Anti-inflammatory activity
The anti-inflammatory property of flavokawain B was assessed by a study conducted by Lin et al. in 2009 [25]. The study tested this compound against LPS-induced RAW 264.7 cells [25]. It was found that flavokawain B had an impressive effect in inhibiting nitric oxide production with an IC50 of 9.8 μM, lesser than the positive control used, curcumin [25]. Additionally, flavokawain B also inhibited PGE2 in a dose-dependent manner [25]. Similarly, these effects were also seen in the inhibition of TNF-α, a cytokine that is regularly involved in inflammation [25]. Furthermore, further analysis on the alteration of iNOS and COX-2 protein were also measured. Flavokawain B was found to inhibit the expression of both proteins depending on the concentration. To further clarify the activated inflammatory pathway upon the exposure of flavokawain B, NF- κ B – related proteins were tested [25]. Results showed that flavokawain B inhibited κ activation by degrading the inhibitory subunit IkBα and inhibiting the activation of Iκκ[25]. Furthermore, flavokawain B has been found to significantly inhibit COX-I at 100 μg/ml, as well as COX-II enzyme [26].
Antinociceptive activity
Two studies by Mohamad et al. [27, 28] proved that flavokawain B has the potential to be developed into an antinocicepetive drug. Through the acetic acid-induced abdominal writhing test, it was discovered that at 128.6 mg/kg, the positive control, acetylsalicylic acid exhibited the same effect as flavokawain B when the dose was 10 mg/kg [27]. This indicates that flavokawain B was 68 fold more effective than acetylsalicylic acid [27]. This indictment also suggests that the activity of flavokawain B may involve inhibition of cyclooxygenases or lipooxygenases [27]. Moreover, flavokawain B was more effective when it is being administered intraperitoneally rather than orally by seven-fold [27]. To further elucidate the antinociceptive activity of flavokawain B, Mohamad et al. conducted another study and suggested that flavokawain B operates via the activation of the NO-cGMP-PKC-ATP-sensitive K + channels pathway [28].
Anti-cancer properties
Researches in treating cancer, and better yet, preventing this disease has been significantly expanding. To date, the number of treatments varies and ranges from radiotherapy to herbal, alternative medicine. The most opted way of treating neoplasm is by administering drugs to the patient [29]. Flavokawain B has been found to be cytotoxic towards several important cancer cell lines. The most recent study was done by Ji et al. whom discovered the cytotoxic effects of flavokawain B on osteosarcoma cell lines [30]. This study revealed that flavokawain B exuded an apoptotic effect on these osteosarcoma cancer cell lines via the activation of caspase 3/7, caspase 8 and caspase 9 [30]. Furthermore, flavokawain B was also shown to down-regulate several anti-apoptotic proteins such as Bcl-2 and survivin [30]. Consequently, this compound also up-regulated various pro-apoptotic proteins including Bax, Fas and Puma [30]. Moreover, it was also observed, that flavokawain B induced a G2/M arrest by increasing Myt1 levels and concomitantly decreasing cdc2, cyclin B1 and cdc25c levels [30]. Interestingly, it has also been noted that flavokawain B decreased the migration and invasion ability of osteosarcoma cell lines [30]. This attribute is a favorable property especially in treating highly metastastic cancer cells.
Oral carcinoma is becoming more prevalent, especially in the South and Pacific Asia region [31]. Flavokawain B was shown to be cytotoxic towards the HSC-3, A-2058, Cal-27 and A-549 cell lines [19]. In normal gingival fibroblast cell line interestingly, flavokawain B showed very minimal cytotoxic effect [19]. Additionally, this compound was proven to cause a cell cycle arrest in the HSC-3 cell line. The number of cells in the G2/M phase increased in comparison with the control upon 12 hours of post-treatment [19]. The arrest in the G2/M phase may involve the inhibition of several important cyclin/cdk complexes. Hseu et al. in 2012, proposed that flavokawain B was involved in the inactivation of Cdc2, Cdc25c, Cyclin A and Cyclin B1 in HSC-3 cell line. Furthermore, it was also proven that this compound caused a shift in the mitochondrial membrane permeabilization potential, indicating that treated cells had a compromised mitochondria [19]. Furthermore, mitochondrial-related proteins were also regulated upon the induction of flavokawain B. Cytochrome c for instance, was found to be upregulated in the cytosol in a dose-dependent manner [19]. Moreover, flavokawain B also induced apoptosis in human oral adenoid cystic cancer, ACC-2 [32]. Flavokawain B was shown to induce G2/M arrest and induce the release of cytochrome c [32]. This subsequently lead to the activation of caspase 3 and the cleavage of the PARP enzyme [32].
Apart from HSC-3 cell line, flavokawain B can also be cytotoxic towards synovial sarcoma cell lines [33]. A study by Sakai et al. in 2011 [33], used two synovial sarcoma lines, SYO-1 and HS-SY-II to test for the cytotoxic activity of flavokawain B. Upon treatment, both the SYO-1 and HS-SY-II cell lines exhibited pro-apoptotic morphology such as cell membrane blebbing and cell shrinkage [33]. It is suggested that flavokawain B impeded cell growth in a concentration-dependent manner [33]. The increment of expression of caspase 8, 9 and 3/7 implied that flavokawain B activates the death-receptor and mitochondrial-mediated apoptotic mechanism [33]. Moreover in flavokawain B-induced SYO-I and HS-SY-II cells, the expressions of death receptor 5, Bim and Puma were up-regulated while the expression of survivin, an anti-apoptotic protein was down-regulated [33]. Another pro-apoptotic protein, Bak was also up-regulated when treated with flavokawain B at a concentration of 7.5 μg/ml [33].
Flavokawain B was also found to induce apoptosis in several prostate cancer cell lines while having insignificant effects on the normal prostate cell line [34]. This predicament is based on the morphology changes that occur in flavokawain B-treated cells which include, cell shrinkage, cell blebbing, nuclear fragmentation and condensation [34]. Additionally, Tang et al. [34] also discovered that flavokawain B induced apoptosis by the activation of several caspases in DU145 and PC-3 cell lines [34]. The cleavage of PARP, a pro-apoptotic protein was also seen in DU145 and PC-3 cell lines when induced with flavokawain B [34]. Besides, this compound increased the expression of several pro-apoptotic proteins, Bim, Bax and Puma, and conversely decreased the expression, of XIAP and survivin, both are anti-apoptotic proteins [34]. Interestingly, Li et al., 2012, conducted another study regarding the effects of flavokawain B on several other prostate cancer-related cell lines such as LAPC4, C4-2B, LNCaP, 22RV1 and WPMY-1 [35]. This study showed that flavokawain B downregulated AR protein expression in all cell lines [35]. Additionally, flavokawain B also downregulated the expression of the AR target protein, PSA [35]. At the transcription level, flavokawain B significantly decreased the mRNA expression of AR, PSA and TMPSS2 in LNCaP and C4-2B cell lines [35]. It wasl aslo deiscovered that, flavokawain B decreased the level of AR through the downregulation of the SP1 protein expression [35].
Additionally, flavokawain B was also found to inhibit HCT116, a colon cancer cell line [6]. Kuo et al. [6], reported that flavokawain B induced an anti-proliferation state of HCT116 cells when treated with 5–50 μM of the compound. This observation was supported when the level of cleaved-PARP protein was noticeable at 50 μM of treatment [6]. The mitochondrial-induced apoptosis pathway was proposed because of the loss of the mitochondrial potential, release of cytochrome c and translocation of Bak [6]. Kuo et al. also found that GADD153, a marker for endoplasmic reticulum stress, was significantly upregulated. This further confirms the mitochondria-mediated apoptosis [6]. Additionally, BCL-2, an anti-apoptotic protein, downstream of GADD153 was downregulated when induced with flavokawain B [6]. Furthermore, it was discovered that flavokawain B increased the level of ROS and p38, both contributing to the execution of apoptosis [6]. Likewise to Hseu et al. 2012, flavokawain B was also found to induce a G2/M arrest in HCT116 cells [6].
Another study was conducted by An et al. in 2012, addressed the anti-neoplastic activity of flavokawain B on non-small cell lung cancer, H460 cells. The findings were similar to the other studies conducted; flavokawain B induced a mitochondrial-dependent apoptosis pathway [36]. This study showed that flavokawain B induced the release of cytochrome c and deregulated the BcL-xL/Bax ratio [36]. Likewise to other studies, it was also proven that flavokawain B down-regulated the anti-apoptotic proteins, XIAP and survivin [36]. Additionally, it was found that flavokawain B activated the MAPK pathway and subsequently the JNK-mediated apoptotic pathway [36]. The effects of flavokawain B on uterine leiomyosarcoma cell lines were tested by Eskander et al. 2012. Similar to other studies, this study also concluded that flavokawain B induced a G2/M arrest in treated cells [37]. Furthermore, also in accordance with other findings, flavokawain B up-regulated several pro-apoptotic proteins such as death receptor 5, Puma and Bim, and also down-regulated IAP and survivin [37].
Another recent study on the anti-cancer mechanism of flavokawain B was conducted on squamous carcinoma cells, KB cells [38]. Lin et al. in 2012, designed the study to determine the apoptotic, anti-proliferative activity and anti-metastatic activity of flavokawain B on KB cells [38]. This study also prove that flavokawain B induced a G2/M arrest as evidenced by the reduction of cyclin A, cyclin B1, cdc2, cdc25c and increment of p53, p21 and wee1 [38]. The induction of apoptosis on the other hand, was substantiated by the activation of caspase 3,-8 and −9 as well as the cleavage of PARP [38]. Additionally, the levels of Bid and Bax were increased coupled with the decrement of Bcl2 [38]. As an anti-metastatic drug, flavokawain B remains promising as it inhibited the expression of matrix-metalloproteinase 9 and urokinase plasminogen activator [38].
Besides in vitro, analyses on in vivo model were also conducted by some studies [7, 24, 34]. A research by Tang et al. tested the compound flavokawain B on nude mice induced with DU145 cells. Expectantly, flavokawain B significantly reduces the growth of these tumors by approximately 67% [34]. The level of Bim was found to be increased in tumor lysates as compared to the untreated control [34]. Additionally, it was reported that there were no necropsy or growth irregularities in the treated mice [34]. The anti cancer in vivo effects of flavokawain B in KB xenograft models was also tested [38]. Flavokawain B significantly reduced the growth of the tumor as proven by the augmentation of apoptotic DNA fragmentation [38]. Li et al. 2012, also studied the interaction of flavokawain B on two patient-derived prostate cancer xenografts in mice. The study found that flavokawain B inhibited the growth of the tumor, reduced the expression of AR as well as the levels of serum PSA [35].
Other properties
Additionally, a research conducted by Feroz et al. studied the interaction between flavokawin B and human serum albumin [39]. The efficacy of the pharmacokinetics and pharmacodynamics of a certain drug once it is inside the body is greatly dependent on its interaction with plasma protein [39]. It is well known that human serum albumin is a major player in the transport of various ligands [39]. As expected, flavokawain B was found to interact with human serum albumin similarly to other flavonoids [39]. The binding essentially comprises of hydrophobic interactions including hydrogen bonding [39].