I've been trying to do the DPPH assay on Andrographolide for weeks, but the result does not show any antioxidant effect on the compound. I use 0.1 mM of DPPH and the starting concentration is 1000 µM. Many published articles mentioned that Andrographolide has antioxidant activity. Does anyone have any suggestions?
To perform a DPPH assay on Andrographolide, follow these steps:
Preparation
1. *Sample Preparation*: Prepare different concentrations of Andrographolide (e.g., 100-500 µg/ml) in a suitable solvent, such as ethanol or methanol.
2. *DPPH Solution*: Prepare a DPPH solution (typically 0.1-0.2 mM) in the same solvent.
Assay Procedure
1. *Mixing*: Mix the Andrographolide solution with the DPPH solution in a specific ratio (usually 1:1).
2. *Incubation*: Incubate the mixture in the dark for a specified time (usually 30 minutes).
3. *Measurement*: Measure the absorbance of the mixture at 517 nm using a spectrophotometer.
Calculation
1. *Inhibition Percentage*: Calculate the percentage inhibition of DPPH radical using the formula: Inhibition (%) = [(A0 - A1) / A0] x 100, where A0 is the absorbance of the control (DPPH solution without sample) and A1 is the absorbance of the sample.
2. *IC50 Value*: Calculate the IC50 value, which represents the concentration of Andrographolide required to inhibit 50% of the DPPH radicals.
Notes
- *Antioxidant Activity*: Andrographolide has shown significant antioxidant activity in various studies, with IC50 values ranging from 69.32 µg/ml to higher concentrations.
- *Solvent Selection*: Choose a suitable solvent for the assay, as it may affect the results.
- *Standardization*: Use a standard antioxidant, such as ascorbic acid or Trolox, as a reference ¹ ² ³.
H.A Adewuyi Thank you for your suggestion. I tried two of the solvents: ethanol, which has a solubility of 0.2 mg/mL, and DMSO, which has a solubility of 3 mg/mL. Neither solvent showed any antioxidant activity for andrographolide.
Yurii V Geletii Thank you for your answer. Many articles mention andrographolide's antioxidant activity. Here are some that report positive results.
https://journals.sagepub.com/doi/full/10.1177/0960327112459530#fig6-0960327112459530
https://www.mdpi.com/2076-3921/8/12/571
Andrographolide is a diterpene lactone with weak H-atom–donating ability, so it often shows little or no DPPH• scavenging. For a fair test, fully dissolve it (EtOH/MeOH, ≤1% DMSO), use fresh 0.06–0.1 mM DPPH, incubate 30–60 min in the dark at RT, and include Trolox/ascorbate controls and solvent blanks while reading A517 across 1–1000 µM. If inhibition remains low, that result is likely real—try ABTS•+, FRAP/CUPRAC, ORAC, or lipid-peroxidation assays, which better capture non-phenolic antioxidants. Also confirm your sample/solvent doesn’t absorb at 517 nm and that the DPPH solution hasn’t degraded.
Amit Kumar Acharzo , you are right that "Andrographolide is a diterpene lactone with weak H-atom–donating ability, so it often shows little or no DPPH• scavenging."
Sasichai Jaiberkban A whatever "simple" antioxidant assay is based on a simplification of chemistry behind the scene. Therefore, the "false positive" results are commonly obtained. Be critical. Published results are not obligatory correct.
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