Caesalpiniasappan seeds were recently shown to display beneficial effects on human alimentary tract notably thanks to their anti-inflammatory and antioxidant properties [1] which are more probably linked to Caesalpiniasappan seeds content in 4-methyl catechol, a well-known divalent cation chelator [2]. Indeed, this type of chelator is able to reduce extra and intracellular available quantities of divalent cations such as Mg²⁺ and Ca²⁺, which are essential to numerous cellular enzymes (such as catechol 2,3-dioxygenase or lipoxygenase) implicated in oxidant and inflammatory processes [3][4].

In order to go further in the exploration of the beneficial effects of Caesalpiniasappan seeds on human health, we were then prompted to investigate the capabilities of a Caesalpiniasappan seeds extract to also protect human skin by assessing more deeply its anti-oxidant and anti-inflammatory properties. Additionally, as calcium-dependent enzymes are also implicated in the regulation of human melanogenesis [5], we also evaluated the ability of our Caesalpiniasappan seeds extract to act on melanin production to finally be able to reduce the anesthetic impact of human skin dark spots which are often linked to melanogenesis disorders and/or aging (for a review, see [6]).

We then first clarified the intrinsic antioxidant potential of our Caesalpiniasappan seeds extract by using an acellular in vitro model calling for the well-known paramagnetic radical DiPhenyl Picryl Hydrazyl (DPPH) (for a review, see [7]). For the second time, we investigated if our Caesalpiniasappan seeds extract could display a sufficient antioxidant effect to protect skin progenitor cells against oxidative attacks (UV irradiations). To reach this goal, we developed an original model of human skin cell cultures enriched in progenitor cells by helping us of the work of 1) Goodell et al., which have published in 1996 a relatively simple method to select stem cells within murine blood [8], and of 2) Durand and Olive which have published in 1982 a result enabling a further simplification of the protocol of Goodell et al. [9]. Briefly, Goodell et al. used a dye called Hoescht 33342 to identify stem cells (only stem cells possess Multi-Drug Resistance—MDR—proteins allowing them to exclude the dye) and a cell-sorter to harvest them (stem cells are the unmarked ones). Durand and Olive showed in 1982 that Hoescht 33342 is finally cytotoxic for dividing cells. As a consequence, the introduction of Hoescht 33342 in monolayer cell cultures ends up killing non-stem cells after several replications.

Reasoning that an antioxidant effect could logically result in an anti-inflammatory one (for a review, see [10]), we then investigate the ability of our Caesalpiniasappan seeds extract to inhibit an enzyme notably implicated in epidermis inflammation processes, i.e. the lipoxygenase (LOX). In fact, even if LOX are key enzymes in the biosynthesis of a variety of signaling molecules implicated in the regulation of many essential biological functions, they are also thought to play a significant role in inflammatory skin diseases and cancer [11].

In a second part of our study, we reasoned that other calcium-dependent enzymes such as tyrosinase could also be inhibited by our Caesalpiniasappan seeds extract. As many melanogenesis disorders and/or aging could trigger the apparition of anesthetic dark spots on human skin (6), possibly leading to additional psychological problems [12]-[14], we were prompted to evaluate the efficacy of our extract to act on human skin melanogenesis regarding this “perhaps” more cosmetic considerations. We studied the ability of our Caesalpiniasappan seeds extract to inhibit tyrosinase in 1) an acellular in vitro model calling for a mushroom purified enzyme showing 98% of sequence homology with the human enzyme, and 2) an in vitro model of human normal melanocytes. Finally, we performed an in vivotest calling for human volunteers to achieve the demonstration of a significant bleaching effect of our Caesalpiniasappan seeds extract on human skin dark spots.

1) DPPH assay: Antiradical activities were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to determine EC50 values. Briefly, 60 µM solution of DPPH was prepared by dissolving 1.5 mg of DPPH crystalline solid in 62.5 mL of analytical grade methanol and stored at 4˚C. For each tested condition 100 µL of test compound (Caesalpiniasappan seeds extract at final concentrations of 0.01; 0.1 and 1%; v/v) or reference standard solution were mixed with 100 µL of DPPH solution in a well of 96 well plates. After 30 min incubation in the dark at room temperature, the absorbance at 520 nm was recorded using a Spectrophotometer.

Reference standard compound being used was Trolox. A stock solution of 1 mg/mL was prepared by dissolving 2 mg of Trolox in 2 mL of ethanol. Then, serial dilution in distilled water were prepared.

For each concentration tested radical scavenging activity has been calculated using the formula bellow:

2) Stem cells protection assay:Primary human keratinocytes were isolated from an abdominal skin residue coming from plastic surgery. Cells were grown in Keratinocytes Growth Medium 2 (Promocell). Second passage keratinocytes were seeded into 96 well plates at a cell density of 5000 cells per well and were cultured at 37˚C in an atmosphere containing 5% of CO₂ until reaching around 80% of confluency. Hoescht 33342 at 1 µg/ml was incubated with cells for 1 hour. Keratinocytes monolayer were rinsed with PBS and incubated again for 6 days. During this period, cell medium was renewed every 2 days. Caesalpiniasappan seeds extract at 0.00015; 0.0005 and 0.0015% (v/v) was put in contact with cells for 24 hours and then cells were rinsed with PBS and irradiated with UVB at 30 mJ/cm² (this UV dose was determined following preliminary dose-dependence studies; data not shown). Keratinocytes were then incubated again with test compound at 37˚C. From this step every 2 days cell viability was measured in wells by using Alamar blue. Briefly, cells were incubated at 37˚C for 2 hours with 10-fold diluted Alamar blue and then optical density was measured at 560 nm and 600 nm. Cells monolayer were rinsed, and incubated again with test compound at 37˚C. The experimentation is stopped when clear difference of viability is noted between irradiated and non-irradiated control.

3) LOX activity assay:LOX activity assay was performed in an acellular in vitro model by using a dedicated assay kit commercialized by Mybiosource (San Diego, USA).

4) Tyrosinase activity assay (acellularin vitromodel): Tyrosinase inhibition assays were performed with L-DOPA as substrate. The reaction mixture (200 μL) contained 50 μL of mushroom tyrosinase (100 U/mL), 100 μL of test compound solution (Caesalpiniasappan seeds extract at final concentrations of 0.5; 1 and 2%, v/v) and 50 μL of L-DOPA at 0.02% (w/v). Reaction between tyrosinase and L-DOPA was followed during 10 minutes after the addition of the L-DOPA by reading the absorbance of the incubation media at 490 nm. Kojic acid was used as a positive control.

5) Melanogenesis assay (cellularin vitromodel):

(1) Materials: Human normal melanocytes were obtained from a 5 years old Caucasian donor. Melanocyte growth medium 2 PMA free (MGM 2 PMA free) was purchased from Promocell (Heidelberg, Germany). Penicillin and streptomycin came from Dutscher (Bernolsheim, France). Kojic acid, Melanin and NaOH were acquired from Sigma-Aldrich (Saint louis).

(2) Cell culture: Normal human melanocytes were seeded in multiwell culture plates and cultured in MGM PMA free in a humidified incubator at 37˚C under a 5% CO₂/95% air atmosphere, until they reached confluence. Melanocytes were then incubated for a 72 hours period in the absence (control), or in the presence of Kojic acid at 250 µM or of increasing concentrations of our Caesalpiniasappan seeds extract (0.001; 0.0025 and 0.005%, v/v).

(3) Melanin quantification: At the end of the incubation period, the monolayers were rinsed with PBS. Cells monolayers were then lysed and melanin was solubilized using NaOH 1 N. Melanin was quantified in cell lysates by measuring optical density at 450 nm.

(4) Proteins measurement: Total proteins contained in the cell lysates were quantified, by using a spectro-colorimetric method (Bradford method [15]).

6)In vivostudy (skin dark spots analysis): 20 healthy volunteers have applied twice a day a formulation containing 1% (v/v) of our Caesalpiniasappan seeds extract during 28 days. At D0 (before the application of any product) Surface of the spots was analyzed on the images by using a dedicated software.

7)Statistics: As indicated under each figure, results were statistically analyzed by a one way ANOVA analysis followed, when it was required, by a Holm-Sidak test or by using paired Student t-tests.

The recent publication of Sasongko etal. [1] in 2025 showing significant benefits of Caesalpiniasappan seeds on human health through its peros action, prompted us to investigate if Caesalpiniasappan seeds extracts could also display benefic actions in other human organs such as skin. As Sasongko etal. [1] strongly suggest that favorable effects of Caesalpiniasappan seeds are linked to its anti-oxidant properties, we start our study by evaluating the intrinsic anti-oxidant effect of our own Caesalpiniasappan seeds extract resorting to a paramagnetic radical, i.e. the Diphenyl Picryl Hydrazyl (DPPH). As shown in Figure 1, in the selected experimental conditions, our Caesalpiniasappan seeds extract display a significant and very powerful anti-oxidant effect: DPPH reduction raised 33.2; 97.5 and 97.1% for Caesalpiniasappan seeds extract concentrations of 0.01; 0.1 and 1% (v/v), respectively.

In the last decades, the number of researches concerning stem cells have grown in an exponential manner showing the growing interest of scientists for this promising “therapeutic” target. Epidermis progenitor cells so today consist in a very interesting target for the development of innovative and pertinent anti-aging products (for a review, see [16]). We then attempted to examine if our Caesalpiniasappan seeds extract could help human skin to slow its aging by at least protecting cutaneous stem cells against environmental aggressions such as UV-irradiations. To achieve this goal, we profit of our newly developed model of human normal keratinocytes cultures enriched in progenitor cells.

Rapidly, this model took advantage of the capability of stem cells to exclude toxic agents thanks to their Multi-Drug Resistance proteins (MDR proteins), which are absent in the non-progenitor cells. As a consequence, when human normal skin cells were cultured in the presence of a genotoxic product, we obtained after several cell divisions, a culture enriched in the only cells which are able to exclude the genotoxic agent, i.e. the progenitor ones.

As shown in Figure 2, in the selected experimental conditions, our Caesalpiniasappan seeds extract at 0.00015; 0.0005 and 0.0015% (v/v) was able to significantly protect cutaneous progenitor cells against Uv-irradiations reducing their cytotoxic effect by 51.1; 76.9 and 86.2%, respectively.

***: Statistically different from the “not treated” condition (p < 0.001); One Way ANOVA analysis + Holm-Sidak test.

Figure 1. Anti-oxidant activity of Caesalpiniasappan seeds extract (DPPH assay).

***: Statistically different from the “not treated” condition (p < 0.001); One Way ANOVA analysis + Holm-Sidak test.

Figure 2. Protective effect of Caesalpiniasappan seeds extract on cutaneous progenitor cells irradiated by UV.

These results clearly show that our Caesalpiniasappan seeds extract could provide a very efficient shield effect against deleterious effect of environmental aggressions such as UV radiations and the oxidative stress that they trigger. Skin stem cells capital could so be efficiently preserved ensuring a younger and healthier aspect to human skin.

As oxidative bursts are often linked to inflammatory situations, we then choose to test the ability of our Caesalpiniasappan seeds extract to inhibit enzymes implicated in pro-inflammatory responses via the generation of Reactive Oxygen Species (ROS), i.e. lipoxygenases (LOX) [17].

As shown in Figure 3, in the selected experimental conditions, our Caesalpiniasappan seeds extract at 0.01; 0.1 and 1% (v/v) significantly inhibited lipoxygenase by 69.4; 88.1 and 91.1%, respectively. This result so confirm that Caesalpiniasappan seeds could also constitute a good tool to soften inflammatory situations in human skin. Additionally, as LOX is also present in human intestine [18], it could be possible that the beneficial effect of Caesalpiniasappan seeds on human alimentary tract showed by Sasongko etal. in 2025, was at least partially due to an inhibitory effect on LOX.

***: Statistically different from the “not treated” condition (p < 0.001); One Way ANOVA analysis + Holm-Sidak test.

Figure 3. Caesalpiniasappan seeds extract effect on LOX activity.

In a more mechanistic way, as LOX activity needs divalent cations (for a review, see [19]), we can imagine that the 4-methyl catechol (a well-known divalent cation chelator [20]) contained in our Caesalpiniasappan seeds extract, could be responsible for the inhibition of this enzyme. Additionally, as divalent cations are implicated in oxidative reactions which not imply enzymes (we can for example mention the Fenton [21] and the Haber-Weis [22] reactions), it is also possible to suggest that the anti-oxidant activity of the 4-methyl catechol contained in our Caesalpiniasappan seeds extract, could also be understand in these terms.

In another part of our study, we reasoned that other divalent cation-dependent enzymes could cover important functions in human skin homeostasis. It is notably the case of tyrosinase, the main enzyme of melanogenesis. In case a melanogenesis disorders notably, it could be useful to act on its activity to reduce esthetic impact of skin dark spots. We then choose to evaluate the ability of our Caesalpiniasappan seeds extract to modulate tyrosinase activity.

As shown in Figure 4, our Caesalpiniasappan seeds extract significantly and virtually totally inhibit tyrosinase activity for concentrations as low as 0.5% (v/v). More probably thanks to its content in 4-methyl catechol, our Caesalpiniasappan seeds extract could so also constitute a very good tool to fight against human melanogenesis disorders. As a consequence, and before to realize an invivo study, we have decided to evaluate the ability of our Caesalpiniasappan seeds extract to reduce melanogenesis in a model of human normal melanocytes monolayers.

***: Statistically different from the “not treated” condition (p < 0.001); One Way ANOVA analysis + Holm-Sidak test.

Figure 4. Caesalpiniasappan seeds extract effect on tyrosinase activity.

As shown in Figure 5, in the selected experimental conditions, our Caesalpiniasappan seeds extract significantly inhibited melanogenesis in a model of human normal melanocytes by 34.4; 93.7 and 78.2% at the following concentrations: 0.001; 0.0025 and 0.005%, respectively.

Reasoning that this effect may logically lead to a visible in vivo efficacy, we decided to perform an in vivo study calling for healthy volunteers showing dark spots on their faces.

As shown in Figure 6, the use twice a day of a formulation containing 1% of our Caesalpiniasappan seeds extract lead to a significant reduction of volunteer’s face dark spot surface: −22.2%.

Taken together, our study results unambiguously show that Caesalpiniasappan seeds not only display beneficial effects on human health through alimentary

**: Statistically different from the “not treated” condition (p < 0.01); One Way ANOVA analysis + Holm-Sidak test. ***: Statistically different from the “not treated” condition (p < 0.001); One Way ANOVA analysis + Holm-Sidak test.

Figure 5. Caesalpiniasappan seeds extract effect on melanogenesis.

*****: Statistically different from the “not treated” condition (p < 0.00001); Paired Student t-tests.

Figure 6. Skin dark spot surface modification after treatment with a formulation containing 1% (v/v) of Caesalpiniasappan seeds extract.

tract, but could also benefit to other human organs such as skin. 4-methyl catechol content of our Caesalpiniasappan seeds extract seems to be largely implicated in its anti-oxidant effects and in its regulatory activity on inflammatory processes. The ability of this compound to chelate divalent cations is clearly responsible, at least in part, for the effects of our Caesalpiniasappan seeds extract on tyrosinase activity and human melanogenesis.

Caesalpiniasappan seeds extracts could so today consist in a very efficient tool to notably act on melanogenesis disorders and its esthetic impacts, but could also help to preserve cutaneous stem cells capital in order to reach a younger and healthier skin, as suggested by our invitro study.