Youying Tu

Preparation, preliminary characterization, antioxidant, hepatoprotective and antitumor activities of polysaccharides from the flower of tea plant (Camellia sinensis)

In the present study, the crude polysaccharides from the flowers of tea plant (Camellia sinensis) (TFPS) were prepared with hot water and further fractionated on a DEAE-52 cellulose chromatography to afford three purified fractions of TFPS-1, TFPS-2 and TFPS-3. Then, their preliminary structures, antioxidant and antitumor activities in vitro and hepatoprotective activity in vivo were investigated. Compared with TFPS-2 and TFPS-3, TFPS-1 had relative higher content of sulfate and relative complicated monosaccharide composition. In addition, TFPS-1 and TFPS-3 showed relative stronger antioxidant activity and inhibitory activity on the growth of human gastric cancer BGC-823 cells. For hepatoprotective activity in vivo, we demonstrated that crude TFPS significantly prevented the increase of serum alanine aminotransferase and aspartate aminotransferase levels, reduced the formation of malondialdehyde and enhanced the activities of superoxide dismutase and glutathione peroxidase in carbon tetrachloride-induced liver injury mice. The results suggested that TFPS should be a potent natural polymer with antioxidant, hepatoprotective and antitumor activities.

Chaetoglobosin K induces apoptosis and G2 cell cycle arrest through p53-dependent pathway in cisplatin-resistant ovarian cancer cells.

Adverse side effects and acquired resistance to conventional platinum based chemotherapy have become major impediments in ovarian cancer treatment, and drive the development of more selective anticancer drugs. Chaetoglobosin K (ChK) was shown to have a more potent growth inhibitory effect than cisplatin on two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and was less cytotoxic to a normal ovarian cell line, IOSE-364, than to the cancer cell lines. Hoechst 33342 staining and Flow cytometry analysis indicated that ChK induced preferential apoptosis and G2 cell cycle arrest in both ovarian cancer cells with respect to the normal ovarian cells. ChK induced apoptosis through a p53-dependent caspase-8 activation extrinsic pathway, and caused G2 cell cycle arrest via cyclin B1 by increasing p53 expression and p38 phosphorylation in OVCAR-3 and A2780/CP70 cells. DR5 and p21 might play an important role in determining the sensitivity of normal and malignant ovarian cells to ChK. Based on these results, ChK would be a potential compound for treating platinum-resistant ovarian cancer.

Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology

Theaflavins were synthesized from tea polyphenols extracted from green tea using an immobilized polyphenol oxidase system. To optimize the production of theaflavins, response surface methodology was applied to determine the effects of five critical variables and their mutual interactions on theaflavin biosynthesis at five levels. A total of 52 individual experiments were performed and a statistical model predicted that the highest theaflavin concentration was 0.766 mg ml−1 at optimized conditions. Using these optimal parameters under experimental conditions in three independent replicates, the average value of the biosynthesized theaflavin concentration reached 0.75 ± 0.017 mg ml−1 and matched the value predicted by the model

Isolation and identification of spermidine derivatives in tea (Camellia sinensis) flowers and their distribution in floral organs

BACKGROUND Recently, tea (Camellia sinensis) flowers have attracted increasing interest because of their content of bioactive compounds such as catechins. The aim of this study was to investigate the occurrence of some characteristic compounds in tea flowers. RESULTS A principal component analysis of metabolites using ultra-performance liquid chromatography/time-of-flight mass spectrometry showed differences in metabolite profile between flowers and leaves of C. sinensis var. Yabukita. Four spermidine derivatives were isolated from tea flowers. One of them was determined as N(1) ,N(5) ,N(10) -tricoumaroyl spermidine based on NMR, MS and UV data. The other three were identified as feruoyl dicoumaroyl spermidine, coumaroyl diferuoyl spermidine and triferuoyl spermidine based on MS(n) data. Tricoumaroyl spermidine as the major spermidine conjugate was not detected in tea leaves. Furthermore, it decreased during floral development and mainly occurred in anthers. CONCLUSION This study has provided the first evidence that spermidine-phenolic acid conjugates occur in tea flowers in considerable amounts. Their presence should prompt a reconsideration of the ecological role of tea flowers. From an economic point of view, tea flowers might be suitable as a raw material in the healthcare food and pharmaceutical industries.

ISOLATION AND PURIFICATION OF FOUR INDIVIDUAL THEAFLAVINS USING SEMI-PREPARATIVE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3′-gallate (TF2B), and theaflavin-3,3′-digallate (TF3) are the major theaflavins, which mostly contribute to the quality and bioactivity of black tea. In this study, a rapid isolation and purification of the four major individual theaflavins from crude theaflavins mixture was established. The crude theaflavins mixture was prepared by enzymatic oxidation of tea polyphenols using immobilized polyphenol oxidase and then fractionated using a Mitsubishi SP-207 resin chromatography with an elution gradient of 20%, 30%, 40%, 50%, and 70% aqueous ethanol to obtain a mixture of theaflavins with 80% purity (TF80). The TF80 was further purified using a semi-preparative high performance liquid chromatography (HPLC) equipped with a C18 column using isocratic elution with water/acetonitrile/glacial acetic acid (73.5:26:0.5, v/v/v) at a flow rate of 5 mL/min as optimized operating conditions. The purity of the isolated individual theaflavins were 92.48% for TF2A, 90.05% for TF2B, 92.40% for TF3, and 73.02% for TF1, respectively.

Evaluation of the antioxidant effects of four main theaflavin derivatives through chemiluminescence and DNA damage analyses

Theaflavins (TFs) are the dimers of a couple of epimerized catechins, which are specially formed during black tea fermentation. To explore the differences among four main TF derivatives (theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3′-gallate (TF2B), and theaflavin-3,3′-digallate (TF3)) in scavenging reactive oxygen species (ROS) in vitro, their properties of inhibiting superoxide, singlet oxygen, hydrogen peroxide, and the hydroxyl radical, and their effects on hydroxyl radical-induced DNA oxidative damage were systematically analyzed in the present study. The results show that, compared with (−)-epigallocatechin gallate (EGCG), TF derivatives were good antioxidants for scavenging ROS and preventing the hydroxyl radical-induced DNA damage in vitro. TF3 was the most positive in scavenging hydrogen peroxide and hydroxyl radical, and TF1 suppressed superoxide. Positive antioxidant capacities of TF2B on singlet oxygen, hydrogen peroxide, hydroxyl radical, and the hydroxyl radical-induced DNA damage in vitro were found. The differences between the antioxidant capacities of four main TF derivatives in relation to their chemical structures were also discussed. We suggest that these activity differences among TF derivatives would be beneficial to scavenge different ROS with therapeutic potential.

The changes of catechins during the fermentation of green tea.

The dynamics of tea catechins and organic acids in fermented fluid and yeast cells were studied. The concentration of eight kinds of catechins in solution decreased by 29.6–47.6%, respectively; some catechins were absorbed and accumulated by yeast cells, but the amount in the cells was very low during the fermentation process. The investigation of the catechins resolved in four citrate buffers with a pH range of 2.6–5.6 over 18 h showed that most catechins were stable in buffer solutions of pH 4.6 and 5.6, while significant losses took place in solutions of pH 2.6 and 3.6. However, most catechins were released and recovered by adjusting the pH value to 5.6, which suggested that catechins in extremely acidic buffer solutions might reversibly combine each other or with other compounds.

Optimisation of supercritical carbon dioxide extraction of essential oil of flowers of tea (Camellia sinensis L.) plants and its antioxidative activity

BACKGROUND To extract natural volatile compounds from tea (Camellia sinensis) flowers without thermal degradation and residue of organic solvents, supercritical fluid extraction (SFE) using carbon dioxide was employed to prepare essential oil of tea flowers in the present study. Four important parameters--pressure, temperature, static extraction time, and dynamic extraction time--were selected as independent variables in the SFE. RESULTS The optimum extraction conditions were the pressure of 30 MPa, temperature of 50°C, static time of 10 min, and dynamic time of 90 min. Based on gas chromatography-mass spectrometry analysis, 59 compounds, including alkanes (45.4%), esters (10.5%), ketones (7.1%), aldehydes (3.7%), terpenes (3.7%), acids (2.1%), alcohols (1.6%), ethers (1.3%) and others (10.3%) were identified in the essential oil of tea flowers. Moreover, the essential oil of tea flowers showed relatively stronger DPPH radical scavenging activity than essential oils of geranium and peppermint, although its antioxidative activity was weaker than those of essential oil of clove, ascorbic acid, tert-butylhydroquinone, and butylated hydroxyanisole. CONCLUSION Essential oil of tea flowers using SFE contained many types of volatile compounds and showed considerable DPPH scavenging activity. The information will contribute to the future application of tea flowers as raw materials in health-care food and food flavour industries.

Anti-inflammatory activity of total flavonoids from seeds of Camellia oleifera Abel.

Inflammation is the primary response to infection or injury that functions to clear the injurious material or agent and promote tissue repair. However, when inflammation persists, such as chronic inflammation, it can cause tissue damage and loss of function. Persistent inflammation is closely associated with many chronic diseases, such as cancer, arthritis, osteoporosis, asthma, Alzheimer’s disease, obesity, diabetes, and cardiovascular disease [1]. Numerous molecules such as cytokines, prostaglandins, and nitric oxide (NO) are involved in the induction and maintenance of the inflammatory response. Inhibition and/or down-regulation of these pro-inflammatory molecules may exert anti-inflammatory effects. In conventional therapy, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs are used to treat acute inflammation. However, they fail to cure chronic inflammatory diseases, such as rheumatoid arthritis and osteoarthritis. Furthermore, these compounds have several undesired side effects. Recently, anti-inflammatory activity of natural bioactive compounds is attracting growing interest because these compounds may offer a safer and effective treatment for inflammation, especially for long-term use [2]. Camellia oleifera Abel. belongs to the Camellia genus in the Theaceae family, which is widely distributed in the central and southern China. Its seeds have been used as oil material in China for more than 1000 years. Camellia seed oil is not only used as cooking oil, but also traditionally applied as a medicine for stomach ache and burning injury in China. Pharmacological studies indicated that seeds of C. oleifera contain various bioactive substances including unsaturated fatty acids, flavonoids, saponins, polysaccharides, and proteins, and possess many bioactivities such as antioxidation, antibacterial, anticancer, hepatoprotection, and anti-inflammation [3]. Flavonoids are members of a class of natural compounds widely distributed in the plant kingdom, and possess many bioactivities including antioxidation, antibacterial, antiviral, and protective effects from many diseases such as cancer, cardiovascular, and inflammation [4]. Kaempferol and several kaempferol glycosides have been isolated from the seeds of C. oleifera, and kaemferol-3-O-[2-O-b-D-glucopyranosyl-6O-L-rhamnopyranosyl]-b-D-glucopyranoside and kaemferol3-O-[2-O-b-D-xylopyranosyl-6-O-a-L-rhamnopyranosyl]-b-Dglucopyranoside were identified as the two main flavonoids [5–10]. Various flavonoids such as genistein, quercetin, daidzein, flavone, isorhamnetin, naringenin, and pelargonidin from fruits, herbs, and spices have been found to possess important activity on the inflammatory process in vitro and in vivo [11]. However, little is known about the antiinflammatory effects of flavonoids from the C. oleifera seeds. In this study, total flavonoids were prepared from the 80% ethanol extract of C. oleifera seeds by semi-preparative HPLC (Waters, Milford, USA), and the composition was characterized by UPLC–UV–MS (Waters) analysis. The antiinflammatory activity of total flavonoids was evaluated by NO inhibitory assay in RAW 264.7 cells. Moreover, the effects of total flavonoids on the mRNA and protein expression levels of pro-inflammatory enzymes and cytokines including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-a (TNF-a), and macrophage inflammatory protein-1a (MIP-1a) were determined by quantitative real-time-polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Four compounds (1–4) were detected in total flavonoid fraction of C. oleifera seeds by UPLC–UV–MS, and numbered corresponding to their order of elution (Supplementary Fig. S1). Quasi-molecular ions [M2H] for compounds 1–4 were observed at 755.19, 739.12, 725.19, and 593.17 in negative ion mode, respectively (Supplementary Fig. S2). The fragment ions at m/z 285 suggested that the aglycone was kaempferol. These results were consistent with the previous reports [5–8,10], and the four compounds were identified as kaemferol-3-O-[2-O-b-D-glucopyranosyl-6-O-L-rhamnopyranosyl]-b-D-glucopyranoside (compound 1), kaempferol 3-Ob-D-glucopyranosyl-(1!4)a-L-rhamnopyranosyl-7-O-a-Lrhamnopyranoside (compound 2), kaemferol-3-O-[2-O-bActa Biochim Biophys Sin 2014, xx: 1–3 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu071.

Safety evaluation of tea (Camellia sinensis (L.) O. Kuntze) flower extract: Assessment of mutagenicity, and acute and subchronic toxicity in rats

ETHNOPHARMACOLOGICAL RELEVANCE Tea (Camellia sinensis (L.) O. Kuntze, Theaceae) flowers possess many physiological functions and have been used in traditional medicines for deodorization, skin care, cough suppressant and expectorant in China. However, there is a little information about its possible toxicity. AIM OF THE STUDY The present investigation was carried out to evaluate the safety of tea flower extract by mutagenicity and acute and subchronic toxicity studies. MATERIALS AND METHODS Mutagenicity of tea flower extract was evaluated by the Ames test in Salmonella typhimurium strains TA97, TA98, TA100 and TA102 at concentrations of 0.008, 0.04, 0.2, 1.0, 5.0 mg/plate. In the acute toxicity study, Sprague-Dawley rats were administered a single dose of 12.0 g/kg of body weight by gavage, and were monitored for 14 days. In the subchronic toxicity study, tea flower extract was administered by gavage at doses of 1.0, 2.0 and 4.0 g/kg body weight daily for 13 weeks to Sprague-Dawley rats. RESULTS In the Ames test, there was no mutagenic effect of tea flower extract (up to 5.0 mg/plate) towards four tested strains (TA97, TA98, TA100, TA102), with or without metabolic activation (S9). In the acute toxicity study, all animals gained weight and appeared active and normal, so the LD(50) value must be >12.0 g/kg body weight. In the subchronic toxicity study, no dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or pathologic lesions were observed. CONCLUSION These results indicate that tea flower extract does not possess mutagenic potential, and that both acute and subchronic toxicity towards animals is very low. A no-observed adverse-effect level (NOAEL) for tea flower extract is 4.0 g/kg bw/day for rats under the conditions of this study.