In vitro cytotoxic activity
According to the United States National Cancer Institute plant screening program, a plant extract is generally considered to have an active cytotoxicity effect if the IC50 value following incubation between 48 to 72 h, is 20 μg/mL or less .
The relevant tissue(s) (in terms of the tissue(s) used in folk medicine) from 52 species of medicinal plants in Thailand were screened as their crude hexane, DCM, ethanol and water extracts for in vitro cytotoxic activity against four human transformed (cancer) cell lines. The highest cytotoxic activity was obtained from the crude hexane extract obtained from the vines (but not leaves) of B. strychnifolia against the MDA-MB-231 cell line. Moreover, this extract also had potential strong activity against the KB3-1 cell line, whilst the crude DCM extract exhibited cytotoxic activity against the A549 and KB3-1 cell lines. From the results, the derived IC50 values for the crude hexane and DCM extracts from the vines of B. strychnifolia are not that different on each cell line from the respective positive controls (etoposide, doxorubicin, vinblastine and oxaliplatin). Assuming no strong synergy among all components in the extract, it can be implied that the bioactive component(s) in these fractions could be far more potent than the standard reference drugs and so merits their enrichment and further characterization.
Clinically, B. strychnifolia has been applied for the treatment of human food poisoning diarrhea  and also in 2011 as an anti-HIV-1 agent . Nevertheless, the antitumor activity has never been reported. As a consequence, this work appears to be the first report on the antitumor activity of B. strychnifolia.
The crude DCM extract of C. fenestratum showed in vitro cytotoxicity against the KB3-1 cell line, which is consistent with the reported antiproliferative activity against the human colorectal carcinoma (HCT-116) cell line , where it apparently induces expression of the peroxisome proliferator-activated receptor γ and pro-apoptotic genes.
The crude DCM and ethanol extracts of E. longifolia roots exhibited cytotoxic activity against the MDA-MB-231 cancer cell line, which has been reported previously on the human breast cancer cell line MCF-7 where the inhibition was linked to the induction of apoptotic cell death [27, 28]. In addition, from nearly 65 compounds isolated from the roots of E. longifolia, eight were found to demonstrate strong cytotoxicity towards the human lung cancer (A549) cell line and some of these were also strongly cytotoxic against the MCF-7 cell line . Thus, it appears that the crude alcohol extracts from E. longifolia roots may exhibit a preferential or specific cytotoxicity against breast cancer.
The bufadienolide isolated from the methanol extract of K. pinnata has been reported to be a potential cancer chemotherapeutic agent since it inhibits the tumor promoting activity of Epstein-Barr Virus . Here, we found that the crude ethanol extract from the leaves of this plant inhibited the growth of the KB3-1 cell line, which was developed from human papilloma virus infected cells. Hence, it is possible that the ethanol extracts affect the regulation of some viral proteins that control cell division.
Overall, the crude hexane and DCM extracts were more active than the corresponding aqueous and ethanol ones, suggesting that the active compound(s) against the cell lines in these plants are of low polarity. In addition, as mentioned before, since these are crude extracts and may contain many non-active components then the IC50 values reported here may in fact be far higher than those of the actual bioactive component(s) in the extracts, assuming no strong synergy between different components. Therefore, these results support that the bioactivity-guided enrichment of these fractions is merited.
Antioxidant and lipase inhibitory activity
With respect to the antioxidant activity, the crude ethanol and water extracts from only six of the 52 investigated plants showed any effective free radical scavenging activity in the DPPH assay when compared to that of the ascorbic acid reference standard. The hexane and DCM extracts did not exert any detectable antioxidant activity in this study. This result is similar to the study on Fenugreek seeds (Trigonella foenum-graecum) which showed that the highest antioxidant activity was found in the ethanol and methanol extracts followed by the aqueous extract with only low activities in the hexane and DCM extracts . Moreover, most of the 52 plants investigated in this study naturally occur (and were obtained from) within mangrove forest areas, which are typically rich sources of phenolic compounds, such as flavonoids , to protect the plants from UV radiation [33, 34]. As a matter of fact, a linear relationship between the flavonoid content and the antioxidant activity has already been reported .
With respect to the lipase inhibitory activity, only the crude ethanol and water extracts were evaluated. The low polarity compounds including the natural lipids in the tissue(s) could be extracted by hexane and DCM at sufficiently high concentrations as to interfere in the assay by acting as alternative but unquantified substrates for the lipase providing false positives and potentially masking genuine weak and moderate positives. However, all but one of the 110 water and ethanol extracts were found to be inactive. The exception was the ethanol extract from C. fenestratum stems that exhibited a weak lipase inhibitory activity (IC50 value of 160 μg/mL) that had a 17.3-fold lower IC50 value than that for Orlistat, a known lipase inhibitor. Nevertheless, it is a crude extract, and so the actual IC50 of the active component(s) may be significantly higher. The lipase inhibitory activity from this plant (the first report) gives a suggestion of the potential to screen for novel plant compounds with anti-lipase activity. These may be of clinical dietary use in countering the problems of human obesity. Thus, it may be interesting for further studies to fractionate the crude hexane and DCM extracts to remove the natural lipid content and screen the other fractions for anti-lipase activity.
Unfortunately, the crude hexane and DCM extracts, although soluble in DMSO were not soluble in the 5% (v/v) DMSO-nutrient broth media used for the broth micro-dilution assay, where higher DMSO levels are themselves inhibitory in the assay. Therefore, only the 110 crude ethanol and water extracts were screened for antimicrobial activity. Significant antimicrobial activities (inhibition zones ≥ 10 mm) were obtained from the extracts of only four plant species, with MIC values of ≤ 500 μg/mL (as determined by subsequent broth assays).
Of the four plants with antimicrobial activity, the crude ethanol extract of C. fenestratum stems displayed a broad range of antimicrobial effects against all tested microorganisms except P. aeruginosa. Moreover, it has been previously reported to inhibit the growth of Propionibacterium acnes and Staphylococcus epidermidis, whereas the methanol extract of this plant was reported to inhibit the growth of S. aureus, B. subtilis and E. coli.
The aqueous extract from the leaves of S. alba displayed an antimicrobial activity against both tested Gram-negative bacteria (E. coli and P. aeruginosa), one (M. luteus) of the three tested Gram-positive bacteria (M. luteus), and the yeast C. albicans. In agreement with this is that both the methanol and ethyl acetate extracts have been reported to have antimicrobial activity against Gram-positive (Bacillus cereus and S. aureus) and Gram-negative (E. coli) bacteria , but the inhibition activity against P. aeruginosa and C. albicans is newly reported here.
Although the results of this work are consistent with those reported by Tao et al. in that the ethanol extract of S. caseolaris had no antimicrobial activity against B. subtilis, S. aureus, P. aeruginosa and E. coli, our ethanol extract did inhibit the growth of E. coli. In addition, we found that the crude water extract of S. caseolaris leaves could inhibit the growth of C. albicans, which is consistent with the report that not only the ethanol leaf extract but also the methanol extract of the cork of this plant could inhibit C. albicans. Thus, these two species from the family Sonneratiaceae merit further investigation for antimicrobial agents.
Finally, the ethanol extract from the leaves of A. occidentale demonstrated antimicrobial activity against E. coli and P. aeruginosa, which is similar to that previously reported [41–43], but is in contrast with the previous report that the ethanol extract of this plant’s leaves also inhibited M. luteus, S. aureus and B. subtilis[41, 42], which was not observed in the present study.
Overall, the ethanol and aqueous extracts exhibited potential antimicrobial activity and merit further bioactivity guided fractionation to obtain the bioactive component(s). However, since the crude hexane and DCM extracts could not be screened, their initial fractionation and assay for antimicrobial activity may be of value, with further bioactivity guided fractionation as required.