Antimicrobial Activity:

Carthamus tinctorius
Iziz De La Cruz CHEM 4499 - 202
Orlando Berumen Dr. Addo-Mensah
Nicole Cohen April 16, 2020
Perla Medrano
Introduction
● Newly discovered antimicrobial resistance mechanisms of infectious agents have created a
grand, ongoing challenge for drug development.

● There is a pressing need to locate new sources of antimicrobial substances with unprecedented
biological mechanisms.

● Medicinal plant extracts and phytochemicals have been considered

a focal point in the search for antimicrobial drug alternatives by
the pharmaceutical industry (Moneim et. al., 2018).

● For the purpose of this study, Carthamus tinctorius was selected

as the plant of interest due to its use in oriental medicine for its
antibacterial and antifungal properties (Ohama et. al., 2016).

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Background
● Belongs to the Asteraceae family

● First used in the 2nd century BC as traditional Chinese medicine.

● Today, it is traditionally used to treat various medical conditions

such as dysmenorrhea, amenorrhea, postpartum abdominal pain
and mass, trauma and pain of joints.

● Other uses include coloring and food preparation.

● The metabolites that are isolated from Carthamus tinctorius

included oils, proteins, minerals, phenolics, flavonoids, alkaloids,
lignans, carboxylic acids, steroids, polysaccharides, quinochalcone
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C-glycosides, and quinone-containing chalcones.
Carthamus tinctorius
(Safflower)
Objective
Due to the reported antimicrobial activities present in C. tinctorius, this research investigation aimed
to:

● identify antibacterial characteristics present in C. tinctorius through a series of metabolite

extractions and microbial assay experimental procedures

● evaluate the biological potency of C. tinctorius against microbial pathogens

● determine its efficacy as a potential alternative resource for future drug development in the
pharmaceutical industry

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Methods - Soxhlet Extraction
● Soxhlet extraction method was utilized using Ethanol,
Acetone, and Petroleum Ether as extraction solvents.

● Cheese cloth was placed at the bottom of the soxhlet

chamber to act as a filter

● 10g of dried safflower petals were placed into the

soxhlet chamber

● 250 ml of one of our solvents was added to the soxhlet

chamber and multiple cycles were run for 24 hours
before switching solvents.

● After all the solvents were obtained, each of the solvent

extracts collected were then evaporated using a rotovap
to obtain the extracted compounds that was then freeze
dried for 48 hours.
Methods - Preparation of Agar and Nutrient Broth
Agar and Nutrient Broth preparation
● Miller Hinton Agar
○ In a 1000 ml Erlenmeyer flask, 19 grams of agar
powder were dissolved in DI water.
○ Using a hot plate, apply heat and stir using a magnetic
stirrer.
○ Solution was then autoclaved.
○ After autoclaving, pour the agar into petri dishes and
allow the agar to solidify
○ Once it solidifies, store the plates in a refrigerator

● Nutrient Broth
○ Dissolved 1.25 grams of LB broth in 50ml of DI water
○ Add 2ml of solution into test tubes and autoclave.
○ These tubes will be used to prepare the bacteria stock
cultures
Methods - Bacteria Stock Culture

Bacteria Stock Culture

● From the prepared bacteria plates, using a sterile toothpick. Pick a colony and add it
into a test tube containing the nutrient agar and incubate for 24 hours at 37 degrees
celsius. Repeated until 3 test tubes were created and we used 1 test tube for control
● We diluted a 1ml sample of the inoculated bacteria to an absorption reading of 0.132
±0.005 that was then used for the microbial assay procedure.
 Methods - Preparation of Extract
…………………….Concentration Stock

Preparation of extract concentrations

● First, concentration of 50M of the extracts were prepared by the following table.

Extract Calculations

Petroleum Ether Extract 38.9mg of PE extract / .796ml DMSO

Acetone Extract 64.7mg Acetone extract / 1.294ml DMSO

Ethanol Extract 57.3mg Ethanol extract/ 1.146ml DMSO

Methods - Preparation of Extract
…………………….Concentration Stock Cont.

● Then the 50M extract concentration was then diluted to 5M, 10M, 25M, and 50M
concentrations with DMSO in a total volume of 100ml

Concentration Calculations

5M 5µl of extract + 95µl of DMSO

10M 20µl of extract + 80µl of DMSO

25M 50µl of extract + 50µl of DMSO

50M 100µl of extract + 0µl DMSO

Methods - Microbial Assay

Disk Diffusion
● Antibiotic disks were positioned as in the picture.
● Positive control used was penicillin (represented by orange disk)
● Negative control used was DMSO (represented by grey disk)
● 5M, 10M, 25M, and 50M extract concentrations were represented by the green , pink,
purple, and red disks respectively, to test inhibition at different concentrations
● 4 trials were set up for each extract giving us a total of 12 plates.
● Once the positive control, negative control, and the extract concentration were added to
their respective disks, the plates were incubated at 38 degrees celsius for 24 hours.
● After 24 hours, the zone of inhibition of each extract concentration was measured.
Results
Acetone extract of C. tinctorius plated with increasing concentrations
Results cont.
Petroleum Ether extract of C. tinctorius plated with increasing concentrations
Discussion

- First discrepancy: experimented with 8 plates instead of 12; lacked ethanol extract

- Second discrepancy: plates were moved during incubation; discs collided with one another

- Interfered with our data collection and results

- Third discrepancy: DMSO is a negative control; only measured sizes of discs were accounted for with

the use of a caliper

- Fourth discrepancy: caliper is another discrepancy, allows for human error and incorrect measurements

- Fifth discrepancy: due to COVID-19, the research team was unable to carry out the rest of the

experiment and perform a second trial to obtain more precise results

Conclusion
Based on the solvents used (acetone & petroleum ether) and previous studies, we can conclude to have extracted
3 primary metabolites from C. tinctorius:

- Petroleum Ether: extracted alkaloids (nitrogen containing compounds)

- Acetone: lignans (type of phenolic/flavonoid)

- Ethanol: good for extracting flavonoid glycosides and lignans; also contains flavonoid glycosides which
has good antimicrobial properties. The research team was unable to obtain this extract.

- C. tinctorius showed minimal inhibition with both methanol and petroleum ether extracts.

- For future research: We would like to use other solvents, including ethanol, to see if C. tinctorius could
show further inhibition.
References
Al-Snafi , Ali Esmali. The Chemical Constituents and Pharmacological Importance of Carthamus tinctorius -
An Overview. Journal of Pharmaceutical Biology. Journal of Pharmaceutical Biology, 2015, 143–166.
Delshad, E., Yousefi, M., Sasannezhad, P., Rakhshandeh, H., & Ayati, Z. (2018). Medical uses of Carthamus
tinctorius L. (Safflower): a comprehensive review from Traditional Medicine to Modern Medicine.
Electronic Physician, 10(4), 6672–6681. doi: 10.19082/6672
Moneim, A. E.; et al. Evaluation of Antimicrobial Activity of Safflower (Carthamus tinctorius) and
its Synergistic Effect with Antibiotic. EC Microbiology 14.3 (2018): 160-166.
Ohama, P.; Namwong, S.; Kumpun, S. Pigment Extraction of Safflower, Dyeing Properties and
Antimicrobial Study of Dyed Silk. Key Engineering Materials 2016, 675-676, 19–22.
Z. E. (2005). Resurgence of Safflower (Carthamus tinctorius L.) Utilization: A Global View. Journal of
Agronomy, 4(2), 83–87. doi: 10.3923/ja.2005.83.87

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