The close association between inflammation and many diseases, such as Parkinson’s disease, osteoarthritis, cardiovascular events, diabetic nephropathy, cancer, and influenza infection, is well known (40). Previous in vivo and in vitro studies have shown that UA acts against both endogenous and exogenous inflammatory stimuli, with favorable anti-inflammatory effects. UA (25 mg/kg body weight, oral) can prevent the degeneration of dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonian mice (41). UA has been shown to significantly suppress xylene-induced ear edema in vivo as well as protect against lipopolysaccharide (LPS)-induced acute kidney injury by blocking the Toll-like receptor/myeloid differentiation primary response 88 pathway in vitro (42). In addition, UA can alleviate osteoarthritis by inhibiting the nuclear factor kappa B/NOD-like receptor protein-3 (NF-κB/NLRP3) inflammasome pathway (43). Furthermore, UA, at concentrations of 160 and 320 μg/mL, could inhibit inflammatory responses via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and NF-κB signaling pathways, reducing the viability of breast cancer cells (44). Treatment with 50 mg/kg UA in combination with caprylic acid (60 mg/kg) was found to decrease the levels of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), to reverse pentylenetetrazole-induced seizure-like symptoms in a zebrafish model (45).

When used at a concentration of 20 μg/mL, UA, in an influenza A virus (IAV)-treated A549 cell model, resolved both cell injury and the inflammatory response (46). Zhou and Wink (47) also revealed that UA can play an anti-inflammatory role by inhibiting NF-κB nuclear translocation, thereby reducing the expression of inflammation-related factors, such as TNF-α, cyclooxygenase-2, and inducible nitric oxide synthase (iNOS). UA derivatives have also been found to be effective in treating inflammation. In a two-stage skin carcinogenesis mouse model, two weeks of treatment with UA (2 μmol) or its synthetic derivatives significantly inhibited the expression of pro-inflammatory genes, such as interleukin-1-alpha (IL-1α), IL-1β, IL-6, and IL-23. Moreover, a few UA derivatives, such as corosolic acid and 3-epi-corosolic acid, were found to have stronger anti-inflammatory activities than UA (48). Similarly, another UA derivative, β-D-glucopyranosyl ester, was also found to have anti-inflammatory activity (49). Overall, previous studies have established that UA and its derivatives regulate the development of inflammation, and hence may be useful for treating inflammation-related diseases.

Oxidative stress results from excessive production of reactive oxygen species (ROS) in cells and tissues, which further leads to various inflammation-associated diseases (50). The in vitro antioxidant activity of UA was evaluated by inhibiting 2,2-diphenyl-1-picryl-hydroxyl at an inhibitory concentration (IC₅₀) of 59.7 ± 1.0 μg/mL (51). Administration of UA (25 and 50 mg/kg of body weight/day, intragastrically) for 6 weeks was found to reduce CCl₄-induced nephrotoxicity, demonstrating the antioxidant activity of UA and its ability to inhibit the phosphorylation of transcription 3 (STAT3)-NF-κB pathway (52). In human lymphocytes and the hamster V79 lung fibroblast cell line, UA was demonstrated to be a natural antioxidant that prevents DNA damage caused by hydrogen peroxide, causing a 50% decrease in cell viability at 224.85 mM (53). Furthermore, UA has been suggested as a potential candidate for the treatment and prevention of oxidative stress-mediated diseases (54), including neurodegenerative diseases in mouse models (55), obesity/diabetes and cardiovascular diseases in mice, skin carcinogenesis in mouse epidermal cells (56), liver disease in mice with carbon tetrachloride (CCl₄)-induced liver fibrosis (57), and osteoporosis in MG-63 cells (58).

UA plays a vital role in the injury caused by cerebral ischemia in mice by activating the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway (59). Nrf2, encoded by the gene NFE2L2, is a regulatory factor of phase II antioxidant enzymes, protecting the body from oxidative stress and inflammation. Nrf2^(−/−) mice showed obvious symptoms of neurodegeneration and oxidative stress, which were significantly reduced by intraperitoneal injection of UA (100 mg/kg), demonstrating the neuroprotective effects of UA (55). UA (50 mg/kg/day, 6 weeks, oral gavage) was also shown to attenuate CCl₄-induced hepatic oxidative damage and inflammation by increasing the expression levels of NAD(P)H:quinone-oxidoreductase-1, glutathione S-transferases, heme oxygenase-1, B-cell lymphoma-2 (Bcl-2), and nuclear Nrf2 (57). In a Caenorhabditis elegans model used for the evaluation of neurological drugs, UA (100 μM) showed antioxidant activity by upregulating the expression of peroxiredoxin-2 and skn-1 transcription factor (which corresponds to human Nrf2); this also led to an anti-depressant effect (60). Dietary administration of 0.1% UA (for 8 weeks) attenuated the tumor growth of transplanted VCaP (human prostatic cancer cell) xenografts in immunodeficient mice, together with epigenetic CpG methylation reprogramming, suggesting potential applications for the treatment/prevention of human prostate cancer (61). Treatment of JB6 P+ mouse epidermal cells with 2.5 μM UA increased Nrf2 expression by altering the methylation status of the Nrf2 promoter, thus inhibiting the development of skin cancer (56). In cisplatin-resistant HepG2/DDP cells, UA, as a natural adjuvant, increased the sensitivity of hepatocellular carcinoma cells to cisplatin through the Nrf2/antioxidant reaction element (ARE) signaling pathway, thus exhibiting anticancer effects (62). In conclusion, the results of various studies indicate that Nrf2 may be the target of the biological activity of UA.

In terms of its anticancer activity, UA is mainly associated with apoptosis and death of cancer cells. Mitochondria are essential for cell respiration and oxidative phosphorylation, and mitochondrial damage leads to apoptosis (63). UA exerts its anticancer effect by activating mitochondrial-dependent signaling pathways (Figure 2).

For example, UA upregulates and activates the expression of the cell tumor antigen p53 and the AMP-activated protein kinase (AMPK) signaling pathway, respectively, and downregulates the expression of the apoptosis regulator Bcl-2 (64). In a previous study, UA was shown to induce apoptosis of human breast adenocarcinoma (MCF7), osteosarcoma (U2OS), cervical adenocarcinoma (HeLa), and colorectal cancer (HCT116) cells after 24 h of treatment, with lethal dose 50 (LD₅₀) values of 12.8 μM, 7.7 μM, 16.8 μM, and 19 μM, respectively (69). Furthermore, UA (20 μM) could inhibit the expression of AKT mRNA, thereby activating the AMPK signaling pathway to promote autophagy and apoptosis (65). Furthermore, in the presence of 30 μM UA for 48 h, the proliferative activity of human lung cancer cells (A549 and H460) decreased significantly, whereas the expression of caspases 3 and 9 increased, further increasing the levels of the apoptosis-related protein Bax and decreasing those of the anti-apoptotic protein Bcl-2 (66). These results suggest that UA can induce apoptosis through caspase-related signaling pathways. Through the action of UA, AMPK is activated and the mammalian target of the rapamycin (mTOR) signaling pathway is suppressed, thus controlling protein synthesis and cell growth (66). In addition, structurally modified UA may also activate non-apoptotic cell death pathways associated with autophagosome and lysosome accumulation (67). UA also showed therapeutic effects on drug-resistant cancer cell lines. In vitro experiments confirmed that 48 h of UA treatment (16 μM) could reduce the adhesion and infiltration of the human breast cancer adriamycin-resistant cell line MCF-7/ADR to human umbilical vein endothelial cells (HUVECs) and could also reduce migration in MCF-7/ADR cells (68). UA induced cellular DNA damage and initiated G0/G1 phase arrest in embryonic cancer cells, and could be a candidate for inhibiting the recurrence of cancer (70).

When C57BL/6 mice were fed a high-fat diet for 15 weeks, accompanied by oral administration of UA, 80 mg/kg of UA could significantly reduce the total cholesterol (TC) and triglyceride (TG) levels in the liver and plasma, effectively relieve liver steatosis and reduce the number of epididymal fat cells. Meanwhile, in vitro experiments also confirmed that UA (20 μM) significantly reduced TC (37.2%) and TG (50.4%) contents in HepG2 cells and upregulated P-AMPK protein expression (71). Therefore, UA may reduce the lipid content of cells and inhibit lipid synthesis by activating the AMPK signaling pathway. UA improves the richness of beneficial intestinal flora through inhibition of the NOX4/NLRP3 inflammasome pathway, thus alleviating liver fibrosis caused by CCl₄ (72). UA can also reduce the activation of mitogen-activated protein kinases (JNK, p38 MAPK, ERK) and inactivate the immunoregulatory transcription factor NF-κB in the liver after treatment with CCl₄, thereby relieving CCl₄-induced inflammation (73).

In vitro experiments have also shown that UA can reverse the progression of liver fibrosis by inhibiting the activation of the NADPH oxidase (NOX)/ROS signaling pathway in hepatic stellate cells (74). UA protects the intestinal barrier by inhibiting the inflammatory factor TNF-α and increasing the expression of tight junction proteins and antimicrobial peptides. A 16S rRNA gene sequencing study also confirmed that UA treatment increased the abundance of beneficial bacteria, such as Firmicutes, Lactobacillus, and Bifidobacterium, thus alleviating the liver fibrosis process (75). Speculatively, the protective effect of UA on the liver may be related to reducing inflammation and oxidative stress levels or regulating intestinal flora. The possible mechanism underlying the role of UA in hepatoprotection against liver fibrosis, inhibiting the proliferation of cancer cells, and reversing nonalcoholic fatty liver disease, thus promoting liver regeneration by different pathways, is shown in Figure 3.

Antibiotic resistance and the emergence of superbugs have become serious public health problems in both human and veterinary medicine. To treat infections caused by multi-drug resistant pathogens, many different strategies have been used, including phages (76), antimicrobial peptides (77), metal nanoparticles (78), and plant-derived compounds--polyphenols (79), flavonoids (80), terpenoids (81), plant essential oils (82), and alkaloids (83). Traditional Chinese Medicine and Ayurveda have also been used in health care and disease prevention in ancient China and Egypt (84). UA, as a representative triterpenoid, showed significant antibacterial activity against the production of biofilm by Escherichia coli CFT073 (ATCC 700928), Enterobacter cloacae ATCC-BAA 2468, and Pseudomonas aeruginosa ATCC 25000 (81).

Although the antibacterial effects of plant extracts may not be as obvious as those of antibiotics, plant extracts can have synergistic effects when combined with antibiotics. UA showed a strong synergistic effect when combined with β-lactam antibiotics (ampicillin and benzacillin), increasing the sensitivity of Staphylococcus aureus and Staphylococcus epidermidis to antibiotics (85). UA (32 μg/mL) also displayed synergy with colistin against clinical isolates of Klebsiella pneumoniae BC936 and E. coli U3790 (86). Table 3 shows the MIC values of UA against different Gram-positive and Gram-negative pathogens. The MIC value of UA or its oleanolic acid isomer was 31.25 μg/mL against both S. aureus ATCC 6538 and methicillin-resistant S. aureus, whereas the MIC against Listeria monocytogenes ATCC 19115 was 250 μg/mL (91). UA is also known to show antifungal activity against Cryptococcus neoformans H99 with a MIC value of 250 μg/mL (92).

UA exposure led to a decrease in intracellular pH and ATP, downregulated the expression of four biofilm-related genes (pgaA, luxS, wbbM, and wzm), and inhibited biofilm formation of carbapenem-resistant Klebsiella pneumoniae (90). The antimicrobial activity of UA is stronger than that of tetracycline, with reductions of 50.5 and 12.7%, respectively, in staphylococcal membrane integrity (87). The MIC value of UA against E. coli (ATCC 25922), K. pneumoniae (ATCC10031), and Shigella flexella (ATCC12022) was 64 μg/mL. However, the MIC of UA against these bacteria was significantly reduced by modification of C-3 (hydroxyl) in UA to form UA ester analogs. Hence, the modified UA derivatives showed markedly increased antibacterial effects (51). Gram-negative bacteria contain an outer membrane, which is rich in LPS and phospholipids and prevents entry of UA into the bacterial cells, and hence the MICs of UA against Gram-negative bacteria are higher than that against Gram-positive bacteria (81).

UA kills bacteria by altering the structure of the bacterial cell, particularly by interfering with the cell membrane and adhesion proteins (93). UA also affects cell morphology and controls the expression of genes related to virulence factors, such as pili and fritillary (94). UA inhibits bacterial growth by reducing the ability of bacteria to adhere to host cells, as well as disrupting biofilm formation (95). In the presence of 10 mg/L UA from Diospyros dendo leaves/flowers, decreases of 72, 87, and 57% were observed in the biofilm biomass of Escherichia coli (ATCC 25404), Pseudomonas aeruginosa, and Vibrio havii, respectively, after culture for 24 h. Furthermore, 10 and 30 mg/L of UA induced the expression of genes related to chemotaxis (cheA, motAB, tap, and tsr). Overexpression of the motAB genes has been shown to cause increased bacterial activity, decreasing their stability in the biofilm environment and resulting in reduced biofilm formation (96). Experiments, such as measurement of bacterial glucosyltransferase activity, computer simulation, site-directed mutagenesis, and capillary electrophoresis, have revealed that UA competes for occupancy of the active site of glycosyltransferase, a key enzyme required for the synthesis of extracellular polymeric substances (EPS), secreted by S. mutans, thus inhibiting EPS formation and reducing the viability of S. mutans and the structural integrity of its biofilm (97). Therefore, in vitro results support the use of UA for the treatment of bacterial diseases. However, UA is rarely used to prevent or treat bacterial diseases in animal husbandry.