Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth

2.1Hibiscus extract preparation

Dried H. rosa-sinensis flowers were incubated with 10 volumes (w/v) of boiling water for 20 min and cooled to room temperature. This 10% solution was then clarified by centrifugation, sterilized by filtration through 0.2 micron filters (Millipore), frozen, and lyophilized to dryness. This dried extract was suspended in water to a final concentration of 50 mg/ml.

2.2Extract fractionation

To examine molecular weights of bioactive compounds, extract was fractionated as previously described.13 Briefly, components greater than 50 kD were concentrated over centrifugal membranes with a 50 kD nominal molecular weight pore size (EMD Millipore Amicon UFC5050). Filtrates were then concentrated over centrifugal membranes with a 3 kD nominal molecular weight pore size (EMD Millipore Amicon UFC5003) to concentrate components between 3 and 50 kD, and obtain material below 3 kD as filtrates. Size fractionation was verified by SDS-PAGE on 18% gels stained with SilverQuest dye (Invitrogen LC6070). Concentrated material, filtrate, and unfractionated extract were diluted in cell culture medium to achieve final concentrations equivalent to 4 mg/ml of original unfractionated extract.

2.3Cell culture

B16, NIH3T3, and LA25 cells were maintained in DMEM (Hyclone SH30021) supplemented with 25 mM HEPES (Hyclone SH3027) and 10% FBS (Seradigm 1400-500) at 37 °C in 5% CO2 and 100% humidity as described.14, 15, 16, 17, 18 B16 and NIH3T3 cells were plated at 5000 cells/well in standard 24 well culture plates and allowed to adhere to plates for 24 h. Different concentrations of aqueous plant extract were then added and cells were incubated for an additional 72 h. LA25 cells were grown overnight at non permissive temperature (40 °C) before being incubated for 24 h at permissive (33 °C) and nonpermissive (40 °C) temperatures with or without hibiscus extract, and then stained with Trypan blue to distinguish living and dead cells. Cells were analyzed on an inverted Zeiss Axiovert microscope and counted from images with the aid of Zeiss Axiovision software as previously described.14, 15 Cells treated with fractionated extracts were trypsinized and counted with a Coulter counter as previously described.13, 15 Statistics were analyzed with Graphpad Prism Software version 5 as previously described.14, 15

2.4Western blotting

Protein extracted from LA25 cells grown at permissive (33 °C) and nonpermissive (40 °C) temperature was analyzed by Western blotting to detect total v-Src (Millipore 05-185), active Src (phosphorylated at tyrosine 416) (Millipore 04-857), and ß-actin (Sigma A1978) as previously described.16, 17, 18

3.1H. rosa-sinensis flower extract inhibits melanoma cell growth

An aqueous extract of H. rosa-sinensis flowers was prepared according to basic recipes used to make tea. Effects of this extract on cell growth were examined after sterilization by filtration. As shown in Fig. 1, this extract inhibited melanoma cell growth in a dose dependent manner. This extract reduced melanoma cell growth by 2 fold at 1 mg/ml, and 4 fold at 2 mg/ml.

Interestingly, this extract preferentially inhibited growth of melanoma cells over nontransformed fibroblasts. Although there did seem to be a trend for fibroblast growth inhibition, this effect was not significant, even at concentrations up to 8 mg/ml (see Fig. 1). These data are consistent with recent studies indicating that H. sabdariffa leaf extracts contain polyphenols that specifically inhibit the growth of melanoma cells, but not nontransformed cells.10

3.2Bioactive components of H. rosa-sinensis flower extract can be fractionated by size

The aqueous H. rosa-sinensis flower extract was fractionated to examine the nature of its bioactive components. Centrifugal filtration was used to obtain products greater than 50 kD, between 3 kD and 50 kD, and less than 3 kD. The effect of these fractions on melanoma cell growth was then examined at an original concentration of 4 mg/ml, which potently inhibited melanoma cell growth (Fig. 1).

3.3Bioactive components of H. rosa-sinensis flower extract work together to inhibit melanoma cell growth

The combined fractions of this extract inhibited melanoma cell growth by over 60% as shown in Fig. 2. In addition, low molecular weight components below 3 kD also inhibited cell growth, but to a significantly smaller extent of about 30%. Moreover, compounds larger than 3 kD did not inhibit melanoma cell growth. Taken together, these data suggest that while low molecular weight compounds in H. rosa-sinensis flower extract can inhibit melanoma cell growth to some extent, they combine with other higher molecular weight compounds to double their effectiveness.

3.4Hibiscus extract inhibits Src transformed cell growth, but not oncogenic Src kinase activity

The Src kinase has been implicated in melanoma progression,19, 20, 21 and is being pursued as a potential chemotherapeutic target.22, 23 We utilized LA25 cells transformed by a temperature sensitive oncogenic Src kinase construct to investigate the effects of hibiscus extract on Src transformed cells and kinase activity. As shown in Fig. 3a, hibiscus extract was preferentially toxic to these cells transformed by Src kinase when grown at permissive temperature. However, as shown in Fig. 3b, hibiscus treatment did not inhibit Src kinase activity in these cells. These data suggest that hibiscus extract contains compounds that affect elements downstream of the Src kinase, or unrelated pathways that control tumor cell viability.