Results of high performance liquid chromatography (HPLC) analysis of flavonoids and phenolic acids are present in Table 1. Leaf extracts of Malaysian ginger, especially the variety H. Bara contained considerably (p ≤ 0.05) high amounts of rutin (0.893 mg g⁻¹ DW) and apigenin (0.384 mg g⁻¹ DW). SA application reduced rutin production in H. Bara (6.8%) and H. Bentong (21.8%). According to the data obtained, the concentration of some flavonoids (e.g., rutin, apigenin) decreased significantly in plants treated with different concentrations of SA (Table 1). High concentrations of these flavonoids were found in the control plants. Conversely, concentration of naringenin, fisetin and morin increased significantly in both varieties when treated with different concentrations of SA. The interesting finding was that application of SA in both varieties induced synthesis of fisetin and anthocyanin which were not detected in the control plants. Highest levels of anthocyanin (0.442 mg g⁻¹ DW) and fisetin (0.359 mg g⁻¹ DW) were observed in leaf extracts of H. Bara treated with 10⁻⁵ M of SA. Obinat et al. [17] also reported that production of anthocyanin was induced in cultured grape cells following foliar application of SA. Myricetin has antioxidant properties and in vitro research suggests that various concentrations of myricetin can modify LDL cholesterol and enable increased uptake by white blood cells [18]. In the present study production of myricetin was enhanced in ginger varieties treated with SA compared to control plants. High levels of this potent antioxidant compound were observed in H. Bara (0.112 mg g⁻¹ DW) treated with 10⁻⁵ M SA. Morien is a rare yet well known flavonoid component of plants and acts as a chemo-preventive agent in vitro and in vivo against oral carcinogenesis [19,20]. The importance of morin and related compounds as anti-tumour drugs has also been widely recognized [21]. A high content of morin (0.193 mg g⁻¹ DW) was obtained in extracts of H. Bara treated with 10⁻⁵ M SA. According to HPLC analysis, it could be concluded that the application of SA induced synthesis of some flavonoids, while conversely inhibiting production of some other flavonoids in ginger. Our results suggest the ability of SA application to modify or alter both the profile and the concentration of flavonoids in ginger.

According to the results CHS activity was influenced by SA concentration (p ≤ 0.01; Figure 2). In both varieties treated with SA, CHS activity was found to be consistently higher in ginger treated with 10⁻⁵ M SA with values ranging between 5.77 and 6.30 nkat mg protein⁻¹ than in gingers treated with 10⁻³ M SA, which recorded CHS activity of 5.40 to 6.14 nkat mg protein⁻¹. Plants not treated with SA showed the lowest values for CHS activity which were registered between 4.37 and 4.80 nkat mg protein⁻¹. The present study showed that with SA application the activity of CHS was enhanced. This is basically due to the fact that CHS is a precursor to flavonoids biosynthesis [22,23]. The increase in CHS activity is usually followed by an increase in C/N ratio due to the enhanced growth rate using SA. Results of recent studies suggest that increases in the C/N ratio in plants are an indication of increases in the synthesis of secondary plant metabolites, especially flavonoids [24,25].

It is hypothesized that the increase in production of anthocyanin and flavonoids in the present work could be attributed to an increase in CHS activity in SA treated plants. Our results are consistent with Compos et al. [10] who reported SA induced CHS activity in beans. Ozeki et al.[26] pointed out that change in CHS activity rather than PAL activity (also involving enzymes for flavonoid synthesis) was correlated with changes in anthocyanin accumulation under various culture conditions. CHS is the first enzyme to switch from phenylpropanoid metabolism to flavonoid metabolism and is believed to be a key enzyme in this system [27]. These findings together with evidence for channeling between SA concentration and CHS activity in the general phenylpropanoid pathway [16], indicate that the organization of these systems are important in the understanding of how plant metabolism is regulated.

The two ginger varieties were found to express MCF-7 and MDA-MB-231 cancer inhibitory activity when tested at concentrations of 4.6875–300 μg mL⁻¹ (Table 3). Significant tumor inhibition was observed at 37.5 μg mL⁻¹. At a concentration of 37.5 μg mL⁻¹ most of the extracts exhibited strong anticancer activity towards MCF-7 and MDA-MB-231 cells. Maximum MCF-7 and MDA-MB-231 cell line inhibition was observed with H. Bara with values of 61.53% and 59.88%, respectively when treated with 10⁻⁵ M SA, while minimum MDA-MB-231 cell line inhibition was observed with the untreated H. Bentong (with values of 45.30% and 40.64%, respectively). MCF-7 and MDA-MB-231 cell lines treated with tamoxifen (positive control) showed 77.44% and 73.82% inhibition at the same concentration. Based on several in vivo and in vitro studies, many mechanisms of anticancer action may be involved. These include cell cycle arrest, carcinogen inactivation, antiproliferation, inhibition of angiogenesis, induction of apoptosis and differentiation, antioxidation and reversal of multi-drug resistance or a combination of these mechanisms [37–39]. Flavonoids are among the best candidates for mediating the protective effect of diets rich in fruits and vegetables with respect to colorectal cancer. Additional information on their effects on cancer cells and their mechanisms of action were obtained by adding, a series of related flavonoids to cultures of cancer cells; all flavonoid compounds were found to increase growth inhibition and cell loss at concentrations of 1 to 100 mM, with relative effectiveness being fisetin > kaempferol [40]. Hence, flavonoid compounds could probably be responsible for the anticancer activity of Z. officinale. According to the American National Cancer Institute, the criterion of normal cell viability for crude extracts of herbs is 76% [41]. This implies that extracts of herbs that show cell viability lower than this range are suitable for human consumption and are not harmful. Anthocyanin is a potent antioxidant [42–45]. Anthocyanin fractions extracted from different sources, including grape rinds and red rice [46], flower petals [47], Vaccinium species [48], red soyabeans and red beans [49], different cherry and berry extracts [50–52], and purple corn [53], have demonstrated anticancer activity. Katsube et al.[51] reported HCT-116 colon cancer cells were inhibited by anthocyanin-containing berry extracts including cowberry, blueberry, strawberry and bilberry extracts [51]. Similarly, tart cherry anthocyanins were shown to inhibit the growth of human colon cancer cell lines HCT116 and HT-29 [52]. Fisetin is found in several plants, fruits, vegetables, nuts and wine [54] and displays a variety of biological effects including anti-inflammatory, antioxidant [55,56], anti-carcinogenic and in vitro anti-angiogenesis [57]. In vivo, fisetin has recently been shown to possess interesting anticancer activity in mice bearing prostate tumours [58], lung carcinoma [59] and human embryonal carcinoma [60]. The in vivo mechanism of action appears rather complex and may include anti-angiogenic, anti-metastatic and anti-androgenic activities [61].

In the current study the highest values of both fisetin and athocyanin were detected in ginger varieties treated with 10⁻⁵ M SA. Meanwhile, the highest anticancer activity against MCF-7 and MDA-MB-231 cell lines has been observed with extracts of H. Bara treated with 10⁻⁵ M SA. This suggests that high anticancer activity in these varieties may be attributed to the high concentrations of potent anticancer components such as fisetin and anthocyanin. However, more research needs to be undertaken before the association between these flavonoids and anticancer activity in ginger varieties is more clearly understood. In varieties treated with SA the value of IC₅₀ decreased significantly compared to non treated plants. The IC₅₀ values of Halia Bara extract (not treated with SA) against MCF-7 and MDA-MB-231 cells were 39.1 and 42.1 μg mL⁻¹, respectively (Table 4). In H. Bara treated with 10⁻⁵ M SA, the IC₅₀ value decreased to 31.5 and 40.6 μg mL⁻¹ for MCF-7 and MDA-MB-231 respectively; while the IC₅₀ values of tamoxifen as a positive control for MCF-7 and MDA-MB-231 cells were 17.4 and 19.5 μg mL⁻¹, respectively.

The rhizomes of ginger varieties Halia Bentong and Halia Bara (Zingiber officinale) were collected from the ginger planting area in Bentong Village, Malaysia. Voucher specimens were identified by the herbarium of the University Putra Malaysia, Selangor, Malaysia (H. Bentong Stone 6030 (KLU) and H. Bara Stone 7233 (KLU)). Rhizomes were sprouted for two weeks in 10 cm diameter pots filled with peat. They were then transferred to polyethylene bags filled with a soilless mixture consisting of burnt rice husk and coco peat (1:1). The plants were grown in a glasshouse at the Universiti Putra Malaysia (UPM) glasshouse complex. The seedlings were raised in specially constructed growth chambers receiving 12-h photoperiod and average photosynthetic photon flux density of 310 μmol m⁻² s⁻¹. Day and night temperatures were maintained at 30 ± 1.0 °C and 20 ± 1.5 °C, respectively, and relative humidity of between 70% and 80%. At the second leaf stage, the ginger seedlings were sprayed with two concentrations (10⁻³ or 10⁻⁵ M) of salicylic acid solution (SA; 2-hydroxybenzoic acid +100 μL dimethyl sulfoxide +0.02% polyoxyethylenesorbitan monolaurate, Tween 20, Sigma Chemicals; pH 6.5). Control plants were sprayed with the same solution but without SA. Plants were sprayed early in the morning with 10 mL of the respective solutions once a week for four weeks.

Leaf samples (0.25 g) were extracted with 20 mL of methanol, and 5 mL of 6 M HCl was added to each extract to give a total volume of 25 mL. The extracts were then refluxed at 90 °C for 2 h. Aliquots of 500 μL were taken before and after hydrolysis, filtered through a 0.45 μm filter, and analyzed for flavonoids using High Performance Liquid Chromatography (HPLC) [62].

CHS activity was assayed spectrophotometrically as described in Obinata et al.[17]. Enzymes were extracted at 4 °C by homogenizing the frozen harvested cells (0.4 g) in 1 mL of 0.1 M borate buffer (pH 8.8) containing 1 mM 2-mercaptoethanol with a homogenizer (Polytron). The homogenates were treated with 0.1 g of Dowex l × 4 for 10 min and the cell debris and resin were removed by centrifugation at 15,000 rpm for 10 min. A 0.2 g sample of Dowex l × 4 resin was added to the supernatant and treated for another 20 min. The resin was then removed by centrifugation at 15,000 rpm for 15 min. The resultant supernatant was used in the CHS assay. The CHS assay was performed with 100 μL of enzyme extract mixed with 1.89 mL of 50 mM Tris-HCI buffer, pH 7.6, containing 10 mM KCN. The enzyme reaction was allowed to proceed for 1 min at 30 °C after adding 10 mg chalcone to 10 μL ethylene glycol monomethylether. The activity was determined by measuring the absorbance at 370 nm.

Data were analyzed using the Statistical Analysis System (SAS, system 9.0; SAS Institute, Inc.: Cary, NC, USA, 2002). Means separation was performed using the Duncan multiple range test. The experimental results were expressed as mean ± standard deviation of three replicates. Mean differences at p ≤ 0.05 were regarded as significant.