Minoxidil sulfotransferase enzymatical activity in plants: A novel paradigm in increasing minoxidil response in androgenetic alopecia

Minoxidil is the only US FDA approved topical drug for the treatment of androgenetic alopecia (AGA). Minoxidil is effective in hair re‐growth in 30%–40% of patients and 50% of males. To exert its hair growing effect, minoxidil must be sulfonated in the scalp by the minoxidil sulfotransferase enzyme (SULT1A1). Low scalp SULT1A1 correlates with lack of minoxidil response; thus, supplementing the scalp SULT1A1 with naturally occurring minoxidil sulfotransferase enzymes could potentially improve treatment outcomes in AGA patients.

flow through the upregulation of vascular endothelial growth factor receptor (VEGFR), allowing increased oxygen delivery to hair follicles.Finally, the metabolite also functions in activating Potassium positive (K+) channels, promoting the anagen phase of hair growth. 4,5e minoxidil sulfotransferase enzyme (SULT1A1) in the human hair follicles converts minoxidil to its bioactive metabolite, minoxidil sulfate.This enzyme displays individual variability, with higher enzyme activity showing enhanced responses to minoxidil. 6The SULT1A1 enzyme activity in plucked hair follicles has been shown to be a predictor for topical minoxidil response. 6eviously, we have demonstrated increasing SULT1A1 expression via a topical "booster" using an alkalizing agent improves patient response to topical minoxidil. 7,8An alternate method would be to supplement the SULT1A1 with a natural source.Sulfotransferase enzymes are found in animals and plants.Sulfation in the liver of mammals increases the elimination of various compounds, but its function in plants has not been entirely discovered. 9The role of sulfation in plants may be linked to its regulation of metabolites and defense mechanisms.Lackus, N.D., et al., discovered the role of salicinoids as sulfur-storage compounds by studying the activity of the sulfotransferase (SOT) gene family in black cottonwood (Populus trichoparpa). 10Wang, Q., et al. found that the resistant gene of rice stripe virus (RSV) encoded a SOT, enabling the formation of sulphonated salicylic acid (SA).Plants may produce metabolites and undergo chemical modification, such as sulfation, when exposed to environmental stress. 11sufficient levels of scalp SULT1A1 have been linked to a diminished response to minoxidil treatment, posing challenges in achieving satisfactory outcomes for patients with AGA.7][8] The aim of this study is to elucidate the SOT activity of different plants directed to the xenobiotic minoxidil that is, minoxidil sulfotransferase enzyme activity in various plants.

| MATERIAL S
The Minoxidil Response Test (MRT) is a diagnostic test used to evaluate SOT activities in hair follicles.Scientists from Daniel Alain Laboratories in Newport Beach, California, USA created this test.Goren et al. reported a modified colorimetric assay to measure sulfotransferase enzyme activity in human hair, which is based on optical absorbance. 11This assay can quantify the conversion of minoxidilto-minoxidil sulfate, coupled with the conversion of p-nitrophenyl sulfate to p-nitrophenyl.
We selected a total of 10 different plants for testing based on their high flavonoid content since sulfate reaction is an important reaction in the transformation of flavonoid in active form. 11 Note: Cherry juice showed nonsignificant activity toward minoxidil as a substrate (optical density <0.40).Welch Two sample t-tests compare mean differences between cherry juice and each botanical extract.All p-value <0.00001 except for difference between cherry juice and grape seed (p = 0.0004).
Abbreviations: CI, confidence intervals; DF, degree of freedom; N/A, not available.
samples were purchased preprepared from Sigma Aldrich, identifications and voucher numbers are not presented.p-values for mean differences.We considered all statistical results statistically significant at p < 0.05.

| RE SULTS
We have presented our statistical results in Table 1.Table 1 shows the mean OD with standard deviations (SD) for differences between cherry juice and each botanical extract.We compared Cherry juice with other extracts because it showed nonsignificant activity toward minoxidil as a substrate (Optical density 0.40).All differences were statistically significant at p < 0.01.Detailed results on the t-test, degree of freedom, 95% CIs, and p-values for mean differences can be seen in Table 1.
From the 10 common botanical extracts we studied, seven exhibited significant activity (defined as OD or absorbance units (AU) > 0.40 based on the results of the previous studies) 12 toward minoxidil as a substrate (Figure 1).The botanical extracts with the highest OD correlated to an increased potency of the enzyme sulfotransferase.Three botanical extracts in particular, spinach powder, matcha powder, and broccoli powder, displayed the highest activity at 2uL.At a 2uL concentration of extract, spinach powder displayed the highest OD of 13.668, following with matcha powder with at 11.935, and broccoli powder at 6.742.The two extracts with minimal activity toward minoxidil as a substrate included cherry juice and grape seed and the OD for cherry juice did not reach 0.04.

| DISCUSS ION
As AGA continues to burden those globally, developing novel therapeutics to improve hair growth is of key importance.Minoxidil, a potent vasodilator has proven to display therapeutic properties in terms of its effects on stimulating hair follicles.The enzyme, SOT is key to the drugs mechanism of action, as the drug needs to be converted to its sulfated form to carry out its mechanism of action.
However, since individuals display variability in the activity of the minoxidil sulfotransferase enzyme (SULT1A1) in hair follicles, some patients will fail to respond to minoxidil therapy.Thus, the aim of our

| CON CLUS ION
Our study is the first to characterize naturally occurring minoxidil sulfotransferase enzymes in plants.We identified seven botanical extracts with a high flavonoid content that showed significant activity toward minoxidil as a substrate.Despite our limited sample size and limited tests, our results provide insight into a new method to increase minoxidil response.
To determine whether botanical extracts contain minoxidil sulfotransferase enzyme activity, different types of extracts were reacted with the MRT solution.The MRT assay solution consisted of 50 mM phosphate buffer (pH 8), 5 mM potassium p-nitrophenyl sulfate, 20 μM adenosine 3′, 5′-diphosphate, 100 μM minoxidil, and 5 mM MgCl2.0.05 g of ten botanical extracts and 2 mL of deionized water were added in centrifuge tubes 1-10 to create a mixture with a 0.025 g/ mL concentration.The 11th tube was mixed with 0.05 g of all ten substances combined into one solution.Two microlitres of each botanical extract solution was mixed with 20 uL of MRT solution.Each mixture of botanical extract combined with MRT solution was vortexed briefly to ensure adequate mixing.The mixture was left overnight for 24 h.After 24 h, each mix was aspirated and placed in 96-well plates.Each mixture was assigned a column, and the 20uL mixture was placed in rows A-G.The plates were then placed in the BioTek Epoch Plate Reader, and the Optical Density (OD) of each sample was read.This was repeated to obtain an average OD by measuring the color intensity of the solution for each sample of botanical extract.The color of each solution, along with the OD, was recorded.

F I G U R E 1
Optical Density (OD) of 2uL of botanical extract solution in 20uL of MRT.
study was to assess the potential of botanical SOT in sulfating the xenobiotic minoxidil.The sulfotransferase enzymes in these plants have not been previously characterized.This marks the first attempt to investigate and characterize such enzymes in these plant species.To determine the botanical extracts with the highest minoxidil sulfotransferase enzyme activity, the OD of the extracts was recorded.The common botanical extracts studied included broccoli, spinach, kale, grape seed, olive leaf, cabbage, cherry juice, red cabbage, cauliflower, and matcha.Out of these 10 extracts, seven exhibited significant activity toward minoxidil as a substrate, with spinach displaying the greatest activity.Our study has limitations regarding its methodology and cohort size.While we utilized a radiometric SOT assay to measure enzyme activity, more sensitive and specific assays, such as fluorescencebased, photometric, and mass spectrometric assays have recently emerged.Each of these alternative assays offers distinct advantages, including continuous measurement, enzymatic kinetic data determination, and high sensitivity.In our study, the radiometric assay produced discontinuous results due to the need to periodically remove samples from the mixture, leading to decreased accuracy, and limited extrapolation of kinetic data.Moreover, radioactive isotopes used in radiometric assays can exhibit nonspecific binding to assay components or other molecules present in the sample, potentially affecting specificity, and exaggerating the OD measurements.To mitigate these limitations, it is suggested to incorporate a highly sensitive nonradioactive assay alongside the conventional radiometric assay, enabling the use of reduced substrate concentrations and enzyme quantities.12Regarding the sample size, although 7 out of 10 extracts showed significant activity toward minoxidil as a substrate, the small number of the tested botanical extracts in the study is a limitation.Expanding the study to include more extracts from diverse plants may reveal additional instances of naturally occurring minoxidil sulfotransferase activity.More reliable conclusions can be drawn with a larger sample of extracts, enhancing our understanding of naturally occurring minoxidil sulfotransferase in plants.Despite these limitations, it is important to highlight that our study is the first to characterize naturally occurring minoxidil sulfotransferase enzymes in plants, providing valuable insights into this novel area of research.

Broccoli powder Spinach powder Kale Grape Seed Olive leaf powder Cabbage powder Cherry juice Red cabbage Cauliflower powder Matcha powder
Mean differences between cherry juice and each botanical extract.
The list of selected plants included Broccoli Powder, Spinach Powder, Kale, Grape Seed, Olive Leaf Powder, Cabbage Powder, Cherry Juice, Red Cabbage, Cauliflower Power, and Matcha Powder.Since all the TA B L E 1