Isoflavone-enriched soybean leaves (Glycine max) restore loss of dermal collagen fibers induced by ovariectomy in the Sprague Dawley rats (2025)

Keywords: Isoflavone, Soybean leaves, Collagen, Skin, Menopause, Ovariectomy

Phytoestrogen-riched soybean leaves extract

IESLs were obtained by metabolite farming through ethylene treatment to soybean plants at Gyeongsang National University [17]. Metabolite farming is a particular procedure for preharvest or post-harvest plants to enhance the content of bioactive metabolites. IESL has the feature of having around 50-fold high isoflavone content in comparison with typical soybean leaves (Fig. 1A, B, and insert). Target isoflavones were annotated as daidzin and genistein by LC-ESI-Q-TOF–MS spectral data and comparing retention time (t_R) with standard compounds. IESL extracts were concentrated to have an 11mg/g content of isoflavones.

Inducing OVX in SD rats

Female Sprague Dawley (SD) rats, aged six months, were procured from Central Lab Animal Inc. (Seoul, South Korea). Following a seven-day acclimation period, the rats underwent a bilateral ovariectomy. After the surgery, the animals were categorized into four different groups: the first control group consisted of intact rats that were given only a water vehicle (CTL); the second group included ovariectomized rats that received the water vehicle (OVX); the third group was made up of ovariectomized rats received IESL daily containing a lower dosage of isoflavones (OVX/L; 6.25mg/kg/day of isoflavones); finally, the fourth group involved ovariectomized rats given IESL containing a higher dosage of isoflavones (OVX/H; 18.8mg/kg/day of isoflavones). Oral administration of IESL was conducted each day for a total duration of three months. Animals were maintained according to the guidelines of NIH for the care and use of laboratory animals. All experimental protocols and surgical procedures were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (Approval no. GNU-150804-R0037).

Loss of dermal collagen fibers and roles of IESL

In the CTL group, well-developed dense connective tissues are detected in the dermis both in hematoxylin and eosin (H&E) staining and Masson’s trichrome (MT) staining (Fig.2A, E). Upon examination of the magnified images from the CTL group, it is evident that the collagen fibers exhibit a remarkably pronounced staining, indicating a strong intensity (Fig.2I). OVX surgery significantly decreased the relative optical density (ROD) of collagen fibers in the dermis (Fig. B, F, and J), showing a reactive optical density of 76.801% compared to the control group (Fig.2M). In the OVX/L group, a significant change in the ROD values was not observed compared to the OVX group (Fig. C, G, and K). In the OVX/H group, however, the ROD value increased significantly compared to the OVX group (Fig. D, H, and L), with a value of 105.515% compared to the control group (Fig.2M). Dermal thickness exhibited a similar pattern of change as the ROD value in the dermis (Fig.2N). In the control group, the average dermal thickness measured 726.824μm, while in the OVX group, it decreased significantly to an average of 580.167μm. The OVX/L group showed increased average values of 90.507% and 625.292μm for ROD and dermal thickness, respectively, compared to the OVX group, but the significance was not observed when compared to the OVX group. In the OVX/H group, dermal thickness was significantly increased compared to the OVX group, with an average value of 664.75μm observed.

In this experiment, IESL that we used have about 50 times the isoflavone content compared to regular soy leaves and were confirmed to contain isoflavones such as daidzin and genistein (Fig.1). Isoflavones, found in plants, are known to exhibit various physiological effects in the human body by mimicking the action of estrogen [18]. Such isoflavones are also known to be involved in skin protection through the up-regulation of antioxidants and their free radical scavenging activities [19, 20]. They are also closely involved in modulating collagen production and reduction in the skin [21, 22]. In the previous study, we confirmed that treatment of IESL significantly increased COL1A1andCOL3A1 expression in human dermal fibroblasts [21]. Furthermore, there are numerous research findings indicating that isoflavones influence the production of collagen in the skin.

Concerning TGF-β (Transforming Growth Factor-Beta) and Smad proteins, it has been widely reported that isoflavones activate TGF-β receptors, enhancing the phosphorylation of Smad proteins and increasing collagen production [23, 24]. In addition, menopause induces an imbalance between MMPs and their inhibitors [25], and it can result in excessive collagen degradation and tissue damage, contributing to skin aging [26, 27]. Isoflavones can bind to estrogen receptors and mimic the actions of endogenous estrogens, including modulation of MMPs via the MAPK and AP-1 pathways [28].

In this study, we utilized a specially cultivated IESL, which had approximately 50 times more isoflavones, and notably observed a significant increase in collagen density in the dermis of the ovariectomized rat skin. Given these results, IESL could be developed as an effective therapeutic agent in mitigating skin aging and promoting tissue damage recovery.

Ethics approval and consent to participate

All experimental protocols and surgical procedures were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (Approval no. GNU-150804-R0037).

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

There is no competing interest.

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Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.