BIOM 428 | Maxwell Garcia

04/5/2023

Immunomodulatory effects of Glycerolic Echinacea purpurea Stem and Leaf Extract on RAW 264.7 Murine Macrophages

Immune Function

First lines of defense in Immune System

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• Barrier immunity

-Barriers (Skin, mucous, cilia)

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• Innate defenses

-Cells (Macrophages, NK cells, neutrophils)

-Inflammation & Fever

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• Importance of balance

Fig 1. Photo of Echinacea purpurea (Purple Coneflower).

Image retrieved from BayGardens.

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Non-Responsive

Over-Responsive

Wikimedia Commons

Immune System

Echinacea & Medicine

• Echinacea (Purple Coneflower) has been used in traditional herbal remedies for various illnesses or wounds.

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• As of recent, Echinacea purpurea (EP) is used principally for the treatment of upper respiratory tract infections (URI)

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• Is suggested to “strengthen” the immune system

Fig 1. Photo of Echinacea purpurea (Purple Coneflower).

Image retrieved from BayGardens.

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Fig 1. Photo of Echinacea purpurea (Purple Coneflower)

Image retrieved from BayGardens.

Purpose & Hypothesis

• Examine how Echinacea purpurea may modulate (control) macrophage response

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• Hypothesis: Glycerolic E. purpurea extract modulates inflammatory response

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• Chemical agent groups: Alkamides, Caffeic Acids, Glycoproteins

Methods of Project

• Evaluating the effect of an E. purpurea extract on RAW 264.7 macrophages

• Evaluating extraction and solvent on nitric oxide (NO) production

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• Cells will be stimulated with bacterial endotoxin: lipopolysaccharide (LPS) and measured with spectrophotometry

1. Plating cells at 4x10^5 cells/well

2. Treatment

3. Measure NO response

24 hrs

24 hrs

Experimental Design

Controls

Treatments

Fig 2. Basic set-up of 24-well plates for experiments.

-Well template retrieved here

Controls

Negative- No treatment

Positive- 100 ng/mL LPS

Glycerol% w/LPS

Echinacea:Glycerol w/o LPS

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Experiment

Echinacea:Glycerol w/ LPS

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Experimental Design

Fig 3. Exp. 3s 24-well plate design. On the left contains controls. Right is Echinacea with and without LPS added.

Control

• 0 ng/mL LPS
• 100 ng/mL LPS

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Extracts and

Solvent Control

• 0.375%
• 0.75%
• 1.5%
• 3%

Treatment Conditions

• Solvent appears to have an independent effect

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* = Statistically significant difference compared to 100 ng/mL LPS (p<0.05)

• Synergistic effect with solvent?
• Glycerolic EP alone has no effect

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* = Statistically significant difference compared to 100 ng/mL LPS (p<0.05)

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Unstimulated (0 ng/mL LPS)

Stimulated (100 ng/mL LPS)

400x magnification

Stimulated (100 ng/mL LPS)

400x magnification

Stim. w/ 1.5% EP (100 ng/mL LPS)

Conclusions & Directions

Glycerolic E. purpurea extract…

• Direct effect on macrophages
• Anti-inflammatory immunomodulation
• Non-stimulatory
• Potential synergistic effect with glycerol solvent

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Directions

• Compare above ground extract with root extract
• Cytokine assay (TNF-alpha) for further inflammatory response evaluation

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Questions?