties(Baba and Malik 2015).
 VI. RESULT

1) Plant Collection

To prepare Aerva lanata samples for polyphenol extraction, begin by carefully

collecting healthy leaf and stem parts during the peak growing season, ideally in the early
morning or late afternoon to reduce moisture loss and ensure optimal polyphenol content.
After collection, thoroughly rinse the samples with distilled water to eliminate any dirt
and contaminants. Next, dry the samples in a shaded area to prevent degradation from
sunlight, or use a dehydrator set to a low temperature, ensuring they are completely dried,
which may take several days depending on environmental conditions. Once dried, grind
the samples using a mortar and pestle or a mechanical grinder, aiming for a fine powder
with a consistent particle size of less than 1mm to maximize extraction efficiency. Store
the ground samples in airtight containers, protecting them from light and moisture to
preserve their polyphenol content. It's important to label each container clearly with
details such as the collection date, the specific part of the plant and any other relevant
information. Additionally, maintain a detailed log of all collection and preparation
activities, including environmental conditions and observations, to ensure proper
documentation and facilitate future analyses. This thorough approach will enhance the
quality and reliability of the polyphenol extraction process.

2) Extraction

Add a small volume of stem sample and leaf sample into the jar containing fully
water or 50:50 ratio of water and ethanol solvent mixture. This solvent mixture helps to
penetrate the cell which inturn helps to recover the secondary metabolites of the cell. The
solvent selection varries depending upon the sample taken. The extracted product was
precipitated by using centrifugation.
3) Microwave Mediated Extraction

Microwave-assisted extraction (MAE) is considered superior to traditional

methods for polyphenol extraction due to its efficiency and speed. It rapidly heats the
plant material and solvent, leading to faster cell rupture and quicker release of
polyphenols. This results in higher yields with less solvent, making MAE more
environmentally friendly and cost-effective. Unlike traditional methods, which can be
time-consuming and require larger amounts of solvent, MAE offers improved extraction
efficiency and better preservation of sensitive polyphenols, as it minimizes long heat
exposure. Additionally, MAE consumes less energy, making it more sustainable. Overall,
MAE is a faster, more efficient and environmentally conscious approach to polyphenol
extraction compared to conventional methods.
4) Total Phenolic Content

100% water method;

Using 100% water as a solvent in Microwave-Assisted Extraction (MAE)

for polyphenol extraction can be effective for hydrophilic polyphenols due to water's
polar nature. However, its efficiency may be limited when extracting polyphenols that are
more lipophilic, as water alone may not dissolve them well. While water can be
advantageous for eco-friendly extraction, the yield of polyphenols may not be as high
compared to using a solvent mixture, such as water combined with ethanol or methanol.
These mixed solvents help to balance the polarity and improve the extraction efficiency
by better dissolving both hydrophilic and lipophilic compounds. The extraction process is
enhanced by the use of heat and pressure in MAE, which helps to break down cell walls,
but 100% water may be less effective in complex matrices, such as those containing
lipids or proteins, which could interfere with the process. Ultimately, while water is a
safe and environmentally friendly option, solvent mixtures are often preferred for
maximizing extraction yields and covering a broader range of polyphenolic compounds.

I) 100% Water dilution (Leaf)

Sample Water FCR Incubation Na2CO3 Incubation O.D

20µl 1980µl 100µl 8 min 300µl 2 hrs 0.067

ii) 100% Water dilution (Stem)

Sample Water FCR Incubation Na2CO3 Incubation O.D

Blank 1600 µl 100µl 8 min 300µl 2 hrs 0.000
20µl 1580µl 100µl 8 min 300µl 2 hrs 0.483

Hydro-Alcoholic Method

Using a hydro-alcoholic solvent for polyphenol extraction in Microwave-

Assisted Extraction (MAE) is highly effective due to its ability to extract both
hydrophilic and lipophilic polyphenolic compounds. A mixture of water and alcohol
(usually ethanol or methanol) offers a balanced polarity, making it ideal for dissolving a
wide range of polyphenols. Hydrophilic polyphenols, like phenolic acids, are efficiently
extracted by the water component, while lipophilic polyphenols, such as flavonoids, are
better extracted by the alcohol. Alcohol also helps break down plant cell walls and reduce
solvent viscosity, improving the overall extraction process. Additionally, alcohol can
prevent the enzymatic degradation of polyphenols, ensuring higher yields. Ethanol is
most widely used choice due to its safer, more environmentally friendly profile compared
to methanol, which is toxic. The optimal alcohol-to-water ratio varies depending on the
polyphenols and plant material, with common ratios ranging from 50:50 to 70:30
(alcohol:water). Overall, a hydro-alcoholic solvent in MAE enhances the extraction
efficiency and recovery of polyphenols, especially when compared to water-only
methods.

i)Hydro-alcoholic Dilution (Leaf)

Sample Water FCR Incubation Na2CO3 Incubation O.D

Blank 1600 µl 100µl 8 min 300µl 2 hrs 0.000
20 µl 1980 µl 100µl 8 min 300µl 2 hrs 0.161
ii) Hydro-alcoholic Dilution (Stem)

Sample Water FCR Incubation Na2CO3 Incubation O.D

20 1980 100 8min 300 2 hrs 0.402

VII. CONCLUSION

In conclusion, the extraction of polyphenols from Aerva lanata. using microwave-

assisted extraction (MAE), a non-conventional method, has demonstrated significant
potential, particularly with stem samples extracted in 100% water, which yielded the
highest polyphenol content compared to hydroalcoholic methods. This finding suggests
that water may serve as an optimal solvent for extracting bioactive compounds from the
stem, emphasizing the importance of selecting appropriate extraction conditions for
maximizing yield and efficiency.

The results highlight the efficacy of MAE as a rapid and effective extraction
technique while showcasing Aerva lanata. as a rich source of polyphenols. Given the
known health benefits of these compounds—including antioxidant, anti-inflammatory,
and antimicrobial properties—future applications could include the development of
functional foods, dietary supplements, and natural health products derived from this
plant.
 Further research is warranted to explore the specific health benefits associated
with the polyphenolic extracts of Aerva lanata., as well as potential applications in the
pharmaceutical and cosmetic industries. Additionally, investigating the synergistic effects
of combining different extraction methods or exploring other parts of the plant could
provide new insights into maximizing the therapeutic potential of Aerva lanata.. Overall,
the use of non-conventional methods like MAE opens new avenues for the efficient
extraction of valuable phytochemicals, positioning Aerva lanata. as a promising
candidate for further scientific exploration. Moreover, further research is warranted to
explore the specific health benefits associated with the polyphenolic extracts of Aerva
lanata., as well as potential applications in the pharmaceutical and cosmetic industries.
Investigating the synergistic effects of combining different extraction methods or
exploring other parts of the plant could also provide new insights into maximizing the
therapeutic potential of Aerva lanata..

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