3.1.2. Activity: Preclinical Studies

Baicalein and baicalin, and extracts containing them, are proposed for various pharmacological effects, such as anti-inflammatory, antiviral, antibacterial, anticancer, antineurodegenerative and protective, against cardiovascular diseases [65,68,69]. The molecular mechanisms of action of baicalein, as well as those of other flavonoids, are mainly linked to the antioxidant activity, which occurs in different steps of the oxidative process; for example, it has demonstrated a role as scavengers of free radicals already formed in the medium (including lipid peroxyl radicals), as chelators of metal ions and by removing oxidatively altered biomolecules [70,71]. The role of the OH groups in polyphenols is dependent on the global geometry of the molecule and interactions among neighbor groups; a detailed conformational analysis and linked antioxidant mechanisms have been recognized for baicalein [70]. Recent studies on the structure–activity relationship suggest that the baicalein moiety is relevant for bioactivities [72]; however, the molecular mechanisms of baicalein are multiple and follow many molecular pathways that require further evaluation. The data confirm the beneficial role of *Scutellaria baicalensis,* which is widely used in traditional Chinese medicine to treat hypertension, respiratory infections, inflammation and diarrhea [73,74]. A study using three structurally related polyphenols, such as baicalin, baicalein and wogonin, showed the inhibition of endothelial cell barrier disruption, suggesting their protective activity against vascular inflammatory diseases [75]. Recently, studies have suggested that baicalein exhibits potential antidiabetic activities in metabolic syndrome [76]; the effect is related to the inhibition of α–glucosidase activity [77,78].

The in vitro and in vivo data suggest that baicalein is able to reduce vascular inflammation induced by high glucose levels. In human umbilical vein endothelial cells (HUVECs), baicalein (5–10 μM) was able to protect the cells from membrane disruption caused by a 25 mM glucose concentration; the polyphenol (10 μM) reduced the expression of the chemokines MPC–1 and IL–8, as well as ROS formation [75]. Furthermore, the alteration of vascular permeability induced by high glucose administration in mice was counteracted by baicalein (4.5–8.9 μg/mouse, i.v.) [75]. The vascular protective effect of baicalein was also demonstrated in vitro and in vivo as mediated by the inhibition of the high-mobility group box 1 (HMGB1) signaling pathway [79]. The role of baicalein on vascular function was assessed further by an in vitro study on the hybridoma endothelial cell line EA.hy926 [80]. The results indicate that baicalein is able to exploit protective effects against the oxidative stress of the endothelium, which is linked to the risk of diabetic angiopathy.

Baicalein has hypotensive effects on hypertensive rats, improving blood pressure and endothelium function [81]. Zhang et al., using streptozotocin and a high-fat-diet-induced diabetic rats, demonstrated that a 4–week treatment with baicalein (150 mg/kg/day) can reduce the level of blood glucose and improve insulin resistance, dyslipidemia and inflammation [82]. The effects have been attributed to the modulation of the gut microbiota, leading to increased levels of short-chain fatty acids (including acetate, propionate and butyrate), which are capable of improving gut barrier activity by stimulating epithelial growth and innate reactivity to invading bacteria [82].

A computational study revealed that baicalein is among the most promising candidates for the development of useful flavonoid derivatives in the treatment of DM [83]. Bioactivity is believed to derive from the structure that allows free-radical scavenging properties, reduces oxidized compounds, chelates metals and inhibits enzymes [83]. The experimental data suggest that baicalein can reduce oxidative stress, the expressions of iNOS and TGF–β1, as well as counteracting NF–κB activation [84]. Through a molecular modeling approach, together with microscopic and spectroscopic analyses, baicalein has also been proven to contrast the formation of AGEs and amyloid fibrils, phenomena that contribute to the loss of protein function and are connected to tissue damage [24].

In LPS-stimulated HUVECs, baicalein inhibits the expression of inflammatory cytokines IL–lβ, IL–6 and TNF–α, as well as monocyte chemoattractant protein 1 (MCP–1) [85]. The authors also demonstrated that the inhibitory activity of baicalein occurs through the TLR4/NF– κB signaling pathway. The toll-like receptor 4 (TLR4)/NF–κB cascade pathway has been considered as a pivotal mechanism that leads to endothelial inflammation, activated by inflammatory signals, such as bacterial toxins; the fact that this pathway was modulated by baicalein provides an explanation for its potential as an anti-inflammatory agent.

Huan et al. indicated that baicalein exerts an anti-angiogenic effect on the inflammation microenvironment by inhibiting the transcriptional activity of activator protein-1 (AP–1) [86]. Tsai et al. observed that baicalein inhibits the expression of the lectin-like oxidized-LDL receptor 1 (LOX–1) protein in HUVECs, therefore protecting against oxidized-LDL atherogenic effects [87]. In this experimental model, baicalein also reduced ROS formation determined by the exposure to oxidized-LDL, as well as the consequent inflammation, by modulating AMPK/PKC/NADPH oxidase/NF–κB signaling [87].

However, Machha et al. demonstrated that acute exposure to baicalein alters the vascular tone in isolated rat aorta, due to the inhibition of endothelium-derived nitric oxide [88]. A dual effect of baicalein has been suggested, evaluating the endothelium-dependent contraction versus a direct relaxation produced by the substance in a rat mesenteric artery [89]. Conversely, the chronic oral administration of baicalein improves endothelium-dependent relaxation in spontaneously hypertensive rat aorta [90]. Considering vascular protection, baicalein (10 mg/kg/day for two weeks, orally) was shown to attenuate intimal hyperplasia in an in vivo model of vascular injury induced in the carotid artery of a rat [91]. As demonstrated in rat vascular smooth-muscle cells, the effect was due to the inhibition of proliferation via the MAPK, NF–κB, PI 3–kinase pathways, and the interaction with cell cycle machinery [91]. Using RAW264.7, HUVEC and MOVAS cells, Zhang et al. observed the anti-inflammatory activity of baicalein, attributed to the activation of the AMPK/Mfn–2 axis, together with the inhibition of downstream MAPKs/NF–κB signaling transduction [84].

Vascular calcification, often observed in patients with hypertension, atherosclerosis and DM, was also evaluated as a target of the action of baicalein [92]. Experiments on primary rat vascular smooth-muscle cells (VSMCs) indicated that baicalein decreased the mineralization rate, as well as calcium deposition and alkaline phosphatase activity [92]. Furthermore, Runt-related transcription factor 2 (Runx2, a transcription factor associated with osteoblast differentiation, also a regulator of the calcification of vascular smoothmuscle cells) and bone morphogenetic protein 2 (BMP–2, an osteogenic protein implicated in vascular calcification) expressions were negatively regulated in calcified VSMCs treated with baicalein. In vivo experiments, performed on a rat model of vascular calcification, demonstrated that baicalein was capable of inhibiting vascular calcification through multiple mechanisms, including the prevention of apoptosis, suppression of Runx2–BMP–2 signaling pathways and the preservation of the vascular contractility phenotype through the increased production of α–SM22 and α–SMA (vascular smooth-muscle markers) [92].
