**8. Anti‐inflammatory and chemopreventive activity of isoflavonoid genistein alone and incorporated in modern pharmaceutical formulations**

From the main isoflavones reported above, we have studied genistein. One of the most important lines in our research group on this topic involves the analysis of the chemopre‐ ventive effect of the phytoestrogen genistein against malignant melanoma. The isoflavonoid genistein (4′,5,7‐trihydroxyisoflavone) is the aglycone of heteroside genistin. It is the most studied compound from the class of isoflavones together with daidzein, glycitein, formonone‐ tin, equol and biochanin A. It is the major active compound from soy seeds, *Glycine max* (L.) Merr., family *Fabaceae* [127–129]. Regarding the biological activities, recent papers report that: genistein induces apoptosis, inhibits cell proliferation, modulates cell cycle progression on different cancerous cell lines, inhibits angiogenesis, suppress lymphocyte activation and proliferation, stabilizes mast cells and presents mild anti‐inflammatory properties [130, 131]. The phytoestrogen also inhibits the production of reactive oxygen species (ROS) which is directly correlated with DNA modification and tissue damage. Production of ROS, especially by activated cells of the immune system, has been postulated to play an important role in carcinogenesis, particularly in tumor promotion [132, 133].

study on HER2 overexpressing mice has shown that genistein mimicked the estradiol effects, in the presence of estrogen receptor alpha [112], while in postmenopausal women, in the absence of estradiol, genistein directly reduced the anticancer activity of cisplatin, a cytostatic drug commonly used in breast cancer [122]. For instance, according to Tonetti et al. *in vitro* and *in vivo* studies, the concomitant administration of tamoxifen with genistein or daidzein might not be safe because this association has produced bigger size tumors than tamoxifen

Soy isoflavones exhibited *in vivo* protective effects against skin chronic disorders including cancer by reducing pro‐inflammatory cytokines and oxidative stress and through activation of NF‐kB [124]. For example, genistein has suppressed UV‐induced skin carcinogenesis in mice through its moderate inhibitory effect on ornithine decarboxylase activity [125]. Moreover, 7,3′,4′‐trihydroxyisoflavone, a major metabolite of daidzein, has reduced UVB‐induced skin cancer in mice through inhibition of cyclooxygenase‐2 (COX‐2) expression by suppress‐ ing NF‐kB transcription activity [103]. In this regard, genistein loaded‐PLA nanocapsules indicated to be a promising formulation with chemopreventive effects against skin cancer in porcine ear skin not only by increasing the penetration of genistein in skin deeper layers, but

According to Ghaemi et al. study on human papillomavirus (HPV) associated‐cervical can‐ cer in mice, genistein has also indicated immunomodulatory effects through increment of interferon‐gamma (IFN‐gamma) level, lymphocyte proliferation and lactate dehydrogenase

Consequently, the isoflavones from soy products may be considered promising alternative therapies to prevent various types of cancer, more experimental and clinical studies being necessary for establishing the safe dose that can be used especially in patients susceptible to

**8. Anti‐inflammatory and chemopreventive activity of isoflavonoid** 

**genistein alone and incorporated in modern pharmaceutical formulations**

From the main isoflavones reported above, we have studied genistein. One of the most important lines in our research group on this topic involves the analysis of the chemopre‐ ventive effect of the phytoestrogen genistein against malignant melanoma. The isoflavonoid genistein (4′,5,7‐trihydroxyisoflavone) is the aglycone of heteroside genistin. It is the most studied compound from the class of isoflavones together with daidzein, glycitein, formonone‐ tin, equol and biochanin A. It is the major active compound from soy seeds, *Glycine max* (L.) Merr., family *Fabaceae* [127–129]. Regarding the biological activities, recent papers report that: genistein induces apoptosis, inhibits cell proliferation, modulates cell cycle progression on different cancerous cell lines, inhibits angiogenesis, suppress lymphocyte activation and proliferation, stabilizes mast cells and presents mild anti‐inflammatory properties [130, 131]. The phytoestrogen also inhibits the production of reactive oxygen species (ROS) which is directly correlated with DNA modification and tissue damage. Production of ROS, especially

alone [123].

270 Flavonoids - From Biosynthesis to Human Health

(LDH) release [126].

hormone‐dependent tumors.

also by limiting its degradation in time [107].

In a recent complex study employing, the B164A5 and B16F10 murine melanoma cell lines, we have shown testing a wide range of concentrations (150, 100, 50, 30, 15, 5 and 1 μM) that this phytocompound is an active antiproliferative and pro‐apoptotic agent on this two cell lines. MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay has shown an IC50 of 41.1 μM genistein for B164A5 cells and 61.4 μM genistein for B16F10 cells. The carboxyfluo‐ rescein diacetate succinimidyl ester (CFSE) assay has shown that after 24 h of incubation, pro‐ liferation of B16 cells was decreased when treated with 30 μM genistein. Genistein at 100 μM was able to cause G2/M arrest in the cell cycle of these murine melanoma cell lines [134]. DAPI staining was performed in order to detect first signs of apoptosis. When B16 cells were incu‐ bated with 100 μM genistein, this phenomenon could be detected and translated by a reduc‐ tion of the cell number and increase in nuclear fragmentation compared to the control group [134]. Western blot analysis was furtherer conduced, for four important proteins involved in the process of apoptosis, namely caspase 3, poly(ADP‐ribose) polymerase (PARP), Bax and Bcl‐2. Incubation with 100 μM genistein conduced for both B16 cell lines to cleaved caspase‐3 as well as cleaved PARP as responsible for the mechanism of apoptotic events. In another study using all concentrations previously tested (150, 100, 50, 30, 15, 5 and 1 μM genistein) and after a period of incubation of 72 h, we have shown that the phytoestrogen does not induce caspase‐2 activation *in vitro* on B16 melanoma cell lines [135]. In order to get the full pic‐ ture about apoptosis, namely to detect early and late apoptotic cells, annexin V‐FITC/7AAD staining was performed by fluorescence cytometry in parallel for the B16 melanoma cells as well as for the bone marrow‐derived dendritic cells (BMDCs). At its highest concentration, genistein induced slightly more apoptotic events in the B16 cells than in the BMDCs [134]. The aim of the above‐mentioned study was to find a drug that "kills" the cancerous cells and stimulate the immune activity. On this purpose, the potential immune stimulatory activ‐ ity of genistein was tested by measuring the anti‐tumorigenic cytokine IL‐12p70 released by murine primary BMDCs. The phytoestrogen, at the concentration of 5 μM decreased the level of IL‐12p70 of the LPS‐stimulated DCs. These findings were in line with the observation that also the IL‐12p35 mRNA levels were downregulated. T cell activity was further screened by analyzing the concentration of IFN‐γ and IL‐2 cytokine in the supernatant of spleen cells of OT I mice expressing the ovalbumin‐specific transgenic T cell receptors. Results have shown that genistein had no effect on the concentration of IFN‐γ and IL‐2 cytokine [134].

Besides the chemopreventive activity for murine melanoma, our research group have investigated genistein alone and incorporated in randomly methylated β‐cyclodextrin (RAMEB), hydroxypropyl‐β‐cyclodextrin (HPBCD) and hydroxypropyl‐γ‐cyclodextrin (HPGCD) in a molar ratio 1:1 for a range of biological activities. This approach was chosen in order to increase the water solubility of this lipophilic compound. CDs are cyclo‐oligosaccharides presenting a hydrophilic outside and hydrophobic inner side with the ability to form host‐guest inclusion complexes with an increased number of chemical structures [136]. Firstly, quantum chemical calculations were performed analyzing the behavior in gas phase, in water and in dimethyl sulfoxide, the solvent used for the solubilization of active agents for all the mentioned assays. Additionally, it was proofed that incorporation of genistein in the above‐mentioned CDs took place by a series of consecrated techniques such as phase solubility studies, differential scan‐ ning calorimetry (DSC), X‐ray diffraction and scanning electron microscopy (SEM) assays [136]. Genistein and its inclusion complexes were studied *in vitro* on four types of cancer cells lines such as HeLa (cervical adenocarcinoma), MCF‐7 (breast adenocarcinoma), A2780 (human ovarian carcinoma) and A431 (skin epidermoid carcinoma) cell lines using the following con‐ centrations: 1, 3, 10, 30, 60 and 90 μM and a period of incubation of 72 h. A2780 human ovarian carcinoma cell line proofed to be the most sensitive to genistein, followed by HeLa. Proliferation was not significantly affected for the other two cell lines. After incorporation in the above‐men‐ tioned CDs, changes in the antiproliferative action occurred with respect to the tested cell line. Cervical adenocarcinoma HeLa cell line was more sensitive for all three inclusion complexes when compared to pure genistein. The same behavior was found for A2780 cell line, except for the complex with RAMEB. Complexation with RAMEB conduced to an increased IC50 also for MCF‐7 and A431 cell lines. For this two cell lines, complexation of genistein with HPBCD seemed to be the best option [136]. In the same study, genistein and its CD complexes were analyzed by the agar disk‐diffusion method and the dilution method against several bacterial strains: *Bacillus subtilis*, *Enterococcus faecalis, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Pseudomonas aeruginosa* and *Staphylococcus aureus*. Tested compounds at the concentration of 10 mM presented antibacterial activity only for *B. subtilis* [137]. The last line of this study was drawn toward tests for antiangiogenic effects employing the chorioallantoic membrane of the chicken embryo. Pure genistein presented antiangiogenic effects and the HPBCD com‐ plex showed superior activity. Also for the other two complexes, namely HPGCD and RAMEB could be described an antiangiogenic effect but a decreased one as evaluated by applying the 0–5 score [136].

Another attempt to increase the bioavailability of this lipophilic phytoestrogen was directed toward the synthesis and analysis of a genistein ester derivative with myristic acid and complexed with beta cyclodextrin. The successful synthesis of the new compound as well as the successful inclusion in beta cyclodextrin was determined using consecrated assays such as TLC analysis, HPLC analysis, FTIR spectroscopy, MS spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Samples were tested *in vitro*, using the MTT prolifera‐ tion assay on three human cell lines: HeLa—cervix adenocarcinoma, A2780—ovary carcinoma and A431—skin epidermoid carcinoma. Results have shown that, after a period of incubation of 72 h at the concentrations of 10 and 30 μM, respectively, genistein is an active agent on HeLa (cervix adenocarcinoma) and A2780 (ovary carcinoma) cell lines. The new formulations did not decrease the viability of the cancerous cells. This behavior may be explained by the increased stability of the complex within the *in vitro* environment [138].

A new modern formulation explored by the pharmaceutical industry, but not only, is the polyurethane microstructures (PM). What determined us to focus on these compounds? Depending on the structure of the particle, such approach can offer: the possibility of amend‐ ing the lipo‐ or water‐solubility of inclusion structures, protection from external agents such as UV radiation, strong acidic or alkaline environments, drug delivery toward a specific receptor or retard activity of the biologically active compound due to the use of transport vehicles with low speed of degradation [139]. Based on these hypotheses, we have synthesized PM with a yield of encapsulation of 68.3% genistein (w/w). The formulation was tested *in vitro* using the MTT proliferation assay on three human breast cancer cell lines MCF7, MDA‐MB‐231 and T47D‐human breast adenocarcinoma cell lines. Tests were performed also for the antimicrobial and antifungal activity against the following strains: *S. aureus*, *E. coli*, *P. aeruginosa*, *Salmonella enteritidis*, *B. subtilis*, *Bacillus cereus* and *Candida albicans* employing the dilution method. Results made us to conclude that the PM are a bad in *vitro* carrier partner for genistein [137].

place by a series of consecrated techniques such as phase solubility studies, differential scan‐ ning calorimetry (DSC), X‐ray diffraction and scanning electron microscopy (SEM) assays [136]. Genistein and its inclusion complexes were studied *in vitro* on four types of cancer cells lines such as HeLa (cervical adenocarcinoma), MCF‐7 (breast adenocarcinoma), A2780 (human ovarian carcinoma) and A431 (skin epidermoid carcinoma) cell lines using the following con‐ centrations: 1, 3, 10, 30, 60 and 90 μM and a period of incubation of 72 h. A2780 human ovarian carcinoma cell line proofed to be the most sensitive to genistein, followed by HeLa. Proliferation was not significantly affected for the other two cell lines. After incorporation in the above‐men‐ tioned CDs, changes in the antiproliferative action occurred with respect to the tested cell line. Cervical adenocarcinoma HeLa cell line was more sensitive for all three inclusion complexes when compared to pure genistein. The same behavior was found for A2780 cell line, except for the complex with RAMEB. Complexation with RAMEB conduced to an increased IC50 also for MCF‐7 and A431 cell lines. For this two cell lines, complexation of genistein with HPBCD seemed to be the best option [136]. In the same study, genistein and its CD complexes were analyzed by the agar disk‐diffusion method and the dilution method against several bacterial strains: *Bacillus subtilis*, *Enterococcus faecalis, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Pseudomonas aeruginosa* and *Staphylococcus aureus*. Tested compounds at the concentration of 10 mM presented antibacterial activity only for *B. subtilis* [137]. The last line of this study was drawn toward tests for antiangiogenic effects employing the chorioallantoic membrane of the chicken embryo. Pure genistein presented antiangiogenic effects and the HPBCD com‐ plex showed superior activity. Also for the other two complexes, namely HPGCD and RAMEB could be described an antiangiogenic effect but a decreased one as evaluated by applying the

Another attempt to increase the bioavailability of this lipophilic phytoestrogen was directed toward the synthesis and analysis of a genistein ester derivative with myristic acid and complexed with beta cyclodextrin. The successful synthesis of the new compound as well as the successful inclusion in beta cyclodextrin was determined using consecrated assays such as TLC analysis, HPLC analysis, FTIR spectroscopy, MS spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Samples were tested *in vitro*, using the MTT prolifera‐ tion assay on three human cell lines: HeLa—cervix adenocarcinoma, A2780—ovary carcinoma and A431—skin epidermoid carcinoma. Results have shown that, after a period of incubation of 72 h at the concentrations of 10 and 30 μM, respectively, genistein is an active agent on HeLa (cervix adenocarcinoma) and A2780 (ovary carcinoma) cell lines. The new formulations did not decrease the viability of the cancerous cells. This behavior may be explained by the increased

A new modern formulation explored by the pharmaceutical industry, but not only, is the polyurethane microstructures (PM). What determined us to focus on these compounds? Depending on the structure of the particle, such approach can offer: the possibility of amend‐ ing the lipo‐ or water‐solubility of inclusion structures, protection from external agents such as UV radiation, strong acidic or alkaline environments, drug delivery toward a specific receptor or retard activity of the biologically active compound due to the use of transport vehicles with low speed of degradation [139]. Based on these hypotheses, we have synthesized PM with a yield of encapsulation of 68.3% genistein (w/w). The formulation was tested *in vitro* using the

stability of the complex within the *in vitro* environment [138].

0–5 score [136].

272 Flavonoids - From Biosynthesis to Human Health

*In vivo* assays were also performed in order to test the chemopreventive effects of genistein against murine melanoma. During the research in our group, we have observed in a murine model of melanoma, obtained by subcutaneous injection of 0.1 ml of 1\*105 B164A5 cells/mouse that, genistein after a period of 15 days at a dose of 15 mg/kg, body weight decreased tumor vol‐ ume and weigh with about 30% and reduced distance tumors. Noninvasive measurements using the Multiprobe Adapter System (MPA5) from Courage‐Khazaka, Germany, Mexameter® MX 18 showed that genistein reduced the quantity of melanin and the degree of erythema directly cor‐ related with the number of days of treatment [140]. Being very well known, the link between inflammation and cancer, we have analyzed the effect of genistein in an animal model of ear inflammation alone and after incorporation in hydroxypropyl‐beta‐cyclodextrin (HPBCD) and randomly‐methylated‐beta‐cyclodextrin (RAMEB). Cyclodextrins (CDs) are well known agents used to increase the hydro solubility of active lipophilic agents [139]. The study concluded that the phytoestrogen, at a dose of 2 mg can be reconsidered as an active anti‐inflammatory natural compound on C57BL/6 J animal model of inflammation. Additionally, complexation of genistein with the above‐mentioned CDs was done and led to a stronger anti‐inflammatory effect [141]. In a recent study, in order to attempt to increase the bioavailability of this phytoestrogen, we have adopted a new strategy that combines two elements: the formulation and the modality of administration. The formulation was lamellar lyotropic liquid crystal in which genistein was incorporated at the concentration 3% and the formulation was applied local, with or without electroporation (EP), using the Mezoforte Duo Mez 120905‐D device on C57BL6J. Results have shown that tumors appeared later when electroporation was applied. During the 21 days of the experiment, genistein incorporated in the new modern formulation, applied topically classic decreased the tumor volume, the degree of erythema and amount of melanin for mice bearing B16 murine melanoma tumors. When the formulation was applied by electroporation, the prog‐ nosis was even better. However, the new approach had no effect in terms of serum concentrations of the protein S100B and serum neuron‐specific enolase (NSE), or the tissue expression of the platelet‐derived growth factor receptor β (PDGFRβ) antibody [142]. Also, soy total extract incor‐ porated in the new modern lamellar lyotropic liquid crystal formulation was tested *in vitro* on the B164A5 mouse melanoma cell line for its pro‐apoptotic potential, employing two consecrated assays: 4',6‐diamidino‐2‐phenylindole (DAPI) and annexin‐FITC‐7AAD double staining. 200 μg/ ml of soy extract, respectively 200 μg/ml of soy extract incorporated into the lamellar lyotropic liquid crystalline formulation were incubated for 72 h together with this murine melanoma cell line. Results have shown that soy extract has pro‐apoptotic properties and incorporation in the new formulation does not affect in a negative manner this effect thus being a suitable excipient for *in vivo* studies [143]. In a recent study, diffusion and penetration of genistein, respectively, genistein incorporated in lamellar lyotropic liquid crystalline formulation through different membranes (a synthetic membrane *in vitro*, chick chorioallantoic membrane (CAM) *ex ovo*, and excised human epidermis *ex vivo*) were also investigated by conventional treatment without EP, and also with the mediation of EP by the help of a Franz diffusion cell system. *In vivo* ATR‐FTIR and *ex vivo* Raman spectroscopy were applied in order to analyze the effect on mice skin [144]. Results have shown that the new formulation is a suitable carrier for the lipophilic genistein. The formulation with the active agent penetrated the skin, but when electroporation was applied, the transdermal drug transport was more rapid and effective. This observation was validated by both ATR‐FTIR and Raman spectroscopy [144].

The research of our group on the phytoestrogen genistein points toward the clear conclusion that this phytocompound is an active chemopreventive agent against malignant melanoma both *in vitro* and *in vivo*. A series of attempts were made in order to increase the bioavailability of this lipophilic compound. We cannot say that we have found the optimal formulation, but we have managed to improve results compared to pure substance. Further studies will be conducted on this matter.
