Structures of some bioactive phytochemicals in bran extract of the black rice cv. Riceberry that had demonstrated anti-cancer activity in leukemic cell line were investigated. After saponification with potassium hydroxide, separation of the unsaponified fraction by reversed-phase high performance liquid chromatography (HPLC) resulted in four sub-fractions that had a certain degree of anti-proliferation against a mouse leukemic cell line (WEHI-3 cell), this being IC₅₀ at 24 h ranging between 2.80–467.11 μg/mL. Further purification of the bioactive substances contained in these four sub-fractions was performed by normal-phase HPLC. Structural characterization by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) resulted in, overall, the structures of seven phytosterols and four triterpenoids. Four phytosterols, 24-methylene-ergosta-5-en-3β-ol, 24-methylene-ergosta-7-en-3β-ol, fucosterol, and gramisterol, along with three triterpenoids, cycloeucalenol, lupenone, and lupeol, were found in the two sub-fractions that showed strong anti-leukemic cell proliferation (IC₅₀ = 2.80 and 32.89 μg/mL). The other sterols and triterpenoids were campesterol, stigmasterol, β-sitosterol and 24-methylenecycloartanol. Together with the data from in vitro biological analysis, we suggest that gramisterol is a significant anti-cancer lead compound in Riceberry bran extract.

The chemical compositions of essential oils obtained from Ocimum basilicum var. thyrsiflora (1.39 % dry weight) and Ocimum basilicum (0.61 %) were analyzed by GC-MS. Seventy-three constituents representing 99.64 % of the chromatographic peak area were obtained in the O. basilicum var. thyrsiflora oil, whereas 80 constituents representing 91.11 % observed in the essential oil of O. basilicum were obtained. Methyl chavicol (81.82 %), ?-(E)-ocimene (2.93 %) and ?-(E)- bergamotene (2.45 %) were found to be the dominant constituents in O. basilicum var. thyrsiflora oil while O. basilicum contained predominantly linalool (43.78 %), eugenol (13.66 %) and 1,8-cineole (10.18 %). The clear separation of the volatiles in all samples, demonstrated by the application of GC?GC, resulted in significantly different fingerprints for the two types of basil. The O. basilicum oil showed strong antioxidant activity while the oil of O. basilicum var. thyrsiflora exhibited very low activity, which was attributed to the significant differences in linalool and eugenol contents in these essential oils.

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid–liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R² = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.

Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body’s immune system. The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3⁺, CD19⁺, and CD11b⁺, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12β cytokines, but suppressed IL-10 level. At the tumor sites, CD11b⁺ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the genes controlling cell survival and function. Phosphorylation of STAT1 was absent in WEHI-3. Instead, similar treatments significantly decreased pSTAT3 signaling that regulates transcription of genes controlling tumor growth and proliferation. Rice bran gramisterol possesses a promising anti-cancer effect against a tumor of white blood cells and induces the production of anti-cancer immune-related cytokines. Gramisterol induces cell cycle arrest and apoptosis via suppression of pSTAT3 signaling control of tumor cells’ growth and progression. Gramisterol increased IFN-γ production and prevented the dysfunctional immune system of leukemic mice by enhancing pSTAT1 transcription signal controlling proliferation and functions of hematopoietic cells in the spleen. Together with IFN-γ, gramisterol efficiently facilitates leukemic mice immune system modulation leading to improvement of the AML condition. Administration of RBDS containing gramisterol potentiates immune recovery of leukemic mice and extends their survival. This finding encourages the medicinal application of rice bran gramisterol as a palliative treatment or an alternative agent for future drug development against AML.