An-Najah National University
Faculty of Engineering and Information Technology
Chemical Engineering Department
Graduation Project 2
“Drying Rates of Calendula Herb and Antioxidant
Test of Ascorbic Acid”
Prepared by:
Dalal Salah
Leena Khaled
Mahmoud Fashafsha
Malik Mahmoud
Shahd Odeh
Supervised By:
Dr. Husni Odeh
Dr. Majd Shhadi
This project was submitted in partial fulfillment of the requirements for the degree of
Bachelor in Chemical Engineering
June 2020
i
Abstract
Calendula officinalis L. (Pot marigold) is one of the commonly used medicinal plants in
India, China, Europe and US. This plant can be planted easily and successfully in Palestine
in winter. The plant species has been reported to contain a variety of phytochemicals,
including carbohydrates, phenolic compounds, lipids, steroids, tocopherols, terpenoids,
quinones and carotenoids.
The Calendula officinalis extracts possess a wide range of pharmacological effects and are
used as antiseptic, stimulant, diaphoretic, anti-spasmodic and anti-pyretic agents. The flower
extracts of the plant have anti-viral effects on HIV. In-vitro, Calendula officinalis plant
extracts show anti-cancerous activity on various tumor cell lines derived from leukemia’s,
melanomas, breast, cervix, prostate, pancreas and lung. It has also been internally used for
the treatment of gastritis, colitis and bleeding of duodenal ulcers.
The main objective is to examine the present of antioxidant agents in both of Calendula
leaves and flowers, and then compare it with the antioxidant activity of Ascorbic acid. The
ethanolic extracts are prepared from Calendula powders obtained from dried Calendula
leaves and flowers. The chosen antioxidant test is 1,1-diphenyl-2-picrylhydrazyl (DPPH).
DPPH experiments have been started only for the Ascorbic acid, but not completed due to
COVID-19 disease outbreak.
The Graduation Project work is completed by carrying out theoretical analysis of
experimental drying data conducted by Dr. Husni Odeh at room temperature and at 55-60 °C
for Calendula flower. The drying rate equations and drying characteristic curves for
Calendula herb flowers are derived and presented. The increasing drying condition from
20- 22 ºC to 55-60 ºC shows increasing the rate of drying about 150 times.
ii
Contents
Abstract ................................................................................................................................. i
Contents .............................................................................................................................. ii
List of Tables ....................................................................................................................... v
List of Figures ...................................................................................................................... v
List of Figures from Appendices ........................................................................................ vi
Abbreviations ................................................................................................................... vii
Chapter One: Introduction ................................................................................................... 1
1.1: Background ........................................................................................................................... 1
1.2: Problem Statement ................................................................................................................ 5
1.3: Objectives .............................................................................................................................. 5
1.4: Scope of the Work ................................................................................................................. 6
1.5: Significance ........................................................................................................................... 6
1.6: Organization of the Report .................................................................................................... 7
Chapter Two: Difficulties and Challenges .......................................................................... 8
Chapter Three: Literature Review ....................................................................................... 9
3.1: Calendula general uses ......................................................................................................... 9
3.2: Therapeutic Potential of Calendula officinalis .................................................................... 10
3.2.1: Antidiabetic and anti hyperlipidemic activities ................................................................ 10
3.2.2: Cardiovascular activities .................................................................................................. 11
3.2.3: Hepatoprotective activities ............................................................................................... 11
3.2.4: Antioxidant activities ....................................................................................................... 11
3.2.5: Anthelmintic activities ..................................................................................................... 11
3.2.6: Anti inflammatory activities ............................................................................................ 11
3.2.7: Wound healing activities .................................................................................................. 12
3.2.8: Anticancer activities ......................................................................................................... 12
3.2.10: Antibacterial activities ................................................................................................... 12
3.2.11: Sedative drugs ................................................................................................................ 12
iii
3.2.12: Antihypertensive drugs .................................................................................................. 12
3.2.13: Hypoglycemic drugs ...................................................................................................... 12
3.2.14: Cholesterol lowering drugs ............................................................................................ 12
3.3: Phytochemistry of Calendula officinalis ............................................................................. 13
3.3.1: Terpenoids ........................................................................................................................ 13
3.3.2: Flavonoids ........................................................................................................................ 13
3.3.3: Coumarins ........................................................................................................................ 13
3.3.4: Quinones .......................................................................................................................... 13
3.3.5: Volatile oil ........................................................................................................................ 13
3.3.6: Carotenoids ...................................................................................................................... 13
3.3.7: Amino acids ..................................................................................................................... 14
3.3.8: Carbohydrates .................................................................................................................. 14
3.3.9: Lipids ............................................................................................................................... 14
3.3.10: Other constituents........................................................................................................... 14
3.4: Extraction ............................................................................................................................ 15
3.5: Antioxidant Capacity of Calendula..................................................................................... 16
3.5.1: Some methods for evaluating the antioxidant. ................................................................. 16
3.5.2: Antioxidant evaluation of Calendula extract by DPPH and other methods ..................... 20
3.5.3: Comparison between antioxidant activity of Calendula extract and another antioxidant 21
3.5.4: The relationship between antioxidant activity and phenolic compounds ......................... 21
3.6: Calendula side effects and special precautions ................................................................... 22
3.7: Drying of herbs ................................................................................................................... 23
3.7.1: Effect of different drying methods on antioxidant activities and color of Calendula herb.24
3.7.2: Effect of drying on concentrations of several phytochemicals and chemical components of
essential oils. .............................................................................................................................. 25
Chapter Four: Methodology .............................................................................................. 27
4.1: Extraction ............................................................................................................................ 27
4.2: UV-Test for Absorbance ..................................................................................................... 28
iv
4.3: Drying herbs ........................................................................................................................ 31
Chapter Five: Results and Discussion ............................................................................... 34
5.1: Drying of Calendula flowers .............................................................................................. 34
5.2: Absorbance and %inhibition for Ascorbic acid solution..................................................... 44
Chapter Six: Conclusion .................................................................................................... 47
Chapter Seven: Future Work and Recommendation ......................................................... 48
References ......................................................................................................................... 50
Appendices ......................................................................................................................... A
v
List of Tables
Table 1: The amount needed from each solution to achieve a certain diluted concentration
of ascorbic acid. .................................................................................................................... 30
Table 2: Start parameters of dried Calendula flowers. ......................................................... 32
Table 3: Drying data obtained at room temperature. ............................................................ 32
Table 4: Drying data obtained at 55-60° C ........................................................................... 33
Table 5: The value of X at room temperature. ..................................................................... 36
Table 6: The value of X at 55-60 °C. ................................................................................... 37
Table 7: Experimental data for the plot of drying rate vs. moisture at room temperature. .. 40
Table 8: Summary results for data at 20-22 °C drying. ........................................................ 41
Table 9: Summary results for data at 55-60 °C drying. ........................................................ 41
Table 10: Absorbance readings and inhibition calculation for ascorbic acid. ...................... 44
List of Figures
Figure 1: Calendula plant. .................................................................................................... 10
Figure 2: Different colors of Calendula flowers. ................................................................. 10
Figure 4: Different plant drying dynamics: M1- marigold, M2- lemon balm, M3-origanum,
M4- common agrimony, M5- common lavender, and M6- common sage (Aboltins and Kic,
2016). .................................................................................................................................... 24
Figure 5: Drying dynamics of Calendula flowers. ............................................................... 35
Figure 6: Plot of X vs. time at room temperature. ................................................................ 38
Figure 7: Plot of X vs. time at 55-60 °C. .............................................................................. 38
Figure 8: Drying rate vs. free moisture at room temperature. .............................................. 40
Figure 9 : Rate of drying vs free moisture at 55-60 °C. ........................................................ 42
Figure 10: Changes in the moisture content of Calendula flowers as related to drying time
at different degrees of drying air temperature and relative humidity of 40%. ..................... 43
Figure 11: Changes in the moisture content of Calendula flowers as related to drying time
at different degrees of drying air temperature and relative humidity of 60%. ..................... 43
Figure 12: Setting %inhibition vs. Concentration. ............................................................... 45
Figure 13: DPPH test by setting %inhibition vs. Log concentration.................................... 45
vi
List of Figures from Appendices
Figure A 1: Dried Calendula flowers. ................................................................................... A
Figure A 2: Grinded Calendula flowers. ............................................................................... A
Figure A 3 : Water shaker bath. .............................................................................................. B
Figure A 4: The two samples of Calendula flowers and leaves in the shaker. ..................... B
Figure A 5: Vacuum filtration of the flowers. ....................................................................... C
Figure A 6: During the process of vacuum filtration of the flowers. .................................... D
Figure A 7: After collecting the samples from the vacuum filtration. .................................. D
Figure A 8: Scanning of the wavelength of the blank. ........................................................... E
vii
Abbreviations
Calendula Officinalis. L.: Calendula officinalis Linn.
HIV: Human Immunodeficiency Virus.
GC-MS: Gas chromatography–mass spectrometry.
m/z: Mass-to-charge ratio.
ROS: Reactive oxygen species.
SO: Superoxide dismutase.
UV–Vis: Ultraviolet–Visible Spectrophotometry.
Cis and Trans: Are from Latin "this side of" and "the other side of", respectively.
w or wt %: Weight percentage.
OS: Organic solvent extraction.
h: Hour.
min: Minute.
g: Gram.
mg: Milligram.
mmol: Millimol.
μg: Microgram.
v/v: Volume ratio.
HECO: Hydroethanolic extract of Calendula officinalis.
et al.: For the Latin phrase et alia which means "and others."
A: Area.
H: Height.
DPPH: 1,1-diphenyl-2-picrylhydrazyl.
ABTS: 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid.
viii
FRAP: Ferric Reducing Antioxidant Power.
EDTA: Ethylenediaminetetraacetic acid.
BHT: Butylated hydroxytoluene.
IC: Inhibitory concentration.
EC: Effective concentration.
A0: Absorbance of the control reaction.
A1: Absorbance in presence of all of the extract samples and reference.
nm: Nanometer.
R: Rate of drying.
X: Moisture content.
FD: Freeze drying.
HA: Hot air drying.
FIR-HA: Combined far-infrared radiation with hot air convection.
1
Chapter One: Introduction
1.1: Background
Calendula officinalis is also known as garden marigold. It is a member of the Asteraceae
family. Other members of this plant family include daisies, arnica, chamomile, and yarrow.
This bright, flowering herb opens its gold blossoms in the morning and closes them at dusk,
or when rain threatens. Calendula is native to Asia and southern and central Europe. It is
cultivated throughout the world and valued for its culinary and medicinal uses. The first
name, Calendula, is from the Latin kalendae, the word Romans used to indicate that it
bloomed throughout the year in their area. The second name officinalis indicates that
Calendula was included in official lists of medicinal herbs. The common name marigold
refers to the blossoms' association with the Virgin Mary (Health Benefits, 2020).
Calendula plant likes sun and will re-seed from year to year, even in poor soil. The erect,
square and branching stems emerge from a taproot to grow up to 60 cm high. The bushy herb
blooms continuously throughout the summer. Seeds are crescent to horseshoe shaped with a
rough exterior (Health Benefits, 2020).
Calendula flower was grown as an ornamental plant for long times until its medicinal effects
were discovered and thus, it began to be used as a medicinal plant. In Europe, the growth of
this plant began in 12th century. Its flowers have been introduced as drugs in some
pharmacopoeia, to cure stomach and intestine diseases. This plant has bitter taste and has
been used as an efficient pesticide, and the plant is used among other plants to kill insects as
well. On the other hand, the pot marigold plant grown by Egyptians, Arabs, Indians, Greeks
and European has been used for medicinal purposes from 12th century (Kashani and
Mohammad, 2012).
This plant is grown as a medical drug in Germany, Australia, Czech, Austria, Switzer land,
Hungary and recently in Egypt and Syria as well. It is also grown in the Mediterranean
countries such as the Balkan states, the east of Europe, north of America and also in Germany
(Kashani and Mohammad, 2012).
2
Calendula officinalis is globally known for its medicinal importance containing various
phytochemicals including carbohydrates, amino acids, lipids, fatty acids, carotenoids,
terpenoids, flavonoids, quinones, coumarins and other constituents, showing some important
biological activities like wound healing, immuno-stimulant, spasmogenic and spasmolytic,
hepatoprotective, genotoxic and antigenotoxic, anti-amylase, anti-inflammatory, anti
oedematous, anti-bacterial and anti-fungal, antioxidant, antidiabetic, anti-HIV and anti-
cancerous, nephron-protective, prevention of oropharyngeal mucositis, hypoglycemic and
gastroprotective activities with no toxic effect (Raina et al., 2014). Phytochemicals in this
project will assay by the GC-MS.
Distribution in Palestine: Mediterranean Woodlands and Shrublands, Semi-steppe
shrublands, Shrub-steppes, Deserts and extreme deserts, Mediterranean coast, Nablus
mountains, Jerusalem and Dead Sea valley (Mahmiyat.ps, 2020).
1.1.1: Introduction to drying of herbs
Requirements of worldwide progress in cultivation of medicinal and aromatic herbs are
necessary for their processing and safety storage. The need for high quality herb raw material
is increasing. This phenomenon is proven by number of cultivation results and registration
procedures, concerning medicinal and aromatic plant cultivars, recently reported from
different countries. In pharmacy, plant raw materials are important sources of new medicines
and their substitutes.
Natural medicines of plant origin have a wider therapeutic spectrum, milder action and less
frequent side effects compared with synthetic substances. According to the data of the World
Health Organization, about 70 000 plant species are currently used for medicinal purposes;
about 1000 species are used in the European pharmaceutical industry (Bernáth, 2002).
Preservation of production is a very important problem to be solved by producers of these
products. One of the ways of preservation of products is drying. Medicinal plants can be dried
in a number of ways: in the open air (shaded from direct sunlight); placed in thin layers on
drying frames, wire-screened rooms or buildings; by dielectric source as microwave; or
infrared devices (Raila et al., 2009). When possible, temperature and humidity should be
controlled to avoid damage to the active chemical constituents. The method and temperature
3
used for drying may have a considerable impact on the quality of the resulting medicinal
plant materials.
Chemical changes are the most important in the post-harvest of medicinal herbs that can be
influenced by drying. Moreover, drying can promote changes in the product appearance
(color) and smell, modifying the final quality (Aboltins and Kic, 2016).
The drying process is characterized by the existence of transport mechanisms such as surface
diffusion, pure diffusion, capillary flow, evaporation, thermo-diffusion, etc. Many studies
were done to process medical plant drying by small heated air (Aboltins and Kic, 2016).
1.1.2: Free Radicals and Antioxidants
Free radicals are atoms, molecules or ions with at least one unpaired electron in the outermost
shell. Free radicals are highly reactive due to the presence of unpaired electron(s). Free
radicals and oxidants play a dual role as both toxic and beneficial compounds, since they can
be either harmful or helpful to the body. They are produced either from normal cell
metabolisms in situ for example, blood cells destroy parasites, bacteria and viruses by using
oxidants such as nitric oxide, superoxide and hydrogen peroxide, another example is cellular
components called peroxisomes produce hydrogen peroxide as a byproduct of the
degradation of fatty acids and other molecules. Or from external sources (pollution, cigarette
smoke, radiation, medication). Free radicals that can be formed within the body include: the
superoxide anion (O2
-), the hydroxyl radical (OH˙), singlet oxygen (1O2), and hydrogen
peroxide (H2O2) (Lien Ai Pham-Huy, 2008).
Oxidative stress is defined as the state in which the level of toxic reactive free radicals
overcome the endogenous antioxidant defenses of the host. This state results in an excess of
free radicals, which can react with cellular lipids, proteins, and nucleic acids, leading to local
injury and eventual organ dysfunction. Lipids are probably the most susceptible biomolecule
to free radical attack. So, free radicals have been implicated as playing a role in the etiology
of cardiovascular disease, cancer, Alzheimer's disease and Parkinson's disease. (This is why
free radical damage is also called “oxidative damage.”) (Liou, 2011).
https://hopes.stanford.edu/glossary/oxidative-damage/
4
The human body has several mechanisms to counteract oxidative stress by producing
antioxidants, which are either naturally produced in situ known as endogenous antioxidants,
including superoxide dismutase (SOD), catalase, many vitamins (vitamin C, vitamin E, beta-
carotene), minerals and glutathione peroxidase that neutralize many types of free radicals. Or
externally called exogenous antioxidant supplied through foods and/or supplements
Antioxidants that come from outside the body like vitamins and herbs. Herbs, such as
bilberry, turmeric (curcumin), grape seed or pine bark extracts, ginkgo and calendula can
provide powerful antioxidant protection for the body (Biofizyka.p.lodz.pl, 2014).
In this project, antioxidant capacity of calendula extract will evaluate by DPPH method. The
results will compare with data found in previous study. Radicals percent will be scavenging
will measure by ultraviolet–visible spectrophotometry (UV–Vis) device.
1.1.3: Ultraviolet–Visible Spectrophotometry (UV–Vis)
UV-Vis Spectroscopy (or Spectrophotometry) is a quantitative technique used to measure
how much a chemical substance absorbs light. This is done by measuring the intensity of
light that passes through a sample with respect to the intensity of light through a reference
sample or blank. This technique can be used for multiple sample types including liquids,
solids, thin-films and glass. Spectrophotometry is a quantitative measurement of the
absorption/transmission or reflection of a material as a function of wavelength (Instruments
Ltd., 2012).
Using a spectrophotometer and carrying out absorption/transmission measurements we can
determine the amount (or concentration) of a known chemical substance simply, by studying
the number of photons (light intensity) that reach the detector. The more a material absorbs
light at a specific wavelength, the higher the concentration of the known substance )
Instruments Ltd., 2012).
5
1.2: Problem Statement
The medical aspect is one of the areas that fall within the field of chemical engineer work,
and since the manufactured chemicals showed side effects and high cost during manufacture.
Resorting to medicinal herbs seemed to be an appropriate solution in treating many diseases
at a lower cost and without possible toxic effects. Calendula herb was used to extract its
compounds in order to work on dermatology formulation.
Phytochemicals in Calendula including carbohydrates, amino acids, carotenoids, terpenoids,
flavonoids, and other constituents, show some important biological activities like wound
healing, anti-inflammatory, anti-bacterial, anti-fungal, and antioxidant.
Different chemical tests are needed to ensure the safety of the bioactive compounds of
Calendula healing effects. Therefore, the antioxidant tests are necessary tools to make the
road map of solving the uncertainty of Calendula healing effects.
Moreover, all of analytical tests are carried on dried herb powders. Therefore, it is necessary
to examine the behavior of the herb through drying by deriving the plant drying characteristic
curves at different temperatures.
1.3: Objectives
The main objective of this project is to evaluate the antioxidant activity of these extracts by
DPPH method. Which could be used in dermatological treatments, since the antioxidants
may protect the body cells against the effects of free radicals. The results will be compared
with data found in the previous studies.
The preparation of medicinal product of natural ingredients for the treatment of skin diseases
is another objective of this project. This enhance the world’s orientation towards the
exploitation of natural antioxidants in plants and less reliance on manufactured antioxidants;
that meets the human and environmental interest.
6
It is known that plant must be dried and grinded before any test. So, the drying rates of herb
flowers at different temperatures is presented and characterized.
1.4: Scope of the Work
The scope of this project is to evaluate the antioxidant capacity of Calendula extract by DPPH
method. The experimental work is stopped or in another word the antioxidant experiments
are halted, and only the ascorbic acid was tested for its antioxidant activity, due to the
COVID-19 disease outbreak. And instead theoretical study of drying data of Calendula
flowers is completed and presented.
1.5: Significance
Herbs/medicinal plant/homemade remedies are less expensive than the synthetic drugs and
majority peoples in rural/backward area have blind faith on them. They are right because they
can treat any disease by using them without any lethal side effects. Homemade remedies are
not only useful for the treatment of different diseases but are also widely used for enhancing
beauty and for curing skin related issues. On the other hand, synthetic drugs synthesized by
employing different methodologies are expensive with some side effects. Although herbal
medicines are less potent in comparison to synthetic drugs in some cases but still these are
considered less toxic or having less side effect in contrast to synthetic drugs. The ultimate
norm for any medicine (human made or natural) is their nontoxicity, effectiveness,
specificity, stability and potency. Now many chemists switching theirs field from synthetic
to natural side in order to explore nature more and more (Anbudhasan et al., 2014).
The consumer concern regarding the safety of using synthetic antioxidants in convenient food
products has forced and motivated the food processors to seek for natural alternatives. This
leads to a situation where the application of synthetic antioxidants started to decrease
drastically in food products. Hence there has been an increasing global trend towards the use
of natural antioxidants present in fruits and green leafy vegetables. The effects of these
7
natural antioxidants in scavenging the free radicals are well discussed and reported in the
earlier studies. The factors that encourage the use of natural antioxidants are its low cost,
compatibility with diet and less harmful effect in the human body. The strong H-donating
capacities of various phytochemicals make them as effective natural antioxidants. Phenols
present in plant extracts acts as a potential antioxidant by inhibiting the free radical formation
and prevent auto oxidation. Phenolic acids, flavonoids and volatile oils possess higher
antioxidant activity and also acts as the essential part of diet and these claims were supported
by various scientific evidence. The health promoting capacity of these natural antioxidants
help in eradicating chronic diseases such as cancer. Hence, in this review the action of
antioxidants of calendula extract on free radicals was discussed (Nisar, Sultan and Rubab,
2018).
In spite of importance of conduction of antioxidant tests. The knowledge of the rate of drying
of Calendula flowers is still attract the attention of researchers. The rate of drying equations
are derived at different safe temperature. This means that you can get dry powder safely
through three hours instead of one week at ambient temperature.
1.6: Organization of the Report
This report consists of seven main chapters; the first introduces the Calendula plant with
general information about drying herbs and antioxidants. The second chapter presents some
of the difficulties and constraints, the third chapter shows the previous researches and studies
published literature on this subject. The next chapter includes the methodology of our work,
summarizes what has been done. Chapter five shows the results and analysis. Finally,
chapters six and seven show the conclusion, and future work & recommendation
respectively.
8
Chapter Two: Difficulties and Challenges
It was impossible to continue the prescribed stages of our experimental program due outbreak
of pandemic COVID-19 virus. The research direction is changed towards theoretical
calculation of experimental drying data of Calendula flowers.
The rate of drying section was added and prepared during the ban period, as it was not among
the project objectives, and the experimental data are provided by Dr. Husni.
9
Chapter Three: Literature Review
3.1: Calendula general uses
Calendula leaves and petals are edible, it can be used for culinary purposes, the leaves are
added to salads dishes and soups, the petals of this plant are used for decoration, the Greeks
and Romans used it as a culinary garnish (Brush Creek Wool Works, 2019).
Colored substances extracted from the Calendula flowers are used in coloring food products;
it can be used as a saffron substitute for coloring and flavoring rice. Calendula also used in
dying fabrics, cosmetics. In cosmetics, the flowers of this plant are used for washing hair,
soften the skin, and to cure skin spots, Ancient Egyptians used it to rejuvenate their skin
(Brush Creek Wool Works, 2019).
An essential oil from plant used in perfume-making, also Calendula has many medicinal
uses, calendula has been revered as a magical medicinal for centuries, The flower is widely
used as a medicinal plant whose anti-inflammatory, antifungal, antibacterial, antioxidant,
antimicrobial activities, anti HIV activities, anticancer activities properties make it a strong
ingredient for healing (All Good Products, 2019).
The food and drug administration (FDA) has approved Calendula for use as a spice and as
an ingredient in cosmetics, soaps and shampoos, body creams, and wound treatment (All
Good Products, 2019).
One medieval author named Macer described Marigold in his volume on herbs he thought
that merely to look upon the blooms would improve eyesight and draw evil “humors” from
the head (Indigo Herbs, 2019).
Figures 1 and 2 show some kinds of Calendula plants.
10
Figure 1: Calendula plant.
Figure 2: Different colors of Calendula flowers.
3.2: Therapeutic Potential of Calendula officinalis
The use of plants to treat disease is as old as the human. Calendula officinalis has many
therapeutic potential and medicinal uses, such as:
3.2.1: Antidiabetic and anti hyperlipidemic activities
Hydro alcoholic extract of Calendula officinalis has both antidiabetic and antihyperlipidemic
effects. Because the extract increases the total hemoglobin level and the extract is similar to
that of insulin (Ashwlayan et al., 2018).
11
3.2.2: Cardiovascular activities
Calendula officinalis could be cardioprotective. Calendula achieved cardio protection; by
encouraging left ventricular developed pressure and aortic flow, also by reducing myocardial
infarct size and cardiomyocytes apoptosis (Ashwlayan et al., 2018).
3.2.3: Hepatoprotective activities
Calendula flower hot water showed anti hepatoma activity (25-26% inhibition) against five
human liver cancer cells (John and Jan, 2017).
3.2.4: Antioxidant activities
Most of phytochemicals presence in Calendula officinalis have free radical scavenging
activity (John and Jan, 2017). The leaves and petals of Calendula officinalis are a potential
source of natural antioxidants (Ashwlayan et al., 2018).
3.2.5: Anthelmintic activities
Anthelmintic activity presence in flowers and leaves of Calendula officinalis in dried phase.
The aqueous extract of dried flowers and leaves of Calendula officinalis were prepared by
decoction method. Anthelmintic activity due to it contain saponins (Ashwlayan et al., 2018).
3.2.6: Anti inflammatory activities
Calendula is highly effective for the prevention of acute dermatitis of grade 2 or higher and
should be proposed for patients undergoing postoperative irradiation for breast cancer
(Ashwlayan et al., 2018).
12
3.2.7: Wound healing activities
The extract of Calendula flower has important healing activity by increasing hexosamine and
collagen hydroxyproline content. Wound healing activities due to the antioxidant and
antimicrobial activities of Calendula (Ashwlayan et al., 2018).
3.2.8: Anticancer activities
Calendula extracts has a direct mitogenic effect on human lymphocytes or thymocytes and
inhibitory effect on the proliferation of lymphocytes in the presence of
polyhydroxyalkanoates (Ashwlayan et al., 2018).
3.2.10: Antibacterial activities
The growth of the bacteria inhibition by ethanolic and aqueous extracts of Calendula
officinalis, at concentrations ranging from 125 μg/ml to 64 mg/ml. Methanolic extract
inhibited the growth of both Staphylococcus aureus and Escaheriachia coli at 64 mg/ml
(Ashwlayan et al., 2018).
3.2.11: Sedative drugs
Ingested of Calendula considered sedatives (Ashwlayan et al., 2018).
3.2.12: Antihypertensive drugs
High doses of Calendula possess hypertensive effects (Ashwlayan et al., 2018).
3.2.13: Hypoglycemic drugs
The activity of hypoglycemic medications or insulin increased by Calendula (Ashwlayan et
al., 2018).
3.2.14: Cholesterol lowering drugs
Calendula decrease lipids and triglyceride (Ashwlayan et al., 2018).
13
3.3: Phytochemistry of Calendula officinalis
A number of phytochemical studies have well reported about the presence of several classes
of chemical compounds, the main ones being terpenoids, flavonoids, coumarin, quinines,
volatile oil, carotenoids and amino acids in the plant.
3.3.1: Terpenoids
They include sitosterols, stigmasterols, diesters of diols, oleanolic acid saponins, and other
constituents (John and Jan, 2017).
3.3.2: Flavonoids
They include quercetin, isorhamnetin, and isoquercetin (Ashwlayan et al., 2018).
3.3.3: Coumarins
Contain coumarins-scopoletin, umbelliferone and esculetin (Ashwlayan et al., 2018).
3.3.4: Quinones
Some quinones were reported in the chloroplast, mitochondria, and in the leaves (Ashwlayan
et al., 2018).
3.3.5: Volatile oil
Calendula officinalis flowers contain maximum volatile oil at full flowering stage (0.97 wt
%) and minimum during the pre-flowering stage (0.13 wt. %). Various monoterpenes and
sesquiterpenes have been reported in the volatile oil (John and Jan, 2017).
3.3.6: Carotenoids
Total carotenoid (mg/g dry weight) was 7.71% for petals and 1.61% for pollens, and the total
carotenoids (mg/g dry weight) for the leaves is 0.85% and for stems 0.18% (Ashwlayan et
al., 2018).
14
3.3.7: Amino acids
Amino acid content of the leaves is about 5%, stems 3.5% and flowers 4.5%, some reported
amino acids: aspartic acid, glutamic acid, serine, and phenylalanine (Ashwlayan et al., 2018).
3.3.8: Carbohydrates
Contain polysaccharides (Ashwlayan et al., 2018).
3.3.9: Lipids
The amount of neutra lipids in the seeds was15.7%, phospholipids 0.6% and glycolipids 0.9%.
Some fatty acids reported in flowers were: lauric, stearic, oleic, and linolenic acid
(Ashwlayan et al., 2018).
3.3.10: Other constituents
Other phytochemicals include the bitter constituent, loliolide (calendin), calendulin and
paraffins (Ashwlayan et al., 2018).
15
3.4: Extraction
The importance of choosing the extraction method comes from that it influences the yield of
needed compounds in the plant and the quality of extract.
Extraction technique: Organic solvent extraction.
The organic solvent extraction (OS) when a solid material is in contact with a solvent, the
soluble components in the solid material are transferred to the solvent. When applying this
extraction method, the active soluble mass is non-selectively transferred to the solvent due
to a concentration gradient (Salomé-Abarca et al., 2015).
Different methods were done for the extraction. Sana Fatima et al., 2018, have separated the
petals and leaves from the plant, dried it at room temperature, and grinded it using grinding
machine. Ethanol solvent was used for the extraction. It was then carried out using Soxhlet
apparatus. 15 grams of grinded dried (petals/leaves) were added to 300mL of (ethanol) then
conducted for 20 cycles at 70 °C. The extract was then concentrated in rotary evaporator to
recover the solvent and then the extract was kept at refrigerator for further drying (Sana
Fatima et al., 2018).
Other method: Ten grams of dried grinded (flowers/leaves) have been transferred into a flask
containing 150 mL of the solvents (ethanol). The materials were stirred at 350 rpm, 35°C for
24 h using an orbital shaker. The sample was filtered and then evaporated under vacuum
using the rotary evaporator. Then finally the extract was transferred into a small vial and
stored at room temperature (Efstratiou et al., 2012).
16
3.5: Antioxidant Capacity of Calendula
Antioxidants are compounds produced in the body and found in foods. They help defend
cells from damage caused by potentially harmful molecules known as free radicals. When
free radicals accumulate, they may cause a state known as oxidative stress. This may damage
your DNA and other important structures in your cells (Raman, 2018).
Sadly, chronic oxidative stress can increase your risk of chronic diseases such as heart
disease, type 2 diabetes and cancer (Raman, 2018).
Fortunately, eating a diet rich in antioxidants can help increase your blood antioxidant levels
to fight oxidative stress and reduce the risk of these diseases. Calendula plant is one of these
foods (Raman, 2018).
This section will present several studies about antioxidant capacity of calendula. The topics
will be covered are
1. Some methods to evaluating the antioxidant.
2. Antioxidant evaluation of Calendula extract by DPPH and other methods.
3. Comparison between antioxidant activity of Calendula extract and another
antioxidant.
4. The relationship between antioxidant activity and phenolics compounds.
5. Conclusion.
6. DPPH method.
3.5.1: Some methods for evaluating the antioxidant.
ABTS Assay
The 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay that uses ABTS
radicals performed by oxidation of ABTS with potassium persulphate. Thus, this assay
17
becomes time consuming in terms of waiting for the ABTS radicals to be generated as it takes
around 12-16 hours for the reaction of ABTS with potassium persulphate, unlike the DPPH
assay where one does not have to wait for it to be generated. However, once the radicals are
generated, it is a very simple assay in terms of performing the assay. The ABTS radical is
soluble in water and organic solvents, enabling the determination of antioxidant capacity of
both hydrophilic and lipophilic compounds/samples. One major drawback of this assay is
that the radicals formed are not very stable and the results are not reproducible (Shah and
Modi, 2015).
FRAP Assay
Ferric Reducing Antioxidant Power (FRAP) method is based on the reduction of colorless
ferric complex (Fe3+ tripyridyltriazine) to blue-colored ferrous complex (Fe2+
tripyridyltriazine) by the action of electron donating antioxidants at low pH. The FRAP assay
is more tedious and time consuming in terms of preparing the chemicals of the working
solution. It is a simple and inexpensive method and does not require the use of any exclusive
chemicals (Shah and Modi, 2015). According to Shah and Modi (2015), the results obtained
in FRAP are found to be reproducible for all the concentrations. Hence, FRAP is a suitable
method for the determination of antioxidant activity. In another study done by Thaipong et
al. (2006) for estimating antioxidant activity from guava fruit extracts, they concluded that
the FRAP assay showed high reproducibility, was simple and could be rapidly performed.
FRAP method, however, has its own limitations, especially for measurements below non-
physiological pH values i.e. at pH 3.6. Physiologic-ph approximately 7.4 (Moniruzzaman et
al., 2012). Physiologic-pH of Calendula ranges between 4.5-8.3, depends on the pH of the
soil (Plants for a Future, 2007).
DPPH Assay
DPPH is one of the few stable and commercially available organic nitrogen radicals. It is one
of the most widely reported methods for the determination of antioxidant activity (Alam et
al., 2013). DPPH is not a very tedious assay in terms of preparation of chemicals and also in
18
terms of performing the assay and hence can be used for its operational simplicity. DPPH
showed high reproducibility according to Shah and Modi (2015). In one study done by
Katalinic et al. (2006), they suggested that from the methodological point of view, the DPPH
method is easy and accurate with regard to measuring the antioxidant activity of the extracts
and also the results are highly reproducible. According to Shah and Modi (2015), DPPH was
found to be the most suitable method for the determination of antioxidant activity of mycelia
of the three mushroom species because it could be rapidly performed and showed high
reproducibility. Similar comment was also obtained by Kedare and Singh (2011), DPPH
assay is considered rapid, simple and inexpensive. The advantage of this method is that
DPPH may be utilized in aqueous, polar, and nonpolar organic solvents and can be used to
examine both hydrophilic and lipophilic antioxidants compounds for their DPPH scavenging
capacities under the same experimental conditions without use of solubilizing agents such as
the b-cyclodextrin that are necessary in other assays (Kedare and Singh, 2011). In the DPPH
method, the radicals did not have to be generated before the assay which turned out to be the
biggest disadvantage of the ABTS method (Moniruzzaman et al., 2012).
Antioxidants react with free radicals by different mechanisms, hydrogen atom transfer (HAT)
or single electron transfer mechanism (SET); or the combination of both HAT and SET
mechanisms. The HAT reaction is a concerted movement of a proton and an electron in a
single kinetic step, as shown in Figure 3. In HAT mechanisms, the free radical removes one
hydrogen atom of antioxidant, and the antioxidant itself becomes a radical. In this
mechanism, the bond dissociation enthalpy (BDE) is an important parameter in evaluating
the antioxidant action (Mader et al., 2007). The lower the BDE of the H-donating group in
the potential antioxidant, the easier it will be for the reaction of free radical inactivation. In
SET mechanisms, the antioxidant provides an electron to the free radical and itself then
becomes a radical cation. In this mechanism, the ionization potential (IP) of the antioxidant
is the most important energetic factor in evaluating the antioxidant action. The lower the
ionization potential, the easier is the electron abstraction. It is very difficult to distinguish
between HAT and SET reactions. In most situations, these two reactions take place
simultaneously, and the mechanism of the reaction is determined by the antioxidant’s
structure and solubility, the partition coefficient and solvent polarity. In the DPPH assay, an
odd electron displays a strong absorption band at a wavelength of 519 nm, which loses
19
absorption once the odd electron is paired off by a hydrogen or electron-donating antioxidant
(Figure 3). DPPH radical react with antioxidants by SET or HAT mechanism. Examples of
HAT-based assays include ABTS. Examples of SET-based assays include DPPH and FRAP
(Liang and Kitts, 2014).
The DPPH radical is a dark-colored crystalline powder. The reaction between a sample and
a DPPH radical is performed in a solvent such as ethanol/methanol, which facilitates the
extraction of antioxidant compounds from the sample. The DPPH radical has a deep violet
color in solution, and it becomes colorless or pale yellow when neutralized (Kedare and
Singh, 2011).
Figure 3: Reaction mechanism of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) with antioxidant.
R: H = antioxidant radical scavenger; R = antioxidant radical.
Phosphomolybdenum method
The total antioxidant assay based on reduction of Phosphate-Molybdenum (VI) to Phosphate-
Molybdenum (V) by the sample analyte and subsequent formation of a green phosphate Mo
(V) complex at acidic pH. Sample solution is combined with reagent (sulfuric acid, sodium
phosphate and ammonium molybdate) (Untea et al., 2018).
20
3.5.2: Antioxidant evaluation of Calendula extract by DPPH and other
methods
There are many methods to measure the antioxidant capacity of Calendula. This part will
show some research that used different methods to evaluate Antioxidant of Calendula extract
and the results they have achieved. Researchers have conducted various studies on
antioxidant capacity of Calendula by several method. One of these studies is for Untea et al.
(2018), the aim of their research were to calculate the antioxidant activity of Calendula
extract by DPPH, ABTS and phosphomolybdenum method. The results showed that,
Calendula leaves proved to have an important antioxidant activity determined by two
different methods, DPPH and phosphomolybdenum. Antioxidant activity of marigold by
DPPH was 225.14 mmol eq trolox/kg. By phosphomolybdenum method was 135.74 mmol
eq ascorbic acid/kg. On the other hand, by ABTS method the antioxidant activity was lower
than two methods 65.69 mmol eq trolox/kg.
One study has shown that Calendula extract has a biological activity. According to Preethi
et al. (2006), Calendula officinalis extract effectively scavenged superoxide, hydroxyl, and
nitric oxide radicals in vitro. These radicals are generated inside the body during the normal
metabolism. The concentration of the extract needed for 50% scavenging of superoxide
generated by photoreduction of riboflavin (IC50) was 500 µg of extract/mL of superoxide
whereas for the inhibition of hydroxyl radicals generated by Fe+3 /ascorbate/EDTA/H2O2
system it was 480 µg/mL. The IC50 for nitric oxide scavenging was 575 µg/mL. Preethi et.
al (2006), also conducted a study in vivo. They found that Calendula officinalis scavenged
the superoxide generated in vivo in the mice. Moreover, administration of Calendula
officinalis significantly increased the catalase and glutathione levels in blood and liver.
Glutathione reductase was increased in liver of treated groups. These results show that
Calendula officinalis has a profound effect on the antioxidant defense system both in vitro
and in vivo (Preethi et al, 2006).
21
3.5.3: Comparison between antioxidant activity of Calendula extract and
another antioxidant
When an overload of free radicals caused by internal and external condition cannot gradually
be destroyed by antioxidant produced in the body, external antioxidant must be supplied
either by natural antioxidant or by synthetic. Comparison between external synthetic and
natural antioxidant activity is so important. Marinescu et al. (2018) compared the antioxidant
activity of ethanol extracts of Calendula with antioxidant activity of synthetic butylated
hydroxytoluene (BHT). The results showed that Antioxidant activity of ethanol extracts
tenfold higher than that of a 0.05 mg/ml BHT solution (Marinescu et al., 2018).
Deuschle et al. (2015), conducted another study, this study compared between antioxidant
activity of Ascorbic acid (vitamin C) and rutin with hydroethanolic extract (HECO) from the
leaves of Calendula officinalis L. The results show that the HECO at lowest concentration
assayed (7.81 µg/mL) gave a DPPH inhibition of 79.84 %, exhibited an excellent in
vitro antioxidant capacity. With the same concentration, ascorbic acid showed an inhibition
of 14.93%, and rutin showed an inhibition of 62.44 %. The percentage of antioxidant capacity
for this method is proportional to the amount consumed by DPPH, and the greater this
percentage, the greater the ability of the sample in scavenging free radicals. The inhibitory
concentration (IC 50) also was determined in this study, the inhibitory concentration (IC 50)
is the concentration that will inhibit 50% of free radicals. The IC50 values were 16.57 µg/mL
for ascorbic acid, 6.25 µg/mL for rutin, and 5.86 µg/mL for HECO. From these results can
be concluded that antioxidant of Calendula extract is very effective and useful (Deuschle et
al., 2015).
3.5.4: The relationship between antioxidant activity and phenolic compounds
Phenolic compounds are considered the most important antioxidants of plant materials due
to their chemical structures, and that they play an important role in the capture and
neutralization of free radicals (Kumar Verma et al., 2014). Kumar Verma et al. (2014)
conducted a study on ethanolic extract and aqueous extract of Calendula, they found that
22
ethanolic extract have significant high total antioxidant activity as compared to aqueous
extract of Calendula officinalis. This is because ethanolic floral extract has significantly high
total phenolics, flavonoids tannin, β-carotene lycopene and chlorophylls contents as
compared to aqueous extract of Calendula officinalis so, there is a positive correlation
between phenolic content and total antioxidant activity of extracts (Raina, Kumar and
Sultana, 2014). Similarly, in vitro ABTS radical scavenging potential of floral extract of
Calendula officinalis and their relationship with total polyphenolic contents have been also
reported (Habila et al., 2010). On the other hand, some authors showed no correlation
between total phenolic content and antioxidant activity (Sulaiman et al., 2011). The low
correlations might be explained that total antioxidant activity is not due to only one
contributor, the presence of non-phenolic antioxidants (vitamin C, vitamin E and
carotenoids) having accountable antioxidant activity (Kumar Verma et al., 2014).
3.6: Calendula side effects and special precautions
Natural products are not always safe, and it is important to know if they have any side effects
before medical use. It has been shown from the long-term use throughout the ages and
transcontinental of Calendula as a medicinal plant, that it has no negative effect mentioned,
and it is likely safe when taken by mouth or applied to the skin for most people (WebMD,
2015).
However, pregnant women advised to avoid taking Calendula by mouth, or topical use,
because there are some concerns of miscarriage, and for more precautions; breast-feeding
women are advised to avoid it as well, since there is not enough information yet indicating
the safety of using Calendula. Also, people who are sensitive to the Asteraceae/Compositae
family are advised to follow the doctor’s instructions before taking Calendula, because it may
cause an allergic reaction to them. The use of Calendula should be discontinued if it is taken
in conjunction with the medications taken during and after surgery because in this case, it
causes much drowsiness (WebMD, 2015).
23
3.7: Drying of herbs
The researchers investigated the influence of some process parameters (temperature, sample
thickness, layer thickness, air flow rate, etc.). The effect of the used airflow and drying air
temperature on the drying kinetics was studied in [Raila et al., 2009; Čipliene, 2015].
Drying is the most common and fundamental method for post – harvest preservation of
medicinal plants. Natural drying can be considered only for drying of small quantities. In
case of mass production, the use of technical drying applications is indispensable. For
preservation of active ingredients of plant material low drying temperature is recommended
(less than 60°C). It means long drying duration. Drying represents 30-50 % of total costs in
medicinal plant productions. Energy demand of drying represents is a significant cost factor.
It is largely due to the high moisture content of the leaves and flowers, to be dried. Different
parts of the plant and their drying aspects were considered in (Müller and Heindl, 2006).
For indoor drying, the duration of drying, drying temperature, humidity and other conditions
should be determined on the basis of the plant part concerned (root, leaf, stem, bark, flower,
etc.) and volatile natural constituents, such as essential oils.
Figure 4 below, shows calculation was made per 500 g of material in order to compare the
drying dynamics of different types and weights of herbs (Aboltins and Kic, 2016).
24
Figure 4: Different plant drying dynamics: M1- marigold, M2- lemon balm, M3-
origanum, M4- common agrimony, M5- common lavender, and M6- common sage
(Aboltins and Kic, 2016).
Looking at the results it is seen that drying dynamics of all samples are similar except for
garden marigold, where longer sample drying is observed. Other examples dry for five days,
but Calendula flowers dries up to 6-7 days at the same conditions. The difference between
garden marigold and other plant drying can be explained by the fact that flowers of garden
marigold are thicker and inside moisture diffusion affects the drying process more.
3.7.1: Effect of different drying methods on antioxidant activities and color of
Calendula herb.
Drying is one of the most important processes for producing marigold powder. Therefore the
effects of different drying processes, namely freeze drying (FD), hot air drying (HA) and
combined far-infrared radiation with hot air convection (FIR-HA), on the color and
antioxidant activities of marigold flowers were evaluated by (Siriamornpun et al., 2012).
The results presented in this study suggest that the changes in the color of FIR-HA dried were
smaller as compared to FD and HA dried. As the color after FIR-HA drying appeared to be
more like fresh petals, this may imply that this drying method can better preserve their
25
bioactive compounds and activities than do the FD and HA methods. As for the antioxidant
activities, the results were shown Combined FIR-HA drying gave higher values of DPPH
radical scavenging (85%) than did FD drying or fresh petals (67% and 65%, respectively),
while HA drying had the lowest DPPH radical scavenging (52.4%). This indicated that the
increase was induced by the FIR-HA treatment, thereby supporting a previous study that FIR
radiation increases the antioxidant activity of rice-hull extracts (Lee et al., 2003), and
mulberry tea (Wanyo et al., 2011). An increase of antioxidant activities may be explained by
the fact that since FIR creates internal heating with molecular vibrations of materials, it may
have the capability to breaking down the covalent complex molecular structures and release
some antioxidant compound such as flavonoids, carotene, lycopene, tannin, ascorbate,
flavoprotein or polyphenols from polymers. Moreover, the mechanism of far infrared drying
is different from hot air drying. FIR is thought to liberate and activate low-molecular-weight
natural antioxidant compounds, because it heats materials without degrading the constitutive
molecules of the surface and contributes to an even transfer of heat to the center of the
material (Niwa et al., 1988).
3.7.2: Effect of drying on concentrations of several phytochemicals and chemical
components of essential oils.
Many studies have examined the effect of drying on chemical components. Okoh O.O. et al.
(2007) Conducted a study on effect of drying on chemical components of essential oils of
Calendula, the results showed a difference in the proportions of the compounds in fresh and
dried sample at different method. The same results appeared with Siriamornpun et al. (2012).
The results from this study suggest that drying methods differ in their effects on bioactive
compounds. For example, HA gave the highest content of b-carotene (15.5 mg/100 g DW),
while FIR-HA and FD provided the highest levels of lutein and lycopene. Hence, we
recommend that each drying method could be suitable for different products depending on
the type of compounds considered to be the most important.
Martinov, M., and coworkers have carried out drying experiments at different drying heights
and five drying experiments. The experiments include five different drying temperatures and
plant material layer heights, total carotenoid content and total microbial count were
26
measured. The significant differences in moisture content between lower and upper layer of
dried plant material were evident, pointing out the necessity of turning plant material during
drying procedure. The content of total carotenoids was higher in lower layer, in all the
experimental variants and lower by natural drying, pointing out that higher temperatures
positively affected the total carotenoid content. The lowest total microbial count was obtained
with the lower plant material layer while higher values were recorded in natural dried
material. The highest values were recorded in fresh samples (Martinov et al., 2009).
Dorozko, J., and coworkers 2019 have analyzed the influence of various drying methods on
the quality of edible flower petals. The study was carried out using different drying methods
such as hot air-drying, microwave drying, and freeze-drying. Edible petals of garden
marigold (Calendula officinalis L.), and true lavender (Lavandula angustifolia L.). Total
phenolic, total flavonoid content and antioxidant activity were determined in their research.
All three drying methods had adverse effects on biologically active compounds of the
analyzed edible flowers petals. Despite the fact that freeze-drying is the most popular method,
microwave drying had the most positive effect in terms of bioactive component content in
this study (Dorozko et al., 2019).
Freeze-drying would be the best method of water removal, but it is also expensive method.
This method is based on the dehydration by sublimation of frozen sample and the major
advantages are protection of bioactive compounds and original shape, color and flavor of
flowers (Zheng et al., 2015). Microwave drying is alternative to the conventional drying
method that allows the product to preserve its useful qualities and is suitable for almost at
home. Very important that the heat not only on the surface but also inside the food products
or plants. Very high speed of drying gives the quality of the final food product. Shi et al.
(2017) reported that microwave drying helps to remain higher content of flavone, vitamin C
and soluble sugars in medicinal chrysanthemum flowers (Shi et al., 2017).
27
Chapter Four: Methodology
4.1: Extraction
Based on the findings of the literature survey, the antioxidant tests will be performed on the
ethanolic extract of Calendula flowers and leaves using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) method (Do et al., 2014).
Solvent for Calendula extraction: Ethanol was used for the extraction process, according to
the FDA (Food and Drug Administration), ethanol considered as an effective, efficient, safe,
reliable, and consistently producing potent extractions with minimal fuss (High Purity
Extractions, 2016). Furthermore, ethanol can dissolve both polar and non-polar substances
(EasyChem Australia, 2013). The ethanol extract showed total antioxidant activity by using
DPPH higher than water and methanol (Do et al., 2014).
Extraction procedure:
The supervisor Dr. Husni, planted and harvested Calendula plants. He provided the GP
group of fresh leaves and yellow flowers.
Leaves and flowers have been separated from the plant, the green leaves weight 113 gm.,
and the fresh flower weight is 37 gm., then the leaves were dried at room temperature for
approximately a week, in the shadow place.
After drying they were grinded using grinding machine.
The grinded leaves were weighed, and it is recorded 17 gm., only 8.4 gm. was taken and
dissolved in 80 ml ethanol. Where 6 gm of grinded flowers weighed and dissolved in 75
ml ethanol.
Then, the two flasks after tightly closed were put into a water shaker bath for 72 hours,
at 100 rpm and 40°C. The purpose of the shaker is to steadily shake and mix samples
while maintaining a constant temperature.
After the shaker, the samples were transferred from the flasks into two bottles and kept
for a day, before filtration.
Vacuum filtration was done on both samples for solid-liquid separation, and the outcome
of filtration was kept in bottles for a day.
28
The extract samples were concentrated in a rotary evaporator attached with a vacuum
pump for solvent recovery, the solvent (more volatile) evaporated at reduced pressure
and temperature and then condensate and trapped in a flask for recovery. It needed around
2 hours for each sample for complete evaporating.
The extracted material will next be scraped from the flask and will be weighed, to be
ready for the DPPH test.
Figures A 1 to A 7 from Appendices show some of the previous procedures.
4.2: UV-Test for Absorbance
The free radical scavenging activity of the reference and extracts will be evaluated by 1, 1-
diphenyl-2-picryl-hydrazyl (DPPH) according to Shen et al., 2010 method.
According to Shen et al., 2010, a 0.004% (W/V) solution of DPPH in ethanol was prepared
and 3 ml of this solution was added to 3 ml of the solution of all extracts/reference in ethanol
at different concentration (50,100, 200, 400 & 800 μg/mL). The mixtures were shaken
vigorously and allowed to stand at room temperature for 30 minutes. Then the absorbance
was measured at 517 nm using a UV-VIS spectrophotometer. Ascorbic acid was used as the
reference. Lower absorbance values of reaction mixture indicate higher free radical
scavenging activity. The capability of scavenging the DPPH radical can be calculated by
using the following formula.
DPPH scavenging effect (% inhibition) =
A0 – A1
A0
∗ 100%
Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of
all of the extract samples and/or reference (Shen et al., 2010).
Ascorbic acid was used as a reference to compare its antioxidant activity with the antioxidant
of the Calendula plant extract. According to literature, the ascorbic acid stock solution
29
concentration used for the test was 800 μg/ml, however, in practical experience, this
concentration was found to be high, and the absorbance readings at 517 nm for several diluted
concentrations showed relatively close results. This concentration was then diluted to
50 μg/ml.
Test for the absorbance wavelength:
The wavelength of DPPH sample was found equal to 517 nm using ethanol as a blank.
Figure A 8 from Appendices shows the absorbance wavelength.
DPPH preparation:
4 mg of DPPH was dissolved in 100 ml of ethanol, then covered by aluminum foil,
and kept in cool conditions, because DPPH radical is sensitive to light. According to
Ozcelik et al. (2003), the light effect on the absorbance of DPPH.
Ascorbic acid stock solution (10 ml) preparation:
4 mg of ascorbic acid was dissolved in 5 ml of deionized water, this gives a
concentration of 800 μg/ml.
Dilute the stock solution to 50 μg/ml according to the following:
𝑀1 × 𝑉1 = 𝑀2 × 𝑉2
Where:
M1: Concentration of the ascorbic solution (before dilution) (μg/ml).
V1: Volume needed from the ascorbic solution to achieve a diluted solution (ml).
M2: Concentration of the diluted ascorbic solution (μg/ml).
V2: Volume of the diluted ascorbic solution (ml).
800 × 𝑉1 = 50 × 10
𝑉1 = 0.625 𝑚𝑙 = 625 μ𝑚𝑙
625 μml of ascorbic acid solution was withdrawn by a micropipette and then
deionized water was added until having 10 ml of diluted ascorbic solution.
30
For serial dilution of the ascorbic acid (10, 20, 30, 40 & 50 μg/ml) using the same
dilution equation for total 3 ml solution, 5 clean test tubes were needed:
For a concentration of 10 μg/ml:
𝑀1 × 𝑉1 = 𝑀2 × 𝑉2
50 × 𝑉1 = 10 × 3
𝑉1 = 0.6 𝑚𝑙 = 600 μ𝑚𝑙 of ascorbic solution was withdrawn by a
micropipette, and then ethanol was added in a test tube until having 3 ml
solution.
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 𝑛𝑒𝑒𝑑𝑒𝑑 = 3 − 0.6 = 2.4 𝑚𝑙
Table 1: The amount needed from each solution to achieve a certain diluted
concentration of ascorbic acid.
Conc. of diluted
ascorbic acid μg/ml
Volume of ascorbic
acid solution (ml)
Volume of ethanol
needed (ml)
0 0 3
10 0.6 2.4
20 1.2 1.8
30 1.8 1.2
40 2.4 0.6
50 3 0
The remaining calculations are included in the Appendices page F, and the
summary results shown in Table 1.
Preparation of the control solution:
31
3 ml of DPPH solution (0.004% W/V) was added to each one of the five previous test
tubes that containing the Ascorbic acid at different concentrations (10, 20, 30, 40 &
50 μg/ml).
Then the test tubes were kept at room temperature for 30 minutes in the dark (covered
by aluminium foil).
Measuring the absorbance at 517 nm:
The blank solution contains ethanol only was inserted in the UV-VIS
spectrophotometer and the test was run.
Then DPPH solution sample, and the 5 other samples (ascorbic acid at different
concentration with DPPH), were inserted individually in the UV-VIS for running the
test.
Absorbance values were measured for each sample individually.
4.3: Drying herbs
Materials and method
The laboratory measurements were carried out at the Laboratory of the chemical engineering
department. The studied and measured material samples were put in plastic perforated sheets
used in Aluminum window frame.
Two drying experiments are carried out at different temperature one is at room (ambient)
temperature on 20-22 ºC. The other drying experiment is carried out at 55-60 ºC.
Drying at ambient temperature: During the experiment, the average room temperature was
20-22 ºC. The dimension of drying samples are presented in Table 2. The average relative
humidity in Nablus area is about 60%.
32
Table 2: Start parameters of dried Calendula flowers.
Temperature °C
Start
weight (g)
Drying surface
area (cm2)
The average dimension of the drying
plate (cm)
room (20-22) 500 900 30x30x3
55-60 200 177 D=15cm, height 2-3 cm
The air temperature and humidity were measured by thermometer and humidity sensor. The
moisture content was identified by gravimetric measurement in regular time intervals. The
samples were weighed on the digital laboratory balance with maximum load weight 500 g
and with resolution 0.01 g.
The total drying time was adapted to the need for determination of the final moisture content.
Experimental data of Calendula at room temperature is shown in Table 3.
Table 3: Drying data obtained at room temperature.
Time
(h)
Weight
(g)
0 500
10 450
24 360
30 340
40 283
50 224
60 176
70 136
80 114
90 98
100 80
120 74
140 73
160 74
33
Drying at 55-60 ºC. Here the sample is placed on a circular tray on a pan, the diameter of tray
is 15 cm. the heat is provided by hair drier that placed and hanged on about 25- 30 cm from
a sample. The weight is continuously and directly measured. The temperature is measured
by a digital thermometer. The start weight is taken 200 g. Experimental data of Calendula
flowers at 55-60° C is shown in Table 4.
Table 4: Drying data obtained at 55-60° C
Time
(min)
Weight
(g)
0 200
10 179
20 141
30 122
40 83
50 66
60 50
70 40
80 35
90 32
100 29
120 27
140 25
150 26
34
Chapter Five: Results and Discussion
5.1: Drying of Calendula flowers
The aim of drying experiments was to investigate and compare principal theoretical problems
of drying by free convections for medical plants as flowers of garden marigold (Calendula
Officinalis). Determining the drying rates of Calendula flowers at constant and at falling rate
periods, drying equation constants at falling rate periods (R=aX+b) experimentally. The
experiment is carried at room temperature and 55-60 °C.
The falling rate of drying will be given by a linear straight line equation. (R=aX+b). Where
R is the rate of drying, X is free moisture content, a and b are constants that experimentally
determined.
Generally, the medicinal plants should not be collected in wet and rainy weather, but when
they are dry. The plants should be collected as the whole, if possible, and cut after drying.
Most plants should be dried in the shade, where they will not be soiled by dust, birds, insects,
etc. The fresh plants must not be added to dried plants since the herb gets wet again and may
degrade by mold.
Drying aims to inhibit micro-organism growth and prevent its deteriorations and spoilage, by
reducing the water content of the plant, which has a negative effect on the action of enzymes
(Honório et al., 2016). Moreover, it reduces weight and volume so it will minimize packaging
and storage costs. Using drying induces quantitative and qualitative differences in the
components of fresh and dry plant materials (Okoh et al., 2008).
The dried herbs color remained unchanged, but the bulk density decreased significantly
during drying. It can be expected that the drying process was under conditions, which were
adequate for the Calendula flowers.
35
The demonstration of experiment data is shown at different forms as presented in drying
literature. This show at the following Figures 5, 6 & 7. These present how a given property
change with time.
Figure 5: Drying dynamics of Calendula flowers.
In order to find the equations of constant rate and falling rate, a plot of free moisture X vs.
time needed; to find the tangent lines that fit the most possible points in order to get the range
where constant and falling rate periods occurred.
X (Free moisture) determined by:
𝑋 =
𝑊𝑒𝑖𝑔ℎ𝑡 (𝑔)−𝐿𝑜𝑤𝑒𝑠𝑡 𝑊𝑒𝑖𝑔ℎ𝑡
𝐿𝑜𝑤𝑒𝑠𝑡 𝑊𝑒𝑖𝑔ℎ𝑡
……………………………………1
Where weight (g) is the weight at any time (t).
For example: at room temperature, the lowest weight is 73 g, for time equals 140 hr. the value
of X is :
𝑋 =
450−73
73
= 5.16
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250
W
A
TE
R
R
EM
O
V
A
L%
TIME (H)
Removal of Water%
36
It is important to remind that the lowest weight still has about 12% moisture (equilibrium
moisture content) at drying condition. So, it is more exact to take the lowest weight as
(73*0.88=64 g). So, the bone dry is 64 g and not 73 g.
Same calculations were done on all weights, and the obtained results shown in Table 5 and
Table 6.
Table 5: The value of X at room temperature.
Time (h) X
0 5.84932
10 5.16438
24 3.93151
30 3.65753
40 2.87671
50 2.06849
60 1.41096
70 0.86301
80 0.56164
90 0.34247
100 0.09589
120 0.0137
140 0
160 0.0137
37
Table 6: The value of X at 55-60 °C.
Excel was used to plot the data, and to determine the tangent lines as shown in Figure 6 &
Figure 7.
Time
(min)
X
0 7
10 6.16
20 4.64
30 3.88
40 2.32
50 1.64
60 1
70 0.6
80 0.4
90 0.28
100 0.16
120 0.08
140 0
150 0.04
38
Figure 6: Plot of X vs. time at room temperature.
Figure 7: Plot of X vs. time at 55-60 °C.
-1
0
1
2
3
4
5
6
7
0 20 40 60 80 100 120 140 160 180
X
(
g
w
at
re
r/
g
d
ry
s
o
lid
)
Time (hr)
Free moisture vs Time
-1
0
1
2
3
4
5
6
7
8
0 20 40 60 80 100 120 140 160
X
(
g
w
at
er
/g
d
ry
s
o
lid
)
Time (min)
Free Moisture vs Time
39
The orange arrows show the period where the constant rate of drying, while the blue lines
show the period of the falling rate of drying. The intercept between the arrow and the line
determines the X critical (Xc). From the intercept shown that Xc in Figure 6 is at 70 hr., and
it is 60 min (1 hr.) in Figure 7.
To find the constant rate of drying: (McCabe et al., 2001)
𝑅 = −
𝐿𝑠
𝐴
×
∆𝑥
∆𝑡
………………………..………………….2
Where:
Ls: Weight of the dried flowers used (Kg).
A: The exposed area m2.
R: Constant rate of drying (Kg water/ h.m2).
At ambient temperature, the weight of the dried flowers was 73 g, the moisture content in
this stage must be considered, and it was assumed to be approximately 12%, so:
𝐿𝑠 = 73 𝑔 × 0.88 × 0.001 = 0.0642 𝐾𝑔
𝐴 = 30 𝑐𝑚 × 30 𝑐𝑚 × (10−2)2 = 0.09 𝑚2
From Figure 6, the period of the constant rate is at ∆𝑡 = 70 hr. and
so ∆𝑥 = 5.8493 - 0.8630 = 4.9863, the rate constant is:
𝑅 = −
0.0642
0.09
×
4.9863
70
= 0.0508 (Kg water/ h. m2)
And this value is constant for the whole period of constant rate, and it will be used to the plot
of R (rate of drying) vs. X (free water) as in Figure 8, to determine the equation of the falling
rate, an arrow was added manually reached between the end of the constant line and the zero
point.
40
Table 7: Experimental data for the plot of drying rate vs. moisture at room
temperature.
Time
(h)
X R
0 5.84932 0.0508
10 5.16438 0.0508
24 3.93151 0.0508
30 3.65753 0.0508
40 2.87671 0.0508
50 2.06849 0.0508
60 1.41096 0.0508
70 0.86301 0.0508
Figure 8: Drying rate vs. free moisture at room temperature.
0
0.01
0.02
0.03
0.04
0.05
0.06
0 1 2 3 4 5 6 7
R
D
ry
in
g
ra
te
(
K
g
w
at
er
/h
.m
2
)
X (Kg water/ Kg dry solid)
Drying rate vs Free moisture
Falling Rate
41
Equation of the falling rate: 𝑅 = 𝑎𝑋 + 𝑏
Where:
a: is the slope.
b = 0
To find the slope: 𝑎 =
∆𝑅
∆𝑋
=
(0.0508−0)
(0.8630−0)
= 0.0589
Then, R = 0.0589X
Table 8: Summary results for data at 20-22 °C drying.
Start
weight (g)
Dimensio
n of
drying
plate
(cm)
Ls (Kg
of solid
used)
A
exposed
area m2
Ls /A
(Kg/m2)
R (Rate
constant)
Kg
H2O/h.m2
Slope of
falling
500 30x30x 1 0.0642 0.09 0.7138 0.0508 0.05891
Same calculations and procedures were made for the data at 55-60 °C drying temperature,
and the results obtained shown in the following Table 9 and Figure 9.
Table 9: Summary results for data at 55-60 °C drying.
Start
weight (g)
Diameter
of
circular
pan (cm)
Ls (kg
of solid
used)
A
exposed
area m2
Ls /A
(Kg/m2)
R (Rate
constant)
kg
H2O/h.m2
Slope of
falling
200 15 0.022 0.0177 1.25 7.47 7.47
42
Figure 9 : Rate of drying vs free moisture at 55-60 °C.
The tendency of drying curves earned at room temperature 20-22 ºC is similar to that obtained
at 55-60 ºC. The only difference is the drying time is reduced for about 2.5-3 h. While the
drying time at ambient temperature takes few days around 6 to 7 days.
The drying rate at 55-60 ºC is obviously higher than at ambient temperature at both constant
drying rate period and the falling rate of the drying period. At ambient temperature, the
constant drying rate is 0.05 kg water/ (m2.h). While the rate of drying at falling rate period is
calculated from R=0.0508X. The rate of drying at a constant rate period is about 7.5 kg water
(m2.h) at higher temperature or at 55-60 ºC. It means 150 times is faster the drying at 55-60
ºC. The straight-line drying equation for the falling rate of drying at 55-60 °C is read out
directly from R vs. X Figure 9. Which is about R=7.47X.
It is clear that the shape of R vs. X drying curves are the same at different temperature. But
the values of the rate of drying at higher temperature is very high or R55-60ºC = 150 R20-22ºC
An investigation was completed to assess the drying conduct of Calendula flowers utilizing
a laboratory scale dryer with controlled air temperature and relative humidity (Matouk et al.,
2016).
The considered parameters included four distinct degrees of drying air temperature (55, 60,
65, and 70 °C) and three levels of air relative humidity (40, 50, and 60%). All test runs were
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8
R
(
K
g
w
at
er
/
h
r
.m
2
)
X ( Kg H2O/Kg dry solid)
Rate of Drying vs Free Moisture
43
directed at a steady airspeed of (0.6 m/sec) (Matouk et al., 2016). The results are shown in
the following both Figure 10 and Figure 11 (Matouk et al., 2016).
Figure 10: Changes in the moisture content of Calendula flowers as related to drying
time at different degrees of drying air temperature and relative humidity of 40%.
Figure 11: Changes in the moisture content of Calendula flowers as related to drying
time at different degrees of drying air temperature and relative humidity of 60%.
44
It is obvious from Figure 10 and Figure 11 that as the temperature increases the time of drying
decreases. However, when the humidity is increasing the time of drying will increase too.
The moisture content of the Calendula flowers ranged between 83.5 and 87.3 w% (Matouk
et al., 2016), and it was between 85.2 and 87 w% in our experimental work. At the same
conditions of 60% humidity and temperatures ranges between 55-60 ºC, the time of drying
in our experiment was ranged between 2-2.5 h, where in the literature (Matouk et al., 2016),
it was ranged 6.5-10 h, due to different initial weight for drying, the moisture content and the
drying method.
Although the drying rate increased when the heat was raised, some studies showed that drying
by hot drying at high temperatures may cause damage to the chemical compound present in
the plant. There are other drying methods such as far infrared ray combined with air
convection (FIR-HA) mentioned in previous studies, which reduces the drying time or
increases the drying rate, and doesn’t cause damage to the chemical compounds in the herbs,
which may be an alternative to drying hot air.
5.2: Absorbance and %inhibition for Ascorbic acid solution
Table 10 shows the readings of absorbance from UV-Spectrophotometry, with %inhibition
for the ascorbic acid:
Table 10: Absorbance readings and inhibition calculation for ascorbic acid.
Concentration
μg/ml
Absorbance
%inhibition
ratio
IC50
0 0.272 75
10 0.214 80
20 0.184 83
30 0.188 83
40 0.185 83
50 0.238 78
Control 1.096
o A Control sample contains 0.004% W/V DPPH solution.
45
READINGS, AND THUS RESULTS DON’T MAKE SENSE, some offered causes: A
reaction may have occurred between DPPH and light, or there may be a human error
in preparing concentrations of the solutions, or in using the UV spectrophotometry.
Sample of calculation
DPPH scavenging effect (% inhibition) =
A0 – A1
A0
∗ 100%
(% inhibition) =
1.096 – 0.272
1.096
∗ 100% = 75%
Figure 12: Setting %inhibition vs. Concentration.
Figure 13: DPPH test by setting %inhibition vs. Log concentration.
y = 0.1825x + 77.318
R² = 0.7098
74.00
76.00
78.00
80.00
82.00
84.00
86.00
0 5 10 15 20 25 30 35 40 45
%
in
h
ib
it
io
n
Concentration μg/ml
y = 5.2388x + 75.33
R² = 0.9663
74.00
75.00
76.00
77.00
78.00
79.00
80.00
81.00
82.00
83.00
84.00
85.00
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
%
in
h
ib
it
io
n
Log Conc
DPPH Test
46
To find IC50, the equation of the trend line from Figure 13 will be used (y = 5.2388x +
75.33), where y must = 50, and x is the needed concentration for the 50% inhibition. These
results were intended to be compared and calibrated with the results of the extract inhibition.
47
Chapter Six: Conclusion
Since there were no complete results for the antioxidant experiment, an analytical study of
drying flowers at two different temperatures and safe conditions was included as part of this
project. It is suggested to dry the herb flower soon at cover transparent plastic sheets to
conserve energy and to dry fast. The drying rates at constant drying period and at falling
drying period are calculated. The drying characterization curves (X vs. time, R vs. X) are
presented as well. The increasing drying condition from 20-22 ºC to 55-60 ºC increases the
rate of drying about 150 times. The characteristic drying curves calculated by our group and
that are taken by literature are the same.
The antioxidant test has not been accomplished as mentioned before, and so no reliable
results could be mentioned.
48
Chapter Seven: Future Work and Recommendation
Since the desired results to complete the main project goal of testing the antioxidant of
ethanolic extract of Calendula wasn’t accomplished, due to the COVID-19 pandemic, it is
possible in the future to complete the experimental work after the end of the pandemic, using
the following recommendations:
Making sure all tools are perfectly cleaned to avoid turbidity.
Trying to provide as much of Calendula herb as possible for the purpose of
experiments.
For the calibration curve using ascorbic acid as a reference, each working
concentration should be performed in triplicate.
Continue the work on Calendula extracts (leaves and flowers) to measure the
absorption and antioxidant scavenging, using the modified method of Shen et al.,
2010, and compare the results with the reference’s results.
Screening and scouring method could be applied to test the most appropriate solvent
and the antioxidant test method, either through laboratory experience or by inferring
from the literature survey.
Conduct the inhibition concentration IC50 and the effective concentration EC50
calculations.
Studying the components of the herb, which are most affected by drying, and those
which have the highest antioxidant effect such as phenols. If there is an intention to
make a marketing product for a certain disease.
Literature survey experiences several drying methods, it is recommended to test the
best method of drying to achieve a certain purpose. Such as far-infrared ray combined
with air convection (FIR-HA) drying method, according to Niwa et al., 1988, it
showed that it could be a suitable alternative to hot drying, by increasing the rate of
drying without causing damage to the plant compounds, also microwave drying
showed preserving the quality of the components of the plant after drying, besides it
49
has a positive effect in terms of bioactive component, according to Dorozko et al.,
2019.
50
References
Aboltins, A. and Kic, P., 2016. Research in some medical plant drying process.
Engineering for rural development, pp.1145-1148.
All Good Products. (2019). Calendula Benefits & How to Use This All-Purpose Plant.
[online] Available at: https://allgoodproducts.com/all-good-stories/calendula-
benefits/?fbclid=IwAR028uM0oTZWNv-HPK-VvFIDw0i-
1uXPj2oFayRhUXqAxHrdCKNVbxTeUg0 [Accessed on 8 Dec. 2019].
Anton Paar (n.d.). Viscosity of Acetonitrile – viscosity table and viscosity chart: Anton
Paar Wiki. [online] Anton Paar. Available at: https://wiki.anton-paar.com/es-
es/acetonitrilo/ [Accessed on 29 Dec. 2019].
Aryal, S. (2018). Centrifugation- Principle, Types and Applications | Instrumentation
| Microbe Notes [online] Available at: https://microbenotes.com/centrifugation-
principle-types-and-applications/ [Accessed on 29 Dec. 2019].
Ashwlayan, V., Kumar, A., Verma, M., Garg, V. and Gupta, S. (2018). Therapeutic
Potential of Calendula officinalis. Pharmacy & Pharmacology International Journal,
6 (2), pp.149‒155.
Anbudhasan, P., Surendraraj, A., Karkuzhali, S. and Sathishkumaran, S. (2014).
Natural antioxidants and its benefits. International journal of food and nutritional
sciences, 3(6), pp.225-230.
Alam, M., Bristi, N. and Rafiquzzaman, M. (2013). Review on in vivo and in vitro
methods evaluation of antioxidant activity. Saudi Pharmaceutical Journal, 21(2),
pp.143-152.
Bernáth, J., 2002. Strategies and recent achievements in selection of medicinal and
aromatic plants. Acta Horticulturae, (576), pp.115-128.
Brush Creek Wool Works. (2019). Calendula Flowers, Calendula officinalis, Pot
Marigold, Herb and Natural Dye. [online] Available at:
https://www.brushcreekwoolworks.com/products/calendula-pot-marigold-dried-
flowers-herb-and-natural-
51
dye?fbclid=IwAR3tqZwsHZm7nUiBjjQPKltCqkCkeqquol-Nft7wUQvPrISyi7Y8-
gsbCIY [Accessed on 8 Dec. 2019].
Biofizyka.p.lodz.pl. (2014). Division of Biophysics. [online] Available at:
http://biofizyka.p.lodz.pl/prezentacje/IFE1.pdf [Accessed on 6 Dec. 2019].
Čipliene, A., 2015. Solar energy usage in drying technologies of medicinal and
spice plants. Doctoral dissertation. Aleksandras Stulginskis University.
Chromatography Today (2014). Understanding the Difference between Retention
Time and Relative Retention Time. [online] Chromatography Today. Available at:
https://www.chromatographytoday.com/news/autosamplers/36/breaking-
news/understanding-the-difference-between-retention-time-and-relative-retention-
time/31166 [Accessed on 29 Dec. 2019].
Do, Q., Angkawijaya, A., Tran-Nguyen, P., Huynh, L., Soetaredjo, F., Ismadji, S. and
Ju, Y. (2014). Effect of extraction solvent on total phenol content, total flavonoid
content, and antioxidant activity of Limnophila aromatica. Journal of Food and Drug
Analysis, 22(3), pp.296-302.
Dinh Phuc, N., Phuong Thy, L., Duc Lam, T., Hoang Yen, V. and Thi Ngoc Lan, N.
(2019). Extraction of Jasmine Essential Oil by Hydrodistillation method and
Applications on Formulation of Natural Facial Cleansers. [online]
Iopscience.iop.org. Available at: https://iopscience.iop.org/article/10.1088/1757-
899X/542/1/012057/pdf [Accessed on 7 Dec. 2019].
Deuschle, V., Deuschle, R., Piana, M., Boligon, A., Bortoluzzi, M., Dal pra’, V.,
Dolwisch, C., Lima, F., Carvalho, L. and Athayde, M. (2014). Phytochemical
evaluation and in vitro antioxidant and photo-protective capacity of Calendula
officinalis L. leaves. [online] scielo.br. Available at:
http://www.scielo.br/scielo.php?pid=S1516-
05722015000500693&script=sci_arttext [Accessed on 7 Dec. 2019].
Dorozko, J., Kunkulberga, D., Sivicka, I. and Kruma, Z., 2019. The Influence of
Various Drying Methods on the Quality of Edible Flower Petals. FOODBALT,
pp.182-187.
52
DH Abdul Jalill, D. (2014). Scinapse | Academic search engine for paper. [online]
Scinapse. Available at: https://scinapse.io/papers/2189211019 [Accessed on 7 Dec.
2019].
EasyChem Australia. (2013). Production of Materials - Ethanol as a Solvent -
EasyChem Australia. [online] Available at: https://easychem.com.au/production-of-
materials/renewable-ethanol/ethanol-as-a-solvent/ [Accessed on 2 Feb. 2020].
Efstratiou, E., Hussain, A., Nigam, P., Moore, J., Ayub, M. and Rao, J. (2012).
Antimicrobial activity of Calendula officinalis petal extracts against fungi, as well as
Gram-negative and Gram-positive clinical pathogens. Complementary Therapies in
Clinical Practice, 18(3), pp.173-176.
Health Benefits, H., 2020. Calendula Facts and Health Benefits. [online]
Healthbenefitstimes.com. Available at:
[Accessed on
8 June 2020].
High Purity Extractions (2016). Why Choose Ethanol for Botanical Extractions?
[Blog] High Purity Extractions and Equipment. Available at:
http://www.highpurityextractions.com/blog/why-choose-ethanol-for-botanical-
extractions [Accessed on 20 Feb. 2020].
Honório, I. C. G., Bonfim, F. P. G., Montoya, S. G., Casali, V. V. D., Leite, J. P. V.,
& Cecon, P. R. (2016). Growth, development and content of flavonoids in calendula
(Calendula officinalis L.). Acta Scientiarum. Agronomy, 38(1), pp.69-75.
Habila, J., Bello, I., Dzikwi, A., Musa, H. and Abubakar, N. (2010). Total phenolics
and antioxidant activity of Tridax procumbens Linn.. [online] Available at:
https://www.researchgate.net/profile/Jd_Habila/publication/266371914_Total_phen
olics_and_antioxidant_activity_of_Tridax_procumbens_Linn/links/54dda2040cf25
b09b9143a2b.pdf [Accessed on 8 Dec. 2019].
Indigo Herbs. (2019). Marigold Benefits & Information. [online] Available at:
https://www.indigo-herbs.co.uk/natural-health-
guide/benefits/marigold?fbclid=IwAR02cVH9m23H3tDpCMtg7iEilNJWilDV6u_P
WNQhObr5hSMHRaWLynArdL4 [Accessed on 8 Dec. 2019].
53
Instruments Ltd., E. (2012). UV Vis Spectroscopy | UV Vis Spectroscopy
Applications. [online] Edinburgh Instruments. Available at:
https://www.edinst.com/techniques/uv-vis-spectroscopy/ [Accessed on 8 Dec. 2019].
John, R. and Jan, N. (2017). Calendula Officinalis-An Important Medicinal Plant with
Potential Biological Properties. [online] insajournal. Available at:
http://insajournal.in/insaojs/index.php/proceedings/article/view/305 [Accessed on 8
Dec. 2019].
Katalinic, V., Milos, M., Kulisic, T. and Jukic, M. (2006). Screening of 70 medicinal
plant extracts for antioxidant capacity and total phenols. Food Chemistry, 94(4),
pp.550-557.
Kedare, S. and Singh, R. (2011). Genesis and development of DPPH method of
antioxidant assay. Journal of Food Science and Technology, 48(4), pp.412-422.
Kashani, H. and Mohammad, S. (2012). Pot marigold (Calendula officinalis)
medicinal usage and cultivation. Scientific Research and Essays, 7(14), pp.1468-
1472.
Liang, N. and Kitts, D., 2014. Antioxidant Property of Coffee Components:
Assessment of Methods that Define Mechanisms of Action. Molecules, 19(11),
pp.19180-19208.
Lien Ai Pham-Huy, C. (2008). Free Radicals, Antioxidants in Disease and Health.
[online] PubMed Central (PMC). Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614697/ [Accessed on 8 Dec.
2019].
Liou, S. (2011). About Free Radical Damage – HOPES Huntington's Disease
Information. [online] HOPES Huntington's Disease Information. Available at:
https://hopes.stanford.edu/about-free-radical-damage/ [Accessed on 8 Dec. 2019].
Lee, S., Kim, J., Jeong, S., Kim, D., Ha, J., Nam, K. and Ahn, D., 2003. Effect of Far-
Infrared Radiation on the Antioxidant Activity of Rice Hulls. Journal of Agricultural
and Food Chemistry, 51(15), pp.4400-4403.
54
Mader, E., Davidson, E. and Mayer, J., 2007. Large Ground-State Entropy Changes
for Hydrogen Atom Transfer Reactions of Iron Complexes. Journal of the American
Chemical Society, 129(16), pp.5153-5166.
McCabe, W., Smith, J., and Harriott, P., 2001. Unit Operations of Chemical
Engineering. Boston: McGraw-Hill, pp.536-540.
Matouk, A., El-Kholy, M., Tharwat, A. and Sadat, M., 2016. Drying of Pot Marigold
Whole Flowers and Petals under Controlled Drying Air Temperature and Relative
Humidity. Journal of Soil Sciences and Agricultural Engineering, 7(2), pp.221-230.
Müller, J. and Heindl, A., 2006. Drying of medicinal plants. Fronts, 17, pp.237-252.
Mahmiyat.ps. (2019). Field Marigold - Plants of Palestine | Mahmiyat.ps. [online]
Available at: http://www.mahmiyat.ps/en/floraAndFauna/516 [Accessed on 8 Dec.
2019].
Martinov, M., Golub, M., Adamović, D., and Bojić, S., (2009). Multiphase drying of
Calendula officinalis flowers in a batch dryer. Bulletin for hops, sorghum and
medicinal herbs (Serbia), 41(82), pp.25-34.
Moniruzzaman, M., Khalil, M., Sulaiman, S. and Gan, S. (2012). Advances in the
Analytical Methods for Determining the Antioxidant Properties of Honey: A
Review. African Journal of Traditional, Complementary and Alternative Medicines,
9(1), pp.36‐42.
Marinescu, M., Bercu, V., Florin Danet, A., Tecuceanu, V., Lungu, L. and POPA, C.
(2018). Antioxidant capacity of some Calendula extracts by EPR spectroscopy.
[online] Rrp.infim.ro. Available at: http://www.rrp.infim.ro/2019/AN71706.pdf
[Accessed on 8 Dec. 2019].
Nisar, B., Sultan, A. and Rubab, S. (2018). Comparison of Medicinally Important
Natural Products versus Synthetic Drugs-A Short Commentary. Natural Products
Chemistry & Research, 06(02), pp.308.
Niwa, Y., Kanoh, T., Kasama, T. and Negishi, M., 1988. Activation of Antioxidant
Activity in Natural Medicinal Products by Heating, Brewing and Lipophilization. A
New Drug Delivery System. Drugs Exp Clin Res., 14(5), pp.361‐372.
55
Okoh, O., Sadimenko, A., Asekun, O. and Afolayan, A. (2008). The effects of drying
on the chemical components of essential oils of Calendula officinalis L. African
Journal of Biotechnology, [online] 7(10), pp.1500-1502.
Okoh O.O., Sadimenko A.A. and Afolayan A.J., 2007. The Effects of Age on the
Yield and Composition of the Essential Oils of Calendula officinalis. Journal of
Applied Sciences, 7(23), pp.3806-3810.
Ozcelik, B., Lee, J. and Min, D. (2003). Effects of Light, Oxygen, and pH on the
Absorbance of 2,2-Diphenyl-1-picrylhydrazyl. Journal of Food Science, 68(2),
pp.487-490.
Plants for a Future, 2007. Calendula Officinalis Calendula, Pot Marigold PFAF Plant
Database. [online] Pfaf.org. Available at:
[Accessed on
13 June 2020].
Raila, A., Lugauskas, A., Kemzūraite, A., Zvicevicius, E., Ragazinskiene, O. and
Railiene, M., 2009. Different drying technologies and alternation of mycobiots in the
raw material of Hyssopus officinalis L. Ann Agric Environ Med, 16(1), pp.93-101.
Raman, R. (2018). 12 Healthy Foods High in Antioxidants. [online] Healthline.
Available at: https://www.healthline.com/nutrition/foods-high-in-antioxidants
[Accessed on 10 Dec. 2019].
Raina, R., Kumar, P. and Sultana, M. (2014). Phytochemical constituents and
antioxidant potential in floral extracts of Calendula officinalis Linn.. [online]
Available at:
https://www.researchgate.net/publication/265206603_phytochemical_constituents_
and_antioxidant_potential_in_floral_extracts_of_Calendula_officinalis_linn
[Accessed on 7 Dec. 2019].
Sana Fatima, S., U Govekar, S., V Satardekar, D., S Barve, D. and P Dhawal, P.
(2018). In vitro analysis of ethanolic extract of flowers of Calendula officinalis for
antioxidant, antimicrobial and uv-h2o2 induced DNA damage protection activity,
7(5), pp. 2378-2383.
56
Salomé-Abarca, L., Soto-Hernández, R., Cruz-Huerta, N. and González-Hernández,
V. (2015). Chemical composition of scented extracts obtained from Calendula
officinalis by three extraction methods. Botanical Sciences, [online] 93(3), pp.633.
Sulaiman, S., Yusoff, N., Eldeen, I., Seow, E., Sajak, A., Supriatno and Ooi, K.
(2011). Correlation between total phenolic and mineral contents with antioxidant
activity of eight Malaysian bananas (Musa sp.). Journal of Food Composition and
Analysis, 24(1), pp.1-10.
Shen, Q., Zhang, B., Xu, R., Wang, Y., Ding, X. and Li, P. (2010). Antioxidant
activity in vitro of the selenium-contained protein from the Se-enriched
Bifidobacterium animalis 01. Anaerobe, 16(4), pp.380-386.
Shah, P. and Modi, H. (2015). Comparative Study of DPPH, ABTS and FRAP Assays
for Determination of Antioxidant Activity. [online] Semanticscholar.org. Available at:
https://www.semanticscholar.org/paper/Comparative-Study-of-DPPH-%2C-ABTS-
and-FRAP-Assays-of-Shah-Modi/18d1c505b9cad43443ed054d17508aaa4c742225
[Accessed on 8 Dec.2019]
Siriamornpun, S., Kaisoon, O. and Meeso, N., 2012. Changes in colour, antioxidant
activities and carotenoids (lycopene, β-carotene, lutein) of marigold flower (Tagetes
erecta L.) resulting from different drying processes. Journal of Functional Foods,
4(4), pp.757-766.
Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zeval