Limonin, a Component of Immature Citrus Fruits, Activates Anagen Signaling in Dermal Papilla Cells

https://www.mdpi.com/2072-6643/14/24/5358

Abstract
Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss. We evaluated the hair anagen activation effects of limonin, which is abundant in immature citrus fruits. Limonin increased the proliferation of rat dermal papilla cells (rDPC) by changing the levels of cyclin D1 and p27, and increasing the number of BrdU-positive cells. Limonin increased autophagy by decreasing phosphorylated mammalian target of rapamycin levels and increasing the phospho-Raptor, ATG7 and LC3B. Limonin also activated the Wnt/β-catenin pathway by increasing phospho-β-catenin levels. XAV939, a Wnt/β-catenin inhibitor, inhibited these limonin-induced changes, including induced autophagy, BrdU-positive cells, and cell proliferation. Limonin increased the phosphorylated AKT levels in both two-dimensional cultured rDPC and three-dimensional spheroids. Treatment with the PI3K inhibitor wortmannin inhibited limonin-induced proliferation, and disrupted other limonin-mediated changes, including decreased p27, increased BrdU-positive cells, induced autophagy, and increased ATG7 and LC3B levels. Wortmannin also inhibited limonin-induced cyclin D1 and LC3 expression in spheroids. Collectively, these results indicate that limonin can enhance anagen signaling by activating autophagy via targeting the Wnt/β-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.
Keywords: limonin; dermal papilla cells; anagen signaling; autophagy; Wnt/β-Catenin; PI3K/AKT

Wow, these tech essays kill me. I didn't know there was so many technical terms to describe what's happening in our scalps. It's like learning another language lol. So was this limonin extract used as a topical on rats skin. It'd be cheap and easy to buy a bunch of unripe fruit and squeeze out the juice and rub it on our scalps. Actually it may not be that easy. Who sells unripe fruit? You'd have to sneak around lemon and lime orchards and pick them yourself. Or maybe there's enuff limonin in concentrated lemon or lime juice you buy at the grocery store. But you may have to be careful of the acid content if it irritates your skin. Maybe add some buffer or salt to it.

shaftless wrote:Wow, these tech essays kill me. I didn't know there was so many technical terms to describe what's happening in our scalps. It's like learning another language lol. So was this limonin extract used as a topical on rats skin. It'd be cheap and easy to buy a bunch of unripe fruit and squeeze out the juice and rub it on our scalps. Actually it may not be that easy. Who sells unripe fruit? You'd have to sneak around lemon and lime orchards and pick them yourself. Or maybe there's enuff limonin in concentrated lemon or lime juice you buy at the grocery store. But you may have to be careful of the acid content if it irritates your skin. Maybe add some buffer or salt to it.

Not hard to find if you want to take a crack at it:

https://www.alibaba.com/limonin-extract-suppliers.html

Anyway that said, this pathway in the hair loss world is not unique or novel. After reading these papers for over 2 decades, there's a lot more of the "Greek" and lexicon used to describe the mechanisms. The basic take away here is to understand what underpins these processes, and how lifestyle choices, etc., can impact these.

On a further note to discern the quality of the information. One way for example is noting the effects on animals versus humans and the methods section.

Let's have a look:

2. Materials and Methods

2.1. Preparation of Immature Citrus Fruit Extract

The peel of immature citrus fruits native to Jeju Island was washed with distilled water and dried. The sample was homogenized using a grinder and extracted with 70% ethanol at 2000 rpm and 25 °C for 2 h (WIS 20R, WITEG, Wertheim, Germany). After extraction and filtration, the sample was concentrated using a vacuum evaporator (Hei-VAP Advantage, Heldolph, Germany) and lyophilized to a powder.

2.2. Cell Culture

Immortalized rat vibrissa DPC (rDPC) were donated by the Skin Research Institute of the Amore Pacific R&D Center (Yongin, Korea). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) containing 1% antibiotics (Gibco Life Technologies, Grand Island, NY, USA) and 10% heat-inactivated fetal bovine serum (FBS; HyClone). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and sub-cultured every three days.

So they didn't use just *any* rat dermal papilla cells...they used "immortalized" ones, so this also means they are using cloned cells preserved in calf serum. Also very similar culturing methods, frequently mentioned in the virology discussions here.

2.3. Proliferation Assay

The proliferation of rDPC was measured using the water-soluble tetrazolium (WST) assay kit (EZ-Cytox; Daeil Lab Service, Seoul, Korea) according to the manufacturer’s protocol. For 2D culture, rDPC (2000 cells/well) were seeded in 96-well plates in DMEM containing 1% FBS (low-serum condition) for 24 h. The cells were stimulated with immature citrus fruit extract (0.1, 1, 10, and 100 μg/mL), rhoifolin (0.1, 1, and 10 μM; Sigma-Aldrich, St. Louis, MO, USA), hesperidin (0.1, 1, and 10 μM; Sigma-Aldrich), and limonin (0.1, 1, and 10 μM; Sigma-Aldrich) for 72 h. To investigate whether the PI3K/AKT or Wnt/β-catenin pathway affects the limonin-mediated proliferation of 2D rDPC, the cells were pretreated with 10 nM of the PI3K/AKT inhibitor wortmannin (Invitrogen, Carlsbad, CA, USA) or 10 μM of the Wnt/β-catenin inhibitor XAV939 (Tocris Bioscience, Bristol, UK) for 2 h, and then treated with limonin for 72 h. To investigate the effects of limonin on 3D cultured rDPC (3D spheroids), 2D rDPC were cultured in low-attachment 96-well plates for 48 h and treated with limonin for 72 h after 3D spheroid generation. To investigate whether the PI3K/AKT pathway affects limonin-mediated proliferation of 3D spheroids, 3D spheroids were stimulated with 10 nM of wortmannin for 2 h and then treated with or without limonin for 72 h. WST dye (10 μL/well) was added to a 96-well plate and incubated for 3 h. Absorbance (450 nm) was measured using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The experiment was performed thrice, and the changes in the drug-treated groups were compared with the average absorbance value of the dimethyl sulfoxide (DMSO; Sigma-Aldrich)-treated control group. All chemicals were dissolved in DMSO. The final concentration of DMSO used in the experiments is at 0.2% or less.

We can see here where they obtain the immature citrus extract from, including all of its constituents. : Sigma-Aldrich, St. Louis, MO, USA

Getting into the "meat" of the discussion section: Limonin (10 μM) showed a proliferative effect similar to that of minoxidil, a positive control...

I remember a few years ago that an ingredient in wasabi sauce that you put on your sushi could grow hair as well as minoxidil too. True wasabi paste is hard to find at the grocery store cuz it's so potent. It's always diluted with other stuff. Still you wonder if something is that harsh it may not be too good for your skin.