## 4 Biological Activity of Cannabis Metabolites
### Biological Activity of Cannabis Flavonoids
#### 4.3.1 Cannabis Flavonoids In Vitro Activity  
Cannflavin A was tested by [PERSON] et al. (1985) against synovial cells for prostaglandin E2 (PE2) inhibition activity. They used phorbol 12-myristate 13-acetate (PMA) (10 ng/mL) as a PE2-inducing compound and reported an IC\({}_{50}\) = 31 ng/mL, compared to aspirin (IC\({}_{50}\) = 840 ng/mL) [101]. Cannflavin A was reported to inhibit the TLR4-induced production of IL-1\(\beta\) (IC\({}_{50}\) = 12.9 uM) and CXCL10 (IC\({}_{50}\) = 43.4 uM), further corroborating its anti-inflammatory potential [102]. The anti-leishmanial activity of cannflavin A was reported, with an IC\({}_{50}\) = 4.5 ug/mL [103]. Cannflavin B and C were reported to be anti-leishamanial as well, with an IC\({}_{50}\) = 5.0 ug/mL, and cannflavin C with an IC\({}_{50}\) = 17.0 ug/mL [104]. [PERSON] et al. (2011) also tested cannflavin A extracted from _Miumulus bigelovii_ for antileishmanial activity, and for antitrypanosomal activity. They report an IC\({}_{50}\) = 14.6 ug/mL against _Leishmania donovani_, and an IC\({}_{50}\) = 1.9 ug/mL against _T Trypanosoma brucei brucei_[104]. Cannflavin A was tested against PC12 cells by [PERSON] et al. (2019) for the inhibition of amyloid \(\beta\) fibrillization and neurotoxicity. They reported a protective effect of PC12 cells at 10 uM of cannflavin A, but at higher concentrations, the compound exhibited neurotoxic behavior [105]. At 100 uM of cannflavin A, there was a significant reduction in amyloid \(\beta\) fibrils, suggesting a potential utilization in [PERSON]'s disease prevention. Cannflavin A showed antiproliferative activity against two bladder cancer cell lines (T24 and TCCSUP) at 100 uM after 48 h treatment [106]. Treatment with cannflavin A at 2.5 uM for 24 h, whilst not affecting bladder cancer cell viability, was able to induce caspase 3 cleavage, suggesting it could affect bladder cancer cell apoptosis. [PERSON] and colleagues also provided evidence that 2.5 uM of cannflavin A could reduce T24 cell invasion in a Matrigel invasion assay, from 25.3% in the control, to 15.1%. Kynureine 3-monooxygenase (KMO) is a drug target for neuroinflammation and neurodegenerative diseases. Cannflavin A, extracted from hemp aerial parts, was proven as a KMO inhibitor, with an IC\({}_{50}\) = 29.4 uM, compared to the positive control Ro 61-8048 (IC\({}_{50}\) = 5.1 uM), suggesting a possible therapeutical usage in neurodegenerative diseases [107]. Cannflavin A and B diminish lipid peroxidation at concentrations of 1.25 uM and increase cell viability of HaCaT cells treated with erastin, a ferroptosis-inducing molecule, at 5 uM for cannflavin A and 25 uM for cannflavin B [108]. This cytoprotective effect is thought to be linked to cannflavins' antioxidant capabilities. Cannfllavin A and B showed anti-nociceptive activity in _Caenorhabditis elegans_ in a heat-avoidance assay at concentrations of 1 uM (1 h treatment) [109]. Furthermore, [PERSON] et al. (2024) provided evidence that this anti-nociceptive effect dissipated after 6 h, suggesting a rapid onset and offset of cannflavins' effect.