Main Research Findings

1) By inhibiting inflammatory reactions and melanin deposition, treatment with carnosic acid reduces postinflammatory hyperpigmentation.

2) Treatment with carnosic acid was found to reduce free fatty acid-induced insulin resistance throughout adipocytes and myocytes.

 

Selected Data

1) The study performed by the research team of Su et al investigated the therapeutic potential of carnosic acid (CA) on postinflammatory hyperpigmentation (PIH) using a comprehensive array of in vitro and in vivo models. Animal models included juvenile zebrafish for observing melanin deposition and C57BL/6J mice for skin wound healing experiments. Zebrafish embryos were meticulously prepared by random separation of adult zebrafish for mating, embryo collection, and subsequent grouping into 6-well plates with 30 embryos/well for treatment. Mouse experiments utilized 8-week-old female C57BL/6J mice, housed under specific pathogen-free conditions with controlled temperature, humidity, and light-dark cycles. Key cellular models included human skin keratinocyte HaCaT cells, B16F10 mouse melanoma cells, and fibroblast L929 cells, all maintained under standard high-glucose DMEM or RPMI-1640 media supplemented with FBS, penicillin, and streptomycin in a humidified 5% CO2 atmosphere at 37°C [1].

Central to the investigation was carnosic acid (CA), alongside other key reagents such as α-melanocyte stimulating hormone (α-MSH) to induce melanogenesis, 1-Phenyl-2-thiourea (PTU) as a positive control for melanin inhibition, and various components for liposome and hydrogel formulations, all procured from reputable suppliers. Melanogenesis and tyrosinase activity were quantified using several methods. In zebrafish, melanin deposition was visually assessed and quantified by measuring absorbance after dissolution in NaOH. For B16F10 cells, melanin content was determined spectrophotometrically after α-MSH stimulation and CA treatment, normalized to total protein content. Tyrosinase activity in both zebrafish larvae and B16F10 cells was measured using a commercial kit, following specific incubation and spectrophotometric absorbance protocols. Cell viability, crucial for confirming non-toxic effects, was determined using the CCK-8 assay on B16F10, HaCaT, and L929 cells, measuring absorbance at 450 nm after 48 hours of treatment.

To assess melanosome transfer, melanocores were prepared from B16F10 cells and added to HaCaT keratinocytes. Transfer was observed and quantified using Fontana-Masson ammoniacal silver staining, which visualizes melanin as brown/yellowish particles, followed by microscopic analysis. The formulation of CA into a liposome-hydrogel system (CA-LP-GEL) involved a multi-step process: initial dissolution of soybean lecithin, cholesterol, CA, and α-tocopherol acetate in organic solvents, followed by solvent evaporation to form a lipid film. This film was then re-suspended in phosphate buffer saline to create CA liposomes (CA-LP). Hydrogels were prepared from chitosan dissolved in hydrochloric acid and mixed with β-glycerol phosphate. CA-LP-GEL was formed by combining CA-LP with the hydrogel matrix. The physicochemical properties of CA-LP were characterized for particle size distribution, polydispersity index (PDI), and zeta potential. The in vitro release kinetics of CA from CA-LP-GEL were monitored by immersing the hydrogel in the PBS buffer and measuring released CA over time. Cytotoxicity of the LP-GEL and CA-LP-GEL formulations was evaluated on L929 fibroblast cells using the CCK-8 assay [1].

For in vivo wound healing studies, female C57BL/6J mice underwent anesthesia, fur shaving, and the creation of two 1 cm diameter excisional wounds on their backs. To induce and maintain inflammation, the wounds were subjected to UVB irradiation three times a week for 14 days. Mice were then divided into seven treatment groups, receiving immediate and daily topical applications of control solutions, model treatments, LP-GEL, or CA-LP-GEL at 0.2% and 1% concentrations. Wound closure was monitored visually through daily photographic records and quantitatively by calculating the wound healing ratio using imaging software to analyze traced wound areas.

Histological examinations of collected wound tissues involved fixation in paraformaldehyde, paraffin embedding, sectioning (5 µm thickness), and staining with hematoxylin and eosin (H&E) for general morphology and Masson’s trichrome (MT) for collagen visualization. Collagen percentage and epidermal thickness were quantified from these stained sections. Gene expression analysis was performed using real-time quantitative PCR (RT-qPCR). Total RNA was extracted from cells or wound tissues using TRIzol reagent, reverse transcribed into cDNA, and then amplified with SYBR Green premix. Primers for target genes related to melanogenesis (MITF, TYR, TRP-1, TRP-2), melanosome transfer (MLPH, Myova, Rab27a), and inflammatory cytokines (IL-1β, TNF-α, VEGF) were used, with β-actin serving as an internal control. All statistical analyses were conducted using one-way ANOVA followed by Tukey’s post-hoc test with GraphPad Prism software [1].

2) This comprehensive study conducted by researchers Den et al aimed to elucidate the mechanisms by which carnosic acid (CA) attenuates free fatty acid (FFA)-induced insulin resistance in muscle cells and adipocytes. The experimental design primarily relied on in vitro cell culture models, specifically L6 rat skeletal muscle cells, GLUT4myc overexpressing L6 rat skeletal muscle cells, and 3T3-L1 adipocytes. Each cell type was meticulously cultured and differentiated to mimic their physiological states. L6 myoblasts were grown to confluence and differentiated into myotubes in α-Minimum Essential Medium (MEM) supplemented with 2% fetal bovine serum (FBS). 3T3-L1 adipocytes were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 2 mM glutamine, followed by a detailed differentiation protocol involving various hormonal cocktails (IBMX, dexamethasone, insulin, rosiglitazone) over eight days. All treatments were subsequently conducted using serum-free media to minimize confounding factors [2].

The core experimental manipulation involved inducing insulin resistance with palmitate, a common FFA. A specific palmitate stock solution was prepared by conjugating palmitic acid with fatty acid-free bovine serum albumin (BSA) to ensure proper solubility and delivery to cells, maintaining a final molar ratio of free palmitate to BSA of 6:1. Differentiated L6 myotubes and 3T3-L1 adipocytes were exposed to 0.2 mM palmitate for L6 myotubes or 0.4 mM palmitate for 3T3-L1 adipocytes for 16 hours, either in the absence or presence of 2 µM CA for L6 myotubes, or varying concentrations of CA at 2, 10, or 20 µM for 3T3-L1 adipocytes. Following this pre-treatment, cells were acutely stimulated with 100 nM insulin for 30 minutes to assess insulin responsiveness [2].

Glucose uptake, a primary indicator of insulin sensitivity, was measured using a [3H]-2-deoxy-D-glucose uptake assay. After treatments, cells were washed, exposed to HEPES-buffered saline (HBS) containing 10 µM [3H]-2-deoxy-D-glucose for 10 minutes, and then lysed. The radioactivity in the lysates was quantified using a liquid scintillation β-counter. To specifically assess the translocation of glucose transporter 4 (GLUT4) to the plasma membrane, GLUT4myc overexpressing L6 myoblasts were utilized. After treatments, these cells were fixed, blocked with goat serum, and incubated with a primary anti-myc polyclonal antibody, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. The presence of GLUT4myc on the plasma membrane was then quantified by measuring the absorbance at 492 nm after adding O-phenylenediamine dihydrochloride (OPD) reagent, which produces a colored product proportional to GLUT4 levels.

To delve into the intracellular signaling cascades, various protein phosphorylation levels were analyzed using immunoblotting techniques. Following treatment, cells were lysed using an ice-cold lysis buffer containing a cocktail of protease and phosphatase inhibitors to preserve protein integrity. Protein concentrations were determined using the Bradford assay. Equal amounts of protein of 20 µg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were blocked and sequentially incubated with specific primary antibodies targeting phosphorylated and total forms of key signaling proteins, including Akt (Ser473), IRS-1 (Ser307, Ser636/639), JNK (Thr183/Tyr185), mTOR (Ser2448), p70S6K (Thr389), AMPK (Thr172), ACC (Ser79), and raptor (Ser792). HRP-conjugated secondary antibodies were used for detection, and protein bands were visualized using reagents and quantified via densitometry with FluroChem software and imaging. For IRS-1 phosphorylation analysis, immunoprecipitation was performed prior to immunoblotting, where whole-cell lysates were incubated with an IRS-1 antibody conjugated to magnetic beads, and the eluted protein was then processed for SDS-PAGE and immunoblotting [2].

All experiments were conducted with a minimum of three to seven independent replicates, and data were pooled. Statistical analysis was performed using GraphPad Prism software version 5.3. One-way analysis of variance (ANOVA) was employed, followed by Tukey’s post hoc test for multiple comparisons to determine significant differences between groups. These rigorous methodological approaches ensured comprehensive and reliable data collection to understand the therapeutic potential of CA against insulin resistance [2].

Discussion

1) The study performed by Su et al demonstrated that carnosic acid (CA) effectively ameliorates postinflammatory hyperpigmentation (PIH) by concurrently inhibiting inflammatory reactions and melanin deposition, both in vitro and in vivo. Initial experiments in juvenile zebrafish larvae showed that CA dose-dependently decreased melanin deposition and tyrosinase activity. Specifically, CA treatment at 1.25, 2.5, and 5 µM significantly reduced melanin deposition by 27.35%, 47.88%, and 73.22%, respectively, with 5 µM CA also significantly inhibiting tyrosinase activity, without notable toxicity. This indicated CA’s direct suppressive effect on melanin production in a living organism [1].

Moving to cellular models, CA was found to inhibit α-MSH-induced melanin synthesis in B16F10 melanoma cells without causing cytotoxicity at concentrations up to 40 µM. At 5, 10, and 20 µM, CA significantly reduced α-MSH-stimulated melanin content by 48.63%, 26.91%, and 42.38%, respectively, and markedly suppressed tyrosinase activity. Furthermore, CA significantly downregulated the mRNA expression of key melanogenesis genes, including MITF, TYR, TRP-1, and TRP-2, which were upregulated by α-MSH, confirming its inhibitory mechanism on melanin synthesis at the genetic level. Beyond synthesis, CA also demonstrated an ability to inhibit melanosome transfer. It suppressed the α-MSH-induced mRNA upregulation of melanosome transfer-related genes (MLPH, Myova, Rab27a) in B16F10 cells. Concurrently, in HaCaT keratinocytes, CA significantly decreased the number of transferred melanocores and reduced intracellular melanin content, confirming its role in preventing melanin dissemination [1].

 

Figure 1: changes in A) cell viability, B) melanin content, C) TYR activity, D) mRNA transcript levels MTIF, E) mRNA transcript levels of TYR, F) mRNA transcript levels of TRP-1 and G) mRNA transcript levels of TRP-2 following treatment with CA

To translate these findings into a therapeutic application, CA was formulated into a liposome-hydrogel system (CA-LP-GEL). Characterization revealed CA-LP had an average particle size of approximately 221 nm with good stability, and the CA-LP-GEL system exhibited no in vitro cytotoxicity on L929 fibroblast cells. In vitro release studies confirmed a sustained release of CA from the hydrogel. In an in vivo mouse model of excisional wounds with UVB-induced inflammation, both LP-GEL and CA-LP-GEL at 0.2% and 1% w/v significantly accelerated skin wound healing compared to the model group, with healed wounds showing no obvious scarring. Histopathological analysis revealed that both LP-GEL and CA-LP-GEL treatments reversed the model group’s increased epidermal thickness and decreased dermal collagen fiber density, indicating improved structural repair. However, a crucial differentiation emerged in their effects on inflammation and hyperpigmentation [1].

While LP-GEL significantly increased the mRNA levels of inflammatory factors (IL-1β and TNF-α) in healing skin, CA-LP-GEL treatment significantly suppressed these inflammatory markers, bringing them closer to control levels. This suggests CA’s potent anti-inflammatory action within the wound environment. Moreover, LP-GEL treatment sharply increased the mRNA levels of melanogenesis-related genes (MITF, TYR) and melanosome transfer genes (MLPH, Myova, Rab27a) in healing skin, leading to significantly increased melanin content. In stark contrast, CA-LP-GEL suppressed these increases, resulting in significantly lower melanin content in the healing skin compared to LP-GEL alone.

This demonstrates that while the hydrogel formulation itself (LP-GEL) can accelerate wound healing, it can exacerbate hyperpigmentation by promoting inflammation and melanogenesis. The incorporation of CA into the hydrogel effectively ameliorated this adverse effect, leading to improved healing with reduced melanin deposition and suppressed inflammation. Overall, the study concluded that CA effectively reduced melanin deposition by inhibiting both melanogenesis and melanosome transfer, and when formulated as CA-LP-GEL, it accelerated skin wound healing while crucially suppressing inflammation and hyperpigmentation, underscoring its significant potential as a therapeutic agent for PIH [1].

2) The study conducted by the research team of Den et al investigated the impact of carnosic acid (CA) on palmitate-induced insulin resistance in L6 muscle cells and 3T3-L1 adipocytes, revealing significant protective and restorative effects across multiple metabolic and signaling pathways. The primary findings consistently demonstrated that CA counteracted the deleterious effects of palmitate, often restoring cellular functions to levels comparable to or better than insulin-sensitive controls [2].

Firstly, CA effectively restored insulin-stimulated glucose uptake in both palmitate-treated L6 myotubes and 3T3-L1 adipocytes. In L6 myotubes, palmitate significantly blunted insulin-stimulated glucose uptake, indicating insulin resistance, but CA treatment at a concentration of 2 µM resulted in a robust restoration of glucose uptake to 179% of control, comparable to insulin-alone stimulation. Interestingly, CA alone also significantly increased both basal glucose uptake, by 253% of control, and insulin-stimulated glucose uptake, by 269% of control, in L6 myotubes. Similarly, in 3T3-L1 adipocytes, palmitate abolished insulin-stimulated glucose uptake, which was significantly improved by CA treatment and restored to 139% of control. CA alone also increased basal glucose uptake in adipocytes by 152% of control. This direct improvement in glucose utilization is a cornerstone of CA’s anti-diabetic potential [2].

Complementing the glucose uptake data, CA also restored insulin-stimulated GLUT4 translocation to the plasma membrane in palmitate-treated L6 myotubes. Palmitate exposure significantly reduced the insulin-mediated increase in plasma membrane GLUT4 levels, reflecting impaired glucose uptake. However, in the presence of CA, GLUT4 translocation was effectively restored to 194.4% of control, a level similar to that achieved with insulin alone. CA alone had no significant effect on basal or insulin-stimulated GLUT4 translocation, suggesting its role is primarily corrective under insulin-resistant conditions.

The study further elucidated CA’s molecular mechanisms by examining key signaling proteins. In both L6 myotubes and 3T3-L1 adipocytes, palmitate significantly reduced insulin-stimulated Akt phosphorylation at Ser473, a critical step in insulin signaling. CA treatment completely prevented this palmitate-induced decline, restoring Akt phosphorylation levels to those seen in insulin-sensitive cells by 335.6% of control in L6 myotubes. The total levels of Akt remained unchanged across all treatments, confirming that CA specifically influenced Akt activation [2].

Moreover, CA effectively prevented the palmitate-induced serine phosphorylation of IRS-1, a known contributor to insulin resistance. In L6 myotubes, palmitate significantly increased IRS-1 phosphorylation at Ser307 and Ser636/639, but CA completely abolished these increases. Similar results were observed in 3T3-L1 adipocytes, where CA treatment prevented the palmitate-induced increase in IRS-1 phosphorylation at Ser307. Total IRS-1 levels were unaffected by any treatment.

CA also demonstrated inhibitory effects on stress-activated kinases implicated in insulin resistance. Palmitate significantly increased JNK phosphorylation (Thr183/Tyr185) in both L6 myotubes and 3T3-L1 adipocytes. CA treatment completely abolished this palmitate-induced JNK activation in both cell types by reducing it to 62.73% of control in L6 myotubes. Surprisingly, CA alone also significantly reduced JNK phosphorylation in L6 myotubes, indicating a broader suppressive effect on this kinase [2].

Furthermore, palmitate significantly increased the phosphorylation of mTOR (Ser2448) and its downstream target p70S6K (Thr389) in both L6 myotubes and 3T3-L1 adipocytes. These activations are often associated with impaired insulin signaling. CA treatment effectively abolished or significantly reduced these palmitate-induced mTOR and p70S6K phosphorylations. For example, in L6 myotubes, CA completely abolished palmitate-induced mTOR phosphorylation and significantly reduced p70S6K phosphorylation [2].

CA also activated the energy sensor AMP-activated protein kinase (AMPK) and its downstream target ACC. In L6 myotubes, CA significantly increased the phosphorylation of both AMPK (Thr172) and ACC (Ser79) even in the presence of palmitate. Similar dose-dependent increases in AMPK and ACC phosphorylation were observed in 3T3-L1 adipocytes, with CA (20 µM) significantly activating both proteins even under palmitate-induced stress. This activation of AMPK, known for its insulin-sensitizing effects, is a key mechanism underlying CA’s beneficial actions. Additionally, CA increased the phosphorylation of raptor (Ser792), an mTORC1 component, further suggesting AMPK-mediated inhibition of mTOR activity.

In conclusion, the study provides robust evidence that carnosic acid effectively counteracts palmitate-induced insulin resistance in both muscle cells and adipocytes. CA restores insulin-stimulated glucose uptake and GLUT4 translocation, prevents the palmitate-induced impairments in Akt and IRS-1 signaling, and suppresses the activation of stress kinases like JNK, mTOR, and p70S6K. Critically, CA activates AMPK and ACC, positioning it as a potent modulator of cellular energy homeostasis and a promising therapeutic agent for ameliorating insulin resistance and Type 2 diabetes mellitus [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Su H, Yang F, Lu K, et al. Carnosic acid ameliorates postinflammatory hyperpigmentation by inhibiting inflammatory reaction and melanin deposition. Biomed Pharmacother. 2024;180:117522. doi:10.1016/j.biopha.2024.117522

[2] Den Hartogh DJ, Vlavcheski F, Giacca A, MacPherson REK, Tsiani E. Carnosic Acid Attenuates the Free Fatty Acid-Induced Insulin Resistance in Muscle Cells and Adipocytes. Cells. 2022;11(1):167. Published 2022 Jan 5. doi:10.3390/cells11010167

 

Carnosic Acid is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.