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Tea (Camellia sinensis) is a beverage widely drunk across the world, and its extracts have been used as medicinal and dietary supplements in many countries such as China, Japan and the US. Tea contains a variety of bioactive compounds including tea polyphenols (TPP), theanine and tea polysaccharides (TPS), which contribute to the health benefits of tea. A polysaccharide is a high molecular weight (MW) polymer, consisting of at least ten monosaccharides mutually joined by glycosidic linkages. The glycosyl moiety of the hemiacetal or hemiketal, together with the hydroxyl group of another sugar unit, formed the glycosidic linkages. TPS is a group of heteropolysaccharides extracted from leaves, flowers and seed peels of the tea plant. Great advances have been made in chemical and bioactive studies of TPP or catechins and related tea products over the last few decades. However, TPS has received rare attention. There have been studies showing that TPS has many health benefits including antioxidant, anti-aging, antitumor, antibacterial and anti-skin-aging properties, as well as the ability to inhibit diabetes, improve immunity, and alleviate hepatotoxicity.
Polysaccharides in Tea
TPS is a nonstarch protein-bound acidic polysaccharide, which contains 44.2% neutral sugar, 43.1% uronic acid and 3.5% protein. The carbohydrate composition of TPS includes glucose, galactose, arabinose, rhamnose, xylose, galacturonic acid, mannose, ribose and glucuronic acid. TPP is a group of abundant bioactive components in tea, and crude TPS usually contains partial TPP. The carbohydrate, protein and polyphenols are conjugated with each other in the crude TPS. The composition of crude TPS varies with processing methods including extraction and drying.
Bioavailability and Toxicity of TPS
TPS is generally recognized as a safe and non-toxic food additive. An in vitro test on dendritic cells revealed that the cell viability showed no significant difference between TPS-treated cells at concentrations of 0.2–200 μg/mL and media-tread cells, during which TPS did not induce any apoptosis in DCs, showing TPS can be used for a long period without cytotoxicity. An in vivo test by oral administration of TPS (5.0 g/kg BW) in mice showed that TPS had no toxicity to the liver, kidney, heart, thymus, or spleen of the tested mice and none of the mice died throughout the 15 days of experiment. There was no significant difference in the thymus index, spleen index, and liver index of the mice between the test and control groups. Based on the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and OECD (Organization for Economic Co-operation and Development) Test Guideline 420 (fixed dose procedure), TPS was classified as GHS Category 5. Therefore, TPS can be classified as a very low toxicity substance which can thus be used for dietary supplements and as an additive in food processing.
Alleviating Oxidative Stress
TPS alleviates oxidative stress through direct scavenging of free radical species and improving activities of antioxidase enzymes. TPS is a group of heteropolysaccharides bound with proteins which can alleviate oxidative stress. The antioxidant activities of TPS vary with free radical species and molecular size of TPS. The higher the uronic acid level in TPS, the stronger its ability to scavenge hydroxyl and superoxide radicals. An in vivo test in gastric cancer mice showed that TPS fraction with small MW showed stronger promoting effect on stomach antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). When exhausting training mice were orally administrated by TPS (daily dosage 100–300 mg/kg BW) for 30 days, SOD, CAT, GSH-Px activities in blood, liver and heart were significantly increased, whereas malondialdehyde (MDA) level in plasma, liver and heart were reduced, compared to control mice.
Antitumor
Many in vitro tests revealed that TPS showed antitumor potential. TSPS significantly inhibited the growth of human immortalized myelogenous leukemia cell K562 at a concentration of 50 μg/mL, with an inhibition ratio 38.44% ± 2.22%. When the TSPS was further separated into NTPS, ATPS-1 and ATPS-2, they all showed inhibitory effects on K562 cells in a dose-dependent manner, with inhibition ratios of 30.13% ± 3.54% for NTPS, 36.61% ± 2.75% for ATPS-1 and 32.33% ± 2.53% for ATPS-2 at 400 μg/mL, respectively. TPS extracted from Se-enriched “Ziyang” green tea significantly inhibited the proliferation of human osteosarcoma U-2 OS cancer cells in a dose-dependent manner at 25–200 μg/mL. TFPS with a high level of sulfate and complicated monosaccharide composition showed strong inhibitory activity on growth of human gastric cancer BGC-823 cells. After 72 h in vitro incubation, the inhibition rates of TFPS-1 with 2.63% sulfuric radical and TFPS-3 with 1.76% sulfuric radical at a concentration of 200 μg/mL were 82.60% and 80.73%, respectively, which is significantly higher than those of crude TFPS with 1.45% sulfuric radical and TFPS-2 with 0.84% sulfuric radical. An in vitro test showed that TPS (25, 50, 100 and 200 μg/mL) could significantly inhibit the proliferation of human osteosarcoma U-2 OS cells in a concentration-dependent fashion. These experiments suggest that TPS will be a potential candidate for natural antitumor drugs.
The antitumor activity of TPS was also confirmed by in vivo tests. An in vivo test on U-2 OS cancer xenograft model BALB/c athymic mice showed that oral administration at three daily doses of 100, 200 and 400 mg/kg BW for 28 days resulted in obvious tumor regression as compared to model control. In addition, body weights of the mice in control or TPS-treated groups did not differ significantly and no mice died during the experiment, suggesting TPS has cancer-preventive and cancer-therapeutic benefit for human osteosarcoma. Oral administration of TFPS at daily dosages of 75, 150 and 300 mg/kg for 10 days inhibited the growth of transplanted sarcoma 180 tumor (S180) on S180-bearing mice, prolonged the mice survival days, promoted the plasma interleukin-2 and interferon-γ levels, and improved the T-lymphocyte subsets CD4+ and CD4+/CD8+ percentages. In addition, TFPS was found to increase the delayed-type hypersensitivity response and macrophage phagocytosis significantly, indicating TFPS enhanced the host defense response to tumor due in part to the immunomodulatory activity. TPS could inhibit the growth of H22 transplantable hepatocarcinoma (HCC) tumor in mice. An in vivo test showed that TPS significantly inhibited the growth of H22 transplantable tumor in mice, remarkably decreased the spleen index and increased the thymus index compared with that of model group. Furthermore, TPS significantly improved the splenocyte proliferation induced by concanavalin A (ConA) or lipopolysaccharide (LPS), and notably enhanced the macrophage phagocytosis towards neutral red. A test on Wistar rats with H22 HCC cells confirmed that oral administration of TPS (100, 200 and 300 mg/kg BW, once a day for 40 consecutive days) inhibited tumor growth and decreased microvessel density in tumor tissue. The altered amount of serum white blood cells (WBC), interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) in HCC animals were dose-dependently increased, whereas activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were dose-dependently decreased in the TPS-treated animals. The suppressive effect of TPS on tumor growth is also considered to be related to its inhibiting expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in H22 tumor tissue.
Anti-Hyperglycemia
An in vivo test showed that TPS had an inhibitory effect on blood glucose (BG) increase and diabetes mellitus (DM). When seven-week-old C57BL/8 mice were injected with TPS with MW 107 kDa–110 kDa, the BG levels in normal mice and model mice with high BG were significantly decreased by 13.54% and 22.18%, respectively. Four-week oral administration of PTPS (40 mg/kg BW daily) could significantly lower the BG levels in alloxan-induced diabetic mice, accompanying improvement of activities of SOD and GSH-Px as well as MDA levels both in serum and liver. Oral administration of GTPS (200 and 400 mg/kg BW daily) for six consecutive days could also suppress BG increase in alloxan-induced mice.
DM is an endocrine disorder caused by inherited and/or acquired deficiency in the amount of insulin from the pancreas, or by the defects in insulin action. Glucokinase is the first enzyme in glycolysis and glycogenesis; it is also a key enzyme in diabetes management, thereby serving as a signal to both the b-cells and the liver that glucose levels in the blood are high. Glucokinase plays a role in promoting insulin secretion and reducing glucose production by the liver. Glucokinase facilitates phosphorylation of glucose to glucose-6-phosphate, which is regulated by insulin. Glucokinase influences glucose uptake by liver. Increase in glucokinase activity is beneficial to alleviating the symptoms of diabetes. TPS had elements related to reducing blood sugar (ERBS), with inhibitory percentages ranging from 0.03% to 9.57%. The bioactivities of OTPS were proportional to its contents of protein and uronic acid. The protein and uronic acid in TPS had an inhibitory effect on α-glucosidase activities and had potential for prevention of type 2 diabetes (T2D). Pu-erh tea extracts containing TPS had beneficial effects on glucose homeostasis in T2D and in amendment of insulin resistance. TPS improved impaired glucose tolerance and ameliorated retarded insulin response at 60 and 120 min in diabetic db/db mice. An ATPS purified by gel filtration chromatography, which contained 8% galacturonic acid and had MW 60 kDa, showed a significantly stimulating effect on glucokinase activity, resulting in BG reduction and suppression of MD.
Dysfunction of the vascular endothelium contributes to the etiology of diabetic micro- and macro-angiopathy. Excessive increase in intra cellular glucose induces serious loss of vascular endothelial cells and accelerates the occurrence of atherosclerosis in DM patients. Fractions 1–3 of GTPS obtained by extracting low-grade green tea in hot water and precipitating in ethanol, and finally fractionating on DEAE-cellulose DE-52 column showed protective effects on human umbilical vein endothelial (HUVE) cells. Exposure of HUVE cells to high glucose (33 mM) for 12 h significantly decreased cell viability relative to normal glucose control. As compared with the cell injury group, fractions 1–3 of GTPS at three dose levels (50, 150 and 300 μg/mL) showed remarkably protective effects on HUVE cells against impairments induced by high glucose in a dose-dependent manner. The inhibitory effects of GTPS on high glucose-mediated HUVE cell loss were, at least in part, correlated with their potential scavenging potency of reactive oxygen species (ROS).
Type 1 diabetes (T1D) is an autoimmune disorder induced by dysregulation of the immune system. During development of functional regulatory T cells (Treg), interleukin 2 (IL-2) is a necessary second signal after T cell antigen receptor (TCR), signaling that it upregulates Tregs CD25 and Foxp3. IL-2 may not only cause proliferation of Tregs, but also compensate for a genetic defect associated with T1D. IL-1 has a major role in inflammation. The blockade of IL-1 activity (especially IL-1β) is a standard therapy for patients with autoimmune diseases. TPS treatment promoted production of IL-2 in spleen cells but suppressed production of IL-1 in adjuvant arthritis rats in vivo. The hypoglycemic mechanism of TPS is also considered to be involved in its regulation of the PI3K/Akt signal pathway because TPS was found to upregulate the expressions of PI3Kp85/p-Akt/GLUT4 in T2D mice. When diabetic mice were orally gavaged with TPS dissolved in NS at the doses of 200, 400 and 800 mg/kg BW per day for 28 days, the expression of PI3Kp85, p-Akt and GLUT4 increased in a dose-dependent manner, accompanying a dose-dependent decrease in serum glucose level.
Anti-glutamic acid decarboxylase (anti-GAD) antibody is considered to be an important marker for T1D. Daily oral administration of 150 mg/kg green tea water-soluble TPS and alkali-soluble TPS suppressed spontaneous DM in non-obese diabetic (NOD) mice by decreasing the levels of anti-GAD antibody and blood glucose. The hypoglycemic activity of TPS can be further improved by molecular modification such as sulfation. An in vivo test on alloxan-induced diabetic mice showed that BG levels of a sulfated NTPS group and sulfated ATPS group after administration for 7 h were 8.31 mmol/L and 8.18 mmol/L, being significantly lower than those of non-sulfated NTPS and ATPS, respectively.
Improving Immunity
TPS can improve immunity by enhancing the activity of immunocytes such as splenocytes. Splenocytes consist of a variety of cell populations such as lymphocytes, DCs and macrophages, which have different immune functions. TPS significantly improved the splenocyte proliferation induced by ConA or LPS, and notably enhanced the macrophage phagocytosis towards neutral red. TPS promoted both phenotypic and functional maturation of murine bone marrow-derived DCs, achieving potentiation of immune responses to alleviate the diseases. TPS promoted the phagocytic activity of monocyte-macrophage system, resulting in enhancement of self-protection activity and increasing phagocytosis through toll-like receptor 7.
Cytokines form a group of proteins with small MW released by cells that have specific effects on the interactions and communications between cells, or on the behavior of cells. The cytokines include interleukins (IL), lymphokines and cell signal molecules, such as tumor necrosis factor (TNF) and the interferons, which trigger inflammation and respond to infections. An in vivo test on Kunming mice showed that oral administration of TPS could significantly decrease the level of pro-inflammatory cytokines such as TNF-α, but could increase the level of anti-inflammatory cytokines such as serum immunoglobulin A (IgA), IgG, IgM, IL-2, IL-4, IL-10 as well as IL-6 which plays an important role in T cell activation. Oral administration of TFPS could also improve the percentages of T-lymphocyte subsets CD4+ and CD4+/CD8+. The effect of TPS on immune stimulation was superior to that of TPP to some extent. Therefore, TPS can be used as an immunopotentiator.
However, the immunological activities of TPS were differentiated between various sources. The TPS from immature leaves had higher immunostimulating activity than that from mature leaves and its activities depend on the content of strictinin in the leaf extract. A mixture of TPS without polyphenols and catechin did not increase the immunostimulating activity. Crude polysaccharide from tea leaf containing a lot of catechins is a potential immunostimulator, and strictinin might promote the formation of a catechin-polysaccharide complex, indicating that the catechin-polysaccharide complex is a very important molecule in the immunomodulating activity of tea extracts. ATPS showed stronger immunological activity than NTPS at concentrations 0.5–400 μg/mL. The detail mechanisms of immunological activity of TPS have not been clear.
Anti-Hepatotoxicity
TPS plays a role in anti-hepatotoxicity through ameliorating hepatic oxidative injury and improving metabolic syndrome. Oral administration of TFPS for 28 consecutive days protected liver from lipid peroxidation induced by bromobenzene in mice through increasing SOD activity, resulting in reduction of MDA in a dose-dependent manner. In vivo test on exhausting training mice showed that oral administration of TPS (100, 200 and 300 mg/kg BW) for 30 days increased the activities of SOD, catalase (CAT), GHS-Px and reduced MDA level in plasma, liver and heart.
Carbon tetrachloride (CCl4) induced hepatotoxicity, accompanying an increase in serum alanine transaminase (ALT), aspartate transaminase (AST), triglycerides (TG), cholesterol (TC), hepatic MDA and 8-iso-PGF2α (8-iso-prostaglandin F2 alpha). Administration of GTPS or BTPS (200, 400 and 800 mg/kg BW) in mice ahead of CCl4 injection could antagonize the CCl4-induced increases in levels of ALT, AST, TG, TC, hepatic MDA and 8-iso-PGF2α. The TPS-treated mice displayed a better profile of hepatosomatic index and improved GSH-Px and SOD activities. These protective effects can be attributed to TPS enhancing the effects on enzymatic and non-enzymatic antioxidants and restraining lipid peroxidation in liver tissue.
Nitric oxide (NO) is a free radical which can be produced by nitric oxide synthase (NOS) in the body. There are three NOS isoforms identified in the body, i.e., endothelial nitric oxide synthase (eNOS), neural nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). The iNOS is inducible in response to various stimuli, such as LPS which can activate Toll-like receptor 4 (TLR4) signal pathway. Tests showed that PTPS suppressed the increase in level of LPS-induced NO in SD rats by inhibiting iNOS expression through reducing TLR4 signaling. The SD rats fed with PTPS extracted from pu-erh tea at a daily dose 50 mg/kg BW for four weeks had less expression of iNOS mRNA. The relative mRNA unit of PTPS groups was 48% of that in control group (water + LPS).
Anti-Skin Aging
An in vitro test on senescent human diploid fibroblast (HDF) showed that PTPS promoted proliferation of HDF significantly and the anti-aging effect of TPS on HDF was even stronger than vitamin C and TPP. The abilities of TPS and TPP to protect the skin were assessed from four aspects, i.e., moisture absorption and retention, sunscreen, promoting the proliferation of fibroblast cells, and tyrosinase inhibitory ability. Purified TPS had better moisture absorption and retention abilities than TPP. TPP protected skin against the sun’s ultraviolet (UV) radiation, enhanced proliferation of fibroblast cells and had an inhibitory effect on tyrosinase, whereas purified TPS hardly protected the skin from UV rays and showed weak ability to inhibit tyrosinase. TPS and TPP had complementary advantages and they should be appropriately combined to achieve higher performance when applied as active components in cosmetics. A six-month double-blind, placebo controlled, randomized study on healthy post-menopausal females showed that a dietary supplement containing white tea extract and fish protein polysaccharides provided improved condition, structure and firmness of the skin in post-menopausal women, showing improvement of forehead, periocular and perioral wrinkles, mottled pigmentation, laxity, sagging, under eye dark circles and overall appearance.