The pellet was fixed in cold ethanol at 4?C for 1?h. than water extract (IC50 values?=?0.22C0.85?mg/mL) and Jurkat cells were the most sensitive to both extracts (IC50 values?=?0.12C0.69?mg/mL). The underlying mechanism for antiproliferative activity was apoptosis induction, especially in HT29, HCT116, MCF7 and Jurkat cells. HT29 cells were the most sensitive to extract-induced apoptosis. Ethanolic extract was more effective at inducing apoptosis than water extract. Moreover, cell cycle arrest was found to be another mechanism behind growth inhibition in Jurkat and HCT116 cells. However, these extracts were relatively less toxic to non-cancer Vero cells. HPLC analysis demonstrated that the powder mix extracts contained seven identified phenolic acids namely gallic, fermented broth and fruit as an alternative medicine for cancer prevention and treatment. Thunb. (known in Thai as Plu-Kao) is a medicinal herb indigenous to many parts of East and Southeast Asia including Thailand [1, 2]. The plant has been recognized as possessing many biological and pharmacological properties including anticancer, antioxidative, antiviral and immunomodulatory activities . The fermented has been AS-1517499 demonstrated to exhibit cytotoxic effects on human leukemia cells and hepatocellular cancer cells to a considerable extent [4, 5]. Linn. or Gaertn. (known in Thai as Ma-kham-pom) is another medicinal plant used in Asian traditional medicine systems against different ailments including peptic ulcer, inflammation, dyspepsia and cancer, etc. [6, 7]. Its fruits are traditionally consumed for its immense nutritive values as well. Experimental evidence suggested that its fruit had several phytochemicals such as gallic acid, ellagic acid and pyrogallol that possess antineoplastic effects . It has also been reported to possess immunomodulatory, chemomodulatory, radiomodulatory, antioxidative, anti-inflammatory and antimutagenic activities, which directly or indirectly prevent carcinogenesis [6, 7]. A number of in vitro and in vivo experiments point out that fruit extract of has potent anticarcinogenic property [8C11]. A recent study has indicated that extract inhibits ovarian cancer cell proliferation both in vitro and in vivo through inhibiting angiogenesis and inducing autophagy . Furthermore, the bioactive compound gallic acid, present in fruit of causes cell death through induction of apoptosis . The bioactivities of extract are mainly mediated by polyphenols . Many phenolics have been documented to have potent antioxidant, anticancer, antibacterial, antiviral, and anti-inflammatory potentials . The fruit of being rich in natural antioxidants with free radical scavenging potential, might prevent reactive oxygen species from DNA damage and carcinogenesis [10, 11, 15]. The anti-inflammatory activity of extracts might deter inflammation-related cancer . It has also been mentioned that ethanolic extract of fruit showed antiproliferative and antioxidant activities, whereas methanol RNF57 extract showed chemopreventive AS-1517499 potential against hepatocarcinogenesis [16C18]. In recent times, the fermented broth has been widely used in Thailand as a dietary supplement, however, the consumption may be limited due to its unpleasant taste. Therefore, a powdered formula of fermented broth and fruit has been developed. This is very interesting from a health perspective and thus, anticancer effects and chemical composition of this powdered formula needs to be investigated to provide scientific information to the public. In the present study, we evaluated antiproliferative activity of the powdered formula extracts in a panel of cancer cell lines and identified several phenolic compounds from ethanolic and water extracts of the powdered formula with HPLC and LC-MS techniques. Methods Preparation of water and ethanolic extracts The powdered formula of fermented broth and fruit was obtained from the Prolac (Thailand) Co., Ltd., in Lamphun Province, Thailand. Distilled water (50?mL) or absolute ethanol (50?mL) was added to 5.0?g of the powder mix and stirred continuously for 48?h at room temperature. Subsequently, the mixture was centrifuged at 2800 x and filtered through Whatman filter AS-1517499 paper (No. 4). Finally, the water extract was lyophilized, whereas the ethanolic extract AS-1517499 was subjected to evaporation by using rotary evaporator and blowing a stream of N2 gas over the sample. The water (yield?=?10%; fermented broth and fruit for 24, 48 and 72?h. Control groups were treated with double distilled water or a mixture of DMSO and ethanol (1:1). After the indicated time, medium was replaced with 110?l of fresh medium containing MTT (0.5?mg/mL in PBS) (Sigma Chemical Co., St Louis, MO, USA) and incubated for 2?h. Formazan formed after conversion of MTT was dissolved in DMSO. The absorbance of formazan was measured with a microplate reader (Bio-Rad Laboratories, USA) at the wavelength of 550?nm AS-1517499 with a reference wave length of 655?nm. The percentage of viable cells which corresponds to the production of formazan.