ANTIOXIDANT CAPACITY TEST, TOTAL
PHENOLIC, TOTAL ALKALOID, AND TOXICITY OF MARIGOLD FLOWER (TAGETES ERECTA L.)
Hadinata Tenggono Son, David Limanan
Universitas Tarumanagara,
Indonesia
E-mail:
[email protected], [email protected]
|
KEYWORDS alkaloids; BSLT; DPPH; Phenolic; Tagetes erecta L. |
ABSTRACT Antioxidants are a set of
compounds that can fight the effects of reactive oxygen species (ROS) so as
to eliminate their effects in the form of damage to macromolecules of
carbohydrates, fats, proteins, and DNA chains. One source of antioxidants
that are easily available and widely available is through herbal ingredients
such as marigold flowers (Tagetes erecta L.). Marigold flowers are well known
and used as a source of antioxidants, anti-inflammatory, antibacterial,
diuretic and accelerate wound healing. This flower is often consumed as an
edible flower and is also drunk in the form of tea. This study aims to look
for the antioxidant capacity, total phenolic, total alkaloids, and toxicity
of marigold flowers. Marigold flower extract is obtained by evaporating the
results of methanol maceration from dried marigold flowers. Test antioxidant
capacity with DPPH (2,2-diphenyl-1-picrilhydrazyl) using Blois� method, total phenolic using Singleton and
Rossi method, total alkaloid by method by Trivedi et al, and BSLT toxicity by
Meyer method. The antioxidant capacity of marigold flowers is measured in
IC50� as much as 74.5 μg/mL which
includes active antioxidant levels. The total phenolic content of marigold
flowers was obtained as much as 10,350.68 μg/mL. Total alkaloid levels
were obtained as much as 13.05 μg/mL. The BSLT toxicity test in LC50 is
162.82 μg/mL which is categorized as moderate toxicity. From the results
of this study, marigold flowers can be used as a source of antioxidants and
have antimitotic properties. |
INTRODUCTION
Marigold
flowers, which are usually ornamental plants, have also been sold in dried form
to brew and drink as tea. Marigold flowers are already known to contain
carotenoids such as lutein, neoxantin, zeaxantin, β-carotene, etc. In addition the main
compounds in marigold flowers are quercetagetin and
syringic acid (Sing,
Gupta, & Kannojia, 2020). These two
substances belong to the group of flavonoids and phenolics which are powerful
antioxidants. There are also antibacterial, antifungal, anti-inflammatory,
antiendotoxic, hepatoprotective and cytotoxic effects.� (Priyanka,
Shalini, & Navneet, 2013)
(Vallisuta
et al., 2014)
Click
or tap here to enter text. The antibacterial properties of marigold flowers
have been found due to the content of alkaloids (Motamedi,
Seyyednejad, Bakhtiari, & Vafaei, 2015)
Secondary
metabolite compounds such as phenolics and alkaloids are part of herbal plants
that can be utilized. Marigold flowers themselves have the highest phenolic
levels compared to other edible flowers (Socha,
Kałwik, & Juszczak, 2021). A wide
variety of phenolic compounds have been used as anti-inflammatory agents and
recent discoveries have found the presence of�
beneficial effects in the prevention and treatment of type 2 diabetes (Aravind
& Dhanavel, n.d.)
(Dias
et al., 2022).� In medicine, alkaloids are known as a source
of analgesic, anti-inflammatory and cardioprotective drugs such as morphine,
berberine and atropine (Heinrich,
Mah, & Amirkia, 2021). Apart from being
an antioxidant, there are also cytotoxic properties and antimitotic effects of
marigold flowers.� This effect is
important in inducing apoptosis so that cell death occurs and prevents the
presence of unwanted cell division.�
Reactive oxygen species or ROS is one of the compounds that plays a role
in the use of antioxidants and the mitosis process.
ROS
is all reactive compounds that contain oxygen. Oxygen in our bodies has many
essential functions such as its role as the last electron receiver on the
respiratory chain to make energy or ATP in the mitochondria. This process can
also occasionally make much more reactive byproducts such as oxygen radicals,
hydrogen peroxide, and hydroxyl radicals (Zhao,
Jiang, Zhang, & Yu, 2019). This
increase in endogenous ROS can be triggered due to external effects such as
stress, UV radiation, strenuous exercise, poor diet, and malnutrition (Li,
Jia, & Trush, 2016). �ROS has physiological functions needed by the
body, namely in cell signaling, apoptosis, vasodilation, inflammation and also
immunity. Under normal circumstances, ROS is in small amounts but there can be
an increase that will result in disturbances in homeostasis (Jakubczyk
et al., 2020). This
imbalance in homeostasis is referred to as oxidative stress.
Oxidative
stress is a condition where there is an imbalance between proxidan
compounds such as ROS and antioxidants. This state has many consequences in the
systems of our body as in the cardiovascular system can occur atherosclerosis,
in the nervous system can appear dementia and Alzheimer's, in the DNA of cells
there can be damage resulting in mutations to cancer. (Pizzino
et al., 2017)
�Antioxidants can counteract ROS in a way
called free radical scavenging where antioxidants have the ability to
contribute or reproduce electrons. The resulting product can be inactive
ingredients or less reactive ingredients than before (Shastri,
Srivastava, Jyoti, & Gupta, 2016). Antioxidants can
be divided into several groups based on their origin, namely endogenous and
exogenous. Endogenous antioxidants originate in the body such as catalase,
glutathione peroxidase and superanion dismutase while
exogenous antioxidants must be obtained such as vitamin C, vitamin E, selenium
and zinc derived from food (Mohamed,
2015)
With
the results of previous studies, this encourages the author to conduct various
subsequent tests. This study aims to determine the total antioxidant capacity,
total phenolic levels, total alkaloid levels, and toxicity of marigold flowers
RESEARCH METHODS
This
research was conducted at the Laboratory of Biochemistry & Molecular
Biology, Faculty of Medicine, Tarumagarara
University, West Jakarta. All data is processed using the GraphPad Prism 9.0
application.
Extract Creation
A
total of 515 grams of fresh marigold flowers are dried in a place with room
temperature and dark. 52 grams of dried simplisia of
marigold flowers were obtained, which were then macerated using methanol. The
maceration process is carried out for 24 hours, after which methanol is accommodated
and poured new methanol to be re-macerated. This process is carried out three
times. The methanol solvent from maceration was evaporated using� a rotary evaporator so that 19.77
grams of marigold flower extract� was
obtained.
Antioxidant Capacity Test
Test
this antioxidant capacity using the Blois method with DPPH. A total of 9.86
grams of DPPH powder was dissolved in 500 ml of aquades
to obtain a concentration of 50 μM. To find the
optimal gelomang length and absorbance control, 3.5
mL of DPPH solution and 0.5 mL of methanol were taken and mixed on one tube.
The tubes are incubated and read with a spectrophotometer at wavelengths of
400-800 after 30 minutes.
Vitamin
C was used as a comparison standard in this study. Vitamin C solution is
prepared with a concentration of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL and 10 μg/mL. From each concentration was taken as much as 0.5 mL and
3.5 mL of DPPH solution was added. The mixed solution is also left incubated
for 30 minutes and fed to a UV-Vis spectrophotometer for optimal wavelength to
be determined.
Marigold
flower extract was weighed as much as 10 mg and dissolved in 10 mL of methanol
to obtain a 1mg/mL solution consetration. The stock
solution was dissolved again to obtain a concentration of 25 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL, 125 μg/mL. On each tube is added
0.5 mL of each consetration and 3.5 mL of DPPH
solution. The tube was left in a dark room for 30 minutes and checked for
absorbance using a spectrophotometer with the optimal wavelength of the DPPH solution.
This test was carried out twice. All results are recorded for processing.
The
percentage of inhibition is calculated by dividing the value of the control
absorbance that has been reduced by the absorbance of the sample by the
absorbance of the control. The percentage of inhibition is used as the Y-axis
and the concentration as the X-axis on the graph of the linear line equation.
To obtain the IC value of 50, enter the number 50 on the Y axis and look for
the value of X. This value represents the concentration of marigold flower
extract needed to inhibit or neutralize 50% of the free radicals from DPPH.
Total Phenolic Level Test
The
method by Singleton and Rossi was used in this test. Tannins in powder form are
weighed as much as 0.25 grams for use as a standardpem
banding. The weighed tannins were dissolved with 5 mL of 95% ethanol. A total
of 50 mL of distilled water was added so that a concentration of 5 mg / mL was
obtained. The stock solution was re-dissolved to obtain concentrated solutions
of 300 μg/mL, 400 μg/mL,
500 μg/mL, 600 μg/mL,
700 μg/mL. From each
concentration, 0.2 mL of solution is taken and added with 15.8 mL of distilled
water. A total of 1 mL of Folin-Ciocalteu reagent
solution is added to each tube. After homogenization, the tubes are left
stationary in a dark room for 8 minutes. A 20% solution of sodium bicarbonate
is added as much as 3 mL. The contents of the tube
are re-homogenized and left at rest for 2 hours. The absorbance of the solution
is read with a spectrophotometer at a wavelength of 765 nm.
A
solution of a mixture of methanol and distilled water is prepared in a ratio of
1:1. A total of 10 mL of the solution is used to dissolve 0.3 grams of marigold
flower extract. The procedure performed is the same as the procedure for making
a standard solution of tannin comparison. This test was carried out twice. The
absorbance results on the spectrophotometer are recorded for later processing.
Total Alkaloid Level Test
This
test uses a method by Trivedi et al. The berberine chloride stock solution was
prepared using 1 gram of its powder dissolved with 10 mL of methanol as the
comparison standard. The stock solution was diluted to obtain a concentration
of 20 μg/mL, 40 μg/mL,
60 μg/mL, 80 μg/mL,
100 μg/mL. Phosphate
pH 4.7 and bromocrescol green (BCG) were added at 5
mL each. The pre-mixed solution is transferred on the separator flask, covered
and homogenized. After homogeneous, the solution is left at rest until it
separates into two layers. The undercoat is accommodated on a measuring flask
and chloroform is added until its volume reaches 10 mL.
The absorbance of each solution is measured with a spectrofometer.
For
the marigold flower extract test solution, 100 mg of extract dissolved in 3 mL
HCl was used. Testing of alkaloid extract levels was carried out duplo to obtain an average value. All results are recorded
for processing.
BSLT Toxicity Test
Toxicity
testing using the Meyer method. To obtain the larvae of Artemia salina shrimp,
5 grams of eggs are weighed and added to 500 mL of seawater. The tube is left
under the lamp with an aerator for 48 hours until the shrimp larvae can be seen
moving.
After
that a solution of marigold flower extract is made by dissolving 20 mg of
extract with 10 mL of seawater to obtain a stock solution. The stock solution
is dissolved until it reaches a concentration of 100 μg/mL,
150 μg/mL, 200 μg/mL,
250 μg/mL with a volume of 1 mL.
At each concentration are added 10 larvae of Artemia salina shrimp. Each tube
is supplemented with seawater to reach a volume of 2 mL.
This test is carried out duplo. All tubes are left
stationary under the lamp for 24 hours. After 24 hours, the number of shrimp
larvae alive and dead is calculated.
The
mortality percentage of shrimp larvae is calculated by dividing the number of
dead larvae by the number of larvae on each tube. This mortality percentage
value is used as the Y-axis and the concentration of the solution on the X-axis
to graph the linear line equation. The LCvalue of 50
can be calculated by entering the number 50 on the Y axis and calculating the
value of the X axis. This LC value of 50 means that the required concentration
of marigold flower extract kills 50% of the population of Artemia salina shrimp
larvae.
RESULTS AND
DISCUSSION
Preliminary
testing
is carried out to determine the phytochemical content of marigold flowers. From
the qualitative phytochemical test, it was found that there were alkaloid
compounds, betasianin, phenolic, flavonoids,
glycosides, cardioglycosides, quinones, coumarins,
saponins, steroids, sterols, tannins, and terpenoids. Anthocyanin compounds
were found to be negative in phytochemical tests.
Antioxidant
Capacity Test
Didapatkan optimal
wavelength at 516 nm and control absorbance at 0.838. With vitamin C as the
comparison standard, a linear line equation with the formula is obtained Y=
6,934X + 12,52 under R2= 0,9988. IC value50 the
calculated vitamin C is 5.40 μg/mL (Table 1).
The results of the
antioxidant capacity test of marigold flower extract obtained the linear line
equalization formula Y = 0.7112X � 6.539 and R2 = 0.9986. The IC
value of 50 marigold flower extracts is 74.50 μg/mL
(Table 2).
In one study, an ICvalue �of 50 was
obtained of 71.6 μg / mL.
While in other studies that also used the same method IC
Total
Phenolic Level Test
Tannin
solution tested as Standards for obtaining curves by the formula of the linear
line equation Y= 0.00073X � 0.1568 with a result of R2= 0,9727. The
absorbance of the tannin solution was obtained using a spectrophotometer at a
length of 765 nm (Table 3). A standard curve is created with the concentration
of tannins as the X axis and the absorbance result as the Y axis.
Phenolic levels of
marigold flower extract were calculated using a standard curve of tannin
solution. 516.16 μg/mL and 518.90 μg/mL were obtained for phenolic levels. These two
values were averaged to obtain 517.53 μg/mL
(Table 4). Because in the test of 20x dilution, the total phenolic content of
marigold flower extract was 10,350.68 μg / mL.
Previous studies
using marigold flowers and galat acid as comparison
standards obtained total phenolic levels of 57.52 1.42 mgGAE/g
�dry weight
Total
Alkaloid Level Test
This
test uses berberine chloride as a comparison standard. The absorbance of
the solution is measured at a wavelength of 420 nm (Table 5). The standard
curve obtained has a formula Y= 0.09105x � 0.0959 with an R value2=
0,9857.
The
absorbance of marigold flower extract solution is 0.502 and 0.495. This
absorbance value can be fed into the standard curve of berberine chloride
to obtain total alkaloid levels. The mean result of alkaloid levels was 6.53 μg/mL (Table 6). Due to the dilution of two times in
the test, the total alkaloid level obtained was 13.05 μg/mL.
In research on
plants of the Estericeae family with
the genus Senecio, the
BSLT Toxicity
Test
The test result
curve was made with the concentration of marigold flower extract on the X axis
and the mortality percentage on the Y axis. The formula of the BSLT toxicity
linear line equation is Y= 205X � 403.4 with R2= 0.9700. From the
formula, the calculated LCvalue of 50
is 162.82 μg/mL (Table 7).
The LCvalue of 50 obtained is 162.82 μg / mL which means that there are cytotoxic and
antimitotic effects on marigold flower extract. In a study with marigold
flowers, an LC
CONCLUSION
Marigold flowers
contain an antioxidant capacity in IC 50 of 74.50 μg
/ mL. This antioxidant content can be categorized as
active. The total phenolic content was 10,350.68 μg/mL. The total alkaloid level obtained was 13.05 μg/mL. In the BSLT toxicity
test, LC50 was obtained with a value of 162.82 μg
/ mL which means that marigold flowers have stitotoxic
and antimitotic effects. The LC value of 50 can be categorized as medium. It
can be concluded that marigold flowers have the potential to be used as a
source of antioxidants and antimitotics.
REFERENCES
Aravind, S., &
Dhanavel, D. (N.D.). A Laconic Aphorism About
The African Marigold (Tagetes Erecta L.)-. Journal Homepage: Www. Ijrpr. Com
Issn, 2582, 7421.
Dias, Marisol,
Roman�-P�rez, Marina, Roman�, Aloia, De La Cruz, Aimara, Pastrana, Lorenzo,
Fuci�os, Pablo, & Amado, Isabel R. (2022).
Recent Technological Advances In Phenolic Compounds Recovery And Applications:
Source Of Nutraceuticals For The Management Of Diabetes. Applied Sciences,
12(18), 9271.
Heinrich, Michael,
Mah, Jeffrey, & Amirkia, Vafa. (2021). Alkaloids
Used As Medicines: Structural Phytochemistry Meets Biodiversity�An Update And
Forward Look. Molecules, 26(7), 1836.
Jakubczyk, Karolina,
Dec, Karolina, Kałduńska, Justyna, Kawczuga, Dorota, Kochman, Joanna,
& Janda, Katarzyna. (2020).
Reactive Oxygen Species-Sources, Functions, Oxidative Damage. Polski
Merkuriusz Lekarski: Organ Polskiego Towarzystwa Lekarskiego, 48(284),
124�127.
Li, Robert, Jia,
Zhenquan, & Trush, Michael A. (2016).
Defining Ros In Biology And Medicine. Reactive Oxygen Species (Apex, Nc),
1(1), 9.
Mohamed, S. (2015). Antioxidants As Functional Foods In Health And Diseases, Austin J. Nutri.
Food Sci, 3(3).
Motamedi, Hossein,
Seyyednejad, Seyyed Mansour, Bakhtiari, Ameneh, & Vafaei, Mozhan. (2015). Tagetes Erecta, A Potential Medicinal Plant For Discovering A New
Antibacterial Agent.
Pizzino, Gabriele,
Irrera, Natasha, Cucinotta, Mariapaola, Pallio, Giovanni, Mannino, Federica,
Arcoraci, Vincenzo, Squadrito, Francesco, Altavilla, Domenica, & Bitto,
Alessandra. (2017). Oxidative Stress: Harms
And Benefits For Human Health. Oxidative Medicine And Cellular Longevity,
2017.
Priyanka, Dixit,
Shalini, Tripathi, & Navneet, Verma Kumar. (2013). A Brief Study On Marigold (Tagetes Species): A Review. International
Research Journal Of Pharmacy, 4(1), 43�48.
Shastri, Anu,
Srivastava, Rahul, Jyoti, Bhuvan, & Gupta, Manas. (2016). The Antioxidants-Scavengers Of Free Radicals For Immunity Boosting And
Human Health/Overall Well Being. International Journal Of Contemporary
Medical Research, 3(10), 2918�2923.
Sing, Y., Gupta,
Amit, & Kannojia, Pushpendra. (2020).
Tagetes Erecta (Marigold)-A Review On Its Phytochemical And Medicinal
Properties. Curr Med Drugs Res, 4(1), 1�6.
Socha, Robert,
Kałwik, Justyna, & Juszczak, Lesław. (2021). Phenolic Profile And Antioxidant Activity Of The Selected Edible Flowers
Grown In Poland. Acta Universitatis Cinbinesis, Series E: Food Technology,
25(2).
Vallisuta, Omboon,
Nukoolkarn, Veena, Mitrevej, Ampol, Sarisuta, Narong, Leelapornpisid, Pimporn,
Phrutivorapongkul, Ampai, & Sinchaipanid, Nuttanan. (2014). In Vitro Studies On The Cytotoxicity, And Elastase And Tyrosinase
Inhibitory Activities Of Marigold (Tagetes Erecta L.) Flower Extracts. Experimental
And Therapeutic Medicine, 7(1), 246�250.
Xu, Li Wei, Juan,
Chen, Qi, Huan Yang, & Shi, Yan Ping. (2012). Phytochemicals And Their Biological Activities Of Plants In Tagetes L. Chinese
Herbal Medicines, 4(2), 103�117.
Zhao, Ru‑Zhou,
Jiang, Shuai, Zhang, Lin, & Yu, Zhi‑Bin. (2019). Mitochondrial Electron Transport Chain, Ros Generation And Uncoupling. International
Journal Of Molecular Medicine, 44(1), 3�15.
Copyright holders:
Hadinata Tenggono
Son, David Limanan (2023)
First publication right:
Devotion - Journal of Research and Community Service
This
article is licensed under a Creative Commons
Attribution-ShareAlike 4.0 International