Filtration Protocol Improves Botanical Supplement Safety

Botanical dietary supplements (BDS) sales topped $27B in 2020 and the global market continues to grow. ^[1] Considered to be low risk by most regulatory agencies botanical supplements are subject to only minimal levels of regulation, product warning labels are rare to non-existent and the products can be purchased without a prescription. For most people and most products, this is not a problem, but the hidden danger of botanical supplements is their potential for interaction with prescription drugs. Perhaps the most famous of these botanical – prescription interactions is the effect of grapefruit, including extracts or simply drinking the juice, on the activity of a range of medications including statins taken to lower cholesterol, drugs to treat high blood pressure, some anti-anxiety drugs, and drugs taken to treat abnormal heart rhythm, amongst others, with potentially dangerous consequences for patients. ^[2] While more widely known, grapefruit is not alone amongst botanicals in its ability to alter the effect of prescription medications. 

The botanical – drug interaction problem

The interaction problem is compounded by the fact that many, if not the majority, of patients fail to disclose their use of botanicals and other alternative medicines to their doctors. Botanical supplementation is particularly prevalent among cancer patients, even though FDA adverse event reporting data suggests that concomitant use of products such as acai berry with anticancer drugs may increase the risk of adverse events.

Part of the reason it is has been difficult to predict BDS-drug interactions is that the available in vitro data for these supplements has historically not been considered an accurate predictor of their effect in vivo. BDS interactions are often over-estimated because extracts are often solubilized at higher concentrations than those at which they are likely to be present in vivo. Furthermore, the complexity of phytochemical composition compared to manufactured pharmaceuticals makes it difficult to predict to what extent they will be absorbed and metabolized. 

A new filtration-based protocol to assess botanical – drug interaction

In her 2019 Thesis ^[3] at Auburn University Department of Medicinal Chemistry, Yilue Zhang described a new method for predicting the effect of two popular botanical supplements, maca root and acai berry, on CYP3A4, a liver enzyme that mediates the metabolism of about 60% of prescription drugs. The fact that CYP3A4 is so central to drug metabolism makes it an important study target, as up or down-regulation of CYP3A4 will affect the metabolism of more than half of all prescription drugs.

In this study, the measurement of liver enzyme metabolic activity was used to provide a more direct estimate of the degree to which the two botanical supplements affect drug metabolism. Ms. Zhang set out to investigate the potential of passively diffused compounds derived from maca and acai extracts to either induce or inhibit CYP3A4 activity, specifically by chemically profiling extracts that had high abundance, high permeability and exhibited bioactivity.

To prepare maca and acai botanical extracts, a protocol was needed that could accurately identify the most prevalent bioactive compounds. Following methanol extraction and sonication of the botanicals, solubilized supernatants were filtered to remove any potential contaminants using AcrodiscÒ One PSF Syringe Filters with 0.45µm PTFE membranes to obtain plant extract stock solutions. 

Improving the estimation of intestinal epithelial absorption

The prepared plant extract stock solutions were subsequently used to investigate several different aspects of CYP3A4 related drug interaction and metabolism. To mimic in vivo physiological conditions more closely, various dilutions of the BDS extracts were processed through a Parallel Artificial Membrane Permeability Assay (PAMPA) 96-well plate system, which is used to assess the likelihood of absorption through a biological membrane such as the intestinal epithelium.

Both passively diffused and non-passively diffused maca and acai extracts were subsequently screened for CYP3A4 inhibition and induction. To analyze CYP3A4 inhibition, the production of the enzyme’s primary metabolite was monitored in the presence of various high abundance BDS extract constituents. Acai berry extracts showed the highest inhibition of CYP3A4 enzyme activity, with passively diffused compounds having a greater effect. No significant induction or inhibition was observed with passively or non-passively diffused constituents of maca extracts.

To analyze CYP3A4 induction, BDS extract constituents were applied to human primary hepatocytes and quantitative PCR analysis was performed to determine CYP3A4 mRNA expression. Results showed a statistically significant increase in CYP3A4 mRNA expression in response to both passively diffused and non-passively diffused acai berry extracts, with passively diffused compounds having the greater effect. Five biologically active acai berry compounds with high abundance and high permeability were further evaluated by Mass Spectrometry (LC-MS and MS-MS) analysis, resulting in the successful separation and identification of nine separate acai berry metabolites.

The incorporation of PAMPA to improve drug interaction prediction, combined with mass spectrometry-based methodology provides a sensitive and effective strategy for more rapidly screening for and increasing the precision of botanical extract - prescription drug interactions.  

Acrodisc PSF syringe filters are available in a variety of membrane materials and pore sizes and can accommodate a range of flow rates and sample volumes. The PTFE membranes used are specifically designed to be compatible with a wide variety of chemical solvents.

You can learn more about the Acrodisc syringe filters used in this study on the Pall website.